人胎盘型谷胱甘肽S转移酶在胃癌组织中的表达与基因甲基化关系？…

目的：研究胃癌组织中ＧＳＴ－π的表达与其基因甲基化状态的关系。方法：应用Ｓ－Ｐ法检测１１６例胃癌和５３例胃癌前病变的ＧＳＴ－π表达和限制性内切酶及ＰＣＲ、Ｓｏｕｔｈｅｒｎ印迹方法检测１４例胃癌及其相应正常胃粘膜ＧＳＴ－πＤＮＡ５端调我ＣＣＧＧ特定位点的甲基化水平。结果：ＧＳＴ－π阳性率,正常胃癌膜１０％,胃癌７７％,肠上皮化生７６％,不典型增生８９％。ＧＳＴ－π在胃癌及癌前病变中的表达较正常胃粘膜     

CDKN2基因CpG岛异常甲基化与原发性胃癌的关系研究

探讨原发性胃癌中抑癌基因ＣＤＫＮ２的灭活机制。方法ＰＣＲ┐甲基化检测法检测３６例胃癌和２２例正常胃粘膜组织中ＣＤＫＮ２基因外显子１的ＣｐＧ岛异常甲基化情况。结果有１３例（３６．１％）胃癌标本和１例（４．５％）正常胃组织中ＣＤＫＮ２基因５′端ＣｐＧ岛异常甲基化，两者之间有显著性差异（Ｐ＜０．０５）。结论ＣＤＫＮ２基因的５′端ＣｐＧ岛异常甲基化与胃癌的发生发展相关，是该基因在原发性胃癌中的主要灭活机制。     

人胃癌组织中C—myc癌基因甲基化及其表达的研究

目的：研究胃癌及正常胃粘膜ｃ－ｍｙｃ基因外显子ⅢＣＣＧＧ位点的甲基化状态及其ｍＲＮＡ表达及ＳＡＢＣ法检测胃癌４５例、胃溃疡１４例、不典型增生１０例ｐ６７表达。结果：胃癌ｃ－ｍｙｃ基因外显子ⅢＣＣＧＧ位点比其相应正常胃粘膜呈高度去甲基化，ｍＲＮＡ表达升高（ｐ〈０．００１），ＤＮＡ去甲基化与ｍＲＮＡ表达存在相关性（ｐ〈０．０２５），ｐ６７在胃癌、不典型增生、胃溃疡表达率分别为８４．４４％（３８／４５）     

肺癌组织酸性谷胱甘肽S—转移酶基因测定及其临床意义

研究运用ＲＴ－ＰＣＲ方法对５１例手术切除肺癌组织和相应的癌周正常肺组织进行ＧＳＴ－πｍＲＮＡ水平检测。结果表明：ＧＳＴ－πｍＲＮＡ在正常肺组织和肺癌组织中表达阳性率分别为１９．６％（１０／５１）和５１．０％（２６／５１），两者之间具有明显差别。ＧＳＴ－πｍＲＮＡ表达与肿瘤病理类型、组织分化、ＴＮＭ分期等均无明显的相关性（Ｐ＞０．０５）。上述结果表明肺组织细胞在恶变过程中ＧＳＴ－πｍＲＮＡ水平表达明显增加，提示ＧＳＴ－π基因是肺癌肿瘤标志之一，同时在肺癌先天性耐药机制中占有重要的作用     

ＧＳＴ-π和Ｐ-ｇｐ在胃癌中的表达及其临床意义

目的　探讨耐药蛋白ＧＳＴ-π和Ｐ-ｇｐ在胃癌中的表达及其与胃癌临床病理特征的关系。方法　用免疫组化法检测２０９例胃癌组织ＧＳＴ-π和Ｐ-ｇｐ的表达情况,并收集临床病理相关资料作回顾性分析。结果　ＧＳＴ-π表达阳性率为８１.８２％（１７１／２０９）,与淋巴结转移数目有关（Ｐ＜０.０５）;Ｐ-ｇｐ表达阳性率为７９.９０％（１６７／２0９）,在印戒细胞癌中表达率较高（Ｐ＜０.０５）;ＧＳＴ-π和Ｐ-ｇｐ表达具有相关性（ｒ＝０.２４１,P＜０.０５）。结论　ＧＳＴ-π和Ｐ-ｇｐ的表达与胃癌的生物学行为有一定的相关性。     

