摘要研究不同浓度配比的无血清培养基对小鼠胚胎发育的影响。本实验.docVIP

小鼠胚胎无血清培养基系统优化的研究 王丽娜，赵红卫，张凤倩，王宏伟 摘 要：本研究旨在通过添加几种确定的组分来改良无血清培养基以提高小鼠胚胎的发育率。试验以昆明小鼠的原核胚作为研究对象，在基础培养基KSOM中添加不同浓度的葡萄糖、海藻糖、L-谷氨酰胺和谷胱甘肽，对胚胎进行培养，通过单因素试验和正交试验获得如下结果：单因素试验表明，较低或较高浓度的葡萄糖均对胚胎发育有一定的抑制作用 (P0.05)；低浓度海藻糖的胚胎发育率高于高浓度海藻糖和葡萄糖 (P0.01)。高浓度的L-谷氨酰胺比低浓度的L-谷氨酰胺更有利于胚胎的发育 (P0.05)。过高或过低浓度的谷胱甘肽均均导致胚胎发育率降低 (P0.01)。正交试验结果表明，最佳组合方案为A2B2C1D1，通过极差分析进一步表明谷胱甘肽在胚胎发育中具有更加重要的作用。 关键字：无血清；胚胎培养；KSOM；单因素；正交试验 Abstract: In order to improve the development of mouse embryos in vitro, our test ameliorates the serum free medium by adding several different certain components . This test took Kunming mice pronucleus embryo as the research object. The KSOM was used as basic culture medium which was added with glucose, trehalose, L - glutamine, glutathione .Set single factor variable group before orthogonal experimental group to investigate the embryo development rate in the different phase .The analysis of the results :The results of single factor indicate as follows, lower or higher levels of glucose were inhibited embryonic development, but the difference is insignificant (P 0.05).Low concentration trehalose ( 3 mg/mL) is beneficial to the late embryonic development, and the difference was significant (P 0.01). High concentration L - glutamine is more advantageous to embryonic development and the difference was significant (P0.05). Nor higher or lower glutathione was in favour of to the development of embryos,and the difference was significant (P 0.01). Orthogonal experiment results analysis reveals that the optimum scheme is the combination of A2B2C1D1. The range analysis reveals that GSH plays a main role in the process of embryo culture in vitro. Key words: serum free；embryo culture；KSOM；single factor；orthogonal test 引言 哺乳动物胚胎体外培养不仅是哺乳动物胚胎工程的关键技术之一，也是研究早期胚胎发育机制的重要方法。目前早期胚胎体外培养大多是在合成培养基中添加血清 (或牛血清白蛋白) 或与体细胞共培养，但是血清 (或血清白蛋白) 及体细胞分泌物质中均含有许多不确定成分，对揭示影响早胚发育的因素造成较大的困难。所以，本研究旨在对小鼠胚胎体外无血清培养进行研究。多年来，国内外学者对体外胚胎培养的影响因素进行了大量的研究，譬如小鼠胚胎体外培养还存在发育阻断、囊胚发育率低等问题[1]；Schini 等[2]和Seshagiri 等[3]首先发现葡萄糖、磷酸盐是