KR20160084004A - detection markers of differentiation potency of adipocyte-derived stem cells and use thereof - Google Patents

Abstract

The present invention relates to a marker detecting composition for detecting a differentiation potency of adipocyte-derived stem cells, a marker detecting composition for measuring a titer of an adipocyte-derived stem cell treating agent, a marker detecting composition for measuring the aging of adipocyte-derived stem cells, and a use of the same. The marker detecting composition for detecting a differentiation potency of adipocyte-derived stem cells comprises a formulation for measuring a level of one or more gene mRNA molecules or a protein thereof, wherein the gene mRNA molecules are selected from the group consisting of Kynureninase, L-kynurenine hydrolase (KYNU); kazal-type serine peptidase inhibitor domain 1 (KAZALD1); calmegin (CLGN); small cell adhesion glycoprotein (SMAGP); scavenger receptor class A, member 3 (SCARA3); epithelial stromal interaction 1 (EPSTI1); limb bud and heart development homolog (LBH); lipase, endothelial (LIPG); lipopolysaccharide binding protein (LBP); PAWR which is PRKC, apoptosis, WT1, and regulator; and secreted frizzled-related protein 2 (SFRP2).

Description

[0002] The present invention relates to a marker for detecting the differentiation ability of adipocyte stem cells and a use thereof,

The present invention also relates to the use of a compound of formula (I), wherein KYNU (Kynureninase, L-kynurenine hydrolase), KAZALD1 (Kazal-type serine peptidase inhibitor domain 1), CLGN (Calmegin), SMAGP (Small cell adhesion glycoprotein), SCARA3 (LBP), Lipopolysaccharide binding protein (LBP), PAWR (PRKC, apoptosis, WT1, regulator) and SFRP2 (Secreted frizzled-related protein 2 A composition for detecting a marker for detecting the differentiation ability of an adipose derived stem cell, a method for measuring the activity of an adipose stem cell therapeutic agent, A composition for detecting markers for measuring the aging of adipose derived stem cells, and a use thereof.

Regenerative medicine is associated with cell-based therapies that mainly utilize stem cells with multipotential potential as a way to treat damaged or underperformed tissues and organs. Human mesenchymal stem cells are adult stem cells that not only are free from cancer or ethical problems that are generally a problem in embryonic stem cells, but also include adipocytes, osteoblasts, cartilage cells, cardiac cells, It is possible to differentiate into various cells such as muscle cells and nerve cells. In addition, since it has an immunomodulating function, it can be used as an immunosuppressant for the same kind of bone marrow transplantation or autoimmune diseases.

Adipocyte-derived stem cells (ASCs) have been studied for use as biomaterials because they contain large amounts of stem cells that are more than 1,000 times the number of stem cells that can be isolated from the same amount of bone marrow. . Fat-derived stem cells also have differentiation potentials such as bone marrow stem cells, which can differentiate into cartilage, bone, fat, muscle cells, and the like. In addition, the adipose tissue-derived stem cells are similar in the expression pattern of bone marrow-derived stem cells and cell surface markers, and do not induce immune responses in autologous or allogeneic transplantation in vivo or in vitro , but rather function to regulate the immune response And has been attracting attention as an effective means of cell therapy.

However, in the case of stem cell treatment drugs, the clinical results of the stem cells disclosed so far have confirmed the possibility of development as a therapeutic agent, but the evaluation of the therapeutic effect is unsatisfactory. One of the causes of the inadequate therapeutic effect of such a stem cell therapeutic agent is that the quality evaluation through analysis of the molecular biological characteristics of stem cells related to the therapeutic effect is not performed properly. In the case of a cell therapy agent, the quality of the product can be changed even if the preparation is made using the same protocol because the characteristics of the preparation vary with the age and health condition of the cell donor (Kretlow JD et al, BMC Cell Biol. 2008 Oct 28; : 60).

Therefore, in order to develop a stem cell treatment agent as a practical drug having a therapeutic effect of a certain level or more and to produce it reproducibly, there is a strong demand for development of a quality control test method that reflects the effect of stem cells such as therapeutic effect and potency. In order to develop such a cell therapeutic agent titration method, a biomarker is required to represent the therapeutic effect and activity of stem cells.

Accordingly, the inventors of the present invention focused on the differentiation efficiency and therapeutic effect of the low-passage ASCs and the aged adipose-derived stem cells (high passage ASCs) We have searched for gene clusters that can discriminate the cells and tried to use the expression of these marker genes to determine the distribution of early passage ASCs in stem cell therapy drugs. This may have a different meaning from the stem cell markers that divide known stem cells and differentiated cells. As a result, the inventors of the present invention found that ANXA10 (annexin A10), insulin-like growth factor 2 binding protein 3 (IGF2BP3) as markers for measuring the differentiation ability and aging of adipose derived stem cells and measuring the activity of a fat- , MALL (Mal, T cell differentiation protein-like), PTGER2 (prostaglandin E receptor 2), RGS20 (regulator of G-protein signaling 20 transcript variant 1), ITM2A (integral membrane protein 2A), LGI2 LGI family member 2), sulfatase 2 transcript variant 1, CCND2 (cyclin D2) and GADD45G (growth arrest and DNA-damage-inducible, gamma), and compositions and applications thereof (Korean Patent No. 10-2012-0042886).

