【作者】 赵丽霞；

【导师】 杜怡峰；

【作者基本信息】 山东大学 ， 临床医学（专业学位）， 2013， 博士

【摘要】 卒中后抑郁(post-stroke depression, PSD)是脑卒中后最常见的心理行为障碍并发症,在卒中患者中的发病率约为25%-79%。PSD具体发病机制尚未完全清楚,给治疗带来极大的困难。最新研究表明,miRNA可能在抑郁症的发生、发展中扮演着重要的角色,可以作为抑郁症临床治疗的潜在靶点。作为抑郁症中的特殊一种,PSD的发病是否也与miRNA相关呢?脑卒中患者外周血检测发现,有多种miRNA在急性期出现上调或下降,随后在鼠类缺血模型中的研究证实了脑内也出现了相应的miRNA变化。令人感兴趣的是,在脑缺血后下调的miRNA和抑郁症患者下调的miRNA有着部分的重叠,但这些重叠的miRNA在PSD的发生发展中发挥的作用尚无人研究。因此,本实验选取miR-137为对象,研究其对PSD大鼠模型行为学的影响,旨在为PSD发病机制和治疗药物的研究提供新的思路。第一部分大脑中动脉阻塞法结合慢性温和刺激建立卒中后抑郁大鼠模型目的：建立卒中后抑郁大鼠模型,为疾病的研究提供实验基础。方法：筛选体重180～210g的清洁级健康SD大鼠,雌雄各半,正常饲养1周适应环境,监测并记录大鼠体重、蔗糖水消耗量、水平活动得分及垂直运动得分。平均分为3组：对照组、卒中组与PSD(缺血加应激)组,采用大脑中动脉阻塞法(middle cerebral artery occlusion, MCAO)制作脑缺血再灌注模型,模型成功后选取Longa评分≥1并<4的大鼠,通过慢性温和刺激(chronic mild stress, CMS)建立PSD模型。分别采用糖水消耗试验、旷野试验(open-field test, OFT)评估大鼠快感缺失、活动减少、行为绝望等行为。结果：与对照组相比,PSD组大鼠体重的增长明显滞缓,于7d时PSD组大鼠体重开始低于对照组(P<0.05),于14d时体重低于卒中组(P<0.05)。随着时间变化,PSD组大鼠蔗糖水消耗比例逐渐下降。与对照组相比,14d和21d时PSD组大鼠蔗糖水消耗量明显降低(P<0.05),并且在21d时PSD组大鼠蔗糖水消耗比例低于卒中组(P<0.05)。随时间变化,PSD组大鼠水平得分逐渐下降,至21d时仍呈下降趋势,各时间段水平得分均小于对照组(P<0.05)。21d时,PSD组大鼠脑组织中NE、DA和5-HT含量均明显低于对照组(P<0.05)。结论：PSD模型大鼠较充分而持续表现快感缺乏、活动减少等“抑郁”核心症状,可操作性和重复性较好,是研究PSD较为理想的大鼠模型。第二部分miR-137在PSD大鼠脑内的表达及其治疗作用目的：观察microRNA在PSD大鼠脑内的表达及其对PSD大鼠的治疗作用。方法：①取正常大鼠、MCAO大鼠和PSD大鼠处死取脑,microarray检测差异microRNA表达,并对表达差异的microRNA进行qRT-PCR检测验证；②并选取24只SD大鼠入组：6只大鼠不作任何处理(control组)、6只大鼠经MCAO+CMS制作PSD模型(PSD组)、6只大鼠经MCAO+脑室注射agomir-137+CMS (agomir-137组)、6只大鼠经MCAO+脑室注射agomir-NC+CMS (agomir-NC组),对各组大鼠进行行为学检测。结果：①microarray检测结果显示,与正常大鼠相比,PSD大鼠脑内多种microRNA表达上调或下调,如miR-137, miR-124, miR-181和miR-145等。qRT-PCR验证结果与microarray结果相符。PSD大鼠脑内和外周血中miR-137表达水平均明显低于正常大鼠(p<0.05)。②旷野实验结果显示,在脑缺血第3w,经脑室注射agomir-137的PSD大鼠垂直得分以及水平得分均显著高于agomir-NC组大鼠以及普通PSD大鼠(p<0.05)。糖水消耗实验结果显示,在脑缺血后第2w末起,agomir-137组大鼠糖水消耗百分比即显著高于agomir-NC组大鼠以及普通PSD大鼠(p<0.05)。结论：①多种microRNA可能参与PSD的病理生理过程,其中以miR-137作用比较明显；②人为增强内源性miR-137表达可以发挥抗PSD的作用。第三部分miR-137通过抑制Grin2A表达发挥对卒中后抑郁大鼠模型的治疗作用目的：寻找miR-137治疗PSD的作用靶点。方法：①为揭示miR-137对卒中后抑郁大鼠模型的治疗作用机制,我们通过pictar、 microbase和targetscan三个数据库寻找miR-137的作用靶点,结果发现Grin2A基因的3’UTR端包含一个7mer大小的序列与miR-137相匹配；②为验证miR-137影响Grin2A的蛋白表达,我们将agomir-137及其阴性对照agomir-NC、antagomir-137及其阴性对照antagomir-NC转染至PC12细胞中,western blot检测Grin2A水平差异。③为确定Grin2A基因的3’UTR端包含的7mer大小的序列与miR-137结合后可下调Grin2AmRNA的翻译,我们将含此位点的Grin2AmRNA3’UTR构建入luciferase报告系统(Grin2A-3’UTR-wt),同时将突变后的Grin2AmRNA3’UTR也插入luciferase报告系统中(Grin2A-3’UTR-mut)。然后将miR-137与luciferase空质粒(empty vector)或Grin2A-3’UTR-wt质粒或Grin2A-3’UTR-mut质粒共同转染至HEK-293细胞中,检测各组细胞中的荧光强度。④为检测Grin2A是否参与miR-137抗PSD作用,12只大鼠被添加入组：6只大鼠经MCAO+脑室注射AAV9-CMV-Grin2A质粒和agomir-137+CMS (agomir-137+Grin2A),6只大鼠经MCAO+脑室注射空质粒和agomir-137+CMS (agomir-137+vector),并对所有组大鼠进行行为学检测。结果：(DmiR-137mimic可显著降低PC12细胞中Grin2A水平(p<0.05),而miR-137inhibitor可显著升高PC12细胞中Grin2A水平(p<0.05)。②Grin2A-3’UTR-wt组荧光强度明显低于其阴性对照组(miR-con),而Grin2A-3’UTR-mut组或空质粒组荧光强度与其阴性对照无明显差异。③Grin2A过表达组(agomir-137+Grin2A)的大鼠垂直得分以及水平得分均明显低于其阴性对照(agomir-137+vector)和agomir-137组(p<0.05)。并且Grin2A过表达组蔗糖水消耗百分比明显低于其阴性对照和agomir-137组(p<0.05)。结论：miR-137可以通过与Grin2A mRNA3’UTR端结合而调节其蛋白表达水平,过表达Grin2A可阻断miR-137对PSD大鼠行为学的影响,证明其参与了miR-137对PSD大鼠的治疗作用。全文结论：1.PSD模型大鼠较充分而持续表现快感缺乏、活动减少等“抑郁”核心症状,可操作性和重复性较好,是研究PSD较为理想的大鼠模型。2.卒中后抑郁大鼠模型脑内和外周血中的miR-137水平明显下降,上调脑内miR-137水平后可显著改善PSD模型的行为学变化。3.miR-137可以与Grin2A的3’UTR端结合,抑制Grin2A mRNA的翻译从而下调其蛋白的表达,而在卒中后抑郁大鼠模型的脑内过表达Grin2A基因后,miR-137抗卒中后抑郁的作用被显著减弱,表明阻断Grin2A基因表达是miR-137治疗PSD的作用途径之一。更多还原

