反义STAT3对肿瘤细胞增殖抑制和诱导凋亡的作用  

Effects of STAT3 Antisense Oligodeoxynucleotides on Apoptosis and Proliferation of Mouse Melanoma Cell Line B16

作　　者：唐古生[1] 蔡建明[1] 倪瑾[1] 项莺松[1] 崔建国[1] 朱丹[1] 董俊瑞[1] 

机构地区：[1]第二军医大学海医系放射医学教研室,上海200433

出　　处：《癌症》2006年第3期269-274,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30470415)~~

摘　　要：背景与目的:研究表明STAT3蛋白在多种肿瘤组织或细胞中高表达,STAT3蛋白可能参与了肿瘤的形成和发生。本文拟研究STAT3蛋白在鼠黑色素瘤细胞B16、人肝癌细胞SMMC-7721、人肝癌细胞HepG-2、人肺癌细胞A549、人宫颈癌HeLa细胞株中表达和活化情况,研究反义STAT3寡核苷酸对B16细胞的增殖抑制和促凋亡作用,为进一步反义药物设计及应用于辐射领域作前期研究。方法:利用Westernblot检测所选几种肿瘤细胞的STAT3蛋白表达和磷酸化情况、反义干涉前后B16细胞中STAT3蛋白表达和磷酸化活化的变化情况,MTT法检测细胞增殖变化,利用Hoechst33258染色对细胞凋亡作形态学上的观察,用AnnexinⅤ/PI复染结合流式细胞仪检测细胞早期凋亡。结果:所研究的几种肿瘤细胞中均可检测到STAT3蛋白的高表达和磷酸化;反义STAT3转染后,B16细胞中STAT3蛋白的表达量及磷酸化水平均有下降;转染后48h,在0到200nmol/L范围内,反义核酸浓度越高,对B16细胞的增殖抑制效应越强,但是并不能够完全抑制肿瘤细胞的增殖(P<0.01)。寡核苷酸浓度超过250nmol/L时,正义对照也表现出一定的增殖抑制作用(P<0.05);转染后不同时间内检测,结果表明转染后24h反义核酸即开始表现出一定的增殖抑制效应,48h起表现出明显的抑制效应;反义转染后,检测各组细胞早期凋亡率:对照组早期凋亡率为5.52%,反义400、800、2000nmol/L组早期凋亡率分别为8.22%、9.99%,16.97%,正义对照400、800、2000nmol/L组早期凋亡率分别为5.87%、5.36%、13.31%。统计分析表明反义寡核苷酸与B16细胞作用后能够促进细胞的早期凋亡(P<0.01),正义低浓度转染组(400、800nmol/L组)与对照组无明显差别(P>0.05),而对照组与正义2000nmol/L组相比,细胞的早期凋亡率也有统计学差异(P<0.01)。结论:B16、SMMC-7721、HepG-2、A549、HeLa等恶性肿瘤细胞株中STAT3蛋白高表达并且可检测到磷酸化水平的增高BACKGROUND ＆ OBJECTIVE： STAT3 protein has been found constitutively activated in a wide variety of human tumor tissues and cell lines. It may be involved in tumorigenesis and development. This study was to observe the activation of STAT3 protein in mouse melanoma cell line B16, human liver cancer cell lines SMMC-7721 and HepG-2, human lung cancer cell line A549, and human cervical cancer cell line HeLa, and to investigate the effects of STAT3 antisense oligodeoxynucleotides （ASODN） on proliferation and apoptosis of B16 cells. METHODS： B16 cells were transfected with STAT3 ASODN or STAT3 sense oligodeoxynucleotides （STAT3 SODN）, respectively. The expression and phosphorylation levels of STAT3 protein in all the tumor cells were measured by Western blot. The proliferation of B16 cells was detected by MTI- assay. Cell apoptosis was determined by Hoechst33258 staining and Annexin V/PI using flow cytometry. RESULTS： STAT3 was highly expressed and phosphorylated in all the tumor cells. Transfection of STAT3 ASODN suppressed the expression and phosphorylation levels of STAT3 protein in B16 cells. Forty-eight hours after transfection, the proliferation of B16 cells was inhibited-the higher the concentration （0-200 nmol/L） of STAT3 ASODN was, the heavier the inhibition was （P〈0.01）; when transfected with STAT3 SODN over 250 nmol/ L, the proliferation of B16 cells was also inhibited （P〈0.05）. Inhibitory effects appeared 24 h later after transfection of STAT3 ASODN, and became more obvious 48 h later （P〈0.01）. The early apoptosis rate was significantly higher in 400, 800, 2 000 nmol/L STAT3 ASODN groups and 2 000 nmol/L STAT3 SODN group than in blank control group （8.22%, 9.99%, 16.97%, and 13.31% vs. 5.52%, P〈0.01 ）; no significant difference was found among 400 and 800 nmol/L STAT3 SODN groups and blank control group （5.87%, 5.36% and 5.52%, P〉0.05）. CONCLUSIONS： STAT3 is highly expressed and activated in all the tumor cells detected. STAT3 ASODN can abrogate the activit

关 键 词：反义寡核苷酸/药理学 STAT3蛋白 凋亡 肿瘤细胞 增殖抑制 

分 类 号：R730.5[医药卫生—肿瘤]