RNAi靶向抑制p65基因对大鼠骨髓间充质干细胞向神经元分化的影响  

Effect of p65 gene inhibited by siRNA on differention of rat marrow mesenchymal stem cells into neurons

作　　者：赵二义[1] 贾延吉力 王带媚[1] 文国强[1] 关文娟[2] 景黎君[2] 邓益东[1] 

机构地区：[1]海南省人民医院神经内科,海口570311 [2]郑州大学第一附属医院神经内科,河南郑州450052

出　　处：《中国应用生理学杂志》2015年第3期254-258,共5页Chinese Journal of Applied Physiology

基　　金：海南省自然科学基金资助(813208)

摘　　要：目的:探讨RNAi靶向抑制p65基因对骨髓间充质干细胞(MSCs)向神经元分化中的影响。方法:采用RNAi干扰技术,Rn-p65-siRNA转染体外培养的大鼠MSCs,用盐酸法舒地尔诱导MSCs向神经元分化,并以未转染组及Cy3标记的negative control siRNA为对照组。在倒置荧光显微镜下观察经negative control siRNA转染24 h、48h及72 h后MSCs的荧光表达变化;采用MTT比色法检测转染前、转染24 h、48 h及72 h后MSCs的存活率;RTPCR检测p65 mRNA的表达;Western blot法检测3组MSCs中p65蛋白的表达;细胞免疫组化法检测p65蛋白、神经元特异性烯醇化酶(NSE)、神经元微管结合蛋白(MAP-2)和胶质细胞原纤维酸性蛋白(GFAP)的表达。结果:Negative control siRNA转染72 h,MSCs荧光表达最强,转染率可达83.3%±3.8%;Rn-p65-siRNA转染组MSCs的p65 mRNA转录下降,p65蛋白表达减少;Rn-p65-siRNA转染组的MSCs存活率也显著减少,显著高于对照组(P<0.05)。转染72 h后,诱导转染组MSCs向神经元分化的诱导效果最佳,可高效表达NSE、MAP-2等,而不表达胶质纤维酸性蛋白(GFAP),与对照组比较有统计学差异(P<0.05)。结论:靶向抑制p65基因表达可促进骨髓间充质干细胞(MSCs)向神经元分化。Objective： To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells （MSCs）. Methods： The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neu- rons. The non-transfected group and negative control group （transfected with negative control siRNA marked by Cy3） were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTY at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The ex- pressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein （GFAP）were detected by immunocytochemical method after transfection for 6 h. Results： The fluores- cence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile , the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased（ P 〈 0.05） ; MTT displayed that the via- bility of MSCs was also significantly reduced（ P 〈 0.05）. The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group（ P 〈 0.05）. Conclusion： The p65 gene inhibited by siRNA can promote the mar- row mesenchymal stem cells to differentiate into neurons.

关 键 词：盐酸法舒地尔 骨髓间充质干细胞 神经元 Rn-p65-siRNA NF-κB 大鼠 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学]