Application of NLRC3 in preparation of product for diagnosing, screening or evaluating coronary atherosclerotic heart disease

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of NLRC3 in preparation of a product for diagnosing, screening or evaluating coronary atherosclerotic heart disease. The invention provides a novel marker NLRC3 for detecting coronary heart disease, which has excellent stability, brings convenience and low trauma to diagnosis of coronary heart disease, and avoids radiation risk. The expression of NLRC3 in human carotid plaque is obviously reduced compared with that of normal carotid artery; nLRC3 expression in peripheral blood mononuclear cells of a patient with coronary heart disease is obviously reduced compared with that of a patient with normal coronary artery; nLRC3 expression in plasma of a patient with coronary heart disease is obviously reduced compared with that of a patient with normal coronary artery. The AUC value of ROC curve analysis is as high as 0.926; when the diagnosis threshold value of the relative expression quantity of the NLRC3 is set to be 0.012, the sensitivity reaches 85%, and the specificity reaches 80%; the accuracy of screening coronary heart disease patients in chest pain patients is 84.4%; the medicine has excellent popularization and application values clinically.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of NLRC3 in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease. Background Art

[0002] Coronary atherosclerotic heart disease (CAD) is a cardiovascular disease caused by the formation of atherosclerotic plaques in the coronary arteries. Its pathological basis mainly involves damage to the arterial endothelium, inflammatory response, and abnormal lipid metabolism. Recent studies have shown that chronic inflammation plays a key role in the occurrence and development of CAD, especially closely related to the formation of atherosclerosis. In the process of atherosclerosis, damage to endothelial cells leads to increased vascular permeability, which promotes the deposition of lipids such as low-density lipoprotein (LDL) in the vascular wall, forming lipid streaks, and then developing into atherosclerotic plaques. In addition, the infiltration and activation of inflammatory cells will also aggravate this process, promote smooth muscle cell proliferation and matrix remodeling, and form unstable plaques, which may rupture in some cases, leading to the occurrence of acute coronary syndrome. With the change of lifestyle and the increase of cardiovascular disease risk factors, the incidence of coronary atherosclerosis has increased year by year, especially in the young population. Early identification and intervention of CAD can significantly reduce the risk of heart attack and related complications, thereby improving the quality of life of patients. In clinical practice, coronary angiography is a common technique for CAD diagnosis, but it is not suitable for routine screening due to its invasiveness, difficulty in operation, and high cost. Therefore, circulating markers play an important role in the clinical auxiliary diagnosis of coronary heart disease due to their convenience, speed, and easy interpretation of results.

[0003] Nucleotide-binding domain-like receptors (NLRs) are a family of pattern recognition receptors in the cytoplasm that play a key role in identifying pathogens and monitoring changes in body homeostasis. NLRs are crucial for the regulation of inflammatory and immune-related diseases by regulating downstream signaling pathways. NLRC3 is a new member of the NLR family that was found in Jurkat T cells and is widely present in mammalian tissues. Studies have shown that NLRC3 negatively regulates immune inflammatory responses by interacting with TRAF6, reducing its ubiquitination level, and inhibiting NF-κB activation. NLRC3-deficient mice have enhanced inflammatory responses after endotoxin stimulation, confirming its immunoregulatory role. Rajendra et al. revealed that NLRC3 is downregulated in colorectal cancer tissues and inhibits tumor cell proliferation through the PI3K-PKB-mTOR signaling pathway. However, there is currently no research on NLRC3 in coronary atherosclerotic heart disease, and its function and role are still unclear. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide the use of NLRC3 in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease. The present invention uses NLRC3 as a gene marker for diagnosing, screening or evaluating coronary atherosclerotic heart disease, which is timely, specific and sensitive, so that patients can know the risk of the disease in the early stage of the disease, and take corresponding prevention and treatment measures according to the risk level.

[0005] The purpose of the present invention is achieved through the following solutions:

[0006] In a first aspect, the present invention provides the use of NLRC3 in the preparation of a product for diagnosing, screening or evaluating coronary atherosclerotic heart disease (CHD).

[0007] Furthermore, the expression level of NLRC3 in patients with CHD was significantly lower than that in those with normal coronary arteries.

