曲古菌素A上调食管癌CAR的表达及增强溶瘤腺病毒转染效率的体外研究  

Trichostatin A enhances transfection efficiency of oncolytic adenovirus in vitro through up-regulating CAR in human esophageal cancer cell line EC9706

作　　者：冯婷[1] 刘霞[2] 黄幼田[3] 杨红艳[3] 董子明[3] 

机构地区：[1]郑州大学护理学院基础教研室,郑州450052 [2]郑州大学护理学院基础医学院病理生理学教研室,郑州450052 [3]徐州医学院附属第三医院病理科,江苏徐州221003

出　　处：《第三军医大学学报》2010年第22期2404-2408,共5页Journal of Third Military Medical University

基　　金：教育部科技创新工程基金资助重点项目(207150)~~

摘　　要：目的通过观察组氨酸去乙酰化酶(histone deacetylase,HDAC)抑制剂曲古菌素(trichostatin A,TSA)对食管癌细胞株EC9706膜表面柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)表达水平的影响,观察TSA能否通过上调食管癌EC9706CAR表达而增强其对腺病毒基因转染效率。方法采用免疫细胞组织化学、RT-PCR、Western blot检测食管癌EC9706细胞CAR的表达情况,再用上述方法检测TSA作用EC9706细胞后,CAR mRNA和CAR蛋白表达的改变;同时采用流式细胞术测定病毒转染效率,MTT实验评价腺病毒携带的胸苷激酶(Ad-TK)的体外抗瘤效应。结果 0.5μmol/LTSA处理EC9706细胞12、24、48h后,CAR mRNA和蛋白表达量[分别为(0.44±0.01)、(0.37±0.02),(0.66±0.04)、(0.58±0.01),(0.77±0.05)、(0.70±0.03)]与未加TSA处理组[(0.27±0.02)、(0.22±0.03)]相比,CAR mRNA和蛋白表达水平显著提高(P<0.05,P<0.01),呈现出时间依赖关系。流式细胞仪分析Ad-GFP转染后GFP的表达发现,未加TSA处理细胞转染效率为(1.35±0.11)%,0.5μmol/L TSA作用12、24h和48h后,细胞转染效率分别为(2.58±0.17)%、(4.96±0.14)%、(6.36±0.15)%。MTT检测Ad-TK介导的体外杀伤作用结果显示,0.5μmol/L TSA作用48h比未加TSA增强8倍。结论 TSA通过上调CAR表达量从而增强腺病毒对食管癌细胞的腺病毒基因转染效率。Objective To explore the effect of histone deacetylase （HDAC） inhibitor, trichostatin A （TSA）, on the expression of Coxsackie and adenovirus receptor （CAR） in esophageal cancer cell line EC9706. Methods Immunocytochemistry, RT-PCR and Western blotting were used to detect the expression of CAR in EC9706 cells, and its mRNA and protein expressions after the treatment of 0.5 mol/L TSA for 12, 24 and 48 h were also studied. Transfection efficiency of virus Ad-GFP in the EC9706 cells with or without 0.5 mol/L TSA treatment was determined by flow cytometry. Anti-tumor effect of adenovirus carrying thymidine kinase （Ad-TK） was evaluated by MTT assay. Results After 0.5 μmol/L TSA treatment for 12, 24 and 48 h, CAR mRNA and protein expression were 0.44±0.01 and 0.37±0.02, 0.66±0.04 and 0.58±0.01, and 0.77±0.05 and 0.70±0.03 respectively. Compared to those of control group （0.27±0.02 and 0.22±0.03）, the CAR mRNA and protein expressions were significantly increased in EC9706 cells after TSA treatment （P0.05, 0.01）, and the expressions showed a time-dependent manner. Flow cytometry indicated that the transfection efficiency was （1.35±0.11）% in the EC9706 cells, but was （2.58±0.17）%, （4.96±0.14）%, and （6.36±0.15）% respectively in the cells after 0.5 μmol/L TSA treatment for 12, 24 and 48 h. MTT results showed that the Ad-TK-mediated cytotoxicity in the cells with 0.5 μmol/L TSA treatment for 48 h was eight times higher than in the cells untreated. Conclusion TSA can increase gene transfer efficiency of adenovirus into cancer cells by upregulating CAR.

关 键 词：柯萨奇-腺病毒受体 曲古菌素 食管癌细胞株EC9706 腺病毒 基因转染 

分 类 号：R394.2[医药卫生—医学遗传学] R730.53[医药卫生—基础医学]