透明质酸凝胶中肌源性干细胞与透明软骨联合培养的实验研究

 摘　要：目的 探讨肌源性干细胞与软骨细胞混和培养体外成软骨的可行性。方法 分离培养、扩增传代兔MDSCs及关节软骨细胞，二者按一定比例混和，6×1010/L密度接种于5%浓度的透明质酸凝胶为共培养组,以相同密度的单纯MDSCs和单纯软骨细胞作为阴性与阳性对照。倒置相差显微镜下观察，10d后行甲苯胺蓝染色和Ⅱ型胶原免疫细胞化学检测，RT-PCR检测Ⅱ型胶原和aggrecan的mRNA表达。结果 5d后镜下见共培养组MDSCs形态由梭形向多边多角形转化，10d后细胞甲苯胺蓝染色和Ⅱ型胶原免疫组织化学检测与阳性对照组相同，RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达。阴性对照组在体外培养过程中未分化为软骨细胞. 结论:软骨微环境在MDSCs成软骨分化过程中具有重要作用,软骨细胞能有效地诱导MDSCs向软骨样细胞分化，透明质酸凝胶可以作为软骨组织工程的良好载体。

Co-culture of Muscle-derived Stem Cells and Hyaline Cartilage in Hyaluronic Acid Gel

Abstract: Objective To discussion the feasibility of mixed culture muscle-derived stem cells and chondrocytes to chondrogenesis in vitro. Methods Isolated culture and amplification of passage of MDSCs and articular chondrocytes from rabbit. They mixed by a certain percentage. Then they inoculated in 5% hyaluronic acid gel with the density of 6×1010/L as co-culture group. The same density of MDSCs as negative control group, and the same density of articular chondrocytes as positive control group. Observed with inverted microscope. They detected by toluidine blue staining and Ⅱcollagen immunocytochemistry after 10 days. RT-PCR detected the mRNA expression of Ⅱcollagen and aggrecan. Results In co-culture group, the form of MDSCs changed from fusiform to multilateral polygon after 5 days. After 10 days, toluidine blue staining and Ⅱcollagen immunocytochemistry of cells were the same to positive control group, RT-PCR detected the mRNA expression of Ⅱcollagen and aggrecan were positive. They were not differentiated to chondrocytes in negative control group. Conclusion Cartilage microenvironment plays an important role in MDSCs chondrocyte differentiation. Chondrocytes can effective induce MDSCs to cartilage-like cells. Hyaluronic acid gel may be a good vector in cartilage tissue engineering.