【作者】 宋敏；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的肺癌在形成前经历了一系列形态学及分子水平的改变,从正常的支气管肺泡上皮细胞演变成为各种形态学表现的肺癌是一个复杂漫长的多阶段多步骤的癌变过程,支气管上皮细胞增殖／分化的控制失调是其中的根本原因。在致癌剂如苯并芘促进肺部细胞恶化和促进肿瘤成长的过程中,这些对于肺正常细胞如支气管上皮细胞的作用是潜移默化从量变向质变恶性转化进行时的。研究表明酚类等抗氧化剂可以拮抗这一作用。人们致力于寻找良好的化学预防策略及物质,干预致癌物代谢,不使其活化成完成致癌物或者抑制、防止致癌物与细胞DNA结合,抑制癌前病变演变成癌或使其逆转成正常细胞。在本课题组的前期研究中,证实3,4-苯并芘油剂经皮肺内注射构建大鼠肺肿瘤模型是一种简单可靠的肺肿瘤动物模型的构建方法,其中肿瘤的病理组织学类型以非小细胞肺癌为主。我们通过二维凝胶电泳获取未成瘤大鼠3,4-苯并芘注射旁肺组织与成瘤大鼠癌旁肺组织的差异表达蛋白质图谱,从而筛选大鼠肺组织微环境中与3,4-苯并芘诱导大鼠原发肺癌发生相关的蛋白质点。这些蛋白质点是包括DJ-1在内的9个质点,我们认为是与3,4-苯并芘肺内注射后未成瘤大鼠抑制肺癌形成相关的蛋白质点。提示肿瘤微环境中的DJ-1蛋白可以作为化学预防治疗癌症的靶点,化学预防可以在早期对致癌因素进行控制,通过对微环境中多种细胞及细胞因子的干预而影响肿瘤表型。肿瘤微环境靶点众多,从中挑选DJ-1作为分子靶点及寻找其定位细胞是第一步,弄清微环境中蛋白分子水平的变化对肿瘤细胞的影响,从而验证靶点蛋白及定位细胞的作用及与绿茶(酚类)肺癌预防的关系。方法实验第一部分,构建3,4-苯并芘油剂经皮肺内注射大鼠肺肿瘤模型,80只7-8周龄雌性SD大鼠,体重180-220g,随机分为2组,A组与B组各40只。A组接受3,4-苯并芘-玉米油肺内注射,B组接受玉米油肺内注射。两组大鼠采用相同普通饲料饲养,以水为饮料。32周处死大鼠,获取成瘤鼠和未成瘤鼠的苯并芘注射旁肺组织,验证肺组织抑制肺癌蛋白点DJ-1表达水平及主要定位细胞。DJ-1靶向siRNA转染人支气管上皮细胞,从mRNA水平和蛋白水平研究转染后DJ-1基因的表达下调的效应,检测转染DJ-1靶向siRNA转染HBE细胞对细胞增殖及凋亡的影响。实验第二部分,建立人支气管上皮细胞和肺腺癌细胞的共培养模型,分成三组,A组为单独肿瘤细胞培养组即空白组,B组为未处理的上皮细胞和肺腺癌细胞共培养组即对照组,C组为转染靶向DJ-1的siRNA后的上皮细胞和肺腺癌细胞共培养组即实验组。研究各组细胞生长增殖曲线及细胞周期凋亡水平及三组的裸鼠移植瘤实验。第三部分,构建3,4-苯并芘油剂经皮肺内注射大鼠肺肿瘤模型,60只7-8周龄雌性SD大鼠,体重180-220g,随机分为2组,A组与B组各30只。均接受3,4-苯并芘-玉米油肺内注射,两组大鼠采用相同普通饲料饲养,A组以1%绿茶为饮料,B组以水为饮料。16周处死大鼠,从mRNA水平和蛋白水平比较两组大鼠苯并芘注射旁肺组织的DJ-1基因表达差异。用绿茶有效成分(EGCG)处理支气管上皮细胞,从mRNA水平和蛋白水平测定上皮细胞中DJ-1基因表达的时间效应和剂量效应关系。结果成功构建3,4-苯并芘油剂经皮肺内注射大鼠肺肿瘤模型,免疫组化发现DJ-1主要表达于上皮细胞,且组间表达存在明显差异,未成瘤组肺组织表达水平较成瘤组明显降低。定位苯并芘促进肺癌发生过程中,大鼠肺组织微环境防癌作用的位于支气管上皮细胞的靶点DJ-1蛋白。DJ-1靶向siRNA转染HBE细胞,能有效的从mRNA水平和蛋白水平下调DJ-1基因的表达,在一定转染浓度对HBE细胞的增值及凋亡无明显影响。第二部分细胞实验,C组中肺腺癌细胞的增殖受到增殖抑制,细胞周期阻滞及凋亡水平较前2组增高。各组裸鼠移植瘤实验提示C组与A组的成瘤时间及成瘤体积存在差异,C组肿瘤的平均体积指数小于A组。第三部分,A组苯并芘注射旁肺组织DJ-1基因mRNA水平和蛋白水平较B组明显降低。绿茶有效成分(EGCG)能有效地下调支气管上皮细胞中的DJ-1蛋白的表达水平,并呈一定剂量和时间效应。结论苯并芘肺内注射构建大鼠原发肺肿瘤过程中,位于支气管上皮细胞DJ-1蛋白可能是大鼠肺组织微环境抑制肺癌的靶点蛋白之一。下调支气管上皮细胞中的的DJ-1蛋白的表达水平,对共培养的肺腺癌细胞增殖及成瘤性有抑制作用。上皮细胞中这个蛋白水平的改变可能参与抗促癌剂(茶酚类)拮抗促癌剂肺癌预防作用。更多还原

【Abstract】 OBJECTIVEBefore the formation of lung cancer experienced a series of morphological and molecular changes, Bronchoalveolar lavage from normal epithelial cells turn into various morphological features of lung cancer is a complex and lengthy multi-stage multi-step carcinogenesis,and Bronchial epithelial cell proliferation/differentiation control disorders are among the root causes. In the cancer-causing agents such as benzo (a) pyrene compounds for lung cancer process to promote tumor growth, These normal cells, such as the lung bronchial epithelial cells, From quantitative to qualitative change is imperceptible to the time of malignant transformation. Studies show that the polyphenols and other antioxidants could antagonize the effect. People dedicated to finding prevention strategies and excellent chemical substances, interfere with metabolism of carcinogens, do not make it into a complete carcinogen activation or inhibition、prevention of carcinogen DNA binding and cell, Inhibiting cancer or precancerous lesions evolving into its reversal to normal cells. In the