CN111458513A

CN111458513A - Atherosclerosis biomarker and application thereof

Info

Publication number: CN111458513A
Application number: CN201910053770.5A
Authority: CN
Inventors: 曾嵘; 高笑静; 李辰
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2019-01-21
Filing date: 2019-01-21
Publication date: 2020-07-28

Abstract

The invention provides a substance for detecting S L C25A5 and application thereof in preparing a kit for diagnosing atherosclerosis, wherein the AUC value of a ROC curve of the atherosclerosis biomarker S L C25A5 is 1 and is higher than the ACU values of known I L K and DCN, and the condition of atherosclerosis disease can be better characterized by the biomarker compared with the known I L K, DCN.

Description

Atherosclerosis biomarker and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an atherosclerosis biomarker and application thereof.

Background

Atherosclerosis (Atherosclerosis) is a chronic inflammatory disease with high morbidity and mortality in cardiovascular disease in western countries. The development of atherosclerosis is often accompanied by a number of circulatory imbalances, including coronary artery disease and cerebrovascular disease, among others (Falk, 2006). In the case of the heart, atherosclerosis can cause coronary artery stenosis, thereby causing myocardial infarction and heart failure. In the case of the brain, stenosis and rupture of the hardened plaque may lead to transient cerebral ischemia, ischemic stroke, hemorrhagic stroke, and the like. When plaque stenosis affects the renal artery branches, it causes renal damage and general hypertension, while when in other arterial branches of the extremities, atherosclerosis causes peripheral arterial occlusion and severe limb ischemia (Wu et al, 2017).

The hallmark of atherosclerosis is that lipid-containing macrophages accumulate under the endothelium of the arterial wall and promote inflammatory reactions in the arterial wall, leading to a variety of pathogenic consequences such as bleeding, rupture and calcification (L u and Daugherty,2015) hardened plaques can be stable for years, but can also rapidly become unstable, rupture and initiate thrombosis.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a biomarker and its use, which solve the problems of the prior art.

To achieve the above and other related objects, the present invention provides, in one aspect, use of a substance for detecting S L C25a5 in preparing a kit for diagnosing atherosclerosis.

In some embodiments of the invention, the use of a combination of a substance for detecting S L C25a5 and a substance for detecting TPM2 in the preparation of a kit for diagnosing atherosclerosis.

In some embodiments of the invention, the use of a combination of a substance for detecting S L C25a5 and a substance for detecting DAG1 in the preparation of a kit for diagnosing atherosclerosis.

In some embodiments of the invention, the combination of a substance for detecting S L C25a5, a substance for detecting TPM2, and a substance for detecting DAG1 is used in the preparation of a kit for diagnosing atherosclerosis.

In some embodiments of the present invention, the substance for detecting S L C25a5 is a reagent for detecting the expression level of S L C25a 5.

In some embodiments of the invention, the substance for detecting TPM2 is an agent for detecting the expression level of TPM 2.

In some embodiments of the invention, the substance for detecting DAG1 is a reagent for detecting the expression level of DAG 1.

In some embodiments of the present invention, the kit diagnoses whether the subject has atherosclerosis and/or characterizes the progression of atherosclerosis based on the expression level of S L C25a 5.

In some embodiments of the invention, the kit is expressed according to the expression level of S L C25a5 and/or TPM2 and/or DAG 1.

In some embodiments of the invention, the kit is directed to a target organ tissue of an atherosclerotic disease in a subject.

In some embodiments of the invention, the kit is directed to a combination of one or more of aortic tissue, liver tissue, adipose tissue, and blood tissue in a subject.

In some embodiments of the invention, the kit is used for diagnosing early atherosclerosis.

In another aspect, the present invention provides a test kit comprising a substance for detecting S L C25A 5.

In some embodiments of the invention, a substance for detecting the TPM2 is also included.

In some embodiments of the invention, a substance for detecting DAG1 is also included.

In some embodiments of the invention, a combination of a substance for testing S L C25A5 and a substance for testing TPM2 is included.

In some embodiments of the invention, a combination of a substance for detecting S L C25a5 and a substance for detecting DAG1 is included.

In some embodiments of the invention, a combination of a substance for testing S L C25a5, a substance for testing TPM2, and a substance for testing DAG1 is included.

In another aspect, the invention provides a biomarker comprising S L C25a5, optionally further comprising Tpm2, and optionally further comprising DAG 1.

