【摘要】：摘要應(yīng)激是在生物學(xué)上被定義為各種生理學(xué)的改變,包括內(nèi)環(huán)境失穩(wěn)態(tài)及腦下垂體腎上腺軸的激活。在應(yīng)激條件下促腎上腺皮質(zhì)激素釋放因子(Corticotropin releasing Factor, CRF)由下丘腦釋放,激活下丘腦-垂體-腎上腺軸(Hypothalamus pituitarydrenal axis, HPA軸),在正常情況下,負(fù)反饋回路能抑制CRF的進(jìn)一步合成和釋放,而在HPA軸調(diào)節(jié)發(fā)生異常時則會引起負(fù)反饋功能障礙,過度增加的血漿糖皮質(zhì)激素合成釋放,最后會進(jìn)入中樞造成神經(jīng)元的損傷。然而目前的研究發(fā)現(xiàn),CRF除了通過HPA軸引起神經(jīng)元損傷外,還可通過與中樞CRF受體作用引起海馬神經(jīng)元損傷。因此探索CRF對海馬神經(jīng)元的損傷作用及可能機(jī)制,可為應(yīng)激損傷中樞神經(jīng)元的分子機(jī)制提供新理論和實(shí)驗(yàn)依據(jù)。 目的：系統(tǒng)研究CRF通過CRFR1受體后信號通路直接導(dǎo)致中樞神經(jīng)元損傷的分子機(jī)制,旨在揭示與慢性應(yīng)激相關(guān)的神經(jīng)精神疾病的病理生理機(jī)制,為最終闡明慢性應(yīng)激損傷中樞神經(jīng)元的分子機(jī)制提供新理論和實(shí)驗(yàn)依據(jù)。 方法：1、免疫熒光法分析CRF對海馬神經(jīng)元結(jié)構(gòu)的影響：培養(yǎng)原代海馬神經(jīng)元細(xì)胞至第五天,CRF (0.02μM,0.2μM,2μM)處理原代培養(yǎng)的大鼠海馬神經(jīng)元,繼續(xù)培養(yǎng)至第十天。顯微鏡下觀察海馬神經(jīng)元細(xì)胞結(jié)構(gòu)的變化,然后用絲裂原活化蛋白-2(Mitogen Activated Protein, MAP2)標(biāo)記海馬神經(jīng)元樹突,免疫熒光法觀察海馬神經(jīng)元樹突的變化。用CRFR1特異性拮抗劑(DMP696)處理海馬神經(jīng)元細(xì)胞,同樣顯微鏡下觀察對照組、CRF處理組和CRF+特異性拮抗劑(DMP696)海馬神經(jīng)元細(xì)胞結(jié)構(gòu)的變化,然后用MAP2標(biāo)記海馬神經(jīng)元樹突,免疫熒光法觀察海馬神經(jīng)元樹突的變化。 2、磺酰羅丹明B (sulforhodamine B, SRB)法檢測CRF對海馬神經(jīng)元細(xì)胞活力的影響：培養(yǎng)原代海馬神經(jīng)元細(xì)胞至第五天,加入不同濃度的CRF處理海馬神經(jīng)元細(xì)胞,實(shí)驗(yàn)分為：空白組(無細(xì)胞),對照組(不加藥物組),CRF (0.02μM,0.2μM,2μM)處理組。培養(yǎng)至第十天SRB法測細(xì)胞活力。 3、Western Blot分析與神經(jīng)元生長密切相關(guān)的關(guān)鍵分子蛋白水平的變化：培養(yǎng)原代海馬神經(jīng)元細(xì)胞至第五天,CRF (0.02μM,0.2μM,2μM)處理原代培養(yǎng)的大鼠海馬神經(jīng)元,繼續(xù)培養(yǎng)至第十天。Western Blot分析cAMP反應(yīng)元件結(jié)合蛋白(cAMP response element bindingprotein, CREB),微管相關(guān)蛋白(microtubule asso-ciated proteins, Tau)磷酸化水平的變化,及MAP2,突觸后密度蛋白-95(Postsynaptic density-95, PSD95)蛋白水平的變化,觀察CRF是否對海馬神經(jīng)元細(xì)胞的以上幾種蛋白的水平產(chǎn)生影響。