Application of serum MMP-7 protein as a serum marker in the preparation of a kit for determining chronic obstructive pulmonary disease combined with sarcopenia

Abstract

The present application discloses a use of serum MMP-7 protein as a serum marker in the preparation of a kit for determining chronic obstructive pulmonary disease combined with sarcopenia, belonging to the technical field of biological diagnostic products. The kit is used to detect the concentration of MMP-7 protein in serum samples of patients with chronic obstructive pulmonary disease, and determine whether the patient has chronic obstructive pulmonary disease combined with sarcopenia based on the concentration of MMP-7 protein in the serum sample.

Description

Technical Field

[0001] The present application relates to the technical field of biological diagnostic products, and in particular to a kit for determining chronic obstructive pulmonary disease combined with sarcopenia using serum MMP-7 protein as a serum marker and its application. Background Art

[0002] At present, with chronic obstructive pulmonary disease becoming the fourth leading cause of death in the world and the aging of the population, the incidence of chronic obstructive pulmonary disease combined with sarcopenia (sarcopenia) continues to rise, and 15%-21.6% of chronic obstructive pulmonary disease patients have sarcopenia. Sarcopenia leads to decreased diaphragmatic function and decreased exercise tolerance, and the inflammatory response caused by chronic obstructive pulmonary disease aggravates sarcopenia. Chronic obstructive pulmonary disease and sarcopenia affect each other, forming a vicious circle. In the prior art, the method for measuring the skeletal muscle mass of the limbs for sarcopenia is costly, has radiation risks, and is limited to those with metal implants. Summary of the invention

[0003] In view of this, the present application provides a diagnostic kit using serum MMP-7 protein as a serum marker, which can be used to determine whether a patient has chronic obstructive pulmonary disease combined with sarcopenia.

[0004] Relevant data showed that serum MMP-7 protein was highly expressed in the non-sarcopenia group. When the MMP-7 protein concentration in the patient's serum sample was 46.366125 ng / mL, the sensitivity of distinguishing between the sarcopenia population and the non-sarcopenia population in chronic obstructive pulmonary disease was 90.24%, and the specificity was 92.68%.

[0005] In the first aspect, the present application provides the use of serum MMP-7 protein as a serum marker in the preparation of a kit for determining chronic obstructive pulmonary disease combined with sarcopenia, wherein the kit is used to detect the concentration of MMP-7 protein in a serum sample of a patient with chronic obstructive pulmonary disease, and when the concentration of MMP-7 protein in the patient's serum sample is lower than a threshold value m, the patient is determined to have chronic obstructive pulmonary disease combined with sarcopenia, wherein the threshold value m is 47 ng / mL. It can be understood that as long as the concentration of MMP-7 protein in the serum sample of a patient with chronic obstructive pulmonary disease is less than or equal to 47 ng / mL, the patient can be determined to be a patient with chronic obstructive pulmonary disease combined with sarcopenia.

[0006] Preferably, when the MMP-7 protein concentration in the patient's serum sample is 46.366125 ng / mL, the sensitivity of distinguishing chronic obstructive pulmonary disease with sarcopenia from chronic obstructive pulmonary disease without sarcopenia is 90.24%, and the specificity is 92.68%.

[0007] Preferably, the kit is an Elisa kit for detecting serum MMP-7 protein.

[0008] Preferably, the method for detecting the MMP-7 protein concentration in serum samples of patients with chronic obstructive pulmonary disease comprises: collecting 2-3 ml of blood sample from each patient, centrifuging at 4000 rpm for 15 min at 4°C, aspirating 1000 μl of supernatant and placing it in a 1.5 ml EP tube and adding 10 μl of protease inhibitor, followed by protein extraction.

[0009] In a second aspect, the present application provides a kit for determining whether a patient suffers from chronic obstructive pulmonary disease combined with sarcopenia, wherein the kit contains a reagent capable of quantifying serum MMP-7 protein.

[0010] Optionally, the reagent can be adapted to detect the MMP 7 protein content by colloidal gold method, electrophoresis method, immunofluorescence method, direct competitive ELISA method, indirect competitive ELISA method, double antibody sandwich ELISA method, RIA method, flow cytometry method or immunochromatography method.