[0008] Furthermore, the expression level of NLRC3 in carotid plaques is significantly lower than that in normal carotid arteries. By comparing the results of NLRC3 immunofluorescence in situ detection in human carotid plaques and normal carotid arteries, the present invention found that the expression level of NLRC3 in carotid plaques is significantly lower than that in normal carotid arteries.

[0009] Furthermore, the expression level of NLRC3 in single peripheral blood cells of patients with coronary heart disease was significantly lower than that in patients with normal coronary arteries.

[0010] Furthermore, the expression level of NLRC3 in the plasma of patients with coronary heart disease was significantly lower than that in those with normal coronary arteries.

[0011] Furthermore, the present invention reveals through correlation analysis that there is a significant correlation between the expression level of NLRC3 and the coronary artery stenosis score index (Gensini score) of patients with coronary heart disease.

[0012] Furthermore, through ROC curve analysis, NLRC3 had a high diagnostic value, with an AUC value of 0.926; when the diagnostic threshold of NLRC3 relative expression was set at 0.012, the sensitivity of diagnosing CAD was 85% and the specificity was 80%.

[0013] Furthermore, by detecting the expression level of NLRC3, it was further used as a screening tool for coronary heart disease, and compared with the results of coronary angiography, the accuracy was as high as 84.4%.

[0014] Based on this, the present invention proposes for the first time a biomarker NLRC3 for diagnosing coronary heart disease, provides a method for diagnosing coronary heart disease based on the circulating marker NLRC3, and proposes the application of NLRC3 in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease.

[0015] Furthermore, the product includes a chip, a test kit, a test paper or a high-throughput sequencing platform, etc. Among them, the high-throughput sequencing platform is a special diagnostic tool, and the product for detecting the expression of the NLRC3 gene can be applied to the platform to detect the expression of the NLRC3 gene. With the development of high-throughput sequencing technology, the construction of a person's gene expression profile will become a very convenient task. By comparing the gene expression profiles of disease patients and normal people, it is easy to analyze which gene abnormality is related to the disease. Therefore, knowing that the abnormality of the NLRC3 gene is related to coronary atherosclerotic heart disease in high-throughput sequencing also belongs to the use of the NLRC3 gene, which is also within the scope of protection of the present invention.

[0016] Furthermore, the product can diagnose coronary heart disease by detecting the expression of the NLRC3 gene in a sample.

[0017] In a second aspect, the present invention also provides the use of a product for detecting NLRC3 gene expression in the preparation of a product for diagnosing, screening or evaluating coronary atherosclerotic heart disease.

[0018] Furthermore, the product for detecting NLRC3 gene expression includes: a product for detecting NLRC3 gene expression level by RT-PCR, fluorescent quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform.

[0019] Furthermore, the product for detecting the expression level of the NLRC3 gene by RT-PCR includes at least a pair of primers for specifically amplifying the NLRC3 gene.

[0020] Furthermore, the product for detecting the expression level of the NLRC3 gene by fluorescent quantitative PCR includes at least a pair of primers for specifically amplifying the NLRC3 gene.

[0021] Furthermore, the product for detecting the expression level of the NLRC3 gene by immunoassay at least includes an antibody that specifically binds to the NLRC3 protein.

[0022] Furthermore, the product for detecting the expression level of the NLRC3 gene by in situ hybridization at least includes a probe that hybridizes with the nucleic acid sequence of the NLRC3 gene.

[0023] Furthermore, the product for detecting the expression level of the NLRC3 gene by means of a chip comprises at least a protein chip and a gene chip; wherein the protein chip comprises an antibody that specifically binds to the NLRC3 protein, and the gene chip comprises a probe that hybridizes to the nucleic acid sequence of the NLRC3 gene.

[0024] Furthermore, the sources of the NLRC3 gene and its expression products for diagnosing coronary heart disease include, but are not limited to, blood, tissue fluid, urine, saliva, cerebrospinal fluid, and other body fluids from which genomic DNA can be obtained. In a specific embodiment of the present invention, the source of the NLRC3 gene and its expression products for diagnosing coronary heart disease is blood.

[0025] Furthermore, the above-mentioned product was used to detect the expression level of NLRC3 in single peripheral blood cells of patients with coronary heart disease and those with normal coronary arteries for diagnosis. The results showed that the expression level of NLRC3 in patients with coronary heart disease was significantly reduced.

