JP2000014390A - New immortalized liver cell line derived from human - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new immortalized liver cell line, derived from human, which retains an ability of expressing genes coding for enzymes which are involved in the metabolism of xenobiotic substances in the liver, and can be used, for example, for the screening of compounds which inhibit or promote the activity of the enzymes. SOLUTION: This is a new immortalized liver cell line, from human, which retains an ability of expressing genes coding for enzymes, which are involved in the metabolism of xenobiotic substances in the liver, such as NADPH cytochrome P450 reductase, NADPH cytochrome P450, flavin monooxygenase, epoxyhydratase, glucuronyl transferase, sulfotransferase, and glutathione S- transferase, and can be used, for example, of the screening of compounds which inhibit or promote the activity of at least one of the enzymes or which inhibit or promote the expression of at least one of the genes coding for the enzymes. This cell line is obtained by introducing SV40T antigen gene into the liver parenchymal cell isolated from the liver from a dead human embryo.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to (1) a novel human (preferably human fetal) liver-immortalized cell line derived from normal cells, (2) a method for producing the cell line, and (3) use of the cell line. A method for screening or screening a compound or salt thereof that inhibits or promotes the enzyme activity involved in the metabolism of liver xenobiotics, or inhibits or promotes the expression of a gene encoding an enzyme involved in the metabolism of liver xenobiotics, (4) A compound that inhibits or promotes the activity of an enzyme involved in the metabolism of a xenobiotic in the liver or inhibits or promotes the expression of a gene encoding an enzyme involved in the metabolism of a xenobiotic in the liver obtained by using the screening method Or a salt thereof, and (5) a method for analyzing an xenobiotic and / or an enzyme involved in endogenous substrate metabolism using the cell line.

[0002]

2. Description of the Related Art Liver cells have a great many physiological functions. Among them, so-called metabolism of xenobiotics such as drugs, food additives, environmental pollutants and the like is converted into a form easily excreted. It plays a very important role in the metabolism of xenobiotics. This metabolic function of xenobiotics may simultaneously result in mutagenesis, toxic expression, or expression of the efficacy of the xenobiotics, and is being studied very widely. For these reasons, cultured hepatocytes are not only a substitute for laboratory animals, but also a so-called artificial liver that replaces the function of the liver, not only providing a test method for rapidly and inexpensively and accurately examining metabolism in the liver. It has been considered possible to create. However, normal human hepatocytes separated from living tissues cannot be subcultured. Cells that can be established as cell lines often have no intrinsic differentiation trait, and often do not accurately reflect the function of the tissue to which the cell line originally belonged. In particular, enzymes that metabolize so-called xenobiotics in hepatocytes lose their activity in a very short period of time in primary culture, and no cell line that has sufficiently retained its properties has been found so far. (J. Dich et
al., Hepatology, 8, 39-45 (1988)). From such a viewpoint, hepatocytes that retain the metabolic ability of xenobiotics and can be cultured have been widely demanded. Preparation of the human liver cell line is performed by selection of human tumor cells, for example, HepG
2 (Aden et al. Nature, 282, 615-616, 1979) and the like. However, these cells are derived from tumor cells and are not normal cells immortalized. As a method for immortalizing normal cells, that is, for infinitely proliferating, for example, a method of introducing a T antigen gene derived from SV (simian virus) 40 is generally used. However, the immortalization of normal parenchymal cells of the liver, especially the enzymatic activity involved in xenobiotic metabolism, the expression of genes encoding enzymes involved in xenobiotic metabolism, and the induction of the expression of genes encoding enzymes involved in xenobiotic metabolism. There is no known immortalized cell line derived from normal human liver parenchymal cells. Also,
In culture of many cell lines, a serum component is essential in the medium. The need for this serum component not only severely impairs the stability of the characteristics of the cultured cells due to the lack of quality stability of the serum, but also makes the cell line stable, accurate and inexpensive due to the very high price of the serum. Use of such a material was significantly impaired. Therefore, if the established immortalized cell line can be grown in a serum-free medium while maintaining its trait stably, industrially significant benefits can be obtained.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a human metabolic function derived from human normal liver cells (preferably human normal liver parenchymal cells), which can be grown in a serum-free complete synthetic medium, and which has a specific metabolic function in human liver. In particular, it is an object of the present invention to provide a cell line capable of observing the activity of an enzyme involved in metabolism of xenobiotics and the expression of a gene encoding an enzyme associated with metabolism of xenobiotic, and to isolate and manufacture the cell line.

[0004]

Means for Solving the Problems In view of the above-mentioned problems, the present inventors have conducted intensive studies and as a result, they have been derived from human normal liver parenchymal cells and can be grown in a serum-free complete synthetic medium, Succeeded in establishing a cell line in which the enzyme activity related to the metabolism of xenobiotics and the expression of the gene encoding the enzyme related to xenobiotic metabolism among the specific metabolic functions can be observed. As a result, the present invention has been completed.

