Application of REG family proteins in diagnosis, screening or assessment of atherosclerosis

Abstract

The present invention discloses an application of a REG family protein in diagnosing, screening or evaluating atherosclerosis, and belongs to the field of biomedical technology. The present invention found that the Reg family gene showed significant up-regulated expression at the mRNA transcription level, protein level and serum free level in atherosclerosis modeling rats. It was further found that the REG family protein also showed up-regulated expression in the serum of patients with atherosclerosis, providing a more convenient, accurate and sensitive detection index for the clinical diagnosis and course evaluation of atherosclerosis, thereby improving the clinical testing means and evaluation scheme of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of regeneration gene REG family proteins in the diagnosis, screening or evaluation of atherosclerosis. Background Art

[0002] Cardiovascular disease is one of the most common and harmful diseases in humans. Atherosclerosis (AS) is its main cause. The formation of AS is characterized by the accumulation of lipids in the lumen to form atherosclerotic plaques, which causes the narrowing or even blockage of the arterial lumen, leading to ischemia and necrosis of tissues and organs supplied by the arteries. The pathological process mainly includes endothelial cell damage and dysfunction, abnormal proliferation and migration of smooth muscle cells, and foam cell formation. At present, the diagnosis of AS mainly relies on imaging. Compared with imaging, serological testing has the advantages of simplicity, rapidity, easy operation and result interpretation. In addition, the biological manifestations of AS precede the imaging manifestations. Early intervention can be carried out according to the results of serological tests to reduce the occurrence of complications. Traditional AS serological detection indicators, including inflammatory markers, oxidative stress indicators, coagulation function indicators, lipid-related markers, etc., have played a certain role in assisting the diagnosis of AS, but their specificity is poor. Clinically, more specific and effective serum biomarkers are urgently needed for the diagnosis of AS, plaque stability assessment, risk stratification management, and prognosis assessment; at the same time, the development of new biomarkers will help to further reveal the occurrence and development mechanism of AS.

[0003] The regeneration gene REG family proteins were first discovered in the study of pancreatitis and islet proliferation and are highly conserved in humans and rodents. Each REG family protein consists of a signal peptide and a C-type lectin domain and is divided into four groups based on the primary structure of the protein: (i) REG1: rats and mice are Reg1, and humans have two homologous analogs REG1A and REG1B; (ii) REG2, which only exists in mice; (iii) REG3: rats and mice are Reg3α, Reg3β, Reg3γ and Reg3δ, and humans have two homologous analogs REG3A and REG3G; (iv) Reg4. The five REG family proteins expressed by humans, REG1A, REG1B, REG3A, REG3G and REG4, have functions such as promoting cell proliferation, anti-apoptosis and immune regulation; studies have shown that REG family proteins play an important role in diseases such as diabetes, Alzheimer's disease and gastrointestinal tumors, and have the value of becoming potential biomarkers. There is no report on whether the regeneration gene Reg or REG family genes or the REG family proteins they encode are related to atherosclerosis. Summary of the invention

[0004] The purpose of the present invention is to provide a biomarker for diagnosis, screening and evaluation of atherosclerosis and its application, in particular, the application of REG family protein as a biomarker in diagnosis, screening or evaluation of atherosclerosis.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a biomarker for diagnosis, screening and evaluation of atherosclerosis, wherein the biomarker is a REG family protein. Specifically, the REG family protein is REG1A (protein sequence number in NCBI is P05451), REG1B (protein sequence number in NCBI is P48304), REG3A (protein sequence number in NCBI is Q06141) or REG3G (protein sequence number in NCBI is Q6UW15).

