Application of chemokine ligand 19 inhibitor capable of treating systemic sclerosis

Abstract

The invention relates to application of a chemokine ligand 19 inhibitor capable of treating systemic sclerosis, and belongs to the technical field of biological medicine. The invention provides application of a chemokine ligand 19 inhibitor in preparation of a medicine for preventing and / or treating systemic sclerosis. Researches show that the chemotactic factor ligand 19 can promote immune cell infiltration in the skin by recruiting CCR7 + T cells in peripheral blood, aggravate skin fibrosis, and also can promote the immune cell infiltration in the skin by recruiting CCR7 + T cells in the peripheral blood. The anti-chemokine ligand 19 monoclonal antibody has a remarkable treatment effect on systemic sclerosis (specifically, the anti-chemokine ligand 19 monoclonal antibody can relieve infiltration of immune cells in skin of a systemic sclerosis modeling mouse and relieve the fibrosis degree of the skin of the systemic sclerosis modeling mouse by inhibiting expression of the chemokine ligand 19 in fibroblasts). Therefore, the chemokine ligand 19 can be used as a new target for preventing and treating the systemic sclerosis, and has a great application prospect in preparation of medicines for preventing and / or treating the systemic sclerosis.

Description

Technical Field

[0001] The invention relates to the application of a chemokine ligand 19 inhibitor capable of treating systemic sclerosis, and belongs to the technical field of biomedicine. Background Art

[0002] Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive fibrosis caused by excessive deposition of extracellular matrix (ECM) in the skin and internal organs. It is also the prototype of various fibrotic diseases. Since fibrosis is an irreversible global problem and there is no specific treatment, systemic sclerosis will cause irreversible fibrosis and dysfunction of multiple organs such as the skin, blood vessels, lungs, heart, kidneys, digestive tract, liver, etc., which will gradually worsen, causing great pain to patients.

[0003] At present, little is known about the cellular and molecular mechanisms of pathological regulation of systemic sclerosis. Therefore, existing non-specific treatments for systemic sclerosis often lead to extensive killing of body cells and subsequent adverse reactions. Statistics show that systemic sclerosis is the autoimmune disease with the highest mortality rate, with a five-year survival rate of 74.9% and a 10-year survival rate of 62.5%. The quality of life of patients is extremely poor. In order to truly control systemic sclerosis, an in-depth understanding of its precise pathogenic cells and molecular pathological mechanisms is key. Current studies have shown that in the pathogenesis of systemic sclerosis, the most core pathological mechanism is that fibroblasts are regulated to develop into pathogenic subpopulations that promote fibrosis, and excessive secretion of extracellular matrix leads to irreversible fibrosis.

[0004] Fibroblasts are polymorphic cells with multiple physiological functions in organ morphogenesis, tissue homeostasis, and immune response. In fibrotic diseases represented by systemic sclerosis, quiescent fibroblasts develop into profibrotic phenotypes, differentiate and synthesize a large amount of extracellular matrix, leading to fibrosis and organ failure, and are the main pathogenic cells. In the past, it was believed that fibroblasts were a group of spindle cells with highly homogeneous functions and origins, and were not divided into multiple subpopulations like immune cells. However, with the maturity and application of technologies such as lineage tracing and single-cell high-throughput sequencing, more and more evidence shows that fibroblasts have multiple characteristics, and each subpopulation of cells has different markers and functions, but they are still poorly understood and there is no recognized classification. In the process of fibrosis, the fibroblasts that play a pathogenic role are likely to be only a small part of them, and the mechanism that drives fibroblasts to differentiate into pathogenic cells is still unclear. If the mechanism that drives fibroblasts to differentiate into pathogenic cells can be accurately analyzed, it is critical for the prevention and treatment of systemic sclerosis.

[0005] Chemokine ligand 19 (CCL19) is a small molecule cytokine belonging to the CC chemokine family. It exerts multiple biological functions by binding to the chemokine receptor CCR7. At present, there are no reports on the involvement of chemokine ligand 19 in the development of systemic sclerosis by driving fibroblasts to develop into profibrotic pathogenic subpopulations, and there are no reports on the use of chemokine ligand 19 inhibitors to prevent and treat systemic sclerosis. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of a chemokine ligand 19 inhibitor in the preparation of a drug for preventing and / or treating systemic sclerosis.

[0007] In one embodiment of the present invention, the prevention and / or treatment of systemic sclerosis includes reducing the infiltration of immune cells in the skin of systemic sclerosis patients and / or reducing the degree of fibrosis in the skin of systemic sclerosis patients.

