体外心肌微环境下益气活血化瘀方药诱导骨髓间充质干细胞向心肌样细胞分化的研究

Effects of qi supplementing,blood activating,and blood stasis resolving medicinals on differentiation of bone mesenchymal stem cells to cardiomyocyte-like cells in microenvironment of rat myocardium: a laboratory study in vitro

目的探讨体外心肌微环境下不同治法方药诱导骨髓间充质干细胞(BMSC)向心肌样细胞分化的差异性。方法 40只成年SD大鼠随机分为活血化瘀组、益气活血组、益气组、空白组,给相应药物后制备含药血清;另再从60只正常SD大鼠提取、培养及分离纯化BMSC;同时提取、培养SD新生鼠心肌细胞,建立体外模拟大鼠心肌微环境模型。用MTT法选择各给药组的最佳倍比浓度;流式细胞仪检测BMSC中CD105阳性细胞(CD105+-BMSC)分裂增殖情况;免疫细胞化学法检测心肌肌钙蛋白T(CTNT)、心肌细胞转录因子4(GATA-4)分化情况;免疫荧光技术检测心肌特异性肌球蛋白重链(MHC)表达。结果各用药组CD105+-BMSC的群体倍增时间为24 h,活血化瘀组的细胞数于第2天开始明显高于其他用药组和对照组且呈持续状态。活血化瘀组除第1天外各时间点的增殖指数均为最高值,分裂代次明显多于其他组。第2、3、4、5天,活血化瘀组、益气活血组、益气组的MHC、CTNT和GATA-4表达量比空白组明显增加(P<0.05);活血化瘀组明显优于益气活血组、益气组(P<0.05);益气组与益气活血组差别无统计学意义(P>0.05)。结论在体外模拟心肌微环境的条件下,活血化瘀法与益气活血法、益气法均有促进CD105+-BMSC分裂并向心肌样细胞分化的作用,其中活血化瘀法效果最为明显。

Objective To investigate the effects of different therapeutic formulas on the differentiation of bone marrow mesenchymal stem cells （BMSC） to cardiomyocyte-like cells in rat myocardium of in vitro micro-environment. Methods 40 SD rats randomly divided into blood-activating stasis-resolving group, qi supplementing blood-activating group, qi supplementing group, and blank group were administered o- rally Chinese medicinals to obtain madicated serum ; another 60 SD rats were used to extract, incubate, i- solate and purify BMSC; SD newborn rats were used to extract and incubate myocardial cell, then to es- tablish an external myocardial micro-environment. MTI？ assay was used to select the optimal compoundingconcentration of each treatment group. Flow cytometry was used to measure the proliferation of CD105 ＋ - BMSC of each treatment group. Immunocytochemical method was used to test the CD105 ＋-BMSC＇ s ex- pression of myocardium protein CTNT, GATA4 while immunofluorescence technique was used to meas- ure the expression of MHC. Results The doubling time of CD105 ＋-BMSC of each treatment group was 24 h. The cells numbers in blood-activating and stasis-resolving group were significantly higher than those in control group and other treatment groups from the 2nd day, and remained steady. All the proliferation index of blood - activating and stasis-resolving group at every time point were the highest except the 1 st day. The number of cell division was also significantly more than other groups： qi-supplementing and blood-activating group, qi-supplementing group, blood activating and stasis resolving group. From the 2nd day to 5th day, the expressions in the blood-activating stasis-resolving group, qi-supplementing blood-activating group, qi-supplementing group increased markedly with statistical significance compared with the blank group （P 〈 0.05 ）, blood-activating stasis-resolving group was superior to the other two groups（P 〈0. 05）. There were no statistical significance between the qi-supplementing blood-activating group and qi-supplementing group （P 〉 O. 05）. Conclusion Under the external mimic myocardial mi- cro-environment condition, all therapeutic formulas could promote CD105＋-BMSC＇ s differentiation. They also could promote CD105 ＋-BMSC＇ s metaplasia to the cardiomyocyte-like cells, however, the blood-activating stasis-resolving medicinal appears to be most effective.