That is, the present invention provides (1) an immortalized liver cell derived from a normal human cell, which retains the activity of an enzyme involved in the metabolism of a xenobiotic in the liver or the ability to express a gene encoding an enzyme involved in the metabolism of a xenobiotic in the liver. Strain, (2) the enzyme activity is NA
DPH cytochrome P450 reductase activity, gluclosyltransferase activity, ethoxyresorufin dealkylation activity, benzyloxyresorufin dealkylation activity,
(3) The cell line according to (1), which has pentoxyl resorufin dealkylation activity, methoxy resorufin dealkylation activity, flavin monooxygenase activity, epoxyhydratase activity, sulfotransferase activity, or glutathione S-transferase activity. (4) The cell strain according to (1), wherein the enzyme is NADPH cytochrome P450 reductase, NADPH cytochrome P450, flavin monooxygenase, epoxyhydratase, gluclosyltransferase, sulfotransferase or glutathione S-transferase. NADP
H The cell line according to (3) above, wherein the cytochrome P450 is CYP1A1, CYP1A2 or CYP3A, and (5) the cell line is FERM B.
(6) a cell line according to (1), wherein the T antigen gene derived from SV (simian virus) 40 is introduced into normal human liver cells; (7) The method according to (6), wherein the normal human liver cells are liver cells derived from a human fetus, and (8) the living body of the liver, wherein the cell line according to (1) is used. A method for screening a compound or a salt thereof that inhibits or promotes the activity of an enzyme involved in metabolism of a foreign substance or inhibits or promotes the expression ability of a gene encoding an enzyme involved in metabolism of a xenobiotic in the liver, (9) The method according to the above (8). A compound that inhibits or promotes an enzyme activity related to metabolism of a xenobiotic in the liver obtained by using the screening method, or inhibits or promotes expression of a gene encoding an enzyme involved in metabolism of a xenobiotic in the liver Or its salt, (1
0) using the cell line according to (1) above
(a) enzymes involved in xenobiotic and / or endogenous substrate metabolism, (b) xenobiotic and / or endogenous substrate metabolic pathways, (c) xenobiotic and / or endogenous substrate metabolite chemical structure, (d ) Inhibition of xenobiotic and / or endogenous substrate metabolizing enzymes, (e) promotion of xenobiotic and / or endogenous substrate metabolizing enzyme activity, (f) cytotoxicity due to xenobiotic and / or endogenous substrate metabolism, (g ) Genotoxicity due to xenobiotic and / or endogenous substrate metabolism; (h) carcinogenicity due to xenobiotic and / or endogenous substrate metabolism; (i) mutagenicity due to xenobiotic and / or endogenous substrate metabolism; j) hepatotoxicity due to xenobiotic and / or endogenous substrate metabolism, or (k) a method for analyzing xenobiotic and / or endogenous substrate acting on the liver, and (11) xenobiotic and / or endogenous substrate metabolite For preparation method To.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, “normal cells”, “normal liver cells” or “normal tissues” means cells or tissues that are not cancerous. Further, the term “metabolism of xenobiotics” means, for example, the metabolism of drugs, food additives, environmental pollutants, etc., among which drug metabolism is preferably used. Normal human liver cells (preferably human normal liver parenchymal cells) to be used can be isolated from normal tissues such as human adults and human fetuses (preferably human fetuses) by an established method called collagenase perfusion. Immortalization of the so-called primary culture cells obtained in this manner is performed according to various known methods and the like. Specifically, focusing on the fact that cancerous tissue continues to proliferate permanently, a method of introducing a part of an oncogene into individual normal cells and transforming the cells to immortalize them, etc. .
The immortalized cell line thus established includes, for example, oncogenes such as ras and c-myc, or
Oncogenes of DNA-type tumor viruses such as adenovirus EIA, SV (simian virus) 40 virus, and human papilloma virus (HPV16), or their tumor antigens (T antigens)
Introducing a gene or the like into animal cells and subculturing the transformant (E. Ponet et al, Proc. N
atl. Acad., Sci., USA 82 8503, (1985)). Preferably, a method of introducing an SV40-derived T antigen gene or a method analogous thereto can be used (M. Miyazaki
Et al., Experimental Cell Research, 206, 27-35 (1993)).
When culturing (subculturing) these immortalized liver cells, a known medium [for example, a complete synthetic medium (a serum-free complete synthetic medium is preferable (eg, ASF104 medium and the like) (Ajinomoto)),
MEM medium containing about 5 to 20% fetal bovine serum (Seience, 122, 501 (1952)), DM
EM medium [Virology, 8, 396
(1959)], RPMI 1640 medium [Journal
The Journal of the American Medical Association
n) Volume 199, 519 (1967)], Williams Medium (Nissui Pharmaceutical), 199 Medium [Proceeding of the Society for the Biological Medicine (Proceeding of the Society for the Biological Medicine)]
icine), 73, 1 (1950)]]. Among them, a completely synthetic medium (serum-free completely synthetic medium (eg, ASF104 medium) (Ajinomoto)) and the like are preferably used. pH is about 7-7.2
It is preferred that The culture is usually performed at about 37 ° C.

