Lung vascular endothelial barrier injury model as well as construction method and application thereof

Abstract

The invention discloses a pulmonary vascular endothelial barrier injury model and a construction method and application thereof, and relates to the technical field of biologication.The construction method of the pulmonary vascular endothelial barrier injury model comprises the following steps that a healthy mouse is adaptively fed for one week and then anesthetized with a medicine, oncostatin M recombinant protein is pretreated and then instilled into the lung of the mouse through an airway, and then the mouse is subjected to tumor cell apoptosis; obtaining a pulmonary vascular endothelial barrier injury model; the invention further discloses a pulmonary vascular endothelial barrier injury model, application of the model in preparation of a pulmonary vascular disease research model and application of the model in preparation of drugs for treating pulmonary vascular diseases. According to the pulmonary vascular endothelial barrier injury model as well as the construction method and the application thereof provided by the invention, the oncostatin M protein is combined with an oncostatin M receptor type II on an endothelial cell to activate a downstream signal channel, so that the endothelial barrier is damaged, a clear target spot is provided for the endothelial barrier injury, and a pulmonary injury mechanism can be deeply discussed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, it is a lung vascular endothelial barrier injury model, a construction method and an application thereof. Background Art

[0002] Vascular endothelial cells are widely lined in the arteries, veins, microvessels and lymphatic vessels of various tissues and organs in the human body. As the first interface between blood and vascular tissues, they play a key role in maintaining vascular permeability, transmitting vascular information, regulating vascular tone, hemostasis, secreting vasoactive substances and maintaining the oxygen and nutrient supply of all tissues in the body. When stimulated by infection, endotoxin, hypoxia, oxidized low-density lipoprotein, inflammatory cytokines, etc., endothelial cells can change from a quiescent state to an activated state. Activated endothelial cells secrete a series of pro-inflammatory cytokines, chemokines, enzymes and adhesion molecules. Inflammatory cytokines can increase inflammatory proteins and activate multiple signaling pathways, causing endothelial cells to transform into a pro-inflammatory state. Endothelial cells with this inflammatory phenotype can trigger inflammation of the vascular wall, thereby damaging their normal functions, manifested as weakened barrier function, increased permeability and exacerbated inflammation. Lung vascular endothelial cells are the center of the inflammatory storm in acute respiratory distress syndrome (ARDS). The loss of the endothelial barrier leads to pulmonary edema (a characteristic of acute lung injury) and high altitude pulmonary edema. The pathogenesis of these two diseases is still unclear, and so far no drugs have been approved for the treatment of these life-threatening diseases. Endothelial cell barrier injury plays an important role in the pathological processes of acute lung injury, ARDS, atherosclerosis, hypertension, diabetes, cerebrovascular diseases, etc. Therefore, establishing an endothelial barrier inflammatory injury model is of great significance for the research of pulmonary vascular diseases and accelerating the development of effective therapeutic drugs.

[0003] Establishing a pulmonary vascular endothelial injury model is an important means for studying pulmonary vascular diseases. In recent years, with the development of various methods such as chemical induction, gene editing, physical induction, and combined induction, significant progress has been made in the establishment of vascular endothelial injury models. Lipopolysaccharide (LPS) has been widely used in the establishment of sepsis models related to pathogen infection. However, the purity of LPS extracted by different manufacturers varies, and it may be contaminated with bacterial lipoproteins or other bacterial substances. At the same time, LPS mainly triggers an inflammatory response by activating the TLR4 signaling pathway, indirectly damaging endothelial cells, and its direct damage to the endothelial barrier is relatively limited. Its pathogenesis is relatively single and may not be able to fully simulate complex pathophysiological processes. TNF-α is mainly used to construct a preeclampsia vascular endothelial injury model. The endothelial injury induced by ox-LDL is mainly oxidative damage and is applied to establish an endothelial barrier injury model in atherosclerotic diseases. AGEs are important models for studying endothelial injury related to diseases such as diabetes. The Hcy-induced endothelial barrier injury model is used to study vascular diseases related to hyperhomocysteinemia. The Ang II-induced endothelial barrier injury model is of great significance in the study of cardiovascular diseases such as hypertension. Feeding a high-cholesterol diet mainly causes atherosclerosis in rats. None of the above several methods of chemically inducing endothelial injury can be completely and accurately used for the construction of a pulmonary vascular endothelial barrier injury model. H2O2, MCT, and rotenone are highly toxic to endothelial cells, are easy to obtain, inexpensive, and have obvious injury effects. However, H2O2 has poor stability and is prone to decomposition. Different endothelial cells have significant differences in sensitivity to the injury of MCT and rotenone. Therefore, H2O2, MCT, and rotenone can only be used as tool drugs for studying endothelial injury in vitro and cannot ensure safety, effectiveness, and repeatability in in vivo experiments. Although hypoxia and mechanical ventilation can induce endothelial barrier injury, these methods require specific experimental conditions and equipment and are not suitable for most laboratories. Although gene editing technology can precisely regulate gene expression, its operation is complex and the cost is high, making it not suitable for early promotion.

