KR101904677B1 - A use of 5-Hydroxytryptamine receptor 2B and neuropeptide Y as a marker for predicting differentiation of stem cells - Google Patents

Abstract

The present invention discloses the use of 5-Hydroxytryptamine receptor 2B (HTR2B) and neuropeptide Y as stem cell differentiation prediction markers, and more specifically, to a stem cell differentiation prediction including a substance for measuring mRNA expression or protein expression of HTR2B gene A method for predicting the differentiation of stem cells using the marker marker composition; Or neuropeptide Y gene mRNA expression or protein expression, and a method for predicting the differentiation of stem cells using the marker composition.
The present invention confirms that HTR2B and neuropeptide Y can be used as a stem cell differentiation prediction marker. Thus, it is possible to predict the differentiation into various cardiac cells or neurons in various undifferentiated stem cells, The possibility of using undifferentiated stem cells as a selective cell therapy agent can be provided.

Description

A use of 5-Hydroxytryptamine receptor 2B and neuropeptide Y as a marker for predicting differentiation of stem cells}

The present invention has identified the use of 5-Hydroxytryptamine receptor 2B and neuropeptide Y as markers for predicting stem cell differentiation, and more specifically, stem cell differentiation including an agent measuring mRNA expression or protein expression of the 5-Hydroxytryptamine receptor 2B gene. A predictive marker composition and a method for predicting the differentiation of stem cells using the same; Or it relates to a marker composition for predicting stem cell differentiation comprising an agent for measuring neuropeptide Y gene mRNA expression or protein expression, and a method of predicting stem cell differentiation using the same.

Mesenchymal stem cells (MSCs) are often referred to as mesenchymal stromal cells, and exist in the bone marrow (BM), but in response to migration signals to maintain connective tissue homeostasis. Released into the circulatory system. Recently, mesenchymal stem cells have received wide interest in the field of cellular and immunotherapy due to their multifaceted functions including tissue regeneration, hematopoietic support, and immunomodulation. In addition, mesenchymal stem cells are capable of differentiation into various cells such as adipocytes, osteoblasts, chondrocytes, heart cells, muscle cells and nerve cells, and have limited proliferative power unlike embryonic stem cells, so there is no possibility of cancer cell formation. Since it does not induce an immune response even during transplantation, it has been in the spotlight as an effective means of cell therapy in tissue engineering and regenerative medicine.

Neuropeptide Y (NPY) is a 36 amino acid peptide and is abundantly present in the central and peripheral nervous systems of mammals, particularly in the hypothalamus and cortex. Neuropeptide Y potentially exerts a wide range of therapeutic physiological effects, and when administered alone, it is known that it induces vasoconstriction and has the potential to cause angina. In addition, neuropeptide Y is a neurotransmitter distributed in the central and peripheral nervous systems, which increases in starvation and induces increased appetite and decreased energy metabolism. However, there is no research on the action and use of neuropeptide Y in human pluripotent stem cells.

5-Hydroxytryptamine receptor 2B (hereinafter referred to as "HTR2B") represents serotonin receptor 2B. This serves as a mediator of various physiological functions, for example, causing constriction of blood vessels and shape change of platelets due to cardiovascular effects, and nerve sensitization caused by tactile stimulation due to central nervous system effects. In addition, HTR2B is required for heart development and is known to regulate cardiovascular function during embryonic formation and maturity. However, there is no research on whether HTR2B can act as a marker and its use in human pluripotent stem cells.

In the present inventors, stem cells with high expression of HTR2B in undifferentiated stem cells are cells that have the ability to differentiate into cardiac cells, and stem cells with high expression of neuropeptide Y are cells that have the ability to differentiate into neurons. It was confirmed that it can be predicted. In addition, the present inventors found that, compared with other mesenchymal stem cells, the expression of HTR2B was relatively high in chorionic mesenchymal stem cells (CMSCs), and that there is a specificity for differentiating into cardiomyocytes. The present invention was completed by confirming that the expression of neuropeptide Y was relatively high in umbilical cord mesenchymal stem cells (UC-MSCs), and the specificity to differentiate into neural cells was confirmed through this. I did.

Korean Patent Publication No. 20100135197

Serotonin 2B receptor is required for heart development, Luc Maroteaux, 9508-513 PNAS August 15, 2000 vol. 97 no. 17

An object of the present invention is to provide a marker composition for predicting stem cell differentiation, including an agent for measuring 5-Hydroxytryptamine receptor 2B (HTR2B) gene mRNA expression or HTR2B protein expression.

Another object of the present invention is to provide a method for predicting differentiation from stem cells into heart cells by measuring HTR2B gene mRNA expression or HTR2B protein expression.

In addition, it is an object of the present invention to provide a marker composition for predicting stem cell differentiation, including an agent for measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression.

Another object of the present invention is to provide a method for predicting differentiation from stem cells into neurons by measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression.

In order to achieve the above object, the present invention provides a marker composition for predicting stem cell differentiation comprising an agent for measuring 5-Hydroxytryptamine receptor 2B (HTR2B) gene mRNA expression or HTR2B protein expression.

In addition, in an embodiment of the present invention, the stem cells may be chorionic mesenchymal stem cells (CMSc).

In addition, in an embodiment of the present invention, the differentiation may be differentiation into cardiomyocytes.

In addition, in one embodiment of the present invention, the measurement is reverse transcriptase polymerase chain reaction, real-time polymerase chain reaction, Northern blot, Western blot, radioimmunoassay It can be selected from the group consisting of (radioimmunoassay), radioimmunodiffusion, immunoprecipitation assay, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), and microarray chip. However, it is not limited thereto.

In addition, the present invention provides a method for predicting differentiation from stem cells into heart cells by measuring HTR2B gene mRNA expression or HTR2B protein expression in order to achieve the above object.

In addition, in an embodiment of the present invention, the stem cells of the present invention may be chorionic mesenchymal stem cells.

* In addition, the present invention provides a marker composition for predicting stem cell differentiation comprising a material for measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression in order to achieve the above object.

In addition, in an embodiment of the present invention, the stem cells may be umbilical cord mesenchymal stem cells.

In addition, in an embodiment of the present invention, the differentiation may be differentiation into neural cells.

In addition, in one embodiment of the present invention, the measurement is reverse transcription polymerase chain reaction, real-time polymerase chain reaction, Northern blot, Western blot, radioimmunoassay, radioimmuno diffusion method, immunoprecipitation assay, immunohistochemical analysis, ELISA And microarrays, but is not limited thereto.

In addition, the present invention provides a method for predicting differentiation from stem cells into neurons by measuring mRNA expression of neuropeptide Y gene in order to achieve the above object.

In addition, in an embodiment of the present invention, the stem cells of the present invention may be umbilical mesenchymal stem cells.

In the present invention, by confirming that 5-Hydroxytryptamine receptor 2B and neuropeptide Y can be used as markers for predicting stem cell differentiation, differentiation into cardiac cells or neurons targeting various stem cells in an undifferentiated state, not differentiated cells. It is predictable and can provide the possibility of using undifferentiated stem cells as a selective cell therapy.

Figure 1 is a form of amniotic mesenchymal stem cells (AMSCs), chorionic mesenchymal stem cells (CMSCs), umbilical cord mesenchymal stem cells (UC-MSCs) ( morphology).
2 shows the results of gene ontology analysis in each mesenchymal stem cell.
Figure 3 shows the expression of a specific gene originating from mesenchymal stem cells through real-time polymerase chain reaction (real-time PCR).
4 shows the differentiation of chorionic mesenchymal stem cells into cardiac cells, and differentiation from umbilical mesenchymal stem cells into neurons. (A) shows the cell morphology at x 50 magnification, and (B) shows the real-time PCR result. In addition, (C) shows alpha actinin immunofluorescence staining, the picture above is a picture of x 100 magnification, and the picture below is a picture of x 200 magnification.
5 shows the differentiation of chorionic mesenchymal stem cells into heart cells. In (A), black arrows indicate mature focal adhesion, and white arrows indicate dyed stress fibers (x 100 magnification). (B) shows immunofluorescence staining of chorionic mesenchymal stem cells derived from heart cells (x 200 magnification).
6 shows the differentiation of umbilical mesenchymal stem cells into nerve cells. Black arrows in (A) indicate highly refracted cell bodies with process-like structures (A-1,2,3,4,6: x 200 magnification, A-5, 6: x 50 magnification). (B) shows immunofluorescence staining of a neuronal marker for late differentiation of umbilical mesenchymal stem cells (B-1: x 200 magnification, B-2: x 100 magnification).

Hereinafter, a marker composition for predicting stem cell differentiation comprising an agent for measuring 5-Hydroxytryptamine receptor 2B (HTR2B) gene mRNA expression or HTR2B protein expression according to an embodiment of the present invention; Alternatively, a marker composition for predicting stem cell differentiation comprising an agent for measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression will be described in detail.

Hereinafter, terms used in the present invention are defined.

The terms used in the present specification are for describing exemplary embodiments and are not intended to limit the present invention. In this specification, the singular form also includes the plural form unless specifically stated in the phrase. As used herein, "and/or" includes all combinations of each and one or more of the recited elements.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more new cells while having self-replicating ability, and mesenchymal stem cells, depending on the tissue derived from the stem cell, It can be classified into embryonic stem cells and dedifferentiated stem cells.

