【作者】 李世杰；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 尽管体细胞克隆在许多物种中都获得了成功，但克隆效率却非常低，只有低于4％的重组胚胎可以发育成活的个体。若体细胞克隆技术在医药及农业等方面得到广泛应用，必需首先解决克隆效率低的问题。由于供体核支持整个克隆动物的发育，所以当供体核被移入去核的卵母细胞后，必需进行重编程，回到胚胎发育的起始阶段。在重编程过程中，供体核必需正确激活对早期胚胎发育有重要作用的基因，抑制在供体核中表达的、与分化相关的基因。目前认为：供体核的不完全重编程，导致在发育过程中有重要作用的基因没有表达或异常表达，是克隆效率低的主要原因。有关基因在克隆动物中表达的研究目前主要集中在植入前的克隆胚胎中。有一些克隆动物在出生后不久死亡，并表现出某些器官发育异常，但造成克隆动物器官异常和出生死亡的原因并不清楚。应用成纤维细胞进行核移植生产体细胞克隆动物许多优势。首先，从一个有价值的动物中，很容易得到皮肤的活体组织，这样通过体细胞克隆便可以生产大量的、具有相同遗传物质的克隆动物。其次，成纤维细胞很容易培养及冷冻储存，是改变细胞遗传物质从而生产转基因克隆动物很好的材料来源。 本研究应用40日龄荷斯坦奶牛的胎儿成纤维细胞和4岁荷斯坦奶牛的耳组织成纤维细胞作为供体核的来源生产体细胞克隆牛，其囊胚率分别达到了37.9％和27.9％，分别有3.4％(10／294)和3.9％(11／283)的重组胚胎发育到出生个体，这些发育到出生个体的胚胎分别有4头和5头在出生时死亡。对这些克隆牛我们进行了尸检，发现某些克隆牛心脏、肝脏、脾脏、肺脏、肾脏和大脑的发育存在着异常，其中以心脏和肺脏的异常最为严重，分别有7头克隆牛的心脏和肺脏发现异常。我们对克隆牛的组织制作常规石蜡切片进行观察，发现克隆牛的器官在发育过程中存在着严重的病理学变化 为了研究造成克隆动物出生死亡和器官发育异常的可能分子机理，我们用荧光定量RT-PCR技术研究了22个在胚胎发育和器官形成过程中有重要作用的基因在出生死亡的克隆牛心脏、肝脏、脾脏、肺脏、肾脏和大脑等6个组织中的表达，其中包括4个对染色质进行修饰的基因(DNMT、PCAF、MeCP2、EED)、类胰岛素生长因子系统的8个基因(IGF1、IGF2、IGF1R、IGF2R、IGFBP1、IGFBP2、IBGBP3和IGFBP4)、成纤维生长因子(FGF2和FGF10)和成纤维生长因子受体(FGFR1和FGFR2)以及另外6个对发育有重要作用的基因(EGFR、PDGFRa、VEGF、BMP4、Hsp70.1和Xist)。 结果表明：在22个被研究的基因中，有16个在体细胞克隆牛的组织中表达发生异常。其中IGFBP4在体细胞克隆牛的5个组织中表达发生异常：IGF2R和BMP4在体细胞克隆牛的4个组织中表达发生异常；DNMT、PCAF、IGF2、VEGF和Hsp70.1等5个基因在体细胞克隆牛的3个组织中表达发生异常；FGF10、FGFR1和PDGFRa等3个基因在体细胞克隆牛的3个组织中表达发生异常：IGF1R、IGFBP2、IGFBP3、EGFR和Xist等5个基因各在体细胞克隆牛的1个组织中表达发生异常。EED、MECP2、IGF1、IGF1R、FGF2和FGFR2等6个基因在体细中国农业大学博士学位论文中文摘要胞克隆牛的组织中未发现表达异常。 从基因异常表达的器官角度分析，心脏是发现基因异常表达最多的器官，有川个基因在体细胞克隆牛的心脏中表达发生异常，其中9个表现为显著升高。体细胞克隆牛的肝脏中有7个基因表达发生异常，其中5个表现为显著升高。体细胞克隆牛的肺脏、’肾脏和大脑中均有6个基因表达发生异常，其中肾脏中异常表达的6个基因均表现为显著降低，也是6个被研究器官中唯一的一个基因异常表达的趋势表现为一致的器官。脾脏是6个被研究器官中发现基因异常表达最少的器官，有4个基因在体细胞克隆牛的脾脏中表达发生。 比较来自两种不同供体细胞的克隆牛之间的表达差异，发现有8个基因(IGFZR、HsP7()l、Xist、PDGFRa、BMP4、IGFBP4、FGF10及IGFZ)的表达在来自成年成纤维细胞的克隆牛和来自胎儿成纤维细胞的克隆牛的某个组织中存在显著差异。 在16个异常表达的基因中(只有在一个组织中异常表达的基因除外)，有3个基因在体细胞克隆牛组织中表现出相同的异常表达趋势。其中VEGF在3个异常表达的组织中均表现为显著升高，FGFRI在2个异常表达的组织中也均表现为显著升高，而PDGFRa在2个异常表达的组织中均表现为显著降低。在所有异常表达的状态中(39个)，显著升高的有24个，而显著降低的有巧个。 由于这些基因在胚胎发育和器官形成过程中有重要作用，所以它们的异常表达可能是造成克隆动物出生死亡和器官发育异常的原因。更多还原

【Abstract】 Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embryonic ground state. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. Examination of gene expression in cloned animals has largely been limited to preimplantation embryos for a small number of genes important for early embryogenesis. Cloning by somatic nuclear transfer is an inefficient, process in which some of the cloned animals die shortly after birth and display organ abnormalities. In somatic cloning of cattle, fibroblast was often used as donor nuclear. Fibroblasts offer several advantages for future applications of somatic cloning. First, the skin biopsy can be easily gained from a valuable animal, so that to produce large numbers of genetically identical copies of the