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WO2025165052A1 - Method for enhancing cancer cell killing ability of natural killer cells by using electrical stimulation system - Google Patents

Method for enhancing cancer cell killing ability of natural killer cells by using electrical stimulation system

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Publication number
WO2025165052A1
WO2025165052A1 PCT/KR2025/001243 KR2025001243W WO2025165052A1 WO 2025165052 A1 WO2025165052 A1 WO 2025165052A1 KR 2025001243 W KR2025001243 W KR 2025001243W WO 2025165052 A1 WO2025165052 A1 WO 2025165052A1
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WIPO (PCT)
Prior art keywords
electrical stimulation
natural killer
cells
killer cells
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2025/001243
Other languages
French (fr)
Korean (ko)
Inventor
이민선
권순조
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Inha University Research and Business Foundation
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Inha University Research and Business Foundation
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Publication of WO2025165052A1 publication Critical patent/WO2025165052A1/en
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • the present invention relates to a method for enhancing the cancer cell killing ability of natural killer cells (NK cells) using an electrical stimulation system.
  • NK cells play a crucial role in innate immunity without antigen presentation. This immune activity is regulated by the balance of signaling from activating and inhibitory receptors on the cell surface.
  • the antitumor activity of NK cells largely involves target cell recognition, formation of an immune synapse, and ultimately cytolytic degranulation.
  • the underlying mechanisms remain unclear, and NK cell activity is easily inhibited by tumor cells.
  • the inventors of the present invention introduced electrodes by applying platinum wires to a 3D printed plate cover, designed an electrical stimulation system capable of controlling stimulation conditions through a function generator, and observed the correlation between the influx of intracellular calcium ions induced by the electrical stimulation of this system and the activation of natural killer cells, and confirmed the effect of enhancing the efficacy of cell therapy without introducing genetic modification, leading to the present invention.
  • the present invention aims to provide a method for enhancing the cancer cell killing ability of natural killer cells using an electrical stimulation system, and to provide a correlation between the cancer cell killing ability of natural killer cells and the intracellular calcium ion level.
  • a method for activating natural killer cells comprising the step of providing electrical stimulation to natural killer cells.
  • the electrical stimulation can be performed at a voltage of 0.3 to 1.2 V/cm for 40 to 70 minutes.
  • the electrical stimulation can increase the calcium ion concentration of the natural killer cells by 1.1 to 1.5 times.
  • natural killer cells are provided, produced by a method for activating natural killer cells according to the present invention.
  • a pharmaceutical composition for preventing or treating cancer comprising natural killer cells according to the present invention as an active ingredient.
  • Natural killer cells that receive electrical stimulation through the electrical stimulation system according to the present invention not only exhibit 1.2 to 1.6 times higher cytotoxicity toward tumor cells compared to natural killer cells that do not receive electrical stimulation, but also can enhance the immune activity of natural killer cells by activating a calcium ion-mediated mechanism.
  • Figure 1 is a diagram showing a schematic diagram of the electrical stimulation system design process according to Manufacturing Example 2.
  • Figure 2 is a diagram illustrating a schematic of the experimental design process for co-cultivation of NK cells and target tumor cells according to Example 1.
  • Figure 3 is a diagram showing the results of analyzing the effect of electrical stimulation on the cell survival rate of NK cells according to Example 2.
  • Figure 4 is a diagram showing the results of confirming the effect of electrical stimulation on the cancer cell killing ability of NK cells according to Example 3.
  • Figure 5 is a diagram showing the results of confirming changes in gene and protein expression in NK cells after electrical stimulation, according to Experimental Example 4.
  • Figure 6 is a diagram showing the results of confirming changes in intracellular calcium concentration and calcium-mediated signal transmission after electrical stimulation according to Example 5.
  • Figure 7 is a diagram showing the results of Example 6, which confirms the effect of the BAPTA-AM calcium chelator on calcium-mediated gene expression changes and NFAT1 dephosphorylation due to electrical stimulation.
  • Figure 8 is a diagram schematically showing the calcineurin-NFAT signal transduction pathway according to the present invention.
  • a method for activating natural killer cells comprising the step of providing electrical stimulation to natural killer cells, and more specifically, the electrical stimulation is provided through an electrical stimulation system.
  • the above natural killer cells are cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGLs) and play an important role in the innate and adaptive immune systems.
  • the above natural killer cells may include not only mature natural killer cells but also natural killer precursor cells.
  • the above natural killer cells may be of mammalian origin, and the mammal may be a human, monkey, goat, sheep, rat, mouse, etc., and in particular, may be a human, monkey, or mouse.
  • the increase in the cell killing capacity of the above natural killer cells may be due to an increase in the expression or secretion of substances such as perforin, granzyme, and interferon that are involved in killing target cells, or may be due to an increase in the degranulation of granules containing the above substances.
  • the perforin may be perforin-1, perforin-2, etc.
  • the granzyme may be granzyme A, granzyme B, granzyme H, granzyme K, granzyme M, etc.
  • the interferon may be type 1 interferon such as interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , etc., type 2 interferon such as interferon- ⁇ , or type 3 interferon such as interferon-L1.
  • the above-mentioned allogeneic killing may refer to the phenomenon in which natural killer cells exhibit cell killing ability among themselves, in other words, the natural killer cells themselves act as both effector cells and target cells at the same time.
  • the allogeneic killing between the above-mentioned natural killer cells may be increased by TGF- ⁇ , and the decrease in the allogeneic killing of the above-mentioned natural killer cells means that the degree to which the natural killer cells recognize other natural killer cells as target cells is reduced.
  • the increase in the sensitivity of the above-mentioned natural killer cells to target cells means that the natural killer cells can more easily recognize the target cells.
  • the activity of the above natural killer cells may be the killing ability against target cells such as cancer cells or virus-infected cells, the degranulation phenomenon of natural killer cells, or the stimulation of activating receptors of natural killer cells or the inactivation of inhibitory receptors.
  • the activating receptors may be NKG2D, 2B4, DNAM-1, NCRs, etc.
  • the inhibitory receptors may be PD-1, LAG-3, TIM-3, etc.
  • the enhancement of the activity of the above natural killer cells may mean an increase in the cytotoxicity of the natural killer cells against target cells, a decrease in fratricide, and an enhancement in sensitivity to target cells.
  • the natural killer cells used in the present invention may be commonly used natural killer cells, and most suitably may be the human NK cell line KHYG-1.
  • the electrical stimulation system is designed and adjusted to fit a cell culture plate, and includes an electrical system chamber manufactured in the form of a lid that fits the cell culture plate, and a function generator connected to the electrical system chamber.
  • the electrical system chamber was manufactured by printing with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea), and the stimulation conditions were controlled by a function generator.
  • the electrical stimulation system manufactured accordingly was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA).
  • the electrical system chamber consists of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wire with a diameter of 0.3 mm) that are manufactured parallel to each other and spaced 20 mm apart.
  • Pt platinum
  • an electrical system chamber manufactured to fit the size of a standard 6-well plate can be configured such that two platinum (Pt) electrodes are inserted completely across the entire plate well, such that the electrodes controlled by the function generator can influence all cells throughout the well through the medium.
  • Pt platinum
  • the two platinum (Pt) electrodes can be inserted to a depth of 13 to 17 mm or 14 to 16 mm, and suitably inserted to a depth of 15 mm in order to affect all cells in the entire well, thereby providing electrical stimulation to the cells.
  • the electrical stimulation can be performed for 40 to 70 minutes or 50 to 65 minutes at a voltage of 0.3 to 1.2 or 0.4 to 1.1 V/cm, and suitably, in terms of enhancing cell killing ability against target cells while maintaining cell viability, it can be characterized by performing the treatment for 60 minutes at a voltage of 0.5 to 1.0 V/cm.
  • the electrical stimulation may be characterized by increasing the calcium ion concentration of the natural killer cells, the expression of Granzyme B, and the dephosphorylation of nuclear factor of activated T cell 1 (NFAT 1).
  • the calcium ion concentration of the natural killer cells and the expression of Granzyme B can be increased by 1.1 to 1.5 times compared to the control group that did not receive electrical stimulation according to the present invention, and the dephosphorylation of NFAT 1 can be increased by 1.1 to 2.5 times compared to the control group.
  • the electrical stimulation can enhance the gene expression level of a calcium-mediated signaling-related protein of the calcineurin-NFAT pathway, and for example, the calcium-mediated signaling-related protein can be one or more selected from the group consisting of CALM2, CALN, NFAT1, NFAT2, and NFAT4.
  • granzyme B a key apoptosis-inducing granule molecule
  • the electrical stimulation according to the present invention can enhance the immune function of natural killer cells by inducing changes in intracellular calcium ion levels by affecting calcium influx and downstream mechanisms through calcium-mediated signaling proteins.
  • natural killer cells produced by a method for activating natural killer cells according to the present invention are provided.
  • the cytotoxic ability against target cells can be characterized by increasing by 1.2 to 1.6 times.
  • a problem may arise in which cytotoxicity against the intended target cells is not exhibited, and a problem in which there is no practical benefit may arise in which a significant effect on cytotoxicity against target cells is not exhibited compared to an increase in the ratio of natural killer cells.
  • the natural killer cells can be included at a ratio three times that of target cells.
  • a pharmaceutical composition for preventing or treating cancer comprising natural killer cells according to the present invention as an active ingredient.
  • the cancer generally refers to a physiological condition of mammals characterized by uncontrolled cell growth, and refers to a condition in which a problem occurs in the control function of normal division, differentiation, and death of cells, resulting in abnormal excessive proliferation, infiltration into surrounding tissues and organs, formation of a mass, and destruction or deformation of existing structures.
  • it may be a solid cancer or a metastatic cancer, such as colorectal cancer including colon cancer and rectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head and neck cancer, skin melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, color
  • the cancer may refer to human breast cancer cells MDA-MB-231 or MCF-7, and most preferably MCF-7.
  • the above pharmaceutical composition can be formulated and used in various forms according to conventional methods.
  • it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.
  • composition of the present invention may contain one or more known effective ingredients having preventive, improving, and therapeutic effects on cancer or tumors together with natural killer cell-derived endoplasmic reticulum.
  • pharmaceutically acceptable additives may be further included, and pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, and white sugar.
  • pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate
  • the pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight in the composition, but is not limited thereto.
  • it can be administered in various oral or parenteral dosage forms during actual clinical administration, and when formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used, and it is preferable to use suitable formulations known in the relevant technical field disclosed in the literature.
  • diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used, and it is preferable to use suitable formulations known in the relevant technical field disclosed in the literature.
  • the above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.
  • parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.
  • the dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and/or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field.
  • a person having ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment, and therefore the dosage does not limit the scope of the present invention in any way.
  • the route and method of administration of the pharmaceutical composition of the present invention may be independent, and are not particularly limited in their methods, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.
  • the pharmaceutical composition may be administered orally or parenterally.
  • Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.
  • Human breast cancer cells were obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea) and cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2 .
  • KCLB Korean Cell Line Bank
  • FBS fetal bovine serum
  • Gibco penicillin-streptomycin
  • the human NK cell line KHYG-1 was purchased from AcceGen Biotech (cat. # ABC-TC0506; Fairfield, NJ, USA). KHYG-1 cells were cultured in T-25 flasks in RPMI 1640 medium supplemented with heat-treated FBS, 1% penicillin-streptomycin, and 100 Units/mL recombinant human IL-2 (cat. # 200-02; PeproTech, Rocky Hill, NJ, USA) at 37°C and 5% CO 2 , with fresh medium added or subcultured every 2–3 days.
  • the chamber was printed with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea) and the lid was fabricated to fit a standard 6-well plate.
  • the electrical stimulation system consisted of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wires with a diameter of 0.3 mm) positioned 20 mm apart and parallel to each other. Stimulation conditions were controlled by a function generator designed to fully penetrate the electrodes throughout the plate wells, thereby influencing the cells throughout the wells through the medium.
  • the electrical stimulation system was designed and adjusted to fit a cell culture plate, and its performance was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA), and a schematic structure of the designed electrical stimulation system is shown in Fig. 1.
  • DSOX1102A Keysight, Santa Rosa, CA, USA
  • NK cells were exposed to two voltage ranges (0.5 V/cm, 1.0 V/cm) of direct current (denoted as DC) electrical stimulation or biphasic pulse electrical stimulation (symmetric, charge-balanced, denoted as BP) at a frequency of 10 Hz for 1 h.
  • DC direct current
  • BP biphasic pulse electrical stimulation
  • tumor cells that reached saturation in the plate were co-cultured with NK cells that had been electrically stimulated for 1 hour in advance for 24 hours to prepare an experimental group, and cells co-cultured with tumor cells and NK cells that had not been electrically stimulated for 24 hours were set as a control group.
  • KHYG-1 cells were cultured for 30 min before electrical stimulation in medium supplemented with the cell-permeable calcium ion chelator BAPTA-AM (B6769; Invitrogen, Carlsbad, CA, USA) at a final concentration of 5 ⁇ M, or in medium containing an equivalent amount of DMSO as a control.
  • BAPTA-AM cell-permeable calcium ion chelator
  • KHYG-1 cells that received electrical stimulation for 1 h were transferred to a 96-well plate (100 ⁇ L, 150,000 cells), then 10 ⁇ L of CCK-8 was added, and incubated at 37°C for 3 h. The absorbance of each plate well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).
  • DC direct current
  • BP bipolar
  • biphasic electrical stimulation (biphasic BP) significantly reduced cell survival, whereas stimulation under continuous DC conditions did not. Accordingly, subsequent experiments were conducted using continuous electrical stimulation at two different voltages (0.5 V/cm, 1.0 V/cm) under 1-h DC conditions that did not affect cell survival.
  • LDH lactate dehydrogenase
  • cancer cells MDA-MB-231 and MCF-7, were cultured in 12-well plates, and NK cells (KHYG-1 cells): cancer cells (MDA-MB-231 or MCF-7) were mixed at a ratio of 3:1 and cultured as experimental and control groups, depending on whether or not they were exposed to the electrical stimulation of Example 1.
  • LDH lactate dehydrogenase
  • MDA-MB-231 cells did not show a significant increase in cytotoxicity between the control and experimental groups, but MCF-7 cells showed an increase in cytotoxicity ability, with an increase of 1.27 times (0.5 V/cm electrical stimulation) and 1.55 times (1.0 V/cm electrical stimulation) in the experimental group compared to the control group, respectively.
  • the live/dead cell imaging experiment was conducted using control and experimental groups for MCF-7 cells.
  • the experimental and control groups co-cultured for 6 hours were washed three times with DPBS to remove KHYG-1 cells, and then the MCF-7 cells were stained with 2 ⁇ M calcein-AM (live) and 4 ⁇ M Ethidium homodimer-1 (dead) and analyzed by fluorescence microscopy according to the manufacturer's protocol, as shown in Fig. 4B.
  • KHYG-1 cells were seeded in 6-well culture plates and cultured for 4 and 8 h at 37°C and 5% CO2 after stimulation at 0.5 V/cm or 1.0 V/cm for 1 h, respectively.
  • the culture medium was then collected and centrifuged at 600 g for 5 min.
  • Intracellular proteins were extracted using RIPA cell lysis (RIPA) buffer (Elpis Biotech, Daejeon, Korea) with the addition of a protease inhibitor cocktail (Thermo Fisher Scientific) according to the manufacturer's recommended protocol.
  • Intracellular or culture medium-secreted granzyme B protein levels were measured using a Human Granzyme B ELISA kit (3486-1H-6; MABTECH, Nacka Strand, Sweden) according to the manufacturer's instructions.
  • the intracellular granzyme B level of KHYG-1 cells was found to be higher at 0.5 V/cm compared to the control group, and in particular, it was confirmed to increase 1.26-fold at 1.0 V/cm.
  • the level of granzyme B secreted into the cell medium obtained 4 hours and 8 hours after electrical stimulation was measured, and it was confirmed that the level of granzyme B released outside the cells in the medium 4 hours after electrical stimulation under 0.5 V/cm conditions was 1.20 times higher than that of the control group.
  • the relative fluorescence levels of the calcium ion concentration measured for 45 minutes of electrical stimulation were calculated, and it was confirmed that they increased 1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) after electrical stimulation.
  • KHYG-1 cells were washed twice with cold DPBS and lysed with RIPA buffer (Elpis Biotech) containing protease and phosphatase inhibitors provided by Thermo Fisher Scientific. The cells were then stored on ice for 15 minutes and sonicated for 30 seconds. The total cell lysate was centrifuged for 15 minutes and used for further analysis. Each sample (10 ⁇ L) was diluted in Laemmli sample buffer, incubated at 95°C for 5 minutes, and then separated by 10% SDS-PAGE electrophoresis. The protein samples were then transferred to PVDF membranes using a semi-dry transfer method.
  • the membranes were stored in Tris-buffered saline containing skim milk at 25°C for 1 hour to prevent nonspecific binding.
  • the membrane was incubated overnight at 4°C with primary antibodies against NFAT1 (MA1-025; Invitrogen) and GAPDH (2118S; Cell Signaling Technology, Danvers, MA, USA), which served as a loading control.
  • the membrane was specifically labeled with secondary antibodies (anti-rabbit, ab6721, Abcam; anti-mouse, 31430, Thermo Fisher).
  • the upper band (140 kDa) represents phosphorylated NFAT1 (p-NFAT1)
  • the lower band (120 kDa) represents dephosphorylated NFAT1.
  • the dephosphorylation of NFAT1 was quantitatively expressed as the ratio of p-NFAT1 to NFAT1, and it was confirmed that the dephosphorylation ratio of NFAT1 in the experimental group treated with electrical stimulation (1.0 V/cm) increased 2.21-fold compared to the control group.
  • a mechanism schematic diagram for the calcineurin-NFAT signal transduction pathway according to the above embodiment is shown in Fig. 8.
  • this study is expected to be useful as a method to enhance the efficacy of cell therapy without introducing genetic modification.

