脱落酸论文：急性应激对大鼠脑中脱落酸变化的影响

脱落酸论文：急性应激对大鼠脑中脱落酸变化的影响【中文摘要】脱落酸(Abscisic Acid,ABA)是植物中广泛存在的一种调节激素,它在植物种子休眠、幼苗生长发育、多种生物(如病原菌)和非生物(干旱、寒冷和高盐等)应激过程中都起到重要调控作用。

近年来,越来越多的研究发现许多动物中也存在ABA,包括海绵、水螅、人寄生虫和多种哺乳动物组织和细胞,并参与相应的应激反应、组织生长、炎症调节、糖代谢等信号调节通路中。

ABA在这些通路中的信号作用方式与植物类似,首先ABA与细胞膜上的特异性蛋白结合,激活ADP核糖环化酶,从而提升胞内cADPR的表达,增加胞内Ca2+浓度,并进一步激活一系列下游信号反应。

这些发现表明ABA可能作为一个普遍的信号分子,广泛参与调节植物和动物对环境的应激反应。

本实验建立了动物脑组织中ABA的提取和检测方法,研究了大鼠不同脑区ABA含量的特异性分布。

结果发现,ABA在前额叶、左右海马和下丘脑不同脑区的含量不同,其中以下丘脑中含量最高,为18.1±1.9ng/g组织鲜重。

实验进一步以强迫游泳和束缚两种急性应激方式,研究了大鼠大脑中ABA含量随应激的变化。

结果表明,在前额叶和海马中,急性束缚1小时后ABA含量约增加到对照组2倍水平,而下丘脑ABA 含量却明显下降。

另外,不同应激条件下,下丘脑内ABA的变化趋势与前额叶和海马相反。

急性应激下,大鼠血浆皮质酮水平、下丘脑CRF 和c-fos的基因表达上调。

相关性分析显示,c-fos的表达变化与皮质酮的水平变化呈正相关,与下丘脑ABA水平变化呈负相关。

这些结果显示,大鼠大脑中存在ABA,下丘脑可能是ABA的分泌或作用中心,而且ABA信号在下丘脑与其他脑区的可能作用方式或时间尺度存在差异。

生物信息学手段对ABA植物中重要信号蛋白ABI1、ABI3和ABRE/ABF的比对检索发现,人源蛋白PPM1B、RNC3和ATF6与上述分子具有高度相似的氨基酸序列,相似度分别为49%,39%,60%。

