WO2021073017A1

WO2021073017A1 - Application of chemokine ccl8 in preparation of dermatomyositis condition and prognosis evaluation reagent

Info

Publication number: WO2021073017A1
Application number: PCT/CN2020/073989
Authority: WO
Inventors: 孙凌云; 张昕; 陈智勇; 余迪
Original assignee: 南京鼓楼医院
Priority date: 2019-10-14
Filing date: 2020-01-23
Publication date: 2021-04-22
Also published as: CN110554202A; CN110554202B

Abstract

The present invention relates to the field of biological diagnosis and medicine, and in particular, to an application of chemokine CCL8 in the preparation of a dermatomyositis condition and prognosis evaluation reagent. In the present invention, the plasma sample of a patient diagnosed with dermatomyositis is used as a research object, protein chip detection is performed thereon, the sample is expanded, the protein chip result is verified by means of an ELISA method, and the correlation is determined in combination with the patient's biochemical indicators and clinical manifestations, confirming that the plasma CCL8 level of the patient with dermatomyositis is significantly higher than that of healthy people, the expression level is related to the patient's condition and prognosis, the plasma CCL8 level of the effectively treated patient with dermatomyositis is significantly reduced, and CCL8 can aggravate lung lesions in mice with interstitial pneumonia. Therefore, CCL8 can be used in a reagent for evaluating the condition and prognosis of the patient with dermatomyositis as a potential biomarker, and experimental data and a theoretical basis are provided for the application of CCL8 in the evaluation of the condition and prognosis of the patient with dermatomyositis. The present invention also provides a corresponding kit for dermatomyositis condition and prognosis evaluation.

Description

Application of chemokine CCL8 in preparation of dermatomyositis condition and prognosis assessment reagent

Technical field

The present invention relates to the fields of biological diagnosis and medicine, in particular to the application of chemokine CCL8 in preparing dermatomyositis condition and prognosis assessment reagents.

Background technique

Dermatomyositis (DM) is a type of autoimmune inflammatory myopathy. The main clinical manifestations are characteristic skin lesions, non-suppurative inflammatory changes of skeletal muscle, and progressive myasthenia. The pathogenesis is still unclear. Patients with dermatomyositis often have interstitial lung disease (ILD). In some patients, lung disease progresses rapidly and the prognosis is poor. At present, the clinical situation is mainly based on the clinical manifestations of patients and some laboratory test indicators (such as kinase, C-reactive protein, serum ferritin, and myositis-specific antibody titers) to comprehensively evaluate the condition. There have been many studies trying to use biomarkers to help diagnose and evaluate the condition of DM, but there are no specific and sensitive indicators for judging the condition of patients and predicting the outcome of patients.

Chemokines are small molecular proteins with similar functions and a molecular weight of about 8-12kDa. Researchers have reported that chemokines CXCL10, CX3CL1, CXCL8, and CCL2 are related to the disease activity of patients with dermatomyositis. However, there is still a lack of large samples and systematic analysis of plasma chemokines in patients with DM, and no more suitable use has been found. Chemokines for evaluating the condition of DM.

Therefore, if chemokines that can accurately and effectively monitor and evaluate the condition of DM and predict the prognosis of patients can be screened out, it will play a vital role in timely treatment of patients and good doctor-patient communication.

Summary of the invention

The purpose of the present invention is to study the role of chemokine CCL8 in the evaluation of the condition and prognosis of patients with dermatomyositis, provide a kit for evaluating the condition and prognosis of dermatomyositis, and provide theoretical basis and experiments for the clinical treatment and evaluation of the prognosis of DM data.

The chemokine CCL8, also named monocyte chemoattractant protein 2 (MCP-2), can chemoattract and activate a variety of immune cells, such as mast cells, eosinophils and basophils related to allergic reactions Sex granulocytes, monocytes-macrophages, T cells and NK cells related to inflammation. Previous studies have shown that chemokines CXCL10, CX3CL1, CXCL8, and CCL2 are related to the disease activity of patients with dermatomyositis and can be used for the diagnosis or assessment of dermatomyositis. However, detailed studies on the condition and prognosis of patients with CCL8 and DM have not yet been seen Report.

In this study, we took the plasma samples of patients who were diagnosed with dermatomyositis and did not have other immune diseases as the research object, carried out protein chip detection on them, and expanded the samples to verify the protein chip detection results by ELISA, combining the patient's biochemical indicators and Judging the relevance of clinical manifestations. The results of the study showed that the plasma CCL8 level of patients with dermatomyositis was significantly higher than that of healthy people, and its expression level was related to the patient's condition and prognosis; in the bleomycin-induced mouse interstitial pneumonia model, CCL8 aggravated lung lesions. The plasma CCL8 level of patients with clinically effective dermatomyositis is lower than before treatment.

