CN115389770A - Application of fibroblast growth factor 21 in vascular aging biological marker - Google Patents

Abstract

The application relates to the technical field of biological detection, in particular to application of fibroblast growth factor 21 in a biological marker of vascular aging; the application comprises judging the aging degree of blood vessels according to the concentration of the protein expressed by the fibroblast growth factor 21 in the blood, wherein the concentration of the protein in the blood is positively correlated with the aging degree of the blood vessels, and the concentration of the protein in the blood is regulated by positive feedback with the aging degree of the blood vessels; therefore, by determining the correlation and the regulation mode between the concentration of the protein expressed by the fibroblast growth factor 21 in blood and the aging degree of blood vessels, the protein expressed by the fibroblast growth factor 21 in blood can be used as a serum biological marker of vascular aging to make up for the blank of the serum biological marker for vascular aging.

Description

Application of fibroblast growth factor 21 in vascular aging biological marker

Technical Field

The application relates to the technical field of biological detection, in particular to application of fibroblast growth factor 21 in a biological marker of vascular aging.

Background

Vascular aging is a variety of cardiovascular diseases such as: atherosclerosis, vascular calcification, hypertension and other major risk factors, and vascular aging is also an important pathophysiological basis for aging of various organ systems of the human body. It has been found that the indicators of vascular function such as vasodilation and stiffness gradually decrease with age. The vascular aging refers to the physiological and pathological processes of functional aging, structural aging and degeneration of blood vessels along with the aging and under the combined action of cardiovascular risk factors, so that the early evaluation and early diagnosis of the vascular aging are important measures for evaluating and preventing the vascular aging and related diseases, and have important medical value and social significance.

At present, the assessment of vascular aging is mainly focused on the aspects of risk factors, vascular functions, vascular structures and the like, but because the change of substances in serum is usually prior to the change of the structures or functions of blood vessels, the assessment of the vascular functions and the vascular structures is more helpful for early prevention, diagnosis and delay of vascular aging and cardiovascular diseases related to the vascular aging by measuring the substances in serum, and the substances are called serum biological markers, but the serum biological markers for the vascular aging are still blank at present, so how to provide the serum biological markers for the vascular aging to make up the serum biological markers for the vascular aging is still blank, and the technical problem which needs to be solved at present is urgently needed.

Disclosure of Invention

The application provides application of a fibroblast growth factor in a vascular aging molecular marker, so as to fill up the blank of a serum biological marker for vascular aging in the prior art.

In a first aspect, the present application provides a use of fibroblast growth factor 21 in a biological marker of vascular senescence, the use comprising determining the degree of senescence of a blood vessel according to the concentration of a protein expressed by the fibroblast growth factor 21 in the blood of the blood vessel for non-diagnostic purposes;

the concentration of the protein and the aging degree of the blood vessel are positively correlated, and the concentration of the protein and the aging degree of the blood vessel are adjusted through positive feedback.

Optionally, the ratio of the weight gain of the protein in the intravascular blood to the volume of the intravascular blood is from 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of increasing age.

Optionally, the indicator of the degree of aging comprises at least one of Pulse Wave Velocity (PWV), brachial artery blood flow mediated vasodilation Function (FMD), ankle Brachial Index (ABI), and carotid Intimal Media Thickness (IMT).

In a second aspect, the present application provides a kit comprising a reagent for measuring the concentration of a protein expressed by fibroblast production factor 21 in blood, wherein the concentration of the protein expressed by fibroblast production factor 21 in blood is positively correlated with the degree of aging of blood vessels, and the concentration of the protein in blood and the degree of aging of blood vessels are regulated by positive feedback.

Optionally, the ratio of the increased weight of the protein expressed by fibroblast growth factor 21 in the intravascular blood to the volume of the intravascular blood is 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of increasing age.

Optionally, the reagent comprises at least one of an antibody, a protein stain, and a fluorescent label.

In a third aspect, the present application provides the use of a kit according to the second aspect in an agent for assessing or aiding in the assessment of the degree of vascular aging.

In a fourth aspect, the present application provides an active ingredient for inhibiting vascular aging according to the principles of the use of the first aspect, the active ingredient comprising an agent capable of increasing the expression level of fibroblast growth factor 21 or increasing the amount of protein expressed by fibroblast growth factor 21.

Optionally, the agent comprises exogenous fibroblast growth factor 21 and analogs thereof; and/or

The agent comprises a pharmaceutical ingredient that enhances the stability of a protein expressed by fibroblast growth factor 21; and/or

The reagent comprises a nucleic acid molecule capable of expressing fibroblast growth factor 21 and a vector thereof.

In a fifth aspect, the present application provides a use of the active ingredient of the fourth aspect in the preparation of a medicament for delaying vascular aging.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the application of the fibroblast growth factor 21 in the biological marker of vascular aging provided by the embodiment of the application is to use the fibroblast growth factor 21 as the biological marker of vascular aging for the first time, because the fibroblast growth factor 21 protein exists in blood, and can promote a series of functions of vascular endothelial cells, including the functions of promoting glycolysis of cells and Nitric Oxide (NO) secretion, and the application innovatively finds that the concentration of the protein expressed by the fibroblast growth factor 21 in the blood is positively correlated with the vascular aging degree, and the aged blood vessels show a resistance phenomenon to the fibroblast growth factor 21 protein, so that the increase of the concentration of the protein expressed by the fibroblast growth factor 21 in the blood accompanied by vascular aging is a compensatory increase, the influence caused by the reduction of the beneficial metabolic regulation effect of the protein expressed by the fibroblast growth factor 21 on the aged blood vessels is partially compensated, so that the protein can be adjusted between the blood concentration and the vascular aging through positive feedback regulation, and the serum concentration of the protein expressed by the fibroblast growth factor 21 in the blood is clearly determined to be adjusted between the blood serum concentration of the aging, so that the serum regulation effect of the biological marker of the aging can be compensated for the biological marker of the serum of the biological marker of vascular aging, and the biological marker of vascular aging.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a graph showing the relationship between the concentration of FGF21 protein in blood of a healthy person and different vascular age intervals, which is provided in the present application;

FIG. 2 is a graph showing the results of the relationship between the concentration of FGF21 protein in blood of a healthy person and the age of blood vessels, provided in the examples of the present application;

FIG. 3 is a schematic diagram showing the correlation between the concentration of FGF21 protein in blood of a healthy person and the vascular aging indicator PWV, provided by an embodiment of the present application;

FIG. 4 is a graph showing fluorescence comparison of effects of FGF21 on nitric oxide release from endothelial cells, provided in the examples of the present application;

FIG. 5 is a graph showing the fluorescence results of the effects of FGF21 on nitric oxide release from endothelial cells, as provided by examples herein;

