摘要
目的研究MPP~+作用于PC12细胞48小时后蛋白质表达谱的改变,进一步从蛋白质水平上阐明帕金森病的发病机制。方法建立符合国际标准的MPP~+诱导的PC12细胞PD模型,采用MTT法检测细胞存活率,荧光染色法检测细胞凋亡百分率;提取MPP~+作用48小时后的PC12细胞总蛋白,采用差异凝胶电泳(2D-DIGE)技术分析蛋白表达谱的改变,获得蛋白点差异表达的信息;利用质谱(MALDI-TOF)技术鉴定MPP~+引起的差异表达的蛋白点。结果首次发现并鉴定了MPP~+诱导的PC12细胞PD模型中的蛋白质组学改变。MPP~+处理PC12细胞48小时后共有32个蛋白表达发生显著改变。
质谱共鉴定出7个蛋白。所鉴定的差异表达蛋白可分为5类:①具有分子伴侣活性的蛋白:新生多肽相关复合物α-多肽(nascent polypeptide associate-ed complex,α-NAC)、晶体蛋白(crystallin),这两个蛋白的表达显著下降;②细胞骨架蛋白:神经细丝蛋白轻链多肽(neurofilament light polypeptide,NF-L)表达量显著上调;③ERM家族的埃兹蛋白(ezrin),它的表达量显著上调;④与氧化应激、线粒体功能相关的蛋白:硫氧还原蛋白(thioredoxin,Trx)和线粒体加工肽酶(mitochondrial processing peptidases,MPPs),这两种蛋白的表达量均显著上升;⑤免疫炎症相关的蛋白:补体结合糖蛋白(complement binder glycoprotein,gC1qBP),表达量显著下降。这7个蛋白中除硫氧还原蛋白之外,其余均是首次用蛋白质组学方法在MPP~+诱导的PC12细胞PD模型中发现的,揭示了这些蛋白的差异改变可能与PD的发病机制相关。对这些蛋白的深入研究将使我们更好地理解PD的发病机制。这些蛋白也有可能成为新的药物作用靶点。
Part I The cytotoxicity of MPP~+ and the construction of the Parkinson's disease model
Objective: To explore the cytotoxicity of MPP~+ and construct the PD model according to the interenational standard. Methods: PC12 cells were treated with different concentrations of MPP~+(50, 100, 200, 250, 300, 500μM) for 12, 24, 48, 72, 96h, respectively. Cell viability was measured by MTT and apoptotic percentage was determined by fluorescent staining. Results: After treatment with 250μM MPP~+ for 48 hour, the PC12 cell viability decreased to 60.14% and the cell apoptotic rate increased to 9.1%. Conclusion: MPP~+ is a cytotoxic substance, which can down-regulate the cell viability and induce apoptosis. Significance: An interentional standard cell model of PD was established, which sets up a reliable basis for the study of MPP~+ mechanisms at proteomic level.
Part II Proteomic study of PC12 cell model of Parkinson's disease induced by MPP~+
Objective: To explore the mechanism of MPP~+ cytotoxicity at preteome level and find new clues for the pathogensis of PD. Method: PC12 cells were treated with or without 250μM MPP~+ for 48 hours, differential expressions of protein spots from two groups were analysed with two dimensional difference gel electrophoresis(2D-DIGE) , proteins were identified by matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometer(MALDI-TOF MS). Result: About 2000 protein spots were seen in each of 2D-DIGE images. The expressions of 32 proteins were significantly changed in MPP~+ treated PC12 cells compared with the control. Among them, 15 proteins were down-regulated and 17 were up-regulated more than 30%. 7 proteins were identified by MALDI-TOF and can be classied into 5 categories:①proteins with chaperone activity : nascent polypeptide-associated complexα-polypeptide and crystallin, both of them were down-regulated;②cytoskeletal protein: neurofilament light polypeptide, it's expression was up-regulated;③the protein of ERM family, ezrin, which was also up-regulated;④oxidative stress and mitochondrial function related proteins: thioredoxin, mitochondrial processing peptidase, each of them was up-regulated;⑤immunoinflama-tion relaterd protein: complement binder glycoprotein, it's expression was down-regulated. Significance: DIGE and MS technology were used in the investigation the PC12 model of PD induced with MPP~+ for the first time and our findings provide new clues for the etiopathogenesis of PD and the candidates of therapy targets.
