摘要
目的
研究软骨源性形态发生蛋白-2(CDMP-2)对大鼠骨髓间充质干细胞(BMSCs)向软骨分化的作用。
方法
体外培养大鼠骨髓间充质干细胞(BMSCs),贴壁细胞传代,取第3代细胞,对照组(A组)以L-DMEM无血清培养液培养,实验组(B、C、D组)以L-DMEM无血清培养液培养,分别加入CDMP-2(100 ng/ml)、TGF-β1(10 ng/ml)和CDMP-2(100ng/ml)+TGF-β1(10 ng/ml),倒置相差显微镜观察细胞形态,14天后终止培养,行甲苯胺蓝染色,免疫组织化学染色和Western blot检测软骨特异性Ⅱ型胶原的表达。
结果
诱导14天后可见BMSCs形态由梭形向软骨细胞的多边形转变。甲苯胺蓝染色示糖胺聚糖(GAGs)均匀分布于基质中,免疫组织化学染色和Western blot示实验组Ⅱ型胶原表达阳性,且CDMP-2+TGF-β1组细胞表达Ⅱ型胶原水平高于单独给予CDMP-2、TGF-β1组(P＜0.05);对照组未见阳性表达。
结论
CDMP-2能够在体外定向诱导大鼠骨髓间充质干细胞(BMSCs)向软骨方向分化;与TGF-β1相比,CDMP-2诱导能力更强;CDMP-2与TGF-β1在诱导BMSCs向软骨分化时具有协同作用,两者合用成软骨的效应最大。
Objective
To study the cartilage differentiation of mouse bone marrow mesenchymal stem cells(BMSCs) induced by cartilage-derived morphogenetic protein-2 in vitro.
Methods
The BMSCs were isolated from mouse bone marrow and cultured in vitro.The cells in passage 3 were chosen to induce into chondrogenic differerntiation with different culture media.In the control group(group A),the cells were cultured in L-DMEM containing 10%fetal bovine serum.In the experimental group,the cells were cultured in L-DMEM containing 10%fetal bovine serum with CDMP-2 100ng/ml(group B),TGF-β1 10ng/ml(group C) and CDMP-2 100ng/ml+TGF-β1 10ng/ml(group D).Under induction for 14 days,morphology of cells was observed under phase-contrast microscopy.Type-Ⅱcollagen protein was examined by using immunohistochemistry staining and western blot.The sulfate glycosaminoglycan was measured by alkaline phosphatase toluidine blue staining.
Results
Under the induction of culture media in groups B,C,D,the morphology of cells changed from spindle appearance to the typical chondrocyte-like polygon shape.GAGs was distributed evenly in the cytoplasm and type-Ⅱcollagen protein was positive in groups B,C,D and negative in group A.Type-Ⅱcollagen protein in CDMP-2 100ng/ml +TGF-β1 10ng/ml(group D) was more than either CDMP-2 100ng/ml(group B) or TGF-β1 10ng/ml(group C).There was statistically significant difference in groups.
Conclusion
Mouse bone marrow mesenchymal stem cells can be differentiated into chondrogenic phonotype with the induction of CDMP-2 in vitro.By comparison, CDMP-2 was more effectively than TGF-β1 and the combination of CDMP-2 and TGF-β1 was more effectively than either CDMP-2 or TGF-β1 alone.
引文
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