[0004] Although certain progress has been made in the establishment of pulmonary vascular endothelial injury models and they have certain applications, some of their respective disadvantages provide opportunities for exploring new methods and new research. Summary of the Invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a pulmonary vascular endothelial barrier injury model, a construction method and an application thereof. The OSM recombinant protein binds to the type II OSM recombinant protein receptor on endothelial cells, activating the downstream signaling pathway, resulting in damage to the tight junctions of endothelial cells. The OSM recombinant protein can mainly damage the pulmonary vascular endothelial barrier, providing a clear target for endothelial barrier injury; it is low-toxic to tissue cells and has tissue compatibility; it has a clear mechanism of action and reproducibility; the protein preparation process is simple, the cost is controlled, and it is convenient for more laboratories to carry out research and improve the utilization efficiency of resources.

[0006] To solve the above technical problems, the present invention provides the following technical solutions: A method for constructing a pulmonary vascular endothelial barrier injury model, comprising the following steps: After adaptively feeding healthy mice for one week, anesthetize them with drugs, pretreat the oncostatin M recombinant protein and then instill it into the lungs of the mice through the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0007] Furthermore, the drug anesthesia uses ketamine and xylazine, the mass ratio of ketamine is 80 mg / kg, and the mass ratio of xylazine is 10 mg / kg.

[0008] Furthermore, the pretreatment of the oncostatin M recombinant protein is to dilute the oncostatin M recombinant protein to 10 - 50 ng / μL with PBS, and the treatment time of the oncostatin M recombinant protein for mice is 12 - 24 h.

[0009] Furthermore, the pretreatment of the oncostatin M recombinant protein is to dilute the oncostatin M recombinant protein to 20 ng / μL with PBS, and the treatment time of the oncostatin M recombinant protein for mice is 24 h.

[0010] A pulmonary vascular endothelial barrier injury model is prepared by the following steps: After adaptively feeding healthy mice for one week, anesthetize them with drugs, pretreat the oncostatin M recombinant protein and then instill it into the lungs of the mice through the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0011] An application of a pulmonary vascular endothelial barrier injury model in the preparation of a research model for pulmonary vascular diseases is prepared by the following steps: After adaptively feeding healthy mice for one week, anesthetize them with drugs, pretreat the oncostatin M recombinant protein and then instill it into the lungs of the mice through the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0012] An application of a pulmonary vascular endothelial barrier injury model in the preparation of a drug for treating pulmonary vascular diseases is prepared by the following steps: After adaptively feeding healthy mice for one week, anesthetize them with drugs, pretreat the oncostatin M recombinant protein and then instill it into the lungs of the mice through the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0013] The technical solution of the present invention has achieved the following beneficial technical effects: 1. The present invention relates to a pulmonary vascular endothelial barrier injury model, a construction method and an application thereof. The OSM recombinant protein is used as a key factor for constructing the pulmonary vascular endothelial barrier injury model. The OSM recombinant protein can mainly damage the pulmonary vascular endothelial barrier, rather than other functions of endothelial cells in other systemic diseases; it is less toxic to tissue cells and has tissue compatibility; it has a clear mechanism of action and reproducibility; the protein preparation process is simple, the cost is controlled, and it is convenient for more laboratories to carry out research, improving the utilization efficiency of resources.

[0014] 2. The present invention relates to a pulmonary vascular endothelial barrier injury model, a construction method and an application thereof. Using the OSM recombinant protein as a stimulant, the feasibility of establishing C166 and HULEC-5a endothelial barrier injury models with different concentrations of OSM recombinant protein and different action times is studied. It can cause endothelial barrier injury without causing a large number of cell deaths, thus providing a reference for the establishment of subsequent inflammatory pulmonary vascular disease models and helping to explore related pathological processes and regulatory mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the effect of OSM recombinant protein of the pulmonary vascular endothelial barrier injury model, construction method and application thereof of the present invention on vascular endothelium; Among them, A is the immunofluorescence staining of OSM recombinant protein in lung tissue sections of mice in the PA group and the PBS group observed by confocal microscopy. The OSM recombinant protein is labeled in red, and the DAPI-stained cell nucleus is labeled in blue. Scale bar: 20 μm; B is a bar graph of ELISA detection of OSM recombinant protein in lung tissues of control mice and mice infected with PAO1 (**: P < 0.01, n = 4); C is the immunofluorescence staining of VE-cadherin in lung tissue sections of mice in the Ctrl group and the OSM group observed by confocal microscopy. VE-cadherin is labeled in red, and the DAPI-stained cell nucleus is labeled in blue; The bar graph in D shows the analysis results of the FITC-dextran permeability of vascular endothelium in mice in the Ctrl group and the OSM group (***: P < 0.01, n = 4); E is the immunofluorescence staining of lung tissue sections of mice in each group observed by confocal microscopy. VE-cadherin is stained in red, and the cell nucleus is labeled in blue by DAPI. Scale bar: 10 μm; The bar graph in F shows the analysis results of the FITC-dextran permeability of vascular endothelium in mice with different treatments (**: P < 0.01, n = 4); Figure 2 Schematic diagram of the effect of OSM recombinant protein of the present invention on the expression of cadherin in endothelial cells of mice and humans; Among them, the bar graph in A shows the expression of Cdh5 mRNA in C166 cells treated with different concentrations of mOSM for different times detected by RT-qPCR; B shows the expression of VE-cadherin protein in C166 cells treated with 20 ng / mL mOSM for 12 hours observed by fluorescence confocal microscopy. VE-cadherin is labeled with red fluorescence, and DAPI stains the cell nucleus blue. Scale bar: 5 μm; The bar graph in C shows the expression of CDH5 mRNA in HULEC-5A cells treated with different concentrations of hOSM for different times detected by RT-qPCR; D shows the expression of VE-cadherin protein in HULEC-5A cells treated with 20 ng / mL hOSM for 12 h observed by fluorescence confocal microscopy. VE-cadherin is labeled with green fluorescence, and DAPI stains the cell nucleus blue. Scale bar: 5 μm; Figure 3 Schematic diagram of the promotion of endothelial barrier injury by OSM of the present invention; Among them, A is an immunoblot band showing the expression maps of OSMR, VE-cadherin and internal reference β-actin proteins in HULEC-5A treated with different treatments; The bar graph in B shows the quantitative analysis of the immunoblot bands (*: P<0.05, **: P<0.01, n = 3); C is the observation of the expression of VE-cadherin protein in HULEC-5A treated with different treatments by laser confocal microscopy. VE-cadherin is labeled green, and the cell nucleus is labeled blue by DAPI. Scale bar: 5 μm; The bar graph in D shows the analysis results of the relative permeability of FITC-dextran in HULEC-5A cells treated with different treatments (**: P<0.01; ***: P<0.001, n = 3); The line graph in E shows the change trend of the transmembrane electrical impedance values of different endothelial cells over time (**: P<0.01, n = 3). Specific embodiments

