CN110656170A

CN110656170A - Reagent, diagnostic product and therapeutic composition for Alzheimer disease diagnosis, candidate drug screening method and application

Info

Publication number: CN110656170A
Application number: CN201911090385.4A
Authority: CN
Inventors: 张伟; 王明永; 李敏; 白姗姗; 石如玲; 张奇
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-01-07

Abstract

The invention relates to a reagent, a diagnostic product, a therapeutic composition for diagnosing Alzheimer disease, a candidate drug screening method and application, belonging to the technical field of diagnosis and treatment of Alzheimer disease. According to the invention, the expression of the FoxO1 gene in the blood of an AD patient is obviously reduced compared with the blood of a normal person, and the fact that whether the AD patient suffers from the AD can be judged by measuring the expression level of the FoxO1 gene in the blood of the AD patient is shown. Therefore, FoxO1 can be used as a biomarker for detecting AD. Experiments show that the over-expression of the FoxO1 gene can reduce the expression of BACE1 and reduce the generation of A beta, and the A beta is related to the generation and development of AD, so that the FoxO1 gene can be used as a target spot for developing a medicament for treating Alzheimer disease or preventing Alzheimer disease, and has good application prospect.

Description

Reagent, diagnostic product and therapeutic composition for Alzheimer disease diagnosis, candidate drug screening method and application

Technical Field

The invention relates to a reagent, a diagnostic product, a therapeutic composition for diagnosing Alzheimer disease, a candidate drug screening method and application, belonging to the technical field of diagnosis and treatment of Alzheimer disease.

Background

Alzheimer's disease AD is the most common neurodegenerative disease with progressive cognitive, behavioral and functional disorders and is highly heritable (with a genetic rate as high as 76%), but its mechanism is particularly complex from a genetic point of view. As the population ages, the risk of developing disease after the age of 85 years can reach 50%, and dementia will become a major challenge for human medical treatment. AD successfully identified 3 genes involved in early-onset AD, late-onset AD and autosomal dominant genetic disease in early genetic studies and showed a complex genetic pattern.

Amyloid plaques formed by amyloid beta (a β) and neurofibrillary tangles formed by phosphorylation of tau protein are two of the major pathological features of AD. A β plays an important role in the pathological development of AD. A β is produced by enzymatic hydrolysis of β -Amyloid Precursor Protein (APP) by various secretases (secretases). The APP is cut by beta-secretase to generate extracellular segments sAPP beta and beta-CTF (C-terminal Fragment, CTF). sAPP is released and the transmembrane β -CTF is further cleaved by γ -secretase to yield a β. Therefore, blocking the enzymatic cleavage of β -secretase and γ -secretase, thereby reducing the production of a β, is an important approach to the treatment of AD. However, in addition to cutting APP, γ -secretase is also involved in the hydrolytic cleavage of various proteins such as notch, Erb4, etc. Blocking the gamma-secretase enzyme may result in normal cellular dysfunction. BACE1 is a novel aspartic protease with a transmembrane structure. BACE1 was found to be the only β -secretase enzyme that cleaves APP, and is also the most important one. Therefore, BACE1 is an important target in the development of drugs for treating AD.

For AD, symptomatic treatment is currently performed mainly at an early stage, however, clinically diagnosed AD patients are mostly in a middle-to-late stage, and the existing treatment only improves symptoms and cannot organize or reverse the progression of the disease. Therefore, early diagnosis and intervention can remarkably reduce the harm of AD, thereby lightening the burden of society and families.

Disclosure of Invention

The invention aims to provide a reagent for diagnosing Alzheimer disease, which can detect and diagnose Alzheimer disease at an early stage.

The invention also provides an Alzheimer disease diagnostic product which can detect and diagnose Alzheimer disease at an early stage.

The invention also provides a composition for treating alzheimer disease, which can be used for treating alzheimer disease.

The invention also provides a method for screening the candidate drug for treating the Alzheimer disease, which can screen the candidate drug for treating the Alzheimer disease.

The invention also provides application of the reagent and the composition.

In order to achieve the purpose, the invention adopts the technical scheme that:

a reagent for diagnosing Alzheimer's disease, which is a reagent for detecting the mRNA expression level of FoxO1 gene or the expression level of FoxO1 protein.

