KR101447132B1 - Compositon for inducing maturation of dendritic cell comprising Mycobacterium tuberculosis RpfB - Google Patents
Compositon for inducing maturation of dendritic cell comprising Mycobacterium tuberculosis RpfB Download PDFInfo
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- KR101447132B1 KR101447132B1 KR1020120013868A KR20120013868A KR101447132B1 KR 101447132 B1 KR101447132 B1 KR 101447132B1 KR 1020120013868 A KR1020120013868 A KR 1020120013868A KR 20120013868 A KR20120013868 A KR 20120013868A KR 101447132 B1 KR101447132 B1 KR 101447132B1
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- dendritic cells
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Abstract
본 발명은 결핵균의 RpfB 단백질을 유효성분으로 포함하는 수지상 세포의 성숙화 유도용 조성물, 결핵균의 RpfB 단백질을 미성숙 수지상 세포에 처리하여 성숙화를 유도하는 방법, 및 상기 성숙화 유도용 조성물을 포함하는 면역 증강용 조성물에 관한 것이다. 본 발명에 따른 RpfB 단백질은 미성숙 수지상 세포를 효과적으로 성숙시킴으로써 신체의 면역 반응을 효과적으로 증강시킬 수 있다.The present invention relates to a composition for inducing maturation of dendritic cells comprising the RpfB protein of Mycobacterium tuberculosis as an active ingredient, a method for inducing maturation of immature dendritic cells by treating the RpfB protein of Mycobacterium tuberculosis with an immature dendritic cell, ≪ / RTI > The RpfB protein according to the present invention can effectively enhance the immune response of the body by effectively matured immature dendritic cells.
Description
본 발명은 결핵균 (Mycobacterium tuberculosis) 유래의 RpfB 단백질을 유효 성분으로 포함하는 수지상 세포 성숙화 유도 조성물, 및 이를 이용하여 미성숙 수지상 세포를 성숙 수지상 세포로 분화시키는 방법에 관한 것이다.
The present invention relates to a Mycobacterium tuberculosis- derived RpfB protein as an active ingredient, and a method of using the same to differentiate immature dendritic cells into mature dendritic cells.
수지상 세포 또는 수상돌기 세포 (dendritic cell, DC)는 포유동물의 면역계의 일부를 이루는 면역 세포이다. 이 세포들은 항원 물질을 처리하여 그것을 표면에 나타나게 함으로써 면역계의 다른 세포가 인식하게 하는 항원 발현 세포의 역할을 한다. 수지상 세포는 주로 피부, 코, 폐, 위 및 장의 내벽과 같이 외부 환경과 접하는 조직에 소량 존재하며 특히 피부에 있는 세포를 랑게르한스 세포라 한다. 수지상 세포는 혈액 중에서 미성숙한 상태로 발견될 수 있으며, 활성화되면 림프기관으로 이동하여 T 세포 및 B 세포와 상호작용하여 면역반응이 시작된다. 특정 발달 단계에서 그것들은 수상돌기(dendrites)라고 하는 돌기를 뻗는다.Dendritic cells or dendritic cells (DCs) are immune cells that form part of the immune system of mammals. These cells act as antigen-expressing cells that make the other cells of the immune system recognize them by treating the antigenic material and making it appear on the surface. Dendritic cells are present in small amounts in tissues that are in contact with the external environment, such as skin, nose, lung, stomach and intestinal wall, and the cells in the skin are called Langerhans cells. Dendritic cells can be found immature in blood, and when activated, they migrate to the lymphatic organs and interact with T cells and B cells to initiate an immune response. At certain developmental stages, they stretch the dendrites, called dendrites.
수지상 세포는 조혈 골수 전구세포 (hemopoietic bone marrow progenitor cells)로부터 유래한다. 이 전구세포는 처음에는 미성숙 수지상 세포로 변화하며, 높은 엔도시토시스 활성 및 T-세포 활성 능력을 특징으로 한다. 미성숙 수지상 세포는 주변에 있는 바이러스 및 박테리아와 같은 병원체를 끊임없이 탐식한다. 이것은 TLR (toll-like receptor)과 같은 패턴 인식 수용체 (pattern recognition receptor, PRR)를 통해서 가능하다. TLR은 병원체의 서브셋 상에서 발견되는 특정 화학적 특징을 인식하며, 미성숙 수지상 세포는 살아있는 자가세포로부터 니블링 (nibbling)이라는 과정을 통해 세포막을 탐식한다. 이들이 현존하는 항원과 접하게 되면, 성숙 수지상 세포로 활성화되어 림프절로 이동한다. 미성숙 수지상 세포는 병원체를 탐식하고 자신의 단백질을 작은 조각들로 분해해서, 성숙하였을 때 이 조각들이 MHC (Major Histocompatibility Complex) 분자를 이용하여 그 세포 표면에 나타나게 된다. 동시에, 그것은 CD (Cluster of Differentiation)80, CD86, 및 CD40과 같이 T-세포 활성화에서의 공동-수용체로 작용하는 세포 표면 수용체를 증가시킨다. 그것들은 비 항원성 특정 공동자극 신호와 함께 병원체에서 유래한 항원을 나타냄으로써 헬퍼 T-세포, 킬러 T-세포 뿐만 아니라, B 세포를 활성화시킨다. Dendritic cells are derived from hemopoietic bone marrow progenitor cells. These progenitor cells are initially transformed into immature dendritic cells, characterized by high endocytosis activity and T-cell activation capacity. Immature dendritic cells constantly digest surrounding pathogens such as viruses and bacteria. This is possible through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). TLRs recognize specific chemical characteristics found on a subset of pathogens, and immature dendritic cells digest the cell membrane through a process known as nibbling from living autologous cells. When they come into contact with existing antigens, they become activated to mature dendritic cells and migrate to the lymph nodes. Immature dendritic cells digest pathogens and break down their proteins into small pieces that, when mature, appear on the cell surface using MHC (Major Histocompatibility Complex) molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation such as CD (Cluster of Differentiation) 80, CD86, and CD40. They activate helper T-cells, killer T-cells as well as B-cells by showing pathogen-derived antigens with specific non-antigenic co-stimulatory signals.
모든 헬퍼 T-세포는 하나의 특정 항원에 대해 특이적이다. 오직 전문적인 항원 발현 세포(대식세포, B림프구 및 수지상 세포)만이 맞는 항원이 존재할 때 나머지 헬퍼 T-세포를 활성화시킨다. 그러나 대식세포와 B림프구는 메모리 T-세포만 활성화시킬 수 있는 반면에 수지상 세포는 메모리 및 처녀 (naive) T-세포를 모두 활성화시킬 수 있어서 가장 강력한 항원 발현 세포이다. All helper T-cells are specific for one particular antigen. Only specialized antigen-expressing cells (macrophages, B lymphocytes and dendritic cells) activate the remaining helper T-cells when the correct antigen is present. However, macrophages and B lymphocytes can only activate memory T-cells, while dendritic cells are the most potent antigen-expressing cells because they can activate both memory and naive T-cells.
성숙 수지상 세포는 신체를 순환하는 백혈구인 단핵구로부터 유래할 수 있는데, 단핵구는 적절한 신호에 따라 수지상 세포로도 대식세포로도 바뀔 수 있다. 단핵구는 골수의 줄기세포에서 유래한다. 단핵구-유래 수지상 세포는 실험실에서 말초 혈액 단핵 세포 (PBMC)로부터 생성될 수 있다. PBMC를 조직 배양 플라스크에 심어서 단핵구가 부착되게 할 수 있는데 이 단핵구를 IL-4 및 GM-CSF (granulocyte-macrophage colony stimulating factor)로 처리함으로써 미성숙 수지상 세포로 분화시킬 수 있다. 이후에 TNF-a로 처리하면 미성숙 수지상 세포가 성숙 수지상 세포로 분화된다. Mature dendritic cells can originate from monocytes, the circulating white blood cells of the body. Monocytes can be turned into dendritic cells as well as dendritic cells, depending on the appropriate signal. Monocytes originate from the bone marrow stem cells. Monocyte-derived dendritic cells can be generated from peripheral blood mononuclear cells (PBMC) in the laboratory. PBMCs can be implanted in tissue culture flasks to attach monocytes, which can be differentiated into immature dendritic cells by treatment with IL-4 and GM-CSF (granulocyte-macrophage colony stimulating factor). Subsequent treatment with TNF-a differentiates immature dendritic cells into mature dendritic cells.
