WO2020262916A1 - Composition for treatment of peripheral arterial disease - Google Patents

Composition for treatment of peripheral arterial disease Download PDF

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Publication number
WO2020262916A1
WO2020262916A1 PCT/KR2020/008135 KR2020008135W WO2020262916A1 WO 2020262916 A1 WO2020262916 A1 WO 2020262916A1 KR 2020008135 W KR2020008135 W KR 2020008135W WO 2020262916 A1 WO2020262916 A1 WO 2020262916A1
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endothelial cells
human
cells
tooth
stem cells
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PCT/KR2020/008135
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French (fr)
Korean (ko)
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주경민
남현
김충권
편희장
황지윤
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(주)메디노
성균관대학교산학협력단
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Priority claimed from KR1020200076924A external-priority patent/KR102397010B1/en
Publication of WO2020262916A1 publication Critical patent/WO2020262916A1/en

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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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a composition for treatment of peripheral arterial disease, and more particularly, to a composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  • Peripheral artery disease includes a wide range of vascular diseases caused by atherosclerotic, thromboembolic and inflammatory mechanisms that alter the structure and function of arteries.
  • peripheral arterial disease includes pulmonary thromboangiitis (thromboangiitis obliterans), obliterans, and critical limb ischemia, which are induced by vascular inflammation, thrombosis, hyperlipidemia, diabetes, etc.
  • thromboangiitis obliterans pulmonary thromboangiitis
  • obliterans obliterans
  • critical limb ischemia which are induced by vascular inflammation, thrombosis, hyperlipidemia, diabetes, etc.
  • peripheral arterial disease surgical operations such as stent implantation, balloon dilatation, angioplasty, or the use of drugs that expand blood vessels are mostly used, but this is to alleviate the primary symptoms, so it is not a fundamental treatment.
  • the only option is to take antisystemic analgesics to reduce pain, or to perform limb amputation to prevent the progression of gangrene or infection. Therefore, if there is a fundamental treatment method that can inhibit inflammation in blood vessels, inhibit muscle damage and fibrosis, and promote blood vessel formation, not only can various peripheral arterial diseases be effectively treated, but also limb amputation rate, mortality rate, etc. It is expected to be able to significantly lower the value.
  • a tooth-derived stem cell is a stem cell located in a tooth, and it is known that the tooth contains mesenchymal stem cells having a higher self-proliferative ability and differentiation ability than any other tissue at a high density. It is known that tooth-derived stem cells can be separated from gums such as pulp, periodontal ligaments, roots, dental pouches, and root papilla of indwelling or permanent teeth. In addition, it is not only possible to easily separate tooth-derived stem cells from naturally missing teeth or surgically removed teeth, but also its parents and grandparents as well as the owners of the teeth can be used in regenerative medicine, so its application value is very high. Therefore, in recent years, research on a method of using tooth-derived stem cells has been actively conducted (Korean Patent Registration No. 10-1715464).
  • the present invention was conceived to solve the problems of the prior art as described above, as a fundamental treatment method for peripheral arterial disease, a composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients Its purpose is to provide such as.
  • the present invention provides a pharmaceutical composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  • the present invention provides a cell therapy for preventing or treating peripheral arterial disease, comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  • the tooth-derived stem cells are preferably stem cells derived from pulp, periodontal ligaments, roots, dental vesicles, root papillae, gums, etc., more preferably pulp-derived stem cells.
  • the tooth-derived stem cells may not express one or more cell surface antigens selected from the group consisting of CD14, CD31, CD34, and CD45. However, it is not limited thereto as long as it is a stem cell capable of differentiating into adipocytes and bones derived from teeth.
  • the vascular endothelial cells are preferably Bend3 cells, human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (Human dermal microvascular). endothelial cells; HMVEC-D or HDMEC), human cardiac endothelial cells, human aortic endothelial cells (HAEC), human coronary and iliac artery endothelial cells; HIAEC or HCAEC), human cardiac microvascular endothelial cells, human bladder and uterine microvascular endothelial cells, human pulmonary artery endothelial cells; HPAEC or PASMC), human lung microvascular endothelial cells (HMVEC-L), human brachiocephalic artery endothelial cells (HBcAEC), human carotid artery endothelial cells (HCtAEC) , Human dermal lymphatic microvascular endothelial cells (HDLMVEC), human internal thoracic
  • vascular endothelial cells derived from stem cells including embryonic stem cells, mesenchymal stem cells, dedifferentiated stem cells, etc., or vascular endothelial cells produced by direct conversion of stem cells, etc.
  • stem cells including embryonic stem cells, mesenchymal stem cells, dedifferentiated stem cells, etc.
  • vascular endothelial cells produced by direct conversion of stem cells etc.
  • the tooth-derived stem cells and vascular endothelial cells may preferably be included in a number ratio of 1:0.1 to 1:10.
  • the peripheral arterial disease is preferably severe limb ischemia, obstructive thromboangiitis (thromboangiitis obliterans), arteriosclerosis obliterans, polyarteritis nodosa. (polyarteritis nodosa), etc., but is not limited to vascular diseases induced by vascular inflammation, thrombosis, hyperlipidemia, diabetes, etc. in all blood vessels except the central artery.
  • the present invention provides a method for preventing or treating peripheral arterial disease, comprising administering to an individual a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  • the present invention provides a use of a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients to prevent or treat peripheral arterial disease.
  • the present invention provides a use for producing a medicament used in the treatment of peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  • compositions comprising tooth-derived stem cells and vascular endothelial cells according to the present invention as active ingredients to an animal model of peripheral arterial disease.
  • inflammatory reactions are suppressed, muscle damage is prevented, and fibrosis is inhibited from being induced.
  • the formation of new blood vessels can be promoted by increasing the ability to form blood vessels. Therefore, the composition of the present invention can be widely applied not only to the treatment of various peripheral arterial diseases caused by various causes such as atherosclerosis, thromboembolism, and inflammatory mechanisms, but also to alleviate primary symptoms by simply expanding blood vessels.
  • it can simultaneously treat various conditions of peripheral arterial disease, it is expected that it can be effectively used in the treatment of peripheral arterial disease by remarkably increasing treatment efficiency.
  • 1A is a view showing a result of confirming tooth-derived stem cells according to an embodiment of the present invention under an optical microscope.
  • 1B is a view showing a result of confirming the immune phenotype of tooth-derived stem cells according to an embodiment of the present invention by flow cytometry.
  • 1C is a view showing the results of confirming the immune phenotype of vascular endothelial cells (HUVEC) according to an embodiment of the present invention by flow cytometry.
  • VEC vascular endothelial cells
  • 1D is a view showing the results of confirming the bone differentiation ability and adipocyte differentiation ability of tooth-derived stem cells according to an embodiment of the present invention.
  • FIG. 2A is a view showing a result of confirming the effect of treating inflammation and muscle damage of tooth-derived stem cells and vascular endothelial cells by H&E staining in a peripheral arterial disease model according to an embodiment of the present invention.
  • FIG. 2B is a view showing a result of confirming the effect of inhibiting fibrosis induction of tooth-derived stem cells and vascular endothelial cells in a peripheral arterial disease model according to an embodiment of the present invention by Masson's trichrome (MT) staining.
  • MT Masson's trichrome
  • FIG. 3 is a view showing the results of confirming the blood vessel formation ability of tooth-derived stem cells and vascular endothelial cells with a Matrigel plug in a peripheral arterial disease model according to an embodiment of the present invention.
  • FIG. 4A is a schematic diagram showing an overall experimental method for confirming the therapeutic effect of tooth-derived stem cells and vascular endothelial cells for peripheral arterial disease according to an embodiment of the present invention
  • FIG. 4B is a blood flow measurement to confirm the therapeutic effect.
  • FIG. 4C is a diagram showing the result of confirming the degree of ischemia injury.
  • composition comprising tooth-derived stem cells and vascular endothelial cells of the present invention as active ingredients inhibits inflammatory reactions and muscle damage in a peripheral arterial disease model, inhibits the induction of fibrosis, as well as promotes the ability to form blood vessels, thereby creating new blood vessels. Since it can be produced, it is expected that it can be effectively used in the treatment of peripheral arterial diseases caused by various causes.
  • stem cell refers to a concept in a broad sense that collectively refers to undifferentiated cells having the ability to differentiate into various types of body tissue cells, that is, stemness.
  • Stem cells refer to stem cells derived from pulp, periodontal ligaments, roots, dental pouches, root papillae, and gums.
  • dimension refers to a soft tissue-like tissue located in the center of the tooth, and the dimension is surrounded by dentin and includes nerve fibers, blood vessels, and lymphatic vessels.
  • Pulp-derived stem cells refer to stem cells that exist in pulp, and pulp is a tissue from which the neural plate differentiates into a neural crest and originates from it.
  • pulp-derived stem cells are a cell source for the development of cell therapy products for regenerative medicine because pulp is easily accessible and has a high ratio between stem cells and collected tissue volumes. Very useful.
  • the pulp-derived stem cells may be derived from pulp of a human tooth.
  • the teeth are not particularly limited, but teeth or wisdom teeth are preferred. Tooth extraction is a process that must be performed, and wisdom teeth extraction is also very common, and since both teeth are mostly discarded after extraction, it has an advantage in that a separate procedure is not required for collecting tooth stem cells.
  • the pulp-derived stem cells may have the characteristics of mesenchymal stem cells.
  • the pulp stem cells may be those in which the markers are expressed when measuring the expression of markers known to exhibit mesenchymal stem cell characteristics through an analysis method such as FACS, real-time PCR, and immunofluorescence staining.
  • the pulp-derived stem cells may express one or more genes selected from the group consisting of CD29, CD44, CD73, CD90, and CD105, preferably CD29, CD44, CD73, CD90, and CD105 genes It may be to express all of.
  • the pulp-derived stem cells may not express one or more types of hematopoietic stem cell markers selected from the group consisting of CD14, CD34, CD45, CD117, and HLA-DR.
  • the pulp-derived stem cells may not express CD31, which is a vascular endothelial cell marker.
  • peripheral artery disease refers to all vascular diseases induced by inflammation, thrombosis, hyperlipidemia, diabetes, etc. in blood vessels excluding the central artery, preferably severe limb ischemia ( critical limb ischemia), pulmonary thromboangiitis (thromboangiitis obliterans), obliterans atherosclerosis (arteriosclerosis obliterans), polyarteritis nodosa, etc.
  • prevention refers to any action that suppresses or delays the onset of peripheral arterial disease by administration of the composition according to the present invention.
  • treatment refers to any action in which symptoms of peripheral arterial disease are improved or advantageously changed by administration of the composition according to the present invention.
