WO2013162286A1

WO2013162286A1 - Marker for detecting potency of adipose-derived stem cells, and use thereof

Info

Publication number: WO2013162286A1
Application number: PCT/KR2013/003517
Authority: WO
Inventors: 김미형; 정경숙; 안지원; 원미선
Original assignee: 한국생명공학연구원; (주)안트로젠
Priority date: 2012-04-24
Filing date: 2013-04-24
Publication date: 2013-10-31
Also published as: KR101297829B1

Abstract

The present invention relates to: a composition for detecting a marker for detecting the potency of adipose-derived stem cells, containing a preparation measuring one or more gene mRNAs selected from the group consisting of annexin A10 (ANXA10), insulin-like growth factor 2 binding protein 3 (IGF2BP3), MALL (Mal, T-cell differentiation protein-like), prostaglandin E receptor 2 (PTGER2), regulator of G-protein signaling 20 transcript variant 1 (RGS20), integral membrane protein 2A (ITM2A), LGI2 (leucine-rich repeat LGI family, member 2), sulfatase 2 transcript variant 1 (SULF2), cyclin D2 (CCND2) and GADD45G (growth arrest and DNA-damage-inducible, gamma), or the protein level thereof; a composition for detecting a marker for measuring the titer of an adipose-derived stem cell therapeutic agent; a composition for detecting a marker for measuring the senescence of adipose-derived stem cells; and a use thereof.

Description

Adipose-derived stem cell differentiation detection marker and use thereof

The present invention is ANXA10 (annexin A10), IGF2BP3 (insulin-like growth factor 2 binding protein 3), MALL (Mal, T-cell differentiation protein-like), PTGER2 (prostaglandin E receptor 2), RGS20 (regulator of G-protein) signaling 20 transcript variant 1), integral membrane protein 2A (ITM2A), leucine-rich repeat LGI family, member 2 (LGI2), sulfatase 2 transcript variant 1 (SULF2), cyclin D2 (CCND2), and growth arrest and DNA-GADD45G marker-detecting composition for detecting the differentiation ability of adipose derived stem cells, the agent for measuring the level of one or more genes mRNA selected from the group consisting of damage-inducible, gamma), adipose derived stem cell therapy The present invention relates to a composition for detecting a marker for measuring the titer of the composition, a composition for detecting a marker for measuring the aging of adipose derived stem cells, and a use thereof.

Regenerative medicine is a method for treating damaged or impaired tissues and organs, and is mainly related to cell-based therapy using stem cells with multipotency. Human mesenchymal stem cells are adult stem cells that are free from cancerization and ethical issues that are generally a problem for embryonic stem cells, as well as fat cells, osteoblasts, chondrocytes, heart cells, It is reported that differentiation into various cells such as muscle cells and neurons is possible. In addition, since it has an immunomodulatory function, it can be used as an immunosuppressive agent for homologous bone marrow transplantation or autoimmune disease.

Adipose tissue contains more than 1,000 times more stem cells than stem cells that can be separated from the same amount of bone marrow, and various studies are attempting to use adipose-derived stem cells (ASC) as a biotransplant material. Is going on. Adipose-derived stem cells also have the same multipotent as bone marrow stem cells, and can differentiate into cartilage, bone, fat, muscle cells, and the like. In addition, the adipose tissue-derived stromal stem cells have similar expression patterns of bone marrow-derived stem cells and cell surface markers, and do not induce an immune response during autologous or allogeneic transplantation in vivo and in vitro , but rather regulate a immune response. It has been reported to have a spotlight as an effective means of cell therapy.

However, in the case of stem cell therapeutics, the clinical results of stem cells published so far have confirmed the possibility of developing them as therapeutic agents, but the evaluation of the therapeutic effect is not satisfactory. One of the causes of the insufficient therapeutic effect of such stem cell therapy is that the quality assessment through the molecular biological characteristics analysis of stem cells related to the therapeutic effect is not performed properly. In the case of cell therapies, the quality of the result may be different even if the formulation is made using the same protocol due to the various characteristics of the cell donor in terms of age and health condition (Kretlow JD et al, BMC Cell Biol. 2008 Oct 28; 9). : 60).

Therefore, in order to develop and reproducibly produce stem cell therapeutics into actual medicines having a certain level of therapeutic effect and to produce reproducibly, it is strongly required to develop a quality control test method that reflects the effects such as therapeutic effects and potency of stem cells. In order to develop such cell therapeutic titer, biomarkers are needed to represent the therapeutic effect and titer of stem cells.

Therefore, the inventors have noticed that the differentiation efficiency and therapeutic effect of the low passage ASCs and the high passage ASCs are different, so that the young fat-derived stem cells and the aged fat-derived stem cells are different. We tried to find a group of genes that can distinguish cells, and to use them to determine the distribution of early passage ASCs of stem cell therapy through the expression of these marker genes. This may have a different meaning from a stem cell marker that divides known stem cells and differentiated cells. The present invention relates to the differentiation capacity of stem cells as a control gene during passage of stem cells collected from a donor, and is known to decrease with increasing passage, ALPL (Alkaline phosphatase transcript variant 1) and VCAM-1 (Vacular). IGF2BP3, ANXA10, MALL, PTGER2, and RGS20 genes increased adipose derived stem cell passages, confirming that these genes decreased with increasing passage of stem cells using cell adhesion molecule-1 transcript variant 1 (CD106). In addition, it was confirmed that the ITM2A, LGI2, SULF2, CCND2, and GADD45G genes decreased with the increase of adipose derived stem cell passage. Therefore, the present invention has been completed by verifying that these genes can be used as markers for identifying youth or aging states represented by passages of donor stem cells.

One object of the present invention is ANXA10 (annexin A10), IGF2BP3 (insulin-like growth factor 2 binding protein 3), MALL (Mal, T-cell differentiation protein-like), PTGER2 (prostaglandin E receptor 2), RGS20 (regulator) of G-protein signaling 20 transcript variant 1), integral membrane protein 2A (ITM2A), leucine-rich repeat LGI family, member 2 (LGI2), sulfatase 2 transcript variant 1 (SULF2), cyclin D2 (CCND2), and GADD45G (growth It provides a composition for detecting a marker for detecting the differentiation capacity of adipose derived stem cells, including an agent for measuring the level of one or more gene mRNA or protein thereof selected from the group consisting of arrest and DNA-damage-inducible, gamma) It is.

Another object of the invention comprises an agent for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. It is to provide a composition for detecting a marker for measuring the titer of adipose derived stem cell therapy.

Another object of the invention comprises an agent for measuring the level of at least one gene mRNA or protein thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. It is to provide a marker detection composition for measuring the aging of adipose derived stem cells.

