本文選題：精子冷凍保存 + 蛋白降解��； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：據(jù)統(tǒng)計(jì)世界范圍內(nèi)大約有10%-20%的已婚夫婦遭遇生育困難,由于男性生殖能力異常所致的不育接近50%。并且隨著我國(guó)經(jīng)濟(jì)迅速發(fā)展,有害的環(huán)境因素不斷增加以及受不良生活習(xí)慣的影響,人類(lèi)精液質(zhì)量呈現(xiàn)下降趨勢(shì)。人類(lèi)精子庫(kù)是指以治療不育癥以及預(yù)防遺傳病等為目的,利用超低溫冷凍技術(shù),采集、檢測(cè)、保存和提供精子的機(jī)構(gòu)。人類(lèi)精子庫(kù)的建立為那些患有不可逆的無(wú)精子癥、嚴(yán)重少、弱、畸形精子癥及有遺傳性疾病風(fēng)險(xiǎn)的患者提供了一種可供選擇的方法。然而,人類(lèi)精子在冷凍復(fù)蘇后其活力、活率、獲能、頂體反應(yīng)等功能發(fā)生相應(yīng)的減退,目前認(rèn)為在冷凍復(fù)蘇過(guò)程中,精子細(xì)胞內(nèi)外的滲透壓的改變是造成精子超微結(jié)構(gòu)發(fā)生破壞的主要原因,但是超微結(jié)構(gòu)的改變并不能直接解釋其功能的損傷。由于蛋白質(zhì)是細(xì)胞功能的重要執(zhí)行者,了解精子冷凍復(fù)蘇后蛋白質(zhì)的改變對(duì)理解精子冷凍損傷機(jī)制具有重要的意義。因此,我們前期利用蛋白質(zhì)組學(xué)的方法對(duì)我中心精子庫(kù)9名健康志愿者的精液標(biāo)本進(jìn)行冷凍前后的差異蛋白質(zhì)組學(xué)研究,發(fā)現(xiàn)了在三羧酸循環(huán)通路中的烏頭酸水合酶(AC02)可能在人類(lèi)精子冷凍損傷中發(fā)揮了重要作用。通過(guò)免疫印跡實(shí)驗(yàn)的驗(yàn)證證實(shí)了其在復(fù)蘇精子中的表達(dá)下降,與蛋白質(zhì)組中的結(jié)果一致,免疫熒光實(shí)驗(yàn)明確了其主要定位于精子中段線粒體鞘區(qū)域,通過(guò)文獻(xiàn)復(fù)習(xí)及蛋白定位信息提示AC02可能參與了線粒體內(nèi)三羧酸循環(huán)的過(guò)程,其凍后降解使得三羧酸循環(huán)釋放ATP產(chǎn)量降低可能是導(dǎo)致精子復(fù)蘇后活力下降的原因。我們進(jìn)一步檢測(cè)了冷凍前后精子內(nèi)ATP含量的變化、線粒體膜電勢(shì)的變化及三羧酸循環(huán)通路中AC02下游產(chǎn)物異檸檬酸含量的變化,發(fā)現(xiàn)冷凍復(fù)蘇后ATP、異檸檬酸的含量顯著下降,線粒體膜電勢(shì)顯著降低,差異均有統(tǒng)計(jì)學(xué)意義。綜上所述,通過(guò)對(duì)差異蛋白AC02的一系列研究,我們提出了以下觀點(diǎn),AC02作為線粒體蛋白在精子冷凍復(fù)蘇后發(fā)生了蛋白降解,使得下游產(chǎn)物異檸檬酸的產(chǎn)量下降,三羧酸循環(huán)受抑制導(dǎo)致了ATP的產(chǎn)生下降,復(fù)蘇后的精子活力受損。通過(guò)對(duì)該機(jī)制的闡述,為研究人類(lèi)精子冷凍損傷機(jī)制奠定了良好的理論基礎(chǔ),并為進(jìn)一步全面地應(yīng)用蛋白質(zhì)組學(xué)技術(shù)篩選、分離、鑒定精子蛋白提供了系統(tǒng)、且詳細(xì)的研究方法,同時(shí)為臨床改進(jìn)精子冷凍保護(hù)劑改善精子凍后活力提供了改進(jìn)思路和作用靶點(diǎn)。
[Abstract]:According to statistics, about 10 to 20 percent of married couples worldwide experience fertility difficulties, and infertility due to abnormal male reproductive capacity is close to 50 percent. With the rapid development of Chinese economy, the increase of harmful environmental factors and the influence of bad living habits, the quality of human semen is declining. Human sperm bank is a kind of body which uses cryopreservation technology to collect detect preserve and provide sperm for the purpose of treating infertility and preventing genetic diseases. The establishment of human sperm bank provides an alternative method for those with irreversible azoospermia, severe, weak, deformed spermatozoa and risk of hereditary diseases. However, the motility, viability, capacitation, acrosome reaction and other functions of human spermatozoa decreased after cryopreservation. The change of osmotic pressure inside and outside of sperm cells is the main reason for the destruction of sperm ultrastructure, but the change of ultrastructure can not directly explain the damage of sperm function. Since protein is an important executor of cell function, it is important to understand the changes of protein after cryopreservation and resuscitation in order to understand the mechanism of cryopreservation injury. Therefore, we used proteomics to study the differential proteomics of semen samples from 9 healthy volunteers in our central sperm bank before and after freezing. It was found that aconitate hydratase AC02 in the tricarboxylic acid cycle pathway may play an important role in human sperm cryopreservation injury. The results of immunoblotting showed that the expression of the protein in resuscitation spermatozoa was decreased, which was consistent with that in proteome. Immunofluorescence assay showed that it was mainly located in the mitochondrial sheath region in the middle part of spermatozoa. The review of literatures and protein localization information suggested that AC02 might be involved in the process of tricarboxylic acid cycle in mitochondria. The degradation of tricarboxylic acid cycle resulted in the decrease of ATP production after resuscitation, which may be the reason for the decline of sperm motility after resuscitation. We further detected the changes of ATP content, mitochondrial membrane potential and isocitric acid content in the downstream of AC02 in the tricarboxylic acid cycle pathway before and after cryopreservation. It was found that the content of isocitric acid decreased significantly after cryopreservation. Mitochondrial membrane potential decreased significantly, and the differences were statistically significant. To sum up, through a series of studies on differential protein AC02, we put forward the following point of view: as a mitochondrial protein, the protein degradation occurred after cryopreservation and resuscitation of spermatozoa, which resulted in a decrease in the yield of isocitric acid, the downstream product. Inhibition of the tricarboxylic acid cycle resulted in a decrease in ATP production and impaired sperm motility after resuscitation. Through the elaboration of the mechanism, it lays a good theoretical foundation for the study of the mechanism of human sperm freezing injury, and provides a system for the further application of proteomics technology in screening, isolation and identification of sperm proteins. The detailed research methods also provide an improved idea and target for clinical improvement of spermatozoa cryopreservation protection agent for improving sperm viability after freezing.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R698.2

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本文編號(hào)：1959486