Use of detection reagent in preparation of diagnostic tool for diagnosing or monitoring ad

Abstract

The use of a reagent, which detects changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid, in the preparation of a diagnostic tool or a therapeutic tool for diagnosing or monitoring Alzheimer's disease. A method for diagnosing or monitoring Alzheimer's disease, in which a reagent for detecting immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid is used to detect changes in the concentration or number of the immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid. A method for treating Alzheimer's disease, in which immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid are taken as targets for administration so as to reduce or decrease the concentration or number of the immune cells and immune factors.

Description

Use of a detection reagent in the preparation of a diagnostic tool for diagnosing or monitoring AD Technical Field The present invention belongs to the field of biotechnology, and particularly relates to the use of a reagent for detecting changes in the concentration or number of immune cells and immune factors in peripheral circulating cerebrospinal fluid in the preparation of a diagnostic tool for diagnosing or monitoring AD. Background Art Alzheimer's disease (AD), also known as senile dementia, accounts for about 60%-70% of all dementia types and is an age-related central neurodegenerative disease, becoming the seventh leading cause of death globally after lung cancer. Clinically, AD patients present with progressive memory loss, cognitive impairment, language disorders, memory loss, decreased executive ability, and personality changes, accounting for about 60%-70% of all dementia. The latest data shows that there are currently 57.4 million dementia patients globally, and it is expected to reach 160 million by 2040. [1,2] The typical pathological features in the brains of AD patients are the aggregation of oligomeric β-amyloid (Aβ) plaques, the formation of neurofibrillary tangles (NFTs) due to the hyperphosphorylation of Tau protein, and extensive inflammatory responses in the brain. [3,4] Currently, due to the complex etiology and unclear pathological mechanism of this disease, the research progress of its therapeutic drugs has been slow, and there is still a lack of drugs or methods for curing AD globally. All along, immunotherapy against Aβ has also been a research hotspot, but due to the great difficulty in drug research and development, most of the drugs entering phase III clinical trials have ended in failure at the present stage. [2,5,6] Therefore, it is urgent to conduct multi-angle pathological mechanism research on AD and search for rapid detection biological markers. Since the occurrence and development of AD is a dynamic evolution process, the interval between the early stage of the disease with Aβ deposition and neuron death and the appearance of dementia symptoms can be as long as more than 20 years, and it is an irreversible chronic neurodegenerative disease process. Early clinical diagnosis and subsequent treatment of AD are the key points for solving the AD condition. [7-9] Currently, the main diagnostic methods for AD are combined diagnoses, mainly including: neuropsychological assessment, cognitive impairment testing; PET scans of cerebral senile plaques and Tau protein; magnetic resonance imaging (MRI) of the brain and cerebrospinal fluid (CSF) markers, Aβ deposition, and phosphorylated tau protein detection, etc.

