KR102124435B1 - A composition for promoting osteogenesis containing peroxiredoxin-6 activity inhibitor - Google Patents

Abstract

The present invention relates to a composition having a bone formation promoting effect, a composition for promoting bone formation comprising a PRDX6 (peroxiredoxin-6) gene activity inhibitor as an active ingredient.

Description

A composition for promoting bone formation containing a peroxidedoxin 6 activity inhibitor {A COMPOSITION FOR PROMOTING OSTEOGENESIS CONTAINING PEROXIREDOXIN-6 ACTIVITY INHIBITOR}

The present invention relates to a composition for promoting bone formation containing a peroxyredoxin 6 (PRDX6) activity inhibitor.

Bone is an active tissue that constantly changes throughout life. The bone is divided into the external cortical bone (density bone) and the internal small bone (spongy bone, sponge bone) with the naked eye, and the cortical bone has strong physical strength to protect and support the body, and the small bone absorbs shock or absorbs calcium. It serves to keep the change of the constant.

This phenomenon is called remodeling of bones, where old bones are destroyed (bones are absorbed) even after the growth of bones is stopped, and fixation (bone formation) that fills the places where new bones are lost is repeated throughout life.

When the interaction between osteoblasts and osteoclasts is balanced, bone absorption and bone formation must be balanced to maintain bone homeostasis and maintain a constant level of calcium in the blood. Absorption is increased to release the calcium of bone into the blood, and if bone absorption is continued, the bones become weak and diseases such as osteoporosis may occur.

Osteoporosis is post-menopausal osteoporosis, which is caused by increased bone resorption due to the activation of osteoclasts due to rapid changes in hormones according to menopause, and senile osteoporosis, which decreases bone formation due to decreased function of osteoblasts with aging. Can be classified as

Fractures due to osteoporosis lead to severe activity limitations, and in the case of hip fractures associated with a high mortality rate of about 15-35%, the diagnosis and treatment of osteoporosis is important before osteoporotic fractures occur.

Nevertheless, except for parathyroid hormones, the osteoporosis treatments developed so far have only a small effect of preventing the progression of symptoms, rather than a therapeutic effect.

In addition, in the case of a parathyroid hormone agent having a bone-forming effect, it is possible to obtain a therapeutic effect by promoting bone formation, but its use is limited because safety for long-term use is not established. Therefore, in order to effectively treat osteoporosis, a clear understanding of the mechanism of bone formation and fundamental treatment measures based on it are required.

On the other hand, peroxiredoxin (PRDX) is an antioxidant enzyme that regulates the redox balance and has been reported to play an important role in maintaining the potential of self-renewal and stem cells (Cancer Research 77 (13 Supplement)) :4272-4272 July 2017), the association with bone-related diseases is unknown.

The present invention aims to clarify the role of PRDX6 related to bone formation and to provide a method for fundamentally preventing or treating bone disease.

According to an aspect of the present invention, there is provided a composition for promoting bone formation comprising a PRDX6 (peroxiredoxin-6) activity inhibitor as an active ingredient.

In one embodiment, the PRDX6 activity inhibitor may inhibit the activity of glutathione peroxidase (GPx) or calcium-independent phospholipase A2 (iPLA2).

In one embodiment, the PRDX6 activity inhibitor may be selected from one or more of the group consisting of antibodies or antigen-binding fragments thereof, aptamers, siRNA, shRNA, microRNA, stem cell therapeutics, inhibitory compounds, and pharmaceutically acceptable salts thereof. have.

In one embodiment, the therapeutic agent for stem cells It may be a stem cell into which the mutated PRDX6 gene has been introduced.

In one embodiment, the stem cells may be derived from one or more tissues selected from the group consisting of pulp, bone marrow, blood, umbilical cord blood, adipose tissue, liver, skin, gastrointestinal tract, placenta, and uterus, specifically, dimensions It can be a stem cell.

In one embodiment, the composition for promoting bone formation may be for preventing or treating bone disease

In one embodiment, the bone disease may be metabolic bone disease or orthopedic bone disease.

In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, bone dysplasia, osteomalacia, bone mass reduction, fracture, bone defect and hip reduction.