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Abstract

The present invention relates to a method for activating natural killer cells, comprising a step of providing electrical stimulation to natural killer cells. Specifically, electrical stimulation under predetermined conditions is applied to natural killer cells by applying an electrical stimulation system established through a 3D-printed electrical stimulation device and a function generator. Here, cancer cell-killing ability and calcium ion-mediated mechanisms are analyzed among induced intracellular effects. In addition, with respect to the natural killer cells produced according to the present invention, a method capable of enhancing the efficacy of a cell therapeutic agent without introducing genetic modification is presented, and the cells can be used to study the correlation between the cancer cell-killing ability and the intracellular calcium ion levels of electrically stimulated natural killer cells.

Description

전기자극 시스템을 활용한 자연살해세포의 암세포 살상 능력 증진 방법 A method for enhancing the cancer-killing ability of natural killer cells using an electrical stimulation system.

본 발명은 전기자극 시스템을 이용한 자연살해세포(NK세포)의 암세포 살상능력 증진 방법에 관한 것이다. The present invention relates to a method for enhancing the cancer cell killing ability of natural killer cells (NK cells) using an electrical stimulation system.

자연살해(NK) 세포는 항원 프레젠테이션 없이 선천적 면역에서 중요한 역할을 한다. 이 면역 활성은 세포 표면의 활성화 및 억제 수용체의 신호 균형에 의해 조절된다. NK 세포의 항암 활성은 대부분 표적세포 인식 후 면역 시냅스를 형성하고, 최종적으로 세포사멸 과립 (cytolytic degranulation) 분출 과정으로 이루어져 있으나, 관련 기작은 여전히 불분명하며 종양 세포에 의해 NK 세포의 세포 활성이 쉽게 저해받는다. Natural killer (NK) cells play a crucial role in innate immunity without antigen presentation. This immune activity is regulated by the balance of signaling from activating and inhibitory receptors on the cell surface. The antitumor activity of NK cells largely involves target cell recognition, formation of an immune synapse, and ultimately cytolytic degranulation. However, the underlying mechanisms remain unclear, and NK cell activity is easily inhibited by tumor cells.

따라서 자연살해세포의 활성화 기작을 이해하는 것은 현재 항암 면역요법(cancer immunotherapy)에서의 한계점 중 하나인 면역 회피 메커니즘을 극복하는 데 필수적이다. Therefore, understanding the activation mechanism of natural killer cells is essential to overcome the immune evasion mechanism, which is one of the limitations of current cancer immunotherapy.

또한, 면역 과정에서 칼슘 이온이 NK 세포의 중요한 활성화 요소인 것으로 알려져 있어, 전기자극이 세포 내 칼슘 이온 수준의 변화를 유발하여 자연살해세포의 면역 기능을 향상시킬 수 있을 것이라는 가설을 세웠다. In addition, since calcium ions are known to be an important activating factor of NK cells in the immune process, we hypothesized that electrical stimulation could enhance the immune function of natural killer cells by inducing changes in intracellular calcium ion levels.

이에 본 발명자들은 3D 프린팅 된 플레이트 덮개에 백금선을 적용하여 전극을 도입하였고, 함수발생기를 통해 자극 조건을 조절할 수 있는 전기자극 시스템을 설계하였으며 이 시스템의 전기자극에 의해 유발된 세포 내 칼슘 이온의 유입과 자연살해세포의 활성화 간의 상관 관계를 관찰하던 중, 도입된 유전적 변형 없이 세포치료제의 효능을 증진하는 효과를 확인하여 본 발명에 이르게 되었다. Accordingly, the inventors of the present invention introduced electrodes by applying platinum wires to a 3D printed plate cover, designed an electrical stimulation system capable of controlling stimulation conditions through a function generator, and observed the correlation between the influx of intracellular calcium ions induced by the electrical stimulation of this system and the activation of natural killer cells, and confirmed the effect of enhancing the efficacy of cell therapy without introducing genetic modification, leading to the present invention.

본 발명은 전기자극 시스템을 이용한 자연살해세포의 암세포 살상능력 증진 방법을 제공하는 것을 목적으로 하고, 자연살해세포의 암세포 살상능력과 세포 내 칼슘 이온 수준 간의 상관 관계를 제공하는 것을 목적으로 한다. The present invention aims to provide a method for enhancing the cancer cell killing ability of natural killer cells using an electrical stimulation system, and to provide a correlation between the cancer cell killing ability of natural killer cells and the intracellular calcium ion level.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다. However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

본 발명은 일 실시예에 따르면, 자연살해세포에 전기자극을 제공하는 단계를 포함하는, 자연살해세포의 활성화 방법이 제공된다.According to one embodiment of the present invention, a method for activating natural killer cells is provided, comprising the step of providing electrical stimulation to natural killer cells.

본 발명에서 상기 전기자극은 0.3 내지 1.2 V/cm의 전압으로 40분 내지 70분 동안 처리할 수 있다. In the present invention, the electrical stimulation can be performed at a voltage of 0.3 to 1.2 V/cm for 40 to 70 minutes.