As used herein, the term "mesenchymal stem cells (MSCs)" refers to cells that help make cartilage, bone, fat, bone marrow interstitial, muscle, nerve, etc., but generally stay in the bone marrow in adults. It is also present in umbilical cord blood, peripheral blood, and other tissues, and refers to cells that can be obtained from them. "Mesenchymal stem cells" are used interchangeably with "mesenchymal stromal cells".

As used herein, the term "differentiation" refers to a phenomenon in which a cell's structure or function is specialized during growth by division and proliferation. Pluripotent mesenchymal stem cells can differentiate into progenitor cells with a limited lineage (e.g., mesenchymal cells), and then further differentiate into other types of progenitor cells (e.g., osteoblasts, etc.), and then specific tissues (e.g., mesenchymal cells). It can be differentiated into terminally differentiated cells (eg, adipocytes, bone cells, chondrocytes, etc.) that play a characteristic role in bones, etc. In a preferred embodiment, the chorionic mesenchymal stem cells of the present invention have the ability to differentiate into cardiac cells, and the umbilical mesenchymal stem cells into nerve cells.

As used herein, the term "cardiomyocytes" refers to not only precursors of cardiac cells capable of differentiating into all types of cardiac muscle cells, including ventricular, atrial and nodular cells, but also all that can be formed during the differentiation process. It means a cell of the heart cell lineage.

The term "neural cells" used in the present invention is also called a neuron, and is a unit cell constituting the cranial nervous system of an animal, and is composed of a part of the cell body excluding neurites, a nucleus, and the cytoplasm surrounding them. In general, it refers to cells that are larger in size than other somatic cells. The nerve cells can be classified into nonpolar nerve cells, unipolar nerve cells, bipolar nerve cells, gastric unipolar nerve cells, multipolar nerve cells, and the like according to the number of neurites included therein.

The term used in the present invention, "5-Hydroxytryptamine receptor 2B (HTR2B)" refers to the serotonin receptor 2B. HTR2B plays a number of mediators of central and peripheral physiological functions, and typically mediates cardiovascular effects, which indicate constriction of blood vessels and changes in the shape of platelets, and central nervous system effects including nerve sensitization by tactile stimulation. The HTR2B gene sequence of the present invention may be a sequence consisting of the gene sequence of SEQ ID NO: 1.

The term "neuropeptide Y" used in the present invention is a substance having a structure similar to that of Pancreatic Polypeptide (PP) composed of 36 amino acids, and is known to exist mainly in nerve cells. The peptide has an amino acid sequence that is 50% identical to PP produced in pancreatic cells, and 69% identical to PYY (Peptide YY) produced in neurons, and belongs to the so-called PP-based peptide group. NPY exhibits various physiological actions such as secretion and regulation of gastrointestinal movement throughout the gastrointestinal tract. It mainly suppresses gastric acid secretion, reduces gastric emptying, inhibits the secretion of various exocrine enzymes in the pancreas, and secretion of insulin and glucagon. It acts to suppress. The neuropeptide-Y gene sequence may be a nucleotide sequence of SEQ ID NO: 2.

As used in the present invention, the term "marker" is a substance capable of discriminating a cell that has initiated or progressed differentiation into a specific cell or differentiated from a cell before differentiation, and is a cell, tissue or site that has initiated, progressed or completed differentiation. And organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, and the like showing an increase or decrease in In the present invention, for example, HTR2B expression increases when stem cells are differentiated into cardiac cells, and neuropeptide Y expression increases when stem cells differentiate into neurons.

The term "gene mRNA expression", as used herein, is a process of confirming the presence and expression of mRNA of marker genes representing the differentiation capability of stem cells in a biological sample in order to diagnose the differentiation capability of stem cells. In addition, the term "protein expression" as used in the present invention is a process of confirming the presence and level of expression of a protein expressed from a marker gene representing the differentiation ability of stem cells in a biological sample in order to diagnose the differentiation ability of stem cells. . The agent for measuring the expression of HTR2B mRNA may be a primer set consisting of SEQ ID NOs: 3 and 4, and the agent for measuring the expression of neuropeptide-Y mRNA may be a primer set consisting of SEQ ID NOs: 5 and 6.

The term "agent for measuring gene mRNA expression or protein expression" used in the present invention is not limited thereto, but reverse transcriptase polymerase chain reaction, real-time polymerase chain reaction), Northern blot, Western blot, radioimmunoassay, radioimmunodiffusion, immunoprecipitation assay, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA) and micro It is preferable to be selected from the group consisting of an array.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.

[Example 1] Materials and methods

1. Mesenchymal stem cell isolation and culture

Placentas were collected from healthy mothers with consent, and the Institutional Review Board of Seoul St. All experiments were conducted with the approval of Mary's hospital (KC09WZZZ0173). The amnion, chorion, and umbilical cord were separated from the placenta.

Amnionic mesenchymal stromal cells (AMSCs) were cut into small pieces, treated with 0.25% trypsin-EDTA (Gibco), and then 0.2% collagenase IV (Gibco). Separated. Chorionic mesenchymal stromal cells (CMSCs) were also isolated by treatment in the same manner as mentioned above, except that 0.2% collagenase I (Gibco) was used. For umbilical cord mesenchymal stromal cells (UC-MSCs), the umbilical cord is chopped into small pieces, treated with 0.2% collagenase II (Gibco), and 0.25% trypsin-EDTA (Gibco). ) And separated (see FIG. 1).

Each of the isolated mesenchymal stem cells was cultured in an environment of 37°C and 5% carbon dioxide using α-MEM (α-modified minimum essential medium, Gibco) medium.

2. Microarray Analysis

Microarray analysis was performed to investigate the difference in the genetic expression of amniotic mesenchymal stem cells (AMSCs), chorionic mesenchymal stem cells (CMSCs), and umbilical mesenchymal stem cells (UC-MSCs). RNA was extracted from each mesenchymal stem cell to check quality and quantity, and then tested using SurePrint G3 Hmn GE 8X0K V2 Microarray Kit (Agilent, Cedar Creek, TX). 200 ng of RNA was synthesized, synthesized with double-stranded cRNA, amplified, and then reacted to a chip to which a probe was attached to calculate data. After that, data was analyzed with GeneSpring GX 7.3 (Agilent, Cedar Creek, TX) analysis program, GeneCards (http://www.genecards.org/), DAVID (http://david.abcc.ncifcrf.gov/) , QIAGEN`s Ingenuity® Pathway Analysis (QIAGEN Redwood City, www.qiagen.com/ingenuity) programs were used to identify genes characteristically expressed in mesenchymal stem cells derived from each tissue, and to investigate the characteristics of genes. .

3. Real-time polymerase chain reaction (real- time PCR)

Characteristically expressed genes were selected and an experiment was conducted with the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) using Taq®Mangene expression assay (Applied Biosystems, Foster City, CA), and 2-△ Data was derived by the ΔCt method. Genes are 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B) (Hs00168362_m1), bcl-2-like protein 10 (BCL2L10) (Hs00368095_m1), renin (REN) (Hs00982555_m1), amphiphysin (AMPH) (Hs0104720_m1), neuropeptide Y (NPY) (Hs00173470_m1) was selected. The 5-hydroxytryptamine receptor 2B and neuropeptide Y genes, whose expression levels were clearly different in chorionic mesenchymal stem cells and umbilical mesenchymal stem cells, were selected to investigate the function of each gene. It was confirmed whether or not the features by the gene appeared.

4. Confirmation of gene differentiation predictive ability

The 5-hydroxytryptamine receptor 2B (HTR2B) gene, which is characteristically expressed in chorionic mesenchymal stem cells, is related to the formation of heart cells, and the neuropeptide Y gene characteristically expressed in umbilical mesenchymal stem cells is related to the formation of neurons. Was predicted.

Therefore, in order to confirm the predictive ability of HTR2B and neuropeptide Y genes, chorionic mesenchymal stem cells and umbilical mesenchymal stem cells were cultured under conditions capable of differentiating into cardiac cells and neurons to confirm their differentiation capabilities.

After differentiating for 2 weeks using a medium capable of differentiating into heart cells and neurons, it was confirmed by immunofluorescence experiment whether the differentiated cells matched the heart cells and neurons. After fixing the differentiated cells with 4% PFA, the presence of cardiac cells was determined using anti-alpha-actinin antibody (Abcam) and anti-cardiac troponin T (Abcam), and the presence of neurons was determined by NESTIN (Life Technologies, Carlsbad, CA). ) And SOX2 (Life Technologies, Carlsbad, CA) and each stained cell was analyzed by Olympus IX 71 phase contrast microscope.

In addition, in order to examine the change in gene expression during the differentiation process, the cells differentiated into heart cells by extracting RNA from each mesenchymal stem cell on days 1, 3, 7, and 14 are alpha-actinin (ACTN1) (Hs00998100_m1). , transcription factors GATA-4 (Hs00171403_m1) and Troponin T (TNNT1), cells differentiated into neurons nestin (NES) (Hs00707120_s1), neuron-specific class III beta-tubulin (TUBB3) (Hs00801390_s1) and SOX2 (Hs01053049_s1) ) Was measured by real-time PCR, and data was derived by the 2-△△Ct method. For real-time PCR, TaqMan® gene expression assay (Applied Biosystems, Foster City, CA) was used.

