本文關(guān)鍵詞： 睪丸發(fā)育 精子發(fā)生 MIWI蛋白 Chromatoid Body 免疫電鏡　出處：《中國醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 前言 生物通過生殖實現(xiàn)親代與后代個體之間生命的延續(xù)。在雄性哺乳動物的生殖過程中,精子發(fā)生是個高度協(xié)調(diào)的過程。精子發(fā)生被分為三個階段：第一階段是生殖干細胞起始的有絲分裂過程,其通過有絲分裂轉(zhuǎn)變?yōu)榫毎罱K成為精母細胞。第二階段是精母細胞通過減數(shù)分裂轉(zhuǎn)變?yōu)榫蛹毎?導(dǎo)致了單倍體圓形精子細胞的產(chǎn)生。第三個階段是圓形精子細胞經(jīng)過形態(tài)轉(zhuǎn)變而成為精子。目前研究認為,成熟的精子不再有轉(zhuǎn)錄發(fā)生,因此與精子發(fā)生有關(guān)的基因在精子發(fā)生的早期階段即開始轉(zhuǎn)錄。但這些轉(zhuǎn)錄產(chǎn)物并未立即翻譯形成蛋白產(chǎn)物,而是呈抑制狀態(tài)達數(shù)天,直到精子發(fā)生需要時才進行翻譯。在由精原細胞轉(zhuǎn)變?yōu)槌墒炀拥穆L過程中有很多RNA和蛋白小分子物質(zhì)的參與,是一個精確和可調(diào)控的過程。本實驗中所涉及的MIWI蛋白便是最近發(fā)現(xiàn)的一個重要的作用于該過程的小分子物質(zhì)。 MIWI蛋白是Argonaute蛋白家族的重要成員之一。Argonaute蛋白家族是RNA誘導(dǎo)沉默復(fù)合體(RISC)的核心元件,在RNA干擾(RNAi)中發(fā)揮重要作用。當小RNA與具有同源序列的靶mRNA或靶DNA結(jié)合后,Argonaute家族蛋白識別部分或者全部的核酸靶標,借以來調(diào)節(jié)mRNA的穩(wěn)定性、蛋白的合成和基因組的結(jié)構(gòu)。Argonaute蛋白主要包括Ago和PIWI兩個亞家族。其中的PIWI亞家族主要有3個成員,分別是MIWI, MILI和MIWI2,是生殖系細胞中的特異性蛋白,與精子的發(fā)生有非常密切的關(guān)系,敲除Miwi, Mili或Miwi2基因,都會造成小鼠精子產(chǎn)生明顯缺陷,表現(xiàn)為雄性不育。此亞家族與生殖細胞特異性事件相關(guān),并且特異性分布于生殖細胞中,其表達與細胞的生長發(fā)育調(diào)節(jié)相關(guān)聯(lián)。小鼠中的MIWI蛋白是這個亞家族的代表。正是由于生殖細胞表達集中的特異性,尤其是哺乳動物睪丸表達的特異性,對PIWI亞家族蛋白功能的研究主要于精子發(fā)生。 目前的研究表明,在哺乳動物的生殖細胞中,PIWI亞家族蛋白通過與piRNA (piwi-interacting RNA)的結(jié)合形成piRNA復(fù)合物(piRC)來調(diào)控基因沉默途徑。通過對MIWI蛋白的分析研究,有利于我們進一步了解生物配子發(fā)生的分子調(diào)控及其機理,對理解此蛋白的作用機制也有重要意義,還可以為不育癥找到新的靶點,從而為臨床治療開拓思路及提供理論依據(jù)。 實驗材料 各年齡段的SPF級C57BL／6J小鼠睪丸組織；常規(guī)免疫組化試劑盒、MIWI抗體、羊抗兔二抗、鋨酸、2.5%戊二醛、4%多聚甲醛、梯度酒精(70%,80%,90%,95%,100%)、二甲苯、石蠟、蘇木素、中性樹膠 方法 1、提取各年齡段小鼠睪丸組織蛋白,進行免疫印跡實驗,檢測是否存在MIWI蛋白的表達； 2、制作小鼠睪丸組織石蠟切片,進行免疫組化實驗,明確MIWI蛋白在細胞水平的表達定位； 3、進一步采用免疫電鏡技術(shù),檢測MIWI蛋白在亞細胞水平的表達定位。 結(jié)果 1.、Western結(jié)果顯示了MIWI蛋白在小鼠發(fā)育的胚胎的各組織中無表達,而是存在于成年小鼠睪丸組織中,并在小鼠發(fā)育至6周、8周檢測到MIWI蛋白的表達。 2、免疫組化結(jié)果顯示MIWI蛋白在小鼠睪丸發(fā)育的精母細胞與精子細胞時期表達。 3、亞細胞定位顯示MIWI蛋白存在于緊貼核膜處,表明其在核質(zhì)運輸中可能起到重要作用,是Chromtoid Body的重要組成成分。 討論 MIWI蛋白在成熟小鼠睪丸中特異性表達。通過本實驗研究,MIWI蛋白只在成年小鼠的睪丸中表達,在胚胎期以及出生后4周內(nèi)小鼠的各組織中均無表達。MIWI蛋白的定位顯示了生殖細胞中特異結(jié)構(gòu)的存在。Chromatoid body(CB,擬染色體)是一個生殖細胞特異性的胞質(zhì)結(jié)構(gòu)。本實驗中,電鏡結(jié)果顯示CB是由細絲分支形成的不規(guī)則網(wǎng)絡(luò),進而結(jié)合而成的緊密團塊。在很多物種中,細胞中如果存在CB結(jié)構(gòu),表明該細胞為生殖細胞。目前已知有多種蛋白定位于CB中。在精子細胞中,CB以平行或是垂直的方式沿核膜快速的移動,而且有實驗證明CB移動過程中從核中收集基因產(chǎn)物,參與核質(zhì)間RNA的運輸。對于CB這種生殖細胞特異結(jié)構(gòu)的形成和功能的分子機制仍然需要進一步研究。本實驗采用免疫電鏡技術(shù)在亞細胞水平定位MIWI蛋白。本實驗中采用了免疫電鏡技術(shù)在精母細胞中定位MIWI蛋白的表達,可見MIWI蛋白在精母細胞后期聚合,并存在于核膜周圍。免疫染色是免疫電鏡技術(shù)中極為重要的一個步驟,本實驗采用的是包埋前染色。免疫電鏡實驗過程中,即要考慮到保存較好的超微結(jié)構(gòu),又要能很好地體現(xiàn)免疫陽性反應(yīng)。包埋前染色后的樣本制作有一定的難度,但易于獲得良好的免疫反應(yīng)。
[Abstract]:Preface
