中文摘要：

目的：研究转化生长因子-β1（transforming growth factor-β1,TGF-β1）诱导骨髓间充质干细胞（bone marrowderived mesenchymal stem cells,BMSCs）向肌成纤维细胞分化的机制。方法：采用全骨髓贴壁培养法体外培养小鼠原代BMSCs,将对数生长期的P3∽P5代BMSCs作为实验细胞,使用不同浓度的TGF-β1诱导BMSCs向肌成纤维细胞分化,并在此基础上观察加入自由基清除剂N-乙酰-半胱氨酸（N-acetylcysteine,NAC）对其分化的影响。采用实时荧光定量PCR及Western blot技术检测BMSCs分化指标α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、Ⅰ型胶原[collagenα1（Ⅰ）,Colα1（Ⅰ）]和Ⅲ型胶原[collagenα1（Ⅲ）,Colα1（Ⅲ）]的表达情况。使用2’,7’-dichlorohydrofluorescein diacetate（DCFH-DA）预孵育BMSCs 15 min,然后加入TGF-β1处理不同时间,检测TGF-β1刺激下BMSCs中活性氧（reactive oxygen species,ROS）的产生,并使用高内涵方法对BMSCs中产生的ROS进行统计分析。结果：TGF-β1可以剂量依赖地诱导BMSCs向肌成纤维细胞分化,上调α-SMA、Colα1（Ⅰ）和Colα1（Ⅲ）的表达。TGF-β1诱导BMSCs分化的作用可以被NAC阻断。TGF-β1在BMSCs中能够引起ROS的产生,且该过程迅速而短暂,当TGF-β1作用30 min时,其在BMSCs中诱发的ROS达到峰值。结论：TGF-β1通过产生ROS介导BMSCs向肌成纤维细胞分化。

英文摘要：

Objective： The aim of this study was to investigate the mechanism underlying transforming growth factor-β1（ TGF-β1） induced differentiation of bone marrow-derived mesenchymal stem cells（ BMSCs） into myofibroblasts. Methods： Primary mouse BMSCs were isolated from bone marrow by flushing the tibias and femurs of mice,and passage 3 to passage 5 of BMSCs were used in the experiments.BMSCs differentiation into myofibroblast was induced by different doses of TGF-β1. In addition,reactive oxygen species（ ROS） inhibitor（ N-acetylcysteine,NAC） was added to test its effect on the action of TGF-β1. Expressions of BMSCs differentiation parameters,α-smooth muscle actin（ α-SMA）,collagenα1（Ⅰ） [Col α1（Ⅰ） ]and collagen α1（ Ⅲ） [Col α1（ Ⅲ） ]were measured by real-time quantitative PCR（ RT-q PCR） and Western blot analysis. BMSCs were preloaded for 15 min with 2＇,7＇-dichlorohydrofluorescein diacetate（ DCFH-DA）,then stimulated with TGF-β1 for different times,and fluorescence of ROS was measured using high content analysis. Results： TGF-β1 stimulated differentiation of BMSCs into myofibroblasts and up-regulated expression of α-SMA,Col α1（Ⅰ） and Col α1（ Ⅲ） in a dose-dependent manner,which blocked by ROS inhibitor NAC. In addition,TGF-β1 could induce a significant rapid and transient increase in ROS production in BMSCs,and the effect of TGF-β1 on ROS production was peaked at 30 min. Conclusion： TGF-β1 induced differentiation of BMSCs into myofibroblasts via production of ROS.