WO2023201511A1 - Biomarker for early diagnosis of alzheimer's disease and use thereof - Google Patents

Biomarker for early diagnosis of alzheimer's disease and use thereof Download PDF

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WO2023201511A1
WO2023201511A1 PCT/CN2022/087570 CN2022087570W WO2023201511A1 WO 2023201511 A1 WO2023201511 A1 WO 2023201511A1 CN 2022087570 W CN2022087570 W CN 2022087570W WO 2023201511 A1 WO2023201511 A1 WO 2023201511A1
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cells
peripheral blood
interleukin
neurodegenerative diseases
disease
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PCT/CN2022/087570
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French (fr)
Chinese (zh)
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陈宇
屈雪琪
毛梦
柴岳
陈静
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深圳先进技术研究院
中国科学院深圳理工大学(筹)
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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 specifically relates to a biomarker for early diagnosis of Alzheimer's disease and its use.
  • AD Alzheimer’s disease
  • a ⁇ oligomeric ⁇ -amyloid
  • NFTs neurofibrillary tangles
  • pathogenic hypotheses include: A ⁇ aggregation hypothesis, Tau protein abnormality hypothesis, cholinergic hypothesis, neuroinflammation hypothesis, etc. Due to the complex etiology of the disease, none of the above hypotheses can effectively elucidate its pathogenesis, and research on its treatment drugs is progressing slowly. Currently, there is still a lack of drugs or methods to cure AD in the world. Anti-A ⁇ immunotherapy has always been a research and development hotspot. However, due to the difficulty of drug development, most of the drugs currently entering Phase III clinical trials have ended in failure. Therefore, it is urgent to study the pathological mechanisms of AD from multiple angles and to find early biodetection markers.
  • AD Alzheimer's disease
  • the current diagnostic method for AD is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment test; PET scan of brain senile plaques and Tau protein; brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) markers, A ⁇ Deposition, phosphorylated tau protein detection, etc.
  • MRI brain magnetic resonance imaging
  • CSF cerebrospinal fluid
  • AD mild cognitive impairment
  • MCI mild cognitive impairment
  • early molecular screening technologies for AD include positron emission computed tomography (PET) and cerebrospinal fluid A ⁇ molecular level detection.
  • PET positron emission computed tomography
  • cerebrospinal fluid A ⁇ molecular level detection The former requires a certain dose of radioactive material to be injected into the subject; the latter requires a large amount of damage and is easy to cause. Cause surgical infection.
  • the reliability of these diagnostic technologies for early diagnosis of AD is also unstable, making it difficult to be used for early AD screening.
  • the prevention, diagnosis, treatment and rehabilitation of AD are currently recognized problems in the world. The development of new markers and simple detection methods for early diagnosis of AD is one of the important directions for the diagnosis and treatment of AD in the future.
  • the present invention aims to provide a biomarker for early diagnosis of Alzheimer's disease and its use.
  • Neuroinflammation is one of the main pathological features in the brain of AD patients, and the inflammatory process promotes the progression of A ⁇ pathology and Tau pathology. Therefore, neuroinflammation plays a key role in the occurrence and development of AD, and the treatment of neuroinflammation in the brain has become a new hot spot in the treatment of AD at this stage. Targeting this key molecule that regulates early neuroinflammation is regarded as a potential new therapeutic target for AD intervention.
  • a large number of studies have shown that circulating immune cells in the blood play a crucial role in maintaining central nervous system homeostasis and mediating immune responses to neurological diseases.
  • AD adaptive peripheral immune cell subsets
  • cytotoxic molecules a group of adaptive peripheral immune cell subsets are found in the brains of AD patients and in the hippocampus of AD mouse models. They can release inflammatory factors and cytotoxic molecules, promote inflammatory reactions in the brain, and lead to neuronal dysfunction. Increased pro-inflammatory cytokines, immune cell infiltration and glial cell activation are important neuroinflammatory responses in AD.
  • the present invention detects the correlation between the levels of inflammatory factors in peripheral blood, immune cell subpopulation changes and the severity of neuroinflammation in the brain at different stages of AD disease, thereby determining the changes in the expression levels of inflammatory factors in peripheral blood and the number of immune cell subpopulations. Change the directive role in the AD process.
  • a first aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as biomarkers for early diagnosis of neurodegenerative diseases.
  • the neurodegenerative disease is Alzheimer's disease.
  • a second aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells in the preparation of products for early diagnosis of neurodegenerative diseases.
  • the products include: products for detecting the expression level of interleukin-6 in peripheral blood to diagnose neurodegenerative diseases through enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR or RT-PCR; and/or through flow cytometry analysis or immunoassay A product that detects changes in the number of peripheral blood CD8 + T cells and/or peripheral blood CD4 + T cells using fluorescent staining to diagnose neurodegenerative diseases.
  • the neurodegenerative disease is Alzheimer's disease.
  • the third aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as drug targets for neurodegenerative diseases.
  • the neurodegenerative disease is Alzheimer's disease.
  • a fourth aspect of the present invention provides a method for early diagnosis of neurodegenerative diseases, which includes the following steps:
  • Detect the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood based on changes in the expression level of interleukin 6 and/or the number of CD8 + T cells in peripheral blood
  • To determine whether you are suffering from a neurodegenerative disease is based on the increase in the expression of interleukin 6 in peripheral blood and/or the severity of abnormalities in CD4 + T cells and/or CD8 + T cells. Determine the progression of neurodegenerative diseases;
  • the early diagnosis method also includes: detecting the expression level of interleukin 6 and/or the number of CD8 + T cells in the cerebrospinal fluid, and based on the changes in the expression level of interleukin 6 and/or the number of CD8 + T cells in the cerebrospinal fluid To determine whether you are suffering from a neurodegenerative disease, the progression of the neurodegenerative disease can be determined based on the increased expression level of interleukin 6 in the cerebrospinal fluid and/or the severity of CD8 + T cell abnormalities;
  • Neurodegeneration occurs when the expression level of interleukin-6 in the cerebrospinal fluid increases and/or the number of CD8 + T cells increases.
  • diagnosis is made based on changes in the expression level of interleukin-6 and the number of CD8 + T cells in peripheral blood;
  • diagnosis is made based on changes in the expression level of interleukin 6, changes in the number of CD8 + T cells, and changes in the number of CD4 + T cells in peripheral blood;
  • diagnosis is made based on changes in the expression level of interleukin-6 in peripheral blood, changes in the number of CD8 + T cells in peripheral blood, and changes in the number of CD8 + T cells in cerebrospinal fluid;
  • peripheral blood is obtained from humans or mice.
  • the neurodegenerative disease is Alzheimer's disease.
  • the above-mentioned use or the above-mentioned early diagnosis method is used in screening drugs for treating neurodegenerative diseases.
  • the drug for treating neurodegenerative diseases inhibits the expression of interleukin-6 in peripheral blood and/or reduces the number of CD8 + T cells and/or increases the number of CD4 + T cells in peripheral blood.
  • the neurodegenerative disease is Alzheimer's disease.
  • the sixth aspect of the present invention provides the above-mentioned use or the application of the above-mentioned early diagnosis method in screening new biomarkers or new drug targets for neurodegenerative diseases.
  • new biomarkers or new drug targets affect the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood.
  • the neurodegenerative disease is Alzheimer's disease.
  • the present invention achieves diagnosis of early AD based on changes in immune cells and immune molecules in peripheral circulation, and specifically provides inflammatory factor IL-6, peripheral circulation immune cell subgroups CD8 + T cells and helper CD4 + T cells as markers for early diagnosis of AD.
  • Cells can directly detect peripheral blood samples to determine the body's inflammation level and enable early screening of AD.
  • the advantage is that clinical diagnosis of the occurrence and development of AD by detecting biomarkers in blood not only reduces the diagnostic cost, but is also easy to operate, avoids the risk of central infection during brain sampling, is highly acceptable to patients, and is relatively easy to perform. Large-scale screening of diseases improves the efficiency of detection and increases the accuracy of results, and has practical application value.
  • the invention can be used to guide clinical screening, research on the pathogenesis of AD, screening of AD therapeutic drugs, and screening of new biomarkers and potential new drug targets for AD, and has broad application prospects.
  • FIG. 1 IL in peripheral blood of 3-month-old (3M), 6-month-old (6M), 9-month-old (9M) and 12-month-old (12M) wild-type mice (WT) and APP/PS1 mice (AD) -6 expression level changes (*p ⁇ 0.05).
  • A. The position of the brain slice taken in the brain atlas.
  • B. Shows the microglia surface marker Iba1, T cell surface marker CD8, and cell nuclear marker DAPI in brain tissue sections at different ages.
  • C Statistical results of brain infiltration of CD8+ T cells in WT and AD mice of different ages.
  • the present invention is mainly carried out in the APP/PS1 mouse model (hereinafter referred to as the AD mouse model).
  • the AD mouse model is one of the ideal animal models for simulating AD disease and is widely used in research on the pathogenesis of AD.
