KR102596937B1 - Biomarker for diagnosing pannus of rheumatoid arthritis and use thereof - Google Patents
Biomarker for diagnosing pannus of rheumatoid arthritis and use thereof Download PDFInfo
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- KR102596937B1 KR102596937B1 KR1020190141956A KR20190141956A KR102596937B1 KR 102596937 B1 KR102596937 B1 KR 102596937B1 KR 1020190141956 A KR1020190141956 A KR 1020190141956A KR 20190141956 A KR20190141956 A KR 20190141956A KR 102596937 B1 KR102596937 B1 KR 102596937B1
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- plgf
- vegf
- rheumatoid arthritis
- pannus
- serum
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Abstract
본 발명은 류마티스 관절염의 판누스 진단용 바이오마커 및 이의 이용에 관한 것으로, 본 발명에 따른 신생혈관형성 관련 인자 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)는 류마티스 관절염 환자의 활액 및 혈청에서 증가되어 있으며, 활액에서의 PlGF 검출량은 임상적 염증수치, GSUS, 및 PDUS 수준과 유의한 상관관계를 가지고, 혈청에서의 VEGF 검출량은 질병활성도, GSUS, 및 PDUS 수준과 유의한 상관관계를 가지는 것을 확인하였다. 따라서, 본 발명에 따른 PlGF 및 VEGF를 류마티스 관절염의 판누스 진단용 바이오마커로 이용하여 판누스의 증식 정도를 확인함으로써 류마티스 관절염 진단 및 치료제 개발 분야 등에 유용하게 이용할 수 있을 것으로 기대된다.The present invention relates to a biomarker for pannus diagnosis of rheumatoid arthritis and its use. The angiogenesis-related factors PlGF (placental growth factor) and VEGF (vascular endothelial growth factor) according to the present invention are found in the synovial fluid and serum of rheumatoid arthritis patients. The amount of PlGF detected in synovial fluid has a significant correlation with clinical inflammation levels, GSUS, and PDUS levels, and the amount of VEGF detected in serum has a significant correlation with disease activity, GSUS, and PDUS levels. Confirmed. Therefore, it is expected that the PlGF and VEGF according to the present invention can be usefully used in the fields of rheumatoid arthritis diagnosis and treatment development by confirming the degree of pannus proliferation by using it as a biomarker for diagnosing the pannus of rheumatoid arthritis.
Description
본 발명은 류마티스 관절염의 판누스 진단용 바이오마커 및 이의 이용에 관한 것이다.The present invention relates to a biomarker for diagnosing pannus in rheumatoid arthritis and its use.
류마티스 관절염(rheumatoid arthritis, 이하 RA)은 대표적인 자가면역질환으로, 관절 주위를 둘러싸고 있는 활막이라는 조직의 염증 때문에 일어나며, 전체 인구의 약 1~2% 가량이 발병하는 것으로 알려진 질환이다. 류마티스 관절염의 발병은 유전적-환경적 인자가 작용하며, 유전적인 요인이 대략 60% 정도 기여하는 것으로 알려져 있다. 대표적인 증상은 활막관절(synovial joint)에 나타나는 염증으로, 관절이 붓고 통증이 있으며, 질환이 발전하면 영구적으로 관절이 변형되고 관절의 기능에 심각한 손상을 주어 삶의 질이 크게 저하된다. 류마티스 관절염은 질병활성도가 조절되지 않으면 시간 경과에 따라 비가역적 관절변형과 장애가 발생하며 이를 조절하기 위한 면역억제제 및 생물학적 제제 등에 대한 약물반응 정도에 따라 관절파괴가 영향을 받는다.Rheumatoid arthritis (RA) is a representative autoimmune disease that occurs due to inflammation of the synovium surrounding the joints, and is known to affect approximately 1-2% of the total population. The onset of rheumatoid arthritis is influenced by genetic and environmental factors, and genetic factors are known to contribute approximately 60%. The representative symptom is inflammation in the synovial joint, which causes swelling and pain in the joint. As the disease develops, the joint is permanently deformed and the function of the joint is seriously damaged, greatly reducing the quality of life. In rheumatoid arthritis, if disease activity is not controlled, irreversible joint deformation and disability occur over time, and joint destruction is affected by the degree of drug reaction to immunosuppressants and biological agents to control this.
초기 류마티스성 관절염은 처음에 더 작은 관절, 예컨대 손목, 손, 발목 및 발의 관절에 영향을 미치며 질환이 진행됨에 따라, 어깨, 팔꿈치, 무릎, 둔부, 턱 및 목의 관절들에도 영향을 나타낼 수 있다. 또한, 관절 내 부위 또는 관절 주변 부위에만 영향을 미치는 다른 관절염 상태들과는 달리, 류마티스성 관절염은 피부, 혈관, 심장, 폐 및 근육을 포함하는 신체에 걸쳐 관절외 조직에서 염증을 야기할 수 있는 전신성 질환이다. 한번 발병이 시작되면 꾸준히 악화되고 관절에 심각한 염증과 통증이 증가하는 질병인 류마티스성 관절염에 대한 적절한 치료방법이 없기 때문에 류마티스성 관절염의 초기 진단이 매우 중요하나 아직까지 류마티스성 관절염의 발병 및 진행 정도를 조기에 진단하는 기술 또한 미흡하여 이에 대한 연구 및 개발이 필요한 실정이다.Early rheumatoid arthritis initially affects smaller joints, such as those of the wrists, hands, ankles, and feet; as the disease progresses, joints in the shoulders, elbows, knees, hips, jaw, and neck may also be affected. . Additionally, unlike other arthritic conditions that only affect intra-articular or peri-articular areas, rheumatoid arthritis is a systemic disease that can cause inflammation in extra-articular tissues throughout the body, including the skin, blood vessels, heart, lungs, and muscles. am. Early diagnosis of rheumatoid arthritis is very important because there is no appropriate treatment for rheumatoid arthritis, a disease that steadily worsens once the onset begins and causes severe inflammation and pain in the joints. However, the onset and progression of rheumatoid arthritis is still unknown. The technology for early diagnosis is also insufficient, so research and development is needed.
류마티스 관절염의 진단에서는 누구나 인정할 수 있는 생화학적 지표나 병리소견, 영상소견 등의 소위 골드 스탠다드가 없기 때문에 여러 지표의 조합을 사용한 분류기준이라는 방법을 통하여 진단하게 된다. 류마티스 인자는 1987년 이후로 미국 류마티스학회 (American College of Rheumatology, ACR)에서 류마티스 진단에서 혈청학적 검사로 사용되고 있다. 류마티스 인자는 면역글로블린 IgG의 Fc 영역에 결합하는 자가항체이며, 류마티스 관절염 환자의 약 80%에서 증가되어 있지만, 관절염에 대한 특이도가 높지 않으며, 다른 자가 면역 질환이나 만성 염증, 악성종양, 건강한 노인에게서도 나타나는 단점이 있다.In the diagnosis of rheumatoid arthritis, there is no so-called gold standard such as biochemical indicators, pathological findings, or imaging findings that anyone can recognize, so the diagnosis is made through a method called classification criteria using a combination of several indicators. Rheumatoid factor has been used as a serological test in rheumatology diagnosis by the American College of Rheumatology (ACR) since 1987. Rheumatoid factor is an autoantibody that binds to the Fc region of immunoglobulin IgG, and is increased in approximately 80% of rheumatoid arthritis patients, but does not have high specificity for arthritis and is used in other autoimmune diseases, chronic inflammation, malignant tumors, and healthy elderly people. There are also disadvantages that appear in .
한편, 류마티스 관절염 발병 후 질환이 발전하면서 관절의 염증을 증가시키고, 나아가 관절의 파괴를 진행시키는 가장 큰 직접적인 요인은 판누스(pannus)의 증식이라고 할 수 있는데, 정상 관절의 경우 1~2겹 정도의 세포층으로 이루어져 있는 활막(synovial membrane)은 점성을 띄는 세포외 기질(extracellular matrix) 등을 만들어 내면서 관절의 윤활에 필수적인 활액(synovial fluid)을 유지시키는 중요한 역할을 하는 반면, 류마티스 관절염을 통해서 지속적인 염증성 환경에 노출된 활막세포들은 과다하게 증식하여 여러겹의 세포층을 형성하게 되고, 염증성 사이토카인을 생산하며 침윤성을 획득하여 연골을 파괴하고 관절의 파괴에 주요하게 기여하게 된다. 이와 같이 류마티스 관절염 활막관절에서 과다하게 증식된 활막의 덩어리를 일컬어 판누스 (pannus)라고 하며, 판누스의 증식 및 활성 정도는 관절염의 진행 정도를 나타내는 지표라고 할 수 있다. 그러므로, 판누스 관련 인자들은 신뢰성 있는 류마티스 관절염 바이오마커로 충분히 제시될 수 있다.Meanwhile, as the disease develops after the onset of rheumatoid arthritis, the biggest direct factor that increases joint inflammation and further causes joint destruction is the proliferation of the pannus, which in normal joints has about 1 to 2 layers. The synovial membrane, which is composed of a layer of cells, plays an important role in maintaining synovial fluid, which is essential for joint lubrication, by creating a viscous extracellular matrix, while rheumatoid arthritis causes persistent inflammation. Synovial cells exposed to the environment proliferate excessively, forming multiple cell layers, producing inflammatory cytokines, and acquiring invasive properties, destroying cartilage and contributing significantly to the destruction of joints. In this way, the excessively proliferated synovial mass in the synovial joint of rheumatoid arthritis is called the pannus, and the degree of proliferation and activity of the pannus can be said to be an indicator of the degree of progression of arthritis. Therefore, pannus-related factors can be sufficiently presented as reliable rheumatoid arthritis biomarkers.