  • “individual” refers to a subject to which the composition of the present invention can be administered, and for example, human or non-human primates, mice, dogs, cats, horses, and cattle It can mean a mammal, but the subject is not limited.
  • the term "pharmaceutical composition” may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition is intended for humans.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections.
  • Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used.
  • the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier as described above.
  • for oral administration it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc.In the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. have.
  • dragees, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained-release preparations, and slurries may be formulated and used.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like may be used.
  • fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally
  • the route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral or parenteral administration is preferable, for example, oral, intravenous, intramuscular, intra-articular, intrasynovial, intraarterial, intramedullary, It includes intrathecal, intracardiac, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, rectal, intrasternal, intralesional, intracranial, and the like.
  • the dosage of the pharmaceutical composition of the present invention can be varied including the activity of the specific compound used, age, body weight, general health, sex, formula, administration time, route of administration, excretion rate, drug combination and the severity of the specific disease to be prevented or treated. It may vary according to factors, and varies depending on the patient's condition, weight, degree of disease, drug type, route and duration of administration, but may be appropriately selected by those skilled in the art, and from 0.0001 to 500 mg/kg per day or from 0.001 to It can be administered at 500 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
  • the term "cellular therapeutic agent” refers to a drug (US FDA regulation) used for treatment, diagnosis, and prevention with cells and tissues manufactured through isolation, culture and special manipulation from humans, In order to restore the function of cells or tissues, these cells are used to treat, diagnose and prevent diseases through a series of actions such as proliferating and screening living autologous, allogeneic, or xenogeneic cells in vitro, or changing the biological characteristics of cells in other ways. It means a drug used for the purpose of.
  • the administration route of the cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue.
  • Parenteral administration for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto.
  • composition may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy.
  • “Pharmacologically acceptable” refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness.
  • Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • composition may be administered by any device capable of moving a cell therapeutic agent to a target cell.
  • the cell therapy composition of the present invention may contain a therapeutically effective amount of cell therapy for the treatment of a disease.
  • “Therapeutically effective amount” means the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as considered by a researcher, veterinarian, physician or other clinician. , which includes an amount that induces relief of symptoms of the disease or disorder being treated.
  • the optimal cell therapy content can be easily determined by those skilled in the art, and the type of disease, severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient. , Administration time, administration route and secretion rate of the composition, treatment period, it may be adjusted according to various factors including drugs used simultaneously. It is important to include an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors.
  • the daily dose of the stem cells of the present invention is 1.0 ⁇ 10 4 to 1.0 ⁇ 10 11 cells/kg body weight, preferably 1.0 ⁇ 10 5 to 1.0 ⁇ 10 9 cells/kg body weight, divided once or several times. Can be administered.
  • the actual dosage of the active ingredient should be determined in the light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age, and sex of the patient. It is not intended to limit the scope of the present invention to any aspect.
  • the composition comprising the cell therapeutic agent of the present invention as an active ingredient is rectal, intravenous (intravenous therapy, iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular Alternatively, it can be administered in a conventional manner via an intradermal route.
  • the present invention provides a therapeutic method comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the present invention.
  • mammal refers to a mammal that is the object of treatment, observation or experiment, and preferably refers to a human.
  • tooth-derived stem cells had typical morphological characteristics of mesenchymal stem cells.
  • the tooth-derived stem cells of the present invention express mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105, but hematopoietic stem cell markers CD14, CD34, CD45, CD117, HLA It was confirmed that -DR and CD31, a vascular endothelial cell marker, were not expressed.
  • human umbilical vein endothelial cells were purchased from Promocell and used as vascular endothelial cells, and vascular endothelial cells were cultured using endothelial cell growth medium (Promocell) added with 1% penicillin/streptomycin. I did.
  • endothelial cell growth medium Promocell
  • flow cytometric analysis was performed in the same manner as for tooth-derived stem cells, and the results are shown in FIG. 1C.
  • CD29 and CD105 were highly expressed, and the hematopoietic stem cell markers were not expressed as in the tooth-derived stem cells of the present invention.
  • the differentiation ability of the tooth-derived stem cells obtained in the same manner as in Example 1.1 was confirmed.
  • the tooth-derived stem cells in culture reach 80 to 90% confluence, they are replaced with a medium for inducing bone differentiation or a medium for inducing adipocyte differentiation, and then replaced with a new medium for inducing differentiation every 3 days, Incubated for 3 weeks. After 3 weeks, the cells were washed twice with a phosphate buffer solution, and bone differentiation was confirmed by staining with Alzarin solution, and adipocyte differentiation was confirmed by staining with Oil red O solution. After staining, washing was performed once with a phosphate buffer solution, and washed once again with third distilled water to remove all dyeing reagents, and then the stained cells were dried and observed using an optical microscope. The results are shown in FIG. 1D.
  • tooth-derived stem cells were normally differentiated into osteogenic cells and adipogenic cells.
  • the tooth-derived stem cells obtained by the method of Example 1.1 express a marker of mesenchymal stem cells and are capable of differentiating into adipocytes and bone cells.
  • mice were anesthetized by intraperitoneally administering a zoletyl anesthetic. Then, the skin of the hind limb of the anesthetized mouse was excised using a surgical knife, the muscle was removed to expose the femoral artery, and the middle and end of the femoral artery were strongly tied, and the blood vessels between them were removed, and severe limb ischemia. Animal models were prepared.
  • tooth-derived stem cells and vascular endothelial cells exhibits a therapeutic effect on inflammation, muscle damage, and fibrosis, which are symptoms of peripheral arterial disease
  • 1 ) HBSS administration group 2) tooth-derived stem cells alone, 3) umbilical vein endothelial cells alone, 4) tooth-derived stem cells and umbilical vein endothelial cells, four groups, respectively.
  • cells were prepared at a concentration of 1 X 10 6 cells/80 ⁇ L HBSS, put in a syringe, and injected into three muscles of the area in which peripheral arterial disease was induced to perform cell transplantation. Tooth-derived stem cells and umbilical vein endothelial cells were mixed in a ratio of 1:1.
  • the mouse 14 days after transplantation, the mouse was anesthetized using CO 2 , and a paraffin block was prepared after obtaining the tissue at the site where the cells were transplanted. Then, the prepared paraffin block was cut to a thickness of 5 ⁇ m and then subjected to a diparaffin process, followed by Masson's trichrome (MT) and H&E staining. And observed using a microscope. The results are shown in FIG. 2A for H&E staining results and in FIG. 2B for MT staining results.
  • MT Masson's trichrome
  • FIG. 2A it was confirmed that in the negative control group administered with only HBSS, muscle damage was increased due to the induction of peripheral arterial disease, so that the normal muscle shape was not maintained, and the severity of muscle degeneration was very high. In addition, it was confirmed that infiltration of leukocytes was induced, thereby increasing the inflammatory response at the site where peripheral arterial disease was induced. On the other hand, in the experimental group to which tooth-derived stem cells were administered, it was confirmed that muscle damage and infiltration of leukocytes were also partially reduced.
  • tooth-derived stem cells and vascular endothelial cells promotes blood vessel formation in the peripheral arterial disease site
  • 1) Matrigel alone, 2) tooth Derived stem cells alone, 3) umbilical vein endothelial cells alone, and 4) tooth-derived stem cells and umbilical vein endothelial cells, respectively were administered in four groups.
  • cells were prepared at a concentration of 2 ⁇ 10 6 cells/200 ⁇ L Matrigel, placed in a 25G syringe, and injected subcutaneously in the area in which peripheral arterial disease was induced to perform cell transplantation. Tooth-derived stem cells and umbilical vein endothelial cells were mixed in a ratio of 1:1.
  • the mouse was anesthetized using CO 2 , and the implanted Matrigel was separated, and then a paraffin block was prepared. And the prepared paraffin block was cut to a thickness of 4 ⁇ m, subjected to a diparaffin process, and H&E staining was performed. And observed using a microscope. The results are shown in FIG. 3.
  • the degree of capillary formation was low in the group administered with Matrigel alone, vascular endothelial cells alone, and tooth-derived stem cells alone, but the formation of capillaries in the combined administration of tooth-derived stem cells and umbilical vein endothelial cells. It was confirmed that the red blood cells increased significantly due to the infiltration of red blood cells.
  • the tooth-derived stem cells and vascular endothelial cells are administered in combination, the inflammatory response due to peripheral arterial disease is suppressed, muscle damage is prevented, fibrosis is inhibited, and the blood vessel formation ability is increased.
  • the treatment efficiency can be significantly increased because various conditions of peripheral arterial disease can be simultaneously treated differently from the existing treatment methods that alleviate primary symptoms simply by expanding blood vessels.
  • composition comprising tooth-derived stem cells and vascular endothelial cells of the present invention as active ingredients prevents inflammatory reactions and muscle damage in a peripheral arterial disease model, inhibits fibrosis, and increases blood vessel formation ability. It can be effectively used for the treatment of various peripheral arterial diseases caused by various causes such as sclerosis, thromboembolism, inflammatory mechanisms, etc., and is different from existing treatment methods that alleviate primary symptoms by simply expanding blood vessels. It is expected that the success rate of treatment can be remarkably increased because it can treat various conditions of the disease at the same time.

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Abstract

The present invention relates to a composition for the prevention or treatment of peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells, and it has been confirmed that, as a result of cell transplantation of the tooth-derived stem cells and vascular endothelial cells according to the present invention, there are effects of suppressing an inflammatory response, preventing muscle damage, inhibiting the induction of fibrosis, and at the same time, increasing the ability to form blood vessels, thereby promoting angiogenesis.