[Example 2] Analysis of gene ontology through gene expression microarray

Gene Ontology (GO) terms were classified based on genes specifically expressed according to the origin of each tissue, and the results are shown in FIG. 2.

Referring to FIG. 2, it was confirmed that genes related to heart morphogenesis and blood circulation (HTR2B, BCL2L10, and REN) were highly expressed in chorionic mesenchymal stem cells (CMSCs). In addition, it was confirmed that genes related to neurological system processes (AMPH and NPY) were highly expressed in umbilical mesenchymal stem cells (UC-MSCs).

3 shows the expression of specific genes originating from mesenchymal stem cells through real-time PCR, and FIG. 4 shows differentiation from chorionic mesenchymal stem cells to heart cells, and differentiation from umbilical mesenchymal stem cells to nerve cells. It can be seen that the Gene Ontology analysis result and the real-time PCR result are exactly the same. In particular, 5-hydroxytryptamine receptor 2B (HTR2B) was expressed significantly in chorionic mesenchymal stem cells, and neuropeptide Y (NPY) was remarkably high in umbilical mesenchymal stem cells. HTR2B is considered to be involved in cardiac remodeling and cardiac cell formation, and neuropeptide Y is known to play a role in promoting differentiation into neurons by activating amyloid-β.

Moreover, the Ingenuity® pathway analysis (IPA) result showed similar results to the Gene Ontology analysis result of FIG. 2. In other words, it was confirmed that the chorionic mesenchymal stem cells were highly expressed in the development of the cardiovascular system and the hematological system, and the umbilical mesenchymal stem cells were highly expressed in genes related to the development and behavior of the nervous system. .

[Example 3] Analysis of gene differentiation predictive ability

Based on the results of Example 2, in order to evaluate the differentiation ability of chorionic mesenchymal stem cells (CMSCs) and umbilical mesenchymal stem cells (UC-MSCs), differentiation experiments were conducted into cardiac cells and neurons, and the results were It is shown in Figures 5 and 6. 5 shows the differentiation of chorionic mesenchymal stem cells into cardiac cells, and FIG. 6 shows the differentiation of umbilical mesenchymal stem cells into nerve cells.

1. Differentiation ability of chorionic mesenchymal stem cells into heart cells

The chorionic mesenchymal stem cells showed superior differentiation ability into heart cells compared to bone marrow mesenchymal stem cells and umbilical mesenchymal stem cells. In addition, the gene expression of the three gene markers GATA4, TNNT1 and ACTN1, which can identify heart cells during two weeks of differentiation, increased with the passage of differentiation time (see FIG. 4B). Also in the immunofluorescence experiment, alpha actinin in chorionic stem cells differentiated into cardiac cells can be found to have a tendency to create a sarcomeric pattern over time (see Fig. 4C), and cardiac troponin T is gradually accumulated over time. Showed the pattern.

2. The ability of umbilical mesenchymal stem cells to differentiate into nerve cells

Umbilical mesenchymal stem cells had better differentiation ability into nerve cells than other stem cells. In addition, the gene expressions of NES, SOX2 and TUBB3, which are three markers capable of identifying neurons during two weeks of differentiation, were increased with passage of differentiation time (see FIG. 4B). In the immunofluorescence experiment, the umbilical cord stem cells differentiated into neurons showed positive results for Nestin and Sox2 (see FIG. 6B).

That is, it can be seen that the chorionic mesenchymal stem cells and umbilical mesenchymal stem cells have a specific differentiation ability to differentiate into heart cells and neurons, respectively, and this is a result consistent with the gene expression data of Example 2.

From the above results, by measuring the expression pattern of 5-Hydroxytryptamine receptor 2B (HTR2B), differentiation from stem cells to heart cells can be predicted, and by measuring the expression pattern of neuropeptide Y, differentiation from stem cells to neurons can be predicted. Was confirmed. That is, through this, it was found that HTR2B and neuropeptide Y can be used as markers for predicting stem cell differentiation.

