KR20090052704A - Adipocyte differentiation detecting marker and a method for regulating differentiation of mesenchymal stem cells using same - Google Patents

Abstract

The present invention relates to a group consisting of protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11). Marker detection composition for detecting the differentiation of human mesenchymal stem cells into adipocytes, comprising a preparation for measuring the expression level of protein tyrosine phosphatase (PTP) selected from, a kit comprising the composition, the marker protein The present invention relates to a method for detecting differentiation into adipocytes, a method for screening differentiation candidate compounds into adipocytes, and a method for controlling differentiation into adipocytes.

Mesenchymal stem cells, dephosphatase, reverse transcriptase PCR

Description

Adipocyte differentiation detecting marker and a method for regulating differentiation of mesenchymal stem cells using same}

The present invention relates to a group consisting of protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11). Marker detection composition for detecting the differentiation of human mesenchymal stem cells into adipocytes, comprising a preparation for measuring the expression level of protein tyrosine phosphatase (PTP) selected from, a kit comprising the composition, the marker protein The present invention relates to a method for detecting differentiation into adipocytes, a method for screening differentiation candidate compounds into adipocytes, and a method for controlling differentiation into adipocytes.

Phosphorylation-dephosphorylation of the protein tyrosine is used as one of the very important means in the intracellular signaling system. Protein tyrosin phosphatase (PTP) is an enzyme that hydrolyzes phosphate groups in tyrosine residues in proteins, and humans have 107 PTPs, of which about 80 to 90 species show enzymatic activity as actual PTPs. Have been reported (Alonso A., et al., Cell, 117; 699-711, 2004).

It has been known that many PTPs are actually associated with disease because disruption of the intracellular signaling system leads directly to disease, and some PTPs have been targeted for drug development (Current Topics in Med. Chem. 3: 739-835). , 2003). In addition, the cellular signal transduction method involving PTP may be deeply involved in the differentiation of stem cells. Therefore, if a PTP that is specifically involved in the differentiation of stem cells into a specific lineage is found, the PTP is not only used as a differentiation inducing target but also by identifying the specific substrate of the PTP. This will provide a great clue to understanding.

Human mesenchymal stem cells are adult stem cells that are free from cancer and ethical issues that are common to 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, mesenchymal stem cells are in the spotlight as an effective means of cell therapy because they do not cause an immune response during transplantation. However, since mesenchymal stem cells have not yet been established for efficient mass differentiation into specific cells, there are significant limitations in actual clinical and industrial use. Therefore, it is urgent to establish a technique for inducing differentiation to a specific stage in a large amount and efficiently, and to do so, it is urgent to reveal the mechanism of induction signaling for differentiation to a specific stage.

Accordingly, the present inventors have tried to develop a method for controlling the differentiation of human mesenchymal stem cells from adipocytes to adipocytes using PTP. As a result, the expression of specific PTP in the differentiation process from mesenchymal stem cells to adipocytes is significantly reduced. Or increased, thus verifying that such PTP can be used as a marker for differentiation into adipocytes.

Accordingly, an object of the present invention is to provide a protein tyrosin phosphatase receptor type M (PTPRM), a protein tyrosin phosphatase receptor type F (PTPRF), a protein tyrosin phosphatase receptor type Q (PTPRQ), a slingshot homolog 2 (SSH2) and a myotubularin related protein (MTMR11). It is to provide a marker detection composition for detecting the differentiation of human mesenchymal stem cells into adipocytes, comprising an agent for measuring the expression level of protein tyrosine phosphatase (PTP) selected from the group consisting of 11).

Another object of the present invention is to provide a kit for detecting a differentiation marker from human mesenchymal stem cells to adipocytes, comprising the composition.

Another object of the present invention is to provide a method for detecting the differentiation of mesenchymal stem cells into adipocytes by checking whether the protein tyrosine phosphatase (PTP) is expressed.

