CN114231618A - Application of S100A10 detection reagent in preparation of COPD diagnostic product - Google Patents

Application of S100A10 detection reagent in preparation of COPD diagnostic product Download PDF

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CN114231618A
CN114231618A CN202111658715.2A CN202111658715A CN114231618A CN 114231618 A CN114231618 A CN 114231618A CN 202111658715 A CN202111658715 A CN 202111658715A CN 114231618 A CN114231618 A CN 114231618A
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宋亚茹
王辉
李荣凯
翟成凯
韩飞
娄运伟
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Xinxiang First People's Hospital
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of an S100A10 detection reagent in preparation of a COPD diagnostic product. The result of the invention shows that the expression of S100A10 in PBMCs of COPD patients is reduced, and the expression level is in negative correlation with FEV 1% pred, and S100A10 plays an important regulation role in the disease process of COPD by detecting and comparing the expression change of S100A10 in peripheral blood mononuclear cells of a healthy control group and a COPD patient group and the correlation between the expression level and a lung function related index.

Description

Application of S100A10 detection reagent in preparation of COPD diagnostic product
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of an S100A10 detection reagent in preparation of a COPD diagnostic product.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a Chronic respiratory disease with persistent decline in lung function caused by factors such as smoking, and the pathogenesis of COPD is complex, and the COPD has close relationship with genetic factors and interaction with environmental factors. Researches show that the infiltration of various immune cells in the local inflammation process of the airway, especially inflammatory mediators released by innate immune cells play an important role in the pathogenic process of COPD, and the researches show that the levels of cytokines such as IL-6, IL-10, TNF-alpha and the like in peripheral blood and the level of lymphocyte subpopulation are closely related to the severity of COPD diseases, and the dynamic process of the process is very favorable for further elucidation of the pathogenesis of the COPD.
The S100 protein is a multifunctional micromolecular Ca+Binding protein family, S100A10 is one member of the protein family, but S100A10 lacks EF-hand Ca+A binding domain. The S100A10 dimer, consisting of two 11-kDa subunits, belongs to an intracellular protein and, when bound to the ligand Annexin A2, is present on the cell membrane as a (S100A10)2- (Annexin A2)2 tetramer as a receptor for plasminogen. Several studies have shown abnormal expression levels of S100a10 in patients with various inflammation-related disorders, such as antiphospholipid syndrome, systemic lupus erythematosus and various tumor tissues. S100A10 regulates the migration process of macrophages and neutrophils to an inflammation part by regulating the production of the fibrin and the activity of matrix metalloproteinase 9, and S100A10 also influences the infiltration of tumor-related macrophages to tumor tissues so as to participate in the process of tumors. Recently, the S100A10 plays an important role in Toll-like receptor signal transduction and anti-infection immune processes, macrophages with S100A10 gene defects are over-activated and secrete more cytokines such as TNF-alpha, IL-6, IL-12 and IFN-beta, and the results suggest that the S100A10 inhibits the production of various proinflammatory cytokines in the immune response process. However, no changes in expression of S100a10 in COPD patients have been reported, and it is unclear whether S100a10 plays an important role in the progression of COPD.
Disclosure of Invention
In order to solve the technical problem, the invention provides an application of an S100A10 detection reagent in preparing a COPD diagnostic product.
The invention aims to provide an application of an S100A10 detection reagent in preparing a COPD diagnostic product.
Preferably, the above S100a10 detection reagent is used for preparing a COPD diagnostic product, and the detection reagent comprises a reagent for detecting S100a10 by real-time fluorescence quantitative PCR, immunoblotting or flow cytometry.
Preferably, the reagent for detecting S100A10 is applied to the preparation of a COPD diagnostic product, and the reagent for detecting S100A10 by real-time fluorescence quantitative PCR comprises a target gene primer and an internal reference gene primer.
Preferably, the application of the S100A10 detection reagent in the preparation of a COPD diagnostic product, the sense strand of the target gene primer: 5-CCAGGTTTCGACAGACTCTTC-3', antisense strand of target gene primer: 5'-CCGTTCCATGAGCACTCTC-3', respectively;
internal reference beta-actin sense chain: 5'-GGAAATCGTGCGTGACATTAA-3', antisense strand 5'-AGGAAGGAAGGCTGGAAGAG-3'.
Preferably, the S100A10 detection reagent is applied to preparing a COPD diagnostic product, and a sample for detecting COPD is peripheral blood mononuclear cells.
Preferably, the S100A10 detection reagent is used for preparing a product for diagnosing COPD, wherein in the peripheral blood mononuclear cells of COPD, S100A10 is in CD14+The expression level of the monocyte subpopulation is reduced.