【Abstract】 Post-stroke depression (PSD) is one of the most common psychological behavior disorders complications after stroke. The incidence of PSD is about25%to79%of stroke patients. The specific mechanism of PSD is not entirely clear, causing great difficulties in disease remedy. Recent research has suggested that miRNAs likely play an important role in the occurrence and development of depression, and show potential as targets for treatment of clinical depression. However, it is not yet known if miRNAs are associated with post-stroke depression. Recently, Scholars showed altered expression of various miRNAs in pe-ripheral blood, from acute stage stroke patients. A further study confirmed corresponding miRNA changes in the brain, in a mouse ischemia model. Interestingly, there are small overlaps between downregulated miRNA expression after cerebral ischemia, and in depression patients. Nevertheless, the effects of these overlapping miRNAs on the occurrence and development of post-stroke de-pression remains unclear. Therefore, in this study, we used a rat model of post-stroke depression to investigate the effects of miR-137on behavior.Part1:The establishment of post-stroke depression rats modelObjective:To establish rats model of post-stroke depression for providing experimental basis of disease research.Methods:SD rats were randomly divided into three groups:control group, stroke group and PSD. Cerebral ischemia reperfusion model was produced using the brain artery occlusion method (middle cerebral artery occlusion, MCAO). And MCAO models of Longa grade1-4were choosed to establish PSD model by chronic mild stimulation (chronic mild stress, CMS). Sugar water consumption test, the wilderness test (open-field test, OFT) and forced swimming were used respectively to assess the sensation loss, decreased activityand behavioral despair of rats. And all of the rats were sacrificed to detect the neurotransmitters levels in the hypothalamus.Results:Compared with control group, the growth of body weight of rats were obviously lagging in PSD group. At7d, PSD group rats weight were lower than the control group (P<0.05) and lower than the stroke group in14d (P<0.05). As time changes, the sucrose water consumption proportion of PSD group rats gradually declined. Compared with control group in14d and21d, sucrose water consumption of rats in PSD group was decreased obviously (P<0.05), and in21d sucrose water consumption ratio of PSD rat was lower than the stroke group (P<0.05). Change over time, the PSD group rats score level gradually decline, to21d was still declining, and scores are less than the control group at each time (P<0.05). In the21d, NE, DA and5-HT levels of the PSD group rats were significantly lower than the control group (P<0.05).Conclusion:PSD rat model could fully performant core symptoms of PSD such as lacking of sensation, decreased activity and etc. The maneuverability and repeatability of rat model is good and the rat model is an ideal model for investigation of PSD.Part2:miR-137in PSD expression in rats and its therapeutic effectObjective:To observe the expression of miRNAs in PSD rat and its effect on the treatment of PSD rat.Methods:①MCAO rats, PSD rats and normal rats were used to investigate microRNA expression in the brain via microarray detection, and the results were verificated by qRT-PCR.