CN111537733A - Application of CCR1 as COPD diagnostic marker - Google Patents
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Abstract
The present invention relates to the use of CCR1 as a diagnostic marker for COPD. The method comprises the following steps: differences among groups were analyzed by FCM to measure the levels of neutrophil oxidative phagocytosis and related receptors in peripheral blood and sputum (excluding healthy controls) of 30 healthy controls, 30 patients with pneumonia and 30 patients with COPD. As a result: compared with a healthy control group, the COPD patient group has a remarkable reduction in the neutrophil oxidative phagocytic function and the expression of a surface CCR1 receptor in peripheral blood and sputum (P < 0.05); compared with the pneumonia control group, the neutrophil oxidative phagocytosis function has no statistical significance in peripheral blood difference, but in sputum, the COPD patient group is obviously lower than the pneumonia control group, and the expression of the neutrophil surface receptor CCR1 in the sputum of the COPD patient group is obviously higher than that of the pneumonia control group, and the difference has statistical significance (P < 0.05).
Description
Technical Field
The invention relates to the technical field, in particular to application of CCR1 expression as a COPD diagnostic marker.
Background
COPD is a disease characterized by airflow limitation that is not fully reversible, a common, multiple, high-mortality chronic respiratory disease, one of the four most fatal diseases worldwide today. To date, the exact pathogenesis of COPD is not clear, COPD is now generally considered to be characterized by chronic inflammation of airways, lung parenchyma and pulmonary vessels, and an imbalance of proteases and antiproteases, oxidation and antioxidant in the lung also plays an important role in COPD pathogenesis.
Neutrophils (PMNs) are effector cells important to the body's involvement in innate immunity, and PMNs play an important role in the regulation of inflammation and immune responses through chemotactic phagocytosis, respiratory burst, secretion of lytic enzymes and immunologically active substances. The oxidative accumulation of neutrophils is one of the important links in the development of COPD, and both neutrophils and their components are involved in the development process of COPD: 1. chemokine Receptors (CR) are a family of transmembrane receptors with chemokines as ligands, and the CR on the surface of neutrophils (e.g. CCR1) is a key molecule for their migration to sites of pathogen infection or to sites of inflammation. Wang et al confirmed that: CCR1 expression on inflammatory cells correlates with the severity of COPD; 2. toll-like receptors (TLRs) are a class of natural immune receptors that play important roles in the regulation of phagocytosis of cells in a variety of inflammatory responses, cell signaling, and apoptosis. TLRs play a decisive role in the function of neutrophils as pathogen recognition receptors and play an important role in the generation, development and regression of inflammation.
Neutrophils are double-edged sword, and cytotoxic substances in cytoplasm of the neutrophils can damage self tissue cells while killing pathogenic microorganisms invaded by outsiders. Neutrophils play a very important role in the pathogenesis of COPD. Much of the research focuses on whether an increase in neutrophil counts in COPD patients results in an increase in lifespan due to a decrease in apoptosis, however, at present apoptosis studies have shown no clear evidence of an increase in neutrophil lifespan, and even some of the research conclusions are contradictory. In recent years, researchers have gradually shifted the visual field to the neutrophil's own function, Prieto et al have suggested that COPD patients have a defect in PMN phagocytic activity, and in animal experiments it has been demonstrated that cigarette smoke extract impairs neutrophil phagocytosis in a pseudomonas-infected murine model. Sapey et al have suggested that neutrophils in COPD patients may have their own functional defects, and their chemotactic behavior and transitional structure are substantially different from those of other people.
Therefore, we focused on the oxidative phagocytic function of neutrophils in the study. COPD belongs to airway inflammation, and pneumonia is non-airway inflammation, and we can better explain that the conclusion of the experiment is unique to airway inflammation by selecting pneumonia as a control group; meanwhile, the phagocytic function of the neutrophil in COPD peripheral blood and sputum can better reflect the overall and local relationship of COPD.
The method for detecting the phagocytic function of the neutrophil adopts a flow cytometer-DHR method, and is a simple, convenient, rapid and good-repeatability method which is commonly used for clinically evaluating the phagocytic and oxidative functions of the neutrophil. The fluorochrome-free dihydrorhodamine123 (dihydrorhodamine123DHR) is reduced to Rhodamine123 (Rhodamine 123, Rho123) having high green fluorescence in oxidation reaction by respiratory burst when neutrophil phagocytosis is exerted. The performance of the neutrophil in enhanced function can be visually detected after being stimulated through flow cytometry detection, and DHR is firstly phagocytized by activated neutrophil and then oxidized by an oxidation system generated by the oxidized neutrophil. Therefore, the function of the neutrophil detected by the method can reflect two functions of phagocytosis and oxidation of the neutrophil, and the detection result is influenced by the defect of any function.