본 발명에서 상기 전기자극은 상기 자연살해세포의 칼슘 이온 농도를 1.1 내지 1.5배 증가시킬 수 있다. In the present invention, the electrical stimulation can increase the calcium ion concentration of the natural killer cells by 1.1 to 1.5 times.

본 발명의 다른 실시예에 따르면, 본 발명에 따른 자연살해세포의 활성화 방법에 의해 생산된, 자연살해세포가 제공된다. According to another embodiment of the present invention, natural killer cells are provided, produced by a method for activating natural killer cells according to the present invention.

본 발명의 다른 실시예에 따르면, 본 발명에 따른 자연살해세포를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물이 제공된다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising natural killer cells according to the present invention as an active ingredient.

본 발명에 따른 전기자극시스템을 통해 전기자극을 받은 자연살해세포는 전기자극을 받지 않은 자연살해세포와 대비하여, 종양세포에 1.2 내지 1.6배 높은 세포독성을 나타낼 뿐만 아니라, 칼슘 이온 매개 기작을 활성화하여 자연살해세포의 면역 활성을 증진시킬 수 있다. Natural killer cells that receive electrical stimulation through the electrical stimulation system according to the present invention not only exhibit 1.2 to 1.6 times higher cytotoxicity toward tumor cells compared to natural killer cells that do not receive electrical stimulation, but also can enhance the immune activity of natural killer cells by activating a calcium ion-mediated mechanism.

이에 따라 도입된 유전적 변형 없이 세포치료제로 활용할 수 있다. Accordingly, it can be used as a cell therapy agent without introducing genetic modification.

도 1은 제조예 2에 관한 것으로, 전기자극시스템 설계 과정의 도식화를 나타낸 도이다.Figure 1 is a diagram showing a schematic diagram of the electrical stimulation system design process according to Manufacturing Example 2.

도 2는 실시예 1에 관한 것으로, NK 세포 및 대상 종양 세포 공동 배양의 실험적 설계 과정의 도식화를 나타낸 도이다. Figure 2 is a diagram illustrating a schematic of the experimental design process for co-cultivation of NK cells and target tumor cells according to Example 1.

도 3은 실시예 2에 관한 것으로, 전기자극이 NK세포의 세포 생존율에 미치는 영향을 분석한 결과를 나타낸 도이다. Figure 3 is a diagram showing the results of analyzing the effect of electrical stimulation on the cell survival rate of NK cells according to Example 2.

도 4는 실시예 3에 관한 것으로, 전기자극 NK세포의 암세포 살상 능력에 미치는 효과를 확인한 결과를 나타낸 도이다. Figure 4 is a diagram showing the results of confirming the effect of electrical stimulation on the cancer cell killing ability of NK cells according to Example 3.

도 5는 실험예 4에 관한 것으로, 전기자극 이후 NK세포에서 유전자 및 단백질 발현의 변화를 확인한 결과를 나타낸 도이다. Figure 5 is a diagram showing the results of confirming changes in gene and protein expression in NK cells after electrical stimulation, according to Experimental Example 4.

도 6은 실시예 5에 관한 것으로, 전기자극 후 세포 내 칼슘 농도 및 칼슘 매개 신호 전달의 변화를 확인한 결과를 나타낸 도이다. Figure 6 is a diagram showing the results of confirming changes in intracellular calcium concentration and calcium-mediated signal transmission after electrical stimulation according to Example 5.

도 7은 실시예 6에 관한 것으로, BAPTA-AM 칼슘 킬레이터가 전기자극으로 인한 칼슘 매개 유전자 발현 변화 및 NFAT1 탈인산화에 미치는 영향을 확인한 결과를 나타낸 도이다.Figure 7 is a diagram showing the results of Example 6, which confirms the effect of the BAPTA-AM calcium chelator on calcium-mediated gene expression changes and NFAT1 dephosphorylation due to electrical stimulation.

도 8은 본 발명에 따른 calcineurin-NFAT 신호 전달 경로에 대한 도식화를 나타낸 도이다.Figure 8 is a diagram schematically showing the calcineurin-NFAT signal transduction pathway according to the present invention.

이하, 보다 구체적으로 설명하기 위해 실시예를 들어 본 발명을 상세하게 설명하기로 한다. Hereinafter, the present invention will be described in detail by way of examples to explain in more detail.

그러나, 하기 실시예는 예시적인 것으로, 본 발명의 범위가 이에 제한되는 것은 아니며, 달리 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 갖고 있다. However, the following examples are illustrative only and the scope of the present invention is not limited thereto, and unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

또한, 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다. In addition, when describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description is omitted.

본 발명의 일 실시예에 따르면, 자연살해세포에 전기자극을 제공하는 단계를 포함하는 자연살해세포의 활성화 방법이 제공되고, 보다 구체적으로, 상기 전기자극은 전기 자극 시스템을 통하여 제공된다. According to one embodiment of the present invention, a method for activating natural killer cells is provided, comprising the step of providing electrical stimulation to natural killer cells, and more specifically, the electrical stimulation is provided through an electrical stimulation system.

상기 자연살해 세포는 선천성 면역계의 주요 성분을 구성하는 세포독성 림프구로서, 대형 과립 림프구(large granular lymphocyte, LGL)로 정의되고 선천성 면역계와 적응면역계에서 중요한 역할을 담당한다. 상기 자연살해 세포는 성숙한 자연살해 세포뿐만 아니라, 자연살해 전구세포를 포함할 수 있다. 또한, 상기 자연살해 세포는 포유동물 유래일 수 있고, 상기 포유동물은 인간, 원숭이, 염소, 양, 랫, 마우스 등일 수 있고, 특히 인간, 원숭이 또는 마우스일 수 있다.The above natural killer cells are cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGLs) and play an important role in the innate and adaptive immune systems. The above natural killer cells may include not only mature natural killer cells but also natural killer precursor cells. In addition, the above natural killer cells may be of mammalian origin, and the mammal may be a human, monkey, goat, sheep, rat, mouse, etc., and in particular, may be a human, monkey, or mouse.

상기 자연살해 세포의 세포 살상능의 증가는 표적세포를 사멸(cell death)시키는데 관여하는 퍼포린, 그랜자임, 인터페론 등의 물질의 발현이나 분비가 증가되는 것일 수 있고, 또는 상기와 같은 물질들이 포함된 과립(granule)의 탈과립화가 증가되는 것일 수 있다. 특히, 상기 퍼포린은 퍼포린-1, 퍼포린-2 등일 수 있고, 상기 그랜자임은 그랜자임 A, 그랜자임 B, 그랜자임 H, 그랜자임 K, 그랜자임 M 등일 수 있고, 상기 인터페론은 인터페론-α, 인터페론-β, 인터페론-κ, 인터페론-ω 등과 같은 1형 인터페론이나, 인터페론-γ과 같은 2형 인터페론, 또는 인터페론-L1과 같은 3형 인터페론일 수 있다. 또한, 상기 동종살해는 자연살해 세포들 상호간에 세포 살상능을 나타내는 것, 다시 말해서 자연살해 세포 자체가 주효세포(effector cell)이면서 동시에 표적세포(target cell)로서 작용하는 현상을 의미할 수 있다. 상기 자연살해 세포들 간의 동종살해는 TGF-β에 의해 증가할 수 있고, 상기 자연살해 세포의 동종살해의 감소는 자연살해 세포가 다른 자연살해 세포를 표적세포로 인식하는 정도가 감소되는 것을 의미한다. 또한, 상기 자연살해 세포의 표적세포에 대한 민감도의 향상은 자연살해 세포가 표적세포를 더욱 쉽게 인식할 수 있게 되는 것을 의미한다.The increase in the cell killing capacity of the above natural killer cells may be due to an increase in the expression or secretion of substances such as perforin, granzyme, and interferon that are involved in killing target cells, or may be due to an increase in the degranulation of granules containing the above substances. In particular, the perforin may be perforin-1, perforin-2, etc., the granzyme may be granzyme A, granzyme B, granzyme H, granzyme K, granzyme M, etc., and the interferon may be type 1 interferon such as interferon-α, interferon-β, interferon-κ, interferon-ω, etc., type 2 interferon such as interferon-γ, or type 3 interferon such as interferon-L1. In addition, the above-mentioned allogeneic killing may refer to the phenomenon in which natural killer cells exhibit cell killing ability among themselves, in other words, the natural killer cells themselves act as both effector cells and target cells at the same time. The allogeneic killing between the above-mentioned natural killer cells may be increased by TGF-β, and the decrease in the allogeneic killing of the above-mentioned natural killer cells means that the degree to which the natural killer cells recognize other natural killer cells as target cells is reduced. In addition, the increase in the sensitivity of the above-mentioned natural killer cells to target cells means that the natural killer cells can more easily recognize the target cells.

상기 자연살해 세포의 활성은 암 세포 또는 바이러스에 감염된 세포 등과 같은 표적세포에 대한 살상능, 자연살해 세포의 탈과립화 현상 또는 자연살해 세포의 활성화 수용체가 자극되거나 억제성 수용체가 비활성화되는 것일 수 있고, 이때 상기 활성화 수용체는 NKG2D, 2B4, DNAM-1, NCRs 등일 수 있고, 상기 억제성 수용체 PD-1, LAG-3, TIM-3 등일 수 있다.The activity of the above natural killer cells may be the killing ability against target cells such as cancer cells or virus-infected cells, the degranulation phenomenon of natural killer cells, or the stimulation of activating receptors of natural killer cells or the inactivation of inhibitory receptors. In this case, the activating receptors may be NKG2D, 2B4, DNAM-1, NCRs, etc., and the inhibitory receptors may be PD-1, LAG-3, TIM-3, etc.

상기 자연살해세포의 활성이 향상된다는 것은 자연살해 세포의 표적세포에 대한 세포 살상능(cytotoxicity)의 증가, 동종살해(fratricide)의 감소, 표적세포에 대한 민감도(sensitivity)의 향상 등을 의미할 수 있다.The enhancement of the activity of the above natural killer cells may mean an increase in the cytotoxicity of the natural killer cells against target cells, a decrease in fratricide, and an enhancement in sensitivity to target cells.

예를 들어, 본 발명에서 사용되는 자연살해세포는 통상적으로 사용되는 자연살해세포일 수 있고, 가장 적합하게는 인간 NK세포주인 KHYG-1일 수 있다. For example, the natural killer cells used in the present invention may be commonly used natural killer cells, and most suitably may be the human NK cell line KHYG-1.

본 발명에서 상기 전기 자극 시스템은 세포 배양 플레이트에 맞게 설계되고 조정된 것으로, 세포 배양 플레이트에 맞는 뚜껑의 형태로 제작된 전기 시스템 챔버와 전기 시스템 챔버와 연결된 함수 발생기를 포함한다. In the present invention, the electrical stimulation system is designed and adjusted to fit a cell culture plate, and includes an electrical system chamber manufactured in the form of a lid that fits the cell culture plate, and a function generator connected to the electrical system chamber.

이때, 상기 전기 시스템 챔버는 3D 프린터 (3DP-110F; Cubicon, 성남, 한국)로 인쇄하여 제작하였고, 자극 조건은 함수 발생기로 제어하며 이에 따라 제작된 상기 전기 자극 시스템은 오실로스코프 (DSOX1102A; Keysight, Santa Rosa, CA, 미국)를 사용하여 확인하였다. At this time, the electrical system chamber was manufactured by printing with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea), and the stimulation conditions were controlled by a function generator. The electrical stimulation system manufactured accordingly was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA).