FIG. 6 is a graph showing the results of the effect of FGF21 on basal glycolysis in endothelial cells, provided in the examples herein;

FIG. 7 is a graph showing the results of compensatory glycolysis of endothelial cells by FGF21 provided in the examples herein;

FIG. 8 is a graph showing the results of FGF21 reduction in the number of regulatable genes in endothelial cells with endothelial cell senescence provided in the examples of the present application;

FIG. 9 is a graph showing the comparison of FGF 21-related mRNA levels in FGF 21-knockout mice provided in the examples herein;

FIG. 10 is a graph showing the comparison of the concentration of FGF21 protein in blood of a knockout FGF21 mouse provided in the examples herein;

FIG. 11 is a graph showing the comparison of the systolic blood pressure of FGF 21-knockdown mice provided in the examples herein;

FIG. 12 is a graph showing the comparative results of the PWV of FGF21 knockout mice provided in the examples herein;

FIG. 13 is a schematic cross-sectional view of a blood vessel of a normal FGF21 mouse provided in an embodiment of the present application;

FIG. 14 is a schematic section view of a blood vessel of a FGF 21-knockout mouse provided in an embodiment of the present application;

fig. 15 is a graph showing the variation of the vascular lumen diameter of FGF21 knockout mice provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The inventive thinking of the application is that:

vascular aging refers to the physiological and pathological processes of functional, structural aging and degeneration of blood vessels with aging and under the combined action of cardiovascular risk factors. Structurally, vascular aging is accompanied by remodeling of the extracellular matrix, resulting in changes in the composition of elastin and collagen in the large elastic arteries. Elastin cleavage and degradation occur in the media of the intima of arteries by the up-regulation of matrix metalloproteinases. The deposition of collagen replaces the missing elastin molecules, accelerates the formation of advanced glycosylation end products, promotes the cross-linking of structural proteins, exacerbates arteriosclerosis and thus promotes the formation of vascular aging. Functionally, vascular aging is mainly manifested by increased stiffness of the arteries, decreased vascular compliance, and decreased ability of the vessels to repair, regenerate.

Vascular aging is one of common pathogenesis of various chronic diseases of the old people and is an important factor influencing healthy aging. The early evaluation and early diagnosis of vascular aging are important measures for evaluating and preventing vascular aging and related diseases, and have important medical value and social significance.

At present, the assessment of vascular aging mainly focuses on risk factors, vascular functions, vascular structures and the like, wherein common indicators for detecting vascular aging functions include blood flow-mediated vasodilation (FMD), nitroglycerin-induced vasodilation (NID), brachial ankle pulse wave velocity (baPWV), brachial ankle index (ABI) and the like, and the assessment of vascular structures can be performed through Magnetic Resonance Imaging (MRI) and intima-media thickness (IMT). Blood in the circulatory system is the most easily collected and information-rich sample in human tissues and is very important for identifying molecular markers, wherein the separated serum contains metabolic small molecules and proteins which are carriers secreted and communicated by various organs of a human body. Therefore, the change of substances in serum is usually prior to the change of the structure or function of blood vessels, and the measurement of the substances in serum is more helpful for early prevention, diagnosis and delay of vascular aging and cardiovascular diseases related to vascular aging than the evaluation of the vascular function and the vascular structure, and the substances are called serum biological markers.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or can be prepared by an existing method.

The embodiment of the application provides an application of fibroblast growth factor 21 in a biological marker of vascular aging, which comprises the steps of judging the aging degree of blood vessels according to the concentration of protein expressed by the fibroblast growth factor 21 in the blood vessels for non-diagnosis purposes;

the concentration of the protein in the blood and the aging degree of the blood vessels are positively correlated, and the concentration of the protein in the blood and the aging degree of the blood vessels are regulated by positive feedback.

In some alternative embodiments, the ratio of the weight gain of the protein expressed by fibroblast growth factor 21 in the intravascular blood to the volume of the intravascular blood is from 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of increasing age.

In the embodiment of the present application, the positive effect of controlling the ratio of the increased weight of the protein expressed by the fibroblast growth factor 21 in the blood to the volume of the blood in the blood vessel to be 20pg/mL to 30pg/mL is that the influence of the aging change of the blood vessel on the concentration of the protein expressed by the fibroblast growth factor 21 in the blood can be accurately reflected within the range of the weight to volume ratio, so that the degree of vascular aging can be reversely deduced by measuring the increase of the concentration of the protein expressed by the fibroblast growth factor 21 in the blood.

In some alternative embodiments, the indicator of the degree of aging comprises at least one of Pulse Wave Velocity (PWV), brachial artery blood flow-mediated vasodilation Function (FMD), ankle Brachial Index (ABI), and carotid Intimal Medial Thickness (IMT).

In the embodiment of the application, the index type for controlling the aging degree has the positive effects that the actual detection index of the blood vessel can be limited, and the aging degree of the blood vessel can be accurately reflected through the actual physiological index of the blood vessel.

Pulse Wave Velocity (PWV) is one of the common evaluation indexes reflecting the stiffness of arterial blood vessels, and is calculated by detecting the pulse wave propagation distance and time between different arterial segments. Vascular aging is usually accompanied by an increase in vascular stiffness and an increase in pulse wave conduction velocity. Therefore, by measuring the pulse wave velocity, the degree of aging of the blood vessel can be reflected to some extent.

The flow-mediated vasodilatory Function (FMD) of the brachial artery depends on the synthesis and secretion of nitric oxide by vascular endothelial cells. Nitric oxide has powerful functions of dilating blood vessels, controlling inflammation, preventing blood coagulation and the like. The aging of blood vessels is accompanied by the decrease of the nitric oxide synthesis and secretion function of vascular endothelial cells, and the aging and function decrease of vascular endothelial cells can be evaluated by evaluating the blood flow-mediated vasodilation function of brachial artery.

Ankle-brachial index (ABI) refers to the ratio of systolic pressure measured at the ankle (dorsal foot artery or posterior tibial artery) to systolic pressure measured at the brachial artery. ABI is reduced when the artery of the lower limb is narrowed or blocked, so that the blood supply of the artery of the distal ankle is reduced; in the case where arteriosclerosis is manifested, such as significant calcification of the vascular wall, the arterial wall cannot be compressed (non-compressible) during measurement, so that the measured systolic blood pressure is significantly increased and the ABI is abnormally increased. Currently, ABI less than 0.9 or greater than 1.3 is considered abnormal. Thus, ABI values may reflect disease and the degree of aging of the patient's blood vessels. The ABI is more than 1.3, which indicates that the calcified vascular wall of a patient ages and the blood vessel loses the contractile function, and can reflect the aging degree of the blood vessel to a certain extent.

