CN113082209A

CN113082209A - CD4+Application of T cell-derived BACE1, EP2 and EP4 as Alzheimer disease treatment target

Info

Publication number: CN113082209A
Application number: CN202110346276.5A
Authority: CN
Inventors: 申勇; 戴林斌
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2021-07-09

Abstract

The invention relates to the technical field of biological medicines, in particular to CD4⁺Use of T cell derived BACE1 and/or prostaglandin E2 receptors EP2 and/or EP4 as therapeutic targets for alzheimer's disease. The invention discovers for the first timeCD4⁺T cell-derived BACE1 can specifically cleave prostaglandin synthetase precursor protein mPGES2, thereby promoting maturation of mPGES2, and mature mPGES2 promotes synthesis of prostaglandin PGE2, thereby leading to CD4⁺Abnormal activation of T cells; meanwhile, CD4 was also found in AD patients and AD mouse models⁺Abnormal activation of T cells and increased expression of BACE 1; at the same time, antagonists of the receptors for PGE2, EP2/EP4, are able to improve the pathological phenotype of alzheimer's disease. Thus CD4⁺T cell-derived BACE1 and/or EP2 and/or EP4 can be used as a target for treating Alzheimer disease, and the invention provides a new target for researching a novel medicine for treating Alzheimer disease.

Description

CD4+Application of T cell-derived BACE1, EP2 and EP4 as Alzheimer disease treatment target

Technical Field

The invention relates to the technical field of biological medicines, in particular to CD4⁺Use of T cell derived BACE1 and/or EP2 and/or EP4 as therapeutic targets for alzheimer's disease.

Background

Alzheimer's Disease (AD) is the most common neurodegenerative disorder, and also the most predominant type of dementia, manifested primarily as cognitive and memory dysfunction. Although there is increasing evidence that AD pathology is deposited in the brain from middle age, clinical pathological symptoms typically occur after the age of 65. With the aging of the global population becoming more and more severe, the 65-year-old population has increased significantly in proportion to the increase in the incidence of AD. Epidemiological studies predict: from 1997 to 2050, the population aged over 65 will grow globally, with an increase from 6300 to 1.37 million in america, 1800 to 3.8 million in africa, 1.13 to 1.7 million in europe, and 1.72 to 4.35 million in asia. The incidence of dementia is less than 1% in people between 60 and 64 years of age, but the incidence increases exponentially with age, and particularly the incidence of people over 85 years of age in western countries will reach 24% -33%. Currently, the specific etiology of AD is still unknown, and there is still a lack of effective therapeutic approaches for AD.

BACE1(β -site APP cleavage enzyme, β secretase) belongs to the family of aspartic proteases, is predominantly expressed in neurons in the brain, is a type I aspartic protease consisting of 501 amino acids, has two aspartic protease active site motifs, including DTGS and DSGT, and mutation of either of them inactivates BACE 1. Amyloid plaques formed by deposition of beta-Amyloid (Amyloid-beta peptide, a β), including a β 40 and a β 42, are one of the most prominent pathological features of AD, based on which the proposed Amyloid hypothesis suggests that abnormal deposition of a β is the main cause of AD. The generation of A beta is caused by sequential cleavage of Amyloid Precursor Protein (APP) in neurons by BACE1 and gamma-secretase. Therefore, BACE1 is regarded as one of the targets of AD treatment, and clinical experiments using BACE1 as the target of AD treatment are receiving more and more attention, but to date, almost all relevant clinical experiments have declared to fail, one of the possible reasons is that the therapy inhibits BACE1 in a broad-spectrum rather than specific targeting, thereby causing potential side effects and risks.

Studies have shown that BACE1 is also peripheral CD4⁺Expression is present in T cells. BACE1 acts as a cleaving enzyme, with a broad enzymatic cleavage substrate. Membrane-bound Prostaglandin E2 synthetase-2 (Membrane-bound Prostaglandin E2 synthsase-2, mPGES2) is a potential cleavage substrate for BACE1, and BACE1 promotes mPGES-2 maturation by cleaving precursor mPGES-2, thereby promoting the production of Prostaglandin E2(Prostaglandin E2, PGE 2). PGE2 is a key product of lipid metabolism and is also the most abundant prostatic hormone in the body, and acts by specifically binding to receptors EP1, EP2, EP3 and EP4 on target cells in an autocrine or paracrine manner under physiological or pathological conditions. PGE2 in the immune system against CD4⁺T cells have important regulatory roles, and CD4⁺T cells specifically express PGE2 receptors EP2 and EP4 only.

CD4⁺T cells are key cells for the body to generate adaptive immune response, and play a central role in the immune response. CD4⁺T cells can induce enhancement of the pathogen clearance function of macrophages by releasing cytokines and chemokines, recruit neutrophils, eosinophils, and basophils to sites of infection and inflammation, assist B cells in antibody production, suppress excessive immune response, and the like.

The central nervous system is traditionally thought to be the site of immune-privileged sites, but there is increasing evidence that T cells are involved in immune surveillance of the central nervous system. Studies have shown that healthy individuals have approximately 150000 lymphocytes in their Cerebrospinal fluid (CSF) and re-enter the CSF after two refreshments in the blood each day. More than 80% of the T cells in CSF are CD4⁺T cells, one third of which are CD45RA-CD 27-effector memory T cells and two third of which are CD45RA^-CD27⁺Central memory T cells of (a). CD4 in CSF⁺T cells are involved in the development of neuroinflammation by recognizing antigens presented by antigen presenting cells to mediate an immune response and infiltrate into the brain parenchyma.

While neuroinflammation is an important factor in promoting the development of AD, and is generally thought to be mediated by microglia and astrocytes, studies have now shown that T cells can be involved in modulating the inflammatory response in AD in at least two ways. First, during the development of AD, due to the decreased expression of the tight junction molecules ZO-1 and Occludin in vascular endothelial cells, the permeability of the Blood-brain barrier (BBB) will gradually increase, and with the chemokine receptors CCR2, CCR4, CCR5, CXCR2 etc. at the periphery CD4⁺The expression on T cells is up-regulated, and the CD4 is promoted⁺Recruitment of T cells and infiltration into the brain parenchyma. Impregnated CD4⁺T cells cause further development of neuroinflammation through interaction with microglia, astrocytes. Second, peripheral CD4⁺T cells can also secrete proinflammatory cytokines such as IFN-gamma and the like into the central nervous system, and then activate astrocytes and microglia. Studies report peripheral CD4 in AD patients⁺T cells exhibit a higher activation profile than controls, which greatly promotes the secretion of more proinflammatory cytokines such as IL-1, IL-6 and TNF- α by peripheral blood mononuclear cells, which are important regulators in the development of AD.

To date, no study has confirmed CD4⁺T cell-derived BACE1 and/or EP2 and/or EP4 can be used as a therapeutic target of AD, and a method for selectively regulating CD4 is searched⁺Medicaments of T cell derived BACE1 and/or EP2 and/or EP4 genes or proteins are of great interest for the treatment of AD.

Disclosure of Invention

To overcome the deficiencies in the prior art, the inventors of the present invention found for the first time CD4⁺T cell-derived BACE1 can specifically cleave the precursor form of prostaglandin synthetase mPGES2, thereby promoting maturation of mPGES2, and mature mPGES2 promotes synthesis of prostaglandin PGE2, thereby leading to CD4⁺Abnormal activation of T cells; meanwhile, CD4 was also found in AD patients and AD mouse models⁺Abnormal activation of T cells and increased expression of BACE 1; at the same time, antagonists of the receptors for PGE2, EP2 and/or EP4, are able to improve the pathological phenotype of alzheimer's disease, reducing the deposition of a β in the brain and the proliferation of microglia. Thus CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 are new potential targets for the treatment of alzheimer's disease. The inventors of the present invention have completed the present invention on this basis.

To this end, the first aspect of the invention provides CD4⁺Use of a T cell derived BACE1 and/or EP2 and/or EP4 gene or protein in the manufacture of a medicament for treating or ameliorating alzheimer's disease.

In an embodiment of the invention, the medicament comprises at least CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Inhibitors of T cell derived BACE1 and/or EP2 and/or EP4 proteins.

In some embodiments of the invention, the CD4 is a CD-CD⁺Substances with reduced expression levels of T cell derived BACE1 and/or EP2 and/or EP4 genes include targeted CD4⁺T cell derived BACE1 and/or RNAi fragment and/or siRNA fragment of EP2 and/or EP4 gene and/or CD4⁺Specific transcriptional inhibitors of the T cell derived BACE1 and/or EP2 and/or EP4 genes.

In some embodiments of the invention, the CD4⁺Specific transcriptional inhibitor of T cell derived BACE1 and/or EP2 and/or EP4 gene refers to specific inhibition of CD4⁺Inhibitors of the transcriptional process of the T cell derived BACE1 and/or EP2 and/or EP4 genes by inhibiting CD4⁺Transcription of T cell derived BACE1 and/or EP2 and/or EP4 genes to reduce CD4 in patients⁺Expression levels of T cell derived BACE1 and/or EP2 and/or EP4 genes and CD4⁺Levels of T cell derived BACE1 and/or EP2 and/or EP4 proteins, alleviating their effects on CD4⁺Promotion of T cell activation.

In other embodiments of the invention, the inhibitor of BACE1 protein is the β -secretase inhibitor IV (BIV), the EP2 inhibitor is PF-04418948, and the EP4 inhibitor is BGC-20-153.

In a second aspect, the invention provides a medicament for treating or alleviating alzheimer's disease, wherein the medicament comprises an active ingredient and/or a pharmaceutically acceptable carrier, and the active ingredient is the compound that enables CD4 to be absorbed⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Inhibitors of T cell derived BACE1 and/or EP2 and/or EP4 proteins.

