转染真核表达载体pGFAP-IRES2-EGFP-p27对星形胶质细胞增殖的抑制作用

目的:探讨脂质体介导真核表达载体pGFAP-IRES2-EGFP-p27转染体外培养星形胶质细胞后对其增殖的影响。方法:利用脂质体将质粒pGFAP-IRES2-EGFP-p27转染入纯化培养的星形胶质细胞,通过免疫荧光化学标记观察转染质粒pGFAP-IRES2-EGFP-p27后星形胶质细胞内外源基因EGFP、p27的表达情况,并观察其对星形胶质细胞Ki67表达的影响。结果:脂质体介导质粒pGFAP-IRES2-EGFP-p27转染后24 h即开始有EGFP的表达,48-72 h EGFP表达达高峰;通过免疫荧光标记发现,表达EGFP的细胞同时有p27表达水平明显增高;和EGFP阴性细胞相比,EGFP阳性细胞中Ki67的阳性率明显下调(P<0.01)。结论:GFAP启动子能启动目的基因p27和EGFP在星形胶质细胞的表达;连于p27的下游EGFP可作为报告基因,通过观察EGFP的表达可了解外源性p27的表达情况;导入外源性p27能有效抑制星形胶质细胞的增殖。

The Inhibitive Effect of Transfection of Eukaryotic Bicistronic Expression Vector pGFAP-IRES2-EGFP-p27 on Astrocytic Proliferation in vitro

Objective: The effect of transfected liposome-mediated eukaryotic bicistronic expression vector pGFAP-IRES2-EGFP-p27 on astrocytic proliferation was explored in vitro.Methods: The plasmid pGFAP-IRES2-EGFP-p27 was transfected to cultured astrocytes with liposome,then the expressions of EGFP,p27 and Ki67 in astrocytes were observed by immunocytochemistry. Results: Fluorescent microscopy revealed that the expression of EGFP could be detectable at 24 h after the plasmid was transfected to the astrocytes,and the expression of EGFP peaked at 48-72 h.Meanwhile,expression of p27 increased significantly in EGFP-positive cells,while the rate of Ki67-positive cells was much lower in EGFP-positive cells than that in EGFP-negative cells(P<0.01). Conclusions: The vectors containing the GFAP promoter resulted in EGFP and p27 expression in astrocytes.EGFP can be a reporter gene to indicate the expression of p27.Astrocytic proliferation can be inhibited significantly by transduction of exogenous gene p27.