骨髓内皮祖细胞释放的细胞微泡对缺氧-复氧诱导心肌细胞凋亡的影响及机制

Effects of EPCs-MVs on apoptosis of cardiomyocytes induced by hypoxia-reoxygenation

目的观察骨髓内皮祖细胞释放的细胞微泡(EPCs-MVs)对缺氧-复氧诱导心肌细胞凋亡的影响,并探讨其作用机制。方法从小鼠骨髓中分离内皮组细胞(EPCs),收集微泡(MVs)及不含RNA的MVs(rd MVs);将心肌细胞H9c2随机分为4组,EP、DEP、模型组均行缺氧-复氧模型制作,EP、DEP组造模后分别给予EPCs-MVs、EPCs-rd MVs 50μg/m L作用24 h,对照组不造模、不进行干预。采用荧光染色的脂质膜标记法检测EPCs-MVs、EPCs-rd MVs与H9c2细胞的融合程度,MTT实验测算细胞存活率,流式细胞仪测算细胞凋亡率,采用Western blotting法检测PI3K/Akt通路相关蛋白Akt、p-Akt蛋白及PI3K抑制剂(LY294002)阻滞前后的p-Akt蛋白。结果EP、DEP组融合率分别为49.12%±8.79%、47.94%±10.22%,差异无统计学意义(P>0.05)。与对照组比较,各组细胞存活率下降、细胞凋亡率升高、p-Akt蛋白表达量降低(P均<0.05);与DEP组、模型组比较,EP组细胞存活率上升、细胞凋亡率下降、p-Akt蛋白表达量增加(P均<0.05);DEP组、模型组各指标差异均无统计学意义(P均>0.05)。各组细胞Akt蛋白表达量差异无统计学意义(P均>0.05),PI3K抑制剂阻滞后p-Akt蛋白表达量均降低(P均<0.05)。结论 EPCs-MVs可通过激活PI3K/Akt通路减少缺氧-复氧对心肌细胞凋亡的影响。

Objective To observe the effects of cellular microvesicles released by bone marrow endothelial progenitor cells （EPCs-MVs） on cardiomyocyte apoptosis induced by hypoxia-reoxygenation and to explore its mechanism.Methods EPCs were isolated from mouse bone marrow, and MVs and RNA-free MVs （rdMVs） were collected. Myocardial cells H9c2 were randomly divided into four groups. In the EP, DEP, and model groups, we made hypoxia reoxygenation models. After model establishment, EP and DEP groups were treated with EPCs-MVs and EPCs-rdMVs （50 μg/mL） for 24 h, respectively, while the control group was not intervened. The fusion degree of EPCs-MVs, EPCs-rdMVs, and H9c2 cells was detected by fluorescent staining of lipid membrane （PKH26）. MTT assay was used to analyze cell viability, and apoptosis rate was measured by flow cytometry. The expression of PI3K/Akt pathway-related proteins Akt and p-Akt before and after PI3K inhibitor （LY294002） was detected by Western blotting.Results The fusion rates of the EP and DEP groups were 49.12%±8.79% and 47.94%±10.22%, respectively, and the difference was not statistically significant （ P 〉 0.05 ）. Compared with the control group, the survival rate of each group decreased, the apoptosis rate increased, and the expression of p-Akt protein decreased （all P 〈0.05）. Compared with the DEP group and the model group, the cell survival rate increased, the apoptosis rate decreased, and the expression of p-Akt protein increased in the EP group （all P 〈 0.05 ）. There was no significant difference in the indexes between DEP group and model group. There was no significant difference in the expression of Akt protein among these groups, and the expression of p-Akt protein decreased after PI3K inhibitor （all P 〈0.05）.Conclusion EPCs-MVs can activate the PI3K/Akt pathway to reduce the effect of hypoxia-reoxygenation on cardiomyocyte apoptosis.