宫颈癌组织HPV16早期基因E6相关转录本检测方法的建立及意义

Establishment and its signifi cance of the method for the detection of HPV16 early gene E6-related transcripts in cervical cancer

目的:建立宫颈癌组织HPV16早期基因E6相关转录本检测方法,分析宫颈癌发生各时期的HPV16早期基因E6相关转录的情况。方法:留取HPV16阳性的宫颈肿瘤组织63例,包括8例低度鳞状上皮内瘤变(LSIL)、38例高度鳞状上皮内瘤变(HSIL)和17例宫颈癌,提取RNA,利用含保守序列的RT引物将标本中的mRNA反转录成5’端含保守序列的cDNA,并利用HPV16 E6特异性引物(P1)和保守序列(P0)进行PCR扩增,建立特异性扩增HPV16 E6相关转录本的方法,检测标本中HPV16 E6相关的转录本,通过E6特异性探针进行Southern杂交加以验证。结果:成功建立了HPV16 E6相关转录模式分析的方法。经分析HPV16阳性的LSIL、HSIL及宫颈癌组织中E6转录模式结果发现,不同级别HPV16阳性的宫颈肿瘤组织的转录模式差异显著。随着LSIL向宫颈癌发展,肿瘤组织中HPV16 E6转录本种类增多,且优势转录模式逐渐清晰。结论:成功建立HPV16 E6相关转录模式分析的方法,并发现HPV16早期基因E6相关的转录模式与宫颈肿瘤癌变程度密切相关,其可能参与宫颈癌的发生和发展。

Objective： To establish the method for the detection of HPV16 early gene E6-related transcripts and analyze the HPV16 E6-associated transcription during the development of cervical cancer.Methods： Sixty-three HPV16 positive cervical tissues, including 8 LSIL （low-grade squamous intraepithelial lesion, 38 HSIL （high-grade squamous intraepithelial lesion） and 17 cervical cancer, were collected. The extracted RNAs were then reversed by a RT primer containing a conserved sequences at 5’ end. The cDNAs were then used as the templates to amplify the transcripts using the HPV16 E6 speciifc primers （P1） and the conserved sequence （P0）. After separated by 2.5% agarose gel electrophoresis, the ampliifcation products were detected by Southern blot-ting using an E6-speciifc probe to verify the HPV16 E6-related transcripts.Results： The method for the detection of HPV16 E6-related transcripts was established. Further analysis showed that HPV16 oncogenes transcription patterns were signiifcantly different in the tissues of LSIL, HSIL, and cervical cancer. The types of HPV16 E6-associated transcripts increased during the development from LSIL to cervical cancer.Conclusion：The method for the detection of HPV16 E6-related transcripts is successfully established and the patterns of HPV16 E6-associated transcripts are closely associated with the malignant degree of cervical neoplasm and may be involved in the development of cervical cancer.