黄芪、红芪小分子提取物对间充质干细胞体外增殖与遗传稳定性的考察

目的:探讨黄芪、红芪小分子提取物对骨髓间充质干细胞(mesenehymal stem cells, MSCs)体外增殖的影响。方法:利用水提法结合超滤法提取黄芪、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,黄芪、红芪最佳浓度分别培养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;采用四唑蓝(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、计数与原子力显微镜法(atomic force microscope,AFM)分析细胞染色体。结果:黄芪质量浓度60 mg·L^-1、红芪质量浓度20 mg·L^-1为最佳促进MSCs增殖质量浓度(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;黄芪组、红芪组细胞呈长梭形,形态类似于对照组。与对照组比较,黄芪组、红芪组细胞生长速度均显著增快(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);染色体无异常改变(P>0.05)。结论:黄芪、红芪小分子提取物分别为60,20 mg·L^-1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。

Influences of micromolecular-aqueous extracts of Hongqi (Radix Hedysari) and Huangqi (Radix Astragali seu Hedysari) on proliferation and genetic stability of MSCs in vitro

OBJECTIVE To study the effects of micromolecular-aqueous extracts of Huangqi and Hongqi on proliferation of mesenehymal stem cells (MSCs) in vitro. METHODS Water-boiling method combined with ultrafiltration was used to extract micromolecular-aqueous substances of Huangqi and Hongqi. The best concentration was screened for proliferative effects on MSCs. MSCs cultured alone were used as control group, and MSCs cultured respectively with the best concentrations of Huangqi and Hongqi for 72 h were adopted as experimental groups. Morphological changes in cells were observed by phase-contrast microscopy. MTT assay was used to measure cell growth profile. Cell cycles of MSCs were determined by flow cytometry. Chromosomes were colored, counted and determined by using Atomic Force Microscope (AFM). RESULTS Results of MTT assay suggested that 60 mg·L^-1 micromolecular-aqueous extracts of Huangqi and 20 mg/L micromolecular-aqueous extracts of Hongqi were best concentrations to promote growth of MSCs (P<0.05). Cell morphologies were observed by phase-contrast microscopy, cells of MSCs were fibroblast-like, cells of Huangqi and Hongqi were long fusiform, close to MSCs cells in morphology. Compared with MSCs, cells of Huangqi and Hongqi grew faster (P<0.05). Proportion of G₀/G₁ phase cells in Huangqi and Hongqi group reduced, while proportion of S phase and G₂/M phase increased, but there was no statistical significance (P>0.05). Chromosomes of Huangqi and Hongqi did not change abnormally (P>0.05). CONCLUSION Micromolecular-aqueous extracts of Huangqi (60 mg·L^-1) and Hongqi (20 mg·L^-1) after cultured with MSCs for 72 h can improve proliferation of MSCs significantly, and preserve the genetic stability of MSC chromosome in vitro.