Carotid intima-media thickness (cIMT) is detected by carotid ultrasound. The thickness of the intimal media of normal carotid arteries increases linearly with increasing age. In addition, abnormal aging and lesions associated with the carotid artery, such as the formation of atherosclerotic plaques in the carotid artery, also significantly increase the value of cIMT. Thus, a normal or abnormal increase in the value of cIMT may also reflect to some extent the degree of aging and pathology of the blood vessels.

Based on a general inventive concept, the present embodiments also provide a kit for preparing a kit for vascular aging according to the first aspect, the kit comprising a reagent for detecting a concentration of a protein expressed by fibroblast production factor 21 in blood, the concentration of the protein expressed by fibroblast production factor 21 in blood and the degree of aging of the blood vessels are positively correlated, and the concentration of the protein in blood and the degree of aging of the blood vessels are regulated by positive feedback.

The kit is realized based on the principle of the application, the specific detection principle of the kit can refer to the embodiment, and the kit adopts part or all of the technical scheme of the embodiment, so that the kit at least has all the beneficial effects brought by the technical scheme of the embodiment, and the detailed description is omitted.

In the embodiment of the application, the concentration of the protein expressed by the fibroblast production factor 21 in blood is positively correlated with the aging degree of the blood vessel, and according to the principle, the reagent capable of detecting the concentration of the protein expressed by the fibroblast production factor 21 in blood is arranged in the kit, so that the aging degree of the blood vessel can be indirectly detected more accurately.

In some alternative embodiments, the ratio of the weight gain of the protein expressed by fibroblast growth factor 21 in the intravascular blood to the volume of the intravascular blood is from 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of aging.

In the embodiment of the application, the positive effect of controlling the ratio of the increase weight of the protein expressed by the fibroblast growth factor 21 to the volume of the blood in the blood vessel to be 20 pg/mL-30 pg/mL is that in the range of the weight-volume ratio, the influence of the change of the blood vessel aging on the concentration of the protein expressed by the fibroblast growth factor 21 in the blood can be accurately reflected, so that the concentration of the protein expressed by the fibroblast growth factor 21 in the blood can be detected by the kit, and the aging degree of the blood vessel can be reversely deduced by comparing the concentration change before and after the comparison.

In some alternative embodiments, the reagent comprises at least one of an antibody, a protein stain, and a fluorescent label.

In the embodiment of the application, the specific type of the control reagent has the positive effect that the fibroblast growth factor 21 protein in blood exists in the expression product of the fibroblast growth factor 21, so that the blood vessel aging degree can be reversely deduced by accurately detecting the protein expressed by the fibroblast growth factor 21 through the concentration change of the protein expressed by the fibroblast growth factor 21.

Based on one general inventive concept, the embodiments of the present application also provide a use of the kit in a preparation for evaluating or assisting in evaluating the degree of vascular aging.

The application is realized based on the kit, the specific detection principle of the application can refer to the embodiment, and the application adopts part or all of the technical scheme of the embodiment, so that at least all the beneficial effects brought by the technical scheme of the embodiment are achieved, and the detailed description is omitted.

In the embodiment of the application, the kit is used for matching with other reagents, the protein expressed by the fibroblast growth factor 21 is detected by the kit, and the concentration of the protein expressed by the fibroblast growth factor 21 is positively correlated with the vascular aging degree, so that the vascular aging degree can be reversely deduced by detecting the concentration of the protein expressed by the fibroblast growth factor 21 by the kit, and the assessment or auxiliary assessment of the vascular aging degree by the kit is realized.

Based on a general inventive concept, the present embodiments also provide an active ingredient for inhibiting vascular aging according to the principles of the application of the first aspect, the active ingredient including an agent capable of increasing the expression level of fibroblast growth factor 21 or increasing the amount of protein expressed by fibroblast growth factor 21.

The active ingredient is realized based on the principle of the above application, the specific principle of the active ingredient can refer to the above examples, and as the active ingredient adopts part or all of the technical solutions of the above examples, the active ingredient at least has all the beneficial effects brought by the technical solutions of the above examples, and details are not repeated herein.

In the embodiment of the application, according to the point that the concentration of the protein expressed by the fibroblast growth factor 21 is in positive correlation with the vascular aging degree, the concentration of the protein expressed by the fibroblast growth factor 21 and the vascular aging degree are found to be in positive feedback regulation in the long-term research process, the vascular aging degree can be delayed by enhancing the expression capacity of the fibroblast growth factor 21, and therefore, the vascular aging degree can be delayed by limiting the active ingredients to contain the reagent.

In some alternative embodiments, the agent comprises exogenous fibroblast growth factor 21 and analogs thereof; and/or

The agent comprises a pharmaceutical ingredient that enhances the stability of a protein expressed by fibroblast growth factor 21; and/or

The reagent comprises a nucleic acid molecule capable of expressing fibroblast growth factor 21 and a vector thereof.

In the embodiment of the present application, the specific type of the control agent may be a reagent in the active ingredient, as long as the control agent can delay the vascular aging degree by positive feedback regulation as long as the control agent increases the expression level of the fibroblast growth factor 21 or increases the amount of the protein expressed by the fibroblast growth factor 21, so that both the control agent and the control agent can be used as agents in the active ingredient for the protein expressed by the fibroblast growth factor 21 and drugs influencing the expression process of the fibroblast growth factor 21.

Exogenous fibroblast growth factor 21 and analogs thereof refers to substances similar or identical in function and structure to fibroblast growth factor 21, such as pegylated fibroblast growth factor 21, fusion proteins of polypeptides and fibroblast growth factor 21.

The pharmaceutical ingredient for enhancing the stability of the protein expressed by the fibroblast growth factor 21 refers to a drug that can stabilize the protein expressed by the fibroblast growth factor 21 or increase the half-life thereof, such as a small molecule drug of the fibroblast growth factor 21 or an antibody drug of the fibroblast growth factor 21;

the nucleic acid molecule for expressing fibroblast growth factor 21 and the vector thereof refer to a nucleic acid molecule containing a nucleic acid molecule capable of expressing fibroblast growth factor 21 or a vector carrying such nucleic acid molecules, for example: a plasmid, adenovirus or lentivirus containing fibroblast growth factor 21.

Based on one general inventive concept, the embodiments of the present application also provide an application of the active ingredient in preparing a medicament for delaying vascular aging.

The application is realized based on the active ingredients, the specific principle of the application can refer to the above embodiments, and as the application adopts part or all of the technical solutions of the above embodiments, at least all the beneficial effects brought by the technical solutions of the above embodiments are achieved, and no further description is given here.

