Application of receptor protein CD74 as leucoderma molecular marker and therapeutic target

Abstract

The invention belongs to the technical field of biological medicine, particularly relates to application of receptor protein CD74 as a vitiligo molecular marker and a treatment target, provides application of the receptor protein CD74 as a vitiligo related molecular marker in diagnosis and treatment for the first time, and determines the effective effect of the receptor protein CD74 in the aspect of vitiligo diagnosis. The blank that no specific laboratory diagnostic index for leucoderma exists at present is filled, and meanwhile, a new target spot is provided for treatment of leucoderma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a receptor protein CD74 as a molecular marker and therapeutic target of vitiligo. BACKGROUND

[0002] Vitiligo is a postnatal pigment loss disease, and the skin lesions show uneven pigment loss spots. The global prevalence rate of vitiligo is 0.5%-2%. The etiology of vitiligo is complex, and there are many theories such as nerves, heredity and immunity. At present, it is mainly believed that T cells mediated autoimmune reaction activates CD8 + T cells specific to melanocytes, thereby destroying the pigment cells. In addition, vitiligo is also highly comorbid with autoimmune diseases such as diabetes and thyroid diseases, further complicating the complexity of clinical management. Although vitiligo does not directly affect life and health, it often brings psychological pressure and social barriers to patients due to its significant impact on appearance, seriously affecting the quality of life. Therefore, early diagnosis and active treatment of vitiligo are crucial to improve the prognosis of patients.

[0003] At present, the diagnosis of vitiligo mainly depends on clinical manifestations and auxiliary examinations such as Wood lamp examination, dermatoscope examination and skin biopsy. However, these methods have diagnostic difficulties in early lesions, atypical cases or patients with light skin color. In recent years, researchers have tried to find biomarkers related to vitiligo in order to improve the accuracy of diagnosis and the possibility of early detection. For example, the mRNA and protein levels of tyrosinase (TYR) and its isozymes TRP-1 / TRP-2 are used to evaluate the synthetic activity of melanocytes, but the sensitivity is often insufficient due to low expression; autoantibodies against melanocytes (such as anti-TYR, anti-PMEL) have limited specificity and clinical application value due to cross-reactions with other autoimmune diseases; inflammatory factors and chemokines (such as CXCL9, CXCL10, IFNγ) in the epidermis and serum of patients with active vitiligo are significantly increased, which can reflect the immune activation state, but are difficult to standardize and quantify due to the influence of treatment intervention and comorbid diseases; in addition, digital dermatoscope has auxiliary value in detecting "invisible" pigment loss, but its results are highly dependent on the experience of operators, and it is still difficult to convert into molecular level diagnostic markers and widely promote in the clinic.

[0004] CD74, as a high-affinity receptor for macrophage migration inhibitory factor (MIF) and a MHC class II molecule chaperone, plays an important regulatory role in the occurrence and development of inflammation and autoimmune diseases. Previous studies have shown that CD74 is abnormally highly expressed in various autoimmune diseases and is closely related to disease activity. Specifically, in systemic lupus erythematosus, the abnormal activation of the MIF-CD74 axis triggers downstream inflammatory signaling pathways such as PI3K-Akt, induces the release of a large number of inflammatory factors, and drives the production of autoantibodies. In rheumatoid arthritis, abnormal antigen presentation mediated by CD74 and activation of the MIF-CD74 axis lead to synovial inflammation and joint destruction. These findings suggest that CD74 is not only an important inflammatory regulator but also a potential diagnostic marker and therapeutic target for autoimmune diseases. In recent years, some studies have reported that the expression levels of MIF in patients with vitiligo and in vitiligo mouse models are significantly higher than those in the control group, and MIF blockers can effectively promote the repigmentation of vitiligo mice. However, the role of CD74 in the pathogenesis of vitiligo has not been studied.

[0005] Therefore, clarifying whether CD74 is involved in the immune-mediated pathogenesis of vitiligo is a key issue that needs to be addressed, which can provide a theoretical basis for clinical diagnosis and the development of targeted drugs. SUMMARY

[0006] To solve the above problems, the present application provides a use of receptor protein CD74 as a molecular marker and therapeutic target for vitiligo.