[0007] In particular, by using a serum-free complete synthetic medium in the process of establishing the immortalized liver cells of the present invention, immortalized liver cells capable of growing in a serum-free complete synthetic medium can be obtained. From the immortalized liver cells thus obtained, those that retain liver-specific metabolic properties, in particular, enzyme activity, enzymes, gene expression, and gene expression induction related to metabolism of xenobiotics are selected. Enzyme activities related to liver-specific xenobiotic metabolism include, for example, NADPH
Cytochrome P450 reductase activity, gluclosyltransferase activity, mixed function oxidation reaction (MFO) activity (eg, ethoxyresorufin dealkylation activity, benzyloxyresorufin dealkylation activity, pentoxylresorufin dealkylation activity, methoxy) Resolufin dealkylation activity), flavin monooxygenase activity, epoxy hydratase activity, sulfotransferase activity or glutathione S-transferase activity. Among them, NADPH cytochrome P450 reductase activity, gluclosyltransferase activity, mixed function oxidation reaction (MFO) activity (eg, ethoxyresorufin dealkylation activity, benzyloxyresorufin dealkylation activity, pentoxylresorufin dealkylation activity) Activity, methoxyresorufin dealkylation activity, etc.), and in particular, NADPH cytochrome P450 reductase activity is considered to be the most important enzyme activity in terms of its function in metabolism of xenobiotics.

[0008] Examples of enzymes involved in liver-specific metabolism of xenobiotics include NADPH cytochrome P450 reductase,
NADPH cytochrome P450, flavin monooxygenase, epoxy hydratase, gluclosyltransferase, sulfotransferase, glutathione S
Transferase and the like. Among these
NADPH cytochrome P450 is the most important enzyme group in terms of its distribution and function in xenobiotic metabolism. NADP
H cytochrome P450 is a generic name for a number of enzyme proteins, and as individual NADPH cytochrome P450 involved in metabolism of xenobiotics in the human liver, CYP1A1, CYP1A2, CYP2A6, CYP2
B6, CYP2C8, CYP2C9, CYP2C19, CYP3A (specifically,
P3A4, CYP3A5, CYP3A7, etc.), CYP2D6, etc. are known. Among them, the immortalized hepatic cell lines of the present invention include CYP1A1, CYP
P1A2, CYP3A and the like are preferably used. Also, NADPH
From the functional aspect of cytochrome P450, it may be collectively called mixed function oxidation reaction (MFO), ethoxyresorufin dealkylation activity, benzyloxyresorufin dealkylation activity, pentoxylresorufin dealkylation Methoxyresorufin dealkylation activity and the like. Furthermore, the presence of NADPH cytochrome P450 reductase is essential for the expression of the MFO function of NADPH cytochrome P450 protein, and this enzyme can also be classified as a xenobiotic metabolizing enzyme. It is known that many metabolic enzymes of xenobiotics are induced under certain conditions. Examples of this are CYP1A1, CYP1
Benzpyrene, benzanthracene, 3 for A2 expression
-Effects of so-called polycyclic aromatic compounds such as methylcholanthrene and dioxin, effects of phenobarbital and phenobarbitone on CYP2B (eg, CYP2B6) induction, and effects of rifampicin, dexamethasone, phenytoin and phenylbutazone on CYP3A induction (CG Gibson et al., New edition xenobiotic metabolism Kodansha, 1995). The human normal cell-derived immortalized liver cell line of the present invention has an enzyme activity involved in the metabolism of xenobiotics in the liver or an expression ability of a gene encoding an enzyme involved in metabolism of the xenobiotics in the liver. Can be used for screening for a compound having a therapeutic / preventive effect on diseases related to metabolic abnormalities of xenobiotics (for example, liver dysfunction). That is, the present invention provides a human normal cell-derived immortalized liver cell line of the present invention, wherein a test compound is brought into contact with a test compound, and a gene encoding an enzyme activity involved in hepatic xenobiotic metabolism or an enzyme involved in hepatic xenobiotic metabolism is obtained. Inhibits or promotes the activity of enzymes involved in the metabolism of xenobiotics in the liver characterized by observing and measuring changes in expression, or inhibits or promotes the expression of genes encoding enzymes involved in the metabolism of xenobiotics in the liver And a method for screening a compound or a salt thereof. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
Examples thereof include cell extracts, plant extracts, animal tissue extracts, and plasma. These compounds may be novel compounds or known compounds.

Specifically, the human normal cell-derived immortalized liver cell line of the present invention is treated with a test compound and compared with an untreated control human-derived immortalized liver cell line. Therapeutic / preventive effects of test compounds can be tested using changes such as changes in the enzyme activity involved in the metabolism of liver xenobiotic metabolism of the strain or the expression of the gene encoding the enzyme involved in liver xenobiotic metabolism as an index . The compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is used for treatment / prevention of diseases related to abnormal metabolism of xenobiotics in the liver (for example, liver dysfunction). Since it has an effect, it can be used as a medicament such as a safe and low-toxic therapeutic / prophylactic agent for the disease. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner. The compound obtained by the screening method may form a salt, and the salt of the compound includes a physiologically acceptable acid (eg, inorganic acid, organic acid) and a base (eg, alkali metal). And particularly preferably a physiologically acceptable acid addition salt. Such salts include:
For example, inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid,
Sulfuric acid) or organic acids (eg, acetic acid, formic acid,
Salts with propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid and the like are used. The drug containing the compound or a salt thereof obtained by the screening method can be produced by a known production method or a method analogous thereto. The preparation obtained in this way is safe and low toxic, and thus can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered. The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered for the purpose of treating hepatic dysfunction, generally the adult (body weight of 6) is used.
0 kg), from about 0.1 to 100 mg, preferably from about 1.0 to 50 mg of the compound per day,
More preferably, about 1.0 to 20 mg is administered. When administered parenterally, a single dose of the compound should be administered to the subject,
For example, the compound is usually administered in the form of an injection to an adult (60%) for the purpose of treating hepatic dysfunction.
kg)), the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day.
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

