CN111007262A - Kit for auxiliary diagnosis of RA and detection method - Google Patents
Kit for auxiliary diagnosis of RA and detection method Download PDFInfo
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Abstract
The invention provides a kit for auxiliary diagnosis of RA and a detection method. According to the technical scheme, firstly, researches show that the relative expression levels of mRNA of ATG5 and ATG12 genes in peripheral blood mononuclear cells of RA patients in different activity periods show different expression differences, and meanwhile, the contents of ATG5, ATG12 and ATG12-Atg5 protein show corresponding regularity, so that the fact that the expression change of ATG5 and ATG12 genes or the change of the contents of related proteins has certain relevance and directionality on the prognosis and activity of RA can be confirmed. Based on the beneficial findings, the invention develops an RA auxiliary diagnostic kit based on autophagic gene detection, which takes human serum, plasma or other body fluids as biological samples, adopts a competitive inhibition ELISA method to quantitatively determine the content of ATG5, ATG12 and Atg12-Atg5 coupled protein, and can realize the auxiliary detection of RA and evaluate the pathological characteristics and the development process of RA.
Description
Technical Field
The invention relates to the technical field of disease diagnosis kits, further relates to a disease auxiliary diagnosis technology based on gene detection, and particularly relates to a kit and a detection method for auxiliary diagnosis of RA.
Background
Rheumatoid Arthritis (RA) is a chronic, inflammatory synovitis-predominant systemic disease of unknown etiology. The pathology of RA arthritis is mainly synovial lining cell hyperplasia, interstitial massive inflammatory cell infiltration, microvascular neogenesis, pannus formation, cartilage and bone tissue destruction, and the like. The symptoms are mostly manifested by polyarticular, symmetrical and invasive arthritis of hand and foot small joints, and often accompanied by positive serum rheumatoid factor of extraarticular organs, which can cause joint deformity and function loss. The onset of RA may be associated with genetics, infection, sex hormones, etc., and diagnostic criteria include the following set of characteristics: 1) morning stiffness is more than or equal to 30 minutes; 2) arthritis in greater than 3 articular areas; 3) arthritis of the hand; 4) rheumatoid Factor (RF) positive; 5) positive for anti-CCP antibody. The 14 articular zones comprise: bilateral elbows, wrists, metacarpal fingers, proximal interphalangeal joints, knees, ankles, and metatarsophalangeal joints; the above characteristics satisfy 3 or more items and can be diagnosed as RA.
In recent years, with the development of molecular biology and medical imaging, other detection bases have been introduced in the diagnosis of RA, for example, detection of autoantibodies to RA patients is one of the hallmarks of RA being different from other inflammatory arthritis such as psoriatic arthritis, reactive arthritis and osteoarthritis, and currently, autoantibodies commonly used in clinical practice include rheumatoid factor (RF-IgM), anti-Cyclic Citrulline (CCP) antibody, rheumatoid factor IgG and IgA, anti-perinuclear factor, anti-keratin antibody, antinuclear antibody, anti-ENA antibody and the like, and also include anti-RA 33 antibody, anti-glucose-6-phosphate isomerase (GPI) antibody, anti-P68 antibody and the like. In addition, soft tissue swelling, osteoporosis and joint facet cystic changes following disease progression, aggressive bone destruction, joint facet blurring, narrowing of the joint space, joint fusion and dislocation can be seen by X-ray. The MRI examination of the hand joint and the wrist joint by the MRI examination can prompt early synovitis pathological changes, is very helpful for finding early joint destruction of patients with rheumatoid arthritis, and the ultrasonic joint ultrasound is simple noninvasive examination and has identification significance for synovitis, joint effusion and joint destruction.
Currently, RA lacks direct and effective preventive and therapeutic strategies, and lacks highly sensitive, specific diagnostic indicators. Although the joint synovial tissue can be left through operation or puncture in clinic, the application of the joint synovial tissue is limited due to the invasive nature of the material selection. Studies have shown that, in the course of the onset of RA, a change in gene expression is accompanied, and in this case, if the relationship between the gene expression profile and the pathological characteristics of RA can be obtained with certainty, it is expected to be a new basis for the diagnosis of RA.
