WO2019112177A1 - Composition comprising stem cell-derived exosome containing culture as effective ingredient for preventing or treating arthritis - Google Patents

Composition comprising stem cell-derived exosome containing culture as effective ingredient for preventing or treating arthritis Download PDF

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WO2019112177A1
WO2019112177A1 PCT/KR2018/012942 KR2018012942W WO2019112177A1 WO 2019112177 A1 WO2019112177 A1 WO 2019112177A1 KR 2018012942 W KR2018012942 W KR 2018012942W WO 2019112177 A1 WO2019112177 A1 WO 2019112177A1
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stem cells
stem cell
arthritis
cell
cells
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PCT/KR2018/012942
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French (fr)
Korean (ko)
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강명희
최은경
서다움
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주식회사 디자인셀
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Priority to CN201880078641.5A priority Critical patent/CN112601533B/en
Publication of WO2019112177A1 publication Critical patent/WO2019112177A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to a composition for preventing or treating arthritis comprising an exosome-containing culture medium secreted from stem cells as an active ingredient.
  • RA Rheumatoid arthritis
  • OA osteoarthritis
  • ECM extracellular matrix
  • rheumatoid arthritis treatment pain and inflammation to relieve pain and anti-inflammatory agent is mainly prescribed, but can not fundamentally solve the immune response has not developed a causative agent.
  • mesenchymal stem cells can be obtained from various tissues such as bone marrow, fat, umbilical cord blood, and have excellent autolytic and proliferative capacity, and can be differentiated into cartilage tissue in vitro and in vivo, thereby being able to replace damaged joints.
  • mesenchymal stem cells can be obtained from various tissues such as bone marrow, fat, umbilical cord blood, and have excellent autolytic and proliferative capacity, and can be differentiated into cartilage tissue in vitro and in vivo, thereby being able to replace damaged joints.
  • UMBSC umbilical cord blood stem cells
  • ASC adipose tissue stem cells
  • CM conditioned medium
  • GF growth factors
  • NF neurotrophic factors
  • the concentrations of functional components such as cartilage regeneration promoting factors are too low in the culture medium of stem cells. Therefore, there is a problem in that arthritis treatment efficacy is lower than direct stem cell transplantation actually when stem cell culture medium alone is administered. Accordingly, the present inventors have focused on the fact that when the effective functional ingredient is secreted from the stem cells, they are secreted in the form of exosomes in the lipid layer. As a method of culturing normal stem cells, the exosome secretion amount is very small, Amniotic fluid stem cells (AFSC), amniotic membrane stem cells (AMSC), placental stem cells (PSC), and the like.
  • AFSC Amniotic fluid stem cells
  • AMSC amniotic membrane stem cells
  • PSC placental stem cells
  • UMBSC Umbilical cord blood stem cells
  • NSC neural stem cells
  • OPC oligodendrocyte progenitor cells
  • ASC adipose tissue stem cells
  • ERCM Exosome-rich conditioned medium
  • BMSC bone marrow stem cells
  • TNF- ⁇ tumor-necrosis factor- ⁇
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
  • the stem cell-derived exosome-containing culture solution may be obtained by treating a stem cell culture with TNF-? And culturing under an oxygen concentration of 1 to 8%.
  • TNF- ⁇ may be treated at a concentration of 5 to 500 ng / ml, and the culture may be performed for 12 to 72 hours.
  • the stem cells are selected from the group consisting of neural stem cells, rare progenitor precursor cells, adipose stem cells, amniotic stem cells, amniotic stem cells, placental stem cells, cord blood stem cells and bone marrow stem cells It can be selected.
  • the neural stem cell is a CBNU-NSC cell line (accession number: KCTC12635BP) which is an immortalized neural stem cell and the amniotic stem cell is a CBNU-AFSC cell line (accession number: KCTC12634BP)
  • the rare proliferating progenitor cell may be a CBNU-NSC.olig2 cell line (accession number: KCTC12636BP) which is an immortal rare proliferating progenitor cell.
  • the stem cells may be in the range of 1 ⁇ 10 4 to 1 ⁇ 10 6 cells.
  • the arthritis may be selected from the group consisting of osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and lupus.
  • the present invention relates to a composition for preventing or treating arthritis of a stem cell-derived exosome-containing culture medium, which comprises culturing a stem cell culture medium with TNF- ⁇ under the condition of oxygen concentration of 1 to 8%
  • the present invention relates to a pharmaceutical composition for preventing or treating arthritis. Since the stem cell-derived exosome-containing culture medium according to the present invention contains a large amount of exosome containing a functional active ingredient such as a cartilage regeneration promoting factor and the like, the conventional stem cell transplantation method for the treatment of arthritis and the treatment of the culture obtained under general stem cell culture conditions Compared to the control group. Therefore, the stem cell-derived exosome-containing culture solution according to the present invention can be used as a new therapeutic agent for effective arthritis treatment.
  • FIG. 1 is a photograph showing X-ray analysis of the osteoarthritis-induced group, the group treated with the stem cell-derived exosome-rich medium of the present invention and the group treated with the stem cell culture medium of the present invention,
  • the white arrowhead indicates the femur or tibial deformation, and the white arrow indicates the tibial sharpening.
  • FIG. 2 is a photograph showing the degree of osteoarthritis symptoms visually observed in the osteoarthritis-induced group, the group treated with the stem cell-derived exosome-rich culture medium of the present invention, and the group treated with the control stem cell culture medium.
  • the black arrowhead shows erosion and ulceration
  • the white arrowhead shows the meniscus adhesion.
  • FIG. 3 shows microscopic observation results of normal group, osteoarthritis-induced group, treated group of stem cell-derived exosome-rich medium of the present invention, and treated group of stem cell culture medium, and white arrowheads showed hematoxylin Purple dyed chondrocyte layer, and a black arrowhead indicates a peptidoglycans (PG) layer reddish in safranin O.
  • PG peptidoglycans
  • the present invention relates to a method for obtaining effective stem cell-derived active ingredients and enhancing the efficacy of stem cells in the course of research for more effectively using stem cells as a material for treating arthritis, It was confirmed that the cell-derived exosome-containing culture solution can effectively prevent or treat arthritis.
  • the present invention is characterized by providing a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
  • the method for increasing the secretion of exosome from the stem cells comprises culturing the stem cell culture medium under a condition of treating with TNF-? Under a hypoxic condition, preferably TNF-? ng / mL and culturing for 12 to 72 hours under an oxygen concentration of 1 to 8%. More preferably, the step may include culturing the stem cell culture solution at a concentration of 50 ng / mL of TNF-a and culturing for 24 hours under an oxygen concentration of 5%.
  • the term 'stem cell' refers to a cell that has not undergone differentiation into a specific cell, and is capable of differentiating into all kinds of cells constituting the body such as nerve, blood, cartilage and the like.
  • the types of stem cells from which exosomes can be secreted include, but are not limited to, neural stem cells, rare progenitor precursor cells, adipose stem cells, amniotic stem cells, amniotic stem cells, placental stem cells, Or bone marrow stem cells.
  • the neural stem cells used were the CBNU-NSC cell line (accession number: KCTC12635BP), an immortal neural stem cell, and the amniotic stem cell was a CBNU-AFSC cell line (accession number: KCTC12634BP) And the CBNU-NSC.olig2 cell line (accession number: KCTC12636BP), which is an immortalized rare progenitor precursor cell, was used as the rare proliferating progenitor cell.
  • CBNU-NSC cell line accession number: KCTC12635BP
  • the amniotic stem cell was a CBNU-AFSC cell line (accession number: KCTC12634BP)
  • CBNU-NSC.olig2 cell line accession number: KCTC12636BP
  • the primary cultured amniotic stem cells and neural stem cells are susceptible to hypoxic conditions and are easily killed.
  • the present inventors prepared immortalized stem cell lines, respectively, by recombining amniotic stem cells and neural stem cells, which die easily under hypoxic conditions, into immortalized stem cells. The viability of stem cells was increased and enough exosome rich culture medium was obtained.
  • the CBNU-AFSC cell line (KCTC12634BP), an immortalized amniotic stem cell, was used as the amniotic stem cell, and the CBNU-AFSC cell line (accession number: KCTC12634BP)
  • the CBNU-NSC.olig2 cell line (accession number: KCTC12636BP), which is an immortalized rare progenitor precursor cell, was deposited with a deposit number from the depository institution before the date of filing of the present patent application, and the immortalized cell line
  • the contents of each immortalized cell line can be referred to the registered patent documents described in the following embodiments.
  • 'exosome' is a vesicle of small size (30 ⁇ 150 nm) containing sophisticated RNA and protein transport material, and it is a membrane-structured necrosis secreted from various kinds of cells.
  • the precise molecular mechanisms and functions of secretion, absorption, and composition of exosomes have not yet been studied, but they are known to play a role in delivering membrane components, proteins and RNAs that bind to other cells and tissues.
  • the present inventors have found that, in order to increase the absorption rate of the active ingredient contained in the stem cell or stem cell culture liquid, a more effective method than the conventional technique in which liposomes composed of lipids are kneaded with the active ingredient is a method in which a large amount of exosome can be secreted from stem cells
  • stem cells culture medium is treated with TNF- ⁇ and cultured under oxygen concentration of 1 ⁇ 8%.
  • This culture condition is a separate treatment according to the liposome grafting method, foreign substance contamination and liposome grafting It is possible to solve such problems as deterioration of the components of the culture medium at the time of culture.
  • stem cells are cultured under the condition of 1 ⁇ 8% oxygen treatment with TNF- ⁇ in the stem cell culture medium, the secretion of exosome from the stem cells can be increased, and the culture medium containing the exosome- And at the same time can mass-produce exosomes from stem cells.
  • the present inventors have studied accelerators capable of promoting the secretion of exosomes from stem cells, and it has been found that TNF- ⁇ effectively promotes exocrine secretion among many substances, and TNF- the optimal treatment amount of? is preferably 5 to 500 ng / mL.
  • TNF- ⁇ Stem cells, like macrophages, are transported and activated by TNF- ⁇ . Activated macrophages secrete active oxygen radicals to attack microorganisms and tumors that enter the body, whereas activated stem cells have growth factors (GF) and neurotrophic factors NF) to secrete and protect tissue. These GF / NF proteins were found to be encapsulated in exosomes. Thus, in the present invention, when TNF-a is treated in the stem cell culture process, the secretion of a large amount of GF / NF-containing exosomes can be promoted and a large amount of GF / NF-containing exosomes can be obtained.
  • GF growth factors
  • NF neurotrophic factors
  • the stem cell culture solution can be used as long as it is a medium for culturing stem cells.
  • DMEM Denbecco's modified Eagle's medium
  • the stem cells treated with the TNF- ⁇ at a concentration of 5 to 500 ng / mL may preferably be stem cells of 1 ⁇ 10 4 to 1 ⁇ 10 6 cells.
  • the stem cells when the stem cells are cultured under the conditions of the present invention, it is possible to prevent the proliferation of the stem cells and the exosomes containing the physiologically active substances such as cell proliferation, differentiation and regeneration related genes, Secretion can be promoted and promoted, and secretion and production of exosome can be significantly increased as compared with the method for obtaining a culture medium for general stem cell (i.e., the method without culturing under conditions of TNF- ⁇ and hypoxic conditions).
  • the stem cell-derived exosome obtained by the method of the present invention has an effect of preventing or treating arthritis.
  • an osteoarthritis- (Normal culture medium) obtained from the stem cell-derived exosome-rich culture medium of the present invention and the stem cell-derived culture medium (normal culture medium) obtained by culturing under the 21% acidogenic condition were each administered into the joints and then the degree of symptom improvement of arthritis was analyzed Respectively.
  • the group treated with the stem cell-derived exosome-rich culture of the present invention showed better arthritis improvement and treatment effects than those of the normal culture treated group used as the control group, It is possible to inhibit the loss of cells and to prevent joint deformation such as meniscal adhesion, femur and tibial deformation and osteoproliferative formation, and to regenerate damaged cartilage (see Tables 1 to 4 Reference).
