Application of lung tissue sample molecular marker in vascular disease diagnosis

Abstract

The invention provides application of a lung tissue sample molecular marker in vascular disease diagnosis, in particular to application of an NRP1 / EFNB2 / KDR gene in vascular disease diagnosis, and the biological marker comprises one or more of an NRP1 gene, an EFNB2 gene and a KDR gene. A lung tissue sample of a patient is rapidly obtained through biopsy, meanwhile, the vascular remodeling degree is evaluated by detecting the expression level of the NRP1 / EFNB2 / KDR gene, and the defects of PVOD diagnosis and prognosis in the prior art are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of vascular disease diagnosis, and provides the application of lung tissue sample molecular markers in vascular disease diagnosis, and particularly relates to the application of NRP1 / EFNB2 / KDR genes in vascular disease diagnosis, especially in pulmonary veno-occlusive disease. Background Art

[0002] In existing technologies, the diagnosis of vascular diseases, such as pulmonary venous occlusive disease (PVOD), mainly relies on clinical manifestations, arterial blood gas analysis, pulmonary diffusion capacity measurement, and auxiliary diagnosis of chest high-resolution computed tomography. PVOD is a rare disease with poor prognosis, characterized by pulmonary venous obstruction, leading to increased pulmonary artery pressure and right heart failure. The occurrence of this disease is idiopathic, heritable, drug- and toxin-induced, and connective tissue disease-related. First, due to the high similarity in clinical and hemodynamic manifestations between PVOD and pulmonary hypertension, it is not easy to accurately distinguish between the two diseases. This similarity leads to the risk of misdiagnosis and mistreatment, and commonly used PAH treatment drugs such as vasodilators may aggravate the condition of PVOD patients. Secondly, although HRCT can provide some imaging clues, such as pulmonary interstitial thickening, pulmonary edema, and lymphadenopathy, its specificity and sensitivity for PVOD are insufficient and cannot be used as an independent diagnostic tool. In addition, although arterial blood gas analysis and DLCO measurement can reflect the patient's lung function status, they are mainly suitable for monitoring the progression of the disease rather than early diagnosis. Current diagnostic methods often require pulmonary vascular remodeling and symptoms to reach a certain level before effective detection can be performed, which has missed the best time for intervention. PVOD is a rare disease with poor prognosis, characterized by pulmonary venous obstruction, leading to increased pulmonary artery pressure and right heart failure. The occurrence of this disease is idiopathic, heritable, drug- and toxin-induced, and connective tissue disease-related. Most importantly, existing technologies cannot achieve early diagnosis and early warning of PVOD, resulting in patients often being in the late stage of the disease when diagnosed, with poor prognosis, accompanied by right heart failure and high mortality. Since there is currently no specific drug, lung transplantation is still the only cure, but its high cost and donor shortage limit its widespread application. Therefore, there is an urgent need to develop new biomarkers to improve the diagnostic accuracy and early detection capabilities of PVOD, thereby providing patients with earlier intervention and treatment opportunities and improving disease prognosis and quality of life. Summary of the invention

[0003] In order to overcome the deficiencies in the prior art, the present invention provides the application of lung tissue sample molecular markers in the diagnosis of vascular diseases, specifically the application of NRP1 / EFNB2 / KDR genes in the diagnosis of vascular diseases.

[0004] To achieve the above object, the present invention provides the use of biomarkers in diagnosing vascular diseases, wherein: the biomarkers include one or more of the NRP1 gene, EFNB2 gene, and KDR gene.

[0005] Preferably, the expression of one or more of the NRP1 gene, EFNB2 gene, and KDR gene in venous endothelial cells is elevated.

[0006] Preferably, said increased expression characterizes venous arterialization in vascular disease.

[0007] Preferably, the vascular disease is pulmonary veno-occlusive disease.

[0008] The present invention also provides use of one or more inhibitors of NRP1 gene, EFNB2 gene and KDR gene in the preparation of diagnosing or treating vascular diseases.

[0009] In the present invention, tissue samples for diagnosing vascular diseases are obtained by lung biopsy or surgical resection.

[0010] The beneficial effects of the present invention are as follows:

[0011] The present invention discloses the application of lung tissue sample molecular markers in the diagnosis of vascular diseases, and relates to the application of NRP1 / EFNB2 / KDR genes in the diagnosis and treatment of PVOD. Lung tissue samples of patients are quickly obtained through lung biopsy, and the expression levels of NRP1 / EFNB2 / KDR genes are detected to evaluate the condition, thereby solving the defects and shortcomings of PVOD diagnosis in the prior art.