In some embodiments of the invention, the biomarker is selected from the group consisting of S L C25a5 and TPM2 in combination.

In some embodiments of the invention, the biomarker is selected from the group consisting of S L C25a5 and DAG1 in combination.

In some embodiments of the invention, the biomarker is selected from the group consisting of S L C25a5, TPM2, and DAG1 in combination.

In a further aspect the invention provides the use of the biomarker in the manufacture of a kit for the diagnosis of atherosclerosis.

Drawings

FIG. 1 shows total cholesterol (TC, FIG. 1A) and triglyceride levels (TG, FIG. 1B) in plasma of animals (CD 2: 2 week normal diet, CD 6: 6 week normal diet, HFD _ R2: 2 week high fat high cholesterol diet, HFD _ WR 6: 6 week high fat high cholesterol diet resistant group, HFD _ R6: 6 week high fat high cholesterol diet sensitive group).

FIG. 2 shows the flow of proteomics analysis of aortic tissues (CD 2: 2 weeks normal diet, CD 6: 6 weeks normal diet, HFD _ R2: 2 weeks high-fat high-cholesterol diet, HFD _ WR 6: 6 weeks high-fat high-cholesterol diet resistant group, HFD _ R6: 6 weeks high-fat high-cholesterol diet sensitive group).

FIG. 3 shows the global changes in rabbit aortic protein (A) rabbit aortic global proteome PCA profile (1132 proteins) under a high-fat high-cholesterol diet. (B) Analysis of variance and TukeyHSD test pick 162 differential protein heatmaps. (C)162 differential proteins showed 6 different expression profiles. (D) Up-and down-regulation of differential protein biological process GO enrichment. Blue indicates an enrichment of p less than 0.05 and red indicates a p less than 0.05 after BH correction. CD 2: 2-week normal diet, CD 6: normal diet for 6 weeks, HFD _ R2: 2-week high-fat high-cholesterol diet, HFD _ WR 6: 6-week high-fat high-cholesterol diet resistant group, HFD _ R6: and 6 weeks of a high-fat high-cholesterol diet sensitive group.

FIG. 4 shows the expression profile of atherosclerosis biomarkers (A: Tpm2, C: S L C25A5, E: DAG1) and ROC curves (B: Tpm2, D: S L C25A5, F: DAG1), CD 2: 2 week regular diet, CD 6: 6 week regular diet, HFD _ R2: 2 week high-fat high-cholesterol diet, HFD _ WR 6: 6 week high-fat high-cholesterol diet resistant group, HFD _ R6: 6 week high-fat high-cholesterol diet sensitive group.

Detailed Description

The inventor of the invention has conducted extensive research, and has found 162 differential proteins through quantitative proteomics analysis of rabbit aorta under a high-fat diet sensitive group, a high-fat diet resistant group and a normal diet group by feeding new zealand white rabbits with a high-fat diet, wherein S L C25a5 has a significant difference between an atherosclerosis disease group (HFD _ R6) and a normal group (CD6), and can be used as a biomarker of disease progression, thereby completing the invention.

The invention provides the use of a substance for detecting S25A in the preparation of a kit for diagnosing atherosclerosis, Solute carrier family 25member 5, S C25A, Ensembl number ENSOCUP 0011416, the product of this gene, adenine nucleotide transporter 2 (ANT), which is a major component of mitochondrial permeability transition pore complex, catalyzes the exchange of mitochondrial ATP with cytosol ADP, which is a major component of the dietary protein kinase in the study of lipid profile of endothelial cell line 1, which is a functional regulator of endothelial kinase in vivo, which is a functional regulator of endothelial kinase in rat atherosclerosis, which is a functional regulator of endothelial kinase in rat, which is a high-cholesterol kinase, which is a functional regulator of endothelial kinase in rat-cholesterol kinase, which is a high-protein kinase, which is a functional regulator of endothelial kinase in rat-cholesterol kinase, which is a high-protein kinase, which is a high-cholesterol kinase, which is a protein involved in the study of endothelial kinase in endothelial cell line 2, which is a high-cholesterol kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-involved in endothelial protein kinase, which is a high-involved in endothelial protein kinase, which is a high-protein kinase, which is a high-involved in endothelial protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high in endothelial protein kinase, which is a high-protein kinase, which is a high in a high-protein kinase, which is a high in a high-protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-protein kinase, which is a high-protein.

