WO2023272576A1 - 一种阿尔茨海默症的标记物及其应用 - Google Patents

一种阿尔茨海默症的标记物及其应用 Download PDF

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WO2023272576A1
WO2023272576A1 PCT/CN2021/103482 CN2021103482W WO2023272576A1 WO 2023272576 A1 WO2023272576 A1 WO 2023272576A1 CN 2021103482 W CN2021103482 W CN 2021103482W WO 2023272576 A1 WO2023272576 A1 WO 2023272576A1
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disease
alzheimer
mcp1
kit
detecting
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PCT/CN2021/103482
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French (fr)
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陈宇
屈雪琪
毛梦
林力
叶涛
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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

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  • the application belongs to the technical field of molecular detection, and in particular relates to a marker of Alzheimer's disease and its application.
  • AD Alzheimer's disease
  • a ⁇ oligomeric ⁇ -amyloid
  • NFT neurofibrillary tangles
  • AD The etiology of AD is complex, and its pathogenesis is still unclear.
  • pathogenic hypotheses include: A ⁇ aggregation hypothesis, Tau protein abnormality hypothesis, cholinergic hypothesis, neuroinflammation hypothesis, etc. It is worth noting that regardless of the pathogenic hypothesis, the brains of AD patients are accompanied by inflammatory reactions, and studies have shown that neuroinflammation begins before the formation of A ⁇ plaques and NFTs. Due to the complex etiology of AD, unclear pathogenesis, and slow progress in the research of its therapeutic drugs, there is still a lack of drugs or methods to cure AD in the world.
  • AD Alzheimer's disease
  • diagnosis method of AD is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment test, brain senile plaque and Tau protein PET scan, brain magnetic resonance (MRI) and cerebrospinal fluid (CSF) marker detection, ⁇ -amyloid protein detection and phosphorylated Tau protein detection, etc.
  • MRI brain magnetic resonance
  • CSF cerebrospinal fluid
  • This application provides a marker for Alzheimer's disease and its application.
  • chemokine MCP1 By detecting the expression level of chemokine MCP1 in peripheral blood, the level of inflammation in the brain is evaluated, and then assists in the early screening of AD.
  • the present application provides a marker of Alzheimer's disease, and the marker of Alzheimer's disease includes chemokine MCP1.
  • MCP1 is monocyte chemotactic protein 1 (monocyte chemotactic protein 1, MCP1), which has chemotactic activity on monocytes, can activate monocytes and macrophages to release cytokines, and can be used to evaluate The level of inflammation in the body.
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • MCP1 monocyte chemotactic protein 1
  • the present application provides a detection method for Alzheimer's disease for the purpose of non-disease diagnosis and/or treatment, and the detection method includes detection of MCP1 in a sample.
  • the detection of MCP1 in the sample can be used for the early screening of AD, the sample is convenient, the operation is simple, the damage to the sample is small, the risk is low, and it has clinical guiding significance.
  • said sample comprises cerebrospinal fluid and/or serum.
  • the detection of MCP1 in the sample includes detecting the expression level of MCP1 and/or determining the localization of MCP1.
  • the method for detecting the expression level of MCP1 includes real-time fluorescent quantitative PCR detection and/or ELISA detection.
  • the method for determining the localization of MCP1 comprises immunofluorescence staining.
  • the detection method also includes any one or at least two of the activity of regulatory T cells, the activity of helper T cells, the aggregation level of A ⁇ plaques, the expression level of inflammatory factors or the expression level of cytokines in the sample.
  • the steps of detecting the combination of species are any one or at least two of the activity of regulatory T cells, the activity of helper T cells, the aggregation level of A ⁇ plaques, the expression level of inflammatory factors or the expression level of cytokines in the sample.
  • the present application provides a kit for detecting Alzheimer's disease, the kit detects the marker of Alzheimer's disease described in the first aspect.
  • the kit includes reagents for detecting the markers of Alzheimer's disease described in the first aspect.
  • the kit includes any one of an immunofluorescent staining kit, a real-time fluorescent quantitative PCR kit or an ELISA kit.
  • the detection result is more accurate and the operation is easier by using the corresponding kit together, which promotes the popularization of related technologies and methods.
  • the immunofluorescence staining kit includes any one or a combination of at least two of fixative, dehydrating agent, embedding agent, washing solution, blocking agent, primary antibody, secondary antibody or mounting agent.
  • the immunofluorescent staining kit includes a fixative, a dehydrating agent, an embedding agent, a washing solution, a blocking agent, a primary antibody, a secondary antibody and a mounting agent.
  • the real-time fluorescent quantitative PCR kit includes any one or a combination of at least two of RNA extraction reagents, reverse transcriptase, amplification enzyme, buffer, primers, RNase inhibitors, dNTPs or fluorescent dyes .
  • the real-time fluorescent quantitative PCR kit includes RNA extraction reagents, reverse transcriptase, amplification enzyme, buffer, primers, RNase inhibitors, dNTPs and fluorescent dyes.
