M1型巨噬细胞源外泌体增强卵泡膜细胞的活力

Exosomes derived from M1 macrophages enhance viability of theca cells

目的:探讨M1型巨噬细胞源外泌体对卵泡膜细胞活力的影响及其作用机制。方法:采用脂多糖(LPS)处理Raw264.7小鼠巨噬细胞,构建M1型巨噬细胞模型及巨噬细胞-卵泡膜细胞共培养体系。超速离心法分离巨噬细胞分泌的外泌体;通过电镜、Western blot和纳米流式检测仪鉴定外泌体。体外分离培养小鼠卵泡膜细胞,与PKH67荧光标记的外泌体共孵育,观察卵泡膜细胞摄取外泌体的情况。CCK-8法检测细胞活力,流式细胞术分析细胞周期分布,q PCR及Western blot检测细胞周期蛋白依赖性激酶抑制因子1B(CDKN1B)的表达。结果:在巨噬细胞-卵泡膜细胞共培养体系中,LPS诱导的M1型巨噬细胞通过外泌体增强卵泡膜细胞活力。电镜、Western blot及纳米流式检测结果显示,巨噬细胞源外泌体被成功分离。PKH67标记的外泌体与卵泡膜细胞共孵育实验证实卵泡膜细胞能摄取大量巨噬细胞源外泌体。这些外泌体可增强卵泡膜细胞的活力,使卵泡膜细胞G0/G1期比例下降,S期比例升高,且卵泡膜细胞CDKN1B的m RNA及蛋白相对表达量均明显低于对照组。结论:卵泡膜细胞能摄取巨噬细胞分泌的外泌体。M1型巨噬细胞源外泌体通过抑制CDKN1B的表达而增强卵泡膜细胞的活力。

AIM:To investigate the effects of exosomes secreted by M1 macrophages on the viability of ovarian follicular theca cells and the possible mechanism.METHODS:Raw264.7 mouse macrophages were treated with lipopolysaccharide(LPS)to establish a model of M1 macrophages.Exosomes secreted by the macrophages were isolated by ultracentrifugation and identified by electron microscopy,Western blot,and flow nanoanalyzer.Primary mouse ovarian follicular theca cells were co-incubated with PKH67 fluorescence-labeled exosomes to detect whether exosomes secreted by the macrophages were uptaken by the theca cells.The cell viability was measured by CCK-8 assay.Flow cytometric analysis was used to evaluate the cell cycle distribution.The expression of cyclin-dependent kinase inhibitor 1B(CDKN1B)was determined by q PCR and Western blot.RESULTS:The results of electron microscopy,Western blot,and flow nanoanalyzer verified that the macrophages secreted exosomes.The co-incubation of PKH67 fluorescence-labeled exosomes and theca cells confirmed that the theca cells were able to ingest large numbers of exosomes secreted by the macrophages.M1 macrophage-derived exosomes significantly enhanced the viability of theca cells by shifting the cells from G1phase to S phase.The expression of CDKN1B was up-regulated in theca cells treated with M1 macrophage-derived exosomes as compared with the controls.CONCLUSION:M1 macrophages-derived exosomes enhance the viability of theca cells in association with down-regulated expression of CDKN1B.