KR101777590B1 - Detection markers of proliferation and therapeutic potency of adipocyte-derived stem cells cultured in media containing EGF or bFGF, and use thereof - Google Patents

Abstract

The present invention relates to a marker detection composition and a detection method for detecting the proliferative ability or therapeutic ability of adipose derived stem cells cultured in a medium containing EGF (Epidermal Growth Factor) or bFGF (basic fibroblast growth factor).

Description

EGF or bFGF, and the use of the markers for the detection and treatment of adipocyte-derived stem cells in a medium containing EGF or bFGF.

The present invention relates to a composition for detecting marker and a detection method for detecting the therapeutic or proliferative capacity of adipose-derived stem cells cultured in a medium containing EGF (Epidermal Growth Factor) or bFGF (basic fibroblast growth factor) (SMGP), CLGN (Calmegin), RGS17 (Regulator of G-Protein Signaling 17), EPSTI1 (Epithelial Stromal Interaction 1), KYNU (Kynureninase), SCARA3 (Scavenger Receptor Class A, Member 3) A marker for detecting therapeutic or proliferative ability of adipose derived stem cells cultured in a medium containing at least one EGF or bFGF selected from the group consisting of SULF2 (Sulfatase 2) and AFF3 (AF4 / FMR2 Family, Member 3) A composition for detection, and a detection method.

Adipocyte-derived stem cells (ASCs) are used as biomaterials because of the large number of stem cells that contain 1,000 times or more of stem cells compared to stem cells that can be isolated from the same amount of bone marrow. Research is being carried out variously. Fat-derived stem cells also have the ability to multiply, such as bone marrow-derived stem cells, to differentiate into cartilage, bone, fat or muscle cells. In addition, adipose derived stem cells have similar patterns of expression of bone marrow-derived stem cells and cell surface markers, and in vivo and in In vitro , it has been reported that it has a function of regulating the immune response rather than inducing an immune response even during autologous or allograft transplantation, and thus it has attracted attention as an effective means of cell therapy.

On the other hand, when the adipose-derived stem cells are cultured in vitro using the standard cell culture method using the previously described basal medium, it takes a long time to cultivate the cells and it is difficult to obtain clinically effective cell numbers. Therefore, in the case of the standard cell culture method, there is a problem in that it is not effective in actual clinical application. Accordingly, the present inventors have developed a culture method that can show an effective clinical therapeutic method. The inventors have found that the adipose-derived stem cells cultured in the proliferation medium containing EGF (Epidermal Growth Factor) or bFGF (basic fibroblast growth factor) (Korean Patent Laid-Open No. 10-2010-0118491). However, it has been confirmed that the excellent clinical effect of adipose-derived stem cells cultured in the medium containing EGF or bFGF as described above Objective molecular biologic characterization related to efficacy has not yet been established.

Particularly, in order to reproduce a stem cell treatment agent having a therapeutic effect of a certain level or higher while reproducing a stem cell cultured in a proliferation medium and representing a superior cell proliferation and therapeutic effect as compared with stem cells cultured in a basal medium, It is necessary to develop a quality control test method that can reflect the efficacy such as differentiation ability, proliferation ability and potency of stem cells obtained by the improved culture method using the growth medium such as the growth medium. In particular, in order to develop a method for increasing the yield of a cell therapy agent and measuring the quality of a therapeutic agent, a biomarker is needed to represent it.

Under these circumstances, the inventors of the present invention have found that when adipose-derived stem cells are cultured in a proliferation medium as compared with a basal medium, they are excellent in their proliferative ability, and they can not only divide and maintain the characteristics of stem cells even in long- (SMAGP) (Small Cell Adhesion Glycoprotein (SMAGP)), which is a marker for differentiation between adipose-derived stem cells cultured in a basal medium and adipogenic stem cells cultured in a growth medium, ), CLGN (Calmegin), RGS17 (Regulator of G-Protein Signaling 17), EPSTI1 (Epithelial Stromal Interaction 1), KYNU (Kynureninase) and SCARA3 (Scavenger Receptor Class A, Member 3) The expression of SULF2 (Sulfatase 2) and AFF3 (AF4 / FMR2 Family, Member 3) genes was increased in adipose derived stem cells, The present inventors have confirmed that the genes can be used for the measurement of therapeutic activity including proliferation ability, immunosuppressive ability, differentiation ability and the like of the fat derived stem cells cultured in the proliferation medium.

One object of the present invention is to provide a method of screening for small cell adhesion glycoprotein (SMAGP), CLGN (Calmegin), RGS17 (Regulator of G-Protein Signaling 17), EPSTI1 (Epithelial Stromal Interaction 1), KYNU (Kynureninase), SCARA3 , Epidermal growth factor (EGF), which comprises at least one gene mRNA selected from the group consisting of SULF2 (Sulfatase 2), AFF3 (AF4 / FMR2 Family, Member 3) Or bFGF (basic fibroblast growth factor). The present invention also provides a composition for detecting a marker for detecting the therapeutic ability of adipose-derived stem cells cultured in a medium containing bFGF (basic fibroblast growth factor).

Another object of the present invention is to provide a pharmaceutical composition containing EGF or bFGF, which comprises at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3, The present invention provides a composition for detecting a marker for detecting the proliferative capacity of adipose-derived stem cells cultured in a culture medium containing the same.

It is still another object of the present invention to provide a marker detection kit for detecting the therapeutic or proliferative ability of adipose derived stem cells cultured in a medium containing EGF or bFGF, which comprises the marker detecting composition.

It is still another object of the present invention to provide a method for the production of stem cells comprising the step of culturing the stem cells in a medium containing EGF or bFGF, wherein the stem cells have at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 And a method for detecting therapeutic or proliferative capacity of adipose-derived stem cells, comprising the step of measuring the level of protein.

In order to achieve the above object, the present invention provides a method for screening a small cell adhesion glycoprotein (SMAGP), CLGN (Calmegin), RGS17 (Regulator of G-Protein Signaling 17), EPSTI1 (Epithelial Stromal Interaction 1), KYNU ), SCARA3 (Scavenger Receptor Class A, Member 3), SULF2 (Sulfatase 2), and AFF3 (AF4 / FMR2 Family, Member 3) Which is cultured in a medium containing EGF or bFGF, which is capable of detecting the therapeutic ability of a stem cell derived from adipose.

