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目的体外培养脂肪组织源间充质干细胞,诱导该细胞向成骨细胞和心肌细胞分化,为骨和心肌组织工程提供种子细胞。方法取Wistar大鼠脂肪组织,以胰蛋白酶消化分离出脂肪间充质干细胞。第三代细胞用于鉴定细胞表面抗原,同时以成骨诱导培养液诱导细胞,并以10μmol/L5-氮胞苷处理细胞24h,然后培养,用免疫组化法和组织化学法检测这些细胞。结果所分离细胞90%以上表达CD44、CD13、CD29抗原,有些细胞表达肌节型肌动蛋白和结蛋白;经成骨诱导液诱导14d的细胞表达碱磷酸酶,21d后表达骨钙蛋白并有少量胞外钙沉积;经10μmol/L5-氮胞苷处理的细胞均表达NKx2.5、肌节型肌动蛋白和连接蛋白Cx43。结论本实验所培养的细胞为脂肪组织源性间充质干细胞,能向成骨细胞和心肌样细胞分化,可为骨和心肌组织工程提供种子细胞。
Objective To culture adipose tissue-derived mesenchymal stem cells in vitro and induce the cells to differentiate into osteoblasts and cardiomyocytes, providing seed cells for bone and myocardial tissue engineering. Methods Adipose tissue was obtained from Wistar rats, and adipose-derived mesenchymal stem cells were isolated by trypsin digestion. The third generation of cells were used to identify cell surface antigen. Meanwhile, cells were induced by osteogenic induction medium and treated with 10μmol / L 5-azacytidine for 24h, then cultured and detected by immunohistochemistry and histochemistry. RESULTS: More than 90% of cells isolated expressed CD44, CD13 and CD29 antigens. Some cells expressed sarcomeric actin and desmin. The cells induced by osteogenic induction fluid for 14 days expressed alkaline phosphatase and expressed osteocalcin 21 days later A small amount of extracellular calcium deposition; cells treated with 10μmol / L 5-azacytidine expressed NKx2.5, sarcomeric actin and connexin Cx43. Conclusion The cells cultured in this experiment are adipose tissue-derived mesenchymal stem cells that can differentiate into osteoblasts and cardiomyocyte-like cells and provide seed cells for bone and myocardial tissue engineering.