【作者】 王雪贞；

【导师】 唐荣华； 卜碧涛；

【作者基本信息】 华中科技大学 ， 神经病学， 2009， 博士

【摘要】 神经系统的慢性炎症在神经变性疾病中起很关键的作用，与轴索变性和神经元脱失密切相关，包括阿尔茨海默病(AD)，帕金森病(PD)，肌萎缩侧索硬化(ALS)和C型Niemann-Pick病(NPC)等神经变性疾病。越来越多的证据表明，小胶质细胞激活并释放过量的TNF-α参与并加速了神经元变性的级联反应，导致神经元功能失调，突触调节异常以及淀粉样蛋白介导的记忆障碍。有多项研究证实，应用TNF-α抑制剂以及TNF-α-SiRNA降低其水平可以改善临床AD病人的认知功能以及改善AD模型小鼠的神经病理损害。但是TNF-α是否为神经变性疾病发病机理中的关键环节尚不清楚。Npc-l基因自然突变的npc小鼠模拟了人类NPC的病理生理变化，非常适合用来研究NPC以及其他的神经变性疾病。我们前期研究发现npc小鼠脑部胶质细胞活化并分泌大量的TNF-α，但是TNF-α在npc小鼠病理变化以及病情进展过程中的作用尚不明确。因此，我们应用这种动物模型和TNF-α-SiRNA来观察TNF-α对npc小鼠病理学以及行为学的影响，进而探讨TNF-α在神经变性过程中扮演的角色。本实验中，我们首先构建了携带TNF-α-SiRNA的慢病毒载体，感染体外培养细胞，通过RT-PCR以及ELISA检测了其对TNF-αmRNA的沉默作用：同时对4周龄的npc小鼠进行该慢病毒侧脑室注射，通过Real-time-PCR以及免疫组化检测其沉默效率，并运用免疫组化、western-blot、体重测定、衣架悬挂实验及足印实验来评价npc小鼠病理及行为学变化。结果发现，携带TNF-αSiRNA的慢病毒在体外培养细胞能有效下调TNF-αmRNA的表达，减少TNF-α分泌：慢病毒npc小鼠侧脑室注射后，可在其脑部长期稳定的表达并有效下调TNF-αmRNA；慢病毒注射后npc小鼠的神经病理以及行为学均有不同程度的改善。为研究和治疗神经变性疾病以及TNF-α相关疾病提供了有力的工具和治疗新策略。更多还原

【Abstract】 Chronic inflammatory processes are considered to play an important role in theprogression of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinsondisease (PD), Amyotrophic lateral sclerosis (ALS) and Niemann-Pick disease type C (NPC),etc. It is thought that microglia activation and the TNF-αit relased participate andaccelerated the neuronal dysfunction, regulates synaptic mechanisms, and mediatesamyloidinduced disruption of molecular mechanisms involved in memory. Many studiesshowed that downregulation of TNF-αby its blockers and TNF-α-SiRNA resulted inpredominant improvement of the symptom of AD patients, ALS patients and theneuropathology of AD mouse model. It is not know whether TNF-αis essential key for theneurodegeneration. The naturally occurring npc-1 mutant mouse mimics human NPC, indiaplaying activation of mocroglia and over excess TNF-αbut its role in the neuropathologyand the processing of NPC is not clearly. We use this mouse model and lentiviral-deliveredTNF-α-SiRNA to determine whether TNF-αis necessary for NPC neuropathology. In thisstudy, we constructed Lentivector-mediated-TNF-α-SiRNA, and the tite was 2×10~8ifu/L.Interference efficiency of the lentivirus expressing TNF-α-SiRNA, was determined byRT-PCR and Elisa in BV-2 cells and astrocytes. At the same time, the constructedLenti-TNF-α-SiRNA was intracerebroventricularly infused into 4-week old npc mice for a4-week period. By using immunohistochemistry and real-time PCR, the down-regulation ofthe target genes was detected. We observed the neuropathology and the behavior of npc niceby immunohistology, western-blot, body weight, hanger test and foot print. As a result, theLenti-TNF-α-SiRNA downregulated the expression of murine TNF-αgene efficiently invitro and in vivo. Lentivirus could be expressed stably for long-term in the npc mice brainand reduced the hyperphosphorylation of cytoskeleton and improved the behavior of npcmice, which provided a potential tool for studying and treating neurodegenerative diseasesand TNF-α-related diseases.更多还原