WO2023035465A1 - 一种阿尔兹海默症生物标志物牛磺酸及其应用 - Google Patents

一种阿尔兹海默症生物标志物牛磺酸及其应用 Download PDF

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WO2023035465A1
WO2023035465A1 PCT/CN2021/138089 CN2021138089W WO2023035465A1 WO 2023035465 A1 WO2023035465 A1 WO 2023035465A1 CN 2021138089 W CN2021138089 W CN 2021138089W WO 2023035465 A1 WO2023035465 A1 WO 2023035465A1
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alzheimer
disease
sample
taurine
early diagnosis
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陈宇
陈艺菁
樊颖颖
陈岳文
叶涛
许进英
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中国科学院深圳先进技术研究院
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and relates to a kind of Alzheimer's disease biomarker taurine and application thereof.
  • AD Alzheimer's disease
  • senile dementia is a progressive degenerative disease of the central nervous system that occurs in old age, characterized by progressive memory impairment and cognitive function decline and daily life. It is characterized by loss of life ability, accompanied by neuropsychiatric symptoms such as personality changes, which seriously affects social and life functions. Since the pathogenesis of Alzheimer's disease is not fully clear, and its early symptoms are relatively secretive, Alzheimer's disease patients are easily missed or misdiagnosed.
  • diagnosis of AD mainly relies on memory scales, PET, and cerebrospinal fluid and blood.
  • CN106062563A discloses a biomarker and method for early diagnosis of Alzheimer's disease, said AD biomarker is at least four selected from brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), tumor growth factor beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), interleukin 18 (IL-18), and monocyte chemoattractant protein-1 (MCP-1) biomarkers , by analyzing its expression level, it can assist the early diagnosis of AD.
  • BDNF brain-derived neurotrophic factor
  • IGF-1 insulin-like growth factor-1
  • TGF-beta1 tumor growth factor beta 1
  • VEGF vascular endothelial growth factor
  • IL-18 interleukin 18
  • MCP-1 monocyte chemoattractant protein-1
  • AD neurodegenerative diseases
  • the host and intestinal flora produce a large number of metabolites in the process of metabolizing food substances.
  • the diversity and abundance of intestinal flora will have an important impact on the types and concentrations of small molecule metabolites in the body.
  • studying the relationship between the metabolic homeostasis of intestinal flora and the onset of AD and screening new AD biomarkers will help expand the basis for early diagnosis of AD, and can be combined with other markers to improve the diagnosis of AD.
  • the accuracy is helpful for early warning of diseases, pathological typing, and prediction and evaluation of developmental stages.
  • the present invention provides a biomarker of Alzheimer's disease, taurine, and its application.
  • the present invention is based on high-resolution non-targeted metabolomics analysis technology to characterize human feces metabolites Quantitative analysis, for the first time, taurine in feces is used as a marker of Alzheimer's disease, and the detection of taurine in feces can assist in the early diagnosis of Alzheimer's disease, and it has timely, convenient, high specificity and high sensitivity. features.
  • the present invention provides a biomarker of Alzheimer's disease, and the biomarker of Alzheimer's disease is taurine.
  • Taurine is a sulfur-containing amino acid with a simple structure. Its chemical name is 2-aminoethanesulfonic acid. Its molecular formula is C 2 H 7 NO 3 S and its molecular weight is 125.15. It is odorless and has a slightly sour taste. Its dilute solution is neutral , stable to heat, combined with bile acid in the bile of humans and animals, exists in a bound form, and exists in a free form in tissues such as brain, ovary, heart, liver, milk, pineal gland, pituitary gland, retina, adrenal gland, etc. , but does not participate in protein synthesis.
  • Taurine is a conditionally essential amino acid for the human body and plays an important role in the development of the nervous system of fetuses and infants. Taurine can be widely used in medicine, food additives, fluorescent whitening agents, organic synthesis, etc. It can also be used as biochemical reagent, wetting agent and pH buffering agent.
  • the present invention conducts qualitative and quantitative analysis on fecal metabolites based on high-resolution non-targeted metabolomics analysis technology, and detects that the taurine level in Alzheimer's disease feces samples is significantly higher than that in normal feces samples, and taurine in feces is used as Alzheimer's disease biomarker, by detecting the level of taurine in feces can assist in the early diagnosis of Alzheimer's disease.