  • the inventors of the present invention found that the stem cell-derived exosome-containing culture solution according to the present invention is superior to the conventional stem cell itself or the culture medium containing exosome obtained under general stem cell culture conditions (culturing under 21% oxygen conditions) Or can be treated.
  • the present invention can provide a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
  • the arthritis may include osteoarthritis, rheumatoid arthritis Rheumatoid Arthritis, Fibromyalgia, gout or lupus.
  • the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method.
  • carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method . More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like.
  • Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like.
  • Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like.
  • excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like.
  • lubricants such as magnesium stearate and talc may also be used.
  • liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin .
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories.
  • the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
  • the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
  • injectable agents such as injection ampoules such as injection ampoules, injection agents such as injection bags, and aerosol formulations are preferably used as a parenteral administration preparation.
  • injectable ampoules such as injection ampoules, injection agents such as injection bags, and aerosol formulations are preferably used as preferably used.
  • the injectable ampoules can be mixed with injections immediately before use, Physiological saline, glucose, Ringer's solution and the like can be used.
  • compositions of the present invention may be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intra-dermal, or intra-articular injection, and are preferably administered directly to the lesion site Lt; / RTI >
  • the pharmaceutical composition of the present invention may be administered orally or parenterally depending on various factors including the activity, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated, And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and is preferably from 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
  • the pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions.
  • AFSC immortalized amniotic fluid stem cells
  • NSC immortalized neural stem cells
  • OPC immortalized rare proliferative progenitor cells
  • an immortal rare proliferative progenitor cell was used.
  • the content of the registered patent for each cell line can be referred to.
  • the amniotic stem cells were the CBNU-AFSC cell line, the immortal amniotic stem cell, the CBNU-NSC cell line, the immortalized neural stem cell, The CBNU-NSC.olig2 cell line, a rare proliferating progenitor cell, was used for analysis.
  • amniotic membrane stem cells (AMSC), placental stem cells (PSC), and umbilical cord blood stem cells (UCBSC) were collected and used for the consent of the mother during normal birth.
  • Stem cells (BMSC) were obtained from abdominal fat aspiration and bone marrow aspiration from normal 53-year-old and 50-year-old women, respectively, after reviewing the Institutional Review Board (IRB) Respectively.
  • Each of the prepared stem cells (1 x 10 6 cells / mL) was inoculated into Dulbecco's modified Eagle's medium (DMEM) medium and cultured in a 5% carbon dioxide (CO 2 ) feeding incubator at 37 ° C for 24 hours.
  • DMEM Dulbecco's modified Eagle's medium
  • CO 2 carbon dioxide
  • TNF- ⁇ was added to the culture medium at a concentration of 50 ng / mL, the oxygen concentration was lowered to 5%, and cultured for 24 hours Then, the culture solution was sampled, filtered, and concentrated 10 times at a low temperature to obtain a stem cell-derived exosome-rich culture according to the present invention.
  • the treatment with TNF- ⁇ (50 ng / mL) and the concentration of oxygen (O 2 ) (Normal method) to be used as a comparative example (control group) was obtained by carrying out the same procedure except that the cultured cells were maintained in a normal culture medium (CM).
  • CM normal culture medium
  • BDNF hepatocyte growth factor
  • HGF hepatocyte growth factor
  • TGF- ⁇ vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • amniotic fluid stem cell culture medium contained a large amount of HGF, and also BDNF, TGF and VEGF.
  • Amniotic membrane stem cells also secreted a large amount of HGF and a considerable amount of VEGF.
  • Placental stem cells secreted a relatively small amount of protein components other than HGF, whereas cord blood stem cells secreted a large amount of HGF and VEGF.
  • neural stem cells and progenitor precursor cells, neuronal cells secrete a large amount of TGF as well as a large amount of BDNF, NGF, and PDGF.
  • bone marrow stem cells secrete moderate levels of HGF and PDGF.
  • the exosome-rich culture medium of the present invention obtained by treating the stem cells with TNF-? And hypoxia contained effective factors in the content of 2.5 ⁇ 3.5 times higher than that in the normal culture medium (control group) And thus it is possible to exhibit superior efficacy in the treatment of various diseases (see Table 1 below).
  • NZW Male New Zealand white (NZW) rabbits at 8 months of age (3.5 kg) were used after about 2 weeks of laboratory purification. Animals were housed in a rabbit cage at a temperature of 23 ⁇ 2 ° C, relative humidity of 55 ⁇ 10%, ventilation frequency of 12 times / hour, lighting cycle of 12 hours (07:00 - 19:00) The irradiance was adjusted to 150-300 lux. The feed was fed with Purina Rabbit Chow ® , a pelleted solid feed for laboratory animals, and sterile purified water. The experiment was conducted by the Institutional Animal Care and Use Committee of IACUC ) In accordance with the Standard Operating Procedures (SOP) of the Agency.
  • SOP Standard Operating Procedures
  • the rabbits raised in ⁇ 1-1> were injected intramuscularly with ketamine (50 mg / kg) and xylazine (5 mg / kg) to remove the hair around the right knee. Betadine And 70% ethanol, and the operation was started aseptically.
  • the skin was incised, the fascia and the capsular incision were made (the medial joint approach), and the anterior cruciate ligament was exposed as shown in the drawing of the knee osteotomy (patella).
  • the medial meniscotibial ligament (MMTL) and the lateral meniscotibial ligament (LMTL) were sutured and then sutured with a surgical knife.
  • the antibiotic Foxolin (Samjin Pharm, 10 mg / kg) and the analgesic Maritrol (Jeil Pharm, 3 mg / kg) were injected intramuscularly twice daily and then Foxolin was administered once daily for 3 days.
  • ERCM stem cell-derived exosome-rich culture
  • CM normal culture medium
  • F femur
  • T tibia
  • MM medial meniscus
  • ACL anterior cranial cruciate ligament
  • MMTL medial meniscotibial ligament Ligament
  • LMTL lateral meniscotibial ligament (lateral meniscotibial ligament).
  • the KL score which is an index of analysis, was calculated as the ratio of the femur to the medial femoral condyle (score 0-5), the femur or tibia deformity (score 0-5) (score 0 ⁇ 15) of the three indicators (sharpening of tibial spine, score 0 ⁇ 5).
  • the rabbit animal model was evaluated by radiographic examination after osteoarthritis induced osteoarthritis through anterior cruciate ligament and internal and external medial tibial tibial ligament resection, abnormal findings of femoral head osteogenesis, medial tibial deformity, ) Of the total score was 10.1 points out of 15 points, indicating that severe arthritis was induced (see Fig. 1 and Table 2 below).
  • CM 0.1 mL / rabbit
  • neural stem cells mean lesion 6.0 (7.1 points) ⁇ cord blood stem cells (7.3 points)> placental stem cells (8.5 points) ⁇ cord blood stem cells (6.1 points)> adipose stem cells (6.4 points)
  • Point bone marrow stem cells (8.5 points), showing excellent therapeutic efficacy against radiological findings.
  • exosome-rich culture medium (ERCM) of the present invention obtained by adding TNF- ⁇ (50 ng / mL) to a stem cell culture medium and culturing the cells under a hypoxic concentration of 5% was administered, (5.2 points) ⁇ amniotic stem cells (5.4 points)> amniotic stromal cells (5.7 points) ⁇ amniotic stem cells (5.7 points) ⁇ amniotic stem cells (6.8 points) umbilical cord blood stem cells (6.2 points) placenta stem cells (6.8 points) ⁇ bone marrow stem cells (6.9 points).
  • ERCM stem cell-derived exosome-rich culture
  • CM control culture medium
  • O'Driscoll scoring analysis was performed by visual observation of knee joint edema, laceration, bony spur formation, synovial thickening, ie, changes in cartilage defect area, cartilage defect surface area, defect area and newly formed tissue
  • the continuity of the border was observed and photographed and the roughness of surface (score 0-5), adhesion with meniscus (score 0-5), hypertrophy of joint (score 0-5), osteoporosis
  • the treatment efficacy was evaluated by the sum of four indicators (score 0 ⁇ 20) for osteophyte (score 0 ⁇ 5).
  • the stem cell-derived exosome-rich culture medium of the present invention when administered, it was confirmed that the therapeutic effect was superior to that of the normal culture medium used as a control group in all the stem cells.
  • Neuronal stem cells (average 9.9 points in the lesion) Cells (10.0 points)> Adipose stem cells (10.7 points)> Amniotic stem cells (11.2 points)> Amniotic membrane stem cells (12.6 points)> Umbilical cord blood stem cells (13.1 points)> Placental stem cells (13.4 points) 13.5 points), indicating that the severity of the lesion was significantly improved.
  • the rabbit-derived joint tissue obtained in the above Experimental Example was fixed to 10% neutral formalin for 3 days, and then decalcified with EDTA for 60 days to prepare a paraffin section. Thereafter, photographs were taken with Hematoxylin & eosin (H & E) staining and the area of cartilage formation was quantitatively analyzed with a ratio of cartilage regeneration area / section width using ImageJ 5.1, a microscope image analysis program. The integrity of the hyaline cartilage was also analyzed by safranin O / fast green staining for peptidoglycals (PG) in cartilage.
  • H & E Hematoxylin & eosin
  • the degree of damage of the cartilage from the staining results was classified into structure (score 0-11), cartilage number (cellularity, score 0-4), cluster formation (score 0-5), cartilage cell component (PG, (score 0 to 6) (score 0 to 24) (see Table 4, below).
  • the stem cell-derived exosome-rich culture medium of the present invention when administered, the symptom of the lesion was improved better than that of the normal culture medium in all the stem cells.
  • neural stem cells (average lesion score 10.4) (11.5 points)> Amniotic Stem Cells (11.7 points)> Amniotic Stem Cells (11.9 points)> Umbilical Cord Blood Stem Cells (12.8 points)> Bone Marrow Stem Cells (14.0 points)> Placental Stem Cells And cells (14.3 points).
  • the stem cell-derived exosome-rich culture medium of the present invention is very effective for the treatment of arthritis (see FIG. 3 and Table 5).
  • IL-6 interleukin-6
  • IL-8 inflammatory cytokines
  • TNF- ⁇ , IL-6 and IL-8 which are cytokines in arthritis-induced arthritic fluid
  • the normal culture solution which is a control group
  • the amount of inflammatory cytokine was decreased.
  • amniotic stem cells, cord blood stem cells, neural stem cells and adipose stem cells showed an inflammatory cytokine reducing efficacy superior to that of rare proliferative progenitor cells, amniotic stem cells, placental stem cells and bone marrow stem cells.
  • the stem cell-derived exosome-rich culture medium of the present invention when administered, the effect of reducing inflammatory cytokines was significantly better in all of the stem cells than in the control culture medium-treated group.
  • Prodrug precursor cells suppress inflammatory cytokines better than cord blood stem cells, amniotic stem cells, placental stem cells and bone marrow stem cells (see Table 6 below).

Abstract

The present invention relates to a composition of a stem cell-derived exosome-containing culture for preventing or treating arthritis and, more particularly, to a pharmaceutical composition comprising a stem cell-derived exosome-containing culture as an effective ingredient for preventing or treating arthritis, wherein the stem cell-derived exosome-containing culture is obtained by treating a stem cell culture with TNF-α and incubating the stem cell culture in the condition of a 1-8 % oxygen concentration. Containing a large amount of exosomes containing functional effective ingredients such as a cartilage regeneration promoting factor, etc., the stem cell-derived exosome-containing culture according to the present invention was found to have a far higher therapeutic effect on arthritis, compared to conventional stem cell transplantation methods for treatment of arthritis and the application of cultures obtained in general stem cell culturing conditions. Hence, the stem cell-derived exosome-containing culture according to the present invention has the effect of being available as a new therapeutic agent for effective treatment of arthritis.

Description

줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는 관절염의 예방 또는 치료용 조성물Composition for preventing or treating arthritis comprising an exosome-containing culture medium derived from stem cells as an active ingredient
본 발명은 줄기세포로부터 분비된 엑소좀 함유 배양액을 유효성분으로 포함하는 관절염의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating arthritis comprising an exosome-containing culture medium secreted from stem cells as an active ingredient.
