红芪小分子提取物对MSCs体外增殖与染色体的影响

Effect of micromolecule-aqueous extracts of Hongqi( Radix Hedysari) on the proliferation and chromosome of MSCs in vitro

目的探讨红芪小分子提取物对骨髓间充质干细胞(MSCs)体外增殖及染色体的影响。方法利用水提法结合超滤法提取红芪中小分子物质,并筛选其促进MSCs增殖的最佳浓度。设单独培养的MSCs为对照组,红芪最佳浓度(20 mg/L)培养72 h后MSCs为实验组,于倒置相差显微镜下观察各组细胞形态学的改变;采用MTT法检测各组细胞生长曲线;流式细胞术检测细胞周期;染色体显色与计数分析细胞染色体;Western印迹检测各组细胞基质金属蛋白酶(MMP)-2、MMP-9蛋白表达水平的变化。结果红芪浓度20 mg/L为最佳促进MSCs增殖浓度(P<0.05)。倒置相差显微镜下观察对照组细胞呈成纤维细胞样;红芪组细胞呈长梭形,形态类似于对照组。与对照组比较,红芪组细胞生长速度显著增快(P<0.05);细胞周期G0/G1期细胞减少,S期和G2/M期细胞增多,但差异无统计学意义(P>0.05);染色体及MMP-2、MMP-9蛋白表达水平无异常改变(P>0.05)。结论红芪小分子提取物20 mg/L体外培养MSCs 72 h后,对其增殖有明显促进作用,且维持其遗传物质染色体的稳定性,其作用机制可能与调控MSCs的MMP-2、MMP-9等相关蛋白表达有关。

Objective To study the effects of micromolecule-aqueous extracts of Hongqi on proliferation and chromosome of mesenehymal stem cells( MSCs) in vitro. Methods The water-boiling method combined with ultrafiltration were used to extract the micromoleculeaqueous substances of Hongqi and the best concentration was screened for proliferative effect on MSCs. MSCs cultured alone was control group,and MSCs cultured with the best concentration of Hongqi( 20 mg / L) for 72 h was experimental groups. The morphological changes in the cells were observed by phase-contrast microscopy. MTT assay was used to measure the cells changes in growth. The cell cycle of MSCs was determined by flow cytometry. Chromosome was examined by colored and counted. The expressions of MMP-2,MMP-9 proteins were determined by Western blot. Results 20 mg / L micromolecule-aqueous extracts of Hongqi was best concentrations to promote the growth of MSCs( P < 0. 05). The cell morphologies were observed by phase-contrast microscopy,cells of MSCs were fibroblast-like,cells of Hongqi were long fusiform,closed to MSCs cells in morphology. Compared with MSCs,cells of Hongqi grow were faster( P < 0. 05),the proportion of G0 / G1 phase cells was reduced,while the proportion of S phase and G2 / M phase were increased,but there was no statistical significance( P > 0.05),the chromosome and the proteins expressions of MMP-2,MMP-9 did not change abnormally( P > 0. 05). Conclusions Micromoleculeaqueous extracts of Hongqi( 20 mg / L) culturing MSCs for 72 h could improve proliferation of MSCs significantly,and preserve the genetic stability of MSCs’ chromosome in vitro. The mechanism is related with the regulation of the expressions of MMP-2,MMP-9 proteins of MSCs.