JP2015008686A - Method for evaluating state of liver cell or cell derived from progenitor cell thereof, and probe or probe set and micro array used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating a state of a liver cell or a cell derived from a progenitor cell of the liver cell which is used in various types of experiment, evaluation and the like.SOLUTION: An evaluation method according to the present invention is a method for evaluating a state of a liver cell or a cell derived from a progenitor cell of the liver cell, which includes a step of measuring a gene expression amount a liver cell or a cell derived from a progenitor cell of the liver cell which are test subjects by using a probe or a probe set including nucleic acid consisting of a base sequence, which constitutes at least one type of gene selected from the group consisting of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9 and the like, or a part of the nucleic acid, for example.

Description

  The present invention relates to a method for evaluating the state of a cell derived from a liver cell or a precursor cell thereof, and a probe or probe set for evaluating the state of a cell derived from a liver cell or a precursor cell thereof that can be used in the method And a microarray and the like.

  The liver performs a number of vital functions of the body that regulate, synthesize and secrete many substances important for maintaining normal state; glycogen (glucose), Including the storage of vital nutrients such as vitamins and minerals; and the purification, conversion and removal of waste products, drugs and toxins. Hepatocytes (liver parenchymal cells) are responsible for most of these functions in the liver.

  In drug discovery, liver cells are often used for evaluation, but normal hepatocytes rarely divide and proliferate, and the cells used are hepatocytes extracted by surgery, Hepatocytes differentiated from progenitor cells and stem cells such as established cells and fetal cells are used. At that time, the closer the cells to be used are to the liver of the living body, the higher the accuracy as the result of the experiment, so the function as a hepatocyte may be evaluated. The evaluation method has generally evaluated the status of hepatocytes and the superiority or inferiority of differentiation using the activities of some enzymes related to liver function (Patent Document 1), cell surface markers, etc. (Patent Document 2). . For example, when assessing whether differentiated cells can be used for the evaluation of drug metabolism, it is almost always measured only the activity of some proteins of the CYP (P450) family involved in drug metabolism. In liver parenchymal cells, various mechanisms are involved, such as drug uptake, the action of enzymes other than P450, and excretion of metabolized drugs. In recent years, a microarray for detecting genes related to the above-described liver function has also been reported (Patent Document 3).

  However, the functions of the liver (hepatocytes) include functions such as hematopoiesis, sugar metabolism, lipid metabolism, and bile acid synthesis in addition to the functions related to drug metabolism described above, and cells other than liver parenchymal cells such as bile ducts Therefore, in the evaluation method using the conventional methods and means, how much the cell to be evaluated (cell derived from hepatocytes etc.) is a cell that retains the function as a hepatocyte (for example, which cell It is not sufficient to judge whether the state is functionally close to hepatocytes in vivo.

  Also, when looking at the effects of substances (drugs, drugs, etc.) on hepatocytes, conventionally only the effects on drug metabolizing enzymes have been confirmed, and functions other than those related to drug metabolism as described above. No effects on hepatocytes have been confirmed.

Special table 2009-542215 Special Table 2004-531270 JP 2004-135552 JP

  Under such circumstances, for example, a method for evaluating the state of a cell derived from a liver cell or a precursor cell thereof used in various experiments and evaluations in drug discovery, for example, the cell is a liver cell. Therefore, it has been desired to develop a method for objectively and appropriately evaluating whether or not the function is sufficiently retained.

  The present invention has been made in consideration of the above situation, and the following probes or probe sets and microarrays for evaluating the state of cells derived from liver cells or their progenitor cells, and the probes or probes. A method for evaluating the state of a liver cell or a progenitor cell-derived cell using a set or a microarray, and a method for screening a substance useful for maintaining a function as a hepatocyte in the liver cell or a progenitor cell-derived cell, etc. It is.

That is, the present invention is as follows.
(1) A probe or probe set for evaluating the state of a liver cell or a progenitor cell-derived cell containing the nucleic acid of the following (a), (b) or (c) or a part thereof.
(A) CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, AHR, NR1I2, CASR, ESR, NR3C1, ABCA1, CC4, CCB2, CCB1, CC4 , ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A7, SLC17A1, SLC22A1, SLC22ALC, SLC22A2, SLC22A2 , SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A1, SLC47A2, SLCO1A2, SLCO1B1, SLCO1B3, SLCO2A1, SLCO2B1, SLCO4C1, UGT2B4, UGT2B, UGT2T, UG2B15, UGT2A , CES5, GSTM1, GSTT1, GSTA1, SULT1A1, SULT1A3, SULT1A4, SULT1B1, SULT1C2, SULT1C4, SULT1E1, NAT1, ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO1, TP1, C2 , TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, FABP6, SREBF1, SREBF2, PPARA, LDLR, SCARB1, NPC1 L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, HNF1A, CYP3A7, POU5F1, EP, PAY, PAY5, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP, PTPRC, TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC, PDGFB, FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, Nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of JUN, REL, ETS1, BCL2, BCL2L1 and SRC (b) Nucleic acid comprising a base sequence complementary to the nucleic acid of (a) (C) hybridizes with a nucleic acid comprising a base sequence complementary to the nucleic acid (a) or (b) under stringent conditions, and Nucleic acid capable of detecting gene expression of progenitor cells

Here, in the probe set of (1), the nucleic acid of (a) may be a combination of two or more groups of nucleic acids selected from the following groups (i) to (viii), for example. Further, it may be a combination of nucleic acids of the following groups (i) to (viii) (a combination of all the groups).
(i) a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5
(ii) a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of AHR, NR1I2, CASR, ESR and NR3C1
(iii) ABCA1, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC16, LC1 SLC22A12, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC28A1, SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A, SLC47A1, SLC47A, SLC47A1, SLC47A A nucleic acid comprising a base sequence constituting at least one gene
(iv) UGT2B4, UGT2B10, UGT2B15, UGT2B7, UGT2B17, UGT1A1, UGT1A6, UGT1A4, UGT1A9, UGT1A3, CES1, CES2, CES3, CES5, GSTM1, GSTT1, ASTA1, ULT1A1, SULT1A1, SULT1A1S And a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of NAT1
(v) ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO2, CPS1, PCK2, OTC, ASS1, HNF4A, GSTP1, TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, RABP6, SREBF1, PP , LDLR, SCARB1, NPC1L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, and HNF1A Nucleic acid consisting of the base sequence constituting the gene
(vi) Selected from the group consisting of CYP3A7, POU5F1, AFP, THY1, EPCAM, DLK1, CEBPA, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP and PTPRC A nucleic acid comprising a base sequence constituting at least one gene
(vii) From a base sequence constituting at least one gene selected from the group consisting of TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC and PDGFB Nucleic acid
(viii) FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, JUN, REL, ETS1 , A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of BCL2, BCL2L1 and SRC

(2) A probe or probe set for evaluating the state of a liver cell or a progenitor cell-derived cell thereof containing the following nucleic acid (α), (β), (γ) or (δ) or a part thereof.
(Α) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 193 (β) Nucleic acid consisting of a base sequence complementary to the nucleic acid of (α) (γ) Said (α ) Or (β) a nucleic acid comprising a nucleotide sequence having a homology of 70% or more with respect to the nucleic acid sequence, and a nucleic acid capable of detecting a liver cell or its precursor cell expression gene (δ) (α), A nucleic acid comprising a base sequence in which 1 to several bases are added, deleted or substituted in the base sequence of the nucleic acid of (β) or (γ), and capable of detecting a gene expressed in liver cells or their precursor cells

Here, in the probe set of (2), the nucleic acid of (α) may be composed of a combination of two or more groups of nucleic acids selected from the following groups (I) to (VIII), for example. Further, it may be a combination of nucleic acids of the following groups (I) to (VIII) (a combination of all the groups).
The probe set according to claim 1, wherein the nucleic acid (α) is a combination of two or more groups of nucleic acids selected from the following groups (I) to (VIII).
(I) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 10
(II) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 11 to 15
(III) Nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 61
(IV) Nucleic acid comprising at least one base sequence of the base sequences shown in SEQ ID NOs: 62 to 86
(V) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 87 to 130 (VI) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 131 to 151 (VII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 152 to 166 (VIII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 167 to 193

(3) A nucleic acid microarray for evaluating the state of a liver cell or a progenitor cell-derived cell comprising the probe or probe set of (1) or (2) above.

