WO2017105081A1 - Pharmaceutical composition for treating lung diseases or alleviating symptoms of lung diseases - Google Patents

Pharmaceutical composition for treating lung diseases or alleviating symptoms of lung diseases Download PDF

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WO2017105081A1
WO2017105081A1 PCT/KR2016/014654 KR2016014654W WO2017105081A1 WO 2017105081 A1 WO2017105081 A1 WO 2017105081A1 KR 2016014654 W KR2016014654 W KR 2016014654W WO 2017105081 A1 WO2017105081 A1 WO 2017105081A1
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atpase
protein
lung
present
pharmaceutical composition
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PCT/KR2016/014654
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French (fr)
Korean (ko)
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박성우
이지민
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순천향대학교 산학협력단
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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
    • 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

Definitions

  • the present invention relates to a pharmaceutical composition for treating or symptomatic pulmonary disease comprising V-atpase B2 protein as an active ingredient.
  • Vacuolar type H + -ATPase (V- ATPase) has to flow into a hydrogen ion (H + ion) cell is used as a transporter (transporter) of the cell membrane and the function of the intracellular pH at which the sustain acidified maintain homeostasis of the environmental cell It is a protein that plays a role.
  • the V-ATPase protein has isoforms depending on the tissue in which it is located, but basically consists of two different functional domains, VO and V1.
  • the V0 domain is present in the plasma membrane and the V1 domain is present in the cytoplasm.
  • the V0 domain is the portion where H ions are translocated and is composed of three subunits of c, d, and e.
  • the V1 domain consists of eight subunits of A3, B3, C, D, E2, F, G2, H.
  • the site where ATP binds is A3B3, which is known to cause hydrolysis of ATP.
  • V-ATPase The most basic role of V-ATPase in cells is to provide a proton pump that maintains an optimal acidic pH to process or degrade intracellular delivery of endosome and lysosomes. pumping) role.
  • pumping the most basic role of V-ATPase in cells is to provide a proton pump that maintains an optimal acidic pH to process or degrade intracellular delivery of endosome and lysosomes. pumping
  • An object of the present invention is to provide a pharmaceutical composition for treating pulmonary disease or alleviating symptoms comprising V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) as an active ingredient.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to provide a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to provide a method for treating or alleviating pulmonary disease, comprising administering to a subject V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein.
  • Another object of the present invention is a method for suppressing or alleviating lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein.
  • V-atpase B2 V-atpase B2, Vacuolar-type H + -ATPase B2 protein.
  • Another object of the present invention is to provide a use for the use of the V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. will be.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to use a V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein for the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It is to provide a use.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • the pharmaceutical composition of the present invention contains the V-atpase B2 protein as an active ingredient, and is useful for treating lung diseases or alleviating symptoms, and in particular, for treating or alleviating symptoms of acute lung injury, pulmonary fibrosis or emphysema among lung diseases. Can be. In addition, it can be utilized as a pharmaceutical composition for suppressing or alleviating lung damage by smoking or secondhand smoke.
  • FIG. 1 is a diagram showing the sequence information of the V-Etipiase B2 protein represented by SEQ ID NO: 1 of the present invention.
  • Figure 2 is IF staining results confirming whether Vatpase B2 protein expression control of the genetically modified mouse prepared in Example 1 of the present invention.
  • Figure 3 is a Western blotting (Western blotting) results confirming whether the control of Vatpase B2 protein expression of the genetically modified mouse prepared in Example 1 of the present invention.
  • Figure 4 is a schematic diagram illustrating the manufacturing process of a mouse model induced acute lung injury pulmonary fibrosis induced in V-atpase B2 TG mice in Example 2 of the present invention.
  • Figure 5 is a graph showing the results of measuring the total lung inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of acute lung injury pulmonary fibrosis mouse model induced in Example 2 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Figure 6 is a result of H & E staining showing the degree of lung tissue damage in a lung lung fibrosis mouse model induced acute lung injury in Example 2 of the present invention.
  • Figure 7 is a trichrome staining result of the degree of collagen accumulation in the lung tissue of a mouse model of acute lung injury pulmonary fibrosis induced in Example 2 of the present invention.
  • Figure 8 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes count from bronchoalveolar lavage fluid (BALF) of CS-induced emphysema mouse model in Example 3 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Example 9 is a result of measuring lung mechanics (left: lung purity, right: lung elasticity) using the lung tissue of the CS-induced emphysema mouse model in Example 3 of the present invention.
  • Figure 10 is the result of observing the alveolar tissue of the CS-induced emphysema mouse model in Example 3 of the present invention by H & E staining.
  • Figure 11 shows the results confirmed by IF staining the influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
  • Example 12 is a result confirmed by Immunoblotting of the cellular influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
  • Figure 13 shows the results of localization of the V-atpaseB2 protein in 293T cells in Example 4 of the present invention using double immunofluorescence staining.
  • FIG. 14 is a schematic diagram illustrating a procedure for testing the effect of V-atpase B2 protein nasal administration in a mouse lung disease model in Example 4 of the present invention.
  • Example 15 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of the mouse lung disease model induced in Example 4 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Example 16 is an immunofluorescence staining result confirming that Vatpase B2 is introduced into the tissue of the mouse lung disease model induced in Example 4 of the present invention.
  • Figure 17 is the result of observing the tissue of the mouse lung disease model induced in Example 4 of the present invention by H & E staining.
  • Example 18 is a result of confirming the degree of collagen accumulation by massons trichrome stain the tissue of the mouse lung disease model induced in Example 4 of the present invention.
  • Example 19 is a result of confirming and quantifying the amount of collagen fibers accumulated in the tissue of the mouse lung disease model induced in Example 4 of the present invention by a sircol assay (* p ⁇ 0.05 by Mann-Whitney U test).
  • a pharmaceutical composition for treating or symptomatic pulmonary disease is V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein active ingredient Include as.
  • the V-Etipiase B2 protein may be hV-atpase B2 represented by SEQ ID NO: 1.
  • the lung disease may be acute lung injury, pulmonary fibrosis or emphysema.
  • the pharmaceutical composition for inhibiting or alleviating pulmonary injury by smoking or indirect smoking includes V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. Include.
  • V-ATPase B2 protein which is a lung disease, especially acute lung injury, pulmonary fibrosis, emphysema.
  • the present invention was completed by using an animal model to confirm that it is effective in treating and relieving symptoms of lung injury diseases.
  • the pharmaceutical composition according to an embodiment of the present invention may include V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient and may be applied for treating lung disease or alleviating symptoms. have.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • the V-Etipiase B2 protein may be a V-Etipiase B2 protein of a mammal, including human, preferably a human V-Etipiase B2 protein, specifically 511 shown in SEQ ID NO: 1 and FIG. V-Etipiaze B2 protein having 4 amino acid sequences.
  • the lung disease may be acute lung injury, pulmonary fibrosis or emphysema, and in particular, the pharmaceutical composition may be useful for lung disease accompanied by cell damage related to oxidative stress.
  • the lung disease may be emphysema induced by tobacco smoke.
  • treatment of the present invention means any action that improves or advantageously changes the symptoms of the lung disease by administration of the composition according to the present invention.
  • the term “relaxation” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
  • the pharmaceutical composition of the present invention may be applied to suppress or alleviate lung damage by smoking or secondhand smoke, including V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. .
  • the lung injury may be emphysema.
  • Administration of the pharmaceutical composition may be by a method generally accepted as a method of administering the above-mentioned pharmacological activity, and specifically to oral, parenteral or topical administration, systemic administration by other methods, etc. You can.
  • the pharmaceutical composition is preferably administered to the patient by the method of intranasal administration or bronchial administration in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also be formulated as a pharmaceutical composition suitable for parenteral administration to a patient, eg, intravenous, arterial, spinal or intraperitoneal.
  • the pharmaceutical composition may be used in the form of a solid, semi-solid or liquid formulation, preferably for example as a pill, capsule, powder, liquid, suspension, etc., suitable for the administration of the correct dose.
  • the pharmaceutical composition may include conventional pharmaceutical carriers or excipients, and may also include other pharmaceutical agents, pharmaceutical agents, carriers, additives, and the like.
  • the excipient may include, but is not limited to, other proteins such as, for example, human serum albumin or plasma proteins.
  • the pharmaceutical composition may be administered at the same time or separately from the existing therapeutic agent in the treatment of the lung disease.
  • the administered pharmaceutical composition or formulation thereof contains an amount of the active ingredient in an amount effective to achieve a desired effect in the subject being treated, and the amount of the active ingredient administered to the patient is determined by the characteristics of the subject, the severity of the disease, Depending on the mode of administration and the judgment of the doctor, it is better to administer a relatively low concentration of protein.
  • the pharmaceutical composition may be 1 x 10 5 IU / person to 9 x 10 6 IU / person.
  • the present invention provides a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • improvement refers to any action that improves or benefits the suspicion of a disease and the onset of a pulmonary disease to be prevented or treated using a food composition comprising V-Etipiase B2 as an active ingredient.
  • prevention of the present invention means any action that inhibits or delays the onset of the lung disease by administration of the composition according to the present invention.
  • V-Etipiaze B2 of the present invention can be added to food compositions for the purpose of preventing or ameliorating lung diseases.
  • the food composition of the present invention may include the form of pills, powders, granules, acupuncture, tablets, capsules or liquids, etc., and there is no particular limitation on the kind of foods to which V-Etipiaze B2 of the present invention may be added. Examples include various beverages, gums, teas, vitamin complexes, and dietary supplements.
  • V-Etipia agent B2 In addition to the V-Etipia agent B2, other ingredients may be added to the food composition, and the kind thereof is not particularly limited.
  • various herbal extracts, food acceptable food additives, natural carbohydrates, and the like may be included as additional ingredients, such as conventional foods, but are not limited thereto.
  • the term "food supplement” means a component that can be added to food supplements, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation.
  • food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, but is not limited to the kind of food additives of the present invention by the above examples.
  • Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, and as flavoring agents other than those described above, natural flavoring agents (such as taumartin), stevia extract (rebaudioside A, glycyr) Higin and the like) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • monosaccharides such as glucose and fructose
  • Disaccharides such as maltose and sucrose
  • polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol
  • sugar alcohols such as xylitol, sorbi
  • the food composition of the present invention may include a health functional food.
  • the term "health functional food” refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body.
  • functional means to obtain a useful effect for health use such as nutrient control or physiological action on the structure and function of the human body.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art.
  • unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the V-Etipiaze B2 of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, in the manufacture of food, but is not limited thereto.
  • the amount may be used below the above range.
  • Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes and the like, and include all of the dietary supplements in the conventional sense.