In some embodiments of the invention, the "pharmaceutically acceptable carrier" is generally non-toxic to the recipient at the dosages and concentrations employed, including but not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbyl quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly (vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

In a third aspect of the invention, CD4 is provided⁺T cellsUse of a gene or protein derived from BACE1 and/or EP2 and/or EP4 in the preparation of a reagent or kit for treating or alleviating cerebrovascular diseases.

In some embodiments of the invention, the reagent or kit comprises at least CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Inhibitors of T cell derived BACE1 and/or EP2 and/or EP4 proteins.

In other embodiments of the present invention, the CD4 is a CD-CD⁺Substances with reduced expression levels of T cell derived BACE1 and/or EP2 and/or EP4 genes include targeted CD4⁺T cell derived BACE1 and/or RNAi fragment and/or siRNA fragment of EP2 and/or EP4 gene and/or CD4⁺Specific transcriptional inhibitors of the T cell derived BACE1 and/or EP2 and/or EP4 genes.

In a fourth aspect, the present invention provides an agent or kit for treating or ameliorating alzheimer's disease, wherein the agent or kit comprises at least CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Inhibitors of T cell derived BACE1 and/or EP2 and/or EP4 proteins.

In some embodiments of the invention, the CD4⁺The sequence of siRNA fragment of T cell derived BACE1 gene is 5'-GCATGATCATTGGAGGTATTT-3'.

A fifth aspect of the invention provides a CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Use of an inhibitor of T cell derived BACE1 and/or EP2 and/or EP4 protein for the manufacture of a pharmaceutical composition for the treatment or alleviation of alzheimer's disease.

In a sixth aspect, the invention provides Alzheimer's diseaseA disease biomarker, characterized in that said biomarker comprises CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 genes.

According to a seventh aspect of the present invention, there is provided a kit for diagnosing Alzheimer's disease, wherein said kit comprises CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 genes.

An eighth aspect of the present invention provides a method for treating or ameliorating alzheimer's disease, which comprises administering to a subject a therapeutically effective amount of the above-mentioned pharmaceutical composition, agent or kit.

The inventor finds that CD4 is in early research⁺T cell-derived BACE1 protein can specifically cleave prostaglandin synthetase precursor protein mPGES2, resulting in CD4⁺Abnormal activation of T cells. The present invention further provides animal models of BACE1 knock-out and overexpression. Further, using the animal model we obtained BACE1 knock-out and over-expressed CD4, respectively⁺T cells. We found that BACE1 knock-out significantly inhibited the maturation of mPGES2, reduced the production of PGE2, and thereby attenuated CD4⁺Activation of T and its mediated immune response; in contrast, BACE1 overexpression can remarkably promote the maturation of mPGES2 and increase the generation of PGE2, thereby promoting CD4⁺Activation of T and its mediated immune response. CD4 in AD patients and AD model mice⁺T cells show an abnormally activated phenotype with increased expression of BACE 1; inhibition of EP2/EP4, however, alleviated the pathology in the brains of AD mice. Thus CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 can be used as target for treating Alzheimer disease.

The invention has the beneficial effects that:

the invention discovers CD4 for the first time⁺T cell-derived BACE1 can specifically cleave prostaglandin synthetase precursor protein mPGES2, CD4⁺An increase in T cell-derived BACE1 levels cleaves the prostaglandin synthase precursor protein mPGES2, promoting mPGES2 maturation, resulting in increased synthesis of PGE2 and, in turn, CD4⁺Abnormal activation of T cells. CD4 in AD patients and AD model mice⁺T cell-derived BACE1 expression increased significantly and was accompanied by CD4⁺Abnormal activity of TThis will lead to an exacerbation of AD pathology. In addition, inhibition of EP2/EP4 alleviated the pathology in the brains of AD mice. Thus CD4⁺T cell-derived BACE1 and/or EP2 and/or EP4 can be used as a target for treating Alzheimer disease, and the invention provides a new target for researching a novel medicine for treating Alzheimer disease.

Drawings

The present invention will be specifically described below with reference to the drawings.

FIG. 1: western blot results plot of BACE1 specifically cleaving mPGES2 but not mPGES 1;

FIG. 2: a Western blot result graph of BACE1-siRNA inhibiting BACE1 from shearing mPGES 2;

FIG. 3: western blot result chart of intracellular cleavage of mPGES2 by CRISPR-Cas9 BACE1 KO (knockout);

FIG. 4: BACE1 inhibitor BIV inhibits mouse CD4⁺Western blot results of BACE1 shearing mPGES2 in T cells;

FIG. 5: BACE1^-/-Mouse-derived CD4⁺T cells, Western blot graphs in which BACE1 expression was successfully knocked out;

FIG. 6: HUBC-derived CD4⁺T cells, Western blot plot with significantly increased expression of BACE 1;

FIG. 7 CD4⁺CD4 expressing BACE1 following T cell activation⁺Flow analysis and statistical plots of elevated proportion of T cells;

FIG. 8 CD4⁺Western blot result graph of increased expression of BACE1 after T cell activation;

FIG. 9 WT mice and BACE1^-/-Mouse-derived CD4⁺Western blot result chart of mPGES2 expression of T cells under CD3/CD28 antibody stimulation;

FIG. 10 WT mice and HUBC mouse-derived CD4⁺Western blot result chart of mPGES2 expression of T cells under CD3/CD28 antibody stimulation;

FIG. 11: WT mice and BACE1^-/-Mouse-derived CD4⁺Elisa results statistical plot of PGE2 expression under CD3/CD28 antibody stimulation by T cells;

FIG. 12: WT mice and HUBC mice derived CD4⁺Elisa results statistical plot of PGE2 expression under CD3/CD28 antibody stimulation by T cells;

FIG. 13: WT mice and BACE1^-/-Mouse-derived CD4⁺T cells, RNA-seq results statistical plots of mRNA expression of different PGE2 receptors under stimulation with CD3/CD28 antibody;

FIG. 14: 24h after PGE2 was intraperitoneally injected into WT mice, CD4⁺Western blot result graph of T cell TCR access protein expression;

FIG. 15: WT mice and BACE1^-/-Mouse-derived CD4⁺Western blot plot of TCR pathways and downstream proteins of T cells stimulated by CD3/CD28 antibodies;

FIG. 16: WT mice and HUBC mice derived CD4⁺Western blot plot of TCR pathways and downstream proteins of T cells stimulated by CD3/CD28 antibodies;

FIG. 17: WT mice and BACE1^-/-Mouse-derived CD4⁺Flow assay and statistical plots of Ki-67 expression by T cells under stimulation with CD3/CD28 antibody;

FIG. 18: WT mice and BACE1^-/-Mouse-derived CD4⁺Elisa results statistical plot of IL-2 secretion under CD3/CD28 antibody stimulation by T cells;

FIG. 19: CD4⁺Elisa results statistical plot of IL-2 secretion under CD3/CD28 antibody stimulation after T cell BACE1 overexpression;

FIG. 20: WT mice and BACE1^-/-Mouse-derived CD4⁺Flow analysis and statistical plots of CD69 expression in T cells under stimulation with CD3/CD28 antibody;

FIG. 21: WT mice and HUBC mice derived CD4⁺Flow analysis and statistical plots of CD69 expression in T cells under stimulation with CD3/CD28 antibody;

FIG. 22: WT mice and BACE1^-/-Mouse-derived CD4⁺Flow-through and statistical plots of CD69 expression in T cells under ConA stimulation;

FIG. 23: WT mice and HUB mice derived CD4⁺Flow-through and statistical plots of CD69 expression in T cells under ConA stimulation;

FIG. 24: WT mice and BACE1^-/-Mice after subcutaneous injection of KLH 7dSpleen CD4⁺Flow assay statistics of T cell Ki-67 expression;

FIG. 25: WT mice and BACE1^-/-Spleen CD4 after subcutaneous injection of KLH 7d in mice⁺Flow analysis and statistical plots of T cell IL-17A, IFN-gamma expression;

FIG. 26: WT mice and BACE1^-/-Elisa results statistical plots of KLH antibodies IgM and IgG in serum after subcutaneous injection of KLH 7d in mice;

FIG. 27 is a schematic view showing: WT mice and BACE1^-/-Elisa result statistical chart of IL-2 in a culture medium after in vitro KLH stimulation of splenocytes for 48h after subcutaneous injection of KLH 7d in mice;

FIG. 28: WT and HUBC mice were injected subcutaneously with KLH 7d, and spleen CD4⁺Flow assay statistics of T cell Ki-67 expression;

FIG. 29: WT and HUBC mice were injected subcutaneously with KLH 7d, and spleen CD4⁺Flow analysis and statistical plots of T cell IL-17A, IFN-gamma expression;

FIG. 30: elisa results of KLH antibodies IgM and IgG in serum after subcutaneous injection of KLH 7d in WT mice and HUBC mice are shown in the figure;

FIG. 31: elisa result statistical chart of IL-2 in the culture medium after in vitro KLH stimulation for 48h on splenocytes after subcutaneous injection of KLH 7d in WT mice and HUBC mice;

FIG. 32: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow analysis statistical plots of T cell ratios;

FIG. 33: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow analysis statistics of T cell CD69 expression;

FIG. 34: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow assay statistics of T cell Ki-67 expression;

FIG. 35: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow analysis statistical plots of T cell ratios;

FIG. 36: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow analysis statistics of T cell CD69 expression;

FIG. 37: WT mice and BACE1^-/-Mouse-derived CD4⁺T cell transfusion to Rag^-/-After KLH stimulation for 7 days, splenic CD4⁺Flow assay statistics of T cell Ki-67 expression;

FIG. 38: AD patients peripheral CD4⁺T cell expression BACE1 is significantly higher than that of a Western blot result graph and a semi-quantitative analysis graph of a healthy human;

FIG. 39: AD patients peripheral blood CD4⁺T(CD45RA⁺CD45RO^-) Cells and Effect memory CD4⁺T(CD45RA^-CD45RO⁺) Flow and statistical plots of cellular imbalance;

FIG. 40: AD mouse peripheral CD4⁺A Western blot result graph that T cells express BACE1 which is significantly higher than that of WT mice;

FIG. 41: peripheral blood Naive CD4 of AD mouse⁺T(CD62L^highCD44^low) Cells and Effect memory CD4⁺T(CD62L^loeCD44^high) Flow and statistical plots of cellular imbalance;

FIG. 42: AD mouse peripheral blood CD4⁺T cells expressed CD69 significantly higher in the activated state than the flow and statistical plots of WT mice;

FIG. 43: histochemical staining pattern of pathology in the brain after administration of PGE2 receptor EP2/EP4 antagonist 7d in AD mice;

FIG. 44: CD4 stimulated by CD3/CD28 antibody after AD mice are administered PGE2 receptor EP2/EP4 antagonist 7d⁺Western blot results of T cell TCR path protein.