[0016] A method for constructing a pulmonary vascular endothelial barrier injury model includes the following steps: Dilute OSM recombinant protein with PBS to 20 ng / μL, and instill 50 μL of the diluted OSM recombinant protein into the airway of mice for 24 hours.

[0017] After the mice are anesthetized with CO2 and sacrificed by cervical dislocation, the lung tissues are taken for frozen section for later experiments.

[0018] Oncostatin-M (OSM) is one of the members of the cytokine interleukin-6 family. It was first isolated from histiocytic lymphoma cells in 1986 and got its name because it can inhibit the proliferation of melanoma cells. OSM is mainly synthesized and secreted by various cells such as activated macrophages, neutrophils, T cells, and mast cells. It has a variety of biological activities and plays a wide range of roles in hematopoiesis, cell growth and differentiation, inflammatory response, metabolic regulation, tumor formation, and immune regulation. OSM interacts with two independent complexes for cell signal transduction, namely the type I LIFRβ / gp130 receptor complex and the type II OSMRβ / gp130 receptor complex. After OSM binds to the type II OSMR complex, multiple signaling pathways are activated, including the Janus-activated kinase / signal transducer and activator of transcription 3 (JAK / STAT3), mitogen-activated protein kinase / extracellular regulated kinase (MAPK / ERK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol-3-kinase / protein kinase B (PI3K / AKT) pathways. The OSM receptor (OSMR) is mainly expressed by mesenchymal cells such as fibroblasts, endothelial cells, osteoblasts, and epithelial cells, as well as certain cancer cells.

[0019] In the present invention, the OSM recombinant protein is used as a key factor for constructing a model of pulmonary vascular endothelial barrier injury. OSM can mainly damage the pulmonary vascular endothelium rather than the endothelial cells of other systemic diseases. It has low toxicity to tissue cells, tissue compatibility, a clear mechanism of action and reproducibility. The protein preparation process is simple, the cost is controlled, which facilitates more laboratories to carry out research and improves the utilization efficiency of resources.

[0020] Example 1 Verify the effect of the OSM recombinant protein on vascular endothelium in a mouse model of pulmonary infectious disease.

[0021] I. Experimental materials: Mouse OSM recombinant protein (495-MO-025, R&D Systems, USA), OSM antibody (NBP3-16686, Novus, USA), vascular endothelial cadherin antibody (VE-cadherin, Cell Signaling, USA), mouse OSM neutralizing antibody (AF-495-NA, R&D Systems, USA), mouse (G3A1) IgG antibody (5415, CST, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA), DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher, USA), fluorescein dextran, 10,000 MW (D1821, Invitrogen, USA), FV3000 laser confocal microscope (OLYMPUS, Japan), Agilent Cary 60 UV-visible spectrophotometer (Agilent, USA), Innova® 40 / 40R benchtop orbital shaker (New Brunswick, USA), Leica CM1860 cryostat (Leica Biosystems, Germany). The PAO1 strain was provided by the Chengdu Institute of Respiratory Health. Six- to eight-week-old male C57BL / 6J mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the guidelines of the Animal Protection and Utilization Committee of Southwest Jiaotong University.

[0022] II. Establishment of mouse lung infection model Healthy C57BJ / 6J mice were randomly divided into the following groups and treated accordingly.

