BACKGROUND Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice.These cells consist of both epithelial and mesenchymal cells.Patient-derived cell lines that maintain tumor characteristics are...BACKGROUND Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice.These cells consist of both epithelial and mesenchymal cells.Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma.However,cholangiocarcinoma sarcoma cell lines are not available in cell banks.AIM To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line,namely CBC2T-2.METHODS We conducted a short tandem repeat(STR)test to confirm the identity of the CBC2T-2 cell line.Furthermore,we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies.The tumorigenic potential of CBC2T-2 cells was tested in vivo using nonobese diabetic/severe combined immunodeficient(NOD/SCID)mice.The cells were injected subcutaneously and tumor formation was observed.In addition,immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts.The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma.Lastly,whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line.RESULTS The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue.The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology.The cells exhibited a high proliferation ratio with a doubling time of 47.11 h.This cell line has migratory,invasive,and clonogenic abilities.The chromosomes in the CBC2T-2 cells were polyploidy,with numbers ranging from 69 to 79.The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice.CBC2T-2 cells and xenografts were positive for both the epithelial marker,CK19,and the mesenchymal marker,vimentin.These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics.The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma,and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option.CONCLUSION We established the first human cholangiocarcinoma sarcoma cell line,CBC2T-2,with stable biogenetic traits.This cell line,as a research model,has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.展开更多
A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and grow...A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and growth temperature of the cells were detected.SMSP cell line is mainly composed of fibroblast-like cells.Most of the SMSP cells contained 44 chromosomes,and the sequence of COI gene confirmed that the cells were originated from turbot.The optimal culture conditions were 24℃,DMEM+10%FBS.The cell line had high transfection efficiency for siRNA and plasmid.After stimulation with lipopolysaccharide(LPS)or poly(I:C),the expressions of immune-related genes such as TNF-β,IL-12s,IL-10 and IL-1βwere up-regulated significantly in the early stage(P<0.05).This study will provide a model for exploring immune mechanism of turbot against pathogen in vitro.展开更多
BACKGROUND An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have be...BACKGROUND An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported.AIM To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese.METHODS Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat(STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 106 cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7(CK7), cytokeratin 20(CK20), cytokeratin low molecular weight(CKL), Ki67 and carcinoembryonic antigen(CEA) were determined by immunocytochemistry assay.RESULTS DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations;its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient’s primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL;the Ki67 was 50%, and CEA was focally expressed.CONCLUSION Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.展开更多
Micrometric-thin cells(MCs)with alkali vapor atoms have been valuable for research and applications of hyperfine Zeeman splitting and atomic magnetometers under strong magnetic fields.We theoretically and experimental...Micrometric-thin cells(MCs)with alkali vapor atoms have been valuable for research and applications of hyperfine Zeeman splitting and atomic magnetometers under strong magnetic fields.We theoretically and experimentally study the saturated absorption spectra using a 100-μm cesium MC,where the pump and probe beams are linearly polarized with mutually perpendicular polarizations,and the magnetic field is along the pump beam.Because of the distinctive thin chamber of the MC,crossover spectral lines in saturated absorption spectra are largely suppressed leading to clear splittings of hyperfine Zeeman transitions in experiments,and the effect of spatial magnetic field gradient is expected to be reduced.A calculation method is proposed to achieve good agreements between theoretical calculations and experimental results.This method successfully explains the suppression of crossover lines in MCs,as well as the effects of magnetic field direction,propagation and polarization directions of the pump/probe beam on saturated absorption spectrum.The saturated absorption spectrum with suppressed crossover lines is used for laser frequency stabilization,which may provide the potential value of MCs for high spatial resolution strong-field magnetometry with high sensitivity.展开更多
Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer element...Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements.The aim of the study is to investigate the extracellular regulated protein kinases(ERKs)pathway of HBx on glomerular mesangial cell(GMC)proliferation and tumor necrosis factor-α(TNF-α)expression.The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector pCI-neo and confirmed by restriction endonuclease digestion and sequence analysis.PCI-neo containing HBV X gene(pCI-neo-X)was then transfected into cultured GMC line via liposome.GMC proliferation,TNF-αand its mRNA expression were compared in the condition of with or without U0126 in culture media.HBx,ERK1/2 and p-ERK1/2 expression in GMCs was assessed by Western blotting.TNF-αmRNA expression was assessed by semi-quantitative reverse transcription-PCR(RT-PCR).TNF-αlevel in supernatants was measured by ELISA.GMC proliferation was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)kit.The results showed that HBx expression was found in transfected GMCs and became prominent at 36th and 48th h after transfection whether with or without U0126 in culture media.TNF-αmRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in PCI-neo-X transfection without U0126 group were(189.0�18.1)and(172.3�24.3)pg/mL at 36th and 48th h after transfec-tion,respectively.In contrast,TNF-αlevels in supernatants with U0126 were(65.6�11.6)and(84.0�24.6)pg/mL at 36th and 48th h,respectively.The TNF-αlevels in the latter groups were significantly lower than those in the former groups(P<0.05).GMCs proliferation was also lower in added U0126 group at 36th and 48th h after transfection.From above,we can conclude that HBx could induce GMC proliferation and increase TNF-αmRNA expression and its protein production.HBx upregulates TNF-αexpression and induces cell proliferation of GMC line partly through ERK1/2 signal transduction pathway.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
AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malign...AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays.The distribution of the cell cycles were detected by flow cytometry.Expression of acquired multidrug resistance P-glycoprotein(MDR1,ABCB1) and multidrug resistance-associated protein 1(MRP1,ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy.RESULTS:The SK-Hep-1/CDDP cells(IC50 = 70.61 ± 1.06 μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells(IC50 = 5.13 ± 0.09 μg/mL),and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin,5-fluorouracil and vincristine.Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups.The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S(42.2% ± 2.65% vs 27.91% ± 2.16%,P < 0.01) and G2/M(20.67% ± 5.69% vs 12.14% ± 3.36%,P < 0.01) phases in comparison with SK-Hep-1 cells,while the percentage of cells in the G0/G1 phase decreased(37.5% ± 5.05% vs 59.83% ± 3.28%,P < 0.01).The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype.CONCLUSION:Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1.展开更多
INTRODUCTIONVascular endothelial growth factor(VEGF)whichis also known as vascular permeability factor(VPF)is a heparin-binding,dimeric polypeptide growthfactor and a potent mitogen for endothelial cells.VEGF can stim...INTRODUCTIONVascular endothelial growth factor(VEGF)whichis also known as vascular permeability factor(VPF)is a heparin-binding,dimeric polypeptide growthfactor and a potent mitogen for endothelial cells.VEGF can stimulate the endothelial cell growth andenhance the motility through its two knownreceptors flt-1 and KDR.Acting through thesereceptors,VEGF may stimulate angiogenesis展开更多
AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Dox...AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.展开更多
Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liv...Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.展开更多
Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like m...Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like metastatic model of human HCC in nude mice (LCI-D20)and a Iow metastatic model of human HCC in nude mice LCI-D35 ) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding.Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including antiangiogenesis, antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.展开更多
INTRODUCTIONGastrin is atrophic gastrointestinal hormone whichis secreted by G cell.Gastrin has long beenconsidered a growth stimulatory hormone formucosa of the gastrointestinal tract.The growthresponses of certain c...INTRODUCTIONGastrin is atrophic gastrointestinal hormone whichis secreted by G cell.Gastrin has long beenconsidered a growth stimulatory hormone formucosa of the gastrointestinal tract.The growthresponses of certain colorectal cancer cells,andxenografts,can be stimulated by endogenousgastrin.Protein kinase C (PKC) is a family ofisozymes that plays a crucial role in transducingsignals of many hormones,growth peptides,展开更多
AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who ...AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.展开更多
Objective:To investigate anticancer activity of different fractions of Tephrosia purpurea[TP] （Sharapunkha,Fabaceae） and Ficus religiosa[FR]（Peepal,Moraceae）.Methods:The fractions of TP and FR were prepared and te...Objective:To investigate anticancer activity of different fractions of Tephrosia purpurea[TP] （Sharapunkha,Fabaceae） and Ficus religiosa[FR]（Peepal,Moraceae）.Methods:The fractions of TP and FR were prepared and tested for in vitro anticancer activity using human MCF 7 cell line by trypan blue exclusion method.Results:The result showed that among all these fractions of TPI.TPIII.FRI and FRIII showed better anticancer activity compared to other fractions.The IC<sub>50</sub> value for TPI（152.4μM）,TPIII（158.71μM）.FRI（160.3μM） and for FRIII（222.7μM） was observed.Conclusions:The present study shows anticancer potential of TP and FR fractions in MCF 7 cell line.展开更多
AIM: To explore the synergistic effect of docosahexaenoic acid(DHA)/5-fluorouracil(5-FU) on the human gastric cancer cell line AGS and examine the underlying mechanism.METHODS: AGS cells were cultured and treated with...AIM: To explore the synergistic effect of docosahexaenoic acid(DHA)/5-fluorouracil(5-FU) on the human gastric cancer cell line AGS and examine the underlying mechanism.METHODS: AGS cells were cultured and treated with a series of concentrations of DHA and 5-FU alone or in combination for 24 and 48 h. To investigate the synergistic effect of DHA and 5-FU on AGS cells, the inhibition of cell proliferation was determined by MTT assay and cell morphology. Flow cytometric analysis was also used to assess cell cycle distribution, and the expression of mitochondrial electron transfer chain complexes(METCs)?Ⅰ, Ⅱ and Ⅴ in AGS cells was further determined by Western blot analysis. RESULTS: DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS cells in a significant time and dose-dependent manner. DHA markedly strengthened the antiproliferative effect of 5-FU, decreasing the IC50 by 3.56-2.15-fold in an apparent synergy. The morphological changes of the cells were characterized by shrinkage, cell membrane blebbing and decreased adherence. Cell cycle analysis showed a shift of cells into the G0/G1 phase from the S phase following treatment with DHA or 5-FU(G0/G1 phase: 30.04% ± 1.54% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 56.76% ± 3.14% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). Combination treatment of DHA and 5-FU resulted in a significantly larger shift toward the G0/G1 phase and subsequent reduction in S phase(G0/G1 phase: 69.06% ± 2.63% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 19.80% ± 4.30% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). This synergy was also reflected in the significant downregulation of the expression of METCs in AGS cells.CONCLUSION: Synergistic anticancer properties of DHA and 5-FU may involve interference with energy production of AGS cells via downregulation of METCs and cell cycle arrest.展开更多
AIM:To establish an untransfected human corneal stromal(HCS) cell line and characterize its biocompatibility to acellular porcine corneal stroma(aPCS).· METHODS:Primary culture was initiated with a pure populatio...AIM:To establish an untransfected human corneal stromal(HCS) cell line and characterize its biocompatibility to acellular porcine corneal stroma(aPCS).· METHODS:Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media(pH 7.2) containing 20% fetal bovine serum and various necessary growth factors.The established cell line was characterized by growth property,chromosome analysis,tumorigenicity assay,expression of marker proteins and functional proteins.Furthermore,the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light,electron microscopies.· RESULTS:HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far.A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined.Results of chromosome analysis,morphology,combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties.Furthermore,HCS cells have no tumorigenicity,and with excellent biocompatibility to aPCS.· CONCLUSION:An untransfected and non-tumorigenic HCS cell line has been established,and the cells maintained positive expression of marker proteins and functional proteins.The cell line,with excellent biocompatibility to aPCS,might be used for in vitro reconstruction of tissue-engineered HCS.展开更多
基金the National Natural Science Foundation of China,No.82060551and Lanzhou Chengguan District Science and Technology Planning Project,No.2019JSCX0092.
