miR-21对脂肪干细胞移植修复外周神经损伤易发凋亡的干预作用及机制

Adipose derive stem cells (ADSCs) isolated from adipose tissue is one kind of adult stem cell, and it is often served as seed cells for treatment of peripheral nerve injury because ADSCs have the ability to differentiated into Schwann cells. However, ADSCs apoptosis induced by microenvironment after injury has been the main obstacle limiting nerve regeneration. MicroRNAs (miRNAs) as important regulatory molecules in cells have been shown to play important roles in a variety apoptotic signal transduction pathway. We found that the expression of miR-21 in apoptotic ADSCs was reduced about 9.04 fold compared to normal ADSCs. Therefore, we think that miR-21 play an important role in the process of ADSCs apoptosis. Thus, the relationship of miR-21 and ADSCs apoptosis, and the effect of miR-21 on nerve regeneration were definited by over expression or suppression expression miR-21. Then we will predict their targets by bioinformatics method, and verify their regulation roles on targets by dual luciferase report gene system. At the same time, the expression of target genes was compared between ADSCs and apoptotic ADSCs. Finally, the targets of miR-21 were definited. Signal transduction pathways of target gene were predicted by bioinformatics method, and effect of miR-21 on activation of main proteins in apoptotic signal transduction pathways were evaluated. Then we could definite signal transduction pathways of miR-21 in the process of ADSCs apoptosis. ADSCs were transfected with miR-21 alone or together with target gene, and activation of main proteins in apoptotic signal transduction pathways were evaluated. Finally, we could definite miR-21-target gene-apoptotic signal transduction pathway according to above results, and finally reveal molecule mechanism of miR-21 to interfere in the process of ADSCs apoptosis.

脂肪干细胞是来源于脂肪组织的一种成体干细胞，因其能够分化为雪旺细胞常被用于修复外周神经损伤，然而损伤微环境诱导的细胞凋亡制约了神经再生。微RNA作为重要的调控分子在多种凋亡信号通路中发挥关键作用。申请人前期发现脂肪干细胞凋亡后miR-21显著下调了9.04倍，推测其干预了凋亡过程。由此，本课题拟采用过/抑制miR-21表达的方法，确定miR-21与脂肪干细胞凋亡的关系以及对神经再生的影响。利用生物信息学预测并通过双荧光素酶报告基因系统验证miR-21的靶基因，比较脂肪干细胞凋亡前后该基因的表达，确定miR-21的靶基因。通过生物信息学分析靶基因的信号通路，验证miR-21对凋亡相关信号通路中关键蛋白活化的影响，确定信号通路。单独转染miR-21或共同转染靶基因，检测信号通路中关键蛋白的活化情况，确定miR-21-靶基因-凋亡信号通路轴，揭示miR-21干预脂肪干细胞凋亡发生的分子机制。

脂肪干细胞是来源于脂肪组织的一种成体干细胞，因其能够分化为雪旺细胞常被用于修复外周神经损伤，然而损伤微环境诱导的细胞凋亡制约了神经再生。微RNA作为重要的调控分子，在多种凋亡信号通路中发挥关键作用。本研究拟采用过/抑制miR-21表达的方法，明确miR-21对脂肪干细胞凋亡的干预作用。应用qRT-PCR方法检测大鼠ADSCs凋亡发生时miR-21的表达量，再用miR-21 mimics转染大鼠ADSCs，使大鼠ADSCs过表达miR-21后用200 μmol/L H2O2作用4 h，检测细胞的凋亡情况。结果发现，凋亡状态下的ADSCs中miR-21的表达量显著下降（P＜0.05）。用miR-21 mimics转染ADSCs后，细胞凋亡率显著下降了69.7%；凋亡相关蛋白Caspase-3和Bax的表达量显著下降，而Bcl-2蛋白的表达量显著升高。为了阐明miR-21发挥抗凋亡作用的分子机制，本研究检测了过表达miR-21后PDCD4和PTEN基因的mRNA及蛋白表达量，qRT-PCR和WB试验结果显示，PDCD4和PTEN的mRNA表达量没有明显变化，但PDCD4和PTEN的蛋白表达量出现了显著下降（P＜0.05），使用PDCD4 siRNA和Phen分别阻断PDCD4和PTEN基因的转录后，细胞产生了与miR-21 mimics组相似的显著抗凋亡能力，在一定程度上表明miR-21在转录后水平抑制PDCD4和PTEN蛋白的表达。本研究进一步检测了PI3K/AKT信号通路中p-AKT的表达量，在过表达miR-21和PDCD4 siRNA及Phe后p-AKT蛋白表达量显著升高，加入PI3K阻断剂LY294002后miR-21的抗凋亡作用被部分逆转，说明miR-21-PDCD4/PTEN-PI3K-AKT信号通路影响大鼠ADSCs凋亡的发生。