CN103513040B - Application of protein CD38 to preparation of rheumatoid arthritis diagnosing marker - Google Patents
Application of protein CD38 to preparation of rheumatoid arthritis diagnosing marker Download PDFInfo
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- CN103513040B CN103513040B CN201310483384.2A CN201310483384A CN103513040B CN 103513040 B CN103513040 B CN 103513040B CN 201310483384 A CN201310483384 A CN 201310483384A CN 103513040 B CN103513040 B CN 103513040B
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- cell
- rheumatoid arthritis
- monoclonal antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Abstract
The invention discloses a novel rheumatoid arthritis diagnosing marker-protein CD38. Through flow cytometrical experiments, the applicant of the invention proves that compared with normal subjects, CD38+ cells are highly expressed in peripheral blood lymphocytes of a RA (rheumatoid arthritis) patient, and the expression levels of the CD38+ cells are significantly related to the RF (rheumatoid factor) level. In addition, flow cytometrical results show that the ratio of a CD38+ cell subset to a CD56+ cell subset is also significantly increased and the CD56 is a cell surface marker of a natural killer cell (NK cell), which means that the CD38 plays an important role in autoimmune disorder of the RA and can be used as the rheumatoid arthritis diagnosing marker. A kit for detecting the rheumatoid arthritis consists of CD3 marked by FITC, a CD4 monoclonal antibody, a CD56 monoclonal antibody marked by PE, and a CD38 monoclonal antibody marked by Percp-Cy5.5.
Description
Technical field
The present invention relates to PROTEIN C D38 as/prepare application in diagnosis of rheumatoid arthritis label.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of autoimmune disease, and the incidence of disease is 3/1000ths. five.At present, clinical is all resist (anti-CCP) to provide clinical diagnosis foundation for RA by detecting rheumatoid factor (rheumatic factor, RF) in peripheral blood in patients and anti-citrullinated oneself's immunity.Owing to detecting the means of RF and anti-CCP all based on the elisa technique that antigen and antibody specific combines, therefore, many, the consuming time length of experimental procedure, false positive rate are high.In addition, because China is without the intellecture property of above-mentioned two detection marks, this kit market price more than 4000 yuan, the testing cost of the every person-portion of hospital more than 200 ~ 260 yuan.
RA major lesions position is synovium of joint.Applicant of the present invention detects RA Synoviolin mass spectrum with proteomics method.By comparing with ankylosing spondylitis (AS) synovial membrane with osteoarthritis (OA), find PROTEIN C D38 specificity overexpression in RA synovial membrane.CD (Cluster of Differentiation) Chinese is leukocyte differentiation antigen, can be used as all kinds of leukocytic cell surface marker thing.CD38 molecular distribution, in the cell surface of the T of jejune hematopoietic cell and activation, bone-marrow-derived lymphocyte, NK cell, at cell activation, is sticked in the processes such as activity, signal transduction, cell factor generation and is played an important role.Recent research finds, CD38 afunction and immune response are damaged, metabolic disorder is closely related, may participate in the pathogenic process of autoimmune disease.Containing a large amount of T cell and B cell and other lymphocyte in RA synovial membrane, therefore applicant detects that CD and the RA histopathology of specifically expressing is consistent.
According to above discovery, applicant of the present invention adopts flow cytometry to continue to carry out detection to the CD38 lymphocyte subgroup of RA peripheral blood in patients and analyzes, wish to understand the expression of CD38 in RA peripheral blood in patients and synovial membrane in depth, understand the immune mechanism of RA morbidity and potential clinical application further.Flow cytometer (English name: flow cytometer; FCM) technology is the detection technique by the technology such as fluid ejection technique, laser technology, power spectrum art technology, robot calculator and the married a kind of advanced person of microfluorophotometer.Flow cytometer, can high speed analysis thousands of cells by measuring scattered light and the mark fluorescent intensity of cell and other biological particle, and can carry out qualitative or quantitative test to cell characteristic parameter multiple in a cell simultaneously.Because flow cytometer has the features such as speed is fast, precision is high, accuracy is good, easy and simple to handle, current counties and districts level and be mostly equipped with this equipment for detecting peripheral blood in patients to go to the hospital.By the analysis to especially all kinds of CD of various types of cells surface marker, flow cytometer can detect tumour cell, leukemia-lymphoma cell, activated blood platelet, the cell quantity of candidate stem cell, ratio, can also carry out immunophenotyping to these cells.Flow cytometer is common, low cost and detection method fast.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of new diagnosis of rheumatoid arthritis label---PROTEIN C D38, present invention also offers the kit prepared for cell marker with PROTEIN C D38 and the application in clinical detection thereof.
