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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 investigate the effects of trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), on the surface of C9 OECs in the esophageal cancer cell line EC9706, CAR), and to observe whether TSA enhances the transfection efficiency of adenovirus gene by up-regulating the expression of EC9706CAR in esophageal cancer. Methods The expression of CAR in esophageal carcinoma EC9706 cells was detected by immunohistochemistry, RT-PCR and Western blot. The expression of CAR mRNA and CAR protein in TSC-treated EC9706 cells was detected by the above method. Flow cytometry Virus transfection efficiency, MTT test evaluation of adenovirus carrying thymidine kinase (Ad-TK) in vitro anti-tumor effect. Results After treated with 0.5μmol / L TSA for 12, 24 and 48h, the mRNA and protein expressions of CAR were significantly higher in EC9706 cells treated with TSA (0.44 ± 0.01, 0.37 ± 0.02, 0.66 ± 0.04, 0.58 ± 0.01, (P <0.05, P <0.05), (0.70 ± 0.03)], compared with those without TSA [(0.27 ± 0.02), (0.22 ± 0.03) Showing a time-dependent relationship. The expression of GFP after Ad-GFP transfection was analyzed by flow cytometry. The transfection efficiency was (1.35 ± 0.11)% in TSA-treated cells, and the transfection efficiencies of TSA were 12, 24 and 48 h after treated with 0.5μmol / L TSA (2.58 ± 0.17)%, (4.96 ± 0.14)% and (6.36 ± 0.15)%, respectively. MTT assay Ad-TK-mediated in vitro cytotoxicity results showed that 0.5μmol / L TSA effect 48h enhanced without TSA 8 times. Conclusion TSA enhances adenovirus transfection efficiency of esophageal cancer cells by upregulating CAR expression.