簡易檢索 / 詳目顯示

研究生: 馬智剛
Chih-Kang Ma
論文名稱: 以Bβ1 基因轉殖鼠建立並分析其海馬迴的組織切片培養
Establishment and Characterization of hippocampal slice culture from Bβ1 transgenic mice
指導教授: 謝秀梅
Hsieh, Hsiu-Mei
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2010
畢業學年度: 98
語文別: 中文
論文頁數: 69
中文關鍵詞: 脊髓小腦共濟失調症第12型海馬迴成鼠腦切片培養
英文關鍵詞: SCA12, hippocampus, slice culture
論文種類: 學術論文
相關次數: 點閱:117下載:1
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 脊髓小腦共濟失調症第12型(SCA12)是由於PPP2R2B基因5’端未轉譯區(UTR)的CAG重覆片段擴增所造成,該基因是一個腦神經專一性表現的去磷酸化酶protein phosphatase 2A (PP2A)中的調控β次單元。該CAG擴增可能造成PPP2R2B基因的過度表現,因而提高了PP2A的活性,這樣的改變可能因而造成神經細胞的失能與死亡,也是目前推論的SCA12的可能致病機理。我們實驗室已經成功的建立Bβ1過度表現的基因轉殖小鼠,也在這些小鼠中觀察到Purkinje 細胞丟失與神經膠細胞些微的增生(gliosis)。在本研究中,我們繼續的在分子層面分析這些老鼠的致病機轉。我們在行為測試發現,Bβ1過表現小鼠,並沒有明顯呈現出焦慮與運動失調的現象,反而表現出較正常組更為好的成績,唯獨在實驗中的排遺程度遠較對照組有顯著提高的結果。此外我們了解PP2A的受質之ㄧ是維繫微管胞器穩定( microtubule- associated)的重要蛋白,也就是Tau蛋白,在這樣的關係上我們認為Bβ1與阿茲海默氏症(Alzheimer’s disease, AD)可能有某種程度的相關性。有鑒於此,為了未來AD與SCA12相關性的研究方向,我們試著建立成鼠的大腦海馬迴(hippocampus)的切片培養(slice culture)。Bβ1過表現基因轉殖小鼠的腦切片培養可以提供我們便捷的平台以了解SCA12在分子層面的作用機理與篩選治療方式。目前能夠確定的是操作的培養技術上可以穩定的讓成鼠的海馬迴離體培養18天後,亦保有60%以上的細胞存活率,雖然沒有觀測到Bβ1過表現小鼠與正常小鼠的切片培養有差異存在,但在實驗技術的開發上的成果,將能提供我們對於Bβ1失調在離體實驗中更多的了解。

    Spinocerebellar ataxia type 12 (SCA12) is associated with CAG repeat expansion of the 5’UTR of PPP2R2B gene isoform 1 (Bβ1), which encodes a brain-specific regulatory subunit of the protein phosphatase 2A (PP2A). SCA12’s pathogenesis was hypothesized that CAG repeat expansion increased PPP2R2B expression, which altered PP2A activity and induced neuronal dysfunction and death. We have established Bβ1 overexpression transgenic mice and found that Purkinje cell loss, gliosis and some emotional alteration were shown in these mice. In this study, we continued characterize these mice. In addition, substrates of PP2A including microtubule-associated protein tau, which has a close connection with Alzheimer’s disease (AD). To have a quick and easy platform for further study the Bβ1 impact on the SCA12 and AD diseases, we tried to establish an adult or young adult slice culture from mouse hippocampus. Slice culture from the Bβ1 transgenic mice enable us to investigate the molecular pathology and screen potential therapeutic compounds for SCA12. Our behavior analysis showed that Bβ1 transgenic mice (line 20) had a better performance in rotarod and lower anxiety in EPM tasks. The only noticeable abnormality of transgenic mice is that they had significant increasing in fecal output during the rotarod analysis. For slice culture, we have successfully established a stable testing platform by using adult mouse hippocampal tissues. We could maintain the slice survival rate over 60% at DIV18. This ex vivo system should enable us to gain more information about the deregulation of Bβ1.

    摘要 2 前言 5 實驗材料及方法 11 結果 23 討論 52 致謝 55 參考文獻 57

    Arai AC, Orwig N (2008) Factors that regulate KiSS1 gene expression in the hippocampus. Brain Res 1243:10-18.
