【作者】 王丽华；

【导师】 蒋军广；

【作者基本信息】 郑州大学 ， 内科学， 2007， 硕士

【摘要】 肺癌为当今世界各地最常见的恶性肿瘤之一，是一种严重威胁人民健康和生命安全的疾病。半个世纪以来世界各国肺癌的发病率和死亡率均呈明显上升趋势。WHO 1997年报告：肺癌居癌症死因第一位。NSCLC发病率约占肺癌的70％以上。NSCLC患者5年生存率平均为16％，其中局部转移的NSCLC患者5年生存率为23％，而有远处转移患者的5年生存率仅为3％。治疗失败和患者死亡的根本原因是肺癌细胞的转移。因此揭示肺癌细胞的转移机制，预测肺癌细胞的转移潜能，将会对临床医生判断患者的病情、制定合理的治疗方案及判断预后起到积极的作用，将有助于减轻患者的痛苦、延长患者的生存期、提高生活质量。已有研究表明肺癌细胞的侵袭、转移是一个受多因素调控的、十分复杂的生物学过程。涉及肺癌细胞在原发病灶处的恶性增殖、癌细胞脱离原发病灶、在脉管内形成癌栓、在脉管内的迁移、在靶器官种植、新的转移灶形成。其关键步骤是癌细胞脱离原发病灶及黏附到靶器官。细胞黏附参与整个过程并起重要作用。介导细胞黏附的是一大类CAM和连环素（catenin）。CAM包括选择素族（selectins）、免疫球蛋白超家族（IGSF）、钙黏附素族（cadherin）等。近几年来连环素和钙黏附素族引起广大科学工作者的广泛关注，成为肿瘤转移分子机制中的研究热点。Cadherins为依赖Ca²⁺的CAM，是AJ的主要结构成分，是由一系列单链跨膜糖蛋白组成。在细胞黏附及分化，尤其是早期的分化中，起着非常重要的作用。目前已知有四十多种不同的Cadherin，但关于N-cadherin的研究甚少，尤其在NSCLC方面的研究更少。国外有数篇报道，而国内仅有一篇。研究发现，N-cadherin对于肿瘤上皮细胞、血管平滑肌细胞和外皮细胞之间的相互黏附起着重要的作用。在肿瘤形成的过程中，N-cadherin的表达有助于血管形成及上皮细胞-间质细胞的迁移，还可以通过FGF激活MAPKERK信号转导途径，诱导MMP-9基因表达，以上两个方面均有利于肿瘤血管的形成，使肿瘤易于生长和转移。Catenins有α、β、γ及P120-catenin四种亚型。有关γ-catenin与肺癌关系的研究报道较少。如果没有γ-catenin的参与，cadherin／catenins细胞黏附复合体不能维持细胞间的正常黏附，只有当γ-catenin先从细胞黏附复合体脱离时，细胞才能浸润转移，当γ-catenin的酪氨酸残基磷酸化后将影响cadherin／catenins细胞黏附符复合体的稳定性，有可能导致细胞癌变。因此，研究γ-catenin在NSCLC细胞中的表达及其与转移的关系，对于NSCLC的诊断和治疗有重要意义。基于目前的研究现状，本课题旨在进一步探明N-cadherin和γ-catenin在NSCLC中的表达及临床意义，了解他们与肺癌的发生、发展和转移的关系，从而在一定程度上探讨NSCLC的发生、发展和转移的机制，为NSCLC的诊断和治疗提供帮助。对象与方法一、研究对象标本取自2002年9月至2005年10月在郑州大学第一附属医院住院手术切除的肺癌患者，共计61例，男42例，女19例。年龄36岁～81岁，平均57.77岁。所有患者术前均未行放疗和化疗。标本均经病理证实，鳞癌36例，腺癌25例。按1997年UICC标准进行TNM分期：ⅠA期2例，ⅠB期12例，ⅡA期O例，ⅡB期7例，ⅢA期9例，ⅢB期19例，Ⅳ期12例。连续4μm厚切片。另选20例癌旁正常肺组织（均距癌组织3 cm以上）作对照。二、方法采用S-P免疫组织化学方法，严格按照S-P试剂盒说明进行，以PBS代替一抗作为阴性对照，N-cadherin的阳性对照为已知的正常心肌组织，γ-catenin的阳性对照为已知的乳腺癌阳性组织。主要步骤如下：（1）常规脱蜡、水化，高温煮沸抗原修复；（2）过氧化物酶阻断剂阻断内源性过氧化物酶活性；（3）非免疫性动物血清阻断非特异性反应；（4）滴加即用型鼠抗人N-cadherin单克隆抗体或1：100γ-catenin多克隆抗体；（5）加生物素标记的第二抗体；（6）滴加链亲和素-过氧化物酶制剂；（7）DAB显色后自来水充分冲洗；（8）苏木素复染，梯度酒精脱水、透明树胶封片、镜检。三、结果判定1．N-cadherin的结果判定：高倍镜（400倍）下每个视野计数100个细胞，每张切片数10个视野。参照文献的方法，将染色强度分为3级：无着色（1级）评1分，浅黄色（2级）评2分，棕黄色（3级）评3分；依据镜下阳性染色细胞数占癌细胞总数的百分率，阳性细胞数≤25％为1级（分），26％～75％为2级（分），≥75％为3级（分）。每张切片的最后得分为两次评分的乘积（1分～9分），最后得分＞7分为阳性表达（+），＜7分为阴性表达（-）。2．γ-catenin的结果判定：高倍镜（400倍）下每个视野计数100个细胞，每张切片数10个视野。免疫反应染色阳性：细胞内见棕色颗粒，染色均匀，阳性细胞率≥50％。免疫反应染色阴性：所有细胞均不着色或阳性细胞率＜50％。四、统计学处理采用SPSS10.0统计学软件，用x²检验及四格表资料的精确概率法进行统计学分析。P＜0.05有统计学意义。结果一、N-cadherin及γ-catenin在NSCLC组织中的表达免疫组化染色结果显示，N-cadherin及γ-catenin的免疫组化阳性产物主要定位于细胞质，偶见定位于细胞核、膜。61例NSCLC组织中N-cadherin表达的阳性率为37.70％（23／61），20例正常肺组织无一例阳性表达（0％），差异有统计学意义（P＜0.01）。61例NSCLC组织中γ-catenin表达的阳性率为72.13％（44／61），20例正常肺组织无一例阳性表达（0％），差异有统计学意义（P＜0.01）。二、N-cadherin及γ-catenin的表达与临床病理特征的关系高、中分化NSCLC中N-cadherin阳性表达率为31.25％，低分化为61.54％，差异具有统计学意义（P＜0.05）。TNM分期Ⅰ、Ⅱ期NSCLC患者N-cadherin阳性表达率为57.14％，Ⅲ、Ⅳ期患者N-cadherin阳性表达率为27.5％，差异有统计学意义（P＜0.05）。N-cadherin的表达与NSCLC患者的年龄、性别、肿瘤的病理类型、临床分型、大小及有无淋巴结转移、胸腔积液无关（P均＞0.05）。高分化NSCLC中γ-catenin阳性表达率为20.00％，中分化为78.95％，低分化为92.31％，差异具有统计学意义（P＜0.01）。有淋巴结转移的患者中γ-catenin阳性表达率为84.21％，无淋巴结转移者为52.17％，差异具有统计学意义（P＜0.05）。TNM分期Ⅰ、Ⅱ、Ⅲ、Ⅳ期患者γ-catenin阳性表达率分别为42.86％、57.14％、82.14％及91.67％，差异有统计学意义（P＜0.05）。γ-catenin的表达与NSCLC患者的年龄、性别、肿瘤的病理类型、临床分型、大小及有无胸腔积液无关（P均＞0.05）。结论1．N-cadherin和γ-catenin在NSCLC中的阳性表达率较高，提示其与NSCLC的发生密切相关，可能成为一个NSCLC新的诊断标志。2．N-cadherin的表达与与NSCLC的分化程度及TNM分期密切相关，与患者的年龄、性别、肿瘤的病理类型、临床分型、大小及有无淋巴结转移、胸腔积液无关。3．γ-catenin的表达与NSCLC患者的分化程度、TNM分期、有无淋巴结转移密切相关，与患者的年龄、性别、肿瘤的病理类型、临床分型、大小及有无胸腔积液无关。4．N-cadherin和γ-catenin的高表达可能对NSCLC患者病情的综合判断及制定合理的治疗方案提供重要依据。更多还原

