小鼠4细胞胚胎密闭培养体系的建立及胚胎密闭培养条件筛选研究
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摘要
作为空间生命科学重要研究内容之一,胚胎的空间发育研究一直受到国内外研究者的高度关注。但是目前所普遍采用的开放式胚胎培养体系无法在真空、失重的环境中支持早期胚胎的体外发育,因此必须选用密闭培养的方式(包括气密和液密)进行胚胎空间密闭培养。胚胎密闭培养就是在胚胎培养前预先向胚胎培养液中充入含有一定比例O_2,CO_2和N_2的标准气以提供胚胎发育的气相支持。同时,由于密闭培养的特殊性,在胚胎发育的过程中无法向体系中补充更新营养与气相成分,因此适宜的胚胎培养密度对体系能否更长时间支持胚胎体外发育至关重要。本研究通过对胚胎培养液充气标准气,胚胎培养液以及胚胎培养密度的筛选,以建立一种可支持早期胚胎体外发育的密闭培养体系,并应用于空间环境条件对胚胎发育影响的研究。
1.本研究选择了两种O_2比例不同的标准气(5% O_2,5% CO_2,90% N_2和10% O_2,5% CO_2,85% N_2)、两种营养条件和缓冲体系不同的胚胎培养液(添加10%胎牛血清的mDPBS和CZB液),并在培养液体积为200μL的前提下,选用三种培养密度(胚胎数/n∶培养液体积/μL为1:1, 1:2和1:4),进行小鼠4细胞胚胎的密闭培养,同时设置不充气培养组和常规微滴培养对照组。通过观察和分析各组胚胎在密闭培养84 h时的囊胚发育率和胚胎孵化率,判定各培养组的胚胎培养效果。
结果表明,利用5% O_2比例的标准气进行胚胎培养液充气时,mDPBS和CZB液表现出类似的胚胎培养结果。1:1培养组的囊胚发育率与对照组无显著差异(P>0.05),但胚胎孵化率显著低于相同培养密度的对照组(P<0.05);1:2培养组的囊胚发育率与胚胎孵化率与相同培养密度的对照组无显著差异(P>0.05);1:4培养组无论是囊胚发育率还是胚胎孵化率皆显著低于相同培养密度的对照组(P<0.05)。
利用10% O_2比例的标准气进行胚胎培养液充气时,mDPBS和CZB液表现出类似的胚胎培养结果。1:1培养组的囊胚发育率和胚胎孵化率与相同培养密度的对照组无显著差异(P>0.05);1:2培养组和1:4培养组的囊胚发育率和胚胎孵化率皆显著低于相同培养密度的对照组(P<0.05)。
培养液不充气密闭培养时,1:1培养组,1:2培养组和1:4培养组的囊胚发育率与胚胎孵化率皆显著低于相同培养密度的对照组。
证明,在密闭培养体系中,胚胎培养密度过低会导致胚胎发育率和胚胎孵化率下降;培养液不充气密闭培养不能支持小鼠4细胞胚胎体外发育。
2.在上述研究结果的基础上,在5% O_2比例的标准气进行胚胎培养液充气时,利用mDPBS和CZB对小鼠4细胞胚胎进行密闭培养,以筛选小鼠胚胎密闭培养的适宜培养液。结果表明,相同培养密度下在mDPBS培养液中发育的胚胎,囊胚总细胞数显著低于在CZB培养液中发育的胚胎(P<0.05),mDPBS组囊胚细胞的凋亡率显著高于CZB组(P<0.05),而且在mDPBS中发育的胚胎形态学质量较差。证明CZB培养液支持密闭培养小鼠胚胎发育的能力优于mDPBS培养液。
3.在上述研究结果的基础上,以CZB培养液为胚胎培养液,分别利用5% O_2比例和10% O_2比例的标准气进行胚胎培养液充气,比较不同培养密度下囊胚细胞总数以及囊胚细胞凋亡率的差异,并检测胚胎发育早期过氧化物产生情况和密闭培养过程中胚胎缺氧诱导因子1 alpha(HIF-1α)蛋白质表达情况。
结果显示,利用5% O_2比例的标准气进行胚胎培养液充气时,1:1培养组的囊胚细胞总数显著低于相同密度的对照组胚胎(P<0.05),凋亡率显著高于对照组(P<0.05);1:2培养组的囊胚细胞总数以及胚胎细胞凋亡率皆与相同密度对照组胚胎无显著差异(P>0.05)。利用10% O_2比例的标准气进行胚胎培养液充气时,1:1培养组的囊胚细胞总数以及胚胎细胞凋亡率皆与相同密度对照组胚胎无显著差异(P>0.05);1:2培养组的囊胚细胞总数皆显著低于相同密度的对照组胚胎(P<0.05),凋亡率显著高于对照组(P<0.05)。
在胚胎发育早期过氧化物产生情况检测时发现,利用10% O_2比例的标准气进行胚胎培养液充气,密闭培养12 h时1:1培养组和1:2培养组胚胎过氧化物产生量高于对照组,而在利用5% O_2比例的标准气进行胚胎培养液充气的密闭培养组中未见此现象。提示当标准气中O_2比例为10%时,密闭培养的胚胎在发育早期可能出现氧化损伤。
利用5% O_2比例的标准气进行胚胎培养液充气时,1:1培养组在密闭培养60 h时胚胎中可检出HIF-1α表达;1:2培养组在密闭培养84 h时胚胎中可检出HIF-1α表达。利用10% O_2比例的标准气进行胚胎培养液充气时,1:1培养组在密闭培养84 h时胚胎中可检出HIF-1α表达;1:2培养组在密闭培养84 h时胚胎中未检出HIF-1α表达。综上所述,小鼠胚胎密闭培养的适宜培养液为CZB液,将胚胎密度为1:2的小鼠4细胞胚胎密闭培养于经5% O_2标准气充气处理的培养液中,胚胎可较好地发育至囊胚和孵化胚阶段。