The present inventors have also found that the present invention relates to the ability of the stem cells to differentiate into stem cells as a control gene in the process of subculturing the stem cells collected from a donor. The present inventors have found that ALK (Alkaline phosphatase transcript variant 1) and VCAM (Kynureninase, L-kynurenine hydrolase), KAZALD1 (Kazal-type), and KYNL-1 were identified using these cells, confirming that these genes decreased with the increase of the stem cell lineage using the negative cell adhesion molecule-1 transcript variant 1 The expression of EGFR1 (EGFR1) and EGFR1 (EGFR1) were increased with the increase of adipocyte stem cell lineage, while that of serine peptidase inhibitor domain 1), CLGN (Calmegin), SMAGP (Small cell adhesion glycoprotein) and SCARA3 interaction 1), LBH (Limb bud and heart development homolog), LIPG (lipase, endothelial), LBP (lipopolysaccharide binding protein), PAWR (PRKC, apoptosis, WT1, regulator) and SFRP2 zled-related protein 2) gene decreased with the increase of adipose-derived stem cell lineage. Therefore, the present invention has been completed by verifying that these genes can be used as markers for confirming the youth or aging state represented by the donor stem cell lineage.

One object of the present invention is to provide a method of screening for Kynuroninase, L-kynurenine hydrolase, KAZALDl (Kazal-type serine peptidase inhibitor domain 1), CLGN (Calmegin), SMAGP (Small cell adhesion glycoprotein), SCARA3 3), EPSTI1 (Epithelial stromal interaction 1), LBH (Limb bud and heart development homolog), LIPG (lipase, endothelial), Lipopolysaccharide binding protein (LBP), PAWR (PRKC, apoptosis, WT1, regulator) and SFRP2 frizzled-related protein 2), or an agent for measuring the protein level of the mRNA. The present invention also provides a composition for detecting a marker for detecting the differentiation ability of an adipose-derived stem cell.

Another object of the present invention is to provide a method for measuring the level of one or more gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR, and SFRP2 And to provide a composition for detecting a marker for measuring the activity of an adipose-derived stem cell therapeutic agent.

Another object of the present invention is to provide a method for measuring the level of one or more gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR, and SFRP2 And to provide a composition for detecting a marker for measuring the aging of adipose-derived stem cells.

It is still another object of the present invention to provide a marker detection kit for detecting the differentiation ability of adipose derived stem cells, which comprises a composition for detecting a marker for detecting the differentiation ability of adipose derived stem cells.

Yet another object of the present invention is to provide a kit for detecting a marker for measuring the activity of an agent for treating adipose stem cells, which comprises a composition for detecting a marker for measuring the activity of the therapeutic agent for treating adipose derived stem cells.

It is still another object of the present invention to provide a kit for detecting a marker for measuring the aging of adipose derived stem cells, which comprises a composition for detecting a marker for measuring the aging of the adipose derived stem cell treatment agent.

Yet another object of the present invention is to provide a method for detecting the differentiation ability of adipose derived stem cells, comprising the step of measuring the expression level of the gene.

It is still another object of the present invention to provide a method for measuring the activity of an adipose-derived stem cell therapeutic agent, which comprises measuring the expression level of the gene.

It is still another object of the present invention to provide a method for detecting the degree of senescence of adipose derived stem cells by confirming the expression level of the gene.

In one aspect of the present invention, the present invention provides a mRNA encoding at least one gene selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2, The present invention provides a composition for detecting a marker for detecting the differentiation ability of adipose derived stem cells.

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 derived from mammals, including, but not limited to, pigs, cows, primates, and humans, which can be transplanted to humans.

In the present invention, the term "marker for detecting the differentiation ability of adipose derived stem cells" refers to the expression of adipocyte stem cells having a high passage number and fat-derived stem cells having a low passage number It may mean an organic biomolecule showing a significant difference. For the purpose of the present invention, the marker of the present invention means a marker capable of detecting the differentiation ability by increasing or decreasing the expression level in adipose-derived stem cells having high differentiating ability represented by a low passage number, and more specifically, KYNU, Expression of KYNU, KAZALD1, CLGN, SMAGP, or SCARA3 was decreased in adipose-derived stem cells with high differentiation potential as expression amounts of KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 , EPSTI1, LBH, LIPG, LBP, PAWR, or SFRP2 are genes whose expression is increased.

The genes used in the present invention can be obtained from known databases such as GenBank of the National Institutes of Health, such as KYNU (NM_003937.2, NP_001028170.1), KAZALD1 (NM_030929.4, NP_112191.2), CLGN (NM_001130675. 1, NP_001124147.1), SMAGP (NM_001033873.1, NP_001026798.1), SCARA3 (NM_182826.1, NP_057324.2), EPSTI1 (NM_033255.2, NP_001002264.1), LBH (NM_030915.3, NP_112177.2) , LIPG (NM_006033.2, NP_006024.1), LBP (NM_203346.2, NP_004130.2), PAWR (NM_002583.2, NP_002574.2) and SFRP2 (NM_003013.2, NP_003004.1) But is not limited thereto.

The term "passage" in the present invention means a method in which a part of cells is periodically transferred to a new culture container in order to continuously and continuously cultivate the cells in a healthy state, and then the culture medium is continuously cultured while changing the culture medium , Replacing the culture container, or cultivating the cell group separately, and changing the culture container once or culturing the cell group separately is referred to as a passage. In the present invention, low passage ASCs with low passage number or early passage means adipose derived stem cells having differentiation ability or therapeutic effect and high-passage stem cells (high passage ASCs ) May mean, but is not limited to, adipogenic stem cells with differentiation or therapeutic effects. In the present invention, the number of early passage passages may be 0 to 3, preferably 0 to 2.

