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(guó)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(guò)生殖實(shí)現(xiàn)親代與后代個(gè)體之間生命的延續(xù)。在雄性哺乳動(dòng)物的生殖過(guò)程中,精子發(fā)生是個(gè)高度協(xié)調(diào)的過(guò)程。精子發(fā)生被分為三個(gè)階段：第一階段是生殖干細(xì)胞起始的有絲分裂過(guò)程,其通過(guò)有絲分裂轉(zhuǎn)變?yōu)榫?xì)胞最終成為精母細(xì)胞。第二階段是精母細(xì)胞通過(guò)減數(shù)分裂轉(zhuǎn)變?yōu)榫蛹?xì)胞,導(dǎo)致了單倍體圓形精子細(xì)胞的產(chǎn)生。第三個(gè)階段是圓形精子細(xì)胞經(jīng)過(guò)形態(tài)轉(zhuǎn)變而成為精子。目前研究認(rèn)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(kāi)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(dá)數(shù)天,直到精子發(fā)生需要時(shí)才進(jìn)行翻譯。在由精原細(xì)胞轉(zhuǎn)變?yōu)槌墒炀拥穆L(zhǎng)過(guò)程中有很多RNA和蛋白小分子物質(zhì)的參與,是一個(gè)精確和可調(diào)控的過(guò)程。本實(shí)驗(yàn)中所涉及的MIWI蛋白便是最近發(fā)現(xiàn)的一個(gè)重要的作用于該過(guò)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(dāng)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(shí)別部分或者全部的核酸靶標(biāo),借以來(lái)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(gè)亞家族。其中的PIWI亞家族主要有3個(gè)成員,分別是MIWI, MILI和MIWI2,是生殖系細(xì)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(huì)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(xì)胞特異性事件相關(guān),并且特異性分布于生殖細(xì)胞中,其表達(dá)與細(xì)胞的生長(zhǎng)發(fā)育調(diào)節(jié)相關(guān)聯(lián)。小鼠中的MIWI蛋白是這個(gè)亞家族的代表。正是由于生殖細(xì)胞表達(dá)集中的特異性,尤其是哺乳動(dòng)物睪丸表達(dá)的特異性,對(duì)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(dòng)物的生殖細(xì)胞中,PIWI亞家族蛋白通過(guò)與piRNA (piwi-interacting RNA)的結(jié)合形成piRNA復(fù)合物(piRC)來(lái)調(diào)控基因沉默途徑。通過(guò)對(duì)MIWI蛋白的分析研究,有利于我們進(jìn)一步了解生物配子發(fā)生的分子調(diào)控及其機(jī)理,對(duì)理解此蛋白的作用機(jī)制也有重要意義,還可以為不育癥找到新的靶點(diǎn),從而為臨床治療開(kāi)拓思路及提供理論依據(jù)。 實(shí)驗(yàn)材料 各年齡段的SPF級(jí)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(shù)膠 方法 1、提取各年齡段小鼠睪丸組織蛋白,進(jìn)行免疫印跡實(shí)驗(yàn),檢測(cè)是否存在MIWI蛋白的表達(dá)； 2、制作小鼠睪丸組織石蠟切片,進(jìn)行免疫組化實(shí)驗(yàn),明確MIWI蛋白在細(xì)胞水平的表達(dá)定位； 3、進(jìn)一步采用免疫電鏡技術(shù),檢測(cè)MIWI蛋白在亞細(xì)胞水平的表達(dá)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(wú)表達(dá),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(cè)到MIWI蛋白的表達(dá)。 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(xì)胞與精子細(xì)胞時(shí)期表達(dá)。 3、亞細(xì)胞定位顯示MIWI蛋白存在于緊貼核膜處,表明其在核質(zhì)運(yùn)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(dá)。通過(guò)本實(shí)驗(yàn)研究,MIWI蛋白只在成年小鼠的睪丸中表達(dá),在胚胎期以及出生后4周內(nèi)小鼠的各組織中均無(wú)表達(dá)。MIWI蛋白的定位顯示了生殖細(xì)胞中特異結(jié)構(gòu)的存在。Chromatoid body(CB,擬染色體)是一個(gè)生殖細(xì)胞特異性的胞質(zhì)結(jié)構(gòu)。本實(shí)驗(yàn)中,電鏡結(jié)果顯示CB是由細(xì)絲分支形成的不規(guī)則網(wǎng)絡(luò),進(jìn)而結(jié)合而成的緊密團(tuán)塊。在很多物種中,細(xì)胞中如果存在CB結(jié)構(gòu),表明該細(xì)胞為生殖細(xì)胞。目前已知有多種蛋白定位于CB中。在精子細(xì)胞中,CB以平行或是垂直的方式沿核膜快速的移動(dòng),而且有實(shí)驗(yàn)證明CB移動(dòng)過(guò)程中從核中收集基因產(chǎn)物,參與核質(zhì)間RNA的運(yùn)輸。對(duì)于CB這種生殖細(xì)胞特異結(jié)構(gòu)的形成和功能的分子機(jī)制仍然需要進(jìn)一步研究。本實(shí)驗(yàn)采用免疫電鏡技術(shù)在亞細(xì)胞水平定位MIWI蛋白。本實(shí)驗(yàn)中采用了免疫電鏡技術(shù)在精母細(xì)胞中定位MIWI蛋白的表達(dá),可見(jiàn)MIWI蛋白在精母細(xì)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(gè)步驟,本實(shí)驗(yàn)采用的是包埋前染色。免疫電鏡實(shí)驗(yàn)過(guò)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(yáng)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(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李彥清,邵龍江,李森,鄧澤沛,祝誠(chéng);大鼠陰栓的實(shí)驗(yàn)觀察和孕鼠模型鑒定方法的比較[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)雜志;2002年01期

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

，

本文編號(hào)：1461960