【摘要】：目的 應用慢病毒載體系統(tǒng)構建出穩(wěn)定、高效表達堿性成纖維細胞生長因子和細胞毒性T淋巴細胞相關抗原4免疫球蛋白的視網膜色素上皮細胞,并檢測其對視網膜色素上皮細胞表達特性的影響。 方法 1.穩(wěn)定表達bFGF的視網膜色素上皮細胞的構建：將bFGF勺基因克隆到慢病毒表達載體pLVX-IRES-ZsGreenl中,轉染293T細胞,包裝出重組慢病毒,測定病毒滴度,感染視網膜色素上皮細胞,western blot方法檢測各組中bFGF的表達。 2.穩(wěn)定表達CTLA4-Ig的視網膜色素上皮細胞的構建：分離人外周血單個核細胞,提取細胞總RNA,經RT-PCR分別擴增CTLA4胞外段及IgG恒定區(qū)基因片段,將PCR產物克隆入質粒pCDNA3,獲得CTLA4-Ig。將目的基因CTLA4-Ig克隆到慢病毒表達載體pLVX-IRES-ZsGreenl中,構建重組慢病毒載體。轉染293細胞包裝產生重組慢病毒,進一步感染視網膜色素上皮細胞,western blot測定CTLA4Ig表達情況。 3.基因修飾后視網膜色素上皮細胞增殖情況的檢測：MTT法檢測：轉染后RPE細胞克隆接種于96孔培養(yǎng)板中,培養(yǎng)24小時后加入MTT,再以DMSO溶解結晶物,酶聯(lián)免疫檢測儀測定光吸收值(OD值),繪制生長曲線。以未轉染視網膜色素上皮細胞為對照。 結果 1.酶切鑒定及基因測序表明成功了構建攜帶bFGF基因的慢病毒表達載體,包裝后獲得滴度為5×107IU/mL的重組慢病毒,western blot結果證實感染后1周、4周、12周的視網膜色素上皮細胞均可檢測出bFGF的表達。 2.成功獲得目的基因CTLA4-Ig。酶切鑒定及基因測序表明成功了構建攜帶CTLA4-Ig基因的慢病毒表達載體,包裝后獲得滴度為4×107IU/mL的重組慢病毒,western blot結果證實感染后1周、4周、12周的視網膜色素上皮細胞均可檢測出CTLA4-Ig的表達。 3.各時間點轉染bFGF細胞組、正常細胞組及轉染CTLA4Ig細胞組OD平均值進行比較無明顯差異(P0.05)。對各組數(shù)據(jù)應用SPSS11.0進行擬合生長曲線模型,各組模型曲線變化趨勢基本相同。結論 應用慢病毒載體系統(tǒng),可以成功建立穩(wěn)定表達bFGF和CTLA4-Ig的視網膜色素上皮細胞,將有望解決視網膜色素上皮細胞移植治療視網膜色素變性中的供體不足及移植過程中發(fā)生的免疫排斥反應及排斥反應帶來的降低細胞因子表達等不良的影響。轉染細胞產生較低水平的細胞因子未引起視網膜色素上皮細胞的異常增殖。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R774.1

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