本文選題：線粒體融合蛋白-2 + 人類精子��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：背景 線粒體有“細(xì)胞動(dòng)力工廠”之稱，是真核細(xì)胞內(nèi)合成三磷酸腺苷（ATP）的主要場所，為細(xì)胞的活動(dòng)提供了能量。除了合成ATP為細(xì)胞提供能量，線粒體還可以儲(chǔ)存鈣離子，通過與內(nèi)質(zhì)網(wǎng)、細(xì)胞外基質(zhì)等結(jié)構(gòu)的協(xié)同作用，維持細(xì)胞中的鈣離子濃度的動(dòng)態(tài)平衡。線粒體也參與細(xì)胞凋亡時(shí)的鈣離子信號(hào)轉(zhuǎn)導(dǎo)，并通過調(diào)節(jié)膜電位控制細(xì)胞凋亡。線粒體還參與調(diào)控細(xì)胞生長和細(xì)胞周期。 線粒體是一種高度動(dòng)態(tài)的細(xì)胞器，通過不斷融合和分裂來滿足各種生理狀態(tài)的需要。線粒體融合蛋白2（Mfn-2）通過調(diào)節(jié)線粒體融合，維持線粒體形態(tài)，調(diào)節(jié)線粒體功能，而且在細(xì)胞能量代謝、細(xì)胞凋亡、信號(hào)傳導(dǎo)、胚胎發(fā)育、心血管疾病、冷凍保護(hù)等方面也起著重要作用。 能量代謝、細(xì)胞凋亡、信號(hào)傳導(dǎo)、氧化應(yīng)激都是影響精子質(zhì)量的至關(guān)因素，Mfn-2可能通過這些方面來調(diào)節(jié)精子的功能或影響精子的質(zhì)量。但是目前Mfn-2在人類精子中的研究還尚未見報(bào)道，本論文主要研究Mfn-2在人精子中的分布和定位，及在精子冷凍保護(hù)中的作用。 目的 本研究主要是明確Mfn-2在人類精子中的定位與分布，初步探討Mfn-2在人類精子冷凍過程中的保護(hù)作用，為進(jìn)一步分析人類精子在冷凍過程中的保護(hù)機(jī)制提供依據(jù)。 方法 征集精液參數(shù)正常志愿者，精液液化后經(jīng)過涂片、固定、透化，用Mfn-2多克隆抗體以間接免疫熒光通過激光共聚焦顯微鏡進(jìn)行觀察Mfn-2在人精子中的定位。 精液參數(shù)正常精液（n=10），液化后進(jìn)行4℃（實(shí)驗(yàn)組）或37℃（對(duì)照組）1h、2h、4h、8h、12h處理，對(duì)兩組各時(shí)間點(diǎn)進(jìn)行精子活力測定，并用Western Blot比較兩組各時(shí)間點(diǎn)的Mfn-2表達(dá)量。 采用半定量Western Blot比較冷凍復(fù)蘇率＞40%和復(fù)蘇率＜40%的兩組精子的Mfn-2表達(dá)量。 結(jié)果 通過激光共聚焦顯微鏡，可見Mfn-2免疫熒光僅出現(xiàn)在人類精子的尾部中段，且頸段與主段之間的長約5～7μm的尾部中段全部均見陽性著色。經(jīng)過4℃4h處理的精子Mfn-2表達(dá)量明顯高于其他處理組，4℃4h處理組的精子活力也優(yōu)于其他組（P0.05）。復(fù)蘇率40%組的精子Mfn-2表達(dá)量明顯高于復(fù)蘇率40%組的精子Mfn-2表達(dá)量（P0.05）。 結(jié)論 在人類精子，Mfn-2位于尾部中段且整個(gè)中段均有分布，，為研究Mfn-2在人類精子中的生物功能奠定了基礎(chǔ)。精子在4℃4h時(shí)相對(duì)表達(dá)量增加，提示Mfn-2可能在此時(shí)冷凍精子活力的恢復(fù)中起一定作用。復(fù)蘇率＜40%組的精子Mfn-2表達(dá)量明顯高于復(fù)蘇率＜40%組的精子Mfn-2表達(dá)量，Mfn-2可能在人類精液冷凍保護(hù)中起到關(guān)鍵作用，其作用機(jī)制尚待進(jìn)一步研究。
[Abstract]:BackgroundMitochondria, known as "cellular power factory", are the main sites for the synthesis of adenosine triphosphate (ATP) in eukaryotic cells, which provide energy for cell activity.In addition to synthesizing ATP to provide energy for cells mitochondria can store calcium ions and maintain the dynamic balance of calcium concentration by synergistic action with endoplasmic reticulum and extracellular matrix.Mitochondria also participate in calcium signal transduction during apoptosis and control apoptosis by regulating membrane potential.Mitochondria are also involved in regulating cell growth and cell cycle.Mitochondria are highly dynamic organelles that meet the needs of various physiological states through continuous fusion and division.Mitochondrial fusion protein 2Mfn-2 regulates mitochondrial fusion, maintains mitochondrial morphology, regulates mitochondrial function, and regulates cell energy metabolism, apoptosis, signal transduction, embryonic development, cardiovascular disease,Freezing protection and other aspects also play an important role.Energy metabolism, apoptosis, signal transduction and oxidative stress are the most important factors affecting sperm quality. Mfn-2 may regulate sperm function or affect sperm quality through these aspects.However, the study of Mfn-2 in human spermatozoa has not been reported. In this paper, the distribution and localization of Mfn-2 in human spermatozoa and the role of Mfn-2 in the cryopreservation of human spermatozoa are studied.PurposeThe purpose of this study was to clarify the localization and distribution of Mfn-2 in human sperm and to explore the protective role of Mfn-2 in the process of human sperm cryopreservation so as to provide a basis for further analysis of the protective mechanism of human spermatozoa during cryopreservation.MethodThe semen parameters of normal volunteers were collected. After liquefaction, the sperm was smeared, fixed and permeated. The localization of Mfn-2 in human spermatozoa was observed by indirect immunofluorescence with Mfn-2 polyclonal antibody and laser confocal microscope.The semen parameters were normal. After liquefaction, they were treated at 4 鈩

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