  • the present invention provides the application of the Alzheimer's disease biomarker as described in the first aspect in constructing an early diagnosis model of Alzheimer's disease and/or preparing an early diagnosis device for Alzheimer's disease.
  • the present invention provides an early diagnosis model of Alzheimer's disease
  • the input variables of the early diagnosis model of Alzheimer's disease include the peak intensity value of the mass spectrum of taurine described in the first aspect.
  • the output variables of the Alzheimer's disease early diagnosis model include differential expression multiples, and the calculation formula of the differential expression multiples is shown in equation (1):
  • the criteria for judging positive for Alzheimer's disease is that the differential expression multiple is ⁇ 8.51.
  • the present invention by fully comparatively analyzing the peak intensity values of taurine mass spectrum in normal feces samples and AD feces samples, and carrying out rational design, a kind of early diagnosis model of Alzheimer's disease is constructed, and described model uses bovine
  • the peak intensity of sulfonic acid mass spectrometry is the input variable
  • the differential expression fold is the output variable, which can quickly output the results and fully characterize the samples with abnormal taurine levels, thereby assisting the early diagnosis of Alzheimer's disease.
  • the present invention provides a device for early diagnosis of Alzheimer's disease, said device comprising the following units:
  • Sample preparation unit prepare the sample to be tested into a sample solution to be tested that can be used for separation by liquid chromatography;
  • Detection unit using the liquid chromatograph to separate the sample solution to be tested, using a mass spectrometer to perform data processing on the separated sample, and measuring the peak intensity value of the mass spectrum of taurine described in the first aspect in the sample;
  • Analysis unit input the detected peak intensity value of the mass spectrum of taurine into the early diagnosis model of Alzheimer's disease described in the third aspect for data analysis;
  • Evaluation unit output the differential expression multiple corresponding to the sample, and judge whether it is positive for Alzheimer's disease.
  • each unit cooperates effectively, is simple and efficient, and can quickly complete sample processing, detection and obtain differential expression multiples, and at the same time, use rationally designed judgment criteria to detect positive results for Alzheimer's disease. Evaluation is of great significance for the early diagnosis of Alzheimer's disease.
  • the sample to be tested comprises a stool sample.
  • the preparation method of the sample solution to be tested includes adding the sample to be tested into an aqueous acetonitrile solution, centrifuging and collecting the supernatant to obtain the sample solution to be tested.
  • the preparation method of the sample solution to be tested comprises the following steps:
  • the volume ratio of methanol, acetonitrile and water in the methanol/acetonitrile/water solution is (1-2):(1-2):1 including but not limited to 1.2:2:1, 1.2:1:1, 2 :2:1, 1.4:1.5:1, 1.6:1.2:1, 1.8:2:1, 1.9:1.8:1 or 1.1:1.4:1.
  • the volume ratio of acetonitrile and water in the aqueous acetonitrile solution is (1-2):1, including but not limited to 1.1:1, 1.2:1, 1.3:1, 1.5:1, 1.6:1, 1.7:1 , 1.8:1 or 1.9:1.
  • the liquid chromatograph includes an ultra-high performance liquid chromatograph.
  • the ultra-high performance liquid chromatograph comprises Agilent 1290Infinity LC ultra-high performance liquid chromatograph.
  • the mass spectrometer comprises a tandem time-of-flight mass spectrometer.
  • the tandem time-of-flight mass spectrometer includes AB Triple TOF 6600 mass spectrometer.
  • the data processing includes:
  • tandem time-of-flight mass spectrometer uses the tandem time-of-flight mass spectrometer to collect the first-order spectrum and second-order spectrum of the separated sample, and convert the first-order spectrum and second-order spectrum into mzXML format, and then perform peak alignment and retention time Calibrate, extract peak area and identify the structure, and determine the peak intensity value of the mass spectrum of taurine described in the first aspect in the sample.