然后在原代培養(yǎng)的大鼠海馬神經(jīng)元上,分別利用蛋白激酶A (protein kinase A, PKA)、磷脂肌醇(protein kinase C, PKC)、絲裂原活化蛋白激酶(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)的特異性抑制劑H89. Calphostisc、PD98059、2-APB、U-73122與CRF共孵育,Western Blot檢測CREB, Tau磷酸化水平的變化,及MAP2, PSD95蛋白水平的變化,分析參與這種變化的信號通路。 4、RT-PCR法分析與神經(jīng)元生長密切相關(guān)的關(guān)鍵分子mRNA表達(dá)水平的變化：培養(yǎng)原代海馬神經(jīng)元細(xì)胞至第五天,用CRF及DMP696處理原代培養(yǎng)的大鼠海馬神經(jīng)元,繼續(xù)培養(yǎng)至第十天。RT-PCR法檢測CREB、Tau、MAP2、PSD95mRNA表達(dá)的變化。分析以上幾種分子的mRNA變化情況。 5、環(huán)磷酸腺苷(Cyclic Adenosine monophosphate, cAMP)釋放試驗(yàn)檢測海馬神經(jīng)元細(xì)胞cAMP含量的變化：培養(yǎng)原代海馬神經(jīng)元細(xì)胞至第十天,cAMP釋放試驗(yàn)檢測CRF刺激海馬神經(jīng)元細(xì)胞釋放cAMP的含量變化,并檢測DMP696對CRF刺激海馬神經(jīng)元細(xì)胞釋放cAMP含量的影響。分析cAMP是否參與了CRFR1介導(dǎo)的海馬神經(jīng)元的損傷作用。 結(jié)果：1、2μM CRF可引起海馬神經(jīng)元樹突密度減少,用特異性拮抗劑(DMP696)處理細(xì)胞,CRF可引起海馬神經(jīng)元樹突密度減少的作用明顯被拮抗。 2、CRF對海馬神經(jīng)元細(xì)胞活力產(chǎn)生抑制,CRF (0.2μM,2μM)處理組細(xì)胞活力依次為71.6士3.1%,72.6±3.5%(n=3,***P0.001與對照組相比)。 3、2μMCRF可下調(diào)海馬神經(jīng)元細(xì)胞MAP2,P-CREB蛋白水平,上調(diào)PSD95, P-Tau蛋白水平；CRFR1特異性拮抗劑(DMP696)可拮抗CRF引起的MAP2, P-CREB蛋白水平下調(diào)及PSD95, P-Tau蛋白水平上調(diào)；PKA特異性抑制劑H89可拮抗CRF引起的MAP2蛋白水平下調(diào)及P-Tau蛋白水平上調(diào)。 4、海馬神經(jīng)元細(xì)胞中PSD95, MAP2的mRNA表達(dá)水平的變化與蛋白水平的變化一致,Tau與CREB的mRNA表達(dá)水平無明顯變化。 5、cAMP釋放試驗(yàn)表明CRF可濃度依賴性的調(diào)節(jié)海馬神經(jīng)元細(xì)胞cAMP含量的變化(EC50=(3.157±0.133)×10-9M, n=3),并且特異性拮抗劑DMP696可減少2μMCRF引起的海馬神經(jīng)元cAMP釋放水平(n=3,***P0.001,‘P0.05,與-5組相比)。結(jié)論：CRF通過與CRFR1作用引起海馬神經(jīng)元結(jié)構(gòu)損傷,其作用與下調(diào)MAP2及P-CREB的蛋白水平,上調(diào)PSD95, P-Tau蛋白水平相關(guān)。CRF促進(jìn)cAMP的釋放,提示CRFR1偶聯(lián)的G蛋白為Gs。CRF引起的MAP2蛋白水平下調(diào)及P-Tau蛋白水平上調(diào)可能是PKA通路參與調(diào)節(jié)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 鹵 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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

【共引文獻(xiàn)】

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