阅读说明：本技术 新型冠状病毒肺炎分期与疗效评估细胞因子标志物及其应用 (Novel coronavirus pneumonia staging and curative effect evaluation cytokine marker and application thereof ) 是由 徐建华 王大伟 石亚玲 黄若磐 张嘉琪 何金勇 杨雁青 于 2020-09-16 设计创作，主要内容包括：本发明属于生物医药技术领域,公开了新型冠状病毒肺炎分期与疗效评估细胞因子标志物及其应用。本发明探索研究得出41种与新型冠状病毒肺炎分期与疗效评估相关度很高的细胞因子(不同来源变异对总变异贡献大小分析：P<0.05),尤其是其中的12种相关度非常高：Eotaxin-2、IP-10、MCP-2、HCC-4、IL-10、ADAM8、G-CSF、Fas、TARC、IL-18、GDF-15、TRAIL R3(进一步用R软件包“LIMMA”,经验贝叶斯方法分析：adj.P.val<0.10),利用这些因子及其组合可用于开发新型冠状病毒肺炎分期与疗效评估产品,在新型冠状病毒肺炎分期与疗效评估方面,以及对于深入探究COVID-19患者细胞因子水平与疾病发生发展之间的关系、建立细胞因子风暴预警体系方面,具有重要的应用价值和应用前景。(The invention belongs to the technical field of biological medicines, and discloses a novel cytokine marker for staging and curative effect evaluation of coronavirus pneumonia and application thereof. The invention explores and researches to obtain 41 cytokines with high correlation with the stage and curative effect evaluation of the novel coronavirus pneumonia (the contribution of variation from different sources to the total variation is analyzed, and the P is less than 0.05), particularly 12 cytokines are very high in correlation: eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15 and TRAIL R3 (further analyzed by an R software package LIMMA, an empirical Bayesian method: adj. P. val is less than 0.10), and the factors and the combination thereof can be used for developing novel products for stage and curative effect evaluation of coronavirus pneumonia, and have important application value and application prospect in the aspects of deeply exploring the relationship between the level of the cell factors of patients with COVID-19 and the occurrence and development of diseases and establishing a cell factor storm early warning system.)

1. A cytokine marker for staging and/or efficacy assessment of novel coronavirus pneumonia, wherein the cytokine marker is selected from one or a combination of the following cytokines: eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15, TRAIL R3, I-TAC, ENA-78, GCP-2, MMP-7, Flt-3L, PARC, CD23, BDNF, MCP-4, P-Cadherin, OSM, Angiostatin, ErbB3, MMP-9, Pentraxin 3, RAGE, VE-Cadherin, MIP-3a, CEACAM-1, IL-18BPa, IL-6, TRAIL-9, Cancan R1, MPIF-1, IL-1ra, uPAR, MMP-2, SyndeMP-1, TIMP-1.

2. The cytokine marker according to claim 1, wherein the cytokine marker is selected from one or more of the following cytokines: eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15, TRAIL R3.

3. Use of the cytokine marker of claim 1 or 2 as a marker for staging and/or efficacy assessment of novel coronavirus pneumonia.

4. Use of a cytokine marker according to claim 1 or 2 for the preparation of a product for staging and/or efficacy assessment of novel coronavirus pneumonia.

5. Use of a detection reagent for a cytokine marker according to claim 1 or 2 for the preparation of a product for the staging and/or efficacy assessment of novel coronavirus pneumonia.

6. A kit for staging and/or efficacy assessment of coronavirus pneumonia, comprising reagents for detecting the amount of the cytokine marker of claim 1 or 2.

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to a novel coronavirus pneumonia COVID-19 staging and curative effect evaluation cytokine marker and application thereof.

Background

The disease caused by the novel coronavirus (SARS-CoV-2) is named 2019 coronavirus disease (COVID-19) [ Coronavir student Group of the International Committee on Taxomy of Virus. the species viral coronary syndrome: sizing 2019-nCoV and negative SARS-CoV-2.Nat Microbiol. 2020; 5(4) 536-544.doi 10.1038/s 41564-020-0695-z. The novel coronavirus (SARS-CoV-2) is highly infectious.