Description

말초 동맥 질환의 치료용 조성물Composition for treatment of peripheral arterial disease
본 발명은 말초 동맥 질환의 치료용 조성물에 관한 것으로, 보다 자세하게는 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 말초 동맥 질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for treatment of peripheral arterial disease, and more particularly, to a composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
말초 동맥 질환(Peripheral artery disease, PAD)은 동맥의 구조 및 기능을 변형시키는 죽상경화성, 혈전색전성 및 염증성 기작에 의해 유발되는 광범위한 혈관 질환들을 포함한다. 일반적으로 말초 동맥 질환에는 혈관 염증, 혈전증, 고지혈증, 당뇨병 등으로 인해 유도되는 폐색혈전혈관염(버거씨병, thromboangiitis obliterans), 폐쇄성 죽상동맥경화증(arteriosclerosis obliterans), 중증하지허혈(critical limb ischemia) 등이 포함되며, 광범위하게는 중심동맥을 제외한 모든 혈관에서 유발되는 질환을 의미한다. 말초 동맥 질환의 치료 방법으로는 스텐트 삽입술, 풍선 확장술, 혈관 성형술 등의 외과적 수술 또는 혈관을 확장시키는 약물을 사용하는 것이 대부분이나, 이는 일차적인 증상을 완화시키는 것으로써, 근본적인 치료가 되지 않아, 최종적으로는 통증을 감소시키기 위한 항전신성 진통제를 복용하거나, 괴저의 진행 또는 감염을 방지하기 위해서는 사지 절단을 하는 방법 밖에 없다. 따라서, 혈관 내의 염증을 억제하며, 근육 손상 및 섬유화를 억제하는 동시에 혈관 형성을 촉진시킬 수 있는 근본적인 치료 방법이 있다면, 다양한 말초 동맥 질환을 효과적으로 치료할 수 있을 뿐만 아니라, 이로 인하여 사지 절단률, 사망률 등을 현저히 낮출 수 있을 것으로 기대된다.Peripheral artery disease (PAD) includes a wide range of vascular diseases caused by atherosclerotic, thromboembolic and inflammatory mechanisms that alter the structure and function of arteries. In general, peripheral arterial disease includes pulmonary thromboangiitis (thromboangiitis obliterans), obliterans, and critical limb ischemia, which are induced by vascular inflammation, thrombosis, hyperlipidemia, diabetes, etc. And broadly refers to a disease caused by all blood vessels except the central artery. As a treatment method for peripheral arterial disease, surgical operations such as stent implantation, balloon dilatation, angioplasty, or the use of drugs that expand blood vessels are mostly used, but this is to alleviate the primary symptoms, so it is not a fundamental treatment. In the end, the only option is to take antisystemic analgesics to reduce pain, or to perform limb amputation to prevent the progression of gangrene or infection. Therefore, if there is a fundamental treatment method that can inhibit inflammation in blood vessels, inhibit muscle damage and fibrosis, and promote blood vessel formation, not only can various peripheral arterial diseases be effectively treated, but also limb amputation rate, mortality rate, etc. It is expected to be able to significantly lower the value.
한편, 치아 유래 줄기세포란 치아 내에 위치하고 있는 줄기세포로서, 치아에는 다른 어떤 조직보다 높은 자가증식 능력과 분화 능력을 가지는 중간엽줄기세포를 높은 밀도로 포함하고 있는 것으로 알려져 있다. 치아 유래 줄기세포는 유치 또는 영구치의 치수, 치주 인대, 치근, 치낭, 치근유두 등과 잇몸에서 분리할 수 있는 것으로 알려져 있다. 또한 자연적으로 빠진 치아, 수술적으로 제거된 치아에서도 손쉽게 치아 유래 줄기세포를 분리할 수 있을 뿐만 아니라, 치아의 주인인 당사자뿐만 아니라 그 부모나 조부모까지 재생의료에 사용할 수 있어 그 활용가치가 매우 높기 때문에, 최근에는 치아 유래 줄기세포를 이용하는 방법에 대한 연구가 활발히 진행되고 있다(대한민국등록특허 10-1715464).Meanwhile, a tooth-derived stem cell is a stem cell located in a tooth, and it is known that the tooth contains mesenchymal stem cells having a higher self-proliferative ability and differentiation ability than any other tissue at a high density. It is known that tooth-derived stem cells can be separated from gums such as pulp, periodontal ligaments, roots, dental pouches, and root papilla of indwelling or permanent teeth. In addition, it is not only possible to easily separate tooth-derived stem cells from naturally missing teeth or surgically removed teeth, but also its parents and grandparents as well as the owners of the teeth can be used in regenerative medicine, so its application value is very high. Therefore, in recent years, research on a method of using tooth-derived stem cells has been actively conducted (Korean Patent Registration No. 10-1715464).
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 말초 동맥 질환의 근본적인 치료 방법으로서, 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 말초 동맥 질환의 예방 또는 치료용 조성물 등을 제공하는 것을 그 목적으로 한다.The present invention was conceived to solve the problems of the prior art as described above, as a fundamental treatment method for peripheral arterial disease, a composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients Its purpose is to provide such as.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical task to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. will be.
본 발명은 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 말초 동맥 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
또한, 본 발명은 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 말초 동맥 질환의 예방 또는 치료용 세포 치료제를 제공한다.In addition, the present invention provides a cell therapy for preventing or treating peripheral arterial disease, comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
본 발명의 일 구체예에 있어서, 상기 치아 유래 줄기세포는 바람직하게는 치수, 치주 인대, 치근, 치낭, 치근유두, 잇몸 등으로부터 유래된 줄기세포이며, 더욱 바람직하게는 치수 유래 줄기세포일 수 있다. 또한, 상기 치아 유래 줄기세포는 CD14, CD31, CD34, 및 CD45로 이루어진 군으로부터 선택된 하나 이상의 세포표면항원을 발현하지 않는 것일 수 있다. 그러나 치아로부터 유래된 지방세포 및 골로 분화할 수 있는 능력을 가진 줄기세포라면 이에 제한되지 않는다.In one embodiment of the present invention, the tooth-derived stem cells are preferably stem cells derived from pulp, periodontal ligaments, roots, dental vesicles, root papillae, gums, etc., more preferably pulp-derived stem cells. . In addition, the tooth-derived stem cells may not express one or more cell surface antigens selected from the group consisting of CD14, CD31, CD34, and CD45. However, it is not limited thereto as long as it is a stem cell capable of differentiating into adipocytes and bones derived from teeth.
본 발명의 다른 구체예에 있어서, 상기 혈관내피세포(vascular endothelial cell)는 바람직하게는 Bend3 세포, 인간 제대정맥내피세포(Human umbilical vein endothelial cells; HUVEC), 인간 진피 미세 혈관내피세포(Human dermal microvascular endothelial cells; HMVEC-D 또는 HDMEC), 인간 심장 내피세포(Human cardiac endothelial cells), 인간 대동맥 내피세포(Human aortic endothelial cells; HAEC), 인간 관상 및 장골 동맥 내피세포(Human coronary and iliac artery endothelial cells; HIAEC 또는 HCAEC), 인간 심장 미세 혈관내피세포(Human cardiac microvascular endothelial cells), 인간 방광 및 자궁 미세 혈관내피세포(Human bladder and uterine microvascular endothelial cells), 인간 폐동맥 내피세포(Human pulmonary artery endothelial cells; HPAEC 또는 PASMC), 인간 폐 미세 혈관내피세포(Human lung microvascular endothelial cells; HMVEC-L), 인간 완두 동맥 내피세포(Human brachiocephalic artery endothelial cells; HBcAEC), 인간 목동맥 내피세포(Human carotid artery endothelial cells; HCtAEC), 인간 진피 림프 미세 혈관내피세포(Human dermal lymphatic microvascular endothelial cells; HDLMVEC), 인간 내흉 동맥 내피세포(Human internal thoracic artery endothelial cells; HITAEC), 인간 쇄골하 동맥 내피세포(Human subclavian artery endothelial cells; HScAEC), 인간 제대 동맥 내피세포(Human umbilical artery endothelial cells; HUAEC) 등이 일수 있다. 또한, 배아줄기세포, 중간엽줄기세포, 역분화줄기세포 등을 포함하는 줄기세포로부터 유래된 혈관내피세포, 또는 줄기세포의 직접전환분화(direct conversion)로 제조된 혈관내피세포 등일 수 있으나, 혈관의 내막을 구성하는 단층 세포막 유래 세포라면 제한이 없다.In another embodiment of the present invention, the vascular endothelial cells are preferably Bend3 cells, human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (Human dermal microvascular). endothelial cells; HMVEC-D or HDMEC), human cardiac endothelial cells, human aortic endothelial cells (HAEC), human coronary and iliac artery endothelial cells; HIAEC or HCAEC), human cardiac microvascular endothelial cells, human bladder and uterine microvascular endothelial cells, human pulmonary artery endothelial cells; HPAEC or PASMC), human lung microvascular endothelial cells (HMVEC-L), human brachiocephalic artery endothelial cells (HBcAEC), human carotid artery endothelial cells (HCtAEC) , Human dermal lymphatic microvascular endothelial cells (HDLMVEC), human internal thoracic artery endothelial cells (HITAEC), human subclavian artery endothelial cells (Human subcla) vian artery endothelial cells; HScAEC), human umbilical artery endothelial cells (HUAEC), and the like. In addition, vascular endothelial cells derived from stem cells including embryonic stem cells, mesenchymal stem cells, dedifferentiated stem cells, etc., or vascular endothelial cells produced by direct conversion of stem cells, etc. There is no limitation as long as it is a cell derived from a single layer cell membrane constituting the inner membrane of
본 발명의 또 다른 구체예에 있어서, 상기 치아 유래 줄기세포 및 혈관내피세포는 바람직하게는 1 : 0.1 내지 1 : 10의 개수비로 포함될 수 있다.In another embodiment of the present invention, the tooth-derived stem cells and vascular endothelial cells may preferably be included in a number ratio of 1:0.1 to 1:10.
본 발명의 또 다른 구체예에 있어서, 상기 말초 동맥 질환은 바람직하게는 중증하지허혈(critical limb ischemia), 폐색혈전혈관염(버거씨병, thromboangiitis obliterans), 폐쇄성 죽상동맥경화증(arteriosclerosis obliterans), 결절성 다발동맥염(polyarteritis nodosa) 등일 수 있으나, 중심동맥을 제외한 모든 혈관에서 혈관 염증, 혈전증, 고지혈증, 당뇨병 등으로 인해 유도되는 혈관 질환들이라면 이에 제한되지 않는다.In another embodiment of the present invention, the peripheral arterial disease is preferably severe limb ischemia, obstructive thromboangiitis (thromboangiitis obliterans), arteriosclerosis obliterans, polyarteritis nodosa. (polyarteritis nodosa), etc., but is not limited to vascular diseases induced by vascular inflammation, thrombosis, hyperlipidemia, diabetes, etc. in all blood vessels except the central artery.
또한, 본 발명은 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물을 개체에 투여하는 단계를 포함하는 말초 동맥 질환의 예방 또는 치료 방법을 제공한다. In addition, the present invention provides a method for preventing or treating peripheral arterial disease, comprising administering to an individual a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
또한, 본 발명은 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물의 말초 동맥 질환의 예방 또는 치료 용도를 제공한다.In addition, the present invention provides a use of a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients to prevent or treat peripheral arterial disease.
