Serum protein markers for identifying lupus nephritis and application thereof

Abstract

The invention discloses a group of serum protein markers for identifying lupus nephritis, and the serum protein markers are characterized in that the serum protein markers comprise one or more of MMP-10, CSF-1, CX3CL1, IL-17C and IL-10RB. According to the present invention, the ROC curve is adopted to evaluate the diagnosis efficiency of the differential protein, and the novel serum marker capable of distinguishing whether the systemic lupus erythematosus patient is accompanied with nephritis is identified;

Description

Technical Field

[0001] The invention belongs to the technical field of proteomics or lupus nephritis diagnostic markers, and specifically relates to a group of serum protein markers for identifying lupus nephritis and applications thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a typical autoimmune disease characterized by overactivation of autoreactive immune cells and massive production of autoantibodies, accompanied by systemic immune system disorders and target tissue inflammatory damage. Lupus nephritis (Lupus Nephritis, LN) is the most common and most important risk factor for the overall morbidity and mortality of SLE. Despite the use of effective hormone anti-inflammatory and immunosuppressive treatments, the diverse clinical manifestations of SLE during diagnosis and treatment and the recurrence of the disease often lead to delayed diagnosis, resulting in some patients ending up with CKD or ESRD.

[0003] In terms of diagnosis, renal biopsy is the gold standard for the diagnosis of lupus nephritis, but it is an invasive examination with risks of infection and bleeding, and it is not convenient for rheumatologists to dynamically monitor the activity of the disease. In the course of lupus nephritis, there are dynamic changes with alternating active and inactive phases. At present, the clinical indicators routinely monitored for lupus nephritis, such as anti-dsDNA antibodies, urine protein, serum creatinine clearance, complement C3 and C4, etc., all have obvious clinical lag and cannot evaluate the prognosis.

[0004] Proteomics testing is a qualitative and quantitative analysis of proteins in biological samples, which is widely used in biopharmaceutical development, clinical diagnosis and basic biological research. As an important functional molecule in the body, protein is responsible for the regulation and execution of life activities, and inflammatory response also plays an important role in the occurrence of autoimmune diseases. However, most of the current work on systemic lupus erythematosus and proteomics is based on mass spectrometry, which cannot target and identify low-abundance proteins. Compared with other technologies, Olink technology has significant advantages in sensitivity, which can achieve a detection sensitivity of fg / ml, and the detection range can span 10 orders of magnitude. Not only can the required detection sample volume reach uL level, but many low-abundance proteins can be accurately detected. At present, there are relatively few studies on olink proteomics and lupus nephritis. Therefore, the development of lupus nephritis-specific serum protein biomarkers based on olink technology can provide new ideas and possibilities for the prevention, screening and treatment of lupus nephritis, so as to truly transfer it from the laboratory to the clinic.

[0005] Purpose of the Invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art, provide a group of serum protein markers for identifying lupus nephritis and their applications, and provide a useful tool for identifying lupus nephritis.

[0007] In one aspect, the present invention provides a group of serum protein markers for identifying lupus nephritis, wherein the serum protein markers include one or more of MMP-10, CSF-1, CX3CL1, IL-17C, and IL-10RB.

[0008] Preferably, the method for selecting serum protein markers comprises the following steps:

[0009] S1. Collect serum samples: Collect serum samples from patients with systemic lupus erythematosus and lupus nephritis respectively;

[0010] S2, perform olink96 Proteomics 96 inflammation panel protein sequencing on the serum samples collected in S1 to screen for differentially expressed proteins;

[0011] S3. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of differentially expressed proteins in S2 for lupus nephritis.