이에, 본 발명자들은 판누스의 병적 상태를 신속 간편하면서도 즉각적으로 진단할 수 있는 바이오마커를 개발함으로써 류마티스 관절염의 진단 등에 활용하고자 하였다.Accordingly, the present inventors attempted to develop a biomarker that can quickly, easily, and immediately diagnose the pathological condition of the pannus and use it for the diagnosis of rheumatoid arthritis.
본 발명자들은 판누스의 병적 상태를 가장 잘 표현해 낼 수 있는 바이오마커를 발굴해내기 위해 연구를 진행한 결과, 활막세포에서 생산되는 주요 신생혈관형성(angiogenesis)과 관련된 인자 중 활액 PlGF 검출량이 임상적 염증수치, 판누스의 증식 정도를 나타내는 GSUS, 및 혈관형성 정도를 나타내는 PDUS 수준과 유의한 상관관계를 가지는 것을 확인하였으며, 혈청 VEGF 검출량은 질병활성도, GSUS, 및 PDUS 수준과 유의한 상관관계를 가지는 것을 확인하였다.The present inventors conducted research to discover a biomarker that could best express the pathological condition of the pannus. As a result, the amount of synovial PlGF detected among the major factors related to angiogenesis produced by synovial cells was clinically significant. It was confirmed that there was a significant correlation with the level of inflammation, GSUS indicating the degree of pannus proliferation, and PDUS level indicating the degree of angiogenesis, and the amount of serum VEGF detected had a significant correlation with the disease activity, GSUS, and PDUS levels. confirmed.
이에, 본 발명의 목적은 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)로 이루어진 군으로부터 선택되는 하나 이상을 포함하는, 류마티스 관절염의 판누스(pannus) 진단용 바이오마커 조성물 및 이의 이용을 제공하는 것이다.Accordingly, the object of the present invention is to provide a biomarker composition for diagnosing pannus in rheumatoid arthritis, including at least one selected from the group consisting of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), and its use. It is done.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
상기와 같은 목적을 달성하기 위해 본 발명은 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)로 이루어진 군으로부터 선택되는 하나 이상을 포함하는, 류마티스 관절염의 판누스(pannus) 진단용 바이오마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a biomarker composition for diagnosing the pannus of rheumatoid arthritis, comprising at least one selected from the group consisting of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF). to provide.
또한, 본 발명은 상기 PlGF 또는 VEGF의 발현 또는 활성 수준을 측정하는 제제를 포함하는, 류마티스 관절염의 판누스(pannus) 진단용 조성물을 제공한다.Additionally, the present invention provides a composition for diagnosing pannus in rheumatoid arthritis, comprising an agent for measuring the expression or activity level of PlGF or VEGF.
또한, 본 발명은 활액에서의 PlGF(placental growth factor) 발현 또는 활성 수준; 또는 혈청에서의 VEGF(vascular endothelial growth factor) 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스(pannus) 진단을 위한 정보제공방법을 제공한다.In addition, the present invention relates to the level of placental growth factor (PlGF) expression or activity in synovial fluid; Alternatively, a method of providing information for diagnosing pannus of rheumatoid arthritis is provided, including the step of confirming the expression or activity level of vascular endothelial growth factor (VEGF) in serum.
또한, 본 발명은 활액에서의 PlGF 발현 또는 활성 수준; 또는 혈청에서의 VEGF 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스(pannus) 진단 방법을 제공한다.Additionally, the present invention relates to PlGF expression or activity levels in synovial fluid; Alternatively, it provides a method for diagnosing pannus of rheumatoid arthritis, including the step of confirming the level of VEGF expression or activity in serum.
또한, 본 발명은 상기 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)로 이루어진 군으로부터 선택되는 하나 이상을 포함하는 바이오마커 조성물의 류마티스 관절염의 판누스(pannus) 진단 용도를 제공한다.In addition, the present invention provides a use of a biomarker composition containing at least one selected from the group consisting of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) for diagnosing the pannus of rheumatoid arthritis.
또한, 본 발명은 상기 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)로 이루어진 군으로부터 선택되는 하나 이상의, 류마티스 관절염의 판누스(pannus) 진단에 이용되는 바이오마커로서의 용도를 제공한다.In addition, the present invention provides the use of at least one selected from the group consisting of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) as a biomarker used in the diagnosis of pannus in rheumatoid arthritis.
본 발명의 일 구현예로서, 상기 PlGF는 활액에서 WBC counts, %호중구, ESR(erythrocyte sedimentation rate), CRP(C reactive protein), GSUS(Grey scale ultrasonography), 및 PDUS(power-Doppler ultrasonography)로 이루어진 군으로부터 선택되는 하나 이상과 양의 상관관계를 가질 수 있다.In one embodiment of the present invention, the PlGF is composed of WBC counts, % neutrophils, erythrocyte sedimentation rate (ESR), C reactive protein (CRP), gray scale ultrasonography (GSUS), and power-Doppler ultrasonography (PDUS) in synovial fluid. It may have a positive correlation with one or more selected from the group.
본 발명의 다른 구현예로서, 상기 VEGF는 혈청에서 TJC(tender joint count), SJC(swollen joint count), ESR, CRP, DAS 28(disease activity score 28), GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상과 양의 상관관계를 가질 수 있다.In another embodiment of the present invention, the VEGF is selected from the group consisting of tender joint count (TJC), swollen joint count (SJC), ESR, CRP, disease activity score 28 (DAS 28), GSUS, and PDUS in serum. It may have a positive correlation with more than one.
본 발명의 또 다른 구현예로서, 상기 활액에서의 PlGF 발현 또는 활성 수준이 정상 대조군에 비해 증가되어 있고, WBC counts, %호중구, ESR, CRP, GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상의 수준과 양의 상관관계를 나타낼 경우; 또는 In another embodiment of the present invention, the level of PlGF expression or activity in the synovial fluid is increased compared to the normal control, and one or more levels selected from the group consisting of WBC counts, % neutrophils, ESR, CRP, GSUS, and PDUS. If there is a positive correlation with; or
혈청에서의 VEGF 발현 또는 활성 수준이 정상 대조군에 비해 증가되어 있고, TJC, SJC, ESR, CRP, DAS 28, GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상의 수준과 양의 상관관계를 나타낼 경우 판누스 증식 정도가 높은 것으로 진단하는 단계를 더 포함할 수 있다.If the level of VEGF expression or activity in serum is increased compared to normal controls and shows a positive correlation with one or more levels selected from the group consisting of TJC, SJC, ESR, CRP, DAS 28, GSUS, and PDUS, the plate A step of diagnosing that the degree of Nus proliferation is high may be further included.
또한, 본 발명은 류마티스 관절염 환자로부터 활액 또는 혈청을 분리하여 후보물질을 처리하는 단계;In addition, the present invention includes the steps of separating synovial fluid or serum from a patient with rheumatoid arthritis and processing a candidate material;
상기 후보물질 처리 후 류마티스 관절염 환자의 활액에서의 PlGF 발현 또는 활성 수준, 또는 혈청에서의 VEGF 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스 억제용 물질의 스크리닝 방법을 제공한다.Provided is a screening method for a substance for inhibiting pannus in rheumatoid arthritis, comprising the step of confirming the expression or activity level of PlGF in synovial fluid or the level of VEGF expression or activity in serum of a patient with rheumatoid arthritis after treatment with the candidate substance.
본 발명의 일 구현예로서, 상기 후보물질 처리 후 류마티스 관절염 환자의 활액에서의 PlGF 발현 또는 활성 수준이 후보물질 처리 전에 비해 감소한 경우; 또는In one embodiment of the present invention, when the level of PlGF expression or activity in the synovial fluid of a rheumatoid arthritis patient after treatment with the candidate substance is reduced compared to before treatment with the candidate substance; or
혈청에서의 VEGF 발현 또는 활성 수준이 후보물질 처리 전에 비해 감소한 경우, 후보물질을 류마티스 관절염의 판누스 억제용 물질로 선택하는 단계를 더 포함할 수 있다.If the level of VEGF expression or activity in serum is reduced compared to before treatment with the candidate material, a step of selecting the candidate material as a substance for inhibiting pannus in rheumatoid arthritis may be further included.