The term "differentiation ability" in the present invention means the ability of an adipose derived stem cell to differentiate into adipocytes, osteoblasts, chondroblasts, myofibroblasts, osteoblasts, muscle cells or neurons, Phase differentiation capacity can be represented by the number of stem cell lines derived from adipose. That is, a high ability to differentiate means that the number of cells is low and differentiation can easily occur to various cells. In the present invention, the term "adipose-derived stem cell" refers to a cell in which induction of differentiation into adipocytes, osteoblasts, chondroblasts, myofibroblasts, osteoblasts, muscle cells or neurons is facilitated, Preferably a fat-derived stem cell in which induction of differentiation into adipocytes is promoted. In the present invention, the adipose-derived stem cells having high differentiation ability have a decreased expression of KYNU, KAZALD1, CLGN, SMAGP, or SCARA3 gene, and the expression of EPSTI1, LBH, LIPG, LBP, PAWR or SFRP2 Derived stem cells in which the expression of KYNU, KAZALD1, CLGN, SMAGP, or SCARA3 gene is increased or the expression of EPSTI1, LBH, LIPG, LBP, PAWR or SFRP2 is decreased, Derived stem cells that are highly efficient in differentiating into stem cells, osteoblasts, cartilage cells, muscle fiber cells, muscle cells or neurons. In one embodiment of the present invention, the adipose-derived stem cells of the second, seventh, and eighth passages were differentiated into adipocytes. As a result, it was confirmed that the lower the number of passages, the better the differentiation ability (Fig. 2) 8, the expression of KYNU, KAZALD1, CLGN, SMAGP, or SCARA3 gene was increased and expression of EPSTI1, LBH, LIPG, LBP, PAWR or SFRP2 gene was decreased ). The markers of the present invention can effectively separate adipose-derived stem cells having the same early passage or similar differentiation ability from adipose-derived stem cells having various numbers of passage or differentiation ability isolated from transplant recipients, Can be increased.

The level of gene expression in a biological sample can be determined by identifying the amount of mRNA or protein.

The term " mRNA level measurement " in the present invention refers to a process for identifying the presence or absence of mRNA and the expression level of marker genes indicating the differentiation capability 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, and 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 KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 genes are NM_003937.2 and NM_030929, respectively, which are preferably primer pairs or probes, Since NM_001130675.1, NM_001033873.1, NM_182826.1, NM_033255.2, NM_030915.3, NM_006033.2, NM_203346.2, NM_002583.2 and NM_003013.2 have been found in the art, A primer or a probe that specifically amplifies a specific region of a gene can be designed.

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 KYNU, a primer pair of SEQ ID NOs: 5 and 6 for the KAZALD1, a pair of primers of SEQ ID NOs: 7 and 8 for the CLGN, The primers for SMAGP are the primer pairs of SEQ ID NOs: 9 and 10, the primers for SCARA3 are the primer pairs of SEQ ID NOs: 11 and 12, the primers for EPSTI1 are the primer pairs of SEQ ID NOs: 13 and 14, the primer for LBH is SEQ ID NO: And 16, the primers for LIPG are the primer pairs of SEQ ID NOs: 17 and 18, the primers for LBP are the primer pairs of SEQ ID NOs: 19 and 20, the primers for PAWR are the primer pairs of SEQ ID NOs: 21 and 22, or the pair of primers for SFRP2 The primers for the primers may be a primer pair of SEQ ID NOS: 23 and 24, but are 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 carried out using probes complementary to KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 polynucleotides to determine the ability to differentiate adipose- Able to know. 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 that indicates the differentiation 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, the antibody refers to an antibody that specifically binds to a marker protein, and specifically, the marker genes KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR Or an antibody that specifically binds to a protein encoded by SFRP2, and includes both a polyclonal antibody, a monoclonal antibody, and a recombinant antibody. 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 may 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: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. The antibodies of the 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 scFv. 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 gene protein was measured using Western blot (Example 4).

In another embodiment, the present invention provides a method for measuring the level of one or more gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 A composition for detecting a marker for measuring the activity of an adipose-derived stem cell therapeutic agent.

The term "adipose-derived stem cell therapeutic agent" in the present invention refers to an agent for proliferating and screening living autologous, allogenic, or xenogenic cells in vitro for restoring the functions of cells and tissues, A drug used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the biological characteristics of adipose stem cells, adipocytes formed by differentiation of adipose stem cells, osteoblasts, chondroblasts, muscle fibers Means a therapeutic agent intended for regeneration such as a stem cell, a muscle cell, or a nerve cell.

The term "potency" in the present invention means a therapeutic activity of a stem cell therapeutic agent, and it may preferably mean the differentiation ability or therapeutic ability of a fat-derived stem cell therapeutic agent.

In one embodiment of the present invention, the adipose-derived stem cells, which have low protein levels of KYNU, KAZALD1, CLGN, SMAGP and SCARA3 and high expression levels of EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2, (Fig. 5).