[0023] PA group: After 1 week of adaptive feeding, the mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). Each mouse was intranasally administered 30 μL of PAO1 (3×10 7 CFU) to induce lung infection. After 24 hours, the mice were anesthetized with CO2 and sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. PBS group: After 1 week of adaptive feeding, the mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). Each mouse was intranasally administered 30 μL of PBS. After 24 hours, the mice were anesthetized with CO2 and sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. In the OSM group, after 1 week of adaptive feeding of mice, the mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). The OSM recombinant protein was diluted to 20 ng / μL with PBS. 50 μL of the diluted OSM recombinant protein was taken with a microsyringe and instilled into the airway of the mice for 24 hours. After 24 hours, the mice were anesthetized with CO2 and then sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. In the Ctrl group, after 1 week of adaptive feeding of mice, the mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). 50 μL of PBS was instilled into the airway of the mice for 24 hours. After 24 hours, the mice were anesthetized with CO2 and then sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. In the anti-OSM + PA group, after 1 week of adaptive feeding of mice, the mice were intraperitoneally injected with 100 μL of OSM neutralizing antibody at 2 μg / μL (200 μg / mouse). The mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg), and then 30 μL of PAO1 (3×10 7 CFU) was instilled into the nose of each mouse to induce lung infection for 24 hours. After 24 hours, the mice were anesthetized with CO2 and then sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. In the IgG + PA group, after 1 week of adaptive feeding of mice, the mice were intraperitoneally injected with 100 μL of IgG control antibody at 2 μg / μL (200 μg / mouse). The mice were anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg), and then 30 μL of PAO1 (3×10 7 CFU) was instilled into the nose of each mouse to induce lung infection for 24 hours. After 24 hours, the mice were anesthetized with CO2 and then sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments. In the IgG group, after 1 week of adaptive feeding of mice, the mice were intraperitoneally injected with 100 μL of IgG control antibody at 2 μg / μL (200 μg / mouse). After 24 hours, the mice were anesthetized with CO2 and then sacrificed by cervical dislocation. The lung tissues were collected for subsequent experiments.

[0024] Compared with some chemical substances (such as H2O2, MCT, rotenone, etc.), as a natural cytokine, OSM recombinant protein has lower toxicity to cells and good biocompatibility. Using OSM recombinant protein in experiments can reduce direct toxic damage to cells, improve the safety of experiments, and better simulate the in vivo physiological environment, reducing non-specific reactions caused by exogenous chemical substances. The endothelial barrier injury model induced by OSM recombinant protein has good repeatability in different laboratories, which helps to ensure the reliability and consistency of experimental results, improve the accuracy of research. OSM recombinant protein induces endothelial barrier injury by binding to OSMR and activating specific signaling pathways (such as JAK / STAT3, MAPK / ERK, etc.). This clear mechanism of action makes the experimental results more interpretable and can more accurately reflect the pathophysiological process of endothelial barrier injury. At the same time, OSM recombinant protein can act synergistically with other cytokines (such as IL-6, TNF-α, etc.) to enhance the effect of endothelial barrier injury. This synergistic effect provides a new perspective for studying endothelial barrier injury under the combined action of multiple factors. Finally, the preparation of OSM recombinant protein is relatively simple and the cost is low. The use of OSM recombinant protein does not require complex equipment or technology, reducing the resource requirements of experiments. Compared with some complex gene editing technologies or expensive chemical reagents, using OSM recombinant protein to construct a model has higher cost-effectiveness.

[0025] III. Experimental Procedures and Results (1)Enzyme-linked immunosorbent assay (ELISA) detection: Rinse the left lung tissue with cold PBS, cut the lung tissue into small pieces with scissors, put them into a grinding tube for grinding, and then ultrasonically disrupt them with an ultrasonic cell disruptor. Centrifuge at 4°C and 12,000 g for 15 min to collect the supernatant, which is the lung tissue sample. The concentration of OSM in the lung tissue is measured using an ELISA kit provided by a commercial supplier according to the manufacturer's instructions.

[0026] (2)Multiplex immunofluorescence staining of lung tissue: Mouse lung tissue is immersed in 4% paraformaldehyde for 48 hours, and then dehydrated by immersion in 30% sucrose solution for 24 hours. After the lung tissue is embedded in OCT and frozen for 24 hours, frozen sections are made with a thickness of 5 µm. Subsequently, the tissue sections are permeabilized with 0.05% Tween 20 for 10 minutes, blocked with 10% goat serum at room temperature for 1 hour to block non-specific antibodies, and then incubated overnight at 4˚C with antibodies (rabbit monoclonal antibody against VE-cadherin, diluted 1:400; mouse monoclonal antibody against OSM, diluted 1:200). Goat anti-rabbit Alexa Fluor™ 594 antibody (diluted 1:1000) binds to the primary antibody at room temperature and incubates for 2 hours. The nuclei are stained with DAPI, and after mounting with an anti-fluorescence quencher, the sections are observed under a laser confocal microscope and photographed for recording.

