PI3K/Akt信号通路在H_2O_2诱导的PC12细胞凋亡中的作用
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摘要
【目的】为探讨ROS在脊髓损伤病理发生过程中的作用及其分子机制，一定浓度H_2O_2作用于PC12细胞模拟其氧化应激状态，制作细胞凋亡模型；通过P13K特异性抑制剂LY294002预处理，研究PI3K／Akt信号通路活化在H_2O_2诱导的PC12细胞凋亡中的作用及其作用机制，为脊髓损伤临床治疗(如抗氧化治疗、筛选ROS相关信号通路新药物靶点)提供新途径。
【方法】不同浓度H_2O_2作用于PC12细胞24h，及H_2O_2作用前用PI3K特异性抑制剂LY294002(20μM)预处理1h，通过MTT法检测，LY294002预处理前后不同浓度H_2O_2处理对细胞增殖抑制率的影响；分别用100μM和200μMH_2O_2作用于PC12细胞24h，制作细胞凋亡模型；经Hoechst33342／PI核荧光双染、DNA片段化分析和流式细胞术检测LY294002预处理对细胞凋亡的影响，以确定P13K活化在H_2O_2诱导的PC12细胞凋亡的作用；为进一步研究PI3K／Akt信号通路的作用机制，利用Western blot技术检测LY294002预处理前后凋亡相关蛋白CPP32、凋亡调节蛋白Bcl-2家族中Bcl-2、Bad、和Bcl-X_L蛋白表达变化及Bad磷酸化情况。
【结果】1．MTT结果显示，50～200μM H_2O_2处理PC12细胞增殖抑制率随处理浓度增加而递增；LY294002预处理后细胞增殖抑制率明显高于相应的H_2O_2处理组。2．用H_2O_2(100μM或200μM)处理细胞24h，用Hoechst33342／PI核双染，荧光显微镜下观察，部分细胞核呈现典型的凋亡特征；LY294002预处理使凋亡细胞数明显增加，从(17.5±1.3)％和(29±2.1)％增加到(31±2.6)％和(44±3)％(P＜0.05)；DNA凝胶电泳显示特征性“梯状条带”，且LY294002
Objective To investigate the effects and mechanism of phosphatidylinositol
3-kinase(PI3K) signaling pathway on apoptosis induced by hydrogen proxide (H_2O_2) in rat pheochromocytoma(PC12) cells.
Methods In the present study, PC12 cells were preconditioned with 20μM
LY294002(a PI3K-specific inhibitor) for 60 min, followed by exposures to different concentrations (20, 50, 100, 200, 400, 500μM) of H_2O_2 for 24-h respectively, cells growth inhibition rate was assessed by MTT. Using H_2O_2(200μM)-induced apoptosis model in PC12 cells, pretreatment with or without LY294002(20μM) for lh, apoptotic cell death was assessed by Hoechst33342/PI nuclei double staining, DNA ladder and flow cytometry. To further explore the downstream effector of PI3K activation, activation of CPP32, protein expression related with apoptotic regulation including Bcl-2, Bcl-X_L, Bad, and phosphorylation of Bad was examined by Western blotting.
Results After pretreated with LY294002(20μM) for 1h, ① by MTT, cell
growth inhibition rate H_2O_2-caused was increased markedly was examined; ② typically apoptotic nuclei and more stronger DNA ladder was observed, increased in the number of apoptotic cell death was examined. (3) elevated level of CPP32 activation was observed, with decrease in protein expression of Bcl-2、 Bcl-X_L, and declined level in phosphorylation of Bad.
Conclusions The results suggested, transient activation of PI3K prevents from apoptosis H_2O_2-induced in PC12 cells. PI3K signaling pathway delays the
【方法】不同浓度H_2O_2作用于PC12细胞24h，及H_2O_2作用前用PI3K特异性抑制剂LY294002(20μM)预处理1h，通过MTT法检测，LY294002预处理前后不同浓度H_2O_2处理对细胞增殖抑制率的影响；分别用100μM和200μMH_2O_2作用于PC12细胞24h，制作细胞凋亡模型；经Hoechst33342／PI核荧光双染、DNA片段化分析和流式细胞术检测LY294002预处理对细胞凋亡的影响，以确定P13K活化在H_2O_2诱导的PC12细胞凋亡的作用；为进一步研究PI3K／Akt信号通路的作用机制，利用Western blot技术检测LY294002预处理前后凋亡相关蛋白CPP32、凋亡调节蛋白Bcl-2家族中Bcl-2、Bad、和Bcl-X_L蛋白表达变化及Bad磷酸化情况。
【结果】1．MTT结果显示，50～200μM H_2O_2处理PC12细胞增殖抑制率随处理浓度增加而递增；LY294002预处理后细胞增殖抑制率明显高于相应的H_2O_2处理组。2．用H_2O_2(100μM或200μM)处理细胞24h，用Hoechst33342／PI核双染，荧光显微镜下观察，部分细胞核呈现典型的凋亡特征；LY294002预处理使凋亡细胞数明显增加，从(17.5±1.3)％和(29±2.1)％增加到(31±2.6)％和(44±3)％(P＜0.05)；DNA凝胶电泳显示特征性“梯状条带”，且LY294002
Objective To investigate the effects and mechanism of phosphatidylinositol
3-kinase(PI3K) signaling pathway on apoptosis induced by hydrogen proxide (H_2O_2) in rat pheochromocytoma(PC12) cells.
