人骨髓间充质干细胞来源外泌体分离、鉴定及卵巢摄取

Isolation,identification and ovarian uptake of exosomes derived from human bone marrow mesenchymal stem cells

目的提取及鉴定人骨髓间充质干细胞(hBMSC)来源外泌体,探讨颗粒细胞及自身免疫性早发性卵巢功能不全(POI)小鼠卵巢摄取外泌体的情况。方法取P3至P9代hBMSC培养上清液,倒置显微镜下观察P4代hBMSC细胞形态,超高速离心法提取外泌体;纳米流式仪测外泌体粒径;透射电镜观察外泌体形态;Western blot检测外泌体标志蛋白CD63、CD9、肿瘤易感基因(TSG)101表达。外泌体采用DiR进行荧光标记;共聚焦显微镜观察人卵巢颗粒细胞(KGN)共孵育24 h和48 h摄取外泌体的情况;用ZP3蛋白多肽构建自身免疫性POI小鼠模型,在建模成功后的第1、7天腹腔注射150µg DiR标记的外泌体。在第2次注射外泌体后的第14天通过活体成像仪监测小鼠卵巢摄取情况。结果hBMSC呈纺锤状,大小均一,提取出的外泌体平均直径为(81.12±17.23)nm。透射电镜显示外泌体结构为双层膜状;外泌体表面相关特异性蛋白CD63、CD9和TSG101呈阳性。共聚焦显微镜下观察到KGN细胞能够摄取外泌体,并与共孵育24 h比,48 h摄取的外泌体量更多。在自身免疫性POI小鼠模型中观察到小鼠卵巢部位有发光信号。结论本研究成功提取出hBMSC来源外泌体,并观察到KGN细胞及自身免疫性POI小鼠卵巢组织摄取外泌体。

Objective To extract and identify exosomes derived from human bone marrow mesenchymal stem cells(hBMSC),and to investigate the exosome uptake by granulosa cells and the ovaries of autoimmune premature ovarian insufficiency(POI)mouse.Methods hBMSC of generation P3 to P9 were cultured,and the cell supernatant was collected.The morphology of hBMSC at P4 generation was observed under an inverted microscope.The morphology of the exosomes was observed by transmission electron microscopy,and the particle size of the exosomes was measured by nanoflow cytometry.Western blot assay was used to detect the expression of exosome surface-associated specific proteins CD63,CD9 and TSG101.DiR was used for fluorescence labeling of exosomes.The exosome uptake by human ovarian granulosa cells(KGN)was observed by confocal microscopy after 24 h and 48 h co-incubation.The autoimmune POI mouse model was constructed with ZP3 protein polypeptide,and 150µg DiR-labeled exosomes were intraperitoneally injected on the 1st and 7th days after successful modeling.Exosome uptake of mouse ovaries were monitored by an in vivo imager on day 14 after the second injection of exosomes.Results hBMSC cells were spindle-shaped and uniform in size.Transmission electron microscopy showed that exosomes were double-layered membranes with a diameter of(81.12±17.23)nm.Exosome expression marker proteins CD63,CD9 and TSG101 were positive.Under confocal microscopy,it was observed that KGN cells could absorb exosomes,and the volume of exosomes taken up by KGN cells at 48 h was more than that of co-incubation for 24 h.In the autoimmune POI mouse model,luminescent signals were observed in mouse ovaries on the 14th day after the second injection of exosomes.Conclusion In this study,exosomes derived from hBMSC are successfully extracted,and the uptake of exosomes by KGN cells and ovarian tissue of autoimmune POI mice is observed.