KR101432881B1 - Cell protecting composition for toxicity suppression of Natural Killer cell comprising retinal or retinoic acid as an effective component - Google Patents
Cell protecting composition for toxicity suppression of Natural Killer cell comprising retinal or retinoic acid as an effective component Download PDFInfo
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- KR101432881B1 KR101432881B1 KR1020120016400A KR20120016400A KR101432881B1 KR 101432881 B1 KR101432881 B1 KR 101432881B1 KR 1020120016400 A KR1020120016400 A KR 1020120016400A KR 20120016400 A KR20120016400 A KR 20120016400A KR 101432881 B1 KR101432881 B1 KR 101432881B1
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- cells
- natural killer
- retinal
- cell
- retinoic acid
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Abstract
본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 유효성분으로 포함하는 세포보호용 조성물에 관한 것으로서, 보다 구체적으로 본 발명은 레티날 또는 레티노산을 유효성분으로 포함하는 자연살해세포의 세포독성 감소 또는 억제를 통한 세포보호용 조성물에 관한 것이다. 본 발명에 따른 레티날 및 레티노산이 첨가된 조절 T세포는 자연살해세포의 세포독성을 감소시키거나 억제하는 효과가 우수하여, 각종 면역반응의 조절 이상으로 유발되는 자가면역질환, 염증성질환 및 이식거부질환과 같은 면역질환을 예방 또는 치료할 수 있는 세포보호용 조성물로 유용하게 사용할 수 있다.The present invention relates to a cell protection composition comprising retinal or retinoic acid as an active ingredient. More particularly, the present invention relates to a cell protective composition comprising retinal or retinoic acid as an active ingredient, To a composition for protecting cells through reduction or inhibition of toxicity. The regulatory T cells to which retinal and retinoic acid are added according to the present invention are excellent in the effect of reducing or suppressing cytotoxicity of natural killer cells and are useful as an autoimmune disease, an inflammatory disease and a transplant rejection And can be usefully used as a composition for protecting cells which can prevent or treat immune diseases such as diseases.
Description
본 발명은 자연살해세포의 독성을 감소시켜 세포를 보호할 수 있는 레티날 또는 레티노산을 유효성분으로 포함하는 세포보호용 조성물에 관한 것이다.The present invention relates to a composition for protecting cells which contains retinal or retinoic acid as an active ingredient which can protect cells against toxicity of natural killer cells.
각종 병원체에 대한 생체 방어 시스템으로 면역계에서 중심적 역할을 담당하는 세포군의 하나로 T 세포가 있다. T 세포는 인체의 흉선에서 생성되며 일련의 분화 과정을 거치면서 고유의 특성을 지닌 T 세포로 분화하게 되는데, 분화를 완료한 T 세포는 그 기능에 따라 크게 1형 보조 세포(Th1)와 2형 보조 세포(Th2)로 구분된다. 이 중에서 Th1 세포의 주된 기능은 세포 매개성 면역에 관여하고, Th2 세포는 체액성 면역에 관여하며, 면역계에서 이러한 두 세포 집단은 과활성화되지 않도록 서로 견제를 통해 면역계의 균형을 유지하고 있다.T cells are one of the cell groups that plays a central role in the immune system as a bio-defense system for various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain a balance of the immune system through mutual checks so as not to be activated.
따라서 면역질환의 대부분은 이러한 두 면역 세포간의 불균형에 기인하는 것으로 볼 수 있는데, 예를 들어 Th1 세포의 활성이 비정상적으로 증가하는 경우 자가면역질환이 발생할 수 있고, Th2 세포의 활성이 비정상적으로 증가하는 경우 과민반응에 의한 면역질환이 발생하는 것으로 알려져 있다.Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, an autoimmune disease may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.
또한, Th1 세포의 분화에 대한 최근 연구 결과에 따르면, Th1 세포의 활성을 조절할 수 있는 새로운 그룹인 면역조절 T 세포(Treg)의 존재가 알려지면서 이를 이용한 면역질환의 치료에 대한 연구가 대두되고 있는데, Treg 세포는 비정상적으로 활성화된 면역세포의 기능을 억제하여 염증 반응을 제어하는 특성이 있어, Treg 세포의 활성을 증가시키는 작용을 통해 면역질환을 치료하는 실험들이 많이 보고되고 있다.In addition, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Treg) capable of regulating the activity of Th1 cells, , Treg cells have the property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction, and there have been reported many experiments for treating immune diseases through the action of increasing the activity of Treg cells.
한편, 자연살해세포(Natural Killer cell; NK cell)는 형태학적으로 세포질에 큰 과립을 가지는 세포(Large Granular Lymphocyte; LGL)로 혈액 내 림프구의 약 10~20%를 차지하며, T 세포 수용체, CD4, CD5 또는 면역글로불린과 같은 세포표면 수용체를 가지고 있지 않아서, 기존의 T 림프구 및 B 림프구와는 구별되는 독특한 림프구를 말한다(Trinchieri, G., Adv. Immunol., 47, 187, 1989). 이러한 자연살해세포는 혈액 이외에도 비장, 간, 폐, 편도선, 임파선 등에서도 발견되고, 골수 조직이 자연살해세포의 형성과 분화 과정에 중요한 것으로 알려져 있다.Natural killer cells (NK cells) are morphologically large granular lymphocytes (LGL), which account for about 10 to 20% of lymphocytes in the blood. T cell receptors, CD4 , T lymphocytes and B lymphocytes, which do not have a cell surface receptor such as CD5 or immunoglobulin (Trinchieri, G., Adv. Immunol., 47, 187, 1989). These natural killer cells are found in spleen, liver, lung, tonsil and lymph node in addition to blood, and it is known that bone marrow tissue is important for the formation and differentiation process of natural killer cells.
또한, 자연살해세포는 종양세포를 살해할 수 있고(Scala, G. et al., J. Immunol., 134, 3049, 1985), 바이러스가 감염된 세포에 대하여 세포독성을 나타내며, 세균과 진균을 살해하는 능력도 가지고 있다고 보고되어 있어, 항종양 면역과 미생물에 대한 보호면역에 대하여 중요한 역할을 하는 것을 알 수 있으며(Walsh, C. M. et al., Proc. Natl. Acad. Sci. USA., 91, 10854, 1994), 또한 림포카인 등을 생산하여 면역계의 조절과정에 깊은 관련이 있고, CD16 세포 표면 수용체를 통하여 항체의존성 세포매개 세포독성(Antibody-Dependent Cell-mediated Cytotoxicity; ADCC)의 기능을 나타내는 것으로 알려져 있다 (Ortaldo, J. R., Pathol. Immunol. Res., 5, 2203, 1986).Natural killer cells can also kill tumor cells (Scala, G. et al., J. Immunol., 134, 3049, 1985), show cytotoxicity against virus-infected cells, kill bacteria and fungi (Walsh, CM et al., Proc. Natl. Acad. Sci. USA, 91, 10854 (1998)). , 1994), and also produced lymphocaine, which is closely related to the regulation of the immune system and shows the function of Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) through CD16 cell surface receptors (Ortaldo, JR, Pathol. Immunol Res., 5, 2203, 1986).
