WO2023238877A1 - Insulin-resistant disease model cell - Google Patents
Insulin-resistant disease model cell Download PDFInfo
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- WO2023238877A1 WO2023238877A1 PCT/JP2023/021083 JP2023021083W WO2023238877A1 WO 2023238877 A1 WO2023238877 A1 WO 2023238877A1 JP 2023021083 W JP2023021083 W JP 2023021083W WO 2023238877 A1 WO2023238877 A1 WO 2023238877A1
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- insulin resistance
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- gene
- liver
- hepatokine
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 8
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the present invention relates to human hepatocytes rich in intracellular triglycerides (TG).
- TG intracellular triglycerides
- the cells can be used as an insulin resistance model.
- Insulin has the function of promoting sugar metabolism in the liver, skeletal muscles, adipose tissue, etc., and these tissues regulate blood sugar by taking in sugar, using it for energy, and storing it as fat. . Insulin resistance occurs because even though insulin is secreted into the blood from the pancreas, the response to insulin in the liver, skeletal muscle, and adipose tissue is slowed down (reduced sensitivity), meaning that insulin's ability to lower blood sugar is insufficient. It is not fully utilized and the condition develops into diabetes. Obesity is said to be the biggest factor causing this insulin resistance (Non-Patent Document 1: Barbara B. Kahn, Jeffrey S. Flier, Obesity and insulin resistance. J Clin Invest.
- the causes of insulin resistance can be broadly classified into genetic predisposition, lifestyle habits such as obesity, and hyperglycemia.
- Insulin resistance associated with obesity is caused by TNF- ⁇ and free fatty acids secreted from enlarged fat cells. There is. Insulin resistance is a strong factor in the development of type 2 diabetes when the insulin secretion and proliferation abilities of ⁇ cells are impaired (Non-Patent Document 2: Vandana Saini, Molecular mechanisms of insulin resistance in type 2 diabetes). mellitus. 2010 Jul 15; 1(3): 68-75.).
- the present inventors have discovered that hepatocytes derived from non-human animals in which all or part of the liver has been replaced with human hepatocytes can be used as the above insulin resistance model. They discovered this and completed the present invention. That is, the present invention is as follows.
- the bad hepatokine is at least one selected from the group consisting of SePP, LECT2, and FetuinA.
- the cell according to [1] in which gene expression of a patokine (good hepatokine) gene for improving insulin resistance is reduced.
- [6] The cell according to [5], wherein the good hepatokine is at least one selected from the group consisting of FGF21, SHBG, and ANGPTL6.
- [7] Detect the hepatokine gene by contacting the cell described in any one of [1] to [6] with a candidate substance, and treat fatty liver and/or insulin resistance pathological conditions using the obtained detection result as an indicator. How to screen drugs.
- [8] The method according to [7], wherein the insulin resistance disease is type 2 diabetes.
- [9] The method according to [7], wherein the fatty liver is non-alcoholic fatty liver.
- the present invention provides model cells having pathological conditions or characteristics of insulin resistance.
- the present invention makes it possible to screen drugs for diseases related to insulin resistance.
- FIG. 2 is a diagram showing the expression levels of bad hepatokine genes (RT-PCR). It is a figure showing the expression level of a bad hepatokine gene.
- the present invention relates to model cells for fatty liver or insulin resistance pathologies, comprising hepatocytes derived from a non-human animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes.
- the liver controls glucose homeostasis by modifying whole-body insulin sensitivity through the production of secreted proteins called hepatokines.
- hepatokines secreted from fatty liver induce insulin resistance in various tissues. Therefore, we analyzed the expression pattern of hepatokines associated with insulin resistance using model cells that reproduce fatty liver conditions. Furthermore, we investigated the correlation between intracellular neutral fat content and hepatokine expression levels by drug treatment.
- human hepatocyte chimera non-human animals reproduce the fatty liver condition described above. We found that it can be used as an insulin resistance model.
- the animal from which insulin resistance model cells are derived is a human hepatocyte chimera non-human animal, and hepatocytes derived from the animal are used as model cells.
- the "non-human animal” is preferably a mammal, more preferably a rodent. Examples of rodent animals include mice, rats, guinea pigs, squirrels, hamsters, etc., but mice and rats, which are commonly used as experimental animals, are preferred.
- a human hepatocyte chimeric non-human animal can be obtained by transplanting human hepatocytes into a liver-damaged, immunodeficient non-human animal according to known techniques (for example, JP-A-2002-45087).
- liver-impaired, immunodeficient non-human animals are produced by subjecting a genetically immunodeficient non-human animal to a liver-damage-inducing treatment, or by subjecting an genetically liver-damaged animal to an immunodeficiency-inducing treatment. It can be made.
- genetically immunodeficient animals include SCID mice, NUDE mice, RAG2 knockout mice, NOD mice, NOG mice, etc.
- Animals with genetic liver damage include proteins specifically expressed in hepatocytes.
- a transgenic animal into which a liver damage-inducing protein gene (uPA gene, tPA gene, etc.) linked under the control of an enhancer and/or promoter can be used.
- the "human hepatocytes" to be transplanted into a liver-impaired, immunodeficient non-human animal may be human-derived hepatocytes, such as those isolated from human liver tissue according to a conventional method such as collagenase perfusion method.
- Human hepatocytes can be used.
- the human liver tissue may be a liver tissue derived from a healthy person or a liver tissue derived from a patient suffering from a disease such as fatty liver or liver cancer, but preferably a liver tissue derived from a healthy person. be.
- the collected human hepatocytes can be used as they are, they may be purified using a monoclonal antibody that specifically recognizes human hepatocytes or non-human animal hepatocytes.
- Human hepatocytes are transplanted into an immunodeficient non-human animal with liver damage, either by transplanting them into the liver via the spleen of the non-human animal, or by directly transplanting them from the portal vein.
- the non-human animal obtained by the above method is an animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes.
- a commercially available mouse (“PXB Mouse", Phoenix Bio Co., Ltd.) can also be used as a mouse in which at least 70% of the liver has been replaced with human hepatocytes.
- Model Cells The model cells of the present invention are hepatocytes collected from the human hepatocyte chimeric non-human animal described above.
- the model cells of the present invention can be cultured and maintained in the same manner as general animal cell cultures.
- tissue culture dishes tissue culture dishes, multi-dishes, microplates, media commonly used for culturing animal cells (e.g., Dulbecco's modified Eagle's medium (DMEM), Williams medium E, RPMI-1640 medium etc.), and further add fetal bovine serum, a buffer, an antibiotic, a pH adjuster, etc. as necessary for maintenance or subculture.
- DMEM Dulbecco's modified Eagle's medium
- RPMI-1640 medium fetal bovine serum
- the model cells of the present invention can be used 7 to 17 days (Day 7 to 17), preferably 8 to 13 days (Day 8 to 13) after collection from a hepatocyte chimeric non-human animal.
- Hepatocytes collected from the above-mentioned "PXB mouse” are also referred to as "PXB-cells.”
- PXB-cells that have an extended period of accumulating excessive TG within the cells are also referred to as "PXB-cells LA”. Since PXB-cells and PXB-cells LA immediately after isolation from PXB mice have the characteristic of accumulating excessive TG within the cells, it is thought that they reproduce the obese state and furthermore reproduce insulin resistance.
- Excess TG means that TG is present in abundance, and refers to a state in which, for example, distinct lipid droplets composed of TG are observed in the cytoplasm.
- the insulin resistance model cells of the present invention can be characterized using the expression of genes ("insulin resistance related genes") and protein expressions targeting molecules related to insulin resistance (mainly hepatokines) in the cells. I can do it.
- insulin resistance-related gene refers to a gene whose expression level changes (increases or decreases) in the model cells of the present invention compared to healthy hepatocytes.
- Hepatokines include, for example, selenoprotein P (SePP), a secreted protein derived from the liver, leukocyte-derived chemotaxin 2 (LECT2), a cytokine related to hepatitis, and fibroblast growth factor 21 (one of the fibroblast growth factors).
- SePP selenoprotein P
- LECT2 leukocyte-derived chemotaxin 2
- fibroblast growth factor 21 one of the fibroblast growth factors
- FGF21 Fibroblast growth factor 21
- SHBG Fibroblast growth factor 21
- ANGPTL6 Angiopoietin-Like Protein 6
- Fetuin-A which is an inflammatory signal transduction activator.
- hepatokines include both bad hepatokines and good hepatokines.
- Bad hepatokines are hepatokines that induce insulin resistance
- good hepatokines are hepatokines that improve insulin resistance.
- the expression of bad hepatokine genes or proteins is enhanced, and the expression of good hepatokine genes or proteins is decreased.
- “Enhanced” means that the expression of the gene or protein is higher than that of cells used as a control or standard, or that the expression of the gene or protein associated with the hepatokine is significantly changed.
- reduced means lower than the expression of the gene or protein in cells used as a control or standard, or a significant change in the expression of the gene or protein associated with the hepatokine. do.
- Bad hepatokines include SePP, LECT2, Fetuin-A, and the like.
- Examples of good hepatokines include FGF21, SHBG, and ANGPTL6.