    Bahl S, Ahmed I, Mukerji M (2009) Utilizing linkage disequilibrium information from Indian Genome Variation Database for mapping mutations: SCA12 case study. J Genet 88:55-60.
    Brera B, Serrano A, de Ceballos ML (2000) beta-amyloid peptides are cytotoxic to astrocytes in culture: a role for oxidative stress. Neurobiol Dis 7:395-405.
    Butterfield DA, Drake J, Pocernich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide. Trends Mol Med 7:548-554.
    Celerier E, Ahdepil T, Wikander H, Berrendero F, Nyberg F, Maldonado R (2006) Influence of the anabolic-androgenic steroid nandrolone on cannabinoid dependence. Neuropharmacology 50:788-806.
    Chen M, Chen Q, Cheng XW, Lu TJ, Liu HX, Jia JM, Zhang C, Xu L, Xiong ZQ (2009) Zn2+ mediates ischemia-induced impairment of the ubiquitin-proteasome system in the rat hippocampus. J Neurochem 111:1094-1103.
    Chen MX, Cohen PT (1997) Activation of protein phosphatase 5 by limited proteolysis or the binding of polyunsaturated fatty acids to the TPR domain. FEBS Lett 400:136-140.
    Chong YH, Shin YJ, Lee EO, Kayed R, Glabe CG, Tenner AJ (2006) ERK1/2 activation mediates Abeta oligomer-induced neurotoxicity via caspase-3 activation and tau cleavage in rat organotypic hippocampal slice cultures. J Biol Chem 281:20315-20325.
    Crossthwaite AJ, Hasan S, Williams RJ (2002) Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca(2+) and PI3-kinase. J Neurochem 80:24-35.
    Dahmani S, Rouelle D, Gressens P, Mantz J Characterization of the postconditioning effect of dexmedetomidine in mouse organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation. Anesthesiology 112:373-383.
    del Rio JA, Soriano E Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration. Nat Protoc 5:217-226.
    Feeney CJ, Frantseva MV, Carlen PL, Pennefather PS, Shulyakova N, Shniffer C, Mills LR (2008) Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices. Brain Res 1198:1-15.
    Frasca G, Carbonaro V, Merlo S, Copani A, Sortino MA (2008) Integrins mediate beta-amyloid-induced cell-cycle activation and neuronal death. J Neurosci Res 86:350-355.
    Gendron TF, Petrucelli L (2009) The role of tau in neurodegeneration. Mol Neurodegener 4:13.
    Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Haynes A, Irving N, James L, et al. (1991) Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704-706.
    Gotz J, Chen F, van Dorpe J, Nitsch RM (2001) Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science 293:1491-1495.
    Goudreault M, D'Ambrosio LM, Kean MJ, Mullin MJ, Larsen BG, Sanchez A, Chaudhry S, Chen GI, Sicheri F, Nesvizhskii AI, Aebersold R, Raught B, Gingras AC (2009) A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics 8:157-171.
    Hassen GW, Tian D, Ding D, Bergold PJ (2004) A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res Brain Res Protoc 13:135-143.
    Hills RD, Jr., Brooks CL, 3rd (2007) Hydrophobic cooperativity as a mechanism for amyloid nucleation. J Mol Biol 368:894-901.
    Hinners I, Hill A, Otto U, Michalsky A, Mack TG, Striggow F (2008) Tau kinase inhibitors protect hippocampal synapses despite of insoluble tau accumulation. Mol Cell Neurosci 37:559-567.
    Holmes SE, O'Hearn E, Margolis RL (2003) Why is SCA12 different from other SCAs? Cytogenet Genome Res 100:189-197.
    Holmes SE, Hearn EO, Ross CA, Margolis RL (2001) SCA12: an unusual mutation leads to an unusual spinocerebellar ataxia. Brain Res Bull 56:397-403.
    Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, Kwak NG, Ingersoll-Ashworth RG, Sherr M, Sumner AJ, Sharp AH, Ananth U, Seltzer WK, Boss MA, Vieria-Saecker AM, Epplen JT, Riess O, Ross CA, Margolis RL (1999) Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 23:391-392.
    Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274:99-102.