[0027] (3)In vivo vascular permeability experiment: Calculate the dosage for each mouse based on 10 mg / kg body weight of FITC-dextran. After anesthetizing the mice in the IgG group, IgG+PA group, anti-OSM+PA group, Ctrl group, and OSM group, slowly inhale 25 μL of FITC-dextran (10000 MW) into each nostril of the mice, keep the vertical position for 2 min until their breathing is stable, sacrifice the mice after 1 h. Disinfect the mice with 70% ethanol, open the chest cavity with sterile forceps and surgical scissors, expose the apex of the heart. First, apply slight negative pressure to the piston of a 1 mL syringe, insert a 22-gauge needle into the apex of the heart. When blood appears in the syringe, gradually withdraw the piston to collect blood. Inject the collected blood into an EP tube pre-wetted with 10 μL of EDTA (60 mg / mL), centrifuge at 550 g for 10 minutes, collect the upper layer of the liquid. Subsequently, detect the fluorescence OD value of plasma FITC-dextran using a multifunctional microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0028] Lung infections caused by Pseudomonas aeruginosa (PA) infection without effective treatment can progress to ARDS. The important pathological changes in ARDS are pulmonary edema and the formation of hyaline membranes. Both of these pathological changes are related to damaged endothelial barriers and increased cell permeability. After PA infects the lungs, immune cells secrete a large amount of the cytokine OSM. After OSM binds to OSMR on endothelial cells, it destroys the tight junctions of endothelial cells, promotes cell permeability, and causes damage to the pulmonary vascular endothelial barrier. After PA infection, the OSM recombinant protein binds to the type II OSM receptor on endothelial cells, activates the downstream signaling pathway, resulting in endothelial damage. This specific mechanism of action provides a clear target for studying endothelial barrier damage.

[0029] The detection results are as Figure 1 shown. Figure 1 In A and 1B, the red fluorescence labeled with OSM recombinant protein in the lung tissues of mice in the PA group was significantly increased compared with the PBS group, and the OSM recombinant protein in the lung tissue homogenate of the PA group was significantly increased compared with the PBS group (P<0.01).

[0030] Drop the mouse OSM recombinant protein into the lungs of healthy mice. After 24 hours of treatment, detect the expression of the adhesion protein (VE-cadherin) to reflect endothelial continuity and endothelial permeability, so as to detect the damage of endothelial barrier function and reflect the success or failure of the model. The results are as Figure 1 shown in C. Compared with the Ctrl group, the expression of VE-cadherin in the lung tissues of mice in the OSM group decreased, and the endothelial integrity was damaged.

[0031] The change in vascular endothelial permeability was reflected by detecting FITC labeled with FITC-dextran in plasma, and the results were as Figure 1 shown in D. Compared with the Ctrl group, the FITC fluorescence in the plasma of mice in the OSM group increased (P<0.001), indicating an increase in vascular endothelial permeability.

[0032] The endogenous OSM produced after PA infection was neutralized with an antibody against OSM, and then the integrity of vascular endothelium was detected. The results were as Figure 1 shown in E and 1F. Compared with the PA group, the expression of VE-cadherin in the lung tissue of mice in the anti-OSM+PA group increased, the endothelium remained intact, the FITC fluorescence in the plasma of mice decreased (P<0.01), and the vascular endothelial permeability decreased.

[0033] In summary, after PA infection, the OSM in the lung tissue increased. After 1 μg of recombinant OSM protein was inhaled into the lungs of mice via the nose for 24 hours, it could lead to an increase in the vascular permeability of mouse lungs and damage to the endothelial barrier.

[0034] Example 2 Recombinant OSM protein reduced the expression of VE-cadherin in endothelial cells of mice and humans I. Experimental materials: Recombinant human Oncostatin M protein (295-OM-050, R&D Systems, USA), recombinant mouse OSM recombinant protein (495-MO-025, R&D Systems, USA), mouse vascular endothelial cells C166 and human lung microvascular endothelial cells (HULEC-5A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), MCDB131 medium (10372019, Gibco, USA), epidermal growth factor (AF-100-15, PeproTech, USA), L-glutamine (A2916801, Gibco, USA), hydrocortisone (HY-N0583R, MCE, USA), Trizol reagent (Invitrogen, USA), reverse transcription kit (Takara Bio, Japan), real-time fluorescence quantitative polymerase chain reaction kit (RT-qPCR, Applied Biosystems, USA), vascular endothelial cadherin (VE-cadherin, cell signaling, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA) and goat anti-rabbit Alexa Fluor™ 488 antibody (Invitrogen, USA), DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher, USA), laser confocal microscope (OLYMPUS, Japan), CFX Connect™ fluorescence quantitative PCR detection system (Bio-Rad, USA).

[0035] II. Experimental Procedures and Results (1) Cell culture: C166 cells were grown adherently in high-glucose DMEM containing 10% FBS and 1% streptomycin / penicillin antibiotics, and were stably passaged in a constant temperature and humidity cell culture incubator at 5% CO2 and 37°C. 2.0×10 5 cells were evenly seeded in 6-well plates. After treatment with different concentrations of mOSM (mouse-derived OSM recombinant protein) (10 ng / mL, 20 ng / mL), the cells were collected for subsequent experiments.

[0036] HULEC-5A cells were grown adherently in MCDB131 containing 10% FBS, 10 ng / mL EGF, 1 μg / mL hydrocortisone, 10 mM L-glutamine and 1% streptomycin / penicillin antibiotics, and were stably passaged in a constant temperature and humidity cell culture incubator at 5% CO2 and 37°C. 2.5×10 5Cells were evenly seeded in 6-well plates and treated with different concentrations of hOSM (human recombinant OSM) (10 ng / mL, 20 ng / mL, 30 ng / mL), and then the cells were collected for subsequent experiments.

[0037] In the Ctrl group, cells were treated with an equal volume of PBS for the same time, and then the cells were collected for subsequent experiments.

