【作者】 赵云飞；

【导师】 申洪；

【作者基本信息】 南方医科大学 ， 病理学， 2010， 硕士

【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。自20世纪中期以来,肺癌的发病率在多数发达国家呈明显上升趋势,且逐年不断提高,已成为世界各地最常见癌的一种,也是我国最常见的恶性肿瘤之一,其发病率及死亡率占各种恶性肿瘤的首位,已成为严重危害人体健康和生命的主要疾病之一。因此探讨肺癌的发生,发展机制,对于肺癌的防治具有重要的现实意义。PDZ结合激酶/T-LAK细胞来源的蛋白激酶(PDZ-binding-kinase/T-LAK cell-originated protein kinase,简称PBK/TOPK),是一种新发现的丝-苏氨酸激酶。2000年,Gaudet等在Hela细胞cDNA文库中克隆出了一种与hDlg分子PDZ结构域结合的分子。同年,另一研究小组Abe等在T-LAK细胞中也发现了一种类似MAPKK分子的蛋白激酶,后来通过序列分析发现二者是同一种分子,于是统一命名为PDZ-binding-kinase/T-LAK-cell- originated protein kinase,简称PBK/TOPK。最初发现能够与PDZ结构域结合,文献报道它能与抑癌基因hDlg、P53蛋白、raf蛋白等多个分子结合并发生作用,能激活p38MAPK、Erk、JNK等多个下游分子,作为蛋白激酶分子参与信号转导通路。既往研究表明PBK/TOPK在多数正常组织中表达量均较低,而在几种肿瘤中表达升高,因而引起大家的关注。然而以往报道的文献表明,人们对肺癌PBK/TOPK的表达研究甚少,对PBK/TOPK表达的研究为定性观察,未见其在不同类型的肺癌中的表达研究,同时也未见有关其表达的量化特点及基于量化改变的变化规律研究未见其在肺癌淋巴结转移灶中的表达报告。为从蛋白和mRNA水平揭示肺癌组织及正常人体组织中PBK/TOPK的表达特点及规律以及在正常人体组织中的表达情况,我们进行了本项研究。方法1、收集2008.10～2009.8南方医院病理科20例正常成人肺组织和20例胚胎肺组织,104例病理活检肺癌组织及46例相应淋巴结转移癌组织。石蜡包埋,用于组织芯片制备,并分析组织中PBK/TOPK表达与患者性别、肿瘤大体类型、分化程度、有无淋巴结转移及TNM分期之间的关系。2、收集2008.10～2009.8间南方医院病理科尸检的27种正常人体组织包括：肺、喉、气管、支气管、食管、胃、小肠、大肠、肝、肾、肾上腺、甲状腺、脑、脊髓、脾、胸腺、扁桃体、心、血管、软骨、肌肉、睾丸、前列腺、乳腺、卵巢、子宫、胎盘,每种组织各包含10例,探讨PBK/TOPK在不同正常组织中的表达情况。3、采用组织微阵列技术,构建包含20例正常成人肺组织、20例胚胎肺组织、104例肺癌组织和46例相应淋巴结转移癌的760点阵的石蜡组织芯片；采用组织微阵列技术,构建包含27种人正常体组织的540点阵的石蜡组织芯片。4、采用免疫组织化学技术检测760点阵肺癌组织芯片上PBK/TOPK蛋白在不同类型肺组织中的表达,检测540点阵正常组织芯片上PBK/TOPK蛋白在不同正常组织中的表达。应用leicaQ500MC图像分析系统测试PBK/TOPK蛋白的阳性单位(PU)值,分析不同类型肺组织中PBK/TOPK蛋白表达强度的变化规律及其与肺癌的关系。5、采用原位杂交技术检测760点阵组织芯片上PBK/TOPKmRNA在不同类型肺组织中的表达,检测540点阵正常组织芯片上PBK/TOPKmRNA在不同正常组织中的表达。应用leicaQ500MC图像分析系统测试PBK/TOPKmRNA的阳性单位(PU)值,分析不同类型肺组织中PBK/TOPKmRNA强度的变化规律及其与肺癌的关系。结果1、应用免疫组织化学方法检测760点阵组织芯片上PBK/TOPK蛋白的表达强度并进行定量测试。结果表明：(1)胚胎肺泡上皮细胞PBK/TOPK阳性单位(PU)值小于正常成人肺泡Ⅱ型上皮细胞PBK/TOPK的PU值(P=0.000)；(2)肺鳞癌、腺癌、大细胞癌癌细胞PBK/TOPK的PU值均大于胚胎肺泡上皮细胞和正常成人肺泡Ⅱ型上皮细胞PBK/TOPK的PU值(P=0.000)；肺小细胞癌癌细胞PBK/TOPK的PU值小于正常成人肺泡Ⅱ型上皮细胞PBK/TOPK的PU值(P=0.000)；(3)肺鳞癌、肺腺癌、肺大细胞癌癌细胞PBK/TOPK的PU值均大于肺小细胞癌癌细胞PBK/TOPK的PU值(P=0.000)；(4)肺的鳞癌、腺癌和大细胞癌淋巴结转移灶中癌细胞PBK/TOPK的PU值均大于其癌原发灶癌细胞PBK/TOPK的PU值(t=23.938,P=0.000；t=17.688,P=0.000；t=6.582,P=0.000)；肺小细胞癌淋巴结转移灶癌细胞PBK/TOPK的PU值与其原发灶癌细胞PBK/TOPK的PU值基本相同(t=0.122,P=0.908)；(5)有淋巴结转移灶的肺癌原发灶癌细胞PBK/TOPK的PU值大于无淋巴结转移的肺癌原发灶癌细胞PBK/TOPK的PU值(t=3.064,P=0.003); (6) TOMⅡ-Ⅳ期癌细胞PBK/TOPK的PU值大于TNMⅠ期(t=3.002,P=0.003)；(7)癌细胞PBK/TOPK蛋白的PU值与肺癌大体类型(t=0.663,P=0.509)、性别(t=1.226,P=0.223)、分化程度(肺鳞癌t=1.512,P=0.137,肺腺癌t=0.594,P=0.594)无关。2、应用原位杂交方法检测760点阵组织芯片上PBK/TOPKmRNA的表达强度并进行定量测试。结果表明：(1)胚胎肺泡上皮细胞PBK/TOPKmRNA阳性单位(PU)值小于正常成人肺泡Ⅱ型上皮细胞PBK/TOPKmRNA的PU值(P=0.000)；(2)肺鳞癌、腺癌、大细胞癌癌细胞PBK/TOPKmRNA的PU值均大于胚胎肺泡上皮细胞和正常成人肺泡Ⅱ型上皮细胞PBK/TOPKmRNA的PU值(P=0.000)；肺小细胞癌癌细胞PBK/TOPKmRNA的PU值小于正常成人肺泡Ⅱ型上皮细胞PBK/TOPKmRNA的PU值(P=0.000)；(3)肺鳞癌、肺腺癌、肺大细胞癌癌细胞PBK/TOPKmRNA的PU值均大于肺小细胞癌癌细胞PBK/TOPKmRNA的PU值(P=0.000)；(4)肺的鳞癌、腺癌和大细胞癌淋巴结转移灶中癌细胞PBK/TOPKmRNA的PU值均大于其癌原发灶癌细胞PBK/TOPKmRNA的PU值(t=12.936,P=0.000；t=12.161,P=0.000；t=7.581,P=0.000)；肺小细胞癌淋巴结转移灶癌细胞PBK/TOPKmRNA的PU值与其原发灶癌细胞PBK/TOPKmRNA的PU值基本相同(t=0.013,P=0.990)；(5)有淋巴结转移灶的肺癌原发灶癌细胞PBK/TOPKmRNA的PU值大于无淋巴结转移的肺癌原发灶癌细胞PBK/TOPKmRNA的PU值(t=3.665,P=0.000)；(6) TNMⅡ-Ⅳ期癌细胞PBK/TOPKmRNA的PU值大于TNMⅠ期(t=3.050,P=0.003)；(7)癌细胞PBK/TOPKmRNA的PU值与肺癌大体类型(t=0.456,P=0.560)、性别(t=0.594,P=0.554)、分化程度(肺鳞癌t=0.164,P=0.870,肺腺癌t=1.223,P=0.225)无关。