According to another aspect of the invention, (a) contacting a cell and a test agent expressing PRDX6; (b) determining a modulatory effect on PRDX6 expression of the test agent; And (c) comparing the expression level of the PRDX6 in cells contacted with the test agent and a control group not contacted with the test agent.

In one embodiment, the test agent may be selected from one or more of the group consisting of polypeptides, small organic molecules, polysaccharides, polynucleotides.

The present invention provides a method for effectively promoting bone formation based on a clear understanding of the molecular mechanism of the PRDX6 gene, and can be effectively used for treatment, prevention, diagnosis, or research on various bone-related diseases.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1A and 1B show the expression level of PRDX6 according to osteoblast differentiation.
Figure 2 shows the degree of iPLA2 activity (A) and the degree of GPx activity (B) of stem cells into which the mutant PRDX6 gene has been introduced.
3 is an analysis of the effect of PRDX6 on bone formation differentiation and bone formation mRNA expression.
Figure 4 analyzes the expression of PRDX6 iPLA2 and GPx enzyme active variants, localization of cells, and functionality.
5 is an analysis of the effect of iPLA2 and GPx enzymatic activity of PRDX6 in bone formation differentiation.
6 is an analysis of the effect of PRDX6 on hDPSC-based bone regeneration.
7 is an analysis of the effect of PRDX6 on the development of undeveloped bone.

The terms used in the present specification have selected general terms that are currently widely used as possible while considering functions in the present invention, but this may be changed according to the intention or precedent of a person skilled in the art or the appearance of new technologies. In addition, in certain cases, some terms are arbitrarily selected by the applicant, and in this case, their meanings will be described in detail in the description of the applicable invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the contents of the present invention, not simply the names of the terms.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Terms such as those defined in a commonly used dictionary should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

When a part is said to "include" a certain component, this means that other components may be further provided, not excluding other components, unless otherwise specified.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, several methods and materials are described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents because it can be used in various ways.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and thus is not limited to the embodiments described herein.

According to an aspect of the present invention, a composition for promoting bone formation comprising peroxiredoxin-6 (peroxiredoxin-6, PRDX6) activity inhibitor is provided.

The term “Peroxyredoxin” refers to an antioxidant enzyme family protein that regulates the level of peroxide caused by cytokines and mediates signaling through it.

Transformation growth factor β (TGFβ) can induce the expression of RUNX2, a transcription factor that regulates bone differentiation induced by TGFβ by phosphorylating receptor-mediated Smad2 and p38 mitogen-activated protein kinase.

When inhibiting the activity of PRDX6, Smad2 and p38 are phosphorylated to restore expression of the RUNX2 gene (FIG. 4), thereby promoting bone formation.

That is, by inhibiting the activity of the PRDX6 can activate the TGFβ signal transduction pathway, it can promote bone formation.

Although the relationship of the peroxyredoxin in the bone formation mechanism has not been revealed, the present inventors have confirmed that the peroxyredoxin-6 is directly involved in the bone formation process and is a potent factor mediating bone metabolism.

The "comprising as an active ingredient" may exhibit a bone-forming effect, and may mean that it contains an effective amount to suppress the activity of the PRDX6 gene.

The “bone formation” refers to a process in which bone is generated, and may include bone matrix formation by osteoblasts and calcification thereof, and the “promotion of bone formation” allows bone formation to occur within a short time. Of course, it may include inducing bone formation.

The PRDX6 activity inhibitor may inhibit the activity of glutathione peroxidase (GPx) or calcium-independent phospholipase A2 (iPLA2).

The “GPx” corresponds to a major antioxidant functional group in the cell, and the “iPLA2” catalyzes the hydrolysis of glycerophospholipids to sn-2 fatty acyl ester bonds to free fatty acids. (free fatty acid) and lysophospholipid.

The redox imbalance of the redox reaction by extracellular stressors is controlled by various antioxidant defenses, including superoxide dismutases (SOD), glutathione peroxidase (GPx), and peroxiredoxins (PRDXs).

The PRDX is a commonly present enzyme that controls the level of peroxide, and contains a conserved cysteine residue, and can remove phospholipid hydroperoxide to suppress ROS-mediated cell damage or cell death.