Autophagy is a process of degrading cytoplasmic proteins and organelles by depending on a lysosome pathway, and autophagy maintains homeostasis in cells by specifically recognizing and degrading substrates, and is closely related to occurrence and development of many diseases. Rheumatoid Arthritis (RA), a systemic, autoimmune disease, autophagy abnormalities may also have a correlation with pathological processes, however, at present, this correlation is not clear; in addition, the autophagy-related proteins can be more than 30, different autophagy-related proteins can be combined with each other to form a core Atg protein complex, the mechanism of participating in the physiological process of a human body is very complex, and the correlation research in the prior art has not yet achieved ideal results.
Disclosure of Invention
The invention aims to provide a kit and a detection method for auxiliary diagnosis of RA (RA) aiming at the technical defects in the prior art, and aims to solve the technical problem that a gene detection means-based RA auxiliary diagnosis kit is lacked in the prior art.
Another technical problem to be solved by the present invention is that the relationship between the pathological process of RA and the expression of autophagic genes in patients is not clear.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a kit for aiding in the diagnosis of RA, comprising: the pore plates are respectively coated with ATG5 antigen, ATG12 antigen and Atg12-Atg5 coupling protein antigen; positive quality control: ATG5 positive serum, ATG12 positive serum, Atg12-Atg5 coupling protein positive serum; negative quality control: negative serum of healthy people; working fluid R: anti-human ATG5 antibody labeled with HRP, anti-human ATG12 antibody labeled with HRP, anti-human Atg12-Atg5 coupled protein antibody labeled with HRP; diluting liquid: deionized water; a TMB substrate; stopping liquid: 1mol/L of H2SO4A solution; concentrating and cleaning a washing solution: PBS-Tween buffer.
Preferably, the well plate is a 96 well plate.
Preferably, the ATG5 positive serum, ATG12 positive serum, and Atg12-Atg5 coupling protein positive serum are each 50 μ L.
Preferably, the negative serum for healthy people is 50 μ L.
Preferably, the anti-human ATG5 antibody labeled with HRP, the anti-human ATG12 antibody labeled with HRP, and the anti-human Atg12-Atg5 conjugated protein antibody labeled with HRP are each 150. mu.L.
Preferably, the volume of the deionized water is 100 times of the total volume of the working solution R.
Preferably, the TMB substrate is present in 15 parts, each 100 μ L.
Preferably, the stop solution is 15 parts, 50 μ L each.
Preferably, the concentrated washing solution is 20 parts, and each part is 250 μ L.
A method for detecting rheumatoid arthritis by using the kit comprises the following steps:
1) sample adding: dividing the holes on the pore plate into standard holes, sample holes to be detected and blank holes, wherein the standard holes have 3 holes, respectively adding 3 positive quality controls into the 3 standard holes, each positive quality control is 50 mu L, the blank holes are added with 50 mu L negative quality controls, and the sample holes to be detected are added with 50 mu L samples to be detected; adding 150 mu L of working solution into each hole, vibrating and mixing uniformly, adding a film on the hole plate, and incubating for 1 hour at 37 ℃;
2) discarding liquid in the holes, washing each hole with 250 microliter of concentrated cleaning washing liquid, soaking for 1 minute, and then removing all liquid in the holes; repeating the plate washing for 3 times, pouring out the residual concentrated cleaning washing solution after the last washing, reversely buckling the enzyme label plate on the water absorption paper, and completely absorbing the liquid remained in the holes;
3) adding 100 mu L of TMB substrate into each hole, adding a film on an ELISA plate, and developing in a dark place at 37 ℃ for 10-30 min;
4) the reaction was stopped by adding 50. mu.L of stop solution to each well.
The kit applies competitive inhibition enzyme-linked immunoassay to determine the content of ATG5, ATG12, Atg12-Atg5 coupled protein compound in a sample. The monoclonal antibody is coated on a microporous plate to prepare a solid phase carrier, and the biotin-labeled antigen and the antigen to be detected (a standard product or a sample) are simultaneously added into the micropore coated with the antibody, and the antigen to be detected and the biotin-labeled antigen are in competitive combination with a specific antibody. After incubation, unbound material was washed away, HRP-labeled avidin was added, and after incubation and thorough washing, the substrate TMB was added for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The higher the concentration of the specimen to be tested is, the more the combination of the labeled antigen and the antibody is inhibited, and the shallower the color development is. The color depth is positively correlated with the enzyme amount and negatively correlated with the content of the substance to be detected in the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated.
