WO2015017949A1 - 成纤维细胞生长因子2的新用途 - Google Patents

成纤维细胞生长因子2的新用途 Download PDF

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WO2015017949A1
WO2015017949A1 PCT/CN2013/000944 CN2013000944W WO2015017949A1 WO 2015017949 A1 WO2015017949 A1 WO 2015017949A1 CN 2013000944 W CN2013000944 W CN 2013000944W WO 2015017949 A1 WO2015017949 A1 WO 2015017949A1
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growth factor
fibroblast growth
influenza
lung injury
virus
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PCT/CN2013/000944
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English (en)
French (fr)
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王希良
蒋澄宇
杨鹏辉
刘鑫
段跃强
邢丽
赖成材
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中国人民解放军军事医学科学院微生物流行病研究所
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Priority to CN201380078758.0A priority Critical patent/CN105451757B/zh
Priority to PCT/CN2013/000944 priority patent/WO2015017949A1/zh
Priority to US14/910,401 priority patent/US10493129B2/en
Publication of WO2015017949A1 publication Critical patent/WO2015017949A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • the present invention relates to a novel use of fibroblast growth factor 2.
  • the fibroblast growth factor family includes 23 structurally related polymorphic growth factors, of which Fibroblast growth factor-2 (FGF-2) is a member of the FGFs family. It was extracted from the pituitary gland by the American scientist Gospodsrowicz D in 1974. It is widely found in cells derived from mesoderm and neuroectoderm and in various tumor cells, mainly in the form of autocrine or/and paracrine activation on the target cell membrane.
  • the FGF receptor causes a series of intracellular signal transduction and participates in many physiological and pathological processes such as embryonic development, angiogenesis, nerve regeneration, and tumor growth.
  • FGF-2 The isoelectric point of FGF-2 is PI>0.9, also known as Basic fibroblast growth factor (bFGF).
  • the FGF-2 gene is located at 4q26 of the human chromosome, 38 kb in length, and contains three exons and two introns.
  • FGF-2 mRNA has multiple translation initiation sites and can produce a variety of molecular weight FGF-2 subtypes, including 18kd low molecular weight subtypes and 22, 22.5, 24 and 34kd high molecular weight subtypes, among which The low molecular weight FGF-2 containing 155 amino acid residues at 18 kd is mainly used. Low molecular weight subtypes are expressed in the cytoplasm and cell membrane, while high molecular weight subtypes primarily function directly into the nucleus.
  • bFGF Basic fibroblast growth factor
  • Acute lung injury is an injury of alveolar epithelial cells and capillary endothelial cells caused by various direct and indirect injury factors, resulting in diffuse pulmonary interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency.
  • Acute lung injury with pulmonary volume reduction, decreased lung compliance, ventilatory/blood flow imbalance is a pathophysiological feature, clinical manifestations of progressive hypoxemia and respiratory distress, pulmonary imaging showed heterogeneous infiltration Outbreaks, which progress to a severe stage (oxygenation index ⁇ 200), are called acute respiratory distress syndrome.
  • Common causes of acute lung injury are direct and indirect lung injury factors.
  • Direct lung injury factors include severe lung infections such as viruses, bacteria and fungi, stomach contents aspiration, lung contusion, oxygen poisoning, etc.;
  • Injury factors include, for example, sepsis, shock, massive blood transfusion, extracorporeal circulation, and disseminated intravascular coagulation.
  • lung injury The clinical manifestations of lung injury are: (1) acute onset, 12-48 hours after direct or indirect lung injury; (2) hypoxemia after conventional oxygen inhalation is difficult to correct; (3) pulmonary signs Non-specific, acute lungs can be heard and wet rales or respiratory sounds are reduced; (4) early lesions Qualitative, X-ray chest radiographs often have no obvious changes, the disease can progress out of the lung after consolidation, the performance of the double lung field generally increased density, decreased brightness, lung texture increased + thickening, visible scattered patch density Increased shadow; (5) diffuse lung infiltration, no evidence of heart failure.
  • influenza Lung injury caused by respiratory viruses, bacteria, and fungal infections is the most common acute respiratory infection in the clinic.
  • influenza is a common and frequently-occurring disease that affects a wide range of people.
  • the clinical symptoms caused by influenza A (H1N1) virus infection are mild, and most patients have typical flu-like symptoms. , can be restored naturally.
  • the most common symptoms include cough, fever, sore throat, headache, and other discomfort.
  • H1N1N1 multiple lesions can be seen on chest X-ray, which can rapidly progress to ARDS, renal or multiple organ failure.
  • Influenza A combined with ARDS is 100 times more common than normal flu, and lung damage is mainly caused by uncontrolled systemic immune responses.
  • ARDS secondary to viral pneumonia, including diffuse alveolar damage, bronchioles and perivascular lymphatic cell infiltration, proliferative airway changes, and obstructive bronchiolitis.
  • Pathological examinations suggest that the lesions in critically ill patients are mainly in the respiratory system. Pathological examination can show the consolidation of the lungs of critically ill patients, often accompanied by pathological changes such as hemorrhage, exudation and abscess. Serous or fibrinous exudation can be seen in the alveolar space, with varying degrees of clear membrane formation, suggesting diffuse lung tissue damage. It is currently believed that the basic lesions of lung tissue damage caused by influenza A (H1N1) virus are similar to those of other types of influenza, SARS, RSV, adenovirus, parainfluenza, recent SARS-like, and H7N9 avian influenza. Diffuse lung tissue damage of varying severity.
  • influenza A H1N1
  • Lipopolysaccharide is a water-soluble glycosylated lipid complex, which is an important component in the outer membrane of Gram-negative bacteria. It consists of lipid A, core polysaccharide and 0 antigen. The lipopolysaccharide has a molecular weight of more than 10,000 Daltons and has a complicated structure. Lipid A is a glycolipid which constitutes endotoxin activity and is covalently bonded to the heteropolysaccharide chain. The human body is extremely sensitive to bacterial endotoxin. A very small amount (1-5 ng/kg body weight) of endotoxin can cause an increase in body temperature, and the fever reaction gradually subsides after about 4 hours.
  • Endotoxin causes a fever reaction because endotoxin acts on macrophages in the body to produce cytokines such as interleukin 1, interleukin-6 and tumor necrosis factor a. These cytokines act on the hypothalamus of the host. Adjust the center to promote fever and fever.
  • cytokines such as interleukin 1, interleukin-6 and tumor necrosis factor a.
  • the clinical symptoms of endotoxemia mainly depend on the host's resistance to endotoxin. Symptoms and signs include: fever, white blood cell count, bleeding tendency, heart failure, renal dysfunction, liver damage, nervous system symptoms, and shock.
  • Endotoxin can cause the release of histamine, serotonin, prostaglandins, kinins, etc., leading to microcirculation expansion, decreased venous return blood volume, decreased blood pressure, insufficient tissue perfusion, hypoxia and acidosis.
  • Fungi can also infect lung tissue and cause lung damage, mainly as fungal inflammation or related lesions of the lungs and bronchi, and may include the pleura or even mediastinum.
  • Pathogenic fungi are primary pathogens that often result in a primary exogenous infection in a person with normal immune function.
  • Conditional pathogenic fungi, or opportunistic fungi are pathogenic and often cause deep fungal infections in susceptible hosts.
  • Zymosan is a macromolecular polysaccharide complex extracted from the yeast cell wall and consists of protein and carbohydrates.
  • Yeast polysaccharides can be used to induce inflammation in experiments, and the induced responses mainly include the expression of inflammatory cytokines, up-regulation of arachidonic acid, phosphorylation of some proteins, and formation of inositol phospholipids.
  • zymosan can also up-regulate the expression of cyclin D2, indicating that it also plays a role in macrophage activation and proliferation.
  • Septicemia refers to an acute systemic infection in which a pathogenic or conditional pathogen enters the blood circulation and grows in the blood to produce toxins. Sepsis is one of the prone factors of acute lung injury.
  • P-Li polymorphonuclear neutrophils
  • LBP lipopolysaccharide-binding protein
  • LBP lipopolysaccharide-binding protein
  • monocytes phospholipid-A-part of LPS
  • macrophages phospholipid-A-part of LPS
  • CD14 receptor binding on major neutrophils promotes translation of the coding genes for specific inflammatory factors such as TNF-a, IL-1, IL_6.
  • Secretion of cytokines into the circulation is an important biochemical feature in a series of inflammatory processes leading to sepsis and lung injury, such as IL-1, IL-6, IL-8, IL-10, IL-12, etc. These cytokines cause A series of cascade reactions involved in the process of lung injury. Therefore, the combination of lipopolysaccharide and zymosan can mimic septic lung injury.
