Use of senescence-related proteins for assessing senescence status

Abstract

The invention provides an aging-related protein for evaluating the biological age of a subject, the aging degree of the subject, the effect of an aging intervention means, the risk of the subject suffering from an aging-related disease and / or the treatment effect of the aging-related disease, and the aging degree of an age-related blood coagulation system. The invention also relates to preparation of a product for delaying or reversing senescence, a product for treating senescence-related diseases and a product for treating senescence-related blood coagulation system diseases. In addition, the invention also relates to the senescence-related protein as a target for delaying or reversing senescence and preventing and / or treating senescence-related diseases, and the senescence-related protein is used for screening drugs for delaying or reversing senescence and preventing and / or treating senescence-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the use of aging-related proteins to assess the biological age of a subject, the degree of aging of a subject, the effect of aging intervention measures, the risk of a subject suffering from aging-related diseases and / or the therapeutic effect of aging-related diseases. In addition, the present invention also relates to the use of aging-related proteins to screen for drugs that delay or reverse aging, prevent and / or treat aging-related diseases. Background Art

[0002] "Population aging" has become an irresistible global trend. As the degree of population aging gradually deepens, it brings huge challenges to society, economy, culture, and medical care. Aging is a topic that has been around since ancient times. Some people in life always look younger than their peers, while others look older than their peers. We realize that calendar age is not equal to biological age. Whether we are aging prematurely or growing stronger in old age, we need to answer a question: How old are we? Is there a standard for measuring aging? With active health or drug intervention, can we become younger from a biological perspective?

[0003] At present, how to analyze aging and further intervene in aging has become an important direction to be tackled in the current field of aging research. Summary of the invention

[0004] The inventors of this application established a cohort of naturally aging people aged 20-87 in Quzhou, Zhejiang. After a series of questionnaires and health examinations, a total of 289 healthy volunteers with similar living backgrounds and unified dietary management were included. By collecting peripheral blood from volunteers for high-throughput proteomics analysis, a blood proteomics map of the aging population in China was established, and a series of age-related proteins were identified. According to the abundance of these proteins, the inventors used the elastic network machine learning model to predict the actual age of the subjects and established a protein age model for the aging of the Chinese population. The results showed that the model has good predictive performance, and after further screening, protein molecules that are significantly upregulated with age were identified, which can be used as biomarkers for population aging. By detecting the abundance of the protein in plasma, it can assist in measuring a person's biological age, aging status, risk of aging-related diseases, treatment effects of aging-related diseases, and / or the effects of aging interventions.

[0005] Therefore, in a first aspect, the present application provides the use of an agent or a combination of agents in the preparation of a medicament for:

[0006] (1) Assess the biological age of the subjects;

[0007] (2) assess the subject's aging status;

[0008] (3) Evaluate the effectiveness of aging interventions;

[0009] (4) assessing the subject's risk of developing age-related diseases; and / or

[0010] (5) Evaluate the effectiveness of treatments for aging-related diseases;

[0011] (6) Assess the degree of aging of the subjects' coagulation system.

[0012] Wherein, the reagent or reagent combination is capable of detecting protein levels, and the protein is selected from one or more of the following table:

[0013] Protein name Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 VWF TFPI A2M CCN5 THBS4 APMAP F8 F5 PRG4 LTBP2 FBLN5 F11 MCAM CRTAC1 NOTCH1 C5a F10 COMP EFEMP1 LGALS3 F9 NOTCH3 LRP1 ITIH3 FGG CD163 PCSK9 CDH2 .

[0014] The above proteins have the meanings commonly understood by those skilled in the art. In certain embodiments, the exemplary Uniprot numbers corresponding to the above proteins are as follows:

[0015]

[0016]

[0017] In certain embodiments, the assessments described in (1) to (6) are independently auxiliary assessments.

[0018] In certain embodiments, the agent or combination of agents determines the level of one or more proteins as defined above by an in vitro detection method.

[0019] In certain embodiments, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography, or any combination thereof.

[0020] In certain embodiments, the immunological detection method is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

[0021] In certain embodiments, the evaluating comprises the steps of:

[0022] (1) determining the level of one or more proteins as defined above in one or more samples from the subject;

[0023] (2) comparing the level of one or more proteins in (1) with their respective corresponding reference values;

[0024] and,

[0025] (3) Assessing the biological age of the subject, the degree of aging of the subject, the effectiveness of aging interventions, the risk of the subject developing aging-related diseases, the effectiveness of treatments for aging-related diseases, and / or the degree of aging of the subject's coagulation system.

[0026] In certain embodiments, the one or more samples are each independently selected from blood (e.g., whole blood, serum or plasma) sample, urine sample, saliva sample, cell or tissue extract sample. In certain embodiments, the one or more samples are each independently selected from blood sample (e.g., plasma sample).

[0027] In certain embodiments, the reference value is the level (or average level) of the corresponding protein in the same type of sample from an individual (or group) who is in the same chronological age group as the subject and in a normal aging state; alternatively, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level (or average level) of the corresponding protein in the same type of sample from an individual (or group) who is in the same chronological age group as the subject and has not received the aging intervention or treatment for the aging-related disease and is in a normal aging state; alternatively, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level of the corresponding protein in the same type of sample from the subject before receiving the aging intervention or treatment for the aging-related disease.

[0028] In certain embodiments, the reference value of the one or more proteins is calculated according to the following formula:

[0029]

[0030]

[0031] Among them, "X 时序 ” is the subject’s chronological age; “C 参照 " is the reference value; "C 参照(女) " is the reference value for female subjects; "C 参照(男) ” is the reference value for male subjects.