본 발명에 따른 신생혈관형성 관련 인자 PlGF(placental growth factor) 및 VEGF(vascular endothelial growth factor)는 류마티스 관절염 환자의 활액 및 혈청에서 증가되어 있으며, 활액에서의 PlGF 검출량은 임상적 염증수치, GSUS, 및 PDUS 수준과 유의한 상관관계를 가지고, 혈청에서의 VEGF 검출량은 질병활성도, GSUS, 및 PDUS 수준과 유의한 상관관계를 가지는 것을 확인하였다. 따라서, 본 발명에 따른 PlGF 및 VEGF를 류마티스 관절염의 판누스 진단용 바이오마커로 이용하여 판누스의 증식 정도를 확인함으로써 류마티스 관절염 진단 및 치료제 개발 분야 등에 유용하게 이용할 수 있을 것으로 기대된다.The angiogenesis-related factors PlGF (placental growth factor) and VEGF (vascular endothelial growth factor) according to the present invention are increased in synovial fluid and serum of rheumatoid arthritis patients, and the amount of PlGF detected in synovial fluid is determined by clinical inflammation levels, GSUS, and It was confirmed that there was a significant correlation with the PDUS level, and that the amount of VEGF detected in serum had a significant correlation with disease activity, GSUS, and PDUS levels. Therefore, it is expected that the PlGF and VEGF according to the present invention can be usefully used in the fields of rheumatoid arthritis diagnosis and treatment development by confirming the degree of pannus proliferation by using it as a biomarker for diagnosing the pannus of rheumatoid arthritis.
도 1은 본 발명의 일 구현예에 따른 활막염 비대 및 과맥관질을 보여주는 초음파 검사 결과를 나타낸 것으로, 도 1의 A 및 B는 Grey scale US(GSUS) 이미지, C 및 D는 power-Doppler US(PDUS) 이미지를 나타낸 것이다.
도 2a는 본 발명의 일 구현예에 따른 류마티스 관절염(RA) 환자(n=54) 및 골관절염(OA) 대조군(n=30)의 활액(SF) 내 VEGF, PlGF, sFlt-1, 및 IL-6 농도를 나타낸 도면이다. Bar는 평균 및 SD를 나타낸다.
도 2b는 본 발명의 일 구현예에 따른 RA 환자의 활액 내 VEGF, PlGF, sFlt-1, 및 IL-6 농도의 초음파 검사(US)상 활막염 중증도, 헤모글로빈, ESR, CRP, WBC count 및 %호중구와의 상관관계를 나타낸 도면이다(상단 도면: 상관관계 매트릭스 히트맵(correlation matrix heatmap), 하단 도면: 상관관계 그래프(correlation plot)). *p < 0.05, **p < 0.01, ***p < 0.001.
도 3a는 본 발명의 일 구현예에 따른 GSUS 0-1 RA 환자와 비교하여 GSUS 2-3 RA 환자의 활액 내 VEGF, PlGF, sFlt-1, 및 IL-6 농도를 나타낸 도면이다.
도 3b는 본 발명의 일 구현예에 따른 PDUS 1-3 RA 환자 및 PDUS 0 RA 환자에서의 VEGF, PlGF, sFlt-1, 및 IL-6 농도를 나타낸 도면이다.
도 3c는 본 발명의 일 구현예에 따른 진행성 활막염(active synovitis)을 갖는 RA 환자 및 진행성 활막염을 갖지않는 RA 환자에서의 VEGF, PlGF, sFlt-1, 및 IL-6 농도를 나타낸 도면이다. Bar는 평균 및 SD를 나타낸다. *p < 0.05, **p < 0.01.
도 4a는 본 발명의 일 구현예에 따른 RA 환자(n=157) 및 OA 대조군(n=50)의 혈청 내 VEGF, PlGF, sFlt-1, 및 IL-6 농도를 나타낸 도면이다.
도 4b는 본 발명의 일 구현예에 따른 RA 환자의 혈청 내 VEGF, PlGF, sFlt-1, 및 IL-6 농도와 압통 관절 수(tender joint count), 부종 관절 수(swollen joint count), ESR, CRP, DAS28 및 활막염의 US 점수(GSUS, PDUS) 사이의 상관관계를 나타낸 도면이다.
도 4c는 본 발명의 일 구현예에 따른 활막염 비대의 중증도에 따른 VEGF, PlGF, sFlt-1 및 IL-6의 혈청 내 농도를 나타낸 도면이다.
도 4d는 본 발명의 일 구현예에 따른 맥관질의 정도에 따른 VEGF, PlGF, sFlt-1 및 IL-6의 혈청 내 농도를 나타낸 도면이다.
도 4e는 본 발명의 일 구현예에 따른 진행성 활막염에 따른 VEGF, PlGF, sFlt-1 및 IL-6의 혈청 내 농도를 나타낸 도면이다. Bar는 평균 및 SD를 나타낸다. *p < 0.05, **p < 0.01, ***p < 0.001.Figure 1 shows the results of an ultrasound examination showing synovitis hypertrophy and hypervascularity according to an embodiment of the present invention. In Figure 1, A and B are Gray scale US (GSUS) images, and C and D are power-Doppler US ( PDUS) image.
Figure 2a shows VEGF, PlGF, sFlt-1, and IL- in synovial fluid (SF) of rheumatoid arthritis (RA) patients (n=54) and osteoarthritis (OA) controls (n=30) according to one embodiment of the present invention. 6 This is a diagram showing concentration. Bar represents mean and SD.
Figure 2b shows synovitis severity, hemoglobin, ESR, CRP, WBC count, and % neutrophils on ultrasonography (US) of VEGF, PlGF, sFlt-1, and IL-6 concentrations in the synovial fluid of RA patients according to one embodiment of the present invention. This is a drawing showing the correlation with (top drawing: correlation matrix heatmap, bottom drawing: correlation plot). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3a is a diagram showing the concentrations of VEGF, PlGF, sFlt-1, and IL-6 in the synovial fluid of GSUS 2-3 RA patients compared to GSUS 0-1 RA patients according to one embodiment of the present invention.
Figure 3b is a diagram showing the concentrations of VEGF, PlGF, sFlt-1, and IL-6 in PDUS 1-3 RA patients and PDUS 0 RA patients according to one embodiment of the present invention.
Figure 3c is a diagram showing the concentrations of VEGF, PlGF, sFlt-1, and IL-6 in RA patients with progressive synovitis (active synovitis) and RA patients without progressive synovitis according to one embodiment of the present invention. Bar represents mean and SD. *p < 0.05, **p < 0.01.
Figure 4a is a diagram showing the concentrations of VEGF, PlGF, sFlt-1, and IL-6 in the serum of RA patients (n = 157) and OA controls (n = 50) according to one embodiment of the present invention.
Figure 4b shows the concentrations of VEGF, PlGF, sFlt-1, and IL-6 in the serum of RA patients according to one embodiment of the present invention, and the tender joint count, swollen joint count, ESR, This diagram shows the correlation between CRP, DAS28, and US scores (GSUS, PDUS) of synovitis.
Figure 4c is a diagram showing serum concentrations of VEGF, PlGF, sFlt-1, and IL-6 according to the severity of synovitis hypertrophy according to one embodiment of the present invention.
Figure 4D is a diagram showing the serum concentrations of VEGF, PlGF, sFlt-1, and IL-6 according to the degree of vascularity according to one embodiment of the present invention.
Figure 4e is a diagram showing serum concentrations of VEGF, PlGF, sFlt-1, and IL-6 according to progressive synovitis according to one embodiment of the present invention. Bar represents mean and SD. *p < 0.05, **p < 0.01, ***p < 0.001.
본 발명은 PlGF(placenta growth factor) 및 VEGF(vascular endothelial growth factor)로 이루어진 군으로부터 선택되는 하나 이상을 포함하는, 류마티스 관절염의 판누스(pannus) 진단용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for diagnosing the pannus of rheumatoid arthritis, comprising at least one selected from the group consisting of placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF).
본 발명에서 사용되는 용어 "PlGF(placental growth factor)"는 태반 성장인자로, 인간 혈관 내피 성장 인자 (VEGF) 의 혈소판-유래 성장 인자-유사 영역에 대해 매우 상동성이 있으며, VEGF 와 같이, PlGF 는 시험관내 및 생체내 혈관신생 활성을 갖는다. 본 발명에 있어서, 상기 PlGF는 이를 갖는 동물의 PlGF라면 한정되지 않으며, 예컨대 인간 또는 마우스의 PlGF일 수 있다. 상기 PlGF는 폴리펩타이드 길이 전체를 포함할 수도 있고 이의 단편을 포함할 수도 있다. 바람직하게, 상기 PlGF는 서열번호 1로 표시되는 아미노산 서열(genebank: CAA38698.1)을 포함할 수 있다. The term "placental growth factor (PlGF)" as used in the present invention is a placental growth factor, which is highly homologous to the platelet-derived growth factor-like region of human vascular endothelial growth factor (VEGF), and, like VEGF, PlGF Has angiogenic activity in vitro and in vivo. In the present invention, the PlGF is not limited as long as it is the PlGF of an animal having it, and may be, for example, PlGF of a human or mouse. The PlGF may include the entire polypeptide length or may include fragments thereof. Preferably, the PlGF may include the amino acid sequence represented by SEQ ID NO: 1 (genebank: CAA38698.1).
