Transfection of CTGF siRNA inhibits transdifferentiation in human lens epithelium cell line B3 in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression of connective tissue growth factor (CTGF) and α-SMA in human lens epithelium cell (HLEC) line B3 after transfection by liposome-coated siRNA targeting CTGF.METHODS: HLECs were transfected with small interfering RNA (siRNA) targeting CTGF,labeled with 5`-fluorescein isothiocyanate (5`-FITC) and coated with lipofectamine.The transfection ratio was evaluated via fluorescence intensity.Cell counting kit-8 (CCK-8) assay was performed to assess cytoviability of both non-transfected and transfected HLECs.Quantitative RT-PCR,cell immunochemistry and Western blot analysis were conducted to detect the expression changes of CTGF and α-SMA after transfection.RESULTS: A highly effective transfection ratio was observed in siRNA co-transfected with lipofectamine.The transfection ratio reached 95% at 24h.The proliferation of HLECs was inhibited by siRNA after 72h transfection.The expression of CTGF and α-SMA significantly decreased in HLECs after transfected by CTGF siRNA for 24h.This effect was not found in negative control siRNA.CONCLUSIONS: SiRNA targeting CTGF decreased CTGF and α-SMA expression in HLECs,which is a potential therapeutic strategy for posterior capsular opacification.

Comparison of gene transfection reagents between PEI and lipofectamine / 国际检验医学杂志

Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.

Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low‑speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK‑iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low‑speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low‑speed centrifugation significantly enhances the transfection efficiency of BLOCK‑iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time‑saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

Experimental study on transfection of humanβ-NGF recombinant plasmid into BMSCs of GFP transgenic mouse / 重庆医学

Objective To investigate the biological features of the mouse bone marrow stromal stem cells (BMSCs) transfected by humanβnerve growth factor(β-NGF) .Methods BMSCs of GFP transgenic mouse were isolated and cultured .Theβ-NGF re-combinant plasmid vectors were transferred into the cultured BMSCs by LipofectamineTM 2000 .The expression of β-NGF was detec-ted with ELISA .Results The β-NGF recombinant plasmid vectors were successfully transferred into BMSCs of GFP transgenic mouse ,the expression of β-NGF in transfected cells appeared .In addition ,the expression ofβ-NGF could effectively protect the BM-SCs which were injured in transfection process .Conclusion BMSCs of GFP transgenic mouse after transfecton with human β-NGF have the proliferation and differentiation capacity ,and possess the expression ability of β-NGF protein .

Generation of transgenic mesenchymal stem cells expressing green fluorescent protein as reporter gene using no viral vector in caprine.

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. While there are many ways to label and track cells each with strengths and weakness, the green fluorescent protein (GFP) is a reporter gene commonly employed. In the present study, caprine MSC were collected from bone marrow and cells were characterised with MSC specific markers. Passage 10 (P10) MSC cells were transfected using plasmid vector containing GFP as reporter gene with different concentrations of DNA and lipofectamine. Six different concentrations of DNA and lipofectamine as 1 µg DNA: 2 µL lipofectamine, 1 µg DNA: 2.5 µL lipofectamine, 1.2 µg DNA: 2.2 µL lipofectamine, 1.2 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 3 µL lipofectamine were used. After 24 h and 48 h of transfection, caprine MSC were observed under florescent microscope. Highest transfection rate indicating green flourecscent MSC were found when the cells were transfected with 1.2 µg DNA: 2.2 µL lipofectamine and 1.5 µg DNA: 2.5 µL lipofectamine than other combinations. These cells have been propagated beyond 4th passage maintaining GFP expression. The results indicated that stable GFP positive MSC cells can be generated using the above protocol. These cells are being used for transplantation studies.

