發(fā)布時間：2018-05-07 07:35

  本文選題：豬 + 體細胞核移植�。� 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：體細胞核移植技術(shù)(SCNT)是指將一個分化的體細胞與去核的卵母細胞融合形成一個重構(gòu)胚胎,并發(fā)育產(chǎn)生與供體細胞遺傳背景一致的克隆后代的技術(shù)。自1996年多利羊誕生以來,研究人員利用體細胞核移植技術(shù)已經(jīng)成功克隆出很多哺乳動物后代,如小鼠、狗、貓、牛、豬等。但盡管該技術(shù)已經(jīng)發(fā)展了近20年,但體細胞克隆效率還是很低(哺乳動物大約1%-5%),而且體細胞克隆后代常常出現(xiàn)一些異常的表型:表現(xiàn)為胚胎著床率低、胎兒流產(chǎn)率高,克隆動物體型過大并伴隨各種器官發(fā)育缺陷的癥狀,也被稱為大型胎兒綜合癥。但表型異常的克隆動物可以正常繁殖且后代表型都正常,說明克隆動物的異常表型是由異常的表觀遺傳修飾造成的而不是由于遺傳物質(zhì)的改變引起的。目前研究普遍認為體細胞核移植效率低的原因是卵母細胞對體細胞核不完全或異常的重編程導(dǎo)致的。體細胞核移植技術(shù)是一項很有應(yīng)用前景的技術(shù),不僅可以幫助我們了解體細胞重編程的機理,同時可以用于人的器官移植等醫(yī)學(xué)研究。SCNT技術(shù)與基因編輯技術(shù)相結(jié)合,可以生產(chǎn)轉(zhuǎn)基因豬,如抗病型豬,優(yōu)良肉品質(zhì)性狀豬,環(huán)境友好型豬等。鑒于體細胞核移植重要的應(yīng)用價值以及其較低的克隆效率,本研究圍繞如何能提高豬的克隆效率這一基本問題展開,在提高其體外克隆效率的同時,在重編程的分子機理進行了進一步的探索,其主要結(jié)果如下所述:1.通過預(yù)實驗我們發(fā)現(xiàn),在豬合子培養(yǎng)基(PZM-3)中添用一定劑量的組蛋白去乙�；�酶抑制劑Oxamflatin,能顯著提高豬體外克隆胚胎囊胚形成率。然后我們優(yōu)化了Oxamflatin的處理條件(不同的處理濃度和持續(xù)時間),發(fā)現(xiàn)用1μM的組蛋白去乙�；�酶抑制劑Oxamflatin處理激活后的重構(gòu)胚胎15h,顯著提高了其體外囊胚發(fā)育率(未處理組vs.處理組;10.3%vs.25.5%;p0.05)。2.用1μM的Oxamflatin處理豬克隆胚胎15h顯著降低了重構(gòu)胚胎原核時期總的去乙酰化酶的活性,提高了克隆胚胎原核期,2細胞和4細胞時期,總的組蛋白H3K9和H4K5的乙�；�水平。我們檢測了四種類型的豬的組蛋白去乙�；�酶(包括HDAC1-11,和Sirt1,2)在MII期卵母細胞和豬胎兒成纖維細胞中的mRNA表達水平,結(jié)果發(fā)現(xiàn),HDAC1、2和3在MII期卵母細胞的相對表達量比胎兒成纖維細胞高,同時也相對比其他類型的組蛋白去乙�；�酶表達要高。Oxamflatin處理顯著降低了重構(gòu)胚胎原核時期HDAC1的表達,部分上解釋了Oxamflatin處理介導(dǎo)的高乙酰化的組蛋白H3K9和H4K5水平。3.我們同時檢測了一個非組蛋白α-tubulin蛋白的乙酰化水平,發(fā)現(xiàn)用1μM的Oxamflatin處理15h顯著提高了豬重構(gòu)胚胎的中間體和紡錘體在胚胎激活后的前兩個有絲分裂細胞周期過程中乙�；�α-tubulin的水平,這可能是通過抑制HDAC6的表達所介導(dǎo)的。4.通過熒光定量PCR我們發(fā)現(xiàn)DNA甲基化轉(zhuǎn)移酶1(DNMT1)在豬MII期卵母細胞中占主導(dǎo),它的表達量相對于其同源基因DNMT2,DNMT3a和3b的表達量要高出50多倍。通過免疫熒光染色我們檢測了豬克隆胚胎在2細胞和4細胞時期總的5-mC和5-hmC水平,結(jié)果表明,豬SCNT胚胎從2細胞到4細胞時期,總的5-mC和5-hmC水平逐漸降低。用1μM的Oxamflatin處理豬重構(gòu)胚胎15h顯著地降低了豬克隆胚胎2細胞時期總的DNA甲基化水平,同時降低了DNMT1在2細胞時期的表達。5.用1μM的Oxamflatin處理豬重構(gòu)胚胎15h顯著提高了多潛能基因POU5F1在豬克隆胚胎囊胚階段的表達,這可能是通過降低POU5F1啟動子區(qū)域DNA甲基化水平引起的。但是,Oxamflatin處理并沒有改變豬衛(wèi)星DNA序列的甲基化水平。6.低劑量的Oxamflatin處理沒有抑制豬胎兒成纖維細胞的生長狀態(tài),Oxamflatin處理同樣導(dǎo)致了豬胎兒成纖維細胞較高的組蛋白H3K9、H4K5和非組蛋白α-tubulin的乙酰化水平,同時Oxamflatin處理在一定程度上降低了豬胎兒成纖維細胞總的DNA甲基化水平。但是用Oxamflatin預(yù)處理的豬胎兒成纖維細胞作為核供體細胞,并沒有提高重構(gòu)胚胎的體外囊胚發(fā)育率。這表明,Oxamflatin不是通過抑制供體細胞組蛋白去乙�；�酶的活性,而是通過抑制卵母細胞組蛋白去乙�；�酶的活性,從而導(dǎo)致了較高的豬體細胞克隆效率。通過本研究我們對組蛋白去乙�；�酶抑制劑Oxamflatin提高克隆效率的分子機制有了初步的認識,這對今后我們更好的理解體細胞核移植重編程機理有一定的參考價值。
[Abstract]:Somatic cell nuclear transplantation (SCNT) is a technique for the fusion of a differentiated somatic cell with a nucleated oocyte to form a reconstructed embryo and develop a technique to clone offspring that is consistent with the genetic background of donor cells. Since the birth of Dolly sheep in 1996, researchers have successfully cloned a lot of lactation by the technique of body cell nucleus transplantation. The offspring of animals, such as mice, dogs, cats, cattle, pigs and so on. But although