[0008] In one embodiment of the present invention, the prevention and / or treatment of systemic sclerosis includes reducing the infiltration of immune cells in the skin of systemic sclerosis patients and reducing the degree of skin fibrosis in systemic sclerosis patients by inhibiting the expression of chemokine ligand 19 in fibroblasts.

[0009] In one embodiment of the present invention, reducing the infiltration of immune cells in the skin of systemic sclerosis patients includes reducing the infiltration of T cells in the skin of systemic sclerosis patients; reducing the degree of fibrosis in the skin of systemic sclerosis patients includes reducing the skin thickness of systemic sclerosis patients and / or reducing the content of type I collagen in the skin of systemic sclerosis patients.

[0010] In one embodiment of the present invention, the T cells include CD3 + T cells and / or CCR7 + CD3 + T cells.

[0011] In one embodiment of the present invention, the chemokine ligand 19 inhibitor comprises an anti-chemokine ligand 19 antibody and / or a siRNA targeting a gene encoding chemokine ligand 19.

[0012] In one embodiment of the present invention, the chemokine ligand 19 inhibitor comprises an antibody and / or siRNA capable of inhibiting the expression of chemokine ligand 19 in fibroblasts.

[0013] In one embodiment of the present invention, the anti-chemokine ligand 19 antibody comprises an anti-chemokine ligand 19 monoclonal antibody.

[0014] In one embodiment of the present invention, the drug comprises a subcutaneous injection drug.

[0015] In one embodiment of the present invention, the drug comprises a chemokine ligand 19 inhibitor and a pharmaceutically acceptable excipient.

[0016] In one embodiment of the present invention, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0017] The present invention also provides a drug for preventing and / or treating systemic sclerosis, wherein the drug comprises a chemokine ligand 19 inhibitor and pharmaceutically acceptable excipients.

[0018] In one embodiment of the present invention, the prevention and / or treatment of systemic sclerosis includes reducing the infiltration of immune cells in the skin of systemic sclerosis patients and / or reducing the degree of fibrosis in the skin of systemic sclerosis patients.

[0019] In one embodiment of the present invention, the prevention and / or treatment of systemic sclerosis includes reducing the infiltration of immune cells in the skin of systemic sclerosis patients and reducing the degree of skin fibrosis in systemic sclerosis patients by inhibiting the expression of chemokine ligand 19 in fibroblasts.

[0020] In one embodiment of the present invention, reducing the infiltration of immune cells in the skin of systemic sclerosis patients includes reducing the infiltration of T cells in the skin of systemic sclerosis patients; reducing the degree of fibrosis in the skin of systemic sclerosis patients includes reducing the skin thickness of systemic sclerosis patients and / or reducing the content of type I collagen in the skin of systemic sclerosis patients.

[0021] In one embodiment of the present invention, the T cells include CD3 + T cells and / or CCR7 + CD3 + T cells.

[0022] In one embodiment of the present invention, the chemokine ligand 19 inhibitor comprises an anti-chemokine ligand 19 antibody and / or a siRNA targeting a gene encoding chemokine ligand 19.

[0023] In one embodiment of the present invention, the chemokine ligand 19 inhibitor comprises an antibody and / or siRNA capable of inhibiting the expression of chemokine ligand 19 in fibroblasts.

[0024] In one embodiment of the present invention, the anti-chemokine ligand 19 antibody comprises an anti-chemokine ligand 19 monoclonal antibody.

[0025] In one embodiment of the present invention, the drug comprises a subcutaneous injection drug.

[0026] In one embodiment of the present invention, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0027] The present invention also provides a molecular marker for diagnosing systemic sclerosis, wherein the molecular marker comprises chemokine ligand 19-positive fibroblasts.

[0028] In one embodiment of the present invention, the diagnosis includes assessing the severity of systemic sclerosis. The greater the number of chemokine ligand 19-positive fibroblasts in the skin tissue of a systemic sclerosis patient, the higher the infiltration degree of immune cells in the skin of the systemic sclerosis patient, and the higher the severity of the disease in the systemic sclerosis patient.

[0029] The present invention also provides the use of a reagent for detecting the above-mentioned molecular markers in a sample to be tested in the preparation of a product for diagnosing systemic sclerosis.

[0030] In one embodiment of the present invention, the diagnosis includes assessing the severity of systemic sclerosis. The greater the number of chemokine ligand 19-positive fibroblasts in the skin tissue of a systemic sclerosis patient, the higher the infiltration degree of immune cells in the skin of the systemic sclerosis patient, and the higher the severity of the disease in the systemic sclerosis patient.

