●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were tre...●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.展开更多
AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12...AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors,yielding an HCEP cell line which was its growth performance,chromosome morphology,tumorigenicity and expression of marker proteins analyzed.In addition,the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light,electron and slit-lamp microscopies.RESULTS: HCEP cells proliferated to confluence in 3 weeks,which have been subcultured to passage 160.A continuous untransfected HCEP cell line,designated as utHCEPC01,was established with a population doubling time of 45.42 hours as was determined at passage 100.The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3.They,with no tumorigenicity,formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture,maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs.The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original.CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study.The cells maintained expression of marker proteins.The cell line was biocompatible with dAM.It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP,promising for the treatment of diseases caused by corneal epithelial disorders.展开更多
A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferati...A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal展开更多
AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.METHODS: HCE cells cultured in vitro with AF antige...AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.METHODS: HCE cells cultured in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-κB and relative downstream cytokines such as TNF-α, IL-1β, IL-8 and IL-10 in supernatant fluid were measured by ELISA.RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-α and IL-1β reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner.CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-κB pathway. SP-D possibly mediates the recognition to AF mycelium.展开更多
Corneal diseases are a major cause of blindness in the world. Although great progress has been achieved in the treatment of corneal diseases, wound healing after severe corneal damage and immunosuppressive therapy aft...Corneal diseases are a major cause of blindness in the world. Although great progress has been achieved in the treatment of corneal diseases, wound healing after severe corneal damage and immunosuppressive therapy after corneal transplantation remain prob-lematic. Mesenchymal stem cells(MSCs) derived from bone marrow or other adult tissues can differentiate into various types of mesenchymal lineages, such as osteocytes, adipocytes, and chondrocytes, both in vivo and in vitro. These cells can further differentiate into specific cell types under specific conditions. MSCs migrate to injury sites and promote wound healing by secreting anti-inflammatory and growth factors. In ad-dition, MSCs interact with innate and acquired immune cells and modulate the immune response through their powerful paracrine function. Over the last decade, MSCs have drawn considerable attention because of their beneficial properties and promising therapeutic prospective. Furthermore, MSCs have been applied to various studies related to wound healing, autoim-mune diseases, and organ transplantation. This review discusses the potential functions of MSCs in protecting corneal tissue and their possible mechanisms in corneal wound healing and corneal transplantation.展开更多
AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group...AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.展开更多
AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were rec...AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line,and scaffold carriers of denuded amniotic membrane(dAM) in air-liquid interface culture for 3,5,7 and 9 days,respectively.The specimens were examined with hematoxylin-eosin(HE) staining of paraffin-section,immunocytochemical staining,scanning and transmission electron microscopy.· RESULTS:During in vitro reconstruction of TE-HCEP,HCEP cells formed a 3-4,6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3,5 and 7 days,respectively.But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9.And the cells maintained positive expression of marker proteins(keratin 3 and keratin 12),cell-junction proteins(zonula occludens-1,E-cadherin,connexin 43 and integrin β1) and membrane transport protein of Na+-K+ ATPase.The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5.· CONCLUSION:The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo.The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells,including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation.The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.展开更多
AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. M...AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.展开更多
AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHOD...AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P <0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P <0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.展开更多