animal are possible. Secondly, fibroblasts can easily cultured and stored frozen, and which are probably good candidates for genetic modification to produce transgenic animals.In this research, the donor nuclei were obtained from skin fibroblast cells of a female four-year-old elite Holstein cow and from fetal fibroblast cells of a 40-day-old female fetus. About 27.9% (n=283) and 37.9% (n=294) of reconstructed embryos derived from adult fibroblast cell and from fetal fibroblast respectively developed into blastocysts, with 11(14.9%, n=74) and 10 (22.7%, n=44) of the transferred embryos developing into full-term calves. Six of the eleven calves from adult fibroblast cell survived and remained healthy, and five died. Six often calves from the fetal fibroblast cells survived and remained healthy, while four died. All of the deceased cloned cattle died within 48 hours of birth. The abnormalities were observed at necropsy and tissue pathologic section of deceased cloned cattle’ organs, including heart, liver, spleen, lung, kidney and brain, and in which the abnormalities of heart and lung were serious.In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of 22 developmentally important genes in six organs (heart, liver, spleen,lung,kidney and brain )of both neonatal death cloned bovines and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The genes include four genes of modifying chromatin strcture (DNNT, PCAF, MeCP2, EED) eight genes of insulin-like growth factors systems (IGF1, 1GF2, IGF1R, IGF2R, IGFBP1, IGFBP2, IBGBP3 and IGFBP4)s fibroblast growth factor (FGF2 and FGF10) and fibroblast growth factor receptor (FGFR1 FGFR2 )and other six developmentally important genes( EGFR, PDGFRa, VEGF, BMP4, Hsp70.1 andAberrant expressions of sixty genes were found in these clones The aberrant expressions of IGFBP4 were observed in five organs; IGF2R and BMP4 in four organs; five genes of DNMT, PCAF, IGF2, VEGF Hsp70.1 in three organs; three genes of FGF10 FGFR1 and PDGFRa in two organs, and aberrant expressions of five genes ofIGFlR IGFBP2 IGFBP3 EGFR and Xist were only observed in one organs.For the studied genes, spleen was the organ that less affected by gene dysregulaion in which four genes were aberration, whereas heart was the most affected one, in which ten genes were aberration, in liver, seven genes were aberration and five genes were up-regulation. In lung, kidney and brain, six genes were aberration and kidney was the only organ with all dysregulated genesof down-regulationComparing the gene expression between the two types clones with each other and eight gene (IGF2R, Hsp70.1 Xist PDGFRa BMP4 IGFBP4 FGF10 and IGF2) expression were found having significant difference between AF cell-derived and FF cell-derived clones.For the sixty aberrant e更多还原