또한, 전기 시스템 챔버는 두 개의 백금 (Pt) 전극 (음극과 양극, 지름 0.3 mm의 99.9% Pt wire)으로 구성되어 있으며 서로 평행한 거리로 20mm 떨어지게 제작되었다. Additionally, the electrical system chamber consists of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wire with a diameter of 0.3 mm) that are manufactured parallel to each other and spaced 20 mm apart.

예를 들어, 도 1을 참조하면, 표준 6-well 플레이트의 크기에 맞추어 제작한 전기 시스템 챔버를 두 개의 백금 (Pt) 전극이 완전히 플레이트 웰 전체에 걸쳐 삽입하여 함수 발생기에 의해 제어되는 전극이 배지를 통해 웰 전체 모든 세포에 영향을 미치도록 할 수 있다. For example, referring to Figure 1, an electrical system chamber manufactured to fit the size of a standard 6-well plate can be configured such that two platinum (Pt) electrodes are inserted completely across the entire plate well, such that the electrodes controlled by the function generator can influence all cells throughout the well through the medium.

이때, 상기 두개의 두 개의 백금 (Pt) 전극은 13 내지 17mm 또는 14 내지 16mm의 깊이로 삽입할 수 있고, 웰 전체 모든 세포에 영향을 미치도록 하는 측면에서 적합하게는 15mm의 깊이로 삽입하여 세포에 전기자극을 처리할 수 있다.At this time, the two platinum (Pt) electrodes can be inserted to a depth of 13 to 17 mm or 14 to 16 mm, and suitably inserted to a depth of 15 mm in order to affect all cells in the entire well, thereby providing electrical stimulation to the cells.

본 발명에서 상기 전기자극은 0.3 내지 1.2 또는 0.4 내지 1.1 V/cm의 전압으로 40분 내지 70분 또는 50 내지 65분 동안 처리할 수 있고, 세포의 생존율을 유지하면서 표적세포에 대한 세포 살상능을 증진시키는 측면에서 적합하게는 0.5 내지 1.0 V/cm의 전압으로 60분 동안 처리하는 것을 특징으로 할 수 있다. In the present invention, the electrical stimulation can be performed for 40 to 70 minutes or 50 to 65 minutes at a voltage of 0.3 to 1.2 or 0.4 to 1.1 V/cm, and suitably, in terms of enhancing cell killing ability against target cells while maintaining cell viability, it can be characterized by performing the treatment for 60 minutes at a voltage of 0.5 to 1.0 V/cm.

예를 들어, 상기 전기자극을 본 발명의 범위를 벗어나는 전압 또는 시간으로 처리할 경우, 세포 생존율이 감소하거나 표적세포에 대한 목적하는 세포 살상능을 달성하지 못하는 문제점이 발생할 수 있다.For example, if the electrical stimulation is applied at a voltage or time outside the scope of the present invention, problems such as a decrease in cell viability or failure to achieve the desired cell killing ability for target cells may occur.

본 발명에서 상기 전기자극은 상기 자연살해세포의 칼슘 이온 농도, 그랜자임 B(Granzyme B)의 발현 및 활성화된 T-세포의 핵 인자 1(Nuclear factor of activated T cell 1, NFAT 1)의 탈인산화를 증가시키는 것을 특징으로 할 수 있다. In the present invention, the electrical stimulation may be characterized by increasing the calcium ion concentration of the natural killer cells, the expression of Granzyme B, and the dephosphorylation of nuclear factor of activated T cell 1 (NFAT 1).

본 발명에서 상기 자연살해세포의 칼슘 이온 농도와 상기 그랜자임 B(Granzyme B)의 발현은 본 발명에 따라 전기자극을 받지 않은 대조군과 대비하여, 1.1 내지 1.5배 증가될 수 있고, NFAT 1의 탈인산화는 대조군과 대비하여 1.1 내지 2.5배 증가될 수 있다. In the present invention, the calcium ion concentration of the natural killer cells and the expression of Granzyme B can be increased by 1.1 to 1.5 times compared to the control group that did not receive electrical stimulation according to the present invention, and the dephosphorylation of NFAT 1 can be increased by 1.1 to 2.5 times compared to the control group.

구체적으로 상기 전기자극은 calcineurin-NFAT 경로의 칼슘 매개 신호전달 관련 단백질의 유전자 발현 수준을 향상시킬 수 있으며 예를 들어, 칼슘 매개 신호전달 관련 단백질은 CALM2, CALN, NFAT1, NFAT2 및 NFAT4로 이루어진 그룹에서 선택되는 하나이상일 수 있다. Specifically, the electrical stimulation can enhance the gene expression level of a calcium-mediated signaling-related protein of the calcineurin-NFAT pathway, and for example, the calcium-mediated signaling-related protein can be one or more selected from the group consisting of CALM2, CALN, NFAT1, NFAT2, and NFAT4.

또한, GZMB 발현을 상승시켜 핵심 세포 사멸 유도 과립 분자인 granzyme B의 단백질 발현을 증가시킬 수 있다. Additionally, it can increase the protein expression of granzyme B, a key apoptosis-inducing granule molecule, by upregulating GZMB expression.

즉, 본 발명에 따른 전기자극은 칼슘 유입 및 칼슘 매개의 신호 단백질을 통한 하위 기작에 영향을 미침에 따라, 세포 내 칼슘 이온 수준의 변화를 유발하여 자연살해세포의 면역 기능을 향상시킬 수 있다. That is, the electrical stimulation according to the present invention can enhance the immune function of natural killer cells by inducing changes in intracellular calcium ion levels by affecting calcium influx and downstream mechanisms through calcium-mediated signaling proteins.

본 발명의 다른 실시예에 따르면, 본 발명에 따른 자연살해세포의 활성화 방법에 의해 생산된 자연살해세포가 제공된다.According to another embodiment of the present invention, natural killer cells produced by a method for activating natural killer cells according to the present invention are provided.

구체적으로, 본 발명에 따른 상기 자연살해세포 및 표적세포를 3:1의 비율로 포함하여 공배양 할 경우, 표적세포에 대한 세포독성 능력이 1.2 내지 1.6배 증가하는 것을 특징으로 할 수 있다.Specifically, when the natural killer cells and target cells according to the present invention are co-cultured at a ratio of 3:1, the cytotoxic ability against target cells can be characterized by increasing by 1.2 to 1.6 times.

예를 들어, 상기 자연살해세포 및 표적세포의 비율을 벗어날 경우, 목적하는 표적세포에 대한 세포 독성 능력이 나타나지 않는 문제점이 발생할 수 있고, 자연살해세포 비율의 증가대비 표적세포에 대한 세포 독성 능력의 현저한 효과가 나타나지 않아 실익이 없는 문제점이 발생할 수 있다. For example, if the ratio of natural killer cells and target cells is exceeded, a problem may arise in which cytotoxicity against the intended target cells is not exhibited, and a problem in which there is no practical benefit may arise in which a significant effect on cytotoxicity against target cells is not exhibited compared to an increase in the ratio of natural killer cells.

따라서, 도입된 유전적 변형 없이 세포치료제의 효능을 증진시키는 측면에서 상기 자연살해세포를 표적세포 3배의 비율로 포함할 수 있다. Therefore, in terms of enhancing the efficacy of cell therapy without introducing genetic modification, the natural killer cells can be included at a ratio three times that of target cells.

본 발명의 다른 실시예에 따르면, 본 발명에 따른 자연살해세포를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물이 제공된다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising natural killer cells according to the present invention as an active ingredient.

본 발명에서 상기 암은 일반적으로 비조절된 세포 성장의 특징을 갖는 포유동물의 생리학적 상태를 의미하고, 세포의 정상적인 분열, 분화 및 사멸의 조절 기능에 문제가 발생하여 비정상적으로 과다 증식하여 주위 조직 및 장기에 침윤하여 덩어리를 형성하고 기존의 구조를 파괴하거나 변형시키는 상태를 의미한다. In the present invention, the cancer generally refers to a physiological condition of mammals characterized by uncontrolled cell growth, and refers to a condition in which a problem occurs in the control function of normal division, differentiation, and death of cells, resulting in abnormal excessive proliferation, infiltration into surrounding tissues and organs, formation of a mass, and destruction or deformation of existing structures.

예를 들어, 고형암 또는 전이암일 수 있고, 예컨대 결장암 및 직장암을 포함하는 대장암, 유방암, 자궁암, 자궁경부암, 난소암, 전립선암, 뇌종양, 두경부암종, 흑색종, 골수종, 백혈병, 림프종, 위암, 폐암, 췌장암, 간암, 식도암, 소장암, 항문부근암, 나팔관암종, 자궁내막암종, 질암종, 음문암종, 호지킨병, 방광암, 신장암, 수뇨관암, 신장세포암종, 신장골반암종, 골암, 피부암, 두부암, 경부암, 피부흑색종, 안구내흑색종, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직육종, 요도암, 음경암, 중추신경계(central nervous system; CNS) 종양, 1차 CNS 림프종, 척수종양, 뇌간신경교종, 뇌하수체선종 등일 수 있고, 적합하게는 전립선암, 자궁암, 간암, 직장암, 폐암, 유방암, 및 난소암으로 이루어진 군에서 선택되는 어느 하나 이상인 것을 특징으로 할 수 있다. For example, it may be a solid cancer or a metastatic cancer, such as colorectal cancer including colon cancer and rectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head and neck cancer, skin melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, etc. and may be characterized by at least one selected from the group consisting of prostate cancer, uterine cancer, liver cancer, rectal cancer, lung cancer, breast cancer, and ovarian cancer.

보다 구체적으로 상기 암은 인간 유방암 세포 MDA-MB-231 또는 MCF-7를 의미할 수 있고 가장 적합하게는 MCF-7일 수 있다. More specifically, the cancer may refer to human breast cancer cells MDA-MB-231 or MCF-7, and most preferably MCF-7.

상기 약학적 조성물은 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 예컨대, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽 등의 경구형 제형으로 제형화할 수 있고, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The above pharmaceutical composition can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

또한, 본 발명의 약학적 조성물은 자연살해세포 유래 소포체와 함께 암 또는 종양에 대한 예방, 개선 및 치료 효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다.In addition, the pharmaceutical composition of the present invention may contain one or more known effective ingredients having preventive, improving, and therapeutic effects on cancer or tumors together with natural killer cell-derived endoplasmic reticulum.

또한, 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정 셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당 등이 사용될 수 있다. In addition, pharmaceutically acceptable additives may be further included, and pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, and white sugar.

본 발명에 따른 약제학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 ~ 90 중량부 포함되는 것이 바람직하나 이에 한정되는 것은 아니다.The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight in the composition, but is not limited thereto.

또한, 실제 임상투여 시에 경구 또는 비경구의 여러가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있으며, 당해 기술 분야에 알려진 적합한 제제는 문헌에 개시되어 있는 것을 이용하는 것이 바람직하다In addition, it can be administered in various oral or parenteral dosage forms during actual clinical administration, and when formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used, and it is preferable to use suitable formulations known in the relevant technical field disclosed in the literature.

상기 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 카보네이트(Calcium carbonate), 수크로스(Sucrose) 또는 락토오스(Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 또한, 상기 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

상기 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 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, 글리세로제라틴 등이 사용될 수 있다.The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

본 발명의 약학적 조성물의 투여량은 상기 약학적 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 상기 약학적 조성물의 투여로 달성하고자 하는 반응의 종류와 정도, 투여 대상이 되는 개체의 종류, 연령, 체중, 일반적인 건강 상태, 질병의 증세나 정도, 성별, 식이, 배설, 해당 개체에 동시 또는 이시에 함께 사용되는 약물 기타 조성물의 성분등을 비롯한 여러 인자 및 의약 분야에서 잘 알려진 유사 인자에 따라 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있음으로, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and/or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field. A person having ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment, and therefore the dosage does not limit the scope of the present invention in any way.