  • the ELISA kit includes any one or a combination of at least two of MCP1 conjugates, luminescent reagents, positive controls, standards, diluents, washing solutions or stop solutions.
  • the ELISA kit includes MCP1 conjugates, luminescent reagents, positive controls, standards, diluents, washing solutions and stop solutions.
  • the present application provides the Alzheimer's disease marker described in the first aspect, the Alzheimer's disease detection method for the purpose of non-disease diagnosis and/or treatment described in the second aspect, or the Alzheimer's disease marker described in the second aspect.
  • This application selects the chemokine MCP1 as a marker of AD, which can directly detect peripheral blood samples to determine the level of inflammation in the body. It is used in the early screening of AD, and the operation is simple and the risk is low; with the corresponding kit, The efficiency of the detection is improved, the accuracy of the result is increased, and it has practical application value. It can be used to guide clinical screening, research on AD pathogenesis and screening of AD therapeutic drugs, and has broad application prospects.
  • Figure 2A is a statistical diagram of the proportion of different cells in the door in the flow analysis of PBMCs of 3-month-old wild-type mice and APP/PS1 mice in Example 2 of the present application;
  • Fig. 2B is a statistical diagram of the proportion of different cells in the door in the flow cytometry analysis of wild-type mice and APP/PS1 mice PBMCs in Example 2 of the present application;
  • 4A is a picture of the results of MCP1 expression levels in the cerebral cortex and hippocampus of 3-month-old wild-type mice and APP/PS1 mice in Example 4 of the present application;
  • Figure 4B is a picture of the results of the expression levels of MCP1 in the cerebral cortex and hippocampus of 6-month-old wild-type mice and APP/PS1 mice in Example 4 of the present application;
  • Figure 5A is a picture of the results of changes in the expression of MCP1 in the cerebrospinal fluid of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice in Example 5 of the present application;
  • 5B is a picture of the results of the changes in the expression of MCP1 in the peripheral blood of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice in Example 5 of the present application.
  • Wild-type mice and APP/PS1 mouse models were from Jackson Laboratory, USA;
  • Paraformaldehyde was purchased from Sigma-aldrich, item number: 158127;
  • the embedding agent OCT was purchased from SAKURA, item number: 4583;
  • the primary antibody of Iba1 was purchased from Wake Company, Cat. No.: 019-19741;
  • CD8 primary antibody was purchased from Invitrogen, catalog number: 14-0195-82;
  • a ⁇ primary antibody was purchased from Biolegend, Cat. No.: 800717;
  • MCP1 primary antibody was purchased from Abcam, Cat. No.: ab7202;
  • Fluorescent secondary antibodies were purchased from Thermo scientific;
  • Red blood cell lysate was purchased from BD Biosciences, product number: 555899;
  • Horse serum was purchased from Gibco, product number: 26050088;
  • Fetal bovine serum was purchased from Life Technologies, item number: 16050-122;
  • DAPI was purchased from Thermo scientific company, item number: D1306;
  • DPBS was purchased from Sigma Company, article number: D8662-24*500ML;
  • Trizol was purchased from Invitrogen Company, article number: 15596026;
  • the reverse transcription kit was purchased from Thermo scientific, Cat. No. K1622;
  • Real-time fluorescence quantitative PCR kit was purchased from Thermo scientific, catalog number 4368706;
  • the ELISA kit was purchased from R&D, Cat. No.: MJE00B.
  • the immunofluorescence staining kit was used to stain the brain tissues of wild-type mice and APP/PS1 mouse models (AD mouse model) for MCP1 and A ⁇ , and the steps were as follows:
  • mice were anesthetized by intraperitoneal injection of chloral hydrate. After deep anesthesia, they were fixed on a surgical board and placed in a dissection tray.
  • mice perfuse mice with 4°C PBS, 20mL per mouse, and then use 4% paraformaldehyde at 4°C (weigh 40g paraformaldehyde and dissolve in a glass container containing 500mL DEPC water , continue to heat, stir magnetically to 60°C, and form a milky white suspension. Adjust the pH to 7.0 with 1.0mmol/L NaOH to make the solution clear, then add about 500mL 2 ⁇ PBS, mix well, filter and dilute to 1000mL, stored at 4°C for future use) perfusion, 20mL per mouse, until the tissue hardens.
  • the slice thickness is 20 ⁇ m.
  • the cut slices are collected continuously and transferred to a 24-well plate containing 4% paraformaldehyde.
  • Secondary antibody incubation and DAPI staining use PBS to dilute the secondary antibody at 1:500, and incubate at room temperature in the dark for 2 h; DAPI to dilute the stock solution at 1:5000, and incubate at room temperature for 15 min.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs cell suspension prepared in the above steps was adjusted to a cell density of 5 ⁇ 10 6 cells/mL with DPBS containing 2% fetal bovine serum.
