Video display terminal associated dry eye disease diagnostic biomarker based on epithelial-mesenchymal transition and fibrosis, and therapeutic drug

Abstract

Provided are a video display terminal (VDT) associated dry eye disease diagnostic biomarker based on epithelial-mesenchymal transition and fibrosis, and a therapeutic drug. By studying the functional transformation of epithelial cells in VDT associated dry eye disease, the epithelial-mesenchymal transition of ocular surface epithelial cells in VDT associated dry eye disease is disclosed, and in the acute phase of the disease course, the epithelial cells show fiber cell characteristics and pro-inflammatory characteristics, such that the acute phase and the chronic phase of VDT associated dry eye disease are distinguished. Provided is a biomarker for diagnosing VDT associated dry eye disease or monitoring treatment efficacy, and a reagent or kit that is prepared on the basis of the biomarker and used for diagnosing VDT associated dry eye disease, monitoring the treatment efficacy, or distinguishing the acute and chronic phases of VDT associated dry eye disease patients. In addition, intervention of the epithelial-mesenchymal transition and fibrosis process is an effective strategy for relieving VDT associated dry eye disease and improving ocular surface damage, which provides potential intervention directions and prospects for clinical treatment of VDT associated dry eye disease.

Description

Video display terminal-related dry eye diagnostic biomarkers and therapeutic drugs based on epithelial-mesenchymal transition and fibrosis

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to a prior application, patent application number 202311752725.1, filed with the State Intellectual Property Office of China on December 19, 2023, entitled “Diagnostic biomarkers for dry eye based on epithelial-mesenchymal transition and fibrosis and their uses.” The entirety of the prior application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the fields of biomarkers and medicine, and in particular to biomarkers of dry eye associated with video display terminals and therapeutic drugs thereof. Background Art

[0004] Dry eye disease (DED) is a multifactorial, chronic ocular surface disease and one of the most common eye diseases seen in ophthalmology clinics. Most patients with dry eye present with a chronic course, with symptoms that are intermittent rather than persistent. It is widely believed that most patients with chronic dry eye experience acute exacerbations, and that different immunological mechanisms are involved in the acute, chronic, and acute exacerbations of dry eye. With the increasing prevalence of video display terminals (VDTs) and changes in modern lifestyles, VDT-related dry eye has become the most common type of dry eye in recent years, with an increasing incidence and a trend toward younger patients. Unlike other types of dry eye, VDT-related dry eye is primarily caused by adverse visual environmental factors (such as prolonged staring at electronic screens, decreased blinking, and exposure to air-conditioned environments), rather than systemic factors. It is now clear that long-term use of video display terminals is an independent risk factor for the development of dry eye.

[0005] The main symptoms of dry eye include ocular discomfort (such as dryness, stinging, tearing, and eye fatigue) and visual impairment. These symptoms can severely impact patients' quality of life and daily activities (including reading, computer use, and driving). They are also highly correlated with anxiety and depression. In addition to the indirect economic losses caused by decreased work efficiency, the long-term treatment of dry eye also carries a significant direct economic burden, making it a highly public health issue.

[0006] Currently, the diagnosis of dry eye is typically based on subjective symptoms, tear break-up time (evaluation of tear film quality), vital dye staining of the ocular surface (such as corneal fluorescein staining), the Schirmer test (evaluation of tear quality), and other less common clinical tests, including tear osmolarity and Rose Bengal staining (measurement of tear meniscus height). However, poor precision in clinical measurement of DED, the lack of a "gold standard" for determining disease severity, inconsistencies between patient-reported ocular symptoms and clinical signs and between different clinical parameters, and significant patient heterogeneity have been major challenges in the clinical diagnosis of DED and the clinical development of therapeutic management.

[0007] Elucidation of the fundamental inflammatory mechanisms and pathways in DED has advanced our understanding of the role of inflammation in the pathogenesis of the disease. Ocular epithelial cells play a key role in the development and progression of inflammatory responses in dry eye. HLA-DR levels are elevated in the conjunctival epithelium of patients with dry eye, and this has been used as a biomarker in clinical trials. Furthermore, epithelial perturbations induced by a dry environment can trigger innate and subsequent adaptive immune responses, further supporting the key role of epithelial activation in dry eye. However, current research on the regulation of the immune microenvironment and immune responses by epithelial cells remains largely unresolved. Understanding how the proinflammatory effects of epithelial cells are regulated and their interactions with immune cells remains understudied. Further research is needed to identify key targets involved in the development and chronicity of dry eye inflammation, thereby enabling more effective diagnosis and treatment of dry eye. Furthermore, the acute and chronic stages of dry eye involve distinct pathogenesis. Acute dry eye is associated with the hypertonic tear environment stimulating the release of inflammatory cytokines from the ocular epithelium, while chronic dry eye may be associated with disturbances in ocular immune homeostasis. This emphasizes the importance of targeted treatment, and understanding these differences is crucial for developing effective treatment strategies and improving treatment outcomes. In particular, for the subtype of VDT-related dry eye, it is urgent to clarify its unique pathogenesis and develop specialized diagnostic and treatment options. Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present invention, through in-depth research on the functional transformation of epithelial cells in VDT-related dry eye, reveals for the first time the phenomenon of epithelial-mesenchymal transition (EMT) in ocular surface epithelial cells in VDT-related dry eye, and explains for the first time that epithelial cells in the acute phase of dry eye exhibit fibroblast characteristics and pro-inflammatory properties, thereby distinguishing the acute and chronic phases of VDT-related dry eye. Based on epithelial-mesenchymal transition and fibrosis indicators, a more accurate and effective dry eye diagnosis scheme and a method for distinguishing the acute and chronic course of VDT-related dry eye patients are provided. In addition, intervention targeting epithelial-mesenchymal transition and fibrosis processes is an effective strategy to alleviate VDT-related dry eye inflammation and improve ocular surface damage, providing potential intervention directions and prospects for the clinical treatment of VDT-related dry eye.

[0009] The primary objective of the present invention is to provide biomarkers and their use for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment. A second objective is to provide biomarkers for distinguishing between acute and chronic disease progression in patients with VDT-related dry eye. A third objective is to provide the use of anti-fibrotic drugs in the preparation of medicaments for inhibiting ocular epithelial-mesenchymal transition and fibrosis in VDT-related dry eye.

[0010] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows:

[0011] In a first aspect, the present invention provides the use of any one or more of the following genes as biomarkers in VDT-related dry eye in the preparation of a reagent or kit for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment, the genes including: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC;

[0012] Preferably, the genes include TWIST1, SNAI1, and IGFBP2.

[0013] In a second aspect, the present invention provides a use of a reagent for detecting target gene expression in the preparation of a reagent or kit for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment, wherein the target gene includes any one or more of the following: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC;

[0014] Preferably, the genes include TWIST1, SNAI1, and IGFBP2.

[0015] In a third aspect, the present invention provides a reagent or kit for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment;

[0016] The reagents include reagents for detecting the expression level of at least one of the following genes in a biological test sample of a subject: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC;

[0017] The kit contains the aforementioned reagents;

[0018] The biological test sample of the subject is taken from the surface cells of the conjunctiva of the eye;

[0019] Preferably, the genes include TWIST1, SNAI1, and IGFBP2.

[0020] In a fourth aspect, the present invention provides the use of any one or more of the following genes as biomarkers in VDT-related dry eye in the preparation of a reagent or kit for distinguishing between acute and chronic disease courses in patients with VDT-related dry eye, the genes including: AQP3, CCL2, CCL7, CCL8.

[0021] In a fifth aspect, the present invention provides a use of a reagent for detecting target gene expression in the preparation of a reagent or kit for distinguishing acute and chronic disease courses in patients with VDT-related dry eye, wherein the target genes include: AQP3, CCL2, CCL7, CCL8, and MUC5AC;

[0022] Preferably, the genes include AQP3, CCL2, and MUC5AC.