(4) using the probe or probe set of (1) or (2) or the nucleic acid microarray of (3) above, the step of measuring the gene expression level of a liver cell or a progenitor cell thereof to be tested ,
Here, the method of (4) above may be, for example, a method comprising a step of bringing a test substance into contact with a liver cell or a precursor cell derived from the liver cell to be tested in advance, and the measured gene expression level The method may include a step of evaluating the function as a liver cell using the result of multivariate analysis based on as an index.

(5) Using the probe or probe set of (1) or (2) above or the nucleic acid microarray of (3) above, screening for a substance useful for maintaining the function as a liver cell in a liver cell or a precursor cell-derived cell thereof Method.

  According to the present invention, the state of a cell derived from a liver cell or a precursor cell thereof used in various experiments and evaluations is evaluated. Specifically, for example, the cell sufficiently functions as a liver cell. It is possible to provide a method for objectively and appropriately evaluating whether or not the cells are held, and for evaluating the influence of the cells on various substances such as drugs. Moreover, according to this invention, the probe or probe set used for the said evaluation method, and a microarray can be provided. Furthermore, by using these probes or probe sets, and microarrays, it is possible to provide a method for screening a substance useful for maintaining the function as a hepatocyte in a liver cell or a precursor cell-derived cell thereof.

It is the schematic which shows the arrangement | sequence fixing jig for hollow fiber bundle (hollow fiber array body) manufacture. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example. It is a figure which shows each gene expression level of each cell used in the present Example.

2A to 2H, in each graph showing the measurement results for the probes (SEQ ID NOs: 1 to 193), the vertical axis represents Intensity (Cy5 fluorescence intensity), and the horizontal axis represents In order from the left end, the experiment Nos. The measurement result regarding A1-1, A1-2, A1-3, A3-1 (2), A3-2 (2), B1-1, B8-1, C-1, and D-1 is shown.
2A is a probe consisting of the base sequence of SEQ ID NO: 1 to 10, FIG. 2B is a probe consisting of the base sequence of SEQ ID NO: 11 to 15, FIG. 2C-1 is a probe consisting of the base sequence of SEQ ID NO: 16 to 55, 2C-2 is a probe consisting of the base sequence of SEQ ID NOs: 56 to 61, FIG. 2D is a probe consisting of the base sequence of SEQ ID NOs: 62 to 86, FIG. 2E-1 is a probe consisting of the base sequence of SEQ ID NOs: 87 to 126, and FIG. -2 is a probe consisting of the base sequence of SEQ ID NOs: 127 to 130, FIG. 2F is a probe consisting of the base sequence of SEQ ID NOs: 131 to 150, FIG. 2G is a probe consisting of the base sequence of SEQ ID NOs: 151 to 166, and FIG. Each graph showing the measurement results for probes having a base sequence of 167 to 193 is listed.

Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.
In this specification, all publications cited in this specification, for example, prior art documents, and publications, patent publications, and other patent documents are incorporated herein by reference.

1. Summary of the InventionIn the present invention, evaluating the state of a liver cell or a precursor cell-derived cell thereof, measuring the expression level of a predetermined gene extracted from a liver cell or a precursor cell-derived cell to be tested, It means to evaluate and judge the state of the cell based on the result.
Here, gene expression usually means mRNA expression. The gene expression level is the amount of mRNA, or the amount of nucleic acid amplified from DNA or mRNA, or the amount of protein. Measuring the level of gene expression is the amount of mRNA or nucleic acid amplified from DNA or mRNA. This means that the amount of protein is measured.

  In addition, the state of the cell is not particularly limited. For example, the state of the cell means (has) a function as an actual liver cell (a so-called liver cell existing in a living body). The state or the state in the sense of how much the function of the cell as a liver cell is changed compared to the state before the test substance is contacted when the test substance is contacted with the cell in advance. Liver cell functions include, for example, functions related to uptake of drugs, lipids or sugars, functions related to metabolism, excretion, hematopoiesis, bile acid synthesis, or the presence or absence of expression of proteins unique to bile ducts, stellate cells or cancerous cells, etc. Is mentioned.

In the present invention, a liver cell is a cell constituting the liver and a progenitor cell that can differentiate into the liver. For example, hepatocyte (hepatocyte), bile duct epithelial cell, stellate cell, Kupffer cell, smooth muscle cell, blood vessel Endothelial cells, mesothelial cells, bit cells, hepatoblasts, embryonic hepatocytes, liver tumor-derived cells are included.
In addition, the liver cells or progenitor cell-derived cells to be tested in the present invention include primary cells and cell lines derived from the liver of mammals (particularly humans), cells derived from these cells or genes, stem cells, Examples include ES cells and iPS cells, fetal liver cells and their progenitor cells, or cells induced to differentiate from these cell lines.

  In evaluating and judging the state of liver cells or their precursor cell-derived cells, as comparative controls, actual liver cells or their precursor cells (liver cells or their precursor cells directly collected (extracted) from the liver in vivo) Various types of liver cells or progenitor cells thereof can be used.

  In the present invention, in order to evaluate the state of a liver cell or a progenitor cell-derived cell as a test subject, for example, (i) a gene related to hydroxylation of drug metabolism, (ii) related to nuclear receptor transcription Genes, (iii) genes related to drug transporters (uptake, excretion), (iv) genes related to the second phase reaction of drug metabolism, (v) functions such as bile acid synthesis, hematopoiesis, lipid metabolism, sugar metabolism, etc. (Vi) genes related to differentiation from progenitor cells, (vii) genes related to non-parenchymal cells such as bile duct cells, (viii) genes related to cancer cells, etc. Can be used. In the present specification and the like, these genes are referred to as liver cell or precursor cell expression genes. At least one or at least two genes selected from these various genes can be selected, and a nucleic acid comprising a base sequence constituting the gene or a part thereof can be used as a probe or a probe set.

