KR101609335B1 - Pharmaceutical composition for prevention or treatment of osteoporosis comprising dental pulp stem cell conditioned medium - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, comprising a stem cell culture liquid as an active ingredient.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention or treatment of osteoporosis,

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, comprising a stem cell culture liquid as an active ingredient.

Studies are underway to develop stem cells with autoproliferation and multiple lineage differentiation potential as therapeutic agents for regenerating damaged tissues. There are groups of mesenchymal stem cells in teeth that have higher self-proliferating and differentiating capacity than any other tissue. In recent years, interest in the use of adult teeth as a source of adult stem cells has been increasing due to the advantage of easily obtaining mesenchymal cells even in spontaneously missing teeth or surgically removed teeth. The cell population with the characteristics of the stem cells is divided into two groups according to the dimensions of the tooth or permanent teeth, the periodontal ligament connecting between the root and the bone, the growing root, the crown surrounding the unerupted tooth, the apical papilla, Separated from several parts, the tooth stem cells are also separated from the gums. These cells share a common lineage and traits derived from the neural crest cells of the embryo generators. They also express specific gene markers in vitro and in vivo and are capable of differentiating into osteoblasts, adipocytes, chondrocytes, etc., of the mesenchymal cell line and differentiating them into cells of other lines. Dimensional stem cells isolated from human dimensions have high proliferative capacity and can differentiate into nerve cells, adipocyte dentin cells, induce bone formation after in vivo transplantation, produce dentin and cells other than teeth But also the ability to differentiate. Studies have been carried out to treat degenerative neurological diseases by regenerating the tissue removed by infection or by inducing the formation of differentiated neural cell populations using such stem cells.

In recent years, in addition to transplanting stem cells directly into injured tissue, the excellent side secretion effect of mediators contained in the culture medium of stem cells has been demonstrated in vivo or in vitro experiments. Stem cells secrete various growth factors and cytokines related to growth and regeneration of cells in the human body for a lifetime, stem cell cultures contain these protein components, and the immune rejection reaction, which is a side effect of cell transplantation, is avoided Therefore, the stem cell culture fluid has advantages advantageous for development as a therapeutic agent.

Osteoblasts form bones and constitute the extracellular matrix during bone reshaping and restoration. Bones are formed by osteoblasts and are dynamic tissues in which the processes of absorption by osteoclasts are repeated. The cells involved in these processes are involved in activities such as growth factors, cytokines, adhesion-molecules, (Jon et al., J Cell Physiol 1990; 143: 213-21; Hiura et al., Endocrinology 1991; 128: 1630-1637), Suda et al., J Cell Biochem 1992; 49: 53-58, Manolagas et al., J Bone Miner Res 1999; 14: 1061-1066). Clinical studies have also shown that oxidative stress and oxidative enzymes are closely related to the pathogenesis of bone loss such as osteoporosis (Maggio et al., J Clin Endocriol Metab 2003; 88; 1523-1527; Yalin et al. , Clin Exp Rheumatol 2005; 23: 689-692). Osteoblast differentiates from mesenchymal cells, and when damaged by aging or oxidative stress, the proliferation and division ability of the osteoblast are decreased, resulting in imbalance in osteoblast and osteoclast action and osteoporosis. Thus, a substance that inhibits damage to osteoblast cells and promotes proliferation and division ability can be utilized for prevention and treatment of osteoporosis. Although a therapeutic agent for various osteoporosis has been developed and used, it is still required to develop a therapeutic agent effective for prevention and treatment of osteoporosis, which promotes osteoblast activity and does not cause toxicity and side effects even in prolonged administration.

Accordingly, the present inventors have completed the present invention by carrying out a study on a therapeutic agent for osteoporosis which inhibits oxidative damage of osteoblasts, has an ability to promote cell differentiation ability, and does not cause side effects.

The present invention aims to provide a pharmaceutical composition for preventing or treating osteoporosis, which comprises a stem cell culture liquid.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoporosis, which comprises a stem cell culture solution.

Dimension Stem cells refer to stem cells present in the dermal tissue. Dimensional stem cells have high proliferative capacity and high cell colony forming ability and form dense calcified nodules and have the ability to differentiate into dentin, adipocyte, chondrocyte, and osteoblast (Gronthos S, Bull Cell Biol 2010; 11: 309-402, 1999. [0157] 32, d'Aquino R, et al., Cell death Differ 2007; 14: 1162-71, and Grazino A, et al., Stem Cell Rev 2008; 4: 21-6).

As used herein, the term " stem cell culture medium "means the culture medium obtained by culturing the stem cells, or the culture supernatant from which the stem cells have been removed, and the term "Quot; stem cell conditioned medium "or" dimensional stem cell conditioned medium ".

The stem cell culture solution obtained by culturing the stem cell has the activity of inhibiting the oxidative damage of the osteoblast and promoting the cell differentiation ability.

