田洪涛 杨述华 徐亮 张宇坤 (华中科技大学同济医学院附属协和医院骨科 武汉 430022)
摘 要 目的：研究骨形态发生蛋白-13对小鼠骨髓基质干细胞向软骨分化过程的作用。方法：体外培养小鼠骨髓基质干细胞(MSCs),贴壁细胞传代,取第3代细胞,用骨形态发生蛋白-13溶液干预培养1,4,7,14 d取样检测。倒置相差显微镜观察细胞形态;RT-PCR,westernblot和免疫细胞化学法检测不同时期Ⅱ型胶原,SOX9,阿尔辛蓝(Alcian)染色蛋白多糖。结果： Ⅱ型胶原和SOX9之mRNA和蛋白的表达在第4天开始出现并不断升高;Alcian染色结果显示骨形态发生蛋白-13诱导细胞分泌蛋白多糖基质,可见细胞小结区域呈明显的异染性。结论：骨形态发生蛋白-13可以定向诱导小鼠骨髓干细胞的软骨分化。
关键词 骨形态发生蛋白-13；骨髓基质干细胞；软骨分化；小鼠
中图分类号：R969.1
文献标识码：A
文章编号：1008-049X(2007)07-0640-04
Enhances of Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells from Mouse by Bone Morphogenetic Proteins-13 in Vitro
Tian Hongtao, Yang Shuhua, Xu Liang,Zhang Yukun(Department of Orthopedics, Affiliated Union Hospital, Tongji Medical College of Huazhong Science and Technology University, Wuhan, 430022，China)
ABSTRACT Objective:To study the cartilage differentiation of mouse messenchymal stem cells (MSCs)induced by bone morphogenetic protein-13 (BMP-13) in vitro.Method:The MSCs were isolated from mouse bone marrow and cultured in vitro.The cells in passage 3 were chosen to induce into chondrogenic differentiation with different concentration of recombinant human cartilage derived morphogenetic proteins-13.After 1,4,7 and 14 days of induction,morphology of cells was observed under phasecontrast microscopy. Type Ⅱ collagen and SOX9 mRNA and protein were examined by RT-PCR,western blot and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue.Result:According to RT-PCR showed that BMP-13 could promote expression of Type Ⅱ collagen and SOX9 mRNA in time dependant manner.And the expression was not reduced in 2 weeks.Immunocytochemistry and western blot exhibited that the protein had a similar change.Histological staining proteoglycan of Alcian blue revealed deposition of typical cartilage extracellular matrix.Conclusion:These results suggests mouse bone marrow mesencymal stem cells can be differentiated into chondrogenic phonotype with the induction of BMP-13 in vitro, and which provides a basis for further research on the mechanism of BMP-13 in chondrogenesis.
KEY WORDS Cartilagederived morphogenetic proteins-13；Chondrogenic differentiation; Mouse; Bone marrow mesenchymal stem cells
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