骨髓基质干细胞与软骨细胞体外共培养向软骨细胞转化的实验研究

目的 探讨骨髓基质干细胞（BMSCs）与软骨细胞体外共培养成软骨的可行性,以及能有效促进BMSCs的软骨向分化混合培养的比例.方法 以全骨髓法及梯度密度离心法分离幼兔BMSCs、梯度密度离心法分离软骨细胞,并分别对这2种细胞进行培养、扩传,将P2 BMSCs与P3软骨细胞进行体外共培养,分为：BMSCs/软骨细胞为2/1及4/1的2个共培养组,软骨细胞组,BMSCs组,分别于第1,3,5,7,9 d以MTT法检测细胞的增殖情况;分别于第1、2、3周对4组的细胞进行甲苯胺蓝染色及以RT-PCR方法检测蛋白多糖和Ⅱ型胶原表达的变化.结果 2种分离培养方法所得BMSCs及软骨细胞增殖旺盛,细胞形态正常,各组增殖情况良好,1,3,5,7,9 d时各组之间的差异具有统计学意义（P＜0.05）,但2/1与4/1共培养组之间无显著性差异;2/1及4/1共培养组在3周时以软骨样细胞为主,细胞呈均一异染,细胞被染成紫色,核仁染成深蓝色;蛋白多糖及Ⅱ型胶原基因表达：在1,2,3周时各组之间的差异具有统计学意义（P＜0.05）,共培养组和软骨细胞组3组之间蛋白多糖及Ⅱ型胶原表达水平无显著性差异,且均与BMSCs组之间有显著性差异,具有统计学意义（P＜0.05）.结论 将BMSCs与软骨细胞体外共培养,可以被有效地诱导为软骨细胞,软骨微环境在BMSCs分化为软骨细胞的过程中起到了很重要的作用.

Experimental research on chondrogenesis by co-culture of BMSCs and chondrocytes in vitro

Objective This study explored the feasibility of in vitro chondrogenesis by co-culture of bone marrow mesenchymal stem cells (BMSCs) and chondrocytes,and explored on what proportion of the two kinds of cells in the co-culture can be more effective,so as to confirm the hypothesis that the chondrocytes can provide chondrogenic microenvironment inducing chondrogenic diffentiation of BMSCs in vitro;Methods BMSCs were isolated by density gradient centrifugation and whole bone marrow culture method and chondrocytes were isolated from articular cartilage.P2 BMSCs were mixed with P3 chondrocytes in different proportion,and the proliferation of each group were detected on day 1,3,5,7 and 9.Cells were stained with toluidine blue,the changes of proteoglycans and collagen type Ⅱ in expression were detected by RT-PCR in week 1,2 and 3.Results Both BMSCs and chondrocytes proliferated well and kept their own normal shape.MTT test showed that cells in all four groups proliferated well and the disparity between them had statistical significance,while there was no statistical significance between the two co-cutured groups.The cells of both co-cultured groups were stained with toluidine blue,their cytoplasm were stained into purple and nucleoli into dark blue,and the majority cells were chondroid.The changes of proteoglycans and collagen type Ⅱ in expression by RT-PCR showed that the disparity between them was statistical significant,but no statistical significance between the two co-cutured groups and the chondrocytes group.Each of all three groups showed statistical significance to the BMSCs group.Conclusion The BMSCs can be induced chondrogenic diffentiation successfully,which shows that chondrogenic microenviroment played an important role in chondrogenesis of BMSCs.