[0026] In a third aspect, the present invention also provides the use of NLRC3 in preparing a fluorescent quantitative PCR kit for diagnosing coronary heart disease.

[0027] Furthermore, the kit includes at least a pair of primers for specifically amplifying the NLRC3 gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0028] Forward primer 5'-TGAAGGCGTTTGGTGTAGACC-3' (SEQ ID NO. 1);

[0029] Reverse primer: 5'-CGATGCCAACGTCTCCTC-3' (SEQ ID NO. 2).

[0030] In a fourth aspect, the present invention provides a fluorescent quantitative PCR kit for diagnosing coronary heart disease.

[0031] Furthermore, the kit includes at least a pair of primers for specifically amplifying the NLRC3 gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0032] Furthermore, the kit also includes GAPDH primer sequence, SYBR Green fluorescent dye, etc. to ensure the accuracy and reliability of the experiment. Furthermore, the kit is used to diagnose coronary heart disease, aiming to provide an efficient and accurate molecular detection method.

[0033] The GAPDH primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0034] Forward primer 5'-ATGACATCAAGAAGGTGGTG-3' (SEQ ID NO. 7);

[0035] Reverse primer: 5'-CATACCAGGAAATGAGCTTG-3' (SEQ ID NO. 8).

[0036] The present invention provides a new marker NLRC3 for detecting coronary heart disease, which has excellent stability, brings convenience and low trauma to the diagnosis of coronary heart disease, and avoids radiation risks. The present invention finds that the expression of NLRC3 in human carotid plaques is significantly downregulated compared with that in normal carotid arteries; by performing RT-qPCR detection in peripheral blood mononuclear cells (PBMC) of patients with coronary heart disease and normal coronary arteries, it is found that the expression of NLRC3 in PBMC of patients with coronary heart disease is significantly reduced; in patients with coronary heart disease, NLRC3 expression is significantly correlated with the coronary artery stenosis score (Gensini score); ROC curve analysis finds that the marker shows significant value, with an AUC value as high as 0.926 (95% confidence interval: 0.889-0.963); when the diagnostic threshold of the relative expression of NLRC3 is set to 0.012, the sensitivity of the marker reaches 85% and the specificity is 80%; the accuracy of screening patients with coronary heart disease in chest pain patients is 84.4%; these results prove that NLRC3 has good diagnostic value; ELISA is used to detect NLRC3 expression in the plasma of patients with coronary heart disease and normal coronary arteries, and it is found that the plasma NLRC3 level is significantly reduced in patients with coronary heart disease. The present invention provides the use of NLRC3 in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease. The above excellent performance indicators indicate that the NLRC3 marker has excellent clinical promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 NLRC3 expression in carotid plaques and normal carotid arteries.

[0039] Figure 2 Agarose gel electrophoresis was used to detect the amplified products.

[0040] Figure 3 The expression of NLRC3 in peripheral blood mononuclear cells of patients with coronary atherosclerotic heart disease and patients with normal coronary arteries.

[0041] Figure 4 To investigate the relationship between NLRC3 expression and Gensini score in patients with coronary atherosclerotic heart disease.

[0042] Figure 5 The clinical diagnostic value of NLRC3 in coronary atherosclerotic heart disease.

[0043] Figure 6 The expression of NLRC3 in the plasma of patients with coronary atherosclerotic heart disease. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial channels unless otherwise specified. The methods described are conventional methods unless otherwise specified.

[0045] Example 1: Immunofluorescence in situ detection of NLRC3 expression in carotid plaque core and normal carotid artery of patients with coronary atherosclerotic heart disease (CHD)

[0046] 1. Research subjects

[0047] Carotid artery plaques and adjacent normal intimal tissues were collected from patients diagnosed with coronary heart disease in the emergency department of Meizhou People's Hospital, and all patients signed informed consent.

[0048] 2. Experimental Methods

[0049] 2.1 Sample fixation

[0050] The carotid intima of CAD patients undergoing carotid endarterectomy was collected, and the plaque-containing intima and normal intima were separated, washed with normal saline, and fixed with 4% paraformaldehyde.

