CN113621699A

CN113621699A - Fibulin-1 protein as vascular aging molecular marker and application thereof

Info

Publication number: CN113621699A
Application number: CN202110911507.2A
Authority: CN
Inventors: 张存泰; 阮磊
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-10
Filing date: 2021-08-10
Publication date: 2021-11-09

Abstract

The invention relates to the technical field of biological detection, and particularly provides a Fibulin-1 protein serving as a vascular aging molecular marker and application thereof. The inventor unexpectedly finds that the expression level of the Fibulin-1 protein is positively correlated with the vascular aging degree in the research process of vascular aging, and the Fibulin-1 protein can be used as a molecular marker of vascular aging or a potential therapeutic target for delaying vascular aging.

Description

Fibulin-1 protein as vascular aging molecular marker and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a Fibulin-1 protein serving as a vascular aging molecular marker and application thereof.

Background

Currently, it is estimated that 1700 million people die annually from cardiovascular disease, accounting for one-third of all deaths worldwide. Vascular aging is a variety of cardiovascular diseases such as: atherosclerosis, vascular calcification, and hypertension, among other major risk factors. Vascular aging is also an important pathophysiological basis for causing aging of various organ systems of the human body. Research shows that the indicators of the blood vessel functions such as the vasodilation function and the stiffness gradually decline with the age. 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, and structurally, the vascular aging is accompanied with extracellular matrix remodeling to cause the change of the components of elastin and collagen in large elastic arteries. Elastin cleavage and degradation occur in the middle intima of arteries by upregulation 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 arterial stiffness, decreased vascular compliance, and decreased ability to repair and regenerate blood vessels. 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 the aspects of risk factors, vascular functions, vascular structures and the like. Common indicators for vascular aging function testing include blood flow-mediated vasodilation (FMD), nitroglycerin-induced vasodilation (NID), brachial malleolus pulse wave velocity (baPWV), brachial malleolus index (ABI), and the like. Structural assessment of vascular aging can be performed by Magnetic Resonance Imaging (MRI), intima-media thickness (IMT). However, serological biological markers for vascular aging are currently lacking. The change of the serological biomarker is usually prior to the change of the structure and the function, and substances in serum can reflect the whole aging and pathological change level of an organism and the change degree of the vascular structure of the organism, thereby being beneficial to early diagnosis and prevention of diseases. Therefore, the search of a novel serological diagnosis marker for vascular aging is helpful for enriching an assessment system for vascular aging and plays an important role in preventing, diagnosing and delaying vascular aging and cardiovascular diseases related to vascular aging.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide the application of the reagent for detecting the Fibulin-1 gene or the Fibulin-1 protein in preparing a product for diagnosing vascular aging.

The second object of the present invention is to provide a kit for diagnosing vascular aging.

The third purpose of the invention is to use the reagent for reducing the expression of the Fibulin-1 gene or the Fibulin-1 protein in preparing the medicine for delaying vascular aging.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

application of a reagent for detecting a Fibulin-1 gene or a Fibulin-1 protein in preparation of a product for diagnosing vascular aging.

Further, the expression level of the Fibulin-1 gene or the Fibulin-1 protein is positively correlated with the degree of vascular aging.

Further, the expression level of the Fibulin-1 gene or the Fibulin-1 protein is the expression level of the Fibulin-1 gene or the Fibulin-1 protein in human blood.

Further, the product can judge the vascular aging degree of the subject by detecting the expression level of the Fibulin-1 gene or the Fibulin-1 protein in the subject.

Further, the detection method of the Fibulin-1 protein comprises a western blotting method, an enzyme-linked immunosorbent assay, a radioimmunoassay, a radioimmunodiffusion method, an Octocroly immunodiffusion method, a rocket immunoelectrophoresis method, an immunohistochemical staining method, an immunoprecipitation analysis method, a complement fixation analysis method, a flow cell fluorescence edge separation technology or a protein chip.