[0031] In one embodiment of the present invention, the product comprises a detection kit.

[0032] The present invention also provides a product for diagnosing systemic sclerosis, which comprises a reagent for detecting the above-mentioned molecular markers in a sample to be tested.

[0033] In one embodiment of the present invention, the diagnosis includes assessing the severity of systemic sclerosis. The greater the number of chemokine ligand 19-positive fibroblasts in the skin tissue of a systemic sclerosis patient, the higher the infiltration degree of immune cells in the skin of the systemic sclerosis patient, and the higher the severity of the disease in the systemic sclerosis patient.

[0034] In one embodiment of the present invention, the product comprises a detection kit.

[0035] The technical solution of the present invention has the following advantages: 1. The present invention provides the use of chemokine ligand 19 inhibitors in the preparation of drugs for preventing and / or treating systemic sclerosis. Studies have shown that chemokine ligand 19 can promote the infiltration of immune cells in the skin and aggravate skin fibrosis by recruiting CCR7+T cells in peripheral blood; at the same time, studies have shown that anti-chemokine ligand 19 monoclonal antibodies have significant therapeutic effects on systemic sclerosis (specifically, they can inhibit the expression of chemokine ligand 19 in fibroblasts, reduce the infiltration of immune cells in the skin of systemic sclerosis model mice, and reduce the degree of fibrosis in the skin of systemic sclerosis model mice). Therefore, chemokine ligand 19 can be used as a new target for the prevention and treatment of systemic sclerosis, and chemokine ligand 19 inhibitors have great application prospects in the preparation of drugs for preventing and / or treating systemic sclerosis.

[0036] 2. The present invention provides the use of a reagent for detecting chemokine ligand 19-positive fibroblasts in a sample to be tested in the preparation of a product for diagnosing systemic sclerosis. In keloids, a group of fibroblasts expressing inflammatory response and cell chemotaxis gene characteristics have been identified in normal and fibrotic dermis. In addition, the pro-inflammatory phenotype of fibroblasts promotes leukocyte recruitment and aggravates immune response during the repair of reindeer fibrotic skin. Based on the above content, further studies have shown that the number of chemokine ligand 19-positive fibroblasts in the skin tissue of patients with systemic sclerosis is significantly increased compared with healthy controls, and is positively correlated with the infiltration of immune cells and the severity of the disease; at the same time, further studies have shown that chemokine ligand 19 can promote the infiltration of immune cells in the skin by recruiting CCR7+T cells in peripheral blood, aggravating skin fibrosis. Therefore, chemokine ligand 19-positive fibroblasts can be used as molecular markers for the preparation of products (e.g., detection kits) for diagnosing systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Schematic diagram of the process of animal experiments.

[0038] Figure 2 : Representative HE staining and Masson staining images of mouse skin tissues in each group.

[0039] Figure 3 : Quantitative analysis results of skin thickness of mice in each group.

[0040] Figure 4 : Quantitative analysis results of the percentage of collagen volume fraction in the skin tissue of each group of mice to the total skin volume.

[0041] Figure 5 : Relative mRNA expression of Col1a1 gene (which encodes the pre-α1 chain of type I collagen) in the skin tissues of mice in each group.

[0042] Figure 6 : Representative staining images of CD3, F4 / 80 and CCR7 in the skin tissues of mice in each group.

[0043] Figure 7 :CD3 in the skin tissue of mice in each group + T cells and F4 / 80 + The proportion of macrophages.

[0044] Figure 8 :CD3 in the skin tissue of mice in each group + T cells and CCR7 + CD3 + The proportion of T cells.

[0045] Fig. 9 : Representative staining images of chemokine ligand 19 in skin tissues of 21 patients with systemic sclerosis and 12 healthy controls.

[0046] Fig.10 : Changes in the proportion of chemokine ligand 19-positive fibroblasts between 21 systemic sclerosis patients and 12 healthy controls.

[0047] Fig.11 : Correlation between chemokine ligand 19 expression and modified Rodnan skin score (mRSS) in the skin of patients with systemic sclerosis. DETAILED DESCRIPTION

[0048] The following experimental examples are provided for a better understanding of the present invention, but are not limited to the best implementation mode described, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the present invention with the features of other prior arts shall fall within the protection scope of the present invention.