Based on the specific and high expression of plasma CCL8 in DM patients and is closely related to the condition and prognosis, CCL8 can be used as a potential biomarker of DM and applied to the examination of plasma samples of DM patients, providing a certain theoretical basis for the evaluation of the condition and prognosis of DM patients .

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention verifies the high expression of CCL8 in the plasma of DM patients, and the level of CCL8 is related to the patient’s condition and prognosis. The present invention proposes for the first time that plasma CCL8 can be used as a potential biomarker for dermatomyositis and applied to the detection and Disease evaluation and prognostic analysis;

2) The present invention provides experimental data and theoretical basis for CCL8 to aggravate lung diseases;

3) The present invention provides technical enlightenment for improving the prognosis of dermatomyositis by intervening and blocking the CCL8 pathway, which may be used as a specific target for targeted therapy of dermatomyositis in the future, providing new ideas for the treatment of dermatomyositis.

Description of the drawings

Figure 1 is the results of plasma protein chip analysis of healthy people and DM patients, in which: the abscissa is the chemokines selected from the plasma samples of healthy people and DM patients in the AAH-CYT-G5 protein chip, and the ordinate represents each chemokine Fluorescence intensity of, HC stands for healthy people, DM stands for DM patients;

Figure 2 is a graph of ELISA test results of plasma samples from healthy people and DM patients, where HC on the abscissa represents healthy people, DM represents DM patients, and the ordinate represents plasma CCL8 expression;

Figure 3 is a graph showing the plasma CCL8 expression levels of DM patients before and after treatment, where the abscissa represents the plasma CCL8 of DM patients before and after hormone plus immunosuppressant treatment (Before) and after effective treatment (After), and the ordinate represents the plasma CCL8 level. Expression

Figures 4A and 4B are the analysis results of plasma CCL8 levels and clinical manifestations, where: A is the proportion of DM patients with high plasma CCL8 levels (CCL8high) and DM patients with low plasma CCL8 levels (CCL8low), and B is the proportion of positive rashes. The proportion of DM patients with high plasma CCL8 levels (CCL8high) and DM patients with low plasma CCL8 levels (CCL8low) developing Gottron sign;

Figures 5A to 5D are the analysis results of plasma CCL8 levels and dermatomyositis laboratory test indicators. A, B, and C are DM patients with high plasma CCL8 levels (CCL8high) and DM patients with low plasma CCL8 levels (CCL8low), respectively The results of the determination of serum ferritin (SF), C-reactive protein (CRP), and serum albumin (ALB), D is the correlation result between plasma CCL8 level and forced vital capacity (FVC), where: the abscissa is the content of CCL8, the ordinate is Coordinates are FVC predicted values;

Figures 6A and 6B are the results of the analysis of the correlation between plasma CCL8 levels and the survival of DM patients, where A is a pie chart of the causes of death in DM patients, and B is a DM patient with high plasma CCL8 levels (CCL8high) and DM with low plasma CCL8 levels The patient (CCL8low) survival time and survival rate correlation chart;

Figure 7 is a HE staining image of lung tissue of C57BL/6 mice, in which: control group (Ctrl), bleomycin treatment group (BLM), bleomycin + phosphate buffer saline group (BLM+PBS), bleomycin BLM+recombinant CCL8 group (BLM+CCL8).

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further illustrated by specific embodiments below.

The experimental methods used in the following examples, unless otherwise specified, are conventional methods, and the reagents, methods, and equipment used, unless otherwise specified, are conventional reagents, methods, and equipment in the technical field.

Example 1

1. Sample selection and processing

Experimental group (DM): Take fresh peripheral blood from patients with dermatomyositis (DM) and centrifuge to separate the plasma, then quickly put it into a cryotube and store it in a refrigerator at -80°C for later use;

Control group (HC): Take fresh peripheral blood from healthy people (HC) with age and sex matched without immune disease and centrifuge to separate the plasma, then quickly put it into the cryotube and store it in the refrigerator at -80°C for later use.

2. Screening and verification of plasma chemokines in patients with DM

2.1) Screening: In this experiment, we used the AAH-CYT-G5 protein chip purchased from RayBiotech to analyze the plasma samples of 16 healthy people and 16 DM patients with a protein chip, and systematically compared the plasma samples of HC and DM patients. The expression of a variety of chemokines is different; from the protein chip analysis results shown in Figure 1, it can be seen that CXCL5, CXCL2, CXCL1, CXCL7, CCL1, CCL8 and other chemokines are expressed in the plasma of healthy people and DM patients. Among them, the expression is different. The most significant is the chemokine CCL8.

