【作者】 耿燕楠；

【导师】 张惠云；

【作者基本信息】 山东中医药大学 ， 中药学， 2011， 硕士

【摘要】 目的:研究愤怒情绪反应模型大鼠顶区皮层、额区皮层、海马和下丘脑中γ-氨基丁酸B2受体(GABABR2)mRNA及蛋白表达变化及经前平颗粒对其表达水平的调节作用,探讨愤怒反应的发生和致病机理以及经前平颗粒对愤怒反应调节的微观作用机制,并构建GABABR2真核表达质粒,为GABABR2转染神经元细胞提供物质基础,同时为研究其下游信号通路做铺垫。方法:1、居住-入侵配合社会隔离慢性应激法制备愤怒情绪反应大鼠模型,调肝方药经前平颗粒进行药物干预,并作行为学评价,逆转录聚合酶链式反应(RT-PCR)基因扩增技术和蛋白免疫印迹技术(Western blot)分别检测大鼠顶区皮层、额区皮层、海马和下丘脑四个脑区GABABR2 mRNA和蛋白的表达水平。2、RT-PCR法扩增GABABR2 cDNA,克隆入载体PMD18-T,然后酶切,定向克隆到真核表达质粒pcDNA3.1-His-lacZ中,构建pcDNA3.1-His-lacZ -GABABR2重组表达质粒,然后用限制性内切酶和DNA测序鉴定。结果:1、大鼠造模行为学评价结果显示,与正常组大鼠相比,愤怒组大鼠均呈显著性差异(P<0.05),给药后较正常对照组无显著差异。RT-PCR结果显示,与正常组相比,GABABR2 mRNA相对水平在愤怒组大鼠4个脑区均极显著性下降(P<0.01);与模型组相比,愤怒给药组均发生极显著性改善(P<0.01),除额区皮层外均与正常组无统计学差异。Western blot结果显示,与正常组相比,GABABR2蛋白相对水平在愤怒组大鼠4个脑区均极显著性下降(P<0.01);与模型组相比,愤怒给药组均发生显著性改善(P<0.05),与正常组无统计性差异。2、经测序鉴定GABABR2 cDNA序列正确,并经酶切和DNA测序证实pcDNA3.1-His-lacZ -GABABR2重组质粒构建正确。结论:采用居住-入侵造模法可以成功制备愤怒情绪反应大鼠模型,愤怒反应的发生和致病可能与顶区皮层、额区皮层、海马和下丘脑组织中GABABR2表达异常密切相关,成功克隆GABABR2 cDNA,并成功构建pcDNA3.1-His-lacZ -GABABR2重组真核表达质粒。更多还原

【Abstract】 Objective: Our aim is to investigate the expression of gamma-aminobutyric acid (GABA) B receptor 2 in different brain regions of anger-out model rats, as well as to research the effects of Jingqianshu granule on the expression, and construction of GABABR2 eukaryotic expression vector.We can study the mechanisms of action of occurrence and pathogenesis of anger with the effect of Jingqianshu granule. At the same time, the construction of GABABR2 eukaryotic expression vector can provide material foundation for neuron in signal transduction.Methods: 1 We made the anger-out model rats by the method of social isolation plus resident-intruder. The model rat were treated with Jingqianshu granule, and evaluated by behavioral test. We analyzed the expression of GABABR2 in four different brain regions by the method of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. 2 GABABR2 cDNA was amplified with mRNA from rat’s brain as template by RT-PCR. The cDNA was cloned into plasmid PMD18-T, then digesting PMD18-T by restriction enzyme, and the products were cloned into eukaryotic expression plasmid pcDNA3.1-His-lacZ for constructing recombinant plasmid pcDNA3.1-His-lacZ- GABABR2. The accuracy of pcDNA3.1-His-lacZ-GABABR2 was confirmed by restrietion enzyme digestion and DNA sequencing.Results: 1 The behavioral test results: Compared with the normal group, the score of the model group rats display significant difference (P<0.05). Meanwhile, when treating with Jingqianshu graulne, the significant difference disappear .RT-PCR results: The mRNA expression of GABABR2 decreased obviously(P<0.01)in the four different brain regions comparing with the normal group. Compared with the model group, the Jingqianshu graulne group had significant improvement (P<0.01); All of them had no significant difference compared with normal group except in frontal region. Western blot findings: The expression of GABABR2 decreased obviously(P<0.01)in the four different brain regions comparing with the normal control group. Compared with the model group, the Jingqianshu graulne group had significant improvement(P<0.05). All of them had no significant difference compared with normal group. 2 By DNA sequeneing, the sequence of GABABR2 cDNA was crrect. By restriction enzymes digesting and DNA sequencing, recombinant plasmid pcDNA3.1-His-lacZ-GABABR2 was constructed correctly. Conclusion: The anger-out model rats can be prepared successfully by using the methods of resident intruder paradigm. The expressions changes of the GABABR2 in the four different brain regions are in close relation to occurrence and pathogenesis of anger. GABABR2 gene was cloned sueeessuflly, and eukaryotic expression vector pcDNA3.1-His-lacZ-GABABR2 was constructed successfully.更多还原