WO2024065892A1 - 检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和羟基二十二碳六烯酸的应用 - Google Patents

检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和羟基二十二碳六烯酸的应用 Download PDF

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WO2024065892A1
WO2024065892A1 PCT/CN2022/125577 CN2022125577W WO2024065892A1 WO 2024065892 A1 WO2024065892 A1 WO 2024065892A1 CN 2022125577 W CN2022125577 W CN 2022125577W WO 2024065892 A1 WO2024065892 A1 WO 2024065892A1
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eos
pulmonary hypertension
pulmonary
smooth muscle
hdha
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French (fr)
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王婧
邢岩江
舒婷
张佳玮
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中国医学科学院基础医学研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension

Definitions

  • the invention belongs to the field of medical treatment and pharmaceutical technology, and in particular relates to a reagent for detecting eosinophils, and applications of eosinophils and their cytoplasmic contents and lipid metabolite hydroxydocosahexaenoic acid.
  • Pulmonary hypertension is a group of pulmonary circulation diseases caused by a variety of causes and different pathogenesis, with progressive increase in mean pulmonary artery pressure.
  • the diagnostic standard is defined as a mean pulmonary artery pressure greater than or equal to 25 mmHg measured by right cardiac catheter at rest and sea level.
  • dysfunction of pulmonary vascular intrinsic cells and abnormal infiltration of various immune cells often cause pulmonary vasoconstriction and pulmonary vascular remodeling, leading to thickening and stenosis of the patient's pulmonary vessels, increased pulmonary artery pressure, and ultimately right heart failure and right heart dysfunction.
  • the purpose of the present invention is to provide a reagent for detecting eosinophils (EOS), and the use of eosinophils and their cytoplasmic contents and lipid metabolites hydroxydocosahexaenoic acid (HDHA).
  • EOS eosinophils
  • HDHA hydroxydocosahexaenoic acid
  • the present invention utilizes a reagent for detecting eosinophils to achieve the diagnosis of pulmonary hypertension and/or the judgment of the prognosis risk of pulmonary hypertension.
  • the present invention provides the use of a reagent for detecting eosinophils in preparing a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension.
  • the detection includes detecting the proportion of eosinophils in peripheral blood.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic content in preparing a drug for inhibiting the proliferation and/or migration of pulmonary artery smooth muscle.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic content in the preparation of a medicine for maintaining the homeostasis of pulmonary artery smooth muscle.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic contents in preparing medicine for treating pulmonary hypertension.
  • the present invention also provides the use of hydroxydocosahexaenoic acid in preparing medicine for treating pulmonary hypertension.
  • the present invention also provides the use of hydroxydocosahexaenoic acid in preparing a medicine for inhibiting the proliferation of pulmonary vascular smooth muscle.
  • the hydroxydocosahexaenoic acid comprises 14-hydroxydocosahexaenoic acid and/or 17-hydroxydocosahexaenoic acid.
  • the present invention provides the use of a reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension.
  • the present invention finds that EOS is closely related to the occurrence of pulmonary hypertension, and therefore proposes to use EOS as a biomarker indicating PH conditions and apply it to the preparation of related products for diagnosing PH. If the EOS ratio in peripheral blood routine tests is lowered, it indicates the risk of pulmonary hypertension and poor prognosis of pulmonary hypertension.
  • the application of the present invention can realize the diagnosis of pulmonary hypertension and the judgment of the prognosis risk of pulmonary hypertension.
  • EOS has a protective effect against pulmonary hypertension.
  • the contents of EOS can inhibit the proliferation and/or migration of pulmonary artery smooth muscle cells, the most important intrinsic cells in the process of pulmonary vascular remodeling.
  • the cytoplasmic contents of EOS have a protective effect in maintaining the homeostasis of pulmonary artery smooth muscle. EOS and its cytoplasmic contents have a therapeutic effect on pulmonary hypertension.
  • FIG1 is a schematic diagram of the experimental results of the EOS ratio in the peripheral blood of patients with pulmonary hypertension provided by the present invention and its correlation analysis with the severity of the disease; wherein A. a graph of the EOS ratio in peripheral blood samples of patients with pulmonary hypertension and normal healthy people; B. a graph of the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary hypertension and the NYHA cardiac function classification; C. a graph of the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary hypertension and the BMPR2 mutation;
  • FIG2 is a schematic diagram of the experimental results of the proportion of EOS in the animal model of pulmonary hypertension provided by the present invention; wherein A. the proportion of EOS in peripheral blood of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen and a typical flow cytometry graph; B. the proportion of EOS in lung tissue of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen and a typical flow cytometry graph;
  • FIG3 is a schematic diagram of the experimental results of EOS knockout aggravated hypoxia combined with Sugen-induced pulmonary hypertension in mice provided by the present invention; wherein A. a graph showing the right ventricular systolic pressure of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; B. a graph showing the right heart hypertrophy index of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; C. a typical schematic diagram of anti- ⁇ -smooth muscle actin immunofluorescence staining of pulmonary vascular of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; wild-type mice were the experimental control group;
  • FIG4 is a schematic diagram of the experimental results of EOS inhibiting abnormal function of pulmonary artery smooth muscle cells provided by the present invention; wherein A. a graph showing the proliferation of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and administration of EOS conditioned medium; B. a graph showing the migration of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and administration of EOS conditioned medium;
  • FIG. 5 is a schematic diagram of the experimental results of the effects of EOS knockout on fatty acid metabolism in lung tissue of mice induced by hypoxia combined with Sugen provided by the present invention; wherein, A. EOS knockout/wild-type mouse lung tissue targeted metabolomics principal component analysis results under pulmonary hypertension; B. EOS knockout/wild-type mouse lung tissue differential expression heat map under pulmonary hypertension; C. EOS knockout/wild-type mouse lung tissue lipid metabolites quantitative analysis results under pulmonary hypertension; wild-type mice were the experimental control group;
  • FIG6 is a schematic diagram of the experimental results of the inhibitory effect of 14-HDHA and 17-HDHA on the proliferation of pulmonary artery smooth muscle cells provided by the present invention.
  • the present invention provides an application of a reagent for detecting eosinophils in preparing a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension.
  • eosinophils can be used as biomarkers for the preparation of products for identification and/or auxiliary diagnosis of pulmonary hypertension.
  • the detection preferably includes detecting the proportion of eosinophils in peripheral blood.
  • the product preferably includes a kit.
  • the sample to be tested preferably includes peripheral blood.
  • the proportion of EOS in peripheral blood routine test if lowered relative to healthy controls, indicates the risk of pulmonary hypertension and/or poor prognosis of pulmonary hypertension.