[0023] In a sixth aspect, the present invention provides a reagent or kit for distinguishing between acute and chronic disease courses in patients with VDT-related dry eye;

[0024] The reagents include reagents for detecting the expression level of at least one of the following genes in a biological test sample of a subject: AQP3, CCL2, CCL7, CCL8, MUC5AC;

[0025] Preferably, the genes include AQP3, CCL2, and MUC5AC;

[0026] The kit contains the aforementioned reagents;

[0027] The biological test sample of the subject is taken from the surface cells of the conjunctiva of the eye surface.

[0028] In a seventh aspect, the present invention provides a system for diagnosing VDT-related dry eye, the system performing the following steps:

[0029] (1) using the reagent described in the third aspect to detect the expression level of the corresponding gene in the biological test sample of the subject;

[0030] (2) Comparing the detected gene expression level value with the normal or reference expression level value of the gene.

[0031] Preferably, the biological test sample is taken from the surface cells of the conjunctiva of the eye.

[0032] Furthermore, the system for diagnosing VDT-related dry eye or monitoring the effectiveness of its treatment includes a data input module, a data comparison module, and a conclusion output module;

[0033] The data input module is used to input the target gene expression value in the subject's biological test sample obtained by detection;

[0034] The data comparison module is used to compare the target gene expression value in the subject's biological test sample with a control value, wherein the control value is the target gene expression value in the healthy subject sample;

[0035] The conclusion output module is used to output a conclusion according to the following criteria: if the expression value of the target gene in the biological test sample of the subject is greater than or less than the control value, then the subject is or is a candidate for being a VDT-related dry eye patient;

[0036] The target genes include any one or more of the following: SNAI1, TWIST1, IGFBP2, MUC5AC;

[0037] The greater than or less than may specifically be greater than or less than with statistical significance; wherein, the target genes whose expression levels in the subject's biological test sample are greater than the control values ​​are TWIST1, SNAI1, and IGFBP2; and the target gene whose expression levels in the subject's biological test sample are less than the control values ​​is MUC5AC.

[0038] In an eighth aspect, the present invention provides use of an anti-fibrotic drug in the preparation of a drug for treating dry eye.

[0039] Preferably, the dry eye is video display terminal-related dry eye, and the anti-fibrotic drug of the present invention is pirfenidone and its derivatives or tranilast and its derivatives.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This study describes for the first time the epithelial-mesenchymal transition (EMT) phenomenon that occurs in ocular surface epithelial cells in VDT-related dry eye. During the acute phase of VDT-related dry eye, epithelial cells exhibit fibroblastic and proinflammatory characteristics, thereby distinguishing the acute from the chronic phase of VDT-related dry eye. Using single-cell sequencing and related research, the study reveals for the first time biomarkers for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment. These biomarkers can be used to prepare reagents or kits for diagnosing VDT-related dry eye, monitoring the efficacy of its treatment, or distinguishing between the acute and chronic stages of VDT-related dry eye in patients.

[0042] The biomarkers provided by the present invention will help to better understand the pathophysiology of VDT-related dry eye and will provide new opportunities for diagnosis and prognosis, thereby improving clinical services for VDT-related dry eye patients.

[0043] The present invention uses the anti-fibrotic drugs pirfenidone or tranilast to inhibit the epithelial-mesenchymal transition and fibrosis process starting from the baseline of the VDT-related dry eye model in mice, and has a significant effect on alleviating VDT-related dry eye symptoms and reducing ocular surface damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1: A. Uniform Manifold Approximation and Projection (UMAP) plots of epithelial cell subsets in the normal control group (NC) and the ICES dry eye model 1-week (DED1W) and 3-week (DED3W) groups. Coloring by major cell type. B. Violin plots showing the expression of cell-specific marker genes used to identify epithelial cell subsets. C. Flow cytometric analysis of cell populations expressing both the basal epithelial cell marker Cytokeratin Pan and the fibroblast marker CD140a in the mouse conjunctiva and their proportion of non-immune cells. D. Flow cytometric analysis of the proportion of AQP3-positive fibroblast-like cells in the conjunctiva of dry eye model mice. E. Monocle2 cell trajectory analysis results, colored by cell subset. Numbers 1 and 2 indicate potential differentiation nodes between cell subsets.

[0045] Figure 2: A. Gene set signature scores for each epithelial cell subset (left) and different stages of dry eye (right) were calculated using the stemness gene set and the epithelial-mesenchymal transition gene set. P values ​​were calculated using a two-sided Wilcoxon rank-sum test. B. Violin plots showing the expression levels of epithelial-mesenchymal transition markers (CDH2, SNAI1, ZEB1, TWIST1) in epithelial cells at different stages of dry eye. P values ​​were calculated using a two-sided Wilcoxon rank-sum test. C. Real-time quantitative PCR assessment of the expression levels of epithelial-mesenchymal transition markers (Twist1, Snai1, Zeb1, Cdh1, Cdh2) in the conjunctiva at different stages of dry eye. n = 9, mean ± standard error (SEM), P values ​​were calculated using an unpaired two-sided t-test. D. Real-time quantitative PCR assessment of the expression levels of TWIST1 in the conjunctiva at different stages of dry eye. Mean ± standard error (SEM), P values ​​were calculated using a one-way analysis of variance. E. UMAP plot colored according to the activity of TGF-β signaling pathway and chemokine signaling pathway.

[0046] Figure 3: Immunofluorescence analysis of conjunctival tissue cryosections at three stages of dry eye: E-cadherin (red), N-cadherin (green), DAPI (blue). Arrows indicate the migration of conjunctival epithelial cells into the stroma.

[0047] Figure 4: Violin plots showing the expression levels of key biomarkers in epithelial cells at different stages of dry eye. P values ​​were calculated using a two-sided Wilcoxon rank-sum test.

[0048] Figure 5: A. Pathway activity differences among epithelial cell subsets in dry eye assessed using GSVA based on the Hallmark gene set. B. Gene set signature scores for the acute inflammatory response in each epithelial cell subset during the course of dry eye. Scores for each subpopulation are shown from left to right for normal, 1-week dry eye, and 3-week dry eye. Gene sets are from the GO database (GO:0002526). C. Violin plots showing the expression of MHC-II molecules in epithelial cell subtypes at different stages of dry eye. D. Violin plots showing the expression of representative genes within the CCL / CXCL signaling pathway in fibroblast-like epithelial cells. E. The number of intercellular interactions among immune cells, epithelial cells, and stromal cells overall. Epithelial cells (epithelial cells); stromal cells (stromal cells); Immune cells (stromal cells). F. Dot plots showing ligand-receptor pairs upregulated in 1-week dry eye with CD4+ T cells as receivers. Epithelial and myeloid cell subsets act as transmitters. P values ​​for all signals are <0.01. G. CCL2-CCR2 signaling network in dry eye disease. Nodes represent major cell types, and line thickness indicates the strength of the interaction.

[0049] Figure 6: A. Immunofluorescence detection of conjunctival tissue sections from dry eye mice using anti-AQP3 (labeling basal epithelial cells, red) and anti-CCL2 (green) antibodies. BC. Flow cytometry analysis of CCR2-expressing cell populations in the mouse conjunctiva. The figure shows a schematic diagram of the population (B) and the proportion of each cell type (C). D. Ligand-target gene prediction based on NicheNet. The heat map shows the expression of ligands sent by lymphocytes, myeloid cells, and stromal cells in the dry eye group for 1 week and the normal group, as well as the expression of corresponding target genes in epithelial cells. E. Real-time quantitative PCR assessment of the expression levels of TGF-β1 and IGFBP2 in the conjunctiva of dry eye mice. Mean ± standard error (Mean ± SEM), P values ​​calculated by unpaired two-sided t-test.

[0050] Figure 7: A. Schematic diagram of the experimental design for the effects of long-term VDT use on the ocular surface microenvironment. B. Corneal sodium fluorescein scores and the number of eyes with the scores and grading at each time point. C. Tear breakup time and the number of eyes with the grading at each time point. D. EMT signature scores for the three groups calculated based on GSVA. P values ​​were calculated using paired t-tests. The EMT gene set was derived from the GO database (GO:0010718). E. CCL2 expression levels in the three groups.

[0051] Figure 8: Changes in the expression levels of key biomarkers in the ocular surface microenvironment at various time points following prolonged VDT. P values ​​were calculated using paired t-tests.

