Application of detection reagent in preparation of diagnostic tool for diagnosing or monitoring AD

Abstract

The invention belongs to the technical field of biology, and provides application of a reagent for detecting the concentration of immune cells and immune factors in peripheral circulating cerebrospinal fluid in preparation of a diagnostic tool or a treatment tool for diagnosing or monitoring AD (Alzheimer's disease). Clinical diagnosis of AD occurrence and development is performed by detecting biomarkers in blood, so that the diagnosis cost is reduced, the operation is easy, and the risk of central infection in the intracerebral sampling process of a subject is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of a reagent for detecting the concentration or number change 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

[0002] Alzheimer's disease (AD), also known as senile dementia, accounts for about 60%-70% of all dementias among all dementia types. It is an age-related central nervous system degenerative disease and has become the seventh leading cause of death globally after lung cancer. Clinically, AD patients show progressive memory loss, cognitive impairment, language disorders, memory decline, decreased executive ability, and personality changes, accounting for about 60%-70% of all dementias. 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 protein (Aβ) plaques, the formation of neurofibrillary tangles (NFTs) due to the hyperphosphorylation of Tau protein, and extensive inflammatory reactions 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 is 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 extremely urgent to conduct multi-angle pathological mechanism research on AD and search for rapid detection biomarkers.

[0003] Since the occurrence and development of AD is a dynamic evolution process, the interval from the early stage of the disease with Aβ deposition and neuronal death to 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 tests; 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.

[10] 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 discover the 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 subjects; the latter has a large operational injury and is prone to cause surgical infections. The reliability of these diagnostic techniques for the early diagnosis of AD is also not very stable, so it is difficult to be used for the early screening of AD. Currently, the prevention, diagnosis, treatment, and rehabilitation of AD are recognized as difficult problems in the world. Therefore, the development of new markers for the early diagnosis of AD and simple detection methods is one of the important directions for the future diagnosis and treatment of AD. Summary of the Invention

[0004] In order to solve the problems of complex detection and inaccurate detection results in the prior art for the early diagnosis of AD, the present invention provides the use of a reagent for detecting the concentration or number change 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.

[0005] Further, the immune molecules include IL - 1α and IFN - γ.

[0006] Further, the immune cells are subsets of peripheral circulatory immune cells.

[0007] Further, the immune cells include CD8 + T cells.

[0008] The present invention selects the inflammatory factor IFN - γ and subsets of peripheral circulatory immune cells as markers for AD, which can directly detect peripheral blood samples, judge the body's inflammatory level, and be used in the diagnostic tool and therapeutic tool for the early screening of AD. The operation is simple and the risk is low; it improves the detection efficiency, increases the accuracy of the results, has practical application value, and can be used to guide clinical screening, the study of the pathogenic mechanism of AD, and the screening of AD treatment drugs, with broad application prospects. Brief Description of the Drawings

[0009] Figure 1 Brain tissue immunofluorescence staining pictures of wild - type mice and APP / PS1 mice provided for Example 1;

[0010] Figure 2Flow cytometry analysis of adaptive immune cell subsets in peripheral blood of wild-type mice (WT) and APP / PS1 mice (AD) provided in Example 2;

[0011] Figure 3 The expression levels of inflammatory factors in the peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 3;

[0012] Figure 4 The expression level of inflammatory factor IL-1α in peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 4;

[0013] Figure 5 The expression level of inflammatory factor IFN-γ in peripheral blood plasma of wild-type mice and APP / PS1 mice provided in Example 4;

[0014] Figure 6 The expression level of IFN-γ in the mouse cerebrospinal fluid of wild-type mice and APP / PS1 mice provided in Example 5;

[0015] Figure 7 The expression level of IL-1α in the mouse cerebrospinal fluid of wild-type mice and APP / PS1 mice provided in Example 5;

[0016] Figure 8 The expression levels of IL-1α in the cerebral cortex and hippocampus of wild-type mice and APP / PS1 mice provided in Example 6.

[0017] Figure 9 The IFN-γ expression levels in the cerebral cortex and hippocampus of wild-type mice and APP / PS1 mice provided in Example 6. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0019] The present invention uses mouse models of different months of age as research objects, and through brain tissue immunofluorescence staining technology, total RNA extraction technology, qRT-PCR technology, enzyme-linked immunosorbent assay (ELISA) of peripheral serum, etc., by comparing the wild-type littermates (wild-type littermates) and the diseased group (APP / PS1 transgenic mice, AD), a clear conclusion is obtained that abnormalities in peripheral blood inflammatory factors (IL-1α, IFN-γ, IL-10, IL-12p70 and IL-17A) and natural killer cells (NK cells) and B cells in the peripheral circulation of AD transgenic mice are related to the occurrence and development of brain inflammation.