In addition, metabolites of the phospholipid membrane caused by PLA2 are important negative mediators for bone formation and can inhibit osteoblast differentiation and bone formation.

The present inventors analyzed the effect of PRDX6 on GPx or iPLA2 and found that it can affect the TGFβ pathway through iPLA2 and GPX activity.

Bone disease may be prevented or treated by administering the PRDX6 activity inhibitor.

Since the PRDX6 gene is located above the bone formation-related signaling pathway or directly affects the TGFβ pathway, the activity of the PRDX6 gene can be controlled to ultimately prevent or treat bone disease.

The "prevention" includes all actions of inhibiting or delaying the onset of bone disease by administering the composition for promoting bone formation of the present invention, and the "treatment" improves or beneficially alters the symptoms caused by bone disease. It can include any act.

According to another aspect of the invention, there is provided a composition for promoting bone formation comprising a PRDX6 activity inhibitor as an active ingredient.

The PRDX6 activity inhibitor may be one or more selected from the group consisting of antibodies or antigen-binding fragments thereof, aptamers, siRNAs, shRNAs, microRNAs, stem cell therapeutics, inhibitory compounds, and pharmaceutically acceptable salts thereof.

The “antibody” includes both a monoclonal antibody and a chimeric antibody, a humanized antibody, and a human antibody, and other antibodies known in the art may be included in addition to the novel antibody. The antibody includes functional fragments of the antibody molecule as well as a complete form with the full length of two heavy and two light chains, as long as it has the properties of binding specifically recognizing the PRDX6 gene. A functional fragment of the molecule of an antibody means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

The “aptamer” is a single-stranded DNA or RNA molecule, which is high in a specific chemical molecule or biological molecule by an evolutionary method using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). It can be obtained by separating oligomers that bind with affinity and selectivity. Aptamers can specifically bind to targets and modulate the activity of the targets, such as blocking the ability of the target to function through binding.

The “siRNA” refers to short double chain RNA capable of inducing RNA interference (RNAi) phenomenon through cleavage of a specific mRNA. It consists of a sense RNA strand with a sequence homologous to the mRNA of the target gene and an antisense RNA strand with a sequence complementary thereto. Since the siRNA can suppress the expression of the target gene, it can be provided as an efficient gene knockdown method or gene therapy method. The siRNA is not limited to the complete pairing of double-stranded RNA portions paired with RNA, and is mismatched (the corresponding base is not complementary), bulge (there is no base corresponding to one chain), and the like. Parts that do not form may be included. The siRNA terminal structure can be either blunt or cohesive as long as it can suppress the expression of the target gene by RNAi effect. The adhesive end structure may be both a 3'end protruding structure and a 5'end protruding structure. The number of protruding bases is not limited.

The siRNA may include low-molecular RNA (eg, natural RNA molecule such as tRNA, rRNA, or viral RNA or artificial RNA molecule) at a protruding portion of one end in a range capable of maintaining the effect of suppressing the expression of the target gene. The siRNA terminal structure does not need to have a cleavage structure on both sides, and may be a step loop structure in which a terminal region of a double chain RNA general is connected by a linker RNA.

The siRNA itself is a complete form having polynucleotide pairing, that is, a form introduced into cells through two transformation processes in which siRNA is directly synthesized in vitro, or a single chain oligo so as to have such a form after administration in vivo. Transforms nucleotide fragments and their reverse complements into a form that can be derived from single chain polynucleotides separated by spacers, such as siRNA expression vectors or PCR-derived siRNA expression cassettes prepared for siRNA expression in cells Or it may be a form that is introduced into the cell through an infection process.

The determination of how the siRNA is prepared and introduced into a cell or animal may differ depending on the purpose and cellular biological function of the target gene product.

The “shRNA” is for overcoming shortcomings such as high cost of biosynthesis of siRNA and short-term maintenance of RNA interference effects due to low cell transfection efficiency. Adenovirus, lentivirus and plasmid expression vector systems from promoters of RNA polymerase III It can be expressed by introducing it into a cell by using, and it is widely known that the shRNA is converted into siRNA having a precise structure by an siRNA processing enzyme (Dicer or Rnase III) present in the cell, leading to silencing of the target gene. .

