【作者】 顾其华；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 目的：肺癌的发病率在工业化国家里持续居高不下，发展中国家的肺癌发病率仍在不断上升。因而人们转而注重肺癌的预防，希望通过化学预防策略降低肺癌的发病率。最佳的化学预防策略是通过日常饮食降低肺癌的发病风险。茶为全球最普遍的饮料之一，年消费量仅次于水，其中绿茶的防癌作用较好，有可能是最佳的肺癌预防饮料。但目前绿茶与肺癌的关系仍存在一定的争议，绿茶预防肺癌的机制尚未完全明确。另外，肺肿瘤动物模型是评价肺癌防治方案必不可少的工具，而目前所采用的肺癌裸鼠移植瘤模型以及采用致癌剂吸入、支气管内灌注、腹腔注射等方法构建的原发性肺肿瘤模型等均存在不同程度的不足。因此，本课题试以苯并芘油剂经皮穿刺肺内注射的方法建立大鼠肺肿瘤动物模型，并以此模型评价绿茶对肺癌的预防作用，在此基础上观察绿茶对大鼠肺肿瘤组织以及对苯并芘所致大鼠肺损伤的p53和Bcl-2基因表达的影响，以初步阐明绿茶预防肺癌的机制。方法：每次以含3，4-苯并芘2mg的苯并芘-玉米油混合剂0.2mL经皮肺穿刺注射于经戊巴比妥钠麻醉后的SD大鼠右肺，每2周处理1次，共4次。并以纯玉米油肺内注射4次作为阴性对照，苯并芘-玉米油皮下注射处理2次作为阳性对照。观察大鼠肿瘤的发生情况。在绿茶预防肺肿瘤的实验中，以3，4-苯并芘油剂经皮穿刺肺内注射构建大鼠原发性肺肿瘤模型，并以1％的绿茶作为大鼠的唯一饮料干预处理，对照组以水为唯一饮料，观察绿茶处理对大鼠成瘤率的影响，并以原位杂交和免疫组织化学方法检测绿茶对大鼠肺肿瘤组织p53和Bcl-2基因表达的影响。在此基础上，进一步采用原位杂交和免疫组织化学方法观察绿茶干预处理对肺内注射3，4-苯并芘油剂后第1周、第4周、第8周及第16周大鼠肺组织p53和Bcl-2基因表达的影响。结果：所有接受玉米油肺内注射的大鼠一年内均未发生肿瘤；皮下注射组局部恶性肿瘤发生率达100％，平均晚期肿瘤摘除时间为15.83周，中位时间为16周；苯并芘-玉米油肺内注射组大鼠一年内肺部恶性肿瘤发生率为70％左右，平均晚期肿瘤摘除时间为31.54周，中位时间为30周，苯并芘肺内注射较皮下注射更难以形成肿瘤。1％的绿茶使大鼠肺恶性肿瘤发生率下降为30％，绿茶干预组大鼠肺肿瘤组织p53表达上调、Bcl-2基因表达下调，且与对照组比较Bcl-2基因表达差异有统计学意义(P＜0.05)。肺内注射3，4-苯并芘油剂后第1周、第4周、第8周及第16周大鼠肺、支气管上皮细胞、粘膜下层细胞、血管内皮细胞、淋巴细胞以及肺间质细胞等组织p53和Bcl-2基因均呈过量表达，但绿茶对苯并芘处理后大鼠各个时期肺组织的p53和Bcl-2基因表达均有影响，上调p53表达，下调Bcl-2表达，差异均有统计学意义(P＜0.05)。结论：3，4-苯并芘油剂大鼠肺内注射是一种简便可靠的肺肿瘤模型构建方法。绿茶对苯并芘所致的肺恶性肿瘤具有明显的预防作用，上调p53和下调Bcl-2的表达是绿茶预防肺癌的重要机制。此外，肺自身具有较强的肿瘤防卫能力，绿茶很有可能通过另一种机制即通过调节肺“防卫细胞”的功能预防肺癌的发生。更多还原

【Abstract】 Objective The incidence of Lung cancer continues to be high inindustrialized countries and is still increasing in many developingcountries of the world. The 5-year survival following treatment has notimproved significantly over the past decades.So the strategy ofchemopreventive intervention has been emphasized to prevent lungcancer. One of the most promising strategies for cancer chemopreventionis prevention by daily used food and beverages. Tea is the second only towater as the most consumed beverage in the world.Green tea,which ismore effective than black tea in cancer prevention,may be the mostpromising chemopreventive agent against lung cancer.But controversiesare still emerged in some studys of tea and tea extracts against lungcancer, and the mechanism of integrated green tea other than green teaextracts in lung cancer prevention remains to be unknown.In addition,asatisfactory animal model plays a important role in evaluation ofprevention and treatment of lung cancer.But various of animal models incurrent lung cancer study are imperfect. The purpose of the current studyis to explore a simple and convenient and reliable method for lungneoplasm model establishing induced by 3,4-benzopyrene-com oilsolution pulmonary injection in rats and to evaluation the effect oftreament with 1%green tea on 3,4-benzopyrene pulmonary injection induced lung tumorigenesis in Sprague Dawley (SD) rats and modulationof p53,Bcl-2 expression in lung neoplasm tissues,and to furtherinvestigation the effect of green tea on p53 and Bcl-2 expression in lungtissues injuring by 3,4-benzopyrene. methods The study was carriedout in three parts. In the first part, The prepared female SD rats,with therange from 180 to 220g of body weight,were divided into three grop atrandom.In the model group of lung neoplasm,the rats were given 2 mg of3,4-benzopyrene soluting in 0.2 mL corn oil fortnightly pulmonaryinjection for 4 times through right middle-chest percutaneous punctureunder control of anaesthesia by pentobarbital sodium i.p. The negativecontrols were given 0.2 mL corn oil pulmonary injection only. Thepositive controls were given 2 mg of 