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

doi:10.3969/j.issn.1006-9771.2017.09.009

《中国康复理论与实践》 ›› 2017, Vol. 23 ›› Issue (9): 1032-1036.doi: 10.3969/j.issn.1006-9771.2017.09.009

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

赵鑫^{1, 2}, 郭永强^{1, 2}, 寇江力^{1, 2}, 丁宁^{1, 2}, 周开升^{1, 2}, 南伟^{1, 2}, 张海鸿¹

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。

收稿日期:2017-05-25 修回日期:2017-06-22 出版日期:2017-09-25 发布日期:2017-10-10
通讯作者: 张海鸿,主任医师,副教授,硕士研究生导师,主要研究方向：脊柱外科。E-mail: zhanghaihong1968@sina.com。
作者简介:赵鑫(1991-),男,汉族,山东潍坊市人,硕士研究生,主要研究方向：脊柱脊髓损伤。
基金资助:
1.国家自然科学基金项目(No.30772299); 2.兰州大学第二医院科研基金项目(No.sdkyjj-04)

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

ZHAO Xin^{1, 2}, GUO Yong-qiang^{1, 2}, KOU Jiang-li^{1, 2}, DING Ning^{1, 2}, ZHOU Kai-sheng^{1, 2}, NAN Wei^{1, 2}, ZHANG Hai-hong¹

1. Department of Orthopedics, Second Clinical Medical College of Lanzhou University, Lanzhou, Gansu 730030, China;
2. Key Laboratory of Orthopedics of Gansu Province, Lanzhou, Gansu 730030, China

Received:2017-05-25 Revised:2017-06-22 Published:2017-09-25 Online:2017-10-10
Contact: Correspondence to ZHANG Hai-hong. E-mail: zhanghaihong1968@sina.com

摘要/Abstract

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-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。

关键词: ski, RNA干扰, 增殖, 星形胶质细胞, 大鼠

Abstract: 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.

Key words: ski, small interfering RNA, proliferation, astrocyte, rats

中图分类号:

R338.2

赵鑫, 郭永强, 寇江力, 丁宁, 周开升, 南伟, 张海鸿. 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[J]. 《中国康复理论与实践》, 2017, 23(9): 1032-1036.

ZHAO Xin, GUO Yong-qiang, KOU Jiang-li, DING Ning, ZHOU Kai-sheng, NAN Wei, ZHANG Hai-hong. Effects of Knocking Down ski on Proliferation of Astrocytes and Expression of Cyclin D1 in Rats[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2017, 23(9): 1032-1036.

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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

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