Methods In the present study, PC12 cells were preconditioned with 20μM
LY294002(a PI3K-specific inhibitor) for 60 min, followed by exposures to different concentrations (20, 50, 100, 200, 400, 500μM) of H_2O_2 for 24-h respectively, cells growth inhibition rate was assessed by MTT. Using H_2O_2(200μM)-induced apoptosis model in PC12 cells, pretreatment with or without LY294002(20μM) for lh, apoptotic cell death was assessed by Hoechst33342/PI nuclei double staining, DNA ladder and flow cytometry. To further explore the downstream effector of PI3K activation, activation of CPP32, protein expression related with apoptotic regulation including Bcl-2, Bcl-X_L, Bad, and phosphorylation of Bad was examined by Western blotting.
Results After pretreated with LY294002(20μM) for 1h, ① by MTT, cell
growth inhibition rate H_2O_2-caused was increased markedly was examined; ② typically apoptotic nuclei and more stronger DNA ladder was observed, increased in the number of apoptotic cell death was examined. (3) elevated level of CPP32 activation was observed, with decrease in protein expression of Bcl-2、 Bcl-X_L, and declined level in phosphorylation of Bad.
Conclusions The results suggested, transient activation of PI3K prevents from apoptosis H_2O_2-induced in PC12 cells. PI3K signaling pathway delays the
引文
[1] Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg, 1991, 75: 15～26.
[2] Thannickal V.J. and Fanburg B.L. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L1005～L1028.
[3] Mc Cord JM. Oxygen-derived free radicals in postischemic tissue injury. New Eugl J Meal, 1985, 321:159
[4] 高颖，许彩民，杨洋．氧化诱导K652细胞凋亡机制的初步探讨．中国生物化学与分子生物学学报，1998，14(3)：295～299．
[5] Ishisaka R, Utsumi K, Utsumi T. Involvement of lysosomal cysteine protease in hydrogen peroxide-induced apoptosis in HL-60 cells. Biosci Biotechnol Biochem, 2002, 66(9):1865～1872.
[6] 王爱国，冉鹏，周明辉等．氟中毒氧化应激与细胞凋亡关系的研究．中国公共卫生，2002，18(6)：681～682．
[7] Murakawa M, Jung SK, Lijima K, et al. Apoptosis inducing protein AIP, from parasite-infected fish induces apoptosis in mammaliam cells by two different molecular mechanisms. Cell Death Differentation, 2001, 8: 298～307.
[8] Emanuele S, Galvaruso G, Lauricella M, et al. Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3. Int J Oncok, 2002, 21(4): 857～865.
[9] Tian B, Liu J, Bitterman PB, et al. Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis. Am J Physiol Heart Cite Physiol, 2002, 283(5): H1958～H1967
[10] Kerr, J.F., Wyllie, A.H. and Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Caner, 1972, 26: 239～257.
[11] Thornberry NA, Lazebnik Y. Caspase: enemies within. Science, 1998, 281(5381):1312～1316.
[12] Alnemri E.S., Livingston D.J.,Nicholson D.W. et al. Human ICE/CED-3 protease nomenclature. Cell, 1996, 87(2): 171.
[13] Nagata S. Apoptosis by death factor. Cell, 1997, 88(3): 355～365.
[14] Li G.L., Brodin G., Farooque M. et al. Apoptosis.and expression of Bcl-2 after compression trauma to rat spinal cord. J. Neuropathol. Exp. Neurol., 1996, 55: 280～289.
[15] Tsujimoto Y., Finger L.R., Yunis J. et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science, 1984, 226(4678): 1097～1099.
[16] Li GI, Brodin G, Farooque M, et al. Apoptosis and expression of bcl-2 after compression trauma to rat spinal cord. J Neuropathol Exp Neurol., 1996, 55: 280～289.
[17] Ray SK, Matzeue DD, Wilford GG, et al. Cell death in spinal cord injury(SCl) requires denovo protein synthesis. Ann N YAcad Sci., 2001, 939: 436～449.
[18] 傅强，侯铁胜，鲁凯伍等．大鼠脊髓急性损伤后神经细胞凋亡及相关基因表达．中国脊柱脊髓杂志，2001，11：92～94．
[19] Finkel T and Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Nature, 2000, 408: 239～247.
[20] Houle, F., Rousseau, S., Mortice, N., Lue, M. et al. Extracellular Signal-regulated Kinase Mediates Phosphorylation of Tropomyosin-1 to Promote Cytoskeleton Remodeling in Response to Oxidative Stress: Impact on Membrane Blebbing. Mol. Biol. Cell, 2003, 14:1418～1432.
[21] Sonoda, Y., Watanabe, S., Matsumoto, Y., et al. Fak iis the upstream signal protein of the phosphatidylinositol 3-kinase/Akt survival pathway in hydrogen proxide-induced apoptosis of a human glioblastoma cell line. J. Biol. Chem., 1999, 274: 10566～10570.
[22] Storz, P., Doppler, H. and Toker A. Protein kinase C selectively reglates protein kinase D-dependent activation of NF-κB in oxidative stress signaling. Mol. Cell. Biol., 2004, 24: 2614～2626.