[0007] The present application found through a number of bioinformatics techniques that the gene expression level of CD74 in patients with vitiligo is positively correlated with the pathological process and the number of CD8 + T cells in the lesion. In vitro experiments found that inhibiting CD74 can effectively reduce the expression of pro-inflammatory maturation markers (CD86, CCR7) and the upregulation of cDC1 subpopulation-specific transcription factors (BATF3, IRF8) in DCs under inflammatory conditions, thereby inhibiting the abnormal activation and migration function of DCs and effectively improving the inflammatory state of DCs. Therefore, receptor protein CD74 is expected to become a new target for the diagnosis and treatment of vitiligo.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a use of receptor protein CD74 as a molecular marker for the diagnosis or treatment of vitiligo.

[0010] In a second aspect, the present application provides a use of receptor protein CD74 as a therapeutic target.

[0011] In a third aspect, the present application provides a use of a detection reagent for expression level of CD74 gene or a protein encoded by the same in preparation of a product for auxiliary diagnosis of vitiligo.

[0012] Further, the diagnosis product detects the expression level of CD74 gene or a protein encoded by the same in a sample by real-time quantitative PCR, a chip, a high-throughput sequencing platform, flow cytometry, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0013] Further, the diagnosis product contains specific primers for amplifying CD74 gene, a probe hybridized with the nucleotide sequence of CD74 gene or an antibody specifically binding to CD74 protein.

[0014] Still further, the antibody is a monoclonal antibody or a polyclonal antibody.

[0015] Further, the product is a chip, a preparation or a kit.

[0016] Further, the sample is a tissue, serum or cell.

[0017] The present application also provides a use of an inhibitor for expression level of CD74 gene in preparation of a medicine for treating vitiligo.

[0018] Further, the inhibitor for expression level of CD74 gene comprises a CD74 antagonist.

[0019] Still further, the medicine further comprises a pharmaceutically acceptable carrier and / or excipient, and is prepared into an internal dosage form for oral administration or an injection for non-oral administration or an external dosage form according to a conventional method.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application firstly proposes a use of receptor protein CD74 as a vitiligo-related molecular marker in diagnosis and treatment of vitiligo, and determines the effective role of receptor protein CD74 in diagnosis of vitiligo. The present application fills the blank of no specific laboratory diagnosis index for vitiligo at present, and also provides a new target for treatment of vitiligo. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 shows the expression level of CD74 and its relationship with pathological process and the number of CD8 + T cells.

[0023] Figure 2 Figure 2 shows that DCs are the main receivers in MIF-CD74 communication axis through cell communication analysis.

[0024] Figure 3Cell viability of DCs was analyzed using CCK8 after 24 hours of treatment of DCs with inhibitor (C36L1) and LPS-IFN.

[0025] Figure 4 Activation markers of DCs were analyzed using flow cytometry after 24 hours of treatment of DCs with inhibitor (C36L1) and LPS-IFN.

[0026] Figure 5 Proportion of subtypes of DCs was analyzed using flow cytometry after 24 hours of treatment of DCs with inhibitor (C36L1) and LPS-IFN.

[0027] Figure 6 Expression of transcription factors in DCs was analyzed using qRT-PCR after 24 hours of treatment of DCs with inhibitor (C36L1) and LPS-IFN.

[0028] Figure 7 Photos of phenotypes of mice in different groups after 5 weeks of treatment. DETAILED DESCRIPTION

[0029] The application will be described in greater detail below with specific reference being made to the following examples. The following examples are intended to be illustrative only and are not intended to limit the application. The methods of operation in the examples are conventional methods of operation in the art.