the technology has been developed for nearly 20 years, the efficiency of somatic cell cloning is still very low (mammalian about 1%-5%), and the cloned progeny of somatic cells often appear some abnormal phenotypes: low embryo implantation rate, high fetal abortion rate, too large cloned animals with various organs and various organs. The symptoms of developmental defects are also known as large fetal syndrome. However, the abnormal phenotype of cloned animals can reproduce normally and the later representative type is normal, indicating that the abnormal phenotype of the cloned animal is caused by abnormal epigenetic modification, not due to the change of genetic material. The low reason is that the oocyte is reprogrammed with incomplete or abnormal somatic cell nuclei. Somatic cell nuclear transplantation is a promising technology. It can not only help us understand the mechanism of reprogramming of somatic cells, but also can be used in the combination of.SCNT technology and gene editing technology in human organ transplantation. In order to produce transgenic pigs, such as disease resistant pigs, pigs with good meat quality, environment friendly pigs and so on. In view of the important application value of somatic cell nuclear transplantation and its low cloning efficiency, this study focuses on how to improve the cloning efficiency of pigs, while improving the efficiency of cloning in vitro, the reprogrammed molecular machine The main results are as follows: 1. through pre experiment, we found that adding a certain dose of histone deacetylase inhibitor Oxamflatin to the porcine zygote medium (PZM-3) could significantly increase the rate of the blastocyst formation of the porcine in vitro cloned embryo. Then we optimized the treatment conditions of Oxamflatin (different treatments). Concentration and duration), it was found that the reconfigurable embryo 15h was treated with 1 M histone deacetylase inhibitor Oxamflatin, which significantly increased the development rate of the blastocyst in vitro (the untreated group vs. treatment group; 10.3%vs.25.5%; P0.05).2. with 1 micron M in the Oxamflatin treatment Zhu Kelong embryo 15h significantly reduced the total deb of the reconstructive embryo during the prokaryotic period. The activity of acylase increased the acetylation level of the total histone H3K9 and H4K5 in the prokaryotic, 2 and 4 cell stages of the cloned embryo. We detected the mRNA expression level of the histone deacetylase (including HDAC1-11, and Sirt1,2) in four types of pig's histone (including HDAC1-11, and Sirt1,2) in MII oocytes and pig fetal fibroblasts. The results were found, HDAC1,2 The relative expression of and 3 in MII oocytes was higher than that of fetal fibroblasts, and the higher.Oxamflatin treatment compared with other types of histone deacetylase expression significantly