Reagent for diagnosing or treating pulmonary arterial hypertension and application thereof

Abstract

The invention provides a reagent for diagnosing and / or treating pulmonary arterial hypertension and application of the reagent, and relates to the technical field of pulmonary artery diagnosis and / or treatment. The key genes for diagnosis and treatment of pulmonary arterial hypertension comprise HBG2, PRKAB1 and THBD, potential targets related to PH diagnosis are found, and the key genes are of great significance to PH management and treatment. Meanwhile, the correlation between the key gene and the PH disease and NO is further verified through in-vivo experiments of peripheral blood of a patient and an animal model, and a new thought is further provided for diagnosis and treatment of PH.

Description

Technical Field

[0001] The present invention relates to the technical field of pulmonary artery treatment, and in particular to a reagent for diagnosing and treating pulmonary hypertension and application thereof. Background Art

[0002] Pulmonary Hypertension (PH) is a global disease. Epidemiological statistics show that the incidence of PH is about 1% of the world's population, and even reaches 10% in the elderly over 65 years old. Pulmonary hypertension is a progressive and fatal disease characterized by increased pulmonary artery pressure, followed by right ventricular failure and even death, which requires continuous treatment.

[0003] Currently, the drugs approved for the treatment of pulmonary hypertension include endothelin receptor antagonists, prostacyclin analogs and PDE5 inhibitors, which treat PH by regulating pulmonary artery vascular tension. Although these drugs and therapies can alleviate the symptoms of PH, they cannot reverse the progression of the disease or cure the disease, let alone reduce the mortality rate of patients with pulmonary hypertension. The 5-year mortality rate of PH is still as high as 40%.

[0004] Therefore, finding potential diagnostic and therapeutic targets for PH, achieving early diagnosis of PH, and discovering effective drugs are still urgent issues to be solved in the treatment of PH. Summary of the invention

[0005] 1. Technical issues to be resolved In view of the deficiencies of the prior art, the present invention provides a reagent for diagnosing and treating pulmonary hypertension and its application.

[0006] (II) Technical solution To achieve the above objectives, the present invention is implemented through the following technical solutions: In one aspect, the present invention provides a use of HBG2, PRKAB1 and / or THBD gene as a target in diagnosing and / or treating pulmonary arterial hypertension in a subject.

[0007] In one aspect, the present invention provides the use of a substance for detecting HBG2, PRKAB1 and / or THBD gene in a subject in the preparation of a reagent for diagnosing pulmonary arterial hypertension.

[0008] In one embodiment, the material for detecting HBG2, PRKAB1 and / or THBD genes comprises a primer pair for detecting HBG2, PRKAB1 and / or THBD.

[0009] In one embodiment, the forward primer HBG2-F of the primer pair for detecting HBG2 is a nucleotide sequence as shown in SEQ ID NO: 1; the reverse primer HBG2-R is a nucleotide sequence as shown in SEQ ID NO: 2.

[0010] In one embodiment, the forward primer PRKAB1-F of the primer pair for detecting PRKAB1 is a nucleotide sequence as shown in SEQ ID NO: 3; the reverse primer PRKAB1-R is a nucleotide sequence as shown in SEQ ID NO: 4.

[0011] The forward primer THBD-F of the primer pair for detecting THBD is the nucleotide sequence shown in SEQ ID NO: 5; the reverse primer THBD-R is the nucleotide sequence shown in SEQ ID NO: 6.

[0012] In one aspect, the present invention provides a primer pair for detecting the HBG2 gene of a subject, wherein the nucleotide sequence of the forward primer HBG2-F of the primer pair is shown in SEQ ID NO: 1; and the nucleotide sequence of the reverse primer HBG2-R is shown in SEQ ID NO: 2.

[0013] In one aspect, the present invention provides a primer pair for detecting the PRKAB1 gene of a subject, wherein the nucleotide sequence of the forward primer PRKAB1-F of the primer pair is shown in SEQ ID NO: 3; and the nucleotide sequence of the reverse primer PRKAB1-R is shown in SEQ ID NO: 4.

[0014] In one aspect, the present invention provides a primer pair for detecting the THBD gene of a subject, wherein the nucleotide sequence of the forward primer THBD-F of the primer pair is shown in SEQ ID NO: 5; and the nucleotide sequence of the reverse primer THBD-R is shown in SEQ ID NO: 6.

