目的研究ski基因对大鼠星形胶质细胞增殖的影响及其分子机制。方法分离3日龄Sprague-Dawley大鼠脑皮质星形胶质细胞,体外培养。合成ski和阴性对照小干扰片段。设置ski-siRNA组、阴性对照组和空白对照组,采用脂质体法将特异性针对ski基因的siRNA和阴性对照siRNA转入ski-siRNA组、阴性对照组星形胶质细胞中。Western blotting分析ski、胶质纤维酸性蛋白(GFAP)和Cyclin D1蛋白表达;CCK8法检测星形胶质细胞增殖活力;流式细胞仪检测细胞周期。结果ski-siRNA组星形胶质细胞ski蛋白表达明显降低(F=38.611, P<0.01),GFAP (F=7.547, P<0.05)和Cyclin D1蛋白(F=3.901, P<0.05)表达降低;培养12 h后,ski-siRNA组增殖活力明显降低(F>30.507, P<0.01),G1期细胞比例明显增加,S期明显减少(F>48.425, P<0.01)。结论沉默ski基因可能通过下调Cyclin D1表达,抑制星形胶质细胞增殖。
ObjectiveTo investigate the role of ski in proliferation of astrocytes and the molecular mechanisms in rats. MethodsAstrocytes were obtained from cerebral cortex of a three-day old rat and cultured in vitro. siRNA targeted to ski and negative control sequences were prepared. The astrocytes were divided into ski-siRNA group, siRNA negative control group and untreated control group, while the specific siRNA targeting ski negative control sequences were transfected into astrocytes with Lipofectamine® RNAiMAX Reagent. The protein levels of ski, glial fibrillary acidic protein (GFAP) and Cyclin D1 were determined with Western blotting. The proliferation of astrocytes were measured with CCK8 assay. The cell-cycle of astrocytes were analyzed with flow cytometer. ResultsThe protein level of ski (F=38.611, P<0.01), GFAP (F=7.547, P<0.05) and Cyclin D1 (F=3.901, P<0.05) reduced in ski-siRNA group, the proliferation of astrocyte was significantly inhibited since twelve hours after culture (F>30.507, P<0.01), and less cells were in S phase and more in G1/G0 phase (F>48.425, P<0.01), compared with the control groups. Conclusionski knocking down by siRNA significantly inhibits the proliferation of astrocytes, which may associate with the down-regulation of Cyclin D1 expression.
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