즉, 본 발명의 약학적 조성물의 투여 경로 및 투여 방식은 각각 독립적일 수 있으며, 그 방식에 있어 특별히 제한되지 아니하며, 목적하는 해당 부위에 상기 약학적 조성물이 도달할 수 있는 한 임의의 투여 경로 및 투여 방식에 따를 수 있다.That is, the route and method of administration of the pharmaceutical composition of the present invention may be independent, and are not particularly limited in their methods, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

상기 약학적 조성물은 경구 투여 또는 비경구 투여 방식으로 투여할 수 있다. 상기 비경구 투여 방식으로는 예를 들어 정맥 내 투여, 복강 내 투여, 근육 내 투여, 경피 투여 또는 피하 투여 등이 포함된다.The pharmaceutical composition may be administered orally or parenterally. Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

또한, 암의 예방과 암으로부터 유래하는 여러 질환들을 예방 또는 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.Additionally, it can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to prevent cancer and prevent or treat various diseases arising from cancer.

이에 따라, 암의 예방 또는 치료용 약학적 조성물 또는 세포치료제로서 활용될 수 있으며, 본 발명이 목적하는 효과를 유지하는 측면에서 이에 제한은 없다. Accordingly, it can be used as a pharmaceutical composition or cell therapy agent for preventing or treating cancer, and there is no limitation thereto in terms of maintaining the effect intended by the present invention.

중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are equally applicable to each other as long as they are not mutually contradictory, and it is also included in the scope of the present invention for a person skilled in the art to make appropriate changes and implement the present invention.

이하 실시예를 통하여 본 발명을 더욱 상세하게 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것으로 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.The present invention will be described in more detail through the following examples. These examples are intended merely to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

실시예Example

제조예 1: 세포배양Manufacturing Example 1: Cell Culture

인간 유방암 세포인 MCF-7 및 MDA-MB-231은 한국 세포주은행(KCLB; 서울, 한국)에서 얻었으며, RPMI 1640 (Gibco, Carlsbad, CA, 미국)에 10% 소태아혈청 (FBS; Gibco) 및 1% 페니실린-스트렙토마이신 (Gibco)을 보충하여 37℃ 및 5% CO2에서 배양하였다. Human breast cancer cells, MCF-7 and MDA-MB-231, were obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea) and cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2 .

인간 NK세포주인 KHYG-1은 AcceGen Biotech (cat. # ABC-TC0506; Fairfield, NJ, 미국)에서 구매하였다. KHYG-1 세포는 RPMI 1640 배지에 열처리된 FBS, 1% 페니실린-스트렙토마이신 및 100 Units/mL의 재조합된 인간 IL-2 (cat. # 200-02; PeproTech, Rocky Hill, NJ, 미국)으로 보충되어, 37℃ 및 5% CO2의 조건에서 T-25 플라스크에 배양되었으며, 매 2-3일마다 신선한 배지를 추가하거나 계대 배양(subculture) 되었다.The human NK cell line KHYG-1 was purchased from AcceGen Biotech (cat. # ABC-TC0506; Fairfield, NJ, USA). KHYG-1 cells were cultured in T-25 flasks in RPMI 1640 medium supplemented with heat-treated FBS, 1% penicillin-streptomycin, and 100 Units/mL recombinant human IL-2 (cat. # 200-02; PeproTech, Rocky Hill, NJ, USA) at 37°C and 5% CO 2 , with fresh medium added or subcultured every 2–3 days.

제조예 2: 전기자극시스템Manufacturing Example 2: Electrical Stimulation System

전기 시스템 중 챔버는 3D 프린터 (3DP-110F; Cubicon, 성남, 한국)로 인쇄되었고 표준 6-well 플레이트에 맞게 뚜껑이 제작되었다.Among the electrical systems, the chamber was printed with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea) and the lid was fabricated to fit a standard 6-well plate.

전기자극 시스템은 두 개의 백금 (Pt) 전극 (음극과 양극, 지름 0.3 mm의 99.9% Pt wire)으로 구성되어 서로 평행한 거리로 20 mm 떨어지게 제작되었다. 자극 조건은 함수 발생기에 의해 제어되었으며, 이는 전극을 완전히 플레이트 웰 전체에 걸쳐 삽입하여 배지를 통해 웰 전체 세포에 영향을 미치도록 설계하였다. The electrical stimulation system consisted of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wires with a diameter of 0.3 mm) positioned 20 mm apart and parallel to each other. Stimulation conditions were controlled by a function generator designed to fully penetrate the electrodes throughout the plate wells, thereby influencing the cells throughout the wells through the medium.

본 발명에서 전기자극 시스템은 세포 배양 플레이트에 맞게 설계되고 조정되었고, 그 성능은 오실로스코프 (DSOX1102A; Keysight, Santa Rosa, CA, 미국)를 사용하여 확인하였으며 도 1에 설계된 전기자극 시스템의 도식적인 구조를 나타내었다. In the present invention, the electrical stimulation system was designed and adjusted to fit a cell culture plate, and its performance was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA), and a schematic structure of the designed electrical stimulation system is shown in Fig. 1.

실시예 1. 전기자극의 노출Example 1. Exposure to electrical stimulation

NK 세포와 종양 세포 간의 공동 배양을 통해 전기 자극이 NK 세포의 세포 독성 활동에 미치는 효과를 확인하기 위하여 상기 제조예 1에 따라 배양된 종양 세포 및 인간 NK세포주 KHYG-1와 상기 제조예 2의 전기자극 시스템을 이용하여 실험을 설계하였으며, 이에 대하여 도 2에 나타내었다. In order to confirm the effect of electrical stimulation on the cytotoxic activity of NK cells through co-culture between NK cells and tumor cells, an experiment was designed using tumor cells cultured according to the above Preparation Example 1 and human NK cell line KHYG-1 and the electrical stimulation system of the above Preparation Example 2, and this is shown in Fig. 2.

구체적으로, 전기 자극 시스템에서 NK 세포는 직류(DC로 표기) 전기자극 또는 10 Hz 주파수에서 1시간 동안의 이상성 펄스 전기 자극 (대칭, 전하 균형 조절, BP로 표기)의 두 전압 범위 (0.5 V/cm, 1.0 V/cm)에 노출되었다. Specifically, in the electrical stimulation system, NK cells were exposed to two voltage ranges (0.5 V/cm, 1.0 V/cm) of direct current (denoted as DC) electrical stimulation or biphasic pulse electrical stimulation (symmetric, charge-balanced, denoted as BP) at a frequency of 10 Hz for 1 h.

다음으로, 플레이트 내 포화 상태에 도달한 종양 세포를 미리 1시간 동안 전기 자극을 받은 NK 세포와 24시간 동안 공동 배양하여 실험군을 제조하였으며, 종양세포와 전기자극을 받지 않은 NK세포를 24시간 공동 배양한 세포를 대조군으로 설정하였다. Next, tumor cells that reached saturation in the plate were co-cultured with NK cells that had been electrically stimulated for 1 hour in advance for 24 hours to prepare an experimental group, and cells co-cultured with tumor cells and NK cells that had not been electrically stimulated for 24 hours were set as a control group.

또한, 세포 생존 능력 평가 (CCK-8)를 제외한 모든 실험에서 DC(직류) 조건의 전기 자극이 적용되었다. 즉, 'ES' 약어에 대한 이후 언급은 모두 DC(직류) 조건에서의 전기자극을 나타낸다. Additionally, in all experiments except for the cell viability assessment (CCK-8), electrical stimulation under direct current (DC) conditions was applied. That is, all subsequent references to the abbreviation 'ES' indicate electrical stimulation under direct current (DC) conditions.

또한, 일부 칼슘 킬레이팅 실험에서는 KHYG-1 세포가 5μM의 최종 농도로 세포 침투 가능한 칼슘 이온 킬레이터인 BAPTA-AM (B6769; Invitrogen, Carlsbad, CA, 미국)로 보충된 배지에서, 전기자극 전 30분 동안 배양되었거나 대조군에서는 동등한 양의 DMSO를 포함한 배지에서 배양되었다. Additionally, in some calcium chelation experiments, KHYG-1 cells were cultured for 30 min before electrical stimulation in medium supplemented with the cell-permeable calcium ion chelator BAPTA-AM (B6769; Invitrogen, Carlsbad, CA, USA) at a final concentration of 5 μM, or in medium containing an equivalent amount of DMSO as a control.

실시예 2. 세포 생존율 평가 Example 2. Cell viability evaluation

전기자극이 NK세포의 세포 생존율에 미치는 영향을 알아보기 위하여, 1시간 동안의 전기자극이 세포 생존율에 미치는 영향을 세포 생존 능력 평가(cell counting kit-8 (CCK-8) 실험 (Dojindo Laboratories, Kumamoto, 일본)로 측정하여 도 3에 결과로 나타내었다. To determine the effect of electrical stimulation on the cell viability of NK cells, the effect of electrical stimulation for 1 hour on cell viability was measured using a cell counting kit-8 (CCK-8) experiment (Dojindo Laboratories, Kumamoto, Japan), and the results are shown in Figure 3.

구체적으로, 1시간 동안의 전기자극을 받은 KHYG-1 세포는 96-well 판 (100 μL, 150,000 세포)으로 옮겨진 후 10μL의 CCK-8을 첨가하여 37℃에서 3시간 동안 배양하였다. 각 플레이트 웰의 흡광도는 마이크로플레이트 리더 (Thermo Fisher Scientific)를 사용하여 450 nm에서 측정되었다. Specifically, KHYG-1 cells that received electrical stimulation for 1 h were transferred to a 96-well plate (100 μL, 150,000 cells), then 10 μL of CCK-8 was added, and incubated at 37°C for 3 h. The absorbance of each plate well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

도 3A은 1시간 동안 지속적인 직류 전류 (DC) 또는 양극성 (BP) 정사각파형 (10 Hz 주파수, 대각선이 그어진 열)에 노출된 후의 결과(n=3)를 나타낸 것으로, 연속적인 DC 전기자극을 가한 직후의 세포 생존율은 약간 감소했지만 통계적으로 유의미하지 않는 것을 확인할 수 있었다. Figure 3A shows the results (n=3) after exposure to continuous direct current (DC) or bipolar (BP) square waveform (10 Hz frequency, diagonally drawn columns) for 1 hour, and it was confirmed that cell viability was slightly reduced immediately after continuous DC electrical stimulation, but this was not statistically significant.

도 3B는 일정한 DC 전기자극 후 1시간 동안 RT-qPCR을 사용하여 자가 세포사멸 관련 유전자 BAX (pro-apoptotic) 및 BCL2 (anti-apoptotic)의 상대적인 mRNA 발현 비율을 평가(n = 3)한 것으로, 연속적인 DC 전기 자극 후 BAX/BCL2 비율은 약간 감소했지만 이 감소는 통계적으로 유의하지 않음을 확인할 수 있었고 더불어, 전기자극 이후 24시간, 48시간 시점에서의 장기적인 영향도 없는 것을 확인할 수 있었다. Figure 3B shows the relative mRNA expression ratios of the apoptosis-related genes BAX (pro-apoptotic) and BCL2 (anti-apoptotic) using RT-qPCR for 1 hour after continuous DC electrical stimulation (n = 3). It was confirmed that the BAX/BCL2 ratio slightly decreased after continuous DC electrical stimulation, but this decrease was not statistically significant. In addition, it was confirmed that there were no long-term effects at 24 and 48 hours after electrical stimulation.

