KR20190060964A - Biomarker for predicting apoptosis of mesenchymal stromal cells and uses thereof - Google Patents

Abstract

The present invention relates to a method for providing information necessary for predicting or discriminating apoptosis of mesenchymal stem cells, which comprises a step of measuring the expression level of CDKN2A gene in mesenchymal stem cells; and a composition and kit for predicting apoptosis of mesenchymal stem cells used therefor.

Description

[Biomarker for predicting apoptosis of mesenchymal stromal cells and uses thereof]

The present invention relates to a method for providing information necessary for predicting apoptosis of mesenchymal stem cells by measuring the expression level of a cyclin-dependent kinase inhibitor 2A (CDKN2A) gene in mesenchymal stem cells, and a composition and kit used therein. .

Mesenchymal stromal cells (MSCs) have self-renewal ability, immunomodulatory ability, and differentiation into mesenchymal lines. Recently, mesenchymal stem cells are recognized as an important resource in the field of regenerative medicine, that is, in the field of medicine that replaces or regenerates human cells, tissues, or organs to restore their original functions. Mesenchymal stem cells can be obtained from various tissues such as bone marrow, lung, fat, liver, umbilical cord blood, amniotic fluid and placenta.

Mesenchymal stem cells can also be obtained from postnatal tissues such as amnion, chorion and umbilical cord, and these tissues are discarded after childbirth and can be studied and clinically applied without ethical issues. And, since it is provided in a relatively large size, it is useful as a source of a large amount of mesenchymal stem cells. However, since the characteristics of mesenchymal stem cells derived from these tissues have not been sufficiently studied, more studies are needed for clinical application.

The present inventors, while studying the characteristics of mesenchymal stem cells derived from amnion, chorion and umbilical cord, selected and confirmed markers capable of predicting or discriminating apoptosis of mesenchymal stem cells from the results of their gene expression analysis. The present invention has been completed.

Korean Patent Publication No. 10-2016-0013214 (2016. 2. 3.)

The present invention is to provide the use of CDKN2A (cyclin-dependent kinase inhibitor 2A) as a marker for predicting apoptosis of mesenchymal stem cells.

Accordingly, one object of the present invention is to provide a method of providing information necessary for predicting apoptosis of mesenchymal stem cells, comprising the step of measuring the expression level of the CDKN2A gene in mesenchymal stem cells.

Another object of the present invention is to provide a composition for predicting apoptosis of mesenchymal stem cells, comprising a substance for measuring the expression level of the CDKN2A gene.

Another object of the present invention is to provide a kit for predicting apoptosis of mesenchymal stem cells, comprising the composition.

Postnatal tissues, such as amnion, chorion, and umbilical cord, can be a rich source of mesenchymal stromal cells and have the advantage of being free from ethical issues, but they still have the advantage of being free from them. The characteristics of the derived mesenchymal stem cells have not been elucidated in detail. The present inventors have succeeded in isolating and culturing amniotic membrane-derived mesenchymal stem cells (AMSCs), chorionic mesenchymal stem cells (CMSCs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs), respectively, and these mesenchymal stem cells are It was confirmed that the proliferative capacity was different, and the amnion-derived mesenchymal stem cells (AMSCs) showed the lowest proliferation rate due to increased annexin V levels and senescence-related beta-galactosidase (SA-β-gal) positive cells. Accordingly, the present inventors investigated the gene expression characteristics in these tissues through gene expression analysis, and as a result, it was confirmed that the expression of apoptosis and aging-related genes including the CDKN2A gene was increased in the amniotic membrane-derived mesenchymal stem cells. In addition, the degree of gene expression was confirmed through quantitative PCR, and CDKN2A was finally selected as a marker capable of predicting or discriminating apoptosis of mesenchymal stem cells.

Accordingly, by measuring the expression level of CDKN2A, apoptosis of mesenchymal stem cells can be predicted or discriminated, and through this, mesenchymal stem cells more suitable for application to a specific disease can be selected and used for disease treatment.

In one aspect, the present invention provides a composition for predicting apoptosis of mesenchymal stem cells, comprising a substance for measuring the expression level of the CDKN2A gene.

In another aspect, the present invention provides a kit for predicting apoptosis of mesenchymal stem cells, comprising the composition.

According to an example of the present invention, the substance for measuring the expression level of the gene is for detecting any one or more of the presence, the abundance, and the presence pattern of the mRNA transcribed by the gene, the protein encoded by the gene, or both. It is characterized by being a material.

According to an example of the present invention, the substance for measuring the expression level of the gene specifically binds to at least one selected from the group consisting of the nucleotide sequence of the gene, a sequence complementary thereto, a fragment of the nucleotide, and a sequence complementary thereto. It is characterized in that at least one of a primer, a probe, an aptamer, and an antisense oligonucleotide.

According to an example of the present invention, the substance for measuring the expression level of the gene is any one or more of a polypeptide encoded in the nucleotide sequence, a polypeptide encoded in the complementary sequence, and a polypeptide encoded in a fragment of the nucleotide sequence. Oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, antibody fragments, ligands, peptide nucleic acids (PNA), aptamers, and avidity multimers that specifically bind to And it is characterized in that any one or more selected from peptidomimetics.

According to an example of the present invention, the substance for measuring the expression level of the gene is one or more of the presence, amount, and pattern of the presence of a conjugate of the protein encoded by the CDKN2A gene and the messenger RNA (mRNA) transcribed by the gene. It is characterized in that it is a substance to detect.

According to an example of the present invention, the substance for measuring the expression level of the gene is reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method. , DNA microarray method, Northern blot, Western blot, ELISA (Enzyme Linked Immuno Sorbent Assay), radiation immunoassay, immune diffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry (Mass spectrometry) and protein microarrays.

According to an example of the present invention, the mesenchymal stem cells may be mesenchymal stem cells isolated from the amniotic membrane, chorion or umbilical cord.

According to an example of the present invention, the kit is characterized in that at least one selected from a microarray, a gene amplification kit, an immunoassay kit, a luminex assay kit, a protein microarray kit, and an ELISA kit.

In another aspect, the present invention provides a method of providing information necessary for predicting apoptosis of mesenchymal stem cells, comprising measuring the expression level of the CDKN2A gene in mesenchymal stem cells.

According to an example of the present invention, the method comprises the steps of measuring the expression level of the CDKN2A gene from a mesenchymal stem cell sample, and when the expression level of the CDKN2A gene is higher than a threshold value (cut-off), the cell of the mesenchymal stem cell Predicting that there is a possibility of death may be included.

According to an example of the present invention, the mesenchymal stem cells may be mesenchymal stem cells isolated from the amniotic membrane, chorion or umbilical cord.

According to an example of the present invention, the measurement of the expression level of the gene may be performed using a primer, probe or antisense oligonucleotide specific for the gene.

According to an example of the present invention, the measurement of the expression level of the gene is reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA Microarray method, Northern blot, Western blot, ELISA (Enzyme Linked Immuno Sorbent Assay), radiation immunoassay, immune diffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry And it may be performed by a method selected from a method of using a protein microarray.

According to an example of the present invention, the cut-off value may be 0.1.

In the present invention, by measuring the expression level of CDKN2A, apoptosis of mesenchymal stem cells can be predicted or discriminated, and through this, mesenchymal stem cells more suitable for application to a specific disease can be selected and used for disease treatment.

Figure 1a is a cell shape, proliferation degree and aging-related beta-galactosid according to the number of passages of amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical cord mesenchymal stem cells (UC-MSC). It shows the activity of the enzyme (SA-β-gal).
FIG. 1B is a graph showing the change in PDT (population doubling time) values during passage in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical mesenchymal stem cells (UC-MSC).
1C is a graph showing the proportion of annexin V-positive cells in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical cord mesenchymal stem cells (UC-MSC).
1D shows the ratio of aging-related beta-galactosidase (SA-β-gal) in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical cord mesenchymal stem cells (UC-MSC). This is the graph shown.
1E shows adipocyte differentiation and chondrocyte differentiation of amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), umbilical cord mesenchymal stem cells (UC-MSC) and bone marrow mesenchymal stem cells (BM-MSCs). And it is the result of confirming the bone cell differentiation ability.
2A shows the results of confirming the mRNA expression levels of stem cell-related marker molecules in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical cord mesenchymal stem cells (UC-MSC) through RT-PCR. Show.
2B shows the results of a clustering analysis of gene expression in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical mesenchymal stem cells (UC-MSC).
3 shows the results of gene expression analysis in amniotic mesenchymal stem cells (AMSC), chorionic mesenchymal stem cells (CMSC), and umbilical cord mesenchymal stem cells (UC-MSC).
4 is a result of confirming that apoptosis occurs in cells when the expression of the CDKN2A gene is released.
5 is a result of microscopic analysis of the phase difference of cells stained in Puson color by performing SA-β-Gal that apoptosis occurs in cells when the expression of the CDKN2A gene is released.
6 shows the ROC curve at the threshold value of 0.1 of the CDKN2A gene.

The present invention is a composition for predicting apoptosis of mesenchymal stem cells comprising a substance for measuring the expression level of the CDKN2A gene, a kit for predicting apoptosis of mesenchymal stem cells, a method for predicting apoptosis of mesenchymal stem cells, and mesenchymal It provides a method for detecting a biomarker for predicting apoptosis of stem cells. Hereinafter, the present invention will be described in detail with reference to the drawings.

In the present invention, the term "(bio)marker, predictive marker or diagnosis marker" is a substance capable of predicting and selecting the apoptosis potential of mesenchymal stem cells at an early stage, and has a specific criticality in mesenchymal stem cells. It includes organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit a value greater than or equal to the value. For the purpose of the present invention, the marker for predicting apoptosis of mesenchymal stem cells is a nucleotide of the CDKN2A gene (including a fragment thereof) or a protein encoded therein (including a fragment thereof), and has a value greater than or equal to a specific threshold in mesenchymal stem cells. It is a gene that represents. These markers may be mRNA for any one gene or any one protein encoded by the gene, and may be a complex marker including two or more of these markers.

The CDKN2A gene is composed of the nucleotide sequence represented by SEQ ID NO: 1, and the amino acid sequence of the protein encoded by it can be identified from a known gene database (eg, NCBI Gene ID: 1029).

As used herein, "polypeptide" (or protein) is construed as including an amino acid sequence that exhibits substantial identity to the corresponding amino acid sequence. The actual identity of the above is at least 60% when the amino acid sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to an amino acid sequence that exhibits homology, more preferably at least 80% homology, and most preferably at least 90% homology.

