目录
摘要 Ⅱ
关键词 Ⅱ
Abstract Ⅲ
引言
引言 1
1 材料与方法 2
1.1 实验动物与样品采集 2
1.2 主要试剂及溶液配制 2
1.3 总RNA提取与qRTPCR检测基因表达 3
1.4 基因组DNA 提取 4
1.5 DNA Dot Blot 4
1.6 鸡胚PGCs冰冻切片制作及免疫荧光染色 4
1.7 数据分析 5
2 结果与分析 5
2.1 维生素C对鸡PGCs甲基化相关基因表达的影响 5
2.1.1 维生素C对Tet1表达的影响 5
2.1.2 维生素C对DNA甲基转移酶表达的影响 6
2.2 维生素C对PGCs多能性基因表达的影响 6
2.3 维生素C对鸡PGCs全基因组甲基化水平的影响 7
2.3.1 Dot blot检测PGCs 5mC表达水平 7
2.3.2 SSEA1免疫荧光染色鉴定PGCs 7
2.3.3免疫荧光染色检测PGCs甲基化及羟甲基化水平 8
3 讨论 9
3.1 维生素C对鸡PGCs全基因组甲基化水平的影响 9
3.2 维生素C对鸡PGCs甲基化相关基因表达的影响 9
3.2 维生素C对多能性基因mRNA表达的影响 10
4 结论 11
致谢 12
参考文献 13
维生素C对鸡原始生殖细胞甲基化及多能性基因表达的影响
摘 要
原始生殖细胞（primordial germ cells, PGCs）是成熟精子和卵子的始祖细胞，具有与胚胎干细胞类似的全能性及自我更新能力，是动物生存及繁衍后代的重要细胞。鸡PGCs在E34的胚胎发育初期，大量PGCs会通过血液循环逐渐迁移至生殖脊部位，最终分化为精原细胞或卵原细胞；E4.56.5左右时，PGCs会进行广泛的基因组DNA去甲基化与重新甲基化过程，对性腺的发育进程影响极大。本实验旨在探究早期鸡胚 *51今日免费论文网|www.jxszl.com +Q: ￥351916072￥ 
注射维生素C对不同时期甲基化相关调控基因（Tet1、Dnmt1、Dnmt3a、Dnmt3b）及多能性调控相关基因（Sox2和PouV）mRNA表达、PGCs数量、全基因组甲基化及羟甲基化水平的影响。结果发现：相较于对照组，维生素C促进早期鸡胚PGCs去甲基化过程，同时也促进PGCs多能性的保持。此外，经综合分析本实验所设三种浓度处理，发现10 μgmL1的维生素C对E3.58时期的鸡PGCs甲基化状态及多能性基因表达等的影响效果更佳。
EFFECTS OF VITAMIN C ON METHYLATION AND PLURIPOTENT GENE EXPRESSION IN PRIMORDIAL GERM CELLS OF CHICKEN
ABSTRACT
Primordial germ cells (PGCs) are the progenitor cells of mature gametes sperm and eggs. Similar to embryonic stem cells, they have totipotency and selfrenewal ability and are important cells for animal survival and reproduction. During the early embryonic development of chicken PGCs at E34, a large number of PGCs will migrate to the genital ridge through the blood circulation and eventually differentiate into spermatogonial cells or oogonocytes. At the early stage of embryonic development of E4.56.5, poultry PGCs will undergo a wide range of genomic DNA demethylation and remethylation processes, which has a great influence on the development and differentiation of gonadal stage. The purpose of this study was to explore the chicken embryo injections of vitamin C for early PGCs the relative mRNA expression of methylation related genes (Tet1, Dnmt1, Dnmt3a, Dnmt3b) and pluripotency related genes (Sox2, PouV), genomewide methylation and hydroxyl methylation and the number of PGCs. We found that compared with the control group, Vitamin C inhibited methylation and promote demethylation of early chicken embryo PGCs to some extent, and also promote the maintenance of pluripotency of early chicken embryo PGCs. In addition, after a comprehensive analysis of the overall performance of the three concentrations treatment in this experiment, it was found that 10 μgmL1 vitamin C had a better effect on the methylation condition and pluripotent gene expression of chicken PGCs on E3.58.

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