The application of the expression of CCR1 in the invention as a diagnostic marker of COPD has not been reported at present.
Disclosure of Invention
The first purpose of the invention is to provide the application of the peripheral blood and sputum neutrophilic granulocyte and CCR1 aiming at the defects of the prior art.
The second purpose of the invention is to provide a kit for distinguishing airway inflammation and non-airway inflammation aiming at the defects of the prior art.
The third purpose of the present invention is to provide a kit for discriminating between COPD and pneumonia, which is directed to the shortcomings of the prior art.
A fourth object of the present invention is to provide a COPD diagnosis kit for overcoming the disadvantages of the prior art.
In order to achieve the first purpose, the invention adopts the technical scheme that:
the application of CCR1 as a diagnostic marker for the positive rate of oxidative killing and oxidation of neutrophils in sputum of COPD patients, wherein the expression amount of CCR1 is inversely proportional to the positive rate of oxidative killing and oxidation of neutrophils in sputum of COPD patients.
Use of sputum neutrophils in combination with CCR1 as a diagnostic marker for the identification of pneumonia and COPD.
Preferably, patients with COPD have a lower positive rate of oxidative phagocytosis and oxidation of neutrophils in sputum than patients with pneumonia, and patients with COPD have a higher expression of CCR1 in neutrophils in sputum than patients with pneumonia.
Use of peripheral blood and sputum neutrophilic granulocytes in combination with CCR1 as a diagnostic marker for COPD.
Preferably, the positive rates of oxidative phagocytosis and oxidation of peripheral blood and sputum neutrophils and the expression level of CCR1 in COPD patients are lower than normal values.
Use of neutrophil sputum in combination with CCR1 as a diagnostic marker for the identification of airway inflammation and non-airway inflammation.
Preferably, patients with airway inflammation have a lower positive rate of oxidative phagocytosis and oxidation of neutrophils in the sputum than patients with non-airway inflammation, and patients with airway inflammation have a higher expression of CCR1 in neutrophils in the sputum than patients with non-airway inflammation.
Application of CCR1 in preparation of medicines for improving oxidation function of neutrophils.
Use of a CCR1 inhibitor for the manufacture of a medicament for increasing the oxidative function of neutrophils.
Preferably, the CCR1 inhibitor is a substance that reduces the expression level of CCR 1.
Preferably, the CCR1 inhibitor is selected from a small molecule compound or a biological macromolecule.
Preferably, the biomacromolecule is small interfering RNA, dsRNA, shRNA, microRNA or antisense nucleic acid which takes CCR1 protein or a transcript thereof as a target sequence and can inhibit CCR1 protein expression or gene transcription; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
In order to achieve the second object, the invention adopts the technical scheme that:
a kit for identifying airway inflammation and non-airway inflammation comprises a reagent for detecting the positive rate of oxidative phagocytosis and oxidation of sputum neutrophil granulocytes and the expression amount of CCR 1.
In order to achieve the third object, the invention adopts the technical scheme that:
a kit for identifying COPD and pneumonia comprises a reagent for detecting the positive rate of oxidative phagocytosis and oxidation of sputum neutrophils and the expression quantity of CCR 1.
In order to achieve the fourth object, the invention adopts the technical scheme that:
a COPD diagnostic kit comprises a reagent for detecting the oxidative phagocytosis oxidation positive rate of peripheral blood and sputum neutrophils and the expression quantity of CCR 1.
The invention has the advantages that:
1. differences between groups were analyzed by FCM to detect the levels of neutrophil oxidative phagocytosis and expression of the associated receptors [ CCR1(CD191) and TLRs (2,4) ] in peripheral blood and sputum (except healthy controls) of 30 healthy controls, 30 patients with pneumonia and 30 patients with COPD. The results show that compared with a healthy control group, the expression of neutral oxidative phagocytosis function and surface CCR1 receptor in peripheral blood and sputum of a COPD patient group is remarkably reduced (P < 0.05); compared with a pneumonia control group, the neutrophil oxidative phagocytic function has no statistical significance in peripheral blood difference, but in sputum, a COPD patient group is obviously lower than that of the pneumonia control group (0.3360 +/-0.3173 vs 0.5157 +/-0.3650), the expression of a neutrophil surface receptor CCR1 in the sputum of the COPD patient group is obviously higher than that of the pneumonia control group, and the difference has statistical significance (P < 0.05); the positive rate of oxidative phagocytosis of granulocytes in the sputum of COPD patients is only negatively correlated with the expression of the surface receptor CD191 (P < 0.01, r ═ 0.548) and has no significant correlation with the expression of the other two receptors TLR2 and TLR4 (r values of-0.206, -0.219, P >0.05, respectively).
