KR102526197B1 - Composition for diagnosis, preventing or treating bone diseases comprising cotl1 - Google Patents

Abstract

The present invention relates to a composition for diagnosing, preventing, or treating bone disease comprising a COTL1 protein or a gene encoding the same, wherein the suppression of the expression of the COTL1 protein or its gene inhibits the differentiation activity of osteoclasts, thereby enhancing bone density compared to a normal control group. and can cause bone diseases such as osteosclerosis or osteoarthritis by increasing factors that cause joint inflammation and cartilage degeneration. and can be prevented or treated.

Description

A composition for diagnosing, preventing or treating bone diseases containing COTL1 as an active ingredient {COMPOSITION FOR DIAGNOSIS, PREVENTING OR TREATING BONE DISEASES COMPRISING COTL1}

The present invention relates to a composition for diagnosing, preventing or treating bone disease comprising a COTL1 protein or a gene encoding the same.

Osteoclasts, which are multinucleated large cells, have functions of destroying and resorbing bone tissue, and are known to play a role in destroying bone matrix and decomposing bone minerals. Activated osteoclasts have three or more nuclei. In order to differentiate from osteoclast progenitors into mature multinucleated osteoclasts, M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of nuclear factor-kappa B ligand) are required. It requires various hormones and factors.

The osteoclasts cause destruction and resorption of abnormal bone tissue due to an imbalance with osteoblasts in the bone, resulting in osteoporosis in which bone mass and bone density are reduced, and osteomalacia in which calcium is lost from bone ( osteomalacia), fibrous ostitis in which bone marrow becomes fibrotic, periodontitis in which alveolar bone is lost, and rheumatoid arthritis in which destruction and deformation of joints occur.

Among them, osteoarthritis is a disease characterized by loss of proteoglycan (PG), a component of articular cartilage, which is a representative disease that causes joint dysfunction by causing destruction of cells and constituent tissues of articular cartilage. am. It is known that the expression of MMP-3, MMP-9, and MMP-13 increases during the onset of osteoarthritis, and this increase in MMPs damages the collagen matrix constituting cartilage, exacerbating degenerative arthritis.

Currently, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, hyaluronan, glucosamine, and chondroitin are mainly used for the treatment of osteoarthritis, but they are only effective in relieving symptoms rather than fundamental treatment. In particular, In the case of some NSAIDs, it is known that they inhibit the synthesis of proteoglycan in articular cartilage, and rather tend to exacerbate the symptoms of osteoarthritis.

Therefore, there is an urgent need to develop an alternative method that can protect articular cartilage itself or directly treat damage.

Republic of Korea Patent Publication No. 10-2016-0048442 (published on May 4, 2016)

An object of the present invention is to provide a biomarker composition for diagnosing bone disease using a factor confirmed to be significantly related to bone disease, a composition for diagnosing bone disease, and a kit for diagnosing bone disease including the same.

Another object of the present invention is to provide a method for providing information necessary for diagnosing bone disease by measuring the above factors.

Another object of the present invention is to provide a composition for preventing, improving or treating bone disease, including the factor or an activator thereof.

Another object of the present invention is to provide a composition for promoting osteoclast differentiation comprising the above factors.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing osteosclerosis or osteoarthritis comprising a COTL1 protein or a gene encoding the same.

The present invention provides a composition for diagnosing osteosclerosis or osteoarthritis, including an agent for measuring the expression level of a COTL1 protein or a gene encoding the same, and a diagnostic kit for osteosclerosis or osteoarthritis including the same.

The present invention provides a method for providing information necessary for diagnosing osteosclerosis or osteoarthritis, comprising measuring the expression level of a COTL1 protein or a gene encoding the same in a biological sample isolated from a subject.

The present invention provides a pharmaceutical composition for preventing or treating osteosclerosis or osteoarthritis, comprising a COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof as an active ingredient.

The present invention provides a health functional food composition for preventing or improving osteosclerosis or osteoarthritis, comprising a COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof as an active ingredient.

In addition, the present invention provides a reagent composition for promoting osteoclast differentiation comprising COTL1 protein or a gene encoding the same as an active ingredient.

Inhibition of the expression of the COTL1 protein or the gene encoding it according to the present invention inhibits the differentiation activity of osteoclasts, thereby enhancing bone density compared to the normal control group, and increasing factors inducing joint inflammation and cartilage degeneration, such as osteosclerosis or osteoarthritis. Since it can cause diseases, the COTL1 protein or the gene encoding it can be usefully utilized for diagnosis of such bone diseases.