  • the present invention provides a novel use of fibroblast growth factor 2.
  • the present invention provides a novel use of fibroblast growth factor 2, namely, the application of fibroblast growth factor 2 in the preparation of a medicament; the use of the medicament is as follows (a) and / or (b) and / or (c) :
  • the fibroblast growth factor 2 may be human fibroblast growth factor 2.
  • the fibroblast growth factor 2 may be the following (A) or (B): a protein represented by SEQ ID NO: 1 in the Sequence Listing; (B) a substitution and/or deletion of (A) through one or several amino acid residues. And/or a protein derived therefrom and having the same activity.
  • the lung injury can be a lung injury caused by a virus and/or bacteria and/or fungi.
  • the virus may be an influenza virus, specifically an influenza A H1N1 virus, more specifically an influenza A H1N1 virus strain BJ501 strain or a H1N1 influenza virus PR8 strain.
  • the bacterium may be a Gram-negative bacterium, specifically Escherichia coli, more specifically Escherichia coli 0111: B4.
  • the fungus may be a yeast, more specifically a Saccharomyces cerevisiae.
  • the lung injury may be a lung injury caused by sepsis.
  • the lung injury can be lung damage caused by lipopolysaccharide and zymosan A.
  • influenza may be influenza A, specifically the influenza caused by the influenza A (H1N1) virus, and the influenza A H1N1 influenza virus may specifically be the influenza A H1N1 influenza virus BJ501 strain or the influenza A H1N1 influenza virus PR8 strain.
  • the influenza virus may be an influenza A H1N1 virus, more specifically an influenza A H1N1 influenza virus BJ501 strain or an influenza A H1N1 influenza virus PR8 strain.
  • the present invention also protects a medicament whose active ingredient is fibroblast growth factor 2; the use of the medicament is as follows) and/or (b) and/or (c): (a) prevention and/or treatment of lung injury ;
  • the fibroblast growth factor 2 may be human fibroblast growth factor 2.
  • the fibroblast growth factor 2 may be the following (A) or (B): a protein represented by SEQ ID NO: 1 in the Sequence Listing; (B) a substitution and/or deletion of (A) through one or several amino acid residues. with/ Or a protein derived therefrom and having the same activity.
  • the lung injury can be a lung injury caused by a virus and/or bacteria and/or fungi.
  • the virus may be an influenza virus, specifically an influenza A H1N1 virus, more specifically an influenza A H1N1 virus strain BJ501 strain or a H1N1 influenza virus PR8 strain.
  • the bacterium may be a Gram-negative bacterium, specifically Escherichia coli, more specifically Escherichia coli 01 1 1 : B4.
  • the fungus may be a yeast, more specifically a Saccharomyces cerevisiae.
  • the lung injury may be a lung injury caused by sepsis.
  • the lung injury can be lung damage caused by lipopolysaccharide and zymosan A.
  • the dosage form of the medicament may be an injection, a spray, a nasal drop, an inhalant or an oral preparation.
  • influenza may be influenza A, specifically the influenza caused by the influenza A (H1N1) virus, and the influenza A H1N1 influenza virus may specifically be the influenza A H1N1 influenza virus BJ501 strain or the influenza A H1N1 influenza virus PR8 strain.
  • the influenza virus may be an influenza A (H1N1) virus, more specifically an influenza A H1N1 influenza virus.
  • the invention also protects the use of fibroblast growth factor 2 in the prevention and/or treatment of lung injury.
  • the fibroblast growth factor 2 may be human fibroblast growth factor 2.
  • the fibroblast growth factor 2 may be the following (A) or (B): a protein represented by SEQ ID NO: 1 in the Sequence Listing; (B) a substitution and/or deletion of (A) through one or several amino acid residues. And/or a protein derived therefrom and having the same activity.
  • the lung injury can be a lung injury caused by a virus and/or bacteria and/or fungi.
  • the virus may be an influenza virus, specifically an influenza A H1N1 virus, more specifically an influenza A H1N1 virus strain BJ501 strain or a H1N1 influenza virus PR8 strain.
  • the bacterium may be a Gram-negative bacterium, specifically Escherichia coli, more specifically Escherichia coli 01 1 1 : B4.
  • the fungus may be a yeast, more specifically a Saccharomyces cerevisiae.
  • the lung injury may be a lung injury caused by sepsis.
  • the lung injury may be lung damage caused by lipopolysaccharide and zymosan A.
  • the present invention also protects the application of fibroblast growth factor 2 for the prevention and/or treatment of influenza, or for the prevention and/or treatment of diseases caused by influenza viruses.
  • the fibroblast growth factor 2 may be human fibroblast growth factor 2.
  • the fibroblast growth factor 2 may be the following (A) or (B): a protein represented by SEQ ID NO: 1 in the Sequence Listing; (B) a substitution and/or deletion of (A) through one or several amino acid residues. And/or a protein derived therefrom and having the same activity.
  • influenza may be influenza A, specifically the influenza caused by the influenza A (H1N1) virus, and the influenza A H1N1 influenza virus may specifically be the influenza A H1N1 influenza virus BJ501 strain or the influenza A H1N1 influenza virus PR8 strain.
  • the influenza virus may be an influenza A (H1N1) virus, more specifically an influenza A H1N1 influenza virus.
  • the invention also protects the application of fibroblast growth factor 2 as a marker of lung injury, or the use of a substance for detecting said fibroblast growth factor 2 in the diagnosis of lung injury, or for detecting said The use of a substance of fibroblast growth factor 2 in the preparation of a product for assisting in the diagnosis of lung injury.
  • the fibroblast growth factor 2 may be human fibroblast growth factor 2.
  • the fibroblast growth factor 2 may be the following (A) or (B): a protein represented by SEQ ID NO: 1 in the Sequence Listing; (B) a substitution and/or deletion of (A) through one or several amino acid residues. And/or a protein derived therefrom and having the same activity.
  • the fibroblast growth factor 2 is specifically fibroblast growth factor 2 in serum, blood plasma or lung lavage fluid.
  • the lung injury can be a lung injury caused by a virus and/or bacteria and/or fungi.
  • the virus may be an influenza virus, specifically an influenza A H1N1 virus, more specifically an influenza A H1N1 virus strain BJ501 strain or a H1N1 influenza virus PR8 strain.
  • the bacterium may be a Gram-negative bacterium, specifically Escherichia coli, more specifically Escherichia coli 0111:B4.
  • the fungus may be a yeast, more specifically a Saccharomyces cerevisiae.
  • the lung injury may be a lung injury caused by sepsis.
  • the lung injury may be lung damage caused by lipopolysaccharide and zymosan A.
  • the present invention utilizes a FGF-2 knockout mouse model and a mouse model infected with influenza A (H1N1 influenza virus) to demonstrate that FGF-2 plays an important role in the pathological damage and death of acute lung tissue caused by influenza A (H1N1) influenza virus infection in mice. Role, the intervention of FGF-2 molecules plays an important role in the treatment of lung injury, especially the damage caused by influenza A (H1N1) virus infection.
  • the present invention uses FGF-2 for the treatment of a mouse model of influenza A (H1N1) virus infection, and the results show that FGF-2 plays an important protective role in acute lung tissue pathological damage caused by influenza A (H1N1) influenza virus. Therefore, the present invention demonstrates for the first time that FGF-2 plays an important role in the pathological process of influenza A and that FGF-2 can prevent or slow the serious consequences caused by influenza A virus infection.
  • the present invention also utilizes a combination of a lipopolysaccharide of Gram-negative bacteria and a yeast polysaccharide A derived from yeast, and finds that the intervention against FGF-2 is in the treatment of lipopolysaccharide from Gram-negative bacteria and zymosan from Saccharomyces cerevisiae.
  • a lung injury caused by co-infection may play an important role.
  • the present invention utilizes FGF-2 for the treatment of a mouse model of combined infection of lipopolysaccharide and zymosan A, and the results show that FGF-2 is involved in acute lung histopathological damage caused by a composition of lipopolysaccharide and zymosan A in mice. Played an important protective role. Therefore, the present invention demonstrates for the first time that the FGF-2 recombinant protein can prevent or slow down the serious consequences caused by the combined infection of lipopolysaccharide and zymosan A.
  • FIG. 1 shows the results of Example 1.
  • Fig. 2 shows the results of section dyeing in Example 2.
  • Fig. 3 shows the results of the wet-to-dry ratio in Example 2.
  • Fig. 4 shows the results of the survival rate statistics in Example 3.
  • Fig. 5 shows the results of the change in body weight in Example 3.
  • Fig. 6 shows the results of section dyeing in Example 3.
  • Figure ⁇ shows the results of the wet-to-dry ratio in Example 3.
  • Fig. 8 shows the results of the survival rate statistics in Example 4.
