Substances for detecting human ATP5D, CD69 and CXCR4 genes and their applications

Abstract

The present application discloses the use of a substance for detecting human ATP5D, CD69 and CXCR4 genes in the preparation of a product for detecting human Alzheimer's disease. The present application also discloses a product for detecting human Alzheimer's disease, the product comprising a substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to substances for detecting human ATP5D, CD69 and CXCR4 genes and their applications. Background Art

[0002] According to recent statistics, about 50 million people worldwide are suffering from dementia, and this number is expected to double every 20 years, reaching about 152 million cases by 2050. Among them, Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly, characterized by progressive cognitive decline, with the main symptoms being memory loss and the inability to generate new memories. In particular, after Alzheimer's disease develops to a certain extent, it can only delay the progression of the disease but cannot be cured, so early diagnosis and judgment of the disease are very important.

[0003] According to current reports, Alzheimer's disease is induced under a variety of complex pathological and physiological conditions, including amyloid cascade, Tau protein phosphorylation, neurotransmitters, excitotoxicity or oxidative stress. Therefore, the onset and progression of Alzheimer's disease are very difficult to prevent, so the current focus has shifted to early diagnosis and treatment to slow down disease progression and improve cognitive decline.

[0004] The main pathological feature of Alzheimer's disease (AD) is the accumulation of β-amyloid (Aβ) peptide or neurofibrillary tangles (NFTs) of Tau protein in the brain, which is closely related to the neurodegenerative mechanism caused by the toxicity and destruction of neurons and synapses during its onset.

[0005] As current diagnostic methods, there are brain imaging, cognitive function tests, and cerebrospinal fluid detection methods, but these methods can only be applied after a large part of the symptoms of Alzheimer's disease appear, so these methods are not enough for early diagnosis. In addition, it is also economically expensive to diagnose, and there are potential dangers in invasive examination steps. Such biomarker tests are not always feasible and have limitations.

[0006] In contrast, blood-based diagnostics help overcome these shortcomings because they are non-invasive, inexpensive, and capable of large-scale, multiple sampling. Therefore, in many recent studies, the correlation between blood-based Alzheimer's disease biomarkers and pathological changes in the brain has been studied. However, despite these efforts, there is still a lack of blood-based Alzheimer's disease biomarker detection methods that can directly reflect the onset and progression of Alzheimer's disease. Summary of the invention

[0007] The present application provides the use of materials for detecting human ATP5D, CD69 and CXCR4 genes in the preparation of products for detecting human Alzheimer's disease.

[0008] The present application also provides a product for detecting human Alzheimer's disease, wherein the product comprises a substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes.

[0009] The beneficial effects brought by the present application include but are not limited to: 1. The substance for detecting Alzheimer's disease provided by the present application can detect a decrease in the number of B lymphocytes and the expression levels of their specific genes ATP5D, CD69 and CXCR4 in the peripheral blood or brain slices of Alzheimer's patients, with strong specificity and high accuracy; 2. The kit prepared according to the above substances can realize diagnosis or prediction through blood test, overcoming the technical obstacle of the prior art that only cerebrospinal fluid can be used for diagnosis during the asymptomatic period, greatly alleviating the suffering of patients, and promoting the treatment policy of early detection and early diagnosis and treatment of the disease, which is of great significance for the diagnosis, treatment and research of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include:

[0011] FIG1 is a single-cell RNA sequencing analysis diagram of ATP5D, CD69 and CXCR4 expression in B lymphocytes of AD patients according to some embodiments of the present application; Figure 1A : Heat map of differentially expressed genes in B lymphocytes of normal, early and late AD patients. Red represents high expression and blue represents low expression; Figure 1B : Volcano plot of up-regulated and down-regulated differential proteins in B lymphocytes of AD patients and normal subjects. Green and red dots represent down-regulated and up-regulated genes, respectively. Figure 1C : RT-qPCR was used to detect the expression changes of ATP5D, IL32, JUNB, LGALS1, HLA-DRB5, MT-CYB, MT-ND2, CCL5, CD69, CXCR4, S100A4, and S100A6 in normal individuals (n=41) and AD patients (n=43). The original data were analyzed using independent sample t test; Figure 1D :Comparison of single-cell RNA sequencing and RT-qPCR results, screening genes with the same trend, ATP5D, CD69 and CXCR4, for subsequent research; Figure 1E-1F : t-SNE and quantitative line graphs show the expression changes of ATP5D, CD69 and CXCR4 in 18 cell populations, respectively. Red shades indicate different expression levels, green and red indicate downregulation and upregulation, respectively. AD: Alzheimer's disease.

[0012] FIG2 shows the expression changes of ATP5D, IL32, JUNB, LGALS1, HLA-DRB5, MT-CYB, MT-ND2, CCL5, CD69, CXCR4, S100A4, and S100A6 under different CDR scores, ages, and genders according to some embodiments of the present application. Figure 2A Normal subjects and patients with CDR of 1 (n=15), 2 (n=16), 3 (n=12), Figure 2B Patients younger than 78 years (n=16) or older than 78 years (n=20), and Figure 2C Expression in male (n = 11) and female (n = 22) patients. The raw data were analyzed by one-way ANOVA followed by post hoc test ( Figure 2A ), or analyzed by independent sample t test ( Figure 2B , Figure 2C ). CDR: Clinical Dementia Rating.