Further, the detection method of the Fibulin-1 gene comprises polymerase chain reaction, micro-digital polymerase chain reaction, fluorescent polymerase chain reaction, loop-mediated isothermal amplification reaction, nucleotide or amino acid sequence sequencing, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, high performance liquid chromatography, in situ hybridization, biological mass spectrometry, high resolution dissolution curve analysis, single-strand conformational isomerism polymorphic analysis or probe amplification retardation mutation system analysis.

A kit for diagnosing vascular aging, comprising a reagent for detecting the Fibulin-1 gene or the Fibulin-1 protein.

Further, the reagent comprises a primer for detecting the Fibulin-1 gene or an antibody for detecting the Fibulin-1 protein.

Use of an agent which reduces the expression of the Fibulin-1 gene or the Fibulin-1 protein in the preparation of a medicament for delaying vascular aging.

Further, the agent for reducing the expression of the Fibulin-1 gene or the Fibulin-1 protein includes any one of the following:

(a) drugs which are combined with the Fibulin-1 gene or the Fibulin-1 protein in a targeted manner, including small molecule drugs or antibody drugs;

(b) a nucleic acid molecule that interferes with or silences Fibulin-1, comprising an siRNA, shRNA, or sgRNA capable of targeting a full-length or truncated sequence of the Fibulin-1 gene.

Compared with the prior art, the invention has the beneficial effects that:

the inventors found that the expression level of Fibulin-1 protein was positively correlated with the degree of vascular aging during the course of a study of vascular aging, which was surprising because Fibulin-1 protein known to be associated with diabetes or atherosclerosis is not predicted to be associated with vascular aging. The experimental results show that: fibulin-1 is up-regulated in plasma of vascular aging population and is positively correlated with the vascular aging indicator PWV. In a model of a naturally aging mouse, Fibulin-1 is up-regulated in the plasma and vascular tissues of aging mice. Therefore, the Fibulin-1 gene or the Fibulin-1 protein can be used as a blood vessel aging serum marker, can predict the occurrence of blood vessel aging and related diseases, diagnoses the blood vessel aging, stratifies dangers, delays the blood vessel aging, and prevents and treats the diseases related to the blood vessel aging.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1A is a plasma proteomic thermogram of the angiosclerosis population and the normal vascular stiffness population of example 1;

FIG. 1B is a graph showing the plasma proteomics results of the human with arteriosclerosis and the human with normal vascular hardness in example 1;

FIG. 2 is a standard curve of the concentration of Fibulin-1 protein in example 2;

FIG. 3A is a graph comparing the expression of Fibulin-1 in plasma of the normal population and the arteriosclerotic population in example 2;

FIG. 3B is a graph showing the results of analysis of the correlation between the Fibulin-1 protein and the vascular aging indicator PWV in example 2;

FIG. 4A is a graph showing the result of Western blotting of Fibulin-1 plasma from the spontaneously aging mouse in example 3;

FIG. 4B is a western blot analysis result of Fibulin-1 on the vascular wall of the naturally aging mouse in example 3;

FIG. 4C is a graph showing the detection results of Fibulin-1 protein by immunohistochemical staining of the naturally aging mouse in example 3.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The inventor unexpectedly finds that the expression level of the Fibulin-1 protein is positively correlated with the vascular aging degree in the research process of vascular aging, in particular that the expression level of the Fibulin-1 protein is obviously increased in the plasma of people with vascular sclerosis compared with the people with normal vascular hardness, and the Fibulin-1 protein in a natural aging mouse model shows an obvious high expression level trend.

Based on this, the present invention provides the following scheme:

In the present invention, "vascular aging" means aging of blood vessels in a healthy state with age, which is different from pathological aging. For example, one of the manifestations of vascular aging is vascular sclerosis, characterized by intima-media walls, freedom from atheromatous plaque formation and luminal dilatation; while lesion-aged atherosclerosis is characterized by intimal thickening, intimal atheromatous plaque formation and luminal narrowing.