In the use provided by the first aspect of the invention, the substance for detecting S L C25A5 is optionally combined with a substance for detecting Tpm2 Tpm2, β -tropomyosin isoform 2, belonging to the Tropimomycin (TM) gene family, EMB L accession number AIC82815.1, a coiled-coil dimeric protein with important functions in striated, smooth and non-muscle cells in vertebrate striated muscle, actin, TM, troponin (Tn) complexes (Tn-I, Tn-C, Tn-T) and tropimodulin (Tmod) together constitute a filament responsible for mediating Ca Tmod²⁺Controlled muscle contraction and relaxation (Dube et al, 2014). Under normal conditions, the main function of vascular smooth muscle cells is to maintain vascular homeostasis by active contraction and relaxation, while in atherosclerosis, this function is inhibited by the surrounding environment (e.g., lipid retention, inflammatory factors, etc.) (Steucke et al., 2015). In addition, early lesions of atherosclerosis cause endothelial cell damage, leading to migration, proliferation and phenotypic modulation of medial smooth muscle cells, but the relationship of vascular smooth muscle contraction to disease is not known in this process, and the role of Tpm2 in it is unknown. In the data disclosed in the embodiment of the invention, the protein level of the Tpm2 is reduced after 2 weeks of induction of high-fat and high-cholesterol diet, the sensitive group at 6 weeks is further reduced, the resistant group at 6 weeks is closer to the control group, and meanwhile, the AUC value of the Tpm2 protein ROC curve is 1, so that the atherosclerosis disease and the normal state can be well distinguished, and the Tpm2 can be used as an effective biomarker.

In the uses provided by the first aspect of the invention, the substances used to detect S L C25a5 optionally include combinations of substances used to detect DAG1 Dystroglycan (DG or DAG1) is considered as a key link between basement membrane and cytoskeleton in various tissues, widely distributed among endothelial cells and epithelial cells, EMB L No. caa 45732.1. dag1 consists of two subunits, an extracellular α -subunit bound to laminin and other basement membrane components and a transmembrane β -subunit, and functions as a cell adhesion receptor (Jarad et al, 2011) Dystrophin-glycoprotein (C) complex is a multicomponent complex linking actin cytoskeleton and extracellular matrix, important for myocardial cell homeostasis, Yuka morokawady et al found that strophin1(DAG1) binds directly to Hippo pathway effector Yap, thereby inhibiting transmembrane proliferation of molecules, and further inhibiting the development of vascular cell proliferation in mice, as well as a peripheral vascular sclerosis marker after reaching a smooth muscle proliferation, as well as a high cholesterol proliferation marker for inducing high cholesterol proliferation in mice, high cholesterol proliferation in peripheral vascular atherosclerosis, high cholesterol proliferation in peripheral vascular atherosclerosis resistant mice, high cholesterol proliferation, endothelial cell proliferation marker 1, and endothelial cell proliferation marker after reaching a smooth muscle proliferation.

In another embodiment of the present invention, there may be a use of a combination of a substance for detecting S L C25A5 and a substance for detecting Tpm2 in the preparation of a kit.

In the use provided by the first aspect of the invention, the substance for detecting S L C25A5 may be a reagent for detecting S L C25A5, more specifically, a reagent for detecting the expression of S L C25A5, and a reagent for detecting the expression of S L C25A5, which are known to those skilled in the art, for example, a reagent for detecting methods such as quantitative or non-labeled quantitative analysis methods based on liquid chromatography mass spectrometry markers, immunological methods (e.g., E L ISA, etc.), the substance for detecting Tpm2 may be a reagent for detecting Tpm2, more specifically, a reagent for detecting the expression of Tpm2, and the reagent for detecting the expression of Tpm2 may be known to those skilled in the art, for example, a reagent for detecting methods such as quantitative or non-labeled quantitative analysis methods based on liquid chromatography mass spectrometry markers, an immunological method (E L ISA, etc.), the substance for detecting the expression of Tpm 1 may be a reagent for detecting the quantitative detection of a substance, a L, a reagent for detecting the expression of a DAG 72, a L, a method of a sample, a method for detecting a labeled dag72, a quantitative analysis method of a specimen, a method of the present invention, a method for detecting a labeled substance, a L, a method for detecting a quantitative detection, a method for detecting a labeled substance, a L, a method for detecting a labeled substance, a method for detecting a method, a method for detecting a labeled substance, or a method for detecting a labeled substance, a labeled quantitative detection, a labeled substance for detecting a labeled substance, a method for detecting a method for.

