【作者】 郝军；

【导师】 王恩华；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2002， 硕士

【摘要】 前言 肺癌是一种严重危害人类健康的疾病，其发病率和死亡率居高不下且呈逐年升高趋势。与其它实体恶性肿瘤一样，化疗仍是控制肺癌术后复发、转移的主要手段。近年来实体肿瘤药物治疗效果多不理想，主要是由于肿瘤细胞接触了一种化疗药物后产生耐药性，同时对其它多种结构和作用机制不同的化疗药物亦产生耐药性，即多药耐药。多药耐药表型可分为两种：一种是先天性耐药，指肿瘤细胞固有的对化疗不敏感；另一种是获得性耐药，指肿瘤细胞初始对化疗药物敏感，但经过几个疗程化疗后逐渐出现耐药。目前已知的相关耐药机制包括MDR1基因编码的P糖蛋白、多药耐药相关蛋白MRP、肺相关蛋白LRP的增多；拓扑异构酶Ⅱ(Topo-Ⅱ)活力降低；谷胱甘肽-S-转移酶以及还原型谷胱甘肽GSH升高；肿瘤某些生化特征的改变：膜离子通道、蛋白激酶PKC；其中自1992年Cole等人发现MRP后，与其相关的问题引起了人们的注意。 近年来国内对MRP在肾癌和血液疾病中的异常表达均有大量研究，但在肺癌中的研究刚刚起步且结果差异也很大。本实验采用免疫组织化学的方法检测非小细胞肺癌(Non-Small Cell Lung Cancer，NSCLC)中MRP的表达情况，旨在探讨其与非小细胞肺癌组织学分型、淋巴结转移和预后的关系。同时采用Western Blot并结合免疫组织化学探讨MRP在正常肺组织和非小细胞肺癌中表达的差异性。为临床肺癌的治疗提供理论基础，使治疗更具针对性。 材料和方法 1．材料 62例非小细胞肺癌组织蜡块来自中国医科大学附属第一临床医院1989－1992年外科手术切除并有随访资料的标本，标本经10％中性福尔马林固定、石蜡包埋。随访日期以患者手术日期起计算，终止日期为患者死亡日期。另外30例新鲜非小细胞肺癌和相应的正常肺组织标本由辽宁省肿瘤医院和中国医科大学附属第一临床医院提供。 2．主要试剂和来源 小鼠抗人单克隆抗体MRPm6由荷兰阿姆斯特丹Free大学医学院病理科R．J．SchePer教授惠赠，S－P超敏免疫组织化学试剂盒（KIT－97 10）和DAB酶底物显色试剂盒（DAB－0031）均购自福州迈新生物技术公司。碱性磷酸酶标记的羊抗鼠二抗和显色试剂盒购自华美公司。 3．方法 3．IWestern Blot检测 MRP在正常肺组织中和非小细胞肺癌中的表达。取新鲜样品门－2克功5倍湿重的裂解缓冲液，剪碎、匀浆超声处理，高速低温离心提取上清蛋白，电泳、转印，经封闭后加相应的一抗、二抗及显色试剂检测。图像扫描人计算机，进行灰度值的测定。 3．二 免疫组织化学检测MRP蛋白表达采用链菌素抗生物素蛋白－过氧化物酶免疫组化法门ps－P法八结果判定：每张切片随机选取10个高倍视野，每个视野计数100个瘤细胞，计数阳性细胞百分比。0分：＜10％；I分：）10％－＜40啪；2分：＞40％－＜刀％；3分：主川阮。按着色强度计分，0分为无着色；1分为淡黄色；2分为棕黄色。将着色细胞百分数计分与着色强度计分 ·2·相乘，总计分 0分为阴性（刁；二－3分为弱阳性（＋入4分以上为强阳性（－）。 4．统计学分析 采用SPSS for Windows 10．0统计分析软件，对生存数据采用Kaplan－Meter分析并绘制生存曲线，用Log－rank检验差异性；用COX模型进行多因素分析；对MRP表达与临床病理特征的关系采用卡方检验和确切概率法。Western Blot图像扫描后进行灰度值测定，采用配对 t检验统计分析。P＜0．05为有统计学意义。 实验结果 1．免疫组织化学结果 1．1 正常肺组织和非小细胞肺癌中MRP表达 MRP在非小细胞肺癌及正常肺组织中均定位于细胞膜或细胞浆，细胞核无着色，呈浅黄色或棕黄色。 卡方分析30例肺癌患者的材料发现正常肺组织和非小细胞肺癌中MRP的表达存在显著性差异。 1．2 MRP蛋白在非小细胞肺癌中的表达与生存期的关系 62例非小细胞肺癌中MRP表达阴性者的术后平均生存期为69．81。17．41月，5年生存率为 50％；而 MRP表达阳性者的术后平均生存期为 25．38。4．46月，5年生存率为 9．62％，二者经＊叶ank检验差异具有显著性汀＝0．0156八按组织学类型分析：发现鳞癌中MRP表达与生存期存在同样相关性，而腺癌无关。 1．3 多因素分析影响生存期的指标 C似多因素分析表明：术后生存期与淋巴结转移的有无（P＝0．038厂MRP的表达汀＝0．035）显著相关。 1．4 非小细胞肺癌组织中MRP的表达与临床病理特征的关系 ·3· 非小细胞肺癌中MRP表达与患者性别、组织分型、组织分化程度、淋巴结转移均无统计学意义瞩＞0．05人 2·Western Blot的结果 正常肺组织中大部分未见特异性条带的出现，而在非小细胞肺癌中可见到特异性染色带门90kD八对染色条带灰度值分析发现正常肺组织和非小细胞肺癌中Mny含量存在显著性差异。 讨 论 1．MRP在亚更多还原