The invention provides a kit for auxiliary diagnosis of RA and a detection method. According to the technical scheme, the relation between the gene expression condition and pathological characteristics of the RA patient is firstly researched, the research finds that the relative expression levels of mRNA of ATG5 and ATG12 genes in peripheral blood mononuclear cells of the RA patient show different expression differences in different degrees in different activity periods, and meanwhile, the protein contents of ATG5, ATG12 and Atg12-Atg5 show corresponding regularity, so that the fact that the expression change of ATG5 and ATG12 genes or the change of the related protein contents thereof has certain relevance and directivity to the prognosis and activity of RA can be confirmed. Based on the beneficial findings, the invention develops an RA auxiliary diagnostic kit based on autophagic gene detection, which takes human serum, plasma or other body fluids as biological samples, adopts a competitive inhibition ELISA method to quantitatively determine the content of ATG5, ATG12 and Atg12-Atg5 coupled protein, and can realize the auxiliary detection of RA and evaluate the pathological characteristics and the development process of RA. The method is simple and easy to implement, the detection process has small wound to the human body, and the evaluation basis is more stable and reliable.
Drawings
FIG. 1 is a graph showing the correlation between the expression level of Atg5 gene and DAS28 score in the present embodiment;
FIG. 2 is a graph showing the correlation between the expression level of Atg12 gene and DAS28 score in the present embodiment;
FIG. 3 is a western blot of Atg5, Atg12, Atg12-Atg5 in an embodiment of the invention;
FIG. 4 shows the WB statistics of Atg5, Atg12, Atg12-Atg5 proteins in the present invention (P <0.05, P < 0.01).
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Approximating language, as used herein in the following examples, may be applied to identify quantitative representations that could permissibly vary in number without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1
1. Materials and methods
1.1 study object
Case groups: 60 patients, 20 men and 40 women in blood rheumatism internal medicine (1 month in 2014-6 months in 2015) affiliated to Zhangzhou city hospital of Fujian medical university are selected, the average age of the patients is (46.75 +/-13.52), and the diagnosis standard of RA is determined according to the United states rheumatology Association (ACR) and European antirheumatic alliance (RULAR) combined RA diagnosis standard:
according to the RA disease activity index (DAS28), the low-activity period (DAS28<3.2), the medium-activity period (DAS 283.2-5.1) and the high-activity period (DAS28>5.1) are divided into 22 cases in the low-activity period L group, 20 cases in the medium-activity period M group and 18 cases in the high-activity period H group. Control group: the patients were outpatient health examiners of the same period, 25, 7 men and 18 women, and had the average age of (45.35 +/-14.02) years, and the autoimmune diseases were excluded.
1.2 instruments and reagents
High speed centrifuge model (model 5424R, Thermo Corp.); ultrapure water systems (model Direct-Q5, Millipore Corp.); real-time PCR instrument (model 7500, ABI Co.); electrophoresis apparatus and membrane transfer apparatus (Bio-Rad, USA); Bio-Rad Chemi Doc MP imaging system gel imaging System (Bio-Rad, Inc., USA); RNA extraction kit, reverse transcription kit, fluorescent quantitative PCR kit (Beijing Quanzijin Biotechnology Co., Ltd.); related antibodies such as Atg5 and Atg12 (Cell Signaling Technology corporation); the main primer sequences are shown in Table 1 and synthesized by Shanghai Biotech.
TABLE 1 primer sequences
1.3 methods
2ml of venous blood was collected and placed in anticoagulant-treated vacuum blood collection tubes for separation of control and experimental PBMCS. Total RNA extraction was performed according to the instructions of the TransZol Up Plus RNA Kit (Cat. No.: ER501-01) of the RNA extraction Kit of Beijing Quanyu gold Biotechnology Ltd (TransGen). Fluorescent quantitative PCR kit 2 XTransStarTMTop Green qPCR SuperMix (Cat. No.: AQ131-02, Beijing Quanjin Biotechnology Co., Ltd.) was subjected to Real-time qPCR to detect mRNAs such as ATG5 and ATG12, and GAPDH was used as an internal reference gene for the analysis of the results, 2-△△CtThe method analyzes the difference of the expression level of each gene.