  • the present invention takes AD mouse models of different months of age as the research object, and uses brain tissue immunofluorescence staining technology, total RNA extraction technology, qRT-PCR technology, enzyme-linked immunosorbent assay (ELISA) of peripheral serum, etc. Comparison between the type (wild type littermate) and the diseased group (APP/PS1 transgenic mice, AD), the inflammatory factor IL-6 and cytotoxic CD8 + T cells and helper CD4 + T cells in peripheral blood were compared.
  • AD transgenic mice are related to the development of brain inflammation. Based on this, the present invention provides reliable evidence that changes in the expression level of the inflammatory factor IL-6 and changes in the number of cytotoxic CD8 + T cells and helper CD4 + T cells in peripheral blood can be used as molecular markers for early diagnosis of AD: 3-month-old AD
  • the mice correspond to the patient's disease course and are about to enter the mild intellectual impairment (MCI) stage.
  • MCI mild intellectual impairment
  • the 6-month-old AD mice have clear characteristics of AD symptoms.
  • mice and APP/PS1 mouse models were from Jackson Laboratory in the United States;
  • Paraformaldehyde was purchased from Sigma-aldrich, item number: 158127;
  • the embedding agent OCT was purchased from SAKURA, item number: 4583;
  • Iba1 primary antibody was purchased from Wake Company, product number: 019-19741;
  • CD8 primary antibody was purchased from Invitrogen, product number: 14-0195-82;
  • DAPI was purchased from Thermo Scientific Company, item number: 62248;
  • 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 Company, product number: 26050088;
  • Fetal bovine serum was purchased from Life Technologies, product number: 16050-122;
  • DAPI was purchased from Thermo Scientific Company, item number: D1306;
  • DPBS was purchased from Sigma Company, item number: D8662-24*500ML;
  • Trizol was purchased from Invitrogen Company, item number: 15596026;
  • the reverse transcription kit was purchased from Thermo scientific, product number K1622;
  • Real-time fluorescence quantitative PCR kit was purchased from Thermo scientific, catalog number 4368706;
  • the ELISA kit was purchased from R&D, product number: M6000B.
  • Example 1 Detection of the expression level of inflammatory factor IL-6 in peripheral blood
  • This example uses an ELISA kit to detect the expression levels of interleukin 6 (IL-6) in the peripheral blood of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice respectively.
  • the steps are 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. The mice were killed head-on, and the serum was isolated as follows:
  • washing liquid dilute 1:25 with deionized water to the working concentration.
  • IL-6 standard Dilute the 5000pg/mL standard provided in the kit with calibration diluent 1:10 in a new EP tube to a 500pg/mL standard, and then dilute to the respective concentrations. Standards for 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.81pg/mL.
  • PBMCs peripheral blood mononuclear cells
  • This example performs flow cytometry analysis on peripheral blood mononuclear cells (PBMCs) of 6-month-old and 9-month-old wild-type mice and APP/PS1 mice. The steps are as follows:
  • PBMCs peripheral blood mononuclear cells
  • this example selected mice at the early stage of AD: 3 months old (3M) and 6 months old (6M) to conduct PBMCs T lymphocyte subpopulation flow cytometry. Analysis, the results are shown in Figure 2.
  • Flow cytometry analysis showed that compared with WT mice, in the peripheral blood of 6-month-old AD mice, the expression of helper T cells (CD4 + T cells) was significantly reduced, and the expression of cytotoxic T cells (CD8 + T cells) was significantly reduced. Increased levels indicate that immune cells in the peripheral circulation can exert an immunoregulatory effect on neuroinflammation in the AD brain. Revealing that CD8 + T cells can be used as peripheral circulating biomarkers for early AD diagnosis during AD progression.
  • This example uses an immunofluorescence staining kit to stain the brain tissue of wild-type mice and APP/PS1 mouse models (AD mouse models) for CD8, Iba1 and DAPI.
  • the steps are 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. The brains were removed from the posterior end and soaked and fixed with paraformaldehyde for 24 hours.
  • mice Use PBS at 4°C to perfuse mice, 20mL each, and then use 4% paraformaldehyde at 4°C (weigh 40g of paraformaldehyde and dissolve it in a glass container filled with 500mL of DEPC water) , continue to heat and magnetically stir to 60°C to form a milky white suspension.
  • Example 4 Detection of expression level of inflammatory factor IL-6 in cerebrospinal fluid
  • mice After the mice were anesthetized, their heads were fixed on the orientation device.
  • When collecting cerebrospinal fluid wipe the skin on the back of the rat's neck with wet gauze, cut off the back hair, expose the skin and disinfect it, use a scalpel to make a longitudinal incision (about 1cm) along the longitudinal axis, and use scissors to bluntly separate the back of the neck. muscle.
  • the deepest layer of muscle attached to the bone was scraped away with the back of a scalpel to expose the atlanto-occipital membrane.
  • a needle is inserted through the foramen magnum to directly extract cerebrospinal fluid. After the extraction is complete, the outer muscles and skin are sutured. Sulfonamide powder can be sprinkled on the incision to prevent infection.
  • an equal amount of sterilized saline should be injected to maintain the original pressure in the cerebrospinal cavity.
  • ELISA was performed on the cerebrospinal fluid of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) to determine the level of IL-6 expression as the disease progresses.
  • the experimental steps are the same as those in Example 1.
  • IL-6 expression levels in the cerebrospinal fluid of 3-month-old (3M) and (6M) wild-type mice (WT) and APP/PS1 mice (AD) are shown in Figure 4.
  • WAT wild-type mice
  • AD mice showed significantly elevated IL-6 expression levels compared with WT mice at both 2 months of age. This shows that IL-6 is involved in the disease process caused by AD in the early stages of AD and can be used as a cerebrospinal fluid detection biomarker for early AD diagnosis.
  • Example 5 Detection of expression levels of inflammatory factor IL-6 in cerebral cortex and hippocampus
  • This example uses a real-time fluorescence quantitative PCR kit to measure the expression levels of IL-6 in the cerebral cortex (cortex) and hippocampus (hippocampus) of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice respectively. To perform the test, the steps are as follows:
  • reaction mixture II according to the following system.
  • the system is as follows:
  • reaction mixture I to reaction mixture II, mix quickly for 5 seconds, incubate at 70°C for 5 minutes, then incubate on ice for 2 minutes, and perform reverse transcription according to the following procedure:
  • the obtained cDNA template was stored at -20°C for later use.
  • reaction system is as follows:
  • the sequence of the forward primer is shown in SEQ ID No.1
  • the sequence of the reverse primer is shown in SEQ ID No.2.
  • SEQ ID No.2 ctgtgaagtctcctctccgg.
  • Cyclic amplification 95°C, 15s; 60°C, 1min; 70°C, 1min; cycle 40 times;
  • the rate of heating and cooling during the entire process is 1.6°C/s.

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Abstract

A biomarker for the early diagnosis of Alzheimer's disease (AD) and a use thereof. In particular, one or more of peripheral blood interleukin 6, peripheral blood CD8+ T cells and peripheral blood CD4+ T cells act as biomarkers for the early diagnosis of neurodegenerative diseases. A method for the early diagnosis of neurodegenerative diseases, comprising: determining whether an individual is affected by a neurodegenerative disease according to a change in the expression levels of interleukin 6 in peripheral blood and/or a change in the number of CD8+ T cells and/or a change in the number of CD4+ T cells; and determining the progression of the neurodegenerative disease according to the extent of the elevation of the expression levels of interleukin 6 and/or the severity of abnormalities in CD4+ T cells and/or CD8+ T cells in the peripheral blood. AD occurrence and progression is clinically diagnosed by means of detecting biomarkers in the blood, which is low-cost, easy to operate, and low-risk, and which facilitates large-scale screening, thus possessing practical application value.

Description

一种用于早期诊断阿尔茨海默症的生物标志物及其用途A biomarker for early diagnosis of Alzheimer's disease and its use 技术领域Technical field
本发明属于生物技术领域，具体涉及一种用于早期诊断阿尔茨海默症的生物标志物及其用途。The invention belongs to the field of biotechnology, and specifically relates to a biomarker for early diagnosis of Alzheimer's disease and its use.
背景技术Background technique
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病人以进性记忆减退、认知功能障碍、语言障碍、记忆力减退、执行能力下降和人格方面的改变等表现，约占所有痴呆的60％-70％。最新数据表明，目前全球有5000万痴呆患者，到2050年，在全世界范围内将增加3倍。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。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。由于该病的病因复杂，上述假说均不能有效阐明其发病机制，且其治疗药物研究进展缓慢，目前全球仍缺乏治愈AD的药物或方法。一直以来，抗Aβ的免疫治疗是也研发热点，但因其药物研发难度大，现阶段进入Ⅲ期临床的药物大都以失败告终。因此，对AD进行多角度的病理机制研究和寻找早期生物检测标记物迫在眉睫。Alzheimer’s disease (AD) is an age-related degenerative disease of the central nervous system and the most common type of Alzheimer’s disease. Clinically, AD patients present with progressive memory loss, cognitive dysfunction, language impairment, memory loss, executive function decline, and personality changes, accounting for approximately 60% to 70% of all dementia cases. The latest data shows that there are currently 50 million dementia patients in the world, and by 2050, the number will triple worldwide. Typical pathological features in the brains of AD patients are the accumulation of oligomeric β-amyloid (Aβ) plaques, neurofibrillary tangles (NFTs) formed by hyperphosphorylation of Tau protein, and extensive inflammatory reactions in the brain. Currently, widely accepted pathogenic hypotheses include: Aβ aggregation hypothesis, Tau protein abnormality hypothesis, cholinergic hypothesis, neuroinflammation hypothesis, etc. Due to the complex etiology of the disease, none of the above hypotheses can effectively elucidate its pathogenesis, and research on its treatment drugs is progressing slowly. Currently, there is still a lack of drugs or methods to cure AD in the world. Anti-Aβ immunotherapy has always been a research and development hotspot. However, due to the difficulty of drug development, most of the drugs currently entering Phase III clinical trials have ended in failure. Therefore, it is urgent to study the pathological mechanisms of AD from multiple angles and to find early biodetection markers.
