KR20200064865A - Screening methods for therapeutic agents of arthritis through cytokine analysis - Google Patents
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Abstract
Description
본 발명은 사이토카인 분석을 통한 관절염 치료제 스크리닝 방법에 관한 것으로, 보다 상세하게는 관절염과 관련된 사이토카인의 분비 패턴 분석 결과를 바탕으로 처리 물질에 의한 관절염 치료 가능성을 예측하는 방법에 관한 것이다.The present invention relates to a method for screening for the treatment of arthritis through cytokine analysis, and more particularly, to a method for predicting the possibility of arthritis treatment by a treatment substance based on the analysis result of a cytokine secretion pattern related to arthritis.
관절염과 관련된 사이토카인은 많은 연구가 진행되어 있는 분야이며, 염증을 조절할 수 있는 사이토카인의 균형은 관련 질병을 치유하는데 있어서 중요한 요소이다.Cytokines related to arthritis have been studied a lot, and the balance of cytokines that can control inflammation is an important factor in healing related diseases.
특히, 최근 연구결과에서는 질병을 치료하기 위한 전략으로 염증을 억제하는데 치중하기 보다 면역반응을 조절할 수 있는 염증조절 사이토카인의 균형에 초점을 맞추는 방법들을 제시하고 있다.In particular, recent research suggests methods to focus on the balance of inflammatory regulatory cytokines that can regulate the immune response rather than focus on suppressing inflammation as a strategy for treating disease.
구체적으로, 상기 연구결과에서는 관절염에서 염증조절 사이토카인으로 IL-4, IL-5, IL-13을 사용하는 방법, IL-9과 IL-33을 사용하는 방법, IL-27, IL-10을 사용하는 방법에 의한 관절염 치료 가능성을 제안하고 있다(Chen Z. et al. Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis.Nat Rev Rheumatol. 2018 Oct 19.).Specifically, in the above study results, IL-4, IL-5, and IL-13 are used as inflammation control cytokines in arthritis, IL-9 and IL-33 are used, and IL-27 and IL-10 are used. The possibility of treating arthritis by the method used is proposed (Chen Z. et al. Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis. Nat Rev Rheumatol. 2018 Oct 19.).
또 다른 연구 결과는, 인터페론 감마(Interferon gamma, IFN-γ)에 의하여 관절염 진행에 있어서 중요한 과정인 파골세포형성(osteoclastogenesis)을 억제할 수 있는 기전을 제시하고 있다(Tang M. et al. Interferon-Gamma-Mediated Osteoimmunology. Front Immunol. 2018 Jun 29;9:1508.).Another study suggests a mechanism by which interferon gamma (IFN-γ) can inhibit osteoclastogenesis, an important process in the development of arthritis (Tang M. et al. Interferon- Gamma-Mediated Osteoimmunology.Front Immunol. 2018 Jun 29;9:1508.).
한편, IL-6와 IL-21은 관절염 진행에 관여하는 사이토카인으로 매우 잘 알려져 있다(Roeleveld DM. et al. Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis.PLoS One. 2017 Feb 3;12(2).).Meanwhile, IL-6 and IL-21 are very well known cytokines involved in the progression of arthritis (Roeleveld DM. et al. Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis.PLoS One. 2017 Feb 3;12(2).).
그러나 상기 연구 결과들은 관절염에 있어 사이토카인의 작용들을 제시하고 있을 뿐, 관련 사이토카인 분비 패턴 분석을 통한 관절염 치료 효능 예측 방법에 대해서는 제시하고 있지 않다.However, the results of the above study only show the actions of cytokines in arthritis, and not how to predict the efficacy of arthritis treatment through analysis of related cytokine secretion patterns.
특히, 염증성 사이토카인인 IL-6, IL-21와 염증조절 사이토카인인 IFN-γ, IL-27, IL-10의 분비 패턴 분석을 통하여 관절염 치료 효능을 예측하는 방법에 관해서는 연구되어진 바 없다.In particular, the method of predicting the efficacy of arthritis treatment by analyzing the secretion patterns of inflammatory cytokines IL-6, IL-21 and inflammatory regulatory cytokines IFN-γ, IL-27, IL-10 has not been studied. .