【Abstract】 Backgrounds and AimsLung cancer is one of the best common malignant tumor in the world.It is a kind of disease that threats severely the health and safety of people.The disease incidence and death rate of lung cancer of every country in the world all offer obvious and adscendent tendency since half century.The report of WHO in 1997:The death rate of lung cancer is the first one cancers. The disease incidence of NSCLC occupies more than 70%.The five years survival rate of patients with NSCLC is average 16%.Among the total .The five years survival rate of patients whose tumours have partly metastasised is average 23%, but it is only 3% that patient’s tumour has metastasised to distant place.The basic reason of cure failure and patient death is the metastasis.So revealing the mechanism of metastasis of cancer cell and predicting the potentiality of metastasis of cancer cell will conduce clinician to judge patient’s condition、establish reasonable treatment plan and judge prognosis and conduce to mitigate patient’s distress、extend patient’s life span and raise quality of life.Studies completed disclose that the invasion and metastasis of lung cancer cell is a very complicated biology course that is controled by many factors.It relates to the malignant multiplication of lung cancer cell in the primary focus of infection cancer cell breaking away from the primary focus of infection、forming cancer embolia and migration in vessel、implantating in target organ and forming new metastases.The key is cancer cell breaking away from the primary focus of infection and how to adhere target organ.Cell adhesion participates the whole course and acts a important role.CAM and catenins mediate cell adhesion.CAM include Selectins、IGSF、Cadherins and so on.In the recent years , many scientises have paied close attention to Catenins and Cadherins.They have become the hot spot among the molecule mechanism of metastasis.Cadherins is a group of CAM depending on Ca²⁺、the main constituent of AJ structure and made up with a series of single strand transmembrane glycoprotein.They possess very important effect in the processes of cell adhesion and differentiation, especially the earlier period of differentiation.We have known there are more than 40 varietases Cadherin now. Study about N-cadherin is very poor, especially in NSCLC.There are several reports about it in foreign country,but only one in our country. Study discover N-cadherin acts a important role in the adhesion of tumour epithelium tissue cell、blood vessel smooth muscle cell and cutaneous cell.In the process of tumour formation, N-cadherin encourages blood vessel formation and the transference between epithelium tissue cell and stroma cell.It can activate MAPKERK system through FGF,then induce the expression of MMP-9 gene .These two factors profit tumour vessel formation and make tumour growth and