【作者】 周春辉；

【导师】 申洪；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 研究背景与目的肺癌是世界范围内最常见的恶性肿瘤,其发病率及死亡率占各种恶性肿瘤的首位,同时也是引起恶性胸腔积液的主要原因之一。大约10%的肺癌患者是以胸腔积液为首发症状,约30%-40%的肺癌患者在病程进展中会出现胸腔积液,而出现胸腔积液的肺癌患者预后差,生存期短。临床上对出现恶性胸腔积液的患者需要及时准确的诊断,以便采取有效的治疗方法。因此探讨肺癌的发生、发展及转移机制,建立一组科学有效的诊断方法,是提高肺癌早期诊断阳性率的重要途径,也是提高肺癌患者生存率与治愈率的关键,对于肺癌的防治具有重要的现实意义。甲状腺转录因子-1（thyroid transcription factor-1,TTF-1）是新近发现的一种表达于甲状腺、肺和脑的转录因子,与这些器官的组织发生、功能维持、肿瘤发生及其它相关疾病的发生密切相关。TTF-1定位于人类染色体14q13,含有371个氨基酸。人类TTF-1与大鼠TTF-1的cDNA核酸序列具有98%的同源性。人类TTF-1基因全长大约3.3kb,包含2个外显子和1个内含子,在第160-220区域有由60个氨基酸组成的具有绝对保守性的同源序列。在正常肺组织中,TTF-1表达于成熟的肺泡Ⅱ型上皮细胞和肺导气部的支气管上皮细胞的亚段,而Ⅰ型肺泡上皮细胞始终不表达。TTF-1控制着表面蛋白A（SP-A）、表面蛋白B（SP-B）、表面蛋白C（SP-C）和Clara细胞分泌蛋白（CCSP）的表达。到目前为止,TTF-1是唯一控制4个肺特异性基因的转录因子。Willemsen等的研究发现,一23岁男子因TTF-1基因在859—860位发生杂合性插入突变,导致舞蹈症、智力发育迟缓、原发性甲状腺机能减退以及慢性肺疾病。他们称此疾病为脑-甲状腺-肺综合征。此患者最终死于肺大细胞癌。Kang Y等在小鼠模型中发现,TTF-1表达减少与鼠肺癌的发生有关,但癌变的具体机制尚不清楚。因此,肺癌发生是否与TTF-1基因异常表达有关受到广大学者的关注。TTF-1在肺癌中的研究表明,肺癌类型不同,TTF-1的表达也各异。目前认为,TTF-1是肺腺癌和肺小细胞癌的标志物。研究表明,除甲状腺癌和肺腺癌外,TTF-1很少表达于其它部位的原发腺癌或转移腺癌。因此TTF-1对于胸腔积液中转移性肺腺癌的鉴别诊断具有很高的应用价值。但是关于TTF-1在胸腔积液转移性肺腺癌中表达的量变规律,其基因突变与原发灶TTF-1基因突变位点及类型是否相同,与肺腺癌的发生、发展和转移是否相关,其能否作为肺癌患者胸水中的肿瘤标志物尚不十分清楚。因此本研究拟通过检测TTF-1蛋白及mRNA在恶性胸腔积液不同组织来源的转移性腺癌细胞中的表达情况,通过定量测试TTF-1蛋白和mRNA的表达强度,从蛋白及mRNA水平探讨TTF-1作为胸腔积液中转移性肺腺癌特异性标志物的临床应用价值;应用PCR-SSCP法与DNA测序相结合分析胸水转移性肺腺癌细胞TTF-1第l、2外显子基因突变情况,探讨TTF-1基因突变在肺腺癌发生、发展中的作用;构建绿色荧光蛋白真核细胞表达载体pEGFP-TTF-1,通过转染人肺腺癌细胞系GLC-82,观察外源性TTF-1基因表达对GLC-82细胞生物学特性的影响。方法1、收集南方医院2006年6月至2007年2月癌性胸腔积液48例,其中肺腺癌37例,胃癌4例,乳腺癌3例,卵巢癌2例,子宫内膜癌1例,结肠癌1例。每例均经组织学、细胞学或结合临床资料证实,均制备巴氏、Giemsa和PAS染色细胞涂片和离心沉渣石蜡包埋细胞蜡块。2、采用免疫组织化学和原位杂交技术检测胸水细胞蜡块切片中TTF-1蛋白及mRNA的表达情况。应用Leica Q500 MC图像分析系统测试TTF-1蛋白及TTF-1mRNA的阳性单位（PU）值,分析胸水中不同组织来源转移性腺癌细胞TTF-1蛋白及mRNA表达强度的量变规律及其作为胸腔积液中转移性肺腺癌标志物的临床应用价值。3、应用聚合酶链-单链构象多态性（PCR-SSCP）和DNA测序相结合检测TTF-1基因突变情况:首先合成TTF-1外显子1和外显子2区特异性引物（共3对引物）,PCR扩增待测DNA片段;用PCR-SSCP对PCR产物进行检测,确定突变的基因片段。对经PCR-SSCP检测出有异常电泳条带改变的DNA片段进行测序,确定突变类型,分析基因功能改变。4、应用免疫细胞化学、RT-PCR和Western-blot技术检测人肺腺癌细胞系GLC-82中TTF-1蛋白及TTF-1mRNA表达情况。5、外源性TTF-1基因表达对人肺腺癌细胞系GLC-82生物学特性的影响:构建含TTF-1基因全长cDNA序列的真核表达载体,通过Lipofectamine^TM2000阳离子脂质体介导转染GLC-82细胞;采用G418筛选,挑取抗性单克隆进行扩大培养,应用RT-PCR和Western-blot检测各克隆细胞中TTF-1基因的表达,以TTF-1表达量最高的克隆为下一步实验的研究对象:利用HE染色、扫描电镜和透射电镜观察转染后细胞形态和超微结构的改变;采用MTT增殖实验、平板克隆形成实验、划痕实验和流式细胞术检测外源性TTF-1基因表达对肺腺癌细胞体外增殖的影响;裸鼠皮下成瘤,利用整体成像系统观察外源性TTF-1基因表达对肺腺癌细胞致瘤能力的影响。结果1、胸腔积液中转移性腺癌细胞的形态特点:转移性腺癌患者胸水细胞巴氏染色涂片显示:肿瘤细胞聚集成团或散在分布,细胞体积增大,胞浆内可见粘液空泡,细胞核增大、偏位,异型性明显;在其细胞蜡块HE染色的切片中可见肿瘤细胞排列成腺腔样结构,这些细胞学表现缺乏组织特异性。2、应用免疫组织化学方法检测48例胸腔积液细胞蜡块切片中不同组织转移性腺癌细胞TTF-1蛋白的表达强度并进行定量测试。