또한, 본 발명은 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 말초 동맥 질환의 치료에 이용되는 약제를 생산하기 위한 용도를 제공한다.In addition, the present invention provides a use for producing a medicament used in the treatment of peripheral arterial disease comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
본 발명에 따른 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물을 말초 동맥 질환 동물 모델에 투여한 결과, 염증 반응이 억제되고, 근육 손상을 방지하고, 섬유화가 유도되는 것을 억제하는 동시에 혈관 형성능을 증가시켜 신규 혈관 형성을 촉진시킬 수 있는 것을 확인하였다. 따라서, 본 발명의 조성물은 죽상경화성, 혈전색전성, 염증성 기작 등 다양한 원인에 의해 유발되는 다양한 말초 동맥 질환의 치료에 폭넓게 적용할 수 있을 뿐만 아니라, 단순히 혈관을 확장하는 것으로 일차적인 증상을 완화시키는 기존의 치료 방법과는 상이하게 말초 동맥 질환의 다양한 병증을 동시에 치료할 수 있기 때문에 치료 효율을 현저히 증가시켜 말초 동맥 질환의 치료에 효과적으로 사용될 수 있을 것으로 기대된다.As a result of administering a composition comprising tooth-derived stem cells and vascular endothelial cells according to the present invention as active ingredients to an animal model of peripheral arterial disease, inflammatory reactions are suppressed, muscle damage is prevented, and fibrosis is inhibited from being induced. It was confirmed that the formation of new blood vessels can be promoted by increasing the ability to form blood vessels. Therefore, the composition of the present invention can be widely applied not only to the treatment of various peripheral arterial diseases caused by various causes such as atherosclerosis, thromboembolism, and inflammatory mechanisms, but also to alleviate primary symptoms by simply expanding blood vessels. Unlike conventional treatment methods, since it can simultaneously treat various conditions of peripheral arterial disease, it is expected that it can be effectively used in the treatment of peripheral arterial disease by remarkably increasing treatment efficiency.
도 1a는 본 발명의 일 실시예에 따른 치아 유래 줄기세포를 광학 현미경 하에서 확인한 결과를 나타낸 도면이다.1A is a view showing a result of confirming tooth-derived stem cells according to an embodiment of the present invention under an optical microscope.
도 1b는 본 발명의 일 실시예에 따른 치아 유래 줄기세포의 면역 표현형을 유세포 분석으로 확인한 결과를 나타낸 도면이다.1B is a view showing a result of confirming the immune phenotype of tooth-derived stem cells according to an embodiment of the present invention by flow cytometry.
도 1c는 본 발명의 일 실시예에 따른 혈관내피세포(HUVEC)의 면역 표현형을 유세포 분석으로 확인한 결과를 나타낸 도면이다.1C is a view showing the results of confirming the immune phenotype of vascular endothelial cells (HUVEC) according to an embodiment of the present invention by flow cytometry.
도 1d는 본 발명의 일 실시예에 따른 치아 유래 줄기세포의 골 분화능 및 지방세포 분화능을 확인한 결과를 나타낸 도면이다.1D is a view showing the results of confirming the bone differentiation ability and adipocyte differentiation ability of tooth-derived stem cells according to an embodiment of the present invention.
도 2a는 본 발명의 일 실시예에 따른 말초 동맥 질환 모델에서 치아 유래 줄기세포 및 혈관내피세포의 염증 및 근육 손상 치료 효과를 H&E 염색으로 확인한 결과를 나타낸 도면이다.2A is a view showing a result of confirming the effect of treating inflammation and muscle damage of tooth-derived stem cells and vascular endothelial cells by H&E staining in a peripheral arterial disease model according to an embodiment of the present invention.
도 2b는 본 발명의 일 실시예에 따른 말초 동맥 질환 모델에서 치아 유래 줄기세포 및 혈관내피세포의 섬유증 유발 억제 효과를 Masson's trichrome(MT) 염색으로 확인한 결과를 나타낸 도면이다.FIG. 2B is a view showing a result of confirming the effect of inhibiting fibrosis induction of tooth-derived stem cells and vascular endothelial cells in a peripheral arterial disease model according to an embodiment of the present invention by Masson's trichrome (MT) staining.
도 3은 본 발명의 일 실시예에 따른 말초 동맥 질환 모델에서 치아 유래 줄기세포 및 혈관내피세포의 혈관 형성능을 마트리겔 플러그(Matrigel plug)로 확인한 결과를 나타낸 도면이다.3 is a view showing the results of confirming the blood vessel formation ability of tooth-derived stem cells and vascular endothelial cells with a Matrigel plug in a peripheral arterial disease model according to an embodiment of the present invention.
도 4a는 본 발명의 일 실시예에 따른 치아 유래 줄기세포 및 혈관내피세포의 말초 동맥 질환의 치료 효과를 확인하기 위한 전반적인 실험 방법을 나타낸 개략도이고, 도 4b는 치료 효과를 확인하기 위하여 혈류량을 측정한 결과를 나타낸 도면이며, 도 4c는 허혈 손상 정도를 확인한 결과를 나타낸 도면이다.4A is a schematic diagram showing an overall experimental method for confirming the therapeutic effect of tooth-derived stem cells and vascular endothelial cells for peripheral arterial disease according to an embodiment of the present invention, and FIG. 4B is a blood flow measurement to confirm the therapeutic effect. A diagram showing one result, and FIG. 4C is a diagram showing the result of confirming the degree of ischemia injury.
본 발명의 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물은 말초 동맥 질환 모델에서 염증 반응 및 근육 손상을 억제하며, 섬유화가 유도되는 것을 저해할 뿐만 아니라 혈관 형성능이 촉진되어 새로운 혈관들을 생성시킬 수 있기 때문에, 다양한 원인에 의한 말초 동맥 질환의 치료에 효과적으로 사용될 수 있을 것으로 기대된다.The composition comprising tooth-derived stem cells and vascular endothelial cells of the present invention as active ingredients inhibits inflammatory reactions and muscle damage in a peripheral arterial disease model, inhibits the induction of fibrosis, as well as promotes the ability to form blood vessels, thereby creating new blood vessels. Since it can be produced, it is expected that it can be effectively used in the treatment of peripheral arterial diseases caused by various causes.
본 명세서에 있어서, "줄기세포(stem cell)"란 여러 종류의 신체 조직 세포로 분화할 수 있는 능력, 즉, 줄기세포성(stemness)을 가진 미분화 세포를 총칭하는 광의의 개념을 말하며, 치아 유래 줄기세포는 치수, 치주 인대, 치근, 치낭, 치근유두, 잇몸 등으로부터 유래된 줄기세포를 의미한다. 이 중 “치수”는 치아 내부의 중심부에 있는 연조직성 고유조직을 말하며, 상기 치수는 상아질로 둘러싸여 있고 내부에 신경섬유, 혈관, 림프관을 포함하고 있다. 치수 유래 줄기세포는 치수에 존재하는 줄기세포를 말하며, 치수는 배아발생 초기에 신경판(neural plate)이 신경능선(neural crest)으로 분화되고 이로부터 발생이 기원하는 조직이기 때문에, 치수 내부에는 신경세포를 이룰 수 있는 외배엽성의 줄기세포가 존재할 개연성이 매우 높다. 또한, 치수는 쉽게 접근가능하고, 줄기세포 및 수집된 조직 부피간의 높은 비율을 갖는 생물학적 지위(niche)를 가지므로, 치수 유래 줄기세포는 재생 의료를 위한 세포치료제 개발의 세포원천(cell source)으로 매우 유용하다. 상기 치수 유래 줄기세포는 인간의 발치한 치아의 치수로부터 유래된 것일 수 있다. 치아는 특별히 한정되지 않으나, 유치 또는 사랑니가 바람직하다. 유치의 발치는 반드시 이루어지는 과정이고, 사랑니의 발치 또한 매우 흔하여 두 치아 모두 발치 후 대부분 버려지는 기관이므로, 치아 줄기세포 수집을 위해 별도의 시술이 필요 없다는 점에서 장점을 갖는다. 상기 치수 유래 줄기세포는 중간엽 줄기세포의 특성을 가질 수 있다. 예컨대, 상기 치수줄기세포는 FACS, real-time PCR, 면역형광염색법 등의 분석방법을 통해 중간엽줄기세포 특성을 나타내는 것으로 공지된 마커들의 발현 측정 시, 상기 마커들이 발현되는 것일 수 있다. 일례로, 상기 치수 유래 줄기세포는 CD29, CD44, CD73, CD90, 및 CD105로 이루어진 군으로부터 선택되는 1종 이상의 유전자를 발현하는 것일 수 있으며, 바람직하게는 CD29, CD44, CD73, CD90, 및 CD105 유전자를 모두 발현하는 것일 수 있다. 또한, 상기 치수 유래 줄기세포는 CD14, CD34, CD45, CD117, 및 HLA-DR로 이루어진 군으로부터 선택되는 1종 이상의 조혈모세포 표지 마커를 발현하지 않는 것일 수 있다. 또한, 상기 치수 유래 줄기세포는 혈관내피세포 마커인 CD31을 발현하지 않는 것일 수 있다.In the present specification, the term "stem cell" refers to a concept in a broad sense that collectively refers to undifferentiated cells having the ability to differentiate into various types of body tissue cells, that is, stemness. Stem cells refer to stem cells derived from pulp, periodontal ligaments, roots, dental pouches, root papillae, and gums. Among these, “dimension” refers to a soft tissue-like tissue located in the center of the tooth, and the dimension is surrounded by dentin and includes nerve fibers, blood vessels, and lymphatic vessels. Pulp-derived stem cells refer to stem cells that exist in pulp, and pulp is a tissue from which the neural plate differentiates into a neural crest and originates from it. It is highly probable that ectodermal stem cells that can form cells exist. In addition, pulp-derived stem cells are a cell source for the development of cell therapy products for regenerative medicine because pulp is easily accessible and has a high ratio between stem cells and collected tissue volumes. Very useful. The pulp-derived stem cells may be derived from pulp of a human tooth. The teeth are not particularly limited, but teeth or wisdom teeth are preferred. Tooth extraction is a process that must be performed, and wisdom teeth extraction is also very common, and since both teeth are mostly discarded after extraction, it has an advantage in that a separate procedure is not required for collecting tooth stem cells. The pulp-derived stem cells may have the characteristics of mesenchymal stem cells. For example, the pulp stem cells may be those in which the markers are expressed when measuring the expression of markers known to exhibit mesenchymal stem cell characteristics through an analysis method such as FACS, real-time PCR, and immunofluorescence staining. As an example, the pulp-derived stem cells may express one or more genes selected from the group consisting of CD29, CD44, CD73, CD90, and CD105, preferably CD29, CD44, CD73, CD90, and CD105 genes It may be to express all of. Further, the pulp-derived stem cells may not express one or more types of hematopoietic stem cell markers selected from the group consisting of CD14, CD34, CD45, CD117, and HLA-DR. Further, the pulp-derived stem cells may not express CD31, which is a vascular endothelial cell marker.