즉, 양극성 전기자극(biphasic BP)이 세포 생존을 유의하게 감소시키는 반면, 연속적인 DC 조건의 자극은 그렇지 않았음을 확인할 수 있었고, 이에 따라, 이후의 실험은 세포 생존율에 영향이 없는 1시간 DC 조건 하에서 두 가지 다른 전압 (0.5 V/cm, 1.0 V/cm)으로 연속적인 전기자극을 사용하여 진행되었다. That is, it was confirmed that biphasic electrical stimulation (biphasic BP) significantly reduced cell survival, whereas stimulation under continuous DC conditions did not. Accordingly, subsequent experiments were conducted using continuous electrical stimulation at two different voltages (0.5 V/cm, 1.0 V/cm) under 1-h DC conditions that did not affect cell survival.

실시예 3. 암세포 살상능력 평가 Example 3. Evaluation of cancer cell killing ability

전기자극이 NK 세포의 암세포 살상 능력에 미치는 효과를 평가하기 위하여, 젖산 탈수소 효소 (lactate dehydrogenase, LDH) 세포 독성 실험 및 Live/dead 세포 이미징 실험을 수행한 결과를 도 4에 나타내었다. To evaluate the effect of electrical stimulation on the cancer cell killing ability of NK cells, lactate dehydrogenase (LDH) cytotoxicity test and live/dead cell imaging test were performed, and the results are shown in Figure 4.

먼저, 암세포인 MDA-MB-231 및 MCF-7 세포는 12-well 플레이트에서 배양하고, 실시예 1의 전기자극 노출 유무에 따라 NK세포(KHYG-1 세포): 암세포(MDA-MB-231 또는 MCF-7)를 3:1의 비율로 혼합하여 실험군 및 대조군을 배양하였다. 다음으로 배양된 실험군 및 대조군의 각 웰에서 배지를 수집하고 600g에서 5분 동안 원심분리기를 통해 세포를 분리한 다음, 상층액 배지 샘플로 젖산 탈수소 효소 (lactate dehydrogenase, LDH) 세포 독성 실험(n=3)을 수행하여 도 4A에 나타내었다. 키트 제조업체의 설명에 따라 세포 독성을 측정 및 계산하였다. First, cancer cells, MDA-MB-231 and MCF-7, were cultured in 12-well plates, and NK cells (KHYG-1 cells): cancer cells (MDA-MB-231 or MCF-7) were mixed at a ratio of 3:1 and cultured as experimental and control groups, depending on whether or not they were exposed to the electrical stimulation of Example 1. Next, the medium was collected from each well of the cultured experimental and control groups, and the cells were separated by centrifugation at 600 g for 5 minutes, and then a lactate dehydrogenase (LDH) cytotoxicity test (n=3) was performed with the supernatant medium sample, which is shown in Fig. 4A. Cytotoxicity was measured and calculated according to the kit manufacturer's instructions.

도 4A를 참조하면, MDA-MB-231 세포는 대조군 및 실험군의 세포 독성에서 유의한 증가를 보이지 않았지만, MCF-7 세포는 대조군 대비 실험군에서 각각 1.27배(0.5 V/cm 전기 자극) 및 1.55배(1.0 V/cm 전기 자극)로 증가하여 세포 독성능력이 증가한 것을 확인할 수 있었다. Referring to Figure 4A, MDA-MB-231 cells did not show a significant increase in cytotoxicity between the control and experimental groups, but MCF-7 cells showed an increase in cytotoxicity ability, with an increase of 1.27 times (0.5 V/cm electrical stimulation) and 1.55 times (1.0 V/cm electrical stimulation) in the experimental group compared to the control group, respectively.

다음으로, Live/dead 세포 이미징 실험은 MCF-7 세포에 대한 대조군 및 실험군을 이용하였으며, 6시간 동안 공배양된 실험군 및 대조군은 DPBS로 세 번 washing하여 KHYG-1 세포를 제거한 다음, MCF-7 세포는 2 μM calcein-AM (live) 및 4 μM Ethidium homodimer-1 (dead)로 염색한 후 제조업체의 프로토콜에 따라 형광 현미경으로 분석하여, 도 4B에 나타내었다. Next, the live/dead cell imaging experiment was conducted using control and experimental groups for MCF-7 cells. The experimental and control groups co-cultured for 6 hours were washed three times with DPBS to remove KHYG-1 cells, and then the MCF-7 cells were stained with 2 μM calcein-AM (live) and 4 μM Ethidium homodimer-1 (dead) and analyzed by fluorescence microscopy according to the manufacturer's protocol, as shown in Fig. 4B.

도 4B를 참조하면, 전기자극을 받은 KHYG-1 세포와 함께한 MCF-7인 실험군의 경우 전기자극을 받지 않은 KHYG-1 세포와 함께한 MCF-7인 대조군과 대비하여, 죽은 세포가 더 많이 나타나는 것을 확인할 수 있었다. 즉, 전기자극이 MCF-7 세포에 대한 NK 세포의 암세포 살상능력을 향상시킬 수 있음을 알 수 있었다. Referring to Figure 4B, it was confirmed that the experimental group of MCF-7 cells with electrically stimulated KHYG-1 cells showed more dead cells compared to the control group of MCF-7 cells with KHYG-1 cells that did not receive electrical stimulation. In other words, it was found that electrical stimulation could enhance the cancer cell killing ability of NK cells against MCF-7 cells.

실시예 4. 유전자 및 단백질 발현의 변화 Example 4. Changes in gene and protein expression

1) 유전자 발현 분석1) Gene expression analysis

전기자극 후 KHYG-1 세포의 암세포 살상능과 관련된 유전자 발현의 변화를 확인하기 위하여 세포 독성 활동 및 granzyme B 생성과 관련된 유전자 발현 수준의 변화를 확인하여 도 5A에 결과로 나타내었다. In order to confirm the change in gene expression related to the cancer cell killing ability of KHYG-1 cells after electrical stimulation, the change in gene expression level related to cytotoxic activity and granzyme B production was confirmed and the result is shown in Figure 5A.

구체적으로, 전기자극이 세포사멸 과립, 사이토카인 단백질, 그리고 칼슘 신호전달 단백질의 발현에 미치는 효과를 실시간 역전사 중합효소 연쇄반응 (RT-qPCR)을 통하여 분석하였다. 이때. 전기 자극된 세포 샘플에서 총 RNA은 TRIzol 시약을 사용하여 추출되었으며 (Life Technologies, Carlsbad, CA, USA), 제조업체의 권장 프로토콜을 따라 진행되었다. 각 샘플의 mRNA 농도는 미세분광계 (DS-11; DeNovix, Wilmington, DE, USA)를 사용하여 측정되었으며, cDNA는 PrimeScript RT 시약 키트를 사용하여 합성하였다 (Takara, Shiga, Japan). 또한, TB Green Premix Ex Taq II (RR810A; Takara)를 사용하여 CFX96 분석 시스템 (Bio-Rad)을 사용하여 실시간 분석을 진행하였으며, 타겟 유전자는 NK세포의 면역활성과 관련된 perforin (PRF1), granzyme B (GZMB), interferon-gamma (IFNG) 및 tumor necrosis factor alpha (TNFA)와 칼슘 신호와 관련된 다른 요소에는 inositol trisphosphate receptor (IP3R), calmodulin (CALM2), calcineurin (CALN), calmodulin-dependent protein kinase II (CaMK II) 및 활성화 T 세포 핵 인자 (NFAT)가 포함되었으며, 이들은 칼슘 이온 신호 전달에 관여하는 요소들이다. 또한, GAPDH는 housekeeping 유전자로 사용되었고, 상대적인 mRNA 발현 수준은 대조군과 비교하여 2(-△△CT) 방법을 사용하여 계산하였다. 이때, 타겟 유전자에 대한 프라이머 서열은 하기 표 1에 나열하였다.Specifically, the effects of electrical stimulation on the expression of apoptotic granules, cytokine proteins, and calcium signaling proteins were analyzed using real-time reverse transcription-polymerase chain reaction (RT-qPCR). Total RNA was extracted from electrically stimulated cell samples using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's recommended protocol. The mRNA concentration of each sample was measured using a microspectrophotometer (DS-11; DeNovix, Wilmington, DE, USA), and cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Shiga, Japan). In addition, real-time analysis was performed using the CFX96 analysis system (Bio-Rad) with TB Green Premix Ex Taq II (RR810A; Takara), and the target genes were perforin (PRF1), granzyme B (GZMB), interferon-gamma (IFNG), and tumor necrosis factor alpha (TNFA), which are related to the immune activity of NK cells, and other factors related to calcium signaling, including inositol trisphosphate receptor (IP3R), calmodulin (CALM2), calcineurin (CALN), calmodulin-dependent protein kinase II (CaMK II), and nuclear factor of activated T cells (NFAT), which are factors involved in calcium ion signaling. In addition, GAPDH was used as a housekeeping gene, and the relative mRNA expression level was calculated using the 2(-△△CT) method compared to the control group. The primer sequences for the target genes are listed in Table 1 below.

도 5A를 참조하면, degranulation 마커 (PRF1, GZMB) 및 사이토카인 (IFNG, TNFA)의 상대적인 mRNA 발현 수준을 분석하고 GAPDH와 자극되지 않은 대조군에 비교 분석 (n = 3)한 결과, GZMB 유전자 발현의 경우, 전기자극 조건 0.5 V/cm 및 1.0 V/cm에서 각각 1.36배, 1.58배로 유의하게 증가한 것을 확인할 수 있었다.Referring to Figure 5A, the relative mRNA expression levels of degranulation markers (PRF1, GZMB) and cytokines (IFNG, TNFA) were analyzed and compared with GAPDH and the unstimulated control group (n = 3). As a result, it was confirmed that GZMB gene expression significantly increased by 1.36-fold and 1.58-fold under electrical stimulation conditions of 0.5 V/cm and 1.0 V/cm, respectively.

2) 효소 연결 면역 흡착 분석 (ELISA)2) Enzyme-linked immunosorbent assay (ELISA)

다음으로, 유전자 발현 변화가 단백질 수준에도 영향을 미쳤는지 확인하기 위해 ELISA를 사용하여 granzyme B 단백질 발현 수준을 분석한 결과를 도 5B에 나타내었다.Next, to determine whether the gene expression changes also affected the protein level, the results of analyzing the granzyme B protein expression level using ELISA are shown in Figure 5B.

구체적으로, KHYG-1 세포는 6-well 배양 플레이트에 세포 접종되었으며, 각각 0.5 V/cm 또는 1.0 V/cm에서 1시간 자극 후 37℃ 및 5% CO2에서 4시간 및 8시간 동안 배양되었다. 다음으로 배양액 수집하여 600g에서 5분 동안 원심 분리되었다. 세포 내 단백질은 리파 세포 용해 (RIPA) 버퍼 (Elpis Biotech, 대전, 한국)를 사용하여 제조업체의 권장 프로토콜에 따라 단백질 분해 효소 저해 칵테일 (Thermo Fisher Scientific)을 첨가하여 추출되었다. 세포 내 또는 배양액으로 분비된 granzyme B 단백질 수준은 Human Granzyme B ELISA kit (3486-1H-6; MABTECH, Nacka Strand, Sweden)을 사용하여 제조업체의 지침에 따라 측정되었다. Specifically, KHYG-1 cells were seeded in 6-well culture plates and cultured for 4 and 8 h at 37°C and 5% CO2 after stimulation at 0.5 V/cm or 1.0 V/cm for 1 h, respectively. The culture medium was then collected and centrifuged at 600 g for 5 min. Intracellular proteins were extracted using RIPA cell lysis (RIPA) buffer (Elpis Biotech, Daejeon, Korea) with the addition of a protease inhibitor cocktail (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Intracellular or culture medium-secreted granzyme B protein levels were measured using a Human Granzyme B ELISA kit (3486-1H-6; MABTECH, Nacka Strand, Sweden) according to the manufacturer's instructions.

