JP2007175010A - Rbp-j CONDITIONAL KNOCKOUT MOUSE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a genetically modified non-human mammal having artificially suppressed expression of Rbp-j gene in the differentiation process of pancreatic cell, a method for screening a pharmaceutical composition for the treatment or prevention of diseases caused by excessive secretion or deficient secretion of insulin; and also to provide a test method and a test agent for diseases caused by the deficient secretion of insulin. <P>SOLUTION: A mouse having specific deletion of Rbp-j in the pancreas of developmental stage is prepared by using Cre/loxP DNA recombination system, and the character of the mouse is observed. The mouse exhibits typical characteristics of insulin-deficient diabetes accompanying pancreatic hypoplasia such as weight reduction, hyperglycemia, plasma insulin reduction and increase in intake (diabetic polyphagia), and it has been found that Rbp-j is necessary for the maintenance of pancreatic precursor cells and the formation of a proper amount of pancreatic β cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a genetically modified non-human mammal characterized in that expression of the Rbp-j gene is artificially suppressed in the early stage of pancreatic cell differentiation. The present invention also relates to a method for screening a pharmaceutical composition for treating or preventing a disease associated with insufficient insulin secretion.

  Pancreatic β cells play a central role in the regulation of glucose homeostasis by secreting insulin. Type 1 diabetes is caused by destruction of β cells (Non-patent Document 1). Data from recent islet transplantation studies suggest that diabetes can be treated if a sufficient supply of islets is available, but the number of donors is much less than the number of diabetics ( Non-patent documents 2 to 4). Many cell sources capable of physiologically regulated insulin secretion other than the pancreas removed from the cadaver have been studied (Non-patent Document 5). In order to reproduce the process of islet development in vivo in vitro, it is important to further study the mechanisms that regulate the process of pancreatic cell development. In mice, the pancreas arises from the dorsal and ventral regions of the foregut endoderm around day 9 of gestation (E9). Since the pancreatic primordium expresses the homeodomain protein Pdx1, all pancreatic cell types are derived from Pdx-1 positive progenitor cells (Non-patent Documents 6 and 7). The pancreatic progenitor cell population generates pancreatic β cells, and cell lineage tracing analysis has revealed that new β cells can be differentiated throughout embryogenesis (Non-Patent Documents 7 to 10). .

  Notch signaling regulates cell fate decisions in various tissues, and mutations in genes encoding Notch receptors or their ligands have been attributed to various human diseases (11). To 21). Interaction of Notch receptor with its ligand induces cleavage of the receptor's intracellular domain (Notch IC), which translocates to the nucleus and binds to Rbp-j and is associated with cell differentiation or proliferation Transactivate transcription of target genes (Non-Patent Documents 22 to 24). Rbp-j is an important mediator of Notch signaling because it binds to all four types of Notch receptors and is widely expressed (Non-patent Document 25).

  Various Notch-related genes are known to be expressed in developing pancreatic cells (Non-patent Document 26). However, the exact role of Notch signaling during pancreatic cell development in mice with homozygous deletion of genes such as Dll1, Notch1, Notch2, Jagged1, Rbp-j or Hes1 shows multiple abnormalities and early embryonic lethality Has not yet been clarified (Non-Patent Documents 12, 14, 29 to 32). In Dll1 or Hes1 systemic knockout mice, excess α (glucagon production) cell differentiation in the pancreas was reported around E10 (Non-patent Documents 29 and 32), but β cells grew to E13.5 in mice. First, since differentiation of α and β cells occurs independently of the Pdx1-expressing epithelium (Non-patent Document 8), it is necessary to elucidate the effect of Notch signaling on β cells in the future. It is also possible that the phenotype in Dll1 or Hes1 systemic knockout mice occurs following changes in other external factors implicated in pancreatic cell development.

Prior art document information related to the invention of the present application is shown below.
Gale EA, Diabetes, 50, 217-226 (2001) Weir GC, and Bonner-Weir, S., Diabetes, 46, 1247-1256 (1997) Shapiro, AM, et al., N Engl J Med., 343, 230-238 (2000) Hirshberg, B., et al., Rev. Endocr. Metab. Disord., 4, 381-389 (2003) Rother KI, and Harlan DM, J. Clin. Invest., 114, 877-883 (2004) Offield, MF, et al., Development, 122, 983-995 (1996) Gu, G., et al., Development, 129, 2447-2457 (2002) Jensen, J., et al., Diabetes, 49, 163-176 (2000) Edlund, H., Nat. Rev. Genet., 3, 524-532 (2002) Wilson, ME, et al., Mech. Dev., 120, 65-80 (2003) Artavanis-Tsakonas, S., et al., Science, 268, 225-232 (1995) Ishibashi, M., et al., Genes Dev., 9, 3136-3148 (1995) de la Pompa, JL, et al., Development, 124, 1139-1148 (1997) Hrabe de Angelis, M., et al., Nature, 386, 717-721 (1997) Morrison, SJ, et al., Cell, 101, 499-510 (2000) MacDonald, HR, et al., Trends Immunol., 22, 155-160 (2001) McCright, B., et al., Development, 128, 491-502 (2001) Han, H., et al., Int Immunol., 14, 637-645 (2002) Tanigaki, K., et al., Nat. Immunol., 3, 443-450 (2002) Yamamoto, N., et al., Curr Biol., 13, 333-338 (2003) Tanigaki, K., et al., Immunity, 20, 611-622 (2004) Jarriault, S., et al., Nature, 377, 355-358 (1995) Tamura, K., et al., Curr Biol., 5, 1416-1423 (1995) Satoh et al., J. Biol. Chem., 279, 24986-24993 (2004) Kato, H., et al., FEBS Lett., 395, 221-224 (1996) Lammert, E., et al., Mech Dev., 94, 199-203 (2000) Swiatek, PJ, et al., Genes Dev., 8, 707-719 (1994) Oka, C., et al., Development, 121, 3291-3301 (1995) Apelqvist, A., et al., Nature, 400, 877-881 (1999) Hamada, Y., et al., Development, 126, 3415-3424 (1999) Xue, Y., et al., Hum Mol Genet., 8, 723-730 (1999) Jensen, J., et al., Nat. Genet., 24, 36-44 (2000)

  Although the role of the Rbp-j gene has been considered to be important in the early stages of pancreatic cell differentiation, the exact role of the Rbp-j gene has not been elucidated so far.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a non-genetically modified gene characterized in that the expression of the Rbp-j gene is artificially suppressed in the early stage of pancreatic cell differentiation. It is to provide a human mammal. Another object of the present invention is to provide a method for screening a pharmaceutical composition for treating or preventing a disease associated with excessive or insufficient secretion of insulin. Furthermore, it is also an object to provide a test method and a test drug for diseases associated with insufficient insulin secretion.

  In order to solve the above problems, the present inventors have prepared a Cre / loxP-mediated DNA recombination system (Metzger, D., et al., Proc. Natl. Acad. Sci., 92, 6991-6995 (1995). )) Was used to create mice with specific deletions of Rbp-j in the developing pancreas and observe their traits.

  Rbp-j deficiency early in pancreatic development caused downregulation of Hes1 in pancreatic buds and at the same time increased the number of neurogenin 3 positive cells. Although promotion of differentiation of glucagon-expressing cells and pancreatic polypeptide-expressing cells was observed at around 11.5 of gestational age (E), insulin-expressing cells were hardly differentiated. Later in development, pancreatic duct differentiation and elongation were promoted, but epithelial branching and acinar cell differentiation were suppressed. The resulting mice showed typical features of insulin-deficient diabetes with pancreatic insufficiency such as weight loss, hyperglycemia, decreased plasma insulin concentration, and increased intake (diabetic polyphagia).

  Since Pdx1-positive progenitor cells are the basis of all mature cells in the pancreas, the Rpb1-j knockout of the mouse of the present invention prevented the inhibition of α cells, PP cells, and pancreatic duct differentiation by Notch / Rpb1-j signaling. It is considered that hyperplasia was caused, and as a result, epithelial progenitor cells were depleted, so that pancreatic β-cells that subsequently differentiate decreased, and typical features of diabetes were expressed. That is, it was suggested that Notch / Rpb1-j signaling regulates α cell, PP cell, and pancreatic duct differentiation and maintains a pool of epithelial progenitor cells.