[0051] 2.1 Sample testing

[0052] (1) Sample preparation: First, place the tissue sections in environmentally friendly dewaxing solutions I, II, and III in sequence, with each step lasting 10 minutes to completely remove the paraffin. Then, treat with anhydrous ethanol I, II, and III for 5 minutes each, and finally wash with distilled water to prepare for the subsequent antigen retrieval step;

[0053] (2) Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 8.0) and perform antigen repair in a microwave oven. Follow the procedure of medium heat for 8 minutes, off heat for 8 minutes, and medium-low heat for 7 minutes. At the same time, pay attention to prevent excessive evaporation of the buffer and avoid drying of the sections. After the repair is completed, let the sections cool naturally and wash them in PBS (pH 7.4) 3 times for 5 minutes each time to remove excess repair solution.

[0054] (3) Circle drawing and blocking: Shake the slide dry and use a histochemical pen to draw circles around the tissue to prevent antibody loss; add BSA to the circled area for blocking and incubate for 30 minutes;

[0055] (4) Primary antibody incubation: After removing the blocking solution, NLRC3 rabbit polyclonal antibody (DF13411, Affinity Biosciences) was added to the slices, and the slices were placed flat in a humidified chamber and incubated overnight at 4°C to ensure effective binding of the antibody to the antigen;

[0056] (5) Secondary antibody incubation: Wash the slides three times in PBS (pH 7.4) for 5 minutes each time, and spin dry the slides. Add goat anti-rabbit secondary antibody (8889S, Cell Signaling Technology) to the circled area and incubate at room temperature for 60 minutes;

[0057] (6) Nuclear staining: Shake the slide dry, add DAPI staining solution to stain the nuclei, and incubate at room temperature for 10 min in the dark;

[0058] (7) Autofluorescence quenching: Shake the slide dry, add autofluorescence quencher, incubate for 5 minutes, and then rinse under running water for 10 minutes to reduce the interference of autofluorescence;

[0059] (8) Sealing and microscopic examination: The slides were washed three times in PBS (pH 7.4) for 5 minutes each time. After drying, they were sealed with anti-fluorescence quenching sealing agent to protect the fluorescence signal. The slides were placed under a confocal fluorescence microscope to collect images.

[0060] 3 Experimental results

[0061] Figure 1Immunofluorescence was used to detect the expression of NLRC3 in carotid artery plaques and normal carotid intima of patients with coronary heart disease. The results showed that the expression level of NLRC3 in carotid artery plaques was significantly lower than that in normal carotid arteries.

[0062] Example 2: Expression of NLRC3 in peripheral blood mononuclear cells (PBMCs) of patients with coronary atherosclerotic heart disease and patients with normal coronary arteries.

[0063] 1. Research subjects

[0064] All patients, including patients with coronary heart disease and those with normal coronary arteries, signed informed consent.

[0065] All patients with coronary atherosclerotic heart disease (CAD) underwent coronary angiography for confirmation. The diagnosis of CAD followed the diagnostic criteria established by the American Heart Association (AHA) and the American College of Cardiology (ACC). All CAD patients were excluded from the study. Exclusion criteria: ① cardiomyopathy, myocarditis, aortic dissection, various acute and chronic infectious diseases, pulmonary embolism, and other non-coronary artery-related thrombotic diseases and autoimmune diseases; ② patients with malignant tumors, recent major trauma or surgery, severe liver and kidney damage, critical conditions (such as cardiogenic shock), and patients using intraaortic balloon pump; ③ individuals who did not agree to participate in the study.

[0066] Normal coronary artery patients were defined as patients whose coronary artery was normal after coronary angiography. Patients with normal coronary artery were matched with CAD patients by age, gender, hypertension, and diabetes. In the end, peripheral blood samples of 80 CAD patients and 80 patients with normal coronary artery (NCA) were collected. The baseline data of the patients are shown in Table 1. There was no statistically significant difference in the clinical baseline data between the two groups of patients (all P values ​​were > 0.05).

[0067] Table 1

[0068]

[0069] 2. PBMC Isolation and Total RNA Extraction

[0070] 2.1 PBMC Isolation

[0071] (1) Specimen collection: 3 mL of peripheral venous blood was collected from all patients using EDTA anticoagulant tubes when they were admitted to the emergency department;

[0072] (2) Cell separation: Peripheral venous blood was placed at room temperature for 30 min, then centrifuged at 3000 g for 15 min at room temperature, and the upper plasma was discarded;

[0073] (3) Blood sample dilution: After separating the plasma, transfer the blood cells to a 15 mL centrifuge tube, add PBS according to the ratio of PBS: blood cells = 1:2, and mix gently to form a cell suspension;

[0074] (4) Density gradient centrifugation: Add 5 mL of lymphocyte separation solution (Mede Pacific) to a new 15 mL centrifuge tube. Use a pipette to draw up the cell suspension from the previous step and slowly add it to the surface of the lymphocyte separation solution. Centrifuge at 600 g and 20°C for 25 min.