[0011] Optionally, the kit is selected from any one of the kits involved in any of the uses described above.

[0012] In a third aspect, the present application provides a method for detecting the concentration of MMP-7 protein in serum samples of patients with chronic obstructive pulmonary disease, the method comprising at least the following steps:

[0013] S100, 2-3 ml of blood sample was collected from each patient, centrifuged at 4000 rpm for 15 min at 4°C, 1000 μl of supernatant was aspirated and placed in a 1.5 ml EP tube and 10 μl of protease inhibitor was added, which was recorded as the standard sample;

[0014] S200, transfer the standard sample into a 50 ml centrifuge tube, add 5 times the volume of pre-cooled acetone, place at -20°C for 2 hours, centrifuge at 25000g at 4°C for 15 minutes, and discard the supernatant;

[0015] S300, air-dry the precipitate, then add protein lysis buffer without SDS to dissolve, centrifuge at 25000g at 4°C for 15min, and take the supernatant;

[0016] S400, add 10 mM dithiothreitol, incubate in a water bath at 37°C for 30 min, then add 55 mM iodoacetamide, and place in dark for 45 min;

[0017] Centrifuge at S500, 25000g at 4°C for 15 min to obtain the supernatant, which is the protein solution.

[0018] Optionally, the method is used to obtain the MMP-7 protein concentration in serum samples of patients with chronic obstructive pulmonary disease involved in any of the above uses.

[0019] The beneficial effects brought about by the technical solutions provided by some embodiments of the present application include at least:

[0020] The blood test method is easy to obtain, widely applicable, low-invasive, and relatively low-cost. There is no definite diagnostic biomarker for sarcopenia. This application discovered for the first time that serum MMP-7 protein can be used as a serum marker to determine whether a patient has chronic obstructive pulmonary disease with sarcopenia. According to statistical calculations, when the MMP-7 protein concentration in the patient's serum sample is 46.366125 ng / mL, its sensitivity for distinguishing chronic obstructive pulmonary disease with sarcopenia and chronic obstructive pulmonary disease without sarcopenia is 90.24%, and its specificity is 92.68%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Bradford quantitative protein concentration results and standard curve in iTRAQ experiment;

[0023] Figure 2 The expression of MMP-7 in the sarcopenia group and the non-sarcopenia group was detected by ELISA;

[0024] Figure 3 The diagnostic efficacy of MMP-7 for chronic obstructive pulmonary disease combined with sarcopenia. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0026] The technical concept of the present application is to use serum MMP-7 protein as a serum marker, and to determine whether the patient has chronic obstructive pulmonary disease combined with sarcopenia by detecting the MMP-7 protein concentration in serum samples of patients with chronic obstructive pulmonary disease.

[0027] First, the present application provides the use of serum MMP-7 protein as a serum marker in the preparation of a kit for determining chronic obstructive pulmonary disease with sarcopenia, including the following: detecting the concentration of MMP-7 protein in the serum sample of a patient with chronic obstructive pulmonary disease by an Elisa kit for detecting serum MMP-7 protein, and determining that the patient has chronic obstructive pulmonary disease with sarcopenia when the concentration of MMP-7 protein in the patient's serum sample is lower than 47 ng / mL. In particular, when the concentration of MMP-7 protein in the patient's serum sample is 46.366125 ng / mL, the sensitivity of distinguishing chronic obstructive pulmonary disease with sarcopenia from chronic obstructive pulmonary disease without sarcopenia is 90.24%, and the specificity is 92.68%.

[0028] Secondly, the present application provides a kit for determining whether a patient has chronic obstructive pulmonary disease combined with sarcopenia, wherein the kit contains a reagent capable of quantifying serum MMP-7 protein, and the reagent can be adapted to colloidal gold method, electrophoresis method, immunofluorescence method, direct competitive ELISA method, indirect competitive ELISA method, double antibody sandwich ELISA method, RIA method, flow cytometry method or immunochromatography method for detecting MMP 7 protein content. Preferably, double antibody sandwich ELISA method.