[0012] First, traditional PVOD diagnostic methods rely on changes in pulmonary vascular remodeling and the appearance of obvious symptoms, which are clinically regarded as irreversible pathological processes, usually indicating that the disease has entered the mid-to-late stage. Therefore, existing methods often miss the best time for intervention. Based on in-depth research on changes in endothelial cell arterialization during pulmonary vascular remodeling, the present invention has developed a new diagnostic method by detecting changes in the expression of specific marker genes (NRP1 / EFNB2 / KDR). The selection of this gene combination is highly specific and can provide reliable early warning information for the choice of treatment methods in the irreversible stage of the disease.

[0013] Secondly, the present invention provides an unprecedented diagnostic strategy for people at high risk of PVOD. By obtaining lung tissue samples through biopsy, detecting and monitoring changes in gene expression of pulmonary vascular endothelial cells, doctors can identify patients who have developed irreversible terminal PVOD at a stage when the treatment method of the disease is unclear. This prospective diagnostic method enables clinical intervention to be carried out more promptly, thereby greatly improving the effectiveness of treatment. In addition, the benefits of this diagnosis are not only timely intervention and treatment, but also include improving the patient's quality of life and disease prognosis. By identifying and managing the PVOD process, the progression of the disease can be slowed or stopped, the incidence of complications due to right heart failure can be reduced, and the quality of life of patients can be improved. In short, the innovation of the present invention lies in its efficient and accurate diagnostic capabilities, which not only fills the deficiencies of the existing technology, but also opens up a new direction for clinical intervention in PVOD. This will be beneficial to the patient's prognosis and improvement of quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Cluster analysis diagram of single-cell transcriptomic sequencing data of PVOD patients;

[0015] Figure 2 Violin plots of NRP1 / EFNB2 / KDR expression in venous endothelial cells of healthy subjects and PVOD patients;

[0016] Figure 3 Five-color immunofluorescence co-staining images of lung tissue sections of healthy people and PVOD patients;

[0017] Figure 4 Identify mutation sites in PVOD patients;

[0018] Figure 5 This is a WB identification diagram of GCN2 expression in HUVEC;

[0019] Figure 6 Comparison of gene expression of NRP1 / EFNB2 / KDR in control cells and GCN2 knockout HUVECs. DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0021] Example 1: Detection of NRP1 / EFNB2 / KDR gene expression in lung tissue of PVOD patients using single-cell sequencing

[0022] Lung tissue samples from patients with PVOD were processed. Before enzymatic dissociation, the tissue was first minced with scissors and then incubated with collagenase. After incubation, the cells were filtered using a 70-μm cell strainer and then washed with red blood cell lysis buffer. Subsequently, the cell suspension with a cell viability greater than 80% was loaded into the Chromium microfluidic chip equipped with 3'v3 chemical reagents and barcoded by the Chromium controller of 10X Genomics. Subsequently, the RNA from the barcoded cells was reverse transcribed using the reagents of the Chromium Single Cell 3'v3 Kit according to the manufacturer's instructions of 10X Genomics, and the sequencing library was constructed. The sequencing of the library was performed using the Illumina HiSeq 2000 platform and strictly followed the instructions of Illumina. The data were aligned using the Cell Ranger software v6.0.0 of 10XGenomics and mapped to the GRCh38 reference genome.

[0023] The single-cell data obtained by sequencing were combined and analyzed with the public single-cell transcriptomics data of healthy people in published literature (Travaglini KJ et al, Nature, 2020). Figure 1 The results showed that by cluster analysis, the venous endothelial cell population was separated according to the markers of venous endothelial cells, and the transcriptional expression levels of the three genes NRP1 / EFNB2 / KDR (three genes were detected separately) in healthy people and PVOD patients were further compared. Figure 2 The violin plot shows that in the single-cell transcriptomics sequencing data, the expression of NRP1 / EFNB2 / KDR genes in the venous endothelial cells of the lung tissue of PVOD patients was increased, and there were characteristics of venous arterialization.

[0024] Example 2: Detection of NRP1 / EFNB2 / KDR genes in tissue samples of PVOD patients

[0025] Multicolor immunofluorescence staining was used to detect the expression levels of three genes, NRP1 / EFNB2 / KDR, in tissue samples of PVOD patients.

