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Mfn-2在人类精子中的定位及在精子冷冻中的作用初探

发布时间:2018-04-17 14:31

  本文选题:线粒体融合蛋白-2 + 人类精子 ; 参考:《华中科技大学》2012年硕士论文


【摘要】:背景 线粒体有“细胞动力工厂”之称,是真核细胞内合成三磷酸腺苷(ATP)的主要场所,为细胞的活动提供了能量。除了合成ATP为细胞提供能量,线粒体还可以储存钙离子,通过与内质网、细胞外基质等结构的协同作用,维持细胞中的钙离子浓度的动态平衡。线粒体也参与细胞凋亡时的钙离子信号转导,并通过调节膜电位控制细胞凋亡。线粒体还参与调控细胞生长和细胞周期。 线粒体是一种高度动态的细胞器,通过不断融合和分裂来满足各种生理状态的需要。线粒体融合蛋白2(Mfn-2)通过调节线粒体融合,维持线粒体形态,调节线粒体功能,而且在细胞能量代谢、细胞凋亡、信号传导、胚胎发育、心血管疾病、冷冻保护等方面也起着重要作用。 能量代谢、细胞凋亡、信号传导、氧化应激都是影响精子质量的至关因素,Mfn-2可能通过这些方面来调节精子的功能或影响精子的质量。但是目前Mfn-2在人类精子中的研究还尚未见报道,本论文主要研究Mfn-2在人精子中的分布和定位,及在精子冷冻保护中的作用。 目的 本研究主要是明确Mfn-2在人类精子中的定位与分布,初步探讨Mfn-2在人类精子冷冻过程中的保护作用,为进一步分析人类精子在冷冻过程中的保护机制提供依据。 方法 征集精液参数正常志愿者,精液液化后经过涂片、固定、透化,用Mfn-2多克隆抗体以间接免疫荧光通过激光共聚焦显微镜进行观察Mfn-2在人精子中的定位。 精液参数正常精液(n=10),液化后进行4℃(实验组)或37℃(对照组)1h、2h、4h、8h、12h处理,对两组各时间点进行精子活力测定,并用Western Blot比较两组各时间点的Mfn-2表达量。 采用半定量Western Blot比较冷冻复苏率>40%和复苏率<40%的两组精子的Mfn-2表达量。 结果 通过激光共聚焦显微镜,可见Mfn-2免疫荧光仅出现在人类精子的尾部中段,且颈段与主段之间的长约5~7μm的尾部中段全部均见阳性着色。经过4℃4h处理的精子Mfn-2表达量明显高于其他处理组,4℃4h处理组的精子活力也优于其他组(P0.05)。复苏率40%组的精子Mfn-2表达量明显高于复苏率40%组的精子Mfn-2表达量(P0.05)。 结论 在人类精子,Mfn-2位于尾部中段且整个中段均有分布,,为研究Mfn-2在人类精子中的生物功能奠定了基础。精子在4℃4h时相对表达量增加,提示Mfn-2可能在此时冷冻精子活力的恢复中起一定作用。复苏率<40%组的精子Mfn-2表达量明显高于复苏率<40%组的精子Mfn-2表达量,Mfn-2可能在人类精液冷冻保护中起到关键作用,其作用机制尚待进一步研究。
[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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