  • the ratio of the lowering is preferably 25% or more, more preferably 30% or more.
  • the average value of male normal controls is 2.668, and the average value of male patients is 1.78, which is lowered by 33%; the average value of female normal controls is 2.024, and the average value of female patients is 1.404, which is lowered by 30%.
  • the pulmonary hypertension preferably includes pulmonary hypertension induced by hypoxia combined with Sugen.
  • the results of the embodiments of the present invention show that the proportion of EOS in the peripheral blood of patients with pulmonary hypertension is reduced, and the lower the proportion of EOS in peripheral blood, the more severe the condition of patients with pulmonary hypertension, and EOS can assist in the diagnosis of pulmonary hypertension.
  • the proportion of EOS in peripheral blood can also indicate the risk of BMPR2 mutation. The lower the proportion of EOS in peripheral blood, the greater the risk of BMPR2 mutation and the greater the risk of poor prognosis.
  • Animal experiments have shown that with the development of pulmonary hypertension, the proportion of EOS in peripheral blood of mice decreases, and the infiltration of EOS in lung tissue increases. EOS has the effect of protecting pulmonary hypertension and can assist in the diagnosis or judgment of pulmonary hypertension.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic content in preparing a drug for inhibiting the proliferation and/or migration of pulmonary artery smooth muscle.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic content in preparing a medicine for maintaining the homeostasis of pulmonary artery smooth muscle.
  • the present invention also provides the use of EOS and/or EOS cytoplasmic contents in preparing medicine for treating pulmonary hypertension.
  • EOS basic research believes that the lysate contains all the cytoplasmic contents of EOS.
  • the present invention embodiment can prove the role of EOS cytoplasmic contents by adding lysate.
  • the test results show that EOS plays a protective role in the progression of pulmonary hypertension; the contents of EOS can inhibit the proliferation and/or migration of pulmonary artery smooth muscle, and the cytoplasmic contents of EOS have a protective effect in maintaining the homeostasis of pulmonary artery smooth muscle, thereby playing a role in the treatment of pulmonary hypertension.
  • the present invention also provides the use of lipid metabolites hydroxydocosahexaenoic acid (HDHA) in the preparation of drugs for treating pulmonary hypertension.
  • HDHA can effectively inhibit the proliferation of smooth muscle cells, the most important intrinsic cells in the process of pulmonary vascular remodeling, in vitro, and inhibit the infiltration of inflammatory cells such as neutrophils; in vivo experiments also show that pulmonary vascular remodeling and peripheral inflammation are aggravated after knocking out eosinophils; HDHA is a natural metabolite of DHA, which has fewer side effects and is safer than other drugs. It is also found that it can simultaneously maintain immune homeostasis and inhibit smooth muscle proliferation.
  • the hydroxydocosahexaenoic acid preferably includes 14-hydroxydocosahexaenoic acid (14-HDHA) and/or 17-hydroxydocosahexaenoic acid (17-HDHA).
  • 14-HDHA 14-hydroxydocosahexaenoic acid
  • 17-HDHA 17-hydroxydocosahexaenoic acid
  • the present invention also provides the use of lipid metabolites hydroxydocosahexaenoic acid (HDHA) in the preparation of drugs for inhibiting the proliferation of pulmonary vascular smooth muscle.
  • the hydroxydocosahexaenoic acid preferably includes 14-hydroxydocosahexaenoic acid and/or 17-hydroxydocosahexaenoic acid.
  • 14-HDHA and 17-HDHA are EOS downstream lipid metabolites, and the levels of 14-HDHA and 17-HDHA are significantly reduced after EOS knockout. Stimulation with a smaller concentration of 14-HDHA and/or 17-HDHA can effectively inhibit the proliferation of pulmonary artery smooth muscle cells.
  • the smaller concentration of the present invention is preferably 10 to 20 nM, more preferably 10 nM or 20 nM. Giving EOS downstream metabolites 14-HDHA and/or 17-HDHA can effectively inhibit the proliferation of pulmonary vascular smooth muscle, and therefore can be used to prepare therapeutic drugs for PH.
  • the proportion of EOS in peripheral blood is reduced in patients with pulmonary hypertension and animal models, and is correlated with the severity of the disease in patients with pulmonary hypertension.
  • the subjects were a single-center clinical cohort of 123 patients with idiopathic or hereditary pulmonary hypertension and 119 age- and sex-matched healthy controls enrolled in the Fuwai Hospital of the Chinese Academy of Medical Sciences.
  • the diagnostic criteria for patients with idiopathic or hereditary pulmonary hypertension were as follows: mean pulmonary artery pressure ⁇ 25 mmHg and pulmonary arteriolar wedge pressure ⁇ 15 mmHg detected by right cardiac catheterization at sea level. Exclusions: 1 patients with allergies, infections, and autoimmune diseases; 2 patients with lung diseases (tumors, infections, fibrosis, etc.), autoimmune diseases, and 3 adolescents under 18 years old.
  • Venous blood was collected from fasting patients and healthy controls, and ethylenediaminetetraacetic acid was used for anticoagulation.
  • the present invention was first approved by the Institutional Ethics Committee of Peking Union Medical College (2018043) and Fuwai Hospital (approval number: 2017-877). All subjects signed informed consent before inclusion in the cohort.
  • mice C57/B6J male mice aged 8 to 10 weeks and weighing more than 25g were placed in a hypoxic chamber with an oxygen concentration of 10%.
  • Sugen5416 was subcutaneously injected once a week at a dose of 20mg/kg/time for a total of 3 injections. The mice were kept in the hypoxic chamber for 3 weeks. The right ventricular pressure of the anesthetized mice was measured. If the right ventricular pressure rose to more than 30mmHg, the model was established.
  • Peripheral blood was collected from healthy controls and mice with pulmonary hypertension and anticoagulated with ethylenediaminetetraacetic acid.
  • Neutral protease (5U/mL, Worthington) + type I collagenase (200U/mL, Vetec) + elastase (0.02U/mL, Sigma) + DNA enzyme (0.3U/mL, Progema) was used to prepare lung tissue digestion solution. The first lobe of the right lung tissue was cut into pieces in the digestion solution. After shaking on a shaker at 37°C for 25 minutes, the digestion was terminated with an equal volume of complete culture medium (basal culture medium with 10% FBS added). After red lysis, the cells were centrifuged and resuspended.
  • Figure 1 is a schematic diagram of the experimental results of the EOS proportion in the peripheral blood of patients with pulmonary hypertension and its correlation with the severity of the disease; A. The proportion of EOS in peripheral blood samples of patients with pulmonary hypertension and normal healthy people; B. The relationship between the proportion of EOS in peripheral blood samples of patients with pulmonary hypertension and NYHA heart function classification; C. The relationship between the proportion of EOS in peripheral blood samples of patients with pulmonary hypertension and BMPR2 mutation.
