培养液充气时间对小鼠4细胞胚胎密闭培养效果的影响

Effects of reference gas aeration time to culture medium on mouse 4 cell embryo development in sealed-culture system

【目的】研究标准气充气时间对密闭培养的小鼠4细胞胚胎体外发育的影响,为太空环境下哺乳动物的早期胚胎发育研究奠定基础。【方法】用气体组成为体积分数5%O 2、5%CO 2、90%N 2的高纯标准气对胚胎培养液进行充气处理,充气时间设为30,60,90,120,150,180和240 min,用充气培养液对小鼠4细胞胚胎进行密闭培养,以常规体外微滴培养的胚胎为对照,分别在培养12,36,60,84 h时检测各组体外培养胚胎过氧化物的积累情况和缺氧诱导因子(HIF-1α)表达的变化,并于培养84 h统计囊胚发育率、孵化率和囊胚总细胞数。【结果】当充气时间≥180 min时,胚胎发育早期存在明显的过氧化物积累现象;当充气时间≤120 min时,胚胎发育过程中可以检测到HIF-1α,表明出现缺氧现象。密闭培养条件下,培养液充气120~180 min时的小鼠胚胎囊胚发育率、孵化率和囊胚总细胞数较高,分别为93.38%~95.68%,61.03%~65.27%和102.33~111.83,其中囊胚发育率和囊胚总细胞数与对照组差异不显著(P>0.05),而囊胚孵化率显著高于对照组(P<0.05)。【结论】小鼠4细胞胚胎密闭培养体系适合的培养液充气方案为:用气体组成比例为体积分数5%O 2、5%CO 2、90%N 2的高纯标准气充气120~150 min,此方案下进行密闭培养的小鼠胚胎可获得较高的发育率,并可有效排除缺氧对早期胚胎体外发育的影响,同时保护胚胎免受过氧损伤。

【Objective】For the purpose of establishing an early embryo culture system of mammal in space,this study investigated the effects of reference gas aeration time to culture medium on mouse 4 cell embryos development in a sealed-culture system.【Method】The reference gas containing 5%O 2,5%CO 2 and 90%N 2 was used to treat the culture medium for 30,60,90,120,150,180 and 240 min before 4 cell embryos were sealed-cultured in the medium immediately.Embryos cultured in microdrops were used as control.The reactive oxygen species(ROS)levels and hypoxia-inducible factor 1 alpha(HIF-1α)accumulation were detected at 12,36,60 and 84 h after incubation.The blastocyst rates,hatching rates,and total cell numbers of blastocyst were calculated at 84 h incubation.【Result】The accumulation of ROS was found in the groups pretreated with reference gas longer than 180 min.However,the accumulation of HIF-1αwas detected in the groups pretreated shorter than 120 min,indicating starving of oxygen.In the sealed-culture system,mouse embryos in the groups pretreated with reference gas for 120-180 min were developed better than other groups with blastocyst rates of 93.38%-95.68%,hatching rates of 61.03%-65.27%,and total cell numbers of 102.33-111.83.There were no significant differences between sealed-culture groups(120-180 min)and control in blastocyst rates and total cell numbers(P>0.05).The hatching rates in sealed-culture groups(120-180 min)were significantly higher than in control(P<0.05).【Conclusion】The optimal aeration time of reference gas containing 5%O 2,5%CO 2 and 90%N 2 pretreated to culture medium was 120 to 150 min.In the sealed-culture system,embryos could acquire high developmental rate and avoid ROS/hypoxia injury.