肾细胞癌细胞P—糖蛋白和谷胱甘肽—S—转移酶的表达及临床意义

目的 研究Ｐ－糖蛋白（Ｐ－ｇｐ）的谷胱甘肽－Ｓ－转移酶（ＧＡＴ－π）在肾细胞癌组织中的表达及临床意义。方法 用免疫组化方法检测４０例肾细胞癌术前进行化疗的和１５例正常肾组织中的Ｐ－ｇｐ和ＧＳＴ－π表达。结果 Ｐ－ｇｐ和ＧＳＴ－π在１５例正常肾组织中的表达率均为１００％,在４０例肾细胞癌组织中分别为６７．５％和５７．５％,Ｐ－ｇｐ和（或）ＧＳＴ－π阳性率为７５％,Ｐ－ｇｐ和ＧＳＴ－π的表达与其组织学     

多药耐药基因产物表达和细胞调亡对胃癌影响的研究

目的 探讨多药耐药（ＭＤＲ）的基因产物和细胞凋亡指数（ＡＩ）表达与胃癌分型、分期、预后的关系。方法 应用免疫组化及原位末端标记法（ＴＵＮＥＬ），对８０例胃癌标本进行ＭＤＲ指标（Ｐ－ｇｐ、ＧＳＴ－π、ＴＯＰＯⅡ）及ＡＩ检测。结果 Ｐ－ｇｐ表达或临床分期有关（Ｐ〈０．０５），正常组织Ｐ－ｇｐ表达高者生存期长（Ｐ〈０．０５）；ＧＳＴ－π表达强度与预后有关，高表达者生存期短（Ｐ〈０．０５）；ＴＯＰＯⅡ表达与分型有关（Ｐ〈０．０５），低分化癌表达高于高中分化癌；凋亡指数表达与临床分期有关（Ｐ〈０．０５），高表达者其临床分期越晚。结论 ＭＤＲ与细胞凋亡都属细胞正常基因组表达的一部分，在正常组织及癌组织均可有表达。正常组织Ｐ－ｇｐ、ＧＳＴ－π的表达与预后有关，不同生存期的ＭＤＲ与细胞凋亡表达差异无显著性（Ｐ〉０．０５），但Ｍ     

RT—PCR定量检测GST—πmRNA的表达

目的：建立一个用于测定临床样本中胎盘型谷胱甘肽Ｓ－转移酶（ＧＳＴ－π）ｍＲＮＡ表达水平的逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）定量技术。方法：根据共扩增ＰＣＲ定量原理，通过多对引物的特异性分析和动力学筛选，建立了以β－ａｃｔｉｎ作内参，底片扫描定量的ＲＴ－ＰＣＲ法，并对３０例食管癌组织和癌旁组织ＧＳＴ－πｍＲＮＡ表达水平进行了检测。结果：本方法灵敏度高、重复性好（批内ＣＶ＜１６％；批间ＣＶ＜３０％）、不用同位素标记，ＧＳＴ－π／β－ａｃｔｉｎ比值不受肝素、ＳＤＳ等的影响。２３例食管癌组织的ＧＳＴ－πｍＲＮＡ表达水平（１．９８±０．７６）明显高于２１例癌旁组织表达水平（１．０２±０．５８）（Ｐ＜０．０１）。结论：所建立的方法能反映ＧＳＴ－πｍＲＮＡ表达的微小差异并能作批量分析，ＧＳＴ－πｍＲＮＡ可作为食管癌的肿瘤基因标志。     

胃癌组织中多药耐药性的研究

研究胃癌组织中ＤＮＡ拓扑异构酶（ｔｏｐｏｉｓｏｍｅｒａｓｅ，ＴｏｐｏⅡ），谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅｓ，ＧＳＴ－π）和Ｐ－糖蛋白（Ｐ－ｇｌｙｃｏｐｒｏｔｅｉｎ，Ｐ－ｇｐ）表达的意义。方法应用免疫组织化学Ｓ－ＰＦ法对９８例胃癌进行检测。结果在９８例胃癌中，７４例（７５．５％）ＧＳＴ－π－表达阳性，５６例（５７．１％）Ｐ－糖蛋白表达阳性，ＴｏｐｏⅡ计数（Ｔｏｐ     

胃癌及癌前病变组织中MUC5AC基因的异常表达及意义

目的：揭示正常胃粘膜、癌前病变和胃癌组织中ＭＵＣ５ＡＣ基因的表达与其临床病理分型之间的联系。方法：应用免疫组织ＳＰ法检测人正常胃粘膜、癌前病变粘膜以及胃癌组织中ＭＵＣ５ＡＣ核蛋白的表达。结果：人正常胃中的浅表１／３范围内广泛分布ＭＵＣ５ＡＣ基因产物（１００％）,肠化、不典型增生和胃癌组织中的表达阳性率分别是２９．６％、１００％和４０．０％。胃癌组织中ＭＵＣ５ＡＣ核粘蛋白的表达与胃癌患者的性别、肿瘤     