  • the Alzheimer's disease early diagnosis device includes the following units:
  • Sample preparation unit prepare the sample to be tested into a sample solution to be tested that can be used for separation by liquid chromatography;
  • Detection unit use the liquid chromatograph to separate the sample solution to be tested, use a tandem time-of-flight mass spectrometer to collect the first-order spectrum and second-order spectrum of the separated sample, and collect the first-order spectrum
  • the figure and the secondary spectrogram are converted into mzXML format, then carry out peak alignment, retention time correction, extraction of peak area and structural identification, and measure the peak intensity value of the taurine mass spectrum described in the first aspect in the sample;
  • Analysis unit input the detected peak intensity value of the mass spectrum of taurine into the early diagnosis model of Alzheimer's disease described in the third aspect for data analysis;
  • Evaluation unit output the differential expression multiple corresponding to the sample, and judge whether it is positive for Alzheimer's disease.
  • the detection of taurine level in feces samples can be used as a diagnostic basis, combined with other detection results, to assist in the early diagnosis of Alzheimer's disease, and it is expected to improve the accuracy of Alzheimer's disease diagnosis.
  • it cannot be used alone as a diagnostic indicator for 100% diagnosis of Alzheimer's disease.
  • Taurine belongs to the ABC transporters pathway in the membrane transport pathway (Membrane transport pathway).
  • the present invention provides the application of the Alzheimer's disease biomarker described in the first aspect in screening drugs for treating and/or preventing Alzheimer's disease.
  • the Alzheimer's disease biomarker described in the first aspect is used as a target to screen a drug for treating and/or preventing Alzheimer's disease.
  • the present invention has the following beneficial effects:
  • the present invention detects for the first time that the level of taurine in feces samples of Alzheimer's disease is significantly higher than that of normal feces samples, uses taurine in feces as a biomarker of Alzheimer's disease, and provides an early diagnosis model of Alzheimer's disease And the device can assist in the early diagnosis of Alzheimer's disease by detecting the level of taurine in feces, which is helpful for non-invasive and rapid detection, and has the characteristics of timeliness, convenience, high specificity and high sensitivity.
  • Fig. 1 is the level figure of taurine in the stool sample of AD model mouse and wild-type mouse;
  • Figure 2 is a map of the levels of mannose in the cerebral cortex samples of AD model mice and wild-type mice;
  • Figure 3 is a graph showing the level of myo-inositol in the cerebral cortex samples of AD model mice and wild-type mice;
  • Fig. 4 is a graph showing glycine levels in cerebral cortex samples of AD model mice and wild-type mice.
  • the feces of 10 AD model mice and 10 wild-type mice cultured under the same conditions were collected respectively, and the samples of wild-type mice were numbered FWT-1-1 ⁇ FWT-1-10 in turn, and the samples of AD model mice were The samples are sequentially numbered FTG-1-1 ⁇ FTG-1-10, and the taurine level in the samples is detected by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry.
  • the specific methods include:
  • HILIC column is used for separation, column temperature is 25°C; flow rate is 0.5mL/min; injection volume is 2 ⁇ L; mobile phase composition includes: Phase A: aqueous solution mixed with ammonium acetate and ammonia water (The final concentrations of ammonium acetate and ammonia water are both 25mM), phase B: acetonitrile; the gradient elution program is as follows: 0 ⁇ 0.5min, 95% phase B; 0.5 ⁇ 7min, phase B changes linearly from 95% to 65%; 7 ⁇ 8min, phase B changes linearly from 65% to 40%; 8-9min, phase B maintains at 40%; 9-9.1min, phase B linearly changes from 40% to 95%; 9.1-12min, phase B maintains at 95% ;Samples were placed in an autosampler at 4°C throughout the analysis process;
  • AB Triple TOF 6600 mass spectrometer is used to collect the first-order and second-order spectra of the samples separated by ultra-high performance liquid chromatography in step (2).