For example, the polypeptide is about 60% or more, 80% or more, 90% or more, 95% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more with the specific amino acid sequence described. , A polypeptide having an amino acid sequence having 99.5% or more, or 99.9% or more identity and functioning as a biomarker for predicting apoptosis of mesenchymal stem cells. In general, the higher the percent identity, the more preferable.

In addition, the polypeptide having the same identity includes the amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and/or added in the polypeptide of the specific amino acid sequence described, and is related to the beta-adipate pathway. Includes. In general, the fewer the number of deletions, substitutions, insertions, and/or additions, the more preferable.

As used herein, "polynucleotide" (or nucleotide, nucleic acid) is meant to encompass DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugars. Or, it also includes analogs with modified base sites (analogues).

The polynucleotide of the present invention is not limited to a nucleic acid molecule encoding the specific amino acid sequence (polypeptide) described above, and as described above, an amino acid sequence exhibiting substantial identity to a specific amino acid sequence or a polynucleotide having a function corresponding thereto. It is interpreted to include a nucleic acid molecule encoding a peptide. The above substantial identity is at least 60% homology when the amino acid sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. , More preferably, it means an amino acid sequence showing at least 80% homology, and most preferably at least 90% homology.

Polypeptides having the corresponding function include, for example, a polypeptide of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and/or added. Such a polypeptide includes a polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and/or added as described above, and is involved in the synthesis of 3-hydroxypropionic acid, and deletion and substitution of amino acid residues It is preferred that the number of, insertions, and/or additions is small. In addition, the polypeptide includes a polypeptide having an amino acid sequence having about 60% or more identity to the specific amino acid sequence described above and functioning as a biomarker for predicting apoptosis of mesenchymal stem cells, and has high identity. The more preferable.

As used herein, the term "complementary" or "complementary" refers to the ability of purine and pyrimidine nucleotides to bind through hydrogen bonds to form double-stranded polynucleotides, and includes partially complementary cases. The following base pairs are associated with complementarity: guanine and cytosine; Adenine and thymine; And adenine and uracil. "Complementary" applies substantially to all base pairs in which the above-mentioned relationship comprises two single-stranded polynucleotides over the full length of the molecule. "Partially complementary" refers to the relationship that one of the two single-stranded polynucleotides is short, so that some of the molecules remain single-stranded.

Expression measurement or detection in the present invention includes quantitative and/or qualitative analysis, and includes detection of presence and absence and detection of expression levels, and such methods are known in the art, and those skilled in the art for the implementation of the present application You will be able to choose the appropriate method.

In the present invention, the substance for measuring the expression level of the gene may be a substance that detects any one or more of the presence, amount, and pattern of the presence of the mRNA transcribed by the gene, the protein encoded by the gene, or both. .

The present invention can be used to predict apoptosis of mesenchymal stem cells through quantitative and/or qualitative detection of the gene at the nucleic acid level, particularly at the mRNA level. In the present invention, the substance for measuring the expression level of the gene is a primer or probe that specifically binds to at least one selected from the group consisting of the nucleotide sequence of the gene, a sequence complementary thereto, a fragment of the nucleotide, and a sequence complementary thereto. , Aptamer and antisense oligonucleotide may be any one or more.

For example, in order to measure the presence and amount or pattern of mRN of the gene by RT-PCR, a probe and/or primer pair specific to the mRNA of the gene is included. The primer or probe refers to a nucleic acid sequence having a free 3'hydroxyl group capable of binding complementarily to the template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template. The gene expression measurement material used herein may be labeled with a color, luminescence, or fluorescent material for signal detection. For example, Northern blot or reverse transcription PCR (polymerase chain reaction) is used to detect mRNA. In the latter case, a specific gene in the sample is detected using a specific primer or a combination of primers and probes after separating the RNA of the sample, particularly mRNA, and then synthesizing cDNA therefrom. Or it is a method that can determine the expression level.

In the present invention, the substance for measuring the expression level of the gene is specifically for any one or more of the polypeptide encoded in the nucleotide sequence, the polypeptide encoded in the complementary sequence, and the polypeptide encoded in the fragment of the nucleotide sequence. Binding oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, antibody fragment, ligand, PNA (Peptide nucleic acid), aptamer, avidity multimer, and peptidomimetic It may be any one or more selected from sieve (peptidomimetics).

In the present invention, the substance for measuring the expression level of the gene is a substance that detects any one or more of the presence, amount, and pattern of presence of a protein encoded by the CDKN2A gene or a conjugate of messenger RNA (mRNA) transcribed by the gene. Can be

In the present invention, the substance for measuring the expression level of the gene may include substances used in various methods for detecting genes (biomarkers) known in the art, for example, reverse transcription polymerase chain reaction, competitive polymerase chain Reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray method, Northern blot, Western blot, ELISA (Enzyme Linked Immuno Sorbent Assay), radioimmunoassay, immune diffusion method , Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, and protein microarray.

That is, the present invention measures the expression of a corresponding gene using a quantitative/qualitative analysis method for various nucleic acids/proteins known in the art. For example, reverse transcription polymerase chain reaction (RT-PCR)/polymerase chain reaction, competitive RT-PCR, real-time RT-PCR, Nuclease protection analysis (NPA) for detection at the RNA level, expression level or pattern detection For example, RNase, S1 nuclease analysis, in situ hybridization, DNA microarray or a method using a chip or Northern blot may be used, and such an analysis method is known and may be performed using a commercially available kit, and those skilled in the art If so, you can select one suitable for the practice of the present application.

In addition, the present invention provides a kit or system for predicting apoptosis of mesenchymal stem cells, comprising the above-described composition for predicting apoptosis of mesenchymal stem cells.

The details are as described above. The kit according to the present invention may use a variety of kits known in the art, for example, any selected from a microarray, a gene amplification kit, an immunoassay kit, a luminex assay kit, a protein microarray kit, and an ELISA kit. There can be more than one.

In addition, the present invention provides a method of providing information necessary for predicting apoptosis of mesenchymal stem cells, comprising the step of measuring the expression level of the CDKN2A gene in mesenchymal stem cells.

The method comprises the steps of measuring the expression level of the CDKN2A gene from the mesenchymal stem cell sample, and predicting that there is a possibility of apoptosis of the mesenchymal stem cells when the expression level of the CDKN2A gene is higher than the cut-off value. It may include steps.

The cut-off value is preferably 0.1, and according to the present embodiment, when a ROC curve is drawn based on a value of 0.1, a sensitivity of 100% and a specificity of 100% are shown (FIG. 6). However, the threshold value may be appropriately changed within a range that does not significantly impair sensitivity and specificity.

In the method for predicting apoptosis of mesenchymal stem cells according to the present invention, the expression level of the gene may be measured using a quantitative/qualitative method for measuring various nucleic acids/proteins known in the art. For example, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method, Northern blot, Western blot, ELISA ( Enzyme Linked Immuno Sorbent Assay), radiation immunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, and protein microarray use. I can.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Isolation and primary culture of mesenchymal stem cells

Postpartum tissue was provided from a healthy donor, and the amniotic membrane, chorionic membrane, and umbilical cord were separated from the placenta. For the isolation of amniotic mesenchymal stem cells (AMSCs), the amniotic membrane sections were treated with 0.25% trypsin-EDTA (Gibco) at 37°C and then 0.2% collagenase IV (Gibco). The same method was used for chorionic mesenchymal stem cells (CMSCs), but 0.2% collagenase I (Gibco) was used. The umbilical cord was separated, treated with 0.2% collagenase II (Gibco) at 37°C, and then treated with 0.25% trypsin-EDTA (Gibco). Bone marrow mesenchymal stem cells (BM-MSCs) were used as a control. The isolated cells were cultured in alpha-modified minimal essential medium (α-MEM; Gibco) in T25 flasks (BD Falcon) at 37° C. and a humidified atmosphere containing 5% carbon dioxide, and until 70% confluency was achieved. The medium was changed every other week.

Example 2. Analysis of proliferation ability

When the mesenchymal stem cells reached 70% confluency, cells were collected using 0.25% trypsin-EDTA and counted using a disposal hemocytometer C-Chip (SystemBukerTurk; Incyto, cheonan, Korea). It was seeded at a concentration of 200 cells/cm ² and subcultured, and the proliferation capacity was calculated by PDT (population doubling time) using an algorithm known online (http://www.doubling-time.com).

Example 3. Analysis of apoptosis through annexin V/Propidium Iodide

Cells were collected at passage 3 (P3) and apoptosis was analyzed using Annexin V-300 fluorescein isothiocynate (FITC) apoptosis detection kit (Abcam, Cambridge, UK). A cell suspension containing 1 X 10 ⁵ cells was cultured in the presence of FITC-conjugated Annexin V and propidium iodide (PI) at dark conditions and room temperature. Samples were analyzed with a FACSCalibur cytometer (BD Biosciences, San 304 Jose, CA ^{) and data were processed with CellQuest™} Pro version 6.0 software (BD Biosciences).

Example 4. Senescence-associated β-galactosidase (SA-β-gal) activity assay

In order to analyze cellular senescence, the expression level of SA-β-gal was analyzed. SA-β-gal was stained using a SA-β-gal staining kit (Cell Signaling Technology, Boston, MA, USA). Specifically, mesenchymal stem cells of ^{300 cells/cm 2} were seeded and cultured in a 6-well culture dish. After 3 days, the cells were stained with β-galactosidase staining solution and cultured in a culture dish at 37°C. Senescent cells were stained blue and observed with an inverted microscope.

Example 5. In vitro mesoderm differentiation

Amniotic membrane, chorionic membrane and umbilical cord mesenchymal stem cells were collected in P3 and seeded at a concentration of ^{1×10 5} per well in a 6-well tissue culture test plate (Nunc; PuDong, Shanghai, China). When the cells reached 70% confluency, the medium was replaced with a differentiation medium. The cells were cultured at 37° C. for 3 weeks and the medium was changed every other week. To induce adipocyte differentiation, culture in basal medium (Gibco) and after 3 weeks, the cells were fixed in 4% paraformaldehyde (PFA) and stained with Oil Red-O (Sigma-Aldrich, St. Louis, MO). ^{To induce chondrocyte differentiation, 2.5 X 10 5} cells were centrifuged at 150 xg for 5 minutes in a 15 mL conical tube (BD Biosciences), and cultured in basal medium (Gibco) for chondrocyte differentiation. After 3 weeks, 4% paraformaldehyde ( PFA) and embedded in paraffin. Sections were stained with 1% alcian blue solution (ScienCell, Carlsbad, CA), washed, and then the nuclei were stained with 0.1% nuclear fast red solution (ScienCell). In order to induce bone cell differentiation, it was cultured in bone cell differentiation medium, fixed in 4% PFA after 3 weeks, and stained with 2% Alizarin Red Solution (ScienCell).