In addition, siRNAs of genes KYNU, KAZALD1, CLGN, SMAGP and SCARA3, which have low expression levels in the low passages, were introduced and subcultured while lowering the level of gene expression. Cells of P5 and P10 were subcultured in differentiation and differentiation maintenance media After 12 days of culture, the cells were identified by oil-red O staining to confirm their differentiation potential. Thus, markers indicating that the low expression level of KYNU, KAZALD1, CLGN, SMAGP or SCARA3 protein is one of the therapeutic activities of adipose- (Fig. 6A).

In addition, siRNAs of LBH, LIPG, LBP, PAWR and SFRP2 genes with low expression levels were transfected by subculturing while lowering the gene expression level, and cells of P3 and P7 were subcultured in differentiation and differentiation maintenance medium After 12 days of culture, the cells were identified by oil-red O staining to confirm their differentiation potential. As a result, low expression level of LBH, LIPG, LBP, PAWR or SFRP2 protein was shown to be one of the therapeutic activities of adipose- (Fig. 6 (b)).

Measurement of the gene mRNA or its protein level is the same as described above.

In another embodiment, the present invention provides a pharmaceutical composition comprising at least one gene mRNA selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2, A composition for detecting a marker for measuring the aging of adipose derived stem cells is provided.

The term "aging" in the present invention means that the differentiation ability or proliferative capacity of adipose-derived stem cells is decreased by an increase in the individual age or the number of passages. For the purpose of the present invention, aged adipose-derived stem cells have increased expression of genes selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, and SCARA3, or EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 Derived stem cells in which expression of a gene selected from the above-mentioned genes is decreased. In one embodiment of the present invention, the adipose-derived stem cells with reduced expression of KYNU, KAZALD1, CLGN, SMAGP and SCARA3 protein and EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 proteins are expressed in KYNU, KAZALD1 , CLGN, SMAGP, and SCARA3, and that induction of differentiation into adipocytes was more evident than that of adipose-derived stem cells, where expression of EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 proteins was decreased (Example 4 and Fig. 5) .

Measurement of the gene mRNA or its protein level is the same as described above.

In another aspect, the present invention provides a kit for detecting a marker for detecting the differentiation ability of adipose derived stem cells, comprising a composition for detecting a marker for detecting the differentiation ability of adipose derived stem cells.

The marker detection kit of the present invention may include a marker gene or a primer or an antibody capable of selectively recognizing the marker gene whose expression is increased or decreased depending on the number of the passage number or the differentiation ability represented by the marker, And tools and reagents commonly used in the art. 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 having 2, 5, and 8 segments was confirmed using a microarray (Example 2 and Fig. 3).

In another aspect, the present invention provides a kit for detecting a marker for measuring the activity of a therapeutic agent for stem cell of fat origin, which comprises a composition for detecting a marker for measuring the activity of the therapeutic agent for treating a fat-derived stem cell.

The marker detection kit that can be used is the same as that described above.

In another aspect, the present invention provides a kit for detecting a marker for measuring the aging of an adipose derived stem cell, which comprises a composition for detecting a marker for measuring the aging of the adipose derived stem cell.

The marker detection kit that can be used is the same as that described above.

In another embodiment, the present invention provides a method for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 And a method for detecting the differentiation ability of adipose-derived stem cells.

Preferably, if the amount of at least one gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, and SCARA3 is smaller than that of the control group, it is determined that the differentiation ability is higher than that of the control, The method further comprises the step of determining that the differentiation ability is higher than that of the control group when the amount of at least one gene mRNA or protein thereof selected from the group consisting of EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 is larger than that of the control group Derived stem cells from the differentiated stem cells. The measurement of the level of mRNA or its protein is the same as described above.

In one embodiment of the present invention, the adipose-derived stem cells with reduced expression of KYNU, KAZALD1, CLGN, SMAGP, and SCARA3 and EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 are expressed in KYNU, KAZALD1, CLGN , SMAGP and SCARA3, and that the differentiation into adipocytes was more pronounced than in adipose-derived stem cells, where expression of EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 was decreased (FIG. 5).

In another embodiment, the present invention provides a method for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 And a method for measuring the activity of an adipose-derived stem cell therapeutic agent.

In another embodiment, the present invention provides a method for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 And a method for measuring the degree of senescence of adipose-derived stem cells.

Determining whether the level of one or more genes mRNA or protein selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, and SCARA3 is decreased or low in comparison with the control group, And that the level of at least one gene mRNA or protein thereof selected from the group consisting of EPSTI1, LBH, LIPG, LBP, PAWR, and SFRP2 is increased or higher than that of the control, As shown in FIG.

In another embodiment, the present invention provides a method of increasing or decreasing the level of one or more gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR, and SFRP2 A method for regulating differentiation from adipose-derived stem cells into adipocytes is provided.

It is possible to promote or suppress the differentiation from adipose-derived stem cells into adipocytes by increasing or decreasing the mRNA or protein level of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR or SFRP2 gene, Differentiation can be controlled.

The gene mRNA or its protein level can be increased by using various methods known in the art that can increase the gene expression rate in a cell. Preferably, a vector containing the genes is prepared, To increase the expression rate of these genes, but the present invention is not limited thereto. In addition, the level of the gene mRNA or protein thereof may be decreased by using a method known in the art that can delete the gene in the cell or lose the function of the gene through gene mutation. Preferably, (ShRNA) (small hairpin RNA), and transfecting them into cells to lower the expression of these genes. However, the present invention is not limited thereto. For the purpose of the present invention, shRNA for one or more genes selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, and SCARA3 is prepared in order to increase the differentiation of adipose derived stem cells into adipocytes, Expression of these genes can be lowered and a vector containing one or more genes selected from the group consisting of EPSTI1, LBH, LIPG, LBP, PAWR, and SFRP2 can be prepared and transfected into cells to increase the expression rate.

