交变电场协同慢速冷冻对大鼠卵巢组织冻存效率影响的研究

Cryopreservation has great implication for the long-term storage of stem cells, human blood, and rare germplasm resources, as well as the transplant of human organs. The low-temperature injury is considered as the most critical factor that limits the post-cryostorage viability。Generally, it includes the mechanical injury by intracellular ice formation and growth, the “solute injury" and so forth. Previous studies have shown that alternating electric field was applied to reduce crystallization of 0.9% NaCl-H2O binary solution, reduce low temperature injury of cell or tissue during the cryopreservation process at a slow cooling rate.Therefore, the proposal first study the influence on crystallization characteristics and mechanism of x%DMSO-0.9%NaCl-H2O ternary solution of alternating electric field at a slow freezing rate. Second, study the slow freezing coupled with alternative electric field exposure affect cryopreservation efficiency of rat ovarian tissues, the evaluation factors including morphological changes, follicular cell survival, proliferation and apoptosis. Third, to explore an optimized multi-factors combination to reduce cryo-injury and improve cryopreservative efficiency. Finally, to establish heat and mass transfer model of ovarian tissue during the slow freezing process coupled with alternating electric field, further to reveal the potential mechanism and to predict and control the freezing process reasonably. This proposal will help to solve low temperature damage scientific problems during cryopreservation, and is expected to develop a new electric field controlled cryopreservation technologies to improve the efficiency.

低温保存技术对于胚胎干细胞、血液和珍稀动植物种质资源的长期保存及人体器官移植等均具有重要意义。制约低温保存效果最关键因素是冷冻保存对生物组织产生的低温损伤，如冰晶损伤和溶质损伤等。前期研究表明在慢速降温过程中施加交变电场能够降低0.9%NaCl-H2O二元溶液结晶度，减少细胞或组织冷冻保存过程中的低温损伤。因此，本项目首先研究交变电场协同慢速冷冻x%DMSO-0.9%NaCl-H2O三元溶液过程结晶特性及影响机理；其次研究交变电场协同慢速冷冻对大鼠卵巢组织冻融后组织形态学改变，卵泡细胞存活率、增殖、凋亡等影响规律；探索出能够减少大鼠卵巢组织低温损伤，提高冻存效率的多因子优化组合；建立交变电场协同慢速冷冻大鼠卵巢组织过程的热质耦合传递模型，揭示作用机理并预测和合理地控制冷冻过程。本项目有助于解决低温冷冻保存过程中低温损伤的科学难题，并有望开发出能够提高冻存效率的电场控制低温保存新技术。

随着近代医学诊断和治疗技术的飞跃发展，越来越多的疾病可以通过基因治疗以及细胞和组织器官的移植而得到根治，但细胞组织的来源供不应求已成为治疗的瓶颈。大规模的生物材料和组织的长期冷冻保存，正是解决细胞组织来源问题的最有效方法。交变电场协同慢速冷冻将作为一种潜在的细胞组织长期保存方法在恶性肿瘤患者恢复生育能力和内分泌功能发挥着非常重要的作用。本课题结结合运用电介质物理学、热力学、细胞生物学等方法与技术，提出了根据低温保护剂溶液的电磁特性，采用交变电场控制其结晶过程，并以大鼠卵巢组织为模型，将该技术在低温冷冻保存领域加以应用。本项目将实验研究和理论计算结合，建立了交变电场协同慢速冷冻大鼠卵巢组织过程中热质耦合传递模型，可为生物材料电场控制冷冻保存技术的应用提供理论指导。本技术重点针对了目前已有冷冻保存技术的两大不足之处：大量低温保护剂的使用和降温速度高，做出了有效的改善。在不要求高浓度低温保护剂或者快速降温条件下，有效防止冷冻损伤。.项目资助发表SCI论文2篇，中文2篇，专利1部，软件著作权1件，待发表1篇。协助培养硕士生3名，均已毕业。项目投入经费28万元，目前支出24.2661万元（包括应付未付的3.85万元），各项支出基本与预算相符。剩余经费3.7339万元，剩余经费计划用于本项目研究后续支出。..