目录
摘要 III
关键词 III
Abstract IV
引言
引言 1
1 实验材料 2
1.1 抗体与试剂 2
1.1.1 抗体 2
1.1.2 试剂 2
1.2 抗体与试剂的制备 2
1.2.1 抗体的稀释 2
1.2.2 试剂的配制 2
1.3 主要仪器设备及耗材 3
2 实验方法 3
2.1 小鼠卵巢RNA的提取 3
2.2 RNA变性及反转录 3
2.3 聚合酶链式反应（PCR）体系及条件 4
2.4 聚合酶链式反应（PCR）产物的检测 5
2.5 质粒（NipblmRFP融合荧光蛋白载体）的构建 5
2.6 小鼠卵母细胞的收集和体外培养 6
2.7 显微注射Nipbl的siRNA 7
2.8 免疫蛋白印迹法（Westernblot技术） 7
2.9 免疫荧光染色和激光共聚焦显微镜观察 8
2.10 统计分析 8
3 结果与分析 9
3.1 Nipbl基因在小鼠卵母细胞成熟过程中的亚细胞定位 9
3.1.1 质粒（NipblmRFP融合荧光蛋白）载体构建 9
3.1.2 Nipbl蛋白在减数分裂过程中发育过程中的定位 9
3.2 敲低Nipbl对小鼠卵母细胞减数分裂进程的影响 10
3.2.1 敲低效果检验（Westernblot检验） 10
3.2.2 敲低Nipbl影响小鼠卵母细胞发育进程 11
3.2.3 敲低Nipbl影响小鼠卵母细胞中纺锤体组装和染色体排列 12
3.2.4 敲低Nipbl影响小鼠卵母细胞中微管的稳定性 13
4 讨论 13
致谢 14
参考文献 16
附录 17
Nipbl调控小鼠卵母细胞纺锤体组装的作用研究
摘 要
减数分裂过程中姐妹染色单体的精确分 *51今日免费论文网|www.51jrft.com +Q: ^351916072# 
离依赖于黏合素介导的染色体黏合，而黏合素在染色体上的精准装载又受到一种名为Kollerin蛋白复合物的调控。研究发现Kollerin由Nipbl和Mau2两种蛋白分子组成，但它们在卵母细胞减数分裂成熟中的功能还未知。因此对它们的研究将有利于建立卵子质量评价分子体系，预防卵子成熟障碍和非整倍体卵子的产生，减少动物生产过程中的胚胎损失，最终为提高动物繁殖力提供理论依据。本课题选取实验动物ICR小鼠为研究对象，采用卵母细胞体外培养、融合荧光表达载体构建、质粒体外转录、siRNA和mRNA显微注射、免疫荧光染色及免疫蛋白印迹等技术，研究染色体黏合素装载因子Nipbl在卵母细胞减数分裂过程中各个时期的定位模式，及其对卵母细胞的发育进程的影响。实验结果显示敲低Nipbl导致纺锤体组装、染色体排列和微管稳定性均发生显著异常，从而造成卵母细胞排极体率下降和减数分裂进程阻滞。这一发现为后续进一步阐释Nipbl调控卵母细胞纺锤体组装的作用机制奠定了基础。
INVESTIGATION OF THE ROLE OF NIPBL IN SPINDLE ASSEMBLY DURING MOUSE OOCYTE MEIOSIS
ABSTRACT
During meiosis，the precise separation of sister chromatids depends on the cohesinmediated chromosome cohesion, and the loading of cohesin on the chromosome is regulated by a protein complex called kollerin. It has been reported that kollerin is composed of two protein molecules Nipbl and Mau2, which form a dimer. However, the functional roles of these molecules during oocyte meiotic maturation are still not clear. Thus, the investigation of them will contribute to the establishment of oocyte quality evaluation system, prevention of oocyte maturation arrest and aneuploidy, as well as reduction of embryonic loss, ultimately providing theoretical basis for the improvement of animal reproduction. The present study selected ICR mice as the experimental animal and took advantage of in vitro maturation of oocytes, the construction of fluorescencefused expression vector, in vitro transcription, microinjection of siRNA and mRNA, immunofluorescence and immunoblotting techniques to investigate the localization pattern of chromosome loading factor Nipbl and its role during oocyte meiotic progression. The results revealed that knockdown of Nipbl led to the aberrant spindle assembly, chromosome alignment and microtubule stability, consequently resulting in the reduced rate of polar body extrusion and arrested oocyte progression. These findings provide scientific basis for further uncovering the molecular mechanism of Nipbl in modulating spindle assembly in oocytes.

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