密闭培养条件对小鼠早期胚胎发育及胚胎Igf2/H19印迹调控区甲基化影响的研究
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摘要
空间胚胎发育研究已成为空间生命科学研究的重要内容之一,而胚胎密闭培养是空间胚胎发育研究的基本条件。由于气密和液密的限制,使密闭培养必须选择最适的哺乳动物早期胚胎长期体外培养的密度条件、气相条件、培养液成分等。因此能够满足胚胎发育的营养物质和代谢废物平衡的适宜的胚胎培养密度(培养胚胎数和培养液体积之比)成为首要研究内容之一。其次由于小鼠早期胚胎对环境的敏感性以及胚胎中与发育相关基因的表观遗传模式易受发育环境影响而改变,特别是Igf2/H19印迹调控区(Imprinting Control Region,ICR)的甲基化印迹容易受到体外操作和体外培养条件的影响而发生变化。因此本研究主要围绕密闭培养条件对小鼠2-细胞胚胎发育和Igf2/H19 ICR甲基化水平的影响进行初步探讨,并对密闭培养体系的优化进行进一步的深入研究和探索,从而为哺乳动物胚胎空间生物学研究提供新的研究手段和地基数据资料。
1.在本研究设立的充有5%O2,5%CO2,90%N2高纯标准气于200μL的CZB培养液的密闭培养条件下,分析在培养胚胎数:培养液体积为1:4、1:2、1:1的3个密度条件下小鼠2-细胞胚胎分别进行4个时间点(24 h, 48 h, 72 h, 96 h)培养的胚胎发育率,以相应培养密度的常规体外培养各期胚胎发育率作为评定密闭培养条件的标准。本研究证实密闭培养和常规体外培养条件下,1:2的培养密度都比1:1和1:4更适合小鼠2-细胞胚胎培养;而且虽然密闭培养的胚胎发育率和发育速度都低于常规体外培养,但是培养96 h的孵化胚发育率几乎相同(75.1%,78.1%),可见该密闭培养条件可以用于小鼠2-细胞胚胎体外培养。
2.应用亚硫酸氢盐PCR测序法(BSP)分析密闭培养条件下小鼠2-细胞胚胎分别培养24 h、48 h、72 h时相对应的8-细胞胚胎、桑葚胚、囊胚中Igf2/H19印记调控区(ICR)的甲基化水平,以常规体外培养条件下相应胚胎的甲基化水平作为对照,以体内发育的相应胚胎的甲基化水平作为标准。本研究证实密闭培养条件下各胚胎期Igf2/H19 ICR的甲基化水平都低于常规体外培养的结果,更低于体内发育的结果,可见密闭培养会引起小鼠早期胚胎Igf2/H19 ICR的甲基化水平降低。同时证实Igf2/H19 ICR的甲基化水平可以作为监测哺乳动物早期胚胎发育状况的一个分子指标。
The development study of embryos in space has become an important part in the research of life in space. Embryo cultured in sealed is the basic condition for the embryo development studies in space. As the gas-tight and liquid-tight restrictions, the sealed-culture should choose the most suitable density conditions, gas phase, medium composition et al, which could maintain long-term cultured for mammalian early embryos. Therefore the appropriate density of embryos cultured (embryo number/ culture medium volume), which will meet the balance between the nutritional and metabolic waste in the development of embryos, is the primary research topic. Second, because the sensitivity of mouse early embryos to the environment and the epigenetics model of genes related to development are easily changed with the development environment, especially the methylation imprinting of Igf2/H19 imprinting control region are easy to change for the impaction of manipulation in vitro and the culture condition in vitro. So this main study focus to approach initially the influence of the development of 2-cell mouse embryos and the methylation levels of Igf2/H19 imprinting control region under sealed culture condition, and to study and explorate the optimal condition of sealed culture system for further in-depth, so as to supply the new research tools and the mew data on earth for the space biology study of mammalian.
1. On the condition of sealed culture set in the study which is composed by 200μL CZB culture medium filled the high pure reference gas of 5%O2, 5%CO2, 90%N2, analysis the development rates of 2-cell mouse embryos respectively cultured for the four time point (24 h, 48 h, 72 h, 96 h), under the 3 different densities that embryo numbers : culture medium volumes is respective 1:4, 1:2, 1:1, and using the development rates of the corresponding stages embryos by general culture in vitro as a standard. The study confirm that the culture destiny of 1:2 is much fitter for 2-cell mouse embryos, comparing 1:1 and 1:4; meanwhile, the development rates and speeds of embryos in sealed culture are lower than the results of embryos in general culture, but the development rates of hatching embryos are almost same(75.1%,78.1%), so this sealed culture condition could be used to culture 2-cell mouse embryos in vitro.