对筛选出的假定人ABA相关蛋白的mRNA表达研究显示,24小时20μM ABA培养神经元母细胞瘤细胞系BE(2)-C能显著降低以上蛋白的表达。

我们推测这些候选蛋白和ABA的信号通路存在某种直接或间接的交叉关联。

为探索ABA在组织和细胞中的定位情况,进行了ABA免疫组织化学和细胞免疫荧光实验。

巨噬细胞ABA免疫荧光染色结果显示,ABA 主要定位于细胞膜和胞浆,无特异性胞核着色。

这与前述细胞内ABA 的作用方式和早期玉米中ABA免疫组化结果吻合。

【英文摘要】Abscisic Acid （ABA） is an important universal phytohormone which regulates plants seeds dormancy, development of seedling and response to stresses including pathogens, drought, cold and salty.It was discovered recently that not only in plants, fungi, cyanobacteria and algae but also ABA exists and functions in a wide range of lower animals, such as sponges, as well as mammals including various human tissues and cells. ABA can also be produced and released from these animal cells and as a regulator on stress response, tissue regeneration, inflammation and glucose metabolism. The signalpathway in animal is remarkably similar to that in plants which ABA first binds to the ABA-binding protein located in the plasma membrane and activates intracellular ADP-ribose cyclase which would promote the production of the cyclic ADP-ribose resulting in increases of intracellular Ca2+ concentrations and then triggers many other correlative signal pathways. These results imply that ABA may act as an endogenous regulator in animals and may serve as a universal signal passenger in plants and animals.In order to investigate the concentration and distribution of ABA in mammalian brain, we successfully build up the method to extract and determine ABA through rat brain tissue. Using this method, we find that ABA concentration differs in different brain area including prefrontal cortex, left or right hippocampus and hypothalamus with the hypothalamus the highest ABA concentration at 18.1 1.9ng/g fresh tissue. We then study the effect of forced swim and immobilized stress on ABA level in rat brain. The results show in the immobilized 1 hour group, ABA level in prefrontal cortex and bilateral hippocampus increases twice compare to control group while decreases greatly in hypothalamus. Moreover, ABA fluctuates toward different direction between hypothalamus and other brain areas. Corresponding with other report,corticosterone in plasma increases after acute stress. Results from Q-PCR indicate the gene expression of CRF and c-fos in hypothalamus significantly up regulated in immobilized 1 hour rats. Pearson Correlation analysis shows positive correlation between changes of c-fos expression and corticosterone while negative correlation between changes of c-fos expression and ABA.We obtain three high sequence homology human proteins PPM1B, ATF6 and RNC3 when comparing by BLAST with important signal proteins ABI1, ABI3 and ABRE/ABF from A. thaliana with Positive Identities 49%,39%,60% respectively. These putative human ABA relative proteins are significantly down regulated in ABA treated BE（2）-C cells. It provides evidence that the three putative human ABA relative proteins may interact with cellular ABA signal pathway in animals.Finally ABA immunocytochemistry experiment has been done in order to discover the localization of ABA in animal brain and cells. The immunofluorescence results demonstrate that ABA is located in macrophage cellular membrane and plasma but not nucleus which is in agreement with the ABA’s celluar function and the early results reported in maize roots.【关键词】脱落酸强迫游泳急性束缚应激促肾上腺皮质激素释放因子免疫组化【英文关键词】Abscisic Acid Forced Swim Acute Immobilized Stress Corticotropin Releasing Factor ImmunoCytochemistry【备注】索购全文在线加好友：1.3.9.9.3.8848同时提供论文写作一对一指导和论文发表委托服务【目录】急性应激对大鼠脑中脱落酸变化的影响摘要4-6ABSTRACT6-7第1章植物激素脱落酸在动物中的研究进展（综述）10-22 1.1 植物激素脱落酸概述10-14 1.1.1 脱落酸介绍10-12 1.1.2 脱落酸的功能和信号通路12-14 1.2 脱落酸在动物中的研究进展14-19 1.2.1 动物中存在脱落酸14-18 1.2.2 脱落酸在动物中的应激通路18-19 1.3 脱落酸的潜在临床应用19-22第2章脱落酸与大鼠急性应激22-40 2.1 引言22 2.2 实验材料与方法22-29 2.2.1 大鼠急性应激及取材22-23 2.2.2 ABA 的组织提取23-24 2.2.3 ABA 放射性免疫分析（RIA）24-25 2.2.4 ABA 酶联免疫分析（ELISA）25-27 2.2.5 组织RNA 提取及实时荧光定量PCR（Q-PCR）27-29 2.2.6 统计与作图29 2.3 实验结果29-33 2.3.1 急性应激大鼠皮血浆质酮变化29-30 2.3.2 不同脑区ABA 含量变化30-31 2.3.3 不同应激大鼠ABA 含量31-32 2.3.4 RNA 抽提质量验证32-33 2.3.5 应激大鼠下丘脑CRF 及c-fos 表达变化33 2.4 实验讨论33-38 2.4.1 ABA 提取和检测方法的建立.33-34 2.4.2 急性应激对大鼠皮质酮水平的影响34-35 2.4.3 ABA 在大鼠脑组织中的区域分布探讨.35-36 2.4.4 新RNA 抽提方法效果讨论36 2.4.5 ABA 与中枢应激信号分子的相关性探讨.36-38 2.5 总结与展望38-40第3章脱落酸的信号通路探索40-46 3.1 引言40 3.2 实验材料与方法40-42 3.2.1 细胞培养与ABA 给药40-41 3.2.2 RNA 提取与实时定量荧光PCR41 3.2.3 BLAST 检索与通路蛋白筛选41-42 3.3 实验结果42-43 3.3.1 ABA 信号相关蛋白比对42 3.3.2 ABA 影响下筛选蛋白的基因表达变化42-43 3.4 实验讨论43-46 3.4.1 ABA 信号通路蛋白检索43-44 3.4.2 假定的人ABA 相关蛋白功能分析44-46第4章脱落酸免疫组化46-56 4.1 引言46 4.2 实验材料与方法46-52 4.2.1 ABA 大鼠石蜡切片46-49 4.2.2 ABA 石蜡切片免疫组化49-51 4.2.3 ABA 细胞免疫荧光51-52 4.3 实验结果52-55 4.3.1 ABA 大鼠石蜡切片免疫组化52-54 4.3.2 ABA 细胞免疫荧光54-55 4.4 实验讨论55-56 4.4.1 ABA 脑组织免疫组化55 4.4.2 ABA 细胞免疫荧光55-56参考文献56-66附录66-68致谢68-70在读期间发表的学术论文与取得的其他研究成果70。