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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 p53 gene is associated with a variety of human cancers. Mutant carcinogenesis is mainly found in exons 5-8. The purpose of this study was to establish a method of high resolution melting (HRM) detection of p53 gene mutations in patients with non-small cell lung cancer (NSCLC) and explore the characteristics of p53 gene mutation and its development in NSCLC In the evolution of law. Methods The mutation of exon 5-8 of p53 gene in 264 NSCLC patients and 54 adjacent non-cancerous lung tissues was detected by HRM. The mutant samples were further analyzed by direct sequencing of PCR products to determine the mutation types. HRM assay was positive and PCR products Negative samples were detected by direct sequencing and confirmed by subcloning sequencing. Results No mutation was found in 54 controls. Among 264 tumor tissues, 104 cases of p53 gene mutations were detected by HRM and 102 cases were confirmed by direct sequencing of PCR products. The mutation rate was 39.4%. 95 cases were point mutations and 7 cases were frame shift caused by base insertion and deletion mutation. The mutation rates of p53 exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively, with no significant difference (P = 0.35). The mutation of p53 gene is related to gender and has nothing to do with other clinicopathological features. Conclusion The method of HRM screening of p53 gene mutation samples is simple, rapid, sensitive and simple to avoid contamination. It is worth promoting. The mutation characteristics of p53 gene suggest that the mutation of p53 gene is a spontaneous mutation, which may be due to random errors in DNA synthesis and repair process.