In the use provided by the first aspect of the invention, it is generally possible to diagnose whether a subject has atherosclerosis and/or a progression of atherosclerosis based on the amount of expression of S L C25a5 and/or Tpm2 and/or DAG1 in a tissue provided by the subject, e.g., when a subject provides a sample having a higher amount of expression of S L C25a5, e.g., at least 1.05 times, 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times higher than the amount of expression of normal tissue, the subject is considered to have a greater likelihood of having atherosclerosis, the higher the fold the greater the likelihood of having atherosclerosis or having a more severe atherosclerotic disease condition, e.g., when a subject provides a sample having a lower amount of expression of Tpm2, e.g., the expression is not higher than 50%, 60%, 70%, 80%, 90%, or 95% of expression of normal tissue, the greater the likelihood of having atherosclerosis or having a greater likelihood of having atherosclerosis, e.g., a greater than the greater the amount of expression of atherosclerosis in the sample, the greater the atherosclerosis or the greater the amount of expression of atherosclerosis, e.g., when a sample provides a sample having a greater likelihood of atherosclerosis, the greater than the greater likelihood of expression of atherosclerosis, the greater than the amount of expression of the atherosclerosis, e.g., the greater of the atherosclerosis, the greater the atherosclerosis, or greater the greater of the greater than the greater of the atherosclerosis, or greater of the atherosclerosis, or.

In the use provided by the first aspect of the present invention, the kit may be a target organ tissue of atherosclerotic disease of a subject, the target organ tissue of atherosclerotic disease usually refers to a tissue organ capable of generating physiopathological changes during the development of atherosclerotic disease, for example, the target organ tissue of atherosclerotic disease may be a combination including but not limited to one or more of aortic tissue, liver tissue, adipose tissue, blood tissue, etc. for another example, the kit may be used to measure the expression level of S L C25a5 and/or Tpm2 and/or DAG1 in the target organ tissue of atherosclerotic disease of a subject, so that the subject may be diagnosed.

In a second aspect, the invention provides a detection kit comprising a substance for detecting S L C25A5, optionally a substance for detecting Tpm2, optionally a substance for detecting DAG1, in a specific embodiment of the invention, the detection kit comprises a combination of a substance for detecting S L C25A5 and a substance for detecting Tpm2, in another specific embodiment of the invention, the detection kit comprises a combination of a substance for detecting S L C25A5 and a substance for detecting DAG1, in another specific embodiment of the invention, the detection kit comprises a specific selection of a substance for detecting S L C25A5, a substance for detecting Tpm2 and a substance for detecting DAG1, a specific selection of a substance for detecting S L C25A5, a substance for detecting Tpm2 and a substance for detecting DAG1, and a specific method of using the kit can be seen in the first aspect of the invention, which is not repeated.

The detection kit provided by the invention can also comprise other reagents, and a person skilled in the art can select and add the required reagents into the detection kit according to the detection principle corresponding to the detection kit.

In a third aspect of the invention there is provided a Biomarker selected from the group consisting of S L C25A5, optionally also including Tpm2, and optionally also DAG 1. a Biomarker (Biomarker) generally refers to a biochemical marker that can mark changes in the structure or function of systems, organs, tissues, cells and sub-cells or changes that may occur, and thus can be used for diagnosis, prognosis or risk assessment of disease etc. in a particular embodiment of the invention, the Biomarker is selected from the group consisting of S L C25A5. in a particular embodiment of the invention, the Biomarker is selected from the group consisting of S L C25A5 and Tpm 2. in another particular embodiment of the invention, the Biomarker is selected from the group consisting of S L C25A5 and DAG 1. in another particular embodiment of the invention, the Biomarker is selected from the group consisting of S L C25A5, Tpm2 and DAG 1. in a subject having a relation to the development of atherosclerosis or not having the relation to the porridge 5A and/or the subject having atherosclerosis.

In a fourth aspect, the invention provides the use of a biomarker provided in the third aspect of the invention in the manufacture of a kit for the diagnosis of atherosclerosis.

