石蜡包埋的石棉肺癌组织中p53基因突变的分析方法

为了利用石蜡包埋的病理组织来研究石棉肺癌的基因突变情况，选用１０例石蜡包埋的石棉肺癌组织进行ＰＣＲ－ＳＳＣＰ分析，检测其抗癌基因ｐ５３的第５、第７和第８外显子的突变情况。经银染检查，发现４个病例的ｐ５３基因的第７或第８外显子片段呈突变阳性。用放射性自显影的ＰＣＲ－ＳＳ－ＣＰ方法分析这１０例病例，检测到７个病例的ｐ５３基因第７或第８外显子片段呈突变阳性。用两种方法检测均未发现这１０个病例的第５外显子呈突变阳性。银染ＰＣＲ－ＳＳＣＰ检测可检出放射性自显影ＰＣＲ－ＳＳＣＰ分析结果的６０％，二者的结果相符合。因而，简便、灵敏而且危害性小的银染检测方法，可以代替放射性自显影用于ＰＣＲ－ＳＳＣＰ分析，研究石棉肺癌的基因突变情况。     

石棉肺癌组织中p53基因突变的PCR-SSCP及部分序列分析研究

为研究石棉致癌的分子机理，本研究首次直接分析石蜡包埋的石棉肺癌病例肺组织中抗癌基因ｐ５３的突变情况。利用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）技术分析１０例石棉肺癌病例，检测到７例（８个片段）存在ｐ５３基因的突变，突变位点位于其第５、７和８外显子区内，突变率为７０％。对其中２例进行ＰＣＲ产物直接序列分析，发现其基因一级结构分别发生Ｃ→Ａ和Ｔ→Ａ转换。     

恶性间皮瘤p16蛋白表达的研究

目的研究恶性间皮瘤与ｐ１６蛋白表达的关系。方法利用微波煮沸脱交联ＳＰ法，对３４例恶性间皮瘤病理标本进行了ｐ１６蛋白表达的研究。结果ｐ１６蛋白阳性表达率５３％（１８／３４），４７％（１６／３４）的病例细胞内缺乏ｐ１６蛋白的表达。多数存在ｐ１６蛋白表达的病例没有发现ｐ５３基因的点突变；反之，存在ｐ５３基因的点突变的病例多数都没有ｐ１６蛋白的表达。ｐ１６蛋白表达与ｐ５３基因点突变之间呈明显的负相关关系。结论恶性间皮瘤可能同时存在ｐ５３和ｐ１６基因的异常，肿瘤组织细胞内缺乏ｐ１６蛋白的表达可能与ｐ１６基因的纯合缺失有关。     

Molecular biology of malignant mesothelioma

Human malignancies develop via a multi-step that involves the accumulation of several key gene alterations with associated genetic and epigenetic events. Although malignant mesothelioma (MM) has been demonstrated to be clearly correlated with asbestos exposure, it remains poorly understood how asbestos fibers confer key gene alterations and induce cellular transformation in normal mesothelial cells, which results in the acquisition of malignant phenotypes, including deregulated cell proliferation and invasion. Malignant mesothelioma presents with the frequent inactivation of tumor suppressor genes of p16 ^INK4a /p14 ^ARF on chromosome 9p21 and neurofibromatosis type 2 (NF2) on chromosome 22q12, with the latter being responsible for the NF2 familial cancer syndrome. In contrast, MM shows infrequent mutation of the p53 gene, which is one of the most frequently mutated tumor suppressor genes in human malignancies. Genetic abnormalities of oncogenes have also been studied in MM, but no frequent mutations have been identified, including the epidermal growth factor receptor (EGFR) and K-RAS genes. Recent studies have suggested the activation of other receptor tyrosine kinases, including Met, and the deregulations of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)-AKT signaling cascades, although the alterations responsible for their activation are still not clear. Thus, further genome-wide studies of genetic and epigenetic alterations as well as detailed analyses of deregulated signaling cascades in MM are necessary to determine the molecular mechanisms of MM, which would also provide some clues for establishing a new molecular target therapy for MM. This article is based upon the Symposium Lecture Series, “To Know Malignant Mesothelioma,” at the 77th Annual Meeting of the Japanese Society for Hygiene held in Osaka, Japan on March 25–28, 2007.     