Example 6. Analysis of stem cell markers through RT-PCR

Total RNA was isolated from P3 amniotic membrane, chorionic membrane, and umbilical mesenchymal stem cells using RNeasy Mini Kit (Qiagen, Hilden, Germany), respectively. CDNA was synthesized using a Transcript or First-strand cDNA synthesis kit (Roche Applied Science, Mannheim, Germany). RT-PCR (Reverse transcription polymerase chain reaction) was performed to evaluate the stemness nature of mesenchymal stem cells. For this, octamer-binding protein 4 (OCT4), homeobox transcription factor nanog (NANOG), stem cell factor (SCF), telomerase reverse transcriptase (TERT), HLA-ABC and HLA-G were analyzed. It was compared with the expression of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The PCR product was observed under ultraviolet light.

Example 7. Derivation of gene markers

The difference in genetic expression of mesenchymal stem cells derived from amniotic membranes (AMSCs), chorionic membranes (CMSCs), and umbilical cords (UC-MSCs) was analyzed through gene expression array. First, RNA was extracted from each mesenchymal stem cell to measure the quality and quantity, and then the experiment was performed using the SurePrint G3 Hmn GE 8Χ60K V2 Microarray Kit (Agilent). 200 ng of RNA was synthesized, synthesized with double-stranded cRNA, amplified, and then reacted to the chip attached to the probe to calculate data. In addition, the data were analyzed with the GeneSpring GX 7.3 analysis program, and genes that were characteristically expressed in mesenchymal stem cells (MSCs) derived from each tissue were identified using GeneCards, DAVID, and QIAGEN's Ingenuity® Pathway Analysis programs. I recognized it.

Example 8. Quantitative measurement method of gene markers

The ABI 7500 Real-Time PCR system was carried out by selecting a characteristic expression of the gene by real-time quantitative PCR (Real-time quantitative PCR) using the TaqMan ^® gene expression assay. Data was derived by the 2-ΔΔCt method. Genes include a total of 10 genes, cyclin-dependent kinase inhibitor 2A ( CDKN2A ) (HS00923894_m1, TaqMan ^® ), pleiomorphic adenoma gene-like 1 ( PLAGL1 ) (HS00414677_m1, TaqMan ^® ), homeobox B2 ( HOXB2 ) (Hs00609873_g1, TaqMan ^® ), a plexin A2 (PLXNA2) (Hs00300697_m1, TaqMan ®) were selected. Three CDKN2A , HTR2B, and NPY genes, whose expression levels were clearly different, were selected to determine the function of each gene, and whether the characteristics of each gene appeared in MSCs derived from each tissue.

Example 9. Determination of a cut off value

Primer (F:5' of cyclin-dependent kinase inhibitor 2A ( CDKN2A ) (HS00923894_m1, TaqMan ^{® ) by TaqMan ®} gene expression assay for each of 5 amniotic membrane (Am), chorionic membrane (Ch) and cord stem cells. -CAGTAACCATGCCCGCATAGA-3' (SEQ ID NO: 2) and R:5'-AAGTTTCCCGAGGTTTCTCAGA-3 (SEQ ID NO: 3)) and probes were used to evaluate CDKN2A gene expression in an ABI 7500 Real-Time PCR system. The experiment was repeated three times, and human GAPDH (Hs99999905_m1) was used as a reference gene. The gene expression level was calculated by the 2-ΔΔCt method.

Example 10. Statistical Analysis

Proliferation data was analyzed by Wilcoxon Signed Rank Test. Gene expression by RT-qPCR was analyzed using the one-way ANOVA test and Kruskal-Wallis method. All tests were performed two-tailed analysis, and a P value of less than 0.05 was considered a significant result. All statistical analyzes were analyzed using MedCalc 12.7.2 software (MedCalc, Mariakerke, Belgium).

Experiment result

1. Results of Isolation and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells were isolated and cultured from the amnion, chorion and umbilical cord. In the primary culture (P0), the amniotic mesenchymal stem cells were a mixture of rod-shaped cells and epithelial-shaped cells, and no epithelial-shaped cells were observed in P1, but rod-shaped cells were maintained until stage P2. (Fig. 1a). In the chorionic mesenchymal stem cells, rod-shaped cells appeared at P1 and were maintained until the P9 or P10 stage. Two amniotic mesenchymal stem cells stopped proliferation at P5 and one amniotic mesenchymal stem cell stopped proliferating at P7. As a result of evaluating the proliferation ability of the amniotic membrane, chorionic and umbilical mesenchymal stem cells during the culture period, the average population doubling time (PDT) at P3 was 68±22.5 hours for amniotic mesenchymal stem cells, and 28.2±3.2 for chorionic mesenchymal stem cells. Time and umbilical mesenchymal stem cells were measured to be 26.1±2.2 hours. The PDT value was found to increase during subculture (Fig. 1b). Amniotic mesenchymal stem cells proliferated more slowly than other mesenchymal stem cells, and chorionic and umbilical mesenchymal stem cells showed similar proliferative capacity. The proportion of annexin V-positive cells was the highest in amnion mesenchymal stem cells (26.7%) compared to chorionic mesenchymal stem cells (3.9%) and umbilical cord mesenchymal stem cells (1.4%) (Fig. 1c).

As a result, the amniotic mesenchymal stem cells showed the lowest proliferation rate and the highest annexin V expression among the mesenchymal stem cells to be tested. Senescence-associated β-galactosidase (SA-β-gal; senescence-associated β-galactosidase) positive cells are amnion mesenchymal stem cells compared to chorionic mesenchymal stem cells (10.5%) and umbilical cord mesenchymal stem cells (11%). Cells (87.5%) were the highest (Fig. 1D).

2. Mesenchymal differentiation ability of mesenchymal stem cells

Mesenchymal stem cells of the amniotic membrane, chorion and umbilical cord were cultured for 3 weeks under conditions for inducing bone formation, adipogenesis, and cartilage formation, showing differentiation into bone cells, adipocytes and chondrocytes, respectively (Fig. 1e).

3. Evaluation of stemness nature

As a result of evaluating stem cellity through analysis of gene mRNA expression through RT-PCR, expressions of OCT4, NANOg, and SCF, which are important for stem cell characteristics, were confirmed, and HLA-ABC and HLA-G were detected in all samples. . TERT was not detected in all samples (FIG. 2A ).

4. Gene ontology analysis result using gene expression analysis data

In gene expression analysis through microarray analysis, genes showing a difference in expression of more than two times in Fisher's combined probability test were selected. Of the 50,682 genes, 1,190 (2.3%) in amniotic mesenchymal stem cells, 509 (1.0%) in chorionic mesenchymal stem cells, and 638 (1.3%) in umbilical mesenchymal stem cells were overexpressed. The clustering analysis results based on the development category are shown in Fig. 2b.

Gene Ontology (GO) terms were classified based on genes specifically expressed according to the origin of each tissue. In particular, the CDKN2A gene was highly expressed in amniotic mesenchymal stem cells (Tables 1 and 3). In particular, CDKN2A inhibited the growth of mesenchymal stem cells by inhibiting CDK4, which is involved in the progression of the cell cycle G1, and this result was consistent with the results observed in annexin V expression and proliferative capacity of amniotic mesenchymal stem cells. Ingenuity® pathway analysis (IPA) results were similar to those of GO analysis. It was confirmed that the amniotic stem cells highly expressed genes related to cell growth and proliferation.

5. Results of the confirmation experiment of specifically expressed genes

In order to confirm that the function of the CDKN2A gene characteristically expressed in amniotic mesenchymal stem cells is related to apoptosis, Annexin V-fluorescein isothiocynate (FITC) was attached to the cells, and apotosis occurred by FACS. By analyzing the cells, it was confirmed that apotosis occurs in the cells when the expression of this gene is high (see FIG. 4).

In addition, senescence-associated β-galactosidase (SA-β-gal) was also performed, and cells stained in blue were analyzed with a phase contrast microscope (see FIG. 5).

6. Determination of the cut off value

Amniotic membrane-derived mesenchymal stem cells (AMSCs), chorionic mesenchymal stem cells (CMSCs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs), each 5 cyclin-dependent kinase inhibitor 2A ( CDKN2A ) ^{by TaqMan ® gene expression assay} (HS00923894_m1, TaqMan ^® ) was used to evaluate the expression of the CDKN2A gene in the ABI 7500 Real-Time PCR system. Primers for amplifying the CDKN2A gene were F: 5'-CAGTAACCATGCCCGCATAGA-3' (SEQ ID NO: 2) and R: 5'-AAGTTTCCCGAGGTTTCTCAGA-3 (SEQ ID NO: 3). The experiment was repeated three times, and human GAPDH (Hs99999905_m1, TaqMan ^® ) was used as a reference gene, and the gene expression level was calculated by the 2-ΔΔCt method.

As a result, the mean (standard deviation) value was measured as 0.705 (0.739) for AMSC, 0.018 (0.013) for CMSC, and 0.020 (0.004) for UC-MSC.

AMSCs CMSCs UC-MSCs 1001 Am 0.2345553 1001 Ch 0.0121576 1001 Cord 0.0152928 1002 Am 2.0139111 1002 Ch 0.008968 1002 Cord 0.0223126 1003 Am 0.4661933 1003 Ch 0.0394727 1003 Cord 0.0174334 1004 Am 0.3269178 1004 Ch 0.0075203 1004 Cord 0.0206584 1005 Am 0.4822991 1005 Ch 0.0205745 1005 Cord 0.0241807 medium 0.7047753 0.0177386 0.0199756 SD value 0.7389413 0.0131615 0.0036056

Therefore, AMSC has a characteristic that apoptosis occurs well, and CDKN2A is provided as a marker capable of predicting or discriminating AMSC apoptosis through the above experiment, and the criterion (threshold) was set to 0.1. When the ROC curve was drawn based on the 0.1 value, the sensitivity was 100% and the specificity was 100% (FIG. 6).

