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

Abstract

The invention provides a video display terminal related dry eye diagnostic biomarker and a therapeutic drug based on epithelial-mesenchymal transition and fibrosis. By studying functional transformation of epithelial cells in the VDT-related dry eye, epithelial-mesenchymal transition of ocular surface epithelial cells in the VDT-related dry eye is revealed, and fibroblast characteristics and proinflammatory characteristics are presented in an acute stage of a disease course, so that the acute stage and the chronic stage of the VDT-related dry eye are distinguished. Disclosed are biomarkers for diagnosing or monitoring the efficacy of a treatment for VDT-related dry eye, and preparation of reagents or kits for diagnosing, monitoring the efficacy of a treatment for a VDT-related dry eye, or differentiating acute and chronic progresses in VDT-related dry eye patients based on the markers. Besides, intervention aiming at the epithelial-mesenchymal transition and fibrosis process is an effective strategy for relieving VDT-related dry eye inflammation and improving ocular surface injury, and a potential intervention direction and prospect are provided for clinical treatment of VDT-related dry eye.

Description

[0001] Cross - reference to related applications

[0002] This invention claims the priority of a prior application with the patent application number 202311752725.1 and the invention title "Dry - eye diagnostic biomarkers based on epithelial - mesenchymal transition and fibrosis and their uses", which was filed with the State Intellectual Property Office of China on December 19, 2023. The full text of this prior application is incorporated herein by reference. Technical field

[0003] This invention relates to the fields of biomarkers and medicine, and particularly to biomarkers related to video display terminal - associated dry eye and their therapeutic drugs. Background art

[0004] Dry Eye Disease (DED) is a multi - factor chronic ocular surface disease and one of the most common eye diseases in ophthalmology clinics. Most dry - eye patients have a chronic course, and the symptoms show an intermittent rather than a continuous pattern. It is widely believed that most chronic dry - eye patients will experience acute attacks, and different immunological mechanisms are involved in the acute phase, chronic phase, and acute exacerbation of the chronic phase of dry eye. With the popularization of video display terminals (VDTs) and the changes in modern lifestyles, in recent years, VDT - related dry eye has become the main type of dry eye, with its incidence increasing year by year and showing a trend of younger patient groups. Different from other types of dry eye, VDT - related dry eye is mainly caused by adverse visual environment factors (such as long - term fixation on electronic screens, reduced blinking frequency, and being in an air - conditioned environment), rather than by systemic factors. It has been clearly established that long - term use of video display terminals is one of the independent risk factors for the occurrence and development of dry eye.

[0005] The main symptoms of dry - eye patients include ocular discomfort (such as dryness, stinging, tearing, eye fatigue) and visual impairment, which can seriously affect the quality of life and daily activities of patients (including reading, using a computer, and driving), and are 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 brings a huge direct economic burden, which has become a social public health problem that has attracted much attention.

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

[0007] Findings elaborating on the basic inflammatory mechanisms and pathways in DED have promoted a deeper understanding of the role of inflammation in the pathogenesis of this disease. The ocular surface epithelial cells play a key role in the occurrence and development of the dry eye inflammatory response. The elevated HLA-DR level in the conjunctival epithelium of dry eye patients has been used as a biomarker in clinical trials. In addition, epithelial disruption caused by a dry environment can trigger innate immune responses and subsequent adaptive immune responses, further supporting the key role of epithelial activation in dry eye. However, there are still many gaps in the existing research on the regulation of the immune microenvironment and immune responses involved by epithelial cells, and there is still a lack of research on how the pro-inflammatory effects of epithelial cells are regulated and their interaction patterns with immune cells. It is necessary to further search for the key targets for the occurrence, development, and chronic persistence of dry eye inflammation, so as to achieve more effective diagnosis and treatment of dry eye. In addition, the acute and chronic courses of dry eye involve different pathogenic mechanisms. Acute dry eye is related to the hyperosmotic environment of tears stimulating the ocular surface epithelium to release inflammatory factors, while chronic dry eye may be related to the disorder of ocular surface immune homeostasis. This emphasizes the importance of targeted treatment. Understanding these differences is crucial for formulating effective treatment strategies and improving treatment effects. Especially for the subtype of VDT-related dry eye, it is urgent to clarify its specific pathogenic mechanism, so as to develop specialized diagnostic and treatment programs. Summary of the Invention

[0008] 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, for the first time reveals the phenomenon of epithelial-mesenchymal transition (EMT) of ocular surface epithelial cells in VDT-related dry eye, and for the first time elaborates that epithelial cells exhibit fibroblast characteristics and pro-inflammatory properties in the acute stage of dry eye, thereby distinguishing the acute stage and chronic stage 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 courses of VDT-related dry eye patients are provided. In addition, the intervention in the process of epithelial-mesenchymal transition and fibrosis is an effective strategy for alleviating VDT-related dry eye inflammation and improving ocular surface damage, providing a potential intervention direction and prospect for the clinical treatment of VDT-related dry eye.

[0009] The primary object of the present invention is to provide biomarkers for the diagnosis of VDT-related dry eye or for monitoring the efficacy of its treatment and their applications. The second object of the present invention is to provide biomarkers for distinguishing the acute and chronic courses of VDT-related dry eye patients. The third object of the present invention is to provide the application of anti-fibrotic drugs in the preparation of drugs for inhibiting the process of epithelial-mesenchymal transition and fibrosis in the ocular surface of VDT-related dry eye.

[0010] To achieve the object of the present invention, the technical solution of the present invention is as follows:

[0011] In the first aspect, the present invention provides the application of any one or more of the following genes as biomarkers in VDT-related dry eye in the preparation of reagents or kits for the diagnosis of VDT-related dry eye or for monitoring the efficacy of its treatment, and the genes include: AQP3, SNAI1, TWIST1, CCL2, CCL7, CCL8, IGFBP2, TGF-β, MUC5AC;

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

[0013] In the second aspect, the present invention provides the application of a reagent for detecting the expression of a target gene in the preparation of a reagent or kit for the diagnosis of VDT-related dry eye or for monitoring the efficacy of its treatment, and 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, IGFBP2.

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

[0016] The reagent includes a reagent 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 reagent;

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

[0019] Preferably, the genes include TWIST1, SNAI1, 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 courses of VDT-related dry eye patients. The genes include: AQP3, CCL2, CCL7, CCL8.