In one embodiment of the present invention, the stem cell may be a cell expressing CD13, CD73, CD105, CD146, CD166, ABCg2, Oct-4, Sox-2, Nanog, Notch-1 and Nestin gene. These genes are genes that are uniquely expressed in mesenchymal stem cells, pluripotent stem cells, and embryonic stem cells, and their expression indicates that the stem cells are stem cells.

In one embodiment of the present invention, the stem cell culture solution may be a culture supernatant obtained after culturing the stem cell and removing the cell, or a concentrate thereof or a lyophilized product thereof.

In one embodiment of the present invention, the stem cell culture solution can be obtained by subculturing the stem cells in a serum medium, followed by culturing in a serum-free medium.

Dimension stem cells can be routinely cultured using a stem cell culture medium. The stem cell culture solution is obtained by subculturing the stem cells obtained from the dental tissues in a serum medium and then subculturing in a serum-free medium. The stem cells are obtained by filtration using centrifuge or filter, The supernatant obtained after removing the molecules can be used. The obtained supernatant can be used as it is or as a concentrate obtained by concentration.

The culture medium and culture conditions for culturing the stem cell are well known in the art and can be appropriately selected or modified by those of ordinary skill in the art.

In one embodiment of the invention, the serum medium may be an alpha-MEM medium supplemented with 5-10% fetal bovine serum and 1% antibiotic.

In one embodiment of the present invention, the serum-free medium may be alpha-MEM medium.

The serum medium may be an alpha-MEM medium supplemented with serum, which is suitable for maintaining and storing the same cell type as the stem cell. The serum may be, but is not limited to, fetal bovine serum (FBS), and may be 1 to 10% by weight based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents and mycoplasma inhibitors may be included, and they may be 1% by weight based on the total weight of the culture medium. Antibiotics include antibiotics commonly used in cell culture such as penicillin-streptomycin, antifungal agents include fungizone, and mycoplasma inhibitors include gentamycin, ciprofloxacin, tylosin and the like , But is not limited thereto.

The culture in the serum-free medium can be performed after removing the culture medium from the serum medium and washing the cells with a phosphate buffer solution.

In one embodiment of the present invention, the stem cell culture medium is prepared by mincing the isolated tissue from the mouth, attaching the tissue to the bottom of the culture plate, adding 10% fetal calf serum and 1% penicillin-streptomycin- fungizone in a culture medium supplemented with alpha-MEM medium at 37 ° C and 5% CO _{2, and} when the cell density reaches 80% confluence, cells are separated and subcultured , Culturing the cells for 5 to 7 days and then washing with phosphate buffer solution, culturing the washed cells in an incubator in an alpha-MEM medium without serum and antibiotics at 37 ° C and 5% CO ₂ ≪ / RTI >

In one embodiment of the present invention, the stem cell culture supernatant can be obtained by removing the cells by centrifugation or filtration of the stem cell culture solution.

In one embodiment of the present invention, the pharmaceutical composition may include a stem cell culture liquid or its supernatant as an active ingredient, and may include a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be formulated into various formulations such as a liquid, a suspension, an emulsion, a lotion, an ointment, and a lyophilizing agent according to a conventional method.

Pharmaceutically acceptable carriers that may be included in the pharmaceutical compositions of the present invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginic acid A lubricant such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, polysorbate, cetyl alcohol, glycerol, and the like, as well as binders such as polyvinylpyrrolidone, But are not limited thereto.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited by these examples.

The stem cell culture solution or its supernatant according to one embodiment of the present invention inhibits oxidative damage of osteoblasts producing osteoblasts, promotes cell differentiation ability, improves osteoblast function, Can be prevented or treated.

FIG. 1 is a graph showing the results of cell culture (A) and cell culture (B) in a state where cells were monolayered (A) (B), and a state where the cells are saturated in the culture dish on the 14th day of cultivation (C).
Fig. 2 shows the results of RT-PCR for detecting the expression of stem cell-specific genes by the stem cell.
FIG. 3A shows that when the osteoblast was treated with 2-deoxy-d-ribose, which is a reducing sugar, the cell survival was decreased in a concentration-dependent manner.
FIG. 3B shows the effect of the stem cell culture medium (CM) on osteoclast damage recovery ability.
FIG. 3c shows that when 20 mM of 2-deoxy-d-ribose (dRib) was treated with osteoblast, the number of cells significantly decreased and degenerated, but the supernatant (CM) , The number of cells is restored.
FIGS. 4A and 4B illustrate the effect of osteoblast apoptosis and production of reactive oxygen species (ROS) on the stem cell culture fluid, respectively.
FIG. 5 shows that when osteoblast cells were treated with dRib 20 mM, the activity of ALP, which is a differentiation marker of osteoblast, and collagen production ability were significantly decreased, and they were recovered when the stem cell culture medium (CM) was added.
FIG. 6 shows a significant increase in the expression level of ALP, collagen, osteocalcin, OPN, osteotelia, AML, FasL, TGF beta 1, FGF2 and TCIRG in osteoblasts when treated with stem cell culture medium (CM) Lt; / RTI >
FIG. 7 shows the growth factors present in the stem cell culture liquids detected using the protein array membrane.