[0029] The preparation method of the kit can refer to the prior art and will not be described in detail in this application.

[0030] Research Methods

[0031] Eligible cases were screened from inpatients in the Department of Respiratory Medicine of the Second Affiliated Hospital of Chongqing Medical University from 2021 to 2022. Blood specimens from two groups of patients (COPD with sarcopenia group and COPD without sarcopenia group) were collected in total, and the two groups of specimens were divided into a discovery cohort (COPD with sarcopenia group and COPD without sarcopenia group) and a validation cohort (COPD with sarcopenia group and COPD without sarcopenia group).

[0032] Inclusion criteria for patients with sarcopenia: 1. All subjects were patients with chronic obstructive pulmonary disease hospitalized in the Department of Respiratory Medicine of the Second Affiliated Hospital of Chongqing Medical University (mainly aged ≥18 years); 2. Diagnostic criteria: limb skeletal muscle mass (ASM): (1). ASM / height 2: DXA (male <7.00kg / m2, female <5.40kg / m2); (2). BIA (male <7.00kg / m2, female <5.7kg / m2) S; muscle strength (MS): grip strength: male <28kg, female <18kg; physical function (PP): 5 sit-ups time ≥12S, SPPB ≤9, walking speed <1m / s. Low ASM + low MS or (and) low PP is considered to have sarcopenia, otherwise it is non-sarcopenia.

[0033] Exclusion criteria: 1. Autoimmune diseases; 2. Tumor diseases; 3. Combination with other serious primary diseases; 4. Those who refused to participate in the study or refused to sign the informed consent form were excluded. Whole blood samples were collected from the ward, placed in an ice-water mixture and sent to the laboratory. After centrifugation and protease inhibitor treatment, they were stored in a -80℃ freezer. After a certain number of serum samples were collected, some serum samples were taken to find the differential proteins between the two groups of samples by isotope labeling relative and absolute quantification (iTRAQ) method. At the same time, samples continued to be collected, and then enzyme-linked immunosorbent assay (ELISA) was performed to quantitatively analyze the target protein. Finally, the data were statistically analyzed using data analysis software to verify the diagnosis of sarcopenia by these biomarkers.

[0034] Experimental Results

[0035] Through proteomics technology and analysis, a total of 275 sarcopenia serum differentially expressed proteins were identified, of which 174 serum proteins were upregulated and 101 serum proteins were downregulated. Combining the function of the protein and clinical practice, proteins that did not meet the requirements were excluded, and finally one key protein was determined: Matrilysin (MMP-7). Analysis of the results of the enzyme-linked immunosorbent assay verified that the above-mentioned MMP-7 was highly expressed in the non-sarcopenia group, and the sensitivity of distinguishing between sarcopenia and non-sarcopenia groups in chronic obstructive pulmonary disease was 90.24%, and the specificity was 92.68%.

[0036] Conclusion: Serum MMP-7 protein has the potential to become a biomarker for sarcopenia. It has certain value in the diagnosis of sarcopenia. If it can be used in clinical practice, early screening of sarcopenia patients and early intervention can improve the quality of life of patients.

[0037] (I) iTRAQ experiment

[0038] 1.1. Specimen collection and processing

[0039] The samples of patients with COPD combined with sarcopenia and COPD without sarcopenia were collected and labeled. A total of 2-3 mL of blood samples were collected from each patient and stored in an ice box with an ice-water mixture for transportation. The samples were centrifuged at 4000 rpm × 4 ° C × 15 min in the laboratory to remove broken tissues and other substances. 1000 μl of the supernatant was aspirated with a pipette and 10 μl of protease inhibitor was added to a 1.5 ml EP tube. The samples were stored in a -80 ° C ultra-low temperature freezer for future use. During storage, the samples should be kept from repeated freezing and thawing to avoid affecting the experimental results.

