【作者】 万顺梅；

【导师】 房殿春；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 前言胃癌是我国最常见的恶性肿瘤之一,我国胃癌发病率也是全球最高的国家之一。胃癌的发生发展是一个多因素多阶段多基因参与的过程,其发生涉及到多种基因的异常改变,归因于环境与遗传因素相互作用的结果,其中幽门螺杆菌感染(Helicobacter pylori,Hp)是胃癌发生的主要危险因素,被WHO列为第一类致癌原。单核苷酸多态性(Single nucleotide polymorphisms,SNP)是指基因组中单个碱基的变异而导致的核苷酸序列多态性,是继微卫星之后的第3代遗传性标志。对SNPs数据库进行大范围评估结果显示,SNPs能够为研究多基因复杂疾病及个体间、不同群体或个体对肿瘤患病风险以及对药物反应的不同提供新的方法。已有对端粒相关基因(TERT、TERC、TERF1、TERF2、TINF2、TERF2IP、POT1和TNKS)共同的SNPs研究认为,这些变异对研究遗传相关疾病如癌症、再生障碍性贫血、自身免疫性疾病及染色体和端粒酶的异常都有重要意义。人端粒保护蛋白1(human protection of telomeres 1, hPOT1)基因是一种端粒单链DNA的结合蛋白,是端粒家族的成员之一,广泛存在于真核细胞中,被称为真核细胞看家基因。其主要作用是维护端粒的功能,调控端粒的长度,保护染色体的稳定。基因组不稳定伴随着端粒功能的异常,端粒功能异常可能促进胃癌的发生。hTERT( Human telomerase reverse transcriptase,hTERT)是端粒酶逆转录酶基因,是端粒酶的调节亚单位,依赖于端粒酶的活性而发挥调节端粒的作用,它能诱发细胞无限增殖及参与肿瘤发生,在肿瘤发生中起着至关重要的作用。端粒酶具有很高的肿瘤特异性,在85%-90%的肿瘤细胞中可以检测到端粒酶活性,而与肿瘤相邻的正常组织或良性病变几乎没有端粒酶活性。目的本研究检测了甘肃西部汉族人群胃癌hPOT1IVS13-98G/T及hTERT Ex2-659A/G位点单核苷酸多态性、hPOT1、hTERT蛋白表达、幽门螺杆菌感染和微卫星不稳,探讨hPOT1和hTERT基因单核苷酸多态性与胃癌患者发病风险的关系,并探讨以上二种基因与幽门螺杆菌感染和微卫星不稳的关系。方法168例胃癌患者来自于甘肃西部三家医院外科手术切除胃的患者,所有病例均经组织病理学确诊,术前未行放疗和化疗。取癌组织及手术切缘的正常组织(距癌5cm以上),立即置于-70℃的冰箱冻存备用。156例正常对照为同期在三家医院查体并行胃镜检查,在胃镜下均排除慢性胃炎、肠化、增生、溃疡和癌变。年龄性别与胃癌组匹配。两组均征得患者的同意,并签署知情同意书。胃癌临床分期按2003年国际抗癌联盟(UICC)TNM标准分期,组织病理按2000年WHO胃癌新的分类标准。全部检查对象均抽静脉血5ml,分离淋巴细胞,-70℃冰箱冻存备用。然后采用常规酚/氯仿/异戊醇抽取法提取DNA。采用聚合酶链反应和限制性片段长度多态性技术(PCR-RFLP)进行SNP基因型分析,采用聚合酶链反应和单链构象多态性技术(PCR-SSCP)检测微卫星不稳。取部分组织固定、石蜡包埋、组织切片HE染色病理证实,免疫组化检测蛋白表达,PCR检测尿素酶基因结合Warthin-Starry银染确诊幽门螺杆菌的感染。结果研究显示,胃癌组hPOT1IVS13-98 T/G位点TT、GG、GT基因型的频率分别为36.90%、41.67%、21.43%, T、G等位基因频率分别为57.74%、42.26%;对照组TT、GT、GG基因型的频率分别为24.36%、51.92%、23.72%,T、G等位基因频率分别为49.68%、50.32%。两组T、G等位基因型频率比较无显著性差异(χ2=3.5853,P= 0.0583)。以T/T基因型作为参照基因型,G/T和G/G两种基因型OR值分别为0.439(95%CI: 0.251-0.767,P=0.004)、0.514(95%CI: 0.264-0.999,P=0.050)。胃癌组hTERTEx2-659A/G位点GG、AG、AA型的频率分别为41.88%、47.5%、10.63%,G、A等位基因频率分别为65.62%、34.38%,对照组GG、AG、AA基因型的频率分别为28.29%、56.58%、15.13%,G、A等位基因的频率为56.58%、43.42%,两组G、A等位基因型频率比较有显著性差异(χ2=5.3734,P=0.0204)。以G/G基因型相比,A/G和A/A两种基因型OR值分别为0.519(95%CI:0.310-0.870,P=0.013)、0.550(95%CI: 0.255- 1.188,P=0.128)。hPOT1蛋白表达的阳性率在胃癌、异型增生、肠化生、正常胃粘膜分别为:91.33%、80.00%、55.00%、28.00%,在胃癌、异型增生、肠化生与正常胃粘膜组织相比差异有显著性( P<0.01)。hPOT1蛋白的表达在浸润较深、分化差的、Ⅲ/Ⅳ期胃癌中显著增高( P <0.05)。hTERT蛋白的阳性率在胃癌、异型增生、肠化生、正常胃粘膜分别为:76.67%、60.00%、25.00%、0.00%,胃癌中hTERT蛋白的表达与胃癌的浸润深度、淋巴结转移、胃癌分期相比差异无显著性(P>0.05),但在低分化胃癌表达的阳性率显著高于高中分化胃癌(P<0.05)。在胃癌、异型增生、肠化生、正常胃粘膜H.pylori的检出率分别为:54.00%、70.00%、55.00%、20.00%。在hPOT1IVS13-98 G/T三种基因型中,H.pylori检出率,GG型为51.52%,GT型为45.16%,TT型为65.45%,三型之间比较差异无统计学意义(χ2 =4.937,P=0.085),提示胃癌患者hPOT1IVS13-98G/T多态性与H.pylori感染无关;在hERTEx2-659A/G三种基因型中,AA型为66.67%,AG型为52.86%,GG型为50.88%,三型之间比较差异无统计学意义(χ2=1.215,P=0.545),提示胃癌组hERTEx2-659A/G多态性与H.pylori感染无关。H.pylori阳性患者中hPOT1蛋白的表达率为96.30%, H.pylori阴性患者中hPOT1蛋白的表达率为85.51%,二者之间比较有显著性差异(P<0.05)。H.pylori阳性患者中hTERT蛋白的表达率为85.19%, H.pylori阴性患者中hTERT蛋白的表达率为71.01%,二者之间比较有显著性差异(P<0.05)。对75例胃癌进行MSI检测,75例胃癌中两个位点MSI阳性29例,检出率为38.67%,其中BAT26位点MSI阳性率为12.00%;D5S346位点MSI阳性率为26.67%。MSI与患者年龄、性别、组织学类型及浸润深度无关(P>0.05),在无淋巴结转移及I、II期胃癌中检出率显著高于有淋巴结转移和III、IV期胃癌(P<0.05)。