  • Figure 2 is a schematic diagram of the experimental results of the EOS proportion in animal models of pulmonary hypertension; A. The proportion of EOS in peripheral blood and a typical flow cytometry graph of a mouse pulmonary hypertension model induced by hypoxia combined with Sugen; B. The proportion of EOS in lung tissue and a typical flow cytometry graph of a mouse pulmonary hypertension model induced by hypoxia combined with Sugen.
  • the present invention finds that the proportion of EOS in the peripheral blood of patients with pulmonary hypertension is reduced (A in Figure 1), and the lower the proportion of EOS in peripheral blood, the more severe the condition of patients with pulmonary hypertension (B in Figure 1). It can be seen that EOS can assist in the diagnosis of pulmonary hypertension. The lower the proportion of EOS in peripheral blood, the higher the risk of illness and the more severe the condition; and the proportion of EOS in peripheral blood of patients carrying BMPR2 mutations is less (C in Figure 1), indicating that the lower the proportion of EOS in peripheral blood, the greater the risk of BMPR2 mutation and the greater the risk of poor prognosis.
  • the present invention finds that with the development of pulmonary hypertension, the proportion of EOS in mouse peripheral blood decreases, and the infiltration of EOS in lung tissue increases (Figure 2). EOS has the effect of protecting pulmonary hypertension and can assist in the diagnosis or judgment of pulmonary hypertension. The lower the proportion of EOS in peripheral blood, the more severe the condition.
  • mice Male mice aged 8 to 10 weeks and weighing more than 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%. Sugen5416 was injected subcutaneously once a week at a dose of 20 mg/kg/time, for a total of 3 injections. The mice were kept in the hypoxic chamber for 3 weeks. The right ventricular pressure of the anesthetized mice was measured. If the right ventricular pressure rose to above 30 mmHg, the model was completed.
  • Control group 1 Normoxic wild type: 4 mice were housed in a normoxic environment for 3 weeks; DMSO was injected subcutaneously once every 7 days starting from day 1, for a total of 3 injections.
  • Control group 2 normoxic EOS knockout: 4 mice were housed in a normoxic environment for 3 weeks; DMSO was injected subcutaneously once every 7 days starting from day 1, for a total of 3 injections.
  • Modeling group 1 Hypoxia combined with Sugen wild type: 7 mice, raised in a hypoxic environment for 3 weeks; Sugen5416 (DMSO as solvent) was injected subcutaneously once every 7 days starting from the first day, for a total of 3 injections.
  • Sugen5416 DMSO as solvent
  • Modeling group 2 hypoxia combined with Sugen EOS knockout: 9 mice were raised in a hypoxic environment for 3 weeks; Sugen5416 (DMSO as solvent) was injected subcutaneously once every 7 days starting from the first day, for a total of 3 injections.
  • Sugen5416 DMSO as solvent
  • a 22-gauge needle connected to the pressure sensor of the PowerLab instrument was inserted into the right ventricle of the mouse anesthetized by 2% tribromoethanol intraperitoneally through the xiphoid angle, and the position of the needle was determined by the waveform displayed by the pressure sensor. After the waveform stabilized, the right ventricular systolic pressure was recorded.
  • RV right ventricular free wall
  • LV + S left ventricle + ventricular septum
  • the left lung tissue was collected, fixed with 10% neutral formaldehyde at 4°C, and then precipitated with sugar for 72 hours, and embedded in OCT. All lung tissues were embedded in the same direction (cross section).
  • the lung tissue was sliced to a thickness of 5 microns. Incubated with the primary antibody against ⁇ -smooth muscle actin at 4°C overnight, washed and stained with the corresponding HRP-conjugated secondary antibody, incubated in a wet box at room temperature for 30 minutes, washed and then DAPI was added to seal the slide and photographed.
  • Figure 3 is a schematic diagram of the experimental results of EOS knockout-aggravated hypoxia combined with Sugen-induced pulmonary hypertension in mice; wherein, A. right ventricular systolic pressure of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; B. right heart hypertrophy index of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; C. typical schematic diagram of anti- ⁇ -smooth muscle actin immunofluorescence staining of pulmonary vascular of EOS knockout/wild-type mice under normal control and pulmonary hypertension conditions; wild-type mice served as the experimental control group.
  • EOS cell lysate After serum starvation for 24 h, pulmonary artery smooth muscle cells were added with growth factor PDGFbb and gradient EOS cell lysate for stimulation and divided into: untreated control group, PDGFbb stimulation group, PDGFbb+10 3 EOS lysate group (marked as 10 3 EOS in the figure), PDGFbb+10 4 EOS lysate group (marked as 10 4 EOS in the figure), PDGFbb+10 5 EOS lysate group (marked as 10 5 EOS in the figure) and PDGFbb+10 6 EOS lysate group (marked as 10 6 EOS in the figure).
  • the culture media corresponding to the above groups are collectively referred to as EOS conditioned medium.
  • the untreated control group contained only smooth muscle cell culture medium
  • the PDGFbb stimulation group contained smooth muscle cell culture medium containing growth factor PDGFbb
  • the other four groups contained smooth muscle cell culture medium containing growth factor PDGFbb and EOS lysate).
  • Pulmonary artery smooth muscle cells were plated into a 96-well plate at 5000 cells/well. After serum starvation was removed for 24 hours, corresponding conditioned medium was added according to the experimental design, 100 ⁇ L of medium per well. After 24 hours of stimulation, 10 ⁇ L of CCK8 stock solution was added to each well and incubated for 2 hours. The absorbance at 450 nm and 570 nm was read with an enzyme reader, and a standard curve was drawn to calculate the cell proliferation percentage.
  • Transwell experiment Pulmonary artery smooth muscle cells were plated into the upper chamber of the Transwell chamber, and 200 ⁇ L serum-free basal medium was added to each well. The corresponding conditioned medium was added to the lower chamber according to the experimental design, with 600 ⁇ L medium per well. After 24 hours of stimulation, the cells were fixed with methanol, stained with crystal violet, and photographed to calculate the number of migrated cells.
  • Figure 4 is a schematic diagram of the experimental results of EOS inhibiting the abnormal function of pulmonary artery smooth muscle cells; wherein, A. the proliferation of pulmonary artery smooth muscle cells after induction by growth factor PDGFbb and administration of EOS conditioned medium, as the concentration of EOS lysate increases, the proliferation of pulmonary artery smooth muscle cells is more strongly inhibited; B. the migration of pulmonary artery smooth muscle cells after induction by growth factor PDGFbb and administration of EOS conditioned medium, as the concentration of EOS lysate increases, the degree of scratch healing decreases and the number of Transwell transmembrane cells decreases.
