人食管癌相关基因cDNA片段的克隆与初步鉴定

目的 分离人原发性食管组织中瓣的相关基因,揭示食管癌的易感性与癌变原理,方法 用高效,灵敏的ｍＲＮＡ差异显示技术,以２例正常食管上皮,１例癌旁上皮,２例高癌家族的食管癌组织互为对照,通过其对其因表达的比较,找出差异条带,进行ＲＴ－ＰＣＲ鉴定和ＤＮＡ序列分析,结果 （１）在实验中,分离,鉴定了１８个差异片段,其中包括正常组织不表达的突变食管基因（ｍｕｔａｔｅｄｅｓｏｐｈａｇｅａｌｇｅｎｅ,ＭＥＧ）５     

Lewis肺癌的RAPD分析及其差异片段克隆

目的 检测Lewis肺癌的遗传改变,寻找与癌变相关的DNA序列或片段。方法 用105条引物对Lewis肺癌及其来源小鼠C57BL／6J正常组织基因组DNA进行随机扩增多态DNA（RAPD）扩增,比较肿瘤组织与正常组织的扩增图,对差异很明显的片段回收、克隆和测序,序列与GenBank数据库进行同源性比较。结果 105条引物中有25条引物扩增出的条带在肿瘤组织与其相应正常组织间存在差异。引物AB7－2和AB7－11扩增所得差异片段L7－2和L7－11回收纯化后克隆测序。L7－2片段长730bp,该序列与已知序列没有任何相关性。L7－11长779bp,与小鼠的V kappa 21-11gene在核酸水平具有90％的同源性。结论 本研究用RAPD技术检测出Lewis肺癌存在一定程度的遗传改变,并发现与Lewis肺癌发生相关的DNA序列L7－2和L7－11,这种改变可能与肺癌的发生相关。     

Analysis of the relationship between p53 immunohistochemical expression and risk factors for lung cancer, with special emphasis on residential radon exposure.

BACKGROUND: Indoor radon exposure has been postulated as the second risk factor of lung cancer after tobacco. The objective of this work is to analyze if there exists any effect on p53 immunohistochemical expression mainly due to radon exposure and other risk factors for lung cancer. PATIENTS AND METHODS: The tumor samples of a case series of 163 lung cancer cases were analyzed to know the p53 staining. The staining was classified into four categories from no staining to intense staining (>60%). This staining was correlated with radon exposure, tobacco consumption, having worked in risk occupations for lung cancer and alcohol consumption. RESULTS: Only 72 samples could be analyzed for immunohistochemistry and some of these samples were sequenced from exons 4-8. No association was observed for staining intensity and radon exposure and also for tobacco and occupation. A slight association with a more intense staining was observed for high alcohol intake. In the four samples with a staining >60% that could be sequenced from exons 4 to 8, no mutation was observed in the p53 gene. CONCLUSION: There is no association between radon exposure and p53 expression, indicating that maybe the effect of radon is not mediated through p53 alterations.     

应用荧光差异显示法对胃癌及癌前病变相关基因的研究

背景与目的：一般认为胃癌是由癌前病变(慢性萎缩性胃炎、肠化生和不典型增生)逐步发展形成的。mRNA差异显示法是筛选肿瘤发生、发展中差异表达基因的有用方法。在胃癌、癌前病变中寻找差异表达的基因将有助于确定胃癌发生前的胃粘膜组织的分子变化。方法：应用荧光mRNA差异显示技术分析胃癌(2例)、癌前病变(2例)和正常胃粘膜(2例)组织,鉴别并分离差异表达的基因片段,进行PCR再扩增。将扩增cDNA片段克隆后进行测序,测序结果提交GenBank,经BLAST软件检索以进行同源性分析。RT-PCR检测血影蛋白基因在胃癌(7例)、癌前病变(7例)和正常胃粘膜(7例)的表达。结果：发现4个差异表达的cDNA片段,其中3个cDNA片段在胃癌中高表达,1个cDNA片段在正常组织和癌前病变组织中高表达。1个在胃癌组织中高表达的cDNA片段与血影蛋白基因(SPTANl)同源,RT-PCR检测也发现血影蛋白基因在胃癌组织中高表达。另外3个与GenBank中已知的基因序列同源,但其功能目前尚不清楚。结论：所发现的4个差异表达的基因有3个在胃癌组织中高表达,其中SPTAN1基因在胃癌组织的表达比正常胃粘膜及胃异型增生组织表达明显高。     

