低浓度TNF-α对大鼠髓核间充质干细胞增殖及迁移的影响

Effects of low concentration of TNF-α on proliferation and migration of nucleus pulposus derived stem cells in rats

[目的]探讨低浓度TNF-α对大鼠髓核间充质干细胞(nucleus pulposus derivedstem cells,NPSCs)增殖及迁移特性的影响。[方法]差速贴壁法从SD大鼠尾椎髓核组织中分离NPSCs,体外培养,镜下观察细胞形态和生长状况;取P3代细胞利用流式细胞仪鉴定干细胞表面抗原;使用诱导培养基进行三系诱导分化,3周后分别用茜素红、油红O、阿利辛蓝染色观察成脂、成骨、成软骨能力。用含不同浓度(0、0.1、1、10 ng/ml)TNF-α的血清培养液培养P3代细胞,0、1、3、5、7 d后CCK-8法检测细胞增殖活力(OD值),划痕实验及Transwell迁移实验比较各组细胞迁移能力。[结果]P0代细胞呈集落样生长,细胞形态呈纺锤状。P3代细胞免疫表型鉴定MSCs表面分子标记CD90、CD29、CD44表达比例分别高达96%、99%、97%以上,低表达CD45、CD34(低于5%和4%)。茜素红、油红O及阿利新蓝染色证实分离的细胞可向骨、脂肪及软骨分化。按照ISCT有关MSCs的判定标准,分离的细胞即NPSCs。用含TNF-α培养液培养髓核间充质干细胞1、3、5、7 d后,与对照组相比,低浓度的TNF-α均促进了NPSCs的增殖。Transwell及划痕实验表明随着TNF-α浓度的增加,NPSCs的迁移能力上升。[结论]低浓度的TNF-α(0.1~10 ng/ml)能促进NPSCs增殖及迁移能力。

[Objective] To investigate the impact of low concentration TNF-α on the proliferation and migration of nucleus pulposus derived stem cells （NPSCs） . [Method] NPSCs were isolated by differential adhesion methods from coccygeal nucleus pulposus tissues of the 3 month SD rat. As the P3 reached, the cells were verified by multiple lineage differentiation tests and flow cytometry, subsequently cultured with TNF-a at different concentrations （0, 0.1, 1 and 10ng/ml）. The proliferation ability was tested by cell counting kit-8 at 0, 1, 3, 5 and 7 days respectively, whereas the migration capacity was evaluated by wound healing assays and Transwell migration tests. [Results] The P0 cells revealed a spindle-shape in appearance with colony-like growth. The expression of CDg0, CD29 and CD44 on the P3 cells were up to 96%, 99% and 97%, respectively, while the expres- sion of CD45 and CD34 was lower than 5% and 4%. The Alizarin red staining, oil red O staining and aleian blue staining con- firmed that the isolated cells possessed the capacity of multiple lineage differentiation. According to ISCT＇ s criteria, the isolat- ed cells were the NPSCs. In addition, the NPSCs were cultured with medium containing TNF-a 0, 0.1, 1, 10 ng/ml for 1, 3, 5 and 7 days, compared with the control group, the different concentrations of TNF-u promoted the proliferation of NPSCs. Tran- swell and wound healing assay showed that the migration ability of NPSCs increased. [Conclusion] Low concentration of TNF-a （0.1-10ng/ml） could promote the proliferation and migration of NPSCs.