WO2014021682A1 - Cd11b+ cx3cr1+ cells, use thereof and method for mass collection of same - Google Patents
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- WO2014021682A1 WO2014021682A1 PCT/KR2013/007002 KR2013007002W WO2014021682A1 WO 2014021682 A1 WO2014021682 A1 WO 2014021682A1 KR 2013007002 W KR2013007002 W KR 2013007002W WO 2014021682 A1 WO2014021682 A1 WO 2014021682A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/5052—Cells of the immune system involving B-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Definitions
- CDllb + CX3CR1 + cells their use and methods for mass collection thereof, [Technical Field]
- the present invention relates to a CDllb + CX3CR1 + cell having an angiogenic function, an angiogenesis-promoting composition comprising the cell, and a composition for preventing or treating ischemic disease comprising the cell. Also.
- the present invention relates to a composition for mass collection of CDllb + CX3CR1 + cells having angiogenic function in vitro, and a method for mass collection of CDllb + CX3CR1 + cells using the same.
- Angiogenesis is the process by which new blood vessels are formed in existing blood vessels, and provides blood vessels that supply nutrients, growth factors and oxygen necessary for hypoxia due to blood circulation disorders or metabolism of ischemic tissues and organs. It is known to functionally improve ischemia-related tissue damage and injury. Excessive angiogenesis is a major cause of disease exacerbation, but blood vessels and aplasia are also a cause of serious disease. E.g,
- the placenta with poor angiogenesis is a significant cause of miscarriage, and necrosis, ulcers, and ischemia due to the formation of blood vessels can cause tissue or organ dysfunction, or even cause death.
- diseases such as atherosclerosis, myocardial infarction, ischemic stroke and angina are caused by poor blood supply.
- attempts have been actively made to apply drugs and techniques for inducing or promoting angiogenesis in the treatment of angiogenesis-dependent diseases such as ischemic diseases.
- VEGF Endothelial Growth Factor
- vascular endother ial growth factor has been used to treat a number of diseases such as myocardial infarction, cerebral ischemic injury, limb ischemia and wound healing.
- VEGF vascular endothelial growth facotr
- Fibroblast growth factor FGF
- Del-1 developmental ly regulated endothelial locus ⁇ 1
- HGF hepathocyte growth factor
- PD-EGF platelet-derived endothelial eel 1 growth factor
- angiopoietin mutant growth factor
- TGF Epidermal growth factor
- these factors are difficult to isolate and purify as proteins, and are difficult for clinical applications. .
- CDllb + CX3CR1 + cells isolated from ischemia-induced muscle tissue of mouse induce angiogenesis through interaction with fractalkine (Fkn), the only ligand (ligancl) of CX3CR1, and induce angiogenesis-related vascular endothelial cells. It has been shown that it promotes angiogenesis by activating migration, infiltration and tube formation. . ''
- CDllb + CX3CR1 + cells can be treated therapeutically.
- the present invention has been completed by developing a method for collecting a large amount of the cells directly from the body.
- One object of the present invention is to provide a cell comprising the immune phenotypes of CDllb + and CX3CR1 + .
- the cells exhibit angiogenic function.
- Still another object of the present invention is to provide a composition for promoting angiogenesis comprising the cell.
- Another object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic disease comprising the cells.
- Another aspect of the present invention is to provide an angiogenic stimulation method comprising administering to the individual an effective amount of the cells.
- Another object of the present invention is to provide a preventing or treating ischemic diseases, comprising the step of administering an "effective amount of the cells in the object. It is another object of the present invention to provide a composition for mass collection of cells comprising an immune phenotype of CDllb + and CXXR1 + , including a granulocyte colony stimulating factor (G-CSF).
- G-CSF granulocyte colony stimulating factor
- Yet another object of the present invention is to provide a method for separating CDNs from the peripheral blood of a subject administered with a composition comprising G-CSF and concentrating the CDNs + CX3CR1 +. It is to provide a method for mass collection of cells comprising an immune phenotype.
- Another object of the present invention is to provide a cell population which is collected in large quantities using the above method and contains at least 10% of cells comprising the immune phenotypes of CDllb + and CX3CR1 + .
- Another object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic disease comprising the cell population.
- the cell of the present invention Since the cell of the present invention has an angiogenic promoting effect, it can be usefully used as an angiogenesis promoter: and furthermore, it can be used for the prevention or treatment of various diseases requiring angiogenesis.
- the cells of the present invention The bulk collection method uihamyeo to collect easily the amount and concentration, which can be scanned several times directly on the ischemic region of the patient CDllb + CX3CRl + cells in large quantities, and this way the CDllb + CX3CRl + cells collected as is of angiogenesis promoting action Therefore, it could be useful as an angiogenesis promoter. Furthermore, it can be used for the purpose of preventing or treating various diseases requiring angiogenesis.
- Fig. La shows the results of comparison of intramuscular CDllb + CX3CRl + cell fractions before and after ischemia induction in mice.
- FIG. Lb is Intramuscular Over Time After Mouse Inferior Ischemia Induction
- Figure lc shows the intramuscular Fkn expression over time after mouse lower limb ischemia induction.
- Id is surface antigen of CDllb + CX3CRl + cells isolated from ischemic muscle. The analysis results are shown.
- Figure 2a shows the comparison of the blood flow over time according to the cell injection.
- Figure 2b shows the comparison of blood flow over time according to the injection of Fractalkine protein.
- Figure 3 is a neutralizing anti-Fkn antibody and after ischemia induction. Blood flow comparison results according to the injection of anti-CX3CR1 antibody are shown.
- Figure 4a shows the result of directly observing the effect of inhibiting angiogenesis in the ischemic muscle after ischemia induction after injection of anti-CX3CR1 antibody.
- Figure 4b shows the results of direct observation of the effect of inhibiting angiogenesis in rat retina after injection of anti-CX3CR1 antibody.
- FIG. 4C shows that VEGF niRNA and protein expression is increased in CDllb + CX3CRl + cells.
- Figure 5 shows the result of the G-CSF prior to administration of peripheral blood (A) and G-CSF 5 ilgan a CDllb + CX3CRl + cell fraction measured in the peripheral blood. Solution and was collected and concentrated monocytes (B) after administration of a FACS analysis .
- Figure 6 shows the results of FACS analysis of CDllb + CX3CRl + cell fractions in peripheral blood and concentrated monocytes after co-administration of G-CSF and AMD3100 ' .
- FIG. 7 shows the results of comparing the number of CDllb + CXXRl + cells per unit peripheral blood (mL) after G-CSF administration and co-administration of G-CSF with AMD3100.
- Monocyte / myeloid lineage cells are known to secrete growth factors that promote angiogenesis and to form blood vessels that increase blood flow to damaged tissues. Yet only it has to do no angiogenesis is known clearly about the cell subset or a mechanism of action of certain monocyte / myeloid system to participate in the forming system of the cell, some of the monocyte / myeloid until now, have been proposed as a target.
- CDllb + Grl dim cells known as myeloid derived suppressor cells
- CDllb + Grl dim cells were monocyte / macrophage-type cells including CX3CR1 + , CCR2 + , CCR5 + , CXCR4 + monocytes, especially CDllb + CX3CRl in ischemic muscle after femoral artery dissection. It was found that the number of + cells increased significantly.
- CDllb + CX3CRl + cell number change were analyzed, and the changes in blood flow when CDllb + CXXRl + cells were injected into the ischemic muscle and the changes in blood flow when pretreated with CDllb + CX3CRl + cells were analyzed.
- CDllb + CX3CRl "had significantly higher cell than the injection group. Furthermore, is verified by the cells increases in a significant level of blood flow in an effective amount range in the group alone introduced into the Fkn protein associated with the movement of, CDllb + CX3CRl + cells It was confirmed that the cells play an important role in the formation of new blood vessels after ischemic injury (FIGS. 2A and 2B).
- CDllb + CX3CRl + cells in large quantities we found that CDllb + CX3CRl + cells in large quantities.
- CDllb + CX3CRl + cells are normally blood, do not almost present in, the G-CSF alone, or with G-CSF and AMD3100.
- Administration separating blood mononuclear cells from the individual When the concentrated, so-called “concentrated monocytes” were made, it was confirmed that a large amount of concentrated CDllb + CX3CRl + cells can be easily obtained in large quantities (Figs. 5 to 7), thereby leading to the present invention.
- the present invention also provides methods and compositions for use in the mass collection of CDllb + CX3CRl + cells.
- the present invention relates to cells comprising the immune phenotypes of CDllb + and CX3CR1 + and exhibiting angiogenic function.
- the present invention relates to a composition for promoting angiogenesis comprising a cell comprising an immune phenotype of CDllb + and CX3CR1 + .
- the present invention relates to a method for promoting angiogenesis comprising administering to the individual an effective amount of the cells.
- the present invention relates to a pharmaceutical composition for treating ischemic disease comprising a cell comprising the immune phenotype of the CDllb + and CX3CR1 + .
- the present invention relates to a method for preventing or treating ischemic disease, comprising administering to a subject an effective amount of said cell.
- the present invention relates to a composition for mass collection of cells comprising an immune phenotype of CDllb + and CX3CR1 + , which contains a granulocyte colony stimulating factor (G-CSF).
- the composition may comprise this or a pharmaceutically acceptable salt of AMD3100 further.
- the present invention also relates to a composition for mass collection of cells comprising an immune phenotype of CDllb + and CX3CR1 + , comprising G ⁇ CSF and AMD3100 or a pharmaceutically acceptable salt thereof.
- the invention provides a CDllb + and CX3CR1 + comprising the steps of isolating the blood monocyte fraction from the peripheral ' blood of the subject to which the composition comprising G-CSF is administered and concentrating the blood monocyte fraction.
- a method of mass collection of cells comprising a phenotype comprising a phenotype.
- the composition may further comprise AMD3100 or a pharmaceutically acceptable salt thereof. Therefore,.
- the present invention also includes the steps of separating the blood monocyte fraction from the peripheral blood of an individual administered with a composition comprising G-CSF and AMD3100 or a pharmaceutically acceptable salt thereof and concentrating the blood monocyte fraction, Immunity of CDllb + and CX3CR1 +
- a method of mass collection of cells comprising a phenotype comprising a phenotype.
- the step of separating the blood monocyte fraction is blood. This can be done using an ingredient collector:
- the step of concentrating the blood monocyte fraction may be performed using a blood ' centrifuge.
- the present invention relates to a cell population which is mass collected using the above method and contains at least 10% of cells comprising the immune phenotypes of CDllb + and CXXR1 + .
- the present inventors dissected the femoral artery of C57BL / 6 mice to develop ischemia . Remove the first CDllb + CX3CRl + cells derived from one leg muscles after, the surface antigens were examined in order to accurately determine the cell whether i than any system cells. As a result, the CDllb + CX3CRl + cells were positive for bone marrow cells (CD45) and macrophages (F4 / 80), but endothelial cells (VEGFR-2).
- CDllb + CX3CRl + cells migrated into ischemic muscle are cells of the monocyte / macrophage lineage .
- CDllb + CX3CRl + cells were positive for all of the monocyte surface antigens VEGFRX1, Tie-2 and CX3CR4, which are known to be involved in angiogenesis.
- VEGFRX1, Tie-2 and CX3CR4 which are known to be involved in angiogenesis.
- the cells of the invention are cells comprising the immune phenotypes of CDllb + and CX3CR1 + .
- the cells of the present invention in addition to the immunophenotypes of CDllb + and CX3CR1 + , include CD45 + , F4 / 80 + , Tie2 + , CXCR4 + , VEGFR1 + , VEGFR2 " , ⁇ 1. ⁇ , CDllc " , CD3 " and CD19 - it may be a cell which further comprises one or more "immune phenotype is selected from the eu.
- cells to which additional additional immune phenotypes or morphological features are added may be included in the scope of the cells of the present invention.
- the cells of the invention artificially cut the lower limb aorta and isolate muscle cells in mice .
- the obtained method is not limited thereto, and may be obtained from ischemic tissues or blood of various mammals including mice, and may be obtained in large quantities by in vitro culture, differentiation and / or amplification using knowledge known in the art. You can get it. For example, in ischemic tissue
- Ficoll-Hypaque separation method Ficoll-Hypaque separation method, nioidfied Ficol 1-Hypaque separation method, 3% gelatin separation method, Per col ⁇ separation method, panning, immunomagnetic field
- the cell of the present invention has an angiogenic promoting effect, it can be usefully used as an angiogenic promoter, and furthermore, can be used for the prevention or treatment of various diseases requiring angiogenesis.
- Ischemic disease is a representative disease that requires angiogenesis. .
- Ischemic disease refers to a disease in which blood is partially deficient due to various causes such as narrowing or contraction of arteries, blood clots or embolism.
- Examples of the ischemic diseases are heart failure, hypertensive heart disease, arrhythmia, nature seok heart disease, myocardial infarction. Stroke, peripheral vascular disease, angina pectoris, cerebrovascular dementia, coronary artery failure. Brain failure, retinal-ischemia and vascular failure, but is not limited thereto.
- ischemic diseases requiring angiogenesis can be included in the scope of the present invention.
- prevention refers to any action that promotes angiogenesis by administration of a composition of the present invention, or any action that causes growth, spread or recurrence of ⁇ angiogenesis-dependent diseases.
- treatment refers to all actions which improve the angiogenesis dependent disease or advantageously changed to tukkyeo of the compositions of the present invention ".
- administration means providing a subject with a composition of the present invention in any suitable manner.
- the term "individual” as used herein refers to an animal suffering from a condition in which angiogenic dependent disease can be improved by administering the composition of the present invention, preferably a vertebrate, more preferably a mammal, such as a human being, a monkey. , dogs, cats ⁇ goats, pigs, and rats. Means all animals such as guinea pig hamsters, chimpanzees or gorillas.
- the term “effective amount” means an amount sufficient to prevent or treat a disease, which is the type of disease of the individual. Severity, drug activity, drug sensitivity, time of administration, route and route of administration, duration of treatment, used concurrently; depending on factors including drug and other well known factors in the scientific field. Can be determined. There is : ' ⁇
- composition of the present invention may be administered orally or parenterally as a cell therapy, and may be intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or It can be administered by intrathoracic injection and can be administered directly to the disease site. ' ,
- the pharmaceutical composition according to the present invention may be administered in the form of an injection to a site intended to induce angiogenesis of a mammal in combination with the composition for injection, and the composition for injection is preferably an isotonic aqueous solution or suspension.
- Pharmaceutical compositions may be sterile or preservatives, stabilizers, wetting agents, emulsifier solution accelerators, auxiliaries such as salts and / or buffers for osmotic pressure control or other It may contain therapeutically useful substances.
- composition of the present invention can be administered directly or indirectly with a pharmaceutically acceptable carrier, such as implanted by surgery to a site requiring repair or expansion, or delivered using a catheter, matrix, or needle.
- a pharmaceutically acceptable carrier such as implanted by surgery to a site requiring repair or expansion, or delivered using a catheter, matrix, or needle.
- Pharmaceutically acceptable carriers may be any suitable organic or organic carrier material which does not adversely react with the cells, compositions or ingredients of the invention, including water, salt solutions (Ringer's solution), alcohols, oils, gelatin, lactose, Carbohydrates such as amylose or starch; Fatty acid ester ,
- Hydroxymethyl cellulose polyvinyl pyrrolidone, and the like. These preparations may be sterile and, where appropriate, lubricants, preservatives: stabilizers, wetting agents, emulsifiers. It may be compatible with auxiliaries such as salts, buffers and coloring agents for osmotic pressure.
- Pharmaceutical carriers suitable for use in the present invention are known in the art.
- compositions of the present invention may optionally be administered together with other beneficial drugs and biomolecules (growth factors, nutritional factors, etc.).
- biological molecules that can be administered include anti-cell killing agents, anti-inflammatory agents, immunomodulators, anti-proliferative agents, corticosteroids.
- Antibody, an anti-oxidant may include, or local anesthetics ⁇ -thrombotic agents, anti. ' '
- the cells may be used at 1 ⁇ 10 4 to 10 ⁇ 10 10 cells / kg, preferably at 2.5 ⁇ 10 7 to 5 ⁇ 10 7 and more preferably at 2.5 ⁇ 10 7 cells / kg. It is not limited to children and can be used for example, from 2x10 9 to 4x10 9 based on 70 kg of adult. However, the weight, age, sex, health status, diet, duration of administration, method of elimination, disease The severity can change depending on , and so on.
- the pharmaceutical composition of the present invention can be used several times a day depending on the half-life . To once a week. The dosage does not limit the scope of the invention in any aspect.
- the present invention is to collect a large amount of the CDllb + CX3CRl + cells in a therapeutically effective amount, it is characterized by using a composition comprising a granulocyte colony stimulating factor (G-CSF) and / or AMD3100.
- G-CSF granulocyte colony stimulating factor
- G-CSF is a factor that proliferates or differentiates neutrophil granulocyte progenitor cells and activates mature neutrophils, mainly bone marrow transplantation, neutropenia due to chemotherapy of cancer, myelodysplastic syndrome, aplastic anemia, congenital and idiopathic neutrophils . It has been used to promote neutrophil increase in attenuated, human immunodeficiency virus (HIV) infections, etc.
- G—CSF is mobilized to CDllb + CX3CRl + cells peripheral blood, and eventually CDllb + CX3CRl + cells are concentrated. To obtain concentrated monocytes.
- G-CSF to be used in the present invention can be obtained through various routes, such as synthesis through known amino acid sequence information, used separately from an individual, or obtained from commercially available agents.
- commercially available G-CSF includes narutograss (trade name Neuap, manufactured by Kyowa Fermentation Industry), filgrasteam (trade name Gran, manufactured by Sankyo; trade name Granulokine, manufactured by Hoffman La Roche;
- the G 'CSF used in the present invention is included in the scope of the present invention as long as it is chemically modified as long as it maintains the equivalent activity.
- a method of a chemical modification the method of international patent publication WO00 / 51626, etc. are mentioned, for example .
- Polypeptides having G-CSF activity for example modified with polyalkylene glycols, for example polyethylene glycol (PEG), can be included.
- the AMD3100 is also called Pier ixafor,
- AMD3100 used in the present invention is synthesized by chemical synthesis methods known in the art, or that available from commercially available preparations. And so on
- AMD3100 can also be used in the form of pharmaceutically acceptable salts.
- suitable acid addition salts are '
- Biocompatible inorganic acids e.g. HCl, HBr, sulfuric acid, phosphoric acid, etc.
