非渗透性保护剂优化卵母细胞冷冻程序及细胞膜关联机制研究

Today, in vitrified oocyte using high concentration of penetrating cryoprotectant(CPA), the potential toxicity and osmotic damage from these chemical reagents put psychologically high stress on the embryologists imagining more poor developed offprings.Whether we could improve the vitrified efficiency by enlarging effects of non-penetrating protectants in low toxicity and achieve the similarity of survival rates and more healthier babies, compared to the vitrified protocols addition of high-concentration penetrated protectants,which is we expected. The objective of our project was to perform a series of physical methods to search a highly efficient non-penetrating CPA with different types, concentrations, including the measurement for solution parameters of the glass transition temperature and devitrification temperature using Differential Scanning Calorimetry (DSC) ,the observation of the crystallization and recrystallization property of the protective agent the aid of cryomicroscope；with the help of image analysis to observe the phase change of oocyte volume to explore cell dehydration rate and final dehydration rate. Comprehensive analysis of three experimental findings reveals the optimal non-penetrating cryoprotectant charaterised by high glass-forming ability and less ice crystals.The combined solution of non-penetrating CPA assessed by physical method with 1～2M Ethylene glycol, compared with a classic protocol involved high concentration(6M) of Ethylene glycol,was to investigate mouse and human oocyte`s survival and developing ability by the survival rate, fertilized rate, blastocysts formation percentage. In the third experiment, cell membrane was investigated by TEM for ultrastructural damage, companied by immunohistochemical technology for assessing the membrane aquaporins-3 expression, and the methylation states of imprinting genes(IGF2R) pre- and post freezing. All the experimental results above all will totally be demostrated, by enlarging the non-penetrated cryoprotectants application accompanied by decrease the concentration of penetrated cryoprotectants,the well survival rates and development percentages and long-term safety of vitrified oocytes.Our experimental results could provide more fundamental theory and research data for designing more safe and efficient vitrified protocols.

高浓度的渗透性冷冻保护剂（CPA）存在化学与渗透损伤,目前已成为影响卵母细胞冷冻技术发展的瓶颈，增加非渗透CPA的应用与机制研究将作为减少高浓度渗透性CPA依赖的新途径。本研究拟采用差示扫描量热仪（DSC）测量不同类型、浓度非渗透性CPA的玻璃化与反玻璃化温度参数；利用低温显微镜观察非渗透保护剂的结晶与重结晶性质；以及结合图像分析观察到的细胞体积在保护剂中的时相变化，得出非渗透保护剂对细胞的脱水速率和最终脱水率，综合判断玻璃化形成能力强、细胞冰晶形成少的非渗透性保护剂的类型及其适宜浓度。然后联合低浓度（1～2M）渗透保护剂保存小鼠及人卵母细胞，以冻融存活率、受精率与囊胚形成率为指标，与高浓度渗透CPA方案比较细胞保存效果与发育潜能。最后，通过冷冻前后细胞膜结构及水通道蛋白AQP3的表达变化，以及印迹基因IGF2R的甲基化状态，揭示非渗透性保护剂优化玻璃化保存效果的关键机制，为更安全、高效的玻璃化法保存卵母细胞提供理论支持。

卵母细胞冷冻是辅助生殖实验室技术中的难题，至今生殖医学界仍未探索出高效的保存方法以减小卵子丢失率。传统方法采用高浓度渗透性保护剂的方法，其存活效率及复苏后的卵子发育潜能均受到不同程度的影响，严重干扰了临床的广泛应用。在此基础上，本项目以“提高非渗透性保护剂”的保存效能为突破点，通过医工交叉科学攻关，在运用一系列的物理学方法，从众多的非渗透性保护剂中寻找到玻璃化形成能力强的、结晶少的、细胞渗透性好的0.7M海藻糖溶液。再联合低浓度的渗透性保护剂，玻璃化保存小鼠卵母细胞与人卵母细胞，通过体外受精与培养技术，验证了0.7M的海藻糖溶液联合低浓度的渗透性保护剂，确实可以达到同高浓度渗透性保护剂相似的受精率与囊胚发育率。本项目的研究成果，不仅丰富了非渗透性保护剂在玻璃化冷冻的理论体系，更是打破了以往采用经验医学的探索模式，本研究成果对提高卵母细胞高效保存具有较高的科学价值，为卵母细胞冷冻技术的完善与临床拓展使用提供了科学的利好信息。