<110> Chatolic universty industry cooperation <120> A use of 5-Hydroxytryptamine receptor 2B and neuropeptide Y as a marker for predicting differentiation of stem cells <130> P-3 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 18089 <212> DNA <213> Artificial Sequence <220> <223> HTR2B whole DNA sequence <400> 1 aattttctga aataaggcaa aaacgtattt tcattacttg tattttctcc aacgtaaagt 60 ctatggggaa aaaaagctgt gaaccactcc taaacccaga gatcatctgt ttaaaaataa 120 gcagtaaaaa agtttctgct aaaacctatg tatacatgta gaattcaggc ctttctgtgc 180 tcttctatat aaggttgcaa ccagagcaaa ttaatggtgt tccacagcaa aggcacaaaa 240 gctagataag aatcttgttt cactgctaga aatcagaact gtagaaatca ctataggctt 300 ttaaaacggc agaatcttca aagaatgaaa cctctaaaat agctagatta tatcttattt 360 tttctcaaat gtgaaaaaca gctcaggctt aaccccaaac aaaactatct gaagctaaaa 420 tgatcaagtt tattacagtc cccccaaata ttaaagagag ctcttaaaaa tataacttga 480 ataagggcca tgaactggtt caacatgctt aacagtggtt ttatgaatta caatgaatgc 540 tgctactctc taaaaatgaa tgtatcaaag aggaaataac ctccttggca tgcttgcagc 600 tatacaacgt atttgtttca ctgctttcaa ccgcctgtgc tggaggctca gaataagtca 660 atgggaggag gatttcagtc acagcagcaa gcaagtctag tgaacagata agatgacatg 720 ctcagcaaaa taacaacgaa accagaggta agcatttaat aaacacatat ggaattaatc 780 taaaggtttt tttttttaat cttcaagaat tctgctatca taatctttca tttctattca 840 gcggtttaaa aatcaaaatg aaaaatttac taatgctaaa ggagagtttt taaaaacgtg 900 atttgtacat ttgatgaaca aataaatggt ctaatcttcc ttttggtttc cctttcttct 960 caaggcaact gggaaggtta aaaaaataaa cactacttaa aacaaaaaaa gaactggttt 1020 gaatttggca aatttagtga ttaaatttag aacacaattg ttgcaaagac ataccatgag 1080 agatgaaatt actaaacaca attatttgga tcctagtaat accttttttt ttccttctct 1140 caaactaaag gaaatctaaa atgacattta tatcagtact cacaaacctg ataagacaaa 1200 gctaattaca tataaggatc cctagctggt aaaacattta tttagaccct tgttgggctt 1260 tgagtgtttc taggtgggtg gccagcgcat actaaaagtt ctattctgaa ctattgtgtt 1320 tcgacttttc tattcttctg taagtaaagg taaaaagaca ttcaagggtc aggaaacacc 1380 taacttttta ttataaccat tgaaaaaatt aatgtgtctt ctgtttcatt aactgagcaa 1440 catcagaaat acagtatatt tggagaattg gctgcattgt cacaaatgca caacagactt 1500 actaagaagg gaagggctat gaagttcaag tgtttttacc actttcagaa atgaataaaa 1560 taatgcagtc tatgtatttt gtatctaggg ggaactctct ggcatgcaag ttcaaacacg 1620 actctacaac tacggcagaa aaagagagag agagaaacta aaaatatata tatatcctat 1680 ttttttcaca gctatcagtt tctttcactg agctttccta aatttaagcc tctagaaaat 1740 aataaatact tggatatctt acctacaaac atggacagat gtgtgtatgc gctcatttta 1800 gagaacttga attttttttt ttaaaggaag gtgtcaactt tggcttttga gtgtttggca 1860 tggttacaat gccttaaaaa aacagatgag cagcttagct actaaccatg ctgaccactg 1920 ttcggaacgg gattgaatca cagaaaaaca gcaaatggct ctctcttaca gagtgtctga 1980 acttcaaagc acaattcctg agcacatttt gcagagcacc tttgttcacg ttatctcttc 2040 taactggtct ggattacaga cagaatcaat accagaggaa atgaaacaga ttgttgagga 2100 acagggaaat aaactgcact gggcagctct tctgatactc atggtgataa tacccacaat 2160 tggtggaaat acccttgtta ttctggctgt ttcactggag aagaagctgc agtatgctac 2220 taattacttt ctaatgtcct tggcggtggc tgatttgctg gttggattgt ttgtgatgcc 2280 aattgccctc ttgacaataa tgtttggtaa gtatttcact ttgtttgtgc catcaaacca 2340 cagaactatg atatactatt agagaatgga cactcattta atctttactt ttcttgaaat 2400 aaaagatgaa cttggactat ttaaagatac ggcaagtaaa cagtcccttg ttttatatca 2460 ttctctatgg ttgatcagca gttttaaaga gcaaaaaata ttttttgtaa actatttttc 2520 tccctcctaa gctatctaat aaaatcattt aaaaagtcat tcaagttagt gatatttata 2580 gattctgaat atattatcta taaattatag taaataggcc tatactttaa cttttctaca 2640 ctgatacttt ttttcctcaa agcaataact aggacacttc cgggacagat atgcgctggg 2700 ttaacgataa ggtctttgaa ttactaaatc ccaatttaaa tcactaaacc ccagtttaaa 2760 attggaactg tccaaaaatt cttattatag ctcttaatag tttgtatatt ataaacttga 2820 acacaattcc tacatatgtg tcatgttaag aaaaattatg tttagttgct gaaggaaatg 2880 gtttggctca gagtgtccaa agagatgatc tatcgattca atttttttat gatatatggg 2940 catctgtgtt cctgataggc ctctaagttg gtaagtggat taaaacctag ttattgcgat 3000 aataaaaggt atagtttgac actgaaatct tagtttgcta taataaaaaa aagatataag 3060 tagttccaga tttctgcata tcatctctag ttataaaatt tggataaaca caaataattc 3120 tgctgatgaa ttatgagggc tcctagaccc ttggaatcag cattaaaagg atatttaact 3180 tattcaatgc ttggtataaa ctgatattcc attcataaac tgcctaagca aaatgattac 3240 ttgtggcaga tatagtttct tagaattata tttacttata tgaagataaa aatattttaa 3300 gttacagaag taaaatttat taaactagaa tccatattag aaggtaaaaa acccctgcta 3360 ttccaactat tatacccagg ttttacacat tacaatttta taagtaataa cagaaagata 3420 tctaaaacta ttatcagata gtaacaggtt tacaccttcc attttaaatc aacatttacc 3480 tgggctttaa aataaatcaa gaaaattatg tggcaggaca agcagtaatg agtaaagttc 3540 tactgcttta attagacttt ctaataatta acagtctttc tatctccctg ttttataaat 3600 ttgtaatctt ttaaataatc taatgctgca tgaaaccatg aaaaataatt aagaaatctt 3660 actataatag agaacataac cctaattctg aataaagctt aaaagaaagt attatgtaga 3720 aatatatatg catatttgat aaatactgta caggcatatg attctagctc tcatctccat 3780 gtgcataata gtagcttaag atgtaaaatc atactcttca aactgtgctt tgctaggcac 3840 acagaaaatg tcaaaatgct tatttcagac taccataaaa tttaatacgt cattcaatac 3900 atgatattga aagaattatg cagaagtgaa ttttagcttt aaaatctttg agtccataaa 3960 tatcaaacat ctattatatg caaagtacca tggtagatac ttaaattatc agctaaatag 4020 ttaaggaatc ttaaaaggca aagtaccata gaattaaagt gagctaacaa acaaaataca 4080 aaaacaggta acaaagattt acctgtttaa tagattccat gtattctata gaaaaatagg 4140 tatccagcag ctgaaaactt taagtgtata tttaaaatgc tggtacccaa catatcctta 4200 gcaatacaag ttagcagaaa acccaacata tgttaacata aaatatgtga aatgtcttca 4260 tatttatgtc ttaaataatt gatagacatt ttaatattat ttcattcttc caaatgttta 4320 ttaactttaa atttattttt atttatttat tttaatagag atggagtctg gcaatgttgc 4380 ctgggctggc ctcaaactcc tgggttcaag tgatcctctg ccttcagcct cttaagtagc 4440 taggattaca ggcacattcc accatgcctg gctcatgttt gttaatttct aattacaaaa 4500 taaaatattt tatagcccca aacactttta ctatcagaaa taagttgtat agtggggttt 4560 gtagattttt aaaaaatgaa caaatctgtg aactactatc taatacatta ttgcattatt 4620 taaagcataa ttcaatttct atgaaaaaat gtaggaaaaa aacacatgtt gccctctaac 4680 aaacttcaaa ggatgaattc ataaattggg aactgcatgt attacaaatg gaaacctgac 4740 ttcaaatacc aagatatttt tataaccttt tgcaagttaa taaacacaaa gatttttcta 4800 aactaatgca tttataagaa cttctgataa ctttaaaagg cagaattaga ctttacactc 4860 ttattgtagt gttaatcttt aggacttcta acgtttgtac aaacaagcgt tcttggttct 4920 atgataaaag attaactatc actatgagca ataattttag ctatcgcagg ggaattcaat 4980 gaaagtaaca caaaaactat actagctcgt agtctaataa aatgaaattt taaaaataag 5040 agcagtaaaa tataaaagtg gaccttctgt ccttatactg agagatattt tattcaacca 5100 ctgatctgta taatatttaa caatatacgg tttgaattca tattttccca agattatgac 5160 aactcaggca caacattgtc tttcaaatga gttgtgctag gtcaattttt ctgccacttc 5220 ttcctatctg ggagcactgg acttgatctt tacagttgta gttttttatt gtcttgtcca 5280 taattcctaa aatccatgat cacaaatcaa cagcttataa cacaaggctt gaaaatacct 5340 acgttagaaa tacttgagca agaaaacaac accttatgct gatataaaaa agacaggatg 5400 ttcaccattt aagagcttct ctgaatcttt gtttcatttt ataagtgatt taaaaagtaa 5460 caggggaggc tgggcacggt ggctcatgcc tgtaacccca gcactttagg