[0015] In one aspect, the present invention provides a PCR reaction solution, which comprises any of the primers shown above.

[0016] In one aspect, the present invention provides a kit for diagnosing pulmonary hypertension in a subject, the kit comprising any of the primers shown above or the above PCR reaction solution.

[0017] In one aspect, the present invention provides a use of any one of the above primer pairs or kits in diagnosing pulmonary hypertension.

[0018] In one embodiment, the subject described above includes a human.

[0019] (III) Beneficial effects The present invention provides a kit for diagnosing and treating pulmonary hypertension and its application. Compared with the prior art, it has the following beneficial effects: 1. The present invention proposes that key genes for the diagnosis and treatment of pulmonary hypertension include HBG2, PRKAB1 and THBD, and finds potential targets for diagnosing PH, which is of great significance for the management and treatment of PH.

[0020] 2. The present invention further verified the correlation between key genes and PH disease and NO through in vivo experiments on patient peripheral blood and animal models, and further provided new ideas for the diagnosis and treatment of PH. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 1A is the PCR differential analysis of PH key genes; 1B is the PCR differential analysis of HBG2 genes; 1C is the PCR differential analysis of THBD genes. Normal is the control group, Patient is the disease group; "*" indicates p < 0.05, "**" indicates p < 0.01.

[0023] Figure 2 The figure is a schematic diagram of pulmonary artery systolic pressure three weeks after MCT injection in the example. SPAP is pulmonary artery systolic pressure, and Model is a rat group with PH after injection of monocrotaline. "***" indicates p < 0.001.

[0024] Figure 3Figure 3A-C are the pressure waveforms measured by the right ventricular catheter in the control group, MCT group and MCT (+NO) group respectively; the ordinate is pressure, unit: mmHg; the abscissa is time. The oscilloscope scanning time factor is 1 s / div (scanning time occupied by each large grid in the horizontal direction). Figure 3D is a model diagram of the right ventricular pressure measurement through the right external jugular vein of rats; the position of the catheter inserted into the external jugular vein in the black box in the figure. Figure E is the right ventricular pressure measured by the right ventricular catheter pressure measurement method in each group. SPAP: pulmonary artery systolic pressure. MCT: oxygen inhalation group after successful modeling of monocrotaline pulmonary hypertension; MCT (+NO): NO inhalation group after successful modeling of monocrotaline pulmonary hypertension. "**" indicates p < 0.01, "***" indicates p < 0.001, and "ns" indicates no statistical difference.

[0025] Figure 4 Figure 4A is a comparison of the right heart hypertrophy index and the lung exudation index in the embodiment of the present invention; Figure 4B is a comparison of the lung exudation index. RV / (LV+S): right heart hypertrophy index; RV: right ventricular free wall; (LV+S): left ventricle plus ventricular septum. LW / BW: lung exudation index; LW: lung mass, BW: rat body weight. MCT group: monocrotaline pulmonary hypertension model group, MCT (+NO) group: MCT model was successfully established and NO was inhaled. "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "ns" indicates no statistical difference.

[0026] Figure 5 5A is the HE staining result of rat lung and the percentage of pulmonary artery wall thickness in the embodiment of the present invention. 5A is the HE staining result of rat pulmonary artery in the control group; 5B is the HE staining result of rat pulmonary artery in the MCT experimental group; 5C is the HE staining result of rat pulmonary artery in the MCT (+NO) experimental group; 5D is the percentage of pulmonary artery wall thickness in rats in each group. Pulmonary artery wall thickness percentage: WT%=(2×WT / ED)×100%, WT: pulmonary artery wall thickness, ED: pulmonary artery outer diameter. MCT: monocrotaline pulmonary hypertension model group, MCT (+NO): MCT modeling successfully followed by NO inhalation group. "**" indicates p<0.01, and "***" indicates p<0.001.

[0027] Figure 6Figure 6A-C are the RT-qPCR results of key genes of rat PH. Figure 6A-C are the qPCR results of key genes HBG, THBD, and PRKAB1 in SD rat tissues. The vertical axis is the relative expression of genes. "***" means p < 0.001, "**" means p < 0.01, "*" means p < 0.05, and "ns" means p > 0.05, with no statistical difference.

