Survivin反义寡核苷酸对喉癌Hep-2细胞放疗的增敏作用

目的探讨SUrvivin在放疗诱导喉癌细胞凋亡中的作用以及Survivin反义寡核苷酸（antisenseoligonucleotide，ASODN）技术对HeP-2细胞株增殖、凋亡及其放疗敏感性的影响。方法人工合成硫代SurvivinASODN，通过脂质体法转染人喉鳞状细胞癌Hep一2细胞株后，采用：①流式细胞法测定SurvivinASODN转染Hep一2细胞的转染率；②RT-PCR法检测基因转染后SurvivinmRNA的表达情况变化；③流式细胞仪检测射线照射后细胞凋亡率及Survivin蛋白的变化。结果①各浓度带FITC标记的SurvivinASODN转染细胞后6小时，取转染细胞制成的单细胞悬液，于流式细胞仪进行检查，发现转染效率均为94％～98％；②各浓度SurvivinASODN作用于Hep-2细胞24小时后，均出现SurvivinmRNA的表达下调，并呈剂量依赖关系，同时伴有细胞凋亡率的升高；③放疗~DSurvivinASODN转染组细胞凋亡率均显著高于单纯放疗组细胞凋亡率（P〈0．05），随着SurvivinASODN剂量的增加，细胞凋亡率逐渐增加（P〈O．01）。放疗加ASODN转染组细胞Survivin蛋白表达率低于单纯放疗各组细胞Survivin蛋白表达率（P〈0．05）。各实验组与对照组中凋亡率的变化与Survivin蛋白荧光指数的变化呈负相关。结论喉癌细胞对放疗的抵抗作用与Survivin蛋白的表达水平有关，Survivin反义寡核苷酸转染Hep-2细胞株能够提高细胞对放疗的敏感性。

Effect of Survivin antisense oligodeoxynucleotides on radiotherapy sensitivity in Hep-2 cells

OBJECTIVE To explore the role of Survivin on radiotherapy inducing apoptosis and the effect of Survivin antisense oligodeoxynucleotides （ASODN） on the growth, apoptosis and radiotherapy sensitivity in Hep-2 cells. METHODS Survivin ASODN was synthesized and thansfected into hep-2 cells with lipofectin. The experiment consisted of three steps. OThe efficiency of transfection was measured with flow cytometry technique. （2）The expression of survivin mRNA was observed with RT-PCR.（3） Apoptosis and protein expression after radiotherapy was analyzed with flow cytometry. RESULTS （1）Six hours after transfection of Survivin ASODN tagged with FITC into the cells, the efficiency of transfection was found to be 94% to 98% with flow cytometry. （2） Twenty four hours after transfection, Survivin mRNA was found decreased with the increasing of apoptosis, which presented a dose-dependent manner. （3）The apoptosis in the radiotherapy combined with Survivin ASODN transfected group was significantly higher than that in radiotherapy group only （P〈0.05） . With the increasing of Survivin ASODN concentration, the apoptosis was increasing too （P〈0.01） . The Survivin protein expression in the radiotherapy combined with Survivin ASODN transfected group was lower than that in radiotherapy group only （P〈0.05） . In the experiment and control groups, the changes of apoptosis and Survivin protein fluorescence index had a negative correlation. CONCLUSION The expression of Survivin protein may be associated with the radiotherapy resistance in laryngeal carcinoma cells, and Survivin ASODN can improve the sensitivity of Hep-2 cells to radiotherapy.