[23] Huang, E.J., and Reichardt, L.F. Neurotrophins: roles in neuronal development and function Annu. Rev. Neurosci., 2001, 24: 677～736.
[24] Miller, F.D., and Kaplan, D.R., Neurotrophin signalling pathways regulating neuronal apoptosis. Cell. Mol. Life Sci., 2001, 58:1045～1053.
[25] Carpenter, C.L. and Cantley, L.C. Phosphoinositide kinases. Current Opinion in Cell Biology, 1996, 8:153～158.
[26] Staal SP, Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcnoma. Proc Nat1 Acad Sci U S A. 1987, 84(14): 5034～5037.
[27] Coffer PJ, Woodgett JR. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991, 201(2): 475～481.
[28] Datta S.R., Brunet A. and Greenberg M.E. Cellular survival: a play in three Akts. Genes Dev., 1991, 13: 2905～2927.
[29] Kandel E.S., Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res., 1999, 253: 210～229.
[30] Matte, B.M. and Downward, J. PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends in biochemical sciences, 1997, 22: 355～358.
[1] 孙大业，郭林燕和马力耕等编著．细胞信号转导．第三版．北京：科学出版社，2001，73～90．
[2] Kandel ES, Hay N. The regulation and activitives of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell. Res., 1999, 253: 210～229.
[3] meier R, Hemmings BA. Regulation of protein kinase B. J. Recept ingual Transduct Res., 1999, 19: 121～128.
[4] Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell, 2000, 103: 211～225.
[5] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system, Int J Dev Neurosci., 2002, 20(3-5): 187～197.
[6] 郑志弦，林玲主编．神经细胞培养理论与实践．第一版．北京：科学出版社，2002．143～147．
[7] Alley MC, Scudiero DA, Monks A, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 1988, 48(3): 589～601.
[8] Yang-ja Lee, Emily Shacter, Oxidative Stress Inhibits Apoptosis in Human Lymphoma Cells. the Journal of Biological Chemistry, 1999, 274(28): 19792～19798.
[9] 郑志竑，林玲主编．神经细胞培养理论与实践．第一版．北京：科学出版社，2002．204～205．
[10] Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J physiol Lung Cell Mol Physiol., 2000, 279: L1005～L1028.
[11] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature, 2000, 408: 239～247.
[12] Mc Cord JM. Oxygen-derived free radicals in postischemic tissue injury. New Eugl J Med, 1985, 321:159
[13] Sugawara T, Lewen A, Gasche Y, et al. Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release. FASEB J. 2002, 16: 1997～1999.
[14] 郑永强，贲昆龙．测定细胞存活和增值的MTF方法的建立．免疫学杂志，1992，8(4)：266～269．
[15] Kerr, J.F., Wyllie, A.H. and Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Canner, 1972, 26: 239～251.
[16] Wyllie, A.H. Apoptosis: an overview, 1997, 53: 451～465.
[17] Sgonc R, Wick G. Methods for the detection of apoptosis, Int Arch Allergy Immunol. 1994, 105(4): 327～332.
[18] 矫毓娟，刘江红综述．细胞凋亡的检测方法，中国神经免疫学和神经病学杂志，2004年1月，11(1)：53～56．
[1] 孙大业，郭林燕和马力耕等编著，细胞信号转导，第三版．北京：科学出版社，2001．73～90．
[2] Kandel ES, Hay N. The regulation and activitives of the multifunctional sefine/threonine kinase Akt/PKB. Exp. Cell. Res., 1999, 253: 210～229.
[3] meier R, Hemmings BA. Regulation of protein kinase B. J. Recept ingnal Transduct Res., 1999, 19: 121～128.
[4] Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell, 2000, 103: 211～225.
[5] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system. Int J Dev Ncurosci, 2002, 20(3-5): 187～197.
[6] Sonoda Y., Watansbe S., Matsumoto Y., et al. FAK is the uptream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathwayin hydrogen peroxide-induce apoptosis of a human glioblastoma cell line. 1999, J. Biol. Chem., 274: 10566～10570.
[7] Wang X., McCullough KD, Franke TF, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. 2000, J. Biol. Chem., 275: 14624～14631.
[8] Klotz LO, Schieke SM, Sies H, et al. Peroxynitdte activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts. Biochem. J., 2000, 352(Ptl): 219～225.
[9] Miura, M., Zhu, H., Totello, R. et al. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.1993, Cell, 75: 653～660.
[10] Nicholson, D.W., Ali, A., Vaillancourt, J.P. et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature, 1995, 376: 37～43.
[11] XQ Tang, JQ Feng, J Chen et al. Protection of oxidative preconditioning against apoptosis induced by H202 in PC12 cells: mechanisms via MMP, ROS, and Bcl-2. Brain Res., 2005, 1057(1-2): 57～64.
[12] BS Mandavilli, Ⅰ Boldogh, and B Van Houten. 3-nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells. Brain Res Mol Brain Res., 2005, 133(2): 215～23.
[13] Christy A. Barlow, Arti Shukla, Brooke T. Mossman and Karen M. Lounsbury. Oxidant-Mediated cAMP Response Element Binding Protein Activation: Calcium Regulation and Role in Apoptosis of Lung Epithelial Cells. Am. J. Respir. Cell Mol. Biol., 2006, 34: 7～14.
