【作者】 王海涛；

【导师】 石玉秀；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 前言创伤后应激障碍(posttraumatic stress disorder, PTSD)是指在强烈的精神创伤后发生的心理、生理的应激反应所表现出的一系列临床综合症,是应激精神疾病中最为典型的一种。其核心症状可以简述为闯入性再体验、警觉性增高及反应麻木。随着战争、社会暴力、重大交通事故和自然灾害等意外事件的逐渐增多,PTSD发生率也愈加增高。现今,PTSD以其发病率高、病程长、难以治愈等特点,严重影响人们身心健康而备受关注。国内外对PTSD进行了大量研究,但其确切的发病机制尚未完全阐明。研究表明,HPA轴负反馈增强是PTSD的神经内分泌一个特征。神经递质5-羟色胺(5-hydroxytryptamine,5-HT)及其受体在HPA轴负反馈调节中起重要作用。5-羟色胺1A受体在情绪和精神调节中起重要作用。但其在PTSD中的作用目前研究不一。ERK1／2在包括神经元在内的许多细胞中均起重要作用,但目前对SPS时脑内神经元中ERK分子磷酸化的研究还只是刚刚起步。单一连续刺激(single prolonged stress, SPS)是PTSD动物模型的理想方法,该方法已经被国际所认同。本研究采用SPS方法建立PTSD大鼠模型,测定大鼠行为学改变和血清皮质醇浓度,验证PTSD模型的准确性。为探讨5-HT1A受体对GR和CRF的影响,我们利用5-HT 1A特异性阻断剂作用于SPS大鼠,观察大鼠海马GR和下丘脑CRF的变化。并观察SPS大鼠1nPFC中磷酸化的ERK1／2和c-fos的变化,探讨ERK通路抑制剂PD98059的作用。为揭示PTSD的发病机制提供实验依据。实验方法1、PTSD大鼠行为学改变将Wistar大鼠随机分为对照组和模型组。采用SPS方法建立PTSD模型。采用旷场(open-field, OF)实验、高架十字迷宫(elevated plus maze, EPM)实验、Morris水迷宫(Morris water mace, MWM)实验进行行为学测定,并测定大鼠的恐惧记忆的行为(僵立行为测定),采用化学发光法检测大鼠血清皮质醇浓度。2、PTSD大鼠5-HT1A受体活性与海马GR和下丘脑CRF表达的关系大鼠分为四组：(1)正常对照组(normal group),大鼠既不给予SPS处理,也不给予药物干预；(2)假手术组(sham group),实验动物不给予SPS处理,但给予等体积的溶剂；(3)模型组(SPS group),实验动物给予SPS处理,处理前给予等体积溶剂；(4)阻断组(blockade group),实验动物给予SPS处理和5-HT1A受体拮抗剂WAY100635。胶体金免疫电镜观察5-HT1A在海马和下丘脑神经元内的分布。免疫组织化学方法分析海马GR和下丘脑CRF表达。Westernblotting分析海马GR表达。RT-PCR分析GR mRNA和CRF mRNA变化。3、PTSD大鼠前额皮质磷酸化细胞外信号转导激酶的变化大鼠被随机分为三组：对照组、SPS组和PD98059+SPS组。SPS组和PD98059+SPS组大鼠给予SPS,并在SPS前30min分别双侧注入生理盐水和PD98059,对照组大鼠给予等体积生理盐水。用SABC法进行mPFC部位的pERK1／2的免疫组织化学染色,将染色后的切片经自动图像分析系统进行半定量分析。Western blotting分析mPFC部位的pERK1/2。RT-PCR分析c-fos mRNA表达结果一、PTSD大鼠行为学改变1、OF实验结果两组大鼠在旷场箱内主要在箱壁区域活动,其运动符合啮齿类动物的趋触性(thigmotaxis)的自然属性,即啮齿类动物趋向于沿壁活动。模型组大鼠中央格停留时间和直立次数显著低于对照组,差异有统计学意义(P<0.05),其余指标两组之间差异没有统计学意义(P>0.05)2、EMP实验结果EMP结果表明,大鼠活动符合大鼠趋暗的自然属性,即大鼠趋向于在光线暗处活动,大鼠在闭臂内活动时间和路程显著多于开臂。随着大鼠对环境的逐渐适应,大鼠向开臂内的探究行为逐渐增多。模型组大鼠在开臂内停留时间所占总臂内停留时间比显著低于对照组,差异具有统计学意义(P<0.01)。模型组大鼠进入开臂次数占总进臂次数的百分比显著低于对照组,差异有统计学意义(P<0.01)。3、MWM实验结果模型组大鼠平均逃避潜伏期显著高于对照组,差异具有统计学意义(P<0.01)。4、僵立行为测定结果模型组大鼠再次放入强迫游泳玻璃缸中(重新面临恐惧情景时)后,大鼠表现为明显的僵立行为。对照组大鼠重新面临恐惧情景时,行为模式与模型组显然不同,表现为自由运动,出现探究、理毛等行为。对照组僵立行为百分比显著低于模型组,两组之间差异有统计学意义(P<0.01)5、血清皮质酮浓度模型组大鼠血清皮质酮浓度显著低于对照组,差异有统计学意义(P<0.01)。二、PTSD大鼠5-HTlA受体活性与海马GR受体和下丘脑CRF表达的关系1、GR和CRF免疫组化分析结果GR的阳性细胞主要分布于海马的CA1区。正常对照组和假手术组之间GR表达没有明显差异。与正常对照组和假手术组相比较,模型组GR表达增高,阳性细胞明显增多(P<0.01),而阻断组阳性细胞数量则比SPS组减少(P<0.05),但仍高于正常对照组和假手术组(P<0.01)。正常对照组和假手术组之间下丘脑CRF表达没有明显差异,模型组CRF表达明显高于正常对照组和假手术组(P＜0.01),而阻断组阳性细胞数量则比SPS组减少(P<0.05)。2、Western blotting分析海马GR表达正常对照组和假手术组均有一定表达,模型组表达明显高于正常对照组和假手术组(P<0.01),阻断组GR表达低于SPS组(P<0.01)。3、RT-PCR分析海马GR mRNA和下丘脑CRF mRNA表达正常对照组和假手术组GR和CRFmRNA表达没有区别,但模型组二者表达显著增高(P<0.01),这种增高可以被WAY100635阻断(P<0.05)三、PTSD大鼠前额皮质磷酸化细胞外信号转导激酶的变化1、pERK1／2免疫组化染色pERK1／2蛋白定位在胞浆,阳性细胞显示棕黄色。对照组阳性细胞的OD值比较小,SPS组大鼠明显增高(P<0.01),PD98059+SPS组大鼠阳性细胞OD值明显比SPS组小(P<0.01)。2、pERK1/2 Western blotting分析SPS组大鼠pERK1/2相对表达明显增高(P<0.01),PD98059+SPS组大鼠pERK1/2比SPS组表达弱(P<0.01)。结果与免疫组化结果相一致。3、RT-PCR分析结果正常大鼠c-fos mRNA有低量表达。SPS大鼠表达增高,注入PD98059后,mPFC c-fos表达减弱(P<0.01)结论1、SPS能使大鼠具有PTSD症状。2、5-HT1A受体活性与海马GR、下丘脑CRF有关。5-HT1A受体活性可能促进海马GR表达,促进下丘脑CRF合成。3、SPS促使大鼠前额皮质ERK磷酸化增高,ERK活性与c-fos表达有关。更多还原