[0030] Example 1

[0031] Single-cell transcriptome data analysis

[0032] 1) Data quality control and normalization: Three filtering criteria were set according to the cellular molecular characteristics, including retaining cells with moderate gene expression (number of detected genes > 500 and < 5,000), strictly controlling mitochondrial gene contamination (< 15%) and hemoglobin gene expression (< 4%), and limiting the total UMI number (< 25,000), effectively eliminating low-quality cells and multiple capture events. Data normalization was performed using logarithmic normalization (scale factor = 10,000) to correct sequencing depth differences, combined with the Harmony algorithm to integrate multiple batches of samples and standardize gene expression;

[0033] 2) Cell clustering and annotation: Based on high-variable gene screening, PCA dimensionality reduction (number of principal components = 30) and Leiden clustering (resolution = 0.9) were performed, combined with the marker genes of each cluster of cells to divide the cell subgroups;

[0034] 3) Differential genes: Disease group-specific genes were screened by Wilcoxon test (|log2FC|≥0.25, FDR<0.01).

[0035] 4) Cell interaction network construction: CellChat tool was used to predict high-activity ligand-receptor pairs and quantify signal pathway communication strength.

[0036] Example 2

[0037] Cell culture

[0038] 1. Thawing

[0039] 1) First, the mouse bone marrow dendritic cell line DC2.4 cell freezing tube was placed in a 37°C water bath and shaken to thaw.

[0040] 2) Transfer to a 15 mL centrifuge tube, add 5 mL of culture solution, centrifuge (1000 rpm, 3 min), discard the supernatant, add 1640 culture medium, and blow evenly before transferring to a T25 cell culture flask. Place in a 37°C, 5% CO2 incubator, and change the medium after 24 hours.

[0041] 2. Cell culture

[0042] When the cell confluence in the culture flask is 70-80%, subculture.

[0043] 1) Collect the culture medium in the culture flask, wash twice with PBS, add 2 mL of 0.25% trypsin-EDTA digestion solution, and incubate at 37°C in a 5% CO2 incubator for 2 min.

[0044] 2) Observe under a microscope, and when the cells float, add 3 mL of 1640 culture medium to stop the digestion.

[0045] 3) Blow with a gun head to detach the cells from the culture flask wall, transfer the cells to the same centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 8 mL of culture medium to resuspend the cells, and transfer to a T75 culture flask for cell subculture.

[0046] Example 3

[0047] Cell grouping and treatment

[0048] 1) NC group: DC2.4 cells without treatment;

[0049] 2) LI group: DC2.4 cells induced by LPS (1 μg / mL) and IFNγ (20 ng / mL);

[0050] 3) LI-3 group: DC2.4 cells induced by LPS-IFNγ and 3 μM C36L1;

[0051] 4) LI-4 group: DC2.4 cells induced by LPS-IFNγ and 4 μM C36L1;

[0052] 5) LI-5 group: DC2.4 cells induced by LPS-IFNy and 5 mM C36L1 combination;

[0053] 6) LI-6 group: DC2.4 cells induced by LPS-IFNy and 6 mM C36L1 combination.

[0054] Example 4

[0055] Detecting the cell viability of DC2.4 by CCK8

[0056] When the cell confluence in the culture flask is 70-80%, the cells are inoculated into the 96-well plate at a density of 1W / well. After 24 hours of culture, the cells are induced by different interventions, and the specific grouping is shown in Example 3. After 24 hours, 10 uL of CCK8 detection solution is added to each well, and the absorbance at 450 nm is measured by the enzyme marker.

[0057] The experimental results show that the CD74 inhibitor C36L1 can inhibit the proliferation of DC2.4 induced by LPS-IFNy, as shown in Figure 2. Figure 3 .

[0058] Example 5

[0059] Detecting the activation markers of DC2.4 by flow cytometry

[0060] When the cell confluence in the culture flask is 70-80%, the cells are inoculated into the 24-well plate at a density of 10W / well. After 24 hours of culture, the cells are induced by different interventions, and the specific grouping is shown in Example 3. After 24 hours, all the cells in the well are collected. Blocking Fc receptors (BD Pharmingen, 553142) are used for incubation at room temperature for 10 minutes. APC anti-mouse CD197 (CCR7) antibodies (BioLegend, 120108) are used for incubation at 37°C for 10 minutes, PE anti-mouse CD80 antibodies (BioLegend, 104707) and APC anti-human CD86 (BioLegend, 374207) antibodies are used for incubation at 2-8°C for 20 minutes. The cells are collected, resuspended after PBS washing, and then stained with 7-AAD dye (BioLegend, 420403) before flow cytometry detection.