[0032] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0033] In certain embodiments, when the level of one or more proteins defined above in one or more samples from the subject is higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, the treatment effect of the aging-related disease is not significant, and / or the subject's coagulation system is in a state of accelerated aging.

[0034] In certain embodiments, the subject is a mammal.

[0035] In certain embodiments, the subject is human, more preferably Asian.

[0036] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0037] In certain embodiments, the subject is a female subject (e.g., a female subject). In certain embodiments, the protein is selected from one or more of the following:

[0038] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 LGALS3 F9 NOTCH3 LRP1 ITIH3 FGG CD163 PCSK9 CDH2 C5a .

[0039] In certain embodiments, the subject is a male subject (e.g., a male subject). In certain embodiments, the protein is selected from one or more of the following:

[0040] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 VWF F9 NOTCH3 LRP1 APMAP FGG CD163 PCSK9 FBLN5 C5a .

[0041] In a second aspect, the present application provides a drug screening method, comprising:

[0042] (1) Provide biological samples and divide them into test group and control group;

[0043] (2) exposing the test group biological samples to the candidate drug;

[0044] (3) analyzing the level of one or more proteins as defined in the first aspect in the biological sample of the test group, and comparing the level of the one or more proteins with the level of the corresponding protein in the biological sample of the control group;

[0045] Wherein, the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient).

[0046] In certain embodiments, if the level of one or more proteins as defined in the first aspect in the biological sample of the test group is lower than the level of the corresponding protein in the biological sample of the control group, it indicates that the candidate drug has the potential to delay or reverse aging in the subject, and / or prevent and / or treat aging-related diseases.

[0047] In certain embodiments, the subject is a mammal.

[0048] In certain embodiments, the subject is human, more preferably Asian.

[0049] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0050] In certain embodiments, the subject is a female subject (e.g., a female subject). In certain embodiments, the protein is selected from one or more of the following:

[0051]

[0052]

[0053] In certain embodiments, the subject is a male subject (e.g., a male subject). In certain embodiments, the protein is selected from one or more of the following:

[0054] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 VWF F9 NOTCH3 LRP1 APMAP FGG CD163 PCSK9 FBLN5 C5a .

[0055] In a third aspect, the present application provides a reagent combination comprising a detection reagent capable of detecting the levels of any two or more proteins selected from the proteins defined in the first aspect.

[0056] In certain embodiments, the detection reagent determines the level of the protein as defined in the first aspect by an in vitro detection method.

[0057] In certain embodiments, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography, or any combination thereof.

[0058] In certain embodiments, the immunological assay is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), or radioimmunoassay (RIA).

[0059] In a fourth aspect, the present application provides a method for assessing the biological age of a subject, comprising:

[0060] (1) determining the level (measured value) of one or more proteins as defined in the first aspect in a sample from the subject;

[0061] and (2) assessing the biological age of the subject.

[0062] In certain embodiments, the assessment is an auxiliary assessment.

[0063] In certain embodiments, the subject is a mammal.

[0064] In certain embodiments, the subject is human, more preferably Asian.

[0065] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0066] In certain embodiments, the biological age is calculated according to the following formula:

[0067]

[0068]

[0069] Among them, "X 生物学 ” is the biological age of the subject; “C 测量 " is the measured value; "X 生物学(女) ” is the biological age of female subjects; “X 生物学(男) ” is the biological age for male subjects.

[0070] In certain embodiments, the method assesses the biological age of the subject based on any combination of the levels of one or more proteins as defined in the first aspect in the subject's plasma sample. In certain embodiments, the method comprises: (a) obtaining the estimated biological age of the subject corresponding to the one or more proteins as defined in the first aspect respectively according to the levels of one or more proteins as defined in the first aspect in the subject's plasma sample according to the above method; (b) combining and evaluating the estimated biological ages in (a) to obtain the final estimated biological age of the subject.

[0071] In a fifth aspect, the present application provides the use of an agent capable of inhibiting protein activity or expression in any one or more of the following (1) to (4), wherein the agent capable of inhibiting protein activity or expression is an agent capable of inhibiting or inactivating the protein activity and / or an agent capable of down-regulating or not expressing the protein, and the protein is one or more selected from the proteins defined in the first aspect:

[0072] (1) Delay or reverse aging in subjects;

[0073] (2) preparing a drug for delaying or reversing aging in a subject;

[0074] (3) preparing a drug for treating an aging-related disease in a subject;

[0075] (4) Prepare a drug for treating aging-related diseases of the coagulation system in a subject.

[0076] In certain embodiments, the agent capable of inhibiting the activity or expression of the protein comprises one or more of the following:

[0077] (a) the protein activity inhibitor;

[0078] (b) an agent for knocking down the protein encoding gene;

[0079] (c) an agent for knocking out the protein encoding gene.

[0080] In certain embodiments, the subject is a mammal.

[0081] In certain embodiments, the subject is human, more preferably Asian.

[0082] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0083] In certain embodiments, the subject is a female subject (e.g., a female subject). In certain embodiments, the protein is selected from one or more of the following:

[0084] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 LGALS3 F9 NOTCH3 LRP1 ITIH3 FGG CD163 PCSK9 CDH2 C5a .

[0085] In certain embodiments, the subject is a male subject (e.g., a male subject). In certain embodiments, the protein is selected from one or more of the following:

[0086] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 VWF F9 NOTCH3 LRP1 APMAP FGG CD163 PCSK9 FBLN5 C5a .

[0087] In the sixth aspect, the present application provides an agent or a combination of agents, which includes an agent capable of inhibiting protein activity or expression, wherein the agent capable of inhibiting protein activity or expression is an agent capable of inhibiting or inactivating the activity of the protein and / or an agent capable of down-regulating or not expressing the protein, and the protein is one or more selected from the proteins defined in the first aspect.

