【作者】 邓镇；

【导师】 姜晓丹； 陈育庆；

【作者基本信息】 南方医科大学 ， 神经外科学， 2010， 博士

【摘要】 脑血管疾病是人类第三死亡原因,其中脑缺血(又称脑梗塞,ischemic stroke)约占其中的70%。脑缺血发病率高,病情严重,临床上缺乏有效的治疗措施,使得死亡率和致残率较高,给个人和社会带来沉重的负担。脑缺血病理情况发展复杂,兴奋性氨基酸中毒、氧化自由基剧增、炎症反应加重等均促进了脑组织的破坏,但就此而设计的药物临床治疗试验均未能成功,因此我们需要以新的角度和方法更深入地理解脑缺血病理发展机制,为临床治疗新措施提供参考。本课题分为两部分,分别对microRNA-497 (miR-497)和过氧化物酶增殖体激活受体δ/β(peroxisome proliferator-activated receptorδ/β, PPARδ/β)调控脑缺血神经损伤的作用机制进行了探讨。microRNA是一类新发现的非转录型小RNA分子,它们在机体生理、病理以及神经系统中的作用越来越受到重视。本课题的第一部分,重点研究了microRNA-497 (miR-497)对脑缺血神经损伤的调控机制。经过脑缺血模型后,我们使用real-time PCR筛选若干已报道的凋亡相关miRNA,发现miR-497在脑缺血前后具有显著变化；并进一步在细胞模拟脑缺血模型(OGD模型)中研究miR-497对神经细胞凋亡的影响,探讨其对凋亡通路的调控及其机制。我们发现,miR-497可促进N2A神经细胞(一种神经细胞瘤细胞系)的凋亡,通过生物信息学的查询、Western Blot检测以及荧光素酶实验证实,miR-497靶基因是抑制线粒体凋亡通路的Bcl-2和Bcl-w基因,miR-497通过下调Bcl-2和Bcl-w而起到促进神经细胞凋亡的作用。为了探讨miR-497在体内对脑缺血神经损伤的调控机制及意义,我们使用寡核苷酸沉默技术设计了沉默miR-497的寡核苷酸链,通过侧脑室给予miR-497沉默寡核苷酸注射,24h后发现miR-497表达呈显著下调,而其他miRNA不受影响,Bcl-2和Bcl-w蛋白水平则明显上升,显示治疗达到了预期效果。注射24h后行脑缺血手术,术后23h行氯化三苯四唑(triphenyltetrazolium chloride, TTC)染色,显示治疗组大脑的总损伤面积、皮层损伤面积和皮层下损伤面积均有减少,神经障碍评估显示障碍水平有所减轻,提示沉默miR-497可起到保护脑缺血所致大脑损伤、改善神经功能障碍的作用。本项目从体外、体内模型入手,探讨了与细胞凋亡相关的miRNAs变化,特别是以miR-497为中心的凋亡机制研究,探索了miR-497对神经细胞线粒体凋亡通路的调控规律,发现miRNA体内干预技术对脑缺血的治疗应用具有显著疗效,从而揭示了miR-497在脑缺血神经损伤中的作用和意义。过氧化物酶增殖体激活受体δ/β(peroxisome proliferator-activated receptorsδ/β, PPARδ/β)是配体激活转录因子型的核受体,对机体能量代谢起有重要调节作用,其激动剂(如GW501516)已经进入临床试验。有报道显示,激活PPARδ/β对内皮细胞有显著的保护功能,由于脑血管内皮细胞对脑缺血后维持血脑屏障起有关键作用的缘故,在第二部分研究中,我们探讨了激活PPARδ/β对脑缺血后微血管内皮细胞损伤的保护作用。给予小鼠侧脑室注射PPARδ/β受体激动剂GW501516后,小鼠在脑缺血后的大脑损伤体积减少；通过尾静脉注射Evans蓝检验大脑微血管通透性,亦显示治疗组血管通透性较低,提示以GW501516治疗、可以更好地保护血脑屏障。为进一步研究激活PPARδ/β对血脑屏障的保护机制,我们检测了脑缺血后大脑微血管内皮细胞的凋亡指标：Caspase-3活性、DNA断裂情况和重要抗凋亡蛋白Bcl-2表达,发现GW501516治疗能降低Caspase-3活性,减轻DNA断裂情况和升高Bcl-2蛋白水平,提示内皮细胞凋亡得到了抑制。根据以往报道,miR-15a(一种凋亡相关miRNA)可通过下调其靶基因Bcl-2而促进凋亡,而我们以使用Real-time PCR检测也观察到,经GW501516治疗后的缺血脑组织微血管内皮细胞miR-15a表达显著下调,因此,激活PPARδ/β、下调miR-15a表达可能是Bcl-2蛋白升高的原因之一。本部分实验从PPARδ/β受体激动剂GW501516减轻脑缺血神经损伤的现象进行深入研究,发现治疗延缓了脑缺血后内皮细胞的凋亡,从而保护了血脑屏障,抑制脑缺血损伤的发展。第一部分miR-497对脑缺血神经细胞凋亡机制的调控研究目的急性脑缺血发病率高,但目前对脑缺血引起神经细胞凋亡的具体机制尚不完全清楚,故未能研发出有效的治疗药物,导致脑缺血总体死亡率和致残率较高。miRNA作为一种新的基因调控方式,逐渐被发现参与体内各种生理病理过程,最近,miRNA在凋亡方面的作用更是引起人们的注意。已有实验报道指出,脑缺血发生后,多种病理机制的发生发展最后将触发神经细胞凋亡通路,是致使大脑损伤不断加重的因素之一。虽然有研究报道脑缺血发生后,大量的miRNA表达发生变化,但这些变化在脑缺血机理中的详细作用尚不清楚。本实验拟通过中脑动脉闭塞(middle cerebral artery occlusion, MCAO)模型,寻找参与调控急性脑缺血病理机制的miRNA,在细胞和动物水平探讨miRNA对神经细胞凋亡的影响。实验方法1.予小鼠行大脑MCAO后23h,生理盐水灌注取大脑损伤和非损伤临界区域的脑组织,提取总RNA后对多个miRNA作real-time PCR并与手术非梗塞组比较,寻找表达有显著差别的miRNA;2.在Neuro2a (N2A)细胞70-80%密度时行氧糖剥夺模型(oxygen glycogen deprivation, OGD);模型后0h,4h,24h分别使用乳酸脱氢酶(lactate dehydrogenase, LDH)和四甲基偶氮噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞凋亡数量,或提取miRNA和蛋白检测miR-497、Bcl-2和Bcl-w表达；3.转染miR-497模拟物或抑制物进N2A细胞,48h后提取蛋白作Western Blot检测Bcl-2和Bcl-w表达；或行OGD,模型后0h,4h,24h使用LDH和MTT法检测细胞凋亡数量；4.构建Bcl-2和Bcl-w正常和突变3’UTR片段荧光素酶质粒,和miR-497模拟物共转染后、使用荧光素酶试剂盒和荧光读板仪读数；5.予小鼠行MCAO模型23h后,生理盐水灌注取大脑损伤和非损伤的临界区域脑组织,作Western Blot检测Bcl-2和Bcl-w表达;6.