The term "adipocyte-derived stem cell (ASC)" in the present invention refers to stem cells isolated from adipocytes capable of differentiating into most mesenchymal cells such as adipocytes, osteoblasts, chondroblasts and myofibroblasts Refers to cells called lipid precursor cells, stromal cells, multipotent adipose-derived cells or adipose derived adult stem cells. The adipose-derived stem cells may be, but are not limited to, adipose-derived stem cells derived from mammals including pigs, cows, primates, and humans that can be transplanted to humans.

In the present invention, the term "marker for detecting the therapeutic ability of adipose derived stem cells cultured in a medium containing EGF or bFGF" refers to a cell cultured in a basal medium not containing EGF or bFGF and a cell comprising EGF or bFGF Refers to an organic biomolecule which shows a significant difference in expression when compared to adipose-derived stem cells cultured in a growth medium. In addition, the adipose-derived stem cells cultured in the proliferation medium are superior to the adipose-derived stem cells cultured in the basal medium, and have excellent therapeutic ability such as immunosuppressive ability or differentiation ability (Korean Patent Laid-Open No. 10-2010-0118491). Therefore, it refers to a marker capable of detecting the therapeutic ability of adipose derived stem cells by confirming the difference in the expression level when compared with the basal medium. Specifically, SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 Derived stem cells, the expression of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 was increased in the adipose-derived stem cells cultured in the growth medium as compared with the adipose-derived stem cells cultured in the basal medium, and SULF2 And AFF3 are genes whose expression is decreased.

The genes used in the present invention can be obtained from known databases such as GenBank of the National Institutes of Health, such as SMAGP (NM_001033873.1, NP_001029045.1), CLGN (NM_001130675.1, NP_001124147.1), RGS17 NM_012419.4, NP_036551.3), EPSTI1 (NM_033255.2, NP_001002264.1), KYNU (NM_003937.2, NP_001028170.1), SCARA3 (NM_182826.1, NP_057324.2), SULF2 (NM_018837.3, NP_061325. 1) and AFF3 (NM_001025108.1, NP_001020279.1).

When the adipose-derived stem cells cultured in the basal medium and the adipose-derived stem cells cultured in the proliferation medium were compared with each other, the above genes showed different expression in the medium. Therefore, based on the difference in expression, Can be used as markers for the detection of the therapeutic potential of the cells.

The term " EGF (epidermal growth factor) " in the present invention means a growth factor capable of promoting cell proliferation, growth and differentiation by binding to its receptor EGFRdp. The EGF has an activity of promoting the proliferation of various epithelial cells, and can also proliferate mouse T cells or human fibroblasts. For the purpose of the present invention, the EGF refers to a protein that is involved in the culture medium of adipose-derived stem cells and has a role of enhancing therapeutic activity such as its proliferation and differentiation ability.

In the present invention, the term " basic fibroblast growth factor (bFGF) " is a growth factor involved in biological processes such as angiogenesis or wound healing. For the purpose of the present invention, the bFGF refers to a protein such as EGF, which is contained in the medium of adipose-derived stem cells and functions to elevate therapeutic activity such as its proliferation and differentiation ability.

In the present invention, the term "basal medium" is a medium for culturing adipose-derived stem cells, particularly a stroma-vascular fraction containing the same. The basal medium may be a medium containing serum in DMEM medium or DMEM / F12 for culturing adipose stem cells. The serum may be FBS (Fetal bovine serum) or the like commonly used for cell culture, About 10%, that is, about 8 to 12%, but it is not particularly limited as long as it is an adipogenic stem cell culture medium commonly used in the art. In one embodiment of the present invention, DMEM medium containing 10% FBS was used as a base medium. In the present invention, the " basal medium " may be mixed with " basic medium ".

In the present invention, the term " proliferation medium " refers to a medium used for improving the proliferative or differentiating ability of adipose derived stem cells. The medium is a medium supplemented with basic fibroblast growth factor (bFGF) or epidermal growth factor . The growth medium may contain EGF or bFGF at a concentration of 0.1 ng / ml to 100 ng / ml, respectively. In the present invention, the " proliferation medium " may be used in combination with " medium containing EGF or bFGF ".

Information on the base medium and the growth medium is disclosed in Korean Patent Laid-Open No. 10-2010-0118491, and the entire contents of the above-mentioned 10-2010-0118491 can be included as a reference material of the present invention, Do not.

The term " therapeutic ability " in the present invention means the therapeutic activity of stem cells, and is also referred to as " potency " when the stem cells of the present invention are used as a cell therapy agent or the like. Refers to both, but not limited to, the proliferative, differentiating, immunomodulating, or ability of adipose-derived stem cells.

The term "differentiation ability" in the present invention means the ability of the adipose derived stem cells to differentiate into adipocytes, osteoblasts, chondroblasts, myofibroblasts, osteoblasts, muscle cells or neurons. In the present invention, the adipose-derived stem cell having high differentiation ability means a cell in which induction of differentiation into adipocytes, osteoblasts, chondroblasts, myofibroblasts, osteoblasts, muscle cells or neurons is facilitated, but is not limited thereto.

In the present invention, " immunomodulating ability " is preferably an immunosuppressive ability, and the immunosuppressive ability means that adipose derived stem cells cultured in a medium containing bFGF or EGF can inhibit the activity of immune cells . In particular, mesenchymal stem cells are known to be involved in immunosuppressive effect by inhibiting APC (antigen presenting cell). Therefore, the immune diseases can be treated by using the adipose-derived stem cells of the present invention as immunosuppressants.

In the present invention, " adipose-derived stem cells having a high therapeutic potential " means that the expression level of at least one gene selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 is higher than that of adipose- derived stem cells cultured in the basal medium Derived stem cells in which the expression of one or more genes selected from the group consisting of SULF2 and AFF3 is reduced and which is superior to the fat-derived stem cells cultured in the basal medium as a result of faster cell proliferation, Derived stem cells having high activity (potency) as a therapeutic agent for high back pain.