【Abstract】 INTRODUCTIONLung neoplasm has done harm to human health for many years. As the other solid tumors, chemotherapy is still the main method for controlling the recurrent and metastasis. The therapy of solid tumor was not very perfect because of the multidrug resistance. The tumor cell lines resisted one kind of anticancer agents and would resist many other drugs that are different from the first one on structure and function. Phenotype of multidrug resistance are classified as two types. One is herent resistance, and the other is acquired resistance. The tumor cells are sensitive to the anticancer drugs firstly, but after several courses they become to resistant the drugs. At present, the related factors included P-gp, MRP, LRP, Topo - H , GST, some changes of biochemical character in tumors. MRP was detected by Cole in 1992, and then the interrelated problems were attended.Recently, the studies on MRP expression and renal tumor, hema-tologic disease, liver tumor were more. But the study in lung neoplasm just began in broad, and the results existed many differences. In the present study we detected the MRP expression in NSCLC by im-munohistochemistry and explored the correlation between MRP expression and clinicopathological parameters, prognosis. At the same time we investigated the difference of MRP expression between the normal lung tissues and NSCLC by Western Blot and immunohistochemistry.Our study provided the theory basis for the clinical therapy of lung cancer, and made the therapy more pertinent.MATERIALS AND METHODS1. MaterialsAll the 62 samples used in immunohistochemical study were obtained from the patients admitted to the 1 st Affiliated Hospital of China Medical University during 1989 - 1992. All the patients were followed up, and follow - up period was from the operation to death of patient. All the samples were fixed in 10% formalin and embedded in paraffin. All the 30 samples used in Western Blot were obtained from the patients admitted to the Liaoning Province Tumor Hospital and the 1st Affiliated Hospital of China Medical University.2. ReagentsThe mouse-anti-human monoclonal antibody of MRP ( MRPm6) was provided by professor R. J. Scheper of Department of Pathology in Free University. The Ultro - sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company. The goat - anti -mouse second antibody was bought from Huamei Biological Company.3. Methods3. 1 Western BlotRapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer (50mM Triscl PH 7.4, 150mM Nacl, 0.1% SDS, 1% Triton -100, ImM EDTA PH8.0, Aprotinin 12ul/10ml, PMSF 60ul/10ml) , Centrifuge the homogenate (13000rpm, 4℃) for 30 minutes to pellet insoluble material. (1) Electrophoresis (2) transfer proteins from gel to membrane (3)blocking (4) incubation with primary antibody (5)enzymeconjugate incubation (6)substrate incubation. (7)scan the results to computer3.2 ImmunohistochemistryDetect the MRP expression by immunohistochemistry S - P method. Scoring of the results: 100 cancer cells were counted in 10 fields which were selected at random. We used a scoring system to evaluate semiquantitatively the immunoexpression, judging the cases with brown plasma/membrane staining as positive. The sum score of positive cell was classified into the following 4 groups: 0, less than 10% positive; 1, more than 10% but less than 40% positive; 2, more than 40% but less than 70% positive; 3 , more than 70% positive. The intensity score was categorized into the following 3 groups; 0, no staining; 1, mild staining; 2, marked staining. The product of sum score and intensity score was classified as following 3 grades: negative, product score was 0; mild positive, product score was 1 - 3; marked positive, product score was 4-6.4. Statistical analysisSPSS for Windows 10. 0 statistical software was used to solve the data and Log ?rank test to analyze the differences. Cox model was used to solve the multivariate analysis. Paired - samples T test was used to solve the da更多还原