【作者】 朱海红；

【导师】 刘克洲； 陈智；

【作者基本信息】 浙江大学 ， 内科学（传染病）， 2001， 博士

【摘要】 胸腺是人体中枢免疫器官之一，是T细胞分化、发育、成熟的主要场所。因此，在淋巴系统发育和维持免疫系统的正常功能中起重要作用。近年来有学者认为，胸腺除了在免疫作用方面的中心地位外，还可能对机体神经-内分泌系统有影响。人体进入青春期以后，胸腺开始生理性萎缩，肿瘤、感染、免疫性疾病等发病率逐渐增高。大量研究表明小牛胸腺的提取物可辅助治疗肿瘤、感染及其他由于胸腺功能低下而引起的疾病。 胸腺组织的网状上皮细胞可产生多种胸腺激素，如胸腺肽、胸腺生成素、胸腺体液因子、淋巴细胞刺激因子、血清胸腺因子等。这些可溶性物质是构成胸腺微环境的主要因素，在T细胞分化和发育成熟过程中起重要的调节作用。近年国外学者将胸腺激素分为几种单独成分。由28个氨基酸组成的胸腺肽α1（thymosinalpha 1，Tα1）是胸腺激素的组成成分之一，属于胸腺肽第五组分，最初由Goldstein等分离。胸腺肽α1具有免疫调节作用，在体外可以促进致敏淋巴细胞分泌细胞因子，如IFN-α，IFN-γ，IL-2；促进高亲和力细胞因子受体的表达；调控骨髓前体细胞和脾细胞的末端脱氧核糖核酸转移酶活性；增强骨髓前体细胞的Thy1和Lyt1，2，3的表达；加速NK细胞的生成，增强NK细胞的活性；通过增强辅助T细胞的活性可以促进同种和/或自身淋巴细胞的反应；拮抗胸腺细胞成熟过程中的凋亡。体内应用结果也表明其在T细胞发育和功能重建中具有重要作用；可促进淋巴细胞分泌IL-2和表达IL-2受体；增强宿主的抗感染能力；促进机体清除病毒；增强免疫功能；有抗氧化；抑制癌细胞生长等重要作用。因此，胸腺肽α1具有增强宿主免疫功能、促进病毒消除和抑制癌细胞生长等重要作用。 临床上常应用于慢性乙型肝炎、丙型肝炎等病毒感染、肿瘤（如黑色素瘤、肺癌、白血病、鳞状上皮细胞癌和结肠癌等）、以及免疫缺陷病人的辅助治疗，效果较为显著。并可作为疫苗增强剂使用。因此，对其作用机制的研究将与其在传染病学、肿瘤学和免疫学领域中的应用密切相关。 目前，国内外对胸腺肽α1的研究多数集中在生物学功能和应用方面，并取得很大进展。但是对胸腺肽of的作川机制尤其是胸腺肽al作用于淋巴细胞后信号传导方面的报道却甚少。 首先，至今尚未克隆到胸腺肽al受体，因此对胸腺肽al受体知之甚少。目前仅有研究表明胸腺肽al可以和细胞表面的一些结构结合。如：Garaci等人用兔疫荧光法观察到小鼠淋巴细胞表面存在高亲和力的受体；MOOdy等人发现胸腺肽o可与 VIP（vasoactive intestinal pCptide）受体结合，但亲和力较低（IC50＝10u M），但胸腺肽 al又不是 VIP受体的拈抗剂，所以推测其是否存在另外的信号接受分子。尽管如此，至少说明存在一种分子可以与胸腺肽al 结合的分子，即胸腺肽。l结合蛋白（thymosin alphalbindng protein，TBP）。所以阐明胸腺肽 al结合蛋白，也就意味着可阐明其发挥生物活性作用的第一级信号接受分子，将对研究胸腺肽al受体，进而研究胸腺肽al的作用机制和进一步探索其功能产生重要影响。 其次，缺乏对胸腺肽al 引起的信号转导分子改变的相关报道。虽然，对胸腺肽al作用的第二信使研究有了一定的进展，但人类迄今尚未明确何种物质首先接受胸腺肽al的信息，并且有哪些分于参与传递胸腺肽al的信息，以及引起哪些信号分子的改变。 为研究胸腺肽al作用的分子机制，也为胸腺肽al的临床应用提供科学的实验依据，本研究利用酵母双杂交系统研究了胸腺肽al 结合蛋白，并利用基因芯片技术探讨Ta信号转导过程中信号分于的变化；对阐明Ta在免疫系统中的重耍作用及其潜在的抗衰老作用具有重要意义。同时对深入认识并合理干预Ta的信号转导过程，从而达到治疗各种相关疾病的目的具有重要的理论价值。并且，为发现新的抗病毒、抗肿瘤和免疫调节的“靶点”奠定基础。 论文一酵母双杂交系统筛选胸腺肽al结合蛋白的初步研究 一、重组体 PGBKT7－T川的构建 为获取人Tal基因，根据文献报道用化学方法合成人Tal基因及其相应的引物，上游带有EcoR及起始密码子（ATG），下游带有BamH酶切位点和终止密码于（TAG）。经聚合酶链反应（PCR）技术扩增得到双链 Tal基因。以 EcoR、BamHI双酶切 pGBKT7载体（Clontech）和 Tal基因，酶切产物用 T4连接酶作粘端连接，得到pGBKT7－Tal重组体。以PCR和酶切方法筛选鉴定重组体，最后经测序证实Tol全序列已完整无误地插入p GBKT7载体。将其中的一个阳性重组体命名为PGT25。 二、鉴定诱饵蛋白无启动wS下游启动子转录报道基因的活性 2 为鉴定重组体PGBKT7－Toil（PGT25）本身是否会启动UAS下游的启动子表达下游基因的活性，小规模乙酸现酵母转化法将pGT25导入酵母AH109。转化菌铺于SD／TrP／X－a－gal的板上，7大后长出的均为白色克隆，证明重组体本身无启动UAS下游的启动于表达厂游基因的活性。大量扩?更多还原