The stem cell therapeutic agent may be introduced using a variant PRDX6 gene transformed into stem cells. The transformation method for introducing the mutant PRDX6 gene can be easily performed according to conventional methods in the art.

The "transformation" refers to a phenomenon in which an external DNA or a viral vector containing an external DNA is introduced into a host, and the DNA artificially causes genetic changes as a factor of the chromosome or by completion of chromosomal integration.

In general, the transformation method uses retrovirus and adenovirus infection method, CaCl ₂ precipitation method of DNA, and the Hanahan method which improves efficiency by using DMSO (dimethyl sulfoxide), which is a reducing material in the CaCl ₂ method, and electroporation (electroporation). , Calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fiber, agrobacterial mediated transformation method, transformation method using PEG, dextran sulfate, liposomes including lipofectamine and dry/inhibition mediated traits And conversion methods.

In the embodiment of the present invention, the pcDNA3.1 vector and the mutant PRDX6 gene were infected with stem cells and transformed.

The “variant PRDX6 gene” means that the expression of PRDX6 is suppressed, interrupted, or the original function is lost due to a variation in the gene sequence, and some or all of the base sequence or amino acid sequence of the PRDX6 gene is deleted, substituted, added, inserted, etc. It may be mutated.

The mutant PRDX6 gene is not limited to the mutant gene of a specific sequence, and is sufficient as long as the original protein function is lost due to the mutagenesis of some sequences, and the gene mutant method may use various methods well known in the art.

The "stem cell" is a cell capable of differentiating into various cells by the characteristics of multi potency when an appropriate signal is given under the influence of the environment in which the cell is located, as well as the ability to self-replicate. As, for example, it may be a stem cell (germline stem cell), an embryonic stem cell (embryonic stem cell), or an induced pluripotent stem cell (induced pluripotent stem cell).

The stem cells are included in fat, bone marrow, umbilical cord blood and placenta, and can be used to treat various cell damage diseases such as spinal cord injury, myocardial infarction, brain infarction, degenerative arthritis and fractures. The stem cells may be autologous or allogeneic stem cells, and may be from any type of animal, including human and non-human mammals.

The stem cells may be derived from one or more tissues selected from the group consisting of pulp, bone marrow, blood, umbilical cord blood, adipose tissue, liver, skin, gastrointestinal tract, placenta, and uterus, preferably pulp stem cells, It is not limited to this.

The “dental pulp stem cell” refers to stem cells present in pulp tissue. The pulp stem cells have a high proliferative ability and a high cell colony forming ability to form dense calcification nodules, and can differentiate into dentin cell, adipocyte, chondrocyte, and osteoblast.

The composition for promoting bone formation may be for preventing or treating bone disease. The bone disease may be metabolic bone disease or orthopedic bone disease

The “metabolic bone disease” refers to a condition or disease that occurs due to excessive production or activity of osteoclasts, and may include a bone mass-lowering disease. The bone loss disease refers to a condition or disease in which a decrease in bone mass accompanied by symptoms such as a decrease in bone density and softening of bone tissue occurs.

The bone disease may be at least one selected from the group consisting of osteoporosis, osteogenic dysfunction, osteomalacia, osteopenia, fractures, bone defects, and hip reduction.

The composition can be used for the prevention and treatment of periodontal disease. The activity of the Runx2 gene has been known to be closely related to periodontal disease as well as to promote bone formation, and it has been reported that the Runx2 gene plays an important role in regulating remodeling of the alveolar bone and maintaining periodontal ligament (Camilleri, S. et al., Eur J Oral Sci. 114(5):361-373, 2006).

Therefore, by activating Runx2 by regulating the activity of the PRDX6 gene, it is possible to prevent or treat periodontal disease as well as bone disease treatment.

The "periodontal disease" is a structure that damages the structure of the teeth and the tissues of the gingiva by the virulence metabolites secreted by the periodontal bacteria infected with the alveolar bone or surrounding tissue of the alveolar bone, and causes lesions that weaken the immunity of the gingival tissue It may mean a disease in which the lesion site is repeatedly expanded due to multiplication of bacteria.

The periodontal disease may include a disease affecting the alveolar bone or the surrounding tissue of the alveolar bone supporting and binding the alveolar bone to the gingiva.

