人食管癌与NMBzA引起的人和猴食管上皮细胞中多种抑癌基因变…

为了研究人原发性食管癌组织中多种抑癌基因突变谱与甲基苄基亚硝胺（ＮＭＢｚＡ）引起的人和猴食管上皮细胞中多种抑癌基因突变谱的相关性。我们应用ＰＣＲ扩增与直接测序技术分析了人原发性食管癌和ＮＭＢｚＡ处理的人和猴食管上皮细胞中多种抑癌基因ｐ５３，Ｒｂ，ＡＰＣ和ＭＣＣ的突变谱；发现４０．９％（９／２２）的人原发性食管癌ｐ５３基因突变与ＮＭＢｚＡ处理的人和猴食管上皮细胞中ｐ５３基因的突变相同，也发现ＮＭＢｚ     

NMBzA对人胎儿食管上皮组织中多种抑癌基因作用的研究

流行病学研究表明，食管癌与环境中亚硝胺致癌物有密切关系。为了从分子水平阐明亚硝胺致癌物诱发食管癌癌变机制，作者应用ＰＣＲ扩增分析发现甲基苄基亚硝胺（ＮＭＢｚＡ）诱发的人胎儿食管癌组织有抑癌基因Ｒｂ、ｐ５３、ＡＰＣ和ＭＣＣ损失。ＮＭＢｚＡ处理２４小时和３周的人胎儿童管上皮细胞中未发现这些基因的缺失；但ＰＣＲ直接测序分析发现ＮＭＢｚＡ体外处理３周的人胎儿食管上皮组织在ｐ５３、Ｒｂ和ＭＣＣ基因突变。以上     

胃癌患者p53基因甲基化的研究

作者应用限制性内切酶ＨｐａⅡ和ＭｓｐⅠ酶切胃癌组织及正常胃组织ＤＮＡ，经ＰＣＲ扩增ｐ５３基因第５外显子，琼脂糖凝胶电泳分析其电泳图谱，比较胃癌组织及正常胃组织ｐ５３基因第５外显子特定序列５′－ＣＣＧＧ－３′位点甲基化差异。结果显示：１５例胃癌组织中１２例ｐ５３基因第５外显子出现高甲基化状态，而１０例正常胃组织为低甲基化状态，结果提示，ｐ５３基因高甲基化状态与胃癌发生有关。     

食管癌组织中Rb基因突变与表达的研究

应用ＰＣＲ直接顺序和Ｎｏｒｔｈｅｒｎ ｂｌｏｔ杂交方法，分析了食管癌组织中Ｒｂ基因的突变和表达状况。应用ＰＣＲ扩增Ｒｂ基因片段，发现５％（１／２０）的食管癌有Ｒｂ基因１７和２１外显子缺失；１／６的食管癌旁组织中，有Ｒｂ基因１７外显子缺失；还发现甲基苄基亚硝胺（ＮＭＢｚＡ）诱发的人胎儿食管癌有Ｒｂ基因１７和２１外显子缺失。ＰＣＲ直接测序，发现４０％（４／１０）癌组织中有Ｒｂ基因的突变，首次证实食管癌     

细胞角蛋白基因13在鼻咽癌中的作用研究

目的 研究细胞角蛋白基因１３（ｃｙｔｏｋｅｒａｔｉｎ １３,ＣＫ１３）在人鼻咽癌组织中的表达,并对其甲基化程度进行了检测。方法 应用Ｎｏｒｔｈｅｒｎ杂交技术检测３２例鼻咽癌活检组织和８例慢性鼻咽炎组织中ＣＫ１３的表达情况。同时运用甲基敏感的限制性内切酶ＨｐａⅡ和ＭｓｐⅠ,结合Ｓｏｕｔｈｅｒｎ杂交,对鼻咽癌细胞 ＨＮＥ１及正常人上皮ＣＫ１３基因的甲基化状态进行检测。     

亚硝胺对人食管上皮中癌基因的作用

用甲基苄基亚硝胺（ＮＭＢｚＡ）诱导胎儿食管上皮（ＨＦＥ）组织，通过Ｓｏｕｔｈｅｍ杂交和免疫组织化学分析发现：ＭＮＢｚＡ诱导２４小时的ＨＦＥ中即有ＥＧＦｒ基因的扩增和高表达；随着诱导时间的延长，在ＮＭＢｚＡ诱导１周和３周的ＨＦＥ中分别发现ｃ－ｍｙｃ和ｉｎｔ－２基因的扩增，组织学观察可发现这些组织中有局部乳头状增生；在ＮＭＢｚＡ诱导的胎儿食管上皮癌中发现Ｒｂ基因的完全丢失及Ｐ５３基因的部分丢失和高表达     