由于AD的发生、发展是一个动态演变的过程，从最早的Aβ沉积到出现痴呆症状之间的间隔，可以长达20多年，并且是一个不可逆的慢性神经退行性病变过程。针对AD的早期临床诊断和后续治疗是解决AD病症的关键点。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，Aβ沉积，磷酸化tau蛋白检测等。但是，由于AD早期症状不明显，当对病症做出明确诊断时，患者多到达病程晚期，多数神经元出现死亡，如果能在病人发病早期进行快速诊断或使潜在病人提前发现风险，针对性地进行治疗或预防，能有助于患者病程滞留在轻微智力损伤阶段(mild cognitive impairment，MCI)而减缓恶化，从而保证病人生活质量和减轻社会负担。现阶段对AD的早期分子筛查技术包括正电子发射型计算机断层显像(PET)和脑脊液Aβ分子水平检测等，前者要对受检者注射一定剂量的放射性物质；后者操作损伤大，易造成外科感染。这些诊断技术针对AD早期诊断的可靠性也不太稳定， 因此很难用于AD早期筛查。AD的预防、诊断、治疗与康复是目前世界上公认的难题，对AD早期诊断新的标志物开发及简便的检测手段是未来对AD诊疗的重要方向之一。Since the occurrence and development of AD is a dynamic evolution process, the interval from the earliest Aβ deposition to the onset of dementia symptoms can be as long as more than 20 years, and it is an irreversible chronic neurodegenerative process. Early clinical diagnosis and subsequent treatment of AD are key points in solving AD symptoms. The current diagnostic method for AD is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment test; PET scan of brain senile plaques and Tau protein; brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) markers, Aβ Deposition, phosphorylated tau protein detection, etc. However, because the early symptoms of AD are not obvious, when a clear diagnosis of the disease is made, the patients often reach the late stage of the disease and most neurons die. If the patient can be quickly diagnosed in the early stage of the disease or potential patients can detect risks in advance, targeted treatment can be achieved. Treatment or prevention can help patients stay in the mild cognitive impairment (MCI) stage and slow down the deterioration, thereby ensuring the patient's quality of life and reducing the burden on society. At present, early molecular screening technologies for AD include positron emission computed tomography (PET) and cerebrospinal fluid Aβ molecular level detection. The former requires a certain dose of radioactive material to be injected into the subject; the latter requires a large amount of damage and is easy to cause. Cause surgical infection. The reliability of these diagnostic technologies for early diagnosis of AD is also unstable, making it difficult to be used for early AD screening. The prevention, diagnosis, treatment and rehabilitation of AD are currently recognized problems in the world. The development of new markers and simple detection methods for early diagnosis of AD is one of the important directions for the diagnosis and treatment of AD in the future.
发明内容Contents of the invention
为了解决现有技术中的问题，本发明旨在提供一种用于早期诊断阿尔茨海默症的生物标志物及其用途。In order to solve the problems in the prior art, the present invention aims to provide a biomarker for early diagnosis of Alzheimer's disease and its use.
神经炎症是AD患者脑内主要病理特征之一，炎症过程促进了Aβ病理学和Tau病理学进展。因此，神经炎症在AD疾病发生和发展过程中起着关键作用，针对脑内神经炎症的治疗已经成为现阶段治疗AD的新的热点。而这种靶向调控早期神经炎症的关键分子被视为有潜力的干预AD新的治疗靶点。大量研究表明，血液中的循环免疫细胞在维持中枢神经系统稳态和介导神经系统疾病的免疫反应方面发挥至关重要的作用。神经炎症过程中，脑内激活的小胶质细胞和星形胶质细胞释放多种细胞因子和趋化因子等炎性介质，有助于血脑屏障的通透性增加并允许外周循环免疫细胞的浸润、粘附和迁移，进一步影响脑内抗神经炎症的免疫反应。近期研究发现，AD患者大脑中存在浸润的外周循环的效应CD8 +T细胞，这群细胞不仅可以激活小胶质细胞诱导剧烈的炎症反应，从而进一步加剧AD脑内神经炎症爆发。这一发现证实了外周循环的免疫细胞在AD发生发展中起着免疫监控的重要作用。最新研究表明，在AD患者脑内和AD小鼠模型海马中发现一群适应性外周免疫细胞亚群，它们可以释放炎症因子和细胞毒性分子，促进脑内炎症反应从而导致神经元功能障碍。促炎细胞因子增加、免疫细胞浸润和神经胶质细胞活化是AD中重要的神经炎症反应。 Neuroinflammation is one of the main pathological features in the brain of AD patients, and the inflammatory process promotes the progression of Aβ pathology and Tau pathology. Therefore, neuroinflammation plays a key role in the occurrence and development of AD, and the treatment of neuroinflammation in the brain has become a new hot spot in the treatment of AD at this stage. Targeting this key molecule that regulates early neuroinflammation is regarded as a potential new therapeutic target for AD intervention. A large number of studies have shown that circulating immune cells in the blood play a crucial role in maintaining central nervous system homeostasis and mediating immune responses to neurological diseases. During the neuroinflammation process, activated microglia and astrocytes in the brain release a variety of inflammatory mediators such as cytokines and chemokines, which help increase the permeability of the blood-brain barrier and allow peripheral circulating immune cells. The infiltration, adhesion and migration further affect the anti-neuroinflammatory immune response in the brain. Recent studies have found that there are infiltrating peripheral circulating effector CD8 + T cells in the brains of AD patients. This group of cells can not only activate microglia to induce a violent inflammatory response, thereby further exacerbating the neuroinflammation outbreak in the AD brain. This finding confirms that peripheral circulating immune cells play an important role in immune surveillance in the development and progression of AD. The latest research shows that a group of adaptive peripheral immune cell subsets are found in the brains of AD patients and in the hippocampus of AD mouse models. They can release inflammatory factors and cytotoxic molecules, promote inflammatory reactions in the brain, and lead to neuronal dysfunction. Increased pro-inflammatory cytokines, immune cell infiltration and glial cell activation are important neuroinflammatory responses in AD.
本发明通过检测AD疾病不同阶段的外周血中炎症因子水平、免疫细胞亚群变化与脑内神经炎症严重程度的相关性，从而确定外周血中炎症因子表达水平的改变和免疫细胞亚群数量的改变在AD进程中的指示作用。The present invention detects the correlation between the levels of inflammatory factors in peripheral blood, immune cell subpopulation changes and the severity of neuroinflammation in the brain at different stages of AD disease, thereby determining the changes in the expression levels of inflammatory factors in peripheral blood and the number of immune cell subpopulations. Change the directive role in the AD process.
本发明的具体技术方案如下：The specific technical solutions of the present invention are as follows:
本发明第一方面提供外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种作为早期诊断神经退行性疾病的生物标志物的用途。 A first aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as biomarkers for early diagnosis of neurodegenerative diseases.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明第二方面提供外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种在制备神经退行性疾病早期诊断的产品中的用途。 A second aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells in the preparation of products for early diagnosis of neurodegenerative diseases.
进一步地，所述产品包括：通过酶联免疫吸附、实时荧光定量PCR或RT-PCR检测外周 血白细胞介素6表达水平以诊断神经退行性疾病的产品；和/或通过流式细胞分析或免疫荧光染色检测外周血CD8 +T细胞和/或外周血CD4 +T细胞数量的变化以诊断神经退行性疾病的产品。 Further, the products include: products for detecting the expression level of interleukin-6 in peripheral blood to diagnose neurodegenerative diseases through enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR or RT-PCR; and/or through flow cytometry analysis or immunoassay A product that detects changes in the number of peripheral blood CD8 + T cells and/or peripheral blood CD4 + T cells using fluorescent staining to diagnose neurodegenerative diseases.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明第三方面提供外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种作为神经退行性疾病药物靶点的用途。 The third aspect of the present invention provides the use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as drug targets for neurodegenerative diseases.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明第四方面提供一种神经退行性疾病的早期诊断方法，包括如下步骤：A fourth aspect of the present invention provides a method for early diagnosis of neurodegenerative diseases, which includes the following steps:
检测外周血中白细胞介素6的表达水平和/或CD8 +T细胞的数量和/或CD4 +T细胞的数量，根据外周血中白细胞介素6表达水平的变化和/或CD8 +T细胞数量的变化和/或CD4 +T细胞数量的变化判断是否罹患神经退行性疾病，根据外周血中白细胞介素6表达上升水平和/或CD4 +T细胞和/或CD8 +T细胞异常情况的严重程度判断神经退行性疾病进程； Detect the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood, based on changes in the expression level of interleukin 6 and/or the number of CD8 + T cells in peripheral blood To determine whether you are suffering from a neurodegenerative disease is based on the increase in the expression of interleukin 6 in peripheral blood and/or the severity of abnormalities in CD4 + T cells and/or CD8 + T cells. Determine the progression of neurodegenerative diseases;
当外周血中白细胞介素6的表达水平增多和/或CD8 +T细胞的数量提高和/或CD4 +T细胞的数量减少，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases and/or the number of CD8 + T cells increases and/or the number of CD4 + T cells decreases, neurodegenerative diseases will occur.