[0020] Based on this, the present 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 the early diagnosis of AD: the disease progression of the patients corresponding to 3-month-old AD mice is about to enter the stage of mild cognitive impairment (MCI), while that corresponding to 6-month-old AD mice is the stage with clear AD disease characteristics. Through specific experimental procedures (see the examples below for details), it is found that the expressions of peripheral blood inflammatory factors IL-1α and IFN-γ in 3-month-old AD mice are significantly increased, and their expression levels are consistent with those in cerebrospinal fluid and brain parenchyma.

[0021] The features and properties of the present invention will be further described in detail below in combination with examples:

[0022] Materials involved in the present invention:

[0023] Littermate wild-type mice and APP / PS1 mouse models are from Jackson Laboratory, USA;

[0024] Multicytokine detection kit is purchased from Millipore, product number: MHSTCMAG-70K;

[0025] Cytokine IL-1α detection kit is purchased from R&D System, product number: MLA00;

[0026] Cytokine IFN-γ detection kit is purchased from R&D System, product number: MIF00;

[0027] Paraformaldehyde is purchased from Sigma-aldrich, product number: 158127;

[0028] Embedding agent OCT is purchased from SAKURA, product number: 4583;

[0029] CD8α primary antibody is purchased from Invitrogen, product number: 14-0195-82;

[0030] DAPI is purchased from Thermo scientific, product number: 62248;

[0031] Fluorescent secondary antibody is purchased from Thermo scientific;

[0032] Red blood cell lysate is purchased from BD Biosciences, product number: 555899;

[0033] Antibodies used for flow cytometry analysis are all purchased from BD Biosciences, and the product numbers are as follows:

[0034] Ms CD45 FITC 30-F11, Catalog No.: 553079;

[0035] Ms CD3 MolCpx PerCP-Cy5.5 17A2, Catalog No.: 560527;

[0036] Ms CD8a PE 53-6.7, Catalog No.: 553032;

[0037] Ms CD19 PE-Cy7 1D3, Catalog No.: 552854;

[0038] Ms CD49b APC DX5, Catalog No.: 560628;

[0039] Horse serum was purchased from Gibco, Catalog No.: 26050088;

[0040] Fetal bovine serum was purchased from Life Technologies, Catalog No.: 16050-122;

[0041] DAPI was purchased from Thermo scientific, Catalog No.: D1306;

[0042] DPBS was purchased from Sigma, Catalog No.: D8662-24*500ML;

[0043] Trizol was purchased from invitrogen, Catalog No.: 15596026;

[0044] Reverse transcription kit was purchased from Thermo scientific, Catalog No. K1622;

[0045] Real-time fluorescence quantitative PCR kit was purchased from Thermo scientific, Catalog No. 4368706;

[0046] ELISA kit was purchased from R&D, Catalog No.: MJE00B.

[0047] Example 1: Detection of the dynamic infiltration of peripheral immune cells into the central nervous system in an AD mouse model

[0048] 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:

[0049] 1.1. Mouse brain tissue sectioning

[0050] (1) Anesthesia and brain tissue perfusion sampling: Mice were anesthetized by intraperitoneal injection of chloral hydrate. After deep anesthesia, they were fixed on a surgical board, placed in a dissection tray, and the brain was removed from the posterior end of the head and immersed in paraformaldehyde for fixation for 24 h.

[0051] (2) Perfusion fixation of mice: Mice were perfused with PBS at 4°C, 20 mL per mouse. Then, they were perfused with 4% paraformaldehyde at 4°C (40 g of paraformaldehyde was weighed and dissolved in a glass container containing 500 mL of DEPC water, continuously heated, and magnetically stirred until 60°C to form a milky white suspension. 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, thoroughly mixed, filtered, and made up to 1000 mL and stored at 4°C for later use), 20 mL per mouse, until the tissue became hard.

[0052] (3) Specimen collection: The brain tissue was carefully dissected and placed in a 15 mL centrifuge tube, and post-fixed with 4% paraformaldehyde (fixative) for 24 h.

[0053] (4) Dehydration: The tissue fixed with paraformaldehyde was washed 3 times with PBS (washing solution), dehydrated with 20% sucrose (dehydrating agent) until the tissue sank to the bottom, and then dehydrated with 30% sucrose at 4°C overnight.

