人骨髓基质干细胞在不同诱导环境下向软骨细胞的分化

Differentiation of human bone marrow stromal cells into chondrocytes in different induction circumstance

目的:探索重组人骨形态发生蛋白-2(rhBMP-2)诱导建系的人骨髓基质干细胞(hBMSC,专利名缩写为MSCxj)生成软骨的可行性、最佳剂量及适宜的诱导环境。方法:rhBMP-2分为六组:空白组、100 ng/m l组、200ng/m l组、400 ng/m l组、600 ng/m l组和800 ng/m l组。血清浓度分为三组:无血清组、2%血清组和5%血清组。根据rhBMP-2和血清不同浓度间的组合,设计18种诱导模式。一部分MSCxj制成微球,模拟三维模式进行细胞爬片培养,另一部分MSCxj直接用96孔板培养,10 d后,检测MSCxj的数量、总蛋白生成量和碱性磷酸酶生成量,并行甲苯胺蓝染色及Ⅱ型胶原mRNA检测,利用MPS60病理图像软件分析各组Ⅱ型胶原mRNA染色后的灰度,比较各组Ⅱ型胶原mRNA的生成量。结果:血清浓度越高细胞生成的数量越多,细胞合成的总蛋白无显著差异,rhBMP-2对细胞数量和总蛋白的合成无明显影响。2%的血清浓度比其他血清浓度更利于软骨生成。在该血清浓度下rhBMP-2为400 ng/m l时,细胞特异性基质Ⅱ型胶原生成最多(F=19.56,P<0.05)。结论:rhBMP-2能诱导MSCxj生成软骨,在血清浓度为2%、自身浓度为400 ng/m l水平下诱导MSCxj生成软骨能力最强。

Objective：To study the possibility, the optimization dose and the best induction circumstance of rhBMP-2 induction human bone marrow stromal cell line （MSCxj） to differentiate into chondrocyte. Methods ： MSCxj were cultured in the style of micromass to imitate three-dimensional culture. The induction medium contained IMDM,FBS and rhBMP-2 etc. FBS was divided into three levels： 0% FBS, 2% FBS and 5%FBS. rhBMP-2 was divided into six levels： 0 ng/ml,100 ng/ml,200 ng/ml,400 ng/ml,600 ng/ml and 800 ng/ml. According to the different combination of FBS and rhBMP-2, eighteen patterns of induction mode were designed. Each pattern was repeated six times. After being cultured for 10 days, analysis system, and matrix quantity was compared semiquantitatively between the patterns. Results： Cell amount went up with serum concentration increasing, but had little connection with rhBMP-2 concentration. Both of serum concentration and rhBMP-2 concentration had little effects on total protein. The pattern containing 2% FBS and 400 ng/ml rhBMP-2 seemed to be the best to promote MSCxj to differentiate into chondrocytes. Conclusion：rhBMP-2 can induce MSCxj to differentiate into chondrocytes. The pattern containing 2% FBS and 400 ng/ml rhBMP-2 seems to be the best mode to promote chondrocytes generation.