文摘BACKGROUND Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice.These cells consist of both epithelial and mesenchymal cells.Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma.However,cholangiocarcinoma sarcoma cell lines are not available in cell banks.AIM To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line,namely CBC2T-2.METHODS We conducted a short tandem repeat(STR)test to confirm the identity of the CBC2T-2 cell line.Furthermore,we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies.The tumorigenic potential of CBC2T-2 cells was tested in vivo using nonobese diabetic/severe combined immunodeficient(NOD/SCID)mice.The cells were injected subcutaneously and tumor formation was observed.In addition,immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts.The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma.Lastly,whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line.RESULTS The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue.The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology.The cells exhibited a high proliferation ratio with a doubling time of 47.11 h.This cell line has migratory,invasive,and clonogenic abilities.The chromosomes in the CBC2T-2 cells were polyploidy,with numbers ranging from 69 to 79.The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice.CBC2T-2 cells and xenografts were positive for both the epithelial marker,CK19,and the mesenchymal marker,vimentin.These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics.The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma,and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option.CONCLUSION We established the first human cholangiocarcinoma sarcoma cell line,CBC2T-2,with stable biogenetic traits.This cell line,as a research model,has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.
基金the National Natural Science Foundation of China(No.31902403)the Young Experts of Taishan Scholars(No.tsqn201909130)+2 种基金the advanced Talents Foundation of QAU grant(No.663-1120029)the Shandong Technical System of Fish Industry(No.SDAIT-12-03)the Breeding Plan of Shandong Provincial Qingchuang Research Team(2019),China。
文摘A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and growth temperature of the cells were detected.SMSP cell line is mainly composed of fibroblast-like cells.Most of the SMSP cells contained 44 chromosomes,and the sequence of COI gene confirmed that the cells were originated from turbot.The optimal culture conditions were 24℃,DMEM+10%FBS.The cell line had high transfection efficiency for siRNA and plasmid.After stimulation with lipopolysaccharide(LPS)or poly(I:C),the expressions of immune-related genes such as TNF-β,IL-12s,IL-10 and IL-1βwere up-regulated significantly in the early stage(P<0.05).This study will provide a model for exploring immune mechanism of turbot against pathogen in vitro.
基金National Natural Science Foundation of China,No.82260555Gansu Provincial Science and Technology Plan,No.1606RJZA139,No.21JR11RA096,No.21JR1RA099,and No.21JR1RA113+3 种基金Gansu Health Industry Project,No.GSWSKY2020-21Traditional Chinese Medicine Scientific Research Project of Gansu Province,No.GZKP-2020-28Talent Innovation and Entrepreneurship Project of Lanzhou,No.2020-RC-46Intra-Hospital Fund of the First Hospital of Lanzhou University,No.ldyyyn2019-97.
文摘BACKGROUND An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported.AIM To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese.METHODS Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat(STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 106 cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7(CK7), cytokeratin 20(CK20), cytokeratin low molecular weight(CKL), Ki67 and carcinoembryonic antigen(CEA) were determined by immunocytochemistry assay.RESULTS DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations;its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient’s primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL;the Ki67 was 50%, and CEA was focally expressed.CONCLUSION Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.61571018 and 61531003).
文摘Micrometric-thin cells(MCs)with alkali vapor atoms have been valuable for research and applications of hyperfine Zeeman splitting and atomic magnetometers under strong magnetic fields.We theoretically and experimentally study the saturated absorption spectra using a 100-μm cesium MC,where the pump and probe beams are linearly polarized with mutually perpendicular polarizations,and the magnetic field is along the pump beam.Because of the distinctive thin chamber of the MC,crossover spectral lines in saturated absorption spectra are largely suppressed leading to clear splittings of hyperfine Zeeman transitions in experiments,and the effect of spatial magnetic field gradient is expected to be reduced.A calculation method is proposed to achieve good agreements between theoretical calculations and experimental results.This method successfully explains the suppression of crossover lines in MCs,as well as the effects of magnetic field direction,propagation and polarization directions of the pump/probe beam on saturated absorption spectrum.The saturated absorption spectrum with suppressed crossover lines is used for laser frequency stabilization,which may provide the potential value of MCs for high spatial resolution strong-field magnetometry with high sensitivity.