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Biomarker for predicting apoptosis of mesenchymal stromal cells and uses thereof <130> PN1603-084 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 28749 <212> DNA <213> Unknown <220> <223> cyclin-dependent kinase inhibitor 2A whole SEQ <400> 1 gcccgcgagg tttaggacgg atccaggcag accgcaggct ccgggtcggg gcaccgggtc 60 agcgcgccgg cctgaaggcg gcgtcctggg ctcgacttcc cgcgcgcgga gagccggcga 120 gcccgcgtcc gagttcctgg acgagagccg agcctcgctt agaccgcgct caggacccgg 180 ctcctccgca ttctccggct gcccctgtgt cctcgactca cccctccttt ctgccgctcc 240 ttcctttcct tgccctgctt ttactgttcc caaacaggac cgcttttcct gtctcccagc 300 tggaaaggag gaagggagag agtccagaaa ggatcggtga tgtggaagaa aaggggagga 360 ggggacatgg agggggagac cggagagaga acgtacgccg aggagtcagg cggcgggatc 420 aaggggagtc ggggtgtctg ggcgcggggc agagcgtgga ggcggcagcg gccaacggtc 480 gccaagacaa ccattctacg cgaggacgcg gcgacaggag gggagcggcc agcaggggag 540 gggagcgcgg gggaagagga aagaggaaga agcgctcaga tgctccgcgg ctgtcgtgaa 600 ggttaaaacc gaaaataaaa atgggctaga cacaaaggac tcggtgcttg tcccagccag 660 gcgccctcgg cgacgcgggc agctgggagg ggaatgggcg cccggaccca gctgggaccc 720 ccgggtgcga ctccacctac ctagtccggc gccaggccgg gtcgacagct ccggcagcgc 780 cagcgccgcg ccgtgtccag atgtcgcgtc agaggcgtgc agcggtttag tttaatttcg 840 cttgttttcc aaatctagaa gaggagcgga gcggctttta gttcaaaact gacattcagc 900 ctcctgattg gcggatagag caatgagatg acctcgcttt cctttcttcc tttttcattt 960 ttaaataatc tagtttgaag aatggaagac tttcgacgag gggagccagg aataaaataa 1020 ggggaatagg ggagcgggga cgcgagcagc accagaatcc gcgggagcgc ggctgttcct 1080 ggtagggccg tgtcaggtga cggatgtagc tagggggcga gctgcctgga gttgcgttcc 1140 aggcgtccgg cccctgggcc gtcaccgcgg ggcgcccgcg ctgagggtgg gaagatggtg 1200 gtgggggtgg gggcgcacac agggcgggaa agtggcggta ggcgggaggg agaggaacgc 1260 gggccctgag ccgcccgcgc gcgcgcctcc ctacgggcgc ctccggcagc ccttcccgcg 1320 tgcgcagggc tcagagccgt tccgagatct tggaggtccg ggtgggagtg ggggtggggt 1380 gggggtgggg gtgaaggtgg ggggcgggcg cgctcaggga aggcgggtgc gcgcctgcgg 1440 ggcggagatg ggcagggggc ggtgcgtggg tcccagtctg cagttaaggg ggcaggagtg 1500 gcgctgctca cctctggtgc caaagggcgg cgcagcggct gccgagctcg gccctggagg 1560 cggcgagaac atggtgcgca ggttcttggt gaccctccgg attcggcgcg cgtgcggccc 1620 gccgcgagtg agggttttcg tggttcacat cccgcggctc acgggggagt gggcagcgcc 1680 aggggcgccc gccgctgtgg ccctcgtgct gatgctactg aggagccagc gtctagggca 1740 gcagccgctt cctagaagac caggtaggaa aggccctcga aaagtccggg gcgcattcgg 1800 cacttgtttt gtttggtgtg atttcgtaaa cagataattc gtctctagcc caggctagga 1860 ggaggaggag ataaccgccg gtggaggctt ccccattcgg gttacaacga cttagacatg 1920 tggttctcgc agtaccattg aacctggacc tcccttcaca cagcccctca atcgtgggaa 1980 actgaggcga acagagcttc taaacccacc tcagaagtca gtgagtcccg aatatcctgg 2040 gtgggaatga ctaagacaca cacacacaca cacacacaca cacacacaca cacacacaca 2100 cagtaggaaa ggtgtatttc aagcacactt tctttctcct tggggagaat tattgctaac 2160 catctaagtt ttctggaggc ggcctttttt ctccccagcc tcccggcggg gtcaccctct 2220 cccaccttcc aggagagtgg aggacccgtg agatacgggg cacgcaggca gcgacttcct 2280 gaaatgctaa caaggatcgt aggatcagtt actgctgcga ggagcaagca cttgcttctt 2340 gggggagttt tgcagccaac agggaaatgg gctttctttg tgagttagag gtagaggtcc 2400 ggcggcctga gtgattgaaa ctgctcggga caatgctcgt atgtttagca aacgacagaa 2460 ctgtagaact gttcctgaga aatcccaact gatagtattt tagtcatctc agacgacagt 2520 tagcacagtt taaaaatgag gcctacttct tgaaaaacag aatccaaggt agttttgtcc 2580 tcacattgac aaatgttgac acagccagtg taatttccta taaccaggaa aactgaaaga 2640 atatatgtac agttaaaata tgtacaatgc taattaaaac ttgtgtaata agtctaaaag 2700 taatttaatg aggcttcact tttatgaccg tccttgtggt atgcttcgcc aggaatatat 2760 agcttcaaaa agcaaaggcc agcggagggg taattatttt tttactgcaa tgttaattgt 2820 ctctttgaca tggaaatata aacctgttaa aactatcagt gtttaattta gtgtctcaat 2880 ttctattagc aaaaatttat aatctatagg ataaatgcac attttatttt ttacttttca 2940 tattatgcaa gttaattttt ttaatttagt caaaggagct tataaaggat ttcagggcct 3000 gttgctggat ttgattttaa ttcattttga aacattgaca agaccctggt tgttgttttt 3060 tttaacagtg gtttatccgt atcagcaaaa gtttagccac tgtgaccggt aactgtatga 3120 atatagttct taatattatt gtctatataa aaatatttat tactctagtt aatattattc 3180 tatataaaat cattttgttt aaattattaa gttgcctctg aaaatctgta gtaacaaagt 3240 agaacatgtc aatgtatata aatgccataa ttatgtattt tttagtttag gcctataaaa 3300 cataacattg tggtgatttt aagttagaga aaatatttta tagtatgtta atgtatatgc 3360 atgaaatgca aaaatattta aatgataggt tcattgaaat agatcatttt ttgttattta 3420 ggtataaatc aattttcagg acgtatgtga aaagcgcaat cttcaggaag tttctcaaga 3480 tagaacacag cttggataga atgtcttgaa atatatgcaa ttttccaatt tcatatgtaa 3540 aatgatatac ataatataaa atctagcggt gttaattata atgatatgta attatatatt 3600 tcacattaat atattttatg cccatggcta tattgatttg ggaatatata tggatactaa 3660 ttatgttagg attcatacaa ttccttgaga ggcacaagtg ctaaaaatta cttgtatgaa 3720 ttatttaata tcattgcaaa taagatgtta ttttaacttt ttttaagttt ctgcaaatat 3780 gtttattatg actttttatt tttatatgat tggaaataca tatactaaaa ttccacgtta 3840 ccagtttctt aaccacagaa acctgaaaaa ttgccatagt tgatttgtta cttctacctt 3900 ggtgcattta caaaatagtc atatttttat tatgaagtta aatattcatt tgtttatagc 3960 tacttcagaa ggctcaggtt atttttttct ttaatagcac agagtcctct caaggtaagc 4020 actgtgcagt tagtataaac cattattccc catgtgtaca tgattcacag tttgtattgt 4080 gttccaagtg aaccatagcc ctttcagaaa tcaagactta tattcatttt acttctttga 4140 gtactcttga attttagaaa gtccattatg atcctaaggt agcaacaaca tagcctatta 4200 ccgtctatga tggtttacag atctattatt ccacgttagt tcatcactat caactaccat 4260 gatagagtta agctaaacca ttttcccaac atatgaaaaa ctcctattac taaagtgata 4320 caaatggtat caaaaatact tttttatagc aaggttcaac agtgggccca gtgcttttac 4380 actttttcaa aagtccttgg agaaacagag aaaatctcac ttgccttctg tactaaaaca 4440 ttctaggccg aactaaaact gaaacttcat agtagaacac tgtaggccag gggtgttcaa 4500 tcttttacct tccctgggcc acataggaag aagaagaatt gtcttgggac acacattaaa 4560 tacactaaca ctaacaatag ctgatgagct taaaaaaaaa aaaaaactca tgatacttta 4620 agaaagtgta tgaatttgtg ttgggcagca ttcaaaacca tcctgggctg catgtgaccc 4680 tcgggcccac aggttggaca agcttgccct agctcctcca tctgctgcaa agcccagcct 4740 gatacaaaaa ccaacgtgat aaaaagtttt tgtggtgctt tattttggca gtttaagtta 4800 tataaacaat gggtacagtt tcattttcta aatataaaat ttttacattg aatatgaatt 4860 tttaagacaa attatctgaa ttctgattct catataccta actactaata tcttctctat 4920 ttgttgccca atgagattaa tccacctctt aaacacttca ccatcaagaa aaacaatttt 4980 gtattttaaa atgaacccat ccactttcat tcagctattt tatattcagg catcatccta 5040 aggaaagaaa ggttctgaca aagattaata cagatggata agtagtagca agaaatcaaa 5100 aactgcataa aattctagca ataaagtgtt aaattatggt acagttacat tctggatcat 5160 caggtatctg aagaatattt catagactgt taaatgattg cattataaag tcaggttttt 5220 ttaaagcaag attccaaaca gtaaacagtt tctctctctc tctctctctc tctctctctc 5280 tctctctctc accaaattag ttataatggt ttccgcagga tgagaggggt tgggaaaaag 5340 tttggtgata tttattttct tcgtttcact tttgagtttt ccaaagtgct atgaccatca 5400 tcagtaaaat atacatttcc aaagcctttg acacacggta acagtcctac acagtggatg 5460 aactaagagc ttctctaccc ttagatgggt agggagggag gaaagacaag gaaactgagt 5520 tgtttaagtg tcatacacga gaacgtggct ttaaggtctg ggaaaacctg cgagggctgt 5580 gacgtcagac tgtgaaatgc acgctatgtc cattcaccaa gacgttccat tttaaaaccc 5640 ataaatccgt agctatacct gtttccaagg tgcctcgtgt taggcctctg gtcacagcac 5700 ttggcgccct tcttgggatc tcttctctcc gcccccacta ccccacccca caagcacact 5760 ctagtcccct ccaatcaatt tcaggcaggt ctcgccgcct ccggagccac gctgggggtg 5820 caagggccct ggacccgaaa