# Patent

## Title
Cell-penetrating fusion protein for regenerating or proliferating stem cell

## Abstract

The present invention relates to a cell permeable fusion protein for strengthening regenerative potential of stem cells, and more particularly to a cell permeable fusion protein for strengthening regenerative potential of stem cells for stimulating the differentiation of stem cells, inhibiting apoptosis, maintaining the functionality of stem cells and restoring the stress-inhibited functionality of stem cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national phase under the provisions of 35 U.S.C. §371 of International Patent Application No. PCT/KR11/09709 filed Dec. 16, 2011, which in turn claims priority of Korean Patent Application No. 10-2011-0051621 filed May 30, 2011. The disclosures of such international patent application and Korean priority patent application are hereby incorporated herein by reference in their respective entireties, for all purposes.
TECHNICAL FIELD
The present invention relates to a cell permeable fusion protein for strengthening regenerative potential of stem cells, and more particularly to a cell permeable fusion protein for strengthening regenerative potential of stem cells for stimulating the differentiation of stem cells, inhibiting apoptosis, maintaining the functionality of stem cells and restoring the stress-inhibited functionality of stem cells.
BACKGROUND ART
The term “mesenchymal stem cells” refers to cells that are involved in the development and growth of the skeletal system in their embryonic stage and have the capability to differentiate into various kinds of skeletal cells, including osteocytes, chondrocytes, myocytes, fibrocytes, etc., after completion of growth. The mesenchymal stem cells are also referred to as bone marrow stromal stem cells. Unlike embryonic stem cells, mesenchymal stem cells do not cause tumors or ethical issues, and thus are highly useful for clinical applications.
However, the problems to be solved in the transplantation of stem cells are developing technology for facilitating engraftment (improving adaptability) and proliferation in vivo after transplantation and ensuring the capability to differentiate selectively into a desired tissue. To improve adaptability in vivo after transplantation, immune-related drugs are administered after transplantation. However, these drugs are administered to improve the adaptability of a subject to which stem cells are transplanted. A substance for improving the adaptability of the cells transplanted has not been reported yet.
The stem cells transplanted by the surrounding tissue environment undergo senescence, die out, and thus cannot exhibit effects of transplantation. In addition, smooth proliferation of the transplanted stem cells may be the first consideration for regeneration of the cells. In addition to engraftment and proliferation, a substance for stimulating selective differentiation into a desired tissue (bone tissue in the present invention) has also not been reported. Transcription factors essential in the differentiation of mesenchymal stem cells into osteoblasts and adipocytes are Runx2 and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These factors induce mesenchymal stem cells to differentiate into osteoblasts which produce bones and adipocytes. Runx2 is a protein which is observed to be localized to bone when injected externally, and in addition to this protein, several proteins are known to stimulate the differentiation of cells into bone tissue. Particularly, Hong et al. demonstrated that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), represses PPAR gamma transcription and activates Runx2 (Hong J. H. et al., Science, 309:1074, 2005). These proteins act to activate genes for osteogenic differentiation in the cytoplasm. However, the molecular weight of these proteins is too large to be introduced into the cytoplasm or tissue for a specific purpose (substances having a molecular weight of 600 or more are almost impossible to pass through the cell membrane). For this reason, the use of carriers to introduce these proteins into cells is acutely required.
Recently, the development of osteogenesis stimulators for repairing bone damaged by osteoporosis, bone fracture or surgery has been required. However, osteogenesis stimulators, such as bisphosphonates, calcitonin, estradiol or vitamin D, developed to date, mainly aim to inhibit bone resorption, and are not significantly effective to the regeneration of lost bone. Thus, many efforts have been made to discover new drugs that can stimulate osteogenesis.
In recent years, it was found that Tat (transactivator of transcription) protein, a kind of human immunodeficiency virus type-1, efficiently passes through the cell membrane and migrates into the cytoplasm. This function appears because of the characteristics of the protein transduction domain (PTD) of the Tat protein, and the exact mechanism thereof is not known yet (Frankel, A. D. and Pabo, C. O., Cell, 55:1189, 1988; Green, M. and Loewenstein, P. M., Cell, 55:1179, 1988; Ma, M. and Nath, A., J. Virol., 71:2495, 1997; Vives, E. et al., B. J. Biol. Chem., 272:16010, 1997).