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로, 본 발명의 목적은 염증성 사이토카인과 염증조절 사이토카인의 분비 패턴 분석을 통해 처리물질의 관절염 치료 효능을 예측하는 관절염 치료제 스크리닝 방법을 제공하는 것이다.The present invention was devised to solve the above problems, and an object of the present invention is to provide an arthritis treatment screening method for predicting the treatment efficacy of a treatment substance through the analysis of secretion patterns of inflammatory cytokines and inflammatory regulatory cytokines. will be.
상기 목적을 달성하기 위하여, 본 발명은 혈액으로부터 분리된 세포에 피검물질을 처리한 시료군과 피검물질을 처리하지 않은 대조군을 준비하는 단계;In order to achieve the above object, the present invention comprises the steps of preparing a sample group treated with a test substance to a cell separated from blood and a control group not treated with the test substance;
상기 시료군과 대조군에서 염증성 사이토카인 및 염증조절 사이토카인의 분비량을 측정하는 단계;Measuring secretion amounts of inflammatory cytokines and inflammatory regulatory cytokines in the sample group and the control group;
상기 시료군에서 측정된 각각의 사이토카인 분비량을 대조군에서 측정된 각각의 사이토카인 분비량으로 나누어 사이토카인 상대비를 얻는 단계; 및Dividing each cytokine secretion amount measured in the sample group by each cytokine secretion amount measured in the control group to obtain a cytokine relative ratio; And
상기 사이토카인 상대비를 분석하여 관절염 치료제를 선별하는 단계를 포함하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법을 제공한다.It provides a method for screening for a treatment for arthritis comprising the step of selecting the therapeutic agent for arthritis by analyzing the relative ratio of the cytokines.
본 발명에 따른 관절염 치료 효능 예측 방법은 염증성 사이토카인 및 염증조절 사이토카인의 변화 패턴 분석을 통해 처리 물질의 관절염 치료 효능을 미리 예측함으로써, 관절염 치료제 개발에 따른 막대한 시간과 비용을 절약할 수 있는 효과가 있다.The arthritis treatment efficacy prediction method according to the present invention is an effect capable of saving enormous time and cost according to the development of an arthritis treatment agent by predicting the arthritis treatment efficacy of a treatment substance in advance through analysis of a change pattern of an inflammatory cytokine and an inflammation-regulating cytokine There is.
도 1은 관절염 치료 효능이 있는 펩타이드(P9)와 일반 펩타이드(AES16-2M)처리에 의한 사이토카인 분비 패턴을 비교한 것이다.1 is a comparison of the cytokine secretion pattern by treatment with a peptide (P9) and a general peptide (AES16-2M) having arthritis treatment efficacy.
이하, 본 발명을 상세히 설명한다. 본 명세서 및 청구범위에 사용되는 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.Hereinafter, the present invention will be described in detail. Terms or words used in the present specification and claims should not be interpreted as being limited to a conventional or dictionary meaning, and the inventor may appropriately define the concept of terms to describe his or her invention in the best way. Based on the principle that it should be interpreted as meanings and concepts consistent with the technical spirit of the present invention.
본 발명은 염증성 사이토카인 및 염증조절 사이토카인 분비 패턴 분석 결과를 바탕으로 처리물질에 의한 관절염 치료 가능성을 예측하는 관절염 치료제 스크리닝 방법에 관한 것이다.The present invention relates to an arthritis treatment screening method for predicting the possibility of treating arthritis by a treatment substance based on an analysis result of an inflammatory cytokine and an inflammation-regulating cytokine secretion pattern.
본 발명은 혈액으로부터 분리된 세포에 피검물질을 처리한 시료군과 피검물질을 처리하지 않은 대조군을 준비하는 단계;The present invention comprises the steps of preparing a sample group treated with a test substance in a cell separated from blood and a control group not treated with the test substance;
상기 시료군과 대조군에서 염증성 사이토카인 및 염증조절 사이토카인의 분비량을 측정하는 단계;Measuring secretion amounts of inflammatory cytokines and inflammatory regulatory cytokines in the sample group and the control group;
상기 시료군에서 측정된 각각의 사이토카인 분비량을 대조군에서 측정된 각각의 사이토카인 분비량으로 나누어 사이토카인 상대비를 얻는 단계; 및Dividing each cytokine secretion amount measured in the sample group by each cytokine secretion amount measured in the control group to obtain a cytokine relative ratio; And
상기 사이토카인 상대비를 분석하여 관절염 치료제를 선별하는 단계를 포함하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법을 제공한다.It provides a method for screening for a treatment for arthritis comprising the step of selecting the therapeutic agent for arthritis by analyzing the relative ratio of the cytokines.