  • Treg regulatory T cells
  • Th helper T cells
  • NK natural killer cells
  • the activity of Treg cells in 3-month-old mice was significantly inhibited, and with the development of the disease, the activity of Treg increased significantly in the peripheral blood of 6-month-old mice. It is suggested that in the early stage of AD, the body's immune response is activated, the activity of Treg is inhibited, and the immunosuppressive effect of Treg on the body is reduced, indicating that Treg cells in peripheral blood in the early stage of AD may inhibit neuroinflammation in the brain. With the aggravation of AD, the activity of Treg is significantly activated, its immunosuppressive effect on the body is enhanced, and the immune response ability of the body is reduced, which may aggravate neuroinflammation in the brain. In addition, Th cell activity was significantly reduced in 6-month-old mice as the course of AD progressed.
  • the brain tissues of 6-month-old wild-type mice and APP/PS1 mice were immunofluorescently stained, and CD8 + T cell markers Iba1, CD8, and A ⁇ were co-stained.
  • the experimental procedure was the same as in Example 1, and the results were as follows Figure 3 shows.
  • CD8 + T cells gather around A ⁇ plaques, which indicates that CD8 + T cells have chemotaxis to neuroinflammation sites in the brain and around A ⁇ plaques, revealing that CD8 + T cells mediate the effect of A ⁇ plaques in the brain The induced neuroinflammation has a regulatory effect.
  • reaction mixture II according to the following system, the system is as follows:
  • reaction mixture I Add the reaction mixture I to the reaction mixture II, mix rapidly for 5s, incubate at 70°C for 5 minutes, then ice-bath for 2 minutes, and perform reverse transcription according to the following procedure:
  • the obtained cDNA template was stored at -20°C for future use.
  • the expression level of MCP1 was detected using a real-time fluorescent quantitative PCR kit, and the reaction system was as follows:
  • sequence of the forward primer is shown in SEQ ID No.1
  • sequence of the reverse primer is shown in SEQ ID No.2.
  • SEQ ID No. 1 aggtgtcccaaagaagctgt;
  • SEQ ID No. 2 acagaagtgcttgaggtggt.
  • Cycle amplification 95°C, 15s; 60°C, 1min; 70°C, 1min; cycle 40 times;
  • the heating and cooling rate during the whole process was 1.6°C/s.
  • ELISA kits were used to detect the expression levels of MCP1 in the cerebrospinal fluid and peripheral blood of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice, and the steps were as follows:
  • mice were anesthetized with isoflurane gas, the blood samples of the mice were collected by fundus blood sampling into 1.5 mL sterilized EP tubes, and the necks were cut off quickly with scissors. Mice were killed by head, and the serum was separated as follows:
  • When collecting cerebrospinal fluid wipe the skin on the back of the neck with wet gauze, cut off the back hair, expose the skin and disinfect it, make a longitudinal incision (about 1 cm) along the longitudinal axis with a scalpel, and bluntly separate the back of the neck with scissors muscle.
  • the deepest bone-attached muscle was scraped away with a scalpel to expose the atlanto-occipital membrane.
  • the cerebrospinal fluid is aspirated directly through the foramen magnum. After extraction, the outer muscle and skin are sutured. Sulfa powder can be sprinkled on the incision to prevent infection.
  • an equal amount of sterile saline should be injected to maintain the original pressure of the cerebrospinal cavity.
  • Positive control preparation Dissolve the MCP1 positive control in 1 mL of deionized water, mix thoroughly, and set aside.
  • MCP1 standard preparation Dilute the 5000pg/mL standard provided in the kit into a new EP tube with a calibration diluent at a ratio of 1:10 to a 500pg/mL standard, and then dilute to a concentration of 250pg /mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.81pg/mL standards.
  • the expression level of chemokine MCP1 in the brain of AD mice was significantly increased, and then caused neuroinflammation; the neuroinflammation mediated by it occurred before 3 months of age, and was accompanied by the body's immune system during the pathogenesis of AD.
  • the changes in the system exacerbate the occurrence of neuroinflammation, which indicates that MCP1 can be used as a marker in the early screening of AD; the detection results of MCP1 in peripheral blood are consistent with the detection results in cerebrospinal fluid, suggesting that it can be directly detected by peripheral blood.
  • the detection can be used for AD screening and intervention, and the operation is simpler, the risk is lower, and the application value is higher.
