【作者】 任惠梅；

【导师】 吕申；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的和背景 胃癌是我国发病率最高的恶性肿瘤之一.临床流行病学调查发现,该病发病具有一定的遗传倾向,并有家族积聚现象,这与欧美人群常见的遗传性非息肉性结肠癌(hereditary nonpolyposiscolorectal cancer, HNPCC)的发病特点相似。目前认为 HNPCC 的发病与错配修复基因突变引起的细胞错配修复功能缺陷有关。错配修复基因突变或功能丧失后,其编码蛋白功能丧失,这就使下游的癌基因和抑癌基因等多种基因的突变因得不到修复而逐渐积累,细胞发生恶变的危险性增高。近年随着饮食习惯的改变,胃癌高发人群的大肠癌发病率有增高趋势。胃癌和大肠癌都是由消化道上皮发生的恶性肿瘤,而消化道上皮细胞的增殖状态比较活跃,我们推测错配修复基因的突变可能也是家系胃癌发生的原因之一,为此,我们对胃癌家系人群的体细胞中错配修复基因 hMLH1 进行了检测,以探讨错配修复基因突变是否在家系胃癌的发生中起作用。 方法 采取辽宁地区 2 个胃癌家系健康人群的 57 份外周血样及正常对照人群的 50 份血样,用小样本血液 DNA 提取试剂盒提取 DNA。以提取的基因组 DNA 为模板,扩增 hMLH1 基因的第 3、8、12、13共 4 个外显子。将 PCR 产物进行热变性后,通过非变性聚丙烯酰胺凝胶电泳,进行单链构象多态性(SSCP)分析。对 SSCP 分析出现异常的 PCR 产物进行测序。 结果 在 2 个胃癌家系健康人群的 57 份外周血样中,hMLH1 基因的突变位点出现在第8和第 12 外显子,第 3 和第 8 外显子没有检测到更多还原

【Abstract】 Background & Objective: Gastric cancer is one of the most commoncancers and a leading cause of cancer death in China with the molecularbiological mechanism of tumorigenesis remaining unclear. Clinical andepidemic studies have shown that gastric cancer occurrence has a tendencyof familial accumulation and several members can be attacked by gastriccancer just in one family, which is similar to hereditary nonpolyposiscolorectal carcinoma (HNPCC). HNPCC is considered being caused byinherited mutation in one of DNA mismatch repair genes, resulting in thedefection of MMR. The MMR system plays an important role in themaintenance of genomic stability. Genomes become rather unstable whenmismatch-repair function is deficient. As errors accumulated, some willinactivate tumor suppress genes or activate cancer promoting genes. In thisway, cells would reach a critical threshold of tumor promoting error. Bothgastric carcinoma and colorectal carcinoma are from glandular epithelia ofdigestion tract, which have a high proliferation rate, and the youngergenerations of these gastric cancer families were found to developcolorectal cancer at a high frequency in recent years because of the changeof food habit. So we estimated that MMR gene mutation or MMRdeficiency may also associate with the coming on of inherited gastriccancer. In order to clarify whether our estimation is correct or not, weselected the hot mutant sequences in hMLH1 gene to detect gene mutationstatus in the family members of patients with gastric cancer. Methods: Peripheral blood samples of 57 members in two gastriccancer families, and 100 control individuals were all from southern districtof Liaoning province, northeast China. Genomic DNA was extracted fromblood using the DNA extraction kit according to the manual. The fragmentsof exon 3, exon 8, exon 12, and exon13 of hMLH1 gene were amplified byPCR followed by SSCP-CE after heat denaturation. The samples suspectedto be mutant were sequenced. Results: Of the 57 blood samples in two gastric cancer families, pointmutations of exon 8 and exon 12 were detected, whereas no mutation wasdetected in exon3 and exon13. Among the samples, 6 cases were detectedmutant in exon8 with mutation frequency being 10.5%. Meanwhile 5 caseswere detected mutant in exon12 with mutation frequency being 8.8%. Themutation frequency difference was not significant of the two exons. Of thetwo kindreds, 1 case was found mutant at exon 8, 4 cases were foundmutant at exon 12 and the total mutation frequency was 25% in one kindred,meanwhile 5 cases were found mutant at exon 8, 1 case was found mutantat exon 12 and the total mutation frequency was 16.2% in another kindred.The total mutation frequency of hMLH1 in the two kindreds was 19.3%.As for the controls, 1 case was found mutant at exon 8, 1 case was foundmutant at exon 12 and the total mutation frequency was 4.0%. pointmutations of exon8 and exon12 were evenly distributed in the two kindredsand the mutation frequency was significantly higher than that the controlsin every kindred. The mutation point of exon 8 was at 219 codon ofhMLH1 gene (A→G), resulting in a substitution of Ile→Val (ATC→GTC),whereas the mutation of exon 12 was at 384 codon of hMLH1 gene (T→A)resulting in a substitution of Asp→Val (GTT→GAT), which were the sameas previously found in hereditary nonpolyposis colorectal carcinoma. Conclusion: 1.hMLH1 gene there are mutant hot points of hMlH1 in exon8 andexon12 in persons of northeast china. 2.The members of gastric cancer families from northern China ave ahigh genetic mutation frequency. 3.The genetic mutation detected in gastric cancer kindreds is similar tothose of hereditary nonpolyposis colorectal carcinoma.更多还原