WO2015199416A1 - Pharmaceutical composition for treating skin or vascular tissue damage and use thereof - Google Patents

Pharmaceutical composition for treating skin or vascular tissue damage and use thereof Download PDF

Info

Publication number
WO2015199416A1
WO2015199416A1 PCT/KR2015/006371 KR2015006371W WO2015199416A1 WO 2015199416 A1 WO2015199416 A1 WO 2015199416A1 KR 2015006371 W KR2015006371 W KR 2015006371W WO 2015199416 A1 WO2015199416 A1 WO 2015199416A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
spheroid
skin
cells
light
Prior art date
Application number
PCT/KR2015/006371
Other languages
French (fr)
Korean (ko)
Inventor
박인수
안진철
정필상
Original Assignee
단국대학교 천안캠퍼스 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020140076416A external-priority patent/KR101816964B1/en
Priority claimed from KR1020140076412A external-priority patent/KR101719870B1/en
Application filed by 단국대학교 천안캠퍼스 산학협력단 filed Critical 단국대학교 천안캠퍼스 산학협력단
Publication of WO2015199416A1 publication Critical patent/WO2015199416A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present invention relates to a pharmaceutical composition for treating skin or vascular tissue damage and its use. More particularly, the present invention relates to a method for treating skin or vascular tissue damage by irradiating light with a red wavelength band using an LED light source.
  • the present invention relates to a therapeutic pharmaceutical composition comprising a spheroid for wound transplantation and to a method for treating skin or vascular tissue damage using the same, which is used to improve a therapeutic effect.
  • the present invention also relates to a method of inducing differentiation of mesenchymal stem cells using light of a red wavelength band.
  • differentiation refers to a process in which early-stage cells have characteristics as individual tissues, a representative example of which can be seen in animal development.
  • 'differentiation' must occur in order for a single cell called fertilized egg made by combining sperm and egg to be made into various tissue cells such as bone, heart, and skin.
  • Stem cells that have such differentiation capacity are the cells before the differentiation into each cell constituting the tissue, and are capable of infinite proliferation in the undifferentiated state and potential to be differentiated into cells of various tissues by specific differentiation stimulation. It means a cell with the possibility.
  • Adipose-derived stem cells as adipose tissue-derived multipotent stem cells are easier and safer to acquire because they can extract a large amount of adipose tissue compared to mesenchymal stem cells. It is attracting attention as a new source of multipotent stem cells because it has advantages in terms of tissue accessibility, stability, efficacy, and economics due to its unlimited limitations and easy in vitro culture.
  • Fat stem cells known to date include human adipose stem cells capable of differentiating into epithelial cells; Human adipose stem cells capable of bone formation and differentiation into adipocytes; Human adipose stem cells capable of differentiating into neurons; Rat adipocytes capable of differentiating into adipocytes; Rat adipocytes capable of differentiating into osteogenic and chondrogenic cells; Human adipose stem cells capable of differentiating into chondrocytes; Rat adipose stem cells capable of differentiating into neurons; And adipose stem cells capable of differentiating into osteocytes, chondrocytes, nerve cells or muscle cells (US Pat. No. 6,777,231).
  • adipose stem cells may be induced to differentiate into vascular endothelial cells in vivo and in vitro.
  • adipose stem cells may be differentiated into vascular endothelial cells when cultured using a medium containing various growth factors in a matrigel-coated culture vessel, a special extracellular matrix component isolated from sarcoma mice.
  • vascular endothelial cells do not interact with inflammatory cells, whereas when vascular endothelial cells are inflamed, the inflammatory cells interact with the inflammatory cells that adhere to the vascular wall, causing an inflammatory reaction.
  • cell therapy using stem cells has attracted attention.
  • adipose stem cells may be differentiated into vascular endothelial cells when cultured using a medium containing various growth factors in a matrigel-coated culture vessel, a special extracellular matrix component isolated from sarcoma mice.
  • US Patent Publication No. 2011-0137385 discloses a method for treating vascular diseases comprising irradiating light at a wavelength of 600 to 1,000 nm to an affected area where ischemic disease develops.
  • vascular endothelial cells damaged by the irradiated light is promoted, thereby regenerating and repairing the damaged blood vessels, and as a result, vascular damage can be treated.
  • the method has the advantage of extremely high safety because it only irradiates the light from the outside and is not administered in the patient's body, while it takes a lot of time for treatment because it promotes self-healing in vivo. .
  • the present inventors can more effectively and more effectively differentiate the adipose derived mesenchymal stem cells into vascular endothelial cells; As a result of intensive research to develop a method for treating skin ulcers and wounds, the stem cells can be effectively differentiated into vascular endothelial cells when the adipose derived mesenchymal stem cells are cultured in a spheroid form and irradiated with red wavelengths.
  • the fat stem cell-derived spheroid is implanted into the skin ulcer and wound, and irradiated with light of the red wavelength band on the blood vessel injury, it was confirmed that the skin ulcer and wound could be more effectively treated, and the present invention was completed. .
  • One object of the present invention to provide a pharmaceutical composition for treating skin or vascular tissue damage, including spheroid derived from adipose stem cells.
  • Another object of the present invention is to provide a method for differentiating adipose derived mesenchymal stem cells into vascular endothelial cells, comprising culturing the spheroid of adipose derived mesenchymal stem cells while irradiating light with a red wavelength band. .
  • the pharmaceutical composition of the present invention can induce the regeneration of damaged blood vessels or skin in vivo, it can be widely used for the treatment of blood vessel damage, skin ulcers or wounds.
  • the method of differentiation of the present invention can induce differentiation of stem cells into vascular endothelial cells without using differentiation-inducing substances, and thus can be widely used for the development of more economical vascular disease therapeutics.
  • Figure 1 is a micrograph showing the results of verifying the stem cells isolated from human adipose tissue.
  • FIG. 2 is a photograph showing an LED irradiation apparatus.
  • Figure 3 is a graph showing the growth rate change of adipose derived mesenchymal stem cells by LED irradiation.
  • Figure 4a is a photograph showing the result of culturing fat-derived mesenchymal stem cells according to the presence of ECM protein coated in the culture vessel.
  • Figure 4b is a photograph showing the change in morphology of fat-derived mesenchymal stem cells with the passage of incubation time while inoculating the fat-derived mesenchymal stem cells in NTCP, and irradiated with light from the LED light source.
  • FIG. 5 is a photograph showing a spheroid formed from adipose stem cells.
  • Figure 6 is a graph showing the expression changes of angiogenesis and skin regeneration factor of adipose derived stem cells by LED irradiation.
  • Figure 7 is a photograph and graph showing the results of measuring the change in the expression level of the angiogenesis-related protein according to whether the LED irradiation and spheroid formation.
  • FIG. 8 is a photograph showing the results of immunofluorescence staining for the adipose tissue-derived mesenchymal stem cells irradiated with LED.
  • the lower photo of Figure 8 is a photograph showing the results confirmed by Western blot analysis whether the expression of the CD31 according to the LED irradiation and the spheroid formation.
  • Figure 9 is a graph showing the results of FACS analysis for the two-dimensional cultured stem cells and spheroid-formed stem cells irradiated with LED.
  • Figure 10 is a photograph and graph showing the results of comparing the therapeutic effect of the spheroid-formed stem cells by examining the LED for the ischemic mouse animal model.
  • Figure 10a is a photograph showing the lower extremities of the mouse animal model taken at 1, 7, 14 and 21 days after the spheroid injection of the LED irradiation
  • Figure 10b is a graph showing the ratio of the mouse according to the degree of injury of the lower extremities after 21 days after the spheroid injection of the LED irradiation.
  • 10C is a photograph showing skin blood flow taken at 1, 7, 14, and 21 days after LED-injected spheroid injection.
  • 10D is a graph showing the percentage of mice treated with lower limb ischemia after 21 days have elapsed.
  • FIG. 11 is a photograph and graph showing the results of comparing the wound treatment effect of each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) using the skin wound mouse model,
  • a is a photograph showing the skin wound mouse model produced.
  • b is a photograph of the wound area of each experimental group over time.
  • c is a graph showing the change of the area of the wound area over time.
  • a is an immunofluorescence staining photograph showing the results of comparing the levels of vascular endothelial cells and vascular epithelial cells in the wounded skin of a mouse model according to the treatment conditions (control, ASCs, LLLT, spheroid and spheroid + LLLT).
  • Green indicates SMAa actin, a marker of vascular endothelial cells
  • red shows a photograph showing CD31, a vascular endothelial cell marker.
  • b is a graph showing the results of comparing the number of microvascular vessels per unit area of the wounded mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
  • c is a graph showing the result of comparing the number of small arteries per unit area in the wounded part of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
  • FIG. 13 is an immunofluorescence staining photograph showing the result of comparing the level of skin cells present in the wound area of the skin wound mouse model according to each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT), and red is skin.
  • Cytokeratin a cell marker.
  • FIG. 14 is a photograph and a graph confirming an effect of increasing angiogenesis and skin regeneration factor expression of a combination of LED irradiation and spheroid transplantation and LED treatment in a skin wound mouse model.
  • a is an immunofluorescence staining photograph showing the results of comparing the levels of angiogenesis and skin regeneration factors in the wounded model of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). Red color represents FGF, VEGF and HGF, respectively, angiogenesis and skin regeneration factors.
  • b is a photograph showing the result of Western blot analysis of the levels of angiogenesis and skin regeneration factors in the wounded area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) to be.
  • c is a graph showing the result of comparing the level of each angiogenesis and skin regeneration factor detected by the Western blot analysis for each treatment condition.
  • 15 is a photograph showing the results of comparing the wound treatment effect using the skin flap mouse model.
  • 16 is a photograph showing the results of comparing the flow of skin blood taken in the skin flap mouse model at the time of 1, 7 and 14 days for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). .
  • the present inventors came to pay attention to a method of irradiating light using an LED light source while conducting various studies to develop a method for more effectively treating skin ulcers and wounds. That is, when the light of the red wavelength band generated by the LED light source is irradiated to the skin ulcer and wound affected area, it is possible to treat the skin ulcer and the wound by enhancing the self-healing ability of the patient.
  • the light of the red wavelength band generated by the LED light source is irradiated to the skin ulcer and wound affected area, it is possible to treat the skin ulcer and the wound by enhancing the self-healing ability of the patient.
  • there is a disadvantage in that it takes a lot of time for treatment because it enhances the self-recovery ability in vivo.
  • studies on the use of stem cells revealed that spheroids formed from stem cells rather than stem cells were studied.
  • the spheroid formed from the adipose stem cells can be used as an active ingredient of the pharmaceutical composition that can assist in the treatment of skin ulcers and wounds using light in the red wavelength band for treating skin ulcers and wounds.
  • One aspect of the present invention to achieve the above object provides a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength band, including a spheroid derived from adipose stem cells as an active ingredient.
  • Adipose-derived stem cells of the present invention is excellent in the self-renewal capacity present in adipose tissue, liver, bone, cartilage, fat, blood vessels, heart, nervous system, etc. It refers to undifferentiated adult stem cells that can differentiate into various cells. Compared with the mesenchymal stem cells, the adipose stem cells are easily and securely obtained in that they can extract a large amount of adipose tissue. And there are advantages in terms of economy. Adipose stem cells in the present invention may form a spheroid (spheroid).
  • the term "spheroid" refers to a cell mass known as a 3D cell mass (3DCM), and refers to a spherical colony formed by gathering a plurality of daughters formed by division of an individual.
  • the spheroid may be formed by culturing adipose stem cells, specifically, may be formed by irradiating the red wavelength band of light to the adipose stem cells.
  • the spheroid for the formation of the spheroid in the present invention, a method such as stop culture, shaking culture, etc. can be used without limitation when culturing fat stem cells, it is possible to form a spheroid more effectively when cultured in a hypoxic environment.
  • the spheroid has a problem that its diameter continuously increases with the incubation time, if the diameter is larger than 1mm, cell necrosis occurs internally and there is a problem that cannot be utilized.
  • the diameter of the spheroid is at least 500 ⁇ m or more, the inside of the spheroid creates a hypoxic environment, and the HIF-1a protein expressed when the hypoxic environment is expressed.
  • angiogenesis and skin regeneration factors are promoted by HIF-1a signal, which is more effective when cultured in hypoxic environment.
  • external stimulation light irradiation
  • angiogenesis and skin regeneration factors FGF, VEGF, HGF, etc.
  • the diameter of a suitable spheroid can be 0.5 to 1 mm.
  • the use of spheroid is not limited due to the diameter range.
  • the spheroid in the present invention in order to form the spheroid in the present invention, it can be cultured by inoculating a culture vessel surface-treated with a polymer that gives hydrophobicity or a culture vessel made of the polymer.
  • the polymer for imparting the hydrophobicity is not particularly limited.
  • the polymer for imparting hydrophobicity is not particularly limited thereto, and specifically, polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chlorite (PVC), polyethylene (PE), Polypropylene (PP), polytetrafluoroethylene (PTFE), aliphatic polyester-based polymer, poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA ), Poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives thereof, copolymers thereof (poly (lactic acid-co-glycolic acid) (PLGA), poly (L-lactic acid-co-caprolactone) (PLCL), poly (glycolic acid-co-caprolactone) (PGCL), or the like, or a combination thereof.
  • PMMA polymethyl methacrylate
  • the culture solution used for the formation of the spheroid in the present invention can be used without limitation as long as it is a medium commonly used for the culture and / or differentiation of fat stem cells, specifically DMEM (Dulbeco's modified eagle medium ), Ham's F12 or the like may be used in a medium containing serum, but may be used in a medium without serum.
  • DMEM Denbeco's modified eagle medium
  • Ham's F12 or the like may be used in a medium containing serum, but may be used in a medium without serum.
  • FBS fetal bovine serum
  • the spheroid formed by irradiating fat stem cells with red wavelength band light may have characteristics of vascular endothelial cells.
  • the light may use light generated from a light emitting diode (LED), and more specifically, may use light indicating a wavelength of 600 to 700 nm and a power of 1 to 30 W generated from the LED. Can be used to generate light from the LEDs representing a wavelength of 660 nm and a power of 5 W.
  • LED light emitting diode
  • LED irradiation means to irradiate light of the red wavelength band.
  • the spheroid can be formed by irradiating the red wavelength band light to the adipose stem cells, and after implanting the spheroid to the skin or vascular tissue damage site, and irradiated with the red wavelength band light in vivo Differentiated into vascular endothelial cells or skin cells, and can assist in vivo self-healing ability promoted by light of the red wavelength band.
  • the spheroid may be used as an active ingredient of a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength band, and as the active ingredient, not only the spheroid, but also a spheroid culture solution and a spheroid cell debris Can also be used.
  • a technique of using the spheroid as an active ingredient of a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength range is not known at all, and was first developed by the present inventors.
  • the spheroid provided by the present invention When the spheroid provided by the present invention is implanted in vivo, when the red wavelength band of light is irradiated, angiogenesis and expression and secretion of skin regeneration factors (FGF, VEGF, HGF, etc.) are promoted, resulting in vascular regeneration and It can promote skin cell differentiation and finally prevent skin necrosis and promote skin regeneration.
  • FGF skin regeneration factors
  • the light of the red wavelength range is not particularly limited thereto, but in particular, light generated from a light emitting diode (LED) may be used. More specifically, the light of the red wavelength band may be generated from the LED to have a wavelength of 600 to 700 nm and 1 to 30 W. Light representing power can be used, and most specifically, light generated from the LED can be used to represent the wavelength of 660nm and the power of 5W.
  • LED light emitting diode
  • the irradiation conditions of the light is not particularly limited as long as it shows an effect of promoting the differentiation of fat stem cells into vascular endothelial cells, specifically, using an LED light source placed at intervals of 1 to 5cm from the implanted site To irradiate for 10 to 15 minutes per day.
  • the adipose stem cells in order to easily form a spheroid from the adipose stem cells, it is preferable to inoculate the adipose stem cells at a density of 1 ⁇ 10 5 to 2 ⁇ 10 6 cells / cm2.
  • a density of 1 ⁇ 10 5 to 2 ⁇ 10 6 cells / cm 2 When inoculating at a density of less than 1 ⁇ 10 5 cells / cm 2, there is a problem that the size of the spheroid is small and the variation is severe.
  • cell death due to excessive cell proliferation May be induced.
  • a spheroid having a diameter of 0.5 to 1 mm detectable with the naked eye can be formed.
  • the pharmaceutical composition of the present invention contains a live spheroid as an active ingredient, it may further include a suitable carrier, excipient or diluent commonly used in the preparation of cell therapy.
  • suitable carrier include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do.
  • organics such as biopolymers, inorganics such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, peptide proteins, ex vivo matrix proteins It may also be formulated in combination with (ECM), gel, and the like.
  • ECM ex vivo matrix proteins
  • physiological saline PBS (phosphate buffered saline) may be used as phosphate buffered saline.
  • the pharmaceutical composition of the present invention is preferably prepared in a dosage form suitable for injection, and for this purpose, the spheroid is preferably dissolved in a pharmaceutically acceptable aqueous solution or kept in frozen form in solution.
  • the composition may further comprise a pharmaceutically acceptable carrier which can be used to suspend or dilute the spheroid.
  • a pharmaceutically acceptable carrier such as distilled water, physiological saline, PBS, etc. are mentioned, for example.
  • Formulation carriers or excipients necessary to take the form of pharmaceutical preparations may further contain stabilizers or anti-adsorption agents, and formulations may be used as injections (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, etc.), Pain-free agents can be used that can reduce pain upon injection, and suitable devices can be used as needed.
  • it may be filled in the form of a final injection in a syringe or device, in the form of a cryoial that can be frozen, or in the form of a pyrogen-free glass bottle and rubber field, which can contain liquid medicine, and an aluminum cap.
  • a syringe In the form of a device, a syringe, a multi-syringe, and the like may be used.
  • a needle In the case of limb ischemic disease, a needle is used to minimize pain without causing damage by shearing the cell while the cell is administered.
  • a material that does not affect the cell viability of the syringe or device.
  • the content of the spheroid included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in an amount of 10 to 50% by weight, more specifically 20 to 40% by weight based on the total weight of the final composition.
  • the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention
  • Sufficient amount means an effective dose level means the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of excretion treatment Period of time, factors including drugs used in combination or coincidental with the compositions of the invention used, and other factors well known in the medical arts.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.
  • the dosage of the pharmaceutical composition of the present invention may be administered to one or a plurality of locations (for example, 2 to 50 locations) to the living muscles (skeletal muscle or myocardium) around the vascular injury site, for example, and the dosage is Specifically, the number may be 1.0 ⁇ 10 5 to 1.0 ⁇ 10 8 cells / kg (body weight), and more specifically 1.0 ⁇ 10 6 to 1.0 ⁇ 10 7 cells / kg (body weight).
  • Another aspect of the invention provides a method of treating skin or vascular tissue damage, comprising administering the pharmaceutical composition to an individual with damaged skin or vascular tissue.
  • the treatment method may further comprise the step of irradiating the subject with light in the red wavelength band.
  • the pharmaceutical composition of the present invention can treat vascular damage, skin ulcers or wounds by itself, but when light of the red wavelength band is irradiated, the differentiation into vascular endothelial cells and skin cells can be promoted to effectively treat skin or vascular tissue damage. Can be.
  • the term "individual” includes without limitation mammals including rats, livestock, humans, and the like, in which vascular damage, skin or vascular tissue damage has occurred.
  • the disease that causes the skin or vascular tissue damage is not particularly limited, but specifically, may be a cardiovascular disease, cerebrovascular disease, ischemic diseases, for example, ischemic skin ulcers, skin wounds, arteriosclerosis, stable And unstable angina, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulatory disorders, myocardial infarction, stroke, transient and ischemic attacks, subarachnoid hemorrhage, or lower limb ischemia.
  • the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention is not particularly limited, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously or intradermally, as desired.
  • the composition may be administered by any device in which the active substance may migrate to the target cell.
  • Another embodiment of the present invention provides a method of differentiating adipose stem cells into vascular endothelial cells by irradiating adipose stem cells with light of a red wavelength band.
  • the proliferation and mobility of stem cells are enhanced to promote spheroid formation, and when the formed spheroid is continuously irradiated with red wavelength band light, Expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) that can promote differentiation into vascular endothelial cells are promoted in the stem cells themselves to form the spheroid, so that angiogenesis related factors or genes are externally added.
  • FGF growth factor
  • VEGF vascular endothelial growth factor
  • the method of differentiating adipose stem cells provided by the present invention into vascular endothelial cells is inoculated and cultured in a culture vessel having a surface hydrophobic adipose stem cells, power of 5 to 15 mW / cm2 and 600 to 700nm Irradiating light to the adipose stem cells.
  • MSCs meenchymal stromal cells
  • adipose-derived stem cells were used as the mesenchymal stem cells.
  • the adipose-derived mesenchymal stem cells can be obtained from adipose tissue which can be extracted in large quantities compared to other types of mesenchymal stem cells, so that the acquisition process is easy and safe, and there is no restriction on tissue supply and in vitro. Easy cultivation has advantages in tissue access, stability, efficacy and economics.
  • the method for differentiating adipose derived mesenchymal stem cells provided by the present invention into vascular endothelial cells essentially includes irradiating the adipose derived mesenchymal stem cells with light in the red wavelength band.
  • a cell mass known as a spheroid or 3D cell mass (3DCM)
  • the continuous irradiation of the light on the spheroid Expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) that can promote the differentiation of the spheroid-forming stem cells into vascular endothelial cells are promoted to accumulate inside the spheroid or Secreted outwardly, the growth factor activates differentiation signaling involved in stem cell differentiation, thereby secreting the spheroid into vascular endothelial cells. Can promote anger.
  • FGF vascular endothelial growth factor
  • the light is not particularly limited as long as it exhibits an effect of promoting differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, but specifically, light generated from a light emitting diode (LED) may be used.
  • LED light emitting diode
  • light generated from the LED can be used to represent the wavelength of 600 to 700nm and power of 5 to 30 mW / cm2, most specifically light generated from the LED to represent a wavelength of 660nm and power of 10 mW / cm2 Can be used.
  • the irradiation conditions of the light is not particularly limited as long as it shows an effect of promoting the differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, specifically, the spacing of 1 to 5 cm from the adipose derived mesenchymal stem cells
  • the method can be used to irradiate for 10 to 15 minutes a day using the LED light source placed.
  • spheroid formation and angiogenesis factors by irradiating light in the red wavelength range without treating or inducing the genetic modification of a substance that induces the differentiation of stem cells into vascular endothelial cells in the culture solution or culture matrix for culturing the adipose stem cells.
  • the culture solution may be used without limitation as long as it is a medium commonly used for culturing and / or differentiating fat stem cells, and specifically, a medium containing serum such as DMEM (Dulbeco's modified eagle medium) or Ham's F12 may be used. Can be used.
  • a medium in which fetal bovine serum (FBS) was added to DMEM / F12 medium in which DMEM and Ham's F12 were mixed at a 1: 1 ratio was used.
  • FBS fetal bovine serum
  • the adipose derived mesenchymal stem cells can be inoculated and cultured in a culture vessel surface-treated with a polymer imparting hydrophobicity or a culture vessel made of the polymer. .
  • the polymer giving the hydrophobicity is as described above.
  • Vascular endothelial cells differentiated by the method can be used for the treatment of vascular damage, skin or vascular tissue damage, the disease causing the skin or vascular tissue damage is not particularly limited, specifically, cardiovascular disease, cerebrovascular disease , Ischemic diseases, skin wounds, and the like, for example, ischemic skin ulcers, skin wounds, arteriosclerosis, stable and unstable angina pectoris, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulatory disorders, myocardial infarction, stroke, transient and It may be a disease such as ischemic attack, subarachnoid hemorrhage or lower limb ischemia.
  • Pluripotent stem cells were isolated from human subcutaneous adipose tissue and observed with a phase contrast microscope (Nikon), observed by DAPI staining, or flow cytometry.
  • flow cytometry CD29, CD90 and CD105 were used as surface antigens to identify mesenchymal stem cells, and CD34, CD31, and KDR (Flk1) were used as surface antigens to observe whether or not other cells were mixed during stem cell isolation and culture.
  • ⁇ -SMA their expression patterns were analyzed using flow cytometry (FIG. 1).
  • Figure 1 is a micrograph showing the results of verifying the stem cells isolated from human adipose tissue. As shown in Figure 1, it was confirmed that the cells isolated from human subcutaneous fat tissue is adipose derived mesenchymal stem cells having a phenotype of mesenchymal stem cells.
  • WST Water-soluble Tetrazolium Salts
  • Figure 2 is a photograph showing the LED irradiation apparatus
  • Figure 3 is a graph showing the growth rate change of fat-derived mesenchymal stem cells by LED irradiation. As shown in FIG. 3, the growth of stem cells was accelerated as the irradiation time of light increased, and the proliferation level of stem cells increased as the amount of energy included in the irradiation light increased.
  • the growth of stem cells was promoted when irradiated with light of 0.5 J / cm2 or more, and the proliferation level of stem cells is proportional to the amount of energy contained in the light until the irradiation of 3 J / cm2.
  • the proliferation level of stem cells rather reduced.
  • Example 3-1 Adhesion Activity of Adipose-derived Mesenchymal Stem Cells to the Culture Vessel Surface and Analysis of Spheroid Formation According to Culture Time
  • adipose-derived mesenchymal stem cells' adhesion activity was inoculated with 4 ⁇ 10 4 cells / cm 2 of adipose derived mesenchymal stem cells per well, and then cultured in DMEM / F12 medium containing 10% FBS for 3 days. After the incubation was completed, the spheroid formation of adipose derived mesenchymal stem cells was observed on each cell adhesion surface (FIG. 4A).
  • Figure 4a is a photograph showing the result of culturing fat-derived mesenchymal stem cells according to the presence of ECM protein coated in the culture vessel.
  • spheroids of adipose-derived mesenchymal stem cells were formed to a size that can be detected by naked eye in NTCP, which is hydrophobic, and has a weak surface cell adhesion.
  • the spheroids have a diameter of about 1 mm or more. Confirmed.
  • adipose-derived mesenchymal stem cells is affected by the adhesion activity to the surface of the culture vessel used from the above results, and cell adhesion is initially weakly induced to form a spheroid having a detectable size with the naked eye.
  • the cell density increased, and it was confirmed that it is preferable to use a culture vessel having a hydrophobic surface, such as polystyrene NTCP, in which cells detach and proliferate in a suspended state.
  • the adipose derived mesenchymal stem cells obtained in Example 1 containing 5% FBS DMEM Each well of NTCP containing / F12 medium was inoculated at a concentration of 0.5 ⁇ 10 4 to 1 ⁇ 10 5 cells / cm 2 and incubated for 3 days while irradiating light from the LED light source for 10 minutes each day (FIG. 4B).
  • Figure 4b is a photograph showing the change in morphology of fat-derived mesenchymal stem cells with the passage of incubation time while inoculating the fat-derived mesenchymal stem cells in NTCP, and irradiated with light from the LED light source. As shown in Figure 4b, it was confirmed that the spheroid of the detectable size with the naked eye was formed at the time 3 days have passed.
  • Each well of a 24-well plate for non-tissue cell culture was inoculated with 5 ⁇ 10 4 cells of adipose stem cells per well and then incubated in DMEM / F12 medium containing 5% FBS for 3 days. After incubation for 3 days, the spheroid formation of adipose stem cells was observed on each cell adhesion surface (FIG. 5).
  • FIG. 5 is a photograph showing a spheroid formed from adipose stem cells. As shown in Figure 5, when the incubation was confirmed that the spheroid having a diameter of about 1mm was formed. Adipocytes cultured for 3 days in monolayer in a 24-well plate for tissue cell culture did not form spheroids.
  • spheroids were formed by irradiating LEDs with two-dimensional cultured adipocyte-derived mesenchymal stem cells (monolayer ASC) Adipose-derived mesenchymal stem cells (spheroid ASC) were analyzed using an ELISA kit (R & D Systems) (Fig. 6). At this time, the expression amount of each factor expressed in the unit cell number (1 ⁇ 10 4 cell number) was measured in pg units.
  • Figure 6 is a graph showing the expression changes of angiogenesis and skin regeneration factor of adipose derived stem cells by LED irradiation. As shown in Figure 6, the three angiogenesis and skin regeneration factors were confirmed that the expression level of the spheroid-forming adipocyte-derived mesenchymal stem cells (spheroid ASC) was increased while irradiating the LED.
  • spheroid ASC spheroid ASC
  • 2D cultured stem cell group control
  • 2D cultured stem cell group Low level light therapy, LLLT
  • spheroid-free stem cell group Spheroid without LED irradiation and spe
  • Figure 7 is a photograph and graph showing the results of measuring the expression level change of the angiogenesis-related protein according to whether the LED irradiation and spheroid formation. As shown in FIG. 7, the expression level of angiogenesis-related proteins in the spheroid-formed stem cells was increased, rather than the two-dimensional cultured stem cells, and among the spheroid-formed stem cells, By examining the LED, it was confirmed that the expression level of angiogenesis-related protein was increased in the spheroid-formed stem cells.
  • Example 6 Confirmation of vascular endothelial cell characteristics of spheroid by LED irradiation
  • Adipose tissue-derived mesenchymal stem cells isolated in Example 1 were inoculated into 24-well NTCP (Non-Treated cell culture plate) at a concentration of 1 ⁇ 10 5 cells / well, and cultured while irradiating LEDs to spheroids. Formed. The formed spheroids were collected, fixed at -70 ° C using an OCT compound, cut into 4 ⁇ m thickness using a microtome, and then the sections were fixed on a slide glass and subjected to immunological staining.
  • NTCP Non-Treated cell culture plate
  • the recovered spheroid was physically disintegrated using a syringe, then placed on a slide glass and attached for 4 hours, and the slide glass was washed several times with PBS, which was immersed in a 4% paraformaldehyde solution. Fixed at room temperature for 30 minutes, then washed again with PBS and subjected to immunological staining.
  • the immunological staining was performed by immersing the slide glass prepared above in PBS containing primary antibodies against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, and washing three times with PBS. After treatment, the secondary antibody capable of binding to the primary antibody was reacted for 1 hour in the dark. After the reaction was completed, the slide glass was washed three times with PBS, mounted (mounted) and analyzed by fluorescence microscope (upper photo of Figure 8).
  • FIG. 8 is a photograph showing the results of immunofluorescence staining for the adipose tissue-derived mesenchymal stem cells irradiated with LED.
  • spheroids formed from adipose derived mesenchymal stem cells by the LED irradiation showed a positive response to CD29, CD34, Flk1 and CD31.
  • CD29 is surface antigen specifically expressed in mesenchymal stem cells and epithelial cells
  • CD34, KDR (Flk1) and CD31 are surface antigens specifically expressed in vascular endothelial cells.
  • spheroids formed by culturing adipose derived mesenchymal stem cells in a red wavelength band and cultured in a culture vessel with a hydrophobic surface were composed of vascular endothelial cells differentiated from adipose derived mesenchymal stem cells. .
  • the lower photo of Figure 8 is a photograph showing the results confirmed by Western blot analysis whether the expression of the CD31 according to the LED irradiation and the spheroid formation.
  • CD31 was expressed only in the stem cells in which spheroids were formed, and among the stem cells in which spheroids were formed, the spheroid-formed stems were irradiated with LEDs rather than the stem cells in which spheroids were formed without LED irradiation. It was confirmed that more CD31 is expressed in the cells.
  • the stem cells of each group were recovered, physically disintegrated using a syringe, placed on a slide glass, and then attached for 4 hours, and then the slide glass was washed several times with PBS, which was immersed in 4% paraformaldehyde solution. The cells were fixed at room temperature for 30 minutes, washed again with PBS, and subjected to immunological staining for CD31, Cd34 and KDR, which are surface antigens specifically expressed in vascular endothelial cells.
  • the immunological staining was performed by immersing the slide glass prepared above in PBS containing primary antibodies against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, and washing three times with PBS. After treatment, the secondary antibody capable of binding to the primary antibody was reacted for 1 hour in the dark. After the reaction was completed, the slide glass was washed three times with PBS, mounted and analyzed by FACS (FIG. 9).
  • Figure 9 is a graph showing the results of FACS analysis for the two-dimensional cultured stem cells and spheroid-formed stem cells irradiated with LED. As shown in FIG. 7, it was confirmed that the spheroid formed by the LED irradiation showed a positive response to CD29, CD34, KDR (Flk1) and CD31, which are surface antigens specifically expressed in vascular endothelial cells.
  • the spheroid irradiated with LED exhibited characteristics of vascular endothelial cells differentiated from adipose derived mesenchymal stem cells.
  • Example 7 Confirmation of the therapeutic effect after spheroid transplantation irradiated with LED on animal model of ischemia
  • the femoral aorta of a 5 week old BALB / c nude mouse (20 g body weight; Narabio, Seoul, Korea) was ligated using 5-0 silk suture to prepare an animal model of the ischemic mouse.
  • FIG. 10A to 10D are photographs and graphs showing the results of comparing the therapeutic effects of spheroid-formed stem cells by examining LEDs for the ischemic mouse animal model.
  • FIG. 10A is 1, 7, 14, and 21 days after injection.
  • Figure 10b is a photograph showing the lower extremity portion of the mouse animal model taken at the elapsed time point
  • Figure 10b is a graph showing the ratio of the mouse according to the degree of damage of the lower extremities among all mice after 21 days
  • Figure 10c is 1, It is a photograph which shows the skin blood flow taken at the time of 7, 14, and 21 days passed
  • FIG. 10D is a graph which shows the ratio of the mouse to which the leg ischemia was treated among all the mice after 21 days.
  • a wound was formed on the skin tissue of the back region using an 8 mm punch, and a splint made of silicone was installed on the wound region to prepare a skin wound mouse model.
  • a control group injected with 600 ⁇ l of PBS in the prepared skin wound mouse model, an experimental group transplanted with adipose tissue-derived mesenchymal stem cells of 1.5 ⁇ 10 6 cells (ASCs), and an LED light source.
  • Low level light thearapy (LLLT) irradiated with light at 50 mW / cm2 and 660 nm wavelengths for 10 minutes each day
  • the experimental group (spheroid) implanted with 15 spheroids and the 15 spheroids were implanted, and the experimental group (spheroid + LLLT) irradiated with LEDs was set, and after 14 days of breeding, confirmed the wound healing effect of damaged skin tissue. (FIG. 11).
  • FIG. 11 is a photograph and a graph showing the results of comparing the wound treatment effect of each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) using the skin wound mouse model
  • Figure 11a is a manufactured skin wound It is a photograph showing a mouse model
  • b in FIG. 11 is a photograph photographing the wound site of each test group with the passage of time
  • FIG. 11 c is a graph which shows the area change of the wound site with passage of time. As shown in FIG.
  • the wound treatment effect of the spheroid-grafted experimental group was superior to that of the adipose tissue-derived mesenchymal stem cells (ASCs) or the LED-irradiated (LLLT) -tested group, and the LED among the spheroid-transplanted experimental groups It was confirmed that the experimental group (spheroid + LLLT) in parallel with the irradiation showed better wound healing effect.
  • ASCs adipose tissue-derived mesenchymal stem cells
  • LLLT LED-irradiated
  • mice of each experimental group set in Example 8 were subjected to vascular endothelial cells.
  • CD31, a cell marker, and SMAa actin, a marker of vascular envelope cells, were immunofluorescent stained.
  • mice of each experimental group were sacrificed to obtain the wound tissue, and the tissue obtained was immersed in 4% paraformaldehyde solution and fixed at room temperature for 30 minutes. It was fixed at 70 °C, and cut into 4 ⁇ m thickness using a microtome to obtain a tissue flakes, the tissue flakes were fixed to the slide glass.
  • the slide glass was immersed in PBS containing a primary antibody against CD31, a vascular endothelial cell marker, and SMAa actin, a marker of vascular endothelial cell, reacted overnight, washed three times with PBS, and then bound to the primary antibody. Secondary antibodies were treated and reacted for 1 hour in the dark to carry out immunofluorescence staining. After the reaction was completed, the slide glass was washed three times with PBS and mounted, and then immunofluorescent stained tissue was analyzed by fluorescence microscopy (Fig. 12a).
  • FIG. 12 a shows immunofluorescence showing the results of comparing the levels of vascular endothelial cells and vascular endothelial cells in the wound region of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
  • green represents SMAa actin, a marker of vascular endothelial cells
  • red CD31, a vascular endothelial marker.
  • FIG. 12a it was confirmed that the expression level of the SMAa actin and CD31 in the tissue of the experimental group (spheroid + LLLT) to which the spheroid was implanted and the LED was shown to be higher than that of the other experimental groups.
  • the number of microvascular and small arteries was counted from the fluorescence micrograph of the immunofluorescent stained tissue, and the number of counted microvascular and small arteries was compared in terms of the number per unit area (mm 2) (b and c of FIG. 12). ).
  • FIG. 12B is a graph showing the results of comparing the number of microvascular vessels per unit area of the wounded mouse model with each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT)
  • FIG. 12c is a graph showing the result of comparing the number of small arteries per unit area of the wounded mouse model with each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
  • cytokeratin which is a skin cell marker
  • FIG. 13 is an immunofluorescence staining photograph showing the result of comparing the level of skin cells present in the wound area of the skin wound mouse model according to each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT), and red is skin.
  • Cytokeratin a cell marker. As shown in FIG. 13, it was confirmed that the expression level of the cytokeratin in the tissue of the experimental group (spheroid + LLLT) irradiated with the spheroid and irradiated the LED showed a higher level than that of the other experimental groups.
  • spheroids formed from adipose tissue-derived mesenchymal stem cells had an effect on the expression levels of FGF, VEGF and HGF known as angiogenesis and skin regeneration factors, immunofluorescence staining and Western blot analysis were performed.
  • Example 11-1 Immunofluorescence Staining Assay
  • Example 8 In order to perform immunofluorescence staining analysis, the wound tissue obtained from the mice of each experimental group set in Example 8 was subjected to immunofluorescence staining for FGF, VEGF and HGF known as angiogenesis and skin regeneration factors. Except that, the same method as in Example 9 was carried out, and immunofluorescent stained tissue was analyzed by fluorescence microscope (Fig. 14a). At this time, HNA was used as the internal control group.
  • Figure 14a is an immunofluorescence staining showing the results of comparing the levels of angiogenesis and skin regeneration factors present in the wound area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) As a photograph, the red color represents FGF, VEGF and HGF, respectively, angiogenesis and skin regeneration factors. As shown in a of FIG. 14, it was confirmed that the expression level of the angiogenesis and skin regeneration factor in the tissue of the experimental group (spheroid + LLLT) implanted with spheroid and irradiated with LED showed higher level than that of other experimental groups. .
  • 14B is a result of Western blot analysis of the levels of angiogenesis and skin regeneration factors present in the wound area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT)
  • 14C is a graph showing the results of comparing the levels of each angiogenesis and skin regeneration factor detected by the Western blot analysis for each treatment condition.
  • the expression level of the angiogenesis and skin regeneration factor in the tissue of the experimental group (spheroid + LLLT) irradiated with a spheroid and irradiated LED shows a higher level than the tissues of other experimental groups Check again.
  • Example 12 Validation of the Combination Effect of Spheroid Implantation and LED Treatment with LED Irradiation through a Skin Flap Mouse Model
  • a group of low level light thearapy (LLLT) irradiated with light of 50 mW / cm 2 and 660 nm wavelength for 10 minutes each day, a spheroid implanted with 15 spheroids irradiated with LED, and 15 spheroids irradiated with LEDs were implanted.
  • Each experimental group (spheroid + LLLT) irradiated with LED was set, and bred for 14 days, and then the wound treatment effect of the damaged skin tissue was confirmed (FIG. 15).
  • FIG. 15 is a photograph showing the results of comparing the wound treatment effect using the skin flap mouse model.
  • spheroid transplanted with adipose tissue-derived mesenchymal stem cells (ASCs), LED irradiation (LLLT), or spheroids showed similar wound healing effects.
  • the experimental group (spheroid + LLLT) implanted with spheroids and irradiated with LEDs showed remarkably excellent wound healing effect.
  • Example 13 Blood flow measurement in a skin flap mouse model
  • FIG. 16 is a photograph showing the results of comparing the flow of skin blood taken in the skin flap mouse model at the time of 1, 7 and 14 days for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). .
  • spheroids implanted with adipose tissue-derived mesenchymal stem cells (ASCs), LEDs (LLLT), or spheroids were measured at similar levels of blood flow, but spheroids were measured.
  • the experimental group (spheroid + LLLT) transplanted and irradiated with LED confirmed that the blood flow was significantly increased. The blood flow decreases when there is a damaged blood vessel, and when the damaged blood vessel is treated, the blood flow increases. Therefore, the experimental group (spheroid + LLLT) implanted with spheroids and irradiated with LEDs showed a high level of treatment of the damaged blood vessels. And it was found.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to: a therapeutic pharmaceutical composition containing spheroids for a diseased area transplant, the composition being used to improve the treatment effect in the treatment of skin or vascular tissue damage by using an LED light source to irradiate a red-wavelength band light; and a method for treating skin or vascular tissue damage using the same. Further, the present invention relates to a method for differentiating mesenchymal stem cells using a red-wavelength band light. The pharmaceutical composition of the present invention can induce the regeneration of the damaged vessel or skin in vivo, and thus can be widely utilized for the treatment of vessel damage, skin ulcers, or wounds. The differentiation method of the present invention can induce the differentiation of stem cells into vascular endothelial cells, even without using a differentiation inducer, and thus can be widely utilized in the development of more economical therapeutic agents for vascular diseases.

