WO2007119799A1 - Marqueur permettant de détecter l'activité de différentiation myocardique d'une cellule - Google Patents

Marqueur permettant de détecter l'activité de différentiation myocardique d'une cellule Download PDF

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WO2007119799A1
WO2007119799A1 PCT/JP2007/058107 JP2007058107W WO2007119799A1 WO 2007119799 A1 WO2007119799 A1 WO 2007119799A1 JP 2007058107 W JP2007058107 W JP 2007058107W WO 2007119799 A1 WO2007119799 A1 WO 2007119799A1
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seq
cell
cells
amino acid
polynucleotide
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PCT/JP2007/058107
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Japanese (ja)
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Yoji Sato
Tetsuya Hasegawa
Teruhide Yamaguchi
Tetsuji Hosono
Mitsutoshi Satoh
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Japan Health Sciences Foundation
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Publication of WO2007119799A1 publication Critical patent/WO2007119799A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a marker useful for accurate and simple detection of myocardial differentiation activity of cells.
  • the present invention also relates to a cell myocardial differentiation regulator useful for the treatment of heart diseases.
  • Cardiomyocytes actively divide and proliferate before birth but with autonomous pulsation. However, cardiomyocytes usually lose their ability to divide quickly as soon as they are born, and unlike liver cells, they usually do not acquire the ability to divide again.
  • cardiomyocyte progenitor cells are also contained in mature myocardial tissue.
  • the number of cardiomyocyte progenitor cells is very small and is not sufficient to fully restore cardiac function by complementing cardiomyocytes when they are damaged. Therefore, when the myocardium is overloaded or necrotic due to ischemic heart disease, cardiomyopathy, etc., cardiomyocytes are adapted mainly in the form of cell hypertrophy rather than cell division.
  • Cardiomyocyte hypertrophy is considered to be a physiological adaptation phenomenon to the load in the initial stage, but coexistence with coexistence of coexistence of cardiac fibroblasts and fibrosis of the stroma leads to a decrease in the heart's own dilation function and a decrease in contraction It becomes associated with heart failure.
  • Treatment of heart failure due to ischemic heart disease, cardiomyopathy, etc. mainly focuses on symptomatic therapies such as enhancement of cardiac contraction force, pressure load with vasodilators, reduction of volume load, and reduction of fluid volume with diuretics.
  • symptomatic therapies such as enhancement of cardiac contraction force, pressure load with vasodilators, reduction of volume load, and reduction of fluid volume with diuretics.
  • the basic treatment for severe heart failure includes heart transplantation.
  • heart transplantation has many problems such as lack of organ donors, difficulty in determining brain death, rejection, and medical expenses. Therefore, for appropriate heart disease treatment, There is a need to develop a method for reacquiring lost myocardium.
  • One method for reacquiring the myocardium is a method for reacquiring the ability to divide and proliferate cardiomyocytes.
  • it is necessary to investigate the mechanism by which cardiomyocytes lose the ability to divide after birth. Then, it is expected that the mechanism that works is clarified, and that gene therapy is expected to regain the ability to divide myocardial cells (Tamamori et al, Critic al role of cyclin Dl nuclear import in cardiomyocyte proliferation, ire Res. 2003). , Jan 10, 92 (1), el2-9).
  • Another method for reacquiring the myocardium is a differentiation induction method in which cells other than cardiomyocytes are converted into cardiomyocytes.
  • Murry et al. Report that non-cardiomyocytes (fibroblasts) can be converted into skeletal muscle cells by introducing MyoD, a master key gene for skeletal muscle differentiation, into the heart in vitro.
  • MyoD a master key gene for skeletal muscle differentiation, into the heart in vitro.
  • MyoD a master key gene for skeletal muscle differentiation
  • cardiomyocytes various cardiomyocyte-specific transcription factors such as Nkx2.5, GATA4, and MEF2C are cloned.
  • Another method for treating heart disease is cardiomyocyte transplantation.
  • stem cells or myocardial progenitor cells have been transplanted into myocardial injury sites to differentiate into myocardial cells, or stem cells or myocardial progenitor cells have been differentiated into cardiomyocytes in vitro and then used as myocardial injury sites.
  • the transplanting method is also attracting attention from the viewpoint of regenerative medicine. This method transplants cells from the patient, so there is no need to worry about rejection!
  • myocardial differentiation activity In order to improve the safety of myocardial cell transplantation treatment, it is essential to appropriately predict the level of cell division into the myocardial cells, that is, “myocardial differentiation activity”.
  • the obtained marker is one of the markers of “the initial process of cardiomyocyte differentiation that has already begun”, “differentiation activity into cardiomyocytes possessed by undifferentiated cells” before differentiation induction It is not a marker for.
  • quality evaluation of undifferentiated cells and isolation of undifferentiated cells with high myocardial capacity are required. Therefore, there is still a need for a marker that can be used to detect the myocardial activity of cells in the separation process, including the undivided stage! / Speak.
  • CMP cardiac myogenesis predictor
  • an object of the present invention is to provide a marker for detecting myocardial differentiation activity of cells.
  • the gene marker for detecting myocardial differentiation activity of the cell according to the present invention is a polynucleotide according to the following (a), (b), (c), (d) or (e): Or its fragment Consists of:
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: A polynucleotide having the nucleotide sequence ability represented by 45,
  • a polypeptide marker for detecting myocardial differentiation activity of a cell consists of the following polypeptide (f), (g) or (h) or a fragment thereof:
  • SEQ ID NO: 14 SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, The amino acid represented by SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46
  • the use of the marker makes it possible to accurately detect the myocardial differentiation activity of cells in various sorting processes including the undifferentiated stage. Therefore, the marker according to the present invention can be advantageously used for quality evaluation of undifferentiated cells and isolation of undifferentiated cells having high myocardial differentiation ability.