본 발명에서 사용되는 용어 "VEGF(vascular endothelial growth factor)"는 상피성장인자 수용체(epidermal growth factor receptor; EGFR/ErbB)의 일원이다. VEGF는 세포 증식 및 분화를 조절하는 데 필수적인 역할을 하는 것으로 알려져 있다. 구체적으로 세포 외 성장 인자가 결합하면 다른 HER 수용체와 함께 동형- 및/또는 이형이합체로 조립되는 강한 경향성을 갖고, 이는 다양한 형태의 신호 전달 경로 활성화를 초래하여, 세포자멸, 생존, 또는 세포 증식을 유도한다. 본 발명에 있어서, 상기 VEGF는 이를 갖는 동물의 VEGF라면 한정되지 않으며, 예컨대 인간 또는 마우스의 VEGF일 수 있다. 상기 VEGF는 폴리펩타이드 길이 전체를 포함할 수도 있고 이의 단편을 포함할 수도 있다. 바람직하게, 상기 VEGF는 서열번호 2로 표시되는 아미노산 서열(genebank: AAA35789.1)을 포함할 수 있다. The term “vascular endothelial growth factor (VEGF)” used in the present invention is a member of the epidermal growth factor receptor (EGFR/ErbB). VEGF is known to play an essential role in regulating cell proliferation and differentiation. Specifically, when extracellular growth factors bind, they have a strong tendency to assemble into homo- and/or heterodimers with other HER receptors, which results in the activation of various types of signaling pathways, leading to apoptosis, survival, or cell proliferation. induce. In the present invention, the VEGF is not limited as long as it is the VEGF of an animal having it, and may be, for example, VEGF of a human or mouse. The VEGF may include the entire polypeptide length or may include fragments thereof. Preferably, the VEGF may include the amino acid sequence represented by SEQ ID NO: 2 (genebank: AAA35789.1).
본 발명에서 사용되는 용어 "진단"은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 모두 포함하는 의미이다.The term "diagnosis" used in the present invention refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or condition, and determining whether an object currently has a specific disease or disorder. It is meant to include both determining the prognosis of an object suffering from , or therametrics (e.g., monitoring the condition of an object to provide information about treatment efficacy).
본 발명에서 사용되는 용어 "바이오마커(biomarker)"는 단백질이나 DNA, RNA, 대사물질 등을 이용해 몸 안의 변화를 알아낼 수 있는 지표를 의미하고, 바이오마커를 활용하면 생명체의 정상 또는 병리적인 상태, 약물에 대한 반응 정도 등을 객관적으로 측정할 수 있다. 암을 비롯해 뇌졸증, 치매 등 각종 난치병을 진단하는데 바이오마커가 활용되고 있으며, 본 발명에 있어서는 류마티스 관절염 또는 류마티스 관절염의 판누스를 진단하는데 이용될 수 있다. 또한, 신약개발과정에서 바이오마커를 활용할 수 있고, 이를 통해서 신약의 안전성을 확보할 수 있고 신약 개발 과정에서 소요되는 비용을 절감할 수 있는 효과도 얻을 수 있다.The term "biomarker" used in the present invention refers to an indicator that can detect changes in the body using proteins, DNA, RNA, metabolites, etc., and when a biomarker is used, the normal or pathological state of a living organism, The degree of response to a drug can be measured objectively. Biomarkers are used to diagnose various incurable diseases such as cancer, stroke, and dementia, and in the present invention, they can be used to diagnose rheumatoid arthritis or pannus of rheumatoid arthritis. In addition, biomarkers can be used in the new drug development process, which can ensure the safety of new drugs and reduce costs during the new drug development process.
또한, 본 발명은 상기 PlGF 또는 VEGF의 발현 또는 활성 수준을 측정하는 제제를 포함하는, 류마티스 관절염의 판누스(pannus) 진단용 조성물을 제공한다.Additionally, the present invention provides a composition for diagnosing pannus in rheumatoid arthritis, comprising an agent for measuring the expression or activity level of PlGF or VEGF.
본 발명에 있어서, 상기 "PlGF 또는 VEGF의 발현 또는 활성 수준을 측정하는 제제"는 PlGF 또는 VEGF의 단백질 또는 이를 코딩하는 mRNA에 특이적으로 결합하여, PlGF 또는 VEGF의 발현 또는 활성 수준을 확인함으로써 PlGF 또는 VEGF의 검출에 사용될 수 있는 물질을 의미한다. 예컨대, PlGF 또는 VEGF에 특이적으로 결합하는 항체, 앱타머, 리간드, PNA(peptide nucleic acid), 올리고펩티드, 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the "agent for measuring the expression or activity level of PlGF or VEGF" binds specifically to the protein of PlGF or VEGF or the mRNA encoding it, thereby confirming the expression or activity level of PlGF or VEGF. Or it refers to a substance that can be used for detection of VEGF. For example, it may be an antibody, aptamer, ligand, PNA (peptide nucleic acid), oligopeptide, antisense oligonucleotide, primer pair, or probe that specifically binds to PlGF or VEGF, but is not limited thereto.
본 발명에서 사용되는 용어 "항체"는 항원에 특이적으로 결합하여 인식하는 면역글로불린 유전자 또는 이의 단편에서 유래하는 프레임워크 영역을 포함하는 폴리펩티드를 의미한다. 인식된 면역글로불린 유전자는 카파, 람다, 알파, 감마, 델타, 엡실론, 및 뮤 불변 영역 유전자를 비롯하여, 무수한 면역글로불린 가변 영역 유전자를 포함한다. 경쇄는 카파 또는 람다로 분류되며, 중쇄는 감마, 뮤, 알파, 델타, 또는 엡실론으로 분류되고, 결과적으로 각각 면역글로불린 부류 IgG, IgM, IgA, IgD 및 IgE를 한정한다. 전형적으로, 항체의 항원-결합 영역은 결합 특이성 및 친화성에서 가장 핵심적이게 된다. 일부 실시형태에서, 항체 또는 항체의 단편은 인간, 마우스, 래트, 햄스터, 토끼, 낙타 등을 포함한, 상이한 유기체에서 유래될 수 있다.The term “antibody” used in the present invention refers to a polypeptide containing a framework region derived from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda, and heavy chains are classified as gamma, mu, alpha, delta, or epsilon, resulting in the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody is critical for binding specificity and affinity. In some embodiments, the antibody or fragment of an antibody may be derived from a different organism, including human, mouse, rat, hamster, rabbit, camel, etc.
본 발명에서 사용되는 용어 "앱타머(aptamer)"는 올리고뉴클레오타이드 물질로서, 그 자체로 안정된 삼차구조를 가지면서 표적분자에 높은 친화성과 특이성으로 결합할 수 있는 특징을 가진 단일가닥 핵산(DNA, RNA 또는 변형핵산)을 의미하며, 화학적으로 합성이 가능하고 열과 pH 변화에 대하여 비교적으로 유연한 특징을 가지고 있고, SELEX(Systematic Evolution of Ligands of Exponential enrichment)라는 방법으로 원하는 다양한 목적 물질 (단백질, 펩타이드, 지질, 무기 화합물, 저분자 유기물질, 당, 염색물질, DNA, 금속이온, 세포, 항생제 등)에 대한 앱타머를 개발할 수 있다.The term "aptamer" used in the present invention is an oligonucleotide material, which is a single-stranded nucleic acid (DNA, RNA) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. or modified nucleic acid), which can be chemically synthesized and has relatively flexible characteristics with respect to heat and pH changes, and a variety of target substances (proteins, peptides, lipids) desired through a method called SELEX (Systematic Evolution of Ligands of Exponential enrichment). , inorganic compounds, low-molecular organic substances, sugars, dyes, DNA, metal ions, cells, antibiotics, etc.).
본 발명에 있어서, 상기 "PlGF 또는 VEGF의 발현 또는 활성 수준을 측정"하는 것은 류마티스 관절염의 판누스를 진단하기 위하여 시료에서 PlGF 또는 VEGF 단백질 또는 이를 코딩하는 mRNA의 존재 여부와 발현 정도 또는 활성 정도를 확인하는 과정일 수 있으며, 예컨대 상기 PlGF 또는 VEGF 단백질 또는 이를 코딩하는 mRNA에 대하여 특이적으로 결합하는 분자를 이용하여 단백질 또는 이를 코딩하는 mRNA의 양 또는 활성 수준을 확인하는 방법이 적용될 수 있다. In the present invention, the "measuring the expression or activity level of PlGF or VEGF" refers to the presence and expression or activity level of PlGF or VEGF protein or mRNA encoding the same in the sample in order to diagnose pannus of rheumatoid arthritis. It may be a confirmation process, and for example, a method of confirming the amount or activity level of the protein or the mRNA encoding it using a molecule that specifically binds to the PlGF or VEGF protein or the mRNA encoding it may be applied.