Example  1. Culture of Human Dimension Stem Cells

The oral cavity was washed three times with sterile physiological saline, the teeth were extracted and washed three times with phosphate buffer containing 1% penicillin and streptomycin. Under aseptic conditions, the teeth were exposed and the pulp was isolated. On the culture plate containing the phosphate buffer solution, the tissue was cut into small pieces of about 0.5 mm x 0.5 mm x 0.5 mm using a surgical knife and a forceps. Dimensional tissues were attached to the bottom of 6-well plates and incubated with alpha-MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-fungizone (Invitrogen, USA Cat # 12561) were dispensed and cultured at 37 ° C in a 5% CO ₂ incubator. The cells were cultured while exchanging the medium at intervals of 3 days. When the density of the cells reached 80% confluence, the cells were separated and the subculture was maintained. The obtained stem cells were kept frozen and subcultured.

To obtain the stem cell culture solution, the cells were inoculated at a cell concentration of 5 × 10 ⁵ on a 100 mm culture plate and cultured in α-MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-HUN at 37 ° C. , 5% CO ₂ for 7 days, and then the culture plate was washed three times with phosphate buffer solution. The obtained cells were inoculated into 5 ml of an alpha-MEM medium containing no fetal bovine serum and antibiotic, and cultured in an incubator at 37 DEG C and 5% CO ₂ for 1 day. The culture broth was collected, sterilized by filtration using a 0.22 μm filter, and stored frozen at -80 ° C. until use.

1 (a) to 1 (c) show a state in which the tissue is attached to a culture plate and cultured for 7 days, and then the cells are cultured as a monolayer around the tissue (a) 50% saturation (confluence) is reached (B), and the cells are saturated on the culture dish for 14 days.

Example  2. Dimension Stem cell gene Marker  Identification

To investigate the characteristics of stem cells as stem cells, the expression of unique gene markers expressed in stem cells was examined.

2-1. RNA  extraction:

The mesenchymal stem cells obtained in Example 1 were subcultured to a concentration of 5 × 10 ⁵ on a 100 mm culture plate and cultured for 4 days. Then, the culture plate was washed with PBS solution. Extraction of RNA was performed as follows by modifying the single step method of Chomczynski et al. (Analytical Biochemistry 162, 156, 1987). The washed cells were homogenized by adding 1 ml of homogenization solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.1 M beta-mercaptoethanol, 0.5% sarkosyl), and 0.1 volume 3 volumes of sodium acetate (pH 4.0), 1 volume of water-saturated phenol and 0.2 volumes of chloroform-isoamyl alcohol (49: 1 v / v) were added. After standing on ice for 15 minutes, the supernatant was separated by centrifugation at 10,000 xg for 20 minutes at 4 ° C, and the supernatant was mixed with 1 volume of isopropanol. After incubation at -70 ° C for 1 hour, the mixture was centrifuged again at 10,000 x g for 20 minutes at 4 ° C to obtain RNA compressed crystals. The cells were washed with 75% ethanol, dried with a vacuum drier, dissolved in sterile distilled water treated with 0.1% DEPC (diethyl pyrocarbonate), and then quantified with a UV spectrophotometer at 260-280 nm.

2-2. cDNA  Produce:

cDNA was synthesized according to the manufacturer's description using a cDNA synthesis kit (Life technologies, USA, Cat # 117520500).

