【作者】 蔡志友；

【导师】 晏勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 目的观察糖尿病大鼠行为学、海马病理组织学特征与β-淀粉样蛋白(β-amyloid protein,Aβ)、β-淀粉样前体蛋白(β-amyloid precursor protein,APP)、淀粉蛋白前体β位分解酶1(β-site APP-cleaving enzyme 1,BACE1)在海马组织中的表达,通过检测糖尿病大鼠海马组织中主要信号通路分子的表达,分析主要信号通路分子与海马BACE1表达的关系,探讨糖尿病糖代谢异常在阿尔茨海默病中的作用机制。方法高脂高糖喂养后腹腔注射链脲佐菌素(streptozocin,STZ)诱发糖尿病大鼠动物模型,随机分为对照组(S组)、4周模型组(M4组)、6周模型组(M6组)、8周模型组(M8组)。Morris水迷宫实验和穿梭箱实验检测大鼠认知功能和行为学改变。刚果红染色检测大鼠脑内淀粉样物质沉着,Bielschowsky氏法银染色检测老年性痴呆特征性病理改变:老年斑和神经纤维缠结。生物化学法检测血糖、血胆固醇和糖化血红蛋白,放射免疫法检测胰岛素血清浓度,蛋白质免疫印迹法、RT-PCR和酶联免疫吸附法检测APP,免疫组织化学法、蛋白质免疫印迹法、RT-PCR和酶联免疫吸附法检测BACE1,酶联免疫吸附法检测Aβ,蛋白质免疫印迹法和RT-PCR检测p38MAPK、PKC、PKA、JAK-2、PI-3K、Akt和ERK1/2,用图象分析仪测定光密度值。结果1.糖尿病模型及行为学检查:与对照组的体重(482.2±16.5)g相比较,模型组的体重明显降低(P＜0.05),模型组内体重无明显差异。血糖浓度从对照组的(6.23±0.62)mmol/dl升至模型组的(23.21±3.46)mmol/dl(P＜0.01),血清糖化血红蛋白从对照组的3.44±0.26%升至模型组的12.56±0.67%(P＜0.01),胆固醇浓度从对照组的(121.6±26.2)mg/dl升至模型组的(291.1±27.5)mg/dl(P＜0.01),血清胰岛素浓度从对照组的(365.3±26.5)pmol/l升至模型组的(406.2±24.5)pmol/l(P＜0.01)。模型组内体重、血糖、血清糖化血红蛋白、胆固醇浓度和血清胰岛素浓度无差异。体重减轻、血糖高于16 mmol/dl以及胰岛素抵抗、胆固醇升高为建模成功。定向航行实验检测大鼠的空间辨别学习能力:模型组大鼠搜索平台的潜伏期比对照组明显延长(P＜0.01),模型鼠组间未见显著性差异(P＞0.05)。空间探索试验检测大鼠的空间记忆能力:模型组大鼠跨越平台次数分比对照组显著减少(P＜0.01)。模型鼠组间未见显著性差异(P＞0.05)。穿梭箱实验测试其条件性学习能力:模型组大鼠遭受电击的次数比对照组明显增加(P＜0.01),从对照组的7.43±2.01次增至模型鼠的15.46±3.62次,模型鼠组间比较未见显著性差异(P＞0.05)。模型鼠被电击的时间比对照组明显延长(P＜0.01),从对照组的7.43±2.01秒增至模型鼠的15.46±3.62秒,模型鼠组间比较未见显著性差异(P＞0.05)。表明模型组有明显的学习、记忆障碍,学习记忆力明显下降。2.病理组织学检测刚果红染色可见对照组大鼠脑内见到轻微淀粉样物质沉着,与对照组比较,模型组大鼠淀粉样物质沉着明显,组织与胞浆中均有红染,模型鼠脑组织内有淀粉样物质沉着,染色阳性。在刚果红染色的3组脑切片上未见刚果红显示的SP散在分布于海马各部的分子层。Bielschowsky氏银染色可见对照组大鼠神经纤维排列整齐、紧密。与对照组比较,模型组大鼠神经纤维增粗、肿胀,排列紊乱、扭曲、稀疏,间隙扩大,但未找到SP和NFT。刚果红染色阳性,Bielschowsky氏法银染色未出现老年斑和神经纤维缠结。糖尿病大鼠脑内未见阿尔茨海默病特征性病理改变。3.APP、Aβ和BACE1检测模型组与假手术组比较,模型组APP、Aβ和BACE1表达明显增高(P＜0.01)。三个模型组比较无显著性差异,Aβ与APP正相关,APP表达水平与学习、记忆损伤负相关,Aβ与学习、记忆损伤正相关。BACE1表达水平与学习、记忆负相关。ELISA法检测Aβ1-40和Aβ1-42显示糖尿病模型鼠脑组织内Aβ1-40水平明显增高,从正常组的(64.13±6.76)pg/mg升至模型组的(86.43±7.03)pg/mg(P＜0.001);Aβ1-42也明显升高,从正常组的(67.43±5.12)pg/mg升至(89.45±5.28)pg/mg(P＜0.001)。ELISA法检测APP显示从正常组的116.65±9.21 pg/mg升至模型组的156.73±8.24pg/mg(P＜0.001),Western blotting法OD值从正常组的0.63±0.12升至模型组的1.56±0.19(P＜0.001),RT-PCR法OD值从正常组的1.68±0.2 1升至模型组的3.54±0.22(P＜0.001)。糖尿病模型鼠脑组织内BACE1水平显著增高,ELISA法从正常组的(116.46±8.10)pg/mg升至模型组的(158.73±6.24)pg/mg(P＜0.001),Western blotting法OD值从正常组的0.61±0.11升至模型组的1.52±0.16(P＜0.001),RT-PCR法OD值从正常组的1.62±0.26升至模型组的3.61±0.32(P＜0.001),免疫组织化学法OD值从正常组的0.81±0.21升至模型组的2.01±11.36(P＜0.001)。BACE1、Ap表达水平与学习、记忆能力负相关。4.胰岛素信号通路主要信号分子检测PKC、PKA、JAK-2结果蛋白质印迹结果均显示:与对照组比较,模型组PKC、PKA、JAK-2蛋白质表达水平无显著差异(P＞0.05);RT-PCR结果显示模型组PKC、PKA、JAK-2 mRNA表达虽然轻微增高,但无统计学意义(P＞0.05)。Werstern blot光密度分析结果显示PI-3K/Akt蛋白表达较对照组下调,从对照组的0.914±0.065降至模型组的0.511±0.051(p=0.000);RT-PCR对PI-3K/Akt分子水平的检测同样显示PI-3K mRNA表达较对照组显著下调,从对照组的1.289±0.072降至模型组的0.821±0.066(P=0.000)。模型组之间差异无统计学意义(P=0.352)。Werstern blot光密度分析结果显示p38MAPK/ERK蛋白表达较对照组下调,从对照组的0.926±0.072降至模型组的0.631±0.056(P=0.000);RT-PCR对p38MAPK/ERK分子水平的检测同样显示p38MAPK/ERK mRNA表达较对照组显著下调,从对照组的1.350±0.071降至模型组的0.96±0.069(P=0.000)。模型组之间差异无统计学意义(P=0.367)。与对照组比较,糖尿病模型组大鼠海马组织内PI-3K/Akt、p38MAPK/ERK表达下调(P=0.000),同时BACE1的表达较对照组上调(P=0.000)。BACE1与PI-3K呈负相关(P＜0.05),BACE1与Akt也呈负相关(P＜0.05);BACE1与p38MAPK无相关(P＞0.05),BACE1与ERK1/2无相关(P＞0.05),Aβ40/42与p38MAPK负相关(P＜0.05),Aβ40/42与ERK1/2负相关(P＜0.05)。结论1.APP、Aβ在糖尿病大鼠脑组织表达增高,糖尿病糖代谢异常增强Aβ表达参与了阿尔茨海默病的发病机,APP具有双重作用机制,既具有脑保护作用,又有增高Aβ表达作用。2.BACE1、Aβ在糖尿病大鼠脑组织表达增高,糖尿病糖代谢异常增强BACE1、Aβ表达参与了阿尔茨海默病的发病机制。3.信号通路MAPK/ERK影响Aβ的代谢、信号通路PI-3K/Akt可以调节BACE1的表达参与了阿尔茨海默病的发病机制。更多还原