【Abstract】 Thymus is one of human central immune organ, which plays an important role in inducing T lymphocytes differentiation and maturation. Thymus also plays main role in the development of peripheral immune organ. So that thymus acts on an important role in the development of lymph system and maintain normal immune function. In recent years some people pointed out that thymus also has a role on nerve-endocrine system. From human puberty, thymus began to atrophy physically and the incidence of tumor, infection and immune deficient diseases increase gradually. A lot of investigations indicated that extract of calf thymus can be used to treat tumor, infection diseases and other diseases caused by the fall of thymus.Thymic epithelial cells may secrete many thymic hormones such as thymosin, thytnopoietin, thymic humoral factor, lymphocytic stimulator, serum thymic factor and so on. They are the main component of thymic microenvironment, and play important role on T lymphocyte differentiation and regulation T cell function. Recently thymic hormone was purified into several components. Thymosin alphal (Ta 1) is one of thymic hormone, which is composed by 28 amino acid’and was first isolated by Goldstein et al. T a 1 has immunoregulating function. It can facilitate sensitized lymphocytes to secrete cytokines such as IFN- a ,IFN- Y, IL-2 in vitro. It can also enhance the expression of cytokine receptor with high affinity, regulate the TdT activity of marrow precusor cells and splenocytes, enhance surface antigen Thy 1 and Lytl,2,3 expression on surface of marrow precusor cells, facilitate the growth of NK cell and enhance its activity, by enhancing T helper cell function to facilitate allo/auto lymphocytes reaction, inhibit apoptosis in process of thymocyte maturation. The results of using T a 1 in vivo also indicated that T a l play important role in T celldevelopment and reconstitute its function. It can promote lymphocytes to secrete IL-2 and to express IL-2 receptor, enhance the ability of anti-infection and clean up virus in vivo, enhance immune function, anti-oxidation and inhibit growth of tumor cells. So that T a 1 plays important role on enhancing host immune function .cleaning up virus and against tumor.Clinically, T a 1 is used to assist the treatment of chronic virus infection such as HBV and HCV infection, tumor such as melanoma, lung cancer, leukemia, squamous epithelial cell carcinoma, colon carcinoma , and immune deficiency patients and took good results. T a 1 can also used as vaccine adjuvant. Study of its mechanism will be osculated with its application on infectious diseases, oncology and immunologic diseases.At present, the study of T a ?l mainly focus on its biologic function and application ,which have make a great progress. But the study covering its mechanism is seldom reported.Firstly, T a 1 receptor is still not be cloned now and we also have little know about it. Only some results showed that Ta 1 can bind with some structure on cell surface. Garaci et al reported that T a 1 receptor with high affinity exist on surface of mice lymphocytes by using immunoflurescent assay. Moody et al found that T a 1 can bind with VIP (vasoactive intestinal peptide) receptor in low affinity (IC50=10 P M), but T a 1 is not an antagonist of VIP receptor, which suggested that other signal accept molecule was exist. The above results at least indicated that an molecule, such as T a 1 binding protein, which can binding T?1 is exist. The elucidation of the To 1 binding protein, which is interacted with T a 1 directly when it exerting its biological function, will make great effect on studying the receptor, mechanisms and function of T a 1 in advance.Secondly, the reports related to the change of signal transduction molecule caused by T a 1 is little. Although some progress have been made in study on second messenger after T a 1 stimulation, we still do not know which molecule firstly recognize Ta land which molecules participated in signal transduction and the changes cause by them.In this research更多还原