[0040] 1.2 iTRAQ specific process

[0041] 1.2.1 Specimen pre-processing information

[0042] Twenty patient specimens were collected, including 10 from the COPD combined with sarcopenia group and 10 from the COPD non-sarcopenia group. 500 μl of each specimen was taken out twice and mixed by group, and finally two non-sarcopenia groups (numbered N1 and N2) and two sarcopenia groups (numbered S1 and S2) with a volume of 5000 μl were obtained.

[0043] 1.2.2 Protein extraction

[0044] a) Transfer the above four specimens into 50 ml centrifuge tubes, add 5 times the volume of pre-cooled acetone, place in a -20℃ refrigerator for 2 hours, centrifuge at 25000g*4℃ for 15 minutes, and discard the supernatant;

[0045] b) Air-dry the precipitate, add an appropriate amount of protein lysis buffer without SDS, use an ultrasonic device to promote protein dissolution, centrifuge at 25000g*4℃ for 15min, and collect the supernatant;

[0046] c) Add DTT (dithiothreitol) to a final concentration of 10 mM and incubate in a water bath at 37°C for 30 min;

[0047] d) Add IAM (iodoacetamide) to a final concentration of 55 mM and place in a dark room for 45 min;

[0048] e) Centrifuge at 25000g at 4℃ for 15 min and take the supernatant, which is the protein solution, and place it in a 4℃ refrigerator for later use.

[0049] 1.2.3 Determination of sample protein concentration

[0050] f) Take 40 μl of BSA standard (5 mg / ml) in a centrifuge tube, add 160 μl of PBS solution to it to prepare a 1 μg / μl standard solution;

[0051] g) Take a 96-well plate and add 20μl, 19μl, 18μl, 16μl, 12μl, 8μl, 4μl, and 0μl of PBS solution to the wells in the first and second columns, respectively, so that the PBS volume of the two wells in the same row is the same and serves as a reference. Add the above standards to the wells that have been added with PBS, with the volume of 0μl, 1μl, 2μl, 4μl, 8μl, 12μl, 16μl, and 20μl, respectively, and gently shake to mix;

[0052] h) Add 10 μl of each of four samples to a 96-well plate, set up one replicate well for each sample, for a total of 8 wells, and then dilute these samples to 20 μl with PBS;

[0053] i) Add 200 μl of Coomassie Brilliant Blue staining solution (G250 staining solution) to each well of the 96-well plate, place at room temperature for 5 minutes, and place in an automatic microplate reader to read the absorbance at 595 nm;

[0054] j) Draw the standard curve as shown below Figure 1 , calculate the sample concentration.

[0055] 1.2.4 iTRAQ sample labeling and mass spectrometry analysis

[0056] I) Serum samples were processed according to the iTRAQ kit manual, and sample labeling and mass spectrometry analysis were completed according to the experimental steps. The sample data obtained from the experimental results were analyzed using ProteinPilot2.0 analysis software, and the comparison database was the Uniprot human protein library;

[0057] II) Selection criteria for differentially expressed proteins: 95% confidence interval and 5% false positive rate; at least 2 or more specific protein recognition peptides; differentially expressed proteins must be screened under the conditions of change fold >1.5 or <0.5 and P-value <0.05.

[0058] The differentially expressed proteins must meet the above conditions in both runs.

[0059] (II) Results Analysis

[0060] 2.1 Comparison of general conditions of the subjects (experimental group and control group)

[0061] According to the above methods and standards, a total of 10 cases were collected in each discovery cohort (chronic obstructive pulmonary disease with sarcopenia group and chronic obstructive pulmonary disease without sarcopenia group). In the discovery cohort, there were 6 males (60%) and 4 females (40%) in the chronic obstructive pulmonary disease with sarcopenia group; there were 4 males (40%) and 6 females (60%) in the chronic obstructive pulmonary disease without sarcopenia group, all of which were mainly males. There was no statistical difference in gender, age, and other aspects among all the enrolled patients (p>0.05). The demographic and clinical characteristics of the subjects are shown in Table 1.

[0062] Table 1

[0063]

[0064] 2.2 Bradford protein quantification standard curve

[0065] Figure 1 The Bradford quantitative protein concentration results and standard curve in the iTRAQ experiment. The R in the standard curve y = 0.75x-0.1271 2 =0.9944>0.99, indicating that the result is relatively accurate.

