Z-VAD-FMK抑制凋亡通路对体外培养玻璃化冷冻未成熟卵母细胞的影响  

Effect of inhibiting apoptosis pathway by Z-VAD-FMK on vitrified immature oocytes in vitro

作　　者：卢美蓉 王静 黄广翅 李桂花 刘秀盈 蔡杏琳 LU Mei-rong;WANG Jing;HUANG Guang-chi;LI Gui-hua;LIU Xiu-ying;CAI Xing-lin(Department of Obstetrics and Gynecology,Hainan Modern Women and Children’s Hospital,Haikou Hainan 570100,China;Clinical Laboratory,Hainan Modern Women and Children’s Hospital,Haikou Hainan 570100,China;Department of Obstetrics,First Affiliated Hospital of Hainan Medical University,Haikou Hainan 570102,China)

机构地区：[1]海南现代妇女儿童医院妇产科,海南海口570100 [2]海南现代妇女儿童医院检验科,海南海口570100 [3]海南医学院第一附属医院产科,海南海口570102

出　　处：《局解手术学杂志》2022年第7期585-590,共6页Journal of Regional Anatomy and Operative Surgery

基　　金：海南省卫生计生行业科研项目(19A200181)。

摘　　要：目的探讨通过泛Caspase抑制剂Z-VAD-FMK抑制凋亡通路对体外培养玻璃化冷冻未成熟卵母细胞(VIOs)的DNA完整性和发育潜能的影响。方法取200只雌性ICR小鼠未成熟卵丘-卵母细胞复合体(COCs)。实验Ⅰ:将未成熟COCs分为阴性对照组(FOs组,n=31,新鲜COCs)、阳性对照组(HPOs组,n=32,过氧化氢暴露COCs约3 h)、2 h VIOs组(n=31,玻璃化复苏COCs 2 h)、18 h VIOs组(n=32,玻璃化复苏COCs 18 h),探讨玻璃化冷冻对未成熟卵母细胞凋亡通路的影响。实验Ⅱ:将未成熟COCs分为VIOs(-/-)组(n=36,未加入Z-VAD-FMK)、VIOs(+/-)组(n=37,仅在玻璃化冷冻复苏培养基中添加Z-VAD-FMK)和VIOs(+/+)组(n=34,在玻璃化冷冻复苏培养基和复苏后培养基中均添加Z-VAD-FMK),复苏后孵育18 h进行染色,探讨Z-VAD-FMK对凋亡标志物[未成熟卵母细胞DNA片段化(TUNEL阳性卵母细胞百分比)、Caspase活性]的影响。实验Ⅲ:将未成熟COCs分为FOs组(n=130)、VIOs(-/-)组(n=129)和VIOs(+/+)组(n=128),分组操作同上,分析Z-VAD-FMK对VIOs体外发育潜能的影响。结果实验Ⅰ结果显示,HPOs组、18 h VIOs组未成熟卵母细胞DNA片段化比例高于FOs组(P<0.05);18 h VIOs组未成熟卵母细胞DNA片段化比例高于2 h VIOs组(P=0.023),低于HPOs组(P=0.004);HPOs组、2 h VIOs组和18 h VIOs组Caspase活性均高于FOs组(P=0.001),但是HPOs组、2 h VIOs组和18 h VIOs组Caspase活性比较,差异无统计学意义(P>0.05)。实验Ⅱ结果显示,与VIOs(-/-)组相比,VIOs(+/-)组未成熟卵母细胞DNA片段化比例较低(P=0.007);VIOs(+/+)组未成熟卵母细胞DNA片段化比例和Caspase活性低于VIOs(+/-)组和VIOs(-/-)组(P<0.001),但VIOs(+/-)组和VIOs(-/-)组Caspase活性比较差异无统计学意义(P=0.588)。实验Ⅲ结果显示,VIOs(+/+)组成熟率、受精率与FOs组和VIOs(-/-)组比较差异无统计学意义(P>0.05),但VIOs(+/+)组和VIOs(-/-)组闭锁率高于FOs组(P<0.001);VIOs(-/-)组和VIOs(+/+)组5~8细胞、9~16细胞胚胎发育率均低于FOs组(P<0.Objective To investigate the effect of inhibiting apoptosis pathway by pan-Caspase inhibitor Z-VAD-FMK on the DNA integrity and development potential of vitrified immature oocytes(VIOs)in vitro.Methods The cumulus-oocyte complexes(COCs)of 200 female ICR mice were obtained.ExperimentⅠ:the immature COCs were divided into the negative control group(FOs group,n=31,fresh COCs),the positive control group(HPOs group,n=32,COCs exposed to hydrogen peroxide for 3 hours),the 2 h VIOs group(n=31,COCs with vitrification resuscitation for 2 hours)and the 18 h VIOs group(n=32,COCs with vitrification resuscitation for 18 hours),to investigate the effect of vitrification on apoptosis pathway of immature oocytes.ExperimentⅡ:the immature COCs were divided into the VIOs(-/-)group(n=36,without Z-VAD-FMK),the VIOs(+/-)group(n=37,with Z-VAD-FMK in the vitrification-warming media)and the VIOs(+/+)group(n=34,with Z-VAD-FMK in the vitrification-warming media and postwarming incubation medium).Staining was conducted after resuscitation and incubation for 18 hours,to investigate the effect of Z-VAD-FMK on apoptosis markers[DNA fragmentation(percentage of TUNEL positive oocytes)and Caspase activity of immature oocytes].ExperimentⅢ:the immature COCs were divided into the FOs group(n=130),the VIOs(-/-)group(n=129)and the VIOs(+/+)group(n=128),the grouping operations were the same as above,and the effect of Z-VAD-FMK on the development potential of VIOs in vitro was analyzed.Results The results of experimentⅠshowed that the DNA fragmentation ratio of immature oocytes in the HPOs group and the 18 h VIOs group were higher than those in the FOs group(P<0.05);the DNA fragmentation ratio of immature oocytes in the 18 h VIOs group was higher than that in the 2 h VIOs group(P=0.023),and lower than that in the HPOs group(P=0.004).The Caspase activities of the HPOs group,the 2 h VIOs group and the 18 h VIOs group were higher than that of the FOs group(P=0.001),but there was no statistically significant difference among the HPOs group,the 2 h VIO

关 键 词：细胞凋亡 超低温保存 玻璃化冷冻 未成熟卵母细胞 DNA完整性 发育潜能 

分 类 号：Q954.4[生物学—动物学]