【作者】 欧阳刚；

【导师】 张惟杰； 李劲；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 食管鳞癌(Esophageal Squamous Cell Carcinoma,ESCC)是世界十大恶性肿瘤之一,中国是世界上食管癌发病率和死亡率最高的国家,每年约有25万新诊断的食管癌病例,占全世界食管癌病例数的一半。食管癌是我国危害严重的重大疾病之一,它严重威胁着人民的健康。本研究在利用现已报道的对食管癌进行基因芯片扫描的结果的基础上,收集和整理与食管癌相关的基因及其SNP信息,利用生物信息学的方法分析其中两个基因(MMP3和KDM4C)及其包含的三个SNP,取得以下研究结果:(1)Chelex100法提取全血基因组DNA方法的建立本研究利用Chelex-100法制备PCR模板并应用于分析人类基因多态性, MMP3外显子2 Lys45Glu(SNP编号: rs679620)PCR特异扩增片段长度为748bp,通过对湖北食管鳞癌散发人群组,河南高发易感移民人群组,河南高发区食管鳞癌人群组和湖北及河南正常人群对照组大量样本进行PCR-RFLP分析,由于人类等位基因的存在,样本中特异扩增的PCR产物经TaqⅠ完全酶切后可产生三种结果:①三种片段748bp、432bp、316bp,即杂合型半数酶切;②两种片段432bp、316bp,即纯和型完全酶切;③一种片段748bp,即纯和型不能酶切。其中,随机抽样上述三种PCR产物的10%进行DNA测序验证,其结果与酶切结果相符合。因此,根据酶切结果产生片段可判断样品的基因型,即在所研究的SNP位点处等位基因有GA、GG、AA三种分型。表明此法适合于大规模的分子流行病学调查及群体遗传学研究。(2)人外周血(血凝块)基因组DNA提取方法的建立有些收集到的样本是临床检验和流行病学调查所采集的血凝块,利用蛋白酶K法和碘化钾法从血凝块中提取的DNA均可获得满意的结果。本研究从血凝块中提取的DNA为模板用于金属基质蛋白酶3(mmp3)基因PCR扩增获得满意效果,凝胶电泳背景清晰,无非特异扩增带。说明从血凝块中提取的DNA完全可以用于常规的PCR分析。(3)DNA聚丙烯酰胺凝胶银染方法的建立聚丙烯酰胺凝胶电泳由于其使用样品量少、不易扩散,具有较高的分辨率且易于观察,目前已经广泛应用于STR、SNP等分子标记研究和遗传图谱构建等方面。如果存在的多态性仅表现为一个或几个bp的差异时,一般都需要采用变性或非变性的聚丙烯酰胺凝胶电泳技术来检测序列的多态性。常规的聚丙烯酰胺凝胶的银染耗时较长,操作繁琐,不宜控制,且染色背景较高,在大批量实验时,费时费力,严重影响实验进展。本研究应用PCR-RFLP和DNA的非变性聚丙烯酰胺凝胶电泳银染法分析人的单核苷酸多态性及SNP与疾病的关联性的工作中,取得了比较满意的效果。(4)KDM4C基因Arg893Trp和MMP3基因Arg248Trp与食管鳞癌遗传易感的关联分析KDM4C外显子19的SNP号为rs41304753(Arg893Trp)PCR产物的片段长度为207bp,经限制性内切酶BciT130Ⅰ(EcoRⅡ)酶切后均产生两个片段分别为177bp,30bp,即基因型为T/T,在已检测的近百份中国人群样本中并未发现C/C和T/C基因型;MMP3基因外显子5的SNP号为rs41357345(Arg248Trp)PCR产物的片段长度为314bp,经限制性内切酶HinfⅠ酶切后均产生两个片段分别为237bp,77bp,即基因型为C/C,在已检测的近百份中国人群样本中并未发现T/T和C/T这两种基因型。因此,这两个SNP位点的基因型分别为T/T(207bp),C/C(314bp),即在本研究人群中只存在一种基因型。因此这两个位点可能与疾病不相关。值得注意的是,这两个SNP位点在NCBI数据库中均为未见报道中国人群的分型比例,所以根据本研究的结果,推测可能中国人群在这两个SNP位点处只存在一种基因型。当然,这一结果需要更大规模的实验证实,并由利用直接测序法验证其结果的可靠性。(5)MMP3 Lys45Glu单核苷酸多态性与食管鳞癌遗传易感性的关联分析本研究旨在探讨基质金属蛋白酶3(Matrix metalloproteinase 3,MMP3)基因外显子2中Lys45Glu(SNP编号: rs679620)单核苷酸多态性与食管癌遗传易感的关系,应用Chelex-100法提取DNA模板和PCR-RFLP方法对1127例包括湖北省、河南省食管鳞癌患者和两地正常人对照及河南移民样本的该SNP进行基因分型,研究表明,河南高发易感移民人群组与湖北正常人群对照组在GG、GA、AA三种基因型频率上均有极显著差异(P<0.01);河南高发区鳞癌组与湖北正常人群对照组在A/A基因型频率上有显著差异(P<0.05);湖北人与河南人在GG和AA基因型频率上差异极显著(P<0.01)。结果提示,MMP3基因外显子2中Lys45Glu单核苷酸多态性与食管鳞癌的遗传易感性似有明显相关。更多还原

【Abstract】 Esophageal squamous cell carcinoma is one of the top ten malignant diseases in the world. Esophageal cancer has the highest incidence and rate of mortality in China. About 250,000 new cases , representing half of the number of cases in the world , are diagnosed each year. Esophageal cancer, a serious threat to the health of the Chinese people, is one of the major diseases in China.Based on the results of this reseach collect and arrange genes which are related with esophageal cancer and relative SNP information by using the method of bioinformatics to analyze the two genes (MMP3 and KDM4C ), as well as three SNP contained in these two genes.The results are the following:1.The establishment of method used to extract genomic DNA in whole blood by using Chelex100By using method of Chelex-100 preparated PCR template , this study analyzea the human gene polymorphism, the SNP of Lys45Glu in exon 2 of MMP3 (SNP ID: rs679620) , which PCR specific amplified fragment is 748bp. Through comparing esophageal squamous cell carcinoma of Hubei Group, Hubei control, Henan migrants group, esophageal squamous cell carcinoma of Henan group and Henan control, we use PCR-RFLP as the chief method to analyse vast number of population samples. Due to the existence of the human alleles, PCR product digested by TaqⅠcan produce three results:①three fragments 748bp, 432bp, 316bp;②two fragments 432bp, 316bp;③a 748bp fragment. In Figure 4.7, respectively, the result displayes by three lanes--2,4,6 bands. Choosing 10% PCR products from the three sample to sequence , the results are in line with the results of digestion. Thus, according to the results from restriction fragments , the genotype of samples, GA, GG, AA three types in SNP allele gene studies by us, can be determined. Consquently ,this shows the method is suitable for large-scale survey of molecular epidemiology and population genetics research. At present, our laboratory is engaged in diseases associated with the SNP analysis and genotyping of large-scale research, the need for large-scale extraction of DNA, so the establishment of a stable economy for large-scale method is very important. 