In the embodiment of the application, according to the level that the expression level of the fibroblast growth factor 21 is improved or the amount of the protein expressed by the fibroblast growth factor 21 can be increased by a reagent, the blood vessel aging degree can be delayed through positive feedback regulation, other medicinal components are mixed by utilizing the active components, and the active components contain the protein which improves the expression level of the fibroblast growth factor 21 or can increase the amount of the protein expressed by the fibroblast growth factor 21, so the blood vessel aging can be effectively delayed through the addition of the active components.

The present application is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer.

The quantitative tests in the embodiment of the application are repeated at least three times, and the results are averaged.

The collection of the subject specimen involved in the embodiment of the application is carried out based on the declaration of helsinki, fully respects the informed consent of the subject and the family members thereof, the experimental means used in the research process is advanced and scientific, the sampling process accords with the medical routine, and the subject protection scheme is formulated and approved by the ethical approval of the medical ethical committee of the affiliated college of science and technology medical college.

The experiments on mice in the examples of the present application were carried out based on the regulations on the management of laboratory animals in Hubei province and the regulations on the ethical Committee of laboratory animals in university of science and technology in Huazhong, and were approved by the ethical examination of the ethical Committee of laboratory animals in the affiliated college of science and technology in Huazhong.

The purchase sources of the reagents in the examples of this application:

non-essential amino acids were purchased from seirui, zhejiang, biotechnology limited;

low serum growth supplement LSGS, 0.05% pancreatin and fetal bovine serum FBS were purchased from Gibco;

sterile PBS, protein loading buffer 5 × purchased from bosch, wuhan;

100 × penicillin streptomycin was purchased from Invitrogen;

m199 medium was purchased from Hyclone;

SYBRGREEN, RNA reverse transcription kit from TOYOBO, japan;

rFGF21 was purchased from PeproTech, USA; FGF21-ELISA kit was purchased from Abcam company (ab 222506);

the Hipure RNA extraction kit is purchased from magenta;

the qPCR primers are designed by Shanghai bioengineering company;

the glycolytic rate kit of SeahorseXF cells (cat # 103344-100);

NO detection kit (bi yun day).

Mouse non-invasive sphygmomanometer (Beijing Zhongshi science and technology);

tissue grinders (***, wuhan);

gas anaesthesia machines (MSS, uk);

small animal non-invasive ultrasound doppler flow meter (INDUS);

384-well PCR instrument (roche, usa);

ultra low temperature refrigerator at-80 deg.C (Sanyo Japan);

CO ₂ incubator (Thermo corporation, usa);

sunrise microplate reader (shimadzu, japan);

laser confocal microscopy (Nikon C2+, tokyo, japan);

seahorse xFp Analyzer System (Agilent, santa Clara, calif.).

Example 1

The method for inspecting the increase of the age of a healthy person along with the increase of the concentration of fibroblast growth factor 21 (FGF 21) protein in blood comprises the following steps:

1. ethical approval:

the study was approved by the ethical committee of the affiliated college of science and technology of Huazhong. The process of administration of this study was in full compliance with ethical and ethical guidelines on human medical research in the world society of medicine, helsinki declaration, and the participants involved in this study signed written informed consent prior to participation.

2. Collection and assay of ex vivo plasma:

the participants were 212 healthy physical examination volunteers aged 20-70 years and having a BMI of 17kg/m ² ～25kg/m ² In addition, metabolic diseases such as hypertension, hyperlipidemia, diabetes, tumor, infection, inflammation or significant vascular sclerosis (PWV value is within normal range) and the like which may affect the index of FGF21 concentration are excluded.

All volunteers were deprived of smoking and drinking for at least one week prior to blood collection, and blood samples were collected from each volunteer in the morning after a night of fasting. Collecting the collected venous blood with a sterile vacuum tube containing heparin, centrifuging at 320g for 20min by a centrifuge, subpackaging the upper layer of plasma obtained after centrifugation, and freezing and preserving at-80 ℃ in a refrigerator until use.

Elisa assay FGF21 concentration in plasma:

(1) The collected cryopreserved plasma samples were removed from the refrigerator, thawed and numbered.

(2) The samples were diluted proportionally into the sample diluent NS.

(3) All reagents, working standards and samples were prepared.

(4) Taking off the redundant micropore strips on the plate frame, putting the plate frame and the drying agent together into an aluminum foil bag, sealing the aluminum foil bag again, and putting the aluminum foil bag back to a storage place at 4 ℃.

(5) 50 μ L of all samples or standards were added to the corresponding test wells.

(6) 50 μ L of the antibody mix was added to each well.

(7) Plates were sealed and incubated for 1h at room temperature on a plate shaker set at 400 rpm.

(8) Each well was washed with 3x350 μ L of 1x Wash Buffer PT, requiring that Wash Buffer PT should remain in the well for at least 10 seconds (the liquid must be thoroughly removed each time for good performance); after the final wash, the plate was inverted and tapped with a clean paper towel to remove excess liquid.

(9) 100 μ L of TMB developer was added to each well, wrapped in tinfoil paper and protected from light, and incubated for 10min on a plate shaker set at 400 rpm. According to the environmental conditions of the laboratory, the incubation is additionally required, and the incubation time is 20min.

(10) 100 μ L of stop solution was added to each well. The plate was shaken on a plate shaker for about 1min to mix thoroughly.

(11) Opening an enzyme-labeling instrument in advance for preheating, setting the OD value of a test wavelength to be 450nm, placing the flat plate into the instrument for testing, recording the OD value at the 450nm position, drawing a corresponding standard curve according to the OD value tested by the standard substance and the concentration of the actual standard substance to obtain a formula of the curve, substituting the OD value tested by the sample into the formula, calculating the actual concentration corresponding to the sample, and finishing and analyzing the physiological concentration range of FGF 21.

As can be seen from the data shown in fig. 1 to 3: the increase in blood concentration of FGF21 protein with aging was accompanied by an increase in blood concentration in healthy persons, as shown in fig. 1, an increase in blood concentration of FGF21 of 25pg/mL per 10 years of aging on average, and as shown in fig. 2, the blood concentration of FGF21 protein positively correlated with the age of healthy persons and the vascular aging indicator PWV as shown in fig. 3.

Example 2

Example 2 is compared with example 1, with the difference between example 2 and example 1 being that:

the effect of FGF21 with the mass concentration of 400pg/mL on promoting the nitric oxide releasing function of HUVEC is investigated, and the specific steps are as follows:

1. endothelial cell isolation step:

(1) Sterile gauze, an infusion set, a 50mL syringe, a 10mL syringe, a 50mL centrifuge tube, a T25 cell culture flask, M199 basal medium, complete medium and endothelial cell apparatus are prepared in advance. And 0.1% collagenase II was preheated at 37 ℃. 50mL of primary endothelial cell complete medium was prepared in advance, containing 84% M199, 10% Fetal Bovine Serum (FBS), 2% growth factor (LSGS), 1% non-essential amino acids, and 1% penicillin-streptomycin.