3、应用免疫组织化学方法检测540点阵正常组织芯片上PBK/TOPK蛋白的表达情况。结果表明：肺、喉、气管、支气管、食管、胃、大肠、肝脏、肾、肾上腺、甲状腺、脑、脊髓、脾、胸腺、扁桃体、心脏、血管、软骨、肌肉、乳腺、睾丸、前列腺、卵巢、子宫、胎盘未见PBK/TOPK阳性细胞表达；小肠粘膜腺上皮细胞有弱阳性表达。4、应用原位杂交的方法检测540点阵正常组织芯片上PBK/TOPKmRNA的表达情况。结果表明：肺、喉、气管、支气管、食管、胃、大肠、肝脏、肾、肾上腺、甲状腺、脑、脊髓、脾、胸腺、扁桃体、心脏、血管、软骨、肌肉、乳腺、睾丸、前列腺、卵巢、子宫、胎盘未见PBK/TOPKmRNA阳性细胞表达；小肠粘膜腺上皮细胞有弱阳性表达。结论1、PBK/TOPK蛋白的表达量在胚胎肺泡上皮细胞、正常成人肺泡Ⅱ型上皮细胞和肺癌细胞中的表达具有差异性,并依次增加(肺小细胞癌除外)；肺癌细胞PBK/TOPK的表达具有癌组织类型差异性,肺鳞癌、腺癌和大细胞癌相对较高,肺小细胞癌极少；PBK/TOPK蛋白高表达的肺癌易发生转移,PBK/TOPK蛋白高表达的肺的鳞癌、腺癌和大细胞癌癌细胞可作为判断该肺癌细胞具有明显转移能力的重要标志之一,且与TNM分期相关。2、PBK/TOPKmRNA的表达量在胚胎肺泡上皮细胞、正常成人肺泡Ⅱ型上皮细胞和肺癌细胞中的表达具有差异性,并依次增加(肺小细胞癌除外)；肺癌细胞PBK/TOPKmRNA的表达具有癌组织类型差异性,肺鳞癌、腺癌和大细胞癌相对较高,肺小细胞癌极少；PBK/TOPKmRNA高表达的肺癌易发生转移,PBK/TOPKmRNA高表达的肺的鳞癌、腺癌和大细胞癌癌细胞可作为判断该肺癌细胞具有明显转移能力的重要标志之一,且与TNM分期相关。3、PBK/TOPK蛋白在正常小肠粘膜组织中弱表达,多数正常组织中未见表达,而在肺癌组织中高表达,从蛋白水平说明PBK/TOPK与肺癌密切相关。4、PBK/TOPKmRNA在正常小肠粘膜组织中弱表达,多数正常组织中未见表达,而在肺癌组织中高表达,从mRNA水平说明PBK/TOPK与肺癌密切相关。更多还原

【Abstract】 Background and ObjectivesLung cancer is increasingly detrimental to people’s health. From the middle of the 20th century,the morbidity and mortality of lung cancer are growing rapidly worldwide. In most developed countries,lung cancer ranks first in males and second or third in females in commonly seen malignant tumors. Therefore, research on occurrence and development of mechanisms of lung cancer to prevent and control has important practical significance.PDZ-binding-kinase/T-LAK cell-originated protein kinase (PBK/TOPK), is a newly discovered silk-threonine kinase. Gaudet, etc. cloned a molecule in the Hela cell cDNA library with the PDZ domain of hDlg molecules combined, In the same year, another research team Abe, etc. found in a protein kinase similaried MAPKK molecules in the T-LAK cells, Later revealed that they were the same type molecule by sequence analysis, so which were unified named PDZ-binding-kinase/T-LAK-cell-originated protein kinase, referred to as PBK/TOPK. Initially found that it could bind with PDZ domain, it was reported in the literature that it could interact the tumor suppressor genes hDlg, P53 protein, raf protein and many other molecules and that could activate p38MAPK, Erk, JNK and other downstream molecules, as molecules involved in protein kinase signal transduction pathway. Past studies indicate that PBK /TOPK show low expression in the normal tissues besides bile duct epithelial cells, but high expression in several tumors, which led to researcher’s attention. However, the research was rarely about lung cancer, studied the expression of PBK/TOPK by qualitative observation. Studies on the quantitative expression of PBK/TOPK in different kinds of lung carcinomas,its expression in lymph node metastases of lung carcinomas (including the qualitative and quantitative analyses) haven’t been investigated.So, this