  • Fig. 9 shows the results of section dyeing in Example 4.
  • Fig. 10 shows the results of the wet-to-dry ratio in Example 4.
  • Figure 11 shows the results of section dyeing in Example 5.
  • Human recombinant FGF-2/basic FGF protein The protein sequence is shown in sequence 1 of the sequence listing, and the coding gene is shown in sequence 2 of the sequence listing; Mi ll ipore, article number 01-106, Dilute to the desired concentration with PBS buffer at the time of use.
  • IL-17 response mediates acute lung injury induced by the 2009 pandemic influenza A (HlNl) virus.
  • Example 1 Influenza A (H1N1) virus caused elevated levels of FGF-2 in lung lavage fluid of mice.
  • Experimental group (5 4-6 weeks old C57 BL/6 mice): Safely fixed mice, each intraperitoneal injection 50-6 ( ⁇ L lg/100mL sodium pentobarbital solution for anesthesia; keep the head of the anesthetized mouse tilted up and down, so that the nasal cavity is upward, and 10 L BJ501 is dropped into each nostril on each side of the pipette Virus strain (10 5 ' 5 TCID 5Q / only), keep the mouse in this position for 15 seconds, let the virus enter the respiratory tract; 24 hours after infection of BJ501 strain, the mice were killed by intraperitoneal injection of excess anesthetic, the dead mice Fix it on the small animal operating table, remove the chest skin and bones, cut the trachea into a small opening, inject 800 ⁇ l of PBS buffer into the mouse from the
  • Control group (5 4-6 weeks old C57 BL/6 mice): Replace the BJ501 strain virus solution with an equal volume of chicken embryo allantoic fluid, and other experimental groups.
  • mice were kept in this position for 15 seconds to allow the virus to enter the respiratory tract; after 5 days of infection with the influenza A virus, the mice were killed by intraperitoneal injection of excess anesthetic; 3 dead mice were fixed on the small animal operating table, and the chest skin was removed.
  • A is the lung tissue of C57BL/6 mice
  • B is the lung tissue of FGF-2 knockout mice.
  • influenza A influenza A
  • severe pathological damage occurred in the lung tissue of C57BL/6 mice the normal structure of lung tissue was destroyed, the texture of lung tissue was disordered, accompanied by hemorrhage, inflammatory exudation and pathological changes of red blood cells and inflammatory cells. damage.
  • the lung tissue of FGF-2 knockout mice infected with the same titer virus showed more significant pathological damage, more significant pathological changes such as hemorrhage, exudation or inflammatory cell infiltration, and the lung tissue texture was not clear and the structure was incomplete.
  • the wet-dry ratio is shown in Figure 3.
  • the lung wet-to-dry ratio can reflect the extent of acute pulmonary edema in mice.
  • FGF-2 knockout mice showed a significant increase in lung wet-dry ratio compared with C57BL/6 mice after infection with influenza A (H1N1) virus (*0.05), indicating that FGF-2 knockdown can aggravate H1N1 Lung edema in mice after influenza virus infection.
  • the results showed that acute lung injury caused by influenza A H1N1 virus in FGF-2 deficient mice was more severe.
  • Example 3 Acute lung injury caused by influenza A H1N1 virus in FGF-2 deficient mice is more serious
  • A is the lung tissue of C57BL/6 mice
  • B is the lung tissue of FGF-2 knockout mice.
  • influenza A influenza A
  • severe pathological damage occurred in the lung tissue of C57BL/6 mice the normal structure of lung tissue was destroyed, the texture of lung tissue was disordered, accompanied by hemorrhage, inflammatory exudation and pathological changes of red blood cells and inflammatory cells. damage.
  • the lung tissue of FGF-2 knockout mice infected with the same titer virus showed more significant pathological damage, more significant pathological changes such as hemorrhage, exudation or inflammatory cell infiltration, and the lung tissue texture was not clear and the structure was incomplete.
  • FGF-2 knockout mice showed a significant increase in lung wet-dry ratio compared with C57BL/6 mice after infection with influenza A (H1N1) virus (* corpse ⁇ 0.05), indicating that FGF-2 knockdown can aggravate Lung edema in mice after infection with H1N1 influenza virus.
  • the results showed that acute lung injury caused by influenza A H1N1 virus in FGF-2 deficient mice was more severe.
  • FGF-2 recombinant protein can alleviate acute lung injury caused by influenza A virus infection
  • mice On the first day of the experiment, each mouse was intravenously injected with ⁇ FGF-2 recombinant protein solution (protein concentration was 0.5 mg/ml); The mice were fixed in a safe manner. Each mouse was intraperitoneally injected with 50-6 ( ⁇ L lg/100 mL of sodium pentobarbital solution for anesthesia with a lmL sterile syringe; the anesthetized mouse was kept tilted up and down.
  • mice The nasal cavity was upward, and 10 L PR8 strain of virus solution (10 5 ⁇ 5 TCID 5 / / only) was instilled into each nostril on each side of the pipette, and the mouse was kept in this position for 15 seconds to allow the virus to enter the respiratory tract;
  • Each mouse Intravenous injection of ⁇ FGF-2 recombinant protein solution (protein concentration of 0.5 mg / ml); on the fifth day of the experiment, each mouse was injected intravenously with ⁇ FGF-2 recombinant protein solution (protein concentration of 0.5 mg / ml) ; The survival rate of the mice was counted.
  • Control group (10 4-6 weeks old C57 BL/6 mice): Replace the FGF-2 recombinant protein solution with an equal volume of PBS buffer, and the other experimental groups.
  • mice On the first day of the experiment, each mouse was intravenously injected with ⁇ FGF-2 recombinant protein solution (protein concentration was 0.5 mg/ml); The mice were fixed in a safe manner. Each mouse was intraperitoneally injected with 50-6 ( ⁇ L lg/100 mL of sodium pentobarbital solution for anesthesia with a lmL sterile syringe; the anesthetized mouse was kept tilted up and down.
  • the nasal cavity was upward, and 10 L PR8 strain of virus solution (10 5 ⁇ 5 TCID 5 / / only) was instilled into each nostril on each side of the pipette, and the mouse was kept in this position for 15 seconds to allow the virus to enter the respiratory tract; 5mg/ ⁇
  • the ⁇ FGF-2 recombinant protein solution protein concentration of 0. 5mg / ph ⁇ FGF-2 recombinant protein solution was injected intravenously with ⁇ ⁇ FGF-2 recombinant protein solution (5 mg / ml).
  • mice dead the three small animals is fixed to the operating table, chest skin and bone is removed to expose the chest, while the lungs removed together with the heart, Wash off the surface blood with sterile PBS buffer, place more The formaldehyde fixative was fixed at room temperature for 48 hours, and then embedded, sliced, HE stained, etc.; the other 3 dead mice were fixed on the small animal operating table, the chest skin and bones were removed, the chest cavity was exposed, the whole lung was taken out, and the surface was removed.
  • Control group (6 4-6 week old C57 BL/6 mice): Replace the FGF-2 recombinant protein solution with an equal volume of PBS buffer, and the other experimental groups.
  • A is the lung tissue of the control group
  • B is the experiment.
  • the lung tissue of the mice infected with the same titer virus showed no significant pathological damage, no significant pathological changes such as hemorrhage, exudation or inflammatory cell infiltration.
  • the lung tissue was clear and structurally intact.
  • the results showed that FGF-2 played an important protective role in acute lung histopathological damage caused by influenza A (H1N1) virus infection.
  • the wet-dry ratio is shown in Figure 10.
  • the lung wet-dry ratio was significantly lower than that of the control group after infection with the influenza A (H1N1) virus (*0.05), indicating that FGF-2 can significantly alleviate the mice caused by influenza A (H1N1) virus infection.
  • Lung edema The results showed that FGF-2 plays an important role in protecting acute lung histopathological damage caused by influenza A virus infection in mice.
  • FGF-2 recombinant protein can alleviate the pathological damage of lung tissue in mice infected by lipopolysaccharide and zymosan A
  • the first group (4 4-6 weeks old C57BL/6 mice):
  • Each mouse was intravenously injected with ⁇ ⁇ FGF-2 recombinant protein solution (protein content 50 ⁇ g) 12 hours before infection with lipopolysaccharide, 1 hour before infection with lipopolysaccharide and 8 hours after infection with lipopolysaccharide.
  • mice After infection with lipopolysaccharide 24, the mice were killed by intraperitoneal injection of excess anesthetic. The dead mice were fixed on the small animal operating table, the chest skin and bones were removed, and the chest cavity was exposed. The lungs were taken out together with the heart, and the surface blood was washed away with sterile PBS buffer, fixed in a paraformaldehyde fixative for 48 h at room temperature, and subjected to embedding, sectioning, HE staining and the like.
