脐带间充质干细胞对角膜内皮细胞增殖能力的影响

Umbilical cord mesenchymal stem cells promote the proliferation of corneal endothelial cells

目的:探讨并优化人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)体外获取及培养增殖的方法并鉴定;观察兔角膜内皮细胞(cornea endothelial cells,CEC)与hUCMSCs共培养后增殖能力及细胞周期的变化。方法:用酶消化法体外分离培养hUCMSCs,流式细胞仪检测hUCMSCs免疫表型;将CEC分别与hUCMSCs、兔角膜基质细胞在Transwell体系中共培养,通过流式细胞术观察内皮细胞增殖能力及细胞周期的变化。结果:采用酶消化法能有效分离纯化hUCMSCs。接种24h,贴壁细胞形态多为长梭形、多边形或成纤维细胞样形态,大小均一。流式细胞仪分析第3代细胞均表达CD29、CD44、CD105,不表达造血干细胞标记CD34、CD45和HLA-DR。与空白对照组CEC周期比较脐带间充质干细胞组与基质细胞组角膜内皮细胞S期细胞比例增加,G1期细胞比例下降,干细胞组增加幅度高于基质细胞组。干细胞组角膜内皮细胞S期细胞比例比空白对照组的平均增加近17%,增殖能力显著提高。结论:经鉴定用酶消化法体外能成功分离培养出hUCMSCs,将其与CEC共培养能促进CEC增殖。

Objective To establish an effective and appropriate method to isolate, culture and identify human umbilical cord mesenchymal stem ceils （hUCMSCs） in vitro and analyze its effects on the proliferation and cell cycle of co-cultured rabbit corneal endothelial cells （CECs）. Methods hUCMSCs isolated with collagenase treatment and cultured in vitro. Phenotype analysis was performed by using flow cytometry. And transwcll co-culture systems and flow cytometry were applied to analyze the effects of bUCMSCs on the proliferation and cell cycle of co-cultured CEC cells. Results hUCMSCs isolation with collagenase treatment was efficient. 24 hours after seeding, adherent cells showed spindle shape, polygonal shape and fibroblast-cell-like shape with homogeneous cell size. Flow cytometry analysis revealed that CD29, CD44, CDI05 were highly expressed on the plasma membrane of passages 3 cells, but negative for CD34, CD45 and HLA-DR. Compared with the CEC cultured alone group, the portions of S-phase CECs in both hUCMSCs and stromal-cells cocultured groups increased while their portions of Gl-phase cells decreased. And changes of cell cycle of CECs in hUCMSCs co-cultured group were more significant than those of stromal-cell co-cultured group. The portion of S- phase CECs in hUCMSCs co-cultured group increased about 17%, indicating a dramatic promotion of cell proliferation. Conclusions hUCMSCs can be isolated with collagenase treatment and cultured successfully in vitro. And hUCMSCs cells promote the proliferation of co-cultured CECs.