본 발명에 따른 관절염 치료제 스크리닝 방법의 첫번째 단계는, 혈액으로부터 분리된 세포에 피검물질을 처리한 시료군과 피검물질을 처리하지 않은 대조군을 준비하는 단계이다.The first step of the screening method for arthritis treatment according to the present invention is a step of preparing a sample group treated with a test substance in cells separated from blood and a control group without treatment with the test substance.
상기 혈액으로부터 분리된 세포는 건강한 성인 혈액으로부터 분리된 말초 혈액 단핵구 세포(Peripheral blood mononuclear cell)를 사용할 수 있다.Cells isolated from the blood may use peripheral blood mononuclear cells isolated from healthy adult blood.
상기 시료군 및 대조군에는 사이토카인 분비 유도 물질을 처리할 수 있고, 바람직하게는 지질다당질(Lipopolysaccharide)을 처리할 수 있다.The sample group and the control group may be treated with a substance inducing cytokine secretion, and preferably, lipopolysaccharide may be treated.
상기 시료군은 혈액으로부터 분리된 세포에 피검물질과 사이토카인 분비 유도 물질을 처리한 후, CO2 인큐베이터에서 10 ~ 30 시간 동안 배양하여 얻은 배양 상청액을 사용할 수 있다.The sample group may use a culture supernatant obtained by incubating for 10 to 30 hours in a CO 2 incubator after treating a test substance and a cytokine secretion-inducing substance in cells separated from blood.
상기 피검물질은 관절염 치료제의 후보물질로, 펩타이드, 단백질, 추출물, 화합물 및 조성물로 구성된 군으로부터 선택된 어느 하나 이상일 수 있다.The test substance is a candidate substance for the treatment of arthritis, and may be any one or more selected from the group consisting of peptides, proteins, extracts, compounds and compositions.
이어지는 단계는, 상기 시료군과 대조군에서 염증성 사이토카인 및 염증조절 사이토카인의 분비량을 측정하는 단계이다.The following step is a step of measuring the secretion amount of inflammatory cytokines and inflammatory regulatory cytokines in the sample group and the control group.
상기 염증성 사이토카인은 염증 반응을 유도하는 사이토카인을 의미하며, 상기 염증조절 사이토카인은 면역반응 조절과 관련된 사이토카인을 의미한다.The inflammatory cytokine refers to a cytokine that induces an inflammatory response, and the inflammatory regulatory cytokine refers to a cytokine associated with the regulation of an immune response.
상기 염증성 사이토카인은 IL-6 및 IL-21 중 선택된 어느 하나 이상인 것을 특징으로 할 수 있다.The inflammatory cytokine may be characterized by at least one selected from IL-6 and IL-21.
상기 IL-6(interleukin-6)는 T 림프구, B 림프구, 대식세포, 섬유아세포 등 여러 세포에서 생산되는 사이토카인으로, 면역응답, 조혈계와 신경계 세포의 증식 및 분화, 급성 반응 등에 관여하며, IL-6의 과잉 생산은 여러 가지 면역이상증, 염증성 질환, 림프계 종양의 발증과 깊은 관련이 있는 것으로 알려져 있다. 특히, IL-6는 류마티스관절염의 병인에 있어서 중요한 역할을 하는 것으로 알려져 있다.The IL-6 (interleukin-6) is a cytokine produced by various cells such as T lymphocytes, B lymphocytes, macrophages, and fibroblasts. It is involved in immune responses, proliferation and differentiation of hematopoietic and nervous system cells, and acute reactions. It is known that the overproduction of IL-6 is closely related to the development of various immunodeficiencies, inflammatory diseases, and lymphoid tumors. In particular, IL-6 is known to play an important role in the pathogenesis of rheumatoid arthritis.