aggctgaaac 5520 ggatggatca cctgaggtca ggagttcgag accagcctgg ccaagatggc gaaaccctgt 5580 ccctactaaa aatacaaaaa ttagctgggc atggtggtac gcgcctgtag tcccagctac 5640 tcgggagact gaggcaggag aatagcttga acccggaaga cagagttgca gtgagccgag 5700 atggtgccac tgcactccag cctgggtgac ggggtgaggc tctgtctcag ggaaaaaaaa 5760 aaaaaaaagt aacacgggaa gatatggcat ctggccatcc attgggtgtt attggtttat 5820 ttataagttt tttttttttt tttttttttg agacggagtc tcgttctgtt gcccagactg 5880 cagggcagtg gcatgatctc agttcactgc aacctccacc cacaggttca aacgattctc 5940 ctgcctcagc ctcccgagta gctgggacta caggcgcagg ccaccacacc tggctaagtt 6000 ttgtattttt agtagagatg gggttttgcc actttggcca ggctggtctt gaactcctga 6060 cctcaggtga tccacctgcc tcggcctccc aaagtgttgg gattacagac atgagccacc 6120 aaacctggct ataacttaag tttcaattga tgcctcatga tctataagtt caaacagtat 6180 ccgtggtata gctgaaagag tatataaaat ggctgaaaga tggctgtttg aagcaagaca 6240 tcccagggtc tgaatcctgg ttctcccgtg ttggatcagg caagtcactt attatttcag 6300 agttcaatca cttattattt cagagctcaa tcaccttgtc aagcagggcc cagcatctta 6360 taagcactaa tagtaactgg tagagtactg catgcaaatt aaatatgatt tagttgagat 6420 gcaagtctaa tcagagatga aacaaatgta tagcagtaag agaaaaatgg cagttattca 6480 tttatgcagt aaaatgttag cctacccaat tctgaaatgg aggagaaaag caatgaatta 6540 aaggcacttc tttttgtaaa gtggaaattt taaaaaccag gaaaaaattc tgtaagcatt 6600 aaaatgctta aaacatttaa aacactatca tattagacac tgaaccccag gataactcat 6660 atgctcaaat gtagtgaaaa aagtaaaaaa caaaacagaa acttttaaac aataatttga 6720 actgaactat gtctttcttt tctggaagat aaagacagaa gcattagaat ttctgagata 6780 aatgtatatt tgaaaattat gttctttttg gacataagaa gtatttatga tttgtcctgc 6840 tgcaataagt tcatgaactg agtacctctg taaactatag gaacagccca cttggaatgc 6900 ctttaaaaat agtcttttac atcctccaaa gggaaacatt ttatgtccca gatttatgga 6960 ctgatgctaa ttattttccg gacaaatagt gtaacctgac ttcccaaact aaaaagctgc 7020 ccaaaggtta agcaggtttc agataaaata agtctgcctc atcaagatac ggggtttcta 7080 gtaaaactcc tcaggttttt gtactaattc aaaagaaagc aaaaacaaac aaaaaaatac 7140 ccaaaccaac aaaaaaccca ggcatgcaat gcatacaggt tattttcctg taccgtatat 7200 aaaataaaca gcagttaaaa ttcaacgtta gtaaaaaaca tacttttatg acaaattcaa 7260 ttgaaagtta aacagagtta actgttctca gcccatcctc cctcaccccc ggaaagtaca 7320 gcttaaagtt ttaagctgaa tcagatgcat gcacttttgc tacagcaggt agaaagacaa 7380 acactcttat ttcatactga tgatttggct tgtttctatg taggagtgtt ttcatggttg 7440 agttagttgg ctagtttctg ttaaacaata gtaataagca ctaacattta ttgagtgttt 7500 tctttatgcc aggaactgtt ctaaacactt cacatgtatt taatcatcat ccatataaac 7560 tctattaaag aaatcctata aaacaggtac tattatcccc actttacaga ggacagagaa 7620 gcagagggat aagtaatctg ttcaagatta caaagtaagt ggcagagaca tgatttaaac 7680 ccaggaggac tgattctaga attcacactc ttaaccttta ttattccgct ttctcaaatg 7740 tcaatgagaa atctgaccag ccaaaactgt catatttggc atacagaacc aacatttata 7800 ccttaaataa ccccatgtaa ctgttcagag gatcacagaa ttttttcccc cctagggaca 7860 agttagaact cagtattctt ttcagttaaa gttatatttc atattaagtg gaaaagaata 7920 cttgtgcgtc aacctatact tgtacaaatc cttcagatga ctgcaagatc tcttttctgt 7980 ggaaggtatt agacctaggc ccatacctat gaagttgttg aaccttgtct gggagacaaa 8040 taaaaattta tataactgaa tgacaactcc taccatccct catgcatggc agaatttaaa 8100 gcagtcttca ttttaaacaa taaaatattt gtgctactga tattctttag ataattaaac 8160 aattgtctat aagacaagac ccatctagga cttgttttgt tgctttaata ttcagtctgt 8220 aattatttca ggcattgaca gaaatgctat tattctttct taattttaat gaaagagtct 8280 aatttagagc cccagaataa cagtaattta gttgaagaac ctagtaactt gtttccctca 8340 gactgaccct tagatacaca tttctttctt tccatttaag tatgtaactg tttcacttca 8400 ttatttcaaa ttttcaatgt cttaagtctt tgtggaagta tattggacag aatacattat 8460 aacaaataat gtaattagaa attcttaaaa ccaaaactcc taaaataaac gttttgaaat 8520 aaagtatttc accataccaa cttaaccttt aatttagtta ttctaaacag aaataaactt 8580 ttctgacttt gagctaaatt aatactaact aaaaaatgat tttagaatga aagtctctag 8640 actaggaaaa gcccctttat aaaacaagtg tgtctagaac tgtttttaat ttctataaat 8700 tcttagatta cccaaatata ttaggcattt aaaaaagcct ctttttcttt tttgtgttgt 8760 atataaggtt tctattaatt taatgcagtt atctttttct tttatattag tttcattaaa 8820 actatccacc agtgtttcac gaaacttgaa atgagaaagg cttggaattg taaagattca 8880 tctctcttat aaatttcagt ctcattcttt cttaaagaaa attctgtatc acatctcaat 8940 atttctgtta acaataaagc actaactgta ataagaaaat tctagtgaat tttaagtggg 9000 aatgagttta taaattctgt ttaaagtgag gctaacaggg cttcataggg ttacttaaat 9060 cagcactaaa aaactttcct tcttagccca caattaggag tatgtggatc agatggtcaa 9120 attccaaaga cacgtacttt ttatatgaat tttcttatgg cttcctgctt atggcaacta 9180 gatttatgac tagaattttc caaaccttaa tcttcagaag tataatccat aactagaata 9240 gctaaataga caggtttaaa tagttggcaa actattttaa aagatgttac agagagagta 9300 aggacttcaa ggaaggaaat ctaagatata acacaataat tacctgcctt ttaaatcctt 9360 aactgcaatc aatatctctg attatagctc tagacgagta ttttcctacc accctactta 9420 cagtcctaaa tctagaactt cagctcaaga tgtaccctca tttttagtag tttccactgc 9480 aaactctaaa atgattcttt tcactatact tagactgtta gttcgctttg aagctgcctg 9540 tgtccaaggt ccttttccac taaatgacaa aaaatctgaa tgctatggct aagagtgaga 9600 aaatccaatt gattagattt tctccaaaca ccagtttctc tgacttaatt tattctgaga 9660 ggcctcccat tagttccttt ccatgagaca ttccaagtgc ctataaccaa ccctacaaca 9720 ggaaacttag gtgcatcttt taagactcag acacaaacct taccagaaag tcttccaaag 9780 cactgcttct caaatgttaa tttgcatacg aatcacctgg aaaccttatt taactgcaga 9840 gtctgataca gtaggtctga gatgagaccc aagagcctgc atatctaagt acccaggtga 9900 tgccgatcct cactctccat cttaagagca aactctgagt ggcagttcta aataacactt 9960 tcagataaca tttacatttc aataaataaa tgcctcccct ccttacctct atattcagac 10020 ttaattatac ctgaatttac tcatctgcaa gatcaatggt aatctctaag gttccttcca 10080 gctcaataaa tctaaaattt accacctcta tcccttccta cccccaattc aacaagcagg 10140 caaagtttat ttacagtaat acactgaaaa gtatgaatga ccctttccac ttgatgcttt 10200 aatccctttt taagaagctg ctgaacatag aactaagtcc caatcattta ttctttctga 10260 gtaaaagttt gtgctgattt tgttattttg ttcctaggaa gaaatatttt aatattctta 10320 ttcagaatgc cacataccaa ctctggaaac agaaggcacc aaaaagtaga tgtgtcagcc 10380 ttgctacctt ctggatactt tgtcaagtgc ccagatattt ttcactgaaa tcctacctga 10440 ctttaaaaaa atctcctact tgatgcttga aaataacagc caaatctatc taaaaactct 10500 gcccctaaat tatgcttttt ttttttttgg tctgagattt taatctctct ccacccccag 10560 gctccttaaa ttttaaacag ttaacaatag aagagtctct tgttgttctg taatcctgag 10620 atgattattc tggttttcct tcaattatga caatcacttt atacctgtca aagtcagata 10680 tacctgcaat agccagaaga aaagtagatt acattttatc agtggacata taacaatcaa 10740 atcctaaaag agacttagat aatctcacat gcatgacatc tgttctctac aggagttaaa 10800 ctggattcta atggaaatat tctaggggga attctaaaat tttagttgtt ttgaaactgt 10860 aaacatgtta agtggtgggt gtaatatata atgcataata tattaaattc acatatgtat 10920 gaacatctgt gtaagtaaat ttattagaaa actatgcaga atattcaata tcacatccac 10980 acctaaaatt tttttaaggt taatatttct aaagtgctta agactatatg aagtatgcag 11040 tgttttataa gagtttgtta aataaattaa gtactacgtc taaccctgag attttctttt 11100 caaatcaaaa gtctgtacta attattttat ctccagtttt tggagtgaat gatgaaatat 11160 agaaggggaa acaatctact tttcttccgg ataatctgaa gccattcctt ttgcaatctt 11220 tatttgagaa ctatttctgt caaaaaaatc