  • A the proliferation of pulmonary artery smooth muscle cells after induction by growth factor PDGFbb and administration of EOS conditioned medium, as the concentration of EOS lysate increases, the degree of scratch healing decreases and the number of Transwell transmembrane cells decreases.
  • the present invention found that the inhibitory effect increased with the concentration gradient of EOS lysate, that is, the higher the concentration of EOS lysate, the stronger the inhibitory effect ( FIG. 4 ), indicating that the contents of EOS can inhibit the proliferation and migration of pulmonary artery smooth muscle, and that the cytoplasmic contents of EOS have a protective effect in maintaining the homeostasis of pulmonary artery smooth muscle.
  • mice Male mice aged 8 to 10 weeks and weighing more than 25g were placed in a hypoxic chamber with an oxygen concentration of 10%.
  • Sugen5416 was subcutaneously injected once a week at a dose of 20mg/kg/time for a total of 3 injections. The mice were kept in the hypoxic chamber for 3 weeks. The right ventricular pressure of the anesthetized mice was measured. If the right ventricular pressure rose to more than 30mmHg, the model was completed. After right ventricular perfusion, the mouse lung tissue was collected and cryopreserved in liquid nitrogen.
  • Wild-type modeling group hypoxia combined with Sugen Wild-type: 9 mice, raised in a hypoxic environment for 3 weeks; Sugen5416 was injected subcutaneously once every 7 days starting from the first day, for a total of 3 injections.
  • EOS knockout modeling group hypoxia combined with Sugen EOS knockout: 9 mice were raised in a hypoxic environment for 3 weeks; Sugen5416 was injected subcutaneously once every 7 days starting from the first day, for a total of 3 injections.
  • Pulmonary artery smooth muscle cells were plated into 96-well plates at 5000 cells/well. After serum starvation was removed for 24 hours, an untreated control group (smooth muscle cell culture medium), a PDGFbb stimulation group (smooth muscle cell culture medium containing growth factor PDGFbb), a PDGFbb+14-HDHA group (smooth muscle cell culture medium containing growth factors PDGFbb and 14-HDHA) and a PDGFbb+17-HDHA group (smooth muscle cell culture medium containing growth factors PDGFbb and 17-HDHA) were set up. Two types of lipid small molecules were set at 10nM and 20nM concentration gradients, respectively, with 100 ⁇ L culture medium in each well. After 24 hours of stimulation, 10 ⁇ L CCK8 stock solution was added to each well and incubated for 2 hours. The absorbance at 450nm and 570nm was read with an enzyme reader, and a standard curve was drawn to calculate the cell proliferation percentage.
  • Figure 5 is a schematic diagram of the experimental results of EOS knockout affecting fatty acid metabolism in lung tissue of mice induced by hypoxia and Sugen;
  • A Targeted metabolomics principal component analysis of lung tissue of EOS knockout/wild-type mice under pulmonary hypertension;
  • B Heat map of differential expression of lipid metabolites in lung tissue of EOS knockout/wild-type mice under pulmonary hypertension;
  • C Quantitative analysis of lipid metabolites in lung tissue of EOS knockout/wild-type mice under pulmonary hypertension; wild-type mice were the experimental control group.
  • Figure 6 is a schematic diagram of the experimental results of the inhibitory effect of 14-HDHA and 17-HDHA on the proliferation of pulmonary artery smooth muscle cells.
  • mice with hypoxia combined with Sugen-induced pulmonary artery after knocking out EOS, there were significant changes in the metabolism of fatty acids such as arachidonic acid, DHA and EPA compared with the wild-type control group (A and B in Figure 5).
  • the present invention found that the levels of small molecule lipid metabolites 14-HDHA and 17-HDHA were significantly reduced after EOS was knocked out (C in Figure 5).
  • 14-HDHA and 17-HDHA were stimulated in vitro to pulmonary artery smooth muscle cells, a small concentration could effectively inhibit the proliferation of pulmonary artery smooth muscle cells (Figure 6).
  • the downstream metabolites of EOS, 14-HDHA and 17-HDHA have a therapeutic effect on pulmonary hypertension.
  • mice Male mice aged 8 to 10 weeks and weighing more than 25g were placed in a hypoxic chamber with an oxygen concentration of 10%.
  • Sugen5416 was subcutaneously injected once a week at a dose of 20mg/kg/time for a total of 3 injections. The mice were kept in the hypoxic chamber for 3 weeks.
  • the right ventricular pressure was measured for anesthetized mice. If the right ventricular pressure rose to more than 30mmHg, the model was completed. Starting from the second week of hypoxia, 14-HDHA and 17-HDHA were injected intraperitoneally every 3 days until the model was completed. At the end of the model, the right ventricular systolic pressure, right heart hypertrophy index and pulmonary artery smooth muscle layer thickness were evaluated.
  • mice (rats) given solvent control group mice (rats) given 14-HDHA or 17-HDHA.
  • mice (rats) given 14-HDHA or 17-HDHA mice (rats) given 14-HDHA or 17-HDHA.
  • the right ventricular systolic pressure and right heart hypertrophy index of mice (rats) given 14-HDHA or 17-HDHA were significantly lower than those of mice (rats) in solvent control group, and the pulmonary artery smooth muscle layer was significantly thinner.
  • mice rats
  • 14-HDHA or 17-HDHA can be used to prepare therapeutic drugs for pulmonary hypertension.