- the gene encoding fatty acid synthase (gene name: FASN), the gene encoding SREBP-1 (gene name: SREBF1), the gene encoding glucose-6-phosphatase (G6PC), Gene encoding cholesterol 7 ⁇ -hydroxylase (CYP7A1), gene encoding cholesteryl ester transfer protein (CETP), gene encoding glucokinase (GCK), gene encoding phosphoenolpyruvate carboxykinase 1 (PCK1), etc.
- FASN fatty acid synthase
- SREBP-1 gene name: SREBF1
- G6PC Gene encoding glucose-6-phosphatase
- CYP7A1 Gene encoding cholesteryl ester transfer protein
- CETP gene encoding glucokinase
- PCK1 phosphoenolpyruvate carboxykinase 1
- the screening method of the present invention involves contacting the model cells of the present invention with a candidate substance, detecting the hepatokine gene expressed by the cells after contact, and using the obtained detection results as an indicator of fatty liver and/or insulin resistance. This is a method for screening therapeutic or preventive drugs for pathological conditions. Specifically, a candidate substance is added to the culture solution of the model cells of the present invention, and then hepatokine or hepatokine gene secreted from the cells is measured and evaluated.
- the fixed period for adding the candidate substance is, for example, about 8 to 10 days after collection, but is not particularly limited.
- Hepatokine can be detected by RT-PCR, Western blotting (WB), immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), intracellular and extracellular lipid content measurement, mass spectrometry, etc. , or a suitable combination.
- WB Western blotting
- ELISA enzyme-linked immunosorbent assay
- intracellular and extracellular lipid content measurement mass spectrometry, etc. , or a suitable combination.
- Candidate substances to be evaluated are not particularly limited, and include, for example, various natural or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA, RNA), oligonucleotides (siRNA, etc.) ), peptide nucleic acids (PNA), etc.), low-molecular or high-molecular organic compounds, and the like.
- the amount of bad hepatokine secreted decreases compared to the control, 2) the amount of bad hepatokine secreted decreases or becomes below the detection limit, and a decrease in the amount of extracellular and extracellular lipids is observed.
- the candidate substance was able to suppress hepatokine secretion, and may be associated with fatty liver, type 2 diabetes, or other diseases. It can be evaluated as a drug that can be used as a combination therapeutic and preventive drug.
- Substances evaluated to be effective in treating or preventing fatty liver or type 2 diabetes by controlling hepatokine production by the screening method of the present invention are drugs for preventing or treating diabetes, or drugs used in combination with other drugs. Can be used as a concomitant drug. Furthermore, since diabetes is a risk factor for angiogenesis, administering drugs discovered through screening during diabetes treatment or during remission can be used to examine the possibility of reducing the incidence of diabetes in the future. It is possible to develop therapeutic drugs for all diseases caused by the secretion of hepatokines, which are discovered in humans.
- ⁇ Method> Gene and protein expression of hepatokines involved in insulin resistance was analyzed in PXB-cells (13 days after collection from PXB mice), unfrozen human-derived hepatocytes (commercially available), HepG2, and HuH7 cells.
- - PXB-cells and PXB-cells LA both 9 days after harvesting from PXB mice) were used to analyze the gene expression of hepatokine involved in insulin resistance in both.
- ⁇ We analyzed lipid metabolism-related and hepatokine gene expression after adding an antidiabetic drug that lowers intracellular fat content in a fatty liver model for 24 hours to PXB-cells (6 days after collection from PXB mice). .
- the evaluation was performed by RT-PCR and WB, and the evaluation items were mainly hepatokines such as SePP, ANGPTL6, LECT2, and FGF21.
- RT-PCR was performed under the following conditions.
- Total RNA was isolated using QuickGene RNA cultured cell kit S.
- Template cDNA was synthesized from total RNA (5 ⁇ g) using Prime-Script RT reagent Kit (reverse transcription sample).
- SsoAdvanced Universal SYBR Green Supermix solution 1/2 diluted with an equal volume of MiliQ water, 21 ⁇ L) containing 0.2 ⁇ M of each primer was added to the reverse transcription sample (4 ⁇ L).
- PCR was performed using a fluorescent temperature cycler (CFX Connect TM Real-Time PCR Detection System). The reaction conditions were 40 cycles of 95°C for 5 seconds (denaturation) and 60°C for 30 seconds (annealing and extension reaction). Normalization was performed based on the expression level of glyceraldehyde-3-phosphate dehydrogenase.
- Table 3 shows the results of gene expression levels (RT-PCR) of hepatokines associated with insulin resistance.
- PXB-cells The expression levels of hepatokine genes associated with insulin resistance in LA were similar to those in PXB-cells.
- Table 4 shows the results of gene expression levels (RT-PCR) of hepatokines related to lipid metabolism and insulin resistance after treatment with antidiabetic drugs.
- both drugs suppressed the expression of LECT2 (bad hepatokine) and enhanced the expression of FGF21 (good hepatokine).
- the gene expression levels of hepatokines associated with insulin resistance in PXB-cells LA are similar to those in PXB-cells, and this indicates that compared to other liver-derived cells, PXB-cells and LA PXB-cells LA was shown to be an excellent cell in the search system for insulin resistance improving drugs targeting the liver.
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Abstract
Provided is a model cell for fatty liver or an insulin-resistant disease state, the model cell comprising a hepatic cell derived from a non-human animal in which at least 70% of the liver is replaced by human hepatic cells.
Description
本発明は、細胞内トリグリセリド（ＴＧ）が豊富なヒト肝細胞に関する。当該細胞は、インスリン抵抗性モデルとして利用できる。
The present invention relates to human hepatocytes rich in intracellular triglycerides (TG). The cells can be used as an insulin resistance model.
インスリンは、肝臓、骨格筋、脂肪組織等で行われる糖代謝を促す機能を有しており、これらの組織が糖を取り込んでエネルギーに利用し、脂肪として蓄えることで血糖の調整を行っている。インスリン抵抗性は、すい臓からインスリンが血液中に分泌されていても肝臓、骨格筋、脂肪組織でのインスリンに対する反応が鈍くなっているために（感受性低下）、インスリンの血糖を下げる働きが十分に発揮されず、糖尿病に発展する状態となっている。このインスリン抵抗性を引き起こす最大の要因は肥満であるといわれている（非特許文献１：Barbara B. Kahn, Jeffrey S. Flier, Obesity and insulin resistance. J Clin Invest. 2000;106(4):473-481.）。
インスリン抵抗性の成因は、遺伝的素因、肥満等の生活習慣、高血糖に大別され、 肥満に伴うインスリン抵抗性は、肥大した脂肪細胞から分泌されるＴＮＦ－αや遊離脂肪酸が関与している。インスリン抵抗性は、β細胞のインスリン分泌能や増殖能に障害のある場合には、２型糖尿病の強力な発症要因となる（非特許文献２：Vandana Saini, Molecular mechanisms of insulin resistance in type 2 diabetes mellitus．2010 Jul 15; 1(3): 68-75.）。 Insulin has the function of promoting sugar metabolism in the liver, skeletal muscles, adipose tissue, etc., and these tissues regulate blood sugar by taking in sugar, using it for energy, and storing it as fat. . Insulin resistance occurs because even though insulin is secreted into the blood from the pancreas, the response to insulin in the liver, skeletal muscle, and adipose tissue is slowed down (reduced sensitivity), meaning that insulin's ability to lower blood sugar is insufficient. It is not fully utilized and the condition develops into diabetes. Obesity is said to be the biggest factor causing this insulin resistance (Non-Patent Document 1: Barbara B. Kahn, Jeffrey S. Flier, Obesity and insulin resistance. J Clin Invest. 2000;106(4):473 -481.).
The causes of insulin resistance can be broadly classified into genetic predisposition, lifestyle habits such as obesity, and hyperglycemia. Insulin resistance associated with obesity is caused by TNF-α and free fatty acids secreted from enlarged fat cells. There is. Insulin resistance is a strong factor in the development of type 2 diabetes when the insulin secretion and proliferation abilities of β cells are impaired (Non-Patent Document 2: Vandana Saini, Molecular mechanisms of insulin resistance in type 2 diabetes). mellitus. 2010 Jul 15; 1(3): 68-75.).
インスリン抵抗性の成因は、遺伝的素因、肥満等の生活習慣、高血糖に大別され、 肥満に伴うインスリン抵抗性は、肥大した脂肪細胞から分泌されるＴＮＦ－αや遊離脂肪酸が関与している。インスリン抵抗性は、β細胞のインスリン分泌能や増殖能に障害のある場合には、２型糖尿病の強力な発症要因となる（非特許文献２：Vandana Saini, Molecular mechanisms of insulin resistance in type 2 diabetes mellitus．2010 Jul 15; 1(3): 68-75.）。 Insulin has the function of promoting sugar metabolism in the liver, skeletal muscles, adipose tissue, etc., and these tissues regulate blood sugar by taking in sugar, using it for energy, and storing it as fat. . Insulin resistance occurs because even though insulin is secreted into the blood from the pancreas, the response to insulin in the liver, skeletal muscle, and adipose tissue is slowed down (reduced sensitivity), meaning that insulin's ability to lower blood sugar is insufficient. It is not fully utilized and the condition develops into diabetes. Obesity is said to be the biggest factor causing this insulin resistance (Non-Patent Document 1: Barbara B. Kahn, Jeffrey S. Flier, Obesity and insulin resistance. J Clin Invest. 2000;106(4):473 -481.).