    Hsu SS, Newell DW, Tucker A, Malouf AT, Winn HR (1994) Adenosinergic modulation of CA1 neuronal tolerance to glucose deprivation in organotypic hippocampal cultures. Neurosci Lett 178:189-192.
    Kadowaki H, Nishitoh H, Urano F, Sadamitsu C, Matsuzawa A, Takeda K, Masutani H, Yodoi J, Urano Y, Nagano T, Ichijo H (2005) Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation. Cell Death Differ 12:19-24.
    Kim EJ, Won R, Sohn JH, Chung MA, Nam TS, Lee HJ, Lee BH (2008) Anti-oxidant effect of ascorbic and dehydroascorbic acids in hippocampal slice culture. Biochem Biophys Res Commun 366:8-14.
    Lebeau G, Maher-Laporte M, Topolnik L, Laurent CE, Sossin W, Desgroseillers L, Lacaille JC (2008) Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells. Mol Cell Biol 28:2896-2907.
    Liu CL, Lin YR, Chan MH, Chen HH (2007) Effects of toluene exposure during brain growth spurt on GABA(A) receptor-mediated functions in juvenile rats. Toxicol Sci 95:443-451.
    Liu F, Iqbal K, Grundke-Iqbal I, Rossie S, Gong CX (2005) Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer's disease. J Biol Chem 280:1790-1796.
    Liu GP, Wei W, Zhou X, Zhang Y, Shi HH, Yin J, Yao XQ, Peng CX, Hu J, Wang Q, Li HL, Wang JZ I(2)(PP2A) regulates p53 and Akt correlatively and leads the neurons to abort apoptosis. Neurobiol Aging.
    Lorivel T, Hilber P (2006) Effects of chlordiazepoxide on the emotional reactivity and motor capacities in the cerebellar Lurcher mutant mice. Behav Brain Res 173:122-128.
    Markesbery WR, Carney JM (1999) Oxidative alterations in Alzheimer's disease. Brain Pathol 9:133-146.
    Markus T, Cronberg T, Cilio C, Pronk C, Wieloch T, Ley D (2009) Tumor necrosis factor receptor-1 is essential for LPS-induced sensitization and tolerance to oxygen-glucose deprivation in murine neonatal organotypic hippocampal slices. J Cereb Blood Flow Metab 29:73-86.
    Mattson MP, Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7:278-294.
    Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT (2008) Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease. Nature 451:720-724.
    Moskowitz SI, Basu SB, Bergold PJ (2001) Chronic and cyclical neuronal loss in hippocampal slice cultures following transient inhibition of the type 1 isoform of superoxide dismutase. Brain Res 913:207-219.
    Nakahara S, Tamura M, Matsuki N, Koyama R (2009) Neuronal hyperactivity sustains the basal dendrites of immature dentate granule cells: time-lapse confocal analysis using hippocampal slice cultures. Hippocampus 19:379-391.
    Naslund J, Haroutunian V, Mohs R, Davis KL, Davies P, Greengard P, Buxbaum JD (2000) Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 283:1571-1577.
    Nicolay NH, Hertle D, Boehmerle W, Heidrich FM, Yeckel M, Ehrlich BE (2007) Inositol 1,4,5 trisphosphate receptor and chromogranin B are concentrated in different regions of the hippocampus. J Neurosci Res 85:2026-2036.
    Pozzo Miller LD, Mahanty NK, Connor JA, Landis DM (1994) Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists. Neuroscience 63:471-487.
    Price DL, Sisodia SS, Borchelt DR (1998) Genetic neurodegenerative diseases: the human illness and transgenic models. Science 282:1079-1083.
    Pringle AK, Angunawela R, Wilde GJ, Mepham JA, Sundstrom LE, Iannotti F (1997) Induction of 72 kDa heat-shock protein following sub-lethal oxygen deprivation in organotypic hippocampal slice cultures. Neuropathol Appl Neurobiol 23:289-298.
    Rahman A, Ting K, Cullen KM, Braidy N, Brew BJ, Guillemin GJ (2009) The excitotoxin quinolinic acid induces tau phosphorylation in human neurons. PLoS One 4:e6344.
    Raval AP, Bramlett H, Perez-Pinzon MA (2006) Estrogen preconditioning protects the hippocampal CA1 against ischemia. Neuroscience 141:1721-1730.
