摘要
目的:研究大肠癌患者粪便c-ki-ras2基因外显子1第12位密码子点突变情况,探讨检测粪便c-ki- ras2基因点突变对筛查大肠癌的价值。方法:提取2005年于广州市第一人民医院就诊的25例大肠癌患者粪便、肿瘤组织和癌旁组织黏膜的DNA,结合限制性内切酶Mva I,用普通聚合酶链反应-限制性片段长度多态性 (PCR-RFLP)及突变富集型聚合酶链反应-限制性片段长度多态性(mutant-enriched PCR-RFLP),分析c-ki-ras2 基因外显子1的12位密码子第一、二位碱基是否存在点突变;用同样方法分析正常对照组17例。特异性分析采用经克隆测序已知突变型(12GGT→AGT)及野生型(12GGT)的c-ki-ras2外显子1片段。取同期住院的166 例胃肠道疾病患者大便潜血试验(FOBT)检查结果作对照。结果:25例大肠癌患者粪便中,突变富集型PCR- RFLP检出7例k-ras基因点突变(28％),其中6例在相应组织中也检出突变,粪便与组织DNA检测结果高度一致(Kappa=0．896,P<0．01)。正常人粪便未检出突变,两组相比差异有显著性(P<0．05)。癌旁黏膜均未发现突变。普通PCR-RFLP只检出其中3例(12％)。c-ki-ras2点突变更多见于左半结肠癌,与结肠其他部位相比差异有显著性(P<0．05)。肠癌患者与其它胃肠道疾病者FOBT阳性率无差异,分别为58％、46％(P> 0．05)。结论:粪便c-ki-ras2基因点突变检测特异性达100%,比FOBT(50．6%)高,但单个基因的检测敏感性较FOBT低(28％ vs 51．2％,P<0．05),粪检基因诊断大肠癌有一定应用前景;突变富集型PCR-RFLP较普通 PCR-RFLP有更高的检出率。
Objective：To investigate the value of stool c-ki-ras2 mutation detection in colorectal cancer screening. Methods： DNA was extracted from stools and tissue samples of 25 colorectal cancer patients and 17 healthy controls. The exon 1 fragments containing eodon 12 were analyzed by one-stcp PCR-RFLP and mutant-enriched PCR-RFLP using Mva Ⅰ. Specificity of the method was determined using the previously known mutant （12GGT→AGT） and wild type samples of c-ki-ras2 exon 1 fragments. Fecal occult blood test （FOBT） was done in 166 patients with gastrointestinal diseases. The results were compared with stool DNA testing. Results： c-ki-ras2 gene was successfully amplified from stool samples. Mutation in the first and second bases of codon 12 was 28% （7/28） by mutant-enriched PCR-RFLP. This result was highly consistent with that of tumor tissue samples with 6 having mutations （ Kappa = 0. 896, P 〈 0.01 ） , Only 3 （ 12% ） of them were found by one-step PCR-RFLP. There was no mutation found in healthy controls. The positive rate of FOBT was not different between colorectal cancers and other gastrointestinal diseases. Conclusions： c-ki-ras2 mutation detcetion in stool samples was more specific than that of FOBT. However, single gene detection had lower sensitivity than FOBT. Stool-basod DNA detection provided a non-invasive screening method for colorectal cancer. Mutant-enriched PCR-RFLP was more sensitive than one-step PCR-RFLP for c-ki-ras2 point mutation analysis.
出处
《广州医学院学报》
2005年第6期40-44,共5页
Academic Journal of Guangzhou Medical College