[0052] Figure 9: Multi-level analysis results of the VDT-related dry eye model before and after treatment;

[0053] AB. Representative images of corneal sodium fluorescein staining (A) and quantitative analysis of staining scores (B) were observed under a slit lamp microscope in mice in the dry eye model at different time points after drug administration in the untreated (Control) group, the dry eye + saline eye drop group, the dry eye + pirfenidone eye drop group, and the dry eye + tranilast eye drop group. *P < 0.05, **P < 0.01, ***P < 0.001.

[0054] C. Real-time fluorescence quantitative PCR was used to examine the relative mRNA expression of Twist1, a key transcription factor in epithelial-mesenchymal transition (EMT), and inflammatory factors IL-1β, IL-17a, IFN-γ, and TNF-α in the conjunctival epithelium of each group of mice. Data are expressed as mean ± SEM and analyzed using one-way analysis of variance.

[0055] D. Flow cytometry analysis was performed to detect the double-positive cell population expressing both the epithelial cell marker CK PAN and the fibroblast marker CD140a and their CCL2 expression levels in the conjunctival tissues of each group of mice.

[0056] E. Flow cytometry analysis was performed to examine the proportion of F4 / 80-positive macrophages in the CD45-positive immune cell population in the conjunctival tissue of mice in each group.

[0057] Figure 10: Immunofluorescence detection results of epithelial-mesenchymal transition and fibrosis-related indicators in the conjunctiva before and after treatment of the VDT-related dry eye model;

[0058] A. Anti-CCL2 (green) and anti-IGFBP2 (red) antibody staining showed that the expression of CCL2 and IGFBP2 in conjunctival tissue was significantly reduced after treatment with two anti-fibrotic drugs; scale bar: 100 μm;

[0059] B. Anti-N-cadherin (green) and anti-E-cadherin (red) antibody staining showed that the expression of N-cadherin, a marker of epithelial-mesenchymal transition induced by dry eye, was downregulated after anti-fibrotic drug treatment; scale bar: 100 μm;

[0060] C. Anti-CD45 (red) antibody staining showed that the infiltration of immune cells in the conjunctival stroma of the dry eye model group was significantly increased, and the infiltration of immune cells was significantly reduced after treatment with anti-fibrotic drugs; scale: 100 μm; cell nuclei were stained with DAPI (blue). DETAILED DESCRIPTION

[0061] definition

[0062] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0063] Unless otherwise indicated, the terms "comprising" and "including" are used herein in their open and non-limiting sense. It should be further understood that where the description of various embodiments uses the terms "comprising" or "comprising," those skilled in the art will understand that, in some specific instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0064] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0065] In the present invention, the term "dry eye (DED)" is a chronic ocular surface disease caused by multiple factors. It is caused by abnormalities in the quality, quantity and dynamics of tears, resulting in tear film instability or imbalance of the ocular surface microenvironment. It may be accompanied by ocular surface inflammatory reactions, tissue damage and neurological abnormalities, and can cause a variety of ocular discomfort symptoms and (or) visual dysfunction.

[0066] In the present invention, "Video Display Terminal (VDT)" refers to a device used for visual display, which can display information stored and electronically processed in the form of symbols, graphics or a combination of both.

[0067] For the purposes of this application, the term "VDT-related dry eye" specifically refers to a chronic ocular surface disease caused by lifestyle factors, such as prolonged use of video display devices, which leads to abnormal tear dynamics (such as decreased blink rate and incomplete blinks) and increased tear evaporation. This leads to tear film instability or an imbalance in the ocular surface microenvironment, which can be accompanied by ocular surface inflammation and tissue damage, resulting in ocular discomfort and visual dysfunction. VDT-related dry eye is one of the major subtypes of dry eye, and its onset is closely related to prolonged exposure to VDT environments.

[0068] In this application, the term "biomarker" refers to a biochemical marker that can identify changes or potential changes in the structure or function of a system, organ, tissue, cell, or subcellular structure. Biomarkers can be used for disease diagnosis, disease staging, and to evaluate the safety and efficacy of new drugs or therapies in a target population.

[0069] In the present invention, "epithelial-mesenchymal transition (EMT)" refers to the process in which epithelial cells lose intercellular adhesion and polarity, acquire migration and invasion capabilities, and thus transform into mesenchymal cells. It plays an important role in embryonic development, chronic inflammation, tissue reconstruction, cancer metastasis and various fibrotic diseases.

[0070] In the present invention, "conjunctival fibrosis" refers to a pathological process in which the conjunctival tissue of the eye undergoes excessive collagen deposition due to tissue damage, chronic inflammation or post-operative reaction, causing normal conjunctival tissue to be replaced by fibrous tissue, thereby leading to impaired conjunctival function.

[0071] In the present invention, "the acute course of VDT-related dry eye" refers to the early stage of VDT-related dry eye, which is usually caused by irritation or pressure on the ocular surface. During this stage, the innate immune system responds rapidly, thereby activating the adaptive immune response, leading to acute discomfort symptoms and / or visual dysfunction in the eyes.

[0072] In the present invention, "chronic course of VDT-related dry eye" refers to the long-term stage of VDT-related dry eye, which may be associated with disturbances in the ocular surface immune homeostasis, leading to a persistent chronic inflammatory response, accompanied by persistent symptoms such as dry eyes, irritation and visual impairment, which may fluctuate in intensity but will not completely subside.

[0073] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0074] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0075] Example 1 Generation of fibroblast-like epithelial cells and differential gene expression during dry eye in mice

[0076] 1. Experimental Materials

[0077] 1. Animals

[0078] SPF-grade wild-type female C57BL / 6J mice (age: 6-8 weeks; weight: 18-25 g) were obtained from Shanghai JST Laboratory Animal Co., Ltd. Inclusion criteria for the experiments were healthy mice with no corneal infection, corneal ulcers, corneal scarring, or leukoplakia under slit-lamp observation, and a corneal sodium fluorescein staining score of less than 10. All experimental procedures complied with the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the use of animals in ophthalmic and visual research and were approved by the Experimental Animal Ethics Committee of Wenzhou Medical University.

[0079] 2. Main instruments

[0080] 3. Main Reagents

[0081] 4. Antibodies and reagents used in flow cytometry:

[0082] APC / Cy7 anti-mouse CD45 (Cat: 557659, clone: 30-F11, BD), Alexa Fluor 488 anti-mouse Cytokeratin Pan Monoclonal (Cat: MA5-18156, clone: C-11, Invitrogen), APC anti-mouse CD140a (Cat: 17-1401-81, clone: APA5, eBioscience), anti-mouse Aquaporin 3 (Cat: ab125219, abcam), Rabbit IgG PE-conjugated Antibody (Cat: F0110, R&D), Brilliant Violet 421 anti-mouse CD68 (Cat: 566388, clone: FA / 11, BD), eFluor 660 anti-mouse LYVE1 (Cat: 50-0443-82, clone: ALY7, eBioscience), PE-Cy7 anti-mouse CD11c (Cat: 558079, clone: HL3, BD), FITC anti-mouse CD72 (Cat: MMCD72101, clone: 10.1.D2, Invitrogen), PE anti-mouse CCR2 (Cat: 568093, clone: Y15-488.rMAb, BD), Brilliant Violet786 anti-mouse CD4 (Cat: 563727, clone: RM4-5, BD), Brilliant Violet 711 anti-mouse γδT (Cat: 563994, clone: GL3, BD), PerCP / Cyanine5.5 anti-mouse CD8a (Cat: 100734, clone: 53-6.7, BioLegend), Brilliant Violet 650 anti-mouse NK1.1 (Cat: 564143, clone: PK136, BD), Brilliant Violet 605 anti-mouse CD3 (Cat: 563004, clone: 145-2C11, BD). The conventional dilution of fluorescently labeled antibodies is 1:100.