완전히 성숙한 수지상 세포는 미숙한 DC와는 정성적 및 정량적으로 상이하다. 완전히 성숙한 DC는 고 수준의 MHC (Major Histocompatibility Complex)I형 및 II형 항원, 및 고수준의 T 세포 공동자극성 분자, 즉 CD80 및 CD86을 발현시킨다. 이들 변화는 수지상 세포의 T 세포 활성화 능력을 증가시키는데, 이는 이들이 세포 표면상의 항원 밀도를 증가시킬 뿐만 아니라, 예를 들어 CD28과 같은 T 세포 상의 공동자극성 분자의 대응물을 통한 T 세포 활성화의 양을 증가시키기 때문이다. 추가로, 성숙한 수지상 세포는 다량의 사이토카인을 생성하며, 이 사이토카인은 T 세포 반응을 자극하고 유도한다. 이들 사이토카인 중 2 가지는 인터루킨 10 (IL-10) 및 인터루킨 12 (IL-12)이다. 이들 사이토카인은 유도된 T 세포 반응의 방향에 대해 정반대의 효과를 갖는다. IL-10이 생성되면 Th-2형 반응이 유도되는 반면, IL-12가 생성되면 Th-1 형 반응이 유도된다. 후자의 반응이 세포 면역 반응이 요구되는 경우, 예를 들면 암 면역요법에서 특히 바람직하다. Th-1형 반응에 의해 세포 면역계의 작동인자 공격수단인 세포독성 T 림프구(CTL)가 유도 및 분극화된다. 이러한 작동인자 공격수단은 종양 성장을 억제하는데 가장 효과적이다. IL-12는 또한 자연살해(NK) 세포의 성장을 유도하고, 항-혈관신생 활성을 갖고, 이는 모두 효과적인 항-종양 공격수단이다. 따라서, IL-12를 생성하는 수지상 세포의 사용은 면역자극에 사용하는데 이론상 최적으로 적합하다.Fully mature DCs differ qualitatively and quantitatively from immature DCs. Fully mature DCs express high levels of MHC (Major Histocompatibility Complex) type I and type II antigens, and high levels of T cell co-stimulatory molecules, CD80 and CD86. These changes increase the T cell activation capacity of dendritic cells, which not only increases the antigen density on the cell surface, but also increases the amount of T cell activation through the counterparts of co-stimulatory molecules on T cells, such as CD28 . In addition, adult dendritic cells produce large amounts of cytokines, which stimulate and induce T cell responses. Two of these cytokines are interleukin 10 (IL-10) and interleukin 12 (IL-12). These cytokines have the opposite effect on the direction of the induced T cell response. When IL-10 is produced, a Th-2 type reaction is induced, whereas when IL-12 is produced, a Th-1 type reaction is induced. The latter response is particularly preferred when a cellular immune response is required, for example in cancer immunotherapy. The Th-1 type response induces and polarizes cytotoxic T lymphocytes (CTLs), which are the means of attacking the working factors of the cell's immune system. This means of action factor attack is most effective in inhibiting tumor growth. IL-12 also induces the growth of natural killer (NK) cells and has anti-angiogenic activity, which are all effective anti-tumor attack means. Thus, the use of dendritic cells to produce IL-12 is theoretically optimal for use in immunostimulation.
수지상 세포는 흔치 않고 분리하기 어렵기 때문에 서로 다른 유형과 서브셋의 수지상 세포의 정확한 생성과 발달 및 그 상호관계에 대해서는 오직 대략적으로만 알려졌다. 수지상 세포는 신체의 다른 세포들과 끊임없는 소통을 한다. 이러한 소통은 세포 표면 단백질의 상호작용에 근거한 직접적인 세포 대 세포 접촉의 형태를 취할 수 있다. Since dendritic cells are rare and difficult to isolate, the precise production and development of dendritic cells of different types and subsets and their interrelationships have only been briefly known. Dendritic cells communicate with other cells in the body. Such communication can take the form of direct cell-to-cell contact based on the interaction of cell surface proteins.
수지상 세포에 의해 생산되는 사이토카인은 세포의 유형에 따라 다르다. 림프구성 수지상 세포는 다량의 타입-1 IFN을 생산할 수 있는데 그것은 더 많은 활성화된 대식 세포를 모아서 탐식 작용을 가능하게 한다. 림프구성 수지상 세포는 중추와 말초 면역조절에 관여하고, 골수성 수지상 세포는 외래성 항원이나 감염에 대한 면역유도에 관여하는 것으로 알려져 있다. 따라서 수지상 세포가 정상 기능을 못할 경우 당뇨병, 류머티스성 관절염, 알레르기성 과민반응과 같은 자가 면역 질환이 나타나거나 감염성 질환이나 암 발생에 대해 정상적인 면역반응이 일어나지 않게 된다. The cytokines produced by dendritic cells differ depending on the type of cell. Lymphoid dendritic cells can produce large amounts of
암환자에서는 수지상 세포의 기능이 저하되어 있어 정상적 항암 면역능을 유도하는 것이 어렵다. 지금까지 수지상 세포의 분화를 조절(활성화)하는 물질들(LPS, TNF-α, IL-1β)은 많이 알려져 있으나 생체독성의 부작용으로 생체에의 직접적인 응용에 문제가 많다. In cancer patients, the function of dendritic cells is deteriorated and it is difficult to induce normal cancer immunity. (LPS, TNF-α, and IL-1β) are known to regulate (activate) dendritic cell differentiation. However, they are a side effect of biotoxicity, which is problematic in direct application to living organisms.
마이코박테리움 (Mycobacterium) 속(屬)에는 결핵, 우형결핵 (牛形結核), 나병 (癩病)과 같이 사람과 동물에 심각한 질병을 일으키는 균종 (species)뿐 아니라, 기회감염균으로 일컬어지는 균종, 그리고 자연환경에서 볼 수 있는 사물 (死物)기생의 균종 (saprophytic species) 등 현재까지 약 72 종(species)이 알려져 있으며, 그 중 인체 질환과 관련된 것이 25종에 이르는 것으로 알려져 있다.The Mycobacterium genus includes not only species that cause serious diseases in humans and animals such as tuberculosis, bovine tuberculosis and leprosy, but also bacteria that are called opportunistic infections, And saprophytic species, which can be found in natural environments. To date, about 72 species have been known, of which 25 are related to human diseases.
마이코박테리아 감염증 가운데 가장 많은 질병은 결핵 (Tuberculosis)으로, 강한 병원성을 갖는 결핵균군(群)(M.tuberculosis complex: TB complex)으로 구분되는 M. tuberculosis , M. bovis , M. africanum , M. microti의 4 종이 원인균이며, 이 중 결핵균 (M. tuberculosis)이 가장 흔하고 중요한 원인균으로 알려져 있다. 결핵은 국내에서 아직까지도 중요시되는 질환이며, 전세계적으로는 해마다 약 8 백만의 새로운 환자가 발생하는 것으로 보고되고 있다. M. tuberculosis , M. bovis , M. africanum and M. microti ( M. tuberculosis ), which are classified as M. tuberculosis complex (TB complex) ( M. tuberculosis ) is the most common and important causative organism. Tuberculosis is still a major disease in Korea and it is reported that around 8 million new cases occur annually worldwide.
상기와 같이 수지상 세포는 신체 자체의 면역 기능을 높이는데 중요한 역할을 하고 있어, 수지상 세포의 분화를 촉진하여 성숙시킴으로써 강력한 면역 반응을 일으키는 무독성의 면역조절제의 개발과 그 물질의 작용기전을 명확히 이해하는 것은 수지상 세포를 이용한 세포 면역 치료에 중요한 과제가 되고 있다As described above, dendritic cells play an important role in enhancing the immune function of the body itself. Therefore, the development of a non-toxic immunomodulatory agent that induces a strong immune response by promoting and matured dendritic cell differentiation and a clear understanding of its mechanism of action Is an important task for cell-mediated immunotherapy using dendritic cells
본 발명자들은 상기와 같은 요구를 충족시키기 위하여 연구를 거듭한 결과 결핵균에서 유래한 RpfB이 수지상 세포를 효과적으로 성숙시킨다는 사실을 발견함으로써 본 발명을 완성하였다.
The inventors of the present invention have completed the present invention by finding out that RpfB derived from Mycobacterium tuberculosis effectively matures dendritic cells.
본 발명은 미성숙된 수지상 세포를 간편하고 효과적으로 성숙시킬 수 있는, RpfB 단백질을 유효성분으로 포함하는 미성숙 수지상 세포의 성숙화 유도용 조성물을 제공하고자 한다. The present invention provides a composition for inducing maturation of immature dendritic cells comprising RpfB protein as an active ingredient, which can mature easily and effectively immature dendritic cells.
또한 본 발명은 미성숙 수지상 세포에 RpfB를 처리하여 성숙 수지상 세포로 성숙화 시키는 것을 특징으로 하는 미성숙 수지상 세포의 성숙화 유도방법을 제공하고자 한다.The present invention also provides a method for inducing maturation of immature dendritic cells characterized by treating immature dendritic cells with RpfB to mature into mature dendritic cells.
또한 본 발명은 상기 수지상 세포 성숙화 유도용 조성물을 포함하는 면역 증강용 조성물을 제공하고자 한다.
The present invention also provides a composition for inducing dendritic cell maturation comprising the composition for immunity enhancement.
상기와 같은 과제를 해결하기 위해, 본 발명은 RpfB 단백질을 유효성분으로 포함하는 수지상의 세포 성숙화 유도용 조성물을 제공한다. In order to solve the above problems, the present invention provides a dendritic cell maturation inducing composition comprising RpfB protein as an active ingredient.
또한, 본 발명은 미성숙 수지상 세포에 RpfB를 처리하여 성숙 수지상 세포로 성숙화 시키는 미성숙 수지상 세포의 성숙화 유도 방법을 제공한다. The present invention also provides a method for inducing the maturation of immature dendritic cells that matured into mature dendritic cells by treating RpfB with immature dendritic cells.
또한, 본 발명은 상기 수지상 세포 성숙화 유도용 조성물을 포함하는 면역 증강용 조성물을 제공한다.