All solutions and reaction solutions were kept on ice to prevent RNA denaturation. 100 mM dATP solution, 100 mM dCTP solution, 100 mM dGTP solution, 100 mM dTTP, 5 mM Biotin-11-dCTP, Biotin-11-dATP 5 mM-Oligo (dT) 12-18 primer, RNase- First-Strand buffer, 0.1 mM DTT, RNaseOut (TM), SuperScript (TM) II Reverse Transcriptase, RNase H, internal standard water mixture-Silverquant and RNA obtained in 2-1 above were prepared on ice. 100 mM dATP and dCTP were dissolved in RNAse-free distilled water to give a final concentration of 16 mM. 10 mM Biotin-dNTP mixture was prepared by mixing 5 mM dTTP, 5 mM dGTP, 800 μM dATP, 800 μM dCTP, 800 μM Biotin-11-dATP and 800 μM Biotin-11-dCTP. 1x Biotin-dNTP mixture, 2 μl of internal standard mixture Sililquant, 2 μl of Oligo (dT) 12-18 Primer (0.5 μg / μl) and the total amount of RNA (10 μg) were added and RNase-free distilled water was added to give a final volume of 10 Lt; / RTI > After shaking gently, it was allowed to react at 70 ° C for 1 minute. The resulting reaction solution was allowed to stand on ice for 50 minutes, and 4 μl of 5 × First-Strand Buffer, 2 μl of 0.1 M DTT, 2 μl of 10 × Biotin-dNTP mixture and 1 μl of RNaseOut ™ (40 U / μl) did. The mixture was allowed to stand on ice for 5 minutes, and 1.5 μl of SuperScript ™ II Reverse Transcriptase (200 U / μl) was added and mixed, followed by reaction at 42 ° C. for 90 minutes. 1.5 μl of SuperScript ™ II Reverse Transcriptase (200 U / μl) was added, mixed, and reacted at 42 ° C for 90 minutes. The reaction was allowed to proceed for 15 minutes at 70 ° C. 1 μl of RNase H (2 U / μl) was added and allowed to react at 37 ° C for 20 minutes after shaking lightly. The mixture was lightly mixed and allowed to stand at 95 DEG C for 3 minutes to stop the reaction. Placed on ice and subjected to occlusion reaction.

2-3. Real-time polymerase chain reaction Real Time - PCR : RT - PCR ):

Using the cDNA obtained in 2-2, the expression of a gene that is uniquely expressed in stem cells was observed in order to characterize human stem cells.

Real-time PCR was performed using the Taqman method using the Roche LightCycler 480 system (Roche Diagnostics, GmbH Mannheim, Germany) and the Roche Universal ProbeLibrary (UPL) kit to analyze differences in the expression of selected genes.

10 μl of the 2 × UPL master mix, 1.0 μl of the 5 'primer (10 pmol / μl), 1.0 μl of the 3' primer (10 pmol / ml), 0.2 μl of the UPL probe, Mu] l and 6.8 [mu] l of sterile water. The PCR was carried out after an initial denaturation at 95 DEG C for 10 minutes, followed by 40 cycles of 94 DEG C for 10 seconds and 60 DEG C for 30 seconds. The primers used for gene expression were designed by the Roche Probe Finder assay tool and have the following nucleotide sequences.

Target gene Primer sequence ABCg2 (ATP-binding cassette, superfamily G, member 2) 5'-GGG TTC TCT TCT TCC TGA CGA CC-3 '(SEQ ID NO: 1)
5'-TGG TTG TGA GAT TGA CCA ACA GAC C-3 '(SEQ ID NO: 2) Oct -4 (POU class 5 homeobox 1) 5'-GAA GGA TGT GGT CCG AGT GT-3 '(SEQ ID NO: 3)
5'-GTG AAG TGA GGG CTC CCA TA-3 '(SEQ ID NO: 4) CD166 (activated leukocyte cell adhesion molecule) 5'-CGT CTG CTC TTC TGC CTC TT-3 '(SEQ ID NO: 5)
5'-TAA ATA CTG GGG AGC CAT CG-3 '(SEQ ID NO: 6) CD105 (endoglin) 5'-CAC TAG CCA GGT CTC GAA GG-3 '(SEQ ID NO: 7)
5'-CTG AGG ACC AGA AGC ACC TC-3 '(SEQ ID NO: 8) CD73 ( 5'-nucleotidase, ecto) 5'-CGC AAC AAT GGC ACA ATT AC-3 '(SEQ ID NO: 9)
5'-CTC GAC ACT TGG TGC AAA GA-3 '(SEQ ID NO: 10) SOX2 (sex determining region Y-box 2) 5'-ACA CCA ATC CCA TCC ACA CT-3 '(SEQ ID NO: 11)
5'-GCA AAC TTC CTG CAA AGC TC-3 '(SEQ ID NO: 12) CD146 (melanoma cell adhesion molecule) 5'-GTC TGC GCC TTC TTG CTC-3 '(SEQ ID NO: 13)
5'-TTC CAC CTC CAC CAG CTC-3 '(SEQ ID NO: 14) CD13 ( alanyl (membrane) aminopeptidase) 5'-GGG CAC AAT CCA CAC GTA G-3 '(SEQ ID NO: 15)
5'-TCA CGG TGG ATA CCA GCA C-3 '(SEQ ID NO: 16) Notch- 1 (neurogenic locus notch homolog protein 1) 5'-GCA CTG CGA GGT CAA CAC-3 '(SEQ ID NO: 17)
5'-AGG CAC TTG GCA CCA TTC-3 '(SEQ ID NO: 18) Nestin 5'-GTG GCA CAC ATG GAG ACG-3 '(SEQ ID NO: 19)
5'-GAG CGA TCT GGC TCT GTA GG-3 '(SEQ ID NO: 20) Nanog 5'-TCT CTC CTC TTC CTT CCT CCA-3 '(SEQ ID NO: 21)
5'-GGA AGA GTA GAG GCT GGG GT-3 '(SEQ ID NO: 22)

