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

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

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殖活性的影响。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、100 U/mL青/链霉素、2 mmol/L L-谷氨酰胺)中加入不同浓度(0、25、50、100、1502、00μg/mL)EDS予以干预,分别于第1、3、5、7天用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 d,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。细胞流式仪鉴定显示,第3代脐带间充质干细胞阳性表达CD44和CD90,阴性表达CD34和CD45。MTT法检测结果显示经EDS干预培养的大鼠脐带间充质干细胞生长状态良好,在细胞活力方面与空白对照组比较差异有统计学意义(P<0.05),EDS 100、150及200μg/mL 3组细胞活性比较差异无统计学意义(P>0.05)。细胞生长曲线同样显示EDS实验组可有效促进脐带间充质干细胞的增殖,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,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。

Objective To investigate the effects of ecdysterone on the proliferation of rat umbilical cord mesenchymal stem cells in vitro. Methods MSCs were isolated from rat umbilical cord with enzyme method and cultured in DMEM medium.Surface antigens ere detected by FACS.Diverse concentrations of EDS（0 μg/mL,25 μg/mL,50 μg/mL,100 μg/mL,150 μg/mL and 200 μg/mL） were added in the culture system for MSCs（DMEM-LG,10%FBS,100 U/mL Streptomycin,100 U/mL penicillin,and 2 mmol/L L-Glutamine）.The cell viability was analyzed by MTT assay. Results MSCs were successfully obtained using enzyme method from rat umbilical cord.CD44CD90-positive and CD34CD45-negative cells were detected in the third passage of obtained cells by flow cytometry.The optical density（OD） differed significantly between the EDS treatment groups and the control group（P〈0.05）,while no significant difference was revealed among 100 μg/mL,150 μg/mL and 200 μg/mL EDS groups（P〉0.05）.Moreover,the growth curves of groups with EDS exhibited a great potential for proliferation. Conclusion EDS promotes the proliferation of mesenchymal stem cells from rat umbilical cord at the optimal concentration of 100 μg/mL in vitro.It is expected to be a new method to induce mesenchymal stem cells to proliferation.