腦卒中,又稱中風,是世界范圍內(nèi)死亡的主要原因之一,也是成人永久性殘疾的主要原因。在全球范圍內(nèi),腦卒中給患者及其親屬和國民經(jīng)濟都帶來了重大負擔。缺血性腦卒中是中風的主要原因。缺血性中風導(dǎo)致神經(jīng)元損傷和死亡,可導(dǎo)致嚴重的殘疾和神經(jīng)功能損傷。腦缺血的細胞和分子機制涉及神經(jīng)元的氧化應(yīng)激、自噬、凋亡、炎癥反應(yīng)和壞死等多種病理過程,這些機制影響缺血性神經(jīng)元損傷的預(yù)后。然而,缺血性神經(jīng)元損傷的確切病理機制尚未完全闡明。迄今為止,FDA批準用于溶栓治療的唯一藥物是重組組織纖溶酶原激活劑（rt-PA）。然而,由于治療時間窗很短和出血并發(fā)癥的高風險,rt-PA的應(yīng)用非常有限。除rt-PA外,多種神經(jīng)保護劑通過挽救缺血灶周圍的半暗帶腦組織,在缺血性腦卒中治療中顯示出良好的應(yīng)用前景。因此,新穎有效的神經(jīng)保護策略可能會抑制梗死核心區(qū)擴張并促進梗死半暗帶存活,為后續(xù)的再灌注治療帶來機會。鑒于腦缺血的復(fù)雜性,開發(fā)治療缺血性中風的神經(jīng)保護藥物是一項重大挑戰(zhàn)。自噬是一種溶酶體介導(dǎo)的細胞內(nèi)分解代謝過程,負責消化多余或受損的細胞質(zhì)大分子和細胞器。自噬過程中,大量細胞質(zhì)和細胞器被一種稱為自噬體的雙層膜泡包裹。自噬體最終與溶酶... 

【文章來源】：浙江大學浙江省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：141 頁

【學位級別】：博士

【文章目錄】：
DEDICATION
ACKNOWLEDGEMENTS
ABSTRACT IN ENGLISH
ABSTRACT IN CHINESE
ABBREVIATIONS
Chapter 1
    1.1 Introduction
    1.2 Materials
        1.2.1 Experimental cell line
        1.2.2 Basic Reagents
        1.2.3 Reagents for N2a Cell Culture
        1.2.4 Reagents for Primary Cortical Neuron Culture
        1.2.5 Reagents for Cell Treatments
        1.2.6 Cell Transfection Reagents
        1.2.7 Extraction Kits
        1.2.8 Protein Extraction Related Reagents
        1.2.9 Antibodies
        1.2.10 Instruments
        1.2.11 Solution Preparation
        1.2.12 Preparation of Cell Culture Medium
        1.2.13 Solution for Protein Extraction
        1.2.14 Solutions for Western blot
        1.2.15 SDS Page Electrophoresis Gel Preparation
        1.2.16 MTT Reagents and Solution Preparation
        1.2.17 Construction of Plasmids
        1.2.18 Mice Handling
    1.3 Methods
        1.3.1 Drugs Administration in Mice
        1.3.2 Middle Cerebral Artery Occlusion(MCAO)Model
        1.3.3 Preoperative Care
        1.3.4 Preparation of Monofilament for MCAO
        1.3.5 Procedure for MCAO
            1.3.5.1 Procedure
            1.3.5.2 Physiological Parameters
            1.3.5.3 Procedure for Sham Surgery
            1.3.5.4 Postoperative Care
            1.3.5.5 Infarct Analysis
            1.3.5.6 Behavioral Measurements
        1.3.6 Primary Cortical Neuron Culture
        1.3.7 Requirement
        1.3.8 Coating and Washing Culture Plates
        1.3.9 Procedure
            1.3.9.1.1 Dissection of Mice
            1.3.9.1.2 Trypsinization
            1.3.9.1.3 Dissociation of Tissues
            1.3.9.1.4 Seeding of Neurons
        1.3.10 N2a Cell culture
            1.3.10.1 Cell Culture Conditions
            1.3.10.2 Procedure for Cell thawing
            1.3.10.3 Cell Passage
            1.3.10.4 Cell Seeding
            1.3.10.5 N2a cell Differentiation
        1.3.11 Oxygen Glucose-Deprivation/reperfusion(OGD/R)Procedures
            1.3.11.1 Preparation
            1.3.11.2 Experimental Procedure
        1.3.12 Reagents Administration in Vitro
        1.3.13 Cell Viability Assay
        1.3.14 LDH Assay
        1.3.15 Plasmid Construction
            1.3.15.1 The Process of Plasmid Construction
            1.3.15.2 The Primers Sequences Used in this Project
            1.3.15.3 pmCherry-LC3 Plasmid Primers
        1.3.16 DNA Transfection Protocol and Image Analysis of N2a cell
        1.3.17 DNA Transfection Protocol and Image Analysis of Primary Cortical Neurons
        1.3.18 Quantitative Real-time PCR
            1.3.18.1 RNA Extraction Protocol
            1.3.18.2 cDNA Extraction Protocol
        1.3.19 Quantitative Real-time PCR
        1.3.20 Protein Extraction and Quantification from neuronal cells
        1.3.21 Protein Extraction and Quantification from mice
        1.3.22 Western Blot Analysis
            Gel Preparation
            Gel Electrophoresis
            Transferring
            Blocking
            Primary Antibody
            Washing
            Secondary Antibody
            Washing
            Digital Image
        1.3.23 Statistical Analysis
    1.4 Results
        1.4.1 Uro-A inhibited OGD/R-induced cell injury in N2a cells and primary cultured neurons
        1.4.2 Uro-A reinforced ischemic reperfusion-induced autophagy both in vitro and in vivo
        1.4.3 Uro-A does not induce mitophagy both in ischemic neuronal cells and in vivo
        1.4.4 Uro-A represses the ER stress by activation of autophagy both in vitro and in vivo
        1.4.5 Uro-A reduced acute ischemic brain injury in mice
    1.5 Discussion
    Conclusion
    REFERENCES
Chapter 2
    2.1 Introduction
    2.2 Materials& Methods
        2.2.1 Reagents and Antibodies
        2.2.2 Primary Cortical Neuron Culture
        2.2.3 N2a Cell culture and Differentiation
        2.2.4 Oxygen Glucose-Deprivation/Reperfusion(OGD/R)Procedures
        2.2.5 Drugs Administration
        2.2.6 Cell Viability Assay
        2.2.7 Detection of Lactate Dehydrogenase(LDH)Activity
        2.2.8 Plasmids and Transfection
        2.2.9 Autophagic Flux Quantification
        2.2.10 DQ Red-BSA Trafficking Assay
        2.2.11 Lysosomal Biogenesis Quantification Assay
        2.2.12 Western blot Analysis
        2.2.13 Statistical Analysis
    2.3 Results
        2.3.1 Tomatidine alleviates OGD/R-induced injury on N2a cell& primary neurons
        2.3.2 Tomatidine enhances autophagic flux by accelerating the autophagic degradation exposed to OGD/R
        2.3.3 Tomatidine enhanced lysosomal activity in OGD/R-treated neuronal cells
        2.3.4 Tomatidine activates TFEB against OGD/R
        2.3.5 Blocking of lysosomes abolishes tomatidine-conferred neuroprotection
        2.3.6 Tomatidine does not induce mitophagy in ischemic neuronal cells
    2.4 Discussion
    Conclusion
    REFERENCES
REVIEW
    Reference
AUTHOR’S BIOGRAPHY AND PUBLICATIONS


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