[0007] The AS model was constructed using APOE knockout (APOE- / -) rats combined with a high-fat diet. Macroscopic O oil red staining showed significant aortic plaque formation in AS rats, and the plaque area was about 30% analyzed using Image J software. In contrast, the aorta of the control group rats (APOE- / -, normal diet) had basically no plaque formation ( Figure 1 ). The transcriptome analysis of the aorta of AS model rats revealed that genes of the regeneration gene Reg family were significantly overexpressed in the aorta of rats with atherosclerosis ( Figure 2 ), mRNA transcription level, protein level and serum free protein concentration are all reflected (see Figures 3 to 5 ). By evaluating the serum levels of four REG family proteins (REG1A, REG1B, REG3A and REG3G) in clinical AS patients and healthy control volunteers, it was found that REG1A and REG3G were significantly overexpressed in AS patients; REG1B and REG3A also showed an increasing trend in AS patients, which was statistically different from the healthy control group; ROC curve analysis revealed that these four REG family proteins have high reliability in the clinical diagnosis of AS patients (see Figure 6 ), indicating that the regeneration gene REG family proteins can be used as potential AS biomarkers, providing more convenient, accurate and sensitive detection indicators for the clinical diagnosis and disease course evaluation of atherosclerosis, and improving the clinical testing methods and evaluation programs of atherosclerosis.

[0008] In a second aspect, the present invention provides the use of a detection reagent for REG family proteins in the preparation of any of the following products:

[0009] 1) Application in the preparation of atherosclerosis diagnostic products;

[0010] 2) Application in the preparation of atherosclerosis screening products;

[0011] 3) Application in the preparation of products for assessing the risk of atherosclerosis;

[0012] 4) Application in the preparation of products for evaluating the prognosis of atherosclerosis.

[0013] Furthermore, the REG family protein is selected from REG1A, REG1B, REG3A and / or REG3G.

[0014] Furthermore, the detection reagent is a reagent for detecting the expression level of REG family proteins in a sample.

[0015] In the above application, the product can detect the expression level of REG family proteins in the sample by, but not limited to, immunohistochemistry or enzyme-linked immunosorbent assay.

[0016] Furthermore, the product contains antibodies that specifically bind to REG family proteins.

[0017] Furthermore, the antibody is a polyclonal antibody or a monoclonal antibody. Preferably, the antibody is a monoclonal antibody, which can be prepared by methods known in the art or purchased commercially.

[0018] In the above application, the test sample may preferably be serum or urine. For example, serum samples of clinical atherosclerosis patients are collected for testing.

[0019] Furthermore, the product may be a chip, a preparation, a test strip or a test kit.

[0020] In a third aspect, the present invention provides a kit, comprising the above-mentioned reagent for detecting the expression level of REG family protein in a sample; and

[0021] The kit is a kit for use in any of the following applications:

[0022] 1) Kits for diagnosing atherosclerosis;

[0023] 2) Kits for screening of atherosclerosis;

[0024] 3) Kits for assessing the risk of atherosclerosis;

[0025] 4) A kit for evaluating the prognosis of atherosclerosis.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention found that the Reg family genes in atherosclerosis model rats showed significant up-regulation in mRNA transcription level, protein level and serum free level. In order to detect whether the up-regulation of serum concentration of Reg family proteins has a role in clinical diagnosis of atherosclerosis, the present invention used ELISA method to measure the concentration of REG1A, REG1B, REG3A and REG3G in fresh blood samples of 30 patients with coronary atherosclerosis before PCI and 29 healthy subjects. The test results are as follows: Figure 6 As shown: the serum concentrations of REG1A, REG1B, REG3A and REG3G in the atherosclerosis group were significantly higher than those in the healthy control group. The receiver operating characteristic curve (ROC) analysis showed that the AUC values ​​of serum REG1A, REG1B, REG3A and REG3G in the clinical diagnosis of atherosclerosis were 0.739, 0.678, 0.695 and 0.892, respectively, indicating that REG family proteins can be effectively used as markers for the clinical diagnosis of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0029] Figure 1 The figure shows the Oil Red O staining of the aorta of rats with atherosclerosis model.

[0030] Figure 2 The volcano plot shows the differentially expressed genes in rats with atherosclerosis model.

[0031] Figure 3 The figure shows the expression of aortic regeneration gene Reg detected by fluorescence quantitative PCR in rats with atherosclerosis model.

[0032] Figure 4 The figure shows the expression of Reg family proteins in rat atherosclerotic plaques detected by immunohistochemistry.

[0033] Figure 5 The figure shows the concentration of Reg3b in the serum of rats with atherosclerosis model determined by enzyme-linked immunosorbent assay.