  In contrast, as a result of observing mice expressing the Cre gene under the control of the promoter of the insulin II gene that is specifically expressed in the differentiated β-cell, the β-cell-specific deficiency of Rbp-j is Does not affect the number and function of pancreatic beta cells.

  That is, the present inventors have found that Rbp-j is necessary for maintenance of pancreatic progenitor cells and generation of an appropriate amount of pancreatic β cells, and thus the present invention has been completed.

More specifically, the present invention provides the following [1] to [23].
[1] A genetically modified non-human mammal, wherein the Rbp-j gene expression is artificially suppressed during pancreatic cell differentiation.
[2] The genetically modified non-human mammal according to [1], wherein a foreign gene is inserted into one or both of the Rbp-j gene pair.
[3] The genetically modified non-human mammal of [1] or [2], wherein Rbp-j gene expression is suppressed only in pancreatic cells.
[4] The genetically modified non-human mammal according to any one of [1] to [3], which is a model animal for diabetes.
[5] A screening method for a pharmaceutical composition that suppresses pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) contacting Rbp-j with a test compound (b) detecting a binding between Rbp-j and the test compound (c) selecting a test compound that binds to Rbp-j.
[6] A screening method for a pharmaceutical composition that suppresses pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) A step of bringing a test compound into contact with a cell expressing the Rbp-j gene (b) A step of measuring the expression level of Rbp-j (c) Rbp as compared with the case where the test compound is not contacted a step of selecting a test compound having a decreased expression level of -j.
[7] A screening method for a pharmaceutical composition that suppresses pancreatic β-cell differentiation, comprising the following steps (a) to (d).
(A) a step of providing a cell or cell extract having DNA to which a reporter gene is operably linked downstream of a promoter region of DNA encoding Rbp-j (b) a test compound in the cell or cell extract (C) measuring the expression level of the reporter gene in the cell or cell extract (d) reducing the expression level of the reporter gene compared to the case where the test compound is not contacted A step of selecting the test compound that has been allowed to enter.
[8] A screening method for a pharmaceutical composition that suppresses pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell expressing the Rbp-j gene (b) a step of measuring the activity of Rbp-j in the cell (c) as compared with the case where the test compound is not contacted A step of selecting a test compound having reduced activity.
[9] The pharmaceutical composition for suppressing or preventing pancreatic β-cell differentiation is a pharmaceutical composition for treating or preventing a disease associated with excessive insulin secretion. The screening method according to any one of the above.
[10] The method according to [9], wherein the disease associated with excessive secretion of insulin is fatty liver, arteriosclerosis, hyperinsulinemia, or metabolic syndrome.
[11] A pharmaceutical composition for treating or preventing a disease associated with excessive secretion of insulin, selected by the screening method according to any one of [5] to [10].
[12] A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) contacting Rbp-j with a test compound (b) detecting a binding between Rbp-j and the test compound (c) selecting a test compound that binds to Rbp-j.
[13] A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) A step of bringing a test compound into contact with a cell expressing the Rbp-j gene (b) A step of measuring the expression level of Rbp-j (c) Rbp as compared with the case where the test compound is not contacted a step of selecting a test compound having an increased expression level of -j.
[14] A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (d):
(A) a step of providing a cell or cell extract having DNA to which a reporter gene is operably linked downstream of a promoter region of DNA encoding Rbp-j (b) a test compound in the cell or cell extract (C) measuring the expression level of the reporter gene in the cell or the cell extract (d) increasing the expression level of the reporter gene compared to the case where the test compound is not contacted A step of selecting the test compound that has been allowed to enter.
[15] A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell expressing the Rbp-j gene (b) a step of measuring the activity of Rbp-j in the cell (c) as compared with the case where the test compound is not contacted A step of selecting a test compound having increased activity.
[16] A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) a step of administering a test compound to a genetically modified non-human mammal characterized in that the expression of the Rbp-j gene is artificially suppressed (b) pancreatic β cells in the genetically modified non-human mammal A step of measuring the amount (c) a step of selecting a compound that increases the amount of pancreatic β cells in the genetically modified non-human mammal as compared with the case where no test compound is administered [17] pancreatic β cell differentiation The screening method according to any one of [12] to [16], wherein the pharmaceutical composition that promotes is a pharmaceutical composition for treating or preventing a disease associated with insufficient insulin secretion.
[18] A pharmaceutical composition for treating or preventing diabetes selected by the screening method according to any one of [12] to [17].
[19] A method of promoting pancreatic β-cell differentiation, comprising a step of promoting Rbp-j gene expression or activity in a cell expressing an Rbp-j gene at an early stage of pancreatic cell differentiation.
[20] A method for treating or preventing a disease associated with insufficient secretion of insulin, comprising a step of promoting Rbp-j gene expression or activity in a cell that expresses an Rbp-j gene at an early stage of pancreatic cell differentiation.
[21] The method according to [20], wherein the disease associated with insufficient insulin secretion is diabetes with decreased insulin secretion.

  In the present invention, pancreatic β-cell differentiation is suppressed by suppressing the expression of Rbp-j gene in the early stage of pancreatic cell differentiation, and Rbp-j gene maintains pancreatic progenitor cells and generates pancreatic β-cells in vivo. It was suggested that it contributed to.

Based on this, screening for drugs for the treatment or prevention of diseases associated with excessive or insufficient secretion of insulin targeting Rbp-j binding, Rbp-j expression level, and Rbp-j activity It becomes possible. In addition, it is possible to examine diseases associated with insufficient insulin secretion using Rbp-j expression level and Rbp-j gene mutation as indices.
Analysis of the Rbp-j conditional knockout mouse of the present invention can be useful in studies to elucidate the physiological role of Rbp-j.

  Furthermore, by observing the symptoms of the genetically modified animal of the present invention and comparing with the symptoms of the disease whose cause has not been clarified so far, it is clear that the cause of the disease is dysfunction in pancreatic cells of Rbp-j It is also possible to do. The genetically modified animal of the present invention is a model mouse that exhibits the same symptoms as diabetes because the expression of Rbp-j is suppressed only in pancreatic cells and pancreatic hypoplasia occurs without causing embryonic lethality.

  The present invention relates to a genetically modified non-human mammal characterized in that expression of the Rbp-j gene is artificially suppressed in the early stage of pancreatic cell differentiation.

  In the present invention, “the expression of the Rbp-j gene is artificially suppressed” usually means a genetic mutation such as nucleotide insertion, deletion, substitution, etc. in one or both of the genomic DNA pairs of the Rbp-j gene. It means a state in which the expression of the gene is suppressed.

  In the present invention, “insertion” means expression of an Rbp-j gene in which a DNA other than the Rbp-j gene is inserted by inserting a DNA having a sequence other than the Rbp-j gene into the Rbp-j gene. This means that the product does not function as Rbp-j. In addition, “deletion” means that the expression product of Rbp-j gene does not function or does not exist by deleting part or all of the genome of Rbp-j gene. Say. In addition, “replacement” means that a part or all of the genome of the Rbp-j gene is replaced by a separate sequence not related to the Rbp-j gene, and the expression product of Rbp-j does not function as Rbp-j or It means not to exist.

  The “suppression” includes not only the case where the expression of the Rbp-j gene is completely suppressed, but also the case where the expression of only one gene of the gene pair of the gene is suppressed. In the present invention, it is preferable that the expression of the Rbp-j gene is specifically suppressed. Further, the site where the gene mutation exists is not particularly limited as long as the gene expression is suppressed, and examples thereof include an exon site and a promoter site.

  In the present invention, the animal to be modified with the Rbp-j gene is usually a non-human animal, preferably a rodent such as a mouse, rat, hamster, rabbit, etc. Among them, a mouse is particularly preferable. In addition, “knockout animals” generally referred to are also included in the genetically modified animals of the present invention.

  The non-human mammal of the present invention is particularly characterized in that it suppresses the expression of the Rbp-j gene in a time-specific and / or tissue-specific manner, that is, in a conditional manner. As general methods for controlling the expression of genes in a time-specific and / or tissue-specific manner, for example, A) a gene recombination-inducing method using a recombinase protein / recombinase target sequence system, B) tetracycline Gene expression control methods using activators, etc., and methods combining A) and B) are known (separate volume experimental medicine The Protocol Series "Latest Technology of Gene Turding" (2000, sheep) Satsha) Conditional Targeting Method p.115-120; Biomanual Series 8 “Gene Targeting” —Generation of Mutant Mice Using ES Cells (1995, Yodosha) p.71-77); Sambrook, et al Molecular Cloning: A LABORATORY MANUAL, 3rd edition, COLD SPRING HARBOR LABORATORY PRESS, 2001, 4.82-4.85).