상기 IL-21(interleukin-21)은 알파-헬릭스(α-helix) 구조를 갖는 사이토카인의 한 종류로서, IL-21의 수용체와 감마쇄(γ-chain)를 이용한 신호전달과정을 통해서 염증성 반응을 일으키며, 자연 살해 세포(NK 세포) 및 세포 독성 T세포를 포함하여 면역계 세포에 강력한 조절 효과가 있는 것으로 알려져 있다.The IL-21 (interleukin-21) is a type of cytokine having an alpha-helix structure, and an inflammatory reaction through signal transduction using a receptor and a gamma chain (γ-chain) of IL-21. It is known to have strong regulatory effects on immune system cells, including natural killer cells (NK cells) and cytotoxic T cells.
또한, 상기 염증조절 사이토카인은 IFN-γ, IL-10 및 IL-27로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 할 수 있다.In addition, the inflammatory regulatory cytokine may be characterized in that at least one selected from the group consisting of IFN-γ, IL-10 and IL-27.
상기 IFN-γ(interferon-γ)는 IFN-α나 IFN-β와는 상동성이 없어 Ⅱ형 인터페론으로 분류되며, T림프구의 분열촉진제 혹은 특이항원 처리에 의해 유발되어 면역인터페론이라고도 한다. IFN-γ는 면역조절기능 뿐만 아니라 항바이러스 및 항종양의 성질이 있으며, 최근 관절염 진행과 관련하여 IFN-γ의 파골세포 형성 억제 기전이 밝혀진 바 있다.The IFN-γ (interferon-γ) is classified as a type II interferon because it has no homology to IFN-α or IFN-β, and is also called an immunointerferon caused by the treatment of a T lymphocyte stimulator or specific antigen. IFN-γ has anti-viral and anti-tumor properties as well as immunomodulatory functions, and recently, a mechanism for inhibiting osteoclast formation of IFN-γ has been revealed in relation to the progression of arthritis.
상기 IL-10(interleukin-10)은 주로 Th2 세포에서 생산되는 사이토카인으로,면역억제와 면역촉진의 양면성을 나타낸다. 즉, Th1세포의 IFN-γ생성을 항원제시세포인 대식세포를 통해 억제하지만, B세포의 생존율을 높이고 비만세포의 증식을 촉진하는 것으로 알려져 있다.The IL-10 (interleukin-10) is a cytokine mainly produced in Th2 cells, and exhibits both sides of immunosuppression and immune promotion. In other words, IFN-γ production of Th1 cells is suppressed through macrophages, which are antigen-presenting cells, but it is known to increase the survival rate of B cells and promote the proliferation of mast cells.
상기 IL-27(Interleukin-27)은 IL-12 계열의 사이토카인으로 EBI3와 p28의 두 서브유닛으로 구성되어 있으며, 면역계에서 T 세포의 다양한 개체군을 분화시키고 IL-10을 상향 조절하는 것으로 알려져 있다.The IL-27 (Interleukin-27) is an IL-12-based cytokine composed of two subunits of EBI3 and p28, and is known to differentiate various populations of T cells in the immune system and up-regulate IL-10. .
상기 사이토카인의 분비량은 시료군 및 대조군에서 각각의 사이토카인에 대해 측정된다.The secretion amount of the cytokine is measured for each cytokine in the sample group and the control group.
상기 사이토카인의 분비량은 웨스턴 블랏(western blotting), ELISA(enzyme linked immunosorbent assay), 면역조직화학염색법(immunohistochemical staining), 면역형광법(immunofluorescence), FACS 분석법(fluorescenceactivated cell sorter analysis), 단백질 칩 분석법(protein chip technology assay) 등을 사용하여 의해 측정될 수 있고, 시중에 판매되는 인간 사이토카인 어레이 키트를 사용하여 측정될 수 있다.The cytokine secretion amount is western blotting, ELISA (enzyme linked immunosorbent assay), immunohistochemical staining, immunofluorescence, FACS analysis, fluorescence activated cell sorter analysis, and protein chip analysis (protein) chip technology assay) and the like, and can be measured using a commercially available human cytokine array kit.