[0034] Figure 6 The results showed that the evaluation of REG1A, REG1B, REG3A and REG3G in human serum has important clinical application value in the clinical diagnosis of coronary atherosclerosis. DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Experimental Materials:

[0037] Reagents and consumables

[0038] The reagents used in the present invention include: Oil Red O staining kit (Biyuntian), Trizol (Invitrogen), PrimeScriptTM RT kit (Takara), TB Premix Ex TagTM II (Takara), ELISA kits (REG1A, REG3G and Reg3β, Wuhan Fine Bio; REG1B and REG3A, AVIVA SYSTEMS BIOLOGY), Reg1antibody (Proteintech), Reg3αantibody (Abcam), Reg3βantibody (Thermo Fisher), Reg3γantibody (Thermo Fisher), DAB color development kit (Maixin Bio) and neutral gum (Biyuntian), etc.; the consumables used in the present invention include: pipette tips, enzyme-free EP tubes, coverslips, slides, transparent 96-well plates, etc.

[0039] Transgenic rats

[0040] ApoE- / - rats and normal SD rats were fed with high-fat diets rich in 1.25% cholesterol and normal diets, and the rats were kept in a pathogen-free environment with controlled temperature and humidity. The rats with aortic atherosclerosis were divided into normal diet group and high-fat diet group, with no less than 5 rats in each group.

[0041] Experimental instruments

[0042] Instruments included spectrophotometer (Thermo Fisher), microplate reader (Thermo Fisher), fluorescence quantitative PCR instrument (ABI), inverted microscope (Leica) and fully automatic digital slide scanner (Leica).

[0043] Example 1: Oil Red O staining of rat aorta

[0044] 1) Anesthesia and perfusion of rats: 2 to 3 rats were selected from the ApoE knockout rats in the normal diet group (KO60NC) and the high-fat diet group (KO60HL) for Oil Red O staining. Rats were intraperitoneally injected with 0.2 ml of 2% sodium pentobarbital per 100 g body weight. After being anesthetized, they were fixed on the dissection table in a supine position, the chest cavity was cut open, a small incision was made in the right atrium using a sterile instrument, the infusion needle was inserted into the left ventricle, and pre-cooled PBS was used for systemic perfusion until clear and transparent liquid flowed out of the incision in the right atrium and the liver turned white.

[0045] 2) Collection of rat aortic tissue: The heart, trachea, esophagus, thymus and excess fat tissue around the aortic arch were removed under a stereomicroscope, and the complete aorta and its branches were stripped and fixed with 4% paraformaldehyde.

[0046] 3) Aorta gross oil red staining and plaque assessment: After being fully fixed, the whole aorta was removed from the fixative and washed with PBS to remove the residual fixative on the surface; because adipose tissue can be stained bright red with oil red and interfere with plaque observation, it is necessary to further remove adipose tissue from the adventitia of the blood vessels using fine forceps under a stereo microscope before staining. Be careful to operate gently to avoid damaging the blood vessels; use dissecting scissors to carefully cut the blood vessels longitudinally along the blood vessel wall, immerse them in oil red stain at 37°C for 60 minutes, then remove them and differentiate them with 75% ethanol until the fat plaques in the lumen are orange-red or bright red, and other parts are almost colorless; the stained gross tissue was spread out and fixed on white filter paper and foam board, and photographed and evaluated under good light conditions and with the focus adjusted. Compared with the control group KO60NC, a large number of aortic plaques were formed in KO60HL, see Figure 1 .

[0047] Example 2: Rat aorta transcriptome sequencing

[0048] 1) Rat aortic tissue collection and lysis: Rat aortic tissue collection was carried out according to the above process. The aortas of 3 rats in the ApoE knockout rat normal diet group and high-fat diet group were removed for transcriptome sequencing. 1.5 ml of Trizol lysis buffer was added to a 2 ml EP tube, and an appropriate amount of tissue sample was quickly ground into powder in liquid nitrogen, and then transferred to the lysis buffer and allowed to stand for 5 minutes to fully lyse the tissue cells.

