[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term su...[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.展开更多
We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant ex...We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene containing the classic estrogen response element(ERE),the C-fos oncogene containing the AP-1 site(a non-classical ER binding site),the epigenetic modifying genes,such as Dnmt1,Dnmt3a,Dnmt3b,and histone methyltransferase(HMT),and the human mismatch repair gene hMLH1.Methylation-specific PCR was used to detect the changes in the methylated sites of the CpG islands in the promoters of the ERβ,PR,and C-fos genes.The results indicated that the human ERβ gene recombinant expression plasmid pEGFP-C1-ERβ was successfully constructed and transfected into Caco-2 cells.As compared with the control group,the mRNA and protein expression of ERβ gene was increased significantly 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.As compared with the control group,the mRNA expression of the PR,C-fos,Dnmt3a and Dnmt3b genes was increased significantly 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells,but the mRNA expression of the Dnmt1,HMT,and hMLH1 genes decreased significantly(P<0.05).As compared with the control group,different degrees of demethylation occurred in the promoters of the ERβ,progesterone receptor(PR),and C-fos oncogene 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.The methylation index of the estrogen signal transfection pathway in Caco-2 cells was decreased significantly following the expression restoration of ERβ gene(P<0.05).It is concluded that the restoration or up-regulation of the ERβ gene in Caco-2 cells may significantly activate the expression of the related target genes in the downstream estrogen signal transfection pathway and may result in the demethylation changes of the pathway.During the process,the expression level and activity of the epigenetic modifying genes and the human mismatch repair gene have changed simultaneously.The regulatory effect of the ERβ gene on the estrogen signal transfection pathway to a certain extent partly involves demethylation.展开更多
AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit...AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.展开更多
Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary ce...Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.展开更多
In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization I...In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.展开更多
Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor del...Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor delivery into targeted cells. We designed and synthesized new amphiphilic amino acid derivatives (cysteine-based) of low molecular weight, formed by the same pentapeptide (AG2: WWCOO) N-acylated, with different hydrophobic chains containing from 12 to 18 carbons, named AG2-Cn (N), which dimerize by oxidation in the presence of pLenti-CMV-GFP Puro plasmid (P) in the respective gemini. We determined transfection efficiency, critical micelle concentration, particle size, ζ-potential and cytotoxicity for the derivatives obtained. We found that all the synthesized compounds were active for DNA delivery and had greater ability to transfect CHO-K1 cells. In particular, AG2-C18 is a promising carrier for gene delivery because it showed no cytotoxicity and its activity was greater than or equal to the commercial actives currently used.展开更多
Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine(PEI) was used for gene therapy successfully. The present study invest...Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine(PEI) was used for gene therapy successfully. The present study investigated the effect of PEI and ultrasound waves on ssD NA delivery into saffron cells. Gel retardation, dynamic light scattering(DLS) and scanning electron microscopy(SEM) assays were employed to determine the physicochemical properties of PEI/f-DNA polyplex(complex of PEI and fluorescently labeled DNA). Moreover, the cytotoxicity of PEI, PEI/f-DNA polyplex and ultrasound were investigated on saffron cells at different concentrations. The gel retardation results indicated that the formation and neutralization of the PEI/f-DNA polyplex were completed at N/P=5. The particle size distribution of the polyplexes was from 50 to 122 nm. The experimental results revealed that the cytotoxicity of the PEI/f-DNA polyplex was lower than that of PEI alone, hence the cells showed both dose-and exposure duration-dependent responses. Furthermore, the viability of saffron cells declined extremely after 5 and 10 min sonication but this reduction was not significant at 2 min exposure duration. The results also indicated that the combined utilization of ultrasound and PEI nanoparticles increased the transfection efficiency of saffron cells up to two times higher than those obtained by PEI or ultrasound separately.展开更多
Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marro...Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method,and purified.Then the recombinant plasmid pEGFP/Ang-1was used to transfect BMSCs and the positive clones were obtained by the screen of C418 and observed under light microscopy inversely.Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.Results:BMSCs cell lines were obtained successfully by adherence screening method and density gradient ccntrifugation.Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs,which can express Ang-1 for 3 d and decreased after 7 d.Conclusions:Adherence screening method und density gradient ceiilrifugation can be effective methods lo obtain BMSCs with high purity and rapid proliferation.Besides,the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.展开更多
In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell ...In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell line with the IL-2 cDNA and explored the changes incell membrane components and function. The genemodified cells releasing 70 - 169 U/106 cells of IL-2 in 24hrs. showed apparent morphological conversion includingthe appearance change from epitheloid form展开更多
Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effec...Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effectorcells but also antigen-presenting cells.They act as abridge connecting tumor cells with immune effector cells.Two cytokines we chosen are closely linkcd with thefunctions of macrophage. IFN-γis a principle factor toactivate macrophages and it incrcases MHC expression ofthem which can improve their antigen presenting ability.M-CSF is an important cytokine to keep theproliferation, differentiation and maturation ofmacrophage progenitor cells. In this study, we used展开更多
The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of ...The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of tumor cells to effector cells, and as aconsequence, the tumorigenicity decreased. Beforekilling tumor cells, effector cells required first torecognize non-specific surface adhesion molecules展开更多
In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT P...In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.展开更多
Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving th...Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application.Therefore,our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells(RECs)using different extracellular matrices and transfection reagents.In this study,we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells,followed by their characterization.Subsequently,we generated iPS-like cells by transfecting renal epithelial cells(RECs)with vectors expressing Oct4,Sox2,L-Myc,Lin-28,and Klf4,and we compared the efficacy of different extracellular matrices and transfection reagents.The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4,Nanog,Lin28,and Klf4.We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested.Moreover,the three matrices are similar in their efficiency of obtaining iPS-like cells.This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.展开更多
This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A an...This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed.After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting.Adhesion ability was evaluated by using MTT.Cell migration was determined by using Boyden chamber method.Tube formation test was conducted on Matrigel.The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased.In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group.ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group.Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively.In ICAP-1α group, the ratio was decreased to 0.1005±0.0073.In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group.In ICAP-1α group, the number was decreased to 12.06±1.72.The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71.In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24.It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect.These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.展开更多
Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are curr...Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are currently being used: conventional (forward;Fw) and reverse (Rev) transfections. Here, to investigate an efficient siRNA transfection method using cationic liposomes, we compared the transfection efficiency of siRNA between Fw-transfection and Rev-transfection methods with various types of cationic liposomes. In Fw-transfection, siRNA/cationic liposomes complex (siRNA lipoplexes) was added to pre-plated cells. In contrast, Rev-transfection was performed by co-incubation of cells with siRNA lipoplexes in suspension. As a result, Rev-transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based or cationic cholesterol derivative-based liposomes could deliver siRNA into the cells via efficient cellular association, and induce an improved gene silencing effect by siRNA compared with Fw-transfection. Furthermore, Rev-transfection did not show increased cytotoxicity compared with Fw-transfection. These findings suggested that Rev-transfection in suspension has better potential for efficient transfection of siRNA into cells with minimal toxicity.展开更多
[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells Hep...[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells HepG2.[Methods]HepG2 cells were cultured for 0,4 and 24 h,respectively and then transfected with ARID4B targeting siRNA.The mRNA expression of ARID4B gene was detected by RT-qPCR,and the protein expression was detected by Western blot.[Results]When HepG2 cells were transfected at 0 h after seeding,the expression of ARID4B was the lowest in both mRNA and protein levels,that is,the inhibition effect was the best.[Conclusions]Transfection at different time points after seeding of HepG2 cells had different inhibition effect on the expression of ARID4B.Transfection at 0 h after seeding showed the best inhibition effect.展开更多
Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent H...Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.展开更多
In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGF...In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/ß-gal lipoplexes complexed at +/- charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar ß-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5°C, 32.5°C and 39°C, respectively. A gel-to-liquid crystalline phase transition temperature below 37°C, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.展开更多
Reducible or imidazolyl polycations of block poly(imidazole/2-dimethyl aminoethyl) are of promising in gene delivery. Dimeric poly(2-dimethyl aminoethyl) methacrylate-block-polyvinylimidazole (rDPDMAEMAIM) and reducib...Reducible or imidazolyl polycations of block poly(imidazole/2-dimethyl aminoethyl) are of promising in gene delivery. Dimeric poly(2-dimethyl aminoethyl) methacrylate-block-polyvinylimidazole (rDPDMAEMAIM) and reducible poly (2-dimethylaminoethyl) methacrylate (rDPDMAEMA) with single disulfide bond in the backbone was synthesized by oxidizing their dithioester-terminated polymers. The polyplexes sizes, rDPDMAEMAIM/pDNA and rDPDMAEMA/ pDNA (plasmid DNA) are in the ranges of 100 nm - 150 nm at the weight ratio of 12:1, and the zeta potential of rDPDMAEMAIM/pDNA from 9.6 mV to 22.7 mV in PBS solutions increases with their weight ratios of 1:1 to 18:1. The results show that the rDPDMAEMAIM/pDNA polyplexes have higher transfection activity and lower cytotoxicity than that of rDPDMAEMAIM/pDNA against 293T cells in vitro in the presence of serum, indicating that the PDMAEMAIM present a promising nonviral gene vector.展开更多
基金Supported by Natural Science Foundation of China(82160386)Guangxi Natural Science Foundation(2023GXNSFAA0261892021GXNSFAA075042)。
文摘[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.
