Rho激酶抑制剂对小鼠胚胎干细胞分离与生物学特性的影响
|
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。
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.
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。
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.
引文
[1] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature, 1981, 292(5819):154-6.
[2] Axelrod HR. Embryonic stem cell lines derived from blastocysts by a simplified technique[J]. Dev Biol, 1984, 101(1): 225-8.
[3] Wobus AM, Holzhausen H, J?kel P, et al. Characterization of a pluripotent stem cell line derived from a mouse embryo[J]. Exp Cell Res, 1984, 152(1):212-9.
[4] Gossler A, Doetschman T, Korn R, et al.. Transgenesis by means of blastocyst-derived embryonic stem cell lines[J]. Proc Natl Acad Sci U S A, 1986, 83(23):9065-9.
[5] Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells[J]. Cell, 1987, 51(3): 503-12
[6] Koller BH, Hageman LJ, Doetschman TC, et al.. Germline transmission of a planned alteration made in the hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells[J]. Proc. Natl. Acad. Sci. USA, 1989, 86, 8927-8931.
[7] Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts[J]. Nature, 1968, 218(5139): 346-8.
[8] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Natl Acad Sci U S A, 1981, 78(12):7634-8.
[9] Joyner, A. L. (1993) IRL PRESS at Oxford University Press[M], p. 109.
[10] Robertson E, Bradley A, Kuehn M, et al.. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector[J]. Nature, 1986, 323(6087): 445-8.
[11] Mitalipov ShM, Mitallipova MM, Ivanov VI. The effect of the duration of culturing on the pluripotency of mouse embryonic stem (ES) cells in vitro and in vivo[J]. Ontogenez, 1994, 25(6): 19-27.
[12] Savatier P, Lapillonne H, van Grunsven LA, et al. Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells[J]. Oncogene, 1996, 12(2), 309-22.
[13] Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine[J]. Genes Dev, 2005, 19(10): 1129-55.
[14] Doetschman TC, Eistetter H, Katz M, et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium[J]. J Embryol Exp Morphol, 1985, 87: 27-45.
[15] Wei H, Juhasz O, Li J, et al. Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses[J]. J Cell Mol Med, 2005, 9(4): 804-17.
[16] Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation , dedifferentiation or self-renewal of ES cells[J]. Nat Genet, 2000, 24(4): 372-76.
[17] Hay DC, Sutherland L, Clark J, et al. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells[J]. Stem Cells, 2004, 22(2): 225-35.
[18] Nichols J, Zevnik B, Anastassiadis K, Niwa H, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[J]. Cell, 1998, 95(3): 379-91.
[19] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog ,a pluripotency sustaining factor in embryonic stem cells[J]. Cell, 2003, 113(5): 643-55.
[20] Mitsui K, Tokuzawa Y, Itoh H, Segawa K, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells [J] . Cell, 2003, 113(5): 631-42.
[21] Fujikura J, Yamato E, Yonemura S, et al. Differentiation of Embryonic stem cells is induced by GATA factors[J]. Genes Dev, 2002, 16(7): 784-9.
[22] Constantinescu S. Stemness, fusion and renewal of hematopoietic and embryonic stem cells[J]. J Cell Mol Med, 2003, 7(2): 103-12.
[23] Chojnacki A, Shimazaki T, Gregg C, et al. Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells[J]. J Neurosci, 2003, 23(5): 1730-41
[24] Gearing DP, Thut CJ, VandeBos T, Gimpel SD, et al. Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130[J]. EMBO J, 1991, 10(10): 2839-48
[25] Cartwright P, McLean C, Sheppard A, et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism[J]. Development, 2005, 132(5): 885-96.
[26] Nakamura T, Arai T, Takagi M, et al. A selective switch-on system for self-renewal of embryonic stem cells using chimeric cytokine receptors[J]. Biochem Biophys Res Commun, 1998, 248(1): 22-7.
[27] Sato N, Meijer L, Skaltsounis L, et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor[J]. Nat Med, 2004, 10(1): 55-63
[28] Haegele L, Ingold B, Naumann H, et al. Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression[J]. Mol Cell Neurosci, 2003, 24(3): 696-708
[29] Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche[J]. Curr Opin Hematol, 2004, 11(2): 88-94.
[30] Cantley LC. The phosphoinositide 3-kinase pathway[J]. Science. 2002;296(5573): 1655-7.
[31] Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream[J]. Cell, 2007, 129(7): 1261-74.
[32] Storm MP, Bone HK, Beck CG, et al. Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells[J]. J Biol Chem. 2007, 282(9): 6265-73.
[33] Paling NR, Wheadon H, Bone HK, et al. Regulation of embryonic stem cell self-renewal byphosphoinositide 3-kinase-dependent signaling[J]. J Biol Chem. 2004, 279(46): 48063-70.