- organic acids e.g. HCl, HBr, sulfuric acid, phosphoric acid, etc.
- Acid 10 for example, acetic acid, propionic acid, butyric acid, etc.
- an acid containing at least one carboxyl group can include salts (such as oxalic acid, glutaric acid, adipic acid, etc.).
- AMD3100 used in the present invention is in the form of a prodrug, ie.
- the compound of the invention may also be prepared in a protective form to release the compound of the invention after administration to a subject.
- the compound when prepared in purified form, the compound may be crystallized in 15 hydrate. .
- the step. (A) is the peripheral of the subject administered a composition comprising G-CSF and / or AMD3100 or 25 pharmaceutically acceptable salts thereof
- the object i is mammalian animal may be "including a vertebrate, more preferably a human.
- Mass Collected cells in the present invention preferably are used as a therapeutic "purpose to promote angiogenesis, to yield the most preferred self-enriched mononuclear cells from 30 patients subject to treatment.
- G-CSF may be administered alone or in combination with AMD3100, and when G100CSF and AMQ3100 are administered together, AMD3100 may be administered after or concurrently with the G—CSF.
- the AMD3100 may be administered 10 hours before blood collection after the addition of G-CSF, but this is not limiting.
- the dosage form of G-CSF and / or AMD3100 is not particularly limited and may be preferably administered parenterally subcutaneously by injection or the like. Also. In consideration of the type and condition of administration, ' CDllb + CX3CRl + cells may be administered once or several times in an amount sufficient to be mobilized to peripheral blood, preferably 1 to 100 zg / kg for G—CSF. It may preferably be administered once or several times at a dose of 10 g / kg, and once or several times at a dose of 100 to 500 ⁇ g / k, preferably 250 / jg / kg for AMD3100. May be administered. But the dosage form , the number of doses. And the dosage may be appropriately changed by the judgment of those skilled in the art.
- Separating the blood monocyte fraction from the peripheral blood in step (a) can be carried out using blood component collection methods known in the art, for example, administering G-CSF to a subject and then dispensing peripheral blood into blood components. It can be collected by passing it through an apheresis machine.
- blood component collectors used in the art can be exemplified by MCS 3p, CS-3000 plus COB Spectra, but the present invention is not limited thereto.
- the heart, blood enters the catheter through the venous blood group apheresis blood mononuclear cell fraction to be collected by the machine remaining blood can "back into Nero body through a catheter. This process can take about 4 to 10 hours and is intended for therapeutics ⁇ purposes.
- step (b) is a step of concentrating the blood monocyte fraction isolated in step (a).
- concentration of the blood monocyte fraction may be performed using a blood centrifuge.
- the centrifugation conditions can be properly adjusted by one skilled in the art according to the situation, and it is possible to remove plasma components and obtain a concentrate containing monocyte components. have.
- the embodiments of the present invention remove plasma components by "centrifugation for 10 minutes at 1200 revolutions per minute to give a concentrated solution containing the components to monocytes, but the invention is not limited to this.
- the ' concentrated monocytes ' contained CDllb + CXXRl + cells in an amount and concentration sufficient to promote angiogenesis when administered directly to the patient's ischemic site, and in a preferred embodiment, CDlib + CX3CRl + cell.
- the fraction may preferably be at least 10%, more preferably at least 15%. Assuming a patient weight of 70 kg when injected from 2.5 X 10 7 to 5 X 10 7 per patient body weight 2 . X 10 9 to 4 X 10 cells and cells are required.
- CDllb + CX3CRl + cell concentrates can be obtained in the amount and concentration which can be injected directly into the ischemic site of the patient from the collected mononuclear cells once collected.
- the monocytes collected in large amounts by the present invention can be stored and mixed with a cell preservative (eg, 7% DMS0) to be shaken (eg Cryomed), and can be stored for a long time without cell damage caused by silver. It can be used for therapeutic purposes because it can be thawed and used as many cells as needed at any time.
- a cell preservative eg, 7% DMS0
- shaken eg Cryomed
- the viability of cells upon thawing is preferably at least 90%.
- the cell population obtained in the present invention has an angiogenic promoting effect, it may be usefully used as an angiogenesis promoter, and furthermore, may be used for the purpose of preventing or treating various diseases in which angiogenesis is required.
- Representative diseases that require angiogenesis include ischemic ⁇ ring.
- the present invention provides a composition for promoting angiogenesis comprising a cell population comprising the immune phenotype of the CDllb + and CX3CR1 + .
- the present invention also provides a method for promoting angiogenesis comprising administering to the subject an effective amount of the cell population. " .
- the present invention provides a pharmaceutical composition for preventing or treating ischemic and hematologic diseases comprising the cytoplasm. "The present invention also provides a prevention or treatment of ischemic disease comprising the step of administering an effective amount of said cell population to an object. [Form for implementation of invention]
- Example 1 Mouse Ischemia Induction and Muscle Cell Isolation
- ischemia was induced by removing veins and arteries in the thighs of 1 to 12-week-old C57BL / 6 mice.
- Single cells were isolated from ischemic muscles using 0.2% collagenase (210 U / mg; Worthi'ngton, Lakewodcl, NJ) and ' dispase (0.95 U / mg; Invitrogen, Grand Island, NY).
- isolated cells were reacted with APC-bound anti-mouse CDllb, biotinylated anti-mouse CX3CR1 antibody at 4 ° C. for 30 minutes.
- VEGF expression was analyzed using FACS. Fkn expression in tissues and VEGF expression in CDllb + CX3CRl + cells were investigated using real-time PCR.
- Fkn expression in tissue was Fkn primer (forward: GGA CAG GAC CTC AGT CCA GA, reverse: TCG GGG ACA GGA GTG ATA AG) and VEGF expression in CDllb + CX3CRl + cells was VEGF primer (forward: CACAGCAGATGTGAATGCAG,. :
- the mouse recombinant protein Fkn (R & D System, Minneapolis, ⁇ ' ) was dissolved in 500 ul of PBS at 0.5, 1, and 5 ug, and injected into the ischemic muscle every day for 100 days at 100 ul immediately after ischemic induction.
- Anti-CX3CR1 neutralizing antibody TP501, Torrey Pines Bio labs, San Diego, Calif.
- Anti-Fkn neutralizing antibodies TP233, Torrey Pines Biolabs
- LDPI laser Doppler perfusion scanner
- CX3CR1 function to block shed, wherein the -CX3CR1 neutralizing antibody was injected into each 0.5 ⁇ g per day and, five days ischemic muscle.
- the same amount of Ig0 was injected in the same manner.
- the OCT compound Tissue ⁇ Tek, Sakur a Fine tek Japan Co., Tokyo, Japan
- 4% parapo Fixed for 15 minutes in aldehydes. It was washed with PBS and then rebounded with 10% normal horse serum for 1 hour to prevent nonspecific reaction.
- G- CSF Choongwae (trade name: Neutrogen) and Dong-A (trade name: Leucost iin) '10 g / kg was the dose five days of subcutaneous injections purchased from.
- G-CSF was injected subcutaneously for 5 days at the dose of, and once subcutaneously at AMD3100 240 / g / kg for 4 days.
- Blood sampler (Cobe® spectra apheresis system, GAMBRO.BCT, Lakewood, USA) at least 10 hours after the last dose of G-CSF or AMD3100 Blood mononuclear fractions were collected continuously for 4 hours, and concentrated monocytes were collected by concentrating the collected blood monocyte fractions using a blood centrifuge (Cobe®2991 cell processor, GAMBRO.BCT, Lakewood, USA).
- Ischemia damage significantly increases CDllb + CX3CRl + cell count in ischemic muscle.
- Fkn which affects CDllb + CX3CRl + cell migration
- Fkn mRNA expression was highest at 4 days after arterial incision (FIG. Lc), indicating that increased Fkn expression in ischemic muscle was associated with an increase in CDllb + CX3CRl + cell number.
- CX3CR1 surface antigens are macrophages, monocytes. Endothelial cells, dendritic cells, natural killer cells (NK cell) and a "cell surface antigens that are variously expressed in T cells, CDllb + CX3CRl + cells ischemia intramuscular and CDllb + to see what i types of cells CX3CRl + After the cells were separated, the surface antigen was examined.
- CDllb + CX3CRl + cells were positive for bone marrow cells (CD45) and macrophages (F4 / 80), Surface antigens for endothelial cells (VEGFR-2), dendritic cells (CDllc), NK cells (NK1.1) and T cells (CD3) were not expressed (Fig. 1 [1]).
- CDllb + CX3CRl + cells were found to be cells of the monocyte / macrophage lineage.
- CDllb + CX3CRl + cells of VEGFR-1, Tie-2 specific, the surface of the monocyte antigen known to be involved in angiogenesis and to this antigen were positive CX3CR4.
- CDllb + CX3CRl + , CDllb + CX3CRl " , and CDilbXX3CRl " cells were isolated from ischemic muscles on day 4 after ischemic induction and injected into ischemic muscles. Changes in blood flow at each time were measured using LDPI (Laser Doppler Perfusion Image). As a result, blood flow in mice injected with CDllb + CXXRl + cells .
- the present inventors measured the blood flow of the ischemic leg by directly injecting Fkn to the ischemic site.
- Fkn protein infusion group at 0.5 days on ischemia did not increase blood flow compared to control group injected with PBS (40.54 ⁇ 2.34%, day 28; 37.69 ⁇ 4.85% .28 days, respectively), but injected Fkn of 1 / jg and 5
- Blocking CX3CR1 function inhibits normal angiogenesis in ischemic muscle and retina.
- CDllb + CX3CRl + cells were not observed in normal tissues ( Figure A, Figure 4a), but a large number of cells migrated into the ischemic muscle at day 7 of ischemia, many of which were CDllb + CX3CRl + cells (arrow; yellow; Gram B, Fig. 4a). On day 14 of the ischemia, a cell cluster consisting of CDllb + CX3CRl + cells was formed inside the ischemic muscle.
- Anti-CX3CR1 antibody was injected into the rat retina at day 2 to block the function of CX3CR1 at the ischemic site.
- Administration of anti-CX3CR1 antibodies in the rat retina Normal retinal vessel formation was confirmed to be aberrant ( Figure C and Figure 4b),
- CDllb + CX3CRl + cells promote the formation of blood vessels at the ischemic site and directly improve blood flow.
- CDllb + CX3CRl + cells ⁇ can express VEGFR mRNA and protein in the cell itself.
- CDllb + CX3CRl + cells are rarely normally present in the blood cells.
- FIG. 5A To be. Appeared (FIG. 5A).
- CDllb + CX3CRl + peripheral blood mobilization G-GSF (10 / ig / kg) to the cells from the bone marrow by CDllb + CX3CRl + cells are 2.8 ⁇ 0.9% of the total white blood cells within 5 days following the injection of peripheral blood obtained. Slightly less than about 1.3% before G-CSF
- the fraction was 16.2 ⁇ 7.2%.
- Cells Total 9.2 ⁇ 1.2X10 9 gae (16 ⁇ 2 ⁇ 7.2 ⁇ 3 ⁇ 4 ⁇
- CDllb + CX3CRl ⁇ Cell number increases.
- CDllb + CX3CRl + cell fraction was approximately 1.1% of the total white blood cell, CDllb + + Three CX3CRl be i was 600 X 10 3 seeds / mL (ll% X54,550 / mm 3).
- CDllb + CX3CRl + cell fraction in the concentrated monocytes was about 12% and the amount collected was 198 mL.
- To calculate the total CDllb + CX3CRl + cell number was 6.3X10 9 (12% X 2.67 X lOVml X 198 ml) (CDllb + CX3CRl + (%) X leukocyte count / ml X vol (ml)). therefore .
- G-CSF and AMD3100 were co-administered, CDllb + CX3CRl + cell concentrates were obtained in the amount and concentration that can be directly injected into the ischemic site of the patient (FIG. 6).
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Abstract
The present invention relates to CD11b+ CX3CR1+ cells having an angiogenesis facilitating function, to a composition comprising the cell for facilitating angiogenesis, and to a composition comprising the cell for preventing or treating ischemic diseases. Further, the present invention relates to a composition for ex vivo mass collection of CD11b+ CX3CR1+ cells having an angiogenesis facilitating function, and to a method for mass collection of CD11b+ CX3CR1+ cells using the composition.
Description
【명세서] 【Specification]
【발명의 명칭】 [Name of invention]
CDllb+ CX3CR1+ 세포, 이의 이용 및 이의 대량 수집 방법 , [기술분야】 CDllb + CX3CR1 + cells, their use and methods for mass collection thereof, [Technical Field]
본 발명은 혈관신생 촉잔 기능이 있는 CDllb+ CX3CR1+ 세포， 상기 세포를 포함하는 혈관신생.촉진용 조성물 및 상기 세포를 포함하는 허혈성 질환 예방 또는 치료용 조성물에 관한 것이다. 또한. 본 발명은 혈관신생 촉진 기능이 있는 CDllb+ CX3CR1+ 세포를 체외에서 대량 수집하기 위한 조성물， 및 이를 이용한 CDllb+ CX3CR1+ 세포 대량 수집 방법에 관한 것이다. 【배경기술】 The present invention relates to a CDllb + CX3CR1 + cell having an angiogenic function, an angiogenesis-promoting composition comprising the cell, and a composition for preventing or treating ischemic disease comprising the cell. Also. The present invention relates to a composition for mass collection of CDllb + CX3CR1 + cells having angiogenic function in vitro, and a method for mass collection of CDllb + CX3CR1 + cells using the same. Background Art
혈관신생 (angiogenesis)은 기존의 혈관에서 새로운 혈관이 생성되는 과정으로， 혈액순환장애에 의한 저산소 상태 또는 허혈성 조직 및 기관의 대사에 필요한 영양소， 성장인자 및,산소 등을 공급하는 혈관을 제공하여 , 허혈 관련 조직 손상 및 상해를 기능적으로 개선하는 것으로 알려져 있다. 과다한 혈관형성은 질병 악화에 있어 주원인이 되기도 하지만, 혈관와 미형성 또한 심각한 질병의 원인이 되고 있다. 예를 들어， Angiogenesis is the process by which new blood vessels are formed in existing blood vessels, and provides blood vessels that supply nutrients, growth factors and oxygen necessary for hypoxia due to blood circulation disorders or metabolism of ischemic tissues and organs. It is known to functionally improve ischemia-related tissue damage and injury. Excessive angiogenesis is a major cause of disease exacerbation, but blood vessels and aplasia are also a cause of serious disease. E.g,
혈관형성이 미발달된 태반은 유산의 증요한 원인이 되며， 혈관의 미형성으로 인한 괴사， 궤양 및 허혈의 경우 조직이나 기관의 기능이상을 유발하거나 심하면 사망의 원인이 될 수 있다. 또한, 동맥경화， 심근경색， 허혈성 뇌졸중 및 협심증과 같은 질병은 원활하지 못한 혈액 공급이 원인이 되고 있다. 아에， 혈관신생을 유도하거나 촉진시키는 약제 및 기술들을 허혈성 질환과 같은 혈관신생에 의존하는 질병의 치료에 적용하려는 시도가 활발히 이루어지고 있다. The placenta with poor angiogenesis is a significant cause of miscarriage, and necrosis, ulcers, and ischemia due to the formation of blood vessels can cause tissue or organ dysfunction, or even cause death. In addition, diseases such as atherosclerosis, myocardial infarction, ischemic stroke and angina are caused by poor blood supply. In the meantime, attempts have been actively made to apply drugs and techniques for inducing or promoting angiogenesis in the treatment of angiogenesis-dependent diseases such as ischemic diseases.
이미 잘 알려진 혈관신생 촉진인자인 혈관내피 성장인자 (VEGF; Endothelial Growth Factor (VEGF), a well-known angiogenesis promoter.
vascular endother 1 ial growth factor)는 심근경색증， 대뇌 허혈손상, 사지허혈 및 상처치유와 같은 여러 질환의 치료제로 사용되어 왔다. 또한, 혈관내피성장인자 (VEGF; vascular endothelial growth facotr), Vascular endother ial growth factor has been used to treat a number of diseases such as myocardial infarction, cerebral ischemic injury, limb ischemia and wound healing. In addition, vascular endothelial growth facotr (VEGF),
섬유아세포성장인자 (FGF; fibroblast growth factor), Del-1
(developmental ly regulated endothelial locusᅳ 1)， HGF (hepathocyte growth factor) , PD-EGF (platelet-derived endothelial eel 1 growth factor) , 안지오포이에틴 (angiopoietin), 변이성 성장인자 (TGF; transforming growth factor), 상피세포 성장민자 (EGF; epidermal growth factor) 등와 Fibroblast growth factor (FGF), Del-1 (developmental ly regulated endothelial locus ᅳ 1), HGF (hepathocyte growth factor), PD-EGF (platelet-derived endothelial eel 1 growth factor), angiopoietin, mutant growth factor (TGF), Epidermal growth factor (EGF)
혈관생성인자 (angiogenic factor) 그 자체 또는 그를 제조하는 유전자를 허혈 부위에 투여하여 측부 혈관의 생성을 촉진시킴으로써 허혈성 질환을 치료하거나， 경피적 동맥성형술 또는 동맥우회술을 시행할 수 없는 심한 허혈성 질환을 치료하기 위한 새로운 방법으로 대두되고 있다.그러나 상기 인자들은 단백질로서 분리정제하기 어렵고， 고가이므로 임상 적용에 어려움이 았다. . Treating ischemic disease by administering angiogenic factor itself or genes thereof to the ischemic site to promote the production of collateral vessels or treating severe ischemic diseases in which percutaneous angioplasty or arterial bypass surgery cannot be performed However, these factors are difficult to isolate and purify as proteins, and are difficult for clinical applications. .
이에, 최근 새로운 신생혈관 유도제의 fl발 및 확립올 위한 연구에 대한 관심이 증가하였다. In recent years, there has been increasing interest in the study of fl development and establishment of new neovascular inducers.
【발명의 상세한 설명] [Detailed Description of the Invention]
[기술적 과제] [Technical Challenges]
본 발명자들은 마우스의 허혈 유도 근육 조직에서 분리한 CDllb+ CX3CR1+세포가 CX3CR1의 유일한 리간드 (ligancl)인 fractalkine (Fkn) 과의 상호작용을 통해 혈관 신생을 유도하고， 혈관신생과 관련된 혈관내피세포의 이동, 침윤 및 관 형성을 활성화시켜 혈관신생을 촉진한다는 것을 확인하게 되었다. . ' ' The present inventors have found that CDllb + CX3CR1 + cells isolated from ischemia-induced muscle tissue of mouse induce angiogenesis through interaction with fractalkine (Fkn), the only ligand (ligancl) of CX3CR1, and induce angiogenesis-related vascular endothelial cells. It has been shown that it promotes angiogenesis by activating migration, infiltration and tube formation. . ''
나아가， 본 발명자들은 상가 CDllb+ CX3CR1+ 세포를 치료학적 . In addition, the present inventors have found that CDllb + CX3CR1 + cells can be treated therapeutically.
