CN114966048A - 血小板AβPP比值在阿尔茨海默病筛查中的应用 - Google Patents
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Abstract
本发明公开了血小板淀粉样体(AβPP)比值作为阿尔茨海默病(AD)的外周血生物标志物及其在AD早期快速筛查中的应用，属于生物科学领域。当前研究发现，在AD的生物学诊断框架下，血小板AβPP比值在正常对照人群、非AD病理改变人群和AD疾病谱系人群血浆中呈差异性表达(仅在AD疾病谱系人群血浆中特异性降低)。此外，单独使用该指标用于区分AD疾病谱系人群和其他人群，具有显著的诊断准确性。因此，血小板AβPP比值可用于AD疾病谱系人群的快速筛查，具有较高的临床应用价值。
Description
技术领域
本发明涉及生物科学领域，具体涉及血小板淀粉样β蛋白前体比值在阿尔茨海默病筛查中的应用。
背景技术
阿尔茨海默病(AD)是一种神经退行性疾病，其特征是进行性认知障碍和记忆力下降(Joe E,Ringman JM(2019):Cognitive symptoms of Alzheimer's disease:clinicalmanagement and prevention.BMJ.367:l6217)。中国的痴呆症患者约占全球病例总人数的25％，目前全国约有900万该病患者(Jia L,Quan M,Fu Y,Zhao T,Li Y,Wei C,et al.(2020):Dementia in China:epidemiology,clinical management,and researchadvances.The Lancet Neurology.19:81-92；Jia J,Wei C,Chen S,Li F,Tang Y,Qin W,et al.(2018):The cost of Alzheimer's disease in China and re-estimation ofcosts worldwide.Alzheimer's&dementia:the journal of the Alzheimer'sAssociation.14:483-491)。2018年美国国立衰老研究院和阿尔茨海默病协会(NationalInstitute of Aging and Alzheimer’s Association，NIA-AA)提出了AD的生物学定义，也称之为ATN标准。ATN标准中的生物标志物包括:Aβ(A)；病理性Tau,包括总Tau和磷酸化Tau(T)和神经变性(N)。按生物标志进行的AD生物学，可分为三大类和8种状态：生物学标志物正常(A-T-(N)-)、AD疾病谱系(A+T-(N)-、A+T+(N)-、A+T+(N)+、A+T-(N)+)和非AD病理改变(A-T+(N)-、A-T-(N)+、A-T+(N)+)。NIA-AA框架在AD的早期诊断方面具有很大的优势。然而，目前检测ATN标准中生物标志物的方法因其特点而使用受限，如PET检查价格昂贵且具有辐射，脑脊液检查具有侵入性。因此，开发价格便宜、检测便捷的血液生物标志物对AD的早期诊断具有重要临床意义。
发明内容
针对现有技术的不足，本发明提出了血小板淀粉样β蛋白前体(AβPP)比值在阿尔茨海默病筛查中的应用。
本发明的目的可以通过以下技术方案实现：
一种阿尔茨海默病筛查的试剂盒，包括：能够检测血小板AβPP比值的试剂与检测器件。
可选地，所述试剂为蛋白质印迹法中使用的试剂。
可选地，所述试剂包括一抗、二抗、SDS-PAGE试剂与缓冲液。
可选地，所述一抗包括：单克隆抗体22C11、Phospho-Tau231、PHF1和甘油醛-3-磷酸脱氢酶。
可选地，所述二抗为辣根过氧化物酶欧联的山羊抗鼠/兔lgG。
可选地，所述检测器件包括电泳模块、免疫反应模块与化学发光模块。
本发明的有益效果：
与现有技术相比，本发明的有益效果在于：本申请提出了血小板AβPP比值作为特异性诊断AD的生物标志物的用途，为AD快速筛查提供了一条全新的途径；采用血小板AβPP比值作为指标，从正常对照人群和非AD病理改变人群中区分AD疾病谱系人群的ROC曲线下的面积值分别为0.846和0.768。
附图说明
下面结合附图对本发明作进一步的说明。
图1为正常对照人群、非AD病理改变人群和AD疾病谱系人群的血小板AβPP比值和其他两种血小板蛋白的蛋白质印迹结果图及蛋白定量柱状图。图1A、1B和1C分别为血小板AβPP比值、P-tau231和Ser396/404P-tau的蛋白质印迹结果图及蛋白定量柱状图。
图2为血小板AβPP比值和其他两种血小板蛋白分子区分正常对照人群和非AD病理改变人群、正常对照人群和AD疾病谱系人群、非AD病理改变人群和AD疾病谱系人群的ROC曲线。A：血小板AβPP比值的蛋白质印迹结果图及蛋白定量柱状图；B：血小板P-tau231的蛋白质印迹结果图及蛋白定量柱状图；C：血小板Ser396/404P-tau的蛋白质印迹结果图及蛋白定量柱状图。图2A、2B和2C为血小板AβPP比值、P-tau231和Ser396/404P-tau分别区分正常对照人群和AD疾病谱系人群、非AD病理改变人群和AD疾病谱系人群和正常对照人群和非AD病理改变人群的ROC曲线。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
本申请的实施例中，共招募68名同意进行腰椎穿刺的主诉记忆力下降的受试者。所有参与者都接受了标准化的临床相关访谈和血样采集。此外，受试者均进行了常规血液检查、脑部磁共振成像扫描和脑脊液检查。受试者根据ATN诊断框架进行分组，包括18例正常对照组(NC)，24例非AD病理改变组(Non-AD)和26例AD疾病谱系组(AD)。
