WO2013111969A1 - Composition containing tanshinone as active ingredient, for increasing differentiation or activity of natural killer cells - Google Patents

Composition containing tanshinone as active ingredient, for increasing differentiation or activity of natural killer cells Download PDF

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Publication number
WO2013111969A1
WO2013111969A1 PCT/KR2013/000556 KR2013000556W WO2013111969A1 WO 2013111969 A1 WO2013111969 A1 WO 2013111969A1 KR 2013000556 W KR2013000556 W KR 2013000556W WO 2013111969 A1 WO2013111969 A1 WO 2013111969A1
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cells
tanshinone
natural killer
differentiation
cancer
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PCT/KR2013/000556
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French (fr)
Korean (ko)
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최인표
김원삼
권병목
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한국생명공학연구원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B3/00—Window sashes, door leaves, or like elements for closing wall or like openings; Layout of fixed or moving closures, e.g. windows in wall or like openings; Features of rigidly-mounted outer frames relating to the mounting of wing frames
    • E06B3/54—Fixing of glass panes or like plates
    • E06B3/58—Fixing of glass panes or like plates by means of borders, cleats, or the like
    • E06B3/5807—Fixing of glass panes or like plates by means of borders, cleats, or the like not adjustable
    • E06B3/5821—Fixing of glass panes or like plates by means of borders, cleats, or the like not adjustable hooked on or in the frame member, fixed by clips or otherwise elastically fixed
    • E06B3/5828—Fixing of glass panes or like plates by means of borders, cleats, or the like not adjustable hooked on or in the frame member, fixed by clips or otherwise elastically fixed on or with auxiliary pieces
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B3/00—Window sashes, door leaves, or like elements for closing wall or like openings; Layout of fixed or moving closures, e.g. windows in wall or like openings; Features of rigidly-mounted outer frames relating to the mounting of wing frames
    • E06B3/04—Wing frames not characterised by the manner of movement
    • E06B3/06—Single frames
    • E06B3/08—Constructions depending on the use of specified materials
    • E06B3/12—Constructions depending on the use of specified materials of metal
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B3/00—Window sashes, door leaves, or like elements for closing wall or like openings; Layout of fixed or moving closures, e.g. windows in wall or like openings; Features of rigidly-mounted outer frames relating to the mounting of wing frames
    • E06B3/54—Fixing of glass panes or like plates
    • E06B3/58—Fixing of glass panes or like plates by means of borders, cleats, or the like
    • E06B3/5807—Fixing of glass panes or like plates by means of borders, cleats, or the like not adjustable
    • E06B3/5842—Fixing of glass panes or like plates by means of borders, cleats, or the like not adjustable fixed by a tongue-and-groove or mortise-and-tenon connection substantially parallel to the pane

Definitions

  • the present invention relates to a composition for differentiating or activating natural killer cells containing tanshinone as an active ingredient, and a method for inducing differentiation from hematopoietic stem cells to natural killer cells.
  • NK cells are immune cells that play an important role in innate immune reactions by destroying cancer or virally infected cells (J. Immunol., 136: 3910-3915, 1986). Melanoma Res., 13: 349-356, 2003; Lung Cancer, 35: 23-28, 2002).
  • indirect immune response may be performed by releasing cytokines through stimulation to mobilize other immune systems.
  • These NK cells can be used for the treatment of solid tumors using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TILs), or donor lymphocyte infusions. bone marrow transplantation by performing immunotherapy (Ti lden. AB et al., J.
  • NK cells are known to be derived from hematopoietic stem cells (HSCs) of bone marrow.
  • HSCs hematopoietic stem cells
  • methods for separating hematopoietic fungal cells, treating cytokines, and culturing them into NK cells have been reported (I ⁇ unity 3: 459-473, 1995; Blood 87: 2632-). 2640, 1996; Eur JJunol. 33: 3439-3447, 2003; Blood 108: 3824-3833, 2006). That is, by adding Flt-3L, IL-7, SCF and IL-15 to hematopoietic stem cells, the cells can be differentiated into general NK cells by culturing for a certain period of time.
  • NK cells undergo differentiation in the bone marrow and the microenvironment of the bone marrow is essential for the differentiation of NK cells.
  • NK cells are characterized by sequentially expressing various surface molecules as they undergo differentiation.
  • NK cells sequentially express CD122, NK1.1, NKG2 family, Ly49 family, DX5 (CD49b) and CD43 in differentiation process (Nat Immunol, 3: 523-528, 2008). .
  • IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice lacking the transcription factor interferon (IFN) -regulatory factor 1 required for IL-15 production (Kouetsu et al., Nature 391, 700-703, 1998), NK cells were not found in mice lacking IL-15 or IL-15Ra. It has been reported that IL ⁇ 15 directly enhances the growth and differentiation of NK cells through IL-15 receptors expressed in K cells (Mrozek E et al., Blood 87, 2632-2640, 1 ⁇ 996).
  • IFN transcription factor interferon
  • Tanshinone family compounds affect the differentiation of acute promyelocyte leukemia (APL) cells (Zhonghua Xueyexue Zazhi 2000, 21: 23-26). It is also known to inhibit the proliferation of cancer and induce apoptosis (Chin J Cancer 2003,22: 1363-1366; Int J Mol Med 2010 Sep, 26 (3): 379-85) . However, it is not yet reported whether these tanshinone-based compounds play a role in inducing differentiation and immune response of lymphocytes including NK cells.
  • tansinone which induces differentiation of certain cells If the action of the compound is applied to dry cells, it can be actively used in the treatment of diseases such as inflammation, infection and cancer by obtaining NK cells more efficiently than the conventional method of differentiating NK cells. Therefore, the present inventors studied to examine the effect of tanshinone on the differentiation and immune response of NK cells, Cryptotanshinone or tanshinone nA (tanshinone ⁇ ) to the differentiation of NK cells in hematopoietic cells Tansy by inducing and promoting, increasing the expression of ID2, a gene involved in NK cell differentiation, having a cytomegalovirus against YAC-1, a cancer cell, and increasing the production of IFN-Y, thereby enhancing the activity of NK cells.
  • the present invention was completed by revealing that the paddy field can be used as an active ingredient of NK cell differentiation or activity enhancing composition. ⁇
  • An object of the present invention is to provide a composition for differentiating or enhancing the activity of natural killer cells (NK cells) containing tanshinone as an active ingredient.
  • NK cells natural killer cells
  • Another object of the present invention is to provide a method for inducing differentiation from hematopoietic stem cells to natural killer cells, comprising administering tanshinone to the natural killer cell precursor cells.
  • Another object of the present invention is to provide a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic stem cells.
  • the present invention is a natural killer cell (NK cell) differentiation containing tanshinone as an active ingredient It provides a composition for induction.
  • NK cell natural killer cell
  • the present invention provides a composition for enhancing natural killer cell activity containing tanshinone as an active ingredient.
  • the present invention provides a composition for inducing natural killer cell differentiation containing tanshinone and IL-15 (interleukin-15) as an active ingredient.
  • the present invention provides a composition for enhancing natural killer cell activity containing tanshinone and IL-15 as an active ingredient.
  • the present invention provides a method for inducing differentiation from hematopoietic cells to natural killer cells, comprising administering tanshinone to the natural killer cell precursor cells of step 1).
  • the present invention provides a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic stem cells.
  • the present invention provides a pharmaceutical composition for preventing and treating cancer containing the composition according to the present invention as an active ingredient.
  • the present invention also provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of the composition according to the present invention.
  • the present invention also provides a method for treating cancer comprising administering to a subject having cancer a pharmaceutically effective amount of the composition according to the present invention.
  • the present invention also provides a composition containing tanshinone for use in inducing differentiation of natural killer cells.
  • the present invention also provides a composition containing tanshinone for use in enhancing the activity of natural killer cells.
  • the present invention also provides a composition containing tanshinone and IL-15 for use in inducing differentiation of natural killer cells.
  • the present invention is tanshinone for use in enhancing the activity of natural killer cells and Provided are compositions containing IL-15.
  • IL-15 Provided are compositions containing IL-15.
  • the present invention provides a composition for inducing differentiation or enhancing activity of natural killer cells (NK cells) containing tansyone (tanshinone) as an active ingredient.
  • the present invention also provides a composition for inducing differentiation or activity of natural killer cells containing tanshinone and IL-15 (interleukin-15) as an active ingredient.
  • tanshinone and IL-15 interleukin-15
  • the tanshinone is specifically a chemical extracted from the root of a Chinese medicinal plant called SaJvia iniotiorrhiza bunge A ⁇ , and it is cryptotanshinone or tanshinone ⁇ , and more specifically, kryptan
  • the structural formula of cryptonshinone is Ci 9 H 20 0 3 and the chemical formula is
  • the structural formula is C 19 H 18 0 3 and the chemical formula is It is cytotoxic and anti-allergic to various human cancer cell lines, but is not limited thereto.
  • the tanshinone induces or promotes the differentiation of hematopoietic stem cells into NK cells, increases the expression of ID2, a gene associated with NK cell differentiation, Not only have cytotoxicity against YAC-1 cells, which are cells, but also increase the production of IFN-Y, but are not limited thereto.
  • the tanshinone is specifically treated with hematopoietic stem cells together with IL-15 (interleukin-15), and more specifically, the Cryptotansinone or tanshinone ⁇ A is at a concentration of 2 uM to lOuM, 15 is 10 to 50ng / i, most specifically the Cryptotansinone is at a concentration of 5uM, the tanshinone ⁇ at a concentration of lOuM, the IL-15 is treated at a concentration of 25ng / ⁇ It is not limited to this.
  • IL-15 interleukin-15
  • NK cells In order to differentiate NK cells from hematopoietic stem cells, the present inventors have isolated hematopoietic stem cells from mouse bone marrow and recovered intermediate precursor cells incubated for 7 days for differentiation into NK cells. After incubation in a medium containing kryptotanshi none (crptotanshi none) or tanshinone ⁇ ACtanshinone ⁇ , antimicrobial (3! 0- ⁇ 1.1 (?
  • NK1.1 + CD3—NKNK cells using (FITC) antibody and flow cytometry analysis of NK cell differentiation using antibodies to various NK cell surface molecules , IL-15 (if 25ng / m «was added, about 3OT differentiated into CD3—NK + cells when differentiated from hematopoietic cells to NK cells, whereas Cryptotansinone (5uM) or tanshinone nA (crptotanshinone / tanshinone ⁇ ) (10 ⁇ ) approx. 65%, 623 ⁇ 4 It was confirmed that the differentiation into NK cells (see A in Fig. 1).
  • the present inventors treated with concentrations of 2, 5 and lOuM, respectively, in order to know the appropriate concentration of Cryptotansinone / Tansinone ⁇ that induces NK cell differentiation from hematopoietic stem cells, Cryptotanshinone (cryptotanshinone)
  • Cryptotanshinone cryptotanshinone
  • the differentiation rate into NK cells was the highest at about 56% when treated with 5uM
  • the tancinone ⁇ ACtanshinone ⁇ showed the highest at about 62% when treated with lOuM (Fig. 1). See B). Therefore, when the appropriate concentration of Cryptotansinone or tanshinone ⁇ was treated with IL-15, it was found that the induction and proliferation of NK cells increased.
  • NK cell number was calculated from the total cell number using NK cell differentiation (%), IL-15-treated control group, IL-15 alone treatment group, Cryptotansinone alone treatment group, and IL-15
  • the total cell number decreased with no significant difference over time, but compared with NK cell number, the group treated with IL-15 and Cryptotansinone It was confirmed that the number of NK cells increased significantly compared to the case of IL-15 alone treatment, and showed a similar pattern in the group treated with IL-15 and tanshinone ⁇ (Fig. 2A and 2 of B reference ).
  • the present inventors measured the expression of genes related to conventional NK cell differentiation by real-time PCR (date-to-date PCR) in order to find out a target gene expressed in the process of differentiating into NK cells.
  • TRAF, IRF2 and PU.1 did not show significant difference in gene expression in each treatment condition, but ID2 expression level was only in the group treated with Cryptotansinone alone, IL-15 and Cryptotansinone. It was confirmed that it was maintained high (see FIG. 3). Therefore, the addition of Cryptotansinone or tanshinone nMcryptotanshinone / tanshinone ⁇ ) is thought to affect NK cell differentiation by increasing the expression level of ID2.