[34] Welham MJ, Storm MP, Kingham E, et al. Phosphoinositide 3-kinases and regulation of embryonic stem cell fate[J]. Biochem Soc Trans, 2007, 35: 225-8.
[35] Cross DA, Alessi DR, Cohen P, et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B[J]. Nature, 1995, 378(6559): 785-9.
[36] Gardner RL, Brook FA. Reflections on the biology of embryonic stem (ES) cells[J]. Int. J Dev Biol, 1997, 41(2): 235-43.
[37] Livy DJ, Wahlsten D. Retarded formation of the hippocampal commissure in embryos from mouse strains lacking a corpus callosum[J]. Hippocampus,1997, 7(1): 2-14.
[38] Royce JR. Avoidance conditioning in nine strains of inbred mice using optimal stimulus parameters[J]. Behav Genet, 1972, 2(1): 107-10.
[39] Bryja V, Bonilla S, Cajánek L, et al. An efficient method for the derivation of mouse embryonic stem cells[J]. Stem Cells, 2006, 24(4): 844-9.
[40] Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse[J]. Proc Natl Acad Sci USA. 1997, 94(11): 5709-12.
[41] Keskintepe L, Norris K, Pacholczyk G, et al. Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line[J]. Transgenic Res, 2007, 16(6): 751-8.
[42] Ledermann B, Bürki K. Establishment of a germ-line competent C57BL/6 embryonic stem cell line[J]. Exp Cell Res, 1991, 197(2): 254-8.
[43] Kawase E, Suemori H, Takahashi N, et al. Strain difference in establishment of mouse embryonic stem (ES) cell lines[J]. Int J Dev Biol, 1994, 38(2): 385-90.
[44] Buehr M, Smith A. Genesis of embryonic stem cells[J]. Philos Trans R Soc Lond B Biol Sci, 2003, 358: 1397-1402.
[45] Batlle-Morera L, Smith A, Nichols J. Parameters Influencing Derivation of Embryonic Stem Cells From Murine Embryos[J]. Genesis, 2008, 46(12): 758-67.
[46] Coucouvanis E, Martin GR. Signals for death and survival: A two step mechanism for cavitation in the vertebrate embryo[J]. Cell, 1995, 83(2): 279-87.
[47] Coucouvanis E, Martin GR. BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo[J]. Development, 1999, 126(3): 535-46.
[48] Thomas P, Beddington R. Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo[J]. Curr Biol, 1996, 6(11):1487-96.
[49]孟国良,汤富酬,尚克刚,等. 5个品系小鼠胚胎干细胞系建立的方法学比较[J].遗传学报, 2003, 30(10): 933-42.
[50] Kim C, Amano T, Park J, et al. Improvement of embryonic stem cell line derivation efficiency with novel medium, glucose concentration, and epigenetic modifications[J]. Cloning Stem Cells, 2009, 11(1): 89-100.
[51]余树民,严兴荣,陈冬梅,等.不同添加物对分离昆明系小鼠胚胎干细胞的影响[J].农业生物技术学报, 2008, 16(1): 71-6.
[52] Shimokawa H, Takeshita A. Rho-kinase is an important therapeutic target in cardiovascular medicine[J]. Arterioscler Thromb Vasc Biol, 2005, 25(9): 1767-75.
[53] Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells[J]. Trends Pharmacol. Sci, 2001, 22(1): 32-9.
[54] Nobes C, Hall A. Regulation and function of the Rho subfamily of small GTPases[J]. Curr Opin Genet Dev, 1994, 4(1): 77-81.
[55] Van Aelst L, D'Souza-Schorey C. Rho GTPases and signaling networks[J]. Genes Dev, 1997, 11(18): 2295-322.
[56] Ishizaki T, Maekawa M, Fujisawa K, et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase[J]. EMBO J. 1996, 15(8): 1885-93.
[57] Takahashi N, Tuiki H, Saya H, et al. Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24[J]. Genomics. 1999, 55(2): 235-7.
[58] Nakagawa O, Fujisawa K, Ishizaki T, et al. ROCK-I and ROCK-II, two isoforms of Rhoassociated coiled-coil forming protein serine/threonine kinase in mice[J]. FEBS Lett, 1996, 392(2): 189-93.
[59] Chang J, Xie M, Shah VR, et al. Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis[J]. Proc Natl Acad Sci U S A, 2006, 103(39): 14495-500.
[60] Sebbagh M, Hamelin J, Bertoglio J, et al. Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner[J]. J Exp Med, 2005, 201(3): 465-71.
[61] Sapet C, Simoncini S, Loriod B, et al. Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2[J]. Blood, 2006, 108(6): 1868-76.
[62] Thumkeo D, Keel J, Ishizaki T, et al. Targeted disruption of the mouse rhoassociated kinase 2 gene results in intrauterine growth retardation and fetal death[J]. Mol Cell Biol, 2003, 23(14): 5043-55.
