Liu D B, Han X S, Huang W L, Deng J, She R F
Department of Orthopedics, Zigong Forth People's Hospital, Zigong, Sichuan 643000, China.
Zhonghua Yi Xue Za Zhi. 2017 Sep 26;97(36):2860-2865. doi: 10.3760/cma.j.issn.0376-2491.2017.36.017.
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 and search for a reliable seed cell for the regeneration and repair of articular cartilage in osteoarthritis. 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. 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 (<0.05). 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.
探讨转化生长因子(TGF)-β3诱导大鼠骨髓间充质干细胞(BMMSCs)向软骨细胞分化的方法,寻找骨关节炎关节软骨再生修复的可靠种子细胞。采用全骨髓贴壁法培养SD大鼠股骨和胫骨BMMSCs,然后通过流式细胞术鉴定纯度。用含TGF-β3的诱导分化培养基诱导P3代细胞向软骨细胞分化,并检测不同诱导时间点细胞的软骨形成分化能力。原代及传代培养的BMMSCs呈纺锤形,部分呈三角形。传代后,P3代细胞增殖速度快,呈鱼群样或漩涡状生长。软骨诱导后,细胞呈多边形和三角形,呈簇状生长,类似软骨细胞形态,细胞增殖下降。免疫荧光染色和蛋白质印迹法显示细胞有大量Ⅱ型胶原荧光表达,阿利新蓝染色细胞及细胞外基质呈蓝色,第7天和第14天无明显差异。使用Wnt信号阻断剂后,Ⅱ型胶原蛋白表达明显降低,差异有统计学意义(<0.05)。TGF-β3能快速诱导BMMSCs向软骨细胞分化,为BMMSCs移植和关节软骨修复提供了良好的载体,从而治疗骨关节炎。