TGF-β3诱导大鼠骨髓间充质干细胞成软骨细胞分化的实验观察  

Study on the differentiation of rat bone marrow mesenchymal stem cells into chondrocytes induced by TGF-beta 3

作　　者：刘登榜 韩小松[2] 黄文良[2] 邓江[2] 佘荣峰[3] 

机构地区：[1]自贡市第四人民医院骨科,四川自贡643000 [2]贵州省遵义市第一人民医院骨科 [3]贵州省人民医院骨科

出　　处：《中华医学杂志》2017年第36期2860-2865,共6页National Medical Journal of China

基　　金：贵州省社发攻关（黔科合SY字[2013]3047号）;贵州省自然科学基金（81660367）;贵州省科技项目（黔科合基础[2016]1420）

摘　　要：目的探索转化生长因子-β3（TGF-β3）体外诱导大鼠骨髓间充质干细胞成软骨细胞分化的培养方法，为后续骨性关节炎中软骨细胞再生修复寻找一种可靠的种子细胞。方法采用全骨髓贴壁法培养SD大鼠股、胫骨骨髓间充质干细胞，并采用流式细胞术鉴定其纯度；用含TGF-β3的成软骨细胞诱导分化培养液对P3代细胞进行诱导分化培养，在不同诱导时间下检测细胞向成软骨细胞的分化能力。结果原代及传代培养的骨髓间充质干细胞多呈梭形，部分呈三角形，传代后，P3代细胞增殖速度快，呈鱼群样或漩涡样生长；经含TGF-β3的培养基诱导培养后，可见细胞呈多角形和三角形，类似软骨细胞样形态，各细胞有集聚生长现象，细胞增殖能力减弱；免疫荧光化学染色及蛋白印迹法可见细胞有大量col Ⅱ的荧光表达，阿尔新蓝染色可见细胞及细胞外基质蓝染，第7天和第14天无明显差别；加入Wnt信号阻断剂后，col Ⅱ蛋白表达明显减低，差异有统计学意义（P〈0．05）。结论TGF-β3能快速地诱导骨髓间充质干细胞成软骨细胞分化，为今后骨髓间充质干细胞移植再生修复关节软骨进而治疗骨性关节炎提供了良好的载体。Objective To explore the methods for the differentiation of rat bone marrow mesenchymal stem cells （BMMSCs） into chondrocytes induced by transforming growth factor （TGF）-beta 3 in vitro, and search for a reliable seed cell for the regeneration and repair of articular cartilage in osteoarthritis. Methods SD rat femoral and tibial BMMSCs were cultured by the whole bone marrow adherent method, and then the purity was identified by flow cytometry. P3 generation cells were induced and differentiated into chondrocytes by the induction of differentiation medium containing TGF-beta 3, and cell chondrogenic differentiation ability at different induction time points was detected. Results The primary and passage cultured BMMSCs were spindle-shaped, and partly triangular. After passage, the proliferation rate of P3 generation cells was fast, like the growth of fish shoal or eddy. After chondrogenic induction, the cells were polygonal and triangular in the form of cluster growth, which was similar to chondrocyte morphology, and the cell proliferation was decreased. Immunofluorescence staining and Western blotting method showed that the cells had a large number of col Ⅱ fluorescent expression, and cells and extracellular matrix was stained blue by Alcian blue staining, with no significant difference at day 7 and day 14. After using Wnt signal blocker, col Ⅱ protein expression was significantly reduced, with statistically significant difference （P 〈 0. 05 ）. Conclusion TGF-beta 3 can rapidly induce the differentiation of BMMSCs into cartilage cells, which provides a good carrier for BMMSCs transplantation and the repair of articular cartilage, and thus to treat osteoarthritis.

关 键 词：骨髓间充质干细胞 转化生长因子-Β2 软骨分化 

分 类 号：R684.3[医药卫生—骨科学]