(2) Human Umbilical Vein Endothelial Cells (HUVECs) are from healthy full-term fetuses without infection history, recent infection and family genetic diseases, are separated from umbilical cords of newborn within 4h after the birth of the fetuses, umbilical cords with the length of about 9-16 cm are cut at the position close to the placenta, the residual blood in umbilical cord blood vessels is squeezed out, the umbilical cord blood vessels are placed in PBS liquid with sterile precooling, samples are moved to a laboratory clean bench, and the process of separating cells is finished within 4 h.

(3) The umbilical cord is clamped by tweezers and moved to a culture dish, the residual blood in the umbilical cord is cleaned by PBS in the culture dish, the part of the umbilical cord which is subjected to edema after being clamped by the tweezers is cut off, the tail end of the umbilical cord is provided with three blood vessels, the thicker one of the three blood vessels is an umbilical vein blood vessel, the blood clot on the umbilical cord at the tail end is cleaned (the umbilical cord can be directly cleaned by hands), and the two ends of the umbilical cord are cut off by an ophthalmic scissors.

(4) The method comprises the steps of placing a needle of an infusion apparatus into a thick umbilical vein (keeping a needle sleeve head to serve as a blunt needle head), clamping the umbilical vein blood vessel containing the blunt needle head by using vascular forceps, sucking 10mL of preheated PBS by using a 5mL injector, injecting the PBS into the infusion apparatus to wash the umbilical vein blood vessel, repeating for 2 to 3 times, and clamping the other end of the umbilical cord by using the vascular forceps when the finally-flowing liquid is clear and transparent.

(5) Sucking residual gas in vein blood vessel with syringe to form vacuum negative pressure state in blood vessel cavity, slowly adding 10mL preheated 0.1% collagenase II until the umbilical vein is filled with red collagenase liquid, at this time, the blood vessel is in swelling and filling state, placing umbilical cord in palm center, and hot compressing for about 13 min. The umbilical cord is gently kneaded several times during this process to allow endothelial cells to fall out sufficiently.

(6) After the hot compress time is over, the vascular clamps on the umbilical cord at the tail end are loosened, about 30mL of M199 cell culture medium is sucked by a 50mL syringe, and the cell digestive juice is collected into a 50mL centrifuge tube after the cell digestive juice is injected into an infusion apparatus.

(7) The collection was centrifuged at 1000rpm for 7min and the supernatant was gently aspirated off with a pasteur pipette. To avoid resuspending the cell pellet for diffusion.

(8) Adding about 3-4 mL of prepared complete culture medium, gently blowing and uniformly mixing by using a Pasteur pipette, and adding the mixture to 25cm ² Resuspending in a culture flask, shaking uniformly, placing at 37 deg.C, containing 5% CO ₂ Culturing in a cell culture box.

(9) After the cells adhere to the wall in about 6 hours, the cells can be gently washed by sterile PBS (to prevent the adherent cells from being washed down), and after a fresh complete culture medium is added, the cells are put into an incubator to be continuously cultured. After that, the liquid is changed every 48 hours, and the cell density reaches 80 to 90 percent, and then the cell can be passaged.

(10) When cells are passaged, firstly discarding the supernatant, adding 3mL sterile PBS to wash the cells, discarding the PBS, adding 1mL trypsin-EDTA solution with the mass concentration of 0.05%, placing the solution into a cell culture box for 1-2 min, taking out the solution, slightly beating the periphery of the culture dish by hands, placing the culture dish into an operation table, adding 4mL complete culture medium, blowing and beating the solution uniformly by a Pasteur pipette, respectively adding the solution into 2 new culture dishes of 10cm, supplementing the complete culture medium to 7mL, and placing the culture dish into the cell culture box.

2. Endothelial cell NO detection

HUVECs were evenly seeded in confocal dishes at an initial cell density of 40% and medium containing different concentrations of rFGF21 (0, 400 pg/mL) was changed every 24h before the experiment. After 48h of cell treatment, DAF-FM DA was diluted with DAF-FM DA diluent provided in the kit to a final concentration of 2.5. Mu. Mol/L according to a ratio of 1. The cell culture was removed, diluted DAF-FM DA was added in an appropriate volume (the volume added was preferably sufficient to cover the cells, usually 500. Mu.L in a confocal dish) and incubated at 37 ℃ for 20min in a cell culture chamber. Cells were washed three times with Hanks' solution (pH 7.4) to remove the DAF-FM DA well around the cells. The intensity of fluorescence before and after stimulation was measured using an excitation wavelength of 495nm and an emission wavelength of 515 nm.

The results of fig. 4 and 5 show that: as shown in fig. 4, compared with the control group, the mean fluorescence intensity of the cells after the intervention treatment with FGF21 of 400pg/mL is significantly increased, and the comparison of the data in fig. 5 shows that the capability of the endothelial cells to release nitric oxide is significantly improved.

Example 3

Example 3 is compared with example 2, which differs from example 2 in that:

investigating the promoting effect of FGF21 with different concentrations on the glycolysis function of HUVEC, the specific steps are as follows:

HUVECs were seeded 3 days prior to the experiment at a density of 10000 cells/well in 24-well hippocampal XF cell culture microplates (Agilent, santa Clara, calif.). The medium containing different concentrations of rFGF21 (0, 100, 400, 5000 pg/mL) was changed every 12h before the experiment.

One day before the experiment, the sensor cartridge was at 37 ℃ non-CO ₂ In an incubator, hydrated overnight in Seahorse XF Calibrant, next day cells were in non-CO ₂ Incubate for 1h in the incubator. The culture medium was changed to a hippocampal culture medium containing glucose, pyruvic acid, glutamine and HEPES buffer. The real-time extracellular acidification rate (ECAR) and Oxygen Consumption Rate (OCR) of the cells were measured at 37 ℃ using a Seahorse xFp analyzer system (Agilent, santa Clara, calif.) according to the manufacturer's instructions to determine the glycolytic rate of HUVECs.

As shown in FIGS. 6 and 7, the basal glycolysis rate of endothelial cells after FGF21 treatment with 400pg/mL addition in FIG. 6 was significantly increased compared to the compensatory glycolysis rate in FIG. 7, compared to the control group.

Example 4

Example 4 is compared with example 3, and example 4 differs from example 3 in that:

the specific steps for examining whether the gene which can be significantly regulated by FGF21 with 400pg/mL in HUVEC is significantly reduced along with the replicative senescence of cells are as follows:

1. culture of primary endothelial cells and treatment with rFGF 21:

HUVECs were cultured and Population Doubling Levels (PDLs) were calculated, the generation number of cell growth and the date of passage were recorded, when cells grew from young to old to different PDLs (T0, T1, T2, T3, T4, T5), the cells were plated evenly in a six-well plate, after cell attachment, the cells were starved for 12h with a medium containing 2% FBS, the cells were cultured for 48h with a medium containing 10% FBS and the same concentration of rFGF21 (400 pg/mL), then the supernatant was removed, and the cells were lysed with 350. Mu.L of RIPE solution in HI Pure RNA Extract Kit. The lysate was added to a 1.5mL enzyme-free EP tube, and the operating time and sample information were recorded and stored in a-80 ℃ freezer.

