【作者】 蒋超；

【导师】 王建平；

【作者基本信息】 郑州大学 ， 神经病学， 2010， 博士

【摘要】 随着人口老龄化的进展,缺血性脑血管病已成为严重危害人类健康的疾病之一,但在治疗上除超早期溶栓及卒中单元外,尚缺乏有效的手段。为实现脑梗死治疗上的突破,研究者对众多临床试验失败的原因进行分析,认为可能与脑梗死复杂的病理变化及治疗药物的作用靶点单一有关,并提出“神经血管单元”的概念。即脑梗死发生后胶质细胞功能的紊乱、微血管结构的破坏均会加重神经元的损伤,仅针对其中的某个环节进行治疗可能收效甚微；因此,寻找与研究新的、多靶点的治疗药物意义重大。在脑外伤及周围神经损伤上的研究发现,黄体酮具有抑制氧自由基、兴奋性氨基酸释放及促进轴突生长、髓鞘形成的作用；提示黄体酮能够通过多种途径发挥神经保护作用。脑梗死与脑外伤具有相似的病理变化,为实现脑梗死治疗上的突破,已有研究观察了黄体酮对脑梗死的治疗价值,研究发现其可以明显改善脑梗死大鼠的神经功能,但是机制不清。因过度活化的小胶质细胞与氧自由基、兴奋性氨基酸、炎症因子等的释放均有关系,且另有研究表明自体神经干细胞的动员亦有助于神经功能的恢复。为进一步研究黄体酮对脑梗死的治疗价值,本研究拟首先研究黄体酮对小胶质细胞活化及相关炎症反应的影响,然后进一步评价黄体酮对自体神经干细胞动员、血脑屏障结构、脑梗死体积及神经功能的影响；研究内容主要涉及到小胶质细胞、血脑屏障的紧密连接、神经元等。第一部分体外研究黄体酮对小胶质细胞活化及相关炎症反应的影响目的：体外研究黄体酮对小胶质细胞活化及与之相关炎症反应的抑制作用,以探讨黄体酮神经保护作用的可能机制。方法：采用改良的McCarthy混合胶质细胞培养法从新生Sprague-Dawley (SD)大鼠(出生24 h内)皮层组织内分离、培养出小胶质细胞。经纯度鉴定后,取培养第一代细胞,用脂多糖(LPS)诱导其活化,并用黄体酮进行干预。采集培养上清液,用ELISA技术检测其内TNF-α、IL-1β的含量；并用MTT法检测LPS刺激及黄体酮干预对小胶质细胞活力的影响。结果：经CD11b/c鉴定,该方法分离的小胶质细胞纯度≥95%。ELISA法检测培养上清液内TNF-α、IL-1β的结果表明,LPS刺激显著增加了小胶质细胞上清液内TNF-α、IL-1β的含量,经黄体酮干预后培养上清液内TNF-α、IL-1β的含量明显下降(均有P<0.05)。MTT分析结果显示,在与对照组比较的基础上,LPS刺激及黄体酮干预对小胶质细胞的活力影响不大(P>0.05)。结论：用LPS刺激小胶质细胞活化能够很好地模拟脑缺血缺氧后的炎症变化。而黄体酮能够有效抑制与LPS刺激有关的炎症反应,说明抑制与小胶质细胞活化相关的炎症反应可能是其发挥脑保护作用的机制之一。第二部分黄体酮对脑梗死大鼠小胶质细胞活化及血脑屏障结构的影响目的：体内研究黄体酮对小胶质细胞活化及相关炎症反应的影响,并探讨其对神经血管单元保护作用的可能机制。方法：成年雄性SD大鼠120只,体重为250-300g。按随机化原则将所用实验动物分对照组(Control),模型组(Ischemia),溶剂治疗组(Vehicle),黄体酮治疗组(PROG)。通过线栓法制作脑梗死(MCAO)动物模型。将黄体酮溶于25% 2-羟丙基-β-环糊精(HBC)内,分别于成模后1h、6h腹腔注射黄体酮,随后每天行皮下注射,以利于逐渐吸收,每次注射剂量为15mg/Kg体质量。分别于成模后24h、72h通过免疫组织化学、Western blot技术检测离子钙接头蛋白(Ionized Calcium-Binding Adapter Molecule 1, Ibal)的表达情况,以反映脑梗死后小胶质细胞的活化情况；并通过上述技术检测与之有关的炎症因子TNF-α、IL-1β、MMP-9及血脑屏障结构蛋白Claudin5的表达情况,最后通过干湿重法检测脑组织含水量。结果：免疫组化染色表明脑梗死大鼠皮层脑组织内均有Iba1、TNF-α、IL-1β、MMP-9与Claudin5的表达；Western blot定量检测结果表明,脑梗死发生后Ibal、TNF-α、IL-1β、MMP-9的表达水平明显升高(P<0.05),血脑屏障结构蛋白Claudin5的表达水平明显下降(P<0.05)。而黄体酮治疗24h或72h后,Ibal、TNF-α、IL-1β、MMP-9蛋白的表达水平均明显下降(P<0.01)。黄体酮治疗24h对皮层脑组织内Claudin5蛋白表达及脑组织含水量的影响较小(P>0.05),在72h时可以显著减轻Claudin5蛋白的丢失程度并抑制脑水肿的严重程度(P<0.01)。结论：脑梗死发生后,通过检测Ibal的表达可以较好地反映小胶质细胞的活化情况,与小胶质细胞活化相关的炎症反应可能与神经血管单元完整性的破坏有关；经黄体酮治疗后小胶质细胞的活化程度及神经血管单元核心结构(血脑屏障)的破坏程度均明显减轻。说明抑制小胶质细胞活化及与之相关的炎症反应可能是黄体酮神经保护作用的机制之一。第三部分黄体酮对脑梗死大鼠脑部BDNF表达及自体神经干细胞增殖的影响目的：研究黄体酮的神经营养作用,进一步探讨黄体酮对神经血管单元保护作用的可能机制。方法：成年雄性SD大鼠84只,体重为250-300g。实验动物分组及给药方法同第二部分。线栓法制作脑梗死动物模型,分别于脑梗死后24h、72h通过免疫组化染色、实时荧光定量PCR、Western blot技术检测脑部脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)的表达情况；并通过BrdU/Nestin免疫荧光双染技术检测脑梗死7d时室管膜下区神经干细胞的增殖情况。结果：脑梗死发生后,脑部BDNF的表达在基因及蛋白水平均轻度增高；经黄体酮治疗24h、72h后,其表达水平明显增高,与模型组及溶剂治疗组比较,差异有显著性(P<0.01)。治疗7d后,黄体酮亦促进了室管膜下区神经干细胞的增殖,与模型组以及溶剂治疗组比较,差异亦有统计学意义(P<0.01)结论：黄体酮可以在基因及蛋白水平促进脑梗死大鼠脑部BDNF的表达及自体神经干细胞的动员。黄体酮的神经营养作用可能亦为其神经保护作用的机制之一。第四部分黄体酮对脑梗死大鼠脑梗死体积及神经功能的影响目的：研究黄体酮治疗对脑梗死体积及行为学的影响,评价其对脑梗死大鼠的治疗价值。方法：成年雄性SD大鼠24只,体重为250-300g。实验动物分组及给药方法同第二部分。分别于脑梗死后1d、2d、3d用Zea-Longer评分评价大鼠神经功能的缺损程度,并通过TTC染色于脑梗死后3d时检测脑梗死体积。结果：Zea-Longer评分显示,造模前所有大鼠均没有神经功能缺损症状,评分为0。成模后,除假手术组外,其余大鼠均有不同程度的神经功能缺损；经黄体酮治疗后,其神经功能明显改善,与模型组及溶剂治疗组比较差异均有显著性(P<0.01)。TTC染色表明,脑梗死体积在模型组及溶剂治疗组均比较大,且差异无统计学意义；经黄体酮治疗后脑梗死体积明显缩小,较模型组及溶剂治疗组差异均有显著性(P<0.01)结论：黄体酮治疗可以显著减小脑梗死体积,这可能与其促进脑梗死大鼠神经功能的恢复有关。更多还原