유효량으로 확보하기 위하여 체내로부터 직접 상기 세포를 대량 수집할 수 있는 방법을 개발하여 본 발명을 완성하게 되었다. 【기술적 해결방법】 In order to secure an effective amount, the present invention has been completed by developing a method for collecting a large amount of the cells directly from the body. Technical Solution
본 발명의 하나의 목적은 CDllb+ 및 CX3CR1+의 면역 표현형을 포함하며.혈관신생촉진 기능을 나타내는 세포를 제공하는 것이다. One object of the present invention is to provide a cell comprising the immune phenotypes of CDllb + and CX3CR1 + . The cells exhibit angiogenic function.
본 발명의 또 하나의 목적은 상기 세포를 포함하는 혈관신생 촉진용 조성물을 제공하는 것이다.
본 발명의 또 하나의 목적은 상기 세포를 포함하는 허혈성 질환 예방 또는 치료용 약학 조성물을 제공하는 것이다. Still another object of the present invention is to provide a composition for promoting angiogenesis comprising the cell. Another object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic disease comprising the cells.
본 발명의 또 하나의 목작은 상기 세포의 유효량을 개체에 투여하는 단계를 포함하는 혈관신생 촉,진 방법을 제공하는 것이다. Another aspect of the present invention is to provide an angiogenic stimulation method comprising administering to the individual an effective amount of the cells.
본 발명의 또 하나의 목적은 상기 세포의 '유효량을 개체에 투여하는 단계를 포함하는 허혈성 질환 예방 또는 치료 방법을 제공하는 것이다. 본 발명의 또 하나의 목적은 G-CSF (granulocyte colony stimulating factor)를 포함하는, CDllb+ 및 CXXR1+의 면역 표현형을 포함하는 세포의 대량 수집용 조성물을 제공하는 것이다. Another object of the present invention is to provide a preventing or treating ischemic diseases, comprising the step of administering an "effective amount of the cells in the object. It is another object of the present invention to provide a composition for mass collection of cells comprising an immune phenotype of CDllb + and CXXR1 + , including a granulocyte colony stimulating factor (G-CSF).
본 발명의 또 하나의 목적:은 G-CSF를 ί함하는 조성물을 투여한 개체의 말초 혈액으로부터 혈액 단핵구 분획을 분리하는 단계 및 상기 혈액 단핵구 분획을 농축하는 단계를 포함하는， CDllb+ 및 CX3CR1+의 면역 표현형을 포함하는 세포의 대량 수집 방법을 제공하는 것이다. Yet another object of the present invention is to provide a method for separating CDNs from the peripheral blood of a subject administered with a composition comprising G-CSF and concentrating the CDNs + CX3CR1 +. It is to provide a method for mass collection of cells comprising an immune phenotype.
본 발명의 또 하나의 목적은 상기 방법을 이용하여 대량 수집되며， CDllb+ 및 CX3CR1+의 면역 표현형을 포함하는 세포가 10% 이상 포함된 세포 집단을 제공하는 것이다 . Another object of the present invention is to provide a cell population which is collected in large quantities using the above method and contains at least 10% of cells comprising the immune phenotypes of CDllb + and CX3CR1 + .
본 발명의 또 하나의 목적은 상기 세포 집단을 포함하는 혈관신생 촉진용조성물을 제공하는 것이다.. It is another object of the present invention to provide an angiogenesis promoting composition comprising the cell population.
본 발명의 또 하나의 목적은 상기 세포 집단을 포함하는 허혈성 질환 예방 또는 치료용 약학 조성물을 제공하는 것이다. Another object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic disease comprising the cell population.
본 발명의 또하나의 목적은 상기 세포 집단의 유효량을 개체에 투여하는 단계를 포함하는 혈관신생 촉진 방법을 제공하는 것이다. It is another object of the present invention to provide a method for promoting angiogenesis comprising administering to a subject an effective amount of said cell population.
본 발명의 또 하나의 목적은 상기 세포 집단의 유효량을 개체에 투여하는 단계를 포함하는 허혈성 질환 예방 또는 치료 방법을 제공하는 것이다. / ᅳ It is another object of the present invention to provide a method for preventing or treating ischemic disease, comprising administering to a subject an effective amount of said cell population. / ᅳ
【유리한 효과】 Advantageous Effects
본 발명의 세포는 혈관신생 촉진 작용을 가지므로, 혈관신생 촉진제로 유용하게 사용될 수 있으며: 나아가 혈관신생이 필요한 각종 질환의 예방 또는 치료 목적으로 사용될 수 있다. 또한， 본 발명의 세포
대량 수집 방법에 의하며 환자의 허혈 부위에 직접 여러 번 주사할 수 있는 양과 농도의 CDllb+CX3CRl+세포를 대량으로 손쉽게 수집할 수 있으며， 이와 같이 수집된 CDllb+CX3CRl+세포는 혈관신생 촉진 작용을 가지므로， 혈관신생 촉진제로 유용하게 사용될 수 았고. 나아가 혈관신생이 필요한 각종 질환의 예방 또는 치료 목적으로 사용될 수 있다. Since the cell of the present invention has an angiogenic promoting effect, it can be usefully used as an angiogenesis promoter: and furthermore, it can be used for the prevention or treatment of various diseases requiring angiogenesis. In addition, the cells of the present invention The bulk collection method uihamyeo to collect easily the amount and concentration, which can be scanned several times directly on the ischemic region of the patient CDllb + CX3CRl + cells in large quantities, and this way the CDllb + CX3CRl + cells collected as is of angiogenesis promoting action Therefore, it could be useful as an angiogenesis promoter. Furthermore, it can be used for the purpose of preventing or treating various diseases requiring angiogenesis.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 la는 마우스 하지 허혈 유도 전과 허혈 유도 4알후의 근육 내 CDllb+CX3CRl+ 세포 분획을 비교한 결과를 나타낸다. Fig. La shows the results of comparison of intramuscular CDllb + CX3CRl + cell fractions before and after ischemia induction in mice.
도 lb는 마우스 하지 허혈 유도 후 시간경과에 따른 근육 내 FIG. Lb is Intramuscular Over Time After Mouse Inferior Ischemia Induction
CDllb+CX3CRl+ 세포 수 변화를 나타낸다. CDllb + CX3CRl + cell number change.
도 lc는 마우스 하지 허혈 유도 후 시간경과에 따른 근육 내 Fkn 발현을 나타낸다. Figure lc shows the intramuscular Fkn expression over time after mouse lower limb ischemia induction.
도 Id는 허혈 근육에서 분리한 CDllb+CX3CRl+세포의 표면항원. 분석 결과를 나타낸다. Id is surface antigen of CDllb + CX3CRl + cells isolated from ischemic muscle. The analysis results are shown.
도 2a는 세포 주입에 따른 시간별 혈류량 비교 결과를 나타낸다. 도 2b는 Fractalkine단백질 주입에 따른 시간별 혈류량 비교 결과를 나타낸다. Figure 2a shows the comparison of the blood flow over time according to the cell injection. Figure 2b shows the comparison of blood flow over time according to the injection of Fractalkine protein.
도 3은 하지 허혈 유도 후 중화 항 -Fkn항체 및. 항 -CX3CR1 항체의 주입에 따른 혈류량 비교 결과를 나타낸다. Figure 3 is a neutralizing anti-Fkn antibody and after ischemia induction. Blood flow comparison results according to the injection of anti-CX3CR1 antibody are shown.
도 4a는 항ᅳ CX3CR1항체 주입 후 '허혈 유도 후 허혈 근육 내에서 혈관 형성 저해 효과를 직접적으로 관찰한 결과를 나타낸다. Figure 4a shows the result of directly observing the effect of inhibiting angiogenesis in the ischemic muscle after ischemia induction after injection of anti-CX3CR1 antibody.
도 4b는 항ᅳ CX3CR1항체 주입 후 래트 망막 내에서 혈관 형성 저해 효과를 직접적으로.관찰한 결과를 나타낸다. Figure 4b shows the results of direct observation of the effect of inhibiting angiogenesis in rat retina after injection of anti-CX3CR1 antibody.
도 4c는 CDllb+CX3CRl+세포 내에서 VEGF niRNA 및 단백질 발현이 증가 된다는 결과를 나타낸다. ' 도 5는 G-CSF투여 전 말초 혈액 (A)과 G-CSF 5일간 투여 후 말초 혈.액 및 수집한 농축 단핵구 (B)에서 측정한 CDllb+CX3CRl+세포 분획을 FACS 분석한 결과를 나타낸다.
도 6은 G-CSF와 AMD3100 의 병용 투여 후 말초 혈액과 농축 단핵구 내의 CDllb+CX3CRl+ 세포 분획을 FACS분석한 결과를 나타낸다'. 4C shows that VEGF niRNA and protein expression is increased in CDllb + CX3CRl + cells. "Figure 5 shows the result of the G-CSF prior to administration of peripheral blood (A) and G-CSF 5 ilgan a CDllb + CX3CRl + cell fraction measured in the peripheral blood. Solution and was collected and concentrated monocytes (B) after administration of a FACS analysis . Figure 6 shows the results of FACS analysis of CDllb + CX3CRl + cell fractions in peripheral blood and concentrated monocytes after co-administration of G-CSF and AMD3100 ' .
도 7은 정상인과 G-CSF투여 및 G— CSF와 AMD3100의 병용 투여 후 단위 말초 혈액 (mL)당 CDllb+CXXRl+세포 수를 비교한 결과를 나타낸다. FIG. 7 shows the results of comparing the number of CDllb + CXXRl + cells per unit peripheral blood (mL) after G-CSF administration and co-administration of G-CSF with AMD3100.
'. '.
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
. 단핵구 /골수성 계통의 세포들은 혈관신생을 촉진하는 성장 인자들을 분비하고 혈관을 형성하여 손상된 조직에 혈류 공급을 증가시킬 수 있는 것으로 알려져 있다. 그러나 아직까지 신생혈관형성에 참여하는 특정 단핵구 /골수성 계통의 세포 아집단 또는 이들의 작용 기전에 대해 명확히 알려져 있지 않으며，' 현재까지는 몇 가지 단핵구 /골수성'계통의 세포들이 대상으로 제안되고 있을 뿐이다. . Monocyte / myeloid lineage cells are known to secrete growth factors that promote angiogenesis and to form blood vessels that increase blood flow to damaged tissues. Yet only it has to do no angiogenesis is known clearly about the cell subset or a mechanism of action of certain monocyte / myeloid system to participate in the forming system of the cell, some of the monocyte / myeloid until now, have been proposed as a target.
； 본 발명자들은 이전의 연구에서 C57BL/6 마우스의 근육에서 허혈을 유도하였을 때. 골수 억제 세포 (myeloid derived suppressor cell)로 알려진 CDllb+Grldim 세포가허혈 근육 내에서 증가하며， 이 세포를 허혈 근육에서 분리하여 다시 주사하면, 허혈 근육에서의 혈류량이 현저하게 증가한다는 것을 확인한 바 있다. 이후 연구를 계속한 결과, CDllb+Grldim세포가 CX3CR1+, CCR2+, CCR5+, CXCR4+단핵구를 포함하는 단핵구 /대식세포 계열의 세포임을 확인하였으며， 특히 대퇴부 동맥 절개 후 허혈 근육에서 CDllb+CX3CRl+ 세포의 수가 눈에 띄게 증가하는 것을 발견하였다. When we induced ischemia in the muscle of C57BL / 6 mice in a previous study. CDllb + Grl dim cells, known as myeloid derived suppressor cells, increase in ischemic muscle, and when isolated from the ischemic muscle and injected again, the blood flow in the ischemic muscle was significantly increased. . Subsequent studies confirmed that CDllb + Grl dim cells were monocyte / macrophage-type cells including CX3CR1 + , CCR2 + , CCR5 + , CXCR4 + monocytes, especially CDllb + CX3CRl in ischemic muscle after femoral artery dissection. It was found that the number of + cells increased significantly.
- 본 발명자들은 상기 CDllb+CX3CRl+세포와 혈관 신생 사이의 연관성을 조사하기 위해. C57BL/6마우스의 하지 허혈 모델을 이용하여， 마우스의 대퇴부 동맥을 절개하여 허혈 * 유도한다리에서 시간이 지남에 따라 To investigate the association between the CDllb + CX3CRl + cells and angiogenesis. Using the C57BL / 6 mouse lower limb ischemia model, the femoral artery of the mouse is incised to induce ischemia * inducing limbs over time.
CDllb+CX3CRl+세포 수가 변화하는 패턴을 분석하였고, CDllb+CXXRl+세포를 허혈 근육 내로 주입할 경우 혈류량의 변화와， CDllb+CX3CRl+세포에 대한 항체를 전처리한 경우 혈류량의 변화를 분석하였다. The patterns of CDllb + CX3CRl + cell number change were analyzed, and the changes in blood flow when CDllb + CXXRl + cells were injected into the ischemic muscle and the changes in blood flow when pretreated with CDllb + CX3CRl + cells were analyzed.
그 결과， 허혈 유도.4일까지 CDllb+CX3CRl+세포의 수가 급격하게 증가하다가 점점 감소하여 허혈 유도 14일째에는 허혈 유도 전과 유사한 수준으로 세포 수가 감소되는 것을 확인하였으며ᅳ 이를 통해 CDllb+CX3CRl+ 세포 수가 혈관 재생이 왕성하게 일어나는 시기에 현저하게 증가되는 것을
알 수 있었다. 또한 CX3CR1+세포 이동에 영향을 주는 것으로 알려진 Fkn (fractalkine; CX3CL1)의 발현의 변화가 CDllb+CX3CRl+세포 수 변화 패턴과 유사하게， 허혈 유도 4일째 발현이 최고로 나타났다가 점차 감소하는 것을 확인할 수 있었다 (도 la 내지 lc). . As a result, by the day ischemia induced CDllb + .4 ischemia induction on day 14 while the number sharply increased to more reduction in CX3CRl + cells was confirmed that the cell count is reduced to roughly before ischemia induction eu this CDllb + CX3CRl + cell number with Significantly increased when blood vessel regeneration occurs vigorously Could know. In addition, the expression of Fkn (fractalkine; CX3CL1), which is known to affect CX3CR1 + cell migration, was similar to the CDllb + CX3CRl + cell number change pattern. (Figures la to lc). .
- 또한, 마우스 허혈'근육에서 분리한 CDllb+CX3CRl+세포를 다시 허혈 근육에 직접 주사하면， 시간이 지남에 따라 혈류량이 점차 증가하는 것을 발견하였으며 , 이러한 혈류량 증자 효과는 CDllbXX3CRl_세포 또는 - Also, when injected directly into the mouse ischemia, ischemia of CDllb + CX3CRl + cells isolated from muscle back muscle was found that blood flow is increasing over time, these effects increase blood flow to the cells or CDllbXX3CRl _
CDllb+CX3CRl" 세포를 주입한그룹보다 월등히 높게 나타났다. 나아가, 이 세포의 이동과 관련된 Fkn 단백질을 단독으로 주입한 군에서도 유효량 범위에서 유의한 수준의 혈류량 증가가 확인되어， CDllb+CX3CRl+세포가 허혈성 손상 후 새로운 혈관 형성에 중요한 역할을 하는 세포임을 확인할 수 있었다 (도 2a 및 2b) · CDllb + CX3CRl "had significantly higher cell than the injection group. Furthermore, is verified by the cells increases in a significant level of blood flow in an effective amount range in the group alone introduced into the Fkn protein associated with the movement of, CDllb + CX3CRl + cells It was confirmed that the cells play an important role in the formation of new blood vessels after ischemic injury (FIGS. 2A and 2B).
아울러， 중화 항 -CX3CR1.항체 또는 중화 항 -Fkn항체를 마우스에 전 처리하여 CDllb+CX3CRl+세포 또는 Fkn 의 기능을 차단할 경우 CDllb+CX3CRl+ ' 세포의 혈류량 개선 기능이 떨어지는 것으로 나타나, CX3CR1과 Fkn의 반웅이 신생 혈관 생성에 중요한 역할을 하는 것을 알 수 있었다 (도 3). 또한， 항 CX3CR1 항체를 허혈 근육 및 생후 2일째 래트 망막에 주사하였다. 정상적으로는 허혈 부위 근육에 새로운 혈관 구조들이 형성되나， 항 -CX3CR1 항체를 주사하면 허혈 부위 근육에 신생 혈관이 생성되지 않는 것을 확인하였다 (화살표.; 그림 F, 도 4a). 또, 래트 망막혈관 형성 시에도 항 CX3CR1 항체를 주사하면, 정상적으로 형성도는 망막 혈관과 In addition, blocking the function of CDllb + CX3CRl + cells or Fkn by pretreatment of neutralizing anti-CX3CR1. Antibody or neutralizing anti -Fkn antibody to mice was found to impair the blood flow improvement of CDllb + CX3CRl + ' cells. It was found that the reaction played an important role in the generation of neovascularization (FIG. 3). In addition, anti-CX3CR1 antibody was injected into the ischemic muscle and rat retina at day 2 of birth. Normally, new blood vessel structures are formed in the ischemic muscle, but the injection of anti-CX3CR1 antibody did not produce new blood vessels in the muscle of the ischemic muscle (arrow F; Fig. 4A). In addition, when anti-CX3CR1 antibody is injected during rat retinal blood vessel formation,
비교하여 (그림 A와 B, 도 4b) 조직화된 혈관 형성이 저해된다는 것을 (그림 C와 D, 도 4b) 면역 형광 염색을 통해 확인하였다. 이러한 결과를 종합하여 CDllb+CX3CRl+세포가 새로운 혈관 형성을 촉진하고, 신생 혈관 구조 형성을 조절하는 기능이 있는 세포로 직접적으로 혈류량을 개선하는 효과가 있다는 결론에 이르게 되었다. 이러한 결과들과 더불어， Cr)llb+CX3CR1+세포는 VEGF의 mRNA와 단백질을 발현하는 세포로 확인되었으며， 이는 이 세포가 허혈 부위의 혈관내피세포의 증식을 촉진하여 혈관 생성을 증가 시키는 효과가 있을 부가적으로 있을 가능성을 시사 하였다 (도 4c).
