低氧预处理脐带间充质干细胞源外泌体抑制肺动脉平滑肌细胞的增殖

Umbilical cord mesenchymal stem cell-derived exosomes treated with hypoxic preconditioning inhibits proliferation of pulmonary artery smooth muscle cells

背景:已有研究表明外泌体可以改善低氧性肺动脉高压,而且不同来源、不同环境的外泌体功能存在显著差别。低氧预处理脐带间充质干细胞来源外泌体对大鼠肺动脉平滑肌细胞增殖的影响尚不清楚。目的:探讨低氧预处理人脐带间充质干细胞来源外泌体对低氧诱导的大鼠肺动脉平滑肌细胞增殖的影响。方法:采用组织贴壁法分离培养原代人脐带间充质干细胞,用超滤法提取人脐带间充质干细胞来源外泌体;采用组织消化法分离大鼠肺动脉平滑肌细胞,用CCK-8法测定不同质量浓度外泌体干预肺动脉平滑肌细胞不同时间后的细胞增殖抑制率,确定外泌体作用的适宜质量浓度和干预时间。将第3代肺动脉平滑肌细胞分为常氧对照组、低氧对照组、低氧+常氧外泌体处理组和低氧+低氧外泌体处理组,EdU法检测各组细胞增殖情况,Western blot检测各组细胞核增殖抗原蛋白表达水平。结果与结论:①与常氧对照组相比,低氧对照组肺动脉平滑肌细胞增殖能力明显增加;与低氧对照组相比,低氧+常氧外泌体组细胞增殖能力下降,且低氧+低氧外泌体组增殖能力下降更明显;②与常氧对照组相比,低氧对照组细胞的细胞核增殖抗原蛋白表达升高;与低氧对照组相比,低氧+常氧外泌体组细胞核增殖抗原蛋白表达水平稍微降低,低氧+低氧外泌体组细胞核增殖抗原蛋白表达水平明显降低;③结果表明,低氧预处理人脐带间充质干细胞来源外泌体具有抑制肺动脉平滑肌细胞增殖的能力。

BACKGROUND:Studies have shown that exosomes can improve hypoxic pulmonary hypertension.There are significant differences in the function of exosomes from different sources and different environments.The effect of hypoxic preconditioning with umbilical cord mesenchymal stem cell-derived exosomes on the proliferation of rat pulmonary artery smooth muscle cells is unclear.OBJECTIVE:To explore the effects of umbilical cord mesenchymal stem cell-derived exosomes pretreated with hypoxia on the hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells.METHODS:Primary human umbilical cord mesenchymal stem cells were isolated and cultured by tissue adhesion method,and the exosomes of human umbilical cord mesenchymal stem cells were extracted by ultrafiltration.Rat pulmonary artery smooth muscle cells were isolated by tissue digestion.The inhibition rate of cell proliferation after the intervention of exosomes with different concentrations was measured by cell counting kit-8 assay to determine the appropriate mass concentration and intervention time for the action of exosomes.Passage 3 pulmonary artery smooth muscle cells were divided into normoxic control group,hypoxic control group,hypoxic+normoxic exosome group and hypoxic+hypoxic exosome group.5-Ethynyl-2'-deoxyuridine method was used to detect the proliferation of cells in each group.The expression level of nuclear proliferation antigen protein was detected by western blot assay.RESULTS AND CONCLUSION:(1)Compared with the normoxic control group,the proliferation of pulmonary artery smooth muscle cells in the hypoxic control group was significantly increased.Compared with the hypoxic control group,the cell proliferation ability of the hypoxic+normoxic exosome group decreased,and the proliferation ability of the hypoxic+hypoxic exosome group decreased more significantly.(2)Compared with the normoxic control group,the expression level of nuclear proliferation antigen protein was increased in the hypoxic control group.Compared with the hypoxic control group,the expression level of nuclear proliferation antigen protein was slightly reduced in the hypoxic+normoxic exosome group.The expression level of nuclear proliferation antigen protein was significantly diminished in the hypoxic+hypoxic exosome group.(3)The results showed that the exosomes derived from human umbilical cord mesenchymal stem cells pretreated with hypoxia had the ability to inhibit the proliferation of pulmonary artery smooth muscle cells.