[0010] However, due to the non - obvious early symptoms of AD, when a clear diagnosis of the disease is made, most patients have reached the late stage of the disease, and most neurons have died. If rapid diagnosis can be carried out in the early stage of the patient's illness or potential patients can detect risks in advance and receive targeted treatment or prevention, it can help the patient's condition stay in the stage of mild cognitive impairment (MCI) and slow down the deterioration, thus ensuring the patient's quality of life and reducing the social burden. Among them, for the early molecular screening techniques of AD such as positron emission tomography (PET) and cerebrospinal fluid Aβ molecular level detection, the former requires injecting a certain dose of radioactive substances into the subject; the latter has great operational damage and is prone to surgical infections. The reliability of these diagnostic techniques for early diagnosis of AD is also not very stable, so it is difficult to be used for early screening of AD. Currently, the prevention, diagnosis, treatment, and rehabilitation of AD are recognized difficult problems in the world at present. Therefore, the development of new markers for early diagnosis of AD and simple detection methods are one of the important directions for future diagnosis and treatment of AD. Summary of the Invention In order to solve the problems of complex detection and inaccurate detection results in the early diagnosis of AD in the prior art, the present invention provides the application of a reagent for detecting changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid in the preparation of a diagnostic tool or a treatment tool for diagnosing or monitoring Alzheimer's disease. Further, the immune molecules include IL - 1α and IFN - γ. Further, the immune cells are subsets of peripheral circulatory immune cells. Further, the immune cells include CD8 + T cells. An object of the present invention is to provide a method for diagnosing or monitoring Alzheimer's disease. The method for diagnosing or monitoring Alzheimer's disease uses the reagent for detecting changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid in the above - mentioned application to detect the changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid. An object of the present invention is a method for treating Alzheimer's disease. The method for treating Alzheimer's disease takes immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid as the drug administration targets to reduce or decrease the concentration or quantity of the immune cells and the immune factors. Further, the immune molecules include IL - 1α and IFN - γ. Further, the immune cells are subsets of peripheral circulatory immune cells. Further, the immune cells include CD8 +T cells. In the present invention, the inflammatory factor IFN-γ and the peripheral circulating immune cell subsets are selected as markers for AD, which can directly detect peripheral blood samples, judge the inflammatory level of the body, and be used in diagnostic tools and treatment tools for early screening of AD. The operation is simple and the risk is relatively low; the detection efficiency is improved, the accuracy of the results is increased, and it has practical application value. It can be used to guide clinical screening, research on the pathogenic mechanism of AD, and screening of AD treatment drugs, and has broad application prospects. Brief Description of the Drawings Figure 1 is an immunofluorescence staining picture of the brain tissues of wild-type mice and APP / PS1 mice provided in Example 1; Figure 2 is a flow cytometry analysis of adaptive immune cell subsets in the peripheral blood of wild-type mice (WT) and APP / PS1 mice (AD) provided in Example 2; Figure 3 is the expression level of inflammatory factors in the peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 3; Figure 4 is the expression level of the inflammatory factor IL-1α in the peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 4; Figure 5 is the expression level of the inflammatory factor IFN-γ in the peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 4; Figure 6 is the expression level of IFN-γ in the cerebrospinal fluid of wild-type mice and APP / PS1 mice provided in Example 5; Figure 7 is the expression level of IL-1α in the cerebrospinal fluid of wild-type mice and APP / PS1 mice provided in Example 5; Figure 8 is the expression level of IL-1α in the cerebral cortex and hippocampus of wild-type mice and APP / PS1 mice provided in Example 6; Figure 9 is the expression level of IFN-γ in the cerebral cortex and hippocampus of wild-type mice and APP / PS1 mice provided in Example 6; Detailed Embodiments In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be made in conjunction with the accompanying drawings, but it should not be construed as a limitation on the scope of the present invention that can be implemented. This invention uses mouse models of different