【Abstract】 With the progress of population aging, ischemic cerebrovascular disease has become one of the serious diseases, which is harmful to human health, but there is still lack of effective treatment except for ultra-early thrombolytic treatment and stroke unit. To achieve a breakthrough in the treatment of cerebral infarction, some researchers analyze the causes of why so many drugs failed in their clinical trials for the treatment of cerebral infarction. They concluded that which may relate with the complex pathological changes of cerebral infarction and the single target of these drugs. After analysis, scientists put forward the concept of "neurovascular unit". That means that dysfunction of glial cells and the damage of microvascular structural are more serious than neuronal damage, and drugs only with a single target may achieve little, therefore, it is significant to find and research new, multi-target drugs for the treatment of stroke. Studies on traumatic brain injury and peripheral nerve injury revealed that progesterone (PROG) can sinificantly inhibit the release of oxygen free radicals and excitatory amino acid. In addition, PROG also promoted the growth of axon and myelination, which suggests PROG may exext neuroprotective effects through multiple targets. In order to achieve a breakthrough in the treatment of cerebral infarction, some scientists have observed the therapeutic value of PROG on cerebral infarction for there is similar pathological changes between cerebral infarction and traumatic brain injury. The results revealed that PROG can significantly improve the neurological functions of rats with cerebral infarction, but the mechanism is still unclear. Since excessive activation of microglial cells are associated with the release of oxygen free radicals, excitatory amino acids and inflammatory factors, and other studies also show that the autologous mobilization of neural stem cells help function recovery. To further study the therapeutic value of PROG on cerebral infarction, the influence of PROG on microglial activation and the associated inflammatory response was researched firstly, and then the influence of PROG on the mobilization of neural stem cell, the integrity of blood-brain barrier, infarct size and neurological functions was also further evaluated. The content of this research is mainly associated with microglia, tight junction of blood-brain barrier (BBB), neurons and so on.PartⅠResearch on the influence of PROG on the activation of microglia and the related inflammatory response in vitroObjective:To further exploit the neuroprotective mechanisms of PROG, this study was designed to research the inhibitory effects of PROG on the activation of microglia and the related inflammatory response in vitro.Methods:Microglia was dissociated, and purified from the cortex of neonatal Sprague-Dawley rats within 24h after born with modified McCarthy methods. After the assessment of the purity of microglia, lipopolysaccharide (LPS) was used to induce the activation of primary cultured microglia. The aliquots of incubation medium were taken and analyzed for the concentration of TNF-α, IL-1βafter the intervention of PROG with ELISA technique. In addition, MTT