[0049] If no specific experimental steps or conditions are specified in the following experimental examples, the conventional experimental steps or conditions described in the literature in this field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0050] Experimental Example 1: Effects of chemokine ligand 19 inhibitor on systemic sclerosis model mice In this study, the experimental animals were 8-week-old female C57BL / 6J wild-type mice, which were supplied by Beijing Weitongda Company. All mice were housed in autoclaved cages and provided with sterile food and water. All animal experiments were performed in accordance with the ethical guidelines of the Peking University Animal Care and Use Committee.

[0051] The specific steps of the experiment are as follows: The mice were divided into three groups: anti-chemokine ligand 19 (CCL19) antibody treatment group, isotype control antibody treatment group, and PBS control group, with 4 mice in each group. After the grouping, the mice in different groups were treated as follows (see the treatment process Figure 1 ): Anti-CCL19 antibody treatment group: A 1 cm × 1 cm area was taken from the back of the mice, the hair was shaved, and 300 μL of bleomycin solution (concentration 0.5 mg / mL, solvent PBS buffer) was subcutaneously injected into the skin every other day, and anti-chemokine ligand 19 monoclonal antibody (injection dose 50 μg / kg, with 100 μL PBS buffer as solvent) (antibody model AF880, purchased from R&D) was subcutaneously injected once every four days, continuing until the 14th day.

[0052] Isotype control antibody treatment group: A 1 cm × 1 cm area was taken from the back of the mice, the hair was shaved, and 300 μL of bleomycin solution (concentration 0.5 mg / mL, solvent PBS buffer) was subcutaneously injected into the skin every other day, and the isotype control antibody of anti-chemokine ligand 19 monoclonal antibody (injection dose 50 μg / kg, with 100 μL PBS buffer as solvent) (antibody model AF880, purchased from R&D) was subcutaneously injected once every four days, continuing until the 14th day.

[0053] PBS control group: A 1 cm × 1 cm area was taken from the back of the mouse, the hair was shaved, and 300 μL PBS buffer (purchased from Solebow Biotechnology Co., Ltd.) was subcutaneously injected into the skin every other day until the 14th day.

[0054] After treatment, the mice were killed and the injured skin was collected for histological analysis, multiple immunohistochemical staining and qRT-PCR assay. The experimental results are shown in Figure 2~Figure 8 .

[0055] Among them, the histological analysis is as follows: (1) Paraffin embedding of mouse skin tissue: The removed mouse skin tissue was immersed in a 4% (w / v, g / 100 mL) paraformaldehyde aqueous solution for 24 hours for fixation, then the mouse skin tissue was removed from the paraformaldehyde aqueous solution, placed in a fume hood and smoothed with a scalpel to obtain fixed mouse skin tissue; the fixed mouse skin tissue was immersed in 70%, 80%, 90%, 95%, 100%, and 100% (v / v) alcohol solutions for 2 hours in sequence for dehydration, then the mouse skin tissue was removed from the alcohol solution to obtain dehydrated mouse skin tissue. Tissue; after the dehydrated mouse skin tissue is immersed in a transparent agent xylene for 10 minutes to remove the alcohol (the removal process is performed twice), the mouse skin tissue is taken out from the xylene to obtain the cleaned mouse skin tissue; the cleaned mouse skin tissue is placed in melted paraffin, and after the paraffin penetrates into the mouse skin tissue, the mouse skin tissue is placed in an embedding machine, and the melted paraffin is first poured into the embedding frame, and the mouse skin tissue is placed in the embedding frame before the paraffin solidifies and a corresponding label is attached, and after the paraffin is cooled and solidified, the wax block is taken out and trimmed with a scalpel to obtain the embedded mouse skin tissue.

[0056] (2) HE staining: The embedded mouse skin tissue was cut into 5 μm thick tissue sections using a microtome; the tissue sections were placed in a 60°C oven for 2 hours to ensure that the sections were tightly attached to the glass slides to obtain baked tissue sections; the baked tissue sections were immersed in xylene for 10 minutes to remove the paraffin in the sections (the removal process was performed twice) to obtain dewaxed tissue sections; the dewaxed tissue sections were immersed in a 95% (v / v) alcohol solution for 5 minutes to remove the xylene (the removal process was performed twice) to obtain washed tissue sections; the washed tissue sections were transferred to a hematoxylin stain solution (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and immersed for 10 minutes for staining, and then rinsed with clean water until the water flow was clear to obtain a tissue section that had been stained once; the tissue sections that had been stained once were immersed in ethanol containing 0.7% (v / v) hydrochloric acid The differentiated tissue sections were transferred to 95% (v / v) alcohol solution and alcohol eosin (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and immersed for 30 seconds for staining, and then rinsed with water to obtain secondary stained tissue sections; the secondary stained tissue sections were transferred to 95% and 100% (v / v) alcohol solutions and immersed for 2 hours for dehydration to obtain dehydrated tissue sections; the dehydrated tissue sections were immersed in xylene for 10 minutes to remove the alcohol, and then sealed with neutral gum to obtain HE stained sections; the HE stained sections were observed under a microscope, and the skin thickness of each group of mice was quantitatively analyzed based on the observation results of the HE stained sections, and the dermal thickness was defined as the distance from the epidermal-dermal junction to the dermal-subcutaneous junction.

