【摘要】：目的淀粉樣蛋白級(jí)聯(lián)假設(shè)學(xué)說(shuō)認(rèn)為淀粉樣蛋白beta(Amyloid beta,Aβ)是導(dǎo)致阿爾茨海默病(Alzheimer’s Disease,AD)疾病發(fā)生、發(fā)展的重要原因。腦組織皮質(zhì)區(qū)和海馬區(qū)神經(jīng)元細(xì)胞外形成以Aβ為主要成分的老年斑塊,已成為AD診斷標(biāo)志物。目前,以Aβ為靶向指標(biāo)的檢測(cè)手段主要包括神經(jīng)影像學(xué)方法和腦脊液生物標(biāo)志物學(xué)檢測(cè)。然而,神經(jīng)影像學(xué)診斷方法,雖然可以對(duì)AD的診斷提供依據(jù),但所采用示蹤劑能否代謝清除尚未確定,對(duì)人體的副作用難以評(píng)估;采用非標(biāo)記技術(shù),檢測(cè)缺乏特異性,易導(dǎo)致假陽(yáng)性結(jié)果,同時(shí)疾病診斷漏診率較高,給臨床診斷帶來(lái)困難。本文旨在通過(guò)建立檢測(cè)外周血中Aβ的簡(jiǎn)易方法,探討外周血中Aβ在AD診斷及致病機(jī)制中的作用。與神經(jīng)影像學(xué)標(biāo)志物和腦脊液標(biāo)志物檢測(cè)相比較,外周標(biāo)本簡(jiǎn)單易得,且能反映腦組織疾病轉(zhuǎn)歸,在疾病的診斷、治療、及致病機(jī)制研究中具有重大優(yōu)勢(shì)。方法1.根據(jù)嚴(yán)格的納入與排出標(biāo)準(zhǔn),選取天津醫(yī)科大學(xué)總醫(yī)院神經(jīng)內(nèi)科2012年3月至2014年10月AD患者(AD組)50例;性別、年齡相匹配的健康體檢者50例(HC組)。抽取靜脈全血,EDTA-2Na抗凝備用。2.采用蛋白免疫印跡(Western blot,WB)技術(shù)和免疫熒光實(shí)驗(yàn)證實(shí)血漿中和紅細(xì)胞膜上存在Aβ纖維/聚集物。3.通過(guò)硫磺素T(Thioflavin-T,Th T)特異性實(shí)驗(yàn),比較分析AD組和HC組血漿淀粉樣變熒光強(qiáng)度。4.AD組和HC組血液物理學(xué)指數(shù)、血脂水平檢測(cè)分析。5.AD組和HC組外周血體積增大紅細(xì)胞數(shù)量比較,并分析AD組體積增大紅細(xì)胞數(shù)量和簡(jiǎn)易智能狀態(tài)檢查(Simple mental state examination,MMSE)的相關(guān)性。6.探討Th T特異性實(shí)驗(yàn)中,血漿對(duì)Th T識(shí)別紅細(xì)胞膜上Aβ纖維/聚集物的影響。7.檢測(cè)AD組和HC組Th T特異性實(shí)驗(yàn)陽(yáng)性個(gè)體數(shù)占總體研究對(duì)象百分比,及個(gè)體Th T陽(yáng)性紅細(xì)胞數(shù)占總體紅細(xì)胞數(shù)量百分比。8.通過(guò)Leica Application Suit 2.0.1軟件,對(duì)Th T陽(yáng)性紅細(xì)胞細(xì)胞形態(tài)進(jìn)行分析。結(jié)果1.AD組血漿淀粉樣變熒光強(qiáng)度為(16.63±5.67)U/ml,HC為(12.83±0.27)U/ml。AD組血漿淀粉樣變熒光強(qiáng)度顯著高于對(duì)照組(P0.05)。2.AD組及HC組全血粘度(低切1.00 1/s)分別為(18.16±3.6)、(17.92±0.4)m Pa.s,兩組比較P0.05;全血粘度(低切5.00 1/s)分別為(8.64±1.45)、(8.40±0.37)m Pa.s,兩組比較P0.05;全血粘度(低切30.00 1/s)分別為(5.32±0.76)、(5.09±0.16)m Pa.s,兩組比較P0.05;全血粘度(高切200.00 1/s)分別為(4.16±0.54)、(3.74±0.15)m Pa.s,兩組比較P0.05;血漿濃度分別為(1.40±0.10)、(1.33±0.07)m