引文
[1] Often D, Ziv I, Barzilai A, et al. Dopamine-melanin induces apoptosis in PC12 cells; possible implications for the etiology of Parkinson's disease. Neurochem. 1997; 31: 207-216.
[2] Olanow CW, Tatton WG. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci.1999; 22: 123-144.
[3] Brooks AI, Chadwick CA, Gelbard HA, et al. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res.1999; 823:1-10.
[4] Javitch JA, D'Amato RJ, Strittmatter SM, et al. Parkinsonism inducing neurot-oxin, 1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity. Proc Natl Acad Sci USA. 1985; 82:2173-2177.
[5] Hartley A, Stone JM, Heron C, et al. Complex Ⅰ inhibitors induce dose dependent apoptosis in PC12 cells: relevance to Parkinson's disease. Neurochem. 1994; 63:1987-90.
[6] Jha N. Glutathione decreases in dopaminergic PC12 cells interfere with the ubiquitin protein degradation pathway: relevance for Parkinson's disease? J. Neurochem.2002; 80: 555-561.
[7] Wang J. Doxycycline-regulated coexpression of GDNF and TH in PC12 cells. Neurosci Lett. 2006; 401: 142-145.
[8] Beal MF. Energetics in the pathogenesis ofneurodegenerative disease. Trends Neurosci. 2000; 23: 298.
[9] Fiskum G, Starkov A, Polster BM, et al.Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease. Ann NY Acd Sci. 2003; 991: 111-119.
[10] Tompkins MM, Basgall EJ, Zanrini E, et al. Apoptotic like changes in Lewy body associated disorders and normal aging in substantia nigral neurons. Am Pathol. 1997; 150: 119-131.
[11] Hartmann A, Michel PP, Troadec JD, et al. Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?. Neurochem. 2001; 76: 1785-1793.
[12] Anglade P, Vyas S, Javoy A F, et al. Apoptosis andautophagy in nigral neurons of patients with Parkinson's disease. Histol Histopathol. 1997; 12: 25-31.
[13] Wasinger VC, Cordwell SJ, Cerpa-Poljak A, et al. Progress with gene product mapping of the molicutes: Mycoplasma genitalium. Electrophoresis. 1995; 16: 1090-1094.
[14] Abbott A. Brain protein project enlists mice in'dry run'. Nature. 2003; 425:110-110.
[15] Beatrix B, Sakai H. and Wiedmann M. The alpha and beta subunit of the nascent polypeptide-associated complexhave distinct functions. Biol Chem. 2000; 275: 37838-37845.
[16] Wiedmann B, Sakai H, Davis T A, et al. A protein complex requireed for signal-sequence specific sorting and translocation. Nature. 1994; 370:434-440.
[17] Wang S, Sakai H. and Wiedmann M. NAC covers ribosome associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidy transferase center. Cell Biol. 1995; 130: 519-528.
[18] Lauring B, Kreibich G. and Weidmann M. The intrinsicability of ribosomes to bind to endoplasmic reticulum membranesis regulated by signal recognition particle and nascent polypeptide associated complex. Proc Natl Acad Sci USA.1995; 92: 9435-9439.
[19] Rospert S, Dubaquie Y, Gautschi M, et al. Nascent-polypeptide associateed complex. Cell Mol Life Sci. 2002; 59: 1632-1639.
[20] Kim SH, Shim KS, Lubec G., et al. Human brain nascent polypeptide associated complex alpha subunit is decreased in patients with Alzheimer's disease and Down syndrome. Investig Med. 2002; 50: 293-301.