Still another object of the present invention is to provide a marker detection kit for detecting a differentiation capacity of adipose derived stem cells, including a composition for detecting a marker for detecting the differentiation capacity of the adipose derived stem cells.

Still another object of the present invention is to provide a marker detection kit for measuring the titer of adipose derived stem cell therapeutic agent, comprising a composition for detecting a marker for measuring the titer of the adipose derived stem cell therapeutic agent.

Still another object of the present invention is to provide a marker detection kit for measuring aging of adipose derived stem cells, including a composition for detecting a marker for measuring aging of the adipose derived stem cell therapeutic agent.

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

Still another object of the present invention is to provide a method for measuring the titer of adipose derived stem cell therapeutic agent, which comprises measuring the expression level of the gene.

Still another object of the present invention is to provide a method for screening a substance capable of regulating differentiation capacity of adipose derived stem cells, including measuring the expression level of the gene.

Still another object of the present invention is to provide a method of controlling differentiation from adipose derived stem cells to adipocytes, the method comprising controlling the expression level of the gene.

The present invention provides useful data in verifying the efficacy of adipose derived stem cells by providing markers capable of determining the differentiation capacity of adipose derived stem cells. In addition, the markers may be useful for measuring the activity and titer of adipose derived stem cell therapeutics.

Figure 1a is a graph comparing the cell growth of adipose derived stem cells in basal medium and proliferation medium.

FIG. 1B is a cell photograph of passage 2 (P2), passage 5 (P5) and passage 8 (Passage 8, P8) in basal medium and proliferation medium.

Figure 2a is a test result to determine the differentiation capacity of P2 (Passage 2), P8 (Passage 8) and P14 (Passage 14) adipose derived stem cells. 12-day incubation in differentiation and differentiation medium followed by Oil-red O staining.

Figure 2b is a micrograph of the cells of Figure 2a, fat accumulation was confirmed by Oil-red O staining.

FIG. 2C is a graph showing the results of the experiment of FIG. 2A.

Figure 3a shows the results of microarray of three sets of P2, P5, P8 adipose derived stem cells using Illumina 24K chip.

3b to 3d are graphs showing expression changes of genes showing significance through microarray analysis. The ALPL and VCAM1 genes of FIG. 3b show the same results as the previous studies as control genes. 3c shows a gene whose expression decreases with increasing passage, and FIG. 3d shows a gene whose expression increases with increasing passage.

Figure 4 shows the change in the expression amount of the genes selected in Figure 3 in adipose derived stem cells collected from 17 donors by RT-PCR and real-time PCR, RPL13A is a control gene. Figure 4a is the result of the control genes ALPL and VCAM1 genes, Figures 4b and 4c shows the results of the genes ANXA10, IGF2BP3, PTGER2, MALL, RGS20 increased in P8 compared to P2, Figures 4d and 4e compared to P2 P8 It shows the result of decreasing genes ITM2A, LGI2, SULF2, CCND2, and GADD45G at.

Figure 5 shows the protein changes in P2 and P8 of the genes, and also verified the protein expression results and cell differentiation capacity.

Figure 6a is to classify and differentiate the cells expressing the representative gene PTGER2 and non-expressing cells, the differentiation capacity of the cells. It is shown by verifying the relationship between cell growth rate and passage gene expression.

Figure 6b is to classify and distinguish the cells expressing the representative gene ITM2A and non-expressing cells, and then verify the differentiation capacity, cell growth rate and passage-specific gene expression relationship of the cells.

Figure 7a is a representative gene in the group consisting of ANXA10, IGF2BP3, PTGER2, MALL, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G genes as a representative gene, and the TRNA-treated TNF-α after introducing the corresponding siRNA Next, as a result of examining the expression changes of NF-kB, Cox-2 and Erk1 / 2 related to inflammation, ALPL siRNA was used as a control.

Figure 7b shows a schematic diagram showing the relationship between the regulation of inflammation, mucosal regeneration and the like by the adipose stem cell therapy by the Cox-2 gene changes by the genes.

In one embodiment, the invention comprises an agent for measuring at least one gene mRNA or protein level thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. It provides a marker detection composition for detecting the differentiation capacity of adipose derived stem cells. The composition is one, two, three, four, five, six, seven, eight, selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. It may include agents that measure the mRNA or protein levels of dogs, nine or ten genes.

As used herein, the term "adipocyte-derived stem cell (ASC)" refers to stem cells isolated from adipose tissue capable of differentiating into most mesenchymal cells such as fat cells, osteoblasts, chondrocytes, and myofibroblasts. By means of adipose progenitor cells, stromal cells, multipotent adipose-derived cells or adipose derived adult stem cells (adipose derived adult stem cells). The adipose derived stem cells are not particularly limited thereto, but may be derived from mammals including pigs, cattle, primates, humans, and the like, which can be transplanted into humans.

In the present invention, the term "marker for detecting the differentiation capacity of adipose derived stem cells" refers to whether the expression of adipose derived stem cells with a high number of passages and adipose derived stem cells with a low passage, It may mean an organic biomolecule showing a significant difference. For the purposes of the present invention, the marker of the present invention refers to a marker capable of detecting differentiation capacity by increasing or decreasing the expression level in adipose derived stem cells having high differentiation capacity represented by a low passage number, more specifically ANXA10, The expression levels of IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G.AnXA10, IGF2BP3, MALL, PTGER2 and RGS20 are reduced in ICT-derived adipose derived stem cells, and ITM2A, LGI2, SULF2, CCND2 and GADD45G are genes with increased expression.

Information on the genes used in the present invention can be obtained from known databases such as GenBank of the National Institutes of Health, for example ANXA10 (NM_007193.3, NP_009124.2), IGF2BP3 (NM_006547.2, NP_006538.2), MALL (NM_005434.3, NP_005425.1), PTGER2 (NM_000956.2, NP_000947.2), RGS20 (NM_170587.1, NP_733466.1), ITM2A (NM_001171581.1, NP_001165052.1), LGI2 (NM_018176.2, NP_060646.2), SULF2 (NM_018837.2, NP_061325.1), CCND2 (NM_001759.3, NP_001750.1) and GADD45G (NM_006705.2, NP_006696.1), but are not limited thereto.

In the present invention, the term "passage" refers to a method of continuously culturing a cell stage while periodically transferring a portion of the cell to a new culture vessel in order to continuously cultivate the cell in a healthy state for a long period of time. In other words, it means replacing the culture vessel or dividing the cell group, and once replacing the culture vessel or dividing the cell group is called one passage. In the present invention, low passage ASCs of low or early passage mean adipose derived stem cells having high differentiation ability or therapeutic effect, and high passage ASCs of high passage or late passage. ) May refer to adipose derived stem cells having low differentiation or therapeutic effects, but are not limited thereto. In the present invention, the initial passage number may be 0 to 3, and preferably 0 to 2.

