CRF对大鼠海马神经元结构的直接效应及机制研究

发布时间：2018-10-22 18:40

【摘要】：摘要应激是在生物学上被定义为各种生理学的改变,包括内环境失稳态及脑下垂体肾上腺轴的激活。在应激条件下促肾上腺皮质激素释放因子(Corticotropin releasing Factor, CRF)由下丘脑释放,激活下丘脑-垂体-肾上腺轴(Hypothalamus pituitarydrenal axis, HPA轴),在正常情况下,负反馈回路能抑制CRF的进一步合成和释放,而在HPA轴调节发生异常时则会引起负反馈功能障碍,过度增加的血浆糖皮质激素合成释放,最后会进入中枢造成神经元的损伤。然而目前的研究发现,CRF除了通过HPA轴引起神经元损伤外,还可通过与中枢CRF受体作用引起海马神经元损伤。因此探索CRF对海马神经元的损伤作用及可能机制,可为应激损伤中枢神经元的分子机制提供新理论和实验依据。目的：系统研究CRF通过CRFR1受体后信号通路直接导致中枢神经元损伤的分子机制,旨在揭示与慢性应激相关的神经精神疾病的病理生理机制,为最终阐明慢性应激损伤中枢神经元的分子机制提供新理论和实验依据。方法：1、免疫荧光法分析CRF对海马神经元结构的影响：培养原代海马神经元细胞至第五天,CRF (0.02μM,0.2μM,2μM)处理原代培养的大鼠海马神经元,继续培养至第十天。显微镜下观察海马神经元细胞结构的变化,然后用丝裂原活化蛋白-2(Mitogen Activated Protein, MAP2)标记海马神经元树突,免疫荧光法观察海马神经元树突的变化。用CRFR1特异性拮抗剂(DMP696)处理海马神经元细胞,同样显微镜下观察对照组、CRF处理组和CRF+特异性拮抗剂(DMP696)海马神经元细胞结构的变化,然后用MAP2标记海马神经元树突,免疫荧光法观察海马神经元树突的变化。 2、磺酰罗丹明B (sulforhodamine B, SRB)法检测CRF对海马神经元细胞活力的影响：培养原代海马神经元细胞至第五天,加入不同浓度的CRF处理海马神经元细胞,实验分为：空白组(无细胞),对照组(不加药物组),CRF (0.02μM,0.2μM,2μM)处理组。培养至第十天SRB法测细胞活力。 3、Western Blot分析与神经元生长密切相关的关键分子蛋白水平的变化：培养原代海马神经元细胞至第五天,CRF (0.02μM,0.2μM,2μM)处理原代培养的大鼠海马神经元,继续培养至第十天。Western Blot分析cAMP反应元件结合蛋白(cAMP response element bindingprotein, CREB),微管相关蛋白(microtubule asso-ciated proteins, Tau)磷酸化水平的变化,及MAP2,突触后密度蛋白-95(Postsynaptic density-95, PSD95)蛋白水平的变化,观察CRF是否对海马神经元细胞的以上几种蛋白的水平产生影响。然后在原代培养的大鼠海马神经元上,分别利用蛋白激酶A (protein kinase A, PKA)、磷脂肌醇(protein kinase C, PKC)、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)、1,4,5-三磷酸肌醇(Inositol1,4,5-triphophate, IP3)、磷脂酶C (Phospholipase C, PLC)的特异性抑制剂H89. Calphostisc、PD98059、2-APB、U-73122与CRF共孵育,Western Blot检测CREB, Tau磷酸化水平的变化,及MAP2, PSD95蛋白水平的变化,分析参与这种变化的信号通路。 4、RT-PCR法分析与神经元生长密切相关的关键分子mRNA表达水平的变化：培养原代海马神经元细胞至第五天,用CRF及DMP696处理原代培养的大鼠海马神经元,继续培养至第十天。RT-PCR法检测CREB、Tau、MAP2、PSD95mRNA表达的变化。分析以上几种分子的mRNA变化情况。 5、环磷酸腺苷(Cyclic Adenosine monophosphate, cAMP)释放试验检测海马神经元细胞cAMP含量的变化：培养原代海马神经元细胞至第十天,cAMP释放试验检测CRF刺激海马神经元细胞释放cAMP的含量变化,并检测DMP696对CRF刺激海马神经元细胞释放cAMP含量的影响。分析cAMP是否参与了CRFR1介导的海马神经元的损伤作用。结果：1、2μM CRF可引起海马神经元树突密度减少,用特异性拮抗剂(DMP696)处理细胞,CRF可引起海马神经元树突密度减少的作用明显被拮抗。 2、CRF对海马神经元细胞活力产生抑制,CRF (0.2μM,2μM)处理组细胞活力依次为71.6士3.1%,72.6±3.5%(n=3,***P0.001与对照组相比)。 3、2μMCRF可下调海马神经元细胞MAP2,P-CREB蛋白水平,上调PSD95, P-Tau蛋白水平；CRFR1特异性拮抗剂(DMP696)可拮抗CRF引起的MAP2, P-CREB蛋白水平下调及PSD95, P-Tau蛋白水平上调；PKA特异性抑制剂H89可拮抗CRF引起的MAP2蛋白水平下调及P-Tau蛋白水平上调。 4、海马神经元细胞中PSD95, MAP2的mRNA表达水平的变化与蛋白水平的变化一致,Tau与CREB的mRNA表达水平无明显变化。 5、cAMP释放试验表明CRF可浓度依赖性的调节海马神经元细胞cAMP含量的变化(EC50=(3.157±0.133)×10-9M, n=3),并且特异性拮抗剂DMP696可减少2μMCRF引起的海马神经元cAMP释放水平(n=3,***P0.001,‘P0.05,与-5组相比)。结论：CRF通过与CRFR1作用引起海马神经元结构损伤,其作用与下调MAP2及P-CREB的蛋白水平,上调PSD95, P-Tau蛋白水平相关。CRF促进cAMP的释放,提示CRFR1偶联的G蛋白为Gs。CRF引起的MAP2蛋白水平下调及P-Tau蛋白水平上调可能是PKA通路参与调节的。