The expression level of such a gene can be confirmed by measuring the amount of mRNA or protein.

In the present invention, the term " mRNA level measurement " refers to a process of confirming the presence and expression level of mRNAs of marker genes showing the therapeutic ability of adipose-derived stem cells in a biological sample in order to diagnose the differentiation ability of adipose- Is measured. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) , Northern blotting, or DNA chips, but are not limited thereto. In one embodiment of the present invention, the level of the gene mRNA was confirmed by RT-PCR and real-time PCR (Example 3).

The nucleic acid sequences of the genes SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 are NM_001033873.1, NM_001130675.1 and NM_012419.4, respectively, which are preferably primer pairs or probes, , NM_033255.2, NM_003937.2, NM_182826.1, NM_018837.3, NP_061325.1 and NM_001025108.1, a person skilled in the art will be able to identify primers or probes which specifically amplify a specific region of these genes based on the sequence You can design.

The term "primer " as used herein refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, a short nucleic acid sequence capable of forming base pairs with a complementary template and serving as a starting point for template strand copy . In the present invention, the primer is preferably a primer pair of SEQ ID NOS: 3 and 4 for SMAGP, a pair of primers of SEQ ID NOS: 5 and 6 for primers for CLGN, a pair of primers of SEQ ID NOS: 7 and 8 for primers for RGS17, The primers for EPSTI1 are the primer pairs of SEQ ID NOS: 9 and 10, the primers for KYNU are the primer pairs of SEQ ID NOs: 11 and 12, the primers for SCARA3 are the primer pairs of SEQ ID NOs: 13 and 14, the primer for SULF2 is SEQ ID NO: And 16, or the primer for AFF3 may be a primer pair of SEQ ID NOS: 17 and 18, but is not limited thereto.

 The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few bases or several hundreds of bases which can specifically bind to mRNA, and is labeled so that presence or absence of a specific mRNA Can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization is performed using probes complementary to SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 polynucleotides, and the ability to differentiate adipose-derived stem cells can be determined through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The primers of the present invention can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences as a primer specific to each marker gene. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid, The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.

The term " measurement of protein level " in the present invention refers to a process for determining the presence and expression level of a protein expressed from a marker gene showing the therapeutic ability of adipose-derived stem cells in a biological sample in order to detect the differentiation ability of adipose- The amount of the protein can be confirmed by using an antibody that specifically binds to the protein of the gene.

As used herein, the term " antibody " refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and more specifically, a protein encoded by the marker genes SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 of the present invention Quot; antibody " includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the marker protein of the present invention has been identified, the production of an antibody using the marker protein can be easily performed using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injecting the marker protein antigen into an animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows and dogs. Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: Biol., 222: 58, 1-597, 1991) techniques, and the like. The antibody prepared by the above method can be isolated and purified by a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography or affinity chromatography. The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv. Methods for measuring protein levels include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip are examples of the immunoassay method, , But is not limited thereto. In one embodiment of the present invention, the level of the protein was measured using Western blot (Example 4).

In one embodiment of the present invention, the adipose-derived stem cells cultured in the medium containing EGF or bFGF are more effective than the adipose-derived stem cells cultured in the basal medium containing no factor, such as proliferation ability, immunosuppression, Based on the excellent activity (Korean Patent Laid-Open No. 10-2010-0118491), there was a significant difference in expression between adipose-derived stem cells cultured in medium containing EGF or bFGF and adipose-derived stem cells cultured in the basal medium As a result, the expression of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 was increased and the expression of SULF2 and AFF3 was decreased in the fat-derived stem cells cultured in the medium containing EGF or bFGF (Figs. 3 to 5). In addition, the overexpression of EPSTI1, KYNU and SCARA3 genes in adipose-derived stem cells revealed that the adipocyte differentiation was much better than that in the control group. From these results, it is suggested that adipogenic stem cells overexpressing the gene have excellent therapeutic ability such as differentiation ability (FIG. 6). Taken together, this suggests that high expression levels of SMAGP, CLGN, RGS17, EPSTI1, KYNU, and SCARA3 proteins or low expression levels of SULF2 and AFF3 proteins may be markers for the therapeutic activity of adipose-derived stem cells .

In another aspect, the present invention provides a pharmaceutical composition comprising EGF or bFGF, which comprises at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3, The present invention provides a composition for detecting a marker for detecting the proliferative capacity of adipose-derived stem cells cultured in a culture medium containing the same.

The measurement and genes of the fat-derived stem cell, EGF, bFGF, mRNA or its protein level are as described above.

In the present invention, the term "proliferative ability" refers to the ability of a cell to autonomously or to increase the number of cells accompanied by DNA synthesis or cell division by external stimulation such as hormone, This includes cases where the potential is maintained. Stem cells in In vitro cultivation has self-renewal capacity, which has proliferative capacity. However, as the subculture progresses, the proliferative capacity decreases, resulting in a limit to obtain a pure and large amount of stem cells. Therefore, in order to effectively utilize stem cells as therapeutic agents, a culture method capable of obtaining a large amount of stem cells in a short period of time is needed.

In the present invention, it has been confirmed that adipose-derived stem cells cultured in a medium containing EGF or bFGF are superior in proliferative capacity to adipose derived stem cells cultured in a basal medium, and a basal medium or a proliferation medium Derived stem cells were identified.

Specifically, the expression of at least one gene selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 in the adipose-derived stem cells cultured in the proliferation medium is increased as compared with the adipose-derived stem cells cultured in the basal medium, The expression of one or more genes selected from the group consisting of SULF2 and AFF3 is decreased as compared with the fat-derived stem cells cultured in the basal medium. The adipose-derived stem cells showing the above-described expression characteristics have a decreased expression of adipose stem cells cultured in the basal medium, particularly SMAGP, CLGN, RGS17, EPSTI1, KYNU, or SCARA3 gene, or expression of SULF2 or AFF3 gene In addition, the cell proliferation is higher than that of the fat-derived stem cells, and thus the cell yield is high, and the clinical therapeutic effect such as cell proliferation, immunosuppression and differentiation ability is high.

