阿尔茨海默病外周血淀粉样蛋白beta检及临床应用研究
发布时间：2018-08-05 20:49
【摘要】：目的淀粉样蛋白级联假设学说认为淀粉样蛋白beta(Amyloid beta,Aβ)是导致阿尔茨海默病(Alzheimer’s Disease,AD)疾病发生、发展的重要原因。脑组织皮质区和海马区神经元细胞外形成以Aβ为主要成分的老年斑块,已成为AD诊断标志物。目前,以Aβ为靶向指标的检测手段主要包括神经影像学方法和脑脊液生物标志物学检测。然而,神经影像学诊断方法,虽然可以对AD的诊断提供依据,但所采用示踪剂能否代谢清除尚未确定,对人体的副作用难以评估;采用非标记技术,检测缺乏特异性,易导致假阳性结果,同时疾病诊断漏诊率较高,给临床诊断带来困难。本文旨在通过建立检测外周血中Aβ的简易方法,探讨外周血中Aβ在AD诊断及致病机制中的作用。与神经影像学标志物和脑脊液标志物检测相比较,外周标本简单易得,且能反映脑组织疾病转归,在疾病的诊断、治疗、及致病机制研究中具有重大优势。方法1.根据严格的纳入与排出标准,选取天津医科大学总医院神经内科2012年3月至2014年10月AD患者(AD组)50例;性别、年龄相匹配的健康体检者50例(HC组)。抽取静脉全血,EDTA-2Na抗凝备用。2.采用蛋白免疫印迹(Western blot,WB)技术和免疫荧光实验证实血浆中和红细胞膜上存在Aβ纤维/聚集物。3.通过硫磺素T(Thioflavin-T,Th T)特异性实验,比较分析AD组和HC组血浆淀粉样变荧光强度。4.AD组和HC组血液物理学指数、血脂水平检测分析。5.AD组和HC组外周血体积增大红细胞数量比较,并分析AD组体积增大红细胞数量和简易智能状态检查(Simple mental state examination,MMSE)的相关性。6.探讨Th T特异性实验中,血浆对Th T识别红细胞膜上Aβ纤维/聚集物的影响。7.检测AD组和HC组Th T特异性实验阳性个体数占总体研究对象百分比,及个体Th T阳性红细胞数占总体红细胞数量百分比。8.通过Leica Application Suit 2.0.1软件,对Th T阳性红细胞细胞形态进行分析。结果1.AD组血浆淀粉样变荧光强度为(16.63±5.67)U/ml,HC为(12.83±0.27)U/ml。AD组血浆淀粉样变荧光强度显著高于对照组(P0.05)。2.AD组及HC组全血粘度(低切1.00 1/s)分别为(18.16±3.6)、(17.92±0.4)m Pa.s,两组比较P0.05;全血粘度(低切5.00 1/s)分别为(8.64±1.45)、(8.40±0.37)m Pa.s,两组比较P0.05;全血粘度(低切30.00 1/s)分别为(5.32±0.76)、(5.09±0.16)m Pa.s,两组比较P0.05;全血粘度(高切200.00 1/s)分别为(4.16±0.54)、(3.74±0.15)m Pa.s,两组比较P0.05;血浆浓度分别为(1.40±0.10)、(1.33±0.07)m Pa.s,两组比较P0.05;血沉分别为(11.8±6.18)、(8.30±6.09)mm/h,两组比较P0.05;红细胞压积分别为(0.43±0.61)、(0.45±0.31),两组比较P0.05;全血高切相对指数分别为(2.94±0.22)、(2.74±0.26),两组比较P0.05;全血低切相对指数分别为(12.79±1.78)、(14.24±1.32),两组比较P0.05;血沉方程k值分别为(39.61±13.08)、(32.26±15.27),两组比较P0.05;红细胞聚集指数分别为(4.34±0.36)、(4.13±0.14),两组比较P0.05;全血低切还原粘度分别为(38.36±2.77)、(38.81±2.50)m Pa.s,两组比较P0.05;全血高切还原粘度分别为(6.33±0.40)、(6.19±0.49)m Pa.s,两组比较P0.05;红细胞刚性指数分别为(4.50±0.34)、(4.70±0.50)m Pa.s,两组比较P0.05;红细胞变形指数TK分别为(0.82±0.08)、(0.81±0.07),两组比较P0.05。3.AD组及HC组总胆固醇分别为(4.71±1.24)、(3.95±0.22)mmol/L,两组比较P0.05;高密度脂蛋白分别为(1.21±0.35)、(1.40±0.17)mmol/L,两组比较P0.05;低密度脂蛋白分别为(2.70±0.84)、(2.78±0.36)mmol/L,两组比较P0.05。4.AD组全血粘度(高切部分)、血浆淀粉样变程度、血清总胆固醇含量存在相关性,其相关性可用回归方程Y?=2.798+0.070X1+0.027X2(Y?,全血粘度高切;X1,血浆淀粉样变程度;X2,总胆固醇)表示。5.AD组体积增大红细胞数量(16.8%)显著高于HC组(6.7%)(P0.01),并且AD组红细胞体积增大数量与MMSE呈负相关性(r=-0.773)。6.Th T能够特异识别红细胞膜上Aβ纤维/聚集物,并且染色结果不受血浆干扰。7.AD组98%的研究对象Th T特异性实验阳性,其Th T阳性红细胞百分比范围为(2%-30%);HC组Th T特异性实验阳性个体百分比为38%,阳性率波动范围为(2%-3.4%)。8.Aβ纤维/聚集物与红细胞结合导致红细胞体积增大,形态发生改变,主要表现为4种类型。结论1.分析AD患者血浆淀粉样变、总胆固醇水平和血液物理指数,结果三项生物标志物明显高于HC组,并且血浆淀粉样变水平与总胆固醇水平和血浆物理指数全血粘度(高切部分)相关,可能作为辅助检查指标。2.探索一种检测血细胞淀粉样变的方法,实现检测血红细胞、粒细胞、单核细胞的淀粉样变,该方法可用于检测血细胞淀粉样变。3.首次报道AD患者血红细胞淀粉样变的临床范围,确定AD患者淀粉样变红细胞为百分比为(2%-30%),可能作为AD患者辅助检查指标。4.发现Th T能够特异性识别外周循环中红细胞膜上Aβ纤维/聚集物。Th T阳性红细胞细胞形态发生改变,提示Aβ纤维/聚集物可能导致红细胞形态改变,预示细胞淀粉样变可能参与AD病理机制。
[Abstract]:Objective amyloid protein cascade hypothesis suggests that amyloid beta (Amyloid beta, A beta) is an important cause for the development of Alzheimer's disease (Alzheimer 's Disease, AD). The formation of senile plaques with A beta as the main component of the neuronal cells in the cortical and hippocampal regions of the brain tissue has become a diagnostic marker for AD. At present, A However, the neuroimaging diagnosis method can provide evidence for the diagnosis of AD, but the metabolic clearance of the tracer is not yet determined, and it is difficult to evaluate the side effects of the human body. The purpose of this paper is to establish a simple method for detecting A beta in peripheral blood and to explore the role of A beta in the diagnosis and pathogenesis of AD in peripheral blood. Compared with the detection of neuroimaging markers and cerebrospinal fluid markers, the peripheral specimen is simple. It has a great advantage in the diagnosis, treatment, and pathogenesis of the disease. Method 1. according to the strict inclusion and discharge standards, 50 cases of AD patients (group AD) of General Hospital Affiliated to Tianjin Medical University neurology from March 2012 to October 2014, 50 cases of sex and age matched healthy persons (Group HC) were selected. The EDTA-2Na anticoagulant.2. used protein immunoblotting (Western blot, WB) and