  • the present inventors confirmed that the differentiation rate into NK cells increased when IL-15 and Cryptotansinone or tanshinone ⁇ were co-treated with precursors in the middle stage of differentiation.
  • the cytotoxicity against YAC-1 cells which are cancer cells, was evaluated, and the production of IFN-Y, which is a major characteristic that can confirm the activity of NK cells, was measured.
  • the highest cytotoxicity was obtained under the conditions of IL-15 (25ng / m «and cryptotanshinone (5uM) together (see FIG. 4A), and IL-15 (like cytotoxicity).
  • Cryptotansinone and tansinone ⁇ of the present invention when treated with IL-15, induces and promotes differentiation of ⁇ cells in hematopoietic cells, increases the expression of ID2, a gene associated with ⁇ ocell differentiation, and IFN-Y. Since it increases the amount of production of N cells to increase the activity can be usefully used as a composition for the differentiation or activity of ⁇ cells.
  • the present invention when treated with IL-15, induces and promotes differentiation of ⁇ cells in hematopoietic cells, increases the expression of ID2, a gene associated with ⁇ ocell differentiation, and IFN-Y. Since it increases the amount of production of N cells to increase the activity can be usefully used as a composition for the differentiation or activity of ⁇ cells.
  • ID2 a gene associated with ⁇ ocell differentiation
  • IFN-Y IFN-Y
  • the natural killer cell precursor inducer of step 1) is specifically one selected from the group consisting of SCFC stem 1 factor, Flt3L (Fms-l ike tyrosine kinase 31 igand) and IL-7 (interleukin-7).
  • SCFC stem 1 factor SCFC stem 1 factor
  • Flt3L Flt3L
  • IL-7 interleukin-7
  • the tanshinone of step 2) is specifically cryptotanshinone or tanshinone ⁇ , but is not limited thereto.
  • IL-15 is administered to the natural killer cell progenitor cells of step 2), but is not limited thereto.
  • the Cryptotansinone or tanshinone ⁇ is treated at a concentration of 2 uM to lOuM, and the IL-15 is treated at 10 to 50 ng / m £, more specifically, the concentration of Cryptotansinone is 5 uM.
  • the tanshinone ⁇ is treated at a concentration of lOuM and the IL-15 is treated at a concentration of 25 ngA, but is not limited thereto.
  • Cryptotansinone and tanshinone ⁇ of the present invention are treated with IL-15 Induces and promotes differentiation of NK cells in hematopoietic stem cells, increases the number of NK cells and increases the expression of ID2, an important gene related to NK cell differentiation, and thus can be useful in the method of inducing differentiation from hematopoietic cells have.
  • the present invention provides a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic enjoyment cells.
  • the tanshinone is specifically treated with Cryptotansinone or tanshinone ⁇ and is treated at a concentration of 2 uM to lOuM, and more specifically the Cryptotansinone is treated at a concentration of 5 uM, and the tanshinone ⁇ is treated at a concentration of lOuM. It is not limited thereto.
  • the tanshinone is treated with hematopoietic stem cells together with IL-15 (interleukin-15), more specifically, the IL-15 is treated with 10 to 50ng /, and most specifically, a concentration of 25ng /. It is treated with, but is not limited thereto.
  • IL-15 interleukin-15
  • Cryptotansinone or tanshinone ⁇ of the present invention is treated with IL-15 to induce and promote differentiation of NK cells in hematopoietic stem cells, increase the expression of ID2, a gene associated with M cell differentiation, and produce IFN-Y. Since it increases the activity of NK cells by increasing the can be usefully used as a method for enhancing the activity of NK cells.
  • the present invention provides a pharmaceutical composition for preventing and treating cancer, which contains the composition for differentiating or activating natural killer cells of the present invention as an active ingredient.
  • the present invention also provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of the composition according to the invention.
  • the present invention also provides a method for treating cancer comprising administering to a subject having cancer a pharmaceutically effective amount of the composition according to the present invention.
  • the cancer is specifically selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer and lung cancer, but is not limited thereto.
  • Cryptotansinone and tanshinone ⁇ of the present invention induce and promote differentiation of NK cells in hematopoietic stem cells, have cytotoxicity against YAC-1, a cancer cell, and increase the production of IFN ⁇ y to increase the activity of NK cells. Since it enhances, it can be usefully used as a pharmaceutical composition for anti-cancer cell prevention and treatment.
  • composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function in addition to the NK cells.
  • it may be prepared further comprising one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers may be used in combination of saline, sterile water, Ringer's solution, buffered saline, textose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients, Other conventional additives such as layer solution and bacteriostatic agent can be added.
  • diluents such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules or injection solutions.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules or injection solutions.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules or injection solutions.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules or injection solutions.
  • it may be specifically formulated according to each disease or component using a suitable method in the art or using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, East on PA, 18th, 1990).
  • composition of the present invention can be specifically administered parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically), and the dosage is weight, age, sex, health condition, diet, time of administration, administration of the patient. The range varies depending on the method, the rate of excretion and the severity of the disease.
  • the daily dosage of the composition according to the present invention is specifically 0.01 to 5000 mg / kg, more specifically 0.01 to 10 mg / kg, and most specifically administered once or several times a day, but is not limited thereto no.
  • the present invention provides a tanshinone for use in inducing differentiation of natural killer cells A composition containing is provided.
  • the present invention also provides a composition containing tansy for use in enhancing the activity of natural killer cells.
  • the present invention also provides a composition containing tanshinone and IL-15 for use in inducing differentiation of natural killer cells.
  • the present invention provides a composition containing tanshinone and IL-15 for use in enhancing the activity of natural killer cells.
  • the present invention relates to crypt otanshi none or tanshinone ⁇ A tanshinone
  • ⁇ induces and promotes the differentiation of ⁇ cells in hematopoietic enjoyment cells, increases the expression of ID2, a gene associated with ⁇ cell differentiation, is cytotoxic to cancer cells YAC-1, and increases the production of IFN-Y. Since it enhances the activity of ⁇ cells, it can be usefully used as an active ingredient of ⁇ cell differentiation or activity enhancing composition.
  • Figure 1 is a diagram showing the result of differentiation into ⁇ cells from hematopoietic stem cells of the mouse.
  • Figure 2 is a diagram showing the results of analysis of NK cells differentiated by treatment of compounds from hematopoietic enjoyment cells of the mouse.
  • NK cells whole cell of NK cells (CD3—NK1.1 + ) differentiated with Cryptotansinone in combination with control, IL-15, cryptotanshinone (CRY) and IL-15 Analysis of the change in number;
  • FIG. 3 is a diagram showing the results of measuring the gene expression associated with maturation (real-time PCR) in NK cells treated with compounds from hematopoietic enjoyment cells and differentiated.
  • Figure 4 is a diagram showing the results of analyzing the cytotoxicity (cytotoxicity) and IFN- ⁇ production capacity of NK cells cultured in various conditions from hematopoietic stem cells of the mouse.
  • Cryptotansinone (5 uM) was treated with control, IL-15 (25 ng), cryptotanshinone (CRY) (5 uM) and IL-15 (25 ng) from stem cells, respectively. Cytotoxicity against YAC-1 with differentiated mature NK mature NK, mNK) cells;
  • Example 1 Isolation of Hematopoietic Stem Cells from Mouse Bone Marrow
  • the calf bone, thigh bone, and pelvic bone of the experimental mouse were isolated and total bone marrow cells were extracted from the method by methods known in the art.
  • Cells extracted from bone marrow were treated with ACK solution to remove red blood cells
  • B220, CD2, N 1.1, CDllb, Gr-1, and TER—119 antibodies are treated and attached to the corresponding B cells, T cells, NK / NKT cells, monocytes, granulocytes, and red blood cells, respectively, and MACSGnagnetic ⁇ activated .
  • the cells were removed by negative selection using a Cell Separat ion Kit (Mitenyi Biotec, Bergisch Gladbach, Germany).
  • hematopoietic stem cells were positively selected using micro-bead and MACS for CD117 (c-kit), a specific surface molecule of hematopoietic stem cells. Separated.
  • CD117 c-kit
  • Hematopoietic stem cells isolated from bone marrow were treated with 10% bovine cells in a 24-well plate (Becton and Dickinson Bioscience, San Diego, Calif.) At a concentration of 1.0 x 10 6 cells / well (eel ls / wel 1). Fetal bovine serum (FBS), 2 g / ⁇ i indometacin (Sigma—Aldrich, St.
  • NK cells Seven days of intermediate precursor cells were harvested for differentiation into NK cells and added for 7 days in a medium containing various concentrations of rat IL-15 and Cryptotanshinone / tanshinone nA (crptotanshinone / tanshinone ⁇ ). Incubated. After 3 days half of the medium was replaced with fresh medium containing cytokines of the same composition. 2 days, 4 days and 6 days by anti (anti) -NKl.l (PE) and anti (anti) -CD3 (FITC) using the antibody analysis the purity of the R _ ⁇ 1.13 ⁇ 403 cells and NK cells, various eu Differentiation of NK cells was analyzed by flow cytometry using antibodies to surface molecules.
  • PE anti (anti) -NKl.l
  • FITC anti (anti) -CD3
  • Cryptotansinone / tansinone also induces NK cell differentiation from hematopoietic stem cells
  • NK cell number was calculated using the percentage of NK cell differentiation from the total cell number.
  • target gene expressed in the process of differentiation into NK cells was measured by real-time PCR by date.
  • RNA from 1 to 3 was reacted for 1 hour at 37 ° C with 2.5 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Roche Diagnostics, Basel, Switzerland) for cDNA Was synthesized.
  • M-MLV Moloney murine leukemia virus
  • Transcripts of the synthesized cDNA were quantified using SYBR Premix Ex Taq (Takara Bio, Tokyo, Japan) and Real-time system TP 800 (Takara Bio), and GAPDH was used for standardization of samples.
  • primer was prepared using the Prier 3 software. The primers used are as follows:
  • TRAF, IRF2 and PU.1 did not show a significant difference in the amount of gene expression under each treatment condition, but in the case of ID2, the group Cryptotansinone (5uM) alone, IL-15 ( 25 ng) and Cryptotansinone (5 uM) together with the group was confirmed that the expression level was maintained high (Fig. 3).
  • a target cell A 4-h 51 Cr- release assay (51 Cr-release assay) method to measure the cytotoxicity (cytotoxicity) were used.
  • mIL-12 (20 ng) of NK cells (2 ⁇ 10 5 cells / well) differentiated by the method of Example 2 to measure the secretion amount of IFN-Y, which is another method of determining the function of NK cells, was measured.
  • the supernatants were measured with an ELISA (enzyme-linked ' immunosorbent assay) kit (R & D systems., CA).
  • IL-15 25 ng / m
  • Cryptotansinone 5 uM
  • IL_15 25 ng / i
  • tanshinone nA 10 uM
  • Tablets were prepared by tableting according to the preparation method of the tablets.
  • Soybean Extract 50 rag Glucose 100 nig starch 100 mg
  • the solution was filled into a Type I ampoule of transparent glass, sealed under the upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution. .
  • the present invention can be usefully used in the development of NK cell differentiation inducing agent, NK cell activity enhancer, and cancer prevention and treatment.

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Abstract

The present invention relates to a composition containing tanshinone as an active ingredient, for increasing a differentiation or activity of natural killer (NK) cells. More particularly, the present invention relates to a composition in which cryptotanshinone or tanshinone IIA induces and promotes a differentiation of NK cells and increases an expression of ID2 which is a gene relating to an NK cell differentiation in hematopoietic stem cells. The cryptotanshinone or tanshinone IIA has cytotoxicity to YAC-1, which is a cancer cell, and also increases generation of IFN-γ, thus increasing the activity of NK cells. Therefore, the cryptotanshinone or tanshinone IIA can be effectively used as an active ingredient for a composition for increasing a differentiation or activity of NK cells.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
¾시논을 유효성분으로 함유하는 자연살해세포 분화 또는 활성 증진용 조성물  Natural killer cell differentiation or activity enhancing composition containing ¾synon as active ingredient
【기술분야】 Technical Field
본 발명은 탄시논 (tanshinone)을 유효성분으로 함유하는 자연살해세포 분화 또는 활성 증진용 조성물， 및 조혈줄기세포로부터 자연살해세포로의 분화를 유도하는 방법에 관한 것이다.  The present invention relates to a composition for differentiating or activating natural killer cells containing tanshinone as an active ingredient, and a method for inducing differentiation from hematopoietic stem cells to natural killer cells.