  • the polynucleotide constituting the gene marker according to the present invention it becomes possible to control myocardial differentiation of cells. Therefore, the polynucleotide in the present invention can be advantageously used in elucidation of the details of the cardiac muscle differentiation mechanism and in the production of cardiomyocytes. Therefore, it can be said that the provision of a powerful invention is significant in the treatment of heart diseases.
  • FIG. 1A is a graph showing the time course of the number of contractile nodules in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 1B is a graph showing the time course of contraction nodule size in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2A is a diagram showing the time course of expression of MLC2a gene in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2B is a diagram showing the time course of MLC2v gene expression in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2C is a diagram showing the time course of a MHC gene expression in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2D shows the time course of jS MHC gene expression in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2E shows the time course of Nkx2.5 gene expression in cell lines (P19, CL6, G26, G36, G45 and G52).
  • FIG. 2F is a diagram showing the time course of GA TA4 gene expression in cell lines (P19, CL6, G26, G36, G45 and G52).
  • Figure 2G shows cell lines (P19, CL6, G26, It is a figure which shows the time course of the expression of MEF2C gene in G36, G45, and G52).
  • FIG. 3 shows a screen plot of the principal component analysis of the mRNA expression data of cardiomyocyte marker genes in cell lines that induced cardiomyocyte differentiation (P19, CL6, G26, G36, G45 and G52)
  • FIG. Figure 3B shows a variable plot of the principal component analysis of the mRNA expression data of the cardiomyocyte marker gene in the cell lines that induced cardiomyocyte differentiation (P19, CL6, G26, G36, G45, and G52). It is.
  • FIG. 5A is a graph showing changes in the expression level of CMP2 gene in G52 cells by RNAi treatment.
  • FIG. 5B shows changes in the expression level of CMP13 gene in G52 cells by RNAi treatment.
  • FIG. 6 is a graph showing the time course of cardiac muscle activity of G52 cells when the expression of CMP2 or CMP13 gene was suppressed by RNAi.
  • FIGS. 7A and B are diagrams showing changes in the expression level of CMP1-24 genes in G52 cells by RNAi treatment.
  • FIGS. 8D to 8F are diagrams showing the time course of cardiac muscle activity of G52 cells when the expression of CMP4-6 gene is suppressed by RNAi.
  • FIGS. 8G to I are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP7-9 gene was suppressed by RNAi.
  • FIGS. 8J-L are diagrams showing the time course of cardiac muscle activity of G52 cells when the expression of CMP10-13 gene was suppressed by RNAi.
  • Fig. 8M-0 shows G52 cells when the expression of CMP14-16 gene was suppressed by RNAi. It is a figure which shows the time passage of myocardial irritation activity.
  • FIGS. 8P to R are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP17-20 gene was suppressed by RNAi.
  • FIGS. 8S to U are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP21-24 gene was suppressed by RNAi.
  • the genetic marker according to the present invention comprises CMP genes described below as CMP1-24.
  • CMP1 is an accession number in the ReSeq database of the National Center for Biotechnology Information (NCBI) in the United States.
  • CMP2 corresponds to the same session number NM_178737
  • CMP3 corresponds to the same session number NM_144888
  • CMP4 corresponds to the same session number NM_182991
  • CMP5 corresponds to the same session number NM_029537
  • CMP6 corresponds to the same session number NM_009982
  • CMP7 corresponds to the same session number NM_010169
  • CMP8 corresponds to the same session number NM_011348
  • CMP9 corresponds to the same session number NM_030700
  • CMP10 corresponds to the same session number NM—025582
  • CMP11 corresponds to the same session number NM_133362
  • CM P12 corresponds to the same session number NM_025422
  • CMP10 corresponds to the same session number NM—025582
  • CMP11 corresponds
  • nucleotide sequence of the CMP gene include the following sequences. That is, CMP1 is represented by SEQ ID NO: 1, CMP2 is represented by SEQ ID NO: 3, CMP3 is represented by SEQ ID NO: 5, and CMP4 is represented by SEQ ID NO: 7. CMP5 is represented by SEQ ID NO: 9, CMP6 is represented by SEQ ID NO: 11, CMP7 is represented by SEQ ID NO: 13, and CMP8 is represented by SEQ ID NO: 15. CMP9 is represented by SEQ ID NO: 17, CMP10 is represented by SEQ ID NO: 19, CMP11 is represented by SEQ ID NO: 21, and CMP12 is sequenced.
  • CMP23 is represented by SEQ ID NO: 25
  • CMP14 is represented by SEQ ID NO: 27
  • CMP15 is represented by SEQ ID NO: 29
  • CMP17 is represented by SEQ ID NO: 33
  • CMP18 is SEQ ID NO: CMP19 is represented by SEQ ID NO: 36
  • CMP20 is represented by SEQ ID NO: 37
  • CMP21 is represented by SEQ ID NO: 39
  • CMP23 is represented by SEQ ID NO: 43
  • CMP24 is represented by SEQ ID NO: 45.
  • the genetic marker 1 is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, sequence SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.
  • the gene marker is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 45
  • SEQ ID NO: 1 SEQ ID NO: 3
  • SEQ ID NO: 5 SEQ ID NO: 7
  • SEQ ID NO: 9 SEQ ID NO: 11
  • SEQ ID NO: 15 SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 45
  • CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 are characterized in that the expression level in cells decreases when the myocardial differentiation activity of the cells increases.
  • the gene marker is a nucleotide sequence represented by SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 43.
  • the expression level in the cell decreases.
  • the gene marker according to the present invention also includes a polynucleotide functionally equivalent to CMP1-24.