胃癌前病变APC抑癌基因原位杂交光镜及电镜观察

目的 探讨胃癌前病变ＡＰＣ基因异常表达及其意义。方法 应用光镜及电镜原位杂交对胃癌前病变ＡＰＣ基因进行细胞和超微水平观察。结果 （１）ＡＰＣ基因阳笥率以正常胃黏膜最高,为８３．３％,轻、中、重度异型增生分别为７７．８％、６２．５％和２５．９％,重度者明显低于前两者,癌组织则更低,为６．７％～８．０％。（２）肠上皮化生中,ＡＰＣ基因阳性率大肠型高于小肠型,水完全性高于完全性,大肠型不完全性最高。（３     

Analysis of Inactivation of hMLH1 by Promoter Hypermethylation and Microsatellite Instability in Gastric Carcinogenesis

OBJECTIVE To study the microsatellite instability (MSI) and methylation state of the hMLH1 gene promoter and their mechanisms underlying the development of gastric cancer. METHODS Forty-one gastric cancer samples were obtained from patients undergoing surgery and 46 chronic atrophic gastritis tissues with dysplasia or intestinal metaplasia (IM) were obtained from patients undergoing gastro-endoscopy. Fourteen normal gastric mucosal samples were used as controls. Genomic DNA was extracted from the samples and 5 microsatellite markers were used to measure MSI. Methylation-specific PCR (MSP) was used to screen the methylation state of the samples. DNA sequencing and immunohistochemistry were performed to verify the results. RESULTS MSI was identified in 22 out of the 41 (53.7%) gastric cancers, of which 8 cases showed high-level MSI (2 or more loci altered) and 14 showed low-level MSI (1 locus altered). MSI was also detected in 12 out of 46 (26.1%) pre-cancerous lesions of the stomach, whereas it was not seen in the normal tissue. Moreover, hMLH1 hypermethylation was detected in 17 out of the 41 (41.5%) gastric cancers, 9 out of the 46 (19.6%) pre-cancerous lesions and 0 out of the 14 normal tissue. Significant differences in frequency of MSI and hMLH1 promoter methylation were observed among gastric cancers, precancerous lesions and normal gastric tissue. Gastric samples with MSI had a tendency to be hypermethylated in the hMLH1 promoter. DNA sequencing and immunohistochemistry results also confirmed that hMLH1 promoter methylation could lead to loss of the hMLH1 protein and gene silence which sequentely resulted in gene mismatch and MSI. CONCLUSION Accumulation of MSI and hMLH1 promoter methylation may be important early molecular events during gastric carcinogenesis and may contribute to the acquisition of a transformed cell phenotype and the development of gastric cancer.     

抑癌基因PTEN在胃癌及癌前病变中的缺失和突变

目的:探讨胃癌及癌前病变中抑癌基因PTEN的缺失和突变.方法:收集中国医科大学2001～2002年胃镜中心活检标本,萎缩性胃炎、肠上皮化生、不典型增生及对应的正常胃粘膜各30例,收集同期肿瘤外科术后大体标本,早期胃癌及进展期胃癌各30例,饱和氯化钠法提取组织DNA,PCR-SSCP变性聚丙烯酰氨凝胶电泳银染法检测PTEN杂合性缺失(LOH),PCR-SSCP测序法检测PTEN基因突变.结果:在胃癌癌前期病变萎缩性胃炎、肠上皮化生、不典型增生中,PTEN的杂合性缺失率分别为10%、10%、13.3%,在早期胃癌中,PTEN的杂合性缺失率为20%,进展期胃癌中PTEN的杂合性缺失率为33.3%,而PTEN基因突变在癌前期病变及早期胃癌中无1例出现,在进展期胃癌中出现比率为10%,而且所有发生PTEN基因突变的病例均为LOH阳性病例.结论:PTEN基因缺失或失活与胃癌的浸润和转移的关系更密切.     