actccactat ttcaccaaac tagagtatgc 11280 tataaacaag gatcttgctg ctagttattg ctagaagtga gcccaaaacc caggtgtcac 11340 agagactccc attctaatac gctttttgtc atactgtttc ctctacatgg taaaatgttt 11400 tctttttata caacagctac ttaaaaataa attttattta aataccaagg ttaagttcca 11460 gtcacaatac tacataaatt ttaaaaagat cacaaatgga atcgacaaaa gagcaaaggt 11520 cataataatg ctaaaaagtt aaactgctgc tgacttagaa aaaaggcaca attattatgc 11580 aaaatagaaa atgtatttaa aagttaccat ccacagaaat attgaattta atgttgtctc 11640 cattatctga agttaaacat tatgttgtta aagttataaa atttcagatg agtaacaaaa 11700 aagacaaaag atgtatcacc tgaaaaactg tagactgaaa gttgccattt tatagaataa 11760 aatgtttgtc ttgtttttgt ttctttcaga ggctatgtgg cccctcccac ttgttctatg 11820 tcctgcctgg ttatttcttg acgttctctt ttcaaccgca tccatcatgc atctctgtgc 11880 catttcagtg gatcgttaca tagccatcaa aaagccaatc caggccaatc aatataactc 11940 acgggctaca gcattcatca agattacagt ggtgtggtta atttcaatag gtatgtagag 12000 aatgccagag tgtgggtggt atacaaaatc tttggttcca cttggctagg agaataccag 12060 gtctggtagg caatgaaact cagcatgaga ggtcaagcca gcctactgat acattaacat 12120 aagctcctta tgtcactgca tttatcatac gccttctaaa acatactgtt ggtaaaaagt 12180 gtgacaagta acaaaactca tgtcctcaga aagaaccatc tgcacagtgt ttattgagat 12240 ttctttctgc ttagatttct atatgcagaa aggttaagtt tgagaacagt cttcatattt 12300 cccaacagat aattatggtc tagctagcat ttggaatcct gtaattacat taaaataaca 12360 agaaatgcac cttaaatcac attttaaaag attaataaga gaaaaaacaa aaataacaac 12420 tcaaacattt tgtgggcagg tatcaaagaa cacaaattca tcttggaagc caacatttct 12480 gtacatatgt gtacagatta aaggcagtta agtgtctgct tcagtgggtt tttttggttt 12540 gttttttctt ttttggagac agggtcttgc tctgtcaccc aggctagagt gcaatggcac 12600 aatcatggtt cactgctgcc tcgacttccc gggctcaagc gatcctccca cctcagcctc 12660 ccaggtagct gggactacag acacaccccc catgcctggc taatttttgt atttttggta 12720 gaggtggggt tttgtcatgt tgcccaggct ggtctcaaac tcttgagctc tggcgatccc 12780 cctgccccag cctcccaaag tgctgggatt acaggcgtga gtcactgcac ttaagtgttt 12840 tgagtatgtt taattcaaag ttttaagaga gatgcaaaga aaacataccg atgttcagaa 12900 attatgtata aaagaaatcc tttttcttct aaatcgtctt acggccctgt actcatctat 12960 aatactcaaa gctatacttc ttatcatgaa gtatgcgtaa atcattcaaa actcttatat 13020 gttagtatca gaaaaacaaa tagatttgca aaaattacaa tggtttgacc agcagattct 13080 cttcttcctt gtaaaaatat ttcaaagagg caaaaaatta agagacctgg aatacttaaa 13 140 ttactcctcc ctaatctgag taggggcact ggggcaaaaa cagcaagaaa gcaaaacaat 13200 gagattgacc ttcatattcc tgaaatgatg atataagggc ttgatctctt tgaaaaagtt 13260 tggaggtaaa agcaagctgt gttactcctc tggtatggga agaaaacagg gtcaagaaaa 13320 agtctattca gaatgaagag atttaaaaac agtgagaaaa ggctagatga gggggcagat 13380 gccgacagag cagaaccagc cccccaaaag cagctacaga aagggaatgg gagacaatgg 13440 gtgaaaatta aaatttgaag tgcagaggag gaaaactata ataaagtcat ttatgccaga 13500 tgactcacat ttggtaagct cactgcaatg agggagaggg caagggagag tgtgaggtat 13560 gtgtatattg gggtggggtg ggagagttgg acagagaaag ggaaaaaaag ttgaagaaat 13620 ggatccttaa gaataattaa aatatttgaa actgtgaaat gtgatcaagc taataaggta 13680 agcagcctgc tggtataaat ggcatctaga ttaaggctgg ataccatcaa tttataaatt 13740 gaccacattt ttattgagtg tttataatgt gctaggtact aagaatacac agatgatcaa 13800 gaaaaagaat ctttgcattc aagaaaggcg gacatataaa caaattacac taacacaaag 13860 tgatgagtga tgcaggagaa aaaagattaa aaggaagact catgatatat agatttagaa 13920 atccacttct tccttttgta ggtaagaaaa aagaaaccta gaggttttaa tcagttggct 13980 taagaactct tacagctttt cagcaccaag gccaaaatca acgctctttc acttcattgc 14040 actttgtctt gtagaaccga tcacaaatta ataactttga aaaaatgaaa atgcagatga 14100 caataagctc agttctgaaa ttctacaaaa tgctgaagag agctcactct cttcttttgg 14160 atttacagtt attctaggta atacctggca acagaaagta taataatata acagggtaag 14220 ggcataaaaa gtaccagtga aaacttttac agttattgac caacaagctg gataggaaag 14280 tagtgtgagt cctgatatta acagaatgta aaaactgaga tgaatgaagt ctcattaata 14340 gaatgagtta atagaatgtg aaaactgaga tgaatgaatg aactagagat aaaaataaca 14400 ggaaacagaa gttctctagg agtgaggggt agcaggacaa ggaagggaag tattagagtt 14460 cataatctta gggattgttc taagtagact tgtagatggc tttagtgcaa ataataataa 14520 ttagagtcaa agagttcaag aaatataaaa ttaaggtctt agaagggtca cgaatgggat 14580 gaagtagaaa atgctggcac tggaacagtg gagagaagac tatagcaggt actgaagcca 14640 ttgatgaaga tgaacaaatg tcatggaggt taacagaaaa gcaagatgga gaagaaagta 14700 ttaagtagat aaaataaact tcaaaaggca agtgttttgg gggagaaata aacagtggaa 14760 tggtgatctg aaagtaacag ggtaaagcag gggtcagcaa actttttcta taaagggtaa 14820 gacagcaaat attttgggct tgcagggcgt atatggtttc tgtcacagct cctccattct 14880 gctgcagtag aacaaaagca gccacagaca atatgttact aacaaatggg agtggcagta 14940 tgccaataaa acttttacaa aaatagaatg tgggccaatg gtcctcaaac tgtagtataa 15000 tgacccctgg tgtagaacaa gtatacagaa tcctctttct ggtggtgtac atgaggaaaa 15060 gtagagaaag taatgacctc tgttagaaag ggtttgagga acatattctt gtcaatggct 15120 ctttagctac cataaaaagg gtgtgtagat gaggggtgct cttggattca caaaacaaca 15180 caaggattcc agaggtttct aaagatcaat taattcagac atttaaaaaa tatctgctgt 15240 ttcaaaggaa aaagattcag gttaacccct gtattatttg tctttctaat aagctacttc 15300 actaagaaac atcatattta aatacttcat ggtatttaaa gatcagaaaa ctaacatgag 15360 gtatatttct ttcccaggtc ataaaagaac tttttttttg agacagagtc ttactttgtc 15420 gctgagcgtg gagggcagtg gtgtgatctc ggctcactgc aacctcggcc tcctgggttc 15480 aagtgattct cccaccttag cctcctgagt agctgggagt acaggcgcgt gccaccatgc 15540 ctggctaatt ttcatatttt tagtagagat ggggtttcgc catgttggcc aggcttgtct 15600 caaactcctg acctcaagta atccaccccc acttggcctc ccaaagtgct ggaattacag 15660 gcatgagcgc cactgtgccc aggcttctac agaagaagaa agattaagaa ctcataatct 15720 ttcaacatca tgattcacac acactcaaat tttaagtagc actttttaat gaactttagt 15780 tttccctaag aagttagatg gaaaggatta aaagacattc acacatatca aaaggaaaaa 15840 ctgttaaatg aaactgtaac ctcaaagaca aaactgagta aagatattga ataaatgtac 15900 tctgaagtgc ctctagatca actacactgg ataataagaa aaaagcaggt tctcttctga 15960 atttctcttc cttaatctga gatagcttaa aaaacaaaaa tagattatga gaaatgtaga 16020 ataaatgaag acattctcca ctgtatcatg gatatactat agctttaggg gagctttcat 16080 tataaatctg aaattactaa ccaatctccg tttaaaaaca gatttgctca cttaggttct 16140 gtttgtcaaa tcttcactga tgaatgcttc gacaaatcct gcattgtggg tcccaggaat 16200 tataatgcac caacaagcat aaagttataa ggatacaatc caaaaggatc taatcgaaga 16260 gttacaggtc ataaaatgac tcaatttatc ttgttttcct cttccttagg cattgccatt 16320 ccagtcccta ttaaagggat agagactgat gtggacaacc caaacaatat cacttgtgtg 16380 ctgacaaagg aacgttttgg cgatttcatg ctctttggct cactggctgc cttcttcaca 16440 cctcttgcaa ttatgattgt cacctacttt ctcactatcc atgctttaca gaagaaggct 16500 tacttagtca aaaacaagcc acctcaacgc ctaacatggt tgactgtgtc tacagttttc 16560 caaagggatg aaacaccttg ctcgtcaccg gaaaaggtgg caatgctgga tggttctcga 16620 aaggacaagg ctctgcccaa ctcaggtgat gaaacactta tgcgaagaac atccacaatt 16680 gggaaaaagt cagtgcagac catttccaac gaacagagag cctcaaaggt cctagggatt 16740 gtgtttttcc tctttttgct tatgtggtgt cccttcttta ttacaaatat aactttagtt 16800 ttatgtgatt cctgtaacca aactactctc caaatgctcc tggagatatt tgtgtggata 16860 ggctatgttt cctcaggagt gaatcctttg gtctacaccc tcttcaataa gacatttcgg 16920 gatgcatttg