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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(P<0.01)。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(P<0.01)。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。
Senescence-associated secretory phenotype (SASP) refers to the secretion of hyperthyroidism with cell senescence, the secretion of factors involved in cellular aging, immune regulation, angiogenesis, cell proliferation and tumor invasion process. Compared with human cells, the in vitro SASP model of mouse cells is still lacking, and the establishment of this model will facilitate the study of the mechanism and biological role of SASP. Therefore, in this study, mouse NIH-3T3 cell line deficient in INK4a locus (encoding p16INK4a protein) and mouse embryonic fibroblasts (MEF) were used as research objects. Mitomycin C C and MMC) were used to induce cell DNA damage. Cell morphology, senescence related β-gal activity staining, Ed U integration rate, Western blot, quantitative RT-PCR and ELISA were used to observe the cell proliferation Aging and SASP factor expression and secretion levels. The results showed that the volume of NIH-3T3 cells became larger and the percentage of β-gal-positive (blue-stained) cells increased significantly after treated with MMC (1μg / ml) for 12 h or 24 h and cultured for 8 days. Reached 77.4% and 90.4% respectively, accompanied by the up-regulation of P21 protein and the decrease of Ed U integration rate (P <0.01). At the same time, m RNA expression levels of common SASP genes such as IL-6, TNF-α, IL-1α and IL-1β were significantly up-regulated. ELISA assay also showed that secretion of IL-6 was significantly increased (P <0.01). In contrast, MMC-induced increase of cell volume, up-regulation of β-gal staining (71.7% and 80.2%, respectively) and P21 expression were induced by MMC treatment for 12 or 24 h No significant increase in secretion. This study shows that MMC can induce cell senescence in NIH-3T3 and wild-type MEFs, but only typical NIH-3T3 cells exhibit SASP. Aging NIH-3T3 cells may be a suitable model for studying mouse SASP.