Protein related to rapid progression type interstitial lung disease, detection kit and application thereof

Abstract

The invention relates to a protein related to a rapid progression type interstitial lung disease, a detection kit and application thereof, and provides a biomarker of connective tissue disease (CTD) combined with rapid progression type pulmonary interstitial fibrosis (RPILD), which can be used for diagnosis of rapid progression type pulmonary interstitial fibrosis (RPILD) combined with connective tissue diseases such as myositis, scleroderma and the like.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a protein associated with rapidly progressive pulmonary interstitial fibrosis, a detection kit and applications thereof. Technical Background

[0002] Some connective tissue diseases (CTDs) develop rapidly progressive pulmonary interstitial fibrosis (RPILD), characterized by a rapid deterioration of respiratory function and lung damage, resulting in a high mortality rate. These patients are difficult to diagnose before the onset of dyspnea. Imaging studies are typically performed after symptoms develop to determine whether the disease has developed. Some patients also undergo bronchoalveolar lavage fluid (BAL) testing for autoantibodies and infection markers to aid diagnosis. However, these methods are only implemented after the onset of obvious symptoms: Imaging changes are not obvious in patients with mild early symptoms, and obtaining BAL fluid is invasive and traumatic, making it impossible to perform large-scale BAL fluid testing and screening in patients with mild early symptoms. Therefore, these two traditional diagnostic techniques objectively make it difficult to achieve early diagnosis and screening for RPILD.

[0003] Testing patient serum markers is a minimally invasive procedure. Early detection and screening of CTD-RPILD as a diagnostic aid using serological markers would facilitate early diagnosis and treatment. However, pulmonary interstitial fibrosis progresses rapidly after symptom onset in patients with CTD-RPILD, often leading to death before appropriate treatment. Therefore, there is an urgent need to explore and validate sensitive, specific, and easily sampled and tested serological biomarkers to improve the early diagnosis of CTD-RPILD. Currently, serum levels of KL-6 and anti-MDA5 autoantibodies are commonly used to diagnose or assess the risk of CTD-RPILD, but their sensitivity and specificity remain insufficient. New serological markers, combined with these two, could improve the diagnostic efficacy of RPILD and have a significant impact on the clinical diagnosis of RPILD. However, no studies have yet confirmed the potential of serum CCL8 for the diagnosis of CTD-RPILD and its prognostic implications. Summary of the Invention

[0004] The purpose of the present invention is to provide a molecular marker associated with rapidly progressive pulmonary fibrosis complicated by connective tissue disease. More preferably, the present invention provides a molecular marker for evaluating the prognosis of rapidly progressive pulmonary fibrosis.

[0005] In order to achieve the above object, the present invention adopts the following technical means:

[0006] The present study isolated bronchoalveolar lavage fluid (BALF) cells from patients with CTD and RPILD, patients with CTD without RPILD, and healthy controls (HC) for transcriptome sequencing, comparing the expression levels of 210,503 genes. The results revealed that CCL8 was included in the top five significant categories of 20 GO functional categories, and CCL8 was also included in categories such as extracellular region, protein binding, phospholipase activator activity, and protein kinase activity. Next, validation was performed using bronchoalveolar lavage fluid (BALF) cells. The results showed that CCL8 transcript levels in BALF cells derived from the lungs were significantly elevated in patients with RPILD. To further verify whether CCL8 protein levels were specifically elevated in the BALF of patients with RPILD, we measured CCL8 levels in BALF using an enzyme-linked immunosorbent assay (ELISA). The results showed that CCL8 levels were significantly elevated in the BALF of patients with CTD-RPILD compared with those of patients with CTD and chronic pulmonary fibrosis (CILD) (P = 0.0003). To further demonstrate the potential of serum CCL8 as a biomarker for diagnosing CTD-RPILD, we expanded our patient validation cohort and confirmed that serum CCL8 levels were significantly elevated in patients with RPILD. We further evaluated the diagnostic efficacy of serum CCL8 levels for CTD-RPILD using a receiver operating characteristic (ROC) curve. The area under the ROC curve was 0.817 (95% confidence interval (CI) 0.704-0.930, P < 0.001), with an optimal cutoff value of 71.4% sensitivity, 86.7% specificity, and a cutoff value of 111.3 pg / mL. These results demonstrate that serum CCL8 can be used for the diagnosis or risk assessment of CTD patients with concurrent RPILD.

[0007] Furthermore, the present invention first proposes the use of serum CCL8 as a molecular marker in the preparation of drugs or reagents for predicting rapidly progressive pulmonary fibrosis.

[0008] Furthermore, the present invention also proposes the use of a reagent for detecting serum CCL8 in the preparation of a drug or reagent for predicting rapidly progressive pulmonary fibrosis.

[0009] Among them, preferably, the reagent for detecting serum CCL8 includes a reagent for detecting serum CCL8 using an enzyme-linked immunosorbent assay (ELISA).

[0010] Further preferably, the reagent for detecting serum CCL8 is an anti-CCL8 antibody.