[21] Hoffrogge R, Beyer S, Hubner R, et al. 2-DE profiling of GDNF over expression related proteome changes in differentiating ST14A rat progenitor cells. Proteomics. 2007; 7:33-46
[22] Munz B, Wiedmann M, Lochmuller H, et al. Cloning of novel injury regulated genes. Implications for animportant role of the muscle specific protein skNAC in muscle repair. Biol Chem. 1999; 274:13305-13310.
[23] Krzysztof L and Dorota W. The distinctive role of small heat shock proteins in oncogenesis. Arch Immunol Ther Exp. 2006; 54:103-111.
[24] De Jong WW, Caspers G.J. and Leunissen JA. Genealogy of the alpha-crystallin small heat shock protein superfamily. Biol Macromol. 1998; 22: 151-162.
[25] Lowe J, McDermott H, Pike I, et al. Alpha B crystallin expression in non-lenticular tissues and selective presence in ubiquitinated inclusion bodies in human disease. Pathol. 1992; 166: 61-68.
[26] Reddy GB, Kumar PA, Kumar MS, et al.Chaperone like activity and hydropho-bicity of alpha-crystallin. IUBMB Life. 2006; 58:632-641.
[27] Ganadu ML, Aru M, Mura GM, et al. Effects of divalent metal ions on the alpha B-crystallin chaperone-like activity: spectroscopic evidence for a complex between copper(Ⅱ) and protein. Inorg Biochem. 2004; 98: 1103-1109.
[28] Deepa V, Dabir M, John Q, et al. Expression of the Small Heat-Shock Protein α B-Crystallin in Tauopathies with Glial Pathology, Am J. Pathol. 2004; 164: 155-166.
[29] Bajramovic JJ,Lassmann H,van Noort JM, et al. Expression of α B-crystallin in glia cells during lesional development in multiple sclerosis. Neuroimmun 1977; 78: 143-151.
[30] Braak H, Del Tredici K, Sandmann-Kiel D,et al. Nerve cells expressing heat shock proteins in Parkinson's disease. Acta Neuropathol. 2001; 102: 449-454.
[31] Schultz C, Dick E J, Cox AB, et al. Expression of stress proteins alpha B-crystallin, ubiquitin, and hsp27 in pallido nigral spheroids of aged rhesus monkeys. Neurobiol Aging. 2001; 22: 677-682.
[32] Renkawek K, Stege GJ and Bosman GJ. Dementia, gliosis and expression of the small heat shock proteins hsp27 and alpha B-crystallin in Parkinson's disease. Neuroreport. 1999; 10: 2273-6.
[33] Lariviere RC. and Julien JP. Functions of intermediate filaments in neuronal development and disease. Neurobiol. 2004; 58:131-148.
[34] Shaw G 0 Neurofilament proteins. In: The Neuronal Cytoskeleton. Burgoyne R. D. (ed.), Wiley, New York. 1991; pp 5-74.
[35] Hsieh ST, Kidd GJ, Crawford TO, et al. Regional modulation of neurofilament organization by myelination in normal axons. Neurosci. 1994; 1:6392-6401
[36] Liu Q, Xie F, Siedlak SL, et al. Biomedicine and Diseases: Review Neurofilament proteins in neurodegenerative diseases. CMLS. Cell. Mol. Life Sci. 2004; 61: 3057-3075.
[37] Weinreb O, Drigues N, Sagi Y, et al. The Application of Proteomics and Genomics to the Study of Age-Related Neurodegeneration and Neuroprotection. Antioxid Redox Signal. 2007; Jan 1. [Epub ahead of print].
[38] Yabe JT, Wang FS, Chylinski T, et al. Selective accumulation of the high molecu-lar weight neurofilament subunit within the distal region of growing axonal neurites. Cell Motil Cytoskeleton. 2001; 50: 1-12.
[39] Hill WD, Arai M, Cohen JA, et al. Neurofilament mRNA is reduced in Parkinson's disease substantia nigra pars compacta neurons. Comp. Neurol. 1993; 329: 328-336.