[0028] Figure 7 Schematic diagram of the immunohistochemical results of PRKAB1 protein in the embodiment of the present invention. 7A-C are the expression level and distribution of PRKAB1 protein in the lung tissue of rats in the control group, MCT group and MCT (+NO) group, respectively; 7D is the result of immunohistochemical analysis. "***" indicates p < 0.001, "**" indicates p < 0.01, and "*" indicates p < 0.05.

[0029] Figure 8 Schematic diagram of the immunohistochemical results of THBD protein in the embodiment of the present invention. 8A-C are the expression level and distribution of THBD protein in the lung tissue of rats in the control group, MCT group and MCT (+NO) group, respectively; 8D is the result of immunohistochemical analysis. "***" means p < 0.001, "**" means p < 0.01, and "*" means p < 0.05. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiments of the present application provide a kit for diagnosing and treating pulmonary hypertension and its application, identify key genes related to inhaled NO treatment of PH, and provide new ideas for the diagnosis and treatment of PH.

[0032] In the embodiments of the present application, the functions and mechanisms of the related terms are explained as follows: Peripheral blood refers to blood other than bone marrow, which can be obtained through superficial peripheral blood vessels of the limbs or through deep blood vessel puncture in the neck, groin, or subclavian.

[0033] Real-time fluorescence quantitative PCR (Quantitative Real-time PCR) is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. It is a method for quantitatively analyzing specific DNA sequences in the sample to be tested through internal or external reference methods.

[0034] Monocrotaline (MCT) is a dipyrrole alkaloid that is converted by P450 monooxygenase in the liver and reaches the lungs through the blood circulation, causing irreversible damage to the pulmonary blood vessels. Pulmonary vascular endothelial cells are considered to be the target cells of MCT, and endothelial cell damage plays a key role in the process of pulmonary vascular reconstruction.

[0035] Right ventricular hypertrophy index (RVHI) is the ratio of right ventricular wall thickness to left ventricular wall thickness. Most patients with pulmonary hypertension eventually develop right ventricular hypertrophy, dilatation, and right ventricular failure.

[0036] Immunohistochemistry is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. It uses chemical reaction to make the color developer (fluorescein, enzyme, metal ion, isotope) labeled with antibody develop color to determine the antigen (peptide and protein) in tissue cells, and conduct localization, qualitative and relative quantitative research on it. This is called immunohistochemistry or immunocytochemistry.

[0037] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0038] Example 1: Patient RNA extraction and quantitative RT-PCR

[0039] Peripheral blood total RNA was extracted and reverse transcribed using TRIzol reagent according to the instructions. RT-PCR was performed using the LightCycler480II qRT-PCR detection system (Roche, USA). The -2−ΔΔCt method was used to calculate the expression of related genes. Actin was selected to regulate the expression level of target genes. All experiments were performed independently in triplicate.

[0040] like Figure 1 As shown, the diagnostic marker RT-qPCR validation results showed that THBD and HBG2 were significantly upregulated, and PRKAB1 was significantly downregulated in PH patients compared with the control group.

