宫颈癌癌前病变中人乳头状瘤病毒16整合状态的检测

Detection of integration status of human papillomavirus 16 in cervical precancerous lesions

目的 探讨宫颈癌癌前病变中人乳头状瘤病毒(HPV)16DNA整合入宿主基因组的发生情况。方法 选取 108例细胞学为宫颈癌癌前病变的患者,应用多重聚合酶链反应 (PCR)同时检测液基细胞标本中的HPV16L1、E_2和E_6基因。采用通用引物GP_(5+) /GP_(6+)扩增HPVL1基因保守区的一个长 150bp的片断,以检测HPV的存在。E_2PCR引物目标是HPV整合时最常缺失的E_2开放阅读框(ORF)的特殊区域,E_6引物目标是E6ORF。在游离型中,E_2、E_6拷贝数比例相等;在整合型中,E_2缺失;在游离、整合的混合型中,E2的拷贝数少于E_6。对E_2、E_6的PCR产物凝胶电泳条带的面积灰度值进行半定量分析,从而确定HPV16的整合状态。结果 HPV感染者 62例 (57. 41% );HPV16感染者 32例 ( 29. 63% ),其中 15例 ( 46 .88% )为纯游离型; 13例 ( 40 .62% )为混合型; 4例(12. 50% )为纯整合型。HPV16DNA整合和 /或混合型的比例随宫颈病变级别升高而增加,不同病变之间差异有统计学意义(P<0 .01)。结论 HPV16整合入宿主基因组在部分宫颈上皮内瘤变中即已发生。多重PCR同时检测HPVL1、HPV16E_2、E_6基因以及E_2 /E_6比值,是一种在宫颈细胞学标本中同时检测HPV感染、HPV16感染和HPV16整合状态的简便方法。它可以作为细胞学筛查的一种补充手段。

Objective To investigate the prevalence of integration of human papillomavirus 16 (HPV16) DNA into the host genome in cervical squamous intraepithelial lesions (SIL). Methods Multiplex PCR was used to detect the HPV/HPV16 infection and integration status of HPV16 in the surplus cells from liquid based cytological samples from 108 patients with cervical cancer precursor lesions. Consensus primers GP5+/GP6+ were used to amplify a 150 bp long fragment in the conserved region of the HPV L1 gene so as to detect the presence pf HPV. Scion Image 4 0 electrophoresis image analysis soft was used to calculate the E2/E6 ratio so as to evaluate the episomal and integrated status of HPV16 infection: in episomal form, both targets should be equivalent, and in integrated form, E2 gene would be Absent, while in mixed form of episomal/integrated mixed form, the copy number of E2 would be less than that of E6. Results Sixty two out of the 108 patients (57 41%) had HPV infection. HPV16 were found in 32 of the 108 samples (29 63%). Among the 32 cases HPV16 DNA was exclusively episomal in 15 cases (46 88%), concomitant in 13 cases (40 62%), and integrated in 4 cases (12 50%). The prevalence of integrated and/or concomitant forms of HPV 16 DNA increased with progression of cervical disease. The prevalence of integrated form was 54 55% in the patients of CIN3 type, 50 00% in CIN2 type, 28 07% in CIN1 type, and 11 54% in the inflammatory type with significant differences between any 2 groups (all P <0 01). Conclusions HPV16 integration into the host genome is already present in some of CIN lesions. The multiplex PCR estimation of the HPV L1, HPV16 E2, E6 genes and E2/E6 ratio could be a simple method for detecting HPV/HPV16 infection and its integration status in liquid based residual samples. It would be a helpful complementary tool for cytological screening to identify those patients at high risk of developing high grade squamous intraepithelial lesions and cervical cancer.