设计沉默miR-497的寡核苷酸链；7.通过立体定位注射仪定位侧脑室,注射miR-497沉默寡核苷酸链,同时另设注射人工脑脊液(空白组)、增强型绿荧光蛋白(enhanced green fluorescin protein, eGFP)寡核苷酸链(对照组)；8.为检测寡核苷酸链的沉默效率,注射后24h、以Real-time PCR检测脑组织miR-497表达,同时检测miR-21表达作用阴性对照；9.为检测沉默miR-497后对脑组织内源性蛋白的影响,Western Blot检测脑组织Bcl-2和Bcl-w蛋白表达变化；10.注射miR-497沉默寡核苷酸链后24h,予小鼠行MCAO模型23h后取大脑切作8层面,TTC染色后扫描,使用Metamorph软件计算总损伤面积、皮层损伤面积和皮层下损伤面积；11.注射miR-497沉默寡核苷酸链后24h,予小鼠行MCAO模型23h后使用5分6级法作神经障碍评分。12.统计学分析方法：两组比较使用双尾T检验,One-Way Anova作方差分析后,组间比较使用Bonferroni’s post-hoc检验,神经功能评分使用非参数Kruskal Wallis检验。P值小于0.05视为有显著性差异。实验结果1. MCAO模型后,在大脑损伤和非损伤的临界区域脑组织,miR-497表达显著升高；2.N2A细胞在OGD模型后,miR-497表达升高,同时,随着细胞凋亡情况的加重,Bcl-2和Bcl-w表达下降；3.在N2A细胞中过表达miR-497使得Bcl-2和Bcl-w表达下降,抑制miR-497则Bcl-2和Bcl-w表达升高；另外,过表达miR-497能增加细胞在OGD模型中的死亡,抑制miR-497则减轻细胞在OGD模型中的死亡；4.MiR-497模拟物转染使得Bcl-2和Bcl-w 3’UTR荧光读数降低,而突变后荧光读数不变；5.小鼠MCAO模型23h后,Western Blot显示Bcl-2和Bcl-w表达下降；6.注射miR-497沉默寡核苷酸链24h后,和空白组和对照组比较,miR-497表达下降;7.注射miR-497沉默寡核苷酸链24h后, Bcl-2和Bcl-w蛋白水平上升；8.注射miR-497沉默寡核苷酸链24h后行MCAO模型,23h后作TTC染色,和空白组和对照组比较,治疗组在总损伤面积、皮层损伤面积和皮层下损伤面积均比其余两组较小。9.注射miR-497沉默寡核苷酸链24h后行MCAO模型,23h后予小鼠行神经障碍评估,和空白组和对照组比较,治疗组神经障碍较轻。结论1. MCAO模型和OGD模型均能引起脑组织或N2A细胞miR-497表达升高；2.miR-497促进神经细胞在OGD模型中的凋亡发展,机制为miR-497降解其靶基因Bcl-2和Bcl-w而产生促进神经细胞凋亡的功能；3.抑制miR-497在脑组织的表达能增加Bcl-2和Bcl-w蛋白水平,从而保护脑缺血引起的大脑损伤体积以及改善脑缺血引起的神经功能障碍。第二部分激活过氧化物酶增殖体激活受体6/p保护脑缺血内皮细胞损伤研究目的过氧化物酶增殖体激活受体(peroxisome proliferator-activated receptors, PPAR)属于核受体第1亚家族C群第2位,也称NR1C2(nuclear receptor subfamily 1,group C, member 2)。已鉴定的三个亚型α、γ和δ/β在体内的分布和功能各不相同。其中PPARδ/β分别最为广泛,能被体内各类脂肪酸及脂肪酸代谢物激活,目前发现的功能主要为促进脂肪酸代谢、增加能量消耗以及改善胰岛素抵抗,其激活剂GW501516已进入高血脂症临床试验治疗,但PPARδ/β在循环系统和神经系统中的作用尚未完全清楚。已有的实验报道表明,激活PPARδ/β能促进内皮细胞增殖,抑制内皮细胞在多种病理模型下的凋亡。脑缺血发生后,大脑微血管内皮细胞的凋亡意味着血脑屏障遭到破坏,血管通透性剧增,使得脑组织失去正常的生理环境,促进神经细胞凋亡。因此,保护内皮细胞,稳定血脑屏障,对抑制脑缺血神经损伤有关键意义。在本部分实验中,我们通过给予PPARδ/β激动剂GW501516,探讨激活PPARδ/β对脑缺血神经损伤的保护作用,研究其对大脑微血管内皮细胞凋亡的调控,揭示PPARδ/β在脑缺血病理机制的意义。实验方法1.小动物头部立体定向仪定位侧脑室钻孔,颈部埋入微量泵,含10ug/ul的GW501516100ul,通过导管往侧脑室注射1ul/h,设对照组注射人工脑脊液(artificial cerebrospinal fluid, aCSF);2.24h后行MCAO手术,术后23h麻醉小鼠快速取大脑切8层面,TTC染色后扫描,使用图形分析软件计算损伤面积,相乘以厚度后得损伤体积,和对照组比较；3. MCAO模型23h后从尾静脉注射Evans蓝,1h后PBS彻底灌注,使用酶标仪分别测量大脑两侧Evans蓝含量,值=(脑缺血侧读数—未缺血侧读数),计算比较两组差别；4. MCAO模型23h后提取脑组织微血管内皮细胞作Caspase-3活性测量；5. MCAO模型23h后提取脑组织微血管内皮细胞作DNA断裂电泳；6. MCAO模型23h后提取脑组织微血管内皮细胞,Western Blot检测Bcl-2蛋白水平,与对照组比较；7. MCAO模型23h后提取脑组织微血管内皮细胞作Real-time PCR检测miR-15a表达水平。8.统计学分析方法：两组比较使用双尾T检验,One-Way Anova作方差分析后,组间比较使用Bonferroni’s post-hoc检验。P值小于0.05视为有显著性差异。实验结果1.与对照组相比,PPARδ/β激动剂GW501516能减少MCAO模型后小鼠大脑损伤体积；2.GW501516治疗后,治疗组Evans蓝读数比对照组少;3. MCAO模型后,GW501516治疗组的脑组织微血管细胞Caspase-3活性下降,DNA断裂情况有所缓解,Bcl-2表达升高,以Bcl-2为靶基因的miR-15a表达受到抑制。结论1. PPARδ/β激动剂GW501516治疗保护了脑缺血引起的大脑损伤；2.和对照组比较,治疗组控制了脑缺血后大脑微血管通透性的急剧增加,保护了血脑屏障；3.激活PPARδ/β可能通过抑制有促进凋亡功能的miR-15a表达而减少微血管内皮细胞凋亡。更多还原