In a fifth aspect, the present invention provides a method for diagnosing, comprising obtaining an expression level of a subject S L C25A5, thereby diagnosing whether the subject suffers from atherosclerosis and/or characterizing the progression of atherosclerosis, optionally obtaining an expression level of a subject Tpm2, optionally obtaining an expression level of a subject DAG1, for example, obtaining an expression level of a subject S L C25A5, for example, obtaining expression levels of a subject S L C25A5 and Tpm2, for example, obtaining expression levels of a subject S L C25A5 and DAG1, for example, obtaining expression levels of a subject S L C25A5, Tpm2 and DAG 686 8, and obtaining an expression level of a subject-related biomarker, which should be known to a person skilled in the art, for example, the expression level of a subject biomarker in a tissue provided by the second aspect of the present invention, obtaining an expression level of a subject-related biomarker 356 and/or a progression of atherosclerosis, for diagnosing whether the subject suffers from atherosclerosis and/or not characterizing the progression of atherosclerosis.

The AUC values of ROC curves of the biomarkers S L C25A5, Tpm2 and DAG1 of the atherosclerotic disease provided by the invention are all 1, and are higher than the ACU values of known I L K and DCN, which shows that compared with the known I L K, DCN, the biomarkers provided by the invention can better represent atherosclerotic disease states.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise stated, the experimental METHODS, detection METHODS, AND preparation METHODS disclosed herein are based on conventional techniques IN the art, such as molecular biology, biochemistry, Chromatin structure AND analysis, analytical chemistry, cell culture, recombinant DNA techniques, AND related fields, which are well described IN the prior art, see, IN particular, Sambrook et al MO L ECU L AR C L ONING: A L ABORATORATOR MANUA 8652, Second edition, Cold Spring Harbor L analysis Press, 1989AND Third edition, 2001, Ausubel et al, CURRENT PROTOCO L S INMO L ECU L AR BIO L OGY, John Wiley & Sons, New York, 1987AND biological orders, the devices will be used, METHODS of DIDIDISY 2 IN L, Actic, Experimental & Sons, Sandwith et al, sample # Press, AND Press, sample # 1998, sample # Press, AND P, sample # Press et al, AND sample # Press et al.

Example 1

1. Animal and aorta Collection

In the examples, a total of 41 healthy male New Zealand white rabbits were used and kept for 4.5 months before the experiment. In the experimental group, animals (n ═ 25) were fed a high fat high cholesterol diet (fat content 6%, cholesterol content 1% in the first week, followed by 0.5%, 21.1% calories from fat) for 2 weeks (n ═ 8, HFD _ R2) and 6 weeks (n ═ 17). Under 6 weeks high-fat high-cholesterol diet, one part of animals showed atherosclerosis characteristic, i.e. disease-sensitive Group under high-fat high-cholesterol diet (HFD _ R6, n ═ 8), while the other part of animals were not prone to form plaques, i.e. disease-resistant Group under high-fat high-cholesterol diet (week stress Group, HFD _ WR6, n ═ 9). The differentiation between the sensitive and resistant groups was dependent on the total cholesterol content (TC) in rabbit plasma. In the control group, animals (n-16) were fed normal diet for 2 weeks (n-8, CD2) and 6 weeks (n-8, CD 6).

The plasma of animals was analyzed for lipid content at 0w, 2 weeks, 4 weeks, and 6 weeks on a high-fat high-cholesterol diet or a normal diet, respectively. Blood samples were collected from the rabbits after a 16-hour fast and Total Cholesterol (TC) and Triglyceride (TG) levels were measured using a Wako assay kit (methods see the official gazette TC, 439-17501, TG, 290-63701).

Conditioned rabbits were removed at the prescribed time and fasted for 16 hours, animals were anesthetized with injection of excess pentobarbital sodium solution and euthanized, aortic root was rapidly harvested and placed in liquid nitrogen for temporary storage, and then transferred to-80 ℃ for storage.

