艾滋痴呆综合征(AIDSdementiacomplex，ADC)是由HIV-1感染所引起的以认知功能障碍、运动能力减退和行为改变为特征的常见综合征，是基底节神经节、大脑皮质以及海马区域神经损伤或凋亡的结果。HIV-1感染小胶质细胞和巨噬细胞，不直接感染神经元，却出现了神经元的损伤，其损伤可能与HIV-1Tat有关。Tat为HIV-1反式激活蛋白，其反式激活需要menin的参与，提示了menin可能参与了ADC的发病机制，但是，其具体机制尚未阐明。我们研究发现，在SIV感染恒河猴ADC动物模型中menin表达上调，尤其是在大脑皮质神经元核内menin表达明显增加，统计学分析menin的表达与神经...Various types of neuronal damage have been reported in AIDS dementia complex (ADC). Human immunode&#64257;ciency virus (HIV) infected microglial/macrophage, not neuron directly in the brain. However, the damage of neuron existed after the infection, in which the regulatory protein Tat of HIV-1 could be the critical factor. It has been recently reported that HIV-1 Tat transactivation requires menin...学位：理学硕士院系专业：医学院_微生物学学号：2452010115332

王俊

Xiamen University Institutional Repository

厦 门 大 学 博 硕 士 论 文 摘 要 库  学校编码：10384                                 分类号     密级     . 学号：24520101153328                                        UDC     .         硕  士  学  位  论  文     SIV 感染恒河猴大脑额叶皮质 menin 表达上升与神经元损伤的相关性 Increased menin expression associated with neural damage in the frontal cortex of SIV infected macaques   王 俊 指导教师姓名： 邢   惠  琴 专  业 名 称： 微 生 物 学 论文提交日期： 2013年 4月 论文答辩时间： 2013年  月 学位授予日期： 2013年  月     答辩委员会主席：   评阅人：    2013年 4月   厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库 厦门大学学位论文原创性声明  本人呈交的学位论文是本人在导师指导下,独立完成的研究成果。本人在论文写作中参考其他个人或集体已经发表的研究成果，均在文中以适当方式明确标明，并符合法律规范和《厦门大学研究生学术活动规范（试行）》。 另外，该学位论文为（                            ）课题（组）的研究成果，获得（               ）课题（组）经费或实验室的资助，在（               ）实验室完成。（请在以上括号内填写课题或课题组负责人或实验室名称，未有此项声明内容的，可以不作特别声明。）  声明人（签名）：           年   月   日           厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库 厦门大学学位论文著作权使用声明  本人同意厦门大学根据《中华人民共和国学位条例暂行实施办法》等规定保留和使用此学位论文，并向主管部门或其指定机构送交学位论文（包括纸质版和电子版），允许学位论文进入厦门大学图书馆及其数据库被查阅、借阅。本人同意厦门大学将学位论文加入全国博士、硕士学位论文共建单位数据库进行检索，将学位论文的标题和摘要汇编出版，采用影印、缩印或者其它方式合理复制学位论文。 本学位论文属于： （     ）1.经厦门大学保密委员会审查核定的保密学位论文，于   年  月  日解密，解密后适用上述授权。 （     ）2.不保密，适用上述授权。 （请在以上相应括号内打“√”或填上相应内容。保密学位论文应是已经厦门大学保密委员会审定过的学位论文，未经厦门大学保密委员会审定的学位论文均为公开学位论文。此声明栏不填写的，默认为公开学位论文，均适用上述授权。）                                声明人（签名）： 年   月   日  厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库摘  要 I 摘  要 艾滋痴呆综合征(AIDS dementia complex，ADC) 是由 HIV-1 感染所引起的以认知功能障碍、运动能力减退和行为改变为特征的常见综合征，是基底节神经节、大脑皮质以及海马区域神经损伤或凋亡的结果。HIV-1 感染小胶质细胞和巨噬细胞，不直接感染神经元，却出现了神经元的损伤，其损伤可能与 HIV-1Tat 有关。Tat 为 HIV-1 反式激活蛋白，其反式激活需要 menin 的参与，提示了 menin 可能参与了 ADC 的发病机制，但是，其具体机制尚未阐明。我们研究发现，在 SIV感染恒河猴 ADC 动物模型中 menin 表达上调，尤其是在大脑皮质神经元核内menin 表达明显增加，统计学分析 menin 的表达与神经元总数呈负相关性。并且，在神经元中 menin 与 caspase3 的共定位，提示 menin 过表达可能与神经元损伤有关。我们进一步研究发现，menin 相关细胞因子 TGF-β 主要在椎体神经元中表达增加，并且在 TGF-β 表达上调的神经元中 Tat 与 TGF-β 共定位。已有报道指出TGF-β 能够上调 menin 的表达，menin 的表达又更进一步的促进了 TGF-β 对细胞的调控作用。据此我们推测在 ADC 中神经元内 TGF-β 的激活可能与 Tat 蛋白有关；其机制可能为 Tat 蛋白激活 TGF-β 表达，激活的 TGF-β 上调 menin 的表达，进而导致神经元损伤。但是，更具体的调控机制有待进一步在细胞水平进行深入细致地研究。   关键词：ADC；Tat；menin；TGF-β厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库Abstract II Abstract Various types of neuronal damage have been reported in AIDS dementia complex (ADC). Human immunodeficiency virus (HIV) infected microglial/macrophage, not neuron directly in the brain. However, the damage of neuron existed after the infection, in which the regulatory protein Tat of HIV-1 could be the critical factor. It has been recently reported that HIV-1 Tat transactivation requires menin，which hints that menin may be involved in the pathogenesis of ADC. However, the mechanism has not been elucidated completely. In this study, we report the up-regulated menin expression in the frontal cortex of SIV-infected macaques, especially in neuronal nucleus by the qRT-PCR and Western blot assay. The co-localization of menin and caspase3 and the negative correlation between numbers of neuron and numbers of menin-positive neurons are also demonstrated in the frontal cortex of our macaques. TGF-β, a cytokine associated with menin expression, is also detected increased in the frontal cortex of SIV-infected macaques. Furthermore, co-localization of SIV Tat and TGF-β appeared in increased TGF-β expressed vertebral neuron. In conclusion, these results indicate that menin may be involved in the pathogenesis of ADC and the mechanism may be related to the pro-apoptosis of menin and the increased expression of TGF-β induced by SIV Tat. However, the precise mechanism needs to be further explored.   