Preferably, the S100A10 detection reagent is used for preparing a COPD diagnostic product, and the expression level of S100A10 in the peripheral blood mononuclear cells of COPD is in negative correlation with FEV 1% pred.
Preferably, the S100A10 detection reagent is applied to preparing a COPD diagnostic product, and the S100A10 detection reagent is applied to preparing a lung function diagnostic product.
Compared with the prior art, the invention has the following beneficial effects:
the invention discusses the potential role of S100A10 in the COPD disease process by detecting and comparing the expression change of S100A10 in peripheral blood mononuclear cells of a healthy control group and a COPD patient group and the correlation between the expression level and a lung function related index. The results show that: the mRNA expression level of S100A10 in PBMCs in the COPD group is obviously lower than that in the control group (1.13 plus or minus 0.12 to 0.47 plus or minus 0.11, P<0.05), the protein expression level of S100A10 in PBMCs in the COPD group is obviously lower than that in the control group (1.72 +/-0.25 to 0.71 +/-0.14, P)<0.05), S100A10 in CD14 among PBMCs in COPD group+The expression level in the cell subset is obviously reduced (51.72 +/-5.34 is compared with 31.05 +/-2.98, P<0.05); the expression level of S100a10 in PBMCs in the COPD group was negatively correlated with FEV 1% pred (r-0.610, P)<0.001; ) (ii) a In conclusion, the expression of S100a10 is reduced in PBMCs of COPD patients, and its expression level is negatively correlated with FEV 1% pred, S100a10 plays an important regulatory role in the disease process of COPD.
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FIG. 1 is a graph showing the expression level of S100A10 in PBMCs of a healthy control group and a COPD group;
a, detecting the expression change of S100A10 in two groups of PBMCs by real-time fluorescence quantitative PCR; b, detecting expression changes of S100A10 in the two groups of PBMCs by Western blot; c is a histogram of B after quantification of band fluorescence intensity, P <0.05 compared to control;
FIG. 2 is a graph showing the expression level of S100A10 in each cell subset of PBMCs in the control group and COPD group;
a, S100A10 CD19 in PBMCs+Changes in expression of B lymphocytes;
a1 is a quantitative graph of the mean fluorescence intensity of A;
b, S100A10 CD4 in PBMCs+Changes in expression of T lymphocytes;
b1 is a quantitative plot of the mean fluorescence intensity of B;
c, S100A10 CD8 in PBMCs+Changes in expression of T lymphocytes;
c1 is a quantitative plot of the mean fluorescence intensity of C;
d, S100A10 CD14 in PBMCs+(ii) a change in expression of monocytes;
d1 is a quantitative plot of the mean fluorescence intensity of D;
in FIG. 2, MFI, Mean fluorescence intensity; p <0.05 compared to control group.
FIG. 3 is a graph showing the correlation of the expression level of S100A10 in PBMCs in the COPD group with FEV 1% pred.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.
In the description of the present invention, reagents used are commercially available and methods used are conventional in the art, unless otherwise specified.
Example 1 case sources and Experimental methods
1.1 study object
The COPD group: cases collected from COPD patients who were breathed in the first civilian hospital and hospitalized in the critical care medical department in the new country city from 8 months to 6 months in 2021 in 2020 total 42 cases, 29 of which were male and 13 of which were female. The patients are diagnosed as COPD for the first time, and the selection criteria of cases all accord with the COPD diagnosis criteria established by the respiratory disease division chronic obstructive pulmonary disease group of the Chinese medical society (the respiratory disease division chronic obstructive pulmonary disease group of the Chinese medical society, the diagnosis and treatment guideline for chronic obstructive pulmonary disease (revised 2021 year) [ J ]. Chinese tuberculosis and J. breathe, 2021, 44(3):170-205.), and the immunomodulator and the hormone preparation are not taken for at least half a year. Exclusion criteria: the patients with the history of other respiratory diseases and the patients with serious dysfunction of other organs except the respiratory diseases are excluded.
Healthy control group: the medical examination center of the first people in New countryside selects 31 healthy volunteers with no visceral diseases such as heart, lung and brain, no recent respiratory tract infection history and normal lung function, wherein 20 male patients and 11 female patients are selected.
All the candidates were informed and signed with informed consent, and approved by the ethical committee of the first people hospital in the new country, and the lot number: 2020072001.
1.2 Experimental methods
1.2.1 pulmonary function assay: all subjects tested for lung function, typically at least 30 minutes prior to testing, followed by testing of lung function under the direction of the same lung function testing physician, as indicated by conventional first Second Forced Expiratory Volume In One Second (FEV 1) as a predicted percentage (FEV 1% pred) and first Second Forced Expiratory Volume as a percentage of Forced Vital Capacity (FVC 1/FVC (%)), etc.