2. Protein extraction and sample preparation

Frozen aorta was homogenized using a fully automated sample freeze grinder (JX-FSTPRP, Shanghai Jingxin technology), tissues were lysed using a lysate containing SDS (4% SDS, 100mM Tris-HCl, 0.1M DTT, pH 7.6), centrifuged at 12000g for 10min at 4 ℃, intermediate clarified fractions were retained, boiled for 5min for denaturation and disulfide bond reduction, and the supernatant was removed after brief centrifugation. The protein concentration was determined by tryptophan fluorometry with 295nm as excitation wavelength and 350nm as emission wavelength (Thakuret al, 2011). The protein samples were subjected to tryptsin enzymatic digestion using a slightly modified filtration-assisted sample preparation (FASP method see literature (Wisniewski et al, 2009)) using a TEAB system compatible with TMT10 labeling (100mM TEAB, triethlamonium bicarbonate). The different channel samples were labeled using TMT10(Thermo Scientific), the labeling method referring to kit instructions. To avoid technical variation between different reagents of TMT10, the same Mix was added to one channel of each TMT10, facilitating comparison between subsequent normalization processes and different TMT10 experiments. The peptide fragment purification method was StageTip desalting (Rappsilber et al, 2007).

3. Liquid chromatography-tandem mass spectrometry identification

After the sample is prepared, the sample firstly enters C18 reversed phase high performance liquid chromatography EASY-n L C1000 (Thermo Fisher scientific), is further split and then enters mass spectrum (Q active)^TMHF-X) analysis. The liquid phase A is 0.1 percent of FA/H₂O, B solution is 0.1% FA/ACN, column 75 μm x 150mm, 3 μm packing. Liquid flow rate 300nl/min, total 120min, chromatographic gradient (% B): time, (2% -5%): 2min, (5% -30%): 96min, (30% -45%): 12min, (45% -90%): 2min, (90%): and 8 min. The mass spectrum is collected at a first-order full scan of 300-1500m/z, a resolution of 120000@ m/z of 200, an AGCtarget of 3E6, and a maximum IT of 50 ms. The secondary scan was data dependent acquisition mode (Top 25), HCD fragmentation with a resolution of 60,000@ m/z 200, AGC target set to 1E5, maximum IT of 45ms, isolation window of 1.2m/z, 35.0% NCE, orbitrap detector detection. The dynamic exclusion settings are: repetition times, 1; dynamic exclusion time, 30 s. All data were collected by Xcalibur software.

4. Database search

All Raw files were data retrieved by MaxQuant 1.5.2.8 software (Cox et al, 2009), database Uniprot rabbit database (2018, download 7 months). Fixing and modifying: carbamidomethyl, variable modification: oxidation (M), Acetyl (Protein N-term), reposer ion MS2TMT10, reporter masspleance 0.04ppm, and the mass tolerance of the peptide first search and main search were set to 20ppm and 4.5ppm, respectively. The FDR of the peptide fragment and the protein was set to 0.01. Enzymatic cleavage site and miss site number: trypsin KR/miss-clean 2.

5. Biometric analysis

And (3) respectively carrying out longitudinal median correction on the protein quantitative result, carrying out Mix correction on the transversely identical participating samples, and completing data analysis and statistical test on software R and Excel. The pathway enrichment was done with DAVID software, with count number set to 2, and enrichment analysis performed using the original p-value and setting the threshold to 0.05 against the rabbit whole protein database as background.

Variance analysis was performed on 5 groups with corrected pvalue (bh)0.05 as the threshold, and any group TukeyHSD test p of CD2& HFD _ R2, HFD _ R6& HFD _ WR6, CD6& HFD _ R6, CD6& HFD _ WR6 was less than 0.05, and was considered a difference protein. After the differential protein is classified by the language R fuzzy c-means, a class of proteins can be found as biomarkers of atherosclerosis.

6. Analysis of results

(1) The rabbit has different reaction to atherosclerosis disease under high fat diet