[0075] (5) Collecting and washing cells: Use a pipette to aspirate the milky white mononuclear cell layer (PBMC) in a ring shape and transfer it into another 15 mL centrifuge tube. Add 10 mL PBS to mix the cells and centrifuge at 250 g for 10 min at room temperature. Discard the supernatant and add 5 mL PBS to resuspend the cells. Centrifuge at 250 g for 10 min at room temperature. Discard the supernatant and add 0.2 mL PBS to transfer the cell suspension into a 1.5 mL EP tube and store at -80 °C for later use.

[0076] 2.2 Total RNA extraction

[0077] The total RNA of cells was extracted by Trizo method. The specific steps are as follows:

[0078] (1) Cell lysis: Add 1 mL of Trizol reagent (65307-12-2, Invitrogen) to the EP tube containing PBMCs, vortex mix for 3 min to ensure that the cells are fully lysed, and let stand for 10 min; add 200 μL of chloroform, invert and mix, let stand for 3 min, and centrifuge at 12,000 g, 4°C for 15 min;

[0079] (2) RNA precipitation: aspirate the supernatant aqueous phase (about 0.5 mL) and transfer it to a new 1.5 mL EP tube. Add isopropanol in the same volume as the water. Mix by inversion and let stand for 10 min to promote the formation of RNA precipitation. Centrifuge at 12,000 g, 4°C for 10 min.

[0080] (3) RNA rinsing and dissolving: discard the supernatant, retain the white RNA precipitate at the bottom of the tube, add 1 mL of pre-cooled 75% ethanol, wash the precipitate, and centrifuge at 12000g, 4°C for 5 min; aspirate the ethanol, and dry the RNA precipitate at room temperature for 10 min; add 30-50 μL of 56°C preheated DEPC water to dissolve the RNA, depending on the size of the precipitate;

[0081] (4) RNA quality control: Take 1.5 μL RNA sample and use a UV spectrophotometer (IMPLEN-NP80) to detect the concentration and purity. The quality of RNA is determined by measuring the A260 / 280 ratio. A ratio between 1.8 and 2.0 indicates that the RNA sample is of qualified quality.

[0082] 3. Reverse Transcription

[0083] Reverse transcription kit (TAKARA PrimeScript TM RT Master Mix) for reverse transcription reaction. According to the instructions of the kit, prepare 10μL of reverse transcription reaction system, add 500ng of total RNA template to the reaction system, add 2μL buffer (5X PrimeScript RT Master Mix), and use nuclease-free water to make up the volume if it is less than 10μL, and mix gently; after mixing, use a PCR instrument (ABI7500) for reverse transcription reaction to obtain cDNA template. The reverse transcription reaction conditions are as follows: 37℃15min (reverse transcription reaction); 85℃5s (inactivation reaction of reverse transcriptase).

[0084] 4. Template dilution

[0085] Add 90 μL of RNase-free water to the obtained 10 μL of cDNA template to dilute the cDNA template.

[0086] 5. Real-time fluorescence quantitative PCR primer design and screening

[0087] 5.1 Primer design

[0088] According to the NLRC3 transcript sequence (NM 178844.4), a total of 3 pairs of NLRC3 primers and 1 pair of GAPDH primers were designed. The primers were synthesized by Sangon Biotech Co., Ltd. The specific primer sequences are shown in Table 2:

[0089] Table 2

[0090]

[0091] 5.2 Primer screening

[0092] ① Analysis of primer specificity by fluorescence quantitative PCR reaction

[0093] Five patients' cDNA templates were randomly selected and fluorescent quantitative PCR was performed using the NLRC3 primers of group 1, group 2, and group 3, respectively. TM Premix Ex Taq TM II amplification kit for fluorescence quantitative PCR reaction, according to the kit instructions, prepare 10 μL of reaction buffer system. TM Premix Ex Taq TMII amplification kit for fluorescence quantitative PCR reaction. According to the kit instructions, 10 μL of reaction buffer system (including 5 μL TB Green buffer, 0.4 μL forward primer, 0.4 μL reverse primer, 0.2 μL ROX reference dye, 2 μL cDNA template, 2 μL deionized water) was prepared. Amplification reaction conditions: 95 ° C 10 min, 1 cycle (pre-denaturation); 95 ° C 5 s, 60 ° C 60 s, 40 cycles (PCR reaction). Amplification was performed using ABI7500 fluorescence quantitative PCR instrument, and the Ct value of NLRC amplified by each primer was analyzed to evaluate the primer specificity.

