| 摘要: |
| 【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% O2、5% CO2、90% N2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% O2、5% CO2、90% N2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。 |
| 关键词: 氧自由基 缺氧诱导因子-1α 密闭培养 小鼠胚胎 |
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| 基金项目:中国科学院战略性先导科技专项(XDA04020222) |
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| Effects of reference gas aeration time to culture medium on mouse 4 cell embryo development in sealed-culture system |
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LIU Jie,WANG Dandan,CHEN Fang,et L
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| Abstract: |
| 【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% O2,5% CO2 and 90% N2 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-1α was 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% O2,5% CO2 and 90% N2 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. |
| Key words: reactive oxygen species hypoxia inducible factors-1α sealed-culture mouse embryo |