As one of the most important parts of the research for life in space, the study of embryo development in space has received particular attention. But the general culture system couldn’t support the embryo development in a vacuum and weightless situation. So we must choose a sealed-culture method (include gas-tight and liquid-tight) to support embryos’development in space. Sealed-culture means culture medium was treated by definite proportion reference gas with O_2, CO_2 and N_2 before embryo culture. One of the most important factors in sealed-culture system is the density of embryo in the medium because we can’t refresh gas and nutrition during the culture. This study mainly concern on the screening of reference gas, culture medium and embryo density to build a system that can support embryo development in vitro and for the research of the influence on embryo develop in space.
1. On the condition of sealed-culture set in the study, two kinds of medium (mDPBS and CZB) were chose to be culture medium and two kinds of reference gas composed of 5% O_2, 5% CO_2, 90% N_2 or 10% O_2, 5% CO_2, 85% N_2 to culture mouse 4 cell stage embryos under the liquid volume of 200μL. Through the analysis of development rates of the embryos under 3 different densities that embryo numbers : culture medium volumes were 1:1, 1:2 and 1:4 to judge the culture effect in different sealed-culture group.
When culture medium treated by the reference gas with 5% O_2 proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate in 1:1 culture group has no significant difference from the control group (P>0.05). But the hatching rate has significant lower than the control group (P<0.05). The blastosphere growth rate and the hatching rate in 1:2 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:4 culture group are significant lower than the control group (P<0.05).
When culture medium treated by the reference gas with 10% O_2 proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate and the hatching rate in 1:1 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:2 and 1:4 culture groups are significant lower than the control groups (P<0.05).
When culture medium not treated by the reference gas, the blastosphere growth rate and the hatching rate in 1:1, 1:2 and 1:4 culture groups are significant lower than the control group (P<0.05).
This result proves that embryo couldn’t develop well in sealed-culture system with low density, and embryo couldn’t develop well in the medium which not treated by the reference gas.