The Fibulin-1 Gene maps to human chromosome 22, has a full length of 98253bp, encodes the Fibulin-1 protein, and has the following Gene IDs queried at the NCBI: gene ID: 2192.

In some embodiments, the amount of Fibulin-1 gene or Fibulin-1 protein expressed is directly correlated with the degree of vascular senescence. Wherein the expression level of the Fibulin-1 gene or the Fibulin-1 protein is the expression level of the Fibulin-1 gene or the Fibulin-1 protein in human blood, and is preferably human plasma.

Blood in the circulatory system is the most easily collected and informative sample in human tissues and is very important for identifying molecular markers. The separated serum contains metabolic small molecules and proteins which are carriers secreted and communicated by various organs of a human body. The subtle changes in the vital activities, which act as actors of the biological function, are directly reflected by proteins. Therefore, the development of molecular markers of vascular aging in blood is of great significance.

It is understood that since the expression level of the Fibulin-1 gene or the Fibulin-1 protein is positively correlated with the degree of vascular aging, the Fibulin-1 protein can also be used to evaluate risk stratification.

In some embodiments, the product determines the degree of vascular aging in a subject by detecting the amount of Fibulin-1 gene or Fibulin-1 protein expressed in the subject.

In some embodiments, the method for detecting the concentration of the vascular aging molecular marker of Fibulin-1 protein comprises western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, oxter immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis, flow cytofluorimetry, or a protein chip, preferably an enzyme-linked immunosorbent assay.

In some embodiments, the method of detecting the Fibulin-1 gene comprises polymerase chain reaction, micro-digital polymerase chain reaction, fluorescent polymerase chain reaction, loop-mediated isothermal amplification reaction, nucleotide or amino acid sequence sequencing, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, high performance liquid chromatography, in situ hybridization, biological mass spectrometry, high resolution melting curve analysis, single-stranded conformational isomeric polymorphism analysis, or probe amplification-retarded mutation system analysis.

The invention also provides a kit for diagnosing vascular aging, which comprises a reagent for detecting the Fibulin-1 gene or the Fibulin-1 protein. Preferably, the reagent comprises a primer for detecting the Fibulin-1 gene or an antibody for detecting the Fibulin-1 protein.

Finally provides the application of the reagent for reducing the expression of the Fibulin-1 gene or the Fibulin-1 protein in preparing the medicine for delaying vascular aging.

In a preferred embodiment, the agent that reduces the expression of the Fibulin-1 gene or Fibulin-1 protein comprises any one of the following:

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1 proteomics analysis of blood samples

(I) detection method

1. Clinical sample collection

The whole collection and subsequent test process meets the medical ethical moral requirements and strictly follows the case data confidentiality principle, and the sampling, sub-packaging and storage conditions of the research samples are consistent. The physical examination population data is collated, PWV is taken as the standard for evaluating the blood vessel hardness, the blood vessel hardness is divided into a standard group and a hardening group, the sex and the age are matched, and plasma samples of 6 cases of PWV low/non-arteriosclerosis and 6 cases of PWV high/arteriosclerosis patients are selected.

Pulse Wave Velocity (PWV) is the most commonly used method for measuring stiffness of large elastic arteries, and it is easy to use and highly repeatable. PWV not only reflects the degree of arterial stiffness, but also is an independent predictor of vascular aging-related cardiovascular disease, and is an important index for evaluating vascular aging. Arteriosclerosis and PWV are increased in people with vascular aging.