Meanwhile, since it was found that a fusion protein obtained by linking PTD to other peptides or proteins is efficiently transported into cells, various applications using PTD has been attempted (Korean Patent Registration No. 10-0568457). However, there has not yet been an attempt to apply the transcription factor NF-Ya or antioxidant protein (e.g., SOD, thioredoxin, etc.) linked to a cell-penetrating peptide to tissue engineering for the purposes of promoting the engraftment of mesenchymal stem cells and inducing the proliferation thereof.
Accordingly, the present inventors have made extensive efforts to develop a cell permeable fusion protein for strengthening regenerative potential of stem cells, and as a result, have prepared a fusion protein by linking a cell-penetrating protein to the amino terminal of an antioxidant protein or a protein having the ability to improve cell proliferation, and have found that the fusion protein can increase the success rate of transplantation of stem cells, can be used to prevent and treat bone disease by selectively inducing the regeneration of bone tissue, can efficiently deliver a protein that inhibits adipocytes into cells, is easily synthesized, and causes no toxicity problem, thereby completing the present invention.
DISCLOSURE OF INVENTION
Technical Problem
It is an object of the present invention to provide a cell permeable fusion protein for strengthening regenerative potential of stem cells.
Technical Solution
To achieve the above object, the present invention provides a cell permeable fusion protein for strengthening regenerative potential of stem cells, wherein a cell permeable peptide is linked to the amino terminal of an antioxidant protein or a protein having an ability to improve cell proliferation.
The present invention also provides a cell permeable fusion protein for strengthening regenerative potential of stem cells, wherein a cell permeable peptide is linked to the amino terminal of the fusion protein of an antioxidant protein and a protein having the ability to improve cell proliferation.
The present invention also provides a vector for expressing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the vector comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines.
The present invention also provides a recombinant microorganism transformed with a vector for expressing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the vector comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines.
The present invention also provides a method for preparing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the method comprising the steps of: culturing a recombinant microorganism transformed with a vector for expressing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the vector comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines; and recovering the expressed fusion protein.
The present invention also provides a method for regeneration of stem cells, by the method for preparing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the method comprising the steps of: culturing a recombinant microorganism transformed with a vector for expressing a cell permeable fusion protein for strengthening regenerative potential of stem cells, the vector comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines; and recovering the expressed fusion protein.
The present invention also provides a cell permeable fusion protein for promoting osteogenesis, wherein a cell permeable peptide is linked to the terminal of the fusion protein of a transcription factor protein for inducing osteogenic differentiation and an antioxidant protein or a protein having the ability to improve cell proliferation.
The present invention also provides a pharmaceutical composition for treating bone diseases, wherein the composition comprising a cell permeable fusion protein for promoting osteogenesis, wherein a cell permeable peptide is linked to the terminal of the fusion protein of a transcription factor protein for inducing osteogenic differentiation and an antioxidant protein or a protein having the ability to improve cell proliferation, as an active ingredient.
The present invention also provides a health functional food for treating bone diseases, wherein the food comprising a cell permeable fusion protein for promoting osteogenesis, wherein a cell permeable peptide is linked to the terminal of the fusion protein of a transcription factor protein for inducing osteogenic differentiation and an antioxidant protein or a protein having the ability to improve cell proliferation, as an active ingredient.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of examining the intracellular penetrability of a LMWP-Thioredoxin-1 conjugate.
FIG. 2 shows the results of examining the in vivo functionality of a LMWP-Thioredoxin-1 conjugate.
FIG. 3 shows the results of examining the ability to inhibit senescence of a LMWP-SOD fusion protein.
FIG. 3A shows a cellular morphological change caused by treatment of a LMWP-SOD fusion protein, and FIGS. 3B and 3C show the results of staining with senescence associated beta-galactosidase.
FIG. 4 shows the examining the ability to inhibit senescence of a LMWP-SOD fusion protein.
FIGS. 4A and 4B show the results of RT-PCR of p53 gene and p21 gene, and FIGS. 4C and 4D show the results of western blotting of p53 protein and p21 protein.
FIG. 5 shows the results of examining the restoration of functionality of stem cell by a LMWP-SOD fusion protein.
FIGS. 5A and 5B show a change in calcification ability, and FIGS. 5C and 5D show the results of RT-PCR of a hard-tissue marker (ALP, type I collagen, osteopontin).



