HPV16E6对不同p53基因型宫颈癌细胞 凋亡及化疗敏感性的影响

目的：探讨HPV16E6对不同p53基因型的宫颈癌细胞化疗药物敏感性的影响。方法：应用AO/EB染色结合荧光显微镜技术及Annexine V-FITC/PI双标法流式细胞仪检测不同浓度DDP干预前后宫颈癌细胞凋亡情况；Western印迹检测DDP干预后对HPV16E6和p53mt蛋白表达的影响；通过差异RT-PCR检测不同浓度DDP干预C33A，C33A-E6，C33A-P和CaSki 4组细胞24和48 h后HPV16E6基因mRNA表达的变化。结果：AO/EB染色和流式细胞术检测的C33A，C33A-E6，C33A-P，CaSki细胞随着DDP干预剂量的增大，凋亡率亦明显增加(P＜0.05)。Western印迹结果示C33A-E6和CaSki细胞中HPV16E6蛋白的表达随着DDP干预剂量的增大及作用时间的延长而逐渐降低甚至难以检测到表达(P＜0.01)，而未干预的对照组C33A-E6和CaSki细胞中HPV16E6的表达量稍有增加。C33A-E6，C33A和C33A-P 3组细胞中均检测到p53mt的表达，但随着DDP干预剂量的增大及作用时间的延长，3组细胞总蛋白中p53mt的表达量均无明显改变(P＞0.05)。差异RT-PCR结果表明，在未予DDP干预的情况下，C33A-E6与CaSki细胞在24 h时HPV16E6 mRNA的表达量无差异，而在48 h时则C33A-E6细胞的HPV16E6 mRNA表达量显著高于CaSki细胞(P＜0.05)，且两组细胞48 h的HPV16E6 mRNA表达均较24 h显著增高(P＜0.05)；在24和48 h两时间段内，随着DDP干预浓度的增加，两组细胞的HPV16E6 mRNA表达量均逐渐减少(P＜0.01)；但在同样浓度的DDP干预时，C33A-E6与CaSki的HPV16E6 mRNA表达量无统计学差异(P＞0.05)。结论：HPV16E6基因对宫颈癌细胞株化疗敏感性的影响与p53的基因型(p53mt/p53wt )无明显关系，其可能通过其他作用机制对C33A细胞的凋亡和化疗敏感性产生作用。

Effect of HPV16E6 on sensitivity of chemotherapy for cervical carcinoma in different p53 genotype cell lines

ObjectiveTo investigate the effect of human papillomavirus types 16E6 on the sensitivity of chemotherapy for cervical carcinoma in different p53 genotype cell lines.MethodsThe apoptosis rates of each group were detected by AO/EB, immunofluorescence and Annexin V/PI stained methods. The expressions of protein HPV16E6 and p53mt after the treatments of different concentration of DDP were detected by Western blot. HPV16E6 mRNA in C33A, C33A-E6, C33A-P, and CaSki cell lines under different DDP treatments were detected by RT-PCR.ResultsAO/EB and Annexin V/PI stained tests showed that the apoptosis rates of C33A, C33A-E6, C33A-P, and CaSki cells were significantly increased when DDP concentration increased. Western blot showed that the HPV16E6 protein could be detected only in C33A-E6 and CaSki cell lines. The expression of HPV16E6 protein in C33A-E6 and CaSki cell lines gradually decreased and was hardly detected with increased dosage of DDP and the prolonged treatment time (P＜0.01), and slightly increased in C33A-E6 and Caski cell lines without the treatment, but there was no significant difference between them (P＞0.05). Protein p53mt persistently expressed in C33A-E6, C33A, and C33A-P cell lines following the increased dosage of DDP and the prolonged treatment time(P＞0.05), while it couldn’t be found in CaSki cell line. RT-PCR showed that without DDP intervention, there was no significant difference of HPV16E6 mRNA in C33A-E6 and CaSki cell lines within 24 h.The HPV16E6 mRNA in C33A-E6 cell line expressed much higher than that in CaSki (P＜0.05), and HPV16E6 mRNA of 2 cell lines expressed much higher at 48 h than at 24 h (P＜0.05).The expression of HPV16E6 mRNA in C33A-E6 and CaSki cell lines gradually decreased with the increased DDP and prolonged treatment time (P＜0.01), while there was no significant difference between C33A-E6 and CaSki cell lines under the same DDP concentration (P＞0.05). ConclusionEffect of HPV16E6 on the sensitivity of chemotherapy for cervical carcinoma cell lines is not markedly related with the different p53  genotype forms(p53mt/p53wt ). HPV16E6 may affect the proliferation and sensitivity of  chemotherapy in C33A cell line through other mechanism.