뿐만 아니라 항종양 면역과 미생물에 대한 보호면역의 자연살해세포 역할 이외에도 골수이식의 거부반응과 자가면역질환 등에 자연살해세포가 관여하는 것이 밝혀지고 있어 자연살해세포의 살상기능 조절이 한층 더 중요하게 인식되어지고 있다(Yu, Y. Y. L. et al., Annu. Rev. Immunol., 19, 189, 1992). 특히, 사이토카인과 항체에 의한 자연살해세포의 살상기능 조절이 주로 연구되어 왔는데, 사이토카인의 예로는 IL-2, IL-12, IFN-γ 등을 들 수 있으며, 이들 사이토카인들은 세포표면 수용체의 발현과 살상유도 물질의 분비를 증가시킴으로써 자연살해세포의 살상능력을 향상시키는 것으로 알려져 있고, 항체로는 CD16에 대한 항체가 대표적인데, 이 항체는 자연살해세포의 CD16 수용체에 결합한 다음 표적세포에 Fc 부분이 결합함으로써 역 세포독성(reverse ADCC)을 통해 살상능력을 증가시킨다. In addition to anti-tumor immunity and protection against microorganisms, it has been shown that natural killer cells play a role in the rejection of bone marrow transplantation and autoimmune diseases in addition to the role of natural killer cells. (Yu, YYL et al., Annu. Rev. Immunol., 19, 189, 1992). Particularly, the control of killing function of natural killer cells by cytokines and antibodies has been mainly studied. Examples of cytokines include IL-2, IL-12 and IFN-γ. These cytokines include cell surface receptors And it is known to enhance the killing ability of natural killer cells by increasing the secretion of kill inducers. Antibodies to CD16 are representative antibodies that bind to the CD16 receptor of natural killer cells, The Fc part binds and increases killing ability through reverse cell toxicity (reverse ADCC).
한편, 자연살해세포는 암세포를 공격할 수 있는 유익한 작용 이면에 allogenic graft 이식 전에 수용자(Host)의 자연살해세포에 의해 공여 세포가 거부 반응을 증가시키는 등 자연살해세포가 가지는 독성으로 인해 정상세포의 손상 및 이식과 같은 치료 과정시 여러 가지 부작용이 발생할 수 있는 문제점이 있다. On the other hand, the natural killer cell is a beneficial action to attack the cancer cell. However, because of the toxicity of the natural killer cell such that the donor cell increases the rejection reaction by the natural killer cell of the host before the allogenic graft, There are problems that various side effects may occur during the treatment process such as injury and transplantation.
따라서 면역계에서 중추적인 역할을 하고 있는 자연살해세포의 활성을 조절하는 것은 매우 중요한 일이며, 특히 면역거부질환과 같은 질병 치료를 위한 다른 방법으로 자연살해세포의 세포 독성을 감소시키는 연구가 필요하다고 할 수 있다.Therefore, it is very important to regulate the activity of natural killer cells, which play a pivotal role in the immune system. In particular, studies to reduce the cytotoxicity of natural killer cells are needed as other methods for the treatment of diseases such as immunodeficiency diseases .
그러나 지금까지 진행된 연구들은 자연살해세포의 세포독성을 이용한 항암제 관련 연구가 대부분을 차지하고 있을 뿐, 자연살해세포의 세포 독성을 감소시킬 수 있는 물질 및 그 기작에 대한 내용에 대해서는 전혀 보고된 바가 없다.However, studies carried out so far have been limited to studies on anticancer drugs using cytotoxicity of natural killer cells, and there is no report on the substances and mechanisms that can reduce the cytotoxicity of natural killer cells.
이에 본 발명자들은 레티날(retinal) 또는 레티노산(retinoic acid)이 자연살해세포의 세포 독성을 저해시킨다는 사실을 규명함으로써 이식 거부 관련 질환과 같이 자연살해세포의 세포 독성으로 인해 유발될 수 있는 질환의 치료제로서 유용하게 사용할 수 있음을 확인하고 본 발명을 완성하였다.Accordingly, the inventors of the present invention have found that retinal or retinoic acid inhibits cytotoxicity of natural killer cells, and thus can prevent the cytotoxicity of natural killer cells such as graft rejection-related diseases And thus the present invention has been completed.
따라서 본 발명의 목적은 자연살해세포로 인해 유발되는 세포 독성을 효과적으로 억제할 수 있도록, 레티날(retinal) 또는 레티노산(retinoic acid)을 유효성분으로 포함하는 세포보호용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for protecting cells which comprises retinal or retinoic acid as an effective ingredient so as to effectively inhibit cytotoxicity caused by natural killer cells.
본 발명의 다른 목적은 레티날(retinal) 또는 레티노산(retinoic acid)을 처리하여 분화시킨 조절 T 세포(Treg 세포) 및 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 억제하는 활성을 갖는 세포보호용 조성물을 제공하는 것이다.Another object of the present invention is to provide a natural killer cell comprising regulatory T cells (Treg cells) treated with retinoic acid or retinoic acid and human adipose derived mesenchymal stem cells (Natural Killer Cell) which has an activity of inhibiting cytotoxicity of a cell.
나아가 본 발명의 또 다른 목적은 레티날(retinal) 또는 레티노산(retinoic acid)을 미분화된 조절 T 세포(Regulatory T cell: Treg)에 처리하여 조절 T 세포를 분화 및 활성화시키는 단계 및 분화 및 활성화된 조절 T 세포를 자연살해세포(Natural Killer Cell)와 함께 배양하는 단계를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법을 제공하는 것이다.It is yet another object of the present invention to provide a method of treating retinal or retinoic acid in an undifferentiated regulatory T cell (Treg) to differentiate and activate regulatory T cells, The present invention provides a method for reducing or suppressing the cytotoxicity of a natural killer cell, which comprises culturing a regulatory T cell together with a natural killer cell.
상기 목적을 달성하기 위하여, 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 유효성분으로 포함하는 세포보호용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for cell protection comprising retinal or retinoic acid as an active ingredient.
본 발명의 일실시예에 있어서, 상기 레티날 또는 레티노산은 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 것일 수 있다.In one embodiment of the present invention, the retinal or retinoic acid may be one which reduces or inhibits the cytotoxicity of natural killer cells.
본 발명의 일실시예에 있어서, 상기 레티날 또는 레티노산은 0.1 ~ 5μM 의 농도로 포함되는 것일 수 있다.In one embodiment of the present invention, the retinal or retinoic acid may be contained at a concentration of 0.1 to 5 μM.
또한, 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 처리하여 분화시킨 조절 T 세포(Treg 세포) 및 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 억제하는 활성을 갖는 세포보호용 조성물을 제공한다.The present invention also relates to natural killer cells comprising regulatory T cells (Treg cells) treated with retinoic acid or retinoic acid and human adipose derived mesenchymal stem cells Natural Killer Cell) which is capable of inhibiting cytotoxicity.
본 발명의 일실시예에 있어서, 상기 분화된 조절 T 세포(Treg 세포)와 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)는 1:0.01~1:0.03 비율로 포함되어 있는 것일 수 있다.In one embodiment of the present invention, the differentiated regulatory T cells (Treg cells) and the adipose-derived mesenchymal stem cells may be contained at a ratio of 1: 0.01 to 1: 0.03.
본 발명의 일실시예에 있어서, 상기 조성물은 자가면역질환, 염증성질환 및 세포, 조직 또는 기관의 이식거부(transplantation rejection)질환으로 이루어진 군 중에서 선택된 질환을 예방 또는 치료하는 효과가 있는 것일 수 있다.In one embodiment of the present invention, the composition may be effective for preventing or treating diseases selected from the group consisting of autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues or organs.