The present invention also provides a composition for enhancing immunity comprising the composition for inducing dendritic cell maturation.
본 발명에 따른 RpfB는 미성숙 수지상 세포의 분화를 촉진함으로써 수지상 세포를 효과적으로 성숙시킬 수 있으며, 이를 통해 신체의 면역 반응을 효과적으로 증강시키는 효과가 있다.
RpfB according to the present invention promotes the differentiation of immature dendritic cells, thereby effectively matured dendritic cells, thereby effectively enhancing the immune response of the body.
도 1은 클로닝을 통해 분리한 재조합 단백질 RpfB를 나타낸 도이다
도 2는 RpfB를 처리한 수지상 세포의 생존율을 유세포 분석기로 측정한 실험 결과를 나타낸 도이다.
도 3은 RpfB를 처리한 수지상 세포에서 특이적인 단백질표면인자인 CD80, CD86과 MHC class I, II의 발현량(A) 및 이를 수치(B)로 나타낸 도이다.
도 4는 RpfB를 처리한 수지상 세포의 사이토카인(TNF-α, IL-6, IL-1β, IL-12 p70, IL-10) 분비량(A) 및 IL-12p70와 IL-10의 분비량(B)의 측정 결과를 나타낸 도이다.
도 5는 RpfB에 의해 유도되는 수지상 세포의 TNF-α 및 IL-6 방출에 대한 열변성(A), 프로테인아제 K (Protease K) 소화(B), 및 PMB(C) 영향 분석 결과를 나타낸 도이다.
도 6은 RpfB를 처리한 수지상 세포의 덱스트란-FITC 탐식능력 측정 결과를 나타낸 도이다.
도 7은 RpfB를 처리한 수지상 세포에서 신호전달계의 변화를 ERK, JNK, p38, IκB-α, NF-κB p65의 인산화 및 분해 여부로 확인한 도이다.
도 8은 수지상 세포에 MAPK 및 NF-kB 억제 물질들을 처리한 후, 항-CD80-PE, 항-CD86-PE 세포 표면인자 항체를 이용하여 염색하여 유세포분석기 FACs Canto로 분석한 결과(A), 수지상 세포의 성숙 여부에 따라 분비되는 사이토카인 (TNF-α, IL-6, IL-1β)을 ELISA 키트로 측정한 결과(B) 및 핵 안에서의 NF-κB p65의 발현량 측정 결과(C)를 나타낸 도이다.
도 9는 RpfB를 처리한 수지상 세포의 T세포 자극에 의한 활성 효과(A) 및 T세포의 사이토카인의 분비량(B)을 측정한 결과를 나타낸 도이다.1 shows a recombinant protein RpfB isolated through cloning
FIG. 2 is a graph showing the results of an experiment in which the survival rate of dendritic cells treated with RpfB was measured with a flow cytometer.
FIG. 3 is a graph showing the expression levels (A) and the numerical values (B) of CD80, CD86 and MHC class I and II as specific protein surface factors in dendritic cells treated with RpfB.
FIG. 4 shows the amount (A) of secretion of cytokines (TNF-α, IL-6, IL-1β, IL-12 p70 and IL-10) and the secretion amount of IL-12p70 and IL- Fig.
FIG. 5 is a graph showing the results of thermal analysis (A), proteinase K digestion (B), and PMB (C) effects of TNF-α and IL-6 release on dendritic cells induced by RpfB to be.
FIG. 6 is a graph showing the result of measuring the dextran-FITC phagocytic ability of dendritic cells treated with RpfB.
FIG. 7 is a graph showing changes in signal transduction system in dendritic cells treated with RpfB by phosphorylation and degradation of ERK, JNK, p38, IκB-α, and NF-κB p65.
FIG. 8 shows the results of analysis of the dendritic cells with FACs Canto (A), staining with anti-CD80-PE and anti-CD86-PE cell surface factor antibodies after treatment with MAPK and NF- (B) and (C) the expression level of NF-κB p65 in the nucleus, as measured by an ELISA kit, of the cytokines secreted by dendritic cells (TNF-α, IL-6, and IL- Fig.
FIG. 9 is a graph showing the results of measuring the activity effect (A) by T cell stimulation and the secretion amount (B) of cytokine of T cells in dendritic cells treated with RpfB.
본 발명은 RpfB를 유효성분으로 포함하는 수지상 세포의 성숙화 유도용 조성물을 제공한다.The present invention provides a composition for inducing maturation of dendritic cells comprising RpfB as an active ingredient.
RpfB는 362개의 아미노산 서열로 이루어진 37 KDa 세포외 항원이다. RpfB는 박테리아와 그람 양성 세균에서 발견되며, 휴면 상태의 박테리아가 소생할 수 있도록 도와주는 작은 단백질이다. Mycobacterium tuberculosis H37Rv 표준균주 (NCBI GenBank ID:CAB00972.1) 전체 유전자 지도 해석을 통하여 부여된 RpfB의 단백질의 고유 ID 번호는 2052146이다. RpfB의 염기서열을 서열번호 1로, 아미노산 서열을 서열번호 2로 각각 나타내었다. RpfB is a 37 KDa extracellular antigen consisting of 362 amino acid sequences. RpfB is found in bacteria and gram-positive bacteria and is a small protein that helps dormant bacteria regenerate. Mycobacterium tuberculosis H37Rv standard strain (NCBI GenBank ID: CAB00972.1) The unique ID number of the protein of RpfB imparted through the entire gene map analysis is 2052146. The nucleotide sequence of RpfB is shown in SEQ ID NO: 1 and the amino acid sequence is shown in SEQ ID NO: 2, respectively.
본 발명은 결핵균 (M. tuberculosis)에 대한 유전자를 클로닝한 후 대장균 발현시스템을 이용하여 분리, 정제하여 수지상 세포 성숙화를 유도할 수 있다. The present invention can induce dendritic cell maturation by isolating and purifying a gene for M. tuberculosis using an E. coli expression system after cloning a gene for M. tuberculosis .
미숙한 수지상 세포는 성숙하여 성숙한 수지상 세포를 형성한다. 성숙한 수지상 세포는 항원을 포획하고 동시 자극하는 세포 표면 분자 및 각종 사이토카인의 상향-조절된 발현을 나타내는 능력을 상실한다. 특히, 성숙한 수지상 세포는 MHC I형 및 II형 항원을 미숙한 수지상 세포보다 더 높은 수준으로 발현시키고, CD (Cluster of Differentiation) 80+, CD83+, CD86+ 및 CD14-을 조절한다. 더 많은 MHC 발현으로 수지상 세포 표면상에서 항원 밀도의 증가를 유도하는 반면, 동시-자극의 분자 CD80 및 CD86의 상향 조절로, T 세포 상에 CD28과 같은 동시-자극의 분자 상응물을 통해서 T 세포 활성 신호를 강화한다.Immature dendritic cells mature to form mature dendritic cells. Adult dendritic cells lose the ability to capture up-regulated expression of cell surface molecules and various cytokines that capture and co-stimulate the antigen. In particular, mature dendritic cells express MHC type I and type II antigens at higher levels than immature dendritic cells and regulate Cluster of Differentiation (CD) 80+, CD83 +, CD86 + and CD14-. Induces an increase in antigenic density on the dendritic cell surface with more MHC expression, whereas upregulation of the co-stimulatory molecules CD80 and CD86 leads to T cell activation through co-stimulatory molecular counterparts such as CD28 on T cells Enhance the signal.
본 발명은 RpfB를 포함하는 조성물을 통해 성숙한 수지상 세포로의 분화를 촉진할 수 있다. 유효한 재조합 방법으로 제조된 RpfB는 이에 제한되는 것은 아니나 0.1 μg/ml 내지 5/ml, 바람직하게는 0.5 μg/ml 내지 2 μg/ml농도로 조성물에 포함될 수 있다.
The present invention is capable of promoting differentiation into mature dendritic cells through a composition comprising RpfB. RpfB produced by an effective recombinant method may be included in the composition at a concentration of from 0.1 μg / ml to 5 μg / ml, preferably from 0.5 μg / ml to 2 μg / ml, though not limited thereto.
또한, 본 발명은 미성숙 수지상 세포에 RpfB를 처리하는 것을 특징으로 하는 미성숙 수지상 세포의 성숙화 유도방법을 제공할 수 있다. In addition, the present invention can provide a method for inducing maturation of immature dendritic cells, characterized in that immature dendritic cells are treated with RpfB.
본 발명의 RpfB 단백질은 미성숙 수지상 세포에 0.1 μg/ml 내지 5μg/ml, 바람직하게는 0.5 μg/ml 내지 2 μg/ml 농도로 처리될 수 있다. 본 발명은 미성숙 수지상 세포에 RpfB를 처리하여 성숙화를 유도하기 위하여 RpfB가 처리된 세포를 배양할 수 있다. 적절한 성숙 수지상 세포로의 분화 유도를 위하여, 상기 세포를 바람직한 일 예로써 12~36시간 동안 배양하여 성숙시킬 수 있으나, 이에 제한되는 것은 아니다.The RpfB protein of the present invention can be treated to immature dendritic cells at a concentration of 0.1 μg / ml to 5 μg / ml, preferably 0.5 μg / ml to 2 μg / ml. The present invention can cultivate RpfB-treated cells to induce maturation by treating RpfB with immature dendritic cells. For induction of differentiation into suitable mature dendritic cells, the cells may be cultured for 12 to 36 hours as a preferred example, but not limited thereto.