RNA extraction, quality inspection, mRNA library establishment and differential expression gene screening:

(1) Extraction of RNA: extracting total RNA in the HUVECs treated by the FGF21 by using a high-purity RNA extraction kit.

(2) Quality inspection of RNA: RNA integrity, purification and quality was checked by a NanoDrop 2000 spectrophotometer (thermoelectric corporation, USA) at 260/280nm and by bioanalyzer 4200 (Agilent, santa Clara, calif.) and agarose gel analysis.

(3) mRNA library construction: the RNA samples of each group of umbilical cords are sent to China Dagen Limited (Shenzhen, china) and mRNA deep sequencing is carried out on an Illumina HiSeq X sequencing platform. Total RNA was extracted from the samples using miRNeasy Micro kit (Qiagen, hilden, germany) according to the manufacturer's instructions. Use of

(Vazyme, nanjing, china) was used to prepare the NGS library using the kit for the preparation of the VAHTS mRNA seq v2 library. To the above system was added a buffer reaction system of "A" at the 3' end. And (3) complementary pairing and accurate connection with a single T-shaped protrusion at the 5' end of a next sequencing joint, connecting the sequencing joint, carrying out PCR amplification on mRNAs, and further purifying a PCR product and using the PCR product for sequencing.

(4) Screening of differentially expressed genes: data quantile normalization, gene screening using the R software package, differentially expressed genes with P <0.05, and results are shown in table 1 and fig. 8.

TABLE 1

As shown in FIG. 8 and Table 1, 400pg/mL of FGF21 gradually reduced the regulation of genes in senescent cells, resulting in FGF21 resistance.

Example 5

Example 5 is compared with example 4, the difference between example 5 and example 4 being:

examining whether the specific knockout of FGF21 (CKO) in the liver in example 4 could reduce the concentration of FGF21 in blood to a baseline level, the specific steps were as follows:

1. construction of transgenic mice: since Fgf21 gene has 1 transcript, exon1-exon3 of Fgf21-201 (ENSMUST 00000033099.5) transcript is recommended as a knockout region, which contains all coding sequences, depending on the structure of Fgf21 gene, which would lead to disruption of protein function.

The Fgf21 gene is modified by using a CRISPR/Cas9 technology, and the process flow is as follows: in vitro transcription of sgRNA and construction of donor vector;

micro-injecting Cas9, sgRNA and Donor into fertilized eggs of a C57BL/6JGpt mouse;

f0 positive mice are obtained by transplanting fertilized eggs and are verified by PCR and sequencing;

mating the F0 positive mouse with a C57BL/6JGpt mouse to obtain a stable F1 generation mouse model;

flox mice are knocked out after mating with mice expressing Cre recombinase, resulting in loss of function of the target gene in liver tissue.

Qpcr detection of expression level of Fgf21 mRNA in mouse liver tissue:

(1) The table was wiped with 75% alcohol.

(2) RNase free EP tubes, pipettes, and RNA extraction kits (magenta Hipure total RNA kits) were prepared.

(3) DEPC treatment water containing 70% ethanol is prepared in advance, and precooled in advance at 4 ℃ by a centrifugal machine.

(4) The RNA extraction operation process comprises the following steps:

1) Taking out the liver tissue from a refrigerator at the temperature of-80 ℃, weighing 10mg of the liver tissue by using an electronic balance, and placing the liver tissue in a 1.5mL enzyme-free Ep tube; adding 600 mu L of buffer RL lysate, adding 2-3 particles of non-enzyme grinding beads (2 mm), placing the mixture into a refrigerator at the temperature of minus 80 ℃ for precooling for 30min, wherein the grinding frequency is 6m/s, the grinding time is 20s, the pause time is 15s, and circulating twice. Standing for 3-5 min after grinding, centrifuging at 4000rpm for 5min, and collecting supernatant.

2) When extracting RNA, precooling at 4 ℃ in advance by a centrifuge, loading a gDNA filter column (blue) into a 2mL collecting pipe, marking the serial number of the filter column, correspondingly adding cell lysate into the filter column, setting the centrifuge as 14000g, and then centrifuging for 2min.

3) After centrifugation, the gDNA filter column was discarded, an equal volume of DEPC water containing 70% ethanol was added to a 2mL collection tube, and the mixture was blown and beaten by a 1mL pipette 4 to 5 times.

4) Putting HiPure RNA Mini column into a new 2mL collecting tube, transferring the mixed liquid containing 70% ethanol into a filter column after marking the sequence, centrifuging at 12000g for 1min, removing protein, and adsorbing RNA on the filter column.

5) After the centrifugation, the liquid in the collection tube was discarded, 500. Mu.L of Buffer RW1 was added to the filtration column, and the mixture was centrifuged at 12000g for 1min. And washing the filter column, and cleaning and removing the protein and other matrixes on the filter column.

6) The Buffer RW2 was diluted with ethanol in advance, the filtrate in the collection tube was discarded after centrifugation, the filter columns were placed in the collection tube, 500. Mu.L of Buffer RW2 was added to each filter column, and centrifugation was carried out at 12000g for 1min in order to remove the salts from the filter columns.

7) After centrifugation, the filtrate in the collection tube was decanted, and the collection tube was returned to the filtration column, 500. Mu.L of Buffer RW2 was added to each filtration column, and the mixture was centrifuged at 12000g for 1min.

8) Discarding the liquid in the collecting pipe, putting the filter column into the collecting pipe again, throwing the filter column, and centrifuging at 12000g for 2min to remove the residual ethanol in the filter column.

9) And taking out the filter column after centrifugation, transferring the filter column to a new 1.5mL enzyme-Free EP tube, marking the number of the EP tube, adding 20-30 mu L of RNase Free water into the center of a filter column membrane, standing for 2min, and centrifuging for 2min under the condition of 12000g, wherein the aim of the operation is to elute the RNA.

10 The liquid after centrifugal elution can be sucked out finally, and then is added into the filter column again for centrifugal elution again.

11 The extracted RNA was placed on an ice box, and the RNA concentration was measured.

(5) Determination of RNA concentration:

1) The computer was turned on in advance, and the Nano drop 2000 software was spotted and then the RNA determination option was selected.

2) The measuring head is cleaned with enzyme-free water and calibrated and zeroed.