[0012] On the other hand, the present invention provides a kit for identifying lupus nephritis, wherein the kit comprises the serum protein marker or a detection reagent thereof.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention provides a group of serum protein markers for identifying lupus nephritis, which can effectively identify whether patients with systemic lupus erythematosus are accompanied by nephritis;

[0015] 2. The present invention provides a kit for identifying lupus nephritis, which provides a new idea for the targeted treatment of lupus nephritis from the perspective of serum protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0017] Figure 1 The number and distribution of differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0018] Figure 2 The clustering heat map of differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0019] Figure 3This is the differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0020] Figure 4 This is the differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0021] Figure 5 This is the differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0022] Figure 6 This is the differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX);

[0023] Figure 7 This is the differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX); Figure 8 This is an integrated differential analysis of the top 5 differentially expressed proteins between the lupus nephritis group (LX) and the systemic lupus erythematosus group (SX). DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0025] Example 1: Sequencing screening of differential serum protein markers

[0026] 1.1 Collection of serum samples:

[0027] A total of 51 patients with systemic lupus erythematosus with or without nephritis diagnosed by the Department of Rheumatology and Immunology of Drum Tower Hospital Affiliated to Nanjing University Medical School were collected, and fresh serum specimens (without any anticoagulant) were collected for routine testing and immediately stored in a -80℃ refrigerator for later use. Diagnosis Inclusion criteria: All of the following criteria must be met at the same time: at least 4 of the 11 systemic lupus erythematosus classification criteria established by the American College of Rheumatology (ACR) in 1997; and clear renal puncture pathology or in accordance with the "2019 Chinese Guidelines for the Diagnosis and Treatment of Lupus Nephritis".

[0028] 1.2 Serum Olink Proteomics Sequencing:

[0029] 1.2.1 Sample randomization: Use the RAND function in the Excel table to output the numerical value corresponding to each sample, and then arrange the values ​​from small to large so that the samples are randomly distributed in the 96-well plate.

[0030] 1.2.2 Sample sampling: According to the randomized sample distribution table, take 20 μL of each sample into a 96-well plate as an external control; add 5 μL of mixed plasma sample to wells A12 and B12, add 5 μL of Negative Control (NC) to wells C12, D12, and E12, and add 5 μL of Interplate Control (IPC) to wells F12, G12, and H12, and mix well.

[0031] 1.2.3 Hybridization incubation: Prepare hybridization mixture first, take out the reagent and thaw it at room temperature, vortex and centrifuge, take a nuclease-free 1.5ml centrifuge tube, add 280μL of Incubation Solution, 40μL of IncubationStabilizer, 40μL of A-probes, and 40μL of B-probes in sequence, vortex and centrifuge. Take out an 8-tube strip and add 47μL of hybridization mixture to each well. Take out a 96-well plate, mark it as an incubation plate, use an 8-channel pipette to add 3μL of hybridization mixture to each well, and then add 1μL of sample to each well one by one. After sticking the sealing film, centrifuge at 400g for 1 minute, put it in the PCR instrument, set it to 4℃ infinity, record the time of putting it into the PCR instrument, and the incubation time must meet 16-24 hours.

[0032] 1.2.4 Extension and amplification: Set the PCR program to 50℃ for 20 minutes; 95℃ for 5 minutes; 95℃ for 30 seconds, 54℃ for 1 minute, and 60℃ for 1 minute for 17 cycles; maintain at 10℃. After the settings are completed, run the PCR instrument and press the pause button to maintain the temperature of the PCR instrument at 50℃. Prepare the extension and amplification mixture. Take a 15mL centrifuge tube and add 9385μL of High Purity Water, 1100μL of PEASolution, 55μL of PEAEnzyme, and 22μL of PCR Polymerase in sequence, vortex and centrifuge. Take out the incubation plate from the PCR instrument and centrifuge. Use an 8-channel pipette to add 96μL of extension and amplification mixture to each well. After sealing the film, vortex and centrifuge, place it on the PCR instrument preheated at 50℃, and then resume the running program.

[0033] 1.2.5 Chip pretreatment: Unpack and take out the chip, check whether the chip and syringe are damaged. Take out the syringe, press down the piston hole, and push the liquid in the syringe. Click the instrument Target 96 option, the instrument pushes out the sample stage, puts in the chip and cover, and automatically returns to the sample stage. Click Start, and the chip pretreatment begins.