The causes of insulin resistance can be broadly classified into genetic predisposition, lifestyle habits such as obesity, and hyperglycemia. Insulin resistance associated with obesity is caused by TNF-α and free fatty acids secreted from enlarged fat cells. There is. Insulin resistance is a strong factor in the development of type 2 diabetes when the insulin secretion and proliferation abilities of β cells are impaired (Non-Patent Document 2: Vandana Saini, Molecular mechanisms of insulin resistance in type 2 diabetes). mellitus. 2010 Jul 15; 1(3): 68-75.).
今後、２型糖尿病をはじめとするインスリン抵抗性疾患のより良い治療又は予防薬を開発するには、ヒトと同じ遺伝子背景を持つインスリン抵抗性の病態を有するモデルや肝細胞モデルを作製することが求められる。
In the future, in order to develop better treatments or preventive drugs for insulin resistance diseases such as type 2 diabetes, it will be necessary to create a model with the same genetic background as humans and a hepatocyte model with the pathology of insulin resistance. Desired.
本発明者は、上記課題を解決するために鋭意検討を行った結果、肝臓の全部又は一部がヒト肝細胞に置換された非ヒト動物由来の肝細胞が、上記インスリン抵抗性モデルとして利用できることを見出し、本発明を完成するに至った。
すなわち、本発明は以下の通りである。 As a result of intensive studies to solve the above problems, the present inventors have discovered that hepatocytes derived from non-human animals in which all or part of the liver has been replaced with human hepatocytes can be used as the above insulin resistance model. They discovered this and completed the present invention.
That is, the present invention is as follows.
すなわち、本発明は以下の通りである。 As a result of intensive studies to solve the above problems, the present inventors have discovered that hepatocytes derived from non-human animals in which all or part of the liver has been replaced with human hepatocytes can be used as the above insulin resistance model. They discovered this and completed the present invention.
That is, the present invention is as follows.
[１] 肝臓の全部又は一部がヒト肝細胞に置換された非ヒト動物由来の肝細胞を含む、脂肪肝又はインスリン抵抗性病態のモデル細胞。
[２] 非ヒト動物がマウスである[１]に記載の細胞。
[３] インスリン抵抗性惹起へパトカイン（悪玉ヘパトカイン）遺伝子の遺伝子発現が亢進した、[１]に記載の細胞。
[４] 悪玉ヘパトカインが、ＳｅＰＰ、ＬＥＣＴ２及びＦｅｔｕｉｎＡからなる群から選ばれる少なくとも１種である、[２]に記載の細胞。
[５] インスリン抵抗性改善へパトカイン（善玉ヘパトカイン）遺伝子の遺伝子発現が低下した、[１]に記載の細胞。
[６] 善玉ヘパトカインが、ＦＧＦ２１、ＳＨＢＧ及びＡＮＧＰＴＬ６からなる群から選ばれる少なくとも１種である、[５]に記載の細胞。
[７] [１]～[６]のいずれか１項に記載の細胞に候補物質を接触させてヘパトカイン遺伝子を検出し、得られる検出結果を指標として脂肪肝及び／又はインスリン抵抗性病態の治療薬をスクリーニングする方法。
[８] インスリン抵抗性疾患が２型糖尿病である[７]に記載の方法。
[９] 脂肪肝が非アルコール性脂肪肝である[７]に記載の方法。 [1] Model cells for fatty liver or insulin resistance pathology, including hepatocytes derived from non-human animals in which all or part of the liver has been replaced with human hepatocytes.
[2] The cell according to [1], wherein the non-human animal is a mouse.
[3] The cell according to [1], in which gene expression of a patokine (bad hepatokine) gene that induces insulin resistance is enhanced.
[4] The cell according to [2], wherein the bad hepatokine is at least one selected from the group consisting of SePP, LECT2, and FetuinA.
[5] The cell according to [1], in which gene expression of a patokine (good hepatokine) gene for improving insulin resistance is reduced.
[6] The cell according to [5], wherein the good hepatokine is at least one selected from the group consisting of FGF21, SHBG, and ANGPTL6.
[7] Detect the hepatokine gene by contacting the cell described in any one of [1] to [6] with a candidate substance, and treat fatty liver and/or insulin resistance pathological conditions using the obtained detection result as an indicator. How to screen drugs.
[8] The method according to [7], wherein the insulin resistance disease is type 2 diabetes.
[9] The method according to [7], wherein the fatty liver is non-alcoholic fatty liver.
[２] 非ヒト動物がマウスである[１]に記載の細胞。
[３] インスリン抵抗性惹起へパトカイン（悪玉ヘパトカイン）遺伝子の遺伝子発現が亢進した、[１]に記載の細胞。
[４] 悪玉ヘパトカインが、ＳｅＰＰ、ＬＥＣＴ２及びＦｅｔｕｉｎＡからなる群から選ばれる少なくとも１種である、[２]に記載の細胞。
[５] インスリン抵抗性改善へパトカイン（善玉ヘパトカイン）遺伝子の遺伝子発現が低下した、[１]に記載の細胞。
[６] 善玉ヘパトカインが、ＦＧＦ２１、ＳＨＢＧ及びＡＮＧＰＴＬ６からなる群から選ばれる少なくとも１種である、[５]に記載の細胞。
[７] [１]～[６]のいずれか１項に記載の細胞に候補物質を接触させてヘパトカイン遺伝子を検出し、得られる検出結果を指標として脂肪肝及び／又はインスリン抵抗性病態の治療薬をスクリーニングする方法。
[８] インスリン抵抗性疾患が２型糖尿病である[７]に記載の方法。
[９] 脂肪肝が非アルコール性脂肪肝である[７]に記載の方法。 [1] Model cells for fatty liver or insulin resistance pathology, including hepatocytes derived from non-human animals in which all or part of the liver has been replaced with human hepatocytes.
[2] The cell according to [1], wherein the non-human animal is a mouse.
[3] The cell according to [1], in which gene expression of a patokine (bad hepatokine) gene that induces insulin resistance is enhanced.
[4] The cell according to [2], wherein the bad hepatokine is at least one selected from the group consisting of SePP, LECT2, and FetuinA.
[5] The cell according to [1], in which gene expression of a patokine (good hepatokine) gene for improving insulin resistance is reduced.
[6] The cell according to [5], wherein the good hepatokine is at least one selected from the group consisting of FGF21, SHBG, and ANGPTL6.
[7] Detect the hepatokine gene by contacting the cell described in any one of [1] to [6] with a candidate substance, and treat fatty liver and/or insulin resistance pathological conditions using the obtained detection result as an indicator. How to screen drugs.
[8] The method according to [7], wherein the insulin resistance disease is type 2 diabetes.
[9] The method according to [7], wherein the fatty liver is non-alcoholic fatty liver.
本発明により、インスリン抵抗性の病態又は特徴を有するモデル細胞が提供される。本発明により、インスリン抵抗性に関する疾患に対する医薬のスクリーニング等が可能となる。
The present invention provides model cells having pathological conditions or characteristics of insulin resistance. The present invention makes it possible to screen drugs for diseases related to insulin resistance.
１．概要
本発明は、肝臓の全部又は一部（例えば少なくとも７０％）がヒト肝細胞に置換された非ヒト動物由来の肝細胞を含む、脂肪肝又はインスリン抵抗性病態のモデル細胞に関する。 1. Overview The present invention relates to model cells for fatty liver or insulin resistance pathologies, comprising hepatocytes derived from a non-human animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes.
本発明は、肝臓の全部又は一部（例えば少なくとも７０％）がヒト肝細胞に置換された非ヒト動物由来の肝細胞を含む、脂肪肝又はインスリン抵抗性病態のモデル細胞に関する。 1. Overview The present invention relates to model cells for fatty liver or insulin resistance pathologies, comprising hepatocytes derived from a non-human animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes.
肝臓は、ヘパトカインと呼ばれる分泌タンパク質の産生を介して全身のインスリン感受性を修飾することで糖の恒常性を制御している。ヒト臨床では、脂肪肝から分泌されるヘパトカインは、種々の組織におけるインスリン抵抗性を誘発する。そこで、脂肪肝状態を再現するモデル細胞を用いて、インスリン抵抗性に関わるヘパトカインの発現パターンを解析した。さらに、薬剤処理により細胞内の中性脂肪含量とヘパトカインの発現量の相関を調べた。
The liver controls glucose homeostasis by modifying whole-body insulin sensitivity through the production of secreted proteins called hepatokines. In human clinical practice, hepatokines secreted from fatty liver induce insulin resistance in various tissues. Therefore, we analyzed the expression pattern of hepatokines associated with insulin resistance using model cells that reproduce fatty liver conditions. Furthermore, we investigated the correlation between intracellular neutral fat content and hepatokine expression levels by drug treatment.
その結果、肝臓における肝細胞の一部又は全部がヒト肝細胞に置換されている非ヒト動物（「ヒト肝細胞キメラ非ヒト動物」という）から採取された細胞が、上記脂肪肝状態を再現しており、インスリン抵抗性モデルとして利用できることを見出した。
As a result, cells collected from non-human animals in which some or all of the hepatocytes in the liver have been replaced with human hepatocytes (referred to as "human hepatocyte chimera non-human animals") reproduce the fatty liver condition described above. We found that it can be used as an insulin resistance model.
