大鼠胚胎肝干细胞的分离、鉴定及转染绿色荧光蛋白基因的研究

Isolation,identification and transfection of green fluorescent protein gene of rat embryonic hepatic stem cells

目的分离、培养大鼠胚胎肝干细胞，检测肝干细胞表面标志CD49f，c-Met，建立携带示踪标记的大鼠肝干细胞。方法Percoll梯度离心法分离孕14d大鼠胚胎肝细胞，体外培养后采用流式细胞术（FACS）检测CD49f和c-Met，pAcGFP1-N1质粒提取并经电泳鉴定后用脂质体法转染细胞。结果成功分离并纯化了孕14d大鼠胚胎肝细胞；CD49f和c—Met阳性细胞的比例分别为65．0％和85．9％；电泳鉴定质粒正确，成功转染细胞后荧光显微镜观察到大量表达绿色荧光蛋白（GFP）的肝干细胞。结论从孕14d大鼠胎肝中成功分离并鉴定肝干细胞，成功转染pAcGFP1-N1质粒，建立携带稳定表达GFP示踪标记的大鼠肝干细胞。

To isolate and culture rat embryonic hepatic stem cells, detect the markers of hepatic stem cells CD49f and c-Met, and establish a rat hepatic stem cell line with trace labeling. Methods The embryonic day 14 fetal rat liver ceils were isolated by density gradient centrifugation. After cells cultured in vitro, two cell markers CD49f and c-Met were detected by fluorescence activated cells sorting （FACS）. The plasmid DNA was extracted from pAcGFP1-N1 and identified by electrophoresis. By using liposome-mediated transfection, the green fluorescent protein gene was transferred into rat embryonic hepatic stem cells. Results In the cells isolated from rat embryonic liver, the proportion of CD49f and c-Met positive cells was 65.0% and 85.9% ,respectively. The plasmid DNA was identified correctly by electrophoresis. Green fluorescence was observed in a great quantity of stem cells which has been transferred by pAcGFP1-N1. Conclusion The rat embryonic hepatic stem cells were isolated successfully from the embryonic day 14 fetal rat liver. When the plasmid DNA of pAcGFP1-N1 was transferred into the rat hepatic stem cells ,the cells expressed green fluorescent protein stably, and a rat hepatic stem cell line with trace labeling was established successfully.