[0094] ②Agarose gel electrophoresis to verify primer specificity

[0095] Use 1x TBE buffer to prepare 50mL of 3% agarose gel, heat at 100℃ for 2min until the agarose is dissolved, cool to 60℃, add 5μL of GelRed indicator, and after the gel solidifies, add 1μL of 10x DNA loading buffer to each tube of PCR product. Use DL500 DNA Marker as a reference, perform electrophoresis at 100V for 90min, image the gel using a high-sensitivity imaging system, identify the PCR products, and evaluate the primer specificity.

[0096] 6. Fluorescence quantitative PCR reaction to detect NLRC3 expression in samples

[0097] 6.1 Reaction system

[0098] Using TB Green TM Premix Ex Taq TM II amplification kit for fluorescence quantitative PCR reaction. According to the instructions of the kit, prepare 10 μL of reaction buffer system. For the specific preparation method, refer to Table 3. Vortex the reaction buffer and use real-time fluorescence quantitative PCR instrument for amplification reaction. Amplification reaction conditions: 95℃10min, 1 cycle (pre-denaturation); 95℃5s, 60℃60s, 40 cycles (PCR reaction).

[0099] Table 3 Reaction buffer system

[0100]

[0101] 6.2 Analysis of Fluorescence Quantitative PCR Reaction Results

[0102] GAPDH was used as the internal reference gene and 2 -△Ct The relative quantitative analysis of NLRC3 was performed by the method.

[0103] 7. Correlation analysis: Coronary stenosis scores (modified Gensini score) were performed based on the results of coronary angiography in patients with coronary heart disease. The scoring method is as follows: the extent and severity of proximal coronary artery lesions are scored by scoring each lesion site: less than 50% luminal diameter stenosis, 1 point; 50%-74% stenosis, 2 points; 75%-99% stenosis, 3 points; complete occlusion, 4 points. The scores of each lesion in the proximal coronary circulation are summarized to obtain the final total score of the severity of coronary atherosclerosis (Gensini score).

[0104] 8 Experimental results

[0105] 8.1 Fluorescence quantitative PCR reaction and agarose gel electrophoresis analysis of primer specificity

[0106] The results of the amplification of 5 samples by different primer groups are shown in Table 4: the Ct value of NLRC3 amplified by primer group 1 is 25.2±0.5, the Ct value of NLRC3 amplified by primer group 2 is 25.2±0.6, and the Ct value of NLRC3 amplified by primer group 3 is 24.2±2.5. It can be seen that the efficiency of NLRC amplification by the three primer groups is good. Figure 2 As shown, the agarose gel electrophoresis results showed that the amplified product of group 1 had only one band, the amplified product of group 2 primers had non-specific bands in sample 5, and the amplified product of group 3 primers had non-specific bands in samples 1 / 2 / 4 / 5. Based on the above results, the primers of group 1 were more specific, and the primers of group 1 were selected for subsequent experiments.

[0107] Table 4 Expression of NLRC amplified by primers in different groups

[0108]

[0109] 8.2 Detection of NLRC3 expression in patients with coronary heart disease by quantitative PCR

[0110] like Figure 3 As shown in the figure, the results of fluorescence quantitative PCR showed that the relative expression of NLRC3 in peripheral blood PBMC of patients with coronary heart disease (0.0067±0.0006) was significantly lower than that of the normal coronary artery group (0.0223±0.0014). The Student's t test was used to calculate the P value and the difference was found to be statistically significant (P<0.001). Figure 4 As shown in the data, in patients with coronary heart disease, the Spearman test was used to calculate the correlation between NLRC3 expression and Gensini score. The results showed that NLRC3 expression was significantly correlated with Gensini score (r=-0.7933, P<0.001), suggesting that NLRC3 expression is correlated with the degree of coronary artery stenosis in patients with coronary heart disease.