관절염은 통증, 강직, 운동기능 부전을 일으키는 가장 흔한 질병 중의 하나이다. 류마티스 관절염(rheumatoid arthritis, RA)은 콜라겐 II (collagen II)에 대한 자가면역 반응으로 인한 염증성 질환으로 관절의 부종과 통증을 동반한다. 이에 비해 골관절염(osteoarthritis, OA)은 관절의 퇴행성 변성을 특징으로 하며, 연골세포 및 조직의 파괴로 인한 통증과 관절변형을 유발한다. 특히 골관절염에서는 노화 및 세포사멸에 따라 연골세포(chondrocytes)의 수 및 기능저하로 peptidoglycans (PG), collagen II 및 aggrecan과 같은 정상적인 세포외기질(extracellular matrix, ECM)의 성분과 구조(architecture)의 소실이 가속화된다. 한편, 현재까지 류마티스 관절염 치료에는 통증과 염증완화를 위한 진통소염제가 주로 처방되고 있을 뿐 면역반응을 근본적으로 해결할 수 있는 원인치료제가 개발되지 못하고 있다. 또한 골관절염의 진행을 멈추고 회복시켜 줄 수 있는 치료제 역시 제시되어 있지 않으며, 단지 일시적으로 통증을 완화시켜 주거나 수술치료에 의존하고 있다.Arthritis is one of the most common diseases causing pain, stiffness, and motor deficits. Rheumatoid arthritis (RA) is an inflammatory disease caused by an autoimmune reaction to collagen II, which is accompanied by joint edema and pain. In contrast, osteoarthritis (OA) is characterized by degenerative degeneration of the joints and causes pain and joint deformation due to destruction of cartilage cells and tissues. In particular, in osteoarthritis, the number and function of chondrocytes are degraded by aging and apoptosis, resulting in loss of components and architecture of normal extracellular matrix (ECM) such as peptidoglycans (PG), collagen II and aggrecan Is accelerated. Meanwhile, until now, rheumatoid arthritis treatment pain and inflammation to relieve pain and anti-inflammatory agent is mainly prescribed, but can not fundamentally solve the immune response has not developed a causative agent. There is also no cure for stopping and restoring the progression of osteoarthritis, and it is only temporarily relieving pain or relying on surgical treatment.
이러한 관점에서 최근 중간엽줄기세포(mesenchymal stem cells, MSC)를 활용한 관절의 보호 및 재생을 도모할 수 있는 새로운 치료 전략이 큰 관심을 불러일으키고 있다. 특히 중간엽 줄기세포는 골수, 지방, 제대혈 등 여러 조직으로부터 얻을 수 있고, 자가분열과 증식능이 우수하며 체외 및 체내에서 연골조직으로 분화될 수 있어 손상된 관절을 대체할 수 있는 것으로 보고되고 있다. 실제로, 최근 제대혈 줄기세포(umbilical cord blood stem cells, UCBSC)와 지방줄기세포(adipose tissue stem cells, ASC)가 골관절염 동물모델과 임상시험에서 큰 부작용 없이 관절의 통증완화 및 연골세포 재생을 통해 관절손상을 개선시켜 줌이 입증된 바 있다. 하지만 자가줄기세포(autologous stem cells)에 비해 동종줄기세포(allogeneic stem cells)나 이종줄기세포(heterogenic stem cells)를 고용량으로 주사할 경우 종종 관절강 내 삼출물이 저류되는 문제점이 발생하고 있고 이를 해결하기 위한 방법으로 줄기세포의 직접 이식방법 보다 배양액(conditioned medium, CM)만의 주입으로 연골손상을 회복시켜 줄 수 있는 기술 연구가 진행 중에 있다. 이는 줄기세포가 다양한 생체기능을 매개 및 조절할 수 있는 사이토카인(cytokines), 케모카인(chemokines), 성장인자(growth factors, GF), 신경영양인자(neurotrophic factors, NF) 등의 많은 분비인자(secretory factors)를 방출함으로써 손상조직의 회복을 촉진시켜 주는 곁분비효과(paracrine effect)를 발휘한다는 장점을 지니고 있으며, 더욱이 제대혈줄기세포, 지방줄기세포, 골수줄기세포(bone marrow stem cells, BMSC)와 같은 중간엽 줄기세포로부터 유리된 트롬보스폰딘2 (thrombospondin 2, TSP2)는 줄기세포를 연골세포로 분화시키고 연골세포 증식을 촉진시킴으로써 골관점염을 회복시켜 주는 것으로 알려져 있다.In this respect, a new therapeutic strategy for protecting and regenerating joints using mesenchymal stem cells (MSCs) has recently been of great interest. In particular, mesenchymal stem cells can be obtained from various tissues such as bone marrow, fat, umbilical cord blood, and have excellent autolytic and proliferative capacity, and can be differentiated into cartilage tissue in vitro and in vivo, thereby being able to replace damaged joints. Indeed, recently, umbilical cord blood stem cells (UCBSC) and adipose tissue stem cells (ASC) have been shown to reduce joint pain Has been proven to improve. However, when injected with a high dose of allogeneic stem cells or heterogenic stem cells compared with autologous stem cells, there is a problem that the exudates in the joints are often stored. To solve this problem, In this study, we are investigating the possibility of repairing cartilage damage by injection of conditioned medium (CM) rather than direct stem cell transplantation. This is because many stem cells secretory factors such as cytokines, chemokines, growth factors (GF), neurotrophic factors (NF) and the like which can mediate and control various biological functions. (Paracrine effect) that promotes the recovery of damaged tissue by releasing the stem cells, stem cells, and bone marrow stem cells (BMSC). It is known that thrombospondin 2 (TSP2) liberated from stem cells differentiates stem cells into chondrocytes and promotes chondrocyte proliferation, thereby restoring bony point of view salt.
하지만 줄기세포 배양액 내에는 연골재생 촉진인자 등 기능성 성분의 농도가 너무 낮아 줄기세포 배양액만의 투여로는 실제적으로 관절염 치료 효능이 줄기세포를 직접 이식하는 것에 비해 저조하다는 문제점이 있다. 이에 본 발명자들은 줄기세포로부터 유효 기능성분이 분비될 때 지질층 내 엑소좀(exosomes) 형태로 분비된다는 사실에 착안하여, 정상적인(일반적인) 줄기세포의 배양방법으로는 엑소좀 분비량이 매우 적기 때문에 엑소좀 분비량을 높일 수 있는 새로운 배양 조건을 확립하였고, 이를 위해 본 발명에서는 양수줄기세포(amniotic fluid stem cells, AFSC), 양막줄기세포(amniotic membrane stem cells, AMSC), 태반줄기세포(placental stem cells, PSC), 제대혈줄기세포(umbilical cord blood stem cells, UCBSC), 신경줄기세포(neural stem cells, NSC), 희소돌기교전구세포(oligodendrocyte progenitor cells, OPC), 지방줄기세포(adipose tissue stem cells, ASC) 및 골수줄기세포(bone marrow stem cells, BMSC)를 tumor-necrosis factor-α (TNF-α) 처리와 저산소 배양 조건 하에서 배양하여 엑소좀 풍부 배양액(exosome-rich conditioned medium, ERCM)을 수득하였으며, 상기 배양액의 우수한 관절염 치료효과를 확인함으로써 본 발명을 완성하였다.However, the concentrations of functional components such as cartilage regeneration promoting factors are too low in the culture medium of stem cells. Therefore, there is a problem in that arthritis treatment efficacy is lower than direct stem cell transplantation actually when stem cell culture medium alone is administered. Accordingly, the present inventors have focused on the fact that when the effective functional ingredient is secreted from the stem cells, they are secreted in the form of exosomes in the lipid layer. As a method of culturing normal stem cells, the exosome secretion amount is very small, Amniotic fluid stem cells (AFSC), amniotic membrane stem cells (AMSC), placental stem cells (PSC), and the like. , Umbilical cord blood stem cells (UCBSC), neural stem cells (NSC), oligodendrocyte progenitor cells (OPC), adipose tissue stem cells (ASC) Exosome-rich conditioned medium (ERCM) was prepared by culturing bone marrow stem cells (BMSC) under tumor-necrosis factor-α (TNF-α) Was beneficial, the present invention has been completed by confirming the excellent arthritis treatment effects of the culture medium.
따라서 본 발명의 목적은 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는, 관절염의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는, 관절염의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
또한 본 발명의 일실시예에 있어서, 상기 줄기세포 유래 엑소좀 함유 배양액은 줄기세포 배양액에 TNF-α를 처리하고 1~8%의 산소농도 조건 하에서 배양하여 수득한 것일 수 있다. In one embodiment of the present invention, the stem cell-derived exosome-containing culture solution may be obtained by treating a stem cell culture with TNF-? And culturing under an oxygen concentration of 1 to 8%.
또한 본 발명의 일실시예에 있어서, TNF-α는 5~500 ng/ml의 농도로 처리하고, 상기 배양은 12~72시간 배양하는 것일 수 있다.In one embodiment of the present invention, TNF-α may be treated at a concentration of 5 to 500 ng / ml, and the culture may be performed for 12 to 72 hours.
또한 본 발명의 일실시예에 있어서, 상기 줄기세포는 신경줄기세포, 희소돌기교전구세포, 지방줄기세포, 양막줄기세포, 양수줄기세포, 태반줄기세포, 제대혈 줄기세포 및 골수 줄기세포로 이루어진 군 중에서 선택되는 것일 수 있다.In one embodiment of the present invention, the stem cells are selected from the group consisting of neural stem cells, rare progenitor precursor cells, adipose stem cells, amniotic stem cells, amniotic stem cells, placental stem cells, cord blood stem cells and bone marrow stem cells It can be selected.
또한 본 발명의 일실시예에 있어서, 상기 신경줄기세포는 불멸화 신경줄기세포인 CBNU-NSC 세포주 (기탁번호: KCTC12635BP)이고, 상기 양수줄기세포는 불멸화 양수줄기세포인 CBNU-AFSC 세포주 (기탁번호: KCTC12634BP)이고, 상기 희소돌기교전구세포는 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주 (기탁번호: KCTC12636BP)일 수 있다.In another embodiment of the present invention, the neural stem cell is a CBNU-NSC cell line (accession number: KCTC12635BP) which is an immortalized neural stem cell and the amniotic stem cell is a CBNU-AFSC cell line (accession number: KCTC12634BP) , And the rare proliferating progenitor cell may be a CBNU-NSC.olig2 cell line (accession number: KCTC12636BP) which is an immortal rare proliferating progenitor cell.
또한 본 발명의 일실시예에 있어서, 상기 줄기세포는 1x104~1x106 세포수일 수 있다.In one embodiment of the present invention, the stem cells may be in the range of 1 × 10 4 to 1 × 10 6 cells.
또한 본 발명의 일실시예에 있어서, 상기 관절염은 골관절염(Osteoarthritis), 류마티스 관절염(Rheumatoid Arthritis), 섬유 근육통(Fibromyalgia), 통풍(gout) 및 루퍼스(lupus)로 이루어진 군 중에서 선택되는 것일 수 있다.In one embodiment of the present invention, the arthritis may be selected from the group consisting of osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and lupus.
본 발명은 줄기세포 유래 엑소좀 함유 배양액의 관절염 예방 또는 치료용 조성물에 관한 것으로, 줄기세포 배양액에 TNF-α를 처리하고 1~8%의 산소농도 조건 하에서 배양하여 수득한 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는 관절염 예방 또는 치료용 약학적 조성물에 관한 것이다. 본 발명에 따른 줄기세포 유래 엑소좀 함유 배양액은 연골 재생 촉진인자 등 기능성 유효성분을 함유한 엑소좀을 다량 함유하고 있어 종래 관절염 치료를 위한 줄기세포 이식 방법 및 일반적인 줄기세포 배양 조건 하에서 수득한 배양액 처리에 비해 월등히 우수한 관절염 치료 효과가 있음을 확인하였다. 따라서 본 발명에 따른 줄기세포 유래 엑소좀 함유 배양액은 효과적인 관절염 치료를 위한 새로운 치료제로 사용할 수 있는 효과가 있다.The present invention relates to a composition for preventing or treating arthritis of a stem cell-derived exosome-containing culture medium, which comprises culturing a stem cell culture medium with TNF-α under the condition of oxygen concentration of 1 to 8% The present invention relates to a pharmaceutical composition for preventing or treating arthritis. Since the stem cell-derived exosome-containing culture medium according to the present invention contains a large amount of exosome containing a functional active ingredient such as a cartilage regeneration promoting factor and the like, the conventional stem cell transplantation method for the treatment of arthritis and the treatment of the culture obtained under general stem cell culture conditions Compared to the control group. Therefore, the stem cell-derived exosome-containing culture solution according to the present invention can be used as a new therapeutic agent for effective arthritis treatment.