  • the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

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Abstract

本申请提供了一种阿尔茨海默症的标记物，其包括单核细胞趋化蛋白-1（MCP1）。本申请还提供了对阿尔茨海默症的标记物进行检测的方法和检测阿尔茨海默症的试剂盒，以及标记物、检测方法和试剂盒在筛选治疗阿尔茨海默症药物中的应用。

Description

一种阿尔茨海默症的标记物及其应用 技术领域
本申请属于分子检测技术领域，尤其涉及一种阿尔茨海默症的标记物及其应用。
背景技术
阿尔茨海默症(Alzheimer’s disease，AD)是和年龄相关的中枢神经退行性疾病，是老年痴呆中最常见的类型。在临床上，AD病人具有记忆减退、认知功能障碍、语言障碍、执行能力下降和人格方面的改变等表现。AD患者脑内典型的病理特征是寡聚β淀粉样蛋白(Aβ)斑块聚集、Tau蛋白过度磷酸化形成的神经元纤维缠结(NFT)和脑内广泛的炎症反应。
AD病因复杂，其发病机制至今尚不清楚。目前，被广泛接受的致病假说有：Aβ聚集假说、Tau蛋白异常假说、胆碱能假说、神经炎症假说等。值得关注的是，无论是那种致病假说，AD患者脑部均伴随炎症反应，且有研究表明，神经炎症始于Aβ斑块和NFT形成之前。由于AD病因复杂，发病机制不清楚，其治疗药物研究进展缓慢，全球仍缺乏治愈AD的药物或方法。
AD病程是一个不可逆的过程，因此，AD治疗的关键是早期诊断，在疾病早期对AD进行干预并延缓病程进展。但至今尚未有一种精准的方法可对AD进行早期预测或诊断。目前AD的诊断方法主要是联合诊断，主要包括：神经心理学评估、认知损伤测试、脑部老年斑块和Tau蛋白PET扫描、脑部核磁共振(MRI)和脑脊液(CSF)标志物检测、β淀粉样蛋白检测以及磷酸化Tau蛋白检测等。然而，这些技术或花费昂贵，或具有侵入性感染风险，将这些方法应用于疾病常规筛查中不切实际。
因此，如何提供一种操作简便、成本较低、更为安全的AD早期筛查方法，已成为亟待解决的问题。
发明内容
本申请提供了一种阿尔茨海默症的标记物及其应用，通过对外周血中趋化因子MCP1的表达水平进行检测，评估脑内炎症水平，进而辅助AD的早期筛 查。
第一方面，本申请提供了一种阿尔茨海默症的标记物，所述阿尔茨海默症的标记物包括趋化因子MCP1。
本申请中，MCP1为单核细胞趋化蛋白-1(monocyte chemotactic protein 1，MCP1)，其对单核细胞具有趋化活性，可以激活单核细胞和巨噬细胞释放细胞因子，可以用来评价机体的炎症水平。研究显示AD患者体内的炎症水平显著提高，这表明外周免疫系统对AD的发病可能有重要作用。因此，可以将MCP1的表达水平作为AD的筛查标记物。以其作为标记物，可以直接对外周血进行检测，无需配合专业的仪器，操作简单，成本较低，更为安全，具有实际应用的意义。
第二方面，本申请提供了一种以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，所述检测方法包括对样本的MCP1进行检测。
本申请中，通过对样本中MCP1进行检测，可用于AD的早期筛查中，取样方便，操作简单，对样本的损害较小，风险较低，具有临床指导意义。
优选地，所述样本包括脑脊液和/或血清。
优选地，所述对样本的MCP1进行检测包括检测MCP1的表达水平和/或确定MCP1的定位。
优选地，所述检测MCP1的表达水平的方法包括实时荧光定量PCR检测和/或ELISA检测。
优选地，所述确定MCP1的定位的方法包括免疫荧光染色。
优选地，所述检测方法还包括对样本的调节性T细胞的活性、辅助性T细胞的活性、Aβ斑块的聚集水平、炎症因子表达水平或细胞因子表达水平中的任意一种或至少两种的组合进行检测的步骤。
第三方面，本申请提供了一种检测阿尔茨海默症的试剂盒，所述试剂盒对第一方面所述的阿尔茨海默症的标记物进行检测。所述试剂盒包括用于检测第一方面所述的阿尔茨海默症的标记物的剂。
所述试剂盒包括免疫荧光染色试剂盒、实时荧光定量PCR试剂盒或ELISA试剂盒中的任意一种。
本申请中，配合使用相应的试剂盒，检测结果更加准确，操作也更为简便，促进了相关技术与方法的推广。
优选地，所述免疫荧光染色试剂盒包括固定剂、脱水剂、包埋剂、清洗液、封闭剂、一抗、二抗或封片剂中的任意一种或至少两种的组合。
优选地，所述免疫荧光染色试剂盒包括固定剂、脱水剂、包埋剂、清洗液、封闭剂、一抗、二抗和封片剂。
优选地，所述实时荧光定量PCR试剂盒包括RNA提取试剂、反转录酶、扩增酶、缓冲液、引物、RNA酶抑制剂、dNTPs或荧光染料中的任意一种或至少两种的组合。
优选地，所述实时荧光定量PCR试剂盒包括RNA提取试剂、反转录酶、扩增酶、缓冲液、引物、RNA酶抑制剂、dNTPs和荧光染料。
优选地，所述ELISA试剂盒包括MCP1偶联物、发光试剂、阳性对照、标准品、稀释剂、洗涤液或终止液中的任意一种或至少两种的组合。