胃癌组hPOT1IVS13-98 G/T位点TT、GT、GG基因型在MSI组和MSS组中的频率比较差异无统计学意义(P>0.05)。hTERTEx2-659 A/G位点GG、AG、AA基因型在MSI组和MSS组中的频率比较差异无统计学意义(P>0.05)。H.pylori感染在MSI组和MSS组中的频率比较差异无统计学意义(P>0.05)。结论1.hPOT1IVS13-98 G/T及hTERTEx2-659A/G位点单核苷酸多态性可能与甘肃西部汉族人群的胃癌有关,为胃癌的保护因素。2. hPOT1表达水平随着胃癌恶性程度的增加而增加,检测胃癌中hPOT1蛋白的表达可判断胃癌的恶性程度及预后;3.hTERT表达水平随着胃癌恶性程度的增加而增加,检测胃癌中hTERT的表达可作为判断胃癌的恶性生物学行为的指标之一。4. H.pylori感染与hPOT1IVS13-98 G/T和hTERTEx2-659A/G单核苷酸多态性无关;H.pylori感染与hPOT1、hTERT蛋白表达密切相关,在hPOT1、hTERT阳性表达的患者H.pylori的感染率较高。5. MSI与hPOT1IVS13-98 G/T和hTERTEx2-659A/G单核苷酸多态性无关,与H.pylori感染也无关。更多还原

【Abstract】 Gastric cancer(GC) is one of the most common malignant tumors in china, which is a multi-gene based disease caused by the interaction between enviromental and genetic factors. A large amount of epidemiological evidence has accumulated indicating a significant relationship between Helicobacterpylori (H.pylori,Hp) infection and gastric adenocarcinoma development. In 1994, the World Health Organization/International Agency for Research on Cancer concluded that Hp is a definite carcinogen based on the epidemiological findings.Single nucleotide polymorphisms(SNPs) is nucleotide sequent polymorphism caused by single-base mutation of genome that has been generally thought to be the third era mark of heredity following microsatellite. Vast investigation of the SNPs data has shown that it can provide us with new approaches to understand multi-gene involved diseases and to evaluate the inter-individual and inter-groups difference in tumor risks and treatment response.Through the common study of SNPs in seven genes of telomere (TERT,TERC, TERF1,TERF2,TINF2,TERF2IP,POT1 andTNKS), it can be presumed that these variants will be of significant meaning in study of gene-associated diseases as cancer, aplastic anemia,autoimmune diseases,and degenerative disorders,as well as chromosomal abnormalities and/or abnormal telomeres. Human protection of telomeres 1(hPOT1), a member of the telomeric family, is a telomeric single-strand DNA binding protein that widely exist in eucaryotic cell as a house-keeping gene. It plays an important role in regulating telomeric length, attendancing telomeric function and protecting chromosome stability. Telomeric disfunction is a common occurrence in genom instability, which may trigger the development of gastric cancer. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of human telomerase, which regulates the telomer length by taking part in telomerase activity. An average of over 85 percent of human malignant tumors has telomerase expression, which can induce the infinite generation of cells and participate in tumorigenesis. However, in normal adult cells and tissues, there is no telomerase activity or telomerase expression. Therefore, it draws increasingly attention at its role in tumorigenesis.In current study we investigated the SNPs of hPOT1IVS13-98G/T and hTERT Ex2-659A/G, the expression of hPOT1,hTERT protein and