进一步地，所述早期诊断方法还包括：检测脑脊液中白细胞介素6的表达水平和/或CD8 +T细胞的数量，根据脑脊液中白细胞介素6表达水平的变化和/或CD8 +T细胞数量的变化判断是否罹患神经退行性疾病，根据脑脊液中白细胞介素6表达上升水平和/或CD8 +T细胞异常情况的严重程度判断神经退行性疾病进程； Further, the early diagnosis method also includes: detecting the expression level of interleukin 6 and/or the number of CD8 + T cells in the cerebrospinal fluid, and based on the changes in the expression level of interleukin 6 and/or the number of CD8 + T cells in the cerebrospinal fluid To determine whether you are suffering from a neurodegenerative disease, the progression of the neurodegenerative disease can be determined based on the increased expression level of interleukin 6 in the cerebrospinal fluid and/or the severity of CD8 + T cell abnormalities;
当脑脊液中白细胞介素6的表达水平增多和/或CD8 +T细胞的数量提高，则罹患神经退行性。 Neurodegeneration occurs when the expression level of interleukin-6 in the cerebrospinal fluid increases and/or the number of CD8 + T cells increases.
进一步地，根据外周血中白细胞介素6表达水平的变化和CD8 +T细胞数量的变化进行诊断； Further, diagnosis is made based on changes in the expression level of interleukin-6 and the number of CD8 + T cells in peripheral blood;
当外周血中白细胞介素6的表达水平增多和CD8 +T细胞的数量提高，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases and the number of CD8 + T cells increases, neurodegenerative diseases will occur.
进一步地，根据外周血中白细胞介素6表达水平的变化、CD8 +T细胞数量的变化和CD4 +T细胞数量的变化进行诊断； Further, diagnosis is made based on changes in the expression level of interleukin 6, changes in the number of CD8 + T cells, and changes in the number of CD4 + T cells in peripheral blood;
当外周血中白细胞介素6的表达水平增多，CD8 +T细胞的数量提高和CD4 +T细胞的数量减少，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases, the number of CD8 + T cells increases and the number of CD4 + T cells decreases, neurodegenerative diseases will occur.
进一步地，根据外周血白细胞介素6表达水平的变化、外周血CD8 +T细胞数量的变化和 脑脊液CD8 +T细胞数量的变化进行诊断； Further, diagnosis is made based on changes in the expression level of interleukin-6 in peripheral blood, changes in the number of CD8 + T cells in peripheral blood, and changes in the number of CD8 + T cells in cerebrospinal fluid;
当外周血白细胞介素6的表达水平增多、外周血CD8 +T细胞的数量提高和脑脊液CD8 +T细胞的数量提高，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases, the number of peripheral blood CD8 + T cells increases, and the number of cerebrospinal fluid CD8 + T cells increases, neurodegenerative diseases will occur.
进一步地，所述外周血取自人或小鼠。Further, the peripheral blood is obtained from humans or mice.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明第五方面上述用途或上述早期诊断方法在筛选神经退行性疾病治疗药物中的应用。In the fifth aspect of the present invention, the above-mentioned use or the above-mentioned early diagnosis method is used in screening drugs for treating neurodegenerative diseases.
进一步地，所述神经退行性疾病治疗药物抑制外周血中白细胞介素6的表达和/或减少外周血中CD8 +T细胞的数量和/或增加CD4 +T细胞的数量。 Further, the drug for treating neurodegenerative diseases inhibits the expression of interleukin-6 in peripheral blood and/or reduces the number of CD8 + T cells and/or increases the number of CD4 + T cells in peripheral blood.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明第六方面提供上述用途或上述早期诊断方法在筛选神经退行性疾病的新生物标志物或新药物靶点中的应用。The sixth aspect of the present invention provides the above-mentioned use or the application of the above-mentioned early diagnosis method in screening new biomarkers or new drug targets for neurodegenerative diseases.
进一步地，新生物标志物或新药物靶点影响外周血中白细胞介素6表达水平和/或CD8 +T细胞数量和/或CD4 +T细胞数量。 Further, new biomarkers or new drug targets affect the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood.
进一步地，所述神经退行性疾病为阿尔茨海默症。Further, the neurodegenerative disease is Alzheimer's disease.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明通过基于外周循环中免疫细胞和免疫分子的改变实现诊断早期AD，具体提供作为早期诊断AD的标志物炎症因子IL-6、外周循环免疫细胞亚群CD8 +T细胞和辅助性CD4 +T细胞，可以直接对外周血样本进行检测，判断机体的炎症水平，实现AD的早期筛查中。其优势在于，通过检测血液中生物标志物进行AD发生发展的临床诊断不仅降低了诊断成本，而且容易操作，避免了受检者脑内取样过程中感染中枢风险，患者接受度高且比较容易进行疾病的大规模筛查，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。本发明可用于指导临床筛查、AD致病机理的研究、AD治疗药物的筛选以及AD的新生物标志物和潜在的新用药靶点的筛选中，具有广阔的应用前景。 The present invention achieves diagnosis of early AD based on changes in immune cells and immune molecules in peripheral circulation, and specifically provides inflammatory factor IL-6, peripheral circulation immune cell subgroups CD8 + T cells and helper CD4 + T cells as markers for early diagnosis of AD. Cells can directly detect peripheral blood samples to determine the body's inflammation level and enable early screening of AD. The advantage is that clinical diagnosis of the occurrence and development of AD by detecting biomarkers in blood not only reduces the diagnostic cost, but is also easy to operate, avoids the risk of central infection during brain sampling, is highly acceptable to patients, and is relatively easy to perform. Large-scale screening of diseases improves the efficiency of detection and increases the accuracy of results, and has practical application value. The invention can be used to guide clinical screening, research on the pathogenesis of AD, screening of AD therapeutic drugs, and screening of new biomarkers and potential new drug targets for AD, and has broad application prospects.
附图说明Description of the drawings
图1. 3月龄(3M)、6月龄(6M)、9月龄(9M)和12月龄(12M)野生型小鼠(WT)和APP/PS1小鼠(AD)外周血中IL-6表达量变化(*p<0.05)。Figure 1. IL in peripheral blood of 3-month-old (3M), 6-month-old (6M), 9-month-old (9M) and 12-month-old (12M) wild-type mice (WT) and APP/PS1 mice (AD) -6 expression level changes (*p<0.05).
图2. 3月龄(3M)和6月龄(6M)野生型小鼠(WT)和APP/PS1小鼠(AD)外周血 中适应性免疫细胞亚群的改变(*p<0.05)。Figure 2. Changes in adaptive immune cell subsets in peripheral blood of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) (*p<0.05).
图3. 3月龄(3M)和6月龄(6M)野生型小鼠(WT)和APP/PS1小鼠(AD)小鼠的脑组织免疫荧光染色的结果图片(比例尺＝50μm)和不同月龄CD8 +T细胞细胞浸润的统计结果(*p<0.05)。A.脑片在脑图谱中拍摄的位置。B.为不同月龄脑组织切片中小胶质细胞表面标记物Iba1，T细胞表面标记物CD8，细胞核标记物DAPI。C.为不同月龄WT和AD小鼠脑内浸润CD8+T细胞统计结果。 Figure 3. Pictures of the results of immunofluorescence staining of brain tissue of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) (scale bar = 50 μm) and different Statistical results of CD8 + T cell infiltration at month-old age (*p<0.05). A. The position of the brain slice taken in the brain atlas. B. Shows the microglia surface marker Iba1, T cell surface marker CD8, and cell nuclear marker DAPI in brain tissue sections at different ages. C. Statistical results of brain infiltration of CD8+ T cells in WT and AD mice of different ages.
图4. 3月龄(3M)和6月龄(6M)野生型小鼠(WT)和APP/PS1小鼠(AD)脑脊液内IL-6表达水平的结果(*p<0.05)。Figure 4. Results of IL-6 expression levels in cerebrospinal fluid of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) (*p<0.05).
图5. 3月龄(3M)和6月龄(6M)野生型小鼠(WT)和APP/PS1小鼠(AD)大脑皮层和海马体内的IL-6基因表达水平的结果(*p<0.05，**p<0.01，***p<0.001)。Figure 5. Results of IL-6 gene expression levels in the cerebral cortex and hippocampus of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) (*p< 0.05, **p<0.01, ***p<0.001).