优选地，所述ELISA试剂盒包括MCP1偶联物、发光试剂、阳性对照、标准品、稀释剂、洗涤液和终止液。
第四方面，本申请提供了第一方面所述的阿尔茨海默症的标记物、第二方面所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法或第三方面所述的检测阿尔茨海默症的试剂盒中的任意一种或至少两种的组合在筛选阿尔茨海默症治疗药物中的应用。
相比于现有技术，本申请具有如下有益效果：
本申请选用趋化因子MCP1作为AD的标记物，可以直接对外周血样本进行检测，判断机体的炎症水平，用于AD的早期筛查中，操作简单，风险较低；配合相应的试剂盒，提高了检测的效率，增加了结果的准确性，具有实际应用的价值，可用于指导临床筛查、AD致病机理的研究以及AD治疗药物的筛选中，具有广阔的应用前景。
附图说明
图1为本申请实施例1中6月龄、9月龄和12月龄的野生型小鼠和APP/PS1小鼠的脑组织免疫荧光染色的结果图片(比例尺＝50μm)；
图2A为本申请实施例2中3月龄的野生型小鼠和APP/PS1小鼠PBMCs流式分析中门内不同细胞占比的统计图；
图2B为本申请实施例2中6月龄的野生型小鼠和APP/PS1小鼠PBMCs流 式分析中门内不同细胞占比的统计图；
图3为本申请实施例3中6月龄野生型小鼠和APP/PS1小鼠的脑组织免疫荧光染色的结果图片(比例尺＝50μm)；
图4A为本申请实施例4中3月龄野生型小鼠和APP/PS1小鼠大脑皮层和海马体内的MCP1的表达水平的结果图片；
图4B为本申请实施例4中6月龄野生型小鼠和APP/PS1小鼠大脑皮层和海马体内的MCP1的表达水平的结果图片；
图5A为本申请实施例5中3月龄和6月龄野生型小鼠和APP/PS1小鼠脑脊液中MCP1表达量变化的结果图片；
图5B为本申请实施例5中3月龄和6月龄野生型小鼠和APP/PS1小鼠外周血中MCP1表达量变化的结果图片。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。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申请，而非对本申请的限定。
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。
材料：
野生型小鼠和APP/PS1小鼠模型来自美国Jackson实验室；
多聚甲醛购自Sigma-aldrich，货号：158127；
包埋剂OCT购自SAKURA，货号：4583；
Iba1一抗购自Wake公司，货号：019-19741；
CD8一抗购自Invitrogen，货号：14-0195-82；
Aβ一抗购自Biolegend公司，货号：800717；
MCP1一抗购自Abcam公司，货号：ab7202；
荧光二抗购自Thermo scientific；
红细胞裂解液购自BD Biosciences公司，货号：555899；
流式分析使用的抗体均购自BD Biosciences公司，货号依次为：
Ms CD45 FITC 30-F11，货号：553079；
Ms CD3 MolCpx PerCP-Cy5.5 17A2，货号：560527；
Ms CD4 APC-H7 GK1.5，货号：560181；
Ms CD8a PE 53-6.7，货号：553032；
Ms CD19 PE-Cy7 1D3，货号：552854；
Ms CD49b APC DX5，货号：560628；
马血清购自Gibco公司，货号：26050088；
胎牛血清购自Life Technologies公司，货号：16050-122；
DAPI购自Thermo scientific公司，货号：D1306；
DPBS购自Sigma公司，货号：D8662-24*500ML；
Trizol购自invitrogen公司，货号：15596026；
反转录试剂盒购自Thermo scientific，货号K1622；
实时荧光定量PCR试剂盒购自Thermo scientific，货号4368706；
ELISA试剂盒购自R&D，货号：MJE00B。
实施例1
本实施例使用免疫荧光染色试剂盒，对野生型小鼠和APP/PS1小鼠模型(AD小鼠模型)的脑组织进行MCP1和Aβ染色，步骤如下：
1、小鼠脑组织切片
(1)麻醉及脑组织灌注取样：向小鼠腹腔注射水合氯醛麻醉，深度麻醉后，固定在手术木板上，置于解剖盘中，后端头取脑，并用多聚甲醛浸泡固定24h。
(2)小鼠的灌注固定：使用4℃的PBS灌注小鼠，每只20mL，后使用4℃的4％多聚甲醛(称取40g多聚甲醛溶于装有500mL DEPC水的玻璃容器中，持续加热，磁力搅拌至60℃，形成乳白色悬液。用1.0mmol/L的NaOH调节pH为7.0，使溶液呈清亮状，再加入约500mL 2×PBS，充分混匀，过滤后定容至1000mL，4℃保存备用)灌注，每只小鼠20mL，至组织变硬。
