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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 differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells induced by different prescriptions in vitro under myocardial microenvironment. Methods 40 adult SD rats were randomly divided into Huoxuehuayu group, Yiqihuoxue group, Yiqi group and blank group. Drug-containing serum was prepared after the corresponding drugs were given. Another 60 SD rats were extracted, cultured and separated Purify BMSC; Simultaneously extract and culture SD neonatal rat cardiomyocytes to establish myocardial microenvironment model in vitro. MTT method was used to select the optimal concentration for each administration group; the proliferation and proliferation of CD105 positive cells (CD105 + -BMSC) in BMSCs were detected by flow cytometry; the expressions of cardiac troponin T (CTNT), cardiomyocytes Transcription factor 4 (GATA-4) was detected by immunohistochemistry. Myocardial specific myosin heavy chain (MHC) expression was detected by immunofluorescence. Results The population doubling time of CD105 + -BMSCs in each drug group was 24 h. The number of cells in the drug group of activating blood circulation and removing blood stasis group was significantly higher than that of the other drug groups and the control group on the second day and continued. The proliferation index of blood-activating and removing-blood-stasis group except for the first day was the highest, and the division generation was more than other groups. On the 2nd, 3rd, 4th and 5th days, the expressions of MHC, CTNT and GATA-4 in Huoxuehuayu, Yiqihuoxue and Yiqi groups were significantly higher than those in blank group (P <0.05) There was no significant difference between Yiqi group and Yiqihuoxue group in Yiqihuoxue group and Yiqi group (P <0.05). Conclusion Under the condition of simulating myocardial microenvironment in vitro, the methods of promoting blood circulation and removing blood stasis, invigorating qi and promoting blood circulation and invigorating qi both promote the division of CD105 + -BMSC and differentiate into cardiomyocyte, and the effect of promoting blood circulation and removing blood stasis is the most obvious.