【배경기술】 Background Art
NK세포 (natural killer cell, 이하 NK세포라 약칭함)는 암이나 바이러스 감염이 된 세포를 파괴함으로서 선천성 면역반웅에서 증요한 역할을 수행하는 면역세포이다 (J. Immunol. , 136： 3910-3915, 1986； Melanoma Res. , 13： 349-356, 2003； Lung Cancer , 35： 23-28, 2002). 또한, 자극 (stimulation)을 통해 사이토카인 (cytokine)을 분비하여 다른 면역 체계 (immune system)를 동원 (recruiting)함으로서 간접적인 면역반웅을 수행하기도 한다. 이러한 NK 세포의 파괴능은 림포카인 활성세포 (lymphokine activated killer cell, LAK)및 종양침윤림프구 (tumor infiltration lymphocytes, TIL)를 이용하여 고형암 (solid tumor) 치료에 이용하거나， 공여자 임파구 주입 (donor lymphocyte infusion)을 통한 면역치료법 (Ti lden. A. B. et al. , J. Immunol . , 136： 3910-3915 1986； Bordignon C, et al . , Hematologia 84： 1110-1149, 1999)을 수행함으로써 , 골수이식이나 장기 이식시 발생하는 거부반웅을 방지하기 위한 새로운 세포치료 요법으로 웅용이 시도되고 있다. 또한, NK 세포의 분화와 활성의 결함은 유방암 (Konjevic G, et al . , Breast Cancer Res. Treat., 66: 255-263, 2001)， 혹색종암 (Ryuke Y, et al . , Melanoma Res., 13： 349-356, 2003)， 폐암 (Villegas FR, et al . , Lung Cancer , 35： 23-28, 2002)등 다양한 암 질환과 관련되어 있음이 보고되어 상기와 같은 질환들을 치료하기 위해 NK 세포 치료법이 대두되고 있다. NK cells (abbreviated as NK cells) are immune cells that play an important role in innate immune reactions by destroying cancer or virally infected cells (J. Immunol., 136: 3910-3915, 1986). Melanoma Res., 13: 349-356, 2003; Lung Cancer, 35: 23-28, 2002). In addition, indirect immune response may be performed by releasing cytokines through stimulation to mobilize other immune systems. These NK cells can be used for the treatment of solid tumors using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TILs), or donor lymphocyte infusions. bone marrow transplantation by performing immunotherapy (Ti lden. AB et al., J. Immunol., 136: 3910-3915 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999). Grafting has been attempted as a novel cell therapy to prevent rejection during organ transplantation. In addition, defects in the differentiation and activity of NK cells have been described in terms of breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), and melanoma cancer (Ryuke Y, et al., Melanoma Res., 13: 349-356, 2003), lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), and have been reported to be associated with various cancer diseases. Treatment is emerging.
또한, NK세포는 골수 (bone marrow)의 조혈줄기세포 (hematopoietic stem cell, HSC)로부터 유래된다고 알려져 있다. 생체 외 (7 vitro) 배양의 방법으로는 조혈즐기세포를 분리하여 사이토카인을 처리하여 배양함으로서 NK 세포로 분화시키는 방법들이 보고되었다 (I瞧 unity 3： 459-473, 1995； Blood 87:2632-2640, 1996； Eur J I瞧 unol .33： 3439-3447 , 2003； Blood 108： 3824-3833, 2006) . 즉， 조혈줄기세포에 Flt-3L, IL-7, SCF 및 IL-15 등을 첨가하여 일정기간을 배양함으로서 일반적인 NK 세포로 분화시킬 수 있다. 생체 내 (//? vivo) NK 세포는 골수에서 분화 과정을 거치며 골수의 미세 환경은 NK 세포의 분화에 필수적이다. NK 세포는 분화 과정을 거침에 따라 다양한 표면 분자들을 순차적으로 발현하는 것을 특징으로 한다. 마우스 모델 (model)에서 NK 세포는 분화 과정 증에 CD122, NK1.1, NKG2 패밀리 (family), Ly49 패밀리， DX5(CD49b) 및 CD43을 순차적으로 발현한다 (Nat Immunol, 3： 523-528, 2008) .  In addition, NK cells are known to be derived from hematopoietic stem cells (HSCs) of bone marrow. As a method of in vitro culture, methods for separating hematopoietic fungal cells, treating cytokines, and culturing them into NK cells have been reported (I 瞧 unity 3: 459-473, 1995; Blood 87: 2632-). 2640, 1996; Eur JJunol. 33: 3439-3447, 2003; Blood 108: 3824-3833, 2006). That is, by adding Flt-3L, IL-7, SCF and IL-15 to hematopoietic stem cells, the cells can be differentiated into general NK cells by culturing for a certain period of time. In vivo NK cells undergo differentiation in the bone marrow and the microenvironment of the bone marrow is essential for the differentiation of NK cells. NK cells are characterized by sequentially expressing various surface molecules as they undergo differentiation. In mouse models, NK cells sequentially express CD122, NK1.1, NKG2 family, Ly49 family, DX5 (CD49b) and CD43 in differentiation process (Nat Immunol, 3: 523-528, 2008). .
IL-15는 NK 세포 분화에 관여하고, 이것은 IL-15 생성에 요구되는 전사인자 인터페론 (transcription factor interferon, IFN)-조절 인자 1이 결핍된 쥐에서는 NK 세포가 결핍되며 (Kouetsu et al. , Nature 391, 700-703, 1998), IL-15 또는 IL-15Ra가 결핍된 쥐에서는 NK 세포가 발견되지 않는다는 것에 의해 알게 되었다. 이로써 ILᅳ 15는 K세포에서 발현되는 IL-15수용체를 통해서 NK 세포의 성장과 분화를 직접적으로 증진시킨다는 것이 보고되었다 (MrozekE et al . , Blood 87, 2632-2640, 1Π 996) .  IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice lacking the transcription factor interferon (IFN) -regulatory factor 1 required for IL-15 production (Kouetsu et al., Nature 391, 700-703, 1998), NK cells were not found in mice lacking IL-15 or IL-15Ra. It has been reported that IL 보고 15 directly enhances the growth and differentiation of NK cells through IL-15 receptors expressed in K cells (Mrozek E et al., Blood 87, 2632-2640, 1Π 996).
그 동안의 연구들에 의하면 탄시논 (Tanshinone) 계열의 화합물들은 급성 전골수성 백혈병 (acute promyelocyte leukemia, APL) 세포의 분화에 영향을 준다는 것이 밝혀졌다 (Zhonghua Xueyexue Zazhi 2000,21:23-26). 또한， 암 (cancer)의 증식을 억제하며 세포자멸사 (apoptosis)를 유도한다는 것이 알려져 있다 (Chin J Cancer 2003,22： 1363-1366； Int J Mol Med 2010 Sep, 26(3) :379-85). 그러나 아직까지 이러한 탄시논 계열의 화합물들이 NK 세포를 비롯한 림프구 (lymphocyte)의 분화 및 면역반웅을 유도하는 역할을 수행할지에 대해서는 보고가 되지 않았다. 따라서 특정 세포의 분화를 유도하는 탄시논 계열 화합물의 작용이 服세포에도 적용이 된다면 기존의 NK세포를 분화시키는 방법보다 효율적으로 NK세포를 획득함으로써 염증， 감염 , 암 등의 질환 치료에 적극적으로 웅용이 가능하다. 이에 본 발명자들은 탄시논이 NK 세포의 분화 및 면역반웅에 미치는 영향을 알아보기 위하여 연구한 결과， 크립토탄시논 (cryptotanshinone) 또는 탄시논 nA(tanshinone ΠΑ)가 조혈즐기세포에서 NK 세포의 분화를 유도 및 촉진시키며 NK 세포 분화와 관련된 유전자인 ID2의 발현을 증가시키고， 암세포인 YAC-1에 대하여 세포뜩성을 지니며 IFN-Y의 생성 또한 증가시켜 NK 세포의 활성을 증진시키는 것을 확인함으로써， 탄시논을 NK 세포 분화 또는 활성 증진용 조성물의 유효성분으로 사용할 수 있음을 밝힘으로써 본 발명을 완성하였다. ■ Previous studies have shown that Tanshinone family compounds affect the differentiation of acute promyelocyte leukemia (APL) cells (Zhonghua Xueyexue Zazhi 2000, 21: 23-26). It is also known to inhibit the proliferation of cancer and induce apoptosis (Chin J Cancer 2003,22: 1363-1366; Int J Mol Med 2010 Sep, 26 (3): 379-85) . However, it is not yet reported whether these tanshinone-based compounds play a role in inducing differentiation and immune response of lymphocytes including NK cells. Thus tansinone, which induces differentiation of certain cells If the action of the compound is applied to dry cells, it can be actively used in the treatment of diseases such as inflammation, infection and cancer by obtaining NK cells more efficiently than the conventional method of differentiating NK cells. Therefore, the present inventors studied to examine the effect of tanshinone on the differentiation and immune response of NK cells, Cryptotanshinone or tanshinone nA (tanshinone ΠΑ) to the differentiation of NK cells in hematopoietic cells Tansy by inducing and promoting, increasing the expression of ID2, a gene involved in NK cell differentiation, having a cytomegalovirus against YAC-1, a cancer cell, and increasing the production of IFN-Y, thereby enhancing the activity of NK cells. The present invention was completed by revealing that the paddy field can be used as an active ingredient of NK cell differentiation or activity enhancing composition. ■
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명의 목적은 탄시논 (tanshinone)을 유효성분으로 함유하는 자연살해세포 (natural killer cell, NK세포) 분화 또는 활성 증진용 조성물을 제공하는 것이다.  An object of the present invention is to provide a composition for differentiating or enhancing the activity of natural killer cells (NK cells) containing tanshinone as an active ingredient.
본 발명의 다른 목적은 자연살해세포 전구체 세포에 탄시논을 투여하는 단계를 포함하는 조혈줄기세포로부터 자연살해세포로의 분화 유도 방법을 제공하는 것이다.  Another object of the present invention is to provide a method for inducing differentiation from hematopoietic stem cells to natural killer cells, comprising administering tanshinone to the natural killer cell precursor cells.
본 발명의 다른 목적은 조혈줄기세포에서 분화된 자연살해세포 전구체 세포에 탄시논을 처리하는 단계를 포함하는 자연살해세포의 활성 증진 방법을 제공하는 것이다.  Another object of the present invention is to provide a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic stem cells.
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위하여， 본 발명은 탄시논 (tanshinone)을 유효성분으로 함유하는 자연살해세포 (natural killer cell, NK 세포) 분화 유도용 조성물을 제공한다. In order to achieve the above object, the present invention is a natural killer cell (NK cell) differentiation containing tanshinone as an active ingredient It provides a composition for induction.
또한， 본 발명은 탄시논을 유효성분으로 함유하는 자연살해세포 활성 증진용 조성물을 제공한다.  In addition, the present invention provides a composition for enhancing natural killer cell activity containing tanshinone as an active ingredient.
또한， 본 발명은 탄시논 및 IL—15(interleukin-15)를 유효성분으로 함유하는 자연살해세포 분화 유도용 조성물을 제공한다.  In addition, the present invention provides a composition for inducing natural killer cell differentiation containing tanshinone and IL-15 (interleukin-15) as an active ingredient.
또한， 본 발명은 탄시논 및 IL-15를 유효성분으로 함유하는 자연살해세포 활성 증진용 조성물을 제공한다.  In another aspect, the present invention provides a composition for enhancing natural killer cell activity containing tanshinone and IL-15 as an active ingredient.
또한， 본 발명은  In addition, the present invention
1) 조혈줄기세포에서 자연살해세포 전구체 유도제를 첨가하여 자연살해세포 전구체 세포로의 증식을 유도하는 단계; 및  1) inducing proliferation of natural killer cell precursor cells by adding natural killer cell precursor inducers in hematopoietic stem cells; And
2) 단계 1)의 자연살해세포 전구체 세포에 탄시논을 투여하는 단계를 포함하는 조혈즐기세포로부터 자연살해세포로의 분화 유도 방법을 제공한다. 또한, 본 발명은 조혈줄기세포에서 분화된 자연살해세포 전구체 세포에 탄시논을 처리하는 단계를 포함하는 자연살해세포의 활성 증진 방법을 제공한다.  2) It provides a method for inducing differentiation from hematopoietic cells to natural killer cells, comprising administering tanshinone to the natural killer cell precursor cells of step 1). In another aspect, the present invention provides a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic stem cells.