본 발명에서의 RpfB 단백질 처리를 통해 성숙이 촉진된 성숙된 수지상 세포는 성숙된 수지상 세포가 가지는 일반적인 특징을 발현할 수 있다. 즉, 본 발명에 따르면, RpfB를 미성숙 수지상 세포에 처리함으로써 미성숙 수지상 세포가 분화되어 성숙한 수지상 세포로 변화하며, 보다 바람직하게는 TNF-α, IL-12p70, IL-6, IL-1ß 의 생성이 증가된 성숙 수지상 세포로 변화할 수 있다.
Matured dendritic cells promoted by RpfB protein treatment in the present invention can express general characteristics of mature dendritic cells. That is, according to the present invention, immature dendritic cells are differentiated into mature dendritic cells by treating RpfB with immature dendritic cells, and more preferably TNF-α, IL-12p70, IL-6 and IL- Can be converted to increased mature dendritic cells.
또한, 본 발명은 RpfB를 유효성분으로 포함하는 수지상 세포 성숙화 유도용 조성물을 포함하는 면역 증강용 조성물을 제공할 수 있다. 본 발명에 따르면 미성숙 수지상 세포에 RpfB를 처리함으로써 미성숙의 수지상 세포가 성숙되며, 수지상 세포의 성숙으로 표면 단백질 표시인자의 발현이 증가될 수 있다. 보다 구체적으로는 미성숙 수지상 세포에 RpfB를 처리함으로써 수지상 세포를 성숙시키고 결과적으로 T 세포의 활성을 유도하여 면역 반응을 증가시킬 수 있다. In addition, the present invention can provide a composition for immuno-enhancement comprising a composition for inducing dendritic cell maturation comprising RpfB as an active ingredient. According to the present invention, immature dendritic cells are matured by treating RpfB with immature dendritic cells, and the expression of surface protein markers can be increased by dendritic cell maturation. More specifically, immature dendritic cells can be treated with RpfB to mature dendritic cells and consequently induce T cell activity, thereby increasing the immune response.
수지상 세포의 성숙은 당해 기술분야에 공지된 방법으로 모니터할 수 있으며 세포 표면 마커를 유세포 분석기(flow cytometry) 및 면역조직화학법 등과 같은 당해 기술분야에 친숙한 검정으로 검출할 수 있다. 또한 상기 세포를 사이토카인 생성 (예, ELISA, FACS 및 다른 면역 검정)을 통해 모니터할 수 있다.
The maturation of dendritic cells can be monitored by methods known in the art and cell surface markers can be detected by assays familiar to the art, such as flow cytometry and immunohistochemistry. The cells can also be monitored via cytokine production (e.g., ELISA, FACS, and other immunoassays).
이하, 본 발명을 실시 예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예들은 본 발명을 더욱 쉽게 이해할 수 있도록 예시하는 것으로 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.
실시예 1. 수지상 세포의 분리 및 재조합 RpfB의 클로닝Example 1. Isolation of dendritic cells and cloning of recombinant RpfB
1. 1 수지상 세포의 분리 및 유도 1. Isolation and induction of dendritic cells
C57BL/6마우스로부터 골수 채취용 주사를 이용해 대퇴부 골수를 채취하였다. 채취한 골수를 세척한 후 적혈구를 염화암모늄을 이용하여 제거하였다. 분리한 세포를 6-웰 플레이트에서 RPMI 1640 (10% FBS(Fetal bovine serum, 송아지 혈청), 2 mM L-글루타민, 100 U/ml 페니실린/스트렙토마이신, 50μM 머캅토에탄올, 0.1 mM 비필수 아미노산, 1 mM 피루브산 나트륨, 20 ng/ml GM-CSF, 20 ng/ml IL-4)을 첨가하여 8 일 동안 배양하였다. GM-CSF 및 IL-4은 수지상 세포로의 분화를 유도하기 위하여 사용하였다.
Femur bone marrow was collected from C57BL / 6 mice using injection for bone marrow harvesting. The collected bone marrow was washed and the red blood cells were removed with ammonium chloride. Separated cells were resuspended in 6-well plates in RPMI 1640 (10% FBS (Fetal bovine serum, calf serum), 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 50 μM mercaptoethanol, 1 mM sodium pyruvate, 20 ng / ml GM-CSF, 20 ng / ml IL-4) for 8 days. GM-CSF and IL-4 were used to induce differentiation into dendritic cells.
1.2 재조합 RpfB의 클로닝1.2 Cloning of recombinant RpfB
결핵균 게놈 DNA로부터 RpfB를 분리하여 클로닝하였다. 정방향 프라이머 (5’-CATATGTGCAAAACGGTGACGTTGACC-3’, 서열번호 3) 및 역방향 프라이머 (5’-AAGCTTGCGCGCACCCGCTCGTGCAGC-3’, 서열번호 4)를 이용해 RpfB 부위를 증폭시켰다. PCR 산물을 NdeI 과 HindIII 효소들로 절단하고, 발현벡터인 pET-22b(+)벡터 (Novagen, Madison, WI)에 삽입하였다. RpfB 유전자가 삽입된 pET-22b(+) 벡터로 형질 전환시킨 E. coli BL21을 37℃에서 600 nm에서의 흡광도 (OD)가 0.4 내지 0.5가 되도록 배양한 후에 1 mM의 이소프로필-D-티오갈락토피라노사이드 (IPTG)를 첨가하고 6시간 동안 배양하였다. 발현된 단백질은 니켈-니트릴로트라이아세트산 (Ni-NTA, Invitrogen, Carlsbad, CA, USA) 아가로즈를 이용하여 제조사의 방법에 준하여 정제하였다. 최종적으로 정제한 재조합 단백질은 SDS-PAGE로 분석하여 확인하였다. RpfB was isolated from M. tuberculosis genomic DNA and cloned. The RpfB site was amplified using a forward primer (5'-CATATGTGCAAAACGGTGACGTTGACC-3 ', SEQ ID NO: 3) and a reverse primer (5'-AAGCTTGCGCGCACCCGCTCGTGCAGC-3', SEQ ID NO: 4). The PCR product was digested with NdeI and HindIII enzymes and inserted into the expression vector pET-22b (+) vector (Novagen, Madison, WI). E. coli BL21 transformed with the pET-22b (+) vector into which the RpfB gene was inserted was cultured at 37 DEG C at an absorbance (OD) at 600 nm of 0.4 to 0.5, followed by the addition of 1 mM isopropyl-D-thio Galactopyranoside (IPTG) was added and incubated for 6 hours. The expressed protein was purified using nickel-nitrilotriacetic acid (Ni-NTA, Invitrogen, Carlsbad, CA, USA) agarose according to the manufacturer's method. The final purified recombinant protein was identified by SDS-PAGE analysis.
결과를 도 1에 나타내었다.The results are shown in Fig.
도 1에서 나타낸 바와 같이, 재조합 RpfB 단백질은 37 kDa 위치에서 나타났다. As shown in Figure 1, the recombinant RpfB protein appeared at 37 kDa position.
실시예 2. RpfB의 세포 독성 확인Example 2. Cytotoxicity of RpfB
RpfB 를 처리한 수지상 세포의 세포 생존율을 측정하기 위해서, 수지상 세포를 0.5, 1, 2 μg/ml 농도의 RpfB 또는 대조군으로 100 ng/ml 농도의 LPS (lipopolysaccharide)로 24 시간 동안 처리하였다. 이 후 요오드화 프로피듐 (propidium iodide, PI) 및 아넥신-V로 이중 염색하고 유세포분석기 (flow cytometry)를 통해 분석하였다.To measure the cell viability of dendritic cells treated with RpfB, dendritic cells were treated with RpfB at a concentration of 0.5, 1, 2 μg / ml or LPS (lipopolysaccharide) at a concentration of 100 ng / ml as a control for 24 hours. Then, double staining with propidium iodide (PI) and annexin-V was performed and analyzed by flow cytometry.
결과는 도 2에 나타내었다. The results are shown in Fig.
도 2에 나타낸 바와 같이, 0.5μg/ml농도의 RpfB 뿐만 아니라 2 μg/ml의 RpfB 역시 수지상 세포에서 독성을 나타내지 않았다.
As shown in Fig. 2, not only RpfB at a concentration of 0.5 μg / ml but also RpfB at 2 μg / ml did not show toxicity in dendritic cells.