Detailed Description

The technical solution of the present invention will be described below by specific examples. However, these examples are for illustrative purposes only, and are not meant to limit the scope of the present invention thereto.

The experimental procedures used in the following examples are conventional unless otherwise specified.

The test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 BACE 1-specific cleavage of the mPGES2 precursor form

1. Procedure of experiment

To confirm the relationship between BACE1 and mPGES2, the present invention constructed HEK293 cells stably transfected with HA-BACE1 and Flag-mPGES2 plasmids, and the changes in the precursor form (molecular weight of 43kDa) and the mature form (molecular weight of 34kDa) of mPGES2 were examined by Western blotting (Western blot) technique (FIG. 1). Meanwhile, to confirm that BACE1 is specific to the regulation of mPGES2, another HEK293 cell stably expressing prostaglandin synthetase mPGES1(GFP-mPGES1 plasmid) and BACE1 plasmid was constructed, and Western blot detection was also performed (FIG. 1). On the other hand, the invention utilizes siRNA and CRISPR-Cas9 technology to construct stable BACE1 knock-down (knockdown) and knock-out (KO, knockout) cell lines, and the cell lines are transfected with Flag-mPGES2 plasmid, and Western blot technology is used to detect the change of precursor form (molecular weight of 43kDa) and mature form (molecular weight of 34kDa) of mPGES2 (see attached figures 2 and 3).

2. Experimental Material

HEK293 cells, cell culture reagents were purchased from gibioc (Carlsbad, CA); Flag-mPGES2(EX-Mm35377-M35, Genencopoeia), GFP-mPGES1(EX-V0007-M98, Genencopoeia), HA-PKH3 (cat # 12555) were purchased from Addgene; siRNA was purchased from GenePharma;

the antibodies were as follows:

a first antibody: anti-BACE1(5606S, CST); anti-GFP (66002, proteintech); anti-Flag (F1804, sigma); anti-Actin (60008, proteintech).

Secondary antibody: Anti-Rabbit IgG (H + L), HRP Conjugate (W4011, promega); Anti-Mouse IgG (H + L), HRP Conjugate (W4021, promega).

The nucleotide sequence is as follows:

si-BACE1:5’-GCATGATCATTGGAGGTATTT-3’；

si-control (as a negative control for si-BACE 1) 5'-UUCUCCGAACGUGUCACGUTT-3';

CRISPR-Cas9 BACE1 knockout sgRNA sequence: 5' -GGATCCGGAGCCCGCTACAT-3, (sgBACE1-1)

3. Detailed description of the invention

The cells used in this study were all cultured in DMEM medium containing 2mM L-glutamic acid, 10% FBS, 100. mu.g/ml penicillin and 100. mu.g/ml streptomycin at 37 ℃ with 5% CO₂. HA-BACE1 and Flag-mPGES2 or GFP-mPGES1 were transfected into HEK293 cells using Lipofectamine 2000. Lipofectamine RNAiMAX (Life Technologies) was used for siRNA transfection.

(1) Construction of HA-BACE1 plasmid: HA-BACE1 was constructed using cDNA of BACE1(Gene ID 23621) and purchased HA-PKH 3;

(2) obtaining a BACE1 knockout HEK293 cell line by using CRISPR-Cas9 technology;

(3) experiment of co-transfection of HEK293 cells with BACE1 and mPGES2 or mPGES 1: control group: HEK293 cells were co-transfected with HA-PKH3 and Flag-mPGES 2; experimental groups: HEK293 cells were co-transfected with HA-BACE1 and Flag-mPGES 2.② comparison group: HEK293 cells were co-transfected with HA-PKH3 and GFP-mPGES 1; experimental groups: HEK293 cells were co-transfected with HA-BACE1 and GFP-mPGES 1.

(4) Experiment of the Effect of si-RNA knockdown of BACE1 in HEK293 cells on mPGES2 expression: because HEK293 itself expresses mPGES2 less, when treated for its si-RNA, Flag-mPGES2 was simultaneously transfected, with control: transfecting Flag-mPGES2+ si-control; experimental groups: Flag-mPGES2+ si-BACE1 was transfected.

(5) Experiment of influence of HEK293 cell CRISPR-Cas9 technology on mPGES2 expression of BACE 1: Flag-mPGES2 was transfected separately with the established BACE1 knockout HEK293 cell line (experimental group) and normal HEK293 cells (control group).

The above cell samples were lysed and collected with cell lysate 1% Nonidet P-40(11332473001, Roche), 0.5% sodium deoxycholate (89905, Thermo), 1M Tris-HCl, 150mM NaCl, pH7.5, protease inhibitor cocktail (Roche)); cells were lysed at room temperature and then an equal volume of 2 x SDS electrophoresis loading buffer was added. The sample was boiled in boiling water for 10min to prepare an SDS electrophoresis sample.

Changes in the precursor form (molecular weight 43kDa) and the mature form (molecular weight 34kDa) of mPGES2 were detected by protein immunoblotting (Western blot) technique.

The operation method of the protein immunoblotting method comprises the following steps:

SDS-PAGE electrophoresis: preparing SDS electrophoresis gel with proper concentration according to the size of the needed protein, and performing electrophoretic separation after equivalently loading a sample to be detected (the constant voltage of upper concentrated gel is 80 volts, and the constant voltage of lower separated gel is 120 volts);

film transfer: after SDS-PAGE electrophoresis is finished, membrane conversion is carried out (PVDF membrane is used after being activated for 3min by methanol), the membrane is converted for different time (about 90-180min) according to the size of protein under the constant current of 240 amperes, and the membrane conversion process is carried out in ice-water bath;

and (3) sealing: after the membrane transfer is finished, placing the membrane loaded with the protein into 5% skimmed milk prepared by TBST buffer solution, and sealing the shaking table for 1 hour at room temperature;

primary antibody incubation: preparing primary antibody with proper concentration according to the use instruction or pre-experimental result of the protein antibody, and transferring the transfer membrane to 4 ℃ overnight after incubating the transfer membrane for 3 hours at room temperature after sealing;

washing a primary antibody: shaking and cleaning with TBST buffer solution at room temperature for 10min for 3 times;

and (3) secondary antibody incubation: incubating the membrane for 90min with a primary antibody and a secondary antibody in a shaking table at room temperature;

washing a secondary antibody: shaking and cleaning with TBST buffer solution at room temperature for 5min for 3 times;

and (3) color development test: and (3) carrying out chromogenic detection on the protein content signal on the membrane by using an ECL developing kit.

4. Analysis of results

As can be seen from the results in FIG. 1, the precursor form of mPGES2 (molecular weight 43kDa) is specifically cleaved by BACE1 to the mature form (molecular weight 34 kDa).

As can be seen from the results in FIG. 2, knocking down the protein expression level of BACE1 can reduce the shearing of precursor form (molecular weight 43kDa) of mPGES2 by BACE1 (p < 0.05).

As can be seen from the results in FIG. 3, knocking out the protein expression level of BACE1 can reduce the shearing of precursor form (molecular weight 43kDa) of mPGES2 by BACE1 (p < 0.05).

Example 2 CD4⁺T cell-derived BACE1 similarly cleaves the mPGES2 precursor form, thereby modulating the PGE2 signaling pathway

1. Procedure of experiment

To explore CD4⁺Whether T cell-derived BACE1 (BACE 1 may have different selectivity for substrate cleavage under different physiological conditions or in different cells) is also able to cleave the precursor form of mPGES2, the present invention utilizes a BACE1 knockout mouse, i.e., BACE1^-/-Mice and BACE1 overexpressing mice, i.e., HUBC mice. The invention uses the magnetic bead sorting technology to separate BACE1^-/-CD4 in mice and HUBC mice⁺T cells were isolated and tested by Western blot for BACE1 expression (FIGS. 4 and 5). The experiment of the invention proves that CD4⁺T cell-derived BACE1 was able to cleave the precursor form of mPGES2, and the inhibitor of BACE1, BIV, was able to significantly inhibit this cleavage (figure 6). Further experiments of the invention found that CD3/CD28 antibody or ConA is used for stimulating CD4⁺CD4 expressing BACE1 after T cells have activated it⁺The T cell ratio and the expression level of BACE1 protein are both increased remarkably (figure 7 and figure 8). Corresponding to it is CD4⁺The significant increase in the mature form of T cells mPGES2 (fig. 9 and 10) and upregulation of PGE2 (fig. 11 and 12) but did not affect the expression of the PGE2 receptors EP2 and EP4 (fig. 13).