The periodontal disease may be divided into gingivitis and periodontitis according to the region of the lesion site, for example, the gingivitis refers to a disease in which the lesion site develops symptoms in soft tissue, and periodontitis is the It may mean a disease that occurs in a region where the lesion site extends to the gums and around the gum bone.

The composition for promoting bone formation may be administered in the form of oral delivery, parenteral delivery. The composition for promoting bone formation may be administered through any general route as long as it can reach the target tissue.

For example, the composition for promoting bone formation is administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrapulmonarily, intrarectally, intranasally, intraperitoneally, intrathecalally. May be made, but is not limited thereto.

The effective dosage of the composition for promoting bone formation is gender, body surface area, type and severity of disease, age, sensitivity to drugs, administration route and discharge rate, administration time, treatment period, target cells, expression level, and other medical fields. It can be varied according to various factors well-known to, and can be easily determined by experts in the field.

The composition for promoting bone formation may be formulated with an appropriate amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form, and may further include a carrier, excipients, and diluents used in the manufacture of pharmaceutical compositions. .

The carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, but is not limited thereto.

In addition, the composition may be used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate and talc may be used in addition to the excipients.

As a liquid preparation for oral administration, a suspension, an intravenous solution, an emulsion, or a syrup may be used. In addition to simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives may be included. .

As a preparation for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories may be used. As the non-aqueous solvent and suspension, injectable esters such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

Another aspect of the invention comprises the steps of (a) contacting a cell and a test agent expressing PRDX6; (b) determining a modulatory effect on PRDX6 expression of the test agent; And (c) comparing the expression level of the PRDX6 in cells contacted with the test agent and a control group not contacted with the test agent.

The term “contact” is a conventional meaning, and may mean combining two or more agents (eg, two polypeptides) or binding agents and cells (eg, protein and cells).

For example, two or more agents may be combined in a test tube or other container, or a test agent may be combined with a cell or cell lysate and a test agent, and a recombinant polynucleotide encoding two polypeptides may be cells. Two polypeptides can be contacted in a cell or cell lysate by coexpresion within.

The "agent" or "test agent" may include any substance, molecule, element, compound, entity, or combinations thereof. Can be. For example, it may include proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like, and may be natural products, synthetic compounds or chemical compounds, or a combination of two or more substances. have. Unless otherwise defined, the agents, materials and compounds can be used interchangeably.

The screening method can utilize various biochemical and molecular biological techniques known in the art [Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Editions] ; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1987-1999)], such as in vitro protein-protein binding assay (in vitro pull-down assay), EMSA, Immunoassays for protein binding, functional assays (such as phosphorylation assays), yeast-2 hybrid assays, non-immunoprecipitation assays, immunoprecipitation western blot assays, and immuno-co-localization assays It can be performed in a variety of known ways.

The compound used for the screening may be a low molecular weight compound having a therapeutic effect. For example, the low molecular weight compound may have a weight of about 1000 Da, such as 400 Da, 600 Da or 800 Da. Depending on the purpose, the compound may constitute a part of the compound library, and the number of compounds constituting the library may also vary from tens to millions. The compound library includes peptides, peptoids and other cyclic or linear oligomeric compounds, and template-based low molecular compounds such as benzodiazepine, hydantoin, viaryl, carbocycle and polycycle compounds (e.g. naphthalene, phenothi) Azine, acridine, steroid, etc.), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (eg, triazine, indole, thiazolidine, etc.), but is not limited thereto.

Biologics may be used in the screening method. The biologics refers to a cell or a biomolecule, and refers to a protein, nucleic acid, carbohydrate, lipid, or a substance produced using a cell system in vivo and in vitro. The biomolecule may be used alone or in combination with other biomolecules or cells. The biomolecule may include, for example, polynucleotide, peptide, antibody, or other protein or biological organic material found in plasma.

The type and concentration of cells used in the screening method and the amount and type of test substances may be different depending on the specific experimental method and type of test agent used, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance that increases or inhibits the activity of PRDX6 in the presence of the test substance can be selected as a candidate substance as compared to a control group not in contact with the test agent. Compared to the control group, about 99% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60 It means less than %, about 55% less, less than about 50% less, and less than 45% less, but does not exclude ranges beyond this.

