【作者】 刘楠；

【导师】 房学东；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 研究背景：阿特拉津（ATR）是一种选择性内传导型的除草剂，适用于去除玉米、高梁、甘蔗、果园和茶园内的杂草，主要可防除一年生禾本科杂草以及阔叶杂草，同时对某些多年生杂草也具有一定的抑制作用。2005年，吉林省东辽河流域的旱田及非旱田水域中，ATR的平均值分别9.70μg/L和8.85μg/L。ATR难降解，可致畸变、突变和癌症，其进入土壤和河流后很难被微生物分解，因而在环境中不断蓄积，极大威胁人类健康和生态平衡。ATR的特点是使用量大、残留期长、污染范围广。相关实验研究发现，ATR可使仓鼠染色体发生断裂，影响精子成熟过程，从而影响鼠的正常繁殖；以ATR处理小鼠，小鼠血液中白细胞减少，免疫系统发生异常，并发生肌肉痉挛；以ATR处理体外培养的人淋巴细胞，当ATR浓度为1×10-3μg/L时，可使核染色体发生轻微损坏，而ATR达到5×10-3μg/L时，染色体损伤显著。研究证实，ATR有致癌作用，长期暴露于ATR的实验动物可发生卵巢癌及乳腺癌。Su等的研究表明，ATR可作用于体内CYP19酶，改变其活性，继而干扰内分泌平衡，诱发癌症。关于ATR诱发肿瘤的机制，有报道称是由于ATR产生过氧化反应，导致正常细胞的氧化应激水平增加，由于细胞内氧自由基增加，导致线粒体和细胞核发生DNA损伤，继而正常细胞发生恶变，癌基因被激活。另外，ATR还具有其他生物学毒性，如生殖系统毒性、神经系统毒性、内分泌系统毒性和免疫系统毒性。肝癌是一种恶性程度高、具有高转移性的肿瘤，原发性肝癌是全世界最常见恶性肿瘤之一，其死亡率在胃癌、食道癌之后，位居恶性肿瘤的第三位。我国是原发性肝细胞癌的高发区，每年死于肝癌的人数约占全世界肝癌死亡人数的50%。近20年来，虽然我国的肝癌治疗水平取得了长足进步，但肝癌的发病率和死亡率仍位于癌症的第二位。肝癌的发病原因较多，其中环境因素诱发的肝细胞恶变较为常见，主要是由于环境中的危险因子长期作用，刺激肿瘤的发生，如酒精和香烟等刺激可诱发肝炎，肝炎的长期作用易诱发肝癌。除草剂ATR作为肿瘤的危险分子，可能与肝癌的发生发展有关，其依据是：ATR难于降解并难于被土壤中的微生物所分解，因此导致农作物中的大量残留，未降解的ATR和作物中残留的ATR可被人体吸收并长期蓄积，造成肝脏负担加重，促进病变，继而可能导致肝细胞发生癌变。环境剂量的阿特拉津对肝癌细胞增殖、侵袭的影响尚未见报道。本研究主要探索ATR对肝癌细胞增殖及侵袭能力的影响，并探讨其机制，为农药ATR的正确使用及有效预防ATR的致瘤毒性提供实验基础，进而为ATR的环境预警与临床防治其生物毒性提供理论依据。目的：通过体内外实验，观察除草剂阿特拉津对肝癌细胞H22增殖、侵袭和迁移能力的影响，并探讨其作用机制。方法：选择鼠源性、恶性度高的肝腹水瘤H22细胞作为研究对象，构建小鼠肝原位移植瘤模型。将H22细胞或肝原位移植瘤模型小鼠分别暴露于不同浓度ATR，应用MTT法检测H22细胞的增殖能力，观察移植瘤的体积；应用免疫组化方法检测核增殖抗原PCNA的表达；应用流式细胞仪检测细胞周期，应用Real time PCR，Western blot方法检测周期相关基因P21、P53和Cyclin D1的转录和表达，分析ATR影响细胞周期的机制；应用Real time PCR， Western blot方法检测MMP9、MMP2及VEGF的表达，分析ATR影响细胞侵袭的机制；应用Western blot和Real time PCR检测STAT3信号通路及其下游基因C-myc的表达，分析ATR影响肿瘤细胞和原位移植瘤的信号通路。并利用免疫组化方法进一步验证上述基因和蛋白的表达及定位。结果：0.01mmol/L、0.1mmol/L和1mmol/L的ATR作用H22细胞48h后，MTT结果显示ATR可促进肝癌细胞增殖，流式细胞仪检测结果显示ATR可促进细胞通过G0-G1期检查点，增加S期细胞的比率。成功构建肝脏原位移植瘤C57BL/6动物模型，给予20mg/kg和100mg/kg ATR灌胃处理小鼠后，小鼠肝脏原位移植瘤体积和重量增加，65%的肝脏原位移植瘤转移为腹水瘤，免疫组化结果显示细胞核中PCNA表达上调。体内外实验提示ATR可能通过下调肝癌细胞P53和P21基因的转录与表达，上调肝癌细胞Cyclin D1基因的转录和表达，促进细胞周期的进程。ATR作用后细胞的侵袭能力增强，肝脏原位移植瘤向腹水瘤转移的百分率增加，该作用可能是通过上调MMP2、MMP9和VEGF表达而实现的。而上述作用可能是通过ATR上调STAT3的表达，从而增加下游基因C-myc的表达。结论：ATR可促进肝癌腹水瘤H22细胞的增殖、侵袭和迁移，并增加肝癌H22细胞存活率；促进原位移植瘤生长，增加原位肿瘤的重量，加速肝脏原位移植瘤向腹水瘤的转移。ATR促进肝癌生长转移的作用机制可能是通过调节P21、 P53及Cyclin D1的表达，促进细胞周期进程，从而促进细胞的增殖；通过上调MMP2、MMP9和VEGF表达，促进H22细胞及其原位移植瘤的侵袭和转移，并促进新生血管形成。上述改变可能是通过激活STAT3信号通路及其下游基因C-myc而实现的。更多还原

【Abstract】 Background: atrazine (ATR) is conducting the selective herbicide for corn,sugarcane, sorghum, tea plantations and orchards, etc. ATR can control annual grassesand broadleaf weeds, perennial certain miscellaneous grass also has a certain extent.2005statistics showed that the East Liaohe River in Jilin and non-upland uplandwaters, the annual average ATR were9.70μg/L and8.85μg/L. Atrazine is difficult todegrade, mutagenic, and carcinogenic distortion caused by chemical contaminants.ATR enters the soil and rivers difficult to balance the degradation and decomposed bymicroorganisms, resulting in their accumulation in the environment constantly, so agreat threat to human health and ecology. ATR is characterized by the use of a greatamount of residual period is very long, very wide range of pollution. Experimentalstudy found that in mice experiments, ATR hamster chromosome breakage can affectthe sperm maturation process, thus affecting the normal breeding rats. Another studyshowed ATR-treated mice, mouse blood leukopenia, abnormal immune and musclespasms, muscle