The present invention provides useful data for verifying the efficacy of adipose derived stem cells by providing markers capable of judging the differentiation ability of adipose derived stem cells. In addition, the markers can be usefully used for measuring the activity and activity of a therapeutic agent for treating adipose-derived stem cells.

FIG. 1A is a graph comparing cell growth of adipose-derived stem cells in a basal medium and a proliferation medium. FIG.
Fig. 1B is a photograph of cells in the basal medium and the growth medium in Passage 2, P2, passage 5, P5, and Passage 8, P8.
FIG. 2A is an experimental result for examining the differentiation ability of P2 (Passage 2), P8 (Passage 8) and P14 (Passage 14) adipose derived stem cells. This is a photograph of a plate cultured in differentiation and differentiation medium for 12 days and then stained with Oil-red O.
Fig. 2b is a microscopic photograph of the cells of Fig. 2a, showing accumulation of fat by Oil-red O staining and graphical representation of the results.
3A shows microarray results of three sets of P2, P5, P8 adipose-derived stem cells using Illumina 24K chip.
FIG. 3B is a graph showing changes in the expression of genes showing a significant difference through microarray analysis. The ALPL and VCAM1 genes are the same as the results of previous studies as control genes.
FIGS. 4A to 4K show RT-PCR and real-time PCR of the expression levels of the genes selected in FIG. 3 from adipose-derived stem cells collected from 17 donors, and RPL13A is a control gene. 4A to 4E show the results of the genes KYNU, KAZALD1, CLGN, SMAGP and SCARA3 which increase at P8 compared to P2. In FIG. 4f, the gene corresponding to 4k is a gene that decreases at P8 EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2.
FIG. 5 shows the results of protein changes in P2 and P8 of the genes, and also confirmed protein expression results and cell differentiation ability.
FIG. 6A is a graph showing the results of the sub-culture of P5 and P10 cells after lowering the expression of genes by introducing siRNA of KYNU, KAZALD1, CLGN, SMAGP, or SCARA3 gene having a low expression level in a low passage, This is a photograph of a plate that has been cultured for 12 days and then stained with Oil-red O.
FIG. 6B is a graph showing the results of the sub-culture of P3 and P7 cells after lowering the expression of genes by introducing siRNAs of genes LBH, LIPG, LBP, PAWR or SFRP2 having a high expression level in a low passage. And then stained with Oil-red O for 12 days.

Hereinafter, the present invention will be described more specifically in the following examples. However, these embodiments are only for the purpose of helping understanding of the present invention, and the present invention is not limited thereto.

Example  One: Adipose-derived stem cells  Passage ( passage )

(1) Culture of human adipose-derived stromal stem cells

Adipose tissue was isolated from the donor (Antrozen, Gyeonggi-do, Korea). From the obtained adipose tissue, adipose-derived stromal stem cells were isolated. Adipose tissue was washed 3 to 4 times with the same volume of 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. Substrate media were suspended and centrifuged at 20 ° 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 substrate medium, inoculated into a culture vessel, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After removal of the culture medium, the cells are washed with a phosphate buffer solution, and the substrate medium or medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml is added to the substrate 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 with the growth medium at a ratio of 1: 3 to 1: 4 and subcultured (Korean Patent Laid-Open No. 10-2010-0118491).

In order to analyze the difference of gene expression according to the composition of the culture medium and subculture, 5 subculture cells cultured in DMEM medium supplemented with 10% FBS and 2 subcultures cultured in a growth medium containing 1 ng / , 5th passage, 8th passage cell were collected.

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

(2) induction of differentiation of human adipose-derived stem cells into the 2nd passage (P2), 8 passage (P8), and 14 passage (P14)

The adipose stem cells were suspended in the proliferation medium, each 90,000 cells were inoculated into a 24-well culture vessel, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. When the cells were filled with a single layer on the bottom of the culture vessel, the culture medium was changed to substrate medium, cultured in a 5% CO ₂ incubator at 37 ° C for 2-3 days, changed into 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 medium was DMEM / F12, 10% fetal bovine serum, 5 ng / ml EGF, 0.25 ng / ml bFGF, 0.25 ng / ml DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% / Ml TGF-? 1, 0.1% antibiotics. The differentiation medium contained DMEM / F12, 3% FBS, 33 μM biotin, 17 μM pantothenate, 1 μM insulin, 1 μM dexamethasone, 250 μM IBMX (isobutylmethylxanthine), 100 μM indomethacin, Include DMEM / F12, 3% Bovine Serum, 33 [mu] M Biotin, 17 [mu] M Pantothenate, 100 nM Insulin and 1 [mu] M Dexamethasone.

(3) Confirmation of adipocyte differentiation pattern (Oil-Red O staining)

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. As a result, the ability to differentiate the stem cells of the 2nd, 8th, and 14th lines was confirmed.

FIG. 2A shows an example of a plate stained with Oil-red O, and FIG. 2B shows a microscopic photograph of the cells stained with Oil-red O and quantitative measurement results of four samples. Stem cells of the second line showed the highest ability to differentiate, and it was confirmed that the differentiation ability was higher in the fat stem cells of the early passage.