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Abstract

提供一种检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和脂质代谢产物羟基二十二碳六烯酸的应用，属于医疗和医药技术领域。提供了检测嗜酸性粒细胞的试剂在制备诊断肺动脉高压和/或肺动脉高压预后风险判断的产品中的应用。通过对外周血中EOS的占比进行检测，可以实现肺动脉高压的诊断以及肺动脉高压预后风险的判断。

Description

检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和羟基二十二碳六烯酸的应用 技术领域
本发明属于医疗和医药技术领域，具体涉及检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和脂质代谢产物羟基二十二碳六烯酸的应用。
背景技术
肺动脉高压(Pulmonary Hypertension,简称PH)是一组由多种病因及不同发病机制所致的平均肺动脉压力进行性升高的肺循环疾病，目前将静息、海平面状态下经右心导管测得平均肺动脉压力大于或等于25mmHg定义为其诊断标准。肺动脉高压进展过程中，肺血管固有细胞的功能紊乱及各类免疫细胞的异常浸润常引起肺血管收缩和肺血管重塑，导致患者肺血管增厚、狭窄，肺动脉压力升高，最终发展为右心衰竭及右心功能不全。
目前临床使用的靶向药物多通过扩张血管来降低肺血管阻力、进而缓解肺动脉高压，但无法从根本上逆转血管重塑的情况，因此仍然急需筛选有效改善血管重构，能够尽快进入临床的药物用于肺动脉高压的治疗。
发明内容
本发明的目的在于提供检测嗜酸性粒细胞(Eosinophil，简称EOS)的试剂、嗜酸性粒细胞及其胞质内容物和脂质代谢产物羟基二十二碳六烯酸(hydroxy docosahexaenoic acid，HDHA)的应用。本发明利用检测嗜酸性粒细胞的试剂，可以实现肺动脉高压疾病的诊断和/或肺动脉高压预后风险的判断。
本发明提供了检测嗜酸性粒细胞的试剂在制备诊断肺动脉高压和/或肺动脉高压预后风险判断的产品中的应用。
优选的是，所述检测包括检测外周血中嗜酸性粒细胞的占比。
本发明还提供了EOS和/或EOS胞质内容物在制备抑制肺动脉平滑肌增殖和/或迁移的药物中的应用。
本发明还提供了EOS和/或EOS胞质内容物在制备维持肺动脉平滑 肌稳态的药物中的应用。
本发明还提供了EOS和/或EOS胞质内容物在制备治疗肺动脉高压的药物中的应用。
本发明还提供了羟基二十二碳六烯酸在制备治疗肺动脉高压的药物中的应用。
本发明还提供了羟基二十二碳六烯酸在制备抑制肺血管平滑肌增殖的药物中的应用。
优选的是，所述羟基二十二碳六烯酸包括14-羟基二十二碳六烯酸和/或17-羟基二十二碳六烯酸。
本发明提供了检测嗜酸性粒细胞的试剂在制备诊断肺动脉高压和/或肺动脉高压预后风险判断的产品中的应用。本发明发现EOS与肺动脉高压发生密切相关，因此提出将EOS作为提示PH病情的生物标志物，应用到制备诊断PH的相关产品中。外周血血常规检测EOS占比，如果下调的话，提示有肺动脉高压以及肺动脉高压预后不良的风险。本发明所述应用能够实现肺动脉高压疾病的诊断以及肺动脉高压预后风险的判断。
试验结果表明，随着肺动脉高压的发展，小鼠外周血EOS占比减少，肺组织EOS浸润增多，EOS具有保护肺动脉高压的作用；EOS的内容物能抑制肺血管重塑过程中最重要的固有细胞——肺动脉平滑肌细胞的增殖和/或迁移，EOS胞质内容物存在维持肺动脉平滑肌稳态的保护性作用。EOS及其胞质内容物具有治疗肺动脉高压的作用。
说明书附图
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明提供的肺动脉高压患者外周血中的EOS占比及其与疾病严重程度的相关性分析实验结果示意图；其中，A.肺动脉高压患者和正常健康人群外周血样本中的EOS占比图；B.肺动脉高压患者外周血样本中EOS占比与NYHA心功能分级的关系图；C.肺动脉高压患者外周血样本中EOS占比与BMPR2突变的关系图；
图2为本发明提供的肺动脉高压动物模型中的EOS占比实验结果示意图；其中，A.低氧联合Sugen诱导的小鼠肺动脉高压模型外周血中的EOS占比及流式细胞术典型图；B.低氧联合Sugen诱导的小鼠肺动脉高压模型肺组织中的EOS占比及流式细胞术典型图；
图3为本发明提供的EOS敲除加重低氧联合Sugen诱导小鼠肺动脉高压的实验结果示意图；其中，A.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠右心室收缩压结果图；B.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠右心肥厚指数结果图；C.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠肺血管抗α-平滑肌肌动蛋白免疫荧光染色典型示意图；野生型小鼠为实验对照组；
图4为本发明提供的EOS抑制肺动脉平滑肌细胞异常功能的实验结果示意图；其中，A.生长因子PDGFbb诱导和给予EOS条件培养基后的肺动脉平滑肌细胞增殖情况结果图；B.生长因子PDGFbb诱导和给予EOS条件培养基后的肺动脉平滑肌细胞迁移情况结果图；
图5为本发明提供的EOS敲除影响低氧联合Sugen诱导小鼠肺组织脂肪酸代谢的实验结果示意图；其中，A.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织靶向代谢组学主成分分析结果图；B.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织脂质代谢产物差异表达热图；C.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织脂质代谢产物定量分析结果图；野生型小鼠为实验对照组；
图6为本发明提供的14-HDHA及17-HDHA对肺动脉平滑肌细胞增殖的抑制作用实验结果示意图。
具体实施方式
本发明提供了一种检测嗜酸性粒细胞的试剂在制备诊断肺动脉高压和/或肺动脉高压预后风险判断的产品中的应用。
在本发明中，嗜酸性粒细胞可作为生物标志物用于鉴别和/或辅助诊断肺动脉高压的产品的制备。在本发明中，所述检测优选包括检测外周血中嗜酸性粒细胞的占比。在本发明中，所述产品优选包括试剂盒。