恶性卵巢上皮肿瘤的P53基因点突变及其意义

目的 为揭示P53抗癌基因与卵巢上皮肿瘤发病学的关系，对P53基因5—8外显子突变进行了检测。方法 采用聚合酶链反应—DNA单链构象多态性分析法，以同期良性卵巢上皮肿瘤作对照组。结果 30例卵巢良性上皮肿瘤中P53基因突变3例(10％)；而50例恶性卵巢上皮肿瘤中发生突变19例(38％)，经x^2检验，差异有显著性(P＜0．05)。但不同组织学分级时P53基因点突变差异不显著(P＞0．05)。结论 恶性卵巢上皮肿瘤P53基因5—8外显子点突变发生率，明显高于卵巢良性上皮肿瘤，该种突变可能与恶性卵巢上皮肿瘤的发生有关。但P53基因5—8外显子点突变与组织学分级天关。     

Occupational cancer genetics: infrequent ras oncogenes point mutations in lung cancer samples from chromate workers.

BACKGROUND: Chromium carcinogenicity and mutagenicity are no longer disputed. However, although chromium has various genetic effects that induce cancer, its mechanism of inducing lung cancer in humans is still not fully understood. p53, a tumor suppressor gene, was found to be infrequently mutated in samples of lung cancer in workers with long occupational exposure to chromium, suggesting other cancer-related genes to be targeted in such tumors. METHODS: To assess the contribution of the ras oncogenes in the pathogenesis of chromate-related lung cancer, we studied point mutations at the critical positions of codons 12, 13, and 61 of the Ha-ras and Ki-ras oncogenes in 38 lung cancer samples derived from Japanese patients who worked in the chromate industry for long periods. We used both radioactive isotope and non-radioisotope PCR-SSCP techniques. RESULTS: The results of this study demonstrated that activation of ras genes due to point mutations in chromate-related lung cancer is a rare event. CONCLUSIONS: Ras oncogenes activated by point mutations do not have a major role in the process of tumorigenesis of chromate-related lung cancer.     

石英的人类致癌性在DNA分子水平的证据

收集３６例石英粉尘作业工人肺癌（石英肺癌）的组织石蜡包埋物,提取其 Ｄ Ｎ Ａ,对 ｐ５３基因和 Ｋ ｒａｓ 基因进行了 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ、 Ｐ Ｃ Ｒ Ｒ Ｆ Ｌ Ｐ及 Ｄ Ｎ Ａ 测序分析,发现具有完全有别于普通肺癌的特异基因突变谱。小细胞肺癌的 ｐ５３基因突变多发于第８外显子,腺癌的 ｐ５３基因突变率高。石英肺癌的 Ｋ ｒａｓ 基因突变未见一例发生于普通肺癌突变热点区第１２密码子上,相反发现多处非热点区突变,突变类型以 Ｇ→ Ｃ 突变为主。     

镍化合物诱发细胞恶变过程中的P53基因的变化

目的 探讨三种镍化合物在诱发细胞恶变过程中不同阶段P53基因突变情况，并比较它们之间的差异。方法 三种镍化合物转化细胞接种BALB／c裸鼠，应用PCR－SSCP进行肿瘤细胞和转化细胞P53基因第5－8外显子检测。结果 硫化镍组一个经软琼脂筛选的转化细胞系和相应的肿瘤细胞系P53基因第8外显子检出突变。氯化镍组的肿瘤细胞系第6外显子检出突变。结论 本文说明了镍化合物诱导细胞恶变的晚期发生P53基因突变。     

PCR-SSCP技术检测矽肺合并肺癌癌组织p53基因突变的研究

用ＰＣＲ－ＳＳＣＰ分析法对３６例矽肺患者的石蜡包埋的原发性肺癌组织ｐ５３基因第５、７、８外显子进行了检测，检出突变１５例。突变在第５、７、８外显子上都有发生，但以第８外显子上发现的阳性突变最多。对肿瘤类型和ｐ５３基因突变的关系进行了分析，发现矽肺病例肺腺癌ｐ５３基因阳性突变率最高，为５３．９％，高于普通型肺癌（３３．０％）。进一步对其中１例样本进行核苷酸序列直接测定，结果显示第５外显子非突变热点区的第１４４位密码子核苷酸由ＣＡＧ突变为ＡＡＧ，氨基酸由谷氨酰胺突变为赖氨酸，以上结果与非职业肺癌明显不同，提示ｐ５３基因突变在矽肺病例肺癌发生中起着重要作用，可能与矽尘作业环境中含有的某些化学致癌物有关。     