人喉癌新的相关基因cDNA片段的克隆与鉴定

目的:寻找喉癌新的相关基因,阐明喉癌发生的分子机制,为喉癌的基因诊治提供研究的靶基因。方法:应用mRNA差异显示方法对喉癌患者的癌旁、癌及颈部转移淋巴结组织差异cDNA进行了筛选、克隆和测序,并对其进行了鉴定和同源性分析。结果:筛选出7个差异cDNA片段,克隆并鉴定了其中两个与喉癌发生相关的cDNA片段,它们与多种肿瘤相关基因的cDNA序列同源。结论:克隆的两个cDNA片段可能是喉癌新的相关基因cDNA片段,全长cDNA克隆及深入研究其功能有助于发现喉癌新的相关基因。     

云南曲靖肺癌高发区氡气浓度与肺癌发病率的关系

目的:探讨肺癌高发区氡气浓度与肺癌发病率是否存在相关性.方法:采用中国辐射防护研究所生产的RLM-L型连续测氡仪,分别对处于宣威、富源肺癌高发区的7个农民肺癌家庭进行氡气监测.同期对宣威肺癌高发区的3个办公区、肺癌低发区的2个农民肺癌家庭以及曲靖麒麟区肺癌低发区的4个健康人口家庭共16个点进行氡气浓度监测.结果:宣威、富源肺癌高发区中6个点氡气浓度均远远超过国家环境标准的200 Bq/L,赋有"世界肺癌村"之称的来宾镇虎头村,其村北、村西、村南3个肺癌家庭氡气浓度也相对较高,但均小于200 Bq/L;其余7个监测点包括1个肺癌高发区、3个肺癌低发区的肺癌家庭以及麒麟区3个健康家庭氡气浓度均低于50 Bq/L.结论:宣威、富源不同地区氡气浓度与肺癌发病率存在一定相关性,氡气浓度高则肺癌发病率也高.宣威、富源肺癌高发病率除了与其特有的环境地理因素、遗传、基因易感性等相关外,也与氡气污染有关.     

胃癌相关基因gcI的克隆及其功能预测

目的:应用生物信息学平台对胃癌相关基因表达片段进行cDNA全长克隆及序列分析和蛋白功能预测.方法:选用人胃癌、癌前病变及正常胃粘膜组织经mRNA差异显示技术筛选获得的差异表达片段,对所获得的1条功能未知基因表达片段"Ⅰ",利用5'cDNA末端快速扩增方法扩增全长cDNA,结合Northern杂交印迹方法进行验证分析;利用NCBI、EMBL数据库资源分析其编码氨基酸的生物学特征以及预测其编码蛋白的功能.结果:扩增得到序列Ⅰ的全长cDNA,命名为gcI.分析显示gcI含有2个跨膜区,定位于胞膜;含有3个蛋白激酶C磷酸化位点和3个N端豆蔻酰化作用位点.结论:克隆的胃癌相关基因表达全长序列gcI、编码与信号转导有关的跨膜蛋白,可能是参与胃癌发生过程中的相关基因.     

核糖体蛋白S24基因在胃癌及其癌前病变中差异表达的研究

背景与目的:将胃癌组织、正常胃黏膜及胃癌前病变的基因表达进行比较,找出与人胃癌密切相关的基因片段,为进一步探讨胃癌的发生机制、胃癌的早期诊断和治疗方案提供重要的理论根据。材料与方法:应用荧光mRNA差异显示技术(FluorescentmRNAdifferentialdisplay,FDD)分析3例胃癌、3例正常胃粘膜、3例胃癌前病变组织,分析基因差异表达情况。分离差异表达基因片段,进行PCR扩增。将cDNA片段测序,测序结果提交Genbank,经BLAST软件检索与同源性分析。选取其中差异表达的核糖体蛋白S24(RibosomalproteinS24,RPS24)基因,应用Northern杂交进行验证,并进一步对RPS24基因进行基因表达系列分析和组织表达谱分析。结果:mRNA差异显示发现RPS24基因在胃癌组织中的表达明显高于癌前病变和正常胃黏膜组织,并且Northern印迹验证阳性。基因表达系列分析和组织表达谱分析发现RPS24基因在多种肿瘤组织中表达增高以及分布广泛。结论:与非癌组织相比,RPS24基因在胃癌中表达明显增高,RPS24的高表达可能与胃癌的发生、恶性进展有关。     