The biological mother and offspring between individuals through the continuation of life. In the process of male reproductive reproduction in mammals, spermatogenesis is a highly coordinated process. Spermatogenesis is divided into three stages: initial germline stem cell mitosis in the first stage, through mitosis into spermatogonia eventually become spermatocytes. Second stage spermatocytes by meiosis into sperm cells, resulting in haploid round spermatids. The third stage is the round spermatids transfers into SPERMATOA. The present study suggests that mature sperm has no transcription to occur, and therefore spermatogenesis related genes in the early stage of sperm there is transcription. But these transcripts did not immediately translate into proteins, but were inhibited for several days, until the spermatogenesis only when needed In the translation. By spermatogonial cells into a lot of RNA and small molecular proteins participate in the long process of mature sperm, is an accurate and controllable process involved in this experiment. The MIWI protein is an important role in the recently discovered small molecules in the process.
MIWI protein is an important protein family member of the Argonaute family is the.Argonaute protein RNA induced silencing complex (RISC) of the core components in RNA interference (RNAi) plays an important role. When the small RNA with homologous sequence targeted mRNA or DNA binding, Argonaute family protein identification of some or all of the nucleic acid targets. By the regulation of mRNA stability, structure and synthesis of.Argonaute genome proteins including Ago and PIWI two subfamily. PIWI subfamily include 3 members, namely MIWI, MILI and MIWI2, is a germ cell specific protein, has close relationship with spermatogenesis knockdown of Miwi, Mili, or Miwi2 gene will cause the mouse sperm produced obvious defects, for male sterility. This subfamily and germ cell specific events, and specific distribution in germ cells, the Expression and cell growth regulation associated MIWI protein in mice. Representatives of this subfamily. It is because of germ cell specific expression of focus, especially the specific expression of the mammalian testis, the research of PIWI subfamily function mainly in spermatogenesis.
The present study shows that in mammalian germ cells, PIWI protein with piRNA (piwi-interacting RNA) with the formation of the piRNA complex (piRC) to regulate gene silencing pathway. According to the analysis of MIWI protein, help us to further understand the molecular regulation and mechanism of biological gametogenesis, and also have important significance to understand the mechanism of this protein, can also find new targets for infertility, so as to provide a theoretical basis for clinical treatment and explore new ideas.
Experimental materials
The SPF level C57BL / 6J mice testis tissues in all age groups, routine immunostaining kit, MIWI antibody, Goat anti rabbit two anti osmium, 2.5% glutaraldehyde, 4% paraformaldehyde, gradient alcohol (70%, 80%, 90%, 95%, 100%), xylene, paraffin, hematoxylin and neutral gum.
Method
1, the mice testis tissue protein was extracted from all ages, and the immunoblotting experiment was carried out to detect the expression of MIWI protein.