[0061] The experimental results show that LPS-IFNy combination treatment can significantly induce the up-regulation of the expression of co-stimulatory molecules CD86 and chemokine receptor CCR7 on the surface of DC2.4, and C36L1 intervention effectively inhibits the abnormally high expression of these two molecules, as shown in Figure 3. Figure 4 .

[0062] Example 6

[0063] Detection of the proportion of subtypes of DC2.4 by flow cytometry

[0064] When the cell confluence in the culture flask reached 70-80%, the cells were seeded into a 24-well plate at a density of 10W / well. After 24 hours of culture, the cells were induced by different interventions, and the specific grouping is shown in Example 3. After 24 hours, all the cells in the wells were collected. Blocking Fc receptors (BD Pharmingen, 553142) was used for incubation at room temperature for 10 minutes. PE / Cyanine7 anti-mouse CD103 (BioLegend, 121425) and FITC anti-mouse / human CD11b (BioLegend, 101205) were used for incubation at 2-8°C for 30 min. After collection and PBS washing, the cells were resuspended and stained with 7-AAD dye (BioLegend, 420403), and then detected by flow cytometry.

[0065] The experimental results showed that the combination of LPS-IFNγ significantly up-regulated the proportion of the CD103+CD11B- subpopulation (i.e. classical dendritic cells cDC1) in DC2.4 cells, and down-regulated the proportion of the CD103-CD11B+ subpopulation (cDC2). After C36L1 intervention, the up-regulation of the proportion of cDC1 induced by LPS-FNγ was inhibited, and the proportion of cDC2 was slightly up-regulated. See Figure 2. Figure 5 .

[0066] Example 7

[0067] Verification of the expression of transcription factors by qRT-PCR method

[0068] When the cell confluence in the culture flask reached 70-80%, the cells were seeded into a 24-well plate at a density of 10W / well. After 24 hours of culture, the cells were induced by different interventions, and the specific grouping is shown in Example 3. After 24 hours, all the cells in the wells were collected. Blocking Fc receptors (BD Pharmingen, 553142) was used for incubation at room temperature for 10 minutes. PE / Cyanine7 anti-mouse CD103 (BioLegend, 121425) and FITC anti-mouse / human CD11b (BioLegend, 101205) were used for incubation at 2-8°C for 30 min. After collection and PBS washing, the cells were resuspended and stained with 7-AAD dye (BioLegend, 420403), and then detected by flow cytometry.

[0069] 1. Total RNA extraction

[0070] 1) Add 1 mL TRIzol (Invitrogen, 15596026) to each EP tube, and let it stand at room temperature for 5 min;

[0071] 2) Add 100 μL of chloroform substitute (Sevyl, G3014) to each EP tube, and operate in a fume hood under light protection, shake vigorously for 15 s, stand at room temperature for 2-3 min, centrifuge at 4°C at 12000 g for 15 min;

[0072] 3) Carefully pipette the supernatant into a new EP tube;

[0073] 4) Add equal volume of isopropanol to each EP tube, mix well by inverting the tube, let it stand at room temperature for 2-3 min, centrifuge at 12000g for 15 min at 4°C;

[0074] 5) Carefully pipette the supernatant, add 500μL of absolute ethanol to each EP tube, mix well by pipetting, centrifuge at 5000g for 5 min at 4°C;

[0075] 6) Carefully pipette the supernatant, add 10-20μL of RNase-free water to each EP tube, after the precipitate is completely dissolved, take 1μL of sample to spectrophotometer, get the sample concentration and A260 / A280 value. A260 / A280 value should be between 1.8-2.0. If not used immediately, store at -80°C refrigerator for later use

[0076] 2. Reverse transcription

[0077] According to the instruction of PrimeScript TM RT Master Mix (Takara, RR036Q), reverse transcribe RNA to generate cDNA.