In one embodiment of the present invention, when the same amount of adipose-derived stem cells were grown for 5 passages in the basal medium and the proliferation medium, it was confirmed that the cell proliferation in the proliferation medium was increased more than 2 times on average (Example 1 and Fig. 2) .

In another aspect, the present invention provides a method for detecting a marker for detecting the differentiation ability of adipose stem cells cultured in a medium containing EGF or bFGF, which comprises culturing the medium containing EGF or bFGF in a medium containing EGF or bFGF A kit for detecting a differentiation ability of stem cells derived from a subject is provided.

The composition for detecting markers for detecting the differentiation ability of adipose derived stem cells cultured in a medium containing EGF or bFGF is as described above.

The marker detection kit of the present invention may be used for immunological analysis as well as a primer or an antibody capable of selectively recognizing a marker gene whose expression is increased or decreased as a result of culturing in a growth medium containing EGF or bFGF Tools and reagents commonly used in the art, and the like. Such tools and reagents include, but are not limited to, suitable carriers, labeling agents capable of generating a detectable signal, solubilizers, cleaning agents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring the activity of the enzyme and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

The marker detection kit of the present invention can be preferably an RT-PCR kit, a DNA kit or a protein chip kit. When an antibody against the protein encoded by the gene is provided on the protein chip, the antigen-antibody complex formation for two or more antibodies can be observed, which is more advantageous in detecting the differentiation ability of adipose derived stem cells.

 The RT-PCR kit may comprise a respective pair of primers specific for the marker gene, and may include other test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq Enzymes such as polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.

The DNA chip kit may include a substrate on which a cDNA or an oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. The substrate may be a control gene Or cDNA or oligonucleotide corresponding to the fragment thereof.

The protein chip kit may be a kit in which one or more antibodies against a marker are arranged at predetermined positions on a substrate and fixed at high density. In the method using a protein chip, a protein is separated from a sample, and a separated protein is hybridized with a protein chip to form an antigen-antibody complex, and the presence or the expression level of the protein can be confirmed by reading it.

In one embodiment of the present invention, the expression of marker genes of adipose-derived stem cells cultured in a basal medium and a proliferation medium was confirmed using a microarray (Example 2 and Fig. 3).

In another aspect, the present invention relates to a method for detecting a proliferative capacity of an adipose-derived stem cell cultured in a culture medium containing EGF or bFGF, which comprises culturing a medium containing EGF or bFGF A kit for detecting a marker for detecting the proliferative capacity of stem cells derived from the subject is provided.

The composition for detecting a marker for detecting the proliferative capacity of adipose derived stem cells cultured in the medium containing EGF or bFGF and the kit for detecting a marker which can be used are as described above.

In another embodiment, the present invention relates to a method for producing at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 of fat derived stem cells cultured in a medium containing EGF or bFGF Or measuring the protein level thereof. The present invention also provides a method for detecting the differentiation ability of adipose derived stem cells.

The medium containing the EGF or bFGF, the mRNA of the gene or the protein level thereof and the differentiation ability of adipose stem cells have been described above.

In addition, the method may further comprise determining the amount of at least one gene mRNA or protein thereof selected from the group consisting of SULF2 and AFF3 of adipose derived stem cells cultured in a medium containing EGF or bFGF, A step of determining that the differentiation ability is higher than that of the control group when the value is smaller than the value measured in stem cells, or the step of determining the amount of one or more gene mRNA or protein thereof selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 Derived stem cells including the step of determining that the differentiation ability is higher than that of the control, when the number of the stem cells is greater than the value measured in the adipose-derived stem cells cultured in the basal medium as the control group. At this time, it is preferable that the adipose-derived stem cells cultured in the basal medium as the control group and the adipose-derived stem cells cultured in the proliferation medium have the same or less than one passage number.

In one embodiment of the present invention, the adipose-derived stem cells cultured in the medium containing EGF or bFGF are superior to the adipose-derived stem cells cultured in the basal medium lacking the factors, such as proliferation and differentiation ability (Korean Patent Laid-Open No. 10-2010-0118491), a gene showing significant differences in expression between adipose-derived stem cells cultured in a medium containing EGF or bFGF and adipose derived stem cells cultured in a basal medium was identified As a result, the expression of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 was increased in adipose derived stem cells cultured in the medium containing EGF or bFGF, compared with the adipose derived stem cells cultured in the basal medium, and SULF2 and AFF3 (Fig. 4 and Fig. 5). Thus, the expression of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3, or decreased expression of SULF2 and AFF3 in adipose-derived stem cells, decreased expression of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 Suggesting that the differentiation ability of SULF2 and AFF3 may be superior to that of adipose derived stem cells.

In another embodiment, the present invention relates to a method for producing at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 of fat derived stem cells cultured in a medium containing EGF or bFGF Or measuring the level of the protein of the stem cell. The present invention also provides a method for detecting the proliferative capacity of adipose-derived stem cells.

The medium containing the EGF or bFGF, the mRNA of the gene or the protein level thereof and the proliferative capacity of the adipose-derived stem cell have been described above.

Preferably, the amount of at least one gene mRNA or protein thereof selected from the group consisting of SULF2 and AFF3 of the adipose-derived stem cells cultured in the medium containing EGF or bFGF is not less than that of the adipose-derived stem cells The amount of at least one gene mRNA or protein thereof selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 is determined to be higher than that of the control group, Derived stem cells, the method further comprising the step of determining that the proliferative capacity of the stem cells is higher than that of the control, when the amount of the stem cells is higher than the value measured in the adipose-derived stem cells cultured in the basal medium.

The present invention can provide more useful data in verifying the efficacy of adipose derived stem cells by providing markers that can be used to determine the increased clinical efficacy of adipose derived stem cells cultured in the proliferation medium. And can be usefully used as a marker for detecting the therapeutic activity represented by the proliferative ability and differentiation ability of adipose-derived stem cells cultured using the improved culture method.