  • the present invention provides a method of treating or alleviating pulmonary disease, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. do.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • the term "individual” means all animals including a human having a lung disease or a lung disease. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung diseases are treated or alleviated by the protein of the present invention.
  • the present invention provides a method for inhibiting lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. Or provide mitigation.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • lung damage is as described above.
  • the term "individual” means all animals including a human in which lung injury has occurred or a lung injury has occurred. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung injury is inhibited or alleviated by the protein of the present invention.
  • the present invention provides the use of the V- atpase B2 (Vacuolar-type H + -ATPase B2) protein for use in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. to provide.
  • V- atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • the present invention uses the V-atpase B2 (Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It provides a use for.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • lung damage is as described above.
  • Example 1 alveoli (type II pneumocyte )on Vatpase Preparation of engineered mice overexpressing B2 protein
  • type II pneumocyte Vatpase B2 protein was overexpressed, and double transgenic mice were prepared to control the overexpression time by DOXY.
  • hVatpase B2 construct was constructed by subcloning the VATPaseB2 cDNA construct containing 1x flag sequene in the TRE-tight vector using RT-PCR, and it was possible to accurately detect V-atpase B2 protein expression as Flag Ab.
  • Double transgenic mice which were made by injecting two constructs into the eggs of C57 / BL6 mice, were applied to the following experiments.
  • mice were confirmed by IF staining and Western blotting using Flag Ab. Whether Vatpase B2 protein expression was regulated by DOXY, and the results are shown in FIGS. 2 and 3, respectively.
  • mice prepared for the following experiments were well expressed in the flag tagged hVatpase B2 protein depending on whether DOXY was introduced.
  • Example 2 Acute induction with engineered mice Lung injury Experimental Effects of Overexpressed V-atpase B2 in a Mouse Model
  • Acute lung injury / pulmonary fibrosis was induced in experimental animals by administering bleomycin into the bronchus (intratracheal) of V-atpase B2 TG mice prepared above. Specifically, as shown in FIG. 4 below, 5 days before bleomycin administration, Doxy water was administered to induce overexpression of V-atpase B2 and sacrifice at 14 days after bleomycin administration to bronchoalveolar lavage fluid (BALF), bronchus and pesos. After the organization was conducted, the experiment was conducted.
  • BALF bronchoalveolar lavage fluid
  • Bronchoalveolar labage fluid (BALF) in mice was obtained by carefully inhaling 1000 ⁇ l of PBS into the airway of the disease-induced mice repeatedly with a syringe at 37 ° C. After washing with Han's balanced salt soulution (HBSS) containing 2% FCS (fetal calf serum), erythrocytes were removed using lysis buffer, and the supernatants were collected separately by centrifugation.
  • HBSS Han's balanced salt soulution
  • FCS fetal calf serum
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples.
  • the tissue samples were deparaffinized and stained with 200 ⁇ l of hematocyline for 1 minute and washed with water for 10 minutes. Subsequently, 1 minute staining with 200 ⁇ l of Eosin was followed by washing with water for 10 minutes, followed by mounting through a dehydration process. The results are shown in FIG. 6.
  • tissue damage was significantly reduced in samples of the experimental group overexpressed with Doxy (+) V-atpase B2 protein.
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples.
  • Tissue samples were washed with running water after deparaffinization and treated with 200 ⁇ l of Bouin solution for 1 hour in a 56 ° incubator. It was washed with running water and stained with Weigrt iron hematoxylin 200 ⁇ l at room temperature for 10 minutes.
  • the treated tissue samples were washed with running water, dehydrated, and then mounted, and the results are shown in FIG. 7.
  • Example 3 Experimental Effects of V-atpase B2 Overexpressed in a Mouse Model of Cigarette Smoke Induced Emphysema
  • mice Genetically modified mice (Vatpase B2 overexpressing mice) prepared in Example 1 above were exposed to cigarette smoke five times a week for three months for five months to prepare a CS (cigarette smoke) induced emphysema mouse model.
  • CS cigarette smoke
  • Vatpase B2 overexpressing mice were exposed to CS in a plexiglass chamber (16x 25x 16cm) for 6 months using a smoking tester system (ThreeShineCom, Daejeon, Korea). Overexpression of V-atpase B2 was induced by the administration of Doxy containing water (50mg / ml) from the first day of exposure to CS. CS was used with a 3R4F burning reference cigarette for 5 hours, 3 times a week, for 6 months. Exposure to tobacco smoke.
  • VaTPase B2 overexpression test group VATPaseB2 air
  • WT_CS Vatpase B2 overexpression test group
  • the CS-induced emphysema mouse model was prepared, and then sacrificed to obtain bronchoalveolar lavage fluid (BALF), bronchial and alveolar tissues, and then experiments were performed.
  • BALF bronchoalveolar lavage fluid
  • Vatpase B2 overexpression may be the result of treating or preventing CS-induced emphysema.
  • Emphysema is characterized by increased lung compliance (static compliance) and reduced elasticity (elastance), the lung mechanics of the sacrificed mouse lung tissue was measured using a Flexi-vent device, the results are shown in FIG.
  • Observations of hematoxylin and eosin (H & E) staining were performed using lung tissue of a cigarette smoke induced emphysema mouse model as described above, and the results are shown in FIG. 10.
  • Doxy (+) / CS that is, normal alveoli without air space enlargement (Doxy (-) CS) does not appear when smoking Vatpase B2 overexpressing mice. It was confirmed that the appearance of.
  • Vatpase B2 significantly reduced animal emphysema induced by CS.
  • VatpaseB2 has a pulmonary protective action against CS and can be used for the prevention or treatment of emphysema. Is thought to be the result showing.
  • Example 4 V- in mouse lung disease model atpase B2 protein nasal administration and acute lungs Damage / Fibrosis Treatment Effect
  • Vatpase B2 protein (ORIGENE, USA, flag tagged recombinant protein, 56K.D.) was administered to 293T cells to confirm that the protein was introduced into the cell during the endocytosis process as follows.
  • 293T cells (ATCC No .: CRL-3216) were treated with 10% (v / v) heat-inactivated fetal bovine serum, 10,000 U / mL Penicillin, 10,000 ⁇ g / mL Streptomycin at 37 ° C. in a humidified atmosphere of 5% CO 2 . Incubated in Dulbecco's modified Eagle's medium, and the cultured 293T cells were administered with Vatpase B2 at 2 ng / well, followed by incubation for 17 hours under the same conditions, followed by IF staining.
  • the culture medium was removed, washed with DPBS, fixed with 15% of 4% paraformaldehyde for 15 minutes, washed with water, and then subjected to permiabilization using Triton X-100. Washed with DPBS and blocked for 1 hour at room temperature with 5% goat normal serum, the first anti-flag was diluted in a blocking solution at 10ug / ml and incubated at 4 °C 17 hours. Subsequently, the secondary antibody (Anti-Rabbit FITC) was diluted 1: 1000, treated, incubated at room temperature for 1 hour while blocking light, and nuclear stained with DAPI. The samples thus treated were mounted with a mounting solution for fluorescence staining, and the results were confirmed with a fluorescence microscope, and are shown in FIG. 11.
  • Vatpase B2 protein was well introduced into the cytoplasm of 293T cells by the method.
  • Vatpase B2 protein (2 ⁇ g / ml) was introduced into the same 293T cells as used in the above experiments, lysis buffer was added to prepare cell lysates, and proteins were extracted. The protein was developed using 10% SDS-PAGE, broken with 5% skim milk and then anti-FLAG antibody (SIGMA, 1: 5000) as the primary antibody and anti-rabbit as the secondary antibody. After the reaction using IgG (1: 5000) to perform a detecting process, the results are shown in Figure 12 below. Beta actin was applied as a positive control.
  • mice Male C57BL / 6 mice were distributed in Oriental Bio, followed by a five-day acclimation period in the laboratory, followed by induction of pulmonary damage with bleomycine and then applied to experiments confirming the therapeutic effect of Vatpase B2 protein (FIG. 14).
  • mice undergoing the adaptation period were administered bleomycin into the airways to induce acute lung injury and fibrosis, and 500 ng of V-atpase B2 protein was administered through the nasal cavity from 4 days to 7 days.
  • mice Two weeks after the administration of bleomycin, mice were sacrificed, and bronchial alveolar lavage fluid (BALF), bronchial and alveolar tissues were obtained, and then experiments were performed.
  • BALF bronchial alveolar lavage fluid
  • bronchoalveolar lavage fluid obtained from the above experimental animals. Obtaining BALF and measuring the number of pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes and the like was applied to the same method as described above, the results are shown in Figure 15 below.
  • FIG. 15 it was confirmed that inflammation due to lung injury induced by v-atpase B2 protein administration was alleviated or suppressed. Specifically, the number of total pulmonary inflammatory cells, neutrophils, and macrophages was induced in the lung injury-inducing group. Compared with the experimental group was significantly reduced.
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples and subjected to immunofluoroscence staining.
  • Tissue samples were subjected to deparaffinization, washed with running water, and subjected to permiabilization using Triton X-100. Washed with DPBS, blocked for 1 hour at room temperature with 5% goat normal serum, diluted the anti-flag at 10 ug / ml in a blocking solution and incubated at 4 ° C for 17 hours, followed by secondary antibody ( Anti-Rabbit FITC) was diluted 1: 1000, incubated for 1 hour at room temperature with light blocking, and nuclear stained with DAPI. Mounting with a mounting solution for fluorescence staining and observing with a fluorescence microscope to confirm the results are shown in Figure 16.
  • Vatpase B2 was well introduced into the bronchus and alveolar cells of the lung through an immunofluoroscence stain using Flag-FITC Ab.
  • Tissue samples were prepared from the sacrificed mouse lung tissues as described above, followed by staining with hematoxylin and eosin (H & E), and the results are shown in FIG. 17.
  • Trichrome staining was used to determine the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue. Specifically, the sacrificed mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples. Tissue samples were washed with running water after deparaffinization and treated with 200 ⁇ l of Bouin solution and embedded in an incubator at 56 ° C. for 1 hour. It was washed with running water and stained with 200 ⁇ l of Weigrt iron hematoxylin at room temperature for 10 minutes.
  • the Sircol assay confirmed the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue.
  • pulmonary tissue lysate was prepared by adding lysis buffer to the sacrificed mouse lung tissue, and the protein was extracted. Protein lysate and standard solution were added to each test tube, sircol dye was added, and reacted at room temperature for 30 minutes. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was washed with wasing buffer. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was sufficiently released with alkari reagent.
  • the collagen amount was measured at a wavelength of 540 nm with an ELISA reader machine, and the value quantified and corrected for the protein is shown in FIG. 19.
  • V-atpase B2 protein is introduced into the intracellular lysosome through endocytosis when administered in vitro or in mice.
