体外条件下蜕皮甾酮对大鼠脐带间充质干细胞增殖的实验研究

目的:研究体外条件下蜕皮甾酮(ecdysterone，EDS)对于大鼠脐带间充质干细胞增值活性的影响。方法：采用胶原酶消化法分离培养大鼠脐带间充质干细胞，并通过动态观察细胞形态，流式细胞术检测细胞表面抗原加以鉴定；取第3代脐带间充质干细胞分别在基础培养基(DMEM-LG培养基、10%的胎牛血清、100U/ml青/链霉素、2mmol/L L-谷氨酰胺)中加入不同浓度（0、25、50、100、150、200μg/ml）EDS予以干预，分别于第1、3、5、7天MTT法检测细胞增殖活性；另外，设置相同分组EDS干预培养大鼠脐带间充质干细胞7天，绘制细胞生长曲线。结果：采用酶消化法能有效分离纯化大鼠脐带间充质干细胞。细胞细胞流式仪鉴定显示，第3代细胞阳性表达CD44、CD90，阴性表达CD34、CD45。实验采用MTT法检测大鼠脐带间充质干细胞活力，结果显示经EDS干预培养的大鼠脐带间充质干细胞生长状态良好，在细胞活力方面较空白对照组有明显差异(P<0.05)，EDS 100μg/ml、150μg/ml及200μg/ml三组细胞活性比较无显著差异(P>0.05)，其他实验组之间无统计学意义(P>0.05)。细胞生长曲线同样显示EDS实验组可有效促进干细胞的增殖，缩短细胞倍增时间，此作用在EDS浓度为100μg/ml时达到最佳。结论：体外条件下，EDS对大鼠脐带间充质干细胞具有促增殖作用，该作用在EDS浓度为100μg/ml时较为显著，不再随浓度的增加而增强。这有望成为促进脐带间充质干细胞增殖的新方法。

Effects of ecdysterone on the proliferation of rat umbilical cord mesenchymal stem cells in vitro

Objective To investigate the effects of ecdysterone on rat umbilical cord mesenchymal stem cells’ proliferation in vitro. Methods MSCs were separated from rat umbilical cord with enzyme method and then cultured in DMEM medium. Surface antigens of these cells were detected by FACS. The various concentrations of EDS (0μg/ml、25μg/ml、50μg/ml、100μg/ml、150μg/ml、200μg/ml) were added in the culture system for MSCs(DMEM-LG,10%FBS,100U/ml Streptomycin, 100U/ml penicillin, 2mmol/L L-Glutamine). Then the cell number viability were analyzed by MTT assay. Results Enzyme method could obtain plenty of MSCs from rat umbilical cord. The third generation cells were CD44、CD90 positive, CD34 and CD45 negative as determined by flow cytometry. The optical density(OD) differed significant between the EDS treatment groups and the control group(P<0.05)，there are no significant differences among 100μg/ml 、150μg/ml 、200μg/ml EDS groups (P>0.05). Also the growth curves of groups with EDS exhibited a great potential for proliferation .Conclusion: EDS can promote the proliferation of mesenchymal stem cells from rat umbilical cord at the optimal concentration of 100μg/ml in vitro. It is expected to be a new method to induce mesenchymal stem cells to proliferation .