大鼠骨髓间充质干细胞培养鉴定及复方扶芳藤合剂含药血清对细胞增殖的影响

目的:建立SD大鼠骨髓间充质干细胞(BMSCs)的分离培养方法,观察不同浓度的复方扶芳藤合剂大鼠含药血清对大鼠BMSCs增殖的影响。方法:通过全骨髓贴壁法体外分离、培养、纯化BMSCs,对其进行细胞形态学观察,将BMSCs进行成骨、成脂定向诱导分化,采用流式细胞仪检测其表面标志物,分析其细胞周期,进行BMSCs鉴定;采用不同浓度(20%、10%和5%)的复方扶芳藤合剂含药血清和空白血清连续7 d对大鼠BM SCs进行干预,以CCK-8法检测其吸光度值,对比各组细胞生长情况。结果:BMSCs具有典型的成纤维样细胞形态,集落生长呈漩涡状。BMSCs成脂、成骨诱导后,油红O染色和茜素红染色均呈阳性。第3代BMSCs表型CD29、CD44、CD34、CD44表达分别为99.645%、99.677%、0.016%、0.133%;不同浓度的复方扶芳藤合剂含药血清和空白血清连续干预7 d后,CCK-8法检测显示:20%浓度的复方扶芳藤合剂含药血清组,其1~7 d的吸光度均值均高于20%浓度的空白血清组(P<0.05);10%的含药血清组的吸光度均值在3~7 d,高于10%的空白血清组(P<0.05);5%的含药血清组与5%的空白血清组比较无明显差异(P>0.05)。结论:全骨髓贴壁法分离、培养BM SCs,操作简单,方法稳定,可大量扩增BMSCs。扩增后的BMSCs具有间充质干细胞的生物学特性,具有多向分化潜能。复方扶芳藤合剂含药血清干预后的BMSCs增殖能力增强,其中20%浓度的复方扶芳藤合剂含药血清在本实验中为最佳干预浓度。

Culture and Identification of Rat Bone Marrow Mesenchymal Stem Cells and Effects of Compound Fufangteng Mixture Serum on Cell Proliferation

Objective:To establish a method of isolation and culture of bone marrow mesenchymal stem cells(BMSCs) in SD rats;to observe the effect of different concentrations of compound fufangteng mixture containing serumon the proliferation of BMSCs.Methods:BMSCs were isolated, cultured and purified in vitro by whole bone marrowadherence method. The morphology of BMSCs was observed. The BMSCs were induced to differentiate into osteoblastsand adipocytes. The surface markers and cell cycle of BMSCs were detected by flow cytometry for the identification ofBMSCs. The BMSCs in rats were intervened with compound fufangteng mixture containing serum and blank serum indifferent concentrations(20%, 10% and 5%)respectively for 7 days continuously. The values of absorbance weretested by CCK-8 method to compare the proliferation of BMSCs from all the groups.Results:BMSCs display typicalfibroblast like cell morphology and dense growth in the form of whirlpool. The Oil red O staining and alizarin redstaining were positive after BMSCs were induced into osteoblasts and adipocytes. The expression of CD29, CD44, CD34 and CD44 were 99.645%, 99.677%, 0.016% and 0.133% respectively in the 3 rdgeneration of BMSCs. After intervened by different concentrations of compound fufangteng mixture containing serum and blank serum respectively for sevendays continuously all values tested by CCK-8 show that 20% compound fufangteng mixture containing serum groupwas higher than 20% blank serum group(P<0.01)(1~7 d), 10% medicated serum group was higher than 10% blankserum group(P<0.05)(3~7 d), 5% medicated serum group and 5% blank serum group were no significant difference(P>0.05).Conclusion:It is a simple and stable method that BMSCs can be isolated, purified and amplified by wholebone marrow adherent method. BMSCs cultured show the general biological characteristics of mesenchymal stem cellsand the potential of multidirectional differentiation;BMSCs proliferation capability was enhanced after the interventionof compound fufangteng mixture containing serum. Among the different concentrations, 20% fufangteng mixture serumis the best in this study.