法舒地尔和RhoA沉默对神经干细胞增殖的影响

Effects of fasudil and RhoA silencing on neural stem cell proliferation

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,5,10,15,20μmol/L法舒地尔组,siRNA沉默RhoA基因组。干预后第3天,采用RT-PCR,Westernblot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:15,20μmol/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5,10μmol/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5,10μmol/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20μmol/L时对细胞的作用并非随浓度的增加而增强,与15μmol/L组的差异无显著性意义(P>0.05)。15,20μmol/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15μmol/L。

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.