나아가, 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 미분화된 조절 T 세포(Regulatory T cell: Treg)에 처리하여 조절 T 세포를 분화 및 활성화시키는 단계 및 분화 및 활성화된 조절 T 세포를 자연살해세포(Natural Killer Cell)와 함께 배양하는 단계를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법을 제공한다.Further, the present invention relates to a method of treating retinal or retinoic acid in an undifferentiated regulatory T cell (Treg) to differentiate and activate regulatory T cells and to differentiate and activate regulated T cells (Natural Killer Cell), which comprises the step of culturing the cell with a natural killer cell.
본 발명의 일실시예에 있어서, 상기 배양하는 단계에서 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 처리하여 함께 배양하는 단계를 추가로 포함할 수 있다.In one embodiment of the present invention, the step of culturing may further include a step of treating and culturing adipose derived mesenchymal stem cells with adipose-derived mesenchymal stem cells.
본 발명의 일실시예에 있어서, 상기 레티날 또는 레티노산은 미분화된 조절 T 세포(Regulatory T cell: Treg)에 0.1 ~ 5μM 의 농도로 처리하여 조절 T 세포를 분화 및 활성화시키는 것일 수 있다.In one embodiment of the present invention, the retinal or retinoic acid may be treated with a concentration of 0.1 to 5 μM to differentiate regulatory T cells (regulatory T cells) to differentiate and activate regulatory T cells.
본 발명에 따른 레티날 및 레티노산이 첨가된 조절 T 세포는 자연살해세포의 세포독성을 감소시키거나 억제하는 효과가 우수하여, 각종 면역반응의 조절 이상으로 유발되는 자가면역질환, 염증성질환 및 이식거부질환과 같은 면역질환을 예방 또는 치료할 수 있는 세포보호용 조성물로 유용하게 사용할 수 있다. 또한, 약물에 대한 독성 및 부작용도 없어 장기간 복용 시에도 안심하고 사용할 수 있으며, 체내에서도 안정한 효과가 있다.The regulatory T cells to which retinal and retinoic acid are added according to the present invention are excellent in the effect of reducing or suppressing cytotoxicity of natural killer cells and are useful as an autoimmune disease, an inflammatory disease and a transplant rejection And can be usefully used as a composition for protecting cells which can prevent or treat immune diseases such as diseases. In addition, there is no toxicity and side effects to drugs, and it can be safely used even when taken for a long time, and it is also stable in the body.
도 1은 미분화 T세포에 레티날과 레티노산을 처리한 다음 Treg의 발현을 비교하기 위해 수행한 유세포 분석의 결과를 나타낸 그래프이다.
도 2는 레티날과 레티노산에 의해 분화 유도된 T 세포에 의한 자연살해세포의 독성 조절 효과를 나타낸 그래프로, (a)는 자연살해세포(NK)와 T 세포의 비를 1:1로 처리한 것이고, (b)는 자연살해세포(NK)와 T 세포의 비를 10:1로 처리하여 나타낸 것이다.
도 3은 지방유래 간엽줄기세포(MSCs)와 레티날에 의해 유도된 조절 T 세포의 자연살해세포(NK) 독성 조절 효과를 측정한 그래프이다.
도 4는 조절 T세포와 간엽줄기세포에 의한 자연살해세포의 사멸정도를 annexin V와 PI 염색하여 확인한 결과이다. FIG. 1 is a graph showing the results of flow cytometry performed to compare the expression of Tregs after treating undifferentiated T cells with retinal and retinoic acid.
FIG. 2 is a graph showing the effect of regulating toxicity of natural killer cells by T cells differentiated by retinal and retinoic acid. FIG. 2 (a) shows the ratio of NK to T cells treated at 1: 1 (B) shows the ratio of natural killer cells (NK) to T cells treated at a ratio of 10: 1.
FIG. 3 is a graph showing the effect of regeneration-induced mesenchymal stem cells (MSCs) and regulatory T cells on natural killer cell (NK) toxicity.
FIG. 4 shows the degree of death of natural killer cells by regulatory T cells and mesenchymal stem cells by annexin V and PI staining.
본 발명은 레티날 또는 레티노산이 자연살해세포의 세포독성으로부터 세포를 보호할 수 있는 효과가 있음을 최초로 규명하였으며, 따라서 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 유효성분으로 포함하는 세포보호용 조성물을 제공함에 그 특징이 있다.The present invention firstly confirmed that retinal or retinoic acid has an effect of protecting cells from the cytotoxicity of natural killer cells. Therefore, the present invention includes retinal or retinoic acid as an active ingredient The present invention provides a composition for protecting cells.
일반적으로 레티날(retinal)은 레티놀(retinol 또는 vitamin A)의 환원체로 주로 망막의 시자홍(rhodopsin)의 구성요소로서 시각기관의 생리적 과정에 관여하며, 레티노산(retinoic acid)은 레티놀(retinol 또는 vitamin A)의 활성본체로 상피세포의 점액(mucus)분비와 성장에 관계한다. 최근 이들 기능 중, 분화유도를 통하여 백혈병(APL)의 치료 및 식육고기의 대리석빛화(고급 육질), AP-1의 활성억제를 통한 항염증 및 피부노화억제 기능을 이용하여 의약, 축산, 식품, 화장품 등에 널리 사용하고 있으며, 레티날(retinal)의 화학명은 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-Nonatetraenal로 알려져 있다.In general, retinal is a reduced form of retinol (retinol or vitamin A). It is mainly involved in the physiological process of the visual organs as a component of rhodopsin of the retina, and retinoic acid is retinol Vitamin A) is the active body of mucus secretion and growth of epithelial cells. In recent years, the use of APL-1 as an anti-inflammatory and anti-aging agent has been used to induce differentiation, to treat leukemia (APL), to improve marbling (meat quality) Cosmetics, etc. The chemical name of retinal is 3,7-dimethyl-9- (2,6,6-trimethyl-1-cyclohexen-1-yl) -2,4,6,8-Nonatetraenal .
본 발명에서는 이러한 레티날 또는 레티노산이 자연살해세포의 세포독성을 억제할 수 있는 효과를 갖는다는 사실을 규명하였는데, 보다 구체적으로는 레티날(retinal) 또는 레티노산(retinoic acid)을 처리하여 분화시킨 면역조절 T 세포(Regulatoru T cell; Treg 세포)에 의해 자연살해세포의 세포독성을 감소시킬 수 있다.In the present invention, it has been clarified that retinal or retinoic acid has an effect of inhibiting cytotoxicity of natural killer cells. More specifically, retinal or retinoic acid is treated to differentiate Immunoregulatory T cells (Regulator T cells; Treg cells) can reduce the cytotoxicity of natural killer cells.
