WO2018164385A1 - Composition for diagnosing sjögren's syndrome - Google Patents

Abstract

Provided in the present invention are a diagnostic composition for differentiating dry eye from dry eye caused by Sjögren's syndrome by using ATG5 and LC3B-II as biomarkers, a diagnostic method, and a screening method thereof. It is ascertained that when ATG5 and LC3B-II are used as biomarkers, only Sjögren's syndrome, excluding non-Sjögren's syndrome, can be effectively differentiated from dry eye, and thus the present invention can be usable in related industries.

Description

Composition for the diagnosis of Sjogren's syndrome

The present invention relates to a diagnostic composition, kit, diagnostic method and screening method for diagnosing Sjogren syndrome dry eye in a dry eye using a biomarker, and more specifically, using ATG5 or LC3B-II as a biomarker, Sjogren syndrome in dry eye Only dry eyes can be diagnosed effectively.

Sjogren's syndrome can exist as a systemic autoimmune disease of the exocrine gland, as a primary disease or as a secondary disease for other well-known autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma and multiple myositis. It is characterized by lymphocytic infiltration of the exocrine glands and mucosal epithelium, causing dry eye as well as dry mouth. The pathological mechanism of Sjogren's syndrome leads to severe signs of dry eye compared to non-Sjogren's syndrome dry eye.

Specifically, dry eye syndrome is a multifactorial disease of the tear and ocular surface that causes discomfort, visual disturbances and tear film instability that can potentially damage the ocular surface. This involves increased osmolality of the tear film and inflammation of the ocular surface. The causes of the dry eye may be divided into general dry eyes, that is, non-Sjogren's syndrome and Sjogren's syndrome, and in the case of non-Sjogren's syndrome, a disease causing lacrimal gland disease, closure of the tear tube, and reduction of reflex tears.

Its common feature is the lack of "basal tears" that protect the eyes. The non-Sjogren's syndrome protects the eye when the eye is dry (lack of basal tear) or the eye is irritated by the external environment, causing "reflex tears". That is, there is a lack of "basal tears", but "reflex tears" play a role in the occurrence of eye damage. On the other hand, in case of Sjögren's syndrome, the cornea lacks a "basal tear", and even when the eye is irritated by the external environment, "reflex tear" is also not secreted. The damage is very bad.

Recent studies have reported that enhanced autophagy and apoptosis are associated with Ro / SAA and La / SSB redistribution in secretory epithelial cells of salivary glands. At the cellular level, localization and production of autoantibodies is followed by relocalization of autoantigens to the cell surface. Autodigestion is a lysosomal-mediated catabolism that maintains cell homeostasis through the degradation and reuse of cellular components and organelles. In addition to its traditional role in response to cellular stress, autodigestion is associated with the development of autoimmune diseases. In the immune system, autodigestion regulates processes such as antigen uptake and presentation, elimination of pathogens, survival of short- and long-lived immune cells and cytokine-dependent inflammation. Recent studies have shown that polymorphism in autophagy-related genes is linked to susceptibility to systemic lupus erythematosus and Crohn's disease.

Therefore, the present inventors studied to accurately diagnose non-Sjogren's syndrome and Sjogren's syndrome in dry eyes. When using ATG5 and LC3B-II, which are used as autodigestion markers as biomarkers, Sjogren except dry-sjogren syndrome in dry eyes Confirming that only the syndrome can be diagnosed effectively, the present invention has been completed.

The present invention aims to effectively diagnose Sjögren's syndrome dry eye in dry eye using ATG5 or LC3B-II as a biomarker.

In order to achieve the above object, the present invention provides a composition for diagnosing Sjogren's syndrome in dry eyes, comprising a substance measuring the expression level of ATG5 gene or LC3B-II gene.

The present invention also provides a kit for diagnosing dry eye of Sjogren's syndrome in a dry eye comprising the composition.

In addition, the present invention comprises the steps of measuring the expression level of the ATG5 gene or LC3B-II gene from a biological sample; And comparing the expression level of the gene with the expression level of the gene from a normal control sample. The method provides information for diagnosing Sjogren syndrome dry eye in dry eye.

In addition, the present invention comprises the steps of treating a test substance to a biological sample obtained from a Sjogren's syndrome patient; Analyzing the effect of the test substance on the expression of ATG5 gene or LC3B-II gene; And it provides a method for screening a dry eye treatment for Sjogren's syndrome in dry eye comprising the step of selecting a test substance that reduced the expression level of the gene compared to the control group not treated with the test substance.