Still another object of the present invention is to provide a method for screening candidate compounds for differentiation into adipocytes using the protein tyrosine phosphatase (PTP).

It is another object of the present invention to provide a method of controlling differentiation of mesenchymal stem cells to adipocytes by increasing or decreasing the expression of the protein tyrosine phosphatase (PTP).

Still another object of the present invention is to provide a composition for regulating differentiation of mesenchymal stem cells to adipocytes, including the inhibitor or promoter of the protein tyrosine phosphatase (PTP).

As one embodiment for achieving the above object, the present invention is a protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type M (PTPRQ), slingshot homolog 2 (SSH2) And a composition for measuring the expression level of protein tyrosine phosphatase (PTP) selected from the group consisting of MTMR11 (myotubularin related protein 11), the composition for detecting markers for detecting the differentiation of human mesenchymal stem cells into adipocytes It is about.

In the present invention, the term "marker for detecting the differentiation of human mesenchymal stem cells from adipocytes" means a significant difference in the expression in the experimental group that induced differentiation as compared to the control group which did not induce differentiation into adipocytes. Refers to the organic biomolecule. For purposes of the present invention, the marker of the present invention means a protein marker capable of detecting differentiation by increasing or decreasing only in cells differentiated into adipocytes, and more specifically, PTPRM (protein tyrosin phosphatase receptor type M), PTPRF (protein tyrosin phosphatase receptor type F), PTPRQ (protein tyrosin phosphatase receptor type Q), SSH2 (slingshot homolog 2) and MTMR11 (myotubularin related protein 11) protein tyrosin phosphatase (PTP) selected from the group consisting of it means.

In the present invention, "protein tyrosin phosphatase (PTP)" is an enzyme that hydrolyzes the phosphate group in the tyrosine residues in the protein, there are 107 kinds of PTP in humans, about 80 to 90 species of the actual PTP It has been reported to exhibit enzymatic activity as (Alonso A. et al. Cell, 117; 699-711, 2004).

In the present invention, in order to identify PTP involved in the differentiation of human mesenchymal stem cells into adipocytes, primers of 107 PTP genes present in humans were synthesized to profile expression patterns of PTPs in the differentiation process.

Specifically, induction of differentiation by treating the adipocyte-derived mixture to the mesenchymal stem cells derived from human bone marrow (bone marrow) was confirmed using Oil-Red-O staining, a lipid specific staining method (Fig. 1). After extracting the mRNA sample from the experimental group that induced differentiation into adipocytes, cDNA was synthesized, and specific nucleotides were added to the synthesized cDNA to 107 PTP genes present in humans, followed by polymerase chain reaction (PCR). After agarose gel electrophoresis was performed to analyze the difference in expression (FIGS. 2 to 5). As a result, PTP with increased expression during mesenchymal stem cell differentiation into adipocytes was identified as PTPRM (protein tyrosin phosphatase receptor type M), and PTP with decreased expression was expressed as PTPRF (protein tyrosin phosphatase receptor type F), Protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2), and myotubularin related protein 11 (MTMR11) were identified (FIG. 6). Therefore, the five PTP can be usefully used as a marker for detecting the differentiation by measuring the increase or decrease in expression during differentiation into adipocytes.

In particular, receptor type PTPRF and PTPRQ are known to modulate the mechanism of action of insulin, which plays the most important role in the synthesis of fat, in the present invention by insulin during the differentiation of fat cells by insulin It was confirmed that the expression decreased.

Such marker proteins may be in various modified forms, in which amino acid residues of a natural amino acid sequence are substituted, deleted and / or added to other residues as long as they can be used to identify whether they are differentiated from human mesenchymal stem cells to adipocytes. It may also be in a form in which modifications such as phosphorylation, saccharification, methylation and panesylation have occurred.