  The gene recombination-inducing method using the recombination protein / recombination target sequence system of A) indicates that a specific recombination protein recognizes the recombination target sequence and causes DNA recombination at that part. This is the method used. The recombination target sequence does not recombine unless a specific recombination is present. Therefore, a genetically modified non-human mammal in which a recombinant protein is expressed in a time-specific and / or tissue-specific manner is crossed with the same genetically-modified non-human mammal into which a recombinant target sequence is introduced, and a combination by recombination is performed. Replacement can occur in a time-specific and / or tissue-specific manner, allowing the desired gene to be controlled in a time-specific and / or tissue-specific manner. A preferred method in the present invention is a Cre / loxP system (Metzger, D) utilizing a Cre recombinase protein derived from bacteriophage P1 and a 34 base pair loxP sequence recognized by Cre protein, which were actually applied in the examples. , et al., Proc. Natl. Acad. Sci., 92, 6991-6995 (1995)), but is not limited to this method. In addition, a yeast-derived FLP / FRT system (Jung, S. et al., Science, 265, 103 (1994)) (mammalian cells and Drosophila also produce homologous recombination), Zygosaccharomyces rouxii pSR1 recombination ( And the like that can function in the yeast S. cerevisiae).

  The gene expression control type method of B) utilizes a method in which a deficient gene is rescued by introducing a modified gene into a specific gene deficient mammal. When the gene expression system is rescued, the deficient gene itself is expressed under the control of a transactivator such as a tetracycline activator (TFT-A). At that time, since the TFT-A gene needs to have the same expression pattern as the defective gene, the TFT-A gene is introduced by knock-in during gene targeting. The knocked-in TFT-A gene is expressed using a gene-deficient gene expression system, and TFT-A acts on the transgenic tet promoter to express the defective gene. That is, a mouse that produces specific gene expression via TFT-A and the tet promoter is prepared. TFT-A cannot act on the tet promoter by adding tetracycline from the outside, so that a specific gene can be deleted in the tissue and time when tetracycline is added. This method can control gene deletion by devising artificial introduction of tetracycline, and can restore gene expression by stopping the addition of tetracycline. Is possible.

  The present invention can include a conditional knockout non-human mammal in which the expression of Rbp-j gene is artificially suppressed in the early stage of pancreatic cell differentiation. A preferred example of the present invention includes the above-mentioned conditional knockout non-human mammal in which expression of the Rbp-j gene is suppressed only in pancreatic cells. These can be obtained, for example, by using a recombinant protein / recombinase target sequence system and using a genetically modified animal that expresses recombinase in an early stage of pancreatic cell differentiation or in a pancreatic cell-specific manner.

  The present invention also provides a cell line established from the genetically modified non-human animal of the present invention. As a method for establishing a cell line derived from the genetically modified animal of the present invention, a known method can be used. For example, in rodents, a method of primary culture of fetal cells can be used.

  The genetically modified animal of the present invention, a cell line established from the animal, and an ES cell line can be used for analysis of detailed functions of the Rbp-j gene. For example, it can be used to infer side effects of Rbp-j inhibitors such as anti-Rbp-j antibodies or Rbp-j antagonist small molecules. Since the conditional knockout mouse obtained in the present invention grew normally and did not die at least in the embryonic stage, the use of an Rbp-j inhibitor (antagonist) for pancreatic cells in the early stage of pancreatic cell differentiation There are no fatal side effects. By examining the genetically modified animals of the present invention in detail, the side effects of Rbp-j inhibitors can be estimated. Further, by using a cell line established from a tissue of a genetically modified animal, it is possible to examine in detail the side effects of the Rbp-j inhibitor in each tissue.

  In addition, by observing the symptoms of the genetically modified animals of the present invention and comparing with the symptoms of the disease whose cause has not been clarified so far, it is clear that the cause of the disease is dysfunction of Rbp-j in pancreatic cells It is also possible to do. For example, phenotypes that appear characteristically in genetically modified mice or mouse-derived cells are observed and compared with various symptoms of human disease. If more than half of the symptoms of the human disease are observed in the genetically modified mouse of the present invention, it can be presumed that the cause of the disease is Rbp-j dysfunction in pancreatic cells. The genetically modified animal of the present invention can also be used as a model animal for diabetes.

  The present invention relates to a method for screening a pharmaceutical composition for suppressing or promoting pancreatic β cell differentiation.

  In the present invention, the pharmaceutical composition that suppresses pancreatic β-cell differentiation may have the same meaning as a pharmaceutical composition for treating or preventing a disease associated with excessive secretion of insulin. In the present invention, diseases associated with excessive insulin secretion may include, but are not limited to, fatty liver, arteriosclerosis, hypersecretion diabetes (such as type 2 diabetes with obesity), or metabolic syndrome. It is not a thing.

  In the present invention, the pharmaceutical composition that promotes pancreatic β-cell differentiation may have the same meaning as a pharmaceutical composition for treating or preventing a disease associated with a relative lack of insulin secretion. .

In the first embodiment of the screening method of the present invention, first, Rbp-j is contacted with a plurality of test compounds.
The nucleotide sequence of human-derived Rbp-j cDNA used in the method of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of Rbp-j encoded by the DNA is shown in SEQ ID NO: 2. In addition, the nucleotide sequence of Rbp-j cDNA derived from mouse is shown in SEQ ID NO: 3, and the amino acid sequence of Rbp-j encoded by the cDNA is shown in SEQ ID NO: 4. In this specification, Rbp-j refers to all of human Rbp-j and mouse Rbp-j unless otherwise specified.

  Further, Rbp-j used in the method of the present invention includes proteins functionally equivalent to the above known Rbp-j. Examples of such proteins include, but are not limited to, Rbp-j mutants, alleles, variants, homologs, Rbp-j partial peptides, or fusion proteins with other proteins.

  As a variant of Rbp-j in the present invention, a naturally occurring protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 or 4 In addition, a protein functionally equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 2 or 4 can be exemplified. A protein encoded by a naturally-occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3, and the amino acid sequence set forth in SEQ ID NO: 2 or 4 Proteins functionally equivalent to the protein consisting of can also be cited as Rbp-j mutants.

  In the present invention, the number of amino acids to be mutated is not particularly limited, but is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids). The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids having amino acids (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains The amino acids (H, F, Y, W) that can be used can be listed (the parentheses indicate the single letter of the amino acid). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes.

  In the present invention, “functionally equivalent” means that the target protein has the same biological function or biochemical function as the Rbp-j protein. In the present invention, the biological function and biochemical function of the Rbp-j protein include a function as a mediator of Notch signaling, and the involvement in maintenance of pancreatic progenitor cells and generation of an appropriate amount of pancreatic β cells. Biological properties include the specificity of the site to be expressed and the expression level.

  Methods well known to those skilled in the art for preparing DNA encoding a “functionally equivalent protein” of the target protein include methods using hybridization techniques and polymerase chain reaction (PCR) techniques. It is done. That is, for those skilled in the art, oligonucleotides that specifically hybridize to Rbp-j (SEQ ID NO: 1 or 3) using Rbp-j base sequence (SEQ ID NO: 1 or 3) or a part thereof as a probe. It is usually possible to isolate DNA having high homology with Rbp-j using as a primer. Thus, DNA encoding a protein having a function equivalent to Rbp-j that can be isolated by hybridization technology or PCR technology is also included in the DNA of the present invention.

  In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5xSSC or equivalent stringency. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 × SSC. The isolated DNA is considered to have high homology with the amino acid sequence of the target protein at the amino acid level. High homology means a sequence of at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence. Refers to the identity of The identity of amino acid sequences and nucleotide sequences is determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  The biological species from which Rbp-j used in the method of the present invention is derived is not limited to a specific biological species. For example, human, monkey, mouse, rat, guinea pig, pig, cow, yeast, insect and the like can be mentioned.

  The state of Rbp-j used in the first embodiment is not particularly limited, and may be, for example, a purified state, a state expressed in a cell, a state expressed in a cell extract, or the like.