본 발명에 있어서, 상기 PlGF 또는 VEGF의 발현 또는 활성 수준은 종래에 개발된 다양한 정량적 또는 정성적 면역분석 프로토콜에 따라 확인할 수 있다. 예컨대, 상기 면역분석 포맷은 효소면역분석법(ELISA), 방사능면역분석법(radioimmnoassay, RIA), 샌드위치 측정법(sandwich assay), 웨스턴 블롯팅, 면역침강법, 면역조직화학염색법(immnohistochemical staining), 유체 세포 측정법(flow cytometry), 형광활성화 세포분류법(FACS), 효소기질발색법 및 항원-항체 응집법을 포함한다. 본 발명의 일 구현예에 따르면 효소면역분석법(ELISA)을 이용하여 PlGF 또는 VEGF의 발현 또는 활성 수준을 확인할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the expression or activity level of PlGF or VEGF can be confirmed according to various quantitative or qualitative immunoassay protocols developed in the past. For example, the immunoassay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, and fluid cytometry. (flow cytometry), fluorescence activated cell sorting (FACS), enzyme matrix colorimetric method, and antigen-antibody agglutination method. According to one embodiment of the present invention, the expression or activity level of PlGF or VEGF can be confirmed using enzyme-linked immunosorbent assay (ELISA), but is not limited thereto.
또한, 본 발명은 활액에서의 PlGF(placental growth factor) 발현 또는 활성 수준; 또는 혈청에서의 VEGF(vascular endothelial growth factor) 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스(pannus) 진단을 위한 정보제공방법을 제공한다.In addition, the present invention relates to the level of placental growth factor (PlGF) expression or activity in synovial fluid; Alternatively, a method of providing information for diagnosing pannus of rheumatoid arthritis is provided, including the step of confirming the expression or activity level of vascular endothelial growth factor (VEGF) in serum.
또한, 본 발명은 활액에서의 PlGF 발현 또는 활성 수준; 또는 혈청에서의 VEGF 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스(pannus) 진단 방법을 제공한다.Additionally, the present invention relates to PlGF expression or activity levels in synovial fluid; Alternatively, it provides a method for diagnosing pannus of rheumatoid arthritis, including the step of confirming the level of VEGF expression or activity in serum.
본 발명에 있어서, 상기 방법은 활액에서의 PlGF 발현 또는 활성 수준이 정상 대조군에 비해 증가되어 있고, WBC counts, %호중구, ESR, CRP, GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상의 수준과 양의 상관관계를 나타낼 경우; 또는 In the present invention, the method involves increasing the level of PlGF expression or activity in synovial fluid compared to the normal control group, and at least one level and amount selected from the group consisting of WBC counts, % neutrophils, ESR, CRP, GSUS, and PDUS. If it shows a correlation of; or
혈청에서의 VEGF 발현 또는 활성 수준이 정상 대조군에 비해 증가되어 있고, TJC, SJC, ESR, CRP, DAS 28, GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상의 수준과 양의 상관관계를 나타낼 경우 판누스 증식 정도가 높은 것으로 진단하는 단계를 더 포함할 수 있다.If the level of VEGF expression or activity in serum is increased compared to normal controls and shows a positive correlation with one or more levels selected from the group consisting of TJC, SJC, ESR, CRP, DAS 28, GSUS, and PDUS, the plate A step of diagnosing that the degree of Nus proliferation is high may be further included.
본 발명에서 사용되는 용어 "상관관계"는 두 변량 중 한 쪽이 증가함에 따라 다른 한 쪽이 증가 또는 감소할 때 두 변량의 관계를 의미한다. 한 쪽이 증가함에 따라 다른 한 쪽이 증가하는 경우 양의 상관관계, 한 쪽이 증가함에 따라 다른 한 쪽이 감소하는 경우 음의 상관관계라고 한다.The term "correlation" used in the present invention refers to a relationship between two variables when one of the two variables increases or decreases as the other increases. If one side increases and the other side increases, it is called a positive correlation. If one side increases and the other side decreases, it is called a negative correlation.
본 발명에 있어서, PlGF는 류마티스 관절염 환자의 활액에서 발현 또는 활성 수준이 증가하며, 상기 PlGF의 발현 또는 활성 수준이 증가함에 따라 활액에서의 WBC(white blood cell) counts, %호중구, ESR(erythrocyte sedimentation rate), CRP(C reactive protein), GSUS(Grey scale ultrasonography), 및 PDUS(power-Doppler ultrasonography)로 이루어진 군으로부터 선택되는 하나 이상의 수준이 증가하여 PlGF와 양의 상관관계를 가질 수 있다. In the present invention, the expression or activity level of PlGF increases in the synovial fluid of patients with rheumatoid arthritis, and as the expression or activity level of PlGF increases, the white blood cell (WBC) counts, % neutrophils, and erythrocyte sedimentation (ESR) in the synovial fluid increase. Rate), C reactive protein (CRP), gray scale ultrasonography (GSUS), and power-Doppler ultrasonography (PDUS) levels may increase and have a positive correlation with PlGF.
또한, VEGF는 류마티스 관절염 환자의 혈청에서 발현 또는 활성 수준이 증가하며, 상기 VEGF의 발현 또는 활성 수준이 증가함에 따라 혈청에서의 TJC(tender joint count), SJC(swollen joint count), ESR, CRP, DAS 28(disease activity score 28), GSUS, 및 PDUS로 이루어진 군으로부터 선택되는 하나 이상의 수준이 증가하여 VEGF와 양의 상관관계를 가질 수 있다.In addition, the expression or activity level of VEGF increases in the serum of rheumatoid arthritis patients, and as the expression or activity level of VEGF increases, tender joint count (TJC), swollen joint count (SJC), ESR, CRP, One or more levels selected from the group consisting of DAS 28 (disease activity score 28), GSUS, and PDUS may increase and have a positive correlation with VEGF.
본 발명에 있어서, 상기 "판누스의 증식"은 활막에 염증이 있다고도 표현하는데, 이를 활막염(synovitis)이라고 한다. 이 활막염의 중증도(severity)를 측정함에 있어서 초음파(ultrasonography, US)를 이용할 수 있으며, 예컨대 GSUS(Grey scale ultrasonoraphy)를 이용하여 활막염의 중증도, 즉 판누스의 증식 정도를 나타낼 수 있다. 또한, 도플러 효과를 이용하여 유체의 흐름을 추가 확인할 수 있는 PDUS(power-Doppler ultrasonography)는 증식된 활막에서 혈관형성 정도를 나타낼 수 있다.In the present invention, the "proliferation of the pannus" is also expressed as inflammation of the synovial membrane, which is called synovitis. Ultrasonography (US) can be used to measure the severity of this synovitis. For example, GSUS (Grey scale ultrasonoraphy) can be used to indicate the severity of synovitis, that is, the degree of proliferation of the pannus. Additionally, PDUS (power-Doppler ultrasonography), which can further confirm fluid flow using the Doppler effect, can indicate the degree of vascularization in the proliferated synovium.
또한, 본 발명은 류마티스 관절염 환자로부터 활액 또는 혈청을 분리하여 후보물질을 처리하는 단계; 및In addition, the present invention includes the steps of separating synovial fluid or serum from a patient with rheumatoid arthritis and processing a candidate material; and
상기 후보물질 처리 후 류마티스 관절염 환자의 활액에서의 PlGF 발현 또는 활성 수준, 또는 혈청에서의 VEGF 발현 또는 활성 수준을 확인하는 단계를 포함하는, 류마티스 관절염의 판누스 억제용 물질의 스크리닝 방법을 제공한다.Provided is a screening method for a substance for inhibiting pannus in rheumatoid arthritis, comprising the step of confirming the expression or activity level of PlGF in synovial fluid or the level of VEGF expression or activity in serum of a patient with rheumatoid arthritis after treatment with the candidate substance.
본 발명에서 사용되는 용어 “후보물질”은 류마티스 관절염 환자의 판누스 억제 여부를 확인하기 위하여 스크리닝에 사용되는 미지의 물질을 의미하며, 본 발명의 일 구현예에 따르면, 상기 류마티스 관절염 판누스 억제용 후보물질은 뉴클레오티드, DNA, RNA, 아미노산, 앱타머, 단백질, 화합물, 천연물, 및 천연추출물로 이루어진 군으로부터 선택되는 하나 이상일 수 있으나, 이에 제한은 없다.The term “candidate substance” used in the present invention refers to an unknown substance used in screening to confirm whether or not the pannus is inhibited in rheumatoid arthritis patients. According to one embodiment of the present invention, the drug for inhibiting the pannus of rheumatoid arthritis patients The candidate material may be one or more selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts, but is not limited thereto.
본 발명에 있어서, 상기 후보물질 처리 후 류마티스 관절염 환자의 활액에서의 PlGF 발현 또는 활성 수준이 후보물질 처리 전에 비해 감소한 경우; 또는In the present invention, when the level of PlGF expression or activity in the synovial fluid of a rheumatoid arthritis patient after treatment with the candidate substance is reduced compared to before treatment with the candidate substance; or
혈청에서의 VEGF 발현 또는 활성 수준이 후보물질 처리 전에 비해 감소한 경우, 후보물질을 류마티스 관절염의 판누스 억제용 물질로 선택하는 단계를 더 포함할 수 있다.If the level of VEGF expression or activity in serum is reduced compared to before treatment with the candidate material, a step of selecting the candidate material as a substance for inhibiting pannus in rheumatoid arthritis may be further included.