As used herein, the term "differentiation capacity" means the ability of the adipose derived stem cells to differentiate into adipocytes, osteoblasts, chondrocytes, myofibroblasts, osteoblasts, muscle cells or neurons, and the like. Phase differentiation capacity can be represented by the passage number of adipose derived stem cells. In other words, high differentiation capacity means that the passage number is low, so that differentiation into various cells can easily occur. Adipose derived stem cells having high differentiation capacity in the present invention are not limited thereto, and mean cells that easily induce differentiation into adipocytes, osteoblasts, chondrocytes, myofibroblasts, osteoblasts, muscle cells or neurons. Preferably, it refers to adipose derived stem cells which induce differentiation into adipocytes well. In the present invention, adipose derived stem cells having high differentiation ability are reduced in expression of one or more genes selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20, and selected from the group consisting of ITM2A, LGI2, SULF2, CCND2 and GADD45G. Adipose-derived stem cells with increased expression of one or more genes, which have increased expression of ANXA10, IGF2BP3, MALL, PTGER2, or RGS20 genes, or reduced expression of ITM2A, LGI2, SULF2, CCND2, or GADD45G. It refers to fat-derived stem cells having high efficiency of differentiation into adipocytes, osteoblasts, chondrocytes, myofibroblasts, muscle cells or neurons compared to cells. In one embodiment of the present invention, as a result of differentiating fat-derived adipose-derived stem cells of two passages, eight passages and 14 passages into adipocytes, it was confirmed that the lower the passage number, the better the differentiation ability (FIG. It was confirmed that the expression of ANXA10, IGF2BP3, MALL, PTGER2 or RGS20 genes was increased in the 8th generation cells, and that the expression of ITM2A, LGI2, SULF2, CCND2 or GADD45G genes was decreased (Figs. 3 to 5). The markers of the present invention can effectively separate fat-derived stem cells having the same initial passage or similar differentiation ability from adipose derived stem cells having various passage numbers or different differentiation capacity isolated from transplant donors, thereby treating adipose derived stem cells. The potency as can be raised.

Gene expression levels in biological samples can be confirmed by identifying the amount of mRNA or protein.

In the present invention, the term “mRNA level measurement” refers to a process of confirming mRNA presence and expression level of marker genes representing differentiation ability of adipose derived stem cells in a biological sample to diagnose differentiation ability of adipose derived stem cells. Measure the amount of. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and 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 agent for measuring mRNA level of the gene is preferably a primer pair or probe, and the nucleic acid sequences of the ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G genes are NM_007193.3, NM_006547.2, respectively. , NM_005434.3, NM_000956.2, NM_170587.1, NM_001171581.1, NM_018176.2, NM_018837.2, NM_001759.3, and NM_006705.2 are known to those skilled in the art based on these sequences A primer or probe can be designed to amplify the protein.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, which forms a base pair with a complementary template and functions as a starting point for template strand copying. Means. Examples of primers in the present invention include a primer pair for ANXA10 with

SEQ ID NOs

7 and 8, a primer pair for IGF2BP3 with

SEQ ID NOs

9 and 10, a primer pair for MALL with

SEQ ID NOs

11 and 12, PTGER2 with

SEQ ID NOs

13 and 14 Primer pair for RGS20, SEQ ID NOs: 15 and 16, primer pair for ITM2A, SEQ ID NOs: 17 and 18, primer pair for LGI2, SEQ ID NOs: 19 and 20, for SULF2, SEQ ID NOs: 21 and 22 Primer pairs, primer pairs for CCND2 with SEQ ID NOs: 23 and 24, or primer pairs for GADD45G with SEQ ID NOs: 25 and 26, but are not limited thereto.

As used herein, the term “probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases or hundreds of bases that can form specific binding with mRNA, and is labeled to indicate the presence or absence of a specific mRNA. You can check it. The probe may be manufactured 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 a complementary probe with ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 or GADD45G polynucleotides, and it is possible to know the differentiation ability of adipose derived stem cells through hybridization. have. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Primers of the present invention can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules and biotin.

In the present invention, the term "protein level measurement" refers to a process for confirming the presence and expression level of a protein expressed from a marker gene representing a differentiation capacity of adipose derived stem cells in a biological sample in order to detect the differentiation ability of adipose derived stem cells. Preferably, the amount of protein can be confirmed 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 purposes of the present invention, an antibody means an antibody that specifically binds to a marker protein, and specifically, the marker gene of the present invention, IGF2BP3, ANXA10, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 or GADD45G Refers to an antibody that specifically binds to the protein to be encoded, and includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the marker proteins of the present invention have been identified, the production of antibodies using them can be readily prepared using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for injecting the marker protein antigen into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow and dog. Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having 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 antigen binding function, and includes Fab, F (ab '), F (ab') ₂ and scFv. Protein levels can be measured by Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion. , Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chip. However, the present invention is not limited thereto. In one embodiment of the present invention, the Western blot was used to measure the level of the gene protein (Example 4).

In another embodiment, the invention comprises an agent for measuring at least one gene mRNA or protein level thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. It provides a composition for detecting a marker for measuring the titer of adipose derived stem cell therapeutic agent.

As used herein, the term "fat-derived stem cell therapeutic agent" refers to the proliferation and selection of live autologous, allogenic, or xenogenic cells in vitro, or other methods for restoring the function of cells and tissues. By using a series of actions such as changing the biological characteristics of fat-derived stem cells, medicines used for the purpose of treatment, diagnosis and prevention, or fat cells, osteoblasts, chondrocytes, and muscle fibers formed by the differentiation of fat-derived stem cells It means a therapeutic agent for the purpose of regeneration of parental cells, muscle cells or nerve cells.

In the present invention, the term "titer" refers to the therapeutic activity of the stem cell therapy, and preferably may refer to the differentiation or therapeutic ability of the adipose derived stem cell therapy.

In one embodiment of the present invention, the protein level of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 is low, and the expression level of ITM2A, LGI2, SULF2, CCND2 and GADD45G, P2 fat derived stem cells are excellent in differentiating ability to adipocytes (FIG. 5), cells with low expression level of PTGER2 and high expression levels of ITM2A were isolated to induce differentiation into adipocytes. As a result, cells with low expression level of PTGER2 were found to be adipocytes as compared to cells with high expression level. Differentiation occurs well, and it was confirmed that differentiation into adipocytes was better than that of cells having high ITM2A expression level (FIG. 6). In addition, when PTGER2 siRNA was used to lower the expression level of PTGER2 in adipose derived stem cells, the expression level of NF-kB and its target gene COX-2 were also reduced. It was confirmed that it can be a marker that can indicate the inhibitory therapeutic activity, which is one of the therapeutic activities of adipose derived stem cells (FIG. 7).