2. Quantitative analysis of plasma proteome iTRAQ

(1) Protein extraction and sample processing: taking a proper amount of each sample to carry out an extraction experiment; adding protein lysate (7M Urea/2M Thiourea/4% SDS/40mM Tris-HCl, pH8.5/1mM PMSF/2mM EDTA) into the sample, uniformly mixing, incubating on ice for 5min, adding DTT with the final concentration of l 0mM, carrying out ice bath ultrasonic treatment for 15min, then carrying out 13000g centrifugation at 4 ℃ for 20min, and taking the supernatant and transferring into a new centrifuge tube; the tubes were charged with 4 volumes of cold acetone and allowed to stand overnight at-20 ℃. And centrifuging to collect protein precipitate, and airing in the air. The protein was redissolved by adding 8M urea/100mM TEAB (pH 8.0), DTT was added to a final concentration of 10mM, and the reduction was carried out in a 56 ℃ water bath for 30 min. Subsequently, IAM was added to a final concentration of 55mM, and the mixture was left at room temperature in the dark for 30min for alkylation. Protein concentration was determined by the Bradford method.

(2) Enzymolysis and desalting: equal amounts of protein from each sample were used for trypsin digestion. 100 μ g of protein was used for trypsin digestion. After the protein solution was diluted 5-fold with 100mM TEAB, trypsin was added in a mass ratio of 1:50 (pancreatin: protein), and the mixture was subjected to enzymatic hydrolysis at 37 ℃ overnight. Desalting the peptide segment after enzymolysis by using a C18 column, and freeze-drying the desalted peptide segment in vacuum.

(3) iTRAQ label and packet division: by 05M TEAB-solubilized polypeptides, labeled according to the instruction of the iTRAQ-8 Standard kit (SCIEX), samples were labeled and mixed, and the mixed peptides were fractionated using an Ultimate 3000HPLC system (Thermo DINOEX, USA). The column used was a Durashell C18 column (5 μm,

4.6X 250 mm). The separation of peptide fragments was achieved by using increasing ACN concentration under alkaline conditions at a flow rate of 1ml/min and one tube was collected per minute. A total of 42 secondary fractions were collected and combined into 15 fractions, and the combined fractions were desalted on a Strata-X column and vacuum dried.

(4) Liquid chromatography-mass spectrometry analysis: mass spectrometry data was collected using TripleTOF 5600+ liquid chromatography-mass spectrometry System (SCIEX). Polypeptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analyzed using a TripleTOF 5600plus mass spectrometer coupled to an ekstrigent nanoLC system (SCIEX, USA). The polypeptide solution was applied to a C18 capture column (5 μm, 100 μm.times.20 mm) and eluted in a 90min time gradient at a flow rate of 300 nL/min on a C18 analytical column (3 μm, 75 μm.times.150 mm). The two mobile phases were buffer A (2% acetonitrile/0.1% formic acid/98% H2O) and buffer B (98% acetonitrile/0.1% formic acid/2% H)₂O). For IDA (information dependent acquisition), a scan of the primary mass spectrum was taken at an ion accumulation time of 250ms, and secondary mass spectra of 30 precursor ions were acquired at an ion accumulation time of 50 ms. MS1 spectra were collected in the range of 350-1500 m/z and MS2 spectra were collected in the range of 100-1500 m/z. The precursor ion dynamic exclusion time was set to 15 s.

(5) Protein identification, wherein the basic process of proteome identification based on a mass spectrum method is adopted in the experiment, namely, MS/MS mass spectrum data is subjected to serial optimization treatment and then is subjected to similarity comparison scoring with a database so as to carry out protein identification, and the method is a high-throughput protein identification method which is most widely applied at present and is accepted in the industry. The method has the advantages of high identification accuracy, high flux, no need of manual sequence analysis and the like. We used the search engine ProteinpilotTM V4.5, which was matched to AB Sciex 5600 plus. The Proteinpilot considers all possible modification types during searching, and meanwhile, the automatic fault-tolerant matching function is added, so that more results can be retrieved than similar software on the premise of ensuring the reliability of the identification result.

