复制缺陷型逆转录病毒载体介导基因工程化视网膜色素上皮细胞的制备

Preparation of genetically engineered retinal pigment epithelial cells capable of secreting nerve growth factor

目的 制备稳定高表达生物活性神经生长因子 (nervegrowthfactor,NGF)工程视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞 ,拟将转基因基因治疗与视网膜移植技术结合 ,用于视网膜变性疾病的治疗。方法 首先经亚克隆和克隆构建复制缺陷性逆转录病毒质粒重组体pLXSN NGF ,然后经包装细胞pT6 7包装 ,获得含有NGF基因的复制缺陷性逆转录病毒重组体 ;NIH3T3细胞测定其滴度 ,然后转导培养RPE细胞 ;反转录聚合酶链反应和Westernblot分析NGF基因在RPE细胞中的表达 ;PC12细胞测定表达产物的生物活性。结果 复制缺陷性逆转录病毒滴度为1 2× 10 7cfu/ml,在转导RPE细胞及其条件培养液中 ,可检测出NGFmRNA和蛋白的存在 ,PC12细胞经转导RPE细胞条件培养液刺激长出轴突样突触。结论 RPE细胞能高效地被复制缺陷性逆转录病毒转导 ,高水平分泌表达了生物活性NGF ,RPE细胞能成为一种理想的工程细胞。

Objective To develop genetically engineered retinal pigment epithelial (RPE) cells which are able to express bioactive nerve growth factor (NGF) steadily and efficiently and to combine gene therapy with retinal transplantation techniques to treat retinal degeneration. Methods Replication deficient retrovirus plasmid recombinant pLXSN NGF was constructed via subclone and clone, and then packaged into pT67 cells to obtain virion containing a secretable form of NGF gene. We evaluated titer of the retroviral recombinant with NIH 3T3 cells and then transduced NGF gene into cultured RPE cells by the retroviral recombinant. We also analyzed the expression of NGF gene in transduced and untransduced RPE cells via reverse transcription (RT) PCR and Western blot. The bioactivity of expressed NGF were evaluated by stimulating PC 12 cells with conditioned medium from transduced and untransduced RPE cells. Results The titer of replication deficient retroviral recombinant reached 1 2×10 7 cfu/ml. NGF protein and mRNA were detected in the conditioned medium from transduced RPE cells, but not from the normal RPE cells. Marked neurite process outgrowth from PC 12 cells was observed after being stimulated with the conditioned medium from transduced RPE cells. Conclusions RPE cells are able to be transduced by replication deficient retrovirus with high efficiency, and secret bioactive NGF at a high level, which might be an ideal candidate for engineered cells.