Applicant of the present invention is proved by Flow cytometry experiments, compared with normal person, and CD38+ cell high expressed in RA patient peripheral blood lymphocyte, and its expression and the horizontal significant correlation of RF.In addition, flow cytometer detection result shows, the ratio of CD38+CD56+ cell subsets also obviously raises, and CD56 is the cell surface marker of natural killer cell (NK cell), this prompting CD38 plays an important role in RA autoimmune disorder, contributes to immunologic mechanism and disease activity situation that we more clearly understand RA morbidity.The more important thing is, applicant finds that CD38 is well potential RA mark, can be used for the clinical detection of this disease.In view of above-mentioned reason, the fluidic cell the detection kit that applicants have invented using CD38 as RA label of the present invention.This kit is changed and is relied on conventional RF and anti-CCP based on elisa technique to detect reagent, utilizes the advantage of convenient, fast, the sensitive and low cost of flow cytometry.Due to the flow cytometer only needing a CD38 antibody and each hospital to be all equipped with, inspection price can be down to 25 ~ 50 yuan/every person-portion, and detection time is also down to 40 minutes.
For a kit for detection type rheumatic arthritis, CD3, CD4 monoclonal antibody marked by FITC, the CD56 monoclonal antibody of PE mark and the CD38 monoclonal antibody composition of Percp-Cy5.5 mark.
Further, in described kit, the consumption of each antibody is 20 μ l.
Described kit, when applying, using method is: the 200 μ l peripheral bloods and each 20 μ l of corresponding streaming antibody that get person to be detected put in streaming loading pipe, 4 DEG C of lucifuges hatch 30min, utilize Q-Prep Workstation(Beckman company, the U.S.) carry out blood preparation and prepare rearmounted FACSAria
tMthe upper machine testing of II flow cytometer (BD Biosciences).Invention has been relevant experimental study, result shows, the ratio of the CD38+ cell in RA patient peripheral blood lymphocyte is 17.29 ± 5.93%, the ratio of the CD38+ cell in healthy human peripheral blood lymphocyte is that the ratio of CD38+ cell in 10.15 ± 3.3%, RA patient peripheral blood lymphocyte is apparently higher than Healthy People (p=1.92E-09).In other indexs, in RA group peripheral blood in patients, the ratio of CD38+CD56+ cell is 8.04 ± 5.26%, the ratio of the CD38+CD56+ cell in Healthy People person week blood is that the ratio of the CD38+CD56+ cell in 0.0556 ± 0.0264%, RA patient peripheral blood lymphocyte is apparently higher than Healthy People (p=0.007).Therefore, by the result of experiment of the present invention, can be sure of that PROTEIN C D38 can be used as diagnosis of rheumatoid arthritis label and applies, checkout and diagnosis standard is: if the ratio of the CD38+ cell in patient peripheral's blood lymphocyte is higher than 15%, then detected patient should be diagnosed as and suffer from rheumatoid arthritis.
The present invention is based on and find can detect CD38 high expressed in RA peripheral blood by flow cytometry, provide a kind of new diagnosis of rheumatoid arthritis label---PROTEIN C D38, additionally provide a kind of kit being cell marker with PROTEIN C D38, there is easy, quick, low cost and other advantages.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 is by the expression of Flow cytometry CD38 in RA peripheral blood
Applicant of the present invention carries out venipuncture collecting blood sample to 103 RA patients of 20 ~ 74 years old, and wherein 75 people are women.The collected vacuum test tube to EDTA-K2 anti-freezing of peripheral blood.Choosing of all patients meets Americanism diseases caused by dampness association (American College of Rheumatology, ACA) to the diagnostic criteria of RA: patient articular all exists the chronic inflammation of more than 3 years, its erythrocyte sedimentation rate (ESR) (erythrocyte sedimentation rate, ESR), c reactive protein (C-reactive protein, and rheumatoid factor (rheumatoid factor CRP), RF) detected value is all apparently higher than normal range, be respectively 19 ~ 22mm/H, 8 ~ 196U/ml and 10.5 ~ 2,090U/ml.Research artificially contrasts with 120 health of 24 ~ 58 years old comprising 80 women.
Applicant of the present invention gets 200 μ l peripheral bloods and each 20 μ l of corresponding streaming antibody put in streaming loading pipe, and 4 DEG C of lucifuges hatch 30min, utilize Q-Prep Workstation(Beckman company, the U.S.) carry out blood preparation and prepare rearmounted FACSAria
tMthe upper machine testing of II flow cytometer (BD Biosciences).The streaming antibody used in this experiment comprises: CD3, CD4, CD22 monoclonal antibody (Beckman) of FITC mark, CD56, CD25, CD19 monoclonal antibody (Beckman) of PE mark and the CD38 monoclonal antibody (Biolegend) of Percp-Cy5.5 mark.