[63] Shimizu Y, Thumkeo D, Keel J, Ishizaki T, et al. ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles[J]. J Cell Biol, 2005, 168(6): 941-53.
[64] Riento K, Guasch RM, Garg R, et al. RhoE binds to ROCK I and inhibits downstream signaling[J]. Mol Cell Biol, 2003, 23(12): 4219-29.
[65] Ward Y, Yap SF, Ravichandran V, et al. The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway[J]. J Cell Biol 2002, 157(2): 291-302.
[66] Pinner S, Sahai E. PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE[J]. Nat Cell Biol 2008, 10(2): 127-37.
[67] Lowery DM, Clauser KR, Hjerrild M, et al. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate[J]. EMBO J 2007, 26(9): 2262-73.
[68] Scott RW, Olson MF. LIM kinases: function, regulation and association with human disease[J]. JMol Med, 2007, 85(6): 555-68.
[69] Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour[J]. Nat Rev Mol Cell Biol, 2003, 4(6): 446-56.
[70] Hagerty L, Weitzel DH, Chambers J, et al. ROCK1 phosphorylates and activates zipper-interacting protein kinase[J]. J Biol Chem, 2007, 282(7): 4884-93.
[71] Narumiya S, Ishizaki T, Uehata M. Use and properties of ROCK-specific inhibitor Y-27632[M]. Methods Enzymol. 2000, 325: 273-84.
[72] Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension[J]. Nature, 389(6654): 990-4.
[73] Ishizaki T, Uehata M, Tamechika I, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases[J]. Mol Pharmacol, 57(5): 976-83
[74] Davies SP, Reddy H, Caivano M, et al. Specificity and mechanism of action of some commonly used protein kinase inhibitors[J]. Biochem J. 2000, 351: 95-105.
[75] Higashi M, Shimokawa H, Hattori T, et al. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo. Effect of endothelial NAD(P)H oxidase system[J]. Circ Res, 2003, 93(8): 767-75.
[76] Rikitake Y, Kim HH, Huang Z, et al. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection[J]. Stroke, 2005, 36(10): 2251-7.
[77] Shimokawa H, Seto M, Katsumata N, et al. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm[J]. Cardiovasc Res, 1999, 43(4): 1029-39.
[78] Hirooka Y, Shimokawa H. Therapeutic potential of rho-kinase inhibitors in cardiovascular diseases[J]. Am J Cardiovasc Drugs, 2005, 5(1): 31-9.
[79] Tamura M, Nakao H, Yoshizaki H, et al. Development of specific Rho-kinase inhibitors and their clinical application[J]. Biochim Biophys Acta. 2005, 1754(1-2): 245-52.
[80] Bourguignon LY, Zhu H, Shao L, et al. Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells[J]. Cell Motil Cytoskeleton, 1999, 43(4): 269-87.
[81] Yoshioka K, Nakamori S, Itoh K. Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells[J]. Cancer Res, 1999, 59(8): 2004-10.
[82] Li Z, Dong X, Wang Z, et al. Regulation of PTEN by Rho small GTPases[J]. Nat Cell Biol, 2005, 7(4): 399-404.
[83] Thorlacius K, Slotta JE, Laschke MW, et al. Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury[J]. J Leukoc Biol, 2006, 79(5): 923-31.
[84] Itoh K, Yoshioka K, Akedo H, et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells[J]. Nat Med, 1999, 5(2): 221-5.
[85] Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelial permeability[J].Vascul Pharmacol. 2002, 39(4-5):187-99.
[86] Wójciak-Stothard B, Potempa S, Eichholtz T, et al. Rho and Rac but not Cdc42 regulate endothelial cell permeability[J]. J Cell Sci, 2001, 114(Pt 7): 1343-55.
[87] Farah S, Agazie Y, Ohan N, et al. A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling[J]. J Biol Chem, 1998, 273(8): 4740-6.
[88] Sordella R, Classon M, Hu KQ, Matheson SFet al. Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development[J]. Dev Cell, 2002, 2(5): 553-65.
[89] O'Cochlain DF, Perez-Terzic C, Reyes S, et al. Transgenic overexpression of human DMPK accumulates into hypertrophic cardiomyopathy, myotonic myopathy and hypotension traits of myotonic dystrophy[J]. Hum Mol Genet, 2004, 13(20): 2505-18.
[90] Pan J, Singh US, Takahashi T, et al. PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes[J]. J Cell Physiol, 2005, 202(2): 536-53.
[91] Sordella R, Jiang W, Chen GC, et al. Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis[J]. Cell, 2003, 113(2): 147-58.
[92] Lobe CG, Nagy A. Conditional genome alteration in mice[J]. Bioessays, 1998, 20(3): 200-8.
[93] Nagy A, Rossant J, Nagy R, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells[J]. Proc Natl Acad Sci U S A, 1993, 90(18): 8424-8.