2. A method for detecting the oxidative phagocytic function of the sputum neutrophils of a COPD patient by using a clinical flow cytometry is established; the oxidative killing function of the neutrophils in the sputum of a COPD patient and the expression of a surface CCR1 receptor are remarkably reduced compared with those of a pneumonia patient and a healthy person, the positive rate of oxidative phagocytosis of the neutrophils in the sputum of the COPD patient is negatively related to the expression of the surface CCR1, and the condition that the oxidative phagocytosis function of the neutrophils in the sputum of the COPD patient is damaged is suggested to be one of possible mechanisms of COPD pathogenesis.
3. The invention provides a kit for identifying airway inflammation and non-airway inflammation and identifying COPD and pneumonia for the first time, provides a new treatment scheme for the patients, and has good prospect.
Drawings
FIG. 1 is a FCM assay antigen scatter plot, note: r1 is a neutrophil population.
FIG. 2 is a schematic diagram showing the measurement of the oxidative phagocytic activity of neutrophils.
FIG. 3 is a flow chart of the oxidative phagocytic function of peripheral blood neutrophils in COPD group, pneumonia group and normal control group, wherein, (a) the normal control group; (b) pneumonia group; (c) the COPD group. Note: p <0.05 compared to healthy controls; compared with the pneumonia group, # P < 0.05.
FIG. 4 is a flow histogram of the expression of peripheral blood neutrophil surface CCR1, TLR2 and TLR4 of COPD, Pneumoniae and normal control groups, wherein (a) to (d) are CD282 detection results of isotype control, normal control group, Pneumoniae and COPD respectively; (e) CD284 detection results of an isotype control group, a normal control group, a pneumonia group and a COPD group respectively; (i) the results of CD191 detection of isotype control, normal control group, pneumonia group and COPD group respectively.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
The COPD patient group comprises 30 COPD acute attack patients, wherein 16 men and 14 women are aged 35-82 years, and the patients are treated by clinical diagnosis hospital admission in Putuo district people hospital in Shanghai city from 1 month in 2018 to 12 months in 2019. Clinical diagnosis includes physical signs, X-ray and laboratory related item detection, and the diagnosis meets the diagnostic criteria in the Global initiative for chronic obstructive pulmonary disease (GOLD) guide 2017. 30 pneumonia patients (pneumonia groups) which are confirmed to be diagnosed and treated in hospital in Putuo district in Shanghai at the same period are selected as disease controls, wherein 12 men and 18 women are selected, and the age is 32-88 years old. 30 people in the Putuo area of Shanghai of the same period (normal control group) are selected for physical examination of healthy people in hospital, wherein 14 men and 16 women are aged 27-79 years, the count of peripheral blood White Blood Cells (WBC) is (5.9-9.9) multiplied by 109/L, the percentage of neutrophils (NEUT%) is 55-68.7%, and the indexes of liver function, kidney function and cardiovascular system are all abnormal. The difference of the clinical data such as sex, age, etc. among the COPD group, the pneumonia group and the normal control group has no statistical significance (P > 0.05).
Inducing sputum: before examination, the patient should be thoroughly rinsed with boiled water or normal saline, swallowed, and cleared of nasal and oral secretions. Then atomizing and sucking 3% sterilized hypertonic sodium chloride solution for 20min, and leaving the sputum in a sterile sputum cup. When the symptoms of wheezing, difficult breathing and the like appear in the atomization process of the subject, the atomization is stopped immediately. The sputum is collected and treated in 2 hours.