이에， 본 발명자들은 CDllb+CX3CRl+세포를 혈관 형성 촉진제 및 허혈성 질환 치료제로 제공하는 본 발명을 완성하게 되었다. In comparison (Fig. A and B, Fig. 4b) it was confirmed by immunofluorescence staining that the formation of organized blood vessels is inhibited (Fig. C and D, Fig. 4b). These results led to the conclusion that CDllb + CX3CRl + cells have the effect of directly improving blood flow to cells that have the function of promoting new blood vessel formation and regulating the formation of new blood vessel structure. In addition to these results, Cr) llb + CX3CR1 + cells have been identified as cells expressing VEGF mRNA and protein, which may have the effect of promoting the proliferation of vascular endothelial cells in the ischemic area to increase angiogenesis. Additionally suggested the possibility (FIG. 4C). Accordingly, the present inventors have completed the present invention for providing CDllb + CX3CRl + cells as an angiogenesis promoting agent and an ischemic disease therapeutic agent.
한편. 상기. CDllb+CX3CRl+세포는 안위적으로 대동맥을 절단하고 근육세포를 분리하여 얻은 세포로， 실제 환자의 허혈 부위에 투여하는 세포 치료제로 사용하기 위해서는 상기 세포를 치료학적 유효량으로 대량으로 , 수집할 수 있어야.한다. 이에, 본 발명자들은 CDllb+CX3CRl+세포를 대량으로Meanwhile. remind. CDllb + CX3CRl + cells in cells obtained by cutting the aorta to the comfort enemy and remove the muscle cell, for use as a cell therapeutic agent for administration to the ischemic site of the actual patient in large quantities of the cells in a therapeutically effective amount, to be able to collect .do. In this regard, we found that CDllb + CX3CRl + cells in large quantities.
' 수집하기 위한 연구를째속한 결과， CDllb+CX3CRl+세포는 정상적으로 혈액 ' 내에는 거의 존재하지 않지만, G-CSF를 단독으로, 또는 G-CSF 및 AMD3100을 함께.투여한 개체로부터 혈액 단핵구를 분리 후 농축한, 소위 "농축 단핵구' '를 만들었을 때, 다량의 농축된 CDllb+CX3CRl+세포를 손쉽게 대량으로 얻을 수 있다는 것을 확인하여 (도 5내지 도 7)， 본 발명에 이르게 되었다.. "Result belongs Me research to collect, CDllb + CX3CRl + cells are normally blood, do not almost present in, the G-CSF alone, or with G-CSF and AMD3100. Administration separating blood mononuclear cells from the individual When the concentrated, so-called "concentrated monocytes" were made, it was confirmed that a large amount of concentrated CDllb + CX3CRl + cells can be easily obtained in large quantities (Figs. 5 to 7), thereby leading to the present invention.
따라서 , 본 발명은 또한, CDllb+CX3CRl+세포를 대량 수집할.수 있는 방법 및 이에 이용되는 조성물을 제공한다. Accordingly, the present invention also provides methods and compositions for use in the mass collection of CDllb + CX3CRl + cells.
이하, 본 발명을 보다 상세하게 설명한다. Hereinafter, the present invention will be described in more detail.
하나의 양태 ^서 본 발명은 CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하며 혈관신생촉진 기능을 나타내는 세포에 관한 것이다. FIELD OF THE INVENTION The present invention relates to cells comprising the immune phenotypes of CDllb + and CX3CR1 + and exhibiting angiogenic function.
또 하나의 양태로서 본 발명은 CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하는 세포를 포함하는 혈관신생 촉진용 조성물에 관한 것이다. As another aspect, the present invention relates to a composition for promoting angiogenesis comprising a cell comprising an immune phenotype of CDllb + and CX3CR1 + .
또 하나의 양태로서 본 발명은 상기 세포의 유효량을 개체에 투여하는 단계를 포함하는 혈관신생 촉진 방법에 관한 것이다. As another aspect, the present invention relates to a method for promoting angiogenesis comprising administering to the individual an effective amount of the cells.
또 하나의 양태로서 본 발명은 상기 CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하는 세포를 포함하는 허혈성 질환 치료용 약학 조성물에 관한 것이다. As another aspect, the present invention relates to a pharmaceutical composition for treating ischemic disease comprising a cell comprising the immune phenotype of the CDllb + and CX3CR1 + .
또 하나의 양.태로서 본 발명은 상기 세포의 유효량을 개체에 투여하는 단계를 포함하는 허혈성 질환 예방 또는 치료 방법에 관한 것이다.. In another aspect, the present invention relates to a method for preventing or treating ischemic disease, comprising administering to a subject an effective amount of said cell.
또 하나의 양태로서， 본 발명은 G-CSF (granulocyte colony stimulating factor)를 ΐ함하는, CDllb+ 및 CX3CR1+의 면역 표현형을 포함하는 세포의 대량 수집용 조성물에 관한 것이다.
바람직한 양태로서,, 상기 조성물은 AMD3100또는 약학적으로 허용되는 이와 염을 추가적으로 포함할 수 있다. 따라서 , 본 발명은 또한， Gᅳ CSF 및 AMD3100또는 약학적으로 허용되는 이의 염을 포함하는， CDllb+및 CX3CR1+의 면역 표현형을 포함하는 세포의 대량 수집용 조성물에 관한 것이다. In another aspect, the present invention relates to a composition for mass collection of cells comprising an immune phenotype of CDllb + and CX3CR1 + , which contains a granulocyte colony stimulating factor (G-CSF). In a preferred embodiment, the composition may comprise this or a pharmaceutically acceptable salt of AMD3100 further. Accordingly, the present invention also relates to a composition for mass collection of cells comprising an immune phenotype of CDllb + and CX3CR1 + , comprising G ᅳ CSF and AMD3100 or a pharmaceutically acceptable salt thereof.
또 하나의 양태로서 , 본 발명은 G-CSF 를 포함하는 조성물을 투여한 개체의 말초'혈액으로부터 혈액 단핵구 분획을 분리하는 단계 및 상기 혈액 단핵구 분획을 농축하는 단계를 포함하는, CDllb+ 및 CX3CR1+의 면역 As another aspect, the invention provides a CDllb + and CX3CR1 + comprising the steps of isolating the blood monocyte fraction from the peripheral ' blood of the subject to which the composition comprising G-CSF is administered and concentrating the blood monocyte fraction. Immunity
표현형을포함하는 세포의 대량 수집 방법에 관한 것이다. A method of mass collection of cells comprising a phenotype.
바람직한 양태로서，상기 조성물은 AMD3100또는 약학적으로 허용되는 이의 염을 추가적으로 포함할 수 있다. 따라서，. 본 발명은 또한, G-CSF 및 AMD3100또는 약학적으로 허용되는 이의 염을 포함하는 조성물을 투여한 개체의 말초 혈액으로부터 혈액 단핵구 분획을 분리하는단계 및 상기 혈액 단핵구 분획을 농축하는 단계를 포함하는， CDllb+ 및 CX3CR1+의 면역 In a preferred embodiment, the composition may further comprise AMD3100 or a pharmaceutically acceptable salt thereof. therefore,. The present invention also includes the steps of separating the blood monocyte fraction from the peripheral blood of an individual administered with a composition comprising G-CSF and AMD3100 or a pharmaceutically acceptable salt thereof and concentrating the blood monocyte fraction, Immunity of CDllb + and CX3CR1 +
표현형을 포함하는 세포의 대량 수집 방법에 관한 것이다. A method of mass collection of cells comprising a phenotype.
바람직한 양태로서， 상기 혈액 단핵구 분획을 분리하는 단계는 혈액 . 성분 채집기를 이용하여 수행될 수 있다： In a preferred embodiment, the step of separating the blood monocyte fraction is blood. This can be done using an ingredient collector:
또한 바람직한 양태로서， 상기 혈액 단핵구 분획을 농축하는 단계는 혈액' 원심분리기를 이용하여 수행될 수 있다. Also in a preferred embodiment, the step of concentrating the blood monocyte fraction may be performed using a blood ' centrifuge.
또 하나의 양태로서, 본 발명은 상기 방법을 이용하여 대량 수집되며， CDllb+ 및 CXXR1+의 면역 표현형을 포함하는 세포가 10% 이상 포함된 세포 집단에 관한 것이다. In another aspect, the present invention relates to a cell population which is mass collected using the above method and contains at least 10% of cells comprising the immune phenotypes of CDllb + and CXXR1 + .
- 본 발명자들은 C57BL/6 마우스의 대퇴부 동맥을절개하여 허혈을 . 유도한 다리 근육에서 처음 CDllb+CX3CRl+세포를 분리한 후， 이 세포가ᅵ어떤 계통의 세포인지를 보다 정확히 확인하기 위하여 표면 항원을 조사하였다. 그 결과, 상기 CDllb+CX3CRl+ 세포는 골수세포 (CD45)와 대식세포 (F4/80) 양성이었으나， 내피세포 (VEGFR-2). 수지상세포 (CDllc),服세포 " (NK1.1) 및 T세포 (CD3)에 대한 표면 항원은 발현되지 않아， 허혈 근육 내로 이동한 CDllb+CX3CRl+세포는 단핵구 /대.식세포 계통의 세포임을 알 수 있었다. 특히， CDllb+CX3CRl+세포는 현재까지 혈관형성에 관여하는 것으로 알려진 단핵구의 표면 항원인 VEGFRᅳ 1, Tie— 2 그리고 CX3CR4항원이 모두 양성으로 이 세포는
본 발명자들이 처음으로 분리 및 제공하는 혈관생성에 관여하는 기능성 세포에 해당한다 (도 Id). The present inventors dissected the femoral artery of C57BL / 6 mice to develop ischemia . Remove the first CDllb + CX3CRl + cells derived from one leg muscles after, the surface antigens were examined in order to accurately determine the cell whether i than any system cells. As a result, the CDllb + CX3CRl + cells were positive for bone marrow cells (CD45) and macrophages (F4 / 80), but endothelial cells (VEGFR-2). Surface antigens for dendritic cells (CDllc), dry cells " (NK1.1) and T cells (CD3) were not expressed, suggesting that CDllb + CX3CRl + cells migrated into ischemic muscle are cells of the monocyte / macrophage lineage . In particular, CDllb + CX3CRl + cells were positive for all of the monocyte surface antigens VEGFRX1, Tie-2 and CX3CR4, which are known to be involved in angiogenesis. Corresponds to the functional cells involved in the angiogenesis we first isolate and provide (FIG. Id).
따라서， 바람직하게, 본 발명의 세포는 CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하는 세포이다. Thus, preferably, the cells of the invention are cells comprising the immune phenotypes of CDllb + and CX3CR1 + .
보다 바람직하게， 본 발명의 세포는 CDllb+및 CX3CR1+의 면역 표현형 외에， CD45+, F4/80+, Tie2+, CXCR4+, VEGFR1+, VEGFR2", ΝΚ1.Γ, CDllc", CD3" 및 CD19-에서 선택되는 하나 이상의 '면역 표현형을 추가로 포함하는 세포일 수 있다. ᅳ . More preferably, the cells of the present invention, in addition to the immunophenotypes of CDllb + and CX3CR1 + , include CD45 + , F4 / 80 + , Tie2 + , CXCR4 + , VEGFR1 + , VEGFR2 " , ΝΚ1.Γ, CDllc " , CD3 " and CD19 - it may be a cell which further comprises one or more "immune phenotype is selected from the eu.
나아가， 상기 면역 표현형의 특징을 나타내며 혈관신생 촉진 작용을 나타내는 한， 추가적인 다른 면역 표현형 또는 형태학적 특징이 부가된 세포들도 본 발명의 세포의 범위에 포함될 수 있다. Furthermore, as long as they exhibit the characteristics of the immune phenotype and exhibit angiogenesis-promoting action, cells to which additional additional immune phenotypes or morphological features are added may be included in the scope of the cells of the present invention.
¾ 발명의 세포는 마우스에 인위적으로 하지 대동맥을 절단하고 근육세포를 분리하여 .수득한 것이나, 그 수득 방법이 이에 한정되는 것은 아니며， 마우스를 포함한 각종 포유동물의 허혈 조직 또는 혈액으로부터 수득할 수 있으며， 당업계에 알려진 지식을 이용한 체외 배양， 분화 및 /또는 증폭에 의하여 대량으로 얻을 수 있다. 예를 들어, 허혈 조직에 ¾ The cells of the invention artificially cut the lower limb aorta and isolate muscle cells in mice . The obtained method is not limited thereto, and may be obtained from ischemic tissues or blood of various mammals including mice, and may be obtained in large quantities by in vitro culture, differentiation and / or amplification using knowledge known in the art. You can get it. For example, in ischemic tissue
콜라게나아제 및 /또는 단백질분해효소 등을 처리하여 조직을 해리하고， Dissociate tissue by treating collagenase and / or protease,
Ficoll-Hypaque 분리 방법， nioidfied Ficol 1-Hypaque 분리방법， 3% 젤라틴 분리방법, Per col Γ분리방법, 패닝 (panning), 면역자장 Ficoll-Hypaque separation method, nioidfied Ficol 1-Hypaque separation method, 3% gelatin separation method, Per col Γ separation method, panning, immunomagnetic field
분리방법 ('Immun에 lagnet ic eel 1 sorting), '유세포 분석방법 (Fluorescence activated cell sorting) 등에 따라'분리할 수 있다. 상기 제시한 방법은 : 대표적인 예시에 불과하며， 본 발명의 세포는 당업계에 공지된 지식을 It can be separated according to the separation method ('lagnetic eel 1 sorting on immun ' ), ' fluorescence activated cell sorting ' . The above-mentioned method is proposed: a mere typical example, the cells of the present invention the known knowledge in the art
활용하여 다양한 방법으로 수득할 수 있다. Can be obtained by various methods.
본 발명의 세포는 혈관신생 촉진 작용을 가지므로, 혈관신생 촉진제로 유용하게 사용될 수 있으며, 나아가 혈관신생이 필요한 각종 질환의 예방.또는 치료 목적으로 사용될 수 있다. Since the cell of the present invention has an angiogenic promoting effect, it can be usefully used as an angiogenic promoter, and furthermore, can be used for the prevention or treatment of various diseases requiring angiogenesis.
혈관신생이 필요한 대표적인 질환으로 허혈성 질환을 들 수 있다. . 허혈성 질환이란 동맥의 협착 또는 수축， 혈전 또는 색전 등 다양한 원인에 의하여 부분적으로 혈액이 부족해 나타나는 질환을 말한다. 허혈성 질환의 ' 예로는 심부전， 고혈압성 심장질환, 부정맥， 섟천성 심장질환, 심근경색, .
뇌졸중， 말초혈관질환， 협심증,,뇌혈관성 치매， 관상동맥부전. 뇌부전， 망막 - 허혈 및 혈관부전 등을 들 수 있으나, 이에 제한되는 것은 아니며， Ischemic disease is a representative disease that requires angiogenesis. . Ischemic disease refers to a disease in which blood is partially deficient due to various causes such as narrowing or contraction of arteries, blood clots or embolism. "Examples of the ischemic diseases are heart failure, hypertensive heart disease, arrhythmia, nature seok heart disease, myocardial infarction. Stroke, peripheral vascular disease, angina pectoris, cerebrovascular dementia, coronary artery failure. Brain failure, retinal-ischemia and vascular failure, but is not limited thereto.
. 혈관신생이 필요한 모든 허혈성 질환이 본—발명의 범위에 포함될 수 있다. . All ischemic diseases requiring angiogenesis can be included in the scope of the present invention.
본 발명에서、사용되는 용어 "예방 "은 본 발명의 조성물의 투여로 혈관신생을 촉진시키는 모든 행위， 또는 혈관신생 의존성 질환의 성장， 확산 또는 재발 ^ 저해 또는지연시키는모든 행위를 의미한다. As used herein, the term "prevention" refers to any action that promotes angiogenesis by administration of a composition of the present invention, or any action that causes growth, spread or recurrence of ^ angiogenesis-dependent diseases.
본 발명에서 사용되는 용어 "치료" 및 "개선"은 본 발명의 조성물의 투껴로 혈관신생 의존성 질환이 호전 또는 이롭게 변경되는 모든 행위를 " 의미한다 . The present invention the term "treatment", and "improvement" as used refers to all actions which improve the angiogenesis dependent disease or advantageously changed to tukkyeo of the compositions of the present invention ".
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에 소정의 본 발명의 조성물을 제공하는 것을 의미한다. As used herein, the term "administration" means providing a subject with a composition of the present invention in any suitable manner.
본 발명에서 사용되는 용어 "개체 "는 본 발명의 조성물을 투여하여 혈관신생 의존성 질환이 호전될 수 있는 질환에 걸린 동물로， 바람직하게는 척추동물，보다 바람직하게는 포유동물이며, 예컨대 인간， 원숭이, 개， 고양이^ 염소， 돼지， 쥐，. 기니아피그 햄스터， 침팬지 또는 고릴라 등 모든 동물을 -의미한다. The term "individual" as used herein refers to an animal suffering from a condition in which angiogenic dependent disease can be improved by administering the composition of the present invention, preferably a vertebrate, more preferably a mammal, such as a human being, a monkey. , dogs, cats ^ goats, pigs, and rats. Means all animals such as guinea pig hamsters, chimpanzees or gorillas.