[94] Nagy A, Gócza E, Diaz EM, et al. Embryonic stem cells alone are able to support fetal development in the mouse[J]. Development, 110(3): 815-21.
[95] Kaufman MH, Webb S. Postimplantation development of tetraploid mouse embryos produced by electrofusion[J]. Development, 1990, 110(4): 1121-32.
[96] Kubiak JZ, Tarkowski AK. Electrofusion of mouse blastomeres[J]. Exp Cell Res, 1985, 157(2): 561-6.
[97] Nagy A, Rossant J. Production of ES-cell aggregation chimeras. Gene Targeting: A Practical Approach (Joyner, A., ed.) IRL Press at Oxford University[M], Oxford, UK, 1999; pp. 177-205.
[98] Ueda O, Jishage K, Kamada N, et al. Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos[J]. Exp Anim, 1995, 44(3): 205-10.
[99] Eakin GS, Hadjantonakis AK. Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos[J]. Nat Protoc, 2006, 1(3): 1145-53.
[100] Wood SA, Allen ND, Rossant J, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras[J]. Nature, 1993, 365(6441): 87-9.
[101] B Hogan, R Beddington, F Costantini, et al. in‘‘Manipulating the Mouse Embryo: A Laboratory Manual’’[M]. Cold Spring Harbor Press 1994; pp. 260-262.
[102] Amano T, Nakamura K, Tani T, et al. Production of mice derived entirely from embryonic stem cells after injecting the cells into heat treated blastocysts[J]. Theriogenology, 2000, 53(7):1449-58.
[103] Kim SU, Han YH, Lee TH, et al. Effective production of microinjectable blastocysts for germ-line transmission of embryonic stem cells[J]. Exp Anim. 2004 Oct;53(5):475-7.
[104] Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation[J]. Proc Natl Acad Sci U S A, 2001, 98(11): 6209-14.
[105] Eakin GS, Hadjantonakis AK, Papaioannou VE, et al. Developmental potential and behavior of tetraploid cells in the mouse embryo[J]. Dev Biol, 2005, 288(1): 150-9.
[106] Gridley T, Woychik R. Laser surgery for mouse geneticists[J]. Nat Biotechnol, 2007, 25(1): 59-60.
[107] Poueymirou WT, Auerbach W, Frendewey D, et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses[J]. Nat Biotechnol, 2007, 25(1): 91-9.
[108] Tokunaga T, Tsunoda Y. Efficacious production of viable germ-line chimeras between embryonic stem (ES) cells and 8-cell stage embryos[J]. Dev Growth Differ, 1992, 34(5): 561-6.
[109] Huang J, Deng K, Wu H, et al. Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation[J]. Stem Cells, 2008, 26(7): 1883-90.
[110] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse[J]. Biol Reprod, 1995, 52(4): 709-20.
[111] Wakayama T, Perry AC, Zuccotti M, et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J]. Nature, 1998, 394(6691): 369-74.
[112] Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei[J]. Science, 2000, 289(5482): 1188-90.
[113] Munsie MJ, Michalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei[J]. Curr Biol, 2000, 10(16): 989-92.
[114] Shoji S, Yoshida N, Amanai M, et al. Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20[J]. EMBO J, 2006, 25(4): 834-45.
[115] Coleman ML, Olson MF. Rho GTPase signalling pathways in the morphological changes associated with apoptosis[J]. Cell Death Differ, 2002, 9(5): 493-504.
[116] Coleman ML, Sahai EA, Yeo M, et al. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I[J]. Nat Cell Biol, 2001, 3(4): 339-45.
[117] Ongusaha PP, Kim HG, Boswell SA, et al. RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress[J]. Curr Biol, 2006, 16(24): 2466-72.
[118] Mi?ambres R, Guasch RM, Perez-AragóA, et al. The RhoA/ROCK-I/MLC pathway is involved in the ethanol-induced apoptosis by anoikis in astrocytes[J]. J Cell Sci, 2006, 119(Pt 2): 271-82.
[119] Watanabe K, Ueno M, Kamiya D, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells[J]. Nat Biotechnol, 2007, 25(6): 681-6.
[120] Koyanagi M, Takahashi J, Arakawa Y, et al. Inhibition of the Rho/ROCK pathway reducesapoptosis during transplantation of embryonic stem cell-derived neural precursors[J]. J Neurosci Res, 2008, 86(2): 270-80.
[121] Smith AG. Embryo-derived stem cells: of mice and men[J]. Annu Rev Cell Dev Biol. 2001, 17: 435-62.
[122] Bajpai R, Lesperance J, Kim M, et al. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells[J]. Mol Reprod Dev, 2008, 75(5): 818-27.
[123] Wachs FP, Couillard-Despres S, Engelhardt M, et al. High Efficacy of Clonal Growth and Expansion of Adult Neural Stem Cells[J]. Lab Invest,2003, 83(7): 949-62.