[0054] (5) OCT embedding medium was dropped onto the specimen stage and placed in a cryostat until it turned white. Then, it was taken out and the surface was quickly trimmed flat with a single-sided blade.

[0055] (6) After trimming the bottom of the specimen flat with a safety blade, it was adhered to the specimen stage, then placed in the freezing stage of a -24°C cryostat. When the tissue became slightly white, a thin layer of OCT was applied to the surface of the specimen, and it was frozen for another 20 min.

[0056] (7) After adjusting the section thickness, sectioning was started. The section thickness was 20 μm. The cut sections were continuously collected and transferred into a 24-well plate containing 4% paraformaldehyde.

[0057] (8) The cut sections were stored at 4°C for later use.

[0058] 1.2 Immunofluorescence staining

[0059] (1) Sections at appropriate positions were carefully selected and placed into a 24-well plate containing 1 mL of pre-cooled PBS, and washed 3 times with pre-cooled PBS, 10 min each time.

[0060] (2) Punching and blocking: 0.2% Triton X-100 (diluted with PBS), 0.1% BSA, and 5% horse serum (diluted with PBS), incubated at room temperature for 40 min, placed on a shaker and shaken slowly.

[0061] (3) Wash 3 times with PBS at room temperature, 5 min each time.

[0062] (4) Primary antibody incubation: Dilute the primary antibody at a ratio of 1:100 using an antibody diluent (0.01% BSA and 5% horse serum in PBS), add 200 μL to each well, and incubate overnight at 4°C with slow shaking.

[0063] (5) Recover the primary antibody and wash the wells 3 times with PBS at room temperature for 10 minutes each time.

[0064] (6) Block the brain slices with 3% horse serum at room temperature for 30 minutes.

[0065] (7) Secondary antibody incubation and DAPI staining: Dilute the secondary antibody at a ratio of 1:5000 using PBS and incubate for 2 hours at room temperature in the dark; dilute the DAPI stock solution at a ratio of 1:5000 and incubate for 15 minutes at room temperature.

[0066] (8) Wash with PBS 3 times at room temperature for 15 minutes each time.

[0067] (9) Mounting: Take a sticky glass slide, mark the specific information with a pencil on the frosted surface on the right side. Place a drop of PBS in the middle of the slide, pick up the section and place it on the PBS droplet, then suck out the PBS solution. Spread 160 μL of mounting medium horizontally in the center of the slide, cover the section with a long coverslip, avoiding the formation of air bubbles and wrinkles.

[0068] (10) Let it dry flat in a dark place.

[0069] 1.3. Experimental results

[0070] Staining pictures of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3M), 6 months (6M), 9 months (9M), and 12 months (12M) are as Figure 1 shown. Result pictures of immunofluorescence staining of brain tissues of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3-month-old), 6 months (6-month-old), 9 months (9-month-old), and 12 months (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.

[0071] As can be seen from the figure, there is infiltration of CD8 + T cells of peripheral origin into the cortex and hippocampus of 6-month-old AD mice, 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, CD8 +The occurrence and development of T cell-mediated neuroinflammation occur after 3 months of age, at 6 months of age or earlier. This indicates that the change in the number of CD8 + T cells can be used as an early biomarker for AD.

[0072] Example 2: Dynamic changes and flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) in an AD mouse model

[0073] 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 and 9 months of age. The steps are as follows:

[0074] 2.1 Preparation of highly active peripheral blood mononuclear cells (PBMCs)

[0075] (1) Add 3 volumes of red blood cell lysate to 200 μL of anticoagulated whole blood, mix gently, and let stand at room temperature for 10 min. During this period, mix gently 2 times to lyse red blood cells.

[0076] (2) Centrifuge at 800×g for 2 min, discard the supernatant, collect the cells, and wash the sample once with 1 mL of PBS.

[0077] (3) Resuspend with 500 μL of buffer, filter through a 300-mesh cell strainer, incubate with antibodies, and then analyze on the machine.

[0078] 2.2 Flow cytometry analysis

[0079] (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.

[0080] (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.

[0081] (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 washing the cells once.

[0082] (4) Resuspend the cells with 500 μL of pre-cooled PBS and prepare for analysis on the machine.

[0083] 2.3 Experimental results

[0084] Based on the changes in the infiltration level of CD8+ cells in the brains of AD mice of different months of age, in this example, the early stage of AD: 3-month-old (3M) and 6-month-old (6M) mice were selected for flow cytometry analysis of PBMCs T lymphocyte subsets, and the results are as 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).