  Rbp-j can be purified by a well-known method. Examples of cells expressing Rbp-j include cells expressing endogenous Rbp-j and cells expressing exogenous Rbp-j. Examples of the cells expressing the endogenous Rbp-j include, but are not limited to, cultured cells. The cultured cells are not particularly limited, and for example, commercially available cells can be used. The biological species from which the cells expressing endogenous Rbp-j are derived is not particularly limited, and includes humans, monkeys, mice, rats, guinea pigs, pigs, cows, yeasts, insects and the like. The exogenous Rbp-j-expressing cell can be prepared, for example, by introducing a vector containing DNA encoding Rbp-j into the cell. Introduction of the vector into the cells can be carried out by a general method, for example, calcium phosphate precipitation method, electric pulse perforation method, lipophetamine method, microinjection method and the like. The cells having the exogenous Rbp-j can be prepared, for example, by inserting a DNA encoding Rbp-j into a chromosome by a gene transfer method utilizing homologous recombination. The biological species from which the cell into which such exogenous Rbp-j is introduced is derived is not limited to mammals, and any biological species that has established a technique for expressing a foreign protein in the cell may be used.

  Examples of cell extracts expressing Rbp-j include those obtained by adding a vector containing a DNA encoding Rbp-j to a cell extract contained in an in vitro transcription / translation system. The in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit can be used.

  There is no restriction | limiting in particular as "test compound" in the method of this invention, For example, a single compound, such as a natural compound, an organic compound, an inorganic compound, protein, a peptide, and an expression product of a compound library and a gene library And cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, animal cell extracts, and the like. The test sample can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label. In addition to the test sample, a mixture obtained by mixing a plurality of these test samples is also included.

  In the present invention, “contact” is performed according to the state of Rbp-j. For example, if Rbp-j is in a purified state, it can be carried out by adding a test sample to the purified sample. Moreover, if it is the state expressed in the cell or the state extracted in the cell extract, it can be carried out by adding a test sample to the cell culture solution or the cell extract, respectively. When the test sample is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell in which Rbp-j is expressed, or a cell extract in which the vector is expressed in Rbp-j It is also possible to carry out by adding to. Also, for example, a two-hybrid method using yeast or animal cells can be used.

  In the first embodiment, the binding between Rbp-j and the test compound is then detected. There is no restriction | limiting in particular as a detection method. The binding between Rbp-j and the test compound can be detected by, for example, a label attached to the test compound bound to the Rbp-j protein (for example, a label capable of quantitative measurement such as a radiolabel or a fluorescent label). . It is also possible to detect the change in Rbp-j activity caused by the binding of the test compound to Rbp-j as an indicator.

  In this embodiment, a test compound that binds to Rbp-j is then selected. Selected compounds include compounds that promote or suppress the activity of Rbp-j or compounds that increase or decrease the expression of Rbp-j, which results in the suppression or promotion of pancreatic β-cell differentiation. It is what causes.

  In the second embodiment of the screening method of the present invention, first, a test compound is contacted with a cell expressing Rbp-j.

  In the second embodiment, the expression level of Rbp-j is then measured. The expression level of Rbp-j can be measured by methods known to those skilled in the art. For example, the level of transcription of the gene can be measured by extracting the mRNA of the gene according to a standard method and performing Northern hybridization or RT-PCR using the mRNA as a template. Furthermore, it is also possible to measure the expression level of the gene using DNA array technology.

  In addition, the fraction containing Rbp-j encoded from the gene is collected according to a standard method, and the expression level of Rbp-j is detected by electrophoresis such as SDS-PAGE to measure the translation level of the gene. You can also. Moreover, it is also possible to measure the translation level of a gene by performing Western blotting using an antibody against Rbp-j and detecting the expression of Rbp-j. The antibody against Rbp-j is described below.

  In the second embodiment, a test compound that decreases or increases the expression level of Rbp-j is then selected as compared to the case where no test compound is contacted. Selected compounds include compounds that increase or decrease the expression of Rbp-j, which result in inhibiting or promoting pancreatic β-cell differentiation.

  As a third embodiment of the screening method of the present invention, first, a cell or a cell extract having DNA to which a reporter gene is operably linked downstream of a promoter region of DNA encoding Rbp-j is provided.

  In the third embodiment, “functionally linked” means that the promoter region of the Rbp-j gene is induced such that expression of the reporter gene is induced by binding of a transcription factor to the promoter region of the Rbp-j gene. And the reporter gene. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, the fusion protein is bound by the transcription factor binding to the promoter region of the Rbp-j gene. Any expression that induces expression is included in the meaning of “functionally linked”.

  The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) and a GFP that are commonly used by those skilled in the art. A gene etc. can be mentioned. The reporter gene also includes DNA encoding Rbp-j.

  A cell or cell extract having a DNA to which a reporter gene is operably linked downstream of the promoter region of DNA encoding Rbp-j can be prepared by the method described in the first embodiment.

  In the third embodiment, the test sample is then brought into contact with the cells or the cell extract. Next, the expression level of the reporter gene in the cells or the cell extract is measured.

The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a β-glucuronidase gene (GUS), the luminescence of Glucuron (ICN) or 5-bromo-4-chloro-3-indolyl- By detecting the color development of β-glucuronide (X-Gluc), and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.
When the Rbp-j gene is used as a reporter, the expression level of the gene can be measured by the method described in the second embodiment.

  In the third embodiment, the test compound that decreases or increases the expression level of the reporter gene is then selected as compared to the case where the test compound is not contacted. Selected compounds include compounds that increase or decrease the expression level of the Rbp-j gene, which compounds result in suppression or promotion of pancreatic β-cell differentiation.

  In the fourth embodiment of the screening method of the present invention, first, a test compound is brought into contact with a cell expressing the Rbp-j gene.

  In the fourth embodiment, the Rbp-j activity is then measured. Examples of the activity of Rbp-j include luciferase activity using a Hes promoter. Subsequently, the test compound which reduced or increased the said activity compared with the case where a test compound is not made to contact is selected. Selected compounds include compounds that increase or decrease the activity of Rbp-j, which result in inhibiting or promoting pancreatic β-cell differentiation.

  It is also possible to screen for a pharmaceutical composition that promotes pancreatic β-cell differentiation using the gene-modified non-human mammal. In the present invention, to promote pancreatic β-cell differentiation is to improve the state in which pancreatic β-cell differentiation has decreased to a normal state (a state in which pancreatic β-cell differentiation is normally performed). Also good.

  In this method, first, a test compound is administered to a genetically modified non-human mammal characterized in that the expression of the Rbp-j gene is artificially suppressed in the early stage of pancreatic cell differentiation. Administration of the test compound to the genetically modified animal can be performed orally or parenterally.

  Next, the amount of pancreatic β cells in the genetically modified non-human mammal is measured. The amount of pancreatic β cells is measured by treating the tissue of the test cells with primary and secondary antibodies, staining the pancreatic β cells, and then defining endocrine cells (islets as endocrine cells containing at least five visible nuclei). Can be calculated as the ratio of the area of each hormone positive cell to the total area of the pancreatic slice.

  Rabbit anti-Pdx1 antibody (Guz, Y., et al., Development, 121. 11-18 (1995)), 1: 1500 dilution (provided by C. Wright) as the primary antibody that treats the tissue of test cells Rabbit anti-Hes1 antibody (Jensen, J., et al., Nat. Genet., 24, 36-44 (2000)), 1: 10000 dilution (provided by R. Kageyama); rabbit anti-neurogenin-3 antibody ( Schwitzgebel et al., 2000), 1: 500 dilution (provided by M. German); guinea pig anti-insulin antibody; 1: 500 dilution (DakoCytomation); rabbit anti-glucagon antibody, 1: 500 dilution (DakoCytomation); rabbit anti-somatostatin antibody , 1: 500 dilution (DakoCytomation); rabbit anti-pancreatic polypeptide antibody, 1: 500 dilution (DakoCytomation); rabbit anti-phospho-histone H3 antibody, 1:50 dilution (Cell Signaling Technology); rabbit anti-pancytokeratin antibody, 1 : 200 dilution (Santa Cruz Biotechnology); rabbit anti-amylase antibody, 1: 1000 dilution (Sigma-Aldrich); GI anti-Glut2 antibody (Thorens, B., et al., J. Clin. Invest., 90, 77-85 (1992)), 1: 200 dilution (provided by B. Thorens), etc. Is not to be done. Secondary antibodies include biotinylated goat anti-guinea pig IgG antibody, 1: 200 dilution; biotinylated goat anti-rabbit IgG antibody, 1: 200 dilution; and biotinylated rabbit anti-goat IgG, 1: 200 dilution (all Vector Laboratories ) And the like, but are not limited thereto.

