新型抗冻蛋白玻璃化保存液保护完整睾丸生育力的作用和机制研究

The fertility preservation of prepubertal tumor patients is urgent to be solved in the current pediatric oncology and urogenital area. Cryopreservation of intact testis might be an ideal approach to achieve fertility preservation for these patients. Antifreeze proteins (AFPs) are specific proteins found in psychrotolerants and can suppress the formation of ice crystals in the organism, thereby help them survive in subfreezing environment. Our previous studies confirmed that AFPⅢ has cryoprotective effect on endothelial cells. It could improve cell survival rate, having the potential value on cryopreservation of intact organ.. On the basis of our patent “Organ vitrified cryopreservation fluid”, our constructed animal experimental platform, and the biological features of AFPs (e.g. thermal hysteresis, ice recrystallization inhibition and interaction with membranes), we adopted AFPs as protectant, try to develop new antifreeze protein vitrified fluid, perform testis cryopreservation and transplantation on experimental animals and evalute the spermatogenic and secretion function. We will further explore and elucidate the cryoprotective mechanism of new antifreeze protein vitrified fluid (e.g., inhibiting ice crystals formation/ Ca2+ overload/ER stress/apoptosis signaling pathway). This study will provide experimental evidencesand and doable recipe for AFPs’ application in fertility preservation of prepubertal tumor patients, as well as other organs’ cryopreservation.

青春期前肿瘤患者生育力保存是小儿肿瘤及泌尿生殖领域亟待解决的难题，完整睾丸的深低温长期保存是实现该类患者生育力保存的理想途径，有效克服冷冻损伤是成功保存的关键。我们前期研究发现耐寒生物体内抑制冰晶形成的特殊功能蛋白——抗冻蛋白，在深低温条件下可提高内皮细胞存活率，具有潜在的器官保护应用价值。.本项目基于授权专利“器官玻璃化冷冻保存液”和已构建实验平台，从抗冻蛋白生物学特性出发（热滞活性、重结晶抑制活性、稳定细胞膜），开展新型抗冻蛋白玻璃化保存液保护完整睾丸生育力的作用和机制研究：①根据低温生物学原理，研发新型保存液，改进深低温保存技术；②进行完整睾丸的深低温保存移植，评估术后近、远期生精及分泌功能；③研究和阐明新型保存液的保护作用机制（如抑制冰晶形成、抑制钙超载/内质网应激/凋亡信号通路）。本项目成果可为青春期前男性肿瘤患者生育力保存及其他组织器官的深低温保存提供实验依据及可行保存液配方。

目前青春期前男性肿瘤患儿的生育力保存是泌尿生殖领域的热点和难点问题。不同于青春期启动后男性及成人，青春期前患儿只能通过睾丸组织移植、精原干细胞移植和睾丸组织体外成熟等方法实现生育可能，但目前以上方法均处于实验阶段。自体睾丸深低温保存移植为该类患儿的生育力保存提供了新的思路，如何有效克服冷冻损伤是成功保存的关键。在本项目中，我们在前期授权专利“器官玻璃化冷冻保存液”基础上结合抗冻蛋白的特殊生物学特性，研发新型抗冻蛋白玻璃化保存液，采用程序控温、保存液梯度灌注等低温生物学操作流程，进行睾丸的深低温保存研究。项目团队证实在-80℃条件下，与对照组相比，采用新型器官深低温保存液保存复温后，睾丸组织曲细精管生精上皮呈轻微浊肿样改变，曲细精管轮廓清晰可见，睾丸间质细胞核形态正常，核膜完整。深低温保存后睾丸组织体外生物学活性测定显示，采用含抗冻蛋白的新型器官深低温保存液保存复温后，睾丸组织睾酮释放量高于不含抗冻蛋白组及DMSO培养基对照组。进一步研究证实新型抗冻蛋白玻璃化保存液通过抑制组织细胞内外冰晶形成、减轻细胞内钙超载对冷冻损伤发挥其深低温保护作用。本项目为青春期前肿瘤患者的生育力保存提供可行保护液配方，也为抗冻蛋白的开发利用提供实验依据及理论支撑。