1.2.2 blood specimen collection: all subjects were fasted in the morning, 3ml of peripheral venous blood was drawn, placed in EDTA anticoagulation tubes, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll density gradient centrifugation in a super clean bench under sterile conditions.
1.2.3 RNA extraction and reverse transcription: adding 1ml of cell RNA extract (RNAioso Plus, 9108, TaKaRa) to PBMCs, shaking to lyse the cells, standing at room temperature for 5min, then adding 0.2ml of chloroform, shaking vigorously in the hands for 15s, standing at room temperature for 3min, centrifuging at 12000rpm for 10min at 4 ℃, taking the upper aqueous phase, placing in a new EP tube, adding 0.5ml of isopropanol, standing at room temperature for 10min, centrifuging at 12000rpm for 10min at 4 ℃, discarding the supernatant, adding 1ml of 75% ethanol for washing, vortex mixing, centrifuging at 7500rpm for 5min at 4 ℃, discarding the supernatant, allowing the RNA precipitate to dry naturally, and finally adding 50. mu.l of RNase-free deionized water to dissolve the RNA precipitate. The RNA concentration was determined spectrophotometrically and the total cellular RNA was reverse transcribed into cDNA following the strict kit (RT Master mix, RR036A, TaKaRa).
1.2.4 real-time fluorescent quantitative PCR: and (3) carrying out real-time fluorescence quantitative Polymerase Chain Reaction (PCR) by taking the reverse transcribed cDNA as a template and beta-actin as an internal reference.
The amplification length of the target gene primer S100A10 is 135bp, and the sense strand of the target gene primer is as follows: 5-CCAGGTTTCGACAGACTCTTC-3' (SEQ ID NO.1), the antisense strand of the target gene primer: 5'-CCGTTCCATGAGCACTCTC-3' (SEQ ID NO. 2).
The amplification length of the internal reference beta-actin is 189bp, and a sense strand: 5'-GGAAATCGTGCGTGACATTAA-3' (SEQ ID NO.3), antisense strand 5'-AGGAAGGAAGGCTGGAAGAG-3' (SEQ ID NO. 4).
The primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc.
The PCR amplification adopts TB Green Premix Ex Taq II kit (TaKaRa), and the used instruments are as follows: applied Biosystems 7500Real Time PCR System, S100a10 and internal reference reaction conditions were: the first step is as follows: pre-denaturation, 30s, second step: PCR reactions, 40 cycles, 95 ℃ 5S → 60 ℃ 30S, 3 replicates of S100A10 and internal controls were performed for each specimen. The relative expression level of the S100A10 gene was calculated by the 2-. DELTA.ct method.
1.2.5 immunoblotting (Western blot): adding 100 mul of mixed solution of protein lysate and protease inhibitor into a sample to be detected, performing ice lysis for 30 minutes, centrifuging at 4 ℃ at 12000rpm for 15 minutes, taking supernatant, taking 5 mul, determining protein concentration by adopting a BCA method (Biyun day), adding a proper volume of 5 x protein loading buffer into the rest sample, heating at 100 ℃ for 10 minutes, collecting the sample, and storing in a refrigerator at-20 ℃. The samples were electrophoresed on SDS-PAGE and transferred to PVDF membrane, and 5% skimmed milk powder was blocked for 1 hour and primary antibodies were incubated, wherein S100A10(66227-1-Ig) and Actin (66009-1-Ig) antibodies were purchased from Proteitech, and after overnight incubation at 4 ℃ the corresponding secondary antibodies were added the next day before exposure to color using Amersham Imager 600RGB instrument.
1.2.6 Flow cytometry (Flow cytometry): frozen PBMCs were thawed quickly and thawed for resuscitation and then fixed for well-punch (BD Cytofix/Cytoperm), stained with intracellular S100A10 using a mouse anti-human monoclonal antibody (2.5. mu.g/ml, clone 148, BD Transduction Laboratories), isotype control using a mouse IgG monoclonal antibody (2.5. mu.g/ml, clone MOPC-21, Biolegend), followed by recognition of the primary antibody with an APC fluorescently labeled rabbit anti-mouse secondary antibody (eBioscience). Cells were washed extensively, blocked with 1% mouse serum and 1% fetal bovine serum and stained with an antibody cocktail (antibody cocktail includes CD4, CD8, CD14 and CD19, all from eBioscience) after 30 minutes, and tested with a flow cytometer (facscan).
1.3 statistical analysis: the experimental data are expressed as mean ± standard error (means ± SEM), mean differences between the two groups were analyzed using a two-sided unpaired t-test, and correlations were compared using Spearman rank correlation analysis. Statistical analysis was performed using GraphPad Prism 8.0 statistical software, with P <0.05 indicating that the difference was statistically significant.
Example 2S 100A10 correlation with COPD
2.1 study subject clinical data analysis comparison
Table 1 shows the clinical data characteristics of two groups of subjects, and it can be seen that there is no difference in gender and age composition between the two groups (P > 0.05). Patients in the COPD group presented airflow limitation to different degrees compared to healthy controls, and the difference between the pulmonary function test-related indicators FEV 1% pred and FEV1/FVC was statistically significant (P < 0.01). It should be noted that the Body Mass Index (BMI) was also significantly lower in the COPD group than in the normal group (P < 0.05).