In addition, bone diseases can be more effectively prevented, improved, or treated by using the COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof.

In addition, differentiation of osteoclasts can be promoted using the COTL1 protein or a gene encoding the same.

1 confirms osteoclast differentiation activity in COTL1 expression suppression (COTL1 knock-out) mice, (a) shows changes in the activity of osteoclast differentiation markers, and (b) shows the staining results thereof.
Figure 2 is a result of confirming changes in bone density, etc. of COTL1 expression-suppressed mice, (a) is the bone density measurement result of COTL1 expression-suppressed mice and normal mice, (b) is a micro-CT photograph of the excised femur, (c ) is bone volume (% bone volume, BV), (d) is trabecular thickness (Tb.Th), (e) is trabecular number (Tb.N), and (f) is This is the result of quantifying and analyzing the trabecular spacing (Tb.Sp).
Figure 3 is the observation of osteoclasts in the cartilage of COTL1 expression-suppressed mice and normal mice, (a) is confirmed through immunostaining and electron microscopy (SEM), (b) shows the number of stained cells .
4 shows the expression of COX-2, MMP-3, and MMP-13, which are factors inducing joint inflammation and cartilage degeneration, in osteoarthritis cell models of COTL1 expression-suppressed mice and normal mice.

Hereinafter, the present invention will be described in detail.

The present inventors confirmed that osteoclast differentiation activity was inhibited in COTL1 expression suppression (COTL1 knock-out) mice, and factors inducing joint inflammation and cartilage degeneration were increased, thereby providing the present invention as a composition for diagnosing and treating bone diseases using the same. completed.

The present invention provides a biomarker composition for diagnosing osteosclerosis or osteoarthritis comprising a COTL1 protein or a gene encoding the same.

As used herein, "COTL1" is a Coactosin-like protein, and its gene encodes one of the actin binding proteins that regulates the actin cytoskeleton, and its protein binds F-actin and 5-lipox It is stabilized by interacting with oxygenase (5-lipoxygenase, ALOX5). Also referred to as "coactosin like F-actin binding protein" or "CLP", it may include a physiologically active fragment thereof having substantially the same activity as COTL1 or a fusion protein thereof, but , but is not limited thereto.

In the present specification, "osteosclerosis" is a disease in which most of bone tissue becomes abnormally dense and bone marrow strength is narrowed, and is also referred to as Albers-Schonberg disease. When osteosclerosis occurs, bones become hard but rather brittle, resulting in fractures, spleen and enlargement, severe anemia, decreased platelets, bleeding does not stop, and the number of white blood cells decreases, but there is no effective treatment yet. am.

in this specification. "Osteoarthritis", as described above, is a disease caused by damage to articular cartilage and underlying tibial tissue, and is the most common form of arthritis. As a representative degenerative disease, symptoms of joint pain and stiffness appear, and at first, pain is felt only when moving, but when it becomes chronic, pain continues to be felt.

As used herein, "diagnosis" means confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, whether or not a bone disease develops or progresses, preferably, whether or not osteosclerosis or osteoarthritis develops or progresses. it may be

In the present specification, a "biomarker" is an index that can detect changes in the body, and is a substance that can determine the normal or pathological state of a living organism, whether or not there is a change thereof, such as a polypeptide, nucleic acid, lipid, glycolipid, glycoprotein , sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like, and the like, and the like can be used to diagnose bone diseases such as osteosclerosis or osteoarthritis, as in the present invention.

In the biomarker according to the present invention, when the expression of the COTL1 protein or the gene encoding it is decreased, the differentiation activity of osteoclasts is inhibited and bone density can be increased, COX-2, MMP-3, MMP-13 The expression of factors that cause joint inflammation and cartilage degeneration of the back may be increased. Thus, osteosclerosis or osteoarthritis can be diagnosed through changes in the expression or activity of the protein or its gene.

Since the biomarker according to the present invention shows the same results even in repeated experiments, and the change in its expression level shows significant results, it can be regarded as a highly reliable marker, and thus the predicted results can be reasonably trusted.

The present invention provides a composition for diagnosing osteosclerosis or osteoarthritis comprising an agent for measuring the expression level of a COTL1 protein or a gene encoding the same.

The agent may include primers or probes that specifically bind to the COTL1 gene; Alternatively, it may be any one of an antibody, peptide, aptamer or compound that specifically binds to the COTL1 protein, but is not limited thereto.

As used herein, a "primer" is a nucleic acid sequence having a short free 3' hydroxl group, capable of forming base pairs with a complementary template and serving as a starting point for template strand copying. Refers to a short nucleic acid sequence. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleotide triphosphates in an appropriate buffer and temperature.

