周期性动态压力对海藻酸钠培养间充质干细胞成软骨分化的作用

Cyclic dynamic compressive stress stimulates chondrogenesis of mesenchymal stem cells in alginate gel three dimentional culture

目的观察延迟性周期性动态压缩应力对立体培养条件下骨髓间充质干细胞成软骨分化的影响,探讨力学因素在软骨分化过程中的作用机制。方法 KM小鼠骨髓间充质干细胞分离,体外培养至第3代,与海藻酸钠混匀制作成海藻酸钠凝胶。根据实验设计,进行7 d的相应的预培养。实验分为4组:空白对照组:普通培养液组,静态培养;实验组:完全成软骨诱导液,后7 d加力培养;单纯诱导组:完全成软骨诱导液,静态培养14 d;阴性对照组:普通培养液,后7 d加力培养。于培养第7和14天分离各组细胞,采用RT-PCR分析SOX9、ROCK1及Col2的表达,应用免疫荧光技术对各组处理前后的Ⅱ型胶原蛋白(Col2)表达进行检测,Image J软件进行图像定量检测。正态计量资料采用均数±标准差表示,组间计量资料采用单因素方差分析,组间多重比较采用SNKq检验法。结果预培养7 d后,实验组SOX9、Col2及ROCK1的mRNA表达与对照组比较有统计学差异(P<0.01)。14 d后,实验组中压力干预组SOX9及Col2的mRNA表达与未受力组比较有统计学差异(P<0.01),ROCK1的mRNA表达与未受力组比较有统计学差异(P<0.01)。免疫荧光显示压力组Col2蛋白表达高于未受力组。结论延迟性周期性动态压缩应力可促进间充质干细胞成软骨分化,ROCK信号通路可能参与有关调控。

Objective To observe the effect of delayed cyclical dynamic compressive load on marrow mesenchymal stem cells（ MSCs） of mice differentiation in three dimentional（ 3D） culture systems,and to explore the mechanism of the mechanical factors in the process of cartilage differentiation. Methods The MSCs were isolated from the femurs and tibias bone marrow of mice. After subculture and proliferation,the cells were encapsulated into 2% alginate hydrogel,pre-cultured for seven days in the mesenchymal stem cell chondrogenesis differentiation medium（ MCDM）,kept under an common medium condition as a control. Accroding to the different treatments,the cultured cells were divided into four groups： the blank control group： common medium,static culture; the experimental group： MCDM for 14 days,mechanical stimulation was performed seven days later; the simple induction group： MCDM for 14 days,static culture; the negative control group： common medium,mechanical stimulation was performed seven days later. The reverse transcription polymerase chain reaction（ RT-PCR） was used to analyze the expression of SOX9,collagen Ⅱ（ Col2） and ROCK1 at the 7^th and 14^th day. Immunofluorescence was used to detect the expression of Col2 protein,and use Image J to quantify the pictures. The normal distribution data were expressed by means ± standard deviation. The single factor analysis of variance was analyzed between groups,and the Student-Newman-Keuls（ SNK）-q method was used in the multiple comparisons among the groups. Results Compared to the normal culture group,the mRNA levels of SOX9,Col2 and ROCK1 of MCDM group increased significantly at the 7thday（ P 0. 01）. In the MCDM group,compared to free-swelling conditions,the mRNA levels of SOX9,Col2 and protein level of Col2 in the delayed cyclical dynamic compressive load group increased significantly at the 14thday（ P 0. 01）,but the ROCK1 mRNA levels decreased（ P 0. 01）. Conclusion Delayed cyclical dynamic compressive load can promote chondrogenesis of MSCs by up-regulating SOX9 expression; the ROCK signaling pathway may play an important role in this progression.