Key Words: ADC; Tat; menin; TGF-β   厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库目  录 III 目  录 摘  要 ····················································· I ABSTRACT ··············································· II 目  录 ··················································· III TABLE OF CONTENTS ···································· V 第一章  前言 ·············································· 1 1.研究背景······················································· 1 2.研究现状······················································· 2 2.1 临床病理特征·············································· 2 2.2 发病机制 ·················································· 3 2.2.1 血脑屏障················································· 3 2.2.2 HIV 感染神经系统 ········································· 5 2.2.3 HIV Tat 对神经系统的损伤 ·································· 6 3.本文研究的目的及内容·········································· 11 3.1 Menin 的结构与功能 ······································· 11 3.2 Menin 与细胞凋亡 ········································· 12 3.3 Menin 与中枢神经系统 ····································· 12 4.展望·························································· 13 第二章  材料与方法 ······································· 14 1.材料·························································· 14 1.1 动物模型 ················································· 14 1.2 所需试剂 ················································· 14 1.3 溶液配制 ················································· 15 1.4 主要仪器 ················································· 17 2.实验方法······················································ 18 厦 门 大 学 博 硕 士 论 文 摘 要 库目  录 IV 2.1 免疫组织化学检测 ········································· 18 2.2 免疫组织化学多重染色 ····································· 19 2.3 免疫荧光双标 ············································· 22 2.4 TUNEL ··················································· 23 2.5 实时荧光定量 PCR（Real-time PCR）定量分析 menin 和 TGF-β 的表达·························································· 24 2.5.1 总 RNA 的提取 ·········································· 24 2.5.2 CDNA 单链的合成 ········································ 25 2.5.3 实时荧光定量 PCR ······································· 26 2.6 免疫印迹（Western blot） ··································· 27 2.6.1 蛋白样品的制备·········································· 27 2.6.2 分离胶制备 ············································· 28 2.6.3 浓缩胶制备 ············································· 29 2.6.4 上样 ··················································· 29 2.6.5 SDS-聚丙烯酰胺凝胶电泳 ································· 29 2.6.6 蛋白质的电转移及膜的封闭 ······························· 29 2.6.7 抗体使用液的配制 ······································· 30 2.6.8 抗原抗体反应 ··········································· 30 2.6.9 ECL 显色 ··············································· 30 2.7 结果判定 ················································· 31 2.8 统计分析 ················································· 31 第三章  实验结果与分析 ··································· 32 3.1 SIV 感染的恒河猴的大脑额叶皮质中 menin 表达上升 ··········· 32 3.2 Menin 主要表达于神经元细胞核 ····························· 34 3.3 Menin 与神经元损伤的相关性 ······························· 36 3.4 Menin 表达增加与 SIV Tat 的相关性·························· 39 3.5 神经元细胞中 SIV Tat 与 TGF-β 表达增加的相关性 ············· 40 第四章 讨论 ·············································· 43 4.1 Menin 与 ADC ············································· 44 厦 门 大 学 博 硕 士 论 文 摘 要 库目  录 V 4.2 Menin 与大脑中细胞表达类型 ······························· 44 4.3 Menin 与神经元损伤 ······································· 45 4.4 Tat 与 menin ··············································· 45 4.5 Tat 与 TGF-β ·············································· 46 4.6 Tat 与 menin、TGF-β ······································· 46 第五章 结论与展望 ········································ 48 参考文献 ················································· 50                 厦 门 大 学 博 硕 士 论 文 摘 要 库厦 门 大 学 博 硕 士 论 文 摘 要 库Table of Contents VI Table of Contents Chapter 1 Introduction ······································· 1 Part I Research Background ·········································· 1 Part II Research Status ·············································· 2 2.1 Clinicopathologic Feature ····································· 2 2.2 Pathogenesis ················································ 3 2.2.1 Different types of cells in the brain ····························· 3 2.2.2 Infection of HIV in the nervous system ························· 5 2.2.3 The damage of HIV Tat to the nervous system ···················· 6 Part Ⅲ Research prospects ·········································· 11 3.1 The structure and function of Menin···························· 11 3.2 Menin and Apoptosis ········································ 12 3.3 Menin and the nervous system ································· 12 Part Ⅳ Purposes and contents of this project ·························· 13 Chapter 2 Materials and Methods ···························· 14  Part I Materials ···················································· 14 1.1 Experimental Animal Models ································· 14 1.2 Reagents in need ··········································· 14 1.3 Solution Preparation········································· 15 1.4 Experiment Equipment ······································ 17 Part II Methods ···················································· 18 2.1 Immunohistochemical examination ····························· 18 2.2 Multiple immunohistochemical staining························· 19 2.3 Immunofluorescence ········································ 22 2.4 TUNEL ·················································· 23 2.5 Real-time PCR ············································· 24 2.6 Western blot ··············································· 27 2.7 Evaluation of the result ······································ 31 2.8 Statistical analysis ·········································· 31 Chapter 3 Results and analysis ·······························32 厦 门 大 学 博 硕 士 论 文 摘 要 库Table of Contents VII 3.1 Menin are Increased in the Frontal Cortex of SIV-infected Macaques ·· 32 3.2 Menin Mainly Expresses in Neuronal Nucleu of Frontal Cortex with SIV-infected Macaques ········································· 34 3.3 Menin Correlates with Neuronal Damage in SIV-Infected Animals ···· 36 3.4 SIV Tat Correlates with Increased Menin in SIV-Infected Animals ···· 39 3.5 Related SIV Tat with Increased TGF-β in Vertebral Neurons of Frontal Cortex with SIV-infected Macaques ······························· 40 Chapter 4 Discussion ·······································43 4.1 Menin and ADC ············································ 44 4.2 Menin and cell types in brain ·································· 44 4.3 Tat and Menin and neuronal damage ···························· 45 4.4 Tat and menin ·············································· 45 4.5 Tat and TGF-β ············································· 46 4.6 Tat and Menin / TGF-β ······································ 46 Chapter 5 Conclusion and Prospects ··························48 References ················································50               厦 门 大 学 博 硕 士 论 文 摘 要 库Degree papers are in the “Xiamen University Electronic Theses and Dissertations Database”. Fulltexts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submitrequests online, or consult the interlibrary loan department in your library. 2. For users of non-CALIS member libraries, please mail to etd@xmu.edu.cn for delivery details.

Increased menin expression associated with neural damage in the frontal cortex of SIV infected macaques

http://dspace.xmu.edu.cn/bitstream/handle/2288/97725/SIV%e6%84%9f%e6%9f%93%e6%81%92%e6%b2%b3%e7%8c%b4%e5%a4%a7%e8%84%91%e9%a2%9d%e5%8f%b6%e7%9a%ae%e8%b4%a8menin%e8%a1%a8%e8%be%be%e4%b8%8a%e5%8d%87%e4%b8%8e%e7%a5%9e%e7%bb%8f%e5%85%83%e6%8d%9f%e4%bc%a4%e7%9a%84%e7%9b%b8%e5%85%b3%e6%80%a7.pdf?sequence=1&isAllowed=y

Increased menin expression associated with neural damage in the frontal cortex of SIV infected macaques

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