本文關(guān)鍵詞： 隨機光重建顯微 卵母細胞 動力蛋白 微管蛋白 染色體　出處：《深圳大學》2017年碩士論文　論文類型：學位論文

【摘要】：非整倍性(aneuploidy)是人類染色體異常中最常見的類型,生殖細胞非整倍性是引起人類智力低下、先天畸形和妊娠失敗的主要原因,體細胞非整倍性與腫瘤發(fā)生密切相關(guān)。雖然人們多年來對非整倍性的發(fā)生進行了大量研究,但確切機制至今尚未闡明。非整倍性患兒和腫瘤發(fā)病往往給家庭和社會帶來沉重負擔,因此,深入探討非整倍性形成的分子調(diào)控機制,對降低非整倍性患兒出生風險和腫瘤發(fā)生有重要意義。人類常染色體非整倍性約90%是卵母細胞成熟過程中減數(shù)分裂錯誤所致,卵母細胞成熟與胞質(zhì)密切相關(guān),而胞質(zhì)中動力蛋白(Dynein)因獨特的生理功能在染色體分離和紡錘體形成關(guān)卡中起重要作用。但是,目前,傳統(tǒng)熒光成像方法對Dynein和微管蛋白以及染色體成像研究受到衍射極限的限制,分辨率較低,本研究嘗試利用共聚焦顯微和隨機光學重建顯微(random optical reconstruction microscopy,STORM)成像方法,觀察卵母細胞減數(shù)分裂過程中動力蛋白、微管蛋白以及染色體的形態(tài)結(jié)構(gòu),為高空間分辨水平下研究卵母細胞非整倍性形成的分子調(diào)控機制提供了技術(shù)支持。針對此研究,本論文基于三維隨機光學重建超分辨熒光顯微鏡開展研究工作,具體內(nèi)容如下:1、介紹了本課題的研究背景,國內(nèi)外現(xiàn)狀及分析,重點介紹了遠場超分辨熒光顯微術(shù),包括受激輻射損耗顯微術(shù)、飽和結(jié)構(gòu)光照明顯微術(shù)、光激活定位顯微術(shù)以及隨機光重建顯微術(shù)的工作原理以及優(yōu)缺點。2、闡述了與本課題密切相關(guān)的隨機光學重建超分辨熒光顯微術(shù)的基本理論與技術(shù)問題,并詳細介紹了實驗室現(xiàn)有的自行搭建的三維超分辨熒光顯微成像系統(tǒng)。3、研究了體細胞熒光超分辨成像的標記方法,挑選了微管結(jié)構(gòu)豐富、伸展開來的小鼠成肌細胞(C2C12)、人胚胎成纖維細胞(MRC-5)和小鼠成纖維細胞(NIH-3T3)進行熒光標記,針對超分辨成像過程中出現(xiàn)的熒光分子不連續(xù)等問題,從固定液、抗體濃度以及抗體等方面進行優(yōu)化,并獲得了穩(wěn)定的標記方法和成像條件。4、探索了卵母細胞熒光超分辨成像過程,首先構(gòu)建了卵母細胞體外正常成熟體系和原釩酸鈉(SOV)作用下的異常成熟體系,標記了減數(shù)分裂過程中兩種重要蛋白以及染色體,通過共聚焦顯微觀察了Dynein分布、微管形成的紡錘體以及染色體形態(tài)與結(jié)構(gòu)熒光定位,明確了減數(shù)分裂過程中不同時期共定位情況,隨機光重建超分辨顯微結(jié)果清晰的展現(xiàn)了紡錘體結(jié)構(gòu)信息。5、最后進行了總結(jié)與展望,對論文內(nèi)容進行了簡單的總結(jié)和概括,并且根據(jù)現(xiàn)有實驗結(jié)果,提出了優(yōu)化實驗設(shè)計的建議和展望。本研究的熒光顯微成像結(jié)果表明,在正常成熟體系中,Dynein分布、微管形成的紡錘體以及染色體形態(tài)與結(jié)構(gòu)熒光定位能反映卵母細胞發(fā)育的不同階段,而在SOV作用下,紡錘體結(jié)構(gòu)異常率增加,出現(xiàn)桶狀、細長、團縮、雜亂、梨狀、多極等典型異常紡錘體結(jié)構(gòu),Dynein與紡錘體定位基本一致,染色體結(jié)構(gòu)異常率增加,出現(xiàn)掉隊、散亂分布等異常情況。STORM顯微結(jié)果更清晰的展現(xiàn)了紡錘體結(jié)構(gòu)信息,以及異常紡錘體的紊亂情況,為高空間分辨水平下研究卵母細胞非整倍性形成機制提供了技術(shù)支持。
[Abstract]:Aneuploidy (aneuploidy) is the most common type of human chromosome abnormalities in reproductive cells, aneuploidy is the main reason causing human mental retardation, congenital malformations and pregnancy failure, somatic aneuploidy and tumorigenesis. Although for many years on the occurrence of aneuploidy were large study, but the exact mechanism has not been elucidated. Aneuploidy and cancer patients often brought a heavy burden on the family and society, therefore, in-depth study of the non molecular mechanism of aneuploidy formation, to reduce non significant aneuploidy and tumorigenesis. The risk of the birth of human chromosome aneuploidy about 90% is the error caused by meiosis during oocyte maturation, oocyte maturation and cytoplasm are closely related, and cytoplasmic dynein (Dynein) because of their unique physiological function in chromosome segregation and spindle formation level Play an important role. However, at present, the traditional fluorescence imaging method by the diffraction limit of Dynein and tubulin and chromosome imaging constraints, low resolution, this study attempts to use confocal microscopic and stochastic optical reconstruction (random optical reconstruction microscopy, STORM) imaging method, dynein observation of oocyte meiosis tubulin, and the morphology of chromosomes, molecular regulation mechanism of aneuploidy formation provides technical support