【Abstract】 Cerebral vascular diseases are the third leading cause of death in the world, among which over 70% are ischemic stroke. Because of limited on treatment, the prevalence of ischemic stroke bears a burden individually and universally by severe outcome. Lots of factors including exitotoxicity, increasing of free radical spices, and the inflammation are believed to contribute to the pathology of stroke. Therefore, new therapeutic methods are valuable for clinical service.There are two parts in the thesis, which shows the study about the regulation of miR-497 and the effects of peroxisome proliferator-activated receptorδ/β(PPARδ/β) on neural injury after brain ischemia.MicroRNA (miRNA) is a group of short and non-coded RNA that involves in physiology, pathology and diseases of central nervous system (CNS) especially. In Part I, the regulation of miR-497 on neural injury after brain ischemia was studied. By real-time PCR, we identified the miR-497 was increased dramatically after middle cerebral artery occlusion (MCAO) model in mice. In order to study the effect of miR-497 in cells apoptosis, we employed oxygen glycogen deprivation (OGD) model to induce cells death. We determined that miR-497 contributed to neruo2a (N2A) cellular line apoptosis in OGD model by down regulating Bcl-2 and Bcl-w. With luciferase assay, we proofed that miR-497 bond to the 3’UTR of Bcl-2 and Bcl-w. To conclusion, in the in vitro data, we confirmed that miR-497 accelerated apoptosis after OGD model by degrading Bcl-2 and Bcl-w, which are key anti-apoptosis genes in neural protection. In order to study the role of miR-497 in neural injury after brain ischemia, we silenced the miR-497 in brain by using miR-497 antagomir. Through the lateral ventricle injection, miR-497 antagomir effectively inhibited the expression level of miR-497 in brain, but the proteins of Bcl-2 and Bcl-w increased. MCAO models were performed 24h after injection of miR-497 antagomir, then the brain was incised to 8 slices at the 23rd hour following the model operation. All of the section samples were stained with triphenyltetrazolium chloride (TTC). By calculating total infarct volume, cortex infarct volume and subcortex volume, we found that the treatment of miR-497 antagomir decreased all of these parameters. At the same time, the treatment improved the neurobiological deficit on mice after MCAO. In this part, we investigated the role of miR-497 in neural apoptosis with in vitro and in vivo models and discovered its pro-apoptosis function. The regulation of miR-497 on Bcl-2 and Bcl-w, which are the key genes regulating the mitochondrial apoptosis pathway, was meaningful in treatment of brain ischemia.Peroxisome proliferator-activated receptorδ/β(PPARδ/β) is a nuclear receptor by acting as a transcriptional factor. PPAR8/P plays an important role in energy regulation as well as it is shown that activating of PPARδ/βrescues endothelial cells on several apoptosis models. Considering