具体实施方式Detailed ways
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。In order to understand the present invention more clearly, the present invention will be further described with reference to the following examples and accompanying drawings. The examples are for explanation only and do not limit the invention in any way. In the examples, each original reagent material is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
本发明主要在APP/PS1小鼠模型(以下简称AD小鼠模型)进行，AD小鼠模型是模拟AD疾病理想的动物模型之一，广泛应用于AD致病机制的研究。本发明以不同月龄AD小鼠模型作为研究对象，通过脑组织免疫荧光染色技术、总RNA提取技术、qRT-PCR技术、外周血清的酶联免疫吸附实验(ELISA)等，通过对同窝野生型(同窝野生型)和患病组(APP/PS1转基因小鼠，AD)组间对比，得到了外周血中炎症因子IL-6和细胞毒性CD8 +T细胞、辅助性CD4 +T细胞在AD转基因小鼠外周循环发生异常与脑内炎症发生发展相关的明确结论。基于此，本发明提供了外周血中炎症因子IL-6表达水平改变和细胞毒性CD8 +T细胞、辅助性CD4 +T细胞数量变化可作为AD早期诊断分子标志物的可靠证据：3月龄AD小鼠对应病人的病程发展为即将迈入轻微智力损伤(MCI)阶段，6月龄AD小鼠则AD病症特征明确。以下将结合实例对本发明的特征和性能进行进一步的详细描述。 The present invention is mainly carried out in the APP/PS1 mouse model (hereinafter referred to as the AD mouse model). The AD mouse model is one of the ideal animal models for simulating AD disease and is widely used in research on the pathogenesis of AD. The present invention takes AD mouse models of different months of age as the research object, and uses brain tissue immunofluorescence staining technology, total RNA extraction technology, qRT-PCR technology, enzyme-linked immunosorbent assay (ELISA) of peripheral serum, etc. Comparison between the type (wild type littermate) and the diseased group (APP/PS1 transgenic mice, AD), the inflammatory factor IL-6 and cytotoxic CD8 + T cells and helper CD4 + T cells in peripheral blood were compared. A clear conclusion that peripheral circulation abnormalities in AD transgenic mice are related to the development of brain inflammation. Based on this, the present invention provides reliable evidence that changes in the expression level of the inflammatory factor IL-6 and changes in the number of cytotoxic CD8 + T cells and helper CD4 + T cells in peripheral blood can be used as molecular markers for early diagnosis of AD: 3-month-old AD The mice correspond to the patient's disease course and are about to enter the mild intellectual impairment (MCI) stage. The 6-month-old AD mice have clear characteristics of AD symptoms. The features and performance of the present invention will be described in further detail below with reference to examples.
本发明所涉及的材料：Materials involved in the invention:
同窝野生型小鼠和APP/PS1小鼠模型来自美国Jackson实验室；Litter wild-type mice and APP/PS1 mouse models were from Jackson Laboratory in the United States;
多聚甲醛购自Sigma-aldrich，货号：158127；Paraformaldehyde was purchased from Sigma-aldrich, item number: 158127;
包埋剂OCT购自SAKURA，货号：4583；The embedding agent OCT was purchased from SAKURA, item number: 4583;
Iba1一抗购自Wake公司，货号：019-19741；Iba1 primary antibody was purchased from Wake Company, product number: 019-19741;
CD8一抗购自Invitrogen，货号：14-0195-82；CD8 primary antibody was purchased from Invitrogen, product number: 14-0195-82;
DAPI购自Thermo scientific公司，货号：62248；DAPI was purchased from Thermo Scientific Company, item number: 62248;
荧光二抗购自Thermo scientific；Fluorescent secondary antibodies were purchased from Thermo scientific;
红细胞裂解液购自BD Biosciences公司，货号：555899；Red blood cell lysate was purchased from BD Biosciences, product number: 555899;
流式分析使用的抗体均购自BD Biosciences公司，货号依次为：The antibodies used in flow cytometry analysis were all purchased from BD Biosciences, and the order numbers are:
Ms CD45 FITC 30-F11，货号：553079；Ms CD45 FITC 30-F11, item number: 553079;
Ms CD3 MolCpx PerCP-Cy5.5 17A2，货号：560527；Ms CD3 MolCpx PerCP-Cy5.5 17A2, Cat. No.: 560527;
Ms CD4 APC-H7 GK1.5，货号：560181；Ms CD4 APC-H7 GK1.5, item number: 560181;
Ms CD8a PE 53-6.7，货号：553032；Ms CD8a PE 53-6.7, item number: 553032;
Ms CD19 PE-Cy7 1D3，货号：552854；Ms CD19 PE-Cy7 1D3, item number: 552854;
Ms CD49b APC DX5，货号：560628；Ms CD49b APC DX5, item number: 560628;
马血清购自Gibco公司，货号：26050088；Horse serum was purchased from Gibco Company, product number: 26050088;
胎牛血清购自Life Technologies公司，货号：16050-122；Fetal bovine serum was purchased from Life Technologies, product number: 16050-122;
DAPI购自Thermo scientific公司，货号：D1306；DAPI was purchased from Thermo Scientific Company, item number: D1306;
DPBS购自Sigma公司，货号：D8662-24*500ML；DPBS was purchased from Sigma Company, item number: D8662-24*500ML;
Trizol购自invitrogen公司，货号：15596026；Trizol was purchased from Invitrogen Company, item number: 15596026;
反转录试剂盒购自Thermo scientific，货号K1622；The reverse transcription kit was purchased from Thermo scientific, product number K1622;
实时荧光定量PCR试剂盒购自Thermo scientific，货号4368706；Real-time fluorescence quantitative PCR kit was purchased from Thermo scientific, catalog number 4368706;
ELISA试剂盒购自R&D，货号：M6000B。The ELISA kit was purchased from R&D, product number: M6000B.
实施例1：外周血中炎症因子IL-6的表达水平检测Example 1: Detection of the expression level of inflammatory factor IL-6 in peripheral blood
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小鼠外周血中的白细胞介素6(IL-6)的表达水平进行检测，步骤如下：This example uses an ELISA kit to detect the expression levels of interleukin 6 (IL-6) in the peripheral blood of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice respectively. The steps are as follows:
1、血清样品采集1. Serum sample collection
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：After 3-month-old and 6-month-old littermate wild-type and AD 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. The mice were killed head-on, and the serum was isolated as follows:
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；Place the anticoagulated blood sample at 4°C for 4 hours. After the blood coagulates, the serum will naturally precipitate. Centrifuge at 4°C and 4000 rpm for 30 minutes to separate the serum and discard the insoluble matter;
将血清移至新的灭菌EP管，分装后-80℃储存备用。Transfer the serum to a new sterilized EP tube, aliquot and store at -80°C for later use.
2、试剂配置2. Reagent configuration
(1)阳性对照配制：用1mL去离子水溶解IL-6阳性对照，充分混匀，备用。(1) Preparation of positive control: Dissolve IL-6 positive control in 1 mL of deionized water, mix thoroughly, and set aside.
(2)洗涤液配制：用去离子水按1:25稀释至工作浓度。(2) Preparation of washing liquid: dilute 1:25 with deionized water to the working concentration.
(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。(3) Preparation of luminescent reagent: 15 minutes before testing on the machine, mix the luminescent reagent A and reagent B in the kit at a volume of 1:1 and store in the dark.
(4)IL-6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。(4) Preparation of IL-6 standard: Dilute the 5000pg/mL standard provided in the kit with calibration diluent 1:10 in a new EP tube to a 500pg/mL standard, and then dilute to the respective concentrations. Standards for 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.81pg/mL.
3、ELISA检测3. ELISA test
(1)向测试孔中加入50μL测定稀释剂RD1W。(1) Add 50 μL of assay diluent RD1W to the test well.
(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。(2) Add the standard, control sample and sample to be tested to the test well in sequence, seal with the sealing film provided in the kit and incubate at room temperature for 2 hours.
(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。(3) After the incubation, tear off the sealing film, discard the liquid, add 400 μL washing solution to each well to wash the detection plate, wash 4 times, and discard the washing solution.
(4)向测试孔中加入100μL小鼠IL-6偶联物，封口膜封口后，室温孵育2h。(4) Add 100 μL of mouse IL-6 conjugate to the test well, seal with parafilm, and incubate at room temperature for 2 hours.
(5)重复步骤(3)1次。(5) Repeat step (3) once.
(6)向测试孔中加入100μL配制的发光试剂，避光室温孵育39min。(6) Add 100 μL of the prepared luminescent reagent to the test well, and incubate at room temperature for 39 minutes in the dark.
(7)加入100μL终止液，轻弹混匀测试板确保充分混匀。(7) Add 100 μL of stop solution and flick the test plate to ensure complete mixing.
(8)读板：在30min内完成每个孔的光密度检测。(8) Plate reading: Complete the optical density detection of each well within 30 minutes.
(9)计算：根据试剂盒提供的公式进行浓度定量计算。(9) Calculation: Calculate the concentration quantitatively according to the formula provided by the kit.