  • “functionally equivalent” preferably means that it correlates with the myocardial differentiation activity of the cell as well as the CMP gene.
  • the polynucleotide according to the present invention can be used for myocardial differentiation control. Therefore, a polynucleotide functionally equivalent to such a polynucleotide is more preferably one having cell myocardial differentiation control activity equivalent to that of the CMP gene.
  • the functional equivalence between the polynucleotide and the CMP gene is determined by, for example, measuring the expression level of the polynucleotide using a nucleic acid molecule or primer according to the present invention described later, and the number of cells that cause pulsation,
  • the correlation with the size can be easily determined by determining by a known statistical method and comparing it with that of the CMP gene.
  • the gene marker according to the present invention may have sequence homology with the CMP gene as long as it serves as an index of myocardial differentiation activity.
  • the gene marker has a nucleotide sequence power having at least 70% homology with the nucleotide sequence encoding the CMP gene, and also has a polynucleotide power functionally equivalent to the CMP gene.
  • the strong sequence homology is more preferably 75% or more, further preferably 80% or more, and further preferably 85% or more.
  • a part of the gene marker according to the present invention consists of a nucleotide sequence in the protein coding region of the CMP gene. That is, among CMP genes, CMP1-16, 18, 20-24 encode polypeptides.
  • SEQ ID NO: 1 (CMP1: In particular, nucleotide sequence represented by nucleotides 64 to 1863) encodes the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 3 (CMP2: In particular, the nucleotide sequence represented by nucleotides 230 to 2644) encodes the amino acid sequence represented by SEQ ID NO: 4, and SEQ ID NO: 5 (CMP3: In particular, represented by nucleotides 277 to 1788) (SEQ ID NO: 7 (CMP4: in particular, the nucleotide sequence represented by nucleotides 42 to 1055)) is SEQ ID NO: 8 which encodes the amino acid sequence represented by SEQ ID NO: 6.
  • SEQ ID NO: 9 (CMP5: in particular, nucleotide sequence represented by nucleotides 99 to 779) encodes the amino acid sequence represented by SEQ ID NO: 10.
  • SEQ ID NO: 11 (CMP6: in particular, the nucleotide sequence represented by nucleotides 70 to 1458) encodes the amino acid sequence represented by SEQ ID NO: 12, and SEQ ID NO: 13 (CMP7: , Nucleotide sequence represented by nucleotides 60 to 1352) encodes the amino acid sequence represented by SEQ ID NO: 14, and SEQ ID NO: 15 (CMP8: in particular, nucleotide sequence represented by nucleotides 610 to 2943) ) Encodes the amino acid sequence represented by SEQ ID NO: 16, and SEQ ID NO: 17 (CMP9: in particular, the nucleotide sequence represented by nucleotides 77 to 1927) is the amino acid sequence represented by SEQ ID NO: 18.
  • CMP13 In particular, the nucleotide sequence represented by nucleotides 392 to 2320 encodes the amino acid sequence represented by SEQ ID NO: 26, and SEQ ID NO: 27 (CMP14: In particular, nucleotides 396 to 2501)
  • the nucleotide sequence represented by SEQ ID NO: 28 encodes the amino acid sequence represented by SEQ ID NO: 28, and SEQ ID NO: 29 (CMP15: in particular, the nucleotide sequence represented by nucleotides 97 to 1962) has the sequence SEQ ID NO: 31 (CMP16: nucleotide sequence represented by nucleotides 470 to 1921 in particular) is an amino acid sequence represented by SEQ ID NO: 32.
  • SEQ ID NO: 34 (CMP18: in particular, the nucleotide sequence represented by nucleotides 92 to 1774) encodes the amino acid sequence represented by SEQ ID NO: 35, and SEQ ID NO: 37 ( CMP20 (in particular, the nucleotide sequence represented by nucleotides 113 to 763) encodes the amino acid sequence represented by SEQ ID NO: 38, and SEQ ID NO: 39 (CMP21: particularly represented by nucleotides 221 to 457) Nucleotide sequence) encodes the amino acid sequence represented by SEQ ID NO: 40, and SEQ ID NO: 41 (CMP22: in particular, the nucleotide sequence represented by nucleotides 14 to 1135) is represented by SEQ ID NO: 42.
  • the gene marker is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 38 or nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 46. It is characterized by doing.
  • the gene marker according to the present invention is an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence, and the amino acid sequence and function A polynucleotide comprising a nucleotide sequence encoding a substantially equivalent amino acid sequence.
  • the “one or several” is preferably 1 to 5, more preferably 1 to 2.
  • the gene marker is an amino acid sequence having at least 60% homology with the amino acid sequence and functionally equivalent to the amino acid sequence.
  • the polynucleotide comprises a nucleotide sequence that encodes the sequence.
  • the homology in the gene marker is more preferably 70% or more, and further preferably 80% or more.
  • the gene marker described above may be DNA or RNA. More specifically, the gene marker may be DNA present in the genome or cDNA generated for its transcription product, mRNA. Further, the present invention in which the above-mentioned mRNA may be directly used as a gene marker also includes a powerful embodiment. Therefore, according to another aspect of the present invention, a genetic marker is a ribonucleic acid obtained by substituting the thymine residue with a uracil residue in the nucleotide sequence encoding the CMP gene or a functionally equivalent gene thereof. It comprises a polynucleotide that also has nucleotide sequence capabilities.
  • the expression product of CMP gene can be used as a marker for detecting myocardial differentiation activity of cells.
  • the marker that can be used include markers made of polypeptides encoded by the above CMP genes 1-16, 18, 20-24.
