动物实验,间充质干细胞 z-bo 2020-09-14 401

　　Objective To investigate whether exosomes (exosome, Exos) secreted by mesenchymal stem cells (MSCs) can regulate cardiac autophagy and affect cardiac function in rats with myocardial ischemia through miR-21-5p.

　　Methods In vitro, the effects of MSCs-Exos on H2O2 stimulated H9C2s were observed; cell viability was detected by CCK-8 assay; apoptosis was detected by flow cytometry; and the production of reactive oxygen species (ROS) in cells was detected by fluorescence microscope ; Western blot analysis of autophagy-related proteins and fluorescent GFP-LC3 to determine autophagosome formation. In the rat MI/RI model, the effects of MSCs-Exos on cell apoptosis, myocardial LC3B expression and cardiac function were examined by TUNEL measurement, immunohistochemical staining and echocardiography.

　　Results In in vitro experiments, MSCs-Exos significantly increased the viability of H9C2 cells stimulated by H2O2 (P<0.05), and reduced the production of ROS and the rate of apoptosis (P<0.05). And compared with the H2O2+MSCs-Exos group, the H2O2+MSC-ExossimiR-21-5p group cell viability was significantly reduced (P<0.01), and the generation of ROS and the rate of apoptosis were significantly increased (P<0.01) . Western blot detection showed that compared with the H2O2 group, the expression of LC3B-Ⅱ/LC3B-Ⅰ and LC3B-Ⅱ in the H2O2 +MSCs-Exos group was significantly increased (P<0.01), and the expression of p62 was significantly reduced (P<0. 01); Compared with the H2O2 +MSCs-Exos group, the expression of LC3B-Ⅱ/LC3B-Ⅰ and LC3B-Ⅱ in the H2O2+MSC-ExossimiR-21-5p group was significantly reduced (P<0.05), and the expression of p62 Significantly enhanced (P<0.05). Autophagy flux results: Compared with the H2O2 group, the number of GFP-LC3 spots in the H2O2 +MSCs-Exos group increased; and compared with the H2O2 +MSCs-Exos group, the H2O2+MSC-ExossimiR-21-5p group cells The number of GFP-LC3 points present was significantly reduced (P<0.05). In vivo, RT-qPCR analysis results showed that the expression of miR-21-5p in MSCs-Exos and myocardial tissue after MI/RI was positively correlated. Compared with other groups, the expression of LC3B in MI/RI+MSCs-Exos group was significantly enhanced (P<0.01); Cardiomyocyte apoptosis was significantly reduced (P<0.01); Score shortening rate (FS%) and The left ventricular ejection fraction (LVEF) was significantly improved (P<0.05).

　　Conclusion MSCs-Exos can improve the cardiac function of MI/RI rats by regulating myocardial autophagy, and its mechanism may be related to the metastasis of miR-21-5p.