본 발명에서 사용되는 용어 "유효량"은 질환을 예방 또는 치료하기에 충분한 양을 의미하며, 이는 개체의 질환의 종류. 중증도， 약물의 활성， 약물에 대한 민감도， 투여 시간, 투여 경로 및 배출비율, 치료기간， 동시에 사용되는;약물을 포함한 요소 및 기타 와학 분야에 잘 알려진 요소에 따라. 결정될 수. 있다： ' ■ As used herein, the term “effective amount” means an amount sufficient to prevent or treat a disease, which is the type of disease of the individual. Severity, drug activity, drug sensitivity, time of administration, route and route of administration, duration of treatment, used concurrently; depending on factors including drug and other well known factors in the scientific field. Can be determined. There is ： '■
본 발명의 조성물은 세포 치료제로서, 경구 또는 비경구 투여할 수 있으며, 비경구 투여시 복강내 주사， 직장내 주사， 피하 주사， 정맥 주사， 근육내 주사， 자궁내 경막 주사， 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고， 직접 질환 부위에 투여될 수 있다. ' , The composition of the present invention may be administered orally or parenterally as a cell therapy, and may be intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or It can be administered by intrathoracic injection and can be administered directly to the disease site. ' ,
. . 바람직하게， 본 발명에 따른 약학적 조성물은 주사용 조성물과 흔합하여 포유동물의 혈관신생을 유도하고자 하는 부위에 주사 형태로 투여할 수 있고， 주사용 조성물은 등장성 수용액 또는 현탁액이 바람직하며， 상기 약학 조성물은 멸균되거나 방부제， 안정화제， 습윤제, 유화제 용액 촉진제， 삼투압 조절을 위한 염 및 /또는 완충제와 같은 보조제 또는기타
치료적으로 유용한 물질을 함유할 수 있다. 이 외에도， 본 발명에 따른 ' ' 약학적 조성물은 통상의 방법에 따라 경구형 제형， 외용제 , 좌제.등의 형태로 제형화하여 사용될 수 있다. ' ᅳ. 또한， 본 발명의 조성물은 복구또는 확장이 필요한 부위에 수술을 통해 이식하거나 , 카테터나 매트릭스， 또는 주사 바늘 등을 이용하여 전달하^ 등 , 약학적으로 허용되는 담체와 함께 직접 또는 간접적으로 투여할 수 있다. 약학적으로 허용되는 담체로는 본 발명의 세포, 그 조성물 또는 성분과 유해하게 반웅하지 않는 적합한 유기 또는 유기 담체 물질을 할 수 있으며， 물， 염 용액 (링거액), 알코올， 오일， 젤라틴， 락토오스， 아밀로오스 또는 전분과 같은 탄수화물; 지방산 에스테르， . . Preferably, the pharmaceutical composition according to the present invention may be administered in the form of an injection to a site intended to induce angiogenesis of a mammal in combination with the composition for injection, and the composition for injection is preferably an isotonic aqueous solution or suspension. Pharmaceutical compositions may be sterile or preservatives, stabilizers, wetting agents, emulsifier solution accelerators, auxiliaries such as salts and / or buffers for osmotic pressure control or other It may contain therapeutically useful substances. In addition, "" a pharmaceutical composition suitable for oral formulations, for external application, suppository by a conventional method according to the invention. It may be used in the form of a formulation. ' ᅳ. In addition, the composition of the present invention can be administered directly or indirectly with a pharmaceutically acceptable carrier, such as implanted by surgery to a site requiring repair or expansion, or delivered using a catheter, matrix, or needle. Can be. Pharmaceutically acceptable carriers may be any suitable organic or organic carrier material which does not adversely react with the cells, compositions or ingredients of the invention, including water, salt solutions (Ringer's solution), alcohols, oils, gelatin, lactose, Carbohydrates such as amylose or starch; Fatty acid ester ，
하이드록시메틸셀를로스 및 폴리비닐 피롤리돈 등을 예시할 수 있다. 이러한 조제물들은 멸균할 수 있으며， 적절한 경 에 윤활제， 보존제： 안정화제， 습윤제， 에멀견화제 . 삼투압을 위한 염， 완충액 및 발색제와 같은 보조제와 흔합할 수 있다 . 본 발명에 이용하기 적합한 약학 담체는 당업계에 공지되어 있다. Hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like. These preparations may be sterile and, where appropriate, lubricants, preservatives: stabilizers, wetting agents, emulsifiers. It may be compatible with auxiliaries such as salts, buffers and coloring agents for osmotic pressure. Pharmaceutical carriers suitable for use in the present invention are known in the art.
또한， 본 발명의 조성물은 선택적으로， 다른 유익한 약물이나 생물 분자 (성장 인자， 영양 인자 등)함께 투여할 수 있다. 함께;투여할 수 있는 생물 분자로는 항-세포사멸제， 항―염증제, 면역조절제, 항-증식제, 코르티코스테로이드. 항체, 항-혈전제, 항-산화제， 또는 국소 마취제를 ■ 포함할 수 있다. ' . ' In addition, the compositions of the present invention may optionally be administered together with other beneficial drugs and biomolecules (growth factors, nutritional factors, etc.). Together; biological molecules that can be administered include anti-cell killing agents, anti-inflammatory agents, immunomodulators, anti-proliferative agents, corticosteroids. Antibody, an anti-oxidant may include, or local anesthetics ■-thrombotic agents, anti. ''
약학작 유효량으로 상기 세포를 1 X 104내지 10 X 1010 cells/kg, 바람직하게는 2.5 X 107내지 5 X 107개, 보다 바람직하게는 2.5 X 107 cells /kg을사용할 수 있으나， 아에 제한 되는 것은 아니며， 예컨대 성인 70 kg을 기준으로 2X109내지 4X109개로 사용될 수 있다.그러나 특정 환자와 체중, 연령，성별，건강상태,.식이,투여기간,투여방법， 제거율， 질환의 중증도,등에 따라 변화될 수 있다. 본 발명의 약학적 조성물은 반감기에 따라 일일 수회. 내지 일주일에 1회 투여할 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.
한편 ;본 발명은 상기 CDllb+CX3CRl+세포를 치료학적 유효량으로 대량 수집하기ᅳ워하여， G-CSF (granulocyte colony stimulating factor) 및 /또는 AMD3100를 포함하는 조성물을 사용함을 특¾으로 한다. " G-CSF 는 호중성 과립구 전구세포를 증식 또는 분화시키고， 성숙 호중구를 활성화하는 인자로서， 주로 골수이식, 암의 화학요법에 의한 호중구 감소증, 골수이형성 증후군， 재생불량성 빈혈， 선천성 ·특발성 호중구' 감소증,. 인간면역결핍 바이러스 (HIV) 감염증 등에 있어서의. 호중구 증가 촉진에 사용되고 있다. 본 발명에서 G— CSF는.， CDllb+CX3CRl+세포 말초 혈액에 가동화 시켜 종국적으로 CDllb+CX3CRl+세포가 농축된 농축 단핵구를 수득할 수 있게 한다. . In a pharmaceutically effective amount, the cells may be used at 1 × 10 4 to 10 × 10 10 cells / kg, preferably at 2.5 × 10 7 to 5 × 10 7 and more preferably at 2.5 × 10 7 cells / kg. It is not limited to children and can be used for example, from 2x10 9 to 4x10 9 based on 70 kg of adult. However, the weight, age, sex, health status, diet, duration of administration, method of elimination, disease The severity can change depending on , and so on. The pharmaceutical composition of the present invention can be used several times a day depending on the half-life . To once a week. The dosage does not limit the scope of the invention in any aspect. On the other hand, the present invention is to collect a large amount of the CDllb + CX3CRl + cells in a therapeutically effective amount, it is characterized by using a composition comprising a granulocyte colony stimulating factor (G-CSF) and / or AMD3100. " G-CSF is a factor that proliferates or differentiates neutrophil granulocyte progenitor cells and activates mature neutrophils, mainly bone marrow transplantation, neutropenia due to chemotherapy of cancer, myelodysplastic syndrome, aplastic anemia, congenital and idiopathic neutrophils . It has been used to promote neutrophil increase in attenuated, human immunodeficiency virus (HIV) infections, etc. In the present invention, G—CSF is mobilized to CDllb + CX3CRl + cells peripheral blood, and eventually CDllb + CX3CRl + cells are concentrated. To obtain concentrated monocytes.
본 발명에서 사용되는 G-CSF 는 공지된 아미노산 서열 정보를 통하여 합성하거나, 개체로부터 분리하여 사용하거나, 시판 제제로부터 입수하는 등, 다양한 경로를 통하여 얻을 수 있다. 예를 들어, 시판되는 G-CSF로는 나루토그라스팀 (상품명 노이압, 교와발효공업 제조), 필그라스팀 (상품명 그란, 산쿄 제조; 상품명 Granulokine,,호프만 라 로슈 제조; 상품명 G-CSF to be used in the present invention can be obtained through various routes, such as synthesis through known amino acid sequence information, used separately from an individual, or obtained from commercially available agents. For example, commercially available G-CSF includes narutograss (trade name Neuap, manufactured by Kyowa Fermentation Industry), filgrasteam (trade name Gran, manufactured by Sankyo; trade name Granulokine, manufactured by Hoffman La Roche;
Neupogen, 암젠 제조; 상품명 Leucostim, 동아제약)， 레노그라스팀 (상품명 노이트로진， 츄가이제약 제조;상품명 Granocyte, 아벤티스 제ᅵ조; 상품명 Neutrogin, 중외제약)， 페그필그라스팀 (상품명 Neulasta, 암젠 제조), . 살그라모스팀 (상품명 Leukine, 쉐링 제조) 등을 들 수 있다.. Neupogen, Amgen Manufacture; Name Leucostim, Dong-A), Reno glass side (trade name: Neu Trojan Jin, Chuo this Pharmaceuticals Ltd .; trade name Granocyte, Aventis The crude i; trade name Neutrogin, Choongwae Pharma Corporation), Peg peel glass side (trade name Neulasta, Amgen, Ltd.). Salgramos team (trade name Leukine, manufactured by Schering).
또한， 본 발명에서 사용되는 Gᅳ CSF 는 동등한 활성을 유지하는 한， 화학적으로 수식되어 있는 것도 본 발명의 범위에 포함돤다. 화학 수식의 방법으로는, 예를 들어 국제특허공개 WO00/51626에 기재된 방법 등을 들 수 있고，. 예를 들어 폴리알킬렌글리콜로 수식, 예를 들어 폴리에틸렌글리콜 (PEG) 로 수식한 G-CSF 활성을 갖는폴리펩티드가 포함될 수 있다. In addition, the G 'CSF used in the present invention is included in the scope of the present invention as long as it is chemically modified as long as it maintains the equivalent activity. As a method of a chemical modification, the method of international patent publication WO00 / 51626, etc. are mentioned, for example . Polypeptides having G-CSF activity, for example modified with polyalkylene glycols, for example polyethylene glycol (PEG), can be included.
AMD3100는 플레릭사포르 (Pier ixafor)라고도 불리우며 , 그 The AMD3100 is also called Pier ixafor,
화합물명이 1，1'_[1,4-페닐렌비스 (메틸렌)]비스 [1，4,8，11-테트라아자사이클 로테트라데칸] 인 화합물로서ᅳ 케모카안 수용체인 _CXCR4의 저해제 ― • (antagonist)로서 기능하는 것으로 알려져 있다 (미국특허 제 5,612,478호 '참조). 본 발명에서 AMD3100은 G-CSF 와 함께 사용되어, CDllb+CXXRl+세포
말초 혈액에 가동화 시켜 종국적으로 CDllb+CX3CRl+세포가 농축된 농축 ： 단핵구를 수득할 수 있게 한다. ' . Compounds were 1, 1 '_ [1,4-phenylene-bis (methylene)] bis [1,4,8,11- tetraaza cycle Rotterdam trad decane] eu in Kane mocha not _CXCR4 inhibitors of the receptor is a compound - • It is known to function as an antagonist (see US Pat. No. 5,612,478). In the present invention, AMD3100 is used in combination with G-CSF, CDllb + CXXRl + cells It is mobilized to peripheral blood to finally obtain a concentrated: monocyte enriched in CDllb + CX3CRl + cells. '
본 발명에서 사용되는 AMD3100는 당업계에 알려진 화학적 합성 방법에 의해 합성하거나 시판 제제'로부터 입수하는. 등， 다양한 경로를 AMD3100 used in the present invention is synthesized by chemical synthesis methods known in the art, or that available from commercially available preparations. And so on
5 통하여 얻을 수 있다. 예를 들어, 시판되는 AMD3100 으로는 샴품명 Can be obtained through 5. For example, the commercially available AMD3100 is Siamese
모조빌주 (사노피 -아벤티스) 등을 들 수 있다. Mojoville (Sanopy-Aventis) and the like.
또한， 본 발명에서 사용도ᅵ는 AMD3100는 약학적으로 허용되는 염의 ¾태로도 사용이 가능하다. 예를 들어， 적합한 산 부가염으로는 ' In addition, in the present invention, AMD3100 can also be used in the form of pharmaceutically acceptable salts. For example, suitable acid addition salts are '
생체적합성인 무기 산 (예: HCl, HBr, 황산, 인산 등).뿐 만 아니라 유기 Biocompatible inorganic acids (e.g. HCl, HBr, sulfuric acid, phosphoric acid, etc.) as well as organic
10 산 (예 아세트산, 프로피온산， 부티르산 등)' 및 하나 이상의 카복실 그룹을 함유하는 산 (예: 옥살산, 글루타르산, 아디프산 등)의 염을 포함할 수 있다.Acid 10 (for example, acetic acid, propionic acid, butyric acid, etc.), and an acid containing at least one carboxyl group: can include salts (such as oxalic acid, glutaric acid, adipic acid, etc.).
■ 또한, 본 발명에서 사용되는 AMD3100는 프로드럭의 형태， 즉 ―. ■ Also, AMD3100 used in the present invention is in the form of a prodrug, ie.
대상체에게 투여한 후에 본 발명의 화합물을 방출시키는 보호 형태로 제조도ᅵ어 사용될 수도 있다. 또한， 정제된 형태로 제조될 경우, 화합물은 15 수화물호서 결정화될 수도 있다. . It may also be prepared in a protective form to release the compound of the invention after administration to a subject. In addition, when prepared in purified form, the compound may be crystallized in 15 hydrate. .
본 발명에서 상기 G-CSF 및 /또는 AMD3100을 포함하는 조성물을 이용하여 상기 CDllb+CXXRl+세포를 대량 수집하는 방법은， 보다 In the present invention, a method for mass collection of the CDllb + CXXRl + cells using the composition comprising the G-CSF and / or AMD3100, more
구체적으로는, ' Specifically, "
(a) G-CSF 및 /또는 AMD3100 또는 약학적으로 허용되는 이의 염을 (a) G-CSF and / or AMD3100 or a pharmaceutically acceptable salt thereof
20 포함하는 조성물을 투여한 7fl체의 말초 혈액으로부터 혈액 단핵구 분획을 분리하는 단계, 및 ᅵ . Separating a blood fraction from peripheral blood mononuclear cells of the body 7fl administering a composition including 20, and i.
(b) 상기 혈액 단핵구 분획을 농축하는 단계를 포함함을 특징으로 한다. ― . (b) concentrating the blood monocyte fraction. ―.
.본 발명에서 , 상기 단계 .(a) 는 G-CSF 및 /또는 AMD3100 또는 25 약학적으로 허용되는 이의 염을 포함하는 조성물을 투여한 개체의 말초 . In the present invention, the step. (A) is the peripheral of the subject administered a composition comprising G-CSF and / or AMD3100 or 25 pharmaceutically acceptable salts thereof
혈액으로부터 혈액 단핵구 분획을 분리하는 단계이다. Separating the blood monocyte fraction from the blood.
상기ᅵ개체는 척추동물, 보다 바람직하게는 인간을 포함한 포유동물일 ' 수 있다. 본 발명에서 대량수집된 세포는 혈관신생 촉진을 위한 치료적' 용도로 사용되므로, 가장 바람직하게는 치료 대상인 환자로부터 자가 농축 30 단핵구를 수득하는 것이 바람직하다.
" 이 단계에서, G-CSF는 단독으로，또는 AMD3100과 조합하여 투여될 수 있으며, Gᅳ CSF 및 AMQ3100을 함께 투여하는 경우 AMD3100 은 G—CSF의 투입 후 또는 동시에. 또는 그 전에 투여될 수 있다. 바람직하게 AMD3100 은 G-CSF의 투입 후 혈액 채집 10시간 전에 투여될 수 있으나， 이는 제한되는 것은 아니다. The object i is mammalian animal may be "including a vertebrate, more preferably a human. Mass Collected cells in the present invention preferably are used as a therapeutic "purpose to promote angiogenesis, to yield the most preferred self-enriched mononuclear cells from 30 patients subject to treatment. "At this stage, G-CSF may be administered alone or in combination with AMD3100, and when G100CSF and AMQ3100 are administered together, AMD3100 may be administered after or concurrently with the G—CSF. Preferably, the AMD3100 may be administered 10 hours before blood collection after the addition of G-CSF, but this is not limiting.
G-CSF 및 /또는 AMD3100의 투여 형태는 특별한 제한은 없으며， 바람직하게는 주사 등으로 비경구 피하 투여될 수 있다. 또한. 투여 대상의 종류와 상태 등을 고려하여' CDllb+CX3CRl+세포 말초 혈액에 가동화시키기에 충분한 양으로 1회 또는 수회에 걸쳐 투여될 수 있으며， 바람직하게는 G— CSF의 경우 1내지 100 zg/kg，바람직하게는 10 g/kg의 용량으로 1회 또는 수회에 걸쳐 투여될 수 았고 , AMD3100의 경우 100 내지 500 ^g/k , 바람직하게는 250 /jg/kg의 용량으로 1회 또는 수회에 걸쳐 투여될 수 있다. 그러나 상기 투여 형태， 투여 횟수. 및 투여량은 당업자의 판단에 의하여 적절히 변경될 수 있다. The dosage form of G-CSF and / or AMD3100 is not particularly limited and may be preferably administered parenterally subcutaneously by injection or the like. Also. In consideration of the type and condition of administration, ' CDllb + CX3CRl + cells may be administered once or several times in an amount sufficient to be mobilized to peripheral blood, preferably 1 to 100 zg / kg for G—CSF. It may preferably be administered once or several times at a dose of 10 g / kg, and once or several times at a dose of 100 to 500 ^ g / k, preferably 250 / jg / kg for AMD3100. May be administered. But the dosage form ， the number of doses. And the dosage may be appropriately changed by the judgment of those skilled in the art.
상기 단계 (a) 에서 말초 혈액으로부터 혈액 단핵구 분획을 분리하는 것은 당업계에 알려진 혈액성분 채집 방밥을 이용하여 수행할 수 있으며, 예를 들어， 개체에 G-CSF 를 투여한 다음 말초 혈액을 혈액 성분 채집기 , (apheresis machine)에 통과시켜 수집할 수 있다. 당업계에 많이 사용되는 혈액 성분 채집기로는 MCS 3p,CS-3000 plus.COBE Spectra 등을 예시할 수 있으나, 본 발명이 이에 제한되는 것은 아니다. 중심 정맥 카테터를 통해 혈액이 혈액 성분 채집기로 들어가면 혈액 단핵구 분획이 기계에 의해 채집이 되고 남은 혈액들은 카테터를 통해 다시 체네로 들어갈 '수 있다. 이 과정은 약 4 내지.10시간 정도 걸릴 수 있으며 , 치료학 ^ 목적으로 Separating the blood monocyte fraction from the peripheral blood in step (a) can be carried out using blood component collection methods known in the art, for example, administering G-CSF to a subject and then dispensing peripheral blood into blood components. It can be collected by passing it through an apheresis machine. Examples of blood component collectors used in the art can be exemplified by MCS 3p, CS-3000 plus COB Spectra, but the present invention is not limited thereto. The heart, blood enters the catheter through the venous blood group apheresis blood mononuclear cell fraction to be collected by the machine remaining blood can "back into Nero body through a catheter. This process can take about 4 to 10 hours and is intended for therapeutics ^ purposes.