본 발명에서 상기 면역조절 T 세포세포는 T 세포의 일종으로, 비정상적으로 활성화된 면역세포의 기능을 억제하여 염증 반응을 제어하는 특성이 있는 세포로 알려져 있으며, 이러한 면역조절 T 세포는 크게 자연성(natural) Treg와 적응성(adaptive) Treg 세포로 나눌 수 있고, 자연성 Treg인 CD+ CD25+ T 세포는 이 세포가 흉선에서 새로 만들어질 때부터 면역억제기능을 부여받게 되며, 정상개체의 말초 CD4+ T 림프구 중 5~10%의 빈도로 존재한다. 아직까지 이 세포의 면역억제 기전은 정확히 파악되지 못하고 있지만, Foxp3라는 유전자의 발현 제어 인자가 이 세포의 분화와 활성에 중요한 역할을 수행한다는 사실이 최근에 밝혀졌다. 또한 말초 자연성 T 세포는 특정 환경 하에서 자가 또는 외부항원의 자극을 받으면 면역억제효과를 나타내는 세포로 분화될 수 있는데, 이를 적응성 Treg 또는 유도성(inducible) Treg로 부르며, IL-10을 분비하는 Tr1, TGF-β를 분비하는 Th3 및 CD8 Ts 등이 여기에 해당한다.In the present invention, the immunoregulatory T cell is a type of T cell, and is known as a cell having a property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction. Such immunoregulatory T cells are natural ) Treg and adaptive Treg cells. CD + CD25 + T cells, which are natural Tregs, are immunosuppressed from the time when the cells are newly formed in the thymus. In the CD4 + T lymphocytes of normal individuals, It is present at a frequency of 10%. Although the immunosuppressive mechanism of this cell has not yet been elucidated yet, it has recently been shown that the expression control gene of Foxp3 plays an important role in the differentiation and activity of this cell. In addition, peripheral T cells can be differentiated into cells that exhibit immunosuppressive effects when stimulated by a self or external antigen under specific circumstances. These cells are referred to as adaptive Tregs or inducible Tregs, and Tr1, And Th3 and CD8 Ts that secrete TGF-beta.
한편, 본 발명자들은 레티날 또는 레티노산이 자연살해세포의 세포독성으로부터 세포를 보호할 수 있는 효능이 있는지 확인하기 위해, 본 발명의 일실시예에서 마우스의 비장 세포에서 분리한 CD4+ T세포를 분화시키기 전에 레티날 또는 레티노산을 처리하고, anti-CD3, anti-CD28 항체 및 TGF-β로 자극하여 조절 T세포를 분화시킨 후, 자연살해세포를 작동세포(effector cell)로 하고, 51Cr labeling YAC-1세포를 표적세포(target cell)로 하여, effector-to-target(E/T) 세포 비를 달리 한 다음, 감마선 계측기(Gamma counter)를 이용하여 51Cr의 방사선량을 측정함으로써 자연살해세포의 세포독성도(cytotoxicity)를 측정하였다.In order to confirm whether retinal or retinoic acid has an effect of protecting cells against cytotoxicity of natural killer cells, the inventors of the present invention have found that, in one embodiment of the present invention, CD4 + T cells isolated from mouse spleen cells are differentiated Previously, retinal or retinoic acid was treated and stimulated with anti-CD3 antibody, anti-CD28 antibody and TGF-beta to differentiate regulatory T cells. Natural killer cells were used as effector cells and labeled with 51Cr labeling YAC- 1 cell was used as a target cell and the effector-to-target (E / T) cell ratio was varied. Then, the radiation dose of 51Cr was measured using a gamma counter, The cytotoxicity was measured.
그 결과, 레티날 또는 레티노산을 첨가하여 유도된 조절 T 세포(Retinoic acid induced Treg)은 자연살해세포의 세포독성도(cytotoxicity)를 감소시켰으며, 세포독성 감소 효과는 레티날 또는 레티노산에 의해 유도된 조절 T 세포의 처리양과 농도 의존적(dose-dependent)으로 비례하는 것으로 나타났다(도 2 참조).As a result, retinoic acid induced Treg induced by the addition of retinal or retinoic acid decreased the cytotoxicity of natural killer cells, and cytotoxicity was reduced by retinal or retinoic acid Dose-dependent manner with the amount of treatment of the induced regulatory T cells (see FIG. 2).
따라서 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 유효성분으로 포함하는 세포보호용 조성물을 제공할 수 있다.Therefore, the present invention can provide a composition for protecting cells which comprises retinal or retinoic acid as an active ingredient.
나아가 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 처리하여 분화시킨 조절 T 세포(Treg 세포) 및 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 억제하는 활성을 갖는 세포보호용 조성물을 제공할 수 있다.Furthermore, the present invention relates to natural killer cells (natural cells) comprising regulatory T cells (Treg cells) treated with retinoic acid or retinoic acid and human adipose derived mesenchymal stem cells Killer Cell) capable of inhibiting cytotoxicity of a cell can be provided.
일반적으로 간엽줄기세포(Mesenchymal Stem Cell; MSC)는 골수(bone marrow), 혈액, 진피 및 골막에서 분리되는 줄기세포로서, 다양한 세포 예컨대 지방세포, 연골세포 및 뼈세포 등으로 분화할 수 있는 전능성(pluripotent) 또는 다능성(multipotent) 세포를 의미한다.In general, Mesenchymal Stem Cell (MSC) is a stem cell which is isolated from bone marrow, blood, dermis and periosteum, and is a stem cell capable of differentiating into various cells such as adipocytes, chondrocytes and bone cells pluripotent or multipotent cells.
본 발명에서 사용되는 상기 간엽줄기세포는, 동물의 간엽줄기세포를 사용할 수 있으며, 바람직하게는 인간의 간엽줄기세포, 보다 바람직하게는 인간의 지방유래 간엽줄기세포를 사용할 수 있다.The mesenchymal stem cells used in the present invention may be mesenchymal stem cells of an animal, preferably human mesenchymal stem cells, more preferably human adipose mesenchymal stem cells.
또한, 간엽줄기세포는 골수 등에 매우 적은 양으로 존재하지만, 이를 분리 및 배양하는 과정은 당업계에 잘 알려져 있으며(예컨대, 미국특허 제5,486,359호에 개시되어 있으며), 상기 간엽줄기세포는 공지된 방법에 따라 분리한 후 분화능력을 잃지 않은 상태에서 증식시켜 얻을 수 있다.In addition, mesenchymal stem cells are present in very small amounts in bone marrow and the like, and the process of isolating and culturing them is well known in the art (for example, as disclosed in U.S. Patent No. 5,486,359) Followed by proliferation without losing the ability to differentiate.
간엽줄기세포를 얻는 과정을 구체적으로 설명하면 다음과 같다: (1) 인간 또는 마우스를 포함하는 포유동물, 바람직하게는, 인간의 간엽줄기세포 소스, 예컨대, 혈액 또는 골수 바람직하게는 지방조직으로부터 간엽줄기세포를 분리하는 단계(상기 골수는 경골, 대퇴골, 척수 또는 장골로부터 추출할 수 있다.); (2) 분리된 세포를 적합한 배지에서 배양하는 단계; 및 (3) 배양과정에서 부유 세포를 제거하고 배양 플레이트에 부착된 세포들을 계대 배양하여, 최종적으로 구축된 간엽줄기세포를 수득하는 단계를 통하여 얻을 수 있다.Specifically, the process for obtaining mesenchymal stem cells is as follows: (1) a mesenchymal stem cell source, such as blood or bone marrow, preferably from adipose tissue, from a mammalian animal, preferably a human, Separating the stem cells (the bone marrow can be extracted from the tibia, femur, spinal cord or iliac bone); (2) culturing the isolated cells in a suitable medium; And (3) removing floating cells in the culture process and subculturing the cells attached to the culture plate, thereby obtaining finally formed mesenchymal stem cells.