또한， 본 발명은 본 발명에 따른 조성물을 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.  In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer containing the composition according to the present invention as an active ingredient.
또한， 본 발명은 본 발명에 따른 조성물을 약학적으로 유효한 양으로 개체에 투여하는 단계를 포함하는 암 예방 방법을 제공한다.  The present invention also provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of the composition according to the present invention.
또한， 본 발명은 본 발명에 따른 조성물을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법을 제공한다.  The present invention also provides a method for treating cancer comprising administering to a subject having cancer a pharmaceutically effective amount of the composition according to the present invention.
또한， 본 발명은 자연살해세포의 분화 유도에 사용하기 위한 탄시논을 함유하는 조성물을 제공한다.  The present invention also provides a composition containing tanshinone for use in inducing differentiation of natural killer cells.
또한， 본 발명은 자연살해세포의 활성 증진에 사용하기 위한 탄시논을 함유하는 조성물을 제공한다.  The present invention also provides a composition containing tanshinone for use in enhancing the activity of natural killer cells.
또한, 본 발명은 자연살해세포의 분화 유도에 사용하기 위한 탄시논 및 IL-15을 함유하는 조성물을 제공한다.  The present invention also provides a composition containing tanshinone and IL-15 for use in inducing differentiation of natural killer cells.
또한， 본 발명은 자연살해세포의 활성 증진에 사용하기 위한 탄시논 및 IL-15을 함유하는 조성물을 제공한다. 이하， 본 발명을 상세히 설명한다. 본 발명은 탄시 fe(tanshinone)을 유효성분으로 함유하는 자연살해세포 (natural killer cell, NK 세포) 분화 유도 또는 활성 증진용 조성물을 제공한다. In addition, the present invention is tanshinone for use in enhancing the activity of natural killer cells and Provided are compositions containing IL-15. Hereinafter, the present invention will be described in detail. The present invention provides a composition for inducing differentiation or enhancing activity of natural killer cells (NK cells) containing tansyone (tanshinone) as an active ingredient.
또한， 본 발명은 탄시논 및 IL-15(interleukin-15)를 유효성분으로 함유하는 자연살해세포 분화 유도 또는 활성 증진용 조성물을 제공한다.  The present invention also provides a composition for inducing differentiation or activity of natural killer cells containing tanshinone and IL-15 (interleukin-15) as an active ingredient.
상기 탄시논은 구체적으로 ^^ SaJvia iniotiorrhiza bunge A^고 불리는 중국 약초 (Chinese medicinal plant)의 뿌리로부터 추출한 화학물질 (chemical)이고 크립토탄시논 (cryptotanshinone) 또는 탄시논 ΠΑ이며， 보다 구체적으로 크립토탄 시논 (cryptotanshinone)의 구조식은 Ci9H2003이고 화학식은 The tanshinone is specifically a chemical extracted from the root of a Chinese medicinal plant called SaJvia iniotiorrhiza bunge A ^, and it is cryptotanshinone or tanshinone ΠΑ, and more specifically, kryptan The structural formula of cryptonshinone is Ci 9 H 20 0 3 and the chemical formula is
Figure imgf000007_0001
퀴노이드-디테르펜 (quinoid— diterpene) 구조로 활성산소의 발생에 의한 항균성, 항돌연변이성을 갖는 것이고， 탄시논 ΠΑ의
Figure imgf000007_0001
It is a quinoid-diterpene structure and has antibacterial and antimutagenicity due to the generation of free radicals.
구조식은 C19H1803이고 화학식은
Figure imgf000007_0002
다양한 인간 암 세포주 (human cancer cell line)에 대해 세포독성을 가지며 항알레르기성이 있는 것이나， 이에 한정되는 것은 아니다. The structural formula is C 19 H 18 0 3 and the chemical formula is
Figure imgf000007_0002
It is cytotoxic and anti-allergic to various human cancer cell lines, but is not limited thereto.
또한， 상기 탄시논은 조혈줄기세포에서 NK 세포로의 분화를 유도하거나 촉진시키고， NK 세포 분화와 관련된 유전자인 ID2의 발현을 증가시키며 암 세포인 YAC-1 세포에 대하여 세포독성을 가질 뿐만 아니라 IFN-Y의 생성을 증가시키는 것이나， 이에 한정되는 것은 아니다. In addition, the tanshinone induces or promotes the differentiation of hematopoietic stem cells into NK cells, increases the expression of ID2, a gene associated with NK cell differentiation, Not only have cytotoxicity against YAC-1 cells, which are cells, but also increase the production of IFN-Y, but are not limited thereto.
또한， 구체적으로 상기 탄시논을 IL-15(interleukin-15)과 함께 조혈줄기세포에 처리하는 것이고, 보다 구체적으로 상기 크립토탄시논 또는 탄시논 Π A는 2uM 내지 lOuM의 농도로， 상기 IL-15는 10 내지 50ng/i 으로 처리하는 것이며， 가장 구체적으로 상기 크립토탄시논은 5uM의 농도로, 상기 탄시논 ΠΑ는 lOuM의 농도로， 상기 IL— 15는 25ng/ ^의 농도로 처리하는 것이나 이에 한정되는 것은 아니다.  In addition, the tanshinone is specifically treated with hematopoietic stem cells together with IL-15 (interleukin-15), and more specifically, the Cryptotansinone or tanshinone Π A is at a concentration of 2 uM to lOuM, 15 is 10 to 50ng / i, most specifically the Cryptotansinone is at a concentration of 5uM, the tanshinone ΠΑ at a concentration of lOuM, the IL-15 is treated at a concentration of 25ng / ^ It is not limited to this.
본 발명자들은 조혈줄기세포로부터 NK 세포를 분화시키기 위하여 마우스 골수로부터 조혈줄기세포를 분리하여 NK 세포로의 분화를 위해 7일간 배양한 중간단계의 전구체 세포들을 회수하여 다양한 농도의 쥐의 IL-15과 크립토탄시논 (crptotanshi none) 또는 탄시논 Π ACtanshinone ΠΑ)를 포함하는 배지에서 배양한 후 2일， 4일 및 6일 별로 항(3! 0-服1.1(?£) 및 항 (ant으 CD3(FITC) 항체를 이용하여 NK1.1+CD3—인 NK세포의 순도를 분석하고, 다양한 NK 세포 표면 분자에 대한 항체를 이용하여 NK 세포의 분화 정도를 유동세포분석법 (flow cytometry)으로 분석한 결과， 조혈즐기세포로부터 NK 세포로 분화시켰을 때 IL-15(25ng/m«만을 첨가한 경우 약 3OT가 CD3—NK+세포로 분화된 반면， 크립토탄시논 (5uM) 또는 탄시논 nA(crptotanshinone/tanshinone ΠΑ)(10υΜ)를 함께 첨가한 경우 약 65%, 62¾가 NK세포로 분화되었다는 것을 확인하였다 (도 1의 A참조). In order to differentiate NK cells from hematopoietic stem cells, the present inventors have isolated hematopoietic stem cells from mouse bone marrow and recovered intermediate precursor cells incubated for 7 days for differentiation into NK cells. After incubation in a medium containing kryptotanshi none (crptotanshi none) or tanshinone Π ACtanshinone ΠΑ, antimicrobial (3! 0- 服 1.1 (? £)) and anti- Purity of NK1.1 + CD3—NKNK cells using (FITC) antibody and flow cytometry analysis of NK cell differentiation using antibodies to various NK cell surface molecules ， IL-15 (if 25ng / m «was added, about 3OT differentiated into CD3—NK + cells when differentiated from hematopoietic cells to NK cells, whereas Cryptotansinone (5uM) or tanshinone nA (crptotanshinone / tanshinone ΠΑ) (10υΜ) approx. 65%, 62¾ It was confirmed that the differentiation into NK cells (see A in Fig. 1).
또한， 본 발명자들은 조혈줄기세포로부터 NK 세포 분화를 유도하는 크립토탄시논 /탄시논 ΠΑ의 적정 처리 농도를 알기 위하여 각 2， 5 및 lOuM의 농도별로 처리한 결과， 크립토탄시논 (cryptotanshinone)의 경우, NK 세포로의 분화율이 5uM을 처리하였을 때 약 56%로 가장 높았고 탄시논 Π ACtanshinone ΠΑ)의 경우에는 lOuM을 처리하였을 때 약 62%로 가장 높게 나왔다는 것을 확인하였다 (도 1의 B 참조). 따라서， 적정 농도의 크립토탄시논 또는 탄시논 ΠΑ를 IL— 15과 함께 처리를 했을 경우， NK 세포의 분화 유도 및 증식이 증가됨을 알 수 있었다. 또한， 본 발명자들은 NK 세포의 분화율 증가가 실제로 NK 집단 (population) 변화와 연관성이 있는지를 알기 위하여 분화 중간단계인 전구체에 IL-15을 처리한 시점부터 날짜 ^로 수거한 세포의 수를 세고, 전체 세포 수로부터 NK 세포 분화도 (%)를 이용하여 NK 세포 수를 계산한 결과， IL-15를 처리하지 않은 대조군， IL-15단독 처리군， 크립토탄시논 단독 처리군, 및 IL-15 및 크립토탄시논을 함께 처리한 군에서 시간이 경과함에 따라 전체 세포 수는 전반적으로 큰 차이 없이 감소하는 양상을 보였지만 NK 세포 수를 비교한 결과 IL-15 및 크립토탄시논을 함께 처리한 군에서 IL-15만을 처리한 경우보다 NK세포 수가 크게 증가한 것을 확인할 수 있었고， IL-15 및 탄시논 ΠΑ를 처리한 군에서도 상기와 유사한 양상을 보였다는 것을 확인하였다 (도 2의 A 및 도 2의 B참조). In addition, the present inventors treated with concentrations of 2, 5 and lOuM, respectively, in order to know the appropriate concentration of Cryptotansinone / Tansinone ΠΑ that induces NK cell differentiation from hematopoietic stem cells, Cryptotanshinone (cryptotanshinone) In the case of, the differentiation rate into NK cells was the highest at about 56% when treated with 5uM, and the tancinone Π ACtanshinone ΠΑ) showed the highest at about 62% when treated with lOuM (Fig. 1). See B). Therefore, when the appropriate concentration of Cryptotansinone or tanshinone ΠΑ was treated with IL-15, it was found that the induction and proliferation of NK cells increased. In addition, the present inventors counted the number of cells collected by date ^ from the time of treatment of IL-15 to intermediate precursors in order to know whether increased NK cell differentiation rate was actually associated with NK population change. , NK cell number was calculated from the total cell number using NK cell differentiation (%), IL-15-treated control group, IL-15 alone treatment group, Cryptotansinone alone treatment group, and IL-15 In the group treated with and Cryptotansinone, the total cell number decreased with no significant difference over time, but compared with NK cell number, the group treated with IL-15 and Cryptotansinone It was confirmed that the number of NK cells increased significantly compared to the case of IL-15 alone treatment, and showed a similar pattern in the group treated with IL-15 and tanshinone ΠΑ (Fig. 2A and 2 of B reference ).
또한, 본 발명자들은 NK 세포로 분화되는 과정에서 발현되는 표적 유전자 (target gene)를 알아내기 위하여 기존의 NK 세포 분화와 관련된 유전자들의 발현을 날짜별로 실시간 PCR(real-time PCR)로 측정한 결과， TRAF, IRF2 및 PU.1은 각 각의 처리조건에서 유전자 발현량이 크게 차이를 보이지 않았지만, ID2의 경우 크립토탄시논 단독 처리군， IL-15 및 크립토탄시논을 함께 처리한 군에서만 발현량이 높게 유지되고 있다는 것을 확인하였다 (도 3 참조)ᅳ 따라서, 크립토탄시논 또는 탄시논 nMcryptotanshinone/tanshinone ΠΑ)를 첨가한 경우 ID2의 발현양이 증가함으로서 NK 세포분화에 영향을 미칠 것으로 생각된다.  In addition, the present inventors measured the expression of genes related to conventional NK cell differentiation by real-time PCR (date-to-date PCR) in order to find out a target gene expressed in the process of differentiating into NK cells. TRAF, IRF2 and PU.1 did not show significant difference in gene expression in each treatment condition, but ID2 expression level was only in the group treated with Cryptotansinone alone, IL-15 and Cryptotansinone. It was confirmed that it was maintained high (see FIG. 3). Therefore, the addition of Cryptotansinone or tanshinone nMcryptotanshinone / tanshinone ΠΑ) is thought to affect NK cell differentiation by increasing the expression level of ID2.