[0078]

[0079] Run the machine at 37°C for 15 min, 85°C for 5 s, 4°C.

[0080] 3. RT-PCR reaction

[0081] According to the instruction of TB Premix Ex Taq TM (Takara, RR820Q), perform RT-PCR reaction.

[0082] Reagent Amount Final concentration TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) 10 μL 1X PCR Forward Primer (10 μM) 0.8 μL 0.4 μM PCR Reverse Primer (10 μM) 0.8 μL 0.4 μM ROX Reference Dye II (50X) 0.4 μL 1X RT reaction solution 2 μL Sterile water 6 μL Total 20 μL

[0083] The primer sequence (BioTNT) is as follows:

[0084]

[0085] The experimental results show that LPS-IFNγ combined treatment significantly up-regulates the mRNA expression levels of BATF3 and IRF8 in DC2.4 cells, and IRF4 shows a downward trend but does not reach statistical significance; after C36L1 intervention, the up-regulation of BATF3 and IRF8 induced by LPS / IFNγ is specifically inhibited, but the expression of IRF4 is not significantly affected, see the following table: Figure 6 .

[0086] Example 8

[0087] In vivo experiment to evaluate the effect of CD74 inhibitors

[0088] 1. Constructing the animal model of vitiligo: 10 C57BL / 6 female mice aged 7 weeks were subcutaneously injected with melanoma cells B16F10 on the right lower back, and then anti-CD4 antibody (BioXcell, BE0003) was intraperitoneally injected at a dose of 10 ug / g on the 4th day and the 10th day, respectively. The melanoma was surgically removed after 12 days. The operation was continued to be observed for 14 days, and the animals with white hair on the operation site were selected as the successful vitiligo mice, i.e. 8 mice were divided into 2 groups.

[0089] 2. Synthesis of CD74 small molecule peptide inhibitor C36L1 (Shanghai Biotech). Starting from the 15th day after the operation, 2 vitiligo mice were intraperitoneally injected with pure water and C36L1, respectively, 3 times a week for 5 weeks. The depigmentation area of the hair of the mice in the drug administration group was significantly smaller than that in the model group, and the mouse phenotype pictures are shown in the attached Figure 7 .

[0090] Based on the analysis of the above research results, it is shown that CD74 on the surface of DCs plays an important role in the pathogenesis of vitiligo. CD74 receptor protein is a key molecule that mediates the inflammatory activation and differentiation of DCs, and drives the abnormal activation and migration function of DCs by promoting the expression of pro-inflammatory maturation markers (CD86, CCR7) and the up-regulation of cDC1 subpopulation-specific transcription factors (BATF3, IRF8). Its inhibitor can significantly weaken the over-activation of the cDC1 subpopulation induced by LPS / IFNγ by targeting the protein, and provides a molecular target for intervening the over-activated Th1 / CTL immune response in vitiligo.

Claims

1. The receptor protein CD74 is used as a molecular marker for the diagnosis or treatment of vitiligo.

2. The receptor protein CD74 serves as a therapeutic target.

3. Application of reagents for detecting the expression level of the CD74 gene or its encoded protein in the preparation of products for the diagnosis of vitiligo.

4. The application according to claim 3, characterized in that, The product is used to detect the expression level of the CD74 gene or its encoded protein in samples by real-time quantitative PCR, microarray, high-throughput sequencing platform, flow cytometry, immunohistochemical staining or enzyme-linked immunosorbent assay.

5. The application according to claim 3, characterized in that, The product contains specific primers for amplifying the CD74 gene, probes for hybridizing with the nucleotide sequence of the CD74 gene, or antibodies that specifically bind to the CD74 protein.

6. The application according to claim 5, characterized in that, The antibody is a monoclonal antibody or a polyclonal antibody.

7. The application according to claim 3, characterized in that, The product is a chip, formulation, or reagent kit.

8. The application according to claim 4, characterized in that, The sample may be tissue, serum, or cells.

9. The application of an inhibitor of CD74 expression level in the preparation of drugs for treating vitiligo.

10. The application according to claim 9, characterized in that, The inhibitors include CD74 antagonists.

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