사용하기에 충분한 세포 분획을 채집하려면 2 내지 10일간 연속적으로 ' 반복할 수 있다. ■ 一 본 발명에서ᅳ 상기 단계 (b) 는 단계 (a) 에서 분리한 혈액 단핵구 분획을 농축하는 단계이다. To collect enough cell fractions for use, it may be repeated ' 2-10 days in a row. In the present invention, step (b) is a step of concentrating the blood monocyte fraction isolated in step (a).
바람직하게, 혈액 단핵구 분획의 농축은 혈액 원심분리기를 이용하여 수행될 수 있다. 원심분리 조건은 당업자가 상황에 따라 적절하게 조절할 수 있으며， 혈장 성분을 제거하고 단핵구 성분들이 포함된 농축액을 수득할 수
있다. 본 발명의 구체적인 실시 예에서는 분당 회전수 1200으로 10분 동안 '원심분리하여 혈장 성분을 제거하고 단핵구 성분들이 포함된 농축액을 수득하였으나， 본 발명이 이에 제한되는 것은 아니다. Preferably, concentration of the blood monocyte fraction may be performed using a blood centrifuge. The centrifugation conditions can be properly adjusted by one skilled in the art according to the situation, and it is possible to remove plasma components and obtain a concentrate containing monocyte components. have. Although the embodiments of the present invention remove plasma components by "centrifugation for 10 minutes at 1200 revolutions per minute to give a concentrated solution containing the components to monocytes, but the invention is not limited to this.
이와 같이 '수득된 농축 단핵구 내에는 환자의 '허혈 부위에 직접 투여했을 때 혈관신생을 촉진하기에 층분한 양과 농도의 CDllb+CXXRl+ 세포가 포함되어 있으며， 바람직한 예로, 전체 농축된 세포 중 CDlib+CX3CRl+ 세포.분획이 바람직하게는 10%이상， 보다 바람직하게는 15%이상일 수 있다. 환자 몸부게당 2.5 X 107내지 5 X 107개로 주사 시 환자 몸무게를 70kg으로 가정한다면 2. X 109내지 4 X 10 개와 세포가 필요한데 , 본 발명에 의할 경우 1회 수집된 농축 단핵구로부터 환자의 허혈 부위에 직접 주사할 수 있는 양과 농도의 CDllb+CX3CRl+ 세포 농축액을 얻을 수 있다 As such, the ' concentrated monocytes ' contained CDllb + CXXRl + cells in an amount and concentration sufficient to promote angiogenesis when administered directly to the patient's ischemic site, and in a preferred embodiment, CDlib + CX3CRl + cell. The fraction may preferably be at least 10%, more preferably at least 15%. Assuming a patient weight of 70 kg when injected from 2.5 X 10 7 to 5 X 10 7 per patient body weight 2 . X 10 9 to 4 X 10 cells and cells are required. According to the present invention, CDllb + CX3CRl + cell concentrates can be obtained in the amount and concentration which can be injected directly into the ischemic site of the patient from the collected mononuclear cells once collected.
.또한, 본 발명에 의해 대량 수집된 단핵구는 세포보존제 (예. 7%의 DMS0)와 흔합하여 넁동 (예, Cryomed)시켜 보관할 수 있으며， 은도 차에 의한 세포 손상 없이 오랜 기간 보관이 가능하며， 언제든지 필요할 때 필요한 세포 수만큼 해동시켜 사용할 수 있으므로 치료적 목적으로 활용아 In addition, the monocytes collected in large amounts by the present invention can be stored and mixed with a cell preservative (eg, 7% DMS0) to be shaken (eg Cryomed), and can be stored for a long time without cell damage caused by silver. It can be used for therapeutic purposes because it can be thawed and used as many cells as needed at any time.
용이하다. 해동 시 세포의 생존률 (viability)은 평균 90% 이상인 것이 바람직하다. It is easy. The viability of cells upon thawing is preferably at least 90%.
본 발명에서 수득된 세포 집단은 혈관신생 촉진 작용을 가지므로， 혈관신생 촉진제로 유용하게 사용될 수 있으며, 나아가 혈관신생이 필요한 각종 질환의 예방 또는 치료 목적:으로 사용될 수 있다. 혈관신생이 필요한 대표적인 질환으로 허혈성 ^환을 들 수 있다. Since the cell population obtained in the present invention has an angiogenic promoting effect, it may be usefully used as an angiogenesis promoter, and furthermore, may be used for the purpose of preventing or treating various diseases in which angiogenesis is required. Representative diseases that require angiogenesis include ischemic ^ ring.
따라서 , 본 발명은 상기 CDllb+ 및 CX3CR1+의 면역 표현형을 포함하는 세포 집단을 포함하는 혈관신생 촉진용 조성물을 제공한다. Accordingly, the present invention provides a composition for promoting angiogenesis comprising a cell population comprising the immune phenotype of the CDllb + and CX3CR1 + .
또한, 본 발명은 상기 세포 집단의 유효량을 개체에 투여하는 단계를 포함하는 혈관신생 촉진 방법을 제공한다. " . The present invention also provides a method for promoting angiogenesis comprising administering to the subject an effective amount of the cell population. " .
' 또한， 본 발명은 상기 세포 질단을 포함하는 허,혈성 질환 예방 또는 치료용 약학 조성물을 제공한다. ' 또한， 본 발명은 상기 세포 집단의 유효량을 개체에 투여하는 단계를 포함하는 허혈성 질환 예방 또는 치료 방법을 제공한다.
【발명의 실시를 위한 형태] In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic and hematologic diseases comprising the cytoplasm. "The present invention also provides a prevention or treatment of ischemic disease comprising the step of administering an effective amount of said cell population to an object. [Form for implementation of invention]
• 이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을쎄시하는 것일 뿐, ；본 발명이 하기 ：실시예에 의해 한정되는 것은 아니다. , ' 실시예 1. 마우스하지 허혈 유도 및 근육세포분리 • Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples only care about the present invention; the present invention is not limited to the following Examples. Example 1 Mouse Ischemia Induction and Muscle Cell Isolation
가를릭대학교 동물 윤리위원회의 규정에 준수하여， 1으 12주령의 C57BL/6마우스의 대퇴부에 정맥과 동맥을 제거하여 허혈을 유도하였다. 허혈 근육으로부터 0.2%콜라게나제 (210U/mg; Worthi'ngton, Lakewodcl, NJ)와 '디스파아제 (0.95 U/mg; Invitrogen, Grand Island, NY)를 이용하여 단일 세포를 분리하였다. FACS분석을 위해， 분리한 세포는 APC-결합된 항-마우스 CDllb, 비오틴화된 항—마우스 CX3CR1항체를 30분간 4°C에서 반웅시켰다. 그 후, FTTC—결합된 스트렙타비딘 (Molecular Probes, Eugene, OR)으로 30분간 더 반응시켰다. CDllb+CX3CIU+세포는 유세포분석기를 이용하여 분석하고 , 분리하였다. (MoFlo™XDP Cell Sorter (Beckman Coulter, Brea. CA) 실시예 2. FACS분석과실시간중합효소반웅 In compliance with the regulations of the Garlic University Animal Ethics Committee, ischemia was induced by removing veins and arteries in the thighs of 1 to 12-week-old C57BL / 6 mice. Single cells were isolated from ischemic muscles using 0.2% collagenase (210 U / mg; Worthi'ngton, Lakewodcl, NJ) and ' dispase (0.95 U / mg; Invitrogen, Grand Island, NY). For FACS analysis, isolated cells were reacted with APC-bound anti-mouse CDllb, biotinylated anti-mouse CX3CR1 antibody at 4 ° C. for 30 minutes. Thereafter, the reaction was further reacted with FTTC-bound streptavidin (Molecular Probes, Eugene, OR) for 30 minutes. CDllb + CX3CIU + cells were analyzed using flow cytometry and isolated. (MoFlo ™ XDP Cell Sorter (Beckman Coulter, Brea. CA) Example 2. FACS Analysis and Real-Time Polymerase Response
CDllb+CX3CRl+세포의 표면항원과 CDllb+CX3CRl+세포에서의 Of CDllb + CX3CRl + Cell Surface Antigens and CDllb + CX3CRl + Cells
VEGF발현은 FACS를 이용하여 분석하였다. 조직에서의 Fkn과 CDllb+CX3CRl+ 세포에서의 VEGF발현은 실시간 PCR을 이용하여 조사하였다. VEGF expression was analyzed using FACS. Fkn expression in tissues and VEGF expression in CDllb + CX3CRl + cells were investigated using real-time PCR.
분리된 CDllb+CX3CRl+세포에 F4/80 , VEGFR2, NK1.1, Tie2, 그리고 CXCR4항체 (eBiosciences, San Diego, CA). CD45, CD3, CD19그라고 CDllc항체 (BD Pharmigen. San Jose , CA) VEGFRl항체 (R&D Systems, Minneapolis, MN), VEGF( Abeam, Cambridge, UK) 이용하여 이들의 발현 정도를 유세포분석기를 이용하여 조사하였다. (MoFlo™XDP Cell Sorter) . F4 / 80, VEGFR2, NK1.1, Tie2, and CXCR4 antibodies (eBiosciences, San Diego, CA) on isolated CDllb + CX3CRl + cells. CD45, CD3, CD19 and CDllc antibodies (BD Pharmigen. San Jose, Calif.) VEGFRl antibodies (R & D Systems, Minneapolis, MN), VEGF (Abeam, Cambridge, UK) were used to investigate their expression level using flow cytometry. . (MoFlo ™ XDP Cell Sorter).
조직에서의 Fkn발현은 Fkn프라이머 (정방향： GGA CAG GAC CTC AGT CCA GA, 역방향: TCG GGG ACA GGA GTG ATA AG)를, CDllb+CX3CRl+세포에서의 VEGF발현은 VEGF프라이머 (정방향: CACAGCAGATGTGAATGCAG， . 역방향: Fkn expression in tissue was Fkn primer (forward: GGA CAG GAC CTC AGT CCA GA, reverse: TCG GGG ACA GGA GTG ATA AG) and VEGF expression in CDllb + CX3CRl + cells was VEGF primer (forward: CACAGCAGATGTGAATGCAG,. :
CTCACAGTGAACGCTCCAGG)로 MyiQ Real Time PCR Detection Systems (Bio-Rad,
Hercules, Canada) 기기를 이용하여 측정하고 /분석을 위해 Bio— Rad IQ5 소프트웨어를 사용하였다. ； 실시예 3.혀혈 근육에서 분리한 CDllb+CX3CRl+세포, Fkn및 항 ~CX3CR1 중화 항체와항 -Fkn중화 항체 투여와 혈관 형성과의 관계 CTCACAGTGAACGCTCCAGG) with MyiQ Real Time PCR Detection Systems (Bio-Rad, Hercules, Canada) using the Bio— Rad IQ5 software for measurement / analysis. Example 3 Relationship between CDllb + CX3CRl + Cells, Fkn and Anti-CX3CR1 Neutralizing Antibody and Anti-Fkn Neutralizing Antibody Isolated from Tongue Muscle and Angiogenesis
허혈 근육으로부터 분라한 CDllb+CX3CRl+, CDllb+CXXRl—그리고 CDllbXX Rl—세포를 하지 허혈을 유도한지 48시간째 마우스의 허할 근육에 ' 주입하였다 (5X105 eel Is/마우스). 마우스 재조합 단백질 Fkn (R&D System, Minneapolis, ΜΝ')은 0.5， 1， 5 ug씩 PBS 500 ul에 녹이고， 허혈 유도 수술 직후부터 100 ul씩 매일 5일간 허혈 근육으로 주입하였다. -CDllb + CX3CRl + , CDllb + CXXRl—and CDllbXX Rl—cells isolated from ischemic muscles were 'injected' into the hypoxic muscles of mice 48 hours after induction of lower limb ischemia ( 5 × 10 5 eel Is / mouse). The mouse recombinant protein Fkn (R & D System, Minneapolis, ΜΝ ' ) was dissolved in 500 ul of PBS at 0.5, 1, and 5 ug, and injected into the ischemic muscle every day for 100 days at 100 ul immediately after ischemic induction. -
CX3CR1이나 Fkn의 기능을 차단하기 위해 , 항 -CX3CR1 중화 항체와 항 -Fkn 증화 항체를 사용하였다. 항 -CX3CR1 중화 항체 (TP501,Torrey Pines Bio labs, San Diego, CA)를 0.5 ig씩 매일ᅳ 5일간 허혈'근육에 주입하였다. 항 -Fkn 중화 항체 (TP233, Torrey Pines Biolabs)와 대조군도 같은 양의 IgG를 같은 방법으로 주사하였다. 허혈 유도 직후부터 허혈 근육에서의 혈류량을수술하지 않은 반대편 다리의 혈류량과 비교하여 측정하였고, 이를 위해 laser Doppler perfusion scanner (LDPI , Moore instruments , Devon , , UK)를 사용하였다. 실시예 4. 조직 면역형광염색 In order to block the function of CX3CR1 or Fkn, anti-CX3CR1 neutralizing antibody and anti-Fkn-enhancing antibody were used. Anti-CX3CR1 neutralizing antibody (TP501, Torrey Pines Bio labs, San Diego, Calif.) Was injected into ischemia ' muscles every 0.5 days for 5 days. Anti-Fkn neutralizing antibodies (TP233, Torrey Pines Biolabs) and controls were injected with the same amount of IgG in the same way. Immediately after induction of ischemia, the blood flow in the ischemic muscle was compared with that of the other leg without surgery. For this purpose, a laser Doppler perfusion scanner (LDPI, Moore instruments, Devon , UK) was used. Example 4. Tissue Immunofluorescence Staining
CX3CR1기능흘 차단하기 위해 ,항 -CX3CR1중화 항체를 0.5 μ g씩 매일， ' 5일간 허혈 근육에 주입하였다. 대조군도 같은 양의 Ig0를 같은 방법으로 주사하였다ᅳ 허혈 유도 21일 후 허혈 다리에사 근육을 분리하여 OCT 화합물 (Tissue一 Tek, Sakur a Fine tek Japan Co. , Tokyo , Japan)에 포미]하고. 0.5 로 절단하여 슬라이드 글라스에 부착하였다. 4% 파라포.름알데히드에서 15 분간 고정하고，. PBS로 세척한 다음 10% 정상 말혈청으로 1시간 동안 반옹시켜 비특이적 반옹을 막았다. CX3CR1 function to block shed, wherein the -CX3CR1 neutralizing antibody was injected into each 0.5 μ g per day and, five days ischemic muscle. In the control group, the same amount of Ig0 was injected in the same manner. After 21 days of ischemia induction, the ischemic leg muscles were separated, and the OCT compound (Tissue 一 Tek, Sakur a Fine tek Japan Co., Tokyo, Japan) was used. Cut to 0.5 and attached to the slide glass. 4% parapo . Fixed for 15 minutes in aldehydes. It was washed with PBS and then rebounded with 10% normal horse serum for 1 hour to prevent nonspecific reaction.
생후 2일¾ 래트 (P2)를 Isoflurane로 마취 후 오른쪽 안구에 0.1 의 항一 CX3CR1.중화 .항체를 Hami lton syringe (Hami lton Company, Reno , NV)를 이용하여 주사하였다. 대조군도 같은 양의 IgG를 같은 방법으로
주사한다. 3일후 (P5), 래트 안구를 적출하여 . 4% 파라포름알데히드에서 1~2시간 고정하고, -20°C에서 30% sucrose 용액에 넣어 보존한다. 염색 전 PBS로 세척하고， 0.5% triton X-100에 반응시킨 뒤 10% 정상 말혈청으로 2시간동안 반웅시켜 비특.이적 반웅을 막았다. Two days after birth, ¾ rats (P2) were anesthetized with Isoflurane and 0.1 anti-CX3CR1 in the right eye . Neutralizing antibodies were injected using a Hami lton syringe (Hami lton Company, Reno, NV). The same amount of IgG in the control Inject. After 3 days (P5), the rat eye was removed. Fix in 1-2% in 4% paraformaldehyde and store in 30% sucrose solution at -20 ° C. Before staining, the cells were washed with PBS, reacted with 0.5% triton X-100, and reacted with 10% normal horse serum for 2 hours to prevent nonspecific transfer reactions.
' 각 조직은 토끼-항 -CX3CR1 항체로 4°C에서 16시간 동안 면역 반웅 시킨 다음 3회 세척하고 .비오틴화된 항 -토끼 항체 (Vector Labs Bur 1 ingame CA)를 이용하여 실은에서 1~2시간 동안 반웅시켰다. 다시 Each tissue was immunized with rabbit-anti-CX3CR1 antibody for 16 hours at 4 ° C., washed three times, and then incubated with biotinylated anti-rabbit antibody (Vector Labs Bur 1 ingame CA). Reaction for hours. again
Cy3-스트랩타비딘 (Jacson Labs, Bar Harbor , Maine )과 이차 반웅을 시킨 뒤, 세척 후 항 -CDllb 항체를 이용하여 면^ 반웅시키고, APC가 결합된 항체를 실온에서 1시간 동안 반웅시켰다. 또., 혈관구조를 관찰하기 위해 FITC가 부착된 isolectin 으로 4°C에서 16시간 동안 면역 반웅 시킨 다음 3회 세척하였다. 핵^ 디아미디노 -2-페닐인돌 (DAPI. Sigma)로 염색한 다음 형광 현미경 (Zeiss fluorescence microscope)으로 관찰하였다. 실시예 5. 통계분석 Second reaction was performed with Cy3-straptavidin (Jacson Labs, Bar Harbor, Maine), followed by washing with anti-CDllb antibody, and APC bound antibody was reacted for 1 hour at room temperature. In addition, to observe the vascular structure was immunized with FITC attached isolectin for 16 hours at 4 ° C and then washed three times. Stained with nuclear ^ diamidino-2-phenylindole (DAPI. Sigma) and observed with a Zeiss fluorescence microscope. Example 5 Statistical Analysis
모든 수행한 실험 결과 분석은 평균土 SD 값으로 나타내었다. 허혈성 상해 이후 세포 개체 간의 차이는 unpaired t-test로 분석하였다. 반복측정 분산 분석 (repeated measures AN0VA)으로 다양한 그룹간의 결과값의 차아를 분석하였다. P값이 0.05 이하의 것을 유의한 것으로 판단하였다. Analysis of all experimental results performed is expressed as mean 土 SD value. The difference between cell populations after ischemic injury was analyzed by unpaired t-test. Repeated measures AN0VA analyzed differences in outcomes between the various groups. It was judged that the P value was 0.05 or less.