【Abstract】 ObjectiveTo measure praxiological and pathological change progress of diabetes mellitus of Wistar rats,to investigate expression of APP,BACE1 and Aβin brain for diabetes mellitus of Wistar rats,to investigate expression of BACE1 and molecules of signal pathway in brain for diabetes mellitus of Wistar rats,to study pathophysiological mechanism of Alzheimer’s disease from diabetic metabolic disorder.MethodsAnimal model of diabetes mellitus was established by high fat and suger and streptozocin with intraperitoneal injection.Wistar rats were randomly divided into control group(S),4 week diabetes mellitus model group(M4),6 week diabetes mellitus model group(M6) and 8 week diabetes mellitus model group(M8).Behaviour was tested with Morris water maze task and shuttle box.Congo red detected deposition of beta-amyloid in the brain tissues.Bielschowsky stained silver determined senile plaques and neurofibillary tangles in the brain tissues,biochemical assay for blood glucose,blood cholesterol and glycosylated hemoglobin, serum concentration of insulin by radioimmunoassay detection,Expression of Aβwas measured by enzyme linked immunosorbent assay and APP by enzyme linked immunosorbent assay,Western blotting and RT-PCR, BACE1 by immunohistochemistry,enzyme linked immunosorbent assay, Western blotting and RT-PCR,p38MAPK,PKC,PKA,JAK-2,PI-3K,Akt and ERK1/2 by Western blotting and RT-PCR.The light density value was measured by imaging analysis.ResultsAnimal model and praxiologicai disorderThe model group in weighting significantly decreased compared with the control group(482.2±16.5) g(P＜0.05).Blood glucose concentration increased from the control group(6.23±0.62) mmol/ dl to the model group (23.21±3.46) mmol/dl(P＜0.01).Glycosylated hemoglobin levels increased from the control group to 3.44±0.26%of the model group 12.56±0.67% (P＜0.01).Cholesterol concentration increased from the control group (121.6±26.2) mg/dl the model group(291.±27.5) mg/dl(P＜0.01).Serum insulin concentration increased from the control group(365.3±26.5) pmol/l to the model group(406.2±24.5) pmol/l(P＜0.01).Weight,blood glucose, serum glycosylated hemoglobin,cholesterol concentration and serum insulin concentration is no difference in model groups.Weight loss,higher than 16mmol/dl blood sugar,insulin resistance and elevated cholesterol stand for success modeling.Navigation experiments in rats to identify the spatial learning ability:in the morris water maze test,the sample rats’ latency of searching for the platform is obviously longer than the control rats(P＜0.01),model group mice no significant difference(P＞0.05).Space exploration