【摘要】：胚胎著床期妊娠障礙是導(dǎo)致奶牛繁殖失敗的最主要原因之一。胚胎植入和胚胎的早期發(fā)育是妊娠過(guò)程中最為關(guān)鍵的因素,胎盤作為聯(lián)系母體與胎兒的母胎界面,可通過(guò)在滋養(yǎng)層細(xì)胞和相關(guān)組織表達(dá)某些免疫因子來(lái)保護(hù)胎兒免受母體免疫系統(tǒng)的排斥,即免疫耐受,這種免疫耐受即妊娠的建立與維持依賴于MHC-I的正確表達(dá)。牛MHC-I稱為Bo LA-I,具有高度多態(tài)性,其經(jīng)典(Bo LA-Ia)和非經(jīng)典的I類(Bo LA-Ib)分子均于特定時(shí)期在牛胚胎滋養(yǎng)層和子宮內(nèi)膜上皮細(xì)胞中表達(dá)且與妊娠免疫密切相關(guān)。IFN-τ于胚胎植入期由胚胎滋養(yǎng)層細(xì)胞分泌,為反芻動(dòng)物妊娠識(shí)別因子,可上調(diào)表達(dá)Bo LA-I重鏈,對(duì)胚胎植入有一定的調(diào)節(jié)作用。mi RNA是一類短的內(nèi)源性非編碼單鏈RNA,長(zhǎng)度約為22 nt,能與靶m RNA結(jié)合位點(diǎn)互補(bǔ)配對(duì)來(lái)調(diào)節(jié)基因的表達(dá)。mi RNA在許多細(xì)胞活動(dòng)的調(diào)節(jié)過(guò)程中具有關(guān)鍵作用,也有研究發(fā)現(xiàn)mi RNA參與MHC-I的蛋白表達(dá),但目前關(guān)于其在IFN-τ調(diào)節(jié)子宮內(nèi)膜上皮細(xì)胞中Bo LA-I表達(dá)的過(guò)程中發(fā)揮的作用還不了解。本試驗(yàn)以分離培養(yǎng)的奶牛子宮內(nèi)膜上皮細(xì)胞為基礎(chǔ),采用200ng/m L IFN-τ作用于細(xì)胞;通過(guò)高通量測(cè)序技術(shù)獲得IFN-τ處理不同時(shí)間的細(xì)胞中mi RNA的表達(dá)情況,篩選出差異表達(dá)的mi RNA,同時(shí)預(yù)測(cè)差異表達(dá)mi RNA的靶基因;對(duì)其靶基因進(jìn)行基因功能注釋(GO)和信號(hào)通路注釋(Pathway)分析,以期預(yù)測(cè)與IFN-τ調(diào)節(jié)奶牛子宮內(nèi)膜上皮細(xì)胞差異表達(dá)Bo LA-I相關(guān)的重要mi RNA,為進(jìn)一步研究奶牛妊娠免疫機(jī)制提供試驗(yàn)依據(jù)。本研究主要結(jié)果如下:1.奶牛子宮內(nèi)膜上皮細(xì)胞總RNA的制備及質(zhì)檢本實(shí)驗(yàn)設(shè)定200ng/m L IFN-τ作用于奶牛子宮內(nèi)膜上皮細(xì)胞,時(shí)間分別為0h、6h和12h,并分別設(shè)置各時(shí)間的對(duì)照組,提取各組細(xì)胞總RNA,通過(guò)1%瓊脂糖凝膠電泳、Nanodrop超微量分光光度計(jì)、Qubit 2.0 Fluorometer熒光計(jì)、Agilent 2100檢測(cè)各組樣品總RNA的降解程度、純度、完整性等,結(jié)果表明各組樣品總RNA純度高、完整性好、無(wú)明顯蛋白污染,符合建庫(kù)要求。2.奶牛子宮內(nèi)膜上皮細(xì)胞mi RNA測(cè)序分析以上樣品用于構(gòu)建c DNA文庫(kù),文庫(kù)經(jīng)質(zhì)檢合格后即按照數(shù)據(jù)量需求進(jìn)行RNA-seq測(cè)序,分析mi RNA在各組樣品中的表達(dá)模式。通過(guò)對(duì)測(cè)序數(shù)據(jù)進(jìn)行質(zhì)量評(píng)估發(fā)現(xiàn),各組樣品RNA文庫(kù)中堿基錯(cuò)誤識(shí)別率均為0.01%,GC含量在47.34%~48.53%之間,位于正常范圍且較為均一。對(duì)原始測(cè)序序列過(guò)濾后,獲得的純凈序列占原始序列的比例位于97.38%~98.76%;對(duì)純凈序列進(jìn)行長(zhǎng)度分析發(fā)現(xiàn),在0h,22 nt所占比例最大,約為37.75%,在6和12h,23 nt占有的比例最大,約為42.72%。牛基因組定位分析結(jié)果表明,各樣品RNA文庫(kù)中有約60%的序列能夠比對(duì)到�；蚪M,注釋較為完整�；谛蛄械耐葱院捅Ｊ匦�,本研究共鑒定出574個(gè)已知mi RNA,并發(fā)現(xiàn)109個(gè)新mi RNA。在已知mi RNA中,有338個(gè)在各時(shí)間的實(shí)驗(yàn)組和對(duì)照組中都有表達(dá),但表達(dá)量有差異;新發(fā)現(xiàn)的mi RNA表達(dá)量均較低,但有14個(gè)在所有樣品中有表達(dá)。對(duì)以上683個(gè)mi RNA進(jìn)行表達(dá)分析發(fā)現(xiàn)bta-mi R-21-5p在各組中的表達(dá)量都是最高的,mir-148家族成員也在各組樣品中以中等豐度表達(dá),而novel_1在109個(gè)新發(fā)現(xiàn)的mi RNA中表達(dá)量整體最高。