The primer is a primer specific to the gene, and may be a sense and antisense nucleic acid having a sequence of usually 7 to 50 nucleotides, as long as the basic properties of the primer serving as the starting point of DNA synthesis are not changed. can be mixed. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

In the present specification, "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to as short as several bases to as long as hundreds of bases that can specifically bind to mRNA, and is labeled so that a specific mRNA The presence or absence of and the expression level can be confirmed. The probe may be formed in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the protein, and the antibody can be prepared according to a conventional method in the art. The type of antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies may be included. The antibody is meant in its complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody may also include a special antibody such as a humanized antibody.

In the present specification, "peptide" has the advantage of high binding force to a target substance, and does not undergo denaturation even during heat/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.

In this specification, "aptamer" is a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. It means a type of polynucleotide composed of. As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simple structure, and are easy to synthesize, so they can be used as replacements for antibodies. can

In the diagnostic composition according to the present invention, when the expression of the COTL1 protein or the gene encoding it is decreased, the differentiation activity of osteoclasts is inhibited and bone density can be increased, COX-2, MMP-3, MMP-13 Since the expression of factors inducing joint inflammation and cartilage degeneration of the back can be increased, osteosclerosis or osteoarthritis can be diagnosed by measuring the expression level of the COTL1 protein or its gene using the agent.

Corresponding features may be substituted in the foregoing.

The present invention provides a diagnostic kit for osteosclerosis or osteoarthritis comprising the composition for diagnosing osteosclerosis or osteoarthritis.

The kit may be a primer kit, a DNA chip kit, or a protein chip kit, but is not limited thereto. The kit may include an antibody that optionally recognizes a marker for diagnosing osteosclerosis or osteoarthritis, as well as one or more other component compositions, solutions, or devices suitable for an assay method.

For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like for immunological detection of the antibody. The substrate may include a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass. It may include patase (alkaline phosphatase) and the like, and FITC, RITC, etc. may be used as the fluorescent material. In addition, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylene diamine), or TMB (tetramethyl benzidine) may be used as the color-developing substrate solution.

Corresponding features may be substituted in the foregoing.

The present invention provides a method for providing information necessary for diagnosing osteosclerosis or osteoarthritis, comprising measuring the expression level of a COTL1 protein or a gene encoding the same in a biological sample isolated from a subject.

In the present specification, a "subject" is an individual to determine whether or not to develop osteosclerosis or osteoarthritis or to predict the risk of developing osteosclerosis or osteoarthritis. Specifically, it may be a mammal, for example, it may be a human (Homo sapiens).

The biological sample may be selected from the group consisting of blood, serum, serum-derived exosomes, tissue, urine, and saliva, which have different expression levels of the protein or its gene from the normal control group, but are not limited thereto.

When the expression level of the COTL1 protein or the gene encoding it measured in the biological sample isolated from the subject is lower than that of the normal control group, osteosclerosis or osteoarthritis can be diagnosed.

The expression level of the gene is next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase It may be measured through one or more methods selected from the group consisting of RNase protection assay (RPA), microarray, and northern blotting, but is not limited thereto.

In addition, the expression or activity level of the protein can be measured by western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, or okteroney. (Ouchterlony) At least one method selected from the group consisting of immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and flow cytometry (FACS) It can be measured through, but is not limited thereto.

The present invention comprises the steps of treating a test substance to a biological sample separated from a subject; Measuring the expression level of the COTL1 protein or the gene encoding it in the sample treated with the test substance; and selecting a test material having an increased expression level of the COTL1 protein or its gene in the sample treated with the test material compared to a control group not treated with the test material; to provide.

The screening method is a method of comparing an increase or decrease in the expression or activity of the COTL1 protein or its gene in the presence or absence of a candidate substance for treating bone diseases such as osteosclerosis or osteoarthritis. It can be usefully used for disease improvement or screening for therapeutic agents.

That is, a substance that increases the expression level of the COTL1 protein or its gene may be selected as a therapeutic agent for osteosclerosis or osteoarthritis.

Corresponding features may be substituted in the foregoing.

The present invention provides a pharmaceutical composition for preventing or treating osteosclerosis or osteoarthritis, comprising a COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof as an active ingredient.

The expression promoter or activator may be a known expression promoter or activator of the COTL1 protein or its gene, but is not limited thereto, and includes all substances capable of directly or indirectly enhancing the expression or activity of the COTL1 protein or its gene. can do

By enhancing the expression or activity of the COTL1 protein or its gene, osteosclerosis or osteoarthritis can be prevented or treated.