  • Animal models of injury and pulmonary fibrosis were found to have a function of effectively inhibiting inflammation and fibrosis of the lungs.

Abstract

A pharmaceutical composition for treating lung diseases or alleviating symptoms of lung diseases, according to the present invention, comprises a vacuolar-type H+-ATPase B2 (V-atpase B2) protein as an active ingredient. The pharmaceutical composition is useful for the treatment of lung diseases, particularly acute lung injury, pulmonary fibrosis or pulmonary emphysema, or alleviating symptoms thereof.

Description

폐질환 치료용 또는 증상완화용 약학조성물Pharmaceutical composition for treating or relieving symptoms of lung disease
본 발명은 V-atpase B2 단백질을 유효성분으로 포함하는 폐질환 치료용 또는 증상완화용 약학조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for treating or symptomatic pulmonary disease comprising V-atpase B2 protein as an active ingredient.
Vacuolar type H+-ATPase(V-ATPase)는 세포막의 트랜스포터(transporter)로서 수소이온(H+ ion)을 세포 내로 유입하여 세포 내 pH를 산성으로 유지를 하는 작용을 하며 세포 내 환경의 항상성을 유지하는 역할을 하는 단백질이다.Vacuolar type H + -ATPase (V- ATPase) has to flow into a hydrogen ion (H + ion) cell is used as a transporter (transporter) of the cell membrane and the function of the intracellular pH at which the sustain acidified maintain homeostasis of the environmental cell It is a protein that plays a role.
V-ATPase 단백질은 위치하는 조직에 따라서 아이소폼(isoform)이 있으나 기본적으로 V0 및 V1 의 두 가지 다른 기능성 도메인(functional domain)로 구성된다. V0 도메인은 원형질막(plasma membrane)에 존재하며 V1 도메인은 세포질(cytoplasm)에 존재한다. V0 도메인은 H ion이 translocation 되는 부분으로 c, d, e의 3가지 서브유닛(subunit)으로 구성되어 있다. V1 도메인은 A3, B3, C, D, E2, F, G2, H의 8가지 서브유닛(subunit)으로 구성이 된다. ATP가 결합(binding)되는 부위는 A3B3로서 이 부위에서 ATP의 분해(hydrolysis)가 발생되는 것으로 알려져 있다.The V-ATPase protein has isoforms depending on the tissue in which it is located, but basically consists of two different functional domains, VO and V1. The V0 domain is present in the plasma membrane and the V1 domain is present in the cytoplasm. The V0 domain is the portion where H ions are translocated and is composed of three subunits of c, d, and e. The V1 domain consists of eight subunits of A3, B3, C, D, E2, F, G2, H. The site where ATP binds is A3B3, which is known to cause hydrolysis of ATP.
V-ATPase의 세포 내에서 가장 기본적인 역할은 엔도솜(endosome), 라이소좀(lysosome) 등에서 세포 내 전달된 물질을 처리(process or degradation)할 수 있는 최적의 산성 pH를 유지하도록 하는 양성자 펌프(proton pumping) 역할을 한다. 그러나, 각 subunit의 구체적인 역할이나 그 활용에 대한 연구는 아직 명확하지 않다.The most basic role of V-ATPase in cells is to provide a proton pump that maintains an optimal acidic pH to process or degrade intracellular delivery of endosome and lysosomes. pumping) role. However, the research on the specific role of each subunit and its use is not yet clear.
본 발명의 목적은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는 폐질환 치료용 또는 증상완화용 약학조성물을 제공하는 것이다.Disclosure of Invention An object of the present invention is to provide a pharmaceutical composition for treating pulmonary disease or alleviating symptoms comprising V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) as an active ingredient.
본 발명의 다른 목적은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 흡연 또는 간접흡연에 의한 폐손상 억제 또는 완화용 약학 조성물을 제공하는 것이다.It is another object of the present invention to provide a pharmaceutical composition for inhibiting or alleviating pulmonary injury by smoking or indirect smoking, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. It is.
본 발명의 다른 목적은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 폐질환 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
본 발명의 다른 목적은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 개체에 투여하는 단계를 포함하는, 폐질환의 치료 또는 증상완화 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating or alleviating pulmonary disease, comprising administering to a subject V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein.
본 발명의 다른 목적은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 개체에 투여하는 단계를 포함하는, 흡연 또는 간접흡연에 의한 폐손상의 억제 또는 완화방법을 제공하는 것이다.Another object of the present invention is a method for suppressing or alleviating lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein. To provide.
본 발명의 다른 목적은, V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 폐질환의 치료 또는 증상을 완화하기 위한 의약품의 제조에 사용하기 위한 용도를 제공하는 것이다.Another object of the present invention is to provide a use for the use of the V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. will be.
본 발명의 다른 목적은, V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 흡연 또는 간접흡연에 의한 폐손상을 억제 또는 완화하기 위한 의약품의 제조에 사용하기 위한 용도를 제공하는 것이다.Another object of the present invention is to use a V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein for the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It is to provide a use.
본 발명의 약학조성물은 V-atpase B2 단백질을 유효성분으로 포함하여, 폐질환 치료 또는 증상완화용으로 유용하며, 특히 폐질환 중에서 급성폐손상, 폐섬유화증 또는 폐기종의 치료 또는 증상 완화용으로 활용될 수 있다. 또한, 흡연 또는 간접흡연에 의한 폐손상 억제 또는 완화용 약학 조성물로 활용될 수 있다.The pharmaceutical composition of the present invention contains the V-atpase B2 protein as an active ingredient, and is useful for treating lung diseases or alleviating symptoms, and in particular, for treating or alleviating symptoms of acute lung injury, pulmonary fibrosis or emphysema among lung diseases. Can be. In addition, it can be utilized as a pharmaceutical composition for suppressing or alleviating lung damage by smoking or secondhand smoke.
도 1은 본 발명의 서열번호 1로 표시되는 V-에이티피아제 B2 단백질의 서열 정보를 보여주는 그림.1 is a diagram showing the sequence information of the V-Etipiase B2 protein represented by SEQ ID NO: 1 of the present invention.
도 2는 본 발명의 실시예 1에서 제조하는 유전자조작 마우스의 Vatpase B2 단백 발현 조절 여부를 확인한 IF staining 결과.Figure 2 is IF staining results confirming whether Vatpase B2 protein expression control of the genetically modified mouse prepared in Example 1 of the present invention.
도 3은 본 발명의 실시예 1에서 제조하는 유전자조작 마우스의 Vatpase B2 단백 발현 조절 여부를 확인한 웨스턴 블로팅(Western blotting) 결과.Figure 3 is a Western blotting (Western blotting) results confirming whether the control of Vatpase B2 protein expression of the genetically modified mouse prepared in Example 1 of the present invention.
도 4는 본 발명의 실시예 2에서 V-atpase B2 TG mice에 유도 급성 폐손상 폐섬유화를 유도한 마우스 모델의 제조 과정을 설명하는 모식도.Figure 4 is a schematic diagram illustrating the manufacturing process of a mouse model induced acute lung injury pulmonary fibrosis induced in V-atpase B2 TG mice in Example 2 of the present invention.
도 5는 본 발명의 실시예 2에서 유도한 급성 폐손상 폐섬유화 마우스 모델의 기관지폐포세척액(BALF)로부터 전체 폐염증세포, 대식세포, 호중구, 호산구, 림프구 수를 측정한 결과를 보여주는 그래프.Figure 5 is a graph showing the results of measuring the total lung inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of acute lung injury pulmonary fibrosis mouse model induced in Example 2 of the present invention.
도 6은 본 발명의 실시예 2에서 유도한 급성 폐손상 폐섬유화 마우스 모델의 폐 조직 손상 정도를 보여주는 H&E 염색 결과.Figure 6 is a result of H & E staining showing the degree of lung tissue damage in a lung lung fibrosis mouse model induced acute lung injury in Example 2 of the present invention.
도 7은 본 발명의 실시예 2에서 유도한 급성 폐손상 폐섬유화 마우스 모델의 폐 조직 내에 콜라겐 축적 정도를 트리그롬 염색 결과.Figure 7 is a trichrome staining result of the degree of collagen accumulation in the lung tissue of a mouse model of acute lung injury pulmonary fibrosis induced in Example 2 of the present invention.
도 8은 본 발명의 실시예 3에서 CS 유도 폐기종 마우스 모델의 기관지폐포세척액(BALF)로부터 전체 폐염증세포, 대식세포, 호중구, 호산구, 림프구 수를 측정한 결과를 보여주는 그래프.Figure 8 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes count from bronchoalveolar lavage fluid (BALF) of CS-induced emphysema mouse model in Example 3 of the present invention.
도 9는 본 발명의 실시예 3에서 CS 유도 폐기종 마우스 모델의 폐조직을 이용하여 lung mechanics(좌: 폐 유순도, 우: 폐탄성)를 측정한 결과.9 is a result of measuring lung mechanics (left: lung purity, right: lung elasticity) using the lung tissue of the CS-induced emphysema mouse model in Example 3 of the present invention.
도 10은 본 발명의 실시예 3에서 CS 유도 폐기종 마우스 모델의 폐포 조직을 H&E 염색으로 관찰한 결과.Figure 10 is the result of observing the alveolar tissue of the CS-induced emphysema mouse model in Example 3 of the present invention by H & E staining.
도 11은 본 발명의 실시예 4에서 293T세포를 이용한 Vatpase B2 protein의 세포 내 유입을 IF staining으로 확인한 결과.Figure 11 shows the results confirmed by IF staining the influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
도 12는 본 발명의 실시예 4에서 293T세포를 이용한 Vatpase B2 protein의 세포 내 유입을 Immunoblotting으로 확인한 결과.12 is a result confirmed by Immunoblotting of the cellular influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
도 13은 본 발명의 실시예 4에서 293T세포 내의 V-atpaseB2 단백의 localization을 이중 면역형광 염색을 이용하여 확인한 결과.Figure 13 shows the results of localization of the V-atpaseB2 protein in 293T cells in Example 4 of the present invention using double immunofluorescence staining.
도 14는 본 발명의 실시예 4에서 마우스 폐질환 모델에서의 V-atpase B2 단백 비강 투여의 효과를 시험하기 위한 과정을 설명하는 모식도.14 is a schematic diagram illustrating a procedure for testing the effect of V-atpase B2 protein nasal administration in a mouse lung disease model in Example 4 of the present invention.
도 15는 본 발명의 실시예 4에서 유도한 마우스 폐질환 모델의 기관지폐포세척액(BALF)로부터 전체 폐염증세포, 대식세포, 호중구, 호산구, 림프구 수를 측정한 결과를 보여주는 그래프.15 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of the mouse lung disease model induced in Example 4 of the present invention.