[14] Yang E, Zha J, Jockel J, Boise L.H, et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell, 1995, 80: 285～291.
[15] Venugopalan D.N., Tony Yuen, C. Warren Olanow, et al. Early single cell bifurcation of proand antiapoptotic states during oxidative stress. J. Bio. Chem., 2004, 279(26): 27494～27501.
[16] Hongbo R.L, Hidenori H. Mir A.H., et al. Akt as a mediator of cell death. PNAS, 2003, 100(20): 11712～11717.
[17] Yin X.M. and Korsmeyer S.J., Molecular thanatopsis: a discourse on the Bcl-2 family and cell death. Blood, 1996,.88(2): 386～401.
[18] Yin X.M., Oltvai Z.N. and Korsmeyer S.J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature, 1994, 369(6478): 321～323.
[1] Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg, 1991, 75: 15～26.
[2] Jennifer L.Martindale, Nikkl LHolbrook. Cellular Response to Oxidativ Stress.Signaling for Suicide and Survival. Journal of Cellular Physiology, 2002,192:1-15.
[3] Tator CH, Fehlings MG Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosury, 1991,75:15-26.
[4] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system. Int J Dev Neurosci, 2002,20(3-5): 187～197.
[5] Hogan, EL., Banik, NL. and Hsu, CY. Calcium activated mediators in secondary injury in experimental spinal cord injury. CNS Trauma, 1986,3:175～180.
[6] Tator, CH. and Fehlings, MG Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J. Neurosurg., 1991,75:15～26.
[7] Feigin, I. The pathological changes produced by focal cerebral anoxia. Res. Publ. Assoc. Res.Nerv. Menu Dis., 1966,41:23～29.
[8] Alclala, H., Gado, M. and Torack. R.M. The effect of size histological elements and water content in the visualization of cerebral infarcts. Arch. Neurol., 1978,35:1～7.
[9] Balentine. J.D. Hypotheses in spinal cord trauma research. In Central Nervous System Trauma Status Report, 1985, pp. 455～461.
[10] Young, W. Secondary CNS injury. J. Neurotrauma, 1988,5:219～221.
[11] Xu, J., Hsu, C.Y., Liu, T.H., et al. Leukotriene B4 release and polymorphonuclear cell infiltration in spinal cord injury. J. Neurochem., 1990,55:907～912.
[12] Hamada, Y., lkata, T., Katoh, S.et al. Involvement of an intracellulat adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats. J. Neutochem., 1996,66:1525～1531.
[13] Tator, C.H. and Fehlings, M.G Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J. Neurosurg., 1991,75:15～26.
[14] Harlan, J.M. Consequences of leukocytes vessel wall interactions in inflammatory and immune reactions. Semin. Thromb. Hemost., 1987,13:425～433.
[15] Abe, H., Okajima, K. and Okabe, H. Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. J.Lab. Clin. Med.,1994,23: 874～881.
[16] Katoh, S., lkata, T, Tsubo, M., et al. Inducton and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci. Lett., 1996, 216: 9～12.
[17] Ong C, Arnold PM, Zoubine MN, et al. apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma, 1998,15:459～472.
[18] Akurai M, Hayashi T, Abek, et al. Delayed and selective motor neuron death after transient spinal cord ischemia:a role of apoptosis? J Thorac Cardiovasc Surg., 1998,115:1310～1315.
[19] Abe Y, Yamamoto T, Sugiyama Y, et al. Apoptotic cells associated with Walleri and egeneration after experimental spinal cord injury: apossible mechanism of oligodendroglial death. J Neurotrauma, 1999,16:945～952.
[20] Liu, X. Z., Xu, X. M., Hu, R. et al. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci., 1997, 17: 5395～5406.
[21] Lennon SV, Martin SJ, Cotter TG. Does-dependent induction of apoptosis in human tumor or cell lines by widely diverging stiumuli. Cell prolif., 1991, 24: 203～214.
[22] 高颖，许彩民，杨洋．氧化诱导K652细胞凋亡机制的初步探讨．中国生物化学与分子生物学学报，1998，14(3)：295～299．
[23] Ishisaka R, Utsumi K, Utsurni T. Involvement of lysosomal cysteine protease in hydrogen peroxide-induced apoptosis in HL-60 cells. Biosci Biotechnol Biochem., 2002, 66(9):1865～1872.
[24] 王爱国，冉鹏，周明辉等．氟中毒氧化应激与细胞凋亡关系的研究．中国公共卫生，2002，18(6)：681～682．
[25] Murakawa M, Jung SK, Lijima K, et al. Apoptosis inducing protein AIP, from parasite-infected fish induces apoptosis in mammaliam cells by two different molecular mechanisms. Cell Death Differentation, 2001, 8: 298～307.
[26] Emanuele S, Galvaruso G, Lauricella M, et al. Apoptosis induced in hepatoblastoma HepG2 cells by the protcasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3. Int J Oncok., 2002, 21(4): 857～865.
[27] Tian B, Liu J, Bitterman PB, et al. Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis. Am J Physiol Heart Cire Physiol., 2002, 283(5): H1958～H1967.