상기 측정 결과가 최종적으로 화학 발광 분석 방법에 의해 얻어질 경우, 이미지 분석 소프트웨어를 사용하여 각 스팟(spot)의 normalized volume값을 구하고 각 사이토카인에 해당하는 스팟(spot)의 normalized volume 평균값을 사용하여 분석될 수 있다.When the measurement result is finally obtained by a chemiluminescence analysis method, the normalized volume value of each spot is obtained using image analysis software, and the normalized volume average value of the spot corresponding to each cytokine is used. Can be analyzed.
다음 단계는, 상기 시료군에서 측정된 각각의 사이토카인 분비량을 대조군에서 측정된 각각의 사이토카인 분비량으로 나누어 사이토카인 상대비를 얻는 단계이다.The next step is to obtain a cytokine relative ratio by dividing each cytokine secretion measured in the sample group by each cytokine secretion measured in the control group.
구체적으로, 시료군에서 측정된 어느 하나의 사이토카인 분비량을 대조군에서 측정된 동일한 사이토카인 분비량으로 나누어 사이토카인 종류에 따라 각각의 상대비를 얻는다.Specifically, one cytokine secretion measured in the sample group is divided by the same cytokine secretion measured in the control group to obtain each relative ratio according to the type of cytokine.
본 발명에 따른 스크리닝 방법은 각 사이토카인의 상대비를 사용하여 분석함으로써, 측정 조건이나 측정 방법에 영향을 받지 않으며 염증성 사이토카인 및 염증조절 사이토카인의 분비 패턴을 동시에 분석할 수 있는 장점이 있다.The screening method according to the present invention has an advantage of simultaneously analyzing secretion patterns of inflammatory cytokines and inflammatory regulatory cytokines without being affected by measurement conditions or measurement methods, by analyzing using a relative ratio of each cytokine.
이어지는 단계는, 상기 사이토카인 상대비를 분석하여 관절염 치료제를 선별하는 단계이다.The following step is a step of selecting a therapeutic agent for arthritis by analyzing the relative ratio of the cytokines.
상기 사이토카인 상대비는 레이다 그래프(Radar graph)로 패턴화하여 분석하는 것을 특징으로 할 수 있다.The cytokine relative ratio may be characterized by patterning and analyzing a radar graph.
상기 레이다 그래프(Radar graph)는 평가항목이 여러 개일 때 항목 수에 따라 원을 같은 간격으로 나누고, 중심으로부터 일정 간격으로 동심으로 척도를 재는 칸을 나누어 각 평가항목의 정량화된 점수에 따라 그 위치에 점을 찍고 평가항목간 점을 이어 선으로 만들어 항목 간 균형을 한눈에 볼 수 있도록 해주는 도표이다.The radar graph divides the circle into equal intervals according to the number of items when there are multiple evaluation items, divides the cells concentrically from a center to a predetermined interval, and positions them according to the quantified score of each evaluation item. It is a chart that allows you to see the balance between items at a glance by dividing the points and making a line between the items to be evaluated.
상기 각각의 사이토카인 상대비를 레이다 그래프로 패턴화할 경우, 각 패턴의 형태가 직관적으로 파악되어 패턴 분석이 용이하며, 관절염 치료제군과의 패턴 비교를 통해 신속하게 관절염 치료제를 선별할 수 있는 장점이 있다.When each of the cytokine relative ratios is patterned with a radar graph, the shape of each pattern is intuitively understood to facilitate pattern analysis, and the advantage of being able to quickly select an arthritis treatment by comparing patterns with the arthritis treatment group have.
본 발명에 따른 관절염 치료제 스크리닝 방법은 상기 염증성 사이토카인의 상대비가 상기 염증조절 사이토카인 상대비보다 큰 경우, 피검물질을 관절염 치료제로 선별하는 것을 특징으로 할 수 있다.The screening method for arthritis treatment according to the present invention may be characterized in that when the relative ratio of the inflammatory cytokine is greater than the relative ratio of the inflammatory cytokine, the test substance is selected as a therapeutic agent for arthritis.