아을러， 본 발명자들은 분화 중간단계의 전구체에 IL-15과 크립토탄시논 또는 탄시논 ΠΑ를 함께 처리하였을 때 NK 세포로의 분화율이 증가됨을 확인하였고， 상기와 같은 과정으로 분화된 NK 세포가 제대로된 면역 반웅을 수행하는지 확인하기 위하여 암세포인 YAC-1 세포에 대한 세포독성을 알아보고 NK 세포의 활성을 확인할 수 있는 주요한 특성인 IFN-Y의 생성을 측정하였다. 그 결과， IL-15(25ng/m« 및 크립토탄시논 (cryptotanshinone)(5uM)을 함께 처리한 조건에서 세포독성이 가장 높게 나왔고 (도 4의 A 참조)， 세포독성과 마찬가지로 ILᅳ 15(25ng/m£)과 크립토탄시논 (5uM) 또는 IL-15(25ng/ )과 탄시논 ΠΑ(ΙΟιιΜ)를 함께 처리한 조건에서 IFN-Y의 분비량이 가장 높게 나왔다는 것을 확인하였다 (도 4의 Β 참조). In addition, the present inventors confirmed that the differentiation rate into NK cells increased when IL-15 and Cryptotansinone or tanshinone ΠΑ were co-treated with precursors in the middle stage of differentiation. In order to confirm whether the proper immune response is performed, the cytotoxicity against YAC-1 cells, which are cancer cells, was evaluated, and the production of IFN-Y, which is a major characteristic that can confirm the activity of NK cells, was measured. As a result, the highest cytotoxicity was obtained under the conditions of IL-15 (25ng / m «and cryptotanshinone (5uM) together (see FIG. 4A), and IL-15 (like cytotoxicity). 25 ng / m £) with Cryptotansinone (5 uM) or IL-15 (25 ng /) It was confirmed that the secretion amount of IFN-Y was the highest under the conditions treated with tanshinone ΠΑ (ΙΟιιΜ) (see Β of Figure 4).
그러므로， 본 발명의 크립토탄시논 및 탄시논 ΠΑ는 IL-15와 함께 처리하여 조혈즐기세포에서 ΝΚ 세포의 분화를 유도 및 촉진시키며 服 세포 분화와 관련된 유전자인 ID2의 발현을 증가시키고 IFN-Y의 생성량을 증가시켜 Ν 세포의 활성을 증진시키므로 ΝΚ 세포의 분화 또는 활성 증진용 조성물로 유용하게 사용될 수 있다. ■ 또한， 본 발명은 Therefore, Cryptotansinone and tansinone ΠΑ of the present invention, when treated with IL-15, induces and promotes differentiation of ΝΚ cells in hematopoietic cells, increases the expression of ID2, a gene associated with 服 ocell differentiation, and IFN-Y. Since it increases the amount of production of N cells to increase the activity can be usefully used as a composition for the differentiation or activity of ΝΚ cells. ■ In addition, the present invention
1) 조혈줄기세포에서 자연살해세포 전구체 유도제를 첨가하여 자연살해세포 전구체 세포로의 증식을 유도하는 단계; 및  1) inducing proliferation of natural killer cell precursor cells by adding natural killer cell precursor inducers in hematopoietic stem cells; And
2) 단계 1)의 자연살해세포 전구체 세포에 탄시논을 투여하는 단계를 포함하는 조혈줄기세포로부터 자연살해세포로의 분화 유도 방법을 제공한다. 상기 방법에 있어서， 단계 1)의 자연살해세포 전구체 유도제는 구체적으로 SCFCstem eel 1 factor) , Flt3L(Fms-l ike tyrosine kinase31 igand)및 IL-7(interleukin-7)로 구성된 군으로부터 선택되는 어느 하나이나， 이에 한정되는 것은 아니다.  2) It provides a method for inducing differentiation from hematopoietic stem cells to natural killer cells comprising the step of administering tanshinone to the natural killer cell precursor cells of step 1). In the above method, the natural killer cell precursor inducer of step 1) is specifically one selected from the group consisting of SCFC stem 1 factor, Flt3L (Fms-l ike tyrosine kinase 31 igand) and IL-7 (interleukin-7). However, the present invention is not limited thereto.
상기 방법에 있어서， 단계 2)의 탄시논은 구체적으로 크립토탄시논 (cryptotanshinone) 또는 탄시논 ΠΑ이나， 이에 한정되는 것은 아니다.  In the above method, the tanshinone of step 2) is specifically cryptotanshinone or tanshinone ΠΑ, but is not limited thereto.
상기 방법에 있어서， 단계 2)의 자연살해세포 전구체 세포에 IL-15를 함께 투여하는 것이나， 이에 한정되는 것은 아니다.  In the above method, IL-15 is administered to the natural killer cell progenitor cells of step 2), but is not limited thereto.
상기 방법에 있어서， 상기 크립토탄시논 또는 탄시논 ΠΑ는 2uM 내지 lOuM의 농도로， 상기 IL-15는 10 내지 50ng/m£으로 처리하는 것이며, 보다 구체적으로 상기 크립토탄시논은 5uM의 농도로， 상기 탄시논 ΠΑ는 lOuM의 농도로， 상기 IL-15는 25ngA 의 농도로 처리하는 것이나, 이에 한정되는 것은 아니다.  In the above method, the Cryptotansinone or tanshinone ΠΑ is treated at a concentration of 2 uM to lOuM, and the IL-15 is treated at 10 to 50 ng / m £, more specifically, the concentration of Cryptotansinone is 5 uM. As such, the tanshinone ΠΑ is treated at a concentration of lOuM and the IL-15 is treated at a concentration of 25 ngA, but is not limited thereto.
본 발명의 크립토탄시논 및 탄시논 ΠΑ는 IL-15와 함께 처리하여 조혈줄기세포에서 NK 세포의 분화를 유도 및 촉진시키고 NK 세포의 수를 증가시키며 NK 세포 분화와 관련된 중요한 유전자인 ID2의 발현을 증가시키므로 조혈즐기세포로부터 자연살해세로의 분화 유도 방법에 유용하게 사용될 수 있다. 또한 본 발명은 조혈즐기세포에서 분화된 자연살해세포 전구체 세포에 탄시논을 처리하는 단계를 포함하는 자연살해세포의 활성 증진 방법을 제공한다. Cryptotansinone and tanshinone ΠΑ of the present invention are treated with IL-15 Induces and promotes differentiation of NK cells in hematopoietic stem cells, increases the number of NK cells and increases the expression of ID2, an important gene related to NK cell differentiation, and thus can be useful in the method of inducing differentiation from hematopoietic cells have. In another aspect, the present invention provides a method for enhancing the activity of natural killer cells comprising the step of treating tanshinone to natural killer cell precursor cells differentiated from hematopoietic enjoyment cells.
상기 탄시논은 구체적으로 크립토탄시논 또는 탄시논 ΠΑ이고 2uM 내지 lOuM의 농도로 처리하는 것이고, 보다 구체적으로 상기 크립토탄시논은 5uM의 농도로， 상기 탄시논 ΠΑ는 lOuM의 농도로 처리하는 것이나， 이에 한정되는 것은 아니다.  The tanshinone is specifically treated with Cryptotansinone or tanshinone ΠΑ and is treated at a concentration of 2 uM to lOuM, and more specifically the Cryptotansinone is treated at a concentration of 5 uM, and the tanshinone ΠΑ is treated at a concentration of lOuM. It is not limited thereto.
또한， 구체적으로 상기 탄시논을 IL-15(interleukin-15)와 함께 조혈줄기세포에 처리하는 것이고， 보다 구체적으로 상기 IL-15는 10 내지 50ng/ 으로 처리하는 것이고， 가장 구체적으로 25ng/ 의 농도로 처리하는 것이나, 이에 한정되는 것은 아니다.  In addition, in particular, the tanshinone is treated with hematopoietic stem cells together with IL-15 (interleukin-15), more specifically, the IL-15 is treated with 10 to 50ng /, and most specifically, a concentration of 25ng /. It is treated with, but is not limited thereto.
본 발명의 크립토탄시논 또는 탄시논 ΠΑ는 IL-15와 함께 처리하여 조혈줄기세포에서 NK세포의 분화를 유도 및 촉진시키며 M세포 분화와 관련된 유전자인 ID2의 발현올 증가시키고 IFN-Y의 생성량을 증가시켜 NK 세포의 활성을 증진시키므로 NK세포의 활성 증진 방법으로 유용하게 사용될 수 있다. 또한， 본 발명은 본 발명의 자연살해세포 분화 또는 활성 증진용 조성물을 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.  Cryptotansinone or tanshinone ΠΑ of the present invention is treated with IL-15 to induce and promote differentiation of NK cells in hematopoietic stem cells, increase the expression of ID2, a gene associated with M cell differentiation, and produce IFN-Y. Since it increases the activity of NK cells by increasing the can be usefully used as a method for enhancing the activity of NK cells. In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer, which contains the composition for differentiating or activating natural killer cells of the present invention as an active ingredient.
또한, 본 발명은 본 발명에 따른 조성물을 약학적으로 유효한 양으로 개체에 투여하는 단계를 포함하는 암 예방 방법을 제공한다.  The present invention also provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of the composition according to the invention.
또한， 본 발명은 본 발명에 따른 조성물을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법을 제공한다. 상기 암은 구체적으로 유방암， 혹색종암, 위암, 간암 및 폐암으로 구성된 군으로부터 선택되는 것이나， 이에 한정되는 것은 아니다. The present invention also provides a method for treating cancer comprising administering to a subject having cancer a pharmaceutically effective amount of the composition according to the present invention. The cancer is specifically selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer and lung cancer, but is not limited thereto.
본 발명의 크립토탄시논 및 탄시논 ΠΑ는 조혈줄기세포에서 NK 세포의 분화를 유도 및 촉진시키고 암세포인 YAC-1에 대하여 세포독성을 지니며 IFNᅳ y의 생성량을 증가시켜 NK 세포의 활성을 증진시키므로 항암 세포 예방 및 치료용 약학적 조성물로서 유용하게 사용될 수 있다.  Cryptotansinone and tanshinone ΠΑ of the present invention induce and promote differentiation of NK cells in hematopoietic stem cells, have cytotoxicity against YAC-1, a cancer cell, and increase the production of IFN ᅳ y to increase the activity of NK cells. Since it enhances, it can be usefully used as a pharmaceutical composition for anti-cancer cell prevention and treatment.
본 발명의 조성물은 상기 NK 세포에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액， 완충 식염수, 텍스트로스 용액， 말토 덱스트린 용액， 글리세를, 에탄올 및 이들 성분 중 1 성분 이상을 흔합하여 이용할 수 있으며， 필요에 따라 항산화제, 완층액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제， 계면활성게， 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액， 유탁액 등과 같은 주이용 제형, 산제， 정제， 캡슐제, 환， 과립 또는 주사액제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science (Mack Publishing Company , East on PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 구체적으로 제제화할 수 있다.  The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function in addition to the NK cells. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination of saline, sterile water, Ringer's solution, buffered saline, textose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients, Other conventional additives such as layer solution and bacteriostatic agent can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules or injection solutions. Furthermore, it may be specifically formulated according to each disease or component using a suitable method in the art or using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, East on PA, 18th, 1990).
