【作者】 刘人恺；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 研究背景：阿尔茨海默病(AD)是一种原因不明,以进行性认知功能障碍、行为和人格障碍等为主要临床特征的中枢神经系统退行性疾病。Aβ是构成神经炎性斑的核心和血管沉积物的主要成分,被认为是AD最重要的致病物质之一。AD的病因和发病机制至今尚不清楚,越来越多的证据提示炎症反应在AD的病因和病理过程中具有重要作用,同时也是AD动物模型致病的一个主要原因。胰高血糖素样肽1(GLP-1)是由肠道内分泌L细胞分泌的肠促胰岛素,具有亲神经性,对神经元轴突的生长,神经元的营养有一定的调节作用,能防护谷氨酸诱导的凋亡和氧化应激损伤,可能成为具有潜在价值的治疗AD的新方法。量子点(QDs)是一种新型的荧光分子探针,具有荧光强度高、激发光谱连续而宽、光学稳定性好等优势。应用量子点探针活体成像观察Aβ的动态变化及药物对AD的疗效,对阿尔茨海默病的早期诊断与早期治疗具有十分重要的意义。研究目的：本研究通过水迷宫和分子生物学方法探讨GLP-1及衍生物对AD可能保护作用及机制,利用量子点-抗体探针活体追踪GLP-1干预前后AD转基因模型Aβ的变化,探讨GLP-1治疗AD的疗效及其神经保护作用机制。研究方法：1.将正常C57BL小鼠随机分3组：QDs组、QDs-Ap-Ab组、对照组,各组小鼠分别行量子点、量子点-Ap-Ab探针、PBS侧脑室注射后,采用组织形态学和动物行为学观察,应用石墨炉原子吸收光谱法检测血、尿中镉含量,全自动生化检测仪测定肝肾功能,系统考察量子点和制备的探针的生物毒性作用。2.将饲养10月和16月的APP转基因鼠、正常C57BL小鼠随机分为：T10组、GLP-1A组、GLP-1±ExA组、正常组和T16组、GLP-1B组、GLP-1+ExB组、正常组,侧脑室连续注药后行水迷宫实验,并取材进行形态学观察,利用ELISA方法检测TNF-α和IL-1p蛋白含量,采用免疫组织化学和量子点荧光组织化学方法检测GFAP、Aβ、GLP-1R蛋白的表达,Real-time PCR检测GLP-1R mRNA基因的表达,Western blot检测p-p38MAPK蛋白含量的变化。3.利用动物活体整体荧光成像系统和体视显微镜监测T10组、T16组、GLP-1A组和正常组量子点探针标记Aβ的变化,检测3天后断头取脑组织冰冻切片,荧光显微镜观察,验证Aβ的变化。研究结果：1、琼脂糖凝胶电泳检测到目的条带,证明量子点-Ap-Ab探针制备成功。毒性检测HE染色示QDs-Ap-Ab组海马CA1区细胞形态、结构与对照组相似；尼氏染色示探针组海马CA1区神经元形态正常,尼氏体清晰可见,与对照组无差异；石墨炉原子吸收光谱法检测示侧脑室QDs与QDs-Ap-Ab探针注射后12h,尿镉含量较对照组明显增加,1-7dQDs组、探针组的尿镉含量与对照组相比,差异无统计学意义；行为学观察7d,三组小鼠进食进水正常,二便正常,无举尾、皮肤毛发颜色改变,无步态异常、兴奋抑制反应,均无死亡；QDs组和QDs-Ap-Ab组血清ALT、AST、BUN和CRE水平与对照组无明显差异(P>0.05)。2、T10组、T16组空间学习记忆能力、思考应变能力明显下降；海马神经元受损明显,线粒体结构不清；IL-1p、TNFα含量明显增多,GFAP、Aβ、GLP-1R蛋白表达明显增高,Aβ、GLP-1R蛋白表达区域部分重叠；GLP-1RmRNA水平增高明显；p-p38蛋白含量增多。侧脑室连续5d注射GLP-1后,GLPA组小鼠空间学习记忆能力、思考应变能力明显改善,GLPB组小鼠空间学习记忆能力、思考应变能力改善不明显；GLPA组L-1p、TNFα含量较T10组明显下降,GFAP、Aβ、GLP-1R蛋白表达明显减少,GLP-1RmRNA水平明显降低；p-p38蛋白含量减低。用GLP-1和Exendin Fragment9-39同时干预T10组,其空间学习记忆能力、IL-1p、TNFα含量、GFAP、Aβ、GLP-1R蛋白表达量、GLP-1RmRNA水平、p-p38蛋白含量与T10组无明显差异。3、NH2-QDs-605脑内非特异性积聚较QDs-605明显NH2-QDs-605在脑脊液中光学特性稳定,生物相容性好。量子点-Ap-Ab探针标记16月和10月转基因鼠Ap,发现前者荧光范围较后者扩大,连续观测3天,量子点荧光强度没有衰减；GLP-1干预10月APP转基因鼠5天后,其荧光范围减小,荧光亮度降低；脑组织冰冻切片观测验证了上述结果。研究结论：1.成功构建了量子点-Ap-Ab探针,在一定的条件下,量子点及量子点-Ap-Ab探针对小鼠整体毒性作用小,生物相容性好。2.GLP-1R在APP转基因模型海马组织、大脑皮层中表达增高,证实GLP-1可能通过与GLP-1R结合,参与减缓炎症反应、改善APP转基因鼠的学习和记忆能力。3.GLP-1抑制脑组织中p38MAPK活化是减轻APP转基因鼠脑内的炎症反应的可能机制之一。4.量子点-Ap-Ab探针可应用于活体动物脑内研究,并能有效识别APP转基因鼠脑内的Ap沉积。5.量子点-Ap-Ab探针荧光成像可以应用于AD转基因模型抗p淀粉样蛋白斑块干预的疗效检测。更多还原

【Abstract】 Objective:Alzheimer’s disease(AD) is a neurodegenerative disease of the nervous system which is characterized by irreversible memory impairment, continuous cognitive decline,and behavioral disturbances.Amyloid-βpeptide (Aβ)is the main component of blood-vessel and core part of neuritic plaques,which is regarded as one of the most important disease-causing material.The etiology and pathogensis of AD are still unknown to date, but a growing body of evidence suggests that neuroinflammation may play a significant role in etiology and pathogenesis of AD and it is also the main case in animal paradigms of the disease. Glucagon-like peptide 1 (GLP-1)is a 30-amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells, in response to meal intake.It has been documented that GLP-1 can induce neurite outgrow, neurotropic properties, and can protect cells against excessive glutamate and other toxic insults.GLP-1 is probably a promising agent in the therapy of AD.Compared with traditional fluorescent dye, Quantum Dots(QDs)is a new fluorescence probe which has significant advantages in fluorescent intensity and ray stability.Thus, molecular imaging of A 0 in Vivo by Quantum Dot can provide not only highly sensitive monitoring of the course of AD at the early stage, which will be of great help in early diagnosis, and also evaluation of the curative effect of GLP-1 used in early treatment.This study was designed to evaluate the biocompatibility and get the preliminary data of A in Vivo labeled by Quantum Dots-probe for