The gene mRNA or protein level measurement thereof is the same as described above.

In another embodiment, adipose derived, comprising an agent that measures one or more gene mRNAs or protein levels thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G It provides a composition for detecting markers for measuring the aging of stem cells.

As used herein, the term "aging" means that the differentiation capacity or proliferation capacity of adipose derived stem cells is decreased due to an increase in the age of an individual or an increase in the number of passages. For the purposes of the present invention, aged adipose derived stem cells have increased expression of one or more genes selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 compared to the control group, or ITM2A, LGI2, SULF2, CCND2 and GADD45G. Expression of one or more genes selected from the group consisting of can refer to adipose derived stem cells are reduced compared to the control. In one embodiment of the present invention, the expression of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 proteins is reduced, and the adipose derived stem cells having increased expression of ITM2A, LGI2, SULF2, CCND2 and GADD45G proteins are ANXA10, IGF2BP3, MALL , Induction of differentiation into adipocytes was better compared to adipose derived stem cells having increased expression of PTGER2 and RGS20 proteins and decreased expression of ITM2A, LGI2, SULF2, CCND2 and GADD45G proteins (Example 4 And FIG. 5) . In addition, in one embodiment of the present invention as a result of comparing the proliferative capacity of adipose derived stem cells with different expression level of PTGER2 in 10 passages or more, in the case of cells with low expression level of PTGER2, the cells with high expression level of PTGER2 Compared to the cells with high proliferative capacity and resulted in greater proliferation, the proliferative capacity of cells with high ITM2A expression was superior to those with low ITM2A expression (Example 5 and FIG. 6).

As another aspect, the present invention provides a marker detection kit for detecting a differentiation capacity of adipose derived stem cells, including a composition for detecting a marker for detecting the differentiation capacity of the adipose derived stem cells.

The marker detection kit of the present invention is used in immunological analysis as well as primers or antibodies capable of selectively recognizing marker genes or proteins thereof whose expression is increased or decreased depending on the number of passages or their differentiation capacity. Tools and reagents commonly used in the art. Such tools or reagents include, but are not limited to, suitable carriers, labeling materials capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. If the label is an enzyme, it may include a substrate and a reaction terminator that can measure the activity of the enzyme. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

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

The RT-PCR kit may comprise individual primer pairs specific for the marker gene, as well as other test tubes or other suitable containers, reaction buffers (variable pH and magnesium concentrations), 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 oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, an agent, an enzyme, etc. for preparing a fluorescent probe, and the substrate may be a control gene. Or cDNA or oligonucleotide corresponding to fragments thereof.

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

In an embodiment of the present invention, the expression difference of marker genes of adipose derived stem cells having

passage numbers

2, 5 and 8 was confirmed using a microarray (Examples 2 and 3).

As another aspect, the present invention provides a marker detection kit for measuring the titer of adipose derived stem cell therapeutic agent, comprising a composition for detecting a marker for measuring the titer of the adipose derived stem cell therapeutic agent.

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

As another aspect, the present invention provides a marker detection kit for measuring the aging of adipose derived stem cells, including a marker detection composition for measuring the aging of the adipose derived stem cells.

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

In another embodiment, the invention comprises measuring the level of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. To provide a method for detecting the differentiation ability of adipose derived stem cells. The method comprises one, two, three, four, five, six, seven, eight, selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. Measuring the mRNA or protein level of the dog, nine or ten genes.

The method preferably determines that if the amount of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 is less than the amount of the control group, the differentiation capacity is higher than that of the control group. Or if the amount of one or more gene mRNAs or proteins thereof selected from the group consisting of ITM2A, LGI2, SULF2, CCND2, and GADD45G is greater than the amount of the control group, determining that the differentiation capacity is higher than that of the control group. It may be a method for detecting the differentiation ability of the adipose derived stem cells, including. Measurement of the level of mRNA or its protein is the same as described above.

In one embodiment of the present invention, the expression of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 is reduced, and the adipose derived stem cells with increased expression of ITM2A, LGI2, SULF2, CCND2 and GADD45G are ANXA10, IGF2BP3, MALL, PTGER2 And it was confirmed that differentiation into adipocytes is better than adipose derived stem cells having increased expression of RGS20 and decreased expression of ITM2A, LGI2, SULF2, CCND2 and GADD45G (FIG. 5). In addition, when adipose derived stem cells with low expression of PTGER2 induced differentiation into adipocytes, it was confirmed that differentiation into adipocytes was better than that of cells with high expression of PTGER2 (FIG. 6A), and ITM2A expression. The high fat-derived stem cells were isolated and compared with the cells with low expression of ITM2A to adipocytes. As a result, it was confirmed that the cells with high expression of ITM2A had better differentiation capacity (FIG. 6B).

In another embodiment, the invention comprises measuring the level of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. To provide a method for measuring the titer of adipose derived stem cell therapy.

Determining that the level of one or more genes mRNA or protein selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 decreases or shows a low level compared to the control group, and judges that it has a high differentiation capacity It may include, ITM2A, LGI2, SULF2, CCND2 and GADD45G one or more genes selected from the group consisting of a high differentiation ability, while confirming that the level of the protein is increased or higher than that of the control group And determining that it is.

As another aspect, the present invention comprises the steps of treating a differentiation capacity control candidate substance to adipose derived stem cells; And measuring an increase or decrease in one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G in adipose derived stem cells. In addition, the present invention provides a method of screening a substance for regulating the differentiation capacity of adipose derived stem cells.

In the present invention, the term "differentiating ability of adipose derived stem cells" is a substance that indirectly or directly inhibits or induces a change in the amount of gene expression significantly occurring according to the passage of adipose derived stem cells or a differentiation ability represented by the same. Means. Differentiation capacity modulators can further differentiate by increasing the expression of a group of proteins (eg, ITM2A, LGI2, SULF2, CCND2 and GADD45G) that have increased expression in adipose derived stem cells with low passage numbers or high differentiation capacity. Can be induced and controlled to inhibit differentiation by reducing its expression. In addition, in the case of proteins whose expression is reduced (eg, ANXA10, IGF2BP3, MALL, PTGER2 and RGS20), the expression may be further reduced to induce differentiation or increase its expression to inhibit differentiation. Therefore, the differentiation capacity of the adipose derived stem cells can be controlled by comparing the increase or decrease in the expression level of the differentiation detection marker gene of the adipose derived stem cells in the presence and absence of the candidate for differentiation control, and furthermore, It can be usefully used for screening.