본 발명의 조성물은 구체적으로 비경구 투여 (예를 들어 정맥 내, 피하， 복강 내 또는 국소에 적용)할 수 있으며， 투여량은 환자의 체중, 연령， 성별， 건강상태, 식이, 투여시간， 투여방법， 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 구체적으로 0.01 내지 5000 mg/kg이며， 보다 구체적으로 0.01 내지 10 mg/kg 이며， 가장 구체적으로 하루 일 회 내지 수회에 나^어 투여하는 것이나 이에 한정되는 것은 아니다. 또한， 본 발명은 자연살해세포의 분화 유도에 사용하기 위한 탄시논을 함유하는 조성물을 제공한다. The composition of the present invention can be specifically administered parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically), and the dosage is weight, age, sex, health condition, diet, time of administration, administration of the patient. The range varies depending on the method, the rate of excretion and the severity of the disease. The daily dosage of the composition according to the present invention is specifically 0.01 to 5000 mg / kg, more specifically 0.01 to 10 mg / kg, and most specifically administered once or several times a day, but is not limited thereto no. In addition, the present invention provides a tanshinone for use in inducing differentiation of natural killer cells A composition containing is provided.
또한， 본 발명은 자연살해세포의 활성 증진에 사용하기 위한 탄시는을 함유하는 조성물을 제공한다.  The present invention also provides a composition containing tansy for use in enhancing the activity of natural killer cells.
또한， 본 발명은 자연살해세포의 분화 유도에 사용하기 위한 탄시논 및 IL-15을 함유하는 조성물을 제공한다.  The present invention also provides a composition containing tanshinone and IL-15 for use in inducing differentiation of natural killer cells.
아을러， 본 발명은 자연살해세포의 활성 증진에 사용하기 위한 탄시논 및 IL-15을 함유하는 조성물을 제공한다.  In addition, the present invention provides a composition containing tanshinone and IL-15 for use in enhancing the activity of natural killer cells.
【유리한 효과】 Advantageous Effects
본 발명은 크립토탄시논 (crypt otanshi none) 또는 탄시논 Π A tanshinone The present invention relates to crypt otanshi none or tanshinone Π A tanshinone
ΠΑ)가 조혈즐기세포에서 ΝΚ세포의 분화를 유도 및 촉진시키며 ΝΚ세포 분화와 관련된 유전자인 ID2의 발현을 증가시키고， 암세포인 YAC-1에 대하여 세포독성을 지니며 IFN-Y의 생성 또한 증가시켜 ΝΚ 세포의 활성을 증진시키므로， ΝΚ세포 분화 또는 활성 증진용 조성물의 유효성분으로 유용하게 사용될 수 있다. ΠΑ) induces and promotes the differentiation of ΝΚ cells in hematopoietic enjoyment cells, increases the expression of ID2, a gene associated with ΝΚ cell differentiation, is cytotoxic to cancer cells YAC-1, and increases the production of IFN-Y. Since it enhances the activity of ΝΚ cells, it can be usefully used as an active ingredient of ΝΚ cell differentiation or activity enhancing composition.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 생쥐의 조혈줄기세포로부터 ΝΚ 세포로 분화한 결과를 나타낸 도이다.  Figure 1 is a diagram showing the result of differentiation into ΝΚ cells from hematopoietic stem cells of the mouse.
A: IL-15(25ng/mi),크립토탄시논 (cryptotanshinone, C Y)(5uM)및 탄시논 A: IL-15 (25 ng / mi), cryptotanshinone (C Y) (5 uM) and tanshinone
Π ACtanshinone ΠΑ, TAKlOuM)로 분화시킨 세포를 유동세포분석법 (Flow cytometry)으로 분석한 결과; 및 Cells differentiated with Π ACtanshinone ΠΑ, TAKlOuM) were analyzed by flow cytometry; And
&: 크립토탄시논 또는 탄시논 ΠΑ의 각 농도별로 처리하여 분화시킨 NK 세포 (CD3— NK1.1+)를 유동세포분석법으로 분석한 결과.  &: Results of analysis of flow cytometry of NK cells (CD3—NK1.1 +) differentiated by treatment with each concentration of Cryptotansinone or tanshinone ΠΑ.
도 2는 생쥐의 조혈즐기세포로부터 화합물을 처리하여 분화한 NK 세포를 분석한 결과를 나타낸 도이다.  Figure 2 is a diagram showing the results of analysis of NK cells differentiated by treatment of compounds from hematopoietic enjoyment cells of the mouse.
A: 대조군 (control), IL-15, 크립토탄시논 (cryptotanshinone, CRY) 및 IL-15과 함께 크립토탄시논으로 분화시킨 NK 세포 (CD3— NK1.1+)의 전체 세포 수의 변화를 분석한 결과; 및 A: whole cell of NK cells (CD3—NK1.1 + ) differentiated with Cryptotansinone in combination with control, IL-15, cryptotanshinone (CRY) and IL-15 Analysis of the change in number; And
B: 대조군 (control), IL-15, 탄시논 Π A(tanshinone ΠΑ, TA) 및 IL-15과 함께 탄시논 ΠΑ로 분화시킨 NK 세포 (CD3— NK1.1+)의 세포 수의 변화를 분석한 결과. B: Analysis of changes in cell number of NK cells (CD3—NK1.1 + ) differentiated with tanshinone ΠΑ with control, IL-15, tanshinone ΠΑ, TA-15 and IL-15 One result.
도 3은 조혈즐기세포로부터 화합물을 처리하여 분화시킨 NK 세포에서 성숙 (maturation)과 관련된 유전자 발현 (gene expression)을 실시간 PC ( real -time PCR)로 측정한 결과를 나타낸 도이다.  3 is a diagram showing the results of measuring the gene expression associated with maturation (real-time PCR) in NK cells treated with compounds from hematopoietic enjoyment cells and differentiated.
도 4는 생쥐의 조혈줄기세포로부터 다양한 조건으로 배양시킨 NK세포의 세포독성 (cytotoxicity) 및 IFN-γ 생성능을 분석한 결과를 나타낸 도이다.  Figure 4 is a diagram showing the results of analyzing the cytotoxicity (cytotoxicity) and IFN-γ production capacity of NK cells cultured in various conditions from hematopoietic stem cells of the mouse.
A: 줄기세포로부터 각 각 대조군 (control), IL-15(25ng) , 크립토탄시논 (cryptotanshinone, CRY)(5uM) 및 IL-15(25ng)과 함께 크립토탄시논 (5uM)을 처리하여 분화시킨 성숙한 NK mature NK, mNK) 세포를 가지고 YAC-1에 대한 세포독성을 측정한 결과; 및  A: Cryptotansinone (5 uM) was treated with control, IL-15 (25 ng), cryptotanshinone (CRY) (5 uM) and IL-15 (25 ng) from stem cells, respectively. Cytotoxicity against YAC-1 with differentiated mature NK mature NK, mNK) cells; And
B: 조혈줄기세포로부터 각 각 대조군 (control), IL-15(25ng) , 크립토탄시논 (5uM), 탄시논 ΠΑ(ΙΟιιΜ), IL-15(25ng)과 함께 크립토탄시논 (5uM) 및 IL-15(25ng)과 함께 탄시논 ΠΑ(ΙΟιιΜ)을 처리하여 분화시킨 성숙한 Κ 세포를 가지고 IL-12(20ng)로 활성화시킨 후 IFN-γ 생성량을 ELISA로 측정한 결과. 【발명의 실시를 위한 형태】  B: Cryptotansinone (5 uM) with control, IL-15 (25 ng), Cryptotansinone (5 uM), tanshinone ΠΑ (ΙΟιιΜ), IL-15 (25 ng) from hematopoietic stem cells And IFN-γ production was measured by ELISA after activating with IL-12 (20ng) with mature Κ cells differentiated by treatment with tanninone ΠΑ (ΙΟιιΜ) together with IL-15 (25ng). [Form for implementation of invention]
이하, 본 발명을 실시예 및 제조예에 의해 상세히 설명한다.  Hereinafter, the present invention will be described in detail by examples and production examples.
단， 하기 실시예 및 제조예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예 및 제조예에 한정되는 것은 아니다. <실시예 1>마우스 골수로부터 조혈줄기세포의 분리  However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples. Example 1 Isolation of Hematopoietic Stem Cells from Mouse Bone Marrow
실험용 마우스 (중앙실험동물 (주) , 한국)의 종아리뼈， 넓적다리뼈， 골반뼈를 분리하고 이로부터 당 업계에 알려진 방법에 의해 총 골수 세포를 추출하였다. 골수에서 추출한 세포에 ACK 용액을 처리하여 적혈구를 제거한 후， B220, CD2, N 1.1, CDllb, Gr-1, 및 TER— 119 항체를 처리하여 각각에 해당하는 B세포 , T세포， NK/NKT세포,단핵구，과립구 및 적혈구에 붙이고， 이를 MACSGnagnetic一 activated . cell sorting) 세포 분리 키트 (Cell Separat ion Kit)(Mi ltenyi Biotec, Bergisch Gladbach, Germany)를 이용하여 음성 선택 (negative selection)으로 제거하였다. 이와 같이 분화된 면역세포들을 제거한 후， 조혈줄기세포의 특이 표면 분자인 CD117(c-kit)에 대한 마이크로 -비드 (micro-bead) 및 MACS를 이용하여 양성 선택 (positive selection)으로 조혈줄기세포를 분리하였다. <실시예 2>골수로부터 분리된 조혈줄기세포로부터 NK세포로의 분화 유도 The calf bone, thigh bone, and pelvic bone of the experimental mouse (Central Animal Co., Ltd., Korea) were isolated and total bone marrow cells were extracted from the method by methods known in the art. Cells extracted from bone marrow were treated with ACK solution to remove red blood cells Subsequently, B220, CD2, N 1.1, CDllb, Gr-1, and TER—119 antibodies are treated and attached to the corresponding B cells, T cells, NK / NKT cells, monocytes, granulocytes, and red blood cells, respectively, and MACSGnagnetic 一 activated . Cell sorting) The cells were removed by negative selection using a Cell Separat ion Kit (Mitenyi Biotec, Bergisch Gladbach, Germany). After removing the differentiated immune cells, hematopoietic stem cells were positively selected using micro-bead and MACS for CD117 (c-kit), a specific surface molecule of hematopoietic stem cells. Separated. Example 2 Induction of Differentiation of NK Cells from Hematopoietic Stem Cells Isolated from Bone Marrow
골수에서 분리한 조혈줄기세포를 24-웰 (well) 플레이트 (plate)(Becton and Dickinson Bioscience, San Diego, CA)에 1.0 x 106세포 /웰 (eel ls/wel 1 )의 농도로 10% 소태아혈청 (fetal bovine serum, FBS), 2 g/\\\i 인도메타신 (indometacin)(Sigma—Aldrich, St. Louis) , 2 fig/ mi 젠타마이신 (gentamicinXSigma— Aldridi), 30 ng/m^ 쥐의 (murine) SCF(PeproTech Rocky Hill, NJ), 50 ng/ml 쥐의 Flt3L(PeproTech)， 0.5 ng/mi 쥐의 IL-7(PeproTech)을 포함하는 RPMI 1640배지를 사용하여 37°C, 5¾)∞2에서 7일간 배양하였다. 최초 배양 3일 후 절반의 배양 상층액을 같은 조성의 사이토카인 (cytokine)이 함유된 새로운 배지로 교체하였다. NK 세포로의 분화를 위해 7일간 배양한 중간단계의 전구체 세포들을 회수하여 다양한 농도의 쥐의 IL-15 및 크립토탄시논 /탄시논 nA(crptotanshinone/tanshinone ΠΑ)를 포함하는 배지에서 7일간 추가 배양하였다. 3일 후 절반의 배지를 같은 조성의 사이토카인이 함유된 새로운 배지로 교체하였다. 2일， 4일 및 6일 별로 항 (anti)-NKl.l(PE) 및 항 (anti )-CD3(FITC) 항체를 이용하여 服1.1¾03_인 R 세포의 순도를 분석하고ᅳ 다양한 NK 세포 표면 분자에 대한 항체를 이용하여 NK 세포의 분화 정도를 유동세포분석법 (flow cytometry)으로 분석하였다. Hematopoietic stem cells isolated from bone marrow were treated with 10% bovine cells in a 24-well plate (Becton and Dickinson Bioscience, San Diego, Calif.) At a concentration of 1.0 x 10 6 cells / well (eel ls / wel 1). Fetal bovine serum (FBS), 2 g / \\\ i indometacin (Sigma—Aldrich, St. Louis), 2 fig / mi gentamicin (gentamicinXSigma— Aldridi), 30 ng / m ^ 37 ° C, using RPMI 1640 medium containing murine SCF (PeproTech Rocky Hill, NJ), 50 ng / ml rat Flt3L (PeproTech), 0.5 ng / mi rat IL-7 (PeproTech) 5¾) was incubated for 7 days. After 3 days of initial culture, half of the culture supernatant was replaced with fresh medium containing cytokines of the same composition. Seven days of intermediate precursor cells were harvested for differentiation into NK cells and added for 7 days in a medium containing various concentrations of rat IL-15 and Cryptotanshinone / tanshinone nA (crptotanshinone / tanshinone ΠΑ). Incubated. After 3 days half of the medium was replaced with fresh medium containing cytokines of the same composition. 2 days, 4 days and 6 days by anti (anti) -NKl.l (PE) and anti (anti) -CD3 (FITC) using the antibody analysis the purity of the R _服1.1¾03 cells and NK cells, various eu Differentiation of NK cells was analyzed by flow cytometry using antibodies to surface molecules.