[124] Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors[J]. Nature, 2008, 451(7175):141-6.
[2] Axelrod HR. Embryonic stem cell lines derived from blastocysts by a simplified technique[J]. Dev Biol, 1984, 101(1): 225-8.
[3] Wobus AM, Holzhausen H, J?kel P, et al. Characterization of a pluripotent stem cell line derived from a mouse embryo[J]. Exp Cell Res, 1984, 152(1):212-9.
[4] Gossler A, Doetschman T, Korn R, et al.. Transgenesis by means of blastocyst-derived embryonic stem cell lines[J]. Proc Natl Acad Sci U S A, 1986, 83(23):9065-9.
[5] Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells[J]. Cell, 1987, 51(3): 503-12
[6] Koller BH, Hageman LJ, Doetschman TC, et al.. Germline transmission of a planned alteration made in the hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells[J]. Proc. Natl. Acad. Sci. USA, 1989, 86, 8927-8931.
[7] Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts[J]. Nature, 1968, 218(5139): 346-8.
[8] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Natl Acad Sci U S A, 1981, 78(12):7634-8.
[9] Joyner, A. L. (1993) IRL PRESS at Oxford University Press[M], p. 109.
[10] Robertson E, Bradley A, Kuehn M, et al.. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector[J]. Nature, 1986, 323(6087): 445-8.
[11] Mitalipov ShM, Mitallipova MM, Ivanov VI. The effect of the duration of culturing on the pluripotency of mouse embryonic stem (ES) cells in vitro and in vivo[J]. Ontogenez, 1994, 25(6): 19-27.
[12] Savatier P, Lapillonne H, van Grunsven LA, et al. Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells[J]. Oncogene, 1996, 12(2), 309-22.
[13] Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine[J]. Genes Dev, 2005, 19(10): 1129-55.
[14] Doetschman TC, Eistetter H, Katz M, et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium[J]. J Embryol Exp Morphol, 1985, 87: 27-45.
[15] Wei H, Juhasz O, Li J, et al. Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses[J]. J Cell Mol Med, 2005, 9(4): 804-17.
[16] Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation , dedifferentiation or self-renewal of ES cells[J]. Nat Genet, 2000, 24(4): 372-76.
[17] Hay DC, Sutherland L, Clark J, et al. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells[J]. Stem Cells, 2004, 22(2): 225-35.
[18] Nichols J, Zevnik B, Anastassiadis K, Niwa H, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[J]. Cell, 1998, 95(3): 379-91.
[19] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog ,a pluripotency sustaining factor in embryonic stem cells[J]. Cell, 2003, 113(5): 643-55.
[20] Mitsui K, Tokuzawa Y, Itoh H, Segawa K, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells [J] . Cell, 2003, 113(5): 631-42.
[21] Fujikura J, Yamato E, Yonemura S, et al. Differentiation of Embryonic stem cells is induced by GATA factors[J]. Genes Dev, 2002, 16(7): 784-9.
[22] Constantinescu S. Stemness, fusion and renewal of hematopoietic and embryonic stem cells[J]. J Cell Mol Med, 2003, 7(2): 103-12.
[23] Chojnacki A, Shimazaki T, Gregg C, et al. Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells[J]. J Neurosci, 2003, 23(5): 1730-41
[24] Gearing DP, Thut CJ, VandeBos T, Gimpel SD, et al. Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130[J]. EMBO J, 1991, 10(10): 2839-48
[25] Cartwright P, McLean C, Sheppard A, et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism[J]. Development, 2005, 132(5): 885-96.
[26] Nakamura T, Arai T, Takagi M, et al. A selective switch-on system for self-renewal of embryonic stem cells using chimeric cytokine receptors[J]. Biochem Biophys Res Commun, 1998, 248(1): 22-7.
[27] Sato N, Meijer L, Skaltsounis L, et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor[J]. Nat Med, 2004, 10(1): 55-63
[28] Haegele L, Ingold B, Naumann H, et al. Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression[J]. Mol Cell Neurosci, 2003, 24(3): 696-708
[29] Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche[J]. Curr Opin Hematol, 2004, 11(2): 88-94.
[30] Cantley LC. The phosphoinositide 3-kinase pathway[J]. Science. 2002;296(5573): 1655-7.
[31] Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream[J]. Cell, 2007, 129(7): 1261-74.
[32] Storm MP, Bone HK, Beck CG, et al. Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells[J]. J Biol Chem. 2007, 282(9): 6265-73.
[33] Paling NR, Wheadon H, Bone HK, et al. Regulation of embryonic stem cell self-renewal byphosphoinositide 3-kinase-dependent signaling[J]. J Biol Chem. 2004, 279(46): 48063-70.
[34] Welham MJ, Storm MP, Kingham E, et al. Phosphoinositide 3-kinases and regulation of embryonic stem cell fate[J]. Biochem Soc Trans, 2007, 35: 225-8.
