构建靶向性高尔基体膜蛋白高尔基体蛋白73修饰脂质体介导单纯疱疹病毒胸苷激酶诱导肝癌细胞凋亡

Construction of a Golgi protein 73 modified liposome that mediates HSVtk induced liver cancer cell apoptosis

目的构建一种具有靶向性的非病毒基因治疗载体高尔基体蛋白73(GP73)-脂质体lipoplexes,并检测其对肝癌细胞Hep G2和正常肝细胞HL-7702的转染效率和凋亡的影响。方法以水溶性碳二亚胺为交联剂将GP73与lipoplexes偶联。用GP73-lipoplexes运载标记有绿色荧光蛋白(green fluorescent protein,GFP)荧光报告基因的PGenesil-l质粒转染肝癌细胞组Hep G2和肝正常细胞组HL-7702,通过荧光显微镜观察被转染细胞GFP的表达情况,流式细胞术检测转染效率。并用GP73-lipoplexes运载含治疗基因HSVtk的质粒PBI-SUR-TK转染肝癌细胞和肝细胞,经RT-PCR和Western blot检测细胞中HSVtk表达情况,流式细胞术和CCK-8检测细胞凋亡情况。结果经GP73修饰的lipoplexes转染的肝癌细胞组[(26.37±0.76)%]比肝细胞组[(9.68±0.57)%]有更高的转染效率和更多的HSVtk基因的表达,肝癌细胞增殖能力明显减弱,凋亡显著。结论 GP73修饰的lipoplexes介导的HSVtk自杀基因系统对肝癌细胞有治疗作用,可以尝试作为一种靶向性杀伤肝癌细胞的非病毒基因转运载体。

Objective To construct a non-viral gene therapy vector（GP73 modified lipoplexes） and to assess its delivery efficiency and impact on apoptosis in hepatoma cell line Hep G2 and normal liver cell line HL-7702. Methods Lipoplexes were conjugated with human GP73 using water-soluble carbodidimide, a crossing agent. Then, GP73-lipoplexes were used to carry p Genesil-l plasmid containing EGFP gene and transfect hepatoma cell line Hep G2 and normal liver cell line HL-7702. Fluorescence microscopy and flow cytometry were used to assess the transfection efficiency. GP73-lipoplexes were then used to carry PBI-SUR-TK plasmid containing the therapeutic gene HSVtk and transfect hepatoma cells and normal hepatocytes. RT-PCR and Western blot were used to detect the expression of HSVtk in cells, and flow cytometry and CCK-8 assay were used to detect apoptosis. Results Compared with HL-7702 cells, Hep G2 cells had higher transfection efficiency [（9.68±0.57）% vs（26.37±0.76）%], obvious HSVtk gene production, and significant apoptosis. Conclusion GP73 modified lipoplexes can be an effective tool to improve the transfection efficiency and therapeutic effects of exogenous genes in hepatoma cells.