(6) And (3) protein quantification, namely the iTRAQ labeling method can realize the simultaneous relative quantification of a plurality of samples, and the quantification has higher accuracy. We used the Proteinpilot software to achieve proteomic iTRAQ quantification. Aiming at the experimental design containing biological repetition or technical repetition, firstly, the mean value of the ratio of pairwise comparison between repeated samples is used as the difference multiple of the samples to be compared, and secondly, the P _ value of the single sample Students t test of pairwise comparison between the repeated samples is used as the significance difference test P value of the samples to be compared. And finally, screening out differential proteins according to the difference multiples and the P _ value. When the difference multiple reaches 1.5 times or more (i.e. up _ rule is more than or equal to 1.5 and down _ rule is less than or equal to 0.67), and the Pvalue value is less than or equal to 0.05 through the significance statistical test, the protein is regarded as the significant difference protein.

(II) the result of the detection

Plasma proteomics (fig. 1A, fig. 1B) showed that the expression level of Fibulin-1 in the plasma of patients with vascular sclerosis was up-regulated by 3.17-fold compared to the vascular stiffness normal group.

EXAMPLE 2 protein expression level of Fibulin-1 in blood sample

(I) detection method

1. Clinical sample collection

The whole collection and subsequent test process meets the medical ethical moral requirements and strictly follows the case data confidentiality principle, and the sampling, sub-packaging and storage conditions of the research samples are consistent. By organizing the data of physical examination population, dividing the physical examination population into a standard group and a hardening group by taking PWV as a standard for evaluating the hardness of blood vessels, matching the sex and the age, and selecting plasma samples of 118 patients with PWV low/non-arteriosclerosis and 72 patients with PWV high/arteriosclerosis.

2. Detection of Fibulin-1 protein content in blood

(1) And (3) standard product configuration: the 600ng/mL Fibulin-1 protein standard mother liquor was diluted by the fold-ratio dilution method to prepare standard proteins with concentrations of 60ng/mL, 30ng/mL, 15ng/mL, 7.5ng/mL, 3.75ng/mL, 1.88ng/mL, 0.94ng/mL and 0ng/mL, and the specific information is shown in Table 1 below.

TABLE 1

(2) Diluting a blood sample: plasma was collected using a heparin anticoagulant tube. Within 30 minutes after collection, centrifuge at 1000 Xg for 15 minutes at 4 ℃. Samples were immediately tested or stored in aliquots at-70 ℃. The samples were subjected to 1: 5000 dilution;

(3) preparing a cleaning solution: 100ml of 10 XWash was added to 900ml of ddH₂O, uniformly mixing, and preparing a cleaning working solution; long-term storage at 4 ℃ or-20 ℃;

(4) a detection step:

the appropriate number of pre-coated 8-tubes was placed in an 8-tube holding plate and the remaining 8-tubes were returned to 4 ℃ for storage. Diluting the sample with a sample diluent;

adding 100 μ L/well of 60ng/mL, 30ng/mL, 15ng/mL, 7.5ng/mL, 3.75ng/mL, 1.88ng/mL and 0.94ng/mL of human Fibulin-1 standard protein to the pre-coated 96-well plate; 100ul of sample dilution buffer was added to the control wells. 100ul of the appropriately diluted plasma sample was added per empty well. The standard and the sample are provided with a plurality of holes.

Seal the 96-well plate with the adhesive lid provided and incubate for 1h with shaking on a horizontal shaker at 300rpm at room temperature (25 ℃); the lid is removed, the liquid in the tray is discarded, and the tray is sucked onto a paper towel or other absorbent material. But at no time is the liquid allowed to dry completely. Add 350. mu.l of washing solution to each well and wash 4 times.

Adding 100 mu L of biotinylated anti-Fibulin-1 antibody working solution into each well, covering a new adhesive cover, and shaking and incubating for 1h at room temperature (25 ℃) by a horizontal shaking table at 300 rpm; the lid is removed, the liquid in the tray is discarded, and the tray is sucked onto a paper towel or other absorbent material. But at no time is the liquid allowed to dry completely. 350ul of cleaning solution was added to each well and cleaned 4 times.