study aims to detect the expression of PBK/TOPK protein and mRNA in lung cancer and normal human tissues.Quantitative analyses of the intensities of PBK/TOPK protein and mRNA were performed to revel the characteristics of PBK/ TOPK protein and mRNA level and research expression of PBK/TOPK normal human tissues.Methods1、20 cases of paraffin embeded normal lung tissues,20 cases of embryonic lung tissues,104 cases of paraffin embeded lung carcinomas and 46 cases of corresponding lymph node metastases were collected from the pathology department of Nanfang Hospital and the autopsy floor of Nanfang Hospital in 2008.10～2009.8. Lung carcinomas were analysed with respect to the relationship between PBK / TOPK expression and patient’s sex,tumor general types,differentiation,lymph node metastasis and TNM stage.2、27 kinds of normal tissue,10 specimens of each type of organizations that include:lung, larynx, trachea, bronchus, esophagus, stomach, small intestine, large intestine, liver, kidney, adrenal gland, thyroid gland, brain, spinal cord, spleen, thymus, tonsils, heart, blood vessels, cartilage, muscle, testis, prostate, breast, ovarian, uterus,placenta were collected from the pathology department of Nanfang Hospital and the autopsy floor of Nanfang Hospital in 2008.10～2009.8. Investigation on expression of PBK/TOPK in different normal tissues.3、Tissue microarray technique was used to construct a tissue chip with 760 disks containing 20 cases of normal lung tissues,20 cases of embryonic lung tissues,104 cases of paraffin embeded lung carcinomas and 46 cases of corresponding lymph node metastases and to construct a tissue chip with 540 disks containing 27 kinds of normal tissue,10 specimens of each type of organizations.4、Immunohistochemical staining was used to detect PBK/TOPK protein expression in different kinds of lung carcinomas on the tissue chip with 760 disks and in different normal tissues on the tissue chip with 540 disks.Leica Q500MC image analysis system was used to dectect the intensity of PBK/TOPK protein expression. The relationship between PBK/TOPK protein expression in fifferent kinds of lung carcinomas was analysed.5、mRNA in situ hybridisation was used to detect PBK/TOPKmRNA expression in different kinds of lung carcinomas on the tissue chip with 760 disks and in different normal tissues on the tissue chip with 540 disks. Leica Q500MC image analysis system was used to dectect the intensity of PBK/TOPKmRNA expression. The relationship between PBK/TOPKmRNA expression in fifferent kinds of lung carcinomas was analysed.Results1、Immunohistochemical staining was used to dectect PBK/TOPK protein expression in lung carcinoma on the chip with 760 disks. (1) Embryonic alveolar epithelial cytoplasm of PBK/TOPK positive unit (PU) is less than the normal adult typeⅡalveolar epithelial cytoplasm of PBK/TOPK PU (P=0.000); (2) Lung squamous cell carcinoma, adenocarcinoma, large cell carcinoma cytoplasm