[0088] In certain embodiments, the agent or combination of agents comprises an agent capable of inhibiting the activity or expression of any two or more proteins selected from the proteins defined in the first aspect.

[0089] In certain embodiments, the agent capable of inhibiting the activity or expression of the protein comprises one or more of the following:

[0090] (a) the protein activity inhibitor;

[0091] (b) an agent for knocking down the protein encoding gene;

[0092] (c) an agent for knocking out the protein encoding gene.

[0093] In certain embodiments, the subject is a mammal.

[0094] In certain embodiments, the subject is human, more preferably Asian.

[0095] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0096] In certain embodiments, the subject is a female subject (e.g., a female subject). In certain embodiments, the protein is selected from one or more of the following:

[0097] Protein name Protein name Protein name Protein name F13B C5 ITLN1 HAVCR2 TFPI A2M CCN5 THBS4 F8 F5 PRG4 LTBP2 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 LGALS3 F9 NOTCH3 LRP1 ITIH3 FGG CD163 PCSK9 CDH2 C5a .

[0098] In certain embodiments, the subject is a male subject (e.g., a male subject). In certain embodiments, the protein is selected from one or more of the following:

[0099]

[0100]

[0101] Definition of terms

[0102] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0103] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0104] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0105] As used herein, the term "aging" has the meaning commonly understood by those skilled in the art, and is a common biological phenomenon in the growth and development of the body. Aging can be considered as a process of body function decline accompanied by aging, which is usually accompanied by a continuous decline in physical health and an increase in the risk of aging-related diseases, functional disorders and death.

[0106] As used herein, the term "aging-related diseases" refers to aging-related diseases whose risk of occurrence increases significantly with aging. In certain embodiments, the aging-related diseases include, but are not limited to, neurodegenerative diseases (such as Alzheimer's disease), diabetes, heart disease, cardiovascular and cerebrovascular diseases, hypertension, hyperlipidemia, osteoporosis, osteoarthritis, chronic kidney disease, cancer, and vision loss.

[0107] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., aging, aging-related diseases) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable.

[0108] As used herein, unless the context clearly indicates, otherwise, the protein described herein has the meaning generally understood by those skilled in the art. In certain embodiments, the protein described herein comprises its wild type and natural or artificially produced mutant type. In certain embodiments, the protein described herein and its corresponding Uniprot number are shown in Table 2. In certain embodiments, the protein described herein has: (i) as the amino acid sequence corresponding to its corresponding Uniprot number, or, (ii) compared with (i) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity amino acid sequence.

[0109] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0110] Advantageous Effects of the Invention

[0111] The protein markers (or combinations of protein markers) provided in this application for assessing the aging state of the body can more accurately and specifically assess the aging state of the Chinese population, and have the advantages of small sample dosage, simple processing, short time course, and easy-to-read results. In addition, this application also provides the use of the protein markers in delaying or reversing aging in subjects, preparing drugs for delaying or reversing aging in subjects, preparing drugs for treating aging-related diseases in subjects, and / or, preparing drugs for treating aging-related diseases of the coagulation system in subjects.

[0112] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 The dot plots show the age-related changes in the levels of complement and coagulation system proteins in males and females of different ages. Confidence intervals are indicated by gray shading. Pearson correlation analysis was used to calculate correlation coefficients and P values.

[0114] Figure 2 The dotted line graphs show the changes in the levels of age-related proteins in males and females of different ages. Confidence intervals are indicated by gray shading. Pearson correlation analysis was used to calculate the correlation coefficient and P value.

[0115] Figure 3 The dot-line graph shows the changes in the levels of age-related proteins in female populations of different ages. The confidence intervals are indicated by gray shading. Pearson correlation analysis was used to calculate the correlation coefficient and P value.

[0116] Figure 4 The dot-line graph shows the changes in the levels of age-related proteins in male populations of different ages. The confidence intervals are indicated by gray shading. Pearson correlation analysis was used to calculate the correlation coefficient and P value. DETAILED DESCRIPTION

[0117] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0118] The technical route of the present invention mainly involves Volunteer recruitment and sample collection , Data Collection , Data preprocessing and Age-related protein analysis 3 parts.

[0119] Example 1: Volunteer recruitment and sample collection

[0120] The "Preliminary Screening Questionnaire" was published through the WeChat Mini Program to collect basic information of potential volunteers who are interested in joining this study based on the inclusion criteria, and to screen volunteers who meet the inclusion criteria; the inclusion criteria are as follows:

[0121] 1) Enrollment conditions

[0122] a) Aged 18 or above, with no major bad habits;

[0123] b) The applicant is in good health and has not undergone any major surgery within the past six months. Routine conditions related to the normal aging process are permitted, including:

[0124] i. Hypertension, well controlled (taking no more than 3 medications);

[0125] ii. Osteoporosis, osteopenia and / or osteoarthritis;

[0126] iii. Benign prostatic hypertrophy;

[0127] iv. Cataract, glaucoma, macular degeneration;

[0128] v.Dyslipidemia;

[0129] vi. Hypothyroidism;

[0130] vii. Prediabetes or abnormal fasting blood sugar (fasting blood sugar 100-126 mg / dL).

[0131] c) The subjects can communicate well with the researchers and cooperate with all tasks during the study;

[0132] i. Subjects must voluntarily sign a written informed consent.