실시예 3. RpfB에 의한 수지상 세포의 표면 인자 발현 분석Example 3. Analysis of surface expression of dendritic cells by RpfB
먼저 모든 세포의 성숙도를 균일하게 하기 위하여 GM-CSF 와 IL-4가 함유된 OptiMEM 배지에서 유지한 후, 수지상 세포에 0.5, 1, 2 μg/ml농도의 RpfB 및 100 ng/ml 농도의 LPS를 각각 24시간 처리한 후 수지상 세포를 회수하였다. 비 특이적인 결합을 억제하기 위하여 1μg/ml의 Fc I/III(BD bioscience)을 4℃에서 20분간 처리한 후, 수지상 세포 분석을 위해 CD11c-FITC(BD bioscience)로 4℃에서 20분간 염색하였다. 이 후 항-CD80-PE(BD bioscience), 항-CD86-PE(BD bioscience), 항-MHC I 및 II-PE(BD bioscience)와 같은 세포 표면 인자 항체를 이용하여 염색한 후 유세포 분석기 FACs Canto (BD Biosciences)로 분석하였다. The cells were maintained in OptiMEM medium containing GM-CSF and IL-4 to homogenize the maturation of all cells, and RpfB and 100 ng / ml LPS at concentrations of 0.5, 1 and 2 μg / ml were added to the dendritic cells After each treatment for 24 hours, dendritic cells were recovered. To inhibit nonspecific binding, 1 μg / ml Fc I / III (BD bioscience) was treated at 4 ° C. for 20 minutes and then dyed with CD11c-FITC (BD bioscience) for 20 minutes at 4 ° C. for dendritic cell analysis . The cells were then stained with anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience) (BD Biosciences).
결과는 도3에 나타내었다.The results are shown in FIG.
도 3에 나타낸 바와 같이, RpfB는 수지상 세포의 단백질 표면 인자인 CD80, CD86과 MHC class I, MHC class II의 발현을 용량 의존적으로 증가시켰다. 이를 통해 RpfB에 의해 수지상 세포의 성숙이 효과적으로 이루어졌음을 확인할 수 있었다.
As shown in FIG. 3, RpfB dose-dependently increased the expression of CD80, CD86, MHC class I, and MHC class II, the protein surface factors of dendritic cells. This suggests that the dendritic cells were effectively maturated by RpfB.
실시예 4. RpfB 가 수지상 세포의 사이토카인 분비에 미치는 영향 분석 Example 4 . Effects of RpfB on the secretion of cytokines from dendritic cells
수지상 세포의 성숙 여부에 따라 분비되는 사이토카인의 종류가 달라진다. 따라서, 이를 확인하기 위해 수지상 세포에 0.5, 1, 2 μg/ml 농도의 RpfB 또는 대조군으로 100ng/ml의 LPS 를 처리한 후 24 시간 동안 배양하였다. 상층액을 TNF-α, IL-6, IL-1β, IL-10 및 IL-12 p70 에 대한 ELISA 키트에 적용하여 사이토카인 분비량을 측정하였다. The type of cytokine secreted depends on the maturation of dendritic cells. To confirm this, dendritic cells were treated with 0.5, 1, 2 μg / ml of RpfB or 100 ng / ml of LPS as a control group and cultured for 24 hours. The supernatant was applied to an ELISA kit for TNF-alpha, IL-6, IL-1 beta, IL-10 and IL-12 p70 to determine the amount of cytokine secreted.
IL-12 p70과 IL-10은 수지상 세포에서 분비되는 주요 사이토카인으로 각각 T 세포가 Th1과 Th2로 분화되는데 중요한 역할을 한다. IL-12는 대표적인 Th1형 사이토카인이며, IL-10은 활성화 된 단핵구, NK 세포 및 Th1 세포에 의해 생산되는 사이토카인 (IL-12 p70포함)의 활성을 방해하는 것으로 알려져 있다. 이러한 내용들을 바탕으로 세포내의 IL-12 p70과 IL-10의 분비량을 측정하기 위해 항-IL-12 p70-APC (BD bioscience)와 항-IL-10-APC를 염색하여 유세포 분석기 FACScallibur (Beckson Dikinson, USA)로 분석하였다. IL-12 p70 and IL-10 are major cytokines secreted from dendritic cells, and play important roles in differentiating T cells into Th1 and Th2, respectively. IL-12 is a typical Th1-type cytokine, and IL-10 is known to inhibit the activity of activated monocytes, NK cells, and cytokines (including IL-12 p70) produced by Th1 cells. IL-12 p70-APC (BD bioscience) and anti-IL-10-APC were stained with FACScallibur (Beckson Dikinson , USA).
결과는 도 4에 나타내었다. The results are shown in Fig.
도 4에 나타낸 바와 같이, IL-12 p70이 RpfB 용량 의존적으로 뚜렷하게 증가하였고, 전염증 사이토카인인 TNF-α, IL-6, IL-1β 또한 뚜렷하게 증가하였다. 따라서, RpfB이 수지상 세포를 성숙하게 하여 사이토카인의 양에 변화를 준다는 것을 알 수 있다. 반면 IL-10의 분비에는 유의성 있는 증가를 보이지 않아, RpfB이 수지상 세포를 Th1형 세포로 편향할 수 있는 가능성을 확인할 수 있었다.
As shown in FIG. 4, IL-12 p70 was significantly increased in an RpfB dose-dependent manner, and the proinflammatory cytokines TNF-a, IL-6 and IL-1β were also significantly increased. Thus, it can be seen that RpfB matures dendritic cells and changes the amount of cytokine. On the other hand, there was no significant increase in the secretion of IL-10, confirming the possibility that RpfB could deflect dendritic cells into Th1 cells.
실시예 5. 수지상 세포의 TNF-a 및 IL-6 방출에 대한 열변성, 프로테인아제 K 소화 및 Example 5: Thermal denaturation of TNF-a and IL-6 release of dendritic cells, proteinase K digestion and
PMBPMB
에 대한 영향 분석Impact Analysis
5.1 열변성 영향 분석 5.1 Thermal Degeneration Impact Analysis
LPS 및 RpfB에 의해 유도되는 수지상 세포의 TNF-α 및 IL-6 방출에 대한 열변성의 영향을 분석하기 위해, C57BL/6 마우스의 미성숙 수지상 세포를 100 ng/ml의 LPS 또는 2 μg/ml의 RpfB, 또는 끓인 LPS 또는 RpfB로 처리하거나, 처리하지 않았다. 24시간 후에 상기 수지상 세포의 상층액내 TNF-α, IL-6의 양을 ELISA를 이용하여 분석하였다. In order to analyze the effect of the thermodynamic effects of TNF-α and IL-6 release on dendritic cells induced by LPS and RpfB, immature dendritic cells of C57BL / 6 mice were treated with 100 ng / ml LPS or 2 μg / ml RpfB , Or treated with boiled LPS or RpfB, or not treated. After 24 hours, the amount of TNF-a and IL-6 in the supernatant of the dendritic cells was analyzed by ELISA.
결과는 도 5A에 나타내었다. The results are shown in Figure 5A.
도 5A에 나타낸 바와 같이, RpfB은 단백질이므로 끓이면 분해되기 때문에 끓인 RpfB의 경우 TNF-α, IL-6의 분비가 억제되었다. 반면, LPS의 경우에는 끓이더라도 TNF-α, IL-6의 분비에 차이가 없었다.
As shown in FIG. 5A, since RpfB is a protein, RpfB inhibited the secretion of TNF-.alpha. And IL-6 in the case of boiled RpfB because it decomposes when boiled. On the other hand, in the case of LPS, there was no difference in secretion of TNF-α and IL-6 even when boiled.
5.2 프로테아제 K 영향 분석 5.2 Analysis of Protease K Effect
또한, 100 ng/ml LPS 또는 2 μg/ml RpfB에 프로테인아제 K (Protease K)를 처리한 후 미성숙 수지상 세포의 배양물에 첨가하였다. 프로테아제 K의 농도를 각각 LPS와 RpfB 농도의 1/20로 처리한 후 50에서 1시간 활성화시키고 100℃에서 비활성화시켰다. 상기 수지상 세포의 상층액내 TNF-α, IL-6양을 ELISA를 이용하여 분석하였다. In addition, 100 ng / ml LPS or 2 μg / ml RpfB was treated with proteinase K and then added to the culture of immature dendritic cells. The protease K concentration was 1/20 of the LPS and RpfB concentrations, respectively, then activated at 50 for 1 hour and deactivated at 100 ° C. The amount of TNF-α and IL-6 in the supernatant of the dendritic cells was analyzed by ELISA.
결과를 도 5B에 나타내었다. The results are shown in Fig. 5B.
도 5B에 나타낸 바와 같이, RpfB는 단백질이므로 단백질 분해효소인 프로테아제 K로 처리하면 분해되기 때문에 프로테아제 K를 처리한 RpfB 처리 수지상 세포의 경우 TNF-α, IL-6의 분비가 억제되었다. 반면 LPS의 경우에는 프로테아제 K를 처리하여도 TNF-α, IL-6의 분비에 차이가 없었다.
As shown in FIG. 5B, since RpfB is a protein, decomposition is caused by protease K, which is proteolytic enzyme, and thus the secretion of TNF-α and IL-6 is suppressed in RpfB-treated dendritic cells treated with protease K. On the other hand, in the case of LPS, there was no difference in the secretion of TNF-α and IL-6 by protease K treatment.
5.3 LPS 억제제를 이용한 LPS 오염 영향 여부 확인5.3 Confirmation of LPS contamination by using LPS inhibitor
RpfB에 의한 수지상 세포 활성화의 효과가 LPS의 오염에 의한 것이 아닌지 확인하기 위해, C57BL/6 마우스의 미성숙 수지상 세포를 LPS 억제제인 폴리믹신 B (polymyxin B, PMB)로 전처리한 후 또는 전처리 없이 100ng/ml LPS 또는 10 μg/ml RpfB로 24시간 동안 처리하였다. 24시간 후에 상기 수지상 세포의 상층액내 TNF-α, IL-6, IL-1β의 양을 ELISA를 이용하여 분석하였다.To confirm whether the effect of dendritic cell activation by RpfB was due to contamination of LPS, immature dendritic cells of C57BL / 6 mice were pretreated with LPS inhibitor polymyxin B (PMB) ml LPS or 10 μg / ml RpfB for 24 hours. After 24 hours, the amount of TNF-α, IL-6, and IL-1β in the supernatant of the dendritic cells was analyzed by ELISA.