BEST MODE FOR CARRYING OUT THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods which will be described later are those well known and commonly employed in the art.
As used herein, the term “antioxidant protein” refers to a protein that inhibits the denaturation and loss of function of the proteins by inhibiting reactive oxygen species caused by various factors or converting the oxidation state of proteins caused by oxidative stress into a reduction state. Examples of the antioxidant protein include SOD (superoxide dismutase), thioredoxin, catalase, glutathion peroxidase, heam-containing peroxidase, and analogues thereof.
SOD (superoxide dismutase; SEQ ID NO: 1) functions to protect cells from toxicity by converting superoxide ions, including free radical anions that adversely affect cells, into oxygen and hydrogen peroxide. An antioxidant defense mechanism by this SOD is important in almost all cells that are exposed to oxygen, and some lactic acid bacteria are known to use other defense mechanisms.
Thioredoxin (SEQ ID NO: 2) refers to a low-molecular-weight protein having a molecular weight of 10,000-13,000 and acts as a proton donor when ribonucleotide is reduced by ribonucleotide reductase. It is isolated from E. coli. A pair of cysteine residues present in the active center is conserved in prokaryotes and eukaryotes, and thioredoxin has the capability to reduce and cleave the disulfide bond of a target protein in the presence of NADPH and thioredoxin reductase. Human TRX/ADF (human thioredoxin/adult T cell leukemia-derived factor) is involved in cell proliferation or the control of transcription factors.
As used herein, the phrase “protein having the ability to improve cell proliferation” refers to a protein that improves the adaptability of stem cells by engraftment with existing cells after transplantation and facilitates the proliferation of the stem cells. Examples of this protein include HOX4 protein and NF-Ya protein.
HOX4 (homeobox-leucine zipper protein; SEQ ID NO: 3) is a transcription factor that plays an important role in embryo formation. Transcription factors play an important role in the regulation of the expression and differentiation of genes in stem cells and convert various cellular processes by binding to a specific gene target.
NF-Ya (nuclear transcription factor Y subunit alpha; SEQ ID NO: 4) is a major transcription activator of a CCAAT promoter gene and regulates the cell cycle by cellular stress through the p53-depterminalent mechanism of transcriptional inhibition.
The cell-penetrating peptide (CPP) that is used in the present invention is a positively charged peptide and may be selected from the group consisting of (a) LMWP (low molecular weight protamine (SEQ ID NO: 5), (b) TAT (trans activator of transcription (SEQ ID NO: 6), (c) penetratin (SEQ ID NO: 7), (d) polyarginine (6 or more arginines), (e) polylysine (6 or more lysines), (f) a protamine fragment, (g) antennapedia (ANTP), and (h) an oligopeptide comprising 70% or more of histidine, arginine, lysine, or a mixture thereof. In addition, the cell-penetrating peptide (CPP) comprises a protein transduction domain (PTD) and may also be a cell permeable protein.
Although most intracellular drug delivery techniques rely on terminalocytosis that uses receptors on the cell surface, the above peptides carry positive charges, unlike terminalocytosis that is one of intracellular mass transport mechanism, and thus they are attached and transferred depterminaling on the charges of the negatively charged cell membrane and can efficiently introduce desired substances directly into cells within a short time.
As used herein, the phrase “regeneration of stem cells” is meant to include stimulating the differentiation of stem cells, inhibiting apoptosis, improving the ability of stem cells to proliferate, maintaining the functionality of stem cells, and restoring the stress-inhibited functionality of stem cells.
In one aspect, the present invention is directed to a cell permeable fusion protein for intensifying the ability of stem cells to regenerate, the fusion protein comprising a cell permeable peptide linked to the amino terminal of an antioxidant protein or a protein having the ability to improve cell proliferation.
In another aspect, the present invention is directed to a cell permeable fusion protein for improving the ability of stem cells to regenerate or proliferate, the fusion protein comprising a cell permeable peptide linked to the amino terminal of a fusion protein of an antioxidant protein and a protein having the ability to improve cell proliferation.
The inventive fusion protein for improving the ability of stem cells to regenerate or proliferate can be prepared by a chemical or biological method, but is preferably prepared by a biological fusion method that produces fewer by-products. In the chemical fusion method, the amino group of the transcription factor protein is linked to the cell-penetrating peptide by an S—S bond using a crosslinking agent selected from the group consisting of 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4), 1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethyl cyclohexane-1-carboxy-[6-amidocaproate]] (SMCC) and sulfo-SMCC, succimidyl 6-[3-(2-pyridyldithio)-ropionamido]hexanoate (SPDP) and sulfo-SPDP, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and sulfo-MBS, succimidyl[4-(p-maleimidophenyl)butyrate] (SMPB) and sulfo-SMPB. In this case, the peptide binds non-specifically to the amino group of the proteins, causing byproducts comprising a plurality of peptides introduced into one protein. The biological fusion method is designed such that one peptide molecule is introduced in the construction of a plasmid, and thus there is an advantage in that the possibility of production of by-products is low.