In the present invention, the term "agent for measuring the expression level of protein tyrosine phosphatase (PTP)" is a marker protein by identifying the expression level of the protein tyrosine phosphatase, a marker protein that increases or decreases the expression of adipocytes as described above. Means a molecule that can be used for detection, preferably refers to an antibody or primer specific for a marker protein.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For purposes of the present invention, the antibody is a marker protein of the present invention protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) Or it refers to an antibody that specifically binds to myotubularin related protein 11 (MTMR11), and includes both polyclonal and monoclonal antibodies. Since the marker protein of the present invention has been identified as described above, the production of antibodies using the same can be easily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the adipocyte differentiation marker protein antigen described above into an animal and collecting blood from the animal to obtain a 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 can be made by fusion methods well known in the art (Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519). One of the two cell populations that are fused to make a "hybridoma" secreting monoclonal antibodies utilizes cells from an immunologically suitable host animal, such as a mouse injected with adipocyte differentiation marker protein antigen, and the other Cancer or myeloma cell lines are used as the population of. These two populations of cells are fused by methods well known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a homogeneous cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the marker protein are in vitro or in vivo according to standard techniques in large quantities. Incubate. The monoclonal antibodies produced by the hybridomas may be used without purification, but in order to obtain the best results, the monoclonal antibodies are preferably purified and used according to methods well known in the art.

As the purification technique of the antibody for use in the present invention, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like can be used.

Antibodies used in the detection of adipocyte differentiation markers of the invention 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 Fv.

As another aspect, the present invention relates to a kit for detecting differentiation markers from human mesenchymal stem cells to adipocytes, comprising the composition for detecting adipocyte differentiation markers.

The marker detection kit of the present invention includes not only antibodies that selectively recognize marker proteins whose expression is increased or decreased in the differentiation of adipocytes, but also tools, reagents, and the like generally used in the art for immunological analysis. . Such tools / reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. If the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. 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, fluorine resin, 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 adipocyte differentiation marker of the present invention is not limited thereto, but is in the form of an ELISA plate, a dip-stick device, an immunochromatography test strip and a radiation split immunoassay device, and a flow-through device. It can have In addition, in the case where an antibody against an adipocyte differentiation marker protein is provided in a protein chip to which a large number of proteins can be immobilized, antigen-antibody complex formation against two or more antibodies can be observed, which is more suitable for detecting differentiation into adipocytes. It is advantageous.

As another aspect, the present invention relates to a method for detecting the differentiation of mesenchymal stem cells to adipocytes by confirming the expression of adipocyte differentiation marker protein. In the present invention, the marker protein may be identified at the gene level or at the protein level, and methods widely used in the art may be used without limitation.

As a specific embodiment, in order to confirm the expression of the marker protein of the present invention at the gene level, reverse transcriptase polymerase chain reaction (RT-PCR) and electrophoresis may be used. It can be seen that the marker protein of the present invention increases or decreases only in cells differentiated into adipocytes (FIGS. 2 to 6).

In addition, the method for confirming the expression of the marker protein of the present invention at the protein level, the step of contacting the antibody against the protein tyrosine phosphatase (PTP) with mesenchymal stem cells to form an antigen-antibody complex and the antigen-antibody And comparing the increase in the amount of formation of the complex with the amount of formation of the control group.

As used herein, the term “antigen-antibody complex” refers to a combination of an adipocyte differentiation marker protein and an antibody that recognizes the adipocyte differentiation protein for detecting the differentiation into adipocytes.

The detection method compares the formation of the antigen-antibody complex in the control group with the formation of the antigen-antibody complex in the differentiation-inducing group to measure the increase or decrease in the expression level of the adipocyte differentiation markers. By the differentiation into adipocytes is meant the presence of markers with increased expression, the decrease in the expression amount means the presence of markers with reduced expression by differentiation into adipocytes.

In the detection method, the amount of antigen-antibody complex formed can be quantitatively measured through the magnitude of a signal of a detection label. Such a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. If an enzyme is used as the detection label, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase, GDPase, RNase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase and β -Latamase and the like, but is not limited to this. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2 ⁺ , [RU (bpy) 3] 2 ⁺ or [MO (CN) 8] 4 ^- and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I or ¹⁸⁶ Re.

Colorimetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and the like to detect antigen-antibody complex formation. A method selected from the group consisting of scintillation counting methods may be used, but is not limited thereto. Preferably, the antigen-antibody complex can be detected using western bloting and enzyme-linked immunosorbent assay (ELISA).

In a specific embodiment of the present invention, as a result of Western blotting in the control group and the differentiation-inducing group, PTPRQ of the marker protein of the present invention compared to the control group (tubulin) in the process of differentiation of mesenchymal stem cells to adipocytes It was confirmed that the expression was significantly reduced, and thus it was verified that the expression level of PTPRQ could be detected to differentiate into adipocytes (FIG. 7).

As another aspect, the present invention comprises the steps of treating the mesenchymal stem cells with the candidate compound for regulating differentiation into adipocytes; And protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11) in mesenchymal stem cells The present invention relates to a method for screening a candidate compound for regulating differentiation into adipocytes, the method comprising measuring an increase or a decrease in a marker protein selected from the group consisting of:

As another aspect, the present invention relates to a method for controlling differentiation of mesenchymal stem cells to adipocytes by increasing or decreasing the adipocyte differentiation marker proteins.

Since the marker proteins of the present invention are closely related to the differentiation regulators into adipocytes, and are mainly located in the cell membrane, the inhibitory proteins or promoters of the marker proteins of the present invention can be efficiently or simply increased or inhibited differentiation. have.

As used herein, the term “differentiation control candidate compound to adipocytes” refers to a compound that indirectly or directly inhibits or induces a change in the amount of marker protein expression significantly occurring in cells upon differentiation into adipocytes. Differentiation regulators can be regulated to inhibit differentiation by inducing differentiation by decreasing its expression by further increasing its expression in the case of proteins (eg, PTPRM) whose expression is increased during differentiation into adipocytes. In addition, in the case of proteins whose expression is reduced (eg, PTPRF, PTPRQ, SSH2 and MTMR11), the expression may be further reduced to induce differentiation by increasing the expression or to suppress the differentiation by increasing the expression.

Thus, by comparing the increase or decrease in the amount of adipocyte differentiation marker protein expression in the presence and absence of candidate compounds, it is possible to control the differentiation of mesenchymal stem cells to adipocytes and furthermore be useful for screening differentiation control factors. have.

As described above, a number of PTPs are involved in the differentiation of human mesenchymal stem cells into adipocytes through the present invention, and the use of such PTP may promote or inhibit the differentiation into adipocytes. Therefore, PTP verified as an adipocyte differentiation marker in the present invention can be used as a new target enzyme for differentiation control and development of obesity treatment.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1. Stem Cell Culture stem cell culture )

    The present inventors purchased and used mesenchymal stem cell lines (Cambrex, USA) collected from bone marrow of normal people for fast and efficient study of mesenchymal stem cells (MSCs) present in trace amounts in bone marrow. It was. Cultures were cultured in α-minimum essential medium (α-MEM) medium containing fetal bovine serum for subcultured mesenchymal stem cells. Culture conditions were maintained at a temperature of 37 ℃, humidity more than 95% in a 5% carbon dioxide incubator, the culture was exchanged every 2-3 days. After incubation, the cells were 100% confluent, trypsin treatment, and subcultivation was performed.

