应用PCR和单克隆抗体方法从人粪便及肿瘤组织中检测大肠癌基因突变的研究

目的：检测大肠癌病人粪便中脱落细胞与肿瘤组织细胞的DNA，对比两者K—ras基因序列和突变位点的变化，为临床非介入性的、简便的大肠癌诊断提供理论和实验依据。方法：分离和提纯大肠癌组织及粪便中脱落细胞的DNA，用PCR(Polymerase chain reaction)法扩增K—ras基因，克隆K-ras基因PCR产物并进行DNA序列的测定。免疫组化检测大肠癌相应抗原的表达：采用ELISA法，用大肠癌单克隆抗体对大便样品进行检测。结果：同一病人的大肠癌组织细胞与粪便中的脱落细胞的K—ras基因的序列完全一致，并检测到1例K—ras基因点突变。K-ras基因突变与否与大肠癌病理类型和大肠癌单克隆抗体检测大便样品中脱落细胞的癌抗原表达水平有一定的相关性。结论：以大肠癌分子发病机理为基础的非介入性检测粪便中K-ras突变是可行的，有望成为正常人群大肠癌筛查以及诊断的新方法之一，可以为大肠癌的早期发现及监测大肠癌的发生、发展和预后提供理论和实验依据。

Detection of gene mutations with PCR and anti-colon cancer monoclonal antibody in tissue and stool of patients with colorectal cancer

AIM: To detect sequence and mutation of K-ras oncogene in tissue and stool DNA of patients with colorectal cancer in order to provide a method of noninvasive and simple colorectal cancer diagnosis. METHODS: DNA was separated and purified from colorectal cancer tissue or stool of patient with colorectal cancer, then the K-ras gene was amplified by PCR and PCR products were cloned, the K-ras gene was sequenced, and the mutation was identified. The expression of color/colorectal cancer antigen was inspected by immunohistochemical technique. Stool sample of patient with colorectal cancer was detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: K-ras gene sequence of the stool was completely same as that of the tissue of the patient;K-ras mutation was detected in one case. There was relativity between the mutation of K-ras gene and the pathology type of colorectal cancer and the expression level of colorectal cancer antigen in stool sample. CONCLUSION: It is feasible that colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of the disease. Detecting K-ras gene mutations of stool DNA can provide bases for the screening, early detection, and prognosis to patients with colorectal cancer.