胚胎发育

Porcine was acted as the experimental animal. It was researched on problem of the origin of obtaining early embryonic cells, methods of separating blastomere cells, best parameter and order of ECNT embryo electric activation and system of ECNT embryo IVC, which was to find a scientific method of fitting ECNT and a procedure of fitting the lab producing embryos largely, optimize the procedure of ECNT and improve the ECNT embryo in vitro.

本实验以猪为实验动物，对猪胚胎细胞核移植供体的来源、卵裂球细胞分离方法、核移植重建胚的电激活最佳参数、核移植受体的激活时序以及核移植重建胚体外培养系统相关问题进行了研究，旨在探索一种适用于猪胚胎细胞核移植的科学方法，提高猪胚胎细胞核移植胚胎的体外发育率，优化胚胎细胞核移植程序。

At the same time,there were not marked difference in the numbers of biastomere and mitotic index of control group and all treatment groups, suggesting that 2 -celt embryos were not only most sensitive to exogenous H2O2 but also rasiest to occur to development block.

但是经H2O2处理之后，度过2-细胞期发育阻滞的胚胎能在以后的发育中发生补偿性生长，其胚细胞数和分裂指数都和体外正常发育的胚胎没有差异。

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。

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

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，在囊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。

The three types of reconstructed embryos(reconstructed embryo of goat-rabbit, goat-bovine and goat-goat) were produced respectively by nuclear transfer using goat ear firbroblast cells as donors and rabbit, bovine and goat ooctyes as recipients, and the factors of cytoplast environments influencing cloned embryo development in vitro, cytoplast effect in nuclear transfer, the methods of transferring cloned embryo and the effects of pregnancy factor on implantation of cloned embryo were studied in this experiment, in order to supply basis for solving the difficulty of implantation of inter-species cloned embryo in recipient and improving the efficiency of cloning.

本试验以波尔山羊耳成纤维细胞为供体，分别以牛、羊、兔的卵母细胞为受体进行体细胞核移植，构建了山羊-兔、山羊-牛和山羊-山羊三种重构胚。研究了不同的胞质环境对重构胚体外发育的影响与核移植中的胞质效应，并对异种克隆胚胎采用了去透明带移植和配种后再移植的方法，探讨移植方法和妊娠因子对异种克隆胚胎在受体动物体内发育的影响，旨在为解决异种克隆胚移植不易着床发育的难题和为提高动物克隆效率提供理论依据。

There is increasing evidence that development of preimplantation embryo is regulated by maternally and embryonically derived growth factors.

大量研究证据表明，许多生长因子和受体存在于早期胚胎和雌性生殖道中，哺乳动物早期胚胎的发育受到母源和胚胎自身生长因子的调节，生长因子以旁分泌、自分泌和内在分泌的方式作用于胚胎，调节胚胎细胞的功能。

Previous studies have demonstrated that primary spermatocyte can undergo meiotic divisions in MI oocytes and the resulting embryos can develop to term. However, the success rate is very poor.

已经证明初级精母细胞在显微融入MI卵母细胞后可以完成减数分裂并且产生的胚胎可以发育到期，只是发育率还非常低，作为一种形式的核移植，细胞周期的同步化处理对于胚胎的发育具有重要意义。

The results of study show:1. White pulp and red pulp in parenchyma of spleen of embryo can be obviously discerned after 18 days. Periarterial lymphoid sheath and ellipsoid periarterial lymphoid sheath also can be obviously discerned in spleen of 4 days chicken. T, B lymphocytes in appendix basement of embryo emerge after 20 days. It is the initial shape of cecal tonsil. Crypt structure of conjunction of esophago and stomachus glandularis form obviously at 4 days. It is the initial shape of esophago tonsil. The germinal center firstly emerges in these three organs at 14 days. With the increase of day age, the characteristic structure peripheral immune organs gradually develop mature. Spleen achieve mature at 21 days and cecal tonsil at 35 days.2. IgM~+ and IgA~+ cells in spleen of embryo emerge at 15 days. IgG~+ cell, CD3~+ and CD8~+T lymphocytes of embryo emerge at 20 days. CD3~+, CD8~+ and IgM~+ cells in cecal tonsil of embryo emerge at 20 days. However CD4~+, IgG~+ and IgA~+ cells all emerge in 1 day age chicken out of crust. CD3~+, CD4~+, CD8~+, IgM~+ and IgG~+ cells in esophago tonsil of embryo all emerge at 20 days. However IgA~+ cells emerge in 1 day age chicken out of crust.3. The amount of T, B lymphocytes in peripheral immune organs increase follow with the increase of day age, and hold an upgrade tendency. The amount of T, B lymphocytes in spleen achieved stabilization at 21 days, and in tonsil of esophago and appendix at 35 days.

研究结果表明：1、在组织结构方面，脾脏实质内的白髓与红髓在胚胎18日龄后明显可辨，4日龄雏鸡脾脏中形成明显可辨的动脉周围淋巴鞘和椭球周围淋巴鞘；盲肠基部T、B淋巴细胞在胚胎20日龄时开始出现，即初步形成盲肠扁桃体；食管与腺胃结合处在4日龄时形成明显的隐窝结构，即食管扁桃体初步形成；14日龄时，三种器官中首次出现生发中心；随着日龄的增长，外周免疫器官特征结构不断发育成熟，脾脏在21日龄时达到成熟水平，盲肠扁桃体和食管扁桃体在35日龄时达到成熟水平。2、在T、B淋巴细胞出现时间方面，脾脏中IgM~+和IgA~+细胞在胚胎15日龄时开始出现，IgG~+细胞、CD3~+和CD8~+T淋巴细胞在胚胎18日龄时出现，CD4~+细胞在胚胎20日龄时出现；盲肠扁桃体中CD3~+、CD8~+和IgM~+细胞在胚胎20日龄时开始出现，而CD4~+、IgG~+和IgA~+细胞均在雏鸡出壳后1日龄时出现；食管扁桃体中CD3~+、CD4~+、CD8~+、IgM~+和IgG~+细胞均在胚胎20日龄时开始出现，而IgA~+细胞则在雏鸡出壳后1日龄时出现。3、在T、B淋巴细胞数量变化方面，外周免疫器官中T、B淋巴细胞的数量随日龄增长，整体均呈上升趋势。

Results: NKA-I cells was first found in the duodenum mucose on the 14th day of fetus, earlier than in the jejunum ( 16th day ) and ileum ( 17th day ), and its Positive reaction and the number of cells increased with further development, after birth the 30th day, it became quite identical to that of adult rat; NKA-I cells were seen in the colon mucosa on the 17th day of fetus, later on the number kept decreasing till grown up stage; No change was observed in the stomach mucosa except that few cells showed up on the 15th day of fetus; No positive cell was found on the oesphagus wall.

结果： NKA-I细胞首先见于胚胎14 d的十二指肠粘膜，早于空肠(胚胎16 d)和回肠(胚胎17 d)，其阳性反应强度及细胞量随发育而增加，30 d时与成年鼠相似；结肠粘膜内于胚胎17 d见细胞，但此后数量较少直至成年；胃粘膜于胚胎15 d见个别细胞外，以后无明显变化；食道壁始终未见阳性细胞。

I can not make it blossom and suits me

我不能让树为我开花

When temperatures are above approximately 80 °C discolouration of the raceways or rolling elements is a frequent feature.

当温度高于 80 °C 左右时，滚道或滚动元件褪色是很常见的特征。