[0041] The specific primer sequences are as follows: serial number name sequence SEQ ID NO: 1 HBG2-F 5′-CCAGAGGTTCTTTGACAGCTTTG-3′ SEQ ID NO: 2 HBG2-R 5′-ACTATTCCCAGGAGCTTGAAGTT-3′ SEQ ID NO: 3 PRKAB1-F 5′-GACGCCGACCTCTTCCACTC-3′ SEQ ID NO: 4 PRKAB1-R 5′-GGGCTTTACATTCACTTCCAG-3′ SEQ ID NO: 5 THBD-F 5′-CACAGGCAGTCAATGCGTG-3′ SEQ ID NO: 6 THBD-R 5′-GAGCGCACTGTCATCAAATGT-3′ SEQ ID NO: 7 ACTB-F 5′-GTGACGTTGACATCCGTAAAGA-3′ SEQ ID NO: 8 ACTB-R 5′-GCCGGACTCATCGTACTCC-3′ The specific steps of patient RNA extraction and quantitative RT-PCR are as follows: (1) Extraction of total RNA 1) Experimental preparation: chloroform, Trizol lysis buffer, isopropanol, DEPC water, sterile PBS, 1.5 mL enzyme-free EP tube, absorbent paper, pipettes of different specifications and enzyme-free Tip heads, etc.; 2) Collect human whole blood samples using heparin anticoagulant tubes. Lyse the red blood cells using red blood cell lysis buffer, centrifuge at 12,000 r, 4°C for 15 min, take the middle layer of white blood cells, and dispense into 1.5 mL enzyme-free EP tubes; 3) Add 1 mL of lysis buffer to the enzyme-free EP tube, incubate for 5 minutes, and gently pipette more than 30 times; 4) Add 200 μL of chloroform to the enzyme-free EP tube, shake vigorously for 15-20 seconds, and place on ice for 10 minutes; 5) Centrifuge at 12000 r, 4 ℃ for 15 min. There are 3 layers in the tube from bottom to top: organic layer, protein layer and aqueous layer containing RNA. Use a blunt tip to gently take 500 μL of the aqueous layer liquid and add it to the enzyme-free EP tube. Do not touch the tube wall when taking the liquid. Add 500 μL of isopropanol to the tube, shake it gently, and let it stand on ice for 15 min. 6) Centrifuge at 12000 r, 4 ℃ for 15 min, remove the supernatant, add 1 mL of 75% ethanol to the tube to dissolve the precipitate. The precipitated flocs are RNA, which should be dispersed by gently blowing several times; 7) Centrifuge at 12000 r, 4 ℃ for 15 min, remove ethanol, open the EP tube and invert it on absorbent paper, and dry it for 10 min; 8) 20 μL of DEPC water was dropped into each EP tube to dissolve the precipitate and obtain total RNA; 9) After zeroing the spectrophotometer, drop 1 μL of total RNA, close the test arm, start the test, and register the A260 / A280 ratio and RNA concentration.

[0042] (2) Reverse transcription of RNA cDNA was synthesized using a reverse transcription kit. The reverse transcription conditions were set as follows: 37°C for 15 min; 85°C for 5 s; 10 µL system.

[0043] Configure the reaction system: Prepare a mixed solution in an RNase-free centrifuge tube: 5×HiScript ⅡqRT SuperMix 2 μL + template RNA (total RNA 1 pg-1 μg), and dilute to 10 μL with enzyme-free sterile water. Finally, pipette gently and evenly.

[0044] (3) Real-time fluorescence quantitative PCR cDNA amplification was performed using a fluorescent quantitative kit with a 20 µL reaction system. A three-step procedure was used to perform the amplification reaction. A careful plan was designed before the start to determine the order in which each reagent was added and the positions of the main and secondary wells for amplification.

[0045] 1) Configure the reaction system: 50×ROX Reference Dye1 0.4 μL + template cDNA 1 μL + upstream and downstream primers (10 μM) 0.4 μL each + 2×AceQ qPCR SYBR Green Master Mix 10 μL, and dilute to 20 μL with enzyme-free sterile water. Finally, mix gently with a pipette.

[0046] 2) Design of target gene primer sequence: Primers were designed based on the primerbank website (https: / / pga.mgh.harvard.edu / primerbank / ) and synthesized by Qingke Biotechnology Co., Ltd.

[0047] 3) Calculation process a) Record the mean Ct values ​​of the internal reference and target genes in the control group and the experimental group. The ΔCt is the CT of the target gene minus the CT of the internal reference. b) ΔΔCt is the difference between ΔCt of the experimental group and ΔCt of the control group: 2 -ΔΔCt This is the amplification factor.

[0048] Example 2: Pulmonary hypertension animal model experiment

[0049] 1. Modeling (1) Forty 6-week-old female Sprague-Dawley rats (160-200 g) were randomly divided into a blank control group of 12 rats and a monocrotaline (MCT) experimental group of 28 rats.

[0050] (2) Monocrotaline was dissolved in a mixture of physiological saline and ethanol (8:2) to a 2% solution. The rats in the monocrotaline group were subcutaneously injected with MCT at a dose of 60 mg / kg. The rats in the control group were subcutaneously injected with physiological saline based on their body weight.

[0051] (3) Two groups of rats were kept in the same SPF breeding room with sufficient water and food, free movement, and observation every day.

[0052] like Figure 2 As shown in the figure, the pulmonary artery systolic pressure was measured 3 weeks after MCT injection, and the pulmonary artery systolic pressure of SD rats in the MCT experimental group was significantly higher than that in the control group, indicating that the monocrotaline-induced PH model was successfully established.