도 16은 본 발명의 실시예 4에서 유도한 마우스 폐질환 모델의 조직에 Vatpase B2가 유입되었음을 확인한 면역형광염색 결과.16 is an immunofluorescence staining result confirming that Vatpase B2 is introduced into the tissue of the mouse lung disease model induced in Example 4 of the present invention.
도 17은 본 발명의 실시예 4에서 유도한 마우스 폐질환 모델의 조직을 H&E 염색으로 관찰한 결과.Figure 17 is the result of observing the tissue of the mouse lung disease model induced in Example 4 of the present invention by H & E staining.
도 18은 본 발명의 실시예 4에서 유도한 마우스 폐질환 모델의 조직을 Massons trichrome stain 하여 콜라겐 축적 정도를 확인한 결과.18 is a result of confirming the degree of collagen accumulation by massons trichrome stain the tissue of the mouse lung disease model induced in Example 4 of the present invention.
도 19는 본 발명의 실시예 4에서 유도한 마우스 폐질환 모델의 조직에 축적된 콜라겐 섬유의 양을 sircol assay를 통해 확인하고 정량화한 결과(* p<0.05 by Mann-Whitney U test).19 is a result of confirming and quantifying the amount of collagen fibers accumulated in the tissue of the mouse lung disease model induced in Example 4 of the present invention by a sircol assay (* p <0.05 by Mann-Whitney U test).
상기 목적을 달성하기 위하여, 본 발명의 일 실시예에 따른 폐질환 치료용 또는 증상완화용 약학조성물은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함한다.In order to achieve the above object, a pharmaceutical composition for treating or symptomatic pulmonary disease according to an embodiment of the present invention is V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein active ingredient Include as.
상기 V-에이티피아제 B2 단백질은 서열번호 1로 표시되는 hV-atpase B2일 수 있다. The V-Etipiase B2 protein may be hV-atpase B2 represented by SEQ ID NO: 1.
상기 폐질환은 급성폐손상, 폐섬유화증 또는 폐기종일 수 있다.The lung disease may be acute lung injury, pulmonary fibrosis or emphysema.
본 발명의 다른 일 실시예에 따른 흡연 또는 간접흡연에 의한 폐손상 억제 또는 완화용 약학 조성물은, V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함한다.According to another embodiment of the present invention, the pharmaceutical composition for inhibiting or alleviating pulmonary injury by smoking or indirect smoking includes V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. Include.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 V-ATPase를 구성하는 서브유닛 중 하나인 V-ATPase B2 단백질이, 폐(lung) 질환, 특히 급성폐손상(acute lung injury), 폐섬유화증(pulmonary fibrosis), 폐기종(emphysema) 등의 폐 손상 질환의 치료와 증상 완화에 효과가 있음을 동물모델을 이용하여 확인하여 본 발명을 완성하였다.The inventors of the present invention found that one of the subunits constituting V-ATPase is V-ATPase B2 protein, which is a lung disease, especially acute lung injury, pulmonary fibrosis, emphysema. The present invention was completed by using an animal model to confirm that it is effective in treating and relieving symptoms of lung injury diseases.
본 발명의 일 실시예에 따른 약학조성물은, V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하여 폐질환 치료용 또는 증상완화용으로 적용될 수 있다.The pharmaceutical composition according to an embodiment of the present invention may include V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient and may be applied for treating lung disease or alleviating symptoms. have.
상기 V-에이티피아제 B2 단백질은 인간을 포함하는 포유동물의 V-에이티피아제 B2 단백질일 수 있고, 좋게는 인간 V-에이티피아제 B2 단백질일 수 있으며, 구체적으로 서열번호 1과 도 1에 표시되는 511개의 아미노산 서열을 가지는 V-에이티피아제 B2 단백질일 수 있다.The V-Etipiase B2 protein may be a V-Etipiase B2 protein of a mammal, including human, preferably a human V-Etipiase B2 protein, specifically 511 shown in SEQ ID NO: 1 and FIG. V-Etipiaze B2 protein having 4 amino acid sequences.
상기 폐질환은 급성폐손상, 폐섬유화증 또는 폐기종일 수 있고, 특히 산화적 스트레스와 관련된 세포손상이 동반되는 폐질환에 상기 약학조성물이 유용할 수 있다. 또한, 상기 폐질환은 담배연기로 유도된 폐기종일 수 있다.The lung disease may be acute lung injury, pulmonary fibrosis or emphysema, and in particular, the pharmaceutical composition may be useful for lung disease accompanied by cell damage related to oxidative stress. In addition, the lung disease may be emphysema induced by tobacco smoke.
본 발명의 용어 "치료"란, 본 발명에 따른 조성물의 투여로 상기 폐질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 의미한다.The term "treatment" of the present invention means any action that improves or advantageously changes the symptoms of the lung disease by administration of the composition according to the present invention.
본 발명의 용어 "완화"란, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "relaxation" means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
본 발명의 약학조성물은, V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하여 흡연 또는 간접흡연에 의한 폐손상 억제 또는 완화용으로 적용될 수 있다.The pharmaceutical composition of the present invention may be applied to suppress or alleviate lung damage by smoking or secondhand smoke, including V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. .
상기 폐손상은 폐기종 일 수 있다.The lung injury may be emphysema.
상기 약학조성물의 투여는, 위에서 언급한 약리 활성을 나타내는 약제의 투여 방법으로 일반적으로 인정되는 방식에 의할 수 있고, 구체적으로 경구, 비경구 또는 국소 투여, 다른 방법에 의한 전신 투여 등의 방법에 의할 수 있다. 상기 약학조성물은 약학적으로 허용 가능한 담체 내에서의 비강내 투여 또는 기관지 투여의 방법으로 방법으로 환자에게 투여되는 것이 좋다. 또한, 상기 약학조성물은 환자에게 비경구적 투여, 예를 들어 정맥, 동맥, 척추 또는 복강 내 투여를 하기에 적합한 약제학적 조성물로서 제형화 될 수도 있다.Administration of the pharmaceutical composition may be by a method generally accepted as a method of administering the above-mentioned pharmacological activity, and specifically to oral, parenteral or topical administration, systemic administration by other methods, etc. You can. The pharmaceutical composition is preferably administered to the patient by the method of intranasal administration or bronchial administration in a pharmaceutically acceptable carrier. The pharmaceutical composition may also be formulated as a pharmaceutical composition suitable for parenteral administration to a patient, eg, intravenous, arterial, spinal or intraperitoneal.
상기 약학조성물은, 바람직하게는 정확한 용량의 투여에 적합한 단위 제제로서, 예를 들면, 알약, 캡슐, 분말, 액상, 현탁액 등과 같은 고체, 반고체 또는 액체 제제의 형태로 사용될 수 있다. 상기 약학조성물은 통상적인 약학적 담체 또는 부형제를 포함하며, 게다가 다른 의약제제, 약학제제, 담체, 부가제 등을 포함할 수 있다. 상기 부형제는 예를 들면, 인간 혈청 알부민 또는 플라즈마 단백질과 같은 다른 단백질을 포함할 수 있으나 이에 한정되는 것인 아니다.The pharmaceutical composition may be used in the form of a solid, semi-solid or liquid formulation, preferably for example as a pill, capsule, powder, liquid, suspension, etc., suitable for the administration of the correct dose. The pharmaceutical composition may include conventional pharmaceutical carriers or excipients, and may also include other pharmaceutical agents, pharmaceutical agents, carriers, additives, and the like. The excipient may include, but is not limited to, other proteins such as, for example, human serum albumin or plasma proteins.
바람직하게 상기 약학조성물은 상기 폐 질환의 치료시 기존의 치료제와 동시에 또는 별도로 투여되는 것일 수 있다.Preferably the pharmaceutical composition may be administered at the same time or separately from the existing therapeutic agent in the treatment of the lung disease.
상기 투여되는 약학조성물 또는 이의 제형물은, 치료받고 있는 피검자에게서 바람직한 효과를 달성하는데 효과적인 양으로 유효성분의 양을 함유하며, 상기 환자에게 투여되는 활성성분의 양은, 피검자의 특성, 병의 심각성, 투여의 방식, 의사의 판단 등에 의하여 달라지나, 비교적 저농도의 단백질을 투여하는 것이 좋다. 예를 들어, 상기 약학조성물은 1 x 105 IU/사람 내지 9 x 106 IU/사람일 수 있다.The administered pharmaceutical composition or formulation thereof contains an amount of the active ingredient in an amount effective to achieve a desired effect in the subject being treated, and the amount of the active ingredient administered to the patient is determined by the characteristics of the subject, the severity of the disease, Depending on the mode of administration and the judgment of the doctor, it is better to administer a relatively low concentration of protein. For example, the pharmaceutical composition may be 1 x 10 5 IU / person to 9 x 10 6 IU / person.
다른 하나의 양태로, 본 발명은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 폐질환 예방 또는 개선용 식품조성물을 제공한다.In another embodiment, the present invention provides a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
본 발명에서, 용어 "V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2)", "폐질환"에 대한 설명은 전술한 바와 같다. In the present invention, the description of the term "V-atpase B2 (Vacuolar-type H + -ATPase B2)", "pulmonary disease" is as described above.
본 발명에서의 용어, "개선"은 V-에이티피아제 B2을 유효성분으로 포함하는 식품조성물을 이용하여 예방 또는 치료되는 폐질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 되는 모든 행위를 말한다.As used herein, the term " improvement " refers to any action that improves or benefits the suspicion of a disease and the onset of a pulmonary disease to be prevented or treated using a food composition comprising V-Etipiase B2 as an active ingredient.
본 발명의 용어 "예방"이란, 본 발명에 따른 조성물의 투여로 상기 폐질환의 발병을 억제 또는 지연시키는 모든 행위를 의미한다The term "prevention" of the present invention means any action that inhibits or delays the onset of the lung disease by administration of the composition according to the present invention.
구체적으로, 본 발명의 V-에이티피아제 B2을 폐질환의 예방 또는 개선을 목적으로 식품 조성물에 첨가할 수 있다.Specifically, V-Etipiaze B2 of the present invention can be added to food compositions for the purpose of preventing or ameliorating lung diseases.
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함할 수 있으며, 본 발명의 V-에이티피아제 B2을 첨가할 수 있는 식품의 종류에는 별다른 제한이 없으며, 예를 들어 각종 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있다.The food composition of the present invention may include the form of pills, powders, granules, acupuncture, tablets, capsules or liquids, etc., and there is no particular limitation on the kind of foods to which V-Etipiaze B2 of the present invention may be added. Examples include various beverages, gums, teas, vitamin complexes, and dietary supplements.