Negative control (without template) was included in the assay to confirm target gene-specific amplification. For quantitative PCR analysis, Light Cycler 480 software version 1.2 (Roche) was used. The values obtained from each sample were corrected to reflect the amount of HPRT gene (hypoxanthine guanine phosphoribosyl transferase) expression. The primer sequences for HPRT amplification are as follows:

5'-TCC TCC TCA GAC CGC TTT T-3 '(SEQ ID NO: 23)

5'-CCT GGT TCA TCA TCG CTA ATC-3 '(SEQ ID NO: 24).

PCR analysis revealed that human dermal stem cells express genes that are uniquely expressed in mesenchymal stem cells, pluripotent stem cells, and embryonic stem cells, whereby the cells obtained in Example 1 are derived from the dimensions of human teeth And that it has the characteristics of stem cells. Fig. 2 shows the results of RT-PCR for detecting the expression of stem cell-specific genes by the stem cell.

Example  3. Dimension The osteoblast of stem cell culture fluid ( MC3T3 - E1 osteoblast )of On survival  Effect on

The effect of stem cell culture media on the viability of osteoblast cells in oxidative injury was investigated.

Osteoclasts were purchased from American Type Culture Collection (USA) (ATCC, CRL-2593). The osteoblast cells were maintained in a 100 mM culture plate at 37 ° C in 5% CO ₂ condition using an alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 unit / 100 mg / , The medium was exchanged at 3-day intervals, and an appropriate subculture period of 5-7 days was maintained for the experiment.

3-1. Experiment of survival ability of osteoblast

The present inventors have found that a reducing sugar, 2-deoxy-d-ribose (dRib), produces a reactive oxygen species (ROS) through autoxidation and protein glycosylation (Suh et al., Biol Pharm Bull, 2009; 32: 746-749, Lee et al, Phytother Res 2010; 24: 419-423, Suh et al. 2012; 35: 121-126). In this example, the effect of dermal fibroblast cultures on the survival of osteoblast cells that were oxidatively damaged by dRib was investigated.

Cell viability was determined by the CCK-8 assay (Suh et al., Biol Pharm Bull, 2012; 35: 121-126). A cell culture in a culture medium containing 10% FBS in a 24-well plate at 2 × 10 ⁴ was dispensed at a concentration of cells / well. After incubation for 48 h in the incubator, the cells were washed three times with phosphate buffered saline (PBS) and cultured in α-MEM containing 0.3% bovine serum albumin (BSA) -Deoxy-d-ribose was incubated for 24 hours. 2-deoxy-d-ribose was added to the culture medium at 60 minutes after the addition of the stem cell supernatant. After adding 50 μl of CCK-8 reagent to each well and incubating for 2 hours, the resulting soluble formazan was measured by absorbance at 450 nm.

FIG. 3A shows that the osteoblast survival rate was decreased in a concentration-dependent manner when osteoblast was treated with 2-deoxy-d-ribose, which is a reducing sugar.

When osteoblast cells were treated with 2-deoxy-d-ribose (dRib) at a concentration of 20 mM, the cell viability was about 50% as compared with the control. However, when 20% to 50% of the stem cell cultures (CM) were treated with each, the survival of the cells was restored in a concentration-dependent manner.

FIG. 3b shows the effect of the stem cell culture fluid on the osteoclast damage recovery ability.

FIG. 3c shows that when 20 mM of 2-deoxy-d-ribose (dRib) was treated with osteoblast, the number of cells was remarkably decreased and degenerated, but when the supernatant of the stem cell culture liquid was added, Of the population recovered.

Example  4. Dimension of osteoblast of stem cell culture solution Apoptosis  And oxidation damage recovery ability

In this example, the effect of osteoblast cell apoptosis and oxidative damage recovery on the ability of stem cell cultures was investigated.

4-1. Apoptosis ( Apoptosis ) Measure

Cell death detection kit (Cat death detection ELISA, Cat # 11774425001, Roche Molecular Biochemicals, Germany) was used for quantification of apoptosis.

Well plate to a concentration of 2 x 10 ⁴ cells / well. Osteoblast cells were cultured for 2 days under the conditions described in Example 3, treated with the stem cell culture solution, and cultured for 24 hours. Thereafter, 100 占 퐇 of the lysis buffer was added and left for 10 minutes. The supernatant obtained by centrifugation at 200 xg for 10 minutes was transferred to a streptavidin-coated well. Histone-biotin and anti-DNA-peroxidase was added, followed by incubation for 2 hours to bind the anti-histone antibody to the histone component of the nucleosome. At the same time, the immunocomplex was immobilized on the streptavidin-coated microplate via biotinylation and the anti-DNA-peroxidase antibody reacted with the DNA component of the nucleosome. After washing the unbound antibody by washing with buffer, the amount of nucleosomes was quantitated and compared with the absorbance at 450 nm of the peroxidase retained in the immunocomplex.