the function of cerebral vascular endothelial cells after stroke, which maintain blood-brain barrier, we investigated the role of PPARδ/βin ischemia-induced endothelial injury in Part II. After lateral ventricle injection of GW501516 (a kind of PPARδ/βagonist), the infarct volume of brain in mice was significantly decreased comparing with sham group. By intravenous injection of Evans blue, the experiments showed that the permeability of microvascular vessels in modeled brain was protected following the GW501516 treatment, which meant that the brain-blood barrier (BBB) was maintained by activating of PPARδ/β. The mechanism was suggested that the endothelial cells in microvascular vessels were less apoptosis comparing with sham group since the functional protein of gene Bcl-2 was increased whereas the activity of Capase-3 and DNA fragmentation were down-regulated. Moreover, the expression of miR-15a, of which target gene is Bcl-2, was decreased after GW501516 treatment. Therefore, it was implied that the activating of PPARδ/βprotect brain tissue after MCAO neural-injury by inhibiting the apoptosis of endothelial cells.PartⅠThe regulation of miR-497 on neural-injury after brain ischemiaObjectiveCurrently, the mechanism of neural apoptosis following the brain ischemia is unclear yet. Therefore, the effective treatment of brain ischemia is limited. It was reported that the miRNAs were involved in multi physiology and pathology processes including apoptosis, especially the miRNAs changed dramatically after cerebral ischemia. However, the detail mechanism of these changes and what kinds of specific miRNA involved in stroke are not well known until now. In this part, we investigated the regulation mechanism of miRNA on brain ischemia.Methods1. At the 23rd hour after MCAO, the brain tissue on the penumbra was detected by Western Blot with multi apoptosis-related miRNAs, which were compared with sham group.2. Oxygen and glucose deprivation (OGD) model was performed when the Neuro2A (N2A) cells were in 70-80% confluence. At Oh,4h and 24h after OGD, the cells apoptosis were detected by LDH (lactate dehydrogenase) and MTT (3-[4,5-dimethythiazol-2-y1]-2,5-diphenyltetrazolium bromide), and also the miR-497 or Bcl-2/Bcl-w were respectively detected by real-time PCR or Western Blot.3. Transferred the miR-497 mimic or miR-497 inhibitor into the N2A, the protein level of Bcl-2 and Bcl-w in the cells was then examined. Or the cells were in exposure to OGD and then the apoptosis situation were assayed by LDH and MTT at Oh,4h and 24h following the OGD.4. Plasmids included 3’UTR and mutant of 3’UTR on Bcl-2 or Bcl-w were constructed into a Luciferase vector. After Co-transfer the vector with miR-497 mimic, fluorescence were examined 48h later.5. At the 23rd hour after MCAO, the brain tissue on penumbra were accessed for