Adding 100 μ L of luminescent substrate solution into each well, covering with new adhesive, keeping out of the sun, and incubating for 10-20min at room temperature (25 deg.C) with shaking at 300rpm of horizontal shaker; the incubation time can be extended to 30min if the room temperature is lower than 20 ℃; adding 100 mul of reaction stop solution into each hole; within 30 minutes after the addition of the reaction termination solution, the absorbance od was read at 450/540nm with a microplate reader.

The standard curve may be expressed as the relative od value (450) of each standard solution to the respective concentration (X) of the standard solution, from which the Fibulin-1 concentration of the sample may be calculated;

relative o.d.450/540 ═ test well o.d.450/540 — control well o.d.450/540;

if the concentration of the diluted sample is measured, the actual concentration of the sample is equal to the concentration of the diluted sample multiplied by the dilution factor;

a typical standard curve is shown in figure 2.

(II) detection results:

statistical analysis of the results was performed using SPSS20.0 statistical software, and it was found that the levels of Fibulin-1 protein differentially expressed in plasma of normal blood vessel stiffness group and hardened group were detected using elisa, and the results showed that Fibulin-1 was positively correlated with PWV in all groups of arteriosclerosis and control group (fig. 3A), where r is 0.2 and P is < 0.05. Whereas in the arteriosclerotic population Fibulin-1 was more strongly correlated with PWV (fig. 3B), r is 0.638 and P < 0.05.

Example 3

The detection method comprises the following steps:

1. immunohistochemical staining

Paraffin sections of mouse vascular tissues were dewaxed and hydrated for antigen retrieval using the microwave method. PBS was washed 3 times for 5 minutes each. Then incubated for another 10 minutes at 37 ℃ with 3% hydrogen peroxide solution. PBS was washed 3 times, blocked with 10% goat serum for 10 minutes, then serum was aspirated, diluted primary antibody working solution was added in appropriate ratio, and incubated overnight at 4 ℃. PBS wash 3 times, 1: the secondary biotin-labeled antibody was diluted at 200 ℃ and incubated at 37 ℃ for 30 minutes. Washing with PBS for 3 times, adding appropriate amount of horseradish peroxidase-labeled streptavidin working solution, and incubating at room temperature. After incubation for 30 minutes, PBS was washed 3 times, and DAB color reagent was added to develop for 15 minutes. Repeatedly washing with tap water, washing off staining agent, and re-staining with hematoxylin staining solution for 2 min. And sealing and storing after washing. White light photographing is carried out on the mouse blood vessel immunohistochemical section by using a common upright optical microscope.

2、Westernblot

(1) Protein extraction and concentration determination

Adding a proper amount of lysate into the separated mouse aorta, grinding the separated mouse aorta on a tissue grinder for 120 Sx 2 times/cell protein extraction, washing the collected cell sediment for 1 time by using precooled PBS (PBS), and then adding a proper amount of lysate (the ratio of the lysate to the PMSF to the protease inhibitor to the phosphatase inhibitor is 100: 1: 1: 1). The sample to be tested was lysed on ice for 30 minutes, placed in a 4 ℃ centrifuge at 12000rpm, and centrifuged for 20 minutes. A standard curve was prepared, and a standard protein (1mg/mL) and ultrapure water were added to the microplate in the order of Table 2 below. The sample to be detected is added into the ELISA plate after being uniformly mixed according to 18 mu L of ultrapure water and 2 mu L of protein to be detected in each hole. BCA working solution is prepared according to the following solution A: solution B is 50: 1, adding 200 mu L of BCA working solution into each hole of the sample group to be detected and the standard group, incubating for 30 minutes at 37 ℃, and measuring the absorbance of each hole by an enzyme-labeling instrument. And calculating the concentration of the sample to be detected according to the standard curve. The protein concentration of each set of samples was adjusted to be the same with ultrapure water, and 5X protein loading buffer was added to the samples at a ratio of 1:4 to the sample volume. Oscillating and mixing. The protein was denatured by heating in water at 100 ℃ for 5 minutes. (plasma preparation of protein samples, directly taking a proper amount of plasma, adding 5 Xprotein loading buffer solution to the plasma at a ratio of 1:4 to the volume of the sample, shaking, mixing, heating in 100 ℃ water for 5 minutes to denature the protein.)