According to one embodiment, it is possible to primarily assay whether the test agent is capable of modulating the biological activity of PRDX6. Specifically, the primary assay can identify a modulating agent that modulates the biological activity of the polypeptide by analyzing the biological activity of PRDX6 isolated in the presence of the test agent.

The primary assay may be assayed for control of PRDX6 for various biological activities. For example, the test agent can be assayed for activity that modulates the expression level of PRDX6, such as transcription or translation. In addition, the test agent can be assayed for activity that modulates the intracellular level or stability of PRDX6, such as post-translational modification or hydrolysis.

After identifying the modulatory agent associated with the biological activity of PRDX6 through the first assay, the test agent can be tested secondarily to relieve neuropathic pain. For example, the test agent is neuropathic pain. It can be further tested for its prophylactic or therapeutic activity.

The primary and secondary assays may use intact PRDX6 and fragments thereof, analogues, or functional equivalents. Fragments that can be used in these assays can generally possess one or more biological activities of PRDX6. In addition, fusion proteins comprising such fragments or analogs can be used for screening for test agents. The functional equivalent of PRDX6 includes amino acid deletions, insertions, or substitutions, but retains the same bioactivity as PRDX6 and can be used to implement the screening method of the present invention.

Various assays commonly practiced in the art can be used to identify agents that modulate PRDX6. Preferably, the agent can be screened with a cell based assay system. For example, in a typical cell-based assay for screening (i.e., secondary assay), reporter gene activity (e.g., enzyme activity) in the presence of the test agent is measured and compared to the activity of the reporter gene in the absence of the test agent. You can.

The reporter gene can encode any detectable polypeptide known in the art (reaction or reporter polypeptide), such as a polypeptide detectable by fluorescence or phosphorescence or a polypeptide detectable by enzyme activity possessed by it. have. Detectable reponse polypeptides include, for example, luciferase, alpha-glucuronidase, alpha-galactosidase, chloramphenicol acetyl transferase, green fluorescent protein, enhanced green fluorescent protein and human It may be a secreted alkaline phosphatase.

In the cell-based assay, the test agent (eg, peptide or polypeptide) can be expressed by other vectors present in the host cell. In some methods, a library of test agents can be encoded by the vector's library (eg, cDNA library). The library can be prepared using methods known in the art (Sambrook et al. and Ausubel et al., supra), or can be obtained from a variety of commercial sources.

In addition to the cell-based assay, it can also be screened by non-cell based methods. Such methods include, for example, mobility shift DNA-binding assays, methylation and uracil interference assays, DNase and hydroxyl radical footprinting analysis. ), fluorescence polarization and UV crosslinking or chemical cross-linkers.

A general overview is disclosed in Ausubel et al. (Ausubel et al., supra, chapter 12, DNA-Protein Interaction). Techniques for separating co-associating proteins, including nucleic acids and DNA/RNA binding proteins, include the cleavable crosslinking agents dithiobis (sucinimidylpropionate) and 3,3'-dithiobis (sulfosuccinimidyl-propionate). Including UV crosslinking or chemical crosslinking agents (McLaughlin, Am. J. Hum. Genet., 1996; Tang, 1996; Lingner, 1996; Chodosh, 1986).

Hereinafter, the present invention will be described in more detail by examples. However, the embodiments of the present invention are provided only to facilitate understanding of the invention, and the protection scope of the invention is not limited by the following examples.

Example : Mutation PRDX6 Preparation of stem cells with gene introduction

In the case of processing stem cells whose PRDX6 gene activity was suppressed, stem cells into which the mutant PRDX6 gene was introduced were prepared to confirm the effect on osteoblast differentiation and function.

Specifically, the mutant PRDX6 gene was cut with EcoR I and Xho I, and then subcloned into a pcDNA3.1 vector (Invitrogen) to prepare a pcDNA3.1 vector into which the mutant PRDX6 gene was introduced. Stem cells were introduced by introducing the above-described vector into pulp stem cells and introducing the mutant PRDX6 gene.

Since the PRDX6 gene is known as a bifunctional enzyme of the GPx and iPLA2 genes, expression of GPx and iPLA2 genes was confirmed through Western blot.