Detection by protein immunoblot analysis (Western blot)
The operation steps comprise that ① protein samples are prepared for single nuclear separation, PBS is washed, lysate containing PMSF is cracked, ② protein content is stored at 80 ℃ below zero, ③ SDS-PAGE electrophoresis is measured by a TU-1901 double-beam ultraviolet spectrophotometer, 10% SDS-PAGE is used for electrophoresis, 200V electrophoresis is carried out for 45min, PVDF membrane is used for membrane transfer, 200mA membrane transfer is carried out for 2h, 5% skimmed milk or 3% BSA (diluted by 0.5% TBST) is used for blocking, room temperature blocking is carried out for 2h, primary antibody is diluted by blocking liquid for primary antibody incubation, shaking incubation is carried out at 4 ℃ overnight, TBST is used for washing for 3 times, 10min is used for washing, secondary antibody is diluted by 0.5% TBST for secondary antibody for incubation, shaking incubation is carried out at room temperature for 1-2 h, TBST is used for washing for 3 times, 10min is carried out for washing, exposure detection of an exposure imager and gel.
1.4 statistical analysis
Data pairs by statistical SPSS softwareProcess and analyze the data and measure the data
The results show that the groups are statistically different by Kruskal Wallis H test, then compared with each other by Nemenyi test, and the correlation analysis between the indexes is linear regression, the test level α is 0.05, the difference is statistically significant when P is less than 0.05, and the association between the indexes and different activity groups of the disease is discussed.
2. Results
2.1 detection results of mRNA of Atg5 and Atg12 genes in RA patients
The mRNA of Atg5 and Atg12 genes in RA patients is significantly higher than that of healthy controls, and with the increase of RA disease activity index (DAS28), the mRNA of Atg5 and Atg12 genes is positively correlated with DAS28 (r is 0.85,0.92), (see table 2, fig. 1 and fig. 2).
TABLE 2 mRNA detection results of Atg5 and Atg12 genes
2.2 Atg5, Atg12, Atg12-Atg5 protein expression in RA patients.
The expression of Atg5, Atg12 and Atg12-Atg5 proteins in RA patients is obviously higher than that of healthy controls, and the difference is statistically significant (P <0.05) and gradually increases. (see FIG. 3, FIG. 4)
3. Conclusion
The relevance of the autophagy genes Atg12 and Atg5 and related proteins thereof to the activity index of rheumatoid arthritis patients is discussed through an experimental method. 60 patients with rheumatoid arthritis of different active periods and 25 healthy controls were selected, and the autophagy-related genes Atg5 and Atg12 and their corresponding proteins in blood were analyzed by extracting mononuclear cells from their blood and assaying. The results show that the mRNA of Atg5 and Atg12 genes in RA patients is obviously higher than that of healthy controls, and is in positive correlation with DAS28 (r is 0.85, 0.92); meanwhile, the expression of Atg5, Atg12 and Atg12-Atg5 proteins in RA patients is obviously higher than that of healthy controls, the difference has statistical significance (P <0.05), and the trend is gradually increased along with the activity index. From this, it was confirmed that the autophagy gene Atg12-Atg5 and its related autophagy protein have a certain correlation with rheumatoid arthritis.
4. Discussion of the related Art
Rheumatoid Arthritis (RA) is a multi-systemic, chronic, invasive autoimmune disease that can be seriously complicated in the later stages and affect the quality of life of patients if not treated systemically. If the patient can be diagnosed and treated early in time, the disease condition can be controlled, and the prognosis of the patient is greatly improved. Dysregulation of autophagy readily induces the development of many diseases, autophagy plays a major role in innate and adaptive immunity, and autophagy often requires the coordinated involvement of autophagy-related proteins (ATGs).
Atg5 is an autophagy-related gene mainly bound by coupling to Atg12, which activates LC3-I to LC3-II by promoting the extension, coating and lipidation of autophagosome membrane LC3, then forms autophagosomes, and also removes wrong proteins by binding to lysosomes using degradation of autophagosomes and phagocytosis of damaged organelles and cytoplasmic proteins. In this study, the mRNA of Atg5 gene in RA patients was significantly higher than that in healthy controls, and the mRNA of Atg5 gene was positively correlated with activity index DAS28 (r ═ 0.85), which indicates that it is likely that the body of RA patients activated the corresponding autophagic activity in vivo to maintain homeostasis in the body as the disease progressed. And Atg12 is an important ubiquitin-like protein and can mediate development of autophagosome membranes, and the BH 3-like domain of the protein is combined with MCL1 to influence apoptosis. Meanwhile, the regulation effect of Atg5 on autophagy is mainly realized by combining with Atgl2 protein to form Atg12-Atg5 coupled protein, and the composite protein is an important protein participating in the early stage of autophagy formation. In the research, the mRNA and the protein of the Atg12 gene are both higher than the level of a healthy control group, and the difference has statistical significance. Since the Atg12-Atg5 protein is an upstream protein in autophagosome formation, it is a related protein in autophagosome initiation. The results of this study show that the Atg12-Atg5 protein also presents an increasing trend with the increase of the activity index of RA, which may be an epidemic prevention response of the body.