[40] Han F, Bulman DE, Panisset M, et al. Neurofilament M gene in a French-Cana-dian population with Parkinson's disease. Can Neurol Sci. 2005; 32: 68-70.
[41] Xu Z, Cork LC, Griffin J. et al. Increased expression of neurofilament subunit NF-L produces morphological alerations that resemble the pathology of human motor neuron disease. Cell. 1993; 73: 23-33.
[42] Zhu Q, Couillard DS, Julien JP. Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol. 1997; 148: 299-316.
[43] Levavasseur F, Zhu Q, Julien JP, et al. No requirementof a internexin for nervous system development and for radialgrowth of axons. Mol Brain Res.1999; 69: 104-112.
[44] Chalabi A, Christopher C, Miller J. Neurofilaments and neurological disease. Bio Essays. 25; 4: 346-355.
[45] Giovannoni G.. Multiple sclerosis cerebrospinal fluid biomarkers. Dis Markers. 2006; 22: 187-196.
[46] Fabiana CM, YT, Erica LK, et al. Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia. Proc Natl Acad Sci U S A. 2004; 101: 17705-17710.
[47] Takeshi M, Masato M, Yoshinori D, et al. Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association. Cell Biol. 1998; 140: 647-657.
[48] Barret C, Roy C, Montcourrier P, et al. Mutagenesisof the phosphatidylinositol 4,5-bisphosphate (PIP(2)) bindingsite in the NH(2)-terminal domain of ezrin correlates with its alteredcellular distribution. Cell Biol. 2000; 151: 1067-1080.
[49] Pietromonaco SF, Simons PC, Altman A, et al. Protein kinase C-theta phosphory-lation of moesin in the actin-binding sequence. Biol Chem. 1998; 273: 7594.
[50] Fais S. A role for ezrin in a neglected metastatic tumor function. Trends Mol Med. 2004; 10: 249-50.
[51] Kreimann EL, Morales FC, de Orbeta-Cruz J. Cortical stabilization of beta cate-nin contributes to NHERF1/EBP50 tumor suppressor function. Oncogene. 2007; 2: 26.[Epub ahead of print].
[52] Wick W, Wild-Bode C, Frank B, et al. BCL-2-induced glioma cell invasiveness depends on furin-like proteases. Neurochem. 2004; 91: 1275-1283.
[53] Arner ES and Holmgren A. Physiological functions of thioredoxin and thiore-doxin reductase. Eur Biochem. 2000; 267: 6102-6109.
[54] Bjornstedt M, Kumar S, Bjorkhem L, et al. Selenium and the thioredoxin and glutaredoxin systems. Biomed Environ Sci. 1997; 10:271-279.
[55] Yoshioka J, Schreiter ER, Lee RT, et al.Role of thioredoxin in cell growth through interactions with signaling molecules. Antioxid Redox Signal. 2006; 8: 2143-2151.
[56] James M, Charles E C, Shalu Mendiratta, et al. Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase. Biol Chem. 1998; 273: 23039-23045.
[57] Mafsukio K. Elevation of antioxidant potency in the brain of mice by low-dose gamma irradiation and its effect on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP) induced brain damage. Free Radic Bio Med. 1999; 26: 388-395.
[58] Takagi Y and Hattori I. Excitotoxic hippocampal injury is attenuated in thioredoxin transgenic mice. Cereb Blood Flow Metab. 2000; 20: 829-833.
[59] Bai J, Nakamura H, Hattori I, et al.Thioredoxin suppresses 1-methyl-4-phenyl pyridinium induced neurotoxicity in rat PC12 cells. Neurosci Lett. 2002; 321: 81-84.
[60] Calabrese V, Guagliano E, Sapienza M, et al.Redox regulation of cellular stress response in neurodegenerative disorders. Biochem. 2006; 55:263-82
[61] Uehara T, Nakamura T, Yao D, et al. S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature. 2006; 441: 513-517.
[62] Landino LM, Skreslet TE, Alston JA, et al. Cysteine oxidation oftau and microtubule associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system. Biol Chem. 2004; 279: 35101-35105.