immunofluorescence experiments to confirm the presence of A beta fiber / aggregation.3. on the erythrocyte membrane and the specificity of the sulphur T (Thioflavin-T, Th T) on the plasma and erythrocyte membrane. The fluid physics index and blood lipid level were detected and analyzed in group.5.AD and group HC, the volume of peripheral blood increased red blood cells, and the volume of red blood cells increased in AD group and the correlation.6. of simple intelligence state (Simple mental state examination, MMSE) in Th T specificity test. The number of Th T specific test positive individuals in group AD and HC group accounted for the percentage of the total subjects, and the percentage of Th T positive red cells accounted for the percentage of the total red cell number.8. through Leica Application Suit 2.0.1 software, the morphology of the positive erythrocytes was analyzed by the Leica Application Suit 2.0.1 software. Results the plasma amyloidosis fluorescence intensity of the group was strong. The plasma amyloid fluorescence intensity of the plasma (16.63 + 5.67) U/ml and (12.83 + 0.27) U/ml.AD was significantly higher than that of the control group (P0.05) and the whole blood viscosity (18.16 + 3.6) (18.16 + 3.6), (17.92 + 0.4) m Pa.s, and two groups of P0.05, and the whole blood viscosity (8.64 + 5 1/s) were respectively (8.64 + 1.45) and m 18.16 respectively. The whole blood viscosity (low cut 30 1/s) was (5.32 + 0.76), (5.09 + 0.16) m Pa.s, and the two group was P0.05, and the whole blood viscosity (200 1/s) was (4.16 + 0.54), (3.74 + 0.15) m Pa.s, and the two group was P0.05, and the plasma concentration was respectively (1.40 +), m Pa.s and P0.05; erythrocyte sedimentation, respectively, mm/h, respectively. The group was compared with P0.05, the hematocrit was (0.43 + 0.61), (0.45 + 0.31), and the two groups were compared with P0.05, the whole blood high cutting relative index was (2.94 + 0.22), (2.74 + 0.26), and two group was P0.05, and the whole blood low shear relative index was (12.79 + 1.78), (14.24 + 0.45) and P0.05, and the K value of the erythrocyte sedimentation equation was respectively. The erythrocyte aggregation index was (4.34 + 0.36), (4.13 + 0.14), and the two group was compared with P0.05, and the low reduction viscosity of the whole blood was (38.36 + 2.77), (38.81 + 2.50) m Pa.s, and two group was P0.05, and the whole blood high reduced viscosity was (6.33 + 0.40), (6.19 + 0.49) m Pa.s and P0.05, and the erythrocyte rigidity index was respectively (4.70 + 0.50) m Pa.s, two groups were compared with P0.05, erythrocyte deformation index TK was (0.82 + 0.08), (0.81 + 0.07). The total cholesterol in group P0.05.3.AD and HC group was respectively (4.71 + 1.24), (3.95 + 0.22) mmol/L, two group was P0.05, and high density lipoprotein was (two) mmol/L, P0.05 and low density lipoprotein. Don't be (2.70 + 0.84), (2.78 + 0.36) mmol/L, two groups compared the whole blood viscosity (high cut part) of group P0.05.4.AD, plasma amyloidosis degree, serum total cholesterol content correlation, its correlation can be used regression equation Y? =2.798+0.070X1+0.027X2 (Y?, whole blood viscosity high cut; X1, plasma amyloidosis degree; X2, total cholesterol) indicated.5.AD volume increase The number of large erythrocytes (16.8%) was significantly higher than that in the HC group (6.7%) (P0.01), and the number of red blood cells in the AD group was negatively correlated with MMSE (r=-0.773).6.Th T can specifically identify the A beta fibers / aggregates on the erythrocyte membrane, and the staining results were not subject to the plasma interference of.7.AD group 98%, the Th T specificity test was positive, and its Th positive red cells were 100%. The ratio range was (2%-30%); the percentage of Th T specific test positive individuals in group HC was 38%, and the positive rate fluctuated in (2%-3.4%).8.A beta fiber / aggregation and red blood cells resulting in increased erythrocyte volume and