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

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

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

[0024] The reagent includes 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;

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

[0026] The kit contains the aforementioned reagent;

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

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

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

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

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

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

[0033] The data input module is used to input the expression level value of the target gene in the biological test sample of the subject detected;

[0034] The data comparison module is used to compare the expression level value of the target gene in the biological test sample of the subject with the control value, and the control value is the expression level value of the target gene in the sample of healthy subjects;

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

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

[0037] The greater than or less than specifically may be greater than or less than with statistical significance; among them, the target genes with the expression level value in the biological test sample of the subject greater than the control value are TWIST1, SNAI1, IGFBP2; the target gene with the expression level value in the biological test sample of the subject less than the control value is MUC5AC.

[0038] In an eighth aspect, the present invention provides the 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 beneficial effects of the present invention are:

[0041] The present invention first expounds the phenomenon of epithelial-mesenchymal transition (EMT) in the ocular surface epithelial cells in VDT-related dry eye. In the acute stage of VDT-related dry eye, epithelial cells exhibit fibroblast characteristics and pro-inflammatory properties, thereby distinguishing the acute stage from the chronic stage of VDT-related dry eye. Using single-cell sequencing and related research, the present invention first reveals biomarkers for diagnosing VDT-related dry eye or monitoring the efficacy of its treatment. With these biomarkers, reagents or kits can be prepared for diagnosing VDT-related dry eye, monitoring the efficacy of its treatment, or distinguishing the acute and chronic courses of VDT-related dry eye patients.

[0042] The biomarkers provided by the present invention help to better understand the pathophysiology of VDT-related dry eye, and will provide new opportunities for diagnosis and prognosis, thereby improving the 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 mouse VDT-related dry eye model, and has a significant effect on relieving the symptoms of VDT-related dry eye and reducing ocular surface damage. Brief Description of the Drawings

[0044] Figure 1 : A. Uniform Manifold Approximation and Projection (UMAP) maps of epithelial cell subsets in the normal group (NC), the 1-week group (DED1W), and the 3-week group (DED3W) of the ICES dry eye model. Colored by major cell type. B. Violin plots showing the expression of cell-specific marker genes used to identify the identity of epithelial cell subsets. C. Flow cytometry analysis of the cell population that simultaneously expresses the basal epithelial cell marker Cytokeratin Pan and the fibroblast marker CD140a in the mouse conjunctiva and its proportion in non-immune cells. D. Flow cytometry analysis of the proportion of AQP3-positive fibroblast-like cells in the conjunctiva of dry eye model mice. E. Results of cell trajectory analysis by Monocle2 colored according to cell subsets. Numbers 1 and 2 are potential differentiation nodes between cell subsets.

[0045] Figure 2: A. Gene set signature scores were calculated for each subpopulation of epithelial cells (left) and different stages of dry eye (right) using cell stemness gene sets and epithelial-mesenchymal transition gene sets. P values were calculated by two-sided Wilcoxon rank-sum test. B. Violin plots show 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 by two-sided Wilcoxon rank-sum test. C. Real-time quantitative PCR was used to evaluate 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 ± SEM, P values were calculated by unpaired two-sided t-test. D. Real-time quantitative PCR was used to evaluate the expression level of TWIST1 in the conjunctiva at different stages of dry eye. Mean ± SEM, P values were calculated by one-way ANOVA. E. UMAP plot colored according to the activities of TGF-β signaling pathway and chemokine signaling pathway.

[0046] Figure 3 : Representative immunofluorescence images of frozen sections of conjunctival tissue 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 show the expression levels of key biomarkers in epithelial cells at different stages of dry eye. P values were calculated by two-sided Wilcoxon rank-sum test.

[0048] Figure 5: A. Use GSVA to evaluate the differences in pathway activities of epithelial cell subsets in dry eye based on Hallmark gene sets. B. Gene set characteristic scores of acute inflammatory response gene sets in each epithelial cell subset during the course of dry eye. The scores of each subset are shown from left to right for normal, 1-week dry eye, and 3-week dry eye. The gene set is from the GO database (GO:0002526). C. Violin plots show the expression of MHC-II molecule genes in epithelial cell subtypes at different stages of dry eye. D. Violin plots show the expression of representative genes within the CCL / CXCL signaling pathway in fibroblast-like epithelial cells. E. The overall number of cell-cell interactions among immune cells, epithelial cells, and stromal cells. Epithelial cells, epithelial cells; Stromal cells, stromal cells; Immunecells, stromal cells. F. Dot plot shows ligand-receptor pairs upregulated in 1W dry eye with CD4+ T cells as the receiving signalers. Among them, epithelial cells and myeloid cell subsets are the sending signalers. The P value of all signals is < 0.01. G. CCL2-CCR2 signaling network during the course of dry eye. Nodes represent major cell types, and the thickness of the lines represents the strength of the interaction.

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

[0050] Figure 7 : A. Schematic diagram of the experimental design for the impact of long-term VDT use on the ocular surface microenvironment. B. The number of eyes with corneal fluorescein sodium scores and score grades at each time point. C. The number of eyes with tear film break-up time and grades at each time point. D. EMT characteristic scores calculated based on GSVA for three groups. P values were calculated by paired t-tests. The EMT gene set is from the GO database (GO:0010718). E. Expression levels of CCL2 in three groups.

[0051] Figure 8 : Changes in the expression levels of key biomarkers in the ocular surface microenvironment at each time point of long-term VDT use. P values were calculated by paired t-tests.

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

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

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

[0055] D. Flow cytometry was used to analyze the double-positive cell population that simultaneously expressed the epithelial cell marker CK PAN and the fibroblast marker CD140a and its CCL2 expression level in the conjunctival tissue of mice in each group.

[0056] E. Flow cytometry was used to analyze 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 :Results graph of immunofluorescence detection of epithelial-mesenchymal transition and fibrosis-related indicators in the conjunctiva before and after treatment of the VDT-related dry eye model;

[0058] A. Staining with anti-CCL2 (green) and anti-IGFBP2 (red) antibodies showed that the expression of CCL2 and IGFBP2 in the conjunctival tissue decreased significantly after treatment with the two anti-fibrotic drugs; Scale bar: 100μm;

[0059] B. Staining with anti-N-cadherin (green) and anti-E-cadherin (red) antibodies showed that the expression of the epithelial cell mesenchymalization marker N-cadherin induced by dry eye decreased after treatment with the anti-fibrotic drug; Scale bar: 100μm;

[0060] C. Staining with anti-CD45 (red) antibody showed that the infiltration of immune cells in the conjunctival stroma of the dry eye model group increased significantly, and the infiltration of immune cells decreased significantly after treatment with the anti-fibrotic drug; Scale bar: 100μm; The cell nuclei were stained with DAPI (blue). Detailed implementation manners

[0061] Definition

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

[0063] Unless otherwise specified, 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 "including", 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 disclosed content supports the definition of the term "or" as being only alternatives and "and / or", unless explicitly stated as being only alternatives or mutually exclusive between alternatives, the term "or" in the claims refers to "and / or".

