肿瘤坏死因子-α对人髓核间充质干细胞衰老的影响△

Effects of TNF-α on the senescence of human nucleus pulposus mesenchymal stem cells

摘要:

［目的］探索肿瘤坏死因子-α（tumor necrosis factor -α, TNF-α）对人髓核间充质干细胞的衰老的影响。［方法］分离培养第三代正常人髓核间充质干细胞 （human nucleus pulposus mesenchymal stem cells, HNPMSCs），分为空白对照组和 TNF-α 组，分别加入无血清培养基与 TNF-α 浓度为 100 ng/ml 的无血清培养基干预 48 h。镜下观察细胞形态，并用细胞衰老 β 半乳糖苷酶试剂盒染色，对细胞衰老情况进行观察；CCK-8 检测第 1、3、5、7、9、11、13、15 d 后的细胞增殖活性；Western Blot 检测衰老相关蛋白 P53、P16 表达情况。［结果］ 镜下观察及 β 半乳糖苷酶染色均可见 TNF-α 组细胞呈衰老状态。CCK-8 检测，随时间推移两组细胞 CCK-8 检测光密度 （optimal density, OD） 值均显著增加 （P<0.05）。相应时间点两组间比较，第 1~7 d，两组间的人 NPMSCs 增殖活性 OD 值的差异无统计学意义（P>0.05）；而第 9~15 d，TNF-α 组的人 NPMSCs 的 OD 值均显著低于空白对照组 （P<0.05）。Western Blot 检测显示 TNF-α 组衰老相关蛋白 P53、P16 的表达显著高于空白对照组 （P<0.05）。 ［结论］TNF-α 能够抑制人 NPMSCs 的生物学活性，加速细胞衰老。

Abstract:

[Objective] To explore the effect of TNF-α on the senescence of human nucleus pulposus mesenchymal stem cells. [Methods] The normal human nucleus pulposus mesenchymal stem cells (hNPMSCs) were isolated from human undegenerated lumbar disc and cultured to the third generation, and then divided into the normal blank control group and TNF-α group, which were treated with serumfree medium, and serum-free medium contenting TNF-α of 100 ng/ml for another 48 h respectively. The cell morphology was observed un- der the microscope and then stained by senescence β-galactosidase kit. CCK-8 assay was used to assess the cell viability at 1, 3, 5, 7, 9, 11, 13, and 15 days after treatment, while western blot assay was performed to detect the expression of aging-related protein p53 and p16. [Results] The microscopic observation and β-galactosidase staining showed that the hNPMSCs treated with TNF-α were considerably more senescent than those in the blank control group. As results of CCK-8 assay, the optical density (OD) presenting cell proliferation viability ramped up significantly in both groups over time (P＜0.05) . Although there were not statistically significant differences between the two groups from 1 day to 7 days (P＞0.05) , the TNF-α group had significantly lower OD than the control group from 9 days to 15 days (P＜0.05) . Regarding to western blot detection, the TNF-α group presented significantly higher expression of aging-related proteins p53 and p16 than the control group (P＜0.05) . [Conclusions] In this study, the TNF-α does inhibit cell proliferation of hNPMSCs, whereas accelerate senes- cence of the cells.