[63] Lovell MA, Xie C, Gabbita SP, et al. Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain. Free Radic Biol Med. 2000; 28: 418-27.
[64] Ishikawa A, Kubota Y, Murayama T, et al. Cell death by 1-chloro-2,4-dinitroben zene, an inhibitor of thioredoxin reductase and itsdual regulation by nitric oxide in rats. Neurosci Lett. 1999; 277: 99-102.
[65] Yamagata KT, Agamin M. Altered gene expressions during hypoxia and reoxygenation in cortiacal neuronsisolated from storke porne spontaneously hypertensiverates. Neurosici Let. 2000; 284:131-134.
[66] Saitoh M, Nishitoh H, Fujii M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal regulating kinasel(ASK)1. EMBO. 1998; 17: 2596-2606.
[67] Oleksandr G, Patrizia C, Grazia I. Mitochondrial processing peptidases. Biochem Biophys Acta. 2002; 1592: 63-77.
[68] Akio Ito. Mitochondrial Processing Peptidase: Multiple-Site Recognition of Precursor Proteins. Biochim Biophys Res Commun 1999; 265: 611-616.
[69] Arretz M, Schneider H, Guiard B, et al. Characterizationof the mitochondrial processing peptidase of Neurospora crassa. Biol Chem. 1994; 269: 4959-4967.
[70] Taylor AB, Smith BS, Kitada S J, et al. Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences. Struc Camb. 2001; 9:615-625.
[71] Luciano P, Tokatlidis K, Chambre I, et al. The mitochondrial processing peptidase behaves as a zinc metallopeptidase. Mol Biol. 1998; 280: 193-199.
[72] Babcock M, Silva de D, Oaks R, et al. Regulation of mitochondrial iron accumulation by Yfhlp, a putative homolog of frataxin. Science. 1997; 276: 1709-1712.
[73] Branda SS, Cavadini P, Adamec J, et al. Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase. Biol Chem. 1999; 74: 22763-22769.
[74] Koutnikova H, Campuzano V, Koenig M. Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase. Hum Mol Genet. 1998; 1485-1489.
[75] Nicholas J, Lynch P, Kenneth BM, et al. Characterisation of the rat and mouse homologues of gClqBP, a 33 kDa glycoprotein that binds to the globular "heads' of C1q. FEBS Letters. 1997; 418: 111-114.
[76] Ghebrehiwet B, Lim BL, Peerschke EIB, et al. Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" ofClq. Exp Med. 1994; 179:1809-1821
[77] Krainer A.R, Mayeda A, Kozak D, et al. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U170K, and Drosophila splicing regulators. Cell. 1991; 66: 383-394.
[78] Yamada T, Megeer PI, Megeer EG., et al. Relationship of complement activated oligodendrocytes to reactive microglial and neuronalpathology in neurodegenear tive disease. Dementia. 1991; 2:71-77.
[79] Mogi M, Harada M, Riederer P, et al.Tumor necrosis factor-α(TNF-α) increase both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett. 1994; 165: 208-221.
[80] Mochizuki H, Goto K, Mori H, et al. Histochemical detection of apoptosis in Parkinson's disease. Neurol Sci. 1996; 137: 120-123.
[81] Anglade P, Vyas S, Javoy-Agid F, et al. Apoptosis andautophagy in nigral neurons of patients with Parkinson's disease. Histol Histopathol, 1997;12: 25-31.
[82] Zhang W, Wang T, Qin L, et al. Neuroprotective effect of dextrome-thorphan in the MPTP Parkinson's disease model: role of NADPH oxidase. FASEB J. 2004; 18: 589-591.
[83] Fonck C, Baudry M. Rapid reduction of ATP synthesis and lack of free radical formation by MPP~+ in rat brain synaptosomes and mitochondria. Brain Res. 2003; 975: 214-221.
