Let-7b对骨髓基质干细胞诱导分化为神经元的作用研究

Effect of Let-7b on the Differentiation of Bone Marrow Mesenchymal Stem Cells into Nerve Cells

目的探讨Let-7b在大鼠骨髓间充质干细胞(MSCs)分化为神经元过程中的作用。方法 2013年1—12月将80只SPF级SD大鼠随机分为空白对照组、阴性对照组、Let-7b-up慢病毒载体(LV-rno-Let-7bup)组、Let-7b-inhibition慢病毒载体(LV-rno-Let-7b-inhibition)组,每组20只。将阴性对照组、LV-rnoLet-7b-up组、LV-rno-Let-7b-inhibition组大鼠分别感染EGFP空病毒,采用颈椎脱臼法处死4组大鼠,并获取MSCs,传代培养4代后备用。取传代培养的MSCs感染慢病毒,具体为:空白对照组、阴性对照组不进行感染,LVrno-Let-7b-up组、LV-rno-Let-7b-inhibition组分别感染LV-rno-Let-7b-up、Let-7b-inhibition,采用MTT法检测感染前、感染后24 h、感染后5 d MSCs存活率。采用法舒地尔对4组进行诱导实验,观察MSCs分化为神经元的分化效率。采用免疫组化法检测4组神经元烯醇化酶(NSE)、神经微管结合蛋白(MAP-2)、微管相关蛋白(Tau)水平,RT-PCR法检测4组NSE、MAP-2、Tau mRNA水平。结果 LV-rno-Let-7b-up组MSCs于LVrno-Let-7b-up感染后第3天荧光表达最强,LV-rno-Let-7b-inhibition组MSCs于LV-rno-Let-7b-inhibition感染后第5天荧光表达最强。感染第5天时,阴性对照组、LV-rno-Let-7b-up组、LV-rno-Let-7b-inhibition组MSCs感染率分别为87.46%、86.81%、87.12%。感染后24 h、5 d阴性对照组、LV-rno-Let-7b-up组、LV-rno-Let-7b-inhibition组MSCs存活率低于空白对照组(P<0.05);阴性对照组、LV-rno-Let-7b-up组、LV-rno-Let-7b-inhibition组感染后5 d MSCs存活率高于感染后24 h(P<0.05)。阴性对照组和空白对照组部分MSCs呈现神经元样改变;LV-rno-Let-7b-up组极大部分MSCs出现典型的神经元样结构;LV-rno-Let-7b-inhibition组仅有少量MSCs出现典型的神经元样结构。LV-rno-Let-7b-up组NSE、MAP-2、Tau及其mRNA水平高于空白对照组、阴性对照组(P<0.05);LV-rno-Let-7b-inhibition组NSE、MAP-2、Tau及其mRNA水平低于空白对照组、阴性对照组(P<0.05)。结论感染LV-rno-Let-7b-up后大鼠MSCs分化为神经元的分化效率增加,提示Let-7b可促进MSCs向神经元分化。

Objective To investigate the role of Let-7 b in the differentiation of rat bone marrow mesenchymal stem cells（ MSCs） into neurons. Methods This study was conducted in 2013. Eighty SPF grade SD rats were randomly divided into blank control group,negative control group,Let-7 b-up lentiviral vector（ LV-rno-Let-7 b-up） group,Let-7 b-inhibition lentiviral vector（ LV-rno-Let-7 b-inhibition） group,20 rats in each group. The negative control group,LV-rno-Let-7 b-up group,LV-rno-Let-7 b-inhibition group were injected EGFP blank virus respectively. Four groups of rats were sacrificed by cervical dislocation,and MSCs were obtained and cultured for 4 generations. Subculture of MSCs were infected with lentivirus,that is,the blank control group and the negative control group were not infected,LV-rno-Let-7 b-up group,LV-rno-Let-7 b-inhibition group were infected with LV-rno-Let-7 b-up, Let-7 b-inhibition,respectively. The survival rate of MSCs was detected by MTT before infection, at 24 h after infection, on the fifth day after infection. Four groups were induced by fasudil,and the differentiation efficiency of MSCs into neurons was observed. The levels of NSE,MAP-2 and Tau were detected by immunohistochemistry. The levels of NSE mRNA,MAP-2 mRNA and Tau mRNA were detected by RT-PCR. Results The MSCs of LV-rno-Let-7 b-up group had the strongest fluorescence expression on the third day after LV-rno-Let-7 b-up infection. The MSCs of LV-rno-Let-7 b-inhibition group had the strongest fluorescent expression on the fifth day after LV-rno-7 b-inhibition infection. On the fifth day after infection, the MSCs infection rates of the negative control group,LV-rno-Let-7 b-up group and LV-rno-Let-7 b-inhibition group were87. 46%,86. 81% and 87. 12%,respectively. At 24 h after infection,and on the fifth day after infection the blank control group presented higher survival rate of MSCs than the other three groups（ P 0. 05）. The survival rate of MSCs on the fifth day after infection was found to be higher than that at 24 h after infection in the negative control group,LV-rno-Let-7 b-up group and LV-rno-Let-7 b-inhibition group（ P 0. 05）. Some MSCs showed neuronal changes in the negative control group and the blank control group,almost all the MSCs showed typical neuron-like structures in LV-rno-Let-7 b-up group,but only a small number of MSCs showed a typical neuron-like structure in the LV-rno-Let-7 b-inhibition group. The protein and mRNA levels of NSE,MAP-2,Tau in LV-rno-Let-7 b-up group were higher than those in blank control group and negative control group（ P 0. 05）; the protein and mRNA levels of NSE,MAP-2,Tau in LV-rno-Let-7 b-inhibition group were lower than those in blank control group and negative control group（ P 0. 05）. Conclusion We found that the differentiation efficiency of MSCs into neurons was increased after infection with LV-rno-Let-7 b-up,which suggests that Let-7 b promotes the differentiation of MSCs into neurons.