[0066] 2.3 iTRAQ experimental results

[0067] Through proteomics technology and analysis, a total of 275 sarcopenia serum differentially expressed proteins were identified, of which 174 serum proteins were upregulated and 101 serum proteins were downregulated. The conditions of peptide ≥ 2, multiple ratio > 1.5 times and P-value < 0.05 screened 16 upregulated proteins; the conditions of peptide ≥ 2, multiple ratio < 0.5 times and P-value < 0.05 screened 1 downregulated protein. Combined with the function of the protein and clinical practice, proteins that did not meet the requirements were excluded, and finally one key protein was determined: Matrilysin (MMP-7). Analysis of the results of the enzyme-linked immunosorbent assay verified that the above-mentioned MMP-7 was highly expressed in the non-sarcopenia group. The results of iTRAQ quantitative analysis are shown in Table 2.1 (upregulated proteins) and Table 2.2 (downregulated proteins).

[0068] Some Chinese and English correspondences in Table 2.1 and Table 2.2: Keratin, type II cytoskeletal2epidermal human keratin type II cytoskeletal2; CREB-binding protein cyclic adenosine monophosphate response element binding protein; Hemoglobin subunit delta hemoglobin δ antibody; Hemoglobin subunit beta hemoglobin β subunit; Thyroglobulin; Hemoglobin subunit alpha hemoglobin Α1 / Α-GLOBIN antibody; Mast cell-expressed membrane protein 1 mast cell expressed membrane protein 1 antibody; Carbonicanhydrase 1 recombinant human carbonic anhydrase 1.

[0069] Table 2.1

[0070] Name Peptides S:N Keratin,type II cytoskeletal 2epidermal 37 4.182106 Hornerin protein (HRNR) 5 3.338287 CREB-binding protein 164 2.303864 Hemoglobin subunit delta 24 2.097201 Hemoglobin subunit beta(HBB) 129 2.033687 Keratin, type I cytoskeletal 9 107 1.922378 Thyroglobulin 2 1.826143 Hemoglobin subunit alpha 229 1.79068 Hemoglobin subunit gamma-1 17 1.740906 Inactive C-alpha-formylglycine-generating enzyme 2 2 1.711854 Mast cell-expressed membrane protein 1 2 1.666995 Immunoglobulin heavy variable 3-49 52 1.642723 Immunoglobulin lambda variable 2-14 6 1.625648 Keratin, type II cytoskeletal 6A 7 1.561449 Keratin, type I cytoskeletal 10 44 1.516143 Carbonic anhydrase 1 58 1.505834

[0071] Table 2.2

[0072] Name Peptides S:N Apolipoprotein A-II 1 0.452777 Pleckstrin homology domain-containing family G member 2 1 0.449891 Matrilysin 5 0.383805 Protocadherin-8 1 0.151259

[0073] Note: S:N refers to the multiple change between S (sarcopenia group) and N (non-sarcopenia group).

[0074] Based on the differentially expressed proteins obtained above, combined with the functions of proteins and clinical practice, after excluding proteins that did not meet the requirements, a key protein, Matrilysin (MMP7), was finally identified.

[0075] The key proteins screened above were further verified to evaluate their diagnostic value in sarcopenia.

[0076] (III) Enzyme linked immunosorbent assay (ELISA) quantitative determination of target protein

