绿色荧光蛋白逆转录病毒及其介导的视网膜色素上皮细胞基因转移研究

Preparation of green fluorescent protein retrovirus and its application in mediating gene transfer into retinal pigment epithelial cells

目的 制备携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒 ,感染原代与建株的人视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞 ,评价逆转录病毒对人RPE细胞的感染能力 ,为视网膜病变的基因治疗奠定基础。方法 分离编码GFP的cDNA片段并插入逆转录病毒载体pLNCX ,借助脂质体将重组逆转录病毒载体pLNCX GFP转入单嗜性及双嗜性包装细胞 ,G418筛选抗性克隆 ,分离GFP表达水平最高的克隆 ,收集含病毒颗粒的上清液感染NIH3T3及体外培养的人原代与建株的RPE细胞。结果 重组逆转录病毒载体pLNCX GFP转染各种包装细胞后 ,可产生GFP逆转录病毒。由双嗜性包装细胞产生的GFP逆转录病毒能够感染原代与建株的人RPE细胞 ,GFP基因可持续在RPE细胞内表达。结论 逆转录病毒能够简便、快速、稳定将目的基因转入RPE细胞 ,可作为眼底病变基因治疗介导目的基因转移的重要工具。

Objective To determine whether human retinal pigment epithelial (RPE) cells can be infected by retrovirus and modified by retrovirus mediated gene transfer. Methods Recombination retroviral vector pLNCX GFP (green fluorescent protein, GFP) was generated by inserting 780 bp GFP cDNA fragment into the MCS site of pLNCX. pLNCX GFP was transfected into ecotropic packaging cell line ΦX Eco and amphotropic packaging cell line ΦX Ampho and PA317. Retroviral titer was tested by counting GFP expression of NIH3T3 cells. Then RPE cells were infected by using GFP retrovirus containing supernatant. Results GFP was expressed and retrovirus was produced upon pLNCX GFP being transfected into packaging cell line. The GFP retrovirus was able to infect primary cultured human RPE cells and immortalized RPE cell line. Conclusion The retrovirus can introduce a foreign gene into RPE cells efficiently, thereby it can be used as an important tool to deliver gene into RPE for therapy of fundus diseases.