method was used to detect the viability of microglia after treatment with LPS and PROG.Results:After identification with the stain of CD11b/c, the purity of microglia is more than 95%. The results of ELISA showed that LPS significantly increased the release of TNF-αand IL-1β. The concentration of TNF-α, IL-1βwas significantly reduced after the intervention of PROG (P<0.05). MTT analysis showed that when based on the control group, PROG and LPS has little effects on cell viability (P>0.05).Conclusions:LPS induced the activation of microglia and increased the release of inflammatory factors, which analogue the inflammatory response associated with the activation of microglia after ischemic and anoxyaemia. PROG significantly inhibited the inflammatory response associated with the activation of microglia, which may be one of the neuroprotective mechanisms of PROG.PartⅡThe influence of PROG on the activation of microglia and the structure of BBB after stroke in ratsObjective:To further exploit the neuroprotective effects of PROG on neurovascular unit, this study was designed to research the influence of PROG on the activation of microglia and the structure of BBB after stroke in vivo. Methods:One hundred and twenty adult male Sprague-Dawley rats weighing 250-300g were used for the experiments and were randomly divided into four groups: sham operated (control) group, ischemic group, vehicle-treated group and PROG-treated group. Rats underwent permanent middle cerebral artery occlusion (pMCAO) and received PROG (15 mg/kg) or vehicle by intraperitoneal injection 1 h,6h post-MCAO. Additional injections of 15 mg/kg were administered subcutaneously once a day after pMCAO. The expression of Ionized Calcium-Binding Adapter Molecule 1 (Ibal) was detected to reveal the activation of microglia with immunohistochemistry and western blot technique at 24h and 72h after cerebral infarction. In addition, TNF-α, IL-1β,MMP-9 and Claudin5 was also measured by immunohistochemistry and western blot technique at 24h and 72h after cerebral infarction. Brain water content was determined by the dry-wet weight method at 24h and 72h after cerebral infarction to explore the influence of PROG on inflammatory response and the structure of BBB.Results:Histochemistry staining revealed that there is the expression of Ibal, TNF-α, IL-1β, MMP-9 and Claudin5 in the brain after stroke. Western blot revealed that the expression of Iba1、TNF-α、IL-1β、MMP-9 is significantly increased after stroke (P<0.05). But the expression of Claudin5 was significantly reduced. After treatment of PROG, the expression of Ibal, TNF-α, IL-1β, MMP-9 is significantly reduced after stroke (P<0.05). The influence of PROG on brain water content and the integrity of BBB is not very significant at 24h after stroke (P>0.05), but PROG significantly increased the expression of Claudin5 and reduced brain water content at 72h after stroke (P<0.01). Conclusions:It is better to detect the activation of microglia by detecting the expression of Ibal after stroke. Inflammatory response associated with the activation of microglia may play important roles