Here, examples of the liver cell or progenitor cell expression gene described above include the following.
<Liver cell or its precursor cell expression gene>
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, AHR, NR1I2, CASR, ESR, NR3C1, ABCA1, ABC4, ABC1, ABCC4, ABCB11, CC4 ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A28, SLC15A1, SLC15A2, SLC16A1, SLC16A7, SLC17A1, SLC22A1, SLC22A12, SLC22S, LC22A3, SLC22A3 SLC29A1, SLC29A2, SLC31A1, SLC47A1, SLC47A2, SLCO1A2, SLCO1B1, SLCO1B3, SLCO2A1, SLCO2B1, SLCO4C1, UGT2B4, UGT2B10, UGT2B15, UGT2B17, UGT2B17, TGT GSTM1, GSTT1, GSTA1, SULT1A1, SULT1A3, SULT1A4, SULT1B1, SULT1C2, SULT1C4, SULT1E1, NAT1, ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO2, CPS1, TP1, CPS1, C NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, FABP6, SREBF1, SREBF2, PPARA, LDLR, SCARB1, NPC1L1, SLC2 7A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, HNF1A, CYP3A7, POU5F1, AFP, THY1, EPCAM, PA1, PAY CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP, PTPRC, TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC, PDGFB, FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, JUN, REL, ETS1, BCL2, BCL2L1, SRC

  The nucleotide sequence information of each of these genes can be obtained from NCBI (National Center for Biotechnology Information Search term Search database). As the notation of each gene name, the official symbol of NCBI is written in capital letters, but this does not limit the species. For example, genes of various mammals such as mice, rats, hamsters, pigs, guinea pigs, monkeys, dogs, cats, etc. can be used in addition to humans.

2. The probe or probe set probe generally captures a nucleic acid (mRNA or cDNA or cRNA, or an antisense strand thereof (aRNA or aDNA)) of a target gene in a specimen (test sample) by hybridization, The term used for detecting a target nucleic acid. The probe is usually a nucleic acid probe, but as a “nucleic acid” constituting the probe, DNA, RNA, PNA or the like can be generally used, and DNA is particularly preferable.
In the present invention, examples of probes (or probe sets) for evaluating the state of liver cells or their precursor cell-derived cells include the following nucleic acids (a), (b) and (c) or a part thereof: Including.

(A) CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, AHR, NR1I2, CASR, ESR, NR3C1, ABCA1, CC4, CCB2, CCB1, CC4 , ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A7, SLC17A1, SLC22A1, SLC22ALC, SLC22A2, SLC22A2 , SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A1, SLC47A2, SLCO1A2, SLCO1B1, SLCO1B3, SLCO2A1, SLCO2B1, SLCO4C1, UGT2B4, UGT2B, UGT2T, UG2B15, UGT2A , CES5, GSTM1, GSTT1, GSTA1, SULT1A1, SULT1A3, SULT1A4, SULT1B1, SULT1C2, SULT1C4, SULT1E1, NAT1, ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO1, TP1, C2 , TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, FABP6, SREBF1, SREBF2, PPARA, LDLR, SCARB1, NPC1 L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, HNF1A, CYP3A7, POU5F1, EP, PAY, PAY5, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP, PTPRC, TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC, PDGFB, FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, Nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of JUN, REL, ETS1, BCL2, BCL2L1, SRC (b) Nucleic acid comprising a base sequence complementary to the nucleic acid of (a) (C) hybridizes with a nucleic acid comprising a base sequence complementary to the nucleic acid of (a) or (b) under stringent conditions, and a liver cell or its Nucleic acid capable of detecting progenitor cell expression gene

  Here, the nucleic acid (a gene expressing liver cells or progenitor cells thereof) of (a) can be classified as shown in the following groups (i) to (viii) based on the function and type of the gene. In the present invention, the nucleic acid (a) is preferably a nucleic acid comprising a combination of two or more groups of nucleic acids selected from the following groups (i) to (viii). Those consisting of a combination of all nucleic acids of viii) are more preferred.

(i) constitute at least one gene selected from the group consisting of genes related to hydroxylation of drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) Nucleic acid consisting of base sequence
(ii) Nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of genes related to nuclear receptor transcription (AHR, NR1I2, CASR, ESR, and NR3C1)
(iii) Genes related to drug transporters (uptake, excretion) (ABCA1, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A7, SLC17A1, SLC22A1, SLC22A12, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC28A1, SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A1, SLC47A2, SLCO1A2, SLCO1B1, A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of SLCO1B3, SLCO2A1, SLCO2B1 and SLCO4C1)

(iv) Genes related to the second phase reaction of drug metabolism (UGT2B4, UGT2B10, UGT2B15, UGT2B7, UGT2B17, UGT1A1, UGT1A6, UGT1A4, UGT1A9, UGT1A3, CES1, CES2, CES3, CES5, GSTM1, GSTM1, GSTM1, TT , SULT1A3, SULT1A4, SULT1B1, SULT1C2, SULT1C4, SULT1E1 and NAT1) nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of
(v) Genes related to functions such as bile acid synthesis, hematopoiesis, lipid metabolism, sugar metabolism (ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO2, CPS1, PCK2, OTC, ASS1, HNF4A, GSTP1, TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, FABP6, SREBF1, SREBF2, PPARA, LDLR, SCARB1, NPC1L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC3, APOC3, AGPTL, APOC3, ANGTTL A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of GSK3A, GSK3B, FOXO1, COMT and HNF1)
(vi) Genes related to differentiation from progenitor cells (CYP3A7, POU5F1, AFP, THY1, EPCAM, DLK1, CEBPA, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of ITGB1, ANPEP and PTPRC)

(vii) Selected from the group consisting of genes related to non-parenchymal cells such as bile duct cells (TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC and PDGFB) Nucleic acid comprising a base sequence constituting at least one gene
(viii) Genes related to cancer cells (FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of FOS, JUN, REL, ETS1, BCL2, BCL2L1 and SRC)

Moreover, the nucleic acid of said (b) can use the state of the liver cell in this invention or its precursor cell origin cell for evaluation etc. similarly to the nucleic acid of said (a). Regarding the nucleic acid of (b) above, the explanation regarding the nucleic acid of (a) described above can be similarly applied except that it consists of the base sequence of the complementary strand of the nucleic acid of (a) above.
Furthermore, the nucleic acid of the above (c) can also be used for evaluating the state of the liver cell or its precursor cell-derived cell in the present invention, like the nucleic acid of the above (a) and (b).

  Here, in the nucleic acid of (c) above, the “nucleic acid hybridizing under stringent conditions” means, for example, a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid of (a) or (b). A nucleic acid obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or a part of the probe as a probe. Examples of hybridization methods include "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997". Can be used.

  The “stringent conditions” may be any of low stringent conditions, medium stringent conditions, and high stringent conditions. Examples of “low stringent conditions” include conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. Examples of “medium stringent conditions” include conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. Examples of “high stringent conditions” include conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 50 ° C. In the present invention, specifically, the “stringent condition” is that when the base chain length is 65 bases, the monovalent cation concentration of the buffer is 97.5-3200 mM, and the temperature is 37-80 ° C. The conditions are preferable, more preferably, the monovalent cation concentration is 97.5 to 800 mM and the temperature is 50 to 70 ° C., and the more preferable buffer is the monovalent cation concentration of 195 mM and the temperature is 65 ° C. However, the present invention is not limited to these conditions.

  Under such “stringent conditions”, nucleic acids having high homology can be efficiently obtained as the temperature is raised. However, factors affecting the stringency of hybridization may include multiple factors such as temperature, probe concentration, probe length, reaction time, ionic strength, salt concentration, and those skilled in the art will select these factors as appropriate. It is possible to achieve the same stringency.