[0026] 1. Lung tissue samples from PVOD patients were fixed in fixative, embedded in paraffin, and then sectioned for staining.

[0027] 2. Place tissue sections in environmentally friendly dewaxing solution for 30 minutes, anhydrous ethanol for 10 minutes, and wash with distilled water.

[0028] 3. Antigen repair: Dilute EDTA antigen repair solution (pH 9.0) 1:20, place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH9.0) and heat in a microwave oven, 2 times for 4 minutes on high heat, 2 times for 4 minutes on medium heat. Each time interval is 2 minutes. During this period, the solution is continuously replenished to prevent excessive evaporation of the buffer. After natural cooling, wash with PBS 3 times, 5 minutes each time.

[0029] 4. Add 3% H2O2 to the slices and incubate at room temperature in the dark for 25 minutes to eliminate endogenous peroxidase activity, and wash with PBS three times, 5 minutes each time.

[0030] 5. Block the cells with 3% BSA in PBS at room temperature for 30 minutes.

[0031] 6. Shake off the blocking solution, add the first primary antibody α-SMA at a dilution ratio of 1:1000, and incubate at 4°C overnight.

[0032] 7. Wash with PBS 3 times, 5 minutes each time. Add HRP-labeled secondary antibody, dilute the antibody with PBS 1:400. Incubate at room temperature for 50 minutes.

[0033] 8. Wash with PBS 3 times, 5 minutes each time. Add iF440 staining working solution (prepared as 1 mL of Tyramide diluent, 10 μl of 0.3% H2O2, 2 μl of iF440-Tyramide), incubate at room temperature in the dark for 10 minutes. After incubation, wash with TBST 3 times, 5 minutes each time.

[0034] 9. Microwave treatment: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 9.0) and heat in a microwave oven, 2 times for 4 minutes on high heat, 2 times for 4 minutes on medium heat, with a 2-minute interval between each time. During this process, excessive evaporation of the buffer should be prevented and the sections should not be dried.

[0035] 10. Block the cells with 3% BSA in PBS at room temperature for 30 minutes.

[0036] 11. Discard the blocking solution, add the second primary antibody NRP1 at a dilution ratio of 1:1000, and incubate at 4°C overnight.

[0037] 12. Wash with PBS 3 times, 5 minutes each time. Add HRP-labeled secondary antibody, dilute the antibody with PBS 1:400. Incubate at room temperature for 50 minutes.

[0038] 13. Wash with PBS 3 times, 5 minutes each time. Add iF594 staining working solution (prepared as 1 mL of Tyramide diluent, 10 μl of 0.3% H2O2, 2 μl of iF594-Tyramide), incubate at room temperature in the dark for 10 minutes. After incubation, wash with TBST 3 times, 5 minutes each time.

[0039] 14. Repeat steps 9-13, replacing the primary antibody with NR2F2 / CD31 each time, and replacing the staining working solution with iF647 / 488-TSA staining working solution each time.

[0040] 15. After the last antibody fluorescent labeling is completed, add DAPI staining solution, incubate at room temperature in the dark for 10 minutes, and wash with PBS three times, 5 minutes each time.

[0041] 16. Drop autofluorescence quencher on the tissue, incubate at room temperature in the dark for 5 minutes, and rinse with running water for 10 minutes.

[0042] 17. Wash with PBS 3 times, 5 minutes each time. Shake the slices slightly dry and seal with anti-fluorescence quenching sealing agent. Collect images with a fluorescence confocal microscope.

[0043] When the tissue slide is to be stained with EFNB2 or KDR, replace NRP1 in step 11 with EFNB2 or KDR. Finally, three five-color immunofluorescence staining combinations are generated, namely α-SMA / NRP1 / NR2F2 / CD31 / DAPI co-staining ( Figure 3 2 rows in the lower middle), α-SMA / EFNB2 / NR2F2 / CD31 / DAPI co-staining ( Figure 3 2 rows in the upper middle), α-SMA / KDR / NR2F2 / CD31 / DAPI co-staining ( Figure 3 middle 2 rows).

