间充质干细胞来源微囊泡对卵巢颗粒细胞的作用

Effect of mesenchymal stem cell-derived microvesicles on ovarian granular cells

目的探讨间充质干细胞来源微囊泡（MVs）对卵巢颗粒细胞的作用。方法取传代第8代（P8）前的人脐带间充质干细胞进行无血清培养,超速离心结合蔗糖密度梯度离心法从培养液上清中分离纯化MVs,透射电镜形态学观察,Bradford法蛋白定量。将PKH26标记的不同浓度MVs（0、10、15、20、25μg/ml）与3周龄雌性SD大鼠卵巢颗粒细胞共培养,3h后荧光显微镜观察颗粒细胞对MVs的内化作用;48h检测培养细胞周期各时相的变化及培养液上清中E2浓度。结果随培养时间的延长,MVs释放量显著增加,8×105个细胞大约释放10μg左右的MVs,电镜下MVs呈圆形或椭圆形,直径介于30~1 000nm之间,中间为低电子密度区。与MVs共培养3h后,颗粒细胞胞浆中可见呈红色荧光信号的MVs;不同浓度的MVs均可显著增加颗粒细胞的增殖指数（PI）、S期细胞比率（SPF）及E2水平,且呈先上升后下降趋势（P〈0.05）。结论MVs能够被颗粒细胞摄取;人脐带间充质干细胞来源的MVs能够促进颗粒细胞增殖及分泌E2功能。

Objective： To investigate the effect of mesenchymal stem cell-derived microvesicles（MSC-MVs）on ovarian granular cells（GCs）.Methods： The human cord blood MSCs before passage 8were cultured in serum-free medium and the medium supernatant were collected after 24,48 and 72hours respectively.Then MVs were extracted from supernatant and purified with the methods of ultracentrifugation and density gradient centrifugation.The morphological observation and protein quantification of MVs were carried out with the methods of transmission electronic microscope and Bradford respectively.GCs were separated from 3weeks female SD rats by mechanical method and identified by FSHR immunohistochemical staining.The effects of MSCMVs on GCs were carried out by co-culture of GCs with different concentration of MSC-MVs（0,10,15,20,25μg/ml）.The internalization of MVs in GCs was observed under fluorescent microscopy after 3hours co-culture of GCs and PKH26 labeled MVs,and cell cycle phases of GCs and the concentrations of E2 in medium supernatants were detected by chemiluminescence and FCM 48 hours later.Results： The MVs looked like circular or elliptic micro vesicles with low electron density shadow in the middle under the transmission electron microscope with diameter ranged from 30 nm to 1 000 nm.The amount of MVs were significantly increased along with the co-culture time,and approximately 8×105 cells released 10μg MVs.GCs of rats were separated successfully with the purity over 95% proved by FSHR immunohistochemical staining.The intensive red fluorescence signal was observed in cytoplasm of GCs after 3hours co-culture with PKH26 labeled MVs,and the proliferation index（PI）,S-phase fraction（SPF）of GCs and the concentration of E2 in medium supernatants were significantly increased when GCs were cocultured with MVs（P〈0.05）,with a trend of increasing first and then descending.Conclusions： MSC-MVs can be ingested by GCs.Mesenchymal stem cell-derived microvesicles can promote the proliferation and secretion function of GCs.