[84] Cassarino DS,Parks JK, Parker WD Jr, et al. The parkinsonian neurotoxin MPP~+ opens the mitochondrial permeability transition pore and releases cytochrome-c in isolated mitochondria via an oxidative mechanism. Biochim Biophys Acta. 1999; 1453:49-62.
[85] Takeyama N, Miki S, Hirakawa A, et al. Role of the mitochondrial permeability transition and cytochrome-c release in hydrogen peroxide induced apoptosis. Exp Cell Res. 2002; 274: 16-24.
[86] Kim JS, He L, Lemasters JJ, et al. Mitochondrial permeability transition: a com-mon pathway to necrosis and apoptosis. Biochem Biophys Res. 2003; 304: 463-470.
[87] Swerdlow RH, Parks JK, Miller SW, et al. Origin and functional conesquences of the complex Ⅰ defect in Parkinson's disease. Ann Neurol. 1996; 40: 663-671.
[88] 高枫,陈清棠,李险峰.帕金森病患者线粒体功能缺陷的研究.中华神经科杂志.1999;32:199-201.
[89] Mandavilli BS, Ali SF, Van Houten B, et al. DNA damage in brain mitochondria caused by aging and MPTP treatment. Brain Res. 2000; 885: 45-52.
[90] Krishnan S, Kamire SP, Michael R, et al. Evidence for generation of oxidative stress in brain by MPTP:in vitro and in vivo studies in mice. Brain Res. 1997; 749:44-52
[91] Wiener HL, Hashim A, Lajtha A, et al. (-)-2-oxo-4-thiazolidine carboxylic acid attenuates 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine induced neurotoxicity. Rcs. Cotntttun Subsr Abrrsr. 1988; 9: 53-60.
[92] Przedborski S, Kostic V, Jackson-Lewis V, et al. Transgenic mice with increased Cu/Zn-superoxide dismutase activity are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. Neurosci. 1992; 5: 1658-1667.
[93] Naoi M, Maruyam W. Cell death of dopamine neurons in aging and Parkinson's disease. Mech Ageing Dev. 1999; 111: 175-188.
[94] Ilic TV, Jovanovic M, Jovicic A, et al. Oxidative stress indicators are elevated in de novo Parkinson's disease patients. Funct Neurol. 1999; 14: 141-147.
[95] Teismann P, Tieu K, Choi DK, et al. Cyclooxygenase-2 is instrumental in Par- kinson's disease neurodegeneration. Proc Natl Acad Sci USA. 2003; 9: 5473-5478.
[96] Przedborski S, Jachsonlewis V, Yokoyama R, et al. Role of neuronal nitricoxide in 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine induced dopami- nergic neurotoxicity. Proc Natl Acad Sci U S A. 1996; 93: 4565-4571.
[97] Unot S, Boissiere F, Faucheux B, et al. Nitricoxide synthase and neuronal vulnerability in parkinson's disease. Neuroscience. 1996; 72: 355-363.
[98] Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the Bcl-2 family and cell death. Blood. 1996; 88: 386-401.
[99] Often D, Ziv I, Barzilai A, et al. Dopamine melanin induces apoptosis in PC12 cells; possible implications for the etiology of Parkinson's disease. Neurochem. 1997; 31: 207-216.
[100] Hassouna I, Wickert H, Zimmermann M, et al. Increase in bax expression in substantia nigra following 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP) treatment of mice. Neurosci Lett. 1996; 204: 85-88.
[101] Garl JM and Rudin C. Cytochrome-c induces caspase dependent apoptosis in intact hematopoietic cells and over rides apoptosis suppress mediated by bcl-2, growth factor signaling MAP kinase and malignant hange. Blood. 1998; 92: 1235-1246.
[102] Susin SA, Zamzami N, Larochette N, et al. A cytofluorometric assay of nuclear apoptosis induced in a cell free system: application to ceramide induced apoptosis. Exp Cell Res. 1997; 236: 397-403.
[103] Patterson SD, Spahr CS, Daugas E, et al. Mass spectrometric identification of proteins released from mitochondria under going permeability transition. Cell Death Differ. 2000; 7: 137-144.