. ' , . ' ,
실시예 6. CDllb+CX3CRl+세포 대량 수득 방법 Example 6 CDllb + CX3CRl + Cell Mass Gain Method
조혈모 세포 이식 치료를 위하여 조혈모세포를 말초혈액으로 가동화하기 위해， 동의서를 받은 환자나 조혈모세포 기증자로부터 ' 수득하였다. 조혈모세포를 말초혈액으로 가동화 하기 위해 , G— CSF는 중외제약 (상품명 : Neutrogen)이나 동아제약 (상품명 : Leucost iin)으로 부터 ' 구입하여 10 g/kg의 용량으로 5일간 피하주사였다. G-CSF와 AMD3100 "It was obtained from peripheral blood stem cell to stem cell mobilization to the peripheral blood, received the agreement patient or donor hematopoietic stem cells for transplantation therapy. For mobilization of stem cells in peripheral blood, G- CSF is Choongwae (trade name: Neutrogen) and Dong-A (trade name: Leucost iin) '10 g / kg was the dose five days of subcutaneous injections purchased from. G-CSF and AMD3100
(상품명 :모조빌， 사노피 -아벤티스)의 병용 투여 시에는 G— CSF는 의 용량으로 5일간 피하주사하고, 4일째 AMD3100 240 /g/kg 로 1회 피하주사 하였다. 마지막 G-CSF 혹은 AMD3100 투여 후 적어도 10 시간 후 혈액 성분 채집기 (Cobe® spectra apheresis system, GAMBRO.BCT, Lakewood , USA)를
이용하여 4시간 등안 지속적으로 혈액 단핵구 분획을 수집하고,수집된 혈액 단핵구 분확을 혈액 원심분리기 (Cobe®2991 cell processor, GAMBRO.BCT, Lakewood, USA)를 이용하여 농축하여 농축 단핵구를 수집하였다. (Commodity name: Mozobil, Sanofi-Aventis) G-CSF was injected subcutaneously for 5 days at the dose of, and once subcutaneously at AMD3100 240 / g / kg for 4 days. Blood sampler (Cobe® spectra apheresis system, GAMBRO.BCT, Lakewood, USA) at least 10 hours after the last dose of G-CSF or AMD3100 Blood mononuclear fractions were collected continuously for 4 hours, and concentrated monocytes were collected by concentrating the collected blood monocyte fractions using a blood centrifuge (Cobe®2991 cell processor, GAMBRO.BCT, Lakewood, USA).
말초 혈액과 농축 단핵구는 iySis buffer를 이용하여 RBC를 제거한 후 IX 106 의 세포에 FITC-결합된 항 -CDllb 항체 (eBioscience)와, APC-결합된 항 -CX3CR1 항체 (Biolegend)를 각각 넣어 4°C에서 30분간 반웅시키고 세척하여 준비하였다. 유세포분석기를 이용하여 측정 및 분석하였다. 실험결과 Peripheral blood and enriched monocytes were removed with iy S is buffer, and then FITC-bound anti-CDllb antibody (eBioscience) and APC-bound anti-CX3CR1 antibody (Biolegend) were added to IX 10 6 cells. Prepared by reaction at 4 ° C for 30 minutes and washed. Measurements and analyzes were performed using a flow cytometer. Experiment result
1. 허혈 손상후허혈 근육내에서 CDllb+CX3CRl+세포수가현저하게 증가된다. 1. Ischemia damage significantly increases CDllb + CX3CRl + cell count in ischemic muscle.
C57BL/6 마우스 허혈—하지 모델에서 허혈 근육 조직을 박리하여, CDllb+CX3CRl+세포 수의 변화를 유세포 측정기를 이용하여 측정하였다. 그 결과 근육 내의 CDllb+CX3CRl+세포와 %는 수술 전에 전체, 세포의 In the C57BL / 6 mouse ischemia—lower leg model, the ischemic muscle tissue was detached and the change in CDllb + CX3CRl + cell number was measured using a flow cytometer. The result is CDllb + CX3CRl + cells within the muscle and% of the total, cells before surgery
으1±0.04%이었고， 허혈. 유도 4일 후는 4.05±0.07%로 증가하였다 (각 n=5. p<0.0001) (도 la). 또한， 허혈 근육에서 분라한 CDllb+CX3CRl+ 세포 수도 수술 전보다 수술 4일 후에 180배 증가하였고 (각 n=5; 수술 전 조직에서 0.01土 0.02X105/g, 수술 4일 후 조직에서 1.84±0.9X105/g; p=0.03), 14 일째는 수술하기 전과 유사한 수준으로 감소되어 (조직에서 Eq. 1 ± 0.04%, ischemia. Four days after induction, it increased to 4.05 ± 0.07% (each n = 5. P <0.0001) (FIG. La). In addition, the number of CDllb + CX3CRl + cells separated from the ischemic muscle was increased 180 times after 4 days after surgery (each n = 5; 0.01 土 0.02X10 5 / g in tissue before surgery, and 1.84 ± 0.9 in tissue after 4 days). X10 5 / g; p = 0.03), on day 14 it is reduced to a similar level as before surgery (in tissue
0:24土 0.33X105/g), CDllb+CX3C l+ 세포 수가 1관 재생이 왕성하게 일어나는 시기에 현저하게 증가되는 것을 알：수 있었다 (도 lb). / 0:24 土 0.33X10 5 / g), it was found that CDllb + CX3C1 + cell number increased significantly at the time of vigorous one-tube regeneration (Fig. Lb). Of
CDllb+CX3CRl+세포 이동에 영향을 주는 Fkn의 발현 정도를 실시간 PCR을 이용하여 .분석하였다. Fkn mRNA 발현은 동맥 절개 .수술 후 4일째 최고로 나타나 (도 lc), 허혈 근육 내의 증가된 Fkn 발현이 CDllb+CX3CRl+ 세포 수 증가와 관련된 것을 알 수 있었다. The expression of Fkn, which affects CDllb + CX3CRl + cell migration, was analyzed using real-time PCR . Analyzed. Fkn mRNA expression was highest at 4 days after arterial incision (FIG. Lc), indicating that increased Fkn expression in ischemic muscle was associated with an increase in CDllb + CX3CRl + cell number.
CX3CR1 표면 항원은 대식세포， 단핵.구. 내피세포， 수지상 세포， 자연살해 세포 (NK cell)와 T세포에서 다양하게 발현되는 '세포 표면 항원으로, CDllb+CX3CRl+세포가 어떤ᅵ종류의 세포인지 확인하기 위해 허혈 근육 내와 CDllb+CX3CRl+세포를 분리 한 뒤 표면 항원을 조사하였다. CX3CR1 surface antigens are macrophages, monocytes. Endothelial cells, dendritic cells, natural killer cells (NK cell) and a "cell surface antigens that are variously expressed in T cells, CDllb + CX3CRl + cells ischemia intramuscular and CDllb + to see what i types of cells CX3CRl + After the cells were separated, the surface antigen was examined.
CDllb+CX3CRl+세포는 골수세포 (CD45)와 대식세포 (F4/80) 양성이었으나，
내피세포 (VEGFR-2),수지상세포 (CDllc), NK세포 (NK1.1)및 T세포 (CD3)에 대한 표면 항원은 발현되지 않아 (도 1〔1)， 허혈 근육 내로 이흥한 CDllb + CX3CRl + cells were positive for bone marrow cells (CD45) and macrophages (F4 / 80), Surface antigens for endothelial cells (VEGFR-2), dendritic cells (CDllc), NK cells (NK1.1) and T cells (CD3) were not expressed (Fig. 1 [1]).
CDllb+CX3CRl+세포는 단핵구 /대식세포 계통의 세포임을 알 수 있었다. 특히 , CDllb+CX3CRl+세포는 현재까지 혈관형성에 관여하는 것으로 알려진 특정 '단핵구의 표면 항원인 VEGFR-1, Tie-2 그리고 CX3CR4항원이 양성이었다. CDllb + CX3CRl + cells were found to be cells of the monocyte / macrophage lineage. In particular, CDllb + CX3CRl + cells of VEGFR-1, Tie-2 specific, the surface of the monocyte antigen known to be involved in angiogenesis and to this antigen were positive CX3CR4.
2. 근육에서 분리한 CDllb+CX3CRl+세포나 Fkn단백질을 허혈 근육에 주사하면 혈류량이 증가된다. 2. Injection of CDllb + CX3CRl + cells or Fkn protein isolated from muscle into ischemic muscle increases blood flow.
근육 유래 CDllb+CX3CRl+세포가 혈관 형성을 증가시키는지 알아보기 위해 허혈 유도 후 4일째 허혈 근육에서 CDllb+CX3CRl+, CDllb+CX3CRl" , 및 CDilbXX3CRl"세포 (대조군)를 분리해 허혈 근육에 주입하고 각 시기별로 혈류량의 변화를 LDPI (Laser Doppler Perfusion Image)를 이용하여 측정하였다. 그 결과， CDllb+CXXRl+세포를 주입한 마우스의 혈류량 . To determine whether muscle-derived CDllb + CX3CRl + cells increase angiogenesis, CDllb + CX3CRl + , CDllb + CX3CRl " , and CDilbXX3CRl " cells (control) were isolated from ischemic muscles on day 4 after ischemic induction and injected into ischemic muscles. Changes in blood flow at each time were measured using LDPI (Laser Doppler Perfusion Image). As a result, blood flow in mice injected with CDllb + CXXRl + cells .
(78.03±3.16¾, 28일째)은 대조군인 )11ᅵ3 乂30?1-세포(38.99土0.26%， (78.03 ± 3.16¾, 28 days) was the control group) 11 ᅵ 3 乂 30? 1-cell (38.99 土 0.26%,
28일째)를 주사한 그룹이나 수술만시행한 그룹 (34ᅳ26±4.76%； p=0.001; n=5)보다 유의하게 증가되었다 (도 2a). CDllb+CX3CRl" 세포 (50.8±2.5%, 28일째 ; n=5)를 주입한 그룹에서도 CDllb"CX3CRr세 ΐ나 수술만 시행한 그룹에 비해 약간의 혈류량 증가가 있었으나， CDllb+CX3CRl+세포를 주입한 군과 비교하여서는 유의하게 낮았다 (P=0.001) (도 2a). 이 결과를 통해서 CDllb+CX3CRl+세포가 허혈성 손상 후 새로운 혈관 형성에 중요한 역 ¾을 하는 세포임을 확인할 수 있었다. 28 days) was significantly increased compared to the group injected with the operation (34 × 26 ± 4.76%; p = 0.011; n = 5) (Fig. 2a). In the group injected with CDllb + CX3CRl " cells (50.8 ± 2.5%, day 28; n = 5), there was a slight increase in blood flow compared with the group treated with CDllb " CX3CRr or surgery only, but CDllb + CX3CRl + cells were injected. Compared with one group was significantly lower (P = 0.001) (FIG. 2A). These results confirm that CDllb + CX3CRl + cells are inverse ¾ important for new blood vessel formation after ischemic injury.
또, 허혈 손상후 CX3CR1+세포를 끌어들이는 Fkn단백질 직접주사로 혈류량이 증가된다면 . 이는 임상에서 보다 쉽게 사용할 수 있는 치료책이 될 수 있을 것으로 생각하여 , 본 발명자들은 허혈 부위에 Fkn을 직접 주사하여 허혈 다리의 혈류량을 측정하였다. 허혈 28일째 0.5 의 Fkn 단백질 주입군은 PBS를 주입한 대조군에 비해 혈류량이 증가되지 않았으나 (각각 40.54±2.34%, 28일째; 37.69±4.85%.28일째), 1 /jg과 5 의 Fkn을주입한 군은 허혈 이후 28일째 혈류량이 각각 정상 다리와 비교하여 51.72±0.4%와 77.83±11.99%로 증가되어, 대조군과 대비하여 유의한수준와 혈류량 증가가 있는 것을 확인하였다 (대조군 대비 P=0.01, P=0.012) (도 2b). '
3..CX3CR1과 Fkn의 기능을차단하면 혈류량 개선이 저하된다. In addition, if the blood flow is increased by direct injection of Fkn protein that draws CX3CR1 + cells after ischemic injury. Since this may be a treatment that can be easily used in the clinic, the present inventors measured the blood flow of the ischemic leg by directly injecting Fkn to the ischemic site. Fkn protein infusion group at 0.5 days on ischemia did not increase blood flow compared to control group injected with PBS (40.54 ± 2.34%, day 28; 37.69 ± 4.85% .28 days, respectively), but injected Fkn of 1 / jg and 5 In one group, blood flow increased to 51.72 ± 0.4% and 77.83 ± 11.99% compared to the normal leg at 28 days after ischemia, respectively, and showed a significant level and blood flow increase compared to the control group (P = 0.01, P compared to the control group). = 0.012) (FIG. 2B). ' 3..Blocking CX3CR1 and Fkn functions impairs blood flow improvement.
Fkn-CX3CR1 반웅과 혈관 생성과의 관계를 알아보고자 허혈을 유도한 . C57BL/6 마우스의 근육에 중화 항 -CX3CRr항체 , 중화 항 -Fkn항체 흑은 대조군 IgG를 주입하고 LDPI를 측정하였다. 28일째 근육에서와혈류량은 - 중화 항 -CX3CR1 항체나 중화 항 -Fkn항체를 주입한 마우스의 근육에서 각각 25.94±4.89%와 29.41±4.59%로 측정되어 (각각 p=0.037 와 p=0.01), 대조군의 LDPI 46.25 ±3.23%보다 현저하게 감소되어 있었다 (도 3). 이 결과로 Fkn-CX3CR1의 상호 작용이 허혈 근육 내의 새로운 혈관 형성에 기여함을 알 수 있었다 .. To determine the relationship between Fkn-CX3CR1 reaction and angiogenesis, we induced ischemia. The muscles of C57BL / 6 mice were injected with neutralizing anti-CX3CRr antibody and neutralizing anti-Fkn antibody black control IgG and LDPI was measured. At 28 days, muscle and blood flow were measured to be 25.94 ± 4.89% and 29.41 ± 4.59%, respectively, in the muscles of mice injected with neutralizing anti-CX3CR1 antibody or neutralizing anti-Fkn antibody (p = 0.037 and p = 0.01, respectively). It was significantly reduced than the LDPI 46.25 ± 3.23% of the control group (Fig. 3). These results indicate that Fkn-CX3CR1 interaction contributes to the formation of new blood vessels in the ischemic muscle.
4. CX3CR1기능을차단하면 허혈부위근육과망막에서 정상적인 혈관생성이 저해된다. 4. Blocking CX3CR1 function inhibits normal angiogenesis in ischemic muscle and retina.
허혈 유도 후 새로운 혈관 형성 시， CDllb+CX3CRl+세포의 역할을 알아보기 위해, 허혈 근육에 항 -CDllb항체 (흰색)， 항ᅳ CX3CR1항체 (붉은색) 그리고 isQlectin (초록색)을 이용하여 면역 형광 염색을 실시하였다. 정상 조직에서는 CDllb+CX3CRl+세포 관찰되지 않지만 (그림 A, 도 4a)허혈 7일째 많은 수의 세포가 허혈 근육 내로 이동하였고.그 중 상당수가 CDllb+CX3CRl+ 세포였다 (화살표;노란색; 그람 B,도 4a).허혈 14일에는 허혈 근육 내부에 CDllb+CX3CRl+세포로 구성된 세포 무리 (cell cluster)가 형성되었다 To determine the role of CDllb + CX3CRl + cells in the formation of new blood vessels after ischemia induction, immunofluorescence staining using anti-CDllb antibody (white), anti-CX3CR1 antibody (red) and isQlectin (green) in ischemic muscle Was carried out. CDllb + CX3CRl + cells were not observed in normal tissues (Figure A, Figure 4a), but a large number of cells migrated into the ischemic muscle at day 7 of ischemia, many of which were CDllb + CX3CRl + cells (arrow; yellow; Gram B, Fig. 4a). On day 14 of the ischemia, a cell cluster consisting of CDllb + CX3CRl + cells was formed inside the ischemic muscle.
(화살표; 그림 D, 도 4a). 허혈 21일 후에는 새로 생긴 혈관 구조 (vascular structure)가 관찰되었고， 이 구조물 밖으로 CDllb+CX3CRl+세포가 일정한 간격으로 위치하는 것을 알 수 있었다 (화살표; 노란색; 그림 E, 도 4a). 허혈 근육에서는 항 -CX3CR1 항체를.주사하면 , 허혈 7일째 허혈 근육에 CDllb+CX3CRl+세포 침윤대신에 CDllb+CXXRl—세포 만 관찰되었으며 (화살표; . 흰색 ; 그림: C, 도 4a) , 허혈 21일 후에는 허혈 근육조직에 새로운 ¾관 형성도 관찰되지 않았다 (화살표; 그림 F, 도 4a). (Arrow; Figure D, Figure 4A). After 21 days of ischemia, a new vascular structure was observed and CDllb + CX3CRl + cells were located at regular intervals out of the structure (arrow; yellow; Fig. E, Fig. 4a). . When ischemic muscle in the injected anti -CX3CR1 antibody, was observed only ischemia 7 days after ischemia CDllb + CXXRl- cells instead of CDllb + CX3CRl + cell infiltration in the muscle (arrow; white; Fig:. C, Fig. 4a), ischemic 21 After days, no new ¾ tube formation was observed in the ischemic muscle tissue (arrow; Fig. F, Fig. 4a).