결과는 도 5C에 나타내었다. The results are shown in Figure 5C.
도 5C 에 나타낸 바와 같이, LPS의 경우 LPS에 의한 TNF-α, IL-6의 분비가 폴리믹신 B에 의해 감소되는 반면, RpfB의 경우 폴리믹신 B를 처리하더라도 TNF-α, IL-6의 분비에는 영향이 없었다. 이는 RpfB에 의한 수지상 세포 활성화의 효과가 LPS 오염에 의한 것이 아님을 보여주었다.
As shown in FIG. 5C, the secretion of TNF-α and IL-6 by LPS in the case of LPS is reduced by polyamicin B, whereas the secretion of TNF-α and IL-6 by RMP- . This indicates that the effect of dendritic cell activation by RpfB is not due to LPS contamination.
실시예 6. RpfB에 의한 수지상세포의 항원 취득 능력 분석Example 6. Analysis of ability of dendritic cells to acquire antigen by RpfB
미성숙 수지상세포는 항원취득을 잘하지만, 성숙한 수지상세포는 항원취득 능력이 감소한다고 알려져 있다. 따라서 RpfB 단백질이 수지상세포의 대식 활동 (endocytic activity)에 미치는 영향을 확인하기 위한 실험을 수행하였다. 미성숙 수지상 세포를 0.5, 1, 2 μg/ml의 RpfB 또는 100 ng/ml LPS를 처리한 후 24시간 동안 배양하였다. 배양된 수지상 세포에 1μg/ml의 플루오레신 결합 덱스트란(fluoresceinconjugated dextran, 분자량 40,000; Molecular Probes, Eugene, OR)을 1시간 동안 첨가하였다. 37℃와 4℃ 에서 각각 30분 동안 배양한 후 콜드 스테이닝 완충액을 첨가하여 반응을 정지시킨 후 세포를 세척하였다. 이 후 PE (Phycoerythrin)-결합 CD11c (BD bioscience)로 염색한 후에 유세포분석기 FACs Canto (BD Biosciences)를 이용하여 덱스트란-FITC(Fluorescein isothiocyanate) 탐식능을 측정하였다. Immature dendritic cells are good for antigen acquisition, but mature dendritic cells are known to decrease antigen acquisition capacity. Therefore, an experiment was conducted to confirm the effect of RpfB protein on dendritic cell endocytic activity. Immature dendritic cells were treated with 0.5, 1, 2 μg / ml RpfB or 100 ng / ml LPS and cultured for 24 hours. Fluorescein conjugated dextran (molecular weight 40,000; Molecular Probes, Eugene, OR) was added to the cultured dendritic cells at 1 ug / ml for 1 hour. After incubation at 37 ° C and 4 ° C for 30 minutes, the cells were washed with cold staining buffer to stop the reaction. After staining with PE (Phycoerythrin) -binding CD11c (BD bioscience), dextran-FITC (Fluorescein isothiocyanate) staining was measured using a flow cytometer FACs Canto (BD Biosciences).
결과는 도 6에 나타내었다. The results are shown in Fig.
도 6에 나타낸 바와 같이, 37℃에서 RpfB를 처리한 수지상 세포가 RpfB를 처리하지 않은 수지상 세포보다 덱스트란(항원) 포식 능력이 감소하였다. 이러한 결과에 따라, RpfB이 기능적인 면에서 수지상 세포를 성숙시킴을 알 수 있었다. 덱스트란의 수지상 세포에 대한 비특이적 결합을 측정하기 위해서 4 ℃에서도 측정하여 결과값을 보정하였다. 이를 통해 RpfB 처리에 의해 수지상세포의 성숙도가 증가하였음을 기능적으로 확인할 수 있었다.
As shown in Fig. 6, dendritic (antigen) predisposing ability of dendritic cells treated with RpfB at 37 占 폚 was lower than that of dendritic cells not treated with RpfB. These results suggest that RpfB matures DCs in terms of function. In order to measure the non-specific binding of dextran to dendritic cells, the result was also measured at 4 ° C. This confirms that the dendritic cell maturation was increased by RpfB treatment.
실시예 7.Example 7. RpfB에 의한 수지상 세포의 신호전달계 활성화Activation of dendritic cell signaling system by RpfB
수지상세포는 성숙과정 중에 ERK, JNK, p38 등의 MAPK(mitogenactivated protein kinases) 및 NF-κB(nuclear factor-kappa B) 신호전달계가 활성화된다고 알려져 있다. 따라서, 미성숙 수지상 세포에 RpfB 2μg/ml을 0분 5 분, 15 분, 30 분, 60 분간 처리한 후, 세포를 파괴하여 단백질을 모두 분리하고, MAPK (mitogen-activated protein kinases) 및 NF-κB (nuclear factor-kappa B) 신호전달계와 관련된 인자인 ERK, JNK, p38, IκB-α, NF-B p65 의 인산화 및 분해 여부를 확인하였다. 단백질양은 특정 항체를 이용하는 웨스턴 블랏 방법으로 측정하였다.It is known that dendritic cells activate MAPK (mitogen activated protein kinases) such as ERK, JNK, p38 and NF-κB (nuclear factor-kappa B) signal transduction systems during maturation. Thus, immature dendritic cells were treated with 2 μg / ml of RpfB for 0 min, 5 min, 15 min, 30 min and 60 min, followed by cell disruption to isolate all proteins, followed by mitogen-activated protein kinases (MAPK) phosphorylation and degradation of ERK, JNK, p38, IκB-α, and NF-B p65, which are related to the nuclear factor-kappa B signaling pathway, The amount of protein was measured by a Western blotting method using a specific antibody.
결과는 도 7에 나타내었다. The results are shown in Fig.
도 7에서 나타낸 바와 같이, RpfB 처리에 의해 인산화되어 있는 p-p38, p-ERK, p-JNK의 양이 증가하였음을 확인하였다. 또한 p-IκB-a가 증가하고 IκB-a는 분해되는 것을 확인하였다. 최종 신호전달 인자인 NF-κB p65도 RpfB 처리에 의해 핵 안으로 전사된 것을 확인하였다. 이를 통해 RpfB는 수지상세포의 MAPKs 및 NF-κB 신호 전달 체계에 관여한다는 것을 확인할 수 있었다.
As shown in FIG. 7, it was confirmed that the amounts of p-p38, p-ERK and p-JNK phosphorylated by RpfB treatment were increased. It was also confirmed that p-IκB-a increased and IκB-a degraded. The final signal transduction factor, NF-κB p65, was also confirmed to be transferred into the nucleus by RpfB treatment. This suggests that RpfB is involved in the MAPKs and NF-κB signaling pathways of dendritic cells.
실시예 8. RpfB에 의한 수지상 세포의 활성에 있어 MAPK 및 NF-B 의 영향 분석Example 8. Effect of MAPK and NF-B on the activity of dendritic cells by RpfB
RpfB에 의해 활성화된 수지상세포가 MAPK 및 NF-κB의 신호 전달 체계에 의해 활성화 유도되었는지를 알아보기 위하여, U0126 (ERK1/2), SB203580 (p38), SP600125 (JNK), Bay 11-0782 (NF-κB)와 같은 MAPK 및 NF-κB 억제 물질을 이용하여 분석하였다. 구체적으로는, 수지상 세포에 MAPK 및 NF-κB 억제 물질을 1시간 동안 전 처리한 후 2 μg/ml 농도의 RpfB 또는 대조군으로 100ng/ml의 LPS 를 처리한 후 24 시간 동안 배양하였다. 수지상세포의 표면인자 발현 영향분석을 위해 CD11c-FITC (ebioscience)로 4℃에서 30분간 염색하였다. 이 후, 항-CD80-PE (ebioscience), 항-CD86-PE (ebioscience)와 같은 세포 표면인자 항체를 이용하여 염색한 후 유세포분석기 FACs Canto (BD Biosciences)로 분석하였다. 또한 수지상 세포의 성숙 여부에 따라 분비되는 사이토카인을 분석하기 위해 상층액을 통하여 TNF-α, IL-6, IL-1β에 대한 ELISA 키트 (ebioscience)를 적용하여 사이토카인 분비량을 측정하였으며, MAPK 및 NF-κB 억제 물질을 이용하여 핵 안에서의 NF-κB p65 발현양을 측정하였다.(ERK1 / 2), SB203580 (p38), SP600125 (JNK), and Bay 11-0782 (NF-κB) in order to investigate whether dendritic cells activated by RpfB were activated by MAPK and NF- -KB) and MAPK and NF-κB inhibitors. Specifically, dendritic cells were pretreated with MAPK and NF-κB inhibitor for 1 hour and then treated with 2 μg / ml of RpfB or 100 ng / ml of LPS as control, followed by incubation for 24 hours. To analyze the effect of surface expression of dendritic cells on the surface, the cells were stained with CD11c-FITC (ebioscience) at 4 ° C for 30 minutes. The cells were then stained with anti-CD80-PE (ebioscience), anti-CD86-PE (ebioscience), and analyzed with a flow cytometer FACs Canto (BD Biosciences). In addition, cytokine secretion was measured by ELISA kit (ebioscience) for TNF-α, IL-6 and IL-1β through the supernatant to analyze secreted cytokines according to the maturation of dendritic cells. The amount of NF-κB p65 expression in the nucleus was measured using an NF-κB inhibitor.