In another embodiment, the invention provides fat-derived by increasing or decreasing one or more gene mRNAs or protein levels thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G. Provided are methods for controlling differentiation of stem cells to adipocytes.

The gene mRNA or its protein level can be increased using various methods known in the art that can increase the gene expression rate in a cell, preferably to construct a vector comprising the genes and transduce them into the cell. It is possible to increase the expression rate of these genes, but is not limited thereto. In addition, the gene mRNA or protein level thereof may be reduced using methods known in the art that can delete genes in a cell or cause loss of gene function through gene mutation, preferably in the genes. Small hairpin RNAs (shRNAs) can be produced and transduced into cells to lower the expression of these genes, but are not limited thereto. For the purposes of the present invention, to increase the differentiation of adipose derived stem cells into adipocytes, shRNAs for one or more genes selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 are prepared and transduced into cells. Expression of these genes can be lowered, and vectors comprising one or more genes selected from the group consisting of ITM2A, LGI2, SULF2, CCND2, and GADD45G can be prepared, and these cells can be transduced to increase the expression rate.

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

실시예 1: 지방유래줄기세포 계대(passage)에 따른 분화능력 검증Example 1 Verification of Differentiation Ability According to Adipose-Derived Stem Cell Passage

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

Adipose tissue was isolated from donors (Antrogen, Gyeonggi-do, Korea). Adipose derived stromal stem cells were isolated from the obtained adipose tissue. Adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. The same volume of collagenase solution as adipose tissue was added and reacted in a 37 ° C. water bath. This was transferred to a centrifuge tube and centrifuged at 20 ° C. and 1200 rpm for 10 minutes. The supernatant fat layer was removed, and the lower collagenase solution was carefully separated so as not to shake. After the substrate medium was suspended and centrifuged for 5 minutes at 20 ℃, 1200 rpm. At this time, since the sinking below is the stromal-vascular fraction, the supernatant was removed. The stromal-vascular fractions were suspended in substrate media and inoculated in culture vessels and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After removal of the culture solution, the cells were washed with phosphate buffer solution and 5 ng of epidermal growth factor (EGF) was added to the substrate medium or the medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml. Proliferation was performed using the medium contained at a / ml concentration. When adipose derived stromal stem cells were grown to about 80-90% of the culture vessel, they were obtained by separating into single cells by treating with trypsin. The obtained cells were diluted 1: 3 to 1: 4 with proliferation medium to carry out passage culture (Korean Patent Publication No. 10-2010-0118491).

To analyze the gene expression differences according to the composition and passage of culture medium, two passages cultured with basal medium of DMEM containing 10% FBS and growth medium containing 1 ng / ml bFGF in basal medium Cells from 5 passages and 8 passages were collected.

As a result of the culture of 5 lots, the growth rate was higher and the fibroblast morphology was stably maintained when cultured with the growth medium than when the basal medium was cultured. FIG. 1A shows an example of cell population doubling levels (CPDL) of adipose derived stem cells cultured in proliferation medium, and FIG. 1B shows a morphology picture of adipose stem cells cultured in proliferation medium in one sample.

(2) Induction of differentiation of 2nd (P2), 8th (P8), 14th (P14) cells of human adipose derived stem cells

Adipose stem cells were suspended in proliferation medium to inoculate each of 90,000 cells in a 24-well culture vessel, incubated for 24 hours in a 37%, 5% CO ₂ incubator and attached to the bottom. When the cells were filled with a single layer at the bottom of the culture vessel, the cells were changed to a substrate medium, incubated for 2-3 days in a 37 ° C., 5% CO ₂ incubator, and then changed into differentiation medium and cultured for 5 days. After that, the differentiation medium was removed and changed into differentiation maintenance medium (fat cell medium, adipocyte media) and cultured for 12 days while changing to new differentiation maintenance medium every 3 days.

Substrate medium is DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% bovine serum and 0.1% antibiotic and proliferation medium is DMEM / F12, 10% bovine serum, 5 ng / ml EGF, 0.25 ng / ml bFGF, 0.25 ng / Ml TGF-β1, 0.1% antibiotic. Differentiation medium contains DMEM / F12, 3% FBS, 33 μM biotin, 17 μM pantothenate, 1 μM insulin, 1 μM dexamethasone, 250 μM isobutylmethylxanthine, 100 μM indomethacin and differentiation maintenance medium DMEM / F12, 3% bovine serum, 33 μM biotin, 17 μM pantothenate, 100 nM insulin and 1 μM dexamethasone.

(3) Confirmation of fat cell differentiation form (Oil-Red O staining)

Oil-red O staining was performed to morphologically confirm the differentiation of adipose derived stem cells into adipocytes. Differentiated cells were rinsed with PBS solution (phosphate buffered saline) and fixed with 10% formalin. After fixing for 12 hours, the mixture was reacted for 20 minutes by adding 60% isoprophenol and stained with 0.5% Oil Red O solution (SIGMA 00625) at room temperature for 1 to 3 hours. After staining, the cells were washed once with 60% isoprophenol, three times with distilled water, dried, and then measured in the microscope to determine the percentage of cells that had undergone adipogenesis. Thereafter, 100% isoprophenol was added to elute the Oil Red O dye from the dyed fat drops, and the absorbance was measured at 520 nm. As a result, the differentiation ability of adipose derived stem cells of 2nd, 8th and 14th passages was confirmed.

An example of a plate stained with Oil-red O is shown in FIG. 2A, and an example of a microscopic picture of cells stained with Oil-red O is shown in FIG. 2B. FIG. 2C is a quantitative measure of the differentiation result of the sample. The mean value is shown. It was confirmed that the fat-derived stem cells of the second passage had the highest differentiation ability, and that the fat-derived stem cells of the early passage had higher differentiation capacity.

실시예 2: 마이크로 어레이 실험에 의한 지방유래줄기세포의 계대별 유전자 발현 증감 확인Example 2: Confirmation of gene expression change by passage of adipose derived stem cells by microarray experiment

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

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

(2) total RNA isolation

Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantified using Experion RNA StdSens (Bio-Rad) chip. First, the cultured adipose derived stem cells were lysed in 15 ml of digestion buffer in a kit to which 150 µl of beta mercapto ethanol was added. 15 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After two washes, total RNA was isolated by adding 1.2 ml of RNase-free water.

