用单层分化法进行胚胎干细胞神经分化的研究

用单层分化法研究了 ES细胞从 0～ 316 h(第 14天 )的神经分化过程。无饲养层培养的 ES细胞在无血清的N2 B2 7培养基中开始神经分化。从 72 h开始 ,神经外胚层的特异性转录因子 Sox1表达 ,在克隆的边缘逐渐出现向外迁移的细胞 ,形状呈圆形或椭圆形 ,有 1～ 2个细胞突起。在 191h(第 8天 )之后 ,细胞可迁移至克隆边缘的 2 0 0 μm之外 ,这些有细长突起的细胞表达神经元特异性蛋白 Tau。以后 ,迁移细胞的突起开始交叠成网状。 316 h(第 14天 )时 ,细长的纤维可跨越在不同的克隆之间 ,长达上千微米。此时 ,免疫染色的结果表明 ,在克隆的中央有较多的圆形 Nestin阳性细胞 ,而克隆的边缘有大量的 β- tubulin 阳性细胞 ,它们交织成复杂的网状图像。阶段性表达基因的表达时序研究表明 ,ES细胞的特异性转录因子 Oct4开始减弱时 ,Sox1表达开始 ,此时细胞由全能状态进入早期神经分化状态 ;Tau的表达是 Sox1表达的下游事件。这一时序与胚胎发育中神经发生的时序相符。单层分化法能直观地展示 ES细胞的神经分化过程 ,可作为研究细胞分化、胚胎发育及药物筛选的体外模型     

胚胎干细胞及种系嵌合体的研究进展

胚胎干细胞是着床前的囊胚内细胞团或早期胎儿的原始生殖细胞经体外分化抑制培养建立的多能性细胞系 ,具有与胚胎细胞相似的形态特征和分化潜能 ,体外培养时保持未分化状态 ,可以传代增殖。改变维持胚胎干细胞不分化的培养条件 ,胚胎干细胞可自发分化成多细胞结构。在一定诱导下 ,胚胎干细胞可向多个方向分化 ,并生成多种功能细胞。胚胎干细胞注入到胚泡期胚胎或与桑椹期胚胎聚合 ,可以参与包括性腺在内的各种组织的嵌合体的形成。胚胎干细胞在细胞分化与调控 ,胚胎发育 ,遗传病 ,肿瘤 ,免疫和组织或器官移植等研究中显示着广泛的应用前景。而种系嵌合体的获得是实现 ES细胞途径的决定步骤 ,低的种系嵌合率则是制约 ES细胞应用的关键。提高供体 PGCs在受体生殖腺中的比例 ,缩短 ES细胞的体外培养时间 ,以及注入早期发育阶段的受体胚胎等都能提高种系嵌合率。文章从多个方面综述了胚胎干细胞的最新研究成果 ,并着重以禽类 ES细胞为例论述了种系嵌合体的检测方法 ,种系嵌合率的影响因素以及提高种系嵌合率的方法     

哺乳动物附植前胚胎的基因表达调控

哺乳动物胚胎附植前期包括:合子的形成,胚胎基因组的激活和细胞分化的开始。在这个时期,发育由母源物质控制转为合子基因控制,在此过程,同时形成染色质介导的转录抑制时期,要解除抑制必须经过胚胎基因组的激活。通过对体内、外附植前胚胎的mRNA的表达特点以及它们与成功发育联系的研究,可以筛选出最佳的体外培养条件,设计最佳的核移植方案。     

猪胚胎死亡的影响因素及其预防措施

猪胚胎死亡直接影响着母猪的产仔数，并进而影响着养猪生产的经济效益。因此，在深入研究猪胚胎死亡规律及其影响因素的基础上，通过有效的措施降低胚胎死亡率，对提高母猪窝产仔数和经济效益具有十分重要的意义。本文综述了影响猪胚胎死亡的遗传、环境、营养和疾病等因素，并对生产实践中如何通过综合预防措施降低猪胚胎死亡率进行了初步探讨。     

高海拔地区鸡胚发育关键因素研究

对藏鸡(T)、饲养在北京的藏鸡(TB)、矮小隐性白(D)、农大小型蛋鸡商品代种蛋(M)、寿光鸡(S)、藏鸡×矮小隐性白(TD)、藏鸡×寿光鸡(TS)7组种蛋在西藏林芝进行孵化研究,通过阶段死亡率、出雏率、失水率数据分析了影响高海拔地区胚胎发育的关键因素。结果表明胚胎本身的高原生活力是鸡蛋高海拔孵化主要因素,其次是蛋壳传导力。     