On the other hand, stem cells that did not introduce PRDX6 gene into pulp stem cells were used as a negative control, and stem cells that introduced PRDX6 gene into pulp stem cells were used as a positive control.

Referring to FIG. 2, the experimental group in which the activity of the GPx and iPLA2 genes were significantly reduced compared to the positive control group was hDPSC/myc-PRDX6(C47S), although there was no significant change in the activity of the GPx gene, but the activity of the iPLA2 gene was The significantly reduced experimental group was hDPSC/myc-PRDX6 (S32A) (P<0.05).

Experimental Example One : PRDX6 Gene expression measurement

Real-time PCR (real-time) to determine whether the peroxy-ledoxin gene is expressed in human dental pulp stem cells (hDPSC) from six types of peroxy-redoxin PRDX1 to PRDX6 proteins to determine their role in bone formation PCR).

Referring to FIG. 1, the expression pattern of peroxyredoxine was unchanged in addition to PRDX3 and PRDX6, and PRDX3 is known as a mitochondrial protein required for mitochondrial function. As a result, PRDX6 mediates bone metabolism of pulp stem cells It suggests that it is the most influential factor.

Experimental Example 2: osteoblast differentiation ability measurement

In order to investigate whether the stem cells of Examples 1 and 2 affect the differentiation of osteoblasts, the effect of the stem cell treatment of Examples 1 and 2 in the bone formation supplementation medium (OS) was observed.

As an initial osteoblast differentiation marker, alkaline phosphatase enzyme activity (ALP enzymatic activity) was measured.

Alkaline phosphatase is a biomarker secreted by osteoblasts and is most commonly used as an indicator of bone formation.

Referring to FIG. 3, compared to the negative control group, the ALP activity was significantly decreased in the positive control group, whereas in Examples 1 and 2, the activity of the ALP was significantly increased (P<0.05).

The degree of mineralization was measured as a terminal osteoblast marker by alizarin red staining. hDPSC/myc-PRDX6 reduced the formation of mineralized crystals on days 7, 14 and 21 (Figures 3C and 3D).

Cells overexpressing PDRX6 reduced early and late markers, so the effect of mRNA expression of bone-forming specific genes was analyzed.

3E and 3F, PRDX6 down-regulated mRNA levels of ALP and OCN (osteocalcin) over 21 days, and the results suggest that PRDX6 plays an important role in osteogenic differentiation.

Experimental Example 3: PRDX6 And TGFβ Signal Path Association Test

To analyze whether the effect of inhibiting bone formation of PRDX6 is related to dual catalytic activity for iPLA2 and GPx, a variant lacking GPx activity (C47A) or a variant lacking iPLA2 activity was constructed (S32A).

Through the above variants, hDPSC/myc-PRDX6 (S32A) and hDPSC/myc-PRDX6 (C47S) were prepared, respectively.

Genetically engineered hDPSC was verified by Western blot and immunofluorescence, and all variants showed similar cytosolic expression (FIGS. 4A and 4B).

In addition, the mutant hDPSC/myc-PRDX6 (S32A) did not affect GPx activity and reduced iPLA2 activity, whereas the mutant hDPSC/myc-PRDX6 (C47S) compared to hDPSC/myc-PRDX6, GPx and iPLA2 activity Was effectively reduced (Figures 4C and 4D).

With the activity of PRDX6, PRDX6(WT) reduced ROS levels, while PRDX6(C47S) and PRDX6 (S32A) variants blocked ROS reduction (Figures 4E and 4F).

In hDPSCs under the same conditions, PRDX6(WT) decreased the ALP activity of hDPSCs, whereas PRDX6(S32A) and PRDX6(C47S) variants increased ALP activity during osteogenic differentiation (Figure 5A).

In order to analyze the network connection between the genes involved in PRDX6 and bone formation differentiation, related genes were predicted using GeneMANIA (https://genemania.org/). GeneMANIA evaluated that TGFβ, RUNX2, MAP3K4, and PRDX6 are highly correlated in humans (FIG. 5B).

Based on these predictions, functional associations of PRDX6 and TGFβ pathways were identified.

PRDX6(WT) reduced phosphorylation of Smad2 and p38 and was associated with the expression of RUNX2.