Reagent 1:
1) monoclonal antibodies: alexa Fluor 488-labeled mouse anti-human CD282(TLR2) antibody (cat # 558318, clone # 11G7), Alexa Fluor 488-labeled mouse IgG1 isotype control (cat # 557702, clone # MOPC-21), Phycoerythrin (PE) -labeled mouse anti-human CD284(TLR4) (cat # 564215, clone # TF901), PE-labeled mouse IgG1 isotype control (cat # 554680), Alexa Fluor 647-labeled mouse anti-human CD191(CCR1) antibody (cat # 557914 clone No. 53504), Alexa
647 labeled mouse anti-IgG 2b isotype control (cat No. 557903, clone No. 27-35), Peridinin-chlorophyl-protein complex (PerCP) labeled mouse anti-human CD45 antibody (cat No. 347464, clone No. 2D1), PerCP labeled mouse IgG1 isotype control (cat No. 550672, clone No. MOPC-31C), lysine Solution hemolytic agent, and FASC Flow buffer were all purchased from BD company, USA. The nonfluorescent dyes dihydrorhodamine123 (DHR 123; working solution concentration 30. mu.g/mL), phorbol ester (PMA; working solution concentration 10. mu.g/mL) were purchased from Sigma, USA. Dithiothreitol (DTT) was purchased from Biotechnology engineering (Shanghai) GmbH.
2. The main apparatus is as follows: flow cytometer FACS Calibur (BD corporation, usa); a table type 80-2 multipurpose centrifuge (Shanghai and Xin scientific and education Equipment Co., Ltd., China); LA-920-3 vertical laminar flow clean bench (LA-920-3, Shanghai clean Equipment Co., Ltd., China); vortex mixer (QL-901, Jiangsu Haimen medical instruments factory, China); an electric heating constant temperature water bath (HSS, Chongqing Sida test instrument factory, China).
Second, Experimental methods
1. Sample collection
Collecting 2mL of venous blood of all subjects, anticoagulating ethylene diamine tetraacetic acid dipotassium-K2 (EDTA-K2), completing the oxidative phagocytosis function of peripheral blood neutrophils and the detection of related receptors CD282, CD284 and CD191 within 24h, and mixing uniformly before detection. Pneumonia patients and COPD patients are rinsed with normal saline in the morning, and then the first deep sputum is collected in the morning. Sputum plugs were removed with forceps and microscopically observed for squamous epithelial cells < 10/high power lens field (HP), WBCs > 25/HP as qualified sputum specimens.
Taking an empty test tube, weighing, selecting 100-500 mg sputum specimens without saliva, putting the sputum specimens into the weighed test tube, adding 0.1% DTT solution with 4 times volume of sputum, and sucking the sputum by using a disposable pipettePlacing in a 37 deg.C water bath box for 15min, filtering with 48 μm nylon net, centrifuging the filtrate for 5min at 150 × g, discarding supernatant, resuspending the precipitate with Phosphate Buffer (PBS), centrifuging at 150 × g, discarding supernatant, resuspending the precipitate with PBS, and adjusting cell number to 1 × 10 under microscope6and/mL as a sample to be tested.
2 detection of oxidative phagocytic function of neutrophil
2.1. Pre-processing a sample on a machine: taking two special flow-type tubes, respectively marking the tubes as a control tube and a stimulation tube, adding 50 mu L PBS into the control tube, adding 50 mu L PMA into the stimulation tube, taking 2mL EDTA anticoagulation blood, mixing uniformly, adding 50 mu L whole blood or resuspended sputum into each tube, mixing uniformly, and incubating for 15min in a 37 ℃ water bath box. Adding DHR 25. mu.L, mixing, and incubating in 37 deg.C water bath for 5min (in dark). 1mL hemolysin (sputum specimen does not need hemolysis step) is added into each tube, and the tube is protected from light for 10min at room temperature. Centrifuging at 3000r/min for 5 min. The supernatant was discarded, resuspended in 300. mu.L PBS and examined by flow cytometry.
2.2. Sample on-machine detection: control tubes were set up and adjusted with Forward Scatter (FSC) and Side Scatter (SS) to ensure that neutrophils were clearly visible. The fluorescence intensity of the control tube was adjusted to 10 on the histogram by setting the neutrophil gate to R1 and the control tube to the negative tube1And recording as M1, collecting cells in the stimulation tube, and arranging the cells on the corresponding histogram in a range of 100-101(M1) negative cells, DHR123 fluorescence Signal 101The above region was positive (M2), see FIG. 2. The positive rate of oxidative phagocytosis of neutrophils is recorded as the Gate% value of the M2 area, and 10000 cells are collected from each sample R1 Gate.