Description

피부 또는 혈관 조직 손상 치료용 약학 조성물 및 이의 이용Pharmaceutical compositions for treating skin or vascular tissue damage and their use
본 발명은 피부 또는 혈관조직 손상 치료용 약학 조성물 및 이의 이용에 관한 것으로, 보다 구체적으로 본 발명은 LED 광원을 이용하여 적색 파장대의 빛을 조사함으로써 피부 또는 혈관조직 손상을 치료하는 방법에 있어서, 이의 치료효과를 향상시키기 위하여 사용되는, 환부 이식용 스페로이드를 포함하는 치료용 약학 조성물 및 이를 이용한 피부 또는 혈관 조직 손상의 치료방법에 관한 것이다.The present invention relates to a pharmaceutical composition for treating skin or vascular tissue damage and its use. More particularly, the present invention relates to a method for treating skin or vascular tissue damage by irradiating light with a red wavelength band using an LED light source. The present invention relates to a therapeutic pharmaceutical composition comprising a spheroid for wound transplantation and to a method for treating skin or vascular tissue damage using the same, which is used to improve a therapeutic effect.
또한, 본 발명은 적색 파장대의 빛을 이용한 중간엽 줄기세포의 분화유도 방법에 관한 것이다.The present invention also relates to a method of inducing differentiation of mesenchymal stem cells using light of a red wavelength band.
일반적으로 분화란 초기 단계의 세포가 각 조직으로서의 특성을 갖게 되는 과정을 말하는데, 그 대표적인 예는 동물의 발생 과정에서 볼 수 있다. 즉, 정자와 난자가 결합하여 만들어진 수정란이라는 하나의 세포가 뼈, 심장, 피부 등의 다양한 조직 세포로 만들어지기 위해서는 '분화'가 일어나야 하는 것이다. 이러한 분화능력을 가지고 있는 줄기세포(stem cell)는 조직을 구성하는 각 세포로 분화되기 전단계의 세포로서, 미분화 상태에서 무한 증식이 가능하며 특정 분화 자극에 의해 다양한 조직의 세포로 분화될 수 있는 잠재적 가능성을 가진 세포를 의미한다.In general, differentiation refers to a process in which early-stage cells have characteristics as individual tissues, a representative example of which can be seen in animal development. In other words, 'differentiation' must occur in order for a single cell called fertilized egg made by combining sperm and egg to be made into various tissue cells such as bone, heart, and skin. Stem cells that have such differentiation capacity are the cells before the differentiation into each cell constituting the tissue, and are capable of infinite proliferation in the undifferentiated state and potential to be differentiated into cells of various tissues by specific differentiation stimulation. It means a cell with the possibility.
최근에, 인간 지방조직(adipose tissue)에 자가 재생능력이 뛰어나고, 간, 골, 연골, 지방, 혈관, 심장, 신경계 등과 같은 다양한 세포로 분화될 수 있는 미분화 세포군이 포함되어 있음이 보고되었다. 이러한 지방조직 유래 다분화능 줄기세포로서 지방줄기세포(adipose-derived stem cells; ASCs)는 중간엽계 줄기세포와 비교하여 지방조직을 대량으로 추출할 수 있다는 점에서 취득과정이 용이하고 안전하며, 조직수급상의 제한이 없는 장점과 체외배양이 용이하여 조직 접근성, 안정성, 유효성, 그리고 경제적 측면에서 이점을 가지고 있어 다분화능 줄기세포의 새로운 공급원으로서 주목받고 있다.Recently, it has been reported that human adipose tissues contain undifferentiated cell populations that have excellent self-renewal ability and can be differentiated into various cells such as liver, bone, cartilage, fat, blood vessels, heart, nervous system and the like. Adipose-derived stem cells (ASCs) as adipose tissue-derived multipotent stem cells are easier and safer to acquire because they can extract a large amount of adipose tissue compared to mesenchymal stem cells. It is attracting attention as a new source of multipotent stem cells because it has advantages in terms of tissue accessibility, stability, efficacy, and economics due to its unlimited limitations and easy in vitro culture.
지금까지 알려진 지방줄기세포로는 상피세포로 분화 가능한 인간 지방줄기세포; 골 형성 및 지방세포로 분화 가능한 인간 지방줄기세포; 신경세포로 분화 가능한 인간 지방줄기세포; 지방세포로 분화 가능한 쥐 지방줄기세포; 골 형성 및 연골 형성 세포로 분화 가능한 쥐 지방줄기세포; 연골세포로 분화 가능한 인간 지방줄기세포; 신경세포로 분화 가능한 쥐 지방줄기세포; 및 골세포, 연골세포, 신경세포 또는 근육세포로 분화가능한 지방줄기세포(미국 등록특허 제6,777,231호) 등이 있다. Fat stem cells known to date include human adipose stem cells capable of differentiating into epithelial cells; Human adipose stem cells capable of bone formation and differentiation into adipocytes; Human adipose stem cells capable of differentiating into neurons; Rat adipocytes capable of differentiating into adipocytes; Rat adipocytes capable of differentiating into osteogenic and chondrogenic cells; Human adipose stem cells capable of differentiating into chondrocytes; Rat adipose stem cells capable of differentiating into neurons; And adipose stem cells capable of differentiating into osteocytes, chondrocytes, nerve cells or muscle cells (US Pat. No. 6,777,231).
아울러, 상기 지방줄기세포가 생체 내 및 시험관 내에서 혈관내피세포로 분화가 유도될 수 있음이 보고되었다. 예를 들어, 지방줄기세포를 육종마우스로부터 분리된 특수한 세포외 기질 성분인 마트리겔(matrigel)이 코팅된 배양용기에서 다양한 성장인자가 첨가된 배지를 이용하여 배양하면 혈관내피세포로 분화될 수 있고(Cao Y, 등, Biochem. Biophys. Res. Commun. 332: 370-379, 2005), 무혈청 배지에서 지방줄기세포를 배양하여 스페로이드(구상체, spheroid)를 형성한 후 이를 조혈모세포 배양배지에서 일주일간 배양하여 Flk-1을 다량 발현하는 혈관내피세포를 획득할 수 있음이 보고되었다(Martinez-Estrada O.M., 등, Cardiovasc. Res. 65(2): 328-333, 2005). 그러나 상기 방법들은 분화에 3주 이상의 시간이 소요되어 장기간의 세포 배양으로 오염이 발생할 수 있고 특정 세포로의 분화를 위해 성장인자와 같은 특수 배지 성분을 사용해야 하기 때문에 배지 제조비용이 상승하여 산업적으로 이용하기에 어렵다는 단점이 있다. 또한, 종래에는 중간엽 줄기세포를 혈관 세포로 유도하기 위해 혈관세포 유도성분이 함유된 배지 또는 배양 메트릭스에서 배양하였을 뿐 혈관세포의 분화를 촉진시키는 장비가 없어 분화시간이 길어지는 문제점이 있었다.In addition, it has been reported that the adipose stem cells may be induced to differentiate into vascular endothelial cells in vivo and in vitro. For example, adipose stem cells may be differentiated into vascular endothelial cells when cultured using a medium containing various growth factors in a matrigel-coated culture vessel, a special extracellular matrix component isolated from sarcoma mice. (Cao Y, et al., Biochem. Biophys. Res. Commun. 332: 370-379, 2005), cultured adipose stem cells in serum-free medium to form spheroids and then cultured hematopoietic stem cells It has been reported that vascular endothelial cells expressing a large amount of Flk-1 can be obtained by culturing for one week at (Martinez-Estrada OM, et al., Cardiovasc. Res. 65 (2): 328-333, 2005). However, these methods can take more than three weeks to differentiate, which can cause contamination by long-term cell culture, and use of special medium components such as growth factors for differentiation into specific cells increases the production cost of the medium. The disadvantage is that it is difficult to do. In addition, conventionally, in order to induce mesenchymal stem cells to vascular cells, there was a problem in that differentiation time is long because there is no equipment for stimulating differentiation of vascular cells, but only cultured in a medium or culture matrix containing vascular cell inducing components.
한편, 당뇨성 질환 등에 의한 피부궤양의 초기병변은 염증반응에 의한 혈관내피세포의 손상이 주된 원인으로 알려져 있다. 정상 혈관내피세포들은 염증세포와 상호작용이 일어나지 않는 반면, 혈관내피세포에 염증이 일어나면 염증세포와 상호작용이 일어나 염증세포가 혈관벽에 달라붙어 염증반응이 일어나게 된다. 이러한 피부궤양 및 피부상처를 치료하는 방안으로서, 줄기세포를 이용한 세포치료제가 주목받고 있다. 예를 들어, 지방줄기세포를 육종마우스로부터 분리된 특수한 세포외 기질 성분인 마트리겔(matrigel)이 코팅된 배양용기에서 다양한 성장인자가 첨가된 배지를 이용하여 배양하면 혈관내피세포로 분화될 수 있고(Cao Y, 등, Biochem. Biophys. Res. Commun. 332: 370-379, 2005), 무혈청 배지에서 지방줄기세포를 배양하여 스페로이드(spheroid)를 형성한 후 이를 조혈모세포 배양배지에서 일주일간 배양하여 Flk-1을 다량 발현하는 혈관내피세포를 획득할 수 있음이 보고되었다(Martinez-Estrada O.M., 등, Cardiovasc. Res. 65(2): 328-33, 2005). 그러나, 줄기세포를 이용한 치료법은 줄기세포를 목적하는 조직세포로 분화시키는 것이 용이하지 않고, 목적하는 조직세포로 분화된 경우에도 면역거부반응이 일어날 수 있다는 문제점이 있다. 이러한 문제점을 해결하기 위하여, 줄기세포를 직접 생체내에 이식하는 방안이 연구되고 있으나, 아직까지는 생체내에서 목적하는 조직세포로 줄기세포의 분화를 유도하는 기술의 개발수준이 낮아 추가적인 시간과 비용이 필요한 실정이다.On the other hand, the early lesions of skin ulcers due to diabetic diseases are known to be the main cause of damage to vascular endothelial cells due to inflammatory reactions. Normal vascular endothelial cells do not interact with inflammatory cells, whereas when vascular endothelial cells are inflamed, the inflammatory cells interact with the inflammatory cells that adhere to the vascular wall, causing an inflammatory reaction. As a method for treating such skin ulcers and skin wounds, cell therapy using stem cells has attracted attention. For example, adipose stem cells may be differentiated into vascular endothelial cells when cultured using a medium containing various growth factors in a matrigel-coated culture vessel, a special extracellular matrix component isolated from sarcoma mice. (Cao Y, et al., Biochem. Biophys. Res. Commun. 332: 370-379, 2005), culturing fat stem cells in serum-free medium to form spheroids, and then culturing them in hematopoietic stem cell culture medium for one week. It has been reported that vascular endothelial cells expressing a large amount of Flk-1 can be obtained by culturing (Martinez-Estrada OM, et al., Cardiovasc. Res. 65 (2): 328-33, 2005). However, the therapy using stem cells is not easy to differentiate the stem cells into the desired tissue cells, there is a problem that even if the differentiation of the desired tissue cells may cause an immune rejection reaction. In order to solve this problem, a method of directly injecting stem cells into a living body has been studied. However, the development of a technique for inducing differentiation of stem cells into target tissue cells in vivo is still low, requiring additional time and cost. It is true.
한편, 줄기세포와는 달리 생체내의 재생효과를 극대화 시켜서, 손상된 혈관을 자가치료하는 방법이 모색되기도 하였다. 예를 들어, 미국 특허공개 제2011-0137385호에는 허혈성 질환이 발병된 환부에 600 내지 1,000nm 파장의 빛을 조사하는 단계를 포함하는 혈관질환 치료방법이 개시되어 있는데, 상기 빛을 혈관이 손상된 환부의 외부에서 조사하면, 상기 조사된 빛에 의하여 손상된 혈관내피세포의 증식이 촉진되어 손상된 혈관을 재생 및 복구됨으로써, 결과적으로는 혈관손상을 치료할 수 있다. 상기 방법은 외부에서 빛을 조사할 뿐, 환자의 생체내에 어떠한 것도 투여되지 않기 때문에, 안전성이 극히 높다는 장점이 있는 반면, 생체내의 자가 회복력을 증진시키는 것이기 때문에 치료에 많은 시간이 소요된다는 단점이 있다.On the other hand, unlike stem cells, by maximizing the regenerative effect in vivo, a method for self-healing damaged blood vessels has been sought. For example, US Patent Publication No. 2011-0137385 discloses a method for treating vascular diseases comprising irradiating light at a wavelength of 600 to 1,000 nm to an affected area where ischemic disease develops. When irradiated from the outside, the proliferation of vascular endothelial cells damaged by the irradiated light is promoted, thereby regenerating and repairing the damaged blood vessels, and as a result, vascular damage can be treated. The method has the advantage of extremely high safety because it only irradiates the light from the outside and is not administered in the patient's body, while it takes a lot of time for treatment because it promotes self-healing in vivo. .
본 발명자들은 보다 효과적으로 보다 효과적으로 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시킬 수 있는 방법; 및 피부궤양 및 상처를 치료하는 방법을 개발하고자 예의 연구노력한 결과, 지방유래 중간엽 줄기세포를 스페로이드 형태로 배양하면서 적색 파장대의 빛을 조사할 경우, 상기 줄기세포를 효과적으로 혈관내피세포로 분화시킬 수 있으며, 지방줄기세포 유래 스페로이드를 피부궤양 및 상처에 이식하고, 상기 혈관손상부에 적색 파장대의 빛을 조사하면, 피부궤양 및 상처를 보다 효과적으로 치료할 수 있음을 확인하고, 본 발명을 완성하였다.The present inventors can more effectively and more effectively differentiate the adipose derived mesenchymal stem cells into vascular endothelial cells; As a result of intensive research to develop a method for treating skin ulcers and wounds, the stem cells can be effectively differentiated into vascular endothelial cells when the adipose derived mesenchymal stem cells are cultured in a spheroid form and irradiated with red wavelengths. When the fat stem cell-derived spheroid is implanted into the skin ulcer and wound, and irradiated with light of the red wavelength band on the blood vessel injury, it was confirmed that the skin ulcer and wound could be more effectively treated, and the present invention was completed. .
본 발명의 하나의 목적은 지방줄기세포에서 유도된 스페로이드를 포함하는 피부 또는 혈관 조직 손상 치료용 약학 조성물을 제공하는 것이다.One object of the present invention to provide a pharmaceutical composition for treating skin or vascular tissue damage, including spheroid derived from adipose stem cells.
본 발명의 다른 하나의 목적은 지방유래 중간엽 줄기세포의 스페로이드에 적색 파장대의 빛을 조사하면서 배양하는 단계를 포함하는, 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for differentiating adipose derived mesenchymal stem cells into vascular endothelial cells, comprising culturing the spheroid of adipose derived mesenchymal stem cells while irradiating light with a red wavelength band. .
본 발명의 또 다른 하나의 목적은 지방유래 중간엽 줄기세포의 스페로이드에 적색 파장대의 빛을 조사하면서 배양하는 단계를 포함하는, 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 방법을 제공하는 것이다.It is another object of the present invention to provide a method of differentiating adipose derived mesenchymal stem cells into vascular endothelial cells, comprising culturing the spheroids of adipose derived mesenchymal stem cells while irradiating light with a red wavelength band. will be.
본 발명의 약학 조성물을 이용하면 생체내에서 손상된 혈관 또는 피부의 재생을 유도할 수 있으므로, 혈관 손상, 피부궤양 또는 상처 치료에 널리 활용될 수 있다. Since the pharmaceutical composition of the present invention can induce the regeneration of damaged blood vessels or skin in vivo, it can be widely used for the treatment of blood vessel damage, skin ulcers or wounds.
또한, 본 발명의 분화시키는 방법은 분화유도물질을 사용하지 않고서도, 줄기세포를 혈관내피세포로 분화유도할 수 있으므로, 보다 경제적인 혈관질환 치료제의 개발에 널리 활용될 수 있다.In addition, the method of differentiation of the present invention can induce differentiation of stem cells into vascular endothelial cells without using differentiation-inducing substances, and thus can be widely used for the development of more economical vascular disease therapeutics.
도 1은 인간의 지방조직으로부터 분리한 줄기세포를 검증한 결과를 나타내는 현미경 사진이다.Figure 1 is a micrograph showing the results of verifying the stem cells isolated from human adipose tissue.
도 2는 LED 조사장치를 나타내는 사진이다.2 is a photograph showing an LED irradiation apparatus.
도 3은 LED 조사에 의한 지방유래 중간엽 줄기세포의 성장율 변화를 나타내는 그래프이다.Figure 3 is a graph showing the growth rate change of adipose derived mesenchymal stem cells by LED irradiation.
도 4a는 배양용기에 코팅된 ECM 단백질의 존재여부에 따른, 지방유래 중간엽 줄기세포를 배양한 결과를 나타내는 사진이다.Figure 4a is a photograph showing the result of culturing fat-derived mesenchymal stem cells according to the presence of ECM protein coated in the culture vessel.
도 4b는 지방유래 중간엽 줄기세포를 NTCP에 접종하고, LED 광원으로부터 빛을 조사하면서 배양시간의 경과에 따른 지방유래 중간엽 줄기세포의 형태변화를 나타내는 사진이다.Figure 4b is a photograph showing the change in morphology of fat-derived mesenchymal stem cells with the passage of incubation time while inoculating the fat-derived mesenchymal stem cells in NTCP, and irradiated with light from the LED light source.
도 5는 지방줄기세포로부터 형성된 스페로이드를 나타내는 사진이다. 5 is a photograph showing a spheroid formed from adipose stem cells.
도 6은 LED 조사에 의한 지방유래 줄기세포의 혈관신생 및 피부재생인자 발현량 변화를 나타내는 그래프이다.Figure 6 is a graph showing the expression changes of angiogenesis and skin regeneration factor of adipose derived stem cells by LED irradiation.
도 7는 LED의 조사여부 및 스페로이드의 형성여부에 따른 혈관신생 관련 단백질의 발현수준 변화를 측정한 결과를 나타내는 사진 및 그래프이다.Figure 7 is a photograph and graph showing the results of measuring the change in the expression level of the angiogenesis-related protein according to whether the LED irradiation and spheroid formation.
도 8의 위측 사진은 LED가 조사된 지방조직 유래 중간엽 줄기세포에 대한 면역형광염색 결과를 나타내는 사진이다.8 is a photograph showing the results of immunofluorescence staining for the adipose tissue-derived mesenchymal stem cells irradiated with LED.
도 8의 아래측 사진은 LED의 조사여부 및 스페로이드의 형성여부에 따른 CD31의 발현여부를 웨스턴 블럿 분석을 통해 확인한 결과를 나타내는 사진이다.The lower photo of Figure 8 is a photograph showing the results confirmed by Western blot analysis whether the expression of the CD31 according to the LED irradiation and the spheroid formation.
도 9는 LED가 조사된 2차원 배양된 줄기세포와 스페로이드가 형성된 줄기세포에 대한 FACS 분석결과를 나타내는 그래프이다.Figure 9 is a graph showing the results of FACS analysis for the two-dimensional cultured stem cells and spheroid-formed stem cells irradiated with LED.
도 10은 하지허혈 마우스 동물모델에 대한 LED를 조사하여 스페로이드가 형성된 줄기세포의 치료효과를 비교한 결과를 나타내는 사진 및 그래프이다. Figure 10 is a photograph and graph showing the results of comparing the therapeutic effect of the spheroid-formed stem cells by examining the LED for the ischemic mouse animal model.
도 10a는 LED 조사한 스페로이드 주입후 1, 7, 14 및 21일이 경과한 시점에서 촬영한 마우스 동물모델의 하지부위를 나타내는 사진이다Figure 10a is a photograph showing the lower extremities of the mouse animal model taken at 1, 7, 14 and 21 days after the spheroid injection of the LED irradiation
도 10b는 LED 조사한 스페로이드 주입후 21일이 경과한 후, 전체 마우스 중에서 하지의 손상정도에 따른 마우스 비율을 나타내는 그래프이다.Figure 10b is a graph showing the ratio of the mouse according to the degree of injury of the lower extremities after 21 days after the spheroid injection of the LED irradiation.
도 10c는 LED 조사한 스페로이드 주입후 1, 7, 14 및 21일이 경과한 시점에서 촬영한 피부혈류를 나타내는 사진이다.10C is a photograph showing skin blood flow taken at 1, 7, 14, and 21 days after LED-injected spheroid injection.
도 10d는 21일이 경과한 후, 전체 마우스 중에서 하지허혈이 치료된 마우스의 비율을 나타내는 그래프이다.10D is a graph showing the percentage of mice treated with lower limb ischemia after 21 days have elapsed.
도 11은 피부 창상 마우스 모델을 이용한 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT)의 상처치료효과를 비교한 결과를 나타내는 사진 및 그래프로서, 11 is a photograph and graph showing the results of comparing the wound treatment effect of each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) using the skin wound mouse model,
a는 제작된 피부 창상 마우스 모델을 나타내는 사진이다.a is a photograph showing the skin wound mouse model produced.
b는 각 실험군의 상처부위를 시간의 경과에 따라 촬영한 사진이다.b is a photograph of the wound area of each experimental group over time.
c는 시간의 경과에 따른 상처부위의 면적 변화를 나타내는 그래프이다.c is a graph showing the change of the area of the wound area over time.
도 12는 피부 창상 마우스 모델에서 LED 조사한 스페로이드 이식 및 LED 처리 조합의 혈관재생 효과를 확인한 사진 및 그래프로서, 12 is a photograph and a graph confirming the blood vessel regeneration effect of the spheroid implantation and LED treatment combination of the LED irradiation in the skin wound mouse model,
a는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관내피세포 및 혈관외피세포의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 녹색은 혈관외피세포의 마커인 SMAa 액틴을 나타내고, 적색은 혈관내피세포 마커인 CD31을 나타낸 사진이다.a is an immunofluorescence staining photograph showing the results of comparing the levels of vascular endothelial cells and vascular epithelial cells in the wounded skin of a mouse model according to the treatment conditions (control, ASCs, LLLT, spheroid and spheroid + LLLT). , Green indicates SMAa actin, a marker of vascular endothelial cells, and red shows a photograph showing CD31, a vascular endothelial cell marker.
b는 피부 창상 마우스 모델의 상처부위에 존재하는 단위면적당 미세혈관의 수를, 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 그래프이다.b is a graph showing the results of comparing the number of microvascular vessels per unit area of the wounded mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
c는 피부 창상 마우스 모델의 상처부위에 존재하는 단위면적당 소동맥의 수를, 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 그래프이다.c is a graph showing the result of comparing the number of small arteries per unit area in the wounded part of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