  In the hybridization, when a commercially available kit is used, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol attached to the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. The hybridized nucleic acid can be detected.

  As nucleic acids that can hybridize in addition to the above, when calculated using homology search software such as FASTA and BLAST using default parameters, the nucleic acid base sequence of (a) above is 70% or more and 71%. 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% More than 97%, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more A nucleic acid consisting of a base sequence having homology can be mentioned.

  The homology of the nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, vol. 87, p. 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, vol. 90, p. 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al., J. Mol. Biol., Vol. 215, p. 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and word length = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

Furthermore, in the nucleic acid of (c) above, “the gene expressing liver cells or their precursor cells can be detected” means either the nucleotide sequence of the gene listed in (a) above or the nucleotide sequence of its complementary strand. Is capable of being captured by hybridization.
The nucleic acids (a), (b) and (c) are not limited to the full length of the nucleic acid, and a part of the nucleic acids can be used as a probe. Examples of the part of the nucleic acid include a nucleic acid composed of 30 to 5000 bases, a nucleic acid composed of 40 to 1000 bases, and a nucleic acid composed of 50 to 500 bases, preferably a nucleic acid composed of 60 bases to 200 bases. .

  In the present invention, the probes (or probe sets) for evaluating the state of liver cells or their precursor cell-derived cells include, for example, the following (α), (β), (γ) and (δ) The thing containing these nucleic acids is also mentioned.

(Α) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 193 (see Tables 1 and 2 below) (β) from a nucleotide sequence complementary to the nucleic acid of (α) (Γ) a nucleic acid comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid (α) or (β) and capable of detecting a gene expressed in a liver cell or its precursor cell (Δ) A nucleic acid expressed in the base sequence of the nucleic acid (α), (β) or (γ), wherein one to several bases are added, deleted or substituted, and expressed in a liver cell or its precursor cell Nucleic acids that can detect

  The base sequences shown in SEQ ID NOs: 1 to 193 listed in (α) above are base sequences corresponding to a part of the base sequence of each gene in the nucleic acid (a) described above. Therefore, the nucleic acid (α) above is also useful as a probe (or probe set) for the evaluation of the state of hepatocytes or precursor cell-derived cells in the present invention.

  The nucleic acids (α) can be classified as the following groups (I) to (VIII). In the present invention, the nucleic acid of (α) is preferably a nucleic acid comprising a combination of two or more groups of nucleic acids selected from the following groups (I) to (VIII). More preferred is a combination of all nucleic acids of VIII). Here, the base sequences indicated by the respective sequence numbers in the following groups (I) to (VIII) are bases corresponding to a part of the base sequence of each gene in the nucleic acids of the groups (i) to (viii) described above, respectively. Is an array.

(I) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 10
(II) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 11 to 15
(III) Nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 61
(IV) Nucleic acid comprising at least one base sequence of the base sequences shown in SEQ ID NOs: 62 to 86
(V) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 87 to 130 (VI) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 131 to 151 (VII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 152 to 166 (VIII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 167 to 193

In addition, the nucleic acid (β) can also be used for evaluation of the state of the liver cell or its precursor cell-derived cell in the present invention, as with the nucleic acid (α). With respect to the nucleic acid (β) above, the explanation regarding the nucleic acid (α) described above can be similarly applied except that it comprises the base sequence of the complementary strand of the nucleic acid (α).
In addition, the nucleic acid (γ) can be used for the evaluation of the state of the liver cell or its precursor cell-derived cell in the present invention, similarly to the nucleic acid (α) and (β).

  The nucleic acid (γ) is a nucleic acid having a base sequence having a homology of 70% or more with respect to the base sequence of the nucleic acid (α) or (β). More than 73%, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% More than 98%, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more A nucleic acid consisting of a base sequence is preferred. In addition, about the description regarding the homology of a base sequence, the description in the nucleic acid of (b) mentioned above can be applied similarly.

In the nucleic acid (γ) above, “the gene expressing liver cells or their precursor cells can be detected” means that the nucleotide sequence of each gene listed in (a) above or the nucleotide sequence of its complementary strand It means that either can be captured by hybridization.
Furthermore, the nucleic acid (δ) can also be used for the evaluation of the state of the liver cell or its precursor cell-derived cell in the present invention, like the nucleic acids (α), (β) and (γ).

  The nucleic acid (δ) is one to several (for example, 1 to 15, 1 to 10, or 1 to 5, or 1 to 5 in the base sequence of the nucleic acids (α), (β), and (γ), or A base sequence in which 1 to 2 bases are added, deleted or substituted. For example, the base sequence of the nucleic acid (α), (β), or (γ) above is modified with a linker base (such as poly T), or another base is inserted into part of the base sequence Or a part of the base sequence is deleted, or a part of the base sequence is replaced with another base.

As described above, for a mutant sequence in which a desired base is added, deleted or substituted, particularly a mutation-substituted nucleic acid, for example, “Moleculer cloning, A Laboratory Manual 3rd ed.” Or “Current Protocols in Molecular Biology, John” It can be prepared according to the site-specific displacement induction method described in “Wiley & Sons (1987-1997)”. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, and examples of the kit include QuickChange ^TM Site. -Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basal kit, Mutan (registered trademark) -Super Express Km Etc .: manufactured by Takara Bio Inc.).

In the nucleic acid (δ) above, the description of the nucleic acid (γ) can be similarly applied to “the gene expressing liver cells or its precursor cells can be detected”.
The nucleic acids of (α), (β), (γ), and (δ) are not limited to the full length of the nucleic acid, and a part thereof, as with the nucleic acids of (a), (b), and (c) described above. Can be used as a probe.
The method for obtaining various nucleic acids described in the present specification is not particularly limited, and can be obtained by a known genetic engineering technique or a known synthesis technique. For example, a commercially available DNA synthesizer is used. Can be obtained.

  In addition, various nucleic acids that can be used as probes may be appropriately modified, for example, those that have been terminal vinylated (acryloylated or methacryloylated) or terminal aminated, or bases that serve as linkers ( And those modified with poly-T). In addition, another base may be inserted into a part of the base sequence of various nucleic acids, a part of the base sequence may be deleted, replaced with another base, or replaced with a substance other than the base. Can also be used. Here, examples of the substance other than the base include dyes (fluorescent dyes, intercalators), quenching groups, and base crosslinking agents.

3. Nucleic acid microarray A nucleic acid microarray is obtained by immobilizing a large number of nucleic acid probes on a carrier independently at high density. The nucleic acid microarray is a system used to comprehensively analyze the expression level related to a plurality of nucleobase sequences and the sequence of a specific nucleobase sequence itself.
The nucleic acid microarray of the present invention is equipped with the above-described probe (or probe set) of the present invention. The nucleic acid microarray of the present invention is not limited as long as the probe of the present invention is immobilized on a support. For example, a probe capable of hybridizing with each mRNA, cDNA or cRNA (or these aDNA or aRNA) derived from the aforementioned liver cell or its precursor cell expression gene (each gene listed in (a) above) By fixing each to a support, the expression and quantification of a plurality of the genes can be simultaneously performed.

  The form of the support for the nucleic acid microarray is not particularly limited, and any form such as a flat plate, a rod, or a bead can be used. When a flat plate is used as the support, a predetermined probe can be fixed on the flat plate with a predetermined interval for each type (spotting method, etc .; see Science 270, 467-470 (1995)). . It is also possible to sequentially synthesize a predetermined probe for each type at a specific position on the flat plate (see, for example, photolithography method; Science 251, 767-773 (1991)).