previous studies, study confirmed 3,4-benzo (a) pyrene in oil injection percutaneous pulmonary rat lung tumor model building is a simple and reliable animal model of lung cancer method comfirmed, one type of tumor pathology mainly in non-small cell lung cancer. In our study, By unpaired two-dimensional gel electrophoresis to obtain tumors in rats 3,4-Benzo (a) pyrene injection into the tumor adjacent lung tissue and adjacent lung tissue in rats differentially expressed protein profiles, To screening in lung tissue microenvironment and 3,4-benzo (a) pyrene induced lung cancer associated with primary protein points. These proteins include the DJ-1, including nine particle, We believe that with 3,4-benzo (a) pyrene in lung tumors after injection of immature rats inhibited the formation of lung cancer-related proteins. Tumor microenvironment suggest DJ-1 protein can serve as a target for cancer chemoprevention, Chemoprevention against cancer-causing factors in the early control, micro-environment through a variety of cells and cytokines affect tumor phenotype intervention. Many targets the tumor microenvironment, Selecting the DJ-1 as a molecular target and find its place cells is the first step, Understand the micro-environment, changes in the level of protein molecules on tumor cells, To verify the target protein and localization of cells and with green tea (polyphenols) the relationship between lung cancer prevention.METHODS AND RESULTSThe study was carried out in three parts.In the first part,80 female SD rats were randomized into two groups(each group has 40 rats).40 rats(group A)were treated with 3,4-benzopyrene-corn oil(2mg 3,4-benzopyrene,dissolved in 0.2mL corn oil);meanwhile 40 rats(group B)were treated with corn oil only.The injection were given in every 2 weeks for 8 weeks through left middle-chest percutaneous puncture.All rats received deionized water as their sole source of drinking fluid.After 32 weeks following the first injection,the rats were killed.In group A,the rats those developed lung tumors were separated into group C and the others were separated into group D.Portions of tumor-adjacent lung tissues(group C)and 3,4-benzopyrene injection-adjacent lung tissues(group D) which might be associated with the carcinogenesis process of lung cancer. Verified by immunohistochemistry in lung tissue of lung cancer proteins inhibit the expression levels of DJ-1 and the main place cells. DJ-1 targeted siRNA transfection of human bronchial epithelial cells, from the mRNA level and protein level of DJ-1 after transfection reduced the effect of gene expression, detected by the DJ-1 HBE cells transfected with targeted siRNA on cell proliferation and apoptosis. In the second part, the establishment of human bronchial epithelial cells and lung adenocarcinoma cells were co-cultured model. Divided into three groups, group A of tumor cell culture as a separate group or the control group, group B untreated epithelial cells and lung adenocarcinoma cells were