contracture performance of acute poisoning, such as the nervoussystem. Furthermore, in vitro with human lymphocytes ATR using ATR concentration0.001μg/L, the resulting lymphocyte chromosome minor damage, the concentration0.005μg/L of chromosomal damage occurs significantly. Studies show that the foreignpart, ATR can make human carcinogen, long-term exposure can cause animals ATRovarian and breast cancer. Su et al found, ATR can affect the body CYP19enzymeactivity, thereby interfering with the endocrine balance, damages the body’s endocrinesystem, causing a series of adverse symptoms, which will induce cancer, therefore,ATR has a priming effect of the tumor. ATR-induced mechanism of tumor has beenreported that due to the ATR produce peroxide, resulting in increased oxidative stressin normal cells, the cells due to the increase of oxygen free radicals, creating a viciouscycle, leading to mitochondrial and nuclear DNA damage in cells, normal cells turnmalignant oncogene is activated. In addition, other biological toxicity studies ATRalso includes nervous system toxicity, reproductive toxicity, endocrine system,immune system toxicity and toxicity. HCC is a highly malignant, with high metastatic tumors, and primary liver cancer is one of the most common malignancies worldwide,and its mortality rate, after qualifying in malignant gastric cancer, esophageal cancer,bit ranks third. Ours is a high incidence of hepatocellular carcinoma, China’s annualdeaths from liver cancer, accounting for about50%of liver cancer deaths worldwide.The past20years, although the level of China’s treatment of liver cancer has madeconsiderable progress, but liver cancer incidence and mortality of cancer is still insecond place. Its causes are many, including environmental factors induced malignanttransformation of liver cells, more common, mainly environmental risk factors in thelong-term effects, stimulate tumorigenesis, such as the liver, such as alcohol andcigarettes being stimulated after, it will induce the occurrence of hepatitis, andlong-term hepatitis will induce liver cancer. ATR herbicide molecules as dangerousdigestive tumors may be associated with the development of HCC. ATR is difficultdue to the degradation and decomposition by microorganisms in soil, in time to giveagricultural pesticide, resulting in residual ATR and other crops in a large number ofresidues, undegraded ATR and vegetables, will be absorbed by the body, the long-termaccumulation, causing liver burden, promote disease, which in turn is likely to makethe liver cells become cancerous. Environmental dose but whether ATR promotesproliferation and invasion of ovarian cancer cells has not been reported. In this study,to explore the effects of ATR on proliferation and invasion of ovarian cancer cells andto explore its mechanisms to provide a theoretical basis and experimental basis for theproper and effective use of preventive ATR ATR tumorigenic toxicity, and thus asatrazine and environmental warning prevention and treatment of its toxicity to providea scientific basis.Objective: In vivo study outside herbicide atrazine on H22hepatoma cellproliferation, invasion and migration capabilities.Methods: To study the effects of ATR on the body, we can choose to buildmurine orthotopic xenografts in mice, a higher degree of ascites