3,4-benzopyrene soluting in 0.2 mLcorn oil injection under skin of scapular area fortnightly for 2 times.Therats were sacrificed after suffering from dyspnea,and the survived rat 1year after the first 3,4-benzopyrene toxicosis were sacrificed at narcotism.Lung,brain,liver, oesophagus and stomach of all cases were anatomizedin search of tumor. In the second part,the animal model of lung neoplasmwas established as previous description. The rats in the green teaadministration group were given 1% green tea drinking only beginningfrom 2 weeks before 3,4-benzopyrene treatment to the end of one-year’sobservation, the controls were given water drinking only.The incidence oflung neoplasm in rats per group were counted. Each case of lung neoplasm was examined for expression of p53 and Bcl-2 withinsituhybridization analysis and immunohistochemistry staining. In thethird part,animals were treatment with 1% green tea drinking beginningfrom 2 weeks before the first 3,4-benzopyrene-com oil solutionpulmonary injection to the end of the 16-week’s observation. The ratswere sacrificed at the begining and 1,4,8,16 weeks after 3,4-benzopyreneinjection respectively. Insituhybridization and immunohistochemistrywere performed to analyse p53 and Bcl-2 expression in lung tissues ingreen tea administration groups and in controls. Results No lungneoplasm was found in the negative controls within 1 year’s observation.Malignant neoplasm was found in every rat under skin in the positivecontrol within 17 weeks,and the cancerogenic rate with 3,4-benzopyreneinjection under skin was 100%. The average time of tumor removing inthis positive control was 15.83 week with a median of 16 weeks.It wasfound that approximately 70% of rats emerged carcinoma at the local ofright lung during 1 year. No tumour was found in brain,liver, oesophagusand stomach in rats. The average time of tumor removing in this groupwas 31.54 week with a median of 30 weeks.It is more difficult thatmalignant neoplasm induced by 3,4-benzopyrene in the lung in rats thanunder skin. Green tea administration reduced lung carcinoma to 30%.Expression of p53 gene was slight upregulation by green teaadministration in lung carcinoma tissues,however expression of Bcl-2 gene was significantly downregulation in lung carcinoma tissues in ratswith green tea administration (P＜0.05).Further study showed thatoverexprssion of p53 and Bcl-2 were appeared in bronchial epithelialcells,cells under mucosa, vascular endothelial cells, lymphocytes andinterstitial cells in 1,4,8,16 weeks after 3,4-benzopyrene pulmonaryinjection. Green tea significantly enhanced p53 gene expression andsignificantly inhibited Bcl-2 gene expression in lung tissues during thewhole term beginning the 1th week to the 16th week after3,4-benzopyrene treatment (P＜0.05). Conclusion Carcinogen3,4-benzopyrene pulmonary injection by percutaneous puncture is anefficiency method for lung neoplasm model establishing in rats. Green teainhibit lung carcinogenesis and can upregulate expression of p53,downregulate expression of Bcl-2 in lung cancer induced by benzopyreneand in lung tessues injuring by benzopyrene attack, and this may berelated to the mechanism of lung cancer prevention. In addition,there mayhave a strong "defend cell" prevention mechanism in lung tissue toresisting canceration,and this may be play an important role in lungcarcinogenesis inhibiting. Green tea probably improve the function of"defend cell" during the process of lung cancer chemoprevention.更多还原