2. According to the result above, culture mouse 4 cell stage embryo by mDPBS and CZB when the culture medium treated by the reference gas with 5% O_2 proportion to screening suitable culture medium for sealed-culture. The result indicates that the total blastomere numbers of embryo cultured in mDPBS was significant lower than embryo cultured in CZB. Embryos The morphology of embryos cultured in mDPBS had worse than in CZB. It proves that embryos could develop better in CZB than in mDPBS.
3. According to the result above, use CZB as the culture medium, which treated by the reference gas of 5% O_2 proportion or 10% O_2 proportion, to culture the 4 cell stage embryo in vitro. Comparing the total blastomere numbers and the apoptosis rate of the embryos, then detecting the peroxide production and the express of hypoxia-inducible factor 1 alpha (HIF-1α) protein in embryos cultured with1:1 and 1:2 density to screening suitable embryo density for sealed-culture.
When culture medium treated by the reference gas with 5% O_2 proportion, the result indicates that the embryo has significant lower total blastomere numbers of 1:1 culture group than that in control group with same density (P<0.05), and apoptosis rate is significant higher than control group (P<0.05). The embryo cultured in 1:2 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). When culture medium treated by the reference gas with 10% O_2 proportion, the embryo cultured in 1:1 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). The embryo cultured in 1:2 group have significant difference of total blastomere numbers and apoptosis rate from control group with same density (P<0.05).
When detecting the peroxide production in embryos during the early period of sealed-culture., we found out that 1:1 and 1:2 culture groups have a higher production of peroxide than control group when cultured in medium treated by the reference gas with 10% O_2 proportion in 12 h. But this situation has not been found in groups cultured in medium treated by the reference gas with 5% O_2 proportion. We can infer that when culture medium treated by the reference gas with 10% O_2 proportion, embryos developed in sealed-culture system may show peroxy-injury in the early culture period.
When culture medium treated by the reference gas with 5% O_2 proportion, HIF-1αprotein express in embryos was detected in 60 h in 1:1 culture, and detected out in 84 h in1:2 culture group. When culture medium treated by the reference gas with 10% O_2 proportion, HIF-1αprotein express in embryo was detected in 60 h in 1:1 culture group, and HIF-1αprotein express was not detected in 1:2 culture group.
In conclusion, in the study of the sealed culture of mouse 4 cell stage embryo, we have determined CZB as optimal culture medium, and 1:2 density of embryos cultured in 5% O_2 reference gas treated medium as the suitable culture condition.
