疱疹性口腔炎病毒G蛋白/胸苷激酶逆转录病毒载体转移人视网膜色素上皮细胞及其表达

目的　应用新型逆转录病毒载体介导单纯疱疹病毒胸苷激酶 (herpessimplexvirus thymidinekinase ,HSV TK)基因转移 ,探讨丙氧鸟苷 (ganciclovir,GCV)对HSV TK基因修饰人视网膜色素上皮细胞 (retinalpigmentepithelialcells,RPE)的杀伤抑制作用。方法　生产制备高滴度疱疹性口腔炎病毒G蛋白 (vesicularstomatitisvirus Gprotein ,VSV G) /胸苷激酶 (thymidinekinase ,TK)和VSV G/LacZ病毒 ,对NIH3T3细胞进行VSV G/TK滴度测定 ;VSV G/LacZ感染CRL2 30 2细胞后 ,以X gal染色估计感染率 ;对CRL2 30 2细胞、分离培养的RPE细胞及NIH3T3细胞感染不同病毒滴度的VSV G/TK ,结合GCV作用 ,观察 3种细胞生长抑制率。结果　VSV G/TK滴度为 1 2× 10 8克隆形成单位 /ml;VSV G/LacZ感染CRL2 30 2细胞的X gal染色蓝染细胞率为 5 8% ;当感染复数为 2 0 0时可最大的抑制细胞生长 ,抑制率为 4 5 %。结论　VSV G逆转录病毒可以介导LacZ、HSV TK基因在离体NIH3T3细胞和人RPE细胞中高效转移和表达。被HSV TK基因修饰的细胞对GCV作用敏感 ,其生长受到抑制。

Vesicular stomatitis virus G-protein retrovector mediated a herpes simplex virus thymidine kinase gene transduction and expression in the human retinal pigment epithelial cells

OBJECTIVE: The herpes simplex virus-thymidine kinase suicide gene (HSV-TK) was delivered to the human retinal pigment epithelial cell (RPE) by a new vesicular stomatitis virus G protein (VSV-G) retroviral vector. The growth inhibitory effects of ganciclovir on VSV-G/HSV-TK transfected RPE were studied. METHODS: A VSV-G retrovirus-packaging cell line 293GPG was transferred with retroviral vector plasmas bearing HSV-TK (G1NaCTK) or galactosidase (LacZ gene, G1BgSvNa) to produce a 293GPG/TK cell line or a 293GPG/LacZ cell line, respectively. LacZ activity was assessed following transduction of CRL2302 cells. HSV-TK transfected RPE and NIH3T3 cells (at different titer levels) were treated with ganciclovir. The growth inhibitory effects of ganciclovir on various cell lines were studied. RESULTS: The titer of VSV-G/HSV-TK concentration was 1.2 x 10(8) cfu/ml. At a high titer (MOI = 200), the efficiency of LacZ gene transfer in CRL2302 cells was 58%. Ganciclovir inhibited the growth of cells transfected by HSV-TK. The maximum inhibitory rate (45%) was obtained at cells with a high titer (MOI = 200). CONCLUSION: Our results suggested that VSV-G/LacZ and VSV-G/HSV-TK retroviral vectors are highly efficient for in vitro delivery and stable expression of genes in NIH3T3 cells and human RPE cells. The cells modified by HSV-TK gene are very sensitive to ganciclovir and the cell growth can be inhibited by ganciclovir.