【作者】 张艳军；

【导师】 郝峻巍；

【作者基本信息】 天津医科大学 ， 神经病学， 2017， 硕士

【摘要】 背景及目的:血管性痴呆(Vascular dementia,Va D)是一种发病率高且危害严重的疾病,是一种因出血性或缺血性脑损伤导致的认知功能障碍的临床综合征。在未来的几十年里,Va D可能会影响越来越多的患者。因血管性痴呆复杂的病理生理变化,使得对其认识非常的有限。小胶质细胞功能紊乱,部分归因于免疫微环境的紊乱,最终导致了认知功能的损害。考虑到雷帕霉素靶蛋白复合体1(mammalian target of rapamycin complex1,m TORC1)在胶质细胞功能中的关键作用,我们的工作重点是讨论是否雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)抑制剂依维莫司(Everolimus,RAD001)能够抑制有害的小胶质细胞功能,从而改变其表现型和改善因慢性脑灌注不足引起的认知障碍。通过利用弹簧圈套扎双侧颈总动脉造成狭窄建立小鼠因血管因素造成的认知障碍模型,利用水迷宫实验(Morris water maze,MWM)对小鼠进行认知功能的检测,以评估各组小鼠的学习和记忆功能。采取组织学观察评估小鼠海马区的变化,评估RAD001对认知障碍小鼠的改善作用。另通过测定海马区M1标记物CD16/32、M2标记物CD206、M1型相关炎症因子(CD16,i NOS,TNF-α)、M2型相关炎症因子(IL-10,TGF-β,Arg1)以及CA1区VACh T,Ch AT和ACh E的变化,探索RAD001的脑保护作用,为改善认知障碍提供新的治疗策略。方法:我们的实验对象为雄性成年C57BL/6J小鼠,它们被随机分为双侧颈动脉狭窄(BCAS)组、RAD001+BCAS组、假手术组。应用双侧颈总动脉狭窄法制备小鼠血管性痴呆模型。我们给双侧颈动脉狭窄的小鼠0.5mg/kg剂量的依维莫司,假手术组的小鼠仅予磷酸盐缓冲溶液(PBS),作为对照。各组取材前分别进行水迷宫实验;取材后,通过HE染色对海马区的组织结构进行初步评价;通过免疫荧光染色法去评价小鼠的胆碱能活性和lba-1(小胶质细胞/巨噬细胞特异性蛋白),CD16/32,CD206的表达情况;通过蛋白质免疫印迹分析进一步检测M1/M2平衡的机制;通过q RT-PCR检测M1/M2相关炎症因子m RNA在海马的表达情况。结果:1、水迷宫实验获得的结果。在逃避潜伏期的实验中,假手术组和BCAS组分别为:第1天45.1±12s,49.2±12s,P>0.05;第2天40.2±8s,58.1±11s,P<0.05;第3天29.2±8s,57.2±7s,P<0.05;第4天25.1±7s,47.2±6s,P<0.05;第5天20.1±6s,48.3±5s,P<0.01。在目标象限游泳时间百分比的实验中,假手术组和BCAS组分别为:43.52±6.17%,19.85±3.21%,P<0.01。水迷宫检测结果说明双侧颈总动脉狭窄造成了小鼠认知功能障碍。RAD001治疗组与BCAS模型组相比,治疗组逃避潜伏期时间:第3天35.2±5s,第4天26.3±8s,P<0.05;第5天22.2±8s,P<0.01。RAD001治疗组目标象限游泳时间百分比43.59±5.12%,P<0.01。这些结果充分的证明RAD001治疗能够明显改善因慢性脑灌注不足引起的空间障碍。2、HE染色观察到结果:通过HE染色法能够对神经元的缺失进行评估,显示在对小鼠进行双侧颈动脉狭窄处理的最初五周,小鼠海马的CA1区的神经元广泛改变,显微镜下能够清楚地看到神经细胞凋亡,细胞核收缩,染色变深(图.2)。而RAD001能明显减轻这些因长期脑灌注不足导致的在小鼠海马区出现的病理改变和细胞凋亡现象。3、通过免疫荧光法观察到结果:在假手术组中可见海马区中存在大量的Ch AT、VACh T、ACh E和少量的CD16/32、Iba-1的阳性细胞表达,而在BCAS模型组中VACh T、Ch AT的阳性细胞数则显著的减少,CD16/32和Iba-1的阳性细胞数则增加显著,同时可以看到ACHE的荧光强度显著增强,但细胞的数目则减少。RAD001治疗组与BCAS模型组相比,RAD001治疗组的CD16/32和Iba-1的阳性细胞数减少显著,而VACh T、Ch AT的表达增加明显,两组相比P<0.05。另一方面,ACh E的变化没有统计学的差异(图.4)。4、q RT-PCR结果显示:氧糖剥夺(OGD)后2小时、4小时M1表型(CD16,i NOS,TNF-α)基因及M2表型(IL-10,TGF-β,Arg1)基因的表达达到顶峰(P<0.01),而RAD001治疗后,M1表型(CD16,i NOS,TNF-α)基因的相对表达量下降,M2表型(IL-10,TGF-β,Arg1)基因的相对表达量上升。差异有统计学意义(P<0.01,图.5)。5、蛋白电泳的结果显示:三组中T-Akt无明显差异,RAD001治疗组与模型组比较,P-Akt蛋白量明显增多,三组中T-p70S6k无明显差异,模型组P-p70S6k蛋白量明显增多,而治疗组P-p70S6k的蛋白量显著减少。差异有统计学意义(P<0.05,图6)。结论:RAD001能够通过有效抑制m TORC1途径而减轻因慢性脑灌注不足引起的认知障碍。这些影响源于调节小胶质细胞由M1表型向M2表型的转化。我们的发现认为,RAD001有望作为因颈动脉狭窄而导致痴呆的一种辅助治疗方法。更多还原

