A detection method for LAMP1 + Application of B-cell level reagents in rheumatoid arthritis

Abstract

The application provides a kind of detection LAMP1 + The application provides a kind of detection LAMP1 + The application provides a kind of detection LAMP1 + The application provides a kind of detection LAMP1 + The application provides a kind of detection LAMP1 + The application provides a kind of detection LAMP1

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to a method for detecting LAMP1. + Application of B-cell level reagents in rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis and progressive bone erosion. In my country, the number of RA patients exceeds 5 million. Without standardized treatment, the disease can lead to joint structural destruction, functional loss, and even disability, severely impacting patients' quality of life. Therefore, elucidating the pathogenesis of RA and establishing sensitive and reliable methods for assessing disease activity are of great significance for guiding clinical treatment and improving prognosis.

[0003] Clinically, the most commonly used assessment of RA disease activity is the Disease Activity Composite Score (such as DAS28, CDAI, and SDAI). These scoring systems integrate the count of tender and swollen joints, the overall assessment of the patient and the assessor, and often incorporate C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, traditional laboratory indicators such as CRP and ESR have limited sensitivity in the early stages of the disease, and all of the above methods lack a direct reflection of the functional status of immune cells, making it difficult to comprehensively assess the degree of disease activity. Therefore, the search for more precise novel immunological biomarkers has become a current research hotspot.

[0004] Recent studies have shown that B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Besides producing autoantibodies, B cells participate in the regulation of inflammatory responses through various pathways, including antigen presentation and cytokine secretion. Lysosome-associated membrane protein 1 (LAMP1) is an important protein located on the lysosomal membrane and has traditionally been used as a marker of cytotoxic lymphocyte degranulation. Recent studies have found that LAMP1 is upregulated in senescent cells and can serve as a membrane surface marker of cellular senescence. However, systematic research and application of LAMP1 as an indicator of disease activity assessment are currently lacking. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting LAMP1, which addresses the shortcomings of the prior art. + Application of B-cell level reagents in rheumatoid arthritis: LAMP1 was detected by flow cytometry using readily available peripheral blood samples. +The proportion of B cells is positively correlated with disease activity indicators. The sampling is convenient and non-invasive, the detection method is simple, low-cost and efficient, and it can dynamically reflect disease progression, thus improving the accuracy of disease monitoring.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a detection method for LAMP1. + Application of reagents at the B-cell level in rheumatoid arthritis, detection of LAMP1 + Reagents at the B-cell level were used to prepare kits for detecting rheumatoid arthritis; LAMP1 was also detected. + Reagents at the B-cell level include: CD19 antibody and LAMP1 antibody.

[0007] Preferably, the LAMP1 + B cell level refers to LAMP1 in peripheral blood. + B cells.

[0008] Preferably, LAMP1 in the peripheral blood + A B cell percentage ≥35% is used as a marker of rheumatoid arthritis activity.

[0009] Compared with the prior art, the present invention has the following advantages: This invention uses readily available, non-invasive, and information-rich patient blood samples as biological samples and employs flow cytometry to detect LAMP1. + Detecting rheumatoid arthritis at the B-cell level is not only simple, low-cost, and efficient, but also convenient to sample, causes minimal damage to the body, and has high diagnostic sensitivity and accuracy. It can more sensitively and dynamically reflect disease progression and improve the accuracy of disease monitoring.

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 LAMP1 in peripheral blood of healthy controls (HC) and rheumatoid arthritis (RA) patients in Example 1 of this invention. + Flow cytometry analysis of B cells.

[0012] Figure 2 The LAMP1 in the peripheral blood of rheumatoid arthritis (RA) patients in Embodiment 1 of the present invention + Correlation analysis diagram of B cell levels and disease activity-related indicators.

[0013] Figure 3 The LAMP1 in the synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) in Embodiment 2 of the present invention. + Immunofluorescence assay of B cells.

[0014] Figure 4 These are flow cytometry analysis and immunofluorescence detection images of spleen and synovial tissue from healthy control Ctrl mice and collagen-induced arthritis CIA mice in Example 3 of the present invention; wherein, Figure A is flow cytometry analysis; Figure B is immunofluorescence detection. Detailed Implementation

[0015] Example 1

[0016] This example focuses on LAMP1 in peripheral blood. + Detection and correlation analysis of B cell levels.

