核糖核酸酶T2调节精子运动能力的相关研究

发布时间：2018-03-05 06:50

  本文选题：核糖核酸酶T2　切入点：蛋白纯化　出处：《上海交通大学》2014年硕士论文　论文类型：学位论文

【摘要】：核糖核酸酶T2是广泛表达在原核生物、真核生物上的一类底物非特异性核糖核酸内切酶,它参与细胞核糖核酸分解、细胞免疫应答、细胞增殖与运动等多种功能。此外,它还有肿瘤抑制作用,而这种抑制作用并不是基于它的核糖核酸酶活性,有研究发现核糖核酸酶T2的家族成员ACTIBIND可以通过与血管生成素竞争性的结合肿瘤细胞中的肌动蛋白,从而抑制细胞的生长与迁移。哺乳动物的精子中表达有丰富的肌动蛋白,并且主要表达在精子头部、颈部和尾部鞭毛部位,其对精子运动能力的产生具有重要的作用。因此我们设想核糖核酸酶T2在哺乳类精子,如小鼠和人精子上是否有表达?如有表达,情况如何?是否通过与肌动蛋白相互作用从而影响精子的运动能力?如果是,那么体外加入纯化的核糖核酸酶T2蛋白是否会影响精子的运动?鉴于上述的推测,我们通过蛋白质免疫印迹、激光共聚焦显微镜分析以及酶联免疫吸附法检测核糖核酸酶T2在雄性小鼠生殖器官表达、及其在精子上与肌动蛋白的表达关系,结果发现核糖核酸酶T2在雄性生殖系统主要定位在附睾头部和前列腺以及输精管管腔,并与肌动蛋白共表达在雄性小鼠精子的顶体部位、颈部以及尾部鞭毛区域。这说明表达在小鼠精子中的核糖核酸酶T2可能与精子活动力的调节有着密不可分的联系。因此,根据上述研究结果,我们通过从黑曲霉菌的培养液中分离、纯化出具有生物活性的核糖核酸酶T2,并将核糖核酸酶T2蛋白与小鼠附睾尾部的精子共孵育后,对小鼠精子的活动力及前向运动等指标进行检测,结果证实核糖核酸酶T2可以明显抑制小鼠精子活动力(p0.05)及前向运动(p0.05),且抑制效果具有时间及剂量依赖性。最后,我们通过免疫荧光技术对核糖核酸酶T2在正常生育男性精子上的表达定位以及其与actin表达共定位进行了鉴定,发现核糖核酸酶T2表达在人精子的顶体部位、颈部以及尾部鞭毛部位,与actin具有部分共定位,尤其是在与精子运动相关的尾部,并且从黑曲霉菌的培养液中分离、纯化出具有生物活性的核糖核酸酶T2同样可以明显抑制人精子活动力及前向运动力且具有时间和剂量依赖性。因此,明确核糖核酸酶对精子活动力的抑制作用可为我们进一步研究其作用的分子机制及发现临床因精子活动力低下导致的男性不育的病理机制奠定重要的理论基础并提供必要的实验依据。
[Abstract]:Ribonuclease T2 is a class of nonspecific ribonucleic acid endonucleases widely expressed in prokaryotes and eukaryotes. It is involved in cell ribonucleic acid decomposition, cellular immune response, cell proliferation and movement. It also has tumor inhibition, which is not based on its ribonuclease activity. It has been found that ACTIBIND, a member of the ribonuclease T2 family, can bind actin in tumor cells by competing with angiopoietin. This inhibits cell growth and migration. Mammalian sperm express a wealth of actin, mainly in the head, neck and tail of the flagellum. It plays an important role in the production of sperm motility. Therefore, we assume that ribonuclease T2 is expressed in mammalian sperm, such as mouse and human spermatozoa. If so, what is the situation? Does it affect sperm motility by interacting with actin? If so, will the addition of purified ribonuclease T2 in vitro affect sperm motility? In view of the above conjecture, we detected the expression of ribonuclease T2 in male mouse reproductive organs by Western blot, laser confocal microscopy and enzyme-linked immunosorbent assay (Elisa), and the relationship between ribonuclease T2 and actin expression in spermatozoa. The results showed that ribonuclease T2 was mainly located in the head of epididymis, prostate and vas deferens, and co-expressed with actin in the acrosome of male mouse spermatozoa. Neck and tail flagellum regions. This suggests that ribonuclease T2 expressed in mouse spermatozoa may be inextricably linked to the regulation of sperm motility. We purified the bioactive ribonuclease T2 from the culture medium of Aspergillus Niger and incubated the ribonuclease T2 protein with the mouse epididymal tail sperm. The results showed that ribonuclease T2 could significantly inhibit the sperm motility of mice (p0.05) and forward motility (p0.05), and the inhibitory effect was time-and dose-dependent, the results showed that RNase T2 could inhibit the motility of spermatozoa in a time-and dose-dependent manner. We identified the localization of ribonuclease T2 in normal fertile male spermatozoa by immunofluorescence technique and its co-localization with actin expression. We found that ribonuclease T2 was expressed in the acrosome of human spermatozoa. The neck and tail flagellum, partially co-located with actin, especially in the tail associated with sperm movement, and isolated from the culture medium of Aspergillus Niger. The purification of ribonuclease T2 with biological activity can also inhibit the motility and forward motility of human spermatozoa in a time-and dose-dependent manner. The determination of the inhibitory effect of ribonuclease on sperm motility may provide an important theoretical basis for the further study of the molecular mechanism of its action and the discovery of the pathological mechanism of male infertility caused by low sperm motility. To provide the necessary experimental basis.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2
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本文编号：1569185