[0013] FIG3 shows the expression of ATP5D, CD69 and CXCR4 in PBMCs, cortex and hippocampus of healthy subjects and AD patients according to some embodiments of the present application, Figure 3A : Expression of ATP5D, CD69, and CXCR4 in healthy subjects (n=41) and AD patients with CDR scores of 1 (n=15), 2 (n=16), and 3 (n=12). Raw data were analyzed by one-way ANOVA followed by post hoc test; Figure 3B : Correlation analysis between the expression of ATP5D, CD69 and CXCR4 and CDR scores (CDR 0 (healthy subjects) n = 41; CDR 1, n = 15; CDR 2, n = 16; CDR 3, n = 12). The data were analyzed using Pearson's correlation coefficient; Figure 3C-Figure 3D : Immunofluorescence staining and quantitative analysis of the expression of CD19 and ATP5D (n=5 for healthy, n=6 for early AD, n=7 for late AD), CD19 and CD69 (n=7 for healthy, n=8 for early AD, n=7 for late AD), CD19 and CXCR4 (n=7 for healthy, n=7 for early AD, n=8 for late AD) in cell smears from PBMCs of healthy subjects, early and late AD patients. Scale bar = 50 μm. Raw data were analyzed by one-way ANOVA followed by post hoc test; Figure 3E-3H: Immunofluorescence staining and quantitative analysis of CD19 and ATP5D, CD19 and CD69, CD19 and CXCR4 expression in hippocampus (CD19: healthy n=6, AD n=7; ATP5D: healthy n=7, AD n=7; CD69: n=4 for healthy, n=5 for AD; CXCR4: n=6 for healthy, n=5 for AD) and cortex (CD19: n=5 for healthy, n=5 for AD; ATP5D: healthy n=5, AD n=5; CD69: n=5 for healthy, n=3 for AD; CXCR4: healthy n=5, AD n=4) from 3 healthy individuals and 5 AD patients. Raw data were presented and analyzed using independent sample t test. White arrows indicate positive cells, green indicates CD19 positive cells (B lymphocyte marker), red indicates ATP5D, CD69 and CXCR4, respectively, blue indicates DAPI (cell nucleus), CDR: clinical dementia grade; AD: Alzheimer's disease.

[0014] FIG4 is a diagram showing the expression and bioinformatics analysis of ATP5D, CD69 and CXCR4 in the cerebral cortex, hippocampus and B lymphocytes of WT and AD mice according to some embodiments of the present application, Figure 4A-4C : Expression of ATP5D, CD69, and CXCR4 in B lymphocytes of WT mice at 3 months (n=6), 10 months (n=6), and 18 months (n=6) and AD mice at 15 months (n=6), and WT mice at 3 months (n=6) and AD mice at 15 months (n=6). The raw data were analyzed by one-way ANOVA followed by post hoc test or independent sample t test; Figure 4D :PPI diagram of B lymphocyte-related differentially expressed genes, green circles indicate genes associated with ATP5D, red circles indicate genes associated with CD69, and yellow circles indicate genes associated with CXCR4. The larger the circle and the thicker the line, the more significant the correlation. Protein interaction analysis of differentially expressed genes was performed on STRING (https: / / string-db.org / ) and displayed with Cytoscape (https: / / cyto scape.org / ). (EF) KEGG and GO (BP, CC, and MF) analysis of B lymphocyte-related genes, red boxes indicate the most relevant pathways as well as BP, CC, and MF, R software, “clusterProfiler” package was used for KEGG and GO analysis, and further visualized by “enrichplot” package, M: month, WT: wild type, AD: Alzheimer’s disease.

[0015] Figure 5The differences in the expression of ATP5D, CD69 and CXCR4 in B cells between the control group and AD patients and the diagnostic efficacy of the combination of the three factors are shown in some embodiments of the present application. (A) Box scatter plot of ATP5D expression in B cells between the control group (n=29) and the AD group (n=33) patients, which is statistically significant (P<0.05). (B) Box scatter plot of CD69 expression in B cells between the control group and the AD group patients, which is statistically significant (P<0.05). (C) Box scatter plot of CXCR4 expression in B cells between the control group and the AD group patients, which is statistically significant (P<0.05). (D) The ROC curve of ADP5D predicting AD was independently analyzed, showing an AUC of 0.6520, P<0.05. (E) The ROC curve of CD69 predicting AD was independently analyzed, showing an AUC of 0.7482, P<0.05. (F) The ROC curve of CXCR4 for diagnosing AD was analyzed independently, showing an AUC of 0.6729, P < 0.05. (G) Using the predicted value in the logistic analysis, the ROC curve was used to analyze the predictive ability of ATP5D, CD69 and CXCR4 combined with the three factors, showing an AUC of 0.7461, P < 0.05. (H) The ROC curve of ATP5D, CD69 and CXCR4 for diagnosing AD was combined and the AUCs were 0.6520, 0.7482 and 0.6729, respectively.

[0016] Figure 6The differences in the expression of ATP5D, CD69 and CXCR4 in B cells at different ages and the diagnostic efficacy of the combination of the three factors are shown in some embodiments of the present application. (A) Box scatter plot of ATP5D expression in B cells of AD patients younger than 78 years old (14 people) and older than 78 years old (19 people), which is statistically significant (P < 0.05). (B) Box scatter plot of CD69 expression in B cells of AD patients younger than 78 years old and older than 78 years old, which is statistically significant (P < 0.05). (C) Box scatter plot of CXCR4 expression in B cells of AD patients younger than 78 years old and older than 78 years old, which is statistically significant (P < 0.05). (D) The ROC curve of ADP5D in diagnosing AD patients younger than 78 years old and older than 78 years old was independently analyzed, showing an AUC of 0.7331, P < 0.05. (E) The ROC curve of CD69 in diagnosing AD patients younger than 78 years old and older than 78 years old was analyzed independently, showing an AUC of 0.9586, P < 0.05. (F) The ROC curve of CXCR4 in diagnosing AD patients younger than 78 years old and older than 78 years old was analyzed independently, showing an AUC of 0.8083, P < 0.05. (G) Using the predicted value in the logistic analysis, the ROC curve was used to analyze the diagnostic ability of ATP5D, CD69 and CXCR4 combined with the three factors, showing an AUC of 0.9586, P < 0.05. (H) The ROC curve of ATP5D, CD69 and CXCR4 in diagnosing AD patients younger than 78 years old and older than 78 years old was combined, showing an AUC of 0.7331, 0.9586 and 0.8083, respectively.

