【作者】 刘国兴；

【导师】 于录；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 硕士

【摘要】 华支睾吸虫(Clonorchis sinensis,Cs)又称东方肝吸虫,是一种危害非常严重的鱼源性寄生虫,它不但给渔业生产造成了巨大的经济损失,而且对人类身体健康也构成了极大的威胁。近年来,越来越多的实验和流行病学数据证明华支睾吸虫的感染与胆管癌的发生存在着病因学联系,世界卫生组织下属国际癌症研究署已于2009年将华支睾吸虫列为了第Ⅰ类致癌物质,成为11种Ⅰ类致癌病原生物的一员。尽管有众多的证据证明华支睾吸虫的致癌作用,然而对于华支睾吸虫诱发胆管癌的发病机制仍然不是很清楚,其中该吸虫排泄/分泌代谢产物(ES)对宿主的作用被认为是最主要的直接性因素,它们除了具有较高的免疫原性,能引起宿主免疫反应外,还可能会对宿主细胞直接施加毒性或者与其相互作用。为了进一步验证它们所发挥的作用,我们收集了这些代谢产物,并将其分别作用于胆管癌和肝癌细胞,利用CCK-8法检测和分析了不同浓度ES及不同作用时间对两种细胞增殖的影响,结果表明该吸虫代谢产物对两种细胞的增殖具有明显的促进作用,从而进一步证实了这些代谢中可能存在某些促细胞增殖的相关因子。为了寻找华支睾吸虫成虫与胆管癌细胞相互作用的蛋白,本研究构建了华支睾吸虫成虫T7噬菌体展示cDNA文库,并利用全细胞淘洗的方法对文库进行了筛选,将筛选得到的噬菌体进行体外扩增,随后提取其基因组DNA,并进行了测序和序列分析。最终获得了6种细胞互作阳性克隆噬菌体。希望通过该方法筛选获得与胆管癌细胞相互作用的多肽或蛋白,为进一步确定华支睾吸虫成虫排泄/分泌代谢产物中促细胞增殖的有效成分和相关机制的研究奠定基础。更多还原

【Abstract】 The Oriental liver fluker, C. sinensis, is a serious fish-borne parasites, not only caused the huge economic losses to fishery production, but also made a great threat to human’s health. These flukes are predominantly endemic in the East and Southeast Asia countries, such as China, Korea, Vietnam and Laos, and is the highly prevalent in China. According to the epidemiological survey, C. sinensis has been reported for most provinces in China, except Inner Mongolia, Ningxia, Qinghai, Tibet, and Xinjiang, and over 15 million people are infected with C. sinensis in our country now, especially in Guangdong, Guangxi and the three northeast provinces. Clonorchiasis is a serious food-borne parasitic zoonosis, which is mainly exhibited as hepatobiliary lesions caused by parasitic C. sinensis in human bile ducts. The liver appears grossly normal in light infections, but in heavy infections there is the enlargement of the liver and loss of appetite with acute pain in the right upper quadrant. In the chronic stages, the liver might eventually become malfunctioning. Biliary obstruction, intrahepatic stone formation, cholangitis, cholecystitis, cholelithiasis, biliary and liver abscesses, and pancreatitis are further severe symptoms. In recent years, both experimental and epidemiologic evidence strongly implicates C. sinensis infection in the aetiology of one of the liver cancer subtypes—cholangiocarcinoma (CCA), or cancer of the bile ducts. Like O. viverrini, C. sinensis is also classified as Group 1 carcinogenic to humans by the International Agency for Research on Cancer for cholangiocarcinoma in 2009.The exact mechanisms of the carcinogenesis, however, are not clearly elucidated. The mechanical damage, parasite secretions and immunopathology may enhance cholangiocarcinogenesis. The ?uke excretory–secretory several metabolic products (ES), which was released from the tegument and excretory opening of C. sinensis during host infection, apart from inducing host immune response, may be toxic to or interact with biliary epithelium. In order to further identify that ES from the liver ?ukes play pivotal roles in development of cholangiocarcinoma, we collected the amounts of ES products of C. sinensis, and treated the cholangiocarcinoma cells lines and hepatocellular carcinoma cell lines in different time and in different concentration. We used the CCK-8 method to measure cell proliferation, and the result showed that cells treated with ES products significantly promote the proliferation of two cells compared to control cells. These findings support a role for ES products of C. sinensis in establishment of a tumorigenic environment.The phage display technique has been widely used in many research areas of the life sciences, such as the reaction of antigen-antibody, interactions of protein-protein and protein-drug, because it has many advantages, for example high library capacity, screening of convenient and flexible. In order to biopan the interaction protein with cholangiocarcinoma cells from adult of C. sinensis, we constructed successfully a T7 phage display cDNA library of adult worms, and the primitive library capacity was 3.7×105pfu, recombinant ratio was 98%, and the title of amplification library was 4.3×1012pfu/ml. Then, the display library was screened by whole-cell selection method. The phages binding on the cells were stable after 5 turn’s biopanning. Finally, we obtained 6 proteins which can interact with cholangiocarcinoma cells by sequencing and analyzing, and separately named Cs-CCA-p8、4、13、21、7、14. They are highly homologous with C. sinensis, S. mansoni and S. japonicum, therefore, this implies that they may have similar biological functions. There were not transmembrane domains in the six proteins through TMHMM Analyzing, however, Cs-CCA-p4 and Cs-CCA-p7 contain a signal peptide, therefore, they may be transported through the way of secretion to play a role in extracellular. There provide a more comprehensive solution to obtain the carcinogenic-related genes by biopanning of display library combination with other screening methods, for example the electronic screening of EST sequence.更多还原