In the present invention, the cell permeable fusion protein may be a cell permeable fusion protein for intensifying the regeneration of stem cells, which further comprises a transcription factor protein for inducing osteogenic differentiation, linked to the cell permeable peptide.
As used herein, the phrase “transcription factor protein for inducing osteogenic differentiation” refers to a transcription factor playing a key role in forming skeletal cells from mesenchymal stem cells. Examples of the transcription factor protein include TAZ, Runx2, LMP-1, and derivatives thereof.
TAZ (Tafazzin; SEQ ID NO: 8 or 9) can activate the transcription factor Runx2, which inhibits PPAR-gamma, a transcription factor that induces differentiation into adipocytes, and is essential in the differentiation of mesenchymal stem cells into osteoblasts. In the present invention, TAZ may also mean a gene encoding the TAZ protein.
Runx2 (Runx domain transcription factor 2; SEQ ID NO: 10 or 11) is a transcription factor essential in the differentiation of mesenchymal stem cells into osteoblasts and adipocytes. It induces mesenchymal stem cells to differentiate into osteoblasts or adipocytes. Runx2 is a protein observed to be localized to bone when injected externally, and in addition to this protein, several proteins are known to stimulate the differentiation of cells into bone tissue.
LMP-1 (LIM mineralization protein-1; SEQ ID NO: 12 or 13) also promotes the osteogenic differentiation in cells. Particularly, the present inventors previously confirmed that a specific region of the amino acid sequence (AADPPRYTFAPSVSLNKTARPPGAPPPADSAPQQNG, ADPPRYTFAP, KPQKASAPAADPPRYTFAP) of the LMP-1 protein is associated directly with osteogenic differentiation.
In still another aspect, the present invention is directed to a expression vector of a fusion protein having the ability of stem cells to intensifying regeneration of stem cells, comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines.
The vector that is used in the present invention may be a conventional TA vector or the like, and the expression of the vector is under the control of T7 promoter and LacO-operator.
In yet another aspect, the present invention is directed to a recombinant microorganism transformed with a expression vector of a fusion protein having the ability of stem cells to intensifying regeneration of stem cells, comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines.
A microorganism for transformation that is used in the present invention may be a conventional microorganism, and typical examples thereof, including E. coli and the like.
In a further aspect, the present invention is directed to a method for preparing a fusion protein having the ability of stem cells to intensifying regeneration of stem cells, the method comprising the steps of: culturing a recombinant microorganism transformed with a expression vector of a fusion protein having the ability of stem cells to intensifying regeneration of stem cells, comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines, thereby expressing a fusion protein having the ability of stem cells to intensifying regeneration of stem cells; and recovering the expressed fusion protein.
In a still further aspect, the present invention is directed to a method for regeneration of stem cells, the method comprising introducing a fusion protein prepared by a method comprising the steps of: culturing a recombinant microorganism transformed with a expression vector of a fusion protein having the ability of stem cells to intensifying regeneration of stem cells, comprising: a cDNA encoding the transduction domain of a cell permeable peptide; a cDNA encoding the transduction domain of an antioxidant protein and/or a cDNA encoding the transduction domain of a protein having the ability to improve cell proliferation; a cDNA encoding a transcription factor protein for inducing osteogenic differentiation; and a cDNA for 6 histidines, thereby expressing a fusion protein having the ability of stem cells to intensifying regeneration of stem cells; and recovering the expressed fusion protein into stem cells.
In a yet further aspect, the present invention is directed to a cell permeable fusion protein for promoting osteogenesis, the fusion protein comprising a cell permeable peptide linked to the terminal of a fusion protein of an antioxidant protein or a protein having the ability to improve cell proliferation and a transcription factor protein for inducing osteogenic differentiation.
In another further aspect, the present invention is directed to a pharmaceutical composition for treating bone disease, the composition comprising a cell permeable fusion protein for promoting osteogenesis as an active ingredient, the fusion protein comprising a cell permeable peptide linked to the terminal of a fusion protein of an antioxidant protein or a protein having the ability to improve cell proliferation and a transcription factor protein for inducing osteogenic differentiation.
In the present invention, the bone disease may be any one selected from the group consisting of osteoporosis, osteogenesis imperfecta, periodontal disease, and bone fracture.
The pharmaceutical composition for treating bone disease can be administered orally or non-orally (intramuscular, intravenous, suppository, etc.). The preferred dosage of the pharmaceutical composition for treating bone disease of the present invention can be suitably adjusted depterminaling on the patient's conditions, for example, age and the severity of disease. In order to achieve the desired effects, however, for adults, the pharmaceutical composition of the present invention may be administered at a daily dose of from 100 to 1,000 mg/day, and preferably 300 to 1,000 mg/day within a dose range of from 100 to 50 mg/day. The pharmaceutical composition may be administered in a single dose per day or in multiple doses per day.
When the pharmaceutical composition for treating bone disease according to the present invention is formulated as an oral dosage form, the formulation can be prepared by adding an excipient, optionally a binder, a disintegrant, a lubricant, a colorant, a flavor enhancer or the like to the composition, and then preparing the mixture into a tablet, a coated tablet, a granule formulation, a capsule or the like according to a conventional method.
Examples of carrier, excipient or diluent that can be used in the pharmaceutical composition for treating bone disease may include lactose, corn starch, white sugar, glucose, lactose, dextrose, sucrose, sorbitol, crystalline cellulose, mannitol, xylitol, erythritol, maltitol, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. Examples of the binder which can be used in the pharmaceutical composition for treating bone disease may include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, Arabic gum, gelatin, (shellac), hydroxyprophyl cellulose, hydroxyprophyl starch, and polyvinyl pyridine. In addition, Examples of the disintegrant which can be used in the pharmaceutical composition for treating bone disease may include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextran, and pectin. Examples of the lubricant which can be used in the pharmaceutical composition for treating bone disease may include stearic acid, magnesium, talc, polyethylene glycol, silica, and vegetable oils. A colorant that can be used in the pharmaceutical composition for treating bone disease may be one approved for use in medical applications, and a flavor enhancer that can be used in the pharmaceutical composition may be cocoa powder, menthol, aromatic acid, mint oil, borneol, cinnamon powder or the like.
In addition, when the pharmaceutical composition for treating bone disease according to the present invention is formulated as an injectable dosage form, the formulation can be prepared by adding a pH adjusting agent, a buffer, a stabilizer, a preservative or the like the composition and formulating the mixture into an injectable liquid for subcutaneous, intramuscular or intravenous injection according to a conventional method.
The pharmaceutical composition for treating bone disease of the present invention may be administered by various routes into mammals, including rats, mice, livestock and humans. All routes of administration can be contemplated and include, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, intrathecal or intracerebrovascular injections.
In another still further aspect, the present invention is directed to a health functional food for treating bone disease, comprising a cell permeable fusion protein for promoting osteogenesis, the fusion protein comprising a cell permeable peptide linked to the terminal of a fusion protein of an antioxidant protein or a protein having the ability to improve cell proliferation and a transcription factor protein for inducing osteogenic differentiation as an active ingredient and further comprising an acceptable food additive.
As used herein, the term “functional food” refers to a food, the functionality of which has been improved by adding thereto a cell permeable fusion protein for promoting osteogenesis (CPP-TAZ) according to the present invention, to conventional food.
In addition, the health functional food for treating bone disease of the present invention may further contain various nutrients, vitamins, minerals (electrolytes), seasonings (artificial seasonings and natural seasonings), coloring agents and improving agents (cheese, chocolate and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH controllers, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like as acceptable food additives. In addition, the health functional food for treating bone disease of the present invention may further contain fruit flesh for preparation of natural fruit juice beverages, fruit juice beverages and vegetable beverages. These additives may be used indepterminalently or in combination. Although the content of these additives in the health functional food for treating bone disease of the present invention is not particularly limited, it is generally selected within the range of 0.01-20 parts by weight based on 100 parts by weight of the cell permeable fusion protein for promoting osteogenesis of the present invention.
In particular, an example of the present example illustrated that, when stem cells are treated with a fusion protein of LMWP with thioredoxin or SOD, the ability of the stem cells to regenerate or proliferate was improved. However, it will be obvious to one skilled in the art from the disclosure of the present invention that a fusion protein of a conventional cell permeable peptide with an antioxidant protein improves the ability of stem cells to regenerate or proliferate, and thus falls within the scope of the present invention.
EXAMPLES
Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.
Example 1
Preparation of Recombinant LMWP-Thoredoxin-1 Fusion Protein and Examination of the Ability Thereof to Penetrate Cells
An LMWP peptide was synthesized, and then conjugated with thioredoxin (1 mg/ml in PBS) using SMCC (Pierce). The LMWP-thioredoxin conjugate was purified using a heparin-agarose bead (Sigma).
A C2C12 model cell line (ATCC) was cultured in serum-free medium for 24 hours under the conditions of 95% air, 5% CO2 and 37° C. Next, the model cell line (mouse C2C12) was treated with the LMWP-Trx-1 (WT and C3134S) conjugates and incubated in serum-free medium for 30 min, and then an experiment for comparison of the conjugates with non-conjugated thioredoxin-1 was performed. As a result, it was shown that the ability of the conjugates to penetrate the cells was excellent compared to that of thioredoxin-1 alone, the ability of WT conjugate to penetrate the cells was superior to that of the C3134S conjugate (see FIG. 1).