[35] Cross DA, Alessi DR, Cohen P, et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B[J]. Nature, 1995, 378(6559): 785-9.
[36] Gardner RL, Brook FA. Reflections on the biology of embryonic stem (ES) cells[J]. Int. J Dev Biol, 1997, 41(2): 235-43.
[37] Livy DJ, Wahlsten D. Retarded formation of the hippocampal commissure in embryos from mouse strains lacking a corpus callosum[J]. Hippocampus,1997, 7(1): 2-14.
[38] Royce JR. Avoidance conditioning in nine strains of inbred mice using optimal stimulus parameters[J]. Behav Genet, 1972, 2(1): 107-10.
[39] Bryja V, Bonilla S, Cajánek L, et al. An efficient method for the derivation of mouse embryonic stem cells[J]. Stem Cells, 2006, 24(4): 844-9.
[40] Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse[J]. Proc Natl Acad Sci USA. 1997, 94(11): 5709-12.
[41] Keskintepe L, Norris K, Pacholczyk G, et al. Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line[J]. Transgenic Res, 2007, 16(6): 751-8.
[42] Ledermann B, Bürki K. Establishment of a germ-line competent C57BL/6 embryonic stem cell line[J]. Exp Cell Res, 1991, 197(2): 254-8.
[43] Kawase E, Suemori H, Takahashi N, et al. Strain difference in establishment of mouse embryonic stem (ES) cell lines[J]. Int J Dev Biol, 1994, 38(2): 385-90.
[44] Buehr M, Smith A. Genesis of embryonic stem cells[J]. Philos Trans R Soc Lond B Biol Sci, 2003, 358: 1397-1402.
[45] Batlle-Morera L, Smith A, Nichols J. Parameters Influencing Derivation of Embryonic Stem Cells From Murine Embryos[J]. Genesis, 2008, 46(12): 758-67.
[46] Coucouvanis E, Martin GR. Signals for death and survival: A two step mechanism for cavitation in the vertebrate embryo[J]. Cell, 1995, 83(2): 279-87.
[47] Coucouvanis E, Martin GR. BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo[J]. Development, 1999, 126(3): 535-46.
[48] Thomas P, Beddington R. Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo[J]. Curr Biol, 1996, 6(11):1487-96.
[49]孟国良,汤富酬,尚克刚,等. 5个品系小鼠胚胎干细胞系建立的方法学比较[J].遗传学报, 2003, 30(10): 933-42.
[50] Kim C, Amano T, Park J, et al. Improvement of embryonic stem cell line derivation efficiency with novel medium, glucose concentration, and epigenetic modifications[J]. Cloning Stem Cells, 2009, 11(1): 89-100.
[51]余树民,严兴荣,陈冬梅,等.不同添加物对分离昆明系小鼠胚胎干细胞的影响[J].农业生物技术学报, 2008, 16(1): 71-6.
[52] Shimokawa H, Takeshita A. Rho-kinase is an important therapeutic target in cardiovascular medicine[J]. Arterioscler Thromb Vasc Biol, 2005, 25(9): 1767-75.
[53] Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells[J]. Trends Pharmacol. Sci, 2001, 22(1): 32-9.
[54] Nobes C, Hall A. Regulation and function of the Rho subfamily of small GTPases[J]. Curr Opin Genet Dev, 1994, 4(1): 77-81.
[55] Van Aelst L, D'Souza-Schorey C. Rho GTPases and signaling networks[J]. Genes Dev, 1997, 11(18): 2295-322.
[56] Ishizaki T, Maekawa M, Fujisawa K, et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase[J]. EMBO J. 1996, 15(8): 1885-93.
[57] Takahashi N, Tuiki H, Saya H, et al. Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24[J]. Genomics. 1999, 55(2): 235-7.
[58] Nakagawa O, Fujisawa K, Ishizaki T, et al. ROCK-I and ROCK-II, two isoforms of Rhoassociated coiled-coil forming protein serine/threonine kinase in mice[J]. FEBS Lett, 1996, 392(2): 189-93.
[59] Chang J, Xie M, Shah VR, et al. Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis[J]. Proc Natl Acad Sci U S A, 2006, 103(39): 14495-500.
[60] Sebbagh M, Hamelin J, Bertoglio J, et al. Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner[J]. J Exp Med, 2005, 201(3): 465-71.
[61] Sapet C, Simoncini S, Loriod B, et al. Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2[J]. Blood, 2006, 108(6): 1868-76.
[62] Thumkeo D, Keel J, Ishizaki T, et al. Targeted disruption of the mouse rhoassociated kinase 2 gene results in intrauterine growth retardation and fetal death[J]. Mol Cell Biol, 2003, 23(14): 5043-55.
[63] Shimizu Y, Thumkeo D, Keel J, Ishizaki T, et al. ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles[J]. J Cell Biol, 2005, 168(6): 941-53.