AIM:To explore the significance of corneal epithelial thickness analysis in diagnosing early keratoconus.METHODS:There were 26 clinical keratoconus,21 forme fruste keratoconus,40 high corneal astigmatism(ΔK)and 40 l...AIM:To explore the significance of corneal epithelial thickness analysis in diagnosing early keratoconus.METHODS:There were 26 clinical keratoconus,21 forme fruste keratoconus,40 high corneal astigmatism(ΔK)and 40 low ΔK eyes involved in the study.Fourierdomain optical coherence tomography was used to measure the corneal epithelial thickness of four groups.The morphological features of topographic map and the thickness of corneal epithelial thinnest point were analyzed.The distribution curve of corneal epithelial thickness at 45°,90°,and 135° axial directions that are through the pupil center was also analyzed.One-way ANOVA was performed to compare the data.RESULTS:The topographic map of forme fruste keratoconus corneal epithelial thickness was uniformity shape;crater shape existed only in clinical keratoconus group;and central island shape mainly existed in highΔK group.The thinnest point of corneal epithelial thickness of forme fruste keratoconus group was significantly lower than that of low ΔK group(P=0.022).The thickness of corneal epithelium in the forme fruste keratoconus at 90°was thinner than that in the low astigmatism group at -1,and -2 mm points(P_(-1mm)=0.015,P_(-2mm)=0.036).CONCLUSION:The analysis of the thinnest point in forme fruste keratoconus corneal epithelium appears earlier than corneal epithelial remodeling.The topographic map of corneal epithelium in high ΔK eyes appears in central island shape,and can be used for the differential diagnosis of early keratoconus.展开更多
AIM: To evaluate the effects of epidermal growth factor(EGF) on transforming growth factor-beta1(TGF-β1)-induced epithelial-mesenchymal transition(EMT) in human corneal epithelial cells(HCECs). METHODS: HCECs were cu...AIM: To evaluate the effects of epidermal growth factor(EGF) on transforming growth factor-beta1(TGF-β1)-induced epithelial-mesenchymal transition(EMT) in human corneal epithelial cells(HCECs). METHODS: HCECs were cultured and treated with TGF-β1 for establishing the model of EMT in vitro. Biological effect of EGF on TGF-β1-induced EMT was evaluated. Proteins and m RNAs expression changes of E-cadherin, N-cadherin and Fibronectin(EMT-relative markers) after TGF-β1 or TGF-β1 combined EGF treatment were detected by Western blot and RT-PCR, respectively. Viability and migration of HCECs were measured by CCK-8, transwell cell migration assay and cell scratch wound healing assay. Activation of Smad2, ERK, p38, JNK and Akt signaling pathways were evaluated by Western blot. Inhibitors of relevant signaling pathways were added to the HCECs to explore the key signal mechanism.RESULTS: With treatment of TGF-β1 only, three EMTrelative proteins and m RNA expression showed that EMT up-regulated in a concentration-dependent and time-dependent manner, with significantly decreasing cell viability(TGF-β1≥5 ng/m L, P<0.05) and increasing cell migration(TGF-β1≥5 ng/m L, P<0.01). The phosphorylation of Smad2 and p38 was a key process of TGF-β1-induced EMT. Meanwhile, EMT-relative proteins and m RNA expression showed that EGF inhibited TGF-β1-indued EMT, with significantly increasing cell viability(EGF≥10 ng/m L, P<0.01). It was noteworthy that EGF significantly enhanced cell migration although EMT was inhibited(EGF≥10 ng/m L, P<0.01), and the blockage of p38(by SB202190, a p38 inhibitor) was a potential mechanism of this phenomenon. CONCLUSION: EGF inhibits TGF-β1-induced EMT via suppressive p38, and promotes cells proliferation and migration in a non-EMT process by inhibiting p38 pathway.展开更多
AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs wer...AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.展开更多
AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.ME...AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.METHODS: HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074(an inhibitor of Raf-1) group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1(Dectin-1)] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS: In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION: Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.展开更多
AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized hum...AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells(S-ihCECs).METHODS:hAECs were isolated by enzyme digestion,and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR.Recovered and cultured S-ihCECs,immunocytochemistry was used to detect the expression of CK3/12.The proliferation of S ihCECs handled by different concentrations of mitomycin was detected by CCK-8.The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8.After filtered out the optimal conditions,we collected S-ihCECs culture media for 5 days,then prepared conditioned medium to incubate hAECs,inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs.Quantitative real-time reverse transcription-polymerase chain reaction(QRT PCR) was carried out to evaluate the expression of Oct4,NANOG,PAX6,and CK12 in the differentiation period.Immunocytochemistry and western bloting were used to detect the expression of CK3/12.RESULTS:The culture media collected every 12h,from 20μg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs.In the period of differentiation,the morphology of differentiated hAECs was obviously different compared with the control group,and the distinctive CK3/12 for corneal epithelial cells was detected.CONCLUSION:This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment,using the culture media collected from S-ihCECs,and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.展开更多
AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM)in vitro.·METHODS:The scaffold was pr...AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM)in vitro.·METHODS:The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate(SDS)solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin(HE)staining and 4',6-diamidino-2-phenylindole(DAPI)staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·RESULTS:Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION:Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vivo.展开更多
In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cell...In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.展开更多
AIM:Toevaluatetheroleofumbilical cord blood serum(CBS) therapy in cases with persistent corneal epithelial defects(PED).METHODS:Sixteen eyes of 14 patients with PED who were resistant to conventional treatment were tr...AIM:Toevaluatetheroleofumbilical cord blood serum(CBS) therapy in cases with persistent corneal epithelial defects(PED).METHODS:Sixteen eyes of 14 patients with PED who were resistant to conventional treatment were treated with 20% umbilical cord serum eye drops. Patients were followed-up weekly until epithelization was complete.The collected data included the grade of corneal lesion(Grade I: epithelial defect +superficial vascularization,Grade II: epithelial defect +stromal edema, Grade III:corneal ulcer +stromal melting), the size of epithelial defect(pretreatment, 7th, 14 thand 21stdays of treatment),and follow-up time was evaluated retrospectively.RESULTS:The mean size of epithelial defect on two perpendicular axes was 5.2×4.6-mm2(range: 2.5-8 mm×2.2-9 mm2). Mean duration of treatment was 8.3 ±5wk.CBS therapy was effective in 12 eyes(75%) and ineffective in 4 eyes(25%). The epithelial defects in 4ineffective eyes were healed with amniotic membrane transplantation and tarsorrhaphy. The rate of complete healing was 12.5% by 7d, 25% by 14 d, and 75% by 21 d.The healing time was prolonged in Grade III eyes in comparison to eyes in Grade I or Grade II.CONCLUSION:The results of the current study indicated the safety effectiveness of CBS drops in the management of PED. The grade of disease seems have a role on the healing time.展开更多
Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered b...Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.展开更多
●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,50...●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,500,800,and 1000 ng/mL of netrin-1,respectively.The cells viability was detected by cell counting kit-8(CCK-8).The wound-healing assay was applied to assess the migration proficiency of HCE cells.Flow cytometry was used to analyze the cell-cycle distribution and apoptosis.In vivo,normal c57(6 wk)mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound.Mice corneas were inflicted no epithelium with a 3-mm wound displayed,but remained the limbal epithelium intact.A blunt scalpel blade was used to remove the corneal epithelian cells,followed by topical netrin-1 application(200 ng/mL),and the group treated by PBS as control.The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma.Mouse corneal inflammation and neovascularization were observed under slit lamp microscope.The apoptosis of corneal cells was determined by TUNEL staining.●RESLUTS:A concentration of 200 ng/mL netrin-1 enhanced 25%of the HCE viability.The relative migration rates were 76.3%and 100%in control and netrin-1 treated group after cultured 72 h.Treated with netrin-1(200 ng/mL)decreased the apoptosis of HCE cells,as well as decreased their percentage from 19.3%±0.57%to 12.7%±0.42%of the total.The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group.Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice.TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3%and 16.7%respectively.●CONCLUSION:Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration.Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells.These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.展开更多
<Abstract>Purpose:To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. Methods: The fresh rabbit cornea was cul...<Abstract>Purpose:To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. Methods: The fresh rabbit cornea was cultured to get the primary RCECs,and RCECs of passage 2 were used for the research. The cells were divided into experimental groups,the cells in which were incubated with different concentrations(18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. Results:MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs,and the inhibition rate was increasing along with the increase of the concentration of diclofenac sodium and the incubation time(P<0.05). Flow cytometer showed that after incubation with diclofenac sodium, the cells in G0/G1 phase were obviously increased, the apoptosis cusp and apoptotic rate were increased. Conclusion: Diclofenac sodium has obvious inhibitory effect on RCECs, which was dosage-dependent,and it may function by inducing cell apoptosis and ceasing cells cycles.展开更多
基金Supported by the National Natural Science Foundation of China(No.81770889)Zhuhai Science and Technology Program(No.ZH22036201210134PWC).
文摘●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.
