bFGF和CTLA4-Ig基因修饰的RPE细胞建立及其表达特性研究
发布时间：2019-05-20 03:28
【摘要】：目的 应用慢病毒载体系统构建出稳定、高效表达碱性成纤维细胞生长因子和细胞毒性T淋巴细胞相关抗原4免疫球蛋白的视网膜色素上皮细胞,并检测其对视网膜色素上皮细胞表达特性的影响。 方法 1.稳定表达bFGF的视网膜色素上皮细胞的构建：将bFGF勺基因克隆到慢病毒表达载体pLVX-IRES-ZsGreenl中,转染293T细胞,包装出重组慢病毒,测定病毒滴度,感染视网膜色素上皮细胞,western blot方法检测各组中bFGF的表达。 2.稳定表达CTLA4-Ig的视网膜色素上皮细胞的构建：分离人外周血单个核细胞,提取细胞总RNA,经RT-PCR分别扩增CTLA4胞外段及IgG恒定区基因片段,将PCR产物克隆入质粒pCDNA3,获得CTLA4-Ig。将目的基因CTLA4-Ig克隆到慢病毒表达载体pLVX-IRES-ZsGreenl中,构建重组慢病毒载体。转染293细胞包装产生重组慢病毒,进一步感染视网膜色素上皮细胞,western blot测定CTLA4Ig表达情况。 3.基因修饰后视网膜色素上皮细胞增殖情况的检测：MTT法检测：转染后RPE细胞克隆接种于96孔培养板中,培养24小时后加入MTT,再以DMSO溶解结晶物,酶联免疫检测仪测定光吸收值(OD值),绘制生长曲线。以未转染视网膜色素上皮细胞为对照。 结果 1.酶切鉴定及基因测序表明成功了构建携带bFGF基因的慢病毒表达载体,包装后获得滴度为5×107IU/mL的重组慢病毒,western blot结果证实感染后1周、4周、12周的视网膜色素上皮细胞均可检测出bFGF的表达。 2.成功获得目的基因CTLA4-Ig。酶切鉴定及基因测序表明成功了构建携带CTLA4-Ig基因的慢病毒表达载体,包装后获得滴度为4×107IU/mL的重组慢病毒,western blot结果证实感染后1周、4周、12周的视网膜色素上皮细胞均可检测出CTLA4-Ig的表达。 3.各时间点转染bFGF细胞组、正常细胞组及转染CTLA4Ig细胞组OD平均值进行比较无明显差异(P0.05)。对各组数据应用SPSS11.0进行拟合生长曲线模型,各组模型曲线变化趋势基本相同。结论 应用慢病毒载体系统,可以成功建立稳定表达bFGF和CTLA4-Ig的视网膜色素上皮细胞,将有望解决视网膜色素上皮细胞移植治疗视网膜色素变性中的供体不足及移植过程中发生的免疫排斥反应及排斥反应带来的降低细胞因子表达等不良的影响。转染细胞产生较低水平的细胞因子未引起视网膜色素上皮细胞的异常增殖。
[Abstract]:Objective to construct retinal pigment epithelial cells with stable and high expression of basic fibroblast growth factor and cytotoxicity T lymphocytes associated antigen 4 immunoglobulin by lentivirus vector system. The effect of retinal pigment epithelial cells on the expression of retinal pigment epithelial cells was detected. Method 1. Construction of retinal pigment epithelial cells stably expressing bFGF: the bFGF spoon gene was cloned into lentivirus expression vector pLVX-IRES-ZsGreenl, transfected into 293T cells, packaged with recombinant lentivirus, the virus titer was determined, and the retinal pigment epithelial cells were infected. The expression of bFGF in each group was detected by western blot. 2. Construction of retinal pigment epithelial cells stably expressing CTLA4-Ig: human peripheral blood mononuclear cells were isolated and total RNA, was extracted and the extracellular segment and IgG constant region gene fragment of CTLA4 were amplified by RT-PCR, and the PCR product was cloned into plasmid pCDNA3,. Get CTLA4-Ig. The target gene CTLA4-Ig was cloned into lentivirus expression vector pLVX-IRES-ZsGreenl and the recombinant lentivirus vector was constructed. The recombinant lentivirus was produced in the package of 293cells, and the expression of CTLA4Ig in retinal pigment epithelial cells (RPE) was detected by, western blot. 3. Detection of proliferation of retinal pigment epithelial cells after gene modification: MTT assay: the cloned RPE cells were inoculated in 96-well culture plate after 24 hours of culture, then MTT, was added and DMSO was used to dissolve the crystals. The light absorption value (OD value) was measured by enzyme-linked immunosorbent assay (Elisa), and the growth curve was drawn. Untransfected retinal pigment epithelial cells were used as control. Result 1. Restriction enzyme digestion and gene sequencing showed that the lentivirus expression vector carrying bFGF gene was successfully constructed. The recombinant lentivirus, western blot with titer of 5 脳 107IU/mL was obtained after packaging. The results confirmed that 1 week and 4 weeks after infection. The expression of bFGF was detected in retinal pigment epithelial cells at 12 weeks. 2. The target gene CTLA4-Ig. was successfully obtained. Restriction endonuclease digestion and gene sequencing showed that the lentivirus expression vector carrying CTLA4-Ig gene was successfully constructed. The recombinant lentivirus, western blot with titer of 4 脳 107IU/mL was obtained after packaging. The results confirmed that 1 week and 4 weeks after infection. The expression of CTLA4-Ig was detected in retinal pigment epithelial cells at 12 weeks. 3. There was no significant difference in the average value of OD between bFGF cell group, normal cell group and CTLA4Ig cell group at each time point (P 0.05). SPSS11.0 was used to fit the growth curve model, and the change trend of each model curve was basically the same. Conclusion Retinal pigment epithelial cells stably expressing bFGF and CTLA4-Ig can be successfully established by lentivirus vector system. It will be expected to solve the adverse effects of retinal pigment epithelial cell transplantation on the lack of donors in the treatment of retinitis pigmentosa and the reduction of cytokine expression caused by immune rejection and rejection during transplantation. The production of low levels of cytokines did not cause the abnormal proliferation of retinal pigment epithelial cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