3) Sucking 1.5 mu L of RNA sample to be detected by a liquid shifter, detecting each sample for 2 times, and finally calculating and taking an average value.

4) Before each sample adding, the measuring head is wiped clean by wiping paper, and then a sample to be detected is dripped for detection.

(6) Adjusting the RNA concentration of the sample:

after the test is completed, the minimum value of the RNA concentration in this batch of samples is selected, and the total RNA amount at a volume of 14. Mu.L is calculated. According to the value of the total RNA quantity, calculating the total RNA volume Vn of other samples, and the volume of the enzyme-free water to be supplemented is (14-Vn) mu L, so that the final RNA concentration and the final volume of all the samples are consistent.

(7) Reverse transcription of mRNA into cDNA:

samples adjusted to be consistent in concentration are added into 200 μ L enzyme-free EP tubes again, then 1 μ L primer mix is added to form a 15 μ L system, the system is placed in a PCR instrument, RNA denaturation is carried out at 65 ℃ for 5min, mix preparation is continued, calculated by the 20 μ L system, 5 μ L mix reagent is added into each tube sample, and the preparation is as follows: 4. Mu.L of 5 × RT buffer, 1. Mu.L of RT Enzyme mix. Placing in a PCR instrument at 37 ℃ for 15min and 98 ℃ for 5min, and carrying out reverse transcription to obtain cDNA. And finally, marking a sample, and storing the sample in a refrigerator at the temperature of-20 ℃.

(8) Fluorescent quantitative PCR:

1) qPCR experiments were performed using TOYOBO SYBR green mix, formulated according to the reaction system as in table 2:

TABLE 2

10 uL reaction system	Volume of
		SYBR green	5μL
Enzyme-free water	2.5μL
		cDNA	0.1μL
Primer and method for producing the same	2.4μL

2) And (3) computer detection: the reagents are mixed evenly and added into a 384-pore plate, 3 multiple pores are arranged for each sample, and then the 384-pore plate is placed into a 384-fluorescence quantitative PCR instrument to set programs. Time and cycle number settings were made based on primer Tm and amplified fragment size.

3) After the reaction was completed, data analysis was performed using 2 ^-ΔΔCT The relative expression level of mRNA was calculated, and the sequences of the mouse primers are shown in Table 3.

TABLE 3

Gene	Forward	Reverse
			Fgf21	CGGTTACAATGTGTACCAGTCT	GTAAAGGCTCTACCATGCTCAG
β-Actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
			Gapdh	AATGGTGAAGGTCGGTGT	GTGGAGTCATACTGGAACATGTAG
18sRNA	ATGCGGCGGCGTTATTCC	GCTATCAATCTGTCAATCCTGTCC

ELISA for detecting the expression change of mouse plasma FGF 21: as indicated in the detection method of example 1.

As shown in fig. 9 and 10, both mouse liver FGF21 mRNA levels and plasma FGF21 protein levels were significantly reduced.

Example 6

Example 6 is compared with example 5, the difference between example 6 and example 5 being:

the specific steps of examining that the CKO can obviously increase the systolic pressure and the PWV of the mouse are as follows:

1. noninvasive blood pressure measurement of mice:

(1) The mouse is fixed by using a mouse fixer with a proper size, the tail part of the mouse is completely exposed, the mouse is fixed in the middle position of the fixer without moving, and the nose part of the mouse can breathe normally. The heating plate was opened and warmed to 37 ℃ in advance;

(2) Placing the mouse fixer on the heating plate, sleeving the blocking sleeve on the tail root, and sleeving the sensor (VPR) on the tail at a distance of about 0.5cm from the blocking sleeve;

(3) The measurement option is started and the machine is automatically inflated until the occlusion sleeve completely blocks the blood flow from the rat tail, and then deflation is started.

(4) After pressure calibration, the tail artery of the rat is slowly opened, the machine recognizes the starting point of pressure change as systolic pressure, deflation is maintained until blood flow enters the vein from the artery, and a receptor record value at the moment of vein opening is diastolic pressure.

Detecting the change of the PWV value of the mice by a PWV instrument:

after the mouse is anesthetized stably by using isoflurane gas (the flow =5 and the concentration = 1.5), the mouse is placed on a heating electrocardio-conduction flat plate in a supine manner, the tail ends of the limbs are pasted at the electrode positions by using conductive paste, and the electrocardiogram of the mouse is observed to be normal without interfering clutter. PWV measurements were made using Doppler ultrasound (Mouse Doppler). The couplant is evenly smeared on the chest and the lower abdomen of the mouse, the 20MHz probe is used for measuring the walking part of the chest and the lower abdomen of the mouse close to the midline abdominal aorta, the couplant is stored when the upper marked vertebra pressure waveform of the display screen appears, and the vertical distance between the chest and the lower abdomen probe is recorded.

PWV＝distance between probe tips(mm)/(time between start of wave at position 1 and start of wave at position 2)(ms)

The results of fig. 11 and 12 show that: the systolic blood pressure and PWV of CKO group mice were significantly increased.

Example 7

Example 7 is compared with example 5, which differs from example 5 in that:

whether the CKO can obviously increase the vessel lumen diameter of the mouse is examined, and the specific steps are as follows:

1. preparing experimental money: after the mice are fed with normal diet for 40 weeks, the mice are anesthetized by 4% chloral hydrate, when the anesthesia is stable, the eyeballs are removed to take blood, the blood is placed in a 1.5mL Ep tube heparinized in advance, a centrifuge is used for separating plasma and blood cells (3000rpm, 15min), the separated plasma on the upper layer is sucked and placed in a 200 mu L Ep tube, and the plasma is stored at the low temperature of-80 ℃. The thoracic cavity and the abdominal cavity of a mouse are quickly opened, a laceration is cut on the right atrium by using an ophthalmic scissors, an intravenous infusion needle is inserted from the left ventricle part of the cardiac apex part, pre-cooled PBS solution is used for perfusion, and the condition that the liver of the mouse is changed from red to grayish yellow to prompt complete perfusion is observed. The thoracic aorta of mice was rapidly isolated and extracted, fixed with 4% paraformaldehyde and dehydrated with 30% sucrose aqueous solution at 4 ℃ and then embedded in paraffin.

