【作者】 崔英；

【导师】 张丽生；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2005， 博士

【摘要】 肝癌是世界上最常见的恶性肿瘤之一,肝癌的防治研究成为最富有挑战性的课题。绿茶及其主要组分茶多酚的抗癌作用日益引起广泛关注。茶多酚具有广泛的生物学特性和药理效应,能够抑制多种肿瘤的发生和发展。但茶多酚抗癌的确切机制尚未清楚,本课题从体外细胞实验、人肝癌细胞SMMC-7721裸鼠移植瘤模型、以及茶多酚预防人类肝癌的基础实验三个方面研究茶多酚的抗癌作用,应用DNA凝胶电泳、流式细胞仪、端粒酶检测、电镜、基因芯片、RT-PCR、免疫组化等现代分子生物学技术多方面探讨其作用机制。 体外细胞实验中,MTT法和集落形成实验显示,茶多酚可以明显的抑制Hep3B肝癌细胞的增殖和集落形成,随着茶多酚的浓度逐渐增加,其抑瘤作用递增,茶多酚对Hep3B肝癌细胞的半数抑制剂量IC₅₀值为143μg/ml。肝癌细胞经茶多酚处理后,细胞形态发生变化。电镜观察:细胞出现凋亡现象。提取茶多酚处理的Hep3B肝癌细胞的DNA进行琼脂糖凝胶电泳显示典型的DNA梯状条带。流式细胞仪检测发现:茶多酚能引起细胞周期（G₁期）阻滞,阻止受损细胞进入S期而抑制细胞过度增殖;茶多酚可以引起Hep3B凋亡,并呈现剂量—效应和时间—效应关系。采用茶多酚诱导Hep3B细胞分化后,端粒酶活性下降。用茶多酚处理24、48小时的Hep3B肝癌细胞及对照组细胞制备cDNA探针,进行Atlas cDNA array杂交,分析发现茶多酚上调死亡受体介导的凋亡通路相关基因TNF、TNFR、Caspase-9和Caspase-10,下调bcl—2等基因。用RT-PCR的方法验证部分结果,与Atlas cDNA array的分析结果一致。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The research on prevention and treatment of HCC is the most challenging topic .Green tea and its key component green tea polyphenols (GTP) have been paid great attention for its anticancer effects.GTP has extensive biologic features and pharmacology effects. It can inhibit the genesis and development of many tumor. But the exact mechanism of the GTP remains unknown. This study explore the anticancer effects of GTP through three following experiments ,they are cell experiments in the vitro, nude mice mode of HCC cell line SMMC-7721 and the basic experiment study in prevention of HCC with GTP. DNA agarose gel electrophoresis, Flow cytometer(FCM), the examination of telomerase, Atlas cDNA array, RT-PCR and electron microscope, immunohistochemistry were used in the study.In vitro cell experiments ,MTT and colony formation experiments show that GTP can apparently inhibit the proliferation and colony formation of HCC cell line Hep3B,with the increase in GTP’s concentration, its anticancer effects augments .the GTP’s IC50 is 143μg/ml. After being processed with GTP, the shape of HCC cell changes. Through electron microscope ,apoptosis of cell can be watched. The cell DNA extracted display ladder bands in DNA agarose gelelectrophoresis. Through the analysis of flow cytometry.,GTP can block cell cycle Gi, preventing the damaged cell entering S phase ,so it can suppress the excess proliferation ,GTP can induce the apoptosis of Hep3B. It showed dose -time and time -effect relations . When Hep3B cell was induced by GTP ,The telomerase activation decrease . Atlas cDNA array was conducted .The results showed that GTP can upgrade related gene TNF,TNFR,Caspase -9 and Caspase-10 which can induce apoptosis while down regulating bcl-2. RT-PCR confirm the result which is in accordance with the result of Atlas cDNA array.Establishing the transplantation tumor in nude mice using HCC cell line SMMC-7721. The studies showed that GTP can inhibit the transplantation tumor in nude mice. Through electron microscope ,we can see that GTP can induce the cell apoptosis of the transplantation tumor in nude mice .Through flow cytometry ,the apoptosis rate in positive group and high ,medium, low dose group of GTP is much higher than that from control group.and the difference is significant (P<0.05). In the experiment of immunohistochemistry,the protein expression of gene Bcl-2 in positive group and high ,medium ,k>w dose group of GTP decrease compared with that from control group . Also the difference is significant(P<0.05)., No difference was found in the protein expression of gene 53 in transplantation tumor of negative control group and high ,medium ,low dose group of GTP (P>0.05).The basic experiment study in prevention of HCC with GTP assess the effects of Long-tern taking GTP on the immune functions and anti-oxidation among people with high risks of suffering from HCC. 1200 people with high risks of更多还原