Pa.s,兩組比較P0.05;血沉分別為(11.8±6.18)、(8.30±6.09)mm/h,兩組比較P0.05;紅細(xì)胞壓積分別為(0.43±0.61)、(0.45±0.31),兩組比較P0.05;全血高切相對(duì)指數(shù)分別為(2.94±0.22)、(2.74±0.26),兩組比較P0.05;全血低切相對(duì)指數(shù)分別為(12.79±1.78)、(14.24±1.32),兩組比較P0.05;血沉方程k值分別為(39.61±13.08)、(32.26±15.27),兩組比較P0.05;紅細(xì)胞聚集指數(shù)分別為(4.34±0.36)、(4.13±0.14),兩組比較P0.05;全血低切還原粘度分別為(38.36±2.77)、(38.81±2.50)m Pa.s,兩組比較P0.05;全血高切還原粘度分別為(6.33±0.40)、(6.19±0.49)m Pa.s,兩組比較P0.05;紅細(xì)胞剛性指數(shù)分別為(4.50±0.34)、(4.70±0.50)m Pa.s,兩組比較P0.05;紅細(xì)胞變形指數(shù)TK分別為(0.82±0.08)、(0.81±0.07),兩組比較P0.05。3.AD組及HC組總膽固醇分別為(4.71±1.24)、(3.95±0.22)mmol/L,兩組比較P0.05;高密度脂蛋白分別為(1.21±0.35)、(1.40±0.17)mmol/L,兩組比較P0.05;低密度脂蛋白分別為(2.70±0.84)、(2.78±0.36)mmol/L,兩組比較P0.05。4.AD組全血粘度(高切部分)、血漿淀粉樣變程度、血清總膽固醇含量存在相關(guān)性,其相關(guān)性可用回歸方程Y?=2.798+0.070X1+0.027X2(Y?,全血粘度高切;X1,血漿淀粉樣變程度;X2,總膽固醇)表示。5.AD組體積增大紅細(xì)胞數(shù)量(16.8%)顯著高于HC組(6.7%)(P0.01),并且AD組紅細(xì)胞體積增大數(shù)量與MMSE呈負(fù)相關(guān)性(r=-0.773)。6.Th T能夠特異識(shí)別紅細(xì)胞膜上Aβ纖維/聚集物,并且染色結(jié)果不受血漿干擾。7.AD組98%的研究對(duì)象Th T特異性實(shí)驗(yàn)陽(yáng)性,其Th T陽(yáng)性紅細(xì)胞百分比范圍為(2%-30%);HC組Th T特異性實(shí)驗(yàn)陽(yáng)性個(gè)體百分比為38%,陽(yáng)性率波動(dòng)范圍為(2%-3.4%)。8.Aβ纖維/聚集物與紅細(xì)胞結(jié)合導(dǎo)致紅細(xì)胞體積增大,形態(tài)發(fā)生改變,主要表現(xiàn)為4種類(lèi)型。結(jié)論1.分析AD患者血漿淀粉樣變、總膽固醇水平和血液物理指數(shù),結(jié)果三項(xiàng)生物標(biāo)志物明顯高于HC組,并且血漿淀粉樣變水平與總膽固醇水平和血漿物理指數(shù)全血粘度(高切部分)相關(guān),可能作為輔助檢查指標(biāo)。2.探索一種檢測(cè)血細(xì)胞淀粉樣變的方法,實(shí)現(xiàn)檢測(cè)血紅細(xì)胞、粒細(xì)胞、單核細(xì)胞的淀粉樣變,該方法可用于檢測(cè)血細(xì)胞淀粉樣變。3.首次報(bào)道AD患者血紅細(xì)胞淀粉樣變的臨床范圍,確定AD患者淀粉樣變紅細(xì)胞為百分比為(2%-30%),可能作為AD患者輔助檢查指標(biāo)。4.發(fā)現(xiàn)Th T能夠特異性識(shí)別外周循環(huán)中紅細(xì)胞膜上Aβ纖維/聚集物。Th T陽(yáng)性紅細(xì)胞細(xì)胞形態(tài)發(fā)生改變,提示Aβ纖維/聚集物可能導(dǎo)致紅細(xì)胞形態(tài)改變,預(yù)示細(xì)胞淀粉樣變可能參與AD病理機(jī)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R749.16;R446.1

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