[0044] α-SMA is a marker of smooth muscle cells, CD31 is an endothelial cell marker, and NR2F2 is a specific marker of venous endothelial cells. According to the structural characteristics of pulmonary blood vessels, α-SMA / CD31 was first used to find the pulmonary blood vessels on the immunofluorescence image, and then NR2F2 / CD31 was used to locate the pulmonary vein endothelial cells, and the expression differences of NRP1 / EFNB2 / KDR on pulmonary vein endothelial cells between healthy people and PVOD patients were compared. Figure 3 The results showed that the fluorescence intensity of NRP1 / EFNB2 / KDR on the pulmonary vein endothelial cells of PVOD was increased, and the protein expression level was significantly higher than that of the pulmonary veins of healthy people, indicating that the characteristics of arterialization exist in the pulmonary vein endothelial cells of PVOD patients.

[0045] Example 3: Changes in the expression of NRP1 / EFNB2 / KDR genes in human umbilical vein endothelial cells (HUVEC) cells with consistent mutation sites in PVOD patients

[0046] 1. Preparation of Lentiviral Infection Vectors

[0047] In order to simulate the mutations of PVOD patients, we designed sgRNAs for the mutation sites of PVOD patients according to the target design principles of the CRISPR / Cas9 system through the online tool http:crispor.tefor.net / . Figure 4 The results showed that the mutation region of the patient was exon 33 of the gene EIF2AK4 (NCBI No. 440275), which encodes GCN2 protein. The obtained sgRNA sequence was GCAGACAGAGAAGCGTGTGC. It was connected to the pLentiCrisprV2 vector, and sequencing confirmed that the vector pLentiCrisprV2-gRNA was correctly connected. The day before, inoculate HEK-293T cells of appropriate density into a 10 cm culture dish, prepare two EP tubes, add 0.5 ml Opti-MEM and 10 μg target vector pLentiCrisprV2-gRNA, 10 μg lentiviral packaging vector PSPAX, and 4 μg lentiviral packaging vector VSVG to one tube, mix well, add 0.5 ml Opti-MEM and 48 μl PEI to the other tube, mix well, then mix the two tubes of liquid, let stand for 20 minutes, add dropwise to the HEK-293T cells, shake gently to mix, change the liquid after 8 hours, and collect the supernatant after 48 hours. The supernatant was filtered and allowed to stand overnight. A concentrate was prepared by adding 3 ml 5% PEG, 1.28 ml 4M NaCl, and 1.37 ml PBS per 10 ml volume. The concentrate was mixed and allowed to stand overnight. Centrifuged at 4°C, 4000 g for 40 minutes. The supernatant was discarded and the virus precipitate was resuspended in endothelial cell culture medium ECM to form a lentiviral suspension for infecting the target cells.

[0048] 2. Construction of knockout lines

[0049] HUVECs were subcultured with ECM complete medium (containing 5% FBS and 100× EGCs). The lentiviral suspension resuspended in ECM was transfected into endothelial cells and screened with puromycin. The expression of GCN2, a protein edited by EIF2AK4, was detected by WB to determine the knockout efficiency. The expression differences of the three genes NRP1 / EFNB2 / KDR in HUVECs transfected with empty vector sgNC and transfected with knockout plasmid sgRNA were compared by qRT-PCR.

[0050] Figure 5 The results showed that HUVECs transfected with sgRNA successfully knocked out GCN2 protein. Figure 6 The results showed that in the HUVEC knockout line carrying the consistent mutations of PVOD patients, the expression of NRP1 / EFNB2 / KDR genes was increased, indicating that the HUVECs with GCN2 knockout had arterialization, which is indicative for the diagnosis of the disease.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The use of biomarkers in the diagnosis of vascular diseases, characterized in that: The biomarkers include one or more of the NRP1 gene, the EFNB2 gene, and the KDR gene.

2. The biomarker for diagnosing vascular diseases according to claim 1, characterized in that: The expression of one or more of the NRP1 gene, EFNB2 gene, and KDR gene in venous endothelial cells is elevated.

3. The biomarker for diagnosing vascular diseases according to claim 2, characterized in that: Such elevated expression characterizes venous arterialization in vascular disease.

4. The biomarker for diagnosing vascular diseases according to claim 1, characterized in that: The vascular disease is pulmonary veno-occlusive disease.

5. Use of one or more inhibitors of NRP1 gene, EFNB2 gene and KDR gene in the preparation of diagnosis or treatment of vascular diseases.

6. Tissue samples in the diagnosis of vascular disease are obtained through lung biopsy or surgical resection.

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