구체적으로, 상기 염증성 사이토카인의 상대비는 1 ~ 100 이고, 상기 염증조절 사이토카인 상대비는 0.01 ~ 10 인 경우, 피검물질을 관절염 치료제로 선별할 수 있다.Specifically, when the relative ratio of the inflammatory cytokines is 1 to 100, and the relative ratio of the inflammatory regulatory cytokines is 0.01 to 10, the test substance may be selected as a therapeutic agent for arthritis.
바람직하게는, 상기 염증성 사이토카인의 상대비는 5 ~ 50 이고, 상기 염증조절 사이토카인 상대비는 0.01 ~ 5 인 경우, 피검물질을 관절염 치료제로 선별할 수 있다.Preferably, when the relative ratio of the inflammatory cytokines is 5 to 50, and the relative ratio of the inflammatory regulatory cytokines is 0.01 to 5, the test substance may be selected as a therapeutic agent for arthritis.
또한, 본 발명에 따른 관절염 치료제 스크리닝 방법은 염증성 사이토카인인 상기 IL-6의 상대비가 15 ~ 50 인 경우, 피검물질을 관절염 치료제로 선별하는 것을 특징으로 할 수 있다.In addition, the screening method for arthritis treatment according to the present invention may be characterized in that when the relative ratio of the IL-6, which is an inflammatory cytokine, is 15 to 50, the test substance is selected as a treatment for arthritis.
바람직하게는, 상기 IL-6의 상대비가 20 ~ 50 인 경우 피검물질을 관절염 치료제로 선별할 수 있고, 바람직하게는 IL-6의 상대비가 40 ~ 45인 경우 피검물질을 관절염 치료제로 선별할 수 있다.Preferably, when the relative ratio of IL-6 is 20 to 50, the test substance may be selected as a therapeutic agent for arthritis, and preferably, when the relative ratio of IL-6 is 40 to 45, the test substance may be selected as a therapeutic agent for arthritis. have.
이하, 본 발명을 구체적으로 설명하기 위해 실시예 및 실험예를 들어 상세하게 설명하기로 한다. 그러나 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, examples and experimental examples will be described in detail to specifically describe the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<실시예> 펩타이드 처리에 따른 사이토카인 분석<Example> Cytokine analysis according to peptide treatment
(1) 펩타이드 처리(1) peptide treatment
세포에서 분비하는 사이토카인을 측정하기 위하여 먼저 건강한 성인 혈액으로부터 말초 혈액 단핵구 세포(Peripheral blood mononuclear cell, PBMC)를 분리하고 1 X 106/mL의 농도로 준비하였다. 이후, 사이토카인 분비를 유도하기 위하여 지질다당질(Lipopolysaccharide,LPS)를 10 ng/mL의 농도로 처리하였다. To measure cytokines secreted by cells, peripheral blood mononuclear cells (PBMC) were first isolated from healthy adult blood and prepared at a concentration of 1
이때, 처리 물질인 펩타이드에 의한 변화를 보기 위해 100 ng/mL의 농도로 관절염 치료 효과가 있는 펩타이드(P9)을 LPS와 함께 처리하였다. 24시간 동안 CO2 incubator에서 배양한 후, 배양 상청액(culture supernatant)을 수거하여 사이토카인 분석을 위한 시료로 이용하였다.At this time, in order to see the change due to the treatment material peptide, a peptide (P9) having an arthritis treatment effect at a concentration of 100 ng/mL was treated with LPS. After incubation in a CO 2 incubator for 24 hours, the culture supernatant was collected and used as a sample for cytokine analysis.
비교 물질로, 일반 펩타이드(AES16-2M)를 동일하게 처리하여 분석 시료로 사용하였으며, 사용된 펩타이드의 아미노산 서열을 표 1에 나타내었다.As a comparative substance, a general peptide (AES16-2M) was treated identically and used as an analysis sample, and the amino acid sequence of the used peptide is shown in Table 1.
(2) 사이토카인 분비 측정(2) Cytokine secretion measurement
수거한 배양 상청액(culture supernatant)은 Human cytokine array(R&D Systems_ARY005B)를 제공하는 프로토콜에 따라 수행하였다. The collected culture supernatant was performed according to a protocol providing a human cytokine array (R&D Systems_ARY005B).