[0065] In the present invention, the term "dry eye (DED)" is a chronic ocular surface disease caused by multiple factors, which is caused by the abnormal quality, quantity, and dynamics of tears, resulting in unstable tear film or imbalance of the ocular surface microenvironment. It may be accompanied by ocular surface inflammatory reaction, tissue damage, and nerve abnormalities, and can cause various ocular discomfort symptoms and / or visual function disorders.

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

[0067] In the present invention, the term "VDT-related dry eye" specifically refers to a chronic ocular surface disease caused by lifestyle factors such as long-term operation of video display terminals, resulting in abnormal tear dynamics (such as reduced blink frequency, incomplete blinking, etc.) and increased tear evaporation, causing unstable tear film or imbalance of the ocular surface microenvironment. It may be accompanied by ocular surface inflammatory reaction and tissue damage, resulting in ocular discomfort symptoms and visual function disorders. VDT-related dry eye is one of the main subtypes of dry eye, and its onset is closely related to long-term exposure to the VDT environment.

[0068] In the present invention, the term "Biomarker" refers to a biochemical index that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell, and subcellular structure. It can be used for disease diagnosis, determining disease stages, or evaluating the safety and effectiveness of new drugs or new therapies in the target population, etc.

[0069] In the present invention, "Epithelial Mesenchymal Transition (EMT)" refers to the process by which epithelial cells lose cell-cell adhesion and polarity, acquire migratory and invasive capabilities, and thus transform into mesenchymal cells, playing an important role in embryonic development, chronic inflammation, tissue remodeling, cancer metastasis, and various fibrotic diseases.

[0070] In the present invention, "conjunctival fibrosis" refers to the pathological process in which the conjunctival tissue of the eye undergoes excessive collagen deposition due to tissue damage, chronic inflammation, or postoperative reaction, resulting in the replacement of normal conjunctival tissue by fibrous tissue, thereby impairing the function of the conjunctiva.

[0071] In the present invention, the "acute course of VDT-related dry eye" refers to the initial stage of the onset of VDT-related dry eye, usually caused by irritation or stress on the ocular surface. In this stage, the innate immune system produces a rapid response, which in turn activates the adaptive immune response, leading to acute discomfort symptoms and / or visual dysfunction in the eyes.

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

[0073] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only illustrative of and explanatory 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 covered within the scope of protection intended by the present invention.

[0074] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products 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] I. Experimental materials

[0077] 1. Animals

[0078] Shanghai Jieshijie Laboratory Animal Co., Ltd. obtained SPF-grade wild-type female C57BL / 6J mice (age: 6-8 weeks; weight: 18-25 g). Inclusion criteria for experimental mice: healthy mice without corneal infection, corneal ulcer, corneal scar, and leukoma under slit lamp and with a corneal fluorescein sodium staining score less than 10. All experimental procedures complied with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) on the use of animals in ophthalmic and vision research, and the protocol was approved by the Experimental Animal Ethics Committee of Wenzhou Medical University.

[0079] 2. Main instruments

[0080]

[0081] 3. Main reagents

[0082]

[0083]

[0084] 4. Antibodies and reagents used in flow cytometry:

[0085] APC / Cy7 anti-mouse CD45 (Cat: 557659, clone: 30-F11, BD), Alexa Fluor488 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), BrilliantViolet 711 anti-mouse γδT (Cat: 563994, clone: GL3, BD), PerCP / Cyanine5.5 anti-mouseCD8a (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.

[0086] 5. PCR primer sequences:

[0087] GAPDH: sense strand, 5’-ATGTTCGTCATGGGTGTGAA-3’,

[0088] antisense strand, 5’-GGTGCTAAGCAGTTGGTGGT-3’;

[0089] Cdh1: sense strand, 5’-CAGGTCTCCTCATGGCTTTGC-3’,

[0090] antisense strand, 5’-CTTCCGAAAAGAAGGCTGTCC-3’;

[0091] Cdh2: sense strand, 5’-AGCGCAGTCTTACCGAAGG-3’,

[0092] antisense strand, 5’-TCGCTGCTTTCATACTGAACTTT-3’;

[0093] Snai1: sense strand, 5’-CACACGCTGCCTTGTGTCT-3’,

[0094] antisense strand, 5’-GGTCAGCAAAAGCACGGTT-3’;

[0095] Zeb1: sense strand, 5’-GCTGGCAAGACAACGTGAAAG-3’,

[0096] antisense strand, 5’-GCCTCAGGATAAATGACGGC-3’;

[0097] Twist1: sense strand, 5’-TCGCAAGAGACGCAGCAGTC-3’,

[0098] antisense strand, 5’-CCTCCGCCCGCAGATTTCTT-3’.

[0099] 6. Antibodies used for immunofluorescence staining:

[0100] anti-E-Cadherin antibody (dilution: 1:200, Cat: 14-3249-82, Thermo), anti-N-cadherin antibody (dilution: 1:200, Cat: PA5-85341, Thermo), Alexa Fluor488 cross-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).

[0101] II. Experimental methods

[0102] 1. Establishment of dry eye animal models

[0103] To induce VDT-related dry eye models, mice were placed in an Intelligent Controlled Environment System (ICES) to simulate the VDT environment (humidity 15 ± 3%, wind speed 2 m / s, temperature 22 ± 1°C) for 1 week and 3 weeks, serving as the 1-week and 3-week VDT-related dry eye groups. Control group mice were fed in a standard environment (humidity 60% - 80%, temperature 22 ± 1°C) without any treatment. At weeks 0, 1, and 3, corneal epithelial staining was graded in a single-blind manner according to the standard grading system (National Eye Institute, Bethesda, MD, USA) to evaluate the condition of the corneal epithelium.