The present invention is the first to identify that autophagy is involved in the development of dry eye, one of the symptoms of Sjogren's syndrome, and thus provides a biomarker use for the diagnosis of Sjogren's syndrome of ATG5 and LC3B-II, which have been used as autophagy markers. can do. The present invention can easily diagnose dry Sjoren's syndrome except dry non-Sjogren's syndrome in dry eyes, while being easily available from relatively easy biological samples such as tears, conjunctival epithelial cells, and the like.

Figure 1 shows the number of subjects in one embodiment of the present invention.

Figure 2 is a graph of (a) Western blot results and (b) relative concentration measurement results showing the conversion ratio of ATG5 protein expression and LC3B-II / I in tears in one embodiment of the present invention.

Figure 3 is a graph showing the expression level of ATG5 and LC3B-II mRNA in conjunctival epithelial cells in one embodiment of the present invention.

Figure 4 is a coordination image of ATG5 and LC3B-II immunostained conjunctiva in one embodiment of the present invention.

FIG. 5 is an image showing immunofluorescence staining results of ATG5 and LC3B-II in lacrimal glands of Sjogren's syndrome animal model (a) NOD / LtJ and (b) IκB-ζ deficient mice according to one embodiment of the present invention.

Figure 6 is a graph of (a) Western blot results and (b) relative concentration results showing the expression of ATG5 and LC3B-II proteins in tears before and after corticosteroid treatment in one embodiment of the present invention.

Figure 7 is a graph showing the expression level of ATG5 and LC3B-II mRNA in conjunctival epithelial cells before and after corticosteroid treatment in one embodiment of the present invention.

Figure 8 is an ATG5 and LC3B-II immunostaining image of the conjunctiva before and after corticosteroid treatment in one embodiment of the present invention.

9 is a view showing the results of the ROC curve analysis for ATG5 in one embodiment of the present invention.

The present invention will be described in detail. However, the present invention may be implemented by various changes or modifications, and is not limited to the embodiments described herein.

The present invention provides a composition for diagnosing dry eye of Sjogren's syndrome in a dry eye, comprising a substance measuring the expression level of an ATG5 gene or LC3B-II gene.

In the present invention, the term ATG5 (Autophagy related gene 5) is an autophagy protein necessary for maturation of the autophagy membrane and forms a junction with ATG12.

In the present invention, the term LC3B-II is an LC3-phosphatidylethanolamine conjugate, in which LC3-I, a cytosol form of the initial LC3, is conjugated with phosphatidylethanolamine through ubiquitination-like enzyme action to form LC3-II, and autophagy It is responsible for convening body members.

As used herein, the term autodigestion refers to lysosomal-mediated catabolism that maintains cell homeostasis through degradation and reuse of cytoplasmic components and organelles.

In one embodiment of the present invention, when the Sjogren syndrome dry eye diagnosis in the dry eye, the cut-off value may be 4.002023.

In one embodiment of the present invention, the ROC curve analysis of the protein digestion markers extracted from the tears of patients with dry and non-Sjogren syndrome dry eyes, AUC is 0.98, the cut-off value is It was confirmed that 4.002023, ATG5 was found to be very useful as a biomarker for the diagnosis of Sjögren's syndrome, except for non-Sjogren's syndrome that causes dry eye.

In one embodiment of the invention, the material for measuring the expression level of the gene may be used without limitation as long as it is a material capable of measuring the amount of mRNA transcribed mRNA or the protein encoded by the gene, for example, Antibodies, primers, probes, and the like, specific for ATG5 or LC3B-II.

In one embodiment of the present invention, the expression level of the gene is measured by measuring the amount of mRNA encoded by the ATG5 gene or LC3B-II gene or the protein encoded by the ATG5 gene or LC3B-II gene or LC3B-II / It may be to measure the LC3B-I conversion ratio. The expression level of the gene was measured by reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method, Northern blot, Choose from Western blots, Enzyme Linked Immuno Sorbent Assay (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry and protein microarray It may be carried out in a manner that is.

In one embodiment of the invention, the expression level of the gene can be measured in biological samples obtainable from Sjogren's syndrome patients, for example in tears, conjunctival epithelial cells, lacrimal gland samples, saliva or salivary gland epithelial cells. can do.