基金Supported by National High Technology Research and Development Program ("863" Program) of China(No. 2006AA02A132)
文摘AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors,yielding an HCEP cell line which was its growth performance,chromosome morphology,tumorigenicity and expression of marker proteins analyzed.In addition,the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light,electron and slit-lamp microscopies.RESULTS: HCEP cells proliferated to confluence in 3 weeks,which have been subcultured to passage 160.A continuous untransfected HCEP cell line,designated as utHCEPC01,was established with a population doubling time of 45.42 hours as was determined at passage 100.The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3.They,with no tumorigenicity,formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture,maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs.The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original.CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study.The cells maintained expression of marker proteins.The cell line was biocompatible with dAM.It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP,promising for the treatment of diseases caused by corneal epithelial disorders.
基金Supported by Save Sight Society New Zealanduckland Medical Research Foundation
文摘A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal
基金National Natural Science Foundation of China(No.81170825)
文摘AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.METHODS: HCE cells cultured in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-κB and relative downstream cytokines such as TNF-α, IL-1β, IL-8 and IL-10 in supernatant fluid were measured by ELISA.RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-α and IL-1β reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner.CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-κB pathway. SP-D possibly mediates the recognition to AF mycelium.
文摘Corneal diseases are a major cause of blindness in the world. Although great progress has been achieved in the treatment of corneal diseases, wound healing after severe corneal damage and immunosuppressive therapy after corneal transplantation remain prob-lematic. Mesenchymal stem cells(MSCs) derived from bone marrow or other adult tissues can differentiate into various types of mesenchymal lineages, such as osteocytes, adipocytes, and chondrocytes, both in vivo and in vitro. These cells can further differentiate into specific cell types under specific conditions. MSCs migrate to injury sites and promote wound healing by secreting anti-inflammatory and growth factors. In ad-dition, MSCs interact with innate and acquired immune cells and modulate the immune response through their powerful paracrine function. Over the last decade, MSCs have drawn considerable attention because of their beneficial properties and promising therapeutic prospective. Furthermore, MSCs have been applied to various studies related to wound healing, autoim-mune diseases, and organ transplantation. This review discusses the potential functions of MSCs in protecting corneal tissue and their possible mechanisms in corneal wound healing and corneal transplantation.
基金Supported by National Natural Science Foundation of China(No.81170825No.81300730)
文摘AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.
基金Supported by National High Technology Research and Development Program("863" Program) of China(No.2006AA02A132)
文摘AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line,and scaffold carriers of denuded amniotic membrane(dAM) in air-liquid interface culture for 3,5,7 and 9 days,respectively.The specimens were examined with hematoxylin-eosin(HE) staining of paraffin-section,immunocytochemical staining,scanning and transmission electron microscopy.· RESULTS:During in vitro reconstruction of TE-HCEP,HCEP cells formed a 3-4,6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3,5 and 7 days,respectively.But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9.And the cells maintained positive expression of marker proteins(keratin 3 and keratin 12),cell-junction proteins(zonula occludens-1,E-cadherin,connexin 43 and integrin β1) and membrane transport protein of Na+-K+ ATPase.The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5.· CONCLUSION:The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo.The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells,including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation.The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.
基金Supported by the National Natural Science Foundation of China (No.81770927)the Natural Science Foundation of Hunan Province, China (No.2015JJ4093)the Science and Technology Project of Changsha, China (No. kq1701079)
文摘AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.
基金Supported by Guangdong Administration of Traditional Chinese Medicine (No.2007095)
文摘AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P <0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P <0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.
