实时荧光定量PCR法检测人乳头状瘤病毒的实验研究

Detection of human papilloma virus 16/18 by the quantitative fluorescent PCR

目的通过研究病变宫颈中人乳头状瘤病毒（HPV）16／18型的表达，探讨HPV16／18型病毒感染与宫颈病变发生发展之间的关系。方法结合病理切片诊断，以免疫组化作对照。运用实时荧光定量PCR技术检测病变宫颈中HPV16／18型DNA拷贝数，以及HPV16／18型E7基因mRNA表达量。结果慢性宫颈炎患者中HPV16／18型感染率低（7．4％）。宫颈管上皮内瘤样变（CIN）组HPV16型感染率较高为69．6％，宫颈癌患者中为72．7％。HPV16型DNA的拷贝数在宫颈上皮内瘤样变患者中与病理分级没有明显的相关性。但在宫颈癌患者中，病毒DNA的拷贝数明显升高，二者差异明显。CIN轻度（Ⅰ）、中度（Ⅱ）、高度（Ⅲ）组和宫颈癌患者中，HPV16 E7基因的表达率分别为0、37．5％、42．9％、63．6％。统计学分析表明，HPV16 E7 mRNA的拷贝数与病情呈明显的正相关性。结论感染者中主要以HPV16型为主，HPV18型较少。宫颈癌患者中HPV16 DNA拷贝数明显高于CINⅡ、Ⅲ组，HPV16型E7 mRNA在宫颈癌中表达率及表达量明显增加并与宫颈癌变呈正相关。实时荧光定量PCR适合临床宫颈病变病毒的筛查与检测。

Objective To study the relation between the HPV6/18 virus infection and the development of pathological changes of cervix. Methods The number of HPV16/18 DNA copies and the expression rate of HPV16/18 E7 mRNA in the pathological cervix were examined by the quantitative fluorescent PCR combined with pathological diagnosis and immunohistochemistry staining. Results The HPV16 infection rates in chronic cervicitis group were much lower （7.4%） than that in the cervical intraepithelial neoplasia （CIN） groups and the cervical cancer group （69.6% and 72.7% ）, respectively. Statistical analysis showed that the difference of HPV16 DNA copies was not significant between the chronic cervicitis group and CIN groups. In contrast to the above mentioned result, the number of HPV DNA copies between the CIN groups and the cervical cancer group was significantly different. The HPV16 E7 gene expression rates in CIN Ⅰ , Ⅱ , Ⅲ and cervical cancer groups were 0, 37.5% , 42.9%, 63.6% , respectively. Conclusion Infection with HPV16 is more common than that with HPV18. The number of HPV16 DNA copies in cervical cancer tissues is markedly higher than that in CIN Ⅱ , Ⅲ groups. The HPV16 E7 mRNA expression is significantly increased in the cervical cancer, and it is more closely correlated to this pathological changes. The quantitative fluorescent PCR can be used to reflect the activity of HPV, and it is a useful method for the screening examination of HPV and for the early diagnosis and treatment of cervical caner.