As used herein, the term “detect” or “measure” means to quantify the concentration of a detected or measured object. In addition, the term “diagnosis” as used herein refers to determining the susceptibility of a subject to a particular disease or condition, determining whether the subject currently has a particular disease or condition, Determining the prognosis of the disease or diseased subject, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy). For the purpose of the present invention, the diagnosis is to diagnose only Sjogren syndrome dry eye except non-Sjogren syndrome dry eye in the dry eye, the expression level of the ATG5 gene or LC3B-II gene is increased in the biological sample, Sjogren syndrome dry eye diagnosis in dry eye Can be utilized.

The term gene, as used herein, means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

As used herein, the term "biomarker" is a substance that can be determined by distinguishing a cell or tissue of a subject having a disease to be diagnosed from a normal cell or tissue, and shows an increase in a diseased cell compared to a normal cell. Organic biomolecules such as polypeptides or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.

As used herein, "a substance which measures the expression level of ATG5 gene or LC3B-II gene" means the mRNA level or protein level or LC3B- of the ATG5 gene or LC3B-II gene by confirming the expression level of ATG5 gene or LC3B-II gene. It refers to a substance or molecule capable of detecting the II / I conversion ratio, for example an antibody, primer or probe specific for ATG5 or LC3B-II.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 hydroxyl group, which can form complementary templates and base pairs and is a starting point for template strand copying. Short nucleic acid sequence that functions. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, PCR amplification using the sense and antisense primers of the ATG5 gene or LC3B-II gene can be used to determine whether the desired product is produced and the level of expression of the ATG5 gene or LC3B-II gene. The lengths of the PCR conditions, sense and antisense primers can be modified based on those known in the art, and thus the present invention is not particularly limited thereto.

As used herein, the term "probe" refers to nucleic acid fragments such as RNA or DNA, which are short to several bases to hundreds of bases, which are capable of specific binding with mRNA, and are labeled to identify the presence of a specific mRNA. Can be. Probes may be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. In the present invention, hybridization may be performed using a probe complementary to the ATG5 gene or the LC3B-II gene, and the degree of expression of the ATG5 gene or the LC3B-II gene may be diagnosed through hybridization. The selection and hybridization conditions of the appropriate probe can be modified based on what is known in the art, and thus the present invention is not particularly limited thereto.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoro) Amidate, carbamate, etc.) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to a protein expressed in the ATG5 gene or LC3B-II gene, which is a marker of the present invention, and the method for preparing the antibody is prepared using a well-known method. can do. This includes partial peptides that can be made from such proteins. The form of the antibody of the present invention is not particularly limited and any part thereof, including polyclonal antibodies, monoclonal antibodies, or antigen-binding agents, is included in the antibodies of the present invention and all immunoglobulin antibodies are included. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

The present invention also provides a kit for diagnosing dry eye of Sjogren's syndrome in a dry eye comprising the composition.

As used herein, the term “kit” may include one or more other component compositions, solutions or devices suitable for analytical methods.

In addition, the kit may include all biological or chemical reagents necessary for the selection of antibody producing cell lines, essential elements necessary for carrying out PCR, and a guide. In addition to the above primer sets, PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNases, RNAse inhibitors, DEPC-water, sterile water and the like.

The guide is a printed document which explains how to use the kit, e.g. the reaction conditions presented. The instructions may include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet. For the purposes of the present invention, when a tear sample is obtained from a dry eye patient and applied to the kit, the tear sample is contacted with the anti-ATG5 or anti-LC3B-II antibody of the kit to determine the binding between the ATG5 or LC3B-II and the antibody. The amount of protein expression of ATG5 or LC3B-II in the tear sample can be confirmed. In addition, the primer expression or probe of the kit and the tear sample may be contacted to confirm the gene expression amount of ATG5 or LC3B-II.

In addition, the present invention comprises the steps of measuring the expression level of the ATG5 gene or LC3B-II gene from a biological sample; And comparing the expression level of the gene with the expression level of the gene from a normal control sample. The method provides information for diagnosing Sjogren syndrome dry eye in dry eye.

In one embodiment of the present invention, the expression level of the gene is measured by measuring the amount of mRNA encoded by the ATG5 gene or LC3B-II gene or the protein encoded by the ATG5 gene or LC3B-II gene or LC3B-II / It may be to measure the LC3B-I conversion ratio. The expression level of the gene was measured by reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method, Northern blot, Choose from Western blots, Enzyme Linked Immuno Sorbent Assay (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry and protein microarray It may be carried out in a manner that is.