2. Experimental Material

BACE 1^-/-Mice were purchased from Jackson Laboratories; HUBC mice entrusted with college university construction; cell culture reagents were all purchased from gibioc (Carlsbad, CA); cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); canavalin A (Concanavalin A, Con A) was purchased from Sigma; BACE1 Inhibitor (beta-Secretase Inhibitor IV, 565788, which can specifically inhibit the enzyme cutting activity of BACE 1) was purchased from Merk corporation; Ultra-LEAF^TMPurified anti-mouse CD28(102115) and Ultra-LEAF^TMBoth Purified anti-mouse CD3(100340) were purchased from Biohedgend; prostagladin E2 ELISA Kit (514010) kits were purchased from Cayman Chemical company; 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; trizol reagent (15596026) was purchased from Thermo Fisher.

BACE1 inhibitors (β -secretase inhibitor IV) specific information is as follows:

alias: BACE inhibitor C3

CAS number: 797035-11-1

Empirical formula (hilt representation): c₃₁H₃₈N₄O₅S

Molecular weight 578.72

Structural formula (xvi):

the antibodies were as follows:

a first antibody: anti-BACE1(5606S, CST); anti-GAPDH (60004, proteintech); anti-Actin (60008, proteintech); anti-mPGES2 (mature form 34kDa) (10881, proteintech); anti-PTGES2 (precursor form 43kDa) (sc-514224, santa); PE anti-mouse CD4(100512, Biohedgend).

Secondary antibody: Anti-Rabbit IgG (H + L), HRP Conjugate (W4011, promega); Anti-Mouse IgG (H + L), HRP Conjugate (W4021, promega); alexa Fluor 488-anti-rabbit IgG (H + L) (A27034, Invitrogen).

3. Detailed description of the invention

The cells used in this study were all cultured in RPMI 1640 medium containing 10% FBS, 100. mu.g/ml penicillin and 100. mu.g/ml streptomycin at 37 ℃ with 5% CO₂(ii) a MACs buffer: namely, the magnetic bead sorting buffer solution contains 2mM EDTA and 0.5% bovine serum albumin in 1 xPBS.

(1) Sorting of CD4+ T cells: 1) after separating spleen or lymph node of mouse to obtain mononuclear cell, 300 μ l MACs buffer resuspends cell precipitation, adds 5 μ l PE-anti mouse CD4 flow antibody +45 μ l rat serum, incubate in dark at 4 deg.C for 30min, add 10ml 1XPBS to stop staining, 500g, centrifuge for 8min at 4 deg.C; 2) discarding the supernatant, resuspending the cell pellet with 350. mu.l MACs buffer, adding 20. mu.l anti-PE magnetic beads, incubating at 4 deg.C in dark for 30min, adding 10ml 1 × PBS to terminate incubation, centrifuging at 500g and 4 deg.C for 8 min; 3) discarding the supernatant, resuspending the cell pellet with 500. mu.l MACs buffer, filtering with 200 mesh nylon net, and discarding impurities; 4) fixing the MS sorting column on a sorting frame, rinsing the sorting column by 500 mul MACs buffer, and adding cell resuspension after the liquid drops completely flow out; 5) after the liquid drops completely flow out, adding 500 mul MACs buffer to wash twice; 6) after the liquid drops completely flow out, transferring the sorting column to a 15ml centrifuge tube containing 1ml of MACs buffer, adding 1ml of MACs buffer to rapidly wash the cells combined on the sorting column, and centrifuging for 8min at 500g and 4 ℃; 7) the appropriate volume of 1 × PBS or 1640 medium resuspended cells.

(2) Detection of BACE1^-/-Mouse and HUBC mouse CD4⁺Experiment of T cell BACE1 expression: isolation of BACE1 Using the method in (1)^-/-Mouse and HUBC mouse CD4⁺T cells.

The above cell samples were lysed and collected with cell lysate (1% Nonidet P-40, 0.5% sodium deoxycholate, 1M Tris-HCl, 150mM NaCl, pH7.5, protease inhibitor cocktail (Roche)); cells were lysed at room temperature and then an equal volume of 2 x SDS electrophoresis loading buffer was added. The sample was boiled in boiling water for 10min to prepare an SDS electrophoresis sample.

The detection of BACE1 by Western blot technique is carried out in the same manner as in example 1.

(3)CD4⁺Experiment of T cell-derived BACE1 cleavage of mPGES2 precursor form: isolation of BACE1 Using the method in (1)^-/-Mouse and HUBC mouse CD4⁺T cells were plated on cell culture plates, and after setting a control group (added with 1 XPBS) and an experimental group (added with the BACE1 inhibitor BIV 25. mu.M) and culturing for 24 hours, the cells were collected.

The precursor form and the mature form of mPGES2 were detected by Western blot technique, and the procedure was the same as in example 1.

(4)CD4⁺Flow cytometric experiments in which T cell activation affects BACE1 expression: isolation of CD4 of WT mice by the method of (1)⁺T cells are paved into a cell culture plate, CD3/CD28 antibodies or ConA with different concentrations are added at the same time for culturing for 24h, and then CD4 is collected⁺T cells.

BACE1 expression was detected by flow cytometry. The specific method comprises the following steps:

1) after obtaining the cell suspension, the cell concentration was adjusted to about 1X 10 in 100. mu.l⁶Placing the cells in a 96-well round bottom plate, adding 10 μ l of rat serum to seal the nonspecific binding, and incubating on ice for 15 min; 2) Adding appropriate amount of flow antibody (usually with final concentration of 1 μ g/ml), keeping out of the sun, and incubating on ice for 30 min; 3) adding 100 μ l of 1XPBS, 500g, centrifuging at 4 deg.C for 5min, discarding the supernatant, adding 200 μ l of 1XPBS, and washing again; 4)200 μ l 1XPBS resuspended cells and CD4 expressing BACE1 detected using a flow cytometer Fortessa (BD)⁺Positive rate of T cells.

(4)CD4⁺Western blot experiments in which T cell activation affected BACE1 expression: isolation of CD4 of WT mice by the method of (1)⁺T cells are plated on a cell culture plate, and are cultured for 24h by adding CD3/CD28 antibodies or ConA with different concentrations, and then the cells are collected.

The expression of BACE1 was detected by Western blot technique, and the procedure was as in example 1.

(5)CD4⁺Western blot experiments affecting the expression of the mature form of mPGES2 after T-cell knock-out or overexpression of BACE 1: isolation of BACE1 by the method in (1)^-/-Mouse and HUBC mouse CD4⁺T cells, plated in cell culture plates with the addition of CD3 or CD2Antibody 8 (1. mu.g/ml) was incubated for 24h, after which the cells were harvested.

The expression of the mature form of mPGES2 was detected by Western blot technique, and the specific procedure was the same as in example 1.

(6)CD4⁺Experiments affecting PGE2 expression following T cell knockout or overexpression of BACE 1: isolation of BACE1 by the method in (1)^-/-Mouse and HUBC mouse CD4⁺T cells were plated on cell culture plates and incubated for 24h or 72h with addition of CD3/CD28 antibody (1. mu.g/ml), after which the cells were harvested and frozen at-80 ℃.

The cell culture medium is taken out from minus 80 ℃, thawed and used for detecting the content of PGE2 by enzyme-linked immunosorbent assay (ELISA) technology. The specific method comprises the following steps:

1) corresponding solutions and standards were prepared as described, and all solutions were used after returning to room temperature (all reagents described below were included in the purchased ELISA kit).

2) Add 100. mu.l ELISA buffer to NSB wells and 50. mu.l ELISA buffer to B0 wells.

3) Add 50. mu.l PGE2 monoclonal antibody to wells (except TA, NSB and Blk wells) and incubate at room temperature for 1 h.

4) Add 50. mu.l of the different concentration gradient standards and test samples to the remaining wells, add 50. mu.l of PGE2 AchE trap wells (except for TA and Blk wells), and incubate overnight at 4 ℃.

5) The plate was decanted and washed 5 times with wash buffer.

6) Add 200. mu.l Ellman's Reagent to each well and add 5. mu.l PGE2 AchE trap to the TA wells, protected from light, and incubate at room temperature for 60-90 min.

7) The microplate reader reads at 410nm, a standard curve is prepared, and the concentration of the sample PGE2 is calculated.

(7)CD4⁺Effect of T cells upon BACE1 knockout PGE2 receptorExperiments with somatic expression: isolation of BACE1 by the method in (1)^-/-Mouse and WT mouse CD4⁺T cells were plated on cell culture plates and cultured for 24 hours with addition of CD3/CD28 antibody (1. mu.g/ml), after which the cells were harvested.

The cells were treated with Trizol (1ml per sample) and then submitted to the sequencer for RNA-Seq experiments. The data were then analyzed for expression of the different receptors of PGE 2.