[0017] (1) Experimental subjects and sample acquisition: Patients with rheumatoid arthritis (RA) and healthy controls (HC) were selected. 3 mL of peripheral venous blood was collected, placed in EDTA anticoagulant tubes, stored at 4°C, and tested within 4 hours.

[0018] (2) Isolation of peripheral blood mononuclear cells (PBMCs): Anticoagulated blood was diluted 1:1 with PBS and slowly added to Ficoll separation buffer. The mixture was centrifuged at 2000 rpm for 20 min at room temperature. The mononuclear cell layer was collected, washed twice with PBS, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 The cells / mL were used to obtain a cell suspension.

[0019] (3) Flow cytometry antibody staining: Take 100 μL of cell suspension and add the following antibodies: anti-human CD19 antibody (PE-conjugated anti-human CD19 antibody) and anti-human LAMP1 antibody (APC-conjugated anti-human LAMP1 antibody). Incubate at 4°C for 30 min in the dark. After incubation, wash twice with PBS and resuspend in 300 μL of PBS for detection.

[0020] (4) Flow cytometry detection: Flow cytometry was used for detection. First, lymphocyte populations were selected by gating based on forward scattered light (FSC) and side scattered light (SSC), and then CD19 cells were analyzed. + LAMP1 expression was analyzed in a cell population, and LAMP1 levels were calculated. + CD19 + The proportion of B cells.

[0021] Flow cytometry results as follows Figure 1 As shown, compared with healthy controls (HC), patients with rheumatoid arthritis (RA) had significantly higher levels of LAMP1 in their peripheral blood.+ The proportion of B cells was significantly increased (**p<0.01), when LAMP1 in the peripheral blood of the subjects was significantly increased. + A B-cell ratio higher than the healthy control range (≥35%) indicates increased disease activity in rheumatoid arthritis; this indicator can be used for disease stratification, efficacy evaluation, and dynamic monitoring.

[0022] This embodiment also addresses LAMP1. + The correlation between B cell levels and clinical indicators was analyzed.

[0023] Statistical analysis was performed using GraphPad Prism software, LAMP1 + Correlation analysis was performed on B cell levels with clinical indicators RF (rheumatoid factor), anti-CCP (anti-cyclic citrullinated peptide antibody), CRP (C-reactive protein), and ESR (erythrocyte sedimentation rate) using Pearson correlation analysis.

[0024] The results are as follows Figure 2 As shown, LAMP1 + The proportion of B cells was positively correlated with C-reactive protein (CRP), anti-cyclic citrullinated peptide (CCP) antibody, rheumatoid factor (RF), and erythrocyte sedimentation rate (ESR) in RA patients.

[0025] Example 2

[0026] This example describes LAMP1 in the synovial tissue of a RA patient. + B cell detection.

[0027] Synovial tissue obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) controls was immediately fixed in 4% paraformaldehyde for 24 hours. After fixation, the tissue was dehydrated by a gradient of ethanol (75% ethanol for 4 hours → 85% ethanol for 2 hours → 90% ethanol for 2 hours → 95% ethanol for 1 hour → anhydrous ethanol I for 30 minutes → anhydrous ethanol II for 30 minutes), cleared with xylene, embedded in paraffin, and sectioned.

[0028] After being dried at 60°C for 1 hour, the paraffin sections were dewaxed in xylene (twice, 10 minutes each time), and then rehydrated with a gradient of ethanol (100%, 95%, 85%, 75%) to distilled water. Antigen retrieval was then performed: the sections were placed in sodium citrate buffer, heated to boiling in a microwave and maintained for 10 minutes, and then allowed to cool naturally to room temperature.

[0029] After rinsing three times with PBS, add 0.3% Triton X-100 and permeabilize at room temperature for 15 min, then wash with PBS, and then block non-specific binding sites with 5% bovine serum albumin (BSA) and incubate at room temperature for 30 min.

[0030] After discarding the blocking solution, add the primary antibody mixture: anti-human CD19 antibody and anti-human LAMP1 antibody, diluted to the recommended concentration (1:100) in the instructions, and incubate overnight in a humidified chamber at 4°C.

[0031] The next day, the sample was brought to room temperature, washed three times with PBS (5 min each time), and then incubated with the corresponding fluorescently labeled secondary antibodies (CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L), catalog number: SA00013-4; CoraLite488-conjugated Goat Anti-Mouse IgG (H+L), catalog number: SA00013-1) for 1 h in the dark (room temperature).