  • the ESI source conditions are as follows: Ion Source Gas1 (Gas1): 60 , Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600°C, IonSapary Voltage Floating (ISVF) ⁇ 5500V (positive and negative two modes); TOF MS scan m/z range: 60- 1000Da, product ion scan m/z range: 25-1000 Da, TOF MS scan accumulation time 0.20s/spectra, product ion scan accumulation time 0.05s/spectra; the secondary mass spectrum is obtained by information dependent acquisition (IDA) and high Sensitivity mode, Declustering potential (DP): ⁇ 60V (both positive and negative modes), Collision Energy: 35 ⁇ 15eV, IDA settings are as follows Exclude isotopes within 4 Da, Candidate
  • the metabolite structure identification was carried out on the data extracted by XCMS, and the taurine level in the sample was analyzed.
  • R Language tools R package (ropls)
  • PCA Principal Component Analysis
  • OPLS-DA Orthogonal Partial Least Squares Discriminant Analysis
  • T Test Student's t-test
  • taurine belongs to the ABC transporters pathway in the Membrane transport pathway.
  • the ATP-driven pump consists of two transmembrane domains and two cytoplasmic ATP-binding domains.
  • the ABC transporter is a transporter of sugars, amino acids, phospholipids and peptides on the bacterial plasma membrane, and a transporter of phospholipids, lipophilic drugs, cholesterol and other small molecules on the plasma membrane of mammalian cells.
  • the plasma membranes of cells in organs such as kidneys and kidneys are abundant, which can remove natural poisons and metabolic wastes from the body.
  • the cerebral cortex samples of 10 AD model mice and 10 wild-type mice cultured under the same conditions were collected respectively.
  • the mouse cerebral cortex samples were sequentially numbered CTG-1-1 ⁇ CTG-1-10, and the metabolites were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry, and the specific methods included:
  • the VIP value and p-value are comprehensively considered to screen the significant differential metabolites.
  • the results are shown in Figure 2- Figure 4 and Table 2, AD
  • the levels of mannose (D-Mannose), myo-Inositol (myo-Inositol) and glycine (Glycine) in the cerebral cortex of model mice were significantly higher than those of wild-type mice, and the above metabolites belong to the ABC transporters pathway in the Membrane transport pathway , indicating that there is a correlation between the ABC transporters pathway in the Membrane transport pathway and Alzheimer's disease, and the present invention finds that taurine also belongs to the ABC transporters pathway in the Membrane transport pathway, indicating from another aspect that taurine in feces metabolites Level changes may reflect the abnormality of related metabolic pathways in the AD brain, which is of great significance for early clinical diagnosis.