The plasma lipid composition (TC and TG) of the animals were examined at 0w, 2 weeks, 4 weeks, 6 weeks under the condition treatment, and it was found that there was a difference in the reaction of rabbits on the high-fat and high-cholesterol diet, and that some of the animals exhibited atherosclerosis characteristics, i.e., disease-sensitive Group (stress Group, HFD _ R6, n ═ 8) under the high-fat and high-cholesterol diet, while some of the animals exhibited less plaque formation, i.e., disease-resistant Group (Weak stress Group, HFD _ WR6, n ═ 9) under the high-fat and high-cholesterol diet, specifically, the TC exhibited as resistant Group was about 50% of the sensitive Group, as shown in fig. 1, in particular, in which total cholesterol (TC, left graph) and triglyceride content (TG, right graph) in the plasma of the animals (CD 2: 2 weeks normal diet, CD 6: 6 weeks normal diet, HFD _ R2: 2 weeks high-fat and high-cholesterol diet, HFD _ WR 6: 6 weeks high-lipid diet, HFD _ R6: group sensitive to 6 weeks high fat high cholesterol diet). Therefore, the experiment was divided into 5 conditions, namely 2 weeks normal diet (CD2, n ═ 8), 6 weeks normal diet (CD6, n ═ 8), 2 weeks high fat and high cholesterol diet (HFD _ R2, n ═ 8), 6 weeks high fat and high cholesterol diet resistant group (HFD _ WR6, n ═ 9) and 6 weeks high fat and high cholesterol diet sensitive group (HFD _ R6, n ═ 8).

(2) Experimental procedure and data overview

The sample has 5 groups, 8 ~ 9 rabbits in each group, and 41 rabbit aorta samples altogether divide into 5 TMT10 and mark, and every TMT10 has 1 ~ 2 the same Mix samples, thereby the technique deviation of the different TMT10 of the later stage correction of being convenient for is eliminated. The marking efficiency of the peptide fragments is more than 99 percent, and 5 groups of TMT10 identify 1761 proteins which correspond to 11332 peptide fragments. The protein quantified by 41 samples is 1132, 6 rabbits are selected for subsequent differential analysis and disease biomarker screening according to the phenotype, the lipid content in plasma and the proteome, the overall flow of the aortic tissue proteomics analysis is shown in fig. 2, wherein the ratio of CD 2: 2-week normal diet, CD 6: normal diet for 6 weeks, HFD _ R2: 2-week high-fat high-cholesterol diet, HFD _ WR 6: 6-week high-fat high-cholesterol diet resistant group, HFD _ R6: and 6 weeks of a high-fat high-cholesterol diet sensitive group.

(3) Differential proteomics analysis

After correction, pvalue (bh)0.05 was used as a threshold for analysis of variance for 5 groups, and CD2& HFD _ R2, HFD _ R6& HFD _ WR6, CD6& HFD _ R6, CD6& HFD _ WR6 were tested for TukeyHSD p less than 0.05, which was used as a criterion to pick up 162 difference proteins. Under high-fat and high-cholesterol diet, the proteomics of new zealand white rabbits are obviously different, wherein fig. 3A shows the clustering condition (1132 proteins) of the whole proteome, and fig. 3B shows the clustering condition of 162 different proteins. Dietary induction for 2 weeks had produced more significant protein recombination, with a further increase in 6 weeks difference. In order to study the mechanism of early atherosclerosis, 6 weeks were selected as experimental nodes, at which time no significant plaque was produced in the aorta, but high-fat and high-cholesterol diet induced significant increases in plasma cholesterol and triglyceride levels in rabbit susceptible groups. Rabbits resistant to atherosclerosis on a high-fat high-cholesterol diet would be useful for us to find better disease markers.

The 162 differential protein expression profiles show different change trends, wherein Cluster3 and Cluster5 contain early atherosclerosis characteristic markers, namely Cluster3 is a disease protection factor and totally 31, Cluster5 is a disease driving factor and totally 9 (figure 3C). the up-regulated Cluster2, Cluster4 and Cluster5 are mainly enriched in biological processes such as lipoprotein metabolism, cholesterol flow, phospholipid flow, lipid transportation, cholesterol metabolism, V L D L particle clearance negative control and angiogenesis negative control, and the down-regulated Cluster1 and Cluster3 and Cluster6 are mainly enriched in biological processes such as glutathione metabolism, ATP synthesis coupling proton transport, cellular redox homeostasis, tricarboxylic acid cycle, cytoskeleton anchoring at a plasma membrane and actin filament depolymerization negative control (figure 3D).

Focal endothelial shedding stimulates the adhesion and local release of platelet-derived growth factor, causing migration, proliferation and phenotypic modulation of medial smooth muscle cells, leading to the formation of fibrous plaques, and finally to the production of atherosclerosis plaques (Gimbrone and Garcia-Cardena, 2016.) in our data, lipid metabolism up-regulation of the atherosclerosis-producing group (HFD _ R2, HFD _ R6), including cholesterol flow, phospholipid flow, lipid transport and metabolism, and inhibition of negative regulation of the V L D L particle clearance pathway, indicating that V L D L clearance is hindered, circulating low density lipoprotein content is increased, leading finally to lipid retention in endothelial cells, while cellular redox homeostasis is also disrupted when atherosclerosis occurs.