基金supported by a grant from the National Natural Sciences Foundation of China(No.30500488)
文摘We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene containing the classic estrogen response element(ERE),the C-fos oncogene containing the AP-1 site(a non-classical ER binding site),the epigenetic modifying genes,such as Dnmt1,Dnmt3a,Dnmt3b,and histone methyltransferase(HMT),and the human mismatch repair gene hMLH1.Methylation-specific PCR was used to detect the changes in the methylated sites of the CpG islands in the promoters of the ERβ,PR,and C-fos genes.The results indicated that the human ERβ gene recombinant expression plasmid pEGFP-C1-ERβ was successfully constructed and transfected into Caco-2 cells.As compared with the control group,the mRNA and protein expression of ERβ gene was increased significantly 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.As compared with the control group,the mRNA expression of the PR,C-fos,Dnmt3a and Dnmt3b genes was increased significantly 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells,but the mRNA expression of the Dnmt1,HMT,and hMLH1 genes decreased significantly(P<0.05).As compared with the control group,different degrees of demethylation occurred in the promoters of the ERβ,progesterone receptor(PR),and C-fos oncogene 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.The methylation index of the estrogen signal transfection pathway in Caco-2 cells was decreased significantly following the expression restoration of ERβ gene(P<0.05).It is concluded that the restoration or up-regulation of the ERβ gene in Caco-2 cells may significantly activate the expression of the related target genes in the downstream estrogen signal transfection pathway and may result in the demethylation changes of the pathway.During the process,the expression level and activity of the epigenetic modifying genes and the human mismatch repair gene have changed simultaneously.The regulatory effect of the ERβ gene on the estrogen signal transfection pathway to a certain extent partly involves demethylation.
基金Supported by the National Natural Science Foundation for Youth of China(No.81901765)。
文摘AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.
文摘Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.
文摘In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.
基金supported by grants from Universidad Nacional del Litoral(U.N.L)(CAI+D,2011),República Argentina.
文摘Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor delivery into targeted cells. We designed and synthesized new amphiphilic amino acid derivatives (cysteine-based) of low molecular weight, formed by the same pentapeptide (AG2: WWCOO) N-acylated, with different hydrophobic chains containing from 12 to 18 carbons, named AG2-Cn (N), which dimerize by oxidation in the presence of pLenti-CMV-GFP Puro plasmid (P) in the respective gemini. We determined transfection efficiency, critical micelle concentration, particle size, ζ-potential and cytotoxicity for the derivatives obtained. We found that all the synthesized compounds were active for DNA delivery and had greater ability to transfect CHO-K1 cells. In particular, AG2-C18 is a promising carrier for gene delivery because it showed no cytotoxicity and its activity was greater than or equal to the commercial actives currently used.
基金supported by the University of Mohaghegh Ardabili, India under Grant (51-487)
文摘Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine(PEI) was used for gene therapy successfully. The present study investigated the effect of PEI and ultrasound waves on ssD NA delivery into saffron cells. Gel retardation, dynamic light scattering(DLS) and scanning electron microscopy(SEM) assays were employed to determine the physicochemical properties of PEI/f-DNA polyplex(complex of PEI and fluorescently labeled DNA). Moreover, the cytotoxicity of PEI, PEI/f-DNA polyplex and ultrasound were investigated on saffron cells at different concentrations. The gel retardation results indicated that the formation and neutralization of the PEI/f-DNA polyplex were completed at N/P=5. The particle size distribution of the polyplexes was from 50 to 122 nm. The experimental results revealed that the cytotoxicity of the PEI/f-DNA polyplex was lower than that of PEI alone, hence the cells showed both dose-and exposure duration-dependent responses. Furthermore, the viability of saffron cells declined extremely after 5 and 10 min sonication but this reduction was not significant at 2 min exposure duration. The results also indicated that the combined utilization of ultrasound and PEI nanoparticles increased the transfection efficiency of saffron cells up to two times higher than those obtained by PEI or ultrasound separately.
基金Supported by National Natural Science Foundation of China(Grant No.20114020)
文摘Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method,and purified.Then the recombinant plasmid pEGFP/Ang-1was used to transfect BMSCs and the positive clones were obtained by the screen of C418 and observed under light microscopy inversely.Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.Results:BMSCs cell lines were obtained successfully by adherence screening method and density gradient ccntrifugation.Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs,which can express Ang-1 for 3 d and decreased after 7 d.Conclusions:Adherence screening method und density gradient ceiilrifugation can be effective methods lo obtain BMSCs with high purity and rapid proliferation.Besides,the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.