  • the present invention detects for the first time that the level of taurine in the fecal samples of Alzheimer's disease is significantly higher than that of normal feces samples, and uses taurine in feces as a biomarker of Alzheimer's disease, and provides Alzheimer's disease
  • the early diagnosis model and device of Alzheimer's disease can assist in the early diagnosis of Alzheimer's disease by detecting the level of taurine in feces, which is helpful for non-invasive and rapid detection, and has the characteristics of timeliness, convenience, high specificity and high sensitivity.
  • the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

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Abstract

一种阿尔兹海默症生物标志物牛磺酸及其应用。阿尔兹海默症生物标志物为牛磺酸。首次检测到阿尔兹海默症粪便样本中牛磺酸水平显著高于正常粪便样本，将粪便中牛磺酸作为阿尔兹海默症生物标志物，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。

Description

一种阿尔兹海默症生物标志物牛磺酸及其应用 技术领域
本发明属于生物技术领域，涉及一种阿尔兹海默症生物标志物牛磺酸及其应用。
背景技术
阿尔兹海默症(Alzheimer disease，AD)，又称老年痴呆症，是一种发生于老年期的进行性发展的中枢神经系统退行性变性疾病，以渐进性记忆障碍及认知功能下降和日常生活能力丧失为特征，伴随人格改变等神经精神症状，严重影响社交与生活功能。由于阿尔兹海默症发病机制尚未完全明确，加上其早期症状比较隐秘，阿尔兹海默症患者容易被漏诊或错诊，目前对于AD的诊断主要依靠记忆量表、PET以及脑脊液、血液中Aβ、磷酸化tau蛋白等病理指标的水平检测，然而这些诊断指标在临床中的检测结果仍存在一定争议，而且对于AD发病早期的症状尚缺乏有效的检测证据，因此，对AD早期诊断新的标志物开发是AD诊疗领域的重要研究方向之一。
CN106062563A公开了一种用于阿尔兹海默症的早期诊断的生物标志物及方法，所述AD生物标志物是至少四种选自脑源性神经营养因子(BDNF)、胰岛素样生长因子-1(IGF-1)、肿瘤生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)、白介素18(IL-18)和单核细胞趋化蛋白-1(MCP-1)中的生物标志物，通过分析其表达水平，能够辅助AD的早期诊断。
有研究发现多种神经精神疾病如帕金森、抑郁症、自闭症等与肠道菌群失衡有关，并且80％以上AD患者存在肠道菌群失衡的现象，提示肠道菌群稳态与AD等神经退行性疾病的发病进程密切相关。宿主和肠道菌群在代谢食物物 质的过程中产生大量的代谢物，肠道菌群的多样性以及丰度的改变会对机体中小分子代谢物的种类和浓度产生重要影响，有数据显示AD患者肠道菌群组成与健康同龄人不同，且越来越多证据表明各种代谢途径的紊乱可能介导AD的病理发生和发展，AD中的菌群失衡可能与机体代谢紊乱的发生有重要关联，而机体外周代谢改变又可能通过血液循环进一步导致中枢神经系统代谢紊乱。
综上所述，研究肠道菌群代谢稳态与AD发病的关系，筛选新的AD生物标志物，有助于扩充AD早期诊断的判断依据，可与其他标志物检测相互结合，提高AD诊断的准确性，有助于疾病的早期预警、病理分型以及发展阶段的预测评估等。
发明内容
针对现有技术的不足和实际需求，本发明提供一种阿尔兹海默症生物标志物牛磺酸及其应用，本发明基于高分辨非靶向代谢组学分析技术对人粪便代谢物进行定性定量分析，首次将粪便中牛磺酸作为阿尔兹海默症标志物，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊断，且具备及时、方便、高特异性及高灵敏度的特点。
为达上述目的，本发明采用以下技术方案：
第一方面，本发明提供一种阿尔兹海默症生物标志物，所述阿尔兹海默症生物标志物为牛磺酸(Taurine)。
牛磺酸是一种结构简单的含硫氨基酸，化学名称为2-氨基乙磺酸，分子式为C 2H 7NO 3S，分子量为125.15，无臭，味略酸，其稀溶液呈中性，对热稳定，在人和动物胆汁中与胆酸结合，以结合形式存在，而在脑、卵巢、心脏、肝、乳汁、松果体、垂体、视网膜、肾上腺等组织中，以游离形式存在，但不参与 蛋白质的合成，牛磺酸是人体的条件必需氨基酸，对胎儿、婴儿神经系统的发育有重要作用，牛磺酸可广泛应用于医药、食品添加剂、荧光增白剂、有机合成等领域，也可用作生化试剂、湿润剂和pH缓冲剂等。