[0017] Figure 7The differences in the expression of ATP5D, CD69 and CXCR4 in B cells according to different genders and the diagnostic efficacy of the combination of three factors as shown in some embodiments of the present application. (A) Box scatter plot of ATP5D expression in B cells of males (11 people) and females (22 people) in AD patients, with no statistical significance (P>0.05). (B) Box scatter plot of CD69 expression in B cells of males and females in AD patients, with no statistical significance (P>0.05). (C) Box scatter plot of CXCR4 expression in B cells of males and females in AD patients, with no statistical significance (P>0.05). (D) The ROC curve of ATP5D in diagnosing male and female AD patients was independently analyzed, showing an AUC of 0.5455, P>0.05. (E) The ROC curve of CD69 in diagnosing male and female AD patients was independently analyzed, showing an AUC of 0.5744, P>0.05. (F) The ROC curve of CXCR4 in the diagnosis of male and female AD patients was analyzed independently, showing an AUC of 0.5826, P>0.05. (G) Using the predicted value in the logistic analysis, the diagnostic ability of ATP5D, CD69 and CXCR4 combined with the three factors was analyzed by ROC curve, showing an AUC of 0.9586, P>0.05. (H) The ROC curve of ATP5D, CD69 and CXCR4 in the diagnosis of male and female AD patients was combined, showing AUCs of 0.5455, 0.5744 and 0.5726, respectively. DETAILED DESCRIPTION

[0018] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0019] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0020] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0021] Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease with high morbidity, high medical costs, and heavy mental burden. It is an important health problem faced by the world. Dementia, represented by AD, affects 47 million patients worldwide, and this number will triple by 2050. Research on clinical diagnostic indicators of Alzheimer's disease has important clinical value for early diagnosis of Alzheimer's disease, early intervention, and treatment.

[0022] The present application provides the use of materials for detecting human ATP5D, CD69 and CXCR4 genes in the preparation of products for detecting human Alzheimer's disease.

[0023] In some embodiments, the ATP5D, CD69 and CXCR4 genes may be B lymphocyte-specific genes.

[0024] The Gene ID of the ATP5D gene in the NCBI GenBank database is 513. The Gene ID of the CD69 gene in the NCBI GenBank database is 969. The Gene ID of the CXCR4 gene in the NCBI GenBank database is 7852.

[0025] In some embodiments, the B lymphocytes may be CD19 positive cells.

[0026] In some embodiments, preferably, the B lymphocytes may be derived from peripheral blood and / or the central nervous system.

[0027] In some embodiments, the Alzheimer's disease patient has a reduced number of B lymphocytes in the peripheral blood and / or central nervous system.

[0028] In some embodiments, the expression levels of B lymphocyte-specific genes ATP5D, CD69 and CXCR4 in the peripheral blood and / or central nervous system of the Alzheimer's disease patient are all reduced.

[0029] In some embodiments, the expression of ATP5D, CD69 and CXCR4 may decrease as the clinical dementia score increases;

[0030] In some embodiments, the relative expression levels of ATP5D, CD69, and CXCR4 may be negatively correlated with clinical dementia scores.

[0031] In some embodiments, the substance for detecting the expression of ATP5D, CD69 and CXCR4 genes may include at least one of a primer pair, a probe or an antibody. In some embodiments, preferably, the primer pair for detecting ATP5D is as follows:

[0032] Forward primer: 5'-CCCCAACCAGATGTCCTTCA-3' (SEQ ID NO. 3), and reverse primer: 5'-TCTTCGGCCAACAACTGCA-3 (SEQ ID NO. 4);

[0033] In some embodiments, preferably, the primer pair for detecting CD69 is as follows:

[0034] Forward primer: 5'-ATGGTGCTACTCTTGCT-3' (SEQ ID NO. 7), and reverse primer: 5'-ATGCTGCTGACCTCTGT-3' (SEQ ID NO. 8);

[0035] In some embodiments, preferably, the CXCR4 primer pair is as follows:

[0036] Forward primer: 5'-AAATCTTCCTGCCCACCAT-3' (SEQ ID NO. 9), and reverse primer: 5'-CGCCAACATAGACCACCTT-3' (SEQ ID NO. 10).

[0037] The present application also provides a product for detecting human Alzheimer's disease, wherein the product comprises a substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes.

[0038] In some embodiments, the product can be any one of a kit, a chip, a membrane strip, a protein array, a composition or a detection system.

[0039] In some embodiments, the ATP5D, CD69 and CXCR4 genes can be B lymphocyte-specific genes. In some embodiments, preferably, the B lymphocytes can be CD19 positive cells. In some embodiments, further preferably, the B lymphocytes can be derived from peripheral blood and / or the central nervous system.

[0040] In some embodiments, the substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes can be at least one of a primer pair, a probe or an antibody.

[0041] In some embodiments, the detection primer of ATP5D can be:

[0042] Forward primer: 5'-CCCCAACCAGATGTCCTTCA-3' (SEQ ID NO. 3), and reverse primer: 5'-TCTTCGGCCAACAACTGCA-3 (SEQ ID NO. 4).

[0043] In some embodiments, the detection primers for CD69 may be:

[0044] Forward primer: 5'-ATGGTGCTACTCTTGCT-3' (SEQ ID NO. 7), and reverse primer: 5'-ATGCTGCTGACCTCTGT-3' (SEQ ID NO. 8).

[0045] In some embodiments, the CXCR4 detection primers may be:

[0046] Forward primer: 5'-AAATCTTCCTGCCCACCAT-3' (SEQ ID NO. 9), and reverse primer: 5'-CGCCAACATAGACCACCTT-3' (SEQ ID NO. 10).

[0047] The term "primer" as used herein refers to a naturally occurring oligonucleotide (e.g., a restriction fragment) or a synthetically produced oligonucleotide that is capable of serving as a starting point for the synthesis of a primer extension product that is complementary to a nucleic acid strand (a template or target sequence) when placed under appropriate conditions (e.g., buffer, salt, temperature, and pH) and in the presence of nucleotides and reagents for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase). Typically, a set of primers will consist of at least two primers, an "upstream primer" and a "downstream primer" that together define an amplicon (a sequence to be amplified using the primers).

[0048] The term "probe" refers to any molecule that can selectively bind to a target biomolecule (e.g., a nucleic acid sequence that hybridizes to the probe). In some embodiments, the probe can be labeled, for example, with a fluorescent group and a quenching group. In some embodiments, the probe can be a Taqman probe with a fluorescent reporter group added to the 5' end and a fluorescent quenching group added to the 3' end.