(4) Discovery of disease biomarkers

To obtain more effective and reliable biomarkers, t-tests of CD6 and HFD _ R6 were performed on 40 proteins in the expression profile of atherosclerosis signature markers (Cluster3 and Cluster5) with a p-value threshold of 0.05. to ensure that the biomarkers were well characterized for disease status, ROC tests were performed on each protein and AUC values were calculated for the two sets of differentiation between CD6 and HFD _ R6. finally, we screened 5 significantly different proteins, including 2 known atherosclerosis-related proteins (table 1), except S L C25a5, 4 other proteins were atherosclerosis-protective proteins, whereas S L C25a5 is a disease-driver protein (fig. 4A), Tpm2, S L C25a5, DAG 38, all had AUC curve values of 1, which were higher than the ACU values of known I L K and DCN (I L K is 0.94, and both these were considered as normal atherosclerosis-generating biomarkers (HFD 2) and HFD 3. these were separated into a porridge-like group.

TABLE 1 characterization of Atherosclerosis disease 5 characteristic proteins

As can be seen, the t test and the ROC test of CD6 and HFD _ R6 are carried out on 40 proteins in the expression profiles of the atherosclerosis characteristic markers (Cluster3 and Cluster5) to screen 5 proteins with remarkable difference, wherein 2 known atherosclerosis-related proteins are included, the ROC curve AUC values of Tpm2, S L C25A5 and DAG1 are all 1 and are higher than the ACU values of known I L K and DCN (the AUC of I L K is 0.94 and the DCN is 0.89), which indicates that the Tpm2, S L C25A5 and DAG1 can better characterize the atherosclerosis disease state compared with the known I L K, DCN.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. Use of a substance for detecting S L C25a5 in the preparation of a kit for diagnosing atherosclerosis.

2. Use according to claim 1, characterized by the use of a combination of a substance for detecting S L C25a5 and a substance for detecting Tpm2 in the preparation of a kit;

and/or use of a combination of a substance for detecting S L C25a5 and a substance for detecting DAG1 in the preparation of a kit;

and/or the use of a combination of a substance for detecting S L C25a5, a substance for detecting Tpm2, and a substance for detecting DAG1 in the preparation of a kit.

3. The use according to claim 1, wherein the substance for detecting S L C25a5 is a reagent for detecting the expression level of S L C25a 5;

and/or the substance for detecting the Tpm2 is a reagent for detecting the expression amount of the Tpm 2;

and/or the substance for detecting DAG1 is a reagent for detecting the expression level of DAG 1.

4. The use according to claim 1, wherein the kit diagnoses whether the subject has atherosclerosis and/or characterizes the progression of atherosclerosis based on the expression level of S L C25a5, preferably further comprising the expression level of Tpm2 and/or DAG 1.

5. Use according to claim 4, wherein the kit is directed against a target organ tissue of an atherosclerotic disease of a subject, preferably a combination selected from one or more of aortic tissue, liver tissue, adipose tissue, blood tissue;

and/or, the kit is used for diagnosing early atherosclerosis.

6. A detection kit comprising a substance for detecting S L C25a 5;

optionally, a substance for detecting Tpm 2;

optionally, a substance for detecting DAG1 is also included.

7. The test kit of claim 6, comprising a combination of a substance for testing S L C25A5 and a substance for testing Tpm 2;

and/or, a combination comprising a substance for detecting S L C25a5 and a substance for detecting DAG 1;

and/or a combination comprising a substance for detecting S L C25a5, a substance for detecting Tpm2, and a substance for detecting DAG 1.

8. A biomarker comprising S L C25A5, optionally further comprising Tpm2, and optionally further comprising DAG 1.

9. The biomarker of claim 8, selected from the group consisting of a combination of S L C25a5 and TPM 2;

and/or, the biomarker is selected from the group consisting of a combination of S L C25a5 and DAG 1;

and/or, the biomarker is selected from the group consisting of S L C25a5, TPM2, and DAG 1.

10. Use of a biomarker according to any of claims 8 to 9 in the manufacture of a kit for the diagnosis of atherosclerosis.