Example 2
Examination of the Ability of Recombinant LMWP-Thoredoxin-1 Fusion Protein to Stimulate the Differentiation of Stem Cells
In order to examine the influence of the LMWP-thioredoxin-1 fusion protein on the differentiation of stem cells (Lonza) into myocytes, stem cells were treated with the LMWP-thioredoxin-1-WT conjugate according to the concentration and incubated under the conditions of 95% air, 5% CO2 and 37° C. Then, the expression levels of MYH, Myogenin and MyoD proteins (myocyte differentiation markers) in the cells were analyzed by a Western blotting technique. In this experiment, thioredoxin-1 not conjugated with LMWP was used as a control. As a result, it was shown that, when the cells were treated with the LMWP-thioredoxin-1-WT conjugate, the differentiation of the cells into myocytes increased in a concentration-depterminalent manner (see FIG. 2).
Example 3
Preparation of Recombinant LMWP-SOD Fusion Protein and Examination of the Ability Thereof to Inhibit Apoptosis
A LMWP peptide was synthesized, and then conjugated with SOD (Sigma, 1 mg/ml in PBS) using SMCC (Pierce). The LMWP-SOD conjugate was purified using a heparin-agarose bead (Sigma).
Analysis was carried out to examine the influence of the LMWP-SOD fusion protein having the ability to penetrate cells on apoptosis. Specifically, human dental pulp stem cells (primary culture) were seeded into a 6-well plate at a density of 1×104 cells per well, and then cultured under the conditions of 95% air, 5% CO2 and 37° C. for 24 hours. Next, the cells were treated with varying concentrations of the LMWP-SOD conjugate and incubated for 2 hours. Then, the cells were treated with a low concentration (150 uM) of hydrogen peroxide in serum-free medium for 2 hours to artificially induce cell senescence, and then incubated in general medium for 3 days. The degree of senescence of the cells was examined by a cellular morphological change (see FIG. 3A) and senescence-associated beta-galactosidase staining (see FIGS. 3B and 3C). As a result, it could be seen that, in the test group pretreated with LMWP-SOD, the flatten morphology of the cells was partially restored and SA-beta-gal staining definitely decreased.
Example 4
Examination of the Ability of Recombinant LMWP-SOD Fusion Protein to Maintain the Functionality of Stem Cells and Examination of Proteins that are Involved in the Maintenance
Analysis was performed to examine the influence of the LMWP-SOD conjugate having the ability to penetrate cells on the functionality of stem cells. Specifically, human dental pulp stem cells were seeded into a 100-mm dish at a density of 1×106 cells, and then cultured for 24 hours. Next, the cells were treated with varying concentrations of the LMWP-SOD conjugate and incubated for 2 hours under the conditions of 95% air, 5% CO2 and 37° C. Then, the cells were treated with a low concentration (150 uM) of hydrogen peroxide in serum-free medium for 2 hours to artificially induce cellular stress, and then incubated in general medium for 3 days. Changes in the p53 gene and protein known to be involved in cellular senescence, and the p21 gene and protein whose activity is regulated by p53, were analyzed by RT-PCR (see FIGS. 4A and 4B) and a Western blotting technique (see FIGS. 4C and 4D). As a result, it could be seen that, in the test group pretreated with LMWP-SOD, the expression levels of the p53 and p21 genes decreased and the expression levels of the proteins also significantly decreased. In this experiment, it was found that the introduction of the physiologically active peptide, which is not toxic and can function to efficiently deliver a specific protein into cells, increases the expression of the SOD protein required to inhibit cellular senescence, thereby inhibiting cellular stress caused by hydrogen peroxide, and p53/p21 signaling pathways are involved in this inhibition process.
Example 5
Restoration of Functionality of Stem Cells Stress-Inhibited by Recombinant LMWP-SOD Fusion Protein
When stem cells undergo stress due to a change in the surrounding environment, the stem cells lose their functionality. In this experiment, from this viewpoint, the degree of mineralization was measured by Alizarin red S staining to determine whether the LMWP-SOD conjugate can penetrate into aged stem cells (Lonza) to restore the functionality of the cells. Specifically, stem cells were treated with the LMWP-SOD conjugate, and after 2 hours, treated with hydrogen peroxide, followed by incubation under the conditions of 95% air, 5% CO2 and 37° C. for 14 days. Following this, the degree of mineralization of the cells was measured by Alizarin red S staining. As a result, it was shown that the mineralization of the group treated with LMWP-SOD was restored, unlike the group treated with SOD alone or the control group (see FIGS. 5A and 5B). In order to confirm this effect of restoring the functionality of stem cells, the mRNA levels of ALP, type I collagen, osteopontin, which are hard-tissue differentiation genes, were analyzed. As a result, it was shown that the expression levels of these genes in the group treated with LMWP-SOD were restored in a pattern similar to that of FIGS. 5A and 5B (see FIGS. 5C and 5D).
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appterminaled claims and equivalents thereof.
INDUSTRIAL APPLICABILITY
According to the present invention, when stem cells are treated with the cell permeable fusion protein for intensifying the regeneration of stem cells, it easily penetrates the stem cells to stimulate the differentiation of the stem cells, to inhibit apoptosis, to maintain the functionality of the cells and to promote the restoration of stress-inhibited functionality of the stem cells. Thus, the fusion protein is useful for the regeneration of stem cells.