microsatellite instability, with the hope of understanding the correlation between GC risk and SNPs of hPOT1、hTERT.MATERIALS AND METHODSOne hundred and sixty-eight cancer and corresponding normal tissues were obtained from surgically resected gastric carcinoma in three hospitals of west Gansu,China. A 5cm section was cut from each tissue and stained with hematoxylin/ eosin in order to ascertain whether the cancer cells in tissues were predominant or not. Normal mucosa was collected from local residents who come for regular health examining, exclused chronic gastritis, intestinal metaplasia, atypical hyperplasia, ulceration and canceration under gastroscopy. These samples were frozen immediately and stored at -70℃until analyzed.Age and sex of two groups has been matched and ethical approval has been informed consent. All carcinomas were staged according to UICC criteria and typed according to WHO 2000 criteria of histopathological classification. Genomic DNA was isolated by standard proteinase-K digestion and phenol-chloroform extraction protocols. SNPs were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP),microsatellite instability were detected by PCR-single strand conformation polymorphism (PCR-SSCP). The expression of hPOT1 and hTERT protein was detected by immunohistochemistry method. H.pylori infection was detected by PCR of urea enzyme gene of H.pylori and Warthin- Starry.RESULTSThe frequencies of TT,GT and GG genotypes of hPOT1IVS13-98 G/T were 36.90%,41.67% and 21.43 % in patients with gastric cancer 24.36%, 51.92% and 23.72% in controls,respectively. The OR for any GT and GG genotype were 0.439 (95 %CI : 0.251-0.767,P=0.004) and 0.514(95%CI: 0.264-0.999,P=0.050) when compared with TT genotype, respectively. There were no significantly difference in genotype distribution and allele frequency of hPOT1IVS13-98 G/T polymorphism (χ2=3.5853,P= 0.0583). The frequencies of GG, AG and AA genotypes of hTERTEx2-659A/G were 41.88%, 47.5% and 10.63% in patients with gastric cancer and 28.29%, 56.58% and 15.13% in controls,respectively. The OR for any AG and AA genotype were 0.519 (95%CI:0.310-0.870, P=0.013), 0.550(95%CI: 0.255-1.188, P=0.128) when compared with GG genotype,respectively. There were significant difference in genotype distribution and allele frequency of hTERTEx2-659A/G polymorphism (χ2=5.3734, P=0.0204).The positive expression ratio of hPOT1 in neoplasms, gastric atypical hyperplasia, intestinal metaplasia, normal mucosa were respectively 91.33%, 80.00%, 55.00% and 28.00%, and hTERT positive expression rate were 76.67%, 55.00%, 25.00%, and 0.00% , respectttively.All the three parameters significantly increased in atypical hyperplasia, neoplasms and intestinal metaplasia compared to normal mucosa( P < 0.05). Expression of hPOT1 protein increased significantly inⅢ/Ⅳstaging gastric cancer with deep invading and bad differentiation (P <0.01), Expression of hTERT protein increased significantly in poorly