[0085] 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 reveals 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.

[0086] Example 3: Changes in the expression levels of inflammatory factors in the plasma of peripheral blood of AD

[0087] In this example, the plasma inflammatory factors of 3-month-old and 6-month-old wild-type mice and APP / PS1 mice were detected. In this example, a multi-cytokine detection kit was used The specific implementation steps are briefly described as follows:

[0088] 3.1. Sample collection

[0089] (1) Plasma sample collection:

[0090] After anesthetizing 3-month-old and 6-month-old littermate wild-type and AD mice with isoflurane gas, collect the blood samples of the mice by fundus blood collection method into 1.5 mL sterilized EP tubes anticoagulated with EDTA-Na2, decapitate the mice after breaking their necks and quickly cut off their heads with scissors, and separate the plasma according to the following method;

[0091] (2) Place the anticoagulated blood sample at 4°C for 4 h, wait for the blood to coagulate and then naturally separate the serum, centrifuge at 4000 rpm for 30 min at 4°C to separate the serum, and discard the insoluble matter;

[0092] (3) Transfer the plasma to a new sterilized EP tube, aliquot and store it at -80°C for later use.

[0093] 3.2. Reagent preparation and configuration

[0094] (1) Preparation of magnetic beads: Ultrasonicate the required magnetic beads for 30 seconds, vortex for 1 minute, then take out 60 μl of each and add it to the mixed solution tube. Make up the remaining volume to 3 ml with analysis buffer, mix thoroughly and set aside.

[0095] (2) Quality control preparation: Dissolve control 1 and control 2 in 250 μl of distilled water respectively, invert several times to mix thoroughly, let stand for 5-10 minutes, then transfer to two test tubes respectively and set aside.

[0096] (3) Preparation of standard: Dissolve the standard in 250 μl of distilled water, invert several times to mix thoroughly, let stand for 5-10 minutes, and then transfer to a test tube labeled S6. Then take 5 test tubes, label them S5 / S4 / S3 / S2 / S1, add 200 μl of assay buffer to each tube, and finally take out 50 μl from S6 for gradient dilution and set aside.

[0097] (4) Preparation of washing solution: Place 10 times concentrated washing solution at room temperature to allow the salt therein to fully dissolve. Add 60 ml of 10 times concentrated washing solution to 540 ml of distilled water to prepare it to the working concentration and set aside.

[0098] (5) Preparation of plasma matrix: Add 2 ml of analytical buffer to the plasma matrix, allow it to fully dissolve, let it stand for 10 min, then transfer it to a test tube and store it at -20°C for one month.

[0099] 3.3 Cytokine detection

[0100] (1) Add 200 μl of washing buffer to each well of a 96-well plate, shake at room temperature for ten minutes to rinse, then pour out and wipe dry.

[0101] (2) According to the instructions, add 25 μl of assay buffer, standards, positive controls, and plasma matrix to the negative control, standards, positive controls, and sample wells, respectively.

[0102] (3) Add magnetic beads to each well and incubate with shaking at 4°C in the dark overnight.

[0103] (4) Wash twice with washing buffer, 5 minutes each time.

[0104] (5) Add 25 μl of detection antibody to each well and shake at room temperature in the dark for 1 hour.

[0105] (6) Add 25 μl of SAPE to each well and shake at room temperature in the dark for 30 minutes.

[0106] (7) Wash twice with washing buffer, 5 minutes each time.

[0107] (8) Finally, add 150 μl of sheath fluid to each well and shake for 5 minutes before testing on the instrument.

[0108] 3.4. Experimental Results

[0109] 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 (3M) and 6 months (6M) are as Figure 3 shown.

[0110] As can be seen from the figure, compared with the WT group of mice, the expression levels of all 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. This 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, and can be used as peripheral blood biomarkers for the early diagnosis of AD.

[0111] Example 4: Detection of the Expression Levels of Plasma Inflammatory Factors IFN-γ and IL-1α

[0112] 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 and 6 months. The steps are as follows:

[0113] 4.1. Collection of Plasma Samples

[0114] The method for plasma collection is the same as that in Example 3.

[0115] 4.2. Reagent Preparation

[0116] (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.

[0117] (2) Preparation of washing solution: Dilute it to the working concentration with deionized water at a ratio of 1:25.

[0118] (3) Preparation of luminescent reagent: 15 minutes before the 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.

[0119] (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 obtain a 500 pg / mL standard, and then further dilute it to 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.

