摘要
目的 体外分离、培养和纯化大鼠骨髓间充质干细胞(MSC),并研究中药单体丹酚酸B对MSC增殖和分泌血管内皮生长因子(VEGF)的影响.方法 用贴壁培养法分离、培养和纯化MSC.免疫荧光法检测细胞抗原CD44和CD34.用含不同浓度丹酚酸B的低糖DMEM培养液培养MSC 24 h后,用MTT法检测细胞增殖水平.RT-PCR检测培养24 h、48 h、72 h 后VEGF mRNA的表达.结果 获得纯化的MSC,免疫荧光检测细胞表面标记CD44呈阳性,CD34呈阴性.MTT结果 显示,0.3 mg/L和3 mg/L丹酚酸B均可促进MSC的增殖,与空白对照组差异均有统计学意义(P<0.01,P<0.05),而003 mg/L和30 mg/L丹酚酸B组细胞的增殖与空白对照组差异均无统计学意义(P>0.05).随着培养时间的增加,0.03、0.3、3和30 mg/L丹酚酸B均可明显促进VEGF mRNA的表达,与空白对照组相比差异均有统计学意义(P<0.05),而空白对照组的VEGF mRNA表达无明显变化.结论 用贴壁培养法可获得纯化的MSC.丹酚酸B可促进MSC增殖和VEGF mRNA的表达.
Objective To isolate,culture and purify rat bone marrow mesenchymal stem cells (MSCs) and observe the effect of salvianolic acid B on MSCs proliferation and vascular endothelial growth factor (VEGF) expression in vitro. Methods MSCs were isolated,cultured and purified in vitro by adherent method and identified by immunofluorescence detection of cell markers CD44 and CD34. MSCs were cultured in five groups of lower glucose DMEM media containing different concentrations of salvianolic acid B. The growth ability of cells was assayed with methyl thiazolyl tetrazolium(MTT) method. The expression of VEGF mRNA in the MSCs harvested at following 24 h,48 h and 72 h were analyzed with reverse transcription-polymerase chain reaction. Results Pure MSCs were obtained. The cultured cells marked with CD44 were positive and those with CD34 were negative by immunofluorescence detection,so it could be identified as MSCs. The cell proliferation was promoted by 0.3 mg/L and 3 mg/L salvianolic acid B as compared with control group (P<0.01,P<0.05),while 0.03 mg/L and 30 mg/L salvianolic acid B did not affect the proliferation of MSCs. The expression of VEGF mRNA of MSCs treated with salvianolic acid B at concentrations of 0.03,0.3,3 and 30 mg/L were increased significantly as compared with control group (P<0.05). Conclusion Pure MSCs can be obtained by adherent method and salvianolic acid B can promote the proliferation and the expression of VEGF mRNA of MSCs.
出处
《中华生物医学工程杂志》
CAS
2008年第1期-,共4页
Chinese Journal of Biomedical Engineering
