【作者】 张磊；

【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】 1997年克隆羊“多莉”诞生之后，大批的克隆动物如鼠、牛、羊和猪等相继问世，但遗憾的是克隆的成功率迄今为止仍然很低。大量的研究表明，克隆动物在核移植后其染色质的再程序化表现异常，使细胞不能成功地通过调节转录活性而有效地控制其发育分化。这些染色质的再程序化包括DNA的甲基化和核小体组蛋白的乙酰化等。组蛋白乙酰化在染色质结构调节、DNA复制、基因转录、细胞分化及癌细胞发育都有研究，但在克隆动物中却是一个有待深入探索的领域。在报道的克隆牛发育异常中，肺脏的异常比较普遍和明显，而在我们的克隆牛工作中也发现了类似问题，所以我们选用克隆牛肺脏组织作为实验材料，进行组蛋白乙酰化研究。 在我们的克隆牛组齿白乙酰化实验中，使用组蛋白乙酰化免疫沉淀反应技术和定量PCR技术，共分析了18个样品利15个基因，这些样品包括9N1，9N3，9N4，8C1，8C2，8C3，8C4，9C1，9C2，9C3，9C4，9C5，9C6，9C8，9C12，Jiumei，Benben和Xiaobai等；这些基因包括β-Actin，BMP4，EGF，FGF，FGFBP，FGFR，GH，GHR，Gli2，HGF，HGFR，IGF1，RAR，SHH和VEGF等。我们发现：(1)在克隆牛中组蛋白乙酰化水平基本上都有所降低；(2)克隆牛Xiaobai乙酰化水平比正常牛低，而基本上是高于或接近死亡克隆牛水平的；(3)在我们所研究的三个供体细胞系(卵丘细胞，成体成纤维细胞和胎儿成纤维细胞)中，乙酰化水平无明显差异。 我们还分析了相关生长因子在克隆牛中的表达状况，共分析了13个样品和7个基因，这些样品包括9N1，9N3，9N4，8C1，9C3，9C4，9C5，9C6，9C8，9C12，Jiumei，Benben和Xiaobai等；基因包括BMP4，FGF10，FGFBP，FGFR，IGF1，SPA和VEGF等。我们发现基因表达的水平变化较大，有些基因的表达水平提高了，如BMP4，IGF1，SPA和VEGF，而有些有所降低，如FGF10，FGFBP和FGFR等。 对克隆牛中组蛋白乙酰化水平和基因在克隆牛中的表达水平对比分析，我们发现两者并没有某种一致的联系。组蛋白乙酰化在基因调节中只是提供了一个微环境，属于粗略调节，只要具备一定的水平即可进一步引发其他因子的参与，来精确调节基因的表达。而在克隆牛中组蛋白乙酰化水平虽然有所降低，但如果这种降低仍然为基因的表达提供了足够的基础，这就对基因的正常表达造不成很大影响；但如果组蛋白乙酰化水平过高或过低，可能要影响到其他因子的作用，从而影响了基因表达的调节，最终导致生物个体发育异常。 为研究基因不同区域的组蛋白乙酰化水平，我们选取了正常牛4N1，6N1，9N3，克隆牛Xiaobai和9C4共五个样品，IGF1和GH两个基冈作为研究对象，在它们的基因序列上分别设计了14对和8对引物来进行定量分析。通过我们的实验，我们认为无论基因表达与否，都首先在其基因组上维持了一定的组蛋白乙酰化水平，这种乙酰化水平同时也是一个整体的概念，而且在不同的基因区域上，组蛋白乙酰化水平是有一定的波动性，另外这种乙酰化水平可以随着发育阶段的不同根据基因表达的要求发生变化。更多还原

【Abstract】 The first mammal have been successfully cloned from a differentiated animal cell was the sheep "Dolly" in 1997, and other subsequent success with a range of mammal somatic cloned animal, such as mice, cattle, goat, pig, horse and mule etc. Despite this, the mammal somatic cloning is still inefficient. Clones are lost from the earliest developmental stages and throughout pregnancy. Now more and more studies support the evidence on the failure of cloning: chromatin inappropriate epigenetic reprogramming, and then result in abnormal genes expression. Epigenetic modification of the genome ensures proper gene activation during development and involves: genome methylation changes; the assembly of histon and histone variants into nucleosomes and remodeling of other chromatin-associated proteins such as linker histones, polycomb group, nuclear scaffold protein, and transcription factors. Histon acetylation plays an important role in regulation chromatin structure and transcriptional activity. However, there is a little investigation in cloned cattle about histone acetylation. Andmore, in many dead cloned cattle, the lung abnormality is mostly obvious. So, in our studies, we want to know how about the histone acetylation in the lung of cloned cattle.In the histone acetylation study, we used the histone immunoprecipitation assay and real-time quantity PCR method. We analyzed 18 samples (including 9N1, 9N3, 9N4, 8C1, 8C2, 8C3, 8C4, 9C1, 9C2,9C3,9C4, 9C5, 9C6, 9C8,9C12, Jiumei, Benben and Xiaobai) with 15 genes (including b-Actin, BMP4, EGF, FGF, FGFBP, FGFR, GH, GHR, GIi2, HGF, HGFR, 1GF1, RAR, SHH and VEGF). We get to the conclusions: (1) the histon acetylation level has some derease in the lung of cloned cattle; (2) in the cloned cattle Xiaobai, the histone acetylation level is lower than that in normal control, however, higher or adjacent to that in other dead cloned cattle; (3) there is no significant difference during those cattle from the cumulus, fetal fibroblast and adult fibroblast cell donor.We also analyzed some genes’ expression, including 13 samples (9N1, 9N3, 9N4, 8C1, 9C3, 9C4, 9C5, 9C6, 9C8, 9C12, Jiumei, Benben and Xiaobai et al) and 7 genes (BMP4, FGF10, FGFBP, FGFR, 1GF1, SPA and VEGF et al). We find some genes’ expression level increase, such as BMP4, IGF1, SPA and VEGF, and others decrease, such as FGF10, FGFBP and FGFR.However, it is difficult to relate the histone acetylation level with the gene expression level. We indicate, although histone acetylation acts to enhance the access of transcription-associated protein to DNA, the activation of gene expression only need containing some acetylation level, but the hyperacetylation and underacetylation would effect the gene expression, so as to the abnormal development.In order to investigate the histone acetylation in gene different regions, we used normal cattle 4N1, 6N1, 9N3, cloned cattle Xiaobai and 9C4, with the IGF1 and GH genes. We designed 14 and 8 pairs of primers on their DNA sequences, respectively. We suggest: (1) histone must maintain some acetylation level whether the associating-gene is activated or inactivated; (2) acetylation and deacetylation are global in these genes’ chromosomal domains; (3) but uneven; (4) and the level will change according to the development stage.更多还原