[0057] (3) Masson staining: Use a microtome to cut the embedded mouse skin tissue into 5 μm thick tissue sections; bake the tissue sections in a 60°C oven for 2 hours to ensure that the sections are tightly attached to the slides to obtain baked tissue sections; soak the baked tissue sections in xylene for 10 minutes to remove the paraffin in the sections (the removal process is performed twice) to obtain dewaxed tissue sections; soak the dewaxed tissue sections in a 95% (v / v) alcohol solution for 5 minutes to remove the xylene (the removal process is performed twice). The washed tissue sections were transferred to Weigert iron hematoxylin staining solution (Weigert iron hematoxylin staining solution, purchased from Solebow Biotechnology Co., Ltd., A solution and B solution were mixed in equal volumes before use) and soaked for 10 minutes for staining, and then rinsed with clean water until the water flow was clear to obtain tissue sections that had been stained once; the tissue sections that had been stained once were soaked in ethanol (blueing solution) containing 1% (v / v) hydrochloric acid for 10 seconds for differentiation, and then rinsed with clean water to obtain differentiated tissue sections; the differentiated tissue sections were transferred to After being stained with acidic fuchsin-ponceau solution (purchased from Solebao Biotechnology Co., Ltd.) for 5 minutes, the sections were first rinsed with clean water and then rinsed with a 0.1% (v / v) acetic acid aqueous solution to obtain a second-stained tissue section; the second-stained tissue section was transferred to a phosphomolybdic acid solution (purchased from Solebao Biotechnology Co., Ltd.) and soaked for 5 minutes for staining (observing the tissue differentiation into different degrees of red under a microscope), and then rinsed with a 0.1% (v / v) acetic acid aqueous solution to obtain a third-stained tissue section; the third-stained tissue section was transferred to a phosphomolybdic acid solution (purchased from Solebao Biotechnology Co., Ltd.) and soaked for 5 minutes for staining (observing the tissue differentiation into different degrees of red under a microscope), and then rinsed with a 0.1% (v / v) acetic acid aqueous solution to obtain a third-stained tissue section; The slides were transferred to aniline blue solution (purchased from Solebow Biotechnology Co., Ltd.) and soaked for 5 minutes for staining, and then rinsed with 0.2% (v / v) acetic acid aqueous solution to obtain tissue sections stained four times; the tissue sections stained four times were transferred to 100% (v / v) anhydrous alcohol and soaked for 2 hours for dehydration to obtain dehydrated tissue sections; the dehydrated tissue sections were soaked in xylene for 10 minutes to remove the alcohol, and then sealed with neutral gum to obtain Masson-stained sections; the Masson-stained sections were observed under a microscope, and the Masson-stained sections were scanned using a NanoZoomer S360 full-slide scanner, and the scanned images were analyzed using qupath software to analyze the percentage of fibrosis area / tissue area.