结果表明:37例肺腺癌胸水标本中TTF-1蛋白表达的阳性率达75.67%,11例肺外腺癌胸水标本TTF-1免疫组化染色均为阴性,即阳性率为0%（0/11）,与胸腔积液中肺源性转移性腺癌癌细胞核TTF-1表达的阳性率相比,差异具有显著性（χ²=19.978,p=0.000）;胸腔积液中肺源性转移性腺癌癌细胞核TTF-1的PU值也显著大于非肺源性腺癌细胞核TTF-1的PU值,差异具有显著性（t=13.208,D=0.000）。3、应用原位杂交方法检测48例胸腔积液细胞蜡块切片中不同组织转移性腺癌细胞TTF-1mRNA的表达强度并进行定量测试。结果表明:其中37例肺腺癌胸水标本中有21例标本TTF-1mRNA呈不同程度的阳性表达,阳性率达56.75%,11例肺外腺癌胸水标本均未检测出TTF-1mRNA,即阳性率为0%（0/11）,与胸腔积液中肺源性转移性腺癌癌细胞核TTF-1mRNA表达的阳性率相比,差异具有显著性（χ²=11.099,p=0.001）。癌性胸水中转移性肺腺癌细胞核TTF-mRNA的PU值明显大于乳腺癌、胃癌、卵巢癌、子宫内膜癌及结肠癌细胞核TTF-1mRNA的PU值,即胸腔积液中肺源性转移性腺癌癌细胞核TTF-1mRNA的PU值与非肺源性腺癌细胞核TTF-1mRNA的PU值相比差异具有显著性（t=9.901,D=0.000）。4、应用PCR-SSCP和DNA双向测序技术检测37例转移性肺腺癌胸腔积液脱落细胞中TTF-1基因的突变情况。结果显示:所检测的37例标本中TTF-1基因外显子1区引物1A、1S所扩增片断a均未出现电泳迁移率的改变,4例转移性肺腺癌TTF-1外显子2区引物2A、2S所扩增片断b出现明显电泳迁移率的改变,经DNA双向测序,均出现单碱基缺失,2例同时出现单碱基置换,突变率为10.8%。5、人肺腺癌细胞系GLC-82中TTF-1蛋白及TTF-1mRNA表达情况免疫细胞化学及Western-blot检测结果显示,GLC-82细胞中TTF-1蛋白及TTF-1mRNA表达均为阴性,适合于进行外源性TTF-1基因表达细胞系的建立。6、外源性TTF-1基因表达对人肺腺癌细胞系GLC-82生物学特性的影响（1）利用Lipofectamine ^TM2000阳离子脂质体介导转染技术建立外源性TTF-1基因表达的细胞株。构建pEGFP-TTF-1融合蛋白真核表达载体,利用Lipofectamine ^TM2000阳离子脂质体介导转染人肺腺癌细胞系GLC-82,通过G418筛选抗性克隆,约14天后,挑取建立抗性单克隆并扩大培养,利用RT-PCR和Western-blot技术检测各克隆的TTF-1表达情况,筛选TTF-1表达最强的克隆为后续实验的研究对象。（2）外源性TTF-1基因表达对人肺腺癌细胞系GLC-82生物学特性的影响MTT法观察TTF-1基因表达后体外细胞的增殖情况,与GLC-82-EGFP细胞和GLC-82细胞相比,其细胞的增殖速度差异无显著性（F=0.388,P=0.863）。平板克隆形成实验显示GLC-82-EGFP-TTF-1细胞的克隆形成能力与GLC-82-EGFP细胞和GLC-82细胞相比,差异无显著性（χ²=1.029,P=0.598）。划痕实验显示GLC-82-EGFP-TTF-1细胞的运动能力与GLC-82-EGFP细胞和GLC-82细胞相比,差异无显著性（F=0.650,P=0.690）。流式细胞术S期及G2/G1期检测结果也显示GLC-82-EGFP-TTF-1细胞的增殖速度与GLC-82-EGFP细胞和GLC-82细胞相比差异无显著性（F=0.960,P=0.435:F=0.542,P=0.608）。因此外源性TTF-1基因在肺腺癌细胞系GLC-82中表达后,肿瘤细胞的体外生长情况并无显著改变。将GLC-82-EGFP-TTF-1细胞和对照细胞接种于裸鼠皮下,通过30天的连续观察,发现外源性TTF-1基因表达后GLC-82细胞皮下成瘤能力及体内增殖能力并无显著差异（F=0.099,P=0.986）。结论1.在排除甲状腺癌的可能性后,TTF-1蛋白作为胸腔积液肺腺癌细胞的诊断性标志物具有较高的特异度和敏感度;胸水中腺癌细胞TTF-1蛋白的PU值可作为判断肺源性与非肺源性腺癌细胞的特异性诊断指标;癌性胸水中肺源性腺癌细胞和非肺源性腺癌细胞TTF-1蛋白PU值的频数分布无重叠,可以PU值7作为两者间的判别阈值。2.TTF-1mRNA在胸腔积液肺腺癌细胞中亦有较高的表达;虽然其表达强度略低于TTF-1蛋白的表达强度,但仍可作为胸水中肺腺癌细胞诊断及鉴别诊断的指标之一;癌性胸水中肺源性腺癌细胞和非肺源性腺癌细胞TTF-1mRNAPU值的频数分布无重叠,可以PU值5作为两者间的判别阈值。3.胸水转移性肺腺癌中存在TTF-1基因外显子2区的基因突变。虽然肺腺癌组织与胸水转移性肺腺癌细胞TTF-1基因突变情况有所不同,但突变都位于第二外显子区。4.外源性TTF-1基因的表达并未能影响人肺腺癌细胞系GLC-82体内外细胞的生长及基本的肿瘤增殖方面的生物学特性。本研究的创新之处1、首次对胸水脱落细胞蜡块切片中TTF-1蛋白的表达进行了定量分析,揭示了癌性胸水中肺源性腺癌细胞和非肺源性腺癌细胞TTF-1蛋白PU值的频数分布特点,发现二者间无重叠,可以PU值7作为两者间的判别阈值。2、首次应用原位杂交的方法检测胸水脱落细胞蜡块切片中TTF-1mRNA的表达并进行了定量分析,发现TTF-1mRNA检测可作为胸水中肺腺癌细胞诊断及鉴别诊断的指标之一;癌性胸水中肺源性腺癌细胞和非肺源性腺癌细胞TTF-1mRNAPU值的频数分布无重叠,可以PU值5作为两者间的判别阈值。3、首次揭示了胸水转移性肺腺癌中存在TTF-1基因外显子2区的基因突变,并认为这一突变可能与部分肺腺癌的发生、发展和转移等有关。4、利用基因转染技术建立了外源性TTF-1基因稳定表达的人肺腺癌细胞系GLC-82-TTF-1,初步证实外源性TTF-1基因表达对于TTF-1基因表达阴性的肺腺癌细胞GLC-82体内外生长及肿瘤增殖方面的生物学行为无显著影响。更多还原