肺癌组织中错配修复基因hMLH1 启动子甲基化状态分析

目的 探讨肺癌组织中错配修复基因ｈＭＬＨ１启动子甲基化状态及其与蛋白表达的关系。方法 用ＨｐａⅡ酶切ＰＣＲ及ＳＰ免疫组织化学方法,分析５０例肺癌标本ｈＭＬＨ１启动子甲基化状态及蛋白表达。结果 ｈＭＬＨ１启动子甲基化有１６例,占３２．０％（１６／５０）。ｈＭＬＨ１启动甲基化与性别、吸烟状态及临床病理无明显的相关性。ｈＭＬＨ１蛋白表达阴性的１４例中,１１例有甲基化（７８．６％）;而３２例蛋白表达阳性者中,只有５例甲基化（１５．６％）。结论 ｈＭＬＨ１启动子区在肺癌组织中有中等频率的甲基化,是ｈＭＬＨ１蛋白表达降低的主要原因之一。     

子宫内膜癌中p16基因甲基化与表达的研究

目的 研究ｐ１６基因甲基化状态及其ｐ１６基因表达异常与子宫内膜癌发生发展的关系。方法 采用限制性内切酶酶切、ＰＣＲ及ＲＴ－ＰＣＲ检测子宫内膜检测ｐ１６基因５’ＣｐＧ岛甲基化状态及ｐ１６基因ｍＲＮＡ表达。结果 ８例正常子宫内膜无甲基化,且ｐ１６ ｍＲＮＡ表达正常。６例子宫内膜非典型增生中,有１例甲基化;３８例子宫内膜癌中,１３例甲基化,占３４．２％。６例子宫内膜单纯及复合增生中,有５例ｐ１６ ｍＲＮ     

人肝细胞癌c—myc,c—N—ras癌基因甲基化与病理学改变的关系

目的探讨人肝细胞癌发生的分子生物学机理以及相关癌基因的甲基化变化。方法以限制性内切酶ＭｓｐＩ、ＨｐａＩ分别消化,Ｓｏｕｔｈｅｒｎｂｌｏｔ法分析ｃ－ｍｙｃ、ｃ－Ｎ－ｒａｓ两种癌基因的甲基化状况,并与病理学改变比较。结果３３例原发性肝细胞癌中,３０．３％癌区及１２．１％的癌旁区的ｃ－ｍｙｃ基因片段和６０．１％的癌区及３０．３％癌旁区的ｃ－Ｎ－ｒａｓ基因片段呈低甲基化状况。也发现伴卫星灶的肝癌患者其ｃ－ｍｙｃ癌基因低甲基化发生率较高（Ｐ＜０．０５）,并且肿瘤直径越大,ｃ－Ｎ－ｒａｓ基因低甲基化倾向越明显（Ｐ＜０．０１）。结论在人肝细胞肝癌发生发展过程中存在着ＤＮＡ甲基化水平的降低,其中ｃ－ｍｙｃ和ｃ－Ｎ－ｒａｓ两种癌基因的低甲基化与肝癌的浸润程度及转移倾向呈现出相关性。     

非小细胞肺癌pl6/CDKN2基因失活的研究

目的：分析中国人非小细胞肺癌中ｐ１６／ＣＤＫＮ２基因失活的情况,探讨该基因在肺癌发生中的作用。方法：选取与ｐ１６基因紧密连锁的Ｄ９Ｓ１７４８位点,对１７例临床切除的原发性肺癌标本进行微卫星不稳定性分析,用甲基化特异性ＰＣＲ（ｍｅｔｈｙｌａｔｉｏｎ－ｓｐｅｃｉｆｉｃ ＰＣＲ, ＭＳＰ）检测 ｐｌ６基因启动子区 ＣｐＧ岛甲基化状况,用免疫组织化学法检测Ｐ１６蛋白表达情况。结果：微卫星不稳定性分析结果表明,在１３例Ｄ９Ｓ１７４８位点存在多态性的肿瘤ＤＮＡ中有 ９例（６９ ．２％）发生了杂合性缺失（ｌｏｓｓ ｏｆ ｈｅｔｅｒｏｚｙｇｏｓｉｔｙ, ＬＯＨ）。 ＭＳＰ的结果显示,有 ７０． ６％（１２／１７）的肿瘤组织存在ｐ１６启动子区的异常高甲基化。在本研究中,８２．４％（１４／１７）的肿瘤组织可以检测出一种或两种ｐ１６基因的异常改变。对此１７例肿瘤标本进行的免疫组织化学分析显示,有 １３例 Ｐ１６蛋白表达阴性,其中 ９２ ３％（１２／１３）存在一种或两种ｐ１６基因的异常改变。免疫组化结果与ｐ１６基因分子遗传学改变情况基本吻合。结论：作为一种抑癌基因,ｐ１６在多种肿瘤组织中都有异常改变。我们的研究表明,ｐ１６基因失表达是非小细胞肺癌     

