新式分层共培养环境下人骨髓间充质干细胞诱导髓核细胞的分化

Differentiation of human bone marrow mesenchymal stem cells-induced nucleus pulposus cells under new-style layer coculture

背景:诱导分化的骨髓间充质干细胞或髓核细胞移植都存在一定的局限性,抑制自体髓核细胞的退行性变有望成为未来治疗椎间盘退变的有效方法。目的:通过建立骨髓间充质干细胞和髓核细胞分层共培养模型,观察骨髓间充质干细胞对髓核细胞分化的影响。方法:取传至第4代的骨髓间充质干细胞,分为2组:新式分层培养组将髓核细胞接种于去掉挂臂的Transwell小室,膜孔0.4μm,其与骨髓间充质干细胞比例为1∶1;传统分层培养组同法将髓核细胞接种于带有挂臂的Transwell小室。同时设立单独髓核细胞培养组,各组均培养7d。免疫组织化学法检测Ⅱ型胶原的表达,35S放射标记渗入放免定量检测蛋白多糖的合成。结果与结论:与单独髓核细胞培养组比较,传统分层培养组、新式分层培养组Ⅱ型胶原阳性细胞数、蛋白多糖合成量均明显增加(P<0.05,P<0.01),且新式分层培养组增高幅度大于传统分层培养组(P<0.05)。提示骨髓间充质干细胞与髓核细胞接触共培养后,能显著增加髓核细胞分化增殖和特征性物质的表达,且新式分层培养环境优于传统分层培养。

BACKGROUND： There are some limitations in induced differentiation of bone-marrow-derived mesenchymal stem cells （hBMSCs and transplantation of nucleus pulposus cells （NPCs）. Inhibition of autologous NPCs degeneration is expected to become an effective way for treatment of disc degeneration in the future. OBJECTIVE： To establish a layered coculture model of hBMSCs and NPCs in vitro, and to observe the effect of hBMSCs on the differentiation of NPCs. METHODS： The fourth passage of hBMSCs was divided into 2 groups. NPCs in the new-style layer culture group were incubated in Transwell cabin without arm, 0.4-μm membrane well; the proportion to BMSCs was 1： 1. NPCs in the traditional culture group were incubated in Transwell cabin with arm. NPC culture group was set. Each group was incubated for 7 days. Collagen Ⅱ was detected by immunohistochemical method. Aggrecan expression was detected by [35S ]-sulfate uptake. RESULTS AND CONCLUSION： Compared with NPC culture group, collagen Ⅱ and aggrecan expression of NPCs were upregulated most evidently in new-style culture and traditional culture groups （P 0.05, P 0.01）. The increased range of new-style culture group was greater than the traditional culture group （P 0.05）. These results suggested that coculture of BMSCs and NPCs can significantly increase NPC proliferation and specific substance expression. The surrounding of new-style culture surpassed the traditional culture.