months of age as research objects. Through techniques such as immunohistofluorescence staining of brain tissue, total RNA extraction, qRT-PCR, and enzyme-linked immunosorbent assay (ELISA) of peripheral serum, by comparing between littermate wild-type (littermate wild-type) and diseased groups (APP / PS1 transgenic mice, AD), it has obtained a clear conclusion that the abnormal occurrence of inflammatory factors (IL-1α, IFN-γ, IL-10, IL-12p70, and IL-17A), natural killer cells (Nature killer cell, NK cell), and B cells in the peripheral blood of AD transgenic mice is related to the occurrence and development of brain inflammation. Based on this, this invention provides reliable evidence that changes in the levels of inflammatory factors (IL-1α and IFN-γ) in peripheral plasma, cerebrospinal fluid, and brain, as well as the numbers of natural killer cells and B cells in peripheral blood, can be used as molecular markers for early diagnosis of AD: The disease course of patients corresponding to 3-month-old AD mice is about to enter the stage of mild cognitive impairment (MCI), and 6-month-old AD mice correspond to the stage with clear AD disease characteristics. Through specific experimental steps (see examples below), it is obtained that the expressions of peripheral blood inflammatory factors IL-1α and IFN-γ in 3-month-old AD mice are both significantly increased, and their expression levels are consistent with those in cerebrospinal fluid and brain parenchyma. The following will further describe the features and properties of this invention in detail in combination with examples: Materials involved in this invention: Littermate wild-type mice and APP / PS1 mouse models are from Jackson Laboratory in the United States; The multi-cytokine detection kit is purchased from Millipore, product number: MHSTCMAG-70K; The cytokine IL-1α detection kit is purchased from R&D System, product number: MLA00; The cytokine IFN-γ detection kit is purchased from R&D System, product number: MIF00; Paraformaldehyde is purchased from Sigma-aldrich, product number: 158127; The embedding agent OCT is purchased from SAKURA, product number: 4583; The CD8α primary antibody is purchased from Invitrogen, product number: 14-0195-82; DAPI is purchased from Thermo scientific, product number: 62248; The fluorescent secondary antibody is purchased from Thermo scientific; The red blood cell lysis buffer is purchased from BD Biosciences, product number: 555899; The antibodies used for flow cytometry analysis were all purchased from BD Biosciences, and the product numbers are as follows: Ms CD45 FITC 30-F11, product number: 553079; Ms CD3 MolCpx PerCP-Cy5.5 17A2, product number: 560527; Ms CD8a PE 53-6.7, product number: 553032; Ms CD19 PE-Cy7 1D3, product number: 552854; Ms CD49b APC DX5, product number: 560628; Horse serum was purchased from Gibco, product number: 26050088; Fetal bovine serum was purchased from Life Technologies, product number: 16050-122; DAPI was purchased from Thermo scientific, product number: D1306; DPBS was purchased from Sigma, product number: D8662-24*500ML; Trizol was purchased from invitrogen, product number: 15596026; The reverse transcription kit was purchased from Thermo scientific, product number K1622; The real-time fluorescence quantitative PCR kit was purchased from Thermo scientific, product number 4368706; The ELISA kit was purchased from R&D, product number: MJE00B. Example 1: Detection of the dynamic infiltration of peripheral immune cells into the central nervous system in an AD mouse model In this example, an immunofluorescence staining kit was used to perform CD8, Iba1, and DAPI staining on the brain tissues of wild-type mice and APP / PS1 mouse models (AD mouse models). The steps are as follows: 1.1. Mouse brain tissue sectioning (1) Anesthesia and brain tissue perfusion sampling: Inject chloral hydrate into the abdominal cavity of the mouse for anesthesia. After deep anesthesia, fix it on the surgical board, place it in the dissection tray, remove the brain from the posterior end of the head, and soak it in paraformaldehyde for fixation for 24 h. (2) Perfusion fixation of the mouse: Perfuse the mouse with PBS at 4°C, 20 mL per mouse, and then use 4% paraformaldehyde at 4°C (weigh 40 g of paraformaldehyde and dissolve it in a glass container containing 500 mL of DEPC water, continuously heat, and stir magnetically until At 60 °C, a milky white suspension was formed. The pH was adjusted to 7.0 with 1.0 mmol / L NaOH to make the solution clear. Then, about 500 mL of 2×PBS was added, mixed well, filtered, and made up to 1000 mL, and stored at 4 °C for later use) perfusion, 20 mL per mouse, until the tissue became hard. (3) Sampling: Carefully dissect the brain tissue and place it in a 15 mL centrifuge tube, and post-fix it with 4% paraformaldehyde (fixative) for 24 h. (4) Dehydration: Wash the tissue fixed with paraformaldehyde 3 times with PBS (washing solution), dehydrate it with 20% sucrose (dehydrating agent) until