[28] Woo SH, Park IC, Park MJ, et al. Arsenic trioxide induces apoptosis through a reactive oxygen species-dependent pathway and loss of mitochondrial membrane potential in Hela cells. Int J Oncol., 2002, 21(1): 57～63.
[29] Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radio Biol Med., 2001, 31(3): 367～373
[30] Steinman HM, The Bcl-2 oncoprotein functions as a pro-oxidant. J Biolchem., 1995, 270:3487～3490.
[31] Lotharius J, Dugan LL, O'Malley KL. Distinct mechanisms underlieneurotoxin mediated cell death in cultured dopaminergic neurons. J Neuro Sci., 1999, 19(4): 1284～1293.
[32] Angkeow P, Deshpande SS, Qi B, et al. Redox fatorl: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ., 2002, 9: 717～725.
[33] Deshpande SS, Angkeow P, Huang J, et al. Racl inhibs TNF-alpha induced endothelial cell apoptosis: dual regulation by reactive oxygen species, FASB J., 2000, 14: 1705～1714.
[34] Zamzarni N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med., 1995, 181: 1661～1672.
[35] Carmody RJ, Cotter TG, Oxidative stress induces caspase-independent retinal apoptosis in vitro. Cell Death Differentiation, 2000, 7: 282～291.
[36] Kulms D, Schwarz T. Independent contribution of three different pathways to ultraviolet B-induced apoptosis. Biochem Pharmacol., 2002, 64(5-6): 837～847.
[37] Kulms D, Schwarz T. Molecular mechanisms involved in UV-indued apoptotic cell death. Skin pharmacol Appl Skin Physiol., 2002, 15(5): 342～347.
[38] Green D, Kroemer G.The central executioners of apoptosis: caspase or mitochondria? Trends Cell Biol., 1998, 8: 267～271.
[39] Matzo Ⅰ, Susin SA, Petit PX, et al. Caspases disrupt mitochondrial membrane barrier functin. FEBS Lett., 1998, 427: 198～202.
[40] Mirella T, Marco G, Angelo C, et al. A P53-P66 signaling pathway controls intracellular redox status, levels of oxidation damaged DNA and oxidative stress induced apoptosis. Oncogene, 2002, 21: 3872～3878.
[41] Schmitt CA, Lowe SW. Apoptosis and therapy. J Patho., 1999, 187: 127～137.
[42] Martin D, Salinas M, Fujita N, et al. ceramide and reactive oxygen species generated by H202 induce caspase-3 independent degradation ofAkt/PKB. J Bio Chem., 2002, 3: 18.
[43] Seoane J, Le HV, Massague J. Myc suppression of P21(Cipl) Cdk inhibitor influences the outcome of the P53 response to DNA damage. Nature, 2002, 419(6908): 729～734.
[44] Liu G, Chen X. The ferredoxin reductase gene is regulated by the P53 family and sensitizes cells to oxidative stress-induced apoptosis. Oncogene, 2002, 21(47): 7195～7204.
[45] Ma XL, Kumar S, Gao F, et al. Inhibition of P38 mitogen activated prtein kinase decrease cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation, 1999, 99(13): 1685～1691.
[46] Inoshita S, Takeda K, Hatai T, et al. Phosphorylation and inactivation of Mcl-1 by c-Jun N-teminal kinase(JNK) in response to oxidative stress. J Biol Chem., 2002, 9: 168.
[2] Thannickal V.J. and Fanburg B.L. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L1005～L1028.
[3] Mc Cord JM. Oxygen-derived free radicals in postischemic tissue injury. New Eugl J Meal, 1985, 321:159
[4] 高颖，许彩民，杨洋．氧化诱导K652细胞凋亡机制的初步探讨．中国生物化学与分子生物学学报，1998，14(3)：295～299．
[5] Ishisaka R, Utsumi K, Utsumi T. Involvement of lysosomal cysteine protease in hydrogen peroxide-induced apoptosis in HL-60 cells. Biosci Biotechnol Biochem, 2002, 66(9):1865～1872.
[6] 王爱国，冉鹏，周明辉等．氟中毒氧化应激与细胞凋亡关系的研究．中国公共卫生，2002，18(6)：681～682．
[7] Murakawa M, Jung SK, Lijima K, et al. Apoptosis inducing protein AIP, from parasite-infected fish induces apoptosis in mammaliam cells by two different molecular mechanisms. Cell Death Differentation, 2001, 8: 298～307.
[8] Emanuele S, Galvaruso G, Lauricella M, et al. Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3. Int J Oncok, 2002, 21(4): 857～865.
[9] Tian B, Liu J, Bitterman PB, et al. Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis. Am J Physiol Heart Cite Physiol, 2002, 283(5): H1958～H1967
[10] Kerr, J.F., Wyllie, A.H. and Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Caner, 1972, 26: 239～257.
[11] Thornberry NA, Lazebnik Y. Caspase: enemies within. Science, 1998, 281(5381):1312～1316.
[12] Alnemri E.S., Livingston D.J.,Nicholson D.W. et al. Human ICE/CED-3 protease nomenclature. Cell, 1996, 87(2): 171.
[13] Nagata S. Apoptosis by death factor. Cell, 1997, 88(3): 355～365.
