發(fā)布時間：2018-03-04 05:19

  本文選題：癌基因　切入點：II型表皮因子受體　出處：《第二軍醫(yī)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：人表皮生長因子受體(Human epidermal growth factor receptor,EGFR)超家族是一大類在生物體生命活動中發(fā)揮重要作用的生長因子受體超家族。該家族有四個成員,第一個成員即廣為人知的EGFR受體。其中,II型表皮生長因子受體(Human epidermal growth factor receptor II,HER2)是近年來腫瘤學(xué)及腫瘤治療學(xué)中的研究熱點。在多種腫瘤中,如乳腺,胃腸道,以及生殖腺體腫瘤中均有HER2受體過表達(dá)、過度激活以及異常突變激活等方式的報道。在乳腺癌中,HER2作為獨立的預(yù)后生物標(biāo)志物已經(jīng)引起廣泛的關(guān)注。在乳腺癌和胃癌的靶向治療領(lǐng)域,針對HER2的抗體藥物及小分子治療手段已經(jīng)成為目前主流的放化療聯(lián)合手段。HER2為分子量大約為180KDa的單次跨膜I型酪氨酸激酶受體,其胞內(nèi)段的酪氨酸激酶活性相較其他三個家族成員為最強(qiáng)。和其他EGFR受體家族成員相比較,HER2具有比較明顯的生理及病理特征:一是目前尚未鑒定出HER2的特異性可溶性配體。二是在該家族內(nèi)的受體進(jìn)行異元二聚化時,HER2為最常見的二聚化對象。三是早期的晶體結(jié)構(gòu)發(fā)現(xiàn)HER2的單獨蛋白質(zhì)結(jié)構(gòu)并沒有在其他家族成員中報道中那樣,具有閘門封閉態(tài)到激活態(tài)的結(jié)構(gòu)基礎(chǔ),而是直接處于二聚化臂暴露的激活態(tài)。但目前對于HER2的激活方式,二聚化的模型仍然存在很多模糊的爭議。爭議的核心在于目前尚無EGFR家族成員異源二聚體的分子模型以及HER2同源二聚體的分子模型。而僅利用目前報道的EGFR二聚化模型來推測其他成員的二聚化機(jī)制具有無法突破的理論限制性。在本課題中,我們首先報道了三種不影響HER2同源二聚體的抗體Fab片段與HER2的共結(jié)晶復(fù)合物。對三種HER2/Fab復(fù)合物的數(shù)據(jù)解析最終的結(jié)果為3.3?,3.5?和3.1?。針對相位問題,本文利用分子置換的方法,成功的解析三種晶體復(fù)合物的三維晶體結(jié)構(gòu)。對三種復(fù)合物的分析確認(rèn)了一種HER2同源相互作用�；趯θN復(fù)合物中的HER2結(jié)構(gòu)提示,通過體外點突變實驗,細(xì)胞膜表面化學(xué)交聯(lián)實驗,免疫共沉淀實驗以及磷酸化檢測試驗,我們揭示并證實了HER2同源二聚體的分子結(jié)構(gòu)模型。并通過對多種治療性抗體對HER2二聚體的影響,從闡述了不同表位治療性抗體對HER2同源二聚體影響的分子機(jī)制。最后,在基于對EGFR同源二聚體及HER2同源二聚體深入的分析比較,課題最終建立了一種新的EGFR家族受體的二聚方式模型,即新的“頭靠背”的分子模型,此模型補(bǔ)充了EGFR家族受體二聚化的機(jī)制,并對EGFR在細(xì)胞膜上的相對位置以及相互作用力方式提出了新的分子模型和理論,并對傳統(tǒng)觀點提出挑戰(zhàn)。本論文的工作對于理解HER2基本的生理過程二聚化具有重要的意義。
[Abstract]:The human epidermal growth factor receptor superfamily is a large class of growth factor receptor superfamilies that play an important role in the life of organisms. There are four members of the superfamily. The first member, known as the EGFR receptor, Human epidermal growth factor receptor II HER2, has been a hot topic in oncology and oncology in recent years. In many kinds of tumors, such as mammary gland and gastrointestinal tract, And the overexpression of HER2 receptor in gonadal tumors. Overexpression and abnormal mutation activation are reported. As an independent biomarker for prognosis, HER2 has attracted wide attention in breast cancer and gastric cancer. Antibody drugs and small molecule therapy for HER2 have become the mainstream combination of radiotherapy and chemotherapy. Her2 is a single transmembrane type I tyrosine kinase receptor with molecular weight of about 180KDa. The activity of tyrosine kinase in the intracellular segment is stronger than that of the other three family members. Compared with other members of the EGFR receptor family, the activity of cytosolic tyrosine kinase has obvious physiological and pathological characteristics. One is that the specificity of HER2 has not been identified. The second is that HER2 is the most common dimerization object when the receptor in the family is dimerized. Third, the early crystal structure found that the individual protein structure of HER2 has not been reported in other family members. It has the structural basis from the gate closed state to the active state, but is directly exposed to the active state of the dimer arm. However, for the current activation mode of HER2, The core of the controversy lies in the absence of a molecular model of heterodimer for members of the EGFR family and a molecular model for the homologous dimer of HER2. Only the currently reported EGFR dimer is used. The model is used to speculate that the dimerization mechanism of other members has theoretical limitations that cannot be broken through. We first report three cocrystalline complexes of Fab fragments with HER2 which do not affect the HER2 homologous dimer. The results of data analysis for the three HER2/Fab complexes are 3.3? ,3.5? And 3.1? In this paper, we use the method of molecular permutation to solve the phase problem. The three dimensional crystal structures of the three crystal complexes were successfully analyzed. An HER2 homologous interaction was confirmed by the analysis of the three kinds of complexes. Based on the HER2 structure cues of the three kinds of complexes, the point mutation experiment in vitro was carried out. The molecular structure model of HER2 homologous dimer was revealed and confirmed by chemical cross-linking of cell membrane, immunoprecipitation and phosphorylation assay. The effects of various therapeutic antibodies on HER2 dimer were also studied. The molecular mechanism of the effect of different epitope therapeutic antibodies on HER2 homologous dimer was discussed. Finally, based on the analysis and comparison of EGFR homologous dimer and HER2 homologous dimer, Finally, a new model of dimerization of EGFR family receptors is established, that is, a new molecular model of "head back", which complements the mechanism of dimerization of EGFR family receptors. A new molecular model and theory about the relative position of EGFR in the cell membrane and the mode of interaction force are proposed, and the traditional view is challenged. The work of this paper is of great significance for understanding the dimerization of the basic physiological process of HER2.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R73-3
，

本文編號：1564295