尿石酸A及番茄碱的抗缺血性神经损伤作用及自噬相关机制研究

发布时间：2021-11-15 22:42

　　脑卒中,又称中风,是世界范围内死亡的主要原因之一,也是成人永久性残疾的主要原因。在全球范围内,脑卒中给患者及其亲属和国民经济都带来了重大负担。缺血性脑卒中是中风的主要原因。缺血性中风导致神经元损伤和死亡,可导致严重的残疾和神经功能损伤。脑缺血的细胞和分子机制涉及神经元的氧化应激、自噬、凋亡、炎症反应和坏死等多种病理过程,这些机制影响缺血性神经元损伤的预后。然而,缺血性神经元损伤的确切病理机制尚未完全阐明。迄今为止,FDA批准用于溶栓治疗的唯一药物是重组组织纤溶酶原激活剂（rt-PA）。然而,由于治疗时间窗很短和出血并发症的高风险,rt-PA的应用非常有限。除rt-PA外,多种神经保护剂通过挽救缺血灶周围的半暗带脑组织,在缺血性脑卒中治疗中显示出良好的应用前景。因此,新颖有效的神经保护策略可能会抑制梗死核心区扩张并促进梗死半暗带存活,为后续的再灌注治疗带来机会。鉴于脑缺血的复杂性,开发治疗缺血性中风的神经保护药物是一项重大挑战。自噬是一种溶酶体介导的细胞内分解代谢过程,负责消化多余或受损的细胞质大分子和细胞器。自噬过程中,大量细胞质和细胞器被一种称为自噬体的双层膜泡包裹。自噬体最终与溶酶... 

【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校

【文章页数】：141 页

【学位级别】：博士

【文章目录】：
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


【参考文献】：
期刊论文
[1]Inactivation of TFEB and NF-kB by marchantin M alleviates the chemotherapy-driven pro-tumorigenic senescent secretion[J]. Huanmin Niu,Lilin Qian,Bin Sun,Wenjian Liu,Fang Wang,Qian Wang,Xiaotian Ji,Yanhai Luo,Effat Un Nesa,Hongxiang Lou,Huiqing Yuan.  Acta Pharmaceutica Sinica B. 2019(05)
[2]Neuroprotective effects of genistein on SH-SY5Y cells overexpressing A53T mutant α-synuclein[J]. Huan-Cheng Wu,Qun-Liang Hu,Shi-Jun Zhang,Yan-Min Wang,Zhan-Kui Jin,Ling-Fu Lv,Sai Zhang,Zhen-Lin Liu,Hong-Lian Wu,Ou-Mei Cheng.  Neural Regeneration Research. 2018(08)
[3]Regulation of mitophagy in ischemic brain injury[J]. Yang Yuan,Xiangnan Zhang,Yanrong Zheng,Zhong Chen.  Neuroscience Bulletin. 2015(04)
[4]中药甘草的药代动力学以及药物相互作用研究进展[J]. 陈江飞,徐萍,朱素燕,胡毅坚.  中国临床药理学与治疗学. 2010(10)



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