[14] Li G.L., Brodin G., Farooque M. et al. Apoptosis.and expression of Bcl-2 after compression trauma to rat spinal cord. J. Neuropathol. Exp. Neurol., 1996, 55: 280～289.
[15] Tsujimoto Y., Finger L.R., Yunis J. et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science, 1984, 226(4678): 1097～1099.
[16] Li GI, Brodin G, Farooque M, et al. Apoptosis and expression of bcl-2 after compression trauma to rat spinal cord. J Neuropathol Exp Neurol., 1996, 55: 280～289.
[17] Ray SK, Matzeue DD, Wilford GG, et al. Cell death in spinal cord injury(SCl) requires denovo protein synthesis. Ann N YAcad Sci., 2001, 939: 436～449.
[18] 傅强，侯铁胜，鲁凯伍等．大鼠脊髓急性损伤后神经细胞凋亡及相关基因表达．中国脊柱脊髓杂志，2001，11：92～94．
[19] Finkel T and Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Nature, 2000, 408: 239～247.
[20] Houle, F., Rousseau, S., Mortice, N., Lue, M. et al. Extracellular Signal-regulated Kinase Mediates Phosphorylation of Tropomyosin-1 to Promote Cytoskeleton Remodeling in Response to Oxidative Stress: Impact on Membrane Blebbing. Mol. Biol. Cell, 2003, 14:1418～1432.
[21] Sonoda, Y., Watanabe, S., Matsumoto, Y., et al. Fak iis the upstream signal protein of the phosphatidylinositol 3-kinase/Akt survival pathway in hydrogen proxide-induced apoptosis of a human glioblastoma cell line. J. Biol. Chem., 1999, 274: 10566～10570.
[22] Storz, P., Doppler, H. and Toker A. Protein kinase C selectively reglates protein kinase D-dependent activation of NF-κB in oxidative stress signaling. Mol. Cell. Biol., 2004, 24: 2614～2626.
[23] Huang, E.J., and Reichardt, L.F. Neurotrophins: roles in neuronal development and function Annu. Rev. Neurosci., 2001, 24: 677～736.
[24] Miller, F.D., and Kaplan, D.R., Neurotrophin signalling pathways regulating neuronal apoptosis. Cell. Mol. Life Sci., 2001, 58:1045～1053.
[25] Carpenter, C.L. and Cantley, L.C. Phosphoinositide kinases. Current Opinion in Cell Biology, 1996, 8:153～158.
[26] Staal SP, Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcnoma. Proc Nat1 Acad Sci U S A. 1987, 84(14): 5034～5037.
[27] Coffer PJ, Woodgett JR. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991, 201(2): 475～481.
[28] Datta S.R., Brunet A. and Greenberg M.E. Cellular survival: a play in three Akts. Genes Dev., 1991, 13: 2905～2927.
[29] Kandel E.S., Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res., 1999, 253: 210～229.
[30] Matte, B.M. and Downward, J. PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends in biochemical sciences, 1997, 22: 355～358.
[1] 孙大业，郭林燕和马力耕等编著．细胞信号转导．第三版．北京：科学出版社，2001，73～90．
[2] Kandel ES, Hay N. The regulation and activitives of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell. Res., 1999, 253: 210～229.
[3] meier R, Hemmings BA. Regulation of protein kinase B. J. Recept ingual Transduct Res., 1999, 19: 121～128.
[4] Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell, 2000, 103: 211～225.
[5] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system, Int J Dev Neurosci., 2002, 20(3-5): 187～197.
[6] 郑志弦，林玲主编．神经细胞培养理论与实践．第一版．北京：科学出版社，2002．143～147．
[7] Alley MC, Scudiero DA, Monks A, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 1988, 48(3): 589～601.
[8] Yang-ja Lee, Emily Shacter, Oxidative Stress Inhibits Apoptosis in Human Lymphoma Cells. the Journal of Biological Chemistry, 1999, 274(28): 19792～19798.
[9] 郑志竑，林玲主编．神经细胞培养理论与实践．第一版．北京：科学出版社，2002．204～205．
[10] Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J physiol Lung Cell Mol Physiol., 2000, 279: L1005～L1028.
[11] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature, 2000, 408: 239～247.
[12] Mc Cord JM. Oxygen-derived free radicals in postischemic tissue injury. New Eugl J Med, 1985, 321:159
[13] Sugawara T, Lewen A, Gasche Y, et al. Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release. FASEB J. 2002, 16: 1997～1999.
[14] 郑永强，贲昆龙．测定细胞存活和增值的MTF方法的建立．免疫学杂志，1992，8(4)：266～269．
[15] Kerr, J.F., Wyllie, A.H. and Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Canner, 1972, 26: 239～251.
[16] Wyllie, A.H. Apoptosis: an overview, 1997, 53: 451～465.
[17] Sgonc R, Wick G. Methods for the detection of apoptosis, Int Arch Allergy Immunol. 1994, 105(4): 327～332.
[18] 矫毓娟，刘江红综述．细胞凋亡的检测方法，中国神经免疫学和神经病学杂志，2004年1月，11(1)：53～56．
[1] 孙大业，郭林燕和马力耕等编著，细胞信号转导，第三版．北京：科学出版社，2001．73～90．
[2] Kandel ES, Hay N. The regulation and activitives of the multifunctional sefine/threonine kinase Akt/PKB. Exp. Cell. Res., 1999, 253: 210～229.