도 1은 골관절염을 유발시킨 군, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 처리한 군 및 대조군 줄기세포 배양액을 처리한 군에 대한 X-ray 분석 사진을 나타낸 것으로, 흰색 화살표는 대퇴골두 골증식체를 나타낸 것이고, 흰색 화살표머리는 대퇴골 또는 경골 변형을 나타낸 것이며, 흰색 화살표는 경골추(골극) 날카로워짐을 나타낸 것이다.FIG. 1 is a photograph showing X-ray analysis of the osteoarthritis-induced group, the group treated with the stem cell-derived exosome-rich medium of the present invention and the group treated with the stem cell culture medium of the present invention, The white arrowhead indicates the femur or tibial deformation, and the white arrow indicates the tibial sharpening.
도 2는 골관절염을 유발시킨 군, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 처리한 군 및 대조군 줄기세포 배양액을 처리한 군에 대한 골관절염 증상 정도를 육안으로 확인한 것을 나타낸 사진이다. 도 3에서 검정색 화살표머리는 미란 및 궤양을 나타낸 것이고, 흰색 화살표머리는 반월판 유착을 나타낸 것이다.FIG. 2 is a photograph showing the degree of osteoarthritis symptoms visually observed in the osteoarthritis-induced group, the group treated with the stem cell-derived exosome-rich culture medium of the present invention, and the group treated with the control stem cell culture medium. In Fig. 3, the black arrowhead shows erosion and ulceration, and the white arrowhead shows the meniscus adhesion.
도 3은 정상군, 골관절염을 유발시킨 군, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 처리한 군 및 대조군 줄기세포 배양액을 처리한 군에 대한 현미경 관찰 결과를 나타낸 것으로, 흰색 화살표 머리는 hematoxylin에 보라색으로 염색된 연골세포층을 나타낸 것이고, 검정색 화살표 머리는 safranin O에 붉게 염색된 peptidoglycans (PG) 층을 나타낸 것이다.FIG. 3 shows microscopic observation results of normal group, osteoarthritis-induced group, treated group of stem cell-derived exosome-rich medium of the present invention, and treated group of stem cell culture medium, and white arrowheads showed hematoxylin Purple dyed chondrocyte layer, and a black arrowhead indicates a peptidoglycans (PG) layer reddish in safranin O.
본 발명은 줄기세포를 관절염 치료제의 소재로 보다 효과적으로 사용하기 위한 연구를 진행하던 중, 줄기세포 유래 유효성분의 수득과 효능 증진을 위한 방법으로써, 줄기세포로부터 엑소좀의 분비를 증가시켜 수득한 줄기세포 유래 엑소좀 함유 배양액이 관절염을 효과적으로 예방 또는 치료할 수 있음을 확인하였다.The present invention relates to a method for obtaining effective stem cell-derived active ingredients and enhancing the efficacy of stem cells in the course of research for more effectively using stem cells as a material for treating arthritis, It was confirmed that the cell-derived exosome-containing culture solution can effectively prevent or treat arthritis.
따라서 본 발명은 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는, 관절염의 예방 또는 치료용 약학적 조성물을 제공함에 특징이 있다. Therefore, the present invention is characterized by providing a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
본 발명에 따른 줄기세포로부터 엑소좀의 분비를 증가시킬 수 있는 방법은, 줄기세포 배양액에 TNF-α를 처리하고 저산소 농도 조건 하에서 배양하는 단계를 포함하며, 바람직하게는 TNF-α를 5~500 ng/mL의 농도로 처리하고 1~8%의 산소농도 조건 하에서 12~72시간 배양하는 단계를 포함한다. 더욱 바람직하게는 줄기세포 배양액에 TNF-α를 50 ng/mL의 농도로 처리하고 5%의 산소농도 조건 하에서 24시간 배양하는 단계를 포함할 수 있다.The method for increasing the secretion of exosome from the stem cells according to the present invention comprises culturing the stem cell culture medium under a condition of treating with TNF-? Under a hypoxic condition, preferably TNF-? ng / mL and culturing for 12 to 72 hours under an oxygen concentration of 1 to 8%. More preferably, the step may include culturing the stem cell culture solution at a concentration of 50 ng / mL of TNF-a and culturing for 24 hours under an oxygen concentration of 5%.
본 발명에서 상기 ‘줄기세포’는 특정 세포로 분화가 진행되지 않은 세포로서 필요한 경우, 신경, 혈액, 연골 등 신체를 구성하는 모든 종류의 세포로 분화할 능력을 가진 세포를 말한다.In the present invention, the term 'stem cell' refers to a cell that has not undergone differentiation into a specific cell, and is capable of differentiating into all kinds of cells constituting the body such as nerve, blood, cartilage and the like.
본 발명에서 엑소좀이 분비될 수 있는 줄기세포의 종류로는 이에 제한되지는 않으나, 신경줄기세포, 희소돌기교전구세포, 지방줄기세포, 양막줄기세포, 양수줄기세포, 태반줄기세포, 제대혈 줄기세포 또는 골수 줄기세포일 수 있다.In the present invention, the types of stem cells from which exosomes can be secreted include, but are not limited to, neural stem cells, rare progenitor precursor cells, adipose stem cells, amniotic stem cells, amniotic stem cells, placental stem cells, Or bone marrow stem cells.
본 발명의 일실시예에서, 상기 신경줄기세포는 불멸화 신경줄기세포인 CBNU-NSC 세포주 (기탁번호: KCTC12635BP)를 사용하였고, 상기 양수줄기세포는 불멸화 양수줄기세포인 CBNU-AFSC 세포주 (기탁번호: KCTC12634BP)를 사용하였으며, 상기 희소돌기교전구세포는 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주 (기탁번호: KCTC12636BP)를 사용하였다.In one embodiment of the present invention, the neural stem cells used were the CBNU-NSC cell line (accession number: KCTC12635BP), an immortal neural stem cell, and the amniotic stem cell was a CBNU-AFSC cell line (accession number: KCTC12634BP) And the CBNU-NSC.olig2 cell line (accession number: KCTC12636BP), which is an immortalized rare progenitor precursor cell, was used as the rare proliferating progenitor cell.
이는 초대 배양 양수줄기세포와 신경계 줄기세포(신경줄기세포, 희소돌기교전구세포)는 저산소 환경에 취약하여 쉽게 사멸하는 문제점이 있고, 줄기세포로부터 기능성 성분을 포집하고 있는 엑소좀을 다량 분비하기 위해서는 줄기세포를 저산소 하에서 배양해야 하기에, 이를 해결하기 위한 방법으로 본 발명자들은 저산소 환경 하에서 쉽게 사멸하는 양수줄기세포와 신경계 줄기세포를 불멸화 줄기세포로 재조합하여 불멸화 특성을 갖는 줄기세포주를 각각 제조하였으며, 따라서 줄기세포의 생존성을 높일 수 있게 하였고 충분한 엑소좀 풍부 배양액을 얻을 수 있도록 하였다. This is because the primary cultured amniotic stem cells and neural stem cells (neural stem cells, rare progenitor precursor cells) are susceptible to hypoxic conditions and are easily killed. In order to secrete a large amount of exosomes collecting functional components from stem cells, In order to solve this problem, the present inventors prepared immortalized stem cell lines, respectively, by recombining amniotic stem cells and neural stem cells, which die easily under hypoxic conditions, into immortalized stem cells. The viability of stem cells was increased and enough exosome rich culture medium was obtained.
상기 기재된 불멸화 줄기세포인 불멸화 신경줄기세포인 CBNU-NSC 세포주 (기탁번호: KCTC12635BP)를 사용하였고, 상기 양수줄기세포는 불멸화 양수줄기세포인 CBNU-AFSC 세포주 (기탁번호: KCTC12634BP)를 사용하였으며, 상기 희소돌기교전구세포는 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주 (기탁번호: KCTC12636BP)는 본 발명자가 본 특허 출원일 이전에 기탁기관으로부터 기탁번호를 부여받았고, 이들 불멸화 세포주들에 대해서는 이미 본 발명자가 특허 출원을 진행하여 특허 등록을 받은 상태로서, 각 불멸화 세포주의 제조에 대한 내용은 하기 실시예에 기재된 등록특허 문헌을 참조할 수 있다.The CBNU-AFSC cell line (KCTC12634BP), an immortalized amniotic stem cell, was used as the amniotic stem cell, and the CBNU-AFSC cell line (accession number: KCTC12634BP) The CBNU-NSC.olig2 cell line (accession number: KCTC12636BP), which is an immortalized rare progenitor precursor cell, was deposited with a deposit number from the depository institution before the date of filing of the present patent application, and the immortalized cell line As the inventor has patented and registered the patent, the contents of each immortalized cell line can be referred to the registered patent documents described in the following embodiments.
한편, ‘엑소좀(exosome)’은 정교한 RNA 및 단백질 운반물질이 들어있는 작은크기(30~150 nm)의 소포로서, 여러 종류의 세포들로부터 분비되는 막 구조의 소낭체이다. 엑소좀의 분비, 흡수, 조성에 대한 정확한 분자기전과 기능은 아직까지 연구가 미비한 실정이며 다만, 다른 세포 및 조직에 결합하는 막 구성요소, 단백질 및 RNA를 전달하는 역할을 하는 것으로 알려져 있다.On the other hand, 'exosome' is a vesicle of small size (30 ~ 150 nm) containing sophisticated RNA and protein transport material, and it is a membrane-structured necrosis secreted from various kinds of cells. The precise molecular mechanisms and functions of secretion, absorption, and composition of exosomes have not yet been studied, but they are known to play a role in delivering membrane components, proteins and RNAs that bind to other cells and tissues.
이에 본 발명자들은 줄기세포 또는 줄기세포 배양액에 함유된 유효성분의 체내 흡수율을 높이기 위해 지질로 구성된 리포좀을 유효성분과 접목시키는 종래 기술에 비해 더욱 효과적인 방법으로서 줄기세포로부터 엑소좀이 다량 분비될 수 있는 조건을 규명하였는데 즉, 줄기세포 배양액에 TNF-α를 처리하고 1~8%의 산소농도 조건 하에서 배양하는 조건으로서, 이러한 배양 조건은 리포좀 접목 방법에 따른 별도의 처리과정, 외부 물질의 오염 및 리포좀 포접 시 배양액 성분의 변질과 같은 문제점을 해결할 수 있음을 알 수 있었다.Accordingly, the present inventors have found that, in order to increase the absorption rate of the active ingredient contained in the stem cell or stem cell culture liquid, a more effective method than the conventional technique in which liposomes composed of lipids are kneaded with the active ingredient is a method in which a large amount of exosome can be secreted from stem cells This means that stem cells culture medium is treated with TNF-α and cultured under oxygen concentration of 1 ~ 8%. This culture condition is a separate treatment according to the liposome grafting method, foreign substance contamination and liposome grafting It is possible to solve such problems as deterioration of the components of the culture medium at the time of culture.
따라서 줄기세포 배양액에 TNF-α를 처리하고 1~8%의 산소농도 조건 하에서 줄기세포를 배양할 경우, 줄기세포로부터 엑소좀의 분비를 증가시킬 수 있어 줄기세포로부터 엑소좀이 풍부하게 함유된 배양액을 생산할 수 있고, 동시에 줄기세포로부터 엑소좀을 대량생산할 수 있다.Therefore, when stem cells are cultured under the condition of 1 ~ 8% oxygen treatment with TNF-α in the stem cell culture medium, the secretion of exosome from the stem cells can be increased, and the culture medium containing the exosome- And at the same time can mass-produce exosomes from stem cells.