硫化镍对16HBE细胞中基因的影响

目的 检测结晶型硫化镍(NiS)对K-ras基因和P15基因的改变及基因组不稳定性的影响，从而进一步探讨镍化合物致癌的分子机制。方法 采用限制性片段长度多态性分析和聚合酶链反应一单链构象多态性分析方法探查结晶型NiS在诱导16人气管上皮细胞(HBE)恶变过程中的K-ras基因Exonl第12密码子及第12密码子以外的密码子改变情况。采用PCR-SSCP分析方法探查结晶型NiS在诱导16HBE细胞恶变过程中的P15基因Exon2存在状况。采用随机扩增多态性技术来对结晶型NiS在诱导16HBE细胞恶变过程中的基因组不稳定性进行分析。结果 K-ras基因Exonl和P15基因Exon2未发生改变。本实验所选用的7条随机引物均能扩增出清晰、明显的条带。其中P4、P5、P7两条引物扩增的片段在实验组和对照组之间无差异，其余4条引物均有差异。对于同一随机引物他们都具有特异的带型。结论 P15基因第2外显子和K-ras基因第1外显子(包括第12、13密码子)可能不是结晶型NiS作用的靶部位。在结晶型NiS诱发细胞恶性转化过程中，基因组变得逐渐不稳定。     

食管癌P53基因第5外显子突变分析

目的　分析不同地区食管癌组织中 p5 3基因第 5外显子的突变谱。 方法　采用PCR扩增产物纯化后直接DNA序列测定技术对陕西省西安市 4 2例和河南省林州市 4 3例食管癌标本 p5 3基因第 5外显子突变情况进行检测。结果　西安和林州市食管癌标本中 p5 3基因第 5外显子突变率分别为 14 3% (6 /42 )和 18 6 % (9/43) ,两地突变率比较差异无统计学意义 (P >0 0 5 )。西安 6个突变位点中 4个为点突变 ,2个为缺失突变 ;林州 10个突变位点中9个为点突变 ,1个为插入突变。西安市有 4例突变发生在 12 6～ 12 8位点 ,林州市仅有 2例发生在该区域 (2 /10 ) ,但两者相比差异无统计学意义 (P >0 0 5 )。结论　西安市食管癌 p5 3基因第 5外显子的突变位点相对集中 ,林州市突变位点比较分散 ,可能与该地区多种危险因素的暴露有关。     

Malignant mesothelioma in Lynch syndrome: A report of two cases and a review of the literature

Malignant mesothelioma is a rare and aggressive cancer most typically associated with prior asbestos exposure. The nature of the relationship between asbestos exposure and hereditary familial syndromes predisposing to malignancy has not been determined. We report two Lynch syndrome patients with paraoccupational asbestos exposure who developed diffuse malignant mesothelioma of the pleura or peritoneum. Interestingly, one showed a separate focus of pleural well-differentiated papillary mesothelioma. It is likely that Lynch syndrome patients are at increased risk for the development of mesothelioma in the setting of exposure to asbestos, even at what is generally considered to be low levels. In the presence of a documented history of low-level asbestos exposure, patients with genetic predisposition disorders (including Lynch syndrome) should be considered to have an independent risk factor modifying the effects of asbestos exposure.     