[0034] 1.2.6 Operation on the machine: Take out a 1.5ml centrifuge tube, add 550μL of Detection Solution, 230μL of High Purity Water, 7.8μL of Detection Enzyme, and 3.1μL of PCR Polymerase in sequence, vortex and centrifuge. Take out another strip tube and dispense 95μL per well. Take out a 96-well plate, add 7.2μL of the mixture to each well, and after the extension amplification program is completed, add 2.8μL of the product, vortex and mix well for use. Prepare the diluted sample plate, primer plate and pre-treated chip, add 5μL of primers to the left side of the chip, and add 5μL of diluted product to the right side in sequence to avoid bubbles. After the sample is added, put it into the sample loading station, set the run name, and click Run.

[0035] 1.2.7 Data analysis and export: Export the run name file after the instrument is shut down.

[0036] Example 2: Clinical application of serum differential protein markers

[0037] 2.1 Identification and analysis of olink sequencing results

[0038] The differential expression protein quantitative analysis was performed with 1.5-fold as the threshold of differential expression change and t-test p-value < 0.05 as the significance threshold. Figure 1 The results showed that compared with the healthy subjects, 19 proteins were significantly upregulated in patients with lupus nephritis and 1 protein was significantly downregulated in patients with lupus nephritis.

[0039] Quantitative proteomics analysis was used to determine the expression of the above differential proteins in 22 SLE groups and 29 LN group samples. The clustering heat map analysis of differential proteins between the different groups is shown in Figure 2. Figure 2 shown.

[0040] 2.2 Analysis of the diagnostic efficacy of differentially expressed proteins in lupus nephritis

[0041] The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. The AUC of a completely worthless diagnostic test is 0.5, and the AUC of an ideal diagnostic test is 1. It is generally believed that an AUC between 0.7 and 0.9 has a certain diagnostic value.

[0042] The areas under the AUC curves of the proteins CSF-1, IL-17C, MMP-10, CX3CL1, SCF, ST1A1, PD-L1, IL-10RB, TNF, CST5, and LAP TGF-beta-1 provided by the present invention are all greater than 0.7, indicating that the above markers can better distinguish systemic lupus erythematosus from lupus nephritis. The specific results are shown in Table 1. The top five proteins are CSF-1, IL-17C, MMP-10, CX3CL1, and SCF, and the p values ​​are all <0.01. The difference in proteins between the groups is analyzed as follows Figure 3-6 As shown in Figure 2, the integrated difference analysis is as follows Figure 8 As shown, the areas under the AUC curve were 0.8574, 0.7978, 0.7947, 0.7837, and 0.7586, respectively, indicating that the above markers have good sensitivity and specificity.

[0043] Table 1 Summary of differentially expressed proteins with an area under the AUC curve greater than 0.7

[0044] Protein name Area under the AUC curve CSF-1 0.86 IL-17C 0.80 MMP-10 0.79 CX3CL1 0.78 SCF 0.76 ST1A1 0.74 PD-L1 0.74 IL-10RB 0.74 TNF 0.73 CST5 0.71 LAP TGF-beta-1 0.71

[0045] The embodiments of the present invention are only to introduce the specific implementation methods thereof, and are not intended to limit the protection scope thereof. The technicians in the industry may make some modifications inspired by the embodiments, so any equivalent changes or modifications made according to the scope of the present invention are within the scope of the patent claims of the present invention.

Claims

1. A group of serum protein markers for identifying lupus nephritis, characterized in that: The serum protein markers include one or more of MMP-10, CSF-1, CX3CL1, IL-17C, and IL-10RB.

2. A set of serum protein markers for identifying lupus nephritis according to claim 1, characterized in that: The method for selecting the serum protein marker comprises the following steps: S1. Collect serum samples: Collect serum samples from patients with systemic lupus erythematosus and lupus nephritis respectively; S2, perform olink96 Proteomics 96 inflammation panel protein sequencing on the serum samples collected in S1 to screen for differentially expressed proteins; S3. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of differentially expressed proteins in S2 for lupus nephritis.

3. A kit for identifying lupus nephritis, characterized in that: The kit comprises the serum protein marker or a detection reagent thereof according to claim 1.

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