differentiated group ( P <0.01). There were significant relationships among the two parameters in neoplasms and dysplasia,respectively(rGC=0.212,P =0.009;rDYS=0.504,P =0.023).The ratio of H.pylori infection in gastric cancer, atypical hyperplasia, intestinal metaplasia and normal mucosa were 54.00%,70.00%,55.00% and 20.00,% respectively. Among three genotypes of hPOT1IVS13-98G/T, the ratio of H.pylori infection were 51.52% in GG, 45.16% in GT and 65.45% in TT, with no difference among the three genotype (χ2= 4.937,P=0.085).It strongly implied that hPOT1IVS13-98 G/T polymorphism was not correlated with H.pylori infection in gastric cancer. Of the three genotype of hTERT Ex2- 659A/G, the ratio of H.pylori infection were 66.67% in AA, 52.86% in AG and 50.88% in GG, there were no significant difference among three genotype (χ2=1.215,P=0.545).This results suggested that hTERTEx2-659A/G polymorphism was not correlated with H.pylori infection in gastric cancer. The positive rate of hPOT1 protein in patients with H.pylori infection (96.30%) was significantly higher than that of patients with H.pylori infection negative (85.51%,P < 0.05).The positive ratio of hTERT protein of patients with H.pylori infection positive (85.19%) was significantly higher than that of patients with H.pylori infection negative (71.01%, P < 0.05) .Seventy-five cases of gastric carcinoma were studied for MSI by using two microsatellite markers. The positive ratio of MSI was detected in 29 of 75(38.67%) in two microsatellite markers, among which 9(12.00%) was detected at BAT26 locus and 20 (26.67%) was detected at D5S346 locus. MSI is not to related to sex, age,invading to serosa and histological type (P >0.05). However,the frequence of MSI in groups without lymph node metastasis and I and II staging is significantly higher than that of groups with lymph node metastasis and staging III and IV(P<0.01). MSI was not correlated with hPOT1 IVS13-98G/T and TERTEx2-659A/G polymorphism in gastric cancer; MSI was not correlated with H.pylori infection in gastric cancer.CONCUSIONOur results indicate:①IVS13-98 G/TSNP of hPOT1 gene and Ex2-659A/GSNP of hTERT gene are probably associated with reduced risk for gastric carcinoma of west Gansu,China.②Expressions of hPOT1 and hTERT are up-regulated at an early stage of the gastric carcinonogenesis and they may play an important role in activity of telomerase. It may be useful to detect expression of hPOT1 and hTERT for predicting the biological behevor of gastric carcinoma.③H.pylori infection is not associated with IVS13-98 G/T SNP of hPOT1 and Ex2-659A/G SNP of hTERT, but associated with expression of hPOT1 and hTERT. There were no significant relationships between the IVS13-98G/TSNP and expression of hPOT1 in gastric carcinoma. There were no significant relationships between in gastric carcinoma.④MSI pathway is not related to hPOT1 IVS13-98 G/T, hTERTEx2- 659A/G polymorphism and H.pylori infection in gastric cancer.更多还原