[0104] 2. Preparation of single-cell suspensions

[0105] After euthanizing the mice, the palpebral conjunctiva and bulbar conjunctiva were excised to obtain the conjunctiva. The conjunctiva from three mice in each group was pooled as a sample and stored in MACS tissue storage solution (Miltenyi Biotec). The sample was washed in phosphate-buffered saline containing 20 mM EDTA and shaken at 37 °C for 15 minutes. Then the tissue was minced and digested in RPMI1640 medium (Thermo Fisher Scientific) containing collagenase IV (Gibco; 1000 U / mL) at 37 °C for 30 minutes, and then filtered through a 70-μm cell strainer. Red blood cells and dead cells were removed using red blood cell lysis buffer (Sigma Aldrich) and dead cell removal kit (Miltenyi Biotec), respectively. The 10-μl suspension was counted using a hemocytometer under an inverted microscope. The cells were observed under a microscope after staining with trypan blue.

[0106] 3. Library Preparation and Single-Cell Sequencing

[0107] 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 a Chromium Single Cell Controller instrument (10×Genomics, Pleasanton, CA, USA) to generate single-cell gel bead in emulsion (GEMs). Subsequently, reverse transcription, cDNA amplification, fragmentation, end repair, adapter ligation, library amplification, circularization, and DNA nanoball (DNB) generation were performed. The constructed libraries were sequenced in 100-bp paired-end mode using combinatorial probe anchor synthesis (cPAS) on the BGI DNBseq platform (BGI, Shenzhen, China).

[0108] 4. Raw Data Processing and Quality Control

[0109] Transcripts were mapped to the mouse reference genome (mm10 - 2020 - A) and reads were quantified using Cell Ranger (version 7.0.1, 10x Genomics) to generate a gene expression matrix for each sample. The expression matrix was then imported into the Seurat R package (version 4.1.0) and subjected to preliminary quality control with the following exclusion criteria: cells expressing fewer than 200 or more than 7,500 genes, cells with more than 80,000 detected molecules, and cells with a mitochondrial gene proportion exceeding 15%. The Scrublet package in Python was used to identify potential doublets, with the expected doublet rate 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 3,000 highly variable genes to obtain the top 50 principal components. These principal components were input into the Harmony package (version 0.1.0) to remove batch effects between different samples.

[0110] 5. Clustering, Annotation, and Visualization

[0111] The Seurat's ElbowPlot function was used to determine the optimal number of principal components (PCs) for clustering. Unsupervised clustering with a resolution of 1.0 was performed on the top 20 PCs using the shared nearest neighbor (SNN) algorithm. The clustering results were visualized using uniform manifold approximation and projection (UMAP). The FindMarkers function was used to identify marker genes for each cluster and compared with established cell - type markers for cell annotation. In addition, specific strategies were adopted for each cell subset dataset to remove clusters that did not meet the set criteria or had over - representation of certain markers: for epithelial cells, the mitochondrial gene count 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.

[0112] 6. Single - cell Sequencing - Enrichment Analysis and Gene Set Scoring

[0113] To characterize the biological processes of different cell subsets, enrichment analysis of differentially expressed genes was performed using the ClusterProfiler package (version 3.18.1) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Gene Ontology (GO) database, and Hallmark database. Gene set variation analysis (GSVA) was performed using the GSVA package (version 1.38.2), and the gsva function was used to estimate the pathway activity score. Pathways with significant differences (adjusted p-value < 0.05) were visualized using the pheatmap package (v1.0.12) and ggplot2 package (v3.2.1) in R. The AddModuleScore function of Seurat was used to calculate the feature scores of gene sets, and the gene sets were downloaded from the Molecular Signatures Database (MSigDB).

[0114] 7. Cell trajectory and RNA velocity analysis

[0115] The cell developmental trajectories of epithelial cells were inferred using Monocle2 (version 2.26.0). The gene expression matrix of all cells from these cell subsets was used as the input for Monocle2. After quality control and selection of highly variable genes, dimensionality reduction was performed using the DDRTree algorithm, and the cell trajectories were visualized according to cell subsets and cell states. The loom file containing mRNA splicing and unspliced information was generated using the velocyto package in python based on the bam file obtained by aligning Cell Ranger with the genome, and then input into scVelo to calculate the RNA velocity. The parameter settings were as follows: counts = 30, n_top_genes = 2000, n_pcs = 30, and n_neighbors = 30.

[0116] 8. Flow cytometry

[0117] The preparation of single-cell suspension was as described above. For cell surface staining, cells were blocked with CD16 / 32 FcR-block (Cat: 101302, BioLegend) for 10 minutes. After staining with Fixable Viability Stain (Cat: 564406, BD), the obtained cells were stained with surface markers at 4 °C for 30 minutes. For intracellular staining, cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (Cat: 00-5523-00, Invitrogen), and then intracellular staining was performed with appropriate fluorescently labeled antibodies in permeabilization buffer at 4 °C for 45 minutes. Cells were acquired on a flow cytometer (Attune Nxt V6, Thermo). Flow cytometer analysis was performed using Flowjo software (version 10.5.3, BD).

[0118] 9. Real-time quantitative PCR

[0119] Total RNA of the conjunctiva was extracted according to the manufacturer's instructions (Cat: 74106, RNeasy mini kit, Qiagen, Crawley, U.K.). 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 an Applied Biosystems Quant Studio 6 Real-Time PCR System (Applied Biosystems, Paisley, UK). Results were analyzed using the comparative threshold cycle (CT) method and normalized using GAPDH as an endogenous reference.

[0120] 10. Immunofluorescence staining

[0121] For each group, the conjunctiva and eyeballs of three mice were taken, embedded with optimal cutting temperature compound (Cat: 4583, SAKURA), and then frozen with liquid nitrogen. These samples were subsequently cut into 10-mm thick sections and stored at -80 °C. When staining, the sections were thawed at room temperature. After thawing, they were fixed in 4% formaldehyde for 15 minutes. Then they were blocked with blocking buffer (Cat: ab64226, Abcam) containing 0.4% Triton X-100 (Cat: T8787, Sigma Aldrich) at room temperature for >60 minutes. After that, the sections were incubated with the primary antibody and the corresponding fluorescently conjugated secondary antibody in the recommended order by the manufacturer. 4',6-Diamidino-2-phenylindole (Cat: S36938, DAPI, Invitrogen) was added for 5 minutes. Images were taken using a laser scanning confocal microscope (LSM880, Carl Zeiss Meditec, Sartrouville, Germany).

