非病毒载体介导Mn-SOD基因在小鼠肝脏的高表达

目的:研究非病毒载体介导Mn-SOD基因在小鼠肝脏的转染效率。方法:构建Mn-SOD真核表达质粒gWiz/Mn-SOD,通过肠系膜上静脉分支注射阳离子脂质体/质粒混合物,采用RT-PCR、Westernblot、SOD活力测定以及免疫组织化学染色等方法,观察小鼠肝脏Mn-SOD基因表达情况。结果:经肠系膜上静脉分支注射质粒/脂质体混合物几乎不对肝脏造成损伤,注射后8h即可检测到小鼠肝脏有明显的外源性Mn-SODmRNA和蛋白表达;转染前及转染后72h肝细胞线粒体SOD活性分别是(29±17)U/g和(272±56)U/g(P<0.01);免疫组化显示小鼠肝细胞转染率达70%。结论:阳离子脂质体可介导非病毒载体gWiz/Mn-SOD在小鼠肝脏高效安全表达。

Non-viral vector-mediated high efficiency expression of manganese superoxide dismutase gene in mouse liver

AIM: To investigate the transfection efficiency of mouse liver with non-viral vector containing manganese superoxide dismutase (Mn-SOD) gene. METHODS: The eukaryotic expression vector, gWiz/Mn-SOD, encoding human manganese superoxide dismutase was constructed. The plasmids of gWiz/Mn-SOD were mixed with cationic lipids, followed by injection into mice via branch of superior mesenteric vein, to induce Mn-SOD over-expression in murine liver detected by RT-PCR, Western blotting, SOD activity and immunohistochemical staining. RESULTS: gWiz/Mn-SOD transfection resulted in the obvious expression of exogenous Mn-SOD mRNA and protein in hepatic tissues at 8 hours after injection, and elevated mitochondria SOD activity 8.4 times in transfected hepatocytes than that in non-transfected cells at 72 hours after injection. It was showed that nearly 70% of mouse hepatocytes was obviously Mn-SOD positive after transfection. CONCLUSION: High expression efficiency of Mn-SOD gene in mouse liver is achieved safely by injection of gWiz/Mn-SOD and cationic lipid mixture into branch of superior mesenteric vein.