결과를 도 8에 나타내었다[(A) 유세포분석기 FACs Canto 분석 결과, (B) ELISA 키트 분석 결과, (C) 핵 안에서의 NF-κB p65 발현양 측정 결과].The results are shown in FIG. 8. [(A) Results of FACs Canto analysis of flow cytometry, (B) Analysis of ELISA kit, (C) Results of measurement of NF-κB p65 expression in the nucleus.
도 8에 나타낸 바와 같이, MAPK 및 NF-κB 억제 물질들은 RpfB에 의한 수지상 세포의 보조자극인자인 CD80과 CD86 발현을 감소시켰고(A), 염증성 사이토카인인 TNF-α, IL-6, IL-1β들을 감소시켰으며(B), 핵에서의 NF-B p65 발현양을 뚜렷하게 감소시켰다(C). As shown in FIG. 8, MAPK and NF-κB inhibitors decreased CD80 and CD86 expression, which are complementary stimulants of dendritic cells induced by RpfB (A), and inflammatory cytokines TNF-α, IL- (B) and markedly decreased the amount of NF-B p65 expression in the nucleus (C).
이를 통해 RpfB에 의한 수지상세포의 활성은 MAPKs (ERK1/2, p38, JNK) 및 NF-κB 신호 전달 체계에 의존적이며, 이러한 신호 전달 체계에 의하여 전 염증성 사이토카인의 생성을 유도한다는 것을 확인하였다.
These results demonstrate that dendritic cell activation by RpfB is dependent on MAPKs (ERK1 / 2, p38, JNK) and the NF-κB signaling pathway and induces the production of proinflammatory cytokines by these signaling pathways.
실시예Example 9. 9. RpfBRpfB 에 의한 On by 수지상세포의Dendritic T 세포 활성 분석 T cell activity assay
성숙 수지상 세포의 생체 내에서의 가장 중요한 특징은 T 림프구의 활성을 유도하는 것이다. RpfB 로 성숙이 유도된 수지상 세포가 항원 특이적 세포독성 T 림프구 (Cytotoxic T Lymphocyte, CTL) 반응을 강화하고, Th1형 세포로의 분화에 효과적이라는 것을 밝히기 위하여 실험을 수행하였다. 활성화된 T 림프구는 증식 속도가 증가하며 인터페론 감마의 분비가 증가되어 주변의 다른 T 림프구의 활성을 돕고 면역 작용을 활발하게 일으켜 항원을 제거한다. 오브알부민 형질전환(Ovalbumin transgenic) 마우스인 OT-I과 OT-II 마우스 (BALB/c 마우스)의 비장으로부터 분리된 CD4+및 CD8+T세포를 C57BL/6로부터 유래된 RpfB 처리 수지상 세포와 함께 배양한 후 T 세포의 증식 정도를 측정하였다. 또한, T 세포에서 분비하는 사이토카인(IFN-, IL-2, IL-4)의 분비량을 ELISA 키트(BD bioscience)를 이용하여 분석하였다.The most important characteristic of mature dendritic cells in vivo is the induction of T lymphocyte activity. Experiments were conducted to demonstrate that dendritic cells induced by RpfB enhance antigen - specific cytotoxic T lymphocyte (CTL) responses and are effective at differentiating into Th1 - type cells. Activated T lymphocytes increase the rate of proliferation and increase the secretion of interferon gamma, which helps the activity of other T lymphocytes in the surrounding area and actively removes the antigen. CD4 + and CD8 + T cells isolated from the spleen of ovalbumin transgenic mice OT-I and OT-II mice (BALB / c mice) were cultured with RpfB-treated dendritic cells derived from C57BL / 6 The degree of proliferation of T cells was measured. In addition, the secretion amount of cytokines (IFN-, IL-2, IL-4) secreted from T cells was analyzed using an ELISA kit (BD bioscience).
결과를 도 9에 나타내었다.The results are shown in Fig.
도 9에 나타낸 바와 같이, RpfB로 처리한 경우 대조군에 비해 T 세포의 증식 정도가 높은 것을 확인할 수 있었다. 이를 통해, RpfB는 수지상세포를 성숙시켜 T 세포의 증식에 의한 면역 활성 능력을 증가시킬 수 있음을 알 수 있었다(A). 또한 T세포에서 분비하는 사이토카인(IFN-γ, IL-2, IL-4) 분비량을 통해 RpfB 처리가 세포를 Th1형 세포로 편향적으로 분화되도록 함을 알 수 있었다(B). 앞서 실시예 4에서 확인한 바와 같이 RpfB 단백질 처리에 의하면 IL-12p70 등의 Th1형 사이토카인의 분비가 편향되어 증가되므로, 이러한 결과들을 종합하면, RpfB로 처리하여 성숙된 수지상 세포는 T 세포를 Th1형 세포로 분화되도록 유도할 수 있으며, 이를 통해 면역 활성을 증가시킬 수 있음을 알 수 있었다. As shown in Fig. 9, it was confirmed that when treated with RpfB, the degree of T cell proliferation was higher than that of the control group. This suggests that RpfB can increase immune activation by T cell proliferation by matured dendritic cells (A). In addition, the secretion of cytokines (IFN-γ, IL-2, and IL-4) secreted from T cells revealed that RpfB treatment biased cells differently into Th1 cells (B). As confirmed in Example 4 above, the secretion of Th1-type cytokines, such as IL-12p70, is increased by RpfB protein treatment. Taken together, these results suggest that mature dendritic cells treated with RpfB can induce T- Cell differentiation, thereby increasing the immune activity.
<110> Industry-Academic Cooperation Foundation, Yonsei University Industry & Academic Cooperation in Chungnam National University (IAC) <120> Compositon for inducing maturation of dendritic cell comprising Mycobacterium tuberculosis RpfB <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1089 <212> DNA <213> Mycobacterium tuberculosis <400> 1 atgttgcgcc tggtagtcgg tgcgctgctg ctggtgttgg cgttcgccgg tggctatgcg 60 gtcgccgcat gcaaaacggt gacgttgacc gtcgacggaa ccgcgatgcg ggtgaccacg 120 atgaaatcgc gggtgatcga catcgtcgaa gagaacgggt tctcagtcga cgaccgcgac 180 gacctgtatc ccgcggccgg cgtgcaggtc catgacgccg acaccatcgt gctgcggcgt 240 agccgtccgc tgcagatctc gctggatggt cacgacgcta agcaggtgtg gacgaccgcg 300 tcgacggtgg acgaggcgct ggcccaactc gcgatgaccg acacggcgcc ggccgcggct 360 tctcgcgcca gccgcgtccc gctgtccggg atggcgctac cggtcgtcag cgccaagacg 420 gtgcagctca acgacggcgg gttggtgcgc acggtgcact tgccggcccc caatgtcgcg 480 gggctgctga gtgcggccgg cgtgccgctg ttgcaaagcg accacgtggt gcccgccgcg 540 acggccccga tcgtcgaagg catgcagatc caggtgaccc gcaatcggat caagaaggtc 600 accgagcggc tgccgctgcc gccgaacgcg cgtcgtgtcg aggacccgga gatgaacatg 660 agccgggagg tcgtcgaaga cccgggggtt ccggggaccc aggatgtgac gttcgcggta 720 gctgaggtca acggcgtcga gaccggccgt ttgcccgtcg ccaacgtcgt ggtgaccccg 780 gcccacgaag ccgtggtgcg ggtgggcacc aagcccggta ccgaggtgcc cccggtgatc 840 gacggaagca tctgggacgc gatcgccggc tgtgaggccg gtggcaactg ggcgatcaac 900 accggcaacg ggtattacgg tggtgtgcag tttgaccagg gcacctggga ggccaacggc 960 gggctgcggt atgcaccccg cgctgacctc gccacccgcg aagagcagat cgccgttgcc 1020 gaggtgaccc gactgcgtca aggttggggc gcctggccgg tatgtgctgc acgagcgggt 1080 gcgcgctga 1089 <210> 2 <211> 362 <212> PRT <213> Mycobacterium tuberculosis <400> 2 Met Leu Arg Leu Val Val Gly Ala Leu Leu Leu Val Leu Ala Phe Ala 1 5 10 15 Gly Gly Tyr Ala Val Ala Ala Cys Lys Thr Val Thr Leu Thr Val Asp 20 25 30 Gly Thr Ala Met Arg Val Thr Thr Met Lys Ser Arg Val Ile Asp Ile 35 40 45 Val Glu Glu Asn Gly Phe Ser Val Asp Asp Arg Asp Asp Leu Tyr Pro 50 55 60 Ala Ala Gly Val Gln Val His Asp Ala Asp Thr Ile Val Leu Arg Arg 65 70 75 80 Ser Arg Pro Leu Gln Ile Ser Leu Asp Gly His Asp Ala Lys Gln Val 85 90 95 Trp Thr Thr Ala Ser Thr Val Asp Glu Ala Leu Ala Gln Leu Ala Met 100 105 110 Thr Asp Thr Ala Pro Ala Ala Ala Ser Arg Ala Ser Arg Val Pro Leu 115 120 125 Ser Gly Met Ala Leu Pro Val Val Ser Ala Lys Thr Val Gln Leu Asn 130 135 140 Asp Gly Gly Leu Val Arg Thr Val His Leu Pro Ala Pro Asn Val Ala 145 150 155 160 Gly Leu Leu Ser