[0122] III. Experimental Results

[0123] Figure 1 A Analysis based on single-cell sequencing results found that conjunctival epithelial cells were abnormally increased in VDT-related dry eye, especially in the acute course, including basal epithelial cells highly expressing basal epithelial cell markers KRT15, KRT6A, and KRT14, fibroblast-like epithelial cells (FLEpi) co-expressing basal epithelial cell markers and fibroblast marker CD140a, and endothelial-like epithelial cells (ELEpi) co-expressing basal epithelial cell markers and endothelial cell marker Pecam1. In addition, superficial epithelial cells (Sup Epi) and goblet cells were decreased. Figure 1 B These increased epithelial cell subsets specifically expressed aquaporin AQP3, and the same phenomenon was not found in other epithelial cell subsets and stromal cells. Figure 1 C Detection of the fibroblast-like epithelial subset by flow cytometry confirmed the existence of this subset, and it was also found that this subset was significantly increased during VDT-related dry eye and highly expressed AQP3( Figure 1 D). Figure 1 E Cell trajectory analysis found that the fibroblast-like epithelial subset was amplified and differentiated from basal epithelial cells. These results indicate that epithelial cells proliferate and differentiate during VDT-related dry eye and generate a new cell subset with fibroblast characteristics.

[0124] Figure 2 Gene set scoring revealed that epithelial cell stemness was significantly upregulated at both 1 week and 3 weeks in VDT-related dry eye, while the epithelial-mesenchymal transition (EMT) pathway was specifically upregulated at 1 week in VDT-related dry eye. Among them, fibroblast-like epithelial cells obtained the highest EMT score in the epithelial cell subset, indicating that its generation may be related to the EMT process. Figure 2 B showed that EMT-related markers (CDH2, SNAI1, ZEB1, TWIST1) were all significantly upregulated at 1 week in VDT-related dry eye, with TWIST1 being the most significant. Real-time quantitative PCR also found that Twist1, Snai1, and Zeb1 were significantly upregulated in VDT-related dry eye at 1 week ( Figure 2 C), where Twist1 was specifically upregulated in acute dry eye, while there was no significant difference compared with the normal group in chronic dry eye ( Figure 2 D). Figure 2 E Gene set characteristic scoring of epithelial cell subsets found that the TGF-β signaling pathway was significantly upregulated in fibroblast-like epithelial subsets at 1 week in VDT-related dry eye, which is an important pathway for inducing EMT. Compared with the control group, during VDT-related dry eye, the conjunctival epithelium showed a transition from E-cadherin to N-cadherin expression and migration to the conjunctival stromal layer, demonstrating a classic epithelial-mesenchymal transition (EMT) phenomenon ( Figure 3 ). On the other hand, fibroblast-like epithelium became the main source of chemokines in epithelial cells at 1 week in VDT-related dry eye and was slightly downregulated at 3 weeks in VDT-related dry eye ( Figure 2 E). This may mean that fibroblast-like epithelium mainly plays an important role in recruiting immune cells and promoting inflammatory responses in VDT-related dry eye.

[0125] From the above results, it can be seen that in the ICES model of mice simulating VDT-related dry eye, conjunctival epithelial cells strongly expressed the specific marker AQP3 and exhibited EMT phenomenon. Among them, fibroblast-like epithelial cells obtained the highest EMT score in the epithelial cell subset, indicating that its generation is related to the EMT process. Figure 2 B and Figure 4 showed that EMT-related markers (CDH2, SNAI1, ZEB1, TWIST1) were all significantly upregulated at 1 week in VDT-related dry eye, remained at a high level at 3 weeks, and were the most significant with TWIST1 and SNAI1. 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.

[0126] Example 2 Fibroblast-like epithelial cells are closely related to the inflammatory response

[0127] I. Experimental materials

[0128] The following antibodies and reagents were used in 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

[0129] 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).

[0130] II. Experimental methods

[0131] 1. Single-cell sequencing - cell communication analysis

[0132] Other analysis methods of single-cell sequencing were the same as those in Example 1.

[0133] The present invention uses the CellChat package (version 1.5.0) to infer the intercellular interaction network between different cell types based on ligand-receptor pairs in the CellChatDB mouse database. The number of communications between groups was compared, and the results were visualized using the netVisual_bubble function. Important ligand-receptor pairs of specific pathways were extracted, and 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 specific signaling pathways, and the main signaling roles were determined using network centrality scores and the netAnalysis_signalingRole_network function. 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 regulating epithelial cell differentiation during dry eye. The ligand-target gene connections were inferred using the minimum LFC method with a cut-off value of 0.75, and the upregulated receptor-ligand pairs in dry eye were visualized. The ligand-target gene heatmap showed the potential regulatory scores of the top-ranked ligands and their target genes.

[0134] 2. Flow cytometry

[0135] The specific steps are as described in Example 1.

[0136] 3. Immunofluorescence

[0137] The mouse conjunctiva and eyeballs were excised and embedded in OCT (optimal cutting temperature compound, Cat: 4583, Sakura), frozen in liquid nitrogen, and then cut into 10-μm sections. The sections were stored at -80 °C. When staining, the sections were thawed at room temperature (RT). After thawing, the sections were fixed in 4% formaldehyde for 15 minutes. Then, they 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 the primary antibody and the corresponding fluorescently labeled secondary antibody in the order recommended by the manufacturer. 4',6-Diamidino-2-phenylindole (Cat: S36938, DAPI, Invitrogen) was added for 5 minutes. Images were taken using a laser scanning confocal microscope (LSM880, Carl Zeiss Meditec, Sartrouville, Germany).

[0138] III. Experimental Results

[0139] Figure 5 A showed that fibroblast-like epithelial cells in VDT-related dry eye were enriched in the "IL-2 / STAT-5 signaling pathway", "IL-6 / JAK / STAT3 signaling pathway", and "inflammatory response" through gene set variation analysis. Meanwhile Figure 5 B showed that this group had the highest acute inflammatory response score and was at the highest value at 1 week. This means that this subgroup plays an important role in promoting the inflammatory response during the acute phase of VDT-related dry eye. Figure 5 C showed that MHC-II molecules were significantly upregulated in epithelial cells during VDT-related dry eye, which would help activate pathogenic T cells and initiate adaptive immune responses. Meanwhile, fibroblast-like epithelial cells showed widespread chemokine upregulation at 1 week of VDT-related dry eye ( Figure 5 D), such as CCL2, CCL7, and CCL8, which can bind to CCR2 and mediate monocyte / macrophage recruitment. Figure 5 E showed that the interaction between epithelial cells and immune cells in VDT-related dry eye increased significantly through cell communication analysis. Systematic analysis of the receptor-ligand interactions between epithelial cells and myeloid cells and CD4+ T cells showed that the most abundant enhanced chemokine-related ligand-receptor pairs were demonstrated in fibroblast-like epithelial cells during VDT-related dry eye, including Ccl2-Ccr2, Ccl7-Ccr2, Cxcl13-Cxcr3, and Cxcl10-Cxcr3 ( Figure 5 F), and enhanced interactions of fibronectin Fn1 and integrin were also observed, which are common molecules involved in cell adhesion. Figure 5 G detected enhanced communication of fibroblast-like epithelial cells with multiple immune cells (CD4+ T cells, γδ T cells, ILC2, NK, and macrophages) through CCL2 expression at 1 week of VDT-related dry eye, indicating their recruitment effect on these immune cells.