test of spatial memory in rats:The times of crossing platform within 120 seconds of sample rats are obviously less than the control ones (P＜0.01),model groups no significant difference(P＞0.05).Shuttle box to test the learning ability for condition reflex:the number of rats subjected to electric shocks increased in model groups than the control group significantly(P＜0.01) from 7.43±2.01 times in control group to 15.46±3.62 times,the model rats no significant differences(P＞0.05).The time of electric stimulation in model groups was significantly longer than the control group from 7.43±2.01 second in control group to 15.46±3.62 second in model.These results show that the model group capacity of learning and memory significantly decreasd in diabetes mellitus model groups and has a clear learning and memory impairment.Histopathological changesCongo Red staining forms non-polar hydrogen bonds with amyloid when viewed by polarised light due to parallel alignment of the dye molecules on the linearly arranged amyloid fibrils.The Alkaline Congo Red Technique uses high concentrations of sodium chloride which act as ionic competitors for the dye thus eliminating background electrochemical (polar) staining while enhancing the hydrogen(non-polar) binding of Congo Red and amyloid.The results by Congo Red staining showed that deposition of amyloid in model group is more significant than control group,but amyloid plaque.Bielschowsky’s silver staining shows that nerve fibers in the control group of rats were arranged regularly and closely. Compared with the control group,model groups of nerve fibers is thickening,swelling and disorder,but SP and NFT.Congo red staining and Bielschowsky’s silver staining method does not appear senile plaques and neurofibrillary tangles.Diabetic rat brain has no characteristic pathological changes of Alzheimer’s disease.Measurement of APP,Aβand BACE1The expression of APP,Aβand BACE1 in MT groups is higher than that of in S group(P＜0.01).The level of Aβand BACE1 has positive correlation with cognitive impairment.The level of APP has negative correlation with cognitive impairment.Three model groups have no significant difference in the expression of APP,Aβand BACE1.The expression of Aβ1-40 increases from(64.13±6.76) pg/mg in normal group to(86.43±7.03) pg/mg in model group by ELISA(P＜0.001) and Aβ1-42 from(67.43±5.12) pg/mg in normal group to(89.45±5.28) pg/mg(P＜0.001) in model group.ELISA assay showed that the expression of APP increases from(116.65±9.21) pg/mg in normal group to(156.73± 8.24) pg/mg in model group(P＜0.001),Western blotting method from 0.63±0.12 in normal group to 1.56±0.19 in model group(P＜0.001),RT-PCR method from 1.68±0.21 in normal group to 3.54±0.22 in model group (P＜0.001).The expression of BACE1 increases from(116.46±8.10)pg/mg in normal group to(158.73±6.24)pg/mg in model group by ELISA and from 0.61±0.11 in normal group to 1.52±0.16 by Western blotting OD valule and from 1.62±0.26 in normal group to 3.61±0.32 by RT-PCR OD valule and from 0.81±0.21 in normal group to 2.01±0.36 by immunohistochemistry OD valule(P＜0.001)。