Autophagy has a dual role in affecting the survival or death of cells, and can maintain the survival of cells by ensuring the supply of metabolism or clearing organelles. RA, a common autoimmune disease, in which autophagy plays a natural role. The research on autophagy-related genes and proteins is helpful for researching the pathogenic mechanism of RA.
Based on the above research results, the present invention provides a kit for auxiliary diagnosis of RA, which uses ATG5, ATG12, ATG12-ATG5 coupled protein as a detection object, the kit comprising: the pore plates are respectively coated with ATG5 antigen, ATG12 antigen and Atg12-Atg5 coupling protein antigen; positive quality control: ATG5 positive serum, ATG12 positive serum, Atg12-Atg5 coupling protein positive serum; negative quality control: negative serum of healthy people; working fluid R: anti-human ATG5 antibody labeled with HRP, anti-human ATG12 antibody labeled with HRP, anti-human Atg12-Atg5 coupled protein antibody labeled with HRP; diluting liquid: deionized water; a TMB substrate; stopping liquid: 1mol/L of H2SO4A solution; concentrating and cleaning a washing solution: PBS-Tween buffer.
Example 2
A kit for auxiliary diagnosis of RA, the reagent is used for detecting the content of ATG5, ATG12, Atg12-Atg5 coupling protein complex in human serum, plasma or other body fluids; the components of the kit are shown in the following table 3:
TABLE 3 kit Components List
The use method of the kit is as follows:
1) sample adding: respectively provided with a standard hole, a sample hole to be detected and a blank hole.
Setting 3 standard holes, and sequentially adding 50 mu L of positive quality control with different concentrations;
II, adding 50 mu L of negative quality control in blank holes;
and adding 50 mu L of the sample to be detected into the hole III to be detected, immediately adding 150 mu L of the working solution into each hole, slightly vibrating, uniformly mixing, paying attention to no air bubbles, adding a film on the ELISA plate, and incubating for 1 hour at 37 ℃.
2) The well contents were discarded, each well was washed with 250. mu.L of washing solution, soaked for 1 minute, and the microplate was tapped on absorbent paper to remove all the contents of the wells.
The plate washing was repeated 3 times. And after the last washing, pouring out the residual washing buffer solution, reversely buckling the enzyme label plate on the absorbent paper, and completely sucking the residual liquid in the holes.
3) The substrate solution was added in an amount of 100. mu.L per well, a cover film was applied to the microplate, and color development was carried out in the dark at 37 ℃ for 10 to 20 minutes, not more than 30 minutes. The standard wells were terminated when a distinct blue gradient was observed).
4) The reaction was stopped by adding 50. mu.L of stop solution to each well, at which point the blue color turned to yellow. The order of addition of the stop solution should be as consistent as possible with the order of addition of the substrate solution.
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A kit for aiding in the diagnosis of RA, comprising:
the pore plates are respectively coated with ATG5 antigen, ATG12 antigen and Atg12-Atg5 coupling protein antigen;
positive quality control: ATG5 positive serum, ATG12 positive serum, Atg12-Atg5 coupling protein positive serum;
negative quality control: negative serum of healthy people;
working fluid R: anti-human ATG5 antibody labeled with HRP, anti-human ATG12 antibody labeled with HRP, anti-human Atg12-Atg5 coupled protein antibody labeled with HRP;
diluting liquid: deionized water;
a TMB substrate;
stopping liquid: 1mol/L of H2SO4A solution;
concentrating and cleaning a washing solution: PBS-Tween buffer.
2. The kit for aiding in the diagnosis of RA according to claim 1, wherein the well plate is a 96 well plate.
3. The kit for the aided diagnosis of RA according to claim 1, wherein said ATG 5-positive serum, ATG 12-positive serum, and Atg12-Atg 5-conjugated protein-positive serum are each 50 μ L.