(3)取材：小心剥离脑组织，置于15mL离心管中，用4％多聚甲醛(固定剂)后固定24h。
(4)脱水：将多聚甲醛固定好的组织用PBS(清洗液)洗3次，20％蔗糖(脱水剂)脱水至组织沉底，30％蔗糖4℃下脱水过夜。
(5)将包埋剂OCT滴加到标本台，放入恒冷箱切片机内至变白，然后取 出，迅速将表面用单面刀片修平。
(6)用安全刀片将标本底部修平后粘附于标本台，然后置入-24℃恒冷箱切片机的冷冻台中，待组织略微发白时用OCT在标本表面涂一薄层，继续冷冻20min。
(7)调好切片厚度后开始切片，切片厚度20μm，切好的片子连续收集，移入含4％多聚甲醛的24孔板。
(8)将切好的片子放4℃保存备用。
2、免疫荧光染色
(1)小心挑出合适位置的切片到预先放有1mL预冷PBS的24孔板中，用预冷PBS洗3次，每次10min。
(2)打孔及封闭：0.2％Triton X-100(PBS稀释)、0.1％BSA及5％马血清(PBS稀释)，室温孵育40min，放置摇床上慢速摇晃。
(3)PBS室温下洗3次，每次5min。
(4)一抗孵育：使用抗体稀释液(PBS中含0.01％BSA和5％马血清)按1:100进行稀释，每孔中加入200μL，4℃慢速摇晃孵育过夜。
(5)回收一抗，PBS室温下洗3次，每次10min。
(6)3％马血清室温下封闭脑片30min。
(7)二抗孵育及DAPI染色：使用PBS按1:500稀释二抗，室温避光孵育2h；DAPI按1:5000稀释储存液，室温孵育15min。
(8)用PBS室温下洗3次，每次15min。
(9)封片：取粘性载玻片，在右侧磨砂面用铅笔标记具体信息，在载玻片中间滴一滴PBS，沾取切片放于PBS液滴上，吸除PBS溶液，在载玻片中央横铺160μL封片剂，用长条盖玻片盖住切片，避免气泡以及褶皱产生。
(10)平放于避光处晾干。
6月龄(6M)、9月龄(6M)和12月龄(12M)的野生型小鼠和APP/PS1小鼠的染色图片如图1所示。由图可知，在6月龄AD小鼠皮层和海马部位有MCP1出现，随着病情发展，9月龄和12月龄AD小鼠脑组织中MCP1表达增多，Aβ斑块数目增多增大，且成聚集状态，而3月龄小鼠没有出现这一现象。这一结果表明，在AD小鼠模型中，MCP1介导的神经炎症发生在3月龄和6月龄之间或者更早阶段。这说明可将MCP1作为AD的早期标记物。
实施例2
本实施例对6月龄和9月龄的野生型小鼠和APP/PS1小鼠的外周血单个核细胞(Peripheral blood mononuclear cells，PBMCs)进行流式细胞分析，步骤如下：
1、高活性的外周血单个核细胞(PBMCs)制备
(1)在200μL抗凝全血中加入3倍体积的红细胞裂解液轻轻混匀，室温静止10min，其间轻混匀2次，裂解红细胞。
(2)在800×g下离心2min，弃上清，收集细胞，用1mL PBS清洗样品1次。
(3)使用500μL缓冲液重悬，300目细胞滤网过滤后，孵育抗体，后上机分析。
2、流式分析
(1)将上述步骤制备的PBMCs细胞悬液用含有2％胎牛血清的DPBS调整细胞密度为5×10 6个/mL。
(2)取40μL细胞悬液加入预先装有50μL荧光标记的特异性抗体的塑料离心管中，再加入50μL灭活正常马血清(按1:20用DPBS稀释)，4℃下孵育30min。
(3)加入2mL含有2％胎牛血清的DPBS充悬混匀细胞洗涤，1000rpm在4℃下离心5min，重复洗涤细胞1次。
(4)加入500μL预冷PBS重悬细胞，准备上机分析。
对不同细胞门中调节性T细胞(Treg)、辅助性T细胞(Th)、B细胞和自然杀伤细胞(NK)的占比分别进行统计，统计结果如图2A和图2B所示。
由图可知，3月龄小鼠Treg细胞活性显著被抑制，随着病程发展，Treg的活性在6月龄的小鼠外周血中显著增加。提示在AD发病早期，机体免疫应答反应被激活，Treg活性被抑制，Treg对机体的免疫抑制作用降低，表明AD早期外周血中Treg细胞可能对脑内神经炎症起抑制作用。随着AD病程加剧，Treg活性被显著激活，其对机体的免疫抑制作用增强，机体的免疫应答能力下降，可能会加剧脑内神经炎症。另外，随着AD病程进展，在6月龄小鼠中Th细胞活性显著降低。
实施例3
本实施例对6月龄野生型小鼠和APP/PS1小鼠的脑组织进行免疫荧光染色，将CD8 +T细胞标记物Iba1、CD8与Aβ共染色，实验步骤与实施例1相同，结果如图3所示。
由图可知，CD8 +T细胞聚集在Aβ斑块周围，这说明CD8 +T细胞向脑内神经炎症部位和Aβ斑块周围进行了趋化，揭示了CD8 +T细胞对脑内Aβ斑块介导的神经炎症具有调控作用。
实施例4
本实施例使用实时荧光定量PCR试剂盒，分别对3月龄和6月龄野生型小鼠和APP/PS1小鼠的大脑皮层(cortex)和海马体(hippocampus)内的MCP1的表达水平进行检测，步骤如下：
1、RNA提取
(1)将3月龄及6月龄同窝野生型和AD小鼠用异氟烷(气体)麻醉后，断颈后用剪刀迅速断头处死，将头放于冰上，迅速分离大脑皮质并分离小鼠的大脑皮层以及海马体。将分离到的组织在含有4U/mL的蛋白酶抑制剂和RNA酶抑制剂的DPBS洗涤两次。