２．インスリン抵抗性モデル細胞の由来となる動物
本発明において、インスリン抵抗性モデル細胞の由来となる動物は、ヒト肝細胞キメラ非ヒト動物であり、当該動物由来の肝細胞をモデル細胞として使用する。「非ヒト動物」は、哺乳動物であることが好ましく、げっ歯類であることがより好ましい。げっ歯類動物としては、マウス、ラット、モルモット、リス、ハムスター等が挙げられるが、実験動物として汎用されているマウス又はラットが好ましい。
ヒト肝細胞キメラ非ヒト動物は、公知の手法（例えば特開２００２－４５０８７号公報）に準じて、肝障害免疫不全非ヒト動物にヒト肝細胞を移植することにより得ることができる。 2. Animal from which insulin resistance model cells are derived In the present invention, the animal from which insulin resistance model cells are derived is a human hepatocyte chimera non-human animal, and hepatocytes derived from the animal are used as model cells. The "non-human animal" is preferably a mammal, more preferably a rodent. Examples of rodent animals include mice, rats, guinea pigs, squirrels, hamsters, etc., but mice and rats, which are commonly used as experimental animals, are preferred.
A human hepatocyte chimeric non-human animal can be obtained by transplanting human hepatocytes into a liver-damaged, immunodeficient non-human animal according to known techniques (for example, JP-A-2002-45087).
本発明において、インスリン抵抗性モデル細胞の由来となる動物は、ヒト肝細胞キメラ非ヒト動物であり、当該動物由来の肝細胞をモデル細胞として使用する。「非ヒト動物」は、哺乳動物であることが好ましく、げっ歯類であることがより好ましい。げっ歯類動物としては、マウス、ラット、モルモット、リス、ハムスター等が挙げられるが、実験動物として汎用されているマウス又はラットが好ましい。
ヒト肝細胞キメラ非ヒト動物は、公知の手法（例えば特開２００２－４５０８７号公報）に準じて、肝障害免疫不全非ヒト動物にヒト肝細胞を移植することにより得ることができる。 2. Animal from which insulin resistance model cells are derived In the present invention, the animal from which insulin resistance model cells are derived is a human hepatocyte chimera non-human animal, and hepatocytes derived from the animal are used as model cells. The "non-human animal" is preferably a mammal, more preferably a rodent. Examples of rodent animals include mice, rats, guinea pigs, squirrels, hamsters, etc., but mice and rats, which are commonly used as experimental animals, are preferred.
A human hepatocyte chimeric non-human animal can be obtained by transplanting human hepatocytes into a liver-damaged, immunodeficient non-human animal according to known techniques (for example, JP-A-2002-45087).
典型的には、肝障害免疫不全非ヒト動物は、遺伝的に免疫不全の非ヒト動物に肝障害誘発処理を行うか、又は遺伝的に肝障害を有する動物に免疫不全誘発処理を施すことにより作製することができる。遺伝的に免疫不全の動物としては、例えばＳＣＩＤマウス、ＮＵＤＥマウス、ＲＡＧ２ノックアウトマウス、ＮＯＤマウス、ＮＯＧマウス等が挙げられ、遺伝的に肝障害を有する動物としては、肝細胞特異的に発現するタンパク質のエンハンサー、及び／又はプロモーターの支配下に連結された肝障害誘発タンパク質遺伝子（ｕＰＡ遺伝子、ｔＰＡ遺伝子等）が導入されたトランスジェニック動物を利用することができる。
Typically, liver-impaired, immunodeficient non-human animals are produced by subjecting a genetically immunodeficient non-human animal to a liver-damage-inducing treatment, or by subjecting an genetically liver-damaged animal to an immunodeficiency-inducing treatment. It can be made. Examples of genetically immunodeficient animals include SCID mice, NUDE mice, RAG2 knockout mice, NOD mice, NOG mice, etc. Animals with genetic liver damage include proteins specifically expressed in hepatocytes. A transgenic animal into which a liver damage-inducing protein gene (uPA gene, tPA gene, etc.) linked under the control of an enhancer and/or promoter can be used.
本発明において肝障害免疫不全非ヒト動物に移植される「ヒト肝細胞」は、ヒト由来の肝細胞であればよく、例えば、ヒト肝組織から、コラゲナーゼ灌流法等の常法に従って単離されたヒト肝細胞を利用することができる。ヒト肝組織は健常人由来の肝組織であってもよいし、脂肪肝や肝臓がん等の疾患に罹患した患者由来の肝組織であってもよいが、好ましくは健常人由来の肝組織である。回収したヒト肝細胞はそのまま用いることができるが、ヒト肝細胞又は非ヒト動物の肝細胞を特異的に認識するモノクローナル抗体を用いて、ヒト肝細胞を精製してもよい。
In the present invention, the "human hepatocytes" to be transplanted into a liver-impaired, immunodeficient non-human animal may be human-derived hepatocytes, such as those isolated from human liver tissue according to a conventional method such as collagenase perfusion method. Human hepatocytes can be used. The human liver tissue may be a liver tissue derived from a healthy person or a liver tissue derived from a patient suffering from a disease such as fatty liver or liver cancer, but preferably a liver tissue derived from a healthy person. be. Although the collected human hepatocytes can be used as they are, they may be purified using a monoclonal antibody that specifically recognizes human hepatocytes or non-human animal hepatocytes.
肝障害免疫不全非ヒト動物へのヒト肝細胞の移植は、当該非ヒト動物の脾臓を経由して肝臓へ移植するか、あるいは、直接門脈から移植する。
上記手法により得られる非ヒト動物は、肝臓の全部又は一部（例えば少なくとも７０％）がヒト肝細胞に置換された動物である。肝臓の少なくとも７０％がヒト肝細胞に置換されたマウスとして、市販品（「ＰＸＢマウス」、株式会社フェニックスバイオ）を使用することもできる。 Human hepatocytes are transplanted into an immunodeficient non-human animal with liver damage, either by transplanting them into the liver via the spleen of the non-human animal, or by directly transplanting them from the portal vein.
The non-human animal obtained by the above method is an animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes. A commercially available mouse ("PXB Mouse", Phoenix Bio Co., Ltd.) can also be used as a mouse in which at least 70% of the liver has been replaced with human hepatocytes.
上記手法により得られる非ヒト動物は、肝臓の全部又は一部（例えば少なくとも７０％）がヒト肝細胞に置換された動物である。肝臓の少なくとも７０％がヒト肝細胞に置換されたマウスとして、市販品（「ＰＸＢマウス」、株式会社フェニックスバイオ）を使用することもできる。 Human hepatocytes are transplanted into an immunodeficient non-human animal with liver damage, either by transplanting them into the liver via the spleen of the non-human animal, or by directly transplanting them from the portal vein.
The non-human animal obtained by the above method is an animal in which all or a portion (eg, at least 70%) of the liver has been replaced with human hepatocytes. A commercially available mouse ("PXB Mouse", Phoenix Bio Co., Ltd.) can also be used as a mouse in which at least 70% of the liver has been replaced with human hepatocytes.
３．モデル細胞
本発明のモデル細胞は、上記ヒト肝細胞キメラ非ヒト動物から採取された肝細胞である。 3. Model Cells The model cells of the present invention are hepatocytes collected from the human hepatocyte chimeric non-human animal described above.
本発明のモデル細胞は、上記ヒト肝細胞キメラ非ヒト動物から採取された肝細胞である。 3. Model Cells The model cells of the present invention are hepatocytes collected from the human hepatocyte chimeric non-human animal described above.
本発明のモデル細胞は、一般的動物細胞の培養と同様に培養し、維持することができる。例えば、細胞培養用デッシュ、組織培養用デッシュ、マルチデッシュ、マイクロプレート中において、動物細胞の培養に一般的に用いられる培地（例えば、ダルベッコ改変イーグル培地（ＤＭＥＭ）、ウイリアムス培地Ｅ、ＲＰＭＩ－１６４０培地等）を用いて、必要に応じてさらにウシ胎児血清、緩衝剤、抗生物質、ｐＨ調整剤等を適宜添加し、維持又は継代することができる。
The model cells of the present invention can be cultured and maintained in the same manner as general animal cell cultures. For example, in cell culture dishes, tissue culture dishes, multi-dishes, microplates, media commonly used for culturing animal cells (e.g., Dulbecco's modified Eagle's medium (DMEM), Williams medium E, RPMI-1640 medium etc.), and further add fetal bovine serum, a buffer, an antibiotic, a pH adjuster, etc. as necessary for maintenance or subculture.
本発明のモデル細胞は、肝細胞キメラ非ヒト動物から採取後７～１７日（Ｄａｙ７～１７）、好ましくは８～１３日（Ｄａｙ８～１３）のものを使用することができる。
上記「ＰＸＢマウス」から採取された肝細胞を「ＰＸＢ－ｃｅｌｌｓ」ともいう。さらにＰＸＢ－ｃｅｌｌｓの細胞内に過剰なＴＧを蓄積する期間を延長した細胞を「ＰＸＢ－ｃｅｌｌｓ ＬＡ」ともいう。ＰＸＢマウスから分離した直後のＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡは細胞内に過剰なＴＧを蓄積する特徴を有することから、肥満状態を再現し、さらにインスリン抵抗性も再現していると考えられる。「過剰なＴＧ」とは、ＴＧが豊富に存在することを意味し、例えばＴＧなどで構成される明瞭な脂肪滴を細胞質内に認める状態をいう。 The model cells of the present invention can be used 7 to 17 days (Day 7 to 17), preferably 8 to 13 days (Day 8 to 13) after collection from a hepatocyte chimeric non-human animal.