[0133] 2) Exclusion criteria

[0134] a) Age does not meet the inclusion criteria;

[0135] b) Participants are currently undergoing other clinical trials or are receiving any research drugs or devices for treatment within 30 days prior to enrollment;

[0136] c) The investigator reports a history or current diagnosis of significant chronic medical conditions, including:

[0137] i. Any cancer (except polycythemia vera, basal cell or squamous cell skin cancer);

[0138] ii. Coronary artery disease / myocardial infarction / clinically significant congestive heart failure;

[0139] iii. Stroke / transient ischemic attack;

[0140] iv. Deep vein thrombosis / pulmonary embolism;

[0141] v. Serum creatinine>1.5mg / dl (male);

[0142] vi. Poorly controlled hypertension (significant hypertension (systolic blood pressure > 160

[0143] mmHg, or diastolic blood pressure>100mmHg);

[0144] vii. History of active liver disease or metabolic acidosis;

[0145] viii. Chronic kidney disease / hemodialysis treatment, history of severe renal impairment and / or

[0146] eGFR≤45ml / min / 1.73m2;

[0147] ix. Severe autoimmune / inflammatory diseases, such as rheumatoid arthritis, lupus, Crohn's disease, etc.;

[0148] x. Neurological diseases such as dementia, such as Alzheimer's / Parkinson's, etc.;

[0149] xi. Diabetes (hemoglobin A1C>6.5% or fasting blood sugar>126 mg / dL or taking diabetes

[0150] medication or insulin therapy);

[0151] xii. Recent (within 3 months) cardiovascular disease events (myocardial infarction, coronary artery intervention, coronary artery bypass grafting);

[0152] xiii. Infectious diseases such as HIV, hepatitis, tuberculosis, etc.

[0153] d) Currently taking the following medications regularly:

[0154] i. Chemotherapy drugs (such as tamoxifen, doxorubicin, mitoxantrone, bleomycin);

[0155] ii. Antiplatelet drugs (e.g., clopidogrel / Plavix, dipyridamole / Angeleno, ticlopidine /

[0156] Ticlopidine, except aspirin);

[0157] iii. Cholinesterase inhibitors for Alzheimer's disease (donepezil / alicept).

[0158] e) Continuous alcohol or drug abuse;

[0159] f) Unable to provide informed consent;

[0160] g) The researcher believes that the physical factors of the participants may have an adverse effect on the research process or results.

[0161] 3) Termination criteria

[0162] a) Failure to follow the diet, work and rest schedule and physical examination requirements in accordance with the research content;

[0163] b) Experiencing abnormal physical conditions such as colds or fever during the study;

[0164] c) Accidental injury or physical discomfort during the research process;

[0165] d) Comply with the subject’s subjective wishes to terminate the program.

[0166] All volunteers who meet the inclusion criteria and are confirmed to be included in the group signed an informed consent form and joined the volunteer WeChat group for management and information communication. Within one month before the start of the project, volunteers need to maintain a normal schedule, eat a proper diet, avoid strenuous exercise, and refrain from excessive smoking or drinking. The day before the project, volunteers received a unified standardized diet (menu see Table 1) at the implementation hospital and were no longer allowed to eat other foods.

[0167] On the day of sample collection and physical examination, volunteers are required to wear clothes and shoes that are convenient for movement, are not allowed to wear makeup, and must bring identification documents (such as ID cards, etc.) to the hospital for physical examination and biological sample collection.

[0168] Table 1 Unified standardized diet menu

[0169]

[0170] Example 2: Data Collection

[0171] 1) Biological sample processing

[0172] Blood samples were collected from volunteers after they fasted overnight. The collected blood was placed in two anticoagulant tubes covered with EDTA (for the separation of plasma and peripheral mononuclear cells, respectively), and immediately transported to the laboratory's 4°C refrigerator for subsequent processing. One tube of blood sample (approximately 5 ml) was centrifuged (400 g, 15 min, 4°C) to separate the plasma sample, which was stored in a cryopreservation tube and placed in a -80°C refrigerator for long-term storage.

[0173] 2) Plasma proteome quantification

[0174] a) Protein extraction: The sample was taken out from -80℃ and centrifuged at 12000g for 10 minutes at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube and centrifuged using Pierce TM Top 14 Abundant Protein Depletion Spin Columns Kit (Thermo Scientific) Instructions Remove high-abundance proteins. Protein concentration was determined using the BCA kit.

[0175] b) Trypsin hydrolysis: Take an equal amount of each sample protein for hydrolysis, add an appropriate amount of standard protein, adjust the volume to the same with lysis buffer, and then add dithiothreitol (DTT) to a final concentration of 5mM, and reduce at 56°C for 30min. Then add iodoacetamide (IAA) to a final concentration of 11mM, and incubate at room temperature in the dark for 15min. Transfer the alkylated sample to an ultrafiltration tube, centrifuge at 12000g for 20min at room temperature, replace with 8M urea 3 times, and then replace urea with replacement buffer 3 times, add trypsin at a ratio of 1:50 (protease: protein, m / m), and hydrolyze overnight. Centrifuge at 12000g for 10min at room temperature to recover the peptides, and then use ultrapure water to recover the peptides once, and combine the two peptide solutions.

[0176] c) HPLC fractionation: Agilent 300Extend C18 column was used to separate the samples. The column was 4.6×250 in size and the wavelength was 214 nm. The column oven temperature was 35°C. The column was equilibrated with 95% buffer A for 30 min. After the baseline was leveled, the graded gradient method was used. The peptide samples were loaded onto the HPLC and the method was started to separate the samples. The samples were collected at 1 min / tube. Tubes 11 to 46 were collected, a total of 36 tubes, which were combined into 12 components and dried under vacuum.