(3) Microarray

The extracted total RNA was hybridized using an Illumina TotalPrep RNA Amplification Kit (Ambion). CDNA was synthesized using T7 Oligo (dT) primers and biotin-UTPin vitro Warrior(in vitro transcription) to prepare biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. Manufactured from adipose derived stem cells cRNA was hybridized to a Human-6 V2 (Illumina) chip. After hybridization, DNA chips were washed using Illumina Gene Expression System Wash (Illumina) to remove nonspecific hybridization, and the washed DNA chips were labeled with streptavidin-Cy3 (Amersham) fluorescent dye. Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina, Inc.) to obtain data of fluorescence present in each spot and stored as TIFF image files. TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantified results were corrected using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.

The results are shown in FIG. Figure 3a shows the difference in expression of the genes measured in passage 2 cells and passage 5, passage 8 cells cultured in proliferation medium. IGF2BP3, ANXA10, MALL, PTGER2, and RGS20 genes show a significant increase in P8 compared to P2, and ITM2A, LGI2, SULF2, CCND2, and GADD45G are genes with reduced expression in P8 compared to P2. ALPL and VCAM1 are known to decrease in relation to differentiation capacity in the literature as control genes, and also showed significant decrease in this experiment. From these results, it can be seen that the genes can be used for diagnosing the differentiation and therapeutic ability of stem cells because the genes show a large expression difference when comparing P2 and P8. However, expression in differentiated cells is independent of expression changes according to predifferentiation passages. For example, IGF2BP3 increases with passage in the stem cell state before differentiation, but rapidly decreases when differentiation begins. In the case of ITM2A, it decreases with passage in the stem cell state but increases when differentiation begins. Figure 3a shows the micro array results, Figures 3b to 3d graphically shows the difference in the expression level of the genes by passage.

실시예 3: 개별 공여자의 지방유래 줄기세포로부터 초기 계대(early Passage, < 2)를 대변할 수 있는 상기 유전자 발현 확인Example 3 Identification of the Gene Expression That Can Represent Early Passage (<2) from Adipose-Derived Stem Cells from Individual Donors

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

(1) cDNA synthesis and concentration correction of template

2 μg of total RNA of each sample, 1 μl of 50 μM Olgo (dT) primer and 2.5 μl of 10 mM dNTP were added, and 25 μl of sterase water containing DEPC, an RNase inhibitor, was prepared so that the total mixture was 25 μl. After reacting at 65 ° C. for 5 minutes, the mixture was transferred to 55 ° C. and stored. Next, 5 µl of 10X 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 were added to make 25 µl. The RNA / primer mixture was stored at 55 ° C. After mixing with, it was reacted at 55 ℃ for 50 minutes. Thereafter, the reaction was carried out at 85 ° C. for 5 minutes to inactivate the RT enzyme, and then placed on ice to terminate the reaction. PRL13A was used as a standard gene for quantifying marker genes. RT-PCR reaction was performed using primers of the standard gene, and the concentration of cDNA was corrected so that the expression level of the standard gene RPL13A was the same. First, each cDNA was diluted 20-fold, and then PCR reaction was performed using 2 μl of the diluted sample. PCR was performed 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. 20, 23, and 25 cycles were performed. RT-PCR reaction conditions were performed at 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minutes. The PRL13A primers used were forward 5'-CATCGTGGCTAAACAGGTACTG-3 '(SEQ ID NO: 1), reverse 5'-GCACGACCTTGAGGGCAGC-3' (SEQ ID NO: 2). Each product was loaded into a 2% agarose gel, electrophoresed, electrophoresed, and gel images were taken. The images were quantified using the TotalLab v1.0 program (Nonlinear Dynamix), and then corrected for PCR. The concentration of was equally corrected.

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

CDNA diluted to the same amount was subjected to PCR using the sense and antisense primers of the genes. cDNA was mixed with 3µl, 2x premix 10µl, primer 2µl (20 pmole), and 2µl of distilled water to make 20µl of total solution.The PCR reaction was 94 ℃ 1min, 54 ℃ 30sec, 72 ℃ Cycle was different for each gene. In order to confirm the PCR product, it was electrophoresed using 2% agarose gel and analyzed using an imaging apparatus. Realtime RT-PCR was used with DNASYBR®GreenI reagent and LightCycler (Roche) from Qiagen (CA, USA). Melt Curve analysis was used to evaluate the quality of PCR products and gene expression levels were analyzed using LightCycler version 3.5 software (Roche). The specific primers of the genes used in the RT-PCR / Real-Time PCR and the sequence of the control primer (SEQ ID NO: 1 to SEQ ID NO: 26) are shown in Table 1. The results are shown in FIG.

As a result, it was confirmed that the above genes were significantly increased or decreased in P8 compared to P2 (control gene ALPL (64.7%, decreased in 11 of 17 samples), and control gene VCAM (58.8%, in 10 samples). Decrease) (FIG. 4A), ANXA10 (94.1%, increased in 16 samples), IGF2BP3 (76.4%, increased in 13 samples), MALL (58.8%, increased in 10 samples), PTGER2 (58.8%, 10) Increase in sample), RGS20 (64.7%, increase in 11 samples) (FIGS. 4B and 4C), ITM2A (94.1%, decrease in 16 samples), LGI2 (76.4%, decrease in 13 samples), SULF2 (82.4) %, Decreased in 14 samples), CCND2 (82.4%, decreased in 14 samples) and GADD45G (76.4%, reduced in 13 samples) (FIGS. 4D and 4E), the genes representing the aging of fat stem cells It may be used as a diagnostic marker or a marker for screening drugs related to stem cell differentiation.

