小鼠子宫内膜间充质干细胞的分离、培养及鉴定

Isolation,culture and identification of stem cells from mice endometrium

目的 建立一种分离、纯化、培养及鉴定小鼠子宫内膜间充质干细胞的方法。方法 根据不同来源间充质干细胞特性相似,用小鼠骨髓间充质干细胞富集培养液对C57BL/6J小鼠子宫细胞群进行间充质干细胞培养,经过传代达到较高的纯度。倒置显微镜观察子宫内膜干细胞形状特点。流式细胞术分析相关表面标记物表达。CCK-8法进行增殖曲线绘制。新赛美无血清细胞冻存液重悬细胞后,- 80 ℃冻存,37 ℃水浴复苏,台盼蓝染色观察细胞冷冻复苏后情况。结果 获得的子宫内膜干细胞呈梭形,融合后极性排列,可见大小不等漩涡状集落出现。培养1-3 d细胞增殖缓慢,4-6 d进入对数生长期,6-7 d进入平台期。流式细胞术分析第5代细胞CD29、CD73、CD105阳性,CD45、CD117阴性。细胞冷冻复苏后存活率达到96%以上,生长状态良好,增殖能力强,与冻存前细胞活力无明显差异。结论 本实验成功分离、培养和鉴定小鼠子宫内膜干细胞。

Objective To establish a method for isolating, purifying and identifying the stem cells from a mouse endometrium. Methods According to the similar characteristics of mesenchymal stem cells from different sources, the stem cells of the C57BL/6J mouse endometrium were cultured with culture fluid enriched by mouse bone marrow mesenchymal stem cells. Then the higher purity of the mouse endometrial mesenchymal stem cells was achieved by passage culture, and the shapes of the stem cells were observed under inverted microscope. The related antibody expression was analyzed by flow cytometry. The proliferation was detected with CCK8 method. After the cells were suspended in the serum-free cryopreservation solution of neo-Saimei, they were frozen at - 80 ℃ and resuscitated in water bath at 37 ℃. After refrigerant recovery, cells were observed by dyeing with trypan blue. Results The obtained endometrial stem cells were spindle,and arranged in polarity, with vortex-shaped colonies of different sizes. Cell proliferation was slowly in the prophase of culture at 1-3 d, and entered logarithmic growth stage at 4-6 d, then was in plateau stage at 6-7 d. CD29, CD73 and CD105 were positive in the stem cells of the fifth generation by flow cytometry, while CD45, CD117 were negative. The survival rate of cell after cryopreservation was more than 96%, the growth was in good condition and the proliferation ability was strong. The difference was not obvious between the normal cells and cryopreserved ones. Conclusion The method for separation and culture of mouse endometrium stem cells is established successfully.