补肾活血方含药血清对小鼠肢芽细胞增殖和分化的影响

Effects of medicated serum of Bushen Huoxue Recipe on proliferation and differentiation of mouse limb bud cells

目的探讨补肾活血方对小鼠肢芽细胞向软骨细胞分化增殖的影响。方法分离12.5天小鼠胚胎肢芽细胞进行微团培养,同时用补肾活血方含药血清进行干预。采用MTT法检测肢芽细胞的增殖活性,阿尔新蓝染色检测肢芽细胞分化为软骨细胞能力,RT-PCR法检测肢芽细胞中Col2a1、Col10a1、Mmp9、β-catenin、Runx2 mRNA的表达水平。结果与空白对照组比较,补肾活血方含药血清组在24 h、48 h和72 h 3个时间点均能显著促进小鼠肢芽细胞增殖。微团培养6 d后,阿尔新蓝染色结果显示补肾活血方含药血清组软骨细胞集落形成增加。RT-PCR结果显示补肾活血方含药血清组肢芽细胞Col2a1 mRNA表达水平增加,而Col10a1、Mmp9、β-catenin、Runx2 mRNA表达下调。结论补肾活血方含药血清可能通过β-catenin信号通路促进小鼠胚胎肢芽细胞的增殖与分化。

Objective To discuss the effects of Bushen Huoxue Recipe on the differentiation and proliferation of mouse limb bud cells into chondrocytes. Methods The limb bud cells of 12. 5-day mouse embryo were isolated and cultured,and then treated with medicated serum of Bushen Huoxue Recipe. The proliferation activity of limb bud cells was detected by MTT,the differentiation capacity of limb bud cells into chondrocytes was observed by alcian blue staining,and the mRNA expression levels of Col2a1,Col10a1,Mmp9,β-catenin and Runx2 in limb bud cells were detected by RT-PCR. Results Compared with the blank control group,the proliferation of mouse limb bud cells was promoted in medicated serum group of Bushen Huoxue Recipe at timepoints of 24 h,48 h and 72 h. After micromass culture for 6 days,the alcian blue staining showed that the cartilage formation was increased in the medicated serum group of Bushen Huoxue Recipe.The RT-PCR results showed that the mRNA expression level of Col2a1 in limb bud cells was up-regulated in the medicated serum group of Bushen Huoxue Recipe,while the mRNA expression levels of Col10a1,Mmp9,β-catenin and Runx2 were down-regulated. Conclusion The medicated serum of Bushen Huoxue Recipe may promote the proliferation and differentiation of mouse embryo limb bud cells through β-catenin signaling pathway.