[0058] Multiple immunohistochemical staining was performed as follows: The embedded mouse skin tissue was cut into tissue sections with a thickness of 5 μm using a microtome; the tissue sections were baked in a 60°C oven for 2 hours to ensure that the sections were tightly attached to the glass slides to obtain baked tissue sections; the baked tissue sections were immersed in xylene for 10 minutes to remove the paraffin in the sections (the removal process was performed twice) to obtain dewaxed tissue sections. After the dewaxed tissue sections were immersed in citric acid buffer (purchased from Solebao Biotechnology Co., Ltd.), they were placed in a microwave oven and heated at high temperature (100°C) for 15 minutes for antigen repair to obtain antigen-repaired tissue sections; after the antigen-repaired tissue sections were naturally cooled to room temperature (25°C), they were first washed with TBST buffer solution (purchased from Solebao Biotechnology Co., Ltd.) for 5 minutes, and then transferred to a 5% (w / v, g / 100mL) bovine serum albumin aqueous solution and incubated at room temperature (25°C) for 10 minutes for blocking to obtain blocked tissue sections; after the blocked tissue sections were transferred to the first primary antibody (CD3 was used as the first primary antibody, the primary antibody model was ab828, purchased from abcam, and used after dilution 100 times with PBS buffer) and incubated at 37°C for 1 hour, they were washed with TBST buffer solution for 5 minutes (the washing process was performed three times) to obtain tissue sections incubated with the primary antibody; the tissue sections incubated with the primary antibody were transferred to the Opal Multiplex mIHC kit (Akoya Biosciences, After incubation at 37°C for 10 minutes in the secondary antibody provided by Akoya Biosciences (USA), the sections were washed with TBST buffer solution for 5 minutes (the washing process was performed three times) to obtain tissue sections incubated with the secondary antibody; the tissue sections incubated with the secondary antibody were transferred to the Opal 520 fluorescein working solution provided by the Opal Multiplex mIHC Kit (Akoya Biosciences, USA) (the working solution was diluted 100 times with PBS buffer before use) and incubated at room temperature (25°C) for 10 minutes for fluorescent labeling, and then washed with TBST buffer solution for 5 minutes (the washing process was performed three times) to obtain tissue sections labeled once. F4 / 80 was used as the second primary antibody (primary antibody model ab300421, purchased from abcam, diluted 1000 times with PBS buffer before use), and Opal 570 fluorescein working solution provided by the Opal Multiplex mIHC Kit was used for fluorescent labeling, and the above-mentioned antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation and fluorescent labeling processes were repeated to obtain tissue sections labeled twice.CCR7 was used as the third primary antibody (primary antibody model ab258137, purchased from abcam, and used after dilution 200 times with PBS buffer), and Opal 690 fluorescein working solution provided by the Opal Multiplex mIHC kit was used for fluorescent labeling. The above antigen repair, blocking, primary antibody incubation, secondary antibody incubation and fluorescent labeling process were repeated to obtain three-time labeled tissue sections. The three-time labeled tissue sections were transferred to the DAPI stain provided by the Opal Multiplex mIHC kit and incubated at room temperature (25°C) for 5 minutes for staining, and then washed with TBST buffer solution for 5 minutes (the washing process was operated three times), and then covered with anti-fluorescence quenching mounting medium (purchased from Solebao Biotechnology Co., Ltd.) to obtain multiple immunohistochemical staining sections; the multiple immunohistochemical staining sections were scanned using the Vectra Ploris system to capture and analyze multi-spectral immunofluorescence images, using the fluorescence spectrum with a wavelength from 480 nanometers to 780 nanometers, and the fluorescence intensity information was obtained from the image, and the obtained images were analyzed using qupath software for fluorescence image analysis.

[0059] The qRT-PCR assay was performed as follows: (1) Take 50 mg of skin tissue, cut it into pieces, grind it, and add 1 mL TRIzol (purchased from Tiangen Biochemical Technology Co., Ltd.) was repeatedly blown several times until the solution became transparent to obtain a homogenate sample; the homogenate sample was first placed at room temperature (25°C) for 5 minutes to completely separate the nucleic acid-protein complex, and then centrifuged at 4°C and 12,000rpm for 5 minutes. After the centrifugation, the supernatant was transferred to a new RNase-free centrifuge tube; after adding 200μL of chloroform (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.) to the centrifuge tube, the tube was covered and then violently shaken for 15 minutes, and then allowed to stand at room temperature (25°C) for 3 minutes. After the standing period, it was centrifuged at 4°C and 12,000rpm for 10 minutes (the sample in the tube was divided into three layers at this time: the bottom yellow organic phase, the middle layer, and the upper colorless aqueous phase, and RNA was mainly in the aqueous phase). After the centrifugation, the aqueous phase was transferred to a new RNase-free centrifuge tube; after adding 500μL of anhydrous ethanol to the centrifuge tube, it was first blown to mix, and then transferred to the adsorption column CR3 (purchased from Tiangen Biochemical Technology Co., Ltd.), and then centrifuged at 4°C and 12,000rpm for 1 minute. After the centrifugation, the waste liquid in the collection tube was discarded; 500μL of deproteinization solution RD (purchased from Tiangen Biochemical Technology Co., Ltd., ethanol was added before use) was added to the adsorption column, and centrifuged at 4°C and 12,000rpm for 1 minute. After the centrifugation, the waste liquid in the collection tube was discarded; 500μL of rinsing solution RW (purchased from Tiangen Biochemical Technology Co., Ltd., ethanol was added before use) was added to the adsorption column, and centrifuged at 4°C and 12,000rpm for 1 minute. After the centrifugation, the waste liquid in the collection tube was discarded. This operation was repeated twice; the adsorption column was centrifuged at 4°C and 12,000rpm for 2 minutes. After the centrifugation, the waste liquid in the collection tube was discarded; the adsorption column was placed on a clean bench for ventilation for 3 minutes, and after being fully dried, it was transferred to a new 1.5mL RNase-free centrifuge tube; 35μL After adding RNase-Free ddH2O (purchased from Tiangen Biochemical Technology Co., Ltd.), place it at room temperature (25°C) for 2 minutes, and then centrifuge it at 4°C and 12,000 rpm for 2 minutes. The liquid obtained by centrifugation is the RNA solution.

