丝裂霉素C诱导小鼠NIH-3T3细胞出现衰老相关分泌表型

发布时间：2018-09-11 18:19

【摘要】：衰老相关分泌表型(senescence-associated secretory phenotype,SASP)是指随细胞衰老而出现的分泌功能亢进,其分泌的因子参与细胞衰老、免疫调节、血管生成、细胞增殖及肿瘤侵袭等过程。与人类细胞相比,目前小鼠细胞的体外SASP模型尚十分缺乏,而建立该模型将为研究SASP的发生机制及生物学作用提供便利。为此,本文以INK4a基因座缺失(编码p16INK4a蛋白)的小鼠NIH-3T3细胞系及野生型小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEF)为研究对象,用丝裂霉素C(mitomycin C,MMC)诱导细胞DNA损伤,并通过细胞形态、衰老相关β-半乳糖苷酶(β-gal)活性染色、Ed U整合率、Western blot、定量RT-PCR及ELISA等检测方法,观察细胞的衰老情况及SASP因子的表达和分泌水平。结果显示,MMC(1μg/m L)处理12 h或24 h,并继续培养到第8天后,NIH-3T3细胞体积变大,β-gal染色阳性(蓝染)细胞的百分率明显升高,分别达到77.4%和90.4%,并伴有P21蛋白表达上调及Ed U整合率下降(P0.01)。同时,IL-6、TNF-α、IL-1α和IL-1β等常见SASP基因的m RNA表达水平显著上调,ELISA检测证明IL-6的分泌量亦显著升高(P0.01)。相反,尽管MMC处理12 h或24 h也能诱导野生型MEF出现细胞体积增大、β-gal染色阳性率升高(分别达71.7%和80.2%)及P21表达上调,但其IL-6的分泌量无显著上升。本研究表明,MMC能诱导NIH-3T3和野生型MEF出现细胞衰老,但只有NIH-3T3细胞出现了典型的SASP现象。衰老NIH-3T3细胞可能是研究小鼠SASP的合适模型。
[Abstract]:Senescence related secretory phenotype (senescence-associated secretory phenotype,SASP) is a kind of hypersecretion associated with cell senescence. The factors secreted by senescence-associated secretory phenotype,SASP are involved in the process of cell senescence, immune regulation, angiogenesis, cell proliferation and tumor invasion and so on. Compared with human cells, the SASP model of mouse cells in vitro is still scarce, and the establishment of this model will facilitate the study of the pathogenesis and biological role of SASP. Therefore, the murine NIH-3T3 cell line (encoding p16INK4a protein) with INK4a locus deletion (encoding p16INK4a protein) and the wild-type mouse embryonic fibroblast (mouse embryonic fibroblasts,MEF) were used to induce DNA damage by mitomycin (C (mitomycin). Senescence related 尾 -galactosidase (尾 -gal) activity staining method was used to detect the integration rate of Ed U by Western blot, quantitative RT-PCR and ELISA, and to observe the senescence of cells and the expression and secretion level of SASP factor. The results showed that MMC (1 渭 g / mL) was treated for 12 h or 24 h, and then cultured for 8 days, the volume of NIH-3T3 cells became larger, the percentage of 尾 -gal staining positive (blue staining) cells increased significantly, reaching 77.4% and 90.4%, respectively, accompanied by the up-regulation of P21 protein expression and the decrease of Ed U integration rate (P0.01). At the same time, the expression level of m RNA in common SASP genes such as TNF- 伪 IL-1 伪 and IL-1 尾 was significantly up-regulated. Elisa assay showed that the secretion of IL-6 was also significantly increased (P0.01). On the contrary, although MMC treatment for 12 or 24 hours also induced the increase of cell volume, the positive rate of 尾 -gal staining (71.7% and 80.2%) and the up-regulation of P21 expression in wild-type MEF, the secretion of IL-6 did not increase significantly. The results showed that MMC could induce the senescence of NIH-3T3 and wild-type MEF cells, but only NIH-3T3 cells had typical SASP phenomenon. Aging NIH-3T3 cells may be a suitable model for the study of SASP in mice.
【作者单位】：广东医科大学东莞科研中心衰老研究所;广东医科大学第二临床学院;广东省医学分子诊断重点实验室;广东医科大学医学检验学院;生物化学与分子生物学研究所;
【基金】：supported by grants from the Medical Scientific Research Foundation of Guangdong Province,China(No.A2012420) the Doctoral Initial Funding of Guangdong Provincial Medical University,China(No.B2011005) the Social Development Funding of Dongguan Municipality,Guangdong Province,China(No.2014108101047) the Special Funds for the Cultivation of Guangdong Provincial College Students’Scientific and Technological Innovation,China(No.pdjh2016b0216) the Innovation and Entrepreneurship Program for College Students of Guangdong Province,China(No.201410571018,201510571008,2014ZYDS017)
【分类号】：R339.38

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