【Abstract】 ObjectivePost-traumatic stress disorder (PTSD), an anxiety disorder, develops after a life-threatening, traumatic experience, and is characterized by symptoms that often endure for years including intrusive memories (flashbacks), avoidance of stimuli associated with the trauma, numbing of general responsiveness, and increased arousal. Currently, PTSD with the characteristics of high incidence, long course, difficult to be cured and seriously impact on people’s physical and mental health. So they have attracted a lot of attention. The exact mechanism of PTSD is still elusive. Negative feedback of the enhanced hypothalamic-pituitary-adrenal (HPA) axis is a putative neuroendocrinological hallmark of PTSD.As a kind of neurotransmitter, serotonin (5-hydroxytryptamine,5-HT) plays an important role in controlling the complex neuronal communication, such as negative feedback of the HPA axis via the serotonergic receptor. It was proposed that serotonergic type 1A (5-HT 1 A) receptor has an important effect on mood and anxiety modulation, but keeps the role of serotonin receptor on PTSD contradicative. It is well established that extracellular signal-regulated kinase 1 and 2 (ERK1/2) play an important role in many cell types, including neurons.Single-prolonged stress (SPS) was shown to induce enhanced inhibition of the hypothalamic-pituitary-adrenal (HPA) axis. And SPS paradigms have been extensively applied in the investigation of PTSD. In this study, we built animal PTSD model using SPS-method, and observed the behavioral changes and determined the hydrocortisone level in rats blood serum. To investigate the effect of 5-HT1A receptor on GR and CRF, we exposed rats to SPS with or without prior treatment with the receptor antagonist and observed the GR level in the hippocampus and CRF level in the hypothalamus. Also, we determined the changes of phosphorylated ERK1/2 (pERKl/2) and c-fos levels in the mPFC of SPS rats, and assessed the effect of PD98059, an inhibitor of the ERK pathway.Methods1, Behavioral changes of PTSD ratsWistar rats were divided randomly into control group and model group. The model of PTSD was prepared by SPS. The behavioral changes of rats were observed with the open field test, elevated plus-maze, and Morris water maze. Hydrocortisone level in rat blood serum was determined with chemiluminescence. And the freezing behavior was measured too.2, Relationship between the activity of 5-HT1A receptor and GR in hippocampus and CRF in hypothalamus in PTSD rats.Rats were randomly divived into normal group, sham group (vehicle administration), SPS group (SPS+vehicle administration), and blockade group (SPS+ WAY100635 administration). GR levels in the hippocampus and CRF levels in the hypothalamus by immunohistochemistry, Western blotting and RT-PCR3, Change of ERK in mPFC of PTSD ratsRats were randomly divided into three groups:control group, SPS group, and PD98059 group. The SPS group rats and PD98059 group rats were treated with SPS procedure, and respectively injected solvent or PD98059 into the bilateral mPFC 30 min before exposure to SPS. Brains were removed for immunohistochemical staining and Western blotting of pERKl/2. And RT-PCR was employed to detect the c-fos mRNA. Results1, Behavioral changes of PTSD rats(1) Results of OF testThe result showed that detention time in central grille and erect quantity of model rats were less than that of control rats (P<0.05). There were no significantly difference in other index.