高发区食管癌患者p16基因甲基化及其表达的比较研究

目的:比较p16基因甲基化在食管癌高发区河南林州(北方组)和广东揭阳(南方组)之间的异同,探讨p16基因甲基化在不同气候环境条件下的两地食管鳞癌(ESCC)发生中的作用。方法:采用甲基化特异性PCR方法(MSP)分别检测两地食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用EnVision免疫组化法检测两地食管癌组织及癌旁组织的p16蛋白的表达。结果:南方组75例标本中,食管癌组织、癌旁组织和切缘组织p16基因甲基化率分别为41.3%(31/75)、13.3%(10/75)和6.67%(5/75);癌组织和癌旁组织p16蛋白的阳性表达率分别为29.3%(22/75)和56.7%(17/30);31例p16基因甲基化阳性标本中有2例(6.4%)检测到p16蛋白的表达,而44例p16基因甲基化阴性标本中有20例(45.5%)检测到p16蛋白的表达。北方组65例标本中,食管癌组织、癌旁组织、切缘组织p16基因甲基化率分别为52.3%(34/65)、16.9%(11/65)和7.69%(5/65);食管癌组织和癌旁组织p16蛋白的阳性表达率分别为32.3%(21/65)和66.7%(20/30);34例p16基因甲基化阳性标本中有4例(11.8%)检测到p16蛋白的表达,而31例p16基因甲基化阴性标本中有17例(54.8%)检测到p16基因的表达。两地组内癌组织p16甲基化率均显著高于癌旁组织和切缘组织,p16蛋白表达与p16基因甲基化呈负相关(P<0.01)。两地同类组织比较p16甲基化率和p16蛋白表达率均无显著性差异(P>0.05)。结论:p16基因异常甲基化后功能失活可能是南、北两地食管癌癌变过程的重要事件,在我国南北环境气候条件不同的两地高发区食管癌的发生中均起着重要的作用。本研究为环境因素和p16基因功能之间的生物学关联在食管癌的发生中提供了一定的证据。     

食管癌组织中抗癌基因Rb的研究

Retinoblastoma gene (Rb) is a tumor suppressor gene. In 21 esophageal cancer and 43 pericancerous non-tumor samples obtained from patients who had undergone surgery for esophageal cancer in Linxian County, a high incidence area of esophageal cancer, Rb gene was studied. DNAs were extracted from the above esophageal tissues, then were separately digested with Hind III, EcoRI, BamHI, PstI, MspI, HpaII and subjected to Southern analysis. The Southern blots were sequentially probed with two fragments of Rb cDAN clone: 0.9 kb and 3.8 kb. Of 43 adjacent non-tumor tissues examined, 15 were found to have structural anomaly (34.9%). Seven of 21 esophageal cancers also showed structural anomaly (33.3%). In order to study the cause of Rb gene deletion, esophageal carcinoma of human fetal origin induced by N-methyl-N-benzylnitrosamine (NMBzA) in nude mice were similarly examined and found to be deleted of Rb gene. Thus, deletional inactivation of Rb gene may play an important role in the pathogenesis of esophageal cancer in this high-risk area. N-nitrosamine may play a causative role in deletion of Rb gene. The above data for the first time demonstrate structural anomaly of Rb gene in esophageal cancer and confirm that chemical carcinogens, such as N-nitrosamines, can delete the antioncogene.     