gagcgcccgc ccggcgacaa gagatgagat gcacgctgct 5880 cctccactcc tcagccccca ccatcctcct cctggatcct aacttcccca ctctctcaat 5940 tcctagagac gctgcggatc ccagaggctt aactggcagc tggaacgagg tcctccaaca 6000 agaatttaga cgctaggtcc aattatcact ccaccgcgcg cactttccgc aggagcgatg 6060 tgatccgtta tcataactgc ggacctgggg ttccacgtgg aagacgattg ggatttcact 6120 ggccgcggtg ggggtgggag cagacagagt ctgagtgggg ttagtggact cgagacgaaa 6180 ggcaggacat gacagaaggc aactctgggt cacctctcca gcttggaact ggctaggcct 6240 tgttttggag gggatgggta gatgaaaagt gagtcagggt tacccggagg aaccacgggg 6300 aaagtgcgct tctgagactc ttgacagcca tttcgttccc ttccaagcca gatggagacc 6360 caagagtgtt gaaaggccac gacttccctc agtttctcca tctgggggtg caggatggta 6420 tagagagtgg cccgtagtat ttttccagtg acgatgtctc tccattgttt tcttcttata 6480 ttgcagcttt ccccatgttt gaaaattttc ttttcaaatg aaatcattga ttagaataaa 6540 aaaaagtaag tagctattaa aacaagatca atttccatga cagtaagcca accgatggag 6600 aaaaccttgg gaattaataa atgaaggatt tgtttggtag atgataaaag gtccttttaa 6660 agggtctgac tcttcctaga aaaacccacc aacttgggac cgcaacagat ttaccatatc 6720 ctaattcatg ctattttaat gtgtattcag caaacccaca tgtgtttaca attgtcgaag 6780 ctaccaaatg tcaatagcgt tttttttcta tttgttgaat gtgaatctct tgtacgaagc 6840 catataaaca gaagaaatta caggaatgat tttaaatcac atacaaaacc aatagtattg 6900 ctagaggaga gttagtcaag gacggcatta tgaagaaagt gagggagaat ttccaaagag 6960 cagaacgata gggcttggtg gaccaaagaa cgtttccatc taaagggaat ggcaaatact 7020 tagagtctct gaacccactg aatcttggac tatttaacta atatttgtag ttccagatat 7080 agcacagtgc cttgtacata gtggtatttt taaaaatata gtgcctcgta gatttttttt 7140 caacttttat ttaggaggag agggcacatg tgcaggttaa ttacaaaggt atattgcacc 7200 atgctgaggt ttcgagtacg actgaatctg tcactcaagt agtgagcaca gtacccacag 7260 taggtagtat ttcagccctc gctcattccc tttctcctcc atctagtagt ccccaatgtc 7320 tattgttctc atatttatgt ccaattagca tttgtttttt aaaaagggtg gttgaagaaa 7380 ttctcagtgc ttgtcagtgt ctctcagtgc attcatttaa ttcatgagcc ctggaatgat 7440 ggtttcattt gggcagaact ctacaatcaa aaagaagtaa taaaagggaa aaaaaagtga 7500 aagccatcaa ctacaggatt gaaattccca aagcatcaga ggtcctttca aaaaatagta 7560 tgttgatttt taatttttat gacttattgg ctttgttcat gaaaatataa acatgttatc 7620 acaaaggatt ttttaattca actatttctc agttttctct ttcaccttca aaataaaata 7680 tcataaatta tttaaatggt tgtgaaggca gtaggatttt tttaagagag aaaagtttta 7740 tagaggttca gaattacatg aacaaagaca tgtaatctct taagcaaatt gaaactaata 7800 aaatcgtaca atcaaggtaa cgtaaataaa aaagcctctg ctttcttaat tgaattatgt 7860 gagtaactag aaattttaaa agtatggcaa aggttaacaa cagcattatt acctgggctg 7920 cctttaaaaa tacatatttc tggggttcac gttcagaaaa tttgattcag atttgctgtg 7980 ggtcccagaa atctgcattt taaataaaca cttgaaggag atactaatac aagtggccca 8040 ttgggacaca atttgacaaa tatgaccaat tttacttttt aaaccttatt tctgcttctt 8100 tatctttgaa ttgaggtcca ggattttagg taagatttta agtttagagt cagtttactg 8160 gatcccaggg aggagagtct gagtaatcag tggaggagtt atttcaccaa atgaaggaga 8220 ccctttatta ttatgtgacc ctttgtatga attggaaaag aatgtcttgt agataccaca 8280 tttttacagt cagaacatag tttgagagaa aaaaatataa caagatatat ttgtgtttta 8340 aagcttacag aaccagacag aaaatttcca cataagctat ataagatacg ttgtcttttt 8400 aaaacactat atacacttct ttctgttcgt gcaggatgaa tggatctctc tctctctctc 8460 tctctctctg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttgtaa taaggggttt 8520 ctttcatttt atgatccaga ccaggctcgt aataaacatg acaacctaaa attatgtaaa 8580 aaagaaaaat caaagcacaa gtgtttcaca ggtttaactt atgcttatct aagatcaggg 8640 caagattgca ggaaaatgta gccataacag aataaagcat ttatggacaa aatgatgggt 8700 ctttatgtct ctgtaaaagc acagtgatgg ggggggaaat atagatgaaa aatgtaagct 8760 aaaaagtaac aattataaga aaaactaaaa tatcatgcct ttcaaatgat catttttctg 8820 cttttaagct aaaatttgtc taatattaca ccagtgactt tgctgatgta ttaggaaaaa 8880 gcttgttttg ctttcttttc tcgagtgcca ccattttctt gctctcattc tctttcaggc 8940 tgccagatca tctgactcag caattgtata actctctcac ccaatttaaa gaaacagcag 9000 ctgtctagag aacaatgact cccccagttg aacatctaat tgttaaatgt ccaacatcgg 9060 acactttgaa ttttactcca tgcaatttac atgctgaata gttgaagttg aatatattat 9120 atttaacatt taatttttaa aagcttattg aaactttctt cctaaatcac atggtaaagt 9180 tattgttttc ttcaaaaaca attaggagga gcttaacaat aataggacac ttcaacttcc 9240 attatctaat ttaattatca caatatcctt atgttttcaa tgtttcattt tttcattttg 9300 tagatctgga gactgaggct cagataggtt gcatggccta ccaaaagtca ttgactagta 9360 attcatatat agttgaactt ggttgcccat ggagtgctat aaatatgtat atggtttcag 9420 ttccatctct tttagttaac tattattttg aaagtcgctt aacccctttg ggcctctact 9480 atactcaagc atcagccgta taagtcacag taaatattta ttggttgaaa ggaggttaac 9540 atctttcaaa aatttatttt ttgaccaaaa taaaaccagt gaaaaattct catatgactg 9600 tacatataaa ttacttattc ctaccttaat ttaaaagcaa taagtgggat acctattcac 9660 cagcacagga accacttgaa gcgtgcagtt gaaagattac tttctttagc attcacatga 9720 cctgtgagca gattctattt cttttgctta ttagctgtca tggtaccaga atgaagtatg 9780 agaaactctc agtgctttca tgttctcatc tgtaaacctg agaccctatg gtagtcccgt 9840 aataagaggt agataaaata gtatgtgtga agagtcactg taaactttta cacagtgtac 9900 gtttgtcagt tattatagtg cctaattaaa ctatgccctt aagaaagcac attagttttt 9960 tacagtaaat acctacttca ttataatttt tcagtgtagc tagaaatttc taaactccac 10020 tttaaaaata tacatatcat aataaaaata tatttatgta ttcagactcc tggtatgttc 10080 caaggtgtta ggtaaaatca gtgtaaattt gcatactttt aaattcacat ctgtacagaa 10140 gatctatatg gtggccttta gggtatacct ctaagctatt ctagtattca taatcattaa 10200 agagatatta agcagtgttt gtgaacccct gttttctaag acaggaaatc aaggtagctt 10260 tagaaaactg gaaaaaaagt tattagtcta tctatctaat aacccagaat aataatttcc 10320 aaaggaatca ctgaagataa ctggattttt aattccttca gaatggttgt cacagtctga 10380 atatctgaat caacagtttt gaccaaaaca attttctaaa aattctttag tataaaaaat 10440 tatgtgtgtg tgtgtctgtg tgatgaaagg aatgataggc agaaacatta ctgtcatcct 10500 tacgacattc aaaatgccta ccttggaggg tgaccttcag ttatttttat gcaaatgtga 10560 agaagttatt tagaagtagg atatcaaaga gtaacacaaa atacactaaa tagtatgctt 10620 tcttaaggct aaattgactt gggggtttta aatcagtaca gagtaaacat acagtatatt 10680 ctgttatcat tgcctttttg aaaaattaat tatggaagtt atcatcttaa ccgtaacaac 10740 acaaaagata aaactctacc ctcaacccag agactcaaag gaaaacatga gtggaaatgt 10800 taaatctgta tgtgaaaagt gctaaaacat gaataggaag cagttactta tttaatcaaa 10860 gttgattata tttcatcaag aagttgattc ccttgagtgg agttgaatca catatcaggt 10920 gaagaatgtg atttggggaa gaatggtcta acacaagaaa attttcttgc aatctttaat 10980 aatatcagag gggagattgg cttcagaact ctcctaagtt caggaaagga cacagaaaat 11040 tgaacataac agtaagacta tagagtccca agaaagcaag ctacttttaa aggatagttt 11100 tttagagggg caaaaggggg acaaccattc tccatttgat gagaaaagct tccatgtaga 11160 tggtgcccct gaaattagag tatcctaaac cagtgttaaa cctatcagtg aaacatgaat 11220 attaaacctc cactcccagt agtgaaaacc gaatacatta ttatttatct gtgactttca 11280 acattatctc agaactctaa cagcacatgc gtacatcagc agcataagca gaaatgagat 11340 attatatatg cttgtgttag caattaaaaa ggacagcata tttgagaggg gaaaatctgt 11400 cctatcaaga atgaaaaaga gggaggttag gaaaagtagt ttagagaaag taaattttgc 11460 aattcctcag ttttaactgt agtttctcca ttgtaccttc cacttgaaat gcactccaag 11520 cagtggaggt gggtagcaat gaatgcagag gaaacactga acacagtgac actctccagt 11580 gtcacttctc atgatttaat gaggggtttt ttttggaaat tcttctgtca taacatggga 11640 aactttgtta caaagaagct gttttttcag agggttagaa ttcagaggta gcatcatacc 11700 ttttagaaga gaatttgctt gttgaaacca cagatacctg ctagaatgta caggaattaa 11760 tgaaaaatta ctcaaaagga catttatttt gatgacctaa atgaataact tcatagtaaa 11820 tgtcatatat attctcaaaa aattaaaaag caccatttat tgagagccta ccgtgcaccc 