도 13은 피부 창상 마우스 모델의 상처부위에 존재하는 피부세포의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 적색은 피부세포 마커인 사이토케라틴을 나타낸다.FIG. 13 is an immunofluorescence staining photograph showing the result of comparing the level of skin cells present in the wound area of the skin wound mouse model according to each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT), and red is skin. Cytokeratin, a cell marker.
도 14는 피부 창상 마우스 모델에서 LED 조사한 스페로이드 이식 및 LED 처리 조합의 혈관신생 및 피부재생 인자 발현량 증가 효과를 확인한 사진 및 그래프로서, FIG. 14 is a photograph and a graph confirming an effect of increasing angiogenesis and skin regeneration factor expression of a combination of LED irradiation and spheroid transplantation and LED treatment in a skin wound mouse model.
a는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관신생 및 피부재생인자의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 적색은 각각의 혈관신생 및 피부재생인자인 FGF, VEGF 및 HGF를 나타낸다.a is an immunofluorescence staining photograph showing the results of comparing the levels of angiogenesis and skin regeneration factors in the wounded model of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). Red color represents FGF, VEGF and HGF, respectively, angiogenesis and skin regeneration factors.
b는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관신생 및 피부재생인자의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 웨스턴 블럿 분석에 의해 측정한 결과를 나타내는 사진이다.b is a photograph showing the result of Western blot analysis of the levels of angiogenesis and skin regeneration factors in the wounded area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) to be.
c는 상기 웨스턴 블럿 분석에 의해 검출된 각 혈관신생 및 피부재생인자의 수준을 각각의 처리조건별로 비교한 결과를 나타내는 그래프이다.c is a graph showing the result of comparing the level of each angiogenesis and skin regeneration factor detected by the Western blot analysis for each treatment condition.
도 15는 skin flap 마우스 모델을 이용한 상처치료효과를 비교한 결과를 나타내는 사진이다.15 is a photograph showing the results of comparing the wound treatment effect using the skin flap mouse model.
도 16은 skin flap 마우스 모델에서 촬영된 피부혈류의 흐름을 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로, 1, 7 및 14일이 경과된 시점에서 비교한 결과를 나타내는 사진이다.16 is a photograph showing the results of comparing the flow of skin blood taken in the skin flap mouse model at the time of 1, 7 and 14 days for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). .
본 발명자들은 보다 효과적으로 피부궤양 및 상처를 치료할 수 있는 방법을 개발하고자 다양한 연구를 수행하던 중, LED 광원을 이용하여 빛을 조사하는 방법에 주목하게 되었다. 즉, LED 광원에서 생성된 적색 파장대의 빛을 피부궤양 및 상처 환부에 조사하면 환자의 자가 회복력을 증진시켜서 피부궤양 및 상처를 치료할 수 있다. 다만, 생체내의 자가 회복력을 증진시키는 것이기 때문에 치료에 많은 시간이 소요된다는 단점이 있는데, 이러한 단점을 극복하기 위하여, 줄기세포를 이용하는 방안을 연구한 결과, 줄기세포가 아닌 줄기세포로부터 형성된 스페로이드를 이용할 경우, 상기 적색 파장대의 빛을 이용한 치료법의 단점인 시간의 과다한 소요라는 점을 해소하여, 보다 신속하게 피부궤양 및 상처를 치료할 수 있음을 확인하였다. 구체적으로, 줄기세포에서 생성된 스페로이드를 생체에 이식하고, 이식된 부위에 지속적으로 적색 파장대의 빛을 조사하면, 상기 스페로이드로부터 피부궤양 및 상처를 치료를 촉진할 수 있는 다양한 혈관생성 및 피부재생인자(FGF, VEGF, HGF 등)의 발현 및 분비가 촉진되어, 상기 스페로이드가 혈관재생 및 피부재생이 촉진될 수 있다. The present inventors came to pay attention to a method of irradiating light using an LED light source while conducting various studies to develop a method for more effectively treating skin ulcers and wounds. That is, when the light of the red wavelength band generated by the LED light source is irradiated to the skin ulcer and wound affected area, it is possible to treat the skin ulcer and the wound by enhancing the self-healing ability of the patient. However, there is a disadvantage in that it takes a lot of time for treatment because it enhances the self-recovery ability in vivo. To overcome these disadvantages, studies on the use of stem cells revealed that spheroids formed from stem cells rather than stem cells were studied. When used, it was confirmed that it is possible to cure skin ulcers and wounds more quickly by eliminating the excessive time required, which is a disadvantage of the treatment using light in the red wavelength band. Specifically, when the spheroids generated from stem cells are implanted into a living body and continuously irradiated with light in the red wavelength band, various angiogenesis and skin that can promote the treatment of skin ulcers and wounds from the spheroids Expression and secretion of regenerative factors (FGF, VEGF, HGF, etc.) are promoted, so that the spheroid can promote blood vessel regeneration and skin regeneration.
따라서, 지방줄기세포로부터 형성된 스페로이드는 피부궤양 및 상처를 치료하기 위한 적색 파장대의 빛을 이용한 피부궤양 및 상처 치료법을 보조할 수 있는 약학 조성물의 유효성분으로서 사용될 수 있다.Therefore, the spheroid formed from the adipose stem cells can be used as an active ingredient of the pharmaceutical composition that can assist in the treatment of skin ulcers and wounds using light in the red wavelength band for treating skin ulcers and wounds.
상기 목적을 달성하기 위해 본 발명의 하나의 양태는 지방줄기세포로부터 유래된 스페로이드를 유효성분으로 포함하는, 적색 파장대의 빛을 이용한 피부 또는 혈관 조직 손상 치료용 약학 조성물을 제공한다.One aspect of the present invention to achieve the above object provides a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength band, including a spheroid derived from adipose stem cells as an active ingredient.
본 발명의 용어 "지방줄기세포(adipose-derived stem cells; ASCs)"란, 지방조직(adipose tissue)에 존재하는 자가 재생능력이 뛰어나고, 간, 골, 연골, 지방, 혈관, 심장, 신경계 등과 같은 다양한 세포로 분화될 수 있는 미분화된 성체줄기세포를 의미한다. 상기 지방줄기세포는 중간엽 줄기세포와 비교하여 지방조직을 대량으로 추출할 수 있다는 점에서 취득과정이 용이하고 안전하며, 조직수급상의 제한이 없는 장점과 체외배양이 용이하여 조직 접근성, 안정성, 유효성, 그리고 경제적 측면에서 장점이 있다. 본 발명에서 지방줄기세포는 스페로이드(spheroid)를 형성할 수 있다. The term "adipose-derived stem cells (ASCs)" of the present invention is excellent in the self-renewal capacity present in adipose tissue, liver, bone, cartilage, fat, blood vessels, heart, nervous system, etc. It refers to undifferentiated adult stem cells that can differentiate into various cells. Compared with the mesenchymal stem cells, the adipose stem cells are easily and securely obtained in that they can extract a large amount of adipose tissue. And there are advantages in terms of economy. Adipose stem cells in the present invention may form a spheroid (spheroid).
본 발명의 용어 "스페로이드(spheroid)"란, 3차원 세포집합체(3D cell mass, 3DCM)라고 알려진 세포괴로서, 하나의 개체가 분열하여 생긴 다수의 딸개체가 모여 형성된 구형 군체를 의미한다. 본 발명에서 상기 스페로이드는 지방줄기세포를 배양하여 형성될 수 있으며, 구체적으로, 지방줄기세포에 적색 파장대의 빛을 조사하여 형성될 수 있다.As used herein, the term "spheroid" refers to a cell mass known as a 3D cell mass (3DCM), and refers to a spherical colony formed by gathering a plurality of daughters formed by division of an individual. In the present invention, the spheroid may be formed by culturing adipose stem cells, specifically, may be formed by irradiating the red wavelength band of light to the adipose stem cells.
구체적으로, 본 발명에서 상기 스페로이드의 형성을 위해, 지방줄기세포의 배양시 정지배양, 진탕배양 등의 방법을 제한없이 사용할 수 있으며, 저산소 환경에서 배양시 더 효과적으로 스페로이드를 형성할 수 있다. 또한, 상기 스페로이드는 배양시간의 경과에 따라 그의 직경이 지속적으로 증가하는데, 직경이 1mm 이상으로 커지면, 내부적으로 세포괴사가 발생하여 이를 활용할 수 없는 문제점이 있다. 또한, 상기 스페로이드의 직경이 적어도 500 ㎛ 이상일 때 스페로이드 내부가 저산소 환경이 만들어지고 저산소 환경일 때 발현하는 HIF-1a 단백질이 발현한다. HIF-1a 시그널에 의해 혈관생성 및 피부재생인자(FGF, VEGF, HGF 등)의 발현 및 분비가 촉진되어 지는데 저산소 환경에서 배양시 더 효과적이다. 또한 외부에서 혈관생성 및 피부재생인자(FGF, VEGF, HGF 등)의 발현 및 분비가 촉진할 수 있는 외부적인 자극(광조사)을 가하면 더욱 효과적일 수 있다. 따라서, 적절한 스페로이드의 직경은 0.5 내지 1mm가 될 수 있다. 그러나, 본 발명에서 피부 또는 혈관 조직 손상을 치료할 수 있다면, 상기 직경범위로 인하여 스페로이드의 사용이 제한되지는 않는다.Specifically, for the formation of the spheroid in the present invention, a method such as stop culture, shaking culture, etc. can be used without limitation when culturing fat stem cells, it is possible to form a spheroid more effectively when cultured in a hypoxic environment. In addition, the spheroid has a problem that its diameter continuously increases with the incubation time, if the diameter is larger than 1mm, cell necrosis occurs internally and there is a problem that cannot be utilized. In addition, when the diameter of the spheroid is at least 500 μm or more, the inside of the spheroid creates a hypoxic environment, and the HIF-1a protein expressed when the hypoxic environment is expressed. The expression and secretion of angiogenesis and skin regeneration factors (FGF, VEGF, HGF, etc.) are promoted by HIF-1a signal, which is more effective when cultured in hypoxic environment. In addition, it may be more effective if external stimulation (light irradiation) can be promoted by external expression and secretion of angiogenesis and skin regeneration factors (FGF, VEGF, HGF, etc.). Thus, the diameter of a suitable spheroid can be 0.5 to 1 mm. However, if the present invention can treat skin or vascular tissue damage, the use of spheroid is not limited due to the diameter range.
구체적으로, 본 발명에서 상기 스페로이드의 형성을 위해, 소수성을 부여하는 고분자로 표면 처리된 배양용기 또는 상기 고분자로 제조된 배양용기에 접종하여 배양할 수 있다. 상기 소수성을 부여하는 고분자는 특별히 제한되지 않는다. Specifically, in order to form the spheroid in the present invention, it can be cultured by inoculating a culture vessel surface-treated with a polymer that gives hydrophobicity or a culture vessel made of the polymer. The polymer for imparting the hydrophobicity is not particularly limited.
상기 소수성을 부여하는 고분자는 특별히 이에 제한되지 않으나, 구체적으로는 폴리스티렌(polystyrene), 폴리메틸메타크릴레이트(PMMA), 폴리에틸렌 테레프탈레이트(PET), 폴리비닐클로라이트(PVC), 폴리에틸렌(PE), 폴리프로필렌(PP), 폴리테트라플루오르에틸렌(PTFE), 지방족 폴리에스테르계 고분자로서, 폴리(L-락트산)(PLLA), 폴리(D,L-락트산）(PDLLA), 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸렌카보네이트, 이들의 유도체, 이들의 공중합체 (폴리(락트산-co-글리콜산)(PLGA), 폴리(L-락트산-co-카프로락톤)(PLCL), 폴리(글리콜산-co-카프로락톤)(PGCL) 등) 또는 이들의 조합 등을 사용할 수 있다.The polymer for imparting hydrophobicity is not particularly limited thereto, and specifically, polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chlorite (PVC), polyethylene (PE), Polypropylene (PP), polytetrafluoroethylene (PTFE), aliphatic polyester-based polymer, poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA ), Poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives thereof, copolymers thereof (poly (lactic acid-co-glycolic acid) (PLGA), poly (L-lactic acid-co-caprolactone) (PLCL), poly (glycolic acid-co-caprolactone) (PGCL), or the like, or a combination thereof.
구체적으로, 본 발명에서 상기 스페로이드의 형성을 위해 사용되는 배양용액은 지방줄기세포의 배양 및/또는 분화에 통상적으로 사용되는 배지라면 제한되지 않고 사용할 수 있고, 구체적으로는 DMEM(Dulbeco's modified eagle medium), Ham's F12 등에 혈청이 포함된 배지를 사용할 수 있으나 혈청이 첨가되지 않은 배지에서 사용하여도 무방하다. 예를 들어, 본 발명의 실시예에서는 DMEM과 Ham's F12가 1:1 비율로 혼합된 DMEM/F12 배지에 우 태아 혈청(fetal bovine serum, FBS)이 첨가된 배지를 사용하였다.Specifically, the culture solution used for the formation of the spheroid in the present invention can be used without limitation as long as it is a medium commonly used for the culture and / or differentiation of fat stem cells, specifically DMEM (Dulbeco's modified eagle medium ), Ham's F12 or the like may be used in a medium containing serum, but may be used in a medium without serum. For example, in the embodiment of the present invention, a medium in which fetal bovine serum (FBS) was added to DMEM / F12 medium in which DMEM and Ham's F12 were mixed at a 1: 1 ratio was used.
구체적으로, 본 발명에서 지방줄기세포에 적색 파장대의 빛을 조사하여 형성된 스페로이드는 혈관내피세포의 특징을 가질 수 있다. 구체적으로, 상기 빛은 LED(light emitting diode)로부터 발생된 빛을 사용할 수 있고, 더욱 구체적으로는 LED로부터 발생되어 600 내지 700nm의 파장 및 1 내지 30W의 전력를 나타내는 빛을 사용할 수 있으며, 더욱더 구체적으로는 LED로부터 발생되어 660nm의 파장 및 5W의 전력을 나타내는 빛을 사용할 수 있다.Specifically, in the present invention, the spheroid formed by irradiating fat stem cells with red wavelength band light may have characteristics of vascular endothelial cells. Specifically, the light may use light generated from a light emitting diode (LED), and more specifically, may use light indicating a wavelength of 600 to 700 nm and a power of 1 to 30 W generated from the LED. Can be used to generate light from the LEDs representing a wavelength of 660 nm and a power of 5 W.
본 발명에서 "LED 조사"는 적색 파장대의 빛을 조사하는 것을 의미한다.In the present invention, "LED irradiation" means to irradiate light of the red wavelength band.
본 발명에 있어서, 상기 스페로이드는 상기 지방줄기세포에 적색 파장대의 빛을 조사하여 형성될 수 있으며, 상기 스페로이드를 피부 또는 혈관 조직 손상 부위에 이식한 후, 적색 파장대의 빛을 조사하면 생체내에서 혈관내피세포 또는 피부세포 등으로 분화되어, 상기 적색 파장 대의 빛에 의하여 촉진되는 생체내 자가 회복력을 보조할 수 있다. In the present invention, the spheroid can be formed by irradiating the red wavelength band light to the adipose stem cells, and after implanting the spheroid to the skin or vascular tissue damage site, and irradiated with the red wavelength band light in vivo Differentiated into vascular endothelial cells or skin cells, and can assist in vivo self-healing ability promoted by light of the red wavelength band.
따라서, 상기 스페로이드를 적색 파장대의 빛을 이용한 피부 또는 혈관 조직 손상 치료용 약학 조성물의 유효성분으로 사용할 수 있으며, 상기 유효성분으로는 상기 스페로이드 뿐만 아니라, 스페로이드 배양액, 스페로이드 세포 파쇄물 등을 사용할 수도 있다. 이처럼 상기 스페로이드를 적색 파장대의 빛을 이용한 피부 또는 혈관 조직 손상 치료용 약학 조성물의 유효성분으로 사용하는 기술은 지금까지 전혀 알려지지 않았으며, 본 발명자들에 의하여 최초로 개발되었다.Therefore, the spheroid may be used as an active ingredient of a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength band, and as the active ingredient, not only the spheroid, but also a spheroid culture solution and a spheroid cell debris Can also be used. As such, a technique of using the spheroid as an active ingredient of a pharmaceutical composition for treating skin or vascular tissue damage using light in the red wavelength range is not known at all, and was first developed by the present inventors.
본 발명에서 제공하는 스페로이드는 생체내에 이식된 후, 적색 파장대의 빛이 조사되면, 혈관생성 및 피부재생인자(FGF, VEGF, HGF 등)의 발현 및 분비가 촉진되어, 결과적으로는 혈관재생 및 피부세포 분화를 촉진하여 최종적으로 피부괴사를 막고 피부재생을 촉진시킬 수 있다. When the spheroid provided by the present invention is implanted in vivo, when the red wavelength band of light is irradiated, angiogenesis and expression and secretion of skin regeneration factors (FGF, VEGF, HGF, etc.) are promoted, resulting in vascular regeneration and It can promote skin cell differentiation and finally prevent skin necrosis and promote skin regeneration.
이때, 상기 적색 파장대의 빛은 특별히 이에 제한되지 않으나, 구체적으로는 LED(light emitting diode)로부터 발생된 빛을 사용할 수 있고, 보다 구체적으로는 LED로부터 발생되어 600 내지 700nm의 파장 및 1 내지 30W의 전력를 나타내는 빛을 사용할 수 있으며, 가장 구체적으로는 LED로부터 발생되어 660nm의 파장 및 5W의 전력을 나타내는 빛을 사용할 수 있다.At this time, the light of the red wavelength range is not particularly limited thereto, but in particular, light generated from a light emitting diode (LED) may be used. More specifically, the light of the red wavelength band may be generated from the LED to have a wavelength of 600 to 700 nm and 1 to 30 W. Light representing power can be used, and most specifically, light generated from the LED can be used to represent the wavelength of 660nm and the power of 5W.
아울러, 상기 빛의 조사조건 역시 지방줄기세포를 혈관내피세포로 분화시키는 것을 촉진시키는 효과를 나타내는 한 특별히 이에 제한되지 않으나, 구체적으로는 이식된 부위로부터 1 내지 5cm의 간격을 두고 위치한 LED 광원을 이용하여 1일 10 내지 15분 동안 조사하는 방법을 사용할 수 있다.In addition, the irradiation conditions of the light is not particularly limited as long as it shows an effect of promoting the differentiation of fat stem cells into vascular endothelial cells, specifically, using an LED light source placed at intervals of 1 to 5cm from the implanted site To irradiate for 10 to 15 minutes per day.
아울러, 상기 지방줄기세포로부터 스페로이드를 용이하게 형성하기 위하여는, 상기 지방줄기세포를 1 × 105 내지 2 × 106 세포/㎠의 밀도로 접종함이 바람직하다. 1 × 105 세포/㎠ 미만의 밀도로 접종할 경우에는 스페로이드의 크기가 작고, 편차가 심하다는 문제점이 있고, 2 × 106 세포/㎠ 이상의 밀도로 접종할 경우에는 과다한 세포증식에 의한 세포사가 유발될 수 있다. 그러나, 1 × 105 내지 2 × 106 세포/㎠ 이상의 밀도로 접종할 경우에는, 육안으로 검출가능한 0.5 내지 1 ㎜의 직경을 갖는 스페로이드를 형성할 수 있다.In addition, in order to easily form a spheroid from the adipose stem cells, it is preferable to inoculate the adipose stem cells at a density of 1 × 10 5 to 2 × 10 6 cells / ㎠. When inoculating at a density of less than 1 × 10 5 cells / cm 2, there is a problem that the size of the spheroid is small and the variation is severe. When inoculating at a density of 2 × 10 6 cells / cm 2 or more, cell death due to excessive cell proliferation May be induced. However, when inoculated at a density of 1 × 10 5 to 2 × 10 6 cells / cm 2 or more, a spheroid having a diameter of 0.5 to 1 mm detectable with the naked eye can be formed.
한편, 본 발명의 약학 조성물은 살아있는 스페로이드를 유효성분으로 포함하므로, 세포치료제의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 예를 들어, 멸균수, 생리식염수, 관용의 완충제(인산, 구연산, 그 밖의 유기산 등), 안정제, 염, 산화방지제(아스코르브산 등), 계면활성제, 현탁제, 등장화제, 또는 보존제 등을 포함할 수 있다. 국소 투여를 위하여는, 바이오 폴리머 등의 유기물, 히드록시아파타이트 등의 무기물, 구체적으로는 콜라겐 매트릭스, 폴리락트산 폴리머 또는 코폴리머, 폴리에틸렌글리콜 폴리머 또는 코폴리머 및 그의 화학적 유도체, 펩타이드 단백질, 생체외 기질 단백질 (ECM), 겔 (gel) 등과 조합시켜서 제형화 할 수도 있다. 상기 스페로이드를 유지하기 위한 기제로는 생리식염수, PBS(phosphate buffered saline) 등을 사용할 수 있다. On the other hand, since the pharmaceutical composition of the present invention contains a live spheroid as an active ingredient, it may further include a suitable carrier, excipient or diluent commonly used in the preparation of cell therapy. Examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, organics such as biopolymers, inorganics such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, peptide proteins, ex vivo matrix proteins It may also be formulated in combination with (ECM), gel, and the like. As a base for maintaining the spheroid, physiological saline, PBS (phosphate buffered saline) may be used.
본 발명의 약학 조성물은 주사에 적당한 제형으로 조제됨이 바람직하고, 이를 위해서는, 상기 스페로이드를 약학적으로 허용되는 수용액 중에 용해되어 있거나 또는 용액상태에서 동결된 형태로 유지함이 바람직하다. 상기 조성물에는 스페로이드를 현탁 또는 희석하기 위해 사용할 수 있는, 약학적으로 허용할 수 있는 목적으로 하는 담체를 추가로 포함할 수 있다. 이러한 담체로서는, 예를 들면 증류수, 생리식염수, PBS 등을 들 수 있다.The pharmaceutical composition of the present invention is preferably prepared in a dosage form suitable for injection, and for this purpose, the spheroid is preferably dissolved in a pharmaceutically acceptable aqueous solution or kept in frozen form in solution. The composition may further comprise a pharmaceutically acceptable carrier which can be used to suspend or dilute the spheroid. As such a carrier, distilled water, physiological saline, PBS, etc. are mentioned, for example.
의약제제로서의 형태를 취하기 위해서 필요한 제제담체나 부형제를, 나아가서는 안정화제나 흡착방지제를 함유할 수 있고, 제형도 주사제 (피하, 피내, 근육 내, 정맥 내, 복강 내 등)등을 사용할 수 있으며, 주사 시 통증을 감소시킬 수 있는 무통화제를 사용할 수 있고, 필요에 따라 적당한 디바이스를 사용할 수 있다.Formulation carriers or excipients necessary to take the form of pharmaceutical preparations may further contain stabilizers or anti-adsorption agents, and formulations may be used as injections (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, etc.), Pain-free agents can be used that can reduce pain upon injection, and suitable devices can be used as needed.