[0120] 4.3 ELISA Detection

[0121] (1) Add 50 μL of assay diluent RD1W to the test wells.

[0122] (2) Add standards, control samples, and test samples to the test wells in sequence. After sealing with the sealing film provided in the kit, incubate at room temperature for 2 h.

[0123] (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.

[0124] (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.

[0125] (5) Repeat step (3) once.

[0126] (6) Add 100 μL of the prepared luminescence reagent to the test wells and incubate in the dark at room temperature for 39 min.

[0127] (7) Add 100 μL of the termination solution and gently flick the test plate to ensure thorough mixing.

[0128] (8) Read the plate: Complete the optical density detection of each well within 30 min.

[0129] (9) Calculation: Perform concentration quantification calculation according to the formula provided in the kit.

[0130] 4.4 Experimental Results

[0131] 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 (3M) and 6 months (6M) are as Figure 4 and Figure 5 shown. Figure 4 . The results of IL-1α expression levels in the peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3M) and 6 months (6M) (*p < 0.05, ***p < 0.001). Figure 5. IFN-γ expression levels in peripheral blood plasma of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3M) and 6 months (6M) of age (*p<0.05).

[0132] As can be seen from the figure, compared with the WT group, the expression levels of IFN-γ ( Figure 5 ) and IL-1α ( Figure 4 ) in the peripheral blood plasma of AD mice at 3 months and 6 months of age were significantly increased. It indicates that IFN-γ and IL-1α are involved in the disease process caused by AD at the early stage of AD development, and play a role in promoting the inflammatory defense of the immune system as the disease progresses. Therefore, IFN-γ and IL-1α in peripheral blood can be used as peripheral blood biomarkers for early AD diagnosis.

[0133] Example 5: Detection of expression levels of cerebrospinal fluid inflammatory factors IFN-γ and IL-1α

[0134] 5.1 Collection of cerebrospinal fluid samples

[0135] After the mice were anesthetized, their heads were fixed 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 muscle attached to the bone with the back of the scalpel to expose the atlanto-occipital membrane. Insert a needle directly into the foramen magnum to 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 sterile saline should be injected to maintain the original pressure of the cerebrospinal cavity.

[0136] 5.2 The reagent preparation and ELISA detection process are the same as in Example 4.

[0137] 5.3 Experimental results

[0138] To further confirm that the expression levels of peripheral blood IFN-γ and IL-1α 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 (3M) and 6 months (6M) of age. The results are as Figure 6 and Figure 7 shown. Figure 6 . IL-1α expression levels in cerebrospinal fluid of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3M) and 6 months (6M) of age (***p<0.001). Figure 7. Expression levels of IFN-γ in the cerebrospinal fluid of wild-type mice (WT) and APP / PS1 mice (AD) at 3 months (3M) and 6 months (6M) of age (**p<0.01, ***p<0.001).

[0139] As can be seen from the figure, compared with the mice in the WT group, the expression of IFN-γ in the cerebrospinal fluid of 3-month-old AD mice was inhibited ( Figure 7 ), and as the disease progressed, the IFN-γ in the cerebrospinal fluid of 6-month-old AD mice ( Figure 7 ) showed a significant increase in the expression level, and IL-1α ( Figure 6 ) showed a significant increase in the cerebrospinal fluid of 3-month-old mice. It 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 development. It further proves that IFN-γ and IL-1α in peripheral blood can be used as peripheral blood biomarkers for early AD diagnosis.

[0140] Experiment 6: Total RNA extraction from brain tissue and qPCR detection

[0141] 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 and hippocampus of wild-type mice and APP / PS1 mice at 3 months and 6 months of age, and the steps are as follows:

[0142] 6.1. RNA extraction

[0143] (1) After anesthetizing 3-month-old and 6-month-old littermate wild-type and AD mice with isoflurane (gas), decapitate them quickly with scissors after breaking their necks, place the heads on ice, and quickly separate the cerebral cortex and the hippocampus of the mice. Wash the separated tissues twice with DPBS containing 4 U / mL of protease inhibitor and RNase inhibitor.

[0144] (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.

[0145] (3) Let the homogenized samples stand at room temperature for 5 min to completely separate the nucleic acid-protein complexes.

[0146] (4) Add 0.2 mL of chloroform to every 1 mL of Trizol, shake vigorously for 15 s, and let it stand at room temperature for 3 min.

[0147] (5) Centrifuge at 10000×g for 15 min at 4°C (the tabletop refrigerated centrifuge is purchased from Eppendorf, model: 5425R).