Fig. 1 is a diagram showing the cell shape of adipose-derived stem cells in the basal medium and the proliferation medium.
FIG. 2 is a graph showing the difference in cell growth of adipose derived stem cells in the basal medium and the proliferation medium. FIG.
FIG. 3 shows microarray results of adipocyte stem cells cultured in three sets of basal medium using Illumina 24K chip and in growth medium. FIG.
FIG. 4 shows RT-PCR and real-time PCR of changes in the expression level of genes that are increased in the proliferating medium selected from FIG. 3 in adipose-derived stem cells from 17 donors, and RPL13A is a control gene . Fig. 4A shows the results of SMAGP, Fig. 4B shows the result of increasing SMAGP, Fig. 4B shows CLGN, Fig. 4C shows RGS17, Fig. 4D shows EPSTI1, Fig. 4E shows KYNU, Fig. 4F shows SCARA3, Fig. 4G shows SULF2, The results are shown.
FIG. 5 is a graph showing the differences in protein expression between the basal medium and the growth medium of CLGN, KYNU, SCARA3 and SULF, which are typical genes of the marker of the present invention.
FIG. 6 shows the results of confirming the proliferative effect and the differentiation ability of adipose-derived stem cells caused by overexpression of the gene. FIG. 6A shows the results of the cloning of EPSTI1, KYNU and SCARA3 genes and the expression of cloned genes in the proliferation medium. 6B is a graph showing cell proliferation improvement in the basal medium of adipose stem cells overexpressing the gene. FIG. 6c is a result of examining the degree of differentiation into adipocytes after oil cell culturing in a differentiation medium by Oil Red O staining. The overexpression of the gene indicates that the cell proliferation and differentiation ability are improved.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example  One: Base badge On the growth medium  Of cell proliferation of adipose-derived stem cells

Example  1-1: derived from human fat Stroma  Culture of stem cells

Adipose tissue was isolated from the donor (Anthrozen, Gyeonggi-do, Korea) and the adipose-derived stromal stem cells were isolated from the obtained adipose tissue. Adipose tissue was washed 34 times with the same volume of Krebs-Ringer Bicarbonate (KRB) solution to remove blood. A volume of collagenase solution such as adipose tissue was added and reacted at 37 ° C in a water bath. This was transferred to a centrifuge tube and centrifuged at 1200 rpm for 10 minutes at 20 ° C. The upper layer of fat layer was removed and the lower layer of collagenase solution was carefully removed to avoid shaking. After suspending in the basal medium, centrifugation was carried out at 20 DEG C and 1200 rpm for 5 minutes. At this time, the supernatant was removed because it was the stroma-vascular fraction that subsided below. The stroma-vascular fraction was suspended in a basal medium, inoculated into a culture vessel, and cultured in a 5% CO ₂ incubator at 37 캜 for 24 hours. After removal of the culture medium, the cells were washed with phosphate buffer solution, and the basal medium or medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml in the basal medium or 5 ng of epidermal growth factor (EGF) / Ml. ≪ / RTI > When the adipose-derived stromal stem cells were grown to about 80-90% of the culture vessel, trypsin was treated and isolated into single cells. The obtained cells were diluted 1: 31: 4 with the growth medium and subcultured (Korean Patent Publication No. 10-2010-0118491). In order to analyze the difference in gene expression according to the composition of the culture medium, 5 subcellular cells cultured in a basal medium of DMEM containing 10% FBS and 5 subcellular cells cultured in a growth medium containing 1 ng / ml bFGF in the basal medium Respectively.

As a result, the growth rate was higher and the fibroblast morphology was stably maintained when cultured in the growth medium. Fig. 1 shows morphological photographs of adipose-derived stem cells cultured in the basal medium and the proliferation medium. FIG. 2 shows an example of the CPDL (cell population doubling level) of the adipose-derived stem cells cultured in the basal medium and the proliferation medium.

As shown in Fig. 2, adipocyte-derived stem cells cultured in the proliferation medium were higher in cell growth than those cultured in the basal medium.

Example  2: Identification of gene expression changes in adipose-derived stem cells by microarray

Example  2-1: derived from human fat Stroma  Culture of stem cells

Adipose tissue was isolated from 17 donors (Antrozen, Gyeonggi-do, Korea) and adipose-derived stem cells were cultured as in Example 1.

Example  2-2: Total RNA  detach

Total RNA was isolated using a QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantitated using an Experion RNA StdSens (Bio-Rad) chip. First, the cultured adipose-derived stem cells were dissolved in a 15 ml kit of digestion buffer supplemented with 150 μl of beta mercaptoethanol. 15 ml of 70% ethanol was added thereto, followed by centrifugation at 3000 g for 5 minutes to adhere the total RNA to the membrane. After two washes, total RNA was isolated by adding 1.2 ml of RNase-free water.

Example  2-3: Microarray  practice

The extracted total RNA was hybridized using Illumina TotalPrep RNA Amplification Kit (Ambion). T7 Oligo (dT) to synthesize cDNA using a primer, and using the biotin-UTP in performing vitro transcription (in vitro transcription) to prepare a biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. The cRNA produced in the adipose-derived stem cells was hybridized with Human-6 V2 (Illumina) chip. After hybridization, the DNA chip was washed with Illumina Gene Expression System washing solution (Illumina) to remove non-specific hybridization, and the washed DNA chip was labeled with streptavidin-Cy3 (Amersham) fluorescent dye. The fluorescence-labeled DNA chip was scanned using a confocal laser scanner (Illumina) to obtain fluorescence data in each spot and stored as a TIFF image file. TIFF image file was quantified with BeadStudio version 3 (Illumina), and the fluorescence value of each spot was quantified. The quantified results were calibrated using 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.

The results are shown in Fig. FIG. 3 shows the difference in expression of the above genes measured in 5 cells cultured in the basal medium and 5 cells cultured in the growth medium. As shown in FIG. 3, SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 genes were significantly increased in the proliferation medium compared to the basal medium, and SULF2 and AFF3 were decreased in the proliferation medium compared with the basal medium It looked. From these results, it is suggested that the genes can be used as an application for judging adipose stem cells cultured in a proliferation medium with high clinical efficacy, since the expression levels of the genes are greatly different when the basal medium and the proliferation medium are compared. However, these results may be different from known markers that differentiate stem cells from differentiated stem cells, and may show different patterns depending on the passage number and differentiation.