2. Using the bisulfite sequencing PCR (BSP) to analysis the methylation levels of Igf2/H19 ICR in 8-cell embryos, morula and blastocyst, these embryos are respectively come from 2-cell mouse embryos after cultured 24 h, 48 h and 72 h in sealed culture, using the methylation levels of the corresponding stages embryos after general culture in vitro as control and the methylation levels of the corresponding stages embryos in vivo as a standard. The study confirmed that the methylation levels of Igf2/H19 ICR of all stages embeyos in sealed culture condition all were lower than the results of general cultured in vitro, and significantly lower than the results of development in vivo, so the sealed culture could make the methylation levels of Igf2/H19 imprinting control region of mouse preimplantation embryos decreased. And the study confirmed that the methylation level of Igf2/H19 imprinting control region could be as a molecular target to detect the development status of the mammal early embryos.
1.在本研究设立的充有5%O2,5%CO2,90%N2高纯标准气于200μL的CZB培养液的密闭培养条件下,分析在培养胚胎数:培养液体积为1:4、1:2、1:1的3个密度条件下小鼠2-细胞胚胎分别进行4个时间点(24 h, 48 h, 72 h, 96 h)培养的胚胎发育率,以相应培养密度的常规体外培养各期胚胎发育率作为评定密闭培养条件的标准。本研究证实密闭培养和常规体外培养条件下,1:2的培养密度都比1:1和1:4更适合小鼠2-细胞胚胎培养;而且虽然密闭培养的胚胎发育率和发育速度都低于常规体外培养,但是培养96 h的孵化胚发育率几乎相同(75.1%,78.1%),可见该密闭培养条件可以用于小鼠2-细胞胚胎体外培养。
2.应用亚硫酸氢盐PCR测序法(BSP)分析密闭培养条件下小鼠2-细胞胚胎分别培养24 h、48 h、72 h时相对应的8-细胞胚胎、桑葚胚、囊胚中Igf2/H19印记调控区(ICR)的甲基化水平,以常规体外培养条件下相应胚胎的甲基化水平作为对照,以体内发育的相应胚胎的甲基化水平作为标准。本研究证实密闭培养条件下各胚胎期Igf2/H19 ICR的甲基化水平都低于常规体外培养的结果,更低于体内发育的结果,可见密闭培养会引起小鼠早期胚胎Igf2/H19 ICR的甲基化水平降低。同时证实Igf2/H19 ICR的甲基化水平可以作为监测哺乳动物早期胚胎发育状况的一个分子指标。
The development study of embryos in space has become an important part in the research of life in space. Embryo cultured in sealed is the basic condition for the embryo development studies in space. As the gas-tight and liquid-tight restrictions, the sealed-culture should choose the most suitable density conditions, gas phase, medium composition et al, which could maintain long-term cultured for mammalian early embryos. Therefore the appropriate density of embryos cultured (embryo number/ culture medium volume), which will meet the balance between the nutritional and metabolic waste in the development of embryos, is the primary research topic. Second, because the sensitivity of mouse early embryos to the environment and the epigenetics model of genes related to development are easily changed with the development environment, especially the methylation imprinting of Igf2/H19 imprinting control region are easy to change for the impaction of manipulation in vitro and the culture condition in vitro. So this main study focus to approach initially the influence of the development of 2-cell mouse embryos and the methylation levels of Igf2/H19 imprinting control region under sealed culture condition, and to study and explorate the optimal condition of sealed culture system for further in-depth, so as to supply the new research tools and the mew data on earth for the space biology study of mammalian.
1. On the condition of sealed culture set in the study which is composed by 200μL CZB culture medium filled the high pure reference gas of 5%O2, 5%CO2, 90%N2, analysis the development rates of 2-cell mouse embryos respectively cultured for the four time point (24 h, 48 h, 72 h, 96 h), under the 3 different densities that embryo numbers : culture medium volumes is respective 1:4, 1:2, 1:1, and using the development rates of the corresponding stages embryos by general culture in vitro as a standard. The study confirm that the culture destiny of 1:2 is much fitter for 2-cell mouse embryos, comparing 1:1 and 1:4; meanwhile, the development rates and speeds of embryos in sealed culture are lower than the results of embryos in general culture, but the development rates of hatching embryos are almost same(75.1%,78.1%), so this sealed culture condition could be used to culture 2-cell mouse embryos in vitro.
2. Using the bisulfite sequencing PCR (BSP) to analysis the methylation levels of Igf2/H19 ICR in 8-cell embryos, morula and blastocyst, these embryos are respectively come from 2-cell mouse embryos after cultured 24 h, 48 h and 72 h in sealed culture, using the methylation levels of the corresponding stages embryos after general culture in vitro as control and the methylation levels of the corresponding stages embryos in vivo as a standard. The study confirmed that the methylation levels of Igf2/H19 ICR of all stages embeyos in sealed culture condition all were lower than the results of general cultured in vitro, and significantly lower than the results of development in vivo, so the sealed culture could make the methylation levels of Igf2/H19 imprinting control region of mouse preimplantation embryos decreased. And the study confirmed that the methylation level of Igf2/H19 imprinting control region could be as a molecular target to detect the development status of the mammal early embryos.
引文
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