【Abstract】 Background and Objective:Vascular dementia(Va D)is a widely prevalent and devastating disease.Va D is a clinical syndrome of cognitive decline caused by ischemic,hemorrhagic,or oligemic injury to the brain as a consequence of cardiovascular disease or cerebrovascular disease.Va D will likely affect an increasing number of patients in the coming decades.Despite the tremendous complexity that limits understanding of the pathophysiology of Va D,microglial dysfunction has been attributed,in part,to immune microenviroment disorder and finally leads to cognitive deficits.Considered the mammalian target of rapamycin complex 1(m TORC1)is a key player in regulation of glial function,our work focused on whether the m TOR inhibitor everolimus(RAD001)could overcome the destructive microglial function,change the phenotype and improve cognitive dysfunctions mediated by cerebral hypoperfusion.This study aimed to assess the underlying role of RAD001 in neuroprotective effects and determine the mechanisms in Va D,all mice were subjected routinely established the bilateral common carotid artery stenosis(BCAS).The Morris water maze(MWM)was used to assess behavioral tests.The expression of the M1 marker CD16/32、 M2 marker CD206 and Iba1 cholinergic function and proinflammatory cytokines were measured.Methods: Male,C57BL/6J mice(12-week-old,weighing 30 to 50 grams)mouse were used and subjected to modified BCAS.All the mice were randomly divided into three groups: sham group,BCAS group and the RAD001 treatment group.After 2 weeks of BCAS,we used RAD001 at a dose of mice animals were injected(s.c.)four times with a total of 0.5 mg/kg RAD001 on BCAS dissolved in 200 μL of PBS.The mice was administrated RAD001 twice injection during the first week.Afterthat,RAD001 was injected total twice eveay 7 days.MWM was used to assess cognitive function,and electrophysiological recordings assessed the synaptic plasticity.Immunofluorescence staining was used to evaluate the central cholinergic activity and Iba-1,CD16/32,CD206 in mice.Western immunoblotting was used to further examine mechanisms mediating the modulation of M1/M2 balance.Real-time PCR was used to detect m RNA expressions of M1/M2.Results: 1.The following results were determined by MWM testing.The longer latency times were compared between BCAS group and sham-operated group: on day 149.2±12s,45.1±12s,P>0.05;on day 2 58.1±11s,40.2±8s,P<0.05,day 3 57.2±7s,29.2±8s,P<0.05,day 4 47.2±6s,25.1±7s,P<0.05 and day 548.3±5s,20.1±6s,P<0.01.The percentage in target quadrant was longer in the sham group compared with he BCAS group 43.52±6.17%,19.85±3.21%,P<0.01.All of this suggested that the mice have significant cognitive impairement induced by the surgery of BCA.However,the mice was dramatically improved in cognitive dysfunctions after the treatment of RAD00.As shown by the lantcey time: on day 3 35.2±5s,P<0.05,day 4 26.3±8s,P<0.05 and day 5 22.2±8s,P<0.01.Meanwhile,the target quadrant time was significantly improved.All these data indicate that RAD001 could improved cognitive impairment.2.The results of H&E staining displayed the change of hippocampal neurons after 5 weeks under the surgery of BCAS.The widely neuronal damage was observed in the hippocampal CA1 area in the BCAS group,whih was manifested by neuronal cell loss,nuclei shrinkage and dark staining of neurons.While these damage were significantly improved after the treatment of RAD001 in the hippocampal CA1 area.3.The results of immunofluorescence showed that the activated microglia determined by Iba-1 and CD16 / 32 positive cell was significantly elevated in the BCAS group compare witn the sham-operated group.The expression of Ch AT and VACh T were declined in the BCAS group.However,all abrove results were reversed by RAD001.On the other hand,the relative expression of ACh E was no change.4.The results of q RT-PCR were used to reflect the expression of inflammatory cytokines.Generally,the expression of these markers increased soon after OGD and peaked after 2 h(i NOS,IL-10,TGF-β)or 4 h(TNF-α,CD16,Arg1).However,in the presence of RAD001,the dramatic increase was blunted from 2 h after OGD,and the effect continued to 6 h.5.Western blots showed the increased expressin of P-Akt in the hippocampus after the RAD001 treatment compared with that in BCAS group.The expression of T-Akt was no change among three groups.However,the expression of P-p70S6 k was decreased under RAD001 treatment.Conclusions The major discovery of the current study is that RAD001 is effective in lessening the cognitive deficits induced by chronic cerebral hypoperfusion and that this beneficial effect is mediated through inhibition of the m TORC1 pathway.These effects stemmed from a balancing adjustment away from the microglial M1 phenotype and toward M2.Our finding suggests that RAD001 is a likely candidate as an adjunct therapy for carotid stenosis and the resulting dementia.更多还原