人宫颈癌DNA与HPV16型核酸分子杂交的研究

本文以人乳头状瘤病毒(HPV)16型-PBR_(322)重组质粒,对大肠杆菌HB101进行转化实验。经筛选、扩增、酶切、电泳鉴定。获得满意的电泳图谱及kb值,HPV-16Bam HI DNA为7.2kb,其260/280O.D比值为1.9-2。纯度合乎要求,可用以制备探针。以HPV-16DNA7.2kb片段为探针,应用~(32)P(α)-dATP标记进行了点杂交及Southern印迹杂交实验。结果表明38例宫颈癌组织DNA,其点杂交阳性率为76.3％,Southern杂交阳性率为65.7％。点杂交与Southern杂交结果基本符合。上述结果说明了在人宫颈癌组织中,存在着HPV-16相关的基因组,Southern杂交表明其以整合形式存在。说明宫颈癌的发病可能与HPV-16型的感染有密切关系。从分子水平进一步探讨了宫颈癌的病毒病因。

A Study on Nucleic Acid Hybridization with ~(32)P-Labelled HPV 16 DNA Probe in Human Carcinoma of Cervix Uteri

The human papillomavirus (HPV) 16 DNAs were prepared and purified from HPV-PBR_(322) recombinant plasmid. Agarose electrophoresis pattern revealled that HPV_(16) DNA BamH_1 fragrnet was 7.2kb. The 7.2kb fragment of HPV DNA, as probe, was ~(32)PdATP labelled by nick translation method. The DNAs extracted from 56 cases of human uterine cervix tissues were detected homologous sequences with ~(32)P-labelled HPV 16 probe by dot and Southern molecular hybridization. The results of dot hybridization showed that 19 out of 25 cases of cervical carcinoma of uterus were positive and positive rate was 76.3%(29/38). Only 2 in 10 cases of chronic inflammation of cervix uteri and 1 in 8 cases of normal uterine cervix tissues showed positive, respectively. The autoradiography of Southegn hybridization showed that the DNAs of cervical carcinoma of uterus had similar pattern of BamH_1 restriction enzyme and that the presence of 7.2kb hybridizing band was observed. The positive hybridizing band had a genetic sequence homologous to HPV-16 BamH_1 fragment and positive rate was 65.7% (25/38). The positive hybridizing band was not observed in the DNAs of inflammatory and normal uterine cervix tissues. There are different extent of length polymorphism for pSt 1 restriction enzyme in DNA of cervical carcinoma of uterus. And the presence of 2.9, 2.4, 1.8 and 1.5kb hybridizing banA in autoradiograph were observed. The results of dot and Southern molecular hybridization were correspondent. These experiments demonstrated that the genome of the HPV-16 type actually existed in the tissues of the cervical carcinoma of uterus. The close relationship between infection of HPV-16 type and the carcinogenesis of cervical carcinoma of uterus was further proved by molecular hybridization techniques This study may provide some experimental evidence for the elucidation of the mechanism of carcinogenesis and pathogeny of cervical carcinoma of uterus.