在本发明中，所述检测的样品优选包括外周血。外周血血常规检测EOS占比，相对于健康对照，如果下调的话，提示有肺动脉高压和/或肺 动脉高压预后不良的风险。在本发明中，所述下调的比例优选为25％以上，更优选为30％以上。如本发明临床试验数据表明，男性正常对照平均值为2.668，男性患者平均值为1.78，下调33％；女性正常对照平均值为2.024，女性患者平均值为1.404，下调30％。
在本发明中，所述肺动脉高压优选包括低氧联合Sugen诱导的肺动脉高压。
本发明实施例的结果表明，肺动脉高压患者外周血中EOS的占比降低，且EOS外周血占比越低，肺动脉高压患者病情越重，EOS能够辅助诊断肺动脉高压疾病。外周血EOS占比还能提示BMPR2突变风险，外周血EOS占比越低，提示BMPR2突变风险大，预后不良的风险大。动物试验表明，随着肺动脉高压的发展，小鼠外周血EOS占比减少，肺组织EOS浸润增多，EOS具有保护肺动脉高压的作用，能够辅助诊断或判断肺动脉高压，外周血EOS占比越低，病情越严重。EOS敲除后，小鼠右心室收缩压和右心肥厚指数明显升高，肺动脉平滑肌层明显增厚，提示EOS在肺动脉高压进展过程中为保护作用，EOS可作为辅助诊断指标。嗜酸性粒细胞的占比的检测方法采用血常规检测即可。
本发明还提供了EOS和/或EOS胞质内容物在制备抑制肺动脉平滑肌增殖和/或迁移的药物中的应用。
本发明还提供了EOS和/或EOS胞质内容物在制备维持肺动脉平滑肌稳态的药物中的应用。
本发明还提供了EOS和/或EOS胞质内容物在制备治疗肺动脉高压的药物中的应用。
EOS基础研究认为裂解液中包含EOS全部胞质内容物。本发明实施例通过裂解液的添加能够证明EOS胞质内容物的作用。试验结果表明，EOS在肺动脉高压进展过程中为保护作用；EOS的内容物能抑制肺动脉平滑肌增殖和/或迁移，EOS胞质内容物存在维持肺动脉平滑肌稳态的保护性作用，进而能够起到对肺动脉高压治疗的作用。
本发明还提供了脂质代谢产物羟基二十二碳六烯酸(HDHA)在制备治疗肺动脉高压的药物中的应用。HDHA可以有效在体外抑制肺血管重塑过程中最重要的固有细胞——平滑肌细胞的增殖，并抑制中性粒等炎细 胞浸润；体内实验也表明敲除嗜酸性粒细胞之后肺血管重构及周围炎症加重；HDHA为DHA的天然代谢产物，相比于其他药物副作用更小更安全，同时也发现其可以同时维持免疫稳态并且抑制平滑肌增殖。在本发明中，所述羟基二十二碳六烯酸优选包括14-羟基二十二碳六烯酸(14-HDHA)和/或17-羟基二十二碳六烯酸(17-HDHA)。EOS的下游代谢产物14-HDHA和17-HDHA对肺动脉高压有治疗作用。
本发明还提供了脂质代谢产物羟基二十二碳六烯酸(HDHA)在制备抑制肺血管平滑肌增殖的药物中的应用。在本发明中，所述羟基二十二碳六烯酸优选包括14-羟基二十二碳六烯酸和/或17-羟基二十二碳六烯酸。14-HDHA和17-HDHA属于EOS下游脂质代谢产物，14-HDHA与17-HDHA的水平在EOS敲除后出现明显降低。较小浓度的14-HDHA和/或17-HDHA刺激即可有效抑制肺动脉平滑肌细胞增殖，本发明所述较小浓度优选为10～20nM，更优选为10nM或20nM。予以EOS下游代谢产物14-HDHA和/或17-HDHA能够有效地抑制肺血管平滑肌增殖，因此可用于制备PH的治疗药物。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和脂质代谢产物羟基二十二碳六烯酸的应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
肺动脉高压患者及动物模型外周血EOS占比降低，并与肺动脉高压患者疾病严重程度相关。
1.1受试人群及样本收集
受试对象来自中国医学科学院阜外医院纳入的123名特发性或遗传性肺动脉高压患者和119名年龄和性别匹配的健康对照组成的单中心临床队列。特发性或遗传性肺动脉高压患者的诊断标准：海平面静息状态下，右心导管检测肺动脉平均压≥25mmHg，肺小动脉楔压≤15mmHg。排除：①过敏、感染及自身免疫病患者；②肺部疾病(肿瘤、感染、纤维化等)、自身免疫性疾病和③18岁以下青少年。
收集空腹患者、健康对照静脉血，乙二胺四乙酸抗凝。本发明均首先 获得北京协和医学院机构伦理委员会(2018043)和阜外医院(批准号：2017-877)的批准。所有受试者在纳入队列前均签署知情同意书。
1.2收集低氧联合Sugen诱导的肺动脉高压小鼠外周血
将8～10周龄体重25g以上的C57/B6J雄鼠置于10％氧气浓度的低氧仓中，每周进行一次皮下注射Sugen5416，剂量为20mg/kg/次，共3次注射，在低氧仓中持续饲养3周，麻醉小鼠进行右心室压力测定，如右心室压力升至30mmHg以上即完成建模。收取健康对照及肺动脉高压小鼠外周血，采用乙二胺四乙酸抗凝。
1.3小鼠肺组织单细胞悬液制备及流式细胞术分析
采用中性蛋白酶(5U/mL，Worthington)+I型胶原酶(200U/mL，Vetec)+弹性蛋白酶(0.02U/mL，Sigma)+DNA酶(0.3U/mL，Progema)配制肺组织消化液，取肺组织右侧第一叶在消化液中剪碎，37℃摇床充分摇晃25min后以等体积完全培养基(加入10％FBS的基础培养基)终止消化，裂红后离心、重悬，并取100μL加入荧光素偶联的目的抗体(CD45-FITC，CD11b-APC，SiglecF-PE)，室温避光孵育30min。孵育完成后加入PBS洗去多余抗体并离心，样品过细胞筛后上机BDAccuri C6。
1.4结果分析
结果见图1和图2，图1为肺动脉高压患者外周血中的EOS占比及其与疾病严重程度的相关性分析实验结果示意图；其中，A.肺动脉高压患者和正常健康人群外周血样本中的EOS占比；B.肺动脉高压患者外周血样本中EOS占比与NYHA心功能分级的关系；C.肺动脉高压患者外周血样本中EOS占比与BMPR2突变的关系。图2为肺动脉高压动物模型中的EOS占比实验结果示意图；其中，A.低氧联合Sugen诱导的小鼠肺动脉高压模型外周血中的EOS占比及流式细胞术典型图；B.低氧联合Sugen诱导的小鼠肺动脉高压模型肺组织中的EOS占比及流式细胞术典型图。