[0011] Furthermore, the present invention provides the use of serum CCL8 as a molecular marker in the preparation of a drug or reagent for the diagnosis of rapidly progressive pulmonary fibrosis in combination with KL-6 and / or anti-MDA5 antibodies.

[0012] Furthermore, the present invention also proposes the use of a reagent for detecting serum CCL8, KL-6, and / or anti-MDA5 antibodies in the preparation of a drug or reagent for predicting rapidly progressive pulmonary fibrosis.

[0013] Among them, preferably, the reagent for detecting serum CCL8, KL-6, and / or anti-MDA5 antibodies includes a reagent for detecting serum CCL8 using an enzyme-linked immunosorbent assay (ELISA).

[0014] Further preferably, the reagent for detecting serum CCL8, KL-6, and / or anti-MDA5 antibodies is an anti-CCL8 antibody, an anti-KL-6 antibody and / or an anti-MDA5 antibody.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention proposes serum CCL8, an indicator of connective tissue disease (CTD) combined with RPILD, which achieves the purpose of predicting and evaluating CTD-RPILD through high specificity and sensitivity. The CCL8 of the present invention refers to human CC motifchemokine ligand 8, also known as MCP-2, with a protein ID of NP_005614.2 and a transcript ID of NM_005623.3 in the NCBI database. The use of serum CCL8, KL-6, and / or anti-MDA5 antibodies alone or in combination is a biomarker with good sensitivity and specificity for evaluating CTD-RPILD. Detecting the levels of the above three markers can assist in the screening and timely diagnosis of CTD-RPILD in clinical practice, provide new targets for the treatment strategy of CTD-RPILD, and improve the clinical precision treatment of CTD-RPILD. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of gene differential display results, where (a) is a heat map of the top 19 differentially expressed genes, with red blocks representing higher expression and blue blocks representing lower expression; (b) is the GO function enrichment of the top 123 differentially expressed genes, including chemokine activity: 10 factors including CCL8; cytokine activity: 12 factors including CCL8; heparin binding: 11 factors including CCL8; CCR chemokine receptor binding: 5 factors including CCL8; and CCR2 chemokine receptor binding: 3 factors including CCL8.

[0018] Figure 2 Comparison of CCL8 levels in patients with CTD-RPILD, CTD-CILD, and HCs. A: Gene expression of the CCL8 cytokine in bronchoalveolar lavage fluid (BALF) cells is consistent with transcript levels. B: Comparison of serum CCL8 levels in patients with CTD-RPILD, CTD-CILD, and HCs.

[0019] Figure 3 The diagnostic efficacy of serum CCL8, KL-6, and anti-MDA5 antibodies for CTD-RPILD;

[0020] Figure 4 Survival curves of CTD-RPILD serum CCL8-positive and -negative patients; DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1 Screening of proteins associated with rapidly progressive pulmonary fibrosis and application of serum CCL8 as a diagnostic marker for rapidly progressive pulmonary fibrosis

[0023] 1) Transcriptome sequencing: We isolated bronchoalveolar lavage fluid (BALF) cells from patients with CTD combined with RPILD, patients with CTD without RPILD, and healthy controls (HC) for transcriptome sequencing, and compared the expression levels of 210,503 genes in total. Since the number of cells in the BALF of each individual patient is small, each biological replicate sample used for sequencing consists of a mixture of BALF cells from 5 individuals, including a total of 15 CTD-RPILD patients, 5 patients without CTD-RPILD, and 5 healthy controls. The gene transcription level of each biological sample represents the average gene transcription level of the individual cells of the patient it constitutes. Compared with the control group, there are 123 genes with an expression difference of 6 times or more. Among the top 19 genes with expression differences, 13 genes have increased expression and 6 genes have decreased expression. The results show that ( Figure 1 ), among the 20 GO functional categories, CCL8 was included in the top 5 significant categories, and CCL8 was also included in the categories of extracellular region, protein binding, phospholipase activator activity, and protein kinase activity.

[0024] 2) Validation of CCL8 in Bronchoalveolar Lavage Fluid (BALF):Although the transcription level of CCL8 in BALF cells from the lungs was significantly increased in RPILD patients, if it is used as a biomarker, a feasible detection scheme should be to detect its protein level in BALF and serum. To further verify whether the protein level of CCL8 is specifically increased in BALF of RPILD patients, we used enzyme-linked immunosorbent assay (ELISA) to detect the level of CCL8 in BALF. The results are as follows: Figure 2 As shown in (A), the level of CCL8 in BALF of CTD-RPILD patients was significantly increased compared with that of CTD-CILD patients (P=0.0003). The results showed that the level of CCL8 protein in BALF of CTD-RPILD patients was significantly increased in CTD-RPILD patients.