  • the second group FGF-2 recombinant protein solution was not injected 12 hours before infection of lipopolysaccharide, 1 hour before infection with lipopolysaccharide and 8 hours after infection with lipopolysaccharide, and the same as the first group.
  • Group 3 Replace the lipopolysaccharide solution with an equal volume of PBS buffer, replace the zymosan A solution with an equal volume of PBS buffer, and the same as the first group.
  • Fig. 1 The results are shown in Fig. 1 1.
  • A is the first group
  • B is the second group
  • C is the third group.
  • the lung tissue of the second group of mice showed significant pathological damage, significant pathological changes such as hemorrhage, exudation or inflammatory cell infiltration, and the lung tissue texture was unclear and the structure was incomplete.
  • the lung tissue of the first group of mice showed no significant pathological damage, no significant pathological changes such as hemorrhage, exudation or inflammatory cell infiltration, and the lung tissue was clear in texture and structurally intact.
  • FGF-2 played an important protective role in the pathological damage of acute lung tissue caused by the combination of lipopolysaccharide and zymosan A in mice.
  • the present invention discloses the use of FGF-2 in the preparation of a medicament for the treatment and/or prevention of lung injury, the use in the prevention and/or treatment of influenza, and the use in the prevention and/or treatment of diseases caused by influenza virus.
  • the present invention is of great value for the treatment and prevention of the above diseases.

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Abstract

本发明提供了一种成纤维细胞生长因子2的新用途，即成纤维细胞生长因子2在制备药物中的应用，所述药物的用途为如下（a）和/或（b）和/或（c）：（a）预防和/或治疗肺损伤；（b）预防和/或治疗流感；（c）预防和/或治疗流感病毒引起的疾病。

Description

成纤维细胞生长因子 2的新用途 技术领域
本发明涉及成纤维细胞生长因子 2的新用途。
背景技术
成纤维细胞生长因子家族包括 23 种结构相关的多态性生长因子， 其中 成纤维细胞生长因子 2 (Fibroblast growth factor- 2， FGF-2 ) 是 FGFs 家 族成员之一。 1974 年由美国科学家 Gospodsrowicz D从牛脑垂体中提取得到， 广泛存在于来源于中胚层及神经外胚层的细胞及多种肿瘤细胞中， 主要以自 分泌或 /和旁分泌的方式激活靶细胞膜上 FGF 受体， 引起一系列细胞内信号 转导，参与胚胎发育、血管生成、神经再生、肿瘤生长等多项生理病理过程。
FGF-2的等电点 PI>9. 0， 也称作碱性成纤维细胞生长因子 （Basic fibroblast growth factor, bFGF) 。 FGF- 2基因定位于人类染色体的 4q26， 全长 38kb， 含有 3个外显子和 2个内含子。 FGF-2的 mRNA有多个翻译起始位 点，可产生多种分子量的 FGF-2 亚型，包括 18kd的低分子量亚型和 22、22. 5、 24和 34kd的高分子量亚型， 其中以 18kd含 155个氨基酸残基的低分子量 FGF-2为主。 低分子量亚型在细胞质和细胞膜中表达， 高分子量亚型则主要 直接进入细胞核中发挥作用。
急性肺损伤 (acute lung injury, ALI)是各种直接和间接致伤因素导致 的肺泡上皮细胞及毛细血管内皮细胞损伤， 造成弥漫性肺间质及肺泡水肿， 导致急性低氧性呼吸功能不全。 急性肺损伤以肺容积减少、 肺顺应性降低、 通气 /血流比例失调为病理生理特征，临床上表现为进行性低氧血症和呼吸窘 迫， 肺部影像学上表现为非均一性的渗出性病变， 其发展至严重阶段 （氧合 指数 <200 ) 被称为急性呼吸窘迫综合征。 常见的导致急性肺损伤的因素分直 接和间接肺损伤因素， 直接肺损伤因素包括例如病毒、 细菌和真菌导致的严 重的肺部感染、 胃内容物误吸、 肺挫伤、 氧中毒等； 间接肺损伤因素包括例 如脓毒症、 休克、 大量输血、 体外循环及弥漫性血管内凝血等。
肺损伤在临床上的表现为： （1 ) 急性起病， 在直接或间接肺损伤后 12-48小时内发病； （2 ) 常规吸氧后低氧血症难以纠正； （3 ) 肺部体征 无特异性， 急性期双肺可闻及湿罗音或呼吸音减低； （4 ) 早期病变以间 质性为主， X线胸片常无明显改变， 病情进展后可出肺内实变， 表现为双 肺野普遍密度增高， 透亮度减低， 肺纹理增多 +增粗， 可见散在斑片状密 度增高阴影； （5 ) 弥漫性肺浸润影， 无心功能不全证据。
肺损伤的临床诊断标准为： （1 )急性起病； （2 )氧合指数（Pa02 / Fi02) ^200 mm Hg ( ( 1 mm Hg=0. 133kPa, 不管呼气末正压 （PEEP) 水平） ； （3 ) 正位 X线胸片显示双肺均有斑片状阴影； （4) 肺动脉嵌顿压 18 匪 Hg， 或 无左心房压力增高的临床证据， 如 (Pa02 I Fi02^300 匪 Hg且满足上述其 他标准， 可诊断急性肺损伤。
呼吸道病毒、 细菌、 真菌感染致肺损伤是临床最为常见的急性呼吸道传 染病。 其中， 流感是一种影响人群极其广泛的常见病、 多发病， 目前流感病 毒跨物种感染形势严峻， 甲型 H1N1流感病毒感染所导致的临床症状， 多数患 者较轻， 表现为典型的流感样症状， 可自然恢复。 最常见症状包括咳嗽、 发 热、 咽喉痛、 头痛及其他不适感。 严重肺炎患者 X线胸片可见多发性病灶浸 润， 可快速进展为 ARDS、 肾或多器官功能衰竭。 甲型流感合并 ARDS的发生 率是普通流感的 100倍， 肺部损坏主要来源于失控的全身免疫反应。 与继发 于病毒性肺炎的 ARDS—致， 包括弥漫性肺泡损伤、细支气管和血管周围淋巴 细胞浸润、 增生的气道改变和梗阻性细支气管炎。