상기 식품 조성물에는 V-에이티피아제 B2 이외에도 다른 성분을 추가할 수 있으며, 그 종류는 특별히 제한되지 않는다. 예를 들어, 통상의 식품과 같이 여러 가지 생약 추출물, 식품학적으로 허용가능한 식품보조첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있으며, 이에 제한되지 않는다.In addition to the V-Etipia agent B2, other ingredients may be added to the food composition, and the kind thereof is not particularly limited. For example, various herbal extracts, food acceptable food additives, natural carbohydrates, and the like may be included as additional ingredients, such as conventional foods, but are not limited thereto.
본 발명에서 용어, "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.In the present invention, the term "food supplement" means a component that can be added to food supplements, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, but is not limited to the kind of food additives of the present invention by the above examples.
상기 천연 탄수화물의 예는 포도당, 과당 등의 단당류; 말토스, 수크로스 등의 이당류; 및 덱스트린, 시클로덱스트린 등의 다당류와, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 있으며, 상기한 것 이외의 향미제로서 천연 향미제(타우마틴 등), 스테비아 추출물(레바우디오시드 Ａ, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, and as flavoring agents other than those described above, natural flavoring agents (such as taumartin), stevia extract (rebaudioside A, glycyr) Higin and the like) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에서 사용된 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능성 식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The food composition of the present invention may include a health functional food. As used herein, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, functional means to obtain a useful effect for health use such as nutrient control or physiological action on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.
유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 V-에이티피아제 B2는 0.1 내지 50 중량%, 바람직하게는 1 내지 10 중량%의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the V-Etipiaze B2 of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, in the manufacture of food, but is not limited thereto. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount may be used below the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes and the like, and include all of the dietary supplements in the conventional sense.
다른 하나의 양태로, 본 발명은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 개체에 투여하는 단계를 포함하는, 폐질환의 치료 또는 증상완화 방법을 제공한다. In another embodiment, the present invention provides a method of treating or alleviating pulmonary disease, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. do.
본 발명에서, 용어 "V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2)", "폐질환"에 대한 설명은 전술한 바와 같다. In the present invention, the description of the term "V-atpase B2 (Vacuolar-type H + -ATPase B2)", "pulmonary disease" is as described above.
본 발명에서 용어 "개체"란 폐질환이 발생하였거나 폐질환이 발생한 인간을 포함한 모든 동물을 의미한다. 소, 돼지, 양, 닭, 개, 인간 등을 포함하는 포유동물, 조류 등을 포함하며, 본 발명의 상기 단백질에 의해 폐질환이 치료되거나, 증상이 완화되는 개체는 제한없이 포함한다.In the present invention, the term "individual" means all animals including a human having a lung disease or a lung disease. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung diseases are treated or alleviated by the protein of the present invention.
다른 하나의 양태로, 본 발명은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 개체에 투여하는 단계를 포함하는, 흡연 또는 간접흡연에 의한 폐손상의 억제 또는 완화방법을 제공한다.In another embodiment, the present invention provides a method for inhibiting lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. Or provide mitigation.
본 발명에서, 용어 "V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2)", "폐손상"에 대한 설명은 전술한 바와 같다. In the present invention, the description of the term "V-atpase B2 (Vacuolar-type H + -ATPase B2)", "lung damage" is as described above.
본 발명에서 용어 "개체"란 폐손상이 발생하였거나 폐손상이 발생한 인간을 포함한 모든 동물을 의미한다. 소, 돼지, 양, 닭, 개, 인간 등을 포함하는 포유동물, 조류 등을 포함하며, 본 발명의 상기 단백질에 의해 폐손상이 억제되거나 완화되는 개체는 제한없이 포함한다.In the present invention, the term "individual" means all animals including a human in which lung injury has occurred or a lung injury has occurred. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung injury is inhibited or alleviated by the protein of the present invention.
다른 하나의 양태로, 본 발명은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 폐질환의 치료 또는 증상을 완화하기 위한 의약품의 제조에 사용하기 위한 용도를 제공한다.In another aspect, the present invention provides the use of the V- atpase B2 (Vacuolar-type H + -ATPase B2) protein for use in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. to provide.
본 발명에서, 용어 "V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2)", "폐질환"에 대한 설명은 전술한 바와 같다. In the present invention, the description of the term "V-atpase B2 (Vacuolar-type H + -ATPase B2)", "pulmonary disease" is as described above.
다른 하나의 양태로, 본 발명은 V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 흡연 또는 간접흡연에 의한 폐손상을 억제 또는 완화하기 위한 의약품의 제조에 사용하기 위한 용도를 제공한다.In another embodiment, the present invention uses the V-atpase B2 (Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It provides a use for.
본 발명에서, 용어 "V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2)", "폐손상"에 대한 설명은 전술한 바와 같다.In the present invention, the description of the term "V-atpase B2 (Vacuolar-type H + -ATPase B2)", "lung damage" is as described above.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.
실시예Example 1: 폐포(type II  1: alveoli (type II pneumocytepneumocyte )에 )on VatpaseVatpase B2 단백이 과발현되는 유전자조작 마우스의 제조 Preparation of engineered mice overexpressing B2 protein
폐질환인 급성 폐손상(acute lung injury), 폐섬유화증(lung fibrosis), 폐기종(cigarette smoke induced emphysema)에 대한 동물모델에서의 Vatpase B2 단백의 치료 효과를 검증하기 위하여, 폐포(type II pneumocyte)에 Vatpase B2 단백이 과발현되며, 과발현시기를 DOXY로 조절할 수 있는 double transgenic mice를 제작하였다. 구체적으로, hVatpase B2 construct는 RT-PCR을 이용하여 TRE-tight vector에 1x flag sequene가 포함된 VATPaseB2 cDNA construct을 subcloning하여 제작하였으며, V-atpase B2 단백발현을 Flag Ab.로 정확히 detection할 수 있도록 하였다. 두 가지 construct를 C57/BL6 마우스의 난자에 주입하여 교배하여 만든 double transgenic mice를 이하 실험에 적용하였다.To verify the therapeutic effect of Vatpase B2 protein in animal models on acute lung injury, lung fibrosis, and cigarette smoke induced emphysema, type II pneumocyte Vatpase B2 protein was overexpressed, and double transgenic mice were prepared to control the overexpression time by DOXY. Specifically, hVatpase B2 construct was constructed by subcloning the VATPaseB2 cDNA construct containing 1x flag sequene in the TRE-tight vector using RT-PCR, and it was possible to accurately detect V-atpase B2 protein expression as Flag Ab. . Double transgenic mice, which were made by injecting two constructs into the eggs of C57 / BL6 mice, were applied to the following experiments.
제조된 유전자조작 마우스는 DOXY로 Vatpase B2 단백 발현이 조절되는지 여부를 Flag Ab.를 이용하여 IF staining과 Western blotting으로 확인했고 그 결과를 각각 도 2와 도 3에 나타내었다.The prepared genetically modified mice were confirmed by IF staining and Western blotting using Flag Ab. Whether Vatpase B2 protein expression was regulated by DOXY, and the results are shown in FIGS. 2 and 3, respectively.
상기 도 2와 도 3을 참조하면, 이하 실험 진행을 위해서 제조된 유전자조작 마우스는 DOXY 도입 유무에 따라서 flag tagged hVatpase B2 단백이 잘 발현되는 것을 확인할 수 있었다.Referring to FIG. 2 and FIG. 3, the genetically engineered mice prepared for the following experiments were well expressed in the flag tagged hVatpase B2 protein depending on whether DOXY was introduced.
실시예Example 2: 유전자조작 마우스를 이용한 유도 급성  2: Acute induction with engineered mice 폐손상Lung injury 마우스 모델에서 과발현된 V-atpase B2의 효과 실험 Experimental Effects of Overexpressed V-atpase B2 in a Mouse Model
1) 실험과정1) Experiment process
위에서 제조한 V-atpase B2 TG mice의 기관지(intratracheal) 내로 블레오마이신(Bleomycin)을 투여하여 실험동물에 급성 폐손상/폐섬유화증을 유도하였다. 구체적으로, 아래 도 4에 나타난 것처럼 블레오마이신 투여 5일 전에 Doxy water를 투여하여 V-atpase B2의 과발현을 유도하고 블레오마이신을 투여한 후 14일째에 희생하여 기관지 폐포세척액(BALF), 기관지 및 페소 조직 등을 확보하여 이후 실험을 진행했다.Acute lung injury / pulmonary fibrosis was induced in experimental animals by administering bleomycin into the bronchus (intratracheal) of V-atpase B2 TG mice prepared above. Specifically, as shown in FIG. 4 below, 5 days before bleomycin administration, Doxy water was administered to induce overexpression of V-atpase B2 and sacrifice at 14 days after bleomycin administration to bronchoalveolar lavage fluid (BALF), bronchus and pesos. After the organization was conducted, the experiment was conducted.
1. 대조군: Doxy(-) + Bleomysin(3 mg/kg) 투여군1.Control group: Doxy (-) + Bleomysin (3 mg / kg) group
2. 실험군: Doxy(+) + Bleomysin(3 mg/kg) 투여군2. Experimental group: Doxy (+) + Bleomysin (3 mg / kg) group
2) 기관지 폐포세척액(BALF) 분석2) Bronchoalveolar lavage fluid (BALF) analysis
마우스의 기관지 폐포 세척액(bronchoalveolar labage fluid; BALF)은 37℃에서 주사기로 PBS 1000 μl를 상기 질환이 유도된 마우스의 기도에 반복하여 주의 깊게 넣고 흡입시킴으로써 수득하였으며. 2% FCS(fetal calf serum)을 함유한 HBSS(Hank’s balanced salt soulution)으로 세척하고 lysis buffer를 이용하여 적혈구를 제거한 후, 원심분리하여 상층액을 따로 모았다.Bronchoalveolar labage fluid (BALF) in mice was obtained by carefully inhaling 1000 μl of PBS into the airway of the disease-induced mice repeatedly with a syringe at 37 ° C. After washing with Han's balanced salt soulution (HBSS) containing 2% FCS (fetal calf serum), erythrocytes were removed using lysis buffer, and the supernatants were collected separately by centrifugation.
BALF에 함유된 전체 폐염증세포를 계산한 후, 상기 수득된 폐포 세척액에 포함된 각종 면역 세포를 사이토스핀 챔버(cytospin chamber)를 사용하여 1000 rpm에서 슬라이드 글라스 위에 도말하고 Diff-Quik로 염색한 다음, 대식세포, 림프구, 중성구 및 호산구의 세포수를 400배 비율의 광학현미경(Nicon, Japan)으로 관찰하였으며, 500개의 세포를 무작위로 선택하여 각각의 세포수를 계산하였고, 그 결과를 도 5에 나타내었다.After counting the total pulmonary inflammatory cells contained in BALF, various immune cells contained in the obtained alveolar lavage fluid were plated on a slide glass at 1000 rpm using a cytospin chamber and stained with Diff-Quik. The number of cells of macrophages, lymphocytes, neutrophils and eosinophils was observed under an optical microscope (Nicon, Japan) at a 400-fold ratio, and 500 cells were randomly selected to calculate the respective cell numbers. Indicated.