通过对肺动脉高压患者外周样本及其病情相关指标进行分析，本发明发现肺动脉高压患者外周血中EOS的占比降低(图1中的A)，且EOS外周血占比越低，肺动脉高压患者病情越重(图1中的B)，可见，EOS能够辅助诊断肺动脉高压疾病，外周血中EOS占比越低，表明患病风险 越高，病情越严重；而携带BMPR2突变的患者EOS外周血占比更少(图1中的C)，说明外周血EOS占比越低，提示BMPR2突变风险大，预后不良的风险大。同时，通过对低氧联合Sugen诱导的小鼠肺动脉高压疾病模型外周血及肺组织样本进行检测，本发明发现随着肺动脉高压的发展，小鼠外周血EOS占比减少，肺组织EOS浸润增多(图2)，EOS具有保护肺动脉高压的作用，能够辅助诊断或判断肺动脉高压，外周血EOS占比越低，病情越严重。
实施例2
EOS敲除小鼠的肺动脉高压模型构建及功能评估
2.1低氧联合Sugen诱导的小鼠PH模型的建立
将8～10周龄体重25g以上的雄鼠置于10％氧气浓度的低氧仓中，每周进行一次皮下注射Sugen5416，剂量为20mg/kg/次，共3次注射，在低氧仓中持续饲养3周，麻醉小鼠进行右心室压力测定，如右心室压力升至30mmHg以上即完成建模。
(1)对照组1：常氧野生型：4只小鼠，常氧环境饲养3周；从第1天起每7天皮下注射一次DMSO，共注射3次。
(2)对照组2：常氧EOS敲除：4只小鼠，常氧环境饲养3周；从第1天起每7天皮下注射一次DMSO，共注射3次。
(3)建模组1：低氧联合Sugen野生型：7只小鼠，低氧环境饲养3周；从第1天起每7天皮下注射一次Sugen5416(DMSO为溶剂)，共注射3次。
(4)建模组2：低氧联合Sugen EOS敲除：9只小鼠，低氧环境饲养3周；从第1天起每7天皮下注射一次Sugen5416(DMSO为溶剂)，共注射3次。
2.2小鼠右心室收缩压的测定
在不暴露小鼠胸腔条件下，通过剑肋角入针，将一根与PowerLab仪器的压力传感器相连的22号针头插入经2％三溴乙醇腹腔麻醉的小鼠右心室，并用压力传感器显示的波形确定针头的位置。待波形稳定后，记录右心室收缩压。
2.3右心室肥厚指数测定
血流动力学测量完成后，开胸，用预冷生理盐水灌洗后，取出整个心脏和肺脏。将心脏沿着房室沟去除心房及大血管根部，沿后室壁间沟将右心室游离壁分离，吸去多余水分后称量右心室游离壁(RV)及左心室+室间隔(LV+S)的重量，并计算右心室肥厚指数＝RV/LV+S。
2.4肺组织免疫荧光染色
收集左肺组织，10％中性甲醛4℃固定后沉糖72h，OCT包埋。所有肺组织均包埋于同一方向(横切面)。将肺组织切片，厚度5微米。在4℃下用抗α-平滑肌肌动蛋白的一抗孵育过夜，洗片后滴染相应的HRP偶联二抗，室温湿盒孵育30min，洗去后滴加DAPI封片，拍照。
2.5结果分析
结果见图3，图3为EOS敲除加重低氧联合Sugen诱导小鼠肺动脉高压的实验结果示意图；其中，A.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠右心室收缩压；B.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠右心肥厚指数；C.正常对照及肺动脉高压条件下EOS敲除/野生型的小鼠肺血管抗α-平滑肌肌动蛋白免疫荧光染色典型示意图；野生型小鼠为实验对照组。
在正常条件下，野生型(对照组1)和EOS敲除组(对照组2)小鼠在右心室收缩压(图3中的A)、右心肥厚指数(图3中的B)和肺动脉平滑肌层厚度(图3中的C)上无差异。在低氧联合Sugen诱导肺动脉高压建模条件下，敲除EOS后，与建模组1相比，建模组2小鼠右心室收缩压和右心肥厚指数明显升高(图3中的A和B)。同时本发明发现与建模组1相比，建模组2小鼠肺动脉平滑肌层明显增厚(图3中的C)。根据图3可知，缺少EOS后，小鼠肺动脉高压加重，提示EOS在肺动脉高压进展过程中为保护作用，EOS可作为辅助诊断指标。
实施例3
肺血管平滑肌细胞的增殖及迁移功能测定
3.1肺动脉平滑肌细胞刺激
肺动脉平滑肌细胞撤去血清饥饿24h后，添加生长因子PDGFbb，及梯度EOS细胞裂解液进行刺激，分为：未处理对照组、PDGFbb刺激组、PDGFbb+10 3EOS裂解液组(图中标记为10 3EOS)、PDGFbb+10 4EOS裂 解液组(图中标记为10 4EOS)、PDGFbb+10 5EOS裂解液组(图中标记为10 5EOS)和PDGFbb+10 6EOS裂解液组(图中标记为10 6EOS)，上述各组对应的培养基统称为EOS条件培养基，未处理对照组仅含平滑肌细胞培养基，PDGFbb刺激组为包含生长因子PDGFbb的平滑肌细胞培养基，其它四组为包含生长因子PDGFbb和EOS裂解液的平滑肌细胞培养基)。
3.2肺动脉平滑肌细胞CCK8增殖测定
肺动脉平滑肌细胞按5000个/孔铺入96孔板中，撤去血清饥饿24h后，根据实验设计加入相应的条件培养基，每孔100μL培养基，刺激24h后每孔加入10μL CCK8原液，孵育2h，用酶标仪读取450nm和570nm的吸光度并绘制标准曲线、计算细胞增殖百分比。
3.3肺动脉平滑肌细胞划痕及Transwell迁移实验
划痕实验：肺动脉平滑肌细胞铺入6孔板中，撤去血清饥饿24h。显微镜下观察细胞密度约100％时，用P200枪头垂直于孔板进行划痕，PBS冲洗去除漂浮细胞后，根据实验设计加入相应的条件培养基，每孔1mL培养基，刺激0h及24h进行拍照并计算划痕愈合面积。
Transwell实验：肺动脉平滑肌细胞铺入Transwell小室上室，每孔加入200μL无血清基础培养基，下室中根据实验设计加入相应的条件培养基，每孔600μL培养基，刺激24h后进行甲醇固定、结晶紫染色并拍照计算迁移细胞数。
3.4结果分析
结果见图4，图4为EOS抑制肺动脉平滑肌细胞异常功能的实验结果示意图；其中，A.生长因子PDGFbb诱导和给予EOS条件培养基后的肺动脉平滑肌细胞增殖情况，随着EOS裂解液浓度增大，肺动脉平滑肌细胞增殖受抑制作用越强；B.生长因子PDGFbb诱导和给予EOS条件培养基后的肺动脉平滑肌细胞迁移情况，随着EOS裂解液浓度增大，划痕愈合程度降低、Transwell穿膜细胞减少。
在PDGFbb诱导肺动脉平滑肌细胞异常增殖及迁移后，给予梯度EOS裂解液，可明显抑制细胞增殖及迁移，同时本发明发现抑制效果随EOS裂解液浓度梯度增强，即EOS裂解液浓度越高，抑制效果越强(图4)， 表明EOS的内容物能抑制肺动脉平滑肌增殖和迁移，EOS胞质内容物存在维持肺动脉平滑肌稳态的保护性作用。
实施例4
EOS下游脂质代谢产物差异表达及作用检测