상기 과정에서 이용되는 배지로는, 줄기세포의 배양에 이용되는 일반적인 어떠한 배지도 이용할 수 있다. 바람직하게는, 혈청(예컨대, 우태아 혈청, 말 혈청 및 인간 혈청)이 함유된 배지이다. 본 발명에서 이용될 수 있는 배지는, 예를 들어, RPMI 시리즈, Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, C.P. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 배지(Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1(1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53:288(1965)), F10 (Ham, R.G. Exp. Cell Res. 29:515(1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8:396(1959)), DMEM과 F12의 혼합물(Barnes, D. et al., Anal. Biochem. 102:255(1980)), Way-mouth's MB752/1 (Waymouth, C. J. Natl. Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T.A., et al., Proc. Soc. Exp. Biol. Med. 100:115(1959)) 및 MCDB 시리즈(Ham, R.G. et al., In Vitro 14:11(1978))를 포함하나, 이에 한정되는 것은 아니다. 상기 배지에는, 다른 성분, 예를 들어, 항생제 또는 항진균제(예컨대, 페니실린, 스트렙토마이신) 및 글루타민 등이 포함될 수 있다.As the medium used in the above process, any medium generally used for culturing stem cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum). Mediums which can be used in the present invention include, for example, RPMI series, Eagles ' s MEM (Eagle's minimum essential medium, Eagle, H. Science 130: Proc. Soc. Exp (1986)), 199 Iscove's MEM (Iscove, N. et al., J. Exp. Med. 1963), F12 (Ham, Proc. Natl. Acad < / RTI > Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1963)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 1959) and McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp. Biol. Med. )), But is not limited thereto. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin) and glutamine.
간엽줄기세포의 확인은 유세포 분석을 통하여 할 수 있다. 이러한 유세포 분석은, 간엽줄기세포의 특이한 표면 마커를 이용하여 실시된다. 예컨대, 간엽줄기세포는 CD44, CD29 및/또는 MHC 클래스 I에 대하여 양성 반응을 나타내므로, 이를 통해 간엽줄기세포를 검증할 수 있다.Identification of mesenchymal stem cells can be done by flow cytometry. Such flow cytometry analysis is carried out using a specific surface marker of mesenchymal stem cells. For example, since mesenchymal stem cells are positive for CD44, CD29 and / or MHC class I, mesenchymal stem cells can be verified.
본 발명에서는 이러한 간엽줄기세포를 레티날(retinal) 또는 레티노산(retinoic acid)으로 처리되어 분화된 조절 T 세포(Treg 세포)와 함께 자연살해세포에 처리하여 배양하였을 경우, 자연살해세포의 세포독성 감소 효과가 매우 우수하다는 사실을 확인하였고, 그 효과는 레티날(retinal) 또는 레티노산(retinoic acid)만을 처리하어 분화시킨 조절 T 세포(Treg 세포)의 경우에 비해 더 우수하다는 사실을 확인하였다(도 3 참조). In the present invention, when these mesenchymal stem cells are treated with natural killer cells together with regulatory T cells (Treg cells) treated with retinal or retinoic acid and differentiated, the cytotoxicity of natural killer cells (Treg cells) treated with retinoic acid or retinoic acid alone (Fig. 1B). The results are shown in Table 1 below. 3).
또한, 본 발명에서 상기 분화된 조절 T 세포(Treg 세포)는 지방유래 간엽줄기세포와 1:0.01~1:0.03의 비율로 혼합할 수 있으며, 보다 바람직하게는 1:0.025의 비율로 혼합하여 자연살해세포(Natural Killer Cell)와 함께 배양할 수 있으며, 이로서 자연살해세포의 세포 독성을 더 효과적으로 억제할 수 있다.In the present invention, the differentiated regulatory T cells (Treg cells) may be mixed with the adipose-derived mesenchymal stem cells at a ratio of 1: 0.01 to 1: 0.03, more preferably at a ratio of 1: 0.025 It can be cultured with Natural Killer Cell, which can more effectively inhibit the cytotoxicity of natural killer cells.
그러므로 본 발명에 따른 레티날 또는 레티노산은 미분화된 T 세포에 첨가되어 분화된 면역조절 T 세포를 이용하여 자연살해세포의 세포독성으로 유발될 수 있는 질환을 예방 또는 치료할 수 있으며, 또한, 레티날(retinal) 또는 레티노산(retinoic acid)을 처리하여 분화시킨 조절 T 세포(Treg 세포) 및 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)의 혼합물을 이용하여 자연살해세포의 세포독성으로 유발될 수 있는 질환을 예방 또는 치료할 수 있다.Therefore, retinal or retinoic acid according to the present invention can be used to prevent or treat diseases that can be induced by cytotoxicity of natural killer cells by using the differentiated immunoregulatory T cells added to undifferentiated T cells, (Treg cells) and human adipose-derived mesenchymal stem cells treated with retinoic acid or retinoic acid to induce cytotoxicity of natural killer cells The disease can be prevented or treated.
본 발명에서 상기 자연살해세포의 독성으로 유발할 수 있는 질환으로는 이에 제한되지는 않으나, 자가면역질환, 염증성질환 및 세포, 조직 또는 기관의 이식거부(transplantation rejection)질환일 수 있으며, 바람직하게는 이식거부 질환일 수 있다.In the present invention, the diseases that may cause toxicity of the natural killer cells include, but are not limited to, autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues or organs, It may be a rejection disease.
상기 자가면역질환이란 자기관용을 유도하거나 계속 유지하는데 있어서 문제가 생기게 되면 자기항원에 대하여 면역반응이 일어나게 되고, 이로 인하여 자신의 조직을 공격하는 현상이 발생하는데 이러한 과정에 의해 발생되는 질환을 말하며, 염증성 질환이란 염증유발인자 또는 방사선조사 등 유해한 자극으로 인해 인체 면역체계를 과도하게 항진시켜 대식세포와 같은 면역세포에서 분비되는 TNF-α(tumor necrosis factor-α), IL-1(interleukin-1), IL-6, 프로스타글란딘(prostagladin), 루코트리엔(luecotriene) 또는 산화질소(nitric oxide, NO)와 같은 염증유발물질(염증성 사이토카인)에 의해 유발되는 질환을 말한다.The autoimmune disease refers to a disease caused by an immune response to self-antigen when a problem occurs in inducing self-tolerance or maintaining self-tolerance, thereby attacking the self-organism. IL-1 (interleukin-1), TNF-α (tumor necrosis factor-α) secreted by immune cells, such as macrophages, is exaggerated by the harmful stimuli such as inflammatory factors or radiation, Inflammatory substances such as inflammatory cytokines such as IL-6, prostagladin, luecotriene or nitric oxide (NO).
한편, 성공적인 장기 이식을 위해서는 이식할 세포 및 장기에 대한 수혜자의 면역 거부반응을 극복해야 한다. 이식면역거부반응의 주요 매개체는 T 세포로서, 이식편(graft)에 발현되어져 있는 주조직적합성분자(major histocompatibility complex, MHC)를 T 세포 수용체(T cell receptor)가 인지함으로써 면역반응이 유도되어 이식거부반응이 발생하게 된다. 따라서 이러한 이식면역거부반응을 감소시키기 위해 면역억제제들이 사용되고 있는데 이러한 면역억제제들의 공통된 목적은 이식편에 대한 T 세포-매개 면역반응을 억제하는 것이며, 자연살해세포의 비정상적인 세포 독성으로 인한 면역반응을 억제함으로써 이식거부질환을 치료하려는 방법이 시도되고 있다.On the other hand, in order to successfully transplant the organ, the recipient's immune rejection response to the transplanted cells and organs must be overcome. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) Reaction occurs. Immunosuppressants have been used to reduce these transplant immune rejection reactions. A common goal of these immunosuppressants is to inhibit T cell-mediated immune responses to grafts and to inhibit immune responses due to abnormal cytotoxicity of natural killer cells Methods to treat graft rejection disease have been attempted.