Other preferred support forms include those using hollow fibers. When using hollow fibers as a support, a nucleic acid microarray obtained by fixing probes to each hollow fiber for each type, converging and fixing all hollow fibers, and then repeating cutting in the longitudinal direction of the fibers is preferable. It can be illustrated. This nucleic acid microarray can be described as a type in which a probe is immobilized on a through-hole substrate, and is also referred to as a so-called “through-hole type microarray” (see, for example, Japanese Patent No. 3510882 and FIG. 1 of this application).
The method for immobilizing the probe to the support is not particularly limited, and any binding mode may be used. Moreover, it is not limited to immobilizing directly to a support body, For example, a support body can be previously coated with polymers, such as polylysine, and a probe can also be fixed to the support body after a process. Furthermore, when a tubular body such as a hollow fiber is used as the support, the tubular body can hold a gel-like material, and the probe can be immobilized on the gel-like material.

Hereinafter, the through-hole type nucleic acid microarray using hollow fibers will be described in detail. This microarray can be manufactured through the following steps (step i) to (step iv), for example.
(Step i) Step of producing an array by arranging a plurality of hollow fibers in three dimensions so that the longitudinal direction of the hollow fibers is the same direction (Step ii) Embedding the array, Step of manufacturing (Step iii) Step of introducing a gel precursor polymerizable solution containing a probe into the hollow portion of each hollow fiber of the block body to perform a polymerization reaction, and holding the gel-like material containing the probe in the hollow portion ( Step iv) Cutting in a direction intersecting the longitudinal direction of the hollow fiber to make the block body into a thin piece The material used for the hollow fiber is not limited, but for example, in JP-A No. 2004-163211 The materials described are preferred.

  The hollow fibers are arranged three-dimensionally so that the lengths in the longitudinal direction are the same (step i). As an arrangement method, for example, a plurality of hollow fibers are arranged in parallel at a predetermined interval on a sheet-like material such as a pressure-sensitive adhesive sheet, and the sheet is formed into a sheet, and then the sheet is wound up in a spiral shape (Japanese Patent Laid-Open No. Hei 11- No. 108928), or two perforated plates each having a plurality of holes provided at predetermined intervals are overlapped so that the holes coincide with each other, and the hollow fibers are passed through the holes, and then the two porous Examples include a method of temporarily fixing the plate with a gap between the plates, filling the periphery of the hollow fiber between the two porous plates with a curable resin material (see JP 2001-133453 A), and the like.

The manufactured array is embedded so that the array is not disturbed (step ii). Examples of the embedding method include a method in which a polyurethane resin, an epoxy resin, or the like is poured into a gap between fibers, and a method in which fibers are bonded together by heat fusion.
The embedded array is filled with a gel precursor polymerizable solution (gel-forming solution) containing a probe in the hollow part of each hollow fiber, and a polymerization reaction is performed in the hollow part (step iii). Thereby, the gel-like thing with which the probe was fixed can be hold | maintained in the hollow part of each hollow fiber.

The gel precursor polymerizable solution is a solution containing a reactive substance such as a gel-forming polymerizable monomer, and the solution can be converted into a gel by polymerizing and crosslinking the monomer. Say. Examples of such monomers include acrylamide, dimethylacrylamide, vinyl pyrrolidone, methylene bisacrylamide and the like. In this case, the solution may contain a polymerization initiator or the like. After fixing the probe in the hollow fiber, the block body is cut into a thin piece in a direction intersecting with the longitudinal direction of the hollow fiber (preferably a direction orthogonal) (step iv). The flakes thus obtained can be used as a nucleic acid microarray. The thickness of the array is preferably about 0.01 mm to 1 mm. The block body can be cut by, for example, a microtome and a laser. Preferred examples of the through-hole type microarray include a nucleic acid microarray (Genopal ^™ (Genopal (registered trademark))) manufactured by Mitsubishi Rayon Co., Ltd.

  In the case of the above Genopal (registered trademark), oligonucleotide probes complementary to each corresponding sequence are immobilized at high density, and double-stranded cDNA synthesis is performed from RNA derived from a biological sample using T7 oligo dT primer. Then, aRNA is synthesized by in vitro transcription. Biotin is incorporated during aRNA synthesis and the sample is labeled. Biotin-labeled aRNA is fragmented and hybridized to the DNA array. After hybridization, aRNA is detected with streptavidin or the like modified with a fluorescent dye. The fluorescence can be read with a fluorescence detector, the array image obtained can be digitized, and the expression level of each gene can be compared and quantified. The nucleic acid microarray to be used is not limited to the above-described method, and the same analysis can be performed using a plurality of types of nucleic acid microarrays available to those skilled in the art.

4). Method for Evaluating State of Liver Cell or its Progenitor Cell Derived In the present invention, the expression level (control) of a normal liver cell (liver cell extracted from an organ) and the test subject cell (liver cell or The status of the liver cell or the precursor cell-derived cell is evaluated by comparing the measurement result with the gene expression level of the precursor cell-derived cell) and evaluating the difference (increase or decrease) in the gene expression level. be able to.
In addition, by evaluating the state of liver cells or progenitor cell-derived cells in the same manner, when any test substance (for example, various compounds, drugs, drug candidates, food ingredients) is contacted with the cells ( For example, the influence on the cells when added to the culture medium can also be evaluated.

(1) Test cells The cells (liver cells or progenitor cell-derived cells) used in the present invention are not limited, and primary cells and cell lines derived from the liver of a mammal (particularly human), those cells. And cell induced by gene or drug, stem cells, ES cells and iPS cells, and fetal liver cells and their precursor cells or cells derived from these cell lines. The cell used as a comparison control in the evaluation is preferably a cell collected directly from the liver in order to more reflect the liver function. Moreover, when evaluating the state before and after culture | cultivation of a test cell, and preservation | save, you may use the test cell itself before the said culture | cultivation or preservation | save as a control cell. The liver cells referred to in the present invention are as described in the above item 1.

(2) Nucleic acid extraction and reaction solution preparation method The amount of mRNA in a biological sample extracted from cells is measured by extracting nucleic acid from the biological sample. The detailed conditions for nucleic acid extraction and the processing method for the extracted nucleic acid are performed by methods suitable for the expression level measurement method.
As a general method for RNA extraction, a 4M guanidine thiocyanate solution is added to an organ or cells frozen at −80 ° C. or liquid nitrogen so as to be 0.5 g / 10 ml and homogenized. Add 1 ml of 2M sodium acetate, 10 ml of phenol and 10 ml of chloroform, and mix well. It is centrifuged at 10,000 rpm for 10 minutes, and the aqueous layer is recovered. An equal amount of isopropanol is added thereto, and the mixture is further centrifuged at 10,000 rpm for 10 minutes to precipitate RNA. Again, dissolve in 10 ml of 4M guanidine thiocyanate, add 1 ml of 2M sodium acetate, 5 ml of phenol and 1 ml of chloroform, and mix well. The aqueous layer is recovered by the same operation as the previous aqueous layer recovery, and an equal amount of isopropanol is added to deposit RNA. The precipitate is suspended by adding 20 ml of 70% ethanol, and centrifuged to precipitate RNA. This precipitate was dissolved in 5 ml of TNES buffer (0.1 M Tris-HCl (pH 7, 4), 50 mM NaCl, 10 mM EDTA, 0.2% SDS), and Proteinase K was added to a concentration of 200 μg / ml. It reacts at 37 degreeC for 30 minutes, acid phenol extraction and chloroform extraction are performed, and it precipitates with ethanol.

