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Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明:利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程,在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样,呈现高度甲基化状态;在细胞核开始融合的时候,甲基化水平急速下降,在细胞核完全融合的时候甲基化水平达到最低点;随着胚胎继续分裂,胚胎甲基化水平逐渐增加,在桑葚胚期甲基化水平最高;但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别,这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cyto-plasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methy-lation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribu-tion of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明:利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程,在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样,呈现高度甲基化状态;在细胞核开始融合的时候,甲基化水平急速下降,在细胞核完全融合的时候甲基化水平达到最低点;随着胚胎继续分裂,胚胎甲基化水平逐渐增加,在桑葚胚期甲基化水平最高;但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别,这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Neuroectoderm; neural crest; neural plate; neural tube; neuraxis; neuroblasts; neural stem cells; neuroprogenitor cells; stem cells; differentiation; ontogeny; morphogenesis; histogenesis; organogenesis; synaptogenesis; gangliogenesis; embryogenesis; axonogenesis; retinogenesis; gliogenesis; glial progenitor cells; oligodendrocyte progenitor cells; retinal progenitor cells; nerve growth factor; neurotrophic factors; trophic factors; growth factors; neural tube defects; anencephaly; spina bifida; neuroblastoma cells; cell migration; neurogenesis; development; developmental stages

神经外胚层;神经脊;神经板;神经管;轴索;成神经细胞;神经干细胞;neuroprogenitor细胞;干细胞;分化;个体发生;形态发生;组织发生;器官发生;突触发生;神经节发生;胚胎发生;轴突发生;视网膜发生;神经胶质发生;神经胶质祖细胞;少突神经胶质细胞祖细胞;视网膜祖细胞;神经生长因子;神经营养因子;营养因子;生长因素;神经管缺陷;无脑;脊柱裂;成神经细胞瘤细胞;细胞迁移;神经发生;发展;发育阶段

Neuroectoderm; neural crest; floor plate; neural tube; neuraxis; neuroblasts; neural stem cells; neuroprogenitor cells; stem cells; differentiation; ontogeny; morphogenesis; histogenesis; organogenesis; synaptogenesis; gangliogenesis; embryogenesis; axonogenesis; retinogenesis; gliogenesis; glial progenitor cells; oligodendrocyte progenitor cells; retinal progenitor cells; nerve growth factor; neurotrophic factors; trophic factors; growth factors; neural tube defects; anencephaly; spina bifida; neuroblastoma cells; cell migration; neurogenesis; development; developmental stages

神经外胚层;神经脊;底板;神经管;轴索;成神经细胞;神经干细胞;神经母细胞;干细胞;分化;个体发生;形态发生;组织发生;器官发生;突触发生;神经节发生;胚胎发生;轴突发生;视网膜发生;神经胶质发生;神经胶质祖细胞;少突神经胶质细胞祖细胞;视网膜祖细胞;神经生长因子;神经营养因子;营养因子;生长因素;神经管缺陷;无脑;脊柱裂;成神经细胞瘤细胞;细胞迁移;神经发生;发展;发育阶段

Neural crest; neural plate; floor plate; neural tube; neuraxis; neuroblasts; neural stem cells; neuroprogenitor cells; stem cells; differentiation; ontogeny; morphogenesis; histogenesis; organogenesis; synaptogenesis; gangliogenesis; embryogenesis; axonogenesis; retinogenesis; gliogenesis; glial progenitor cells; oligodendrocyte progenitor cells; retinal progenitor cells; nerve growth factor; neurotrophic factors; trophic factors; growth factors; neural tube defects; anencephaly; spina bifida; neuroblastoma cells; cell migration; neurogenesis; development; developmental stages

神经脊;神经板;底板神经管;轴索;成神经细胞;神经干细胞;神经母细胞;干细胞;分化;个体发生;形态发生;组织发生;器官发生;突触发生;神经节发生;胚胎发生;轴突发生;视网膜发生;神经胶质发生;神经胶质祖细胞;少突神经胶质细胞祖细胞;视网膜祖细胞;神经生长因子;神经营养因子;营养因子;生长因素;神经管缺陷;无脑;脊柱裂;成神经细胞瘤细胞;细胞迁移;神经发生;发展;发育阶段

Neuroectoderm; neural plate; floor plate; neural tube; neuraxis; neuroblasts; neural stem cells; neuroprogenitor cells; stem cells; differentiation; ontogeny; morphogenesis; histogenesis; organogenesis; synaptogenesis; gangliogenesis; embryogenesis; axonogenesis; retinogenesis; gliogenesis; glial progenitor cells; oligodendrocyte progenitor cells; retinal progenitor cells; nerve growth factor; neurotrophic factors; trophic factors; growth factors; neural tube defects; anencephaly; spina bifida; neuroblastoma cells; cell migration; neurogenesis; development; developmental stages

神经外胚层;神经板;底板;神经管;轴索成神经细胞神经干细胞;神经母细胞;干细胞分化;个体发生;形态发生;组织发生;器官发生;突触发生;神经节发生;胚胎发生;轴突发生;视网膜发生;神经胶质发生;神经胶质祖细胞;少突神经胶质细胞祖细胞;视网膜祖细胞;神经生长因子;神经营养因子;营养因子;生长因素;神经管缺陷;无脑;脊柱裂;成神经细胞瘤细胞;细胞迁移;神经发生;发展;发育阶段

And there were no evidence changes in histopathologic analysis of brain tissues in other ETU-injected and in control rat embryos. 2. Results of In situ cell apoptosis detection In E20 cerebral cortex, more Tunel cells were found in encephalocoele group than control group,and the results were significant.

