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

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

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,10 d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 5 d后镜下见共培养组MDSCs形态由梭形向多边多角形转化,10 d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。

Objective To explore the feasibility of in vitro co-culture of muscle-derived stem cells（MDSCs） and chondrocytes to form cartilage.Methods MDSCs and chondrocytes were isolated,amplified,and passaged.These two kinds of cells were mixed in a certain percentage and inoculated in 5% hyaluronic acid gel with a density of 6×1010/L in co-culture group.MDSCs and chondrocytes of the same density were used as negative and positive control group,respectively.The cells were observed under inverted microscope.Toluidine blue staining and immunocytochemistry for collagen type Ⅱ were performed after 10 days.The expressions of collagen type Ⅱ and aggrecan mRNA were detected by RT-PCR.Results In co-culture group,the shape of MDSCs changed from fusiform to polygon after 5 days.After 10 days,the results of toluidine blue staining and immunocytochemistry for collagen type Ⅱ in co-culture group were the same as those in positive control group.Collagen type Ⅱ and aggrecan mRNA were positively expressed.No chondrocytes were found in negative control group.Conclusion Cartilage microenvironment plays an important role in the differentiation of MDSCs into chondrocytes.Chondrocytes can effectively induce the differentiation of MDSCs to cartilage-like cells.Hyaluronic acid gel could be used as a good vector for cartilage tissue engineering.