As another extraction method, when isolating total RNA, a commercially available reagent kit, RNeasy mini kit (QIAGEN), can be used. In that case, RNA should be extracted according to the attached protocol. You can also.
Furthermore, in some cases, when aRNA is amplified, amplified aRNA can be prepared according to the attached protocol using Message AmpII-Biotin Enhanced kit (Applied Biosystems), which is a commercially available kit. .

(3) Method for measuring the amount of mRNA or the amount of nucleic acid amplified from DNA or mRNA In the present invention, the expression level of gene means the amount of mRNA, the amount of nucleic acid amplified from DNA or mRNA, or the amount of protein. Yes, measuring the expression level of a gene means measuring the amount of mRNA, the amount of nucleic acid amplified from DNA or mRNA, and the amount of protein. Specific examples of the measurement method are shown in the following (3-1) to (3-3).

(3-1) Reverse transcriptase-polymerase chain reaction method (RT-PCR method)
In the case of the RT-PCR method, cDNA is synthesized from mRNA extracted from a biological sample by a reverse transcription reaction, and one of the genes to be measured is detected by the PCR method using two types of DNA fragments complementary to the gene sequence to be measured as primers. The amount of mRNA can be quantified by amplifying the partial sequence and detecting the amplified DNA fragment. The primer to be used can be designed and synthesized so that the base sequence of the gene to be measured can be amplified by a nucleic acid amplification method such as the PCR method. Nucleic acid amplification methods are well known, and primer design and synthesis in nucleic acid amplification methods can be easily performed by those skilled in the art. For example, in the PCR method, one of two primers is paired with the plus strand of the double-stranded DNA of the gene to be measured, the other primer is paired with the minus strand of the double-stranded DNA, and one primer The primer can be selected such that the other primer is paired with the extended strand. The length of the primer is, for example, at least 10 bases, preferably at least 15 bases, more preferably at least 20 bases. Specifically, the primer can be designed and chemically synthesized based on one or more base sequences registered by the accession number of each gene from databases such as Genbank and NCBI. Primers can be prepared according to a conventional method. DNA fragments amplified by the PCR method can be visualized and quantified by labeling with a fluorescent substance such as ethidium bromide or SYBER GREEN that specifically binds to DNA after agarose gel electrophoresis.

(3-2) Real-time PCR method By performing PCR using a real-time PCR device such as ABI PRISM 7500 (Applied Biosystems), the amplification product is detected and monitored in real time, and the gene expression level (mRNA level) to be measured Can be measured more quantitatively. The amplified DNA can be detected by labeling with a fluorescent substance such as SYBER GREEN described above. In the real-time PCR method, a method using a TaqMan probe in combination with two normal primers is more preferable as a method for increasing the specificity of nucleic acid amplification and the sensitivity of measurement. By using three types of DNA fragments including TaqMan (registered trademark) probes that are pre-quenched and labeled with fluorescent substances such as FAM and VIC, the gene sequence to be measured is specifically amplified and highly sensitive. Can be detected. The real-time PCR method using the TaqMan probe is well known, and selection of the probe and primer to be used can be easily performed by those skilled in the art. Gene-specific probes and primers can be designed by using production software such as Primer Express (manufactured by Applied Biosystems) based on mRNA or cDNA sequence information of each gene. In addition, probes and primers that have already been specifically designed and commercialized to detect specific genes can also be used.

(3-3) Northern blotting and dot blotting It can be measured using a DNA fragment that specifically hybridizes under stringent conditions to the gene to be measured. The length of the DNA fragment used in this analysis method is not particularly limited, but is a length sufficient to specifically hybridize under stringent conditions to the gene to be measured.

  The length of the DNA fragment is, for example, a sequence of at least 15 bases, preferably at least 100 bases, more preferably at least 200 bases. Here, the stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. That is, a pair of polynucleotides having high homology (homology or identity is 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, most preferably 95% or more). The conditions for hybridization. More specifically, such conditions are employed in conventional techniques well known in the art, such as Northern blotting, dot blot, colony hybridization, plaque hybridization, or Southern blot hybridization. Conditions can be set. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1M NaCl using a membrane on which a polynucleotide was immobilized, and then 0.1 to 2 times the concentration of SSC (Saline Sodium Citrate; 150mM sodium chloride, 15mM sodium citrate) solution and washing the membrane at 65 ° C.

  Under these conditions, it can be expected that a polynucleotide having high homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. . In addition, the DNA fragment used in this analysis method may be a base sequence that specifically hybridizes to mRNA or cDNA of a gene included in the gene group, and most preferably a gene included in the gene group. The base sequence can be designed as a completely complementary base sequence.

  In addition, if the DNA fragment used in this analysis method specifically hybridizes to the mRNA or cDNA of the gene included in the above gene group, the DNA fragment is complementary to the complementary strand of the base sequence of the gene included in the above gene group. And designed as a base sequence having high homology (homology or identity of 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, most preferably 95% or more). You can also. Here, specifically hybridizing can be rephrased as the above-mentioned stringent conditions. Furthermore, although the DNA fragment used in this analysis method is designed based on the base sequence of the above-mentioned gene, it is preferable to design so as not to include polymorphisms such as SNP known in these genes. Thereby, the detection error of the gene using a DNA fragment can be suppressed low. Furthermore, the means for measuring the gene expression level is not particularly limited, and a conventionally known means such as an RNA protection assay using a membrane filter or riboprobe immobilized with cDNA derived from a biological sample is applied. Can do.

(4) Analysis of measurement result It is preferable to use only a value equal to or higher than a judgment value as data used for evaluation in the measurement result. As the determination value, a method according to each measurement method can be used. For example, the average value X of the negative control can be used, and further, a value obtained by adding X to the standard deviation σ, further X + 2σ, and further X + 3σ is desirable. The negative control is a gene that should not be detected in the sample from the test cell, for example, a gene of another cell type different from the test cell, and a gene that does not interact with the detection target gene is preferable. . The absence of interaction means that, for example, primers used for PCR and probes used for detection do not hybridize under stringent conditions. The error between each measurement of the obtained data is corrected with the value of the housekeeping gene (gapdh, actin, arbp, etc.), and the fluctuation of the mRNA amount is determined using the corrected data. Variations in the amount of mRNA are determined statistically by testing. Obtain n = 3 or more samples from each test cell and perform t-test. When the P value is 0.05 or less, and further 0.01 or less, it is determined that there is a significant change (increase or decrease).

(5) Evaluation of cell state In the present invention, the state of a cell derived from a liver cell or the like is evaluated by, for example, gene expression level profile in a liver cell extracted from an organ and a cell derived from a liver cell or the like (eg, cultured liver cell). Etc.) can be performed by comparative analysis with the gene expression level profile. The gene expression level profile that serves as a control for comparison may be of the same origin, may be of a plurality of different test cells, or may be prestored in a database. In the method of the present invention, the level of mRNA may be measured using samples derived from a plurality of test cells. Therefore, the mRNA amount is measured in a predetermined number of test cells (primary population), and the obtained measurement value is used as basic data, and the basic data and a single or a plurality of test cells to be detected. It is possible to compare the amount of mRNA of the sample derived from the sample. The comparison includes not only comparing the increase / decrease in the amount of mRNA but also comparing the presence / absence of mRNA expression. For example, comparing each sample for mRNA that is not expressed in the basic data and expressed in the test cell.