[0038] (2)RT-qPCR experiment: Cells were collected, and total RNA was extracted using Trizol. Complementary DNA (cDNA) was synthesized according to the instructions of the TAKARA reverse transcription kit, and RT-qPCR was performed using cDNA as a template. The reaction conditions were as follows: 95 °C for 30 s, followed by denaturation at 95 °C for 5 s and annealing at 60 °C for 34 s, for a total of 40 cycles. β-actin was used as an internal reference, and the relative expression level of CDH5 was calculated using the 2–ΔΔCt formula. The primer sequences were as follows: Mouse β-actin forward 5’-GGCTGTATTCCCCTCCATCG-3’, mouse reverse 5’-CCAGTTGGTAACAATGCCATGT-3’; Human β-actin forward 5’-AAATCTGGCACCACACCTTC-3’, reverse 5’-GGGGTGTTGAAGGTCTCAAA-3’; Human CDH5 forward 5’-TTGGAACCAGATGCACATTGAT-3’, reverse 5’-TCTTGCGACTCACGCTTGAC-3’.

[0039] (3)Cell immunofluorescence staining: HULEC-5A / C166 cells were evenly seeded on coverslips in 24-well plates at a cell density of 10×10 4 / well. After infection, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.05% tween20 for 10 minutes, blocked with 10% goat serum at room temperature for 1 hour, and then incubated overnight at 4 °C with the antibody (VE-cadherin rabbit monoclonal antibody, diluted 1:300). Then, goat anti-rabbit Alexa Fluor™ 594 antibody or goat anti-rabbit Alexa Fluor™ 488 antibody (diluted 1:1000) was incubated at room temperature for 2 hours. The nuclei were stained with DAPI. After mounting with an anti-fluorescence quencher, the sections were observed using a laser confocal microscope and photographed.

[0040] Different species of endothelial cells were selected, including mouse vascular endothelial cells C166 and human lung microvascular endothelial cells (HULEC-5A). They were treated with different concentrations of OSM for different times. VE-cadherin (a protein encoded by the CDH5 gene) is the core that controls the opening, closing, and permeability changes of the endothelial barrier. The mRNA of CDH5 and the expression of VE-cadherin protein on the two types of cells were detected by RT-qPCR and immunofluorescence. C166 cells were treated with different concentrations of mOSM (10 ng / mL, 20 ng / mL) for 12 h and 24 h.

[0041] Combined Figure 2 , the expression of Cdh5 mRNA detected by RT-qPCR was as Figure 2 shown in A. Compared with the Ctrl group, after treatment with 10 ng / mL mOSM for 12 and 24 hours, the expression of Cdh5 mRNA showed no significant change; after treatment with 20 ng / mL mOSM for 12 hours, the expression of Cdh5 mRNA decreased significantly (P<0.05), while after treatment for 24 hours, the expression of Cdh5 mRNA had no statistical significance.

[0042] The expression of VE-cadherin protein detected by immunofluorescence was as Figure 2 shown in B. Compared with the Ctrl group, after treatment of C166 cells with 20 ng / mL mOSM for 12 hours, the fluorescence of VE-cadherin decreased significantly, and its integrity was damaged.

[0043] After HULEC-5A cells were treated with different concentrations of hOSM (10 ng / mL, 20 ng / mL, 30 ng / mL) for 12 h and 24 h, the expression of CDH5 mRNA was detected by RT-qPCR. The results were as Figure 2 shown in C. Compared with the Ctrl group, after treatment with 10 ng / mL hOSM for 12 h, the expression of CDH5 mRNA showed no significant change. After treatment with 20 ng / mL hOSM for 12 h, the expression of CDH5 mRNA decreased significantly (P<0.05). After treatment with 30 ng / mL hOSM for 12 h, the expression of CDH5 mRNA decreased significantly (P<0.05); compared with the Ctrl group, after treatment of HULEC-5A cells with 3 different concentrations of hOSM for 24 hours, the change in the expression of CDH5 mRNA had no statistical significance.

[0044] The expression of VE-cadherin protein was detected by immunofluorescence. The results were as Figure 2 shown in D. Compared with the Ctrl group, after treatment of HULEC-5A cells with 20 ng / mL hOSM for 12 h, the fluorescence of VE-cadherin decreased significantly, and the endothelial integrity was damaged.

[0045] In summary, treatment of C166 cells with mouse OSM recombinant protein at a concentration of 20 ng / mL for 12 hours can lead to a decrease in the expression of cell VE-cadherin; treatment of HULEC-5A cells with human OSM recombinant protein at a concentration of 20 ng / mL for 12 hours can lead to a decrease in the expression of cell VE-cadherin. OSM recombinant protein reduces the expression of VE-cadherin in endothelial cells of mice and humans.