食管癌中DNA修复基因MGMT启动子区CpG岛过甲基化研究

目的：探索O~0-甲基鸟嘌呤-DNA甲基转移酶(O~6-methylguanine-DNA methyltransferase,MGMT)基因启动子区过甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)分析食管癌、癌旁和正常食管黏膜细胞中MGMT基因启动子区过甲基化状态。用免疫组织化学SP法检测上述组织中MGMT蛋白表达情况。结果：76例食管癌组织中有26例(34.2%)MGMT基因启动子过甲基化,相应的癌旁组织中有8例发生甲基化,而正常食管黏膜细胞中均无甲基化。所有甲基化的癌组织中均无MGMT蛋白表达,而所有非甲基化的癌组织、相应癌旁组织和8例正常组织中均有MGMT蛋白表达。结论：MGMT基因启动子区过甲基化而使其蛋白表达缺失,可能是食管癌发生的重要因素。     

Hypermethylation-associated inactivation of retinoic acid receptor beta in human esophageal squamous cell carcinoma.

PURPOSE: The purpose of this study was to investigate the mechanism of altered retinoic acid receptor beta (RARbeta) expression during esophageal squamous carcinogenesis. EXPERIMENTAL DESIGN: Samples were collected from Linzhou, China. The hypermethylation of CpG islands in the promoter region of the RARbeta gene was examined by methylation-specific PCR in human esophageal squamous cell carcinoma (ESCC) samples, as well as in neighboring tissues with normal epithelium, basal cell hyperplasia, and dysplasia. RARbeta mRNA expression was determined by in situ hybridization. The DNA methyltransferase inhibitor 2'-deoxy-5-azacytidine was used to treat the ESCC cell line, and the DNA hypermethylation status and mRNA expression level were examined. RESULTS: Two of 17 (12%) normal, 9 of 21 basal cell hyperplasia (43%), 7 of 12 dysplasia (58%), and 14 of 20 ESCC (70%) samples had hypermethylation of the RARbeta promoter region. The loss of RARbeta mRNA expression was highly concordant with RARbeta promoter CpG island hypermethylation when individual samples were considered in the correlation analysis. Good statistical correlation between hypermethylation and loss of RARbeta expression was revealed. Frequencies of hypermethylation appeared to increase with the progression of carcinogenesis. In samples from the same patients, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, 2'-deoxy-5-azacytidine partially reversed CpG island hypermethylation and restored RARbeta mRNA expression. CONCLUSIONS: The results suggest that hypermethylation of RARbeta promoter region is an important mechanism for RARbeta gene silencing in esophageal squamous carcinogenesis.     

新疆哈萨克族食管癌survivin启动子区CpG岛甲基化状态及其临床意义

目的： 检测消化道肿瘤早期相关基因survivin mRNA在哈萨克族食管癌组织中的表达及其启动子区CpG岛甲基化状态,并进一步探究survivin启动子甲基化是否参与了食管癌的发生。方法：提取哈萨克族食管癌患者癌组织及远端无癌组织RNA,RT-PCR检测组织中survivin mRNA的表达水平,甲基化特异性PCR (methylation specific PCR,MSP)检测survivin启动子区CpG 岛的甲基化状态。结果：在20对食管癌组织标本中,癌组织中survivin mRNA阳性表达率 (95%)明显高于远端无癌组织 (65%) (P＜0.05)。癌组织及远端无癌组织中survivin基因启动子区CpG岛多为杂合型甲基化,且两种组织间甲基化状态无明显差异 (P＞0.05)。结论：survivin mRNA表达水平的增高在哈萨克族食管癌的发生、发展过程中起到了一定的作用,但其表达与启动子区CpG岛甲基化无关,提示甲基化并未直接调控该基因的表达并促进哈萨克族食管癌的发生发展。     

新疆哈萨克族和汉族食管癌患者FHIT基因甲基化及其与预后的关系

目的：探讨和比较新疆哈萨克族和汉族食管癌患者FHIT基因甲基化状态及其与食管癌发生及预后之间的关系。方法：采用甲基化特异性PCR法（methvlation-specific polymerase chain reaction,MSF）测定哈、汉两民族共71例食管癌及癌旁组织标本FHIT基因启动子区甲基化状况,建立Cox回归模型分析临床病理指标及FHIT基因甲基化水平等因素与预后的关系。结果：36例哈族食管癌患者癌组织和癌旁组织的FHIT基因启动子甲基化率分别为58.3%和19.4%,35例汉族食管癌患者癌组织和癌旁组织的FHIT基因启动子甲基化率分别为51.4%和14.3%,两民族癌组织FHIT基因的甲基化率均明显高于癌旁组织（P〈0.01）;哈、汉民族食管癌患者癌组织之间、癌旁组织之间FHIT基因启动子甲基化率无显著性差异（P〉0.05）。预后因素分析表明肿瘤浸润程度、TNM分期、淋巴结转移区域数是影响哈、汉民族食管癌预后的独立危险因素,女性性别是保护因素。结论：FHIT基因启动子区甲基化状态与新疆哈族、汉族食管癌的发生有密切的关系,但可能与食管癌预后无关。     

