肝特异性非病毒基因治疗载体介导增强型绿色荧光蛋白基因在小鼠肝细胞及肝脏中的表达

Hepatocyte specific gene therapy vector mediates expression of EGFP in mouse hepatocytes and liver

目的 观察肝细胞特异性基因治疗载体蛋白介导增强型绿色荧光蛋白 (EGFP)在体外培养小鼠肝细胞和小鼠肝脏中表达。方法 将基因工程制备的肝细胞特异载体蛋白与真核表达质粒以所带电荷比为 1∶1的比例混合 ,两者结合形成蛋白 DNA复合物 ,将该复合物加入体外培养的小鼠肝细胞培养基中或经门静脉注射小鼠体内 ,用激光共聚焦显微镜观察EGFP在体外培养小鼠肝细胞及活体小鼠肝脏组织中的表达。结果 在体外培养的小鼠肝细胞和小鼠肝脏切片中均可以观察到明显的绿色荧光 ,而对照组未能观察到绿色荧光蛋白。结论 构建的肝细胞特异性载体蛋白可以有效的将真核表达质粒载体pcDNA3 EGFP带入肝细胞和活体肝脏组织。

Objective To investigate hepatocyte specific gene therapy vector mediates eukaryotic expression plasmid pcDNA3-EGFP to express EGFP in mouse hepatocytes in vitro and mouse liver in vivo.Methods Hepatocyte specific binding vector protein was expressed in E.Coli BL21 (DE3) and purified by immobilized metal affinity expanded bed adsorption (IMAEBA),then vector protein was blended with plasmid pcDNA3-EGFP to form polyplex.The polyplex was added into the hepatocyte medium or injected through mouse portal vein.The EGFP expression was detected by a confocal laser scanning microscope.Results Obvious green fluorescence was observed in cultured mouse hepatocytes and mouse liver,but not in the control groups.Conclusion The hepatocyte specific vector protein can effectively deliver the plasmid pcDNA3-EGFP into cultured mouse hepatocytes and mouse liver,which may provide an effective vector for hepatocyte gene therapy.