Ala Ala Gly Val Pro Leu Leu Gln Ser Asp His Val 165 170 175 Val Pro Ala Ala Thr Ala Pro Ile Val Glu Gly Met Gln Ile Gln Val 180 185 190 Thr Arg Asn Arg Ile Lys Lys Val Thr Glu Arg Leu Pro Leu Pro Pro 195 200 205 Asn Ala Arg Arg Val Glu Asp Pro Glu Met Asn Met Ser Arg Glu Val 210 215 220 Val Glu Asp Pro Gly Val Pro Gly Thr Gln Asp Val Thr Phe Ala Val 225 230 235 240 Ala Glu Val Asn Gly Val Glu Thr Gly Arg Leu Pro Val Ala Asn Val 245 250 255 Val Val Thr Pro Ala His Glu Ala Val Val Arg Val Gly Thr Lys Pro 260 265 270 Gly Thr Glu Val Pro Pro Val Ile Asp Gly Ser Ile Trp Asp Ala Ile 275 280 285 Ala Gly Cys Glu Ala Gly Gly Asn Trp Ala Ile Asn Thr Gly Asn Gly 290 295 300 Tyr Tyr Gly Gly Val Gln Phe Asp Gln Gly Thr Trp Glu Ala Asn Gly 305 310 315 320 Gly Leu Arg Tyr Ala Pro Arg Ala Asp Leu Ala Thr Arg Glu Glu Gln 325 330 335 Ile Ala Val Ala Glu Val Thr Arg Leu Arg Gln Gly Trp Gly Ala Trp 340 345 350 Pro Val Cys Ala Ala Arg Ala Gly Ala Arg 355 360 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 catatgtgca aaacggtgac gttgacc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 aagcttgcgc gcacccgctc gtgcagc 27 <110> Industry-Academic Cooperation Foundation, Yonsei University Industry & Academic Cooperation in Chungnam National University (IAC) <120> Compositon for inducing maturation of dendritic cell Mycobacterium tuberculosis RpfB <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 1089 <212> DNA <213> Mycobacterium tuberculosis <400> 1 atgttgcgcc tggtagtcgg tgcgctgctg ctggtgttgg cgttcgccgg tggctatgcg 60 gtcgccgcat gcaaaacggt gacgttgacc gtcgacggaa ccgcgatgcg ggtgaccacg 120 atgaaatcgc gggtgatcga catcgtcgaa gagaacgggt tctcagtcga cgaccgcgac 180 gacctgtatc ccgcggccgg cgtgcaggtc catgacgccg acaccatcgt gctgcggcgt 240 agccgtccgc tgcagatctc gctggatggt cacgacgcta agcaggtgtg gacgaccgcg 300 tcgacggtgg acgaggcgct ggcccaactc gcgatgaccg acacggcgcc ggccgcggct 360 tctcgcgcca gccgcgtccc gctgtccggg atggcgctac cggtcgtcag cgccaagacg 420 gtgcagctca acgacggcgg gttggtgcgc acggtgcact tgccggcccc caatgtcgcg 480 gggctgctga gtgcggccgg cgtgccgctg ttgcaaagcg accacgtggt gcccgccgcg 540 acggccccga tcgtcgaagg catgcagatc caggtgaccc gcaatcggat caagaaggtc 600 accgagcggc tgccgctgcc gccgaacgcg cgtcgtgtcg aggacccgga gatgaacatg 660 agccgggagg tcgtcgaaga cccgggggtt ccggggaccc aggatgtgac gttcgcggta 720 gctgaggtca acggcgtcga gaccggccgt ttgcccgtcg ccaacgtcgt ggtgaccccg 780 gcccacgaag ccgtggtgcg ggtgggcacc aagcccggta ccgaggtgcc cccggtgatc 840 gcggaagca tctgggacgc gatcgccggc tgtgaggccg gtggcaactg ggcgatcaac 900 accggcaacg ggtattacgg tggtgtgcag tttgaccagg gcacctggga ggccaacggc 960 gggctgcggt atgcaccccg cgctgacctc gccacccgcg aagagcagat cgccgttgcc 1020 gaggtgaccc gactgcgtca aggttggggc gcctggccgg tatgtgctgc acgagcgggt 1080 gcgcgctga 1089 <210> 2 <211> 362 <212> PRT <213> Mycobacterium tuberculosis <400> 2 Met Leu Arg Leu Val Val Gly Ala Leu 1 5 10 15 Gly Gly Tyr Ala Val Ala Cys Lys Thr Val Thr Leu Thr Val Asp 20 25 30 Gly Thr Ala Met Arg Val Thr Thr Met Lys Ser Arg Val Ile Asp Ile 35 40 45 Val Glu Glu Asn Gly Phe Ser Val Asp Asp Arg Asp Asp Leu Tyr Pro 50 55 60 Ala Ala Gly Val Gln Val His Asp Ala Asp Thr Ile Val Leu Arg Arg 65 70 75 80 Ser Arg Pro Leu Gln Ile Ser Leu Asp Gly His Asp Ala Lys Gln Val 85 90 95 Trp Thr Thr Ala Ser Thr Val Asp Glu Ala Leu Ala Gln Leu Ala Met 100 105 110 Thr Asp Thr Ala Pro Ala Ala Ala Ser Ala Ser Ala Val Val Leu 115 120 125 Ser Gly Met Ala Leu Pro Val Val Ser Ala Lys Thr Val Gln Leu Asn 130 135 140 Asp Gly Gly Leu Val Arg Thr Val His Leu Pro Ala Pro Asn Val Ala 145 150 155 160 Gly Leu Leu Ser Ala Ala Gly Val Pro Leu Leu Gln Ser Asp His Val 165 170 175 Val Pro Ala Ala Thr Ala Pro Ile Val Glu Gly Met Gln Ile Gln Val 180 185 190 Thr Arg Asn Arg Ile Lys Lys Val Thr Glu Arg Leu Pro Leu Pro Pro 195 200 205 Asn Ala Arg Arg Val Glu Asp Pro Glu Met Asn Met Ser Arg Glu Val 210 215 220 Val Glu Asp Pro Gly Val Gly Thr Gln Asp Val Thr Phe Ala Val 225 230 235 240 Ala Glu Val Asn Gly Val Glu Thr Gly Arg 245 250 255 Val Val Thr Pro Ala His Glu Ala Val Val Arg Val Gly Thr Lys Pro 260 265 270 Gly Thr Glu Val Pro Pro Val Ile Asp Gly Ser Ile Trp Asp Ala Ile 275 280 285 Ala Gly Cys Glu Ala Gly Gly Asn Trp Ala Ile Asn Thr Gly Asn Gly 290 295 300 Tyr Tyr Gly Gly Val Gln Phe Asp Gln Gly Thr Trp Glu Ala Asn Gly 305 310 315 320 Gly Leu Arg Tyr Ala Pro Arg Ala Asp Leu Ala Thr Arg Glu Glu Gln 325 330 335 Ile Ala Val Ala Glu Val Thr Arg Leu Arg Gln Gly Trp Gly Ala Trp 340 345 350 Pro Val Cys Ala Ala Arg Ala Gly Ala Arg 355 360 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 catatgtgca aaacggtgac gttgacc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 aagcttgcgc gcacccgctc gtgcagc 27
Claims (8)
A composition for inducing maturation of dendritic cells comprising RpfB protein as an active ingredient.
The method of claim 1, wherein the RpfB protein is Mycobacterium tuberculosis , and a composition for inducing maturation of dendritic cells.
The composition for inducing maturation of dendritic cells according to claim 1 or 2, wherein the RpfB protein is contained at a concentration of 0.1 μg / ml to 5 μg / ml.
A method for inducing maturation of immature dendritic cells, characterized in that immature dendritic cells isolated from human body are treated with RpfB to differentiate into mature dendritic cells.
5. The method according to claim 4, wherein the immature dendritic cells are cultured for 12 to 36 hours after RpfB treatment.
5. The method of claim 4, wherein the RpfB increases the production of TNF-a, IL-12p70, IL-6 and IL-l [beta] during the maturation of immature dendritic cells.
5. The method according to claim 4, wherein the RpfB protein is treated at a concentration of 0.1 μg / ml to 5 μg / ml.
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| KR20090017928A (en) * | 2007-08-16 | 2009-02-19 | 동아대학교 산학협력단 | Composition for inducing maturation of dendritic cell comprising 3-bromo-fascaplysine or 10-bromo-fascaplysine |
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| KR20090017928A (en) * | 2007-08-16 | 2009-02-19 | 동아대학교 산학협력단 | Composition for inducing maturation of dendritic cell comprising 3-bromo-fascaplysine or 10-bromo-fascaplysine |
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