Example  2: by microarray experiment Of adipose-derived stem cells Categorical  Identification of gene expression changes

(1) Culture of human adipose-derived stromal stem cells

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

(2) Total RNA Isolation

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.

(3) Microarray implementation

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. Produced from adipose-derived stem cells cRNA 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. 3A shows the expression of the above genes measured in cells having a passage number of 2 cultured in a propagation medium and cells having a passage number of 5 and a passage number of 8. KYNU, KAZND1, CLGN, SMAGP, and SCARA3 genes show a marked increase in P8 compared to P2. EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 are genes whose expression is decreased in P8 compared to P2. It is known that ALPL and VCAM1 decrease in relation to the differentiation ability as a reference gene, and also showed a significant decrease in this experiment. From these results, it can be seen that the above genes can be used to diagnose the differentiation and therapeutic ability of stem cells, because the genes show a large difference in expression when comparing P2 and P8. However, the expression in the differentiated cells differs regardless of the expression change according to the passage before the differentiation. FIG. 3A shows the result of microarray, and FIG. 3B is a graph showing a difference in gene expression level of the genes.

Example  3: Stem cells from adipose-derived stem cells of individual donors early  Passage, ≤ 2).

The expression levels of the genes identified in Example 2 were analyzed by RT-PCR using 17 pairs of donor adipose stem cell samples (P2 and P8). Total RNA was isolated through the method of Example 2. [

(1) cDNA synthesis and calibration of template concentration

1 μl of total RNA, 50 μM Olgo (dT), and 2.5 μl of 10 mM dNTP were added to each sample, and 25 μl of the solution was added to sterilized water containing DEPC as an RNase inhibitor to prepare an RNA / primer mixed 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 SuperScript III RT enzyme to make a total of 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. PRL13A 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 the PCR reaction was performed using 2 μl of the diluted sample. 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 PRL13A primer used is the forward 5'-CATCGTGGCTAAACAGGTACTG-3 '(SEQ ID NO: 1) and the 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.

(2) Expression analysis using RT-PCR / Real-Time PCR

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. Cycle was different for each gene. PCR products were analyzed by electrophoresis using 2% agarose gel and image analysis. Realtime RT-PCR was performed using DNASYBR® Green I 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 NO: 1 to SEQ ID NO: 24) are shown in Table 1.

The results are shown in Fig. As a result, the above genes were confirmed to increase or decrease at P8 compared with P2 (KYNU (76.4%, increase in 13 samples), KAZALD1 (82.4%, increase in 14 samples), CLGN ), SMAGP (82.4%, increased in 14 samples), SCARA3 (94.1% in 16 samples), EPSTI1 (82.4% in 14 samples), LBH (88.2% , Decrease in LIPG (88.2%, decrease in 15 samples), LBP (76.4%, decrease in 13 samples), PAWR (82.4%, decrease in 14 samples) and SFRP2 ), The genes may be used as diagnostic markers representing the degree of senescence of adipose stem cells or as markers for drug screening related to stem cell differentiation.

The 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 KYNU (NM_003937.2) F: 5'-tctttaagcaagcgacaatg-3 ' SEQ ID NO: 3 R: 5'-tggttgctgctttatctttg-3 ' SEQ ID NO: 4 KAZALD1 (NM_030929.4) F: 5'-gatgtgatctttggctgtga-3 ' SEQ ID NO: 5 R: 5'-acccctaaactgcacagaga-3 ' SEQ ID NO: 6 CLGN (NM_001130675.1) F: 5'-aagtgaacctgcccaaatag-3 ' SEQ ID NO: 7 R: 5'-atccgtgtcttcattccagt-3 ' SEQ ID NO: 8 SMAGP (NM_001033873.1) F: 5'-acagcactcattgcagttgt-3 ' SEQ ID NO: 9 R: 5'-actgggctcaccttctgtag-3 ' SEQ ID NO: 10 SCARA3 (NM_182826.1) F: 5'-agtgcaccagatcaacttca-3 ' SEQ ID NO: 11 R: 5'-cgtgtgtagttctgccagtc-3 ' SEQ ID NO: 12 EPSTI1 (NM_033255.2) F: 5'-agagcaagaaagagccaaaa-3 ' SEQ ID NO: 13 R: 5'-atattccaacagcctccaga-3 ' SEQ ID NO: 14 LBH (NM_030915.3) F: 5'-ctgctgcaaactgaaggac-3 ' SEQ ID NO: 15 R: 5'-cttcgcagttatcttgctca-3 ' SEQ ID NO: 16 LIPG (NM_006033.2) F: 5'-ccaacaccttcctggtctac-3 ' SEQ ID NO: 17 R: 5'-ctccttccacaggttgtacc-3 ' SEQ ID NO: 18 LBP (NM_203346.2) F: 5'-aatctgtctgtggaccccta-3 ' SEQ ID NO: 19 R: 5'-ttcaggaacccagtgatctt-3 ' SEQ ID NO: 20 PAWR (NM_002583.2) F: 5'-caggagccacctagaacagt-3 ' SEQ ID NO: 21 R: 5'-tacctgaaacatttgcatcc-3 ' SEQ ID NO: 22 SFRP2 (NM_003013.2) F: 5'-aaaatcatcctggagaccaa-3 ' SEQ ID NO: 23 R: 5'-tgtcgttcatctcctcacag-3 ' SEQ ID NO: 24

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

Using the donor fat stem cell samples (P2 and P8), the expression levels of the genes identified in Example 3 were analyzed by Western blot analysis using protein quantification. The P2 and P8 cells used were differentiated at the same time as the protein analysis, and their differentiation ability was also verified.