【Abstract】 Background and ObjectivesLung cancer is a common malignant tumor in the worldwide.The morbidity and mortality of lung cancer rank first in commonly seen malignant tumors.Meanwhile it is the main cause resulting in malignant pleural effusion.About 10%lung cancer patients have pleural effussion as their first clinical symptom and 30%-40%lung cancer patients have pleural effussion in their disease progression.Lung cancer patients with pleural effussion have bad prognosis and survial.In order to take effective measures for patients with pleural effussion accurate and immediate diagnosis are necessary.So deep research into the carcinogenesis,development and metastasis of lung cancer to establish scientifically and effectively diagnostic methods is of great significance to the early diagnosis,improvement of survial and cure rate,judgement of prognosis of lung cancer.Thyroid transcription factor-1（TTF-1）,with the gene name TITF-1/NKX2.1,is a homeodomain-containing nuclear transcription protein of Nkx2 gene family that is expressed in thyroid,lung and brain.It is closely correlated with the histogenesis,functional maintenance of these organs,tumorigenesis and development of other diseases.TTF-1 is localized to 14q 13 and has 371-amino acid which is 98%identical to the rat sequence.By genomic sequence analysis the TTF-1 gene spans approximately 3.3 kb and contains 2 exons and 1 intron.There is a conserved homeodomain region of 60 amino acids between region 160 and 220.In normal lung tissue,TTF-1 is expressed in mature typeⅡalveolar epithelial cells and epithelial cells of the developing airway but not expressed in typeⅠalveolar epithelial cells.TTF-1 mediates the expression of surfactant protein A,B,C and Clara cell secretory protein（CCSP）.By now,it is the only transcription factor that mediates these four lung special genes.Willemsen et al.found that a de novo heterozygous insertion mutation 859-860insC in the TTF-1 gene of a 23-year-old man exhibited unexplained combinations of chorea,mental retardation,primary hypothyroidism,and chronic lung disease.Introduction of a name for the disorder,e.g.Brain-Thyroid-Lung syndrome. The man died from large cell lung carcinoma at the age of 23.Kang Y et al.found that the decreasded expressin of TTF-1 was associated with mouse lung cancer but the mechanism was not clear.Whether the TTF-1 gene mutation predisposed to the development of lung cancer remains speculative and needs further investigation.Studies show that TTF-1 is expressed in different kinds of lung carcinomas with different intensities.TTF-1 is a marker of adenocarcinoma and small