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目的:探讨环磷腺苷葡胺(MCA)诱导骨髓间充质干细胞(BMSCs)向心肌细胞分化后对自噬的影响及作用机制。方法:通过全骨髓培养法建立体外培养体系,取P3代细胞通过流式细胞仪鉴定细胞表面抗原的表达。将细胞分为3组:正常组、MCA组、5氮胞苷(5-Aza)组,采用western blotting检测心肌特异蛋白表达情况。将诱导后的心肌细胞通过葡萄糖剥夺(GD)建立细胞损伤模型,收集3h、12h、24h细胞,Western blotting法检测自噬蛋白的表达情况。将细胞分为5组:正常组、GD组、MCA预处理组(M+G组)、LY294002+MCA预处理组(M+G+L组)、LY294002组(L组)。利用Western-blotting法检测各组自噬蛋白及相关信号通路蛋白的表达情况。结果:1P3代细胞表面抗原CD44、CD90、CD45的阳性率完全符合BMSCs的标准;2MCA组Ctnt、Cx43蛋白的相对表达量明显高于5-Aza组(P<0.01);3GD处理3h后自噬蛋白Beclin 1、LC3Ⅱ/Ⅰ比值明显升高(P<0.01);4经MCA预处理后自噬蛋白Beclin 1、LC3Ⅱ/Ⅰ比值下降(P<0.01),提高了PI3K的活性,上调了AKT、mTOR磷酸化水平(P<0.01),加入阻断剂后通路蛋白水平下降。结论:MCA可以诱导BMSCs向心肌细胞分化,并在一定程度上抑制了诱导后心肌细胞的自噬,其机制可能与PI3K/AKT/mTOR信号通路相关。
Objective: To investigate the effect of cyclic adenosine monophosphate (MCA) on autophagy induced by bone marrow mesenchymal stem cells (BMSCs) differentiated into cardiomyocytes and its mechanism. Methods: The in vitro culture system was established by whole bone marrow culture. P3 generation cells were identified by flow cytometry for cell surface antigen expression. The cells were divided into 3 groups: normal group, MCA group and 5-Aza group. The expression of myocardial specific protein was detected by western blotting. The induced cardiomyocytes were induced to establish cell injury model by glucose deprivation (GD). The cells were collected for 3h, 12h and 24h, and the expression of autophagy protein was detected by Western blotting. The cells were divided into 5 groups: normal group, GD group, MCA pretreatment group (M + G group), LY294002 + MCA pretreatment group (M + G + L group) and LY294002 group (L group). Western-blotting was used to detect the expression of autophagy protein and related signal pathway proteins in each group. Results: The positive rates of CD44, CD90 and CD45 on the surface of 1P3 cells were completely consistent with the standard of BMSCs. The relative expression of Ctnt and Cx43 in 2MCA group was significantly higher than that in 5-Aza group (P <0.01) Beclin 1, LC3Ⅱ / Ⅰ ratio increased significantly (P <0.01); 4 Beclin 1, LC3Ⅱ / Ⅰ ratio decreased after pretreatment with MCA (P <0.01), increased PI3K activity, increased AKT, mTOR phosphorylation levels (P <0.01), the addition of blocking agent decreased the level of the protein pathway. CONCLUSION: MCA can induce the differentiation of BMSCs into cardiomyocytes and inhibit the autophagy of induced cardiomyocytes to a certain extent. The mechanism may be related to the PI3K / AKT / mTOR signaling pathway.