  The amount of pancreatic β cells can be evaluated by the method of Bouwen et al. (Bouwens L, et al., Diabetes 43, 1279-1283 (1994)). Specifically, the whole pancreatic tissue image and all pancreatic islet images on each tissue specimen are captured into a computer with a microscope via a CCD camera, and then captured using software such as NIH Image1.60 (NIH, USA). Pancreatic tissue image and pancreatic β cell are manually analyzed for the shape area. The amount of pancreatic β cells can also be determined as the ratio of pancreatic β cells in the entire pancreatic tissue. This measurement can also be performed by the method specifically described in the examples.

  The method then selects a compound that promotes pancreatic β-cell differentiation in the genetically modified non-human mammal compared to when no test compound is administered. The selected compound includes a compound that promotes pancreatic β-cell differentiation, and is considered to suppress or promote pancreatic β-cell differentiation.

  The present invention relates to a method for suppressing or promoting pancreatic β-cell differentiation, comprising a step of suppressing or promoting Rbp-j gene expression or activity in a cell expressing an Rbp-j gene at an early stage of pancreatic cell differentiation. It also treats or prevents diseases associated with excessive or insufficient secretion of insulin, including the step of suppressing or promoting Rbp-j gene expression or activity in cells that express the Rbp-j gene in the early stages of pancreatic cell differentiation On how to do.

  In the present invention, the method for suppressing the expression or activity of the Rbp-j gene includes RNA complementary to the transcription product of DNA encoding Rbp-j, or the target of a ribozyme that specifically cleaves the transcription product. Can be mentioned. As DNA encoding Rbp-j, DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 3, DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4, and SEQ ID NO: 2 or 4 Or a naturally-occurring DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted.

  The description “suppressing Rbp-j expression” of the present invention includes suppression of gene transcription and suppression of protein translation. Also included is a decrease in expression as well as complete cessation of DNA expression.

  One embodiment of the “RNA complementary to the transcription product of the DNA encoding Rbp-j” of the present invention is an antisense RNA complementary to the transcription product of the DNA encoding the enzyme.

  There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit the expression of target genes by inhibiting transcription, splicing, or translation processes.

  The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the Rbp-j gene is designed, it is considered effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA thus prepared can be transformed into a desired plant by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the plant to be transformed, but may be not completely complementary as long as the gene expression can be effectively inhibited. Good. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

  Another embodiment of the “RNA complementary to the transcription product of DNA encoding Rbp-j” is dsRNA complementary to the transcription product of DNA encoding Rbp-j. RNAi is a phenomenon in which, when a double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both suppressed. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, a RNaseIII-like nuclease called Dicer with a helicase domain is present in the presence of ATP, and dsRNA is about 21 to 23 base pairs from the 3 ′ end. Cut out and produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the mRNA of the target gene at the center of siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  The RNA of the present invention can be expressed from an antisense coding DNA that encodes an antisense RNA to any region of the target gene mRNA and a sense code DNA that encodes a sense RNA of any region of the target gene mRNA. . Moreover, dsRNA can also be produced from these antisense RNA and sense RNA.

  When the dsRNA expression system of the present invention is retained in a vector or the like, the antisense RNA and sense RNA are expressed from the same vector, and the antisense RNA and sense RNA are expressed from different vectors, respectively. There is. For example, antisense RNA and sense RNA can be expressed from the same vector as antisense RNA in which an antisense coding DNA and a promoter capable of expressing a short RNA such as polIII are linked upstream of the sense coding DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction. It is also possible to construct an expression system in which the antisense code DNA and the sense code DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, it is preferable that the two promoters are of different types.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the sense coding DNA. An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.

  In the RNAi of the present invention, siRNA may be used as dsRNA. “SiRNA” means a double-stranded RNA consisting of short strands that are not toxic in cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 Can be ~ 30 base pairs. Alternatively, the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. be able to.

  The DNA used for RNAi need not be completely identical to the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more sequence homology. .

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

  The “suppression of Rbp-j expression” of the present invention can also be performed using a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type.

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, A or U is also shown to be cleaved. If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. It is. For example, there are a plurality of sites that can be targets in the coding region of the present invention that is an inhibition target.

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (J. M. Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.

  A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. However, at that time, if an extra sequence is added to the 5 ′ end or 3 ′ end of the transcribed RNA, the ribozyme activity may be lost. In such a case, in order to accurately cut out only the ribozyme part from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts in cis for trimming is placed on the 5 'side or 3' side of the ribozyme part. (K. Taira et al. (1990) Protein Eng. 3: 733, AMDzianottand JJ Bujarski (1989) Proc. Natl. Acad. Sci. USA. 86: 4823, CAGrosshansand RTCech (1991) Nucleic Acids Res. 19: 3875, K. Taira et al. (1991) Nucleic Acids Res. 19: 5125). In addition, such structural units can be arranged in tandem so that a plurality of sites in the target gene can be cleaved, thereby further enhancing the effect (N. Yuyama et al. Biochem. Biophys. Res. Commun. 186: 1271, 1992). Such a ribozyme can be used to specifically cleave the transcription product of the target gene in the present invention, thereby suppressing the expression of the gene. In addition, a pharmaceutical composition in which the expression or activity of the Rbp-j gene isolated by the screening method of the present invention is suppressed may be added to the subject.

  In the present invention, examples of the method for promoting the expression or activity of the Rbp-j gene include a method in which DNA encoding the Rbp-j protein is administered to a subject and expressed in the subject. The DNA encoding Rbp-j protein is as described above. In addition, a pharmaceutical composition that is isolated by the screening method of the present invention and that promotes the expression or activity of the Rbp-j gene may be added to the subject.

  When the compound that can be isolated by the screening of the present invention, the DNA encoding Rbp-j protein, or the above compound that decreases the expression or activity of Rbp-j is used as a medicine for humans or other animals, these In addition to directly administering the compound itself to a patient, it is also possible to formulate and administer by a known pharmaceutical method. For example, tablets, capsules, elixirs, microcapsules as required, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

  Examples of additives that can be mixed in tablets and capsules include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration to patients are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

  The dose of the compound varies depending on the symptom, but in the case of oral administration, generally for an adult (with a body weight of 60 kg), about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 To 20mg.

  When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, and administration method. For example, in the form of an injection, it is usually an adult (with a body weight of 60 kg), usually per day. It will be convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, an amount converted per 60 kg body weight or an amount converted per body surface area can be administered.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Generation of pancreatic cell-specific Rbp-j knockout mice To investigate the role of Rbp-j in pancreatic development and function, we conditionalize the Rbp-j gene in developing pancreatic cells. Was deleted. In the floxed allele of the Rbp-j gene (referred to herein as Rbp-j ^f ), loxP sites were introduced upstream of exon 6 and downstream of exon 7 of the Rbp-j gene (FIG. 1A). Exons 6 and 7 encode DNA binding and Notch binding domains, and when these exons are deficient, Rbp-j-mediated Notch signaling is completely abolished. Rbp-j ^f is a very effective target for Cre recombinase in multiple tissues (Han, H., et al.,. Int Immunol., 14, 637-645 (2002); Tanigaki, K., et al , Nat. Immunol., 3, 443-450 (2002); Yamamoto, N., et al., Curr Biol., 13, 333-338 (2003)). We crossed Rbp-j ^f mice with Pdx.cre mice (denoted herein as Pdx.cre) whose Cre recombinase is under the transcriptional control of the mouse Pdx1 promoter (Gu, G. , et al., Development, 129, 2447-2457 (2002)).

  In the above, generation of mice having floxed alleles of Rbp-j exons 6 and 7 was performed based on a known method (Han, H., et al., Int Immunol., 14, 637-645 (2002) ). Pdx.cre mice with Cre recombinase under the transcriptional control of the mouse Pdx1 promoter were kindly provided by Dr Guoqiang Gu and Dr Douglas A Melton (Gu, G., et al., Development, 129, 2447-2457 (2002) ).