the tissue sinks to the bottom, and dehydrate it with 30% sucrose at 4 °C overnight. (5) Drop the embedding agent OCT onto the specimen stage, put it into the cryostat until it turns white, then take it out and quickly trim the surface with a single-sided blade. (6) After trimming the bottom of the specimen flat with a safety blade, adhere it to the specimen stage, then place it in the freezing stage of the cryostat at -24 °C. When the tissue turns slightly white, apply a thin layer of OCT on the surface of the specimen and continue to freeze for 20 min. (7) After adjusting the section thickness, start sectioning. The section thickness is 20 μm. The cut sections are continuously collected and transferred into a 24-well plate containing 4% paraformaldehyde. (8) Store the cut sections at 4 °C for later use. 1.2. Immunofluorescence staining (1) Carefully pick out the sections at the appropriate position and transfer them to a 24-well plate pre-filled with 1 mL of pre-cooled PBS, and wash them 3 times with pre-cooled PBS, 10 min each time. (2) Punching and blocking: 0.2% Triton X-100 (diluted with PBS), 0.1% BSA, and 5% horse serum (diluted with PBS), incubate at room temperature for 40 min, and place it on a shaker and shake slowly. (3) Wash 3 times with PBS at room temperature, 5 min each time. (4) Primary antibody incubation: Dilute the antibody with an antibody diluent (containing 0.01% BSA and 5% horse serum in PBS) at a ratio of 1:100, add 200 μL to each well, and incubate at 4 °C with slow shaking overnight. (5) Recover the primary antibody, wash 3 times with PBS at room temperature, 10 min each time. (6) Block the brain sections with 3% horse serum at room temperature for 30 min. (7) Secondary antibody incubation and DAPI staining: Dilute the secondary antibody with PBS at a ratio of 1:5000, incubate at room temperature in the dark for 2 h; dilute the DAPI stock solution at a ratio of 1:5000 and incubate at room temperature for 15 min. (8) Wash 3 times with PBS at room temperature, 15 min each time. (9) Coverslipping: Take a sticky glass slide, mark specific information with a pencil on the frosted surface on the right side. Drop a drop of PBS in the middle of the glass slide, pick up the section and place it on the PBS droplet, then suck out the PBS solution. Horizontally spread 160 μL of coverslipping agent in the center of the glass slide, and cover the section with a long coverslip, avoiding the generation of air bubbles and wrinkles. (10) Place it flat in a dark place to dry. 1.3. Experimental Results Staining pictures of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M), 6 months old (6M), 9 months old (9M), and 12 months old (12M) are shown in Figure 1. Result pictures of immunofluorescence staining of brain tissues of wild-type mice (WT) and APP / PS1 mice (AD) at 3-month-old, 6-month-old, 9-month-old, and 12-month-old (scale bar = 50 μm). In the figure are brain tissue sections of different months, T cell surface marker CD8, and nuclear marker DAPI. As can be seen from the figure, at 6 months old, there are CD8 + T cells of peripheral origin infiltrating into the AD brain, playing an immunomodulatory role. As the disease progresses, the infiltration of CD8 + T cells in the brain tissues of 9-month-old and 12-month-old AD mice increases, and the number of activated microglia cells increases and shows an aggregated state, while this phenomenon does not occur in 3-month-old mice. This result indicates that in the AD mouse model, the occurrence and development of CD8 + T cell-mediated neuroinflammation occurs after 3 months old, at 6 months old or earlier stages. This shows that the change in the number of CD8 + T cells can be used as an early biomarker for AD. Example 2: Dynamic changes and flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) in an AD mouse model In this example, flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) of wild-type mice and APP / PS1 mice at 6 months old and 9 months old. The steps are as follows: 2.1. Preparation of highly active peripheral blood mononuclear cells (PBMCs) (1) Add 3 volumes of red blood cell lysis buffer to 200 μL of anticoagulated whole blood, gently mix well, and let it stand at room temperature for 10 min, gently mixing 2 times during this period to lyse red blood cells. (2) Centrifuge at 800×g for 2 min, discard the supernatant, collect the cells, and wash the sample once with 1 mL of PBS. (3) Resuspend with 500 μL of buffer, filter through a 300-mesh cell strainer, incubate with antibodies, and then analyze by flow cytometry. 2.2 Flow cytometry analysis (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 bovine serum. (2) Take 40 μL of the cell suspension and add it to a plastic centrifuge tube pre-loaded with 50 μL of fluorescently labeled specific antibody, then add 50 μL of inactivated normal horse serum (diluted 1:20 with DPBS), and incubate at 4°C for 30 min. (3) Add 2 mL of DPBS containing 2% fetal bovine serum to resuspend and mix the cells, centrifuge at 1000 rpm at 4°C for 5 min, and repeat the cell washing once. (4) Add 500 μL of pre-cooled PBS to resuspend the cells and prepare for flow cytometry analysis. 