探讨骨髓间充质干细胞来源的外泌体是否通过miR-99b-5p/PCSK9轴修复受损的子宫内膜基质细胞。通过超速离心分离源自BMSCs的外泌体(BMSC-exos),并使用透射电子显微镜和纳米流式细胞术表征。建立了米非司酮体外诱导的EnSC损伤模型,并评估BMSC-exos的摄取。将EnSCs分为三组:正常组(CTRL),EnSC损伤组(模型)和BMSC-exo治疗组。BMSC-exos对EnSC增殖的影响,凋亡,通过将MSC-exos与子宫内膜细胞共培养来评估血管内皮生长因子(VEGF)的表达。此外,高通量测序用于鉴定差异表达基因(DEGs).通过生物信息学分析,RT-qPCR,西方印迹,CCK8测定,免疫组织化学和双荧光素酶实验,研究了BMSC-exos来源的miRNA修复EnSC损伤的潜在机制。BMSC-exos表达标记蛋白CD9和CD63。激光共聚焦显微镜显示BMSC-exos可以进入受损的EnSC。BMSC-exos-EnSC共培养组与模型组比较,BMSC-exos以剂量依赖的方式显着增加受损的EnSC的增殖并抑制细胞凋亡。BMSC-exos-EnSC共培养组Caspase-3、Caspase-9、Bax和VEGFmRNA表达水平显著下调,而Bcl-2表达上调。我们确定了模型和CTRL组之间的28个重叠DEG,以及BMSC-exo组和模型组之间。转染miR-99b-5p模拟物显著降低PCSK9基因的表达,抑制自噬相关蛋白Beclin-1和LC3-II/I的表达和凋亡,从而促进EnSC增殖。用miR-99b-5p抑制剂转染显示出相反的作用。Beclin-1、LC3-II/I和PCSK9在薄型子宫内膜中表达显著增高。miR-99b-5p通过靶向PCSK9促进细胞增殖。BMSC-exos促进子宫内膜增殖,miR-99b-5p通过靶向PCSK9抑制细胞凋亡,促进EnSC增殖,为薄型子宫内膜的治疗提供新的靶点。
The aim of this article was to investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells (EnSCs) through the miR-99b-5p/PCSK9 axis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos was assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model), and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction, western blotting, the CCK8 assay, immunohistochemistry, and dual-luciferase experiments, the potential mechanism by which BMSC-exos-derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSCs and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax, and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, whereas Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy-related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I, and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.