子宫肌瘤致病相关基因的初步研究

背景与目的：已知子宫肌瘤的发生与某些基因的表达异常有关,但其分子生物学机制尚未完全阐明,本研究通过比较子宫肌瘤及正常子宫肌组织在mRNA分子水平上的表达差异,筛选子宫肌瘤的致病相关基因。方法：应用荧光标记的mRNA差异显示技术,包括4种锚定引物和3种随机引物,共12种组合,比较子宫肌瘤及正常子宫肌组织基因表达的差异,对获得的差异基因片段进行克隆、测序及同源性分析,并对其中一条差异片段在子宫肌瘤及正常子宫肌组织中的表达情况进行RT-PCR分析。结果：从子宫肌瘤中分离出差异基因片段27条,克隆及测序显示N568片段与G(3RG114基因序列同源性达96％,RT-PCR证实该基因的表达水平在90．0％(9／10)的肌瘤组织中显著低于正常子宫肌组织。结论：N568片段与GCRG114基因序列有高度同源性,可能与子宫肌瘤的发病相关。     

蛋白质芯片筛选香港非吸烟女性肺腺癌组织的肿瘤标志物

背景与目的:吸烟虽为肺癌的重要病因,但香港女性肺腺癌患者中逾半数是从不吸烟。本研究应用高通量蛋白质芯片技术,以冀筛选其相关特异性标志蛋白分子作为临床诊断指标。方法:采用表面增强激光解吸电离-飞行时间质谱仪对29例肺癌组织及其配对的癌旁正常组织进行蛋白质指纹图谱检测。结果:针对非吸烟女性肺腺癌与其配对之癌旁正常组织进行检测分析,筛选出52个有明显表达差异的标志蛋白,其中差异性最大的10个标志蛋白之差异率为60%～213%。而非吸烟患者之肺癌亦与吸烟患者之肺癌作蛋白质指纹图谱检测,发现有84个明显表达差异的标志蛋白,其中差异性最大的10个标志蛋白之应用受试者特征曲线下面积为0.82～0.89。把女性肺癌组织与男性肺癌组织蛋白质指纹图谱作比较,则发现有69个明显表达差异的标志蛋白,其中差异性最大的10个标志蛋白之应用受试者特征曲线下面积为0.81～0.86。结论:本研究成功利用蛋白质芯片技术筛选香港非吸烟女性肺腺癌组织中的标志蛋白,更通过与吸烟及性别相关之标志分子的比较,找到非吸烟女性肺腺癌的特异性肿瘤标志物。     

瘦素和瘦素受体在肺癌组织中的表达

目的探讨瘦素及其受体在肺癌发生、发展中的作用。方法采用免疫组化S-P法检测68例肺癌组织、相应癌旁组织和正常肺组织中瘦素及其受体的表达。结果瘦素及其受体在肺癌组织中的表达率分别为72.06%(49/68)、64.71%(44/68),相应癌旁组织分别为42.65%(29/68)、33.83%(23/68),正常肺组织分别为26.47%(18/68)、29.41%(20/68)。瘦素及其受体在肺癌组织的表达高于相应癌旁组织及正常肺组织(P<0.05)。在肺癌组织中瘦素与瘦素受体的表达呈正相关关系(r=0.754,P<0.01)。结论瘦素及其受体的表达在肺癌的发生、发展中起一定的促进作用。     

人胃癌差异表达基因片段的分离与测序

目的:肿瘤差异表达基因在肿瘤的发生发展中起重要作用,胃癌在我国全部恶性肿瘤死亡构成比中居第一位,本文拟钓取人胃癌差异表达基因片段,探讨胃癌发生发展的机制.方法:应用最新分离差异基因的mRNA差异显示技术,优化反应条件对人胃癌组织中差异表达的基因片段进行分离,测序和Northem验证.结果:获得经Northem杂交证实的在胃癌组织中表达的两个cDNA片段scg1和scg2,序列分析表明长度分别为194bp和343bp,同源性比较结果显示scg1和scg2与GenBank中已发表基因序列均无同源性.结论:本研究初步证实scg1和scg2可能是人胃癌组织中表达的新基因片段,推测与人胃癌发生发展相关.检测全长cDNA序列的深入工作正在进行中.     