4. Analysis of results

As can be seen from the results in FIG. 4, BACE1^-/-Mouse CD4⁺T cells with significantly reduced BACE1 expression (p)<0.05)；

As can be seen from the results in FIG. 5, CD4 was observed in HUBC mice⁺T cell BACE1 expression is remarkably increased (p)<0.05)；

As can be seen from the results of FIG. 6, CD4⁺T cell-derived BACE1 cleaves the mPGES2 precursor form, and the BACE1 inhibitor BIV treatment significantly inhibited this cleavage (p)<0.05)；

As can be seen from the results of FIG. 7, with CD4⁺Activation of T cells, CD4 expressing BACE1⁺The proportion of T cells increases significantly (p)<0.05)；

As can be seen from the results of FIG. 8, with CD4⁺Activation of T cells, and the expression level of BACE1 protein is obviously increased (p)<0.05)；

As can be seen from the results of FIG. 9, CD4⁺Expression of the mature form of mPGES2 was significantly reduced following T cell BACE1 knock-out (p)<0.05) in parallel with the results of FIG. 8 (CD 4)⁺Significantly increased BACE1 expression following T cell activation) consistent with CD4⁺Expression of the mature form of mPGES2 was also significantly increased following T cell activation, further suggesting cleavage of the precursor form of mPGES2 by BACE 1;

as can be seen from the results of FIG. 10, CD4⁺After T cell BACE1 is over-expressed, the expression of mPGES2 mature form is remarkably promoted (p)<0.05) in parallel with the results of FIG. 8 (CD 4)⁺Significantly increased BACE1 expression following T cell activation) consistent with CD4⁺Expression of the mature form of mPGES2 was also significantly increased following T cell activation, further suggesting cleavage of the precursor form of mPGES2 by BACE 1;

as can be seen from the results of FIG. 11, CD4⁺Following T cell BACE1 knock-out, expression of PGE2 was also significantly reduced (p) as expression of the mature form of PGE2 synthetase mPGES2 was reduced<0.05)；

As can be seen from the results of FIG. 12, CD4⁺After T cell BACE1 is over-expressed, the expression of PGE2 is also obviously reduced and increased along with the increase of the expression of the mature form of synthetase mPGES2 of PGE2 (p)<0.05)；

As can be seen from the results in FIG. 13, CD4⁺T cells predominantly express the PGE2 receptors EP2 and EP4 but not EP1 and EP3, and BACE1 knockdown does not affect expression of EP2 and EP4 (p)<0.05)；

EXAMPLE 3 BACE1 Regulation of CD4 by PGE2⁺T cell TCR pathway activation

1. Procedure of experiment

To investigate whether BACE1 modulates CD4⁺T function, the present invention demonstrates that PGE2 promotes CD4⁺Activation of T cell TCR pathways and their downstream pathways (figure 14), and knockout of BACE1 was able to significantly inhibit CD4⁺Activation of the T cell TCR pathway (FIG. 15), in contrast, BACE1 overexpression promoted CD4⁺Activation of the T cell TCR pathway (figure 16).

2. Experimental Material

BACE 1^-/-Mice were purchased from Jackson Laboratories; HUBC mice entrusted with college university construction; cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); Ultra-LEAF^TMPurified anti-mouse CD28(102115) and Ultra-LEAF^TMBoth Purified anti-mouse CD3(100340) were purchased from Biohedgend; 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; PGE2(P0409) was purchased from Sigma.

The antibodies were as follows:

a first antibody: anti-Actin (60008, proteintech); t Cell Signaling Antibody Sampler Kit (14541, CST); Anti-IkB alpha (4814, CST) Anti-p-IkB alpha (9246S, CST); Anti-I κ β (11930, CST); Anti-p-Ikappa beta (2697, CST); anti-p38(8690, CST); anti-p-p38(4511, CST); anti-p-AKT (4060, CST); anti-AKT (4685, CST); anti-ZAP (3165, CST); anti-Src (2109, CST); anti-LAT (9166, CST); PE anti-mouse CD4(100512, Biohedgend).

Secondary antibody: Anti-Rabbit IgG (H + L), HRP Conjugate (W4011, promega); Anti-Mouse IgG (H + L), HRP Conjugate (W4021, promega); goat Anti-Armenian hamster (IgG) (ab5738, Abcam).

3. Detailed description of the invention

(1) PGE2 promotes CD4⁺Experiments on T cell TCR pathway activation: WT mice were injected intraperitoneally with PGE2 at different concentrations for 24h, and CD4 was isolated⁺T cells were isolated as in example 2.

CD4⁺Rapid activation of T cell TCR pathways: the CD3/CD28 antibody treatment of the cell samples is carried out to rapidly activate CD4⁺T cell TCR pathways, the specific method is as follows:

1) mu.l of precooled 1XPBS (with 0.5% BSA) were resuspended and anti-CD3/anti-CD28 (final concentration: 1 μ g/ml), incubated on ice for 15 min;

2) add 500. mu.l 1XPBS (with 0.5% BSA), stop incubation, mini-bench centrifuge 6500rpm, 2min, centrifuge, discard supernatant;

3) the same number of cells (2X 10)⁶) Centrifuging, resuspending at 200uL, and uniformly distributing to 2 EP tubes for later use;

4) the EP tube was placed in a 37 ℃ water bath, stimulated with Goat anti-hamsterIg (40. mu.g/ml) or an equal volume of 1XPBS, after 10min, stimulated with 1 XPDS, and boiled at 95 ℃ for 10min to prepare SDS electrophoresis samples.

The Western blot technique is used to detect the changes of the TCR-pathway-associated proteins, and the specific operation method is the same as that in example 1.

(2)CD4⁺Experiments for T cell knockout BACE1 inhibition of TCR pathway activation: first isolating BACE1^-/-Mouse and WT mouse CD4⁺T cells were isolated as in example 1.

Isolated CD4⁺T cells were rapidly activated with CD3/CD28 antibody in the same manner as described in example (1), wherein the stimulation time was 10min or 30min after the addition of Goat anti-hamsterIg (40. mu.g/ml).

(3)CD4⁺Experiments in which T cells overexpress BACE1 promoting TCR pathway activation: first, HUBC mice and WT mice CD4 were isolated⁺T cells were isolated as in example 2.

Isolated CD4⁺T cells were rapidly activated using CD3/CD28 antibody in the same manner as described in example (1), wherein the stimulation time was 10min or 30min after the addition of Goat anti-hamsterIg (40. mu.g/ml).

4. Analysis of results

As shown in the results of FIG. 14, PGE2 promoted CD4⁺Phosphorylation expression of T cell TCR pathway related proteins, i.e., promotion of CD4⁺T cell TCR pathway activation;

as can be seen from the results of FIG. 15, CD4⁺After T cell knockdown of BACE1, phosphorylation expression of TCR pathway related protein is reduced, namely BACE1 knockdown inhibits CD4⁺T cell TCR pathway activation (p)<0.05)；

As can be seen from the results in FIG. 16, CD4⁺After T cells over-express BACE1, phosphorylation expression of TCR (T cell receptor) pathway related protein is up-regulated, namely BACE1 over-expression promotes CD4⁺T cell TCR pathway activation (p)<0.05)；

Example 4 BACE1 promotes CD4⁺T cell in vitro activation

1. Procedure of experiment

The TCR pathway is CD4⁺The most important signaling pathway for T cells, activation of which is also predictive of CD4⁺Activation of T cells. To investigate whether BACE1 indeed modulates CD4⁺Activation of T cells, the present invention utilizes CD4⁺T cell-related activation indicators experiments were conducted to find that BACE1 knockouts are followed by CD4⁺The proliferation level of T was inhibited (FIG. 17), the level of secreted IL-2 was significantly reduced (FIG. 18), while BACE1 overexpression promoted the secretion of IL-2 (FIG. 19); at the same time, CD4⁺Expression of the activation marker CD69 for T cells was also inhibited with BACE1 knock-out (FIG. 20), with BACE1 overexpressionUp-regulated (fig. 21).

2. Experimental Material

BACE 1^-/-Mice were purchased from Jackson Laboratories; HUBC mice entrusted with college university construction; cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); Ultra-LEAF^TMPurified anti-mouse CD28(102115) and Ultra-LEAF^TMBoth Purified anti-mouse CD3(100340) were purchased from Biohedgend; 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; mouse IL-2Elisa kit (1210202) was purchased from Daco.

The antibodies were as follows:

a first antibody: FITC anti-mouse Ki-67(652410, Biohedgend); PE anti-mouse CD4(100512, Biohedgend); pacific Blue^TM anti-mouse CD69(104524 Bioledgend)。

3. Detailed description of the invention

(1)CD4⁺Experiments in which T cells knock out BACE1 affected its proliferation: first isolating BACE1^-/-Mouse and WT mouse CD4⁺T cells were isolated as in example 2.

The cells were cultured for 24 hours after collection, and then the cells were collected and the expression level of the proliferation marker Ki-67 was measured by flow cytometry, which was carried out in the same manner as in example 2.

(2)CD4⁺Experiments in which T cell-derived BACE1 modulates IL-2 expression: first isolating BACE1^-/-Mouse and HUBC mouse CD4⁺T cells were isolated as in example 2.

After the above cells were collected, the cells were incubated for 24 hours with the addition of CD3/CD28 antibody (1. mu.g/ml) or 1XPBS, and the cell culture broth was collected and assayed for IL-2 content by ELISA. The specific method comprises the following steps:

corresponding solutions and standards (all reagents described below are included in the ELISA kit) were prepared as described in the specification and all solutions were used after returning to room temperature.

1) Add 100. mu.l of different concentration gradient standards and samples to be tested into the wells.

2) Add 50. mu.l of biotinylated antibody to the wells and incubate for 90min at 37 ℃.

3) The plate was decanted and washed 5 times with wash buffer.

4) Add 100. mu.l of Streptavidin-HRP to the wells and incubate for 90min at 37 ℃.

5) The plate was decanted and washed 5 times with wash buffer.

6) Adding 100. mu.l of TMB, keeping out of the light, incubating at 37 ℃ for 5-30 min.

7) The reaction was stopped by adding 100. mu.l of Stop solution according to the shade of the color in the wells.

8) Reading at 450nm and 610nm by a microplate reader, making a standard curve, and calculating the IL-2 concentration of the sample.

(3)CD4⁺Experiment of T cell-derived BACE1 to modulate CD69 expression: first isolating BACE1^-/-Mouse and HUBC mouse CD4⁺T cells were isolated as in example 2.

After the above cells were collected, the cells were cultured for 24 hours with addition of CD3/CD28 antibody (1. mu.g/ml) or 1XPBS, and then the cells were collected and examined for expression of CD69 by flow cytometry. The flow cytometry was performed in the same manner as in example 2.