Applicant's research and utilization flow cytometry of the present invention detects the CD38 in RA patient and healthy human peripheral blood and other immune markers.Found that, 70% patient peripheral's blood lymphocyte all high expressed CD38, the ratio of the CD38+ cell in RA patient peripheral blood lymphocyte is 17.29 ± 5.93%, the ratio of the CD38+ cell in healthy human peripheral blood lymphocyte is that the ratio of CD38+ cell in 10.15 ± 3.3%, RA patient peripheral blood lymphocyte is apparently higher than Healthy People (p=1.92E-09).In other indexs, in RA group peripheral blood in patients, the ratio of CD38+CD56+ cell is 8.04 ± 5.26%, the ratio of the CD38+CD56+ cell in Healthy People person week blood is 0.0556 ± 0.0264%, the ratio of the CD38+CD56+ cell in RA patient peripheral blood lymphocyte is apparently higher than Healthy People (p=0.007), and up-regulated is comparatively obvious.And in Healthy People, nearly all human lymphocyte low expression CD38.Flow cytometer detection is the results detailed in Table 1.Applicant of the present invention also have detected the expression of CD38 in 38 routine AS peripheral blood in patients, nearly all AS patient lymphocytes low expression CD38, only has each two routine AS patient peripheral blood lymphocyte moderates to express CD38.
Applicant of the present invention is to CD38 expression and ESR in RA peripheral blood in patients, the relation of CRP and RF is also studied, find that the table level of CD38 positive cell becomes significant correlation (p=0.026) with RF level, and and ESR(p=0.102), CRP(p=0.112) correlativity little.
Table 1. flow cytometry lymph subgroup analysis result
Claims (1)
1. the application of PROTEIN C D38 in the kit of preparation diagnostics classes rheumatic arthritis, CD3 and the CD4 monoclonal antibody that described kit is marked by FITC, the CD56 monoclonal antibody of PE mark, and the CD38 monoclonal antibody composition of Percp-Cy5.5 mark;
Applying flow cytometry detects the expression of CD38 in peripheral blood lymphocyte, the CD38 in patients with rheumatoid arthritis lymphocyte
+the ratio of cell is apparently higher than Healthy People; CD38 in patients with rheumatoid arthritis lymphocyte
+cD56
+the ratio of cell is apparently higher than Healthy People.
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| CN103513040B true CN103513040B (en) | 2015-03-11 |
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| CN109568585B (en) * | 2018-12-29 | 2021-04-23 | 常晓天 | Application of CD38 inhibitor in preparation of anti-rheumatoid arthritis medicine |
| CN113912727B (en) * | 2021-11-16 | 2023-05-02 | 福州迈新生物技术开发有限公司 | anti-CD 38 protein monoclonal antibody, cell line, preparation method and application thereof |
Citations (4)
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| CN1976950A (en) * | 2004-02-06 | 2007-06-06 | 莫佛塞斯公司 | Anti-CD38 human antibodies and uses therefor. |
| CN101287764A (en) * | 2005-10-12 | 2008-10-15 | 莫佛塞斯公司 | Generation and profiling of fully human hucal gold-derived therapeutic antibodies specific for human cd38 |
| CN101616933A (en) * | 2006-10-19 | 2009-12-30 | 赛诺菲-安万特 | The novel anti-cd 38 antibodies that is used for the treatment of cancer |
| CN102597268A (en) * | 2009-09-03 | 2012-07-18 | 弗·哈夫曼-拉罗切有限公司 | Methods for treating, diagnosing, and monitoring rheumatoid arthritis |
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| WO2006090096A1 (en) * | 2005-02-28 | 2006-08-31 | Sheffield Teaching Hospitals Nhs Foundation Trust | Composition, kit and method for fixing cells |
| DK2580243T3 (en) * | 2010-06-09 | 2020-01-13 | Genmab As | ANTIBODIES AGAINST HUMAN CD38 |
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Patent Citations (4)
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|---|---|---|---|---|
| CN1976950A (en) * | 2004-02-06 | 2007-06-06 | 莫佛塞斯公司 | Anti-CD38 human antibodies and uses therefor. |
| CN101287764A (en) * | 2005-10-12 | 2008-10-15 | 莫佛塞斯公司 | Generation and profiling of fully human hucal gold-derived therapeutic antibodies specific for human cd38 |
| CN101616933A (en) * | 2006-10-19 | 2009-12-30 | 赛诺菲-安万特 | The novel anti-cd 38 antibodies that is used for the treatment of cancer |
| CN102597268A (en) * | 2009-09-03 | 2012-07-18 | 弗·哈夫曼-拉罗切有限公司 | Methods for treating, diagnosing, and monitoring rheumatoid arthritis |
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| Maria J. Leandro et al.Reconstitution of Peripheral Blood B Cells After Depletion With Rituximab in Patients With Rheumatoid Arthritis.《ARTHRITIS & * |
| RHEUMATISM》.2006,第54卷(第2期), * |
| sCD38在类风湿关节炎和系统性红斑狼疮患者外周血中的变化及意义;王莉莉;《中国医科大学硕士学位论文》;20090921;摘要 * |
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