[3] meier R, Hemmings BA. Regulation of protein kinase B. J. Recept ingnal Transduct Res., 1999, 19: 121～128.
[4] Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell, 2000, 103: 211～225.
[5] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system. Int J Dev Ncurosci, 2002, 20(3-5): 187～197.
[6] Sonoda Y., Watansbe S., Matsumoto Y., et al. FAK is the uptream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathwayin hydrogen peroxide-induce apoptosis of a human glioblastoma cell line. 1999, J. Biol. Chem., 274: 10566～10570.
[7] Wang X., McCullough KD, Franke TF, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. 2000, J. Biol. Chem., 275: 14624～14631.
[8] Klotz LO, Schieke SM, Sies H, et al. Peroxynitdte activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts. Biochem. J., 2000, 352(Ptl): 219～225.
[9] Miura, M., Zhu, H., Totello, R. et al. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.1993, Cell, 75: 653～660.
[10] Nicholson, D.W., Ali, A., Vaillancourt, J.P. et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature, 1995, 376: 37～43.
[11] XQ Tang, JQ Feng, J Chen et al. Protection of oxidative preconditioning against apoptosis induced by H202 in PC12 cells: mechanisms via MMP, ROS, and Bcl-2. Brain Res., 2005, 1057(1-2): 57～64.
[12] BS Mandavilli, Ⅰ Boldogh, and B Van Houten. 3-nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells. Brain Res Mol Brain Res., 2005, 133(2): 215～23.
[13] Christy A. Barlow, Arti Shukla, Brooke T. Mossman and Karen M. Lounsbury. Oxidant-Mediated cAMP Response Element Binding Protein Activation: Calcium Regulation and Role in Apoptosis of Lung Epithelial Cells. Am. J. Respir. Cell Mol. Biol., 2006, 34: 7～14.
[14] Yang E, Zha J, Jockel J, Boise L.H, et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell, 1995, 80: 285～291.
[15] Venugopalan D.N., Tony Yuen, C. Warren Olanow, et al. Early single cell bifurcation of proand antiapoptotic states during oxidative stress. J. Bio. Chem., 2004, 279(26): 27494～27501.
[16] Hongbo R.L, Hidenori H. Mir A.H., et al. Akt as a mediator of cell death. PNAS, 2003, 100(20): 11712～11717.
[17] Yin X.M. and Korsmeyer S.J., Molecular thanatopsis: a discourse on the Bcl-2 family and cell death. Blood, 1996,.88(2): 386～401.
[18] Yin X.M., Oltvai Z.N. and Korsmeyer S.J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature, 1994, 369(6478): 321～323.
[1] Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg, 1991, 75: 15～26.
[2] Jennifer L.Martindale, Nikkl LHolbrook. Cellular Response to Oxidativ Stress.Signaling for Suicide and Survival. Journal of Cellular Physiology, 2002,192:1-15.
[3] Tator CH, Fehlings MG Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosury, 1991,75:15-26.
[4] Rodgers EE, Theibert AB. Functions of PI 3-kinase in development of the nervous system. Int J Dev Neurosci, 2002,20(3-5): 187～197.
[5] Hogan, EL., Banik, NL. and Hsu, CY. Calcium activated mediators in secondary injury in experimental spinal cord injury. CNS Trauma, 1986,3:175～180.
[6] Tator, CH. and Fehlings, MG Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J. Neurosurg., 1991,75:15～26.
[7] Feigin, I. The pathological changes produced by focal cerebral anoxia. Res. Publ. Assoc. Res.Nerv. Menu Dis., 1966,41:23～29.
[8] Alclala, H., Gado, M. and Torack. R.M. The effect of size histological elements and water content in the visualization of cerebral infarcts. Arch. Neurol., 1978,35:1～7.
[9] Balentine. J.D. Hypotheses in spinal cord trauma research. In Central Nervous System Trauma Status Report, 1985, pp. 455～461.
[10] Young, W. Secondary CNS injury. J. Neurotrauma, 1988,5:219～221.
[11] Xu, J., Hsu, C.Y., Liu, T.H., et al. Leukotriene B4 release and polymorphonuclear cell infiltration in spinal cord injury. J. Neurochem., 1990,55:907～912.
[12] Hamada, Y., lkata, T., Katoh, S.et al. Involvement of an intracellulat adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats. J. Neutochem., 1996,66:1525～1531.
[13] Tator, C.H. and Fehlings, M.G Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J. Neurosurg., 1991,75:15～26.
[14] Harlan, J.M. Consequences of leukocytes vessel wall interactions in inflammatory and immune reactions. Semin. Thromb. Hemost., 1987,13:425～433.
[15] Abe, H., Okajima, K. and Okabe, H. Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. J.Lab. Clin. Med.,1994,23: 874～881.
[16] Katoh, S., lkata, T, Tsubo, M., et al. Inducton and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci. Lett., 1996, 216: 9～12.
[17] Ong C, Arnold PM, Zoubine MN, et al. apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma, 1998,15:459～472.