특히 본 발명자들은 줄기세포로부터 엑소좀의 분비를 촉진할 수 있는 촉진제를 연구하던 중, 많은 물질들 중에서 TNF-α가 효과적으로 엑소좀 분비를 촉진하는 것을 알 수 있었고, 최대 엑소좀 분비를 위한 TNF-α의 적정 처리량은 5~500 ng/mL의 농도로 처리하는 것이 바람직하다는 것을 알 수 있었다.In particular, the present inventors have studied accelerators capable of promoting the secretion of exosomes from stem cells, and it has been found that TNF-α effectively promotes exocrine secretion among many substances, and TNF- the optimal treatment amount of? is preferably 5 to 500 ng / mL.
상기 범위를 초과하여 처리할 경우, 줄기세포에 손상을 주어 세포가 정상적인 기능을 발휘하지 못하는 문제점이 생길 수 있고, 반면 상기 범위에 미치지 못하는 양으로 처리할 경우 엑소좀의 분비량이 충분치 못하는 문제점이 생긴다.In the case of exceeding the above range, there is a problem that the stem cells are damaged and the cells can not exert their normal functions. On the other hand, when the treatment is carried out in an amount not exceeding the above range, there arises a problem that the exosome secretion amount is not sufficient .
줄기세포는 대식세포(macrophages)와 마찬가지로 TNF-α에 의해 이동하고 활성화된다. 활성화된 대식세포는 활성산소 라디칼(active oxygen radicals)을 분비하여 체내에 유입된 미생물과 종양을 공격하는 데 비해, 활성화된 줄기세포는 성장인자(growth factors, GF)와 신경성장인자(neurotrophic factors, NF)를 분비하여 조직을 보호하고 재생한다. 이러한 GF/NF 단백질은 엑소좀에 싸여 분비되는 것으로 밝혀졌다. 따라서 본 발명에서는 줄기세포 배양과정에서 TNF-α를 처리할 경우, 다량의 GF/NF 함유 엑소좀의 분비를 촉진시킬 수 있으며, 다량의 GF/NF 함유 엑소좀을 수득할 수 있다.Stem cells, like macrophages, are transported and activated by TNF-α. Activated macrophages secrete active oxygen radicals to attack microorganisms and tumors that enter the body, whereas activated stem cells have growth factors (GF) and neurotrophic factors NF) to secrete and protect tissue. These GF / NF proteins were found to be encapsulated in exosomes. Thus, in the present invention, when TNF-a is treated in the stem cell culture process, the secretion of a large amount of GF / NF-containing exosomes can be promoted and a large amount of GF / NF-containing exosomes can be obtained.
또한, 줄기세포로부터 관절염 치료 효능을 갖는 엑소좀의 다량 분비 유도를 위한 요건으로 정상농도(약 21% 산소)보다 현저히 낮은 산소농도 하에서의 배양이 필요함을 확인하였는데, TNF-α 처리 후 1~8%의 저산소 조건 하에서 12~72시간 배양하는 것이 바람직함을 알 수 있었다. 산소농도 조건이 1% 미만일 경우 세포의 정상적인 증식 및 성장에 문제를 발생시킬 수 있으며, 8%를 초과할 경우 엑소좀 분비가 충분하지 않을 수 있다. 따라서 1~8%의 저산소 조건 하에서 수행하는 것이 바람직하고 더욱 바람직하게는 5%의 산소 농도로 배양하는 것이 좋다.In addition, it was confirmed that culturing under the oxygen concentration which is significantly lower than the normal concentration (about 21% oxygen) is required as a requirement for the induction of the mass secretion of exosome having arthritis treatment effect from stem cells. Of hypoxic conditions for 12 to 72 hours. If the oxygen concentration is less than 1%, it may cause problems in normal cell proliferation and growth, and if it exceeds 8%, exosome secretion may not be sufficient. Therefore, it is preferable to conduct under the condition of 1 to 8% hypoxic, more preferably 5% of the oxygen concentration.
또한, 본 발명에 따른 방법에서 상기 줄기세포 배양액은 줄기세포를 배양하는 배지라면 모두 사용가능하며, 본 발명의 일실시예에서는 DMEM (Dulbecco’s modified Eagle’s medium) 배지를 사용하였다. In addition, in the method according to the present invention, the stem cell culture solution can be used as long as it is a medium for culturing stem cells. In one embodiment of the present invention, DMEM (Dulbecco's modified Eagle's medium ) medium is used.
상기 TNF-α를 5~500 ng/mL의 농도로 처리되어지는 줄기세포는 바람직하게 1x104~1x106 세포수의 줄기세포일 수 있다.The stem cells treated with the TNF-α at a concentration of 5 to 500 ng / mL may preferably be stem cells of 1 × 10 4 to 1 × 10 6 cells.
이와 같이 본 발명의 조건으로 줄기세포를 배양할 경우, 줄기세포가 증식하는 과정에서 분비되는 줄기세포의 세포증식, 분화, 재생관련 유전자, 단백질, 성장인자 등 생리활성 물질들을 함유하고 있는 엑소좀의 분비를 촉진 및 증진시킬 수 있으며, 일반 줄기세포 배양액 수득 방법(즉 TNF-α 및 저산소 조건 하에서의 배양을 수행하지 않은 방법)에 비해 엑소좀의 분비 및 생산량을 월등히 증가시킬 수 있음을 알 수 있었다.As described above, when the stem cells are cultured under the conditions of the present invention, it is possible to prevent the proliferation of the stem cells and the exosomes containing the physiologically active substances such as cell proliferation, differentiation and regeneration related genes, Secretion can be promoted and promoted, and secretion and production of exosome can be significantly increased as compared with the method for obtaining a culture medium for general stem cell (i.e., the method without culturing under conditions of TNF-α and hypoxic conditions).
또한, 본 발명에서는 본 발명의 방법으로 수득한 줄기세포 유래 엑소좀이 관절염을 예방 또는 치료하는 효과가 있음을 확인하였는데, 본 발명의 일실시예에 따르면 전방십자인대 절제술로 골관절염을 유발시킨 토끼 동물모델을 대상으로 본 발명의 줄기세포 유래 엑소좀 풍부 배양액 및 21% 산조 조건 하에서 배양하여 수득한 줄기세포 유래 엑소좀 함유 배양액(정상배양액)을 각각 관절강 내로 투여한 후, 관절염의 증상 개선 정도를 분석하였다.In addition, according to the present invention, it has been confirmed that the stem cell-derived exosome obtained by the method of the present invention has an effect of preventing or treating arthritis. According to one embodiment of the present invention, an osteoarthritis- (Normal culture medium) obtained from the stem cell-derived exosome-rich culture medium of the present invention and the stem cell-derived culture medium (normal culture medium) obtained by culturing under the 21% acidogenic condition were each administered into the joints and then the degree of symptom improvement of arthritis was analyzed Respectively.
그 결과, 대조군으로 사용한 정상배양액 처리 군에 비해 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 처리한 군이 줄기세포 종류별 모두 월등히 우수한 관절염 개선 및 치료 효과가 있는 것으로 나타났는데, 관절표면의 손상 및 연골세포의 소실을 억제할 수 있고, 반월판 유착, 대퇴골과 경골 변형 및 골증식체 형성과 같은 관절 변형을 방지할 수 있으며, 손상된 연골을 재생시킬 수 있는 효과가 있음을 확인하였다(표 1 내지 표 4 참조).As a result, the group treated with the stem cell-derived exosome-rich culture of the present invention showed better arthritis improvement and treatment effects than those of the normal culture treated group used as the control group, It is possible to inhibit the loss of cells and to prevent joint deformation such as meniscal adhesion, femur and tibial deformation and osteoproliferative formation, and to regenerate damaged cartilage (see Tables 1 to 4 Reference).
뿐만 아니라 관절염의 유발 및 증상 악화에 원인을 주는 염증성 사이토카인의 억제도 대조군으로 사용한 정상배양액 처리 군에 비해 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 처리한 군이 월등하게 우수한 것으로 나타났다(표 5 참조).In addition, inhibition of inflammatory cytokines that cause arthritis and aggravation of symptoms was superior to that of normal cells treated with the stem cell-derived exosome-rich culture of the present invention Reference).
따라서 본 발명자들은 본 발명에 따른 줄기세포 유래 엑소좀 함유 배양액이 종래 줄기세포 자체 또는 일반적인 줄기세포 배양조건(21% 산소 조건에서 배양)에서 수득한 엑소좀 함유 배양액에 비해 더 우수한 효과로 관절염을 예방 또는 치료할 수 있음을 확인하였다. Therefore, the inventors of the present invention found that the stem cell-derived exosome-containing culture solution according to the present invention is superior to the conventional stem cell itself or the culture medium containing exosome obtained under general stem cell culture conditions (culturing under 21% oxygen conditions) Or can be treated.
그러므로 본 발명은 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는, 관절염의 예방 또는 치료용 약학적 조성물을 제공할 수 있으며, 상기 관절염으로는 이에 제한되지는 않으나 골관절염(Osteoarthritis), 류마티스 관절염(Rheumatoid Arthritis), 섬유 근육통(Fibromyalgia), 통풍(gout) 또는 루퍼스(lupus)일 수 있다. Therefore, the present invention can provide a pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient. The arthritis may include osteoarthritis, rheumatoid arthritis Rheumatoid Arthritis, Fibromyalgia, gout or lupus.
또한, 본 발명에서 상기 약학 조성물은 통상의 방법에 따른 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 메틸히드록시 벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 사용할 수 있으나, 이에 제한되지 않는다. In addition, the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명에 따른 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상세하게는, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 본 발명의 약학 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose), 락토오스 (lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다. 또한, 비경구투여 제제로서 주사용 앰플과 같은 주사제, 주입 백과 같은 주입제, 및 에어로졸 제제와 같은 분무제 등이 바람직하며, 상기 주사용 앰플은 사용직전에 주사액과 혼합 조제할 수 있으며, 주사액으로는 생리 식염수, 포도당, 링거액 등을 사용할 수 있다. 또한, 주입 백은 염화폴리비닐 또는 폴리에틸렌 재질의 것을 사용할 수 있다.The pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method . More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like. In addition, as a parenteral administration preparation, injectable agents such as injection ampoules such as injection ampoules, injection agents such as injection bags, and aerosol formulations are preferably used. The injectable ampoules can be mixed with injections immediately before use, Physiological saline, glucose, Ringer's solution and the like can be used. The injection bag may be made of polyvinyl chloride or polyethylene.
본 발명의 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 관절 내 주사에 의해 투여될 수 있으며, 바람직하게는 치료가 필요한 개체의 병변 부위에 직접 투여할 수 있다. The compositions of the present invention may be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intra-dermal, or intra-articular injection, and are preferably administered directly to the lesion site Lt; / RTI >
본 발명의 약학 조성물은 사용된 특정 유효성분의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50 mg/kg 또는 0.001 내지 50 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 약학적 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally depending on various factors including the activity, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated, And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and is preferably from 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 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. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.