그 결과， 도 1의 A에서 보는 바와 같이 조혈줄기세포로부터 NK 세포로 분화시켰을 때 IL-15(25ng/iiO만을 첨가한 경우 약 30%가 CD3 +세포로 분화되었지만， 크립토탄시논 (5uM)/탄시논 IIACcrptotanshinone/tanshinone IIAXlOuM)를 함께 첨가한 경우 '약 65%, 62%가 NK 세포로 분화되었다는 것을 확인하였다 (도 1의 A). As a result, as shown in Figure 1A from hematopoietic stem cells to NK cells At the time of differentiation, IL-15 (approximately 30% differentiated to CD3 + cells when only 25ng / iiO was added, but when Cryptotansinone (5uM) / tansinone IIACcrptotanshinone / tanshinone IIAXlOuM) was added together, ' about 65%, It was confirmed that 62% differentiated into NK cells (A in FIG. 1).
또한, 조혈줄기세포로부터 NK세포 분화를 유도하는 크립토탄시논 /탄시논 Cryptotansinone / tansinone also induces NK cell differentiation from hematopoietic stem cells
ΠΑ의 적정 처리 농도를 알기 위해 각 2， 5 및 lOuM의 농도별로 처리하였다. 그 결과, 도 1의 B에서 보는 바와 같이 크립토탄시논 (cryptotanshinone)의 경우， NK 세포로의 분화율이 5uM을 처리하였을 때 약 56%로 가장 높았고 탄시논 HACtanshinone ΠΑ)의 경우에는 lOuM을 처리하였을 때 약 62%로 가장 높게 나왔으므로 적정 농도의 크립토탄시논 /탄시논 ΠΑ를 IL-15과 함께 처리를 했을 경우, NK 세포의 분화 유도 및 증식이 증가됨을 확인하였다 (도 1의 B). In order to know the appropriate treatment concentration of ΠΑ, it was treated for each concentration of 2, 5 and lOuM. As a result, as shown in B of FIG. 1, in the case of cryptotanshinone, the differentiation rate into NK cells was the highest at about 56% when 5uM was treated, and lOuM was treated in the case of tanshinone HACtanshinone ΠΑ). When the highest concentration of about 62% of Cryptotansinone / tansinone ΠΑ was treated with IL-15, it was confirmed that differentiation induction and proliferation of NK cells increased (FIG. 1B). .
<실시예 3> N 세포의 분화도 확인 Example 3 Confirmation of Differentiation of N Cells
상기 <실시예 2>에서 분석한 NK 세포의 분화율 증가가 실제로 NK 집단 (population) 변화와 연관성이 있는지를 알기 위하여 분화 중간단계인 전구체에 IL- 15(25 ng)을 처리한 시점부터 날짜별로 수거한 세포의 수를 세었다. 전체 세포 수로부터 NK 세포 분화도 (%)를 이용하여 NK 세포 수를 계산하였다.  In order to know whether the increase in the differentiation rate of NK cells analyzed in <Example 2> is actually associated with the change in NK population, the date from the time of treating IL-15 (25 ng) to the intermediate stage of differentiation, The harvested cells were counted. NK cell number was calculated using the percentage of NK cell differentiation from the total cell number.
그 결과， 도 2의 A 및 도 2의 B에서 보는 바와 같이 IL— 15를 처리하지 않은 대조군， IL-15 단독 처리군 (25 ng), 크립토탄시논 (5uM) 단독 처리군， 및 IL-15(25 ng) 및 크립토탄시논 (5uM)을 함께 처리한 군에서 시간이 경과함에 따라 전체 세포 수는 전반적으로 큰 차이 없이 감소하는 양상을 보였지만 NK 세포 수를 비교한 결과 IL-15 및 크립토탄시논을 함께 처리한 군에서 IL-15만을 처리한 경우보다 세포 수가 크게 증가한 것을 확인할 수 있었고， IL-15(25 ng)및 탄시논 ΠΑ(ΙΟιιΜ)를 함께 처리한 군에도 상기와 유사한 양상올 보였다는 것을 확인하였다 (도 2의 Α 및 도 2의 Β).  As a result, as shown in FIG. 2A and FIG. 2B, a control group not treated with IL-15, a IL-15-only group (25 ng), a crypto-tansinone (5uM) -only group, and IL- In the group treated with 15 (25 ng) and Cryptotansinone (5 uM), the overall cell number decreased with no significant difference over time, but the comparison of NK cell numbers showed IL-15 and Crypto In the group treated with tanshinone, the number of cells increased significantly compared to the case of IL-15 alone treatment, and the same pattern as in the group treated with IL-15 (25 ng) and tanshinone ΠΑ (ΙΟιιΜ). It was confirmed that all appeared (Α in Fig. 2 and Β in Fig. 2).
또한， NK 세포로 분화되는 과정에서 발현되는 표적 유전자 (target gene)를 알아내기 위해 기존의 NK 세포 분화와 관련된 유전자들의 발현을 날짜별로 실시간 PCR( real -time PCR)로 측정하였다. In addition, the target gene expressed in the process of differentiation into NK cells (target In order to determine the genes, expression of genes related to conventional NK cell differentiation was measured by real-time PCR by date.
구체적으로， 분화 과정 중의 NK 세포를 회수하고 트리졸 시약 (Trizol Reagent )(Invitrogen, Carlsbad, CA)/클로로포름 (chloroform)/이소프로필 알코을 (isopropyl alcohol)을 이용하여 총 RNA를 분리하였다. 1 내지 3 의 RNA를 2.5 U의 몰로니 뮤린 백혈병 바이러스 (Moloney murine leukemia virus, M—MLV) 역전사 효소 (reverse transcriptase) (Roche Diagnostics, Basel , Switzerland)와 함께 37°C에서 1시간 동안 반웅시켜 cDNA를 합성하였다. 합성된 cDNA의 전사체는 SYBR Premix Ex Taq(Takara Bio, Tokyo, Japan) 및 Real-time system TP 800(Takara Bio)을 이용하여 정량하였으며， 시료의 표준화를 위하여 GAPDH를 사용하였다, 각각의 프라이머 (primer)는 Pr ier3 소프트웨어 (software)를 사용하여 제작하였다. 사용된 프라이머는 하기와 같다: Specifically, NK cells during differentiation were recovered and total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, Calif.) / Chloroform / isopropyl alcohol (isopropyl alcohol). RNA from 1 to 3 was reacted for 1 hour at 37 ° C with 2.5 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Roche Diagnostics, Basel, Switzerland) for cDNA Was synthesized. Transcripts of the synthesized cDNA were quantified using SYBR Premix Ex Taq (Takara Bio, Tokyo, Japan) and Real-time system TP 800 (Takara Bio), and GAPDH was used for standardization of samples. primer) was prepared using the Prier 3 software. The primers used are as follows:
ID2 정방향 프라이머 5' -GCMMGMAGAGMAGTMGA-3' (서열번호 1) 및 ID2 역방향 프라이머 5 ' -GAACACGGACATCAGCATC-3 ' (서열번호 2)；  ID2 forward primer 5'-GCMMGMAGAGMAGTMGA-3 '(SEQ ID NO: 1) and ID2 reverse primer 5' -GAACACGGACATCAGCATC-3 '(SEQ ID NO: 2);
TRAF정방향 프라이머 5 '-CAGACAGAGGAGACAGCAGGA-3' (서열번호 3)및 TRAF 역방향 프라이머 5'-CACACAGAAGAGGAACTA-3' (서열번호 4);  TRAF forward primer 5'-CAGACAGAGGAGACAGCAGGA-3 '(SEQ ID NO: 3) and TRAF reverse primer 5'-CACACAGAAGAGGAACTA-3' (SEQ ID NO: 4);
IRF2 정방향 프라이머 5'-GCTTCCTCTTGGTTrTGCTCᅳ 3' (서열번호 5) 및 IRF2 역방향 프라이머 5'-CTCTGCTGTGCGGGTGTA— 3' (서열번호 6); 및  IRF2 forward primer 5'-GCTTCCTCTTGGTTrTGCTC '3' (SEQ ID NO: 5) and IRF2 reverse primer 5'-CTCTGCTGTGCGGGTGTA- 3 '(SEQ ID NO: 6); And
PU.1 정방향 프라이머 5'-GGTCATCTTCTTGCGGTTCT-3' (서열번호 7) 및 PU.1 역방향 프라이머 5'-CaTCCAGTTCTCGTCCA-3' (서열번호 8).  PU.1 forward primer 5'-GGTCATCTTCTTGCGGTTCT-3 '(SEQ ID NO: 7) and PU.1 reverse primer 5'-CaTCCAGTTCTCGTCCA-3' (SEQ ID NO: 8).
그 결과， 도 3에서 보는 바와 같이 TRAF, IRF2 및 PU.1은 각 각의 처리조건에서 유전자 발현량이 크게 차이를 보이지 않았지만， ID2의 경우 크립토탄시논 (5uM) 단독 처리군， IL— 15(25ng) 및 크립토탄시논 (5uM)을 함께 처리한 군에서만 발현량이 높게 유지되고 있다는 것을 확인하였다 (도 3).  As a result, as shown in Figure 3, TRAF, IRF2 and PU.1 did not show a significant difference in the amount of gene expression under each treatment condition, but in the case of ID2, the group Cryptotansinone (5uM) alone, IL-15 ( 25 ng) and Cryptotansinone (5 uM) together with the group was confirmed that the expression level was maintained high (Fig. 3).
<실시예 4> 전구체로부터 분화한 NK세포의 기능 확인 Example 4 Confirmation of Function of NK Cells Differentiated from Precursors
표적 세포 (target cell)인 YAOl에 대한 NK 세포의 세포독성 (cytotoxicity)을 측정하기 위하여 4-h 51Cr-방출 분석 (51Cr-release assay) 방법을 이용하였다. 51Cr이 라벨된 표적 (51Cr-labeled target) YAC-1 세포 (5 x 103세포 /웰)에 대하여 각 각 50:1， 25： 1 및 5:1로 희석하여 상기 <실시예 2>의 방법으로 분화된 NK 세포를 처리함으로서 실시하였고， 상층액에 분비된 51Cr을 γ -카운터 -counter)를 사용하여 측정하였다. Of NK cells against YAOl, a target cell A 4-h 51 Cr- release assay (51 Cr-release assay) method to measure the cytotoxicity (cytotoxicity) were used. 51 Cr with respect to the labeled target (51 Cr-labeled target) YAC -1 cells (5 x 10 3 cells / well), respectively 50: 1, 25: 1 and 5: diluted to 1, the <Example 2> It was carried out by treating the differentiated NK cells by the method of, and the 51 Cr secreted in the supernatant was measured using γ-counter-counter.
그 결과， 도 4의 A에서 보는 바와 같이 IL-15(25ng/m« 및 크립토탄시논 (5uM)을 함께 처리한 조건에서 세포독성이 가장 높게 나왔다는 것을 확인하였다 (도 4의 A).  As a result, as shown in FIG. 4A, it was confirmed that the highest cytotoxicity was obtained under the conditions treated with IL-15 (25ng / m «and Cryptotansinone (5uM) together (FIG. 4A).
