Improving effect of exosomes of mesenchymal stem cells with high expression of miR-132-3p on hypoxia/reoxygenation impaired brain microvascular endothelial cell function
10.3969/j.issn.1672-5921.2018.11.005
- VernacularTitle:高表达miR-132-3 p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
- Author:
Donghui DU
1
;
Yan WANG
;
Xiaobing XU
;
Jieyi ZHENG
;
Huiting ZHANG
;
Xiaoli KUANG
;
Xiaotang MA
;
Bin ZHAO
;
Yanfang CHEN
;
Qunwen PAN
Author Information
1. 广东医科大学附属医院神经病学研究所广东省衰老相关心脑血管疾病重点实验室
- Keywords:
MiR-132-3p;
Mesenchymal stem cells;
Exosomes;
Brain microvascular endothelial cells;
Cell function
- From:
Chinese Journal of Cerebrovascular Diseases
2018;15(11):584-591
- CountryChina
- Language:Chinese
-
Abstract:
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. MSCmiR-132-3p was obtained from MSCs infected with lentivirus loaded with miR-132-3p vector. At the same time,MSCNC was obtained by infecting MSCs with control lentivirus loaded with scramble sequence. EXs released from MSCNCand MSCmiR-132-3pwas isolated,and MSC-EXs and MSC-EXsmiR-132-3pwere 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-EXsmiR-132-3p group (MSC-EXsmiR-132-3pco-culture), and MSC-EXsmiR-132-3p+ LY294002 group ( before the cells and MSC-EXsmiR-132-3pwere co-cultured,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-EXsmiR-132-3ptreatment group were increased (14 ±3,0. 444 ± 0.050,357±10μm,and1.67±0.23,respectively,all P<0.01).Apoptosis was significantly reduced (34±1%,all P<0. 01) . Compared with the MSC-EXsmiR-132-3ptreatment group, cell proliferation, migration, angiogenesis abilities,and Akt phosphorylation level in the MSC-EXsmiR-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 PI3K/Akt signaling pathway.