According to the invention, through comparative study of healthy old people and AD patients, the FoxO1 gene expression is found to be related to Alzheimer disease; compared with the normal human blood, the expression of the FoxO1 gene in the blood of the Alzheimer disease patient is obviously reduced, and the difference has statistical significance. Thus, by detecting FoxO1 expression in the blood of a subject, a clinician can be instructed to determine whether the subject has, or is at risk for having, alzheimer's disease, thereby providing a prophylactic or therapeutic regimen to the subject. The reagent is particularly suitable for early diagnosis of Alzheimer's disease.

Preferably, the detection reagent for the mRNA expression level of the FoxO1 gene is a probe which specifically recognizes the FoxO1 gene; or a primer for specifically amplifying the FoxO1 gene. Specifically, the primer sequence of the specific amplification FoxO1 gene is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Preferably, the reagent for detecting the expression level of the FoxO1 protein is an antibody or ligand specifically binding to the FoxO1 protein. Antibodies or ligands to FoxO1 protein can detect the expression status of FoxO1 gene from protein levels.

An Alzheimer's disease diagnostic product comprises the reagent for diagnosing Alzheimer's disease. Specifically, the reagent for diagnosing the alzheimer disease can be prepared into a kit, a chip or a nucleic acid membrane strip, and all the reagents can be used for detecting the alzheimer disease.

A composition for treating alzheimer's disease comprising FoxO1 protein, or a promoter comprising FoxO1 gene, or a promoter comprising FoxO1 protein.

In the invention, FoxO1 can reduce the secretion of A beta by inhibiting the expression of BACE1, thereby influencing the amyloid pathway of APP metabolism; the FoxO1 protein can be directly used as a potential drug for treating Alzheimer disease.

The promoter comprises substances for improving the stability of the FoxO1 gene or an expression product thereof, up-regulating the expression level of the FoxO1 gene or an expression product thereof, and prolonging the effective acting time of the FoxO1 gene or an expression product thereof; the pharmaceutically acceptable carrier and/or adjuvant includes (but is not limited to) diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant; the pharmaceutical composition may further include additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

Preferably, the promoter is a vector containing the FoxO1 gene. A specific vector may be pcDNA3.1, which contains the FoxO1 gene sequence.

A method of screening for a candidate drug for the treatment of alzheimer's disease comprising: treating a culture system containing the FoxO1 gene with a substance to be screened; detecting the expression level of FoxO1 gene or FoxO1 protein in the culture system after treatment, or detecting the expression amount of FoxO1 protein; if the substance to be screened can improve the expression level of the FoxO1 gene or improve the expression level of the FoxO1 protein, the substance to be screened is a candidate drug for treating the Alzheimer disease; the above-mentioned reagent for Alzheimer's disease diagnosis is used in the detection.

In the present invention, the culture system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

In the present invention, the method for screening a candidate drug for treating alzheimer's disease may further comprise: and performing further cell experiments and/or animal experiments on the obtained candidate drugs so as to further select the drug capable of treating the Alzheimer disease from the candidate drugs.

The invention provides the use in any one of:

a. the application of the reagent for diagnosing the Alzheimer disease in the preparation of a product for diagnosing the Alzheimer disease;

b. the application of the composition for treating the Alzheimer disease in the preparation of the medicine for treating the Alzheimer disease;

c. the reagent for diagnosing the Alzheimer disease is applied to the construction of a calculation model for diagnosing the Alzheimer disease.

A β plays an important role in the pathological development of AD. The invention discovers that the generation of A beta can be reduced by changing the expression level of FoxO1 for the first time; suggesting that FoxO1 can be used as a potential molecular target for treating Alzheimer disease.

Drawings

FIG. 1 is a graph showing the results of qPCR detection of the expression of FoxO1 gene in the blood of Alzheimer's disease patients in test example 1 of the present invention;

FIG. 2 is a diagram showing the detection of the over-expression of FoxO1 gene by Western blot in test example 2 of the present invention;

FIG. 3 is a graph showing the results of ELISA detection of the effect of FoxO1 gene expression on A β 40 production in test example 3 of the present invention;

FIG. 4 is a graph showing the results of ELISA detection of the effect of FoxO1 gene expression on A β 42 production in test example 3 of the present invention;

FIG. 5 is a graph showing the results of detecting the effect of over-expression of FoxO1 gene on APP metabolism-related protein using Western blot in test example 4 of the present invention;

FIG. 6 is a graph showing the statistical results of the effects of Western blot on detection of over-expression of FoxO1 gene on APP metabolism-related proteins in test example 4 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The apparatus and reagents used in the examples and test examples were commercially available unless otherwise specified.

