KR20200122258A - Biomarker for diagnosing dysfunction of corneal endothelial cells - Google Patents

Abstract

The present invention relates to a biomarker to diagnose a dysfunction of corneal endothelial cells, and an information providing method to diagnose a dysfunction degree of corneal endothelial cells and various diseases related to the same by using the same. The present invention can measure an expression level of biomarker protein or a gene encoding the same in waterproofing of an eye, and thus can rapidly, easily, and accurately diagnose the dysfunction or the degree thereof of corneal endothelial cells. Accordingly, it is possible to diagnose diseases such as Fuchs corneal dystrophy, external injury, pseudophakic bullous keratopathy, endotheliopathy, or vision loss at an early stage, or it is possible to trace the progress, a prognosis, or a treatment effect of the diseases.

Description

Biomarker for diagnosing dysfunction of corneal endothelial cells

The present invention relates to a biomarker capable of diagnosing dysfunction of corneal endothelial cells, and a method of providing information for diagnosing various diseases related to the degree of dysfunction of corneal endothelial cells using the same.

The cornea is a transparent tissue located on the surface of the eyeball, and the corneal epithelium layer, Bowman's membrane, corneal parenchyma layer, Desme's membrane, and corneal endothelial layer are present in order from the surface. The corneal epithelium layer is the outermost layer of the cornea, and functions as a barrier protecting the cornea from external dust or bacteria. The Bowman's membrane is thought to function as a base for the corneal epithelial layer. The corneal parenchyma is the thickest of the three layers, and plays a role in maintaining the strength of the cornea. Dessme's membrane is a thin film underneath the corneal parenchyma and functions to connect the corneal parenchyma to the corneal endothelium. The corneal endothelial layer is a single-layered cell layer in which hexagonal corneal endothelial cells are regularly arranged in the shape of a pavement, and due to the barrier function and pump function, water from the anterior chamber penetrates the corneal parenchyma. At the same time, it plays an important role in maintaining the transparency of the cornea by maintaining a constant moisture content by discharging moisture from the cornea to the anterior side.

When corneal endothelial cells are damaged due to genetic factors or external factors, and the number of corneal endothelial cells significantly decreases, the above functions are inhibited, resulting in corneal edema. The corneal transparency cannot be maintained due to severe corneal endothelial disorder, and when bullous keratopathy occurs, a marked decrease in visual acuity is caused.

Corneal endothelial cells have extremely poor proliferation ability in humans. Therefore, as a fundamental treatment for severe corneal endothelial disorder, corneal transplantation is currently the most effective means. In fact, bullous keratopathy is the number one adaptive disease for corneal transplantation. Conventionally, for corneal endothelial disorders, full-layer corneal transplantation has been performed, but chronic donor shortages and rejection reactions after transplantation are problematic. In order to alleviate the rejection reaction, a method of transplanting only some tissues including the corneal endothelium into the diseased eye (Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK)) was developed, but DSAEK also cannot overcome the problem of lack of donor. . In addition, a method of proliferating corneal endothelial cells in vitro and using them for treatment has also been attempted, but if the passage is repeated, transformation occurs, resulting in loss of function.

Accordingly, despite the importance of evaluating the function of corneal endothelial cells, the existing test methods capable of evaluating the degree of damage and residual function of the corneal endothelial cells are indeed very insufficient. Conventional tests for dysfunction of corneal endothelial cells remain at the level of indirectly inferring the function of cells after calculating the coefficient of variation and average density of cells through a specular microscopy. In addition, it is a test method that predicts the entire cornea of 11000 μm x 12000 μm by examining only a local area of 300 μm x 400 μm, and has a very inaccurate disadvantage.

An object of the present invention is to provide a composition or kit capable of diagnosing dysfunction of corneal endothelial cells.

Another object of the present invention is to provide a method of providing information for diagnosing dysfunction of corneal endothelial cells.

Another object of the present invention is to provide a method for screening a substance that induces dysfunction of corneal endothelial cells.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, CST4 (Cystatin 4), LCN1 (Lipocalin-1), CD163 (Cluster of Differentiation 163), ITIH3 (Inter-alpha-trypsin inhibitor heavy chain H3), FCGBP (Fc fragment of IgG binding) protein), VCAM1 (Vascular cell adhesion protein 1), VIM (Vimentin), COL5A1 (collagen type V alpha 1 chain), CRYAA (Alpha-crystallin A chain), ANGPTL7 (Angiopoietin-related protein 7), C1RL (complement C1r subcomponent) like), CRYAB(crystallin alpha B), LYZ(Lysozyme), PLXNB2(Plexin B2), SERPINF2(Alpha-2-antiplasmin), C1QB(Complement C1q subcomponent subunit B), SERPIND1(Serpin family D member 1), VTN( Vitronectin), SERPINC1 (Serpin family C member 1), KNG1 (Kininogen 1), APOH (Apolipoprotein H), A1BG (Alpha-1-B glycoprotein), C8A (Complement C8 alpha chain), FETUB (Fetuin B), AFM ( Afamin), SERPINA3(Serpin family A member 3), CPB2(carboxypeptidase B2), C1QC(complement C1q C chain), WFIKKN2(WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 2), TMSB4X(Thymosin beta 4 X -linked), TMSB10 (Thymosin beta 10 ), AHSG (Alpha 2-HS glycoprotein), PROS1 (Protein S), RTBDN (Retbindin), NPVF (Neuropeptide VF precursor), C9 (Complement C9), LRG1 (Leucine rich alpha-2-glycoprotein 1), MST1 (Macrophage stimulating 1), DSC3 (Desmocollin 3), C1R (Complement C1r), C8B (Complement C8 beta chain), CLU (Clusterin), CTGF (Connective tissue growth factor), CFHR1 (Complement Factor H Related 1), F10 (Coagulation factor) X), TIMP1 (TIMP metallopeptidase inhibitor 1), IGFBP2 (Insulin like growth factor binding protein 2), F12 (Coagulation factor XII), PCOLCE (Procollagen C-endopeptidase enhancer), HABP2 (Hyaluronan binding protein 2), SLPI (Secretory leukocyte peptidase inhibitor), DKK3 (Dickkopf WNT signaling pathway inhibitor 3), CRYBB1 (Crystallin beta B1), CA2 (Carbonic anhydrase 2), CRYBA1 (Crystallin beta A1), PGAM1 (Phosphoglycerate mutase 1), CRYBA4 (Crystallin beta A4), (CutA divalent cation tolerance homolog), ARSB (Arylsulfatase B), ALDOC (Aldolase, fructose-bisphosphate C), CRYBB2 (Crystallin beta B2), SERPINI1 (Serpin famil) y I member 1), CREG1 (cellular repressor of E1A stimulated genes 1), SPOCK1 (SPARC (osteonectin), cwcv and kazal like domains proteoglycan 1), SDF4 (Stromal cell derived factor 4), SCG2 (Secretogranin II), COL9A3 ( Collagen type IX alpha 3 chain), LGALS3BP (Galectin 3 binding protein), IGHG3 (Immunoglobulin heavy constant gamma 3), HBB (Hemoglobin subunit beta), MDH1 (Malate dehydrogenase 1), IMPG2 (Interphotoreceptor matrix proteoglycan 2), GPI (Glucose -6-phosphate isomerase), TPI1 (Triosephosphate isomerase 1), APLP2 (Amyloid beta precursor like protein 2), CLSTN1 (Calsyntenin 1), FGG (Fibrinogen gamma chain), VGF (VGF nerve growth factor inducible), CLSTN3 (Calsyntenin 3) ), HSPB1(Heat shock protein family B (small) member 1), GAS6(Growth arrest specific 6), L1CAM(L1 cell adhesion molecule), CRYGS(Crystallin gamma S), S100A9(S100 calcium binding protein A9), SEMA3A( Semaphorin 3A), VCAN (Versican), APP (Amyloid beta precursor protein), SPARC (Secreted protein acidic and cysteine rich), TWSG1 (Twisted gastru) lation BMP signaling modulator 1), ALDOA (Aldolase, fructose-bisphosphate A), SOD1 (Superoxide dismutase 1), HBA1 (Hemoglobin subunit alpha 1), ENO1 (Enolase 1), SORCS1 (Sortilin related VPS10 domain containing receptor 1), GALNT2 (Polypeptide N-acetylgalactosaminyltransferase 2), AGRN(Agrin), ATP6AP1(ATPase H+ transporting accessory protein 1), OPTC(Opticin), FSTL5(Follistatin like 5), C2orf40(Chromosome 2 open reading frame 40), FGA(Fibrinogen alpha chain) ), TPP1 (Tripeptidyl peptidase 1), FSTL4 (Follistatin-like 4), APOB (Apolipoprotein B), TGFBI (Transforming growth factor beta induced), GOLIM4 (Golgi integral membrane protein 4), XYLT1 (Xylosyltransferase 1), ITM2B (Integral membrane protein 2B), FABP5 (Fatty acid binding protein 5), TKT (Transketolase), EFNA5 (Ephrin A5), PPIA (Peptidylprolyl isomerase A), SCG5 (Secretogranin V), CPQ (Carboxypeptidase Q), C3 (Complement C3), B4GAT1 (Beta-1,4-glucuronyltransferase 1), NEO1 (Neogenin 1), HEXB (Hexosaminidase subunit beta), IGHM (Immunoglobulin heavy consta nt mu), APOC3(Apolipoprotein C3), LYNX1(Ly6/neurotoxin 1), PSAP(Prosaposin), ENPP2(Ectonucleotide pyrophosphatase/phosphodiesterase 2), SEMA7A(semaphorin 7A (John Milton Hagen blood group)), MAN1A1(Mannosidase alpha class 1A member 1), CHRDL1 (Chordin like 1), F9 (Coagulation factor IX), HRG (Histidine rich glycoprotein), and SERPINF1 (Serpin family F member 1) at least one protein selected from the group consisting of; Or it relates to a biomarker for diagnosing dysfunction of corneal endothelial cells comprising a gene encoding the same.

In the present invention, the "corneal endothelial cells" are physiologically most important single cell layers in maintaining transparency of the cornea by regulating water movement through a dynamic equilibrium of a leaky barrier function and an active pump action. However, the regeneration of damaged corneal endothelial cells in vivo is extremely limited, and if endothelial cells are damaged, such as in various diseases such as Fuchs corneal dystrophy, trauma, and vesicular keratopathy of the artificial lens, when the endothelial cells are damaged, it causes swelling and clouding of the cornea, leading to severe vision. It may lead to loss of energy.

In the present invention, the "function of corneal endothelial cells" refers to ocular trauma, ocular surgery (eg, cataract surgery or intraocular lens insertion, etc.), viral infection, eye inflammation, aging, diabetes, oxidative stress, oxygen due to wearing contact lenses As corneal endothelial cells fail to function properly due to various causes such as shortage and increase in intraocular pressure, it means that the movement of water is not properly regulated due to the failure to achieve a dynamic equilibrium of leaky barrier function and active pump action. In the present invention, diseases such as Fuchs corneal dystrophy, trauma, vesicular keratopathy of the artificial lens, corneal endothelial inflammation, or vision loss may occur due to the malfunction of the corneal endothelial cells.

In the present invention, the biomarker is present in the corneal endothelial cells or the aqueous humor of the eye, preferably in the aqueous humor of the eye, so that accuracy can be improved in diagnosing the degree of dysfunction of the corneal endothelial cells.

In the present invention, the "aqueous humor" is a transparent liquid like water, provides nutrition to the lens and cornea, structurally supports the eye, and fills the anterior chamber and the posterior chamber.

In a preferred example of the present invention, the biomarkers are ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X, C1R, MDH1, MAN1A, B4GAT1, GPI, ENO1, ALDOA, At least one protein selected from the group consisting of PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Or it may be a gene encoding it. Here, the ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X or C1R proteins or genes encoding them may be involved in an immune response mechanism, The MDH1, MAN1A, B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 or APLP2 proteins encoding the same Genes may be involved in carbohydrate metabolic processes, developmental processes, or ion transport.

In a preferred example of the present invention, the biomarker is at least one protein from TIMP1, FCGBP, and ANGPTL7; Or it may be a gene encoding it.

In the present invention, the "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether or not the corneal endothelial cells are dysfunction or the degree thereof, and through this, the onset of various eye diseases due to the dysfunction of the corneal endothelial cells can be predicted early.

According to another embodiment of the present invention, CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1 , A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, F10, TIGF, CFHR1 , IGFBP2, F12, PCOLCE, HABP2, SLPI, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3, HBB, MDBP, LGALS3 , IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNTRN , ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, APOC3, IGHM , PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Or it relates to a composition for diagnosis of dysfunction of corneal endothelial cells comprising an agent capable of measuring the expression level of the gene encoding the same.

In a preferred example of the present invention, the biomarkers to be measured for the expression level are ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X, C1R, MDH1, MAN1A, At least one protein selected from the group consisting of B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Or it may be a gene encoding it.

In a preferred example of the present invention, the biomarker to be measured for the expression level is at least one protein of TIMP1, FCGBP, and ANGPTL7; Or it may be a gene encoding it.

In the present invention, the agent for measuring the expression level of the biomarker protein is not particularly limited, but, for example, an antibody, oligopeptide, ligand, peptide nucleic acid (PNA) and aptamer that specifically binds to the protein. It may include one or more selected from the group consisting of.

In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the biomarker protein. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibody can be easily prepared using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, including the process of injecting an antigen of the biomarker protein into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog. In addition, the monoclonal antibody is a hybridoma method well known in the art (hybridoma method; see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. The functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized, DNA or RNA-like polymer, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt of Copenhagen University in Denmark in 1991. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by a peptide bond, which greatly increases the binding power and stability to DNA or RNA, resulting in molecular biology. , Diagnostic analysis and antisense therapy. PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.

In the present invention, the "aptamer" is an oligonucleotide or a peptide molecule, and general information of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727(1998)].

In the present invention, the formulation for measuring the expression level of the gene encoding the biomarker protein may include at least one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the biomarker protein. I can.

In the present invention, the "primer" is a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, preferably, a pair of primers that provides an analysis result having specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can give high specificity. .

In the present invention, the "probe" means a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, RNA, or DNA, and most preferably Hagi is PNA. More specifically, the probe is a biomaterial that includes an organism-derived or similar thing or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the term "LNA (Locked nucleic acids)" refers to a nucleic acid analog including 2'-O, 4'-C methylene bridges [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502 ]. LNA nucleosides contain common nucleic acid bases of DNA and RNA, and can form base pairs according to the Watson-Crick base pairing rules. However, due to the'locking' of the molecule due to the methylene bridge, the LNA cannot form an ideal shape in the Watson-Crick bond. When LNA is included in a DNA or RNA oligonucleotide, the LNA can more quickly pair with a complementary nucleotide chain to increase the stability of the double helix.

In the present invention, the "antisense" refers to a sequence of nucleotide bases in which the antisense oligomer is hybridized with the target sequence in RNA by Watson-Crick base pairing, and typically allows the formation of mRNA and RNA: oligomer heterodimer within the target sequence. And an oligomer having a backbone between subunits. Oligomers may have exact sequence complementarity or approximate complementarity to the target sequence.

Since the biomarker protein according to the present invention or the information on the gene encoding it is known, a person skilled in the art will be able to easily design a primer, probe or antisense nucleotide that specifically binds to the gene encoding the protein based on this information. .

In the present invention, the expression level of the biomarker protein or the gene encoding the same is measured for the corneal endothelial cells or the aqueous humor of the eye, preferably the eye waterproofing, in diagnosing the degree of dysfunction of the corneal endothelial cells. It is desirable because it can increase the accuracy.

Corneal endothelial cells are extremely poor in proliferative ability in humans, and thus, when their dysfunction occurs, various eye diseases or even vision loss may occur. However, conventionally, there was no method to directly measure the malfunction of the corneal endothelial cells, and the accuracy was remarkably at the level of indirectly inferring the function only with the shape and density of the cells through a corneal endothelial cell spectroscopic examination. There was a downside to falling.

However, by measuring the expression level of the biomarker protein of the present invention or the gene encoding the same in the waterproofing of the corneal endothelial cells or the eyeball, preferably the waterproofing of the eyeball by using the composition of the present invention, The degree can be quickly and easily but accurately diagnosed, and through this, diseases such as Fuchs corneal dystrophy, trauma, artificial lens vesicular keratosis, corneal endothelial inflammation, or vision loss that can be caused by insufficiency of the corneal endothelial cells can be diagnosed early. It is possible to diagnose or follow the course of the disease, prognosis, or treatment effect.

According to another embodiment of the present invention, it relates to a kit for diagnosing dysfunction of corneal endothelial cells comprising the composition for diagnosing dysfunction of corneal endothelial cells according to the present invention.

In the present invention, the diagnostic kit can be used to diagnose the presence or extent of dysfunction of the corneal endothelial cells, and diagnose the onset or possibility of various diseases that can be induced due to the dysfunction of the corneal endothelial cells, or The course of the disease, prognosis, or therapeutic effect can also be diagnosed.

The definition of the diagnostic composition of the present invention and the dysfunction of corneal endothelial cells and diseases caused thereby are duplicated as described in the diagnostic composition of the present invention, and description thereof will be omitted below in order to avoid excessive congestion in the specification.

In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit, but is not limited thereto.

The diagnostic kit of the present invention may further include one kind or more other component compositions, solutions, or devices suitable for the analysis method.

For example, the diagnostic kit of the present invention may further include essential elements necessary to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains a pair of primers specific for the gene encoding the marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. In addition, a primer specific to the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC. -May include DEPC-water, sterilized water, etc.

In addition, the diagnostic kit of the present invention may include essential elements necessary to perform a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, the diagnostic kit of the present invention may contain essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the protein. Antibodies are antibodies that have high specificity and affinity for a marker protein and have little cross-reactivity with other proteins, and are monoclonal, polyclonal, or recombinant antibodies. In addition, the ELISA kit may contain an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or antibodies capable of binding. Other materials may be included.

According to another embodiment of the present invention, CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB in a biological sample isolated from a subject of interest. , SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1 CRG1, MST1 , CLU, CTGF, CFHR1, F10, TIMP1, IGFBP2, F12, PCOLCE, HABP2, SLPI, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4 , COL9A3, LGALS3BP, IGHG3, HBB, MDH1, IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARCA, SOD1, TWSG1 , HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, C3, BG5, CPQAT , NEO1, HEXB, IGHM, APOC3, LYNX1, PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Or, it relates to a method for providing information for diagnosing dysfunction of corneal endothelial cells comprising measuring the expression level of a gene encoding the same.

In the present invention, the "object of interest" refers to an individual whose corneal endothelial cell dysfunction is uncertain and has a high probability of dysfunction.

In the present invention, the "biological sample" means any substance, biological fluid, tissue or cell obtained from or derived from an individual, preferably corneal endothelial cells or aqueous humor in the eye, more preferably Intraocular waterproofing is preferable because it can improve the accuracy in diagnosing dysfunction of corneal endothelial cells.

In the present invention, the step of measuring the expression level of the biomarker protein listed above or the gene encoding the same in the biological sample separated as described above may be included.

In a preferred example of the present invention, the biomarkers to be measured for the expression level are ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X, C1R, MDH1, MAN1A, At least one protein selected from the group consisting of B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Or it may be a gene encoding it.

In a preferred example of the present invention, the biomarker to be measured for the expression level is at least one protein of TIMP1, FCGBP, and ANGPTL7; Or it may be a gene encoding it.

In the present invention, the agent for measuring the expression level of the biomarker protein is not particularly limited, but preferably antibodies, oligopeptides, ligands, peptide nucleic acids (PNA) and aptamers that specifically bind to the biomarker protein ( aptamer) may include at least one selected from the group consisting of.

In the present invention, methods for measuring or comparing the expression level of the biomarker protein include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, and SELDI- TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radiation immunity analysis, radioactive immunity diffusion method, octeroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis method, two-dimensional electrophoresis analysis, Liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting, or ELISA (enzyme linked immunosorbentassay), but are limited thereto. It does not become.

In the present invention, the formulation for measuring the expression level of the gene encoding the biomarker protein may include at least one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the biomarker protein. I can.

In the present invention, as a process of checking the presence and expression level of the gene encoding the biomarker protein, as an analysis method for measuring the expression level of the gene, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, or DNA chip, but are not limited thereto. .

Predicting that the possibility of dysfunction of corneal endothelial cells is high when the expression level of the biomarker protein or the gene encoding the biomarker measured in the biological sample of the object of the present invention is increased or decreased compared to the normal control It may include.

More specifically, CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1 measured for biological samples of the subject of the present invention , VTN, SERPINC1, KNG1, APOH, A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, CLU, DSC3, DSC3 , CTGF, CFHR1, F10, TIMP1, IGFBP2, F12, PCOLCE, HABP2 and at least one protein selected from the group consisting of SLPI; Alternatively, when the expression level of the gene encoding it is increased compared to the normal control, it can be predicted that the possibility of dysfunction of corneal endothelial cells is high.

As a preferred example of the present invention, one selected from the group consisting of ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X and C1R measured for the biological sample of the object of interest More than one protein; Alternatively, when the expression level of the gene encoding it is increased compared to the normal control, it can be predicted that the possibility of dysfunction of corneal endothelial cells is high.

As a preferred example of the present invention, at least one protein of TIMP1, FCGBP, and ANGPTL7 measured for a biological sample of a subject of interest; Alternatively, when the expression level of the gene encoding it is increased compared to the normal control, it can be predicted that the possibility of dysfunction of corneal endothelial cells is high.

In addition, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3 measured for biological samples of the object of the present invention. , HBB, MDH1, IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1 , GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4 HEXB1, NEO1, IGHM , APOC3, LYNX1, PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Alternatively, when the expression level of the gene encoding it is decreased compared to the normal control, it can be predicted that the possibility of dysfunction of corneal endothelial cells is high.

As a preferred example of the present invention, MDH1, MAN1A, B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, measured for the biological sample of the object of interest, At least one protein selected from the group consisting of APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Alternatively, when the expression level of the gene encoding it is decreased compared to the normal control, it can be predicted that the possibility of dysfunction of corneal endothelial cells is high.

Furthermore, in the case of predicting or diagnosing that the possibility of dysfunction of corneal endothelial cells as described above is high in the present invention, it may further include performing appropriate treatment, such as administering a drug for the disease, to the target individual. I can.

In the case of using the method of the present invention, it is possible to diagnose the presence or degree of dysfunction of the corneal endothelial cells, and diagnose the onset or possibility of various diseases that may be induced due to the dysfunction of the corneal endothelial cells, or The course of the disease, prognosis or treatment effect can also be followed.

The definition of the dysfunction of the corneal endothelial cells of the present invention and the disease caused thereby is duplicated as described in the diagnostic composition of the present invention, and description thereof is omitted below in order to avoid excessive congestion in the specification.

According to another embodiment of the present invention, the step of treating a candidate substance expected to induce dysfunction of corneal endothelial cells in the isolated biological sample; And

In biological samples treated with the candidate substances, CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1 , A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, F10, TIGF, CFHR1 , IGFBP2, F12, PCOLCE, HABP2, SLPI, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3, HBB, MDBP, LGALS3 , IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNTRN , ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, APOC3, IGHM , PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Or it relates to a method for screening a drug that induces dysfunction of corneal endothelial cells, comprising the step of measuring the expression level of the gene encoding the same.

In the present invention, the isolated biological sample may be a biological sample isolated from an individual in which dysfunction of corneal endothelial cells is induced or not, and specifically, it is preferable that it is a corneal endothelial cell or an aqueous humor of the eye. Preferably it may be waterproof of the eye.

In addition, the candidate substances in the present invention include any substance, molecule, element, compound, entity, or a combination thereof. Examples include, but are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound, or a combination of two or more substances.

In a preferred example of the present invention, the biomarkers to be measured for the expression level are ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X, C1R, MDH1, MAN1A, At least one protein selected from the group consisting of B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Or it may be a gene encoding it.

In a preferred example of the present invention, the biomarker to be measured for the expression level is at least one protein of TIMP1, FCGBP, and ANGPTL7; Or it may be a gene encoding it.

In the present invention, the method of measuring the expression level of the biomarker protein and the gene encoding the same is duplicated as described in the information providing method of the present invention, and detailed description thereof will be omitted.

In the present invention, when the expression level of the biomarker protein or the gene encoding the biomarker protein in the biological sample after treatment with the candidate substance is increased or decreased compared to before treatment with the candidate substance, the candidate substance is impaired in function of corneal endothelial cells. It may include the step of determining the triggering agent.

More specifically, CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB measured in the biological sample after treatment of the candidate material in the present invention , SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSCC8B, MST1 , CLU, CTGF, CFHR1, F10, TIMP1, IGFBP2, F12, PCOLCE, HABP2 and at least one protein selected from the group consisting of SLPI; Alternatively, when the expression level of the gene encoding it is increased compared to before treatment, the candidate substance may be determined as an inducer of dysfunction of corneal endothelial cells.

As a preferred example of the present invention, selected from the group consisting of ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X and C1R measured in the biological sample after treatment of the candidate material. One or more proteins; Alternatively, when the expression level of the gene encoding it is increased compared to before treatment, the candidate substance may be determined as an inducer of dysfunction of corneal endothelial cells.

As a preferred example of the present invention, at least one protein of ANGPTL7, FCGBP, and TIMP1 measured in the biological sample after treatment of the candidate material; Alternatively, when the expression level of the gene encoding it is increased compared to before treatment, the candidate substance may be determined as an inducer of dysfunction of corneal endothelial cells.

In addition, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP measured in the biological sample after treatment of the candidate material in the present invention , IGHG3, HBB, MDH1, IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, EDOA , SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ1, C3, B4GAT , IGHM, APOC3, LYNX1, PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Alternatively, when the expression level of the gene encoding it is decreased compared to before treatment, the candidate substance may be determined as an inducer of dysfunction of corneal endothelial cells.

As a preferred example of the present invention, MDH1, MAN1A, B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, measured in the biological sample after treatment of the candidate material. At least one protein selected from the group consisting of COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1 and APLP2; Alternatively, when the expression level of the gene encoding it is decreased compared to before treatment, the candidate substance may be determined as an inducer of dysfunction of corneal endothelial cells.

In the present invention, by measuring the expression level of the biomarker protein of the present invention or the gene encoding the biomarker protein of the present invention in the waterproofing of the eye, it is possible to quickly and easily but accurately diagnose whether or not the degree of dysfunction of the corneal endothelial cells. Early diagnosis of diseases such as Fuchs corneal dystrophy, trauma, artificial lens vesicular keratosis, corneal endothelial inflammation, or vision loss, which can be caused by cell dysfunction, or tracking the course, prognosis, or treatment effect of the disease This is possible.

1 shows the distribution of the identified proteins for each group after removal of the rich protein from the aqueous humor isolated from the corneal endothelial cell failure patient group and the normal control group in an embodiment of the present invention.
FIG. 2 shows a change in the type and expression level of a protein whose expression was significantly increased or decreased in a patient group of corneal endothelial cell failure compared to a normal control group through global proteome profiling in an embodiment of the present invention .
3 is a diagram of a functional group of proteins whose expression is significantly increased or decreased in a patient group with corneal endothelial cell failure compared to a normal control group through a gene ontology biological process (GO-BP) in an embodiment of the present invention. It shows the change in type and expression level.
4 is a diagram of a functional group of proteins whose expression is significantly increased or decreased in a patient group with corneal endothelial cell failure compared to a normal control group through a gene ontology molecular function (GO-MF) in an embodiment of the present invention. It shows the change in type and expression level.
5 shows a significant increase or decrease in expression in the corneal endothelial cell failure patient group compared to the normal control group through gene ontology classification and protein-protein interaction network (PPI network) in an embodiment of the present invention. It shows the protein-protein interaction network map of each function-specific protein.
6 shows an experimental design diagram of Experimental Example 2 of the present invention.
7 shows the distribution of transcripts expressed in corneal endothelial cells isolated from the corneal endothelial cell failure patient group and the normal control group in Experimental Example 2 of the present invention.
8 shows the distribution of transcripts expressed in corneal endothelial cells isolated from the corneal endothelial cell failure patient group and the normal control group in Experimental Example 2 of the present invention.
9 shows a transcript showing a significant increase or decrease in corneal endothelial cells of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
10 shows the results of analyzing the mechanism of the transcriptome showing a significant increase in corneal endothelial cells of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
11 shows the results of analyzing the mechanism of the transcriptome showing a significant decrease in corneal endothelial cells of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
12 shows the distribution of proteins expressed in aqueous humor isolated from the corneal endothelial cell failure patient group and the normal control group in Experimental Example 2 of the present invention.
13 shows the distribution of proteins expressed in aqueous humor isolated from the corneal endothelial cell failure patient group and the normal control group in Experimental Example 2 of the present invention.
14 shows a protein showing a significant increase or decrease in water resistance of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
15 shows the results of analyzing the mechanism of protein showing a significant increase in water resistance of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
FIG. 16 shows the results of analyzing the mechanism of protein showing a significant decrease in water resistance of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
FIG. 17 shows the correlation between the transcript (DEG) expressed in corneal endothelial cells of the corneal endothelial cell failure patient group and the protein expressed in aqueous humor (DEP) in Experimental Example 2 of the present invention.
FIG. 18 shows the types of transcripts (DEG) and proteins (DEP) that are equally increased in expression in corneal endothelial cells and aqueous humor of corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.
FIG. 19 shows the types of transcripts (DEG) and proteins (DEP) that are equally reduced in expression in corneal endothelial cells and aqueous humor of corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.
FIG. 20 shows the types of transcripts (DEG) and proteins (DEP) expressed in corneal endothelial cells and aqueous humor of corneal endothelial cell insufficiency patient group in Experimental Example 2 of the present invention, and the results of analyzing the expression patterns and mechanisms thereof. Here, in the left column, red indicates an increase in expression compared to the normal control, and green indicates a decrease in expression.
FIG. 21 shows an interaction network map of transcripts and proteins for each function whose expression was significantly increased or decreased in corneal endothelial cells and aqueous humor of the corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.
22 is a diagram showing an experimental design of a waterproof proteomic assay for identifying a waterproof biomarker protein capable of detecting corneal endothelial cell failure in Experimental Example 2 of the present invention.
23 shows the expression patterns of proteins expressed in the aqueous humor of the normal control group and the corneal endothelial cell insufficiency patient group in Experimental Example 2 of the present invention.
24 shows a protein showing a significant increase or decrease in water resistance of the corneal endothelial cell failure patient group compared to the normal control group in Experimental Example 2 of the present invention.
FIG. 25 shows a graph comparing the expression level of TIMP1 protein in the aqueous humor of the normal control group and the corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.
26 shows a graph comparing the expression level of FCGBP protein in the aqueous humor of a normal control group and a corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.
27 shows a graph comparing the expression level of ANGPTL7 protein in the aqueous humor of a normal control group and a corneal endothelial cell failure patient group in Experimental Example 2 of the present invention.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

[Experimental Example 1]

1. Experimental method

(1) Patient recruitment and clinical examination

This study was approved as a prospective study after being deliberated by the Institutional Review Board of Gangnam Severance Hospital, and all patients were included in the study only after receiving sufficient explanations and consenting to the study. A total of 10 patients were recruited as shown in Table 1 below, which consisted of 5 patients with corneal endothelial cell failure and 5 normal controls. Corneal endothelial cell insufficiency patients were all subjects who had undergone corneal endothelial cell transplantation, and the normal control group were patients who had undergone general cataract surgery. All patients had no specific systemic history including hypertension, diabetes, and immune disease, and no specific ophthalmic history, such as ocular trauma and uveitis. In all patients, the left and right eyes were not classified, and diseased eyes were included in the study subject, but gender ratio, age, etc. were recruited so that there was no difference between the two groups.

After confirming corneal opacity and swelling in the patient group through slit lamp microscopy, abnormalities of the cornea and corneal endothelial cells using a specular microscope (SP-3000P, Topcon, Tokyo, Japan) and ultrasonic corneal thickness (SP-3000, Tpocon) Failure was confirmed. For statistical analysis of clinical data, SPSS v.21.0 (IBM Corp., Armonk, NY, USA) was used. After confirming the normality of the data using the Kolmogorov-Smirnov test, the Mann-Whitney U test or the Wilcoxon signed rank test was used for the unstructured distribution data. P values of less than 0.05 were considered statistically significant in all statistical tests.

division Normal control Patients with corneal endothelial cell failure Age (year) 64.54±9.22 61.32±14.56 Female: Male 2:3 2:3 Corneal endothelial cell density (/mm2, mean ± standard deviation) 2523±534 423 (only 2 people can check) Corneal thickness (㎛) 546±33 1028

(2) waterproofing

When performing intraocular ophthalmic surgery including cataract surgery and corneal endothelial cell transplantation, after complete disinfection for the operation, a primary incision of 0.5 mm size was first performed into the periphery of the cornea of the eye in the same manner as the general surgical procedure. At this time, the waterproofing that fills the eye flows out of the eyeball.After collecting the waterproofing from the front of the eyeball from the primary incision using a 26-gauge syringe, normal eye surgery that was originally planned is performed. Proceeded. At this time, the amount collected was about 0.05 ~ 0.15cc, and the collected waterproofing was put in a 1.5 ml Eppendorf tube and stored in a -80 ℃ cryogenic freezer until analysis was performed.

(3) waterproof analysis

Five pooled waterproof proteins for each group were digested into peptides in the following manner. 8 M urea (urea) in 100 mM ammonium bicarbonate (Sigma, St. Louis, MO, USA) was mixed in a ratio of 1:3 (sample: urea) so that the final concentration was 6 M or more, and stored at room temperature for 20 minutes. . Then, the protein was alkylated using 10 mM DTT (Dithiothreitol, Sigma) and 30 mM IAA (Iodoacetamide, Sigma) for reduction. Trypsin was added to the sample (1:50 = trypsin: sample) and stored at 37° C. overnight. The activated trypsin reaction was quenched with 0.4% TFA, and the peptide was desalted with a C18 Harvard macro spin column. The resulting peptide was dried and stored at -80 °C.

The peptide was resuspended in 0.1% formic acid and analyzed using a Q Exactive™ orbitrap hybrid mass spectrometer coupled with Nanoacquity UPLC (Waters, Manchester, UK). For proteome profiling analysis, peptides were eluted through an ionized trap column using an NSI system bound to an inner column (100 cm x 75 μm ID) filled with 2 μm C18 particles at a potential of 2.0 V. Became. The entire mass spectrometer data was obtained with a resolution of 70,000 in the scan range of 400 to 1,600 m/z, the gain control value was 1.0 x 10 ⁶ and the maximum ion implantation amount was 20 ms. The maximum ion implantation time was set to 60 milliseconds, and the dynamic exclusion time was set to 30 seconds.

The initial data obtained were retrieved with MaxQuant (v. 1.5.1.2) software against the Uniprot human database. Carbamidomethylation of cysteine as a fixed variant and N-acetylation and methionine oxidation as various modifications were used in each search. Peptide spectral concordance was applied with a false discovery cutoff rate of 1% at the protein level. The initial precursor mass deviation was allowed up to 4.5 ppm and the fragment mass deviation was allowed up to 20 ppm. For protein identification, MaxQuant used the'razor plus unique peptides' setting. Protein was quantified using an XIC-based label-free quantification (LFQ) algorithm in MaxQuant. The'Match between runs' setting was used for nonlinear retention time alignment. In order to prevent the influence of the pollutant data, the reverse database was checked, and the contaminated proteins identified in the field were removed.

Perseus software (v.1.5.0.31) was used for statistical analysis of proteomics data. Data of each group was exported'LFQ intensity' from MaxQuant, and all LFQ intensities were converted to log 2 values. Proteins whose values were not indicated in all three measurements were excluded. Proteins that showed a double (FC, fold change) increase or decrease among the total detected proteins, and whose LFQ intensity expression showed a P value of less than 0.05 in t-test statistical analysis were classified as differentially expressed proteins (DEP). .

Analyzing DEP-related gene ontology biological process (GO-BP) and molecular function (GO-MF) terms using Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics database did. Functional clustering and pathway mapping analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) were also performed. Each bioinformatics-based functional classification was performed as P <0.05. To construct a network model of DEP, we collected protein-protein interaction (PPI) information from the STRING 9.1 public database. The network model was built with ordered protein and interaction data using Cytoscape software.

2. Experiment result

The protein composition of waterproofing is very similar to that of blood, and it is essential to remove abundant proteins such as serum albumin.The researchers established a flow-through concentration method using lyophilization to A pretreatment method capable of peptization without loss was completed. After removing abundant proteins such as serum albumin, transferrin, and immunoglobulin through an established method, it was verified through electrophoresis and mass spectrometry analysis. As a result, about 90% of the abundant protein was removed from the waterproof sample of about 800 μg, and through this, the identification rate of 1021 proteins was more than that of previous reports (up to 636 of the previous reports) (FIG. 1). Among them, a total of 51 significantly increased proteins (UP- Differentially expressed proteins (UP-DEP)) and 78 decreased proteins (DN-DEP) were identified in the experimental group of patients with corneal endothelial cell failure compared to the normal control group.

In FIGS. 2 to 4, profiling of proteins that were significantly increased or decreased in the experimental group of patients with corneal endothelial cell failure compared to the normal control group was confirmed. As a result, the proteins shown in Tables 2 and 4 were significantly reduced in expression in the corneal endothelial cell failure patient experimental group compared to the normal control group, and significantly expressed in the corneal endothelial cell failure patient experimental group compared to the normal control group shown in Tables 3 and 4 below. Has increased. In particular, looking at DN-DEP, which showed a greater number of changes, it was found that the expression level of carbonyl anhydrase 2 (CA2), which is important for the function of human corneal endothelial cells, was reduced by approximately 5 times.

DN-DEPs DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3, HBB, MDH1, IMPG1, APLP2, CLSTN1, GPI FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA1, C2orf40 TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, HEXB, IGHM, APOC3, LYNX1, PSAP, ENPP1A1EMA, CHRD7A, MAN F9, HRG and SERPINF1

UP-DEPs CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, FET, AFM, A1BG SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, CFHR1, F10, TIMP1, IGFBP2, F12, PCOLCEBP2 and F12, PCOLCE SLPI

Differentially expressed proteins (DEP) UP-DEP (51) DOWN-DEP (78) Gene name Log ₂ (Fold Change) P-value Gene name Log ₂ (Fold Change) P-value CST4 4.994 0.000 DKK3 -6.432 0.000 LCN1 4.485 0.004 CRYBB1 -5.232 0.000 CD163 4.080 0.036 CA2 -5.055 0.000 ITIH3 3.258 0.047 CRYBA1 -3.784 0.000 FCGBP 3.177 0.005 PGAM1 -3.764 0.001 VCAM1 3.058 0.005 CRYBA4 -3.763 0.003 VIM 2.634 0.049 CUTA -3.425 0.020 COL5A1 2.613 0.025 ARSB -3.372 0.034 CRYAA 2.548 0.004 ALDOC -3.216 0.019 ANGPTL7 2.495 0.032 CRYBB2 -3.188 0.000 C1RL 2.285 0.000 SERPINI1 -3.163 0.006 CRYAB 2.232 0.003 CREG1 -2.949 0.022 LYZ 2.182 0.042 SPOCK1 -2.881 0.001 PLXNB2 2.138 0.027 SDF4 -2.743 0.001 SERPINF2 2.113 0.000 SCG2 -2.723 0.001 C1QB 2.108 0.024 COL9A3 -2.609 0.006 SERPIND1 1.971 0.018 LGALS3BP -2.608 0.002 VTN 1.966 0.001 IGHG3 -2.586 0.004 SERPINC1 1.917 0.000 HBB -2.559 0.000 KNG1 1.881 0.008 MDH1 -2.496 0.006 APOH 1.724 0.006 IMPG2 -2.434 0.005 A1BG 1.711 0.005 GPI -2.430 0.008 C8A 1.685 0.007 TPI1 -2.401 0.001 FETUB 1.665 0.011 APLP2 -2.376 0.000 AFM 1.658 0.006 CLSTN1 -2.318 0.008 SERPINA3 1.610 0.002 FGG -2.306 0.010 CPB2 1.601 0.044 VGF -2.253 0.014 C1QC 1.579 0.002 CLSTN3 -2.207 0.014 WFIKKN2 1.546 0.008 HSPB1 -2.203 0.043 TMSB4X 1.513 0.003 GAS6 -2.200 0.001 TMSB10 1.512 0.020 L1CAM -2.173 0.001 AHSG 1.476 0.006 CRYGS -2.163 0.005 PROS1 1.471 0.003 S100A9 -2.160 0.044 RTBDN 1.432 0.003 SEMA3A -2.094 0.012 NPVF 1.402 0.013 VCAN -2.076 0.013 C9 1.393 0.000 APP -2.057 0.011 LRG1 1.352 0.002 SPARC -2.047 0.001 MST1 1.325 0.008 TWSG1 -2.035 0.014 DSC3 1.308 0.042 ALDOA -1.972 0.000 C1R 1.274 0.001 SOD1 -1.929 0.041 C8B 1.246 0.001 HBA1 -1.885 0.023 CLU 1.240 0.005 ENO1 -1.885 0.018 CTGF 1.186 0.040 SORCS1 -1.846 0.006 CFHR1 1.179 0.007 GALNT2 -1.839 0.039 F10 1.142 0.020 AGRN -1.836 0.000 TIMP1 1.087 0.018 ATP6AP1 -1.804 0.049 IGFBP2 1.087 0.028 OPTC -1.788 0.005 F12 1.075 0.042 FSTL5 -1.782 0.019 PCOLCE 1.075 0.016 C2orf40 -1.773 0.015 HABP2 1.055 0.018 FGA -1.759 0.000 SLPI 1.028 0.010 TPP1 -1.735 0.023 FSTL4 -1.661 0.007 APOB -1.650 0.011 TGFBI -1.597 0.020 GOLIM4 -1.578 0.002 XYLT1 -1.576 0.004 ITM2B -1.560 0.002 FABP5 -1.507 0.008 TKT -1.474 0.049 EFNA5 -1.424 0.030 PPIA -1.419 0.040 SCG5 -1.381 0.005 CPQ -1.331 0.017 C3 -1.229 0.008 B4GAT1 -1.222 0.001

Looking at the gene ontology biological process (GO-BP) analysis of FIG. 3, the biological processes of proteins (DEPs) significantly increased or decreased in the experimental group of patients with corneal endothelial cell failure compared to the normal control group can be confirmed. First of all, DN-DEP showed that processes such as cell metabolism, extracellular matrix formation, and nervous system development were reduced, as well as the reduction of the water transport process through bicarbonate transport. In addition, in the gene ontology molecular function (GO-MF) analysis of FIG. 4, it was also confirmed that molecular functions such as calcium ion binding and various ionic binding functions and immunomodulatory activities were deteriorated. On the other hand, in UP-DEP, immune response and aging-related processes were increased in GO-BP as well, and molecular functions related to immune response activity were also increased in GO-MF.

More specifically, according to FIG. 5, the function and interaction of proteins (DEP) significantly increased or decreased in the experimental group of patients with corneal endothelial cell failure compared to the normal control group can be intuitively confirmed. In the experimental group of patients with corneal endothelial cell insufficiency, substances involved in cell metabolism (ALDOA, ENO1, ALDOC, GPI, including CA2, which are well known to maintain the transparency of the cornea by controlling water movement through dynamic equilibrium of active pump action) The expression levels of HEXB, TPI1, ARSB, FABP5, PGAM1, MDH1, APOC3) were all reduced, as well as the expression of various factors (FGG, FGA, HRG) involved in cell matrix formation. In addition, the expression of proteins (SEMA3A, EFNA5, APOB, APP, VCAN, CHRDL1, ITM2B, SPOCK1) related to the development of the nervous system was also significantly reduced. In addition, the expression of proteins with immunomodulatory functions such as DKK3, TGFBI, C3, SEMA7A, and S100A9 was also reduced. On the other hand, various inflammatory or immune-related factors such as CD163, which is a macrophage marker, as well as complementary factors (C1R, AHSG, FETUB, PCOLCE, CST4, VCAM1, SLPI, VTN, C1QB, C1QC, C1RL, C8A, C8B, C9, CFHR1) Were increased, and the expression of various proteins related to cell adhesion (CRYAB, PLXNB2, KNG1, COL5A1, TIMP1, CLU) was also increased.

[Experimental Example 2]

1. Experimental method

(1) Patient recruitment and clinical examination

This study was approved as a prospective study after being deliberated by the Institutional Review Board of Gangnam Severance Hospital, and all patients were included in the study only after receiving sufficient explanations and consenting to the study. Corneal endothelial cell dysfunction (CECD) in 5 people for profiling analysis of the proteome of the aqueous humor (AH) as well as the transcriptome of corneal endothelial cells (CEnC). Patient groups were first recruited, and all of them were targeted at patients who had undergone corneal transplantation. The normal control group for the experimental group of the above two analyzes was inevitably configured separately to target human tissues and fluids. First, in the case of transcriptome analysis of corneal endothelial cells, the remaining portions of normal human donated corneas were used, and in the case of human waterproof proteomic analysis, patients undergoing general senile cataract surgery were targeted. Subsequently, in the individual analysis at the verification stage of the detected waterproof marker candidates, 7 additional patients of the normal control group and the corneal endothelial cell failure patient group were recruited and included in the study.

All subjects had no specific systemic history including hypertension, diabetes, and immune disease, and no specific ophthalmic history such as ocular trauma or uveitis. In all patients, the left and right eyes were not classified, and the eyes with diseases were included in the study subject, but the gender ratio, age, etc. were recruited so that there was no difference between groups.

Corneal endothelial cell insufficiency test group confirmed corneal opacity and swelling of the patient group through slit lamp microscopy, and then cornea using a specular microscopy cytometer (SP-3000P, Topcon, Tokyo, Japan) and ultrasonic corneal thickness (SP-3000, Tpocon). Endothelial cell failure was confirmed.

For statistical analysis of clinical data, SPSS v.21.0 (IBM Corp., Armonk, NY, USA) was used. After confirming the normality of the data using the Kolmogorov-Smirnov test, the Mann-Whitney U test or the Wilcoxon signed rank test was used for the unstructured distribution data. P values of less than 0.05 were considered statistically significant in all statistical tests.

(2) Experiment summary

The present inventors devised an independent research method in order to derive a waterproof protein marker that can reflect the state of the corneal endothelial cells in the human body (Fig. 6). In order to treat patients with corneal endothelial cell insufficiency, corneal transplantation is inevitably performed, and the damaged corneal endothelial cells are removed first (Stripping the CECs), and then the donor corneal endothelial cell layer is transplanted. At this time, the corneal endothelial cells of the isolated patient (recipient) were collected, and then their transcriptome analysis was performed. When the corneal endothelial cell insufficiency patients were subjected to corneal transplantation, the aqueous humor in the front of the eye was first collected. The protein composition of waterproofing is very similar to that of blood, and it is essential to remove an abundance protein such as serum albumin.The present inventors established a flow-through concentration method using freeze drying to reduce protein loss. There is no pretreatment method capable of peptization was completed. After removing abundant proteins such as serum albumin, transferrin, and immunoglobulin through an established method, it was verified through electrophoresis and mass spectrometry analysis.

(3) Corneal endothelial cell layer separation and collection

During corneal transplantation in patients with endothelial cell insufficiency, the first aqueous humor was collected, and sterile air was injected into the front of the eyeball. An auxiliary incision of 0.5 mm in size was made around the cornea, and a surgical tool for detachment of the Dessme membrane was inserted at the periphery of the cornea to separate the endothelial cell layer with a diameter of about 8 mm including the central cornea. After making an additional 3.2mm main incision in the periphery of the cornea, it was taken out, placed in an Eppendorf tube containing Trizol, and stored in a cryogenic freezer at -80°C until analysis was performed. In the case of the normal control group, corneal transplantation of patients with corneal endothelial cell insufficiency among human donated corneas was performed and separated from the remaining part, and the same was placed in an Eppendorf tube containing Trizol and stored in a cryogenic freezer at -80°C until analysis was performed. I did.

(4) waterproof sampling

When performing intraocular ophthalmic surgery including cataract surgery and corneal endothelial cell transplantation, after complete disinfection for the operation, in the same manner as the general surgical procedure, a 0.5mm-sized auxiliary incision was first performed around the cornea of the eyeball. At this time, the waterproofing that fills the eye flows out of the eyeball.After collecting the waterproofing from the front of the eyeball at the primary incision using a 26-gauge syringe, the originally planned normal eye surgery Proceeded. At this time, the amount collected was about 0.05 ~ 0.15cc, and the collected waterproofing was placed in a 1.5 ml Eppendorf tube and stored in a cryogenic freezer at -80°C until analysis was performed.

(5) Analysis of transcriptome of corneal endothelial cells

After isolation of Total RNA from corneal endothelial cells, DNA contamination was removed using DNase. RNA was purified using an mRNA purification kit, and fragmented arbitrarily for sequencing by short read. After making cDNA through a reverse transcription process for the fragmented RNA fragment, different adapters were attached to both ends and then ligated. After PCR amplification in an amount capable of sequencing, an insert size of 200-400 bp was secured through a size selection process. When sequencing as long as the read length from both ends of the cDNA fragment, quality control analysis of the obtained raw reads was performed. Pretreatment that has low-quality or removes artifacts such as adapter sequences, contaminant DNA, and PCR duplicates to reduce analysis result bias After going through the process, after mapping to a reference genome using the HISAT2 program considering splice, aligned reads were generated. Using the reference-based alignment read information, transcript assembly was performed through the StringTie program. The expression level obtained through transcript quantification of each sample is determined by FPKM (Fragments Per Kilobase of), which is a normalization value that considers read count, transcript length, and depth of coverage. The expression profile was extracted as the value of transcript per Million mapped reads. After converting to Log2, TMM normalization was performed, and the P value was calculated using the edgeR exactTest program. Genes whose values were not indicated in all three measurements were excluded. Among the detected total genes, genes that showed a double (FC, fold change) increase or decrease in expression and a P value of less than 0.05 in t-test statistical analysis were classified as differentially expressed genes (DEG).

(6) waterproof proteomic analysis

First, for profiling, the proteins contained in the waterproof samples of each group were digested into peptides in the following manner. 8 M urea (urea) in 100 mM ammonium bicarbonate (Sigma, St. Louis, MO, USA) was mixed in a ratio of 1:3 (sample: urea) so that the final concentration was 6 M or more, and stored at room temperature for 20 minutes. . Then, the protein was alkylated using 10 mM DTT (Dithiothreitol, Sigma) and 30 mM IAA (Iodoacetamide, Sigma) for reduction. Trypsin was added to the sample (1:50 = trypsin: sample) and stored at 37° C. overnight. The activated trypsin reaction was quenched with 0.4% TFA, and the peptide was desalted with a C18 Harvard macro spin column. The resulting peptide was dried and stored at -80 °C. The peptide was resuspended in 0.1% formic acid and analyzed using a Q Exactive™ orbitrap hybrid mass spectrometer coupled with Nanoacquity UPLC (Waters, Manchester, UK). For protein identification, MaxQuant used the'razor plus unique peptides' setting. Protein was quantified using an XIC-based label-free quantification (LFQ) algorithm in MaxQuant. Data of each group was exported'LFQ intensity' from MaxQuant, and all LFQ intensities were converted to log 2 values. Proteins whose values were not indicated in all three measurements were excluded. Among the total detected proteins, proteins that show a double (FC, fold change) increase or decrease, and whose LFQ intensity expression is less than 0.05 by t-test statistical analysis are classified as differentially expressed proteins (DEP). I did. In the individual analysis of the verification step, 2 ug of peptide samples were used to undergo the same pretreatment as in the profiling experiment, and then mass spectrometry was performed with the Q-Exactive plus interfaced with an EASY-nLC 1000 UHPLC System. After quantification of the selected DEPs, target analysis was performed in Spectronaut Pulsar using XXX spectral library. Perseus software (v.1.5.0.31) was used for statistical analysis.

(7) Bioinformatics analysis of transcripts and proteins

Using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatics database, the terms of gene ontology biological process (GOBP) and gene ontology molecular function (GOMF) related to DEP were analyzed. Functional clustering and pathway mapping analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) were also performed. Each bioinformatics-based functional classification was performed as P <0.05. In order to construct a network model for each of DEG and DEP, interaction information was collected from the STRING 9.1 public database. The network model was built using Cytoscape software.

2. Experiment result

There was no demographic difference between the corneal endothelial cell insufficiency patient group and the normal control group. The CEC density of the corneal endothelial cell insufficiency patient group was very low on a specular microscope, and corneal edema was severe and thickened by ultrasound corneal thickness test. .

(1) Analysis of transcriptome using corneal endothelial cells from patients with corneal endothelial cell failure

Through the transcriptome analysis study of the present researchers, a total of 25,888 mRNAs were expressed in corneal endothelial cells, and these were found to be due to cell bodies, intracellular substances, and cell membrane substances (Figs. 7 and 8). The number of DEGs showing a significant increase (UP-DEG) or decrease (DN-DEG) in corneal endothelial cells in patients with corneal endothelial dysfunction compared to normal corneal endothelial cells was identified as 2658 and 1832, respectively (FIG. 9). As a result of analyzing their mechanisms using bioinformatics techniques (Figs. 10 and 11), UP-DEG is largely developed (development), extracellular excretion (exocytosis), biological adhesion (biological adhesion), ion transport (ion transport) And the cytokine signaling pathway. DN-DEGs consisted of five clusters, including carbohydrate metabolic process, localization, cellular component organization, and It formed a network that produced mechanisms such as cell death and response to stimulus.

(2) Proteomics analysis using waterproofing of corneal endothelial cell insufficiency as a specimen

About 90% of the abundant protein was removed from about 800 ug of the waterproof sample, and through this, more 1021 proteins were identified than previous reports (Fig. 12), which originated from cellular material transporters, secretory granules, and cell surfaces ( 13). Compared to the normal control, a total of 51 DEP (UP-DEP) significantly increased in water resistance of patients with corneal endothelial cell failure, and 78 total decreased DEP (DN-DEP) were confirmed (FIG. 14 ). As a result of analyzing their mechanisms using bioinformatics techniques (FIG. 15), UP-DEPs were largely composed of three clusters, most of which were immune responses, in addition to protein metabolism and hydrolysis. and proteolysis) and extracellular excretion (exocytosis). In addition, DN-DEPs form a network that creates mechanisms such as carbohydrate metabolic process, developmental process, and cell adhesion.

(3) Exploration of waterproofing proteins that can reflect corneal endothelial cell failure and exploring mechanisms within waterproofing

A human specimen called'waterproof' is in constant contact with the entire corneal endothelial cells, so it can directly reflect the molecular biological state of the corneal endothelial cells. Therefore, as a result of confirming whether DEG expressed by corneal endothelial cells in the eye of corneal endothelial cell insufficiency and their expressed DEP in aqueous humor are related, there is a very close positive correlation between DEG expression in corneal endothelial cells and DEP expression in aqueous humor. It was confirmed that there is (Fig. 17). The 13 substances that increase the same in DEG and waterproof DEP of corneal endothelial cells are ANGPTL7, FCGBP, SERPINF2, F10, PROS1, SERPIND1, HABP2, TMSB10, TIMP1, LYZ, SLPI, TMSB4X and C1R, all of which are immune responses. Involved in the mechanism (Figs. 18 and 20). The 22 substances that equally decrease in DEG of corneal endothelial cells and DEP of waterproofing are MDH1, MAN1A, B4GAT1, GPI, ENO1, ALDOA, PGAM1, TPI1, GALNT2, TKT, TGFBI, LYNX1, PSAP, CPQ, SERPINI1, COL9A3, APP, CLSTN1, LGALS3BP, CA2, ATP6AP1, APLP2, etc., which are related to carbohydrate metabolism processes, developmental processes and ion transport mechanisms (FIGS. 19 and 20). It was very rare that the expression of DEG and waterproof DEP in corneal endothelial cells had a negative correlation (FIG. 20). Accordingly, as shown in FIG. 21, the waterproofing of corneal endothelial cell insufficiency patients was found to be an excellent human specimen that reflects the biological changes of corneal endothelial cells well.

(4) Derivation of biomarker substances that can detect corneal endothelial cell failure using waterproofing

In order to identify a waterproof biomarker protein capable of detecting corneal endothelial cell failure, 7 patients with corneal endothelial cell failure and 7 normal controls were additionally recruited, and then a waterproof protein analysis (DIA analysis) was performed for each patient (Fig. 22). As a result of confirming the expression of protein in the aqueous humor of each corneal endothelial cell patient, the normal control group and the experimental group showed a well-differentiated expression pattern, and mainly increased DEPs were included (FIGS. 23 and 24 ). UP-DEP and DIA waterproofing during LFQ AH profiling As a result of considering the expression of transcriptome of corneal endothelial cells (UP-DEG) among UP-DEP during individual analysis, waterproofing that increases significantly in diseases of corneal endothelial cell failure As my biomarkers, TIMP1, FCGBP, and ANGPTL7 were able to be derived (Figs. 25 to 27).

Claims (24)

At least one protein selected from the group consisting of TIMP1, FCGBP and ANGPTL7; Or a biomarker for diagnosing dysfunction of corneal endothelial cells comprising a gene encoding the same. The method of claim 1,
The biomarker is a biomarker for diagnosing dysfunction of corneal endothelial cells, which is present in the aqueous humor of the eye. The method of claim 1,
The biomarkers are CST4, LCN1, CD163, ITIH3, VCAM1, VIM, COL5A1, CRYAA, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, C8A. SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, CFHR1, F10, IGFBP2, F12, PCOLCE, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3, HBB, MDH1, IMPG1, APLP2, CLSTN1, GPI FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA1, C2orf40 TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, HEXB, IGHM, APOC3, LYNX1, PSAP, ENPP1A1EMA, CHRD7A, MAN At least one protein selected from the group consisting of F9, HRG and SERPINF1; Or a biomarker for diagnosing dysfunction of corneal endothelial cells, further comprising a gene encoding it. At least one protein selected from the group consisting of TIMP1, FCGBP and ANGPTL7; Or a composition for diagnosing dysfunction of corneal endothelial cells comprising an agent capable of measuring the expression level of the gene encoding the same. The method of claim 4,
The composition is CST4, LCN1, CD163, ITIH3, VCAM1, VIM, COL5A1, CRYAA, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, C8A, SERPFM3 , CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, CFHR1, F10, IGFBP2, F12, PCOLCE, HAK3, SLPI , CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3, HBB, MDH1, IMPG2, APLPGG2, CLSTN1 , VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, FSTL5, C2orf40PP, , FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, HEXB, IGHM, APOC3, LYNX1, PSAP, ENPP2, SEMA7A, MAN1A1 , At least one protein selected from the group consisting of HRG and SERPINF1; Or a composition for diagnosing dysfunction of corneal endothelial cells, further comprising an agent capable of measuring the expression level of the gene encoding the same. The method of claim 4,
The agent for measuring the expression level of the protein comprises at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNA) and aptamers that specifically bind to the protein, cornea A composition for diagnosing endothelial cell dysfunction. The method of claim 4,
The agent for measuring the expression level of the gene comprises at least one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene, a composition for diagnosing corneal endothelial dysfunction. The method of claim 4,
The protein or the expression level of the gene is measured for the aqueous humor of the eyeball, a composition for diagnosing dysfunction of corneal endothelial cells. A kit for diagnosing dysfunction of corneal endothelial cells comprising the composition of any one of claims 4 to 8. At least one protein selected from the group consisting of TIMP1, FCGBP, and ANGPTL7 in a biological sample isolated from the subject of interest; Or a method for providing information for diagnosing dysfunction of corneal endothelial cells comprising the step of measuring the expression level of the gene encoding the same. The method of claim 10,
The biological sample is an aqueous humor of the eyeball, a method of providing information for diagnosing dysfunction of corneal endothelial cells. The method of claim 10,
The step of measuring the expression level is CST4, LCN1, CD163, ITIH3, VCAM1, VIM, COL5A1, CRYAA, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1, AP8A, A1BG , FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, CFHR1, F12, PCCEBP2, and IGFBP2 , HABP2, SLPI, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGPIALS3BP, IGHG3, HBB, MDH1 , APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC , C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, HEXB, IGHM, APOC3, LYN2, SEMA7, PSAAP , MAN1A1, CHRDL1, F9, HRG, and one or more proteins selected from the group consisting of SERPINF1; Or, the method of providing information for diagnosing dysfunction of corneal endothelial cells, further comprising the step of measuring the expression level of the gene encoding the same. The method of claim 10, wherein the expression level of the protein is at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNA) and aptamers that specifically bind to the protein. Method for providing information for diagnosing dysfunction of corneal endothelial cells, which is measured by. The method of claim 10,
The expression level of the gene is measured using at least one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene, and information providing method for diagnosing dysfunction of corneal endothelial cells. The method of claim 10,
Information for diagnosing dysfunction of corneal endothelial cells, comprising predicting that the likelihood of dysfunction of corneal endothelial cells is high when the expression level of the protein or the gene encoding it is increased or decreased compared to the normal control Delivery method. The method of claim 12,
CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG measured for the biological sample of the object of interest. , APOH, A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LHRRG1, MST1, DSC3, C1R, C8B, CLU, F10 CF , TIMP1, IGFBP2, F12, PCOLCE, HABP2 and at least one protein selected from the group consisting of SLPI; Or, when the expression level of the gene encoding the same is increased compared to the normal control, a method for providing information for diagnosing dysfunction of corneal endothelial cells, predicting that the possibility of dysfunction of corneal endothelial cells is high. The method of claim 12,
DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3, HBB, measured for the biological sample of the object of interest. , IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNTRN , ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, APOC3, IGHM , PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, at least one protein selected from the group consisting of HRG and SERPINF1; Or, when the expression level of the gene encoding the same is decreased compared to the normal control, a method for providing information for diagnosing dysfunction of corneal endothelial cells, predicting that the possibility of dysfunction of corneal endothelial cells is high. The method of claim 10,
The diagnosis of the dysfunction of the corneal endothelial cells includes the onset or the possibility of a disease induced due to the dysfunction of the corneal endothelial cells, or includes tracking the course of the disease, prognosis, or therapeutic effect. How to provide information for diagnosing dysfunction of the body. The method of claim 18,
The disease induced due to the dysfunction of the corneal endothelial cells is Fuchs corneal dystrophy, trauma, vesicular keratopathy of the artificial lens, corneal endothelial or vision loss, a method of providing information for diagnosing corneal endothelial dysfunction. Treating the isolated biological sample with a candidate substance expected to induce dysfunction of corneal endothelial cells; And
At least one protein selected from the group consisting of TIMP1, FCGBP, and ANGPTL7 in the biological sample treated with the candidate substance; Or a method for screening a drug that induces dysfunction of corneal endothelial cells, comprising the step of measuring the expression level of a gene encoding the same. The method of claim 20,
The biological sample is an aqueous humor of the eye, a method for screening a drug that induces dysfunction of corneal endothelial cells. The method of claim 20,
The step of measuring the expression level is CST4, LCN1, CD163, ITIH3, VCAM1, VIM, COL5A1, CRYAA, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, KNG1, APOH, A1BG, C1BG. FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, CFHR1, F10, IGFOLCE, F12 HABP2, SLPI, DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9API, LGALS3BP, IGHG3, HBB, MDH1, IGHG3, HBB, MDH1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, AGRN, ATP6AP1, OPTC, ATP6AP1, OPTC C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, HEXB, IGHM, APOC3, LYNX1, PSA7, ENPP At least one protein selected from the group consisting of MAN1A1, CHRDL1, F9, HRG and SERPINF1; Or a method for screening a drug that induces dysfunction of corneal endothelial cells, further comprising the step of measuring the expression level of the gene encoding the same. The method of claim 22,
CST4, LCN1, CD163, ITIH3, FCGBP, VCAM1, VIM, COL5A1, CRYAA, ANGPTL7, C1RL, CRYAB, LYZ, PLXNB2, SERPINF2, C1QB, SERPIND1, VTN, SERPINC1, measured in the biological sample after treatment of the candidate material. KNG1, APOH, A1BG, C8A, FETUB, AFM, SERPINA3, CPB2, C1QC, WFIKKN2, TMSB4X, TMSB10, AHSG, PROS1, RTBDN, NPVF, C9, LRG1, MST1, DSC3, C1R, C8B, CLU, CTGF, and CLU At least one protein selected from the group consisting of F10, TIMP1, IGFBP2, F12, PCOLCE, HABP2 and SLPI; Or, when the expression level of the gene encoding the same is increased compared to before treatment, the candidate substance is determined as an inducer of dysfunction of corneal endothelial cells, a method for screening a drug that induces dysfunction of corneal endothelial cells. The method of claim 22,
DKK3, CRYBB1, CA2, CRYBA1, PGAM1, CRYBA4, CUTA, ARSB, ALDOC, CRYBB2, SERPINI1, CREG1, SPOCK1, SDF4, SCG2, COL9A3, LGALS3BP, IGHG3, HBB, measured in the biological sample after treatment of the candidate material MDH1, IMPG2, GPI, TPI1, APLP2, CLSTN1, FGG, VGF, CLSTN3, HSPB1, GAS6, L1CAM, CRYGS, S100A9, SEMA3A, VCAN, APP, SPARC, TWSG1, ALDOA, SOD1, HBA1, ENO1, SORCS1, GALNT2, SORCS1 AGRN, ATP6AP1, OPTC, FSTL5, C2orf40, FGA, TPP1, FSTL4, APOB, TGFBI, GOLIM4, XYLT1, ITM2B, FABP5, TKT, EFNA5, PPIA, SCG5, CPQ, C3, B4GAT1, NEO1, APOC3, IGHM, HEXB, IGHM At least one protein selected from the group consisting of LYNX1, PSAP, ENPP2, SEMA7A, MAN1A1, CHRDL1, F9, HRG and SERPINF1; Or, when the expression level of the gene encoding the same is decreased compared to before treatment, the candidate substance is discriminated as an inducer of dysfunction of corneal endothelial cells.

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