临床和病理学检查均提示重症患者病变主要在呼吸系统。 病理学检查可 见重症患者的肺部出现实变， 常伴有出血、 渗出、 脓肿等病理改变。 肺泡腔 内可见浆液性或纤维素性渗出， 伴有不同程度的透明膜形成， 提示有弥漫性 的肺组织损伤。 目前认为， 甲型 H1N1流感病毒所致肺组织损伤基本病变和其 他类型的流感、 SARS、 RSV、 腺病毒、 副流感、 最近出现的类 SARS和 H7N9人 禽流感重症等病例的肺基本病变相似， 均为轻重不等的弥漫性肺组织损伤。
脂多糖（l ipopolysaccharide, LPS )是一种水溶性的糖基化的脂质复合 物， 是革兰氏阴性菌外膜中的重要成分， 由脂质 A、 核心多糖和 0抗原三部 分组成。 脂多糖分子量大于 10000道尔顿， 结构复杂， 脂质 A (Lipid A) 为 构成内毒素活性的糖脂， 以共价键连接到杂多糖链。 人体对细菌内毒素极为 敏感， 极微量 （1-5纳克 /公斤体重） 内毒素就能引起体温上升， 发热反应持 续约 4小时后逐渐消退。 自然感染时， 因革兰氏阴性菌不断生长繁殖， 同时 伴有陆续死亡、释出内毒素，故发热反应将持续至体内病原菌完全消灭为止。 内毒素引起发热反应的原因是内毒素作用于体内的巨噬细胞等， 使之产生白 细胞介素 1、 白细胞介素 6和肿瘤坏死因子 a 等细胞因子， 这些细胞因子作 用于宿主下丘脑的体温调节中枢， 促使体温升高发热。 内毒素血症临床症状 主要取决于宿主对内毒素的抵抗力， 症状和体征有： 发热、 白细胞数变化、 出血倾向、 心力衰竭、 肾功能减退、 肝脏损伤、 神经系统症状以及休克等。 内毒素可引起组胺、 5-羟色胺、前列腺素、激肽等的释放， 导致微循环扩张， 静脉回流血量减少， 血压下降， 组织灌流不足， 缺氧及酸中毒等。
真菌也同样可以感染肺组织并导致肺损伤， 其主要表现为肺和支气管的 真菌性炎症或相关病变， 也可包括胸膜甚至纵膈。 致病性真菌属原发性病原 菌， 常导致免疫功能正常者的原发性外源性感染。 条件致病性真菌或称机会 性真菌， 其病原性弱， 多在易感宿主引起深部真菌感染。
酵母多糖（zymosan)是从酵母细胞壁提取的大分子多糖复合物， 由蛋白 质和碳水化合物组成。 酵母多糖在实验中可用来诱导炎症发生， 其诱导的反 应主要包括炎症细胞因子的表达， 花生四烯酸的上调， 部分蛋白的磷酸化和 肌醇磷脂的形成。 同时， 酵母多糖还可以上调细胞周期蛋白 D2的表达量， 表 明其在巨噬细胞活化和增殖的过程中也起到了作用。
脂多糖和酵母多糖联合感染可以模拟体内由于败血症引起的急性肺损伤。 败血症 （septicemia) 系指致病菌或条件致病菌侵入血循环， 并在血中生长 繁殖， 产生毒素而发生的急性全身性感染。 败血症为急性肺损伤的易发因素 之一，败血症性肺损伤的一个特征就是多形核中性粒细胞 (P丽)在肺微血管内 的聚集和激活， 引起一系列炎症反应过程和血管损伤。 在这一过程中细菌感 染， 尤其是革兰氏阴性菌感染可能是最初炎症反应的关键因素。 革兰氏阴性 菌和脂多糖 (LPS)进入循环后产生脂多糖结合蛋白（LBP)， LBP与 LPS的磷脂 -A—部分结合， 血浆中 LPS-LBP复合物与单核细胞、 巨噬细胞和主要的中性 粒细胞上的 CD14受体结合， 促使特定的炎性因子（如 TNF-a、 IL-1、 IL_6) 的编码基因翻译。 细胞因子分泌到循环中是导致败血症和肺损伤的一系列炎 症反应过程中重要的生化特征， 如 IL-1、 IL-6、 IL-8、 IL-10、 IL-12等， 这 些细胞因子引起一系列的级联反应， 参与肺损伤的过程。 因此， 使用脂多糖 和酵母多糖联合感染可以模拟败血症性肺损伤。
发现新的治疗和 /或预防肺损伤的药物已成为生命科学的重大迫切需求。 发明公开
本发明提供了一种成纤维细胞生长因子 2的新用途。 本发明提供了成纤维细胞生长因子 2的新用途， 即成纤维细胞生长因子 2 在制备药物中的应用； 所述药物的用途为如下 （a) 和 /或 （b) 和 /或 （c) ：
(a) 预防和 /或治疗肺损伤； （b) 预防和 /或治疗流感； （c) 预防和 /或治 疗流感病毒引起的疾病。
所述成纤维细胞生长因子 2可为人源的成纤维细胞生长因子 2。
所述成纤维细胞生长因子 2可为如下 （A) 或 （B) ： 序列表中序列 1所示 的蛋白质； （B) 将 （A) 经过一个或几个氨基酸残基的取代和 /或缺失和 / 或添加且具有相同活性的由其衍生的蛋白质。
所述肺损伤可为病毒和 /或细菌和 /或真菌引起的肺损伤。 所述病毒可 为流感病毒， 具体可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病 毒 BJ501株或甲型 H1N1流感病毒 PR8株。 所述细菌可为革兰氏阴性菌， 具体可为大肠杆菌， 更具体可为大肠杆菌 0111: B4。 所述真菌可为酵母， 更具体可为酿酒酵母。
所述肺损伤可为败血症引起的肺损伤。
所述肺损伤可为脂多糖和酵母多糖 A引起的肺损伤。
所述流感可为甲型流感， 具体可为甲型 H1N1流感病毒引起的流感， 所 述甲型 H1N1流感病毒具体可为甲型 H1N1流感病毒 BJ501株或甲型 H1N1流感 病毒 PR8株。
所述流感病毒可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病毒 BJ501株或甲型 H1N1流感病毒 PR8株。 本发明还保护一种药物， 其活性成分为成纤维细胞生长因子 2; 所述药 物的用途为如下 ) 和 /或 （b) 和 /或 （c) ： (a) 预防和 /或治疗肺损伤；
(b) 预防和 /或治疗流感； （c) 预防和 /或治疗流感病毒引起的疾病。
所述成纤维细胞生长因子 2可为人源的成纤维细胞生长因子 2。
所述成纤维细胞生长因子 2可为如下 （A) 或 （B) ： 序列表中序列 1所示 的蛋白质； （B) 将 （A) 经过一个或几个氨基酸残基的取代和 /或缺失和 / 或添加且具有相同活性的由其衍生的蛋白质。
所述肺损伤可为病毒和 /或细菌和 /或真菌引起的肺损伤。 所述病毒可 为流感病毒， 具体可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病 毒 BJ501株或甲型 H1N1流感病毒 PR8株。 所述细菌可为革兰氏阴性菌， 具体可为大肠杆菌， 更具体可为大肠杆菌 01 1 1 : B4。 所述真菌可为酵母， 更具体可为酿酒酵母。
所述肺损伤可为败血症引起的肺损伤。
所述肺损伤可为脂多糖和酵母多糖 A引起的肺损伤。
所述药物中还可包括其他的可以与所述成纤维细胞生长因子 2起协同作用 的活性成分。 所述药物中还可包括防腐剂、 稳定剂、 缓冲剂等药用物质。 所 述药物的剂型可为注射剂、 喷雾剂、 滴鼻剂、 吸入剂或口服剂。
所述流感可为甲型流感， 具体可为甲型 H1N1流感病毒引起的流感， 所 述甲型 H1N1流感病毒具体可为甲型 H1N1流感病毒 BJ501株或甲型 H1N1流感 病毒 PR8株。
所述流感病毒可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病毒
BJ501株或甲型 H1N1流感病毒 PR8株。 本发明还保护成纤维细胞生长因子 2在预防和 /或治疗肺损伤中的应用。 所述成纤维细胞生长因子 2可为人源的成纤维细胞生长因子 2。
所述成纤维细胞生长因子 2可为如下 （A) 或 （B ) ： 序列表中序列 1所示 的蛋白质； （B ) 将 （A) 经过一个或几个氨基酸残基的取代和 /或缺失和 / 或添加且具有相同活性的由其衍生的蛋白质。
所述肺损伤可为病毒和 /或细菌和 /或真菌引起的肺损伤。 所述病毒可 为流感病毒， 具体可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病 毒 BJ501株或甲型 H1N1流感病毒 PR8株。 所述细菌可为革兰氏阴性菌， 具体可为大肠杆菌， 更具体可为大肠杆菌 01 1 1 : B4。 所述真菌可为酵母， 更具体可为酿酒酵母。
所述肺损伤可为败血症引起的肺损伤。
所述肺损伤可为脂多糖和酵母多糖 A引起的肺损伤。 本发明还保护成纤维细胞生长因子 2的应用，为预防和 /或治疗流感，或， 预防和 /或治疗流感病毒引起的疾病。
所述成纤维细胞生长因子 2可为人源的成纤维细胞生长因子 2。