Example 1

The reagent for diagnosing the alzheimer disease in the present embodiment is the following PCR primers:

SEQ ID NO. 1: human FoxO1 gene detection forward primer 5'-CGCCGTGCTACTCGTTTG-3';

SEQ ID NO. 2: human FoxO1 gene detected reverse primer 5'-ACTTGGGTCAGGCGGTTCA-3'.

Example 2

The reagent used for the diagnosis of alzheimer' S disease in this example was an antibody specifically binding to FoxO1 protein, specifically FoxO1(C29H4) rabbitmab (2880S) antibody from Cell Signaling Technology.

Example 3

In this embodiment, the diagnostic product for alzheimer's disease is a kit, which comprises the following PCR primers:

SEQ ID NO. 2: human FoxO1 gene detection reverse primer 5'-ACTTGGGTCAGGCGGTTCA-3';

SEQ ID NO. 3: human GAPDH gene detection forward primer 5'-ACGGATTTGGTCGTATTGGGC-3';

SEQ ID NO. 4: human GAPDH gene detects reverse primer 5'-CGCTCCTGGAAGATGGTGAT-3'.

Further comprising: PCR is carried out by using reagents commonly used in PCR, such as dNTP, Taq enzyme, etc.

Example 4

The diagnostic product for alzheimer' S disease in this example is a kit, which comprises an antibody against FoxO1 protein (FoxO 1(C29H4) rabbitmab (2880S) antibody from Cell Signaling Technology), and further comprises a secondary antibody and a color developing solution.

Example 5

In this embodiment, the alzheimer disease diagnostic product is a chip, and the chip includes the following PCR primers:

Example 6

In this embodiment, the diagnostic product for alzheimer's disease is a nucleic acid membrane strip, which contains the following PCR primers:

Example 7

The composition for treating alzheimer's disease in this example comprises FoxO1 protein. It can inhibit BACE1 expression and thus influence amyloid pathway of APP metabolism to reduce A beta secretion, relieve or treat AD.

Example 8

The composition for treating alzheimer's disease in this example comprises a vector containing FoxO1 gene, which can express FoxO1 protein; the FoxO1 protein can inhibit the expression of BACE1 so as to influence the amyloid pathway of APP metabolism to reduce the secretion of A beta, and relieve or treat AD.

Example 9

The method for screening a candidate drug for treating alzheimer's disease in this embodiment comprises: treating a culture system containing FoxO1 gene or FoxO1 protein with a substance to be screened; detecting the expression level of FoxO1 gene in the culture system or detecting the expression level of FoxO1 protein after treatment; if the substance to be screened can improve the expression level of the FoxO1 gene or improve the expression level of the FoxO1 protein, the substance to be screened is a candidate drug for treating the Alzheimer disease.

Example 10

The application of the reagent in the embodiment in preparing a product for diagnosing Alzheimer disease is realized by preparing the reagent in the embodiment 1 into a kit, a chip or a nucleic acid membrane strip for treating Alzheimer disease.

Example 11

The composition of example 7 or 8 is used for preparing a medicament for treating alzheimer's disease.

Example 12

The reagent in the embodiment is applied to the establishment of the calculation model for diagnosing the Alzheimer disease, the reagent in the embodiment 1 is used for detecting the expression difference of the FoxO1 gene or the FoxO1 protein in normal people and AD people, and the calculation model for diagnosing the Alzheimer disease is established according to the difference.

Test example 1 qPCR verification of differential expression of FoxO1 Gene

1. Peripheral blood sample collection

30 patients with AD diagnosis in 10 homes in the new rural downtown were collected. According to the MMSE grading (21-26 are mild, 10-20 are moderate, 0-9 are severe), wherein 22 cases of severe dementia and 8 cases of moderate dementia are included. Meanwhile, 30 healthy old people who are admitted to the home for nursing and live in the old are collected as a normal group, and the MMSE score is more than or equal to 27 points. The cognitive function and the daily life ability are normal, and the cognitive function and the daily life ability are not obviously reduced compared with the prior art and are not obviously different from the ordinary people. All subjects excluded diseases such as blood lipid metabolism, and the age was 60-82 years. All subjects signed informed consent and peripheral blood was provided for gene detection.