4-2. Active oxygen ROS )

As described above, the reducing sugar, 2-deoxy-d-ribose (dRib), produces a reactive oxygen species (ROS) through autoxidation and protein glycosylation, It causes damage. In this Example, osteoblast cells were treated with 2-deoxy-d-ribose to induce damage caused by oxidative stress, and the effect of treating the culture medium of stem cells to inhibit damage and differentiation of osteoblasts was investigated .

Active oxygen was determined by the DCFH-DA (2 ', 7'-dichlorofluorescein diacetate) assay (Suh et al., Biol Pharm Bull, 2012; 35: 121-126). 2 × 10 the cell culture in the alpha -MEM containing 10% FBS in 24-well plates were dispensed at a concentration of ⁴ cells / well. Cells were washed three times with phosphate buffered saline (PBS) after 48 h incubation. After that, the stem cell culture medium (CM) and 2-deoxy-d-ribose were added to the culture medium containing 0.3% BSA and incubated for 24 hours, and the cells were washed with PBS solution. 10 mM 2 ', 7'-dichlorodehydrofluorene (DCFH) solution was dissolved in anhydrous ethanol and stored in a stock solution at -20 ° C. For the measurement of free radicals, 10 [mu] M of DCFH-DA solution was added to the culture plate and incubated at 37 [deg.] C for 20 minutes. The lipid soluble DCFH-DA is deacetylated by the esterase or oxidative hydrolysis to the non-fluorescent DCFH, and DCFH is oxidized to 2 ', 7'-dichlorofluorescein (DCF) by the active oxygen. The resulting 2 ', 7'-dichlorofluorescein (DCF) was measured for fluorescence using a Multiprex reader (Zenyth 3100, Austria) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

When osteoblast cells were treated with dRib 20 mM, the apoptosis and the production of reactive oxides were significantly increased and decreased when the stem cell culture medium (CM) was added. Figures 4A and 4B show apoptosis of osteoblasts and generation of reactive oxides, respectively.

Example  5. Dimension The osteoblast alkaline phosphatase of stem cell culture ( ALP ) Active and collagen ( collagen ) Effect on recovery ability

In this example, to ascertain whether the stem cell culture medium promotes the differentiation ability of osteoblast, the activity of alkaline phosphatase (ALP) and the ability to recover collagen production, which are indicators of bone formation promotion, were measured.

5-1. Alkaline phosphatase ( Alkaline 포스화제 , ALP ) Active measurement

Cells treated with the sample were incubated for 48 hours, washed three times with DPBS (Dulbecco's Phosphate-Buffered Saline) and collected in 1 ml of 10 mM Tris-HCl (pH 7.6) buffer containing 0.1% Triton-X And then centrifuged. To measure ALP activity, the supernatant was taken to determine the production of p-nitrophenol from p-nitrophenyl phosphate.

5-2. Collagen Sum performance  Measure

The cells were washed with DPBS and fixed with Bouin's fluid (saturated picric acid: 35% formaldehyde: glacial acetic acid = 15: 5: 1) for 1 hour. Washed and dried, and stained with Sirius Red reagent (1 mg / ml saturated picric acid) for 1 hour with shaking. After washing with 0.01 N HCl, it was dissolved in 0.2 ml of 0.1 N NaOH and the absorbance was measured at OD 550 nm.

As shown in FIGS. 5A and 5B, when osteoblast cells were treated with dRib 20 mM, the activity of ALP, which is a differentiation marker of osteoblast, and collagen production ability were significantly decreased, and the concentration-dependent Respectively.

Example  6. Effects of differentiation on osteoblast differentiation-related gene expression of stem cell cultures

In this example, it was observed whether the expression of various genes involved in osteoblast differentiation was changed when treated with the stem cell culture solution. The expression levels of the genes OPN ALP, AML, collagen, FasLR, FGF-2, osteocalcin, osteerix, TCIRG and TGF beta, which are involved in the differentiation ability of osteoblasts, were examined using the method described in Example 2 . Polymerase chain reaction was carried out using primers having the following nucleotide sequences.