②Subsequently,24SD rats were divided into4groups, specifically, model, stroke, agomir-137, agomir-NC (negative control). The lasttwo groups were received an injection of agomir-137or agomir-NC into the left lateral ventricle48hours before model induction.21days later, behavioral tests of each group rats were examined.Results:①Microarray detection results showed that compared with normal rats, the levels of a variety of miRNAs expressed in brain of PSD rat increased or decreased, such as miR-137, miR-124, miR-181, miR-145, and etc. QRT-PCR verification results were consistent with the microarray results. The expression level of miR-137in the peripheral blood and brain of PSD rat were significantly lower than normal rats (p<0.05).②Wilderness, according to the results of experiment on cerebral ischemia3w, the PSD rat ventricle injection agomir-137vertical and horizontal score score were significantly higher than that of agomir-NC group of rats and normal PSD rats (p <0.05). The experimental results showed that the sugar water consumption after cerebral ischemia, at the end of the2w agomir-137percentage group rats sugar water consumption is significantly higher than agomir-NC group of rats and normal PSD rats (p<0.05).Conclusion:①Many miRNAs may participate in the pathological processes of PSD and the effect of miR-137was more noticeable.②Artificially enhanced the expression of endogenous miR-137could play a role of resistance to the PSD.Part3:miR-137by inhibiting Grin2A expression of play in the treatment of post-stroke depression model ratsObjective:In order to search the targets of miR-137involving in the treatment of PSD.Methods:①To investigate the mechanism underlying miR-137behavioral improvements in rats with post-stroke depression, potential miR-137targets were identified using the databases, pictar, microbase and targetscan.②To determine if Grin2A participates in miR-137-induced behavioral improvements in post-stroke depression rats, we injected a plasmid containing the Grin2A gene into the brain of post-stroke depression rats, via the brain ventricles.③To determine if the identified sequence downregulates Grin2A mRNA translation following miR-137binding, Grin2A3’UTRs containing the target site (Grin2A-3’UTR-wt) or a mutated site (Grin2A-3’UTR-mut), were constructed in a luciferase reporter system. Subsequently, miR-137and either luciferase empty vector or Grin2A-3’UTR-wt or Grin2A-3’UTR-mut plasmids were co-transfected into HEK-293cells. HEK-293cells were incubated in RPMI1640medium containing10%fetal bovine serum at37℃in an incubator containing5%CO2. Full-length Grin2A3’UTR containing the miR-137binding site, was PCR amplified and cloned into HindⅢ and SacⅠ sites of the pMIR-REPORT miRNA expression reporter vector. The resulting vector was named Grin2A-3’UTR-wt. A point mutation of Grin2A-3’UTR-wt was identified using the Easy Mutagenesis System and subcloned also, with the resulting vector named Grin2A-3’UTR-mut. HEK293T cells were