detecting protein of Bcl-2 and Bcl-2.6. According previous reports, the antisense oligonucleotides for silencing miR-497 was generated and named as miR-497 antagomir.7. Continuous intracerebroventricular injection of miR-497 antagomir lateral ventricle was performed on mice at the 24th hour before MCAO with micro-pump. There were three groups in the experiment including CSF (cerebrospinal fluid, as Sham), eGFP (enhanced green fluorescin protein, as control), and antagomir (as treatment).8. At the 24th hour after injection, brain tissue was isolated for detecting expression of miR-497 and protein level of Bcl-2 and Bcl-w.9. At the 23rd hour after MCAO, the brains of mice were sliced into 8 sections and then they were stained with TTC. Calculating the infarct volume of total area, cortex, and subcortex.10. At the 23rd hour after MCAO, neurobiological deficit was evaluated by method of "5 score 6 grades".11. A two-tail t-test was employed for the statistics evaluation when make a comparison between two group. One way ANOVA following Bonferroni’s post-hoc test was employed when comparison among different groups. Kruskal Wallis test was employed when analysis the neurobiological deficit among groups.Result1. At the 23rd hour after MCAO, the expression of miR-497 increased in the brain penumbra. At the 24th hour after OGD, N2A generated more miR-497 than non-OGD;2. Over-expression of miR-497 contributed to apoptosis of N2A with OGD. In the opposition, repression of miR-497 inhibited apoptosis of N2A.3. The fluorescence value decreased after co-transferring the miR-497 mimic with 3’UTR of Bcl-2 and Bcl-w, however, the mutant of 3’UTR on Bcl-2 and Bcl-w didn’t show the decrease.4. The protein level of Bcl-2 and Bcl-w was down-regulated after MCAO;5. With the miR-497 antagomir treatment, the miR-497 expression was decreased in brain whereas the protein of Bcl-2 and Bcl-w were increased;6. With the miR-497 antagomir treatment, at the 23rd hour after MCAO model, the infarct volumes of total area, cortex, and subcortex in brain were decreased comparing with Sham and control.7. With the miR-497 antagomir treatment, at the 23rd hour after MCAO, the neurobiological deficit was improved in the model mice.Conclusion1. Both the OGD in vitro and MCAO in vivo increased the expression level of miR-497 in N2A neural cells or brain;2. The miR-497 contributed the apoptosis of N2A neural cells in OGD. The mechanism was suggested as the degradation of target genes including Bcl-2 and Bcl-w.3. Repression of miR-497 improved the ethology of mice after MCAO.PartⅡActivation of PPARδ/βprotects ischemia-induced endothelial injuryObjectivePPARδ/βbelongs to the first subfamily of nuclear receptors, and the member 2 in group C (NR1C2). There are three members such as a, y, andδ/βin PPAR family, which show the different function and distribution