gccgatatat cacctgcaat taccgggcca caaagtcagt aaaaactctc 16980 agaaaacgct ccagtaagat ctacttccgg aatccaatgg cagagaactc taagtttttc 17040 aagaaacatg gaattcgaaa tgggattaac cctgccatgt accagagtcc aatgaggctc 17100 cgaagttcaa ccattcagtc ttcatcaatc attctactag atacgcttct cctcactgaa 17160 aatgaaggtg acaaaactga agagcaagtt agttatgtat agcagaactg gcagttgtca 17220 tcaaacataa tgatgagtaa gatgatgaat gagatgtaaa tgtgccaaga atatattata 17280 taaagaattt tatgtcatat atcaaatcat ctctttaacc taagatgtaa gtattaagaa 17340 tatctaattt tcctaatttg gacaagatta ttccatgagg aaaataattt tatatagcta 17400 caaatgaaaa caatccagca ctctggttaa attttaaggt attcgaatga aataaagtca 17460 aatcaataaa tttcaggctt taaaaagaac cactgtcatc ataaaattta tttggtttct 17520 aattatatgc aaagttgtgc taagaatgaa ataaaacaaa acaaacatta gccttgcctt 17580 ccaggtctta aaagcataag gcatgataca gaacaaaagg aacacagagc taaaaataag 17640 tgcacaaaac agtgatcaca tctggaggtg agttaagatt gccatttata gtctgattta 17700 gttgaatgca gtcataattt tcctctgatc aggaaactgt tttgatggaa gggataggga 17760 ggatagtagc tggatatcac acaaaataac aagaacttgg aactagacct gtttgttaaa 17820 tgactgatat tgagcttcta ctcagcacag gtaggtctat tggagaagga ggaaaaagca 17880 aataaaatcg ctcttgggta cctgaaccac attaggtttc ttagagatat taattaacgt 17940 gcacaagcat gggatcttaa tcagggatac aaactttaag tgagatcatg cctgcctatg 18000 tatgcttgtc cagtgactca agtgaacttc cagcacaaaa ttttttattc atcctttcag 18060 tatattattt tgcactgtta gatctcaga 18089 <210> 2 <211> 8903 <212> DNA <213> Artificial Sequence <220> <223> NPY whole DNA sequence <400> 2 cccctgaaac cacgggcggg ggtggggtgg ggagcgcagc tttgggaccc tctagccgga 60 gacttccggc agctgcctcc gacttgttct aagtacagga aaaatctgtg cgcccagttg 120 cctcactcca acagcgcgca gttgtgcccg gcgaggatgc cgcgctagtc gtggagatgc 180 cccaccacaa agaggattca ggtgcttcct actccggcac ccagtgggtt ggtagtcctg 240 ttggcaggag acaagaatcg tctgggctgc tcctatctct ggcaggacta gacggggcgt 300 gaaggaaaga aggaaagaag gaaagcaggg atcgggcact gcccgagggc agatacttgg 360 gctttggtgt tgtccagcgc gctcggagtg cgctgcctcg ctcacgcggt cccaggcccc 420 gcttcttcag gcagtgcctg gggcgggagg gttggggtgt gggtggctcc ctaagtcgac 480 actcgtgcgg ctgcggttcc agccccctcc ccccgccact caggggcggg aagtggcggg 540 tgggagtcac ccaagcgtga ctgcccgagg cccctcctgc cgcggcgagg aagctccata 600 aaagccctgt cgcgacccgc tctctgcacc ccatccgctg gctctcaccc ctcggagacg 660 ctcgcccgac agcatagtac ttgccgccca gccacgcccg cgcgccagcc accgtgagtg 720 ctacgacccg tctgtctagg ggtgggagcg aacggggcgc ccgcgaactt gctagagacg 780 cagcctcccg ctctgtggag ccctggggcc ctgggatgat cgcgctccac tccccagcgg 840 actatgccgg ctccgcgccc cgacgcggac cagccctctt ggcggctaaa ttccacttgt 900 tcctctgctc ccctctgatt gtccacggcc cttctcccgg gcccttcccg ctgggcggtt 960 cttctgagtt accttttagc agatatggag ggagaacccg ggaccgctat cccaaggcag 1020 ctggcggtct ccctgcgggt cgccgccttg aggcccagga agcggtgcgc ggtaggaagg 1080 tttccccggc agcgccatcg agtgaggaat ccctggagct ctagagcccc gcgccctgcc 1140 acctccctgg attcttgggc tccaaatctc tttggagcaa ttctggccca gggagcaatt 1200 ctctttcccc ttccccaccg cagtcgtcac cccgaggtga tctctgctgt cagcgttgat 1260 cccctgaagc taggcagacc agaagtaaca gagaagaaac ttttcttccc agacaagagt 1320 ttgggcaaga agggagaaaa gtgacccagc aggaagaact tccaattcgg ttttgaatgc 1380 taaactggcg gggcccccac cttgcactct cgccgcgcgc ttcttggtcc ctgagacttc 1440 gaacgaagtt gcgcgaagtt ttcaggtgga gcagaggggc aggtcccgac cggacggcgc 1500 ccggagcccg caaggtggtg ctagccactc ctgggttctc tctgcgggac tgggacgaga 1560 gcggattggg ggtcgcgtgt ggtagcagga ggaggagcgc ggggggcaga ggagggaggt 1620 gctgcgcgtg ggtgctctga atccccaagc ccgtccgttg agccttctgt gcctgcagat 1680 gctaggtaac aagcgactgg ggctgtccgg actgaccctc gccctgtccc tgctcgtgtg 1740 cctgggtgcg ctggccgagg cgtacccctc caagccggac aacccgggcg aggacgcacc 1800 agcggaggac atggccagat actactcggc gctgcgacac tacatcaacc tcatcaccag 1860 gcagaggtgg gtgggaccgc gggaccgatt ccgggagcgc cagtgcctgc acaccaggag 1920 atcctgggga tgttagggaa agggattgtt tcttttcctt cgctctatcc cagggcagga 1980 cagtatcagg cacttagtca gctctaggta aatgtttgta cagggcacac tctacacaaa 2040 atgggtacct tccattttgt gcaactacag tcacagagtc gtgatcccca gattcaggtt 2100 ccccaggctg gtaggctggc aatctcctct cactcacctc ttatggtttg ttgtggttct 2160 tacggcagtg gggcccggtc cagaaatctc gaaagtaccc agtgaaaggg gcaagaatgc 2220 gccagagaaa tgctgtaggg ggaaacgcta gcaaggtgtc taggagaaac agaacgacca 2280 ccaaagaaaa ccaaaccaag gagtaaactg cagggttgcc acagacattg tcagactttc 2340 cggcctgccc agggctaatt gaatgacagt atttagaaaa agacaaatag agctattttc 2400 ttttcttctt cactgtgttt tttttgcagt ctcactccca tatttgaaaa ttctagtccc 2460 aataattaga gatttagcct gaaaacagtt tatctaaaat gcagaggtct ccctatttta 2520 tagctggtga attctgaaaa gaaagtgccc acttggtatt tcattaagaa gaaacttacg 2580 ttggtccagt aactttagga tgaattcagg gattacatgg aatctgagaa attgtaaact 2640 ttattctgaa cattccgaaa gtcaaaatga aatgccatat tttcactctc aacattgaca 2700 attaagggaa aaatcaaatc caaactattt tacaaattac ataggattta aattttgttt 2760 tcaaaattgg ttgatcttag ttctaaatca tttaatatct tattttaatt actgagatta 2820 aatgatttta gtcatttgac cgtatctaaa ggataagccc tttagaagca ttaatgttag 2880 ttaacatgtt taaattatat gtttaaagta ttgtggggtt tatatattta tttatttagt 2940 ccatattttt actgtttaca agtcagggag tagttggaaa ggatccaaaa ggggggaaga 3000 tttatacttt taaaataaag tttgcttttt cctgtttcat caaaaaggta tttaacatcc 3060 taaaacactt ttgaaaaagc cccaatttat atagaatctg aaaaacatga aaatgccaga 3120 attaaatagt agttgtttgt gatcccttga aatctgatca ttttaattag caaaaagcaa 3180 taatatttta tattccatac ttcccagaaa gttaattgat tcaaattctt gctatgttat 3240 gccgtcatgc agatcaaaga gaacttgtac aaacaaaaag atattaactg ttttttctag 3300 aaaaaccttt gttagttatt tagctgtagc ataatacaca gtagaactca tttttccgaa 3360 gatgattgcc ataaagcttt accattttac aaagctctag cattttaaaa tgttttccta 3420 ggcattctct cattcagtcg ttacctcaaa gctttgaagg gagtataatt atcttcattt 3480 cacaaatcat gaagctaatg acatttcatg gaagctattt ttataggaat tatggaattg 3540 gagccctcct ttgagtgtct ttggatgttg tcaccaccat caacaggcag tgttatctcc 3600 aacatgtttg atgagaaaaa aacataaagg aaagggactt gggtttcatg gcaagagatt 3660 cagcttagat gggaatgagc tggtctctga ggcagagttt cctgtctcat ctttgtataa 3720 actccttctt agaaaccagc agaggctctt aatgtacgtc catcttctcc ttcccaccac 3780 agacacccac tcctatgtgc gtttctgaaa attacagggt catttgtgga tgtgctttaa 3840 tttagtatag atataatctg atatcattat ctttctttcc ccccgccccc cgccaccagc 3900 attccccctg cactcaattt gtgcttatat tccttttgct gtatatatac ccctggctca 3960 ttgcttctgg aagctgcatt acttattcta gagtgtgggt ctcctacaat ttatttatat 4020 agccccctaa atcagtggtt ctcaactggg ggtgatttta tgctccccag gtgtgatttg 4080 gcagtgtctg gagacatttt tggttttcac aactggaggt gggcactgct ggtacctagg 4140 tgtatagatg ccaaggaggc tgctaaatat cctaccatgc ccaggacagc ccccattaca 4200 cagaatagcc agtcccagag aaactcagcc ttagattagt gtttctcctt cctggctgca 4260 cattaaaatt atcaggggag cttttaaaat acactgatgc tgggatccac ccctgaccaa 4320 ttaaatcaga ccctctggga tggggcctgg gctttagagt gtgtttttaa cactccccag 4380 gtgattctaa tgtgcagcca ggtttgagaa tctttactct aaggactcaa tctttgaaag 4440 ttacagcatt gtagagggga aggatgatcc agaattaatt aaagaaggaa gaagagtgaa 4500 cagtactgaa