[0046] Example 3 OSM promotes vascular endothelial barrier injury through OSMR I. Experimental materials: Recombinant human Oncostatin M protein (295-OM-050, R&D Systems, USA), human lung microvascular endothelial cells (HULEC-5A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), MCDB131 medium (10372019, Gibco, USA), epidermal growth factor (AF-100-15, PeproTech, USA), L-glutamine (A2916801, Gibco, USA), hydrocortisone (HY-N0583R, MCE, USA), LipofectamineRNAiMAX transfection reagent (13778150, Invitrogen, USA), Opti-MEM (31985070, Gibco, USA), VE-cadherin rabbit polyclonal antibody (36-1900, Invitrogen, USA), OSMRβ mouse monoclonal antibody (sc-21797, Santa Cruz, USA), β-actin mouse polyclonal antibody (sc-47778, Santa Cruz, USA), HRP-labeled goat anti-rabbit antibody (7074, cell signaling, USA), HRP-labeled goat anti-mouse antibody (7076, cell signaling, USA), goat anti-rabbit Alexa Fluor™ 488 antibody (Invitrogen, USA), DAPI (4′6-diamidino-2-phenylindole, Thermo Fisher, USA), RIPA lysis buffer, protease phosphatase inhibitor, Triton X 100, 4% paraformaldehyde, sodium dodecylbenzenesulfonate, N,N methylenebisacrylamide, skim milk powder, etc., products of Solarbio; polyvinylidene fluoride membrane (PVDF) membrane, product of Merck, ECL chemiluminescent solution was purchased from Millipore, USA, PageRuler™ Prestained Protein Molecular Weight Marker (26617, thermo fisher, USA), Pierce™ BCA Protein Assay Kit (23227, thermo fisher, USA), dextran fluorescein, 3000MW (D3305, Invitrogen, USA), laser confocal microscope (OLYMPUS, Japan), Bio-Rad vertical electrophoresis apparatus (Bio-Rad, USA), electronic film pressing imaging system, product of e-blot, CellZscope automatic cell transmembrane resistance measuring instrument (NanoAnalytics, Germany).

[0047] II. Experimental Procedures and Results (1)Cell culture and transfection: The cell culture method and reagents used for HULEC-5A cells were the same as those in Example 2. HULEC-5A cells were evenly seeded in a 6-well plate at a cell density of 2.5×10 5 cells per well. After 12 h, according to the product manual of RNAiMAX transfection reagent, 30 pmol of siRNA was mixed and diluted with 150 μL of opti-MEM serum-reduced medium and incubated at room temperature for 5 min. RNAiMAX transfection reagent was diluted with 150 μL of opti-MEM serum-reduced medium and incubated at room temperature for 3 min. Then, the two opti-MEM media containing OSMR siRNA and the transfection reagent were gently mixed and incubated at room temperature for 15 min. Subsequently, they were carefully added to the cell culture medium. After culturing the cells for 6 h, the medium was replaced with normal medium. Subsequent experiments were performed 24 h after transfection.

[0048] The siRNA target sequence of OSMR was: GAAGAGAAGUGUCAAUAUCUU. The negative control group was transfected with Scrambled siRNA, denoted as the Scramble group.

[0049] Cell immunofluorescence staining: HULEC-5A cells were evenly seeded on coverslips embedded with laminin in 24-well plates at a cell density of 1.0×10 5 cells per well. After the treatment of cells with OSM, the excess medium was washed off with pre-cooled PBS, and the cells were fixed with 4% paraformaldehyde for 15 min, rinsed 3 times with PBS, permeabilized with 0.05% tween20 for 10 min, and rinsed 2 times with PBS. Then, the cells were incubated with 10% goat serum at 37 °C for 1 h. After diluting the VE-cadherin rabbit polyclonal antibody at 1:200, the cells were incubated with it overnight at 4 °C. After diluting the Alexa Fluor™ 488-labeled goat anti-rabbit antibody at 1:1000, the cells were incubated with it for 2 h at room temperature. The cell nuclei were stained with DAPI and incubated in the dark at room temperature for 10 min. Then, the cells were rinsed 3 times with PBS. 10 μL of anti-fluorescence quencher was added to the center of the front side of the adhesive coverslip. The cell coverslip was carefully removed from the 24-well plate with forceps and placed with the cell side on the anti-quencher area of the adhesive coverslip, trying to avoid generating air bubbles. Then, a circle was painted around the coverslip with nail polish to prevent sliding. The cells were observed under a laser confocal microscope and photographed.

[0050] Immunoblotting: Cells at the end of treatment were lysed in RIPA lysis buffer containing a protein phosphatase inhibitor for 30 min. After measuring the protein concentration with a BCA protein assay kit, the same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h, and then incubated overnight at 4 °C with VE-cadherin (diluted 1:1000), OSMRβ (diluted 1:1000), and β-actin (diluted 1:10000). After washing three times with TBST, the membrane was incubated with the corresponding HRP-labeled goat anti-mouse / rabbit antibody for 2 h at room temperature and scanned using an eBlot Touch imager. Immunobands were analyzed using Image J software.

[0051] In vitro cell permeability assay: 2×10 5 HULEC-5A cells were evenly seeded in the upper chamber of a transwell in a 24-well plate (3.0 μm pore size). Twelve wells were randomly divided into 4 groups: Scramble group, OSM group, siOSMR group, and siOSMR + OSM group, with 3 wells in each group. The Scramble group and OSM group were transfected with negative control siRNA, and the siOSMR group and siOSMR + OSM group were transfected with siRNA against OSMR. The cells were cultured at 37 °C with constant temperature and humidity until they formed a monolayer. The monolayer endothelial cells in the OSM group and siOSMR + OSM group were treated with 30 ng / mL hOSM for 12 h. Subsequently, 100 μL of PBS containing FITC-dextran (1 μg / mL) was added to each upper chamber, and 500 μL of PBS without FITC-dextran (3000 MW) was added to the lower chamber. After incubation at 37 °C for 5 min, the fluorescence intensity of FITC-dextran transferred to the lower chamber was measured using a multifunctional microplate reader, with the excitation wavelength and emission wavelength being 494 nm and 521 nm, respectively.

