CD40信号通过TNFRⅠ途径抑制肺癌细胞增殖的研究  

Inhibition of lung cancer cell proliferation by CD40 signaling through tumor necrosis factorⅠ

作　　者：卢旭东[1] 朱晓兰[1] 陈成[1] 张光波[2] 张学光[2] 黄建安[1] 

机构地区：[1]苏州大学附属第一医院呼吸内科,江苏苏州215006 [2]苏州大学医学生物技术研究所,江苏苏州215007

出　　处：《癌症》2009年第1期27-32,共6页Chinese Journal of Cancer

基　　金：江苏省高校自然科学基础研究项目(No06KJB320100);江苏省医学重点人才基金项目(NoRC2007075);国家自然科学基金项目(No30770947)~~

摘　　要：背景与目的:CD40活化信号可抑制多种肿瘤细胞体外生长 ,但其分子机制尚不明确,本研究旨在探讨激发CD40信号对肺癌细胞株NCI-H460、A549的增殖及肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)、膜型TNF-α(mTNF-α)表达的影响及相关机制。方法:免疫荧光标记和流式细胞术检测肺癌细胞表面CD40的表达以及激发CD40对细胞表面TNFR和mTNF-α表达谱的影响;Westernblot检测激发CD40对细胞TNFR和mTNF-α蛋白含量表达的影响;采用四氮唑盐(MTT)比色法抗体中和实验分析阻断TNFRⅠ及TNF-α对CD40激发效应的影响;酶联免疫吸附(ELISA)法检测激发CD40对肺癌细胞培养上清中可溶性TNF-α(sTNF-α)含量的影响。结果:(1)肺癌细胞株NCI-H460、A549表面CD40表达分别为(89.0±3.2)%、(62.2±4.5)%。(2)免疫荧光和流式细胞术示,激发NCI-H460和A549细胞表面CD40分子48h后,其表面TNFRⅠ表达率分别为(36.2±4.6)%、(38.5±5.9)%,较对照组分别为(7.2±1.9)%、(15.2±3.1)%明显升高(P<0.05);而其表面TNFRⅡ表达率分别为(5.8±1.2)%、(18.0±1.6)%,较对照组分别为(38.8±4.3)%、(58.1±3.6)%明显降低(P<0.05);其表面TNF-α表达率分别为(7.0±0.9)%、(8.7±1.1)%,较对照组分别为(15.0±2.1)%、(26.5±3.2)%也明显降低(P<0.05)。(3)Western blot示,激发CD40可使NCI-H460和A549细胞TNFRⅠ蛋白表达增强,TNFRⅡ蛋白表达减弱,而mTNF-α蛋白表达无明显变化。(4)CD40激发前后肺癌细胞培养上清中未检测到sTNF-α的存在。(5)激发CD40能明显抑制NCI-H460、A549细胞的增殖(P<0.05),而阻断TNFRⅠ后CD40信号的细胞增殖抑制效应消失。(6)阻断TNF-α亦可明显抑制两肺癌细胞株增殖(P<0.05),而同时激发CD40未见两者存在协同效应。结论:CD40信号是通过mTNF-α/TNFRⅠ途径抑制CD40表达阳性肺癌细胞株的体外增殖。Background and Objective：CD40 signaling induces growth inhibition in some tumor cells in vitro, but the precise molecular mechanism remains unclear. This study was to investigate the biological effects and mechanisms of CD40 stimulation on proliferation of lung cancer cell lines NCI-H460 and A549, changes in tumor necrosis factor receptors （TNFR） and membrane tumor necrosis factor alpha （mTNF-α）. Methods：The immunofiuorescence technique and flow cytometry were applied to detect the expression of CD40 on the cell surface and changes in TNFR and mTNF-α expression after CD40 stimulation. Western blot was used to measure the changes in protein contents of TNFR as well as mTNF-α expression after CD40 stimulation. MTT assay was adopted to determine the cell proliferation rate. ELISA assay was performed to measure the change in the content of soluble TNF-α （sTNF-α） in the supernatant of lung cancer cells. Results： The expression rates of CD40 in NCI-H460 and A549 were （89.0±3,2）% and （62.2±4.5）%. After 48 h of CD40 stimulation, the expression rates of TNFR Ⅰ in NCI-H460 and A549 were （36.2±4.6）% and （38.5±5.9）%, higher than those in the corresponding control cells [（15.2±3.1）% and （7.2±1.9）%] （P〈0.05）; the expression rates of TNFR Ⅱ were significantly lower in the two cell lines than in control cells E（18.0±1.6）% and （5.8± 1.2）% vs. （58.1±3.6）% and （38.8±4.3）%] （P〈0.05）; the expression rates of mTNF-α were significantly decreased in the two cell lines [（8.7±1.1）% and （7.0±0.9）%] as compared to those in control cells [（15.0±2.1）% and （26.5±3.2） % （P〈0.05）. The level of TNFRⅠ protein was elevated with the down-regulation of TNFR Ⅱ protein in NCI-H460 and A549. There was no detectable change in mTNF-α protein in the two cell lines. TNF-α was not detected in the supernatant of lung cancer cells. Moreover, cell proliferation of NCI-H460 and A549 were inhibited after CD40 stimulation （P〈0.05）, but t

关 键 词：CD40 肺肿瘤 NCl-H460细胞 A549细胞 肿 瘤坏死因子受体 细胞增殖 

分 类 号：R734.2[医药卫生—肿瘤]