microrna-9-1慢病毒载体构建及对小鼠骨髓间质 - 中国病理生理杂志.docVIP

MicroRNA-9-1慢病毒载体的构建及其对小鼠骨髓间质干细胞诱导分化为神经细胞的影响* 景黎君，贾永林，鲁晶晶，韩瑞，王舒阳，李尽义，彭涛，贾延劼△（郑州大学第一附属医院神经内科，河南 郑州 450052） [摘 要] 目的：探讨microRNA-9-1在体外诱导小鼠骨髓间充质干细胞（MSCs）向神经细胞分化中的作用。方法: 构建小鼠microRNA-9-1慢病毒载体（microRNA-9-1-LV）并感染小鼠MSCs，筛选最适感染复数（MOI）；实验分为未感染组、感染组（感染microRNA-9-1-LV组）、阴性对照组（感染FU-RNAi-NC-LV）；采用β-巯基乙醇诱导感染后小鼠MSCs分化为神经细胞。倒置荧光显微镜下观察MSCs感染后荧光表达情况；采用免疫细胞化学染色检测神经元烯醇化酶（NSE）、神经微管结合蛋白（MAP-2）和胶质纤维酸性蛋白（GFAP）的表达变化；PT-PCR检测MAP-2 mRNA的表达变化；MTT方法检测细胞存活率。结果：1) 阳性克隆PCR证明小鼠microRNA-9-1慢病毒载体构建成功，孔稀释法测定病毒滴度为1×1012 TU/L。2) 倒置显微镜下观察小鼠microRNA-9-1慢病毒载体感染成功，MOI值为20，感染4 d时感染率最高，细胞存活率较高，感染率可达91.3% ± 4.2%。3) β-巯基乙醇可以诱导MSCs向神经细胞分化，其中以感染组诱导效果最佳，NSE和MAP-2的表达率显著高于其它各组（P0.05）。结论：1) microRNA-9-1可高效感染小鼠MSCs。(2) 感染miRNA-9-1-LV后MSCs经β-巯基乙醇诱导向神经细胞分化比率增加。 [关键词] MicroRNA-9-1；骨髓间充质干细胞；神经元；慢病毒 [中图分类号] R329.21 [文献标识码] A *[基金项目] 国家自然科学基金资助项目（No.；河南省教育厅自然科学研究计划项目（No. 2008A320032） △通讯作者 Tel: 0371 E-mail: jiayanjie1971@ Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons JING Li-jun, JIA Yong-lin, LU Jing-jing, HAN Rui, WANG Shu-yang, LI Jin-yi, PENG Tao, JIA Yan-jie (Department of Neurology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China. E-mail: jiayanjie1971@) [ABSTRACT] AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cells (MSCs) to differentiate into neurons. METHODS: The lentiviral vector of microRNA-9-1 (microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group (transfected with microRNA-9-1-LV) and negative control group (transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol (β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtubulin-associated protein 2 (MAP-2) was detected by RT-PCR. The expression of