본 명세서에 있어서, “말초 동맥 질환(peripheral artery disease; PAD)”은 중심동맥을 제외한 혈관에서 염증, 혈전증, 고지혈증, 당뇨병 등으로 인해 유도되는 모든 혈관 질환을 총징하며, 바람직하게는 중증하지허혈(critical limb ischemia), 폐색혈전혈관염(버거씨병, thromboangiitis obliterans), 폐쇄성 죽상동맥경화증(arteriosclerosis obliterans), 결절성 다발동맥염(polyarteritis nodosa) 등일 수 있다.In the present specification, “peripheral artery disease (PAD)” refers to all vascular diseases induced by inflammation, thrombosis, hyperlipidemia, diabetes, etc. in blood vessels excluding the central artery, preferably severe limb ischemia ( critical limb ischemia), pulmonary thromboangiitis (thromboangiitis obliterans), obliterans atherosclerosis (arteriosclerosis obliterans), polyarteritis nodosa, etc.
본 명세서에 있어서, “예방(prevention)”이란 본 발명에 따른 조성물의 투여에 의해 말초 동맥 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.In the present specification, “prevention” refers to any action that suppresses or delays the onset of peripheral arterial disease by administration of the composition according to the present invention.
본 명세서에 있어서, "치료(treatment)"란 본 발명에 따른 조성물의 투여에 의해 말초 동맥 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used herein, "treatment" refers to any action in which symptoms of peripheral arterial disease are improved or advantageously changed by administration of the composition according to the present invention.
본 명세서에 있어서, “개체(individual)”란 본 발명의 조성물이 투여될 수 있는 대상을 말하며, 일례로 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말, 및 소 등의 포유동물을 의미할 수 있으나, 그 대상에는 제한이 없다.In the present specification, “individual” refers to a subject to which the composition of the present invention can be administered, and for example, human or non-human primates, mice, dogs, cats, horses, and cattle It can mean a mammal, but the subject is not limited.
본 명세서에 있어서, “약학적 조성물(pharmaceutical composition)”이란 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 본 발명의 약학적 조성물은 약제적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용가능한 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용가능한 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 당의정, 겔, 환제, 산제, 과립제, 좌제, 외용제, 용액, 현탁액, 서방형 제제, 슬러리 등으로 제형화하여 사용할 수도 있다. 한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.In the present specification, the term "pharmaceutical composition" may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition is intended for humans. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc.In the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. have. In addition, dragees, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained-release preparations, and slurries may be formulated and used. On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like may be used. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally be included.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 경구 또는 비경구 투여가 바람직하며, 예를 들어, 구강, 정맥내, 근육내, 관절내, 활액낭내, 동맥내, 골수내, 경막내, 심장내, 경피, 피내, 피하, 복강내, 비강내, 장관, 국소, 설하, 직장, 흉골내, 병소내, 두개골내 등이 포함된다. The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral or parenteral administration is preferable, for example, oral, intravenous, intramuscular, intra-articular, intrasynovial, intraarterial, intramedullary, It includes intrathecal, intracardiac, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, rectal, intrasternal, intralesional, intracranial, and the like.
본 발명의 약학적 조성물의 투여량은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 500 mg/kg 또는 0.001 내지 500 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition of the present invention can be varied including the activity of the specific compound used, age, body weight, general health, sex, formula, administration time, route of administration, excretion rate, drug combination and the severity of the specific disease to be prevented or treated. It may vary according to factors, and varies depending on the patient's condition, weight, degree of disease, drug type, route and duration of administration, but may be appropriately selected by those skilled in the art, and from 0.0001 to 500 mg/kg per day or from 0.001 to It can be administered at 500 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
본 명세서에 있어서, "세포 치료제(cellular therapeutic agent)"란, 인간으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 이러한 세포가 질병의 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.In the present specification, the term "cellular therapeutic agent" refers to a drug (US FDA regulation) used for treatment, diagnosis, and prevention with cells and tissues manufactured through isolation, culture and special manipulation from humans, In order to restore the function of cells or tissues, these cells are used to treat, diagnose and prevent diseases through a series of actions such as proliferating and screening living autologous, allogeneic, or xenogeneic cells in vitro, or changing the biological characteristics of cells in other ways. It means a drug used for the purpose of.
본 발명의 세포 치료제 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 비경구 투여, 예를 들어, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여될 수 있으나, 이에 제한되지는 않는다.The administration route of the cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto.
상기 조성물은 세포 치료에 일반적으로 사용되는 약제학적 담체와 함께 적합한 형태로 제형화될 수 있다. “약학적으로 허용되는”이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).The composition may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy. "Pharmacologically acceptable" refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
또한, 상기 조성물은 세포 치료제가 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수도 있다.In addition, the composition may be administered by any device capable of moving a cell therapeutic agent to a target cell.
본 발명의 세포 치료제 조성물은 질환의 치료를 위하여 치료학적으로 유효한 양의 세포 치료제를 포함할 수 있다. “치료학적으로 유효한 양(therapeutically effective amount)”은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다.The cell therapy composition of the present invention may contain a therapeutically effective amount of cell therapy for the treatment of a disease. "Therapeutically effective amount" means the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as considered by a researcher, veterinarian, physician or other clinician. , Which includes an amount that induces relief of symptoms of the disease or disorder being treated.
본 발명의 조성물에 포함되는 세포 치료제는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로 최적의 세포 치료제 함량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 포함하는 것이 중요하다. 예컨대, 본 발명의 줄기세포의 1일 투여량은 1.0×10 4 내지 1.0×10 11 세포/kg 체중, 바람직하게는 1.0×10 5 내지 1.0×10 9 세포/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.It is apparent to those skilled in the art that the cell therapy products included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy content can be easily determined by those skilled in the art, and the type of disease, severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient. , Administration time, administration route and secretion rate of the composition, treatment period, it may be adjusted according to various factors including drugs used simultaneously. It is important to include an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors. For example, the daily dose of the stem cells of the present invention is 1.0×10 4 to 1.0×10 11 cells/kg body weight, preferably 1.0×10 5 to 1.0×10 9 cells/kg body weight, divided once or several times. Can be administered. However, it should be understood that the actual dosage of the active ingredient should be determined in the light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age, and sex of the patient. It is not intended to limit the scope of the present invention to any aspect.
또한, 본 발명의 치료 방법에서 본 발명의 세포 치료제를 유효성분으로 포함하는 조성물은 직장, 정맥내(intravenous therapy, i.v), 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.In addition, in the treatment method of the present invention, the composition comprising the cell therapeutic agent of the present invention as an active ingredient is rectal, intravenous (intravenous therapy, iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular Alternatively, it can be administered in a conventional manner via an intradermal route.
본 발명은 포유동물에게 치료학적으로 유효한 양의 본 발명의 상기 세포 치료제 조성물을 투여하는 것을 포함하는 치료방법을 제공한다. 여기에서 사용된 용어 포유동물은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다. The present invention provides a therapeutic method comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the present invention. The term mammal as used herein refers to a mammal that is the object of treatment, observation or experiment, and preferably refers to a human.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1: 세포의 준비Example 1: Preparation of cells
1.1. 치아 유래 줄기세포의 분리 및 배양1.1. Isolation and culture of tooth-derived stem cells
치과 병원에서 치료를 위해 발치된 치아의 충치 또는 염증과 같은 결손 부위를 제거한 후에, 3 %의 antibiotic-antimycotic 용액이 첨가된 HBSS(Hanks' Balanced Salt solution) 용액에 보관하였다. 그리고 보관된 치아를 수술용 가위 및 blade를 이용하여 기계적으로 분쇄한 후에 Collagenase(1 ㎎/㎖, Gibco)와 Dispase(2.4 ㎎/㎖, Gibco)를 첨가하고, 37 ℃에서 1 시간 동안 반응시켰다. 그리고 상기 효소들과 반응시킨 치아를 피펫을 이용하여 단일세포 수준으로 해리시킨 후에, 70 μm 나일론 메시(nylon mesh) 망을 통과시켜 단일세포를 획득하였다. 그리고 획득된 세포들은 10 % FBS가 첨가된 MEM에 접종하여 계대배양하였으며, 6 회 이상 계대배양을 실시한 후에 실험에 사용하였다. 그리고 획득된 치아 유래 줄기세포(dental pulp stem cell; DPSC)는 광학 현미경 하에서 확인하였으며, 그 결과는 도 1a에 나타내었다.After removing defects such as tooth decay or inflammation of teeth extracted for treatment in a dental clinic, they were stored in HBSS (Hanks' Balanced Salt solution) solution added with 3% antibiotic-antimycotic solution. The stored teeth were mechanically crushed using surgical scissors and blades, and then Collagenase (1 mg/ml, Gibco) and Dispase (2.4 mg/ml, Gibco) were added and reacted at 37° C. for 1 hour. And after dissociating the teeth reacted with the enzymes to the level of single cells using a pipette, single cells were obtained by passing through a 70 μm nylon mesh network. And the obtained cells were inoculated in MEM added with 10% FBS and subcultured, and were used in experiments after subculture at least 6 times. And the obtained dental pulp stem cell (DPSC) was confirmed under an optical microscope, the results are shown in Figure 1a.
도 1a에 나타난 바와 같이, 획득된 치아 유래 줄기세포는 중간엽줄기세포의 전형적인 형태학적 특징을 지님을 확인하였다.1A, it was confirmed that the obtained tooth-derived stem cells had typical morphological characteristics of mesenchymal stem cells.