[0010] Specific examples of the dosage form of the above preparation include tablets (including sugar-coated tablets and film-coated tablets), pills, capsules (including microcapsules),
Granules, fine granules, powders, syrups, emulsions, suspensions, injections, inhalants, ointments and the like are used. These preparations are prepared according to a conventional method (for example, a method described in the Japanese Pharmacopoeia). In the preparation, the content of the compound or a salt thereof obtained by the above screening method varies depending on the form of the preparation, but is usually 0.01 to 100% by weight, preferably 0.1 to 50% by weight based on the whole preparation. weight%,
More preferably, it is about 0.5 to 20% by weight.
Specifically, in the tablet manufacturing method, the drug is used as it is, excipients, binders, disintegrants or other suitable additives are added and uniformly mixed, and then granulated by an appropriate method, Add a lubricant, etc., compression-mold or, as is, the drug as it is, or excipients, binders, disintegrants or other appropriate additives and evenly mixed,
It can also be produced by direct compression molding, or by compression molding after pre-produced granules are mixed as they are or by adding an appropriate additive thereto and mixed uniformly. In addition, the present agent can contain a coloring agent, a flavoring agent, and the like as needed. Further, the agent can be coated with an appropriate coating agent. In the method of manufacturing an injection, a certain amount of a drug is used in the case of an aqueous solvent, water for injection, physiological saline,
In the case of a non-aqueous solvent such as Ringer's solution, it can be usually prepared by dissolving, suspending or emulsifying in a vegetable oil or the like to make a fixed amount, or taking a fixed amount of a drug and sealing it in a container for injection. Oral pharmaceutical carriers include, for example, starch,
Materials commonly used in the pharmaceutical field such as mannitol, crystalline cellulose, and sodium carboxymethylcellulose are used. As the carrier for injection, for example, distilled water, physiological saline, glucose solution, infusion agent and the like are used. In addition, additives generally used in preparations can be appropriately added.

The present invention further provides a method for analyzing a xenobiotic and / or an enzyme involved in endogenous substrate metabolism, which comprises using the above-mentioned immortalized liver cell line derived from a human normal cell, and (b) a xenobiotic. (C) a method for analyzing the chemical structure of a xenobiotic and / or endogenous substrate metabolite, (d) a method for preparing a xenobiotic and / or endogenous substrate metabolite, (e) a biological method. Method for analyzing inhibition of xenobiotic and / or endogenous substrate metabolizing enzyme, (f) Method for analyzing xenobiotic and / or endogenous substrate metabolizing enzyme activity, (g) Expression of cytotoxicity due to xenobiotic and / or endogenous substrate metabolism (H) a method for analyzing the expression of genotoxicity due to xenobiotic and / or endogenous substrate metabolism, (i)
(J) Analysis method of carcinogenicity by xenobiotic and / or endogenous substrate metabolism, (j) Mutagenicity analysis by xenobiotic and / or endogenous substrate metabolism, (k) The present invention relates to a method for analyzing toxicity expression or (1) a method for analyzing xenobiotics and / or endogenous substrates acting on the liver. Hereinafter, each method described in the above (a) to (l) will be described.

(A) Method for analyzing enzymes involved in xenobiotic and / or endogenous substrate metabolism: For example, the structure of xenobiotic and / or endogenous substrate by exposing a test substance to human normal cell-derived immortalized liver cells By analyzing the changes in the enzyme, it is possible to analyze enzymes involved in xenobiotic and / or endogenous substrate metabolism (JL Napoli et al., Methods
in Enzymology vol. 206 pp.491-501 Ed. by MR Wat
erman and others Academic Press 1991, HK Kroemer and others
Methods in Enzymology vol. 272 pp.99-198 Ed. By
MR Waterman et al. Academic Press 1996). Specifically, changes in the structure of xenobiotics and / or endogenous substrates due to exposure of test substances to human normal cell-derived immortalized liver cells are analyzed using inhibitors / antagonists of various enzymes or neutralizing antibodies of various enzymes. Identification of enzymes involved in xenobiotic and / or endogenous substrate metabolism by analyzing the structure of xenobiotics and / or endogenous substrate by exposing the test substance to cells; Analysis of substrate specificity and the like can be mentioned. Test substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc., and these compounds are novel compounds. Or a known compound. (b) Methods for analyzing xenobiotic and / or endogenous substrate metabolic pathways: for example, analyzing changes in the structure of xenobiotic and / or endogenous substrates due to exposure of a test substance to human normal cell-derived immortalized liver cells Xenobiotics and / or
Alternatively, analysis of the endogenous substrate metabolic pathway is possible (JL Na
poli and others Methods in Enzymology vol. 206 pp.491-50
1 Ed. By MR Waterman et al. Academic Press 1991, H.
K. Kroemer et al. Methods in Enzymology vol. 272 p
p.99-198 Ed. byM.R.Waterman and others Academic Press 199
6). As the test substance, the same substances as described above are used. (c) A method for analyzing the chemical structure of a xenobiotic and / or endogenous substrate metabolite: for example, by analyzing a change in the structure of a xenobiotic and / or endogenous substrate caused by exposure of a test substance to cells. It is possible to analyze the chemical structure of xenobiotics and / or endogenous substrate metabolites (JL N
apoli et al. Methods in Enzymologyvol. 206 pp.491-50
1 Ed. By MR Waterman et al. Academic Press 1991, H.
K. Kroemer et al. Methods in Enzymology vol. 272 p
p.99-198 Ed. by MR Waterman et al. Academic Press 1
996). As the test substance, the same substances as described above are used.