[0049] Antibodies refer to proteins with protective effects produced by the body due to antigen stimulation. In some embodiments, antibodies can be used as detection reagents to detect gene expression. In some embodiments, specific antibodies can be used to detect the expression of human ATP5D, CD69 and CXCR4 genes.

[0050] The present application also discloses a method for diagnosing human Alzheimer's disease, which utilizes the above-mentioned product to detect whether the expression levels of ATP5D, CD69 and CXCR4 genes from a sample to be tested are lower than those of a healthy person.

[0051] In some embodiments, the method may include the following steps: (1) collecting a sample to be tested and extracting sample genomic DNA; (2) preparing a reaction system, wherein the reaction system includes sample genomic DNA, DNA polymerase, a deoxynucleotide (dNTP) mixture, and a buffer solution; (3) performing an amplification reaction; (4) analyzing the results to obtain the expression levels of ATP5D, CD69, and CXCR4 genes in the sample to be tested; and (5) determining whether the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0052] By statistically analyzing a large number of samples, the present application obtained that the average expression levels of ATP5D, CD69 and CXCR4 genes in healthy people were 2.26, 2.82 and 6.78 respectively, and the average content of CD19 positive B lymphocytes in PBMCs in healthy people was 7.18%.

[0053] In some embodiments, when the expression level of ATP5D in the sample to be tested is significantly lower than 2.26 (p less than 0.05), it can be determined that the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0054] In some embodiments, when the expression level of CD69 in the sample to be tested is significantly lower than 2.82 (p less than 0.05), it can be determined that the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0055] In some embodiments, when the expression level of CXCR4 in the sample to be tested is significantly lower than 6.78 (p less than 0.05), it can be determined that the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0056] In some embodiments, when the expression levels of ATP5D, CD69 and CXCR4 genes in the sample to be tested are significantly lower than 2.26, 2.82 and 6.78 respectively (p less than 0.05), it can be determined that the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0057] In some embodiments, when the expression levels of ATP5D, CD69 and CXCR4 genes in the sample to be tested are significantly lower than 2.26, 2.82 and 6.78 respectively (p less than 0.05), and the content of CD19 positive B lymphocytes in PBMC is significantly lower than 7.18% (p less than 0.05), it can be judged that the person corresponding to the sample to be tested suffers from Alzheimer's disease.

[0058] The present application also discloses a method for predicting the risk of a subject developing Alzheimer's disease, wherein the method uses the above-mentioned product to detect whether the expression levels of ATP5D, CD69 and CXCR4 genes from a sample to be tested are lower than those of a healthy person.

[0059] In some embodiments, the method may include the following steps: (1) collecting a sample to be tested and extracting sample genomic DNA; (2) preparing a reaction system, wherein the reaction system includes sample genomic DNA, DNA polymerase, a deoxynucleotide (dNTP) mixture, and a buffer solution; (3) performing an amplification reaction; (4) analyzing the results to obtain the expression levels of ATP5D, CD69, and CXCR4 genes in the sample to be tested; and (5) predicting the risk of the subject developing Alzheimer's disease.

[0060] By statistically analyzing a large number of samples, the present application obtained that the average expression levels of ATP5D, CD69 and CXCR4 genes in healthy people were 2.26, 2.82 and 6.78 respectively, and the average content of CD19 positive B lymphocytes in PBMCs in healthy people was 7.18%.

[0061] In some embodiments, when the expression level of ATP5D in the sample to be tested is significantly lower than 2.26 (p less than 0.05), it can be predicted that the subject corresponding to the sample to be tested suffers from Alzheimer's disease.

[0062] In some embodiments, when the expression level of CD69 in the sample to be tested is significantly lower than 2.82 (p less than 0.05), it can be predicted that the subject corresponding to the sample to be tested suffers from Alzheimer's disease.

[0063] In some embodiments, when the expression level of CXCR4 in the sample to be tested is significantly lower than 6.78 (p less than 0.05), it can be predicted that the subject corresponding to the sample to be tested suffers from Alzheimer's disease.

[0064] In some embodiments, when the expression levels of ATP5D, CD69 and CXCR4 genes in the sample to be tested are significantly lower than 2.26, 2.82 and 6.78 respectively (p less than 0.05), it can be predicted that the subject corresponding to the sample to be tested suffers from Alzheimer's disease.

[0065] In some embodiments, when the expression levels of ATP5D, CD69 and CXCR4 genes in the test sample are significantly lower than 2.26, 2.82 and 6.78, respectively (p less than 0.05), and the content of CD19-positive B lymphocytes in PBMC is significantly lower than 7.18% (p less than 0.05), it can be predicted that the subject corresponding to the test sample suffers from Alzheimer's disease.

[0066] In some embodiments, the subject can be a mammal, such as a human.

[0067] In some embodiments, the amplification reaction can be a polymerase chain reaction (PCR). In some embodiments, the amplification reaction can be real-time fluorescence quantitative PCR. Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. Real-time fluorescence quantitative PCR (Quantitative Real-time PCR), also known as qPCR, is a method for detecting the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction by using fluorescent chemicals. The specific DNA sequence in the sample to be tested can be quantitatively analyzed by internal or external reference methods.

[0068] The term "sample" refers to any composition containing nucleic acid isolated from a subject. In some embodiments, the sample can be selected from at least one of blood, tissue, blood cells, bone marrow, ascites, fine needle biopsy samples, body fluids containing cells, free floating nucleic acids, sputum, saliva, urine, semen, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, skin swab, oral swab, nasal swab or lavage. In some embodiments, preferably, the sample can be blood or brain slices.

[0069] In some embodiments, tissue samples may include fresh tissue samples, formalin-fixed paraffin-embedded tissue (FFPET), etc. In some embodiments, the sample may be fresh or frozen (e.g., stored in liquid nitrogen, stored in a -80° refrigerator, stored in a -20° refrigerator). Fresh samples may be obtained from the subject and then directly processed (e.g., RNA extraction, etc.), or the sample may be frozen and stored and then processed when needed.