These results suggest that PRDX6(WT) is a key regulator in bone formation differentiation, while PRDX6(S32A) and PRDX6(C47S) variants can inhibit or block the TGFβ pathway (Figures 5C-5E).

Referring to FIG. 5F, BMP2 expression was not affected by PRDX6(WT), PRDX6(S32A), and PRDX6(C47S), and the results showed that iPLA2 and GPX activity of PRDX6 were TGFβ pathways (smad2, p38 and RUNX2) It suggests that it may affect bone formation.

Experimental Example 4: bone regeneration and bone development in animal experiments

In 12-week-old mice, the stem cell treatment of the example was analyzed to affect the recovery of cranial defects.

Collagen sponges containing DPSC/myc-PRDX6 (WT), hDPSC/myc-PRDX6 (S32A), and hDPSC/myc-PRDX6 (C47S) were placed in the defective site for 6 weeks (Figure 6A).

After 6 weeks, newly formed bones were analyzed through 3D micro-CT images (FIG. 6C ).

hDPSC/myc-PRDX6 (WT) significantly reduced the rate of recovery of cranial defects compared to hDPSC (Figure 6B), and verified the results through H&E staining (Figures 6D and 6E).

On the other hand, hDPSC/myc-PRDX6(S32A) and hDPSC/myc-PRDX6(C47S) effectively recovered delayed bone regeneration unlike hDPSC/myc-PRDX6(WT), and the results showed that iPLA2 and GPX activity of PRDX6 hDPSC -Suggests that it is associated with mediated bone formation.

PRDX6 transgenic mice (Tg) were used to test the association of PRDX6 to postnatal day P1 (P1) on the birth day (FIGS. 7A and 7B ).

In PRDX6 Tg mice, the expression of RUNX2, a molecular determinant of bone formation, was significantly reduced (FIG. 7C ).

In addition, bone formation was compared between PRDX6 Tg mice and wild-type mice using Alizarin Red and Alcian Blue staining (FIG. 7D ).

In the skeleton of the PRDX6 Tg mouse, skeletal elements such as the skull and face, which are the main growth sites, were found to be short and thin.

That is, PRDX6 is functionally closely related to bone formation differentiation, bone regeneration, and bone development, and the results suggest that PRDX6 is an important target in the treatment of diseases related to bone formation (FIG. 7E ).

Experimental Example 5: PRDX6 Test for the effect of promoting the formation of bone by an inhibitor

In order to test whether PRDX6 activity inhibitors have equivalent bone formation promoting effects in addition to stem cells introduced with mutated PRDX6, anti-PRDX6 antibodies, siRNA, and MJ33 (1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol) are used. The experiment was repeated.

As a result of the experiment, each of the inhibitors did not differ from the experimental example, but showed similar or equivalent bone formation relatedness.

The above results suggest that PRDX6 is directly associated with bone formation, and that if it is a means capable of inhibiting PRDX6 activity or expression, it can be used for treatment of diseases related to bone formation.

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all modifications or variations derived from the meaning and scope of the claims and their equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (10)

As a composition for promoting bone formation comprising a PRDX6 (peroxiredoxin-6) activity inhibitor as an active ingredient,
The PRDX6 activity inhibitor is pulp stem cells into which the mutant PRDX6 gene is introduced,
The mutant PRDX6 gene includes PRDX6 in which cysteine (C), the 47th amino acid, is substituted with serine (S); And
A composition for promoting bone formation, wherein the 32nd amino acid serine (S) is at least one of PRDX6 substituted with alanine (A). According to claim 1,
The PRDX6 activity inhibitor is a composition for promoting bone formation that inhibits the activity of GPx (glutathione peroxidase) or iPLA2 (calcium-independent phospholipase A2). delete delete delete The method according to claim 1 or 2,
The composition is a composition for promoting bone formation, which is for preventing or treating osteoporosis, bone disease. The method of claim 6,
The bone disease is a composition for promoting bone formation, which is metabolic bone disease or orthopedic bone disease. The method of claim 6,
The bone disease is osteoporosis, osteogenic dysfunction, osteomalacia, osteopenia, fractures, bone defects and hip joint reduction composition selected from the group consisting of at least one. delete delete

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