[0083] 5. PCR primer sequences:

[0084] GAPDH: positive strand, 5'-ATGTTCGTCATGGGTGTGAA-3',

[0085] Minus strand, 5′-GGTGCTAAGCAGTTGGTGGT-3′;

[0086] Cdh1: positive strand, 5'-CAGGTCTCCTCATGGCTTTGC-3',

[0087] Minus strand, 5'-CTTCCGAAAAGAAGGCTGTCC-3';

[0088] Cdh2: positive strand, 5'-AGCGCAGTCTTACCGAAGG-3',

[0089] minus strand, 5′-TCGCTGCTTTCATACTGAACTTT-3′;

[0090] Snai1: positive strand, 5'-CACACGCTGCCTTGTGTCT-3',

[0091] Minus strand, 5'-GGTCAGCAAAAGCACGGTT-3';

[0092] Zeb1: positive strand, 5'-GCTGGCAAGACAACGTGAAAG-3',

[0093] Minus strand, 5'-GCCTCAGGATAAATGACGGC-3';

[0094] Twist1: positive strand, 5'-TCGCAAGAGACGCAGCAGTC-3',

[0095] Minus strand, 5'-CCTCCGCCCGCAGATTTCTT-3'.

[0096] 6. Antibodies used for immunofluorescence staining:

[0097] anti-E-Cadherin antibody(dilution:1:200,Cat:14-3249-82,Thermo),anti-N-cadherin antibody(dilution:1:200,Cat:PA5-85341,Thermo),Alexa Fluor 488cross-adsorbed secondary antibody,donkey anti-rabbit IgG(H+L)(dilution:1:300,Cat:A-21206,Thermo), Alexa Fluor 594 cross-adsorbed secondary antibody,goat anti-rat IgG(H+L)(dilution:1:300,Cat:A-11007,Thermo).

[0098] 2. Experimental Methods

[0099] 1. Dry Eye Animal Model

[0100] To induce a VDT-related dry eye model, mice were placed in an Intelligent Controlled Environment System (ICES) to simulate a VDT environment (humidity 15±3%, wind speed 2m / s, temperature 22±1°C) for 1 week and 3 weeks, as VDT-related dry eye 1 week and dry eye 3 week groups. The control group mice were fed in a standard environment (humidity 60%-80%, temperature 22±1°C) without any treatment. At 0, 1 and 3 weeks, corneal epithelial staining was graded in a single-blind manner according to a standard grading system (National Eye Institute, Bethesda, MD, USA) to assess the condition of the corneal epithelium.

[0101] 2. Preparation of Single-cell Suspension

[0102] After euthanasia, the mice were excised and the palpebral and bulbar conjunctivae were harvested. The conjunctivae of three mice in each group were pooled into one sample and stored in MACS tissue storage medium (Miltenyi Biotec). The samples were washed in phosphate-buffered saline containing 20 mM EDTA and shaken at 37°C for 15 minutes. The tissue was then cut into pieces and digested in RPMI1640 medium (Thermo Fisher Scientific) containing collagenase IV (Gibco; 1000 U / mL) at 37°C for 30 minutes, then filtered through a 70 μm cell strainer. Red blood cells and dead cells were removed using an erythrocyte lysis buffer (Sigma Aldrich) and a dead cell removal kit (Miltenyi Biotec), respectively. 10 μl of the suspension was counted using a hemocytometer under an inverted microscope. The cells were observed under a microscope after staining with trypsin blue.

[0103] 3. Library Preparation and Single-Cell Sequencing

[0104] Single-cell RNA sequencing libraries were generated using the Chromium Single Cell 3' Kit according to the manufacturer's instructions. The prepared single-cell suspension was processed through the Chromium Single Cell Controller instrument (10× Genomics, Pleasanton, CA, USA) to generate single-cell gel bead structures (GEMs). This was followed by reverse transcription, cDNA amplification, fragmentation, end repair, adapter ligation, library amplification, circularization, and DNA nanoball (DNB) generation. The constructed library was sequenced at 100 bp using combined probe synthesis (cPAS) on the BGI IDNBseq platform (BGI, Shenzhen, China).

[0105] 4. Raw Data Processing and Quality Control

[0106] Cell Ranger (version 7.0.1, 10x Genomics) was used to map transcripts to the mouse reference genome (mm10-2020-A) and quantify reads to generate a gene expression matrix for each sample. The expression matrix was then imported into the Seurat R software package (version 4.1.0) and subjected to preliminary quality control. The specific exclusion criteria were as follows: cells expressing fewer than 200 or more than 7500 genes, cells with more than 80,000 detected molecules, and cells with a mitochondrial gene ratio of more than 15%. The Scrublet package in Python was used to identify potential doublets. The expected doublet rate was set to 0.13, and the threshold was adjusted using the scrub.call_doublets function based on the doublet score histogram for each sample. The quality-controlled dataset was normalized using Seurat's SCTransform, and principal component analysis (PCA) was performed on the resulting 3000 highly variable genes to obtain the top 50 principal components. These principal components were input into the Harmony software package (version 0.1.0) to eliminate batch effects between different samples.

[0107] 5. Clustering, Annotation, and Visualization

[0108] The optimal number of principal components (PCs) for clustering was determined using the ElbowPlot function in Seurat. The top 20 PCs were unsupervisedly clustered at a resolution of 1.0 using the shared nearest neighbor (SNN) algorithm. Clustering results were visualized using the unified manifold approximation and projection (UMAP). Marker genes were identified for each cluster using the FindMarkers function and compared with established cell type markers for cell annotation. In addition, specific strategies were implemented for each cell subset dataset to remove clusters that did not meet the set criteria or were overrepresented in certain markers: for epithelial cells, the number of mitochondrial genes was corrected, and clusters with Cd3 expression exceeding 50% were removed. For immune cells, cell populations lacking Ptprc expression were excluded, and clusters with Lum or Pecam1 expression exceeding 50% were removed.

[0109] 6. Single-cell Sequencing - Enrichment Analysis and Gene Set Scoring

[0110] To characterize the biological processes of different cell subpopulations, differentially expressed genes were enriched using the ClusterProfiler package (version 3.18.1) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Hallmark databases. Gene set variation analysis (GSVA) ​​was performed using the GSVA package (version 1.38.2), and pathway activity scores were estimated using the gsva function. Significantly differentially expressed pathways (adjusted p-value < 0.05) were visualized using the pheatmap package (v1.0.12) and ggplot2 package (v3.2.1) in R. Signature scores for gene sets were calculated using the AddModuleScore function in Seurat. Gene sets were downloaded from the Molecular Signatures Database (MSigDB).

[0111] 7. Cell Trajectory and RNA Velocity Analysis

[0112] Monocle2 (version 2.26.0) was used to infer epithelial cell developmental trajectories. Gene expression matrices from all cells in these subpopulations were used as input to Monocle2. After quality control and selection of highly variable genes, dimensionality reduction was performed using the DDRTree algorithm. Cell trajectories were visualized according to subpopulation and cell state. Loom files containing spliced ​​and unspliced ​​mRNA information were generated from bam files generated after alignment to the genome using the velocyto Python package. These files were then input into scVelo to calculate RNA rates using the following parameters: counts = 30, n_top_genes = 2000, n_pcs = 30, and n_neighbors = 30.

[0113] 8. Flow Cytometry

[0114] The preparation of single cell suspension is as described above. For cell surface staining, cells were blocked for 10 minutes by CD16 / 32FcR-block (Cat: 101302, BioLegend). After staining with a fixable viability stain (Fixable Viability Stain, Cat: 564406, BD), the obtained cells were stained with surface markers at 4 ° C for 30 minutes. For intracellular staining, the cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer (Foxp3 / Transcription Factor Staining Buffer, Cat: 00-5523-00, Invitrogen), and then intracellular staining was performed with appropriate fluorescent-labeled antibodies in the permeabilization buffer, and stained for 45 minutes at 4 ° C. Cells were collected on a flow cytometer (Attune Nxt V6, Thermo). Flow cytometric analysis was performed using Flowjo software (version 10.5.3, BD).

[0115] 9. Real-time Quantitative PCR

[0116] Total RNA was extracted from the conjunctiva according to the manufacturer's instructions (Cat: 74106, RNeasy mini kit, Qiagen, Crawley, UK). cDNA was synthesized from 0.5 μg of total RNA using random primers and M-MLV reverse transcriptase (Cat: 28025013, Applied Biosystems, Paisley, UK). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using Power SYBR Green PCR Master Mix (Cat: A25742, Applied Biosystems, Paisley, UK) and the Applied Biosystems Qua nt Studio 6 Real-Time PCR System (Applied Biosystems, Paisley, UK). Results were analyzed using the comparative threshold cycle (CT) method and normalized to GAPDH as an endogenous reference.