[Abstract]:Stress is defined biologically as a variety of physiological changes, including homeostasis of the internal environment and activation of the pituitary adrenal axis. The corticotropin releasing factor (CRF) is released by the hypothalamus under stress conditions to activate the hypothalamus-pituitary-adrenal axis (HPA axis), in which case the negative feedback loop can inhibit further synthesis and release of the CRF, However, in the case of abnormal HPA axis regulation, negative feedback dysfunction, excessive increase of plasma glucocorticoid synthesis release, and eventually lead to the damage of neurons. However, it has been found that in addition to neuronal damage caused by HPA axis, CRF can also cause damage to hippocampal neurons through interaction with central CRF receptors. Therefore, the damage effect and possible mechanism of CRF on hippocampal neurons can be explored, which can provide new theory and experimental basis for the molecular mechanism of the central nervous system of stress injury. Objective: To study the molecular mechanism of CRFR1 receptor signaling pathway leading to the damage of central nervous system (CNS) and to reveal the pathophysiology of neuropsychiatric disorders associated with chronic stress. The mechanism is to provide new theory and experiment to clarify the molecular mechanism of chronic stress injury central nervous system. Methods: 1. Immunofluorescence method was used to analyze the effect of CRF on hippocampal neuron structure: To culture primary hippocampal neurons to the fifth day, CRF (0.02. mu.M, 0.2. mu.M, 2. mu.M) to treat the primary cultured rat hippocampal neurons, and to continue the culture. The changes of the cell structure of hippocampal neurons were observed under the microscope, and then the hippocampal neurons were labeled with Mitogen Activated Protein-2 (Mitogen Activated Protein, MAP2), and the hippocampal neurons were observed by immunofluorescence. The changes of hippocampal neurons were treated with CRFR1 specific antagonist (DMP696), and the changes of hippocampal neuronal cell structure in the control group, CRF treatment group and CRF + specific antagonist (DMP696) were observed under the same microscope, then the hippocampus was labeled with MAP2. Observation of hippocampal neurons by neuron-derived dendritic and immunofluorescence method The effect of CRF on the activity of hippocampal neurons was determined by the change of dendritic cells. Blank group (no cells), control group (no drug group), CRF (0.002. mu.M, 0.2. mu.M, 2. mu.M) Treatment Group. Culture to Day 10 S The cell viability was measured by RB method. 3. Western blot analysis was closely related to neuronal growth: cultured primary hippocampal neurons to day 5, CRF (0.02. mu.M, 0.2. mu.M, 2. mu.M) treated primary cultured rat hippocampal neurons. The changes of cAMP response element binding protein (CREB), microtubule-associated protein (Tau) phosphorylation level, and postsynaptic density protein-95 (PS) were analyzed by Western blot. D95) Changes in protein levels, observing whether