本文選題：DMAJB13　切入點(diǎn)：精子運(yùn)動(dòng)　出處：《中南大學(xué)》2014年碩士論文

【摘要】：男方因素導(dǎo)致不孕不育所占比例約為50%,其中約19%與精子活力低下(又稱弱精子癥)有直接密切關(guān)系。研究表明弱精癥的病因多樣,特發(fā)性弱精癥是其中數(shù)量較多、病情較重、病因不明的一類。遺傳因素是導(dǎo)致特發(fā)性弱精癥發(fā)生的一個(gè)主要因素。目前臨床對(duì)于這類患者常采用人工輔助生殖技術(shù)治療,這樣就不可避免的導(dǎo)致異�；虬l(fā)生垂直傳遞,到了下一代又會(huì)面臨不育的難題。由此可見,要從根本上治療特發(fā)性弱精癥的問題,就必須了解精子發(fā)生的分子機(jī)制。 前期的實(shí)驗(yàn)研究中我們定位及克隆了人類生精相關(guān)基因DNAJB13及其小鼠同源新基因Dnajbl3。RT-PCR顯示DNAJB13在人睪丸中有高表達(dá),在人成熟精子中亦有表達(dá),原位雜交實(shí)驗(yàn)結(jié)果顯示DNAJB13表達(dá)于各級(jí)生精細(xì)胞。 目的和方法：DNAJB13在人類睪丸組織中有高表達(dá),但目前對(duì)DNAJB13定位、功能及作用的分子機(jī)制均尚不清楚。本研究擬通過Western Blot、免疫熒光共定位、免疫電鏡研究DNAJB13蛋白在人成熟精子中的表達(dá)及定位；通過基因突變篩查研究DNAJB13基因與人類特發(fā)性弱精癥的相關(guān)性；利用免疫共沉淀技術(shù)聯(lián)合液相質(zhì)譜分析研究DNAJB13在成熟精子運(yùn)動(dòng)過程中的相互作用蛋白。 結(jié)果：①Western blot結(jié)果顯示DNAJB13蛋白在成年人成熟精子中有表達(dá)；免疫熒光共定位將DNAJB13蛋白進(jìn)一步定位在人成熟精子尾部；免疫電鏡實(shí)驗(yàn)則將其更精細(xì)的定位在人成熟精子鞭毛中段放射輻、外側(cè)微管、外周致密纖維以及線粒體。②國際上首次在特發(fā)性弱精癥病人中發(fā)現(xiàn)DNAJB13基因第2外顯子存在2個(gè)錯(cuò)義突變(c.857TC、c.912TC),另外檢測(cè)到第2外顯子3個(gè)已知多態(tài)位點(diǎn)(rs199547235,rs10793069,rs10793068)；第3外顯子2個(gè)已知多肽位點(diǎn)(rs65326,rs145909750),第8外顯子3個(gè)已知多態(tài)位點(diǎn)(rs2306820,rs2306819,rs72982975)。③篩查到包括HKI和LDHC在內(nèi)的一批蛋白,在人成熟精子中可能與DNAJB13存在相互作用。 結(jié)論：DNAJB13定位于成熟精子尾部鞭毛中段放射輻、外側(cè)微管、外周致密纖維以及線粒體,提示其功能可能與精子運(yùn)動(dòng)相關(guān)；突變篩查結(jié)果提示到DNAJB13功能可能與人類特發(fā)性弱精癥相關(guān),將其列為導(dǎo)致特發(fā)性弱精癥的候選基因；作用機(jī)制可能是通過在人精子中與HKI和LDHC的相互作用,以糖代謝過程作為其可能的通路之一影響精子運(yùn)動(dòng)中能量的供應(yīng)。
[Abstract]:About 50% of male infertility is caused by male factors, of which about 19% are directly related to low sperm motility (also known as weak spermatozoa).Studies show that the etiology of asthenospermia is diverse, and idiopathic asthenospermia is one of the most serious and unknown etiology.Genetic factors are a major factor leading to idiopathic asthenospermia.At present, this kind of patients are often treated with artificial assisted reproductive technology, which inevitably leads to the vertical transmission of abnormal genes, and will face the problem of infertility in the next generation.Therefore, to fundamentally treat idiopathic azoospermia, we must understand the molecular mechanism of spermatogenesis.In previous experimental studies, we located and cloned the human spermatogenic gene DNAJB13 and its mouse homologous gene Dnajbl3.RT-PCR, which showed that DNAJB13 was highly expressed in human testis and in human mature sperm.The results of in situ hybridization showed that DNAJB13 was expressed in spermatogenic cells of all levels.Objective and methods: DNAJB13 JB13 is highly expressed in human testis, but the molecular mechanism of DNAJB13 localization, function and action is still unclear.The aim of this study was to study the expression and localization of DNAJB13 protein in human mature spermatozoa by immunofluorescence co-localization with Western blot.The relationship between DNAJB13 gene and human idiopathic azoospermia was studied by gene mutation screening.The interaction proteins of DNAJB13 during the motility of mature spermatozoa were studied by immunoprecipitation and liquid mass spectrometry.Results the results of Western blot showed that DNAJB13 protein was expressed in adult spermatozoa, and DNAJB13 protein was further located in the tail of human mature sperm by immunofluorescence.The immuno-electron microscopy showed that the microtubule was located in the middle of the flagellum and the lateral microtubule of the mature human spermatozoa.Two known polypeptide sites, rs65326rs145909750, and three known polymorphic loci in exon 8, rs2306820, rs2306819, rs72982975.3, were screened for a number of proteins, including HKI and LDHC.There may be interaction with DNAJB13 in human mature spermatozoa.Conclusion DNA JB13 is located in the midflagellum, lateral microtubules, peripheral dense fibers and mitochondria of mature spermatozoa, suggesting that its function may be related to sperm motility.Mutation screening results suggest that DNAJB13 function may be associated with human idiopathic azoospermia as a candidate gene leading to idiopathic azoospermia, and its mechanism may be through interaction with HKI and LDHC in human spermatozoa.Glucose metabolism is one of the possible pathways to affect the energy supply in sperm motility.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R698.2

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