所述成纤维细胞生长因子 2可为如下 （A) 或 （B) ： 序列表中序列 1所示 的蛋白质； （B) 将 （A) 经过一个或几个氨基酸残基的取代和 /或缺失和 / 或添加且具有相同活性的由其衍生的蛋白质。
所述流感可为甲型流感， 具体可为甲型 H1N1流感病毒引起的流感， 所 述甲型 H1N1流感病毒具体可为甲型 H1N1流感病毒 BJ501株或甲型 H1N1流感 病毒 PR8株。
所述流感病毒可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病毒
BJ501株或甲型 H1N1流感病毒 PR8株。 本发明还保护成纤维细胞生长因子 2作为肺损伤的标志物的应用，或， 用于检测所述成纤维细胞生长因子 2的物质在辅助诊断肺损伤中的应用， 或 用于检测所述成纤维细胞生长因子 2的物质在制备辅助诊断肺损伤的产品中 的应用。
所述成纤维细胞生长因子 2可为人源的成纤维细胞生长因子 2。
所述成纤维细胞生长因子 2可为如下 （A) 或 （B) ： 序列表中序列 1所示 的蛋白质； （B) 将 （A) 经过一个或几个氨基酸残基的取代和 /或缺失和 / 或添加且具有相同活性的由其衍生的蛋白质。
所述成纤维细胞生长因子 2具体为血清、 血桨或肺灌洗液中的成纤维 细胞生长因子 2。
所述肺损伤可为病毒和 /或细菌和 /或真菌引起的肺损伤。 所述病毒可 为流感病毒， 具体可为甲型 H1N1流感病毒， 更具体可为甲型 H1N1流感病 毒 BJ501株或甲型 H1N1流感病毒 PR8株。 所述细菌可为革兰氏阴性菌， 具体可为大肠杆菌， 更具体可为大肠杆菌 0111 : B4。 所述真菌可为酵母， 更具体可为酿酒酵母。
所述肺损伤可为败血症引起的肺损伤。
所述肺损伤可为脂多糖和酵母多糖 A引起的肺损伤。 本发明利用 FGF-2基因敲除小鼠模型以及利用甲型 H1N1流感病毒感染小 鼠模型证明 FGF-2在甲型 H1N1流感病毒感染导致的小鼠急性肺组织病理损伤、 死亡的过程中发挥重要作用， 针对 FGF-2分子的干预在治疗肺损伤， 特别是 甲型 H1N1流感病毒感染所导致的损伤中， 发挥重要作用。本发明将 FGF-2用 于治疗甲型 H1N1流感病毒感染的小鼠模型， 结果表明， FGF-2对小鼠在感染 甲型 H1N1流感病毒导致的急性肺组织病理损伤中发挥了重要保护作用。因此， 本发明第一次证明了 FGF-2在甲型流感病理过程中发挥重要作用且 FGF-2可 以阻止或减缓甲型流感病毒感染所造成的严重后果。
本发明还利用革兰氏阴性菌的脂多糖和来自酵母菌的酵母多糖 A联合感 染小鼠， 发现针对 FGF-2的干预在治疗来自革兰氏阴性菌的脂多糖和来自酿 酒酵母的酵母多糖 A联合感染所导致的肺损伤中有可能发挥重要作用。 本发 明利用 FGF-2用于治疗脂多糖和酵母多糖 A联合感染的小鼠模型，结果表明， FGF-2对小鼠在感染脂多糖和酵母多糖 A的组合物导致的急性肺组织病理损 伤中发挥了重要保护作用。 因此， 本发明第一次证明了 FGF-2重组蛋白可以 阻止或减缓由脂多糖和酵母多糖 A联合感染所造成的严重后果。
附图说明
图 1为实施例 1的结果。
图 2为实施例 2中切片染色的结果。
图 3为实施例 2中湿干比的结果。
图 4为实施例 3中存活率统计的结果。
图 5为实施例 3中体重变化统计的结果。
图 6为实施例 3中切片染色的结果。
图 Ί为实施例 3中湿干比的结果。
图 8为实施例 4中存活率统计的结果。
图 9为实施例 4中切片染色的结果。
图 10为实施例 4中湿干比的结果。
图 11为实施例 5中切片染色的结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施 例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试 验材料， 如无特殊说明， 均为自常规生化试剂商店购买得到的。 以下实施 例中的定量试验， 均设置三次重复实验， 结果取平均值。 利用 GraphPad Pri sm 5 软件对数据进行分析处理。 存活率统计中， 感染病毒后， 24h 内 发生死亡的小鼠为非特异性死亡， 在进行存活率统计时不予计算在内。
C57 BL/6小鼠 （4周龄） ： 军事医学科学院实验动物中心。 FGF-2基因 敲除小鼠 （背景为 SPF级 C57 BL/6小鼠） ： 美国 Jackson Laboratory, 货 号是 003256。脂多糖（LPS，来自大肠杆菌 0111 : B4的脂多糖）： Sigma, L2630。 酵母多糖 A (Zymosan A，来自酿酒酵母）： Sigma, Z4250。FGF- 2重组蛋白（Human recombinant FGF-2/basic FGF protein) : 蛋白序列如序列表的序列 1所示， 其编码基因如序列表的序列 2所示； Mi ll ipore 公司， 货号 01-106， 使用时 用 PBS缓冲液稀释为所需的浓度。
甲型 H1N1流感病毒株 A/Bei j ing/501/2009 (H1N1) (简称 BJ501株）： http： //www. ncbi . nlm. nih. gov/Taxonomy/Browser/ wwwtax. cgi?mode=In fo&id=648856&lvl=3&keep=l&srchmode=l&imlock&l in=s ; Yang P， Deng J, Li C， Zhang P， Xing L, Li Z, Wang W, Zhao Y， Yan Y， Gu H， Liu X, Zhao Z, Zhang S, Wang X, Jiang C. Characteri zat ion of the 2009 pandemic A/Be i j ing/501/2009 HlNl influenza strain in human airway epithel ial cel l s and ferret s. PLoS One. 2012 ; 7 (9) : e46184. doi : 10. 1371/journal. pone. 0046184. Epub 2012 Sep 26.
甲型 HlNl流感病毒株 A/Puerto Rico/8/1934 ( HlNl ) (简写 PR8株）： http： //www. ncbi . nlm. nih. gov/Taxonomy/Browser/ wwwtax. cgi?mode=In fo&id=211044&lvl=3&l
Figure imgf000009_0001
; Li C， Yang P， Sun Y, Li T, Wang C, Wang Z, Zou Z, Yan Y, Wang W, Wang C, Chen Z, Xing L, Tang C, Ju X, Guo F, Deng J, Zhao Y, Yang P, Tang J, Wang H, Zhao Z, Yin Z, Cao B, Wang X, Jiang C. IL-17 response mediates acute lung injury induced by the 2009 pandemic influenza A (HlNl) virus. Cel l Res. 2012 Mar ; 22 (3) : 528-38. doi : 10. 1038/cr. 2011. 165. Epub 2011 Oct 25.
实施例中所用的 PBS 缓冲液， 如无特殊说明均为 pH7. 2、 0. 01mol/L 的 PBS缓冲液。 实施例 1、 甲型 H1N1流感病毒引起小鼠肺灌洗液中 FGF-2水平升高 实验组 （5只 4-6周龄 C57 BL/6小鼠） ： 安全固定小鼠， 每只腹腔注 射 50-6(^L lg/100mL 的戊巴比妥钠溶液以进行麻醉； 保持麻醉小鼠头部 向上向后倾斜姿势，使其鼻腔向上，用移液管每侧鼻孔各滴入 10 L BJ501 株病毒液（105' 5 TCID5Q/只）， 保持小鼠此体位 15秒， 使病毒进入呼吸道； 感染 BJ501株 24小时后， 通过腹腔注射过量麻醉剂的方法使小鼠死亡， 将死亡小鼠固定于小动物手术台，移除胸部皮肤及骨骼，将气管剪一小口， 从开口处向小鼠注射 800微升 PBS缓冲液， 反复吸取三次后将肺灌洗液吸 出，利用 Bi o-Plex Mouse Cytokine 23-Plex 试剂盒检测肺灌洗液中 FGF-2 的浓度。
