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目的 了解变性梯度凝胶电泳 (DGGE)和自动DNA序列分析方法检测肿瘤基因变异及 p5 3基因、p5 3蛋白在大肠癌发生、转移过程中的动态变化。方法 以DGGE及自动DNA序列分析法检测 41例大肠癌原发病灶和肝转移灶p5 3外显子 5～ 11的基因突变。以免疫组织化学方法检测 p5 3蛋白表达。结果 41例中 2 4例有 p5 3基因突变 (62 % ) ,其中 6例仅在肝转移灶发现p5 3基因突变 ,其余均为原发灶、转移灶有一致性的突变。另有 3例原发灶即有p5 3基因突变的病人 ,在转移灶除保留原有突变外 ,还出现新增加的突变。在原发、转移灶同时有突变的 16例中 ,14例呈现突变的 p5 3碱基峰和正常峰之比在肝转移灶明显高于大肠癌原发灶 (P <0 .0 0 1)。p5 3免疫组织化学染色结果和DGGE、DNA序列分析结果高度一致。但在基因分析呈无义突变的癌灶 ,免疫组织化学显示 p5 3蛋白的过度表达。结论 在大肠癌肝转移过程中 ,p5 3基因突变主要开始于肠癌原发灶 ,并被保持于转移至肝脏的癌细胞内 ,在转移灶其含量或含突变型 p5 3癌细胞量明显增加。p5 3基因突变与 p5 3蛋白过度表达呈正相关关系。DGGE和自动序列分析法只有在于免疫组织化学方法结合使用时 ,才能对基因改变作出最全面的判断
Objective To understand the dynamic changes of tumor gene mutations and p53 and p53 proteins during colorectal carcinogenesis and metastasis by denaturing gradient gel electrophoresis (DGGE) and automated DNA sequencing. Methods DGGE and automated DNA sequence analysis were used to detect 41 cases of colorectal cancer and liver metastasis p53 exon 5-11 gene mutations. The expression of p53 protein was detected by immunohistochemistry. Results Of the 41 cases, 24 cases had p53 mutation (62%), of which 6 cases had p53 mutations only in liver metastases. The rest were all primary tumors and metastases had consistent mutations. Another 3 cases of primary lesions were patients with p53 mutations. In addition to the original mutations, there were also new mutations. Among the 16 cases with primary and metastatic lesions, the proportion of p53 base peaks and normal peaks showing mutations in 14 cases was significantly higher in the liver metastases than in the primary colorectal tumors (P < 0.01). The results of p53 immunohistochemical staining were highly consistent with the results of DGGE and DNA sequence analysis. However, in the case of cancerous lesions with nonsense mutations in gene analysis, immunohistochemistry showed overexpression of p53 protein. Conclusion In the process of liver metastasis of colorectal cancer, p53 mutations mainly originate from the primary foci of colorectal cancer and are retained in the cancer cells that metastasize to the liver. The content of p53 in metastatic foci or the amount of mutant p53 cancer cells increases significantly. . The p53 gene mutation was positively correlated with p53 protein overexpression. DGGE and automated sequence analysis can only make the most comprehensive assessment of genetic changes when combined with immunohistochemical methods.