  • polypeptides encoded by CMP7, CMP12, CMP16, CMP21 and CMP23 have a decreased expression level in cells when the myocardial differentiation activity of the cells increases. sell. Therefore, according to another preferred embodiment of the present invention, the polypeptide marker is a nucleotide encoding the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 or SEQ ID NO: 44. It consists of a sequence, and when the myocardial differentiation activity of the cell increases, the expression level in the cell decreases.
  • a polypeptide encoded by a gene functionally equivalent to the CMP gene can also be used as an indicator of cellular myocardial differentiation activity. Therefore, according to another preferred embodiment of the present invention, the polypeptide marker has an amino acid sequence ability in which one or several amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence, and It comprises a polypeptide functionally equivalent to the polypeptide encoded by the CMP gene.
  • the polypeptide marker is an amino acid sequence having at least 60% homology with the amino acid sequence, and a polypeptide encoded by the CMP gene. It comprises a functionally equivalent polypeptide.
  • polypeptide marker according to the present invention is required to have the entire sequence of the polypeptide encoded by the CMP gene or a functionally equivalent polynucleotide as long as it can be used as an index of myocardial differentiation activity. It may be a partial sequence.
  • a polypeptide marker according to the present invention encompasses fragments of the aforementioned polypeptides. Such fragments have, for example, 13 or more amino acid residues.
  • the cells in the present invention are preferably undifferentiated cells, and more preferably embryonic stem cells, somatic stem cells or cardiomyocyte progenitor cells.
  • the cell in the present invention is preferably a mammal-derived cell, more preferably a human, mouse or monkey-derived cell.
  • the polynucleotide constituting the gene marker according to the present invention or a polynucleotide complementary thereto is targeted and the polynucleotide and the string are targeted.
  • Nucleic acid molecules that can be hybridized under gentle conditions can be used.
  • complementary means that two nucleotides can be paired under hybridization conditions, for example, adenine (A) and thymine (T) or uracil (U ), And the relationship between cytosine (C) and guanine (G).
  • hybridize means that the nucleic acid molecule according to the present invention is a genetic marker under normal hybridization conditions, preferably under stringent hybridization conditions. It means that it does not hybridize to nucleotide molecules other than gene markers. Stringent conditions can be determined depending on the melting temperature Tm (° C) of the duplex of the nucleic acid molecule of the present invention and its complementary strand, the salt concentration of the hybridization solution, and the like. For example, J. Sambrook, EF Frisch, T. Maniatis; Molecular loning 2nd edition, old bpnng Harbor Laboratory (1989) can be referred to.
  • the nucleic acid molecule when hybridization is performed at a temperature slightly lower than the melting temperature of the nucleic acid molecule to be used, the nucleic acid molecule can be specifically hybridized to the gene marker.
  • the nucleic acid molecule that hybridizes to the gene marker according to the present invention does not need to be completely complementary to the gene marker, as long as specific hybridization is possible, but preferably is complementary to the gene marker. A complete or partial sequence of such a nucleotide molecule.
  • nucleic acid molecule is used to mean DNA, RNA, and PNA (peptide nucleic acid). According to a preferred embodiment of the invention, the nucleic acid molecule is DNA or RNA.
  • nucleotide sequence of the nucleic acid molecule according to the present invention can be appropriately designed by those skilled in the art. As described above, the nucleic acid molecule according to the present invention targets a gene marker. However, when the detection target is a genome, the nucleic acid molecule according to the present invention has only a portion that hybridizes to an exon. It can also include a portion that hybridizes to the intron.
  • the nucleic acid molecule according to the present invention can be used as a nucleic acid probe in the detection of the gene marker.
  • the nucleic acid probe may be prepared as a synthetic oligonucleotide using a commercially available oligonucleotide synthesizer or the like, or a certain double-stranded DNA obtained by restriction enzyme treatment or the like. It may be produced as a fragment.
  • the chain length of the nucleic acid molecule is preferably 18 nucleotides or more, and preferably 30 nucleotides or less, more preferably 24 nucleotides or less. ,.
  • nucleic acid molecule according to the present invention when used as a nucleic acid probe, it is preferable to appropriately label the nucleic acid molecule.
  • a labeling method a T4 polynucleotide kinase is used to label the 5 ′ end of the oligonucleotide by phosphorylation with 32 P, and a random hexamer oligo is used using a DNA polymerase such as Talenow enzyme.
  • examples thereof include a method of incorporating a substrate base labeled with an isotope such as 32 P using a nucleotide or the like as a primer, a fluorescent dye, or piotin (such as a random prime method).
  • the nucleic acid molecule according to the present invention can be used as a primer for nucleic acid amplification in the detection of a genetic marker according to the present invention.
  • the chain length of the nucleic acid molecule is preferably 18 to 30 nucleotides, more preferably 18 to 24 nucleotides.
  • the nucleic acid amplification method is usually performed using a pair of primers. Therefore, according to the present invention, there is provided a primer pair for detecting the gene marker, which can amplify all or part of the gene marker according to the present invention.
  • a primer pair for detecting the gene marker which can amplify all or part of the gene marker according to the present invention.
  • the nucleic acid molecule according to the present invention can be used as the two primers constituting such a primer pair.
  • Such a primer can be appropriately designed by those skilled in the art based on the nucleotide sequence of the region to be amplified.
  • the primer in one of the primer pairs has the 5 ′ end sequence in the nucleotide sequence of the amplification target region, and the other primer has the 5 ′ end portion in the nucleotide sequence of the complementary strand in the amplification target region. It can have the arrangement of
  • the method for detecting the gene marker includes: A method known as TaqMan PCR method is used.
  • the probe specifically hybridizes to the gene marker according to the present invention, and the fluorescently labeled substance is at the 5 ′ end.