[0060] (2) Reverse transcription of RNA solution was performed using reverse transcription reagent (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number AT311-03). The specific process is as follows: prepare the reverse transcription reaction system in a 250 μL PCR tube according to the formula in Table 1; place the PCR tube in an Applied Biosystems PCR instrument and react at 65°C for 5 minutes. After the reaction is completed, take out the PCR tube and cool it on ice for 5 minutes to obtain the reverse transcription product; prepare the enzyme mixture according to the formula in Table 2, take 8 μL of the enzyme mixture and add it to the reverse transcription product to obtain a cDNA sample.

[0061] (3) RT-PCR reagent (purchased from Acryl, catalog number AG11701) was used to perform RT-PCR on the test samples to detect the relative mRNA expression level of Col1a1 in each group of mouse tissues. The specific process is as follows: the RT-PCR reaction system was prepared in a 250 μL PCR tube according to the formula in Table 3 (primers used are shown in Table 4); the PCR tube was placed in the CFX384 Touch™ fluorescent quantitative PCR detection system for PCR amplification (amplification program: 95°C pre-denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds, and 40 cycles); after the amplification, the Gapdh gene was used as the internal reference gene for data normalization, and the qPCR data was analyzed by the ΔΔCt method to calculate the relative expression of the Col1a1 gene (three replicates were set for all samples in the experiment).

[0062] The experimental results are as follows: Bleomycin was used to induce systemic sclerosis modeling. Mice received subcutaneous injections of bleomycin from the first day of the experiment, and continued every other day until the mice were killed on the 14th day to observe the pathological changes of skin fibrosis in the mice. In the treatment group, anti-CCL19 antibody or isotype control antibody was subcutaneously injected from the second day, and then injected every four days, that is, repeated injections on the 6th, 10th and 14th days ( Figure 1 ) to observe the therapeutic effect of anti-CCL19 antibody on skin fibrosis, the main pathological feature of systemic sclerosis.

[0063] Among them, after 14 days of continuous treatment, a detailed evaluation of skin fibrosis in the systemic sclerosis mouse model was conducted, and it was found that the mice group using anti-CCL19 antibody had significantly reduced skin fibrosis compared with the mice group receiving isotype control antibody. First, compared with the isotype control antibody group, the skin thickness of the mice in the anti-CCL19 antibody group was significantly reduced ( Figure 2 , Figure 3 ); Secondly, the amount of collagen deposition in the dermis was significantly reduced ( Figure 2 , Figure 4 ); Finally, the skin Col1a1 The relative mRNA expression levels of genes were also significantly reduced ( Figure 5 ), these results indicate the efficacy of anti-CCL19 antibodies in inhibiting skin fibrosis.

[0064] Another important finding was that CCR7 in the skin of mice with systemic sclerosis who received bleomycin injections was significantly higher than that in mice who received PBS buffer injections. + T cell infiltration increased significantly ( Figure 6 , Figure 7 ), which suggests that CCR7 +T cells may play a role in the process of skin fibrosis. Experimental data also showed that CCR7 + The number of macrophages did not change significantly ( Figure 6 , Figure 7 ), suggesting that it plays a minor role in skin fibrosis. Further studies have shown that treatment with anti-CCL19 antibodies can significantly reduce T cells and CCR7 in the skin of a mouse model of bleomycin-induced systemic sclerosis. + Infiltration of T cells ( Figure 6 , Figure 8 ).

[0065] These experimental results confirmed that anti-CCL19 antibodies can reduce CCR7 + Skin infiltration of T cells and treatment of bleomycin-induced skin fibrosis, the main pathological feature of systemic sclerosis.