CRF is above hippocampal neuronal cells Protein kinase A (PKA), inositol (MAPK), 1, 4, 5-triphosphoinositide (MAPK), 1, 4, 5-triphosphoinositide (IP3) and phospholipase C (Pho) were used in primary cultured rat hippocampal neurons. pholipase C, PLC The changes of CREB and Tau phosphorylation were detected by Western blot, and the changes of MAP2 and PSD95 protein levels were detected by Western blot. The change of the expression level of key molecular mRNA closely related to neuronal growth was analyzed by RT-PCR. The primary cultured rat hippocampal neurons were treated with CRF and DMP696. hippocampal neurons continue to be cultured for 10 days. RT-PCR detect CREB, Tau, MAP2, Changes in the expression of PSD95mRNA. The changes of cAMP content in hippocampal neurons were detected by cyclic voltammetry (cAMP) release test. The changes of cAMP content in hippocampal neurons were detected in 5, cylic Adenosine monophate (cAMP) release assay. The primary hippocampal neurons were cultured for the tenth day, and the cAMP release assay was used to detect the CRF stimulating sea. The content of cAMP in hippocampal neurons was changed, and DMP696 was tested to stimulate the sea. The effect of cAMP on the release of cAMP in hippocampal neurons. Results: 1, 2. m CRF can induce the decrease of dendritic density in hippocampal neurons, and the specific antagonist (DMP696) can be used to treat the cells. The inhibitory effect of CRF on the cell viability of hippocampal neurons was inhibited, and the activity of CRF (0.2. mu.M, 2. mu.M) cells was 71. 6 卤 3.1%, 72. 6-3, respectively. 5% (n = 3, ** * P0. 001 vs. control). 3, 2. m MCRF down-regulated MAP2, P-CREB protein levels in hippocampal neurons, up-regulated PSD95, P-Tau protein levels; CRFR1 specific antagonist (DMP696) antagonized the MAP2, P-CR caused by CRF. Down regulation of EB protein and upregulation of PSD95, P-Tau protein; PKA specific inhibitor H89 antagonized CR Down-regulation of MAP2 protein induced by F and upregulation of P-Tau protein level. The expression level of PSD95 and MAP2 in hippocampal neurons was changed with protein water. There was no significant change in the level of mRNA expression of Tau and CREB. 5. cAMP release assay showed that CRF could regulate the cAMP content of hippocampal neurons in concentration-dependent manner (P = (3.157, 0. 133), 10-9M, n = 3), and the specific antagonist, DMP696, could reduce 2. m cAMP Release of hippocampal neurons induced by RF Conclusion: CRF induced hippocampal neuronal structural damage by the action of CRFR1, its function and downregulation MAP2. and the protein level of P-CREB is correlated with the level of PSD95 and P-Tau protein. The CRF promotes the release of cAMP, suggesting that the G protein coupled by CRFR1 is Gs.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【共引文献】