[0053] 2. Model testing and inhaled NO Three weeks after MCT administration, right ventricular systolic pressure was detected by ultrasound and right cardiac catheterization. Rats with successfully modeled increased pulmonary artery pressure were screened out from the experimental group and randomly divided into two groups. One group was the MCT (+NO) group, which was given 1 L / min NO + 4 L / min oxygen for 60 min / d for 3 days; the other group was the MCT group, which was given 4 L / min oxygen for 60 min / d for 3 days.

[0054] Hemodynamic index determination: After anesthetizing the rats, fix them on the operating table. A pulmonary artery catheter filled with heparin water is placed through the free right external jugular vein, through the right atrium and right ventricle to the pulmonary artery, and then connected to the pressure transducer. The rat pulmonary artery systolic pressure and right ventricular systolic pressure (RVSP) are collected using a multi-channel physiological signal acquisition instrument. The pulmonary artery systolic pressure is consistent with the right ventricular systolic pressure when there is no tricuspid regurgitation.

[0055] In the right ventricular catheter pressure measurement experiment, rats with increased pulmonary artery pressure were selected from the monocrotaline experimental group as the model group, and the model group was randomly divided into the MCT group and the MCT (+NO) group. Figure 3 As shown in the figure, according to the right ventricular catheter pressure measurement results, the pulmonary artery systolic pressure in the MCT group was significantly increased compared with that in the control group, indicating that the PH model was successful; compared with the MCT group, the pulmonary artery systolic pressure in the MCT (+NO) group was significantly decreased, indicating that inhaled NO had a therapeutic effect on the PH model established by monocrotaline.

[0056] Example 3: Cardiopulmonary Weighing Experiment

[0057] After the intervention of inhaled NO, the right ventricular systolic pressure was measured again by ultrasound and right cardiac catheterization, and then the heart and lungs were weighed: after the pulmonary hemodynamic index was measured, the chest cavity was opened, normal saline was injected into the pulmonary blood vessels, the blood in the lungs was washed out, the hilum was cut off, the water on the surface of the lungs was absorbed with absorbent paper, the lungs were weighed, and the lung mass (LW) was recorded; the heart was taken out, the atrium was cut off, the left ventricle plus ventricular septum (LV+S) and the right ventricular free wall (RV) were freed, the blood was washed out, and the water was absorbed with absorbent paper, and the masses were weighed respectively. Finally, the right heart hypertrophy index [RV / (LV+S)] and the lung exudation index [LW / BW(%)] (BW is body weight) were obtained.

[0058] like Figure 4 As shown in the figure, the right heart hypertrophy index and lung exudation index were measured by heart and lung weighing. Compared with the control group, the right heart hypertrophy index and lung exudation index of the MCT group were significantly increased, indicating that the MCT modeling was successful. At the same time, the right heart hypertrophy index and lung exudation index of the MCT (+NO) group were reduced compared with the MCT group.

[0059] These results indicate that inhaled NO can improve right ventricular remodeling and reduce pulmonary edema in MCT pulmonary hypertension model rats, and also prove that inhaled NO has a therapeutic effect on MCT pulmonary hypertension model rats.

[0060] Example 4: HE staining and calculation of rat pulmonary artery wall thickness percentage

[0061] In this example, a portion of the heart and lung tissues in the heart and lung weighing were fixed with neutral formaldehyde and subjected to HE staining. At the same time, the software was used to measure and calculate the percentage of pulmonary artery wall thickness (WT%) of each group of rats in the HE staining results. WT%=(2×WT / ED)×100%, where WT is the pulmonary artery wall thickness and ED is the pulmonary artery outer diameter. The results are shown in FIG. Figure 5 shown. Specifically: Compared with the control group, the pulmonary artery wall of rats in the MCT experimental group was significantly thickened, the cells were unevenly distributed, and the arterial lumen was significantly narrowed. This result once again shows that the MCT pulmonary hypertension model was successfully established. Compared with the MCT group, the thickening of the pulmonary artery wall and the narrowing of the arterial lumen of rats in the MCT (+NO) group were significantly improved. This shows that after the model is successfully established, inhalation of NO can significantly treat pulmonary hypertension in rats caused by monocrotaline.