  • a probe (TaqMan probe) in which a quencher (quenching substance) for the fluorescent label is attached to the 3 'end and the 3' end is phosphorylated is used in the PCR reaction solution.
  • the nucleic acid molecule according to the present invention can be used, and the specific nucleotide sequence thereof can be appropriately determined by those skilled in the art.
  • the fluorescent labeling substance, the quencher, and the combination thereof can use those known to those skilled in the art.
  • the fluorescent labeling substance includes, for example, FAM, VIC, etc.
  • the quencher includes MGB, TAMRA etc.
  • the released fluorescent labeling substance Since the released fluorescent labeling substance is not affected by quenching, it emits fluorescence. Therefore, according to this method, each fluorescence having an intensity corresponding to the expression level of the gene marker can be observed, whereby the expression level of the gene marker can be easily determined.
  • kits comprising at least the nucleic acid molecule and Z or a primer pair.
  • the CMP gene or polynucleotide functionally equivalent thereto in the present invention can be used to suppress or induce myocardial differentiation of cells by controlling its expression in cells.
  • RNAi RNA interference
  • RNA interference can suppress or induce myocardial differentiation of cells.
  • RNAi suppresses the expression of a polynucleotide comprising CMP1-6, CMP8-10, CMP13, CMP18, CMP20 or CMP24 or a polynucleotide functionally equivalent thereto.
  • a cell myocardial differentiation inhibitor comprising a double-stranded RNA molecule as an active ingredient, wherein the antisense strand of the double-stranded RNA molecule specifically hybridizes with the above-mentioned polynucleotide in the cell.
  • a myocardial differentiation inhibitor comprising a sequence is provided.
  • CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 expression levels negatively correlated with increased myocardial differentiation activity of cells, and the expression of these genes was suppressed by RNAi. In this case, cell myocardial differentiation can be induced. Therefore, according to one embodiment of the present invention, a double strand capable of suppressing the expression of a polynucleotide consisting of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 or a polynucleotide functionally equivalent thereto by RN Ai.
  • a cell myocardial differentiation inducer comprising an RNA molecule as an active ingredient, wherein the antisense strand of a double-stranded RNA molecule comprises a nucleotide sequence that specifically hybridizes with the polynucleotide in the cell
  • An agent for inducing myocardial differentiation is provided.
  • RNAi refers to a phenomenon in which double-stranded RNA molecules suppress their expression by degrading target RNA containing the same sequence.
  • the above-mentioned double-stranded RNA molecule refers to an RNA molecule obtained by hybridizing over the whole or part of two single-stranded RNA molecular forces.
  • a nucleotide chain having a sequence homologous to the target sequence is referred to as a sense strand! /
  • a nucleotide chain having a sequence complementary to the target sequence is referred to as an antisense strand.
  • “specifically hybridize” means that the CMP gene, a polynucleotide functionally equivalent thereto, and a polynucleotide that is functionally equivalent to the above-mentioned antisense strand strength CMP gene or a polynucleotide functionally equivalent thereto. It means that it does not hybridize to nucleic acid molecules other than these transcripts.
  • Such an antisense strand shall contain a sequence corresponding to the target sequence in the CMP gene or a functionally equivalent polynucleotide, but must have a sequence that is completely complementary to the target sequence. In short, as long as it specifically hybridizes to the target sequence, it may contain nucleotides that are not complementary.
  • the antisense strand in the myocardial differentiation inhibitor is preferably provided with a sequence power complementary to the target sequence.
  • the double-stranded RNA molecule may be composed of different strands, and may be provided by a single RNA stem loop structure. Further, the chain length of the single-stranded RNA molecule in the double-stranded RNA molecule has at least 19 residues, preferably 19 to 27 residues, in view of obtaining an appropriate RNAi effect.
  • the sense strand and the antisense strand may have the same chain length, but by providing an overhang of about 2 bases on the 3 ′ side of each strand, it is possible to enhance the gene expression suppression effect. it can.
  • the double-stranded RNA molecule can be obtained by, for example, a known method using an oligonucleotide synthesizer or the like using a sense strand homologous to a target sequence of a CMP gene or a polynucleotide functionally equivalent thereto and an antisense strand thereof. Can be created by combining and annealing each. It should be noted that a person skilled in the art can try various modifications using the known methods for the target sequence and the length and structure of the double-stranded RNA molecule as described above. However, it is possible to optimize double-stranded RNA molecules.
  • the expression levels of CMP1-6, CNP8-10, CMP13, CMP18, CMP20, and CMP24 positively correlate with an increase in cell myocardial differentiation activity.
  • myocardial differentiation comprising CMP1-6, CNP8-10, CMP13, CMP18, CMP20 and CMP24 or a functionally equivalent polynucleotide thereof in a functional form An inducer is provided.
  • the expression level of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 has a negative correlation with an increase in myocardial differentiation activity of cells.
  • a myocardial differentiation inhibitor comprising a functionally capable of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 or a functionally equivalent polynucleotide.
  • the myocardial differentiation controlling agent that is, myocardial differentiation inducer or inhibitor
  • examples of the myocardial differentiation controlling agent include, for example, a vector into which the polynucleotide is inserted in a functional manner.
  • “comprising functionally” means that the polynucleotide (DNA) can be expressed under the control of appropriate regulatory elements (eg, promoter, enhancer, transcription terminator, etc.). Means that the polynucleotide is inserted into the vector.
  • the above vector can be constructed by standard methods well known in the art. For example, Sam Brook, J. et al., “Molecular loning: a laboratory manual”, Cold 3 ⁇ 4pn ng. Can be constructed as described in Harbor Laboratory Press, New York (1989)
  • the gene marker according to the present invention is used as an index of myocardial differentiation activity of cells. Therefore, according to another aspect of the present invention, there is provided a method for detecting myocardial differentiation activity of a cell, comprising measuring the expression level of the polypeptide marker or gene marker in the cell. A method is provided wherein the expression level correlates with the level of cardiac muscle activity of the cells.
