宫颈癌p16基因甲基化研究新方法

Detection of p16 Promoter Hypermethylation in Cervical Carcinoma

目的建立SYBR GreenⅠ实时荧光定量PCR检测宫颈癌p16基因甲基化的方法,以期达到早期诊断宫颈癌。方法正常基因组DNA经修饰后进行PCR,构建含有DNA甲基化β-actin的重组质粒,克隆阳性质粒作为标准品用SYBR GreenⅠ实时荧光定量PCR进行检测,建立检测的标准曲线。利用建立好的方法进行检测临床60例宫颈癌组织中p16基因启动子区域5’CpG岛甲基化状态。结果结果显示正常对照组织及癌旁p16基因无甲基化,60例宫颈癌标本的甲基化率为28.33%(17/60)。结论SYBR GreenⅠ实时荧光定量PCR用于p16基因甲基化检测为宫颈癌的早期诊断提供了一种有效的方法。

Objective To establish the SYBR Green Ⅰ fluorescent quantitative PCR method for accurate detection of DNA methylamine of the p16 gene in cervical careinoma（CC） ,so to diagnose CC in the early stage. Methods The 133bp bisulfatetreated ACTB gene was amplified and cloned into T vector which was transformed into E. coli DH5α. The positive recombinant plasmid was used as quantitative template to generate standard curve. We used SYBR Green Ⅰ fluorescent quantitative PCR determine the methylation status of 5＇ CpG islands of the p16 gene in 60 sample from patients with CC. Results The results showed absence of methylation of the p16 gene in the control and adjacent tissues of CC. Hypermethylation of the p16 gene was detected in 21.67% （17/60） of the tumor tissues. Detection of aberrant methylation of p16 gene may be useful for diagnosis of CC. Conclusions The SYBR GreenⅠ fluorescent quantitative PCcerivical careinoma;pl6 gene;methylationR method is a rapid sensitive method for detection of DNA methylamine of the p16 gene in the early stage of cervical carcinoma.