[0111] Example 3: Evaluation of the diagnostic value of NLRC3 in patients with coronary heart disease

[0112] like Figure 5 As shown in the figure, the receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of NLRC3 in patients with coronary heart disease. The results showed that the AUC value of the ROC curve was 0.926, and the 95% confidence interval was 0.889-0.963 (P<0.001). When the diagnostic threshold of the relative expression of NLRC3 was set at 0.012, the sensitivity of diagnosing coronary heart disease was 85% and the specificity was 80%, indicating that NLRC3 has a good value as a circulating diagnostic marker for coronary heart disease.

[0113] Example 4: Verify the accuracy of NLRC3 in diagnosing coronary heart disease.

[0114] 1. Research subjects and methods

[0115] Peripheral venous blood samples were collected from 32 patients who were admitted to the emergency department for chest pain. The baseline data are recorded in detail in Table 5. Peripheral blood was collected from these patients at the time of emergency admission, and peripheral blood mononuclear cells (PBMCs) were isolated. Subsequently, the expression level of the NLRC3 gene was detected by fluorescent quantitative PCR technology to screen out patients who may have coronary heart disease. In addition, the NLRC3 expression results of these patients were compared with the results of subsequent coronary angiography to evaluate the accuracy and reliability of NLRC3 as a biomarker for the diagnosis of coronary heart disease. Through this method, it is hoped that new biomarkers can be provided for the clinical diagnosis of patients with chest pain and provide a scientific basis for the early identification and treatment of coronary heart disease.

[0116] Table 5

[0117]

[0118] 2. Blood collection method

[0119] All patients had 3 mL of peripheral venous blood collected when they were admitted to the emergency department.

[0120] 3. Peripheral Blood Mononuclear Cell Isolation and Total RNA Extraction

[0121] Peripheral blood mononuclear cells (PBMCs) were isolated from the patient's peripheral blood samples using density gradient centrifugation following the same procedure as in Example 2. Total RNA was then extracted from these isolated PBMCs using the same Trizol reagent extraction method as in Example 2.

[0122] 4. Accuracy of reverse transcription and qRT-PCR detection of NLRC3 in diagnosing coronary heart disease

[0123] The same experimental method and system as in Example 2 were used to perform reverse transcription and fluorescence quantitative PCR reactions. -△Ct The method used GAPDH as the internal reference gene to perform relative quantitative analysis on the expression level of the NLRC3 gene. The NLRC3 expression value was set below 0.0012 as the diagnostic threshold, and this result was compared with the subsequent coronary angiography results of patients with chest pain to evaluate the accuracy of NLRC3 as a biomarker for diagnosing coronary heart disease.

[0124] 5. Experimental Results

[0125] The experimental results are detailed in Table 6. Using the NLRC3 expression level below 0.012 as the diagnostic threshold, 32 patients with chest pain were diagnosed. According to this standard, 23 patients with coronary heart disease were identified, while 9 patients with chest pain symptoms caused by other diseases. At the same time, the results of coronary angiography showed that among these patients with chest pain, 20 actually had coronary heart disease, while 12 had chest pain symptoms caused by other diseases.

[0126] The relative expression level of NLRC3 was used as a diagnostic test and compared with the results of coronary angiography. The results showed that the diagnostic test could accurately identify 19 "true positive" patients (coronary heart disease) and 8 "true negative" patients (non-coronary heart disease patients). By calculating the proportion of true positive and true negative results in the total number of cases tested, the accuracy (Acc) of the test reached 84.4%. This result shows that the NLRC3 expression level has high accuracy and reliability as a diagnostic tool in diagnosing coronary heart disease.

[0127] Table 6

[0128]

[0129] Example 5: A fluorescent quantitative PCR kit for diagnosing coronary heart disease

[0130] 1. Composition

[0131] NLRC3 and GAPDH primers (10 μM, sequences as shown in Table 7), SYBR Green buffer (2X), ROX reference dye (50X) and deionized water.

[0132] Table 7 Primer sequences

[0133]

[0134] 2. The reaction system preparation method is shown in Table 8

[0135] Table 8

[0136]

[0137] 3. Reaction conditions

[0138] Pre-denaturation: 95°C for 10 min (1 cycle); PCR reaction: 95°C for 15 s, 60°C for 60 s (40 cycles).