구체적인 제형으로서, 주사기나 디바이스에 담겨진 최종 주입 형태, 냉동이 가능한 크라이오바이알(cryovial)의 형태, 또는 액상의약품을 담을 수 있는 파이로젠이 없는 유리병과 고무전, 알루미늄 캡형태로 충진될 수 있다.As a specific formulation, it may be filled in the form of a final injection in a syringe or device, in the form of a cryoial that can be frozen, or in the form of a pyrogen-free glass bottle and rubber field, which can contain liquid medicine, and an aluminum cap.
디바이스의 형태로는 주사기, 멀티실린지 등이 사용될 수 있으며 사지허혈성 질환의 경우에는 세포가 투여되는 동안 세포가 shear 되어 손상을 입히지 않으면서 고통을 최소화 할 수 있는 주사바늘(needle)을 이용하며 구체적으로는 20guage에서 31guage 범위에서 투여할 부위나 근육의 깊이를 고려하여 사용하며, 실린지나 디바이스가 세포 생존력에 영향을 미치지 않는 소재를 사용함이 바람직하다.In the form of a device, a syringe, a multi-syringe, and the like may be used. In the case of limb ischemic disease, a needle is used to minimize pain without causing damage by shearing the cell while the cell is administered. In consideration of the depth of the site or muscle to be administered from 20guage to 31guage range, it is preferable to use a material that does not affect the cell viability of the syringe or device.
본 발명의 약학 조성물에 포함된 상기 스페로이드의 함량은 특별히 이에 제한되지 않으나, 최종 조성물 총중량을 기준으로 10 내지 50 중량%, 보다 구체적으로는 20 내지 40 중량%의 함량으로 포함될 수 있다. The content of the spheroid included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in an amount of 10 to 50% by weight, more specifically 20 to 40% by weight based on the total weight of the final composition.
상기 본 발명의 약학 조성물은 약제학적으로 유효한 양으로 투여될 수 있는데, 본 발명의 용어 "약제학적으로 유효한 양"이란 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적으로 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention Sufficient amount means an effective dose level means the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of excretion treatment Period of time, factors including drugs used in combination or coincidental with the compositions of the invention used, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.
본 발명의 약학 조성물의 투여량은 예를 들어, 혈관손상부위 주변의 생존근(골격근 또는 심근 등)에 1개소 또는 복수 개소(예를 들면 2~50개소)에 투여할 수 있으며, 투여량은 구체적으로는 1.0 × 105 내지 1.0 × 108 세포수/kg(체중), 보다 구체적으로는 1.0 × 106 내지 1.0 × 107 세포수/kg(체중)이 될 수 있다.The dosage of the pharmaceutical composition of the present invention may be administered to one or a plurality of locations (for example, 2 to 50 locations) to the living muscles (skeletal muscle or myocardium) around the vascular injury site, for example, and the dosage is Specifically, the number may be 1.0 × 10 5 to 1.0 × 10 8 cells / kg (body weight), and more specifically 1.0 × 10 6 to 1.0 × 10 7 cells / kg (body weight).
본 발명의 다른 하나의 양태는 상기 약학 조성물을 피부 또는 혈관 조직이 손상된 개체에 투여하는 단계를 포함하는, 피부 또는 혈관 조직 손상의 치료방법을 제공한다. Another aspect of the invention provides a method of treating skin or vascular tissue damage, comprising administering the pharmaceutical composition to an individual with damaged skin or vascular tissue.
또한, 상기 치료방법은 적색 파장대의 빛을 상기 개체에 조사하는 단계를 추가로 포함할 수 있다.In addition, the treatment method may further comprise the step of irradiating the subject with light in the red wavelength band.
본 발명의 약학 조성물은 그 자체로서도 혈관손상, 피부궤양 또는 상처를 치료할 수 있으나, 적색 파장대의 빛이 조사될 경우에는 혈관내피세포 및 피부세포로의 분화가 촉진되어 피부 또는 혈관 조직 손상을 효과적으로 치료할 수 있다.The pharmaceutical composition of the present invention can treat vascular damage, skin ulcers or wounds by itself, but when light of the red wavelength band is irradiated, the differentiation into vascular endothelial cells and skin cells can be promoted to effectively treat skin or vascular tissue damage. Can be.
본 발명에서 용어 "개체"란 혈관손상, 피부 또는 혈관 조직 손상이 발생된 쥐, 가축, 인간 등을 포함하는 포유동물을 제한 없이 포함한다. 이때, 상기 피부 또는 혈관 조직 손상이 유발되는 질환은 특별히 이에 제한되지 않으나, 구체적으로 심혈관 질환, 뇌혈관 질환, 허혈성 질환 등이 될 수 있고, 예를 들어 허혈성 피부 궤양, 피부상처, 동맥경화증, 안정형 및 불안정형 협심증, 말초 심혈관 질환, 고혈압, 심부전증, 말초 순환장애, 심근경색증, 뇌졸중, 일과성 및 허혈성 발작, 지주막하 출혈, 또는 하지허혈 등의 질환이 될 수 있다.As used herein, the term "individual" includes without limitation mammals including rats, livestock, humans, and the like, in which vascular damage, skin or vascular tissue damage has occurred. At this time, the disease that causes the skin or vascular tissue damage is not particularly limited, but specifically, may be a cardiovascular disease, cerebrovascular disease, ischemic diseases, for example, ischemic skin ulcers, skin wounds, arteriosclerosis, stable And unstable angina, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulatory disorders, myocardial infarction, stroke, transient and ischemic attacks, subarachnoid hemorrhage, or lower limb ischemia.
본 발명의 피부 또는 혈관 조직 손상을 치료하는 방법에 있어서, 상기 약학 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여 또는 피내 투여될 수 있다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. In the method of treating skin or vascular tissue damage of the present invention, the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously or intradermally, as desired. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.
본 발명의 다른 하나의 양태는 지방줄기세포에 적색 파장대의 빛을 조사하여 지방줄기세포를 혈관내피세포로 분화시키는 방법을 제공한다.Another embodiment of the present invention provides a method of differentiating adipose stem cells into vascular endothelial cells by irradiating adipose stem cells with light of a red wavelength band.
줄기세포를 배양하면서 LED 광원을 이용하여 적색 파장대의 빛을 조사하면, 줄기세포의 증식 및 이동성이 향상되어 스페로이드의 형성이 촉진되고, 상기 형성된 스페로이드에 지속적으로 적색 파장대의 빛을 조사하면, 혈관내피세포로의 분화를 촉진할 수 있는 다양한 성장인자(FGF, VEGF, HGF 등)의 발현 및 분비가 상기 스페로이드를 형성하는 줄기세포 자체에서 촉진되어, 외부에서 혈관생성 관련인자의 첨가 또는 유전자 조작 없이도 상기 스페로이드가 혈관내피세포로 분화되는 것이 촉진된다. When culturing stem cells and irradiating light with a red wavelength band using an LED light source, the proliferation and mobility of stem cells are enhanced to promote spheroid formation, and when the formed spheroid is continuously irradiated with red wavelength band light, Expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) that can promote differentiation into vascular endothelial cells are promoted in the stem cells themselves to form the spheroid, so that angiogenesis related factors or genes are externally added. The differentiation of the spheroids into vascular endothelial cells is facilitated without manipulation.
한편, 상기 적색 파장대의 빛을 조사하여 지방줄기세포를 혈관내피세포로 분화시키는 분화효율을 향상시키기 위한 조건을 연구한 결과, 600 내지 700nm의 파장 및 5 내지 30 mW/㎠의 전력을 나타내는 빛(에너지량 3 내지 18 J/㎠)을 조사하고, 지방유래 중간엽 줄기세포의 배양용기로는 세포 배양용기에 소수성을 부여하는 고분자로 표면 처리되거나 또는 소수성을 갖는 세포 배양용기(non-tissue culture-treated well plate)를 사용하며, 상기 배양용기에 지방줄기세포를 24웰 플레이트의 각 웰에 1 × 105 세포 이상의 지방줄기세포를 접종함이 바람직함을 확인하였다On the other hand, as a result of studying the conditions for improving the differentiation efficiency of differentiating adipose stem cells into vascular endothelial cells by irradiating light of the red wavelength band, light indicating a wavelength of 600 to 700nm and a power of 5 to 30 mW / ㎠ 3 to 18 J / cm 2) of energy, and the culture vessel of adipose-derived mesenchymal stem cells was treated with a polymer that gives hydrophobicity to the cell culture vessel, or the cell culture vessel having a hydrophobicity (non-tissue culture- treated well plate), and it was confirmed that it is preferable to inoculate the adipose stem cells into the culture vessel and inject the adipose stem cells of 1 × 10 5 cells or more into each well of a 24-well plate.
구체적으로, 본 발명에서 제공하는 지방줄기세포를 혈관내피세포로 분화시키는 방법은 지방줄기세포를 표면이 소수성을 띠는 배양용기에 접종하고 배양하면서, 5 내지 15 mW/㎠의 전력 및 600 내지 700nm의 빛을 상기 지방줄기세포에 조사하는 단계를 포함한다.Specifically, the method of differentiating adipose stem cells provided by the present invention into vascular endothelial cells is inoculated and cultured in a culture vessel having a surface hydrophobic adipose stem cells, power of 5 to 15 mW / ㎠ and 600 to 700nm Irradiating light to the adipose stem cells.
본 발명의 용어 "중간엽 줄기세포(mesenchymal stromal cells; MSCs)"란, 자가 재생능력이 뛰어나고, 간, 골, 연골, 지방, 혈관, 심장, 신경계 등과 같은 다양한 세포로 분화될 수 있는 미분화된 성체줄기세포를 의미한다. The term "mesenchymal stromal cells (MSCs)" of the present invention is an undifferentiated adult that has excellent self-renewal ability and can be differentiated into various cells such as liver, bone, cartilage, fat, blood vessels, heart, nervous system, and the like. Means stem cells.
본 발명에 있어서, 상기 중간엽 줄기세포로서 지방유래 중간엽 줄기세포(adipose-derived stem cells; ASCs)를 사용하였다. 상기 지방유래 중간엽 줄기세포는 다른 종류의 중간엽 줄기세포와 비교하여 대량으로 추출할 수 있는 지방조직으로부터 수득할 수 있다는 점에서 취득과정이 용이하고 안전하며, 조직수급상의 제한이 없는 장점과 체외배양이 용이하여 조직 접근성, 안정성, 유효성, 그리고 경제적 측면에서 장점이 있다.In the present invention, adipose-derived stem cells (ASCs) were used as the mesenchymal stem cells. The adipose-derived mesenchymal stem cells can be obtained from adipose tissue which can be extracted in large quantities compared to other types of mesenchymal stem cells, so that the acquisition process is easy and safe, and there is no restriction on tissue supply and in vitro. Easy cultivation has advantages in tissue access, stability, efficacy and economics.
본 발명에서 제공하는 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 방법은 상기 지방유래 중간엽 줄기세포에 적색 파장대의 빛을 조사하는 단계를 필수적으로 포함하는데, 상기 지방세포에 조사된 빛은 줄기세포의 증식 및 이동을 촉진시켜서, 스페로이드(spheroid) 또는 3차원 세포집합체(3D cell mass, 3DCM)라고 알려진 세포괴의 형성을 촉진시킬 수 있고, 상기 스페로이드에 상기 빛을 지속적으로 조사하면, 상기 스페로이드를 형성하는 줄기세포로부터 혈관내피세포로의 분화를 촉진할 수 있는 다양한 성장인자(FGF, VEGF, HGF 등)의 발현 및 분비가 촉진되어, 스페로이드의 내부에 축적되거나 또는 스페로이드의 외부로 분비되고, 상기 성장인자는 줄기세포 분화에 관여하는 분화 신호전달을 활성화시켜서, 혈관내피세포로의 상기 스페로이드의 분화를 촉진시킬 수 있다. The method for differentiating adipose derived mesenchymal stem cells provided by the present invention into vascular endothelial cells essentially includes irradiating the adipose derived mesenchymal stem cells with light in the red wavelength band. By promoting the proliferation and migration of stem cells, it is possible to promote the formation of a cell mass known as a spheroid or 3D cell mass (3DCM), the continuous irradiation of the light on the spheroid, Expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) that can promote the differentiation of the spheroid-forming stem cells into vascular endothelial cells are promoted to accumulate inside the spheroid or Secreted outwardly, the growth factor activates differentiation signaling involved in stem cell differentiation, thereby secreting the spheroid into vascular endothelial cells. Can promote anger.
이때, 상기 빛은 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 것을 촉진시키는 효과를 나타내는 한 특별히 이에 제한되지 않으나, 구체적으로는 LED(light emitting diode)로부터 발생된 빛을 사용할 수 있고, 보다 구체적으로는 LED로부터 발생되어 600 내지 700nm의 파장 및 5 내지 30 mW/㎠의 전력을 나타내는 빛을 사용할 수 있으며, 가장 구체적으로는 LED로부터 발생되어 660nm의 파장 및 10 mW/㎠의 전력을 나타내는 빛을 사용할 수 있다.In this case, the light is not particularly limited as long as it exhibits an effect of promoting differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, but specifically, light generated from a light emitting diode (LED) may be used. Specifically, light generated from the LED can be used to represent the wavelength of 600 to 700nm and power of 5 to 30 mW / ㎠, most specifically light generated from the LED to represent a wavelength of 660nm and power of 10 mW / ㎠ Can be used.
아울러, 상기 빛의 조사조건 역시 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 것을 촉진시키는 효과를 나타내는 한 특별히 이에 제한되지 않으나, 구체적으로는 지방유래 중간엽 줄기세포로부터 1 내지 5cm의 간격을 두고 위치한 LED 광원을 이용하여 1일 10 내지 15분 동안 조사하는 방법을 사용할 수 있다.In addition, the irradiation conditions of the light is not particularly limited as long as it shows an effect of promoting the differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, specifically, the spacing of 1 to 5 cm from the adipose derived mesenchymal stem cells The method can be used to irradiate for 10 to 15 minutes a day using the LED light source placed.
또한, 상기 지방줄기세포를 배양하는 배양용액 또는 배양 메트릭스에 혈관내피세포로 줄기세포의 분화를 유도하는 물질을 처리하거나 유전자 변형을 하지 않고, 적색 파장대의 빛을 조사하여 스페로이드 형성 및 혈관생성인자 발현을 유도하는 것만으로도 줄기세포를 혈관내피세포로 분화시킬 수 있다. 이때, 사용되는 배양용액은 지방줄기세포의 배양 및/또는 분화에 통상적으로 사용되는 배지라면 제한되지 않고 사용할 수 있고, 구체적으로는 DMEM (Dulbeco's modified eagle medium), Ham's F12 등에 혈청이 포함된 배지를 사용할 수 있다. 예를 들어, 본 발명의 실시예에서는 DMEM과 Ham's F12가 1:1 비율로 혼합된 DMEM/F12 배지에 우 태아 혈청(fetal bovine serum, FBS)이 첨가된 배지를 사용하였다. 하지만, 혈청을 포함하지 않는 배지를 사용하여도 본 발명의 방법을 수행하는 것이 가능하다.In addition, spheroid formation and angiogenesis factors by irradiating light in the red wavelength range without treating or inducing the genetic modification of a substance that induces the differentiation of stem cells into vascular endothelial cells in the culture solution or culture matrix for culturing the adipose stem cells. Just inducing expression can differentiate stem cells into vascular endothelial cells. In this case, the culture solution may be used without limitation as long as it is a medium commonly used for culturing and / or differentiating fat stem cells, and specifically, a medium containing serum such as DMEM (Dulbeco's modified eagle medium) or Ham's F12 may be used. Can be used. For example, in the embodiment of the present invention, a medium in which fetal bovine serum (FBS) was added to DMEM / F12 medium in which DMEM and Ham's F12 were mixed at a 1: 1 ratio was used. However, it is possible to carry out the method of the invention even with a medium which does not contain serum.
아울러, 상기 지방유래 중간엽 줄기세포의 분화를 촉진시키기 위하여, 상기 지방유래 중간엽 줄기세포를 소수성을 부여하는 고분자로 표면 처리된 배양용기 또는 상기 고분자로 제조된 배양용기에 접종하여 배양할 수 있다. 상기 소수성을 부여하는 고분자는 전술한 바와 같다.In addition, in order to promote differentiation of the adipose derived mesenchymal stem cells, the adipose derived mesenchymal stem cells can be inoculated and cultured in a culture vessel surface-treated with a polymer imparting hydrophobicity or a culture vessel made of the polymer. . The polymer giving the hydrophobicity is as described above.
상기 방법에 의해 분화된 혈관내피세포는 혈관손상, 피부 또는 혈관 조직 손상의 치료에 사용될 수 있는데, 상기 피부 또는 혈관 조직 손상이 유발되는 질환은 특별히 이에 제한되지 않으나, 구체적으로 심혈관 질환, 뇌혈관 질환, 허혈성 질환, 피부상처 등이 될 수 있고, 예를 들어 허혈성 피부 궤양, 피부상처, 동맥경화증, 안정형 및 불안정형 협심증, 말초 심혈관 질환, 고혈압, 심부전증, 말초 순환장애, 심근경색증, 뇌졸중, 일과성 및 허혈성 발작, 지주막하 출혈 또는 하지허혈 등의 질환이 될 수 있다.Vascular endothelial cells differentiated by the method can be used for the treatment of vascular damage, skin or vascular tissue damage, the disease causing the skin or vascular tissue damage is not particularly limited, specifically, cardiovascular disease, cerebrovascular disease , Ischemic diseases, skin wounds, and the like, for example, ischemic skin ulcers, skin wounds, arteriosclerosis, stable and unstable angina pectoris, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulatory disorders, myocardial infarction, stroke, transient and It may be a disease such as ischemic attack, subarachnoid hemorrhage or lower limb ischemia.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 인간의 지방유래 중간엽 줄기세포의 분리Example 1 Isolation of Adipose-derived Mesenchymal Stem Cells from Humans
인간 피하 지방조직으로부터 다분화능 줄기세포를 분리하고, 이를 그대로 위상차 현미경(Nikon)으로 관찰하거나, DAPI 염색을 수행하여 관찰하거나 또는 유세포분석을 수행하였다. 유세포분석 시에는 중간엽 줄기세포의 확인을 위한 표면항원으로 CD29, CD90 및 CD105를 사용하였고, 줄기세포의 분리, 배양 중 다른 세포의 혼입 여부를 관찰하기 위한 표면항원으로 CD34, CD31, KDR(Flk1) 및α-SMA를 사용하여, 이들의 발현양상을 유세포 분석기(flow cytometry)를 사용하여 분석하였다(도 1).Pluripotent stem cells were isolated from human subcutaneous adipose tissue and observed with a phase contrast microscope (Nikon), observed by DAPI staining, or flow cytometry. In flow cytometry, CD29, CD90 and CD105 were used as surface antigens to identify mesenchymal stem cells, and CD34, CD31, and KDR (Flk1) were used as surface antigens to observe whether or not other cells were mixed during stem cell isolation and culture. ) And α-SMA, their expression patterns were analyzed using flow cytometry (FIG. 1).
도 1은 인간의 지방조직으로부터 분리한 줄기세포를 검증한 결과를 나타내는 현미경 사진이다. 도 1에서 보듯이, 인간 피하지방 조직에서 분리한 세포가 중간엽 줄기세포의 표현형을 가지는 지방유래 중간엽 줄기세포임을 확인하였다. Figure 1 is a micrograph showing the results of verifying the stem cells isolated from human adipose tissue. As shown in Figure 1, it was confirmed that the cells isolated from human subcutaneous fat tissue is adipose derived mesenchymal stem cells having a phenotype of mesenchymal stem cells.
실시예 2: LED 조사에 의한 지방유래 중간엽 줄기세포의 성장율 변화Example 2 Growth Rate Change of Adipose-derived Mesenchymal Stem Cells by LED Irradiation
지방유래 중간엽 줄기세포의 성장율 변화를 관찰하기 위해서 WST(Water-soluble Tetrazolium Salts) assay(Dojindo laboratories)를 실시하였다. 대략적으로, 96웰 플레이트에 2 x 103 세포수의 줄기세포를 접종하고, 적색 파장대인 660nm의 빛을 조사할 수 있는 LED 조사장치를 이용하여 0 내지 6 J/㎠의 빛을 조사하였다. 이후 본 발명에서 LED 조사는 적색 파장대의 빛을 조사하는 것을 의미한다. 일정한 시간이 경과한 후, 배양액을 제거하고, 20㎕의 WST 용액과 180㎕의 새로운 배양액을 가하고 37℃에서 4시간동안 반응시킨 다음, OD450에서 흡광도를 측정하였다(도 2 및 3).Water-soluble Tetrazolium Salts (WST) assay (Dojindo laboratories) was performed to observe the growth rate of adipose derived mesenchymal stem cells. Approximately, 2 x 10 3 cells were inoculated into 96-well plates and stem cells were irradiated with light of 0 to 6 J / cm 2 using an LED irradiation apparatus capable of irradiating 660 nm light having a red wavelength band. Since the LED irradiation in the present invention means to irradiate light of the red wavelength band. After a certain time, the culture solution was removed, 20 μl of WST solution and 180 μl of fresh culture medium were added and reacted for 4 hours at 37 ° C., and then absorbance was measured at OD450 (FIGS. 2 and 3).
도 2는 LED 조사장치를 나타내는 사진이고, 도 3은 LED 조사에 의한 지방유래 중간엽 줄기세포의 성장율 변화를 나타내는 그래프이다. 도 3에서 보듯이, 빛의 조사시간이 증가할 수록 줄기세포의 증식이 가속화되었고, 조사되는 빛에 포함된 에너지양이 증가할 수록 줄기세포의 증식수준이 증가하는 양상을 나타내었다. Figure 2 is a photograph showing the LED irradiation apparatus, Figure 3 is a graph showing the growth rate change of fat-derived mesenchymal stem cells by LED irradiation. As shown in FIG. 3, the growth of stem cells was accelerated as the irradiation time of light increased, and the proliferation level of stem cells increased as the amount of energy included in the irradiation light increased.
특히, 0.5 J/㎠ 이상의 빛을 조사할 경우에 줄기세포의 증식이 촉진되었고, 3 J/㎠의 빛을 조사할 때까지는 빛에 포함된 에너지양에 따라 줄기세포의 증식수준이 비례하는 양상을 나타내었으나, 6 J/㎠의 빛을 조사할 경우에는 줄기세포의 증식수준이 오히려 감소함을 확인하였다. In particular, the growth of stem cells was promoted when irradiated with light of 0.5 J / ㎠ or more, and the proliferation level of stem cells is proportional to the amount of energy contained in the light until the irradiation of 3 J / ㎠. However, when irradiated with light of 6 J / ㎠ it was confirmed that the proliferation level of stem cells rather reduced.
따라서, 줄기세포의 증식을 촉진시키기 위하여는 0.5 내지 3 J/㎠의 빛을 조사함이 바람직함을 알 수 있었다.Therefore, in order to promote the proliferation of stem cells, it was found that it is preferable to irradiate light of 0.5 to 3 J / ㎠.
실시예 3: 지방유래 중간엽 줄기세포의 구상체(spheroid: 3DCM, 3D cell mass) 형성Example 3 Formation of Spheroids (3DCM, 3D Cell Mass) of Adipose-derived Mesenchymal Stem Cells