[0025] 3) Validation and diagnostic efficacy evaluation of CCL8 in serum: To further validate whether serum CCL8 can be used as a biomarker for diagnosing CTD-RPILD, we expanded the patient validation cohort and collected and measured the levels of CCL8 in serum samples from 53 CTD patients (26 with RPILD and 27 with CILD) and 23 healthy controls (HCs) for comparison. Figure 2 As shown in (B), serum CCL8 levels in RPILD patients (165.4 (130.5, 208.7) pg / mL) were significantly higher than those in CILD patients (122.3 (90.35, 136.7) pg / mL, P = 0.0004) and HCs (123.9 (81.08, 135.9) pg / mL, P = 0.0002). This result indicates that the detection of serum CCL8 levels is feasible for the evaluation of CTD-RPILD. We further used the receiver operating characteristic (ROC) curve to evaluate the diagnostic efficacy of serum CCL8 levels for CTD-RPILD. Figure 3 As shown in the results, the AUC of the receiver operating characteristic (ROC) curve was 0.817 (95% CI 0.704-0.930, P < 0.001), indicating that serum CCL8 levels have a high value in distinguishing CTD from RPILD. The optimal cutoff value was 71.4% for sensitivity, 86.7% for specificity, and 111.3 pg / mL for cutoff. This means that among patients with CTD and pulmonary fibrosis who had serum CCL8 levels above 111.3 pg / mL, 81.7% also had RPILD. This result demonstrates that serum CCL8 can be used for the diagnosis or risk assessment of patients with CTD and RPILD.

[0026] Example 2 Comparison of the diagnostic efficacy of serum CCL8 levels, KL-6, and anti-MDA5 antibodies for CTD-RPILD and their combined diagnosis Applications of CTD-RPILD

[0027] Anti-MDA5 antibody is an antibody known to be associated with RPILD, while KL-6 is a serological indicator of lung injury. Both are currently used as clinical diagnostic CTD -RPILD or a classic serological biomarker for risk assessment. We also tested the serum anti-MDA5 antibody and KL-6 levels in the above serum test cohort and used the ROC curve to evaluate the effect of serum CCL8 level combined with anti-MDA5 antibody and KL-6 on the risk of RPD. CTD -The diagnostic efficacy of RPILD. Figure 3 As shown in Figure 3, compared with serum KL-6 (AUC = 0.687) and anti-MDA5 antibody (AUC = 0.719), the AUC of serum CCL8 level for diagnosing RPILD was higher (0.817 (95% CI 0.668-0.967, P < 0.001)), which means that serum CCL8 level is important for the diagnosis of RPILD. CTD CCL8 demonstrated higher diagnostic efficacy for CTD-RPILD. Furthermore, when CCL8 was combined with KL-6 or anti-MDA5 antibodies for the diagnosis of CTD-RPILD, the AUC values ​​were significantly improved: Specifically, the AUCs for CCL8 combined with KL-6 or anti-MDA5 antibodies were 0.823 (cutoffs: KL-6 > 39.142, CCL8 > 150.997) and 0.841 (cutoffs: anti-MDA5 positive, CCL8 > 80.382), respectively. Notably, the specificity of CCL8 combined with anti-MDA5 antibodies for the diagnosis of CTD-RPILD was 0.923. Furthermore, when CCL8 was combined with KL-6 and anti-MDA5 antibodies, the area under the curve was 0.876 (cutoffs: KL-6 > 53.372, anti-MDA5 positive, and CCL8 > 88.897). The optimal cutoff values ​​achieved a sensitivity of 74.1% and a specificity of 88.5%.

[0028] Serum CCL8 as CTD Impact on patient survival: We investigated the 26 patients CTD-RPILD The patients were followed up for 30 months. During the study, 17 patients survived and 9 died, with a cumulative mortality rate of 34.6%. Among the 9 deaths, the CCL8 levels in the serum samples of all 9 patients were positive. The survival analysis curves of the CCL8 positive and negative groups are shown in Figure 2. Figure 4 The results showed that CCL8-positive CTD-RPILD patients had a higher mortality rate, while CCL8-negative CTD-RPILD patients survived during the follow-up period. This result suggests that serum CCL8 can be used as a follow-up marker for the prognosis of CTD-RPILD patients.

[0029] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Use of serum CCL8 as a molecular marker in the preparation of drugs or reagents for diagnosing rapidly progressive pulmonary fibrosis and / or assessing the prognosis of patients with rapidly progressive pulmonary fibrosis.

2. Use of a reagent for detecting serum CCL8 in the preparation of a drug or reagent for diagnosing rapidly progressive pulmonary fibrosis and / or evaluating the prognosis of patients with rapidly progressive pulmonary fibrosis.

3. Use of serum CCL8 as a molecular marker in the preparation of drugs or reagents for combined diagnosis of rapidly progressive pulmonary fibrosis with KL-6 and / or anti-MDA5 antibodies.

4. The use according to claim 3, wherein the reagent for detecting serum CCL8, KL-6, and / or anti-MDA5 antibodies comprises a reagent for detecting serum CCL8 using an enzyme-linked immunosorbent assay (ELISA).

5. The use according to claim 4, wherein the reagent for detecting serum CCL8, KL-6, and / or anti-MDA5 antibodies is an anti-CCL8 antibody, an anti-KL-6 antibody and / or an anti-MDA5 antibody.

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