4. Analysis of results

As can be seen from the results in FIG. 17, CD4⁺Ki-67 expression was significantly reduced following T cell knock-out of BACE1 (p)<0.05), namely CD4⁺T cell proliferation is inhibited.

As can be seen from the results in FIG. 18, CD4⁺Significant reduction in IL-2 expression following T cell knockout of BACE1 (p)<0.05), namely CD4⁺T cell proliferation is inhibited.

As can be seen from the results in FIG. 19, CD4⁺IL-2 expression is significantly increased (p) after T cells overexpress BACE1<0.05), namely CD4⁺T cell proliferation is up-regulated.

As can be seen from the results of FIG. 20, CD4⁺Significant reduction in CD69 expression following T cell knockout of BACE1 (p)<0.05), namely CD4⁺Survival of T cellsThe formation is suppressed.

As can be seen from the results in FIG. 21, CD4⁺Significant increase in CD69 expression (p) following T cell overexpression of BACE1<0.05), namely CD4⁺T cell activation is upregulated.

Example 5 BACE1 promotes CD4⁺In vivo activation of T cells

1. Procedure of experiment

To investigate whether BACE1 modulates CD4 as well⁺T cell activation in vivo, mice were non-specifically immunized with ConA according to the invention, respectively, and CD4 was then detected⁺T cell activation indicators (fig. 22 and 23); and KLH-specific immunization of mice followed by detection of CD4⁺T cell activation indicators (fig. 24-31). To exclude the effects of BACE1 from other sources, the present invention also used T/B cell deficient immunodeficient murine Rag1^-/-Mice, and BACE1^-/-CD4 in mice and HUBC mice⁺T cells were isolated and transferred to Rag1^-/-Mice were simultaneously immunized with KLH and then tested for CD4⁺T cell activation indices (fig. 32-37).

2. Experimental Material

BACE 1^-/-Mice were purchased from Jackson Laboratories; HUBC mice entrusted with college university construction; rag1^-/-Mice were from Jackson Laboratories. Cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; canavalin A (Concanavalin A, Con A) was purchased from Sigma; complete freund's adjuvant (F5881) available from Sigma company; keyhole limpet hemocyanin (KLH, H7017) was purchased from Sigma company; HRP-labeled streptavidin (N100) was purchased from Thermo Fisher; TMB Super Sensitive One Component HRP Microwell Substrate (TMBS-0100-01) available from Surmodics; mouse IL-2Elisa kit (1210202) available from David as Inc.; mouse

CD4⁺T Cell Isolation Kit (19765) was purchased from Stem Cell Inc.

The antibodies were as follows:

a first antibody: biotin anti-IgM (406504, Biohedgend); biotin anti-IgG (53441, BD), FITC anti-mouse Ki-67(652410, Biohedgend); PE anti-mouse C D4(100512, Biohedgend); pacific Blue^TM anti-mouse CD69(104524，Biol edgend)；PE/Cy7 anti-mouse IL-17A(506921，Bioledgend)；FITC anti-mo use IFN-γ(505806，Bioledgend)，PerCP/Cy5.5 anti-mouse CD3e(100328， 505806，Bioledgend)。

3. Detailed description of the invention

(1) BACE1 knockdown inhibits ConA-induced CD4⁺Experiment of in vivo activation of T cells: first BACE1^-/-Mice and WT mice were injected with Con A (3mg/kg) via tail vein, 24h later, and then mouse spleen cells were isolated and subjected to flow cytometry to detect CD4⁺Expression of T cell CD 69.

The flow cytometry was performed in the same manner as in example 2.

(2) BACE1 overexpression promotes ConA-induced CD4⁺Experiment of in vivo activation of T cells: HUBC mice and WT mice were first injected with Con A (3mg/kg) via tail vein, 24h later, mouse spleen cells were isolated, and CD4 was detected by flow cytometry⁺Expression of T cell CD 69.

The flow cytometry was performed in the same manner as in example 2.

(3) BACE1 knockout inhibits (overexpression promotes) KLH-induced CD4⁺Experiment of in vivo activation of T cells: KLH (1mg/ml) was first emulsified with complete Freund's adjuvant, then 100. mu.l of the emulsified antigen was injected into each hind limb of the mice, and after 7 days, mouse sera were collected and the mouse spleen and inguinal lymph nodes were isolated. A portion of the spleen cells were cultured for a further 48h in vitro stimulated with different concentrations of KLH.

Detection of spleen CD4 by flow cytometry⁺Expression of Ki-67 in T cells and inguinal lymph node CD4⁺The specific procedure of flow cytometry was the same as in example 2 in the case of T cells secreting IL-17A and IFN- γ.

ELISA technology is used for detecting the expression level of IL-2 in the culture medium after spleen cells continue to be stimulated by KLH for 48 hours in vitro, and the specific method is the same as that in example 4.

The titers of the KLH-specific antibodies IgG and IgM in the mouse sera were determined by ELISA technique. The specific method comprises the following steps:

1) preparing a carbonic acid buffer solution: 1.59g Na₂CO₃+2.93g NaHCO₃+ddH₂And (4) metering the volume of O to 1L, and adjusting the pH value to 9.6.

2) Antigen coating: one day before the experiment, KLH was diluted to 10. mu.g/ml with carbonate buffer (antigen concentration can be determined as required), and 100. mu.l was added to each well overnight at 4 ℃ (typically about 16 h).

3) PBST (0.05% Tween) wash, 300. mu.l.times.3 times.

4) Non-specific binding was blocked by addition of 300. mu.l of 1% BSA (in PBS) for 2h at room temperature.

5) PBST (0.05% Tween) wash, 300. mu.l.times.3 times.

6) And adding a 200-time diluted (adjustable) serum sample into the first hole, diluting the ELISA plate 2 times downwards in each hole longitudinally, mixing uniformly, and incubating for 1h at 37 ℃.

7) PBST (0.05% Tween) wash, 300. mu.l.times.3 times.

8) Anti-mouse IgG and IgM antibodies were diluted with PBST 1:2000, added to 100. mu.l per well, and incubated at 37 ℃ for 1 h.

9) PBST (0.05% Tween) wash, 300. mu.l.times.3 times.

10) Mu.l of HRP-labeled streptavidin (1:10000) was added to each well, incubated at room temperature for 30 min.

11) PBST (0.05% Tween) wash, 300. mu.l.times.3 times.

12) Add 100. mu.l TMB substrate, take out in advance and return to room temperature for use, protect from light, room temperature, 15 min.

13) The reader detects the reading of 450nm and calculates the antibody titer.

(4)CD4⁺T cell-derived BACE1 knock-out inhibits (over-expression promotes) KLH-induced CD4⁺In vivo activation of T cellsThe experiment of (2): first isolating BACE1^-/-Of mice and HUBC mice

CD4⁺T cells, isolated as follows:

1) after separating the spleen or lymph node of mouse to obtain mononuclear cell, 1X 10⁸Cells/ml (0.1-2ml 1640 medium resuspended) were transferred to a 5ml BD flow tube. This experiment was typically resuspended in 1ml of medium.

2) Blocking and antibody incubation: add 50. mu.l rat serum + 50. mu.l isolation Cocktail to the cell suspension, mix well using a pipette gun, and incubate at room temperature for 7.5 min.

3) Add 50. mu.l of deletion Cocktail to the cell suspension, mix well using a pipette, and incubate for 2.5min at room temperature.

4) RapidSpheres was shaken for 30sec, 75. mu.l was added to the cell suspension, mixed well using a pipette, and incubated at room temperature for 2.5 min.

5) Adding into 1.5ml 1640 culture medium cell suspension, mixing well with pipette, transferring flow tube to magnetic pole, and incubating at room temperature for 2.5 min.

6) And pouring the cell suspension in the flow tube into a 15ml centrifuge tube (pouring the flow tube together with a magnetic pole integrally), centrifuging for 8min at 500g and 4 ℃, removing supernatant, and adding a proper amount of 1640 culture medium for resuspension for later use.

CD4 isolated as described above⁺T cells were transfused via tail vein to Rag1^-/-Mice were immunized with KLH, and the specific immunization method was the same as that described in (3) of this example; 7d after immunization, mouse spleen CD4 was detected by flow cytometry⁺Proportion of T cells and CD4⁺T cells express CD69 and Ki-67.

4. Analysis of results

As shown in FIG. 22, it can be seen from the results of BACE1 knock-out that ConA non-specifically induces CD4⁺Activation of T cells is inhibited (p)<0.05) as indicated by the inhibition of the expression of the activation protein CD 69.

As shown in FIG. 23, the result shows that after BACE1 is over-expressed, ConA non-specifically induced CD4⁺Activation of T cells is upregulated (p)<0.05), performanceThe expression of CD69 is up-regulated to activate the protein.

As can be seen from the results in FIG. 24, KLH-specific induction of CD4 following BACE1 knock-out⁺Inhibition of T cell activation (p)<0.05) as indicated by the inhibition of the expression of the proliferation marker Ki-67.

As can be seen from the results in FIG. 25, KLH-specific induction of CD4 following BACE1 knock-out⁺Inhibition of T cell activation (p)<0.05) as indicated by the suppression of the expression of the activating factors IL-17A and IFN-gamma.

As can be seen from the results in FIG. 26, KLH-specific induction of CD4 following BACE1 knock-out⁺Inhibition of T cell activation (p)<0.05) expressed as CD4⁺T cell-mediated antibody production is inhibited.

As can be seen from the results in FIG. 27, KLH-specific induction of CD4 following BACE1 knock-out⁺Inhibition of T cell activation (p)<0.05) as indicated by the inhibition of the expression of the activating factor IL-2.