[0148] (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 for every 1 mL of Trizol used and let it stand at room temperature for 10 min.

[0149] (7) Centrifuge at 10000×g for 10 min at 4℃. A gelatinous precipitate will appear on the side and bottom of the tube. Discard the supernatant.

[0150] (8) Wash the RNA precipitate with 75% ethanol. Add 1 mL of 75% ethanol for every 1 mL of Trizol used and centrifuge at 7500×g for 5 min at 4℃. Discard the supernatant.

[0151] (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℃.

[0152] 6.2, Reverse transcription

[0153] Use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA. The steps are as follows:

[0154] Prepare reaction mixture I in a RNase-free centrifuge tube. The system is as follows:

[0155]

[0156] 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:

[0157]

[0158] 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:

[0159] 25℃, 5 min; 42℃, 60 min; 70℃, 5 min.

[0160] Store the obtained cDNA template at -20℃ for later use.

[0161] 6.3, Real-time fluorescence quantitative PCR

[0162] Use a real-time fluorescence quantitative PCR kit to detect the expression levels of IL-1α and IFN-γ. The reaction system is as follows:

[0163]

[0164] Among them, the sequence of the forward primer is as shown in Table 1, as shown by forward primer F, and the sequence of the reverse primer is as shown by R.

[0165]

[0166] Mix the above components, centrifuge at 6000 rpm for 1 min, and perform amplification according to the following procedure:

[0167] Pre-denaturation: 95 °C, 10 min;

[0168] Cyclic amplification: 95 °C, 15 s; 60 °C, 1 min; 70 °C, 1 min; cycle 40 times;

[0169] Form melting curve: 95 °C, 15 s; 60 °C, 1 min;

[0170] The heating and cooling rates during the whole process are 1.6 °C / s.

[0171] 6.4. Experimental results

[0172] 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 detects 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).

[0173] The results show that Figure 8 and Figure 9 It can be seen that in 3-month-old and 6-month-old WT mice, the changes in cytokine expression among wild-type mouse individuals in the same group are relatively concentrated. In AD mice, the differences in cytokine expression among different individuals in the same group are relatively large, and the gene expression level of IL-1α in the cerebral cortex of 3-month-old AD model mice is significantly increased while showing an inhibitory effect in the hippocampal region. This experiment further proves that there is an obvious correlation between the peripheral and central expression of IFN-γ and IL-1α, and further proves that IL-1α in peripheral blood found in the present invention can be used as a diagnostic indicator for early AD.

[0174] The data of the present invention indicate that in AD mice, the immune surveillance of PBMC subsets has significant changes at different disease stages. In particular, the number of cytotoxic T cells increases significantly, and a tendency to aggregate towards the inflammatory sites is found in the brains of 6-month-old AD mice. This suggests that circulating immune cells in the peripheral blood may play an important role in immune regulation of the AD pathological process. Early AD is diagnosed based on the changes in immune cells and immune molecules in the peripheral circulating cerebrospinal fluid, and new biomarkers and potential new drug targets for the treatment of AD are 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 the blood not only reduces the diagnostic cost, but is also easy to operate, avoiding the risk of central nervous system infection during the brain sampling process of the subjects.

[0175] References

[0176] 1. Patterson, C., Alzheimer's Disease International 2018. World Alzheimer Report 2018. pp. (1 - 48)

[0177] 2. Scheltens, P., et al., Alzheimer's disease. The Lancet, 2021.

[0178] 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.

[0179] 4. Heneka, M. T., et al., Neuroinflammation in Alzheimer's disease. Lancet Neurol, 2015. 14(4): p. 388 - 405.

[0180] 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.

[0181] 6. Vandenberghe, R., et al., Bapineuzumab for mild to moderate Alzheimer's disease in two global, randomized, phase 3 trials. Alzheimers Res Ther, 2016. 8(1): p. 18.

[0182] 7. Mufson, E.J., et al., Mild cognitive impairment: pathology and mechanisms. Acta Neuropathol, 2012. 123(1): p. 13 - 30.

[0183] 8. Long, J.M. and D.M. Holtzman, Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell, 2019. 179(2): p. 312 - 339.

[0184] 9. Masters, C.L., et al., Alzheimer's disease. Nat Rev Dis Primers, 2015. 1: p. 15056.

[0185] 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, characterized in that, The immune cells are subsets of peripheral circulatory immune cells.

4. The use according to claim 1, characterized in that, The immune cells include CD8 + T cells.

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