Melatonin reduces apoptotic cells,SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts

Effects of adding different concentrations of melatonin (10^?7, 10^?9 and 10^?11 M) to maturation (Experiment 1; Control, IVM  + 10^?7, IVM  + 10^?9, IVM  + 10^?11) and culture media (Experiment 2; Control, IVC  + 10^?7, IVC  + 10^?9, IVC  + 10^?11) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10^?9 M) from Experiments 1–2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM  + 10^?9, IVC  + 10^?9, IVM /IVC  + 10^?9). In Experiment 1, maturated oocytes from Control and IVM  + 10^?9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC  + 10^?7 (43.5%; 56.7%) and IVC  + 10^?9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC  + 10^?9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC  + 10^?9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM /IVC  + 10^?9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC  + 10^?9 treatment also had a higher mRNA expression of antioxidant gene (SOD 2) compared to the Control, as well as the heat shock protein (HSPB 1) compared to the IVM  + 10^?9. Reactive oxygen species production was greater in the IVM /IVC  + 10^?9 treatment group. In conclusion, the 10^?9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.     

Nutritional properties of dried salmon silage for broiler feeding

The aim of this study was to evaluate the chemical composition, energy and amino acid profile of dried salmon silage (DSS) for broilers. The DSS was obtained by acid digestion of salmon mortalities and subsequently co‐dried with wheat bran in a 70:30 ratio (70 parts silage and 30 parts wheat bran). Samples of DSS were evaluated for chemical composition, gross energy, nitrogen‐corrected true metabolizable energy (TME_n), mineral content, total and digestible amino acids for broilers, and amino acid score. The chemical composition of DSS was (mean ± SD): moisture (12.3 ± 0.8%), crude protein (44.0 ± 1.1%), ether extract (5.0 ± 2.4%), crude fiber (3.3 ± 0.4%) and ash (9.4 ± 0.6%). The gross energy and TME_n for broilers were 4 069 kcal/kg and 2 613 kcal/kg, respectively. The DSS mineral composition showed a high content of calcium (1.01%) and phosphorus (1.08%). The DSS had high levels of digestible methionine (0.74%), lysine (2.27%), and threonine (1.16%), and did not present limiting amino acids for broilers. Nutritional composition of DSS showed high protein content with an amino acid profile considered to be suitable as a protein source for broiler feeding.     

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

Lipid characterization of both wild and cultured eggs of cuttlefish (Sepia officinalis L.) throughout the embryonic development

The present work reports a characterization of mean wet weight and moisture, the lipid class and fatty acid (FA) composition from the total lipids (TL), of both culture and wild eggs of the cuttlefish, Sepia officinalis , throughout the embryonic development. Additionally, reproductive data, such as the number of spawnings, number and mean weight of eggs and duration of spawning period of cultured cuttlefish is provided. Both types of eggs were similar in mean wet weight, moisture content, TL content and lipid composition throughout embryonic development. Females from the cultured group spawned 13 times and laid 8654 eggs in 64 days, with a mean weight of 0.607 ± 0.179 g. A sex ratio of 1.57 (11♀ for 7♂) promoted an individual fecundity of 787 eggs/♀ (the biggest until now on our culture facilities), which might be related to increased bottom areas. The TL increased with day/stage of embryonic development ( P  < 0.05) only in the cultured egg group. However, no differences were found on TL between culture and wild eggs at the same day/stage ( P  > 0.05). Eggs displayed predominant levels of phosphatidylcholine, phosphatidylethanolamine (PE), cholesterol and triacylglycerol at the end of embryonic development. Polar and neutral lipids of both eggs groups remained consistently proportional (∼50% for each lipid fraction) and a significant increase ( P  < 0.05) was observed in phosphatidylserine, PE and free FA throughout the embryonic development. In either egg type and day, 16:0, 18:0, 20:5n-3 and 22:6n-3 accounted for approximately 70 g Kg⁻¹ of all FA and saturated and n-3 totals seemed to have the same proportion in the cuttlefish eggs. The present results suggest that lipids are not used as energetic substrate but as structural components in cuttlefish egg.