그러므로 본 발명에 따른 상기 조성물은 자연살해세포의 독성으로 인해 유발할 수 있는 질환을 예방 또는 치료할 수 있는 약학적 조성물로 사용될 수 있을 뿐만 아니라, 세포치료용 조성물로도 사용될 수 있다.Therefore, the composition according to the present invention can be used not only as a pharmaceutical composition capable of preventing or treating diseases caused by toxicity of natural killer cells, but also as a composition for cell therapy.
또한, 본 발명에서 상기 조성물에 함유되는 레티날은 0.1 ~ 5μM의 농도로 포함될 수 있다.In the present invention, the retinal contained in the composition may be contained at a concentration of 0.1 to 5 μM.
상기 치료란, 달리 언급되지 않는 한, 상기 용어가 적용되는 질환 또는 질병, 또는 상기 질환 또는 질병의 하나 이상의 증상을 역전시키거나, 완화시키거나, 그 진행을 억제하거나, 또는 예방하는 것을 의미하며, 본원에서 사용된 상기 치료란 용어는 치료하는이 상기와 같이 정의될 때 치료하는 행위를 말한다. 따라서 포유동물에 있어서 면역질환의 치료 또는 치료요법은 하기의 하나 이상을 포함할 수 있다:The term treatment refers to reversing, alleviating, inhibiting, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated, As used herein, the term " treatment " refers to an act of treating when the treatment is defined as above. The therapeutic or therapeutic treatment of an immune disorder in a mammal therefore may include one or more of the following:
(1) 자연살해세포의 독성으로 유발할 수 있는 질환의 성장을 저해함, 즉, 그 발달을 저지시킴,(1) inhibiting the growth of diseases that may be caused by the toxicity of natural killer cells, that is,
(2) 자연살해세포의 독성으로 유발할 수 있는 질환의 확산을 예방함, 즉, 전이를 예방함,(2) preventing the spread of diseases that may be caused by toxicity of natural killer cells, that is, preventing metastasis,
(3) 자연살해세포의 독성으로 유발할 수 있는 질환을 경감시킴.(3) alleviate diseases that may be caused by the toxicity of natural killer cells.
(4) 자연살해세포의 독성으로 유발할 수 있는 질환의 재발을 예방함, 및(4) preventing the recurrence of diseases that may be caused by the toxicity of natural killer cells, and
(5) 자연살해세포의 독성으로 유발할 수 있는 질환의 증상을 완화함(palliating)(5) palliating symptoms of diseases that may be caused by toxicity of natural killer cells;
본 발명에 따른 상기 조성물은 약학적으로 유효한 양의 레티날 또는 레티노산을 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함할 수 있다. 상기에서 약학적으로 유효한 양이란 자연살해세포의 독성으로 유발할 수 있는 질환의 증상을 예방, 개선 및 치료하기에 충분한 양을 말한다.The compositions according to the present invention may comprise a pharmaceutically effective amount of retinal or retinoic acid alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat the symptoms of diseases that may be caused by toxicity of natural killer cells.
본 발명에 따른 레티날 또는 레티노산 화합물의 약학적으로 유효한 양은 0.5~100mg/day/체중kg, 바람직하게는 0.5~5mg/day/체중kg이다. 그러나 상기 약학적으로 유효한 양은 자연살해세포의 독성으로 유발할 수 있는 질환 증상의 정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료기간 등에 따라 적절히 변화될 수 있다.The pharmaceutically effective amount of the retinal or retinoic acid compound according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of the disease symptoms that may be caused by the toxicity of natural killer cells, the age, body weight, health condition, sex, administration route and treatment period of the patient.
또한, 상기에서 약학적으로 허용되는이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.Also, the pharmaceutically acceptable hereinabove is physiologically acceptable and refers to a composition which, when administered to a human, does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.
또한, 본 발명에 따른 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있으며, 활성 성분의 투여량은 투여 경로, 환자의 연령, 성별, 체중 및 환자의 중증도 등의 여러 인자에 따라 적절히 선택될 수 있고, 본 발명에 따른 조성물은 증상을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.In addition, the composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may vary depending on the administration route, age, sex, And the composition according to the present invention may be administered in combination with a known compound having an effect of preventing, ameliorating or treating symptoms.
또한, 본 발명은 미분화 T 세포에 레티날 화합물 또는 레티노산을 처리하는 단계를 포함하는 시험관(in vitro)내에서 미분화 T 세포의 분화 및 활성화를 유도하는 방법을 제공할 수 있으며, 분화 및 활성화된 T 세포는 자연살해세포와 함께 배양되어 자연살해세포의 세포독성을 감소 또는 억제시킬 수 있다.In addition, the present invention can provide a method for inducing differentiation and activation of undifferentiated T cells in vitro, comprising the step of treating the undifferentiated T cells with a retinal compound or retinoic acid, T cells may be cultured with natural killer cells to reduce or inhibit cytotoxicity of natural killer cells.
따라서 본 발명은 레티날(retinal) 또는 레티노산(retinoic acid)을 미분화된 조절 T 세포(Regulatory T cell: Treg)에 처리하여 조절 T 세포를 분화 및 활성화시키는 단계 및 분화 및 활성화된 조절 T 세포를 자연살해세포(Natural Killer Cell)와 함께 배양하는 단계를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법을 제공할 수 있다.Accordingly, the present invention relates to a method of treating retinal or retinoic acid in an undifferentiated regulatory T cell (Treg) to differentiate and activate regulatory T cells and to differentiate and activate regulated T cells (Natural Killer Cell) comprising the step of incubating the cells with a natural killer cell.
또한, 시험관(in vitro)내에서 레티날 또는 레티노산을 미분화 T 세포(Regulatory T cell: Treg)에 처리하여 자연살해세포와 함께 배양하는 단계의 경우지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 처리하여 함께 배양하는 단계를 추가로 더 포함할 수 있다.In addition, in the step of culturing retinal or retinoic acid in a non-differentiated T cell (T reg) in vitro and culturing it together with natural killer cells, adipose derived mesenchymal stem cells ), And culturing the cells together.
본 발명에서 레티날 또는 레티노산을 미분화 T 세포에 처리하는 방법은 상기 세포를 배양하는 배지에 레티날 또는 레티노산을 직접 처리하거나 또는 상기 본 발명에 따른 레티날 또는 레티노산을 유효성분으로 하는 조성물을 배양 배지에 처리하는 것일 수 있다. 또한, 이때 상기 배양 배지에 처리할 수 있는 레티날 화합물의 농도는 최종농도가 0.1μM ~ 5μM이 되도록 처리할 수 있으며, 본 발명의 일실시에에서는 레티날 또는 레티노산의 농도가 1μM이 되도록 세포의 배양 배지에 첨가하였다.
In the present invention, the method for treating retinal or retinoic acid into undifferentiated T cells may be performed by treating retinal or retinoic acid directly with a culture medium for culturing the cells or using a composition comprising retinal or retinoic acid according to the present invention as an active ingredient Lt; / RTI > to the culture medium. In this case, the concentration of the retinal compound that can be treated in the culture medium may be adjusted to a final concentration of 0.1 μM to 5 μM. In one embodiment of the present invention, the concentration of retinal or retinoic acid is 1 μM Of culture medium.