2. Blood Total RNA extraction

The RNA stabilizer reagent product of Beijing Tiangen Biotech company comprises: blood RNA stabilizer, suspension RSB, protease K. The Beijing Tiangen biological RNA extraction kit provides the following prepackaged reagents: erythrocyte lysate Buffer RBL, cell lysate Buffer RL, deproteinization solution Buffer RW1, rinsing solution Buffer RW, deoxyribonuclease DNase I, and the like. The following operations are strictly performed according to the requirements of kit experimental steps:

(1) blood samples with the RNA stabilizer added are taken out, frozen at room temperature and centrifuged at 4000 Xg for 10min, and the supernatant is aspirated and left to precipitate.

(2) Adding 1mL of enzyme-inactivating water into the precipitate, and blowing to dissolve the precipitate.

(3) The cryocentrifuge centrifuged at 4000 Xg for 10 minutes and the supernatant was removed by pipetting thoroughly.

(4) Add 240. mu.L of suspension RSB and blow to dissolve the precipitate.

(5) Add 200. mu.L of cell lysate RL and then 20. mu.L of protease K solution to the solution, mix well, incubate at 55 ℃ for 10min, mix 2-3 times by inversion.

(6) Transferring the obtained liquid to a filter column CS filled with a collecting pipe, centrifuging for 3 minutes at 13400 Xg, collecting filtrate, abandoning the filter column, and taking the filtrate supernatant to a new enzyme inactivating EP pipe (taking care to avoid sucking cell debris and precipitating).

(7) Adding 220 μ L anhydrous ethanol into the newly collected filtrate, mixing, transferring to adsorption column CR2, centrifuging at 12000 × g for 1min, and discarding the solution.

(8) mu.L of deproteinized solution Buffer RW1 was added to the adsorption column, centrifuged at 12000rpm for 30s, the waste solution was discarded, and the adsorption column was replaced in the collection tube.

(9) Preparing DNaseI mixed solution: add 10. mu.L of DNase I to 70. mu.L of RDD solution.

(10) Add 80. mu.L DNase I mixture directly to the adsorption column and incubate at 20-30 ℃ for 15 min.

(11) mu.L of Buffer RW1 was added to the adsorption column, centrifuged at 12000rpm for 1min, and the column was discarded.

(12) Adding 500 μ L rinsing solution Buffer RW mixed with anhydrous ethanol in advance into adsorption column, centrifuging at 12000rpm for 2min, and discarding the solution and leaving the column.

(13) Step 12 is repeated.

(14) Centrifuge at 12000rpm for 2 minutes and discard the tube. Standing at room temperature and drying the adsorption column.

(15) Placing the adsorption column in a new enzyme inactivating tube, adding 30 μ L enzyme inactivating water to the middle part of the adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, collecting RNA solution, immediately using NanoDrop2000 ultramicro spectrophotometer to determine RNA concentration and purity, and immediately performing cDNA reverse transcription operation on the standard-reaching person.

3. Reverse transcription:

2. mu.g of total RNA was subjected to reverse transcription, and 2. mu.L of oligo (dT) was added thereto and mixed well. Water bath is carried out for 5 minutes at 70 ℃, and ice bath is carried out for 2-3min immediately; 5 μ L of 5 XTRT, 5 μ L of dNTP (2.5 mM), 40U/μ L of RNase and 200U/μ L of M-MLV were added, and ribozyme-free water was added to the desired volume, followed by 60 minutes of 42 ℃ water bath and 5 minutes of 95 ℃ water bath to inactivate M-MLV.

4. qPCR amplification

(1) Primer design

QPCR amplification primers were designed based on the coding sequences of FoxO1 gene and GAPDH gene in Genbank and synthesized by Shanghai Biotechnology engineering services, Inc. The specific primer sequences are as follows:

human FoxO1 gene:

the forward primer is 5'-CGCCGTGCTACTCGTTTG-3';

the reverse primer was 5'-ACTTGGGTCAGGCGGTTCA-3'.

Human GAPDH gene:

the forward primer is 5'-ACGGATTTGGTCGTATTGGGC-3';

the reverse primer was 5'-CGCTCCTGGAAGATGGTGAT-3'.

(2) Preparing a PCR reaction system:

1 mu L of each of the forward primer and the reverse primer, 12.5 mu L of SYBR Green polymerase chain reaction system and 2 mu L of template, and deionized water is added to make up to 25 mu L.

(3) And (3) PCR reaction conditions:

95 ℃ 10min, (95 ℃ 15s, 60 ℃ 60s) 45 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

5. Statistical method

The experiment was repeated 3 times, the data were expressed as mean ± sd, and the statistical analysis was performed using SPSS13.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

6. Results

The results are shown in fig. 1, the expression of FoxO1 gene was significantly down-regulated in the blood of alzheimer patients compared to normal human blood, with the difference being statistically significant (P < 0.05).