对照组 （5只 4-6周龄 C57 BL/6小鼠） ： 用等体积鸡胚尿囊液代替 BJ501株病毒液， 其它同实验组。
结果见图 1。感染甲型 H1N1流感病毒的小鼠肺灌洗液中 FGF-2的浓度 高于对照组 （* P<0. 05 ) ， 即感染甲型 H1N1 流感病毒的小鼠肺灌洗液中 FGF-2的表达水平显著高于对照组。 结果表明， FGF-2在甲型 H1N1流感病 毒感染导致肺损伤中发挥重要作用， 针对 FGF-2 的干预在治疗甲型 H1N1 流感病毒感染所导致的损伤中发挥重要作用。 实施例 2、 FGF-2缺陷小鼠中甲型 H1N1流感病毒引起的急性肺损伤更 加严重
安全固定 6只 4-6周龄 C57BL/6小鼠 （或 6只 4-6周龄 FGF-2基因敲 除小鼠） ， 用 lmL无菌注射器腹腔注射 50-6( L lg/100mL的戊巴比妥钠 溶液以进行麻醉； 保持麻醉小鼠头部向上向后倾斜姿势， 使其鼻腔向上， 用移液管每侧鼻孔各滴入 BJ501株病毒液（105· 5 TCID /只）， 保持小鼠此体 位 15秒， 使病毒进入呼吸道； 感染甲型流感病毒 5天后， 通过腹腔注射 过量麻醉剂的方法使小鼠死亡； 将 3只死亡小鼠固定于小动物手术台， 移 除胸部皮肤及骨骼， 暴露胸腔， 将肺脏连同心脏同时取出， 用无菌 PBS缓 冲液洗去表面血液， 置于多聚甲醛固定液中室温固定 48h， 进行包埋、 切 片、 HE染色等处理； 将另外 3只死亡小鼠固定于小动物手术台， 移除胸部 皮肤及骨骼，暴露胸腔，将完整肺脏取出，去除表面血液及多余结缔组织， 称量并记录肺脏湿重， 然后将肺脏置于 55°C高温组织干燥器中干烤， 24h 后取出， 待温度降至室温后称量肺脏干重， 湿干比 =肺脏湿重 /肺脏干重。
切片染色结果见图 2( X 200倍）， A为 C57BL/6小鼠肺组织， B为 FGF-2 基因敲除小鼠肺组织。感染甲型 H1N1流感病毒后， C57BL/6小鼠肺组织中 出现严重的病理损伤， 肺组织正常结构被破坏， 肺组织纹理紊乱， 伴随出 血、 炎性渗出及大量红细胞、 炎症细胞浸润等病理损伤。 感染相同滴度病 毒的 FGF-2基因敲除小鼠肺组织出现更加显著病理损伤，更为显著的出血、 渗出或者炎症细胞浸润等病理变化， 肺组织纹理不清晰， 结构不完整。
湿干比结果见图 3。肺脏湿干比可以反映小鼠发生急性肺水肿的程度。
FGF-2 基因敲除小鼠在感染甲型 H1N1 流感病毒后， 其肺脏湿干比相比 C57BL/6小鼠显著增加（* 0. 05 )，说明 FGF-2的敲除可以加重甲型 H1N1 流感病毒感染后小鼠的肺脏水肿。 结果表明， FGF-2缺陷小鼠中甲型 H1N1 流感病毒引起的急性肺损伤更加严重。 实施例 3、 FGF-2缺陷小鼠中甲型 H1N1流感病毒引起的急性肺损伤更 加严重
安全固 20只 4-6周龄 C57BL/6小鼠 （或 20只 4-6周龄 FGF-2基因敲 除小鼠） ， 用 lmL无菌注射器腹腔注射 50-6( L lg/100mL的戊巴比妥钠 溶液以进行麻醉； 保持麻醉小鼠头部向上向后倾斜姿势， 使其鼻腔向上， 用移液管每侧鼻孔各滴入 10 μ L PR8株病毒液（105' 5 TCID5。/只）， 保持小鼠 此体位 15秒， 使病毒进入呼吸道； 14只小鼠进行存活率统计 （感染甲型 流感病毒前记为第 0天，从感染甲型流感病毒开始， 24小时后记为第 1天， 依次类推） 和体重监测； 剩余的 6只小鼠于感染甲型流感病毒 5天后， 通 过腹腔注射过量麻醉剂的方法使小鼠死亡； 将 3只死亡小鼠固定于小动物 手术台， 移除胸部皮肤及骨骼， 暴露胸腔， 将肺脏连同心脏同时取出， 用 无菌 PBS缓冲液洗去表面血液， 置于多聚甲醛固定液中室温固定 48h， 进 行包埋、切片、 HE染色等处理；将另外 3只死亡小鼠固定于小动物手术台， 移除胸部皮肤及骨骼， 暴露胸腔， 将小鼠完整肺脏取出， 去除表面血液及 多余结缔组织， 称量并记录肺脏湿重， 然后将肺脏置于 55°C高温组织干燥 器中干烤， 24h后取出， 待温度降至室温后称量肺脏干重， 湿干比 =肺脏湿 重 /肺脏干重。
感染甲型流感病毒后的存活率统计结果见图 4。 感染相同滴度的甲型 H1N1流感病毒后， C57BL/6小鼠的死亡率明显低于 FGF-2基因敲除小鼠（* P<0. 05 ) 。 感染甲型流感病毒后的体重变化 （即某一天的体重与第 0天的 体重的比值） 的统计结果见图 5。 感染相同滴度的甲型 H1N1流感病毒后， C57BL/6小鼠的体重变化明显低于 FGF-2基因敲除小鼠 （* P<0. 05 ) 。 结 果表明， FGF-2在保护甲型 H1N1流感病毒感染小鼠免于死亡的过程中发挥 重要作用，针对 FGF-2分子的干预在治疗甲型 H1N1流感病毒感染保护中， 有可能发挥重要作用。
切片染色结果见图 6( X 200倍）， A为 C57BL/6小鼠肺组织， B为 FGF-2 基因敲除小鼠肺组织。感染甲型 H1N1流感病毒后， C57BL/6小鼠肺组织中 出现严重的病理损伤， 肺组织正常结构被破坏， 肺组织纹理紊乱， 伴随出 血、 炎性渗出及大量红细胞、 炎症细胞浸润等病理损伤。 感染相同滴度病 毒的 FGF-2基因敲除小鼠肺组织出现更加显著病理损伤，更为显著的出血、 渗出或者炎症细胞浸润等病理变化， 肺组织纹理不清晰， 结构不完整。
湿干比结果见图 7。FGF-2基因敲除小鼠在感染甲型 H1N1流感病毒后， 其肺脏湿干比相比 C57BL/6小鼠显著增加 （*尸<0. 05 ) ， 说明 FGF-2的敲 除可以加重甲型 H1N1流感病毒感染后小鼠的肺脏水肿。 结果表明， FGF-2 缺陷小鼠中甲型 H1N1流感病毒引起的急性肺损伤更加严重。 实施例 4、 FGF-2重组蛋白可以使甲型 m流感病毒感染引起的急性 肺损伤得以减轻
一、 实验一
实验组 （10只 4-6周龄 C57BL/6小鼠） ： 实验第 1天， 每只小鼠静脉 注射 Ιθθμΐ FGF-2重组蛋白溶液 （蛋白浓度为 0. 5mg/ml ) ； 实验第 2天， 安全固定小鼠， 每只小鼠用 lmL 无菌注射器腹腔注射 50-6(^L lg/100mL 的戊巴比妥钠溶液以进行麻醉； 保持麻醉小鼠头部向上向后倾斜姿势， 使 其鼻腔向上， 用移液管每侧鼻孔各滴入 lO L PR8株病毒液（105· 5 TCID5。/ 只）， 保持小鼠此体位 15秒， 使病毒进入呼吸道； 实验第 3天， 每只小鼠 静脉注射 Ιθθμΐ FGF-2重组蛋白溶液 （蛋白浓度为 0. 5mg/ml ) ； 实验第 5 天，每只小鼠静脉注射 Ιθθμΐ FGF-2重组蛋白溶液（蛋白浓度为 0. 5mg/ml ) ; 每天统计小鼠的存活率。
对照组 （10只 4-6周龄 C57 BL/6小鼠） ： 用等体积 PBS缓冲液代替 FGF-2重组蛋白溶液， 其它同实验组。
实验第 1天至第 14天的存活率统计结果见图 8。感染相同滴度的甲型 H1N1 流感病毒后， 对照组的死亡率明显高于实验组的小鼠 （* 0. 05 ) 。 结果表明， FGF-2在甲型 H1N1流感病毒感染小鼠死亡的过程中发挥的治疗 重要作用，针对 FGF-2分子的干预在治疗甲型 H1N1流感病毒感染恢复中， 有可能发挥重要作用。
二、 实验二
实验组 （6只 4-6周龄 C57BL/6小鼠） ： 实验第 1天， 每只小鼠静脉 注射 Ιθθμΐ FGF-2重组蛋白溶液 （蛋白浓度为 0. 5mg/ml ) ； 实验第 2天， 安全固定小鼠， 每只小鼠用 lmL 无菌注射器腹腔注射 50-6(^L lg/100mL 的戊巴比妥钠溶液以进行麻醉； 保持麻醉小鼠头部向上向后倾斜姿势， 使 其鼻腔向上， 用移液管每侧鼻孔各滴入 lO L PR8株病毒液（105· 5 TCID5。/ 只）， 保持小鼠此体位 15秒， 使病毒进入呼吸道； 实验第 3天， 每只小鼠 静脉注射 Ιθθμΐ FGF-2重组蛋白溶液 （蛋白浓度为 0. 5mg/ml ) ； 实验第 5 天，每只小鼠静脉注射 Ιθθμΐ FGF-2重组蛋白溶液（蛋白浓度为 0. 5mg/ml ) ; 实验第 6天， 通过腹腔注射过量麻醉剂的方法使小鼠死亡； 将 3只死亡小 鼠固定于小动物手术台， 移除胸部皮肤及骨骼， 暴露胸腔， 将肺脏连同心 脏同时取出， 用无菌 PBS缓冲液洗去表面血液， 置于多聚甲醛固定液中室 温固定 48h， 进行包埋、 切片、 HE染色等处理； 将另外 3只死亡小鼠固定 于小动物手术台， 移除胸部皮肤及骨骼， 暴露胸腔， 将完整肺脏取出， 去 除表面血液及多余结缔组织， 称量并记录肺脏湿重， 然后将肺脏置于 55°C 高温组织干燥器中干烤， 24h后取出， 待温度降至室温后进行称量肺脏干 重， 湿干比 =肺脏湿重 /肺脏干重。