1.本研究选择了两种O_2比例不同的标准气(5% O_2,5% CO_2,90% N_2和10% O_2,5% CO_2,85% N_2)、两种营养条件和缓冲体系不同的胚胎培养液(添加10%胎牛血清的mDPBS和CZB液),并在培养液体积为200μL的前提下,选用三种培养密度(胚胎数/n∶培养液体积/μL为1:1, 1:2和1:4),进行小鼠4细胞胚胎的密闭培养,同时设置不充气培养组和常规微滴培养对照组。通过观察和分析各组胚胎在密闭培养84 h时的囊胚发育率和胚胎孵化率,判定各培养组的胚胎培养效果。
结果表明,利用5% O_2比例的标准气进行胚胎培养液充气时,mDPBS和CZB液表现出类似的胚胎培养结果。1:1培养组的囊胚发育率与对照组无显著差异(P>0.05),但胚胎孵化率显著低于相同培养密度的对照组(P<0.05);1:2培养组的囊胚发育率与胚胎孵化率与相同培养密度的对照组无显著差异(P>0.05);1:4培养组无论是囊胚发育率还是胚胎孵化率皆显著低于相同培养密度的对照组(P<0.05)。
利用10% O_2比例的标准气进行胚胎培养液充气时,mDPBS和CZB液表现出类似的胚胎培养结果。1:1培养组的囊胚发育率和胚胎孵化率与相同培养密度的对照组无显著差异(P>0.05);1:2培养组和1:4培养组的囊胚发育率和胚胎孵化率皆显著低于相同培养密度的对照组(P<0.05)。
培养液不充气密闭培养时,1:1培养组,1:2培养组和1:4培养组的囊胚发育率与胚胎孵化率皆显著低于相同培养密度的对照组。
证明,在密闭培养体系中,胚胎培养密度过低会导致胚胎发育率和胚胎孵化率下降;培养液不充气密闭培养不能支持小鼠4细胞胚胎体外发育。
2.在上述研究结果的基础上,在5% O_2比例的标准气进行胚胎培养液充气时,利用mDPBS和CZB对小鼠4细胞胚胎进行密闭培养,以筛选小鼠胚胎密闭培养的适宜培养液。结果表明,相同培养密度下在mDPBS培养液中发育的胚胎,囊胚总细胞数显著低于在CZB培养液中发育的胚胎(P<0.05),mDPBS组囊胚细胞的凋亡率显著高于CZB组(P<0.05),而且在mDPBS中发育的胚胎形态学质量较差。证明CZB培养液支持密闭培养小鼠胚胎发育的能力优于mDPBS培养液。
3.在上述研究结果的基础上,以CZB培养液为胚胎培养液,分别利用5% O_2比例和10% O_2比例的标准气进行胚胎培养液充气,比较不同培养密度下囊胚细胞总数以及囊胚细胞凋亡率的差异,并检测胚胎发育早期过氧化物产生情况和密闭培养过程中胚胎缺氧诱导因子1 alpha(HIF-1α)蛋白质表达情况。
结果显示,利用5% O_2比例的标准气进行胚胎培养液充气时,1:1培养组的囊胚细胞总数显著低于相同密度的对照组胚胎(P<0.05),凋亡率显著高于对照组(P<0.05);1:2培养组的囊胚细胞总数以及胚胎细胞凋亡率皆与相同密度对照组胚胎无显著差异(P>0.05)。利用10% O_2比例的标准气进行胚胎培养液充气时,1:1培养组的囊胚细胞总数以及胚胎细胞凋亡率皆与相同密度对照组胚胎无显著差异(P>0.05);1:2培养组的囊胚细胞总数皆显著低于相同密度的对照组胚胎(P<0.05),凋亡率显著高于对照组(P<0.05)。
在胚胎发育早期过氧化物产生情况检测时发现,利用10% O_2比例的标准气进行胚胎培养液充气,密闭培养12 h时1:1培养组和1:2培养组胚胎过氧化物产生量高于对照组,而在利用5% O_2比例的标准气进行胚胎培养液充气的密闭培养组中未见此现象。提示当标准气中O_2比例为10%时,密闭培养的胚胎在发育早期可能出现氧化损伤。
利用5% O_2比例的标准气进行胚胎培养液充气时,1:1培养组在密闭培养60 h时胚胎中可检出HIF-1α表达;1:2培养组在密闭培养84 h时胚胎中可检出HIF-1α表达。利用10% O_2比例的标准气进行胚胎培养液充气时,1:1培养组在密闭培养84 h时胚胎中可检出HIF-1α表达;1:2培养组在密闭培养84 h时胚胎中未检出HIF-1α表达。综上所述,小鼠胚胎密闭培养的适宜培养液为CZB液,将胚胎密度为1:2的小鼠4细胞胚胎密闭培养于经5% O_2标准气充气处理的培养液中,胚胎可较好地发育至囊胚和孵化胚阶段。
As one of the most important parts of the research for life in space, the study of embryo development in space has received particular attention. But the general culture system couldn’t support the embryo development in a vacuum and weightless situation. So we must choose a sealed-culture method (include gas-tight and liquid-tight) to support embryos’development in space. Sealed-culture means culture medium was treated by definite proportion reference gas with O_2, CO_2 and N_2 before embryo culture. One of the most important factors in sealed-culture system is the density of embryo in the medium because we can’t refresh gas and nutrition during the culture. This study mainly concern on the screening of reference gas, culture medium and embryo density to build a system that can support embryo development in vitro and for the research of the influence on embryo develop in space.
