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

犬胚胎干细胞系的建立及其在再生兽医学中的应用

• 批准号: 14360191
• 负责人: INABA Toshio
• 金额: $ 9.47万
• 依托单位: Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14360191/
• 关键词: dog; embryonic stem cell; oocyte; in vitro maturation; in vitro fertilization; embryonic fibroblast cell; estrogen; estrus induction; 上皮成長因子; 精子; EGF; MHC欠損マウス; 胚性幹細胞; 卵丘細胞; エストロジェンレセプター遺伝子; 性腺刺激ホルモン放出ホルモン; アロマターゼ遺伝子

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

胚胎干细胞（ES）是具有产生任何类型细胞能力的多能细胞，本研究的目的是建立用于再生兽医学的狗ES细胞系，并获得以下结果：1.胚胎干细胞数量的增加。下丘脑 P450 芳香酶 mRNA、下丘脑和垂体雌激素受体 α 和 β mRNA 以及血浆雌二醇 17β 的水平与不发情的终止相关。母狗.2. 鼻内给予强效 GnRH 激动剂醋酸亮丙瑞林的长效注射形式是控制不发情母犬发情诱导的有效方法，这似乎不会给动物带来什么压力，而醋酸亮丙瑞林和口服的组合雌二醇-17β可能更有效地诱导发情。3.尽管雌激素培养的犬MI卵母细胞的体外成熟率和表皮生长因子高于仅在培养基中培养的卵母细胞，达到 MII 阶段的比率在两组中没有差异。4.胚胎成纤维细胞的共培养增强了犬卵母细胞的核和细胞质成熟，并且与犬胚胎成纤维细胞共培养胚胎发育至16细胞期，与小鼠胚胎成纤维细胞共培养至桑葚胚期。 5.原代犬ES样细胞孵化囊胚内细胞团形成集落的比例为 67.9%，显着高于囊胚 (25.6%) 和桑葚胚阶段 (0%)。 6. 犬 ES 样细胞具有典型的干细胞样形态，表达特异性标记物，例如碱性磷酸酶活性、阶段特异性胚胎抗原-1和Oct-4，以及EB悬浮培养物中形成的胚状体(EB)；导致囊性EB的形成。当简单的Ms在组织培养板中培养时，它们分化成多种类型的细胞，包括神经元样、上皮样、成纤维细胞样、黑素细胞样和心肌样细胞。7．用胶原酶解离犬类 ES 样细胞集落导致立即分化，但通过机械解聚，这些细胞保持未分化，并且两个 ES 样细胞系传代次数较少。

项目成果

イヌ科動物の樹状細胞の分化誘導方法

诱导犬树突状细胞分化的方法

• 发表时间: 2005

Delayed implantation induced by fadrozole hyrochioride in the rats

盐酸法屈唑诱导大鼠延迟着床

• 发表时间: 2003
• 期刊: Contraception 68・1
• 作者: Hagiwara;S.;Kawai;K.;Anri;A.;Nagahata;H.;Hiromichi Tamada et al.
• 通讯作者: Hiromichi Tamada et al.

Effect of ovarian hormones on periodical changes in immune resistance associated with estrous cycle in the beagle bitch

• DOI: 10.1016/j.imbio.2004.09.003
• 发表时间: 2004-01-01
• 期刊: IMMUNOBIOLOGY
• 影响因子: 2.8
• 作者: Sugiura, K;Nishikawa, M;Inaba, T
• 通讯作者: Inaba, T

Expression of estrogen receptor α and β genes in the mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle

• DOI: 10.1016/s0304-3940(03)00639-6
• 发表时间: 2003-08-21
• 期刊: NEUROSCIENCE LETTERS
• 影响因子: 2.5
• 作者: Hatoya, S;Torii, R;Inaba, T
• 通讯作者: Inaba, T

Detection of transforming growth factor-α and epidermal growth factor receptor mRNA and immunohistochemical localization of the corresponding proteins in the canine uterus during the estrous cycle

发情周期犬子宫内转化生长因子-α和表皮生长因子受体mRNA的检测及相应蛋白的免疫组织化学定位

• 发表时间: 2005
• 期刊: Histology and Histopathology 7:11
• 作者: Hiromichi Tamada et al.