[0070] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0071] The relevant test methods in the examples of this application are as follows:

[0072] 1. Patient recruitment and ethics

[0073] The subjects were enrolled in the West China Hospital of Sichuan University (Sichuan, China) and divided into AD patients and age-matched healthy controls. According to the diagnostic guidelines of the National Institute on Aging (NIA) and the Alzheimer's Association (AA), the subjects were diagnosed with possible AD. The recruited AD patients met the following criteria: Mini-Mental State Examination (MMSE) score was no more than 26 points. In addition, the recruited healthy controls met the following criteria: (1) no history of psychiatric or neurological diseases in the past or present, and (2) MMSE score was higher than 27 points. All patients included in this study were evaluated for age, gender, and Clinical Dementia Rating (CDR). Subjects with HIS scores greater than 7 points were excluded from other dementia syndromes that were unlikely to be AD dementia, such as vascular dementia, Lewy body dementia, paralytic dementia, and dementia syndromes caused by other brain or physical diseases, depressive pseudodementia, delirium, or other acute psychiatric diseases that severely affected cognitive function, as well as patients with severe limb movement disorders, aphasia, visual and hearing impairment, and other reasons for being unable to cooperate with the study.

[0074] All research procedures involving human subjects were approved by the Clinical Trial and Biomedical Ethics Committee of West China Hospital, Sichuan University (approval number 20150236; Chinese Clinical Trial Registration number CHCTR1900022805). This study was conducted under the guidance of the Declaration of Helsinki. Venous blood was first collected from 41 healthy subjects and 43 AD patients at different stages of the disease, and the changes in molecular expression were verified by RT-qPCR, and correlation analysis was performed. Sichuan University provided cadaveric brain specimens from well-diagnosed AD patients (n=5) and age-matched non-demented patients (n=3) to further verify the expression of candidate molecules in B lymphocytes. The sample size was estimated by comparing the mean values ​​of two independent samples using the MedSci sample size tool: α=0.05, POWER=0.8. The primary indicators of the clinical study were scRNA-seq of venous blood mononuclear cells from healthy controls and AD patients at different stages and the verification of differentially expressed genes. Secondary indicators included observing the relationship between the differential molecules obtained from the scRNA-seq data analysis and the severity of the patient's disease (CDR score). Informed consent was obtained from all participants before blood and brain sample collection.

[0075] 2. Single-cell RNA-seq and its data processing

[0076] According to the single-cell RNA-seq results of this experiment (the single-cell RNA sequencing data of this application has been deposited in the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI) of the United States, and can be accessed through the GEO series accession number GSE168522 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE168522)), further analysis was performed. The raw image data files obtained by single-cell RNA-seq are converted into raw sequencing sequences through base calling analysis. It is called raw data or raw reads. The results are stored in the FASTQ (abbreviated as FQ) file format, which contains the sequence information of the sequencing sequence (Reads) and its corresponding sequencing quality information. A large number of analyses were performed on the data, such as tSNE dimensional clustering analysis, heat maps, differential gene screening, differential gene volcano maps, PPI, GO and KEGG analysis.

[0077] 3. Real-time quantitative polymerase chain reaction (RT-qPCR)

[0078] To further verify the differential expression of B lymphocytes, 12 abundant genes were screened to verify the differences in gene expression in PBMCs of AD patients, and RT-qPCR was used to detect the expression of ATP5D, CD69 and CXCR4 in the cerebral cortex and hippocampus of WT (3, 10, 18 months) and AD (15 months) mice, as well as the expression of ATP5D, CD69 and CXCR4 in B lymphocytes extracted from WT (3 months) and AD (15 months) mice. Briefly, total RNA was extracted using RNAiso Plus (Takara, Japan) according to the manufacturer's protocol, and complementary DNA (cDNA) was synthesized by Revetaid First Strand cDNA Synthesis Kit (Thermo, USA). The messenger RNA (mRNA) level of the target gene was then detected by polymerase chain reaction (PCR). CFX96 TM Real-time polymerase chain reaction was performed using a real-time system (Bio-Rad, USA). -ΔΔCT Methods The expression levels of target genes were normalized to the GAPDH value in each sample. The primer sequences are shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] 4. Immunofluorescence staining

[0083] Normal people, early and late AD patients PBMCs cell smears and AD late brain slices were used to further detect the expression of ATP5D, CXCR4 and CD69 in B lymphocytes (CD19). Cell smear pretreatment: After the frozen PBMCs were revived, they were washed with 0.01M PBST and the supernatant was discarded, and then 4% paraformaldehyde was used to fix the PBMCs for 20 minutes. Then centrifuged at 100g for 10 minutes, washed twice with 0.01M PBST, the supernatant was removed and 50μl of 0.01M PBST was added, and evenly smeared on the glass slide. Paraffin section pretreatment was as described above, and then blocked with 5% sheep serum + 0.3% TritonX-100. The primary rabbit anti-CD19 antibody (bs-0079, 1:200) and mouse anti-ATP5D antibody (66673-1-IgG1, 1:200) or mouse anti-CXCR4 (sc-53534-IgG1, 1:200) or mouse anti-CD69 (sc-373799, 1:200) were diluted with 2% goat serum and incubated together at 4°C for 18 hours. After that, the cells were rinsed again with 0.01M PBST for 3 times, 5 minutes each time. The secondary antibodies goat anti-mouse IgG 594 (red fluorescence, abbkine, 1:200) and goat anti-rabbit IgG 488 (green fluorescence, abbkine, 1:200) were diluted with 2% goat serum and incubated at 37°C for 1 hour. After rinsing in 0.01M PBST, DAPI containing anti-fluorescence quencher was added to the coverslips and observed and collected images under a two-photon confocal microscope (LeicaTCS SP8 DIVE) at high magnification (x400). Five fields of view were randomly selected, and the optical density values ​​of CD19, ATP5D, CXCR4, and CD69 factors were analyzed using Image J software.