文摘AIM:To explore the significance of corneal epithelial thickness analysis in diagnosing early keratoconus.METHODS:There were 26 clinical keratoconus,21 forme fruste keratoconus,40 high corneal astigmatism(ΔK)and 40 low ΔK eyes involved in the study.Fourierdomain optical coherence tomography was used to measure the corneal epithelial thickness of four groups.The morphological features of topographic map and the thickness of corneal epithelial thinnest point were analyzed.The distribution curve of corneal epithelial thickness at 45°,90°,and 135° axial directions that are through the pupil center was also analyzed.One-way ANOVA was performed to compare the data.RESULTS:The topographic map of forme fruste keratoconus corneal epithelial thickness was uniformity shape;crater shape existed only in clinical keratoconus group;and central island shape mainly existed in highΔK group.The thinnest point of corneal epithelial thickness of forme fruste keratoconus group was significantly lower than that of low ΔK group(P=0.022).The thickness of corneal epithelium in the forme fruste keratoconus at 90°was thinner than that in the low astigmatism group at -1,and -2 mm points(P_(-1mm)=0.015,P_(-2mm)=0.036).CONCLUSION:The analysis of the thinnest point in forme fruste keratoconus corneal epithelium appears earlier than corneal epithelial remodeling.The topographic map of corneal epithelium in high ΔK eyes appears in central island shape,and can be used for the differential diagnosis of early keratoconus.
基金Supported by the 63th Postdoctoral Science Foundation of China(No.2018M632487)General Natural Science ProjectsDepartment of Education,Zhejiang Province,China(No.Y201636718)
文摘AIM: To evaluate the effects of epidermal growth factor(EGF) on transforming growth factor-beta1(TGF-β1)-induced epithelial-mesenchymal transition(EMT) in human corneal epithelial cells(HCECs). METHODS: HCECs were cultured and treated with TGF-β1 for establishing the model of EMT in vitro. Biological effect of EGF on TGF-β1-induced EMT was evaluated. Proteins and m RNAs expression changes of E-cadherin, N-cadherin and Fibronectin(EMT-relative markers) after TGF-β1 or TGF-β1 combined EGF treatment were detected by Western blot and RT-PCR, respectively. Viability and migration of HCECs were measured by CCK-8, transwell cell migration assay and cell scratch wound healing assay. Activation of Smad2, ERK, p38, JNK and Akt signaling pathways were evaluated by Western blot. Inhibitors of relevant signaling pathways were added to the HCECs to explore the key signal mechanism.RESULTS: With treatment of TGF-β1 only, three EMTrelative proteins and m RNA expression showed that EMT up-regulated in a concentration-dependent and time-dependent manner, with significantly decreasing cell viability(TGF-β1≥5 ng/m L, P<0.05) and increasing cell migration(TGF-β1≥5 ng/m L, P<0.01). The phosphorylation of Smad2 and p38 was a key process of TGF-β1-induced EMT. Meanwhile, EMT-relative proteins and m RNA expression showed that EGF inhibited TGF-β1-indued EMT, with significantly increasing cell viability(EGF≥10 ng/m L, P<0.01). It was noteworthy that EGF significantly enhanced cell migration although EMT was inhibited(EGF≥10 ng/m L, P<0.01), and the blockage of p38(by SB202190, a p38 inhibitor) was a potential mechanism of this phenomenon. CONCLUSION: EGF inhibits TGF-β1-induced EMT via suppressive p38, and promotes cells proliferation and migration in a non-EMT process by inhibiting p38 pathway.
基金Supported by Biomedical Research Institute Grant(No.2009-39)Pusan National University Hospital
文摘AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.
基金Supported by National Natural Science Foundation of China(No.81170825No.81470609+3 种基金No.81500695)Specialized Research Fund for the Doctoral Program of Higher Education(No.20123706110003)the Youth Natural Science Foundation of Shandong Province(No.ZR2013HQ007)the Key Project of Natural Science Foundation of Shandong Province(No.ZR2012HZ001)
文摘AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.METHODS: HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074(an inhibitor of Raf-1) group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1(Dectin-1)] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS: In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION: Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.
基金National Natural Science Foundation of China(No.30872808)
文摘AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells(S-ihCECs).METHODS:hAECs were isolated by enzyme digestion,and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR.Recovered and cultured S-ihCECs,immunocytochemistry was used to detect the expression of CK3/12.The proliferation of S ihCECs handled by different concentrations of mitomycin was detected by CCK-8.The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8.After filtered out the optimal conditions,we collected S-ihCECs culture media for 5 days,then prepared conditioned medium to incubate hAECs,inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs.Quantitative real-time reverse transcription-polymerase chain reaction(QRT PCR) was carried out to evaluate the expression of Oct4,NANOG,PAX6,and CK12 in the differentiation period.Immunocytochemistry and western bloting were used to detect the expression of CK3/12.RESULTS:The culture media collected every 12h,from 20μg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs.In the period of differentiation,the morphology of differentiated hAECs was obviously different compared with the control group,and the distinctive CK3/12 for corneal epithelial cells was detected.CONCLUSION:This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment,using the culture media collected from S-ihCECs,and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.