TABLE 1 clinical data characterization of two groups of subjects
Figure BDA0003446757500000061
Figure BDA0003446757500000071
Note: BMI: body mass index, which is the square of body weight (kg) divided by height (m); FEV1/FVC (%): the first second forced breathing volume as a percentage of forced vital capacity; FEV 1% Pred (%): the first second forced expiratory volume is a percentage of the predicted value; smoking index: the number of smoking cigarettes per day is multiplied by the number of years of smoking, i.e. the number of years.
2.2 decreased expression level of S100A10 in PBMCs in COPD group
After PBMCs are separated, the expression change of S100A10 in the PBMCs is detected by real-time fluorescent quantitative PCR, and the mRNA expression level of S100A10 in the PBMCs of the COPD group is obviously reduced compared with that of a healthy control group (1.13 +/-0.12 is compared with 0.47 +/-0.11, and P is less than 0.05). Western blot was used to detect the expression change of S100A10 in PBMCs, and compared with healthy control group, the protein expression level of S100A10 in PBMCs of COPD group was also significantly reduced, and the quantified value of gray scale analysis (1.72 + -0.25 to 0.71 + -0.14, P <0.05) is shown in FIG. 1.
2.3 COPD group CD14+Decreased expression levels of S100A10 in cell subsets
To elucidate the expression changes of S100A10 in PBMCs in each cell subset, CD4 was first circled by flow cytometry+T lymphocytes, CD8+T lymphocytes, CD14+Monocytes and CD19+B lymphocytes, and the subpopulations were analyzed for mean fluorescence intensity of S100a 10. Andcompared with the healthy control group, the PBMCs in the COPD group have S100A10 in CD14+The expression level in the cell subset is obviously reduced (51.72 +/-5.34 is compared with 31.05 +/-2.98, P<0.05) without significant difference between the two groups in other cell subsets, as shown in figure 2.
Correlation analysis of the expression level of S100A10 with FEV 1% pred
To further investigate the possible role S100a10 may play in COPD disease processes, we analyzed the correlation of mRNA expression levels of S100a10 with FEV 1% pred and the results showed: the expression level of S100A10 in the COPD group was negatively correlated with the lung function index FEV 1% pred, with a correlation coefficient of-0.610 (P <0.001), as shown in FIG. 3.
Studies have reported that S100A10 expression levels in PBMCs in peripheral blood of patients with suicidal idealities are significantly reduced compared to healthy controls and patients with nonselpftic idealities, and S100A10 expression levels in PBMCs can be used as potential biomarkers for assessing suicidal risk (ZHANG L, SU T, CHOI K, et al. P11(S100A10) as a potential biomarker of psychiatric patients with suicidal risk [ J J.]J Psychiator Res,2011,45(4): 435-41). Green et al recently reported significant changes in the expression levels of S100A10 in Parkinson' S Patients (PBMCs), monocytes, NK cells and CD8+The expression level of toxic T lymphocytes S100A10 was positively correlated with the severity of Parkinson' S disease, and the expression level of NK cells S100A10 was also positively correlated with depression score (GREEN H, SU T, TIKLOVA K, et al]Proc Natl Acad Sci U S A,2017,114(10):2735-2740), in the present invention, we found that S100A10 in PBMCs was significantly reduced in expression at both the mRNA and protein levels by detecting the expression level of S100A10 in PBMCs using real-time fluorescence quantitative PCR and Western blot techniques, and further analyzed to find that S100A10 was at CD14+Monocyte subpopulation other than CD4+T lymphocytes and CD8+The expression level of cell subgroups such as T lymphocytes is reduced, finally, the correlation between the expression level of S100A10 and a lung function related index FEV 1% pred is analyzed, and the expression level of S100A10 in PBMCs in a COPD group is found to be presented with FEV 1% predA negative correlation. Under the action of various stimulating factors in the COPD pathogenesis process, such as acute exacerbation phase often accompanied by various bacterial infections of haemophilus influenzae, moraxella catarrhalis and the like, the immune system of COPD patients is abnormally activated, such as macrophage activation releases a large amount of proinflammatory mediators including TNF-alpha, IL-6, IL-1 beta and the like, CD4+The effector T lymphocyte activates and secretes cytokines such as IL-17 and IL-23 to act on PBMCs, thereby inhibiting the expression of S100A 10. Indeed, there are studies reporting CD45 in COPD patients+CD11b+F4/80+Macrophages and CD4+The number and proportion of T cells and Th17 cells increased, while the number and proportion of regulatory T cells decreased. S100a10 is an important anti-inflammatory protein, and its suppressed expression means that its ability to suppress inflammatory responses is impaired. By analyzing the correlation of S100A10 and the lung function index FEV 1% pred, the level of S100A10 and FEV 1% pred are obviously and negatively correlated, and the expression level of S100A10 in PBMCs can predict the condition of the lung function to a certain extent.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
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Claims (8)