cell carcinoma of the lung.TTF-1 is little expressed in other primary or metastatic adenocarcinoma exclude lung adenocarcinoma and thyroid adenocarcinoma.So it is a useful and reliable marker for metastatic pulmonary adenocarcinoma in diagnosis of malignant pleural.But there are no studies on quantitative analysis of TTF-1 expression in malignant pleural.It is not clear whether its gene mutation in malignant pleural is as the same as its primary site.Whether the TTF-1 gene mutation predisposed to the tumorigenesis,development and metastasis of pulmonary adenocarcinoma remains speculative.Wether it can be used as a tumor marker for pulmonary adenocarcinoma diagnosis in supernatant of malignant pleural effusion is still unknown.So,our study aims to detect the expression of TTF-1 protein and mRNA in metastatic adenocarcinoma cells in malignant pleural to reveal the characteristics of TTF-1 expression in adenocarcinoma cells from different origins in malignant pleural. Quantitative analysis of the intensities of TTF-1 protein and mRNA were performed to reveal the charicterristics of TTF-1 for metastatic pulmonary adenocarcinoma in diagnosis of malignant pleural.The.Polymerase chain reaction-single strand conformation polymorphism（PCR-SSCP） and DNA sequencing were used to analyse TTF-1 gene mutation and its relationship with the carcinogenesis of lung cancer.To construct eukaryotic fluotrscent expressin vector pEGFP-N1-TTF-1,induce the vector express in human adenocarcinoma cell line GLC-82 and observe the behavior changes of GLC-82 after TTF-1 transfection.Methods1.A series of pleural specimens were available from the Pathology Department of Nanfang Hospital of Guangzhou in China from 2006,Jun to 2007.Mar.The primary sites of origin were confirmed clinically or histologically in all patients.There were 37 adenocarcinomas from lung,4 adenocarcinomas from stomach,3 adenocarcinomas from breast,2 adenocarcinomas from ovary,1 endometrial carcinoma and 1 metastatic colon carcinoma.Pap,Giemsa and PAS stained smears and cell block were prepared for each case.2.Immunohistochemical staining and in situ hybridisation were used to detect TTF-1 protein and TTF-1mRNA expression on the cell block sections.Leica Q500 MC image analysis system was used to detect the intensity of TTF-1 protein and TTF-1mRNA expression.To explore the clinical usefulness of TTF-1 as a marker for metastatic pulmonary adenocarcinoma cells in diagnosis of malignant pleural. 