도 5의 결과를 참조하면, Doxy (+), 즉 VAtpase B2의 과발현이 블레오마이신 투여로 인한 폐 염증 발생을 감소시킨다는 점을 보여주고 있으며, 특히 폐손상에서 가장 중요한 역할을 하는 호중구의 수를 감소시킴을 확인하였다.5 shows that overexpression of Doxy (+), ie, VAtpase B2, reduces the incidence of pulmonary inflammation due to bleomycin administration, in particular reducing the number of neutrophils that play the most important role in lung injury. Confirmed.
3) H&E 염색을 통한 폐손상 평가3) Evaluation of lung damage through H & E staining
희생된 마우스 폐조직은 4% 파라포름알데하이드로 고정시키고 파라핀 블록으로 제조된 후 4 μm 간격으로 절단하여 조직 샘플로 제조하였다. 조직 샘플은deparaffinization 과정을 거친 후 Hematocyline 200μl로 1분간 염색하고 10분간 수세하였다. 이어서, Eosin 200μl로 1분간 염색하였고 10분간 수세 한 후 Dehydration 과정을 거쳐서 마운팅하여 관찰하였으며, 그 결과를 도 6에 나타내었다.Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 μm intervals to prepare tissue samples. The tissue samples were deparaffinized and stained with 200 μl of hematocyline for 1 minute and washed with water for 10 minutes. Subsequently, 1 minute staining with 200 μl of Eosin was followed by washing with water for 10 minutes, followed by mounting through a dehydration process. The results are shown in FIG. 6.
상기 도 6을 참조하면, Doxy(+)로 V-atpase B2 단백이 과발현된 실험군의 샘플들에서 조직 손상이 현저하게 줄어든 것을 확인할 수 있었다.Referring to FIG. 6, tissue damage was significantly reduced in samples of the experimental group overexpressed with Doxy (+) V-atpase B2 protein.
4) 트리크롬 염색(Trichrome stain)을 이용한 콜라겐 형성 억제능 평가4) Evaluation of collagen formation inhibitory activity using trichrome stain
희생된 마우스 폐조직은 4% 파라포름알데하이드로 고정시키고 파라핀 블록으로 제조된 후 4 μm 간격으로 절단하여 조직 샘플을 제조하였다. 조직 샘플은deparaffinization 과정을 거친 후 흐르는 물에 수세하고 Bouin solution을 200μl 처리하여 56도의 인큐베이터에서 1시간 매염하였다. 흐르는 물에 수세하고 Weigrt iron hematoxylin 200μl로 상온에서 10분간 염색을 진행하였다. Bibrich scarlet 200μl로 상온에서 10분간 염색한 후 수세하고, Phosphomoybdic-phosphotungstic acid를 상온에서 10분간 처리한 후, Aniline blue 200μl로 상온에서 2시간 염색하는 과정을 거쳤다. 이렇게 처리된 조직 샘플은 흐르는 물에 수세하고 Dehydration 과정을 거친 후 마운팅하여 관찰하였으며, 그 결과를 도 7에 나타내었다.Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 μm intervals to prepare tissue samples. Tissue samples were washed with running water after deparaffinization and treated with 200 μl of Bouin solution for 1 hour in a 56 ° incubator. It was washed with running water and stained with Weigrt iron hematoxylin 200μl at room temperature for 10 minutes. Bibrich scarlet 200μl stained at room temperature for 10 minutes, washed with water, and treated with Phosphomoybdic-phosphotungstic acid at room temperature for 10 minutes, and then stained with Aniline blue 200μl at room temperature for 2 hours. The treated tissue samples were washed with running water, dehydrated, and then mounted, and the results are shown in FIG. 7.
도 7을 참조하면, Doxy(+)로 V-atpase B2 단백이 과발현된 실험군의 샘플들에서 파란색으로 나타나는 콜라겐 축적이 현저하게 적게 나타나는 것을 확인할 수 있었으며, 이는 V-atpase B2 단백의 과발현이 콜라겐 섬유 축적에 의한 폐섬유화를 억제할 수 있음을 보여주는 결과이다. Referring to FIG. 7, it was confirmed that collagen accumulation in blue was significantly decreased in samples of the experimental group overexpressing V-atpase B2 protein with Doxy (+), which indicates that the overexpression of V-atpase B2 protein was caused by collagen fiber. The results show that lung fibrosis due to accumulation can be suppressed.
실시예Example 3: 유전자조작 마우스를 이용한 CS(cigarette smoke) 유도 폐기종(emphysema) 마우스 모델에서 과발현된 V-atpase B2의 효과 실험 3: Experimental Effects of V-atpase B2 Overexpressed in a Mouse Model of Cigarette Smoke Induced Emphysema
1) 실험과정1) Experiment process
위의 실시예 1에서 제조한 유전자 조작 마우스(Vatpase B2 과발현 mice)를 6개월 동안 담배 연기에 하루 3시간씩 주 5회 노출시켜 CS(cigarette smoke) 유도 폐기종(emphysema) 마우스 모델을 제작하였다.Genetically modified mice (Vatpase B2 overexpressing mice) prepared in Example 1 above were exposed to cigarette smoke five times a week for three months for five months to prepare a CS (cigarette smoke) induced emphysema mouse model.
구체적으로, Vatpase B2 과발현 mice를 6개월간 plexiglass chamber(16x 25x 16cm)에서 smoking tester system (ThreeShineCom, Daejeon, Korea)을 이용하여 CS에 노출시켰다. CS에 노출시킨 첫날로부터 희생 전까지 Doxy containing water(50mg/ml)를 투여하여 V-atpase B2의 과발현을 유도하였고, CS는 3R4F burning reference cigarette를 이용하였으며, 하루 3시간씩 주 5회, 6개월 동안 담배 연기에 노출시켰다.Specifically, Vatpase B2 overexpressing mice were exposed to CS in a plexiglass chamber (16x 25x 16cm) for 6 months using a smoking tester system (ThreeShineCom, Daejeon, Korea). Overexpression of V-atpase B2 was induced by the administration of Doxy containing water (50mg / ml) from the first day of exposure to CS. CS was used with a 3R4F burning reference cigarette for 5 hours, 3 times a week, for 6 months. Exposure to tobacco smoke.
CS 노출 없이 air에만 노출한 Vatpase B2 과발현 실험군(VATPaseB2 air)과 Vatpase B2 과발현을 유도하지 않고 CS만 위와 동일한 조건으로 노출한 실험군(WT_CS)을 대조군으로 적용하였다.VaTPase B2 overexpression test group (VATPaseB2 air) exposed to air only without CS exposure and Vatpase B2 overexpression test group (WT_CS) exposed under the same conditions as above without inducing Vatpase B2 overexpression were applied as controls.
이렇게 CS 유도 폐기종(emphysema) 마우스 모델을 제조한 후, 희생하여 기관지폐포세척액(BALF), 기관지 및 폐포 조직 등을 확보하여 이후 실험을 진행하였다.The CS-induced emphysema mouse model was prepared, and then sacrificed to obtain bronchoalveolar lavage fluid (BALF), bronchial and alveolar tissues, and then experiments were performed.
2) 기관지 폐포세척액(BALF) 분석2) Bronchoalveolar lavage fluid (BALF) analysis
위의 실험동물로부터 얻은 기관지폐포세척액(BALF)에서 염증 세포들의 분획을 위에서 설명한 것과 동일한 방법으로 측정하였고 그 결과를 도 8에 나타내었다.Fractions of inflammatory cells in the bronchoalveolar lavage fluid (BALF) obtained from the above experimental animals were measured in the same manner as described above, and the results are shown in FIG. 8.
상기 도 8을 참조하면, 기관지폐포세척액에서 염증 세포들의 분획을 측정한 결과, 측정된 대식세포, 호산구, 중성구, 림프구 수치 중에서 Vatpase B2 과발현의 경우 대식세포, 호중구의 수가 흡연군에 비해 현저히 감소되는 것으로 나타났다. CS로 인한 폐기종의 특징은 폐포 내 대식세포와 호중구의 증가라는 점을 고려하면 위의 실험 결과는 VatpaseB2과발현이 CS로 인한 폐기종 발생을 치료 또는 예방하는 결과라 생각된다.Referring to FIG. 8, as a result of measuring the fraction of inflammatory cells in bronchoalveolar lavage fluid, the number of macrophages and neutrophils was significantly reduced in the case of Vatpase B2 overexpression among measured macrophages, eosinophils, neutrophils and lymphocytes. appear. Considering that CS is characterized by an increase in alveolar macrophages and neutrophils, the above results suggest that VatpaseB2 overexpression may be the result of treating or preventing CS-induced emphysema.
3) 폐 조직의 기계적 특성 평가3) Evaluation of mechanical properties of lung tissue
폐기종은 폐유순도(static compliance)증가 및 탄성(elastance)의 감소를 특징으로 하므로, Flexi-vent기기를 이용하여 희생된 마우스 폐조직의 lung mechanics를 측정하였고, 그 결과를 도 9에 나타내었다.Emphysema is characterized by increased lung compliance (static compliance) and reduced elasticity (elastance), the lung mechanics of the sacrificed mouse lung tissue was measured using a Flexi-vent device, the results are shown in FIG.
상기 도 9를 참조하면, CS로 인해 증가된 폐 유순도 및 감소된 폐탄성이 Vatpase B2과발현으로 정상화되는 결과를 보여주었다.Referring to FIG. 9, the increased lung purity and decreased waste elasticity due to CS were shown to be normalized by Vatpase B2 overexpression.
4) H&E 염색을 통한 폐손상 평가4) Evaluation of lung damage through H & E staining
CS(cigarette smoke) 유도 폐기종(emphysema) 마우스 모델의 폐조직을 이용하여 위에서 설명한 것과 동일하게 H&E(hematoxylin and eosin) 염색 관찰을 실시하고 그 결과를 도 10에 나타내었다.Observations of hematoxylin and eosin (H & E) staining were performed using lung tissue of a cigarette smoke induced emphysema mouse model as described above, and the results are shown in FIG. 10.