tumor malignant cellline H22as the research object, which are used to build orthotopic liver tumor. TheH22cells were exposed to different concentrations of0.01mmol/L,0.1mmol/L, aftertreated by1mmol/L ATR for48h, detected by MTT proliferation of H22cells,immunohistochemical detection of cell proliferation antigen expression of PCNA;using flow H22detected the cell cycle, the application of real-time fluorescencequantitative-PCR, Western blotting was used to detect H22cells or liver orthotopic xenograft P21, P53, cyclin D1transcription and expression, and analysis of tumor cellcycle ATR mechanism; using real-time fluorescence quantitative expression-PCR,Western blotting detection MMP9, MMP2and VEGF, analysis ATR affect cellinvasion mechanisms and neovascularization capacity of marker genes and proteins;application of Western blotting and Real time PCR detection of STAT3signalingpathway and its downstream gene C-myc state analysis of the fundamentalmechanisms of tumor cells and in situ ATR xenografts. Similarly, the use ofimmunohistochemical methods in the cytoplasm and the nucleus is further validationof the expression of these genes and proteins.Results: In vitro experiments, the concentration of0.01mmol/L,0.1mmol/L,ATR role1mmol/L of H22cells after48h, MTT showed that ATR can promote livercancer cell proliferation, flow cytometry showed that ATR can promote cell by G0-G1phase checkpoint, increasing the ratio of S phase cells; vitro experiments, orthotopicliver transplantation tumor was successfully constructed C57BL/6animal models,different concentrations20mg/kg and100mg/kg ATR orally treated mice, the resultsin mice orthotopic liver tumor volume and weight increase,65%of orthotopic livertumor metastasis into ascites tumor immunohistochemistry showed upregulation ofexpression of PCNA in the nucleus. ATR in vivo experiments confirmed bydown-regulating transcription and expression may P53and P21genes upregulatedCyclin D1gene and protein expression to promote cell cycle progression; ATR afterthe invasion ability of cells to enhance the role of liver ascites orthotopic xenografttumor metastasis to the percentage increase, which may be regulated by MMP2,MMP9and VEGF expression and realization. The above mechanism may be byupregulating the expression of STAT3, thereby increasing the expression ofdownstream genes C-myc-related genes and proteins.Conclusion: In vitro experiments, the ATR doses did not cause apoptosis, and canpromote the proliferation of H22hepatoma ascites tumor cells, invasion and migration,and increase the survival rate of liver cancer H22cells. In vivo, ATR promoteorthotopic xenograft tumor growth, increase the weight of in situ tumors, acceleratedliver orthotopic xenograft tumor metastasis to ascites.Its mechanism of action is probably vitro and in vivo by regulating the expression ofP53, P21and Cyclin D1promotes cell cycle S increases, thereby contributing to theproliferation of H22cells and by promoting the expression of MMP2and VEGF promote H22cells and orthotopic transplantation tumor invasion and metastasis andpromote the formation of new blood vessels, regulates the expression of PCNA andpromote tumor cells in situ malignant proliferation. These changes may be through theactivation of STAT3signaling pathway and its downstream gene C-myc achieve.更多还原