组蛋白去乙酰化酶抑制剂Oxamflatin对猪体细胞克隆胚胎体外发育率的影响

发布时间：2018-05-07 07:35

本文选题：猪 + 体细胞核移植　；参考：《华中农业大学》2016年博士论文

【摘要】：体细胞核移植技术(SCNT)是指将一个分化的体细胞与去核的卵母细胞融合形成一个重构胚胎,并发育产生与供体细胞遗传背景一致的克隆后代的技术。自1996年多利羊诞生以来,研究人员利用体细胞核移植技术已经成功克隆出很多哺乳动物后代,如小鼠、狗、猫、牛、猪等。但尽管该技术已经发展了近20年,但体细胞克隆效率还是很低(哺乳动物大约1%-5%),而且体细胞克隆后代常常出现一些异常的表型:表现为胚胎着床率低、胎儿流产率高,克隆动物体型过大并伴随各种器官发育缺陷的症状,也被称为大型胎儿综合症。但表型异常的克隆动物可以正常繁殖且后代表型都正常,说明克隆动物的异常表型是由异常的表观遗传修饰造成的而不是由于遗传物质的改变引起的。目前研究普遍认为体细胞核移植效率低的原因是卵母细胞对体细胞核不完全或异常的重编程导致的。体细胞核移植技术是一项很有应用前景的技术,不仅可以帮助我们了解体细胞重编程的机理,同时可以用于人的器官移植等医学研究。SCNT技术与基因编辑技术相结合,可以生产转基因猪,如抗病型猪,优良肉品质性状猪,环境友好型猪等。鉴于体细胞核移植重要的应用价值以及其较低的克隆效率,本研究围绕如何能提高猪的克隆效率这一基本问题展开,在提高其体外克隆效率的同时,在重编程的分子机理进行了进一步的探索,其主要结果如下所述:1.通过预实验我们发现,在猪合子培养基(PZM-3)中添用一定剂量的组蛋白去乙酰化酶抑制剂Oxamflatin,能显著提高猪体外克隆胚胎囊胚形成率。然后我们优化了Oxamflatin的处理条件(不同的处理浓度和持续时间),发现用1μM的组蛋白去乙酰化酶抑制剂Oxamflatin处理激活后的重构胚胎15h,显著提高了其体外囊胚发育率(未处理组vs.处理组;10.3%vs.25.5%;p0.05)。2.用1μM的Oxamflatin处理猪克隆胚胎15h显著降低了重构胚胎原核时期总的去乙酰化酶的活性,提高了克隆胚胎原核期,2细胞和4细胞时期,总的组蛋白H3K9和H4K5的乙酰化水平。我们检测了四种类型的猪的组蛋白去乙酰化酶(包括HDAC1-11,和Sirt1,2)在MII期卵母细胞和猪胎儿成纤维细胞中的mRNA表达水平,结果发现,HDAC1、2和3在MII期卵母细胞的相对表达量比胎儿成纤维细胞高,同时也相对比其他类型的组蛋白去乙酰化酶表达要高。Oxamflatin处理显著降低了重构胚胎原核时期HDAC1的表达,部分上解释了Oxamflatin处理介导的高乙酰化的组蛋白H3K9和H4K5水平。3.我们同时检测了一个非组蛋白α-tubulin蛋白的乙酰化水平,发现用1μM的Oxamflatin处理15h显著提高了猪重构胚胎的中间体和纺锤体在胚胎激活后的前两个有丝分裂细胞周期过程中乙酰化α-tubulin的水平,这可能是通过抑制HDAC6的表达所介导的。4.通过荧光定量PCR我们发现DNA甲基化转移酶1(DNMT1)在猪MII期卵母细胞中占主导,它的表达量相对于其同源基因DNMT2,DNMT3a和3b的表达量要高出50多倍。通过免疫荧光染色我们检测了猪克隆胚胎在2细胞和4细胞时期总的5-mC和5-hmC水平,结果表明,猪SCNT胚胎从2细胞到4细胞时期,总的5-mC和5-hmC水平逐渐降低。用1μM的Oxamflatin处理猪重构胚胎15h显著地降低了猪克隆胚胎2细胞时期总的DNA甲基化水平,同时降低了DNMT1在2细胞时期的表达。5.用1μM的Oxamflatin处理猪重构胚胎15h显著提高了多潜能基因POU5F1在猪克隆胚胎囊胚阶段的表达,这可能是通过降低POU5F1启动子区域DNA甲基化水平引起的。但是,Oxamflatin处理并没有改变猪卫星DNA序列的甲基化水平。6.低剂量的Oxamflatin处理没有抑制猪胎儿成纤维细胞的生长状态,Oxamflatin处理同样导致了猪胎儿成纤维细胞较高的组蛋白H3K9、H4K5和非组蛋白α-tubulin的乙酰化水平,同时Oxamflatin处理在一定程度上降低了猪胎儿成纤维细胞总的DNA甲基化水平。但是用Oxamflatin预处理的猪胎儿成纤维细胞作为核供体细胞,并没有提高重构胚胎的体外囊胚发育率。这表明,Oxamflatin不是通过抑制供体细胞组蛋白去乙酰化酶的活性,而是通过抑制卵母细胞组蛋白去乙酰化酶的活性,从而导致了较高的猪体细胞克隆效率。通过本研究我们对组蛋白去乙酰化酶抑制剂Oxamflatin提高克隆效率的分子机制有了初步的认识,这对今后我们更好的理解体细胞核移植重编程机理有一定的参考价值。
[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.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828

【参考文献】