2.2) Verification:

2.2.1) Expand the sample size for analysis, refer to step 1 to obtain plasma samples of 41 HC and 158 DM patients, and perform ELISA test on the plasma samples of 41 HC and 158 DM patients. The test results shown in Figure 2 confirm that DM patients The plasma CCL8 level was significantly higher than that of HC.

2.2.2) The plasma CCL8 levels of 22 DM patients among the above 158 DM patients were tested multiple times. Refer to step 1 to obtain the plasma samples of the 22 DM patients, and use the ELISA method to detect and record the CCL8 expression level in these plasma samples. Then, the 22 DM patients were treated with hormones and immunosuppressants for 2 months, and 20 patients were treated It is effective and the condition is relieved after treatment. Then refer to step 1 to obtain plasma samples of 20 patients with effective treatment and measure the CCL8 expression level to compare the changes in CCL8 expression level of these 20 patients with effective treatment.

It can be seen from Figure 3 that the plasma CCL8 expression level of DM patients treated with hormones and immunosuppressants effectively decreased, indicating that the plasma CCL8 level is positively correlated with the condition of DM.

3. The correlation between plasma CCL8 level and the condition of DM patients

Taking 66 DM patients with high plasma CCL8 levels (CCL8high) and 63 DM patients with low plasma CCL8 levels (CCL8low) who were successfully followed up as the research objects, the plasma CCL8 levels and clinical manifestations of these 129 DM patients who were successfully followed up ( The integrated analysis of sun rash and Gottron sign) and various laboratory test indicators (serum ferritin, C-reactive protein, albumin, forced vital capacity FVC) verified the correlation between CCL8 and the condition of DM patients.

Among them, the cutoff value of CCL8 level is the mean value of HC plasma CCL8 level + 2SD (ie 67.3 pg/ml), a value higher than this value is regarded as a high level of CCL8, and a value lower than this value is regarded as a low level of CCL8.

It can be seen from Figure 4 that the proportion of DM patients with high plasma CCL8 levels (Figure 4A) and Gottron sign (Figure 4B) is higher than that of DM patients with low plasma CCL8 levels, indicating that plasma CCL8 levels are positive for positive rash and Gottron signs. Related, that is, those with high plasma CCL8 levels are more likely to have sun rash and Gottron sign;

It can be seen from Figure 5 that the clinical data and biochemical indicators of all DM patients with successful follow-up were collected, and the results of biochemical indicators and plasma CCL8 levels were comprehensively analyzed. The serum ferritin (Figure 5A) and C-reactive protein ( Figure 5B) The serum albumin of DM patients with high plasma CCL8 levels was significantly higher than that of patients with low plasma CCL8 levels (Figure 5C) was lower than that of DM patients with low plasma CCL8 levels. CCL8 expression levels were significantly negatively correlated with FVC in DM patients (Figure 5C). 5D);

The above-mentioned series of clinical manifestations (Sunny eruption and Gottron sign) and various laboratory test indicators (serum ferritin, C-reactive protein, albumin, forced vital capacity FVC) results are consistent with the condition of DM patients, and the plasma CCL8 level of DM patients Significantly related to the above-mentioned series of clinical manifestations and various laboratory indicators, so the plasma CCL8 level can reflect the condition of DM patients.

4. The correlation between plasma CCL8 level and survival time of DM patients

The aforementioned 66 DM patients with high plasma CCL8 levels (CCL8high) and 63 DM patients with low plasma CCL8 levels (CCL8low) were followed up for a long time. The first appearance of the characteristic rash of DM (xiangyang rash or Gottron sign) was taken as the starting time. The outcome of death or the one-year period after the plasma CCL8 level was measured was the follow-up endpoint. The main causes of death in DM patients were analyzed, and the difference in survival between patients with high plasma CCL8 levels and patients with low plasma CCL8 levels was compared.

It can be seen from Figure 6A that lung infection is the leading cause of death in DM patients (71.4%); from Figure 6B, it can be seen that the survival time of DM patients with high plasma CCL8 levels is significantly shorter than that of DM patients with low plasma CCL8 levels; the above results indicate that plasma CCL8 levels may be positively correlated with lung infections.

5. CCL8 aggravated lung lesions in mice with interstitial pneumonia

Twenty-four 8-week-old C57BL/6 mice were randomly divided into four groups for modeling. All mice were given a normal diet. On the 7th day, all mice were sacrificed and lung tissues were taken for hematoxylin-eosin staining (HE staining). ), observe the inflammation of the lungs:

1) Control group (Ctrl): On the first day, 6 8-week-old C57BL/6 mice were anesthetized and instilled 50ul of normal saline into the trachea as a control group;

2) Bleomycin treatment group (BLM): On the first day, 6 8-week-old C57BL/6 mice were anesthetized and instilled 5 mg/kg bleomycin into the trachea as the bleomycin treatment group;

3) Bleomycin + phosphate buffer saline group (BLM+PBS): On the first day, 6 8-week-old C57BL/6 mice were anesthetized and 5 mg/kg bleomycin was instilled into the trachea. After that, 50ul of phosphate buffer was administered on the 3rd and 4th day.