본 발명의 일 실험예에서는 활막세포에서 주로 분비되는 혈관신생유발 인자인 PlGF 및 IL-6 수준이 RA 환자의 SF에서 증가되어 있으며, PlGF 및 IL-6 수준과 전신 염증 반응을 나타내는 마커인 혈액 내 ESR 및 CRP 수준, SF 내 WBC counts 및 %호중구, 판누스의 증식 정도를 나타내는 GSUS 및 혈관형성 정도를 나타내는 PDUS의 상관관계를 확인함으로써, PlGF 및 IL-6 수준과 RA 환자에서의 활막염 중증도 및 전신 염증 반응이 상관관계를 가지는 것을 확인하였다(실험예 1 참조).In one experimental example of the present invention, the levels of PlGF and IL-6, which are angiogenic factors mainly secreted from synovial cells, were increased in the SF of RA patients, and the levels of PlGF and IL-6 and blood, which are markers of systemic inflammatory response, were increased. By confirming the correlation between ESR and CRP levels, WBC counts and % neutrophils in SF, GSUS indicating the degree of proliferation of the pannus, and PDUS indicating the degree of angiogenesis, the level of PlGF and IL-6 and the severity and systemic inflammation of synovitis in RA patients were determined. It was confirmed that the inflammatory response was correlated (see Experimental Example 1).
본 발명의 다른 실험예에서는 VEGF 및 IL-6 수준이 RA 환자의 혈청에서 증가되어 있으며, VEGF 및 IL-6 수준과 TJC, SJC, ESR, CRP 및 DAS28 점수, GSUS 및 PDUS의 상관관계를 확인함으로써, 상기 VEGF 및 IL-6의 혈청 수준이 활막 증식 및 초음파 검사(US) 상 과맥관질(hyper-vascularity)을 나타내는 것을 확인하였다(실험예 2 참조).In another experimental example of the present invention, VEGF and IL-6 levels were increased in the serum of RA patients, and by confirming the correlation between VEGF and IL-6 levels and TJC, SJC, ESR, CRP and DAS28 scores, GSUS and PDUS , it was confirmed that the serum levels of VEGF and IL-6 showed synovial proliferation and hyper-vascularity on ultrasonography (US) (see Experimental Example 2).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
실시예Example 1. One. 활액synovial fluid 및 혈청 채취 and serum collection
류마티스 관절염(RA) 환자(n=157)는 2010 ACR/EULAR RA 분류 기준을 충족하였다. 활액(synovial fluid, 이하 SF) 시료는 부은 관절(swollen joint)에 대한 관절강세정술(arthrocentesis)을 시술받은 RA 환자(n=54) 및 골관절염(osteoarthritis, OA) 대조군(n=30)으로부터 채취하였으며, 상기 관절강세정술을 시술받은 RA 환자들의 특징을 하기 표 1에 나타내었다.Rheumatoid arthritis (RA) patients (n=157) met the 2010 ACR/EULAR RA classification criteria. Synovial fluid (SF) samples were collected from RA patients (n=54) and osteoarthritis (OA) controls (n=30) who underwent arthrocentesis for swollen joints. , The characteristics of RA patients who underwent the joint space irrigation are shown in Table 1 below.
상기 표 1에서 데이터는 연속 변수에 대한 평균(표준편차) 또는 중앙값(interquartile range)으로 표시된다. RF: 류마티스성 인자(rheumatoid factor), ACPA: 항-시트룰린화 펩티드 항체(anti-citrullinated peptide antibodies), WBC: 백혈구(white blood cell), ESR: 적혈구 침강 속도(erythrocyte sedimentation rate), CRP: C-반응성 단백질(C-reactive protein), SF: 활액(synovial fluid), PD: prednisone, GSUS: grey scale ultrasound, PDUS: power Doppler ultrasound.In Table 1 above, data are expressed as mean (standard deviation) or median (interquartile range) for continuous variables. RF: rheumatoid factor, ACPA: anti-citrullinated peptide antibodies, WBC: white blood cell, ESR: erythrocyte sedimentation rate, CRP: C- C-reactive protein, SF: synovial fluid, PD: prednisone, GSUS: gray scale ultrasound, PDUS: power Doppler ultrasound.
혈청 시료 역시 연구 개시시점에서 RA 환자(n=157) 및 OA 대조군(n=50)으로부터 채취하였다. 157명의 RA 환자 중에서, 중등도(moderate) 또는 중증(high)의 질병활성도를 갖는 환자는 103명, 낮은 질병활성도를 갖는 환자는 54명으로 분류되었고, 이들의 특징을 하기 표 2에 나타내었다. Serum samples were also collected from RA patients (n=157) and OA controls (n=50) at study initiation. Among 157 RA patients, 103 patients had moderate or high disease activity and 54 patients had low disease activity, and their characteristics are shown in Table 2 below.
DA: 질병활성도(disease activity), BMI: 체질량 지수(body mass index)본 발명은 가톨릭대학교 병원 임상연구심의위원회로부터 승인 받았으며(No.KC14TIMI0697), 연구 참여자로부터 서면으로 동의를 받았다.DA: disease activity, BMI: body mass index This invention was approved by the Clinical Research Review Committee of Catholic University Hospital (No. KC14TIMI0697), and written consent was obtained from the study participants.
실시예Example 2. 2. VEGFVEGF , , PlGFPlGF , , sFltsFlt -1 및 IL-6 측정-1 and IL-6 measurements
증식하는 활막세포에서 주로 분비되는 주요한 혈관신생 유발인자인 VEGF, PlGF 및 IL-6의 농도를 RA 및 OA 환자의 SF 및 혈청에서 알려진 방법(Yoo SA, Kim M, Kang MC, et al. Nat Immunol 2019;20:1348-59.)에 따라 ELISA(enzyme-linked immunosorbent assay)로 측정하였다. 대조군으로, 활막세포에서 생산되는 항-혈관신생 분자인 sFlt-1의 수준을 동일 시료에 대하여 ELISA로 측정하였다. ELISA의 VEGF, PlGF, IL-6 및 sFlt-1 검출한계는 10 pg/ml이었다.The concentrations of VEGF, PlGF, and IL-6, which are major angiogenesis factors secreted mainly by proliferating synovial cells, were measured in SF and serum of RA and OA patients using a known method (Yoo SA, Kim M, Kang MC, et al. Nat Immunol 2019;20:1348-59.) was measured by ELISA (enzyme-linked immunosorbent assay). As a control, the level of sFlt-1, an anti-angiogenic molecule produced in synovial cells, was measured by ELISA on the same sample. The detection limit of ELISA for VEGF, PlGF, IL-6, and sFlt-1 was 10 pg/ml.
실시예Example 3. 활막염 중증도( 3. Synovitis severity ( synovitissynovitis severity) 평가를 위한 관절의 초음파 검사 평가 Ultrasound evaluation of joints to assess severity
상기 실시예 1에서 SF 시료를 채취한 RA 환자(n=54)에 대하여 근골격계 초음파 검사(US)를 실시하였다. 추가적으로, 연구 개시시점에서 관절강세정술 및/또는 관절 내 스테로이드 주사가 필요한 명확한 관절 종창 및 압통을 가진 RA 환자(n=68)의 경우, 혈액을 채취할 때 관절에 대한 초음파 검사 역시 실시하였다. 활막염(synovitis) 점수는 도 1에 나타낸 Grey scale US(GSUS) (도 1의 A 및 B) 및 power-Doppler US(PDUS) (도 1의 C 및 D) 이미지로 결정하였다. 이 때 GSUS 이미지는 근위지간(proximal interphalangeal, PIP) 관절 내 활막염 비대를 나타내며, PDUS 이미지는 PIP 관절 내 활막의 과맥관질을 나타낸다. GSUS 점수(0-3 range)는 다음과 같이 활막 비대(synovial hypertrophy)의 정도에 따라 정의하였다: grade 0 = 활막 비대 없음, grade 1 = 최소 활막 비대, grade 2 = 중등도(moderate) 활막 비대, 및 grade 3 = 중증(severe) 활막 비대. PDUS 점수는(0-3 range) 다음과 같이 관절의 활막 내 맥관질(vascularity)의 정도로 평가하였다: grade 0 = Doppler 활성 없음, grade 1 = 최소 Doppler 활성, grade 2 = 중등도(moderate) Doppler 활성(background 활막의 <50%), 및 grade 3 = 중증(severe) Doppler 활성 (background 활막의 >50%). GSUS가 ≥2이거나, PDUS가 ≥1인 경우, 진행성(active) 활막염을 갖는 환자로 고려하였다.Musculoskeletal ultrasound (US) was performed on RA patients (n=54) from whom SF samples were collected in Example 1. Additionally, for RA patients (n=68) with definite joint swelling and tenderness requiring joint space irrigation and/or intra-articular steroid injection at study entry (n=68), ultrasound examination of the joint was also performed at the time of blood draw. The synovitis score was determined using the Gray scale US (GSUS) (A and B in Figure 1) and power-Doppler US (PDUS) (C and D in Figure 1) images shown in Figure 1. At this time, the GSUS image shows synovitis hypertrophy within the proximal interphalangeal (PIP) joint, and the PDUS image shows hypervascularity of the synovium within the PIP joint. The GSUS score (0-3 range) was defined according to the degree of synovial hypertrophy as follows: grade 0 = no synovial hypertrophy, grade 1 = minimal synovial hypertrophy, grade 2 = moderate synovial hypertrophy, and grade 3 = severe synovial hypertrophy. The PDUS score (range 0-3) evaluated the degree of vascularity within the synovium of the joint as follows: grade 0 = no Doppler activity, grade 1 = minimal Doppler activity, grade 2 = moderate Doppler activity ( <50% of background synovium), and grade 3 = severe Doppler activity (>50% of background synovium). Patients were considered to have active synovitis if GSUS was ≥2 or PDUS was ≥1.