[0177] d) Liquid chromatography-mass spectrometry analysis:

[0178] Construction of spectral library: The peptides were dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC 1200 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile solution containing 0.1% formic acid. Liquid phase gradient settings: 0-55min, 4%~20%B; 55-67min, 20%~32%B; 67-71min, 32%~80%B; 71-75min, 80%B, and the flow rate was maintained at 500nL / min. After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the Capillary ion source for ionization and then into the Orbitrap Exploris TM 480 mass spectrometer for data collection. DIA data collection: Liquid phase parameters are consistent with those used in library construction. After peptides are separated by ultra-high performance liquid phase system, OrbitrapExploris TMThe ion source voltage was set to 1.6 kV, and the peptide parent ion and its secondary fragments were detected and analyzed using TOF. The data acquisition mode used the data-independent parallel accumulation serial fragmentation (dia-PASEF) mode, and the primary mass spectrometry scanning range was set to 390-810 m / z. After one primary mass spectrum was acquired, 10 PASEF mode acquisitions were performed, and the secondary mass spectrometry scan was in the range of 100-1700 m / z, with every 25 m / z as a window.

[0179] Example 3: Data preprocessing

[0180] a) The raw data of mass spectrometry were first imported into MaxQuant software (version 1.6.15.0) and then searched based on the SwissProt human database. The key parameters included:

[0181] i. Lysing enzyme is Trypsin / P

[0182] ii. A maximum of 2 missing cleavages are allowed

[0183] iii. For the precursor ion, the mass tolerance is 20ppm and 5ppm in the first and main searches respectively

[0184] iv. For fragment ions, the mass tolerance is 0.02Da

[0185] v. Fixed modification is carbamidomethylation on Cys

[0186] vi. Variable modifications include acetylation of the protein N-terminus and oxidation of Met

[0187] b) The screening criteria for search results was FDR < 1%;

[0188] c) The raw intensity was first divided by the median value of each protein to remove batch effects;

[0189] d) The results are divided by the median value of each sample to obtain a normalized protein abundance matrix.

[0190] Example 4: Age-related protein screening

[0191] a) First, the protein abundance matrix was subjected to age correlation analysis, that is, the abundance of each protein was subjected to Pearson correlation analysis with the individual's age, and BMI was used as a covariate;

[0192] b) The results were screened and only the results with BH-corrected P values ​​less than 0.05 were selected as age-related proteins.

[0193] Example 5: Plasma ELISA to verify age-related proteins

[0194] Based on the age-related proteins screened in Example 4, the proteins with the highest changes were further selected for ELISA detection, and a total of 31 proteins with aging evaluation capabilities were obtained ( Figure 1-4 ), as shown in Table 2:

[0195] Table 2:

[0196] Protein name Protein name Protein name Protein name Protein name F13B C5a ITLN1 HAVCR2 VWF TFPI A2M CCN5 THBS4 APMAP F8 F5 PRG4 LTBP2 FBLN5 F11 MCAM CRTAC1 NOTCH1 F10 COMP EFEMP1 LGALS3 F9 NOTCH3 LRP1 ITIH3 FGG CD163 PCSK9 CDH2

[0197] Among the above proteins, the levels of several proteins related to complement and coagulation systems have been verified to increase significantly with age, including F13B, TFPI, F8, F11, F10, F9, FGG, C5a, A2M, VWF, and F5. These evidences indicate that the coagulation system is activated with aging, and these proteins have the ability to indicate the aging of the coagulation system related to age. The ELISA detection method of the above proteins will be described below.

[0198] for F9, ITLN1, F8, TFPI, A2M, COMP Protein, the detection steps are as follows:

[0199] (1) All reagents were equilibrated at room temperature for 20 minutes before testing.

[0200] (2) Diluting the standard: Dilute the standard with sample diluent. The final concentrations are shown in Table 3.

[0201] Table 3:

[0202] Protein name Standard concentration F9 80, 40, 20, 10, 5, 2.5, 1.25, and 0 ng / mL ITLN1 40, 20, 10, 5, 2.5, 1.25, 0.63 and 0 ng / mL F8 3000, 1500, 750, 375, 187.5, 93.75, 46.88 and 0 mIU / mL TFPI 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 0 pg / mL A2M 4000, 2000, 1000, 500, 250, 125, 62.5 and 0 ng / mL COMP 40, 20, 10, 5, 2.5, 1.25, 0.63 and 0 ng / mL

[0203] Diluted plasma samples: According to the experimental design, 6 males and 6 females in six age groups (20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old) were selected, totaling 72 plasma samples. The plasma was diluted with sample diluent according to the ratio shown in Table 4.

[0204] Table 4:

[0205] Protein name Dilution ratio F9 1:100 ITLN1 1:25 F8 1:10 TFPI 1:100 A2M 1:3000 COMP 1:10

[0206] (4) Sample addition: Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard & sample diluent to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Apply the film and incubate at 37°C for 90 min.

[0207] (5) Shake off the liquid in the wells without washing. Use a spray gun to add 100 μL of 1× biotinylated antibody working solution to each well, apply the film, and incubate at 37°C for 1 hour.

[0208] (6) Discard the liquid, spin dry, and wash the plate three times with 350 μL of 1× Wash Buffer, soaking for 1 min each time.

[0209] (7) Use a dispenser to add 100 μL of 1× HRP conjugate working solution to each well, cover the plate with a film, and incubate at 37°C for 30 min.

[0210] (8) Discard the liquid, spin dry, and wash the plate five times with 350 μL of 1× Wash Buffer, soaking for 1 min each time.

[0211] (9) Use a spray gun to add 90 μL of substrate solution in sequence and incubate at 37°C in the dark for about 20 min.

[0212] (10) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0213] (11) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation to calculate the detected protein concentration of each sample.