In addition, the above-mentioned measured cell-derived data is incorporated into the population value, and the mRNA level is processed again (averaging, etc.) to increase the number of target cell (population) cases. You can also. By increasing the number of examples, the accuracy of the critical value of the amount of mRNA can be increased, and in some cases, the detection accuracy can be increased by appropriately correcting the critical value.
Furthermore, a database in which measurement results of gene expression levels of the same type of test cells are accumulated can be created, and changes in gene expression levels can be evaluated as patterns. Preferably, it is preferable to create a database in which the effects of known components are measured using the same type of cells, and to evaluate changes in gene expression levels as patterns. Examples of such a method include a method using performing using multivariate analysis as described above. For example, principal component analysis, factor analysis, discriminant analysis, quantification theory (Class I, II, III, IV), cluster analysis, multidimensional scaling (MDS), multiple regression analysis, conjoint analysis, Mahalanobis Pattern comparison using Taguchi system (MT method), prediction of effects, etc. can be mentioned.

The present invention can also provide a database that stores such data, and an analysis device that reads and executes the data and programs necessary for comparative analysis. According to such an analysis apparatus, the state of the eye disease of the test cell can be easily evaluated at any time simply by taking out the accumulated data from the database and comparing it with the measured data of the test cell.
A gene expression level profile is, for example, measuring the amount of mRNA in a collected biological sample and indicating the measured expression level of each gene as an absolute value and / or a relative value.

5. Screening method The present invention can also provide a method for screening a substance useful for maintaining the function of a liver cell or a precursor cell-derived cell thereof using the probe, probe set or nucleic acid microarray of the present invention. .
Specifically, the screening is performed, for example, by culturing or storing (eg, cryopreserving) a liver cell or a progenitor cell-derived cell thereof after contacting or contacting with a candidate substance, and then gene expression in the cell. Screening can be performed by measuring the expression level and comparatively analyzing it with the control gene expression level. Contact here means, for example, adding a candidate substance to the culture solution of cultured cells.

As a control, for example, actual liver cells collected from a living body or their precursor cells themselves, cells when cultured without contacting a candidate substance, cells when contacted with a candidate substance without culturing, or Examples include cells that are not cultured and the candidate substance is not contacted.
Note that candidate substances that can be used for screening include any of various compounds, which may be naturally derived or artificially prepared, and are not limited.

  The expression level data of the control gene may be obtained from the same test subject or may be obtained from a plurality of different test subjects of the same type, and is accumulated in advance in a database. May be. In addition, the expression level data derived from the test subject can be incorporated into the value of the population (test subject) and the expression level can be processed again (averaging, etc.) to increase the number of examples in the population. By increasing the number of examples, the accuracy of the critical value of the expression level can be increased, and in some cases, the accuracy of screening can be increased by appropriately correcting the critical value.

  The concentration and amount of the candidate substance to be brought into contact with the cells can be set and selected as appropriate according to the candidate substance and the type of test subject, etc., but the toxicity of the candidate substance does not affect the nucleic acid extraction. It is preferable to carry out at a concentration or amount that does not occur in the test subject. In the case of cultured cells, that the toxicity does not occur is at least a cell viability of 90% or more.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

1. Nucleic acid microarray In this example, a nucleic acid microarray (Genopal (registered trademark), Mitsubishi Rayon Co., Ltd.) equipped with the probes (SEQ ID NOs: 1 to 198) shown in Tables 1 and 2 was used. Of the SEQ ID NOs: 1 to 198, the nucleotide sequences of SEQ ID NOs: 194 to 198 are sequences corresponding to a part of the housekeeping gene and yeast and E. coli, and were used as probes for positive control or negative control.

2. Cells and culture conditions Tables 3 and 4 below show the types of cells used in this example and the culture conditions. The culture vessel used was a collagen-coated 24-well plate (manufactured by BIOPREDIC), and the culture environment was the same, and the culture was performed in a 37 ° C., 5% CO ₂ incubator. Total RNA was extracted from the cells in Table 3 using a commercially available reagent kit, RNeasy mini kit (QIAGEN). The extraction conditions were in accordance with the liver tissue conditions of the attached protocol.

3. Preparation of specimen solution Antisense RNA using Total RNA extracted from the above-mentioned cells and 1 μg of HepG2 total RNA of hepatoma cells as a reference and using a synthesized Message Amp II-Biotin Enhanced kit (Applied Biosystems) according to the attached protocol (ARNA) was prepared. Put 5 μg of the obtained aRNA in a plastic tube, add 4 μl of 5 × Array Fragmentation Buffer attached to the Message AmpII-Biotin Enhanced kit (Applied Biosystems), mix up to 20 μl, and mix well. Fragmentation was performed by heating for 5 minutes. Sample preparation for reaction with Genopal was prepared by mixing 24 μl of 1M Tris-HCl solution (Invitrogen), 24 μl of 1M NaCl solution (Nacalai Tesque) and 20 μl of 0.5% Tween 20 solution, respectively. After making the volume up to 180 μl with free water, 20 μl of the fragmented solution was mixed to prepare a sample solution.

4). Measurement The nucleic acid microarray was immersed in the prepared sample solution, and a hybridization reaction was performed at 65 ° C. for 16 hours. After removing the sample solution used for hybridization from the nucleic acid microarray, the array was immersed in a 0.12 M TNT solution (0.12 M Tris-HCl, 0.12 M NaCl, 0.5% Tween 20 solution) at 65 ° C. Then, it was washed by immersing in a 0.12 M TN solution (0.12 M Tris-HCl, 0.12 M NaCl) warmed to 65 ° C. for 10 minutes. The signal in the array was detected using a nucleic acid microarray detector (Yokogawa: MB-M3A, laser wavelength: 633 nm) and the fluorescence intensity of Cy5 was measured (exposure time: 0.1 seconds, 1 second, 4 Seconds, 40 seconds).

5. Data analysis Data analysis was performed according to the following procedure.
(I) The average value of the background was subtracted.
(Ii) Correction was performed with positive control genes (β-actin, GAPDH, RPLP0).
(Iii) The corrected average value, expression ratio, and P value were calculated (expression ratio is based on CTR).
(Iv) Significantly fluctuating genes (P value of 0.05 or less) were picked up.

6). Results The measurement results are shown in FIGS. 2A, 2B, 2C-1, 2C-2, 2D, 2E-1, 2E-2, 2F, 2G and 2H. By comparing the results of Group A classified in Table 3 above with the results of Group B, Group C, and Group D, the difference in cell type could be identified by gene. In addition, Experiment No. Results of A1-1 to A1-3 and Experiment No. By comparing the results of A3-1 (2) and A3-2 (2), it was possible to observe the changes in the cells when the drug was added to the cells and cultured with the gene. Furthermore, Experiment No. Results of B1-1 and Experiment No. By comparing with the result of B8-1, it was possible to confirm the decrease in the function as the liver by continuing the culture with the gene.
Further, based on the measurement results obtained in this example and the function of each gene, the eight groups (groups (i) to (viii)) enumerated in the section “2. Probes or probe sets” described above are included. Classified. This classification was confirmed to be a classification that can be a combination including genes that can confirm a difference between cell types (a difference in gene expression level of 1.2 times or more) in any combination of genes. .