Example  3: From individual donor's fat-derived stem cells Base badge On the growth medium  Identification of individual genes with differential expression

The expression levels of the genes identified in Example 2 were analyzed by RT-PCR using 17 pairs of donor adipose stem cell samples (basal medium and proliferation medium culture cells). Total RNA was isolated through the method of Example 2. [

Example  3-1: cDNA  Synthesis and Template Correction

2 μg of total RNA of each sample, 1 μl of 50M Oligo (dT) primer and 2.5 μl of 10 mM dNTP were added and 25 μl of the solution was added with sterilized water containing DEPC as an RNase inhibitor to prepare an RNA / primer mixture solution . The reaction was carried out at 65 ° C for 5 minutes, then transferred to 55 ° C and stored. Next, add 5 μl of 10 × RT buffer, 10 μl of 25 mM MgCl ₂ , 5 μl of 0.1 M DTT, 1 μl of RNase inhibitor and 1 μl of SuperScript III RT enzyme to make the total volume 25 μl, , And the mixture was reacted at 55 DEG C for 50 minutes. The reaction was then terminated at 85 ° C for 5 min to inactivate the RT enzyme and place in ice. RPL13A was used as a standard gene for quantifying marker genes. RT-PCR was performed using a primer of the standard gene and the concentration of the cDNA was corrected so that the expression level of the standard gene RPL13A became equal. First, each cDNA was diluted 20-fold and then 2 μl of the diluted sample was used for PCR reaction. PCR was carried out using 15 μl of 2x PCR premix (Hot start), 2 μl of PRL13A forward primer, 2 μl of PRL13A reverse primer, and 11 μl of distilled water, followed by 20 cycles, 23 cycles, and 25 cycles. RT-PCR was performed at 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute. The RPL13A primer used is forward 5'-CATCGTGGCTAAACAGGTACTG-3 '(SEQ ID NO: 1), reverse 5'-GCACGACCTTGAGGGCAGC-3' (SEQ ID NO: 2). The PCR product was loaded on 2% agarose gel, electrophoresed, and then the gel was photographed. The image was quantified with TotalLab v1.0 program (Nonlinear Dynamix) and then corrected again to perform PCR. Was corrected to be the same.

Example  3-2: RT - PCR / Real - Time PCR  Analysis of expression level using

PCR was performed using the sense and antisense primers of the above-mentioned cDNAs diluted to the same amount. The PCR reaction was performed at 94 ° C for 1 minute, 54 ° C for 30 seconds, and 72 ° C for 1 minute. The PCR reaction was carried out at 94 ° C for 1 minute, 54 ° C for 30 seconds, and 2 minutes at 72 ° C. The cycle was different for each gene. PCR products were analyzed by electrophoresis using 2% agarose gel and image analysis. Realtime RT-PCR was performed with DNASYBRI reagent from Qiagen (CA, USA) and LightCycler (Roche). Melt curve analysis was used to assess the quality of PCR products and gene expression was quantified using LightCycler version 3.5 software (Roche). The specific primers of the genes used in the RT-PCR / Real-Time PCR and the sequences of the control primers (SEQ ID NOS: 1 to 18) are shown in Table 1 and PCR results are shown in FIG.

As a result, it was confirmed that the above genes were significantly increased or decreased in the proliferation medium compared to the basal medium: SMAGP (76.4%, increased in 13 out of 17), CLGN (100%, 16 out of 16 ), RGS17 (100%, increased in 17 of 17 samples), ESPTI1 (82.3%, increased in 14 of 17 samples), KYNU (100%, increased in 16 of 16 samples), SCARA3 94.1%, increase in 16 of 17 samples), SULF2 (93.75%, decrease in 15 of 16 samples), AFF3 (94.1%, decrease in 16 of 17 samples) (Fig.

These results suggest that the genes may be used as markers for judging fat-derived stem cells having increased clinical efficacy cultured in a propagation medium or as markers for detecting the consistency of quality of adipose-derived stem cells cultured using an improved culture method As a result.

gene( REF ) primer SEQ ID NO: RPL13A (NM_012423.02) F: 5'-catcgtggctaaacaggtactg-3 ' SEQ ID NO: 1 R: 5'-gcacgaccttgagggcagc-3 ' SEQ ID NO: 2 SMAGP (NM_001033873.1) F: 5'-acagcactcattgcagttgt-3 ' SEQ ID NO: 3 R: 5'-actgggctcaccttctgtag-3 ' SEQ ID NO: 4 CLGN (NM_001130675.1) F: 5'-aagtgaacctgcccaaatag-3 ' SEQ ID NO: 5 R: 5'-atccgtgtcttcattccagt-3 ' SEQ ID NO: 6 RGS17 (NM_012419.4) F: 5'-catgtatgaagatgcccaac-3 ' SEQ ID NO: 7 R: 5'-gccagcagtactttcaacaa-3 ' SEQ ID NO: 8 EPSTI1 (NM_033255.2) F: 5'-agagcaagaaagagccaaaa-3 ' SEQ ID NO: 9 R: 5'-atattccaacagcctccaga-3 ' SEQ ID NO: 10 KYNU (NM_003937.2) F: 5'-catcacaaaagctggacaag-3 ' SEQ ID NO: 11 R: 5'-aatcaactccccagtcatgt-3 ' SEQ ID NO: 12 SCARA3 (NM_182826.1) F: 5'-agtgcaccagatcaacttca-3 ' SEQ ID NO: 13 R: 5'-cgtgtgtagttctgccagtc-3 ' SEQ ID NO: 14 SULF2 (NM_018837.3) F: 5'-tgatgaatgcagtgaacaca-3 ' SEQ ID NO: 15 R: 5'-tttaagtcccaggtccatgt-3 ' SEQ ID NO: 16 AFF3 (NM_001025108.1) F: 5'-ggcagaaacttgtcttcgat-3 ' SEQ ID NO: 17 R: 5'-cactcgataaacgacaatgc-3 ' SEQ ID NO: 18

Example  4: Investigation of protein expression according to the passage of selected genes

The amount of expression of some of the genes identified in Example 3 was analyzed by Western blot analysis using protein quantification using donor fat stem cell samples (culture in basal medium and proliferation medium).