실시예 3-1: 배양용기 표면에 대한 지방유래 중간엽 줄기세포의 접착활성 및 배양시간에 따른 스페로이드 형성 분석Example 3-1: Adhesion Activity of Adipose-derived Mesenchymal Stem Cells to the Culture Vessel Surface and Analysis of Spheroid Formation According to Culture Time
배양용기 표면에 대한 지방유래 중간엽 줄기세포의 접착활성과 스페로이드 형성의 상관관계를 조사하기 위하여, ECM 단백질로서 피브로넥틴이 코팅되거나 또는 코팅되지 않은 비-조직세포 배양용 24-웰 플레이트(NTCP, 폴리스티렌)에 웰당 4 × 104 세포/㎠의 지방유래 중간엽 줄기세포를 접종한 후 10% FBS 함유 DMEM/F12 배지에서 3일간 배양하였다. 배양이 종료된 후, 각 세포접착 표면에서 지방유래 중간엽 줄기세포의 스페로이드 형성 여부를 관찰하였다(도 4a). In order to investigate the correlation between adipose-derived mesenchymal stem cells' adhesion activity and spheroid formation on the surface of culture vessels, 24-well plates for non-tissue cell cultures with or without fibronectin as ECM protein (NTCP, Polystyrene) was inoculated with 4 × 10 4 cells / cm 2 of adipose derived mesenchymal stem cells per well, and then cultured in DMEM / F12 medium containing 10% FBS for 3 days. After the incubation was completed, the spheroid formation of adipose derived mesenchymal stem cells was observed on each cell adhesion surface (FIG. 4A).
도 4a는 배양용기에 코팅된 ECM 단백질의 존재여부에 따른, 지방유래 중간엽 줄기세포를 배양한 결과를 나타내는 사진이다. 도 4에서 보듯이, 표면이 소수성을 띠어 세포접착이 약하게 유도되는 NTCP에서 육안으로 검출가능한 크기로 지방유래 중간엽 줄기세포의 스페로이드가 형성되었는데, 상기 스페로이드는 약 1 mm 이상의 직경을 갖는 것으로 확인되었다. 이에 반하여, 세포접착이 강하게 유도되는 파이브로넥틴이 코팅된 NTCP에서는 지방유래 중간엽 줄기세포가 2차원적으로 플레이트 표면에 넓게 접착된 상태로 단층 배양되어 세포집합체가 형성되지 않음을 확인하였다.Figure 4a is a photograph showing the result of culturing fat-derived mesenchymal stem cells according to the presence of ECM protein coated in the culture vessel. As shown in FIG. 4, spheroids of adipose-derived mesenchymal stem cells were formed to a size that can be detected by naked eye in NTCP, which is hydrophobic, and has a weak surface cell adhesion. The spheroids have a diameter of about 1 mm or more. Confirmed. On the contrary, in fibronectin-coated NTCP which strongly induced cell adhesion, it was confirmed that adipose derived mesenchymal stem cells were monolayer cultured in a state of being widely adhered to the surface of the plate in two dimensions and cell aggregates were not formed.
상기 결과로부터 사용되는 배양용기의 표면에 대한 접착활성에 따라 지방유래 중간엽 줄기세포의 스페로이드 형성이 영향을 받으며, 육안으로 검출가능한 크기의 스페로이드를 형성하기 위해서는 초기에는 세포접착이 약하게 유도되다가 시간이 경과함에 따라 세포 밀도가 증가하면서 세포들이 탈착되어 부유된 상태로 증식하게 되는 폴리스티렌 재질의 NTCP와 같이 표면이 소수성을 띠는 배양용기를 사용하는 것이 바람직함을 확인하였다.The spheroid formation of adipose-derived mesenchymal stem cells is affected by the adhesion activity to the surface of the culture vessel used from the above results, and cell adhesion is initially weakly induced to form a spheroid having a detectable size with the naked eye. As time went by, the cell density increased, and it was confirmed that it is preferable to use a culture vessel having a hydrophobic surface, such as polystyrene NTCP, in which cells detach and proliferate in a suspended state.
또한, NTCP에서 육안으로 검출가능한 크기의 스페로이드를 형성하는데 요구되는 지방유래 중간엽 줄기세포의 배양시간을 알아보기 위하여, 상기 실시예 1에서 수득된 지방유래 중간엽 줄기세포를 5% FBS 함유 DMEM/F12 배지가 담겨진 NTCP의 각 웰에 0.5 × 104 내지 1× 105 세포/㎠의 농도로 접종하고 매일 10분씩 LED 광원으로부터 빛을 조사하면서 3일동안 배양하였다(도 4b). In addition, in order to determine the incubation time of the adipose derived mesenchymal stem cells required to form a spheroid of the size detectable by the naked eye in NTCP, the adipose derived mesenchymal stem cells obtained in Example 1 containing 5% FBS DMEM Each well of NTCP containing / F12 medium was inoculated at a concentration of 0.5 × 10 4 to 1 × 10 5 cells / cm 2 and incubated for 3 days while irradiating light from the LED light source for 10 minutes each day (FIG. 4B).
도 4b는 지방유래 중간엽 줄기세포를 NTCP에 접종하고, LED 광원으로부터 빛을 조사하면서 배양시간의 경과에 따른 지방유래 중간엽 줄기세포의 형태변화를 나타내는 사진이다. 도 4b에서 보듯이, 3일이 경과된 시점에서 육안으로 검출가능한 크기의 스페로이드가 형성됨을 확인하였다.Figure 4b is a photograph showing the change in morphology of fat-derived mesenchymal stem cells with the passage of incubation time while inoculating the fat-derived mesenchymal stem cells in NTCP, and irradiated with light from the LED light source. As shown in Figure 4b, it was confirmed that the spheroid of the detectable size with the naked eye was formed at the time 3 days have passed.
실시예 3-2: 비-조직세포 배양용 플레이트와 조직세포 배양용 플레이트의 스페로이드 형성 비교Example 3-2: Spheroid Formation Comparison of Non-Tissue Cell Culture Plates and Tissue Cell Culture Plates
비-조직세포 배양용 24웰 플레이트의 각 웰에 웰당 5 × 104 세포수의 지방줄기세포를 접종한 후 5% FBS 함유 DMEM/F12 배지에서 3일간 배양하였다. 3일간 배양 후 각 세포접착 표면에서 지방줄기세포의 스페로이드 형성 여부를 관찰하였다(도 5). Each well of a 24-well plate for non-tissue cell culture was inoculated with 5 × 10 4 cells of adipose stem cells per well and then incubated in DMEM / F12 medium containing 5% FBS for 3 days. After incubation for 3 days, the spheroid formation of adipose stem cells was observed on each cell adhesion surface (FIG. 5).
도 5는 지방줄기세포로부터 형성된 스페로이드를 나타내는 사진이다. 도 5에서 보듯이, 배양이 종료될 때에는 약 1mm의 직경을 갖는 스페로이드가 형성됨을 확인하였다. 조직세포 배양용 24웰 플레이트에서 monolayer로 3일간 배양된 지방줄기세포는 스페로이드를 형성하지 못하였다.5 is a photograph showing a spheroid formed from adipose stem cells. As shown in Figure 5, when the incubation was confirmed that the spheroid having a diameter of about 1mm was formed. Adipocytes cultured for 3 days in monolayer in a 24-well plate for tissue cell culture did not form spheroids.
실시예 4: LED 조사에 의한 지방유래 줄기세포의 혈관신생 및 피부재생인자 발현량 변화 측정Example 4 Measurement of Expression of Angiogenesis and Skin Regeneration Factor of Adipose derived Stem Cells by LED Irradiation
혈관신생 및 피부재생인자로서 알려진 FGF, VEGF 및 HGF의 발현량을 측정하기 위하여, LED를 조사하면서 2차원 배양된 지방세포유래 중간엽 줄기세포(monolayer ASC)와 LED를 조사하면서 스페로이드를 형성한 지방세포유래 중간엽 줄기세포(spheroid ASC)를 대상으로 하여 ELISA kit(R&D Systems)를 사용한 분석을 수행하였다(도 6). 이때, 단위 세포수(1 × 104 세포수)에서 발현되는 각 인자의 발현량을 pg 단위로 측정하였다.In order to measure the expression level of FGF, VEGF and HGF known as angiogenesis and skin regeneration factors, spheroids were formed by irradiating LEDs with two-dimensional cultured adipocyte-derived mesenchymal stem cells (monolayer ASC) Adipose-derived mesenchymal stem cells (spheroid ASC) were analyzed using an ELISA kit (R & D Systems) (Fig. 6). At this time, the expression amount of each factor expressed in the unit cell number (1 × 10 4 cell number) was measured in pg units.
도 6은 LED 조사에 의한 지방유래 줄기세포의 혈관신생 및 피부재생인자 발현량 변화를 나타내는 그래프이다. 도 6에서 보듯이, 3종의 혈관신생 및 피부재생인자는 LED를 조사하면서 스페로이드를 형성한 지방세포유래 중간엽 줄기세포(spheroid ASC)에서 발현량이 증가됨을 확인하였다.Figure 6 is a graph showing the expression changes of angiogenesis and skin regeneration factor of adipose derived stem cells by LED irradiation. As shown in Figure 6, the three angiogenesis and skin regeneration factors were confirmed that the expression level of the spheroid-forming adipocyte-derived mesenchymal stem cells (spheroid ASC) was increased while irradiating the LED.
실시예 5: LED 조사에 의한 지방유래 줄기세포의 혈관신생 관련 단백질 발현량 변화 측정Example 5 Measurement of Changes in Protein Expression of Angiogenesis of Adipose-Derived Stem Cells by LED Irradiation
2차원 배양된 줄기세포 그룹(control), 2차원 배양된 줄기세포에 LED를 조사한 그룹(low level light therapy, LLLT), LED 조사없이 스페로이드가 형성된 줄기세포 그룹(spheroid) 및 LED를 조사하여 스페로이드가 형성된 줄기세포 그룹(L-spheroid)으로부터 얻어진 각 줄기세포를 대상으로 혈관신생 관련 단백질의 발현수준 변화를 비교하기 위하여, Human Angiogenesis Array Kit (R&D Systems, Ltd., Abingdon, UK)를 사용하여 상기 각 줄기세포로부터 혈관신생 관련 단백질의 발현수준을 측정하였다(도 7). 이때, 상기 각 줄기세포로부터 얻어진 단백질 50mg을 15㎕의 바이오틴이 결합된 검출용 항체와 혼합하여 반응시켰고, 검출신호는 image reader LAS-3000(Kodak, Rochester, NY)을 사용하여 측정한 다음, MultiGauge 4.0 software (Kodak)를 사용하여 분석하였다.2D cultured stem cell group (control), 2D cultured stem cell group (Low level light therapy, LLLT), spheroid-free stem cell group (Spheroid) without LED irradiation and spe In order to compare the expression level of angiogenesis-related proteins in each stem cell obtained from L-spheroid, Loid-formed stem cells, we used Human Angiogenesis Array Kit (R & D Systems, Ltd., Abingdon, UK). Expression levels of angiogenesis-related proteins were measured from each stem cell (FIG. 7). At this time, 50 mg of the protein obtained from each of the stem cells were reacted with 15 μl of biotin-coupled detection antibody, and the detection signal was measured using an image reader LAS-3000 (Kodak, Rochester, NY), followed by MultiGauge. Analysis was performed using 4.0 software (Kodak).
도 7은 LED의 조사여부 및 스페로이드의 형성여부에 따른 혈관신생 관련 단백질의 발현수준 변화를 측정한 결과를 나타내는 사진 및 그래프이다. 도 7에서 보듯이, 2차원 배양된 줄기세포 보다는 스페로이드가 형성된 줄기세포에서 혈관신생 관련 단백질의 발현수준이 증가하였고, 상기 스페로이드가 형성된 줄기세포 중에서도, LED 조사없이 스페로이드가 형성된 줄기세포보다는 LED를 조사하여 스페로이드가 형성된 줄기세포에서 혈관신생 관련 단백질의 발현수준이 증가함을 확인하였다.Figure 7 is a photograph and graph showing the results of measuring the expression level change of the angiogenesis-related protein according to whether the LED irradiation and spheroid formation. As shown in FIG. 7, the expression level of angiogenesis-related proteins in the spheroid-formed stem cells was increased, rather than the two-dimensional cultured stem cells, and among the spheroid-formed stem cells, By examining the LED, it was confirmed that the expression level of angiogenesis-related protein was increased in the spheroid-formed stem cells.
실시예 6: LED 조사에 의한 스페로이드의 혈관내피세포 특성 확인Example 6: Confirmation of vascular endothelial cell characteristics of spheroid by LED irradiation
실시예 6-1 : 스페로이드의 면역염색학적 분석Example 6-1: Immunostaining Analysis of Spheroids
상기 실시예 1에서 분리한 지방조직 유래 중간엽 줄기세포를 1 × 105 세포/웰의 농도로 24-웰 NTCP(Non-Treated cell culture plate)에 접종하고, LED를 조사하면서 배양하여 스페로이드를 형성시켰다. 상기 형성된 스페로이드를 회수하고, OCT 화합물을 이용하여 -70℃에서 고정한 후 마이크로톰을 이용해 4 ㎛ 두께로 자른 후 그 절편을 슬라이드 글라스에 고정시키고 면역학적 염색을 수행하였다.Adipose tissue-derived mesenchymal stem cells isolated in Example 1 were inoculated into 24-well NTCP (Non-Treated cell culture plate) at a concentration of 1 × 10 5 cells / well, and cultured while irradiating LEDs to spheroids. Formed. The formed spheroids were collected, fixed at -70 ° C using an OCT compound, cut into 4 μm thickness using a microtome, and then the sections were fixed on a slide glass and subjected to immunological staining.
또 다른 방법으로서, 상기 회수된 스페로이드를 주사기를 이용해 물리적으로 와해시킨 후, 이를 슬라이드 글라스 위에 놓고 4시간 동안 부착시키고, 상기 슬라이드 글라스를 PBS로 수회 세척하였으며, 이를 4% 파라포름알데하이드 용액에 침지하여 30분간 실온에서 고정시킨 다음, 다시 PBS로 세척하고 면역학적 염색을 수행하였다. As another method, the recovered spheroid was physically disintegrated using a syringe, then placed on a slide glass and attached for 4 hours, and the slide glass was washed several times with PBS, which was immersed in a 4% paraformaldehyde solution. Fixed at room temperature for 30 minutes, then washed again with PBS and subjected to immunological staining.
상기 면역학적 염색은 상기에서 준비된 슬라이드 글라스를 다양한 분화마커(CD29, CD34, KDR(Flk1) 및 CD31)에 대한 일차항체를 함유한 PBS에 침지하여, 하룻밤 동안 반응시키고, PBS로 3회 세척한 다음, 다시 상기 일차항체에 결합할 수 있는 이차항체를 처리하고, 암실에서 1시간 동안 반응시켜서 수행하였다. 반응이 종결된 후, 슬라이드 글라스를 PBS로 3회 세척하고 봉입(mounting)한 후 형광현미경으로 분석하였다(도 8의 위측 사진).The immunological staining was performed by immersing the slide glass prepared above in PBS containing primary antibodies against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, and washing three times with PBS. After treatment, the secondary antibody capable of binding to the primary antibody was reacted for 1 hour in the dark. After the reaction was completed, the slide glass was washed three times with PBS, mounted (mounted) and analyzed by fluorescence microscope (upper photo of Figure 8).
도 8의 위측 사진은 LED가 조사된 지방조직 유래 중간엽 줄기세포에 대한 면역형광염색 결과를 나타내는 사진이다. 도 8의 위측 사진에서 보듯이, LED 조사에 의하여 지방유래 중간엽 줄기세포로부터 형성된 스페로이드는 CD29, CD34, Flk1 및 CD31에 대해서는 양성반응을 나타내었다. CD29는 중간엽줄기세포 및 상피세포에서 특이적으로 발현되는 표면항원이고, CD34, KDR(Flk1) 및 CD31은 혈관내피세포에서 특이적으로 발현되는 표면항원이다. 8 is a photograph showing the results of immunofluorescence staining for the adipose tissue-derived mesenchymal stem cells irradiated with LED. As shown in the upper photo of Figure 8, spheroids formed from adipose derived mesenchymal stem cells by the LED irradiation showed a positive response to CD29, CD34, Flk1 and CD31. CD29 is surface antigen specifically expressed in mesenchymal stem cells and epithelial cells, and CD34, KDR (Flk1) and CD31 are surface antigens specifically expressed in vascular endothelial cells.
따라서, 지방유래 중간엽 줄기세포를 적색 파장대의 광을 조사, 표면이 소수성을 띠는 배양용기에서 배양하여 형성된 spheroid가 지방유래 중간엽 줄기세포로부터 분화된 혈관내피세포로 구성되어 있음을 알 수 있었다.Therefore, spheroids formed by culturing adipose derived mesenchymal stem cells in a red wavelength band and cultured in a culture vessel with a hydrophobic surface were composed of vascular endothelial cells differentiated from adipose derived mesenchymal stem cells. .
실시예 6-2 : 스페로이드의 웨스턴 블럿 분석Example 6-2 Western Blot Analysis of Spheroids
2차원 배양된 줄기세포 그룹(control), 2차원 배양된 줄기세포에 LED를 조사한 그룹(LLLT), LED 조사없이 스페로이드가 형성된 줄기세포 그룹(spheroid) 및 LED를 조사하여 스페로이드가 형성된 줄기세포 그룹(spheroid+LLLT)으로부터 얻어진 각 줄기세포를 대상으로 CD31의 발현여부를 확인하기 위하여 웨스턴 블럿 분석을 수행하였다(도 8의 아래측 사진).2D cultured stem cell group (control), 2D cultured stem cell group (LLLT) irradiated with LED, spheroid-free stem cell group (spheroid) without LED irradiation, spheroid-formed stem cell by irradiation Western blot analysis was performed to confirm the expression of CD31 in each stem cell obtained from the group (spheroid + LLLT) (bottom photo of Figure 8).
도 8의 아래측 사진은 LED의 조사여부 및 스페로이드의 형성여부에 따른 CD31의 발현여부를 웨스턴 블럿 분석을 통해 확인한 결과를 나타내는 사진이다. 도 8의 아래측 사진에서 보듯이, 스페로이드가 형성된 줄기세포에서만 CD31이 발현되었고, 상기 스페로이드가 형성된 줄기세포 중에서도, LED 조사없이 스페로이드가 형성된 줄기세포보다는 LED를 조사하여 스페로이드가 형성된 줄기세포에서 CD31이 더 많이 발현됨을 확인하였다.The lower photo of Figure 8 is a photograph showing the results confirmed by Western blot analysis whether the expression of the CD31 according to the LED irradiation and the spheroid formation. As shown in the lower photo of FIG. 8, CD31 was expressed only in the stem cells in which spheroids were formed, and among the stem cells in which spheroids were formed, the spheroid-formed stems were irradiated with LEDs rather than the stem cells in which spheroids were formed without LED irradiation. It was confirmed that more CD31 is expressed in the cells.
실시예 6-3 : 스페로이드의 FACS 분석Example 6-3 FACS Analysis of Spheroids
2차원 배양된 줄기세포에 LED를 조사한 그룹(ASC+LLLT) 및 LED를 조사하여 스페로이드가 형성된 줄기세포 그룹(L-spheroid)으로부터 얻어진 각 줄기세포를 대상으로 CD31, Cd34 및 KDR에 대한 FACS 분석을 수행하였다.FACS analysis of CD31, Cd34, and KDR in each stem cell obtained from the group irradiated with LED (ASC + LLLT) and the spheroid-formed stem cell group (L-spheroid) Was performed.
상기 각 그룹의 줄기세포를 회수하여, 주사기를 이용해 물리적으로 와해시킨 후, 이를 슬라이드 글라스 위에 놓고 4시간 동안 부착시킨 다음, 상기 슬라이드 글라스를 PBS로 수회 세척하였으며, 이를 4% 파라포름알데하이드 용액에 침지하여 30분간 실온에서 고정시키고, 다시 PBS로 세척하였으며, 혈관내피세포에서 특이적으로 발현되는 표면항원인 CD31, Cd34 및 KDR에 대한 면역학적 염색을 수행하였다. The stem cells of each group were recovered, physically disintegrated using a syringe, placed on a slide glass, and then attached for 4 hours, and then the slide glass was washed several times with PBS, which was immersed in 4% paraformaldehyde solution. The cells were fixed at room temperature for 30 minutes, washed again with PBS, and subjected to immunological staining for CD31, Cd34 and KDR, which are surface antigens specifically expressed in vascular endothelial cells.
상기 면역학적 염색은 상기에서 준비된 슬라이드 글라스를 다양한 분화마커(CD29, CD34, KDR(Flk1) 및 CD31)에 대한 일차항체를 함유한 PBS에 침지하여, 하룻밤 동안 반응시키고, PBS로 3회 세척한 다음, 다시 상기 일차항체에 결합할 수 있는 이차항체를 처리하고, 암실에서 1시간 동안 반응시켜서 수행하였다. 반응이 종결된 후, 슬라이드 글라스를 PBS로 3회 세척하고 봉입(mounting)한 후 FACS 으로 분석하였다(도 9).The immunological staining was performed by immersing the slide glass prepared above in PBS containing primary antibodies against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, and washing three times with PBS. After treatment, the secondary antibody capable of binding to the primary antibody was reacted for 1 hour in the dark. After the reaction was completed, the slide glass was washed three times with PBS, mounted and analyzed by FACS (FIG. 9).
도 9는 LED가 조사된 2차원 배양된 줄기세포와 스페로이드가 형성된 줄기세포에 대한 FACS 분석결과를 나타내는 그래프이다. 도 7에서 보듯이, LED 조사에 의하여 형성된 스페로이드는 혈관내피세포에서 특이적으로 발현되는 표면항원인 CD29, CD34, KDR (Flk1) 및 CD31에 대하여 양성반응을 나타냄을 확인하였다.Figure 9 is a graph showing the results of FACS analysis for the two-dimensional cultured stem cells and spheroid-formed stem cells irradiated with LED. As shown in FIG. 7, it was confirmed that the spheroid formed by the LED irradiation showed a positive response to CD29, CD34, KDR (Flk1) and CD31, which are surface antigens specifically expressed in vascular endothelial cells.
따라서, LED가 조사된 스페로이드는 지방유래 중간엽 줄기세포로부터 분화된 혈관내피세포의 특징을 나타냄을 알 수 있었다.Therefore, it was found that the spheroid irradiated with LED exhibited characteristics of vascular endothelial cells differentiated from adipose derived mesenchymal stem cells.
실시예 7: 하지허혈 마우스 동물모델에 LED 조사한 스페로이드 이식 후 치료효과 확인Example 7: Confirmation of the therapeutic effect after spheroid transplantation irradiated with LED on animal model of ischemia
먼저, 5주령의 BALB/c 누드마우스(20 g body weight; Narabio, Seoul, Korea)의 대퇴부 대동맥을 5-0 silk suture을 사용하여 결찰함으로써, 하지허혈 마우스 동물모델을 제작하였다. First, the femoral aorta of a 5 week old BALB / c nude mouse (20 g body weight; Narabio, Seoul, Korea) was ligated using 5-0 silk suture to prepare an animal model of the ischemic mouse.
상기 제작된 하지허혈 마우스 동물모델의 결찰부위에, PBS를 주입하거나(control), 1.5 × 106 세포수의 2차원 배양된 줄기세포를 주입하거나(ASC), LED를 조사하거나(LLLT) 또는 1.5 × 106 세포수의 LED를 조사하여 스페로이드가 형성된 줄기세포를 주입하고(L-spheroid) 21일 동안 사육한 다음, 외관, 하지의 손상정도(하지손실, 발괴사 및 회복) 및 피부혈류(cutaneous blood flow)를 촬영 또는 측정하였다(도 10a 내지 10d). 이때, 피부혈류 사진은 마우스를 37℃ 열판에 위치시키고, 스캔하여 레이저 도트 칼라 이미지를 촬영하는 방식으로 수득하였다.In the ligation region of the prepared ischemic mouse animal model, PBS was injected (control), two-dimensional cultured stem cells of 1.5 × 10 6 cells were injected (ASC), LED was irradiated (LLLT) or 1.5 Stem cells with spheroids were injected by irradiating LEDs of × 10 6 cells (L-spheroid) and bred for 21 days. cutaneous blood flow) was taken or measured (FIGS. 10A-10D). At this time, skin blood flow pictures were obtained by placing a mouse on a 37 ° C. hot plate and scanning to photograph a laser dot color image.