  1. Use of a S100a10 detection reagent in the manufacture of a product for the diagnosis of COPD.
  2. 2. Use of the S100a10 detection reagent according to claim 1 in the preparation of a COPD diagnostic product, wherein the detection reagent comprises a reagent for detecting S100a10 by real-time fluorescent quantitative PCR, immunoblotting or flow cytometry.
  3. 3. The use of the S100A10 detection reagent in the preparation of COPD diagnostic products according to claim 2, wherein the reagents used for real-time fluorescence quantitative PCR detection of S100A10 comprise a target gene primer and an internal reference gene primer.
  4. 4. Use of the S100A10 detection reagent according to claim 3 in the preparation of a COPD diagnostic product,
    the sense strand of the target gene primer is as follows: 5-CCAGGTTTCGACAGACTCTTC-3', antisense strand of target gene primer: 5'-CCGTTCCATGAGCACTCTC-3', respectively;
    internal reference beta-actin sense chain: 5'-GGAAATCGTGCGTGACATTAA-3', antisense strand 5'-AGGAAGGAAGGCTGGAAGAG-3'.
  5. 5. The use of the S100a10 detection reagent according to claim 1 in the preparation of a COPD diagnostic product, wherein a sample for detecting COPD is peripheral blood mononuclear cells.
  6. 6. The use of the S100a10 detection reagent according to claim 5 in the preparation of a COPD diagnostic product, wherein S100a10 is in CD14 in peripheral blood mononuclear cells of COPD+The expression level of the monocyte subpopulation is reduced.
  7. 7. The use of the S100a10 detection reagent in the preparation of a COPD diagnostic product according to claim 5, wherein the expression level of S100a10 in peripheral blood mononuclear cells of COPD is negatively correlated with FEV 1% pred.
  8. 8. The use of the S100a10 test agent according to claim 1 in the preparation of a COPD diagnostic product, wherein the S100a10 test agent is used in the preparation of a lung function diagnostic product.
CN202111658715.2A 2021-12-30 2021-12-30 Application of S100A10 detection reagent in preparation of COPD diagnostic product Pending CN114231618A (en)

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KR102194215B1 (en) * 2019-07-02 2020-12-22 사회복지법인 삼성생명공익재단 Biomarkers for Diagnosing Gastric Cancer And Uses Thereof

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KR102194215B1 (en) * 2019-07-02 2020-12-22 사회복지법인 삼성생명공익재단 Biomarkers for Diagnosing Gastric Cancer And Uses Thereof

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范仁根等: "miR-590-5P通过下调靶基因S100A 10表达、抑制Wnt信号通道调控肝癌细胞增殖", 中国医学创新, vol. 13, no. 16, pages 4 - 8 *

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