Therefore, the search for the physiopathological mechanism to guide staging, the formulation of medication and treatment regimens, and the evaluation of prognosis is urgent.

At present, the COVID-19 staging and curative effect evaluation mainly depends on means such as clinical symptoms, lung images and the like, and a plurality of factors such as large heterogeneity of clinical manifestations of patients, limitation of image result judgment and the like are not favorable for clinical rapid guidance of related treatment. The fast and stable staging and evaluation system is sought, and has important clinical application value.

Disclosure of Invention

The antibody array capable of simultaneously and quantitatively detecting 440 cytokines is adopted, the cytokine levels of common and heavy COVID-19 patients in three different clinical periods in the course of disease are analyzed, the relationship between the cytokine levels of the COVID-19 patients and the occurrence and development of the disease is explored, and a cytokine storm early warning system is established.

The invention aims to provide a novel cytokine marker for staging and evaluating curative effect of coronavirus pneumonia.

The invention also aims to provide application of the cytokine marker in the staging and curative effect evaluation of the novel coronavirus pneumonia.

The above purpose of the invention is realized by the following technical scheme:

the research of the invention finds that the correlation degree between the following 41 cytokines and the novel coronavirus pneumonia stage and the curative effect evaluation is very high: eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15, TRAIL R3, I-TAC, ENA-78, GCP-2, MMP-7, Flt-3L, PARC, CD23, BDNF, MCP-4, P-Cadherin, OSM, Angiostatin, ErbB3, MMP-9, Pentraxin 3, RAGE, VE-Cadherin, MIP-3a, CEACAM-1, IL-18BPa, IL-6, TRAIL-9, Cancan R1, MPIF-1, IL-1ra, uPAR, MMP-2, SyndeMP-1, TIMP-1.

In particular, 12 cytokines: eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15, TRAIL R3, and has a very high correlation with the staging of the novel coronavirus pneumonia and the evaluation of the curative effect (R software package "LIMMA", statistical analysis by empirical Bayesian method: adj. P. val < 0.10).

Therefore, one or more of the above cytokines, and their use in the staging and/or efficacy assessment of a novel coronavirus pneumonia, including their use as markers, and their use in the preparation of a novel coronavirus pneumonia staging and/or efficacy assessment product, are all within the scope of the present invention.

Based on the above, the reagent for detecting the content of the above cytokine markers can be developed into a corresponding novel coronavirus staging and/or curative effect evaluation kit, and shall also fall within the protection scope of the present invention.

The invention has the following beneficial effects:

the invention explores and researches 41 cell factors with high correlation degree with the novel coronavirus pneumonia staging and curative effect evaluation, especially 12 of the cell factors, and the cell factors and the combination of the cell factors can be used for developing novel coronavirus pneumonia staging and curative effect evaluation products, and have high application value and application prospect in the aspects of novel coronavirus pneumonia staging and curative effect evaluation.

Meanwhile, based on the result, the method also has very good value for deeply researching the relation between the level of the cytokine of the COVID-19 patient and the occurrence and development of diseases and establishing a cytokine storm early warning system.

Drawings

FIG. 1 is a graph of the cytokine profiles in early stages of normal and severe patient samples; in the figure, Mo represents the normal type, Se represents the heavy type, and SYM represents the early stage.

FIG. 2 is a graph of the cytokine profiles in terms of remission for samples of normal and severe patients; in the figure, Mo represents the normal type, Se represents the heavy type, and REM represents the remission stage.

FIG. 3 is a graph of cytokine profiles during convalescence for samples of normal and severe patients; in the figure, Mo represents the normal type, Se represents the heavy type, and CON represents the convalescent period.

FIG. 4 is a graph showing the cytokine profiles in different stages (early, remission and convalescent stages) of samples from patients of normal type versus patients of severe type; in the figure, Mo represents the normal type, Se represents the severe type, SYM represents the early stage, REM represents the remission stage, and CON represents the remission stage.