cultured group that controls, group C transfected with the siRNA targeting DJ-1 and lung epithelial cells after co-culture group of cancer cells that the experimental group. Proliferation of cell growth curve of each group and the level of cell cycle and apoptosis in three groups of nude mice experiments. In the third part, construction of 3,4-benzo (a) pyrene in oil percutaneous injection of rat lung tumor model of lung,60 female SD rats were randomized into two groups(each group has 30 rats),one for green tea(group A),and the other for control(group B).All rats were treated with 3,4-benzopyrene-corn oil(as described in Experment 1).16 weeks, rats were killed and protein from the mRNA levels were compared in rats of benzo (a) pyrene injected next to the lung tissue of DJ-1 gene expression differences. With the active ingredients of green tea (EGCG) treatment of bronchial epithelial cells, mRNA and protein levels from the determination of epithelial cells in DJ-1 gene expression in time and dose-response relationship. Successfully constructed and 3,4-benzo (a) pyrene in oil percutaneous injection of rat lung tumor model of lung, immunohistochemistry showed that DJ-1 was mainly expressed in bronchial epithelial cells, and the expression of significant differences between groups, immature lung tumor tissue levels of tumor was significantly lower than that. Location benzo (a) pyrene in the process of promoting lung cancer, lung tissue micro-environment in the anti-cancer effect of bronchial epithelial cells in DJ-1 protein target. DJ-1 HBE cells transfected with siRNA targeting, effective from the mRNA level and reduced levels of DJ-1 protein expression, a certain concentration of HBE cells transfected with the value-added and no significant effect on apoptosis. The second part of the cell experiments, group C, lung cancer cell proliferation was inhibited, the level of cell cycle arrest and apoptosis increased over the previous 2 groups. Nude mice in each group experiment indicated, group C and group A of tumor formation time and tumor volume differences, group C, the average tumor size of less than group A of indices. In the third part, group A of benzo (a) pyrene injected next to the lung tissue levels of DJ-1 gene mRNA and protein levels were significantly lower than group B. Active ingredients of green tea (EGCG) effectively reduced bronchial epithelial cells the expression of DJ-1 protein levels, and a dose and time effect.CONCLUSIONBenzo (a) pyrene in rat lungs injected construct during the primary lung tumor, DJ-1 protein in the bronchial epithelial cells may be inhibited in lung tissue micro-environment of the target protein of lung cancer. Reduced bronchial epithelial cells of the DJ-1 protein expression, On the co-cultured lung cancer cell proliferation and tumorigenesis was inhibited. Epithelial cells, the protein level may be involved in anti-tumor promotion agents (tea polyphenols) against cancer-promoting agent of lung cancer prevention.更多还原