文摘In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell line with the IL-2 cDNA and explored the changes incell membrane components and function. The genemodified cells releasing 70 - 169 U/106 cells of IL-2 in 24hrs. showed apparent morphological conversion includingthe appearance change from epitheloid form
文摘Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effectorcells but also antigen-presenting cells.They act as abridge connecting tumor cells with immune effector cells.Two cytokines we chosen are closely linkcd with thefunctions of macrophage. IFN-γis a principle factor toactivate macrophages and it incrcases MHC expression ofthem which can improve their antigen presenting ability.M-CSF is an important cytokine to keep theproliferation, differentiation and maturation ofmacrophage progenitor cells. In this study, we used
文摘The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of tumor cells to effector cells, and as aconsequence, the tumorigenicity decreased. Beforekilling tumor cells, effector cells required first torecognize non-specific surface adhesion molecules
文摘In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.
基金the Programa de Apoyo a la Investigación Científica y Tecnológica(PAICyT,grant no.SA813-19)from the Universidad Autónoma de Nuevo Leónthe Consejo Nacional de Ciencia y Tecnología(CONACyT,grant no.CB-2015/255725)ORR thanks CONACyT for its support through a scholarship under register number 296354.
文摘Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application.Therefore,our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells(RECs)using different extracellular matrices and transfection reagents.In this study,we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells,followed by their characterization.Subsequently,we generated iPS-like cells by transfecting renal epithelial cells(RECs)with vectors expressing Oct4,Sox2,L-Myc,Lin-28,and Klf4,and we compared the efficacy of different extracellular matrices and transfection reagents.The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4,Nanog,Lin28,and Klf4.We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested.Moreover,the three matrices are similar in their efficiency of obtaining iPS-like cells.This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.
基金supported by a grant from the National Natural Science Foundation of China (No.30670856)
文摘This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed.After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting.Adhesion ability was evaluated by using MTT.Cell migration was determined by using Boyden chamber method.Tube formation test was conducted on Matrigel.The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased.In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group.ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group.Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively.In ICAP-1α group, the ratio was decreased to 0.1005±0.0073.In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group.In ICAP-1α group, the number was decreased to 12.06±1.72.The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71.In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24.It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect.These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.
文摘Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are currently being used: conventional (forward;Fw) and reverse (Rev) transfections. Here, to investigate an efficient siRNA transfection method using cationic liposomes, we compared the transfection efficiency of siRNA between Fw-transfection and Rev-transfection methods with various types of cationic liposomes. In Fw-transfection, siRNA/cationic liposomes complex (siRNA lipoplexes) was added to pre-plated cells. In contrast, Rev-transfection was performed by co-incubation of cells with siRNA lipoplexes in suspension. As a result, Rev-transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based or cationic cholesterol derivative-based liposomes could deliver siRNA into the cells via efficient cellular association, and induce an improved gene silencing effect by siRNA compared with Fw-transfection. Furthermore, Rev-transfection did not show increased cytotoxicity compared with Fw-transfection. These findings suggested that Rev-transfection in suspension has better potential for efficient transfection of siRNA into cells with minimal toxicity.
基金Scientific Research Foundation of Chengde Medical University for High-level Talents(10204030004).
文摘[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells HepG2.[Methods]HepG2 cells were cultured for 0,4 and 24 h,respectively and then transfected with ARID4B targeting siRNA.The mRNA expression of ARID4B gene was detected by RT-qPCR,and the protein expression was detected by Western blot.[Results]When HepG2 cells were transfected at 0 h after seeding,the expression of ARID4B was the lowest in both mRNA and protein levels,that is,the inhibition effect was the best.[Conclusions]Transfection at different time points after seeding of HepG2 cells had different inhibition effect on the expression of ARID4B.Transfection at 0 h after seeding showed the best inhibition effect.
文摘Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.
文摘In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/ß-gal lipoplexes complexed at +/- charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar ß-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5°C, 32.5°C and 39°C, respectively. A gel-to-liquid crystalline phase transition temperature below 37°C, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.
文摘Reducible or imidazolyl polycations of block poly(imidazole/2-dimethyl aminoethyl) are of promising in gene delivery. Dimeric poly(2-dimethyl aminoethyl) methacrylate-block-polyvinylimidazole (rDPDMAEMAIM) and reducible poly (2-dimethylaminoethyl) methacrylate (rDPDMAEMA) with single disulfide bond in the backbone was synthesized by oxidizing their dithioester-terminated polymers. The polyplexes sizes, rDPDMAEMAIM/pDNA and rDPDMAEMA/ pDNA (plasmid DNA) are in the ranges of 100 nm - 150 nm at the weight ratio of 12:1, and the zeta potential of rDPDMAEMAIM/pDNA from 9.6 mV to 22.7 mV in PBS solutions increases with their weight ratios of 1:1 to 18:1. The results show that the rDPDMAEMAIM/pDNA polyplexes have higher transfection activity and lower cytotoxicity than that of rDPDMAEMAIM/pDNA against 293T cells in vitro in the presence of serum, indicating that the PDMAEMAIM present a promising nonviral gene vector.