Hepatocytes collected from the above-mentioned "PXB mouse" are also referred to as "PXB-cells." Furthermore, PXB-cells that have an extended period of accumulating excessive TG within the cells are also referred to as "PXB-cells LA". Since PXB-cells and PXB-cells LA immediately after isolation from PXB mice have the characteristic of accumulating excessive TG within the cells, it is thought that they reproduce the obese state and furthermore reproduce insulin resistance. "Excess TG" means that TG is present in abundance, and refers to a state in which, for example, distinct lipid droplets composed of TG are observed in the cytoplasm.
上記「ＰＸＢマウス」から採取された肝細胞を「ＰＸＢ－ｃｅｌｌｓ」ともいう。さらにＰＸＢ－ｃｅｌｌｓの細胞内に過剰なＴＧを蓄積する期間を延長した細胞を「ＰＸＢ－ｃｅｌｌｓ ＬＡ」ともいう。ＰＸＢマウスから分離した直後のＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡは細胞内に過剰なＴＧを蓄積する特徴を有することから、肥満状態を再現し、さらにインスリン抵抗性も再現していると考えられる。「過剰なＴＧ」とは、ＴＧが豊富に存在することを意味し、例えばＴＧなどで構成される明瞭な脂肪滴を細胞質内に認める状態をいう。 The model cells of the present invention can be used 7 to 17 days (Day 7 to 17), preferably 8 to 13 days (Day 8 to 13) after collection from a hepatocyte chimeric non-human animal.
Hepatocytes collected from the above-mentioned "PXB mouse" are also referred to as "PXB-cells." Furthermore, PXB-cells that have an extended period of accumulating excessive TG within the cells are also referred to as "PXB-cells LA". Since PXB-cells and PXB-cells LA immediately after isolation from PXB mice have the characteristic of accumulating excessive TG within the cells, it is thought that they reproduce the obese state and furthermore reproduce insulin resistance. "Excess TG" means that TG is present in abundance, and refers to a state in which, for example, distinct lipid droplets composed of TG are observed in the cytoplasm.
本発明のインスリン抵抗性モデル細胞は、当該細胞におけるインスリン抵抗性に関連する分子（主にヘパトカイン）を対象とした遺伝子（「インスリン抵抗性関連遺伝子」）の発現及びタンパク質発現を指標として特徴づけることができる。
本発明において「インスリン抵抗性関連遺伝子」は、本発明のモデル細胞において、健康な肝細胞と比べて発現量の変化（増大又は低下）が認められる遺伝子を意味する。 The insulin resistance model cells of the present invention can be characterized using the expression of genes ("insulin resistance related genes") and protein expressions targeting molecules related to insulin resistance (mainly hepatokines) in the cells. I can do it.
In the present invention, the term "insulin resistance-related gene" refers to a gene whose expression level changes (increases or decreases) in the model cells of the present invention compared to healthy hepatocytes.
本発明において「インスリン抵抗性関連遺伝子」は、本発明のモデル細胞において、健康な肝細胞と比べて発現量の変化（増大又は低下）が認められる遺伝子を意味する。 The insulin resistance model cells of the present invention can be characterized using the expression of genes ("insulin resistance related genes") and protein expressions targeting molecules related to insulin resistance (mainly hepatokines) in the cells. I can do it.
In the present invention, the term "insulin resistance-related gene" refers to a gene whose expression level changes (increases or decreases) in the model cells of the present invention compared to healthy hepatocytes.
このようなインスリン抵抗性関連遺伝子としては、ヘパトカイン遺伝子が挙げられる。
ヘパトカインは、例えば、肝臓に由来する分泌タンパク質であるセレノプロテインＰ（ＳｅＰＰ）、肝炎に関連するサイトカインであるＬｅｕｋｏｃｙｔｅ－ｄｅｒｉｖｅｄ ｃｈｅｍｏｔａｘｉｎ ２（ＬＥＣＴ２）、繊維芽細胞増殖因子のひとつであるＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２１（ＦＧＦ２１）、性ホルモン機能制御因子の一つであるＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２１（ＳＨＢＧ）、血管新生因子であるＡｎｇｉｏｐｏｉｅｔｉｎ－Ｌｉｋｅ Ｐｒｏｔｅｉｎ ６（ＡＮＧＰＴＬ６）及び炎症性シグナル伝達活性化因子であるＦｅｔｕｉｎ－Ａ等を例示できる。 Such insulin resistance-related genes include hepatokine genes.
Hepatokines include, for example, selenoprotein P (SePP), a secreted protein derived from the liver, leukocyte-derived chemotaxin 2 (LECT2), a cytokine related to hepatitis, and fibroblast growth factor 21 (one of the fibroblast growth factors). FGF21), Fibroblast growth factor 21 (SHBG), which is a sex hormone function regulator, Angiopoietin-Like Protein 6 (ANGPTL6), which is an angiogenic factor, and Fetuin-A, which is an inflammatory signal transduction activator. can.
ヘパトカインは、例えば、肝臓に由来する分泌タンパク質であるセレノプロテインＰ（ＳｅＰＰ）、肝炎に関連するサイトカインであるＬｅｕｋｏｃｙｔｅ－ｄｅｒｉｖｅｄ ｃｈｅｍｏｔａｘｉｎ ２（ＬＥＣＴ２）、繊維芽細胞増殖因子のひとつであるＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２１（ＦＧＦ２１）、性ホルモン機能制御因子の一つであるＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２１（ＳＨＢＧ）、血管新生因子であるＡｎｇｉｏｐｏｉｅｔｉｎ－Ｌｉｋｅ Ｐｒｏｔｅｉｎ ６（ＡＮＧＰＴＬ６）及び炎症性シグナル伝達活性化因子であるＦｅｔｕｉｎ－Ａ等を例示できる。 Such insulin resistance-related genes include hepatokine genes.
Hepatokines include, for example, selenoprotein P (SePP), a secreted protein derived from the liver, leukocyte-derived chemotaxin 2 (LECT2), a cytokine related to hepatitis, and fibroblast growth factor 21 (one of the fibroblast growth factors). FGF21), Fibroblast growth factor 21 (SHBG), which is a sex hormone function regulator, Angiopoietin-Like Protein 6 (ANGPTL6), which is an angiogenic factor, and Fetuin-A, which is an inflammatory signal transduction activator. can.
これらのヘパトカインには悪玉ヘパトカイン及び善玉ヘパトカインの両者が含まれる。悪玉ヘパトカインとは、インスリン抵抗性を惹起するへパトカインであり、善玉ヘパトカインはインスリン抵抗性を改善するへパトカインである。
そして、本発明の細胞は、悪玉ヘパトカインの遺伝子又はタンパク質の発現が亢進しており、また善玉ヘパトカインの遺伝子又はタンパク質の発現が低下している。「亢進」とは、対照又は標準品として使用される細胞の遺伝子又はタンパク質の発現と比較して高いこと、あるいは当該ヘパトカインと関連する遺伝子又はタンパク質の発現が有意に変化していることを意味し、「低下」とは、対照又は標準品として使用される細胞の遺伝子又はタンパク質の発現と比較して低いこと、あるいは当該ヘパトカインと関連する遺伝子又はタンパク質の発現が有意に変化していることを意味する。 These hepatokines include both bad hepatokines and good hepatokines. Bad hepatokines are hepatokines that induce insulin resistance, and good hepatokines are hepatokines that improve insulin resistance.
In the cells of the present invention, the expression of bad hepatokine genes or proteins is enhanced, and the expression of good hepatokine genes or proteins is decreased. "Enhanced" means that the expression of the gene or protein is higher than that of cells used as a control or standard, or that the expression of the gene or protein associated with the hepatokine is significantly changed. , "reduced" means lower than the expression of the gene or protein in cells used as a control or standard, or a significant change in the expression of the gene or protein associated with the hepatokine. do.
そして、本発明の細胞は、悪玉ヘパトカインの遺伝子又はタンパク質の発現が亢進しており、また善玉ヘパトカインの遺伝子又はタンパク質の発現が低下している。「亢進」とは、対照又は標準品として使用される細胞の遺伝子又はタンパク質の発現と比較して高いこと、あるいは当該ヘパトカインと関連する遺伝子又はタンパク質の発現が有意に変化していることを意味し、「低下」とは、対照又は標準品として使用される細胞の遺伝子又はタンパク質の発現と比較して低いこと、あるいは当該ヘパトカインと関連する遺伝子又はタンパク質の発現が有意に変化していることを意味する。 These hepatokines include both bad hepatokines and good hepatokines. Bad hepatokines are hepatokines that induce insulin resistance, and good hepatokines are hepatokines that improve insulin resistance.
In the cells of the present invention, the expression of bad hepatokine genes or proteins is enhanced, and the expression of good hepatokine genes or proteins is decreased. "Enhanced" means that the expression of the gene or protein is higher than that of cells used as a control or standard, or that the expression of the gene or protein associated with the hepatokine is significantly changed. , "reduced" means lower than the expression of the gene or protein in cells used as a control or standard, or a significant change in the expression of the gene or protein associated with the hepatokine. do.