Specifically, the 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 then transferred to a 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. Information about the antibody is as follows. (Pierce, PA5-26448), anti-CYNU (Pierce, PA5-22379), anti-LIPG (abcam, ab24447), anti-PAWR -LGN (Pierce, PA5-26038), anti-ACTIN (Sigma, A5316). The amount of each protein was determined by using the Immobilon ^™ Western blotting Detection reagents (Millipore).

As a result, it was observed that ALPL, LBP, LIPG and PAWR proteins, which are genes that decrease mRNA level according to the passage through the RT-PCR, show a high expression in P2 and a decrease in expression in P8, The expression of KYNU and CLGN proteins, which are mRNA levels increased, was low in P2 and increased in P8 (FIG. 5). The P2 and P8 cells used were differentiated as in Example 1, and their differentiation ability was also verified. As shown in FIG. 5, P2 cells showed differentiation into adipose stem cells and P8 cells were differentiated but decreased in their differentiation level compared to P2 cells. Therefore, it has been proved that the difference in the expression level of the gene is useful as a marker for detecting young and aging of adipose stem cells.

Example  5: Selection of the gene siRNA  Identification of differentiation potential by induction of fat after induction

Human adipose-derived stem cells (passage 5, P5) were plated on 12-well plates at 1 × 10 ⁵ cells and allowed to attach for 24 hours. Then siRNA and control siRNA (40 nmol) of the gene were transformed into 250 μl of Opti-MEM medium, respectively. The nucleotide sequences of the siRNAs used are shown in Table 2 below.

siRNA Base sequence SEQ ID NO: si-control GUCACAUUCGACGCCCAC SEQ ID NO: 25 si-KYNU CCAUGAACUCAGCACCAGA SEQ ID NO: 26 si-KAZALD1 GAGAAUGACGAUUACUACU SEQ ID NO: 27  si-CLGN CAUUUUGUUGGCCAAGAAA SEQ ID NO: 28 si-SMAGP CCUCCUGAUAAAUGUGCUA SEQ ID NO: 29 si-SCARA3 CGGUGCGGAUUCUUUACCU SEQ ID NO: 30 si-EPSTI1 CAUCAAAAGAGUGAAUUAC SEQ ID NO: 31 si-LBH ACUGAAGGACCGUCUGCCC SEQ ID NO: 32 si-LIPG CUAAUGCAGAUUCCCAGAC SEQ ID NO: 33 si-LBP GACUCUAAUAUCCGACUGA SEQ ID NO: 34 si-PAWR CAGGAGCCACCUAGAACAG SEQ ID NO: 35 si-SFRP2 GUGAAGGAGAUAACCUACA SEQ ID NO: 36

The siRNA transfection reagent, hiperfect (Qiagen), was added to each of the samples as described in the manufacturer's manual, mixed thoroughly with a stirrer, and allowed to stand at room temperature for 15 minutes to allow siRNA transfection complex to form. During this time, the medium in each well was replaced with 750 ml of fresh 10% FBS-DMEM, and 15 minutes later, each sample was slowly added to each well by one drop. After 48 hours of incubation, induction of differentiation of adipose stem cells corresponding to P3 or P5 was carried out. As in Example 1, the medium was first changed to a substrate medium, cultured in a 5% CO ₂ incubator at 37 ° C for 2-3 days, changed to a differentiation medium, and cultured for 3 days or 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.

In the case of adipose stem cells cultured from P5 to P10, the process of injecting the same concentration of siRNA into each cell was repeatedly performed in P7 and P9, and cultured up to passage 10 to induce differentiation. Oil-red O staining was performed to morphologically identify the differentiation of adipocytes from adipocyte stem cells. Figure 6a shows a microscopic photograph of cells stained with Oil-red O and the difference in absorbance (OD ₅₂₀ ) between Oil-red O stain. si-KYNU (custom made, Samchully Pharmaceutical), si-KAZALD1 (1076824, Bioneer co.) si-CLGN (1032614, Bioneer co.), si-SMAGP (1088181, Bioneer co. Bioneer co.) To reduce adipogenesis, we found that adipocyte differentiation was more evident than si-Control-treated adipose stem cells. In other words, it was confirmed that the stem cells derived from P10 subculture treated with si-KYNU, si-KAZALD1, si-CLGN, si-SMAGP or si-SCARA3 possessed more differentiation potential.

In the case of adipocyte stem cells cultured from P3 to P7, the process of injecting the same concentration of siRNA into each cell was repeatedly performed in P5 and P7, and cultured up to passage 7, followed by induction of differentiation. Oil-red O staining was performed to morphologically identify the differentiation of adipocytes from adipocyte stem cells. Figure 6b shows the microscopic photograph of cells stained with Oil-red O and the difference in absorbance (OD ₅₂₀ ) between Oil-red O stain. si-LBP (custom made, Samchully Pharmaceutical, Bioneer co.), si-LIPG (custom made, Samchully Pharmaceutical, Bioneer co.) si-LBP (1083042, Bioneer co.) si-PAWR ) Or si-SFRP2 (custom made, Samchully Pharmaceutical) The results showed that the adipocyte differentiation did not occur more than si-Control-treated adipose stem cells when gene expression was decreased. In other words, the P7 lineage-derived stem cells treated with si-LBH, si-LIPG, si-LBP, si-PAWR or si-SFRP2 were found to have lower differentiation potential.