seeded into24-well plates and divided into three groups. Empty vector (400ng) was added to the control group, Grin2A-3’UTR-wt plasmid (400ng) to the wild-type group, and Grin2A-3’UTR-mut plasmid (400ng) to the mutation group. Plasmids and miR-137were transfected using Lipofectamine2000, and20ng pRL-TK was added as an internal reference. Within each group, parallel wells were used for the addition of mimic-NC, a negative control for miR-137mimic (miR-con). After36hours of transfection, the fluorescence intensity of cells was measured using the Dual-Luciferase Reporter Assay System.④24rats were randomly assigned to4groups, specifically, agomir-137, agomir-NC (negative control), agomir-137+Grin2A and agomir-137+vector groups. The four agomir groups received an injection of agomir-137, a miR-137antagonist into the left lateral ventricle48hours before model induction. Agomir-NC, LV-CMV-Grin2A and-control plasmids were also injected into the left lateral ventricle48hours before model induction.Results:①Moreover, western blot assays showed that in PC12cells, miR-137mimic (mimics of synthesized mir-137, showing similar effects to natural mir-137) significantly reduced (P<0.05), but miR-137inhibitor significantly increased (P<0.05), Grin2A levels. These results suggest that miR-137binds to the3’UTR of Grin2A mRNA and regulates Grin2A protein expression.②Luciferase assays found significantly lower fluorescence intensities in HEK-293cells transfected with Grin2A-3’UTR-wt, compared with negative controls (miR-con)(P<0.05). However, no significant difference was detected between HEK-293cells transfected with Grin2A-3’UTR-mut or empty vector (P>0.05; Figure3B). This suggests miR-137binds to the3’UTR of Grin2A mRNA, and regulates its translation.③We found significantly lower locomotor and rearing activities in the agomir-137+Grin2A group than in the negative control (agomir-137+vector) and agomir-137groups (P<0.05). Moreover, the sucrose consumption percentage was significantly lower in the agomir-137+Grin2A group than in the negative control and agomir-137groups (P<0.05). These results suggest that Grin2A overexpression prevents the improved behavioral effects of miR-137in rats with post-stroke depression, and confirms the involvement of Grin2A in the miR-137therapeutic effect in post-stroke depression rats..Conclusion:miR-137could bind with Grin2A mRNA3’UTR and downregulated its protein expression at the post-transcriptional level. Overexpression of Grin2A could block theinfluence of mi-137on ethology of PSD rat.The conclusions:1. PSD rat model could fully performant core symptoms of PSD such as lacking of sensation, decreased activity and etc. The maneuverability and repeatability of rat model is good and the rat model is an ideal model for investigation of PSD.2. Many miRNAs may participate in the pathological processes of PSD and the effect of miR-137was more noticeable. Artificially enhanced the expression of endogenous miR-137could play a role of resistance to the PSD. 3. miR-137could bind with Grin2A mRNA3’UTR and downregulated its protein expression at the post-transcriptional level. Overexpression of Grin2A could block theinfluence of miR-137on ethology of PSD rat.更多还原