[0052] Transcellular electrical impedance measurement: 12×10 4HULEC-5A cells were evenly seeded in the upper chambers of 24-well transwell plates (with a pore size of 3.0 μm). Fifteen wells were randomly and evenly divided into 3 groups: the Scramble group, the OSM group, and the siOSMR + OSM group, with 3 wells in each group. Cells in the Scramble group and the OSM group were transfected with negative control siRNA; cells in the siOSMR + OSM group were transfected with siRNA targeting OSMR. After the transfection, the upper chambers were transferred to a cell transwell resistance measuring instrument and cultured at 37°C with constant temperature and humidity until the cells formed a monolayer. The monolayer endothelial cells in the OSM group and the siOSMR + OSM group were treated with 30 ng / mL hOSM for 12 h, and the transcellular resistance values of the cells in each well were detected in real time.

[0053] The results are as Figure 3 shown. Figure 3 As shown by the immunoblotting results in A, after treating HULEC-5A with OSM, the expression of OSMR increased significantly. To explore whether OSM damages endothelial cells through the OSMR signal, the OSMR of HULEC-5A was knocked down with siRNA. The immunoblotting band results ( Figure 3 A, 3B) showed that compared with the Scramble group, the expression of VE-cadherin protein in the OSM group decreased. The quantitative analysis results showed that P = 0.0098, and the difference was statistically significant. Compared with the OSM group, the expression of VE-cadherin protein in the siOSMR + OSM group increased, and the protein quantification results showed that P = 0.0428, and the difference was statistically significant.

[0054] The immunofluorescence results are as Figure 3 shown in C. Compared with the Scramble group, the fluorescence of VE-cadherin in HULEC-5A cells in the OSM group weakened and the continuity was disrupted. Compared with the OSM group, the fluorescence of VE-cadherin in the siOSMR + OSM group of cells increased and the continuity was somewhat restored. The in vitro FITC-dextran permeability test of cells can reflect the endothelial permeability through the permeability of FITC-dextran in monolayer endothelial cells.

[0055] The permeability results of the cells are as Figure 3 shown in D. Compared with the Scramble group, the relative permeability of cells in the OSM group increased (P = 0.0005). Compared with the OSM group, the permeability of cells in the siOSMR + OSM group decreased (P = 0.0057).

[0056] The results of the endothelial cell transmembrane impedance experiment are as Figure 3As shown in E, compared with the Scramble group, the transcellular membrane resistance value of the OSM group was significantly decreased (P<0.01). Compared with the OSM group, the transcellular membrane resistance value of the siOSMR+OSM group cells increased (P<0.01), indicating that the integrity of the endothelial barrier was somewhat restored.

[0057] Therefore, all of the above results indicate that OSM promotes the reduction of intercellular adhesion protein expression and the increase of endothelial permeability through the OSMR receptor on endothelial cells, resulting in the destruction of the endothelial barrier.

[0058] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the claims of this patent application.

Claims

1. A method for constructing a pulmonary vascular endothelial barrier injury model, characterized in that, After adaptively feeding healthy mice for one week, they were anesthetized with drugs. After pretreatment with oncostatin M recombinant protein, it was instilled into the lungs of mice via the airway to obtain a model of pulmonary vascular endothelial barrier injury.

2. The construction method according to claim 1, characterized in that The drug anesthesia used ketamine and xylazine, with the mass ratio of ketamine being 80 mg / kg and the mass ratio of xylazine being 10 mg / kg.

3. The construction method according to claim 1, wherein The pretreatment of oncostatin M recombinant protein was to dilute oncostatin M recombinant protein to 10 - 50 ng / μL with PBS, and the treatment time of oncostatin M recombinant protein on mice was 12 - 24 h.

4. The construction method according to claim 3, wherein The pretreatment of oncostatin M recombinant protein was to dilute oncostatin M recombinant protein to 20 ng / μL with PBS, and the treatment time of oncostatin M recombinant protein on mice was 24 h.

5. A lung vascular endothelial barrier injury model, characterized in that, It was prepared by the following steps: After adaptively feeding healthy mice for one week, they were anesthetized with drugs. After pretreatment with oncostatin M recombinant protein, it was instilled into the lungs of mice via the airway to obtain a model of pulmonary vascular endothelial barrier injury.

6. Use of a lung vascular endothelial barrier injury model in the preparation of a research model for pulmonary vascular diseases, characterized in that, It was prepared by the following steps: After adaptively feeding healthy mice for one week, they were anesthetized with drugs. After pretreatment with oncostatin M recombinant protein, it was instilled into the lungs of mice via the airway to obtain a model of pulmonary vascular endothelial barrier injury.

7. Use of a pulmonary vascular endothelial barrier injury model in the preparation of a drug for treating pulmonary vascular diseases, characterized in that, It was prepared by the following steps: After adaptively feeding healthy mice for one week, they were anesthetized with drugs. After pretreatment with oncostatin M recombinant protein, it was instilled into the lungs of mice via the airway to obtain a model of pulmonary vascular endothelial barrier injury.

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