对照组 （6只 4-6周龄 C57 BL/6小鼠） ： 用等体积 PBS缓冲液代替 FGF-2重组蛋白溶液， 其它同实验组。
切片染色结果见图 9 ( X 200倍） ， A为对照组小鼠肺组织， B为实验 组小鼠肺组织。 感染甲型 H1N1 流感病毒后， 对照组小鼠肺组织中出现严 重的病理损伤， 肺组织正常结构被破坏， 肺组织纹理紊乱， 伴随出血、 炎 性渗出及大量红细胞、 炎症细胞浸润等病理损伤。 感染相同滴度病毒的实 验组小鼠肺组织未见显著病理损伤， 无显著的出血、 渗出或者炎症细胞浸 润等病理变化， 肺组织纹理清晰， 结构完整。 结果表明， FGF-2对小鼠在 感染甲型 H1N1 流感病毒导致的急性肺组织病理损伤中发挥了重要的保护 作用。
湿干比结果见图 10。 实验组小鼠在感染甲型 H1N1流感病毒后， 其肺 脏湿干比相比对照组鼠显著降低 （* 0. 05 ) ， 说明 FGF-2可以显著缓解 甲型 H 1N1流感病毒感染引起的小鼠的肺脏水肿。 结果表明， FGF-2对小鼠 在感染甲型 m 流感病毒导致的急性肺组织病理损伤中发挥了重要的保 护作用。 实施例 5、 FGF-2重组蛋白可以缓解由脂多糖和酵母多糖 A联合感染 后小鼠的肺组织病理损伤
第一组 （4只 4-6周龄 C57BL/6小鼠） ：
分别于感染脂多糖前 12小时、 感染脂多糖前 1小时和感染脂多 糖后 8小时， 每只小鼠每次静脉注射 Ι θθμΐ FGF-2重组蛋白溶液 （蛋 白含量为 50微克） ；
感染操作： 每只小鼠用 lmL 无菌注射器腹腔注射 50-6( L l g/ 100mL的戊巴比妥钠溶液以进行麻醉，保持麻醉小鼠头部向上向后 倾斜姿势， 使其鼻腔向上， 滴鼻接种 50微升脂多糖溶液（溶剂为 PBS 缓冲液， 含 100微克脂多糖） ， 保持小鼠此体位 5分钟， 使脂多糖进 入呼吸道； 感染脂多糖 1小时后， 每只小鼠用 lmL无菌注射器腹腔注 射 50-6( L l g/ 100mL 的戊巴比妥钠溶液以进行麻醉， 保持麻醉小鼠 头部向上向后倾斜姿势， 使其鼻腔向上， 滴鼻接种 50微升酵母多糖 A 溶液 （溶剂为 PBS缓冲液， 含 60微克脂多糖） ， 保持小鼠此体位 5 分钟， 使脂多糖进入呼吸道；
感染脂多糖 24后，通过腹腔注射过量麻醉剂的方法使小鼠死亡， 将死亡小鼠固定于小动物手术台， 移除胸部皮肤及骨骼， 暴露胸腔， 将肺脏连同心脏同时取出， 用无菌 PBS缓冲液洗去表面血液， 置于多 聚甲醛固定液中室温固定 48h， 进行包埋、 切片、 HE染色等处理。 第二组： 感染脂多糖前 12小时、 感染脂多糖前 1小时和感染脂多糖 后 8小时均未注射 FGF-2重组蛋白溶液， 其它同第一组。
第三组： 用等体积 PBS缓冲液代替脂多糖溶液， 用等体积 PBS缓冲液 代替酵母多糖 A溶液， 其它同第一组。
结果见图 1 1， 图 11中， A为第一组， B为第二组， C为第三组。 第三 组小鼠的肺组织未见显著病理损伤， 无显著的出血、 渗出或者炎症细胞浸 润等病理变化， 肺组织纹理清晰， 结构完整。 第二组小鼠的肺组织显著病 理损伤， 显著的出血、 渗出或者炎症细胞浸润等病理变化， 肺组织纹理不 清晰， 结构不完整。 第一组小鼠的肺组织而未见显著病理损伤， 无显著的 出血、 渗出或者炎症细胞浸润等病理变化， 肺组织纹理清晰， 结构完整。 结果表明， FGF-2对小鼠在感染脂多糖和酵母多糖 A联合导致的急性肺组 织病理损伤中发挥了重要保护作用。
工业应用
本发明公开了 FGF-2 在制备治疗和 /或预防肺损伤的药物中的用途， 在预防和 /或治疗流感中的用途， 在预防和 /或治疗流感病毒引起的疾病中的 用途。 本发明对于上述疾病的治疗和预防具有重大价值。

Claims

权利要求
1、 成纤维细胞生长因子 2在制备药物中的应用； 所述药物的用途为如下 (a) 和 /或 （b ) 和 /或 （c ) ： (a) 预防和 /或治疗肺损伤； （b ) 预防和 / 或治疗流感； （c) 预防和 /或治疗流感病毒引起的疾病。
2、 如权利要求 1所述的应用， 其特征在于： 所述成纤维细胞生长因子 2 为人源的成纤维细胞生长因子 2。
3、 如权利要求 2所述的应用， 其特征在于： 所述成纤维细胞生长因子 2 为如下 （A) 或 （B) ： 序列表中序列 1所示的蛋白质； （B) 将 （A) 经过一 个或几个氨基酸残基的取代和 /或缺失和 /或添加且具有相同活性的由其 衍生的蛋白质。
4、 如权利要求 1至 3中任一所述的应用， 其特征在于： 所述肺损伤为 病毒和 /或细菌和 /或真菌引起的肺损伤。
5、 如权利要求 1至 3中任一所述的应用， 其特征在于： 所述肺损伤为败 血症引起的肺损伤。
6、 一种药物， 其活性成分为成纤维细胞生长因子 2; 所述药物的用途为 如下 （a) 和 /或 （b ) 和 /或 （c) ： (a) 预防和 /或治疗肺损伤； （b) 预防 和 /或治疗流感； （c ) 预防和 /或治疗流感病毒引起的疾病。
7、 如权利要求 6所述的药物， 其特征在于： 所述成纤维细胞生长因子 2 为人源的成纤维细胞生长因子 2。
8、 如权利要求 7所述的药物， 其特征在于： 所述成纤维细胞生长因子 2 为如下 （A) 或 （B) ： 序列表中序列 1所示的蛋白质； （B) 将 （A) 经过一 个或几个氨基酸残基的取代和 /或缺失和 /或添加且具有相同活性的由其 衍生的蛋白质。
9、 如权利要求 6至 8中任一所述的药物， 其特征在于： 所述肺损伤为病 毒和 /或细菌和 /或真菌引起的肺损伤。
10、 如权利要求 6至 8中任一所述的药物， 其特征在于： 所述肺损伤为 败血症引起的肺损伤。
11、 成纤维细胞生长因子 2在预防和 /或治疗肺损伤中的应用。
12、 如权利要求 11所述的应用， 其特征在于： 所述成纤维细胞生长因 子 2为人源的成纤维细胞生长因子 2。
13、 如权利要求 12所述的应用， 其特征在于： 所述成纤维细胞生长因子 2 为如下 （A) 或 （B) ： 序列表中序列 1所示的蛋白质； （B) 将 （A) 经过一 个或几个氨基酸残基的取代和 /或缺失和 /或添加且具有相同活性的由其 衍生的蛋白质。
14、 如权利要求 11至 13中任一所述的应用， 其特征在于： 所述肺损伤 为病毒和 /或细菌和 /或真菌引起的肺损伤。
15、 如权利要求 11至 13中任一所述的应用， 其特征在于： 所述肺损伤为 败血症引起的肺损伤。
16、 成纤维细胞生长因子 2的应用， 为预防和 /或治疗流感， 或， 预防和
/或治疗流感病毒引起的疾病。
17、 如权利要求 16所述的应用， 其特征在于： 所述成纤维细胞生长因 子 2为人源的成纤维细胞生长因子 2。
18、 如权利要求 17所述的应用， 其特征在于： 所述成纤维细胞生长因子 2 为如下 （A) 或 （B) ： 序列表中序列 1所示的蛋白质； （B) 将 （A) 经过一 个或几个氨基酸残基的取代和 /或缺失和 /或添加且具有相同活性的由其 衍生的蛋白质。
19、 如权利要求 16至 18中任一所述的应用， 其特征在于： 所述流感病 毒为甲型 H1N1流感病毒。
20、 成纤维细胞生长因子 2作为肺损伤的标志物的应用， 或， 用于检 测所述成纤维细胞生长因子 2的物质在辅助诊断肺损伤中的应用， 或用于检 测所述成纤维细胞生长因子 2的物质在制备辅助诊断肺损伤的产品中的应用。
PCT/CN2013/000944 2013-08-09 2013-08-09 成纤维细胞生长因子2的新用途 WO2015017949A1 (zh)

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