도 5B를 참조하면, KHYG-1 세포의 세포 내 granzyme B 수준은 대조군 대비 0.5 V/cm에서 높게 나타났으며, 특히 1.0 V/cm에서는 1.26배 증가하는 것을 확인할 수 있었다. Referring to Figure 5B, the intracellular granzyme B level of KHYG-1 cells was found to be higher at 0.5 V/cm compared to the control group, and in particular, it was confirmed to increase 1.26-fold at 1.0 V/cm.

또한, 전기자극 후 4시간 및 8시간에 획득한 세포 배지 내로 분비된 granzyme B의 수준을 측정한 결과, 0.5 V/cm 조건의 전기자극 후 4시간 시점의 배지에서 세포 외부로 방출된 granzyme B 수준은 대조군 대비 1.20배 높은 것을 확인할 수 있었다. In addition, the level of granzyme B secreted into the cell medium obtained 4 hours and 8 hours after electrical stimulation was measured, and it was confirmed that the level of granzyme B released outside the cells in the medium 4 hours after electrical stimulation under 0.5 V/cm conditions was 1.20 times higher than that of the control group.

따라서 핵심 세포 사멸 유도 과립 분자인 granzyme B의 유전자 발현이 0.5 V/cm 및 1.0 V/cm 전기 자극 후 유의하게 상승하였으며, 이로 인해 단백질 수준도 증가한 것을 알 수 있었으며 GZMB 유전자 발현의 상승은 단백질 발현에도 변화를 일으키는 것을 알 수 있었다. Therefore, it was found that the gene expression of granzyme B, a key apoptosis-inducing granule molecule, was significantly increased after 0.5 V/cm and 1.0 V/cm electrical stimulation, which also increased the protein level, and it was found that the increase in GZMB gene expression also caused changes in protein expression.

실시예 5. 세포 내 칼슘 농도 및 칼슘 매개 신호 전달의 변화 Example 5. Changes in intracellular calcium concentration and calcium-mediated signaling

전기자극이 칼슘 유입에 영향을 미치는 영향을 확인하기 위해 칼슘 이온 특이적으로 결합하는 Fluo-4 형광 물질을 이용하여, 세포 내 칼슘 이온 측정 및 Ca2+-NFAT 경로와 관련된 유전자 발현 수준을 평가하여 도 6에 결과로 나타내었다. To confirm the effect of electrical stimulation on calcium influx, intracellular calcium ion measurement and gene expression levels related to the Ca 2+ -NFAT pathway were evaluated using Fluo-4 fluorescent material that specifically binds to calcium ions, and the results are shown in Figure 6.

구체적으로, 세포 내 칼슘 이온 농도의 변화는 Fluo-4 NW Calcium Assay Kit (F36206; Invitrogen)을 사용하여 정량적으로 감지되었다. KHYG-1 세포는 전기 자극의 각 전압 범위에 노출되면서 Fluo-4 AM 형광 물질과 함께 37℃에서 45분 동안 배양되었으며, 이는 형광 염색을 위한 제조업체의 권장 최대 시간이었다. 다음으로, Varioskan LUX 멀티모드 마이크로플레이트 리더 (Thermo Fisher)를 사용하여 형광 강도 (494 nm Ex; 516 nm Em)를 측정하였고, 100 μL을 96-well 플레이트로 이동하여 형광 강도를 측정하였다. 데이터는 기준선을 기준으로 상대적인 형광으로 계산되었으며, 미처리된 대조군에 대한 백분율 변화로 표시되었다. 제조업체의 권장 프로토콜에 따라 기준선 형광은 측정 시작과 종료 시에 반복적으로 설정되었다. 또한, Fluo-4 AM로 염료로 염색된 KHYG-1 세포는 정량 분석과 독립적으로 형광 현미경으로 이미징 되었다.Specifically, changes in intracellular calcium ion concentration were quantitatively detected using the Fluo-4 NW Calcium Assay Kit (F36206; Invitrogen). KHYG-1 cells were incubated with the Fluo-4 AM fluorophore at 37°C for 45 min while exposed to each voltage range of electrical stimulation, which was the maximum time recommended by the manufacturer for fluorescence staining. Next, fluorescence intensity (494 nm Ex; 516 nm Em) was measured using a Varioskan LUX multimode microplate reader (Thermo Fisher), and 100 μL was transferred to a 96-well plate to measure fluorescence intensity. Data were calculated as relative fluorescence to baseline and expressed as a percentage change relative to the untreated control. Baseline fluorescence was repeatedly established at the beginning and end of the measurements according to the manufacturer's recommended protocol. In addition, KHYG-1 cells stained with Fluo-4 AM were imaged by fluorescence microscopy independently of the quantitative analysis.

도 6A는 Fluo-4 NW 형광을 사용한 칼슘의 정량분석 및 Fluo-4로 염색된 세포의 형광 이미징을 독립적으로 수행하여 전기자극에 의해 유도된 칼슘 이온 유입을 확인(n = 3)한 것으로, 전기자극 45분 동안 측정한 칼슘 이온 농도의 상대적인 형광 수준을 계산한 결과 전기자극 후 1.31배 (0.5 V/cm) 및 1.11배 (1.0 V/cm) 증가한 것을 확인할 수 있었다. Figure 6A shows the quantitative analysis of calcium using Fluo-4 NW fluorescence and fluorescence imaging of cells stained with Fluo-4 independently performed to confirm the calcium ion influx induced by electrical stimulation (n = 3). The relative fluorescence levels of the calcium ion concentration measured for 45 minutes of electrical stimulation were calculated, and it was confirmed that they increased 1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) after electrical stimulation.

도 6B는 전기자극 후 칼슘 신호 전달 마커의 유전자 발현 수준을 1시간 동안 전기자극 후 RT-qPCR을 사용하여 분석(n = 3)한 것으로, 두가지 다른 전압 조건으로 자극된 경우, IP3R를 제외한 calcineurin-NFAT 경로의 모든 칼슘 관련 마커는 유의적으로 상승한 것을 확인할 수 있었다. Figure 6B shows the gene expression levels of calcium signaling markers after electrical stimulation for 1 hour using RT-qPCR (n = 3). When stimulated with two different voltage conditions, all calcium-related markers of the calcineurin-NFAT pathway except IP3R were significantly elevated.

따라서, 전기자극이 칼슘 유입 및 칼슘 매개의 신호 단백질을 통한 하위 기작에 영향을 미친다는 것을 알 수 있었다. Thus, we found that electrical stimulation affects calcium influx and downstream mechanisms through calcium-mediated signaling proteins.

실시예 6. 칼슘 이온의 영향을 확인Example 6. Confirming the effect of calcium ions

BAPTA-AM 칼슘 킬레이터 처리가 전기자극으로 인한 칼슘 농도 증가의 유전자 발현 및 NFAT1 탈인산화에 미치는 영향을 분석하여 도 7에 나타내었다,The effects of BAPTA-AM calcium chelator treatment on gene expression and NFAT1 dephosphorylation in response to electrical stimulation-induced calcium concentration increase are analyzed and shown in Figure 7.

1) 칼슘 농도 증가의 유전자 발현 1) Gene expression for increased calcium concentration

NK세포의 과립 매개 면역 반응에 대한 칼슘 신호 전달 기작의 영향을 더 자세히 살펴보기 위해 세포막 투과성 칼슘 킬레이터인 BAPTA-AM을 사용하여 실험을 진행하였으며, RT-qPCR을 수행하여 칼슘 이온 킬레이션 이후 GZMB 발현 수준 및 calcineurin-NFAT 신호 단백질(CALM, CALN, NFAT1)을 분석하였다. KHYG-1 세포는 DMSO (대조군) 또는 칼슘 킬레이터 (5μM BAPTA-AM)와 함께 전기자극 여부에 따라 배양되었다 (n = 3). 이때, KHYG-1 세포는 전기자극 직전 5μM BAPTA-AM으로 30분 동안 처리되었다. To further investigate the influence of calcium signaling mechanisms on NK cell granule-mediated immune responses, we conducted experiments using BAPTA-AM, a membrane-permeable calcium chelator, and analyzed GZMB expression levels and calcineurin-NFAT signaling proteins (CALM, CALN, NFAT1) after calcium ion chelation by RT-qPCR. KHYG-1 cells were cultured with DMSO (control) or a calcium chelator (5 μM BAPTA-AM) with or without electrical stimulation (n = 3). KHYG-1 cells were treated with 5 μM BAPTA-AM for 30 minutes immediately before electrical stimulation.

도 7A를 참조하면, 5μM BAPTA-AM을 사용한 경우에는 전기자극 여부에 관계없이 세포 생존율에 유의미한 차이가 없었다. 또한, 칼슘 킬레이터 BAPTA-AM과 전기자극을 함께 후에는 세포 내 자유 칼슘을 킬레이팅 함으로써, GZMB 발현이 증가하지 않았음을 알 수 있었다. Referring to Figure 7A, there was no significant difference in cell viability when 5 μM BAPTA-AM was used, regardless of whether electrical stimulation was performed. Furthermore, it was found that GZMB expression was not increased after electrical stimulation by chelating intracellular free calcium when the calcium chelator BAPTA-AM was used together.

또한, BAPTA-AM이 함께 사용된 경우에는 대조군 대비 calcineurin-NFAT 신호 경로와 관련된 다른 표지자의 유전자 발현이 낮은 수준으로 나타났다. Additionally, when BAPTA-AM was used together, the gene expression of other markers related to the calcineurin-NFAT signaling pathway was shown to be at a lower level compared to the control group.

이는 전기자극 후에 세포 내 칼슘 농도의 일시적 변화가 킬레이션을 통해 억제되어, 칼슘 매개 신호 기작이 억제되는 결과로 이어진 것으로 알 수 있다. This suggests that the transient change in intracellular calcium concentration after electrical stimulation is suppressed through chelation, resulting in inhibition of the calcium-mediated signaling mechanism.

2)2) 전사 인자 NFAT1의 transcriptional activity Transcriptional activity of the transcription factor NFAT1

전기자극에 노출된 후 전사 활동으로 이어질 수 있는 전사 인자 NFAT1의 탈인산화 수준을 확인하기 위해 전기자극 (1.0 V/cm) 또는 BAPTA-AM과 함께 전기자극에 노출된 후 NFAT1의 웨스턴 블로팅을 통해 분석되었다. To determine the level of dephosphorylation of transcription factor NFAT1, which can lead to transcriptional activity after exposure to electrical stimulation, Western blotting of NFAT1 was performed after exposure to electrical stimulation (1.0 V/cm) or electrical stimulation with BAPTA-AM.