[0062] Comparison of the percentage of pulmonary artery wall thickness in rats in each group showed that the WT% in the MCT experimental group increased significantly compared with the control group, and the WT% in the MCT (+NO) group decreased significantly after inhalation of NO. This result once again shows that the pulmonary hypertension model in the MCT experimental group was successfully established, and that inhalation of NO after successful establishment can significantly improve the thickening of the pulmonary artery wall in rats caused by monocrotaline, and has a therapeutic effect on the MCT-established pulmonary hypertension model.

[0063] Example 5: RT-qPCR detection

[0064] The other part of the heart and lung tissues from the heart and lung weighing was stored in a -80℃ refrigerator and then subjected to RT-qPCR testing. The frozen rat lung tissues were taken from the tissues near the hilum of the lungs and subjected to RT-qPCR testing. The test results were as follows: Figure 6 shown.

[0065] Compared with the control group, the expression of gene HBG2 in the lung tissues of rats in the MCT group and the MCT (+NO) group was significantly increased; compared with the MCT group, the expression of gene HBG2 in the lung tissues of rats in the MCT (+NO) group was decreased, as shown in Figure 2. Figure 6 As shown in A.

[0066] Compared with the control group, the expression of PRKAB1 gene in the lung tissue of rats in the MCT group and the MCT (+NO) group was significantly decreased; compared with the MCT group, the expression of PRKAB1 gene in the lung tissue of rats in the MCT (+NO) group was increased, as shown in Figure 2. Figure 6 As shown in B.

[0067] Compared with the control group, the expression of THBD gene in the lung tissue of rats in the MCT group was significantly increased; compared with the MCT group, the expression of THBD gene in the lung tissue of rats in the MCT (+NO) group was significantly decreased; in addition, there was no statistically significant difference in the expression of THBD gene in the lung tissue of rats in the control group and the MCT (+NO) group. Figure 6 As shown in C.

[0068] The experimental results showed that compared with the control group, the key genes HBG2 and THBD were expressed more in the lung tissue of MCT-PH model rats, and the PRKAB1 gene was expressed less in the lungs of MCT-PH model rats; and the expression of genes HBG2 and THBD decreased, and the expression of PRKAB1 increased in MCT-PH model rats after inhaling NO. This result is consistent with the previous verification results of this article, and inhaling NO can also improve the expression of target genes, indicating that NO may regulate the expression of these target genes through some way or pathway.

[0069] Example 6: Immunohistochemistry experiment

[0070] In order to further detect the expression level of target genes in the lung tissue of each group of rats, immunohistochemical analysis was performed. A portion of the heart and lung tissues from the heart and lung weighing were fixed with neutral formaldehyde and subjected to immunohistochemistry. Specifically, when conducting the immunohistochemical experiment, the operating steps are as follows: (1) The sections were sequentially placed in xylene I, II, and III for 15 min each, anhydrous ethanol I, II, 85% ethanol, and 75% ethanol for 5 min each, and then washed with distilled water.

[0071] (2) Antigen retrieval on sections to avoid excessive evaporation of buffer and drying of the sections. Wash the slides in PBS on a decolorizing shaker for 6 min, repeat 3 times.

[0072] (3) Incubate the sections in hydrogen peroxide for 20 minutes in the dark. Wash the slides in PBS on a shaker for 6 minutes. Repeat three times.

[0073] (4) Add BSA to fully coat the tissue and block at room temperature for 30 min.

[0074] (5) Remove the blocking solution and add the diluted primary antibody to the sections. Place the sections flat in a humidified chamber and incubate at 4 °C overnight (a small amount of water should be left in the chamber to prevent evaporation of the antibody).

[0075] (6) Wash the slides in PBS on a shaker for 6 min, repeat 3 times. Gently spin dry the sections, add HRP-labeled secondary antibody (same species as the primary antibody) to fully coat the tissue, and incubate at room temperature for 50 min.

[0076] (7) Wash the slides in PBS on a shaker for 6 min, repeat 3 times. Gently shake dry the sections, add freshly prepared DAB colorimetric reagent, and observe the color development under a microscope. Brown-yellow color indicates positive, and the sections are washed with water to stop color development.

[0077] (8) Counterstain the nuclei with hematoxylin for 3 min, wash with running water, differentiate with differentiation solution, wash with running water, re-blue with bluing solution, and wash with running water.