[0077] 3.1 Determination of MMP7 content in serum

[0078] A commercial enzyme-linked immunosorbent assay kit was used to measure the MMP-7 content in serum samples from 82 patients with chronic obstructive pulmonary disease. The detection limit of MMP-7 was 0.75 ng / ml. The Elisa kit used a double antibody sandwich method. Taking MMP-7 as an example, the test was carried out according to the following steps: (1) All tests were carried out at room temperature, with 96 wells, including 6 standard wells and 40 sample wells, and the sample wells were repeated twice; (2) 50 μL of standard solutions with different concentrations were added to each standard well, 40 μL of sample diluent was added to the sample well, and then 10 μL of the sample to be tested was added (the final sample dilution was 5 times); (3) 100 μL of enzyme standard reagent was added to each well except the empty well, and then sealed with a sealing film and placed in an incubator at 37°C for 60 minutes; at the same time, the 20-fold concentrated washing solution was diluted with 20-fold distilled water and set aside. (4) Remove the film of the lid, drain the water, then pour the washing solution into each well, let it sit for 30 seconds, drain the water again, rinse it repeatedly 5 times, and finally wipe it dry; (5) Then, add 50 μL of color developer A to each well, then add 50 μL of color developer B, shake and mix evenly, and color develop for 15 minutes under the shade of 37°C; (6) Then add 50 μL of stop liquid to end the reaction; (7) Finally, put it into the enzyme marker and measure the absorbance (OD value) of each well in turn at a wavelength of 450 nanometers. Finally, repeat the above steps to complete the determination of RBP-4 content.

[0079] 3.2 Statistical analysis

[0080] This study used SPSS25.0 for statistical analysis and GraphPad Prism 8.0.1 for graphical analysis.

[0081] All data passed the normality test, and descriptive results were presented according to normal distribution or non-normal distribution. Normally distributed measurement data were presented as mean ± standard deviation, and non-normally distributed measurement data were presented as median and interquartile range. Independent sample t test was used to compare measurement data between two groups with normal distribution, and Mann-Whitney U test was used to compare measurement data between two groups with non-normal distribution. Chi-square test was used for count data and rank data. When P value was less than 0.05, the difference was considered statistically significant.

[0082] 3.3 Experimental Results

[0083] 3.3.1 Research subjects (experimental group and control group)

[0084] In the validation experiment, the validation cohort (41 cases in the COPD combined with sarcopenia group and 41 cases in the COPD non-sarcopenia group) totaled 82 cases. There was no statistical difference in gender and age among all the enrolled patients (p>0.05). The demographic and clinical characteristics of the validation cohort subjects are shown in Table 3.

[0085] Table 3

[0086]

[0087] 3.3.2 Quantitative analysis results of serum proteins

[0088] By detecting the MMP-7 protein molecule with a commercial ELISA kit (Table 4), and using SPSS25.0 statistical analysis and GraphPad Prism8.0.1 for graphical analysis, we found that the expression of MMP-7 in the non-sarcopenia group was higher than that in the sarcopenia group. After statistical analysis, the protein was statistically significant. Figure 2 Its diagnostic efficacy is shown in Figure 3 .

[0089] Table 4

[0090]

[0091]

[0092] Combination Figure 2 and Figure 3 When the area under the receiver operating curve was 0.962 and the MMP7 serum concentration was 46.366125 ng / ml, the diagnostic efficacy of the sarcopenia population was the highest, with a sensitivity of 90.24% and a specificity of 92.68%.

[0093] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of serum MMP-7 protein as a serum marker in the preparation of a kit for determining chronic obstructive pulmonary disease combined with sarcopenia, characterized in that: The kit is used to detect the concentration of MMP-7 protein in serum samples of patients with chronic obstructive pulmonary disease; When the MMP-7 protein concentration in the patient's serum sample is lower than the threshold value m, the patient is determined to have chronic obstructive pulmonary disease combined with sarcopenia; Wherein, the threshold value m is 47 ng / mL.

2. The use according to claim 1, characterized in that When the MMP-7 protein concentration in the patient's serum sample was 46.366125 ng / mL, its sensitivity for distinguishing between COPD with sarcopenia and COPD without sarcopenia was 90.24% and its specificity was 92.68%.

3. The use according to claim 1 or 2, characterized in that The kit is an Elisa kit for detecting serum MMP-7 protein.

4. The use according to claim 1 or 2, characterized in that: The method for detecting the concentration of MMP-7 protein in serum samples of patients with chronic obstructive pulmonary disease includes: 2-3 ml of blood sample was collected from each patient, centrifuged at 4000 rpm for 15 min at 4°C, 1000 μl of supernatant was aspirated and placed in a 1.5 ml EP tube, and 10 μl of protease inhibitor was added before protein extraction.

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