Quantitative evaluation of the radon and lung cancer association in a case control study of Chinese tin miners

Studies of underground miners have consistently shown an increased risk of lung cancer with cumulative exposure to radon-222 and its decay products. Although the deleterious effects of high radon exposure are clear, questions regarding the shape of the exposure-response relationship, and the effects of time factors such as attained age, time since exposure and early age at first exposure, the effect of exposure rate, and the joint association of radon exposure and tobacco use have not yet been fully clarified. This report considers these questions by fitting various models for the relative odds of disease to 74 male lung cancer cases who were diagnosed between 1981 and 1984 and were alive in 1985 and an equal number of controls. All subjects are current or past employees of the Yunnan Tin Corporation, Gejiu City, China, who reside in the local area. Workers were interviewed to obtain information on work history, from which radon exposure in cumulative working level months and arsenic exposure were estimated, and on tobacco use. Results indicate that excess relative risk increases by 1.7% per cumulative working level month [95% confidence interval (0.5, 5.4)]. The linear exposure response relationship significantly declines with year since last radon exposure (P = 0.02). The risk trend also declines with increasing exposure rate (P = 0.001), indicating that long duration of exposure at a low rate may be more deleterious than short duration of exposure at a high rate. A unique aspect of this study population is the very early ages at first radon exposure for many of the workers, about 37% of the radon-exposed workers were first exposed under the age of 13 years. The analysis shows no modification of the radon lung cancer relationship with age at first exposure. These patterns of risk with radon exposure are generally consistent with those reported in the recent National Academy of Sciences' Biological Effects of Ionizing Radiations IV report. The primary method of tobacco consumption in this area of China is by waterpipe. Lung cancer risk increases with pipe-years of use. The joint analysis of tobacco use and radon exposure supports the Biological Effects of Ionizing Radiations IV conclusion that the most likely model is between additive and multiplicative. The variations of the radon lung cancer relationship by years since last exposure and exposure rate are not affected by adjustment for arsenic exposure.     

Residential radon and lung cancer risk: an updated meta- analysis of case-control studies

Background: Numbers of epidemiological studies assessing residential radon exposure and risk of lung cancerhave yielded inconsistent results. Methods: We therefore performed a meta-analysis of relevant published casecontrolstudies searched in the PubMed database through July 2011 to examine the association. The combinedodds ratio (OR) were calculated using fixed- or random-effects models. Subgroup and dose-response analyseswere also performed. Results: We identified 22 case-control studies of residential radon and lung cancer riskinvolving 13,380 cases and 21,102 controls. The combined OR of lung cancer for the highest with the lowestexposure was 1.29 (95% CI 1.10-1.51). Dose-response analysis showed that every 100 Bq/m3 increment inresidential radon exposure was associated with a significant 7% increase in lung cancer risk. Subgroup analysisdisplayed a more pronounced association in the studies conducted in Europe. Studies restricted to female ornon-smokers demonstrated weakened associations between exposure and lung cancer. Conclusions: This metaanalysisprovides new evidence supporting the conclusion that residential exposure to radon can significantlyincrease the risk of lung cancer in a dose-response manner.     

D10-86 cDNA片段在人食管癌中的表达

目的：D10－86 cDNA片段是通过mRNA差异显示技术分离的在食管癌中低表达的cDNA片段。由于mRNA差异显示技术具有较高的假阳性，故需进一步鉴定其在食管癌中的表达情况。方法：采用RT－PCR方法检测了41对食管癌组织及配对食管癌旁粘膜和食管癌细胞系EC9706和肺癌细胞系GLC82中D10－86 cDNA片段的表达。结果：D10－86 cDNA片段在41．5％（17／41）食管癌组织中的表达明显低于相应的癌旁粘膜，未检测到表达和微弱表达的分别为9．8％（4／41）和31．7％（13／41），食管癌组织中的表达高于相应的癌旁粘膜7．3％（3／41），表达无差别的51．2％（21／41）；在食管癌细胞系EC9706和肺腺癌细胞系GLC82未检测到表达；D10－86 cDNA片段表达下调与食管癌临床病理因素无显著相关性（P＞0．05）。结论：D10－86 cDNA片段在人食管癌中表达下调。     