本文编号：2481308
[Abstract]:Objective to construct retinal pigment epithelial cells with stable and high expression of basic fibroblast growth factor and cytotoxicity T lymphocytes associated antigen 4 immunoglobulin by lentivirus vector system. The effect of retinal pigment epithelial cells on the expression of retinal pigment epithelial cells was detected. Method 1. Construction of retinal pigment epithelial cells stably expressing bFGF: the bFGF spoon gene was cloned into lentivirus expression vector pLVX-IRES-ZsGreenl, transfected into 293T cells, packaged with recombinant lentivirus, the virus titer was determined, and the retinal pigment epithelial cells were infected. The expression of bFGF in each group was detected by western blot. 2. Construction of retinal pigment epithelial cells stably expressing CTLA4-Ig: human peripheral blood mononuclear cells were isolated and total RNA, was extracted and the extracellular segment and IgG constant region gene fragment of CTLA4 were amplified by RT-PCR, and the PCR product was cloned into plasmid pCDNA3,. Get CTLA4-Ig. The target gene CTLA4-Ig was cloned into lentivirus expression vector pLVX-IRES-ZsGreenl and the recombinant lentivirus vector was constructed. The recombinant lentivirus was produced in the package of 293cells, and the expression of CTLA4Ig in retinal pigment epithelial cells (RPE) was detected by, western blot. 3. Detection of proliferation of retinal pigment epithelial cells after gene modification: MTT assay: the cloned RPE cells were inoculated in 96-well culture plate after 24 hours of culture, then MTT, was added and DMSO was used to dissolve the crystals. The light absorption value (OD value) was measured by enzyme-linked immunosorbent assay (Elisa), and the growth curve was drawn. Untransfected retinal pigment epithelial cells were used as control. Result 1. Restriction enzyme digestion and gene sequencing showed that the lentivirus expression vector carrying bFGF gene was successfully constructed. The recombinant lentivirus, western blot with titer of 5 脳 107IU/mL was obtained after packaging. The results confirmed that 1 week and 4 weeks after infection. The expression of bFGF was detected in retinal pigment epithelial cells at 12 weeks. 2. The target gene CTLA4-Ig. was successfully obtained. Restriction endonuclease digestion and gene sequencing showed that the lentivirus expression vector carrying CTLA4-Ig gene was successfully constructed. The recombinant lentivirus, western blot with titer of 4 脳 107IU/mL was obtained after packaging. The results confirmed that 1 week and 4 weeks after infection. The expression of CTLA4-Ig was detected in retinal pigment epithelial cells at 12 weeks. 3. There was no significant difference in the average value of OD between bFGF cell group, normal cell group and CTLA4Ig cell group at each time point (P 0.05). SPSS11.0 was used to fit the growth curve model, and the change trend of each model curve was basically the same. Conclusion Retinal pigment epithelial cells stably expressing bFGF and CTLA4-Ig can be successfully established by lentivirus vector system. It will be expected to solve the adverse effects of retinal pigment epithelial cell transplantation on the lack of donors in the treatment of retinitis pigmentosa and the reduction of cytokine expression caused by immune rejection and rejection during transplantation. The production of low levels of cytokines did not cause the abnormal proliferation of retinal pigment epithelial cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
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