    Rottkamp CA, Atwood CS, Joseph JA, Nunomura A, Perry G, Smith MA (2002) The state versus amyloid-beta: the trial of the most wanted criminal in Alzheimer disease. Peptides 23:1333-1341.
    Sanchez-Ortiz E, Hahm BK, Armstrong DL, Rossie S (2009) Protein phosphatase 5 protects neurons against amyloid-beta toxicity. J Neurochem 111:391-402.
    Sato N et al. (2009) Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet 85:544-557.
    Sayre LM, Perry G, Smith MA (2008) Oxidative stress and neurotoxicity. Chem Res Toxicol 21:172-188.
    Schild A, Isenmann S, Tanimoto N, Tonagel F, Seeliger MW, Ittner LM, Kretz A, Ogris E, Gotz J (2006) Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice. Mech Dev 123:362-371.
    Schrag M, Sharma S, Brown-Borg H, Ghribi O (2008) Hippocampus of Ames dwarf mice is resistant to beta-amyloid-induced tau hyperphosphorylation and changes in apoptosis-regulatory protein levels. Hippocampus 18:239-244.
    Schweers O, Mandelkow EM, Biernat J, Mandelkow E (1995) Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments. Proc Natl Acad Sci U S A 92:8463-8467.
    Selkoe DJ (1991) The molecular pathology of Alzheimer's disease. Neuron 6:487-498.
    Shetty AK, Rao MS, Hattiangady B (2008) Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus. J Neurosci Res 86:3062-3074.
    Stepanichev M, Moiseeva Iu V, Lazareva NA, Guliaeva NV (2004a) [Effects of amyloid beta peptide (25-35) on the behavior of rats in a radial maze]. Zh Vyssh Nerv Deiat Im I P Pavlova 54:382-389.
    Stepanichev M, Zdobnova IM, Zarubenko, II, Lazareva NA, Guliaeva NV (2004b) [Effects of central administration of beta-amyloid peptide (25-35): pathomorphological changes in the hippocampus and impairments of spatial memory]. Zh Vyssh Nerv Deiat Im I P Pavlova 54:705-711.
    Suh EC, Jung YJ, Kim YA, Park EM, Lee KE (2008) A beta 25-35 induces presynaptic changes in organotypic hippocampal slice cultures. Neurotoxicology 29:691-699.
    Tamagno E, Robino G, Obbili A, Bardini P, Aragno M, Parola M, Danni O (2003) H2O2 and 4-hydroxynonenal mediate amyloid beta-induced neuronal apoptosis by activating JNKs and p38MAPK. Exp Neurol 180:144-155.
    Vitale M, Zamai L, Mazzotti G, Cataldi A, Falcieri E (1993) Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes. Histochemistry 100:223-229.
    Vornov JJ, Tasker RC, Coyle JT (1991) Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures. Exp Neurol 114:11-22.
    Xiang Z, Bergold PJ (2000) Synaptic depression and neuronal loss in transiently acidic hippocampal slice cultures. Brain Res 881:77-87.
    Xiang Z, Hrabetova S, Moskowitz SI, Casaccia-Bonnefil P, Young SR, Nimmrich VC, Tiedge H, Einheber S, Karnup S, Bianchi R, Bergold PJ (2000) Long-term maintenance of mature hippocampal slices in vitro. J Neurosci Methods 98:145-154.
    Xu Y, Chen Y, Zhang P, Jeffrey PD, Shi Y (2008) Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation. Mol Cell 31:873-885.
    Xu Y, Xing Y, Chen Y, Chao Y, Lin Z, Fan E, Yu JW, Strack S, Jeffrey PD, Shi Y (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127:1239-1251.
    Yamamoto H, Saitoh Y, Fukunaga K, Nishimura H, Miyamoto E (1988) Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly. J Neurochem 50:1614-1623.
    Zanoli P, Zavatti M, Rivasi M, Brusiani F, Losi G, Puia G, Avallone R, Baraldi M (2007) Evidence that the beta-acids fraction of hops reduces central GABAergic neurotransmission. J Ethnopharmacol 109:87-92.
    Zhu X, Mei M, Lee HG, Wang Y, Han J, Perry G, Smith MA (2005) P38 activation mediates amyloid-beta cytotoxicity. Neurochem Res 30:791-796.

    下載圖示
    QR CODE