悪玉ヘパトカインとしては、ＳｅＰＰ、ＬＥＣＴ２及びＦｅｔｕｉｎ－Ａ等が挙げられる。
善玉ヘパトカインとしてはＦＧＦ２１、ＳＨＢＧ及びＡＮＧＰＴＬ６等が挙げられる。 Bad hepatokines include SePP, LECT2, Fetuin-A, and the like.
Examples of good hepatokines include FGF21, SHBG, and ANGPTL6.
善玉ヘパトカインとしてはＦＧＦ２１、ＳＨＢＧ及びＡＮＧＰＴＬ６等が挙げられる。 Bad hepatokines include SePP, LECT2, Fetuin-A, and the like.
Examples of good hepatokines include FGF21, SHBG, and ANGPTL6.
上記のほか、本発明においては、脂肪酸合成酵素をコードする遺伝子（遺伝子名：ＦＡＳＮ）、ＳＲＥＢＰ－１をコードする遺伝子（遺伝子名：ＳＲＥＢＦ１）、グルコース－６－ホスファターゼをコードする遺伝子（Ｇ６ＰＣ）、コレステロール７α－ヒドロキシラーゼをコードする遺伝子（ＣＹＰ７Ａ１）、コレステリルエステル転送タンパク質をコードする遺伝子（ＣＥＴＰ）、グルコキナーゼをコードする遺伝子（ＧＣＫ）、ホスホエノールピルビン酸カルボキシキナーゼ１をコードする遺伝子（ＰＣＫ１）等が挙げられる。
In addition to the above, in the present invention, the gene encoding fatty acid synthase (gene name: FASN), the gene encoding SREBP-1 (gene name: SREBF1), the gene encoding glucose-6-phosphatase (G6PC), Gene encoding cholesterol 7α-hydroxylase (CYP7A1), gene encoding cholesteryl ester transfer protein (CETP), gene encoding glucokinase (GCK), gene encoding phosphoenolpyruvate carboxykinase 1 (PCK1), etc. can be mentioned.
４．スクリーニング方法
本発明のスクリーニング方法は、本発明のモデル細胞に候補物質を接触させ、接触後、当該細胞が発現するヘパトカイン遺伝子を検出し、得られる検出結果を指標として脂肪肝及び／又はインスリン抵抗性病態の治療又は予防薬をスクリーニングする方法である。具体的には、本発明のモデル細胞の培養液中に候補物質を添加し、その後、細胞から分泌されるヘパトカイン又はヘパトカイン遺伝子を測定し、評価する。候補物質を添加する一定期間は、例えば採取後８～１０日程度であるが特に限定されるものではない。 4. Screening method The screening method of the present invention involves contacting the model cells of the present invention with a candidate substance, detecting the hepatokine gene expressed by the cells after contact, and using the obtained detection results as an indicator of fatty liver and/or insulin resistance. This is a method for screening therapeutic or preventive drugs for pathological conditions. Specifically, a candidate substance is added to the culture solution of the model cells of the present invention, and then hepatokine or hepatokine gene secreted from the cells is measured and evaluated. The fixed period for adding the candidate substance is, for example, about 8 to 10 days after collection, but is not particularly limited.
本発明のスクリーニング方法は、本発明のモデル細胞に候補物質を接触させ、接触後、当該細胞が発現するヘパトカイン遺伝子を検出し、得られる検出結果を指標として脂肪肝及び／又はインスリン抵抗性病態の治療又は予防薬をスクリーニングする方法である。具体的には、本発明のモデル細胞の培養液中に候補物質を添加し、その後、細胞から分泌されるヘパトカイン又はヘパトカイン遺伝子を測定し、評価する。候補物質を添加する一定期間は、例えば採取後８～１０日程度であるが特に限定されるものではない。 4. Screening method The screening method of the present invention involves contacting the model cells of the present invention with a candidate substance, detecting the hepatokine gene expressed by the cells after contact, and using the obtained detection results as an indicator of fatty liver and/or insulin resistance. This is a method for screening therapeutic or preventive drugs for pathological conditions. Specifically, a candidate substance is added to the culture solution of the model cells of the present invention, and then hepatokine or hepatokine gene secreted from the cells is measured and evaluated. The fixed period for adding the candidate substance is, for example, about 8 to 10 days after collection, but is not particularly limited.
ヘパトカインの検出は、ＲＴ－ＰＣＲ、ウェスタンブロット（ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ： ＷＢ）、免疫沈降、酵素結合免疫吸着測定法（ Ｅｎｚｙｍｅ－ ｌｉｎｋｅｄ ｉｍｍｕｎｏ－ ｓｏｒｂｅｎｔ ａｓｓａｙ： ＥＬＩＳＡ）、細胞内外脂質量測定、質量分析等を単独で、又は適宜組み合わせて行うことができる。例えば、ヘパトカインの検出は、ヘパトカインを認識する市販の抗体を用いた免疫ＷＢを行う。
評価する候補物質は特に限定されるものではなく、例えば、天然又は人為的に合成された各種ペプチド、タンパク質（酵素や抗体を含む）、核酸（ポリヌクレオチド（ＤＮＡ， ＲＮＡ）、オリゴヌクレオチド（ｓｉＲＮＡ等）、ペプチド核酸（ＰＮＡ）等）、低分子又は高分子有機化合物等を例示することができる。 Hepatokine can be detected by RT-PCR, Western blotting (WB), immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), intracellular and extracellular lipid content measurement, mass spectrometry, etc. , or a suitable combination. For example, to detect hepatokine, immune WB using a commercially available antibody that recognizes hepatokine is performed.
Candidate substances to be evaluated are not particularly limited, and include, for example, various natural or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA, RNA), oligonucleotides (siRNA, etc.) ), peptide nucleic acids (PNA), etc.), low-molecular or high-molecular organic compounds, and the like.
評価する候補物質は特に限定されるものではなく、例えば、天然又は人為的に合成された各種ペプチド、タンパク質（酵素や抗体を含む）、核酸（ポリヌクレオチド（ＤＮＡ， ＲＮＡ）、オリゴヌクレオチド（ｓｉＲＮＡ等）、ペプチド核酸（ＰＮＡ）等）、低分子又は高分子有機化合物等を例示することができる。 Hepatokine can be detected by RT-PCR, Western blotting (WB), immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), intracellular and extracellular lipid content measurement, mass spectrometry, etc. , or a suitable combination. For example, to detect hepatokine, immune WB using a commercially available antibody that recognizes hepatokine is performed.
Candidate substances to be evaluated are not particularly limited, and include, for example, various natural or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA, RNA), oligonucleotides (siRNA, etc.) ), peptide nucleic acids (PNA), etc.), low-molecular or high-molecular organic compounds, and the like.
候補物質の添加後、１）悪玉ヘパトカインの分泌量がコントロールと比較して低下した場合、２）悪玉ヘパトカインの分泌量が低下又は検出限界以下となり、かつ、細胞内外脂質量減少が認められた場合、３）善玉ヘパトカインの分泌量がコントロールと比較して増加した場合、４）善玉ヘパトカインの分泌量が増加し、かつ、細胞内外脂質量減少が認められた場合、５）悪玉ヘパトカインの分泌量低下及び善玉ヘパトカインの分泌量増加とともに細胞の形態に変化が認められた場合、６）悪玉ヘパトカインの分泌量低下又は善玉ヘパトカインの分泌量増加とともに細胞の形態に変化が認められた場合、７）さらには、細胞内外脂質量が減少し、かつ、細胞の形態に変化が認められた場合には、当該候補物質は、ヘパトカインの分泌を抑えることができたと考えられ、脂肪肝、２型糖尿病、あるいはこれらの組み合わせの治療・予防薬として使用できるものと評価できる。
After the addition of the candidate substance, 1) the amount of bad hepatokine secreted decreases compared to the control, 2) the amount of bad hepatokine secreted decreases or becomes below the detection limit, and a decrease in the amount of extracellular and extracellular lipids is observed. , 3) When the secretion amount of good hepatokine increases compared to the control, 4) When the secretion amount of good hepatokine increases and a decrease in the amount of intracellular and extracellular lipids is observed, 5) When the secretion amount of bad hepatokine decreases and 6) if a change in cell morphology is observed with an increase in the secretion amount of good hepatokines, 6) if a change in cell morphology is observed with a decrease in the secretion amount of bad hepatokines or an increase in the secretion amount of good hepatokines, and 7) further. If the amount of intracellular and extracellular lipids decreases and changes in cell morphology are observed, it is considered that the candidate substance was able to suppress hepatokine secretion, and may be associated with fatty liver, type 2 diabetes, or other diseases. It can be evaluated as a drug that can be used as a combination therapeutic and preventive drug.
