Establishment of embryonic stem cell lines from the dog and their application to regenerative veterinary medicine

胚胎干细胞是一种多能细胞，具有生成任何类型细胞的能力。本研究的目的是建立犬再生兽药ES细胞系，并获得以下结果：1.下丘脑P450芳香酶基因、下丘脑和垂体雌激素受体α和ββ水平的升高以及血浆雌二醇-17 mRNAs水平的升高与母犬的动情终止有关。2.鼻腔注射高效促性腺激素释放激素激动剂醋酸亮丙内酯是一种有效的方法，可以控制对发情母犬的发情诱导，似乎对动物造成轻微的应激。联合应用醋酸亮丙瑞林和17-雌二醇β诱导发情的效果可能更好。3.虽然用雌激素和表皮生长因子培养的犬MI卵母细胞的体外成熟率高于单独用培养液培养的卵母细胞，但用…培养的MII期卵母细胞的成熟率高于单独用EGF培养的卵母细胞。胚胎成纤维细胞共培养可促进犬卵母细胞的核和胞质成熟，与犬胚胎成纤维细胞共培养可发育至16-细胞期，与小鼠胚胎成纤维细胞共培养可发育至桑椹胚期。5.在孵化的囊胚中，67.9%的内细胞团形成原代犬ES样细胞团，显著高于囊胚期(25.6%)和桑胚期(0%)。6.犬ES样细胞具有典型的干细胞样形态，表达碱性磷酸酶等特异性标志，阶段特异性胚胎抗原-1和OCT-4，在悬浮培养中形成类胚体；延长培养时间可形成囊状EBS。单纯MS在组织培养板上培养时，可分化为神经元样、上皮样、成纤维细胞样、黑素细胞样和心肌样细胞。7.犬ES样细胞集落在胶原酶作用下可立即分化，但机械解聚后仍未分化，两株ES样细胞系传代8代。较少

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. The objective of this study was to establish ES cell lines from the dog for regenerative veterinary medicine, and the following results were obtained.1.The rise in the levels of hypothalamic P450 aromatase mRNA, hypothalamic and pituitary estrogen receptor α and β mRNAs, and plasma estradiol-17β was associated with termination of anestrus in the bitch.2.The intranasal administration of a depot-injectable form of the potent GnRH agonist leuprolide acetate was a useful method for controlling the induction of estrus in anestrous bitches that appears to cause the animals little stress, and a combination of leuprolide acetate and oral estradiol-17β might be more effective in inducing estrus.3.Although the in vitro maturation rate of canine MI oocytes cultured with estrogen and epidermal growth factor was higher than for oocytes cultured in culture media only, the rate that reached the MII stage w … More as not different in both groups.4.Co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes, and co-culture with canine embryonic fibroblasts developed the embryo up to the 16-cell stage, and with mouse embryonic fibroblasts up to morula stage.5.Primary canine ES-like cell colonies were formed in 67.9% of inner cell masses from hatched blastocysts, which was significantly higher than from blastocysts (25.6%) and d morulae stage (0%).6.Canine ES-like cells had typical stem cell-like morphology, expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4, and formed embryoid bodies (EBs) in a suspension culture ; extended culture of EBs resulted in the formation of cystic EBs. When the simple Ms were cultured in tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like and myocardium-like cells.7.Dissociation of canine ES-like cell colonies with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remain undifferentiated, and two ES-like cell lines passed 8 passages. Less