실시예Example 4. 임상 파라미터 평가 4. Evaluation of clinical parameters
RA 환자의 ESR과 CRP 수준, 압통 관절 수(tender joint count, TJC) 및 부종 관절 수(swollen joint count, SJC)를 연구 개시시점과 그 이후에 측정하였다. 상기 TJC는 관절의 통증을, SJC는 붓기를 의미한다. 또한, 기존에 혈액에서 측정되던 염증 수치인 ESR 및 CRP 수준도 함께 검토되었으며, 이에 더하여 류마티스 인자(rheumatoid factor, RF) 및 ACPA(anti-citrullinated peptide antibodies)를 포함하는 혈청학적 마커에 대한 데이터를 얻었다. 손과 발에 대한 방사선 촬영 검사를 실시하여, 관절열극(joint space)의 좁은 정도와 침식 정도를 van der Heijde-modified Sharp score (vdH Sharp score)에 따라 점수화 하였다. RA 환자의 질병활성도를 질환(disease) 활성도(activity) 점수 28ESR (DAS28ESR)에 기초하여 결정하였다: DAS28≤3.2 = 낮은 질병활성도; DAS28>3.2 = 중등도(moderate) 또는 중증(high) 질병활성도. RA patients' ESR and CRP levels, tender joint count (TJC), and swollen joint count (SJC) were measured at study initiation and thereafter. TJC refers to joint pain, and SJC refers to swelling. In addition, ESR and CRP levels, which are inflammation levels previously measured in blood, were also reviewed, and in addition, data on serological markers including rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) were obtained. . Radiographic examination of the hands and feet was performed, and the degree of narrowing and erosion of the joint space was scored according to the van der Heijde-modified Sharp score (vdH Sharp score). Disease activity of RA patients was determined based on the disease activity score 28 ESR (DAS28 ESR ): DAS28≤3.2 = low disease activity; DAS28>3.2 = moderate or high disease activity.
실시예Example 5. 통계 분석 5. Statistical analysis
연속 변수의 비교를 student’s unpaired t 테스트 또는 Mann-Whitney U 테스트로 조사하였으며, 범주형 변수의 비교를 Chi-Square 테스트 또는 Fisher’s exact 테스트로 조사하였다. 두 변수 사이의 상관관계를 Pearson’s correlation 테스트로 분석하였다. 동일 대상에서 연구 개시시점과 그 이후의 측정 값 사이의 차이를 조사하기 위하여 paired t test를 적용하였다. 통계 분석은 IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, USA)을 사용하여 실시하였고, 그래프는 GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA)를 사용하여 그렸다. 히트맵 상관관계 이미지(heatmap correlation image)는 R software 3.5.1 (R Project, Vienna, Austria)의 package ‘corrplot’를 사용하여 얻었다.Comparisons of continuous variables were examined using the student’s unpaired t test or Mann-Whitney U test, and comparisons of categorical variables were examined using the Chi-Square test or Fisher’s exact test. The correlation between two variables was analyzed using Pearson’s correlation test. A paired t test was applied to investigate the differences between the measurement values at the start of the study and thereafter in the same subject. Statistical analysis was performed using IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, USA), and graphs were drawn using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Heatmap correlation image was obtained using package ‘corrplot’ of R software 3.5.1 (R Project, Vienna, Austria).
실험예Experiment example 1. One. PlGFPlGF 및 IL-6 수준과 RA and IL-6 levels and RA. 환자에서의in patient 활막염 중증도 및 전신 염증 반응의 상관관계 확인 Confirmation of correlation between synovitis severity and systemic inflammatory response
RA 환자의 SF 내 VEGF, PlGF, sFlt-1 및 IL-6 농도는 도 2a에 나타낸 바와 같이 OA 대조군에 비하여 유의성 있게 높은 것을 확인하였다. 또한, 활막세포에서 주로 분비되는 혈관신생유발 사이토카인인 VEGF, PlGF 및 IL-6 수준이 RA 환자에서 국소 및/또는 전신 염증 반응을 나타내는지 테스트 한 결과, 도 2b 및 상기 표 1에 나타난 바와 같이, SF(n=54) 내 PlGF 농도는 RA에서 전신 염증 반응을 나타내는 마커인 혈액 내 ESR 및 CRP 수준뿐만 아니라, SF 내 WBC counts(r = 0.466 and p = 0.001) 및 %호중구와 상관관계를 나타내었다. 대조군으로 사용한 항-혈관신생 단백질인 sFlt-1의 수준 또한 혈액 내 염증성 마커와 양의 상관관계를 나타내었다(ESR: r = 0.364, p = 0.007; CRP: r = 0.496, p <0.001). 그러나, SF 내 VEGF와 IL-6 수준은 SF 내 WBC counts 및 %호중구와 양의 상관관계를 보였지만, ESR 및 CRP와는 상관관계를 보이지 않았다.The concentrations of VEGF, PlGF, sFlt-1, and IL-6 in the SF of RA patients were confirmed to be significantly higher than those of the OA control group, as shown in Figure 2a. In addition, we tested whether the levels of VEGF, PlGF, and IL-6, which are proangiogenic cytokines mainly secreted from synovial cells, indicate local and/or systemic inflammatory responses in RA patients, as shown in Figure 2b and Table 1 above. , PlGF concentration in SF (n = 54) correlated with ESR and CRP levels in the blood, which are markers of systemic inflammatory response in RA, as well as WBC counts (r = 0.466 and p = 0.001) and % neutrophils in SF. It was. The level of sFlt-1, an anti-angiogenic protein used as a control, also showed a positive correlation with inflammatory markers in the blood (ESR: r = 0.364, p = 0.007; CRP: r = 0.496, p <0.001). However, VEGF and IL-6 levels in SF showed a positive correlation with WBC counts and % neutrophils in SF, but did not show correlation with ESR and CRP.
다음으로, 혈관신생유발 사이토카인이 RA 환자에서 초음파 검사(US)로 결정한 활막염 중증도를 반영하는지 알아보았다. 그 결과, 도 3a에 나타낸 바와 같이 SF 내 PlGF 및 IL-6 농도는 GSUS 상 중증도에서 중증 활막염 비대(grade 2 및 3)를 갖는 환자에서 활막 비대가 없거나 약한(mild) 활막 비대(grade 0 및 1)를 갖는 환자에 비하여 높았다. 또한, PDUS 검사 결과, 도 3b에 나타낸 바와 같이 PlGF 농도는 증가된 맥관질(vascularity, grade 1 to 3)을 갖는 환자에서 그렇지 않은 환자(grade 0)에 비하여 높았으나, 다음 세 분자들은 증가된 맥관질에 따른 유의한 차이를 보이지 않았다. 진행성 활막염의 존재를 기준(GSUS ≥2 또는 PDUS ≥1)으로 환자를 두 그룹으로 분류한 경우, 도 3c에 나타낸 바와 같이 VEGF와 sFlt-1은 아니었지만, PlGF 및 IL-6 수준은 비진행성 활막염을 갖는 환자에 비하여 진행성 활막염을 갖는 환자에서 상당히 높은 것을 확인할 수 있었다.Next, we investigated whether proangiogenic cytokines reflect synovitis severity determined by ultrasonography (US) in RA patients. As a result, as shown in Figure 3a, the concentrations of PlGF and IL-6 in SF were similar to those with no or mild synovial hypertrophy (grades 0 and 1) in patients with moderate to severe synovitis hypertrophy (grades 2 and 3) on GSUS. ) was higher than that of patients with In addition, as a result of PDUS examination, as shown in Figure 3b, the concentration of PlGF was higher in patients with increased vascularity (grade 1 to 3) than in patients without (grade 0), but the following three molecules were associated with increased vascularity. There was no significant difference according to quality. When patients were divided into two groups based on the presence of progressive synovitis (GSUS ≥2 or PDUS ≥1), levels of PlGF and IL-6, but not VEGF and sFlt-1, were significantly higher in patients with non-progressive synovitis, as shown in Figure 3C. It was confirmed that it was significantly higher in patients with progressive synovitis compared to patients with .
