不同冻存保护剂对未性成熟小鼠睾丸生精细胞冻存的影响  

Effects of different cryoprotectants on cryopreservation of immature mouse testis germ cells

作　　者：张啸龙 张杰平 王金玉 于岚 冯科 曲晓伟 夏彦清 郭海彬 万锋 张翠莲 ZHANG Xiao-long;ZHANG Jie-ping;WANG Jin-yu;YU Lan;FENG Ke;QU Xiao-wei;XIA Yan-qing;GUO Hai-bin;WAN Feng;ZHANG Cui-lian(Zhengzhou University People’s Hospital,Henan Provincial People’s Hospital,Zhengzhou 450003;People’s Hospital of Henan University,Zhengzhou 450003;Henan Medical School,Zhengzhou 450003)

机构地区：[1]郑州大学人民医院河南省人民医院,郑州450003 [2]河南大学人民医院,郑州450003 [3]河南大学医学院,郑州450003

出　　处：《生殖医学杂志》2022年第3期366-372,共7页Journal of Reproductive Medicine

基　　金：河南省重点研发与推广专项(科技攻关)(192102310396,202102310130);河南省医学科技攻关计划项目(LHGJ20200040);国家自然科学基金青年科学基金项目(81801519)。

摘　　要：目的探索不同冻存保护剂对2周龄未性成熟小鼠睾丸生精细胞冻存的影响。方法取2周龄雄性小鼠两步酶法制备睾丸细胞悬液(TCS),根据DMEM/F12培养基中不同浓度二甲基亚砜(DMSO)、蔗糖、海藻糖的组合将冻存保护剂分为5组:A组含10%DMSO和10%胎牛血清(FBS);B组含10%DMSO、0.1 mol/L蔗糖和10%FBS;C组含10%DMSO、0.1 mol/L海藻糖和10%FBS;D组含15%DMSO、0.1 mol/L蔗糖0.1 mol/L海藻糖和10%FBS;E组含10%DMSO、0.1 mol/L蔗糖、0.1 mol/L海藻糖和10%FBS。将5组睾丸细胞采用程序化冻存于液氮中,2月后进行复苏。台盼蓝检测细胞活性,计算各组细胞冻存后的细胞活率和复苏率,流式细胞仪检测睾丸生精细胞的凋亡和线粒体膜电位变化,以新鲜的2周龄小鼠睾丸生精细胞作为对照组。结果与冻存前比较,冻存复苏后各组的细胞活率均显著下降(P<0.05),且冻存组中E组细胞活率下降程度最低[(83.03±1.61)%]、细胞复苏率最高[(87.37±1.66)%](P<0.05)。细胞凋亡比较中,冻存复苏后各组细胞凋亡率均显著高于对照组[(18.77±0.95)%](P<0.05),其中A组细胞凋亡率最高[(57.08±2.40)%],E组细胞凋亡率最低[(29.62±1.95)%](P<0.05)。线粒体膜电位比较中,各组复苏后细胞线粒体膜电位均低于冻存前,除E组外,其余4组均显著低于对照组(P<0.05),在冻存的5组中E组下降程度最低,差异有统计学意义(P<0.05)。结论不同冷冻保护剂对未性成熟小鼠睾丸生精细胞冻存效果有显著影响,其中含10%DMSO、0.1 mol/L蔗糖、0.1 mol/L海藻糖及10%HBS组合冷冻剂方案是冻存未性成熟小鼠睾丸生精细胞较适宜的冷冻保护剂方案。Objective:To explore the effects of different cryoprotectant on cryopreservation efficacy of testis germ cells in 2-week-old sexually immature mice.Methods:The testicular cell suspension of 2-week-old male mice was prepared by two-step enzymatic method.According to the combination of dimethyl sulfoxide(DMSO),sucrose and trehalose in different concentrations in DMEM/F12 medium,the cryopreservation protectants were divided into 5 groups:10%DMSO and10%fetal bovine serum(FBS)in group A;10%DMSO 0.1 mol/L sucrose and 10%FBS in group B;10%DMSO,0.1 mol/L trehalose and 10%FBS in group C;15%DMSO,0.1 mol/L sucrose,0.1 mol/L trehalose and 10%FBS in group D;10%DMSO,0.1 mol/L sucrose,0.1 mol/L trehalose and 10%FBS in group E.The testicular germ cells in five groups were programmatically frozen in liquid nitrogen and resuscitated after 2 months.Trypan blue was used to test the cell viability,and the cell viability and recovery rate of each group after frozen-thawed were calculated.Flow cytometry was used to test the apoptosis and the changes of mitochondrial membrane potential of testicular germ cells.And fresh testicular germ cells of 2-week-old mice were used as the control group.Results:Compared with before cryopreservation,the cell viability of each group decreased significantly after frozen-thawed(P<0.05),and the cell viability of group E decreased the least[(83.03±1.61)%]and the recovery rate was the highest[(87.37±1.66)%]in the cryopreservation groups(P<0.05).Compared with the control group[(18.77±0.95)%],the apoptosis rate increased in all cryopreservation groups after frozen-thawed(P<0.05),and the apoptosis rate in group A was the highest[(57.08±2.40)%,P<0.05]and that of group E was the lowest[(29.62±1.95)%,P<0.05].Compared with the control group,the mitochondrial membrane potential of each cryopreservation group decreased after frozen-thawed.Except group E,the mitochondrial membrane potential of the other four groups were significantly lower than the control group(P<0.05),and the decrease degree of group E was th

关 键 词：睾丸生精细胞 冷冻保护剂 生育力保存 凋亡 线粒体膜电位 

分 类 号：R332[医药卫生—人体生理学]