[64] Riento K, Guasch RM, Garg R, et al. RhoE binds to ROCK I and inhibits downstream signaling[J]. Mol Cell Biol, 2003, 23(12): 4219-29.
[65] Ward Y, Yap SF, Ravichandran V, et al. The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway[J]. J Cell Biol 2002, 157(2): 291-302.
[66] Pinner S, Sahai E. PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE[J]. Nat Cell Biol 2008, 10(2): 127-37.
[67] Lowery DM, Clauser KR, Hjerrild M, et al. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate[J]. EMBO J 2007, 26(9): 2262-73.
[68] Scott RW, Olson MF. LIM kinases: function, regulation and association with human disease[J]. JMol Med, 2007, 85(6): 555-68.
[69] Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour[J]. Nat Rev Mol Cell Biol, 2003, 4(6): 446-56.
[70] Hagerty L, Weitzel DH, Chambers J, et al. ROCK1 phosphorylates and activates zipper-interacting protein kinase[J]. J Biol Chem, 2007, 282(7): 4884-93.
[71] Narumiya S, Ishizaki T, Uehata M. Use and properties of ROCK-specific inhibitor Y-27632[M]. Methods Enzymol. 2000, 325: 273-84.
[72] Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension[J]. Nature, 389(6654): 990-4.
[73] Ishizaki T, Uehata M, Tamechika I, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases[J]. Mol Pharmacol, 57(5): 976-83
[74] Davies SP, Reddy H, Caivano M, et al. Specificity and mechanism of action of some commonly used protein kinase inhibitors[J]. Biochem J. 2000, 351: 95-105.
[75] Higashi M, Shimokawa H, Hattori T, et al. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo. Effect of endothelial NAD(P)H oxidase system[J]. Circ Res, 2003, 93(8): 767-75.
[76] Rikitake Y, Kim HH, Huang Z, et al. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection[J]. Stroke, 2005, 36(10): 2251-7.
[77] Shimokawa H, Seto M, Katsumata N, et al. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm[J]. Cardiovasc Res, 1999, 43(4): 1029-39.
[78] Hirooka Y, Shimokawa H. Therapeutic potential of rho-kinase inhibitors in cardiovascular diseases[J]. Am J Cardiovasc Drugs, 2005, 5(1): 31-9.
[79] Tamura M, Nakao H, Yoshizaki H, et al. Development of specific Rho-kinase inhibitors and their clinical application[J]. Biochim Biophys Acta. 2005, 1754(1-2): 245-52.
[80] Bourguignon LY, Zhu H, Shao L, et al. Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells[J]. Cell Motil Cytoskeleton, 1999, 43(4): 269-87.
[81] Yoshioka K, Nakamori S, Itoh K. Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells[J]. Cancer Res, 1999, 59(8): 2004-10.
[82] Li Z, Dong X, Wang Z, et al. Regulation of PTEN by Rho small GTPases[J]. Nat Cell Biol, 2005, 7(4): 399-404.
[83] Thorlacius K, Slotta JE, Laschke MW, et al. Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury[J]. J Leukoc Biol, 2006, 79(5): 923-31.
[84] Itoh K, Yoshioka K, Akedo H, et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells[J]. Nat Med, 1999, 5(2): 221-5.
[85] Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelial permeability[J].Vascul Pharmacol. 2002, 39(4-5):187-99.
[86] Wójciak-Stothard B, Potempa S, Eichholtz T, et al. Rho and Rac but not Cdc42 regulate endothelial cell permeability[J]. J Cell Sci, 2001, 114(Pt 7): 1343-55.
[87] Farah S, Agazie Y, Ohan N, et al. A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling[J]. J Biol Chem, 1998, 273(8): 4740-6.
[88] Sordella R, Classon M, Hu KQ, Matheson SFet al. Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development[J]. Dev Cell, 2002, 2(5): 553-65.
[89] O'Cochlain DF, Perez-Terzic C, Reyes S, et al. Transgenic overexpression of human DMPK accumulates into hypertrophic cardiomyopathy, myotonic myopathy and hypotension traits of myotonic dystrophy[J]. Hum Mol Genet, 2004, 13(20): 2505-18.
[90] Pan J, Singh US, Takahashi T, et al. PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes[J]. J Cell Physiol, 2005, 202(2): 536-53.
[91] Sordella R, Jiang W, Chen GC, et al. Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis[J]. Cell, 2003, 113(2): 147-58.
[92] Lobe CG, Nagy A. Conditional genome alteration in mice[J]. Bioessays, 1998, 20(3): 200-8.
[93] Nagy A, Rossant J, Nagy R, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells[J]. Proc Natl Acad Sci U S A, 1993, 90(18): 8424-8.
[94] Nagy A, Gócza E, Diaz EM, et al. Embryonic stem cells alone are able to support fetal development in the mouse[J]. Development, 110(3): 815-21.