In one embodiment of the present invention, the biological sample can be used without limitation as long as it is a biological sample obtained from Sjogren's syndrome patient, and can be measured in tears, conjunctival epithelial cells, lacrimal gland samples, saliva or salivary gland epithelial cells. Can be.

In addition, the present invention comprises the steps of treating a test substance to a biological sample obtained from a Sjogren's syndrome patient; Analyzing the effect of the test substance on the expression of ATG5 gene or LC3B-II gene; And it provides a method for screening a dry eye treatment for Sjogren's syndrome in dry eye comprising the step of selecting a test substance that reduced the expression level of the gene compared to the control group not treated with the test substance.

In addition, the present invention comprises the steps of obtaining a tear sample from a dry eye patient;

Measuring the amount of protein expression of ATG5 or LC3B-II in the tear sample by contacting the tear sample with an anti-ATG5 or anti-LC3B-II antibody to determine binding between ATG5 or LC3B-II and the antibody,

Measuring the gene expression amount of ATG5 or LC3B-II in the tear sample using a primer or probe that selectively binds the tear sample to the mRNA of ATG5 or LC3B-II. Provided are methods for measuring gene or protein expression.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

EXAMPLE

The prospective cross-sectional contrast-controlled study of this example was constructed in an ophthalmology department at the Catholic University of Korea, St. Mary's Hospital. The research protocol was determined in accordance with the guidelines of the Declaration of Helsinki and was approved by the Bioethics Committee of St. Mary's Hospital, Seoul. Samples were obtained with consent from all subjects.

All described data are expressed as mean and SD. Data was analyzed with SPSS software (version 15.0, SPSS Inc, Chicago, IL) and GraphPad Prism (version 5.0, GraphPad Inc. LaJolla, CA). Shapiro-Wilk test was used to assess the steady state of the data. Groups were compared by one-way analysis of variance (ANOVA ) and T-test. Pearson correlation coefficients between autodigestion markers and clinical parameters were calculated to assess the relationship between expression levels of autodigestion markers and clinical parameters. Two-sided p values <0.05 were considered statistically significant.

Example 1. Clinical Evaluation

Screening for Subjects

In this example, 82 female subjects were studied (FIG. 1): 40 Sjogren's syndrome dry eye (SS DE) patients (78 eyes), 24 non-Sjogren's syndrome non-SS DE patients (48) Eyes), and 16 normal healthy subjects (25 eyes) as controls. Of the 40 Sjogren syndrome dry eye patients, 16 received topical corticosteroid treatment (four times daily for one month, 0.1% fluoromethorone, Sam-Il Pharmaceutics Co. Seoul, Korea). All dry eye patients had a severity level 2 or higher by criteria classified by the International Dry Eye WorkShop (DEWS) severity grading scheme. All Sjogren's syndrome dry eye patients were diagnosed as primary Sjogren's syndrome by a Lumatis specialist under the 2012 Sjogren's International Collaborative Clinical Alliance (SICCA) classification criteria. Sjogren's syndrome patients or subjects who received disease-modifying treatment were excluded from the study. Because of the unique prevalence of females and males found in Sjögren's syndrome (F: M = 9: 1), only female patients were included to prevent data misinterpretation. Exclusion criteria are pregnant or nursing staff, contact lens wearers, patients with tear occlusion, those who have undergone ophthalmic surgery within 6 months, those with acute or chronic eye disease in addition to dry eyes, concomitant allergies, eyelid abnormalities, and grade 2 or higher mibom dysfunction Who has it. Subjects also excluded those who received systemic or other topical treatments in addition to artificial tears within the last three months.

Clinical evaluation

The 82 subjects underwent the following ocular surface examination; Schirmer I test, tear film breakup time (TBUT), ocular surface staining (OSS) score and ocular surface disease index (OSDI) questionnaire.