도 10a 내지 10d는 하지허혈 마우스 동물모델에 대한 LED를 조사하여 스페로이드가 형성된 줄기세포의 치료효과를 비교한 결과를 나타내는 사진 및 그래프로서, 도 10a는 주입후 1, 7, 14 및 21일이 경과한 시점에서 촬영한 마우스 동물모델의 하지부위를 나타내는 사진이고, 도 10b는 21일이 경과한 후, 전체 마우스 중에서 하지의 손상정도에 따른 마우스 비율을 나타내는 그래프이며, 도 10c는 주입후 1, 7, 14 및 21일이 경과한 시점에서 촬영한 피부혈류를 나타내는 사진이고, 도 10d는 21일이 경과한 후, 전체 마우스 중에서 하지허혈이 치료된 마우스의 비율을 나타내는 그래프이다. 이를 통해, LED를 조사하여 스페로이드가 형성된 줄기세포를 사용하면, 하지허혈 마우스에서 발생된 혈관손상을 효과적으로 치료할 수 있음을 알 수 있었다.10A to 10D are photographs and graphs showing the results of comparing the therapeutic effects of spheroid-formed stem cells by examining LEDs for the ischemic mouse animal model. FIG. 10A is 1, 7, 14, and 21 days after injection. Figure 10b is a photograph showing the lower extremity portion of the mouse animal model taken at the elapsed time point, Figure 10b is a graph showing the ratio of the mouse according to the degree of damage of the lower extremities among all mice after 21 days, Figure 10c is 1, It is a photograph which shows the skin blood flow taken at the time of 7, 14, and 21 days passed, and FIG. 10D is a graph which shows the ratio of the mouse to which the leg ischemia was treated among all the mice after 21 days. Through this, it was found that the use of the stem cells formed spheroid by irradiation with LED, it can effectively treat the vascular damage caused in the ischemic mouse.
실시예 8: 피부 창상 마우스 모델을 통해 LED 조사한 스페로이드 이식 및 LED 처리의 조합 효과 검증Example 8 Validation of Combination Effect of Spheroid Implantation and LED Treatment with LED Irradiation through a Skin Wound Mouse Model
먼저, 누드마우스를 마취시킨 후,8mm 펀치를 이용하여 등 부위의 피부조직에 상처를 형성시키고, 상처부위에 실리콘 재질의 부목(splint)을 설치하여 피부 창상 마우스 모델을 제작하였다.First, after anesthetizing nude mice, a wound was formed on the skin tissue of the back region using an 8 mm punch, and a splint made of silicone was installed on the wound region to prepare a skin wound mouse model.
다음으로, 상기 제작된 피부 창상 마우스 모델에 600㎕의 PBS를 주사한 대조군(control), 1.5 × 106 세포수의 지방조직유래 중간엽 줄기세포를 이식한 실험군(ASCs), LED 광원을 이용하여 매일 50 mW/㎠ 및 660nm 파장의 빛을 10분씩 조사한 실험군(low level light thearapy; LLLT), LED를 조사한 15개의 스페로이드를 이식한 실험군(spheroid) 및 상기 15개의 스페로이드를 이식하고, LED를 조사한 실험군(spheroid+LLLT)을 각각 설정하고, 14일동안 사육한 다음 손상된 피부조직의 상처 치료효과를 확인하였다(도 11). Next, a control group injected with 600 μl of PBS in the prepared skin wound mouse model, an experimental group transplanted with adipose tissue-derived mesenchymal stem cells of 1.5 × 10 6 cells (ASCs), and an LED light source. Low level light thearapy (LLLT) irradiated with light at 50 mW / cm2 and 660 nm wavelengths for 10 minutes each day The experimental group (spheroid) implanted with 15 spheroids and the 15 spheroids were implanted, and the experimental group (spheroid + LLLT) irradiated with LEDs was set, and after 14 days of breeding, confirmed the wound healing effect of damaged skin tissue. (FIG. 11).
도 11은 피부 창상 마우스 모델을 이용한 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT)의 상처 치료효과를 비교한 결과를 나타내는 사진 및 그래프로서, 도 11의 a는 제작된 피부 창상 마우스 모델을 나타내는 사진이고, 도 11의 b는 각 실험군의 상처부위를 시간의 경과에 따라 촬영한 사진이며, 도 11의 c는 시간의 경과에 따른 상처부위의 면적 변화를 나타내는 그래프이다. 도 11에서 보듯이, 지방조직유래 중간엽 줄기세포를 이식(ASCs)하거나 LED를 조사(LLLT)한 실험군 보다는 스페로이드를 이식한 실험군의 상처치료 효과가 우수하였고, 스페로이드를 이식한 실험군 중에서도 LED 조사를 병행한 실험군(spheroid + LLLT)이 보다 우수한 상처치료 효과를 나타냄을 확인하였다.11 is a photograph and a graph showing the results of comparing the wound treatment effect of each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) using the skin wound mouse model, Figure 11a is a manufactured skin wound It is a photograph showing a mouse model, b in FIG. 11 is a photograph photographing the wound site of each test group with the passage of time, and FIG. 11 c is a graph which shows the area change of the wound site with passage of time. As shown in FIG. 11, the wound treatment effect of the spheroid-grafted experimental group was superior to that of the adipose tissue-derived mesenchymal stem cells (ASCs) or the LED-irradiated (LLLT) -tested group, and the LED among the spheroid-transplanted experimental groups It was confirmed that the experimental group (spheroid + LLLT) in parallel with the irradiation showed better wound healing effect.
실시예 9: 혈관재생에 대한 LED 조사한 스페로이드 이식 및 LED 처리의 조합 효과 검증Example 9 Validation of Combination Effect of LED Irradiated Spheroid Implantation and LED Treatment on Vascular Regeneration
지방조직유래 중간엽 줄기세포로부터 형성된 스페로이드가 생체내에서 혈관내피세포로 분화되어 혈관재생효과를 나타낼 수 있는지를 확인하기 위하여, 상기 실시예 8에서 설정한 각 실험군의 마우스를 대상으로, 혈관내피세포 마커인 CD31과 혈관외피세포의 마커인 SMAa 액틴을 면역형광염색하였다.In order to confirm whether the spheroids formed from adipose tissue-derived mesenchymal stem cells can differentiate into vascular endothelial cells in vivo and exhibit vascular regeneration effects, mice of each experimental group set in Example 8 were subjected to vascular endothelial cells. CD31, a cell marker, and SMAa actin, a marker of vascular envelope cells, were immunofluorescent stained.
구체적으로, 상기 각 실험군의 마우스를 희생시켜서, 상처부위의 조직을 적출하여 수득하고, 상기 수득한 조직을 4% 파라포름알데하이드 용액에 침지하여 30분간 실온에서 고정시킨 다음, OCT 화합물을 이용하여 -70℃에서 고정시키고, 마이크로톰을 이용해 4 ㎛ 두께로 세절하여 조직박편을 수득하였으며, 상기 조직박편을 슬라이드 글라스에 고정시켰다. 상기 슬라이드 글라스를 혈관내피세포 마커인 CD31과 혈관외피세포의 마커인 SMAa 액틴에 대한 일차항체를 함유한 PBS에 침지하여, 하룻밤 동안 반응시키고, PBS로 3회 세척한 다음, 다시 상기 일차항체에 결합할 수 있는 이차항체를 처리하고, 암실에서 1시간 동안 반응시켜서 면역형광염색을 수행하였다. 반응이 종료된 후, 상기 슬라이드 글라스를 PBS로 3회 세척하고 봉입(mounting)한 다음, 면역형광염색된 조직을 형광현미경으로 분석하였다(도 12의 a). Specifically, the mice of each experimental group were sacrificed to obtain the wound tissue, and the tissue obtained was immersed in 4% paraformaldehyde solution and fixed at room temperature for 30 minutes. It was fixed at 70 ℃, and cut into 4 ㎛ thickness using a microtome to obtain a tissue flakes, the tissue flakes were fixed to the slide glass. The slide glass was immersed in PBS containing a primary antibody against CD31, a vascular endothelial cell marker, and SMAa actin, a marker of vascular endothelial cell, reacted overnight, washed three times with PBS, and then bound to the primary antibody. Secondary antibodies were treated and reacted for 1 hour in the dark to carry out immunofluorescence staining. After the reaction was completed, the slide glass was washed three times with PBS and mounted, and then immunofluorescent stained tissue was analyzed by fluorescence microscopy (Fig. 12a).
도 12의 a는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관내피세포 및 혈관외피세포의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 녹색은 혈관외피세포의 마커인 SMAa 액틴을 나타내고, 적색은 혈관내피세포 마커인 CD31을 나타낸다. 도 12의 a에서 보듯이, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)의 조직에서 상기 SMAa 액틴과 CD31의 발현수준이 다른 실험군의 조직에 비하여 높은 수준을 나타냄을 확인하였다.12 a shows immunofluorescence showing the results of comparing the levels of vascular endothelial cells and vascular endothelial cells in the wound region of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). As the stained picture, green represents SMAa actin, a marker of vascular endothelial cells, and red, CD31, a vascular endothelial marker. As shown in Figure 12a, it was confirmed that the expression level of the SMAa actin and CD31 in the tissue of the experimental group (spheroid + LLLT) to which the spheroid was implanted and the LED was shown to be higher than that of the other experimental groups.
또한, 상기 면역형광염색된 조직의 형광현미경 사진으로부터 미세혈관과 소동맥의 수를 계수하고, 계수된 미세혈관 및 소동맥의 수를 단위면적(㎟)당 수로 환산하여 비교하였다(도 12의 b 및 c).In addition, the number of microvascular and small arteries was counted from the fluorescence micrograph of the immunofluorescent stained tissue, and the number of counted microvascular and small arteries was compared in terms of the number per unit area (mm 2) (b and c of FIG. 12). ).
도 12의 b는 피부 창상 마우스 모델의 상처부위에 존재하는 단위면적당 미세혈관의 수를, 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 그래프이고, 도 12의 c는 피부 창상 마우스 모델의 상처부위에 존재하는 단위면적당 소동맥의 수를, 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 그래프이다.FIG. 12B is a graph showing the results of comparing the number of microvascular vessels per unit area of the wounded mouse model with each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT), FIG. 12c is a graph showing the result of comparing the number of small arteries per unit area of the wounded mouse model with each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT).
도 12의 b 및 c에서 보듯이, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)의 조직에서 미세혈관과 동맥의 수가 가장 많이 생성됨을 확인하였다.As shown in b and c of FIG. 12, it was confirmed that the number of microvascular and arteries was most generated in the tissue of the experimental group (spheroid + LLLT) irradiated with spheroids and irradiated with LEDs.
상기 도 12의 결과를 종합하면, 스페로이드를 이식하고, LED를 조사한 경우에 혈관부위의 상처가 효과적으로 치료되어 혈관재생이 촉진되는 효과를 나타냄을 알 수 있었다.12, it can be seen that when the spheroid is implanted and the LED is irradiated, the wound on the vascular site is effectively treated to promote the revascularization.
실시예 10: 피부재생에 미치는 스페로이드 이식 및 LED 처리의 조합 효과 검증Example 10: Combination Effect of Spheroid Implantation and LED Treatment on Skin Regeneration
지방조직유래 중간엽 줄기세포로부터 형성된 스페로이드가 피부재생에도 효과를 나타내는지를 검증하고자, 피부세포 마커인 사이토케라틴(cytokeratin)을 대상으로 면역형광염색을 수행하는 것을 제외하고는, 상기 실시예 9와 동일한 방법을 수행하고, 면역형광염색된 조직을 형광현미경으로 분석하였다(도 13).In order to verify whether the spheroids formed from adipose tissue-derived mesenchymal stem cells also have an effect on skin regeneration, except for performing immunofluorescence staining on cytokeratin (cytokeratin), which is a skin cell marker, The same method was followed and immunofluorescent stained tissues were analyzed by fluorescence microscopy (FIG. 13).
도 13은 피부 창상 마우스 모델의 상처부위에 존재하는 피부세포의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 적색은 피부세포 마커인 사이토케라틴을 나타낸다. 도 13에서 보듯이, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)의 조직에서 상기 사이토케라틴의 발현수준이 다른 실험군의 조직에 비하여 높은 수준을 나타냄을 확인하였다.FIG. 13 is an immunofluorescence staining photograph showing the result of comparing the level of skin cells present in the wound area of the skin wound mouse model according to each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT), and red is skin. Cytokeratin, a cell marker. As shown in FIG. 13, it was confirmed that the expression level of the cytokeratin in the tissue of the experimental group (spheroid + LLLT) irradiated with the spheroid and irradiated the LED showed a higher level than that of the other experimental groups.
따라서, 스페로이드를 이식하고, LED를 조사한 경우에는 혈관재생도 함께 촉진되는 효과를 나타냄을 알 수 있었다.Therefore, when the spheroid was implanted and the LED was irradiated, the blood vessel regeneration was also promoted.
실시예 11: 혈관 및 피부 재생인자의 발현수준 확인Example 11 Confirmation of Expression Levels of Blood Vessels and Skin Regeneration Factors
지방조직유래 중간엽 줄기세포로부터 형성된 스페로이드가 혈관신생 및 피부재생인자로서 알려진 FGF, VEGF 및 HGF의 발현량에도 효과를 나타내는지를 검증하고자 면역형광염색 분석 및 웨스턴 블럿 분석을 수행하였다.To verify whether the spheroids formed from adipose tissue-derived mesenchymal stem cells had an effect on the expression levels of FGF, VEGF and HGF known as angiogenesis and skin regeneration factors, immunofluorescence staining and Western blot analysis were performed.
실시예 11-1: 면역형광염색 분석Example 11-1: Immunofluorescence Staining Assay
면역형광염색 분석을 수행하기 위하여, 상기 실시예 8에서 설정한 각 실험군의 마우스로부터 얻어진 상처부위 조직을 대상으로 하고, 혈관신생 및 피부재생인자로서 알려진 FGF, VEGF 및 HGF를 대상으로 면역형광염색을 수행하는 것을 제외하고는, 상기 실시예 9과 동일한 방법을 수행하고, 면역형광염색된 조직을 형광현미경으로 분석하였다(도 14의 a). 이때, 내부대조군으로는 HNA를 사용하였다.In order to perform immunofluorescence staining analysis, the wound tissue obtained from the mice of each experimental group set in Example 8 was subjected to immunofluorescence staining for FGF, VEGF and HGF known as angiogenesis and skin regeneration factors. Except that, the same method as in Example 9 was carried out, and immunofluorescent stained tissue was analyzed by fluorescence microscope (Fig. 14a). At this time, HNA was used as the internal control group.
도 14의 a는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관신생 및 피부재생인자의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 비교한 결과를 나타내는 면역형광염색 사진으로서, 적색은 각각의 혈관신생 및 피부재생인자인 FGF, VEGF 및 HGF를 나타낸다. 도 14의 a에서 보듯이, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)의 조직에서 상기 혈관신생 및 피부재생인자의 발현수준이 다른 실험군의 조직에 비하여 높은 수준을 나타냄을 확인하였다.Figure 14a is an immunofluorescence staining showing the results of comparing the levels of angiogenesis and skin regeneration factors present in the wound area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) As a photograph, the red color represents FGF, VEGF and HGF, respectively, angiogenesis and skin regeneration factors. As shown in a of FIG. 14, it was confirmed that the expression level of the angiogenesis and skin regeneration factor in the tissue of the experimental group (spheroid + LLLT) implanted with spheroid and irradiated with LED showed higher level than that of other experimental groups. .
실시예 11-2: 웨스턴 블럿 분석Example 11-2: Western Blot Analysis
웨스턴 블럿 분석을 수행하기 위하여, 상기 상기 실시예 8에서 설정한 각 실험군의 마우스로부터 얻어진 상처부위 조직에서 발현된 혈관신생 및 피부재생인자로서 알려진 FGF, VEGF 및 HGF를 대상으로 웨스턴 블럿 분석을 수행하였다(도 14의 b 및 c).In order to perform Western blot analysis, Western blot analysis was performed on FGF, VEGF and HGF known as angiogenesis and skin regeneration factors expressed in wound tissue obtained from the mice of each experimental group set in Example 8. (B and c of FIG. 14).
도 14의 B는 피부 창상 마우스 모델의 상처부위에 존재하는 혈관신생 및 피부재생인자의 수준을 각각의 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로 웨스턴 블럿 분석에 의해 측정한 결과를 나타내는 사진이고, 도 14의 C는 상기 웨스턴 블럿 분석에 의해 검출된 각 혈관신생 및 피부재생인자의 수준을 각각의 처리조건별로 비교한 결과를 나타내는 그래프이다. 도 14의 b 및 c에서 보듯이, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)의 조직에서 상기 혈관신생 및 피부재생인자의 발현수준이 다른 실험군의 조직에 비하여 높은 수준을 나타냄을 다시한번 확인하였다.14B is a result of Western blot analysis of the levels of angiogenesis and skin regeneration factors present in the wound area of the skin wound mouse model for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT) 14C is a graph showing the results of comparing the levels of each angiogenesis and skin regeneration factor detected by the Western blot analysis for each treatment condition. As shown in b and c of Fig. 14, the expression level of the angiogenesis and skin regeneration factor in the tissue of the experimental group (spheroid + LLLT) irradiated with a spheroid and irradiated LED shows a higher level than the tissues of other experimental groups Check again.
따라서, 스페로이드를 이식하고, LED를 조사한 경우에는 혈관 및 피부 재생인자의 발현수준이 가장 높은 수준을 나타냄을 알 수 있었다.Therefore, when the spheroid was implanted and the LED was irradiated, the expression level of blood vessels and skin regeneration factors showed the highest level.
실시예 12: skin flap 마우스 모델을 통해 LED 조사한 스페로이드 이식 및 LED 처리의 조합 효과 검증Example 12: Validation of the Combination Effect of Spheroid Implantation and LED Treatment with LED Irradiation through a Skin Flap Mouse Model
먼저, 누드마우스를 마취시키고, 이의 등 부위의 피부조직을 가로 2cm, 세포 4cm를 절개한 후 절개한 피부를 다시 봉합하여 skin flap 마우스 모델을 제작하였다.First, anesthetized the nude mouse, and cut the skin tissue of the back region 2cm, the cell 4cm and then sutured the incision skin again to prepare a skin flap mouse model.
다음으로, 상기 제작된 skin flap 마우스 모델에 600㎕의 PBS를 주사한 대조군(control), 1.5 × 106 세포수의 지방조직유래 중간엽 줄기세포를 이식한 실험군(ASCs), LED 광원을 이용하여 매일 50 mW/㎠ 및 660nm 파장의 빛을 10분씩 조사한 실험군(low level light thearapy; LLLT), LED를 조사한 15개의 스페로이드를 이식한 실험군(spheroid) 및 LED를 조사한 15개의 스페로이드를 이식하고, LED를 조사한 실험군(spheroid + LLLT)을 각각 설정하고, 14일동안 사육한 다음 손상된 피부조직의 상처 치료효과를 확인하였다(도 15). Next, a control group injected with 600 μl of PBS in the prepared skin flap mouse model, an experimental group transplanted with adipose tissue-derived mesenchymal stem cells of 1.5 × 10 6 cells (ASCs), using an LED light source A group of low level light thearapy (LLLT) irradiated with light of 50 mW / cm 2 and 660 nm wavelength for 10 minutes each day, a spheroid implanted with 15 spheroids irradiated with LED, and 15 spheroids irradiated with LEDs were implanted. Each experimental group (spheroid + LLLT) irradiated with LED was set, and bred for 14 days, and then the wound treatment effect of the damaged skin tissue was confirmed (FIG. 15).
도 15는 skin flap 마우스 모델을 이용한 상처치료효과를 비교한 결과를 나타내는 사진이다. 도 15에서 보듯이, 지방조직유래 중간엽 줄기세포를 이식(ASCs)하거나, LED를 조사(LLLT)하거나 또는 스페로이드를 이식한 실험군(spheroid)은 상호 유사한 수준의 상처치료 효과가 나타났으나, 스페로이드를 이식하고, LED를 조사한 실험군(spheroid+LLLT)은 현저히 우수한 상처치료 효과를 나타냄을 확인하였다.15 is a photograph showing the results of comparing the wound treatment effect using the skin flap mouse model. As shown in FIG. 15, spheroid transplanted with adipose tissue-derived mesenchymal stem cells (ASCs), LED irradiation (LLLT), or spheroids showed similar wound healing effects. The experimental group (spheroid + LLLT) implanted with spheroids and irradiated with LEDs showed remarkably excellent wound healing effect.
실시예 13: Skin flap 마우스 모델에서 혈류 측정Example 13: Blood flow measurement in a skin flap mouse model
상기 Skin flap 마우스 모델에서 피부혈류(cutaneous blood flow)의 흐름을 촬영하였다. 구체적으로, 상기 실시예 12에서 설정한 각 실험군의 마우스를 37℃ 열판에 위치시키고, Laser Doppler blood flowmeter(Laser Doppler Perfusion Imager System PeriScan PIM 3 System; Perimed AB, Stockholm, Sweden)를 사용하여 스탠함으로써, 레이저 도트 칼라 이미지를 촬영하였다(도 16).In the skin flap mouse model, cutaneous blood flow was photographed. Specifically, by placing the mouse of each experimental group set in Example 12 on a 37 ℃ hot plate, and using a laser Doppler blood flow meter (Laser Doppler Perfusion Imager System PeriScan PIM 3 System; Perimed AB, Stockholm, Sweden), Laser dot color images were taken (FIG. 16).
도 16은 skin flap 마우스 모델에서 촬영된 피부혈류의 흐름을 처리조건(control, ASCs, LLLT, spheroid 및 spheroid+LLLT) 별로, 1, 7 및 14일이 경과된 시점에서 비교한 결과를 나타내는 사진이다. 도 16에서 보듯이, 지방조직유래 중간엽 줄기세포를 이식(ASCs)하거나, LED를 조사(LLLT)하거나 또는 스페로이드를 이식한 실험군(spheroid)은 상호 유사한 수준의 혈류량이 측정되었으나, 스페로이드를 이식하고 LED를 조사한 실험군(spheroid+LLLT)은 현저히 증가된 혈류량이 측정됨을 확인하였다. 상기 혈류량은 손상된 혈관이 존재할 경우 감소되고, 손상된 혈관이 치료되면 혈류량이 증가되므로, 상기 결과로부터 스페로이드를 이식하고 LED를 조사한 실험군(spheroid+LLLT)은 손상된 혈관을 높은 수준으로 치료하는 효과를 나타냄을 알 수 있었다.16 is a photograph showing the results of comparing the flow of skin blood taken in the skin flap mouse model at the time of 1, 7 and 14 days for each treatment condition (control, ASCs, LLLT, spheroid and spheroid + LLLT). . As shown in FIG. 16, spheroids implanted with adipose tissue-derived mesenchymal stem cells (ASCs), LEDs (LLLT), or spheroids were measured at similar levels of blood flow, but spheroids were measured. The experimental group (spheroid + LLLT) transplanted and irradiated with LED confirmed that the blood flow was significantly increased. The blood flow decreases when there is a damaged blood vessel, and when the damaged blood vessel is treated, the blood flow increases. Therefore, the experimental group (spheroid + LLLT) implanted with spheroids and irradiated with LEDs showed a high level of treatment of the damaged blood vessels. And it was found.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (19)