[0066] Table 1 Reverse transcription reaction system formula

[0067] Table 2 Enzyme mixture formulation

[0068] Table 3 Formula of RT-PCR reaction system

[0069] Table 4 Primers and their sequences

[0070] Experimental Example 2: Detection of changes in the proportion of CCL19-positive fibroblasts in skin tissues of systemic sclerosis patients and healthy controls The specific steps of the experiment are as follows: According to the multiple immunohistochemical staining method of Experimental Example 1, the changes in the proportion of CCL19-positive fibroblasts in the skin tissues of 21 patients with systemic sclerosis and 12 healthy controls matched in age and gender were detected to obtain multiple immunohistochemical staining sections; the multiple immunohistochemical staining sections were scanned using the Vectra Ploris system to capture and analyze multi-spectral immunofluorescence images, and the fluorescence intensity information was obtained from the images using a fluorescence spectrum with a wavelength ranging from 480 nanometers to 780 nanometers. The obtained images were analyzed using qupath software for fluorescence image analysis. The staining and analysis results are shown in Figure 9~Figure 10During the staining process, CCL19 was used as the first primary antibody (primary antibody model MAB361, purchased from R&D Company, USA, and diluted 200 times with PBS buffer before use), and the Opal 520 fluorescein working solution provided by the Opal Multiplex mIHC kit was used as a fluorescent marker, and vimentin was used as the second primary antibody (primary antibody model ab92547, purchased from abcam, and diluted 200 times with PBS buffer before use), and the Opal 690 fluorescein working solution provided by the Opal Multiplex mIHC kit was used as a fluorescent marker.

[0071] According to the staining and analysis results, the Spearman correlation analysis method was used to analyze the correlation between the expression of CCL19 in the skin of patients with systemic sclerosis and the modified Rodnan skin score (mRSS). The analysis results are shown in Fig.11 .

[0072] The experimental results are as follows: The staining results showed that the proportion of CCL19-positive fibroblasts in the skin tissue of patients with systemic sclerosis was significantly higher than that in the healthy control group ( Fig. 9 , Fig.10 ). Through correlation analysis, it was found that the expression level of CCL19 in the skin tissue of patients with systemic sclerosis was significantly positively correlated with the skin severity score (modified Rodnan skin score) of patients with systemic sclerosis ( Fig.11 ). The above results suggest that the level of CCL19 and the proportion of CCL19-positive fibroblasts can be used as indicators to assess the severity and progression of systemic sclerosis patients. In addition, the detection of CCL19 and the proportion of CCL19-positive fibroblasts may provide a new method for early diagnosis and monitoring of SSc, help to better understand the pathological mechanism of the disease, and provide a basis for the formulation of personalized treatment strategies.

[0073] Obviously, the above experimental examples are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. Use of a chemokine ligand 19 inhibitor in the preparation of a drug for preventing and / or treating systemic sclerosis.

2. The use according to claim 1, characterized in that The prevention and / or treatment of systemic sclerosis includes reducing the infiltration of immune cells in the skin of systemic sclerosis patients and / or reducing the degree of fibrosis in the skin of systemic sclerosis patients.

3. The use according to claim 2, characterized in that The prevention and / or treatment of systemic sclerosis comprises reducing the infiltration of immune cells in the skin of systemic sclerosis patients and reducing the degree of fibrosis in the skin of systemic sclerosis patients by inhibiting the expression of chemokine ligand 19 in fibroblasts.

4. The use according to claim 3, characterized in that The method of reducing the infiltration of immune cells in the skin of systemic sclerosis patients includes reducing the infiltration of T cells in the skin of systemic sclerosis patients; the method of reducing the degree of fibrosis in the skin of systemic sclerosis patients includes reducing the skin thickness of systemic sclerosis patients and / or reducing the content of type I collagen in the skin of systemic sclerosis patients.

5. The use according to any one of claims 1 to 4, characterized in that: The chemokine ligand 19 inhibitor includes an anti-chemokine ligand 19 antibody and / or an siRNA targeting a gene encoding chemokine ligand 19.

6. The use according to claim 5, characterized in that The chemokine ligand 19 inhibitor includes antibodies and / or siRNA that can inhibit the expression of chemokine ligand 19 in fibroblasts.

7. A drug for preventing and / or treating systemic sclerosis, characterized in that: The drug comprises a chemokine ligand 19 inhibitor and pharmaceutically acceptable excipients.

8. A molecular marker for diagnosing systemic sclerosis, characterized in that: The molecular markers include chemokine ligand 19-positive fibroblasts.

9. Use of a reagent for detecting the molecular marker according to claim 8 in a sample to be tested in the preparation of a product for diagnosing systemic sclerosis.

10. A product for diagnosing systemic sclerosis, characterized in that: The product comprises a reagent for detecting the molecular marker of claim 8 in a sample to be tested.

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