沉默ski基因对大鼠星形胶质细胞活化增殖及Cyclin D1表达的影响  

Effects of Knocking Down ski on Proliferation of Astrocytes and Expression of Cyclin D1 in Rats

作　　者：赵鑫[1,2] 郭永强 寇江力[1,2] 丁宁 周开升 南伟[1,2] 张海鸿 ZHAO Xin;GUO Yong-qiang;KOU Jiang-li;DING Ning;ZHOU Kai-sheng;NAN Wei;ZHANG Hai-hong(Department of Orthopedics, Second Clinical Medical College of Lanzhou University, Lanzhou, Gansu 730030, China;Key Laboratory of Orthopedics of Gansu Province, Lanzhou, Gansu 730030, China)

机构地区：[1]兰州大学第二医院骨科,甘肃兰州市730030 [2]甘肃省骨关节疾病研究重点实验室,甘肃兰州市730030

出　　处：《中国康复理论与实践》2017年第9期1032-1036,共5页Chinese Journal of Rehabilitation Theory and Practice

基　　金：国家自然科学基金项目(No.30772299);兰州大学第二医院科研基金项目(No.sdkyjj-04)

摘　　要：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-si RNA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si RNA和阴性对照si RNA转入ski-si RNA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-si RNA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-si RNA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。Objective To investigate the role of ski in proliferation of astrocytes and the molecular mechanisms in rats.Methods Astrocytes 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 D1were determined with Western blotting.The proliferation of astrocytes were measured with CCK8assay.The cell-cycle of astrocytes were analyzed with flow cytometer.Results The 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/G0phase(F>48.425,P<0.01),compared with the control groups.Conclusion ski knocking down by siRNA significantly inhibits the proliferation of astrocytes,which may associate with the down-regulation of Cyclin D1expression.

关 键 词：SKI RNA干扰 增殖 星形胶质细胞 大鼠 

分 类 号：R338.2[医药卫生—人体生理学]