종합하면, 상기와 같은 결과는 활막세포에서 주로 분비되는 혈관신생유발 인자인 PlGF 및 IL-6 수준이 RA 환자의 SF에서 증가되어 있으며, RA 환자의 국소 및 전신 염증 상태뿐만 아니라 활막염 중증도를 나타냄을 보여준다.In summary, the above results show that the levels of PlGF and IL-6, which are angiogenic factors mainly secreted by synovial cells, are increased in the SF of RA patients, indicating the severity of synovitis as well as the local and systemic inflammatory status in RA patients. It shows.
실험예Experiment example 2. 2. VEGFVEGF 및 IL-6의 and IL-6 순환성circularity 수준과 RA의 질병활성도 및 중증도의 상관관계 확인 Verification of correlation between level and disease activity and severity of RA
낮은 질병활성도를 갖는 54명의 환자 및 중등도 또는 중증 질병활성도를 갖는 103명의 환자로 구성된 총 157명의 RA 환자로부터 상기 실시예 1과 같이 혈청 시료를 채취하였으며, 연구 개시시점에서의 의료정보(demographic) 및 질환 특성을 상기 표 2에 나타내었다. 먼저, 혈청 VEGF, PlGF, sFlt-1 및 IL-6 농도를 확인한 결과, 도 4a에 나타낸 바와 같이 RA 환자에서 상기 인자들의 농도가 증가되어 있음을 확인하였다. 또한, RA 질병활성도에 대한 파라미터와 달리, 도 4b에 나타낸 바와 같이 VEGF 및 IL-6 수준은 TJC, SJC, ESR, CRP 및 DAS28 점수와 상관관계를 보였다. Serum samples were collected as in Example 1 above from a total of 157 RA patients, consisting of 54 patients with low disease activity and 103 patients with moderate or severe disease activity, and medical information (demographic) and Disease characteristics are shown in Table 2 above. First, as a result of checking the serum VEGF, PlGF, sFlt-1, and IL-6 concentrations, it was confirmed that the concentrations of the above factors were increased in RA patients, as shown in Figure 4a. Additionally, unlike parameters for RA disease activity, VEGF and IL-6 levels correlated with TJC, SJC, ESR, CRP, and DAS28 scores, as shown in Figure 4B.
추가적으로, 도 4b의 히트맵 및 상관관계 플롯에 나타난 바와 같이, 혈청 VEGF 및 IL-6는 GSUS 및 PDUS 점수 모두와 양의 상관관계를 보였다. 더욱이, 혈청 VEGF 및 IL-6 농도는 도 4c에 나타낸 GSUS 상 중등도에서 중증 활막 비대를 갖는 환자 및 도 4d에 나타낸 PDUS 상 증가된 맥관질을 갖는 환자에서 그렇지 않은 환자에 비하여 높았으며, 혈청 VEGF 및 IL-6 농도는 도 4e에 나타낸 바와 같이 진행성 활막염과 유의성 있게 관련성을 보였다.Additionally, as shown in the heatmap and correlation plot in Figure 4B, serum VEGF and IL-6 were positively correlated with both GSUS and PDUS scores. Moreover, serum VEGF and IL-6 concentrations were higher in patients with moderate to severe synovial hypertrophy on GSUS shown in Figure 4C and in patients with increased vascularity on PDUS shown in Figure 4D compared to patients without, IL-6 concentration was significantly associated with progressive synovitis, as shown in Figure 4e.
상기 실험예 1의 SF 데이터와는 대조적으로, 혈청 PlGF 수준은 TJC, SJC, CRP 또는 DAS28 점수와 상관관계를 보이지 않았으며(도 4b 참조), 혈청 PlGF 농도는 초음파 검사상 중증도에 따른 차이를 보이지 않았다. 항-혈관신생 단백질인 sFlt-1의 혈청 수준은 ESR 및 DAS28과 단지 보통 정도(modest)의 상관관계만을 보였으며, TJC, SJC, CRP 또는 GSUS와 PDUS 상 활막염 중증도와 어떠한 관련성을 보이지 않았다.In contrast to the SF data of Experimental Example 1, serum PlGF levels did not show any correlation with TJC, SJC, CRP, or DAS28 scores (see Figure 4b), and serum PlGF concentrations did not show differences according to severity on ultrasound examination. didn't Serum levels of the anti-angiogenic protein sFlt-1 showed only a moderate correlation with ESR and DAS28 and no correlation with synovitis severity on TJC, SJC, CRP, or GSUS and PDUS.
종합하면, VEGF 및 IL-6의 혈청 수준은 혈청 PlGF와 혈청 sFlt1과 달리, 활막 증식 및 초음파 검사(US) 상 과맥관질(hyper-vascularity)을 나타내는 것이며, 이는 TJC, SJC, ESR, CRP 및 DAS28 점수로 평가한 RA의 전신 염증 상태를 반영하는 것임을 알 수 있었다.Taken together, serum levels of VEGF and IL-6, unlike serum PlGF and serum sFlt1, are indicative of synovial hyperplasia and hyper-vascularity on ultrasound (US), which is associated with TJC, SJC, ESR, CRP and It was found that it reflects the systemic inflammatory state of RA evaluated by the DAS28 score.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.
<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Biomarker for diagnosing pannus of rheumatoid arthritis and use thereof <130> MP19-289 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 149 <212> PRT <213> Homo sapiens <400> 1 Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp 130 135 140 Ala Val Pro Arg Arg 145 <210> 2 <211> 191 <212> PRT <213> Homo sapiens <400> 2 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190 <110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Biomarker for diagnosing pannus of rheumatoid arthritis and use of that <130> MP19-289 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 149 <212> PRT <213> Homo sapiens <400> 1 Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp 130 135 140 Ala Val Pro Arg Arg 145 <210> 2 <211> 191 <212> PRT <213> Homo sapiens <400> 2 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190
Claims (11)
상기 PlGF 및 VEGF는 GSUS(Grey scale ultrasonography) 점수와 양의 상관관계를 가지고,
상기 피검체로부터 채취된 활액 시료에서의 PlGF 단백질 또는 이를 코딩하는 mRNA의 발현 수준, 또는 혈청 시료에서의 VEGF 단백질 또는 이를 코딩하는 mRNA의 발현 수준이 상기 GSUS 점수가 grade 0 내지 1인 류마티스 관절염 환자에 비해 높을 경우, 진행성 활막염을 가지고 판누스의 증식 정도가 중등도에서 중증인 것으로 판단하는 단계를 더 포함하는 것을 특징으로 하는, 정보제공방법.
Confirming the expression level of PlGF (placental growth factor) protein or mRNA encoding it in a synovial fluid sample collected from the subject, or the expression level of VEGF (vascular endothelial growth factor) protein or mRNA encoding it in a serum sample. As an information provision method for diagnosing the degree of pannus proliferation in rheumatoid arthritis, including,
The PlGF and VEGF have a positive correlation with the GSUS (Grey scale ultrasonography) score,
The expression level of the PlGF protein or the mRNA encoding it in the synovial fluid sample collected from the subject, or the expression level of the VEGF protein or the mRNA encoding it in the serum sample was measured in rheumatoid arthritis patients with a GSUS score of grade 0 to 1. If it is higher than that, the method of providing information further comprises the step of determining that the degree of proliferation of the pannus is moderate to severe with progressive synovitis.
상기 PlGF는 피검체로부터 채취된 활액(synovial fluid)에서 WBC counts, %호중구, ESR(erythrocyte sedimentation rate), 및 CRP(C reactive protein)로 이루어진 군으로부터 선택되는 하나 이상과 양의 상관관계를 가지는 것을 특징으로 하는, 정보제공방법.
According to clause 6,
The PlGF has a positive correlation with one or more selected from the group consisting of WBC counts, % neutrophils, ESR (erythrocyte sedimentation rate), and CRP (C reactive protein) in synovial fluid collected from the subject. Characterized by a method of providing information.
상기 VEGF는 피검체로부터 채취된 혈청(serum)에서 TJC(tender joint count), SJC(swollen joint count), ESR, CRP, 및 DAS 28(disease activity score 28)로 이루어진 군으로부터 선택되는 하나 이상과 양의 상관관계를 가지는 것을 특징으로 하는, 정보제공방법.
According to clause 6,
The VEGF is one or more selected from the group consisting of TJC (tender joint count), SJC (swollen joint count), ESR, CRP, and DAS 28 (disease activity score 28) in the serum collected from the subject. An information provision method characterized by having a correlation of.
상기 발현 수준은 활액 PlGF 및 혈청 VEGF로 이루어진 군으로부터 선택되는 하나 이상의 단백질; 또는 이를 코딩하는 mRNA에 특이적으로 결합하는 항체, 앱타머, 리간드, PNA(peptide nucleic acid), 올리고펩티드, 안티센스 올리고뉴클레오티드, 프라이머 쌍 및 프로브로 이루어진 군으로부터 선택되는 하나 이상을 이용하여 확인하는 것을 특징으로 하는, 정보제공방법.According to clause 6,
The expression level is one or more proteins selected from the group consisting of synovial fluid PlGF and serum VEGF; Or, confirmation using one or more selected from the group consisting of antibodies, aptamers, ligands, PNA (peptide nucleic acids), oligopeptides, antisense oligonucleotides, primer pairs, and probes that specifically bind to the mRNA encoding the same. Characterized by a method of providing information.
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