11880 ggatttttat atatctgaca ttctttattc ctcacagtaa ccttatgggg taaattttat 11940 tttccccact ttgtgaggtg aggaaataaa ggctcagaaa gtttacataa cttattcaag 12000 cccacagagc tggtaaatga gaggtcagtt ctatctgagt ttaaagacta ggcttgtccc 12060 acttgcatat gtgtcatttc caaaattatg attaaggata tggttggcat ttcccgccac 12120 ccacattaag tccaattaag tagctgtggc catagaaaga atggagaatg gagagaggaa 12180 ctgacttcaa cagctacagc aaacatttat tagctgagta accatagcta catagttcct 12240 caatatgtac cactcctcca ttttgttatc tataaatcaa aatggtggct ttttaaaaag 12300 cagttttaca atatattcaa gagccttcta ccctttgaaa aactgcaata ctatttttag 12360 tagcaattag aaacacctta aatatctgac aacagggaca tcattaagta aattataact 12420 ttttccagtg ggatgtgtta cagctgttaa aagtagcatt tatgaagtgt ttttggagaa 12480 gtttggaaaa tgctgtaata agttagaaaa agctcatttc aaaattgcat aatattcaca 12540 atgtaaagat taagcaaaga aaaaggaaga agtatttcaa aatgttaata attattgctt 12600 tgtgtggggt agtttttcat tttctatgtg cagctaattc cttaattatt tttaaatatg 12660 tgagctttaa tcaggaaagc aaatcattca aaaatgaggg gactgaatta agtgactttc 12720 aggggacttt gcgtgtcttt gagttccaaa tttctatcac tatgtattac tactgaagaa 12780 taatcataga agcacagtag tttctgaaaa tggagagtca gtaatcttgg cccaggtttt 12840 gcaacttgct ctaaagcaga gtcctcaaag aaaaggaagc attgatgagt tgtccacaat 12900 gtactggata aattatcatt aggaaaacat attgtagtag ggagagtgag gacctctcaa 12960 acagaactga gaaccttaag tttgaacttt tcttttcctt attacttaag cactctgagc 13020 tttttttttt gtctgcatta tgaagaaaga ataatactct ctatcccatg ggacagctgt 13080 ggaattataa attacacata taaaactgct tgatgcttgt cacatagctg ggggttgaaa 13 140 aaatgatagc cattattttc ttggcaactt ttaatgaatt ttttattatc tctatttctt 13200 tctgcctatc tcctctaatt atgtttatta cttattttgt tcctcaggat gaggtcaatt 13260 ctcaatatct gtgctgtaca taatatacat atataccaaa tatgtgcata tagtatgtac 13320 atacatacat actgtgctaa tcttttagtg ttctcagctg atcaaatagc tacaaataga 13380 tataagtaat tcgccacaag taatttatca acataaaaaa aatttacaaa aaagttaagg 13440 aataattgtc tccatgagct gcaaagatcc ctcatttcac aagagtacac cctagagata 13500 ttttaatagt aaatttctca catagattta aaatcacatt tgttttgcac ataatttaga 13560 aaagatacct gctatataat aagtaatata cttttaagtt tccttcaaaa tattcttggg 13620 aagatgataa taggtactgc taattctata cccagttaac attttggaaa ctaaggttga 13680 aaattgtgac ttaactataa ttatgcatta aatctacaac acatcaaaga attttgcatt 13740 ttgtactcct tactaagatc cagtttgagt aggaagataa attttacagt aattctgaat 13800 gagggaagtt ggcacagagt ttctaaaaga gtaccttcct tatagcaaat actaaataat 13860 tgtgctatat tgaatttaat taaatagaga atagtaaaag ggagaaagaa acatccaatg 13920 ttttgaaact tctagagatc tactcccagg gacacattgt tttttcttag caaatctgtt 13980 tggaggtctg ctctactttc tcagaggtct ccctttcatg ctgaagctat cttttttcct 14040 tgtggaacat aagtaattaa ataccttgca attatttacc taagaaagtg tttctttccc 14100 gtttaaaatg ctcttaccac ccacattgga ctcgattatc agaattttta tccggggcag 14160 cttcaggagc actttggcac ttcggggcta aaccacaatc tgtttttaca tgtttgtgat 14220 tatacccgtt ttgtagatca agacattgaa gctagtaaaa aaaaaaaaaa gtcatttttt 14280 cagggtaaca aagtaggtgg tagaactagg acagggactc taatttcctt acattattgc 14340 ttttctaaat taaagggatg catggaatta ttcctccatt gcctttgcct tcaaataatt 14400 atctattgca cccaacatcc tattctagaa ctcatctatg aaggcttaac acagctgtac 14460 ctgggagctc cattacaggg catatatctc gctctcataa gctacttcct aaggaattct 14520 ctttaattat gggagctttt ccagactctg aaatcttttt ttcctggtaa cacaagtgtg 14580 aggtgtcatt tatcagaatg catcacccca gtcttccctc ctcaaatgat tactgtaggc 14640 tccactcaag agctcatccc agttcaagac caccttcctc ctccagagaa gcaaatatat 14700 atatacacgt atatatatat atacacgtat atatatatat acacgtatat atatatatac 14760 acgtatatat atatatacac gtatatatat atatacacgt atatatatac acgtatatat 14820 atatatacac gtatatatat atacacgtat atatatatat acacgtatat atatatacac 14880 gtatatatat atatacacgt atatatatat acgtgtatat atatatatac attttttttt 14940 tttgagacgg agtctcgctc tgttgcccag gctggagtgc agtggcgcga tctcggctca 15000 ctgcaagctc cgccccccgg gttcacgcca ttctccttcc tcagcctccg gagtagctgg 15060 gactacaggt gcccgccacc tcgcctggct aattttttgt atctttagta gagatggggt 15120 ttcaccgtgt tacctaggat ggtctagatc tcctgacctc gtgatccgcc cgcctcggcc 15180 tcccaaagtg ctgggattac aggcgtgagc caccgcgcct ggcagagaag caaatatatt 15240 gatggttgtt accaatacat gctcttgact aagaaacctt ctttcttaat taatattgac 15300 aactttaagc cgagtgcctg acatatatta ggtactcagt tactcttttt caactaaagt 15360 tatgaatgat gattctaata aaagtaactt atttgtctac tagttttatt atgtttattt 15420 aattcattag aaaggccatg gacatagtac aaaattcaaa caatataaat catggaatgt 15480 gaaaagtaag tcacatgccc atcccagttc ttcatttcct tacctcacag gtaacagctt 15540 ttcctgtatc tccccagaga tattctatgt atattttgtt tttaacacca agctatattt 15600 aaaacaatta tctttaataa taatgttaat attgaaactg gtaaagaaat atgtgtgtat 15660 tatctcacct caagcgtaaa caatagaaca agagagagcc cattttgaaa attatggaca 15720 atgaatctag aaataatctc aaaagatttt gcagtcaaaa aatagttcat tagatacatg 15780 agaactgtca cttggtctca gtgtagagct attgcctcaa ctccctttat tttcctaaca 15840 aaatcatctt gcttatccca tgaaatacgt gcatattgcc aatcctacaa tgccgcatca 15900 gaaccagaac ccaactctgg aacactacct tctcaagtat ctttctgtct ctttatggta 15960 atatgttgaa ttaatattca catctattat gactagtctt tgatttgtag ggttgctgaa 16020 gtagtagcac cactgcaggg ctttctttag tttaaagaaa gtaatcaggt gtccctactg 16080 tgtcatgatc tccaccctca gctgggttct ccagtctggt tttaaagaac aaaacaaaag 16140 gcttctctgt ctgagtctta ctcaacccat cctctctact cataagaggt attccaaacc 16200 tttacgattc tcaaacttcc taaccgacca tcttattttc actctgcaaa caagctaacc 16260 tcctcattca tagaaggaag tgcctcaact tcctccccgt tctgaccttt tctccctccc 16320 aaatctatgt atctcttgtg acaaaatcta taaccaccgc tgtactttga gttctatttc 16380 ttcattattt ttgagggacc tcaagtcctc aaaaatatcc tatcttgcct gtgtacttaa 16440 cttttctttt attcttttct aactttccct tctcttcact tggcacttgc ccttccaggt 16500 atatgtgtgc tcaggtctcc tccaccttcc atctgcctca cttcatggca tagggccttg 16560 aactatcaca accaagctat gaaagagtag tcaacgcagt gtccccactt ccttgccatc 16620 ccattatcct agtttttctt ttggctctct gaggagtcct tcacaggctg gttttcagga 16680 ataagtctaa atgaatcact ttcagttttc ctaaacttct atgcctttgc acatcctctt 16740 acctctgcct agaatatctt tctccttctt ttccatcttt aaactctcac atcattcttc 16800 aagactggga tcagctctca gcatccggaa gcctttgcct actagagaca aatgagaatg 16860 agtttggtca ccttttcatt ttcttgtatc attctgtgct ttattttgct cttctaagag 16920 cgttacatgc ttcatttaat ccctaaacaa ctgtttgagg caagtacagt tattatccta 16980 atcatgcaaa tgagaaaaca gaggcccaga catgttgagt aactttgata aaagttaaag 17040 aaccaataag tggaacagtt gaggtttgaa ccctggcagt ctgactgtag agatactatg 17100 tttgacctac tcccctctgc ccccacccca tgtctgccct tagtttctga gcttgttgaa 17160 tgaatgaaca ggtggtagtc tttttttgtt ataagactga tcagaattaa gacaggttta 17220 aatttcacgt gtagaatttt caaaactgca aaggcagtgc aaatctaaaa aaagaatggc 17280 attctcagga aagaggaaaa gtaagtgtga gaataataat aacaataacc aacaaacttt 17340 agtaaattta gtaaatgtag taaattttta cattaaaagc ttttggacat acattatcat 17400 attttatggc cacatgaaat atattataat cccattttgc acataggaaa tctgagactg 17460 gcataaggag cacagagatc caggacttta tattttcatt cttctaggat tttgcacctc 17520 aggtcgatat gtatgagtaa actgggagta taatgggctc tttaacagaa aaactaggaa 17580 agttttccca ctattattaa ttatttacat aatatttttt taattttatt attatttata 17640 ctttaagttt tagagtacat gtgcacaatg tgcaggtttg ttacatatgt atacatgtgc 17700 catgttggtg tgctgcaccc atcaactcat catttagcat taggtatatc tcctaatgct 17760 atccctcccc cctcccccct acataagatt tataatggat aatggacttc aatttctaga 17820 gcaaaatggc cccacccaag gatgccataa tccttccaga gctctactgc aagatatgag 