(D) A method for preparing a xenobiotic and / or endogenous substrate metabolite: for example, a xenobiotic and / or endogenous substrate conversion substance (so-called metabolite) resulting from exposing a test substance to cells. It is possible to prepare xenobiotics and / or endogenous substrate metabolites by collecting and purifying and separating them by an appropriate method (JLNapoli et al.
s in Enzymology vol. 206 pp.491-501 Ed. by MR W
aterman et al. Academic Press 1991). As the test substance, the same substances as described above are used. (e) Method for analyzing inhibition of xenobiotic and / or endogenous substrate metabolizing enzyme For example, it is possible to analyze inhibition of xenobiotic and / or endogenous substrate metabolizing enzyme activity by exposing a test substance to cells. (JL Napoli et al. Methods
inEnzymology vol. 206 pp.491-501 Ed. by MR Wat
Erman et al. Academic Press 1991). Specifically, it can be detected by inhibiting cytochrome P450 enzyme activity, decreasing the amount of protein, decreasing mRNA, and the like. As a detection method, measurement of enzyme activity corresponding to various P450, various P450
Western blotting for proteins, various P450 m
Northern hybridization or R corresponding to RNA
Known techniques such as the T-PCR method can be used. As the test substance, the same substances as described above are used. (f) Analysis method for promoting the activity of xenobiotic and / or endogenous substrate metabolizing enzyme: for example, exposing a test substance to cells to increase xenobiotic and / or endogenous substrate metabolizing enzyme activity, increasing the amount of enzyme Alternatively, by detecting an increase in the amount of transcription of a gene encoding an enzyme or the like, a xenobiotic and / or
Alternatively, it is possible to analyze the promotion of the activity of endogenous substrate metabolizing enzymes (J. Rueff et al., Mutation Research 353 (1996) 151-).
176). Specifically, an increase in cytochrome P450 enzyme activity,
It is possible by detecting an increase in the amount of protein and an increase in mRNA. As a detection method, a known method such as measurement of enzyme activity corresponding to various P450s, western blotting corresponding to various P450 proteins, Northern hybridization corresponding to various P450 mRNAs, or RT-PCR can be used. As the test substance, the same substances as described above are used.

(G) Method for analyzing cytotoxicity due to xenobiotic and / or endogenous substrate metabolism: For example, it is possible to analyze cytotoxicity due to xenobiotic and / or endogenous substrate metabolism by exposing a test substance to cells. is there. Specifically, analysis is performed by observing changes in cell morphology, changes in the number of living cells, leakage of intracellular enzymes, changes in cell surface structure, or changes in intracellular enzymes due to exposure to test substances.
(D. Wu et al. Journalof Biological Chemistry, 271,
(1996) 23914-23919). As the test substance, the same substances as described above are used. (h) Method for analyzing genotoxicity due to xenobiotic and / or endogenous substrate metabolism: for example, exposing a test substance to cells,
The genotoxicity due to xenobiotic and / or endogenous substrate metabolism can be analyzed by subjecting the cells to a chromosomal aberration test, a micronucleus test, or the like. Furthermore, after the test substance is exposed to the cells, analysis can be performed by subjecting the test substance changed by the cells to a chromosome aberration test, micronucleus test, reversion test, etc. by evaluating it with an appropriate evaluation system. Is
(J. Rueff et al. Mutation Research 353 (1996) 151-176,
ME McManus et al. Methods in Enzymology vol.
206 pp.501-508 Ed. By MR Waterman et al. Academic P
ress 1991)). As the test substance, the same substances as described above are used. (i) Method for analyzing carcinogenicity by xenobiotic and / or endogenous substrate metabolism: for example, exposing a test substance to cells,
Carcinogenicity due to xenobiotics and / or endogenous substrate metabolism can be analyzed by subjecting cells to chromosomal aberration tests, DNA modification, and the like. Furthermore, after exposing the test substance to the cells, analysis can be performed by evaluating the test substance changed by the cells with a carcinogenesis evaluation system using an appropriate chemical substance (J. Rueff et al., Mutation Research 353 (1991)).
6) 151-176, K. Kawajiri et al., Cytochromes P450 metab
olic and toxicological aspects pp77-98 ed. by C.Io
annides CRC press (1996)). As the test substance, the same substances as described above are used. (j) A method for analyzing mutagenicity by xenobiotic and / or endogenous substrate metabolism: for example, exposing a test substance to cells,
By subjecting the cells to a chromosomal aberration test, a micronucleus test, and the like, it is possible to analyze mutagenicity by xenobiotic and / or endogenous substrate metabolism. Furthermore, after the test substance is exposed to the cells, analysis can be performed by subjecting the test substance changed by the cells to a chromosome aberration test, micronucleus test, reversion test, etc. by evaluating it with an appropriate evaluation system. Is
(J. Rueff et al. Mutation Research 353 (1996) 151-17
6). As the test substance, the same substances as described above are used. (k) Method for analyzing hepatotoxicity due to xenobiotic and / or endogenous substrate metabolism: for example, by exposing the test substance to cells and observing the onset of cytotoxicity, or exposing the test substance to cells Thereafter, the test substance changed by the cells is administered to other hepatocytes, liver sections, excised livers, or experimental animals, and the resulting changes in cells, tissues, and living bodies are observed, whereby xenobiotic and / or endogenous substrate metabolism is observed. Hepatotoxicity can be analyzed. As the test substance, the same substances as described above are used. (l) Method for analyzing xenobiotics and / or endogenous substrates acting on the liver: for example, by observing the change in cells due to exposure of the test substance to the cells, or exposing the test substance to the cells After that, the test substance changed by the cells is administered to other hepatocytes, liver sections, isolated liver, or experimental animals, and the changes in cells, tissues, and living bodies are observed, and the expression of the effect on the liver is analyzed. It is possible to
As the test substance, the same substances as described above are used.

