人类生精相关基因DNAJB13在精子运动中的功能和机制研究

发布时间：2018-03-31 08:42

本文选题：DMAJB13　切入点：精子运动　出处：《中南大学》2014年硕士论文

【摘要】：男方因素导致不孕不育所占比例约为50%,其中约19%与精子活力低下(又称弱精子症)有直接密切关系。研究表明弱精症的病因多样,特发性弱精症是其中数量较多、病情较重、病因不明的一类。遗传因素是导致特发性弱精症发生的一个主要因素。目前临床对于这类患者常采用人工辅助生殖技术治疗,这样就不可避免的导致异常基因发生垂直传递,到了下一代又会面临不育的难题。由此可见,要从根本上治疗特发性弱精症的问题,就必须了解精子发生的分子机制。前期的实验研究中我们定位及克隆了人类生精相关基因DNAJB13及其小鼠同源新基因Dnajbl3。RT-PCR显示DNAJB13在人睾丸中有高表达,在人成熟精子中亦有表达,原位杂交实验结果显示DNAJB13表达于各级生精细胞。目的和方法：DNAJB13在人类睾丸组织中有高表达,但目前对DNAJB13定位、功能及作用的分子机制均尚不清楚。本研究拟通过Western Blot、免疫荧光共定位、免疫电镜研究DNAJB13蛋白在人成熟精子中的表达及定位；通过基因突变筛查研究DNAJB13基因与人类特发性弱精症的相关性；利用免疫共沉淀技术联合液相质谱分析研究DNAJB13在成熟精子运动过程中的相互作用蛋白。结果：①Western blot结果显示DNAJB13蛋白在成年人成熟精子中有表达；免疫荧光共定位将DNAJB13蛋白进一步定位在人成熟精子尾部；免疫电镜实验则将其更精细的定位在人成熟精子鞭毛中段放射辐、外侧微管、外周致密纤维以及线粒体。②国际上首次在特发性弱精症病人中发现DNAJB13基因第2外显子存在2个错义突变(c.857TC、c.912TC),另外检测到第2外显子3个已知多态位点(rs199547235,rs10793069,rs10793068)；第3外显子2个已知多肽位点(rs65326,rs145909750),第8外显子3个已知多态位点(rs2306820,rs2306819,rs72982975)。③筛查到包括HKI和LDHC在内的一批蛋白,在人成熟精子中可能与DNAJB13存在相互作用。结论：DNAJB13定位于成熟精子尾部鞭毛中段放射辐、外侧微管、外周致密纤维以及线粒体,提示其功能可能与精子运动相关；突变筛查结果提示到DNAJB13功能可能与人类特发性弱精症相关,将其列为导致特发性弱精症的候选基因；作用机制可能是通过在人精子中与HKI和LDHC的相互作用,以糖代谢过程作为其可能的通路之一影响精子运动中能量的供应。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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