p53-Bax线粒体凋亡通路DNA甲基化与胆管癌病理生物学的关系

目的探讨p53-Bax线粒体凋亡通路抑癌基因启动子区的甲基化状态与胆管癌病理生物学行为的关系。方法采用甲基化PCR检测36例胆管癌患者癌组织和癌旁组织中抑癌基因甲基化诱导静止基因（TMS1／ASC）、死亡相关蛋白激酶（DAPK）和p14启动子区甲基化状态,并对p53基因外显子5～8进行DNA测序,分析以上基因的突变与胆管癌生物学行为的关系。结果24例（66．7％）胆管癌患者癌组织中至少有1个抑癌基因启动子区存在甲基化,p14、DAPK和TMS1／ASC的甲基化率分别为25％、30．6％和36．1％。5例（13．9％）患者的癌旁组织中存在抑癌基因启动子区甲基化,其中TMS1／ASC启动子区甲基化3例（8．3％）,DAPK启动子区甲基化2例（5．6％）。DNA测序显示,22例（61．1％）胆管癌患者癌组织中p53基因外显子5～8存在突变。其中14例（38．9％）p53基因外显子突变伴1个以上抑癌基因启动子区甲基化,与胆管癌的病理类型、浸润深度、分化程度显著相关（P〈0．05）。抑癌基因非甲基化及p53突变阴性组（4例）术后1、2、3年的生存率分别为70％、43％、28％,抑癌基因甲基化及p53突变阳性组（14例）术后1、2、3年的生存率分别为28％、5％、0％,前者的生存率显著高于后者（X^2=9．060,P=0．03）。结论p53-Bax线粒体凋亡通路中抑癌基因启动子过甲基化是胆管癌组织中常见的分子事件,癌旁组织中TMS1／ASC和DAPK基因甲基化程度虽然较低,但可能对胆管癌有早期诊断意义。p53突变伴抑癌基因甲基化与胆管癌的病理生物学行为有关,趋向于较高的恶性程度和较低的生存率。     

帕金森病Parkin,LRRK 2基因序列变异研究

目的 探讨散发性早发性帕金森病患者遗传易感基因突变的形式和分布及易感基因突变在PD发病中的可能作用.方法 病例组由23例散发性早发帕金森患者组成,10例对照组.以基因组DNA为模板,扩增Parkin基因的第1、4、6号外显子和LRRK 2基因的第31号外显子.观察PCR产物测序后的突变情况.结果 发现样本中存在突变以及单核苷酸多态性(Single Nucleotide Polymorphism,SNP).在一例患者Parkin基因Exon6上发现735ntT→C,对应的密码子TGT212CGT,翻译的氨基酸C212R,国内罕有报道.另一例患者Parkin基因Exon6上发现突变833ntG→C,导致第244密码子同义突变.结论 Parkin基因外显子4、6的突变可能是我国散发性早发PD患者的致病原因之一.     

中国人热性惊厥与MASS1基因的分子生物学研究

目的:研究中国人热性惊厥患者MASS1单核苷酸多态性位点(SNP)与基因突变情况。方法:抽取44位热性惊厥患者外周血,提取DNA,设计MASS1基因全部35个编码外显子引物,经PCR扩增,采用Sanger双脱氧链终止法测序。结果:共发现3个单核苷酸多态性位点,其中1个为位于第13外显子尚未有文献报道的新单核苷酸多态性位点2625A>C,致使原来的密码子CGA变成CGC,均编码精氨酸。另有2个单核苷酸多态性位点与已有文献报道相符,分别为第29外显子6666G>A及第33外显子7798T>G单核苷酸多态性位点。未发现新的基因突变。结论:MASS1基因存在多个单核苷酸多态性位点,对进一步研究中国人热性惊厥分子遗传学机制具有理论积累意义。     

聚合酶链反应-单链构象多态法在研究新疆地方性砷中毒患者p53基因突变中的应用

目的 探索砷致癌的基因多态性,为进一步研究砷对人体健康作用的远期影响提供科学依据.方法 于2001年采集新疆奎屯123团4例经病理诊断为皮肤癌的砷中毒患者的6份皮肤癌石蜡包埋组织及其4份血液组织;采集奎屯128团2例经病理诊断为皮肤癌的砷中毒患者和2例癌前砷中毒患者的血液组织;采集对照区新疆奎屯125团2例健康居民的血液组织.采用聚合酶链反应-单链构象多态(PCR-SSCP)银染术测定p53基因外显子(exon,E)5～9突变情况.结果 p53基因中,仅E6、E7发生突变.凝胶电泳成像显示,p53基因E6、E7有异常条带、条带缺失及条带上移现象.结论 砷对p53基因有致突变作用,突变位于E6、E7.     