구체적으로, 전기자극 후에 KHYG-1 세포는 차가운 DPBS로 두 번 씻어서 Thermo Fisher Scientific에서 제공하는 프로테아제 및 인산화제 억제제가 포함된 RIPA 버퍼 (Elpis Biotech)로 세포 용해를 진행하였다. 이어서 15분 동안 얼음에서 보관한 다음, 30초 동안 소니케이션을 수행하였다. 총 세포 용해물은 15분 동안 원심분리 진행 후 추가 분석에 사용하였다. 각 샘플 (10μL)은 Laemmli 샘플 버퍼에 희석되어 95℃에서 5분 동안 반응한 다음, 10% SDS-PAGE 전기영동을 통해 분리된 후 단백질 샘플은 PVDF 멤브레인에 semi-dry transfer 방법을 사용하여 이동하였다. 멤브레인은 25℃에서 1시간 동안 skim milk가 첨가된 Tris-버퍼에서 보관되며 비특이적 결합을 방지하였다. 멤브레인은 NFAT1 (MA1-025; Invitrogen) 및 loading control로 사용되는 GAPDH (2118S; Cell Signaling Technology, Danvers, MA, USA)의 1차 항체와 함께 4℃에서 밤새 반응되었다. 멤브레인은 (Anti-rabbit, ab6721, Abcam; Anti-mouse, 31430, Thermo Fisher)의 2차 항체로 특이적으로 표지 되었다. 이미지는 chemiluminescence 기질(W3651-012; GenDEPOT, Barker, TX, USA)을 사용하여 얻어졌으며, 젤 이미징 시스템 (G:BOX Chemi XRQ; Syngene, Cambridge, UK)과 Syngene GeneTools 소프트웨어를 사용하여 정량화되었다. 탈 인산화된 NFAT1 수준은 탈 인산화-NFAT1 대 인산화된 NFAT1(p-NFAT1)의 비율로 계산되었으며 대조군과 비교하였다. Specifically, after electrical stimulation, KHYG-1 cells were washed twice with cold DPBS and lysed with RIPA buffer (Elpis Biotech) containing protease and phosphatase inhibitors provided by Thermo Fisher Scientific. The cells were then stored on ice for 15 minutes and sonicated for 30 seconds. The total cell lysate was centrifuged for 15 minutes and used for further analysis. Each sample (10 μL) was diluted in Laemmli sample buffer, incubated at 95°C for 5 minutes, and then separated by 10% SDS-PAGE electrophoresis. The protein samples were then transferred to PVDF membranes using a semi-dry transfer method. The membranes were stored in Tris-buffered saline containing skim milk at 25°C for 1 hour to prevent nonspecific binding. The membrane was incubated overnight at 4°C with primary antibodies against NFAT1 (MA1-025; Invitrogen) and GAPDH (2118S; Cell Signaling Technology, Danvers, MA, USA), which served as a loading control. The membrane was specifically labeled with secondary antibodies (anti-rabbit, ab6721, Abcam; anti-mouse, 31430, Thermo Fisher). Images were acquired using a chemiluminescence substrate (W3651-012; GenDEPOT, Barker, TX, USA) and quantified using a gel imaging system (G:BOX Chemi XRQ; Syngene, Cambridge, UK) and Syngene GeneTools software. The level of dephosphorylated NFAT1 was calculated as the ratio of dephosphorylated-NFAT1 to phosphorylated NFAT1 (p-NFAT1) and compared with the control.

도 7B를 참조하면, 상단 밴드 (140 kDa)는 인산화된 NFAT1 (p-NFAT1)을 나타내며, 하단 밴드 (120 kDa)는 탈인산화된 NFAT1을 나타내고 있고, NFAT1의 탈인산화는 NFAT1 대비 p-NFAT1의 비율로 정량적으로 표현되었으며, 이를 통해 전기자극 (1.0 V/cm)에 처리된 실험군의 경우 NFAT1의 탈인산화 비율이 대조군 대비 2.21배 증가한 것을 확인할 수 있었다. 또한, 칼슘 킬레이터 BAPTA-AM 처리는 전기자극이 NFAT1의 칼슘 이온-calcineurin 매개 탈인산화에 미치는 영향을 감소시켜, 탈인산화된 NFAT1의 비율이 대조군 대비 약 0.17배 낮게 나타나는 것을 확인할 수 있었다. Referring to Figure 7B, the upper band (140 kDa) represents phosphorylated NFAT1 (p-NFAT1), and the lower band (120 kDa) represents dephosphorylated NFAT1. The dephosphorylation of NFAT1 was quantitatively expressed as the ratio of p-NFAT1 to NFAT1, and it was confirmed that the dephosphorylation ratio of NFAT1 in the experimental group treated with electrical stimulation (1.0 V/cm) increased 2.21-fold compared to the control group. In addition, it was confirmed that treatment with the calcium chelator BAPTA-AM reduced the effect of electrical stimulation on calcium ion-calcineurin-mediated dephosphorylation of NFAT1, and the ratio of dephosphorylated NFAT1 was approximately 0.17-fold lower than that of the control group.

상기의 모든 실험은 적어도 세 번 독립적으로 반복되었으며, 각 결과는 평균±평균 표준 오차로 표현되었다. 통계 분석은 Microsoft Excel 2016에서 수행되었으며, 각 실험군과 대조군에서 얻어진 결과 간의 통계적 유의성을 확인하기 위해 Student's t-테스트가 사용되었다. (*P <0.05, **P <0.01, 그리고 ***P <0.001). 실험예 1 내지 6에 따른 분석은 모두 적어도 세 차례 독립적인 측정을 진행하였으며, 모든 데이터는 평균 ± 표준 오차로 표시되었다. 두 그룹 간 비교는 양측 Student's t-test를 사용하여 통계적으로 분석되었으며, 유의미한 경우 P 값에 따라 * P < 0.05, ** P < 0.01 및 *** P < 0.001로 나타내었다.All of the above experiments were repeated independently at least three times, and the results were expressed as the mean ± standard error of the mean. Statistical analysis was performed in Microsoft Excel 2016, and Student's t-test was used to determine the statistical significance between the results obtained in each experimental group and the control group (*P <0.05, **P <0.01, and ***P <0.001). All analyses according to Experimental Examples 1 to 6 were performed at least three times independently, and all data were expressed as the mean ± standard error. Comparisons between two groups were statistically analyzed using a two-tailed Student's t-test, and significant results were expressed as *P <0.05, **P <0.01, and ***P <0.001, depending on the P value.

또한, 상기 실시예에 따른 calcineurin-NFAT 신호 전달 경로에 대한 매커니즘 도식도를 도 8에 나타내었다. Additionally, a mechanism schematic diagram for the calcineurin-NFAT signal transduction pathway according to the above embodiment is shown in Fig. 8.

종합적으로 본 발명에 따른 전기자극 시스템을 통해 전기자극을 받은 KHYG-1 세포(자연살해세포)를 유방암 MCF-7 세포에 처리하여 24시간 공배양 하였을 경우(실험군), 자극을 처리하지 않은 NK세포-MCF-7 공배양군(대조군)과 대비하여 1.27배 (0.5 V/cm) 및 1.55배 (1.0 V/cm) 높은 세포 독성을 나타내고, 칼슘 이온 매개 활성화 T 세포 핵 인자 1 (NFAT1)의 증가된 활성에 의해 granzyme B (GZMB) 유전자 발현이 각각 1.36배 (0.5 V/cm) 및 1.58배 (1.0 V/cm) 상승할 뿐만 아니라, 5 μM BAPTA-AM으로 칼슘 이온의 유입을 킬레이팅 (chelating)한 실험의 경우, 전기자극의 효과를 중화시켜 칼슘 이온 신호 전달 단백질 및 세포사멸 과립 내 granzyme B 단백질의 유전자 발현 증진 및 NFAT1의 탈인산화, 즉 활성화를 억제한 것을 알 수 있었다. In summary, when KHYG-1 cells (natural killer cells) that received electrical stimulation through the electrical stimulation system according to the present invention were treated with breast cancer MCF-7 cells and co-cultured for 24 hours (experimental group), compared to the NK cell-MCF-7 co-culture group (control group) that was not treated with stimulation, the cytotoxicity was 1.27 times (0.5 V/cm) and 1.55 times (1.0 V/cm) higher, and granzyme B (GZMB) gene expression was increased by 1.36 times (0.5 V/cm) and 1.58 times (1.0 V/cm), respectively, due to the increased activity of nuclear factor 1 (NFAT1) mediated by calcium ion activation. In addition, in the case of an experiment in which the influx of calcium ions was chelated with 5 μM BAPTA-AM, the effect of electrical stimulation was neutralized, thereby promoting gene expression of calcium ion signaling proteins and granzyme B proteins in apoptotic granules and NFAT1. It was found that dephosphorylation, i.e., inhibition of activation, was observed.

즉, 전기자극의 효과는 칼슘 이온 매개 기작으로 나타났으며, 칼슘 이온은 자연살해세포 내에서 전사인자와 세포사멸 과립 단백질의 발현 변화를 통해 자연살해세포의 면역 활성에도 영향을 주는 것을 알 수 있었다. That is, the effect of electrical stimulation was shown to be mediated by a calcium ion mechanism, and calcium ions were found to affect the immune activity of natural killer cells through changes in the expression of transcription factors and apoptotic granule proteins within natural killer cells.

따라서, 본 연구는 도입된 유전적 변형 없이 세포치료제의 효능을 증진할 수 있는 방법으로 활용 가능할 것으로 사료된다.Therefore, this study is expected to be useful as a method to enhance the efficacy of cell therapy without introducing genetic modification.

본 발명은 한국 정부(과학기술정보통신부)의 재원으로 한국연구재단(NRF)의 지원(RS-2023-00207801) 및 한국 정부(과학기술정보통신부, 보건복지부)의 재원으로 범부처재생의료기술개발사업단(KFRM)의 지원(RS-2024-00333403)을 받아 수행되었습니다.This invention was made possible with the support of the National Research Foundation of Korea (NRF) funded by the Korean government (Ministry of Science and ICT) (RS-2023-00207801) and the Korea Foundation for Regenerative Medicine (KFRM) funded by the Korean government (Ministry of Science and ICT, Ministry of Health and Welfare) (RS-2024-00333403).

Claims (5)

자연살해세포에 전기자극을 제공하는 단계를 포함하는, 자연살해세포의 활성화 방법. A method for activating natural killer cells, comprising the step of providing electrical stimulation to natural killer cells. 제1항에 있어서, In the first paragraph, 상기 전기자극은 13 내지 17mm 깊이의 전극을 이용하여, 0.3 내지 1.2 V/cm의 전압으로 40분 내지 70분 동안 처리하는 것을 특징으로 하는, 자연살해세포의 활성화 방법. A method for activating natural killer cells, characterized in that the electrical stimulation is performed for 40 to 70 minutes at a voltage of 0.3 to 1.2 V/cm using an electrode having a depth of 13 to 17 mm. 제1항에 있어서, In the first paragraph, 상기 전기자극은 상기 자연살해세포의 칼슘 이온 농도, 그랜자임 B(Granzyme B)의 발현 및 활성화된 T-세포의 핵 인자 1(Nuclear factor of activated T cell 1, NFAT 1)의 탈인산화를 증가시키는 것을 특징으로 하는, 자연살해세포의 활성화 방법. A method for activating natural killer cells, characterized in that the electrical stimulation increases the calcium ion concentration of the natural killer cells, the expression of Granzyme B, and the dephosphorylation of nuclear factor of activated T cells 1 (NFAT 1). 제1항 내지 제3항 중 어느 한 항에 따른 자연살해세포의 활성화 방법에 의해 생산된, 자연살해세포. A natural killer cell produced by a method for activating natural killer cells according to any one of claims 1 to 3. 제4항에 따른 자연살해세포를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing or treating cancer, comprising natural killer cells according to Article 4 as an active ingredient.
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