(2)匀浆处理：将组织或细胞在液氮中磨碎，每100mg组织加入1mL Trizol(RNA提取试剂)，用匀浆仪进行匀浆处理。
(3)将匀浆样品在室温放置5min，使核酸蛋白复合物完全分离。
(4)每使用1mL Trizol加入0.2mL氯仿，剧烈振荡15s，室温放置3min。
(5)4℃下10000×g离心15min。
(6)把水相转移到新管中，用异丙醇沉淀水相中的RNA，每使用1mL Trizol加入0.5mL异丙醇，室温放置10min。
(7)4℃下10000×g离心10min，在管侧和管底出现胶状沉淀，弃上清。
(8)用75％乙醇洗涤RNA沉淀，每使用1mL Trizol加1mL 75％乙醇，4℃下7500×g离心5min，弃上清。
(9)室温放置干燥5min，加入50μL无RNase的水，用枪头吸打几次，55℃放置10min使RNA溶解，-70℃保存。
2、反转录
使用反转录试剂盒将提取的总RNA反转录成cDNA，步骤如下：
在无RNA酶的离心管中制备反应混合物I，体系如下：
Figure PCTCN2021103482-appb-000001
将上述组分混合后快速离心5s，在70℃孵育5min后，冰浴2min，再按下述体系制备反应混合物II，体系如下：
Figure PCTCN2021103482-appb-000002
将反应混合物I加入到反应混合物II中，快速混合5s，在70℃孵育5min后，冰浴2min，按一下程序进行反转录：
25℃，5min；42℃，60min；70℃，5min。
得到的cDNA模板置于-20℃保存备用。
3、实时荧光定量PCR
使用实时荧光定量PCR试剂盒对MCP1的表达水平进行检测，反应体系如下：
Figure PCTCN2021103482-appb-000003
其中，正向引物的序列如SEQ ID No.1所示，反向引物的序列如SEQ ID No.2所示。
SEQ ID No.1：aggtgtcccaaagaagctgt；
SEQ ID No.2：acagaagtgcttgaggtggt。
将上述组分混合，6000rpm离心1min，按以下程序进行扩增：
预变性：95℃，10min；
循环扩增：95℃，15s；60℃，1min；70℃，1min；循环40次；
形成熔解曲线：95℃，15s；60℃，1min；
整个过程中升温和降温的速率为1.6℃/s。
检测结果如图4A和图4B所示。
由图可知，在3月龄和6月龄AD小鼠中，同一组中野生型小鼠个体之间细胞因子表达的变化相对比较集中，而AD小鼠中，同一组不同个体之间各个细胞因子表达变化差异比较大，且MCP1在3月龄和6月龄AD模型小鼠的大脑皮层和海马区基因表达水平显著升高。
实施例5
本实施例使用ELISA试剂盒，分别对3月龄和6月龄野生型小鼠和APP/PS1小鼠的脑脊液和外周血中的MCP1的表达水平进行检测，步骤如下：
1、样本采集
(1)血清样品采集：
将3月龄及6月龄同窝野生型和AD小鼠用异氟烷气体麻醉后，眼底采血法收集小鼠的血液样本至1.5mL的灭菌EP管中，断颈后并用剪刀迅速断头处死小鼠，并按照下述方法分离血清：
将抗凝血样品置于4℃静置4h，待血液凝固后自然析出血清，在4℃下4000rpm离心30min，分离血清，弃去不溶物；
将血清移至新的灭菌EP管，分装后-80℃储存备用。
(2)脑脊液样品采集：
小鼠麻醉后，将头部固定于定向仪上。采集脑脊液时，用湿纱布擦试大鼠颈背部皮肤，剪去背毛，暴露皮肤并消毒，用手术刀沿纵轴切一纵行切口(约1cm)，用剪刀钝性分离颈部背侧肌肉。为避免出血，最深层附着在骨上的肌肉用手术刀背刮开，暴露寰枕膜。由枕骨大孔进针直接抽取脑脊液。抽取完毕后缝合外层肌肉、皮肤。刀口处可撒涂磺胺药粉，防止感染。采完脑脊液后，应注入等量的消毒生理盐水，以保持原来脑脊髓腔的压力。
2、试剂配置
(1)阳性对照配制：用1mL去离子水溶解MCP1阳性对照，充分混匀，备用。
(2)洗涤液配制：，用去离子水按1:25稀释至工作浓度。
(3)发光试剂配制：在上机检测前15min，将试剂盒中的发光试剂A和试剂B按照1:1体积混匀，避光保存。
(4)MCP1标准品配制：将试剂盒中提供的5000pg/mL的标准品在新的EP管中用校准稀释剂按照1:10稀释成500pg/mL的标准品，再稀释成浓度分别为250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL和7.81pg/mL的标准品。
3、ELISA检测
(1)向测试孔中加入50μL测定稀释剂RD1W。
(2)向测试孔中依次加入标准品、对照样品和待测样品，用试剂盒中提供的封口膜封口后室温孵育2h。
(3)孵育结束后，撕下封口膜，弃去液体，每孔中加入400μL洗涤液洗涤检测板，洗涤4次，弃去洗涤液。
(4)向测试孔中加入100μL小鼠MCP1偶联物，封口膜封口后，室温孵育2h。
(5)重复步骤(3)1次。