新疆哈萨克族食管癌患者ALDH1L1基因甲基化及其与预后的关系

目的： 探讨新疆哈萨克族食管癌患者ALDH1L1基因甲基化状态及其与食管癌发生及预后之间的关系。方法：采用联合亚硫酸氢钠限制性内切酶分析(COBRA)法检测28例哈萨克族食管癌及癌旁组织标本中ALDH1L1基因启动子区甲基化状况,建立Cox回归模型分析临床病理指标、ALDH1L1基因甲基化水平等因素与预后的关系。结果：28例哈族食管癌患者癌组织和癌旁正常组织的ALDH1L1基因启动子甲基化率分别为71.43% 和39.28%,差异有统计学意义(P<0.05)。预后因素分析表明肿瘤浸润程度、TNM分期、淋巴结转移、癌组织ALDH1L1基因甲基化水平是影响哈萨克族食管癌预后的独立危险因素。癌组织ALDH1L1基因无甲基化患者术后生存时间长于完全甲基化患者(P<0.05)。结论：癌组织ALDH1L1基因启动子区甲基化状态与新疆哈萨克族食管癌的发生及预后有关。     

Loss of heterozygosity of the Rb gene correlates with pRb protein expression and associates with p53 alteration in human esophageal cancer.

To understand the alterations of Rb tumor suppressor gene and the relationship between defects in the Rb and p53 pathways in human esophageal carcinogenesis, we examined the loss of heterozygosity (LOH) of the Rb gene and immunohistochemical staining of pRb protein in 56 esophageal squamous cell carcinoma specimens and related the results to the p53 gene alterations. Using four introgenic polymorphic markers as probes, we observed LOH of the Rb gene in 30 of the 55 informative tumor samples. Immunohistochemical analysis revealed different patterns of pRb expression among the tumor samples. In the 56 cases, 16 displayed extensive pRb staining comparable to that of the adjacent normal epithelia, whereas 33 showed either significantly decreased or no pRb staining and 7 had a focal staining pattern reflecting heterogeneous cancer nests in the tumor with respect to Rb status. In the tumor samples containing Rb LOH, 90% showed low or no pRb expression, whereas in samples without Rb LOH, only 20% had altered pRb expression. There was a strong association between LOH of the Rb gene and alteration of pRb expression in our samples (P < 0.0001), suggesting LOH is a main event leading to Rb inactivation. We found that Rb LOH was more frequent in tumors with p53 mutations (P < 0.05), which occurred in 31 of the 49 cases analyzed. When the status of Rb and p53 alterations was evaluated by the combined results of immunohistochemical and genetic analyses, we found that alteration of Rb and p53 had an even stronger association in our esophageal squamous cell carcinoma samples (P = 0.0015). Among the 51 cases in which both the Rb and p53 status were determined, 31 contained alterations in both genes, and only 5 and 6 cases were altered in only Rb and only p53, respectively. Our results suggest that defects in the Rb and p53 pathways and their potential synergistic effect in deregulating cell cycle and apoptosis are major mechanisms for esophageal carcinogenesis.     

Promoter hypermethylation of RASSF1A in esophageal squamous cell carcinoma.

PURPOSE: The RAS association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is frequently inactivated by hypermethylation of its promoter region in several human cancers. The aim of this study was to evaluate the promoter methylation status of the RASSF1A in esophageal squamous cell carcinoma. EXPERIMENTAL DESIGN: We analyzed the methylation status of RASSF1A promoter by methylation-specific PCR in 23 esophageal squamous cell carcinoma cell lines and 48 primary tumors. RESULTS: Hypermethylation of RASSF1A was found in 74% of cell lines and 52% of primary tumors. The presence of hypermethylation was statistically associated with loss of RASSF1A mRNA expression in both cell lines (P = 0.007) and primary tumors (P = 0.003). There was a statistically significant correlation between the presence of hypermethylation and tumor stage (P = 0.009). CONCLUSIONS: Our findings suggest that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation could play an important role in primary esophageal squamous cell carcinogenesis.