TABLE 2

Numbering	0	1	2	3	4	5	6	7
									H₂O(μl)	20	18	16	14	12	10	8	6
Protein Standard solution (ul)	0	2	4	6	8	10	12	14
									Protein concentration (μ g/. mu.l)	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7

(2) SDS-PAGE gel electrophoresis

1.1 the glass plate for glue preparation is cleaned by detergent and then is put into an oven for drying. And aligning the bottom ends of the thin and thick glass plates, and manufacturing the glue making groove after assembling.

1.2 the separation gel and the concentration gel were prepared as shown in Table 3 below. And adding the prepared separation gel into a gel preparation groove, slowly adding the separation gel to the horizontal line at the upper end of a gel preparation frame, then slowly adding distilled water, flattening the separation gel and removing residual gas. After 30 minutes, when the separation gel solidified, a distinct boundary was seen between the separation gel and the distilled water. The upper layer of distilled water was aspirated, and the remaining liquid was blotted with absorbent paper. Filling the gel preparation tank with the concentrated gel, inserting a comb, paying attention to no bubbles in the process, standing for 30 minutes, and waiting for the concentrated gel to solidify.

TABLE 3

Reagent	Separating glue (10%)	Concentrated gum (5%)
			Ultrapure water	2.7mL	2.7mL
Tris·HCl(PH8.8)	3.3mL	0.67mL
			30％Acr-Bis(29:1)	3.8mL	0.5mL
10% SDS solution	100μL	80μL
			10% ammonium persulfate solution	100μL	80μL
TEMED	4μL	4μL
			Total volume	10mL	4mL

1.3 electrophoresis. The electrophoretic fluid was prepared according to the following Table 4. Cleaning the electrophoresis tank, assembling the electrophoresis tank with SDS (sodium dodecyl sulfate) glue prepared in advance into the electrophoresis tank, slowly pulling out a comb in the concentrated glue, adding electrophoresis liquid into the electrophoresis tank, and checking whether the liquid leaks. And (3) sequentially adding the samples into small holes of the concentrated gel according to the grouping sequence of the protein samples, adding a proper amount of protein Marker into holes on two sides (10-20 mu L/hole), and recording the sequence of the samples. And (4) correctly installing the electrophoresis tank according to the electrode direction and connecting the electrophoresis apparatus. Adjusting the initial voltage to 60V, performing constant voltage 60V electrophoresis until the protein sample enters the separation gel and the protein Marker presents a plurality of clearly separated strips, adjusting the voltage to 120V, performing constant voltage 120V electrophoresis until the protein sample runs to the lower edge of the separation gel, confirming that the target molecular protein is fully separated according to the protein Marker, and stopping the electrophoresis.

TABLE 4

Reagent	H₂O	Trise-baze	Glycine	SDS
					Electrophoretic fluid	1000mL	3.02g	18.8g	1.0g

And 1.4, rotating the film. The spin-on solution was prepared according to table 5 below. Preparing the film transfer liquid in advance, and pre-cooling in a refrigerator at 4 ℃. The PVDF membrane and the filter paper are cut into a proper size. And opening the film rotating clamp, sequentially placing the filter paper, the adhesive tape and the PVDF film on the film rotating clamp, paying attention to the fact that the PVDF film is tightly attached to the adhesive tape, and avoiding bubbles in the middle. The positive and negative electrodes are connected with a membrane converter, 230mA and the membrane is converted for 100 minutes under constant current.