[0140] Figure 6 Immunofluorescence staining in A confirmed the co-localization of CCL2 with epithelial cells highly expressing AQP3, and a basal epithelial cell population with high CCL2 expression and stromal expansion was identified in VDT-related dry eye. Figure 6 Flow cytometry was used in B-C and found that the receptor CCR2 of CCL2 was highly expressed in various immune cells on the ocular surface, and CD68+ macrophages and CD4+ T cells were the main cell types expressing CCR2.

[0141] Figure 6In D, NicheNet analysis was used to link ligands and target genes to predict cell - cell interactions, and it was found that the signals of myeloid cells might promote the differentiation of epithelial cells into fibroblasts. Specifically, at 1 week of VDT - related dry eye, TGF - β1 and other factors produced by myeloid cells (mainly macrophages) 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 6 In E, PCR was also used to confirm the up - regulation of the expression of TGF - β1 and IGFBP2 on the ocular surface in VDT - related dry eye. This indicates that macrophages may enhance the pro - inflammatory effect of epithelial cells and regulate the fate of epithelial cells differentiating into fibroblast - like cells by secreting TGF - β1 and APOE to change the expression pattern of epithelial cells in VDT - related dry eye. Furthermore, as the main central regulator of inflammation and stress signals, epithelial cells chemotax CD4+T cells and myeloid cell populations to infiltrate, thus maintaining the vicious cycle of dry - eye inflammation.

[0142] From the above results, it can be seen that epithelial cells undergo fate changes during VDT - related dry eye, generating a sub - population of fibroblast - like epithelial cells, and playing a pro - inflammatory role by secreting chemokines during the acute phase, actively regulating the immune microenvironment of the ocular surface. The effector molecules related to this process can be used as biomarkers for the diagnosis of VDT - related dry eye, including TGF - β1 that regulates the fate change of epithelial cells and the corresponding target IGFBP2, as well as the key chemokines CCL2, CCL7, and CCL8 secreted by epithelial cells.

[0143] Example 3 Up - regulation of factors related to epithelial cell phenotype changes in dry - eye patients with long - term VDT use

[0144] I. Experimental materials

[0145] Human samples

[0146] A total of 26 healthy subjects aged 18 - 50 years participated. These healthy subjects had no history of eye trauma or surgery (including refractive surgery), no ocular medical history other than refractive errors, no recent contact - lens use, no autoimmune diseases or recent use of immunosuppressants, did not participate in other clinical trials, and had 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 included according to the following inclusion and exclusion criteria.

[0147] Exclusion criteria are as follows: a history of previous eye trauma or eye surgery (including refractive surgery); a confirmed history of other eye diseases except dry eye and refractive errors; those who still wore contact lenses 1 week before the examination; those with a definite autoimmune disease or who had used immunosuppressants within 1 month; patients who were simultaneously participating in any other clinical trials; and those with systemic diseases that the investigator judged might be unsuitable for participating in this study.

[0148] II. Experimental methods

[0149] 1. Study design

[0150] The study will last for 4 weeks (28 days) and is divided into two phases. In the first phase (week 0), the subjects need to keep the daily VDT usage time ≤ 3 hours; in the second phase (weeks 1, 2, and 3), the subjects need to keep the VDT usage time > 8 hours. The subjects need to complete 4 times of questionnaire collection and eye examinations at the end of weeks 0, 1, 2, and 3, and collect 3 times of ocular surface cell samples at the end of weeks 0, 1, and 3.

[0151] 2. Filling out the questionnaire by the subjects

[0152] The subjects complete the Ocular Surface Disease Index (OSDI) according to their own situations. To ensure the accuracy and credibility of the scale, before the subjects fill out the questionnaire, the researchers will explain the questionnaire. For example, in the OSDI questionnaire, the option of "not applicable" means no recent above activities or not in the above environment, while the option of "none" means no symptoms when having the above activities or being in the above environment recently.

[0153] 3. Eye examinations of the subjects

[0154] The examination contents are carried out in the following order according to the principle of non-invasive first and then invasive: meibography, slit lamp biomicroscopy, and collection of ocular surface cell samples. Except for the collection of ocular surface cell samples from the right eye, all other examinations are performed on both eyes.

[0155] 4. Slit lamp biomicroscopy

[0156] 4.1 Corneal fluorescein sodium staining

[0157] After moistening the fluorescein sodium filter paper strip with a drop of tobramycin eye drops, gently apply it to the lateral palpebral conjunctiva of the lower eyelid. Instruct the subject to roll the eyes, and observe under cobalt blue light using a slit lamp microscope. Green-stained corneal epithelial defects can be seen. Record the fluorescein sodium staining scores of the corneal regions at 5 locations: superior, inferior, nasal, temporal, and central. Each region is scored from 0 to 3 according to the severity, with a maximum of 15 points. The specific scoring is as follows: 0 points, no punctate staining; 1 point, 1 - 30 punctate stainings; 2 points, ≥30 punctate stainings but no fusion; 3 points, punctate stainings with fusion or filaments.

[0158] 4.2 Lissamine green staining of the conjunctiva

[0159] After moistening the Lissamine green filter paper strip with a drop of tobramycin eye drops, gently apply it to the lateral palpebral conjunctiva of the lower eyelid. Instruct the subject to roll the eyes, and observe under white light using a slit lamp microscope. Record the Lissamine green staining scores of the conjunctival regions at 2 locations: nasal and temporal. Each region is scored from 0 to 3 according to the severity, with a maximum of 6 points. The specific scoring is as follows: 0 points: 0 - 9 staining points; 1 point: 10 - 32 staining points; 2 points: 33 - 100 staining points; 3 points: ≥100 staining points.

[0160] 4.3 Fluorescein sodium tear film break-up time

[0161] After staining the ocular surface with fluorescein sodium, instruct the subject to blink 3 times and then open the eyes. Use a stopwatch to record the time from when the subject opens the eyes until the first black spot appears on the tear film as the tear film break-up time. Check 3 times in total and take the average as the measurement result.

