大鼠皮层AA-COX2通路参与抑郁症发病机制研究

发布时间：2018-05-13 00:14

本文选题：抑郁 + CUMS　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:观察慢性应激大鼠的抑郁行为与皮层神经元COX2表达变化的相关性,并从皮层COX2-EPs-BDNF通路初步探讨抑郁症发病机制。方法:1.大鼠抑郁模型的建立与皮层中AA-COX2通路改变:雄性SD大鼠20只,随机分为正常组(n=10)和模型组(n=10),正常组大笼饲养,每笼5只,自由摄水进食且不给予任何刺激;模型组孤养并给予慢性不可预见性温和刺激(chronic unpredictable mild stress,CUMS),连续42d,造模完成后进行行为学测试。2.COX2干预对慢性应激大鼠抑郁行为的影响:(1)雄性SD大鼠50只,随机分为5组(n=10),即正常对照组、模型组、阳性药物对照组、美洛昔康1 mg?kg-1组和美洛昔康3 mg?kg-1组,正常组大鼠处理同第一部分,其余四组大鼠孤养并给予42d CUMS,给药组大鼠分别灌胃给予舍曲林(5 mg?kg-1)、美洛昔康(1、3 mg?kg-1),模型组与正常组给予等体积0.5%CMC-Na,每天一次,连续21d,给药完成后进行行为学测试。(2)另取雄性SD大鼠40只,并将其随机分为3组,即空载病毒(control)组(n=10)大笼饲养,自由摄水进食且不给予任何刺激;COX2过表达慢病毒注射+CUMS组(n=10)和空载病毒+CUMS组(n=20)。大鼠施行侧脑室定位注射导管埋管,从术后第5天开始给予cums,并分别在术后第5d,12d,19d进行脑室内慢病毒注射,每只15μl/次。给予cums42d后进行行为学测试,空载病毒+cums组大鼠随机分为两组,即cox2rnai(n=10)和cums组(n=10),分别脑室内注射cox2rnai慢病毒和空载病毒,每7天一次,共3次,每只15μl/次,第3次注射后第7d进行行为学测试。在以上分组和干预的基础上采用糖水实验、旷场实验和强迫游泳实验检测大鼠行为学变化,以苏木精-伊红染色观察大鼠皮层神经元形态变化;elisa和生化酶学法测定大鼠皮层组织pge2、tnf-α、il-1β、camp、mda含量和sod活性;免疫组化检测皮层bdnf蛋白表达情况;rt-pcr检测大鼠皮层cox2mrna表达;westernblot检测cox2、ep2、ep3、pkaⅡαreg、creb、p-creb、bdnf蛋白含量。结果:1.与正常组相比,cums大鼠糖水偏好率下降(p0.01),旷场实验水平(p0.01)和垂直得分(p0.01)减少,强迫游泳静止不动时间增加(p0.01);皮层神经元出现明显核固缩;皮层pge2、tnf-α、il-1β、mda含量增加(p0.01),而sod活性下降(p0.01);皮层cox2mrna和蛋白的表达(p0.01)均增加。2.与单独给予cums的空载病毒大鼠相比,在给予大鼠cums的同时侧脑室注射过表达cox2慢病毒,能进一步降低大鼠糖水偏好率,明显减少大鼠旷场水平和垂直得分(p0.05),增加强迫游泳不动时间(p0.05);增加皮层pge2、tnf-α、il-1β、mda含量(p0.01或P0.05)和降低SOD活性(P0.05),减少c AMP含量(P0.05),显著增加COX2 mRNA(P0.05)表达的同时,COX2、EP2和EP3蛋白表达有上升趋势,PKAⅡαreg蛋白表达显著减少(P0.05),而p-CREB、BDNF蛋白表达有下降趋势。当给予美洛昔康抑制COX2活性或侧脑室注射COX2 RNAi慢病毒,则能明显提高抑郁大鼠糖水偏好率(P0.05),增加大鼠旷场实验水平和垂直得分(P0.01),减少强迫游泳不动时间(P0.05);显著减少皮层PGE2、TNF-α、IL-1β、MDA含量(P0.01)和增加SOD活性(P0.01);明显增加皮层c AMP含量(P0.01)和PKAⅡαreg、p-CREB、BDNF蛋白表达(P0.01或P0.05),下调COX2 mRNA(P0.01)和蛋白(P0.05)以及EP2(P0.01)、EP3(P0.05)蛋白的表达。结论:CUMS致抑郁大鼠皮层中AA-COX2通路明显激活。抑制COX2活性或降低其在大脑中的表达可明显改善抑郁大鼠的抑郁行为;而COX2过表达可增加大鼠对CUMS的易感性,加重抑郁症状。皮层AA-COX2通路激活参与抑郁症发病机制可能涉及下游产物PGE2激动EP2、EP3受体,直接影响c AMP/PKA-CREB-BDNF通路的活化程度,从而改变皮层神经元可塑性。
[Abstract]:Objective: To observe the correlation between the depressive behavior of chronic stress rats and the changes of COX2 expression in cortical neurons, and to explore the pathogenesis of depression from the cortical COX2-EPs-BDNF pathway. Methods: the establishment of the depression model in 1. rats and the change of the AA-COX2 pathway in the cortex: 20 male SD rats were randomly divided into normal group (n=10) and model group (n=10), normal and normal. The group was fed with 5 cages, free of water intake and no stimulation. The model group was isolated and given chronic unpredictability and mild stimulation (chronic unpredictable mild stress, CUMS) and continuous 42d. After the completion of the model, the behavioral test was carried out by.2.COX2 intervention on the depressive behavior of chronic irritable rats: (1) 50 male SD rats were randomly selected. Divided into 5 groups (n=10), namely normal control group, model group, positive drug control group, meloxicam 1 mg? Kg-1 group and meloxicam 3 mg? Kg-1 group, normal group rats were treated with the first part, the other four groups of rats were isolated and given 42d CUMS, and the rats in the drug group were given Seculin (5 mg? Kg-1), meloxicam (1,3 mg? Kg-1), model group and normal Group given equal volume 0.5%CMC-Na, once a day, continuous 21d, after the completion of the drug test. (2) another 40 male SD rats were taken, and they were randomly divided into 3 groups, namely, the empty virus (control) group (n=10) large cage, free intake of water intake and no prickly stimulation; COX2 overexpression lentivirus +CUMS group (n=10) and the empty virus +CUMS group ( N=20). The rats performed the lateral ventricle with intraventricular catheter buried tube, and were given cums from fifth days after the operation. The intraventricular lentivirus was injected at 5D, 12D, and 19d after the operation, each 15 l/ times. After cums42d, the behavior test was carried out. The empty virus +cums group rats were randomly divided into two groups, cox2rnai (n=10) and cums group (n=10), intraventricular injection respectively. Cox2rnai lentivirus and no-load virus were shot, once every 7 days, 3 times, each 15 mu l/ times, and after third injection, the behavior test was tested at 7d. On the basis of the above group and intervention, the behavior changes of rats were detected by the sugar water experiment, the open field experiment and the forced swimming test, and the morphological changes of the cortical neurons in the rats were observed with hematoxylin eosin staining; e Determination of PGE2, tnf- alpha, IL-1 beta, camp, MDA content and SOD activity in rat cortical tissue by Lisa and biochemical enzymology; immunohistochemistry to detect the expression of BDNF protein in cortex; RT-PCR detection of cortical cox2mrna expression in rats; Westernblot detection COX2. The rate decreased (P0.01), the level of open field experiment (P0.01) and the vertical score (P0.01) decreased, the resting time of forced swimming increased (P0.01), the cortical neurons had obvious nuclear pyknosis, the cortex PGE2, tnf- a, IL-1 beta, MDA content increased (P0.01), and SOD activity decreased (P0.01), and the expression of cortical cox2mrna and protein increased and was given alone. Compared with the no-load virus, the injection of COX2 lentivirus in the lateral ventricle of rat cums could further reduce the sugar water preference rate, reduce the level and vertical score (P0.05) of rats and increase the time of forced swimming (P0.05), and increase the PGE2, tnf- alpha, IL-1 beta, MDA content (P0.01 or P0.05) and SOD activity (P0.) (P0.) (P0.) and SOD activity (P0.). 05), decrease the content of C AMP (P0.05), significantly increase the expression of COX2 mRNA (P0.05), while COX2, EP2 and EP3 protein expression has a rising trend, PKA II alpha reg protein expression significantly decreased (P0.05). Rats' sugar water preference rate (P0.05) increased rats' open field experiment level and vertical score (P0.01), reduced forced swimming time (P0.05), decreased cortical PGE2, TNF- alpha, IL-1 beta, MDA content (P0.01) and increased SOD activity (P0.01). MRNA (P0.01) and protein (P0.05) and the expression of EP2 (P0.01), EP3 (P0.05) protein. Conclusion: the AA-COX2 pathway in the cortex of depressive rats is obviously activated by CUMS. Inhibiting the activity of COX2 or decreasing its expression in the brain can obviously improve the depressive behavior of the depressed rats, and COX2 overexpression can increase the susceptibility to CUMS and aggravate the depressive symptoms. The activation of the layer AA-COX2 pathway involved in the pathogenesis of depression may involve the downstream product PGE2 excited EP2, the EP3 receptor, which directly affects the activation of the C AMP/PKA-CREB-BDNF pathway, and thus changes the plasticity of cortical neurons.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.4

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