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 to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Korea Research Institute of Bioscience and Biotechnology          ANTEROGEN CO., LTD. <120> detection markers of differentiation potency of adipocyte-derived          stem cells and use thereof <130> KPA141078KR <160> 36 <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> KYNU-F <400> 3 tctttaagca agcgacaatg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KYNU-R <400> 4 tggttgctgc tttatctttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KAZALD1-F <400> 5 gatgtgatct ttggctgtga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KAZALD1-R <400> 6 acccctaaac tgcacagaga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CLGN-F <400> 7 aagtgaacct gcccaaatag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CLGN-R <400> 8 atccgtgtct tcattccagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAGP-F <400> 9 acagcactca ttgcagttgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAGP-R <400> 10 actgggctca ccttctgtag 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-F <400> 11 agtgcaccag atcaacttca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SCARA3-R <400> 12 cgtgtgtagt tctgccagtc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-F <400> 13 agagcaagaa agagccaaaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EPSTI1-R <400> 14 atattccaac agcctccaga 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LBH-F <400> 15 ctgctgcaaa ctgaaggac 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LBH-R <400> 16 cttcgcagtt atcttgctca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIPG-F <400> 17 ccaacacctt cctggtctac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIPG-R <400> 18 ctccttccac aggttgtacc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LBP-F <400> 19 aatctgtctg tggaccccta 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LBP-R <400> 20 ttcaggaacc cagtgatctt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAWR-F <400> 21 caggagccac ctagaacagt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAWR-R <400> 22 tacctgaaac atttgcatcc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > SFRP2-F <400> 23 aaaatcatcc tggagaccaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SFRP2-R <400> 24 tgtcgttcat ctcctcacag 20 <210> 25 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-control <400> 25 gucacauucg acgcccac 18 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-KYNU <400> 26 ccaugaacuc agcaccaga 19 <210> 27 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-KAZALD1 <400> 27 gagaaugacg auuacuacu 19 <210> 28 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-CLGN <400> 28 cauuuuguug gccaagaaa 19 <210> 29 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-SMAGP <400> 29 ccuccugaua aaugugcua 19 <210> 30 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-SCARA3 <400> 30 cggugcggau ucuuuaccu 19 <210> 31 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-EPSTI1 <400> 31 caucaaaaga gugaauuac 19 <210> 32 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-LBH <400> 32 acugaaggac cgucugccc 19 <210> 33 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-LIPG <400> 33 cuaaugcaga uucccagac 19 <210> 34 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-LBP <400> 34 gacucuaaua uccgacuga 19 <210> 35 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-PAWR <400> 35 caggagccac cuagaacag 19 <210> 36 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-SFRP2 <400> 36 gugaaggaga uaaccuaca 19

Claims (13)

KYNU (Kynureninase, L-kynurenine hydrolase), KAZALD1 (Kazal-type serine peptidase inhibitor domain 1), CLGN (Calmegin), SMAGP (Small cell adhesion glycoprotein), SCARA3 (Scavenger receptor class A, member 3), EPSTI1 interaction 1), Limb bud and heart development homolog (LBH), lipase, endothelial, Lipopolysaccharide binding protein (LBP), PAWR (PRKC, apoptosis, WT1, regulator) and SFRP2 (Secreted frizzled- Wherein the composition comprises at least one gene mRNA selected from the group consisting of the mRNA and the protein of the present invention.
Derived stem cell treatment agent comprising an agent for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 For measuring the activity of the marker.
Derived stem cells, comprising one or more gene mRNAs selected from the group consisting of KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2, A composition for detecting a marker for measuring aging.
4. The composition according to any one of claims 1 to 3, wherein the agent for measuring the level of the gene mRNA comprises a primer that specifically binds to the gene.
4. The composition of any one of claims 1 to 3, wherein the agent that measures the level of the protein comprises an antibody specific for the protein.
A kit for detecting markers for detecting the differentiation ability of adipose derived stem cells comprising the composition of claim 1.
A kit for detecting a marker for measuring the activity of a therapeutic agent for a stem cell derived from an adipose comprising the composition of claim 2.
A kit for detecting a marker for measuring the aging of adipose derived stem cells comprising the composition of claim 3.
9. The kit for detecting a marker according to any one of claims 6 to 8, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
Comprising the step of measuring the level of in vitro (in vitro) in KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, 1 or more gene mRNA or a protein selected from the group consisting of PAWR and SFRP2 A method for detecting the differentiation ability of adipose derived stem cells.
Comprising the step of measuring the level of in vitro (in vitro) in KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, 1 or more gene mRNA or a protein selected from the group consisting of PAWR and SFRP2 A method for measuring the activity of a fat-derived stem cell therapeutic agent.
In vitro comprising the step of measuring the KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 1 or more gene mRNA or a protein level is selected from the group consisting in (in vitro) , A method for detecting the aging of adipose derived stem cells.
The step of ex vivo (in vitro) in KYNU, KAZALD1, CLGN, SMAGP, SCARA3, EPSTI1, LBH, LIPG, LBP, PAWR and SFRP2 increase or decrease in one or more gene mRNA or a protein level is selected from the group consisting of A method for regulating differentiation from adipose-derived stem cells into adipocytes.

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