morphogenesis, mainly in 4 types. Conclusion 1. analysis of plasma amyloidosis, total cholesterol level and blood physical index in AD patients. Results three biomarkers were significantly higher than those in the HC group, and the level of plasma amyloidosis was related to the total cholesterol level and the plasma physical index of the whole blood viscosity (high shear part). It may be used as an auxiliary examination index.2. to explore a method for detecting amyloidosis in blood cells, and to detect the amyloidosis of blood red cells, granulocytes and monocytes. This method can be used to detect the clinical range of amyloidosis in AD patients with amyloidosis.3. for the first time. It is determined that the percentage of amyloid erythrocytes in AD patients is (2%-30%), and may be used as an auxiliary examination of AD patients.4. to find that Th T can specifically identify the.Th T positive of A beta fiber / aggregation on the erythrocyte membrane in the peripheral circulation. The morphological changes of erythrocyte suggest that A beta fibers/aggregates may lead to morphological changes of erythrocyte, indicating that cellular amyloidosis may participate in the pathological mechanism of AD.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.16;R446.1
[Abstract]:Objective amyloid protein cascade hypothesis suggests that amyloid beta (Amyloid beta, A beta) is an important cause for the development of Alzheimer's disease (Alzheimer 's Disease, AD). The formation of senile plaques with A beta as the main component of the neuronal cells in the cortical and hippocampal regions of the brain tissue has become a diagnostic marker for AD. At present, A However, the neuroimaging diagnosis method can provide evidence for the diagnosis of AD, but the metabolic clearance of the tracer is not yet determined, and it is difficult to evaluate the side effects of the human body. The purpose of this paper is to establish a simple method for detecting A beta in peripheral blood and to explore the role of A beta in the diagnosis and pathogenesis of AD in peripheral blood. Compared with the detection of neuroimaging markers and cerebrospinal fluid markers, the peripheral specimen is simple. It has a great advantage in the diagnosis, treatment, and pathogenesis of the disease. Method 1. according to the strict inclusion and discharge standards, 50 cases of AD patients (group AD) of General Hospital Affiliated to Tianjin Medical University neurology from March 2012 to October 2014, 50 cases of sex and age matched healthy persons (Group HC) were selected. The EDTA-2Na anticoagulant.2. used protein immunoblotting (Western blot, WB) and immunofluorescence experiments to confirm the presence of A beta fiber / aggregation.3. on the erythrocyte membrane and the specificity of the sulphur T (Thioflavin-T, Th T) on the plasma and erythrocyte membrane. The fluid physics index and blood lipid level were detected and analyzed in group.5.AD and group HC, the volume of peripheral blood increased red blood cells, and the volume of red blood cells increased in AD group and the correlation.6. of simple intelligence state (Simple mental state examination, MMSE) in Th T specificity test. The number of Th T specific test positive individuals in group AD and HC group accounted for the percentage of the total subjects, and the percentage of Th T positive red cells accounted for the percentage of the total red cell number.8. through Leica Application Suit 2.0.1 software, the morphology of the positive erythrocytes was analyzed by the Leica Application Suit 2.0.1 software. Results the plasma amyloidosis fluorescence intensity of the group was strong. The plasma amyloid fluorescence intensity of the plasma (16.63 + 5.67) U/ml and (12.83 + 0.27) U/ml.AD was significantly higher than that of the control group (P0.05) and the whole blood viscosity (18.16 + 3.6) (18.16 + 3.6), (17.92 + 0.4) m Pa.s, and two groups of P0.05, and the whole blood viscosity (8.64 + 5 1/s) were respectively (8.64 + 1.45) and m 18.16 respectively. The whole blood viscosity (low cut 30 1/s) was (5.32 + 0.76), (5.09 + 0.16) m Pa.s, and the two group was P0.05, and the whole blood viscosity (200 1/s) was (4.16 + 0.54), (3.74 + 0.15) m Pa.s, and the two group was P0.05, and the plasma concentration was respectively (1.40 +), m Pa.s and P0.05; erythrocyte sedimentation, respectively, mm/h, respectively. The group was compared with P0.05, the hematocrit was (0.43 + 0.61), (0.45 + 0.31), and the two groups were compared with P0.05, the whole blood high cutting relative index was (2.94 + 0.22), (2.74 + 0.26), and two group was P0.05, and the whole blood low shear relative index was (12.79 + 1.78), (14.24 + 0.45) and P0.05, and the K value of the erythrocyte sedimentation equation was respectively. The erythrocyte aggregation index was (4.34 + 0.36), (4.13 + 0.14), and the two group was compared with P0.05, and the low reduction viscosity of the whole blood was (38.36 + 2.77), (38.81 + 2.50) m Pa.s, and two group was P0.05, and the whole blood high reduced viscosity was (6.33 + 0.40), (6.19 + 0.49) m Pa.s and P0.05, and the erythrocyte rigidity index was respectively (4.70 + 0.50) m Pa.s, two groups were compared with P0.05, erythrocyte deformation index TK was (0.82 + 0.08), (0.81 + 0.07). The total cholesterol in group P0.05.3.AD and HC group was respectively (4.71 + 1.24), (3.95 + 0.22) mmol/L, two group was P0.05, and high density lipoprotein was (two) mmol/L, P0.05 and low density lipoprotein. Don't be (2.70 + 0.84), (2.78 + 0.36) mmol/L, two groups compared the whole blood viscosity (high cut part) of group P0.05.4.AD, plasma amyloidosis degree, serum total cholesterol content correlation, its correlation can be used regression equation Y? =2.798+0.070X1+0.027X2 (Y?, whole blood viscosity high cut; X1, plasma amyloidosis degree; X2, total cholesterol) indicated.5.AD volume increase The number of large erythrocytes (16.8%) was significantly higher than that in the HC group (6.7%) (P0.01), and the number of red blood cells in the AD group was negatively correlated with MMSE (r=-0.773).6.Th T can specifically identify the A beta fibers / aggregates on the erythrocyte membrane, and the staining results were not subject to the plasma interference of.7.AD group 98%, the Th T specificity test was positive, and its Th positive red cells were 100%. The ratio range was (2%-30%); the percentage of Th T specific test positive individuals in group HC was 38%, and the positive rate fluctuated in (2%-3.4%).8.A beta fiber / aggregation and red blood cells resulting in increased erythrocyte volume and morphogenesis, mainly in 4 types. Conclusion 1. analysis of plasma amyloidosis, total cholesterol level and blood physical index in AD patients. Results three biomarkers were significantly higher than those in the HC group, and the level of plasma amyloidosis was related to the total cholesterol level and the plasma physical index of the whole blood viscosity (high shear part). It may be used as an auxiliary examination index.2. to explore a method for detecting amyloidosis in blood cells, and to detect the amyloidosis of blood red cells, granulocytes and monocytes. This method can be used to detect the clinical range of amyloidosis in AD patients with amyloidosis.3. for the first time. It is determined that the percentage of amyloid erythrocytes in AD patients is (2%-30%), and may be used as an auxiliary examination of AD patients.4. to find that Th T can specifically identify the.Th T positive of A beta fiber / aggregation on the erythrocyte membrane in the peripheral circulation. The morphological changes of erythrocyte suggest that A beta fibers/aggregates may lead to morphological changes of erythrocyte, indicating that cellular amyloidosis may participate in the pathological mechanism of AD.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.16;R446.1
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