상기 도 10을 참조하면, Doxy(+)/CS, 즉 Vatpase B2 과발현쥐가 흡연을 한 경우에, 흡연으로 인한 폐기종(air space enlargement(중간 칸그림, Doxy(-) CS)이 나타나지 않는 정상 폐포의 모습을 나타낸다는 점을 확인할 수 있었다.Referring to FIG. 10, Doxy (+) / CS, that is, normal alveoli without air space enlargement (Doxy (-) CS) does not appear when smoking Vatpase B2 overexpressing mice. It was confirmed that the appearance of.
이상의 실험결과들을 종합하면, Vatpase B2을 과발현시킬 경우, CS로 유도되는 동물 폐기종이 현저히 감소됨을 확인하였으며, VatpaseB2가 CS에 대항하는 폐보호 작용이 있고, 폐기종의 예방 또는 치료 용도로 활용될 수 있음을 보여주는 결과로 생각된다.In conclusion, it was confirmed that overexpression of Vatpase B2 significantly reduced animal emphysema induced by CS. VatpaseB2 has a pulmonary protective action against CS and can be used for the prevention or treatment of emphysema. Is thought to be the result showing.
실시예Example 4: 마우스 폐질환 모델에서의 V- 4: V- in mouse lung disease model atpaseatpase B2 단백 비강 투여와 급성  B2 protein nasal administration and acute 폐lungs 손상/섬유화증 치료효과Damage / Fibrosis Treatment Effect
1) 293T세포를 이용한 Vatpase B2 protein의 세포 내 유입 시험.1) Invasion test of Vatpase B2 protein using 293T cells.
<Immunofluorescence stain 실험><Immunofluorescence stain experiment>
Vatpase B2 단백(ORIGENE, USA, flag tagged recombinant protein, 56K.D.)을 293T세포에 투여하여 상기 단백이 endocytosis과정으로 세포에 유입됨을 아래와 같은 방법으로 확인하였다.Vatpase B2 protein (ORIGENE, USA, flag tagged recombinant protein, 56K.D.) was administered to 293T cells to confirm that the protein was introduced into the cell during the endocytosis process as follows.
293T세포 (ATCC 번호: CRL-3216)를 37℃, 5% CO2의 습윤 분위기 하에서 10 % (v/v) 열-불활성화된 태아 소혈청, 10,000U/mL Penicillin, 10,000 μg/mL Streptomycin을 포함하는 Dulbecco's modified Eagle's 배지에서 배양하였고, 배양된 293T세포에 Vatpase B2 단백을 2 ng/well로 투여한 후 위와 동일한 조건에서 17 시간 배양한 후, IF staining을 실시하였다.293T cells (ATCC No .: CRL-3216) were treated with 10% (v / v) heat-inactivated fetal bovine serum, 10,000 U / mL Penicillin, 10,000 μg / mL Streptomycin at 37 ° C. in a humidified atmosphere of 5% CO 2 . Incubated in Dulbecco's modified Eagle's medium, and the cultured 293T cells were administered with Vatpase B2 at 2 ng / well, followed by incubation for 17 hours under the same conditions, followed by IF staining.
구체적으로, 배양액을 제거하고 DPBS로 수세한 후 4% 파라포름알데히드로 15분간 고정시킨 후 깨끗하게 수세하고 Triton X-100을 사용하여 permiabilization 과정을 수행하였다. DPBS로 수세하고 5% goat normal serum 으로 상온에서 1시간 블로킹하고 1차 안티바디 (anti-flag)를 10ug/ml로 블로킹 용액에 희석하여 4℃에서 17시간 인큐베이션 시켰다. 이어서 2차 안티바디 (Anti-Rabbit FITC)를 1:1000으로 희석하여 처리하고 빛을 차단한 채로 상온에서 1시간동안 인큐베이션 시키고 DAPI로 핵 염색을 수행하였다. 이렇게 처리된 샘플은 형광 염색용 마운팅 용액으로 마운팅하고 형광현미경으로 결과를 확인했으며 도 11에 나타냈다.Specifically, the culture medium was removed, washed with DPBS, fixed with 15% of 4% paraformaldehyde for 15 minutes, washed with water, and then subjected to permiabilization using Triton X-100. Washed with DPBS and blocked for 1 hour at room temperature with 5% goat normal serum, the first anti-flag was diluted in a blocking solution at 10ug / ml and incubated at 4 ℃ 17 hours. Subsequently, the secondary antibody (Anti-Rabbit FITC) was diluted 1: 1000, treated, incubated at room temperature for 1 hour while blocking light, and nuclear stained with DAPI. The samples thus treated were mounted with a mounting solution for fluorescence staining, and the results were confirmed with a fluorescence microscope, and are shown in FIG. 11.
상기 도 11을 참조하면, 방법으로 293T세포의 세포질에 Vatpase B2 단백이 잘 도입되어 있는 것을 확인할 수 있었다.Referring to FIG. 11, it was confirmed that Vatpase B2 protein was well introduced into the cytoplasm of 293T cells by the method.
<Immunoblotting 실험><Immunoblotting Experiment>
위의 실험에서 사용한 것과 동일한 293T세포에 Vatpase B2 단백(2 μg/ml)을 도입한 후, lysis buffer를 가하여 세포용해물을 제조하고, 단백질을 추출하였다. 10% SDS-PAGE를 이용하여 이 단백질을 전개하고, 5% 탈지유(skim milk)로 브로킹한 후 1차 항체로 anti-FLAG 항체(SIGMA, 1:5000)를, 2차 항체로 anti-rabbit IgG(1:5000)을 이용하여 반응시킨 후 detecting 과정을 수행하여, 그 결과를 하기 도 12에 나타내었다. 양성대조군으로 베타액틴을 적용하였다.Vatpase B2 protein (2 μg / ml) was introduced into the same 293T cells as used in the above experiments, lysis buffer was added to prepare cell lysates, and proteins were extracted. The protein was developed using 10% SDS-PAGE, broken with 5% skim milk and then anti-FLAG antibody (SIGMA, 1: 5000) as the primary antibody and anti-rabbit as the secondary antibody. After the reaction using IgG (1: 5000) to perform a detecting process, the results are shown in Figure 12 below. Beta actin was applied as a positive control.
상기 도 12를 참조하면, Vatpase B2 단백이 도입된 실험군(+)에서 Vatpase B2 단백의 크기에 해당하는 56.3kDa 밴드가 대조군(-)과 비교하여 진하게 나타나서, 실험군의 경우, Vatpase B2 단백이 잘 도입되었다는 것을 확인할 수 있었다.Referring to FIG. 12, in the experimental group (+) in which the Vatpase B2 protein was introduced, 56.3kDa band corresponding to the size of the Vatpase B2 protein appeared darker than the control group (-), and in the case of the experimental group, the Vatpase B2 protein was well introduced. It could be confirmed.
< double IF staining 을 이용한 세포 내 Vatpase B2 단백의 localization 확인 실험 > < Confirmation of localization of intracellular Vatpase B2 protein by double IF staining >
293 T cell에 2 μg/ml로 투여한 V-atpaseB2 단백의 세포 내 localization을 확인하기 위하여, FITC-flag 과 PE-LAMP-1으로 이중 면역형광 염색한 V-atpase B2 단백이 도입된 293 T cell을 형광현미경을 이용하여 관찰하였고 그 결과를 도 13에 나타내었다.In order to confirm intracellular localization of V-atpaseB2 protein administered at 2 μg / ml to 293 T cells, 293 T cell into which V-atpase B2 protein was introduced by double immunofluorescence staining with FITC-flag and PE-LAMP-1 Was observed using a fluorescence microscope and the results are shown in FIG.
상기 도 13을 참조하면, PE-LAMP-1으로 표시된 리소좀(lysosome) 위치에 V-atpase B2 단백에 의한 초록색 형광의 발현이 일부 겹쳐서 나타나 Vatpase B2가 세포내 lysosome에 주로 존재함을 확인할 수 있었다.Referring to FIG. 13, green fluorescence expression by the V-atpase B2 protein was partially overlapped at the lysosome position indicated by PE-LAMP-1, indicating that Vatpase B2 was mainly present in the intracellular lysosome.
2) 블레오마이신 유도 폐손상 / 섬유화증 모델에서 Vatpase B2 단백의 치료효과 검증 시험. 2) Validation test of Vatpase B2 protein in bleomycin- induced lung injury / fibrosis model .
<실험동물의 준비 및 실험방법><Preparation and Experiment Method of Experimental Animal>
웅성 C57BL/6 마우스를 오리엔탈 바이오에서 분양받아 실험실에서 5일 동안의 적응기간을 거친 후 이후 블레오마이신(Bleomycine)으로 폐손상을 유도한 후 Vatpase B2 단백의 치료효과를 확인하는 실험에 적용하였다(도 14 참조).Male C57BL / 6 mice were distributed in Oriental Bio, followed by a five-day acclimation period in the laboratory, followed by induction of pulmonary damage with bleomycine and then applied to experiments confirming the therapeutic effect of Vatpase B2 protein (FIG. 14).
적응기간을 거친 마우스는 블레오마이신을 기도 내로 투여하여 급성 폐손상, 섬유화증을 유도하고, 4일 경과 후부터 7일 경과 후까지 비강을 통해서 V-atpase B2 단백을 각각 500 ng 투여하였다.The mice undergoing the adaptation period were administered bleomycin into the airways to induce acute lung injury and fibrosis, and 500 ng of V-atpase B2 protein was administered through the nasal cavity from 4 days to 7 days.
1. 정상대조군(PBS, Phosphate buffer saline 투여, n=8, CTL)Normal control group (PBS, Phosphate buffer saline administration, n = 8, CTL)
2. 폐손상유도군(블레오마이신 3 mg/kg 투여, n=8, BLM)2. Lung induction group (bleomycin 3 mg / kg administration, n = 8, BLM)
3. 실험군(블레오마이신 3 mg/kg, V-atpase B2 단백 0.5 mg/100 μl, n=8, BLM+B2)3. Experimental group (bleomycin 3 mg / kg, V-atpase B2 protein 0.5 mg / 100 μl, n = 8, BLM + B2)
4. B2 단백투여군(V-atpase B2 단백 0.5 mg/100 μl, n=8, B2)4.B2 protein administration group (V-atpase B2 protein 0.5 mg / 100 μl, n = 8, B2)
블레오마이신을 투여한 후 2주가 경과한 시점에서 마우스를 희생하고 기관지 폐포세척액(BALF), 기관지 및 폐포 조직 등을 확보하여 이후 실험을 진행했다.Two weeks after the administration of bleomycin, mice were sacrificed, and bronchial alveolar lavage fluid (BALF), bronchial and alveolar tissues were obtained, and then experiments were performed.