real-time subsurface imaging in APP transgenic mice in Vivo, then to discuss the neuroprotective effect of intracerebroventricular injection of GLP-1 in APP transgenic mice, and revealed its probable mechanism.Methods:1.Normal C57BL mice were randomly divided into control-, QDs-Aβ-Ab-and QDs-group.Observing the response of injecting with respectively 5μL QDs, QDs-Aβ-Ab probe and saline in each group with behavioral comparison,HE staining, nissl staining, electron micrograph. Urine and blood cadmium levels were measured by HPLC.Hepatic and renal functions were detected by using automatic chemistry analyzer.2.10-and 16-month old APP transgenic mice and normal C57BL mice were randomly divided into T10、GLP-1A、GLP-1+ExA、normal groups and T16、GLP-IB、GLP-1+ExB、normal groups, After intracerebroventricular injection of respectively 5μL GLP-1,GLP-1 and Exendin Fragment 9-39, saline,do the Morris water maze tests and use behavioral comparison on each group.The histomorphology of hippocampus were tested by micrograph. TNF-a and IL-1βlevels were measured by enzyme-linked immunosorbent assay (ELISA). The expression of GFAP、Aβand GLP-1R were tested by immuno-stochemistry. The distribution of Aβand GLP-1R were observed by Quantum Dots immunofluorescence double-labeling part.The variable expression of GLP-1 mRNA was detected by Real-time PCR. p-p38MAPK protein levels were investigated by western blot.3.Monitoring the variation of Aβin T10、T16、GLP-1A and normal groups by using bio-imaging system and binocular stereomicroscope.After 3 days, using fluorescence microscope to detect the changes of the Aβin the frozen sections of the brain.Results:1. QDs-Aβ-Ab probe showed only one band as NH2-QDs-605 on the agarose gel electrophoresis.Cell morphology, ultrastructureand and nissl bodies in hippocampal CA1 of probe and QDs group mice are probably normal as control.After intracerebroventricular injecting with QDs and probes, t the urine cadmium levels were apparently higher than that in controls in 12 hours and down to normal as control in 1～7days. But there was little change in the blood cadmium level.The movements of all groups were normal.There was no significant difference in the content of serum ALT、AST、BUN and CRE among the three groups. 2.The learning and memory ability of APP transgenic mice was greatly declined, the ultrastructure in hippocampal CA1 shows neurons damage and is very vague. A part of distribution of Aβand GLP-1R were overlapped. The expression of IL-1β,TNFα, GFAP, Aβ,GLP-1R and p-p 38 are largely increased in APP transgenic mice, but the expressions are obviously decreased after GLP-1 treatment. GLP-1 had improved the spatial learning-memory in mices on a certain extent. GLP-1+ExA group had no significant change after treatment.3.There were some non-specific deposits after NH2-QDs-605 injection in brain, but the optical properties of QDs and the probe remain stable. By using QDs-Aβ-Ab probe to bio-image the Aβ, the spatial width of fluorescence in T16 group were wider than T10.The spatial width of fluorescence in T10 group becomes less after GLP-1 treatment. The frozen section showed the same change as bio-imaging.Conclusion:1.The QDs-Aβ-Ab probe is successfully constructed, which had no toxic effects and good biocompatibility in brain.2.GLP-1R are largely increased in APP transgenic mice. GLP-1 can improve the learning and memory ability combined with GLP-1R.3.GLP-1 can play a part as an inhibitor of p38 MAPK phosphorylation, which might be a mechanism for the inhibition of neuroinflammation4.The QDs-Aβ-Ab probe can be used in the investigation of the animal brain, and can effectively identify Aβ.5.Bio-imaging with QDs can evaluate the curative effect of drugs in AD animal model.更多还原