간략하게 제공하는 array buffer 4를 2 mL 처리하여 멤브레인을 1시간 동안 rocking shaker에서 bloking 하였다. 샘플은 1 mL의 배양 상청액과 제공하는 Array buffer 4 500 uL를 섞고, 제공하는 Detection antibody cocktail 15 uL를 처리하여 상온에서 1시간 동안 pre-binding 시켰다. 멤브레인을 워싱하여 남아있는 blocking buffer를 제거한 후, 샘플을 처리하고 하룻밤 동안 방치해 두었다. 다음날 샘플을 워싱하여 제거한 뒤에 제공하는 Streptavidin-HRP를 2 mL 처리하여 30 분 동안 binding 시켰다. 멤브레인을 워싱하여 남아있는 Streptavidin-HRP를 제거하고 detection을 위한 HCL 웨스턴 블로팅 검출시약(Western blotting detection reagent, GE Healthcare_RPN2232)를 처리한 후, 30초 간격으로 멤브레인을 방출 광원(emission light)에 노출시켜 화학 발광 분석(chemiluminescent detection)에 따른 방법으로 검출하여 Tiff 이미지로 저장하였다(Fuji_LAS-3000). The membrane buffer was bloking in a rocking shaker for 1 hour by processing 2 mL of the briefly provided array buffer 4. The sample was mixed with 1 mL of culture supernatant and 500 uL of the provided Array buffer 4, and treated with 15 uL of the provided Detection antibody cocktail to pre-binding at room temperature for 1 hour. After washing the membrane to remove the remaining blocking buffer, the sample was processed and left overnight. The next day, after washing and removing the sample, the provided Streptavidin-HRP was treated with 2 mL and bound for 30 minutes. Wash the membrane to remove the remaining Streptavidin-HRP, treat the HCL Western blotting detection reagent (GE Healthcare_RPN2232) for detection, and then expose the membrane to emission light at 30 second intervals. It was detected by a method according to chemiluminescent detection and stored as a Tiff image (Fuji_LAS-3000).
(3) 사이토카인 분석(3) Cytokine analysis
저장한 이미지 파일은 Totallab (CLIQS 1D) 프로그램을 이용하여 수치화하여 spot의 normalized volume값을 구하였다.The saved image file was quantified using Totallab (CLIQS 1D) program to obtain the spot's normalized volume value.
이후, 구하고자 하는 사이토카인에 해당하는 spot의 normalized volume의 평균값을 구하고 분석을 위하여 relative ratio로 계산하여 나타내었다. 간략하게는 LPS와 펩타이드를 처리한 샘플의 사이토카인에 대한 normalized volume 값을 LPS만 처리한 샘플의 타겟 사이토카인에 대한 normalized volume 값으로 나누어 ratio를 계산하고 radar graph를 그려 패턴을 비교하였다.Subsequently, the average value of the normalized volume of the spot corresponding to the cytokine to be obtained was obtained and calculated and expressed as a relative ratio for analysis. Briefly, the ratio was calculated by dividing the normalized volume value for the cytokine of the sample treated with LPS and peptide with the normalized volume value for the target cytokine of the sample treated with LPS only, and comparing the patterns by drawing a radar graph.
(4) 사이토카인 array 분석 결과(4) Cytokine array analysis results
실제 관절염 치료에 효과가 있는 펩타이드(P9)을 최종 계산된 ratio 값을 분석하여 치료 효능 예측 가능성을 검증하였다.Peptides (P9), which are effective in treating arthritis, were analyzed by analyzing the final calculated ratio values to verify the predictability of treatment efficacy.
표 1에 나타낸 바와 같이, 염증성 사이토카인의 경우 각각 IL-6는 41.18배, IL-21은 6.75배 증가하였다. 반면에 염증조절 사이토카인의 경우에는 IFN-gamma가 0.61배, IL-10이 0.29배, IL-27이 1.35배 증가한 것으로 나왔다.As shown in Table 1, for inflammatory cytokines, IL-6 increased by 41.18 fold and IL-21 increased by 6.75 fold, respectively. On the other hand, in case of inflammatory regulatory cytokines, IFN-gamma increased by 0.61 times, IL-10 by 0.29 times, and IL-27 by 1.35 times.