respectively. PPAR8/P can be activated by fatty acid and its metabolites. The main functions of PPARδ/βwere discovered as the accelerated fatty acid metabolism, the increased energy expenditure and the improved insulin resistance. GW501516, the agonist of PPARδ/β, has been used as the clinical trials on treatment of hyperlipidemia, but its function in circulation system and CNS is unknown now.After occur of ischemia, the apoptosis of endothelial cells in the cerebral vascular vessel significantly contributed the damage of BBB(brain-blood barrier). Then the permeability increased dramatically, which coursed the unbalance of physiology situation of neurons. There was the report that activation of PPARδ/βcould protect endothelial cells in several apoptosis models. Here, we investigated the effects of GW501516 as activator of PPARδ/βon endothelial injury after brain ischemia.Methods:1. With stereotaxic apparatus, we injected the GW501516 into the lateral ventricle with micro-pump in a concentration of 10μg/μl at speed of 1μl/h. The injection was performed at the 24th hour before MCAO. There were three groups in this experiment, including Non-MCAO surgery as Sham, injection of CSF as control and injection of GW501516 as treatment.2. At the 23rd hour after MCAO, the brain was incised into 8 slices and then stained with TTC. The infarct volume was calculated.3. At the 23rd hour after MCAO,0.1ml of 4% Evans blue was injected intravenously through the mice tail vein. And then, the amount of Evans blue in the mice brain was detected at the 1st hour following injection.4. At the 23rd hour after MCAO, the brain tissue were isolated and protein level of Bcl-2, activity of Caspase-3 and expression of miR-15a were respectively detected.5. A two-tail t-test was employed for the statistics evaluation when make a comparison between two group. One way ANOVA following Bonferroni’s post-hoc test was employed when comparison among different groups.Result1. Comparing with Sham group, the treatment of GW501516 decreased the infarct volume of brain in model mice.2. After the treatment of GW501516, there were less amount of Evans blue in the treated brain than in the control.3. At the 23rd hour after MCAO, the data showed that the activity of Caspase-3 in endothelial cells were decreased accompanying with GW501516 treatment and DNA fragmentation inhibit, comparing with the sham. Also it was found that the expression level of Bcl-2 protein was increased. However, the expression of miR-15a, of which target gene is as Bcl-2, was repressed.Conclusion1. GW501516 protected the brain tissue and the BBB after cerebral ischemia, comparing with the sham group.2. Activation of PPARδ/βrescued the endothelial cells after brain ischemia by increasing the express level of Bcl-2 protein, which maybe was the cause of miR-15a repression.更多还原