基金Supported by the National Natural Science Foundation of China(No.81271716)
文摘AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM)in vitro.·METHODS:The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate(SDS)solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin(HE)staining and 4',6-diamidino-2-phenylindole(DAPI)staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·RESULTS:Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION:Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vivo.
文摘In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
文摘AIM:Toevaluatetheroleofumbilical cord blood serum(CBS) therapy in cases with persistent corneal epithelial defects(PED).METHODS:Sixteen eyes of 14 patients with PED who were resistant to conventional treatment were treated with 20% umbilical cord serum eye drops. Patients were followed-up weekly until epithelization was complete.The collected data included the grade of corneal lesion(Grade I: epithelial defect +superficial vascularization,Grade II: epithelial defect +stromal edema, Grade III:corneal ulcer +stromal melting), the size of epithelial defect(pretreatment, 7th, 14 thand 21stdays of treatment),and follow-up time was evaluated retrospectively.RESULTS:The mean size of epithelial defect on two perpendicular axes was 5.2×4.6-mm2(range: 2.5-8 mm×2.2-9 mm2). Mean duration of treatment was 8.3 ±5wk.CBS therapy was effective in 12 eyes(75%) and ineffective in 4 eyes(25%). The epithelial defects in 4ineffective eyes were healed with amniotic membrane transplantation and tarsorrhaphy. The rate of complete healing was 12.5% by 7d, 25% by 14 d, and 75% by 21 d.The healing time was prolonged in Grade III eyes in comparison to eyes in Grade I or Grade II.CONCLUSION:The results of the current study indicated the safety effectiveness of CBS drops in the management of PED. The grade of disease seems have a role on the healing time.
文摘Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.
基金Supported by the National Natural Science Foundation of China(No.81300729,No.81160118,No.81460092,No.81660158)Natural Science Foundation of Fujian Province(No.2015J05170).
文摘●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,500,800,and 1000 ng/mL of netrin-1,respectively.The cells viability was detected by cell counting kit-8(CCK-8).The wound-healing assay was applied to assess the migration proficiency of HCE cells.Flow cytometry was used to analyze the cell-cycle distribution and apoptosis.In vivo,normal c57(6 wk)mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound.Mice corneas were inflicted no epithelium with a 3-mm wound displayed,but remained the limbal epithelium intact.A blunt scalpel blade was used to remove the corneal epithelian cells,followed by topical netrin-1 application(200 ng/mL),and the group treated by PBS as control.The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma.Mouse corneal inflammation and neovascularization were observed under slit lamp microscope.The apoptosis of corneal cells was determined by TUNEL staining.●RESLUTS:A concentration of 200 ng/mL netrin-1 enhanced 25%of the HCE viability.The relative migration rates were 76.3%and 100%in control and netrin-1 treated group after cultured 72 h.Treated with netrin-1(200 ng/mL)decreased the apoptosis of HCE cells,as well as decreased their percentage from 19.3%±0.57%to 12.7%±0.42%of the total.The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group.Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice.TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3%and 16.7%respectively.●CONCLUSION:Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration.Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells.These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.
文摘<Abstract>Purpose:To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. Methods: The fresh rabbit cornea was cultured to get the primary RCECs,and RCECs of passage 2 were used for the research. The cells were divided into experimental groups,the cells in which were incubated with different concentrations(18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. Results:MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs,and the inhibition rate was increasing along with the increase of the concentration of diclofenac sodium and the incubation time(P<0.05). Flow cytometer showed that after incubation with diclofenac sodium, the cells in G0/G1 phase were obviously increased, the apoptosis cusp and apoptotic rate were increased. Conclusion: Diclofenac sodium has obvious inhibitory effect on RCECs, which was dosage-dependent,and it may function by inducing cell apoptosis and ceasing cells cycles.