本発明のスクリーニング方法により、ヘパトカインの産生を制御することにより脂肪肝や２型糖尿病の治療又は予防に対して効果があると評価された物質は、糖尿病予防又は治療用薬剤、あるいは他薬剤との併用薬として使用できる。また、糖尿病は血管新生のリスクファクターであることから、糖尿病治療期間中、あるいは寛解期に、スクリーニングで見出した薬剤投与は、将来糖尿病罹患率を減少させる可能性を検討することができ、将来的に発見されるヘパトカインの分泌により引き起こされる疾患全般について治療薬の開発を行うことができる。
Substances evaluated to be effective in treating or preventing fatty liver or type 2 diabetes by controlling hepatokine production by the screening method of the present invention are drugs for preventing or treating diabetes, or drugs used in combination with other drugs. Can be used as a concomitant drug. Furthermore, since diabetes is a risk factor for angiogenesis, administering drugs discovered through screening during diabetes treatment or during remission can be used to examine the possibility of reducing the incidence of diabetes in the future. It is possible to develop therapeutic drugs for all diseases caused by the secretion of hepatokines, which are discovered in humans.
実施例
以下、実施例により本発明をさらに具体的に説明する。但し、本発明の範囲はこれらの実施例により限定されるものではない。 Examples Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the scope of the present invention is not limited by these Examples.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明の範囲はこれらの実施例により限定されるものではない。 Examples Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the scope of the present invention is not limited by these Examples.
本実施例では、脂肪肝状態のＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡにおけるインスリン抵抗性に関与するへパトカインの発現パターンを解析し、さらに、ＰＸＢ－ｃｅｌｌｓの薬剤処理によるへパトカイン発現量の影響を調べた。
In this example, we analyzed the expression pattern of hepatokine involved in insulin resistance in PXB-cells in fatty liver state and PXB-cells LA, and further investigated the effect of drug treatment on hepatokine expression level in PXB-cells. Ta.
＜方法＞
・ＰＸＢ－ｃｅｌｌｓ（ＰＸＢマウスから採取後１３日）、非凍結ヒト由来ヘパトサイト（市販）、ＨｅｐＧ２、及びＨｕＨ７細胞におけるインスリン抵抗性に関与するヘパトカインの遺伝子及びタンパク質発現を解析した。
・ＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡ（いずれもＰＸＢマウスから採取後９日）を対象とし、両者のインスリン抵抗性に関与するへパトカインの遺伝子発現を解析した。
・ＰＸＢ－ｃｅｌｌｓ（ＰＸＢマウスから採取後６日）を対象とし、脂肪肝モデルの細胞内脂肪含量を低下させる抗糖尿病薬を２４時間添加した後の脂質代謝関連及びへパトカインの遺伝子発現を解析した。 <Method>
- Gene and protein expression of hepatokines involved in insulin resistance was analyzed in PXB-cells (13 days after collection from PXB mice), unfrozen human-derived hepatocytes (commercially available), HepG2, and HuH7 cells.
- PXB-cells and PXB-cells LA (both 9 days after harvesting from PXB mice) were used to analyze the gene expression of hepatokine involved in insulin resistance in both.
・We analyzed lipid metabolism-related and hepatokine gene expression after adding an antidiabetic drug that lowers intracellular fat content in a fatty liver model for 24 hours to PXB-cells (6 days after collection from PXB mice). .
・ＰＸＢ－ｃｅｌｌｓ（ＰＸＢマウスから採取後１３日）、非凍結ヒト由来ヘパトサイト（市販）、ＨｅｐＧ２、及びＨｕＨ７細胞におけるインスリン抵抗性に関与するヘパトカインの遺伝子及びタンパク質発現を解析した。
・ＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡ（いずれもＰＸＢマウスから採取後９日）を対象とし、両者のインスリン抵抗性に関与するへパトカインの遺伝子発現を解析した。
・ＰＸＢ－ｃｅｌｌｓ（ＰＸＢマウスから採取後６日）を対象とし、脂肪肝モデルの細胞内脂肪含量を低下させる抗糖尿病薬を２４時間添加した後の脂質代謝関連及びへパトカインの遺伝子発現を解析した。 <Method>
- Gene and protein expression of hepatokines involved in insulin resistance was analyzed in PXB-cells (13 days after collection from PXB mice), unfrozen human-derived hepatocytes (commercially available), HepG2, and HuH7 cells.
- PXB-cells and PXB-cells LA (both 9 days after harvesting from PXB mice) were used to analyze the gene expression of hepatokine involved in insulin resistance in both.
・We analyzed lipid metabolism-related and hepatokine gene expression after adding an antidiabetic drug that lowers intracellular fat content in a fatty liver model for 24 hours to PXB-cells (6 days after collection from PXB mice). .
評価はＲＴ－ＰＣＲ及びＷＢにより行い、評価項目は、ＳｅＰＰ、ＡＮＧＰＴＬ６、ＬＥＣＴ２及びＦＧＦ２１等のへパトカインを中心に行った。
The evaluation was performed by RT-PCR and WB, and the evaluation items were mainly hepatokines such as SePP, ANGPTL6, LECT2, and FGF21.
ＲＴ－ＰＣＲは以下の条件で行った。
総ＲＮＡの単離は、ＱｕｉｃｋＧｅｎｅ ＲＮＡ ｃｕｌｔｕｒｅｄ ｃｅｌｌ ｋｉｔ Ｓを使用した。鋳型ｃＤＮＡは、総ＲＮＡ（５μｇ）から、Ｐｒｉｍｅ－Ｓｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔを用いて合成した（逆転写サンプル）。反応液は、逆転写サンプル（４μＬ）に０．２μＭ の各々のプライマーを含むＳｓｏＡｄｖａｎｃｅｄ Ｕｎｉｖｅｒｓａｌ ＳＹＢＲ Ｇｒｅｅｎ Ｓｕｐｅｒｍｉｘ溶液（等量のＭｉｌｉＱ水で１／２希釈したもの、２１μＬ）を加えた。ＰＣＲは、ｆｌｕｏｒｅｓｃｅｎｔ ｔｅｍｐｅｒａｔｕｒｅ ｃｙｃｌｅｒ（ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ）を用いて行った。反応条件は、９５℃で５秒（変性）と６０℃で３０秒保持（アニーリングと伸長反応）を４０サイクル繰り返して行った。ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ発現量を基準にノーマライズした。
RT-PCR was performed under the following conditions.
Total RNA was isolated using QuickGene RNA cultured cell kit S. Template cDNA was synthesized from total RNA (5 μg) using Prime-Script RT reagent Kit (reverse transcription sample). For the reaction solution, SsoAdvanced Universal SYBR Green Supermix solution (1/2 diluted with an equal volume of MiliQ water, 21 μL) containing 0.2 μM of each primer was added to the reverse transcription sample (4 μL). PCR was performed using a fluorescent temperature cycler (CFX Connect ™ Real-Time PCR Detection System). The reaction conditions were 40 cycles of 95°C for 5 seconds (denaturation) and 60°C for 30 seconds (annealing and extension reaction). Normalization was performed based on the expression level of glyceraldehyde-3-phosphate dehydrogenase.
総ＲＮＡの単離は、ＱｕｉｃｋＧｅｎｅ ＲＮＡ ｃｕｌｔｕｒｅｄ ｃｅｌｌ ｋｉｔ Ｓを使用した。鋳型ｃＤＮＡは、総ＲＮＡ（５μｇ）から、Ｐｒｉｍｅ－Ｓｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔを用いて合成した（逆転写サンプル）。反応液は、逆転写サンプル（４μＬ）に０．２μＭ の各々のプライマーを含むＳｓｏＡｄｖａｎｃｅｄ Ｕｎｉｖｅｒｓａｌ ＳＹＢＲ Ｇｒｅｅｎ Ｓｕｐｅｒｍｉｘ溶液（等量のＭｉｌｉＱ水で１／２希釈したもの、２１μＬ）を加えた。ＰＣＲは、ｆｌｕｏｒｅｓｃｅｎｔ ｔｅｍｐｅｒａｔｕｒｅ ｃｙｃｌｅｒ（ＣＦＸ ＣｏｎｎｅｃｔＴＭ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ）を用いて行った。反応条件は、９５℃で５秒（変性）と６０℃で３０秒保持（アニーリングと伸長反応）を４０サイクル繰り返して行った。ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ発現量を基準にノーマライズした。
Total RNA was isolated using QuickGene RNA cultured cell kit S. Template cDNA was synthesized from total RNA (5 μg) using Prime-Script RT reagent Kit (reverse transcription sample). For the reaction solution, SsoAdvanced Universal SYBR Green Supermix solution (1/2 diluted with an equal volume of MiliQ water, 21 μL) containing 0.2 μM of each primer was added to the reverse transcription sample (4 μL). PCR was performed using a fluorescent temperature cycler (CFX Connect ™ Real-Time PCR Detection System). The reaction conditions were 40 cycles of 95°C for 5 seconds (denaturation) and 60°C for 30 seconds (annealing and extension reaction). Normalization was performed based on the expression level of glyceraldehyde-3-phosphate dehydrogenase.
＜結果＞
（１）ＰＸＢ－ｃｅｌｌｓにおける悪玉ヘパトカインの発現
悪玉ヘパトカイン遺伝子発現量（ＲＴ－ＰＣＲ）の結果を図１及び表２－１に示す。
<Results>
(1) Expression of bad hepatokine in PXB-cells The results of bad hepatokine gene expression (RT-PCR) are shown in FIG. 1 and Table 2-1.
（１）ＰＸＢ－ｃｅｌｌｓにおける悪玉ヘパトカインの発現
悪玉ヘパトカイン遺伝子発現量（ＲＴ－ＰＣＲ）の結果を図１及び表２－１に示す。
(1) Expression of bad hepatokine in PXB-cells The results of bad hepatokine gene expression (RT-PCR) are shown in FIG. 1 and Table 2-1.