[0117] 10. Immunofluorescence Staining

[0118] Conjunctiva and eyeballs from three mice per group were embedded in optimal sectioning temperature compound (Cat: 4583, SAKURA) and then frozen in liquid nitrogen. These samples were then cut into 10 mm thick sections and stored at -80°C. For staining, sections were thawed at room temperature. After thawing, they were fixed in 4% formaldehyde for 15 minutes. Sections were then blocked with blocking buffer (Cat: ab64226, Abcam) containing 0.4% Triton X-100 (Cat: T8787, Sigma Aldrich) for >60 minutes at room temperature. Sections were then incubated with primary antibodies and corresponding fluorescent-conjugated secondary antibodies according to the manufacturer's recommended sequence. 4'6-Diamino-2-phenylindole (Cat: S36938, DAPI, Invitrogen) was added for 5 minutes. Images were captured using a laser scanning confocal microscope (LSM880, Carl Zeiss Meditec, Sartrouville, Germany).

[0119] 3. Experimental Results

[0120] Figure 1A, based on single-cell sequencing results, revealed an abnormal increase in conjunctival epithelial cells, particularly during the acute course of VDT-related dry eye. These included basal epithelial cells (BECs) that highly expressed the basal epithelial cell markers KRT15, ​​KRT6A, and KRT14; fibroblast-like epithelial cells (FL EEPs) that co-expressed both the basal epithelial cell marker and the fibroblast marker CD140a; and endothelial-like epithelial cells (EL EEPs) that co-expressed both the basal epithelial cell marker and the endothelial cell marker Pecam1. Furthermore, the number of superficial epithelial cells (SUP EEPs) and goblet cells decreased. Figure 1B showed that these increased epithelial cell subsets specifically expressed the aquaporin AQP3, a phenomenon not observed in other epithelial cell subsets or stromal cells. Figure 1C shows the presence of a fibroblast-like epithelial subpopulation detected by flow cytometry. This subpopulation was also found to increase significantly during VDT-related dry eye and to have high expression of AQP3 (Figure 1D). Cell trajectory analysis in Figure 1E revealed that the fibroblast-like epithelial subpopulation is derived from the expansion and differentiation of basal epithelial cells. These results suggest that epithelial cells undergo proliferation and differentiation during VDT-related dry eye, generating a new cell subpopulation with fibroblast characteristics.

[0121] Gene set scoring in Figure 2A revealed that epithelial cell stemness was significantly upregulated at both 1 and 3 weeks in VDT-related dry eye. The epithelial-mesenchymal transition (EMT) pathway was specifically upregulated at 1 week in VDT-related dry eye. Fibroblast-like epithelial cells achieved the highest EMT score among epithelial cell subpopulations, suggesting that their generation may be involved in the EMT process. Figure 2B shows that EMT-related markers (CDH2, SNAI1, ZEB1, and TWIST1) were all significantly upregulated at 1 week in VDT-related dry eye, with TWIST1 being the most prominent. Real-time quantitative PCR also revealed that Twist1, Snai1, and Zeb1 were significantly upregulated at 1 week in VDT-related dry eye (Figure 2C). Twist1 was specifically upregulated in acute dry eye, while no significant difference was observed in chronic dry eye compared with the normal control group (Figure 2D). Figure 2E shows gene set signature scoring of epithelial cell subsets, revealing that the TGF-β signaling pathway was significantly upregulated in the fibroblast-like epithelial subpopulation during 1 week of VDT-related dry eye, a key pathway for inducing EMT. Compared with the control group, the conjunctival epithelium during VDT-related dry eye showed a shift in E-cadherin to N-cadherin expression and migration to the conjunctival stroma, demonstrating a classic epithelial-mesenchymal transition (EMT) phenomenon (Figure 3). On the other hand, fibroblast-like epithelium became the primary source of chemokines in epithelial cells at 1 week of VDT-related dry eye, and this expression was slightly downregulated at 3 weeks of VDT-related dry eye (Figure 2E). This may indicate that fibroblast-like epithelium plays an important role in recruiting immune cells and promoting inflammatory responses in VDT-related dry eye.

[0122] The above results indicate that in the ICES mouse model of VDT-related dry eye, conjunctival epithelial cells strongly express the specific marker AQP3 and undergo EMT. Fibroblast-like epithelial cells achieved the highest EMT score among epithelial cell subpopulations, indicating that their generation is associated with the EMT process. Figures 3B and 4 show that EMT-related markers (CDH2, SNAI1, ZEB1, TWIST1) were significantly upregulated at 1 week in VDT-related dry eye and remained at high levels at 3 weeks, with TWIST1 and SNAI1 being the most prominent. The goblet cell marker MUC5AC showed a significant decrease in expression during the acute phase of VDT-related dry eye and partial recovery during the chronic phase.

[0123] Example 2 Fibroblast-like epithelial cells are closely related to inflammatory response

[0124] 1. Experimental Materials

[0125] The following antibodies and reagents were used for immunofluorescence staining: anti-CCL2 antibody (dilution: 1:100, Cat: 127-545-160, Thermo), anti-Aquaporin 3 antibody (dilution: 1:100, Cat: ab125219, Abcam), Alexa Fluor 488-conjugated secondary antibody, goat anti-Armenian Hamster IgG (H+L) (dilution: 1:300, Cat: 127-545-160, Jackson), Alexa Fluor 594-conjugated secondary antibody, goat anti-rabbit IgG (H+L) (dilution: 1:300, Cat: ab150080, Abcam).

[0126] 2. Experimental Methods

[0127] 1. Single-cell sequencing-cell communication analysis

[0128] Other analysis methods for single-cell sequencing are the same as those in Example 1.

[0129] The present invention uses the CellChat package (version 1.5.0) to infer the intercellular interaction network between different cell types based on the ligand-receptor pairs in the CellChatDB mouse database. The number of communications between each group was compared, and the results were visualized using the netVisual_bubble function. The important ligand-receptor pairs of specific pathways were extracted, and the key intercellular communications were visualized using the netVisual_individual function. The information flow of each signaling pathway was compared, and the overall information flow was visualized using rankNet. The netVisual_aggregate function revealed the strength of the specific signaling pathway, and the network centrality score and the netAnalysis_signalingRole_network function were used to determine the main signaling effects. NicheNet analysis used Nichenetr (version 1.1.1) to predict ligand-target gene relationships. All epithelial cells were defined as signal receiving cells, and other cell types were defined as signal sending cells to predict potential ligands that regulate epithelial cell differentiation during dry eye. The minimum LFC method was used to infer the connection between ligands and target genes, with a cutoff value of 0.75, and the receptor-ligand pairs that were upregulated in dry eye were visualized. The ligand-target gene heat map shows the potential regulatory scores of the top-ranked ligands and their target genes.

[0130] 2. Flow Cytometry

[0131] The specific steps are as described in Example 1.

[0132] 3. Immunofluorescence

[0133] The mouse conjunctiva and eyeball were excised and embedded in OCT (optimal cutting temperature compound, Cat: 4583, SA KURA), frozen in liquid nitrogen, and then cut into 10 μm sections. The sections were stored at -80°C. For staining, the sections were thawed at room temperature (RT). After thawing, the sections were fixed in 4% formaldehyde for 15 minutes. Then, the sections were blocked with blocking buffer (Cat: ab64226, serum-free protein blocking solution, Abcam) supplemented with 0.4% Triton X-100 (Cat: T8787, Sigma Aldrich) at RT for 60 minutes. The sections were incubated with primary antibodies and corresponding fluorescent-labeled secondary antibodies according to the manufacturer's recommended order. 4'6-diamino-2-phenylindole (Cat: S36938, DAPI, Invitrogen) was added for 5 minutes. Images were captured using a laser scanning confocal microscope (LSM880, Carl Zeiss Meditec, Sartrouville, Germany).