[0049] 2) Sample extraction and purification: centrifuge the ground and crushed tissue samples at 4°C, 12000g for 5 min, transfer the supernatant to an EP tube with 300μl of chloroform:isoamyl alcohol (24:1), invert and shake vigorously to mix, and centrifuge at 4°C, 12000g for 8 min; add 600μl of isopropanol to the supernatant in an EP tube, invert and mix, and let stand at room temperature for more than 30 min; centrifuge at 4°C, 12000g for 10 min, discard the supernatant, wash twice with 75% ethanol, dry for 3-5 min, dissolve in enzyme-free water, and measure the RNA concentration.

[0050] 3) mRNA fragmentation and cDNA synthesis: 2-3 μg of total tissue RNA was taken, denatured at 70°C for 2 min, purified using oligo-dT magnetic beads, fragmented with divalent metal ions, enriched and purified, and reverse transcribed to synthesize cDNA.

[0051] 4) Library construction: Purify cDNA, prepare adapter ligation reaction system, ligate at room temperature for 1 hour, purify with Ampure XP magnetic beads and perform PCR amplification, detect library size with Agilent 2100 analyzer, and perform high-throughput sequencing with Illumina sequencing platform. The regeneration gene Reg family, including Reg1, Reg3α, Reg3β and Reg3γ, is significantly overexpressed in the aorta of atherosclerotic rats, see Figure 2 .

[0052] Example 3: Fluorescence quantitative PCR verification of Reg family gene expression

[0053] 1) Extraction and reverse transcription of RNA from rat aorta tissue: Total RNA from rat aorta was extracted according to Example 2. 2 μl of PrimeScript RT mixture was added to 1 μg of total RNA, reacted at 37°C for 15 min, and denatured at 85°C for 5 s. Oligo dT and 6-base random primers were used as reverse transcription primers.

[0054] 2) Real-time fluorescence PCR: Select the Premix Ex Taq II kit for fluorescence quantification.

[0055] 3) Data analysis: Using 2 -ΔΔCt The calculation method was used for relative quantification of Reg family genes.

[0056] Results Figure 3 The expression levels of Reg1, Reg3α, Reg3β and Reg3γ mRNA in the aorta of ApoE knockout rats in the high-fat diet group were significantly higher than those in the normal diet control group.

[0057] Example 4: Immunohistochemical detection of the expression of Reg family proteins in rat plaques

[0058] 1) Fixation, dehydration and embedding of rat aortic tissue: The rat aortic tissue was collected according to the above process and fixed in 4% paraformaldehyde for 3 to 4 hours. After fixation, the aortic tissue was placed in an embedding box for dehydration, wax infiltration and embedding.

[0059] 2) Tissue sectioning and preparation: Fix the wax block in the slicer and adjust the distance between it and the blade. Adjust the slice thickness to 10 μm. Adjust the blade angle to ensure that the slicing direction is perpendicular to the aortic root. Observe under a microscope while cutting. Keep 10 slices for each sample.

[0060] 3) Immunochemical staining: Tissue sections can be placed in a 60°C incubator for dewaxing for 1 to 2 hours, followed by hydration, antigen retrieval, blocking, and primary antibody incubation overnight.

[0061] 4) Color development: The next day, warm up at room temperature for 30 minutes, wash with PBS, incubate with secondary antibody, and wash with PBS; add two drops of DAB color developing solution, observe under a microscope for 3 to 10 minutes, stop staining when the staining is appropriate and rinse with tap water; counterstain with hematoxylin for 1 to 2 minutes, stop staining when the cell nucleus turns blue, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 seconds, and rinse with tap water for 3 minutes.

[0062] 5) Sealing: dehydrating tissue sections, sealing with neutral resin, air drying, and scanning.

[0063] The results showed that Reg1, Reg3α, Reg3β and Reg3γ proteins were significantly overexpressed in aortic plaques of ApoE knockout rats fed a high-fat diet for 60 weeks. Aortic plaques are marked with dotted lines. Figure 4 .