17880 atatacatat ctaaaacttg ttcttggtat ttccaaagca gtcaactttt acacctgttt 17940 ataatgcatc caaatgttgt ttttatatgg ttgcatctcc catcttcttc accaatagct 18000 atatatattt ttcacaagag ctgaaagagt tcttgatgta ggaatccatg gtagagtttc 18060 agagaaatcc ctgaattcac tgaaagtttt atctagaaat acatgtgcaa gtgaacacat 18120 cttttttaaa aaaaatcatt acctactttc ttttttgaga agaaggtatt tatttcaaca 18180 gactcttgaa ggagcctact cttcccactc tcccaccccc attaagaacc actgtaggcc 18240 gggcacgatg gctcatgcct gtaatcccag cactttggga ggctaaggtg ggtggatcac 18300 ctgaggtcag gagttcgaga caagcctagc caacatagtg aaaccccgtc tctactaata 18360 atacaaaaat tagctgggta tggcagcatg tgcctgtaat cccagctact cgggaggctg 18420 aggcaggaga attgctcgaa cccgggaggc ggaggttgca gtgaaccgag agagatcgtg 18480 cggtgccatt tcactccagc ctgggcaaca gagcgaaact ccatctcaaa aaaacacaca 18540 aaacaaacaa acaaaaagaa agaaccattg tattagtgat ggaaatgtgt tccctccctc 18600 ccatcctggc aaccactttc ttcctcctcc atcataaaat atcttaaact aaactaaaat 18660 aattttattt atcgatagtt tgaattttcc ctatcattgc tacacagcta attgagaggt 18720 accccgagga aaatataaat ggtacagtaa tgcattgtag attttaataa catacttgac 18780 atcccaaatt gttttcattg gcttcatttt aaaaactaca tgttttaaaa tcaagcagac 18840 actaaaagta caagatatac tgggtctaca aggtttaagt caaccaggga ttgaaatata 18900 acttttaaac agagctggat tatccagtag gcagattaag catgtgctta aggcatcagc 18960 aaagtctgag caatccattt tttaaaacgt agtacatgtt tttgataagc ttaaaaagta 19020 gtagtcacag gaaaaattag aacttttacc tccttgcgct tgttatactc tttagtgctg 19080 tttaactttt ctttgtaagt gagggtggtg gagggtgccc ataatctttt cagggagtaa 19140 gttcttcttg gtctttcttt ctttctttct ttcttttttt cttgagacca agtttcgctc 19200 ttgtctccca ggctggagtg caatggcgcg atctcggctc actgcaacct ccgccttctc 19260 ctgggttcaa gcgattctcc tacatcagcc tccgagtagc tgggattaca ggcatgcgcc 19320 accaagcccc gctaattttg tattttttag tagagacagg gtttcgccat gttggtcagg 19380 cttgtctcga actcctggcc tcaggtgatc cgcctgtctc ggcctcccag aatgctggga 19440 ttatagacgt gagccaccgc atccggactt tccttttatg taatagtgat aattctatcc 19500 aaagcatttt tttttttttt tttgagtcgg agtctcattc tgtcacccag gctggagggt 19560 ggtggcgcga tctcggctta ctgcaacctc tgcctcccgg gttcaagcga ttctcctgcc 19620 tcagcctcct gagtagctgg aattacacac gtgcgccacc atggccagct aatttttgta 19680 tttttagtag agacggggtg tcaccatttt ggccaagctg gcctcgaact cctgacctca 19740 ggtgatctgc ccgcctcggc ttcccaaagt gctgggatta caggtgtgag ccaccgcgtc 19800 ctgctccaaa gcattttctt tctatgcctc aaaacaagat tgcaagccag tcctcaaagc 19860 ggataattca agagctaaca ggtattagct taggatgtgt ggcactgttc ttaaggctta 19920 tatgtattaa tacatcattt aaactcacaa caacccctat aaagcagggg gcactcatat 19980 tcccttcccc ctttataatt acgaaaaatg caaggtattt tcagtaggaa agagaaatgt 20040 gagaagtgtg aaggagacag gacagtattt gaagctggtc tttggatcac tgtgcaactc 20 100 tgcttctaga acactgagca ctttttctgg tctaggaatt atgactttga gaatggagtc 20160 cgtccttcca atgactccct ccccattttc ctatctgcct acaggcagaa ttctcccccg 20220 tccgtattaa ataaacctca tcttttcaga gtctgctctt ataccaggca atgtacacgt 20280 ctgagaaacc cttgccccag acagccgttt tacacgcagg aggggaaggg gaggggaagg 20340 agagagcagt ccgactctcc aaaaggaatc ctttgaacta gggtttctga cttagtgaac 20400 cccgcgctcc tgaaaatcaa gggttgaggg ggtaggggga cactttctag tcgtacaggt 20460 gatttcgatt ctcggtgggg ctctcacaac taggaaagaa tagttttgct ttttcttatg 20520 attaaaagaa gaagccatac tttccctatg acaccaaaca ccccgattca atttggcagt 20580 taggaaggtt gtatcgcgga ggaaggaaac ggggcggggg cggatttctt tttaacagag 20640 tgaacgcact caaacacgcc tttgctggca ggcgggggag cgcggctggg agcagggagg 20700 ccggagggcg gtgtgggggg caggtgggga ggagcccagt cctccttcct tgccaacgct 20760 ggctctggcg agggctgctt ccggctggtg cccccggggg agacccaacc tggggcgact 20820 tcaggggtgc cacattcgct aagtgctcgg agttaatagc acctcctccg agcactcgct 20880 cacggcgtcc ccttgcctgg aaagataccg cggtccctcc agaggatttg agggacaggg 20940 tcggaggggg ctcttccgcc agcaccggag gaagaaagag gaggggctgg ctggtcacca 21000 gagggtgggg cggaccgcgt gcgctcggcg gctgcggaga gggggagagc aggcagcggg 21060 cggcggggag cagcatggag ccggcggcgg ggagcagcat ggagccttcg gctgactggc 21120 tggccacggc cgcggcccgg ggtcgggtag aggaggtgcg ggcgctgctg gaggcggggg 21180 cgctgcccaa cgcaccgaat agttacggtc ggaggccgat ccaggtgggt agagggtctg 21240 cagcgggagc aggggatggc gggcgactct ggaggacgaa gtttgcaggg gaattggaat 21300 caggtagcgc ttcgattctc cggaaaaagg ggaggcttcc tggggagttt tcagaagggg 21360 tttgtaatca cagacctcct cctggcgacg ccctgggggc ttgggaagcc aaggaagagg 21420 aatgaggagc cacgcgcgta cagatctctc gaatgctgag aagatctgaa ggggggaaca 21480 tatttgtatt agatggaagt atgctcttta tcagatacaa aatttacgaa cgtttgggat 21540 aaaaagggag tcttaaagaa atgtaagatg tgctgggact acttagcctc caattcacag 21600 atacctggat ggagcttatc tttcttacta ggagggatta tcagtggaaa tctgtggtgt 21660 atgttggaat aaatatcgaa tataaatttt gatcgaaatt attcagaagc ggccgggcgc 21720 ggtgcctcac gccttgtaat cccttcactt tgggagatca aggcgggggg aatcacctga 21780 ggtcgggagt tcgagaccag cctggccaac aggtgaaacc tcgcctctac taaaaataca 21840 aaaagtagcc gggggtggtg gcaggcgcct gtaatcccag ctactcggga ggttgaggca 21900 ggagaatcgc ttgaacccgg gaggctgagg ttgtagtgaa cagcgagatg gagccacttc 21960 actccagcct gggtgacaga gtgagacttt gtcgaaagaa agaaagagag aaagagagag 22020 agaaaaatta ttcagaagca actacatatt gtgtttattt ttaactgagt agggcaaata 22080 aatatatgtt tgctgtagga acttaggaaa taatgagcca cattcatgtg atcattccag 22140 aggtaatatg tagttaccat tttgggaata tctgctaaca tttttgctct tttactatct 22200 ttagcttact tgatatagtt tatttgtgat aagagttttc aattcctcat ttttgaacag 22260 aggtgtttct cctctcccta ctcctgtttt gtgagggagt taggggagga tttaaaagta 22320 attaatacat gggtaactta gcatctctaa aattttgcca acagcttgaa cccgggagtt 22380 tggctttgta gtcctacaat atcttagaag agaccttatt tgtttaaaaa caaaaaggaa 22440 aaagaaaagt ggatagtttt gacaattttt aatggagacg ggagaagaac atgtagaaaa 22500 ggggaaatga tgttggctta gaatcctaac tacattggtg tttaatatag gaacatttat 22560 ttatataaca ttttaaagta ctaaattcat attagtatat tatcaaatgg atatattatc 22620 aaatgggttt aagcatccta cacattttaa ttcaattgat tcattttctt tttgctttgg 22680 atttctatca tgatttaaat atttacatat gggttacttt ttagattttt catactatga 22740 aatataagaa aaacctttaa ggctagtttt atgaccaaga cgaaggactt cattgaatac 22800 acaaaacaat aaatatactg caacattttg tctttctttt tgtagctgca atttggtttg 22860 cttatacttt ctctttgtct ctttgaaaac tgagtcagtt tcactttctc aggacaggat 22920 ttaataacca taatataatt tagtataatt ccttgattta ggcaaattat gcaatttgtg 22980 tttagtatga aatgtaccta aaaataagta actcctcttt aacaccacca tcctcaaact 23040 aatataacaa ataacagtta tcctaaaata aattgtctac ttccaccatg cagcactcaa 23 100 attttaaggt tgctatgact gcagacagta ttttaaaatt cctctctgga aatggctttg 23160 tttccaagat gatttaggaa ccaaagaggt gaccatctct tgtttaatga actctcaaat 23220 cataaacctg ggaagtgttt tagtttccta ctgctgctgt tacaaattat cacaaatgtg 23280 ttagctaaaa caaacacaaa attattattt tacagttcta gagatcagaa gtcaaaaatg 23340 ggtccacaag gtttcattcc ttttggaaac tctaaggggc aatctgtttc cttgtctttt 23400 ccagcttcta gtgaccatca aattccttgg ctcatggtct ctgtattttc tctgtggcct 23460 gtgcttccat tcttgtatct tctctctgac tgtgaccctc taataaaaac acttggggtt 23520 atgttgggcc caccctgaaa attctggata atctccctca agaccattaa ttaaatcaca 23580 tctgcaaagc ctcttttgcc acataagtta atgtattaaa agtttttgag gattaggaca 23640 tagacattgg gggtgggggg gcattattca gcctaccaca ggaaggaatt ttagggttaa 23700 ttaaactagc cttcttattt tatacttgaa gaaattgaag ttttggaatt ggagagcatt 23760 atgctaaatg aaataagcca aacacagaaa gacaaatatc acatgttctc acttatctgt 23820 gaaatataaa acaattacat tcttagcagt aaagagtaga atggtggtta