摘要
目的建立甲基化敏感的限制性内切酶结合半巢式降落PCR方法,研究原发性肝癌P 16基因启动子区甲基化状态。方法针对P 16基因启动子区富含CpG的序列,设计3条引物,进行半巢式降落PCR扩增,检测原发性肝癌P 16基因启动子区甲基化状态;并将P 16基因启动子区340 bp片段克隆到pM D 18-T载体,E.coli JM 109扩增此质粒后经CpG甲基化酶M.S ssⅠ处理,再用Hp aⅡ酶切验证获得P 16基因启动子区甲基化阳性的质粒P 16Pm+并进行特异性和灵敏度实验。以此甲基化敏感的限制性内切酶结合半巢式降落PCR方法检测40例人原发性肝癌和3例正常人肝组织DNA样品的P 16基因启动子区甲基化状态。结果所采用的Hp aⅡ酶切后半巢式PCR方法的特异性强,灵敏度达100 fg;对40例人原发性肝癌组织DNA样品P 16基因的启动子区甲基化状态检测显示甲基化阳性率为30%(12/40),3例正常人肝组织均为阴性(0/3)。结论P 16抑癌基因启动子区甲基化可能与肝癌发生发展有关。本实验方法简便、成本低廉,并有高度的特异性与灵敏度,在大规模检查中有实际应用价值。
Objective To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma. Methods According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I , the methylated plasmid was named P16Pm^＋. This P16Pm^＋ is validated by digestion of Hpa Ⅱ and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue. Results It was confirmed that the specificity and sensitivity of this method are solid and reliable （100 fg）. It was found that 12/40 （30%） of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none （0/3） of the normal tissues was methylated in the promoter region in P16 gene. Conclusion Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2007年第1期53-56,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号39670702)资助