The Schirmer I test was performed without anesthesia before injecting the drug into the eye. A standard Schimmer strip was placed on the lateral third of the lower eyelid and after 5 minutes the wetness of the strip was measured on a millimeter scale of each strip (Eagle Vision, Memphis, TN). Tear strips were cut transversely and placed in cryovial to be frozen and stored for further analysis. To evaluate TBUT, a drop of preservative-free saline was added to the fluorescein strip (Haag-Streit, Koeniz, Switzerland) and applied to the lower eyelid conjunctiva. The patient was blinked 3-4 times in a few seconds to allow sufficient mixing with the dye and then examined using a slit lamp with cobalt blue light. The time until the appearance of a defect in the stained tear film was measured and recorded as TBUT. TBUT was measured three times and averaged. OSS scores, including conjunctival and corneal staining scores, respectively, were evaluated according to the SICCA registry ocular examination protocol. First, a drop of 2% sterile fluorescein was infiltrated into the conjunctival sac, and after 1 minute, the corneal staining score was evaluated by evaluating fluorescein staining using a slit lamp cobalt blue filter. Corneal punctate epithelial erosions (PEE) stained with fluorescein were counted and scored. If there is no PEE, the score is zero. If 1-5 PEE is seen, the corneal score is 1; The score is 2 when 6-30 PEEs are seen; The score is 3 if more than 30 PEEs are seen. Additional points were added if there were one or more fused stained portions, stained at the pupil of the cornea, or at least one filament in any portion on the cornea. The maximum possible score for each cornea is 6. To determine conjunctival staining scores, fluorescein was washed with preservative-free saline and applied with a drop of 1% Lysamine Green Dye (Leiter's Pharmacy San Jose, Calif.) On the lower conjunctiva in both eyes. After several flashes, conjunctival staining scores were evaluated in the nasal and temporal conjunctiva, respectively; Score 0 is defined by 0 to 9 lysamine green staining points; Score 1 is defined as the presence of 10 to 32 points; Score 2 is 33 to 100 points; Score 3 is more than 100 points. The maximum possible score in the conjunctiva (nasal and lateral conjunctival conjunctiva) is 6. The overall OSS score for each eye is the sum of the fluorescein score for the cornea and the Lysamine Green score for the nasal and temporal conjunctival conjunctiva. Therefore, the maximum possible score for each eye is 12.

Table 1 shows the clinical evaluation results of 82 subjects. Schirmer I levels and TBUT were significantly lower in patients with Sjogren's syndrome dry eye compared to patients with non-Sjogren's syndrome dry eye, and conjunctival staining scores and OSS were significantly higher. There were no significant differences in age, OSDI, and corneal staining scores among Sjogren syndrome dry and non-Sjogren syndrome dry eyes patients (P = 0.3602, 0.0938, 0.0579, respectively).

TABLE 1

Example 2. Expression of autophagy markers in tear and conjunctival epithelial cells

Tear Collection and Western Blot Analysis

Tears were collected from the Schimmer strips that absorbed the tear fluid as in Example 1. For tear extraction, Schirmer strips were placed in tubes containing extraction buffer (NaCl 0.5 M, Tween20 0.5% in PBS), respectively. After 1 hour, the strips were transferred to a 0.5 ml tube drilled into the bottom with a cannula. The tube was placed in a larger (1.5 ml) tube and centrifuged for 15 minutes at 12,000 rpm. Lucent fluid was drawn from the strip by centrifugal force and collected into an external 1.5 ml tube through the central hole in the bottom of the small tube. Lubricated solutions were mixed with sample-loading buffer, heated for 10 minutes, centrifuged and protein concentration in the clear isolates was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL). Equal amounts of protein were separated by 15% SDS-PAGE under reducing conditions and electro-migrated to PVDF membranes (Millipore, Billerica, Mass.). PVDF membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 and primary rabbit polyclonal Ab targets LC3B-I and II (Cell Signaling Technology, Boston, MA) or ATG5 (Novus Biologicals, Littleton, CO) Incubated at 4 ° C. for 18 h. After three washes, the membranes were incubated with anti-rabbit horseradish peroxadaase-conjugated secondary Ab (Thermo Fisher Scientific) for 1 hour at room temperature. Finally, the membrane was washed three times and protein bands were detected using an improved chemiluminescent reagent (ECL; Amersham Biosciences, Sweden). All membranes were digested and reprobed with mouse monoclonal anti-β-actin Ab (Santa Cruz Biotechnology, Dallas, TX) to provide a standardized reference. Each experiment includes a colorant for β-actin that is used to normalize the relative level of expression by image analysis. The experiment was conducted 4-6 times.

Impression Cytology (IC)

IC was performed 15 minutes after the last stain eye solution soaked using a polyethersulfone filter (Suopor 200 membrane, Pall Corporation, Port Washington, NY). The filter paper was cut in half (13 × 6.5 mm) and each face adsorbed to the top-side and top-non-nasal conjunctiva. The filter from the upper-side conjunctiva is transferred into a tube containing 2 ml PBS with 0.05% paraformaldehyde (PFA) for immunostaining, and the paper from the upper-nasal conjunctiva is removed from the filter paper within 3 hours for RNA extraction. Collected.

