CN111693707A - Application of ROCK kinase activity in auxiliary diagnosis of vasculitis patients - Google Patents
Application of ROCK kinase activity in auxiliary diagnosis of vasculitis patients Download PDFInfo
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- CN111693707A CN111693707A CN202010684683.2A CN202010684683A CN111693707A CN 111693707 A CN111693707 A CN 111693707A CN 202010684683 A CN202010684683 A CN 202010684683A CN 111693707 A CN111693707 A CN 111693707A
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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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
- G01N2800/32—Cardiovascular disorders
- G01N2800/328—Vasculitis, i.e. inflammation of blood vessels
Abstract
The invention discloses application of ROCK kinase activity in auxiliary diagnosis of vasculitis patients. The invention provides application of a substance for detecting ROCK kinase activity in preparation of products. The product has the function of diagnosing or assisting in diagnosing vasculitis. The product has the function of screening or assisting in screening patients with vasculitis. The activity of the ROCK kinase of a vasculitis patient is obviously higher than that of a healthy volunteer ROCK kinase, and the result indicates that a Rho GTPase/ROCK signal channel participates in the occurrence and development process of vasculitis. The invention has great application value for the auxiliary diagnosis of patients with vasculitis and the auxiliary screening of patients with vasculitis.
Description
Technical Field
The invention relates to application of ROCK kinase activity in auxiliary diagnosis of vasculitis patients.
Background
Vasculitis is a group of autoimmune diseases with inflammation and destruction of each blood vessel of the whole body as main pathological manifestations, and can be divided into large vasculitis, middle vasculitis and small vasculitis according to different affected blood vessels. According to the classification standard of CHCC 2012, single organ vasculitis such as allergic purpura and leucocyte clastic vasculitis is increased; vasculitis associated with systemic diseases such as systemic lupus erythematosus and vasculitis variabilises such as Behcet's disease, etc.
Systemic Lupus Erythematosus (SLE) is a more common autoimmune disease with recurrent episodes that involve multiple organ systems. Behcet's disease is also known as eye, mouth and genital syndrome, and is a multisystem disease based on the pathological condition of small and fine vasculitis. The allergic vasculitis is also called as cutaneous small vessel vasculitis and skin leukocyte fragmented vasculitis, and is necrotizing vasculitis mainly causing cutaneous small vessels, especially capillary rear venules, with unknown etiology. Allergic purpura (anaphaloic purpura), Henoch-Schonlein purpura (Henoch-Schonlein purpura), also known as self-limiting acute hemorrhage, is an allergic vasculitis which invades the tiny arteries and capillaries of skin and other organs, and the pathogenesis of the allergic vasculitis can be pathogen infection, certain drug action, allergy and the like, so that IgA or IgG circulating immune complexes are formed in vivo and deposited on the capillaries on the upper layer of dermis to cause vasculitis.
Disclosure of Invention
The invention aims to provide application of ROCK kinase activity in auxiliary diagnosis of vasculitis patients.
The invention provides an application of a substance for detecting ROCK kinase activity in preparing products; the product has the function of diagnosing or assisting in diagnosing vasculitis.
The invention provides an application of a substance for detecting ROCK kinase activity in preparing products; the product has the function of screening or assisting in screening patients with vasculitis.
In any of the above applications, the substance for detecting ROCK kinase activity is a substance for detecting P-MYPT1 and a substance for detecting MYPT 1.
In any of the above applications, the substance for detecting ROCK kinase activity is a substance for detecting human P-MYPT1 and a substance for detecting human MYPT 1.
In any of the above applications, the substance for detecting P-MYPT1 comprises Phospho-MYPT1 antibody.
In any of the above applications, the substance for detecting P-MYPT1 further comprises horseradish enzyme labeled goat anti-rabbit IgG antibody.
In any of the above applications, the substance for detecting P-MYPT1 further comprises a substance for extracting total cellular protein from peripheral blood mononuclear cells.
In any of the above applications, the substance for detecting P-MYPT1 further includes a substance for extracting peripheral blood mononuclear cells from peripheral blood.
In any of the above applications, the substance for detecting MYPT1 comprises a MYPT1 antibody.
In any of the above applications, the substance for detecting MYPT1 further comprises horseradish enzyme labeled goat anti-rabbit IgG antibody.
