不同浓度转化生长因子-β1对人骨髓间充质干细胞/海藻酸钠复合物体外软骨形成能力的影响

Experimental study of chondrogenic differentiation of human MSCs/ alginate composition in different doses of TGF-β1 in vitro

目的探讨人骨髓间充质干细胞(MSCS)体外定向分化为软骨样组织的可行性以及不同浓度TGF-β1对人MSCs/海藻酸钠复合物体外软骨形成能力的影响。方法将体外培养扩增的人骨髓间充质干细胞与海藻酸钠结合,制成MSCs/海藻酸钠复合物,诱导培养液中添加不同浓度TGF-β1,在培养的不同时间进行Ⅱ型胶原的免疫组织化学,原位杂交,蛋白多糖的特殊染色,以及蛋白多糖的定量检测。结果细胞在海藻酸钠中生长良好,可见细胞分裂增殖,形成同源细胞团。10μg/L诱导组Ⅱ型胶原表达阳性,基质中有红染的黏多糖物质的堆积,蛋白多糖定量检测均高于其他实验组(P<0.05)。结论TGF-β1浓度过低不能诱导软骨形成,而浓度过高则不利于软骨形成,10μg/L是体外诱导软骨形成的适宜浓度。海藻酸钠是运载MSCs的理想载体和支架。

Objective To investigate the feasibility and experimental condition of chondrogenic differentation of human bone marrow-derived mesenchymal stem cells （MSCs） cultured in alginate beads in vitro. Methods MSCs were suspended within alginate beads and treated with different doses of TGF-β1. Immunohistochemistry and in situ hybridization were applied to detect the expression of collagen type Ⅱ. Some sections were stained with AB-PAS, and proteoglycan synthesis was quantified with alcian blue staining. Results Immunohistochemistry and in situ hybridization for type Ⅱ collagen, and AB-PAS staining for proteoglycan showed positive staining in the 10μg/L TGF-β1 group, The proteoglycan quantity in the 10μg/L TGF-β1 group was significantly higher than other experiment groups at different induction time points. Conclusion 10μg/L TGF-β1 can induce the chondrogenesis differentiation of MSCs/Alginate composition effectively. The three-dimensional alginate gel is a potential candidate bioactire scaffold for cartilage tissue engineering applications.