2.3 Experimental results Based on the changes in the infiltration levels of CD8+ cells in the brains of AD mice of different months of age, this example selected the early stage of AD: 3-month-old (3M) and 6-month-old (6M) mice for flow cytometry analysis of PBMCs T lymphocyte subsets. The results are shown in Figure 2. Changes in the subsets of adaptive immune cells in the peripheral blood of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP / PS1 mice (AD) (*p<0.05). Flow cytometry results 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 decreased, and the expression of cytotoxic T cells (CD8 + T cells) was significantly increased, indicating that immune cells in the peripheral circulation can play an immunomodulatory role in the neuroinflammation in the AD brain. It was revealed that during the AD process, the immune response mediated by the peripheral blood circulation system plays an immunomodulatory role in the early stage of AD onset. Therefore, CD8 + T cells can be used as peripheral circulation biomarkers for early AD diagnosis. Example 3: Changes in the expression levels of inflammatory factors in the plasma of peripheral blood of AD In this example, the plasma inflammatory factors of 3-month-old and 6-month-old wild-type mice and APP / PS1 mice were detected. This example used a multi-cytokine detection kit The specific implementation steps are briefly described as follows: 3.1 Sample collection (1) Plasma sample collection: After anesthetizing 3-month-old and 6-month-old wild-type and AD littermate mice with isoflurane gas, collect blood samples from the mice by fundus blood collection method into 1.5 mL sterilized EP tubes anticoagulated with EDTA-Na2. After decapitating the mice by breaking their necks and quickly cutting off their heads with scissors, separate the plasma according to the following method; (2) Place the anticoagulated blood samples at 4 °C for 4 h. After the blood coagulates, serum will separate out naturally. Centrifuge at 4000 rpm for 30 min at 4 °C to separate the serum, and discard the insoluble matter; (3) Transfer the plasma to a new sterilized EP tube, divide it into aliquots and store at -80 °C for later use. 3.2. Reagent preparation and configuration (1) Magnetic bead preparation: Ultrasonic the required magnetic beads for 30 seconds, vortex for 1 minute, then take out 60 μL each and add them to the mixing liquid tube. Make up the remaining volume to 3 mL with the analysis buffer, mix well, and set aside. (2) Quality control preparation: Dissolve control 1 and control 2 with 250 μL of distilled water respectively, invert several times to mix well, let stand for 5 - 10 minutes, and then transfer them into two test tubes respectively, set aside. (3) Standard preparation: Dissolve the standard with 250 μL of distilled water, invert several times to mix well, let stand for 5 - 10 minutes, and then transfer it into a test tube, labeled as S6. Then take another 5 test tubes, labeled as S5 / S4 / S3 / S2 / S1 respectively. Add 200 μL of analysis buffer to each tube. Finally, take out 50 μL from S6 for serial dilution, set aside. (4) Wash buffer preparation: Place the 10-fold concentrated wash buffer at room temperature to fully dissolve the salts in it. Add 60 mL of the 10-fold concentrated wash buffer to 540 mL of distilled water to prepare the working concentration, set aside. (5) Plasma matrix preparation: Add 2 mL of analysis buffer to the plasma matrix, dissolve it well, let stand for 10 min, and then transfer it into a test tube and store at -20 °C for one month. 3.3. Cytokine detection (1) Add 200 μL of wash buffer to each well of the 96-well plate, shake at room temperature for ten minutes for rinsing, then pour it out directly and dry it thoroughly. (2) According to the instructions, add 25 μL of analysis buffer, standard, positive control, and plasma matrix to the negative control, standard, positive control, and sample wells respectively. (3) Add the magnetic beads to each well and incubate at 4 °C overnight with shaking in the dark. (4) Wash twice with wash buffer, 5 minutes for each wash. (5) Add 25 μL of detection antibody to each well and shake at room temperature in the dark for 1 hour. (6) Add 25 μL of SAPE to each well and shake at room temperature in the dark for 30 minutes. (7) Wash twice with washing buffer, 5 minutes for each wash. (8) Finally, add 150 μL of sheath fluid to each well, shake for 5 minutes, and then perform on-machine detection. 