【摘要】：目的 應(yīng)用慢病毒載體系統(tǒng)構(gòu)建出穩(wěn)定、高效表達(dá)堿性成纖維細(xì)胞生長因子和細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4免疫球蛋白的視網(wǎng)膜色素上皮細(xì)胞,并檢測其對視網(wǎng)膜色素上皮細(xì)胞表達(dá)特性的影響。 方法 1.穩(wěn)定表達(dá)bFGF的視網(wǎng)膜色素上皮細(xì)胞的構(gòu)建：將bFGF勺基因克隆到慢病毒表達(dá)載體pLVX-IRES-ZsGreenl中,轉(zhuǎn)染293T細(xì)胞,包裝出重組慢病毒,測定病毒滴度,感染視網(wǎng)膜色素上皮細(xì)胞,western blot方法檢測各組中bFGF的表達(dá)。 2.穩(wěn)定表達(dá)CTLA4-Ig的視網(wǎng)膜色素上皮細(xì)胞的構(gòu)建：分離人外周血單個核細(xì)胞,提取細(xì)胞總RNA,經(jīng)RT-PCR分別擴(kuò)增CTLA4胞外段及IgG恒定區(qū)基因片段,將PCR產(chǎn)物克隆入質(zhì)粒pCDNA3,獲得CTLA4-Ig。將目的基因CTLA4-Ig克隆到慢病毒表達(dá)載體pLVX-IRES-ZsGreenl中,構(gòu)建重組慢病毒載體。轉(zhuǎn)染293細(xì)胞包裝產(chǎn)生重組慢病毒,進(jìn)一步感染視網(wǎng)膜色素上皮細(xì)胞,western blot測定CTLA4Ig表達(dá)情況。 3.基因修飾后視網(wǎng)膜色素上皮細(xì)胞增殖情況的檢測：MTT法檢測：轉(zhuǎn)染后RPE細(xì)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(jié)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(zhuǎn)染視網(wǎng)膜色素上皮細(xì)胞為對照。 結(jié)果 1.酶切鑒定及基因測序表明成功了構(gòu)建攜帶bFGF基因的慢病毒表達(dá)載體,包裝后獲得滴度為5×107IU/mL的重組慢病毒,western blot結(jié)果證實(shí)感染后1周、4周、12周的視網(wǎng)膜色素上皮細(xì)胞均可檢測出bFGF的表達(dá)。 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(gòu)建攜帶CTLA4-Ig基因的慢病毒表達(dá)載體,包裝后獲得滴度為4×107IU/mL的重組慢病毒,western blot結(jié)果證實(shí)感染后1周、4周、12周的視網(wǎng)膜色素上皮細(xì)胞均可檢測出CTLA4-Ig的表達(dá)。 3.各時間點(diǎn)轉(zhuǎn)染bFGF細(xì)胞組、正常細(xì)胞組及轉(zhuǎn)染CTLA4Ig細(xì)胞組OD平均值進(jìn)行比較無明顯差異(P0.05)。對各組數(shù)據(jù)應(yīng)用SPSS11.0進(jìn)行擬合生長曲線模型,各組模型曲線變化趨勢基本相同。結(jié)論 應(yīng)用慢病毒載體系統(tǒng),可以成功建立穩(wěn)定表達(dá)bFGF和CTLA4-Ig的視網(wǎng)膜色素上皮細(xì)胞,將有望解決視網(wǎng)膜色素上皮細(xì)胞移植治療視網(wǎng)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(yīng)及排斥反應(yīng)帶來的降低細(xì)胞因子表達(dá)等不良的影響。轉(zhuǎn)染細(xì)胞產(chǎn)生較低水平的細(xì)胞因子未引起視網(wǎng)膜色素上皮細(xì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R774.1

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