[0214] for NOTCH3, HAVCR2, VWF, F13B, N-Cad(CDH2), FBLN5, FGG, THBS4, CRTAC1, LRP1, PRG4, PCSK9, LTBP2, EFEMP1, WISP2(CCN5), F10, F11 For detection, refer to the method and steps recommended in the kit instructions, as follows:

[0215] (1) All reagents were equilibrated at room temperature for 30 minutes before testing.

[0216] (2) Dilute the standard: Use sample diluent to dilute the standard to the final concentration shown in Table 5.

[0217] Table 5:

[0218]

[0219]

[0220] (3) Diluting plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 male and 6 female samples in each group. The plasma was diluted with sample diluent according to the ratios shown in Table 6 below.

[0221] Table 6:

[0222] Protein name Dilution ratio NOTCH3 1:10 HAVCR2 Original solution VWF 1:100 F13B 1:2000 N-Cad(CDH2) 1:5 FBLN5 Original solution FGG 1:1000 THBS4 1:10 CRTAC1 1:10 LRP1 Original solution PRG4 Original solution PCSK9 1:200 LTBP2 Original solution EFEMP1 Original solution WISP2 1:10 F10 1:2000 F11 1:2000

[0223] (4) Sample addition: Add 100 μL of standard or plasma sample to each well, cover with film, and incubate at 37°C for 2 h.

[0224] (5) Discard the liquid, spin dry, and do not wash.

[0225] (6) Use a dispenser to add 100 μL of 1× Biotin-antibody to each well, apply the film, and incubate at 37°C for 1 h.

[0226] (7) Discard the liquid, spin dry, and wash the plate three times with 200 μL of 1× Wash Buffer, soaking for 2 min each time.

[0227] (8) Use a dispenser to add 100 μL of 1× HRP-avidin to each well, apply the film, and incubate at room temperature for 1 h.

[0228] (9) Wash the plate five times according to the procedure in (7).

[0229] (10) Use a spray gun to add 90 μL of color developing solution in sequence and develop the color for about 20 min in the dark.

[0230] (11) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0231] (12) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation to calculate the protein concentration of each sample.

[0232] for APMAP Protein, the detection steps are as follows:

[0233] (1) All reagents were equilibrated at room temperature for 30 min before testing.

[0234] (2) Diluting the standard: Use sample diluent to dilute the standard. The final concentrations are 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.63 ng / mL, 0.32 ng / mL, 0.16 ng / mL and 0 ng / mL.

[0235] (3) Diluted plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted at 1:100 using sample diluent.

[0236] (4) Sample addition: Add 100 μL of standard or plasma sample to each well, cover with film, and incubate at 37°C for 80 min.

[0237] (5) Discard the liquid in the wells, spin dry, and wash the plate three times with 200 μL of 1× washing solution per well, soaking for 2 min each time.

[0238] (6) Use a dispenser to add 100 μL of biotin antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 50 min.

[0239] (7) Wash the plate according to the procedure in (5).

[0240] (8) Use a dispenser to add 100 μL of enzyme conjugate working solution to each well in turn, cover the ELISA plate with a film, and incubate at 37°C for 50 min.

[0241] (9) Wash the plate five times according to the procedure in (5).

[0242] (10) Use a spray gun to add 90 μL of TMB colorimetric solution in sequence and color for about 10-15 min in the dark.

[0243] (11) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0244] (12) Data processing: The OD values ​​of the standard and samples were subtracted from the OD value of the blank well to draw a curve. The regression equation of the standard curve was calculated with the concentration of the standard as the ordinate and the OD value as the abscissa. The OD value was substituted into the equation to calculate the APMAP concentration of each sample and multiply it by the dilution factor.

[0245] for C5a, MCAM Protein, the detection steps are as follows:

[0246] (1) All reagents were equilibrated at room temperature for 30 min before testing.

[0247] (2) Add 300 μL of 1× Wash Buffer to each well and let stand for about 2 minutes. Aspirate or pour off the liquid and pat dry on a paper towel. Wash twice in this way.

[0248] (3) Dilute the standard: Use 1× dilution buffer to dilute the standard. The final concentration is shown in Table 7:

[0249] Table 7:

[0250]

[0251] (4) Diluting plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted with 1× dilution buffer according to the ratio shown in Table 8.

[0252] Table 8:

[0253] Protein name Dilution ratio C5a Original solution MCAM 1:500

[0254] (5) Sample addition: Add 100 μL of standard or plasma sample to each well, apply the film, and incubate at room temperature for 2 hours.

[0255] (6) Discard the liquid in the wells, spin dry, and wash the plate three times with 300 μL of 1× washing solution per well, soaking for 2 min each time.

[0256] (7) Use a dispenser to add 100 μL of detection antibody working solution to each well in turn, cover the ELISA plate with a film, and incubate at room temperature for 1 hour.

[0257] (8) Wash the plate according to the procedure in (5).

[0258] (9) Use a spray gun to add 100 μL of substrate solution (an equal mixture of colorimetric solutions A and B) in sequence and incubate at room temperature in the dark for about 20 min.

[0259] (10) Use a dispenser to add 100 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0260] (11) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation to calculate the protein concentration of each sample.

[0261] for CD163、NOTCH1 Protein, the detection steps are as follows:

[0262] (1) All reagents should be equilibrated to room temperature before testing.

[0263] (2) Dilute the standard: Use sample diluent to dilute the standard. The final concentration is shown in Table 9:

[0264] Table 9:

[0265] Protein name Standard concentration CD163 4000, 2000, 1000, 500, 250, 125, 62.5 and 0 pg / mL NOTCH1 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 0 pg / mL

[0266] (3) Diluting plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted with 1× assay buffer according to the ratio shown in Table 10.

