人精浆外泌体及以纳米金星输送在弱精子症中的作用机制

发布时间：2018-05-09 06:47

本文选题：弱精子症 + 精浆外泌体　；参考：《中国科学技术大学》2017年博士论文

【摘要】：全世界范围内15%～20%的育龄夫妇存在着不孕不育的问题,其中弱精子症导致的不育率达15%。弱精子症主要表现为精子活力低下。弱精子症很少甚至没有器质性病理改变,这为诊断和治疗带来困难,且严重的弱精子症进行ICSI的成功率也很低,因此研究弱精子症的致病机制具有重要意义。人精浆中存在大量外泌体,其能够调控精子成熟运动,但是精浆外泌体调节精子运动的机制目前尚不明确,需要进一步阐明。本课题通过分离正常人与弱精子症患者精浆外泌体,应用iTRAQ蛋白质组学技术鉴定两者精浆外泌体差异蛋白,共鉴定出3699个精浆外泌体蛋白,其中,弱精子症患者精浆外泌体相比正常人,11个蛋白含量显著上调(1.2),80个蛋白含量显著下调(0.833)。通过免疫印迹实验证实了 TRPV6、ACE、PRSS8和PPAP2A等蛋白在弱精子症患者精浆外泌体中显著下调,验证了本次蛋白组学的检测结果。通过生物信息学分析发现,这些差异蛋白主要集中在跨膜运输和新陈代谢相关的生物学通路。我们在小鼠模型中证实TRPV6在附睾头部开始以附睾小体的形式从附睾转移到精子表面,且通过免疫荧光发现TRPV6定位在人正常精子顶体和颈部位置,TRPV6在弱精子症患者精浆外泌体中较正常人显著下调,提示精浆外泌体的分泌紊乱与弱精子症的发生密切相关。纳米金在生物医药领域受到越来越多的关注和应用,很多纳米金已经被广泛应用于基因运输、药物靶向运输、生物成像、细胞标记和高热治疗。然而生物体内存在多个生物屏障,比如:血脑屏障、血胎屏障和血睾屏障,克服生物屏障是一个困难而不可避免的问题,这限制了大量材料用于体内药物输送,所以寻找一种有效穿透生物屏障的输送载体也势在必行。这里我们主要关注血睾屏障,我们合成五角纳米金星,发现纳米金星不仅在体外可以迅速的进入生殖细胞系GC-2细胞,而且在体内能够克服血睾屏障进入到生殖细胞中,我们利用纳米金星处理GC2细胞和尾静脉注射雄性ICR小鼠,发现纳米金星并不影响GC2的增殖和凋亡,对睾丸曲细精管的结构也无明显影响,说明纳米金星具有很好的生物相容性。此外,我们进一步研究发现在雄鼠青春时期或者43度水浴10min处理后,纳米金星穿透血睾屏障进入到生殖细胞变得更加容易,说明纳米金星可作为一个新的药物载体用于雄性生殖系统疾病(如单基因突变导致的弱精子症)的治疗,这为男性不育药物输送提供一个新的研究方向。
[Abstract]:Worldwide, 15% of couples of childbearing age have infertility problems, of which 15% of infertile couples are caused by asthenospermia. Asthenospermia is mainly characterized by low sperm motility. There are few or no organic pathological changes in asthenospermia, which brings difficulties in diagnosis and treatment, and the success rate of ICSI in severe asthenospermia is very low. Therefore, it is of great significance to study the pathogenesis of asthenospermia. There are a large number of exocrine bodies in human seminal plasma which can regulate the mature motility of spermatozoa. However the mechanism of seminal plasma exosome regulating sperm motility is still unclear and needs further elucidation. In this study, the seminal plasma exocrine bodies were isolated from normal and asthenospermia patients, and the differential proteins of seminal plasma exocrine were identified by iTRAQ proteomics. A total of 3 699 seminal plasma exocrine proteins were identified. In asthenospermia patients, 11 proteins were significantly up-regulated by 1.2U, and 80 proteins were significantly down-regulated by 0.833% compared with normal controls. Western blotting showed that TRPV6 ACESS-8 and PPAP2A were down-regulated in seminal plasma exocrine of patients with asthenospermia, and the results of proteomics were verified. Bioinformatics analysis showed that these differentially expressed proteins were mainly involved in transmembrane transport and metabolism related biological pathways. In a mouse model, we confirmed that TRPV6 began to move from epididymis to sperm surface in the form of epididymal bodies at the head of the epididymis. The localization of TRPV6 in acrosome and neck of human normal spermatozoa was significantly down-regulated in the seminal plasma exocrine of asthenospermia, suggesting that the secretion disorder of seminal plasma exocrine was closely related to the occurrence of asthenospermia. Nanocrystalline gold has received more and more attention and application in biomedical field. Many nanocrystalline gold have been widely used in gene transport, drug targeting transport, biomedical imaging, cell labeling and hyperthermia therapy. However, there are many biological barriers in organisms, such as blood-brain barrier, blood-fetal barrier and blood-testis barrier. Overcoming biological barrier is a difficult and inevitable problem, which limits the use of a large amount of materials for drug delivery in vivo. Therefore, it is imperative to find a carrier that can effectively penetrate the biological barrier. Here we focus on the blood-testis barrier, we synthesize pentagonal nanometer Venus, and we find that nano-Venus not only can quickly enter germ cell line GC-2 cells in vitro, but also can overcome the blood-testis barrier to enter germ cells in vivo. We treated GC2 cells and male ICR mice with Venus nanoparticles. We found that Venus nanoparticles did not affect the proliferation and apoptosis of GC2 and had no obvious effect on the structure of testicular seminiferous tubules, which indicated that Venus nanoparticles had good biocompatibility. In addition, we further found that after 10min treatment in male rats during adolescence or 43 degrees water bath, it became easier for nanometer Venus to penetrate the blood-testis barrier into germ cells. It is suggested that Venus nanoparticles can be used as a new drug carrier for the treatment of male reproductive system diseases, such as weak spermatozoa caused by single gene mutation, which provides a new research direction for drug delivery of male infertility.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R698.2

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