  1. 지방줄기세포로부터 유래된 스페로이드를 유효성분으로 포함하는, 적색 파장대의 빛을 이용한 피부 또는 혈관 조직 손상 치료용 약학 조성물.A pharmaceutical composition for treating skin or vascular tissue damage using light having a red wavelength, comprising a spheroid derived from adipose stem cells as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 스페로이드는 지방줄기세포를 배양하여 형성되는 것인, 조성물.The spheroid is formed by culturing fat stem cells.
  3. 제1항에 있어서,The method of claim 1,
    상기 스페로이드는 지방줄기세포에 적색 파장대의 빛을 조사하여 형성되는 것인, 조성물.The spheroid is formed by irradiating adipose stem cells with light in the red wavelength band.
  4. 제2항에 있어서,The method of claim 2,
    상기 스페로이드의 직경은 0.5 내지 1mm 인 것인, 조성물.The diameter of the spheroid is 0.5 to 1mm, the composition.
  5. 제2항에 있어서,The method of claim 2,
    상기 배양은 표면이 소수성을 띠는 배양용기를 사용하여 수행되는 것인, 조성물.The culturing is carried out using a culture vessel having a hydrophobic surface.
  6. 제5항에 있어서,The method of claim 5,
    상기 배양용기는 소수성을 부여하는 고분자 물질로 표면 처리된 배양용기 또는 상기 고분자 물질로 제조된 배양용기인 것인, 조성물.Wherein the culture vessel is a culture vessel surface-treated with a polymer material to impart hydrophobicity or a culture vessel made of the polymer material, composition.
  7. 제2항에 있어서,The method of claim 2,
    상기 배양시 지방줄기세포의 접종밀도는 1 × 105 내지 2 × 106 세포/㎠인 것인, 조성물.The inoculation density of the adipose stem cells in the culture is 1 × 10 5 to 2 × 10 6 cells / ㎠, the composition.
  8. 제1항에 있어서,The method of claim 1,
    상기 적색 파장대의 빛은 600 내지 700nm의 파장을 갖는 것인, 조성물.The light in the red wavelength range, the composition having a wavelength of 600 to 700nm.
  9. 제1항에 있어서,The method of claim 1,
    상기 스페로이드는 상기 적색 파장대의 빛에 의하여 생체내에서 혈관내피세포로 분화되어, 상기 적색 파장대의 빛에 의하여 촉진되는 생체내 자가 회복력을 보조하는 것인, 조성물.The spheroid is differentiated into vascular endothelial cells in vivo by light of the red wavelength band, to assist in vivo self-healing promoted by light of the red wavelength band.
  10. 제1항에 있어서,The method of claim 1,
    상기 스페로이드는 상기 적색 파장대의 빛에 의하여 생체내에서 피부세포로 분화되어, 상기 적색 파장대의 빛에 의하여 촉진되는 생체내 자가 회복력을 보조하는 것인, 조성물.The spheroid is differentiated into skin cells in vivo by light of the red wavelength band, to assist in vivo self-healing promoted by light of the red wavelength band.
  11. 지방유래 중간엽 줄기세포를 표면이 소수성을 띠는 배양용기에 접종하고 배양하면서, 5 내지 30 mW/㎠ 및 600 내지 700nm 파장의 빛을 상기 지방유래 중간엽 줄기세포 또는 스페로이드를 형성한 상태의 지방유래 중간엽 줄기세포에 조사하는 단계를 포함하는, 지방유래 중간엽 줄기세포를 혈관내피세포로 분화시키는 방법.While inoculating and incubating adipose derived mesenchymal stem cells into a culture vessel having a hydrophobic surface, the light of 5 to 30 mW / cm 2 and 600 to 700 nm wavelengths formed the adipose derived mesenchymal stem cells or spheroids. A method of differentiating adipose derived mesenchymal stem cells into vascular endothelial cells, comprising the step of irradiating adipose derived mesenchymal stem cells.
  12. 제11항에 있어서,The method of claim 11,
    상기 소수성을 띠는 배양용기는 소수성을 부여하는 고분자로 표면 처리된 배양용기 또는 상기 고분자로 제조된 배양용기인 것인, 방법.The hydrophobic culture vessel is a culture vessel surface-treated with a polymer imparting hydrophobicity or a culture vessel made of the polymer.
  13. 제11항에 있어서,The method of claim 11,
    상기 소수성을 부여하는 고분자는 폴리스티렌(polystyrene), 폴리메틸메타크릴레이트(PMMA), 폴리에틸렌 테레프탈레이트(PET), 폴리비닐클로라이트(PVC), 폴리에틸렌(PE), 폴리프로필렌(PP), 폴리테트라플루오르에틸렌(PTFE), 폴리(L-락트산)(PLLA), 폴리(D,L-락트산）(PDLLA), 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸렌카보네이트, 이들의 유도체 및 이들의 공중합체로 구성된 군으로부터 선택되는 하나 이상의 화합물인 것인, 방법.The polymer which gives the hydrophobicity is polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chlorite (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoro Ethylene (PTFE), poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyal) Canoate), polydioxanone (PDS), polytrimethylenecarbonate, derivatives thereof and copolymers thereof.
  14. 제11항에 있어서,The method of claim 11,
    지방유래 중간엽 줄기세포의 접종밀도는 1 × 105 내지 2 × 106 세포/㎠인 것인, 방법.The inoculation density of adipose derived mesenchymal stem cells is 1 × 10 5 to 2 × 10 6 cells / cm 2.
  15. 제11항에 있어서,The method of claim 11,
    상기 빛은 지방유래 중간엽 줄기세포로부터 1 내지 5cm의 간격을 두고 위치한 LED 광원을 이용하여 1일 10 내지 15분 동안 조사하는 것인, 방법.The light is to be irradiated for 10 to 15 minutes a day using an LED light source placed at intervals of 1 to 5 cm from adipose derived mesenchymal stem cells.
  16. 제11항에 있어서,The method of claim 11,
    상기 지방유래 중간엽 줄기세포는 LED 조사에 의하여 스페로이드를 형성한 다음, 혈관내피세포로 분화되는 것인, 방법.The fat-derived mesenchymal stem cells form spheroids by LED irradiation, and then differentiate into vascular endothelial cells.
  17. 제1항의 조성물을 피부 또는 혈관 조직이 손상된 개체에 투여하는 단계를 포함하는, 피부 또는 혈관 조직 손상의 치료방법.A method of treating skin or vascular tissue damage, comprising administering the composition of claim 1 to a subject having damaged skin or vascular tissue.
  18. 제17항에 있어서, The method of claim 17,
    적색 파장대의 빛을 상기 개체에 조사하는 단계를 추가로 포함하는 것인, 치료방법.And irradiating the subject with light in a red wavelength band.
  19. 제17항에 있어서, The method of claim 17,
    상기 피부 또는 혈관 조직 손상은 허혈성 피부 궤양, 피부상처, 동맥경화증, 안정형 및 불안정형 협심증, 말초 심혈관 질환, 고혈압, 심부전증, 말초 순환장애, 심근경색증, 뇌졸중, 일과성 및 허혈성 발작, 지주막하 출혈 또는 하지허혈로 이루어진 군에서 선택되는 하나 이상에 의해서 유발된 것인, 치료방법.The skin or vascular tissue damage may include ischemic skin ulcers, cuts, arteriosclerosis, stable and unstable angina, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulation, myocardial infarction, stroke, transient and ischemic attacks, subarachnoid hemorrhage or lower extremity And is caused by one or more selected from the group consisting of ischemia.
PCT/KR2015/006371 2014-06-23 2015-06-23 Pharmaceutical composition for treating skin or vascular tissue damage and use thereof WO2015199416A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2014-0076416 2014-06-23
KR1020140076416A KR101816964B1 (en) 2014-06-23 2014-06-23 Pharmaceutical adjuvant composition for treating damages of skin or blood vessel tissue
KR10-2014-0076412 2014-06-23
KR1020140076412A KR101719870B1 (en) 2014-06-23 2014-06-23 Mehtod for inducing differentiation of mesenchymal stromal cells using light with red wavelength