Immunofluorescence Staining

IC filter paper with isolated epithelial cells was firmly pressed against silane coated slides (Muto Pure Chemicals co., Ltd. Tokyo, Japan) to transfer epithelial cells to slides and used for immunofluorescence staining according to the Baudouins protocol. Cells were fixed with cold methanol, permeated with 0.1% Triton X-100, and incubated with 10% goat serum for 1 hour to block nonspecific reactions. Cells were then incubated with anti-LC3B-I, II or ATG5 antibodies, washed twice with PBS and then incubated with Alexa Flour 488 conjugated anti-rabbit IgG Abs. Staining was captured by confocal microscopy (LSM 510 Meta (Carl Zeiss Meditec Inc. Dublin, Calif.) And transferred to Photoshop (Adobe systems, Santa Clara, Calif.).

RNA isolation and real-time PCR

IC using TRIzol reagent (Gibco-Invitrogen, Grand Island, NY) according to the modified protocol (Barabino S et al., Immune response in the conjunctival epithelium of patients with dry eye.Experimental eye research 2010; 91: 524-9) RNA was isolated from. Primary strands of complementary DNA (cDNA) were synthesized with random hexamers using SuperScript III ^™ reversetranscriptase (Invitrogen). The SYBR Green I real-time PCR method was used and the average threshold cycle (Ct) values for GAPDH were used for internal measurements to correct the integrity and total RNA by reaction. The 2 ^ΔΔCt method was used for relative quantification. Primers used in this example are LC3B-II (Forward: 5-CTT TGG GTG CGA CTT GAC G-3, Reverse: 5-GTC GAC CCC GCT CCT TTT-3), ATG5 (Forward: 5-AGG AGA GCC TGT ACC TAT GGA-3, Reverse: 5-TTC TCT GTT GCG CTT TTC TGA-3).

Western blot analysis of tears showed a statistically significant increase in ATG5 protein and LC3BII / I conversion rates in Sjogren's syndrome eye dry compared to non-Sjogren's syndrome eye dry and control. Relative concentration measurements showed that the ratio of ATG5 protein (vs β-actin) and LC3B-II / I protein in tears showed non-Sjogren's syndrome dry eye (1.44 ± 0.46 and 1.67 ± 0.40) and control (1.00 ± 0.35 and 1.00 ± 0.35) ) And higher in Sjogren's syndrome dry eye (5.06 ± 1.31 and 3.37 ± 1.08) compared to (P <0.05) (FIG. 2).

From conjunctival IC samples, mRNA levels encoding ATG5 and LC3B-II in the conjunctiva were also compared to non-Sjogren's syndrome dry eye (0.80 ± 0.17 and 2.21 ± 0.61 fold change) and control (1.00 ± 0.25 and 1.00 ± 0.24 fold change) Sjogren's syndrome ocular dryness (8.15 ± 1.38 and 6.14 ± 1.51 fold change) was upregulated (FIG. 3, all P <0.05). There was no significant difference in the expression of autophagy markers from tear and conjunctival IC samples between non-Sjogren's syndrome eye dry and control. Immunofluorescence staining of the conjunctival IC specimens confirmed that a dot cytoplasmic staining pattern of ATG5 exhibiting autodigestion was observed in Sjogren's syndrome eye dry, whereas ATG5 diffused and stained in non-Sjogren's syndrome eye dry and control (FIG. 4). . In addition, the spot staining focus of LC3B-II protein was significantly increased in Sjogren's syndrome eye dry, unlike in non-Sjogren's syndrome dry eye and control group (FIG. 4). Overall, autophagy marker expression is upregulated in the tears and conjunctiva of Sjogren's syndrome eye dry patients, suggesting that it is related to the onset of Sjogren's syndrome eye dryness of autophagy.