[0134] 3. Experimental Results

[0135] Figure 5A, through gene set variation analysis, shows that fibroblast-like epithelial cells in VDT-related dry eye are enriched in the "IL-2 / STAT-5 signaling pathway," "IL-6 / JAK / STAT3 signaling pathway," and "inflammatory response." Figure 5B also shows that this subpopulation has the highest acute inflammatory response score, reaching its highest value at 1 week. This suggests that this subpopulation plays an important role in promoting inflammatory responses during the acute phase of VDT-related dry eye. Figure 5C shows that MHC-II class molecules are significantly upregulated in epithelial cells during VDT-related dry eye, which helps activate pathogenic T cells and initiate adaptive immune responses. At the same time, fibroblast-like epithelial cells exhibited a wide range of chemokine upregulation at 1 week in VDT-related dry eye (Figure 5D), such as CCL2, CCL7, and CCL8, which can bind to CCR2 and mediate monocyte / macrophage recruitment. Cell communication analysis in Figure 5E shows that the interaction between epithelial cells and immune cells is significantly increased in VDT-related dry eye. Systematic analysis of receptor-ligand interactions between epithelial and myeloid cells and CD4+ T cells revealed that during dry eye, fibroblast-like epithelial cells displayed an increase in the most abundant chemokine-related ligand-receptor pairs, including Ccl2-Ccr2, Ccl7-Ccr2, Cxcl13-Cxcr3, and Cxcl10-Cxcr3 (Figure 5F). Enhanced interactions with fibronectin Fn1 and integrins, which are common molecules involved in cell adhesion, were also observed. Figure 5G detected enhanced communication between fibroblast-like epithelial cells and various immune cells (CD4+ T cells, γδ T cells, ILC2, NK cells, and macrophages) through CCL2 expression at 1 week of VDT-related dry eye, indicating their recruitment of these immune cells.

[0136] Figure 6A shows immunofluorescence staining confirming the colocalization of CCL2 with epithelial cells that highly express AQP3. Furthermore, a basal epithelial cell population with high CCL2 expression and stroma expansion was identified in VDT-related dry eyes. Figures 6B-C show that flow cytometry analysis reveals that the CCL2 receptor, CCR2, is highly expressed in various immune cells on the ocular surface, with CD68+ macrophages and CD4+ T cells being the most prevalent CCR2-expressing cells.

[0137] In Figure 6D, NicheNet analysis was used to connect ligands and target genes to predict cell-to-cell interactions, and it was found that signals from myeloid cells may promote the differentiation of epithelial cells into fibroblasts. Specifically, TGF-β1 and other proteins produced by myeloid cells (mainly macrophages) at 1 week of VDT-related dry eye have the potential to induce epithelial cells to express collagen fiber encoding genes such as Col1a1, Col1a2, Col3a1, and Postn. In addition, the macrophage-specific marker APOE has the activity of inducing IGFBP2 in epithelial cells. Figure 6E also confirmed the upregulation of TGF-β1 and IGFBP2 expression on the ocular surface in VDT-related dry eyes using PCR. This suggests that macrophages may enhance the pro-inflammatory effect of epithelial cells by secreting TGF-β1 and APOE to change the expression pattern of epithelial cells and regulate the fate of epithelial cells to differentiate into fibroblast-like cells in VDT-related dry eyes; furthermore, epithelial cells serve as the main regulatory center for inflammatory and stress signals, chemotactic CD4+ T cells and myeloid cell populations to infiltrate, thereby maintaining the vicious cycle of dry eye inflammation.

[0138] These results suggest that epithelial cells undergo fate changes during VDT-related dry eye, generating a fibroblast-like epithelial cell subset that, during the acute phase, exerts proinflammatory effects by secreting chemokines, actively regulating the immune microenvironment of the ocular surface. Effector molecules associated with this process could serve as biomarkers for the diagnosis of VDT-related dry eye. These include TGF-β1 and its corresponding target, IGFBP2, which regulate epithelial cell fate changes, as well as the key chemokines CCL2, CCL7, and CCL8 secreted by epithelial cells.

[0139] Example 3: Upregulation of factors associated with epithelial cell phenotype changes in dry eye patients using long-term VDT

[0140] 1. Experimental Materials

[0141] Human samples

[0142] A total of 26 healthy subjects aged 18 to 50 years were enrolled. These subjects had no history of ocular trauma or surgery (including refractive surgery), no history of ocular disease other than refractive error, no recent contact lens use, no autoimmune disease or recent use of immunosuppressants, no participation in other clinical trials, and no systemic contraindications. All subjects provided informed consent before participation. All experimental procedures were approved by the Research Ethics Office of Wenzhou Medical University (ID: 2022-132-K-101-01) and strictly adhered to the approved guidelines. A total of 26 healthy subjects were enrolled in the study according to the following inclusion and exclusion criteria.

[0143] The exclusion criteria were as follows: history of eye trauma or eye surgery (including refractive surgery); confirmed history of eye diseases other than dry eye and refractive error; patients who still wore contact lenses one week before the examination; patients with comorbid autoimmune diseases or use of immunosuppressants within one month; patients who were participating in any other clinical trials at the same time; and patients who were judged by the researchers to have systemic diseases that made them unsuitable for participation in this study.

[0144] 2. Experimental Methods

[0145] 1. Study Design

[0146] The study will last four weeks, totaling 28 days, and will be divided into two phases. In the first phase (week 0), participants will be required to maintain daily VDT use for ≤3 hours; in the second phase (weeks 1, 2, and 3), participants will maintain VDT ​​use for >8 hours. Participants will complete four questionnaires and eye examinations at the end of weeks 0, 1, 2, and 3, and three ocular surface cell samples will be collected at the end of weeks 0, 1, and 3.

[0147] 2. Subjects fill out the questionnaire

[0148] Participants completed the Ocular Surface Disease Index (OSDI) based on their individual circumstances. To ensure the accuracy and reliability of the questionnaire, researchers explained it to participants before they completed it. For example, the "not applicable" option on the OSDI questionnaire indicated they had not recently engaged in the aforementioned activities or been in the aforementioned environments, while the "none" option indicated they had no symptoms while engaging in the aforementioned activities or being in the aforementioned environments.

[0149] 3. Eye Examination of Subjects

[0150] The examinations were performed in the following order, starting with non-invasive procedures and then progressing to invasive ones: meibography, slit lamp biomicroscope, and ocular surface cell sampling. All examinations were performed on both eyes, except for the ocular surface cell sampling, which was performed on the right eye.

[0151] 4. Slit lamp examination

[0152] 4.1 Corneal fluorescein sodium staining

[0153] After moistening a sodium fluorescein filter paper strip with a drop of tobramycin eye drops, it was gently applied to the lateral conjunctival surface of the lower eyelid. The subject was instructed to rotate their eyeball. Slit-lamp microscopy under cobalt blue light revealed a green-stained corneal epithelial defect. Corneal sodium fluorescein staining scores were recorded for five corneal regions: superior, inferior, nasal, temporal, and central. Each region was scored 0–3 points based on severity, with a maximum of 15 points. Scoring was as follows: 0, no punctate staining; 1, 1–30 punctate staining; 2, ≥30 punctate staining without fusion; and 3, punctate staining with fusion or the presence of filaments.

[0154] 4.2 Conjunctival Lissamine Green Staining

[0155] After moistening a lissamine green filter paper strip with a drop of tobramycin eye drops, it was gently applied to the lateral conjunctival surface of the lower eyelid. The subject was instructed to rotate their eyeballs and observed under white light using a slit lamp microscope. Lissamine green staining scores were recorded for two conjunctival areas, one on the nasal and one on the temporal side. Each area was rated 0-3 points based on severity, with a maximum of 6 points. The specific scores were as follows: 0: 0-9 staining points; 1: 10-32 staining points; 2: 33-100 staining points; 3: ≥100 staining points.

[0156] 4.3 Fluorescein sodium tear film breakup time

[0157] After sodium fluorescein staining of the ocular surface, the subjects were instructed to blink three times before opening their eyes. A stopwatch was used to record the time from eye opening to the first appearance of dark spots on the tear film, which was the tear film breakup time. The average of these three examinations was used as the measurement result.