In any of the above applications, the substance for detecting MYPT1 further includes a substance for extracting total cellular protein from peripheral blood mononuclear cells.
In any of the above applications, the substance for detecting MYPT1 further includes a substance for extracting peripheral blood mononuclear cells from peripheral blood.
In any of the above applications, the product is a kit.
The invention also provides a product comprising a substance for detecting ROCK kinase activity; the function of the product is as follows (a), or (b):
(a) diagnosis or aiding diagnosis of vasculitis;
(b) screening or auxiliary screening of patients with vasculitis.
In the product, the substances for detecting the ROCK kinase activity are substances for detecting P-MYPT1 and substances for detecting MYPT 1.
In any of the above applications, the substance for detecting ROCK kinase activity is a substance for detecting human P-MYPT1 and a substance for detecting human MYPT 1.
In the product, the substance for detecting P-MYPT1 comprises Phospho-MYPT1 antibody.
In the product, the substance for detecting P-MYPT1 also comprises horseradish enzyme labeled goat anti-rabbit IgG antibody.
In the product, the substance for detecting P-MYPT1 also comprises a substance for extracting total cellular protein from peripheral blood mononuclear cells.
In the product, the substance for detecting P-MYPT1 also comprises a substance for extracting peripheral blood mononuclear cells from peripheral blood.
In the product, the substance for detecting MYPT1 comprises MYPT1 antibody.
In the product, the substance for detecting MYPT1 also comprises horseradish enzyme labeled goat anti-rabbit IgG antibody.
In the product, the substances for detecting MYPT1 also include substances for extracting total cellular protein from peripheral blood mononuclear cells.
In the product, the substance for detecting MYPT1 further includes a substance for extracting peripheral blood mononuclear cells from peripheral blood.
The product may specifically be a kit.
The product also comprises a carrier which is described with the following methods:
if ROCK kinase activity > 0.55, the subject is or is candidate for a vasculitis patient.
The product also comprises a carrier which is recorded with the following detection methods:
(1) peripheral venous blood of the subject was taken and peripheral blood mononuclear cells were isolated.
(2) Taking peripheral blood mononuclear cells, and extracting total cell protein.
(3) Carrying out Western-Blot on the total cell protein to develop P-MYPT1 and MYPT1 protein bands;
(4) the gray values of the protein bands in the Western-Blot development image are analyzed to obtain P-MYPT1/MYPT 1.
The invention also protects the application of ROCK kinase activity as a marker of vasculitis patients.
Any one of the above ROCK kinase activities is P-MYPT1/MYPT 1.
Any one of the above ROCK kinase activities is P-MYPT1 abundance/MYPT 1 abundance.
Any one of the above ROCK kinase activities is abundance of human P-MYPT 1/abundance of human MYPT 1.
Any one of the above ROCK kinase activities is P-MYPT1/MYPT1 in the total cellular protein of peripheral blood mononuclear cells.
Any one of the above ROCK kinase activities is P-MYPT1 abundance/MYPT 1 abundance in the total cellular protein of peripheral blood mononuclear cells.
The activity of the ROCK kinase of a vasculitis patient is obviously higher than that of a healthy volunteer ROCK kinase, and the result indicates that a RhoGTPase/ROCK signal channel participates in the occurrence and development process of vasculitis. The invention has great application value for the auxiliary diagnosis of patients with vasculitis and the auxiliary screening of patients with vasculitis.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Each disease met the 2012 CHCC nomenclature system, the American College of Rheumatology (ACR) diagnostic criteria, international diagnostic criteria, and typical clinical and histopathological manifestations in 1990.
The normal group consisted of 95 healthy people. Healthy people group entry standard: selecting 95 healthy volunteers belonging to the Beijing Chaoyang hospital physical examination center of the university of capital medical science for physical examination at the same time; the volunteers need to be excluded from the history of liver and kidney diseases, diabetes or other autoimmune diseases and tumor diseases before being grouped.
The vasculitis group consisted of 77 patients with vasculitis. Vasculitis inclusion criteria: 77 patients with initial vasculitis who visit the Beijing Kogyang Hospital affiliated to the first medical university between 10 and 2019 in 2016, wherein all the patients are from dermatology, rheumatoid immunity department, respiration department, nephrology department outpatient service and hospitalization system of the Beijing Kogyang Hospital affiliated to the first medical university; all patients met the 2012 CHCC nomenclature system, the American College of Rheumatology (ACR) diagnostic criteria, international diagnostic criteria, and typical clinical and histopathological manifestations in 1990; before the patient is taken into the group, the patient needs to obtain consent and sign an informed consent, and patient information and data, such as height, weight, disease onset time, diagnosis and treatment history, laboratory and imaging examination results, need to be collected.