3.Polymerase Chain Reaction-Single Strand Conformation Polymorphism（PCR-SSCP） and DNA sequencing analysis were used to analyze TTF-1 gene mutation.Three specific primers for exon 1 and exon 2 of TTF-1 gene were synthesized and used for PCR amplification.PCR products were detected using PCR-SSCP.DNA sequencing analysis was used to analyze fragments with anomalous migrating bands by PCR-SSCP detection to identify the type of mutation and analyze the changes of gene function.4.Immunocytochemistry,RT-PCR and western blot were used to detect TTF-1 protein and TTF-1 mRNA expression in human adenocarcinoma cell line-GLC-82.5.Effect of TTF-1 expression on the biological behaviors of human adenocarcinoma cell line-GLC-82.TTF-1 cDNA was transfected into GLC-82,which had no endogenous TTF-1 expression.The TTF-1 expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot.The biological behaviors of tranfectant were investigated by MTT assay,Plate colony formation,wound-healing assay and flow cytometry in vitro.Moreover,tumorigenesis of tumor cells after transfection was observed in nude mice models in vivo by whole-body visualizing methods.Results1.Pap,Giemsa and PAS stain were used to observed the morphologic features of metastatic adenocarcinoma cells from different origins in malignant pleural.These tumor cells had the samilar morphologic features for example,forming cell clusters, marked variation in size and shape,marked atipical nuclei,mucus in plasma and so on. Sometimes adenocarcinoma cells can form the glandular structure in HE smears.But these morphologic features can not determine the primary site of metastatic adenocarcinoma cells in serous pleural. 2.Immunohistochemical staining was used to detect TTF-1 protein expression on the cell block sections.TTF-1 was expressed in 28 lung adenocarcinomas while none of the 11 extrapulmonary adenocarcinomas expressed TTF-1.The sensitivity and specificity of the TTF-1 for the adenocarcinomas of lung origin were 75.7%and 100%, respectively and there is a significant difference between them（x²=19.978,p=0.000）. TTF-1 positive unit（PU） is bigger in adenocarcinomas from lung than in those of extrapulmonary adenocarcinomas and there is a significant difference between them （t=13.208,p=0.000）.3.In situ hybridisation was used to detect TTF-1mRNA expression on the cell block sections.TTF-1 mRNA was expressed in 21 lung adenocarcinomas while none of the 11 extrapulmonary adenocarcinomas expressed TTF-1 mRNA.The sensitivity and specificity for the adenocarcinomas of lung origin were 56.75%and 100%, respectively and there is a significant difference between