1.2. 세포의 특성 확인1.2. Checking the characteristics of cells
상기 실시예 1.1과 동일한 방법으로 획득된 치아 유래 줄기세포의 특성을 확인하기 위하여, 세포표면항원을 이용하여 유세포 분석(FACS analysis)을 실시하였다. 배양된 치아 유래 줄기세포를 0.05 % trypsin-0.5 mM EDTA를 이용하여 세포를 회수한 후에, 항원 당 2 X 10 5 cell를 이용하여 유세포 분석을 실시하였다. 보다 자세하게는, 인산염완충용액(phosphate buffered saline, PBS)에 담겨있는 세포를 4 ℃에서 800 rpm으로 5 분간 원심분리한 후에, 항체의 비특이적 결합을 차단하기 위하여 1 % bovine serum albumin(BSA)이 포함된 PBS(PH 7.5)로 30분간 전처리하였다. 그리고 전처리된 세포에 Fluorescein isothiocyanate(FITC) 또는 Phycoerythrin(PE)가 결합된 단일클론 항체(CD29, CD44, CD73, CD90, CD105, HLA-1, CD14, CD31, CD34, CD45, CD117, HLA-DR)를 1 μg/10 6 cells의 농도로 처리하고, 4 ℃에서 30 분간 반응시켰다. 반응이 완료된 세포는 PBS를 이용하여 3회 세척한 후에 유세포 분석을 위하여 5 X 10 4 cells로 희석하였다. 그리고 희석된 세포를 이용하여 유세포 분석기를 이용하여 분석을 실시하였고, FLOJO 소프트웨어를 이용하여 발현 정도를 분석하였다. 그 결과는 도 1b에 나타내었다.In order to confirm the characteristics of the tooth-derived stem cells obtained by the same method as in Example 1.1, FACS analysis was performed using a cell surface antigen. After the cells were recovered from the cultured tooth-derived stem cells using 0.05% trypsin-0.5 mM EDTA, flow cytometry was performed using 2 X 10 5 cells per antigen. In more detail, after centrifuging the cells in phosphate buffered saline (PBS) for 5 minutes at 800 rpm at 4°C, 1% bovine serum albumin (BSA) is included to block non-specific binding of the antibody. It was pretreated for 30 minutes with PBS (PH 7.5). And monoclonal antibodies (CD29, CD44, CD73, CD90, CD105, HLA-1, CD14, CD31, CD34, CD45, CD117, HLA-DR) conjugated to Fluorescein isothiocyanate (FITC) or Phycoerythrin (PE) to pretreated cells. Was treated at a concentration of 1 μg/10 6 cells, and reacted at 4° C. for 30 minutes. After the reaction was completed, the cells were washed 3 times with PBS and then diluted with 5 X 10 4 cells for flow cytometry. Then, the diluted cells were analyzed using a flow cytometer, and the expression level was analyzed using FLOJO software. The results are shown in Fig. 1b.
도 1b에 나타난 바와 같이, 본 발명의 치아 유래 줄기세포는 중간엽줄기세포 표지마커인 CD29, CD44, CD73, CD90, 및 CD105가 발현되나, 조혈모세포 표지마커인 CD14, CD34, CD45, CD117, HLA-DR과 혈관내피세포 표지마커인 CD31은 발현되지 않는 것을 확인하였다.As shown in Fig. 1b, the tooth-derived stem cells of the present invention express mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105, but hematopoietic stem cell markers CD14, CD34, CD45, CD117, HLA It was confirmed that -DR and CD31, a vascular endothelial cell marker, were not expressed.
그리고 Promocell에서 인간 탯줄정맥내피세포(Human umbilical vein endothelial cells; HUVECs)를 구매하여 혈관내피세포로 사용하였으며, 혈관내피세포는 1% penicillin/streptomycin이 첨가된 endothelial cell growth medium(Promocell)을 이용하여 배양하였다. 혈관내피세포의 특성을 확인하기 위하여, 치아 유래 줄기세포와 동일한 방법으로 유세포 분석을 실시하였으며, 그 결과는 도 1c에 나타내었다.And human umbilical vein endothelial cells (HUVECs) were purchased from Promocell and used as vascular endothelial cells, and vascular endothelial cells were cultured using endothelial cell growth medium (Promocell) added with 1% penicillin/streptomycin. I did. In order to confirm the characteristics of vascular endothelial cells, flow cytometric analysis was performed in the same manner as for tooth-derived stem cells, and the results are shown in FIG. 1C.
도 1c에 나타난 바와 같이, 중간엽줄기세포의 표지마커 중에서는 CD29와 CD105의 발현이 높았으며, 본 발명의 치아 유래 줄기세포와 마찬가지로 조혈모세포 표지인자는 발현되지 않음을 확인하였다.As shown in FIG. 1C, it was confirmed that among the markers of mesenchymal stem cells, CD29 and CD105 were highly expressed, and the hematopoietic stem cell markers were not expressed as in the tooth-derived stem cells of the present invention.
1.3. 치아 유래 줄기세포의 분화 확인1.3. Confirmation of differentiation of tooth-derived stem cells
상기 실시예 1.1과 동일한 방법으로 획득된 치아 유래 줄기세포의 분화능을 확인하였다. 보다 자세하게는, 배양 중인 치아 유래 줄기세포가 80 내지 90 %의 confluence에 도달하였을 때, 골 분화 유도용 배지 또는 지방세포 분화 유도용 배지로 교체한 후에 3 일마다 새로운 분화 유도용 배지로 교체해주며, 3 주 동안 배양하였다. 그리고 3 주 후에 세포를 인산염완충용액을 이용하여 2 회 세척하고, 골 분화는 Alzarin 용액으로 염색하여 확인하고, 지방세포 분화는 Oil red O 용액으로 염색하여 확인하였다. 염색을 실시한 후에는 인산염완충용액을 이용하여 1 회 세척하고, 다시 3차 증류수를 이용하여 1 회 세척하여 염색시약을 모두 제거해준 후에, 염색된 세포를 건조시키고 광학 현미경을 이용하여 관찰하였다. 그 결과는 도 1d에 나타내었다.The differentiation ability of the tooth-derived stem cells obtained in the same manner as in Example 1.1 was confirmed. In more detail, when the tooth-derived stem cells in culture reach 80 to 90% confluence, they are replaced with a medium for inducing bone differentiation or a medium for inducing adipocyte differentiation, and then replaced with a new medium for inducing differentiation every 3 days, Incubated for 3 weeks. After 3 weeks, the cells were washed twice with a phosphate buffer solution, and bone differentiation was confirmed by staining with Alzarin solution, and adipocyte differentiation was confirmed by staining with Oil red O solution. After staining, washing was performed once with a phosphate buffer solution, and washed once again with third distilled water to remove all dyeing reagents, and then the stained cells were dried and observed using an optical microscope. The results are shown in FIG. 1D.
도 1d에 나타난 바와 같이, 치아 유래 줄기세포가 골 형성 세포 및 지방 생성 세포로 정상적으로 분화되었음을 확인하였다.As shown in Figure 1d, it was confirmed that the tooth-derived stem cells were normally differentiated into osteogenic cells and adipogenic cells.
상기 결과들을 통하여, 실시예 1.1의 방법으로 획득한 치아 유래 줄기세포는 중간엽줄기세포의 표지인자를 발현하고 있으며, 지방세포 및 골세포로 분화가 가능한 줄기세포임을 확인하였다.Through the above results, it was confirmed that the tooth-derived stem cells obtained by the method of Example 1.1 express a marker of mesenchymal stem cells and are capable of differentiating into adipocytes and bone cells.
실시예 2: 말초 동맥 질환의 치료 효과 확인Example 2: Confirmation of the therapeutic effect of peripheral arterial disease
2.1. 말초 동맥 질환 동물 모델의 구축2.1. Construction of an animal model of peripheral arterial disease
말초 동맥 질환 동물 모델을 구축하기 위하여, 6 내지 7 주령의 누드 마우스에 졸레틸 마취제를 복강 내로 투여하여 마취하였다. 그리고 마취된 마우스의 뒷다리의 피부를 수술용 칼을 이용하여 절제하고, 근육을 제거하여 대퇴부 동맥을 노출시키고 대퇴부 동맥의 중간부와 말단을 강하게 묶어준 후에 그 사이의 혈관을 제거하여, 중증하지허혈 동물 모델을 제조하였다.In order to construct an animal model of peripheral arterial disease, 6 to 7 week old nude mice were anesthetized by intraperitoneally administering a zoletyl anesthetic. Then, the skin of the hind limb of the anesthetized mouse was excised using a surgical knife, the muscle was removed to expose the femoral artery, and the middle and end of the femoral artery were strongly tied, and the blood vessels between them were removed, and severe limb ischemia. Animal models were prepared.
2.2. 말초 동맥 질환 동물 모델에서의 염증, 근육 손상 및 섬유증 치료 효과2.2. Effects of Inflammation, Muscle Damage and Fibrosis Treatment in Animal Models of Peripheral Artery Disease
치아 유래 줄기세포와 혈관내피세포의 투여가 말초 동맥 질환의 증상인 염증, 근육 손상과 섬유증에 대하여 치료 효과를 나타내는지 확인하기 위하여, 실시예 2.1과 동일한 방법으로 구축한 말초 동맥 질환 동물 모델에 1) HBSS 투여군, 2) 치아 유래 줄기세포 단독, 3) 탯줄정맥내피세포 단독, 4) 치아 유래 줄기세포 및 탯줄정맥내피세포, 네 개의 그룹을 각각 투여하였다. 보다 자세하게는, 세포를 1 X 10 6 cells/80 μL HBSS의 농도로 준비하고 주사기에 담아, 말초 동맥 질환이 유도된 부분의 근육의 세 곳에 주사하여 세포 이식을 실시하였다. 치아 유래 줄기세포와 탯줄정맥내피세포는 1 : 1의 비율로 혼합하였다. 이식하고 14 일 후에 마우스를 CO 2를 이용하여 마취시키고, 세포를 이식한 부위의 조직을 획득한 후 파라핀 블록을 제작하였다. 그리고 제작된 파라핀 블록은 5 μm의 두께로 잘라준 후에 디파라핀 과정을 거치고, Masson's trichrome(MT) 및 H&E 염색을 실시하였다. 그리고 현미경을 이용하여 관찰하였다. 그 결과는 H&E 염색 결과는 도 2a에, MT 염색 결과는 도 2b에 나타내었다.In order to confirm whether the administration of tooth-derived stem cells and vascular endothelial cells exhibits a therapeutic effect on inflammation, muscle damage, and fibrosis, which are symptoms of peripheral arterial disease, 1 ) HBSS administration group, 2) tooth-derived stem cells alone, 3) umbilical vein endothelial cells alone, 4) tooth-derived stem cells and umbilical vein endothelial cells, four groups, respectively. In more detail, cells were prepared at a concentration of 1 X 10 6 cells/80 μL HBSS, put in a syringe, and injected into three muscles of the area in which peripheral arterial disease was induced to perform cell transplantation. Tooth-derived stem cells and umbilical vein endothelial cells were mixed in a ratio of 1:1. 14 days after transplantation, the mouse was anesthetized using CO 2 , and a paraffin block was prepared after obtaining the tissue at the site where the cells were transplanted. Then, the prepared paraffin block was cut to a thickness of 5 μm and then subjected to a diparaffin process, followed by Masson's trichrome (MT) and H&E staining. And observed using a microscope. The results are shown in FIG. 2A for H&E staining results and in FIG. 2B for MT staining results.