Specifically, cells grown in a 60 mm dish were rinsed once with PBS, and then washed with cold protein lysis buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10 % Glycerol, 1 mM PMSF, 1 mM DTT, 20 mM NaF, 1 mM EDTA, protease inhibitor) was added to prepare cell proteins. Proteins were separated by size on a 10% or 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel using 30 μg of each prepared protein sample, and transferred to PVDF membrane.

The filter on which the protein was transferred was cut to an appropriate size, and then an antibody corresponding to each protein was attached. Anti-SILF2 (R & D, MAB7087), anti-SCARA3 (abcam, ab96205) -ACTIN (Sigma, A5316).

The amount of each protein was determined in a band using Immobilon (TM) Western blotting detection reagents (Millipore).

As a result, in the case of CLGN, KYNU and SCARA3 genes, in which the mRNA levels in the growth medium were increased by RT-PCR, the protein expression in the basal medium was low and the protein expression was high in the growth medium, SULF2, a gene whose mRNA level decreased with increasing passage in the growth medium, showed high protein expression in the basal medium and decreased protein expression in the growth medium, confirming that it matched the mRNA test result similarly 5).

Example  5: Investigation of cell proliferation and differentiation enhancing effect of adipose-derived stem cells by overexpression of selected genes

Example  5-1: Genes Cloning

The production process of vectors expressing EPSTI1, KYNU and SCARA3 is shown in Fig. 6-1. The EPSTI1 gene was amplified using c-DNA of adipose derived stem cells and primers of SEQ ID NOS: 19 and 20, which are the primer pairs shown in Table 2. The KYNU gene was primers of SEQ ID NOs: 21 and 22, and the SCARA3 gene was primer Amplified using primers of SEQ ID NOS: 23 and 24, and then cleaved with restriction enzymes BglII and HindIII, followed by pure separation. To 20 to 50 ng of each fragment prepared by digesting pDsRed-N1 (Clontech co.), Which is capable of tagging RFP (Red fluorescence protein), with restriction enzymes BglII and HindIII and CIP, One gene fragment and one unit of T4 DNA ligase were mixed and reacted at 16 ° C for one day. 2 ~ 5 ㎕ of the reaction solution was transformed into the bacterial strain DB3.1 (Frozen Stock Method using RbCl). In order to select the transformed strains, the plasmids were purified on an LB solid medium containing antibiotics and cultured in a 37 ° C incubator for one day. The plasmids were purified using a plasmid purification kit (Solgent). The structure was confirmed by digestion with a detectable restriction enzyme. As a result, a vector expressing the RFP-tagged gene was prepared.

gene( REF ) primer SEQ ID NO: EPSTI1
(NM_033255.2) F: 5'-atagatctatgaacacccgcaatagagtggt-3 ' SEQ ID NO: 19 R: 5'-ataagctttataccccagaggttaccgctat-3 ' SEQ ID NO: 20 KYNU
(NM_003937.2) F: 5'-gaaagcttatggagccttcatctcttgagct-3 ' SEQ ID NO: 21 R: 5'-ataagcttattttttgtttctgcagagtcaag-3 ' SEQ ID NO: 22 SCARA3
(NM_182826.1) F: 5'-gaagatctatgaaagtgaggtcggccggcg-3 ' SEQ ID NO: 23 R: 5'-atagatcttatccgattatgtgaaaacaaag-3 ' SEQ ID NO: 24

Example  5-2: Confirmation of gene expression

Fat-derived stem cells were transiently transfected using the lipofectamine plus method to verify that the engineered vectors normally expressed protein.

Specifically, a day before transfection, adipose-derived stem cells were seeded into a basal medium using a 60 mm dish, and for transfection of one petri dish, 2 μg DNA was added to 4 μl plus reagents (Life Technologies ), Mixed and incubated at room temperature for 15 minutes (solution A). Six microliters of the lipofectamine reagent was added to 150 microliters of serum-free basal medium (solution B). Solutions A and B were combined, gently mixed and incubated at room temperature for 15 minutes to form a complex. After 15 minutes, 900 占 퐇 of serum-free basal medium was added to the mixture. The cells were washed once with serum-free basal medium and DNA-lipofectamine plus complex (final volume 1200 μl) was added to the cells and incubated at 37 ° C for 5 hours in a 5% CO ₂ incubator. Subsequently, the basal medium was removed and serum-containing basal medium was added to the cells and cultured in an incubator. After 48 hours, protein expression was confirmed by fluorescence microscopy using RFP fluorescence (Fig. 6A).

Example  5-3: By gene overexpression Increased  Identification of cell proliferation effect of protein

Transformed adipose-derived stem cells were seeded and adhered to the outer wells of a Transwell plate, and the expression vector cloned in Example 5-1 was transformed into an inner well. After the stem cells were added, the effect of promoting the cell proliferation of the overexpressed protein was confirmed in the transformed cells. After the transformed cells were cultured for 2 weeks, the cell shape was photographed with a microscope. Then, the cells were separated by treatment with trypsin / EDTA solution, and the number of cells was observed using a cell counting machine to observe the change in cell proliferation (Fig. 6B).

As a result, as shown in FIG. 6B, the fat-derived stem cells overexpressing the EPSTI1, KYNU and SCARA3 genes, which are representative genes of the present invention, showed superior proliferation ability as compared with the control group.

Example  5-4: By gene overexpression Increased  Verification of the enhancement effect of differentiation ability of protein

The adipose stem cells were suspended in the proliferation medium, each 40,000 cells were inoculated into a 24-well culture vessel, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. The expression vector was transformed by the method described in Example 5-2, cultured in a basal medium for 2-3 days in a 5% CO ₂ incubator at 37 ° C, changed to a differentiation medium, and cultured for 5 days. After that, the differentiation medium was removed and replaced with differentiation maintenance medium (adipocyte media) and cultured for 12 days while changing to a new differentiation maintenance medium every 3 days. The basal medium was DMEM / F12, 3% FBS, 33 μM biotin, 17 μM pantothenate, 1 μM insulin, 1 μM insulin, 1 μM insulin, DMEM / F12, 3% bovine serum, 33 [mu] M biotin, 17 [mu] M pantothenate, 100 nM insulin, and 1 [mu] M insulin and 100 [mu] M indomethacin, And dexamethasone.