The expression of BACE1 and Aβin MT group is higher than that of in S group(P＜0.01).The level of BACE1 and Aβhas positive correlation with cognitive impairment.The major molecules in insulin signal pathwayThe results of PKC,PKA and JAK-2 by Western blotting show that: the level of protein expression of p38MAPK in model groups was no significant difference,compared with the control group(P＞0.05);RT-PCR results showed that the mRNA level of PKC,PKA and JAK-2 in the model group has been slightly increased,but no statistical significance(P＞0.05).Optical density by Werstern blot analysis revealed that protein level of PI-3K/Akt in model groups has lower than the control group,from the control group 0.914±0.065 to the model group 0.511±0.051(P=0.000); RT-PCR for molecular PI-3K/Akt show the same level of detection of PI-3K mRNA expression than in the control group significantly decreased from the control group 1.289±0.072 to the model group 0.821±0.066 (P=0.000),there was no significant difference in model groups(P=0.352). Optical density by Werstern blot analysis revealed that protein expression p38MAPK/ERK in model groups has lower than the control group,from the control group 0.926±0.072 to the model group 0.631±0.056(P = 0.000);RT-PCR for the detection of the molecular level p38MAPK/ERK mRNA expression showed a more significant downward adjustment from the control group 1.350±0.071 to the model group 0.96±0.069(P = 0.000),there was no significant difference in model groups(P = 0.367). Compared with the control group,,the expression of PI-3K/Akt, p38MAPK/ERK in the hippocampal tissue of diabetes mellitus model group decreased significantly(P=0.000),while the expression of BACE1 increased than the control group(P=0.000).BACE1 and PI-3K negatively correlated(P＜0.05),BACE1 and Akt is also negatively correlated(P＜0.05);BACE1 and p38MAPK was no correlation(P＞0.05),BACE1 and ERK1/2 was no correlation(P＞0.05),Aβ40/42 and p38MAPK negative correlation(P＜0.05),Aβ40/42 and ERK1/2 negative correlation(P＜0.05).Conclusions1.The expression of Aβ,APP increased in diabetes mellitus rats. Diabetes mellitus contributes to the pathogenesis of Alzheimer’s disease that diabetic metabolic disorder increases expression of Aβbut APP.APP has a dual mechanism,both the role of cerebral protection, and increased expression of the role of Aβ.2.The expression of BACE1 and Aβincreased in diabetes mellitus rats. Diabetes mellitus contributes to Alzheimer’s disease that Diabetic metabolic disorder increases expression of BACE1 and Aβ.3.Signaling pathway MAPK/ERK affect Aβmetabolism and signaling pathway regulates PI-3K/Akt involved in BACE1 expression in the pathogenesis of Alzheimer’s disease.PI-3K/Akt signaling pathway might effect the expression of BACE1,which demonstrates that impaired signaling pathway shoud make amyloid precursor protein easy to be processed by BACE1 to involve the pathology of Alzheimer’s disease.更多还原