of PBK/ TOPK PU values are greater than Embryonic alveolar epithelial and normal adult alveolar typeⅡepithelial cytoplasm of PBK/TOPK PU (P=0.000); Small cell lung cancer cytoplasm of PBK/TOPK PU is less than the normal adult typeⅡalveolar epithelial cytoplasm of PBK/TOPK PU (P=0.000); (3) Lung squamous cell carcinoma, lung adenocarcinoma,large cell lung cancer cytoplasm of PBK/ TOPK PU are greater than the small cell lung cancer cytoplasm of PBK/TOPK PU (P=0.000); (4) Lung squamous cell carcinoma, lung adenocarcinoma and large cell carcinoma lymph node metastasis of cancer cytoplasm of PBK/TOPK PU are greater than primary tumor cytoplasm of PBK/TOPK PU (t=23.938,P=0.000; t=17.688,P=0.000;t=6.582, P=0.000); Small cell lung cancer lymph node metastasis of cancer cytoplasm of PBK/TOPK PU and the primary tumor cytoplasm of PBK/ TOPK PU is basically the same (t=0.122,P=0.908);(5)PBK/TOPK PU in cancerous cells of lymph node metastases than in those withiout (t=3.064,P=0.003); (6) PBK/TOPK PU was bigger in TNM stageⅡ-Ⅳthan stageⅠ(t=3.002,P=0.003); (7) PBK/TOPK PU was not associated with patients’sex (t=1.226,P=0.223), general types (t=0.663,P=0.509) and differentiation (Squamous cell lung carcinoma: t=1.512,P=0.137; Lung adenocarcinoma:t=0.594,P=0.594).2、mRNA in situ hybridisation was used to dectect PBK/TOPKmRNA expression in lung carcinoma on the chip with 760 disks. (1) Embryonic alveolar epithelial cytoplasm of PBK/TOPKmRNA positive unit (PU) is less than the normal adult typeⅡalveolar epithelial cytoplasm of PBK/TOPKmRNA PU (P=0.000); (2) Lung squamous cell carcinoma, adenocarcinoma, large cell carcinoma cytoplasm of PBK/TOPKmRNA PU are greater than Embryonic alveolar epithelial and normal adult alveolar typeⅡepithelial cytoplasm of PBK/TOPKmRNA PU (P=0.000); Small cell lung cancer cytoplasm of PBK/TOPKmRNA PU is less than the normal adult typeⅡalveolar epithelial cytoplasm of PBK/TOPKmRNA PU (P=0.000); (3) Lung squamous cell carcinoma, lung adenocarcinoma,large cell lung cancer cytoplasm of PBK/TOPKmRNA PU are greater than the small cell lung cancer cytoplasm of PBK/TOPKmRNA PU (P=0.000); (4) Lung squamous cell carcinoma, lung adenocarcinoma and large cell carcinoma lymph node metastasis of cancer cytoplasm of PBK/TOPKmRNA PU are greater than primary tumor cytoplasm of PBK/TOPKmRNA PU (t=12.936,P=0.000;t=12.161, P=0.000;t=7.581, P=0.000); Small cell lung cancer lymph node metastasis of cancer cytoplasm of PBK/TOPKmRNA PU and the primary tumor cytoplasm of PBK/TOPKmRNA PU is basically the same (t=0.013,P=0.990);(5)PBK/TOPKmRNA PU in cancerous cells of lymph node metastases than in those withiout (t=3.665,P=0.000); (6) PBK/TOPKmRNA PU was bigger in TNM stageⅡ-Ⅳthan stageⅠ(t=3.050,P=0.003); (7) PBK/TOPKmRNA PU was not associated with patients’sex (t=0.594,P=0.554), general types (t=0.456,P=0.560) and differentiation (Squamous cell lung carcinoma:t=0.164,P=0.870; Lung adenocarcinoma:t=1.223,P=0.225).3、Immunohistochemical