reduced the expression of HDAC1 in the prokaryotic stage of restructured embryos, partly explaining the high acetylation of histone H3K9 and H4K5 level.3. mediated by Oxamflatin treatment. We also detected the acetylation level of a non histone alpha -tubulin protein. It was found that the use of 1 M Oxamflatin to treat 15h significantly improved the intermediate of porcine reconstituted embryos and the level of acetylated alpha -tubulin during the cycle of the first two mitotic cells after the activation of the embryo, which may be mediated by the inhibition of the expression of HDAC6. .4. guided by fluorescence quantitative PCR we found that DNA methyltransferase 1 (DNMT1) was dominant in porcine MII oocytes, and its expression was more than 50 times higher than that of its homologous gene DNMT2, DNMT3a and 3b. We detected the total 5-mC and 5-hmC levels of porcine cloned embryo fetal in 2 and 4 cells by immunofluorescence staining. The results showed that the total 5-mC and 5-hmC levels of the pig SCNT embryos decreased gradually from 2 to 4 cells. The total DNA methylation level in the 2 cell period of pig cloned embryos was significantly reduced by the Oxamflatin treatment with 1 u M Oxamflatin, and the Oxamflatin treatment of DNMT1 in the 2 cell period of.5. with 1 micron M Oxamflatin treatment of the porcine reconstructed embryo 15h was reduced. The expression of the multipotential gene POU5F1 in the blastocyst stage of the porcine cloned embryo could be significantly increased by reducing the level of DNA methylation in the POU5F1 promoter region. However, Oxamflatin treatment did not alter the methylation level of the DNA sequence of the pig satellite.6. and the low dose of Oxamflatin did not inhibit the growth of porcine fetal fibroblasts. State, Oxamflatin treatment also leads to higher histone H3K9, H4K5 and the level of acetylation of non histone alpha -tubulin in pig fetal fibroblasts, while Oxamflatin treatment reduces the total DNA methylation level in pig fetal fibroblasts to some extent. However, pig fetal fibroblasts treated with Oxamflatin are used as nuclear donor cells. Somatic cells do not increase the development rate of blastocysts in vitro of reconstructed embryos. This indicates that Oxamflatin is not by inhibiting the activity of the donor cell histone deacetylase, but by inhibiting the activity of the oocyte histone deacetylase, which leads to the higher cloning efficiency of the pig somatic cells. The molecular mechanism of the acylase inhibitor Oxamflatin to improve the cloning efficiency has a preliminary understanding, which is of certain reference value for our better understanding of the mechanism of reprogramming of somatic cell nuclear transfer in the future.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S828

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