4.1低氧联合Sugen诱导的小鼠肺动脉高压模型的肺组织取材
将8～10周龄体重25g以上的雄鼠置于10％氧气浓度的低氧仓中，每周进行一次皮下注射Sugen5416，剂量为20mg/kg/次，共3次注射，在低氧仓中持续饲养3周，麻醉小鼠进行右心室压力测定，如右心室压力升至30mmHg以上即完成建模。右心室灌流后，收取小鼠肺组织液氮冻存。
(1)野生型建模组：低氧联合Sugen野生型：9只小鼠，低氧环境饲养3周；从第1天起每7天皮下注射一次Sugen5416，共注射3次。
(2)EOS敲除建模组：低氧联合Sugen EOS敲除：9只小鼠，低氧环境饲养3周；从第1天起每7天皮下注射一次Sugen5416，共注射3次。
4.2小鼠肺组织靶向代谢组学检测
取冷冻组织样本，称重并制备质控样本后，采用安捷伦超高效液相色谱仪和三重四极杆质谱仪进行检测分析，通过MassHunter软件对原始数据进行处理，使用默认参数并辅助人工检查，得到每个化合物以及内标的积分面积数据。采用每个化合物标准品与内标的峰面积比针对该化合物的浓度进行回归分析，得到10个点的定量标准曲线，采用标曲计算并输出各目标物质的上机测试样本含量，并计算原始样本(每克肺组织样本)含量。采用集成数据分析方法进行主成分分析、热图绘制，并进行脂质小分子代谢产物定量分析。
4.3 HDHA抑制增殖效果测定
肺动脉平滑肌细胞按5000个/孔铺入96孔板中，撤去血清饥饿24h后，分别设置未处理对照组(平滑肌细胞培养基)、PDGFbb刺激组(包含生长因子PDGFbb的平滑肌细胞培养基)、PDGFbb+14-HDHA组(包含生长因子PDGFbb和14-HDHA的平滑肌细胞培养基和PDGFbb+17-HDHA组(包含生长因子PDGFbb和17-HDHA的平滑肌细胞培养基，分别设置两类脂质小分子10nM及20nM浓度梯度，每孔100μL培养基，刺激24h后每孔加入10μL CCK8原液，孵育2h，用酶标仪读取 450nm和570nm的吸光度并绘制标准曲线、计算细胞增殖百分比。
4.4结果分析
结果见图5和图6，图5为EOS敲除影响低氧联合Sugen诱导小鼠肺组织脂肪酸代谢的实验结果示意图；其中，A.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织靶向代谢组学主成分分析；B.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织脂质代谢产物差异表达热图；C.肺动脉高压条件下EOS敲除/野生型的小鼠肺组织脂质代谢产物定量分析；野生型小鼠为实验对照组。图6为14-HDHA及17-HDHA对肺动脉平滑肌细胞增殖的抑制作用实验结果示意图。
在低氧联合Sugen诱导肺动脉的小鼠中，敲除EOS后，与野生型对照组相比表现出了花生四烯酸、DHA和EPA等脂肪酸代谢的明显改变(图5中的A和B)。同时本发明发现小分子脂质代谢产物14-HDHA与17-HDHA的水平在EOS敲除后出现明显降低(图5中的C)。体外给予肺动脉平滑肌细胞14-HDHA及17-HDHA刺激后，较小浓度即可有效抑制肺动脉平滑肌细胞增殖(图6)，EOS的下游代谢产物14-HDHA和17-HDHA对肺动脉高压有治疗作用。
实施例5
EOS的下游代谢产物14-HDHA和17-HDHA对肺动脉高压的治疗作用
5.1低氧联合Sugen诱导的小鼠PH模型的建立以及14-HDHA和17-HDHA治疗效果评估
将8～10周龄体重25g以上的雄鼠置于10％氧气浓度的低氧仓中，每周进行一次皮下注射Sugen5416，剂量为20mg/kg/次，共3次注射，在低氧仓中持续饲养3周，麻醉小鼠进行右心室压力测定，如右心室压力升至30mmHg以上即完成建模。在低氧第二周起，每3天腹腔分别注射14-HDHA和17-HDHA，直至建模结束，在建模结束评估右心室收缩压、右心肥厚指数和肺动脉平滑肌层厚度。
5.2低氧联合Sugen诱导的大鼠PH模型的建立和14-HDHA和17-HDHA治疗效果评估
将200g左右的SD大鼠皮下注射一次Sugen5416(剂量为20mg/kg)后，迅速置于10％氧气浓度的低氧仓中，持续饲养3周。3周后，置于常氧饲养2周，完成建模。在低氧3周后的常氧阶段，每3天腹腔分别注射14-HDHA和17-HDHA，直至建模结束，在建模结束评估右心室收缩压、右心肥厚指数和肺动脉平滑肌层厚度。
5.3结果分析
在正常条件下，给予溶剂对照组小鼠(大鼠)和给予14-HDHA或17-HDHA的小鼠(大鼠)在右心室收缩压、右心肥厚指数和肺动脉平滑肌层厚度上无显著差异。在低氧联合Sugen诱导肺动脉高压建模条件下，给予14-HDHA或17-HDHA的小鼠(大鼠)比溶剂对照组小鼠(大鼠)右心室收缩压和右心肥厚指数明显降低，肺动脉平滑肌层明显变薄。可知，补充14-HDHA或17-HDHA后小鼠(大鼠)肺动脉高压得到改善，提示14-HDHA和/或17-HDHA在肺动脉高压中的治疗作用，14-HDHA或17-HDHA可用于制备肺动脉高压的治疗药物。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (8)

  1. 检测嗜酸性粒细胞的试剂在制备诊断肺动脉高压和/或肺动脉高压预后风险判断的产品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述检测包括检测外周血中嗜酸性粒细胞的占比。
  3. EOS和/或EOS胞质内容物在制备抑制肺动脉平滑肌增殖和/或迁移的药物中的应用。
  4. EOS和/或EOS胞质内容物在制备维持肺动脉平滑肌稳态的药物中的应用。
  5. EOS和/或EOS胞质内容物在制备治疗肺动脉高压的药物中的应用。
  6. 羟基二十二碳六烯酸在制备抑制肺血管平滑肌增殖的药物中的应用。
  7. 羟基二十二碳六烯酸在制备治疗肺动脉高压的药物中的应用。
  8. 根据权利要求6或7所述的应用，其特征在于，所述羟基二十二碳六烯酸包括14-羟基二十二碳六烯酸和/或17-羟基二十二碳六烯酸。
PCT/CN2022/125577 2022-09-26 2022-10-17 检测嗜酸性粒细胞的试剂、嗜酸性粒细胞及其胞质内容物和羟基二十二碳六烯酸的应用 WO2024065892A1 (zh)

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