(2) Results of EMP testPercentage of the number of entry into open arms and percentage of time spent were significantly decreased in SPS rats compare to control rats (P＜0.01).(3) Results of MWM testCompared to control group, a striking increase of the latency was observed in SPS rats (P<0.01).(4) Results of freezing behavior The model rats showed freezing behavior when they were faced contextual fear condition again. However, the control rats showed free movement when faced the fear condition again. The percentage of freezing of control rats was less than model rats (P＜0.01).(5) Hydrocortisone level in blood serumHydrocortisone level in model rats blood serum was less than that of control rats (P＜0.01).2, Relationship between the activity of 5-HT1A receptor and GR in hippocampus and CRF in hypothalamus in PTSD rats.(1) Results of immunohistochemical analysis of GR and CRFThere was no statistically significant difference between normal and sham groups. Compared to the normal and sham groups, expression of GR and CRF increased in SPS group (P＜0.01), which were partially blocked by WAY100635 (P<0.05).(2) GR expression detected by Western blotting Similar to the immunohistological staining, there was GR expression in the hippocampus area when rats were under normal conditions. SPS significantly increased the GR expression (P<0.01) which is partially decreased by WAY100635 (P＜0.01).(3) GR and CRF mRNA expression detected by RT-PCRThere was both GR and CRF expression in the normal condition. Compared with the normal group, sham had no effect on the GR and CRF mRNA expression, however, SPS significantly increased their expression (P<0.01) which were partially decreased by WAY100635 (P<0.05).3, Change of ERK in mPFC of PTSD rats(1) Result of pERK1/2 immunohistochemistry stainingThe pERKl/2 protein was located in cytoplasm, and positive cells were brown. In control group, the OD of pERKl/2 positive cells was little, and that of SPS rats were significantly increased (P<0.01), and after the rats were bilaterally infused the PD98059, the pERKl/2 positive cells in the mPFC was significantly abolished (P< 0.01).(2) Result of Western blotting analysis pERKl/2The density of pERKl/2 bands had a significant increase in SPS rats (P<0.01). After the rats were bilaterally infused the PD98059, the SPS-induced ERK1/2 hyperphosphorylation in the mPFC was significantly abolished (P<0.01).(3) Result of RT-PCRVery low level of c-fos mRNA were detected of control rats. However, the expression of c-fos mRNA had a significant increase in SPS rats (P＜0.01). After the rats were bilaterally infused the PD98059, the SPS-induced c-fos mRNA increasing in the mPFC was significantly abolished (P＜0.01).Conclusions1, SPS rats exhibit behavioral abnormalities that mimic the symptoms of PTSD.2, Activity of 5-HT1A receptor is involved in the alteration of GR and CRF in SPS rats.3, SPS promoted the phosphorylated ERK in mPFC and ERK may be related to signal transduction pathway in single-prolonged stress.更多还原