变性凝胶电泳和自动DNA序列分析检测结直肠肝转移癌p53基因突变的研究

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p53基因、p53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p53外显子5～11的基因突变。以免疫组织化学方法检测p53蛋白表达。结果　41例中24例有p53基因突变(62%),其中6例仅在肝转移灶发现p53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p53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p53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＜0.001)。p53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p53蛋白的过度表达。结论　在大肠癌肝转移过程中，p53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p53癌细胞量明显增加。p53基因突变与p53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。

Study the detection of p53 gene mutation on colorectal cancer and liver meta stasis by denaturing gradient gel electrophoresis and automated sequencing

Objective　To study the detection of gene mutation in tumor by denaturing gradient gel electrophoresis (DGGE) and automate d sequencing and the change of p53 gene and p53 protein during the process of de velop ment and metastasis of colorectal carcinoma. Methods　p53 gene (exon 5～11) mutat ion was examined by DGGE and automated sequencing. p53 protein expression was de tected by immunohistochemistry using the monoclonal antibody DO-7. R esults　p53 gene mutations were found in exons 5 through 9 at 24 of 4 1 pat ients (62%). Among them, 6 patients had a mutation in the liver metastasis but n ot in corresponding colorectal tumors. The rest showed consistent mutations on b oth of primary colorectal and hepatic metastatic lesions. There was no mutation found alone in primary colorectal tumor. In addition, additional mutation was fo und in the metastatic lesions of three cases. A highly concordance between the p resence of p53 mutations and p53 protein accumulation was observed (r=0.97 , P＜0.001), but a negative immunohistochemical staining was showed in all patients with nonsense mutations. In 4 of 6 colorectal lesions, which showe d no mutation, p53 overexpression was detected immunohistochemically. Conclusion　p53 mutations, in the patients with colorectal carcinom as fol lowed by hepatic metastases, mostly started in the primary colorectal lesion and then was kept and brought into hepatic organ. Their mutant character does not c hange during metastatic process. Mutated p53 gene amount and the amount of tumor cells containing p53 mutations were increased in hepatic metastatic lesion. P53 mutation was positively correlated with overexpression of p53 protein. Combined use of DGGE or automated sequencing and immunohistochemistry can fully evaluate the gene alterations.