  • cells of CMP1-6, CMP8-10, CMP13, CMP18, CMP20 or CMP24, polynucleotides functionally equivalent thereto, or markers of these gene product potentials The expression level is higher than a preset threshold value with reference to the expression level of the marker in a control cell having no myocardial differentiation activity. Methods are provided that have cardiac muscle activity. According to another preferred embodiment of the present invention, the expression level in cells of CMP7, CMP12, CMP16, CMP17, CMP21 or CMP23, a functionally equivalent polynucleotide, or a marker of these gene products is determined. taking measurement And when the expression level is lower than a preset threshold value with reference to the highest level of expression level in a control cell that does not have myocardial differentiation activity, the cell has myocardial differentiation activity. Is provided.
  • the expression level of the marker can be measured, for example, by collecting a biological sample derived from a cell, extracting a nucleic acid sample such as genomic DNA or mRNA from the obtained sample, CDNA is prepared by reverse transcriptase, the obtained nucleic acid sample is used as a saddle, nucleic acid amplification method is performed using the nucleic acid molecule or primer pair according to the present invention, and the expression level of the obtained amplification product is analyzed. This can be done by quantifying.
  • a nucleic acid amplification method and a marker detection method using the nucleic acid amplification method a method of! /, which is known in the art, may be used.
  • a normal PCR method or the like can be used when genomic DNA or cDNA is in a vertical shape
  • an RT-PCR method, NASBA method, or the like is used when an mRNA is in a vertical shape.
  • the threshold value of the expression level of the marker in the control cell can be set by a known statistical method with reference to the quantitative value of the marker measured in advance by the above method.
  • Specific examples of the method for setting the expression level include (average value standard deviation of marker expression level in control cells) or ROC (Receiver Operating Characteristic) analysis.
  • the above detection method can be used for efficient and simple isolation of cells having myocardial differentiation activity. Therefore, according to another preferred embodiment of the present invention, there is provided a method for isolating cells having myocardial differentiation activity, wherein cells having myocardial differentiation activity are detected and isolated by the above detection method. There is provided a method comprising: Cells isolated by such a method can be advantageously used as transplant cells for the treatment of refractory heart diseases such as cardiomyopathy and ischemic heart disease.
  • the isolation step is appropriately performed under culture conditions known to those skilled in the art.
  • a method for isolating the cells for example, a method of isolating cells by flow cytometry using an antibody against the marker (FACS), or a magnetic bead carrying an antibody, a specific bead is used. Examples include a method of collecting and isolating cells with a magnet (MACS).
  • FACS antibody against the marker
  • MCS magnetic bead carrying an antibody
  • a method for monitoring the myocardial differentiation activity of a cell comprising CMP1-6, CMP8-10, CMP13, CMP18CMP20 or CMP24, functionally equivalent thereto. Measuring the expression level of polynucleotides or markers of these gene products in cells, wherein the expression level of the markers increases with time, and the myocardial differentiation activity of the cells is increased. Is provided. According to another preferred aspect of the present invention, there is provided a method for monitoring myocardial differentiation activity of a cell, comprising:
  • myocardial differentiation of cells can be induced or suppressed. Therefore, according to another aspect of the present invention, there is provided a method for inducing myocardial differentiation of a cell, comprising introducing the myocardial differentiation inducing agent according to the present invention into a cell. Further, according to another aspect of the present invention, there is provided a method for suppressing cardiac muscle myocardial differentiation, comprising introducing a myocardial differentiation inhibitor according to the present invention into a cell. .
  • a method for introducing the myocardial differentiation controlling agent a method known in the art as a transfection or transformation method can be used.
  • the calcium phosphate method the electoral position method Method, microinjection method, DEAE-dextran method, method using a ribosome reagent, lipofusion method using a cationic lipid, and transfusion using a viral vector.
  • a method for controlling myocardial differentiation also includes an embodiment in which the gene product of the polynucleotide constituting the gene marker according to the present invention may be directly introduced into a cell, and the present invention includes a profitable embodiment. .
  • Mouse-derived fetal cancer cells P19 cells were donated by the American Type Culture Collection (ATCC).
  • the sub-line CL6 cells (RCB number: RCB1539) were provided by the RIKEN BioResource Center (RIKEN Cell Bank).
  • CL6 cells were subjected to 5′-directional force by using pcDNA3.1 (+) (Invitrogen), a vector having a neomycin resistance gene, mouse a MHC promoter, EGFP gene (Clontech) and human growth hormone poly A signal.
  • FCS fetal calf serum
  • FCS fetal calf serum
  • MEM medium Minimum Essential Medium, SIGMA
  • SIGMA penicillin G
  • streptomycin sulfate lOOu nit / mL
  • the undifferentiated cells were cultured and grown to about 60 to 70% of the dish area to induce differentiation into cardiomyocytes.
  • the basic medium was first removed and washed twice with 5 mL of sterilized phosphate-buffered saline (FBS, SIGMA).
  • FBS sterilized phosphate-buffered saline
  • G3 ⁇ 4co trypsin-EDTA
  • it was suspended in 10 mL of differentiation medium (( ⁇ medium containing 1% DMSO).
  • the cell suspension was dispensed into a cell culture vessel (Falcon) to obtain differentiation-induced cells. Furthermore, the obtained cells were seeded at a density of IX 10 5 cells / well in a multiwell plate for 6-well cell culture (Costar). This was cultured in a 37 ° C differentiation medium in the presence of 5% C02, and the medium was changed every two days.