Table 1

Gene (REF)	primer	SEQ ID NO:
RPL13A (NM_012423.02)	F: 5'-catcgtggctaaacaggtactg-3 '	SEQ ID NO: 1
RPL13A (NM_012423.02)	R: 5'-gcacgaccttgagggcagc-3 '	SEQ ID NO: 2
ALPL (NM_000478.3)	F: 5'-acagacaagaagcccttcac-3 '	SEQ ID NO: 3
ALPL (NM_000478.3)	R: 5'-gttgtgagcatagtccacca-3 '	SEQ ID NO: 4
VCAM1 (NM_001078.2)	F: 5'-tgcgggagtatatgaatgtg-3 '	SEQ ID NO: 5
VCAM1 (NM_001078.2)	R: 5'-acgagaagctcaggagaaaa-3 '	SEQ ID NO: 6
ANXA10 (NM_007193.3)	F: 5'-cgagacaaaccagcctattt-3 '	SEQ ID NO: 7
ANXA10 (NM_007193.3)	R: 5'-tggtcagcaggtctatttca-3 '	SEQ ID NO: 8
IGF2BP3 (NM_006547.2)	F: 5'-cacctgatgagaatgaccaa-3 '	SEQ ID NO: 9
IGF2BP3 (NM_006547.2)	R: 5'-actttgcagagccttctgtt-3 '	SEQ ID NO: 10
MALL (NM_005434.3)	F: 5'-acaagtacatgccacgattg-3 '	SEQ ID NO: 11
MALL (NM_005434.3)	R: 5'-aggcatggagaatgtagagc-3 '	SEQ ID NO: 12
PTGER2 (NM_000956.2)	F: 5'-cacctcattctcctggctat-3 '	SEQ ID NO: 13
PTGER2 (NM_000956.2)	R: 5'-gaggtcccatttttcctttc-3 '	SEQ ID NO: 14
RGS20 (NM_170587.1)	F: 5'-tcaacagaaacatggtggag-3 '	SEQ ID NO: 15
RGS20 (NM_170587.1)	R: 5'-tctccgataaggactgaagc-3 '	SEQ ID NO: 16
ITM2A (NM_001171581.1)	F: 5'-ggctgacattcgtgaggatg-3 '	SEQ ID NO: 17
ITM2A (NM_001171581.1)	R: 5'-tgaggggcatcagatagcag-3 '	SEQ ID NO: 18
LGI2 (NM_018176.2)	F: 5'-caatgacatcgagctgtttc-3 '	SEQ ID NO: 19
LGI2 (NM_018176.2)	R: 5'-ctcgtgcagtgactggtaag-3 '	SEQ ID NO: 20
SULF2 (NM_018837.2)	F: 5'-tgatgaatgcagtgaacaca-3 '	SEQ ID NO: 21
SULF2 (NM_018837.2)	R: 5'-tttaagtcccaggtccatgt-3 '	SEQ ID NO: 22
CCND2 (NM_001759.3)	F: 5'-gatcgcaactggaagtgtgg-3 '	SEQ ID NO: 23
CCND2 (NM_001759.3)	R: 5'-tggtgatcttagccagcagc-3 '	SEQ ID NO: 24
GADD45G (NM_006705.2)	F: 5'-cgagaacgacatcgacatag-3 '	SEQ ID NO: 25
GADD45G (NM_006705.2)	R: 5'-gttcgaaatgaggatgcagt-3 '	SEQ ID NO: 26

실시예 4: 선별된 유전자의 계대에 따른 단백질 발현 조사 Example 4: Investigation of protein expression according to passage of selected genes

The amount of expression of the genes identified in Example 3 using donor adipose stem cell samples (P2 and P8) was analyzed by protein quantification by Western blot analysis. P2 and P8 cells used were differentiated simultaneously with protein analysis to verify their differentiation potential.

Specifically, after rinsing the cells grown in a 60 mm dish with PBS once, the cold protein lysis buffer (RIPA cell 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 inhibitors) were added to prepare the proteins of the cells. Using 30 μg of each prepared protein sample, 10% or 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel was used to separate proteins by size, and then transferred to PVDF membrane.

After removing the filter to which the protein was transferred to an appropriate size, an antibody corresponding to each protein was attached. Information on antibodies is as follows. Anti-ALPL (Sigma, HPA008765), anti-LGI2 (Santa Cruz, sc-74723), anti-ITM2A (Santa Cruz, sc-134811), anti-SULF2 (R & D, MAB7087), anti-CCND2 (Santa Cruz, sc -181), anti-GADD45G (abcam, ab56802), anti-ANXA10 (Santa Cruz, sc-70009), anti-IGF2BP3 (Santa Cruz, sc-100766), anti-MALL (Santa Cruz, sc-168544), anti -PTGER2 (abcam, ab16151), anti-RGS20 (abcam, ab77009), anti-ACTIN (Sigma, A5316). The amount of each protein was identified by band using Immobilon ^™ Western Blotting Detection reagents (Millipore).

As a result, it was observed that ALPL, LGI2, ITM2A, SULF2, CCND2, and GADD45G proteins, which are genes whose mRNA levels decrease with RT-PCR, are expressed in P2 and decreased in P8. In the case of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 proteins, which are genes whose mRNA levels increase with increasing passage, the expression was low in P2 and increased in P8 (FIG. 5). P2 and P8 cells used were induced differentiation as in Example 3 to verify the differentiation capacity of each passage. As shown in FIG. 5, P2 cells differentiated well from adipose stem cells, and P8 cells were differentiated, but the degree of differentiation was reduced compared to P2 cells. Therefore, the difference in the expression level of the gene proved to be useful as a marker for recognizing the young and aging of adipose stem cells.

실시예 5: 막단백질 유전자인 PTGER2와 ITM2A의 항체를 이용한 세포의 분리; 및 유전자 발현 정도와 분화 증가량 관련성 조사Example 5: Isolation of cells using antibodies of membrane protein genes PTGER2 and ITM2A; Of gene expression and differentiation increase

The cultured cells were separated into single cells using trypsin, suspended in 0.5 ml buffer, and monoclonal antibodies against PTGER2 and ITM2A were added. After mixing well, the mixture was reacted at 4 ° C. for 30 minutes, washed with a buffer, centrifuged at 250 g for 10 minutes, cells were collected, and suspended again in 150 μl of buffer. The secondary antibody labeled with fluorescence was added and reacted at 4 ° C. for 30 minutes, and further reacted for 15 minutes at a place where light at room temperature did not pass. This was washed with a buffer and suspended in 3 ml of phenol red-free medium. Thereafter, cells separated by fluorescence and separated by unlabeled cells were separated using a cell sorter. These isolated cells were used for differentiation induction, cell growth rate and protein expression change.

The separated cells were placed in 24 wells (for differentiation and cell growth investigation) and 12 wells (for protein extraction) Corning plates to induce differentiation using the same method as in Examples 3 and 4, or to express cell growth and protein expression. Etc. were observed.

As a result, it was observed that cells that did not express PTGER2 had better differentiation induction than cells that had high PTGER2 expression. It was found that the rapid growth of cells with low PTGER2 expression was rapidly crossed (FIG. 6A). In the case of ITM2A, in contrast to PTGER2, cells with high ITM2A expression were better differentiated and cell growth was faster after passage 10 (Fig. 6b).

Therefore, the genes of the present invention, as well as the expression changes depending on the passage, the effect of differentiation ability and cell growth depending on whether the expression of the gene is changed, the expression level of the gene represents the degree of aging of the stem cells It can be used as a diagnostic marker or a stem cell therapeutic marker.

