联合survivin反义寡核苷酸和基因重组融合蛋白对人膀胱癌细胞的抑制作用

Inhibitory Effect of Recombinant TGF alpha-PE40 Combined with survivin Antisense Oligonucleotides on Bladder Transitional Cell Carcinoma Cells

目的用体外实验探讨联合应用survivin反义寡核苷酸、基因重组融合蛋白TP40是否对膀胱移行细胞癌T24细胞具有协同抑制作用。方法实验分为对照组、错义寡核苷酸组(MS)、反义寡核苷酸组(AS)、TP40组和联合组(AS+TP40)。RT-PCR和Western blot法检测各组细胞survivin的表达;MTT法检测各组细胞生长情况;流式细胞仪检测各组细胞凋亡率;体外成瘤实验检测各组细胞的体外锚定非依赖性生长能力。处理时间为3天。结果所合成AS(300nM)对survivin mRNA的表达具有明显抑制作用。分别从转录水平和表达水平证明了联合组下调survivin的能力最强。MTT法显示联合组用药后生长抑制更加明显(P<0.0001),第3天细胞存活率仅12.2%,凋亡率达到96.37%。在软琼脂糖体外成瘤实验中,TP40组和AS组的体外锚定非依赖性生长的能力大大降低,联合组(TP40+AS)细胞几乎没有形成克隆。结论采用survivin反义寡核苷酸封闭survivin的表达,可以增强TP40对膀胱移行细胞癌T24细胞的生长抑制作用,呈现协同效应。

Objective To investigate whether the survivin antisense oligonucleotides could decrease the expression of survivin in vitro and sensitize human bladder transitional cell carcinoma T24 cells to recombinant TGF alpha-PE40（TP40）. Methods The experiment cells were divided into five group,such as control group, survivin missense oligonucleotides group （MS）, survivin antisense oligonucleotides group （AS） ,TP40 group and combined group（TP40 ＋ AS）. RT-PCR and Western blot assay was performed to detect the expression of survivin in all groups cells. MTT assay was applied to detect the cell proliferation of all groups. Flow cytometry were performed to detect TP40-triggered apoptosis. The tumor formation experiment of T24 cells in vitro was used to evaluate all groups cells＂ independent ability of anchoring growth. Results The survivin antisense oligonucleotides could down-regulate the expression of survivin at a concentration of 300nM. We have demonstrated from the mRNA level and protein level that the combined group could down-regulate the expression of survivin compared with the non-combined group powerfully. In the third day,the survival rate of the combined group cells was only 12.2% ,the apoptotic rate was 96. 37% ,and the ability of tumor formation in vitro was very poor. Conclusion survivin antisense olignucleotides could sensitize human bladder transitional cell carcinoma T24 cells to TP40 with a cooperative effect.