, 항 -CX3CR1 항체를 생후 2일째 래트 망막에 주사하여， 허혈 부위의 CX3CR1의 기능을 차단하였다. 래트 망막에서도 항 -CX3CR1 항체를 투여하면
정상적인 망막 혈관 형성이 쩌해되는 것이 확인되어 (그림 C와으 도 4b), , Anti-CX3CR1 antibody was injected into the rat retina at day 2 to block the function of CX3CR1 at the ischemic site. Administration of anti-CX3CR1 antibodies in the rat retina Normal retinal vessel formation was confirmed to be aberrant (Figure C and Figure 4b),
CDllb+CX3CRl+세포는 허혈 부위에서 혈관 형성을 촉친하고， 신생 혈관 구조 형성을 조절하는 기능이 있는 세포로 직접적으로 혈류량을 개선하는 효과가 있다는 결론에 이르:게 되었다. It was concluded that CDllb + CX3CRl + cells promote the formation of blood vessels at the ischemic site and directly improve blood flow.
또， CDllb+CX3CRl+세포^ 세포 자체에서 VEGFR mRNA와 단백질을 In addition, CDllb + CX3CRl + cells ^ can express VEGFR mRNA and protein in the cell itself.
발현하였다 (도 4c). 이러한 결과는 CDllb+CX3CRl+세포가 허혈 부위의: Expression (FIG. 4C). These results indicate that CDllb + CX3CRl + cells are in the ischemic site:
. 혈관내피세포의 증삭을 촉진하여 혈관'생성을 증가 시키는 효과가 있을' . Promoting the jeungsak of vascular endothelial cells, it has the effect of increasing the production, vessel
가능성을 시사 하였다. 5. G-CSF투여 시 농축 단핵구에서 )llb+CX3CRl+세포수가증가된다. ¬상적으로 혈액에서 CDllb+CX3CRl+ 세포는 전체 혈액 백혈구 중 ^土으^였고, 그 수는 66.2±8.8X10 fl/mL (1.3±0.1%X5.000/隱 3)이었다. Suggested the possibility. 5. Increased) llb + CX3CRl + cell count in enriched monocytes upon G-CSF administration. Normally, CDllb + CX3CRl + cells in blood were ^^ in total blood leukocytes and the number was 66.2 ± 8.8 × 10 fl / mL (1.3 ± 0.1% × 5.000 / 隱3 ).
： 즉, CDllb+CX3CRl+세포는 정상적으로 혈벡 내에는 거의 존재하지 않는 In other words, CDllb + CX3CRl + cells are rarely normally present in the blood cells.
것으로. 나타났다 (도 5A). To be. Appeared (FIG. 5A).
골수로부터 CDllb+CX3CRl+ 세포를 말초 혈액으로 가동화 시키기 위해 G-GSF (10/ig/kg)를 5일간 주사한 다음 얻은 말초 혈액 내의 CDllb+CX3CRl+ 세포는 전체 백혈구 중 2.8土 0.9%로. G-CSF 투여 전의 약 1.3%보다 약간 The CDllb + CX3CRl + peripheral blood mobilization G-GSF (10 / ig / kg) to the cells from the bone marrow by CDllb + CX3CRl + cells are 2.8土0.9% of the total white blood cells within 5 days following the injection of peripheral blood obtained. Slightly less than about 1.3% before G-CSF
증가하였지만 백혈구 수가 5,000/隱3에서 평균 21，905/瞧3으로 약 4.3배 Although it is increasing on average 21,905 /瞧3 in the number of leukocytes 5,000 /隱3 about 4.3 times
증가하였으며ᅳ CDllb+CX3CRl+세포 수는 단위 '혈액량 niL당 평균적으로 ' 667.3±521.8X103개 /mL (2.8 (21，905/瞧 3)로 거의 10배 증가도 1었고， G-CSF와 Increase was eu CDllb + CX3CRl + cell number was 1 unit "blood volume average per niL, 667.3 ± 3 521.8X10 gae / mL almost 10-fold increase in (2.8 (21,905 /瞧3) , with G-CSF
AMD3100을 병용 투여한 경우에도 CDllb+CX3CRl+ 세포 수가 643X103/nil로 CDllb + CX3CRl + cell count 643X10 3 / nil
증가하여 G-CSF 단독 투여와유사한 결과로 투여 전과 비교하여 약 10배 . Increased to about 10-fold compared to pre-administration, similar to G-CSF alone.
가량 증가를 나타내었다 (도 5B, 도 7).혈액 성분 채집기로 수집하고， 혈액 원심분리기로 농축한 농축 단핵구의 경우 전체 세포 중 CDllb+CX3CRl+세포 The increase was shown (Fig. 5B, Fig. 7). CDllb + CX3CRl + cells of the total cells for the concentrated monocytes collected by the blood component collector and concentrated by blood centrifuge
분획이 16.2±7.2%였다. 수집된 농축 단핵구의 양은 .146 ± 130ml로 농축 ' 단핵구에서 CDllb+CXXR^.세포는 총 9.2±1.2X109개 (16·2±7.2<¾ Χ The fraction was 16.2 ± 7.2%. The amount of the collected concentrate from monocytes, monocyte concentrated to .146 ± 130ml CDllb + CXXR ^. Cells Total 9.2 ± 1.2X10 9 gae (16 · 2 ± 7.2 <¾ Χ
- 5.3±2.2X108/ml X 146±130 ml) (CDllb+CX3CRl+ (%) X 백혈구 수 /ml X vol 5.3 ± 2.2X10 8 / ml X 146 ± 130 ml) (CDllb + CX3CRl + (%) X white blood cell count / ml X vol
(ml))이었다. 환자 몸무게당 2.5 X 107내지 5 X 107개로 주사 시 . 환자 (ml)). When injected between 2.5 X 10 7 and 5 X 10 7 per patient weight. patient
몸무게를 70kg으로 가정한다면， 2 X 109내지 4 X 109개의 세포가 필요한데 ,
1회 수집된 농축 단핵구로부터 환자의 허혈 부위에 작접 주사할 수 있는 양과 농도의 CDllb+CX3CRl+ᅳ세포 농축액을 얻을 수 있었다 (도 5B). Assuming a weight of 70 kg, 2 X 10 9 to 4 X 10 9 cells are required. The amount and concentration of CDllb + CX3CRl + islet cell concentrates that can be directly injected into the ischemic site of the patient from the collected mononuclear cells were obtained (Fig. 5B).
6. G~CSF와 AMD3100을 병용투여 시에도농축 단핵구에서 6. Even when G-CSF and AMD3100 were used in combination,
CDllb+CX3CRl†세포수가증가된다. CDllb + CX3CRl † Cell number increases.
G-CSF와 AMD3100을 병용 투여'한 다음 얻은 말초 혈액 내의 Co-administration of G-CSF with AMD3100 '' followed by
CDllb+CX3CRl+세포 분획이 전체 백혈구 중 약 1.1% 이었고， CDllb+CX3CRl+ 세 i 수는 600 X 103깨 /mL (l.l%X54,550/mm3)였다. CDllb + CX3CRl + cell fraction was approximately 1.1% of the total white blood cell, CDllb + + Three CX3CRl be i was 600 X 10 3 seeds / mL (ll% X54,550 / mm 3).
혈액 성분 채집기로 수집하고. 혈액 원심분리기로 농축한 농축 단핵구의 경우 농축 단핵구 내 CDllb+CX3CRl+세포 분획이 약 12%였고， 수집된 양은 198mL였다. 계산하면 CDllb+CX3CRl+세포 수는 총 6.3X109개 (12% X 2.67 X lOVml X 198 ml) (CDllb+CX3CRl+(%) X 백혈구 수 /ml X vol (ml))었다. 따라서 . G-CSF와 AMD3100을 병용 투여한 경우에도 환자의 허혈 부위에 직접 주사할 수 있는 양과 농도의 CDllb+CX3CRl+세포 농축액을 얻을 수 있었다 (도 6).
Collect blood components with a collector. For concentrated monocytes concentrated by blood centrifuge, the CDllb + CX3CRl + cell fraction in the concentrated monocytes was about 12% and the amount collected was 198 mL. To calculate the total CDllb + CX3CRl + cell number was 6.3X10 9 (12% X 2.67 X lOVml X 198 ml) (CDllb + CX3CRl + (%) X leukocyte count / ml X vol (ml)). therefore . Even when G-CSF and AMD3100 were co-administered, CDllb + CX3CRl + cell concentrates were obtained in the amount and concentration that can be directly injected into the ischemic site of the patient (FIG. 6).
Claims
【청구형 " 1】 . - Billing type "1].
CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하며 혈관신생 촉진 기능을 나타내는'세포ᅳ -CDllb + and including an immune phenotype of CX3CR1 + and "eu cells indicating angiogenesis promoting function -
5 - ' 5- '
[청구항 2】 ᅳ [Claim 2] ᅳ
제 1항에 있어서，추가적으로 CD45+, F4/80+, Tie-2+, CXCR4+, VEGFR-1+, VEGFR-2", ΝΚ1.Γ, CDllc-, CD3" 및 CD19—에서 선택되는 하나 아상의 면역 ■ 표현형을 포함하는 세포. The one sub-phase according to claim 1, additionally selected from CD45 + , F4 / 80 + , Tie-2 + , CXCR4 + , VEGFR-1 + , VEGFR-2 " , ΝΚ1.Γ, CDllc-, CD3 " and CD19— ■ immune cells that contain an expression.
10 ' ' ' . ； ' ' 10 ''' . ''
【청구항 3】 . . [Claim 3]. .
CDllb+ 및 CX3CR1+ 의 면역 표현형을 포함하는 세포를 포함하는. Containing cells comprising an immune phenotype of CDllb + and CX3CR1 + .
혈관신생 촉진용 조성물. Composition for promoting angiogenesis.
15 【청구항 4】 - 제 3항에 있어서， 상기 세포는 추가적으로, CD45+-,'F4/80+, Ti,e_2+, ■ CXCR4+, VEGFR-1+, VEGFR-2", ΝΚ1.Γ, CDllc", CD3—및 CD19—에서 선택되는 하나 이상의 면역 표현형을 포함하는 것인, 혈관신생 촉진용 조성물. 0 [청구항 5】 15 [Claim 4]-The cell according to claim 3, wherein the cell is additionally selected from CD45 + -, ' F4 / 80 + , Ti , e_2 + , CXCR4 + , VEGFR-1 + , VEGFR-2 " , ΝΚ1.Γ, Comprising one or more immune phenotypes selected from CDllc " , CD3—and CD19—, composition for promoting angiogenesis. 0 [claim 5]
제 3항에 있어서, Fkn (fractalkine)단백질을 추가로 포함하는 것인, 혈관신생 촉진용 조성물. . The composition for promoting angiogenesis according to claim 3, further comprising a Fkn (fractalkine) protein. .
【청구항 6】[Claim 6]
5 CDllb+및 CX3CR1+의 면역 표현형을 포함하는 세포를 포함하는, Comprising cells comprising an immune phenotype of 5 CDllb + and CX3CR1 + ,
허혈성 질환 예방 또는 치료용 약학 조'성물. . The pharmaceutical Joe Dangerous for the prevention or treatment of ischemic diseases. .
【청구항 7】 . . ■ 【Claim 7】. . ■
제 6항에 있어서， 상기 세포는 추가,적으로 , CD45+, F4/80+, Tie-2+, 0 CXCR4+, VEGFR-1+, VEGFR-2", N 1.1", CDllc", 003 .및 )19_에서 선택되는 하나
이상의 ：면역 표현형을 포한하는 것인, 허혈성 질환 예방 또는 치료용 약학 조성물. ' - 7. The method of claim 6 wherein the cells are added, typically, CD45 +, F4 / 80 + , Tie-2 +, 0 CXCR4 +, VEGFR-1 +, VEGFR-2 ", N 1.1", CDllc ", 003. And one selected from 19 _ Above: The pharmaceutical composition for preventing or treating ischemic disease, including the immune phenotype. ' -
【청구항' 8】 【Claim Port ' 8】
5 제 6항에 았어서， 상기 허혈성 질환은 심부전 , 고혈압성 심장질환， 부정맥'， 선천성 심장질환， 심근경색， 뇌졸중， 말초혈관질환， 협심증， 뇌혈관성 치매， 관상동맥부전, 뇌부전'. 망막.허할 및 혈관부전으로；이루어진 군에서 선택된 것인, 허혈성 질환 예방 또는 치료용 약학 조성물. 5 Article 6 ateo stand, the ischemic disease, heart failure, hypertensive heart disease, arrhythmias, congenital heart disease, myocardial infarction, stroke, peripheral vascular disease, angina pectoris, cerebrovascular dementia, coronary insufficiency, cerebrovascular insufficiency "in. Retinal. Percutaneous and vascular insufficiency; Pharmaceutical composition for preventing or treating ischemic disease, selected from the group consisting of.
10 10
【청구항 9】 . 【Claim 9】 .
' 제 6항에 있어서， Fkn (fractalkine)단백질을 추가로 포함하는 것인， 허혈성 질환 예방 또는 치료용 약학 조성물; 'According to claim 6, Fkn (fractalkine) A pharmaceutical composition for the, ischemic disease preventing or treating further comprises a protein;
【청구항 10】 , 【Claim 10】 ,
15 G-CSF (granulocyte colony stimulating factor )¾- 포함하는, CDllV및15 G-CSF (granulocyte colony stimulating factor) ¾-containing, CDllV, and
CX3CR1+ 와 면역 표현형을 포함하는 세포의 대량 수집용 조성물. A composition for mass collection of cells comprising CX3CR1 + and an immune phenotype.
[청구항 11】 . . [Claim 11]. .
제 10항에 있어서, 상기 세포는 추가적으로.， CD45+, F4/80+, Tie-2+, 20 CXCR4+, VEGFR-1+, VEGFR-2", NK 1.1", CDllc", CD3'및 CD19—에서 선택되는 하나 이상의 면역 표현형을 포함하는 세포인 조성물. The cell of claim 10, wherein the cells further comprise CD45 + , F4 / 80 + , Tie-2 + , 20 CXCR4 + , VEGFR-1 + , VEGFR-2 “ , NK 1.1 ” , CDllc “ , CD3 ”, and CD19. — A composition that is a cell comprising one or more immune phenotypes selected from.
【청구항 121 . Claims 121.
제 10항에 있어서， AMD3100 또는 약학적으로 허용되는 이의 염을 25. 추가적으로 포함하는 조성.물、 11. The composition of claim 10 further comprising AMD3100 or a pharmaceutically acceptable salt thereof.
【청구항 .13】 【Claims .13】
G-CSF (granulocyte colony stimulating factor )'를 함하는 조성물을 투여한 개체의 말초 혈액으로부터 혈액 단핵구분획을 분리하는 단계， 및 30 상기 혈액'단핵구 분획을 농축하는 단계를 포함하는,
CDllb+ 및 CX3CR1+의 면역 표현형을포함하는 세포의 대량 수집 방법 . ： Separating blood mononuclear fractions from peripheral blood of a subject administered a composition comprising a composition comprising a granulocyte colony stimulating factor (G-CSF), and 30 concentrating the blood ' monocyte fractions, Method for mass collection of cells comprising immune phenotypes of CDllb + and CX3CR1 + . ：
【청구항 14】. [Claim 14].
제 13항에 있어서, 상기 세포는 추가적으로 CD45+, F4/80+, Tie-2+, CXCR4+, VEGFR-1+, VEGFR— 2—， ΝΚΙ'.1", CDllc", CD3" 및 CD19—에서 선택되는 하나 이상의 면역 표현형을 포함하는 세포인 방법. The method of claim 13, wherein the cells further comprise CD45 + , F4 / 80 + , Tie-2 + , CXCR4 + , VEGFR-1 + , VEGFR— 2—, ΝΚΙ ' .1 " , CDllc " , CD3 " and CD19— A cell comprising one or more immune phenotypes selected from.
【청구항 15] [Claim 15]
제 13항에 있어서 , 상기 조성물은 AMD3100또는 약학적으^ 허용되는 이의 염을 추가적으로 포함하는 방법 . The method of claim 13, wherein the composition further comprises AMD3100 or a pharmaceutically acceptable salt thereof.
【청구항 16】' ' . [Claim 16] '' .
제 13항에 있어서， 상기에서 혈액 단핵구 분획을 분리하는 단계는 혈액 성분 채집기를 이용하여 행되는 방법. The method of claim 13, wherein said separating said blood monocyte fraction is performed using a blood component collector.
. .
[청구항 17】 [Claim 17]
제 13항에 있어서， 상기에서 혈액 단핵구 분획을 농축하는 단계는 혈액 원심분리에 의하여 수행되는 방법. ' The method of claim 13, wherein concentrating the blood monocyte fraction in the method is performed by blood centrifugation. '
【청구항 18】 [Claim 18]
제 13항 내지 제 17항 중 어느 한 항의 방법을 이용하여 대량 수집되며， CDllb+및 CX3CR1+의 면역 표현형을 포함하는 세포가 1OT 이상 포함된 세포 집단. ■ 18. A cell population collected in bulk using the method of any one of claims 13-17, wherein the cell population comprises at least one OT cell comprising an immunophenotype of CDllb + and CX3CR1 + . ■
[청구힝" 19] [Claimed Hing "19;
제 18항의 세포 집단을 포함하는, 혈관신생 촉진용 조성물. 19. A composition for promoting angiogenesis, comprising the cell population of claim 18.
【청구항 20】 ' . ' 【Claim 20】 ' . '
^ 제 18항의 세포 집단을 포함하는，허혈성 질환 예방 또는 치료용 약학 조성물. ..
;【청구항' 21】 ^ Pharmaceutical composition for preventing or treating ischemic disease, comprising the cell population of claim 18. . . ; [Claim port '21]
: 제 20항에 있어서， 상기 허혈성 질환은 심부전. 고혈압성'심장질환, -부정맥， .선천성 심장질환'. 심근경색.; 뇌.졸중， 말초혈관질환， 협심증/ . 관상동맥부전,'뇌부천， 망막 허혈 혈관부.전으로 이루어진 군에서 선택된 '：조성물 .
: The ischemic disease according to claim 20, wherein the ischemic disease is heart failure. Hypertensive ' heart disease, arrhythmia,. Congenital heart disease ' . Myocardial infarction; Brain, stroke, peripheral vascular disease, angina . Coronary insufficiency, "Brain Bucheon, retinal ischemia vessel portion selected from the group consisting of I ': the composition.
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| KR1020120085341A KR101518369B1 (en) | 2012-08-03 | 2012-08-03 | CD11b+CX3CR1+ cells and a composition for preventing or treating ischemic disease comprising the same |
| KR10-2012-0085341 | 2012-08-03 | ||
| KR1020120085342A KR101419480B1 (en) | 2012-08-03 | 2012-08-03 | A method for mobilization of CD11b+CX3CR1+ cells |
| KR10-2012-0085342 | 2012-08-03 |
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