In the present specification, when bases and the like are represented by abbreviations, IUPAC-IUB Commision on Biochem.
This is based on the abbreviation by ical Nomenclature or the abbreviation commonly used in the art, and examples thereof are described below. A: adenine T: thymine G: guanine C: cytosine

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] RT performed in Example 3 described later
-Shows the synthetic primer base sequence used for CYP1A1 in the PCT method. [SEQ ID NO: 2] RT performed in Example 3 described later
-Shows the synthetic primer base sequence used for CYP1A1 in the PCT method. [SEQ ID NO: 3] RT performed in Example 3 described later
-Shows the synthetic primer base sequence used for CYP1A2 in the PCT method. [SEQ ID NO: 4] RT performed in Example 3 described later
-Shows the synthetic primer base sequence used for CYP1A2 in the PCT method. [SEQ ID NO: 5] RT performed in Example 3 described later
-Shows the base sequence of a synthetic primer used for CYP3A in the PCT method. [SEQ ID NO: 6] RT performed in Example 3 described later
-Shows the base sequence of a synthetic primer used for CYP3A in the PCT method.

OUMS-29 obtained in Example 1 described later
The strain was deposited on April 21, 1998 with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology (NIBH) under the deposit number FER.
Deposit No. FIFO 50 as MBP-6328 with the Foundation and Fermentation Research Institute (IFO) from April 21, 1998.
487 has been deposited.

[0018]

EXAMPLES Examples of the present invention will be described below in detail, but the present invention is not limited thereto. Unless otherwise specified, individual genetic manipulation techniques are described in Sambrook et al.'S manual ((Molecular Cloning: AL).
aboratory Manual) and Cold Spring Harbor Laboratory Press).

[0019]

[Example 1] Establishment of hepatocyte cell line Establishment of immortalized cells by introducing the SV40 T antigen gene was carried out according to an established method (M. Miyazaki et al., Experim
ental Cell Research, 206, 27-35 (1993)). The liver was removed from a 21-week-old dead human fetus, and primary hepatocyte cells were separated by a known collagenase perfusion method. These cells were added to Williams medium (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum.
The seedling was cultured. After 24 hours of culture, use the lipofection method
Introduction of SV40 T antigen gene was performed. The plasmid pSV3Neo (PJ Southern and P. Be
rg, J. Mol. Appl. Genet., 1, 327-341). For lipofection and thereafter, a serum-free complete synthetic medium (ASF104, Ajinomoto) was used as a culture medium throughout. On the third day after transfection, subculture was performed to promote the proliferation of hepatocytes, and G418 (GIBCO Laboratoyies) at a final concentration of 100 μg / ml was added. The cells were further cultured for 2 days to select neomycin-resistant cells. did. Thereafter, a clone showing clear G418 resistance was obtained by culturing for 30 days.
It was named. This clone is ASF1 for more than 300 generations.
Since the cells continued to grow in the 04 medium, the cells were considered immortalized.

[0020]

Example 2 Measurement of Drug Metabolizing Enzyme Activity of OUMS-29 Strain The OUMS-29 cells which reached the confluence after culturing in ASF104 medium for 5 to 7 days were collected and suspended in 0.1 M phosphate buffer (pH 7.6). It was crushed by an ultrasonic generator and used as an enzyme source for measurement of the following enzyme activities.

