CD73通过Nanog促进脂肪间充质干细胞心肌分化和梗死心肌修复的功能与机制研究

The adipose tissue-derived mesenchymal stem cells (ADMSCs) have different biological characteristics and differentiation potential of different subpopulations. ADMSCs have been one of the most promising stem cells in cardiac tissue engineering, regenerative medicine and cell therapy. And screening advantage subpopulations of ADMSCs to treat myocardial injury has an important significance. Our previous study showed that CD73+ subpopulations have higher cardiac differentiation potential and the expression of VEGF is increased in CD73+ ADMSCs transplanted area of the myocardial infarction model. Meanwhile CD73+ subpopulations have higher expression of Nanog Which plays an important role of maintaining mesenchymal stem cells in the steady state and differentiation potential in vitro than CD73- subpopulations and when the expression of CD73 is inhibited, the expression of Nanog decline. CD73 is the important mesenchymal stem cells marker, but there are rare reports that the effect of CD73 on mesenchymal stem cells and the correlation with Nanog. Our study includes the following aspects. The first is to explore that CD73 can promote ADMSCs to myocardial differentiation and increase the secretion of some cytokines. The second is to prove the regulatory effect of Nanog on CD73 by changing the Nanog expression in CD73+ and CD73- subgroup. The third is to transplant CD73+ ADMSCs to the myocardial infarction model in rats, and to evaluate the curative effect of myocardial infarction. The purpose of our study is to screen the advantage subpopulations of ADMSCs for treatment of myocardial injury, and to explore the molecular mechanisms of CD73 factor promotion ADSMCs to cardiac repair, and to provide the theoretical basis and the key molecular for CD73+ ADMSCs treatment of myocardial injury.

脂肪间充质干细胞（ADMSC）由具有不同生物学特性和分化潜能的亚群组成，是细胞治疗心肌损伤最具前景的干细胞之一，筛选治疗心肌损伤优势亚群可增强应用的针对性。前期工作显示CD73+ADMSC具有较高心肌分化的潜能，其移植区域VEGF表达升高；且CD73+细胞高表达对维持间充质干细胞在体外稳态和分化潜能起重要作用的Nanog，抑制CD73则Nanog下降。CD73作为间充质干细胞重要标记分子，其对ADMSC的作用及与Nanog相关性鲜有报道。本研究拟明确CD73促ADMSC心肌分化和细胞因子分泌的功能；改变Nanog在CD73+/-亚群中的表达，分析Nanog在CD73促ADMSC心肌修复中发挥的调控作用；CD73+细胞移植治疗大鼠心肌梗死，评价疗效。从而筛选ADMSC治疗心肌损伤的优势亚群，探讨CD73促进ADMSC心肌修复的机制，为CD73+ADMSC治疗心肌损伤提供理论依据和质控参考。

脂肪间充质干细胞（ADMSCs）由具有不同生物学特性和分化潜能的亚群组成，是细胞移植治疗心肌损伤最具前景的干细胞之一，筛选治疗心肌损伤优势亚群无疑会增强应用的针对性。本研究旨在筛选ADMSCs治疗心肌损伤的优势亚群，揭示其分子机制，为CD73+ADMSCs治疗心肌损伤提供理论依据和质控的分子参考。首先将流式分选小鼠CD73+/CD73- ADMSCs两个亚群和混群ADMSCs进行DAPI标记，移植大鼠心肌梗死区域，1W、2W、3W和4W多普勒超声检测心功能：CD73+亚群移植组的左室射血分数、左室短轴缩短率较ADMSCs组和CD73-亚群移植组提高(P<0.05)；CD73+亚群移植组的左室舒张末期内径较ADMSCs移植组和CD73-亚群移植组下降(P<0.05)。免疫组化和West Blotting检测移植区域Ⅷ因子、VEGF和心肌特异标志，CD73+亚群能更有效的增加移植区域毛细血管密度、促进血管新生和移植细胞c-TNT和MHC表达。构建CD73干扰载体（CD73-RNAi）和过表达载体（CD73-OE）转染ADMSCs亚群, 使用CD73抑制剂APCP，改变CD73+和CD73-亚群中CD73的表达或活性。采用免疫荧光、流式细胞技术比较各实验组心肌化诱导后cTNT表达，CD73+组心肌化诱导效率高于CD73+-APCP组和CD73-组（P＜0.05）。ELISA法检测各实验组细胞培养上清液中细胞因子表达：VEGF浓度，CD73+组高于CD73-组（P＜0.05）；TNF-α浓度，CD73+组明显低于和CD73-组（P＜0.01）；慢病毒转染后，VEGF浓度CD73+组高于CD73+-CD73RNAi组（P＜0.05）；TNF-α浓度CD73--CD73OE组低于CD73-组（P＜0.05）。基因芯片技术检测各组的差异基因表达，GO分析提示CD73参与VEGF和Actin Cytoskeleton 信号通路调控。. 综上，心肌梗死区域移植CD73+ADMSCs能更有效地促进心功能改善、心肌血管新生和移植细胞向心肌分化，与CD73促进MSC向心肌细胞分化和分泌细胞因子VEGF，且抑制其分泌TNF-α有关，可能因其参与VEGF和Actin Cytoskeleton 通路调控，在移植ADMSCs治疗心肌损伤时推荐CD73可作为亚群分选和质控标准。