3.4. Experimental Results The expression levels of inflammatory factors GM-CSF, IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, LIX, IL-17A, KC, MCP-1, MIP-2, and TNF-α in the peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months of age (3M) and 6 months of age (6M) are shown in Figure 3. As can be seen from the figure, compared with the WT group of mice, the expression levels of all the cytokines in the table showed an increasing trend in the peripheral blood serum of 3-month-old AD mice. With the development of the disease process, in 6-month-old mice, compared with the WT group of mice, IFN-γ, IL-1α, IL-1β, IL-2, IL-7, IL-12 (p70), LIX, and MCP-1 in the peripheral blood plasma of AD mice showed a continuous upward trend. It indicates that the peripheral blood inflammatory factors are involved in the disease process caused by AD in the peripheral blood circulation system at the early stage of AD occurrence and can be used as peripheral blood biomarkers for early AD diagnosis. Example 4: Detection of the Expression Levels of Plasma Inflammatory Factors IFN-γ and IL-1α In this example, an ELISA kit was used to detect the expression levels of IL-1α and IFN-γ in the cerebrospinal fluid and peripheral blood plasma of wild-type mice and APP / PS1 mice at 3 months of age and 6 months of age. The steps are as follows: 4.1. Collection of Plasma Samples The plasma collection method was the same as that in Example 3. 4.2. Reagent Preparation (1) Preparation of positive control: Dissolve the IL-1α or IFN-γ positive control with 1 mL of deionized water respectively, mix well, and reserve. (2) Preparation of washing solution: Dilute with deionized water to the working concentration at a ratio of 1:25. (3) Preparation of luminescent reagent: 15 minutes before on-machine detection, mix the luminescent reagent A and reagent B in the kit at a volume ratio of 1:1, and store in the dark. (4) Preparation of IL-1α and IFN-γ standards: Dilute the 5000 pg / mL standard provided in the kit with calibration diluent at a ratio of 1:10 in a new EP tube to a 500 pg / mL standard, and then dilute it into standards with concentrations of 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, 15.6 pg / mL, and 7.81 pg / mL respectively. 4.3, ELISA detection (1) Add 50 μL of assay diluent RD1W to the test wells. (2) Add standard samples, control samples, and samples to be tested to the test wells in sequence. After sealing with the sealing film provided in the kit, incubate at room temperature for 2 h. (3) After incubation, tear off the sealing film, discard the liquid, add 400 μL of washing solution to each well to wash the detection plate, wash 4 times, and discard the washing solution. (4) Add 100 μL of mouse IL-1α or IFN-γ conjugate to the test wells. After sealing with the sealing film, incubate at room temperature for 2 h. (5) Repeat step (3) once. (6) Add 100 μL of the prepared luminescent reagent to the test wells, and incubate at room temperature for 39 min in the dark. (7) Add 100 μL of the termination solution, and gently flick the test plate to ensure thorough mixing. (8) Read the plate: Complete the optical density detection of each well within 30 min. (9) Calculate: Perform concentration quantification calculation according to the formula provided in the kit. 4.4, Experimental results The expression levels of inflammatory factors IFN-γ and IL-1α in the peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M) are shown in Figures 4 and 5. Figure 4. Results of the expression level of IL-1α in the peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M) (*p < 0.05, ***p < 0.001). Figure 5. Results of the expression level of IFN-γ in the peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M) (*p < 0.05). As can be seen from the figure, compared with the WT group of mice, the expression levels of IFN-γ (Figure 5) and IL-1α (Figure 4) in the peripheral blood plasma of AD mice at 3 months old and 6 months old were significantly increased. It is shown that IFN-γ and IL-1α are involved in the disease process caused by AD in the early stage of AD. With the development of the disease, they play a role in promoting the inflammatory defense of the immune system. Therefore, IFN-γ and IL-1α in the peripheral blood can be used as peripheral blood biomarkers for the early diagnosis of AD. Example 5: Detection of the expression levels of inflammatory factors IFN-γ and IL-1α in cerebrospinal fluid 5.1, Collection of cerebrospinal fluid samples After anesthetizing the mice, fix the head on the stereotaxic apparatus. When collecting cerebrospinal fluid, wipe the skin of the back of the rat's neck with a 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 dorsal muscles of the neck with scissors. To avoid bleeding, scrape the deepest layer of muscle attached to the bone with the back of the scalpel to expose the atlanto-occipital membrane. Insert a needle into the foramen magnum to directly extract cerebrospinal fluid. After extraction, suture the outer muscles and skin. Sulfonamide powder can be sprinkled on the incision to prevent infection. After collecting cerebrospinal fluid, an equal amount of sterilized normal saline should be injected to maintain the original pressure in the cerebrospinal cavity. 5.2. The reagent preparation and ELISA detection process are the same as those in Example 4. 