< 기관지 폐포세척액(BALF) 분석>Bronchoalveolar lavage fluid analysis
위의 실험동물로부터 얻은 기관지폐포세척액(BALF)에서 염증 세포들의 분획을 측정하였다. BALF을 얻고 이로부터 폐염증세포, 대식세포, 호중구, 호산구, 림프구 수 등을 측정하는 방법은 위에서 설명한 방법과 동일한 방법을 적용하였고, 그 결과를 아래 도 15에 나타내었다.Fractions of inflammatory cells were measured in bronchoalveolar lavage fluid (BALF) obtained from the above experimental animals. Obtaining BALF and measuring the number of pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes and the like was applied to the same method as described above, the results are shown in Figure 15 below.
상기 도 15를 참조하면, v-atpase B2 단백 투여로 인하여 유도된 폐손상으로 인한 염증이 완화 또는 억제되는 것을 확인할 수 있었고, 구체적으로 전체 폐염증세포, 호중구, 대식세포의 수가 폐손상유도군에 비해 실험군에서 현저히 저하됨을 관찰할 수 있었다.Referring to FIG. 15, it was confirmed that inflammation due to lung injury induced by v-atpase B2 protein administration was alleviated or suppressed. Specifically, the number of total pulmonary inflammatory cells, neutrophils, and macrophages was induced in the lung injury-inducing group. Compared with the experimental group was significantly reduced.
< 마우스 폐조직에서 세포내 v-atpase B2 단백 유입 확인>Intracellular v-atpase B2 Protein Influx in Mouse Lung Tissue
희생된 마우스 폐조직은 4% 파라포름알데하이드로 고정시키고 파라핀 블록으로 제조된 후 4 μm 간격으로 절단하여 조직 샘플을 제조하고 immunofluoroscence stain을 진행하였다. 조직 샘플은 deparaffinization 과정을 거친 후 흐르는 물에 수세하고 Triton X-100을 사용하여 permiabilization 과정을 수행했다. DPBS로 수세하고 5% goat normal serum으로 상온에서 1시간 블로킹하고 1차 안티바디 (anti-flag)를 10ug/ml로 블로킹 용액에 희석하여 4℃에서 17시간 인큐베이션 시켰으며, 이어서 2차 안티바디 (Anti-Rabbit FITC)를 1:1000으로 희석하여 처리하고 빛을 차단한 채로 상온에서 1시간동안 인큐베이션 시키고 DAPI로 핵 염색을 수행한다. 형광 염색용 마운팅 용액으로 마운팅하고 형광현미경으로 관찰해 결과를 확인했으며 도 16에 나타냈다.Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 μm intervals to prepare tissue samples and subjected to immunofluoroscence staining. Tissue samples were subjected to deparaffinization, washed with running water, and subjected to permiabilization using Triton X-100. Washed with DPBS, blocked for 1 hour at room temperature with 5% goat normal serum, diluted the anti-flag at 10 ug / ml in a blocking solution and incubated at 4 ° C for 17 hours, followed by secondary antibody ( Anti-Rabbit FITC) was diluted 1: 1000, incubated for 1 hour at room temperature with light blocking, and nuclear stained with DAPI. Mounting with a mounting solution for fluorescence staining and observing with a fluorescence microscope to confirm the results are shown in Figure 16.
상기 도 16을 참조하면, Vatpase B2가 폐의 기관지, 폐포세포 내부로 잘 유입되었음을 Flag-FITC Ab를 이용한 immunofluoroscence stain을 통해서 확인할 수 있었다.Referring to FIG. 16, it was confirmed that Vatpase B2 was well introduced into the bronchus and alveolar cells of the lung through an immunofluoroscence stain using Flag-FITC Ab.
< 마우스 폐조직에서 폐손상 완화 효과 확인><Checking the effect of alleviating lung damage in mouse lung tissue>
위에서 기재한 것과 동일하게 희생된 마우스 폐조직으로 조직 샘플을 제조한 후, H&E(hematoxylin and eosin)으로 염색하여 관찰하고 그 결과를 도 17에 나타내었다. Tissue samples were prepared from the sacrificed mouse lung tissues as described above, followed by staining with hematoxylin and eosin (H & E), and the results are shown in FIG. 17.
상기 도 17을 참조하면, V-atpase B2 단백의 투여로 블레오마이신으로 인한 폐손상이 현저히 감소됨을 확인할 수 있었다.Referring to FIG. 17, it was confirmed that lung damage due to bleomycin was significantly reduced by administration of the V-atpase B2 protein.
< 마우스 폐조직에서 폐섬유화 억제 효과 확인><Checking the effect of inhibiting pulmonary fibrosis in mouse lung tissue>
트리크롬 염색(Trichrome stain)을 이용해 마우스 폐조직에서 V-atpase B2 단백의 폐섬유화 억제 효과를 확인했다. 구체적으로, 희생된 마우스 폐조직은 4% 파라포름알데하이드로 고정시켰고, 파라핀 블록으로 제조된 후 4 μm 간격으로 절단하여 조직 샘플을 제조하였다. 조직 샘플은 deparaffinization 과정을 거친 후 흐르는 물에 수세하고 Bouin solution을 200μl 처리하여 56 ℃의 인큐베이터에서 1시간 매염하였다. 흐르는 물에 수세하고 Weigrt iron hematoxylin 200μl로 상온에서 10분간 염색하였다. 이후, Bibrich scarlet 200μl로 상온에서 10분간 염색한 후 수세하고 Phosphomoybdic-phosphotungstic acid를 상온에서 10분간 처리하고 Aniline blue 200μl로 상온에서 2시간 염색하였다. 흐르는 물에 수세하고 Dehydration 과정을 거친 후 마운팅하여 관찰하였으며, 그 결과를 도 18에 나타냈다.Trichrome staining was used to determine the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue. Specifically, the sacrificed mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 μm intervals to prepare tissue samples. Tissue samples were washed with running water after deparaffinization and treated with 200 μl of Bouin solution and embedded in an incubator at 56 ° C. for 1 hour. It was washed with running water and stained with 200 μl of Weigrt iron hematoxylin at room temperature for 10 minutes. Subsequently, after staining with Bibrich scarlet 200μl for 10 minutes at room temperature and washed with water and treated with Phosphomoybdic-phosphotungstic acid for 10 minutes at room temperature and stained with Aniline blue 200μl at room temperature for 2 hours. After washing with running water and dehydration, it was observed by mounting and the result is shown in FIG.
Sircol assay를 이용해서 마우스 폐조직에서 V-atpase B2 단백의 폐섬유화 억제 효과를 확인했다. 구체적으로, 희생된 마우스 폐조직을 lysis buffer를 가하여 폐조직 용해물을 제조하고, 단백질을 추출하였다. 단백질 용해물과 standard 용액을 각각 실험 tube에 넣고 sircol dye를 넣은 후, 30분간 상온에서 반응시켰다. 14000rpm으로 15분간 원심분리하고 상층액을 제거한 후 pellet을 wasing buffer로 깨끗하게 워싱하였다. 다시 14000rpm으로 15분간 원심분리하고 상층액을 제거한 후 alkari reagent로 pellet을 충분히 풀어주었다. ELISA reader 기계로 540nm의 파장에서 collagen 양을 측정하고, protein을 정량하여 보정한 값을 도 19에 나타냈다.The Sircol assay confirmed the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue. Specifically, pulmonary tissue lysate was prepared by adding lysis buffer to the sacrificed mouse lung tissue, and the protein was extracted. Protein lysate and standard solution were added to each test tube, sircol dye was added, and reacted at room temperature for 30 minutes. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was washed with wasing buffer. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was sufficiently released with alkari reagent. The collagen amount was measured at a wavelength of 540 nm with an ELISA reader machine, and the value quantified and corrected for the protein is shown in FIG. 19.
위의 도 18과 도 19의 결과를 참조하면, 블레오마이신으로 인한 폐섬유화로 인해 증가된 폐 콜라겐의 양이 V-atpase B2 단백의 투여로 정상군과 비슷하게 억제된다는 점을 확인할 있었고, sircol assay을 이용한 정량적 평가에서도 통계적으로 유의미한 수준(* p<0.05 by Mann-Whitney U test)으로 폐손상 유도군(BLM)과 비교한 실험군(BLM+B2)의 콜라겐의 양이 억제되어 V-atpase B2 단백이 급성 폐 손상에 의한 폐 섬유화를 억제한다는 결과를 얻을 수 있었다.Referring to the results of FIGS. 18 and 19 above, it was confirmed that the increased amount of pulmonary collagen due to bleomycin-induced pulmonary fibrosis was inhibited similarly to the normal group by administration of V-atpase B2 protein. In the quantitative evaluation, the statistically significant level (* p <0.05 by Mann-Whitney U test) suppressed the amount of collagen in the experimental group (BLM + B2) compared to the lung injury induction group (BLM), resulting in the V-atpase B2 protein. Inhibition of lung fibrosis due to acute lung injury was obtained.
상기 실험 결과들을 종합하면, 1)V-atpase B2 단백은 in vitro 또는 생쥐의 기도를 통한 투여 시 세포 내 lysosome으로 endocytosis를 통해 유입이 되며, 2) V-atpase B2 단백은 bleomycin으로 유도된 급성 폐손상 및 폐섬유화증의 동물모델에서 폐의 염증 및 섬유화를 효과적으로 억제하는 기능이 있음을 확인할 수 있었다.In conclusion, 1) V-atpase B2 protein is introduced into the intracellular lysosome through endocytosis when administered in vitro or in mice. Animal models of injury and pulmonary fibrosis were found to have a function of effectively inhibiting inflammation and fibrosis of the lungs.
이상에서 본 발명의 바람직한 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다.Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims (5)

  1. V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 폐질환 치료용 또는 증상완화용 약학조성물.V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) comprising a protein as an active ingredient, lung disease treatment or symptomatic pharmaceutical composition.
  2. 제1항에 있어서,The method of claim 1,
    상기 폐질환은 급성폐손상, 폐섬유화증 또는 폐기종인 약학조성물.The lung disease is acute lung injury, pulmonary fibrosis or emphysema pharmaceutical composition.
  3. V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 흡연 또는 간접흡연에 의한 폐손상 억제 또는 완화용 약학 조성물.A pharmaceutical composition for inhibiting or alleviating pulmonary injury by smoking or secondhand smoke, comprising V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) as an active ingredient.
  4. V-에이티피아제 B2(V-atpase B2, Vacuolar-type H+-ATPase B2) 단백질을 유효성분으로 포함하는, 폐질환 예방 또는 개선용 식품조성물. V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) comprising a protein as an active ingredient, food composition for preventing or improving lung disease.
  5. 제4항에 있어서, 상기 식품조성물은 건강기능식품인 것인, 식품조성물. The food composition of claim 4, wherein the food composition is a health functional food.
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