摘要
背景与目的 p53基因与人类多种肿瘤相关,突变型具有致癌作用,主要分布在外显子5-8。本研究旨在建立高分辨熔解曲线(high resolution melting,HRM)检测非小细胞肺癌(non-small cell lung cancer,NSCLC)患者p53基因突变的方法,探讨p53基因突变的特点及其在NSCLC发生发展中的演变规律。方法采用HRM法检测264例NSCLC患者肿瘤组织和54例癌旁肺组织p53基因外显子5-8的突变,突变样品进一步使用PCR产物直接测序法分析确定突变类型;HRM法检测阳性而PCR产物直接测序法检测阴性的样品,进一步进行亚克隆测序证实。结果 54例对照未发现突变。264例肿瘤组织中,HRM法检出p53基因突变104例,102例得到PCR产物直接测序法证实,突变率为39.4%;95例为点突变,7例为碱基插入和缺失导致的移码突变。p53外显子5-8的突变率分别为11.7%、8%、12.5%和10.6%,差异无统计学意义(P=0.35)。p53基因突变与性别有关,与其它临床病理特征无关。结论 HRM法筛选p53基因突变样品,具有操作简便、快速、敏感、单管避免污染等优点,值得推广。p53基因的突变特点提示,p53基因突变是自发性突变,可能是DNA合成和修复过程中的随机错误所致。
Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting （HRM） assay to detect p53 mutations from patients with non-small cell lung cancer （NSCLC）, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them （P=0.35）. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.
出处
《中国肺癌杂志》
CAS
2011年第10期767-773,共7页
Chinese Journal of Lung Cancer