本发明基于高分辨非靶向代谢组学分析技术对粪便代谢物进行定性定量分析，检测到阿尔兹海默症粪便样本中牛磺酸水平显著高于正常粪便样本，将粪便中牛磺酸作为阿尔兹海默症生物标志物，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊断。
第二方面，本发明提供如第一方面所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
第三方面，本发明提供一种阿尔兹海默症早期诊断模型，所述阿尔兹海默症早期诊断模型的输入变量包括第一方面所述的牛磺酸质谱的峰强度值。
优选地，所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数，所述差异表达倍数的计算公式如方程式(1)所示：
Figure PCTCN2021138089-appb-000001
优选地，阿尔兹海默症阳性的判断标准为所述差异表达倍数≥8.51。
本发明中，通过对正常粪便样本和AD粪便样本中牛磺酸质谱的峰强度值进行充分对比分析，并进行理性设计，构建了一种阿尔兹海默症早期诊断模型，所述模型以牛磺酸质谱的峰强度值为输入变量，以差异表达倍数为输出变量，能够快速输出结果，且充分表征牛磺酸水平异常的样本，从而辅助阿尔兹海默症早期诊断。
第四方面，本发明提供一种阿尔兹海默症早期诊断装置，所述装置包括如 下单元：
样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行数据处理，测定样本中第一方面所述的牛磺酸质谱的峰强度值；
分析单元：将检测到的牛磺酸质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行数据分析；
评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本发明的阿尔兹海默症早期诊断装置中，各单元间有效配合，简单高效，能够快速完成样本处理、检测及获得差异表达倍数，同时以经过合理设计的判断标准进行阿尔兹海默症阳性评估，对于阿尔兹海默症早期诊断具有重要意义。
优选地，所述待测样本包括粪便样本。
优选地，所述待测样本溶液的配制方法包括将待测样本加入乙腈水溶液中，离心并收集上清液，得到所述待测样本溶液。
优选地，所述待测样本溶液的配制方法包括以下步骤：
(1)取待测样本加入预冷甲醇/乙腈/水溶液中，混合并超声25～35min(例如可以是26min、27min、28min、29min或32min)，置于-20～-15℃(例如可以是-19℃、-18℃、-16℃或-17℃)静置5～15min(例如可以是6min、7min、8min、9min、10min、12min或14min)，于0～4℃(例如可以是1℃、2℃或3℃)、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心15～25min(例如可以是16min、17min、18min、19min、20min、21min、22min、23min或24min)，取上 清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入80～120μL乙腈水溶液中复溶，涡旋，于0～4℃、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心10～20min(例如可以是11min、12min、13min、14min、15min、16min、17min、18min或19min)，取上清液，得到所述待测样本溶液。
优选地，所述甲醇/乙腈/水溶液中甲醇、乙腈和水的体积比为(1～2):(1～2):1包括但不限于1.2:2:1、1.2:1:1、2:2:1、1.4:1.5:1、1.6:1.2:1、1.8:2:1、1.9:1.8:1或1.1:1.4:1。
优选地，所述乙腈水溶液中乙腈和水的体积比为(1～2):1，包括但不限于1.1:1、1.2:1、1.3:1、1.5:1、1.6:1、1.7:1、1.8:1或1.9:1。
优选地，所述液相色谱仪包括超高效液相色谱仪。
优选地，所述超高效液相色谱仪包括Agilent 1290Infinity LC超高效液相色谱仪。
优选地，所述质谱仪包括串联飞行时间质谱联用仪。
优选地，所述串联飞行时间质谱联用仪包括AB Triple TOF 6600质谱仪。
优选地，所述数据处理包括：
利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中第一方面所述的牛磺酸质谱的峰强度值。
作为优选的技术方案，所述阿尔兹海默症早期诊断装置包括如下单元：
样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中第一方面所述的牛磺酸质谱的峰强度值；
分析单元：将检测到的牛磺酸质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行数据分析；
评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本发明中，对粪便样本中牛磺酸水平进行检测，可以作为一种诊断依据，与其他检测结果结合，辅助阿尔兹海默症早期诊断，预期可以提高阿尔兹海默症诊断的准确性，但并不能单独作为能够100％诊断阿尔兹海默症的诊断指标。
本发明中，通过KEGG通路分析发现Taurine属于膜转运通路(Membrane transport pathway)中的ABC transporters途径。
第五方面，本发明提供第一方面所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
即以第一方面所述的阿尔兹海默症生物标志物作为靶点筛选治疗和/或预防阿尔兹海默症的药物。