4、实验结果4. Experimental results
3月龄(3M)、6月龄(6M)、9月龄(9M)和12月龄(12M)的野生型小鼠(WT)和APP/PS1小鼠(AD)外周血血清中的炎症因子IL-6的表达水平如图1所示。由图可知，与WT组小鼠相比，在3月龄AD小鼠的外周血血清中IL-6表达水平显著上升，随着疾病进程的发展，IL-6在外周血血清中出现持续上升的趋势。表明在AD发生早期阶段外周血循环系统参与到AD引起的疾病进程，可以作为早期AD诊断的外周血生物标记物。Inflammation in peripheral blood serum of 3-month-old (3M), 6-month-old (6M), 9-month-old (9M) and 12-month-old (12M) wild-type mice (WT) and APP/PS1 mice (AD) The expression levels of factor IL-6 are shown in Figure 1. It can be seen from the figure that compared with the mice in the WT group, the expression level of IL-6 in the peripheral blood serum of 3-month-old AD mice increased significantly. With the development of the disease process, IL-6 continued to increase in the peripheral blood serum. the trend of. This indicates that the peripheral blood circulatory system is involved in the disease process caused by AD in the early stages of AD and can be used as a peripheral blood biomarker for early AD diagnosis.
实施例2：外周血单个核细胞(Peripheral blood mononuclear cells，PBMCs)的流式细胞分析Example 2: Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs)
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：This example performs flow cytometry analysis on peripheral blood mononuclear cells (PBMCs) of 6-month-old and 9-month-old wild-type mice and APP/PS1 mice. The steps are as follows:
1、高活性的外周血单个核细胞(PBMCs)制备1. Preparation of highly active peripheral blood mononuclear cells (PBMCs)
(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。(1) Add 3 times the volume of red blood cell lysis buffer to 200 μL of anticoagulated whole blood and mix gently, let stand at room temperature for 10 minutes, and mix gently twice during this time to lyse red blood cells.
(2)在800×g下离心2min，弃上清，收集细胞，用1mLPBS清洗样品1次。(2) Centrifuge at 800 × g for 2 minutes, discard the supernatant, collect the cells, and wash the sample once with 1 mL PBS.
(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。(3) Resuspend in 500 μL buffer, filter through a 300-mesh cell strainer, incubate the antibody, and then analyze on the computer.
2、流式分析2. Flow analysis
(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。 (1) Adjust the cell density of the PBMCs cell suspension prepared in the above steps to 5×10 6 cells/mL with DPBS containing 2% fetal calf serum.
(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。(2) Add 40 μL of cell suspension into a plastic centrifuge tube pre-filled with 50 μL of fluorescently labeled specific antibody, then add 50 μL of inactivated normal horse serum (diluted with DPBS at 1:20), and incubate at 4°C for 30 min.
(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。(3) Add 2 mL of DPBS containing 2% fetal bovine serum to resuspend the cells and wash them, centrifuge at 1000 rpm for 5 min at 4°C, and repeat washing the cells once.
(4)加入500μL预冷PBS重悬细胞，准备上机分析。(4) Add 500 μL of pre-cooled PBS to resuspend the cells and prepare for analysis on the machine.
3、实验结果3. Experimental results
基于不同月龄AD外周血中炎症因子IL-6表达水平的变化，本实施例选择AD早期阶段：3月龄(3M)和6月龄(6M)小鼠进行PBMCs T淋巴细胞亚群流式分析，结果如图2。流式结果分析表明，与WT小鼠相比，在6月龄AD小鼠的外周血中，辅助性T细胞(CD4 +T细胞)表达显著降低，细胞毒性T细胞(CD8 +T细胞)显著升高，说明外周循环中的免疫细胞可以对AD脑内的神经炎症发挥免疫调节作用。揭示在AD进程(CD8 +T细胞)可以作为早期AD诊断的外周循环生物标志物。 Based on the changes in the expression levels of the inflammatory factor IL-6 in the peripheral blood of AD at different ages, this example selected mice at the early stage of AD: 3 months old (3M) and 6 months old (6M) to conduct PBMCs T lymphocyte subpopulation flow cytometry. Analysis, the results are shown in Figure 2. Flow cytometry analysis showed that compared with WT mice, in the peripheral blood of 6-month-old AD mice, the expression of helper T cells (CD4 + T cells) was significantly reduced, and the expression of cytotoxic T cells (CD8 + T cells) was significantly reduced. Increased levels indicate that immune cells in the peripheral circulation can exert an immunoregulatory effect on neuroinflammation in the AD brain. Revealing that CD8 + T cells can be used as peripheral circulating biomarkers for early AD diagnosis during AD progression.
实施例3：CD8 +T细胞的细胞浸润检测 Example 3: Cell infiltration detection of CD8 + T cells
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CD8、Iba1和DAPI染色，步骤如下：This example uses an immunofluorescence staining kit to stain the brain tissue of wild-type mice and APP/PS1 mouse models (AD mouse models) for CD8, Iba1 and DAPI. The steps are as follows:
1、小鼠脑组织切片1. Mouse brain tissue sections
(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。(1) Anesthesia and brain tissue perfusion sampling: The 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. The brains were removed from the posterior end and soaked and fixed with paraformaldehyde for 24 hours.
(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。(2) Perfusion fixation of mice: Use PBS at 4°C to perfuse mice, 20mL each, and then use 4% paraformaldehyde at 4°C (weigh 40g of paraformaldehyde and dissolve it in a glass container filled with 500mL of DEPC water) , continue to heat and magnetically stir to 60°C to form a milky white suspension. Use 1.0mmol/L NaOH to adjust the pH to 7.0 to make the solution clear, then add about 500mL 2×PBS, mix thoroughly, filter and dilute to 1000mL, stored at 4°C for later use) perfusion, 20mL per mouse until the tissue becomes hard.
(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。(3) Material collection: Carefully peel off the brain tissue, place it in a 15mL centrifuge tube, and post-fix it with 4% paraformaldehyde (fixative) for 24 hours.
(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。(4) Dehydration: Wash the paraformaldehyde-fixed tissue three times with PBS (cleaning solution), dehydrate with 20% sucrose (dehydrating agent) until the tissue sinks to the bottom, and dehydrate with 30% sucrose at 4°C overnight.
(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。(5) Add the embedding agent OCT dropwise to the specimen table, put it into the cryostat microtome until it turns white, then take it out, and quickly smooth the surface with a single-sided blade.
(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。(6) Use a safety blade to smooth the bottom of the specimen and adhere it to the specimen table, then place it in the freezing stage of the -24°C cryostat microtome. When the tissue turns slightly white, apply a thin layer of OCT on the surface of the specimen and continue freezing. 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。(7) After adjusting the slice thickness, start slicing to a thickness of 20 μm. Collect the slices continuously and move them into a 24-well plate containing 4% paraformaldehyde.
(8)将切好的片子放4℃保存备用。(8) Store the cut slices at 4°C for later use.
2、免疫荧光染色2. Immunofluorescence staining
(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。(1) Carefully pick out the appropriate sections and put them into a 24-well plate pre-placed with 1 mL of pre-cooled PBS, and wash them 3 times with pre-cooled PBS for 10 minutes each time.
(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。(2) Punch and seal: 0.2% Triton X-100 (diluted in PBS), 0.1% BSA and 5% horse serum (diluted in PBS), incubate at room temperature for 40 minutes, and place on a shaker to shake slowly.
(3)PBS室温下洗3次，每次5min。(3) Wash 3 times with PBS at room temperature, 5 minutes each time.
(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。(4) Primary antibody incubation: Use antibody diluent (PBS containing 0.01% BSA and 5% horse serum) to dilute 1:100, add 200 μL to each well, and incubate overnight at 4°C with slow shaking.
(5)回收一抗，PBS室温下洗3次，每次10min。(5) Recover the primary antibody and wash 3 times with PBS at room temperature, 10 minutes each time.
(6)3％马血清室温下封闭脑片30min。(6) Block the brain slices with 3% horse serum at room temperature for 30 minutes.
(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。(7) Secondary antibody incubation and DAPI staining: Dilute the secondary antibody 1:500 in PBS and incubate for 2 hours at room temperature in the dark; dilute the DAPI stock solution 1:5000 and incubate at room temperature for 15 minutes.
(8)用PBS室温下洗3次，每次15min。(8) Wash 3 times with PBS at room temperature, 15 minutes each time.
(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。(9) Sealing the slide: Take a sticky slide, mark the specific information with a pencil on the frosted surface on the right side, drop a drop of PBS in the middle of the slide, dip the section into the PBS drop, suck off the PBS solution, and place on the slide Spread 160 μL of mounting medium across the center of the slice, and cover the slice with a long coverslip to avoid bubbles and wrinkles.
(10)平放于避光处晾干。(10) Lay it flat in a dark place to dry.