胃癌组织runt相关转录因子3基因表达与其甲基化状态的关系

目的 探讨胃癌组织中DNA甲基化调控机制对runt 相关转录因子3(Runx3)基因表达的影响.方法 采用逆转录聚合酶链反应(RT-PCR)法检测70例胃癌组织及其配对的正常胃黏膜组织中Runx3 mRNA表达,Westen blot法检测Runx3蛋白表达,甲基化特异性PCR(MSP)法检测Runx3基因启动子的甲基化状态;应用RT-PCR法检测 DNA甲基转移酶1(Dnmt1)mRNA表达,分析Runx3基因甲基化与Dnmt1 mRNA表达之间的相关性. 结果胃癌组织中Runx3 mRNA表达(0.5740±0.3580)明显低于其配对的正常胃黏膜组织(1.7250±0.4080, P<0.05),胃癌组织Runx3蛋白表达亦明显低于其配对的正常胃黏膜组织(P<0.05).在Runx3 mRNA表达下调的56例胃癌组织中,有28例(50.0%)呈启动子高甲基化状态.胃癌组织中,Runx3甲基化阳性者的Dnmt1 mRNA表达量明显高于Runx3甲基化阴性者(P<0.05),Runx3基因启动子甲基化与Dnmt1转录表达呈正相关(r＝0.64,P<0.05).结论 Runx3基因启动子高甲基化是其基因表达下调的原因之一,可能参与胃癌的发生发展;Dnmt1高表达可能影响Runx3启动子区域甲基化改变.     

Epstein-Barr virus infection as an epigenetic driver of tumorigenesis

Epstein-Barr virus (EBV) establishes latent infection and is associated with tumors, such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancers. We recently reported that EBV(+) gastric cancer shows an EBV(+)/extensively high-methylation epigenotype, and in vitro EBV infection induces extensive DNA methylation with gene repression within 18 weeks. On the basis of the absence of both EBV and high-methylation accumulation in the surrounding mucosa of EBV(+) gastric cancer, it is suggested that an EBV-infected cell acquires extensive methylation to silence multiple tumor suppressor genes in a short time period and transforms into cancer cells, not forming a precancerous field with EBV infection or methylation accumulation. The methylation mechanism induced by EBV infection has not been fully clarified. Differences in EBV genome methylation that are dependent on a different latency status or other epigenomic alterations, such as 3-dimensional conformation and histone modification, may affect host genome methylation. Expressions of viral proteins and small RNAs are also different depending on latency status, and some viral proteins might trigger DNA methylation by inducing DNA methyltransferase overexpression. In this review, we discuss these roles of EBV infection in driving tumorigenesis and their possible association with aberrant DNA methylation.     

食管和贲门癌及癌前病变患者血清中多个自身抗体的检测及其临床意义

目的:探讨食管和贲门癌及癌前病变患者血清中多个肿瘤相关自身抗体的变化特征及其在食管和贲门癌高危人群筛查和早期诊断中的意义。方法:应用间接酶联免疫反应法和肿瘤相关抗原微阵列(包含8个重组的癌抗原蛋白:C-myc、p53、cyclinB1、p16、p62、Koc、IMP1和Survivin)检测376例食管和贲门各级病变患者(正常、癌前病变和癌)血清中的自身抗体。结果:在所检测的八种抗原中,p53、C-myc、cyclinB1、IMP1和p62从正常食管到癌前病变及癌患者血清中和p53、C-myc、p16、p62在正常贲门到癌前病变及癌患者血清中阳性百分率均具有线性升高趋势。单一抗体对食管和贲门癌检出率较低,p53在癌血清中的表达阳性率在所有抗原中最高,在食管癌和贲门癌患者中分别为23%(13/57)和21%(9/43)。但是,当应用8个抗原分析时,至少有1个反应为阳性时,其检出阳性率明显增高,食管和贲门癌的阳性检出率分别提高到63%(36/57)和61%(26/43),上述检测指标在正常食管和贲门与相应各级癌前病变和癌患者阳性表达率差异有统计学意义,P<0·05。结论:联合应用多个肿瘤相关抗原比应用单个肿瘤相关抗原分析食管和贲门癌及癌前病变患者血清中的自身抗体变化能够提高癌和癌前病变患者的检出率。食管和贲门癌多个相关抗原微阵列的进一步优化有可能成为临床上食管和贲门癌和高危人群检测和早期诊断的非侵袭性方法。     

Bmi-1 在正常胃组织和胃癌及其癌前病变中的表达及意义*

目的:检测Bmi-1 在正常胃粘膜、胃癌及癌前病变中的表达,分析探讨Bmi-1 与核增殖抗原ki-67和凋亡抑制基因Bcl- 2 之间的关系,以及Bmi-1+细胞与Bcl- 2+/ki- 67-细胞分布之间的关系,探讨其与胃癌临床病理因素的关系及意义。方法:采用免疫组化Envision法检测162 例胃癌及配对正常胃粘膜中Bmi-1 和ki-67的表达,采用免疫组化双染法观察Bcl- 2+/ki- 67-细胞的分布。结果:胃癌组织中Bmi-1 表达（52.5%）明显高于正常胃粘膜（21.6%）,P<0.05;胃癌中Bmi-1 表达与Lauren 分型、Borrmann 分型和胃癌临床病理分期有关（P<0.05）。 胃癌中ki-67表达与Borrmann 分型有关（P<0.05）。 Bcl- 2 在胃癌中的表达与胃癌Lauren 分型有关（P<0.05）。 在胃癌组织和肠化生粘膜中,Bmi-1 与Bcl- 2 呈正相关（P<0.05）。 结论:胃癌及其癌前病变中Bmi-1 表达可能与细胞增殖、凋亡和癌变有关。在胃癌和肠化生粘膜中Bmi-1 和Bcl- 2 表达的相关性以及Bmi-1+细胞和Bcl- 2+/ki- 67-细胞分布之间的关系提示Bmi-1 与胃癌的发生密切相关。      