5.3. Experimental results To further confirm that the expression levels of IFN-γ and IL-1α in peripheral blood are positively correlated with the level of neuroinflammation in the central nervous system, the present invention detected the expression levels of the inflammatory factors IFN-γ and IL-1α in the cerebrospinal fluid of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M). The results are shown in Figures 6 and 7. Figure 6. Results of the expression level of IL-1α in the cerebrospinal fluid of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M) (***p < 0.001). Figure 7. Results of the expression level of IFN-γ in the cerebrospinal fluid of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months old (3M) and 6 months old (6M) (**p < 0.01, ***p < 0.001). As can be seen from the figure, compared with the WT group of mice, the expression of IFN-γ in the cerebrospinal fluid of 3-month-old AD mice was inhibited (Figure 7). As the disease progressed, the expression level of IFN-γ in the cerebrospinal fluid of 6-month-old AD mice (Figure 7) increased significantly, and the expression of IL-1α (Figure 6) increased significantly in the cerebrospinal fluid of 3-month-old mice. This indicates that IFN-γ and IL-1α are involved in the activation of the peripheral and central immune systems caused by AD in the early stage of AD. It further proves that IFN-γ and IL-1α in peripheral blood can be used as peripheral blood biomarkers for the early diagnosis of AD. Experiment 6: Total RNA extraction from brain tissue and qPCR detection In this example, a real-time fluorescence quantitative PCR kit was used to detect the expression levels of IL-1α and IFN-γ in the cerebral cortex (cortex) and hippocampus (hippocampus) of wild-type mice and APP / PS1 mice at 3 months old and 6 months old, respectively. The steps are as follows: 6.1. RNA extraction (1) After anesthetizing 3-month-old and 6-month-old wild-type and AD littermate mice with isoflurane (gas), decapitate them quickly with scissors after breaking their necks. Place the heads on ice and rapidly isolate the cerebral cortex, as well as the hippocampus of the mice. Wash the isolated tissues twice with DPBS containing 4 U / mL protease inhibitor and RNase inhibitor. (2) Homogenization treatment: Grind the tissues or cells in liquid nitrogen. Add 1 mL of Trizol (RNA extraction reagent) to every 100 mg of tissue and perform homogenization treatment with a homogenizer. (3) Let the homogenized samples stand at room temperature for 5 min to completely separate the nucleic acid-protein complexes. (4) Add 0.2 mL of chloroform to every 1 mL of Trizol used, shake vigorously for 15 s, and let it stand at room temperature for 3 min. (5) Centrifuge at 10000×g for 15 min at 4℃ (the tabletop refrigerated centrifuge is purchased from Eppendorf, model: 5425R). (6) Transfer the aqueous phase to a new tube and precipitate the RNA in the aqueous phase with isopropanol. Add 0.5 mL of isopropanol to every 1 mL of Trizol used and let it stand at room temperature for 10 min. (7) Centrifuge at 10000×g for 10 min at 4℃. A gelatinous precipitate appears on the side and bottom of the tube. Discard the supernatant. (8) Wash the RNA precipitate with 75% ethanol. Add 1 mL of 75% ethanol to every 1 mL of Trizol used. Centrifuge at 7500×g for 5 min at 4℃ and discard the supernatant. (9) Let it stand at room temperature to dry for 5 min. Add 50 μL of RNase-free water, pipette a few times with a pipette tip, and let it stand at 55℃ for 10 min to dissolve the RNA. Store at -70℃. 6.2, Reverse transcription Use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA. The steps are as follows: Prepare reaction mixture I in a RNase-free centrifuge tube. The system is as follows: After mixing the above components, centrifuge quickly for 5 s. Incubate at 70℃ for 5 min, then ice-bath for 2 min. Then prepare reaction mixture II according to the following system. The system is as follows: Add reaction mixture I to reaction mixture II, mix quickly for 5 s, incubate at 70℃ for 5 min, then ice-bath for 2 min. Perform reverse transcription according to the following program: 25℃, 5 min; 42℃, 60 min; 70℃, 5 min. Store the obtained cDNA template at -20℃ for standby. 6.3, Real-time fluorescence quantitative PCR The expression levels of IL-1α and IFN-γ were detected using a real-time fluorescence quantitative PCR kit. The reaction system was as follows: Among them, the sequences of the forward primers are shown in Table 1, as shown by forward primer F, and the sequences of the reverse primers are shown by R. Mix the above components, centrifuge at 6000 rpm for 1 min, and perform amplification according to the following program: Pre-denaturation: 95°C, 10 min; Cyclic amplification: 95°C, 15 s; 60°C, 1 min; 70°C, 1 min; cycle 40 times; Generate a melting curve: 95°C, 15 s; 60°C, 1 min; During the whole process, the heating and cooling rates are 1.6°C / s. 6.4. Experimental results To further prove that the expression