<준비예> <Preparation Example>
줄기세포Stem Cells
하기 실시예에서 관절염 치료 효과 여부를 확인하기 위한 유효성분인 엑소좀 풍부 줄기세포 배양액을 수득하기 위한 줄기세포는 다음과 같은 세포들을 사용하였다. 먼저, 불멸화 양수줄기세포(AFSC), 불멸화 신경줄기세포(NSC) 및 불멸화 희소돌기교전구세포(OPC)는 본 발명자가 이미 불멸화 세포주로 제조하여 확립한 세포주이며, 각 세포주들은 미생물 기탁기관으로부터 기탁번호를 부여받은 줄기세포주로서, 불멸화 양수줄기세포인 CBNU-AFSC 세포주 (KCTC12634BP; 대한민국 등록특허 제10-1719274호), 불멸화 신경줄기세포인 CBNU-NSC 세포주 (KCTC12635BP; 대한민국 등록특허 제10-1719274호) 및 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주를 (KCTC12636BP; 대한민국 등록특허 제10-1740357호 참조) 각각 사용하였다. 상기 불멸화 세포주들의 제조방법은 각 세포주에 대한 등록특허의 내용을 참고할 수 있다. 또한 하기 본 발명의 실시예 및 실험예에서 양수줄기세포는 불멸화 양수줄기세포인 CBNU-AFSC 세포주를 사용한 것이고, 신경줄기세포는 불멸화 신경줄기세포인 CBNU-NSC 세포주를 사용한 것이며, 희소돌기교전구세포는 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주를 사용하여 분석하였다.In the following examples, the following cells were used as stem cells for obtaining an exosome-rich stem cell culture solution, which is an effective ingredient for confirming the therapeutic effect of arthritis treatment. First, immortalized amniotic fluid stem cells (AFSC), immortalized neural stem cells (NSC), and immortalized rare proliferative progenitor cells (OPC) were established by the present inventor as an immortalized cell line. Each cell line was obtained from a microorganism depository (KCTC12634BP; Korean Patent No. 10-1719274), immortalized neural stem cell CBNU-NSC cell line (KCTC12635BP; Korean Patent No. 10-1719274) and stem cell line The CBNU-NSC.olig2 cell line (KCTC12636BP; see Korean Patent No. 10-1740357), an immortal rare proliferative progenitor cell, was used. For the preparation of the immortalized cell lines, the content of the registered patent for each cell line can be referred to. In the following Examples and Experiments of the present invention, the amniotic stem cells were the CBNU-AFSC cell line, the immortal amniotic stem cell, the CBNU-NSC cell line, the immortalized neural stem cell, The CBNU-NSC.olig2 cell line, a rare proliferating progenitor cell, was used for analysis.
또한, 양막줄기세포(AMSC), 태반줄기세포(PSC) 및 제대혈줄기세포(UCBSC)는 정상적인 출산 과정에서 산모의 동의하여 채취한 줄기세포들을 각각 수집하여 사용하였고, 지방줄기세포(ASC)와 골수줄기세포(BMSC)는 각각 53세와 50세의 정상인 여성으로부터 복부 지방흡입술과 골수천자를 통해 각각의 기관윤리위원회(Institutional Review Board, IRB) 심의를 거쳐 본인의 동의서를 받아 채취한 줄기세포를 사용하였다.  In addition, amniotic membrane stem cells (AMSC), placental stem cells (PSC), and umbilical cord blood stem cells (UCBSC) were collected and used for the consent of the mother during normal birth. Stem cells (BMSC) were obtained from abdominal fat aspiration and bone marrow aspiration from normal 53-year-old and 50-year-old women, respectively, after reviewing the Institutional Review Board (IRB) Respectively.
<실시예 1> &Lt; Example 1 >
<1-1> 줄기세포의 배양 및 엑소좀 풍부 배양액의 채취<1-1> Culture of stem cells and extraction of exosome-rich culture medium
상기 준비한 각각의 줄기세포(1 x 106 세포/mL)를 Dulbecco’s modified Eagle’s medium (DMEM) 배지에 접종하고 5% 이산화탄소(CO2) 공급 배양기에서 37℃의 온도로 24시간 동안 배양하였다. 이후 줄기세포로부터 엑소좀이 풍부하게 함유된 배양액(ERCM)을 수득하기 위해, 상기 배양 배지에 TNF-α를 50 ng/mL의 농도로 첨가하고 산소 농도를 5%로 낮춘 후, 24시간 동안 배양한 다음, 배양액을 채취하여 필터로 거른 후 저온에서 10배 농축하여 본 발명에 따른 줄기세포 유래 엑소좀 풍부 배양액을 수득하였다.Each of the prepared stem cells (1 x 10 6 cells / mL) was inoculated into Dulbecco's modified Eagle's medium (DMEM) medium and cultured in a 5% carbon dioxide (CO 2 ) feeding incubator at 37 ° C for 24 hours. In order to obtain a culture medium (ERCM) rich in exosomes from stem cells, TNF-α was added to the culture medium at a concentration of 50 ng / mL, the oxygen concentration was lowered to 5%, and cultured for 24 hours Then, the culture solution was sampled, filtered, and concentrated 10 times at a low temperature to obtain a stem cell-derived exosome-rich culture according to the present invention.
비교예로서, 각각의 줄기세포로부터 엑소좀이 함유된 배양액을 수득하는 과정에서, 상기 실시예 1의 과정 중, TNF-α (50 ng/mL) 처리와 산소(O2) 농도를 21%로 유지한 상태로 수행한 것으로 제외하고는 동일한 방법으로 수행하여, 비교예(대조군)로 사용할 일반적인 방법(정상적인 방법)의 줄기세포 정상배양액(CM)을 수득하였다.As a comparative example, in the course of obtaining the culture medium containing exosome from each stem cell, the treatment with TNF-α (50 ng / mL) and the concentration of oxygen (O 2 ) (Normal method) to be used as a comparative example (control group) was obtained by carrying out the same procedure except that the cultured cells were maintained in a normal culture medium (CM).
<1-2> 배양액 내 유효 분비인자 함량 분석<1-2> Analysis of efficacy factor content in culture medium
줄기세포 배양액을 채취하여 5분간 초음파로 엑소좀을 파쇄한 다음, Sephacryl gel filtration column을 장착한 protein liquid chromatograph를 사용하여 주요 기능성 분비인자[brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), nerve grown factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF)]의 함량을 분석하였다.(BDNF) and hepatocyte growth factor (HGF) were measured by using a protein liquid chromatograph equipped with a Sephacryl gel filtration column. (TGF-β), vascular endothelial growth factor (VEGF)] were analyzed by immunohistochemical staining.
분석 결과, 양수줄기세포 배양액에는 HGF가 다량 함유되어 있고, BDNF, TGF 및 VEGF 역시 상당량 함유되어 있는 것을 확인하였다. 양막줄기세포 역시 다량의 HGF와 상당량의 VEGF를 분비하고 있었으며, 태반줄기세포는 HGF 외에는 상대적으로 적은 량의 단백질 성분을 분비한 데 비해, 제대혈줄기세포는 다량의 HGF와 VEGF를 분비하였다. 흥미롭게도 신경계세포인 신경줄기세포와 희돌기교전구세포는 다량의 BDNF, NGF, PDGF는 물론, 어느 정도의 TGF도 분비하였는데, 희돌기교전구세포는 HGF도 상당량 배출하였다. 이에 비해 골수줄기세포는 중등도의 HGF와 PDGF를 분비하는 것으로 확인되었다.As a result of the analysis, it was confirmed that the amniotic fluid stem cell culture medium contained a large amount of HGF, and also BDNF, TGF and VEGF. Amniotic membrane stem cells also secreted a large amount of HGF and a considerable amount of VEGF. Placental stem cells secreted a relatively small amount of protein components other than HGF, whereas cord blood stem cells secreted a large amount of HGF and VEGF. Interestingly, neural stem cells and progenitor precursor cells, neuronal cells, secrete a large amount of TGF as well as a large amount of BDNF, NGF, and PDGF. In contrast, bone marrow stem cells secrete moderate levels of HGF and PDGF.
한편, 줄기세포를 TNF-α와 저산소 처리하여 얻어진 본 발명의 엑소좀 풍부 배양액 내에는 모든 줄기세포에서 정상적인 배양액(대조군) 내 농도에 비해 2.5~3.5배 높은 함량으로 유효인자가 함유되어 있는 것으로 확인되어 각종 질환의 치료에 더 우수한 효능을 보일 수 있음을 알 수 있었다(하기 표 1 참조).On the other hand, it was confirmed that the exosome-rich culture medium of the present invention obtained by treating the stem cells with TNF-? And hypoxia contained effective factors in the content of 2.5 ~ 3.5 times higher than that in the normal culture medium (control group) And thus it is possible to exhibit superior efficacy in the treatment of various diseases (see Table 1 below).
Figure PCTKR2018012942-appb-T000001
Figure PCTKR2018012942-appb-T000001
<실험예 1><Experimental Example 1>
본 발명의 줄기세포 유래 엑소좀 풍부 배양액의 관절염 치료 효과 분석Analysis of the effect of arthritis treatment of the stem cell-derived exosome-rich culture of the present invention
<1-1> 실험동물용 토끼의 사육 <1-1> Rabbit raising for experimental animals
생후 8개월령(3.5 kg)의 수컷 New Zealand white (NZW) 토끼를 약 2주간 실험실 순화과정을 거친 후 사용하였다. 동물을 토끼용 케이지에 1마리씩 수용하고, 동물실험실의 환경을 온도 23±2℃, 상대습도 55±10%, 환기 횟수 12 회/시간, 조명주기 12시간(07:00 - 19:00), 조도 150-300 lux로 조절하여 사육하였다. 사료는 실험동물용으로 공급되는 펠렛형 고형사료인 Purina Rabbit Chow®와 멸균정제수를 자유롭게 섭취하도록 하였으며, 본 실험은 충북대학교 실험동물연구지원센터의 동물실험윤리위원회(Institutional Animal Care and Use Committee, IACUC)의 승인 하에 동 기관의 표준작업지침서(Standard Operation Procedures, SOP)에 따라 수행하였다.Male New Zealand white (NZW) rabbits at 8 months of age (3.5 kg) were used after about 2 weeks of laboratory purification. Animals were housed in a rabbit cage at a temperature of 23 ± 2 ° C, relative humidity of 55 ± 10%, ventilation frequency of 12 times / hour, lighting cycle of 12 hours (07:00 - 19:00) The irradiance was adjusted to 150-300 lux. The feed was fed with Purina Rabbit Chow ® , a pelleted solid feed for laboratory animals, and sterile purified water. The experiment was conducted by the Institutional Animal Care and Use Committee of IACUC ) In accordance with the Standard Operating Procedures (SOP) of the Agency.
<1-2> 전방십자인대절제술(anterior cruciate ligament transection , ACLT )을 통한 골관절염 유발 및 본 발명의 줄기세포 유래 엑소좀 풍부 배양액 투여 <1-2> Osteoarthritis induction through anterior cruciate ligament transection ( ACLT ) and administration of the stem cell-derived exosome-rich culture of the present invention
상기 <1-1>에서 사육한 토끼를 대상으로 ketamine (50 mg/kg)과 xylazine (5 mg/kg)을 근육 내로 주사하여 전신 마취시키고, 우측 무릎 주변의 털을 제거한 다음 베타딘(Betadine)과 70% 에탄올로 피부를 소독한 후, 무균상태에서 수술을 개시하였다. 피부를 절개하고 근막과 관절낭을 절개(내측 관절 도달법)한 후, 무릎골(슬개골)을 젖혀 하기 도면과 같이 전방십자인대를 노출시켰다. 외과용 수술칼로 전방십자인대의 중간 실질부위와 내측반월판경골인대(medial meniscotibial ligament, MMTL) 및 외측반월판경골인대(lateral meniscotibial ligament, LMTL)를 절단한 후 봉합하였다. 수술 후 3일간 항생제인 Foxolin (삼진제약, 10 mg/kg)과 진통제 Maritrol (제일제약, 3 mg/kg)을 매일 2회 근육 내로 주사하였고, 이후 3일간 Foxolin을 매일 1회 추가로 처치하였다. 관절염 유발수술 8주 후에 상기 실시예 1에서 수득한 본 발명에 따른 줄기세포 유래 엑소좀 풍부 배양액(ERCM)과 대조군인 정상배양액(CM)을 0.1 mL/토끼의 양으로 매주 2일 간격으로 8주간 관절강 내로 투여하였다.The rabbits raised in <1-1> were injected intramuscularly with ketamine (50 mg / kg) and xylazine (5 mg / kg) to remove the hair around the right knee. Betadine And 70% ethanol, and the operation was started aseptically. The skin was incised, the fascia and the capsular incision were made (the medial joint approach), and the anterior cruciate ligament was exposed as shown in the drawing of the knee osteotomy (patella). The medial meniscotibial ligament (MMTL) and the lateral meniscotibial ligament (LMTL) were sutured and then sutured with a surgical knife. Three days after surgery, the antibiotic Foxolin (Samjin Pharm, 10 mg / kg) and the analgesic Maritrol (Jeil Pharm, 3 mg / kg) were injected intramuscularly twice daily and then Foxolin was administered once daily for 3 days. After 8 weeks of arthritis induction surgery, the stem cell-derived exosome-rich culture (ERCM) according to the present invention obtained in Example 1 and a normal culture medium (CM) as a control group were infused with 0.1 mL / rabbits every 2 days for 8 weeks Were administered intraarticularly.