The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition may be formulated with an appropriate pharmaceutically acceptable carrier according to the formulation and, if necessary, may further contain excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, and the like. there is. The suitable carrier and the like do not inhibit the activity or characteristics of the COTL1 protein or the gene encoding the same, or the expression promoter or activator according to the present invention, and may be selected differently depending on the dosage form and formulation.

As used herein, "pharmaceutically acceptable" means that the composition is not toxic to cells or humans exposed to the composition.

The pharmaceutical composition according to the present invention can be applied in any dosage form, and more specifically, it can be formulated and used in parenteral dosage forms such as oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.

Among the oral dosage forms, the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and includes at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. may be prepared by mixing, and lubricants such as magnesium stearate and talc may be included in addition to simple excipients. In addition, in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.

Among the oral formulations, liquid formulations include suspensions, solutions for internal use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogeratin, and the like may be used. It is not limited thereto, and all suitable agents known in the art may be used.

In addition, calcium or vitamins may be further added to the pharmaceutical composition according to the present invention to enhance therapeutic efficacy.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount.

In the present specification, "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects.

The effective dosage level of the pharmaceutical composition depends on the purpose of use, the patient's age, sex, weight and health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment Duration, combination, or factors including drugs used concurrently and other factors well known in the medical arts may be determined differently. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal that may develop osteosclerosis or osteoarthritis, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs. can

The pharmaceutical composition according to the present invention may be administered by an appropriate administration route according to the formulation form, and may be administered through various oral or parenteral routes as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, rectal or intravenous, intramuscular, skin application, intraventricular inhalation, intrauterine dural or intracerebroventricular injection. there is.

The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of osteosclerosis or osteoarthritis, or may be used in combination with surgery or other drug treatment.

The present invention provides a health functional food composition for preventing or improving osteosclerosis or osteoarthritis, comprising a COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof as an active ingredient.

Corresponding features may be substituted in the foregoing.

In the health functional food composition according to the present invention, the health functional food may be prepared into powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving osteosclerosis or osteoarthritis. There is no limit to the form that the health functional food can take, and it can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods.

In the health functional food composition according to the present invention, the health functional food may include all foods in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc., and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further including food chemically acceptable food additives (food additives) commonly used in the art and other appropriate auxiliary components. Unless otherwise specified, suitability as a food additive can be determined according to the standards and standards for the item in accordance with the General Rules of the Code of Food Additives approved by the Ministry of Food and Drug Safety and general test methods. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations; and the like.

The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. may additionally contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be used. As the sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

The effective dose of the COTL1 protein, the gene encoding it, or its expression promoter or activator contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as preventing or improving osteosclerosis or osteoarthritis.

The health functional food composition uses food as a raw material and has the advantage of not having side effects that can occur when taking general medicines for a long time, and has excellent portability, so it can be taken as an adjuvant for preventing or improving osteosclerosis or osteoarthritis.

In addition, the present invention provides a reagent composition for promoting osteoclast differentiation comprising COTL1 protein or a gene encoding the same as an active ingredient.

When the expression of the COTL1 protein or the gene encoding it is suppressed, the differentiation activity of osteoclasts is suppressed, and thus, the differentiation of osteoclasts can be promoted by increasing the expression of the protein or its gene.

Corresponding features may be substituted in the foregoing.

In addition, the present invention provides a reagent composition for inhibiting the expression of one or more osteoarthritis inducing factors selected from the group consisting of COX-2, MMP-3 and MMP-13 containing COTL1 protein or a gene encoding the same as an active ingredient.

When the expression of the COTL1 protein or the gene encoding it is suppressed, the expression of one or more osteoarthritis inducers selected from the group consisting of COX-2, MMP-3 and MMP-13 is increased, and the protein or its gene When the expression of is increased, the expression of the factor can be suppressed.

Corresponding features may be substituted in the foregoing.

In addition, the present invention provides a method for promoting osteoclast differentiation, comprising the step of treating an animal other than human with COTL1 protein or a gene encoding the same, or an expression promoter or activator thereof.

In addition, the present invention is selected from the group consisting of COX-2, MMP-3 and MMP-13, including the step of treating the cell isolated from the subject with the COTL1 protein or the gene encoding it, or its expression promoter or activator. A method for inhibiting the expression of one or more osteoarthritis triggers is provided.