[0139] 4. Results Analysis

[0140] Adoption 2 -△Ct The relative quantitative analysis of NLRC3 was performed using GAPDH as an internal reference.

[0141] Example 6: ELISA detection of NLRC3 expression in plasma of patients with coronary heart disease and control patients

[0142] 1. Research subjects

[0143] Patients with coronary heart disease (100 cases) and patients with normal coronary arteries (92 cases), the specimens were from all patients in Example 2 and Example 4. The baseline data are shown in Table 9 (all P values>0.05), and the two groups of patients are comparable.

[0144] Table 9

[0145]

[0146]

[0147] 2. Sample Type

[0148] All patients had 3 mL of plasma separated from peripheral venous blood collected when they were admitted to the emergency department.

[0149] 3. Detection methods

[0150] The human NLRC3 ELISA kit (AE30906HU, Abebio) was used to detect the plasma NLRC3 concentration in patients with coronary heart disease and those with normal coronary arteries. The operation was carried out according to the manufacturer's instructions. The specific steps are as follows:

[0151] ① Dissolve the standard and dilute the NLRC3 standard using the doubling dilution method;

[0152] ② Add 100 μL of diluted standard and sample to the ELISA plate and incubate at 37°C for 2 h;

[0153] ③ Wash the plate three times using an automatic plate washer, add 100 μL of diluted biotinylated antibody to each well, and incubate at 37°C for 1 h;

[0154] ④ Wash the plate three times, add 100 μL of HRP-labeled streptavidin complex to each well, and incubate at 37°C for 1 h;

[0155] ⑤ Wash the plate 5 times, add 100 μL of color substrate to each well, and incubate at 37°C for 20 min;

[0156] ⑥ Add 50 μL of stop buffer to each well and detect using an enzyme reader at a wavelength of 450 nm.

[0157] 4. Experimental Results

[0158] like Figure 6 As shown, the plasma NLRC3 level in patients with coronary heart disease (1.09±0.04ng / mL) was significantly lower than that in patients with normal coronary arteries (2.30±0.07ng / mL). The Student's t test was used to calculate the P value, and the difference was found to be statistically significant (P<0.001).

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of NLRC3 in the preparation of products for diagnosis, screening or assessment of coronary atherosclerotic heart disease.

2. The use according to claim 1, characterized in that: The expression level of NLRC3 in patients with coronary heart disease was significantly lower than that in patients with normal coronary arteries.

3. The use according to claim 1, characterized in that: The expression level of NLRC3 in carotid plaques was significantly lower than that in normal carotid arteries.

4. The use according to claim 1, characterized in that: The product diagnoses, screens or evaluates coronary atherosclerotic heart disease by detecting the expression of the NLRC3 gene in a sample.

5. Use of products for detecting NLRC3 gene expression in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease.

6. The use according to claim 5, characterized in that: The products for detecting NLRC3 gene expression include: products for detecting NLRC3 gene expression levels by RT-PCR, fluorescent quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform.

7. The use according to claim 6, characterized in that: The product for detecting the expression level of the NLRC3 gene by RT-PCR includes at least a pair of primers that specifically amplify the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by fluorescent quantitative PCR includes at least a pair of primers that specifically amplify the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by immunoassay includes at least an antibody that specifically binds to the NLRC3 protein; the product for detecting the expression level of the NLRC3 gene by in situ hybridization includes at least a probe that hybridizes to the nucleic acid sequence of the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by chip includes a protein chip or a gene chip, the protein chip includes an antibody that specifically binds to the NLRC3 protein, and the gene chip includes a probe that hybridizes to the nucleic acid sequence of the NLRC3 gene.

8. Application of NLRC3 in the preparation of fluorescent quantitative PCR kit for diagnosing coronary heart disease.

9. The use according to claim 8, characterized in that: The kit comprises at least a pair of primers for specifically amplifying the NLRC3 gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2.

10. A fluorescent quantitative PCR kit for diagnosing coronary heart disease, characterized in that: The kit comprises at least a pair of primers for specifically amplifying the NLRC3 gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; the kit also comprises a GAPDH primer sequence; the GAPDH primer sequence is shown in SEQ ID NO.7 and SEQ ID NO.8.

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