(1) Cytochrome P450 reductase activity The measurement was carried out based on the method described in Biological Pharmacology, 37, 4111-4116, 1988. That is, cytochrome C (reduced nicotinamide adenine dinucleotide phosphate) and an enzyme source derived from OUMS-29 were used in the presence of cytochrome C (Wako Pure Chemical Industries) as a substrate, and cytochrome P450 reductase activity was measured based on the reduction of cytochrome C. As a result, protein 1
The enzyme source derived from the OUMS-29 strain showed an enzyme activity of 8 units with the activity of reducing 1 nmol of cytochrome C per milligram per minute as 1 unit.

(2) Glucurosyltransferase activity Measurement was performed based on the method described in Biological Pharmacology, 37, 4111-4116, 1988. That is, using 1-naphthol (sigma) as a substrate, UDP-glucuronic acid (sigma) and OU
The amount of 1-naphthol glucuronide produced was measured in the presence of an MS-29-derived enzyme source. As a result, the enzyme source derived from the OUMS-29 strain showed an enzyme activity of 196 units, with the activity of producing 1 picomole of 1-naphthol glucuronide per milligram of protein per minute as 1 unit.

(3) Mixed Function Oxidation Reaction (MFO) Activity The measurement was carried out based on the method described in Biological Pharmacology, 42, 1307-1313, 1991. That is, using ethoxyresorufin (sigma), pentoxyresorufin (sigma), benzyloxyresorufin (sigma) and methoxyresorufin (sigma) as substrates, coexisting NADPH and an OUMS-29-derived enzyme source to remove each substrate. The amount of product resulting from the alkylation was measured. As a result, the enzyme source derived from the OUMS-29 strain was 0.25 units when ethoxyresorufin was used as the substrate, and pentoxyresorufin was used as the substrate, assuming that the activity to produce 1 pmol product per milligram of protein per minute was 1 unit. The enzyme activity was 0.47 unit when the enzyme was used, 0.38 unit when benzyloxyresorufin was used as a substrate, and 0.32 unit when methoxyresorufin was used as a substrate.

[0024]

Example 3 Expression of Cytochrome P450 Gene Expression of cytochrome P450 in the OUMS-29 strain is analyzed by examining the level of mRNA amount by a known RT-PCR method using DNA primers specific for different cytochrome P450s. be able to. These primers can be prepared from the sequence of each cytochrome P450 available from the Gene Bank database. GeneBank
CYP1A1 is K03191, CYP
1A2 is M55053, CYP2E1 is J02625, CYP3A4 is J04449, CYP3A
5 is J04813 and CYP3A7 is D00408. Individual primers are 5'-ATGCTTTTCCCAATCTCCATGTGC for CYP1A1.
And 5'-TTCAGGTCCTTGAAGGCATTCAGG. For CYP1A2, 5'-GGAAGAACCCGCACCTGGCACTGT and 5'-AAACAGCATC
ATCTTCTCACTCAA. 5'-ATGGCTCTCATCCC for CYP3A
AGACTTG and 5'-GGAAAGACTGTTATTGAGAGA were used respectively. The annealing temperature for RT-PCR was 5 for CYP1A1.
The test was performed at 5 ° C, 65 ° C for CYP1A2, 55 ° C for CYP3A, 65 ° C for CYP2E1, and 28 to 36 cycles. OU
The MS-29 strain was cultured for 5 to 7 days, and confluent cells were collected. From this, RNA was extracted using an RNAeasy kit (Qiagen). This RNA and
The primers specific to each cytochrome P450 determined above are subjected to reverse transcription from mRNA and a PCR reaction using a one-step PCR kit (Takara Shuzo), then separated on an agarose gel, and visualized with ultraviolet light in the presence of ethidium bromide.
The result is shown in FIG. 76 predicted for each CYP1A1
3bp, 1180bp predicted for CYP1A2, 68 predicted for CYP3A
A signal near 0 bp is detected and the corresponding gene OUMS-29
Expression in the strain was confirmed.

[0025]

Example 4 Induction of Cytochrome P450 Gene Expression OUMS-29 Cultured for 5 to 7 Days to Confluence
Cells were given a final concentration of 0-1000 nM 3-methylcholanthrene (3-MC) (FIG. 2), 0-50000 nM benzpyrene (BP) (FIG. 3), 0-25 mM phenobarbitone (P
B) (FIG. 4) or 0 to 1000 nM dexamethasone (DEX) (FIG. 5) was added, and the cells were further cultured for 1 day. After culturing, isolate the cells and use the methods described above to
Was extracted and an RT-PCR reaction was performed. Annealing temperature in RT-PCR method is 55 ℃ for CYP1A1, 65 ℃ for CYP1A2, CYP3
A was carried out under the conditions of 55 ° C. and the number of cycles was 28 to 36. The number of cycles of beta-actin used as a control was 15 cycles. At this time, the total mR of each sample was determined based on the amount of beta actin mRNA expressed in all tissues to the same extent.
To correct the NA amount, an actin-competitive RT-PCR kit (Takara Shuzo) was used. The results are shown in FIGS. CY
P1A1 expression is 3-methylcholanthrene, benzpyrene,
With the addition of phenobarbitone, the expression of CYP1A2
By adding methylcholanthrene and benzopyrene, CYP3
A expression is enhanced by the addition of dexamethasone,
It was confirmed that 29 strains had the ability to express the gene encoding cytochrome P450.