levels of IFN-γ and IL-1α in peripheral blood are correlated with the level of inflammation in the brain parenchyma, the present invention detected the gene expression levels of IFN-γ and IL-1α in the cerebral cortex and hippocampus of 3-month-old and 6-month-old wild-type mice and AD mice. Figure 8. Results of IL-1α expression levels in the cerebral cortex (Cortex) and hippocampus (Hippocampus) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP / PS1 mice (AD) (*p<0.05). Figure 9. Results of IFN-γ expression levels in the cerebral cortex (Cortex) and hippocampus (Hippocampus) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and APP / PS1 mice (AD). As can be seen from Figures 8 and 9, in 3-month-old and 6-month-old WT mice, the changes in cytokine expression among wild-type mouse individuals in the same group were relatively concentrated. In AD mice, the differences in cytokine expression among different individuals in the same group were relatively large, and the gene expression level of IL-1α was significantly increased in the cerebral cortex of 3-month-old AD model mice, while an inhibitory effect was shown in the hippocampal region. This experiment further demonstrated the obvious correlation between the peripheral and central expression of IFN-γ and IL-1α, further proving that IL-1α in peripheral blood discovered in the present invention can be used as a diagnostic indicator for early AD. The data of the present invention indicate that in AD mice, the immune surveillance of PBMC subsets has significant changes at different disease stages, especially the significant increase in the number of cytotoxic T cells, and the tendency of these cells to aggregate towards the inflammatory sites is found in the brains of 6-month-old AD mice. This suggests that circulating immune cells in peripheral blood may play an important role in immunoregulating the pathological process of AD. Early AD can be diagnosed based on the changes in immune cells and immune molecules in peripheral cerebrospinal fluid, and new biomarkers and potential new drug targets for treating AD can be screened through the changes in specific molecules. Its advantage lies in that the clinical diagnosis of the occurrence and development of AD by detecting biomarkers in blood not only reduces the diagnostic cost but also is easy to operate, avoiding the risk of central nervous system infection during the brain sampling process of the subjects. References 1. Patterson, C., Alzheimer's Disease Internation 2018. World Alzheimer Report 2018. pp. (1 - 48) 2. Scheltens, P., et al., Alzheimer's disease. The Lancet, 2021. 3. Hardy, J. and D. J. Selkoe, The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 2002. 297(5580): p. 353 - 6. 4. Heneka, M. T., et al., Neuroinflammation in Alzheimer's disease. Lancet Neurol, 2015. 14(4): p. 388 - 405. 5. Honig, L. S., et al., Trial of Solanezumab for Mild Dementia Due to Alzheimer's Disease. N Engl J Med, 2018. 378(4): p. 321 - 330. 6.Vandenberghe,R.,et al.,Bapineuzumab for mild to moderate Alzheimer's disease in two global,randomized,phase 3trials.Alzheimers Res Ther,2016.8(1):p.18. 7.Mufson,E.J.,et al.,Mild cognitive impairment:pathology and mechanisms.Acta Neuropathol,2012.123(1):p.13-30. 8.Long,J.M.and D.M.Holtzman,Alzheimer Disease:An Update on Pathobiology and Treatment Strategies.Cell,2019.179(2):p.312-339. 9.Masters,C.L.,et al.,Alzheimer's disease.Nat Rev Dis Primers,2015.1:p.15056. 10.Selkoe,D.J.,Alzheimer disease and aducanumab:adjusting our approach.Nat Rev Neurol,2019.15(7):p.365-366.

Claims

1. Use of a reagent for detecting changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid in the preparation of a diagnostic tool or a therapeutic tool for diagnosing or monitoring Alzheimer's disease.

2. The use according to claim 1, wherein The immune molecules include IL-1α and IFN-γ.

3. The use according to claim 1, wherein The immune cells are subsets of peripheral circulating immune cells.

4. The use according to claim 1, wherein The immune cells include CD8 + T cells.

5. A method for diagnosing or monitoring Alzheimer's disease, wherein Use the reagent for detecting changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid in the application according to any one of claims 1-4 to detect changes in the concentration or number of immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid.

6. A method for treating Alzheimer's disease, wherein Using the immune cells and immune factors in the peripheral circulatory system and cerebrospinal fluid as the drug administration targets, reduce or decrease the concentration or quantity of the immune cells and the immune factors.

7. The method according to claim 6, wherein The immune molecules include IL-1α and IFN-γ.

8. The method according to claim 6, wherein The immune cells are subsets of peripheral circulating immune cells.

9. The method according to claim 6, wherein The immune cells include CD8 + T cells.

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