Experimental example 2 overexpression of FoxO1 Gene

1. Cell culture

N2a/APPsw cells (mouse cells) were cultured in DMEM (pH 7.2-7.4) containing 10% bovine serum and 1% penicillin/streptomycin at 37 ℃ in an incubator containing 5% CO2 and 90% relative humidity. Changing the culture solution once every 2 days, carrying out passage when the cells grow to 90% contact, washing with PBS, adding 0.25% -EDTA trypsin for digestion to separate the cells from the bottle wall, stopping pancreatin digestion reaction with DMEM culture solution containing fetal calf serum, centrifuging for 2min at 1000g, discarding supernatant, re-suspending with newly configured culture solution, carrying out passage at a ratio of 1: 3-1: 4, changing the culture solution when the cells enter a logarithmic phase after 24 hours, and carrying out different interventions according to experimental requirements.

2. Transfection

1) Treatment of cells prior to transfection

One day before transfection, 6-well culture plates are seeded with 3-5 multiplied by 10⁵Cells/well, cultured for one day in antibiotic-free medium, at 70-90% cell density at transfection, were replaced with serum-free medium before transfection.

2) Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the sequence of the mouse FoxO1 gene in GeneBank (since the cells used in the experiment were mouse cells, the mouse FoxO1 gene was overexpressed there for validation), and the primer sequences were as follows:

a forward primer: 5'-GGATCCATGGCCGAGGCGCCCCAG-3' (SEQ ID NO. 5);

reverse primer: 5'-CTCGAGTTAGCCTGACACCCAGCTGTGTG-3' (SEQ ID NO. 6).

Restriction sites BamHI and XhoI were added to the 5 '-end primer and the 3' -end primer, respectively. cDNA extracted and reverse transcribed from Alzheimer patients is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by BamHI and XhoI after double enzyme digestion by restriction enzymes BamHI and XhoI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.

3) Transfection

The cells were divided into 2 groups, control (pcDNA3.1-NC transfected) and experimental (pcDNA3.1-FoxO 1 transfected). Transfection of the vector was performed using liposome 3000, with the specific transfection method being performed as indicated in the instructions.

3. Western blot detection of expression level of FoxO1

After culturing for 48-72 hours, collecting cell samples by RIPA protein lysate and protease inhibitor, extracting total protein, measuring protein concentration by BCA method, and carrying out Western blot detection.

Mixing 4 volumes of cell lysate of the sample with 1 volume of 5 xSDS loading buffer (containing 5% beta-mercaptoethanol), boiling in boiling water for 5-10min, and centrifuging to obtain the sample, wherein the sample can be left at room temperature for 20-30min before boiling. Preparing 10% SDS-PAGE gel for electrophoresis, performing electrophoresis at 90V for 3-4h, and performing die transfer by using a wet transfer film method: soaking PVDF membrane in methanol for 2min, sequentially placing filter paper, separating gel and membrane by sandwich method, clamping the membrane rotating frame, correctly placing into membrane rotating groove, filling with 1 × membrane rotating solution, and starting membrane rotating at constant pressure of 90V for 1-2 h. After the membrane transfer is finished, the membrane is put into 5% skimmed milk and sealed for 1-2h at room temperature, primary antibodies resisting FoxO1 and beta-actin are added, and the membrane is incubated overnight at 4 ℃. After primary antibody is finished, washing the cells with an appropriate amount of TTBS buffer solution for 3 multiplied by 10 min/time, and then incubating the cells with a secondary antibody labeled by a primary antibody corresponding to horseradish peroxidase for 1 h at room temperature. After the secondary anticaking agent is finished, washing the secondary anticaking agent by TTBS buffer solution for 3 multiplied by 10 min/time.

4. Results

The Western blot detection result is shown in FIG. 2, compared with the control group, the expression level of the FoxO1 gene of the experimental group is obviously increased, and the expression amount of the FoxO1 protein is obviously increased.

Test example 3 determination of the Effect of cell-level expression of FoxO1 on A.beta.levels

The test example includes the following steps:

1. culturing N2a/APPsw cells, when the fusion degree is about 80%, transfecting pcDNA3.1-NC and pcDNA3.1-FoxO1 plasmids by using a liposome 3000, and culturing for 6h and then changing the liquid;

2. culturing for 48-72h, and collecting supernatant;

3. the supernatant was analyzed for the production of A.beta.40, 42 using an ELISA kit (KHB 3481/3441, Invitrogen).