ctagagctgg 23880 ggggtgggag gaatggggag atggtaatca agatataaag cctcagttaa gatgggagga 23940 ataagtttga ttgttttttt tgagatgtgt ttcatagcat gatgaatata gctaaatagt 24000 aaatcccaaa tgctctcatt tgacaaaaat gtcaaatatt tgagatgatg gataggttac 24060 ttagcttgac ttaataattc cccattgtgt tcaaagatca taacttcata ttgtaccaca 24120 taaatatata caactgtact atcccaatat ataattttaa aactaatata atgaaaaaga 24180 aattgaagtt caacattccc agaagctaag tgtaacttaa aagttttgtg agaatttgtt 24240 ttaacaaaca aacaagtttt ctctttttaa caattaccac attctgcgct tggatataca 24300 gcagtgaaca aaaaaaaaaa aaaaaatctc caggcctaac ataatttcag gaagaaattt 24360 cagtagttgt atctcagggg aaatacagga agttagcctg gagtaaaagt cagtctgtcc 24420 ctgccccttt gctattttgc ccgtgcctca cagtgctctc tgcctgtgac gacagctccg 24480 cagaagttcg gaggatataa tggaattcat tgtgtactga agaatggata gagaactcaa 24540 gaaggaaatt ggaaactgga agcaaatgta ggggtaatta gacacctggg gcttgtgtgg 24600 gggtctgctt ggcggtgagg gggctctaca caagcttcct ttccgtcatg ccggccccca 24660 ccctggctct gaccattctg ttctctctgg caggtcatga tgatgggcag cgcccgagtg 24720 gcggagctgc tgctgctcca cggcgcggag cccaactgcg ccgaccccgc cactctcacc 24780 cgacccgtgc acgacgctgc ccgggagggc ttcctggaca cgctggtggt gctgcaccgg 24840 gccggggcgc ggctggacgt gcgcgatgcc tggggccgtc tgcccgtgga cctggctgag 24900 gagctgggcc atcgcgatgt cgcacggtac ctgcgcgcgg ctgcgggggg caccagaggc 24960 agtaaccatg cccgcataga tgccgcggaa ggtccctcag gtgaggactg atgatctgag 25020 aatttgtacc ctgagagctt ccaaagctca gagcattcat tttccagcac agaaagttca 25080 gcccgggaga ccagtctccg gtcttgcctc agctcacgcg ccaatcggtg ggacggcctg 25 140 agtctcccta tcgccctgcc ccgccagggc ggcaaatggg aaataatccc gaaatggact 25200 tgcgcacgtg aaagcccatt ttgtacatta tacttcccaa agcataccac cacccaaaca 25260 cctaccctct gctagttcaa ggcctagact gcggagcaat gaagactcaa gaggctagag 25320 gtctagtgcc ccctcttcct ccaaactagg gccagttgca tccacttacc aggtctgttt 25380 cctcatttgc ataccaagct ggctggacca acctcaggat ttccaaaccc aattgtgcgt 25440 ggcatcatct ggagatctct cgatctcggc tcttctgcac aactcaacta atctgaccct 25500 cctcagctaa tctgaccctc cgctttatgc ggtagagttt tccagagctg ccccaggggg 25560 ttctggggac atcaggacca agacttcgct gaccctggca gtctgtgcac cggagttggc 25620 tcctttccct cttaaacttg tgcaagagat cgctgagcga tgaaggtaga attatggtcc 25680 tccttgccct tgcctttcct ttttgtgatc tcaaagcatc ctccctccgc ccccattcca 25740 tggccccagt tccctactcc cacagctgtc tgctgaaact gccaacatta ctcaattgtt 25800 tctgggggga ggaacatttt tttttgaaac aaaatagata tatgaaacag tacacgggaa 25860 ttaacacgaa tatttaaggt aaaacatgac cttgaagatt atgaaatcca tcttattttg 25920 gcccagaacg ggggcattgg gctccttggg ccatagggga gctggggagg acagggtgaa 25980 gagttagctc taagccctct gcttggagat gctgtaaata cagaacgcaa aatcaccttc 26040 gaagttaaag acgcgaagtt cttctttact cggcccctcc tcccctcccc cccgccaatt 26100 ccctccagtt acagctagca tccaggtccc gggaggtgaa gaaggagact tcggctccag 26 160 ttacagctag catccgggtc ccgatttaga aggagctgcc aattacagcg cggttccagg 26220 gctgagcaaa aagcctgagg agccaagtgg gagagggagt aaaactactg aattgggcca 26280 caagcaaatg aataaactga acgactctta accaaaccta atatatttaa tccaaacaca 26340 caagtctttc atttcttccc tcctcccttc cttctcttac tccccaacac cccctcttca 26400 agcacaatta attatatggt tagattctac tgcgtgatca gccctgttct aggtggtggg 26460 cacgccaagg tgaatgagac caaacaagag tcttgccctc atggggttta catttggaga 26520 cagagtcgat ctgttgccca acctggagtg cagtggcgcg atcacagctc actgcagcct 26580 caaactccct ggctcaaggg gttctcccac ctgagcctcc cgactagctg ggaccacagg 26640 tgcacgccac gacgcctggg tttgtttgtt tgtttaatag agacgaaggt ctcaccatgt 26700 tatctgggct caagcgatca tcccccctcc tcctcctaaa gtactgggat tacagtccca 26760 agctatcttg cccgacctgg gaaacagacg ttaaggaaga taacaatcta ttttcagaga 26820 gcgagtttat aaaaccaatg caatgggtaa atatgaagtg tgaataggag gagaagctaa 26880 agagtggtcg gagaatctaa tgcaagctac gggagaaaga aactcaagtg caaatgctgc 26940 ctcaggaata aacgtaaaaa gagactttca agtgcaaatg ctccctcagg aataaaataa 27000 tcttgagact ctcaagtgta aatgctgcct cgggagaacc gaacggcgag ctggagccca 27060 tacgcaacga gattagagag gaaggcagaa gccagagcac atgaataaat gagcatccat 27120 tttgtttcag aaatgatcgg aaaccatttg tgggtttgta gaagcaggca tgcgtaggga 27180 agctacggga ttccgccgag gagcgccaga gcctgaggcg ccctttggtt atcgcaagct 27240 ggctggctca ctccgcacca ggtgcaaaag atgcctgggg atgcgggaag ggaaaggcca 27300 catcttcacg ccttcgcgcc tggcattgtg agcaaccact gagactcatt atataacact 27360 cgttttcttc ttgcaaccct gcgggccgcg cggtcgcgct ttctctgccc tccgccgggt 27420 ggacctggag cgcttgagcg gtcggcgcgc ctggagcagc caggcgggca gtggactagc 27480 tgctggacca gggaggtgtg ggagagcggt ggcggcgggt acatgcacgt gaagccattg 27540 cgagaacttt atccataagt atttcaatgc cggtagggac ggcaagagag gagggcggga 27600 tgtgccacac atctttgacc tcaggtttct aacgcctgtt ttctttctgc cctctgcaga 27660 catccccgat tgaaagaacc agagaggctc tgagaaacct cgggaaactt agatcatcag 27720 tcaccgaagg tcctacaggg ccacaactgc ccccgccaca acccaccccg ctttcgtagt 27780 tttcatttag aaaatagagc ttttaaaaat gtcctgcctt ttaacgtaga tatatgcctt 27840 cccccactac cgtaaatgtc catttatatc attttttata tattcttata aaaatgtaaa 27900 aaagaaaaac accgcttctg ccttttcact gtgttggagt tttctggagt gagcactcac 27960 gccctaagcg cacattcatg tgggcatttc ttgcgagcct cgcagcctcc ggaagctgtc 28020 gacttcatga caagcatttt gtgaactagg gaagctcagg ggggttactg gcttctcttg 28080 agtcacactg ctagcaaatg gcagaaccaa agctcaaata aaaataaaat aattttcatt 28 140 cattcactca tttattgtca acatttattg agcacctatt acaacaattt catcgcatgg 28200 aagacagcat cgtttctgac actgttgttt catgtatctc ttagaaaaac gctgctatta 28260 gacatctaac actatttatc ttgaggtgat aaaatatcaa aagccgtgtc tcaagatcga 28320 tgaaatgcgg ttaaaatgat gaatagaaac tctaggggga cctcatatcg atagactcga 28380 gactggcaca tctggagatc cgtatttatc cggcttcccc ttccagatca cgcgaggttt 28440 gggatatttt gctcaccagg cctcagccag gtaactgaat ccagccaacc ctggcccata 28500 gtctcggaat ccgactcggc tcccagtccc cgcctcggcg ttctgagacc cccaggctgg 28560 ggttccagag ggctgtgagg ttgcgaatga ctgctgccaa accggaagga actctgcggt 28620 tctctgccac agtgggattg ttgcaggcac gcggctcaga cttcactgag gttgggagat 28680 gctcctgtcc acgctgcctc atcccgtgct ggagcactgc acctctattt ttttttttag 28740 ggtacacgc 28749 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDKN2A forward primer <400> 2 cagtaaccat gcccgcatag a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CDKN2A reverse primer <400> 3 aagtttcccg aggtttctca ga 22

Claims (1)

(a) culturing cells isolated from the amniotic membrane in vitro to obtain amnion-derived mesenchymal stem cells;
(b) cyclin-dependent kinase inhibitor 2A (CDKN2A) comprising the nucleotide sequence of SEQ ID NO: 1 using a primer set consisting of the nucleotide sequences of SEQ ID NOS: 2 and 3 in amniotic membrane-derived mesenchymal stem cells of step (a) Measuring the expression level of the gene;
(c) When the expression level of the CDKN2A gene in step (b) is higher than the cut-off value of 0.1, the amnion-derived mesenchymal stem cells are likely to die, and when the expression level is lower than 0.1, The method comprising the steps of: determining the possibility of apoptosis of amniotic membrane-derived mesenchymal stem cells,
The gene expression level of step (b) was measured by real-time PCR and calculated by the 2-ΔΔ method,
Wherein the sensitivity and specificity of the amniotic membrane-derived mesenchymal stem cells are 100% when the ROC curve is plotted on the basis of the threshold value of 0.1 in step (c).

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