Livin 蛋白在支气管肺癌中的表达及与 Bcl －2的关系

目的：探讨 Livin 蛋白在原发性支气管肺癌组织的表达及其与 Bcl －2蛋白之间的关系。方法：采用免疫组化 SP 法检测 Livin 与 Bcl －2在支气管肺癌组织和癌旁5cm 以上正常肺组织中蛋白的表达情况。结果：肺癌组的 Livin 蛋白和 Bcl －2蛋白阳性表达率均高于对照组（均 P ＜0．05）。男性肺癌患者 Livin 蛋白的表达阳性率为62．96％,女性为40．00％,两者相比差异有统计学意义（P ＜0．05）。有淋巴结转移的肺组织中Livin 蛋白和 Bcl －2蛋白的阳性表达率较高（78．95％ vs 36．36％,68．42％ vs 27．27％,均 P ＜0．05）。肺鳞癌组织 Bcl －2基因蛋白的阳性表达率为58．82％,显著高于肺腺癌组织中 Bcl －2基因蛋白表达（20．45％）,差异有统计学意义（P ＜0．05）。吸烟指数≥400的肺癌患者 Livin 蛋白及 Bcl －2蛋白表达阳性率均高于吸烟指数＜400的患者,差异有统计学意义（P ＜0．05）。支气管肺癌的组织分化程度、临床分期对 Livin 蛋白和 Bcl －2蛋白的阳性表达率的影响均无统计学差异。Livin 蛋白与 Bcl －2蛋白的表达呈正相关。结论：Livin 蛋白与Bcl －2蛋白在支气管肺癌的异常表达,有望成为肿瘤诊断及基因治疗的新靶点。Livin 蛋白与凋亡相关蛋白Bcl －2的异常表达在支气管肺癌癌变中有协调作用。     

Lung cancer in women and type of dwelling in relation to radon exposure

A case-control study based on interviews with 210 incident female lung cancer patients, 209 age-matched population controls, and 191 hospital controls was carried out in Stockholm county, Sweden. Radon measurements made in a sample of 303 dwellings, in which the study subjects had lived, showed that dwellings with ground contact had an average concentration of approximately 160 Bqm-3, twice the average concentration of other dwellings. A cumulated radon exposure index was calculated for each subject based on data from the interviews and the measurements. For the total group of lung cancer a relative risk (RR), adjusted for smoking, age, and degree of urbanization, of 1.8 (95% confidence interval: 1.2-2.9) and 1.7 (0.9-3.3) associated with "intermediate" and "high" exposure to radon was found. There was also a significant trend to a positive dose-response relationship (Ptrend = 0.03). For small cell cancer the corresponding figures for RRs were 1.9 (0.6-4.5) and 4.7 (1.5-14.2), respectively (Ptrend = 0.01). There seemed to be a positive interaction between radon exposure and smoking in relation to lung cancer. The findings indicate that domestic radon may be of importance for the induction of lung cancer, particularly for some histological types.     

抑制性消减杂交构建肺腺癌及同源正常组织cDNA文库

目的：分离肺腺癌及同源正常组织中的未知特异性基因片段，并建立相应的cDNA文库，为研究肿瘤基因疫苗打下良好的基础。方法：从肺腺癌及同源正常组织中分别提取总RNA，磁珠分离mRNA并逆转录为dsDNA，经RasI消化酶切将Adaptor-1和Adaptor-2R适配子分别与肺腺癌消化后的cDNA连接（作为tester），再与正常同源组织（作为driver）进行正向杂交和逆向杂交。具有上述2种不同适配子的杂交后cDNA分子，通过初次PCR和巢式PCR富集未知的cDNA片段，此PCR产物与TA克隆载体pGEM连接，转化Top10大肠杆菌，获得白色菌落并经菌液PCR扩增出未知基因片段。结果：经正向抑制性消减杂交和逆向抑制性消减杂交分别获取了肺腺癌组织及同源正常组织的特异性cDNA文库。结论：抑制性消减杂交是一种快速、方便、有效的建立cDNA文库的方法。     

沉默S100P基因抑制肺腺癌细胞A549增殖、迁移和侵袭

目的:探讨肺癌组织及细胞中S100P基因表达对增殖、迁移、侵袭的影响及其作用机制。方法:在GEPIA(Gene Expression Profiling Interactive Analysis)数据库和HPA数据库（Human Protein Atlas）得到肺癌数据,分析S100P 基因在不同分型肺癌组织与正常组织中差异表达。使用shRNA -S100P RNA下调肺癌细胞系中S100P基因表达水平;细胞的增殖活性使用MTS比色法检测;细胞周期变化运用流式细胞仪检测;细胞划痕实验和Transwell实验分别检测细胞迁移能力和侵袭能力。RT-qPCR与Western blot分别验证S100P表达及细胞周期、凋亡蛋白表达水平。结果:S100P基因在肺癌组织中表达水平明显高于正常组织(P＜0.01);不同分型的肺癌组织中S100P基因表达存在差异。与对照组相比,下调S100P使A549增殖、迁移和侵袭能力均降低。结论:S100P基因在肺癌中高表达且下调后可抑制A549细胞的增殖、迁移和侵袭能力。