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丝裂霉素C诱导小鼠NIH-3T3细胞出现衰老相关分泌表型

黄炜幸, 郭晓璇, 彭忠智, 翁春亮, 黄春燕, 石本艳, 杨杰, 廖小欣, 李晓毅, 郑慧玲, 刘新光, 孙雪荣 4, *

广东医科大学东莞科研中心衰老研究所；第二临床学院；医学检验学院；广东省医学分子诊断重点实验室，东莞 523808；生物化学与分子生物学研究所，湛江 524023

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、EdU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 (1 μg/mL)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EdU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mRNA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。

关键词： 细胞衰老; 小鼠 ; 衰老相关分泌表型 ; p16INK4a ; 成纤维细胞

分类号：Q255

Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C

HUANG Wei-Xing, GUO Xiao-Xuan, PENG Zhong-Zhi, WENG Chun-Liang, HUANG Chun-Yan, SHI Ben-Yan, YANG Jie, LIAO Xiao-Xin, LI Xiao-Yi, ZHENG Hui-Ling, LIU Xin-Guang, SUN Xue-Rong

1Institute of Aging Research, Dongguan Scientific Research Center； 2The Second Clinical School； 3School of Laboratory Medicine； 4Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, China； 5Institute of Biochemistry &； Molecular Biology, Guangdong Medical University, Zhanjiang 524023, China

Abstract

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.

Key words: senescence; mice ; senescence-associated secretory phenotype ; p16INK4a ; fibroblasts

收稿日期：2016-07-11　　录用日期：2016-10-25

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引用本文：

黄炜幸, 郭晓璇, 彭忠智, 翁春亮, 黄春燕, 石本艳, 杨杰, 廖小欣, 李晓毅, 郑慧玲, 刘新光, 孙雪荣 4, *. 丝裂霉素C诱导小鼠NIH-3T3细胞出现衰老相关分泌表型[J]. 生理学报 2017; 69 (1): 33-40.

HUANG Wei-Xing, GUO Xiao-Xuan, PENG Zhong-Zhi, WENG Chun-Liang, HUANG Chun-Yan, SHI Ben-Yan, YANG Jie, LIAO Xiao-Xin, LI Xiao-Yi, ZHENG Hui-Ling, LIU Xin-Guang, SUN Xue-Rong. Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C. Acta Physiol Sin 2017; 69 (1): 33-40 (in Chinese with English abstract).