[95] Kaufman MH, Webb S. Postimplantation development of tetraploid mouse embryos produced by electrofusion[J]. Development, 1990, 110(4): 1121-32.
[96] Kubiak JZ, Tarkowski AK. Electrofusion of mouse blastomeres[J]. Exp Cell Res, 1985, 157(2): 561-6.
[97] Nagy A, Rossant J. Production of ES-cell aggregation chimeras. Gene Targeting: A Practical Approach (Joyner, A., ed.) IRL Press at Oxford University[M], Oxford, UK, 1999; pp. 177-205.
[98] Ueda O, Jishage K, Kamada N, et al. Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos[J]. Exp Anim, 1995, 44(3): 205-10.
[99] Eakin GS, Hadjantonakis AK. Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos[J]. Nat Protoc, 2006, 1(3): 1145-53.
[100] Wood SA, Allen ND, Rossant J, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras[J]. Nature, 1993, 365(6441): 87-9.
[101] B Hogan, R Beddington, F Costantini, et al. in‘‘Manipulating the Mouse Embryo: A Laboratory Manual’’[M]. Cold Spring Harbor Press 1994; pp. 260-262.
[102] Amano T, Nakamura K, Tani T, et al. Production of mice derived entirely from embryonic stem cells after injecting the cells into heat treated blastocysts[J]. Theriogenology, 2000, 53(7):1449-58.
[103] Kim SU, Han YH, Lee TH, et al. Effective production of microinjectable blastocysts for germ-line transmission of embryonic stem cells[J]. Exp Anim. 2004 Oct;53(5):475-7.
[104] Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation[J]. Proc Natl Acad Sci U S A, 2001, 98(11): 6209-14.
[105] Eakin GS, Hadjantonakis AK, Papaioannou VE, et al. Developmental potential and behavior of tetraploid cells in the mouse embryo[J]. Dev Biol, 2005, 288(1): 150-9.
[106] Gridley T, Woychik R. Laser surgery for mouse geneticists[J]. Nat Biotechnol, 2007, 25(1): 59-60.
[107] Poueymirou WT, Auerbach W, Frendewey D, et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses[J]. Nat Biotechnol, 2007, 25(1): 91-9.
[108] Tokunaga T, Tsunoda Y. Efficacious production of viable germ-line chimeras between embryonic stem (ES) cells and 8-cell stage embryos[J]. Dev Growth Differ, 1992, 34(5): 561-6.
[109] Huang J, Deng K, Wu H, et al. Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation[J]. Stem Cells, 2008, 26(7): 1883-90.
[110] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse[J]. Biol Reprod, 1995, 52(4): 709-20.
[111] Wakayama T, Perry AC, Zuccotti M, et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J]. Nature, 1998, 394(6691): 369-74.
[112] Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei[J]. Science, 2000, 289(5482): 1188-90.
[113] Munsie MJ, Michalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei[J]. Curr Biol, 2000, 10(16): 989-92.
[114] Shoji S, Yoshida N, Amanai M, et al. Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20[J]. EMBO J, 2006, 25(4): 834-45.
[115] Coleman ML, Olson MF. Rho GTPase signalling pathways in the morphological changes associated with apoptosis[J]. Cell Death Differ, 2002, 9(5): 493-504.
[116] Coleman ML, Sahai EA, Yeo M, et al. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I[J]. Nat Cell Biol, 2001, 3(4): 339-45.
[117] Ongusaha PP, Kim HG, Boswell SA, et al. RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress[J]. Curr Biol, 2006, 16(24): 2466-72.
[118] Mi?ambres R, Guasch RM, Perez-AragóA, et al. The RhoA/ROCK-I/MLC pathway is involved in the ethanol-induced apoptosis by anoikis in astrocytes[J]. J Cell Sci, 2006, 119(Pt 2): 271-82.
[119] Watanabe K, Ueno M, Kamiya D, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells[J]. Nat Biotechnol, 2007, 25(6): 681-6.
[120] Koyanagi M, Takahashi J, Arakawa Y, et al. Inhibition of the Rho/ROCK pathway reducesapoptosis during transplantation of embryonic stem cell-derived neural precursors[J]. J Neurosci Res, 2008, 86(2): 270-80.
[121] Smith AG. Embryo-derived stem cells: of mice and men[J]. Annu Rev Cell Dev Biol. 2001, 17: 435-62.
[122] Bajpai R, Lesperance J, Kim M, et al. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells[J]. Mol Reprod Dev, 2008, 75(5): 818-27.
[123] Wachs FP, Couillard-Despres S, Engelhardt M, et al. High Efficacy of Clonal Growth and Expansion of Adult Neural Stem Cells[J]. Lab Invest,2003, 83(7): 949-62.
[124] Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors[J]. Nature, 2008, 451(7175):141-6.