FIG. 5 shows the results of hierarchical clustering analysis performed by the difference factor R software; thermographic analysis of patients with general type (Mo) and severe type (Se) in early clinical stage (A), remission stage (B), and convalescent stage (C).

FIG. 6 is a graph of the difference factor functional enrichment analysis (GO) results.

FIG. 7 is a graph of the difference factor functional enrichment analysis (GO) results.

FIG. 8 is a Wien diagram of a difference factor functional analysis.

FIG. 9 shows the R package "pROC" to construct ROC curves and calculate the area under the ROC curve (AUC) (early clinical phase and convalescent phase).

FIG. 10 shows the R package "pROC" to construct ROC curves and calculate the area under the ROC curve (AUC) (early clinical and remission).

FIG. 11 shows the R package "pROC" to construct the ROC curve and calculate the area under the ROC curve (AUC) (remission and convalescence).

FIG. 12 is a graph of the R software package "pROC" for constructing ROC curves and calculating the area under the ROC curve (AUC) (severe and normal).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1

1. Data and Experimental methods

1.1 study object

The study of the invention analyzes 14 patients infected by Sars-cov-2 (hospitalized patients from the novel coronavirus fixed-point admission hospital in Guangzhou city and Foshan city), all the patients are sras-cov-2 positive by nucleic acid detection, and are classified into 10 common patients and 4 heavy patients according to the novel coronavirus auxiliary diagnosis and treatment guideline issued by the national health committee. The general patients have symptoms of fever, respiratory tract and the like, and the pneumonia can be seen through imaging. Critical patient definition needs to meet one of the following: tachypnea (RR is more than or equal to 30 times per minute), oxygen saturation in a resting state is less than or equal to 93 percent, arterial blood oxygen partial pressure (PaO 2)/oxygen uptake concentration (FiO2) is less than or equal to 300mmHg (1mmHg is 0.133kPa), and pulmonary imaging shows that the lesion is multi-lobal or the lesion progress is more than 50 percent in 48 hours.

1.2 sample Collection

To study the development of covid-19, three different clinical blood samples were collected longitudinally for the study, for a total of 42 samples. Sample groupings are as in table 1:

TABLE 1

	Normal type (Mo)	Heavy type (Se)
			A: early SYM	10	4
B: remission period REM	10	4
			C: convalescence CON	10	4

Wherein the sampling point A is collected in the early clinical stage, namely within 24 hours after the patient is admitted; b, sampling points are collected in a symptom relieving period, and after a patient is effectively treated for a period of time, the condition of the patient is relieved, which is reflected in that pulmonary imaging, symptoms and the like are improved; the sampling point C is collected in the convalescence, the body temperature of the patient is recovered to be normal, the respiratory tract symptoms are obviously improved, the lung imaging shows that the inflammation is obviously absorbed, and the respiratory tract pathogenic nucleic acid detection is negative for two times continuously, namely, the discharge standard specified in the novel coronavirus auxiliary diagnosis and treatment guideline is met.

1.3 antibody array Q440

Coviv 19 Cytokines levels of biomarkers were measured in 42 serum samples from coviv 19 patients using a ribo QAH-CAA-440 protein chip (QAH-CAA-440, RayBiotech, inc., peach red irons, GA, USA), including analytically observing the serum Cytokine levels in samples of different covv 19 classifications (including normal and severe), staging (including early, remission, convalescence) patients.

Specifically, protein screening was performed using a cytokine array Q440(QAH-CAA-440, RayBiotech, Inc, Peachtree Corners, GA, USA) that can quantitatively detect 440 kinds of human serum proteins. Antibody array manipulations were performed exactly according to the manufacturer's instructions. Briefly, the array was dried at room temperature for 2 hours, then plugged by adding 100 μ l of 2-fold diluted sample diluent to each well, and after 30 minutes the sample diluent was aspirated. Mu.l of 2-fold diluted serum samples were added to each well and incubated overnight at 4 ℃. The slides were washed 5 times for 5min with wash buffer I and then 3 times for 5min with wash buffer II. Wash buffer was blotted dry and 80 μ l biotin-conjugated antibody mix was added to each well and incubated for 2 hours at room temperature. After washing the array, 80. mu.l AlexaFluor 555-conjugated streptavidin was added to each well and incubated for 1 hour at room temperature in the absence of light. Finally, the signal was scanned and extracted using an InnoScan310 scanner (Innopsys, carbone, france).