실시예 6: 대표유전자로서 ALPL, PTGER2 및 ITM2A에 해당하는 siRNA 도입에 따른 염증관련 NF-kB, Cox-2 및 Erk1/2의 발현변화 조사 Example 6: Investigation of the expression changes of inflammation-related NF-kB, Cox-2 and Erk1 / 2 according to the introduction of siRNA corresponding to ALPL, PTGER2 and ITM2A as representative genes

Human adipose derived stem cells were plated for 5 hours after placing 5 × 10 ⁵ cells in a 6 well plate. Then, 500 μl of Opti-MEM medium was transformed with siRNA and control siRNA (40 nmol) of the gene, respectively. Each of these samples was added to the siRNA transfection reagent hiperfect (Qiagen) according to the manufacturer's manual, mixed well using a stirrer, and allowed to stand for 15 minutes to form a siRNA transfection complex at room temperature. During this time, the medium in each well was changed to 1.5 ml of fresh 10% FBS-DMEM, and after 15 minutes, each sample was slowly added dropwise to each well. After 48 hours of incubation time, the cell injection of siRNA using hiperfect (Qiagen) was performed again using the same concentration of siRNA, and 500 μl of Trizol was added 72 hours after the addition of the second siRNA. RNA was extracted from each sample, and knock-down of the gene was first confirmed by siRNA.

In order to artificially induce inflammation after treatment with siRNA twice, TNF-α was treated at 5 ng / ml, followed by 120 minutes at 15 to 30 minute intervals. Samples were collected and 200 μl of RIPA cell lysis buffer was added to extract proteins, and the expression changes of NF-kB, Cox-2, and Erk1 / 2 protein were investigated. Antibodies used were anti-phospho-Erk1 / 2 (Pharmingen, 554093), anti-phospho-NF-kB (Cell signaling, # 3037) and anti-Cox-2 (Santa Cruz, sc-6248). The amount of protein used in the experiment was confirmed by blotting through antibodies to GAPDH (anti-GAPDH, cell signaling).

As a result, in the case of ALPL used as a control, there was no change in NF-kB, but the increase in Cox-2 expression was reduced by ALPL knock-down (FIG. 7A). In addition, as a result of processing siRNA of the representative gene ITM2A among genes decreasing with passage, there was no change in F-kB similar to the siRNA treatment of the control gene ALPL, but an increase in Cox-2 expression was caused by ITM2A knock-down. It can be seen that the effect is remarkable compared to ALPL. In addition, the siRNA of the representative gene PTGER2 among the genes increased according to the passage was found to decrease the activity of the NF-kB gene and to significantly reduce the expression of inflammation related Cox-2 gene. This suggests that Cox-2 induces inflammation associated with NF-kB (Fig. 7B), and therefore, the PTGER2 gene is adipose because it is expected that adipose stem cell therapeutics will generally inhibit inflammation in the affected area. It was confirmed that it can function as a marker gene for the passage and therapeutic activity of stem cells (Fig. 7a). In addition, as shown in FIG. 7B, Prostagrandin E2 (PGE2), which is increased by Cox-2, may have different functions such as inflammation and mucosal regeneration according to its level, and thus, the anti-inflammatory effect of fat stem cells may be treated with Cox-2. It can be predicted that it will be regulated by the homeostasis of -2 and PGE2.

Therefore, the genes are expected to be used as markers for predicting the future differentiation ability of adipose derived stem cells, and also proved to be involved in mechanisms such as adipose stem cell growth control, inflammation regulation, and mucosal regeneration.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims

ANXA10 (annexin A10), IGF2BP3 (insulin-like growth factor 2 binding protein 3), MALL (Mal, T-cell differentiation protein-like), PTGER2 (prostaglandin E receptor 2), regulator of G-protein signaling 20 transcript variant 1), integral membrane protein 2A (ITM2A), leucine-rich repeat LGI family, member 2 (LGI2), sulfatase 2 transcript variant 1 (SULF2), cyclin D2 (CCND2), and growth arrest and DNA-damage-inducible , gamma) one or more genes selected from the group consisting of an agent measuring the protein level thereof, comprising a marker detecting composition for detecting the differentiation capacity of adipose derived stem cells.
Titers of adipose derived stem cell therapeutics, including agents measuring levels of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2, and GADD45G Marker detection composition for measuring.
Aging of adipose derived stem cells, including an agent measuring the level of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G Marker detection composition for measurement.
The composition of claim 1, wherein the agent for measuring the level of the gene mRNA comprises a primer that specifically binds to the gene.
According to claim 4, wherein primers for ANXA10 among the primers specifically binding to the gene is a primer pair of SEQ ID NO: 7 and 8, primers for IGF2BP3 is a primer pair of SEQ ID NO: 9 and 10, primers for MALL is a sequence Primer pairs 11 and 12, primers for PTGER2 are primer pairs SEQ ID NO: 13 and 14, primers for RGS20 are primer pairs SEQ ID NO: 15 and 16, primers for ITM2A are primer pairs SEQ ID NOs: 17 and 18, Primers for LGI2 are primer pairs SEQ ID NOs: 19 and 20, primers for SEQ ID NOs: 21 and 22, primer pairs for CCND2, primer pairs SEQ ID NOs: 23 and 24, and primers for GADD45G are SEQ ID NO: 25 And a primer pair of 26.
The composition of claim 1, wherein the agent for measuring the level of the protein comprises an antibody specific for the protein.
Marker detection kit for detecting the differentiation capacity of the adipose derived stem cells comprising the composition of claim 1.
Marker detection kit for measuring the titer of adipose derived stem cell therapy comprising the composition of claim 2.
Marker detection kit for measuring the aging of fat-derived stem cells comprising the composition of claim 3.
The kit for detecting a marker according to any one of claims 7 to 9, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit.
Differentiation ability of adipose derived stem cells, comprising measuring the level of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G How to detect it.
The method of claim 11, wherein if the amount of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2 and RGS20 is less than the amount of the control group, it is determined that the differentiation capacity is higher than that of the control group. Further comprising the step of detecting the differentiation capacity of the adipose derived stem cells.
12. The method of claim 11, wherein if the amount of one or more gene mRNAs or proteins thereof selected from the group consisting of ITM2A, LGI2, SULF2, CCND2, and GADD45G is higher than that of the control group, the differentiation capacity is higher than that of the control group. Further comprising, a method for detecting the differentiation ability of the adipose derived stem cells.
Titer of adipose derived stem cell therapy comprising measuring the level of one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G How to measure it.
Treating adipose derived stem cells with candidate substances for controlling differentiation capacity; And

Measuring an increase or decrease in one or more gene mRNAs or proteins thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G in adipose derived stem cells, A method for screening a differentiation regulating substance of adipose derived stem cells.
Fat tax from adipose derived stem cells, comprising increasing or decreasing one or more gene mRNAs or protein levels thereof selected from the group consisting of ANXA10, IGF2BP3, MALL, PTGER2, RGS20, ITM2A, LGI2, SULF2, CCND2 and GADD45G How to control the differentiation of prisoners.