3. Detection of expression of neutrophil surface receptors (TLRs and CR)
Pre-processing a sample on a machine: taking two special flow tubes, marking as a control tube and a test tube, sequentially adding 5 microliter and 50 microliter of whole blood (or sputum sample) of CD282-Alexa Fluor antibody 10 microliter L, CD191-Alexa Fluor antibody 5 microliter L, CD284-PE antibody 5 microliter L, CD45-PerCP antibody into the test tube, sequentially adding equivalent corresponding isotype control antibody and 50 microliter of whole blood (or sputum sample) into the control tube, uniformly mixing, incubating at room temperature in a dark place for 25min, adding 500 microliter of hemolytic agent into each tube (the sputum sample does not need a hemolysis step), uniformly mixing, standing at room temperature in a dark place for 10min, centrifuging at 126 Xg for 5min, abandoning the supernatant and adding 1ml of PBS, uniformly mixing, centrifuging at 126 Xg for 5min, abandoning the supernatant and then adding 300 microliter of PBS, and detecting by an up-flow cytometer.
Loading a specimen on a machine: the neutrophil population was selected using side scattered light and CD45-PerCP (gated R1), and using side scattered light (SS) and CD45-PerCP (gated R1), (see FIG. 2) to measure the Mean Fluorescence Intensity (MFI) of CD282, CD284, CD191 expressed on the surface of neutrophils. Data are obtained and analyzed by Cell Quest software, 10000 cells are detected by each sample, and the detection on the computer is completed within two hours to ensure accuracy.
4. Statistical treatment
Statistical analysis was performed using SPSS 17.0 software. Data in a normal distribution
Showing that independent samples are taken for comparison among groups and tested by t test. Correlation between items was assessed using Pearson correlation analysis. With P<A difference of 0.05 is statistically significant.
Three, result in
Identification of the neutrophil phylum
On the dot plots obtained by CellQuest software, the neutrophil gate R1 was set according to the parameters of CD45-PerCP and side scatter, see FIG. 1.
(II) detecting the neutrophil oxidative phagocytic function and the related receptor expression positive rate of peripheral blood samples of different groups by using the established method, wherein the detection result is shown in table 1, and the result shows that compared with a healthy control group, the neutrophil oxidative phagocytic function of a pneumonia control group and that of a COPD patient group are reduced, and the difference has statistical significance; and the expression of only CD191 receptor of the three detected receptors is statistically different, so that the pneumonia control group and the COPD patient group have statistical significance compared with the healthy control group, and the pneumonia control group and the COPD patient group also have statistical significance.
TABLE 1 blood samples of each groupSex granulocyte oxidative phagocytosis percentage and relative receptor positive rate comparison
And (III) compared with a normal control group, the neutrophil oxidative phagocytosis function is remarkably reduced in the pneumonia group and the COPD group (P <0.05), and the difference between the pneumonia group and the COPD group has no statistical significance (P > 0.05). The differences in CCR-1(CD191) expression among the COPD group, the pneumonia group and the normal control group are statistically significant (P <0.05), while the differences among the TLR2 and TLR4 expression 3 groups are not statistically significant (P > 0.05). See fig. 3, fig. 4, table 1.
(IV) correlation analysis: correlation of positive rate of neutrophil oxidative phagocytosis with surface receptor (CD282, CD284, CD191) expression in sputum samples from COPD patient groups by Pearson correlation analysis, results show: the positive rate of oxidative phagocytosis of the sputum neutrophil is only negatively related to the expression of a surface receptor CD191 (P is less than 0.01), and the correlation coefficient r is-0.548, so that the positive rate has no obvious correlation with the expression of the other two receptors. See table 2.
Table 2: the oxidation and phagocytosis function of the neutrophils in each group of natural sputum specimens and related receptors
Note:▲compared with the pneumonia control group, P is less than 0.05
And (V) detecting the neutrophil oxidative phagocytosis function and the relevant receptor expression positive rate in the same COPD patient natural sputum and induced sputum specimen by using the established method, wherein the result is shown in Table 3, and the result shows that the differences of various detection indexes of the natural sputum and the induced sputum have no statistical significance, and the result shows that the results are consistent according to the detection result in the natural sputum and the result of the induced sputum.