Target gene Primer sequence ALP 5'-GGC CAG CTA CAC CAC AAC A-3 '(SEQ ID NO: 25)
5'-CTG AGC GTT GGT GTT ATA TGT CTT-3 '(SEQ ID NO: 26) Collagen (Col1A1) 5'-AGA CAT GTT CAG CTT TGT GGA C-3 '(SEQ ID NO: 27)
5'-GCA GCT GAC TTC AGG GAT G-3 '(SEQ ID NO: 28) FasLR (Fas ligand) 5'-ACC GGT GGT ATT TTT CAT GG-3 '(SEQ ID NO: 29)
5'-AGG CTT TGG TTG GTG AAC TC-3 '(SEQ ID NO: 30) FGF2 5'-CGG CTC TAC TGC AAG AAC G-3 '(SEQ ID NO: 31)
5'-TGC TTG GAG TTG TAG TTT GAC G-3 '(SEQ ID NO: 32) OPG 5'-ATG AAC AAG TGG CTG TGC TG-3 '(SEQ ID NO: 33)
5'-CAG TTT CTG GGT CAT AAT GCA A-3 '(SEQ ID NO: 34) OPN 5'-TGA GAT TGG CAG TGA TTT GC-3 '(SEQ ID NO: 35)
5'-ATC TGG GTG CAG GCT GTA AA-3 '(SEQ ID NO: 36) Osterix 5'-CTC CTG CAG GCA GTC CTC-3 '(SEQ ID NO: 37)
5'-GGG AAG GGT GGG TAG TCA TT-3 '(SEQ ID NO: 38) AML 5'-GCC CAG GCG TAT TTC AGA T-3 '(SEQ ID NO: 39)
5'-TGC CTG GCT CTT CTT ACT GAG-3 '(SEQ ID NO: 40) TCIRG 5'-CCA TAT CCC TTT GGC ATT GA-3 '(SEQ ID NO: 41)
5'-GAG AAA GCT CAG GTG GTT CG-3 '(SEQ ID NO: 42) TGF beta 5'-TGG AGC AAC ATG TGG AAC TC-3 '(SEQ ID NO: 43)
5'-CAG CAG CCG GTT ACC AAG-3 '(SEQ ID NO: 44)

FIG. 6 shows a significant increase in the expression level of ALP, collagen, osteocalcin, OPN, osteotelia, AML, FasL, TGF beta 1, FGF2 and TCIRG in osteoblasts when treated with stem cell culture medium (CM) Lt; / RTI >

Example  7. Detection of Growth Factors in Dimension Stem Cell Culture by Protein Array

In order to identify the growth factors present in the culture medium of the stem cell, a growth factor (growth factor) 1 kit (RayBiotech, Inc. USA) capable of measuring 43 kinds of growth factor proteins listed in Table 2 below was used. Respectively.

First, the stem cell culture solution stored at -80 ° C was thawed at room temperature. The array membrane labeled with various growth factor antibodies was allowed to stand at room temperature for 20 minutes. 2 ml of the blocking solution was added to each tray containing the antibody-labeled array membrane labeled with the growth factor, taking care not to allow air bubbles to enter, and incubated at room temperature for 30 minutes. After removing the blocking solution, 1 ml of a sample of the stem cell culture was added, followed by incubation for 2 hours with shaking at 100 rpm at room temperature. 2 ml of washing solution I was added and washed three times for 5 minutes each. It was then washed twice with Wash Solution II at room temperature. The biotin-conjugated antibody was mixed with 2 ml of blocking buffer, and 1 ml of each buffer was added to each tray, followed by incubation at room temperature for 2 hours. Washing was carried out using washing solutions I and II. HRP-conjugated streptavidin diluted 2,000-fold was added to each membrane in an amount of 2 ml, followed by incubation at room temperature for 2 hours. As described above, washing with cleaning solutions I and II was carried out again. The detection buffers C and D were mixed in the same volume and dispensed in the amount of 0.5 ml on the membrane. After 2 minutes, the film was taken out and mounted on a Kodax X-Omat AR film, left in a dark room for 2 minutes, developed and images were obtained.

Table 3 below shows the locations on the protein array film.

In Table 2, POS is a positive control, NEG is a negative control, BLANK is an empty protein, and the 42 proteins contained in the protein array are AR (androgen receptor), basic-fibroblast growth factor (b-FGF) FGF-6, FGF-7, GDSF (glial derived stimulating factor (FGF)), basic growth factor (EGF), epidermal growth factor (EGF) ), Glial derived neurotrophic factor (GDNF), granulocyte-macrophage colony stimulating factor (GM-CSF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin growth factor binding protein 1 IGF-1, insulin growth factor-1, IGF-1 SR, IGF-2, macrophage colony stimulating factor (IGFBP 2), IGFBP 3, IGFBP 4, IGFBP 5, IGFBP 5, IGFBP 6, PDGF-AA, PDGF-AB, PDGF-R, PDGF-R, PDGF-R, , PDGF-BB, PIGF (placental growth factor, SCF (stem cell factor receptor), TGF-alpha (transforming growth factor alpha), TGF-beta, TGF-beta2, TGF-beta3, vascular endothelial growth factor (VEGF) vascular endothelial growth factor receptor 2), VGFR3, and VEGF-D.