(6)向测试孔中加入100μL配制的发光试剂，避光室温孵育39min。
(7)加入100μL终止液，轻弹混匀测试板确保充分混匀。
(8)读板：在30min内完成每个孔的光密度检测。
(9)计算：根据试剂盒提供的公式进行浓度定量计算。
检测结果如图5A和图5B所示。
由图5A可知，趋化因子MCP1的表达量在3月龄AD小鼠的脑脊液中显著升高；由图5B可知，MCP1在3月龄和6月龄AD小鼠外周血中表达水平持续升高。趋化因子主要发挥促炎作用，在免疫应答过程中诱导免疫细胞进入感染部位，因此表达水平升高，提示在AD发展过程中，外周血趋化因子在发挥免疫监视的同时控制免疫细胞向神经炎症部位趋化。上述结果表明，外周血中炎性因子和脑内神经炎症的发生发展具有一定的相关性。
综上所述，趋化因子MCP1在AD小鼠的脑内表达量显著升高，进而引起神经炎症；其介导的神经炎症发生在3月龄之前，且在AD发病过程中伴随着机体免疫系统的变化，加剧了神经炎症的发生，这表明MCP1可作为标记物用于AD的早期筛查中；外周血中MCP1的检测结果与脑脊液中的检测结果一致，提示可直接通过对外周血进行检测从而进行AD的筛查与干预，操作更为简便，风险更低，应用价值更高。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请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，对本申请的任何改进，对本申请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，均落在本申请的保护范围和公开范围之内。

Claims (12)

  1. 一种阿尔茨海默症的标记物，其包括趋化因子MCP1。
  2. 一种以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其包括对样本的MCP1进行检测。
  3. 根据权利要求2所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其中，所述样本包括脑脊液和/或血清。
  4. 根据权利要求2或3所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其中，所述对样本的MCP1进行检测包括检测MCP1的表达水平和/或确定MCP1的定位。
  5. 根据权利要求4所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其中，所述检测MCP1的表达水平的方法包括实时荧光定量PCR检测和/或ELISA检测。
  6. 根据权利要求4所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其中，所述确定MCP1的定位的方法包括免疫荧光染色。
  7. 根据权利要求2～6任一项所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法，其中，所述检测方法还包括对样本的调节性T细胞的活性、辅助性T细胞的活性、Aβ斑块的聚集水平、炎症因子表达水平或细胞因子表达水平中的任意一种或至少两种的组合进行检测的步骤。
  8. 一种检测阿尔茨海默症的试剂盒，其对权利要求1所述的阿尔茨海默症的标记物进行检测；
    其中，所述试剂盒包括免疫荧光染色试剂盒、实时荧光定量PCR试剂盒或ELISA试剂盒中的任意一种。
  9. 根据权利要求8所述的检测阿尔茨海默症的试剂盒，其中，所述免疫荧光染色试剂盒包括固定剂、脱水剂、包埋剂、清洗液、封闭剂、一抗、二抗或封片剂中的任意一种或至少两种的组合；
    任选地，所述免疫荧光染色试剂盒包括固定剂、脱水剂、包埋剂、清洗液、封闭剂、一抗、二抗和封片剂。
  10. 根据权利要求8或9所述的检测阿尔茨海默症的试剂盒，其中，所述实时荧光定量PCR试剂盒包括RNA提取试剂、反转录酶、扩增酶、缓冲液、引物、RNA酶抑制剂、dNTPs或荧光染料中的任意一种或至少两种的组合；
    任选地，所述实时荧光定量PCR试剂盒包括RNA提取试剂、反转录酶、 扩增酶、缓冲液、引物、RNA酶抑制剂、dNTPs和荧光染料。
  11. 根据权利要求8～10任一项所述的检测阿尔茨海默症的试剂盒，其中，所述ELISA试剂盒包括MCP1偶联物、发光试剂、阳性对照、标准品、稀释剂、洗涤液或终止液中的任意一种或至少两种的组合；
    任选地，所述ELISA试剂盒包括MCP1偶联物、发光试剂、阳性对照、标准品、稀释剂、洗涤液和终止液。
  12. 权利要求1所述的阿尔茨海默症的标记物、权利要求2～7任一项所述的以非疾病诊断和/或治疗为目的的阿尔茨海默症的检测方法或权利要求8～11任一项所述的检测阿尔茨海默症的试剂盒中的任意一种或至少两种的组合在筛选阿尔茨海默症治疗药物中的应用。
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