子宫内膜基质干细胞的分离培养研究

Isolation and identification of human endometrial stromal stem cells

目的:通过体外分离、培养子宫内膜基质干细胞,观察其生物学特性,探索一种简便、高效的分离培养人子宫内膜基质干细胞的方法。方法:取10例因子宫肌瘤行全子宫切除术患者的内膜组织标本,磁珠分选及克隆形成实验法分选子宫内膜基质干细胞,传代培养。流式细胞仪检测原代细胞表面标记,MTT法观察基质干细胞体外增殖情况;体外向成骨、成脂诱导分化,茜素红和油红O染色鉴定细胞分化能力,RT-PCR测定骨细胞和脂肪细胞特异性基因表达。同时检测基质干细胞表达胚胎时期标志蛋白OCT-4的情况。结果:通过该方法可获得子宫内膜基质干细胞,细胞易于贴壁,生长良好,传代稳定。细胞CD90和CD105表达阳性而CD45和CD34表达阴性。细胞周期的S期和G2-M期细胞比例高,细胞生长曲线呈"S"形。经成骨及成脂诱导后茜素红和油红O染色分别呈阳性反应,成骨及成脂标志性基因表达阳性。此外,免疫细胞化学染色显示OCT-4表达阳性。结论:子宫内膜基质干细胞具有间充质干细胞特性,来源广、易获取,可作为组织工程种子细胞来源。

Objective： To isolate,culture and identify of human endometrial stromal stem cells.Methods： Endometrial tissue was obtained from 10 women undergoing hysterectomy.Purified stromal stem cell suspensions were then obtained by selecting cells with magnetic bead sorting and colony-forming.The surface markers of stromal cell were identified by flow cytometry.Proliferation of stromal stem cell was observed by MTT methods.The osteogenic potential was evaluated by alizarin red staining,the adipogenic potential by oil red O staining.Then the adipogenic and osteogenic specific markers of differentiated cells assayed by RT-PCR method.Expression of cell surface antigen OCT-4 was detected with immunocytochemical staining.Results： Endometrial stem cells were successfully isolated from human endometrial tissue.They were stably proliferated and subcultured in vitro.Most of the passage cells expressed mesenchymal stem cell markers CD90,CD105 but not hemopoietic markers CD34,CD45.The analysis of cell cycle indicated that the percentage of G2-M phase and S phase cells increased.The growth curves of the third passage presented in ＂S＂ shape.After cultured in differentiation medium,the cells differentiated toward adipoblasts and osteoblasts as verified by positive staining with Oil Red O and alizarin red staining.Under induction,cells expressed osteogenic and adipogenic marker genes.The immunocytochemical staining of OCT-4 was positive.Conclusion： Human endometrium contains endometrial stromal stem cells,which present characteristics of mesenchymal stem cells and may be used as seed cells for tissue engineering.