3、实验结果3. Experimental results
3月龄(3M)、6月龄(6M)、9月龄(9M)和12月龄(12M)的野生型小鼠(WT)和APP/PS1小鼠(AD)的染色图片如图3所示。由图可知，在6月龄AD小鼠皮层和海马部位有外周来源的CD8 +T细胞浸润入脑内病灶部位，随着病情发展，9月龄和12月龄AD小鼠脑组织中CD8 +T细胞浸润增多，小胶质细胞激活数目增多，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，CD8 +T细胞介导的神经炎症发生发展在3月龄和6月龄之间或者更早阶段。这说明可将CD8 +T细胞的数目改变可以作为AD的早期标记物。 Staining pictures of 3-month-old (3M), 6-month-old (6M), 9-month-old (9M) and 12-month-old (12M) wild-type mice (WT) and APP/PS1 mice (AD) are shown in Figure 3 shown. It can be seen from the figure that peripherally derived CD8 + T cells infiltrate into the brain lesions in the cortex and hippocampus of 6-month-old AD mice. As the disease progresses, CD8 + T cells in the brain tissue of 9-month-old and 12-month-old AD mice T cell infiltration increased, and the number of activated microglia increased and formed an aggregation state, but this phenomenon did not occur in 3-month-old mice. This result indicates that in AD mouse models, CD8 + T cell-mediated neuroinflammation develops between 3 months of age and 6 months of age or earlier. This suggests that changes in the number of CD8 + T cells can serve as an early marker for AD.
实施例4:脑脊液中炎症因子IL-6的表达水平检测Example 4: Detection of expression level of inflammatory factor IL-6 in cerebrospinal fluid
1、脑脊液样品采集：1. Cerebrospinal fluid sample collection:
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。After the mice were anesthetized, their heads were fixed on the orientation device. When collecting cerebrospinal fluid, wipe the skin on the back of the rat's neck with wet gauze, cut off the back hair, expose the skin and disinfect it, use a scalpel to make a longitudinal incision (about 1cm) along the longitudinal axis, and use scissors to bluntly separate the back of the neck. muscle. To avoid bleeding, the deepest layer of muscle attached to the bone was scraped away with the back of a scalpel to expose the atlanto-occipital membrane. A needle is inserted through the foramen magnum to directly extract cerebrospinal fluid. After the extraction is complete, the outer muscles and skin are sutured. Sulfonamide powder can be sprinkled on the incision to prevent infection. After collecting the cerebrospinal fluid, an equal amount of sterilized saline should be injected to maintain the original pressure in the cerebrospinal cavity.
2、IL-6表达水平检测2. IL-6 expression level detection
本实施例对3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和APP/PS1小鼠(AD)的脑脊液进行ELISA检测，测定随着疾病进程IL-6表达平的变化，实验步骤与实施例1相同。In this example, ELISA was performed on the cerebrospinal fluid of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP/PS1 mice (AD) to determine the level of IL-6 expression as the disease progresses. The experimental steps are the same as those in Example 1.
3、实验结果3. Experimental results
3月龄(3M)和(6M)的野生型小鼠(WT)和APP/PS1小鼠(AD)的脑脊液中IL-6表达水平变化如图4。在3月龄和6月龄小鼠的脑脊液中，与WT小鼠相比，AD小鼠在两个月龄中均表现出IL-6表达水平显著升高。说明在AD发生早期阶段IL-6参与到AD引起的疾病进程，可以作为早期AD诊断的脑脊液检测生物标记物。The changes in IL-6 expression levels in the cerebrospinal fluid of 3-month-old (3M) and (6M) wild-type mice (WT) and APP/PS1 mice (AD) are shown in Figure 4. In the cerebrospinal fluid of 3-month-old and 6-month-old mice, AD mice showed significantly elevated IL-6 expression levels compared with WT mice at both 2 months of age. This shows that IL-6 is involved in the disease process caused by AD in the early stages of AD and can be used as a cerebrospinal fluid detection biomarker for early AD diagnosis.
实施例5：大脑皮层和海马体内炎症因子IL-6的表达水平检测Example 5: Detection of expression levels of inflammatory factor IL-6 in cerebral cortex and hippocampus
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IL-6的表达水平进行检测，步骤如 下：This example uses a real-time fluorescence quantitative PCR kit to measure the expression levels of IL-6 in the cerebral cortex (cortex) and hippocampus (hippocampus) of 3-month-old and 6-month-old wild-type mice and APP/PS1 mice respectively. To perform the test, the steps are as follows:
1、RNA提取1. RNA extraction
(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。(1) 3-month-old and 6-month-old littermates of wild-type and AD mice were anesthetized with isoflurane (gas), and then killed by rapid decapitation with scissors after breaking their necks. The heads were placed on ice and the cerebral cortex was quickly separated. And isolate the mouse cerebral cortex and hippocampus. The isolated tissue was washed twice in DPBS containing 4 U/mL protease inhibitors and RNase inhibitors.
(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。(2) Homogenization treatment: Grind the tissue or cells in liquid nitrogen, add 1mL Trizol (RNA extraction reagent) for every 100 mg of tissue, and perform homogenization treatment with a homogenizer.
(3)将匀浆样品在室温放置5min，使核酸蛋白复合物完全分离。(3) Place the homogenized sample at room temperature for 5 minutes to completely separate the nucleic acid-protein complexes.
(4)每使用1mL Trizol加入0.2mL氯仿，剧烈振荡15s，室温放置3min。(4) Add 0.2 mL of chloroform for every 1 mL of Trizol used, shake vigorously for 15 s, and leave at room temperature for 3 min.
(5)4℃下10000×g离心15min。(5) Centrifuge at 10,000 × g for 15 minutes at 4°C.
(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。(6) Transfer the aqueous phase to a new tube, use isopropanol to precipitate the RNA in the aqueous phase, add 0.5 mL of isopropyl alcohol for every 1 mL of Trizol used, and leave it at room temperature for 10 minutes.
(7)4℃下10000×g离心10min，在管侧和管底出现胶状沉淀，弃上清。(7) Centrifuge at 10,000 × g for 10 minutes at 4°C. If a gelatinous precipitate appears on the side and bottom of the tube, discard the supernatant.
(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。(8) Wash the RNA pellet with 75% ethanol, add 1mL of 75% ethanol for every 1mL of Trizol used, centrifuge at 7500×g for 5 minutes at 4°C, and discard the supernatant.
(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。(9) Leave to dry at room temperature for 5 minutes, add 50 μL of RNase-free water, pipette several times with a pipette tip, place at 55°C for 10 minutes to dissolve the RNA, and store at -70°C.
2、反转录2. Reverse transcription
使用反转录试剂盒将提取的总RNA反转录成cDNA，步骤如下：Use a reverse transcription kit to reverse-transcribe the extracted total RNA into cDNA. The steps are as follows:
在无RNA酶的离心管中制备反应混合物I，体系如下：Prepare reaction mixture I in an RNase-free centrifuge tube as follows:
Figure PCTCN2022087570-appb-000001
Figure PCTCN2022087570-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：Mix the above components and centrifuge quickly for 5 seconds. After incubating at 70°C for 5 minutes, incubate on ice for 2 minutes. Then prepare reaction mixture II according to the following system. The system is as follows:
Figure PCTCN2022087570-appb-000002
Figure PCTCN2022087570-appb-000002
Figure PCTCN2022087570-appb-000003
Figure PCTCN2022087570-appb-000003
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：Add reaction mixture I to reaction mixture II, mix quickly for 5 seconds, incubate at 70°C for 5 minutes, then incubate on ice for 2 minutes, and perform reverse transcription according to the following procedure:
25℃，5min；42℃，60min；70℃，5min。25℃, 5min; 42℃, 60min; 70℃, 5min.
得到的cDNA模板置于-20℃保存备用。The obtained cDNA template was stored at -20°C for later use.
3、实时荧光定量PCR3. Real-time fluorescence quantitative PCR
使用实时荧光定量PCR试剂盒对IL-6的表达水平进行检测，反应体系如下：Use a real-time fluorescence quantitative PCR kit to detect the expression level of IL-6. The reaction system is as follows:
Figure PCTCN2022087570-appb-000004
Figure PCTCN2022087570-appb-000004
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。Among them, the sequence of the forward primer is shown in SEQ ID No.1, and the sequence of the reverse primer is shown in SEQ ID No.2.
SEQ ID No.1：ctggggatgtctgtagctca；SEQ ID No.1:ctggggatgtctgtagctca;
SEQ ID No.2：ctgtgaagtctcctctccgg。SEQ ID No.2: ctgtgaagtctcctctccgg.
将上述组分混合，6000rpm离心1min，按以下程序进行扩增：Mix the above components, centrifuge at 6000rpm for 1 minute, and amplify according to the following procedure:
预变性：95℃，10min；Pre-denaturation: 95℃, 10min;
循环扩增：95℃，15s；60℃，1min；70℃，1min；循环40次；Cyclic amplification: 95℃, 15s; 60℃, 1min; 70℃, 1min; cycle 40 times;
形成熔解曲线：95℃，15s；60℃，1min；Form a melting curve: 95℃, 15s; 60℃, 1min;
整个过程中升温和降温的速率为1.6℃/s。The rate of heating and cooling during the entire process is 1.6°C/s.
4、实验结果4. Experimental results
由图5可知，在3月龄和6月龄WT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IL-6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。提示，IL-6在外周和中枢表达具有明显的相关性，进一步证明本发明中发现的外周血中IL-6可以作为早期AD的诊断指标。As can be seen from Figure 5, in 3-month-old and 6-month-old WT mice, the changes in cytokine expression between individuals in the same group of wild-type mice are relatively concentrated, while in AD mice, the changes in cytokine expression between different individuals in the same group are relatively concentrated. The differences in cytokine expression changes were relatively large, and IL-6 gene expression levels were significantly increased in the cerebral cortex and hippocampus of 3-month-old and 6-month-old AD model mice. It is suggested that there is a clear correlation between the expression of IL-6 in the periphery and the central nervous system, which further proves that the IL-6 in peripheral blood found in the present invention can be used as a diagnostic indicator of early AD.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (20)

  1. 外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种作为早期诊断神经退行性疾病的生物标志物的用途。 Use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as biomarkers for early diagnosis of neurodegenerative diseases.