[0084] 5. Enrichment analysis and survival analysis

[0085] Protein-protein interaction (PPI): 50 differentially expressed genes (DEGs) were imported into the string database to analyze protein interactions, and Cytoscape software (version 3.7.2) MCODE was used to obtain a subnetwork with higher connectivity. The "betweenness" topology algorithm in CytoNCA was used to further sort the factors and perform visual network analysis.

[0086] Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to functionally annotate the differentially expressed genes (DEGs) among the three groups with adjusted P values ​​as indicators of functional enrichment. The analysis steps were implemented by the “clusterProfiler” package and further visualized by the “enrichplot” package.

[0087] 6. Statistical analysis

[0088] All data were plotted using raw data, error bars represent standard deviations, P < 0.05 indicates statistical significance, and all statistical analyses were performed using SPSS 21.0 software (IBM Corporation, NY, USA). Independent sample T-tests were used to analyze the comparisons between two groups, and one-way ANOVA was used to analyze the comparisons between multiple groups. In addition, Pearson's correlation coefficient was used to analyze the correlation between CDR scores and the expression of ATP5D, CXCR4, and CD69 factors.

[0089] Example 1 - The expression levels of B lymphocytes and their specific genes ATP5D, CD69 and CXCR4 in the peripheral and central nervous systems of AD patients are reduced

[0090] Based on cluster analysis of gene expression data from early single-cell RNA sequencing, we screened for significantly up-regulated and down-regulated genes in B lymphocytes from healthy, early-stage, and late-stage AD patients ( Figure 1A ). Based on the volcano plot, 12 genes (ATP5D, IL32, JUNB, LGALS1, HLA-DRB5, MT-CYB, MT-ND2, CCL5, CD69, CXCR4, S100A4, S100A6) were screened for further study ( Figure 1B RT-qPCR confirmed that the expression of ATP5D, CXCR4, CD69, and S100A4 was downregulated (p=0.026, p=0.048, p=0.019, p=0.032), and 2 genes (MT-CYB, MT-ND2) were upregulated in B lymphocytes ( Figure 1C , p=0.009). In addition, the expression of these 12 genes in CDR was also detected. Except for IL32, LGALS1 and CCL5, they all changed with the increase of CDR score ( Figure 2A In addition, CD69, LGALS1, CXCR4, S100A6, HLA-DRB5, and MT-ND2 were expressed at lower levels in AD patients aged 78 years or older compared with those aged less than 78 years ( Figure 2B In contrast, the expression of JUNB, S100A4, and S100A6 in female AD patients was lower than that in males ( Figure 2C ). Since B lymphocytes in AD patients have down-regulated expression, the down-regulated differentially expressed genes in B lymphocytes were selected for further study. By comparing the sequencing and RT-qPCR results, ATP5D, CD69, and CXCR4 with consistent down-regulation trends were selected for subsequent verification ( Figure 1D). The T-SNE plot showed that ATP5D expression was reduced in all 18 cell clusters of AD patients, while CD69 and CXCR4 were simultaneously reduced in clusters 9 and 12, which are B lymphocyte-specific cell clusters ( Figure 1E -F). In addition, the expressions of ATP5D, CD69, and CXCR4 decreased with the increase of CDR score, and their decrease was most significant, especially when CDR=2 ( Figure 3A , p=0.003, p=0.005, p=0.022). At the same time, the correlation analysis between the relative expression of the three genes and the CDR score showed that the relative expression levels of ATP5D (Y=-0.5742*X+2.942), CD69 (Y=-1.329*X+3.737) and CXCR4 (Y=-3.607*X+11.55) were negatively correlated with the CDR score ( Figure 3B , p=0.0206, p=0.0349, p=0.0267). These results suggest that the specificity of ATP5D, CXCR4, and CD69 in B lymphocytes may play a key role in the progression of AD.

[0091] To verify the expression of ATP5D, CD69, and CXCR4 in B lymphocytes from PBMCs of AD patients, cell smears of PBMCs were prepared and immunofluorescence staining was performed, which showed that the number of CD19-positive cells (B lymphocytes) was significantly decreased in early and late AD patients compared with normal controls ( Figure 3C ). Similarly, the number of ATP5D-positive and CXCR4-positive cells in B lymphocytes of AD patients was significantly less than that in healthy controls ( Figure 3C Interestingly, downregulation of ATP5D expression was observed in early AD (p=0.002), but downregulation of CXCR4 was more pronounced in late AD ( Figure 3D , p<0.001). To further verify the distribution and expression of ATP5D, CD69, and CXCR4 in B lymphocytes in the cerebral cortex and hippocampus of AD patients, brain slice samples of healthy and AD patients provided by West China Hospital of Sichuan University (Sichuan, China) were collected. Immunofluorescence staining of CD19 and ATP5D showed that B lymphocytes in the hippocampus of AD patients were significantly reduced (p=0.001), while there was no significant difference in B lymphocytes in the cerebral cortex of AD patients compared with the control group ( Figure 3E -F). Compared with the healthy control group, the fluorescence intensity of ATP5D in the cerebral cortex and hippocampus of AD patients was significantly weaker ( Figure 3F, p=0.007, p<0.001). Similarly, compared with healthy controls, the fluorescence intensity of CD69 in the cortex and hippocampus and CXCR4 in the hippocampus of AD patients was significantly reduced (p<0.001), while the expression of CXCR4 in the cortex of AD patients was higher than that in healthy controls ( Figure 3G -H, p=0.002). In conclusion, the expression levels of B lymphocytes and ATP5D, CD69, and CXCR4 were decreased in the peripheral and central nervous systems of AD patients.