As can be seen from the results in FIG. 28, KLH-specific induction of CD4 following overexpression of BACE1⁺T cell activation is upregulated (p)<0.05) as expressed by the proliferation marker Ki-67, was up-regulated.

As can be seen from the results in FIG. 29, KLH-specific induction of CD4 following overexpression of BACE1⁺T cell activation is upregulated (p)<0.05) showing up-regulated expression of the activating factors IL-17A and IFN-gamma.

As can be seen from the results in FIG. 30, KLH-specific induction of CD4 following overexpression of BACE1⁺T cell activation is upregulated (p)<0.05) expressed as CD4⁺T cell mediated antibody production is upregulated.

As can be seen from the results in FIG. 31, KLH-specific induction of CD4 following overexpression of BACE1⁺T cell activation is upregulated (p)<0.05) as indicated by up-regulation of the expression of the activator IL-2.

From the results in FIG. 32, it is clear that CD4⁺KLH-specific induction of CD4 following T cell BACE1 knockdown⁺T cell activation is inhibited as evidenced by BACE1 knockout CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺The proportion of T cells is significantly reduced(p<0.05)。

From the results in FIG. 33, it is clear that CD4⁺KLH-specific induction of CD4 following T cell BACE1 knockdown⁺T cell activation is inhibited as evidenced by BACE1 knockout CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺Suppression of CD69 expression by T cells (p)<0.05)。

From the results in FIG. 34, CD4 is shown⁺KLH-specific induction of CD4 following T cell BACE1 knockdown⁺T cell activation is inhibited as evidenced by BACE1 knockout CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺Ki-67 expression in T cells is suppressed (p)<0.05)。

From the results in FIG. 35, it is clear that CD4⁺KLH-specific induction of CD4 following overexpression of BACE1 in T cells⁺T cell activation is upregulated as evidenced by BACE1 overexpressing CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺The proportion of T cells is significantly upregulated (p)<0.05)。

From the results in FIG. 36, CD4 is shown⁺KLH-specific induction of CD4 following overexpression of BACE1 in T cells⁺T cell activation is upregulated as evidenced by BACE1 overexpressing CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺Expression of CD69 by T cells is upregulated (p)<0.05)。

From the results in FIG. 37, CD4 is shown⁺KLH-specific induction of CD4 following overexpression of BACE1 in T cells⁺T cell activation is upregulated as evidenced by BACE1 overexpressing CD4⁺T cells were transfused to Rag1^-/-Post mouse, CD4 in its spleen⁺Ki-67 expression by T cells is upregulated (p)<0.05)。

Example 6 AD patients and AD model mice CD4⁺T cell BACE1 expression is remarkably up-regulated and activation is abnormal

1. Procedure of experiment

Existing studies have shown that BACE1 expression is significantly elevated in brain and peripheral blood of AD patients, but CD4⁺It is not known whether T cells also show up-regulation of BACE1 expression. To explore AD patients and AD model mice CD4⁺T cellsBACE1 expression and activation state, CD4 in the peripheral blood of AD patients and spleen of AD model mice⁺T cells were subjected to relevant experiments. Studies have found CD4 in peripheral blood of AD patients⁺T cells BACE1 expression was significantly upregulated (FIG. 38) (p)<0.05), and activating CD4⁺T cell proportion Up-regulated (FIG. 39) (p)<0.05). Similarly, CD4 in spleen of AD model mouse⁺T cells expression of BACE1 was also significantly upregulated (figure 40) (p)<0.05) of its activating property CD4⁺Upregulation of T cell proportion and expression of the activator protein CD69 (FIGS. 41 and 42) (p)<0.05)。

2. Experimental Material

Peripheral blood of AD patients and cognitive normal controls is from the neurology department of the first hospital affiliated to the university of science and technology in China; 5 × FAD is from Jackson Laboratories. Cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; HISTOPAQUE (10771) from Sigma.

The antibodies were as follows:

a first antibody: PE anti-mouse CD4(100512, Biohedgend); pacific Blue^TM anti -mouse CD69(104524，Bioledgend)；APC anti-mouse CD62L(104412，B ioledgend)；APC/Cy7 anti-mouse/human CD44(103028，Bioledgend)；AP C/Cyanine7 anti-human CD45RO(304228，Bioledgend)；PE anti-human C D4(300508，Bioledgend)；PerCP/Cy5.5 anti-human CD3(317336，Bioled gend)；APC anti-human CD45RA(304112，Bioledgend)anti-BACE1(560 6S，CST)；anti-Actin(60008，proteintech)。

Secondary antibody: Anti-Rabbit IgG (H)⁺L)，HRP Conjugate(W4011，promega)；A nti-Mouse IgG(H+L)，HRP Conjugate(W4021，promega)。

3. Detailed description of the invention

The cell sorting reagent MACs buffer used in this study: namely, the magnetic bead sorting buffer solution contains 2mM EDTA and 0.5% bovine serum albumin in 1 XPBS.

(1) CD4 in peripheral blood of AD patients⁺T cell BACE1 expression up-regulation and activationThe general experiments were as follows: collecting peripheral blood of a patient and separating cells, wherein the specific method comprises the following steps:

1)5ml of blood, directly balancing and centrifuging at 2000rpm for 10min, taking supernatant (blood plasma), subpackaging 200 mul/tube by using an EP tube with low protein adsorption, and transferring to-80 ℃ for storage.

2) After removing the upper plasma, 2ml 1640 medium was added for resuspension;

3) a15 ml centrifuge tube was added with 3ml HISTOPAQUE density gradient separation (used after returning to room temperature in advance), the centrifuge tube was tilted (45 °), 2ml of cell resuspension was slowly added to the upper layer of the separation along the tube wall, 400g was added, centrifuged at room temperature for 20min, and the solution was raised 6 and lowered 0.

4) The middle white cell layer (single nucleus cell) was carefully aspirated by pipette gun into a new 15ml centrifuge tube containing 8ml 1640 medium, 300g, room temperature, and centrifuged for 5 min.

5) The supernatant was discarded, and 2ml of 1640 medium was added to resuspend the pellet, 300g, and centrifuged at room temperature for 5 min.

6) Abandoning the supernatant, resuspending the cells with 500-.

Detection of BACE1 expression by Western blot technique, the concrete operation method is the same as example 1

Simultaneously, the CD4 of the cell sample is detected by flow cytometry⁺Expression of T cell surface molecules. The flow cytometry method was the same as in example 2.

(2) CD4 in spleen of AD model mouse⁺Experiments with abnormal upregulation and activation of T cell BACE 1: isolation of mouse spleen CD4⁺The specific procedure for T cells was the same as in example 1.

4. Analysis of results

As can be seen from the results in FIG. 38, CD4 in the peripheral blood of AD patients⁺T cells expression of BACE1 is significantly upregulated (p)<0.05)。

As can be seen from the results in FIG. 39, CD4 was found in the peripheral blood of AD patients⁺T cell activation is upregulated, and is manifested by activating CD4⁺T cells (CD45 RA)^-CD45RO⁺) Proportional Up-regulation (p)<0.05)。

As can be seen from the results in FIG. 40, CD4 was found in spleen of AD model mouse⁺T cells expression of BACE1 is significantly upregulated (p)<0.05)。

As can be seen from the results in FIG. 41, CD4 was found in spleen of AD model mouse⁺T cell activation is upregulated, and is manifested by activating CD4⁺T cell (CD 4562L)^low CD44^hi) Proportional Up-regulation (p)<0.05)。

As can be seen from the results in FIG. 42, CD4 was found in spleen of AD model mouse⁺T cell activation is upregulated, as is expression of the activator protein CD69 (p)<0.05)。

Example 7 treatment of PGE2 receptor EP2/EP4 inhibitor significantly improves pathology in the brain of AD model mice

1. Procedure of experiment

The above example experiments show that BACE1 can promote maturation of mPGES2 by shearing mPGES2 precursor form, thereby promoting production of PGE2, and further up-regulating CD4⁺Activation of T cells. Meanwhile, the invention also discovers CD4 in AD patients and AD model mice⁺BACE1 was significantly upregulated by T cells, consistent with CD4⁺Abnormal T cell activation. Studies have shown CD4⁺Abnormal activation of T may lead to worsening of AD pathology, therefore, the present invention aims to explore whether intervention in the receptor through PGE2 could improve CD4⁺Activation function of T cells and intracerebral pathology of AD model mice. According to the invention, firstly, the AD model mice are respectively treated by the intragastric administration of EP2 and EP4 inhibitors, and then the pathology in the mouse brain is detected by an immunohistochemical method, so that the EP2 and EP4 inhibitor treatment can obviously reduce the pathology in the AD mouse brain (figure 43) (p)<0.05), and CD4⁺Activation of the T cell TCR pathway was also significantly inhibited (FIG. 44) (p)<0.05)。

2. Experimental Material

5 × FAD is from Jackson Laboratories. Cell sorting reagents Anti-PE beads (130-048-801, Miltenyi Biotec); 20 × phosphate buffered saline (20 × PBS, working solution diluted 50-fold to 1 × use with distilled water) was purchased from Sangon corporation; EP2 inhibitors (PF-04418948, which specifically binds to EP2 and thereby inhibits the binding of PGE2 to EP 2) were purchased from MCE; EP4 inhibitor (BGC 20-1531, which specifically binds to EP4 and thereby inhibits the binding of PGE2 to EP 4) was purchased from abin corporation; VECTASTAIN Elite ABC Kit Components and DAB Peroxidase Substrate Kit were purchased from Vector.