ctggatgcaa gattatgact gtgcaaatcc tcctatttca gatttaggtt 4560 ctttactggt aaattggata aaaagttggc agttctttta ctcactggag catgatgaag 4620 ataaatgagt tacagttgcc ctagactttt acgattagta aaaccaatgg atggacagaa 4680 taaataagta taagcagaca cctatagtct aatatcgcaa ctccagaaaa tctttgagaa 4740 aatctcacac atgatgggag tgggacaggg agttgttttg tttttgcttc ccttgagtct 4800 cagcaaaaga acagtcatag aaaatatttc caaaaaattt agatataatt ttgcttggca 4860 attagaatat accctctgtc agctttgttc agttgttgga cctattaacc aatccatacc 4920 agactaattt atttctaaac aaagtggcga atggtgtcca atgccttttc tgagaaagag 4980 aaaaatggcc tcttcaaaag acgcaatttg gtatagactt gtctggccgt tttgtagtgt 5040 tctggagtgg ggagcatata ctaccctcac aggtaaacga aaccttacct gtatgtattg 5100 tctcacccat atcctgctac aggagaaaaa aaaaaaaaaa aaaaaaagaa catttccatg 5160 gatcagttag attcctttag acttacaggg aggctaaaat cacagcatta ggaaagactt 5220 tagcatttgg caccttgaat gaatatggaa aatataagac ccagggtctg ttggtagcca 5280 gaagaatgga tgcatttcaa cagcagtaat ttgcatgcgt gtatcacttt gtcactttga 5340 aagcattgtg actcacattt cacactggat gttcacaaag actctgattt aagcaggggt 5400 tgttttacaa acgagaaaat tgggattcaa agacacaggt gggtcttgga cggcaatctg 5460 ctgatttttt ttttctagga ctgtctttga aatatcatct ggtctccttg ttaagattta 5520 aactgctgtg tatcatcaga acaatagcca attaactaat tgtggaaaca tttacaatca 5580 tgcttccatg ttttcaaatt tcctgtcacc tagttataaa agtatgtatc tctatacata 5640 ttatttgtgg ttgtttaaga aggcaggaat ttgactagga attttggttc ggtttaaata 5700 acctcctaga ttatttatta tttgagcaaa gtttaaatat atatacaaat aatttgaaat 5760 aatatttatg ataatgtttc gataatatat gcttcataca cctagcttgc ttgttacaga 5820 tgaacacctg acaataatgt ttagttttca tatcccaaat aggagactat cttccttttt 5880 cctaaaggtt tttatgccta ttccaaactt gctttaaaag actttttttt ttccagatat 5940 ggaaaacgat ccagcccaga gacactgatt tcagacctct tgatgagaga aagcacagaa 6000 aatgttccca gaactcggta tgacaaggct tgtgatgggg acattgttgc agagctcaag 6060 gtgcccaggg gagggaagtc agagacaggt gcctggtggg aggcaccacc aggcttctag 6120 actaggggag atttcggcag aaatgaggat gaagaagcag gcagaggaga cctctctttt 6180 catgtactca ttgtcaaatc tctaaacttc tgtggaataa cattttttta gctcaggtta 6240 tttggtgctc ctttgacgat tttaaaaata attgactttt aacttagaaa ttatatagaa 6300 tttctaacat tgttggagtc aggggagcat agagtgagtc cctgactcta aattgaaatg 6360 accaactgtc catctctggc tgggaagatc tgtagatttt atatatgtgg agtgtccaga 6420 tgtcctagga aatgtcccat gtggttttta ctccaacttt cccagtttta catgaagctg 6480 taggtaaatt aggtgctctg gattactttt tgacatattt taattcaact tgtgtcatat 6540 agtgatttat tacattcaac gaatgcacgt tgaatgacag tattcttagg agtttatctg 6600 tagtctcagg actttcttgc agtttgggtc tgtcttggaa agaggtgccc accaatgtct 6660 aagtgttaaa cattgagcta aggttttctg gtgcctcctg gacaaggagt gctgctgaag 6720 aggagcctgg tcctcttcca tcttcataac ctcctgactc ttcagtcctt gcttcctgtc 6780 tctaaccaca gtaagatcat agttcacagg gtccacccac gtctgtggca gcttggctgc 6840 cttaaaccaa gttctacaat gtcaagaacc aagttcccca gaagaggtca ctacaacact 6900 tgctcagtga gtgtgactga cgtggctctc catcaagttc tccctagaga ctcagcaggg 6960 tgacccgctg ggccacccac cttcccttct aacctccctc agttctcttc taaaatggcc 7020 agccagcatg aagggtgtta gtatagagtt gtgagatttg ttttctctaa agggaacaac 7080 gatttgaact gagataaaac tccagtgctc ctgtccctgg aggaaccagc ccagacttaa 7140 atagcaaatg caagggaaac aggcagaaaa tttgtaaggg ttatcttttt ttaattaaat 7200 aataataata ataattatta ttattattca gagacaaggt ctccttctgt cactcaggcc 7260 agagcacagt ggtgcaatca aagatcactg cagcctcgag ctcccagcct caagagatcc 7320 tccctcctca gcctgcccaa gtgctaggat tacagttgtg agccataatg cctggccagg 7380 gttgtcttta acactaagca gtaaatgtct ttccatgttt cactgacatg catatgatgt 7440 ttgccacagt aaaaagttta gggtagacta tatcgactta ctgttagaaa aataacaacg 7500 cagacctact tagaaccttg gtttttgtct ttgttttgtg gcccaaggtc acacagttag 7560 tgcatgaagt aaaattaatt gcaacctgat tgacagactc catcaggtgc agttagaagg 7620 agacaaaact tgacggtctt gaagttgcct atgagtgcta ttttattata aaggtcttat 7680 tatagtctaa taagtagcat cttactacat aataaagctg gcaggaaaag gaagcatatt 7740 tagagttgag gaacaataac tttcctgtca cagcttcatg tgatttctgt aattttgatt 7800 ttcaaaagat cggggtgttc ttcggctcct ttgatacata cagagccttt caaatgctct 7860 ctctgatgtt tgcccaagac gcctgtcaac atcgtggaat gctgctcagc tggggctgtg 7920 catattgtgc ctccgcctca cgatctgata ttccacatgg cttcttattt agaatgccaa 7980 caggaaactt ttcaacagtt cccggtcatc tttcacttca gatctaaatg tctcaccctt 8040 gctcatacct caggaagttg ggcagctttc cttacatgct ttgcttctta tgttttacag 8100 gcttgaagac cctgcaatgt ggtgatggga aatgagactt gctctctggc cttttcctat 8160 tttcagccca tatttcatcg tgtaaaacga gaatccaccc atcctaccaa tgcatgcagc 8220 cactgtgctg aattctgcaa tgttttcctt tgtcatcatt gtatatatgt gtgtttaaat 8280 aaagtatcat gcattcaaaa gtgtatcctc ctcaatgaaa aatctattac aatagtgagg 8340 attattttcg ttaaacttat tattaacaat tagtcattat agtctccctc aactagcaga 8400 ttcagggttc gtttcgctaa tattaaaaca gcggctatga aagatatgaa gatatgatta 8460 ttcatgataa ccaagatcag ctgtgtggtg tgtgtgtatg tgtgtaaaac atatagatgc 8520 atttgaagat attgatcaaa ggaatgctaa tttgggaaat ttttctgagc tttcccccca 8580 ttaacattgg aacccttttg ctaaccttgt taaccagtgc tgtcagcaga agaacttcct 8640 gactaactag agctttctgt ggaatgtcct ttatccctct gtctaatatg gcagccacta 8700 gccacatgtg gtttttgagc ccctgaatgt agtaagtgca aataaagaga tgagttttaa 8760 attttattta attttaatga attttaactt aaatggccat taagtctcta gtggctacca 8820 tatcagaccg ttgaggtcta tacatctatt ttggtcacta ctgttagaat ttgagtgaga 8880 aatacaatga gtgggatgat tat 8903 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for HTR2B <400> 3 aaacaagcca cctcaacgcc t 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for HTR2B <400> 4 tcccgaaatg tcttattgaa gag 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer NPY <400> 5 agagatccag ccctgagaca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer NPY <400> 6 tcaccacatg gaagggtctt 20

Claims (8)

A marker composition for predicting the differentiation of umbilical cord mesenchymal stem cells comprising an agent for measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression. The method according to claim 1,
Wherein the neuropeptide Y gene comprises the nucleotide sequence shown in SEQ ID NO: 2. The method according to claim 1,
Wherein the neuropeptide Y gene is amplified with a primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6. delete The method according to claim 1,
Wherein said differentiation is differentiation into neural cells. The method according to claim 1,
The measurement may be performed using a reverse transcriptase polymerase chain reaction, a real-time polymerase chain reaction, Northern blot, Western blot, radioimmunoassay, radioimmunodiffusion, Wherein the composition is selected from the group consisting of an immunoprecipitation assay, an immunohistochemical analysis, an enzyme-linked immunosorbent assay (ELISA), and a microarray chip. A method for predicting the differentiation of umbilical cord mesenchymal stem cells into neural cells by measuring neuropeptide Y gene mRNA expression or neuropeptide Y protein expression.
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