【作者】 张晓光；

【导师】 苏成芝； 药立波； 俞强； 刘新平；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2000， 博士

【摘要】 Eph受体是已知最大的酪氨酸蛋白激酶受体家族。Eph受体及其膜附着配体ephrins在发育过程中呈现不同的表达模式。功能分析表明，该家族在包括神经网络形成，神经管和轴旁中胚层的成型（patterning），细胞迁移和神经轴突导向、血管发生等方面具有重要功能。Eph受体及其配体也与肿瘤发生有关，因此深入分析这些分子的功能特别是在肿瘤生长中的作用而应用于治疗具有重要的临床意义。EphB2是1994年Kiyokawa等从胃癌组织cDNA文库中筛选出的Eph亚类中又一新基因，在各种肿瘤组织中的表达均高于相应正常组织的几倍。其信号转导途径及作用机理尚需深入研究。 本研究目的是获得与EphB2相互作用并调节其功能的分子。因此，我们首先克隆表达了与EphB2受体结合配体有关的两段cDNA片段（EH and EL）。从胃癌细胞系SGC7901中提取总RNA，RT-PCR扩增出cDNA，测序证实得到与文献报道一致的目的片段。将cDNA分别克隆入载体pRSETB，pET28a，构建重组表达载体pHRA和pLEA，转化大肠杆菌BL21（DE3），经IPTG诱导，表达了N端融合His6的融合蛋白。在非变性及变性条件下，利用Ni-NTA金属螯合亲和层析法对表达的蛋白进行纯化，得到纯度94％以上的融合蛋白。ELISA测定表明，His₆融合蛋白在体外可与其天然配体结合。功能分析表明，Ig结构域（EL）可刺激胃癌细胞内蛋白质发生酪氨酸磷酸化状态的变化。通过脂质体介导将缺失激酶区的ΔEphB2转入胃癌SGC7901细胞，流式细胞仪分析表明胃癌细胞发生G₁期阻断，有13％细胞凋亡，提更多还原

【Abstract】 Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands,the ephrins,show diverse expression patterns during development.Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network,patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfmding,and vascular formation.Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.It is therefore of clinical importance to further. analyze the function of these molecules,particularly their function in tumor cell growth,since manipulation of the activities of these molecules may have therapeutic applications.EphB2 was isolated as a new member of Eph subfamily in 1994 by Kiyokawa from a human gastric cancer cDNA library.Northern blot analysis showed that EphB2 was expressed at higher levels in various tumors of epithelial origin than in corresponding normal tissues. The molecular mechanism by which the EphB2 receptor signaling is propagated, and transmitted remains to be elucidated.The goal of this study is to identify regulatory proteins of EphB2,which can physically interact and regulate the activities of EphB2.To accomplish this goal, we firstly cloned and expressed two extracelluar domains of EphB2 that are related with EphB2 binding to its ligand. Total RNA was isolated from a human更多还原