[0158] 5. Diagnosis of dry eyes

[0159] According to the diagnostic criteria for dry eye proposed in the 2017 Asian Dry Eye Society (ADES) report, "New Perspectives on the Definition and Diagnosis of Dry Eye: A Consensus Report from the ADES," the presence of subjective symptoms and tear film breakup time are diagnostic indicators. In this study, an OSDI score ≥ 13 and a FBUT < 5 seconds were used as the diagnostic criteria for dry eye.

[0160] 6. Collection and Storage of Ocular Surface Cell Samples

[0161] Ocular surface cell samples were collected from the right eye of each subject. One drop of proparacaine hydrochloride eye drops was placed in the conjunctival sac of the lower eyelid, and the subject was instructed to rotate their eyeball. Two throat swabs were used to gently swab the upper and lower eyelid conjunctiva three times each. One throat swab was used to gently swab the superior, inferior, nasal, and temporal bulbar conjunctiva once each. The tips of the three throat swabs were folded down and placed in TRIzol (Thermo Fisher, 15596018) and stored at -80°C for batch RNA sequencing.

[0162] 7. Transcriptome Sequencing

[0163] Total RNA was extracted from the samples using TRIzol according to the manufacturer's instructions. The concentration and integrity of RNA were assessed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511). The cDNA library was isolated and purified using an Illumina NovaSeq TM Sequencing was performed on the 6000 platform using 150 bp paired-end reads. Low-quality reads were filtered out using Cutadapt (v1.9), and sequence quality was verified using FastQC (v0.11.9). Reads were mapped to the human reference genome (UCSC hg38) using HISAT277 (v2.0.4). Mapped reads for each sample were assembled using StringTie (v1.3.4d), and transcriptomes from all samples were compared and merged using gffcompare (v0.9.8). StringTie was used to estimate the expression of all transcripts and generate count tables. Transcript expression differences between samples were quantified using transcripts per million tags (TPM).

[0164] Differential expression analysis between two different groups was performed using the DESeq2R software package (v1.20.0). Genes with a P value < 0.05 and an absolute fold change > 2 were considered differentially expressed genes. GSVA analysis was performed to assess pathway enrichment differences between groups.

[0165] 3. Experimental Results

[0166] To investigate the effects of VDT environmental stress on ocular surface parameters, a self-controlled before-after design was used, prospectively enrolling 26 healthy subjects (see Figure 7A for a flowchart). As shown in Figures 7B-C, corneal staining scores increased with prolonged VDT exposure, while tear breakup time decreased, remaining stable at 3 weeks compared to 2 weeks. According to the 2017 Asian Dry Eye Association diagnostic criteria for dry eye, which include the presence of subjective symptoms combined with a tear breakup time of less than 5 seconds, the prevalence of VDT-related dry eye increased after 1 week of prolonged VDT use, reaching a peak of 32.7% at 3 weeks. Figure 7D shows that EMT characteristic scores increased in VDT-related dry eye, indicating that EMT also occurs on the human ocular surface. Figures 7E and 8 show that the EMT-related markers TWIST1 and SNAI1 were significantly upregulated in conjunctival surface cells during VDT-related dry eye. EMT-related regulatory factors TGF-β1 and IGFBP2 also showed an upregulation trend. In addition, AQP3, a key marker of fibroblast-like epithelial expression, and chemokines CCL2, CCL7, and CCL8 were upregulated during VDT-related dry eye, while the goblet cell marker MUC5AC showed a significant decrease in expression in acute VDT-related dry eye and partial recovery in chronic VDT-related dry eye. The expression changes of these biomarkers are consistent with the trends observed in VDT-related dry eye mice, indicating that the characteristics of epithelial cells acquiring fibroblast-like and proinflammatory phenotypes during the course of VDT-related dry eye are conserved across species. Biomarkers related to this process can help diagnose and accurately stage patients with VDT-related dry eye.

[0167] Example 4 Evaluation of the direct effect of inhibition of epithelial-mesenchymal transition and fibrosis in the treatment of VDT-related dry eye

[0168] 1. Experimental Materials

[0169] 1. Animals

[0170] SPF-grade wild-type female C57BL / 6J mice (age: 6-8 weeks; weight: 18-25 g) were obtained from the Zhejiang Provincial Animal Center. Inclusion criteria for the experiments were healthy mice without corneal infection, corneal ulcers, corneal scarring, or leukoplakia under slit-lamp observation, and with a corneal sodium fluorescein staining score of less than 10. All experimental procedures complied with the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the use of animals in ophthalmic and visual research and were approved by the Experimental Animal Ethics Committee of Wenzhou Medical University.

[0171] 2. Main Reagents

[0172] 1) Preparation of 0.5% anti-fibrosis drug solution:

[0173] (1) Weigh 20 mg of anti-fibrotic drug;

[0174] (2) Dissolve the drug in 40 μL DMSO and dissolve thoroughly;

[0175] (3) Slowly add normal saline to a total volume of 4 mL;

[0176] (4) Place the solution on a shaker overnight to ensure thorough mixing;

[0177] Final concentration: 0.5% (w / v).

[0178] 2) Preparation of control excipient solution:

[0179] (1) Measure 40 μL of DMSO;

[0180] (2) Add normal saline to a total volume of 4 mL;

[0181] (3) Mix gently.

[0182] 3. Antibodies and reagents used in flow cytometry:

[0183] BV711 anti-mouse F4 / 80 (Cat: 565612, clone: ​​T45-2342, BD) and other antibodies and reagents are as described in Example 1.

[0184] 4.PCR primer sequences:

[0185] Twist1: positive strand, 5′-TCGCAAGAGACGCAGCAGTC-3′;

[0186] minus strand, 5′-CCTCCGCCCGCAGATTTCTT-3′;

[0187] IL-1β: positive chain, 5'-GCAACTGTTCCTGAACTCAACT-3',

[0188] minus strand, 5′-ATCTTTTGGGGTCCGTCAACT-3′;

[0189] IL-17a: positive chain, 5'-AAAGCTCAGCGTGTCCAAAC-3',

[0190] Minus strand, 5′-ACGTGGAACGGTTGAGGTAG-3′;

[0191] IFNγ: positive chain, 5'-ATGAACGCTACACACTGCATC-3',

[0192] minus strand, 5′-CCATCCTTTTGCCAGTTCCTC-3′;

[0193] TNFα: positive chain, 5'-AGGCACTCCCCAAAAAGATG-3',

[0194] Minus strand, 5'-CCACTTGGTGGTTTGTGAGTG-3'.

[0195] 5. Antibodies used for immunofluorescence staining:

[0196] Anti-IGFBP2 antibody (dilution: 1:500, Cat: ab188200, abcam), anti-CD45 antibody (dilution: 1:200, Cat: MAB114, R&D Systems) and other antibodies are described in Example 1.

[0197] 2. Experimental Methods

[0198] 1. Study Design

[0199] This example uses an intelligent controlled environment system (ICES) to construct a VDT-related dry eye mouse model. In ICES, mice are placed under adverse conditions simulating a VDT environment for 2 weeks to induce the formation of VDT-related dry eyes. After modeling, the mice continue to be raised under ICES conditions to maintain the VDT-related dry eye state, which is closer to real-world conditions. Subsequently, a 4-week drug eye drop treatment intervention was started on the VDT-related dry eye mice. The drug was 0.5% anti-fibrotic drug pirfenidone or tranilast, twice a day, one drop in each eye each time, each drop was about 6 μL.

[0200] This example sets up four experimental groups:

[0201] (1) Dry eye model without intervention (Control group, n = 9 (a total of 18 eyes);

[0202] (2) Dry eye + saline eye drops group, n = 10 (20 eyes in total);

[0203] (3) Dry eye + pirfenidone eye drops treatment group n = 10 (20 eyes in total)

[0204] (4) Dry eye + Tranilast eye drops treatment group n = 10 (20 eyes in total).