[0092] Example 2—ATP5D, CD69, and CXCR4 are involved in the regulation of B lymphocyte activation in AD disease

[0093] Based on the expression of ATP5D, CD69 and CXCR4 in AD patients, the expression of these three factors in the cortex, hippocampus and B lymphocytes of 3-month-old, 10-month-old, 18-month-old WT mice and 15-month-old AD mice was further detected. It was found that the expression of ATP5D in the cortex of 15-month-old AD mice was significantly lower than that of wild-type 3-month-old, 10-month-old and 18-month-old mice (p<0.001). In addition, the expression of ATP5D in the hippocampus of 15-month-old AD mice and the expression of CXCR4 in the cortex of 15-month-old AD mice were significantly lower than those of 10-month-old WT mice ( Figure 4A , C, p=0.045, p=0.010). In contrast, the expression of CD69 in the cortex and hippocampus of 15-month-old AD mice and CXCR4 in the hippocampus of 15-month-old AD mice showed significant increases compared with WT mice at 3, 10, and 18 months of age ( Figure 4B -C, p = 0.005, p = 0.006, p = 0.018, p = 0.007, p < 0.001). These findings suggest that young B lymphocyte therapy delays AD progression, which may be related to the regulation of ATP5D, CD69, and CXCR4 in B lymphocytes. In addition, it was found that the expression of ATP5D and CXCR4 in B lymphocytes of AD mice was significantly lower than that in B lymphocytes of WT mice, while CD69 showed no difference ( Figure 4A -C, p<0.001). In addition, bioinformatics was used to analyze the differentially expressed genes related to ATP5D, CD69, and CXCR4. The results showed that the genes related to ATP5D were MT-ATP6, MT-CYB, MT-ND2, and MT-ND3, the genes related to CD69 were CD3D, CCL5, GZMA, GNLY, KLRB1, HCST, TYROBP, and CXCR4, and the genes related to CXCR4 were S100A4 and CD69. ( Figure 4D ). Among the top 12 enriched pathways, “Alzheimer’s disease”, “axon guidance”, and “apoptosis” ( Figure 4E). In addition, GO analysis annotated ATP5D, CD69, and CXCR4 involved in various biological functions, such as "positive regulation of B lymphocyte activation", "B lymphocyte activation", "immunoglobulin complex, circulation", and "immunoglobulin receptor binding". These genes may participate in neural pathways such as AD, axonal mediation, and apoptosis by regulating immune-related B lymphocyte activation functions and promoting the release of antibodies and neurotrophic factors by B lymphocyte activation.

[0094] Example 3 - Dataset and Statistical Methods

[0095] 3.1 Study Dataset

[0096] A total of three datasets were included in this study.

[0097] Dataset 1 includes data from venous blood participants of 41 healthy subjects and 43 AD patients at different stages of the disease (after removing discrete values, n=62 cases, including 33 AD patient subjects and 29 healthy controls).

[0098] Dataset 2 is the data of AD patient participants (n=33 cases, including 14 subjects with AD younger than 78 years old and 19 subjects with AD older than 78 years old).

[0099] Dataset 3 is the data of AD patient participants (n=33 cases, including 11 male subjects with AD and 22 female subjects). According to the results, the participants were divided into two groups: healthy control group and clinical AD. Among the 62 subjects, 29 were controls and 33 had clinical AD. We compared the differences in ATP5D, CD69 and CXCR4 expression between clinical AD patients and healthy controls and the diagnostic efficacy of the combination of the three factors, the differences in ATP5D, CD69 and CXCR4 expression between patients under 78 years old and patients over 78 years old in clinical AD and the diagnostic efficacy of the combination of the three factors, and the differences in ATP5D, CD69 and CXCR4 expression between male and female patients in clinical AD and the diagnostic efficacy of the combination of the three factors. To determine whether ATP5D, CD69 and CXCR4 can predict clinical AD.

[0100] All participants or their legal guardians were fully informed and signed written consent. This study was approved by the Institutional Review Board of West China Hospital, Sichuan University.

[0101] 3.2 Statistical analysis

[0102] Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The data for each dataset were analyzed independently. The data were quantitative and were analyzed using the rank sum test because they did not conform to the normal distribution. In datasets 1, 2, and 3, the predicted values ​​were calculated using a binary logistic regression model with qPCR results, age, and sex as covariates, which were then used for ROC curve analysis. All tests were two-tailed, and the significance level was set at P < 0.05.

[0103] 3.3 Results

[0104] Diagnosis using peripheral blood B cell ATP5D, CD69, CXCR4 and their combination

[0105] A relatively large sample (dataset 1) was recruited to evaluate the differential expression of three factors, ATP5D, CD69, and CXCR4, between subjects with AD and the control group. Genes that were significantly upregulated and downregulated in B cells of AD patients. Based on the results of single-cell RNA-seq data analysis and processing, 12 genes were screened for further study. Since B cell expression was downregulated in AD patients, the differentially downregulated genes in B cells were selected for further study. By comparing sequencing and RT-qPCR results, we selected ATP5D, CD69, and CXCR4 with a consistent downregulation trend for subsequent validation. A total of three factors were included as covariates, and diagnosis (AD or control) was used as the dependent variable. Using the predicted values ​​in the logistic analysis, the diagnostic ability of the three factors used alone or in combination was evaluated by receiver operating characteristic (ROC) analysis. Our data showed that ATP5D (P=0.040, Figure 5 A), CD69 (P=0.010, Figure 5 B), CXCR4 (P=0.020, Figure 5 C) were expressed in peripheral blood B cells with statistical significance; ATP5D (AUC = 0.652, P = 0.0401, Figure 5 D), CD69 (AUC=0.7482, P=0.0008, Figure 5 E) and CXCR4 (AUC = 0.6729, P = 0.0196, Figure 5 F) Three factors and three factors combined (AUC = 0.7461, P = 0009, Figure 5G) were all high, indicating that the three factors alone or in combination could successfully distinguish subjects with AD from the control group, indicating that ATP5D, CD69, and CXCR4 were effective factors for diagnosing AD. In addition, the ATP5D (P = 0.024, Figure 6 A), CD69 (P<0.001, Figure 6 B), CXCR4 (P=0.003, Figure 6 C) were expressed in peripheral blood B cells and had statistical significance; ROC curves of age (greater than 78 years old and less than 78 years old) in AD patients showed that ATP5D (AUC = 0.7331, P = 0.0239, Figure 6 D), CD69 (AUC=0.9586, P<0.0001, Figure 6 E) and CXCR4 (AUC = 0.8083, P = 0.0028, Figure 6 F) Three factors and three factor combinations (AUC = 0.9586, P < 0001, Figure 6 G) were all high, indicating that the use of the three factors alone or in combination can be more conducive to screening AD patients in the elderly (over 78 years old), indicating that ATP5D, CD69 and CXCR4 are effective factors for diagnosing AD. We also analyzed the diagnostic ability of the three factors for AD patients of different genders. The results showed that ATP5D (P = 0.674, Figure 7 A), CD69 (P=0.492, Figure 7 B), CXCR4 (P=0.445, Figure 7 C) had no statistically significant expression in peripheral blood B cells; ATP5D (AUC = 0.5455, P = 0.6744, Figure 7 D), CD69 (AUC=0.5744, P=0.4918, Figure 7 E) and CXCR4 (AUC = 0.5826, P = 0.4450, Figure 7 F) Three factors and three factors combined (AUC = 0.5165, P = 8786, Figure 7 G) were all relatively low, indicating that the three factors and their combination had poor diagnostic ability for AD patients of different genders.