The specific information for EP2 inhibitors is as follows:

alias: 1- (4-fluorobenzoyl) -3- [ [ ((6-methoxy-2-naphthyl) oxy ] methyl ] -3-azetidinecarboxylic acid

CAS number: 1078166-57-0

Empirical formula (hilt representation): c₂₃H₂₀FNO₅

Molecular weight 409.41

Structural formula (xvi):

the specific information for EP4 inhibitors is as follows:

alias: 4- [ [4- (5-methoxy-2-pyridinyl) phenoxy ] methyl ] -5-methyl-N- [ ((2-methylphenyl) sulfonyl ] -2-furancarboxamide

CAS number: 1186532-61-5

Empirical formula (hilt representation): c₂₆H₂₅ClN₂O₆S

Molecular weight 492.5

Structural formula (xvi):

the antibodies were as follows:

a first antibody: PE anti-mouse CD4(100512, Biohedgend); t Cell Signaling A ntibody Sampler Kit (14541, CST); Anti-beta-Amyloid, 17-24(800711, Bi oledaged); Anti-Iba1(019-19741, WAKO).

Secondary antibody: Anti-Rabbit IgG (H + L), HRP Conjugate (W4011, promega); a nti-Mouse IgG (H + L), HRP Conjugate (W4021, Promega); biotinylated H organ Anti-Rabbit IgG (BA-1100, Vector); biotinylated Horse Anti-Mouse IgG (BA-2000, Vector).

3. Detailed description of the invention

(1) Experiments in which PGE2 receptor EP2/EP4 inhibitors improved pathology in AD mice brain: 4-month-old 5 XFAD mice were divided into four groups A-D, wherein group A had gastric lavage with PBS for 7 consecutive days; group B was gavaged for 7 consecutive days with PF-04418948, 62.5 mg/kg/day; group C, gastric lavage BGC 20-1531 for 7 consecutive days, 62.5 mg/kg/day; group D was gavaged for 7 consecutive days with PF-04418948(62.5mg/kg/day) and BGC 20-1531(62.5 mg/kg/day). After 7 days, frozen sections of mouse brains were taken and stored at-80 ℃ for immunohistochemical experiments, the specific method was as follows:

1) taking out brain slice in a refrigerator at-80 deg.C, and standing at room temperature for 2 h.

2) Rinsing: rinse 3 times 5min each time with 1 × PBS. The aim is to eliminate the OCT embedding medium used by the frozen section.

3) Blocking endogenous peroxidase: the tissue has 0.3% H prepared from methanol as solvent and endogenous peroxidase capable of reacting with color-developing agent to generate false positive result₂O₂Blocking is performed, and the sections are placed in a moisture-preserving box and incubated in an oven at 37 ℃ for 30 min.

4) Rinsing: rinsing with 1XPBS was performed 3 times for 5min each time to avoid actual cross-reactions.

5) Antigen retrieval: preparing a citric acid repairing solution (solution A: 21.01g citric acid +1L ddH 2O; solution B: 29.41g sodium citrate (2H2O) +1L ddH₂And O. Working solution when in use: 9ml of solution A +41ml of solution B +450ml of ddH₂O pH＝6.0)

6) The water bath heat repairing technology is adopted, the slices are placed into a small beaker filled with repairing liquid, the beaker is placed into a large beaker filled with water, and a preservative film seal (with a plurality of small openings for ventilation) is placed in a microwave oven. Burning the high-grade material to be bubbling, wherein the strength is based on that the bubbling is not stopped during heating, the medium-grade material is subjected to cooling in a microwave oven for 10min, and the medium-grade material is naturally cooled at room temperature (about 20-30 min).

7) Rinsing: rinse 3 times 5min in 1 × PBS.

8) The following steps can be selected: sections were incubated with 88% formic acid at room temperature for 20 min. Rinse 3 times 5min in 1 × PBS.

9) Cell permeation: ttiton X-100 at 0.3% in 1XPBS was incubated at room temperature for 30 min.

10) Rinsing: rinsing: rinse 3 times 5min in 1 × PBS.

11) And (3) sealing: 10% goat serum, PBS) or 10% horse serum, PBS), incubated at 37 ℃ for 20 min.

12) Primary antibody incubation: spin-dry gently and wipe excess serum blocking solution around the section with filter paper, add primary antibody, incubate overnight at room temperature for 1h, 4 ℃.

13) Rewarming: after the primary antibody is incubated at 4 ℃ overnight, the primary antibody is incubated at room temperature for 20min in order to prevent the slices from being washed by 1XPBS directly from 4 ℃ and causing the slices to fall off.

14) Rinsing: rinsing: rinse 3 times 5min in 1 × PBS.

15) And (3) secondary antibody incubation: spin-dry gently and wipe excess serum blocking solution around the section with filter paper, add biotin-labeled secondary antibody, incubate for 30min at 37 ℃ (secondary antibody diluted with vector kit, 10% goat serum 1: 2000). And preparing the ABC compound, and standing for reaction for about 30 min.

16) Rinsing: rinse 3 times 5min in 1 × PBS.

17) ABC incubation: spin-drying gently, wiping the slices with filter paper, and adding ABC complex (1% goat serum 10 μ l A solution and 10 μ l B solution, AB mixed solution is C, so called ABC together)

18) Rinsing: 2 times for 10min each time.

19) Color development: DAB color-developing solution (1ml DAB solution + 16.8. mu.l buffer solution +16. mu. l H) was added dropwise₂O₂) The color development effect is seen, and the color development is stopped by tap water.

20) Hematoxylin counterstain, bluing.

21) And (3) dehydrating and transparency: 75% alcohol-85% alcohol-95% alcohol-absolute ethanol 1-absolute ethanol 2 (the first cylinder of absolute ethanol may be diluted by the last cylinder of 95% alcohol, and two cylinders of absolute ethanol are needed for thorough dehydration), xylene 1-xylene 2 (the first cylinder of xylene may be diluted by the last cylinder of absolute ethanol, and two cylinders of xylene are needed for thorough transparency), each for 3 min.

22) Sealing: dripping neutral gum before xylene drying, air drying in ventilation place, and wiping periphery with alcohol.

23) Full-automatic inverted fluorescence microscope photographing

(2) PGE2 receptor EP2/EP4 inhibitor for improving AD mouse CD4⁺Abnormal activation of T cells: (1) taking spleen of mouse for separating CD4 while taking brain of middle mouse⁺T cells were isolated in the same manner as in example 1. Separating to obtain CD4⁺Rapid activation of CD4 after T-cell activation using CD3/CD28 antibody⁺T cell TCR pathways were stimulated by SDS electrophoresis as described in example 3.

The expression of the TCR access protein is detected by Western blot technology, and the specific operation method is the same as that in example 1.

4. Analysis of results

From the results in FIG. 43, it is clear that treatment with the EP2/EP4 inhibitor significantly improved the pathology in the brains of AD mice, as indicated by a decrease in the deposition of A β and a decrease in the activation of microglia in the brains of AD mice (p < 0.05).

As can be seen from the results in FIG. 44, treatment with the EP2/EP4 inhibitor can significantly improve CD4 in AD mice⁺Abnormal activation of T cells, manifested by their TCR pathway phosphoprotein profilesDecrease (p)<0.05)。

In summary, CD4⁺T cell-derived BACE1 can specifically cleave the precursor form of PGE2 synthetase mPGES2, thereby promoting maturation of mPGES2, further resulting in increased synthesis of PGE2, which will ultimately lead to CD4⁺Abnormal activation of T cells; CD4 of AD patients and AD model mice⁺T cells mediated CD4 due to high expression of BACE1⁺Abnormal activation of T cells, which may lead to exacerbation of AD pathology; after the PGE2 receptor EP2/EP4 receptor inhibitor is used for treating AD model mice, the invention discovers that the intracerebral pathology and CD4 of the AD model mice can be obviously improved⁺Abnormal activation of T cells. Thus, the present invention first discovered CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 can be used as new therapeutic targets for treating AD.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1. A pharmaceutical composition for treating or ameliorating Alzheimer's disease, said pharmaceutical composition being CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 genes or proteins as targets.

2. The pharmaceutical composition of claim 1, wherein the drug comprises at least CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Inhibitors of T cell derived BACE1 and/or EP2 and/or EP4 proteins.

3. The pharmaceutical composition of claim 2, wherein the administration of CD4 is carried out⁺Substances with reduced expression levels of T cell derived BACE1 and/or EP2 and/or EP4 genes include targeted CD4⁺T cell derived BACE1 and/or RNAi fragment and/or siRNA fragment of EP2 and/or EP4 gene and/or CD4⁺Specific transcriptional inhibitors of the T cell derived BACE1 and/or EP2 and/or EP4 genes.

4. The pharmaceutical composition of any one of claims 1-3, wherein the CD4 is targeted⁺The sequence of siRNA fragment of T cell derived BACE1 gene is 5'-GCATGATCATTGGAGGTATTT-3'.

5. The pharmaceutical composition of any one of claims 1-2, wherein said CD4⁺Inhibitors of T cell-derived BACE1 have the structural formula:

6. the pharmaceutical composition of any one of claims 1-2, wherein the EP2 inhibitor has the structural formula:

7. the pharmaceutical composition of any one of claims 1-2, wherein the EP4 inhibitor has the structural formula:

8. make CD4⁺Substance with reduced expression level of T cell derived BACE1 and/or EP2 and/or EP4 genes and/or CD4⁺Use of an inhibitor of T cell derived BACE1 and/or EP2 and/or EP4 protein for the manufacture of a pharmaceutical composition for the treatment or alleviation of alzheimer's disease.

9. Alzheimer's disease biomarker, characterized in that said biomarker comprises CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 genes.

10. Kit for diagnosing alzheimer's disease, characterized in that said kit comprises CD4⁺T cell derived BACE1 and/or EP2 and/or EP4 genes.