[0162] 5. Diagnosis of dry eye

[0163] According to the dry eye diagnostic criteria proposed in the "New Perspectives on the Definition and Diagnosis of Dry Eye: The Consensus Report of the Asian Dry Eye Society (ADES)" published by the Asian Dry Eye Society (ADES) in 2017, the presence of subjective symptoms and the tear film break-up time will be used as diagnostic indicators. In this study, the diagnostic criteria for dry eye are OSDI score ≥13 points and FBUT < 5 seconds.

[0164] 6. Collection and preservation of ocular surface cell samples

[0165] All ocular surface cell samples are collected from the right eye of the subject. Instill a drop of proparacaine hydrochloride eye drops into the conjunctival sac of the lower eyelid, and instruct the subject to roll the eyes. Use 2 pharyngeal swabs to gently wipe the superior and inferior palpebral conjunctiva 3 times each, and use 1 pharyngeal swab to gently wipe the superior, inferior, nasal, and temporal bulbar conjunctiva once each. Break off the tips of the 3 pharyngeal swabs and put them into TRIzol (ThermoFisher, 15596018), and store them at -80 °C for bulk RNA sequencing.

[0166] 7. Transcriptome sequencing

[0167] Total RNA was extracted from the samples using TRIzol according to the manufacturer's instructions. The concentration and integrity of the RNA were evaluated using a Bioanalyzer 2100 and an RNA6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511). The cDNA library was subjected to paired-end sequencing with a read length ≥ 150 bp using a high-throughput sequencing platform. Low-quality reads were filtered out using Cutadapt (v1.9), and the sequence quality was verified using FastQC (v0.11.9). The reads were mapped to the human reference genome (UCSC hg38) using HISAT277 (v2.0.4). The mapped reads of each sample were assembled using StringTie (v1.3.4d), and the transcriptomes of all samples were compared and merged using gffcompare (v0.9.8). StringTie was used to estimate the expression levels of all transcripts and generate a count table. The transcript expression differences between samples were quantified using transcripts per million (TPM).

[0168] Differential expression analysis between two different groups was performed using the DESeq2 R 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 evaluate the pathway enrichment differences between groups.

[0169] III. Experimental Results

[0170] To explore the effects of VDT environmental stress on ocular surface parameters, a self-controlled before-and-after design was adopted, and 26 healthy subjects were prospectively enrolled (flow chart shown in Figure 7 A). As shown in Figure 7 B-C, with the prolongation of VDT exposure time, the corneal staining score increased, and the tear film break-up time decreased, and it remained stable when compared at 3 weeks and 2 weeks. According to the dry eye diagnosis criteria proposed by the Asian Dry Eye Society in 2017, with the presence of subjective symptoms combined with a tear film break-up time less than 5 seconds as the dry eye diagnosis criterion, it was found that the prevalence of VDT-related dry eye increased after 1 week of long-term use of VDT devices and reached the highest of 32.7% at 3 weeks. Figure 7 As shown in D, the EMT characteristic score increased in VDT-related dry eye, indicating that EMT also occurred in the human ocular surface. Figure 7 E and Figure 8It is shown that the EMT-related markers TWIST1 and SNAI1 are significantly upregulated in the conjunctival surface cells during VDT-related dry eye, and the regulatory factors TGF-β1 and IGFBP2 related to the EMT process also show an upward trend. In addition, the key marker AQP3 expressed by fibroblast-like epithelium and the chemokines CCL2, CCL7, and CCL8 are upregulated during VDT-related dry eye, while the goblet cell marker MUC5AC shows a significant decrease in acute VDT-related dry eye and partial recovery in chronic VDT-related dry eye. The expression changes of these biomarkers are the same as the trends observed in VDT-related dry eye mice, indicating that the characteristics of epithelial cells acquiring fibroblast-like and pro-inflammatory phenotypes during the course of VDT-related dry eye are conserved among species, and the biomarkers related to this process contribute to the diagnosis and precise staging of patients with VDT-related dry eye.

[0171] Example 4 Evaluate the direct role of inhibiting epithelial-mesenchymal transition and fibrosis in the treatment of VDT-related dry eye

[0172] I. Experimental materials

[0173] 1. Animals

[0174] SPF-grade wild-type female C57BL / 6J mice (age: 6 - 8 weeks; weight: 18 - 25 g) were obtained from the Zhejiang Animal Center. Inclusion criteria for experimental mice: healthy mice without corneal infection, corneal ulcer, corneal scar, and leukoplakia under slit lamp and with a corneal fluorescein sodium staining score less than 10. All experimental procedures complied with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) regarding the use of animals in ophthalmic and vision research, and the protocol was approved by the Experimental Animal Ethics Committee of Wenzhou Medical University.

[0175] 2. Main reagents

[0176] 1) Preparation of 0.5% anti-fibrotic drug solution:

[0177] (1) Weigh 20 mg of anti-fibrotic drug;

[0178] (2) Dissolve the drug in 40 μL of DMSO and dissolve it completely;

[0179] (3) Slowly add normal saline to a total volume of 4 mL;

[0180] (4) Place the solution on a shaker and shake it overnight to ensure complete mixing;

[0181] Final concentration: 0.5% (w / v).

[0182] 2) Preparation of control excipient solution:

[0183] (1) Measure 40 μL of DMSO;

[0184] (2) Add normal saline to a total volume of 4 mL;

[0185] (3) Gently mix.

[0186] 3. Antibodies and reagents used in flow cytometry:

[0187] BV711 anti-mouse F4 / 80 (Cat: 565612, clone: T45-2342, BD). The remaining antibodies and reagents are as described in Example 1.

[0188] 4. PCR primer sequences:

[0189] Twist1: Forward strand, 5’-TCGCAAGAGACGCAGCAGTC-3’;

[0190] Reverse strand, 5’-CCTCCGCCCGCAGATTTCTT-3’;

[0191] IL-1β: Forward strand, 5’-GCAACTGTTCCTGAACTCAACT-3’,

[0192] Reverse strand, 5’-ATCTTTTGGGGTCCGTCAACT-3’;

[0193] IL-17a: Forward strand, 5’-AAAGCTCAGCGTGTCCAAAC-3’,

[0194] Reverse strand, 5’-ACGTGGAACGGTTGAGGTAG-3’;

[0195] IFNγ: Forward strand, 5’-ATGAACGCTACACACTGCATC-3’,

[0196] Reverse strand, 5’-CCATCCTTTTGCCAGTTCCTC-3’;

[0197] TNFα: Forward strand, 5’-AGGCACTCCCCCAAAAGATG-3’,

[0198] Reverse strand, 5’-CCACTTGGTGGTTTGTGAGTG-3’.