HE staining:

(1) Paraffin sectioning (dewaxing to washing process); sequentially placing the paraffin sections into dimethylbenzene to stand for 20min, then placing the paraffin sections into another fresh dimethylbenzene to stand for 20min, standing the paraffin sections in absolute ethyl alcohol for 5min, then placing the paraffin sections in another fresh absolute ethyl alcohol for 5min, carrying out gradient dewaxing on 75% alcohol for 5min, and finishing water washing;

(2) Hematoxylin staining: staining the prepared section by using hematoxylin working solution for 3-5 min; after dyeing is finished, cleaning redundant dyeing liquid by using clear water; using 1% hydrochloric acid solution to differentiate for several seconds, and quickly washing with clear water; bluing with 0.6% ammonia solution, and washing with clear water for several seconds;

(3) Eosin staining: using 85-95% ethanol solution to perform gradient dehydration on the paraffin section, and putting the paraffin section into eosin staining solution for incubation for 5 minutes;

(4) Dewatering and sealing: sequentially placing the prepared paraffin section into anhydrous ethanol, standing for 5min, further standing for 5min in fresh anhydrous ethanol, further standing for 5min in n-butanol, standing for 5min in xylene, further standing for 5min in fresh xylene until the section is transparent, taking out the section from xylene, slightly drying, sealing with neutral gum

(5) The blood vessel diameters were analyzed using Image pro plus 6 software, and the results are shown in fig. 13 to 15.

As shown in fig. 13 to 15, the vascular lumen diameter of CKO group mice was significantly increased compared to the control group.

Therefore, it is apparent from the above examples that a positive correlation between the concentration of FGF21 protein in blood and vascular aging is observed, and the occurrence of vascular aging or assessment and diagnosis of vascular aging, risk stratification, etc. can be predicted based on this correlation.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

(1) The application of the fibroblast growth factor 21 in the biological marker of vascular aging provided by the embodiment of the application deduces that the concentration of the FGF21 protein is positively correlated with the aging degree of blood vessels and is positively correlated with PWV reflecting the stiffness of the blood vessels by limiting the increase of the level of the FGF21 protein in blood along with the aging of the blood vessels.

(2) According to application of the fibroblast growth factor 21 in the biological marker of vascular aging, through multiple groups of experimental investigation, it is found that with aging of vascular endothelial cells, the regulation and control effect of FGF21 on aging cells is remarkably reduced, and the knockout of FGF21 gene in the liver of a mouse can remarkably reduce the level of FGF21 in the blood of the mouse to a baseline level, and the mouse shows a remarkable vascular aging acceleration phenomenon under the feeding of high-fat feed, including remarkable increase of aging and vascular aging indexes systolic pressure and PWV and remarkable increase of vascular lumen, and the remarkable effect of FGF21 in inhibiting vascular aging is clarified.

(3) According to the application of the fibroblast growth factor 21 in the biological marker of vascular aging, on one hand, the concentration of FGF21 in blood of aging people is up-regulated and is positively correlated with vascular aging index PWV; on the other hand, aging vessels exhibit a resistance phenomenon to FGF21, and an increase in blood FGF21 concentration accompanying aging is a compensatory increase for partially compensating for a decrease in the beneficial metabolic regulation effect of FGF21 on aging vascular cells. In a mouse model of knocking out FGF21 expression, the concentration of FGF21 in the blood of a mouse is obviously reduced, so that the mouse shows obvious phenomenon of accelerating vascular aging under the condition of being fed by high-fat feed, including the significant increase of systolic blood pressure and PWV (vascular endothelial growth factor) of vascular aging indexes and the significant increase of vascular lumen, and the significant effect of FGF21 in inhibiting vascular aging is clarified. Therefore, the FGF21 protein can be used as a blood marker for evaluating or assisting in evaluating vascular aging, can predict the occurrence of vascular aging and related diseases, evaluates and diagnoses the vascular aging, stratifies risks, delays the vascular aging and prevents and treats the vascular aging related diseases.

Various embodiments of the present application may exist in a range of forms; it should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the application; accordingly, the described range descriptions should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, it is contemplated that the description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the stated range, such as 1, 2, 3, 4, 5, and 6, for example, as applicable regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any number (fractional or integer) recited within the range so indicated.

In this application, where the context requires no explicit explanation, the use of directional words such as "upper" and "lower" in particular refers to the direction of the drawing in the figures. In addition, in the description of the present specification, the terms "include", "includes" and the like mean "including but not limited to". In this document, relational terms such as "first" and "second", and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Herein, "and/or" describes an association relationship of associated objects, and means that there may be three relationships, for example, a, and/or B, and/or C, and that there may be any one of a, B, and C alone, or any one of them at the same time, or three of them at the same time. Wherein A, B and C can be a single technical feature or a collection of a plurality of technical features. As used herein, "at least one" means one or more, and "a plurality" means two or more. "at least one," "at least one of the following," or similar expressions, refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (one) of a, b, or c," or "at least one (one) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, and c may be single or plural, respectively.

The above description is merely exemplary of the present application and is presented to enable those skilled in the art to understand and practice the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The use of fibroblast growth factor 21 as a biological marker of vascular aging, which comprises judging the aging degree of blood vessels according to the concentration of a protein expressed by the fibroblast growth factor 21 in the blood vessels, wherein the concentration of the protein is not used for diagnosis;

the concentration of the protein in the blood and the aging degree of the blood vessels are positively correlated, and the concentration of the protein in the blood and the aging degree of the blood vessels are regulated by positive feedback.

2. The use according to claim 1, wherein the ratio of the added weight of the protein to the volume of blood in the blood vessel is from 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of increasing age.

3. The use of claim 1, wherein the indication of the degree of aging comprises at least one of pulse wave velocity, brachial artery blood flow-mediated vasodilation function, ankle brachial index, and carotid intimal media thickness.

4. A kit comprising a reagent for detecting the concentration of a protein expressed by fibroblast production factor 21 in blood, wherein the concentration of the protein expressed by fibroblast production factor 21 in blood is positively correlated with the degree of senescence of blood vessels, and wherein the concentration of the protein in blood and the degree of senescence of blood vessels are regulated by positive feedback.

5. The kit according to claim 4, wherein the ratio of the weight increase of the protein expressed by fibroblast growth factor 21 in the intravascular blood to the volume of the intravascular blood is from 20pg/mL to 30pg/mL based on the length of the blood vessel per 10 years of aging.

6. The kit of claim 4, wherein the reagent comprises at least one of an antibody, a protein stain, and a fluorescent label.

7. Use of a kit according to any one of claims 4 to 6 in a formulation for assessing or aiding in the assessment of the degree of vascular aging.

8. An active ingredient comprising an agent that increases the expression level of fibroblast growth factor 21 or increases the amount of protein expressed by fibroblast growth factor 21.

9. The active ingredient of claim 8, wherein the agent comprises exogenous fibroblast growth factor 21 and analogs thereof; and/or

The agent comprises a pharmaceutical ingredient that enhances the stability of a protein expressed by fibroblast growth factor 21; and/or

The reagent comprises a nucleic acid molecule capable of expressing fibroblast growth factor 21 and a vector thereof.

10. Use of an active ingredient as claimed in claim 8 or 9 for the preparation of a medicament for delaying vascular aging.

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