Example  2. Differentiation into adipocytes differentiation of adipocyte )

The cultured cells were dispensed in a 100 mm culture dish at a cell number of 1 × 10 ⁵ / cm ² in α-MEM medium containing 10% fetal calf serum and cultured at 5% carbon dioxide at 37 ° C. until 100% congregation was performed. After that, the culture medium was exchanged with the adipocyte induction medium and cultured under the same conditions. The composition of adipocyte differentiation medium consisted of differentiation medium (α-MEM medium with 10% fetal calf serum + 10 μM insulin, 1 μM dexamethasone, 0.5 mM methylisobutylxanthine, and 100 μM indomethacin) and growth medium (10% fetal calf serum). Α-MEM medium plus 10 μM insulin) was treated in alternating 3 days with 18 days. In order to grow mature adipocytes, the growth medium was reprocessed for 7 days and the growth medium was replaced every 2-3 days.

In order to observe differentiation into adipocytes, Oil-Red-O staining, a lipid specific staining method, was performed three weeks after differentiation. Specifically, the cells were fixed with 10% formalin for 30 minutes and then reacted with 0.3% Oil-Red-O solution for 30 minutes. After washing twice with distilled water, the lipid spheres were observed under a microscope (FIG. 1).

Example  3. Reverse transcription  Polymerase chain reaction reverse transcription - polymerase chain  reaction, RT - PCR )

In the differentiation process of mesenchymal stem cells into adipocytes, mRNA was extracted on the first 3 days of differentiation to determine cell traits. After separation from each sample using RNeasy mini kit of Qiagen (USA), 2 μl of RNA and 2 μl of Oligo dT primer (500 μg / ml) were reacted at 70 ° C. for 5 minutes, followed by RT of Bioneer (Korea). Complementary cDNA was synthesized using -premix kit.

The polymerase chain reaction was analyzed using a PCR Premix kit from Binoeer (Korea). The reaction was performed at 95 ° C. for 5 minutes with a 20 μl reaction solution containing 5 μl of cDNA, 2 μl of 10 pM primers, and 13 μl of distilled water. After 30 cycles of 30 seconds, 30 seconds, 30 seconds, 72 ℃, 30 seconds, the reaction was confirmed by Etbr (ethidium bromide) staining electrophoresis on 1.5% agarose gel (Fig. 2 to 5).

As a result, PTP with increased expression during mesenchymal stem cell differentiation into adipocytes was identified as PTPRM (protein tyrosin phosphatase receptor type M), and PTP with decreased expression was expressed as PTPRF (protein tyrosin phosphatase receptor type F), Protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2), and myotubularin related protein 11 (MTMR11) were identified (FIG. 6).

Example  4. Weston Blotting  ( Western blotting )

During the differentiation of mesenchymal stem cells to adipocytes, the expression of protein tyrosin phosphatase receptor type Q (PTPRQ), one of the PTP proteins, was confirmed by Western blotting.

Specifically, intracellular lysates (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris) were added to prepare protein samples, and then disrupted using a sonicator at 4 ° C. for 10 seconds or more for 10 seconds. . The lysate of stem cells was centrifuged at 13,000 rpm for 20 minutes at 4 ° C and the supernatant was recovered. Protein content of the supernatant was quantified by the Bradford method.

20 μg of the above-described protein sample was loaded on 11-12% acrylamide gel, followed by electrophoresis (SDS-PAGE), and proteins were separated by size. Proteins separated in 1 × transition buffer solution (20% methanol, 0.025M tris base, 0.19M glycine) were transferred to polyvinylidene fluoride (PVDE) membrane and reacted for 12 hours at 4 ° C. using PTPRQ specific antibody. The PVDF membrane reacted with the primary antibody was thoroughly washed at least 3 times with 1 X TBST for at least 15 minutes and treated with goat anti goat immunoglobulin G HRP (goat anti rabbit lgG-HRP) isolated from serum. It was reacted for a time. Again washed 3 times with 1X TBST for 15 minutes and treated with ECL to develop a film. In addition, in order to clarify the difference in expression was standardized using tubulin (tubulin).

As a result, as shown in Figure 7, it was confirmed that the expression of PTPRQ significantly reduced compared to the control (tubulin) in the process of differentiation of mesenchymal stem cells to adipocytes. Thus, it was once again confirmed at the protein level that the PTPs of the present invention can be used as adipocyte differentiation markers.

