不同浓度转化生长因子β1体外诱导大鼠骨髓间充质干细胞向心肌样细胞分化的研究

Study of different concentrations of transforming growth factor-β1 inducing the differentiation of rat bone mesenchymal stem cells to cardiomyogenic cells in vitro

目的:研究不同浓度转化生长因子-β1(TGF-β1)在体外定向诱导大鼠骨髓间充质干细胞(BMSCs)向心肌样细胞分化的能力。方法:取SD大鼠四肢骨骨髓,密度梯度离心法分离大鼠BMSCs,以TGF-β1 5μg/L(低剂量组)、10μg/L(高剂量组)对第3代BMSCs进行体外诱导,未诱导的骨髓间充质干细胞做对照。相差显微镜观察细胞形态,MTS试剂盒检测细胞活性。分化14天后,流式细胞仪分析三组细胞DNA周期;免疫荧光法鉴定心肌肌钙蛋白I(cardiac troponin,c Tn I)、心肌平滑肌肌动蛋白(aactin)和心肌肌球蛋白重链(myosin heavy chain,MHC)的表达率。结果:三组细胞均呈对数生长趋势,诱导组细胞数量增长较对照组缓慢(P<0.05),诱导组之间比较,细胞增长无明显差异。分析细胞DNA周期显示:诱导14天后,对照组增殖指数明显高于诱导组(P<0.05),不同浓度诱导组之间比较,细胞增殖指数无明显差异。免疫荧光法检测结果显示:诱导14天后,诱导组细胞均部分表达c Tn I、α-actin及MHC,TGF-β1高剂量组细胞转化率高于低剂量组;对照组均不表达c Tn I、α-actin及MHC。结论:10μg/L TGF-β1与5μg/L TGF-β1比较,更有效促进BMSCs体外向心肌样细胞分化,且对细胞增殖活性无明显影响。

Objective：To investigate the feasibility of different concentrations of Transforming growth factor-β1 （TGF-β1） in inducing bone mesenchymal stem cells （BMSCs） to differentiate into cardiomyocyte-like cells directionally in vitro. Methods： BMSCs were obtained from rats＇ bone marrow using density gradient centrifugation. BMSCs of the third generation were divided into 3 groups-control group, TGF-β1 low-dose group （ TGF-β1 5μg/L） and TGF-β1 high-dose group （ TGF-β1 10μg/L）, and cultured; Their morphologic changes were observed under the microscope; the proliferations of BMSCs were detected by MTS kits. After 14- day culturing, DNA cycle was analyzed using Flow cytometry. The expression of cTnI,α-actin and MHC was detected by immumofluorescence method. Results： BMSCs obtained through using density gradient centrifugation was fusiform. In metabohc activity of BMSCs in different groups the cells in each group showed logarithmic growth trend, the proliferations of BMSCs in TGF-β1 low-dose group and TGF-β1 high-dose group were less than those in group control, and there was significant difference （ P 〈 0.05 ）, while there was no significant difference between TGF-β1 high-dose group and low-dose group. After induced differentiation of 14 days, the results of the DNA cycle indicted that the proliferative index of BMSCs in TGF-β1 high-dose group and low-dose group were less than those in group control, and there was significant difference （ P 〈 0. 05 ）, while there was no significant difference between TGF-β1 high-dose group and low-dose group. The result of immumofluorescence method showed： cTnI, α-actin and MHC were partially expressed in BMSCs in TGF-β1 high-dose group and low-dose group partially. Conclusion： That TGF-β1 with a concentration of 101xg/L can promote the differentiation of BMSCs into cardiomyogenic cells in vitro more effectively without affecting the proliferation or metabolic activity of the BMSCs.