4. The kit for the aided diagnosis of RA according to claim 1, wherein said negative serum of healthy human is 50 μ L.
5. The kit for the aided diagnosis of RA according to claim 1, wherein the HRP-labeled anti-human ATG5 antibody, the HRP-labeled anti-human ATG12 antibody, and the HRP-labeled anti-human Atg12-Atg5 conjugated protein antibodies are each 150. mu.L.
6. The kit for auxiliary diagnosis of RA according to claim 1, wherein the volume of the deionized water is 100 times of the total volume of the working solution R.
7. The kit for the aided diagnosis of RA according to claim 1, wherein said TMB substrate is present in 15 portions, each portion being 100. mu.L.
8. The kit for aiding in the diagnosis of RA according to claim 1, wherein the stop solution comprises 15 parts, each of which is 50. mu.L.
9. The kit for aiding in the diagnosis of RA according to claim 1, wherein said concentrated washing solution comprises 20 parts, each of which is 250. mu.L.
10. A method for detecting rheumatoid arthritis using the kit of claim 1, comprising the steps of:
1) sample adding: dividing the holes on the pore plate into standard holes, sample holes to be detected and blank holes, wherein the standard holes have 3 holes, respectively adding 3 positive quality controls into the 3 standard holes, each positive quality control is 50 mu L, the blank holes are added with 50 mu L negative quality controls, and the sample holes to be detected are added with 50 mu L samples to be detected; adding 150 mu L of working solution into each hole, vibrating and mixing uniformly, adding a film on the hole plate, and incubating for 1 hour at 37 ℃;
2) discarding liquid in the holes, washing each hole with 250 microliter of concentrated cleaning washing liquid, soaking for 1 minute, and then removing all liquid in the holes; repeating the plate washing for 3 times, pouring out the residual concentrated cleaning washing solution after the last washing, reversely buckling the enzyme label plate on the water absorption paper, and completely absorbing the liquid remained in the holes;
3) adding 100 mu L of TMB substrate into each hole, adding a film on an ELISA plate, and developing in a dark place at 37 ℃ for 10-30 min;
4) the reaction was stopped by adding 50. mu.L of stop solution to each well.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999028344A2 (en) * | 1997-11-28 | 1999-06-10 | Innogenetics N.V. | Synthetic peptides containing citrulline recognized by rheumatoid arthritis sera as tools for diagnosis and treatment |
| CN107436358A (en) * | 2017-06-28 | 2017-12-05 | 温州医科大学 | Application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared |
| CN109959787A (en) * | 2019-04-18 | 2019-07-02 | 漳州卫生职业学院 | A kind of rheumatoid arthritis auxiliary diagnostic box and detection method |
-
2019
- 2019-12-25 CN CN201911352225.2A patent/CN111007262A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999028344A2 (en) * | 1997-11-28 | 1999-06-10 | Innogenetics N.V. | Synthetic peptides containing citrulline recognized by rheumatoid arthritis sera as tools for diagnosis and treatment |
| CN107436358A (en) * | 2017-06-28 | 2017-12-05 | 温州医科大学 | Application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared |
| CN109959787A (en) * | 2019-04-18 | 2019-07-02 | 漳州卫生职业学院 | A kind of rheumatoid arthritis auxiliary diagnostic box and detection method |
Non-Patent Citations (5)
| Title |
|---|
| 叶文芳等: "新风胶囊对强直性脊柱炎患者疗效及血清免疫球蛋白亚型、外周血淋巴细胞自噬的影响", 《中国中西医结合杂志》 * |
| 孙英焕等: "巨自噬及分子伴侣介导自噬在类风湿关节炎中的相互作用及其联系", 《中国免疫学杂志》 * |
| 王亚黎: "基于PI3K-AKT-mTOR信号通路/Atg/LC3/Beclin1探讨新风胶囊调节IgG治疗类风湿关节炎的机制", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
| 王亚黎等: "新风胶囊对佐剂性关节炎大鼠Beclin1/PI3K-AKT-mTor的影响", 《中国中西医结合杂志》 * |
| 郭月丽等: "PI3K/AKT/mTOR信号通路与类风湿关节炎的相关性", 《广东医学》 * |
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