metastasis easy.Catenins have four subtypes,α、β、γand P120-catenin.The reports about the relationship betweenγ-catenin and NSCLC are few. Cadherin/catenins cell adhesion complex could not maintain normal adhesion,if have not the participation ofγ-catenin.Only whenγ-catenin separates from cell adhesion complex,cell could infiltration and metastasis.After the tyrosine residue ofγ-catenin becomes phosphorylation、will effect the stability of Cadherin/catenins cell adhesion complex. All these could induce canceration. So studing the expression ofγ-catenin in NSCLC and the relationγ-catenin with metastasis is very important to diagnosis and therapy of NSCLC.According to the study state at present, the aim of this topic is to explore more deeply the expression and clinical significance of N- cadherin andγ-catenin in NSCLC and understand the relation between they and the genesis、development and metastasis of NSCLC,then approach in some extent the mechanisms of NSCLC’s genesis、 development and metastasis,in order to provid some Assistance to diagnosis and therapy of NSCLC.Study Object and Methods1.Study object: Samples were collected from patients with primary NSCLC who were operated in the first affiliated hospital of zhengzhou university from September in 2002 to October in 2005.The sum total was 61 examples. Among the total,42 examples were male and 19 examples were female.The age of patients were form 36 years to 81 years,and averaged 57.77 years.All patients were not taken radiotherapy and chemotherapy. The pathological type of all samples were witnessed by pathologists,36 examples were squamous carcinoma and 25 examples were adenocarcinoma.According to UICC standard of 1997,these samples were divided in:ⅠA stage 2 examples,ⅠB stage 12 examples,ⅡA stage 0 example,ⅡB stage 7 examples,ⅢA stage 9 examples,ⅢB stage 19 examples,Ⅳstage 12 examples. The thickness of sections was 4μm.in addition,20 examples normal lung tissue（the distances form cancer are all above 3 cm） by the side of cancer acted as comparison.2.Methods: S-P immunohistochemical method was used.ALL steps were strictly operated according to the directions of S-P kit.Made PBS take the place of the first antibody as negative comparison.The masculine comparison of N-cadherin was normal heart muscle.The masculine comparison ofγ-catenin was breast cancer tissue.The main steps were : （1）deparaffinage and hydration according to common practice and reparating antigen through boiling;（2）Using peroxydase blocking agent to block the activity of endogenous peroxydase;（3）nonimmune animal blood serum blocked non-specificity reflection;（4）dropwising mouse anti-human N-cadherin monoclonal antibody or 1:100γ-catenin polyclonal antibody;（5）dropwising the second antibody;（6）dropwising streptavidin-peroxydase praeparatum;（7）dyeing with DAB,then washing sufficiently with tap water;（8）afterstaining with hematoxylin,then dehydrating、mounting and testing under microscope.3.Assessing result（1）N-cadherin: counting 100 cells every visual field under high power lens（400 times）. Counting 10 visual fields. According