中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (10): 1832-1836.doi: 10.3969/j.issn.1673-8225.2011.10.026

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

法舒地尔和RhoA沉默对神经干细胞增殖的影响

王丙乾1，王  东2，张建军2，汪永新1，朱国华1，杜郭佳1，党木仁1   

  1. 1新疆医科大学第一附属医院神经外科，新疆维吾尔自治区乌鲁木齐市  830054
    2天津第四中心医院神经外科，天津市  300140
  • 收稿日期:2010-11-03 修回日期:2010-11-26 出版日期:2011-03-05 发布日期:2011-03-05
  • 通讯作者: 党木仁，主任医师，博士生导师，新疆医科大学第一附属医院神经外科，新疆维吾尔自治区乌鲁木齐市 830054 damrjab@xj.cninfo.net
  • 作者简介:王丙乾★，男，1983年生，河北省邢台市人，汉族，新疆医科大学在读硕士，主要从事神经外科研究。 ydwangbingqian@126.com
  • 基金资助:

    天津市卫生局科技基金面上课题(2010ky04)。

Effects of fasudil and RhoA silencing on neural stem cell proliferation

Wang Bing-qian1, Wang Dong2, Zhang Jian-jun2, Wang Yong-xin1, Zhu Guo-hua1, Du Guo-jia1, Dang Mu-ren1   

  1. 1Department of Neurosurgery, First Affiliated Hospital, Xinjiang Medical University, Urumqi  830054, Xinjiang Uygur Autonomous Region, China
    2Department of Neurosurgery, Tianjin Fourth Central Hospital, Tianjin  300140, China
  • Received:2010-11-03 Revised:2010-11-26 Online:2011-03-05 Published:2011-03-05
  • Contact: Dang Mu-ren, Chief physician, Doctoral supervisor, Department of Neurosurgery, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China damrjab@xj.cninfo.net
  • About author:Wang Bing-qian★, Studying for master’s degree, Department of Neurosurgery, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China ydwangbingqian@126.com
  • Supported by:

    the General Program of Science and Technology Foundation of Health Bureau of Tianjin City, No. 2010ky04*

PDF

342

被引次数

10

摘要：

背景：Rho及其相关分子在神经轴突生长、分化、延伸及突触形成中起重要作用，阻断和抑制RhoA/ROCK通路可促进神经干细胞的增殖与生长。
目的：观察Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默对大鼠神经干细胞增殖的影响。
方法：体外培养Wistar胎鼠神经干细胞，分6组干预：空白对照组，5，10，15，20 μmol/L 法舒地尔组，siRNA 沉默RhoA基因组。干预后第3天，采用RT-PCR，Western blot检测各组神经干细胞RhoA基因及蛋白的表达。应用MTT比色法观察神经干细胞增殖情况；采用流式细胞术测定神经干细胞周期分布的变化。
结果与结论：15，20 μmol/L 法舒地尔组、siRNA 沉默RhoA基因组神经干细胞RhoA基因及蛋白表达量较5，10 μmol/L 法舒地尔组、空白对照组明显降低(P< 0.05)，细胞的生长速度较5，10 μmol/L 法舒地尔组、空白对照组明显增快(P < 0.05)，细胞周期G0/G1期减少(P< 0.05)，S期细胞数增多(P< 0.05)。当法舒地尔浓度增加到20 μmol/L时对细胞的作用并非随浓度的增加而增强，与15 μmol/L组的差异无显著性意义(P > 0.05)。15，20 μmol/L 法舒地尔组与siRNA 沉默RhoA基因组相比差异无显著性意义(P> 0.05)。说明Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默在体外均能促进神经干细胞增殖，法舒地尔最佳作用浓度为15 μmol/L。

关键词: 法舒地尔, 神经干细胞, RhoA, RNA干扰, 干细胞

Abstract:

BACKGROUND: Rho and its relevant molecules play important roles in growth, differentiation, extension and synapse formation of nerve axons. To block and inhibit RhoA/ROCK pathway can promote the proliferation and growth of neural stem cells.
OBJECTIVE: To observe the effects of Rho kinase inhibitor fasudil and RNAi-mediated RhoA gene silencing on the proliferation of neural stem cells in rats.
METHODS: Neural stem cells of Wistar fetal rats were cultured in vitro, and were divided into six groups: blank control group, 5, 10, 15, 20 μmol/L fasudil groups, and siRNA silencing RhoA gene group. On day 3 following intervention, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were used to assess the expression of RhoA mRNA and RhoA protein of neural stem cells in each group. Cellular proliferation was determined by MTT assay. The cell cycle was analyzed by flow cytometry.
RESULTS AND CONCLUSION: RhoA gene and protein expression was significantly lower in the 15, 20 μmol/L fasudil groups and siRNA silencing RhoA gene group compared with 5, 10 μmol/L fasudil groups and blank control group (P < 0.05). Cell growth speed was significantly faster compared with the 5, 10 μmol/L fasudil groups and blank control group (P< 0.05); cell number was reduced in the cell cycle G0/G1 (P < 0.05) and increased in S phase (P < 0.05). At 20 μmol/L fasudil, cell effect was not enhanced with the increased concentration. No significant difference was determined compared with 15 μmol/L fasudil group (P > 0.05). No significant difference was detected between 15, 20 μmol/L fasudil groups and siRNA silencing RhoA gene group (P > 0.05). Results suggest that Rho kinase inhibitor fasudil and RNAi-mediated RhoA gene silencing can promote the proliferation of neural stem cells in vitro. The optimal concentration of fasudil was 15 μmol/L.

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