复合型AD大鼠模型的建立及其神经细胞凋亡的线粒体通路研究

发布时间：2018-03-04 11:16

本文选题：阿尔茨海默病　切入点：硫氧还蛋白　出处：《苏州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:根据Aβ1-42及D-半乳糖致衰老理论,在亚急性衰老模型基础上侧脑室注射Aβ1-42建立一种复合式阿尔茨海默病(Alzheimer’s disease,AD)动物模型。在此基础上观察神经细胞凋亡的线粒体通路与硫氧还蛋白(Thioredoxin,Trx)的关系,旨在为神经退行性疾病的发病机制、临床防治等提供科学理论依据。方法: (1)侧脑室注射Aβ1-42建立单一因素AD模型(B组)、采用D-半乳糖腹腔注射SD大鼠建立亚急性衰老模型(C组)、腹腔注射D-半乳糖联合侧脑室注射Aβ1-42建立复合式AD模型(D组)、选用正常大鼠作为对照组(A组); (2)行为学采用Morris水迷宫检测大鼠学习和记忆能力; (3)甲苯胺蓝染色观察神经元形态及数量的变化; (4)生化指标检测血清及大脑组织匀浆中CAT、T-AOC及GSH/GSSG; (5)免疫组织化学检测衰老相关蛋白AGEs及AGER的表达变化,脑组织各区Trx,Cyto-C及Caspase-9表达采用免疫组织化学及免疫印迹观察。结果: (1)Morris水迷宫检测显示:与A组相比,B组、C组及D组大鼠学习和记忆能力明显下降,有显著性差异(P0.05或P0.01);与B组、C组相比,D组大鼠学习记忆能力也明显下降,差异有显著性(P0.01); (2)尼氏染色结果显示:与A组相比,B组、C组及D组染色神经元的数量有不同程度的减少,差异具有统计学意义(P0.05或P0.01)。镜下可见核固缩、胞体变小等形态学改变,以D组最为明显,其次是B组,再者是C组; (3)生化指标检测显示:与A组相比,B组、C组及D组血清和脑组织匀浆中CAT、T-AOC和血清中GSH/GSSG显著下降(P0.05或P0.01),与B组、C组相比,D组血清和脑组织匀浆中CAT、T-AOC及匀浆中GSH/GSSG都明显下降(P0.01); (4)免疫组化结果显示:与A组相比,B组、C组及D组AGEs、AGER在皮质、海马、小脑的表达明显增多,有显著性差异(P0.05或P0.01);与B组、C组相比,D组AGEs、AGER在皮质、海马、小脑的表达显著增多(P0.01); (5)免疫组织化学法和免疫印迹检测发现B组、C组及D组与A相比,在皮质、海马、小脑中Trx的表达明显下降(P0.05或P0.01),Cyto-C和Caspase-9的表达明显上升(P0.05或P0.01);D组与B组、C组相比,在皮质、海马、小脑中Trx的表达有所下降(P0.01),Cyto-C和Caspase-9的表达上升(P0.05或P0.01)。结论: (1)较好的在衰老的基础上建立了一种AD复合动物模型,初步探讨了衰老与AD之间存在的潜在关系。 (2)在D-半乳糖亚急性衰老模型基础上可建立Aβ1-42复合式AD动物模型。复合式动物模型在行为学,生化指标及组织学上优于单一因素AD动物模型。此种复合式动物模型不仅可以用于老年痴呆的基础研究;而且也可用于探讨老年痴呆的防治途径。 (3)D-半乳糖联合Aβ1-42建立AD大鼠模型中AGEs和AGER的表达明显增多,AGEs和AGER的增加反过来会加速AD的发病进程。AGEs和AGER二者的抑制剂可能是AD的临床研究药物靶点。 (4)在不同模型组Trx都有不同程度的下降,线粒体凋亡途径中重要蛋白Cyto-C、Caspase-9都有所上降。Trx的表达与Cyto c、caspase-9的表达负相关,Cyto c的表达与caspase-9的表达正相关。提示AD中Trx的下降与细胞凋亡的发生之间有重要的联系,并且有可能是通过Cyto-C的释放和Caspase-9的激活而引起凋亡。
[Abstract]:Objective: according to the A beta 1-42 and D- galactose induced aging theory, in the subacute aging model based on intraventricular injection of A beta 1-42 build a composite type of Alzheimer's disease (Alzheimer 's disease, AD) animal model. Based on the observation of nerve cell apoptosis pathway and mitochondrial thioredoxin (Thioredoxin, Trx) the relation to pathogenesis of neurodegenerative diseases, provide scientific theoretical basis for clinical prevention and treatment.
Method:
(1) intracerebroventricular injection of A beta 1-42 build single factor AD model (group B), established by the subacute aging model induced by D- galactose intraperitoneal injection of SD rats (C group), intraperitoneal injection of D- galactose combined with intracerebroventricular injection of A beta 1-42 build complex AD model (D group), normal rats as for the control group (A group);
(2) the learning and memory ability of rats was detected by Morris water maze.
(3) to observe the changes of morphology and number of neurons toluidine blue staining;
(4) biochemical indexes were used to detect CAT, T-AOC and GSH/GSSG in serum and brain tissue homogenate.
(5) immunohistochemistry was used to detect the expression changes of aging related proteins AGEs and AGER. The expression of Trx, Cyto-C and Caspase-9 in different regions of the brain was examined by immunohistochemistry and Western blotting.
Result:
(1) Morris water maze test showed that compared with group A, the learning and memory ability of B group, C group and D group decreased significantly (P0.05 or P0.01). Compared with B group and C group, the learning and memory ability of the D group decreased significantly, with significant difference (P0.01).
(2) Nissl staining showed that: compared with A group, B group, C group and D group the number of neurons was reduced in different degrees, the difference was statistically significant (P0.05 or P0.01). Under the microscope, nuclear pyknosis, change smaller cell body morphology, the D group was most obvious. Followed by the B group, and C group;
(3) biochemical index test showed that compared with group A, GSH/GSSG, CAT, T-AOC and serum GSH/GSSG in serum and brain homogenate of B group, C group and D group decreased significantly (P0.05 or P0.01), compared with those in B group and C group, there was a significant decrease in serum and brain homogenate in group A.
(4) immunohistochemistry showed that compared with group A, the expression of AGEs and AGER in cortex, hippocampus and cerebellum increased significantly in group B, group C and group D (P0.05 or P0.01). Compared with B group and C group, the expression of B and its expression in cortex, hippocampus and cerebellum increased significantly.
(5) immunohistochemistry and Western blot detection showed that B group, C group and D group compared with A in cortex, hippocampus and cerebellum in the expression of Trx decreased significantly (P0.05 or P0.01), the expression of Cyto-C and Caspase-9 increased significantly (P0.05 or P0.01); D group and B group, C group in the cortex, hippocampus, cerebellum, the expression of Trx decreased (P0.01), increased expression of Cyto-C and Caspase-9 (P0.05 or P0.01).
Conclusion:
(1) a AD compound animal model was established on the basis of aging, and the potential relationship between aging and AD was preliminarily discussed.
(2) based on D- D-galactose subacute aging model can be established on the A beta 1-42 composite AD animal model. The compound in animal models of behavior, biochemistry and histology is better than single factor AD animal model. The combined type animal model not only can be used in basic research of senile dementia; but also can be used to investigate ways to prevent and cure Alzheimer's disease.
(3) the expression of AGEs and AGER increased significantly in the establishment of AD rat model with D- galactose and A beta 1-42. The increase of AGEs and AGER in turn would accelerate the pathogenesis of AD. The inhibitors of.AGEs and AGER two might be the clinical drug targets of AD.
(4) have different degrees of decline in different model group Trx, Cyto-C protein in the mitochondrial apoptotic pathway, Caspase-9 has on the expression of Cyto and.Trx reduced C, negative correlation between the expression of caspase-9, expression of caspase-9 and Cyto C positive correlation. There is an important connection between Trx and AD decreased in tips the occurrence of apoptosis, and might induce apoptosis through activation of Cyto-C release and Caspase-9.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R749.16;R-332

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