耐药喉癌Hep-2/VCR细胞ATP7B表达与顺铂耐药的相关性

The relationship between ATP7B expression in drug-resistant laryngeal carcinoma cell line Hep-2/VCR and cisplatin resistance

目的检测copper transporting P-type adenosine triphosphatase(ATP7B)在人喉癌耐药细胞株中的表达,并研究其与喉癌细胞顺铂耐药的关系。方法人喉鳞癌Hep-2细胞和Hep-2/长春新碱(vincristine,VCR)耐药细胞分别给予顺铂作用24小时和48小时,应用细胞增殖/毒性检查试剂盒(cell counting kit-8,CCK8)细胞活力试验、末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定法[terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling,TUNEL]细胞凋亡实验分别检测细胞增殖和凋亡。分别用免疫荧光染色和实时RT-PCR检测ATP7B蛋白和mRNA在细胞中的表达。结果 50μmol/L和100μmol/L的顺铂作用24小时后,Hep-2细胞活力下降为71.0%,而Hep-2/VCR细胞活力无下降,差异有统计学意义(P<0.01)。50μmol/L顺铂能够诱导Hep-2和Hep-2/VCR细胞表达ATP7B。Hep-2细胞在作用后3小时表达增强,6小时达到峰值,12小时下调(P<0.05);而Hep-2/VCR细胞在3小时表达增强,6小时达到峰值(P<0.05),持续高表达24小时无明显变化。结论喉癌耐药细胞Hep-2/VCR的ATP7B蛋白的高表达与喉癌细胞的顺铂耐药有关。

Objective To examine the expression of copper transporting P-type adenosine triphosphatase（ ATP7B）in drug-resistant human laryngeal carcinoma cells,and to study its relation with cisplatin resistance. Methods Human squamous cell carcinoma cell line Hep-2 and Hep-2 / vincristine（ VCR） were treated with cisplatin for 24 h and 48 h. Then the differences between Hep-2 and Hep-2 / VCR on cisplatin resistance were examined using cell counting kit-8（ CCK8）test and terminal dexynucleotidyl transferase（ Td T）-mediated d UTP nick end labeling（ TUNEL） assay. The expression level of ATP7 B protein and mRNA in both cell lines was analyzed by immunofluorescence staining and RT-PCR. Results After being treated with 50 μmol / L and 100 μmol / L cisplatin for 24 h,the survival rate of Hep-2 cell was declined to 71. 0%,yet no significant alterations of Hep-2 / VCR survival was detected（ P〈0. 01）. Moreover,50 μmol / L cisplatin induced the expression of ATP7 B in both the Hep-2 and Hep-2 / VCR cell lines. In the Hep-2 cell line,the ATP7 B expression started to elevate at around 3 h post cisplatin treatment,which peaked at 6 h and reduced at 12 h（ P〈0. 05）. In the Hep-2 / VCR cell line,the ATP7 B expression also elevated at around 3 h and peaked at 6 h,but persisted at the peak level through 24 h post treatment（ P〈0. 05）. Conclusion High expression of ATP7 B in drug-resistant laryngeal carcinoma cell line Hep-2 / VCR is related with cisplatin resistance of laryngeal squamous cell carcinoma.