[0078] (9) Place the sections in each reagent in the reverse order of the first step to make them transparent for 5 min each, allow them to dry slightly, and then seal the sections.

[0079] (10) View and analyze images using a microscope.

[0080] Since no suitable antibodies for the expression protein of HBG2 were found, only the expression proteins of PRKAB1 and THBD genes were detected here. ImageJ software was used to analyze the results of immunohistochemistry and calculate the average optical density value. The principle is to determine the relative expression of the target protein based on its distribution area and color depth. After immunohistochemistry experiments, the results are as follows Figure 7 and8 shown.

[0081] like Figure 7 The figure shows the immunohistochemical results of PRKAB1 protein. Compared with the control group, the expression of PRKAB1 protein in the lung tissues of rats in the MCT group and the MCT (+NO) group was significantly reduced; compared with the MCT group, the expression of PRKAB1 protein in the lung tissues of rats in the MCT (+NO) group was increased.

[0082] like Figure 8 The figure shows the immunohistochemical results of THBD protein. Compared with the control group, the expression of THBD protein in the lung tissue of rats in the MCT group and the MCT (+NO) group was significantly increased; compared with the MCT group, the expression of THBD protein in the lung tissue of rats in the MCT (+NO) group was significantly decreased.

[0083] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0084] 1. The present invention proposes key genes for the diagnosis and treatment of pulmonary hypertension, including HBG2, PRKAB1 and THBD, finds potential targets related to NO and PH, and verifies the mechanism of key genes for the treatment of PH by inhaled NO, which is of great significance for the management and treatment of PH.

[0085] 2. The present invention further verified the correlation between key genes and PH disease and NO through in vivo experiments on patient peripheral blood and animal models, and further provided new ideas for the diagnosis and treatment of PH.

[0086] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting HBG2, PRKAB1 and / or THBD gene in a subject in the preparation of a reagent for diagnosing pulmonary arterial hypertension.

2. The use according to claim 1, characterized in that: The material for detecting HBG2, PRKAB1 and / or THBD genes comprises a primer pair for detecting HBG2, PRKAB1 and / or THBD.

3. The use according to claim 2, characterized in that: The forward primer HBG2-F of the primer pair for detecting HBG2 has a nucleotide sequence as shown in SEQ ID NO: 1; the reverse primer HBG2-R has a nucleotide sequence as shown in SEQ ID NO: 2; The forward primer PRKAB1-F of the primer pair for detecting PRKAB1 is a nucleotide sequence as shown in SEQ ID NO: 3; the reverse primer PRKAB1-R is a nucleotide sequence as shown in SEQ ID NO: 4; The forward primer THBD-F of the primer pair for detecting THBD is the nucleotide sequence shown in SEQ ID NO: 5; the reverse primer THBD-R is the nucleotide sequence shown in SEQ ID NO:

6.

4. A primer for detecting the HBG2 gene of a subject, characterized in that: The primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer HBG2-F is shown in SEQ ID NO: 1; the nucleotide sequence of the reverse primer HBG2-R is shown in SEQ ID NO:

2.

5. A primer for detecting the PRKAB1 gene of a subject, characterized in that: The primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer PRKAB1-F is shown in SEQ ID NO: 3; the nucleotide sequence of the reverse primer PRKAB1-R is shown in SEQ ID NO:

4.

6. A primer for detecting the THBD gene of a subject, characterized in that: The primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer THBD-F is shown in SEQ ID NO: 5; the nucleotide sequence of the reverse primer THBD-R is shown in SEQ ID NO:

6.

7. A PCR reaction solution for diagnosing pulmonary hypertension in a subject, characterized in that: The PCR reaction solution comprises the primers shown in any one of claims 4-6.

8. A kit for diagnosing pulmonary hypertension in a subject, characterized in that: The kit comprises the primers shown in any one of claims 4 to 6 or the PCR reaction solution according to claim 7.

9. The use according to any one of claims 1 to 3, the primers according to any one of claims 4 to 6, the PCR reaction solution according to claim 7, or the kit according to claim 8, characterized in that: The subject includes humans.

10. Use of the primers as claimed in any one of claims 4 to 6, the PCR reaction solution as claimed in claim 7, or the kit as claimed in claim 8 in the preparation of a reagent or kit for diagnosing pulmonary arterial hypertension.

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