与现有技术相比，本发明具有以下有益效果：
本发明首次检测到阿尔兹海默症粪便样本中牛磺酸水平显著高于正常粪便样本，将粪便中牛磺酸作为阿尔兹海默症生物标志物，并提供阿尔兹海默症早期诊断模型和装置，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊 断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
附图说明
图1为AD模型小鼠和野生型小鼠的粪便样本中牛磺酸水平图；
图2为AD模型小鼠和野生型小鼠的大脑皮层样本中甘露糖水平图；
图3为AD模型小鼠和野生型小鼠的大脑皮层样本中肌醇水平图；
图4为AD模型小鼠和野生型小鼠的大脑皮层样本中甘氨酸水平图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例对9月龄AD模型小鼠(APP/PS1转基因小鼠，由南京大学模式动物研究所提供)与野生型(WT)小鼠的粪便样本进行代谢物定性定量分析。
分别采集在相同条件下培养的10只AD模型小鼠和10只野生型小鼠的粪便，将野生型小鼠样本依次编号为FWT-1-1～FWT-1-10，将AD模型小鼠样本依次编号为FTG-1-1～FTG-1-10，采用超高效液相色谱-串联飞行时间质谱联用检测样本中的牛磺酸水平，具体方法包括：
(1)取粪便样本加入预冷甲醇/乙腈/水溶液(体积比为2:2:1)，涡旋混合，低温超声30min，置于-20℃静置10min，于4℃、14000×g离心20min，取上 清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水＝1:1)中复溶，涡旋，于4℃、14000×g离心15min，取上清液进样分析，采用Agilent 1290Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离，柱温25℃；流速0.5mL/min；进样量2μL；流动相组成包括：A相：乙酸铵和氨水混合的水溶液(乙酸铵和氨水终浓度均为25mM)，B相：乙腈；梯度洗脱程序如下：0～0.5min，95％B相；0.5～7min，B相从95％线性变化至65％；7～8min，B相从65％线性变化至40％；8～9min，B相维持在40％；9～9.1min，B相从40％线性变化至95％；9.1～12min，B相维持在95％；整个分析过程中样本置于4℃自动进样器中；
(3)采用AB Triple TOF 6600质谱仪对步骤(2)超高效液相色谱系分离后的样本进行样本一级、二级谱图的采集，ESI源条件如下：Ion Source Gas1(Gas1)：60，Ion Source Gas2(Gas2)：60，Curtain gas(CUR)：30，source temperature：600℃，IonSapary Voltage Floating(ISVF)±5500V(正负两种模式)；TOF MS scan m/z range：60-1000Da，product ion scan m/z range：25-1000 Da，TOF MS scan accumulation time 0.20s/spectra，product ion scan accumulation time 0.05s/spectra；二级质谱采用information dependent acquisition(IDA)获得，并且采用high sensitivity模式，Declustering potential(DP)：±60V(正负两种模式)，Collision Energy：35±15eV，IDA设置如下Exclude isotopes within 4 Da，Candidate ions to monitor per cycle：10，将采集到的Wiff格式的原始数据经ProteoWizard转换成mzXML格式，然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积，对XCMS提取得到的数据进行代谢物结构鉴定，并分析 样本中的牛磺酸水平，利用R语言工具(R package(ropls))进行变异倍数分析(Fold Change Analysis，FC Analysis)、主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)和T检验(Student's t-test)，结果如图1及表1所示，AD模型小鼠粪便样本中牛磺酸水平显著高于野生型小鼠，表明可将粪便中牛磺酸作为阿尔兹海默症生物标志物，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊断。
表1
  代谢物 VIP 差异表达倍数 pvalue 显著性
AD模型小鼠vs野生型小鼠 牛磺酸 10.18 8.51 0.013 *
注：*为p<0.05。
此外，进一步通过KEGG通路注释与分析发现牛磺酸属于Membrane transport pathway中的ABC transporters途径，ABC transporters(ABC转运蛋白)，即ATP结合盒式蛋白(ATP-binding cassette transporter，ABC)，是一类ATP驱动泵，由两个跨膜结构域及两个胞质侧ATP结合域组成。正常生理条件下，ABC转运蛋白是细菌质膜上糖、氨基酸、磷脂和肽的转运蛋白，是哺乳动物细胞质膜上磷脂、亲脂性药物、胆固醇和其他小分子的转运蛋白，其在肝、小肠和肾等器官细胞质膜分布丰富，能将天然毒物和代谢废物排除体外。
实施例2
本实施例对9月龄AD模型小鼠与野生型(WT)小鼠的大脑皮层样本进行代谢物定性定量分析。
分别采集在相同条件下培养的10只AD模型小鼠和10只野生型小鼠的大脑皮层样本，将野生型大脑皮层样本依次编号为CWT-1-1～CWT-1-10，将AD 模型小鼠大脑皮层样本依次编号为CTG-1-1～CTG-1-10，采用超高效液相色谱-串联飞行时间质谱联用仪进行代谢物定性定量分析，具体方法包括：