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
조절 T 세포로의 분화 유도Induction of differentiation into regulatory T cells
본 발명자들은 레티날 화합물을 처리하여 조절 T 세포(Regulatory T cells, Treg cells)의 분화를 유도하기 위해, C57BL/6 마우스의 비장(spleen) 세포에서 분리된 CD4+ T세포를 1 μg/mL의 anti-CD3를 코팅 처리한 24-웰 플레이트(24-well plate)에 분주하고, anti-CD28 항체와 TGF-β를 각각 1 μg/mL, 5 ng/mL 의 농도로 처리하여 3일 동안 조절 T 세포 분화를 유도하였다. 이 때 조절 T세포를 분화시키기 전에 레티날(Retinal)을 1 μM 의 농도로 처리한 군과 레티노산(Retinoic Acid)을 1 μM 의 농도로 처리한 군을 각각 제작하였으며, 대조군으로는 anti-CD3와 anti-CD28 항체로만 자극한 T 세포(conventional T cell)를 이용하였다.In order to induce the differentiation of regulatory T cells (Treg cells) by treating the retinal compound, the present inventors isolated CD4 + T cells isolated from spleen cells of C57BL / 6 mice with 1 μg / ml of anti -CD3 in a 24-well plate coated with anti-CD28 antibody and TGF-β at a concentration of 1 μg / mL and 5 ng / mL for 3 days, respectively. Differentiation was induced. At this time, pretreatment of regulatory T cells with retinal (Retinal) at 1 μM and retinoic acid at 1 μM were performed, and anti-CD3 And T cells stimulated only with anti-CD28 antibody (conventional T cell).
이후, 유세포 분석을 위해 anti-mouse CD4 PerCP, CD25 APC와 Foxp3 PE형광이 라벨된 항체를 넣고 4℃에서 30분간 반응시킨 다음 PBS로 세척하고, 유세포 분석기(FACs Calibur)를 이용하여 측정하였다.
For analysis of flow cytometry, antibodies labeled with anti-mouse CD4 PerCP, CD25 APC and Foxp3 PE fluorescence were incubated at 4 ° C for 30 min, washed with PBS, and analyzed using a flow cytometer (FACs Calibur).
그 결과, 도 1에 나타낸 바와 같이, Foxp3의 발현은 레티날을 처리한 경우 65.9이고, 레티노산을 처리한 경우 66.4인 것으로 나타났다. 이러한 결과를 통해 레티날 및 레티노산 처리하였을 때 Foxp3의 발현이 증가한다는 사실을 확인하였다.
As a result, as shown in Fig. 1, the expression of Foxp3 was found to be 65.9 when treated with retinal and 66.4 when treated with retinoic acid. These results indicate that the expression of Foxp3 is increased when treated with retinal and retinoic acid.
자연살해세포(Natural killer cells ( NKNK cellcell )에 의한 세포독성도 분석) Cytotoxicity Analysis
자연살해세포(Natural Killer cell; NK cell)에 의한 세포독성도(cytotoxicity)를 측정하기 위해, 자성-활성 세포 분류기(Magnetic-Activated Cell Sorting)를 이용한 NK 세포 분리 키트(MACS: Miltenyi Biotec, Germany)를 이용하여 자연살해세포를 분리한 다음, 분리한 자연살해세포를 RPMI 1640 배지에 현탁시켜 작동세포(effector cell)로 사용하고, 표적세포(target cell)로는 51Cr labeling YAC-1세포를 이용하였다. 51Cr labeling YAC-1 세포수가 5× 103/100 ㎕가 되도록 조정하고, 자연살해세포와 함께 96-웰 U-바닥 배양 플레이트(96-well U-bottom culture plate; Corning Glass Works, Corning, USA)에 배양하였는데, 자연살해세포의 수는 effector-to-target(E/T) 세포 비가 1: 6.25, 1：12.5, 1：25 및 1：50 이 되도록 농도로 달리 하여 세포 수를 맞춰주었다. 그 다음 반응이 이루어지도록 37℃에서 4시간 동안 세포 배양기에 두었다가 원심 분리하고 상층액의 120 ㎕만을 채취하였다. 채취한 상층액에서 감마선 계측기(Gamma counter)를 이용하여 51Cr의 방사선량을 측정함으로써 자연살해세포의 활성정도를 측정하였다. 측정 시, 라벨(Label)된 표적 세포로부터의 51Cr 방사능의 자연적인 유리(Spontaneous Release; SR)는 배양액을 넣어주고, 전체 유리(Total Release; TR)는 triton x-100 용액을 첨가하여 세포가 완전히 용해되도록 배양한 것을 이용하였다. 이 때, 조절 T 세포에 의해 자연살해세포의 활성이 억제될 수 있는지 알아보기 위해, 자연살해세포와 IL-2를 4일간 함께 배양한 군을 또 다른 대조군으로 사용하였으며, 세포독성도(% of cytotoxicity)는 다음과 같은 공식을 이용하였다.NK cell separation kit (MACS: Miltenyi Biotec, Germany) using Magnetic-Activated Cell Sorting was used to measure cytotoxicity by natural killer cells (NK cells) , And the isolated natural killer cells were suspended in RPMI 1640 medium to be used as effector cells and 51Cr-labeled YAC-1 cells were used as target cells. 51Cr labeling YAC-1 cell counts 5 × 10 3 / adjusted to be 100 ㎕, 96- well U- bottom culture plates with NK cells, and (96-well U-bottom culture plate; Corning Glass Works, Corning, USA) . The number of natural killer cells was adjusted to the effector-to-target (E / T) cell ratio of 1: 6.25, 1: 12.5, 1:25 and 1:50. The cells were then placed in a cell incubator at 37 ° C for 4 hours to allow the reaction to proceed, followed by centrifugation and only 120 μl of the supernatant was collected. The activity of natural killer cells was measured by measuring the radiation dose of 51Cr using a gamma counter on the collected supernatant. For measurement, the culture of 51Cr radioactive Spontaneous Release (SR) from the labeled target cells is added, and the total release (TR) is added to the triton x-100 solution And then cultured for dissolution. At this time, in order to investigate whether natural killer cells can be inhibited by regulatory T cells, natural killer cells and IL-2 cultured for 4 days were used as another control group, and the cytotoxicity (% of cytotoxicity) using the following formula.
% cytotoxicity = 100 x (측정된 실험수치 - SR) / (TR - SR)
% cytotoxicity = 100 x (measured experimental values - SR) / (TR - SR)
그 결과, 도 2에 나타낸 바와 같이, 자연살해세포의 세포독성 능력은 레티노산을 첨가하여 유도된 조절 T 세포(Retinoic acid induced Treg)에서도 감소되었지만, 조절 T 세포가 분화되기 전 레티날을 첨가하여 분화를 유도한 조절 T 세포(Retinal induced Treg)에서 가장 현저하게 감소되는 것을 확인할 수 있었다. 또한, 자연살해세포와 T 세포의 비율이 10:1일 때보다 1:1 일 때의 조건에서 효과가 더욱 우수한 것으로 나타나, 조절 T 세포에 의한 자연살해세포의 조절효과가 양에 의존적(dose-dependent)임을 확인할 수 있었다.
As a result, as shown in Fig. 2, the cytotoxic ability of natural killer cells was also decreased in regulatory T cells induced by addition of retinoic acid, but the retinal was added before regulatory T cells were differentiated (Retinal-induced Treg), which is the most prominent marker of T-cell differentiation. In addition, the effect was more excellent in the condition of 1: 1 than in the ratio of natural killer cells and T cells of 10: 1, and the regulatory effect of natural killer cells by regulatory T cells was dose- dependent).