11 hole 21 perforated plate 31 hollow fiber 41 plate

Claims (13)

A probe or probe set for evaluating the state of a liver cell or a progenitor cell-derived cell thereof, which comprises the nucleic acid of the following (a), (b) or (c) or a part thereof.
(A) CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, AHR, NR1I2, CASR, ESR, NR3C1, ABCA1, CC4, CCB2, CCB1, CC4 , ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A7, SLC17A1, SLC22A1, SLC22ALC, SLC22A2, SLC22A2 , SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A1, SLC47A2, SLCO1A2, SLCO1B1, SLCO1B3, SLCO2A1, SLCO2B1, SLCO4C1, UGT2B4, UGT2B, UGT2T, UG2B15, UGT2A , CES5, GSTM1, GSTT1, GSTA1, SULT1A1, SULT1A3, SULT1A4, SULT1B1, SULT1C2, SULT1C4, SULT1E1, NAT1, ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO1, TP1, C2 , TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, FABP6, SREBF1, SREBF2, PPARA, LDLR, SCARB1, NPC1 L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, HNF1A, CYP3A7, POU5F1, EP, PAY, PAY5, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP, PTPRC, TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC, PDGFB, FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, Nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of JUN, REL, ETS1, BCL2, BCL2L1 and SRC (b) Nucleic acid comprising a base sequence complementary to the nucleic acid of (a) (C) hybridizes with a nucleic acid comprising a base sequence complementary to the nucleic acid (a) or (b) under stringent conditions, and Nucleic acid capable of detecting gene expression of progenitor cells The probe set according to claim 1, wherein the nucleic acid (a) comprises a combination of two or more groups of nucleic acids selected from the following groups (i) to (viii).
(i) a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5
(ii) a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of AHR, NR1I2, CASR, ESR and NR3C1
(iii) ABCA1, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, ABCG2, ABCG5, ABCG8, SLC5A1, SLC5A2, SLC7A5, SLC7A8, SLC10A1, SLC10A2, SLC15A1, SLC16, LC1 SLC22A12, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC28A1, SLC28A2, SLC29A1, SLC29A2, SLC31A1, SLC47A, SLC47A1, SLC47A, SLC47A1, SLC47A A nucleic acid comprising a base sequence constituting at least one gene
(iv) UGT2B4, UGT2B10, UGT2B15, UGT2B7, UGT2B17, UGT1A1, UGT1A6, UGT1A4, UGT1A9, UGT1A3, CES1, CES2, CES3, CES5, GSTM1, GSTT1, ASTA1, ULT1A1, SULT1A1, SULT1A1S And a nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of NAT1
(v) ALB, CYP7A1, CYP8B1, CYP27A1, TTR, SERPINA1, TDO2, CPS1, PCK2, OTC, ASS1, HNF4A, GSTP1, TPMT, NR1H4, RXRA, FGFR2, FGFR4, NR5A2, NR0B2, RABP6, SREBF1, PP , LDLR, SCARB1, NPC1L1, SLC27A5, SLC51A, SLC51B, BAAT, SULT2A1, PLTP, APOC2, APOC3, ANGPTL3, G6PC, TRIB3, AKT1, GSK3A, GSK3B, FOXO1, COMT, and HNF1A Nucleic acid consisting of the base sequence constituting the gene
(vi) Selected from the group consisting of CYP3A7, POU5F1, AFP, THY1, EPCAM, DLK1, CEBPA, YBX1, CD34, KIT, MET, KRT18, CD44, VIM, KRT14, PVRL3, SLC36A6, CDH1, ITGB1, ANPEP and PTPRC A nucleic acid comprising a base sequence constituting at least one gene
(vii) From a base sequence constituting at least one gene selected from the group consisting of TECR, ITGA6, NGFR, ASGR1, KRK8, KRT19, ONECUT1, HNF1B, STAB2, DMBT1, NOTCH2, DES, CTNNB1, APC and PDGFB Nucleic acid
(viii) FGF3, FGF4, CSF1R, FLT1, FLT3, EGFR, ERBB2, ERBB4, ABL1, FYN, LYN, RAF1, MOS, HRAS, KRAS, NRAS, MYC, MYCN, MYCL1, MYB, FOS, JUN, REL, ETS1 , A nucleic acid comprising a base sequence constituting at least one gene selected from the group consisting of BCL2, BCL2L1 and SRC   The probe set according to claim 2, wherein the nucleic acid (a) comprises a combination of the nucleic acids of the groups (i) to (viii). A probe or probe set for evaluating the state of a liver cell or a progenitor cell-derived cell, which comprises the following (α), (β), (γ), or (δ) nucleic acid or a part thereof.
(Α) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 1 to 193 (β) Nucleic acid consisting of a base sequence complementary to the nucleic acid of (α) (γ) Said (α ) Or (β) a nucleic acid comprising a nucleotide sequence having a homology of 70% or more with respect to the nucleic acid sequence, and a nucleic acid capable of detecting a liver cell or its precursor cell expression gene (δ) (α), A nucleic acid comprising a base sequence in which 1 to several bases are added, deleted or substituted in the base sequence of the nucleic acid of (β) or (γ), and capable of detecting a gene expressed in liver cells or their precursor cells The probe set according to claim 1, wherein the nucleic acid (α) is a combination of two or more groups of nucleic acids selected from the following groups (I) to (VIII).
(I) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 1 to 10
(II) a nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 11 to 15
(III) Nucleic acid comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 61
(IV) Nucleic acid comprising at least one base sequence of the base sequences shown in SEQ ID NOs: 62 to 86
(V) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 87 to 130 (VI) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 131 to 151 (VII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 152 to 166 (VIII) Nucleic acid consisting of at least one of the base sequences shown in SEQ ID NOs: 167 to 193   The probe set according to claim 5, wherein the nucleic acid (α) comprises a combination of the nucleic acids of the groups (I) to (VIII).   A nucleic acid microarray for evaluating the state of a liver cell or a progenitor cell-derived cell comprising the probe or probe set according to any one of claims 1 to 6.   A liver cell or a progenitor cell thereof, comprising a step of measuring the gene expression level of a liver cell or a progenitor cell derived cell thereof to be tested using the probe or probe set according to any one of claims 1 to 6. A method for evaluating the state of a derived cell.   A method for evaluating the state of a liver cell or a progenitor cell-derived cell, comprising a step of measuring the gene expression level of a liver cell or a progenitor cell-derived cell to be tested using the nucleic acid microarray according to claim 7.   The method of Claim 8 or 9 including the process of making a test substance contact the liver cell or its precursor cell origin cell used as test object previously.   The method of any one of Claims 8-10 including the process of evaluating the function as a liver cell by using as a parameter | index the result of the multivariate analysis based on the measured gene expression level.   A method for screening a substance useful for maintaining a function as a liver cell in a liver cell or a progenitor cell thereof using the probe or probe set according to claim 1.   A method for screening a substance useful for maintaining the function of a liver cell or a precursor cell-derived cell thereof using the nucleic acid microarray according to claim 7.