  2. 根据权利要求1所述的用途，其特征在于，所述神经退行性疾病为阿尔茨海默症。The use according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.
  3. 外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种在制备神经退行性疾病早期诊断的产品中的用途。 Use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells in preparing products for early diagnosis of neurodegenerative diseases.
  4. 根据权利要求3所述的用途，其特征在于，所述产品包括：通过酶联免疫吸附、实时荧光定量PCR或RT-PCR检测外周血白细胞介素6表达水平以诊断神经退行性疾病的产品；和/或通过流式细胞分析或免疫荧光染色检测外周血CD8 +T细胞和/或外周血CD4 +T细胞数量的变化以诊断神经退行性疾病的产品。 The use according to claim 3, characterized in that the product includes: a product for detecting the expression level of interleukin-6 in peripheral blood to diagnose neurodegenerative diseases through enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR or RT-PCR; and/or products for diagnosing neurodegenerative diseases by detecting changes in the number of peripheral blood CD8 + T cells and/or peripheral blood CD4 + T cells by flow cytometry or immunofluorescence staining.
  5. 根据权利要求3所述的用途，其特征在于，所述神经退行性疾病为阿尔茨海默症。The use according to claim 3, wherein the neurodegenerative disease is Alzheimer's disease.
  6. 外周血白细胞介素6、外周血CD8 +T细胞和外周血CD4 +T细胞中的一种或几种作为神经退行性疾病药物靶点的用途。 Use of one or more of peripheral blood interleukin-6, peripheral blood CD8 + T cells and peripheral blood CD4 + T cells as drug targets for neurodegenerative diseases.
  7. 根据权利要求6所述的用途，其特征在于，所述神经退行性疾病为阿尔茨海默症。The use according to claim 6, wherein the neurodegenerative disease is Alzheimer's disease.
  8. 一种神经退行性疾病的早期诊断方法，其特征在于，包括如下步骤：An early diagnosis method for neurodegenerative diseases, characterized by including the following steps:
    检测外周血中白细胞介素6的表达水平和/或CD8 +T细胞的数量和/或CD4 +T细胞的数量，根据外周血中白细胞介素6表达水平的变化和/或CD8 +T细胞数量的变化和/或CD4 +T细胞数量的变化判断是否罹患神经退行性疾病，根据外周血中白细胞介素6表达上升水平和/或CD4 +T细胞和/或CD8 +T细胞异常情况的严重程度判断神经退行性疾病进程； Detect the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood, based on changes in the expression level of interleukin 6 and/or the number of CD8 + T cells in peripheral blood To determine whether you are suffering from a neurodegenerative disease is based on the increase in the expression of interleukin 6 in peripheral blood and/or the severity of abnormalities in CD4 + T cells and/or CD8 + T cells. Determine the progression of neurodegenerative diseases;
    当外周血中白细胞介素6的表达水平增多和/或CD8 +T细胞的数量提高和/或CD4 +T细胞的数量减少，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases and/or the number of CD8 + T cells increases and/or the number of CD4 + T cells decreases, neurodegenerative diseases will occur.
  9. 根据权利要求8所述的早期诊断方法，其特征在于，所述早期诊断方法还包括：检测脑脊液中白细胞介素6的表达水平和/或CD8 +T细胞的数量，根据脑脊液中白细胞介素6表达水平的变化和/或CD8 +T细胞数量的变化判断是否罹患神经退行性疾病，根据脑脊液中白细胞介素6表达上升水平和/或CD8 +T细胞异常情况的严重程度判断神经退行性疾病进程； The early diagnosis method according to claim 8, characterized in that the early diagnosis method further includes: detecting the expression level of interleukin 6 in the cerebrospinal fluid and/or the number of CD8 + T cells, and based on the interleukin 6 in the cerebrospinal fluid. Changes in expression levels and/or changes in the number of CD8 + T cells can be used to determine whether you are suffering from neurodegenerative diseases, and the progression of neurodegenerative diseases can be determined based on the increased expression level of interleukin 6 in the cerebrospinal fluid and/or the severity of CD8 + T cell abnormalities. ;
    当脑脊液中白细胞介素6的表达水平增多和/或CD8 +T细胞的数量提高，则罹患神经退行性。 Neurodegeneration occurs when the expression level of interleukin-6 in the cerebrospinal fluid increases and/or the number of CD8 + T cells increases.
  10. 根据权利要求8所述的早期诊断方法，其特征在于，根据外周血中白细胞介素6表达水平的变化和CD8 +T细胞数量的变化进行诊断； The early diagnosis method according to claim 8, characterized in that diagnosis is made based on changes in the expression level of interleukin 6 and changes in the number of CD8 + T cells in peripheral blood;
    当外周血中白细胞介素6的表达水平增多和CD8 +T细胞的数量提高，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases and the number of CD8 + T cells increases, neurodegenerative diseases will occur.
  11. 根据权利要求8所述的早期诊断方法，其特征在于，根据外周血中白细胞介素6表达水平的变化、CD8 +T细胞数量的变化和CD4 +T细胞数量的变化进行诊断； The early diagnosis method according to claim 8, characterized in that diagnosis is made based on changes in the expression level of interleukin 6, changes in the number of CD8 + T cells, and changes in the number of CD4 + T cells in peripheral blood;
    当外周血中白细胞介素6的表达水平增多，CD8 +T细胞的数量提高和CD4 +T细胞的数量减少，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases, the number of CD8 + T cells increases and the number of CD4 + T cells decreases, neurodegenerative diseases will occur.
  12. 根据权利要求9所述的早期诊断方法，其特征在于，根据外周血白细胞介素6表达水平的变化、外周血CD8 +T细胞数量的变化和脑脊液CD8 +T细胞数量的变化进行诊断； The early diagnosis method according to claim 9, characterized in that diagnosis is made based on changes in the expression level of interleukin 6 in peripheral blood, changes in the number of CD8 + T cells in peripheral blood, and changes in the number of CD8 + T cells in cerebrospinal fluid;
    当外周血白细胞介素6的表达水平增多、外周血CD8 +T细胞的数量提高和脑脊液CD8 +T细胞的数量提高，则罹患神经退行性疾病。 When the expression level of interleukin-6 in peripheral blood increases, the number of peripheral blood CD8 + T cells increases, and the number of cerebrospinal fluid CD8 + T cells increases, neurodegenerative diseases will occur.
  13. 根据权利要求8或9所述的早期诊断方法，其特征在于，所述外周血取自人或小鼠。The early diagnosis method according to claim 8 or 9, characterized in that the peripheral blood is taken from humans or mice.
  14. 根据权利要求8或9所述的早期诊断方法，其特征在于，所述神经退行性疾病为阿尔茨海默症。The early diagnosis method according to claim 8 or 9, characterized in that the neurodegenerative disease is Alzheimer's disease.
  15. 权利要求1所述用途、权利要求3所述用途、权利要求6所述用途或权利要求8所述早期诊断方法在筛选神经退行性疾病治疗药物中的应用。The use of claim 1, the use of claim 3, the use of claim 6 or the application of the early diagnosis method of claim 8 in screening drugs for the treatment of neurodegenerative diseases.
  16. 根据权利要求15所述的应用，其特征在于，所述神经退行性疾病治疗药物抑制外周血中白细胞介素6的表达和/或减少外周血中CD8 +T细胞的数量和/或增加CD4 +T细胞的数量。 The application according to claim 15, characterized in that the drug for treating neurodegenerative diseases inhibits the expression of interleukin-6 in peripheral blood and/or reduces the number of CD8 + T cells in peripheral blood and/or increases CD4 + The number of T cells.
  17. 根据权利要求15所述的应用，其特征在于，所述神经退行性疾病为阿尔茨海默症。The application according to claim 15, wherein the neurodegenerative disease is Alzheimer's disease.
  18. 权利要求1所述用途、权利要求3所述用途、权利要求6所述用途或权利要求8所述早期诊断方法在筛选神经退行性疾病的新生物标志物或新药物靶 点中的应用。The use of claim 1, the use of claim 3, the use of claim 6 or the application of the early diagnosis method of claim 8 in screening new biomarkers or new drug targets for neurodegenerative diseases.
  19. 根据权利要求18所述的应用，其特征在于，新生物标志物或新药物靶点影响外周血中白细胞介素6表达水平和/或CD8 +T细胞数量和/或CD4 +T细胞数量。 The application according to claim 18, characterized in that the new biomarker or new drug target affects the expression level of interleukin 6 and/or the number of CD8 + T cells and/or the number of CD4 + T cells in peripheral blood.
  20. 根据权利要求18所述的应用，其特征在于，所述神经退行性疾病为阿尔茨海默症。The application according to claim 18, wherein the neurodegenerative disease is Alzheimer's disease.
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