2. The establishment of method extract human blood clot genomic DNASome samples are blood clots collected from the clinical examination and epidemiological investigation. To use proteinase K and potassium extract DNA from blood clots may get satisfactory results. In this study, extracting DNA from the blood clot and using it as the template for metal matrix 3 (mmp3) gene PCR amplification can get satisfied results with a clear gel background and no more than specific amplification. DNA extracted from blood clots can be used for conventional PCR analysis.3.The establishment DNA polyacrylamide gel by silver staining methodBescause Polyacrylamide gel electrophoresis needs a little sample and during the process the sample is difficult to spread,at the same time,the results has high resolution and can be observed easily. It has been used widely in STR,SNP and other molecular markers,besides building genetic mapping studies ect..If there is only one or a few bases differences at the performance of the polymorphism,then the denatured or non-denatured polyacrylamide gel electrophoresis will be generally adopted to detect sequence polymorphisms. Conventional polyacrylamide gel by silver staining is time-comsuming, uncontrolled ,operated complicatedly and having a higher background staining, experiments in high-volume needs much more time and more labor,which seriously effects the progress. In this study, PCR-RFLP and DNA non-denaturing polyacrylamide gel electrophoresis by silver staining are used to analyze human single nucleotide polymorphisms and the relevance between SNP and disease,we have got satisfactory results.4. The correlation analysis of GASC1 gene Arg893Trp and MMP3 gene Arg248Trp with genetic susceptibility of esophageal squamous cell carcinomaThe SNP number of KDM4C exon 19 is rs41304753 (Arg893Trp),its PCR product’s fragment length is 207bp, which can generated two fragments of 177bp and 30bp after been digested by restriction endonuclease BciT130Ⅰ(EcoRⅡ),the two fregments’genotype is T/T. C/C and T/C are not detected in the Chinese population of nearly a hundred samples.The SNP number of MMP3 gene exon 5 is rs41357345 (Arg248Trp) ,its PCR product’s fragment length is 314bp,which can generated two fragments of 237bp and 77bp after been digested by restriction endonuclease HinfⅠ, the two fregments’genotype is C/C. T/T and C/T are not detected in the Chinese population of nearly a hundred samples. Therefore,the genotype of these two SNP loci is T/T (207bp), C/C (314bp),in other words, there is only one genotype existing in the study population.So these two loci may be not related to associate with disease.It is worth noting that these two SNP loci in the NCBI database are not reported in Chinese population the proportion of sub-type, so through the results of this study,we can speculate that Chinese people in these two SNP loci existing only a genotype. Of course, this result need further proving, especially using direct sequencing methods, we can prove this result directly and precisely.5. The association of the SNP(Lys45Glu) in exon 2 of the MMP3 gene with ESCC susceptibilityTo study the association of ESCC genetic susceptibility with the SNP rs679620 (-Lys45Glu-) in exon 2 of the MMP3 gene, the MMP3 SNP was genotyped by PCR-RFLP analysis in total 1127 samples, in which there are 317 ESCC cases (227 in Henan and 90 in Hubei) and 810 controls with 432 Hubei population and 197 Henan population plus 181 Henan emigrants. The statistic data show: G/G,G/A and AA genotype frequencies of SNP rs679620 were significant difference in groups of emigrants from the high incidence region of Henan with the healthy population in Hubei (P<0.01). Compared with AA genotype frequencies, the ESCC cases in Henan were significantly different from the healthy controls in Hubei (P<0.05). The difference of GG and AA genetype frequencies between Hubei and Henan populations is significant (P<0.01). This study suggests that the SNP rs679620 (-Lys45Glu-) in exon 2 of the MMP3 gene might be associated with ESCC susceptibility.更多还原