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目的:构建人肌球蛋白2v基因启动子(pMLC2v)驱动的绿色荧光蛋白(GFP)和萤光素酶(Luc)报告基因慢病毒载体并观察其在人心肌细胞系(HCM)和人肺癌细胞株A549中的整合表达特征。方法:应用去毒化的人类I型免疫缺陷病毒和pMLC2v-GFP或pMLC2v-Luc报告基因构建慢病毒示踪载体,转染HCM和A549细胞,激光共聚焦显微镜及生物发光检测仪观察2种报告基因在不同细胞生长进程中的表达特征。非特异性启动子驱动的GFP(GFPC)和红色荧光蛋白(RFPC)报告基因作为对照。结果:2种细胞转染GFPC和RFPC后第3 d,都表达GFP和RFP;而HCM只在转染pMLC2v-GFP和pMLC2v-Luc 21 d后表达GFP和Luc,A549细胞不表达。结论:pMLC2v主要在培养21 d后新增殖的人心肌细胞中驱动GFP和Luc报告基因表达,这为监测干细胞向心肌细胞的分化进程提供了可靠的病理及活体示踪工具。
OBJECTIVE: To construct a lentiviral vector encoding green fluorescent protein (GFP) and luciferase (Luc) reporter gene of human myosin 2 v gene promoter (pMLC2v) and to observe its effect on human cardiomyocyte (HCM) and human lung cancer cells Strains A549 expression characteristics. Methods: The lentivirus tracer vector was constructed by using detoxified human immunodeficiency virus type I and pMLC2v-GFP or pMLC2v-Luc reporter plasmids. HCM and A549 cells were transfected. Two kinds of reporter genes were detected by laser confocal microscopy and bioluminescence detector Expression characteristics in different cell growth. Non-specific promoter-driven GFP (GFPC) and red fluorescent protein (RFPC) reporter genes served as controls. Results: Both GFP and RFP were expressed on the 3rd day after transfected with both GFPC and RFPC. However, HCM expressed GFP and Luc only after transfected with pMLC2v-GFP and pMLC2v-Luc for 21 days, but not in A549 cells. CONCLUSION: pMLC2v mainly drives GFP and Luc reporter gene expression in newly proliferated human cardiomyocytes 21 days after culture, which provides a reliable pathological and in vivo tracking tool for monitoring stem cell differentiation into cardiomyocytes.