[0199] 5. Antibodies used in immunofluorescence staining:

[0200] anti-IGFBP2 antibody (dilution: 1:500, Cat: ab188200, abcam), anti-CD45 antibody (dilution: 1:200, Cat: MAB114, R&D Systems). The remaining antibodies are as described in Example 1.

[0201] II. Experimental methods

[0202] 1. Research design

[0203] In this example, an intelligent controlled environment system (ICES) was used to construct a mouse model of VDT-related dry eye. The mice were placed in adverse conditions simulating the VDT environment in the ICES for 2 weeks to induce the formation of VDT-related dry eye. After the model was established, the mice continued to be raised under ICES conditions to maintain the VDT-related dry eye state, so as to be closer to real-world conditions. Subsequently, a 4-week drug eye drop treatment intervention was started for the mice with VDT-related dry eye. The drug was 0.5% anti-fibrotic drug pirfenidone or tranilast, twice a day, one drop in each eye, about 6 μL per drop.

[0204] A total of four experimental groups were set up in this example:

[0205] (1) Dry eye model without intervention (Control group, n = 9 (18 eyes in total));

[0206] (2) Dry eye + normal saline (Saline) eye drop group, n = 10 (20 eyes in total);

[0207] (3) Dry eye + pirfenidone eye drop treatment group, n = 10 (20 eyes in total)

[0208] (4) Dry eye + tranilast eye drop treatment group, n = 10 (20 eyes in total).

[0209] 2. Corneal fluorescein sodium staining

[0210] Use a pipette to drop 0.5 μL of 5% fluorescein sodium solution into the conjunctival sac of the mouse. After 3 minutes, grade the corneal epithelial staining under cobalt blue light of a slit lamp microscope. The scoring was recorded in a masked manner by fixed personnel. According to the standard grading system of the National Eye Institute of the United States, the cornea was divided into five equal parts in the central, superior, inferior, nasal, and temporal regions. The staining situation in each region was divided into grades 0 - 4 (grade 0: no staining; grade 1: 1 - 5 dots; grade 2: 6 - 15 dots; grade 3: 16 - 30 dots; grade 4: 30 dots and above). The sum of the scores in each region was the corneal fluorescein sodium staining score.

[0211] 3. Real-time quantitative PCR detection

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

[0213] 4. Flow cytometry

[0214] The specific steps are as described in Example 1.

[0215] 5. Immunofluorescence

[0216] The specific steps are as described in Example 1.

[0217] III. Experimental results

[0218] To further evaluate the direct effect of inhibiting epithelial-mesenchymal transition and fibrosis in the treatment of VDT-related dry eye, the present invention intervened in mice starting from the baseline of the VDT-related dry eye model and performed multi-level analysis before and after treatment. The experimental results are as follows:

[0219] Corneal staining and symptom relief:

[0220] The mice in each group were stained with sodium fluorescein in the cornea and observed under a slit lamp microscope at different time points ( Figure 1 A). The results showed that after 1 week of treatment with the anti-fibrotic drug, the corneal staining degree of the mice in the VDT-related dry eye model had been significantly improved ( Figure 1 A - B), and this improvement trend could still be maintained after 4 weeks of continuous treatment. It is suggested that inhibiting epithelial cell fibrosis has good early and lasting curative effects on VDT-related dry eye.

[0221] Changes in the expression of inflammatory and epithelial-mesenchymal transition (EMT)-related markers:

[0222] Real-time quantitative PCR was used to detect the expression levels of the key EMT transcription factor Twist1 and various pro-inflammatory factors (IL-1β, IL-17a, IFN-γ, TNF-α) in the conjunctival tissue. After treatment with pirfenidone and tranilast, Twist1 was significantly down-regulated, and after treatment with tranilast, the expression of the genes of IL-17a, IFN-γ, and TNF-α was significantly down-regulated ( Figure 1 C, n = 4), indicating that by inhibiting the fibrotic-like transformation of epithelial cells, the inflammatory response related to VDT-related dry eye can be effectively reduced.

[0223] Analysis of epithelial-mesenchymal transition and immune cell infiltration:

[0224] Flow cytometry results showed that the proportions of cells highly expressing CK PAN (epithelial marker) and CD140a (fibroblast marker) increased in the VDT-related dry eye model, and were accompanied by an elevated expression level of CCL2. After treatment with anti-fibrotic drugs, the proportion of this fibroblast-like epithelial cell subset and the expression level of CCL2 decreased significantly ( Figure 1 D, n = 3). In addition, the proportion of F4 / 80+ macrophages in the conjunctiva of VDT-related dry eye mice decreased significantly after treatment ( Figure 1 E, n = 3).

[0225] Immunofluorescence detection further supported the above findings ( Figure 2 A-C, n = 3):

[0226] Staining with anti-CCL2 and IGFBP2 showed that the expression of these two molecules in the conjunctival tissue was significantly downregulated after anti-fibrotic drug treatment ( Figure 2 A), indicating that the epithelial-mesenchymal transition and fibrosis-related inflammatory chemotactic signals were effectively inhibited.

[0227] The results of staining with anti-N-cad and E-cad indicated that the EMT-related changes in epithelial cells were alleviated after treatment ( Figure 2 B).

[0228] Staining with anti-CD45 showed that the degree of immune cell infiltration decreased significantly ( Figure 2 C), verifying that the inflammatory microenvironment of VDT-related dry eye was effectively improved.

[0229] In summary, inhibiting the epithelial-mesenchymal transition and fibrosis processes has a significant effect on alleviating VDT-related dry eye symptoms and reducing ocular surface damage. Using anti-fibrotic drugs such as pirfenidone and tranilast can significantly improve corneal damage in VDT-related dry eye mice, reduce the expression of pro-inflammatory and pro-fibrotic key factors such as Twist1 and CCL2 in ocular surface epithelial cells, and reduce immune cell infiltration, thereby alleviating the inflammatory response and promoting the repair of ocular surface damage. This further demonstrates the core idea of the present invention: Intervention targeting the epithelial-mesenchymal transition and fibrosis processes is an effective strategy for alleviating VDT-related dry eye inflammation and improving ocular surface damage, providing a potential intervention direction and prospect for the clinical treatment of VDT-related dry eye.

[0230] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to 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 eyes; 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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