[0267] Table 10:

[0268] Protein name Dilution ratio CD163 1:500 NOTCH1 1:10

[0269] (4) Soak the microplate: Add 300 μl of 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, use the microplate immediately and do not let it dry.

[0270] (5) Sample addition: Add 100 μl of the diluted standard to the standard well, add 100 μl of 1× detection buffer to the blank well, and add 100 μl of the pre-diluted sample to the sample well.

[0271] (6) Add detection antibody: Add 50 μL of 1× detection antibody working solution to each well and seal the plate with a sealing film. Shake at 300 rpm and incubate at room temperature (25°C ± 3°C) for 2 hours.

[0272] (7) Discard the liquid in the wells, spin dry, and wash the plate six times with 300 μL of 1× washing solution per well, soaking for 2 min each time.

[0273] (8) Use a dispenser to add 100 μL of 1× streptavidin working solution to each well, seal the plate with a new sealing film, shake at 300 rpm, and incubate at room temperature (25°C ± 3°C) for 45 minutes.

[0274] (9) Wash the plate according to the procedure in (7).

[0275] (10) Use a spray gun to add 100 μL of TMB colorimetric solution in sequence and develop the color for about 20 min at room temperature in the dark.

[0276] (11) Use a dispenser to add 100 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0277] (12) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation to calculate the protein concentration of each sample.

[0278] for LGALS3 Protein, the detection steps are as follows:

[0279] (1) All reagents should be equilibrated to room temperature before testing.

[0280] (2) Diluting the standard: Dilute the standard with sample diluent to a final concentration of 2000, 1111, 617.3, 342.9, 190.5, 105.8, 58.80, and 0 pg / mL, respectively.

[0281] (3) Diluted plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted at 1:100 using sample diluent.

[0282] (4) Sample addition: Add 50 μL of standard or plasma sample to each well, add 50 μL of Antibody Cocktail, apply the film, shake at 400 rpm, and incubate at room temperature for 1 hour.

[0283] (5) Discard the liquid in the wells, spin dry, and wash the plate three times with 300 μL of 1× washing solution per well, soaking for 2 min each time.

[0284] (6) Use a spray gun to add 100 μL of TMB colorimetric solution in sequence and develop the color for about 20 min at room temperature in the dark.

[0285] (7) Use a dispenser to add 100 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0286] (8) Data processing: The OD value of the blank well was subtracted from the OD value of the standard and sample to draw a curve. The regression equation of the standard curve was calculated with the concentration of the standard as the ordinate and the OD value as the abscissa. The OD value was substituted into the equation to calculate the LGALS3 concentration of each sample and multiplied by the dilution factor.

[0287] for ITIH3 Protein, the detection steps are as follows:

[0288] (1) All reagents were equilibrated at room temperature for 30 minutes before testing.

[0289] (2) Diluting the standard: Use sample diluent to dilute the standard. The final concentrations are 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.3 ng / mL, 78.13 ng / mL and 0 ng / mL.

[0290] (3) Diluted plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted at 1:100 using sample diluent.

[0291] (4) Sample addition: Set up blank wells, standard wells, and sample wells. Do not add any solution to the blank wells. Add 50 μl of the standard or sample to be tested to each of the remaining wells. Immediately add 50 μl of 1× horseradish peroxidase marker working solution (not to the blank wells). Gently shake to mix, cover with a plate sticker, and incubate at 37°C for 30 minutes.

[0292] (5) Discard the liquid, spin dry, and wash the plate five times with 200 μL of 1× Wash Buffer, soaking for 2 min each time.

[0293] (6) Use a spray gun to add 90 μL of substrate solution in sequence and incubate at 37°C in the dark for about 20 min.

[0294] (7) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0295] (8) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation and calculate the ITIH3 concentration of each sample.

[0296] for F5 Protein, the detection steps are as follows:

[0297] (1) All reagents were equilibrated at room temperature for 30 min before testing.

[0298] (2) Dilution of standards: Dilute the standards with sample diluent to final concentrations of 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL and 0 pg / mL.

[0299] (3) Diluted plasma samples: According to the experimental design, 72 plasma samples were selected from six age groups: 20-29 years old, 30-39 years old, 40-49 years old, 50-59 years old, 60-69 years old, and ≥70 years old, with 6 males and 6 females in each group. The plasma was diluted at 1:2000 using sample diluent.

[0300] (4) Sample addition: Add 100 μL of standard or plasma sample to each well, cover with film, and incubate at 37°C for 80 min.

[0301] (5) Discard the liquid in the wells, spin dry, and wash the plate three times with 200 μL of 1× washing solution per well, soaking for 2 min each time.

[0302] (6) Use a dispenser to add 100 μL of biotin antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 50 min.

[0303] (7) Wash the plate according to the procedure in (5).

[0304] (8) Use a dispenser to add 100 μL of enzyme conjugate working solution to each well in turn, cover the ELISA plate with a film, and incubate at 37°C for 50 min.

[0305] (9) Wash the plate five times according to the procedure in (5).

[0306] (10) Use a spray gun to add 90 μL of TMB colorimetric solution in sequence and color for about 10-15 min in the dark.

[0307] (11) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0308] (12) Data processing: Plot the curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value into the equation to calculate the F5 concentration of each sample.

[0309] The names of the above-mentioned protein detection kits and the corresponding manufacturer's product number information are shown in Table 11:

[0310] Table 11

[0311]

[0312]

[0313] Among the proteins tested above, except for LGALS3, ITIH3, and CHD2, which are only applicable to female populations ( Figure 3 ), VWF, APMAP, FBLN5 are only applicable to male population ( Figure 4 ), the remaining proteins showed good aging evaluation capabilities in all populations. The relationship between the concentrations of these proteins and age is shown in Table 12.