  • the appearance date (contraction start date), size and number of contracting nodules were observed every 2 days with a CKX41 or CKX31 culture microscope (OLYMPUS) to evaluate differentiation efficiency.
  • the number of shrinkage nodules is 0.016 / cm 2 or more (when there is more than one shrinkage per plate), 0.098 / cm 2 or more (one well, more than one shrinkage) Defined as “medium” and 157 / cm 2 or more (when there is one or more contractions in one field of view of the microscope (x2 00)).
  • shrinkage nodule size 6.36 X 10- 5 cm 2 or more of a size (contracted to 1/100 or more size punctate microscopic field of view area of (X 200), Ru ones) the "small! /,", 3.18 X 10- 3 cm 2 or more the size of those (microscope field area of (X 200) of about 1/2 or more of those contracts to the size of a sheet-like) was defined as “large”.
  • a score of 0 was given to the “no” shrinkage, 1 to the “small”, and 2 to the “large”.
  • FIG. 1A The result of the number of contraction nodules as an index is as shown in Fig. 1A.
  • the results using the size of the light as an index are shown in Figure 1B.
  • the horizontal axis indicates the number of days after induction of myocardial differentiation. From the results of FIGS. 1A and B, it was confirmed that there was a difference in the start date of contraction and the number and size of contraction nodules depending on the cell line.
  • P19 cells become confluent on day 6 after induction of differentiation, and on day 16, one or more small contractions are observed for each well of the plate, with at least 20 contractions. It was observed to continue until day one.
  • P19 cells are cultured in the presence of DMSO for a relatively long period of time, compared to CL6 cells, so that they do not form embryoid bodies as in CL6 cells. Confirmed to do.
  • FIGS. 2A to 2G The results were as shown in FIGS. 2A to 2G.
  • the vertical axis represents relative values when mRNA / 18S rRNA values are compared with adult mouse myocardium.
  • cardiomyocyte marker gene expression in relation to cardiomyocyte differentiation, it is not clear how much weight should be given to the expression of individual cardiomyocyte marker genes. Therefore, principal component analysis was performed based on the mRNA expression data of the above cardiomyocyte marker gene obtained for cell lines Pl, CL6, G26, G36, G45 and G52. Principal component analysis was performed using statistical software SYSTAT (SYSTAT Software). The mRNA expression level of the cardiomyocyte marker gene was standardized based on the average value and standard deviation of the samples.
  • variable plots for the two principal components were as shown in FIG. 3B.
  • the first principal component was interpreted as an index of myocardial differentiation because the coefficient values of all variables were positive.
  • the coefficient of the cardiomyocyte marker gene functioning relatively early in the development was negative, and the coefficient of the cardiomyocyte marker gene functioning relatively late in the development was positive. Therefore, it was interpreted as an indicator of maturity.
  • FIGS. 4A and 4B the changes over time of the main component scores of each cell line were as shown in FIGS. 4A and 4B. It was confirmed that there were differences in the state of differentiation into cardiomyocytes depending on the type of cell line.
  • the vertical axis represents the first principal component score.
  • the vertical axis represents the second principal component score.
  • RNA was extracted using RNeasy Midi (QIAGEN) with careful attention not to confluent the cells before induction of differentiation.
  • the obtained RNA was tanned up twice using RNeasy Mini (QIAGEN).
  • GeneChip AfiVmetrix
  • synthesize cDNA from each RNA sample, and then synthesize biotinylated cRNA fragment based on the cDNA. did.
  • the biotinylated cRNA fragment was hybridized to GeneChip using GeneChip Hybridizatioin Oven, and the hybridized cRNA was visualized by staining with Streptavidin-Phycoerythrin using GeneChip Fluidics Station, and GeneChip Scanner 3000 Scanned.
  • the obtained fluorescence data was analyzed using GCOS software (Aifymetrix).
  • GeneChip used MOE430A (Sequence Tag number 22,626) and MOE430B (Sequence Tag number 22,511).
  • the extracted gene force was also corrected so that the average value of the remaining Sequence Tag was 500 by default, excluding 2% from the higher signal and 2% from the lower signal.
  • the following filters were applied to extract genes correlated with cardiomyocyte differentiation of each cell line.
  • the signal of each Sequence Tag analyzed by GCOS is the result of Absolute Analysis (analysis to determine the presence or absence of expression) ⁇ Those with expression: P (Present) j, ⁇ Those with expression or not: M (Marginal or Judgment is made as “No expression: A (Absent).”
  • Cell More than half of the 5 strains in each group ie 3 or more) have a sequence tag that is determined to be P! / And if the gene containing the sequence sequence of the sequence tag is expressed in the cell line Judged. On the contrary, it was determined that there was no expression of the gene containing the nucleotide sequence of the Sequence Tag in the cell line in the case where the P determined among the 5 cases in each group was less than 3 cases.
  • Sequence tags that are expressed in at least one of the cell lines were subjected to the following filter, and sequence tags that were not expressed in all cell lines were discarded.
  • Sequence tags that have an average difference in gene expression between cell lines of 50% or more, that is, the difference between the lowest average value of the 6 cell lines and the highest average value is more than 2.5 times. Those less than 2.5 times were rejected.
  • the expression signal of the above-mentioned Sequence Tag before differentiation induction obtained with GeneChip, the number of days until the appearance of automatic pulsation ability, and the score of the number of cell nodules that automatically pulsate Calculate Spearman's rank correlation number between the first principal component score or the second principal component score of myocardial gene expression data obtained by quantitative RT-PCR, and a significant difference under the condition of 5% significance level And a Sequence Tag with a significant correlation was extracted.
