小鼠髓核细胞体外衰老模型的建立及评价

Establishment and evaluation of an in vitro senescence model of mouse nucleus pulposus cells

中文关键词:  髓核细胞  叔丁基过氧化氢  衰老  小鼠

英文关键词:Nucleus pulposus cells  Tert-butyl hydrogenperoxide  Senescence  Mouse

中文摘要:

  【摘要】 目的：建立并评价小鼠髓核细胞（nucleus pulposus cells，NPCs）体外衰老模型。方法：取8周龄C57BL/6雄性小鼠，体外分离培养小鼠NPCs，并采用细胞免疫荧光（immunofluorescence，IF）检测NPCs标志蛋白聚集蛋白聚糖（aggrecan）的表达进行细胞鉴定。取不同浓度（0μmol/L、50μmol/L、100μmol/L、200μmol/L、300μmol/L、400μmol/L、500μmol/L）的叔丁基过氧化氢（tert-Butyl hydroperoxide，TBHP）干预NPCs，采用CCK8法检测干预2h、4h后的细胞活性，筛选TBHP的最佳干预浓度及时间。应用最佳干预浓度TBHP、最佳干预时间干预P1代NPCs后换成完全培养基继续培养24h、48h、72h，采用衰老相关β半乳糖苷酶染色（senescence-associated β-galactosidase staining，SA-β-gal）检测各时间点的衰老NPCs阳性率，观察不同时间点NPCs的衰老变化。将P2代NPCs分为对照组和模型组，对照组在完全培养基中培养，模型组加入最佳干预浓度TBHP干预最佳时间干预后换成完全培养基培养，采用实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）检测两组细胞衰老相关基因p53、p21、p16以及Aggrecan、Ⅱ型胶原蛋白（collagen Ⅱ）、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶-5（a disintegrin and metalloproteinase with thrombospondin motifs-5，ADAMTS-5）、基质金属蛋白酶-3（matrix metalloproteinase-3，MMP-3）、基质金属蛋白酶-13（matrix metalloproteinase-13，MMP-13）mRNA表达，免疫印迹法（Western blot，WB）检测Aggrecan、Collagen Ⅱ、MMP-3、MMP-13、p53、p21蛋白表达水平。结果：分离培养的细胞高表达Aggrecan，为NPCs。TBHP最佳干预浓度为100μmol/L，最佳干预时间为4h。在100μmol/L TBHP干预4h后培养24h、48h、72h，NPCs的SA-β-gal染色阳性率均显著性增加（P＜0.05），三个时间点间无统计学差异（P＞0.05），后续模型组加入最佳干预浓度TBHP干预最佳时间干预后换成完全培养基培养24h，对照组培养相同时间。RT-qPCR检测模型组p53、p21、p16、MMP-3、MMP-13、ADAMTS-5 mRNA表达较对照组显著性升高（P＜0.05），Aggrecan、Collagen Ⅱ mRNA表达较对照组显著性降低（P＜0.05）；WB检测模型组MMP-3、MMP-13、p53、p21蛋白表达较对照组显著性升高（P＜0.05），Aggrecan、Collagen Ⅱ蛋白表达较对照组显著性降低（P＜0.05）。结论：采用TBHP诱导能成功构建小鼠NPCs体外衰老模型，可为椎间盘退行性变的发病机制研究提供良好的研究样本。

英文摘要:

  【Abstract】 Objectives: To establish a senescence model of mouse nucleus pulposus cells(NPCs) in vitro. Methods: NPCs were isolated and cultured in vitro from 8-week-old C57BL/6 male mice, and the expression of the NPCs marker protein-aggrecan was detected by cell immunofluorescence(IF) for cell identification. Different concentration gradients of tert-Butyl hydroperoxide(TBHP) of 0μmol/L, 50μmol/L, 100μmol/L, 200μmol/L, 300μmol/L, 400μmol/L, and 500μmol/L were applied to intervene NPCs, and cholecystokinin octapeptide(CCK8) was used to detect the cell activity after 2h and 4h to screen the optimal intervention concentration and time of TBHP. After intervening the P1 generation NPCs with the optimal intervention concentration of TBHP for optimal intervention time, complete medium was applied to culture for further 24h, 48h and 72h, and senescence-associated β-galactosidase staining(SA-β-gal) was used to detect the positive rate of senescent NPCs at each time point and observe the aging changes of NPCs. P2 generation NPCs were divided into control group(complete medium culture) and model group(complete medium culture after intervention with optimal concentration of TBHP for optimal time), real-time quantitative PCR(RT-qPCR) was used to detect the relative cell aging genes of p53, p21 and p16 as well as aggrecan, collagen Ⅱ and matrix metalloproteinase-3(MMP-3), matrix metalloproteinase-13(MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5) mRNA expressions, and western blot(WB) test was applied to detect the protein expressions of aggrecan, collagen Ⅱ, MMP-3, MMP-13, and p53 and p21. Results: Aggrecan protein was highly expressed in the isolated and cultured cells, the NPCs. The results of CCK8 experiment showed that the optimal intervention concentration of TBHP was 100μmol/L and the optimal intervention time was 4h. After 100μmol/L TBHP intervention for 4h and continued culture with complete medium for 24h, 48h and 72h, the positive rate of SA-β-gal of NPCs significantly increased than that in the control group(P<0.05), but there was no significant difference between the three time points of 24h, 48h and 72h(P>0.05). Therefore, complete medium was used to culture for 24h after the optimal intervention concentration of TBHP was added to the model group for optimal intervention time, while the control group was cultured in complete medium for the same time. The results of RT-qPCR showed that compared with the control, the expressions of p53, p21, p16, MMP-3, MMP-13 and ADAMTS-5 mRNA were increased significantly in the model group(P<0.05), while the expressions of aggrecan and collagen Ⅱ mRNA were decreased in the model group(P<0.05). WB detection showed that the protein expressions of MMP-3, MMP-13, p53 and p21 were increased in the model group(P<0.05), but the protein expressions of aggrecan and collagen Ⅱ were decreased(P<0.05). Conclusions: The senescence model of mouse NPCs can be successfully established by TBHP induction, which provides the basis for the study of the pathogenesis of intervertebral disc degeneration.