基金supported by the National Natural Science Foundation of China(Grant No.30772360)the Natural Science Foundation of Health Department of Hubei Province,China(No.JX4B48).
文摘Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements.The aim of the study is to investigate the extracellular regulated protein kinases(ERKs)pathway of HBx on glomerular mesangial cell(GMC)proliferation and tumor necrosis factor-α(TNF-α)expression.The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector pCI-neo and confirmed by restriction endonuclease digestion and sequence analysis.PCI-neo containing HBV X gene(pCI-neo-X)was then transfected into cultured GMC line via liposome.GMC proliferation,TNF-αand its mRNA expression were compared in the condition of with or without U0126 in culture media.HBx,ERK1/2 and p-ERK1/2 expression in GMCs was assessed by Western blotting.TNF-αmRNA expression was assessed by semi-quantitative reverse transcription-PCR(RT-PCR).TNF-αlevel in supernatants was measured by ELISA.GMC proliferation was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)kit.The results showed that HBx expression was found in transfected GMCs and became prominent at 36th and 48th h after transfection whether with or without U0126 in culture media.TNF-αmRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in PCI-neo-X transfection without U0126 group were(189.0�18.1)and(172.3�24.3)pg/mL at 36th and 48th h after transfec-tion,respectively.In contrast,TNF-αlevels in supernatants with U0126 were(65.6�11.6)and(84.0�24.6)pg/mL at 36th and 48th h,respectively.The TNF-αlevels in the latter groups were significantly lower than those in the former groups(P<0.05).GMCs proliferation was also lower in added U0126 group at 36th and 48th h after transfection.From above,we can conclude that HBx could induce GMC proliferation and increase TNF-αmRNA expression and its protein production.HBx upregulates TNF-αexpression and induces cell proliferation of GMC line partly through ERK1/2 signal transduction pathway.
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 The National Natural Science Foundation of China,No 304708651520 Project of Xinqiao Hospital
文摘AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays.The distribution of the cell cycles were detected by flow cytometry.Expression of acquired multidrug resistance P-glycoprotein(MDR1,ABCB1) and multidrug resistance-associated protein 1(MRP1,ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy.RESULTS:The SK-Hep-1/CDDP cells(IC50 = 70.61 ± 1.06 μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells(IC50 = 5.13 ± 0.09 μg/mL),and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin,5-fluorouracil and vincristine.Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups.The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S(42.2% ± 2.65% vs 27.91% ± 2.16%,P < 0.01) and G2/M(20.67% ± 5.69% vs 12.14% ± 3.36%,P < 0.01) phases in comparison with SK-Hep-1 cells,while the percentage of cells in the G0/G1 phase decreased(37.5% ± 5.05% vs 59.83% ± 3.28%,P < 0.01).The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype.CONCLUSION:Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1.
基金National Distinguished Young Scientist Fund,39525021 State Key Basic Research Program G1998051203
文摘INTRODUCTIONVascular endothelial growth factor(VEGF)whichis also known as vascular permeability factor(VPF)is a heparin-binding,dimeric polypeptide growthfactor and a potent mitogen for endothelial cells.VEGF can stimulate the endothelial cell growth andenhance the motility through its two knownreceptors flt-1 and KDR.Acting through thesereceptors,VEGF may stimulate angiogenesis
文摘AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.
基金supported by Wuhan Municipal Science and Technology Bureau of applied basic research project(No.2013062301010823)Wuhan City health planning medieal research project(No.WX14A11)
文摘Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.
基金Partly supporled by the State Key Basic Research Program Grant of China(G1998051211)Leading Speciality Grant of Shanghai Health Bureau.
