Survivin反义寡核苷酸联合TCS诱导食管癌细胞Eca-109凋亡的研究

Apoptosis of Eca-109 cells treated with antisense oligodeoxynucleotide targeting survivin and trichosanthin

目的:探讨survivin反义寡核苷酸(Antisense oligodeoxynucleotide,ASODN)联合TCS诱导细胞凋亡的作用及其分子机制。方法:以食管癌细胞Eca-109为实验对象,将实验分6组:未处理组、脂质体组、survivin正义寡核苷酸组(Sense oligodeoxyn ucleotide,SODN)、survivin反义寡核苷酸组(Antisense oligodeoxynucleotide,ASODN)、天花粉蛋白组(Trichosan thein,TCS)、survivin反义寡核苷酸联合天花粉蛋白组。各处理因素作用食管癌细胞Eca-10948h,RT-PCR检测Eca-109细胞survivin mRNA表达变化;Western blot检测Survivin、Bcl-2、Bax、Caspase-3蛋白表达变化;Annexin V-FITC/PI双染法检测Eca-109细胞早期凋亡率。结果:ASODN组、TCS组和联合组的survivin mRNA和Survivin蛋白表达水平明显降低,联合组较单独药物组(ASODN组、TCS组)降低,而以上3组的早期凋亡率和Caspase-3蛋白表达水平明显升高,联合组较单独药物组增高(P<0.05);TCS组和联合组细胞的Bcl-2蛋白的表达水平明显降低,Bax蛋白的表达增加。结论:TCS联合survivin的ASODN在诱导Eca-109细胞的凋亡方面,能够发挥协同作用;TCS和survivin的ASODN共同抑制survivin基因表达,TCS下调Bcl-2蛋白表达和上调Bax蛋白表达,分别激活caspase级联反应上游、下游途径,是两药发挥协同作用的分子基础。

Objective：To explore the effects of antisense oligodeoxynucleotide targeting survivin and trichosanthin on the apoptosis of human esophageal carcinoma Eca-109 cells and its molecular mechanisms.Methods：The experimental Eca-109 cells were divided into six groups：untreated group,lipofectamine group,sense oligodeoxynucleotide targeting survivin group（SODN）,antisense oligodeoxy nucleotide targeting survivin group（ASODN）,trichosanthin group,and antisense oligodeoxynucleotide targeting survivin and trichosanthin group.Expression of survivin mRNA was detected by RT-PCR.Expressions of Survivin,Bcl-2,Bax,and Caspase-3 proteins were detected by Western blot.The rate of early apoptosis was detected by AnnexinV-FITC/PI double stain.Results：Expressions of survivin mRNA and protein of the ASODN group,the TCS group,and the combination group were significantly decreased.The combined group had significantly lower expression than the single group（SODN group or the TCS group）.However,the early apoptosis rates and expression of caspase-3 protein in the ASODN group,the TCS group and the combination group were significantly increased,with significantly higher results in the combination group than in the single group.Expression of bcl-2 protein of the TCS group and the combination group decreased.However,expression of the bax protein was increased.Conclusion：Survivin antisense oligodeoxynucleotide targeting survivin and trichosanthin can collaboratively induce apoptosis of the Eca-109 cells.The molecular mechanism may be that two drugs can activate caspase cascade channels due to collaborative suppressing the expression of survivin gene by two drugs and down-regulating the expression of bcl-2 protein and up-regulating the expression of bax protein by TCS.