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-αon the senescence of human nucleus pulposus mesenchymal stem cells

[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1~7d,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。

[Objective]To explore the effect of TNF-αon the senescence of human nucleus pulposus mesenchymal stem cells.[Method]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-αgroup,which were treated with serumfree medium,and serum-free medium contenting TNF-αof 100 ng/ml for another 48 h respectively.The cell morphology was observed under the microscope and then stained by senescenceβ-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.[Result]The microscopic observation andβ-galactosidase staining showed that the hNPMSCs treated with TNF-αwere 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-αgroup had significantly lower OD than the control group from 9 days to 15 days(P<0.05).Regarding to western blot detection,the TNF-αgroup presented significantly higher expression of aging-related proteins p53 and p16 than the control group(P<0.05).[Conclusion]In this study,the TNF-αdoes inhibit cell proliferation of hNPMSCs,whereas accelerate senescence of the cells.