摘要：
The sealed culture of embryos is the fundation of space embryonic development study.In this special culture system,appropriate oxygen content in culture medium is essential to the development of sealed culture embryos.The purpose of present study is to research the influence of two reference gas containing different percentage of O2 and two different aeration time on mouse(Mus rnusculus) 2-cell embryos developmental competence in sealed culture system.We firstly aerated culture medium with high purity reference gas(5％ O2,5％ CO2,90％ N2 or 7.5 ％ O2,5 ％ CO2,87.5 ％ N2) for 120 or 150 min,respectively.Control groups were the embryos cultured in medium of no gas aeration and micro-drop under conventional open condition.Peroxide accumulation in cultured embryos was detected after culture for 24 and 48 h; The expression of hypoxia-inducible factor-1α (HIF-1α) in embryos was detected after culture for 96 and 72 h.Furthermore,the blastocyst rate and hatching rate were statistical analyzed and the total cell number of blastocyst were counted after culture for 72 and 96 h.As the results showed,the peroxide production could be detected in embryos cultured for 24 h in medium aerated with 7.5％ O2 for 120 and 150 min.The expression of HIF-1α was detectable after culture for 96 h in medium aerated with 5％ O2 for 120 min,150 min and 7.5％ O2for 120 min group,while the accumulation of HIF-1 α in no gas group was detectable since cultered for 48 h.Mouse 2-cell embryo could grow well with a pretty high blastocyst rate and hatching rate in 5％ O2 aeration 120 min,5％ O2 aeration 150 min,7.5％ O2 aeration 120 min and 7.5％ O2 aeration 150 min group.The blastocyst rate of mouse 2-cell embryo cultured for 72 h with 5％ O2 aeration 120 min(92.63±0.89)％ was higher than the other three aeration sealed culture group,but no significant difference showed(P＞0.05).The blastocyst rate of no gas group(57.04± 10.04)％ was significantly lower than each aeration sealed culture group(P＜0.05).The blastocyst rate of micro-drop culture group(98.67± 1.33)％ was significantly higher than 7.5％ O2 aeration 120 min group((87.15±2.35)％,P＜0.05).After culture for 96 h,the blastocyst and hatching rate of each aeration sealed culture group and micro-drop culture group had no significant difference(P＞0.05),but all significantly higher than no gas group(P＜0.05).The total cell number of blastocyst of each aeration sealed culture group cultured for 72 h had no significant difference(P＞0.05).The total cell number of blastocyst in 5％ O2 aeration 120 min group(114.12±3.66) at 96 h was significantly higher than other aeration group and no gas group(P＜ 0.05),but there was no significant difference between micro-drop culture group(110.56±5.24,P＞0.05).Taken together,we could conclude that aerating embryo culture medium with high purity reference gas containing 5％ O2,5％ CO2,90％ N2 for 120 ～150 min can support mouse 2-cell embryos developing to blastocyst and hatching in sealed culture system.This study determines the appropriate proportion of O2 in reference gas and appropriate aeration time for sealed culture of mouse 2-cell embryos,and perfects the sealed culture system,and accumulates data for the establishment of a suitable culture system for space embryonic development study.%胚胎密闭培养是空间胚胎发育研究的基本条件,在胚胎密闭培养中,培养液适宜的氧含量对早期胚胎发育至关重要.本研究采用两种氧气比例不同的标准气和两个充气时长,对胚胎培养液进行标准气充气,研究标准气的氧气比例和培养液充气时间对密闭培养小鼠(Mus musculus)2-细胞胚胎体外发育的影响.胚胎密闭培养前,使用由5％O2,5％CO2,90％N2或7.5％O2,5％CO2,87.5％N2组成的高纯标准气对胚胎培养液分别充气120或150 min,以不充气密闭培养和常规微滴培养为对照组.在培养开始后24和48 h检测胚胎过氧化物；培养96和72 h时检测胚胎缺氧诱导因子-1 α(hypoxia-inducible factor-1α,HIF-1α);培养72和96 h时统计囊胚发育率和孵化率,并进行囊胚细胞计数.结果显示,利用氧气比例为7.5％的标准气对胚胎培养液充气120 min组和充气150 min组的胚胎在培养24 h时能够检测出过氧化物累积；5％O2标准气充气120min,5％O2标准气充气150 min和7.5％O2标准气充气120 min组胚胎,在培养96 h时可检测到HIF-1α阳性,而不充气密闭培养组在培养48 h时即可检测到HIF-1α阳性；5％O2标准气充气120或150 min,7.5％O2标准气充气120或150 min时,密闭培养胚胎均能获得较理想的囊胚发育率和孵化率.培养72 h时,5％O2标准气充气120 min组的囊胚发育率(92.63±0.89)％高于其他3个充气密闭培养组,但无统计学差异(P＞0.05),不充气密闭培养组囊胚发育率(57.04±10.04)％显著低于各充气密闭培养组(P＜0.05),微滴培养组囊胚发育率(98.67±1.33)％显著高于7.5％O2的标准气充气120 min组((87.15±2.35)％,P＜o.05).培养96 h时,各充气密闭培养组及微滴培养组囊胚发育率和囊胚孵化率无显著差异(P＞0.05),但均显著高于不充气密闭培养组(P＜0.05).各充气密闭培养组胚胎体外培养72h后,囊胚细胞数差异不显著(P＞0.05).培养96 h时,5％O2标准气充气120 min组密闭培养胚胎的囊胚细胞数(114.12±3.66)显著高于其他充气密闭培养组和不充气密闭培养组(P＜0.05),与微滴组(110.56±5.24)无统计学差异(P＞0.05).研究结果表明,使用由5％O2,5％ CO2和90％N2组成的标准气对胚胎培养液持续充气120～150min时,可较好地支持密闭培养小鼠2-细胞胚胎发育至囊胚阶段,并完成囊胚的孵化.本研究确定了小鼠2-细胞胚胎密闭培养适宜的标准气O2比例和充气时间,完善了胚胎密闭培养体系,为建立适宜于小鼠早期胚胎空间发育研究的密闭培养体系积累基础资料.