## Claims

1. A cell permeable fusion protein for improving an ability to regenerate or proliferate stem cells, wherein a cell permeable peptide is linked to the amino terminal of Thioredoxin antioxidant protein, wherein the cell permeable peptide is LMWP (low molecular weight protamine) (SEQ ID NO: 5).

2. The cell permeable fusion protein for improving an ability to regenerate or proliferate stem cells of claim 1, wherein a transcription factor protein having an functionality of osteogenic differentiation is further linked to the cell permeable fusion protein.

3. The cell permeable fusion protein for improving an ability to regenerate or proliferate stem cells of claim 1, wherein the Thioredoxin is represented by SEQ ID NO: 2.

4. An expression vector of the permeable fusion protein for improving an ability to regenerate or proliferate stem cells according to claim 1, wherein the vector comprises: a cDNA encoding the transduction domain of the LMWP (low molecular weight protamine); a cDNA encoding the transduction domain of the thioredoxin protein.

5. A recombinant microorganism transformed with the expression vector of claim 4.

6. A method for preparing a fusion protein for improving an ability to regenerate or proliferate stem cells, wherein the method comprises the steps of:
culturing the recombinant microorganism of claim 5, thereby expressing the fusion protein for improving an ability to regenerate or proliferate stem cells; and
recovering the expressed fusion protein.

7. A method for regeneration of stem cells, wherein the method comprises introducing the fusion protein prepared by the method of claim 6 into stem cells.

8. A cell permeable fusion protein for promoting osteogenesis, wherein a cell permeable peptide is linked to the terminal of a fusion protein of Thioredoxin antioxidant protein and a transcription factor protein having functionality of osteogenic differentiation, wherein the cell permeable peptide is LMWP (low molecular weight protamine) (SEQ ID NO: 5).

9. A pharmaceutical composition for treating bone disease, the composition comprising the cell permeable fusion protein for promoting osteogenesis of claim 8 as an active ingredient.

10. The pharmaceutical composition for treating bone disease of claim 9, wherein the bone disease is any one selected from the group consisting of osteoporosis, osteogenesis imperfecta, periodontal disease and bone fracture.

11. A health functional food for treating bone disease comprising the cell permeable fusion protein for promoting osteogenesis of claim 8 as an active ingredient.