[18] Akurai M, Hayashi T, Abek, et al. Delayed and selective motor neuron death after transient spinal cord ischemia:a role of apoptosis? J Thorac Cardiovasc Surg., 1998,115:1310～1315.
[19] Abe Y, Yamamoto T, Sugiyama Y, et al. Apoptotic cells associated with Walleri and egeneration after experimental spinal cord injury: apossible mechanism of oligodendroglial death. J Neurotrauma, 1999,16:945～952.
[20] Liu, X. Z., Xu, X. M., Hu, R. et al. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci., 1997, 17: 5395～5406.
[21] Lennon SV, Martin SJ, Cotter TG. Does-dependent induction of apoptosis in human tumor or cell lines by widely diverging stiumuli. Cell prolif., 1991, 24: 203～214.
[22] 高颖，许彩民，杨洋．氧化诱导K652细胞凋亡机制的初步探讨．中国生物化学与分子生物学学报，1998，14(3)：295～299．
[23] Ishisaka R, Utsumi K, Utsurni T. Involvement of lysosomal cysteine protease in hydrogen peroxide-induced apoptosis in HL-60 cells. Biosci Biotechnol Biochem., 2002, 66(9):1865～1872.
[24] 王爱国，冉鹏，周明辉等．氟中毒氧化应激与细胞凋亡关系的研究．中国公共卫生，2002，18(6)：681～682．
[25] Murakawa M, Jung SK, Lijima K, et al. Apoptosis inducing protein AIP, from parasite-infected fish induces apoptosis in mammaliam cells by two different molecular mechanisms. Cell Death Differentation, 2001, 8: 298～307.
[26] Emanuele S, Galvaruso G, Lauricella M, et al. Apoptosis induced in hepatoblastoma HepG2 cells by the protcasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3. Int J Oncok., 2002, 21(4): 857～865.
[27] Tian B, Liu J, Bitterman PB, et al. Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis. Am J Physiol Heart Cire Physiol., 2002, 283(5): H1958～H1967.
[28] Woo SH, Park IC, Park MJ, et al. Arsenic trioxide induces apoptosis through a reactive oxygen species-dependent pathway and loss of mitochondrial membrane potential in Hela cells. Int J Oncol., 2002, 21(1): 57～63.
[29] Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radio Biol Med., 2001, 31(3): 367～373
[30] Steinman HM, The Bcl-2 oncoprotein functions as a pro-oxidant. J Biolchem., 1995, 270:3487～3490.
[31] Lotharius J, Dugan LL, O'Malley KL. Distinct mechanisms underlieneurotoxin mediated cell death in cultured dopaminergic neurons. J Neuro Sci., 1999, 19(4): 1284～1293.
[32] Angkeow P, Deshpande SS, Qi B, et al. Redox fatorl: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ., 2002, 9: 717～725.
[33] Deshpande SS, Angkeow P, Huang J, et al. Racl inhibs TNF-alpha induced endothelial cell apoptosis: dual regulation by reactive oxygen species, FASB J., 2000, 14: 1705～1714.
[34] Zamzarni N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med., 1995, 181: 1661～1672.
[35] Carmody RJ, Cotter TG, Oxidative stress induces caspase-independent retinal apoptosis in vitro. Cell Death Differentiation, 2000, 7: 282～291.
[36] Kulms D, Schwarz T. Independent contribution of three different pathways to ultraviolet B-induced apoptosis. Biochem Pharmacol., 2002, 64(5-6): 837～847.
[37] Kulms D, Schwarz T. Molecular mechanisms involved in UV-indued apoptotic cell death. Skin pharmacol Appl Skin Physiol., 2002, 15(5): 342～347.
[38] Green D, Kroemer G.The central executioners of apoptosis: caspase or mitochondria? Trends Cell Biol., 1998, 8: 267～271.
[39] Matzo Ⅰ, Susin SA, Petit PX, et al. Caspases disrupt mitochondrial membrane barrier functin. FEBS Lett., 1998, 427: 198～202.
[40] Mirella T, Marco G, Angelo C, et al. A P53-P66 signaling pathway controls intracellular redox status, levels of oxidation damaged DNA and oxidative stress induced apoptosis. Oncogene, 2002, 21: 3872～3878.
[41] Schmitt CA, Lowe SW. Apoptosis and therapy. J Patho., 1999, 187: 127～137.
[42] Martin D, Salinas M, Fujita N, et al. ceramide and reactive oxygen species generated by H202 induce caspase-3 independent degradation ofAkt/PKB. J Bio Chem., 2002, 3: 18.
[43] Seoane J, Le HV, Massague J. Myc suppression of P21(Cipl) Cdk inhibitor influences the outcome of the P53 response to DNA damage. Nature, 2002, 419(6908): 729～734.
[44] Liu G, Chen X. The ferredoxin reductase gene is regulated by the P53 family and sensitizes cells to oxidative stress-induced apoptosis. Oncogene, 2002, 21(47): 7195～7204.
[45] Ma XL, Kumar S, Gao F, et al. Inhibition of P38 mitogen activated prtein kinase decrease cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation, 1999, 99(13): 1685～1691.
[46] Inoshita S, Takeda K, Hatai T, et al. Phosphorylation and inactivation of Mcl-1 by c-Jun N-teminal kinase(JNK) in response to oxidative stress. J Biol Chem., 2002, 9: 168.