人卵巢组织慢速程序化冷冻保存方案的初步探讨  

Preliminary Study of Human Ovarian Tissue Cryopreservation by Controlled-rate Freezing

作　　者：刘丽英[1] 曲文玉[1] 蒋丽[1] 张晓丽[1] 刘晓莉[1] 

机构地区：[1]沈阳市妇婴医院生殖中心,沈阳110014

出　　处：《中国医科大学学报》2015年第5期425-428,433,共5页Journal of China Medical University

基　　金：沈阳市科委项目(F14-158-9-13)

摘　　要：目的系统比较不同冷冻保护剂在不同浓度时对卵巢组织慢速程序化冷冻效果的影响。方法卵巢组织来源于15例卵巢良性肿瘤手术的患者。分别采用丙二醇(PROH)、乙二醇(EG)、二甲基亚砜(DMSO)作为冷冻保护剂,在每种冷冻保护剂1.5 mol/L或2.0 mol/L浓度下进行慢速程序化冷冻。每例患者平行进行上述6种冷冻程序,光镜及电镜下分析卵泡的形态正常率,结果与该患者的新鲜对照组织比较。结果各组中不同发育阶段的卵泡分布无统计学差异(P>0.05)。光镜观察显示,1.5mol/L EG组及2.0 mol/L EG组的冷冻效果最好,与新鲜对照组织相比卵泡形态正常率无统计学差异(P>0.05);1.5 mol/L PROH组、2.0 mol/L PROH组、1.5 mol/L DMSO组及2.0 mol/L DMSO组与新鲜对照组织相比,卵泡形态正常率的差异有统计学意义(P<0.05)。电镜观察显示,经程序化冷冻后,卵母细胞质量下降,但由于数据少未进行统计分析;与新鲜对照组织相比,6个冷冻组颗粒细胞的活力均明显降低(P<0.01)。形态正常的颗粒细胞数量在2.0 mol/L PROH组及1.5 mol/L EG组中稍高,但6个冷冻组间两两比较,差异无统计学意义(P>0.05)。结论慢速程序化冷冻采用1.5 mol/L EG作为冷冻保护剂,能较好的保存卵泡中的卵母细胞及颗粒细胞,是一种较好的冷冻卵巢组织的方案。Objective To compare the effect of different cryoprotectants and different concentrations on controlled-rate freezing of human ovarian tissues. Methods Ovarian tissues were sampled from 15 patients undergoing benign ovarian tumor surgery. Cortical slices were frozen by controlled-rate freezing using three cryoprotectants,propanediol,ethanediol,and dimethylsulphoxide,and the concentration of each cryoprotectant was 1.5 mol/L or 2.0 mol/L. Cortical slices obtained from each patient were processed with each cryopreservation procedure simultaneously. Morphology of fol-licles was studied by light and electron microscopy and the normal rate was compared with that of the fresh tissues from the patient. Results There were no significant differences in distribution of follicles of different developmental stages between each group（P〈0.05）. Light microscopy showed 1.5 mol/L EG and 2.0 mol/L EG groups had the best freezing effect,and the difference in the morphologically normal rate of follicles was not statisti-cally significant compared to fresh controls（P〉0.05）. However,the difference was statistically significant for 1.5 mol/L PROH,2.0 mol/L PROH, 1.5 mol/L DMSO and 2.0 mol/L DMSO groups（P〈0.05）. Electron microscopy showed the oocyte quality declined after controlled-rate freezing pro-cedure. However,the statistical analysis was not conducted due to little data. The viability of granulosa cells was significantly declined after all the freezing procedures compared to that of the fresh control tissues（P〈0.01）. The number of morphologically normal granulosa cells was slightly higher in the tissues which had been cryopreserved with 2.0 mol/L PROH and 1.5 mol/L EG,but no significant differences were found between any of two frozen groups（P〉0.05）. Conclusion Controlled-rate freezing using 1.5 mol/L EG as the cryoprotectant can better save oocytes and granulosa cells. It is a preferable freezing procedure for ovarian tissues.

关 键 词：冷冻保存 人卵巢组织 超微结构 慢速程序化冷冻 

分 类 号：R71[医药卫生—妇产科学]