小鼠卵巢组织玻璃化冷冻的初步研究

目的建立一种有效的卵巢组织玻璃化冷冻方法。方法采用乙二醇和蔗糖两步法、小鼠卵巢组织经5m in、10m in、15m in 3种不同的预平衡时间处理,在玻璃化溶液中暴露相同的时间后直接投入液氮进行玻璃化冷冻。复苏后与新鲜的卵巢组织均进行HE染色、提取DNA进行琼脂糖凝胶电泳。结果玻璃化冷冻复苏后,预平衡时间为10m in的实验组小鼠卵巢组织中的卵泡保持了较好的形态结构。提取DNA进行琼脂糖凝胶电泳,各实验组与新鲜对照组的结果相似。结论采用乙二醇和蔗糖两步法、预平衡为10m in、直接投入液氮的玻璃化冷冻方法可能是一种有效的卵巢组织片冷冻保存方法。该玻璃化冷冻方法没有造成细胞DNA的损伤。

Primary study on vitrification of mouse ovarian tissues

Objective:To establish an efficient vitrification of ovarian tissues.Methods : Mouse ovarian tissues were vitrified by being treated with different time of pre-equilibration:5min,10min and 15min,being exposed in the vitrification solution with the same time,then being directely plunged into the liquid nitrogen,with the method of two-steps,and using ethylene glycol(EG) and sucrose as cryoprectant.After thawing,the vitrified-warmed ovarian tissues and fresh ovarian tissues were stained with hematoxylin and eosin.DNA was observed using agarose gel.Results:After vitrification and thawing,the follicle in the surface layer of the ovarian cortex which pre-equaled ten minutes remains better morphology than other groups.The DNA in the agarose gel of all research groups was similar as the fresh-control group.Conclusion:The vitrification which uses ethylene glycol(EG) and sucrose as cryoprectant,has two steps,pre-equals ten minutes and has been directly plunged into liquid nitrogen may be an efficient cryopreservation method of ovarian cortex fragment.This method didn't injury DNA of the cells.