受试者纳入研究标准如下：(1)年龄在45至85岁之间；(2)天然右撇子；(3)满足量表评估的视力和听力。同时，所有受试者的排除标准包括：(1)其他神经系统疾病(如帕金森病)或任何精神疾病(如抑郁症)；(2)严重的脑血管疾病；(3)既往脑外伤史；(4)重大的身体健康问题(例如肝脏或肾脏功能受损)；(5)目前正在服用影响血小板功能的药物(例如抗凝剂，皮质类固醇或抗血小板药物)；(6)药物滥用或药物成瘾；(7)任何腰椎穿刺的禁忌症。本申请的实施例的实验已通过东南大学附属中大医院研究伦理委员会的批准。所有受试者均提供书面知情同意书。
血小板制备
1)全血2ml，4度、200g、10分钟离心，获得上层富含血小板的血浆。
2)将获得的血浆转移到新EP管中，然后立即进行4度、3000g、20分钟离心，弃上清，沉淀为血小板。
3)使用Advanced Tyrode溶液(不含钙)(Solarbio)洗涤EP管底部血小板颗粒两次，并在每次洗涤后进行4℃、1500g、10分钟离心。
4)收集血小板衍生的蛋白质，并使用Bradford法(Solarbio)测量浓度。
蛋白质印迹分析
本申请的实施例中使用的一抗如下：单克隆抗体22C11(Merck，稀释1：2000)，Phospho-Tau231(Abcam，稀释1：5000)，PHF1(Abcam，稀释1：1000)和甘油醛-3-磷酸脱氢酶(GAPDH)(Abcam，稀释1：4000)。22C11抗体可以识别未成熟的～110kDa和成熟的～130kDa的AβPP同种型蛋白。P-Tau231抗体识别threonine-231 phosphorylated tau蛋白，PHF1抗体识别Ser396/404 P-tau。以GAPDH为内部参考，量化了目标波段的灰度值。蛋白质水平的测量一式重复，一式三份。
具体步骤如下：
1)将血小板细胞溶解在100ul放射免疫沉淀分析裂解缓冲液(GenStar)和1ul蛋白酶抑制剂混合物中进行组织裂解，置于冰上，30分钟。
2)0度、10秒声波降解法处理溶解产物。
3)4度、13000r/分钟、10分钟离心溶解产物。
4)使用Bradford法测定血小板衍生蛋白浓度。
5)在蛋白上清液中加入SDS-PAGE上样缓冲液(Bevotime)，然后在沸水中孵育蛋白变质。在8％丙烯酰胺凝胶中，每孔装载20μg蛋白质，在90mV下电泳45min。
6)然后，蛋白质转移到聚偏二氟乙烯膜在276mA下90分钟。用5％脱脂奶粉在室温下封膜1小时，然后用以下一抗在4度下孵育过夜:单克隆抗体22C11(Merck,稀释1:20000)、Phospho-Tau231(Abcam,稀释1:5000)、PHF1(Abcam,稀释1:1000)、甘油醛-3-磷酸脱氢酶(GAPDH)(Abcam,dilution 1:20000)。
7)用含Tween-20(BioFroxx)(TBST)的tris缓冲盐水在室温下洗涤4次，洗涤15分钟后，加入辣根过氧化物酶(HRP)欧联的山羊抗鼠/兔lgG(Bioshap，稀释1:10 000)，室温孵育1h。
8)用TBST冲洗聚偏二氟乙烯膜(15分钟×4次)，用增强化学发光试剂盒(ECL；Millipore)检测蛋白条带。使用LAS400 mini(GE Healthcare,USA)对蛋白条带进行量化。
9)采用Image J软件(http://rsbweb.nih.gov/ij/)对western blot条带进行量化。为了控制系统错误，对每个参与者的血小板衍生蛋白样本进行三份重复测量，当重复之间的测量显示出15％的变异时，对相同样本进行了额外的印迹。
受试者的一般人口统计学特征见表1，血小板AβPP比值、P-tau231和Ser396/404P-tau的蛋白质印迹结果图及蛋白定量柱状图见图1，血小板AβPP比值、P-tau231和Ser396/404P-tau的诊断效能分析见图2。
图1可见，3组人群中，血小板AβPP比值仅在AD疾病谱系人群中显著性降低。相比于正常对照人群，P-tau231和Ser396/404P-tau在AD疾病谱系人群和非AD病理改变人群均降低，而在这两组人群间无差异。图2表明血小板AβPP比值在区分AD疾病谱系人群与正常对照人群、非AD病理改变人群时诊断效能优于P-tau231和Ser396/404P-tau。
表1.一般人口统计学特征
注：数据以均值±标准差的形式表示。a Kruskal-Wallis H检验；b卡方检验；c单因素方差分析。
缩写：AD，阿尔茨海默病谱系组；Non-AD，非阿尔茨海默病病理改变组；NC，正常对照组；MMSE，简易精神状态检查量表；Aβ42，β-淀粉样蛋白42；p-tau,磷酸化tau蛋白；t-tau，总tau蛋白。
在本说明书的描述中，参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。
Claims (6)
1.一种阿尔茨海默病筛查的试剂盒，其特征在于，包括：能够检测血小板AβPP比值的试剂与检测器件。
2.根据权利要求1所述的试剂盒，其特征在于，所述试剂为蛋白质印迹法中使用的试剂。
3.根据权利要求2所述的试剂盒，其特征在于，所述试剂包括一抗、二抗、SDS-PAGE试剂与缓冲液。
4.根据权利要求3所述的试剂盒，其特征在于，所述一抗包括：单克隆抗体22C11、Phospho-Tau231、PHF1和甘油醛-3-磷酸脱氢酶。
5.根据权利要求3所述的试剂盒，其特征在于，所述二抗为辣根过氧化物酶欧联的山羊抗鼠/兔lgG。
6.根据权利要求2所述的试剂盒，其特征在于，所述检测器件包括电泳模块、免疫反应模块与化学发光模块。
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