[0205] 2. Corneal Fluorescein Sodium Staining

[0206] Using a pipette, 0.5 μL of 5% sodium fluorescein solution was instilled into the conjunctival sac of the mice. Three minutes later, corneal epithelial staining was graded under cobalt blue light using a slit lamp microscope. Scoring was performed by a masked observer using the National Eye Institute's standard grading system, which divides the cornea into five equal sections: central, superior, inferior, nasal, and temporal. The staining of each section was graded from 0 to 4 (grade 0: no staining; grade 1: 1-5 staining points; grade 2: 6-15 staining points; grade 3: 16-30 staining points; grade 4: 30 staining points or more). The sum of the scores for each section resulted in the corneal sodium fluorescein staining score.

[0207] 3. Real-time quantitative PCR detection

[0208] The specific steps are as described in Example 1.

[0209] 4. Flow Cytometry

[0210] The specific steps are as described in Example 1.

[0211] 5. Immunofluorescence

[0212] The specific steps are as described in Example 1.

[0213] 3. Experimental Results

[0214] To further evaluate the direct role of inhibiting epithelial-mesenchymal transition and fibrosis in the treatment of VDT-related dry eye, the present invention intervened in mice based on the baseline VDT-related dry eye model and performed multi-level analysis before and after treatment. The experimental results are as follows:

[0215] Corneal staining and symptom relief:

[0216] The corneas of mice in each group were stained with sodium fluorescein and observed under a slit lamp microscope at different time points (Figure 1A). The results showed that after one week of treatment with anti-fibrotic drugs, the corneal staining of VDT-related dry eye model mice had significantly improved (Figure 1A-B). This improvement trend was maintained after four weeks of treatment. This suggests that inhibiting epithelial cell fibrosis has a good early and long-lasting therapeutic effect on VDT-related dry eye.

[0217] Changes in expression of inflammation and epithelial-mesenchymal transition (EMT)-related markers:

[0218] Real-time quantitative PCR was used to examine the expression levels of Twist1, a key transcription factor in EMT, and various proinflammatory cytokines (IL-1β, IL-17a, IFN-γ, and TNF-α) in conjunctival tissue. Twist1 was significantly downregulated after treatment with pirfenidone and tranilast, while tranilast significantly downregulated the expression of IL-17a, IFN-γ, and TNF-α genes (Figure 1C, n = 4), indicating that inhibition of epithelial fibrotic transition can effectively reduce the inflammatory response associated with VDT-related dry eye.

[0219] Epithelial-mesenchymal transition and immune cell infiltration analysis:

[0220] Flow cytometry results showed that the proportion of cells with high expression of CK PAN (epithelial marker) and CD140a (fibroblast marker) in the VDT-related dry eye model increased, accompanied by increased CCL2 expression levels. After treatment with anti-fibrotic drugs, the expression of this fibroblast-like epithelial cell subset and CCL2 was significantly reduced (Figure 1D, n = 3). In addition, the proportion of F4 / 80+ macrophages in the conjunctiva of VDT-related dry eye mice was significantly reduced after treatment (Figure 1E, n = 3).

[0221] Immunofluorescence analysis further supported the above findings (Figure 2A-C, n=3):

[0222] Anti-CCL2 and IGFBP2 staining showed that the expression of these two molecules in conjunctival tissue was significantly downregulated after anti-fibrotic drug treatment (Figure 2A), indicating that epithelial-mesenchymal transition and fibrosis-related inflammatory chemotactic signals were effectively inhibited.

[0223] Anti-N-cad and E-cad staining results showed that EMT-related changes in epithelial cells were alleviated after treatment ( Figure 2B ).

[0224] Anti-CD45 staining showed that the degree of immune cell infiltration was significantly reduced ( Figure 2C ), verifying that the inflammatory microenvironment of VDT-related dry eye was effectively improved.

[0225] In summary, inhibiting epithelial-mesenchymal transition and fibrosis has a significant effect on alleviating VDT-related dry eye symptoms and reducing ocular surface damage. The use of anti-fibrotic drugs such as pirfenidone and quinone can significantly improve corneal damage in VDT-related dry eye mice, reduce the expression of key pro-inflammatory and pro-fibrotic factors such as Twist1 and CCL2 in ocular surface epithelial cells, reduce immune cell infiltration, thereby alleviating inflammatory responses and promoting ocular surface damage repair. This further demonstrates the core idea of ​​the present invention: intervention in epithelial-mesenchymal transition and fibrosis is an effective strategy to alleviate VDT-related dry eye inflammation and improve ocular surface damage, providing potential intervention directions and prospects for the clinical treatment of VDT-related dry eye.

[0226] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of any one or more of the following genes as biomarkers in dry eye in the preparation of reagents or kits for diagnosing dry eye or monitoring the efficacy of its treatment, characterized in that: The genes include: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC; Preferably, the dry eye is video display terminal-related dry eye, and the genes include TWIST1, SNAI1, and IGFBP2.

2. Use of a reagent for detecting target gene expression in the preparation of a reagent or kit for diagnosing dry eye or monitoring the efficacy of its treatment, characterized in that: The target genes include any one or more of the following: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC; Preferably, the dry eye is video display terminal-related dry eye, and the genes include TWIST1, SNAI1, and IGFBP2.

3. A reagent or kit for diagnosing dry eye or monitoring the efficacy of its treatment; characterized in that: The reagents include reagents for detecting the expression level of at least one of the following genes in a biological test sample of a subject: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC; the kit contains the aforementioned reagents; Preferably, the dry eye is video display terminal-related dry eye, and the genes include TWIST1, SNAI1, and IGFBP2.

4. The reagent or kit according to claim 3, characterized in that The biological test sample of the subject is taken from the surface cells of the conjunctiva of the eye surface.

5. Use of any one or more of the following genes as biomarkers in dry eye in the preparation of a reagent or kit for distinguishing between acute and chronic course of dry eye patients, characterized in that: The genes include: AQP3, CCL2, CCL7, CCL8, MUC5AC; Preferably, the dry eye is video display terminal-related dry eye, and the genes include AQP3, CCL2, and MUC5AC.

6. Use of a reagent for detecting target gene expression in the preparation of a reagent or kit for distinguishing acute and chronic course of dry eye patients, characterized in that: The target genes include: AQP3, CCL2, CCL7, CCL8, MUC5AC; Preferably, the dry eye is video display terminal-related dry eye, and the genes include AQP3, CCL2, and MUC5AC.

7. A reagent or kit for distinguishing between acute and chronic course of dry eye in patients; the reagent comprising a reagent for detecting the expression level of at least one of the following genes in a biological test sample of a subject: AQP3, CCL2, CCL7, CCL8, MUC5AC; the kit comprising the aforementioned reagents; Preferably, the dry eye is video display terminal-related dry eye, and the genes include AQP3, CCL2, and MUC5AC.

8. The reagent or kit according to claim 7, characterized in that The biological test sample of the subject is taken from the surface cells of the conjunctiva of the eye surface.

9. A system for diagnosing dry eye or monitoring the effectiveness of its treatment, characterized in that The system performs the following steps: (1) using the reagent or kit described in claim 3 or 4 to detect the expression value of the corresponding biomarker in the biological test sample of the subject; (2) comparing the detected gene expression value with the normal or reference expression value of the biomarker; Preferably, the biological test sample is taken from the surface cells of the conjunctiva of the eye surface.

10. The system for dry eye diagnosis according to claim 9, characterized in that: It includes data input module, data comparison module and conclusion output module; The data input module is used to input the target gene expression value in the subject's biological test sample obtained by detection; The data comparison module is used to compare the target gene expression value in the subject's biological test sample with a control value, wherein the control value is the target gene expression value in the healthy subject's sample; The conclusion output module is used to output a conclusion according to the following criteria: if the expression value of the target gene in the subject's biological test sample is greater than or less than the control value, the subject is or is a candidate for being a dry eye patient; The greater than or less than may specifically be greater than or less than with statistical significance; wherein, the target genes whose expression values ​​in the biological test samples of the subjects are greater than the control values ​​are TWIST1, SNAI1, and IGFBP2; the target gene whose expression values ​​in the biological test samples of the subjects are less than the control values ​​is MUC5AC; preferably, the dry eye is video display terminal-related dry eye.

11. Application of anti-fibrotic drugs in the preparation of drugs for treating dry eye; Preferably, the dry eye is video display terminal-related dry eye, and the anti-fibrotic drug is tranilast and its derivatives or pirfenidone and its derivatives.

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