悪玉ヘパトカインタンパク質発現量（ＷＢ）の結果を図２及び表２－２に示す。
The results of bad hepatokine protein expression level (WB) are shown in Figure 2 and Table 2-2.
ＰＸＢ－ｃｅｌｌｓは、ＰＸＢマウスから採取した後１３日（Ｄａｙ１３）でも、悪玉ヘパトカインの発現量が、他細胞より高いことが示された。
Even on day 13 (Day 13) after being collected from PXB mice, PXB-cells were shown to have a higher expression level of bad hepatokines than other cells.
（２）ＰＸＢ－ｃｅｌｌｓと ＰＸＢ－ｃｅｌｌｓ ＬＡの比較
インスリン抵抗性に関連するヘパトカインの遺伝子発現量（ＲＴ－ＰＣＲ）の結果を表３に示す。 (2) Comparison of PXB-cells and PXB-cells LA Table 3 shows the results of gene expression levels (RT-PCR) of hepatokines associated with insulin resistance.
インスリン抵抗性に関連するヘパトカインの遺伝子発現量（ＲＴ－ＰＣＲ）の結果を表３に示す。 (2) Comparison of PXB-cells and PXB-cells LA Table 3 shows the results of gene expression levels (RT-PCR) of hepatokines associated with insulin resistance.
ＰＸＢ－ｃｅｌｌｓ ＬＡのインスリン抵抗性に関連するヘパトカインの遺伝子発現量はＰＸＢ－ｃｅｌｌｓと同等であった。
PXB-cells The expression levels of hepatokine genes associated with insulin resistance in LA were similar to those in PXB-cells.
（３）インスリン抵抗性改善薬剤の影響
抗糖尿病薬処理後の脂質代謝関連及びインスリン抵抗性に関連するヘパトカインの遺伝子発現量（ＲＴ－ＰＣＲ）の結果を表４に示す。 (3) Effects of insulin resistance improving drugs Table 4 shows the results of gene expression levels (RT-PCR) of hepatokines related to lipid metabolism and insulin resistance after treatment with antidiabetic drugs.
抗糖尿病薬処理後の脂質代謝関連及びインスリン抵抗性に関連するヘパトカインの遺伝子発現量（ＲＴ－ＰＣＲ）の結果を表４に示す。 (3) Effects of insulin resistance improving drugs Table 4 shows the results of gene expression levels (RT-PCR) of hepatokines related to lipid metabolism and insulin resistance after treatment with antidiabetic drugs.
ＰＸＢ－ｃｅｌｌｓに対する抗糖尿病薬の影響を調べた結果、両薬剤ともＬＥＣＴ２（悪玉へパトカイン）の発現を抑制し、ＦＧＦ２１（善玉へパトカイン）の発現を亢進した。
As a result of examining the effects of antidiabetic drugs on PXB-cells, both drugs suppressed the expression of LECT2 (bad hepatokine) and enhanced the expression of FGF21 (good hepatokine).
（５)小括
作用機序が異なる抗糖尿病薬のメトホルミン（ＡＭＰＫを介して）及びロシグリタゾン（ＰＰＡＲ－ｇａｍｍａを介して）とも、脂肪肝様のＰＸＢ－ｃｅｌｌｓの悪玉ヘパトカインを抑制し、善玉へパトカインを上げる等、その効果が確認された。一方、ロシグリタゾンはβ-酸化を活性化が認められたことから、本来のＰＰＡＲ－ｇａｍｍａ活性化（ＦＡＳやＦＳＰ２７遺伝子の亢進作用）以外に、ＰＰＡＲ－ａｌｐｈａ活性化物質として作用している可能性がある。
また先に示した通り、ＰＸＢ－ｃｅｌｌｓ ＬＡのインスリン抵抗性に関連するヘパトカインの遺伝子発現量はＰＸＢ－ｃｅｌｌｓと類似しており、このことから他の肝由来細胞と比較して、ＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡは、肝臓を標的にしたインスリン抵抗性改善薬の探索系において、優れた細胞であることが示された。 (5) Summary Both metformin (via AMPK) and rosiglitazone (via PPAR-gamma), which are antidiabetic drugs with different mechanisms of action, suppress the bad hepatokine in fatty liver-like PXB-cells and promote the good hepatokine. Its effects, such as increasing patocaine, have been confirmed. On the other hand, since rosiglitazone was found to activate β-oxidation, it may act as a PPAR-alpha activator in addition to the original PPAR-gamma activation (FAS and FSP27 gene enhancement). There is.
Furthermore, as shown above, the gene expression levels of hepatokines associated with insulin resistance in PXB-cells LA are similar to those in PXB-cells, and this indicates that compared to other liver-derived cells, PXB-cells and LA PXB-cells LA was shown to be an excellent cell in the search system for insulin resistance improving drugs targeting the liver.
作用機序が異なる抗糖尿病薬のメトホルミン（ＡＭＰＫを介して）及びロシグリタゾン（ＰＰＡＲ－ｇａｍｍａを介して）とも、脂肪肝様のＰＸＢ－ｃｅｌｌｓの悪玉ヘパトカインを抑制し、善玉へパトカインを上げる等、その効果が確認された。一方、ロシグリタゾンはβ-酸化を活性化が認められたことから、本来のＰＰＡＲ－ｇａｍｍａ活性化（ＦＡＳやＦＳＰ２７遺伝子の亢進作用）以外に、ＰＰＡＲ－ａｌｐｈａ活性化物質として作用している可能性がある。
また先に示した通り、ＰＸＢ－ｃｅｌｌｓ ＬＡのインスリン抵抗性に関連するヘパトカインの遺伝子発現量はＰＸＢ－ｃｅｌｌｓと類似しており、このことから他の肝由来細胞と比較して、ＰＸＢ－ｃｅｌｌｓ及びＰＸＢ－ｃｅｌｌｓ ＬＡは、肝臓を標的にしたインスリン抵抗性改善薬の探索系において、優れた細胞であることが示された。 (5) Summary Both metformin (via AMPK) and rosiglitazone (via PPAR-gamma), which are antidiabetic drugs with different mechanisms of action, suppress the bad hepatokine in fatty liver-like PXB-cells and promote the good hepatokine. Its effects, such as increasing patocaine, have been confirmed. On the other hand, since rosiglitazone was found to activate β-oxidation, it may act as a PPAR-alpha activator in addition to the original PPAR-gamma activation (FAS and FSP27 gene enhancement). There is.
Furthermore, as shown above, the gene expression levels of hepatokines associated with insulin resistance in PXB-cells LA are similar to those in PXB-cells, and this indicates that compared to other liver-derived cells, PXB-cells and LA PXB-cells LA was shown to be an excellent cell in the search system for insulin resistance improving drugs targeting the liver.
配列番号１～１０：合成DNA
Sequence number 1-10: Synthetic DNA
Claims (9)
- 肝臓の全部又は一部がヒト肝細胞に置換された非ヒト動物由来の肝細胞を含む、脂肪肝又はインスリン抵抗性病態のモデル細胞。 A model cell for fatty liver or insulin resistance pathology, which contains hepatocytes derived from a non-human animal in which all or part of the liver has been replaced with human hepatocytes.
- 非ヒト動物がマウスである請求項１に記載の細胞。 The cell according to claim 1, wherein the non-human animal is a mouse.
- インスリン抵抗性惹起へパトカイン遺伝子の遺伝子発現が亢進した、請求項１に記載の細胞。 The cell according to claim 1, in which gene expression of a patokine gene that induces insulin resistance is enhanced.
- インスリン抵抗性惹起へパトカインが、ＳｅＰＰ、ＬＥＣＴ２及びＦｅｔｕｉｎＡからなる群から選ばれる少なくとも１種である、請求項２に記載の細胞。 The cell according to claim 2, wherein the insulin resistance-inducing patokine is at least one selected from the group consisting of SePP, LECT2, and FetuinA.
- インスリン抵抗性改善へパトカイン遺伝子の遺伝子発現が低下した、請求項１に記載の細胞。 The cell according to claim 1, wherein the gene expression of the patokine gene is reduced to improve insulin resistance.
- インスリン抵抗性改善へパトカインが、ＦＧＦ２１、ＳＨＢＧ及びＡＮＧＰＴＬ６からなる群から選ばれる少なくとも１種である、請求項５に記載の細胞。 The cell according to claim 5, wherein the patokine for improving insulin resistance is at least one selected from the group consisting of FGF21, SHBG, and ANGPTL6.
- 請求項１～６のいずれか１項に記載の細胞に候補物質を接触させてヘパトカイン遺伝子を検出し、得られる検出結果を指標として脂肪肝及び／又はインスリン抵抗性病態の治療薬をスクリーニングする方法。 A method of detecting a hepatokine gene by contacting the cell according to any one of claims 1 to 6 with a candidate substance, and screening a therapeutic agent for fatty liver and/or insulin resistance pathological conditions using the obtained detection result as an indicator. .
- インスリン抵抗性疾患が２型糖尿病である請求項７に記載の方法。 The method according to claim 7, wherein the insulin resistance disease is type 2 diabetes.
- 脂肪肝が非アルコール性脂肪肝である請求項７に記載の方法。 The method according to claim 7, wherein the fatty liver is non-alcoholic fatty liver.
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