for the high spatial resolution level of oocytes. In this research, the 3D stochastic optical reconstruction of super-resolution fluorescence microscopy to carry out research work based on the specific contents are as follows: 1, introduces the research background of this research and analysis of the situation at home and abroad, focuses on the far-field super-resolution fluorescence microscopy, including stimulated radiation loss of microscopic surgery, saturated structure The light was micro surgery, light activated localization microscopy and working principle of stochastic optical reconstruction microscopy as well as the advantages and disadvantages of.2, expounds the basic theory and technology of stochastic optical reconstruction is closely related with the subject of super-resolution fluorescence microscopy, and introduces the existing self built three-dimensional super-resolution fluorescence microscopy imaging system.3 study on somatic cell fluorescence labeling method of super resolution imaging, selection of microtubule structure rich stretch of mouse myoblasts (C2C12), human embryonic fibroblasts (MRC-5) and mouse fibroblast cells (NIH-3T3) labeled with fluorescence for super resolution fluorescence appeared in the imaging process of discontinuous problems from the fixed liquid, antibody concentration and antibody has been optimized, and the marking method and imaging condition of stable.4, explored oocyte fluorescence superresolution imaging process, the first First constructed in vitro oocyte maturation system and normal sodium orthovanadate (SOV) under the action of the abnormal mature system, marked the meiosis and chromosome two important proteins, by confocal microscopic observation of the Dynein distribution, the spindle microtubule formation and chromosome morphology and structure of positioning clear co localization of fluorescence. In the process of meiosis in different periods, random optical superresolution reconstruction results clearly show the structure of the spindle.5, finally summarizes and prospects, the contents of the paper are summarized and summarized, and according to the experimental results, and puts forward suggestions to optimize the experimental design. The results of fluorescence microscopy imaging show that the distribution of Dynein in the normal mature system, the formation of microtubules, spindle and chromosome morphology and structure of fluorescence localization can reflect the development of oocytes of different Stage, and in the presence of SOV spindle structure abnormal rate increased, barrel, slender, round shrink, messy, pear shaped, multi typical abnormal spindle structure, Dynein and spindle positioning consistent chromosomal structural abnormality rate increases, appear left behind, scattered and other anomalies.STORM microscopic results more clearly show the spindle the structure of information, and the disorder of abnormal spindle, high spatial resolution level of oocyte aneuploidy formation mechanism and provide technical support.

【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q253;TH742

【參考文獻】

相關(guān)期刊論文 前10條

1 魏通達;張運海;楊皓e，

本文編號：1461877