Figure PCTKR2018012942-appb-I000001
Figure PCTKR2018012942-appb-I000001
상기 그림에서, F: femur (대퇴골), T: tibia (경골), MM: medial meniscus (내측반월판), ACL: anterior (cranial) cruciate ligament (전방십자인대), MMTL: medial meniscotibial ligament (내측반월판경골인대), LMTL: lateral meniscotibial ligament (외측반월판경골인대)를 나타낸다. In the above figure, F: femur, T: tibia, MM: medial meniscus, ACL: anterior cranial cruciate ligament, MMTL: medial meniscotibial ligament Ligament) and LMTL: lateral meniscotibial ligament (lateral meniscotibial ligament).
<1-3> 방사선학적 치료효능 평가 <1-3> Evaluation of radiotherapeutic efficacy
8주간의 골관절염 유발기간 및 8주간의 줄기세포 유래 엑소좀 풍부 배양액(ERCM) 및 대조군 배양액(CM)을 투여한 후, X-ray 촬영을 통해 골관절염지수(osteoarthritis scores)를 Modified Kellgren-Lawrence 등급(KL score)으로 분석하였다. 분석지표인 KL score는 대퇴골두 골증식체(osteophytes in medial femoral condyle, score 0~5), 대퇴골 또는 경골 변형(deformity of femur or tibia, score 0~5), 경골추(골극) 날카로워짐(sharpening of tibial spine, score 0~5)의 3가지 지표의 합(score 0~15)으로 치료 효능을 평가하였다.After 8 weeks of osteoarthritis induction and 8 weeks of stem cell-derived exosome-rich culture (ERCM) and control culture (CM), X-ray was taken to determine osteoarthritis scores as Modified Kellgren-Lawrence grade KL score). The KL score, which is an index of analysis, was calculated as the ratio of the femur to the medial femoral condyle (score 0-5), the femur or tibia deformity (score 0-5) (score 0 ~ 15) of the three indicators (sharpening of tibial spine, score 0 ~ 5).
분석 결과, 토끼 동물모델을 대상으로 전방십자인대와 내외측 반월판경골인대 절제를 통한 골관절염 유발 8주 후 방사선 검사 결과, 대퇴골두 골증식체, 내측경골 변형 및 경골추 날카로워짐의 이상소견(병변)이 총점 15점 중 평균 10.1점으로 심한 관절염이 유발된 것을 확인할 수 있었다(도 1 및 하기 표 2 참조).As a result of the analysis, the rabbit animal model was evaluated by radiographic examination after osteoarthritis induced osteoarthritis through anterior cruciate ligament and internal and external medial tibial tibial ligament resection, abnormal findings of femoral head osteogenesis, medial tibial deformity, ) Of the total score was 10.1 points out of 15 points, indicating that severe arthritis was induced (see Fig. 1 and Table 2 below).
이러한 골관절염이 유발된 토끼의 관절강 내로 줄기세포를 21% 산소농도 하에서 배양하여 수득한 대조군의 정상배양액(CM, 0.1 mL/토끼)을 2일 간격으로 8주간 투여했을 때, 신경줄기세포(병변 평균 6.0점) ≥ 희소돌기교전구세포(6.1점) > 지방줄기세포(6.4점) > 양수줄기세포(6.8점) > 양막줄기세포(7.1점) ≥ 제대혈줄기세포(7.3점) > 태반줄기세포(8.5점) = 골수줄기세포(8.5점)의 순으로 방사선 소견에 대해 우수한 치료효능을 보이는 것으로 나타났다.(CM, 0.1 mL / rabbit) obtained by culturing the stem cells in the osteoarthritic rabbit joints of the rabbits under an oxygen concentration of 21% for 8 weeks at intervals of 2 days, neural stem cells (mean lesion 6.0 (7.1 points) ≥ cord blood stem cells (7.3 points)> placental stem cells (8.5 points) ≥ cord blood stem cells (6.1 points)> adipose stem cells (6.4 points) Point) = bone marrow stem cells (8.5 points), showing excellent therapeutic efficacy against radiological findings.
한편, 줄기세포 배양배지에 TNF-α (50 ng/mL)를 첨가하고 5%의 저산소농도 하에서 배양하어 수득한 본 발명의 엑소좀 풍부 배양액(ERCM)을 투여했을 때는 모든 줄기세포에서 대조군 정상배양액 투여 보다 더 우수한 치료 효능을 보였는데, 신경줄기세포(병변 평균 4.8점) ≥ 희소돌기교전구세포(5.0점) ≥ 지방줄기세포(5.2점) ≥ 양수줄기세포(5.4점) > 양막줄기세포(5.7점) > 제대혈줄기세포(6.2점) > 태반줄기세포(6.8점) ≥ 골수줄기세포(6.9점)의 순으로 치료효능을 나타내었다.On the other hand, when the exosome-rich culture medium (ERCM) of the present invention obtained by adding TNF-α (50 ng / mL) to a stem cell culture medium and culturing the cells under a hypoxic concentration of 5% was administered, (5.2 points) ≥ amniotic stem cells (5.4 points)> amniotic stromal cells (5.7 points) ≥ amniotic stem cells (5.7 points) ≥ amniotic stem cells (6.8 points) umbilical cord blood stem cells (6.2 points) placenta stem cells (6.8 points) ≥ bone marrow stem cells (6.9 points).
Figure PCTKR2018012942-appb-T000002
Figure PCTKR2018012942-appb-T000002
*골관절염(ACLT) 유발군에 비해 유의함(P<0.05). #정상배양액에 비해 유의함(P<0.05).* Compared with osteoarthritis (ACLT) induced group, significant ( P <0.05). # Also significantly higher than in normal culture medium (P <0.05).
<1-4> 육안적 치료효능 평가 <1-4> Evaluation of gross therapeutic efficacy
8주간의 줄기세포 유래 엑소좀 풍부 배양액(ERCM) 및 대조군 배양액(CM)의 투여기간 종료 후, 동물을 희생시키고 내측관절 도달법을 통해 무릎관절 실험부위를 노출시킨 다음 연골손상에 주의하면서 무릎관절 주위의 연부조직을 제거하고 실험부위를 포함한 대퇴골의 과상부를 채취하였다. 무릎관절의 부종, 열상, 골극 형성, 활액막 비후를 육안적으로 관찰하여 O'Driscoll scoring 분석을 수행하였는데, 즉, 연골결손 부위의 변화, 연골결손 부위 표면상태, 결손 경계부위와 새롭게 생긴 조직과의 경계부의 연속성을 관찰하고 사진 촬영하여 표면 거침(roughness of surface, score 0~5), 반월판 유착(adhesion with meniscus, score 0~5), 관절 비대(hypertrophy of joint, score 0~5), 골증식체(osteophyte, score 0~5)에 대한 4가지 지표의 합(score 0~20)으로 치료 효능을 평가하였다.After 8 weeks of stem cell-derived exosome-rich culture (ERCM) and control culture medium (CM), animals were sacrificed and the knee joint test site was exposed through medial joint approach, The surrounding soft tissues were removed and overgrowth of the femur, including the experimental site, was obtained. O'Driscoll scoring analysis was performed by visual observation of knee joint edema, laceration, bony spur formation, synovial thickening, ie, changes in cartilage defect area, cartilage defect surface area, defect area and newly formed tissue The continuity of the border was observed and photographed and the roughness of surface (score 0-5), adhesion with meniscus (score 0-5), hypertrophy of joint (score 0-5), osteoporosis The treatment efficacy was evaluated by the sum of four indicators (score 0 ~ 20) for osteophyte (score 0 ~ 5).
상기 방법에 의해 골관절염 유발 8주 후 토끼를 부검하여 관절염 유발부위에 대한 육안적 검사를 수행한 결과, 하기 표 3 및 도 2에 나타낸 바와 같이 표면 거침, 반월판 유착, 관절 비대 및 골증식체 형성의 이상소견(병변)이 총점 20점 중 평균 18.1점으로 심한 손상이 확인되었다. 이러한 골관절염 유발 토끼의 관절강 내로 대조군으로 사용한 정상배양액을 투여했을 때 신경줄기세포(병변 평균 13.4점) > 희소돌기교전구세포(13.8점) > 양수줄기세포(14.3점) > 지방줄기세포(14.7점) > 양막줄기세포(15.4점) > 제대혈줄기세포(15.8점) > 태반줄기세포(16.3점) = 골수줄기세포(16.3점)의 순으로 육안적 소견에 대해 치료 효능이 있는 것으로 나타났다.After 8 weeks of osteoarthritis induction, the rabbits were autopsied and examined grossly for the arthritis-induced areas. As a result, as shown in Table 3 and FIG. 2, surface roughness, meniscus adhesion, joint hypertrophy, The abnormal findings (lesions) were 18.1 points out of 20 points. (13.3 points)> amniotic stem cells (14.3 points)> adipose stem cells (14.7 points) in the osteoarthritis-induced rabbit joints were treated with the normal culture medium used as a control group. (16.4 points), placenta stem cells (16.3 points), and bone marrow stem cells (16.3 points).
한편, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 투여했을 때는 모든 줄기세포에서 대조군으로 사용한 정상배양액 투여군 보다 더 우수한 치료 효능이 있음을 확인하였는데, 신경줄기세포(병변 평균 9.9점) ≥ 희소돌기교전구세포(10.0점) > 지방줄기세포(10.7점) > 양수줄기세포(11.2점) > 양막줄기세포(12.6점) > 제대혈줄기세포(13.1점) > 태반줄기세포(13.4점) ≥ 골수줄기세포(13.5점)의 순으로 나타나, 병변증상 정도가 월등히 개선됨을 알 수 있었다.On the other hand, when the stem cell-derived exosome-rich culture medium of the present invention was administered, it was confirmed that the therapeutic effect was superior to that of the normal culture medium used as a control group in all the stem cells. Neuronal stem cells (average 9.9 points in the lesion) Cells (10.0 points)> Adipose stem cells (10.7 points)> Amniotic stem cells (11.2 points)> Amniotic membrane stem cells (12.6 points)> Umbilical cord blood stem cells (13.1 points)> Placental stem cells (13.4 points) 13.5 points), indicating that the severity of the lesion was significantly improved.
Figure PCTKR2018012942-appb-T000003
Figure PCTKR2018012942-appb-T000003
*골관절염(ACLT) 유발군에 비해 유의함(P<0.05). #정상배양액에 비해 유의함(P<0.05).* Compared with osteoarthritis (ACLT) induced group, significant ( P <0.05). # Also significantly higher than in normal culture medium (P <0.05).