文摘Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like metastatic model of human HCC in nude mice (LCI-D20)and a Iow metastatic model of human HCC in nude mice LCI-D35 ) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding.Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including antiangiogenesis, antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.
文摘INTRODUCTIONGastrin is atrophic gastrointestinal hormone whichis secreted by G cell.Gastrin has long beenconsidered a growth stimulatory hormone formucosa of the gastrointestinal tract.The growthresponses of certain colorectal cancer cells,andxenografts,can be stimulated by endogenousgastrin.Protein kinase C (PKC) is a family ofisozymes that plays a crucial role in transducingsignals of many hormones,growth peptides,
文摘AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.
文摘Objective:To investigate anticancer activity of different fractions of Tephrosia purpurea[TP] （Sharapunkha,Fabaceae） and Ficus religiosa[FR]（Peepal,Moraceae）.Methods:The fractions of TP and FR were prepared and tested for in vitro anticancer activity using human MCF 7 cell line by trypan blue exclusion method.Results:The result showed that among all these fractions of TPI.TPIII.FRI and FRIII showed better anticancer activity compared to other fractions.The IC<sub>50</sub> value for TPI（152.4μM）,TPIII（158.71μM）.FRI（160.3μM） and for FRIII（222.7μM） was observed.Conclusions:The present study shows anticancer potential of TP and FR fractions in MCF 7 cell line.
文摘AIM: To explore the synergistic effect of docosahexaenoic acid(DHA)/5-fluorouracil(5-FU) on the human gastric cancer cell line AGS and examine the underlying mechanism.METHODS: AGS cells were cultured and treated with a series of concentrations of DHA and 5-FU alone or in combination for 24 and 48 h. To investigate the synergistic effect of DHA and 5-FU on AGS cells, the inhibition of cell proliferation was determined by MTT assay and cell morphology. Flow cytometric analysis was also used to assess cell cycle distribution, and the expression of mitochondrial electron transfer chain complexes(METCs)?Ⅰ, Ⅱ and Ⅴ in AGS cells was further determined by Western blot analysis. RESULTS: DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS cells in a significant time and dose-dependent manner. DHA markedly strengthened the antiproliferative effect of 5-FU, decreasing the IC50 by 3.56-2.15-fold in an apparent synergy. The morphological changes of the cells were characterized by shrinkage, cell membrane blebbing and decreased adherence. Cell cycle analysis showed a shift of cells into the G0/G1 phase from the S phase following treatment with DHA or 5-FU(G0/G1 phase: 30.04% ± 1.54% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 56.76% ± 3.14% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). Combination treatment of DHA and 5-FU resulted in a significantly larger shift toward the G0/G1 phase and subsequent reduction in S phase(G0/G1 phase: 69.06% ± 2.63% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 19.80% ± 4.30% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). This synergy was also reflected in the significant downregulation of the expression of METCs in AGS cells.CONCLUSION: Synergistic anticancer properties of DHA and 5-FU may involve interference with energy production of AGS cells via downregulation of METCs and cell cycle arrest.
基金National High Technology Research and Development Program("863" Program) of China(No.2006AA02A132)
文摘AIM:To establish an untransfected human corneal stromal(HCS) cell line and characterize its biocompatibility to acellular porcine corneal stroma(aPCS).· METHODS:Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media(pH 7.2) containing 20% fetal bovine serum and various necessary growth factors.The established cell line was characterized by growth property,chromosome analysis,tumorigenicity assay,expression of marker proteins and functional proteins.Furthermore,the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light,electron microscopies.· RESULTS:HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far.A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined.Results of chromosome analysis,morphology,combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties.Furthermore,HCS cells have no tumorigenicity,and with excellent biocompatibility to aPCS.· CONCLUSION:An untransfected and non-tumorigenic HCS cell line has been established,and the cells maintained positive expression of marker proteins and functional proteins.The cell line,with excellent biocompatibility to aPCS,might be used for in vitro reconstruction of tissue-engineered HCS.