Corresponding features may be substituted in the foregoing.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Confirmation of inhibition of osteoclast differentiation activity in COTL1 knock-out mice

Osteoclasts are derived from hematopoietic stem cells and are responsible for bone resorption that destroys aged bones. remodeling) is maintained.

The inventors of the present invention isolated mononuclear cells from the mouse femur to confirm the effect of inhibiting osteoclast differentiation activity in COTL1 expression suppression (COTL1 knock-out) mice, and 30ng/mL macrophage colony-stimulating factor (M-CSF) and 50ng /mL RANKL (receptor activator of nuclear factor kappa-κΒ ligand) was treated together to induce differentiation for 3 days, and then the osteoclast differentiation marker, tartrate-resistant acid phosphatase (TRAP), was activated. measured. At this time, after washing the medium containing the cells with physiological saline, the TRAP activity was confirmed by measuring the absorbance at a wavelength of 405 nm using the Acid-Phosphatase Kit for the cells in the medium, and TRAP staining was performed through a microscope. Osteoclasts were observed.

As a result, it was confirmed that TRAP activity was reduced in osteoclasts isolated from COTL1 expression suppression (COTL1 knock-out) mice compared to osteoclasts from normal mice, and osteoclast differentiation activity was significantly inhibited (FIG. 1).

<Example 2> COTL1 expression suppression (COTL1 knock-out) confirmation of bone density in mice

For the bone density animal experiment, 24-week-old female (C57BL6N) COTL1 knock-out mice and normal mice were used. At the beginning of the animal experiment, bone mineral density (BMD) was measured with a PIXImus bone densitometer. measured.

After anesthetizing the mouse by injecting 50 μl of a mixed anesthetic of zoletil and rompun (a 1:2 mixed solution diluted with physiological saline at a ratio of 2:3), the mouse was anesthetized, fixed to a bone density measuring frame, and bone density was measured. After the end of the experiment, the femur bone was extracted and micro-CT was taken, and then the bone volume ratio (% bone volume, BV), trabecular thickness (Tb.Th), trabecular number (Tb.N) ) and trabecular spacing (Tb.Sp) were quantified and analyzed.

As a result, as shown in FIG. 2, the bone mineral density (BMD) of COTL1 knock-out mice was increased compared to that of normal mice (FIG. 2a), and the density of bone microstructure on micro-CT images was increased ( Fig. 2b). The volume ratio of bone (Fig. 2c) and the number of cancellous bone trabeculae (Fig. 2e) were significantly increased, and the trabecular space (Fig. 2f) was found to be low.

In addition, after analyzing the femoral bone, the bones were sectioned and analyzed for TRAP immunostaining and electron microscopy (SEM) images, which are osteoclast differentiation markers (FIG. 3).

<Example 3> Cell test for confirming osteoarthritis improvement effect

Osteoarthritis (degenerative arthritis) is a disease in which degeneration of articular cartilage tissue and structural changes of subchondral bone occur. Cartilage abrasion, heredity, damage to cartilage tissue due to impact, pressure due to overweight, and muscle weakness around the knee may be mentioned. There are symptomatic therapies such as pain control and surgery, but no fundamental treatment has been developed yet.

To confirm the effect of COTL1 expression suppression on arthritis, the inventors of the present invention isolated chondrocytes from the cartilage of 5-day-old mice, cultured the cells in DMEM medium, and produced osteoarthritis cell models. .

In order to verify the expression of catabolic factors (COX-2, MMP-3, and MMP-13) by suppressing the expression of COTL1 at the cellular level, catabolic factors secreted by chondrocytes were inhibited by suppressing the expression of COTL1. It was confirmed whether or not it was triggered.

As a result, as shown in FIG. 4 , when the expression of COTL1 was suppressed, it was confirmed that the expression of COX-2, MMP-3 and MMP-13, which are osteoarthritis inducing factors, increased significantly.

Based on these results, it was confirmed that COTL1 can be used in a pharmaceutical composition for preventing, diagnosing, or treating bone diseases including osteosclerosis or osteoarthritis and a method for screening a therapeutic agent for the disease.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (14)

(1) treating COTL1-expressing mice with a candidate substance;
(2) measuring the expression level of COTL1 in COTL1-expressing mice before and after treatment with the candidate substance; and
(3) selecting a candidate material with reduced COTL1 expression before and after treatment with the candidate material; osteoclast differentiation inhibitor screening method comprising the. delete delete delete delete delete delete delete delete delete delete delete delete A reagent composition for promoting osteoclast differentiation comprising a COTL1 protein or a gene encoding the same as an active ingredient.

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