Vasculitis was divided into four subgroups, systemic lupus erythematosus group (69 cases), Behcet disease group (2 cases), allergic vasculitis group (3 cases), and allergic purpura group (3 cases).
The diagnostic criteria for systemic lupus erythematosus are shown in Table 1. Of the 11 items in table 1, those who meet 4 or more items can be diagnosed with systemic lupus erythematosus after exclusion of infection, tumor, and other connective tissue diseases.
TABLE 1 diagnosis of systemic lupus erythematosus (ACR 1997 diagnosis)
The diagnostic criteria for Behcet's disease are shown in Table 2. Having item 1 in Table 2 in addition to any 2 or more items 2 to 5, the diagnosis of Behcet's disease can be made.
International Behcet disease research organization Behcet disease diagnostic criteria in Table 21990
Diagnostic criteria for allergic vasculitis: the allergic cutaneous vasculitis is diagnosed mainly according to the shape and distribution characteristics of skin lesions and by combining histopathological changes; skin damage is mainly manifested as: purpuric macula and maculopapule symmetrically appear at the body hypophysis part and can be used for secondary generation of blood blister, pustule, necrosis and ulcer; histopathology is characterized by leukoclastic vasculitis of the superficial dermal small vessels; in addition, laboratory examinations may show changes in rapid blood sedimentation, reduced complement, etc.
Diagnostic criteria for allergic purpura: allergic purpura does not have a unified clinical diagnosis standard at present, and the diagnosis mainly depends on clinical symptoms, histopathology and immunofluorescence examination; the clinical manifestations are usually: the lower limb recurrent and batch appearing palpable purpura can be accompanied with gastrointestinal tract or joint symptoms and kidney affected manifestations; histopathology is manifested by leucocytic fragmented vasculitis of the upper dermis capillaries and the posterior capillary veins; furthermore, direct immunofluorescence examination of skin surrounding skin lesions revealed the presence of IgA, C3 and cellulose deposits in the dermal vessel wall, which are characteristic manifestations of the disease.
Example 1 detection of ROCK kinase Activity
Study subjects: normal group and vasculitis group.
1. Peripheral venous blood of each of the subjects was collected, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated.
2. And (3) adding the lymphocyte lysate into the peripheral blood mononuclear cells obtained in the step (1), incubating for 20min on ice (blowing every 5 min), centrifuging for 5min at 4 ℃ at 12000r/min, and absorbing supernatant, namely the cell total protein solution. Lymphocyte lysate: 1ml of RIPA lysate, 20. mu.l of 50 XCocktail protease inhibitor, 10. mu.l of PMSF, 10. mu.l of phosphorylase inhibitor A solution, and 10. mu.l of phosphorylase inhibitor B solution.
3. And (3) taking the total cell protein solution obtained in the step (2), and detecting the protein concentration by using a BCA method to determine the loading amount.
4. Western blotting (Western-Blot) was performed to visualize the protein bands of P-MYPT1 and MYPT1 and β -Actin in PBMCs. beta-Actin is an internal reference.
SDS-PAGE parameters: 8% of separation gel and 5% of concentrated gel; the initial voltage is 80V, when the protein runs to the interface of the concentrated gel-separation gel, the voltage is adjusted to 120V, then the electrophoresis is continued for about 30min, the power supply is turned off until the protein marker and the blue protein strip are observed to run to the bottom of the gel, and the electrophoresis is stopped.
For detecting P-MYPT 1: the primary antibody was Phospho-MYPT1 antibody, the primary antibody was diluted 1:1000 fold, Cell Signaling organism, USA, cat # 4563; the secondary antibody is horseradish enzyme labeled goat anti-rabbit IgG antibody, the working concentration of the secondary antibody is 1:1000 times diluted, and the product number of the secondary antibody is ZB-2301 by Beijing Zhonghua Jinqiao biotechnology company.