staining was used to dectect PBK/TOPK protein expression in normal tissue on the chip with 540 disks. There was not PBK/ TOPK protein expression in lung,larynx, trachea, bronchus, esophagus, stomach, small intestine, kidney, adrenal gland, thyroid, brain, spinal cord, spleen, thymus, tonsils, heart, blood vessels, cartilage, muscle, breast, testis, prostate, ovary, uterus and placenta. the glandular epithelial cells of Small intestinal expressed positive.4、mRNA in situ hybridisation was used to dectect PBK/TOPKmRNA expression in normal tissue on the chip with 540 disks. There was not PBK/ TOPK PBK/TOPKmRNA expression in lung,larynx, trachea, bronchus, esophagus, stomach, small intestine, kidney, adrenal gland, thyroid, brain, spinal cord, spleen, thymus, tonsils, heart, blood vessels, cartilage, muscle, breast, testis, prostate, ovary, uterus and placenta. the glandular epithelial cells of Small intestinal expressed positive. Conclusions1、The amount of PBK/TOPK expression differs in the cytoplasm of embryonic alveolar epithelial cells, normal adult alveolar typeⅡepithelial cells and lung cancer cells and increases orderly. The expression of PBK/TOPK differs in different types of lung carcinomas,which is comparatively higher in lung squamous cell carcinoma, adenocarcinoma and large cell carcinoma and lower in small cell carcinoma. The stronger the expression of PBK/TOPK,the easer lung carcinoma metastasizes.Cancer cells of lung squamous cell carcinoma, adenocarcinoma and large cell carcinoma with strong expression of PBK/TOPK in the cytoplasm are one of the important hallmarks of cells with distinct potential of metastasis.2、The amount of PBK/TOPKmRNA expression differs in the cytoplasm of embryonic alveolar epithelial cells, normal adult alveolar typeⅡepithelial cells and lung cancer cells and increases orderly. The expression of PBK/TOPKmRNA differs in different types of lung carcinomas,which is comparatively higher in lung squamous cell carcinoma, adenocarcinoma and large cell carcinoma and lower in small cell carcinoma. The stronger the expression of PBK/TOPKmRNA,the easer lung carcinoma metastasizes.Cancer cells of lung squamous cell carcinoma, adenocarcinoma and large cell carcinoma with strong expression of PBK/ TOPKmRNA in the cytoplasm are one of the important hallmarks of cells with distinct potential of metastasis.3、PBK/TOPK protein expressed in few normal tissues and didn’t express in most normal tissues. PBK/TOPK showed no expression in adult lung tissues and in embryonic tissues, but high expression in lung cancer tissues, which indicates PBK/ TOPK is closely related to lung cancer formation.4、PBK/TOPKmRNA expressed in few normal tissues and didn’t express in most normal tissues. PBK/TOPKmRNA showed no expression in adult lung tissues and in embryonic tissues, but high expression in lung cancer tissues, which indicates PBK/TOPK is closely related to lung cancer formation.更多还原