[0064] Example 5: ELISA to detect REG family proteins in serum

[0065] 1) ELISA method (ER0296, Wuhan Fine Biotechnology) to determine rat serum Reg3β:

[0066] ① ApoE knockout rats fed with a normal diet or a high-fat diet for 60 weeks were selected, and six serum samples from different rats were collected from each group;

[0067] ② Sample addition: Add 100 μl of standard and 50-fold diluted rat serum sample to the corresponding wells, cover with film, and incubate at 37°C for 90 minutes;

[0068] ③ Washing: Remove the cover and wash the plate 3 times with washing buffer, leaving the washing buffer in the well for 1 to 2 minutes each time;

[0069] ④ Biotin-labeled antibody: Add 100 μl of biotin-labeled antibody working solution to the above wells, cover the plate, incubate at 37°C for 60 minutes, and then wash the plate 3 times;

[0070] ⑤ Add HRP-streptavidin conjugate (SABC): Add 100 μl SABC working solution to each well, cover the plate, incubate at 37°C for 30 minutes, and wash the plate 5 times;

[0071] ⑥TMB substrate: Add 90 μl TMB substrate to each well, cover the plate, incubate at 37°C in the dark for 15 minutes, then add 50 μl stop solution to each well;

[0072] ⑦ Use an enzyme reader to measure the OD value at 450nm, draw a standard curve, and calculate the concentration.

[0073] 2) Human serum samples: Fresh serum samples were collected from 30 patients diagnosed with coronary atherosclerosis before PCI and 29 healthy subjects in the Cardiovascular Disease Center of our hospital. Human serum REG1A (EH3707, Wuhan Fine Biotechnology), REG1B (OKEH00380, AVIVA SYSTEMS BIOLOGY), REG3A (OKBB01254, AVIVA SYSTEMS BIOLOGY) and REG3G (EH0847, Wuhan Fine Biotechnology) were determined by ELISA:

[0074] ① Thirty serum samples from the disease group and 29 serum samples from the health examination group were selected;

[0075] ② Sample addition: Add 100 μl of standard and 2-fold diluted human serum samples to the corresponding wells, cover with the membrane, and incubate at 37°C for 90 minutes;

[0076] ③ Washing: Remove the cover and wash the plate 3 times with washing buffer, leaving the washing buffer in the well for 1 to 2 minutes each time;

[0077] ④ Biotin-labeled antibody: Add 100 μl of biotin-labeled antibody working solution to the above wells, cover the plate, incubate at 37°C for 60 minutes, and then wash the plate 3 times;

[0078] ⑤ Add HRP-streptavidin conjugate (SABC): Add 100 μl SABC working solution to each well, cover the plate, incubate at 37°C for 30 minutes and wash the plate 5 times;

[0079] ⑥TMB substrate: Add 90 μl TMB substrate to each well, cover the plate, incubate at 37°C in the dark for 15 minutes, then add 50 μl stop solution to each well;

[0080] ⑦ Use an enzyme reader to measure the OD value at 450nm, draw a standard curve, and calculate the concentration.

[0081] Results Figure 6: The scatter plot represents the ELISA determination of REG1A, REG1B, REG3A and REG3G concentrations in the serum of AS patients and healthy controls. The serum of AS patients has higher REG1A, REG1B, REG3A and REG3G protein concentrations; ROC curve analysis of serum REG1A, REG1B, REG3A and REG3G in AS patients and healthy controls has a high credibility and can be used as potential AS biomarkers.

[0082] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of the detection reagent of REG family proteins in the preparation of any of the following products: 1) Application in the preparation of atherosclerosis diagnostic products; 2) Application in the preparation of atherosclerosis screening products; The REG family protein is one of REG1A, REG1B, REG3G, or a combination of REG1A, REG1B, REG3A and REG3G; The detection reagent is a reagent for detecting the expression level of REG family proteins in a sample; The sample is serum.

2. The use according to claim 1, characterized in that: The product detects the expression level of REG family proteins in a sample by immunohistochemistry or enzyme-linked immunosorbent assay.

3. The use according to claim 2, characterized in that: The product contains antibodies that specifically bind to REG family proteins.

4. The use according to claim 3, characterized in that: The antibody is a polyclonal antibody or a monoclonal antibody.

5. The use according to claim 1, characterized in that: The product is a chip, a preparation, a test strip or a test kit.

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