Oil-red O staining was performed to morphologically identify the differentiation of adipocytes from adipocyte stem cells. The differentiated cells were rinsed with PBS solution (phosphate buffered saline) and fixed with 10% formalin. Fixed for 12 hours and then reacted with 60% isopropanol for 20 minutes and stained with 0.5% Oil Red O solution (SIGMA O0625) for 1 to 3 hours at room temperature. After staining, the cells were washed once with 60% isopropanol, and three times with distilled water, and then dried. Then, the proportion of cells having adipogenesis on the microscope was measured. Then, 100% isopropanol was added to elute Oil Red O dye from the stained fat droplets and absorbance was measured at 520 nm. FIG. 6c shows a microscope photograph of cells stained with Oil-red O and the difference in absorbance (OD ₅₂₀ ) between Oil-red O stain.

As a result, it was confirmed that the differentiation potential of adipose-derived stem cells overexpressing EPSTI1, KYNU and SCARA3 genes of the present invention was more favorable than that of adipose derived stem cells transformed with a control vector .

Therefore, these genes may be used as markers for predicting the future differentiation potential of adipose derived stem cells, and it is proved that they will be involved in the regulation of fat stem cell senescence and regulation of differentiation.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, as well as all changes or modifications derived from the meaning and scope of the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology          ANTEROGEN CO., LTD. <120> Detection markers of proliferation and therapeutic potency of          adipocyte-derived stem cells cultured in media containing EGF or          bFGF, and use thereof <130> PA130675 / KR <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RPL13A-F <400> 1 catcgtggct aaacaggtac tg 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RPL13A-R <400> 2 gcacgacctt gagggcagc 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAGP-F <400> 3 acagcactca ttgcagttgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAGP-R <400> 4 actgggctca ccttctgtag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CLGN-F <400> 5 aagtgaacct gcccaaatag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CLGN-R <400> 6 atccgtgtct tcattccagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RGS17-F <400> 7 catgtatgaa gatgcccaac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RGS17-R <400> 8 gccagcagta ctttcaacaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-F <400> 9 agagcaagaa agagccaaaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-R <400> 10 atattccaac agcctccaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KYNU-F <400> 11 catcacaaaa gctggacaag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KYNU-R <400> 12 aatcaactcc ccagtcatgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-F <400> 13 agtgcaccag atcaacttca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-R <400> 14 cgtgtgtagt tctgccagtc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SULF2-F <400> 15 tgatgaatgc agtgaacaca 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SULF2-R <400> 16 tttaagtccc aggtccatgt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AFF3-F <400> 17 ggcagaaact tgtcttcgat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AFF3-R <400> 18 cactcgataa acgacaatgc 20 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-F <400> 19 atagatctat gaacacccgc aatagagtgg t 31 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-R <400> 20 ataagcttta taccccagct gttaccgcta t 31 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> KYNU-F <400> 21 gaaagcttat ggagccttca tctcttgagc t 31 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> KYNU-R <400> 22 ataagcttat tttttgtttc tgcagagtca ag 32 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-F <400> 23 gaagatctat gaaagtgagg tcggccggcg 30 <210> 24 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-R <400> 24 atagatctta tccgattatg tgaaaacaaa g 31

Claims (10)

Small Cell Adhesion Glycoprotein (SMAGP), CLGN (Calmegin), RGS17 (Regulator of G-Protein Signaling 17), EPSTI1 (Epithelial Stromal Interaction 1), KYNU (Kynureninase), SCARA3 (Scavenger Receptor Class A, Member 3) An epidermal growth factor (EGF) or a basic fibroblast growth factor (bFGF), which comprises an agent for measuring the level of at least one gene mRNA selected from the group consisting of AFF3 (AF4 / FMR2 Family, ) For detecting the differentiation ability of adipose derived stem cells cultured in a medium containing adipocyte-derived stem cells and adipose tissue-derived stem cells.
Derived from a culture medium containing EGF or bFGF, comprising at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 or an agent for measuring the protein level thereof A composition for detecting a marker for detecting the proliferative capacity of a stem cell, wherein the expression level of the marker is different from that of a fat derived stem cell cultured in a basal medium.
3. The marker detection composition according to claim 1 or 2, wherein the agent for measuring the level of the gene mRNA comprises a primer pair or a probe that specifically binds to the gene.
The composition for detecting a marker according to any one of claims 1 to 3, wherein the agent for measuring the level of the protein comprises an antibody specific to the protein.
When compared to the fat-derived stem cells cultured in the basal medium for detecting the differentiation ability of the adipose-derived stem cells cultured in the medium containing EGF or bFGF, which comprises the composition for detecting a marker of claim 1, And a marker detecting unit for detecting the marker.
In order to detect the proliferative capacity of adipose derived stem cells cultured in a medium containing EGF or bFGF, which comprises the composition for detecting a marker as set forth in claim 2, when compared with adipose derived stem cells cultured in a basal medium, And a marker detecting unit for detecting the marker.
The kit according to claim 5 or 6, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
Measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 of adipose derived stem cells cultured in a medium containing EGF or bFGF A method for detecting the differentiation ability of adipose derived stem cells by detecting adipose derived stem cells cultured in a basal medium and markers showing a difference in expression level.
9. The method according to claim 8, wherein the method comprises the step of culturing the adipose tissue culture medium containing at least one gene mRNA selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU and SCARA3 Wherein the amount of mRNA or protein of the at least one gene selected from the group consisting of SULF2 and AFF3 is higher than that measured in the fat-derived stem cells cultured in the basal medium as the control, And determining that the differentiation capacity is lower than that of the control stem cells, and determining that the differentiation ability is lower than that of the control stem cells.
Measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of SMAGP, CLGN, RGS17, EPSTI1, KYNU, SCARA3, SULF2 and AFF3 of adipose derived stem cells cultured in a medium containing EGF or bFGF Derived stem cells and detecting markers showing differences in the expression level between the adipose-derived stem cells cultured in the basal medium and the proliferative ability of the adipose-derived stem cells.

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