For detecting MYPT 1: the primary antibody was MYPT1 antibody, the working concentration of the primary antibody was 1:1000 fold dilution, American CellSignaling organism, cat # 2634; the secondary antibody is horseradish enzyme labeled goat anti-rabbit IgG antibody, the working concentration of the secondary antibody is 1:1000 times diluted, and the product number of the secondary antibody is ZB-2301 by Beijing Zhonghua Jinqiao biotechnology company.
The development method is ECL chemiluminescence development.
5. The grey scale values of the protein bands in the Western-Blot development Image were analyzed using Image J software, and the ROCK kinase activity was represented by P-MYPT1/MYPT 1.
The ROCK kinase activity of each subject in the normal group is shown in Table 3.
The ROCK kinase activity of each subject in the vasculitis group is shown in Table 4.
The ROCK kinase activity of the normal group is: 0.38 + -0.01. The vasculitis group has ROCK kinase activity: 0.75 +/-0.09.
The ROCK kinase activity of the systemic lupus erythematosus subgroup is as follows: 0.75 +/-0.08. The Behcet disease subgroup has ROCK kinase activity of 0.74. + -. 0.21. The ROCK kinase activity of the allergic vasculitis subgroup was 0.67. + -. 0.08. The ROCK kinase activity of the anaphylactoid purpura subgroup is 0.70 +/-0.01.
The results show that the ROCK kinase activity has universality for diagnosing vasculitis.
The threshold value for diagnosing vasculitis is that the ROCK kinase activity is more than 0.55, the false negative rate is 0%, and the false positive rate is 0%.
TABLE 3
TABLE 4
Claims (10)
1. Use of a substance for detecting ROCK kinase activity in the preparation of a product; the product has the function of diagnosing or assisting in diagnosing vasculitis.
2. Use of a substance for detecting ROCK kinase activity in the preparation of a product; the product has the function of screening or assisting in screening patients with vasculitis.
3. Use according to claim 1 or 2, characterized in that: the ROCK kinase activity is P-MYPT1 abundance/MYPT 1 abundance.
4. Use according to any one of claims 1 to 3, wherein: the substance for detecting the ROCK kinase activity is a substance for detecting P-MYPT1 and a substance for detecting MYPT 1.
5. The use of claim 4, wherein: the substance for detecting P-MYPT1 comprises Phospho-MYPT1 antibody, and the substance for detecting MYPT1 comprises MYPT1 antibody.
6. A product comprising a substance for detecting ROCK kinase activity; the function of the product is as follows (a), or (b):
(a) diagnosis or aiding diagnosis of vasculitis;
(b) screening or auxiliary screening of patients with vasculitis.
7. The product of claim 6, wherein: the ROCK kinase activity is P-MYPT1 abundance/MYPT 1 abundance.
8. The product of claim 6 or 7, wherein: the substance for detecting the ROCK kinase activity is a substance for detecting P-MYPT1 and a substance for detecting MYPT 1.
Use of ROCK kinase activity as a marker for patients with vasculitis.
10. The use of claim 9, wherein: the ROCK kinase activity is P-MYPT1 abundance/MYPT 1 abundance.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016064A (en) * | 2007-09-27 | 2011-04-13 | 海因茨-伯恩哈德·克拉茨 | Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate |
| CN105659085A (en) * | 2013-08-28 | 2016-06-08 | 中美冠科生物技术（太仓）有限公司 | Gene expression signatures predictive of subject response to a multi-kinase inhibitor and methods of using the same |
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- 2020-07-16 CN CN202010684683.2A patent/CN111693707A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016064A (en) * | 2007-09-27 | 2011-04-13 | 海因茨-伯恩哈德·克拉茨 | Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate |
| CN105659085A (en) * | 2013-08-28 | 2016-06-08 | 中美冠科生物技术（太仓）有限公司 | Gene expression signatures predictive of subject response to a multi-kinase inhibitor and methods of using the same |
Non-Patent Citations (4)
| Title |
|---|
| YUANYUAN WANG 等: "Y-27632, a Rho-associated protein kinase inhibitor, inhibits systemiclupus erythematosus" * |
| 刘娜;: "糖尿病合并肺结核患者外周血T淋巴细胞Rho激酶活性变化及其意义" * |
| 彭世光 等: "Rho激酶活性与系统性红斑狼疮疾病活动的相关性研究" * |
| 李文华 等: "Rho激酶对急性冠状动脉综合征患者复合心血管不良事件的影响" * |
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