Beta-微管蛋白ⅢASODN对Ec9706/P-1细胞凋亡的影响  

Effect of β-tubulin Ⅲ antisense oligonucleotides on Ec9706/P-1 cells apoptosis

作　　者：卢红[1] 

机构地区：[1]河南大学淮河医院肿瘤科,河南开封475000

出　　处：《河南大学学报（医学版）》2010年第3期164-169,共6页Journal of Henan University:Medical Science

摘　　要：目的:观察β-微管蛋白Ⅲ(β-TubulinⅢ,TUBB3)反义寡核苷酸(Antisense oligonucleotide,ASODN)对Ec9706/P-1细胞凋亡的影响。方法:以体外诱导的人食管癌紫杉醇耐药细胞株Ec9706/P-1为模型,设ASODN组和无关序列寡核苷酸(Nonsense oligonucleotide,NSODN)组,应用免疫细胞化学方法检测NF-κBppabp65、Bcl-2和Caspase-3蛋白的表达;采用逆转录-多聚酶链反应(RT-PCR)检测NF-κBppabp65、bcl-2和caspase-3 mRNA的表达;采用DNA凝胶电泳和吖啶橙荧光染色法检测细胞凋亡变化。结果:免疫细胞化学方法显示ASODN组NF-κBppabp65蛋白表达率与NSODN组比较差异无统计学意义(P>0.05),ASODN组Bcl-2蛋白表达率与NSODN组比较差异有统计学意义(P<0.05),ASODN组Caspase-3蛋白表达率与NSODN组比较,差异有统计学意义(P<0.05);RT-PCR法显示ASODN组caspase-3 mRNA条带亮度强于NSODN组,bcl-2 mRNA条带亮度低于NSODN组,ASODN组NF-κBppab mRNA条带亮度与NSODN组相近;DNA凝胶电泳显示紫杉醇作用后ASODN组有凋亡特征的DNA改变,而NSODN组细胞却未出现;吖啶橙荧光染色法表明ASODN组凋亡率与NSODN组比较,差异有统计学意义(P<0.05)。结论:TUBB3反义寡核苷酸增加了紫杉醇对Ec9706/P-1细胞的诱导凋亡能力,证明了Bcl-2和Caspase-3的表达变化很可能是产生紫杉醇耐药的主要机制之一。Objective： To investigate the effect of β-tubulinⅢ antisense oligonucleotides on Ec9706/P-1 cells apoptosis, study the expression of NF-κBppabp65 ,Bcl-2 and Caspase-3, and to investigate the possible mechanism of acquired resistance of Paclitaxel-resistant human esophageal carcinoma cell line Ec9706/P-1. Methods： There were 2 groups in this study： antisense oligonucleotides（ASODN） group and oligodeoxynueleotides（NSODN） group. Immunohistochemistry assay was performed to detect the expression of Bel 2 protein, caspase-3 protein and NF-κBppab protein in Ec9706/P-1 cells. The expression level of Bcl-2 mRNA, caspase-3 mRNA and NF-κBppabmRNA were detected by semiquantity RT-PCR assay. DNA fragments were resolved by agarose gel electrophoresis to detect cell apoptosis. Apoptosis rates and morphology were detected by arcading orange（AO） staining. Results： The expression rate of Bel-2 protein in ASODN group was lower than that in NSODN group statistically （P〈0.05）. The expression rate of caspase-3 protein in ASODN group was higher than that in NSODN group statistically （P〈0.05）. There was no significant difference in the expression of NF-κBppab protein between ASODN group and NSODN group （P〉0.05）. The expression level of Bcl-2 mRNA in ASODN group was lower than that in NSODN group statistically （P〈0.05）. The expression level of caspase-3 mRNA in ASODN group decreased significantly compared with that of NSODN group （P〈0.05）. There was no significant difference in the expression of NF- κBppab mRNA between ASODN group and NSODN group （P〉0. 05）, There was obvious apoptosis characteristic DNA ladder in ASODN group induced by Paclilaxel but not in NSODN group. The apoptosis rate in NSODN group was lower significantly than that in ASODN group （P〈0.05）.Conclusion： TUBB3 ASODN increases the apoptosis of Ec9706/P-1 cells induced by Paclitaxel. It is proved that Bcl 2 and caspase-3 may be one of the main mechanisms of acquired resistance of Paclitaxel-resistant

关 键 词：β-微管蛋白Ⅲ 食管癌 耐药 凋亡 

分 类 号：R735.1[医药卫生—肿瘤]