Table 3: COPD patient induces neutrophil oxidative phagocytic function in sputum and natural sputum and related receptor
Note:△compared with natural phlegm group, P is less than 0.05
Fourth, discuss
COPD is a disease characterized by airflow limitation that is not fully reversible, a common, multiple, high-mortality chronic respiratory disease, one of the four most fatal diseases worldwide today. To date, the exact pathogenesis of COPD is not clear, COPD is now generally considered to be characterized by chronic inflammation of airways, lung parenchyma and pulmonary vessels, and an imbalance of proteases and antiproteases, oxidation and antioxidant in the lung also plays an important role in COPD pathogenesis.
Neutrophils (PMNs) are important effector cells of the body involved in innate immunity, and PMNs play an important role in the regulation of inflammation and immune responses by chemotactic phagocytosis, respiratory burst, secretion of lytic enzymes and immunologically active substances. The oxidative aggregation of neutrophils is one of the important links in the development of COPD, and both neutrophils and their components are involved in the development of COPD: 1. chemokine Receptors (CR) are a family of transmembrane receptors with chemokines as ligands, and the CR on the surface of neutrophils (e.g. CCR1) is a key molecule for their migration to sites of pathogen infection or to sites of inflammation. Wang et al confirmed that: CCR1 expression on inflammatory cells correlates with the severity of COPD; 2. toll-like receptors (TLRs) are a class of natural immune receptors that play important roles in the regulation of phagocytosis of cells in a variety of inflammatory responses, cell signaling, and apoptosis. TLRs play a decisive role in the function of neutrophils as pathogen recognition receptors and play an important role in the generation, development and regression of inflammation.
Neutrophils are double-edged sword, and cytotoxic substances in cytoplasm of the neutrophils can damage self tissue cells while killing pathogenic microorganisms invaded by outsiders. Neutrophils play a very important role in the pathogenesis of COPD. Much of the research has focused on whether an increase in neutrophil counts in COPD patients results in an increase in lifespan due to a decrease in apoptosis, however, at present apoptosis studies have shown no clear evidence of an increase in neutrophil lifespan, and even some of the findings are contradictory. In recent years, researchers have gradually shifted the visual field to the neutrophil's own function, Prieto et al have suggested that COPD patients have a defect in PMN phagocytic activity, and in animal experiments it has been demonstrated that cigarette smoke extract impairs neutrophil phagocytosis in a pseudomonas-infected murine model. Sapey et al have suggested in their studies that neutrophils in COPD patients may have their own functional deficits, with essential differences in chemotactic behavior and transitional structure from other populations.
Therefore, we focused on the oxidative phagocytic function of neutrophils in the study. COPD belongs to airway inflammation, and pneumonia is non-airway inflammation, and we can better explain that the conclusion of the experiment is unique to airway inflammation by selecting pneumonia as a control group; and meanwhile, the phagocytic function of the neutrophils in the COPD peripheral blood and sputum can better reflect the overall and local relation of the COPD.
The method for detecting the phagocytic function of the neutrophil adopts a flow cytometer-DHR method, and is a simple, convenient, rapid and good-repeatability method which is commonly used for clinically evaluating the phagocytic and oxidative functions of the neutrophil. The fluorochrome-free dihydrorhodamine123 (dihydrorhodamine123DHR) is reduced to Rhodamine123 (Rhodamine 123, Rho123) having high green fluorescence in oxidation reaction by respiratory burst when neutrophil phagocytosis is exerted. The performance of the neutrophil in enhanced function can be visually detected after being stimulated through flow cytometry detection, and DHR is firstly phagocytized by activated neutrophil and then oxidized by an oxidation system generated by the oxidized neutrophil. Therefore, the function of the neutrophil detected by the method can reflect two functions of phagocytosis and oxidation of the neutrophil, and the detection result is influenced by the defect of any function.
The research result shows that in peripheral blood, compared with a healthy control group, the oxidative killing capacity of the granulocytes in the pneumonia control group and the COPD patient group is reduced, but no statistical difference exists between the two groups; in the sputum test, the oxidative killing function of neutrophils in a COPD patient group is lower than that of a pneumonia control group, but the expression of CCR1(CD191) is higher than that of the pneumonia control group. Increased expression of CCR1 may be more neutrophil accumulation in the airways of COPD patients compared to pneumonia patients, but COPD patients are more likely to cause pathological accumulation and tissue damage in the airways compared to pneumonia patients because of the lower oxidative phagocytic function of aggregated neutrophils. This result suggests that neutrophils in COPD have their own functional defects, leading to excessive and less precise chemotaxis upon infection, and that the ability to clear pathogens is reduced despite the increased number of neutrophils in COPD, ultimately leading to pathological accumulation of neutrophils in the lung and tissue damage.
