【作者】 唐桂娥；

【导师】 方南洙；

【作者基本信息】 延边大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 体内、外环境产生过量的活性氧可对胚胎细胞造成毒性损伤，活性氧引起的氧化损伤可导致胚胎发育阻滞，本实验研究活性氧在胚胎体外发育阻滞中的作用。以昆明小鼠的早期胚胎为研究对象，以mCZB培养液为基础培养液，用2’，7’-二氯荧光素双乙酸盐（DCFH-DA）和氢化溴乙非锭（HE）两种染料测定胚胎内部的活性氧（ROS）含量，来探讨活性氧（ROS）与小鼠胚胎体外发育阻滞之间的关系。利用2’，7’-二氯荧光素双乙酸盐（DCFH-DA）来测定过氧化氢（H₂O₂）的含量，以此对小鼠体内和体外不同发育阶段胚胎内部H₂O₂的含量以及相同发育阶段H₂O₂的含量进行比较。我们测量的结果是体内2-细胞早期、2-细胞晚期、4-细胞期、桑椹胚、囊胚胚胎的DCF荧光密度分别是：31.33，32.60，39.55，28.73和25.79，；体外2-细胞早期、2-细胞晚期、4-细胞期、桑椹胚、囊胚胚胎的DCF荧光密度分别是：38.56，45.49，41.13，30.2和29.21。通过结果分析可知：体外培养胚胎中的H₂O₂含量从2-细胞早期到2-细胞晚期之间是逐渐增加的，然而从2-细胞晚期以后又逐渐下降；通过SPSS统计软件分析可知，体外培养的2-细胞早期和晚期胚胎中的H₂O₂含量与体内相同发育阶段的胚胎相比差异显著，而4-细胞期，桑椹胚，囊胚差异不显著。利用氢化溴乙非锭（HE）测定小鼠体内和体外不同发育阶段胚胎内部超氧阴离子(O^2-)的含量。我们测量的结果是体内2-细胞早期、2-细胞晚期、4-细胞期、桑椹胚、囊胚胚胎的EB荧光密度分别是52.92，50.77，48.84，39.96和30.36；体外2-细胞早期、2-细胞晚期、4-细胞期、桑椹胚、囊胚胚胎的EB荧光密度分别是74.85，82.62，82.98，81.28和54.09。体内胚胎的O^2-含量从2-细胞早期到囊胚是逐渐减少的。通过SPSS统计软件分析可知，体内2-细胞早期、2-细胞晚期和4-细胞期与囊胚和桑椹胚之间差异显著，且桑椹胚与囊胚之间差异也显著。体外培养的2-细胞早期，2-细胞晚期，4-细胞期胚胎和桑椹胚中的EB荧光密度较高，且它们之间差异不显著，但它们与囊胚均差异显著。比较相同发育阶段体内和体外的胚胎，它们之间均差异显著。所以我们得出结论：1：体内胚胎中的ROS含量低于体外培养胚胎中的ROS含量；2：体外培养胚胎中H₂O₂含量在2-细胞晚期最高，这可能是导致小鼠2-细胞期胚胎发育阻滞的原因。更多还原

【Abstract】 The excessive Reactive Oxygen Species （ROS） produced in vivo and in vitro conditions have a toxic effect on embryo. The Oxidative damage induced by ROS may result in development block of embryo. We investigate into the effect of ROS on the development block of embryo in vitro. We detect ROS levels within embryos using 2’,7’-dichlorodihydrofluorescein diacetate （DCFH-DA） or hydroethidine （HE）, and search after the relation between ROS and development block of Kunming mouse embryos in vivo and which cultured in mCZB in vitro in the experiments. We compare H₂O₂ levels within embryos on the different stages with on the same stages using DCFH-DA in vivo and in vitro. The DCF fluorescence density of early 2-cell stage, late 2-cell stage, 4-cell stage, morula and blastula are 31.33, 32.60, 39.55, 28.73 and 25.79 in vivo, respectively, and are 38.56, 45.49, 41.13, 30.2 and 29.21 on the same stages in vitro, respectively. The result suggests that H₂O₂ levels rise gradually when cultured in vitro from early 2-cell stage to late 2-cell stage. However, H₂O₂ levels decrease gradually after late 2-cell stage. There are significantly differences on H₂O₂ levels between embryos in vivo and cultured in vitro on the early 2-cell stage and late 2-cell stage, but there are no significantly differences among embryos those on the 4-cell stage, morula and blastula using SPSS statistics software. We also detect superoxide anion (O^2-) levels within embryos on the different stages in vivo and in vitro using HE. The EB fluorescence density of early 2-cell stage, late 2-cell stage, 4-cell stage, morula and blastula are52.92, 50.77, 48.84, 39.96 and 30.36 in vivo, respectively, and are 74.85, 82.62, 82.98, 81.28 and 54.09 on the same stages in vitro, respectively. The result suggests that O^2- levels within embryos decrease gradually from early 2-cell stage to blastula in vivo. There are significantly differences on O^2- levels between embryos on the early 2-cell stage, late 2-cell stage, 4-cell stage and morula, blastula, furthermore there are significantly differences between morula and blastula in vivo, the EB fluorescence density are higher among early 2-cell stage, late 2-cell stage, 4-cell stage and morula in vitro, but there are no significantly differences among those embryos, nevertheless there are significantly differences when compare with blastula, in addition, there are significantly differences among embryos on the same stage in vivo and in vitro using SPSS statistics software. So we conclude that 1) ROS levels within embryos are lower in vivo than those cultured in vitro, 2)更多还原