1. On the condition of sealed-culture set in the study, two kinds of medium (mDPBS and CZB) were chose to be culture medium and two kinds of reference gas composed of 5% O_2, 5% CO_2, 90% N_2 or 10% O_2, 5% CO_2, 85% N_2 to culture mouse 4 cell stage embryos under the liquid volume of 200μL. Through the analysis of development rates of the embryos under 3 different densities that embryo numbers : culture medium volumes were 1:1, 1:2 and 1:4 to judge the culture effect in different sealed-culture group.
When culture medium treated by the reference gas with 5% O_2 proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate in 1:1 culture group has no significant difference from the control group (P>0.05). But the hatching rate has significant lower than the control group (P<0.05). The blastosphere growth rate and the hatching rate in 1:2 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:4 culture group are significant lower than the control group (P<0.05).
When culture medium treated by the reference gas with 10% O_2 proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate and the hatching rate in 1:1 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:2 and 1:4 culture groups are significant lower than the control groups (P<0.05).
When culture medium not treated by the reference gas, the blastosphere growth rate and the hatching rate in 1:1, 1:2 and 1:4 culture groups are significant lower than the control group (P<0.05).
This result proves that embryo couldn’t develop well in sealed-culture system with low density, and embryo couldn’t develop well in the medium which not treated by the reference gas.
2. According to the result above, culture mouse 4 cell stage embryo by mDPBS and CZB when the culture medium treated by the reference gas with 5% O_2 proportion to screening suitable culture medium for sealed-culture. The result indicates that the total blastomere numbers of embryo cultured in mDPBS was significant lower than embryo cultured in CZB. Embryos The morphology of embryos cultured in mDPBS had worse than in CZB. It proves that embryos could develop better in CZB than in mDPBS.
3. According to the result above, use CZB as the culture medium, which treated by the reference gas of 5% O_2 proportion or 10% O_2 proportion, to culture the 4 cell stage embryo in vitro. Comparing the total blastomere numbers and the apoptosis rate of the embryos, then detecting the peroxide production and the express of hypoxia-inducible factor 1 alpha (HIF-1α) protein in embryos cultured with1:1 and 1:2 density to screening suitable embryo density for sealed-culture.
When culture medium treated by the reference gas with 5% O_2 proportion, the result indicates that the embryo has significant lower total blastomere numbers of 1:1 culture group than that in control group with same density (P<0.05), and apoptosis rate is significant higher than control group (P<0.05). The embryo cultured in 1:2 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). When culture medium treated by the reference gas with 10% O_2 proportion, the embryo cultured in 1:1 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). The embryo cultured in 1:2 group have significant difference of total blastomere numbers and apoptosis rate from control group with same density (P<0.05).
When detecting the peroxide production in embryos during the early period of sealed-culture., we found out that 1:1 and 1:2 culture groups have a higher production of peroxide than control group when cultured in medium treated by the reference gas with 10% O_2 proportion in 12 h. But this situation has not been found in groups cultured in medium treated by the reference gas with 5% O_2 proportion. We can infer that when culture medium treated by the reference gas with 10% O_2 proportion, embryos developed in sealed-culture system may show peroxy-injury in the early culture period.
When culture medium treated by the reference gas with 5% O_2 proportion, HIF-1αprotein express in embryos was detected in 60 h in 1:1 culture, and detected out in 84 h in1:2 culture group. When culture medium treated by the reference gas with 10% O_2 proportion, HIF-1αprotein express in embryo was detected in 60 h in 1:1 culture group, and HIF-1αprotein express was not detected in 1:2 culture group.
In conclusion, in the study of the sealed culture of mouse 4 cell stage embryo, we have determined CZB as optimal culture medium, and 1:2 density of embryos cultured in 5% O_2 reference gas treated medium as the suitable culture condition.
引文
陈海燕. 2010.密闭培养条件对小鼠早期胚胎发育及胚胎中Igf2/H19印迹调控区甲基化影响的研究[硕士学位论文].杨凌:西北农林科技大学
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