Oct3/4在体外诱导大鼠骨髓间质干细胞神经分化中的作用

Role of Oct3/4 in differentiation of rat bone marrow mesenchymal stem cells into neurons

目的:探讨Oct3/4在体外诱导大鼠骨髓间充质干细胞(MSCs)分化为神经元中的作用。方法:构建大鼠Oct3/4慢病毒载体(Oct3/4-LV)并感染大鼠MSCs;实验分为感染组(感染Oct3/4-LV)、阴性对照组(感染FU-PGC-NC-LV)和未感染组3组;采用β-巯基乙醇诱导各组大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs感染后形态学变化;MTT法检测细胞存活率;免疫细胞化学法检测神经元烯醇化酶(NSE)、微管相关蛋白2(MAP-2)、胶质纤维酸性蛋白(GFAP)和Oct3/4的表达变化;Western blotting法检测MAP-2和Oct3/4蛋白的表达变化;RT-PCR法检测MAP-2和Oct3/4 mRNA的表达变化。结果:(1)阳性克隆PCR证明大鼠Oct3/4慢病毒载体构建成功,孔稀释法测定病毒滴度为2×10^(11)TU/L。(2)倒置显微镜下观察大鼠Oct3/4慢病毒载体感染成功,感染复数(MOI)值为10,感染48 h时感染率最高,荧光表达最强;感染率可达83.4%±2.2%。感染组中,MSCs形态发生变化;MTT提示感染组细胞存活率显著降低(P<0.05)。(3)β-巯基乙醇可以诱导大鼠MSCs向神经元分化,其中以感染组诱导效果最佳,具有比较典型的神经元形态,NSE和MAP-2的表达率与其它各组相比显著增高(P<0.05)。(4)感染组与其它各组同时点的Oct3/4表达相比均显著增高(P<0.01)。并且随着诱导时间的延长,各组Oct3/4表达持续减少,诱导后5 h与诱导前相比存在显著差异(P<0.05)。结论:Oct3/4在大鼠MSCs分化为神经元的过程中可能起到了重要的调控作用。

AIM ： To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells （MSCs） into neurons in vitro. METHODS： Lentivirus （LV） vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non - transfection group, transfection group （transfected with Oct3/4 - LV） and negative control group （transfected with FU - PCG - NC - LV）. β - mercaptoethanol （ β - ME） was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in trans- fected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin - associated protein 2 （ MAP - 2） at mRNA and protein levels was detected by RT - PCR and Western blotting, The expression of Oct3/ 4 and the neural cell specific markers neuron -specific enolase（ NSE）, MAP- 2 and glial fibrillary acidic protein（GFAP） were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTr assay. RESULTS ： The results of PCR confirmed that the Oct3/4 - LV was successfully constructed and the virus titer was 2 × 10^11 TU/L. The best transfection efficiency and survival rate appeared when multiply of infection（MOI） was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4% ± 2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups （P 〈 0. 05）. MSCs were induced by 13 - ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP - 2 were higher than those in other groups （P 〈 0. 05 ）. Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased （P 〈0.01 ）. Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction （P 〈 0. 05）. CONCLUSION： Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.