또한, 도 1에 나타낸 바와 같이 일반 펩타이드인 AES16-2M(Sci Rep. 2018 Sep 26;8(1):14398)인 경우에는 상기 사이토카인 중, IL-6만 12.71배 증가하여 펩타이드(P9)과는 전혀 다른 패턴을 나타내었다. 이를 통해, 펩타이드(P9)의 관절염 효능을 입증하였다.In addition, in the case of AES16-2M (Sci Rep. 2018 Sep 26;8(1):14398), which is a general peptide, as shown in FIG. 1, among the cytokines, only IL-6 increased by 12.71 times, resulting in a peptide (P9) and Showed a completely different pattern. Through this, the efficacy of the arthritis of the peptide (P9) was demonstrated.
<110> Korea University Industry and Academy Cooperation Foundation <120> Screening methods for therapeutic agents of arthritis through cytokine analysis <130> SP-1903 <150> KR 10-2018-0151344 <151> 2018-11-29 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> P9 <400> 1 Pro Ser Pro Pro Ser Pro Pro Ser Pro 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> AES16-2M <400> 2 Arg Glu Gly Arg Thr 1 5 <110> Korea University Industry and Academy Cooperation Foundation <120> Screening methods for therapeutic agents of arthritis through cytokine analysis <130> SP-1903 <150> KR 10-2018-0151344 <151> 2018-11-29 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> P9 <400> 1 Pro Ser Pro Pro Ser Pro Pro Ser Pro 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> AES16-2M <400> 2 Arg Glu Gly Arg Thr 1 5
Claims (7)
상기 시료군과 대조군에서 염증성 사이토카인 및 염증조절 사이토카인의 분비량을 측정하는 단계;
상기 시료군에서 측정된 각각의 사이토카인 분비량을 대조군에서 측정된 각각의 사이토카인 분비량으로 나누어 사이토카인 상대비를 얻는 단계; 및
상기 사이토카인 상대비를 분석하여 관절염 치료제를 선별하는 단계를 포함하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
Preparing a sample group treated with the test substance in cells separated from the blood and a control group without the test substance;
Measuring secretion amounts of inflammatory cytokines and inflammatory regulatory cytokines in the sample group and the control group;
Dividing each cytokine secretion measured in the sample group by each cytokine secretion measured in the control group to obtain a cytokine relative ratio; And
A method for screening for a treatment for arthritis comprising the step of selecting a therapeutic agent for arthritis by analyzing the relative ratio of the cytokines.
상기 염증성 사이토카인은 IL-6 및 IL-21 중 선택된 어느 하나 이상인 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 1,
The inflammatory cytokine is a screening method for treating arthritis, characterized in that at least one selected from IL-6 and IL-21.
상기 염증조절 사이토카인은 IFN-γ, IL-10 및 IL-27로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 1,
The inflammation control cytokine is IFN-γ, IL-10 and IL-27 screening method for the treatment of arthritis, characterized in that at least one selected from the group consisting of.
상기 사이토카인 상대비는 레이다 그래프(Radar graph)로 패턴화하여 분석하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 1,
The cytokine relative ratio is patterned and analyzed by radar graph (Radar graph) screening method for arthritis treatment.
상기 염증성 사이토카인의 상대비가 상기 염증조절 사이토카인 상대비보다 큰 경우, 피검물질을 관절염 치료제로 선별하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 1,
If the relative ratio of the inflammatory cytokines is greater than the relative ratio of the inflammation-regulating cytokines, the screening method for arthritis therapeutics characterized in that the test substance is selected as a therapeutic agent for arthritis.
상기 IL-6의 상대비가 15 ~ 50인 경우, 피검물질을 관절염 치료제로 선별하는 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 2,
When the relative ratio of IL-6 is 15 to 50, the screening method for arthritis treatment is characterized in that the test substance is selected as a treatment for arthritis.
상기 피검물질은 펩타이드, 단백질, 추출물, 화합물 및 조성물로 구성된 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는 관절염 치료제 스크리닝 방법.
According to claim 1,
The test substance is a screening method for treating arthritis, characterized in that at least one selected from the group consisting of peptides, proteins, extracts, compounds and compositions.
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