高表达miR-132-3p间充质干细胞外泌体对缺氧/复氧受损脑微血管内皮细胞功能的改善作用

Improving effect of exosomes of mesenchymal stem cells with high expression of miR-132-3p on hypoxia/reoxygenation impaired brain microvascular endothelial cell function

目的研究高表达miR-132-3p间充质干细胞(MSCs)释放的外泌体(EXs)对缺氧/复氧(H/R)受损内皮细胞功能的作用。方法原代培养从C57BL/6小鼠骨髓中提取的MSCs,采用装载miR-132-3p载体的慢病毒感染MSCs获得MSC^(miR-132-3p),同时采用装载有scramble序列的对照慢病毒感染MSCs获得MSC^(NC)。分离提取MSC^(NC)及MSC^(miR-132-3p)释放的EXs,分别获得MSC-EXs及MSC-EXs^(miR-132-3p)。将所得EXs与H/R受损小鼠脑微血管内皮细胞(bend3)共培养,根据培养条件将细胞分为正常培养组(细胞正常培养)、H/R组(制作H/R模型)、MSC-EXs处理组(MSC-EXs共培养)、MSC-EXs^(miR-132-3p)处理组(MSC-EXs^(miR-132-3p)共培养)、MSC-EXs^(miR-132-3p)+LY294002组[细胞与MSC-EXs^(miR-132-3p)共培养前加入磷脂酰肌醇(-3)激酶(PI3K)信号通路阻断剂LY294002(20μmol/L)处理]。实时荧光定量聚合酶链反应检测MSCs、MSC-EXs及bend3细胞中miR-132-3p的表达。血管形成试剂盒检测bend3细胞血管形成能力,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测bend3细胞增殖能力,划痕实验检测bend3细胞迁移能力,hochest33258染色显示细胞的凋亡,Western blot检测蛋白激酶B(Akt)的磷酸化水平。结果与H/R组相比,MSC-EXs处理组显著改善H/R诱导下bend3细胞受损的血管形成、增殖、迁移能力及Akt磷酸化水平[H/R组分别为(3±1)条、0. 275±0. 020、(147±8)μm和0. 89±0. 12; MSC-EXs处理组分别为(8±3)条、0. 358±0. 030、(218±10)μm和1. 37±0. 25,均P <0. 01],细胞凋亡明显减少[(47±2)%比(63±2)%,均P <0. 01]。与MSC-EXs处理组比较,MSC-EXs^(miR-132-3p)处理组bend3细胞的血管形成、增殖、迁移能力及Akt磷酸化水平增加[分别为(14±3)条、0. 444±0. 050、(357±10)μm和1. 67±0. 23,均P <0. 01],细胞凋亡明显减少[(34±1)%,P <0. 01]。与MSC-EXs^(miR-132-3p)处理组比较,MSC-EXs^(miR-132-3p)+LY294002组细胞的增殖、迁移、血管形成能力及Akt磷酸化水平明显减少[分别为(5±2)条、0. 304±0. 050、(175±8)μm和0. 95±0. 11,均P <0. 01]。结论高表达miR-132-3p的MSC-EXs能够通过激活PI3K/Akt信号通路改善H/R诱导受损脑血管内皮细胞多种生理功能。

Objective To study the effect of exosomes（ EXs） released from high expression of miR-132-3p mesenchymal stem cells（ MSCs） on hypoxia/reoxygenation（ H/R） injured endothelial cell function. Methods MSCs extracted from bone marrow of C57BL/6 mice were cultured primarily. MSC^（miR-132-3p） was obtained from MSCs infected with lentivirus loaded with miR-132-3p vector. At the same time,MSC^（NC） was obtained by infecting MSCs with control lentivirus loaded with scramble sequence. EXs released from MSC^（NC）and MSC^（miR-132-3p） was isolated,and MSC-EXs and MSC-EXs^（miR-132-3p） were obtained respectively. The obtained EXs and H/R damaged mouse brain microvascular endothelial cells（ bend3） were co-cultured. According to culture conditions,the cells were divided into normal culture group（ normal cell culture）,H/R group（ making a H/R model）,MSC-EXs group（ MSC-EXs co-culture）,MSC-EXs^（miR-132-3p） group（ MSC-EXs^（miR-132-3p） coculture）,and MSC-EXs^（miR-132-3p） ＋ LY294002 group（ before the cells and MSC-EXs^（miR-132-3p） were cocultured,treated by adding phosphatidyl alcohol 3 kinase[PI3K] signaling pathway blocker LY294002[20 μmol/L]）. Quantitative real-time quantitative polymerase chain reaction was used to detect the expression of miR-132-3p in MSCs,MSC-EXs,and bend3 cells. Angiogenesis kit was used to detect angiogenic ability of bend3 cells,and 3-（ 4,5-dimethylthiazole-2）-2,5-diphenyltetrazolium bromide（ MTT） assay was used to detect the proliferative capacity of bend3 cells. Scratch test was used to detect the migration ability of bend3 cells. hochest33258 staining showed cell apoptosis. Western blot was used to detect the phosphorylation level of protein kinase B（ Akt）. Results Compared with the H/R group,the MSC-EXs treatment group significantly improved the angiogenesis,proliferation,migration abilities,and Akt phosphorylation level of bend 3 cell damage induced by H/R（ The H/R group were 3 ± 1,0. 275 ± 0. 020,147 ± 8 μm,and 0. 89 ±0. 12,respectively; the MSC-EXs treatment group were 8 ± 3,0. 358 ± 0. 030,218 ± 10 μm,and 1. 37 ±0. 25 μm,respectively; all P〈0. 01）. Apoptosis was significantly reduced（ 47 ± 2% vs. 63 ± 2%,all P 0. 01）. Compared with the MSC-EXs treatment group,the angiogenesis,proliferation,migration abilities,and Akt phosphorylation level of bend 3 cells in the MSC-EXs^（miR-132-3p） treatment group were increased（ 14 ± 3,0. 444 ±0. 050,357 ±10 μm,and 1. 67 ± 0. 23,respectively,all P 0. 01）. Apoptosis was significantly reduced（ 34 ± 1%,all P 0. 01）. Compared with the MSC-EXs^（miR-132-3p） treatment group,cell proliferation,migration,angiogenesis abilities,and Akt phosphorylation level in the MSC-EXs^（miR-132-3p） ＋ LY294002 group were significantly reduced（ 5 ±2,0. 304 ± 0. 050,175 ± 8 μm and 0. 95 ± 0. 11,respectively,all P 0. 01）. Conclusion MSC-EXs with high expression of miR-132-3p may improve many physiological functions of H/R-induced damaged cerebrovascular endothelial cells by activating PI3 K/Akt signaling pathway.