Our findings were consistent with the findings of other scholars: liu et al report that the main chemokine receptor in COPD contains at least one tyrosine sulfation site, tyrosine sulfation affects signal transduction and further affects related cells such as neutrophils which participate in COPD, and the discovery of the tyrosine sulfation site is helpful for the development of an atopic receptor-ligand antagonist, and has positive significance for the treatment of COPD. Chemokine receptors such as CCR1 play an important role in COPD, and insight into this has helped develop specific receptor-ligand antagonists that are important events in the modulation of airway disease; according to the clinical test results of a plurality of medicines, Gladue et al think that the expression of CCR1 is up-regulated in diseases such as COPD and the like, and suggest that antagonists of CCR1 or ligands thereof have the prospect of providing therapeutic means for the diseases.
The main ligands for TLR2 and TLR4 are gram-positive, fungal and gram-negative bacteria, respectively, and play an important central role in bacterial and fungal infections. The VonScheele et al study found that the down-regulation of TLR2 in PMN expression of bronchoalveolar lavage (BAL) in COPD patients may be associated with a decrease in phagocytic function of neutrophils. Our findings showed no significant difference in TLR2 and TLR4 expression in the sputum of COPD patients compared to the controls of pneumonia, probably because the expression of TLR2 and TLR4 is mainly related to the species of infection, and the species of infection in pneumonia and COPD patients are similar, so there is no difference in the expression of both receptors.
Abnormally active neutrophil basal metabolism in COPD patients is an important cause of the continued progression of the course of COPD. The resolution of neutrophil inflammation is a basic idea for curing COPD. Through experiments, a method suitable for detecting COPD (chronic obstructive pulmonary disease) sputum neutrophils by clinical flow cytometry is successfully established, and the correlation between the oxidization and killing function of the sputum neutrophils of a COPD patient and the expression level of the surface CCR1 of the sputum neutrophils is found, so that the fact that the oxidization and phagocytosis function of the sputum neutrophils of the COPD patient is damaged is suggested, the method is one of possible mechanisms of COPD pathogenesis, and a theoretical basis is provided for the follow-up exploration of a treatment strategy for effectively controlling COPD inflammation by taking the improvement of PMN phagocytosis as a target.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (10)
- The application of CCR1 as a diagnostic marker for the positive rate of oxidative killing and oxidation of neutrophils in sputum of a COPD patient, wherein the expression amount of CCR1 is inversely proportional to the positive rate of oxidative killing and oxidation of neutrophils in sputum of the COPD patient.
- 2. Use of sputum neutrophils in combination with CCR1 as a diagnostic marker for the identification of pneumonia and COPD.
- 3. The use according to claim 2, wherein patients with COPD have a lower positive rate of oxidative phagocytosis of neutrophils in sputum than patients with pneumonia, and patients with COPD have a higher expression of neutrophil CCR1 in sputum than patients with pneumonia.
- 4. Use of peripheral blood and sputum neutrophilic granulocytes in combination with CCR1 as a diagnostic marker for COPD.
- 5. The use according to claim 4, wherein the positive rates of oxidative phagocytosis and oxidation of peripheral blood and sputum neutrophils and the expression level of CCR1 in COPD patients are lower than normal.
- 6. Use of neutrophil sputum in combination with CCR1 as a diagnostic marker for the identification of airway inflammation and non-airway inflammation.
- 7. The use according to claim 6, wherein patients with airway inflammation have a lower positive rate of oxidative phagocytosis of neutrophils in sputum than patients with non-airway inflammation, and patients with airway inflammation have a higher expression of granulocyte CCR1 in sputum than patients with non-airway inflammation.
- 8. A kit for distinguishing airway inflammation from non-airway inflammation is characterized by comprising a reagent for detecting the positive rate of oxidative phagocytosis and oxidation of sputum neutrophils and the expression quantity of CCR 1.
- 9. A kit for identifying COPD and pneumonia is characterized by comprising a reagent for detecting the positive rate of oxidative phagocytosis and oxidation of sputum neutrophil granulocytes and the expression amount of CCR 1.
- 10. A COPD diagnostic kit is characterized by comprising a reagent for detecting the oxidative phagocytosis oxidation positive rate of peripheral blood and sputum neutrophil granulocytes and the expression quantity of CCR 1.
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