The culture broth was treated with an ELISA kit according to the manufacturer's instructions, and its OD at 492nm was measured with a fully automatic microplate reader to calculate the protein content. The results are shown in FIGS. 3 and 4, and it can be seen from FIGS. 3-4 that overexpression was effective in reducing the concentrations of A β 40 and A β 42 in N2a/APPswe cells.

Test example 4 Effect of cellular level expression of FoxO1 on APP Metabolic-related proteins

The test example includes the following steps:

2. culturing for 48-72h, and collecting protein;

3. the 6-well plate was placed on ice and the cells were washed 3 times with 1 × PBS reagent, PBS was removed as clean as possible and 100 μ L RIPA lysate supplemented with 1% PMSF and protease inhibitor was added to each well separately and the proteins were scraped off with a cell scraper. The extracted proteins were separated by SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skimmed milk powder in TBST for 1.5h at room temperature, primary antibody incubated: flAPP, CTF alpha, CTF beta, BACE1 and beta-actin, overnight at 4 deg.C, PVDF membrane was washed 4 times, the first 5 minutes and the second 10 minutes with 1 XTBST reagent. HRP-labeled secondary antibodies were incubated for 1.5 hours at room temperature, and the PVDF membrane was washed 4 times, 5 minutes first, and 10 minutes later with 1 × TBST reagent. And (6) developing.

The detection results are shown in fig. 5 and fig. 6, and it can be seen from fig. 5-6 that over-expressing FoxO1 results in the reduction of the expression levels of flAPP, CTF β and BACE1 compared with the control group, and the results indicate that FoxO1 can reduce the secretion of a β by inhibiting the expression of BACE1, thereby affecting the amyloid pathway of APP metabolism.

From the above results, it can be seen that the over-expression of FoxO1 in cells leads to the reduction of BACE1 expression level and the reduction of a β production, and can be used as a potential drug for treating alzheimer disease.

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Claims

1. A reagent for diagnosing alzheimer's disease, characterized in that: the reagent is a detection reagent for the mRNA expression level of the FoxO1 gene, or a detection reagent for the expression level of the FoxO1 protein.

2. The reagent for the diagnosis of alzheimer's disease according to claim 1, wherein: the detection reagent for the FoxO1 gene mRNA expression level is a probe for specifically recognizing FoxO1 gene; or a primer for specifically amplifying the FoxO1 gene.

3. The reagent for the diagnosis of alzheimer's disease according to claim 2, wherein: the primer sequence of the specific amplification FoxO1 gene is shown as SEQ ID NO.1 and SEQ ID NO. 2.

4. The reagent for the diagnosis of alzheimer's disease according to claim 1, wherein: the detection reagent for the expression level of the FoxO1 protein is an antibody or a ligand which specifically binds to the FoxO1 protein.

5. An alzheimer's disease diagnostic product characterized by: the product contains the reagent for diagnosing Alzheimer's disease as set forth in claim 1.

6. The diagnostic product for alzheimer's disease according to claim 5, wherein: the product is a kit, a chip or a nucleic acid membrane strip.

7. A composition for treating alzheimer's disease, characterized by: an enhancer comprising FoxO1 protein, or comprising FoxO1 gene; or an enhancer comprising FoxO1 protein.

8. The composition for the treatment of alzheimer's disease according to claim 7, wherein: the promoter is a carrier containing FoxO1 gene.

9. A method of screening a candidate drug for the treatment of alzheimer's disease, comprising: the method comprises the following steps: treating a culture system containing FoxO1 gene or FoxO1 protein with a substance to be screened; detecting the expression level of FoxO1 gene in the culture system or detecting the expression level of FoxO1 protein after treatment; if the substance to be screened can improve the expression level of the FoxO1 gene or improve the expression level of the FoxO1 protein, the substance to be screened is a candidate drug for treating the Alzheimer disease;

in the detection, the detection is performed using the reagent for Alzheimer's disease diagnosis as set forth in claim 1.

10. Use in any one of:

a. use of an agent according to any one of claims 1 to 4 for the manufacture of a product for the diagnosis of alzheimer's disease;

b. use of a composition according to any one of claims 7 to 8 in the manufacture of a medicament for the treatment of alzheimer's disease;

c. use of an agent according to any one of claims 1 to 4 for the construction of a computational model for the diagnosis of alzheimer's disease.