A20介导TNF-α炎症微环境下髓核细胞衰老的机制研究

Mechanism research of senescence of NP cells mediated by A20 in TNF-ɑ inflammation microenvironment

中文关键词:  髓核细胞  锌指蛋白A20  肿瘤坏死因子α  炎症反应  细胞衰老

英文关键词:Nucleus pulposus cells  Zinc finger protein A20  TNF-α: Inflammatory response  Cell senescence

中文摘要:

  【摘要】 目的：探讨锌指蛋白A20[也称为肿瘤坏死因子诱导蛋白3（tumor necrosis factor-induced protein 3，TNFAIP3）]在肿瘤坏死因子α（tumor necrosis factor α，TNF-α）炎症微环境下髓核细胞（nucleus pulposus cells，NPCs）衰老发生机制。方法：体外分离培养SD大鼠髓核细胞。实验分为三组：正常对照组（只加入正常培养基）、TNF-α干预组（加入不同浓度的TNF-α，10ng/ml、50ng/ml、100ng/ml）和自然衰老组（在体外通过细胞不断增长传代至P20代）。应用CCK-8细胞增殖实验﹑β半乳糖苷酶染色﹑Western blot（WB）、QT-PCR、immunofluorescence（IF）从基因、蛋白及细胞功能水平评估髓核细胞衰老变化。应用WB、QT-PCR和IF检测锌指蛋白A20﹑NF-?资B（p65）﹑NF-ｋB（p-p65/phospho S536）表达情况。结果：与正常对照组相比，TNF-α干预48h、72h后髓核细胞的增殖能力明显降低；TNF-α干预组和衰老组β半乳糖染色阳性率较对照组明显增高；QT-PCR结果提示，与对照组比，TNF-α干预组p53表达增加（P<0.05），而A20表达随着TNF-α浓度增高而降低；衰老组A20表达较对照组减少，而p53扩增升高。WB检测显示：TNF-α可以诱导p53﹑A20、p-p65表达上调，而A20表达随着TNF-α浓度增加而降低；同时衰老组A20表达较对照组减少，而p-p65和p53表达增加（P<0.05）；IF显示：与对照组对比，TNF-α处理下A20﹑p-p65表达增加，但是A20的表达随着TNF-α增加而降低，衰老组A20较对照组也明显降低，p-p65表达增加。结论：髓核细胞衰老程度与TNF-α干预存在剂量关系，TNF-α可以刺激髓核细胞A20表达升高，并激活NF-?资B信号通路。而A20表达情况受细胞衰老程度影响，随着髓核细胞衰老A20所参与的作用效能逐渐减低。

英文摘要:

  【Abstract】 Objectives: To investigate the mechanism of zinc finger protein A20(also known as tumor necrosis factor-inducible protein 3, TNFAIP3) involved in the senescence of nucleus pulposus cells(NPCs) in the tumor necrosis factor α(TNF-α) inflammatory microenvironment. Methods: Isolated NPCs of sprague-dawley rat (SD) were cultured with or without TNF-α in vitro. The experiment was divided into three groups: normal control group(0ng/ml), TNF-α intervention group(10ng/ml, 50ng/ml, 100ng/ml), and natural aging group(p20 generation). CCK-8 cell proliferation assay, β-galactosidase staining, Western blot(WB), QT-PCR and immunofluorescence(IF) were used to assess senescence changes in NP cells from genes, proteins and cell function levels. The expressions of p53, A20, NF-ｋB(p65) and NF-ｋB (p-p65/phospho S536) were observed by Western Blotting, QT-PCR and IF. Results: Compared with the normal control group, the proliferation ability of NP cells significantly decreased after TNF-α intervention for 48h and 72h. The positive rate of β-galactose staining in the TNF-α intervention group and the aging group was significantly higher than that of the control group. The results of QT-PCR demonstrated that compared with the control group, the expression of p53 in the TNF-α intervention group increased(P<0.05), while the expression of A20 decreased with the increase of TNF-α concentration. The expression of A20 in the aging group decreased compared with that in the control group, while p53 expansion increased. WB showed that TNF-α could induce up-regulated expressions of p53, A20 and p-p65, while the expression of A20 decreased with the increase of TNF-αconcentration. Meanwhile, the expression of A20 in the aging group decreased compared with that in the control group, while the expression of p-p65 and p53 increased(P<0.05). IF showed that compared with the control group, the expressions of A20 and p-p65 increased with TNF-α treatment, but the expression of A20 decreased with the increase of TNF-α. The A20 of aging group also decreased significantly and the expression of p-p65 increased. Conclusions: There is a dose relationship between senescence of NP cells and TNF-α intervention. TNF-α can stimulate the expression of A20 in NP cells and activate NF-?资B signaling pathway. In addition, the expression of A20 is affected by the degree of cellular senescence, with aging of NP cells, the mechanism of action of A20 is decreasing.