PCR扩增石蜡包埋小鼠组织p53基因突变热点片段

目的 扩增石蜡包埋小鼠组织p53基因第5、7外显子。方法 用3种方法提取DNA，比较提取效果，用聚合酶链式反应（PCR）扩增相应片段。结果 用二甲苯脱蜡，蛋白酶K消化120小时可获得较好的DNA提取物。结论 用该提取物为模板扩增小鼠p53基因突变热点片段成功率高。     

MTHFR variants reduce the risk of G:C->A:T transition mutations within the p53 tumor suppressor gene in colon tumors

5,10-Methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in folate-mediated 1-carbon metabolism. Reduced MTHFR activity has been associated with genomic DNA hypomethylation. Methylated cytosines at CpG sites are easily mutated and have been implicated in G:C-->A:T transitions in the p53 tumor suppressor gene. We investigated 2 polymorphisms in the MTHFR gene (C677T and A1298C) and their associations with colon tumor characteristics, including acquired mutations in Ki-ras and p53 genes and microsatellite instability (MSI). The study population comprised 1248 colon cancer cases and 1972 controls, who participated in a population-based case-control study and had been analyzed previously for MSI, acquired mutations in Ki-ras, p53, and germline MTHFR polymorphisms. Multivariable-adjusted odds ratios are presented. Overall, MTHFR genotypes were not associated with MSI status or the presence of any p53 or Ki-ras mutation. Individuals with homozygous variant MTHFR genotypes had a significantly reduced risk of G:C-->A:T transition mutations within the p53 gene, yet, as hypothesized, only at CpG-associated sites [677TT vs. 677CC (referent group) OR = 0.4 (95% CI: 0.1-0.8) for CpG-associated sites; OR = 1.5 (0.7-3.6) for non-CpG associated sites]. Genotypes conferring reduced MTHFR activity were associated with a decreased risk of acquired G:C-->A:T mutations within the p53 gene occurring at CpG sites. Consistent with evidence on the phenotypic effect of the MTHFR C677T variant, we hypothesize that this relation may be explained by modestly reduced genomic DNA methylation, resulting in a lower probability of spontaneous deamination of methylated cytosine to thymidine. These results suggest a novel mechanism by which MTHFR polymorphisms can affect the risk of colon cancer.     

食管癌与p53突变和人乳头状肉瘤病毒感染

目的探讨p53基因突变与人乳头状肉瘤病毒16（HPV16）感染在食管癌发病中的作用及相互关系。方法应用病例-病例研究方法进行分析，采用聚合酶链反应（PCR）、单链构像多态性（SSCP）、测序等分子生物学技术对110例食管癌组织标本中p53基因的突变与HPV16感染进行了检测。结果110例食管癌组织标本p53基因突变率为49．1％，其中外显exon5—6，exon7，exon8—9的突变率分别为19．1％，27．3％，17．2％。食管癌组织HPV16的检出率为49．1％。吸烟患者p53基因突变率（61．4％）明显高于非吸烟患者p53基因突变率（48．2％），差异有统计学意义；淋巴结转移患者p53基因突变率（65．2％）明显高于无淋巴结转移患者p53基因突变率（37．5％）；HPV16阳性者中，p53突变率是40．7％，HPV16阴性者仅为57．1％，两者差异无统计学意义。结论HPV16感染可能是食管癌高发区的危险因素；p53基因突变与吸烟、淋巴结转移有明显相关性；HPV16感染和p53基因突变可能是2个独立的事件。     

Gallium scanning in differentiating malignant from benign asbestos-related pleural disease

In order to assess the utility of 67gallium citrate in delineating malignant pleural mesothelioma from benign asbestos-related pleural disease, 49 patients with malignant mesothelioma and 16 with benign asbestos-related pleural disease were studied. Seven patients with malignant mesothelioma had no history of asbestos exposure, while the remaining 58 patients were exposed. Forty-three of the 49 patients (88%) with malignant mesothelioma had a positive 67gallium scan including 36 of the 42 (86%) patients with asbestos exposure and all 7 patients without a history of asbestos exposure. Three of 16 patients (19%) with benign asbestos-related pleural disease had a positive scan. 67Gallium radionuclide imaging is nonspecific but may be valuable in noninvasive monitoring of asbestos-exposed populations, which have a high risk for the late development of benign and/or malignant pleural disease.