[0032] After incubation, the cells were washed three times with PBS, and DAPI staining solution was added for nuclear counterstaining. The cells were incubated at room temperature in the dark for 5 minutes, then rinsed with PBS. After mounting with anti-fluorescence quenching mounting medium (Beyotime, catalog number P0126-25ml), the cells were observed and images were acquired using a fluorescence microscope.

[0033] Immunofluorescence (IF) detection results as follows Figure 3 As shown, LAMP1 in the synovial tissue of RA patients + B cell counts were higher in patients with osteoarthritis (OA) (scale bar 50 μm).

[0034] Example 3

[0035] This embodiment is used to validate and detect LAMP1 in an animal model. + Application of B-cell level reagents in rheumatoid arthritis.

[0036] A collagen-induced arthritis (CIA) mouse model was established. Eight-week-old male DBA / 1 mice (weighing 20g-25g) were randomly divided into a model group and a control group, with no fewer than six mice in each group.

[0037] The model group underwent immunization induction with a type II collagen (CII) emulsion: bovine type II collagen (2 mg / mL) was emulsified with complete Freund's adjuvant (CFA) at a volume ratio of 1:1 to prepare a stable emulsion. Mice were initially immunized by intradermal injection of 100 μL of the emulsion (containing 100 μg CII) at the base of the tail. On day 21, a booster immunization was performed using a mixture of type II collagen and incomplete Freund's adjuvant (IFA) at a volume ratio of 1:1, with the same injection dosage and method. Control group mice received an equal volume of PBS injected at the base of the tail at the same time point.

[0038] The arthritis score and paw swelling of mice were observed and recorded every 3 days after immunization. The model was considered to have been successfully constructed when mice showed typical signs such as joint redness, swelling and deformity and an arthritis score ≥2.

[0039] After successful modeling, mice were sacrificed on day 45, and spleen and ankle synovial tissue were collected. The spleen was mechanically ground to prepare a single-cell suspension, which was then treated with erythrocyte lysis buffer and washed with PBS. The synovial tissue was minced and digested with type I collagenase (1 mg / mL) at 37°C for 30 min, and then filtered to obtain a single-cell suspension.

[0040] The obtained cells were resuspended and their concentration adjusted. Following the flow cytometry staining method described above, anti-mouse CD19 and anti-LAMP1 antibodies were used for staining to detect CD19 levels. + LAMP1 in B cells + Cell proportion; LAMP1 in mouse synovial tissue + The method for detecting B cells is the same as in Example 2.

[0041] A mouse model of collagen-induced arthritis (CIA) was established. After sacrifice, spleen and synovial tissue were collected to prepare single-cell suspensions, which were then analyzed by flow cytometry. Figure 4 As shown in Figure A, compared with healthy control (Ctrl) mice, CIA mice had higher levels of LAMP1 in their peripheral blood. + The proportion of B cells was significantly increased; IF test results were as follows: Figure 4 As shown in Figure B, LAMP1 in the synovial tissue of CIA mice + B cell count was higher in Ctrl mice.

[0042] In summary, LAMP1 levels in the peripheral blood and synovial tissue of RA patients... + The proportion of B cells was significantly increased and positively correlated with disease activity-related indicators (including RF, anti-CCP, CRP, and ESR), suggesting that LAMP1... + B cells play an important role in RA, and the detection of LAMP1 + Reagents at the B-cell level can be used to prepare kits for detecting rheumatoid arthritis, and LAMP1 can be detected using flow cytometry. + B-cell level diagnostics for rheumatoid arthritis is not only simple, low-cost, and efficient, but also convenient to sample, causes minimal damage to the body, and has high diagnostic sensitivity and accuracy. It can more sensitively and dynamically reflect disease progression and improve the accuracy of disease monitoring.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A detection method for LAMP1 + The application of B-cell level reagents in rheumatoid arthritis, characterized by... Detecting LAMP1 + Reagents at the B-cell level were used to prepare kits for detecting rheumatoid arthritis; LAMP1 was also detected. + Reagents at the B-cell level include: CD19 antibody and LAMP1 antibody.

2. A method for detecting LAMP1 according to claim 1 + The application of B-cell level reagents in rheumatoid arthritis, characterized by... LAMP1 + B cell level refers to LAMP1 in peripheral blood. + B cells.

3. A detection method for LAMP1 according to claim 2 + The application of B-cell level reagents in rheumatoid arthritis, characterized by... LAMP1 in peripheral blood + A B cell percentage ≥35% is used as a marker of rheumatoid arthritis activity.

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