Example  5. Analysis using fluorescence microscope

       The present inventors performed immunostaining using fluorescence microscopy to track the intracellular location of PTPRQ, one of the marker proteins of the present invention, and the migration route upon differentiation into adipocytes.

The cover glass was sterilized and placed on the cell culture vessel, and the cells were inoculated thereon. Cultures of stem cells differentiated into undifferentiated cells and adipocytes were removed, washed three times with PBS, and treated with Cytofix / Cytoperm (BD falcon, USA). After washing four times with PBS again, four drops of Image-iT FX signal enhancer of Invitrogen (USA) was left to stand again for 30 minutes. After washing four times with PBS again, the PTPRQ antibody (Santa Cruz, USA) was diluted 1: 500 and treated and left overnight at 4 ° C. After washing 5 times with PBS, the secondary antibody (anti-Goat, deep red fluorescence) was diluted 1: 500 and treated and left in the dark for 1 hour 30 minutes. After washing five times with PBS, DAPI (nuclear staining) was diluted 1: 2000, left in the dark for 30 minutes, washed again with PBS five times and observed through a light microscope.

As a result, as shown in Figure 5, the conventional PTPRQ was known to exist in the cell membrane, but through the present invention it was confirmed that a large number in the cytoplasm. In particular, after stimulation of differentiation into adipocytes, some of the migration to the cell nucleus was found.

1 is a result of observing differentiation patterns of adipocytes through adipocytes through a lipid-specific staining when the mesenchymal stem cells are differentiated into adipocytes under a microscope.

      2 to 5 show the results of analyzing the expression patterns of 107 PTPs by RT-PCR method while differentiating human mesenchymal stem cells into adipocytes.

      Figure 6 shows the expression patterns of PTPs that significantly increase or decrease the mRNA expression level of PTP during differentiation into adipocytes.

      7 is a result of confirming the change in protein level of PTP-RQ (PTP-RQ), one of the PTP expression decreased during adipocyte differentiation by Western blotting method.

8 is a result of recognizing the position in the cell of PTP-RQ through the fluorescence microscope.

Claims (10)

Protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11) A composition for detecting markers for detecting the differentiation of human mesenchymal stem cells into adipocytes, comprising an agent measuring the expression level of protein tyrosine phosphatase (PTP). The composition of claim 1, wherein said agent is an antibody. The composition of claim 1 wherein said agent is a primer.        A kit for detecting differentiation markers from human mesenchymal stem cells to adipocytes, comprising the composition of any one of claims 1 to 3. Protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11) Method for detecting the differentiation of mesenchymal stem cells into adipocytes by checking the expression of protein tyrosine phosphatase (PTP). The method according to claim 5, which is confirmed by reverse transcription polymerase chain reaction (RT-PCR) and electrophoresis. The method of claim 5, wherein the antibody against the protein tyrosine phosphatase (PTP) is contacted with mesenchymal stem cells to form an antigen-antibody complex and an increase in the amount of the antigen-antibody complex formed is compared with that of the control group. Including a step, detecting whether the differentiation of mesenchymal stem cells into adipocytes. 8. The method according to claim 7, which is detected by western bloting. Treating the mesenchymal stem cells with candidate compounds for controlling differentiation into adipocytes; And protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11) in mesenchymal stem cells A method for screening a candidate compound for regulating differentiation into adipocytes, comprising measuring an increase or a decrease in a marker protein selected from the group consisting of: Protein tyrosin phosphatase receptor type M (PTPRM), protein tyrosin phosphatase receptor type F (PTPRF), protein tyrosin phosphatase receptor type Q (PTPRQ), slingshot homolog 2 (SSH2) and myotubularin related protein 11 (MTMR11) A method of controlling differentiation of mesenchymal stem cells to adipocytes by increasing or decreasing the expression of protein tyrosine phosphatase (PTP).

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