As a result of the analysis of the growth factors secreted from the stem cell supernatant using the protein array membrane, growth factors such as HGF, IGFBP-1, IGFBP-4, IGFBP-6, EGF, FGF and IGF- IGFBP-2, IGF-1, TGF-β2, TGF-β3 and VEGF were secreted in a small amount. It is considered that the growth factors observed in the supernatant of stem cells increase the viability of damaged osteoblasts and promote osteoblast function by promoting differentiation of cells.

7 shows the growth factors detected in the supernatant of the dimensional cell culture fluid detected using the protein array membrane.

<110> RHEE, SANGYOUL <120> Pharmaceutical composition for prevention or treatment of          osteoporosis comprising dental pulp stem cell conditioned medium <130> PN097312 <160> 44 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of ABCg2 <400> 1 gggttctctt cttcctgacg acc 23 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of ABCg2 <400> 2 tggttgtgag attgaccaac agacc 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Oct-4 <400> 3 gaaggatgtg gtccgagtgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Oct-4 <400> 4 gtgaagtgag ggctcccata 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD166 <400> 5 cgtctgctct tctgcctctt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD166 <400> 6 taaatactgg ggagccatcg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD105 <400> 7 cactagccag gtctcgaagg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD105 <400> 8 ctgaggacca gaagcacctc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD73 <400> 9 cgcaacaatg gcacaattac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD73 <400> 10 ctcgacactt ggtgcaaaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of SOX2 <400> 11 acaccaatcc catccacact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of SOX2 <400> 12 gcaaacttcc tgcaaagctc 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD146 <400> 13 gtctgcgcct tcttgctc 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD146 <400> 14 ttccacctcc accagctc 18 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD13 <400> 15 gggcacaatc cacacgtag 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of CD13 <400> 16 tcacggtgga taccagcac 19 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Notch-1 <400> 17 gcactgcgag gtcaacac 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Notch-1 <400> 18 aggcacttgg caccattc 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Nestin <400> 19 gtggcacaca tggagacg 18 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Nestin <400> 20 gagcgatctg gctctgtagg 20 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Nanog <400> 21 tctctcctct tccttcctcc a 21 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Nanog <400> 22 ggaagagtag aggctggggt 20 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of HPRT <400> 23 tcctcctcag accgctttt 19 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of HPRT <400> 24 cctggttcat catcgctaat c 21 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of ALP <400> 25 ggccagctac accacaaca 19 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of ALP <400> 26 ctgagcgttg gtgttatatg tctt 24 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Col1A1 <400> 27 agacatgttc agctttgtgg ac 22 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Col1A1 <400> 28 gcagctgact tcagggatg 19 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Fas ligand <400> 29 accggtggta tttttcatgg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Fas ligand <400> 30 aggctttggt tggtgaactc 20 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of FGF2 <400> 31 cggctctact gcaagaacg 19 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of FGF2 <400> 32 tgcttggagt tgtagtttga cg 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of OPG <400> 33 atgaacaagt ggctgtgctg 20 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of OPG <400> 34 cagtttctgg gtcataatgc aa 22 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of OPN <400> 35 tgagattggc agtgatttgc 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of OPN <400> 36 atctgggtgc aggctgtaaa 20 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Osterix <400> 37 ctcctgcagg cagtcctc 18 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of Osterix <400> 38 gggaagggtg ggtagtcatt 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of AML <400> 39 gcccaggcgt atttcagat 19 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of AML <400> 40 tgcctggctc ttcttactga g 21 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of TCIRG <400> 41 ccatatccct ttggcattga 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of TCIRG <400> 42 gagaaagctc aggtggttcg 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of TGFbeta <400> 43 tggagcaaca tgtggaactc 20 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplification of TGFbeta <400> 44 cagcagccgg ttaccaag 18

Claims (6)

A pharmaceutical composition for preventing or treating osteoporosis, which comprises a stem cell culture solution, wherein the stem cell culture solution is obtained by culturing a stem cell, removing the stem cell from the culture supernatant or a concentrate thereof or a lyophilized product thereof &Lt; / RTI &gt; The pharmaceutical composition according to claim 1, wherein said stem cells express CD13, CD73, CD105, CD146, CD166, ABCg2, Oct-4, Nanog, Notch-1 and Nestin genes. delete [3] The method according to claim 1 or 2, wherein the stem cell culture solution is obtained by subculturing stem cells in a serum medium, culturing in a serum-free medium, removing the stem cells from the culture, &Lt; / RTI &gt; 5. The pharmaceutical composition according to claim 4, wherein the serum medium is an alpha-MEM medium supplemented with 5 to 10% fetal bovine serum and 1% antibiotic. 5. The pharmaceutical composition according to claim 4, wherein the serum-free medium is an alpha-MEM medium.

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