二、应用细胞凋亡原位检测法检测不同组E20胎鼠大脑皮质中细胞凋亡情况 E20胎鼠大脑皮质中Tunel阳性细胞数目在正常组、给药无畸形组及脊柱裂组细胞凋亡数无明显改变,而在脑膨出组胎鼠大脑皮质细胞凋亡数明显多于对照组,计算凋亡细胞百分率,然后用X~2检验进行分析,脑膨出组和对照组的细胞凋亡指数差异有统计学意义(P.05)。

So we selected thevinblastine optimum parameters were 0.1μg/ml and 8 hour cultured duration.The fourth, there were significantly differences percent between 10 hourgroups and other three groups in 0.1μg/ml groups; and there were no significantlydifferences between 8 hour groups and 10 hour groups, but they had significantlydifferences among other two groups in 0.3μg/ml groups; and there weresignificantly differences between 10 hour groups and other three hour groups in0.5μg/ml groups; and there were significantly differences between 10 hour groupsand other three groups in 0.7μg/ml groups on the metaphases (P<0.05), in theexperiment of the effect on the mouse 8- cell embryo stage single blastomere ofvinblastine with different concentrations and duration, by x~2 statistical analysis.

根据实验的实际情况,适宜制备小鼠4-细胞期胚胎单卵裂球染色体标本的长春花碱的处理浓度和时间是0.1μg/ml和8小时。4、在确定长春花碱不同处理浓度和时间对小鼠8-细胞期胚胎单卵裂球中期分裂相数影响的实验中,经统计分析,阻断培养0.1μg/ml浓度组中10小时组与其他三组差异显著;阻断培养0.3μg/ml浓度组中8小时组和10小时组间差异不显著,但与其他两组差异显著;阻断培养0.5μg/ml浓度组中10小时组与其他三组差异显著;阻断培养0.7μg/ml浓度组中10小时组与其他三组差异显著(P<0.05)。

So we selected the colchicine optimumparameters were 0.2μg/ml and 6 hour cultured duration.The third, there were significantly differences percent between 10 hourgroups and other three groups (4 hour groups, 6 hour groups and 8 hour groups) in0.1μg/ml groups; and there were significantly differences between 8 hour groupsand 10 hour groups, but they had no significantly differences among other twogroups in 0.3μg/ml groups; and there were significantly differences between 10hour groups and 6 hour groups, but 10 hour groups had no significantlydifferences among other two groups in 0.5μg/ml groups; and there weresignificantly differences between 8 hour groups and other three groups in0.7μg/ml groups on the metaphases (P<0.05), in the experiment of the effect onthe mouse 4- cell embryo stage single blastomere of vinblastine with differentconcentrations and duration, by x~2 statistical analysis.

根据实验的实际情况,适宜制备小鼠8-细胞期胚胎单卵裂球染色体标本的秋水仙素的处理浓度和时间是0.2μg/ml和6小时。3、在确定长春花碱不同处理浓度和时间对小鼠4-细胞期胚胎单卵裂球中期分裂相数影响的实验中,经统计分析,阻断培养0.1μg/ml浓度组中10小时组与其他三组差异显著(4小时组、6小时组和8小时组);阻断培养0.3μg/ml浓度组中8小时组和10小时组间差异不显著,但与其他两组差异显著;阻断培养0.5μg/ml浓度组中10小时组与6小时组差异不显著,而与其他两组差异显著;阻断培养0.7μg/ml浓度组中8小时组与其他三组差异显著(P<0.05)。

Sowe selected the vinblastine optimum parameters were 0.1μg/ml and 8 hourcultured duration.The fifth, there were significantly differences between 0.45% sodium chloride groups and other two groups (0.5% sodium citrate groups and 0.35%potassium chloride groups) on the metaphases (P<0.05), in the experiment of theeffect on the mouse 4-and 8-cell embryo stage single blastomere of differenthypotonic fluids, by x~2 statistical analysis.

根据实验的实际情况,适宜制备小鼠8-细胞期胚胎单卵裂球染色体标本的长春花碱的处理浓度和时间是0.1μg/ml和10小时。5、在确定不同种类低渗液处理对小鼠4、8-细胞期胚胎单卵裂球中期分裂相数影响的实验中,经统计分析,0.45%氯化钠低渗液组与其他两组(0.5%柠檬酸钠和0.35%氯化钾)差异显著(P<0.05)。

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