Next, the present inventors confirmed the timing of Cre / loxP recombination in the pancreatic epithelium of Rbp-j ^{f / f} Pdx.cre mice. The Rosa26R allele has the lacZ gene, a histochemical marker gene, and is transcribed by the widely expressed Rosa26 promoter. An intervening stop sequence sandwiched between two Lox-P sequences is placed upstream of the lacZ reporter (Rosa26 promoter-loxP- [intervening stop sequence] -loxP- [lacZ = βgalactosidase gene]). Only after the sequence has been recombined with Cre will the transcription of the lacZ = βgalactosidase gene be initiated, and blue color will be observed by X-gal staining. (In this specification, it is described as Rosa26R ^f . Soriano, P., Nat. Genet., 21, 70-71 (1999)). Mating mouse Rbp-j ^{f / +} Rosa26R ^{f / f} with floxed Rbp-j heterozygous and Rosa26R homozygous with double heterozygous mouse Rbp-j ^{f / +} Pdx.cre I let you. In the obtained Rbp-j ^{f / f} Rosa26R ^{f / +} Pdx.cre mouse, Cre recombinase was expressed under the control of the Pdx1 promoter, thereby causing the stop cassette upstream of the LacZ gene and the genome of exons 6 to 7 of the Rbp-j gene. The region is excised, resulting in activation of β-galactosidase expression and inactivation of Rbp-j function.

By E9.5, it became clear that the dorsal and ventral pancreatic buds of the foregut region of the Rbp-j ^{f / f} Rosa26R j ^{f / +} Pdx.cre embryo were stained with X-gal (FIG. 1B). X-gal staining was performed in the following steps. Whole embryos on designated days after pregnancy were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 1 hour at 4 ° C and washed with PBS. The activity of β-galactosidase was detected using X-gal by methods known to those skilled in the art (Wu et al., 1997). After staining with X-gal, E8.5 and E9.5 embryos were dehydrated in methanol and washed with a solution of benzyl benzoate: benzyl alcohol = 2: 1. Macroscopic photographs were taken using a Zeiss Axioskop Microscope (Carl Zeiss).

  Based on the above results, Pdx.cre mice began recombining loxP sites in the pancreatic epithelium before E9.5, which is the basis for all pancreatic cell types including islets, acinar and pancreatic duct cells. Suggested (Gu, G., et al., Development, 129, 2447-2457 (2002); Lammert, E., et al., Curr Biol., 13, 1070-1074 (2003); Murtaugh, LC, et al., Proc. Natl. Acad. Sci., 100, 14920-14925 (2003)).

The floxed Rosa26R allele was then detected using a 1.2 kb ClaI-SacI LacZ fragment as a probe. Specificity and efficiency of deletion in Rbp-j ^{f / f} Pdx.cre mice with genomic DNA obtained from the tail or other tissues was assessed by Southern blot analysis of SphI digested genomic DNA prepared from neonatal tissues.

As a result, in the tail, brain and liver of Rbp-j ^{f / f} Pdx.cre mice, only the non-recombinant floxed allele was detected in the 1.2 kb band and the recombinant null allele was found in the 3 kb band only in the pancreas. Detected (FIG. 1C). Although assessment of deletion efficiency in the pancreas is affected by contamination of blood vessels and connective tissue, we confirmed an efficient deletion of the floxed allele in the pancreas from Rbp-j ^{f / f} Pdx.cre mice did.

  All mice used in the examples of the present invention were kept in polycarbonate cages, and were air-controlled (temperature: 24 ± 2 ° C .; humidity: 50 ± 10%) under light control (12 hours illumination, 12 hours dark place). ) Free of food and drink in an environment free of certain pathogens. All experimental procedures were approved by the Animal Experiment Committee of Kyoto University Graduate School of Medicine and carried out in accordance with the 2000 revision of the Declaration of Helsinki. Followed laboratory animal care policy (NIH publication no. 85-23, revised in 1985) and national legislation.

[Example 2] Confirmation of decreased Hes1 expression and increase of neurogenin-3 positive cells in pancreatic buds of Rbp-j ^{f / f} Pdx.cre mice Drosophila hairy and mammalian homologues of Enhancer of split protein are basic called Hes Helix-loop-helix proteins, which are typically positively regulated by Notch / Rbp-j signaling and function as transcriptional repressors of proneural bHLH factors (Kageyama, R., and Ohtsuka, T., Cell Res., 9, 179-188 (1999)). Of the Hes family members isolated in mammals, only Hes1 is expressed early in pancreatic development (Jensen, J., et al., Nat. Genet., 24, 36-44 (2000)). In Rbp-j ^{f / f} mice, Hes1 was uniformly expressed in the nucleus of E10.5 Pdx-1-positive dorsal pancreatic buds, but in Rbp-j ^{f / f} Pdx.cre mice, The number decreased (Figure 1D). In contrast to this decreased expression of Hes1, the number of neurogenin-3 positive cells increased in the pancreatic buds of Rbp-j ^{f / f} Pdx.cre mice (FIG. 1D).

In the examples of the present invention, histological analysis of Rbp-j ^{f / f} Pdx.cre mice was performed by the following steps.
Whole embryos or isolated pancreas were fixed overnight at 4 ° C. with 4% paraformaldehyde in PBS, then embedded in paraffin, 5 μm sections were cut and mounted on glass slides. Antigen activation was performed by dewaxing the slides, rehydrating, and optionally autoclaving at 121 ° C. for 5 minutes with 10 mM citrate buffer. Endogenous peroxidase was inactivated with 0.3% H ₂ O ₂ in methanol for 30 minutes. Slides were blocked with a reagent containing casein (DAKO Protein Block Serum-Free Solution; DakoCytomation) for 1 hour and then stained overnight with the following primary antibodies: rabbit anti-Pdx1 antibody (Guz, Y., et al., Development, 121. 11-18 (1995)), 1: 1500 dilution (provided by C. Wright); rabbit anti-Hes1 antibody (Jensen, J., et al., Nat. Genet., 24, 36-44 (2000)) , 1: 10000 dilution (provided by R. Kageyama); rabbit anti-neurogenin-3 antibody (Schwitzgebel et al., 2000); 1: 500 dilution (provided by M. German); guinea pig anti-insulin antibody; 1: 500 dilution (DakoCytomation); rabbit anti-glucagon antibody, 1: 500 dilution (DakoCytomation); rabbit anti-somatostatin antibody, 1: 500 dilution (DakoCytomation); rabbit anti-pancreatic polypeptide antibody, 1: 500 dilution (DakoCytomation); rabbit anti-phospho-histone H3 antibody, 1:50 dilution (Cell Signaling Technology); Rabbit anti-pancytokeratin antibody 1: 200 dilution (Santa Cruz Biotechnology); rabbit anti-amylase antibody, 1: 1000 dilution (Sigma-Aldrich); rabbit anti-Glut2 antibody (Thorens, B., et al., J. Clin. Invest., 90, 77- 85 (1992)), 1: 200 dilution (provided by B. Thorens). The next day, the slides were washed with PBS and incubated for 2 hours with the following secondary antibodies: biotinylated goat anti-guinea pig IgG antibody, 1: 200 dilution; biotinylated goat anti-rabbit IgG antibody, 1: 200 dilution; and biotinylated rabbit Anti-goat IgG, 1: 200 dilution (all Vector Laboratories). The slide was then incubated with avidin-biotin complex (ABC) reagent (Vectastain Elite ABC Kit; Vector Laboratories) for 50 minutes, and then diaminobenzidine tetrahydrochloride (DAB) (DakoCytomation) was added as a substrate-chromogen solution. After hematoxylin counterstaining and dehydration, slides were encapsulated in mounting medium (MGK-S; Matsunami Glass) and photographed using a Zeiss Axioskop Microscope (Carl Zeiss). Morphological analysis of pancreas from 10-week-old mice was performed using the Scion Image image analysis program (Scion). Islet was defined as a group of endocrine cells with at least 5 cell nuclei and the number of islets was calculated. The amount of endocrine cells was calculated as the ratio of the respective hormone positive cell area to the total area of the pancreatic section. Alexa488 goat anti-rabbit IgG and Alexa546 goat anti-guinea pig IgG (Molecular Probes) were used as secondary antibodies for immunofluorescence. Images were captured using a Zeiss LSM 5 PASCAL confocal microscope (Carl Zeiss). Using the In situ Apoptosis Detection Kit (Takara Bio) for the TUNEL assay, tissues obtained from the degenerated mammary glands of post-lactating female Wistar rats were used as positive controls for apoptosis (Casey, TM, et al. Cell Biol Int., 20, 763-767 (1996)). These sections were counterstained with methyl green. Hes1 in situ hybridization was performed on E15 embryos using a digoxigenin-labeled cRNA probe as per protocol (Tomita, K., et al., EMBO J., 19, 5460-5472 (2000)). . In addition, E15 embryos were fixed, dehydrated, embedded, cut, and then counterstained with Nuclear Fast Red (Vector Laboratories) or immunohistochemically stained. Cell growth was performed on E18.5 embryos using a commercially available cell growth kit (Amersham Pharmacia Biotech), and 5-bromo-2′-deoxyuridine (BrdU) incorporation into replicating DNA was detected. To label the growing cells, E18.5 pregnant mice were injected intraperitoneally with BrdU (100 μg / g) and sacrificed 2 hours later.

