Let-7c慢病毒载体对骨髓间充质干细胞体外诱导分化为神经细胞的影响

发布时间：2018-08-30 07:34

【摘要】：背景:微小RNA参与调控干细胞增殖、分化、衰老等过程,通过了解Let-7家族成员Let-7c对骨髓间充质干细胞向神经细胞分化的影响可以为干细胞移植治疗提供新思路。目的:探讨Let-7c慢病毒载体在体外诱导大鼠骨髓间充质干细胞向神经细胞分化中的作用。方法:构建Let-7c上调及下调慢病毒载体并体外转染大鼠骨髓间充质干细胞,筛选最适转染复数;实验分为未转染组、阴性转染组(转染空白慢病毒)、转染上调组(转染LV-rno-Let-7c-up)、转染下调组(转染LV-rno-Let-7c-5p-inhibition);采用法舒地尔诱导转染后的大鼠骨髓间充质干细胞分化为神经细胞,倒置荧光显微镜下观察骨髓间充质干细胞转染后荧光表达,免疫细胞化学染色检测神经元标记物NSE和MAP-2的表达,RT-PCR检测MAP-2 m RNA的表达,MTT法检测细胞存活率。结果与结论:倒置荧光显微镜下观察大鼠LV-rno-Let-7c-up和LV-rno-Let-7c-5p-inhibition慢病毒载体转染成功。法舒地尔可以诱导骨髓间充质干细胞分化为神经细胞,与阴性转染组相比,转染上调组的诱导效率升高(P0.05),NSE和MAP-2表达率上升(P0.05),转染下调组的诱导效率下降,NSE和MAP-2表达率下降(P0.05)。转染LV-rno-Let-7c-up后骨髓间充质干细胞经法舒地尔诱导向神经细胞分化比率增加,Let-7c可能具有促进骨髓间充质干细胞向神经细胞分化的作用。
[Abstract]:Background: micro RNA is involved in the regulation of stem cell proliferation, differentiation and senescence. Understanding the effect of Let-7c, a member of Let-7 family, on the differentiation of bone marrow mesenchymal stem cells into neural cells can provide new ideas for stem cell transplantation. Aim: to investigate the role of Let-7c lentivirus vector in inducing the differentiation of rat bone marrow mesenchymal stem cells into neural cells in vitro. Methods: Let-7c up-regulated and down-regulated lentivirus vectors were constructed and transfected into rat bone marrow mesenchymal stem cells in vitro. Negative transfection group (transfection of blank lentivirus), transfection upregulation group (LV-rno-Let-7c-up), transfection down-regulation group (LV-rno-Let-7c-5p-inhibition), induced differentiation of rat bone marrow mesenchymal stem cells into neural cells by fasudil. Fluorescence expression of bone marrow mesenchymal stem cells after transfection was observed under inverted fluorescence microscope. The expression of NSE and MAP-2 were detected by immunocytochemistry. The expression of MAP-2 m RNA was detected by RT-PCR. Results and conclusion: rat LV-rno-Let-7c-up and LV-rno-Let-7c-5p-inhibition lentivirus vectors were successfully transfected with inverted fluorescence microscope. Mesenchymal stem cells were induced to differentiate into neural cells. Compared with negative transfection group, the induction efficiency of transfection up-regulated group was higher (P0.05) and the expression rate of NSE and MAP-2 was higher (P0.05), but the induction efficiency of down-transfected group was lower than that of negative transfection group (P0.05). After transfection of LV-rno-Let-7c-up, the ratio of differentiation of bone marrow mesenchymal stem cells induced by fasudil was increased. Let-7c may promote the differentiation of bone marrow mesenchymal stem cells into neural cells.
【作者单位】：郑州大学第一附属医院神经内科;
【基金】：国家自然科学基金项目(骨髓间充质干细胞神经命运决定中的自噬现象及其m TOR信号通路的作用/81071114;Lin28/let-7调控的骨髓间充质干细胞移植治疗阿尔兹海默病的实验研究/81371385)~~
【分类号】：R457.7

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