Inhibitory effect of vascular endothelial growth factor siRNA on retinal neovascularization in mouse / 中华实验眼科杂志

Background To suppress vascular endothelial growth factor (VEGF) is a researching hot topic for the treatment and prevention of retinal neovascularization.Some detectable efficacy of VEGF small interference RNA (VEGF siRNA) in anti-tumor neovascularization has been well-known.But relevant study on VEGF siRNA on retinal neovascularization is seldom.Objective Present study was to investigate the inhibiting effect of VEGF siRNA on retinal neovascularization.Methods The 48 clean C57BL/6J mice aged 7-day-old were randomly divided into normoxia group,hypoxia control group,vector group and VEGF siRNA group and 12 mice for each.Hypoxia models were established by raising the pups with mother mice in the airtight oxygen-cabin for 5 days.The lipofectamineTM 2000 (LF2000)-mediated vector plasmids or VEGF siRNA recombinant plasmids were then injected intravitreally in 12 12-day-old pup mice respectively.The animals were sacrificed in 1 week after intravitreal injection,and the numbers of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM) were counted by hematoxylin-eosin stain.The expressions of VEGF protein and mRNA in retina were assayed by immunoinfluorescence technique and RT-PCR.Results The numbers of vascular endothelial cell nuclei extending beyond the ILM were 0.19±0.09,24.89±2.03,23.65±2.15 and 8.83±1.12 in normoxia group,model control group,vector group and VEGF siRNA group separately,showing significant decrease in VEGF siRNA group compared with model control group or vector group (q=5.67,q=4.97,P＜0.01).RT-PCR revealed that VEGF mRNA was faintly expressed in mouse retina in normoxia group.However,in model control group and vector group,the level of VEGF mRNA was 52.3 times and 36.7 times more than that of normoxia group respectively and only 3.5 times in VEGF siRNA group,presenting a inhibitory rate of 43.39% of VEGF siRNA on VEGF.Immunofluorescence showed that the expression of VEGF was weaker in normoxia group and strong positive response in model control group and vector group,but the expression intensity of VEGF protein was significantly weaker in VEGF siRNA group.Conclusion VEGF siRNA recombinant plasmids can efficiently inhibit retinal neovascularization in oxygen-induced retinopathy mouse model through intravitreal injection.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

The anti-tumor effect of lipofectamine mediated interferon-? gene therapy on experimental colon cancer model / 中国普通外科杂志

Objective To investigate the anti-tumor effect of interferon-?(IFN-?) gene therapy on colon cancer. Methods BALB/c mice were inoculated subcutaneously with CT26 (murine colon carcinoma cell line) cells to prepare an in vivo tumor model. Eight days after tumor inoculation,the tumor-bearing mice were divided into 3 groups and injected intra-tumorally with one of the following preparations: pcDNA3-IFN-?/Lipofectamine,pcDNA3 /Lipofectamine,and PBS respectively. The expression of IFN-?,the immunity function of mice,the histological changes of the tumor,the tumor volume in tumor-bearing mice were tested after gene therapy. ResultsThe level of IFN-? in the serum and the CTL activity increased significantly( P

Vaccine preparation of dendritic cell transfected with HPV16E6 antigen gene and its biological characteristics / 中国免疫学杂志

Objective:To prepare the vaccine of DC derived from human peripheral blood and transfected with HPV16E6 antigen gene, and to detect its morphological character,surface marker and immunological effect.Methods:DC-enriched populations were prepared from human peripheral blood mononuclear cell(PBMC) with the combination of rhGM-CSF,rhIL-4 and rhTNF-?. The plasmid containing HPV16E6 gene was transfected into DC with lipofectamine. The morphology of DC was observed dynamically, and the expression of surface markers of DC vaccine could be detected using immuno-cytochemical staining and flow cytometry. MTT assay was applied to detect the activity of CTL in vitro.Results:The transfected DC had typical morphologic and phenotypic characteristics, and expressed E6 protein 47.3%, CD80 82.5%, CD86 79.8% and CD83 85.7%. The killing activities of CTL to Caski cells induced by transfected DC were higher evidently than that of control groups(P

Camparision of the transfection characteristic of Dosper and Lipofectamine / 中国普通外科杂志

0.05); in with ser um condition, the transfection efficiency in Dosper group was significant hi gher than that in Lipofectamine group (P