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: a population-based study

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population-based study was conducted in Linqu County, a high-risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation-specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C-urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p<0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90-7.10), and dysplasia (OR, 2.48; 95%CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island.     

Dysregulated expression of stem cell factor Bmi1 in precancerous lesions of the gastrointestinal tract.

PURPOSE: It is important to identify the definitive molecular switches involved in the malignant transformation of premalignant tissues. Cellular senescence is a specific characteristic of precancerous tissues, but not of cancers, which might reflect tumorigenesis-protecting mechanisms in premalignant lesions. Polycomb protein Bmi1, which is a potent negative regulator of the p16INK4 gene, suppresses senescence in primary cells and is overexpressed in various cancers. We hypothesized that Bmi1 expression would also be dysregulated in precancerous lesions in human digestive precancerous tissues. EXPERIMENTAL DESIGN: Bmi1 expression was investigated in cancerous and precancerous tissues of the digestive tract. The expression of p16, beta-catenin, and Gli1 and the in vivo methylation status of the p16 gene were also analyzed in serial sections of colonic precancerous lesions. RESULTS: Bmi1 was clearly overexpressed across a broad spectrum of gastrointestinal cancers, and the expression of Bmi1 increased in a manner that reflected the pathologic malignant features of precancerous colonic tissues (low-grade dysplasia, 12.9 +/- 2.0%; high-grade dysplasia, 82.9 +/- 1.6%; cancer, 87.5 +/- 2.4%). p16 was also strongly expressed in high-grade dysplasia, but not in cancers. p16 promoter methylation was detected only in some Bmi1-positive neoplastic cells. CONCLUSIONS: Bmi1 overexpression was correlated with the malignant grades of human digestive precancerous tissues, which suggests that advanced Bmi1 dysregulation might predict malignant progression. The abnormal Bmi1 expression might link to malignant transformation via the disturbance of orderly histone modification.     

从表观遗传学角度探讨RAR-β2 基因在乳腺癌发生中的作用*

目的:探讨视黄酸受体β 2（RAR-β 2）基因在乳腺癌、乳腺癌前病变组织中的表达改变,分析其表达情况与基因启动子甲基化状态的关系。方法:采用半定量RT-PCR 和甲基化特异PCR（MSP）方法检测120 例乳腺肿瘤组织中RAR-β 2 基因mRNA 表达情况及其启动子甲基化状态。去甲基化药物5- 氮-2'- 脱氧胞苷（5-aza-2'-deoxycytidine,5-aza-dC）处理乳腺癌细胞系MCF-7和T-47D,分别检测RAR-β 2 mRNA 表达改变和甲基化状况。结果:在mRNA 水平上,RAR-β 2 基因在乳腺癌及癌前病变组织中阳性表达率较正常乳腺及腺纤维瘤组织显著降低（P<0.05）。 MSP 结果显示,20例正常乳腺组织均未发现RAR-β 2 基因启动子的甲基化;60例乳腺癌组织中,27例（45%）检测到RAR-β 2 基因启动子的甲基化;40例乳腺癌前病变组织中,14例（35%）检测到RAR-β 2 基因启动子的甲基化;20例腺纤维瘤组织中,2 例检测到RAR-β 2 基因启动子的甲基化。乳腺癌及癌前病变组织中RAR-β 2 基因甲基化发生率均明显高于正常组织（P<0.05）,乳腺癌组织甲基化发生率与癌前病变组织无显著性差异（χ2=0.99,P>0.05）。 RAR-β 2 基因表达和甲基化水平之间存在负相关（P<0.05）。 5-aza-dC 处理后,MCF-7 及T-47D 细胞系均出现RAR-β 2 基因再表达,MSP 法检测证实基因发生了去甲基化。结论:DNA甲基化是乳腺癌中 RAR-β 2 基因表达调控的一种重要机制,RAR-β 2启动子甲基化引起的基因表达下调可能与乳腺癌的发生相关。