Example 3. Expression of autophagy markers in the lacrimal glands of animal models

Animal model

Since human lacrimal biopsy is a relatively invasive process, lacrimal glands of Sjogren's syndrome animal models were studied. Expression of ATG5 and LC3B-II proteins in the lacrimal glands was assessed by immunostaining analysis of NOD / LtJ mice and IκB-ζ KO mice known to exhibit Sjogren's syndrome dry eye-like characteristics. Paraffin sections of lacrimal glands of NOD / LtJ mice and IκB-ζ KO mice were provided by Professor Cho Mira of Catholic University (Seoul, Korea) and Kwon Min Kwon of Asan Seoul Hospital (Seoul, Korea). NOD / LtJ mice were purchased from Charles River Laboratory (Wilmington, Mass.) And IκB-ζ KO mice were provided by Professor Shizuo Akira of Osaka University (Osaka, Japan). The tear glands were removed from 8- and 16-week-old NOD / LtJ mice and 5-week-old IκB-ζ mice (-/-, +/-). All processes of animal experiments were performed according to the standards of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals of Ophthalmic and Vision Research. Protein amounts and mRNA expression of ATG5 and LC3B-II in lacrimal glands of animal models were performed via Western blot analysis and real-time PCR methods as described in Examples 2 and 3

In the lacrimal glands of NOD / LtJ mice, a dot cytoplasmic staining pattern of ATG5 and LC3B-II proteins was observed at 8 weeks of age and developed predominantly during the 16 week gestational period (FIG. 5A). Five-week-old IκB-ζ KO mice also showed distinct spots of ATG5 with increased background intensity, and marked spots of LC3B-II throughout the progenitor cells compared to control mice (IκB-ζ +/-) (FIG. 5B ). From these findings, it was speculated that autodigestion could be induced in the lacrimal glands of Sjogren's syndrome dry eye patients.

Example 4 Correlation Between Autodigestion Markers and Clinical Parameters in Sjogren's Syndrome

Because autodigestion markers are significantly upregulated in Sjogren syndrome dry eyes, not non-Sjogren syndrome dry eyes, the correlation between autodigestion markers and clinical parameters in Sjogren syndrome dry eyes was investigated (Table 2). As LC3B-II / I protein ratios represent autophagy markers and both markers increase in Sjogren's syndrome dry eye, LC3B-II / I protein ratio in tears in Sjogren's syndrome dry eye and ATG5 protein expression and LC3B in conjunctival epithelium The correlation between -II and ATG5 gene coding levels and clinical parameters were investigated. ATG5 expression has a strong association with corneal staining, conjunctival staining, and OSS score in dry Sjogren's syndrome, indicating that it can be used as a biomarker in Sjogren's syndrome dry eyes.

TABLE 2

Example 5 Effect of Topical Corticosteroid Treatment on the Level of Clinical Indicators and Autodigestion Markers in Sjogren's Syndrome Eye Drying

After topical corticosteroid application to Sjogren's syndrome dry eye patients, changes in clinical markers as well as the expression of autophagy markers were evaluated. After 1 month-local corticosteroid treatment with artificial tears, the OSDI score, Schirmer I levels, TBUT, corneal staining, conjunctival staining, and OSS scores were measured as in Example 1. In addition, Western blot and real time PCR were performed as in Examples 2 and 3.

In Sjogren's syndrome dry eye syndrome after corticosteroid treatment, decreased OSDI scores, increased Schirmer I levels, increased TBUT, decreased corneal staining score scores, decreased conjunctival staining scores, and improved OSS scores (Table 3). ).

TABLE 3

According to clinical improvement, representative Western blot images demonstrate LC3B-II / I protein conversion and decrease in ATG5 protein expression after corticosteroid treatment (FIG. 6). Relative concentration measurements showed that ATG5 protein (vs β-actin) and LC3B-II / I protein ratios in tears were approximately 0.88 ± 0.07 and 1.27 ± 0.08, respectively, compared to pre-corticosteroid levels (5.06 ± 0.31 and 1.87 ± 0.10). Statistically significant decrease with fold change (FIG. 6). The gene coding levels of ATG5 and LC3B-II were statistically significantly reduced by approximately 1.23 ± 0.27 and 2.57 ± 0.48 fold changes, respectively, compared to the levels before corticosteroid treatment (13.16 ± 4.36 and 12.93 ± 4.44 fold changes) (Figure 7). . In addition, representative immunostaining images demonstrated that the dot staining pattern of autophagy markers in the conjunctiva was also normalized in Sjogren's syndrome dry eye syndrome after corticosteroid treatment (FIG. 8).

Example 6 ROC Curve Analysis and Cut-off Values of Intraprotein Autodigestion Markers Extracted from Tears of Non-Sjogren Syndrome and Sjogren Syndrome

To verify whether ATG5 markers can be used as diagnostic biomarkers for the effective diagnosis of Sjögren's syndrome, except for non-Sjogren's syndrome, in proteins extracted from tears from patients with non-Sjogren syndrome dry eye and from tears from patients with Sjogren syndrome dry eye Curve analysis and cut-off values were derived. Specifically, 31 patients with non-Sjogren syndrome dry eye were inputted as 0 as a control group, 55 patients with Sjogren's syndrome were inputted as 1, and the response rate of each ATG5 protein was calculated. After that, the 95% confidence interval (CI) value of sensitivity and specificity was calculated, and the ROC curve was displayed while adjusting the cut-off value. (area under curve) was obtained. As shown in Table 4-7 and FIG. 9, when the cut-off value was 4.002, the sensitivity was 94.6 and the specificity was 93.6. In addition, it was confirmed that the AUC is 0.98 and included in 1.00 or more than 0.90 was found to be very useful. Therefore, autodigestion marker ATG5 was found to be very useful as a biomarker for the diagnosis of Sjögren's syndrome, except for non-Sjogren's syndrome, which causes dry eye.

TABLE 4

TABLE 5

TABLE 6

TABLE 7

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (13)

1. A composition for diagnosing Sjogren's syndrome in dry eyes, comprising a substance for measuring the expression level of an ATG5 (Autophagy protein 5) gene or an LC3B-II (light chain 3B-II) gene.
2. The composition of claim 1, wherein the cut-off value is 4.002023 when the Sjogren syndrome dry eye is diagnosed in the dry eye.
3. The composition of claim 1, wherein the substance for measuring the expression level of the gene measures the amount of mRNA to which the gene is transcribed or a protein encoded by the gene.
4. The composition of claim 1, wherein the substance measuring the expression level of the LC3B-II gene measures the amount of LC3B-II mRNA or LC3B-II protein or the ratio of LC3B-II / LC3B-I conversion.
5. The composition of claim 1, wherein the expression level of said gene is measured in tears, conjunctival epithelial cells, lacrimal gland samples, saliva or salivary gland epithelial cells of the subject.
6. The method of claim 1, wherein the expression level of the gene is measured by reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microorganism. Array Method, Northern Blot, Western Blot, Enzyme Linked Immuno Sorbent Assay (ELISA), Radioimmunoassay, Immune Diffusion, Immunoelectrophoresis, Tissue Immunostaining, Immunoprecipitation Assay, Complementary Assay, FACS, Mass Spectrometry and The composition is performed by a method selected from among protein microarrays.
7. A kit for diagnosing dry eye of Sjogren's syndrome in a dry eye comprising the composition of claim 1.
8. Measuring the expression level of ATG5 gene or LC3B-II gene from a biological sample; And

   Comparing the expression level of said gene with the expression level of said gene from a normal control sample.
9. The method of claim 8, wherein the expression level of the gene is measured by reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microorganism. Array Method, Northern Blot, Western Blot, Enzyme Linked Immuno Sorbent Assay (ELISA), Radioimmunoassay, Immune Diffusion, Immunoelectrophoresis, Tissue Immunostaining, Immunoprecipitation Assay, Complementary Assay, FACS, Mass Spectrometry and The method is performed by a method selected from among protein microarrays.
10. The method of claim 8, wherein the expression level of the gene is measured by measuring the amount of mRNA or ATG5 gene or LC3B-II gene, which is transcribed by ATG5 gene or LC3B-II gene, or LC3B-II / LC3B-I. Measuring the conversion rate.
11. The method of claim 8, wherein the biological sample is tear, conjunctival epithelial cell, lacrimal gland sample, saliva or salivary gland epithelial cell.
12. Treating the test substance with a biological sample obtained from a Sjogren's syndrome patient;

    Analyzing the effect of the test substance on the expression of ATG5 gene or LC3B-II gene; And

    Screening method for the treatment of dry eye treatment for Sjogren's syndrome in the dry eye, comprising the step of selecting a test substance to reduce the expression level of the gene compared to the control group not treated with the test substance.
13. Obtaining a tear sample from a dry eye patient;

    Measuring the amount of protein expression of ATG5 or LC3B-II in the tear sample by contacting the tear sample with an anti-ATG5 or anti-LC3B-II antibody to determine binding between ATG5 or LC3B-II and the antibody,

    Measuring the gene expression amount of ATG5 or LC3B-II in the tear sample using a primer or probe that selectively binds the tear sample to the mRNA of ATG5 or LC3B-II. A method of measuring gene or protein expression.

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