另外,在以上683個(gè)mi RNA中共有464個(gè)差異表達(dá)的mi RNA,其中291個(gè)在各實(shí)驗(yàn)組中均有差異表達(dá),而有173個(gè)在各對(duì)照組差異表達(dá)。在差異表達(dá)的mi RNA中,bta-mi R-181c、bta-mi R-181d和bta-mi R-152與Bo LA-I相關(guān),其中,bta-mi R-181c和bta-mi R-181d在6h和12h時(shí)間組均下調(diào)表達(dá);各組bta-mi R-152的表達(dá)與0小時(shí)比較均表現(xiàn)為上調(diào)(除12h實(shí)驗(yàn)組),但在6h實(shí)驗(yàn)組中的表達(dá)量明顯低于6h對(duì)照組。結(jié)果說(shuō)明IFN-τ下調(diào)了bta-mi R-152的表達(dá)。對(duì)差異表達(dá)的mi RNA進(jìn)行靶基因預(yù)測(cè)和GO及KEGG通路分析發(fā)現(xiàn),這些靶基因主要被富集到代謝過(guò)程和催化活性等,也被顯著富集到免疫應(yīng)答(immune response)和免疫過(guò)程(immune system process)。這些靶基因也有被富集到一些經(jīng)典的代謝通路,以及同種移植物排斥、自然殺傷細(xì)胞介導(dǎo)的細(xì)胞毒性等自體免疫相關(guān)通路。在富集到這些免疫相關(guān)的功能注釋和信號(hào)通路的靶基因中均包含Bo LA-I重鏈基因。以上結(jié)果說(shuō)明IFN-τ可通過(guò)影響mi RNA調(diào)控多種基因,進(jìn)而調(diào)節(jié)奶牛妊娠免疫。相關(guān)基因及其作用機(jī)制尚需要進(jìn)一步的研究去驗(yàn)證和闡明。結(jié)論:IFN-τ可通過(guò)影響bta-mi R-152等mi RNA調(diào)控Bo LA-I重鏈的表達(dá)。
[Abstract]:In this study , the expression of mi - RNA in bovine endometrium epithelial cells was studied by means of high - throughput sequencing . The results were as follows : 1 . The expression of the mRNA of Bo LA - I was detected by high - throughput sequencing . The results showed that the total RNA purity was high , the integrity was good , no significant protein contamination was found in each group . The results showed that there were about 60 % of the samples in each group . The expression of bta - mi R - 152 and bta - mi R - 181d were down - regulated in both 6h and 12h . The expression of bta - mi R - 152 in the experimental group was significantly lower than that in the control group . The results showed that IFN - 蟿 downregulated the expression of bta - mi R - 152 . The results showed that IFN - 蟿 downregulated the expression of bta - mi R - 152 . The results showed that IFN - 蟿 downregulated the expression of bta - mi R - 152 . These target genes were mainly enriched in metabolic process and catalytic activity , and were also significantly enriched to immune response and immune system process . Conclusion : IFN - 蟿 can regulate the expression of Bo LA - I heavy chain by affecting the mi RNA of bta - mi R - 152 . Conclusion : IFN - 蟿 can regulate the expression of Bo LA - I heavy chain by affecting the mi RNA of bta - mi R - 152 .
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.23

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