Publications (1)

Publication Number Publication Date
WO2015199416A1 true WO2015199416A1 (en) 2015-12-30

Family

ID=54938428

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/006371 WO2015199416A1 (en) 2014-06-23 2015-06-23 Pharmaceutical composition for treating skin or vascular tissue damage and use thereof

Country Status (1)

Country Link
WO (1) WO2015199416A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521900A (en) * 2022-07-23 2022-12-27 复旦大学附属华山医院 Visible light control angiogenesis method based on diode light modulation technology and application
WO2023214824A1 (en) * 2022-05-04 2023-11-09 (주)에스엔이바이오 Composition for wound treatment comprising stem cell-derived extracellular vesicles with enhanced efficacy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080091832A (en) * 2006-01-30 2008-10-14 유니버시티 오브 버지니아 페이턴트 파운데이션 Methods of preparing and characterizing mesenchymal stem cell aggregates and uses thereof
US20120219572A1 (en) * 2009-11-12 2012-08-30 Prockop Darwin J Spheroidal Aggregates of Mesenchymal Stem Cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080091832A (en) * 2006-01-30 2008-10-14 유니버시티 오브 버지니아 페이턴트 파운데이션 Methods of preparing and characterizing mesenchymal stem cell aggregates and uses thereof
US20120219572A1 (en) * 2009-11-12 2012-08-30 Prockop Darwin J Spheroidal Aggregates of Mesenchymal Stem Cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARBOZA ET AL., LOW-LEVEL LASER IRRADIATION INDUCES IN VITRO PROLIFERATION OF MESENCHYMAL STEM CELLS, vol. 12, no. 1, March 2014 (2014-03-01), Sao Paulo, pages 75 - 81, XP055597405 *
CHENG, NAI-CHEN ET AL.: "Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Sternness, Angiogenesis, and Chemotaxis", STEM CELLS TRANSLATIONAL MEDICINE, vol. 2, no. 8, 1 August 2013 (2013-08-01), pages 584 - 594, XP055340340 *
PARK, IN - SU: "Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation", PLOS ONE, vol. 10, no. 6, e0122776, 11 June 2015 (2015-06-11), pages 1 - 16, XP055597454 *
PARK, IN SU: "A novel three-dimensional adipose-derived stem cell cluster for vascular regeneration in ischemic tissue", CYTOTHERAPY, vol. 16, no. 4, April 2014 (2014-04-01), pages 508 - 522, XP055597391, DOI: 10.1016/j.jcyt.2013.08.011 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023214824A1 (en) * 2022-05-04 2023-11-09 (주)에스엔이바이오 Composition for wound treatment comprising stem cell-derived extracellular vesicles with enhanced efficacy
CN115521900A (en) * 2022-07-23 2022-12-27 复旦大学附属华山医院 Visible light control angiogenesis method based on diode light modulation technology and application

Similar Documents

Publication Publication Date Title
Turner et al. The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells
Marx et al. Adipose-derived stem cells in veterinary medicine: characterization and therapeutic applications
Cerqueira et al. Stem cells in skin wound healing: are we there yet?
WO2018101708A1 (en) Pharmaceutical composition containing mitochondria
Yao et al. Reversing bone loss by directing mesenchymal stem cells to bone
WO2013094988A1 (en) Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
WO2011049414A2 (en) Method for inducing migration of adult stem cells derived from adipose tissue
KR101413048B1 (en) Stem cell fusion model of carcinogenesis
Dunham et al. Adipose stem cells exhibit mechanical memory and reduce fibrotic contracture in a rat elbow injury model
WO2018143552A2 (en) Injectable composition for preventing hair loss or stimulating hair growth
Rashtbar et al. Critical‐sized full‐thickness skin defect regeneration using ovine small intestinal submucosa with or without mesenchymal stem cells in rat model
WO2018117573A1 (en) Multilayer neural crest stem cell sheet and method for manufacturing same
CN107073041A (en) The multi-functional stem cell of Diabetic Skin Ulcer treatment
Marmotti et al. Pulsed electromagnetic fields improve tenogenic commitment of umbilical cord-derived mesenchymal stem cells: a potential strategy for tendon repair—an in vitro study
WO2019004792A9 (en) Preparation method for and use of human-derived cardiac stem cell microspheroid
Koch et al. Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice
WO2015199416A1 (en) Pharmaceutical composition for treating skin or vascular tissue damage and use thereof
Ding et al. Galectin-1-induced skeletal muscle cell differentiation of mesenchymal stem cells seeded on an acellular dermal matrix improves injured anal sphincter
WO2013165120A1 (en) Method for culturing neural crest stem cells, and use thereof
Cancedda et al. Transit Amplifying Cells (TACs): a still not fully understood cell population
WO2015186906A1 (en) Autologous and allogeneic adipose-derived mesenchymal stem cell composition for healing tendon or ligament injury, and preparation method therefor
CN113846064A (en) FGF18 gene modified mesenchymal stem cell and preparation method and application thereof
KR101719870B1 (en) Mehtod for inducing differentiation of mesenchymal stromal cells using light with red wavelength
WO2020067774A1 (en) Synovium-derived mesenchymal stem cells and use thereof
WO2015023147A1 (en) Mtor/stat3 signal inhibitor-treated mesenchymal stem cell having immunomodulatory activity, and cell therapy composition comprising same, for preventing or treating immune disorders

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15811854

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15811854

Country of ref document: EP

Kind code of ref document: A1