TABLE 5

Reagent	H₂O	Trise-baze	Glycine	Methanol
					Electrophoretic fluid	800mL	3.03g	15.1g	200ml

1.5 blocking, antibody incubation. After the membrane transfer is finished, the PVDF membrane is taken out and placed in 5 percent skim milk to be sealed for 1 hour at room temperature. Note that the PVDF membrane should be placed protein side up. After 1 hour blocking was complete, the PVDF membrane was removed and washed 3 times with TBST solution for 5 minutes each. Add primary antibody dilution and incubate overnight at 4 ℃. The primary anti-diluent was recovered and washed 3 times with TBST solution for 5 minutes each. After washing, appropriate secondary antibody dilutions were added and incubated for 1 hour at room temperature. The TBST solution was washed 3 times for 5 minutes each.

1.6 exposure. ECL developer according to solution A: solution B is 1: 1, mixing evenly and using in dark place. And after uniformly incubating the ECL developing solution on the exposed strip, placing the strip on a plastic film, placing the strip in an exposure machine, adjusting the exposure time according to the condition of the strip, scanning and exposing, recording an image, and marking and analyzing the image.

(II) detection results:

a natural aging mouse model is established, the expression of Fibulin-1 protein in the plasma of a 24M aging mouse is found to be higher than that of a 3M young mouse by utilizing western blot detection, and the experimental result is shown in FIG. 4A. Immunohistochemical staining (FIG. 4C) and Western blot (FIG. 4B) detection revealed that Fibulin-1 protein expression was higher in the vascular wall of 24M-aged mice than in 3M-aged mice.

In conclusion, the invention discovers that the occurrence of vascular aging can be predicted, or vascular aging can be diagnosed, risk stratification and the like can be carried out by detecting the concentration of the Fibulin-1 protein in a tissue sample of a patient.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. Application of a reagent for detecting a Fibulin-1 gene or a Fibulin-1 protein in preparation of a product for diagnosing vascular aging.

2. The use according to claim 1, wherein the expression level of Fibulin-1 gene or Fibulin-1 protein is positively correlated with the degree of vascular senescence.

3. The use according to claim 2, wherein the expression level of the Fibulin-1 gene or the Fibulin-1 protein is the expression level of the Fibulin-1 gene or the Fibulin-1 protein in human blood.

4. The use according to any one of claims 1 to 3, wherein the product is used for determining the degree of vascular senescence in a subject by detecting the amount of Fibulin-1 gene or Fibulin-1 protein expressed in the subject.

5. The use of claim 4, wherein the detection method of Fibulin-1 protein comprises Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, Octocloney immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis, flow cytofluorimetry, or protein chips.

6. The use according to claim 4, wherein the method for detecting the Fibulin-1 gene comprises polymerase chain reaction, micro-digital polymerase chain reaction, fluorescent polymerase chain reaction, loop-mediated isothermal amplification reaction, nucleotide or amino acid sequence sequencing, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, high performance liquid chromatography, in situ hybridization, mass spectrometry, high resolution melting curve analysis, single-stranded conformational isomerism polymorphic analysis, or probe amplification-retarded mutation system analysis.

7. A kit for diagnosing vascular aging, which comprises a reagent for detecting the Fibulin-1 gene or the Fibulin-1 protein.

8. The kit according to claim 7, wherein the reagent comprises a primer for detecting the Fibulin-1 gene or an antibody for detecting the Fibulin-1 protein.

9. Use of an agent which reduces the expression of the Fibulin-1 gene or the Fibulin-1 protein in the preparation of a medicament for delaying vascular aging.

10. Use according to claim 9, wherein the agent which reduces the expression of the Fibulin-1 gene or Fibulin-1 protein comprises any of: