Screening methods for drugs that control cellular aging

Abstract

The object of the present invention is to provide a method for analyzing the disorder of collagen in living organisms. [Solution] The above problem can be solved by a screening method for cellular senescence-related substances, which includes the step of contacting a candidate substance with cells expressing procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein of the present invention.

Description

Technical Field

[0001] The present invention relates to a method for screening drugs that control cell senescence. According to the present invention, drugs that suppress cell senescence can be obtained.

Background Art

[0002] Collagen accounts for 30% of the proteins in the living body and is an important protein having functions such as skeletal support and cell adhesion. For example, it is a major component of tissues such as bones, cartilage, ligaments, tendons, corneal stroma, skin, liver, and muscles in the human body. Among collagens, there is fibrous collagen that forms collagen fibers. Fibrous collagen has a triple helix structure (tropocollagen) formed by three polypeptide chains of collagen. The tropocollagen with this triple helix structure self-organizes, shifts by 1 / 4 of the molecular length, and aligns to form collagen protofibrils (collagen fibrils). The present inventors studied the process of collagen biosynthesis in cells using a fusion protein in which a fluorescent protein or a luminescent protein was fused to collagen (Patent Document 1, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] ​In the process of studying diseases caused by molecular abnormalities in collagen using the aforementioned fusion protein, the inventors noticed that the collagen constructed by aging cells becomes disordered. Therefore, the object of the present invention is to provide a method for analyzing the disorder of collagen in living organisms. [Means for solving the problem]

[0006] The inventors, through diligent research into methods for analyzing collagen disorder in living organisms, have surprisingly discovered that the orientation of collagen fibers is disrupted in aged cells. Furthermore, they found that this disruption of collagen fiber orientation occurs because the cleavage of procollagen by collagen processing enzymes is suppressed. In other words, they discovered that normal collagen production is disrupted in aged cells. This invention is based on these findings. Therefore, the present invention is [1] A method for screening cellular senescence-related substances, comprising the step of contacting cells expressing procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein with a candidate substance. [2] A screening method for aging-related substances according to [1], comprising the step of measuring a decrease or increase in procollagen C-proteinase function, a decrease or increase in procollagen C-proteinase enhancer function, a decrease or increase in the function of proteins cleaved by procollagen C-proteinase, a decrease or increase in procollagen N-proteinase function, a decrease or increase in procollagen N-proteinase activator function, or a decrease or increase in the function of proteins cleaved by procollagen N-proteinase. [3] The method for screening aging-related substances according to [2], wherein the decrease or increase in the function is a decrease or increase in genes, a decrease or increase in proteins, or a decrease or increase in protein activity. [4] The method for screening aging-related substances according to [1], wherein the procollagen C-proteinase is BMP-1, mTLD, mTLL1, or mTLL2, the procollagen C-proteinase enhancer is PCPE1 or PCPE2, the protein cleaved by the procollagen C-proteinase is procollagen, the procollagen N-proteinase is ADAMTS-2, ADAMTS-3, or ADAMTS-14, the procollagen N-proteinase activator is furin, and the protein cleaved by the procollagen N-proteinase is procollagen. [5] A method for diagnosing cellular senescence, comprising the step of measuring the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein function, procollagen N-proteinase, procollagen N-proteinase activator function, or procollagen N-proteinase-cleaved protein function in cells. [6] The method for diagnosing cellular senescence as described in [5], wherein the measurement of the function is the measurement of genes, proteins, or protein activity, and it is determined that the cells have senescent when the genes, proteins, or protein activity have decreased, and [7] The method for diagnosing cellular senescence according to [6], wherein the procollagen C-proteinase is BMP-1, mTLD, mTLL1, or mTLL2, the procollagen C-proteinase enhancer is PCPE1 or PCPE2, the protein cleaved by the procollagen C-proteinase is procollagen, the procollagen N-proteinase is ADAMTS-2, ADAMTS-3, or ADAMTS-14, the procollagen N-proteinase activator is furin, and the protein cleaved by the procollagen N-proteinase is procollagen. Regarding. [Effects of the Invention]

[0007] According to the present invention's method for screening drugs that control cellular aging, it is possible to search for substances (compounds) that control cellular aging. [Brief explanation of the drawing]

[0008] [Figure 1] These images show the results of subculturing human fibroblasts and examining SA-b-gal staining, cell proliferation rate, and expression of cellular senescence marker genes at stages 10-15, 21-26, and 35-41. [Figure 2] These are photographs showing the orientation of collagen fibers in human fibroblasts that have been subcultured, examined using picrosilius red staining, immunofluorescence with anti-type I collagen antibody, and transmission electron microscopy. [Figure 3] This image shows the changes in the collagen biosynthesis process associated with aging in human fibroblasts. [Figure 4] This reference example shows photographs and graphs analyzing changes in intracellular procollagen processing associated with aging in human fibroblasts, as analyzed by Western blotting of cleaved C-propeptides. [Figure 5] These are photographs and graphs showing the changes in extracellular secreted collagen due to aging in human fibroblasts, analyzed by Western blotting. [Figure 6] This diagram schematically illustrates the mechanism of procollagen cleavage (processing). [Figure 7] These are graphs and Western blotting images showing changes in BMP-1 mRNA, protein, and protein activity associated with aging in human fibroblasts. [Figure 8] These are graphs and Western blotting images showing changes in PCPE1 mRNA and protein associated with aging in human fibroblasts. [Figure 9] This image shows how inhibiting PCPs in human fibroblasts disrupts the orientation of collagen fibers. [Modes for carrying out the invention]

[0009] [1] Screening method for cellular senescence-related substances The present invention provides a method for screening cellular senescence-related substances, comprising the step of contacting a candidate substance with cells expressing procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein.

[0010] (1) Contact process The contact step in the screening method of the present invention is a step of bringing a candidate substance into contact with cells. The cells used in the screening method of the present invention are not particularly limited as long as they express procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein, but are preferably aged cells. While the cell type is not limited, cells with reduced function (decreased levels) of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen N-proteinase, or procollagen N-proteinase activator are preferred. However, cells without reduced function (decreased levels) of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen N-proteinase, or procollagen N-proteinase activator can also be used in the screening method of the present invention by comparing them with a control that is not exposed to the candidate substance. As another aspect of the cell, although not limited thereto, cells in which the function of a cleavage protein by procollagen C-proteinase or a cleavage protein by procollagen N-proteinase is reduced (decreased) are preferred. However, even cells in which the function of a cleavage protein by procollagen C-proteinase or a cleavage protein by procollagen N-proteinase is not reduced (decreased) can be used in the screening method of the present invention by comparison with a control that does not contact the candidate substance.

[0011] Specific cells include, but are not particularly limited to, for example, fibroblasts, nerve cells (e.g., neurons, glial cells, ganglion cells, or retinal cells, etc.), bone cells, osteoblasts, osteoclasts, chondrocytes, epithelial cells, mesothelial cells, endothelial cells, stromal cells, adipocytes (e.g., white adipocytes, etc.), blood lineage cells (lymphocytes, B cells, T cells, erythrocytes, leukocytes, granulocytes, monocytes, megakaryocytes, macrophages, dendritic cells, or bone marrow cells, etc.), organ-derived cells (hepatocytes, splenocytes, pancreatic cells, renal cells, gastric cells, intestinal cells, pituitary cells, bladder cells, skin cells, Langerhans islets, adrenal medulla cells, ovarian cells, testicular cells, or lung cells, etc.), muscle tissue-derived cells (skeletal muscle cells, smooth muscle cells, myoblasts, or cardiomyocytes, etc.), embryonic stem cells (ES cells), and the like. The origin of the cells is not limited either, and examples include mammals (e.g., humans, chimpanzees, cynomolgus monkeys, common marmosets, pigs, cows, horses, goats, sheep, dogs, cats, rabbits, pandas, mice, or rats), birds (e.g., chickens), amphibians (e.g., Xenopus laevis), reptiles (e.g., green anole), and fish (e.g., salmon).

[0012] The expression of the procollagen C-proteinase, procollagen C-proteinase enhancer, cleavage protein by procollagen C-proteinase, procollagen N-proteinase, procollagen N-proteinase activator, or cleavage protein by procollagen N-proteinase may use those originally expressed in each cell. However, transformed expression cells using vectors containing genes encoding each protein may also be used.

[0013] Senescent cells can be obtained, although not limited to, for example, by cell passage, photoaging, drug stimulation, or physical stimulation. For example, that the obtained cells are senescent can be confirmed, although not limited to, as shown in the examples below, by the decline (decrease) in the function of procollagen C-proteinase, procollagen C-proteinase enhancer, cleavage protein by procollagen C-proteinase, procollagen N-proteinase, procollagen N-proteinase activator, or cleavage protein by procollagen N-proteinase.

[0014] 《Procollagen C-proteinase》 Procollagen C-proteinase (PCPs) is an enzyme that cleaves the C-terminal pro-peptide (C-pp) of procollagen, as shown in Figure 6. Examples of procollagen C-proteinase include, but are not limited to, BMP-1, mTLD, mTLL1, or mTLL2. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in procollagen C-proteinase function is observed. If procollagen C-proteinase function decreases or increases, the candidate substance can be determined to be a substance related to cellular senescence. A decrease or increase in procollagen C-proteinase function may include, but is not limited to, a decrease or increase in the procollagen C-proteinase gene, a decrease or increase in the protein, or a decrease or increase in protein activity.

[0015] Procollagen C-Proteinase Enhancer Procollagen C-proteinase enhancers (PCPEs) are proteins that promote the activity of procollagen C-proteinase. Examples of procollagen C-proteinase enhancers include, but are not limited to, PCPE1 and PCPE2. PCPE1 and PCPE2 are proteins that activate the function of BMP-1. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in procollagen C-proteinase enhancer function is observed. If the procollagen C-proteinase enhancer function decreases or increases, the candidate substance can be determined to be a substance related to cellular senescence. The decrease or increase in procollagen C-proteinase enhancer function may include, but is not limited to, a decrease or increase in the procollagen C-proteinase enhancer gene, a decrease or increase in the protein, or a decrease or increase in protein activity.

[0016] Proteins cleaved by procollagen C-proteinase Proteins cleaved by procollagen C-proteinase are proteins on which procollagen C-proteinase acts, such as procollagen, osteoglycin, biglycan, laminin 5, or (pro)lystl oxidase. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in the function of procollagen, etc., is observed. If the function of procollagen, etc., decreases or increases, the candidate substance can be determined to be a cellular senescence-related substance. The decrease or increase in the function of procollagen, etc., is not limited to, but may include a decrease or increase in gene expression of procollagen, etc., a decrease or increase in protein, or a decrease or increase in protein activity. More specifically, decreases or increases in the function of procollagen, etc., include decreases or increases in the amount of collagen produced from procollagen, decreases or increases in the intracellular transport of procollagen and collagen, decreases or increases in the amount of procollagen and collagen secreted from cells, decreases or increases in the expression level of collagen genes, or decreases or increases in the disorder of collagen orientation. Human procollagen includes fibrous procollagen and non-fibrous procollagen of types I to XXVIII. The procollagen used in the screening method of the present invention can be either fibrous or non-fibrous procollagen, but fibrous procollagen is preferred, and examples include type I procollagen, type II procollagen, type III procollagen, type V procollagen, or type XI procollagen. In this specification, procollagen includes pre-procollagen having a signal peptide at the N-terminus, and pre-procollagen can also be used in the screening of the present invention.

[0017] Procollagen N-proteinase Procollagen N-proteinases (PNPs), as shown in Figure 6, are enzymes that cleave the N-terminal propeptide (N-pp) of procollagen. Examples of procollagen N-proteinases, though not limited to them, include ADAMTS-2, ADAMTS-3, and ADAMTS-14. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in procollagen N-proteinase function is observed. If procollagen N-proteinase function decreases or increases, the candidate substance can be determined to be a substance related to cellular senescence. A decrease or increase in procollagen N-proteinase function may include, but is not limited to, a decrease or increase in procollagen N-proteinase gene expression, a decrease or increase in protein, or a decrease or increase in protein activity.

[0018] Procollagen N-proteinase activator Procollagen N-proteinase activators are proteins that promote the activity of procollagen N-proteinase. Examples of procollagen N-proteinase activators include, but are not limited to, furin. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in procollagen N-proteinase activator function is observed. If the procollagen N-proteinase activator function decreases or increases, the candidate substance can be determined to be a substance related to cellular senescence. The decrease or increase in procollagen N-proteinase activator function may include, but is not limited to, a decrease or increase in the procollagen N-proteinase activator gene, a decrease or increase in the protein, or a decrease or increase in protein activity.

[0019] Proteins cleaved by procollagen N-proteinase Proteins cleaved by procollagen N-proteinase are proteins on which procollagen N-proteinase acts, such as procollagen. In the screening method of the present invention, a candidate substance is brought into contact with the cells, and a decrease or increase in the function of procollagen, etc., is observed. If the function of procollagen, etc., decreases or increases, the candidate substance can be determined to be a cellular senescence-related substance. The decrease or increase in the function of procollagen, etc., is not limited to, but may include a decrease or increase in gene expression of procollagen, etc., a decrease or increase in protein, or a decrease or increase in protein activity. More specifically, decreases or increases in the function of procollagen, etc., include decreases or increases in the amount of collagen produced from procollagen, decreases or increases in the intracellular transport of procollagen and collagen, decreases or increases in the amount of procollagen and collagen secreted from cells, decreases or increases in the expression level of collagen genes, or decreases or increases in the disorder of collagen orientation. The procollagen used can be the same as the procollagen described in "Procollagen C-protein cleavage protein" above.

[0020] The candidate substances (test substances) that can be used in the screening method of the present invention are not particularly limited, but include, for example, various compounds registered in the Chemical File, a group of compounds obtained by combinatorial chemistry technology (Terrett NK et al., Tetrahedron, 51, 8135-73, 1995), a group of random peptides created by applying phage display methods (Felici, F. et al., J.Mol.Biol., 222, 301-310, 1991), etc.; proteins, nucleic acid molecules, peptides, antibodies; culture supernatants or cell extracts of microorganisms or plant or animal cells, natural components derived from plants or marine organisms, animal tissue extracts, bodily fluids in living organisms; and furthermore, compounds obtained by chemically or biologically modifying compounds selected by the screening method of the present invention.

[0021] Contact between screening cells and the test substance can be carried out, for example, by culturing the cells in a culture medium containing the test substance. For example, the contact can be carried out by preparing a microplate with cells pre-cultured in each well, and then adding the test substance to each well and starting the culture.

[0022] In the contact step of the screening method of the present invention, the amount of cells used (e.g., cell count / culture medium volume) and the amount of candidate substance can be determined as appropriate. The concentration of the test substance cannot be specified in general, as it varies depending on factors such as its type, degree of purification, and strength of activity. However, a person skilled in the art can determine it appropriately by conducting preliminary experiments at several different concentrations.

[0023] In the contact step of the screening method of the present invention, in addition to the test group to which the test substance is added, a negative control to which the test substance is not added, and a positive control to which a known positive substance is added instead of the test substance, can be prepared as desired.

[0024] (2) Measurement process The screening method of the present invention may include a step of measuring the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein. To measure the genes encoding these proteins, the mRNA of these genes should be measured. Specifically, this can be done using known mRNA analysis methods, such as PCR using PCR primers (e.g., real-time PCR, RT-PCR), hybridization using nucleic acid probes (e.g., Northern blotting), or single-cell RNA sequencing.

[0025] Real-time PCR methods include the intercalator method, which uses a primer set consisting of forward and reverse primers and adds an intercalator compound, such as SYBR Green I, which fluoresces upon binding to double-stranded DNA, to the PCR reaction system; and the TaqMan method, which adds the aforementioned primer set and a probe (TaqMan probe) modified with a reporter dye at the 5' end and a quencher dye at the 3' end to the PCR reaction system. Such real-time PCR itself is well known, and kits and equipment for it are commercially available, so if a primer set, or a primer set and probe, is synthesized, it can be easily carried out using commercially available kits and equipment.

[0026] The forward primers, reverse primers, and probes can be prepared based on the nucleotide sequences of the BMP-1 gene, mTLD gene, mTLL1 gene, mTLL2 gene, PECP1 gene, PECP2 gene, ADAMTS-2 gene, ADAMTS-3 gene, or ADAMTS-14 gene. The length of the primers is not particularly limited, but is preferably 15mer to 35mer, more preferably 16mer to 30mer, and most preferably 19mer to 25mer. The length of the probes is not particularly limited, but is preferably 12mer to 30mer, more preferably 13mer to 29mer, and most preferably 14mer to 18mer. For example, the following can be used as primers for BMP-1 and PCPE-1. BMP-1 F: 5'-ccagtcctttgagattgagc-3'(Sequence ID 1) R: 5'-tcatcaggcttctcatagcc-3'(Sequence ID 2) PCPE-1 F: 5'-taaaactggaggactggacc-3'(Sequence ID 3) R: 5'-tgactcctttcttcatgggg-3'(Sequence ID 4)

[0027] The aforementioned PCR method, in particular the real-time PCR method, (1) A process of extracting mRNA from a biological sample, (2) Using the extracted mRNA as a template, a step is taken to synthesize cDNA using reverse transcriptase. (3) Amplify DNA using a primer set, or a primer set and probe. Process, and (4) Steps to detect amplified DNA, It can include...

[0028] When measuring the aforementioned protein, it can be measured by known protein analysis methods, such as immunological analysis methods using antibodies (e.g., ELISA, latex agglutination, Western blotting), biochemical analysis methods such as electrophoresis, and mass spectrometry. Automated analyzers for clinical testing can also be used. As the measurement sample, the aforementioned cells or cell extracts, and in some cases, culture supernatant or body fluids (e.g., blood, cerebrospinal fluid, lymph, urine, ascites, pleural fluid, etc.) can be used.

[0029] (Immunological analysis method) When using immunological analytical methods, monoclonal antibodies or polyclonal antibodies that bind to each protein can be used. When using enzyme immunoassay, particularly the sandwich method, as an immunoassay method, the following procedure can be performed. First, an antibody that binds to a protein (capture antibody, or primary antibody) is immobilized on an insoluble carrier such as a microplate or beads. Next, to prevent nonspecific adsorption to the capture antibody or insoluble carrier, the insoluble carrier is blocked with a suitable blocking agent (e.g., bovine serum albumin or gelatin). The test sample containing the protein is added to the plate or beads on which the capture antibody is immobilized, along with the primary reaction solution, and the capture antibody and protein come into contact and bind (primary reaction step). After this, antigens and contaminants that did not bind to the capture antibody are washed with a suitable washing solution (e.g., phosphate buffer containing a surfactant). Next, a labeled antibody (secondary antibody), which is an antibody that binds to the captured protein and an enzyme such as horseradish peroxidase (HRP), is added, and the labeled antibody binds to the captured antigen (secondary reaction step). As a result of this reaction, an immune complex of capture antibody-protein-labeled antibody is formed on the carrier such as a microplate. Unbound labeled antibodies are washed with a washing solution, and a chromogenic or luminescent substrate is added to the enzyme of the labeled antibody. The signal is detected by reacting the enzyme with the substrate. Alternatively, it is possible to detect the signal by labeling the antibody that binds to the secondary antibody, rather than directly labeling the secondary antibody.

[0030] The measurement process allows for the determination of whether the function (gene, protein, or activity) of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein is decreased or increased.

[0031] (3) Judgment process The screening method of the present invention may include a step of determining whether or not a candidate substance (test substance) is a substance related to cellular senescence. To determine whether a substance is related to cellular senescence, the reference value (mean value) of genes, gene transcripts, or proteins in cells without the candidate substance added can be measured. If the amount of the substance increases (or decreases) compared to the reference value, it can be determined to be a substance related to cellular senescence. A cutoff value such as mean ± SD, mean ± 2SD, or mean ± 3SD can also be set. Senescent cells have reduced levels of procollagen C-proteinase or procollagen C-proteinase enhancer. Therefore, although not limited to these, if procollagen C-proteinase or procollagen C-proteinase enhancer increases, it may be determined to be a substance that suppresses aging. Conversely, if procollagen C-proteinase or procollagen C-proteinase enhancer decreases, it may be determined to be a substance that accelerates aging.

[0032] [2] Methods for diagnosing cellular senescence The present invention provides a method for diagnosing cellular senescence, comprising the steps of measuring the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein function, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein function in cells. The "diagnostic method" of this original invention can also be implemented as an auxiliary method for diagnosing cellular senescence. That is, it can be used, for example, as an auxiliary method for a physician's diagnosis.

[0033] In the method for diagnosing cellular senescence of the present invention, the cells to be diagnosed are not particularly limited, as long as they are cells undergoing senescence. The cells to be identified in the diagnostic method of the present invention are not particularly limited, but include fibroblasts, nerve cells (e.g., neurons, glial cells, ganglion cells, or retinal cells), osteocytes, osteoblasts, osteoclasts, chondrocytes, epithelial cells, mesothelial cells, endothelial cells, stromal cells, adipocytes (white adipocytes, etc.), hematopoietic cells (lymphocytes, B cells, T cells, erythrocytes, leukocytes, granulocytes, monocytes, megakaryocytes, macrophages, dendritic cells, or bone marrow cells, etc.), organ-derived cells (hepatocytes, spleen cells, pancreatic cells, kidney cells, gastric cells, intestinal cells, pituitary cells, bladder cells, skin cells, islets of Langerhans, adrenal medulla cells, ovarian cells, testicular cells, or lung cells, etc.), muscle tissue cells (skeletal muscle cells, smooth muscle cells, myoblasts, or cardiomyocytes, etc.), embryonic stem cells (ES cells), etc. These cells may be in vivo cells or in vitro cells. Therefore, when diagnosing cellular aging in a living organism, cells collected from the organism may be measured. Furthermore, the function of procollagen C-proteinase, procollagen C-proteinase enhancer, proteins cleaved by procollagen C-proteinase, procollagen N-proteinase, procollagen N-proteinase activator, or proteins cleaved by procollagen N-proteinase secreted into living cells can also be measured. Therefore, in addition to cells (including cultured cells) or tissues, samples to be measured include urine, blood, serum, plasma, lymph, tissue fluid, cerebrospinal fluid, saliva, or sweat.

[0034] 《Measurement process》 The present invention provides a method for diagnosing cellular senescence, which includes a step of measuring the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein. In the method for diagnosing cellular senescence of the present invention, the procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein is the same as that described in "[1] Method for screening cellular senescence-related substances". Therefore, the aforementioned function refers to genes such as procollagen N-proteinase, gene expression, protein, or protein activity, and in this measurement, these genes, gene expression, protein, or activity are measured.

[0035] When measuring genes or gene expression, the procedure can be carried out in accordance with "(2) Measurement step" of "[1] Method for screening substances related to cellular senescence." Similarly, when measuring proteins or protein activity, the procedure can also be carried out in accordance with "[1] Method for screening substances related to cellular senescence."

[0036] 《Judgment process》 In the cellular senescence diagnostic method of the present invention, cellular senescence can be determined when the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved proteins, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved proteins is reduced compared to normal cells. In other words, cellular senescence can be determined when genes, gene expression, proteins, or protein activity are reduced compared to normal cells.

[0037] Diagnostic kit The present invention provides a diagnostic kit for cellular senescence that measures the gene, gene expression, protein, or protein activity of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein.

[0038] In the case of a kit for measuring genes or gene expression, it includes primers and / or probes as described in "[1] Method for screening cellular senescence-related substances." In the case of a kit for measuring proteins, it may include antibodies, etc., as described in "[1] Method for screening cellular senescence-related substances."

[0039] 《Action》 In this invention, the mechanisms by which cellular senescence-related substances can be screened and the mechanisms by which cellular senescence can be diagnosed have not been analyzed in detail, but can be presumed as follows. However, this invention is not limited by the following presumes. In normal cells that have not undergone aging, collagen forms a triple helix structure. These triple helix collagen fibers align with a 1 / 4 offset, forming well-oriented collagen fibrils (collagen microfibrils). On the other hand, in cells that have aged due to cell passage, photoaging, drug stimulation, or physical stimulation, the orientation of collagen fibrils is disrupted. This disruption of orientation is presumed to be because procollagen proteins, which have an N-propeptide at the N-terminus and a C-propeptide at the C-terminus, are not properly cleaved by procollagen C-proteinase and / or procollagen N-proteinase. In other words, procollagen that is not properly cleaved, even when secreted extracellularly, is presumed to be unable to align with a 1 / 4 offset because it still contains both C-propeptide and N-propeptide. This is thought to be because, when cells age, the gene expression, protein expression, and activity of procollagen C-proteinase and / or procollagen N-proteinase decrease. One possible cause of the decrease in these genes, proteins, and their activity is a reduction in procollagen C-proteinase enhancer and / or procollagen N-proteinase activator. Therefore, it is thought that cellular aging can be detected by measuring the decrease in these genes. Furthermore, procollagen C-proteinase and / or procollagen N-proteinase can also cleave osteoglycin, biglycan, laminin 5, or (pro)lystl oxidase, in addition to collagen N-propeptides and C-propeptides. Therefore, it is thought that cellular aging can also be diagnosed or drugs related to cellular aging can be screened by measuring the decrease in the production of non-procollagen substrates such as osteoglycin, biglycan, laminin 5, or (pro)lystl oxidase. [Examples]

[0040] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0041] 《Reference example 1》 In this reference example, human fibroblasts were subcultured, and the expression of aging markers was examined. By continuously culturing normal human fibroblasts (Chinese, Male, Age 17, Back skin), we created three types of cells: young, moderately aged, and senescent. As markers of cellular senescence, we examined SA-b-gal staining, cell proliferation rate, and the expression of cellular senescence marker genes. As shown in Figure 1, these results indicated that senescence markers (p16 and p21) increased with increasing passage numbers, suggesting that the cells had aged.

[0042] 《Reference example 2》 In this reference example, human fibroblasts were subcultured, and the orientation of collagen fibers was examined. The cell population shown in Figure 1 was cultured long-term on a culture dish, and the collagen fibers constructed by each cell were examined using picrosilius red staining and immunofluorescence with anti-type I collagen antibody. In addition, electron microscopy was performed to obtain detailed information about the collagen structure. As shown in Figure 2, the orientation of collagen fibers was clearly disrupted in the collagen constructed by aged cell populations.

[0043] 《Reference example 3》 In this reference example, the collagen production process during the culture of human fibroblasts was analyzed. To investigate the biosynthesis process of collagen fibers produced by young and senescent cell populations, we performed analysis using visualized type I collagen (Patent No. 6868232). As shown in Figure 3, senescent cells had more procollagen at the tips of their pseudopods than young cells. From this, it was concluded that propeptide cleavage (processing) of procollagen does not occur in senescent cells.

[0044] 《Reference example 4》 In this reference example, we analyzed intracellular processing changes (propeptide cleavage) of collagen due to aging in human fibroblasts. To understand the process of cleavage within cells, cleaved C-propeptides were detected by Western blotting. As shown in Figure 4, the amount of cleaved propeptides was found to be reduced in senescent cells.

[0045] 《Reference example 5》 This reference example analyzes changes in secreted collagen (extracellular collagen) due to aging in human fibroblasts. Furthermore, when extracellular collagen was detected, it was found that processed and matured collagen was reduced in senescent cells. As a result, as shown in Figure 5, it was found that senescent cells secrete procollagen that has not undergone processing.

[0046] Example 1 In this example, we investigated the mRNA, protein, and protein activity of BMP-1 in relation to the aging of human fibroblasts. When the intracellular abundance of proteins that cleave propeptides from procollagen was examined, the amount of gene transcripts and proteins decreased with cellular aging, as shown in Figure 7. Similarly, protein activity also decreased. Here, BMP-1 is shown as an example of a protein with propeptide cleavage activity. The sequences of the PCR primers used are as follows: BMP-1 F: 5'-ccagtcctttgagattgagc-3'(Sequence ID 1) R: 5'-tcatcaggcttctcatagcc-3'(Sequence ID 2)

[0047] Example 2 In this example, we investigated the mRNA and protein of PCPE1 associated with aging in human fibroblasts. When the intracellular abundance of procollagen C-proteinase enhancers was examined, the amount of gene transcripts and proteins decreased with cellular aging. Here, PCPE-1 is presented as an example of a procollagen C-proteinase enhancer. The sequences of the PCR primers used are as follows: PCPE-1 F: 5'-taaaactggaggactggacc-3'(Sequence ID 3) R: 5'-tgactcctttcttcatgggg-3'(Sequence ID 4)

[0048] Example 3 In this example, we investigated the usefulness of the screening method of the present invention using known PCPs inhibitors. Inhibiting the activity of procollagen C-proteinase in young cells disrupted the orientation of collagen fibers, similar to senescent cells. This result suggests that by detecting the disruption of collagen fiber orientation produced by cells exposed to a screening substance, it is possible to search for substances that promote or inhibit cellular senescence. [Industrial applicability]

[0049] The screening method of the present invention can be used to search for substances that promote or suppress cellular senescence.

Claims

1. A step of contacting cells expressing procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein, procollagen N-proteinase, procollagen N-proteinase activator, or procollagen N-proteinase-cleaved protein with a candidate substance. A screening method for cellular senescence-related substances, including [specific substances].

2. A step of measuring a decrease or increase in procollagen C-proteinase function, a decrease or increase in procollagen C-proteinase enhancer function, a decrease or increase in the function of proteins cleaved by procollagen C-proteinase, a decrease or increase in procollagen N-proteinase function, a decrease or increase in procollagen N-proteinase activator function, or a decrease or increase in the function of proteins cleaved by procollagen N-proteinase. A method for screening aging-related substances according to claim 1, comprising:

3. The method for screening aging-related substances according to claim 2, wherein the decrease or increase in the function is a decrease or increase in genes, a decrease or increase in proteins, or a decrease or increase in protein activity.

4. The method for screening aging-related substances according to claim 1, wherein the procollagen C-proteinase is BMP-1, mTLD, mTLL1, or mTLL2, the procollagen C-proteinase enhancer is PCPE1 or PCPE2, the protein cleaved by the procollagen C-proteinase is procollagen, the procollagen N-proteinase is ADAMTS-2, ADAMTS-3, or ADAMTS-14, the procollagen N-proteinase activator is furin, and the protein cleaved by the procollagen N-proteinase is procollagen.

5. A method for diagnosing cellular senescence, comprising the step of measuring the function of procollagen C-proteinase, procollagen C-proteinase enhancer, procollagen C-proteinase-cleaved protein function, procollagen N-proteinase, procollagen N-proteinase activator function, or procollagen N-proteinase-cleaved protein function in cells.

6. The method for diagnosing cellular senescence according to claim 5, wherein the measurement of the function is the measurement of genes, proteins, or protein activity, and it is determined that the cells have aged when the genes, proteins, or protein activity have decreased.

7. The method for diagnosing cellular senescence according to claim 6, wherein the procollagen C-proteinase is BMP-1, mTLD, mTLL1, or mTLL2, the procollagen C-proteinase enhancer is PCPE1 or PCPE2, the protein cleaved by the procollagen C-proteinase is procollagen, the procollagen N-proteinase is ADAMTS-2, ADAMTS-3, or ADAMTS-14, the procollagen N-proteinase activator is furin, and the protein cleaved by the procollagen N-proteinase is procollagen.

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