<1-5> 현미경적 치료효능 평가 <1-5> Evaluation of Microscopic Therapeutic Efficacy
상기 실험예에서 수득한 토끼 유래의 관절조직을 3일간 10% 중성 포르말린에 고정하고 EDTA로 60일간 탈석회화 과정을 거친 후 파라핀 절편을 제작하였다. 이후 Hematoxylin & eosin (H&E)으로 염색하여 사진을 촬영하고 연골생성 면적을 현미경 영상분석 프로그램인 ImageJ 5.1을 이용해 연골재생부위/절단면 넓이의 비율로 정량 분석하였다. 또한 연골 내 peptidoglycals (PG)에 대한 safranin O/fast green 염색을 통해 초자연골(hyaline cartilage)의 온전성을 분석하였다. 즉, 상기 염색결과로부터 연골의 손상 정도를 구조(structure, score 0~11), 연골 세포수(cellularity, score 0~4), 클러스터 형성(cluster, score 0~5), 연골세포 성분(PG, score 0~6)에 대한 4가지 지표의 합(score 0~24)으로 치료효능을 평가하였다(하기 표 4 참조).The rabbit-derived joint tissue obtained in the above Experimental Example was fixed to 10% neutral formalin for 3 days, and then decalcified with EDTA for 60 days to prepare a paraffin section. Thereafter, photographs were taken with Hematoxylin & eosin (H & E) staining and the area of cartilage formation was quantitatively analyzed with a ratio of cartilage regeneration area / section width using ImageJ 5.1, a microscope image analysis program. The integrity of the hyaline cartilage was also analyzed by safranin O / fast green staining for peptidoglycals (PG) in cartilage. That is, the degree of damage of the cartilage from the staining results was classified into structure (score 0-11), cartilage number (cellularity, score 0-4), cluster formation (score 0-5), cartilage cell component (PG, (score 0 to 6) (score 0 to 24) (see Table 4, below).
Figure PCTKR2018012942-appb-T000004
Figure PCTKR2018012942-appb-T000004
분석 결과, 관절염 유발조직을 H&E와 safranin O로 염색하여 현미경으로 검사해 보니, 연골의 구조, 연골세포수, 클러스터 형성 및 peptidoglycans (PG) 함량 등의 충실도 이상소견이 총점 24점 중 평균 17.2점으로 심각한 수준인 것으로 나타났다. 이러한 골관절염 유발 토끼의 관절강 내로 정상배양액을 투여했을 때 신경줄기세포(병변 평균 13.1점) > 희소돌기교전구세포(13.7점) > 지방줄기세포(14.2점) > 양막줄기세포(14.8점) > 제대혈줄기세포(15.5점) ≥ 양수줄기세포(15.6점) > 태반줄기세포(16.3점) = 골수줄기세포(16.3점)의 순으로 현미경 소견에 대해 치료효과가 있는 것으로 나타났다. As a result, the arthritis-induced tissues were stained with H & E and safranin O and examined microscopically. Fidelity abnormalities such as cartilage structure, chondrocyte count, cluster formation and peptidoglycans (PG) content were found to be 17.2 points Which is a serious level. (13.1 points)> Rare embryonic progenitor cells (13.7 points)> Adipose stem cells (14.2 points)> Amniotic stem cells (14.8 points)> Umbilical cord stem (15.5 points) ≥ amniotic stem cells (15.6 points)> placenta stem cells (16.3 points) = bone marrow stem cells (16.3 points).
한편, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 투여했을 때는 모든 줄기세포에서 정상배양액 투여 시보다 병변의 증상이 더 우수하게 개선된 것으로 나타났는데, 특히 신경줄기세포(병변 평균 10.4점) ≥ 지방줄기세포(10.5점) > 희소돌기교전구세포(10.9점) > 양수줄기세포(11.7점) ≥ 양막줄기세포(11.9점) > 제대혈줄기세포(12.8점) > 골수줄기세포(14.0점) > 태반줄기세포(14.3점)의 순으로 나타내어, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액이 관절염 치료에 매우 효과적임을 알 수 있었다(도 3 및 표 5 참조).On the other hand, when the stem cell-derived exosome-rich culture medium of the present invention was administered, the symptom of the lesion was improved better than that of the normal culture medium in all the stem cells. In particular, neural stem cells (average lesion score 10.4) (11.5 points)> Amniotic Stem Cells (11.7 points)> Amniotic Stem Cells (11.9 points)> Umbilical Cord Blood Stem Cells (12.8 points)> Bone Marrow Stem Cells (14.0 points)> Placental Stem Cells And cells (14.3 points). Thus, it was found that the stem cell-derived exosome-rich culture medium of the present invention is very effective for the treatment of arthritis (see FIG. 3 and Table 5).
Figure PCTKR2018012942-appb-T000005
Figure PCTKR2018012942-appb-T000005
*골관절염(ACLT) 유발군에 비해 유의함(P<0.05). #정상배양액에 비해 유의함(P<0.05).* Compared with osteoarthritis (ACLT) induced group, significant ( P <0.05). # Also significantly higher than in normal culture medium (P <0.05).
<1-6> 염증성 사이토카인 분석 <1-6> Inflammatory cytokine analysis
염증인자 분석을 위해 관절강 개구 직전 0.1 mL의 생리식염수를 관절강 내로 주입하고 5회 정도의 피스톤 운동 후 0.1-0.2 mL의 희석된 관절강액을 채취하였다. 채취된 관절강액으로부터 염증성 사이토카인인 TNF-α, interleukin-6 (IL-6) 및 IL-8을 ELISA로 분석하여 항염증 효능을 분석하였다.To analyze the inflammatory factors, 0.1 mL of physiological saline was injected into the joints just before the opening of the joint, and 0.1-0.2 mL of diluted articular fluid was collected after 5 times of piston motion. The anti-inflammatory effects of TNF-α, interleukin-6 (IL-6) and IL-8, which are inflammatory cytokines, were analyzed by ELISA.
분석 결과, 관절염 유발 관절강액 내 사이토카인인 TNF-α, IL-6 및 IL-8의 함량은 정상연골 내 농도보다 2배 이상 증가한 것으로 나타났고, 골관절염 유발 토끼의 관절강 내로 대조군인 정상배양액을 투여했을 때 염증성 사이토카인의 함량이 감소되는 효과가 있는 것으로 나타났다. 상대적인 효과에 대해, 양수줄기세포, 제대혈줄기세포, 신경줄기세포 및 지방줄기세포가 희소돌기교전구세포, 양막줄기세포, 태반줄기세포 및 골수줄기세포보다 우수한 염증성 사이토카인 저감 효능을 보였다. 한편, 본 발명의 줄기세포 유래 엑소좀 풍부 배양액을 투여했을 때는 모든 줄기세포에서 대조군인 정상배양액 투여군에 비해 염증성 사이토카인 저감 효과가 월등히 더 우수한 것으로 나타났고, 구체적으로 양수줄기세포, 신경줄기세포, 희소돌기교전구세포가 제대혈줄기세포, 양막줄기세포, 태반줄기세포 및 골수줄기세포에 비해 우수하게 염증성 사이토카인을 억제하는 것으로 나타났다(하기 표 6 참조).As a result, the content of TNF-α, IL-6 and IL-8, which are cytokines in arthritis-induced arthritic fluid, was increased more than twice that of normal cartilage, and the normal culture solution, which is a control group, was administered into the joints of osteoarthritis- And the amount of inflammatory cytokine was decreased. Regarding the relative effects, amniotic stem cells, cord blood stem cells, neural stem cells and adipose stem cells showed an inflammatory cytokine reducing efficacy superior to that of rare proliferative progenitor cells, amniotic stem cells, placental stem cells and bone marrow stem cells. On the other hand, when the stem cell-derived exosome-rich culture medium of the present invention was administered, the effect of reducing inflammatory cytokines was significantly better in all of the stem cells than in the control culture medium-treated group. Specifically, Prodrug precursor cells suppress inflammatory cytokines better than cord blood stem cells, amniotic stem cells, placental stem cells and bone marrow stem cells (see Table 6 below).
Figure PCTKR2018012942-appb-T000006
Figure PCTKR2018012942-appb-T000006
*골관절염(ACLT) 유발군에 비해 유의함(P<0.05). #정상배양액에 비해 유의함(P<0.05).* Compared with osteoarthritis (ACLT) induced group, significant ( P <0.05). # Also significantly higher than in normal culture medium (P <0.05).
<1-7> 통계학적 분석 <1-7> Statistical analysis
모든 시험결과는 mean±SEM으로 표시하였고, 집단 간 차이는 oneway analysis of variance (ANOVA) (SPSS 12.0 version)을 이용하여 분석하였으며 P value<0.05 미만일 때 통계학적으로 유의성이 있다고 판단하였다.All test results were expressed as mean ± SEM, differences between groups were analyzed using oneway analysis of variance (ANOVA) ( SPSS 12.0 version) were determined to be statistically significant when P value <0.05 is below.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
[수탁번호][Access number]
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC12634BPAccession number: KCTC12634BP
수탁일자 : 20140725Funding date: 20140725
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC12635BPAccession number: KCTC12635BP
수탁일자 : 20140725Funding date: 20140725
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC12636BPAccession number: KCTC12636BP
수탁일자 : 20140725Funding date: 20140725
수탁일자 : 20140725Funding date: 20140725
[규칙 제26조에 의한 보정 03.01.2019]　
Figure WO-DOC-FIGURE-123
[Correction according to Rule 26.01.2019]
Figure WO-DOC-FIGURE-123
[규칙 제26조에 의한 보정 03.01.2019]　
Figure WO-DOC-FIGURE-124
[Correction according to Rule 26.01.2019]
Figure WO-DOC-FIGURE-124
[규칙 제26조에 의한 보정 03.01.2019]　
Figure WO-DOC-FIGURE-125
[Correction according to Rule 26.01.2019]
Figure WO-DOC-FIGURE-125

Claims (7)

  1. 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는, 관절염의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating arthritis, which comprises a stem cell-derived exosome-containing culture medium as an active ingredient.
  2. 제1항에 있어서,The method according to claim 1,
    상기 줄기세포 유래 엑소좀 함유 배양액은 줄기세포 배양액에 TNF-α를 처리하고 1~8%의 산소농도 조건 하에서 배양하여 수득한 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating arthritis is characterized in that the stem cell-derived exosome-containing culture broth is obtained by treating a stem cell culture broth with TNF-? And culturing under an oxygen concentration of 1 to 8%.
  3. 제2항에 있어서, 3. The method of claim 2,
    TNF-α는 5~500 ng/ml의 농도로 처리하고, 상기 배양은 12~72시간 배양하는 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.Wherein the TNF-? Is treated at a concentration of 5 to 500 ng / ml, and the culturing is carried out for 12 to 72 hours.
  4. 제1항에 있어서, The method according to claim 1,
    상기 줄기세포는 신경줄기세포, 희소돌기교전구세포, 지방줄기세포, 양막줄기세포, 양수줄기세포, 태반줄기세포, 제대혈 줄기세포 및 골수 줄기세포로 이루어진 군 중에서 선택되는 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.Wherein said stem cells are selected from the group consisting of neural stem cells, rare progenitor precursor cells, adipose stem cells, amniotic stem cells, amniotic stem cells, placental stem cells, cord blood stem cells and bone marrow stem cells. Or a pharmaceutically acceptable salt thereof.
  5. 제4항에 있어서, 5. The method of claim 4,
    상기 신경줄기세포는 불멸화 신경줄기세포인 CBNU-NSC 세포주 (기탁번호: KCTC12635BP)이고, 상기 양수줄기세포는 불멸화 양수줄기세포인 CBNU-AFSC 세포주 (기탁번호: KCTC12634BP)이고, 상기 희소돌기교전구세포는 불멸화 희소돌기교전구세포인 CBNU-NSC.olig2 세포주 (기탁번호: KCTC12636BP)인 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.Wherein the neural stem cells are CBNU-NSC cell line (accession number: KCTC12635BP) which is an immortalized neural stem cell and the amniotic stem cell is a CBNU-AFSC cell line (accession number: KCTC12634BP) which is an immortal amniotic stem cell, And a CBNU-NSC.olig2 cell line (accession number: KCTC12636BP), which is a rare proliferating progenitor cell, for preventing or treating arthritis.
  6. 제1항에 있어서,The method according to claim 1,
    상기 줄기세포는 1x104~1x106 세포수인 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.Wherein the stem cell is 1 x 10 4 to 1 x 10 6 cells.
  7. 제1항에 있어서, The method according to claim 1,
    상기 관절염은 골관절염(Osteoarthritis), 류마티스 관절염(Rheumatoid Arthritis), 섬유 근육통(Fibromyalgia), 통풍(gout) 및 루퍼스(lupus)로 이루어진 군 중에서 선택되는 것을 특징으로 하는, 관절염의 예방 또는 치료용 약학적 조성물.Wherein said arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and lupus. .
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