them（x²=11.099,p=0.001）. TTF-1 mRNA positive unit（PU） is bigger in adenocarcinomas from lung than in those of extrapulmonary adenocarcinomas and there is a significant difference between them （t=9.901,p=0.000）.4.PCR-SSCP technique was used to detect TTF-1 gene mutation in 37 cases of metastatic adenocarcinomas from lung origin and 20 cases of normal lung tissues. After 3 times of silver staining,no abnormal migrating bands were observed in fragment a amplified from primer 1A,1S of exon 1 of TTF-1 gene in 37 cases of metastatic adenocarcinomas from lung origin and 20 cases of normal lung tissues. Abnormal migrating bands were observed in fragment b amplified from primer 2A,2S of exon 2 of TTF-1 gene in 4 cases of metastatic lung adenocarcinomas.Heterozygous single nucleotide absence and substitutions were conformed by DNA sequence.The mutation rate is 10.8%.5.The TTF-1 expression in human adenocarcinoma cell line GLC-82 was determined by RT-PCR,immunohistochemistry and Western blot.The result shows that there no TTF-1 expression in GLC-82 cell.6.Effect of TTF-1 expression on the biological behaviors of human adenocarcinoma cell line-GLC-82.（1） The pEGFP-N1 plasmid and pEGFP-N1-TTF-1 plasmid were transfected respectively into GLC-82,which had no endogenous TTF-1 expression by cationic liposomes-Lipofectamine ^TM2000.Stable EGFP and TTF-1 expression cells（GLC-82-EGFP and GLC-82-TTF-1） were acqired through G418 selective culture.The TTF-1 expression was detected by RT-PCR and western blot.（2） There was no significant effects of extra TTF-1 expression on cell proliferation which were assessed by MTT（F=0.388,P=0.863）,plate colony formation assay（ x²=1.029,P=0.598）,wound-healing assay（F=0.650,P=0.690） and flow cytometry（F=0.960,P=0.435;F=0.542,P=0.608） in vitro.The result indicated that there was no significant inhibitory of cell proliferation in GLC-82-TTF-1 cell as compared with GLC-82 and GLC-82- EGFP cells.The effect of TTF-1 expression in vivo tumor growth was detected by subcutaneous growth of GLC-82,GLC-82-EGFP and GLC-82-TTF-1 cells in nude mice models for thirty days.Extra expression of TTF-1 had no significant effects on tumor growth and cell proliferation in vivo（F=0.099,P=0.986）.Conclusions1.TTF-1 protein and TTF-1 PU detection can be used as a tissue special marker in distiguishing pulmonary adenocarcinoma cells from extrapulmonary adenocarcinoma cells in pleural effusion.The TTF-1 PU betwwen pulmonary adenocarcinoma cells and extrapulmonary adenocarcinoma cells in pleural effusion has no overlop.So the TTF-1更多还原