4) Bleomycin+recombinant CCL8 group (BLM+CCL8): On the first day, 6 8-week-old C57BL/6 mice were anesthetized and instilled 5 mg/kg bleomycin into the trachea. On the 3rd day and on the 4th day, 400 ng of recombinant mouse MCP-2 (CCL8, purchased by Peprotech) was administered.

As shown in Figure 7, bleomycin can cause acute lung interstitial inflammation (BLM) in mice, and CCL8 can aggravate lung lesions in mice with interstitial pneumonia (BLM+CCL8).

From the above analysis results of "4. The correlation between plasma CCL8 levels and survival of DM patients": plasma CCL8 levels may be positively correlated with lung inflammation, combined with the analysis results of Figure 7: CCL8 can aggravate lung lesions in mice with interstitial pneumonia. It can be speculated that the plasma CCL8 level of DM patients is closely related to the condition and prognosis.

In summary, CCL8 is highly expressed in the plasma of DM patients, and CCL8 can aggravate lung lesions in mice with interstitial pneumonia. The level of CCL8 is closely related to the patient's condition and prognosis. Therefore, CCL8 can be used as a potential biomarker for evaluating the condition of DM and used in dermatomyositis condition assessment reagents. At the same time, it also provides experimental data and theoretical basis for the application of CCL8 in dermatomyositis patients condition and prognosis assessment reagents. It is helpful for the diagnosis and prognosis analysis of patients with dermatomyositis, and can provide important basis and reference value for clinical prediction of patient outcome, and has the value of popularization and application.

Example 2

A kit for evaluating the condition and prognosis of dermatomyositis, the kit comprising:

a) Container;

b) The specific antibody of CCL8 protein contained in the container;

c) a label or instructions for use, the label or instructions for use indicate that the kit is used for dermatomyositis disease assessment and the method for using the kit to predict the prognosis of DM patients.

Further, the label or instructions indicate that: when the plasma CCL8 value of a DM patient is ≥67.3pg/ml, the patient is diagnosed as having a severe condition and poor prognosis; when the plasma CCL8 value of a patient with dermatomyositis is less than 67.3pg/ml, It is diagnosed that the patient's condition is mild and the prognosis is better.

Using the above-mentioned detection kit of the present invention, the plasma samples of 129 DM patients were quantitatively detected (starting with the first appearance of DM characteristic rash (sunny rash or Gottron sign) as the starting time, and the death outcome or the 1-year period after the plasma CCL8 level was detected The content of CCL8 in the follow-up end), 49 patients died during the follow-up, and 80 patients survived to the end of the follow-up.

The results show that when the cut-off point of the protein content of CCL8 is 67.3 pg/ml, the results are shown in Table 1 below. The sensitivity of CCL8 to evaluate the prognosis of DM is 81.6% and the specificity is 67.5%.

Table 1. Diagnosis result table of CCL8 diagnostic kit

The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments. All technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

Application of chemokine CCL8 in preparation of dermatomyositis condition and prognosis assessment reagent.
The application of the chemokine CCL8 in the preparation of dermatomyositis condition and prognosis assessment reagents according to claim 1, wherein the dermatomyositis patient condition and prognosis assessment reagent is a specific antibody of CCL8 protein.
The chemokine CCL8 is used as a biomarker for evaluating the condition and prognosis of patients with dermatomyositis.
A kit for evaluating the condition and prognosis of dermatomyositis, characterized in that, the kit includes:

a) Container;

b) CCL8 protein detection reagent contained in the container;

c) a label or instructions for use, the label or instructions for use indicate that the kit is used for the evaluation of the condition of patients with dermatomyositis and the use method of using the kit to evaluate the prognosis of patients with dermatomyositis.
The kit for assessing the condition and prognosis of dermatomyositis according to claim 4, characterized in that: the label or instructions indicate that: when the plasma CCL8 value of patients with dermatomyositis is greater than or equal to 67.3 pg/ml, then It is diagnosed that the patient's condition is severe and the prognosis is poor; when the plasma CCL8 value of the patient with dermatomyositis is less than 67.3pg/ml, it is diagnosed that the patient's condition is milder and the prognosis is better.
The kit for evaluating the condition and prognosis of dermatomyositis according to claim 4, wherein the CCL8 protein detection reagent is a specific antibody of CCL8 protein.