  • sequence tags correlated with the first principal component 342 sequence tags correlated with the second principal component, correlated with the number of days until the appearance of automatic pulsation ability 122 Sequence Tags were extracted, and 274 Sequence Tags correlated with the number of contraction nodules were extracted.
  • sequence tags there were a total of 24 sequence tags that significantly correlated with the three elements of the first principal component, which is an index of myocardial differentiation, the number of days until the appearance of automatic pulsatile capacity, and the number of contractile nodules.
  • the genes corresponding to these 24 Sequence Tags are designated as CMP1-24. Named.
  • the first principal component (indicator of cardiomyocyte differentiation) and the second principal component (indicator of cardiomyocyte maturation) are significantly correlated with the four elements of the number of days until the appearance of automatic pulsation and the number of contractile nodules. There were 9 genes.
  • Table 2 shows the results of the statistical analysis and functional analysis by the SOSUI system.
  • rs is Spearman's rank correlation coefficient
  • p is the P-value of Spearman's rank correlation coefficient.
  • the genes indicated by * were found to have a significant correlation with the first principal component, the second principal component, the number of days until the appearance of automatic pulsatile activity and the number of contractile nodules. It is.
  • CMP2 and CMP13 were set as RNAi targets.
  • StealthRNAi (Invitrogen) having the nucleotide sequence shown in Table 3 was introduced into G52 cells by the lipofusion method.
  • Reverse primer 5, -TAGTGGCAACCATGCTGAGTGT-3 (SEQ ID NO: 73)
  • Taqman probe 5,-CCGATGGACGTGAGGACAGTAATCTGGA-3 (SEQ ID NO: 74)
  • G52 cells were seeded on a 24-well cell culture multi-well plate (Falcon) at 1 X 104 cells / well without differentiation induction treatment. This was cultured at 37 ° C and 5% C02. Each cell in the well was washed twice with 500 ⁇ L of basic medium without antibiotics, and 400 L of the same medium was added to each well.
  • siRNAs having the nucleotide sequences described in Tables 4 to 5 below obtained using Stealth RNAi (Invitrogen) or siRNA (QIAGEN) were added to each of the wells using Lipofectamine 2000 (Invit rogen). Introduced into cells, CMP1-24 were knocked down.
  • OPTI- MEMI Reduced Serum Medium containing B LOCK-iT (Invitrogen) (final concentration 10 pmol / well) as a fluorescent oligo so that the siRNA force per well is 0 pmol.
  • the siRNA was diluted with Gibco) to obtain a siRNA solution.
  • Stealth RNAi Negative Control Duplexes (Invitrogen), which is a negative control (NC) was also diluted in the same manner. Thereafter, the mixture was diluted with OPTI-MEMI Reduced Serum Medium so that the Lipofectamine 2000 force was 1.5 ⁇ L / well, and incubated at room temperature for 5 minutes to obtain a Lipofectamine 2000 solution.
  • siRNA solution or negative control solution and 50 L of Lipofectamine 2000 solution were mixed and incubated at room temperature for 20 minutes to form a complex. 100 L of this complex was mixed in each well, mixed while gently shaking, and incubated at 37 ° C for 48 hours under 5% CO 2.
  • undifferentiated CL6G52 was cultured in a cell culture dish (Falcon) having a diameter of 100 mm so as not to become confluent, and total RNA extracted from the cells using BioRobot M48 Workstation (QIAGEN) was used. It was diluted to 20ng / 1, 6ng / 1, 2ng / ⁇ 1, 0.6ng / ⁇ 1, 0.2ng / ⁇ 1, 0.06ng / ⁇ 1 and used to create a calibration curve.
  • 18S rRNA was measured as an internal standard. 1 In the 8S rRNA measurements were diluted each measurement sample 0.02 ⁇ ⁇ / L, a sample for calibration curve 0.2ng I [ ⁇ , 0.06ng I [ ⁇ , 0.02ng / ⁇ 1, 0.006g / ⁇ 1, 0.002 ng / ⁇ 1, and 0.0006 ng / ⁇ 1.
  • the PCR primers and Taqman probe used in the above quantitative RT-PCR are as shown in Table 6 and Table 7.
  • FAM was used as the fluorescent labeling substance at the 5 ′ end
  • TAMRA was used as the quencher at the 3 ′ end.
  • the expression level of the existing cardiomyocyte marker genes was measured by quantitative RT-PCR.
  • the expression levels of CMP1-10, CMP12-13, CMP16-18, CMP20-21 and CMP23-24 were compared.
  • Quantitative RT-PCR was performed in the same manner as in Example 2 using the primers shown in Table 1 and TaqMan probe.
  • RNAi Shows that it was significantly knocked down by RNAi (p ⁇ 0.05 vs. NC).
  • any cardiomyocyte marker gene (a MHC, ⁇ MHC, MLC2a or MLC2v) is promoted by RNAi knockdown of CMP7, CMP12, CMP16, CMP17, CMP21, or CMP23, or contraction nodules
  • CMP23 the number of contractile nodules increased and the cardiomyocyte marker remains.
  • MLC2a decreased significantly. Since MLC2a, which characterizes atrial muscle, decreased and MLC 2v, which characterizes ventricular muscle, increased, it was confirmed that CMP23 can be used for specific induction of cells into ventricular muscle.

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Abstract

Le marqueur faisant l'objet de cette invention contient un gène d'élément de prédiction de la cardiomyogenèse (PCM), ou un produit génique dudit gène, démontrant une corrélation avec l'activité de différentiation myocardique d'une cellule.
PCT/JP2007/058107 2006-04-12 2007-04-12 Marqueur permettant de détecter l'activité de différentiation myocardique d'une cellule WO2007119799A1 (fr)

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