[0026]

Industrial Applicability The immortalized liver cell line derived from human normal cells of the present invention, that is, the enzyme activity related to the metabolism of xenobiotics in the liver or the ability to express the gene encoding the enzyme related to metabolism of xenobiotics in the liver is retained. Human-derived immortalized liver cell lines
For example, it is useful for screening a compound having a prophylactic or therapeutic effect on liver dysfunction or a salt thereof.

[0027]

[Sequence list]

 [SEQUENCE LISTING] <110> Takeda Chemical Industries, Ltd. <120> Novel immortalized human hepatic cell line <130> A98087 <150> JP 10-119394 <151> 1998-04-28 <160> <210> 1 <211 > 24 <212> DNA <213> Artificial Sequence <220> <223> <400> 1 ATGCTTTTCC CAATCTCCAT GTGC 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> <400 > 2 TTCAGGTCCT TGAAGGCATT CAGG 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> <400> 3 GGAAGAACCC GCACCTGGCA CTGT 24 <210> 4 <211> 24 <212> DNA < 213> Artificial Sequence <220> <223> <400> 4 AAACAGCATC ATCTTCTCAC TCAA 24 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 5 ATGGCTCTCA TCCCAGACTT G 21 < 210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 6 GGAAAGACTG TTATTGAGAG A 21

[0028]

[Brief description of the drawings]

FIG. 1 shows the results of an RT-PCR method performed in Example 3 (electropherogram). In the figure, Markers 2, 5, and 6 are DNAs, respectively.
The molecular weight marker (made by Nippon Gene) is shown.

FIG. 2 shows the results of RT-PCR after addition of 3-methylcholanthrene (3-MC) performed in Example 4.

FIG. 3 shows the results of RT-PCR performed after adding benzpyrene (BP) in Example 4.

FIG. 4 Phenobarbitone (PB) performed in Example 4
Shows the results of the RT-PCR method after addition of.

FIG. 5: Dexamethasone (DEX) performed in Example 4
Shows the results of the RT-PCR method after addition of.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/566 33/566 C12N 5/00 B (72) Inventor Satoshi Asahi Toyonaka, Osaka 1-424 A-307 Kamishinda (72) Inventor Sumie Yoshitomi 1-27-4 Taishibashi, Asahi-ku, Osaka-shi, Osaka

Claims (11)

[Claims] 1. An immortalized liver cell line derived from a human normal cell, which retains the activity of an enzyme involved in the metabolism of a xenobiotic in the liver or the ability to express a gene encoding an enzyme involved in the metabolism of a xenobiotic in the liver. 2. The enzyme activity is NADPH cytochrome P450 reductase activity, gluclosyltransferase activity, ethoxyresorufin dealkylation activity, benzyloxyresorufin dealkylation activity, pentoxylresorufin dealkylation activity, methoxyresorufin. 2. The cell line according to claim 1, which has dealkylation activity, flavin monooxygenase activity, epoxyhydratase activity, sulfotransferase activity or glutathione S-transferase activity. 3. The cell line according to claim 1, wherein the enzyme is NADPH cytochrome P450 reductase, NADPH cytochrome P450, flavin monooxygenase, epoxyhydratase, gluclosyltransferase, sulfotransferase or glutathione S-transferase. 4. The method of claim 4, wherein NADPH cytochrome P450 is CYP1A1, CYP1A2.
4. The cell line according to claim 3, which is CYP3A. 5. The cell line according to claim 1, wherein the cell line is FERM BP-6328. 6. The method according to claim 1, wherein a T antigen gene derived from SV (simian virus) 40 is introduced into normal human liver cells. 7. The method according to claim 6, wherein the normal human liver cells are liver cells derived from a human fetus. 8. The use of the cell line according to claim 1 for inhibiting or promoting the activity of an enzyme involved in the metabolism of xenobiotics in the liver, or the expression of a gene encoding an enzyme involved in the metabolism of xenobiotics in the liver. A method for screening for a compound that inhibits or promotes or a salt thereof. 9. A method for inhibiting or promoting the activity of an enzyme involved in the metabolism of a xenobiotic in the liver, obtained by using the screening method according to claim 8, or expressing a gene encoding an enzyme involved in the metabolism of a xenobiotic in the liver. Or a salt thereof that inhibits or promotes 10. Use of the cell line according to claim 1, wherein (a) an enzyme involved in xenobiotic and / or endogenous substrate metabolism, (b) xenobiotic and / or endogenous substrate metabolism pathway, (c) chemical structure of xenobiotic and / or endogenous substrate metabolite, (d) inhibition of xenobiotic and / or endogenous substrate metabolizing enzyme, (e) promotion of xenobiotic and / or endogenous substrate metabolizing enzyme activity (F) cytotoxicity due to xenobiotic and / or endogenous substrate metabolism, (g) genotoxicity due to xenobiotic and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotic and / or endogenous substrate metabolism, (i) mutagenicity due to xenobiotic and / or endogenous substrate metabolism,
(j) A method for analyzing hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism or (k) xenobiotics and / or endogenous substrates acting on the liver. 11. A method for preparing a xenobiotic and / or endogenous substrate metabolite, comprising using the cell line according to claim 1.