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题名	猕猴吗啡成瘾的DNA甲基化机制以及MCPH1过表达小鼠吗啡成瘾模型的研究
作者	张蕾
学位类别	硕士
答辩日期	2015-05
授予单位	中国科学院研究生院
授予地点	北京
导师	马原野
关键词	猕猴 吗啡成瘾 DNA 甲基化 小鼠 MCPH1
其他题名	Morphine addiction-related DNA methylation in Macaque and morphine addiction model of MCPH1 over-expression mouse
中文摘要	第一部分 猕猴吗啡成瘾的DNA甲基化机制 药物成瘾是一种不正常的学习记忆，并且形成的记忆持久，提示可能引起细胞和分子方面的改变。本实验采用经典的药物成瘾学习记忆模型—条件化位置偏好（Conditioned Place Preference, CPP）在非人灵长类动物—猕猴上建立吗啡相关学习记忆，然后在不同时间段取脑组织，运用荧光定量PCR（Real-Time Quantitative PCR, RT-qPCR）的方法检测与DNA甲基化相关基因的改变。本实验中，猕猴分为3组，分别为空白对照组、食物CPP组、吗啡CPP组。其中吗啡CPP组又分为CPP形成后3天分离脑组织组和CPP后2-6个月分离脑组织组。我们所测定的脑区为药物成瘾通路上的脑区，包括内侧前额叶（Medial Frontal Cortex, mPFC）、眶额叶（Orbital Frontal Cortex, OFC）、腹侧纹状体(Ventral Striatum）、伏隔核(Nucleus Accumbens, NAc）、海马（Hippocampus, HiP）、杏仁核（Amygdala，Amy），前岛叶（Anterior Insula, AI）和后岛叶（Posterior Insula, PI)。结果显示，我们在猕猴上成功建立了吗啡相关学习记忆，发现与DNA甲基化相关基因的转录水平，具有时间特异性：NAc、mPFC、OFC、HiP和AI中，DNA甲基化转移酶（DNA methyltransferases，DNMTs）的转录水平在吗啡CPP建立后的3天显著升高，在吗啡CPP建立后的2-6个月转录下降。丝氨酸/苏氨酸蛋白磷酸酶-1（serine / threonine protein phosphatase-1 ，PP1c）的转录水平改变发生在CPP建立后的2-6个月，3种异构体中发生变化较明显的是PP1cβ和PP1cγ。此外，在NAc、OFC、Amy中发现fosB（FBJ murine osteosarcoma viral oncogene homolog B）基因有转录改变。 关键词：猕猴，吗啡，条件化位置偏好，荧光定量PCR，DNA甲基化转移酶，丝氨酸/苏氨酸蛋白磷酸酶1，fosB 第二部分 MCPH1过表达小鼠吗啡成瘾模型的建立 药物成瘾是一种不正常的学习记忆，其涉及到的细胞和分子机制仍然没有定论。实验室通过向宿兵实验室引进四种MCPH1过表达小鼠，并且之前已经在这些小鼠上做过水迷宫和Y迷宫等测试，发现MCPH1过表达小鼠有更好的学习记忆，而吗啡成瘾记忆和普通学习记忆不同，因此我们采用吗啡条件化位置偏好（Conditioned Place Preference, CPP）来测试MCPH1过表达小鼠的吗啡相关学习记忆。小鼠经过1天适应期，之后8天交替腹腔注射吗啡和无菌生理盐水，吗啡剂量为10mg/kg，检测期维持1天，检测期后的第3天和第7天作为消退期检测小鼠CPP的消退情况。MCPH1过表达小鼠共4种：转入猕猴的MCPH1基因（M），分别为M10和M14；转入了人的MCPH1基因(Line)，分别为LineA和LineD，野生型（WT）作为对照组。结果显示，WT组的小鼠建立了极显著的CPP行为，并且在CPP后的第3天和第7天仍然具有对吗啡相关环境的明显偏好行为。除了M14未建立起CPP之外，其它三种基因型的小鼠均建立了CPP。但是，转基因型的小鼠，在第3天或第7天之后未见CPP行为，说明它们对吗啡所相关的记忆消退较快。 关键字：小鼠，MCPH1，吗啡，条件化位置偏好
英文摘要	Part 1: Drug addiction is an abnormal process of long-lasting learning and memory, suggesting that it might cause change in cellular and molecular aspects. In the present study, we use conditioned place preference( CPP) to establish morphine-related learning and memory on macaque, and brain structures of interest were dissected at 3d, 2month, 6month after CPP test. We use Real-Time Quantitative PCR(RT-qPCR) to detect gene changes in DNA methylation. The macaques were divided into three groups: control, food CPP, morphine CPP. The macaques in morphine CPP were divided into two groups: one is that they sacrificed 3d after CPP test, and the other is sacrificed 2m and 6m after CPP test. The brain areas we selected are on the addiction pathway, including Medial Prefrontal (mPFC), Orbital Frontal Cortex (OFC), Ventral Striatum, Nucleus Accumbens (NAc), Hippocampus (HiP), Amygdala (Amy), Anterior Insula (AI) , Posterior Insula (PI). The results show that we successfully established morphine CPP in macaques. And the transcription change of DNA methylation-related genes has time-specificity: DNA methyltransferases mRNA were upregulated at post morphine 3 days in NAc、mPFC、OFC、HiP、AI, and serine / threonine protein phosphatase-1 (PP1c) mRNA change at post morphine 2-6 months. PP1cβ and PP1cγ have obvious change in morpnine CPP. For FosB gene, we only found change in NAc、OFC、Amy. Key words: Macaque, Morphine, Conditioned Place Preference，Real-Time Quantitative PCR, DNA methyltransferases, PP1c, FosB Part 2: Drug addiction is an aberrant learning and memory, suggesting that it might cause change in cellular and molecular. We have done water maze and Y-maze test in MCPH1-overexpression mice,and the result show that they have better learning and memory. However, morphine addiction-related memory is different from normal learning and memory. Microcephine 1(MCPH1) is related to brain development, and it is one of the factors which cause microcephaly. Mice were first placed in CPP box for one day. After this Pre-CPP period, CPP conditioning occurred when mice were injected intraperitoneally with morphine and saline on alternative days at 10mg/kg. Morphine and saline treatment lasted 8 days, altogether. CPP was tested for 1 day after CPP training. The extiction period was at third dat and seventh day after CPP test. We bring in MCPH1- overexpression mice to explore the relationship between morphine-related learning and memory and MCPH1. In the present study, we use two different mice: one is transferred into the macaque MCPH1 gene(M),including M10 and M14, and the other is transferred into human MCPH1 gene (Line), including LineA and LineD, and wild-type(WT).The results showed that WT mice establish very significant CPP, and they still have CPP in the third and seventh day. We could not establish CPP behavior in M14 mice, but the other three transgenic mice were established CPP. However, the transgenic mice show less significant CPP in third or seventh day, indicating that their morphine-related learning and memory did not last long. Key words: mice, MCPH1, morphine, Conditioned Place Perference
语种	中文
内容类型	学位论文
源URL	[http://159.226.149.26:8080/handle/152453/10092]
专题	昆明动物研究所_认知障碍病理学
推荐引用方式 GB/T 7714	张蕾. 猕猴吗啡成瘾的DNA甲基化机制以及MCPH1过表达小鼠吗啡成瘾模型的研究[D]. 北京. 中国科学院研究生院. 2015.

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