또한， NK 세포의 기능을 알아보는 또 다른 방법인 IFN-Y의 분비량을 측정하기 위하여 상기 <실시예 2>의 방법으로 분화시킨 NK 세포 (2 X 105세포 /웰)를 mIL-12(20ng/m 및 mIL_15(25ng/m )로 16시간 동안 자극 시킨 후에 상층액을 EL ISA (enzyme- linked' immunosorbent assay) 키트 (kit)(R&D systems., CA)로 측정하였다. In addition, mIL-12 (20 ng) of NK cells (2 × 10 5 cells / well) differentiated by the method of Example 2 to measure the secretion amount of IFN-Y, which is another method of determining the function of NK cells, was measured. After stimulation with / m and mIL_15 (25 ng / m) for 16 hours, the supernatants were measured with an ELISA (enzyme-linked ' immunosorbent assay) kit (R & D systems., CA).
그 결과， 도 4의 B에서 보는 바와 같이 세포독성과 마찬가지로 IL-15(25ng/m )과 크립토탄시논 (5uM), 또는 IL_15(25ng/i )과 탄시논 nA(10uM)를 함께 처리한 조건에서 IFN-y의 분비량이 가장 높게 나왔다는 것을 확인하였다 (도 4의 B). 하기에 본 발명의 조성물을 위한 제조예를 예시한다.  As a result, as shown in FIG. 4B, IL-15 (25 ng / m) and Cryptotansinone (5 uM), or IL_15 (25 ng / i) and tanshinone nA (10 uM) were treated together with cytotoxicity. It was confirmed that the secretion amount of IFN-y was the highest under the conditions (Fig. 4B). The preparation examples for the compositions of the present invention are illustrated below.
<제조예 1> 약학적 제제의 제조 Preparation Example 1 Preparation of Pharmaceutical Formulation
<1-1>산제의 제조  <1-1> Preparation of powder
탄시논 또는 크립토탄시논 25 mg  Tanshinone or Cryptotansinone 25 mg
IL-15 25 rag  IL-15 25 rag
유당 50 nig  Lactose 50 nig
상기의 성분을 흔합한후, 기밀포에 층진하여 산제를 제조하였다.  After mixing the above components, it was laminated on an airtight cloth to prepare a powder.
<1-2>정제의 제조 탄시논 또는 크립토탄시논 25 mg <1-2> Preparation of the tablet Tanshinone or Cryptotansinone 25 mg
IL-15 25 rag  IL-15 25 rag
유 당 50 nig  50 nig lactose
스테아린산 마그네슘 2 rag  Magnesium Stearate 2rag
상기의 성분을 흔합한 후,
Figure imgf000019_0001
정제의 제조방법에 따라서 타정하여 정제를 제조하였다. After mixing the above components,
Figure imgf000019_0001
Tablets were prepared by tableting according to the preparation method of the tablets.
<1-3>캡술제의 제조 <1-3> Preparation of the capsule
탄시논 또는 크립토탄시논  Tanshinone or Cryptotansinone
IL-15  IL-15
유 당  Lactose
스테아린산 마그네슘  Magnesium Stearate
상기의 성분을 흔합한 후， 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.  After mixing the above components, it was filled into gelatin capsules in accordance with a conventional method for producing a capsule to prepare a capsule.
<1-4>환의 제조 <1-4> Preparation of the ring
탄시논 또는 크립토탄시논 25 mg  Tanshinone or Cryptotansinone 25 mg
IL-15 25 rag  IL-15 25 rag
유 당 50 mg  Lactose 50 mg
글리세린 100 mg  Glycerin 100 mg
자일리를 100 mg  100 mg xyl
상기의 성분을 흔합한 후， 통상의 방법에 따라 1 환 당 300 mg이 되도록 제조하였다.  After mixing the above components, it was prepared to 300 mg per ring according to a conventional method.
<1-5>과립의 제조 <1-5> Preparation of granules
탄시논 또는 크립토탄시논 25 mg  Tanshinone or Cryptotansinone 25 mg
IL-15 25 mg  IL-15 25 mg
대두추출물 50 rag 포도당 100 nig 전분 100 mg Soybean Extract 50 rag Glucose 100 nig starch 100 mg
상기 의 성분을 흔합한 후， 30% 에탄을 100 rag을 첨가하여 섭씨 60°C에서 건조하여 과립을 형성한 후 포에 층진하였다 . After mixing the above components, 30% ethane was dried at 60 ° C by adding 100 rag to form granules and layered on the fabric.
<1-6> 주사액제의 제조 <1-6> Preparation of Injection Solution
탄시논 또는 크립토탄시논 25 mg  Tanshinone or Cryptotansinone 25 mg
IL-15 50 ^g/mi  IL-15 50 ^ g / mi
묽은 염산 BP pH 7.6로 될 때까지  Dilute hydrochloric acid BP until pH 7.6
주이용 염화나트륨 BP 최 대 1  Sodium Chloride BP Maximum 1
적당한 용적의 주이용 염화나트륨 BP 중에 탄시논 /크립토탄시논 및 IL-15을 용해시 키고， 생성된 용액의 pH를 묽은 염산 BP를 이용하여 pH 7.6로 조절하고， 주이용 염화나트륨 BP를 이용하여 용적을 조절하고 충분히 흔합하였다 . 용액을 투명 유리로 된 5 타입 I 앰플 증에 충전시 키고， 유리를 용해시 킴으로써 공기의 상부 격자하에 봉입시 키고， 120°C에서 15분 이상 오토클래이브시 켜 살균하여 주사액제를 제조하였다 .  Dissolve tanshinone / kryptotansinone and IL-15 in a suitable volume of main sodium chloride BP, adjust the pH of the resulting solution to pH 7.6 with dilute hydrochloric acid BP, and use volume of main sodium chloride BP And mixed well. The solution was filled into a Type I ampoule of transparent glass, sealed under the upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution. .
• 【산업상 이용가능성】 • Industrial Applicability
본 발명은 NK 세포 분화 유도제， NK 세포 활성 증진제 , 및 암 예방 및 치료제 개발에 유용하게 사용될 수 있다 .  The present invention can be usefully used in the development of NK cell differentiation inducing agent, NK cell activity enhancer, and cancer prevention and treatment.

Claims

【청구의 범위】 [Range of request]
【청구항 11  [Claim 11
탄시논 (tanshinone)을 유효성분으로 함유하는 자연살해세포 (natural killer cell, NK세포) 분화 또는 활성 증진용 조성물.  Natural killer cell (NK cell) differentiation or activity enhancing composition containing tanshinone as an active ingredient.
【청구항 2] [Claim 2]
제 1항에 있어서， 상기 탄시논은 하기 화학식 1로 표시되는 크립토탄시논 (crypt otanshi none) 또는 하기 화학식 2로 표시되는 탄시논 ΠΑ인 것을 특징으로 하는 자연살해세포 분화 또는 활성 증진용 조성물:  The method of claim 1, wherein the tanshinone is a crypt otanshi none (crypt otanshi none) represented by the formula (1) or tanshinone ΠΑ represented by the following formula (2), characterized in that the composition for natural killer cell differentiation or activity enhancement:
[화학식 1]  [Formula 1]
Figure imgf000021_0001
Figure imgf000021_0001
【청구항 3】 제 1항에 있어서， 상기 탄시논은 조혈줄기세포에서 자연살해세포로의 분화를 유도하거나 촉진시키는 것을 특징으로 하는 자연살해세포 분화 또는 활성 증진용 조성물. [Claim 3] The method of claim 1, wherein the tanshinone is a composition for natural killer cell differentiation or activity, characterized in that to induce or promote the differentiation of hematopoietic stem cells to natural killer cells.
【청구항 4】 [Claim 4]
제 1항에 있어서， 상기 탄시논은 ID2의 발현을 증가시키는 것을 특징으로 하는 자연살해세포 분화 또는 활성 증진용 조성물.  The method of claim 1, wherein the tanshinone is a composition for enhancing natural killer cell differentiation or activity, characterized in that to increase the expression of ID2.
【청구항 5】 [Claim 5]
제 1항에 있어서， 상기 탄시논은 YAC-1세포에 대하여 세포독성을 가지며 The method of claim 1, wherein the tanshinone is cytotoxic to YAC-1 cells
IFN-y의 생성을 증가시키는 것을 특징으로 하는 자연살해세포 분화 또는 활성 증진용 조성물. Natural killer cell differentiation or activity enhancing composition, characterized in that to increase the production of IFN-y.
【청구항 6】 [Claim 6]
탄시논 및 IL-15(interleukin-15)를 유효성분으로 함유하는 자연살해세포 분화또는 활성 증진용 조성물.  Natural killer cell differentiation or activity enhancing composition containing tanshinone and IL-15 (interleukin-15) as an active ingredient.
【청구항 7] [Claim 7]
1) 조혈줄기세포에서 자연살해세포 전구체 유도제를 첨가하여 자연살해세포 전구체 세포로의 증식을 유도하는 단계; 및  1) inducing proliferation of natural killer cell precursor cells by adding natural killer cell precursor inducers in hematopoietic stem cells; And
2) 단계 1)의 자연살해세포 전구체 세포에 탄시논을 투여하는 단계를 포함하는 조혈즐기세포로부터 자연살해세포로의 분화 유도 방법 .  2) A method for inducing differentiation from hematopoietic cells to natural killer cells, comprising administering tanshinone to the natural killer cell precursor cells of step 1).
【청구항 8] [Claim 8]
제 7항에 있어서， 단계 2)의 탄시논은 크립토탄시논 또는 탄시논 ΠΑ인 것을 특징으로 하는 조혈줄기세포로부터 자연살해세포로의 분화 유도 방법.  8. The method of claim 7, wherein the tanshinone of step 2) is either Cryptotansinone or Tanshinone ΠΑ.
【청구항 9】 제 7항에 있어서， 단계 2)의 자연살해세포 전구체 세포에 IL-15를 함께 투여하는 것을 특징으로 하는 조혈줄기세포로부터 자연살해세포로의 분화 유도 방법. [Claim 9] 8. The method of claim 7, wherein IL-15 is administered to the natural killer cell precursor cells of step 2).
【청구항 10】 [Claim 10]
조혈줄기세포에서 분화된 자연살해세포 전구체 세포에 탄시논올 처리하는 단계를 포함하는 자연살해세포의 활성 증진 방법.  Method for enhancing the activity of natural killer cells, comprising the step of treating tanninol to natural killer cell precursor cells differentiated from hematopoietic stem cells.
【청구항 11】 [Claim 11]
제 10항에 있어서， 상기 탄시논과 IL-15를 함께 처리하는 단계를 포함하는 것을 특징으로 하는 자연살해세포의 활성 증진 방법.  The method of claim 10, comprising treating the tanshinone and IL-15 together.
【청구항 12] [Claim 12]
제 1항 또는 제 6항의 조성물을 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물.  A pharmaceutical composition for preventing and treating cancer, comprising the composition of claim 1 or 6 as an active ingredient.
【청구항 13】 [Claim 13]
제 12항에 있어서， 상기 암은 유방암， 흑색종암， 위암, 간암 및 폐암으로 구성된 군으로부터 선택되는 것을 특징으로 하는 암 예방 및 치료용 약학적 조성물 .  The pharmaceutical composition of claim 12, wherein the cancer is selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, and lung cancer.
[청구항 14】 [Claim 14]
약학적으로 유효한 양의 제 1항 또는 제 6항의 조성물을 개체에 투여하는 단계를 포함하는 암 예방 또는 치료 방법 .  A method of preventing or treating cancer comprising administering to a subject a pharmaceutically effective amount of the composition of claim 1.
【청구항 15] [Claim 15]
제 14항에 있어서， 상기 암은 유방암, 흑색종암, 위암, 간암 및 폐암으로 구성된 군으로부터 선택되는 것을 특징으로 하는 암 예방 또는 치료 방법. The method for preventing or treating cancer of claim 14, wherein the cancer is selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, and lung cancer.
【청구항 16】 [Claim 16]
자연살해세포의 분화 유도에 사용하기 위한 제 1항 또는 제 6항의 조성물.  The composition of claim 1 or 6 for use in inducing differentiation of natural killer cells.
【청구항 17] [Claim 17]
자연살해세포의 활성 증진에 사용하기 위한 제 1항 또는 제 6항의 조성물.  The composition of claim 1 or 6 for use in enhancing the activity of natural killer cells.
PCT/KR2013/000556 2012-01-26 2013-01-24 Composition containing tanshinone as active ingredient, for increasing differentiation or activity of natural killer cells WO2013111969A1 (en)

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