도 2a에 나타난 바와 같이, HBSS만 투여한 음성 대조군에서는 말초 동맥 질환의 유도로 인하여 근육 손상이 증가되어 정상적인 근육의 형태를 유지하지 못하고, 근육 퇴행성의 중증도가 매우 높은 것을 확인하였다. 또한, 백혈구(leukocyte)의 침윤(infiltration)이 유도되며, 이로 인하여 말초 동맥 질환이 유도된 부위의 염증 반응이 증가된 것을 확인하였다. 반면, 치아 유래 줄기세포를 투여한 실험군에서는 근육 손상이 감소되고, 백혈구의 침윤 또한 일부 감소된 것을 확인하였다. 그러나 치아 유래 줄기세포와 혈관내피세포의 병용 투여군에서는 거의 정상 조직과 동일한 형태를 나타낼 정도로 근육 손상 정도가 감소되어 근육 퇴행성의 중증도가 가장 낮으며, 백혈구의 침윤 또한 거의 관찰되지 않으며, 이로 인한 염증 반응 또한 관찰되지 않은 것을 확인하였다.As shown in FIG. 2A, it was confirmed that in the negative control group administered with only HBSS, muscle damage was increased due to the induction of peripheral arterial disease, so that the normal muscle shape was not maintained, and the severity of muscle degeneration was very high. In addition, it was confirmed that infiltration of leukocytes was induced, thereby increasing the inflammatory response at the site where peripheral arterial disease was induced. On the other hand, in the experimental group to which tooth-derived stem cells were administered, it was confirmed that muscle damage and infiltration of leukocytes were also partially reduced. However, in the group administered with tooth-derived stem cells and vascular endothelial cells, the degree of muscle damage was reduced to the extent that they showed almost the same morphology as normal tissues, so the severity of muscle degeneration was the lowest, and infiltration of leukocytes was hardly observed, resulting in inflammatory reactions. It was also confirmed that it was not observed.
도 2b에 나타난 바와 같이, HBSS만 투여한 음성 대조군에서는 말초 동맥 질환의 유도로 인하여 섬유화(청색으로 도시)가 조직에 전반적으로 유발되었으나, 치아 유래 줄기세포를 투여한 실험군에서는 섬유증이 일부 감소되었고, 치아 유래 줄기세포와 혈관내피세포의 병용 투여군에서는 섬유화가 거의 유발되지 않은 것을 확인하였다.As shown in Figure 2b, in the negative control group administered with only HBSS, fibrosis (shown in blue) was induced overall in the tissues due to induction of peripheral arterial disease, but in the experimental group administered with tooth-derived stem cells, fibrosis was partially reduced, It was confirmed that fibrosis was hardly induced in the group administered with tooth-derived stem cells and vascular endothelial cells.
상기 결과들을 통하여, 치아 유래 줄기세포와 혈관내피세포를 병용 투여하는 경우 말초 동맥 질환으로 인해 유도되는 근육 손상과 섬유증이 현저히 개선되는 것을 확인할 수 있었다.From the above results, it was confirmed that when the tooth-derived stem cells and vascular endothelial cells were administered in combination, muscle damage and fibrosis induced by peripheral arterial disease were significantly improved.
2.3. 말초 동맥 질환 동물 모델에서의 혈관 형성능 확인2.3. Confirmation of blood vessel formation ability in animal model of peripheral arterial disease
치아 유래 줄기세포 및 혈관내피세포의 투여가 말초 동맥 질환 부위에서 혈관 형성을 촉진시키는지 확인하기 위하여, 실시예 2.1과 동일한 방법으로 구축한 말초 동맥 질환 동물 모델에 1) 마트리겔 단독, 2) 치아 유래 줄기세포 단독, 3) 탯줄정맥내피세포 단독, 4) 치아 유래 줄기세포 및 탯줄정맥내피세포, 네 개의 그룹을 각각 투여하였다. 보다 자세하게는, 세포를 2 X 10 6 cells/200 μL 마트리겔의 농도로 준비하고 25G 주사기에 담아, 말초 동맥 질환이 유도된 부분의 피하에 주사하여 세포 이식을 실시하였다. 치아 유래 줄기세포와 탯줄정맥내피세포는 1 : 1의 비율로 혼합하였다. 이식하고 4 일 후에 마우스를 CO 2를 이용하여 마취시키고, 이식된 마트리겔을 분리한 후 파라핀 블록을 제작하였다. 그리고 제작된 파라핀 블록은 4 μm의 두께로 잘라준 후에 디파라핀 과정을 거치고, H&E 염색을 실시하였다. 그리고 현미경을 이용하여 관찰하였다. 그 결과는 도 3에 나타내었다.In order to confirm whether the administration of tooth-derived stem cells and vascular endothelial cells promotes blood vessel formation in the peripheral arterial disease site, 1) Matrigel alone, 2) tooth Derived stem cells alone, 3) umbilical vein endothelial cells alone, and 4) tooth-derived stem cells and umbilical vein endothelial cells, respectively, were administered in four groups. In more detail, cells were prepared at a concentration of 2×10 6 cells/200 μL Matrigel, placed in a 25G syringe, and injected subcutaneously in the area in which peripheral arterial disease was induced to perform cell transplantation. Tooth-derived stem cells and umbilical vein endothelial cells were mixed in a ratio of 1:1. Four days after implantation, the mouse was anesthetized using CO 2 , and the implanted Matrigel was separated, and then a paraffin block was prepared. And the prepared paraffin block was cut to a thickness of 4 μm, subjected to a diparaffin process, and H&E staining was performed. And observed using a microscope. The results are shown in FIG. 3.
도 3에 나타난 바와 같이, 마트리겔 단독, 혈관내피세포 단독, 및 치아 유래 줄기세포 단독 투여 그룹에서는 모세혈관의 형성 정도가 낮으나, 치아 유래 줄기세포 및 탯줄정맥내피세포의 병용 투여군에서는 모세혈관의 형성이 현저히 증가되어 적혈구 세포의 침투로 인한 적색이 현저히 증가된 것을 확인하였다. As shown in FIG. 3, the degree of capillary formation was low in the group administered with Matrigel alone, vascular endothelial cells alone, and tooth-derived stem cells alone, but the formation of capillaries in the combined administration of tooth-derived stem cells and umbilical vein endothelial cells. It was confirmed that the red blood cells increased significantly due to the infiltration of red blood cells.
2.4. 말초 동맥 질환 동물 모델에서의 치료 효과 확인2.4. Confirmation of therapeutic effect in animal model of peripheral arterial disease
치아 유래 줄기세포 및 혈관내피세포의 투여가 염증 반응 억제, 근육 손상 감소, 섬유화 억제, 및 혈관 형성능 증가를 통하여 직접적으로 말초 동맥 질환을 치료할 수 있는지 확인하기 위하여 최종적으로 혈류량과 손상 정도를 확인하였다. 보다 자세하게는, 실시예 2.2와 동일한 방법으로 세포 이식을 실시한 후 레이져 도플러 이미지 장비(Laser Doppler perfusion imager)를 이용하여 혈류량을 측정하였고, 손상 정도를 카메라로 확인하였다. 전반적인 실험 방법은 도 4a에 나타내었고, 혈류량 측정 결과는 도 4b에, 허혈 손상 정도는 도 4c에 나타내었다.In order to confirm whether the administration of tooth-derived stem cells and vascular endothelial cells can directly treat peripheral arterial disease through inhibition of inflammatory response, reduction of muscle damage, inhibition of fibrosis, and increase in blood vessel formation ability, blood flow and degree of damage were finally checked. In more detail, after cell transplantation was performed in the same manner as in Example 2.2, blood flow was measured using a laser Doppler perfusion imager, and the degree of damage was confirmed with a camera. The overall experimental method is shown in Fig. 4a, the blood flow measurement result is shown in Fig. 4b, and the degree of ischemia injury is shown in Fig. 4c.
도 4b에 나타난 바와 같이, HBSS만 투여한 음성 대조군에서는 말초 동맥 질환의 유도로 인하여 혈관 손상이 유도되어 혈류량이 감소되었으나, 치아 유래 줄기세포와 혈관내피세포의 병용 투여군에서는 세포 이식 후 7일째부터 혈류량이 현저히 회복되는 것을 확인하였다.As shown in Figure 4b, in the negative control group administered with only HBSS, blood vessel damage was induced due to the induction of peripheral arterial disease, and blood flow was reduced, but in the group administered with tooth-derived stem cells and vascular endothelial cells, blood flow from the 7th day after cell transplantation. It was confirmed that this remarkably recovered.
또한, 도 4c에 나타난 바와 같이, HBSS만 투여한 음성 대조군과 치아 유래 줄기세포 단독 투여군, 혈관내피세포 단독 투여군에서는 7 일째 이미 괴사가 진행되어 14 일째에는 다리 형태가 거의 유지되지 않는 반면, 치아 유래 줄기세포와 혈관내피세포의 병용 투여군에서는 7 일째까지는 다리 형태가 거의 유지되는 것을 확인하였다.In addition, as shown in Figure 4c, in the negative control group administered with only HBSS, the group administered with only tooth-derived stem cells, and the group administered with only vascular endothelial cells, necrosis has already progressed on the 7th day and the leg shape is hardly maintained on the 14th day, whereas the tooth-derived In the group administered with stem cells and vascular endothelial cells, it was confirmed that the leg shape was almost maintained until the 7th day.
상기 결과들을 통하여, 치아 유래 줄기세포 및 혈관내피세포를 병용 투여하는 경우에는 말초 동맥 질환으로 인한 염증 반응이 억제되고, 근육 손상을 방지하고, 섬유화가 유도되는 것을 억제하는 동시에 혈관 형성능을 증가시켜 신규 혈관 형성을 촉진시킴으로써, 죽상경화성, 혈전색전성, 염증성 기작 등 다양한 원인에 의해 유발되는 다양한 말초 동맥 질환의 치료에 효과적으로 사용될 수 있다는 것을 확인할 수 있었다. 또한, 단순히 혈관을 확장하는 것으로 일차적인 증상을 완화시키는 기존의 치료 방법과는 상이하게 말초 동맥 질환의 다양한 병증을 동시에 치료할 수 있기 때문에 치료 효율을 현저히 증가시킬 수 있다는 것을 확인할 수 있었다.Through the above results, when the tooth-derived stem cells and vascular endothelial cells are administered in combination, the inflammatory response due to peripheral arterial disease is suppressed, muscle damage is prevented, fibrosis is inhibited, and the blood vessel formation ability is increased. By promoting blood vessel formation, it was confirmed that it can be effectively used in the treatment of various peripheral arterial diseases caused by various causes such as atherosclerosis, thromboembolism, and inflammatory mechanisms. In addition, it was confirmed that the treatment efficiency can be significantly increased because various conditions of peripheral arterial disease can be simultaneously treated differently from the existing treatment methods that alleviate primary symptoms simply by expanding blood vessels.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.