[0314] Table 12

[0315]

[0316]

[0317] Among them, "X 时序 ” is the subject’s chronological age; “C 参照 " is the reference value; "C 参照(女) " is the reference value for female subjects; "C 参照(男) ” is the reference value for male subjects.

[0318] When the level of one or more proteins defined above in one or more samples from the subject is higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, and / or the treatment effect of the aging-related disease is not significant.

[0319] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of an agent or a combination of agents in the preparation of a medicament for: (1) Assess the biological age of the subjects; (2) assess the subject's aging status; (3) Evaluate the effectiveness of aging interventions; (4) assessing the subject's risk of developing age-related diseases; and / or (5) Evaluate the effectiveness of treatments for aging-related diseases; (6) Assess the degree of aging of the subjects' coagulation system. in, The reagent or reagent combination is capable of detecting protein levels, and the protein is selected from one or more of the following table: 。 2. The use of claim 1, wherein The evaluations described in (1) to (6) are independently auxiliary evaluations.

3. The use according to claim 1 or 2, wherein The reagent or reagent combination determines the level of one or more proteins as defined in claim 1 by an in vitro detection method; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography or any combination thereof; Preferably, the immunological detection method is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

4. The use according to any one of claims 1 to 3, wherein The assessment includes the following steps: (1) determining the level of one or more proteins as defined in claim 1 in one or more samples from the subject; (2) comparing the level of one or more proteins in (1) with their respective corresponding reference values; and, (3) assessing the biological age of the subject, the degree of aging of the subject, the effect of aging interventions, the risk of the subject developing aging-related diseases, the effect of treatment of aging-related diseases, and / or the degree of aging of the subject's coagulation system; Preferably, the one or more samples are each independently selected from a blood sample (e.g., whole blood, serum or plasma) sample, a urine sample, a saliva sample, a cell or tissue extract sample; Preferably, the one or more samples are each independently selected from a blood sample (e.g., a plasma sample); Preferably, the reference value is the level (or average level) of the corresponding protein in the same type of sample of an individual (or group) who is in the same chronological age group as the subject and in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level (or average level) of the corresponding protein in the same type of sample of an individual (or group) who is in the same chronological age group as the subject and has not received the aging intervention or treatment for the aging-related disease and is in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level of the corresponding protein in the same type of sample of the subject before receiving the aging intervention or treatment for the aging-related disease; Preferably, the degree of aging includes normal aging, accelerated aging, and / or delayed aging; Preferably, when the level of one or more proteins as defined in claim 1 in one or more samples from the subject is higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, the treatment effect of the aging-related disease is not significant, and / or the subject's coagulation system is in a state of accelerated aging.

5. The use according to any one of claims 1 to 4, wherein The subject is a mammal; Preferably, the subject is a human, more preferably an Asian; Preferably, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

6. The use according to any one of claims 1 to 5, wherein The subject is a female subject (e.g., a female subject); Preferably, the protein is selected from one or more of the following: 。 7. The use according to any one of claims 1 to 5, wherein The subject is a male subject (e.g., a male subject); Preferably, the protein is selected from one or more of the following:

8. A drug screening method comprising: (1) Provide biological samples and divide them into test group and control group; (2) exposing the test group biological samples to the candidate drug; (3) analyzing the level of one or more proteins as defined in claim 1 in the biological sample of the test group, and comparing the level of the one or more proteins with the level of the corresponding protein in the biological sample of the control group; wherein the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient); Preferably, if the level of one or more proteins as defined in claim 1 in the biological sample of the test group is lower than the level of the corresponding protein in the biological sample of the control group, it indicates that the candidate drug has the potential to delay or reverse aging in the subject, and / or prevent and / or treat aging-related diseases.

9. The method of claim 8, wherein The subject is a mammal; Preferably, the subject is a human, more preferably an Asian; Preferably, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

10. The method of claim 8 or 9, wherein The subject is a female subject (e.g., a female subject); Preferably, the protein is selected from one or more of the following:

11. The use according to claim 8 or 9, wherein The subject is a male subject (e.g., a male subject); Preferably, the protein is selected from one or more of the following: 。 12. A reagent combination comprising a detection reagent capable of detecting the levels of any two or more proteins selected from the group consisting of proteins as defined in claim 1.

13. The reagent combination of claim 12, wherein The detection reagent determines the level of the protein as defined in claim 1 by an in vitro detection method; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography or any combination thereof; Preferably, the immunological assay is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

14. A method for assessing the biological age of a subject, comprising: (1) determining the level (measured value) of one or more proteins as defined in claim 1 in a sample from the subject; and (2) assessing the biological age of the subject.

15. Use of an agent capable of inhibiting protein activity or expression in any one or more of the following (1) to (4), wherein: The agent capable of inhibiting protein activity or expression is an agent capable of inhibiting or inactivating the activity of the protein and / or an agent capable of down-regulating or not expressing the protein, and the protein is one or more selected from the proteins defined in claim 1: (1) Delay or reverse aging in subjects; (2) preparing a drug for delaying or reversing aging in a subject; (3) preparing a drug for treating an aging-related disease in a subject; (4) Prepare a drug for treating aging-related diseases of the coagulation system in a subject.

16. An agent or a combination of agents, comprising an agent capable of inhibiting protein activity or expression, wherein the agent capable of inhibiting protein activity or expression is an agent capable of inhibiting or inactivating the activity of the protein and / or an agent capable of down-regulating or not expressing the protein, wherein the protein is one or more selected from the proteins defined in claim 1; Preferably, the agent or agent combination comprises an agent capable of inhibiting the activity or expression of any two or more proteins selected from the group consisting of proteins defined in claim 1.

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