[104] Jyoti C, Seth GN Grant. Proteomics in postgenomic neuroscience: the end of the beginning. Nature Neurosci. 2004; 5: 440-445.
[105] Gert L, Kurt K, Michael F, et al. Proteomics in brain research: potentials and limitations. Pro Neurobiol. 2003; 69:193-211
[106] Matthias M and Ole NJ. Proteomic analysis of post translational modification. Nature Biotechnol. 2003; 3: 255-261.
[107] 龙晓辉,莫志宏,张耀洲.基于二维凝胶电泳的蛋白质定量分析技术.化学进展.2006;18:474-481.
[108] Wickware P and Smaglik P. Labs and companies seek their niches as work continues after the draft. Nature. 2001; 409:961-963.
[109] Edgar PF, Douglas JE, Knight C, et al. Proteome map of the human hippocampus. Hippocampus. 1999; 4: 173-178.
[110] Tsuji T, Shiozaki A, Kohno R, et al. Proteomic Profiling and Neurodegeneration in Alzheimer's Disease. Neurochem Res. 2002; 10: 1245-1253.
[111] Mark R, Nicolls, Jason M, et al. Proteomics as a tool for discovery: proteins implicated in Alzheimer's disease are highly expressed in normal ncreatic. Proteome Res 2003; 2: 199-205.
[112] Kim SH, Krapfenbauer K, Cheon MS, et al. Human brain cytosolic histamine-N-methyltransferase is decreased in Down syndrome and increased in Pick's disease. Neurosci Lett. 2002; 321: 169-172.
[113] Lubec G; Nonaka M, Krapfenbauer K, et al. Expression of the dihydropyrimi dinase related protein 2 (DRP-2) in Down syndrome and Alzheimer's disease brain is downregulated at the mRNA and dysregulated at the protein level. Neural Transm Suppl. 1999; 57: 161-177.
[114] Schonberger SJ, Edgar PF, Robert Kydd, et al. Proteomic analysis of the brain in Alzheimer's disease:Molecular henotypeof a complex disease process. Proteomics. 2001; 1: 1519-1528.
[115] Wang Q, Woltjer RL, Cimino PJ, et al. Proteomic analysis of neurofibrillary tangles in Alzheimer's disease identifies GAPDH as a detergent-insoluble paired helical filament tau binding protein. FASEB. 2005; 3: 1-12.
[116] Liao LJ, Cheng DM, Wang J, et al. Proteomic characterization of postmortem amyloid plaques isolataed by laser capture microdissection. Biol Chem. 2004; 279:37061-37068
[117] Yuan X, Russell T, Wood G, et al. Analysis of the human lumbar cerebrospinal fluid proteome. Electrophoresis. 2002; 23:1185-1196.
[118] Relkin N, Folstein M. Neurobiology of primary dementia, 1998; PP. 271-288.
[119] Leila HC, Michael J. Studies of potential cerebrospinal fluid molecular markers for Alzheimer'disease. Electrophoresis. 2003; 23:2247-2251.
[120] Davidsson P, Westman-Brinkmalm A, Nilsson CL, et al. Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients. Clini Neurosci Neuropathol. 2002; 2: 611-615.
[121] Maja P, Sara FH, Nilssonl CL, et al. Proteomic studies of potential cerebrospinal fluid protein markers for Alzheimer's disease. Brain Res. 2003; 118: 140-146.
[122] Kultima K, Scholz B, Alm H, et al. Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: a proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE. BMC Bioinform. 2006; 7: 475-478.
[123] Abdi F, Quinn JF, Jankovic J, et al. Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders. Alzheimers Dis. 2006; 9: 293-348.
[124] Zhou Y, Wang Y, Kovacs M, et al. Microglial activation induced by neurode-generation: a proteomic analysis. Mol Cell Proteomics. 2005; 10: 1471-1479.
[125] Manuela B, Sabrina G, Davide C, et al. Proteome analysis of human substantia nigra in Parkinson's disease. Proteomics. 2004; 4: 3943-3952.