본 발명의 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물은 말초 동맥 질환 모델에서 염증 반응 및 근육 손상을 방지하고, 섬유화가 유발되는 것을 억제하는 동시에 혈관 형성능을 증가시킬 수 있기 때문에 죽상경화성, 혈전색전성, 염증성 기작 등 다양한 원인에 의해 유발되는 다양한 말초 동맥 질환의 치료에 효과적으로 사용될 수 있으며, 단순히 혈관을 확장하는 것으로 일차적인 증상을 완화시키는 기존의 치료 방법과는 상이하게 말초 동맥 질환의 다양한 병증을 동시에 치료할 수 있기 때문에 치료 성공률을 현저히 증가시킬 수 있을 것으로 기대된다.The composition comprising tooth-derived stem cells and vascular endothelial cells of the present invention as active ingredients prevents inflammatory reactions and muscle damage in a peripheral arterial disease model, inhibits fibrosis, and increases blood vessel formation ability. It can be effectively used for the treatment of various peripheral arterial diseases caused by various causes such as sclerosis, thromboembolism, inflammatory mechanisms, etc., and is different from existing treatment methods that alleviate primary symptoms by simply expanding blood vessels. It is expected that the success rate of treatment can be remarkably increased because it can treat various conditions of the disease at the same time.

Claims (12)

  1. 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는, 말초 동맥 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating peripheral arterial disease, comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 치아 유래 줄기세포는 치수, 치주 인대, 치근, 치낭 및 치근유두로 이루어진 군으로부터 선택된 어느 하나 이상으로부터 유래된 줄기세포인 것을 특징으로 하는, 약학적 조성물.The tooth-derived stem cells are stem cells derived from any one or more selected from the group consisting of pulp, periodontal ligaments, tooth roots, dental pouches and tooth root papillae.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 혈관내피세포는 Bend3 세포, 인간 제대정맥내피세포(Human umbilical vein endothelial cells; HUVEC), 인간 진피 미세 혈관내피세포(Human dermal microvascular endothelial cells; HMVEC-D 또는 HDMEC), 인간 심장 내피세포(Human cardiac endothelial cells), 인간 대동맥 내피세포(Human aortic endothelial cells; HAEC), 인간 관상 및 장골 동맥 내피세포(Human coronary and iliac artery endothelial cells; HIAEC 또는 HCAEC), 인간 심장 미세 혈관내피세포(Human cardiac microvascular endothelial cells), 인간 방광 및 자궁 미세 혈관내피세포(Human bladder and uterine microvascular endothelial cells), 인간 폐동맥 내피세포(Human pulmonary artery endothelial cells; HPAEC 또는 PASMC), 인간 폐 미세 혈관내피세포(Human lung microvascular endothelial cells; HMVEC-L), 인간 완두 동맥 내피세포(Human brachiocephalic artery endothelial cells; HBcAEC), 인간 목동맥 내피세포(Human carotid artery endothelial cells; HCtAEC), 인간 진피 림프 미세 혈관내피세포(Human dermal lymphatic microvascular endothelial cells; HDLMVEC), 인간 내흉 동맥 내피세포(Human internal thoracic artery endothelial cells; HITAEC), 인간 쇄골하 동맥 내피세포(Human subclavian artery endothelial cells; HScAEC) 및 인간 제대 동맥 내피세포(Human umbilical artery endothelial cells; HUAEC)로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.The vascular endothelial cells are Bend3 cells, human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMVEC-D or HDMEC), human cardiac endothelial cells (Human cardiac cells). endothelial cells), human aortic endothelial cells (HAEC), human coronary and iliac artery endothelial cells (HIAEC or HCAEC), human cardiac microvascular endothelial cells ), human bladder and uterine microvascular endothelial cells, human pulmonary artery endothelial cells (HPAEC or PASMC), human lung microvascular endothelial cells (HMVEC) -L), human brachiocephalic artery endothelial cells (HBcAEC), human carotid artery endothelial cells (HCtAEC), human dermal lymphatic microvascular endothelial cells (HDLMVEC) ), human internal thoracic artery endothelial cells (HITAEC), human subclavian artery endothelial cells (HScAEC) and human umbilical artery endothelial cells Human umbilical artery endothelial cells; HUAEC), characterized in that at least one selected from the group consisting of, a pharmaceutical composition.
  4. 제 1 항에 있어서,The method of claim 1,
    상기 치아 유래 줄기세포 및 혈관내피세포는 1 : 0.1 내지 1 : 10의 개수비로 포함되는 것을 특징으로 하는, 약학적 조성물.The tooth-derived stem cells and vascular endothelial cells are 1: 0.1 to 1: 10, characterized in that contained in the number ratio of, pharmaceutical composition.
  5. 제 1 항에 있어서,The method of claim 1,
    상기 말초 동맥 질환은 중증하지허혈(critical limb ischemia), 폐색혈전혈관염(버거씨병, thromboangiitis obliterans), 결절성 다발동맥염(polyarteritis nodosa) 및 폐쇄성 죽상동맥경화증(arteriosclerosis obliterans)으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.The peripheral arterial disease is at least one selected from the group consisting of critical limb ischemia, obstructive thromboangiitis (thromboangiitis obliterans), polyarteritis nodosa, and obliterans atherosclerosis (arteriosclerosis obliterans). Characterized in, pharmaceutical composition.
  6. 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는, 말초 동맥 질환의 예방 또는 치료용 세포 치료제.Cell therapy for preventing or treating peripheral arterial disease, comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  7. 제 6 항에 있어서,The method of claim 6,
    상기 치아 유래 줄기세포는 치수, 치주 인대, 치근, 치낭 및 치근유두로 이루어진 군으로부터 선택된 어느 하나 이상으로부터 유래된 줄기세포인 것을 특징으로 하는, 세포 치료제.The tooth-derived stem cells are stem cells derived from any one or more selected from the group consisting of pulp, periodontal ligaments, tooth roots, dental cysts and tooth root papillae.
  8. 제 6 항에 있어서,The method of claim 6,
    상기 혈관내피세포는 Bend3 세포, 인간 제대정맥내피세포(Human umbilical vein endothelial cells; HUVEC), 인간 진피 미세 혈관내피세포(Human dermal microvascular endothelial cells; HMVEC-D 또는 HDMEC), 인간 심장 내피세포(Human cardiac endothelial cells), 인간 대동맥 내피세포(Human aortic endothelial cells; HAEC), 인간 관상 및 장골 동맥 내피세포(Human coronary and iliac artery endothelial cells; HIAEC 또는 HCAEC), 인간 심장 미세 혈관내피세포(Human cardiac microvascular endothelial cells), 인간 방광 및 자궁 미세 혈관내피세포(Human bladder and uterine microvascular endothelial cells), 인간 폐동맥 내피세포(Human pulmonary artery endothelial cells; HPAEC 또는 PASMC), 인간 폐 미세 혈관내피세포(Human lung microvascular endothelial cells; HMVEC-L), 인간 완두 동맥 내피세포(Human brachiocephalic artery endothelial cells; HBcAEC), 인간 목동맥 내피세포(Human carotid artery endothelial cells; HCtAEC), 인간 진피 림프 미세 혈관내피세포(Human dermal lymphatic microvascular endothelial cells; HDLMVEC), 인간 내흉 동맥 내피세포(Human internal thoracic artery endothelial cells; HITAEC), 인간 쇄골하 동맥 내피세포(Human subclavian artery endothelial cells; HScAEC) 및 인간 제대 동맥 내피세포(Human umbilical artery endothelial cells; HUAEC)로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 세포 치료제.The vascular endothelial cells are Bend3 cells, human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMVEC-D or HDMEC), human cardiac endothelial cells (Human cardiac cells). endothelial cells), human aortic endothelial cells (HAEC), human coronary and iliac artery endothelial cells (HIAEC or HCAEC), human cardiac microvascular endothelial cells ), human bladder and uterine microvascular endothelial cells, human pulmonary artery endothelial cells (HPAEC or PASMC), human lung microvascular endothelial cells (HMVEC) -L), human brachiocephalic artery endothelial cells (HBcAEC), human carotid artery endothelial cells (HCtAEC), human dermal lymphatic microvascular endothelial cells (HDLMVEC) ), human internal thoracic artery endothelial cells (HITAEC), human subclavian artery endothelial cells (HScAEC) and human umbilical artery endothelial cells Human umbilical artery endothelial cells; HUAEC), characterized in that at least one selected from the group consisting of, cell therapy.
  9. 제 6 항에 있어서,The method of claim 6,
    상기 치아 유래 줄기세포 및 혈관내피세포는 1 : 0.1 내지 1 : 10의 개수비로 포함되는 것을 특징으로 하는, 세포 치료제.The tooth-derived stem cells and vascular endothelial cells are 1: 0.1 to 1: 10, characterized in that included in the number ratio of, cell therapy.
  10. 제 6 항에 있어서,The method of claim 6,
    상기 말초 동맥 질환은 중증하지허혈(critical limb ischemia), 폐색혈전혈관염(버거씨병, thromboangiitis obliterans), 결절성 다발동맥염(polyarteritis nodosa) 및 폐쇄성 죽상동맥경화증(arteriosclerosis obliterans)으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 세포 치료제.The peripheral arterial disease is at least one selected from the group consisting of critical limb ischemia, obstructive thromboangiitis (thromboangiitis obliterans), polyarteritis nodosa, and obliterans atherosclerosis (arteriosclerosis obliterans). Characterized in, cell therapy.
  11. 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물을 개체에 투여하는 단계를 포함하는, 말초 동맥 질환의 예방 또는 치료 방법.A method for preventing or treating peripheral arterial disease, comprising administering to an individual a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients.
  12. 치아 유래 줄기세포 및 혈관내피세포를 유효성분으로 포함하는 조성물의 말초 동맥 질환의 예방 또는 치료 용도. Use of a composition comprising tooth-derived stem cells and vascular endothelial cells as active ingredients to prevent or treat peripheral arterial disease.
PCT/KR2020/008135 2019-06-24 2020-06-24 Composition for treatment of peripheral arterial disease WO2020262916A1 (en)

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JP2014168714A (en) * 2008-03-12 2014-09-18 Japan Health Sciences Foundation Root canal filler
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JP2014168714A (en) * 2008-03-12 2014-09-18 Japan Health Sciences Foundation Root canal filler
KR20140054206A (en) * 2011-08-10 2014-05-08 디퍼이 신테스 프로덕츠, 엘엘씨 Treatment of peripheral vascular disease using umbilical cord tissue-derived cells
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