2, the paraffin section of the mouse testis tissue was made, and the immuno histochemical experiment was carried out to determine the expression of MIWI protein at the cell level.
3, immuno electron microscopy was used to detect the expression of MIWI protein at subcellular level.
Result
1., Western results showed that MIWI protein was not expressed in the tissues of mouse developing embryos, but existed in adult mouse testis tissues, and the expression of MIWI protein was detected at 8 weeks after developing in mice for 6 weeks.
2, the immunohistochemical results showed that MIWI protein was expressed in spermatocyte and spermatocyte during mouse testicular development.
3, subcellular localization shows that MIWI protein exists at the close to the nuclear membrane, indicating that it may play an important role in the nuclear transport and is an important component of the Chromtoid Body.
discuss
The specific expression of MIWI protein in mature mouse testis. Through this experimental study, MIWI protein was expressed only in the testis of adult mice, during embryonic and postnatal 4 weeks mice in different tissues showed no localization.MIWI protein expression showed the presence of.Chromatoid body specific structure in the germ cells (CB, pseudo chromosome) is a cytoplasmic structure of a germ cell specific. In this experiment, the results of SEM showed that the CB is formed by the filament branch of the irregular network, and then combined into clumps. In many species, if there is a CB cell structure, the cells into germ cells. Now known to have a variety of protein located in CB. In sperm cells, CB in parallel or vertical movement along the membrane quickly, and experiments have proved that CB mobile collected from nuclear gene products involved in nucleocytoplasmic transport. RNA for the CB The molecular mechanism of the formation and function of reproductive cell specific structure still needs further study. The subcellular location of MIWI protein by immune electron microscopy. The expression was used in the experiment of immune electron microscopy localization of MIWI protein in spermatocytes in the visible polymerization of MIWI protein in spermatocyte stage, and exists in the nucleus. Immunostaining is an extremely important step in immune electron microscopy, the experiment uses the pre embedding immunoelectron microscopy staining. In the experimental process, namely to consider the preservation of ultrastructure, but also can well reflect the immunoreactivity. The pre embedding staining after the sample production has a certain degree of difficulty, it is easy to obtain a good immune response.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R321

【參考文獻】

相關(guān)期刊論文 前2條

1 李彥清,邵龍江,李森,鄧澤沛,祝誠;大鼠陰栓的實驗觀察和孕鼠模型鑒定方法的比較[J];中國實驗動物學(xué)雜志;2002年01期

2 余磊,潘樹義,劉大庸,蔣立新;一種獲得適齡胎鼠的方法[J];中國臨床解剖學(xué)雜志;1999年03期

，

本文編號：1461960