【作者】 赵东红；

【导师】 周开亚； 李朝军； 戴祝英；

【作者基本信息】 南京师范大学 ， 动物学， 2002， 博士

【摘要】 肝癌是我国高发性恶性肿瘤，分布广，转移率高，但其发生的分子机制尚不明确。本文选择信号转导及转录活化因子(signal transducers and activators of transcription)STAT3及Wnt信号转导途径中的信号分子β-catenin和TCF4作为研究对象，研究了STAT3、β-catenin和TCF4在肝癌中的异常表达及其在肝癌细胞生长迁移中的作用，试图在细胞水平上寻找治疗肝癌的方法。 1．STAT3对肝癌细胞迁移的抑制作用 为研究STAT3在人肝癌细胞生物学行为中的作用，通过优势负性突变(dominant negative)策略造成肝癌细胞中STAT3缺陷，并利用过表达STAT3造成异常活化，发现STAT3介导了白细胞介素-6(IL-6)对肝癌细胞迁移的抑制。并发现肝癌细胞BEL-7402中存在磷酸化STAT3的组成性活性。 2．肝癌细胞中Wnt异常信号转导途径对细胞增殖及迁移的影响 本文采用免疫组化、RT-PCR及Western Blot检测的方法，调查了β-catenin蛋白在中国人肝细胞癌组织及肝癌细胞BEL-7402中的异常高表达。并首次发现TCF4 mRNA在肝癌细胞BEL-7402中存在高表达。利用TOP荧光素酶报告质粒检测了肝癌细胞中Wnt途径信号传递的异常增强。提示我们在肝癌发生中异常Wnt信号途径可能起到极其重要的作用，TCF4在肝癌细胞中强表达可能是肝癌中Wnt途径异常活化的主要原因。为了证实β-catenin和TCF4在肝癌细胞Wnt信号途径异常活化中的作用，本文采用β-catenin突变型β-catenin S37A造成β-catenin异常活化，及利用Dominant negative TCF4阻断Wnt信号途径，发现β-cateninS37A可明显刺激肝癌细胞生长，而Dominant negative TCF4对肝癌细胞BEL-7402的增殖和迁移有明显抑制作用，并证实异常Wnt信号途径是通过cyclinD1来影响肝癌细胞增殖的。说明特异性的破坏TCF4和β-catenin形成的复合物以阻断Wnt信号通路，对于抑制肝癌细胞生长和迁移是行之有效的。本文为今后利用阻断Wnt信号通路来探索治疗肝癌的方法打下了基础。 博士学位论文3．STAT3与卜catcnin的相互关系研究 STAT3信号转导途径与p｛atenin的信号转导途径很有可能存在着一定的内在联系。我们在肝癌细胞HepGZ中发现STAT3可以和p｛atenin发生免疫共沉淀，并且此免疫沉淀物含量随着细胞密度的增加而减少。STAT3和p－catenin都是转录激活因子，但我们在研究STAT3对p－catenin转录活性的影响中尚未发现可喜的结果。4．中国家蚕抗菌肽基因在杆状病毒表达系统中的表达 抗菌肽是一类具有分子量小，无免疫原性，抗菌谱广等特点的抗菌多肽，有望成为新一代的抗菌、抗癌药物。本文对中国家蚕抗菌肽的天然基因及人工改造基因在 Bac to Bac杆状病毒表达系统中进行了表达，得到了有活性的抗菌肽。通过实验认为C－术端酚氨化对抗菌肽的抗菌活性很重要。本文为进一步探讨抗菌肽的抗菌抗癌机理提供了理想的材料。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. We selected STATS (signal transducer and activator of transcription), P -catenin and TCF4 as research objects. We studied the inappropriate expression and functions of them in HCC so as to try to search the way to treat the HCC.To understand the effect of STAT3 on the HCC, we overexpressed STATS to cause inappropriate activation and used the dominant negative form of STATS to lead to STATS absence. Studies demonstrated that the migration inhibition of BEL-7402 cells in response to IL-6 cytokine was dependent on STATS activation and STATS mediated the inhibitory effect of 11-6 on the liver cancer cell migration. Constitutive activation of STATS was present in BEL-7402 cells.Immuno-histochemistry, RT-PCR and Western blot analysis were carried out to examine the high expression of P -catenin and TCF4 in HCC tissues and HCC cell lines. We found the TCF4 mRNA was high expressed in HCC cell line for the first time. Functional importance of ?-catenin and TCF4 for the Wnt signaling pathway was determined by TOP luciferase reporter assays. The results suggested that TCF4 is the predominant TCP factor present in liver cancer cells and inappropriate activation of Wnt pathway maybe play an important role in the development of HCC.We showed that P -cateninSSTA overexpression stimulated tumor cell proliferation and the dominant negative TCF4 expression inhibited evidently the growth and migration ability of BEL-7402. Wnt pathway governed cellular proliferation by regulating cyclinDl protein level. These results indicated that specifically disrupting the ?-catenin-TCF4 complex could be useful in treating liver cancers and TCF4 may be an effective therapeutic molecule for suppressing growth of hepatocellular cancers.There would be certain crosstalk between STATS pathway and Wnt pathway. We demonstrated that endogenous P -catenin could be coimmunoprecipitated with endogenous STATS in HepG2 cell, and the immunoprecipitates were down-regulatedwith the increase of cell density. As transcriptional factors, no synergized activation of the two pathways was detected.Cecropins are a family cytokines of small, antibacterial peptides from insects. It is potentially to be a new generation of antibacterial and antitumor drugs. We used the baculoviruses system expressing the nature antibacterial peptide gene and the synthesized CMlV-like gene of Chinese silkworm Bombyx mori. The results showed that C-terminal amidation was important for antibacterial peptide’s function. We provided good materials to study the mechanism of their antibacterial and antitumor activity.更多还原