[0106] 3.4 Experimental study results

[0107] In the experiment of this sample (dataset 1), the results of ATP5D, CD69, CXCR4 and their combination showed that there was a significant difference in the expression of B cells between subjects with AD and controls (P>0.05); AD patient samples (dataset 2) were divided into two groups according to age, those under 78 years old and those over 78 years old, and it was found that there was a significant difference in the expression of ATP5D, CD69, CXCR4 and their combination between the two groups (P>0.05); AD patient samples (dataset 3) were divided into males and females according to gender, and it was found that there was no significant difference in the expression of ATP5D, CD69, CXCR4 and their combination between the two groups (P<0.05).

[0108] 3.5 Single-cell sequencing (scRNA-seq) was used to analyze the differential expression of venous blood mononuclear cells in healthy controls and AD patients at different stages, thereby screening out 12 differentially expressed genes. By comparing the sequencing and RT-qPCR results, three genes, ATP5D, CD69 and CXCR4, which had been showing a down-regulated trend, were selected. The correlation analysis between the relative expression of these three genes and the CDR score showed that the relative expression levels of ATP5D (Y = -0.5742*X + 2.942), CD69 (Y = -1.329*X + 3.737) and CXCR4 (Y = -3.607*X + 11.55) were negatively correlated with the CDR score ( Figure 3B , p=0.0206, p=0.0349, p=0.0267). The three genes with the highest prediction accuracy for AD were selected as candidate AD feature genes, and a prediction model for AD and normal subjects was established.

[0109] 3.6 The relative expression levels of the three AD gene markers in the peripheral blood of 33 AD patients and 29 normal subjects were detected by fluorescence quantitative RT-PCR. The AD prediction model was used to classify and predict the above samples (including training set and test set samples). The prediction results were compared with the clinical diagnosis test results of AD, and the sensitivity (Sensitivity), specificity (Specificity) and accuracy (Accuracy) of the three AD gene markers of the present invention for AD prediction were obtained. The specific test results are shown in the ROC curve Figure 5 D-5H and Table 2.

[0110] Table 2

[0111]

[0112] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0113] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0114] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0115] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. Use of a substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes in B lymphocytes in the preparation of a product for detecting human Alzheimer's disease, wherein the expression of ATP5D, CD69 and CXCR4 decreases with the increase of clinical dementia scores, and the substance for detecting the expression of ATP5D, CD69 and CXCR4 genes is at least one of a primer pair, a probe or an antibody.

2. The use according to claim 1, characterized in that The B lymphocytes are CD19 positive cells.

3. The use according to claim 1, characterized in that The B lymphocytes are derived from peripheral blood and / or central nervous system.

4. The use according to claim 1, characterized in that The expression levels of B lymphocyte-specific genes ATP5D, CD69 and CXCR4 in the peripheral blood and / or central nervous system of the Alzheimer's disease patients are all reduced.

5. The use according to claim 1, characterized in that The relative expression levels of ATP5D, CD69 and CXCR4 were negatively correlated with the clinical dementia scores.

6. The use according to claim 1, characterized in that The primer pair for detecting ATP5D is as follows: Forward primer: 5'-CCCCAACCAGATGTCCTTCA-3' (SEQ ID NO. 3), and reverse primer: 5'-TCTTCGGCCAACAACTGCA-3 (SEQ ID NO. 4); And / or, the primer pair for detecting CD69 is as follows: Forward primer: 5'-ATGGTGCTACTCTTGCT-3' (SEQ ID NO. 7), and reverse primer: 5'-ATGCTGCTGACCTCTGT-3' (SEQ ID NO. 8); And / or, the CXCR4 primer pair is as follows: Forward primer: 5'-AAATCTTCCTGCCCACCAT-3' (SEQ ID NO. 9), and reverse primer: 5'-CGCCAACATAGACCACCTT-3' (SEQ ID NO. 10).

7. A product for detecting human Alzheimer's disease, characterized in that: The product is a substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes in B lymphocytes, and the substance for detecting the expression of human ATP5D, CD69 and CXCR4 genes is at least one of a primer pair, a probe or an antibody.

8. The product according to claim 7, characterized in that The product is any one of a kit, a chip, a membrane strip, a protein array, a composition or a detection system; And / or, the ATP5D, CD69 and CXCR4 genes are B lymphocyte-specific genes.

9. The product according to claim 7, characterized in that The B lymphocytes are CD19 positive cells.

10. The product according to claim 7, characterized in that The B lymphocytes are derived from peripheral blood and / or central nervous system.

11. The product according to claim 7, characterized in that The detection primers of ATP5D are: Forward primer: 5'-CCCCAACCAGATGTCCTTCA-3' (SEQ ID NO. 3), and reverse primer: 5'-TCTTCGGCCAACAACTGCA-3' (SEQ ID NO. 4); The detection primers for the CD69 are: Forward primer: 5'-ATGGTGCTACTCTTGCT-3' (SEQ ID NO. 7), and reverse primer: 5'-ATGCTGCTGACCTCTGT-3' (SEQ ID NO. 8); The detection primers for the CXCR4 are: Forward primer: 5'-AAATCTTCCTGCCCACCAT-3' (SEQ ID NO. 9), and reverse primer: 5'-CGCCAACATAGACCACCTT-3' (SEQ ID NO. 10).

Images