【作者】 张鹏；

【导师】 李相运；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 小G蛋白家族成员Rho与细胞内信号关系重大。Rho家族主要参与细胞骨架机化和基因表达的调节。与其它Rho GTP酶一样,Rho通过具有活性的GTP结合状态和无活性的GDP结合状态扮演分子开关的角色。作为最具特征的Rho下游效应器,Rho激酶(ROCK),能够对为数众多的细胞功能进行调节。包括迁移、分裂、凋亡、黏附、吞噬。ROCK包含2种亚型:ROCK I和ROCK II。人的ROCK I和ROCK II的基因表达分别定位在18号染色体和2号染色体上。二者具有高度相似性,在氨基酸序列上具有65%的相似性;在它们的激酶结构域上具有92%的相似性。虽然ROCK具有2种亚型,但在目前的研究中还未显示出其中的差异。吡啶衍生物Y-27632是一种广泛使用的Rho激酶抑制剂。Y-27632作为一种具有细胞穿透性的ATP竞争抑制剂,在体外研究证实,它能够有效抑制ROCK I和ROCK II。因此,Y-27632是一种理想的选择性ROCK抑制剂,成为了研究生理事件中Rho激酶作用的理想工具。本试验以小鼠胚胎干细胞为实验材料,通过ROCK抑制剂Y-27632的使用,从多方面探讨了Y-27632在小鼠ES细胞方面产生的生物学效应。获得了一些基础性的研究资料。Y-27632能够引起ES细胞形态发生改变,通过Y-27632的添加能够使质地紧密的ES细胞集落变得松散、扁平。将Y-27632应用于ES细胞传代,能够显著提高传代效率。并且,在Y-27632长期存在的条件下,ES细胞依旧维持稳定的染色体数目,较强的碱性磷酸酶活性,在体内能够分化形成畸胎瘤,在体外能够分化形成心肌细胞。证明Y-27632不会对小鼠ES细胞的多能性造成影响,能够安全用于小鼠ES细胞的相关操作。在嵌合体小鼠生产中,通过Y-27632对细胞进行短期处理能够获得理想的细胞状态,细胞呈球形且质地柔软,便于注射针进行筛选与收集,从而缩短体外显微操作时间。并且,Y-27632的使用不会影响嵌合体小鼠生产效率。在ES细胞衍生方面,通过accutase结合Y-27632的使用,能够显著提高ES细胞亚克隆的产生,从而建立起了一种简便有效的小鼠ES细胞分离方法。并且,获得的ES细胞系符合生物学检验标准并能够稳定传代。更多还原

【Abstract】 The member of small G-proteins family Rho is substantially involved in intracellular signaling. The Rho regulates both cytoskeletal reorganization and gene expression. As with other Rho GTPases, Rho acts as a molecular switch, cycling between an active GTP-bound state and an inactive GDP-bound state. The best-characterized downstream effector of Rho is Rho-kinase (ROCK), which mediates various cellular functions, including migration, division, apoptosis, adhesion, Phagocytosis. There are two isoforms of ROCK: ROCK I and ROCK II. The genes expressing human ROCK I and ROCK II are located on chromosome 18 and chromosome 2, respectively. ROCK I and ROCK II are highly homologous, sharing 65% homology in amino acid sequence and 92% homology in their kinase domains. Although there are two isoforms, it has not exposed differentiation between these in the resent study.The pyridine derivative Y-27632 ([(+)-(R)-trans-4-(1-aminoethyl)-N-4-pyridyl) cyclohexanecarboxamide dihydrochloride]) is commonly used as a Rho kinase inhibitor. Y-27632 is a cell permeable, ATP competitive inhibitor, that inhibits the activities of ROCK-I and ROCK-II in vitro. Y-27632 is therefore a reasonably selective ROCK-I and ROCK-II inhibitor, and provides a useful pharmacological tool to study the role of Rho kinase in (patho)physiological events, both in vitro and in vivo.In this study, we use mouse embryonic stem cells as experimental materials to investigate biological effects of Y-27632 in mouse embryonic stem cells, obtaining some foundational research datas.Y-27632 could change the morphology of ES colonies. Y-27632 can make tight cell-cell connection into loose and flat connection. In passage, Y-27632 could improve the efficiency. In the Y-27632 under the conditions of long-standing, ES cells remain stability of chromosome number, high alkaline phosphatase activity, and differentiate into teratomas in vivo, cardiomyocytes in vitro. Suggest Y-27632 could not affect self-renewal and pluripotency of embryonic stem cells, moreover, it could be applied to the relative manipulations of embryonic stem cells.In the generation of chimeric mice, we get perfect cell shape via Y-27632 treatment for short term. The cells change as spherical, soft, and enable needle to select, collect easily, reducing the microinjection time in vitro. Moreover, Y-27632 has not affected production efficiency of chimeric mice.For derivation of ES cells, the joint use of accutase and Y-27632 could improve the production of sub-colonies. Therefore we establish a simple and efficient approach to get mouse ES cells. And the obtaining ES cell lines by this means conform biological characteristics and stabilize passage.更多还原