in the destruction of BBB. After treatment with PROG, the activation of microglia and the destruction of the core structure of neurovascular unit (BBB) was significantly inhibited, indicating that PROG may exert neuroprotective effects by inhibiting the activation of microglia and the associated inflammatory response.PartⅢInjection of PROG following stroke induces BDNF expression and neurogenesis in ratsObjective:This study was designed to research the neurotrophic effects of PROG in vivo to further exploit the neuroprotective effects of PROG on neurovascular unit.Methods:Eighty-four adult male Sprague-Dawley rats weighing 250-300g were used in this section. The grouping and medication was similar with sectionⅡ. The expression of BDNF was measured by immunohistochemistry, real-time quantitative PCR and western blot technique at 24h and 72h after cerebral infarction, respectively. The proliferation of neural stem cells in subependymal zone (SVZ) was detected through BrdU/Nestin double immunofluorescence staining at 7d after stroke.Results:Statistical analysis showed that PROG administration significantly increased the expression of BDNF in gene level and protein level after permanent middle cerebral artery occlusion model (pMCAO) in the PROG-treated rats when compared with ischemic and vehicle-treated rats at 24h and 72h (P<0.05). In addition, PROG also significantly increased the proliferation of neural stem cells in SVZ (P<0.05).Conclusions:Treatment with PROG significantly increased the expression of BDNF in gene and protein levels. In addition, PROG also increased the mobilization of neural stem cells, suggesting that PROG may also exert neurotrophic effects after stroke.Part IV The efficacy of PROG on infarct volume and functional outcome after cerebral ischemia in ratsObjective:The influence of PROG on infarct volume and functional outcome was studied to evaluate the therapeutic value of PROG on cerebral infarction.Methods:Twenty-four adult male Sprague-Dawley rats weighing 250-300g were used in this section. The grouping and medication was similar with sectionⅡ. Zea Longa test was used to evaluate their functional outcome at 1d,2d,3d after stroke. TTC staining was used to detect the infarct volume at 3d after stroke.Results:The results of Zea Longa test showed that there were no functional deficits in all animals prior to ischemia. There was no significant changes in motor function in sham-operated animals across the 3 day assessment period confirming that the surgical procedure did not affect motor outcome (P>0.05). Both PROG and vehicle-treated rats experienced significant decline in scores following occlusion. However, PROG-treated rats demonstrated a gradual improvement in scores compared with ischemic and vehicle-treated rats (P<0.05). TTC staining revealed that PROG administration significantly reduced the total infarct volume after pMCAO in the PROG-treated rats compared with ischemic and vehicle-treated rats (P<0.05).Conclusions:PROG significant reduced infarct volume after pMCAO, which may relate with the improvement of functional recovery after stroke in rats.更多还原