發(fā)布時間：2018-03-05 06:50

  本文選題：核糖核酸酶T2　切入點：蛋白純化　出處：《上海交通大學》2014年碩士論文　論文類型：學位論文

【摘要】：核糖核酸酶T2是廣泛表達在原核生物、真核生物上的一類底物非特異性核糖核酸內(nèi)切酶,它參與細胞核糖核酸分解、細胞免疫應答、細胞增殖與運動等多種功能。此外,它還有腫瘤抑制作用,而這種抑制作用并不是基于它的核糖核酸酶活性,有研究發(fā)現(xiàn)核糖核酸酶T2的家族成員ACTIBIND可以通過與血管生成素競爭性的結(jié)合腫瘤細胞中的肌動蛋白,從而抑制細胞的生長與遷移。哺乳動物的精子中表達有豐富的肌動蛋白,并且主要表達在精子頭部、頸部和尾部鞭毛部位,其對精子運動能力的產(chǎn)生具有重要的作用。因此我們設想核糖核酸酶T2在哺乳類精子,如小鼠和人精子上是否有表達?如有表達,情況如何?是否通過與肌動蛋白相互作用從而影響精子的運動能力?如果是,那么體外加入純化的核糖核酸酶T2蛋白是否會影響精子的運動?鑒于上述的推測,我們通過蛋白質(zhì)免疫印跡、激光共聚焦顯微鏡分析以及酶聯(lián)免疫吸附法檢測核糖核酸酶T2在雄性小鼠生殖器官表達、及其在精子上與肌動蛋白的表達關系,結(jié)果發(fā)現(xiàn)核糖核酸酶T2在雄性生殖系統(tǒng)主要定位在附睪頭部和前列腺以及輸精管管腔,并與肌動蛋白共表達在雄性小鼠精子的頂體部位、頸部以及尾部鞭毛區(qū)域。這說明表達在小鼠精子中的核糖核酸酶T2可能與精子活動力的調(diào)節(jié)有著密不可分的聯(lián)系。因此,根據(jù)上述研究結(jié)果,我們通過從黑曲霉菌的培養(yǎng)液中分離、純化出具有生物活性的核糖核酸酶T2,并將核糖核酸酶T2蛋白與小鼠附睪尾部的精子共孵育后,對小鼠精子的活動力及前向運動等指標進行檢測,結(jié)果證實核糖核酸酶T2可以明顯抑制小鼠精子活動力(p0.05)及前向運動(p0.05),且抑制效果具有時間及劑量依賴性。最后,我們通過免疫熒光技術對核糖核酸酶T2在正常生育男性精子上的表達定位以及其與actin表達共定位進行了鑒定,發(fā)現(xiàn)核糖核酸酶T2表達在人精子的頂體部位、頸部以及尾部鞭毛部位,與actin具有部分共定位,尤其是在與精子運動相關的尾部,并且從黑曲霉菌的培養(yǎng)液中分離、純化出具有生物活性的核糖核酸酶T2同樣可以明顯抑制人精子活動力及前向運動力且具有時間和劑量依賴性。因此,明確核糖核酸酶對精子活動力的抑制作用可為我們進一步研究其作用的分子機制及發(fā)現(xiàn)臨床因精子活動力低下導致的男性不育的病理機制奠定重要的理論基礎并提供必要的實驗依據(jù)。
[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.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698.2
，

本文編號：1569185