to literature,divide stain intension in three stages: no stain（1 stage） 1 point,Buff（2 stage） 2 point,brown（3 stage） 3 point; According to the percentage of the positive cells occuping cancer cells, the percentage≤25% 1 stage （point）, 26%～75% 2 stage （point）,≥75% 3 stage （point）, the ultimum score of evry section is the cross product of two counting（1 point～9 point）.Dividing it in two stage: masculine（+） >7 point, negative （-） <7 point. （2）γ-catenin: counting 100 cells every visual field under high power lens（400 times）. Counting 10 visual fields, masculine （+）:We could see brown drop in cell.The drop was stained uniformitily and the positive rate≥50%. negative （-）:A11 cells were not stained or the positive rate≤50%.4.treatment in statistics: Using SPSS 10.0 and thex²test and exact probabilities in 2×2 table treats data. P<0.05Results1.The expression of N-cadherin andγ-catenin in NSCLC:The results of immunohistochemistry displayed that N-cadherin andγ-catenin located on cell envelope or cytoplasmand located perchanc nucleus.N-cadherin was negative in all tumor-adjacent normal tissues.the positive rate of N-cadherin expression in NSCLC tissues was 37.70%（23/61）（P<0.01）.γ-catenin was negative in all tumor-adjacent normal tissues.the positive rate ofγ-catenin expression in NSCLC tissues was 72.13%（44/61）（P<0.01）.2.The relation N-cadherin andγ-catenin with the clinical patho-character : The positive rate of N-cadherin expression （61.54%） in the poorly differentiated NSCLC tissues was significant higher than that（31.25 %） in the well and moderately differentiated NSCLC tissues （P<0.05）. In addition , The expression rate of N-cadherin was related to the tumor stage（ P<0.05）. The expression rate of N-cadherin was not related to histologic type、having or no pleural effusion、lymph node metastasis、age、sex、tumor location and tumor size.The positive rate ofγ-catenin expression （84.21%） in the NSCLC tissues of having lymph node metastasis was significant higher than that（52.17 %） in the NSCLC tissues of no lymph node metastasis （P<0.05）. In addition ,γ-catenin expression rate was related to the grade differentiation and tumor stage（all .P<0.05）. The expression rate ofγ-catenin was not related to histologic type、having or no pleural effusion、age、sex、tumor location and tumor size.Conclusion1.The positive rate of N-cadherin andγ-catenin expression were significant higher than normal tissues.It revealed that they were close correlation and could become a new diagnosis marker of NSCLC.2.The expression rate of N-cadherin was related to the tumor stage and differentiation and was not related to histologic type、having or no pleural effusion、lymph node metastasis、age、sex、tumor location and tumor size.3. The expression rate ofγ-catenin was related to the tumor stage、differentiation and lymph node metastasis and was not related to histologic type、having or no pleural effusion、age、sex、tumor location and tumor size.4.The high expression of N-cadherin andγ-catenin could offer important proof to judge patient’s condition and establish reasonable treatment plan.更多还原