지방유래 Locally derived 간엽줄기세포에On mesenchymal stem cells 의한 효과 분석 Effect analysis
상기 실시예 1에서 제작한 레티날(Retinal)에 의해 유도된 조절 면역 세포와 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells, MSCs)에 의한 세포독성 조절 능력을 평가하기 위해, 상기 실시예 2와 동일한 방법을 이용하되, 지방유래 간엽줄기세포를 단독으로 처리한 군과 지방유래 간엽줄기세포와 레티날에 의해 유도된 조절 면역 세포를 함께 배양한 군으로 나누어 자연살해세포의 세포독성도(% of cytotoxicity)를 측정하였다.To evaluate cytotoxicity of regulatory immune cells and human adipose-derived mesenchymal stem cells (MSCs) induced by Retinal produced in Example 1, , The cytotoxicity of natural killer cells (%) was evaluated by dividing the group treated with adipocyte-derived mesenchymal stem cells and the group cultured with adipose-derived mesenchymal stem cells and retinal-induced regulated immune cells, of cytotoxicity were measured.
그 결과, 도 3에 나타낸 바와 같이, 지방유래 간엽줄기세포를 단독으로 처리한 군에 비해 지방유래 간엽줄기세포와 레티날에 의해 유도된 조절 면역 세포를 함께 배양한 군의 조건에서 자연살해세포의 세포독성 능력이 현저하게 감소되는 것을 확인할 수 있었다.
As a result, as shown in Fig. 3, in comparison with the group treated with the adipose-derived mesenchymal stem cells alone, in the condition of the group cultured with adipose-derived mesenchymal stem cells and retinal-induced regulated immune cells, It was confirmed that the cytotoxic ability was remarkably reduced.
자연살해세포(Natural killer cells ( NKNK cellcell )에 의한 세포사멸도 측정) To measure cell death
자연살해세포(Natural Killer cell; NK cell)에 의한 세포사멸도(cytotoxicity)를 측정하기 위해, 자성-활성 세포 분류기(Magnetic-Activated Cell Sorting)를 이용한 NK 세포 분리 키트(MACS: Miltenyi Biotec, Germany)를 이용하여 자연살해세포를 분리한 다음, 분리한 자연살해세포를 RPMI 1640 배지에 현탁시켜 작동세포(effector cell)로 사용하고, 조절 T세포와 간엽줄기세포에 의해 자연살해세포의 사멸 정도를 조사하기 위해, 자연살해세포(NK cell)와 3일간 IL-2와 함께 배양하는 조건에 자연살해세포(NK cell)를 면역조절세포와 간엽줄기세포(MSC)와 함께 공조배양을 하였다. 공조배양한 뒤 3일 후 세포에 annexin V와 PI를 염색하여 세포사멸정도를 관찰하였다. NK cell separation kit (MACS: Miltenyi Biotec, Germany) using Magnetic-Activated Cell Sorting was used to measure cytotoxicity by natural killer cells (NK cells) , And the isolated natural killer cells were suspended in RPMI 1640 medium to be used as effector cells. The degree of killing of natural killer cells was examined by regulatory T cells and mesenchymal stem cells NK cells were co-cultured with immune-regulated cells and mesenchymal stem cells (MSCs) under conditions of incubation with NK cells for 3 days with IL-2. After 3 days of incubation, the cells were stained with annexin V and PI and the degree of apoptosis was observed.
그 결과, 도 4에 나타낸바와 같이, 자연살해세포(NK cell)의 세포사멸조절 능력은 조절 T세포의 분화조건하에 레티날(Retinal)이 들어가 분화가 유도된 조절 T세포(Retinal induced Treg)에 의해 현저하게 증가된 것을 확인하였으며, 레티노산(Retinoic acid)이 들어가 유도된 조절 T세포(Retinoic acid induced Treg) 또한 자연살해세포(NK cell)의 세포독성 능력이 증가되었지만 레티날(Retinal)이 들어가 분화가 유도된 조절 T세포(Retinal induced Treg) 보다는 덜 효과적이었음을 확인할 수 있었으며 간엽줄기세포(MSC) 단독에 의한 자연살해세포(NK cell)의 세포사멸 또한 증가되는 경향을 보였으나 면역조절세포에 의한 효과에 비해 미비하였음을 확인할 수 있었다.As a result, as shown in FIG. 4, the ability of the natural killer cell (NK cell) to regulate apoptosis is regulated by regenerated T cells (retinal induced T cells) (Retinoic acid induced Treg) induced by retinoic acid also increased the cytotoxic ability of NK cell, but retinal (retinoic acid induced) (MSC) alone, the cell death of NK cells was also increased. However, in immunocompetent cells, the expression of NK cells was decreased The results of this study are as follows.
따라서, 간엽줄기세포(MSC) 조건에 레티날과 레티노산이 들어가 분화가 유도된 조절 T세포를 같이 넣어 준 경우, 더 효과적으로 자연살해세포(NK cell)의 세포사멸을 유도함을 알 수 있었다.
Therefore, when regulatory T cells induced by differentiation into retinal and retinoic acid were added to mesenchymal stem cell (MSC) conditions, the cell death of NK cells was more effectively induced.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (9)
상기 레티날 또는 레티노산은 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein said retinal or retinoic acid reduces or inhibits the cytotoxicity of Natural Killer Cells.
상기 레티날 또는 레티노산은 0.1 ~ 5μM 의 농도로 포함되어 있는 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein the retinal or retinoic acid is contained at a concentration of 0.1 to 5 μM.
상기 분화된 조절 T 세포(Treg 세포)와 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)는 1:0.01~1:0.03의 비율로 포함되어 있는 것을 특징으로 하는 자연살해세포(Natural Killer Cell)의 세포 독성을 억제하는 활성을 갖는 조성물.The method according to claim 1,
Wherein the differentiated regulatory T cells (Treg cells) and human adipose derived mesenchymal stem cells are contained in a ratio of 1: 0.01 to 1: 0.03. Wherein the composition has an activity of inhibiting cytotoxicity.
분화 및 활성화된 조절 T 세포를 자연살해세포(Natural Killer Cell)와 함께 배양하는 단계를 포함하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법.Step of the process to: (Treg Regulatory T cell) differentiation and activation of regulatory T cells; in vitro (in vitro) retinal (retinal) or retinoic acid (retinoic acid) to undifferentiated regulatory T cells And
A method of reducing or inhibiting the cytotoxicity of a natural killer cell comprising culturing the differentiated and activated regulatory T cells together with a natural killer cell.
상기 배양하는 단계에서 지방유래 간엽줄기세포(Human adipose derived mesenchymal stem cells)를 처리하여 함께 배양하는 단계를 추가로 포함하는 것을 특징으로 하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법.8. The method of claim 7,
The method according to claim 1, further comprising the step of culturing human adipose-derived mesenchymal stem cells and culturing them together in the step of culturing, wherein the method further comprises the step of reducing or inhibiting the cytotoxicity of natural killer cells Way.
상기 레티날 또는 레티노산은 미분화된 조절 T 세포(Regulatory T cell: Treg)에 0.1 ~ 5μM 의 농도로 처리하여 조절 T 세포를 분화 및 활성화시키는 것을 특징으로 하는 자연살해세포(Natural Killer Cell)의 세포 독성을 감소 또는 억제시키는 방법.
8. The method of claim 7,
Wherein the retinal or retinoic acid is treated with a concentration of 0.1 to 5 μM in an undifferentiated regulatory T cell (Treg) to differentiate and activate regulatory T cells. The cytotoxicity of the natural killer cell ≪ / RTI >
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