Example 3 E11.5 Rbp-j ^{f / f} Pdx.cre mice histological analysis in ^{- Rbp-j f / f Pdx.cre} α cells and pancreatic polypeptide production in mice (PP) promoting differentiation of the cells Next, histological analysis in E11.5 was performed based on the method described in Example 2, and a small number of dispersed α cells were observed in the protruding epithelial cells of Rbp-j ^{f / f} mice ( Figure 2A). In Rbp-j ^{f / f} Pdx.cre mice, a cap of differentiated endocrine cells expressing glucagon or pancreatic polypeptide was found to surround the pancreatic bud. The number of Pdx-1 positive cells and neurogenin-3 positive cells was decreased in Rbp-j ^{f / f} Pdx.cre mice compared to control mice. The relative number of proliferating cells against Pdx-1 positive epithelium detected by phospho-histone H3 immunostaining was comparable between control and mutant mice. The present inventors detected apoptosis using the TUNEL (dUTP nick end labeling with terminal deoxynucleotide transferase) method. However, no apoptotic cells were observed in the pancreatic epithelium of control mice and mutant mice. Whole tissue X-gal staining at this stage resulted in smaller pancreatic buds in Rbp-j ^{f / f} Rosa26R ^{f / +} Pdx.cre mice compared to control Rbp-j ^{f / +} Rosa26R ^{f / +} Pdx.cre littermates (FIG. 2B). These data indicate that the lack of Notch / Rbp-j signaling promotes endocrine cell differentiation, a substantial decrease in both pancreatic progenitor (Pdx-1 positive) and endocrine progenitor (neurogenin-3 positive) cells in the early pancreatic development Shows that cause.

Example 4 E15 Rbp-j ^{f / f} Pdx.cre mice histological analysis in - Rbp-j ^{f / f} tubular structure extending involves branching decrease in Pdx.Cre mice Next, according to Example 2 Histological analysis in E15 based on this method revealed that Pdx1 is still expressed in the epithelial cells of the developing pancreatic cells, which are the pancreatic progenitor cell pool. In the control pancreas, epithelial tissues with strong positive Pdx-1 and weak positive cytokeratin showed a complex and branched network (FIG. 3A). However, in the mutant mouse pancreas, the branching morphogenesis of epithelial tissue was disrupted, and the tubular structure was prominent (FIG. 3A). The cells lining the cavities of these circular structures showed no signs of apoptosis (Figure 3A) and were actively mitotic (Figure 3B). Glucose transporter 2 (Glut2) is expressed in mature beta cells and is also thought to be a marker for early pancreatic progenitor cells (Pang, K., et al., Proc. Natl. Acad. Sci., 91, 9559) -9563 (1994)). Expression of Glut2 was not observed in the tubular structure observed in pancreatic cells of mutant mice (FIG. 3B). Hes1 was strongly expressed in intestinal crypt cells (as shown in Rbp-j ^{f / f} mouse photo) and renal pretubular aggregates (as shown in Rbp-j ^{f / f} Pdx.cre mouse photo) (Figure 3B). A large number of neurogenin-3 positive cells were also observed in the pancreatic cells of the control group, but substantially no expression of Hes1 or neurogenin-3 was observed in the pancreatic cells of the mutant mice (FIG. 3B). . Analysis of the various differentiation markers and tubular morphology described above indicate that the cells lining these tubular structures are proliferating pancreatic duct cells rather than progenitor cells. By tracing the cell lineage, the present inventors confirmed that the aggregated glucagon-producing cells and the accompanying tubule cells at the tip of the tube bundle were derived from Rbp-j-deficient cells (FIG. 3C).

Example 5 E18.5 Rbp-j ^{f / f} Pdx.cre mice histological analysis at - embryos Late Rbp-j ^{f / f} Pdx.cre mouse pancreatic duct cells dominant and acinar in pancreas catch-up growth Next, histological analysis in E18.5 was performed based on the method described in Example 2, and amylase positive cells were observed in Rbp-j ^{f / f} Pdx.cre mice. Much smaller, cytokeratin-positive pancreatic duct cells were found to occupy a larger area than Rbp-j ^{f / f} mice. High proliferation of acinar cells was observed by BrdU incorporation assay (Figure 4A), but hematoxylin-eosin staining of spaced sections from embryos resulted in much smaller pancreatic cells compared to control mice It became clear (Fig. 4B).

Histological analysis of Rbp-j ^{f / f} Pdx.cre mice in Example 6 postnatal Next, examine the pancreas of postnatal Rbp-j ^{f / f} Pdx.cre mice according to the method described in Example 2 It was. Adult Rbp-j ^{f / f} Pdx.cre mice clearly showed normal gastrointestinal tract and small pancreas (FIG. 5A). Absolute pancreatic weight (Figure 5B) (Rbp-j ^{f / f} Pdx.cre, 209 ± 73 mg vs. Rbp-j ^{f / f} , 685 ± 81 mg, P <0.005) and ratio of pancreatic weight to total body weight (Rbp-j ^{f / f} Pdx.cre, 0.915 ± 0.30% vs. Rbp-j ^{f / f} , 1.83 ± 0.11%, P <0.05) compared to Rbp-j ^{f / f} mice in mutant Rbp-j ^{f / f} Pdx.cre mice It was low. Rbp-j ^{f / f} Pdx.cre pancreas sections had fewer islets per pancreas area than Rbp-j ^{f / f} mice (Rbp-j ^{f / f} Pdx.cre, 0.146 ± 0.060 pair) Rbp-j ^{f / f} , 0.618 ± 0.127 islet / islet area (mm ² ), P <0.05; FIGS. 5C and 5D), islet size was small (FIG. 5C).

  Isolation of islets was performed by the following steps. For islet isolation, 3 mL of 1.5 mg / mL collagenase (Worthington Biochemical) in Hank's solution (HBSS) (Invitrogen) was injected into the pancreas from the pancreatic duct, which was then removed and incubated at 37 ° C. for 20 minutes. Digested pancreas was washed 4 times with HBSS, and islets were manually sorted under a microscope (Gotoh, M., et al., Transplantation, 40, 437-438 (1985)).

The amount of endocrine cells was quantified by estimating the hormone-positive area per total pancreatic area in multiple pancreatic sections. The amount of β cells in Rbp-j ^{f / f} Pdx.cre mice was significantly reduced to about 25% of the amount of β cells in Rbp-j ^{f / f} mice (P <0.001; FIG. 5E). Rbp-j ^{f / f} Pdx.cre mouse pancreas showed a relative increase of α cells compared to β cells, but the absolute amount of α cells was also significantly increased to about 50% of Rbp-j ^{f / f} mice. Decreased (P <0.001; FIGS. 5E and 5G). Pancreatic total insulin content (per 1 mg pancreatic weight) was measured in acid-ethanol extracts of whole pancreas. Rbp-j ^{f / f} Pdx.cre mice had much lower insulin content than Rbp-j ^{f / f} mice (FIG. 5F). In addition to fewer islets, histological examination of the pancreas of adult Rbp-j ^{f / f} Pdx.cre mice revealed that endocrine cells were frequently seen with dilated pancreatic ducts (Figure 5G). . The relative pancreatic duct cell hyperplasia observed in the embryonic stage in Rbp-j ^{f / f} Pdx.cre mice (Figures 3 and 4) was not evident in adult Rbp-j ^{f / f} Pdx.cre mice (Figure 5C). And 5G).