(1)将小鼠断颈处死后用手术剪剖开脑壳，暴露大脑，将大脑沿中间脑缝剖成两半，分别去掉小脑、脑干、丘脑、下皮层和海马体，剩下大脑皮层，收集左右两边完整的大脑皮层作为样本，加入预冷甲醇/乙腈/水溶液(体积比为2:2:1)，涡旋混合，低温超声30min，置于-20℃静置10min，于4℃、14000×g离心20min，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水＝1:1)中复溶，涡旋，于4℃、14000×g离心15min，取上清液进样分析，采用Agilent1290Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离，条件与实施例1相同；
(3)采用AB Triple TOF 6600质谱仪对步骤(2)超高效液相色谱系分离后的样本进行样本一级、二级谱图的采集，条件与实施例1相同，将采集到的Wiff格式的原始数据经ProteoWizard转换成mzXML格式，然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积，对XCMS提取得到的数据进行代谢物结构鉴定，随后进行单变量统计分析、多维统计分析、差异代谢物筛选、差异代谢物相关性分析和KEGG通路分析，其中，OPLS-DA模型得到的变量权重值(Variable Importance for the Projection，VIP)能够用于衡量各代谢物的表达模式对各组样本分类判别的影响强度和解释能力，挖掘具有生物学意义的差异分子，本实施例综合考虑VIP值和p-value来筛选显著性差异代谢物，结果如图2-图4以及表2所示，AD模型小鼠大脑皮层组织的甘露糖(D-Mannose)、肌醇(myo-Inositol)和甘氨酸(Glycine)水平显著高于野生型小鼠，且上述代 谢物均属于Membrane transport pathway中的ABC transporters途径，说明Membrane transport pathway中的ABC transporters途径与阿尔兹海默症存在关联性，而本发明发现牛磺酸也属于Membrane transport pathway中的ABC transporters途径，从另一层面表明粪便代谢物中牛磺酸水平变化可能反映AD脑中相关代谢通路的异常，对临床早期诊断具有重要意义。
表2
  代谢物 VIP 差异表达倍数 pvalue 显著性
AD模型小鼠vs野生型小鼠 甘露糖 2.67 1.55 0.002 **
AD模型小鼠vs野生型小鼠 肌醇 10.80 1.10 0.026 *
AD模型小鼠vs野生型小鼠 甘氨酸 1.93 1.13 0.045 *
注：*为p<0.05，**为p<0.01。
综上所述，本发明首次检测到阿尔兹海默症粪便样本中牛磺酸水平显著高于正常粪便样本，将粪便中牛磺酸作为阿尔兹海默症生物标志物，并提供阿尔兹海默症早期诊断模型和装置，通过检测粪便中牛磺酸水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种阿尔兹海默症生物标志物，其特征在于，所述阿尔兹海默症生物标志物为牛磺酸。
  2. 如权利要求1所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
  3. 一种阿尔兹海默症早期诊断模型，其特征在于，所述阿尔兹海默症早期诊断模型的输入变量包括权利要求1所述的牛磺酸质谱的峰强度值。
  4. 根据权利要求3所述的阿尔兹海默症早期诊断模型，其特征在于，所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数，所述差异表达倍数的计算公式如方程式(1)所示：
    Figure PCTCN2021138089-appb-100001
  5. 根据权利要求4所述的阿尔兹海默症早期诊断模型，其特征在于，阿尔兹海默症阳性的判断标准为所述差异表达倍数≥8.51。
  6. 一种阿尔兹海默症早期诊断装置，其特征在于，所述装置包括如下单元：
    样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行数据处理，测定样本中权利要求1所述的牛磺酸质谱的峰强度值；
    分析单元：将检测到的牛磺酸质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析；
    评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  7. 根据权利要求6所述的装置，其特征在于，所述待测样本包括粪便样本。
  8. 根据权利要求6或7所述的装置，其特征在于，所述数据处理包括：
    利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中权利要求1所述的牛磺酸质谱的峰强度值。
  9. 根据权利要求6-8任一项所述的装置，其特征在于，所述装置包括如下单元：
    样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中权利要求1所述的牛磺酸质谱的峰强度值；
    分析单元：将检测到的牛磺酸质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析；
    评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  10. 权利要求1所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
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