【摘要】：研究目的: 哺乳動(dòng)物精子發(fā)生過(guò)程(Spermatogenesis process)是一被精確調(diào)控的復(fù)雜的細(xì)胞分化過(guò)程。原始生殖細(xì)胞經(jīng)有絲分裂,兩次減數(shù)分裂和變態(tài)過(guò)程最終發(fā)育成成熟精子。該過(guò)程中最顯著的一個(gè)特點(diǎn)就是染色體重建(Chromatin remolding),其機(jī)制主要從兩個(gè)方面來(lái)解讀,第一,精子發(fā)生過(guò)程中有大量不同種類的組蛋白合成;第二,這些組蛋白的翻譯后修飾可能呈現(xiàn)時(shí)間及空間特異性�！吧�(xì)胞特異的組蛋白編碼”這一概念也隨之提出,而目前還沒有這方面系統(tǒng)的研究。 為了更好地理解精子發(fā)生過(guò)程中染色體重建。本研究應(yīng)用質(zhì)譜技術(shù)分析了參與精子發(fā)生的組蛋白H2A.H2B的亞型種類,組蛋白H2A.H2B翻譯后修飾的動(dòng)態(tài)變化,初步探索了相應(yīng)翻譯后修飾的功能。 研究方法: 首先應(yīng)用密度梯度沉降的方法分離精原細(xì)胞,精母細(xì)胞和圓形精子細(xì)胞;分別純化各細(xì)胞的組蛋白,應(yīng)用HPLC進(jìn)一步分離純化組蛋白各組分(H1,H2A,H2B,H3,H4)。純化后的組蛋白H2A和H2B經(jīng)蛋白酶酶切,應(yīng)用LC-MS;LC-MS/MS分析H2A.H2B的亞型種類及其翻譯后修飾。 聯(lián)合應(yīng)用Pull-down和免疫共沉淀的方法來(lái)鑒定識(shí)別組蛋白修飾基團(tuán)的伴侶分子。 研究結(jié)果: 精子發(fā)生過(guò)程中,一共鑒定了7種H2A亞型,睪丸組織特異的組蛋白TH2A首先出現(xiàn)在精原細(xì)胞中。發(fā)現(xiàn)了一些新的位于H2A1 C末端的翻譯后修飾,包括K99和K100的甲基化,K119的二甲基化和K96的乙�；�。 同時(shí)鑒定了6種H2B亞型。在此期間,睪丸組織特異的組蛋白TH2B的翻譯后修飾呈動(dòng)態(tài)變化:精原細(xì)胞中乙�；疶H2B的相對(duì)豐度最高,約有28.9%的TH2B為乙�；癄顟B(tài);精母細(xì)胞乙�；腡H2B降到了最低(8.3%),到了圓形精子細(xì)胞階段乙�；腡H2B約為11.2%。 同時(shí)也鑒定了幾種TH2B的翻譯后修飾,TH2B的N端呈高乙酰化修飾狀態(tài);在TH2B的C端,我們發(fā)現(xiàn)了T116的磷酸化和K117的甲基化(乙�；�),形成了兩個(gè)新的“磷酸化開關(guān)”(phospho switch)。 蛋白-蛋白相互作用的結(jié)果提示,蛋白質(zhì)TRRAP,CENP-E和PTP-BL可能是識(shí)別H2B PhT_(116)/AcK_(117)的蛋白因子,提示該組合修飾可能參與了減數(shù)分裂過(guò)程中的染色體分離。 另外,應(yīng)用質(zhì)譜技術(shù)描述了參與精子發(fā)生的組蛋白H1的多樣性。 研究結(jié)論: 本研主要應(yīng)用質(zhì)譜技術(shù)描述了精子發(fā)生過(guò)程中組蛋白H2A和H2B的亞型種類及其翻譯后修飾;鑒定了識(shí)別其特異修飾基團(tuán)的蛋白質(zhì)。該研究盡管還不盡完善,為進(jìn)一步理解精子發(fā)生過(guò)程中染色體的重建奠定了基礎(chǔ)。
[Abstract]:Objective: mammalian spermatogenesis (Spermatogenesis process) is a complex cell differentiation process regulated precisely. Through mitosis, meiosis and metamorphosis, the primordial germ cells develop into mature spermatozoa. One of the most significant characteristics of this process is that the mechanism of chromosome reconstruction (Chromatin remolding), is interpreted from two aspects: first, there are a large number of different types of histone synthesis during spermatogenesis; second, Posttranslational modifications of these histones may present temporal and spatial specificity. The concept of specific histone coding for spermatogenic cells has been proposed, but no systematic research has been done. In order to better understand the process of spermatogenesis chromosome reconstruction. In this study, the subtypes of histone H2A.H2B involved in spermatogenesis and the dynamic changes of post-translational modification of histone H2A.H2B were analyzed by mass spectrometry, and the function of the corresponding post-translational modification was preliminarily explored. Methods: firstly, spermatogonia, spermatocytes and round spermatocytes were isolated by density gradient sedimentation, histone of each cell were purified and further purified by HPLC. The purified histone H2A and H2B were digested by protease, and the subtypes of H2A.H2B and its posttranslational modification were analyzed by LC-MS / MS / MS. Pull-down and immunoprecipitation were used to identify the chaperones that recognize histone modified groups. Results: during spermatogenesis, seven H2A subtypes were identified, and testis histone TH2A was first found in spermatogonia. Some new post-translational modifications at the C-terminal of H2A1 were found, including the dimethylation of K99 and K100 methylation of K119 and the acetylation of K96. At the same time, 6 subtypes of H2B were identified. During this period, the posttranslational modification of testicular histone TH2B showed dynamic changes: the relative abundance of acetylated TH2B was the highest in spermatogonia, and about 28.9% of TH2B was acetylated; The TH2B of acetylation of spermatocytes was the lowest (8.3%), and the TH2B of acetylation of spermatozoa was about 11.2% at the stage of round spermatozoa. At the same time, several kinds of TH2B were identified as highly acetylated N-terminal modification of TH2B. At the C end of TH2B, we found phosphorylation of T116 and methylation (acetylation) of K117, which formed two new "phosphorylation switches" (phospho switch). The results of protein-protein interaction suggest that CENP-E and PTP-BL may be the protein factors that recognize H2B PhT116 / AcK117, suggesting that the combined modification may be involved in chromosome segregation during meiosis. In addition, the diversity of histone H 1 involved in spermatogenesis was described by mass spectrometry. Conclusion: the subtypes and posttranslational modifications of histone H2A and H2B during spermatogenesis were described by mass spectrometry, and the proteins that recognized their specific modification groups were identified. This study, though imperfect, lays a foundation for further understanding of chromosome reconstruction during spermatogenesis.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R321

【共引文獻(xiàn)】

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本文編號(hào)：2191631