1.4 statistical analysis

And determining the influence of the controllable factors on the research result by analyzing the contribution of the variation from different sources to the total variation in the research. When P <0.05, it means that the difference is statistically significant.

The effect of COVID19 on serum cytokine levels in patients with different stages and different symptoms was analyzed using the R package "LIMMA" using an empirical bayesian approach to establish a linear model, including correlation analysis within individuals. When adj.p.val <0.10, it indicates that the difference is statistically significant. All statistics were analyzed using R software (version 3.6.3).

GO analysis and KEGG enrichment analysis determined significant enrichment of protein function and signaling pathways with P values < 0.05. PCA (R package "ggbilot"), hierarchical cluster analysis (R package "gplots"), KEGG enrichment analysis (R package "clusterProfiler"), and GO enrichment analysis (R package "org.hs.e.db" and "clusterProfiler") were analyzed using R software (version 3.6.3).

2. Results of the experiment

2.1 Linear model analysis Using empirical Bayesian methods

Differential proteins are counted under various factors, the screening condition P.value is less than 0.5, and 41 differential proteins are shown in Table 2. The screening condition adj.P.val is less than 0.1, and 12 differential proteins (the first 12 in Table 2: Eotaxin-2, IP-10, MCP-2, HCC-4, IL-10, ADAM8, G-CSF, Fas, TARC, IL-18, GDF-15, TRAIL R3) are included.

TABLE 2

2.2 the screening conditions were further stringent, i.e., adj. P. val <0.1, and 12 differential cytokines were screened for expression levels in three different clinical stages in patients of normal and severe type, as shown in FIGS. 1-4.

2.3 hierarchical clustering analysis was performed using the R software to analyze the differences between the different groups of samples and proteins according to the differences in protein expression patterns, see FIG. 5, thermographic analysis of early clinical (A), remission (B), and convalescence (C) in patients of general type (Mo) and severe type (Se).

2.4 by the way enrichment analysis (KEGG) of the differential factors, the differential factors are mainly enriched in the ways of 'the interaction of virus proteins and cytokines and receptors thereof', 'chemokine signal paths', and the like. The differential factors are mainly enriched in the functions of lymphocyte migration and chemotaxis through functional enrichment analysis (GO). As shown in fig. 6-7.

2.5 through database query and classification of 12 cytokines to corresponding functions and pathways, producing Wien map, 5 cytokines with lymphocyte migration to chemotactic function and participating in interaction pathways of virus proteins, cytokines and receptor receptors thereof, including HCC-4, IP-10, MCP-2, TARC, Eotaxin-2, see FIG. 8, Table 3.

TABLE 3

2.6 Using the R software package "pROC", we constructed ROC curves and calculated the area under the ROC curve (AUC). For the early clinical stage and remission stage, Eotaxin-2(AUC 0.872), MCP-2(AUC 0.883), HCC-4 (AUC 0.878), IP-10(AUC 0.867), and G-CSF (AUC 0814) have better staged diagnostic efficacy, as shown in fig. 9.

For the early clinical stage and convalescent period, IP-10(AUC 0.918), TARC (AUC 0.913), HCC-4 (AUC 0.913), IL-10(AUC 0.923), Eotaxin-2(AUC 0.898), MCP-2(AUC 0.888), Fas (AUC 0.832), TRAIL R3(AUC 0.817) have better staged diagnostic efficacy, as shown in fig. 10.

For the remission and convalescent phases, TARC (AUC 0.806) has better staged diagnostic efficacy, see fig. 11.

Meanwhile, we explored the possibility of being a recognition factor of the critical cases of patients with new coronary pneumonia, and GDF-15 (AUC ═ 0.856) and IL-18(AUC ═ 0.883) have better staged diagnostic efficacy in recognizing severe cases and general cases, as shown in fig. 12.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.