软骨源性形态发生蛋白2基因转染自体成肌细胞表达软骨细胞表型的实验研究

Study on chondrogenesis of myoblast under transfection of cartilage-derived morphogenetic protein-2 in vitro

目的研究软骨形态发生蛋白-2(CDMP-2)对小鼠成肌细胞向软骨分化的作用。方法体外培养小鼠成肌细胞,贴壁细胞传代,取第3代细胞,对照组(A组)以无血清L-DMEM培养液培养,实验组(B、C、D组)以无血清L-DMEM培养液培养,分别加入透明质酸钠、CDMP-2(100ng/mL)和CDMP-2基因转染的小鼠成肌细胞,14d后终止培养,倒置相差显微镜观察细胞形态,甲苯胺蓝染色糖胺聚糖(GAG),行Ⅱ型胶原免疫组织化学染色、灰度值分析和Western blot检测。结果培养14d后转染组和诱导组可见成肌细胞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基因转染组细胞表达Ⅱ型胶原水平高于单独给予CDMP-2,且具有统计学意义;对照组和透明质酸钠组未见阳性表达。结论 CDMP-2基因转染的小鼠成肌细胞能够更好的表达软骨特异性Ⅱ型胶原。

【Objective】 To study the cartilage differentiation of mouse myoblast cells transferred by cartilage-derived morphogenetic protein-2 in vitro.【Methods】 The myoblast were isolated from mouse muscle and cultured in vitro.The cells in passage 3 were chosen to transfer and 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 coniaining 10% fetal bovine serum with hyaluronic acid sodium（groupB）,the cells were cultured in L-DMEM coniaining 10% fetal bovine serum with CDMP-2 100ng/ml（groupC）,the cells were transfered in L-DMEM coniaining 10% fetal bovine serum with CDMP-2（groupD）.Under transfection and induction for 14days,morphology of cells were observed under phase-contrast microscopy.Type-Ⅱcollagen protein was examined by using immunohistochemistry staining and westen blot.The sulfate glycosaminoglycan was measured by alkaline phosphatase toluidine blue staining.【Results】 Under the transfection and induction of media in groups C,D,the morphology of cells changed from spindle appearance to the typical chondrocyte-like polygon shape.GAGs was distributed enenly in the cytoplasm and type-Ⅱcollagen protein was positive in groups C,D and negative in group A,B.There was statistieally signifieant difference in groups.【Conclusion】 Mouse myoblast cells can be differentiated into chondrogenic phonotype with the transfection and induction of CDMP-2 in vitro.By comparison,transfection was more effectively than induction.