TNF-α激活NF-κB信号通路抑制培养成纤维细胞SDF-1α分泌  

TNF-α inhibits SDF-1α secretion in cultured fibroblasts through activating NF-kappaB signaling pathway

作　　者：张玉龙[1] 毋巨龙[1] 杨忠[2] 丁可[3] 李世荣[1] 

机构地区：[1]第三军医大学西南医院整形美容科,重庆400038 [2]第三军医大学基础医学部神经生物学教研室,重庆市神经科学研究所,重庆400038 [3]第三军医大学西南医院骨科,全军矫形外科中心,重庆400038

出　　处：《第三军医大学学报》2012年第15期1509-1513,共5页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC 2009BB5146)~~

摘　　要：目的探讨肿瘤坏死因子-α(TNF-α)激活核因子-κB(NF-κB)信号通路对成纤维细胞生物学行为的影响。方法小鼠胚胎成纤维细胞(MEF)进行体外培养,将细胞分为TNF-α组、TNF-α+吡咯烷二硫氨基甲酸(PDTC)组及对照组,处理后的细胞采用CCK-8方法检测细胞增殖情况、Western blot和细胞免疫荧光检测IκBα蛋白表达与定位、ELISA检测基质细胞衍生因子-1α(SDF-1α)的分泌。结果 TNF-α在一定浓度范围内对成纤维细胞起到明显的促增殖作用,CCK-8检测显示TNF-α组在不同浓度和不同时相点MEF的光密度值与TNF-α+PDTC组及对照组相比差异显著(P<0.05);免疫荧光染色显示IκBα主要分布于细胞质,TNF-α组与对照组和TNF-α+PDTC组相比,IκBα荧光强度明显减弱。Western blot结果发现TNF-α刺激后30 min,TNF-α组和TNF-α+PDTC组IκBα蛋白表达[(3 070.0±39.0)vs(1 421.7±75.3),(3 212.2±57.5)vs(1 468.2±45.8),P<0.05]均降至最低值,随时间推移逐渐恢复,TNF-α+PDTC组IκBα蛋白表达显著高于TNF-α组[(3 112.3±89.7)vs(2 610.4±26.9),P<0.05];ELISA检测发现TNF-α作用后成纤维细胞SDF-1α的分泌呈减少趋势[(15.9±1.6)vs(12.7±0.6),P<0.05],TNF-α+PDTC组则能显著逆转TNF-α作用[(23.5±3.2)vs(15.0±1.2),(21.6±0.4)vs(12.7±0.6),P<0.05],促进SDF-1α分泌。结论 TNF-α能通过激活NF-κB通路,影响小鼠胚胎成纤维细胞的增殖并抑制SDF-1α分泌。Objective To determine the effects of tumor necrosis factor-α（TNF-α） on the biological behaviors of cultured fibroblasts and its relevant nuclear factor-κB（NF-κB）.Methods Mouse embryonic fibroblasts（MEF） were cultured and treated with TNF-α or TNF-α plus pyrrolidine dithiocarbamate（PDTC） respectively.Cell proliferation were analyzed by CCK-8 method and flow cytometry.The levels of IκBα in the cytoplasm were examined with Western blotting and immunofluorescence staining.The secretion of SDF-1α was measured through ELISA.Results TNF-α promoted the proliferation of MEF in a concentration-dependent manner,the result of CCK-8 showed that the absorbance in TNF-α group was significantly higher than that in TNF＋PDTC group and control group（P〈0.05）.Immunofluorescence staining showed that IκBα was mainly distributed in the cytoplasm,and fluorescent intensity of IκBα in the TNF-α group was decreased significantly when compared with control and the TNF-α＋PDTC group.Western blotting indicated that the expression of IκB both in the TNF-α and TNF-α＋ PDTC group was dropped to a minimum value in 30 min after treatment（3 070.0±39.0 vs 1 421.7±75.3,3 212.2±57.5 vs 1 468.2±45.8,P〈0.05）,then gradually increased and restored to near normal level around 4 h.The expression of IκB in TNF-α＋PDTC group was significantly higher than that of TNF-α group（3 112.3±89.7 vs 2 610.4±26.9,P〈0.05）.The secretion of SDF-1α in MEF was decreased upon the treatment of TNF-α（15.9±1.6 vs 12.7±0.6,P〈0.05）,TNF-α＋PDTC group excerted opposite effect（23.5±3.2 vs 15.0±1.2,21.6±0.4 vs 12.7±0.6,P〈0.05） and improved SDF-1α secretion.Conclusion TNF-α influences the proliferation and secreting function of mouse embryonic fibroblasts through activating NF-κB pathway.

关 键 词：肿瘤坏死因子-α 核因子-ΚB 小鼠胚胎成纤维细胞 吡咯烷二硫氨基甲酸 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R364.3[医药卫生—基础医学]