In the present invention, analysis of metabolic parameters was performed by the following steps.
The blood glucose level was determined from whole venous blood collected from the mouse tail using an automatic blood glucose meter (Glutest Ace, Sanwa Kagaku) or an enzyme colorimetric assay kit (Glucose CII test, Wako Pure Chemical). Blood for insulin and amylase was collected by retroorbital bleeds. Plasma insulin levels were measured using an ELISA kit (Morinaga). For glucagon, blood samples were collected in tubes containing EDTA (final concentration 1 mg / mL) and aprotinin (500 KIE in 1 mL blood; Bayer). For measurement of pancreatic insulin content, the pancreas was quickly removed, weighed and frozen in liquid nitrogen. Insulin was extracted by mechanical homogenization in ice-cold acid ethanol. After 24 hours at 4 ° C, the sample was centrifuged and the supernatant was collected and stored at -20 ° C. Insulin concentration was determined by ELISA. Amylase activity was measured using a kit (Amylase-Test Wako, Wako Pure Chemical) according to the Caraway method (Casey et al., 1996). All values are expressed as mean ± standard error.

[Example 7] Observation of postnatal characteristics of Rbp-j ^{f / f} Pdx.cre mice Next, the postnatal characteristics of Rbp-j ^{f / f} Pdx.cre mice were observed. The growth of Pdx.cre Rbp-j ^{f / f} mice fed with normal chow and control Rbp-j ^{f / f} mice was observed for 4 months. Under continuous observation, Rbp-j ^{f / f} Pdx.cre mice showed a very lean phenotype compared to Rbp-j ^{f / f} mice, with no further weight gain (Figure 6A and 6B). Mutant mice showed polyuria, heavy drinking, and exhibited lethargy before death. These mice develop marked hyperglycemia both in the fasted and fed state (fasting: Rbp-j ^{f / f} Pdx.cre, 348 ± 61 mg / dL vs. Rbp-j ^{f / f} , 98 ± 6mg / dL, P <0.001; After breakfast: Rbp-j ^{f / f} Pdx.cre, 524 ± 59mg / dL vs. Rbp-j ^{f / f} , 124 ± 12mg / dL, P <0.001) (Figure 6C), Plasma insulin levels were significantly reduced (fasting: Rbp-j ^{f / f} Pdx.cre was below the detection limit, whereas Rbp-j ^{f / f} was 0.48 ± 0.07 ng / mL, P <0.001; After breakfast: Rbp-j ^{f / f} Pdx.cre was 0.04 ± 0.04 ng / mL and Rbp-j ^{f / f} was 1.35 ± 0.29 ng / mL (P <0.001) (FIG. 6D). Daily intake of Rbp-j ^{f / f} Pdx.cre mice was increased compared to the control group (FIG. 6E), which corresponded to diabetic polyphagia. Thus, Rbp-j ^{f / f} Pdx.cre mice exhibited characteristics typical of diabetes with a deficiency in insulin secretion. Furthermore, Rbp-j ^{f / f} Pdx.cre mice were found to have lower serum amylase activity than Rbp-j ^{f / f} mice (Rbp-j ^{f / f} Pdx.cre, 711 ± 58 U / dl vs. Rbp -j ^{f / f} , 1121 ± 67 U / dl, P <0.01; FIG. 6G). This was probably due to pancreatic insufficiency and severe diabetes.

Example 8
To evaluate the direct role of Notch / Rbp-j signaling in pancreatic β cells, we next selected Rbp-j ^{f / f} mice, mice expressing Cre under the control of the rat insulin II gene promoter ( In this specification, Rip.cre is described as mouse (Postic, C., et al., J Biol Chem., 274, 305-315 (1999)). Rip.cre mice with Cre recombinase under the control of the rat insulin II promoter were purchased from Jackson laboratory (Postic, C., et al., J Biol Chem., 274, 305-315 (1999)).

Transgene expression of Rip.cre in β cells begins during embryogenesis and causes recombination in about 85% of β cells in adult mice (Kulkarni, RN, et al., Cell, 96, 329-339 (1999) Sund, NJ, et al., Genes Dev., 15, 1706-1715 (2001)). The present inventors confirmed high deletion efficiency of the Rbp-j gene by Southern blot analysis of genomic DNA from the isolated island (FIG. 7A). Rbp-j ^{f / f} Rip.cre mice had normal body size (data not shown). In the postprandial state, blood glucose levels (Rbp-j ^{f / f} Rip.cre, 134 ± 10 mg / dL vs. Rip.cre, 135 ± 12 mg / dL, p = NS) or plasma insulin levels (Rbp-j ^{f / f} Rip. cre, 1.17 ± 0.19 ng / mL vs. Rip.cre, 1.10 ± 0.12 ng / mL, p = NS), no significant difference was detected (FIGS. 7B and 7C). No abnormalities were observed by hematoxylin-eosin staining and immunohistochemistry using glucagon, insulin, Pdx1, somatostatin, pancreatic polypeptide, pancytokeratin and amylase (FIG. 7D). These data, combined with findings from Rbp-j ^{f / f} Pdx.cre mice, Rbp-j is not required to maintain β-cell function or β-cell mass, but is sufficient to generate a sufficient number of β-cells. Indicates that it is necessary.

In the present invention, the inventors focused on the role of Rbp-j in the pancreas. The results of the present invention indicate that Rbp-j function is essential for normal pancreatic development and their normal glucose homeostasis. In Rbp-j ^{f / f} Pdx.cre mice, all types of pancreatic cells differentiate, but both organs and islets are significantly smaller than control littermates. Analysis of fetal pancreas in Rbp-j ^{f / f} Pdx.cre mice revealed early endocrine cell differentiation of E11.5 (Fig. 2), after which residual epithelial cells tended to differentiate into pancreatic duct cells (Figures 3 and 4). From the normal phenotype of Rbp-j ^{f / f} Rip.cre mice, it is also clear that Rbp-j is not required for β-cell function and maintenance after cell differentiation (FIGS. 7A-E).

  Rbp-j is an important mediator of Notch signaling because it binds to all intracellular regions of Notch receptor type 4 (Kato, H., et al.,. FEBS Lett., 395, 221-224 (1996). )). However, the downstream target of Notch / Rbp-j signaling has not been fully identified and is thought to be cell type specific. For example, Northern blot analysis of whole embryos showed that Rbp-J or Notch1 knockout mice have decreased levels of Hes5 expression but not Hes1 or Hes3 expression (de la Pompa, JL, et al ., Development, 124, 1139-1148 (1997)). The present inventors have also reported that Hes1 and Hes5 expression is not significantly changed in Rbp-j-deficient T lymphocytes (Tanigaki, K., et al., Immunity, 20, 611-622 (2004)). ). In the present invention, a partial decrease in Hes1 expression and an increase in the number of neurogenin-3 positive cells were observed (FIG. 1D). These limited effects suggest that several other mechanisms may be involved in maintaining Hes1 expression and neurogenin-3 repression early in pancreatic development. Since our mutant mice show strong pancreatic duct differentiation that was not observed in Hes1 knockout mice (Jensen, J., et al., Nat. Genet., 24, 36-44 (2000)), Rbp-j May have a role unrelated to Hes1 activation. A possible mechanism to explain this involves the basic helix-loop-helix transcription factor Ptf1a. Like Pdx1, Ptf1a is expressed in progenitor cells of all pancreatic cell lineages, and the mutation causes pancreatic aplasia (Kawaguchi, Y., et al., Nat. Genet., 32, 128-134 (2002 )). Since Rbp-j binds to Ptf1a and further stimulates its transcriptional activity (Obata, J., et al., Genes Cells, 6, 345-360 (2001)), Rbp-j is responsible for the full activity of Ptf1a. This is considered necessary.

In Rbp-j ^{f / f} Pdx.cre mice, E10.5 increased the number of neurogenin-3 positive cells (Figure 1D), and E11.5 depleted both Pdx1 and neurogenin-3 positive cells, α and PP cell differentiation was promoted (FIG. 2). This decrease in common pancreatic (Pdx1 positive) and endocrine (neurogenin-3 positive) cell pools may be one explanation for pancreatic dysplasia (FIGS. 5A and 5B). However, not all cells differentiate into endocrine cells due to lack of Notch / Rbp-j signaling (FIGS. 2 and 3). In Rbp-j ^{f / f} Pdx.cre mice, residual Pdx-1-positive epithelial cells that have not differentiated from endocrine cells tend to differentiate into pancreatic duct cells. Although relatively little is currently known about pancreatic duct differentiation, cell lineage tracking studies show that adult pancreatic duct cells arise from Pdx-1-positive neurogenin-3-negative progenitor cells between E9.5 and E12.5 (Gu, G., et al., Development, 129, 2447-2457 (2002)). Since disruption of the Rbp-j gene in Rbp-j ^{f / f} Pdx.cre mice occurred with the initiation of pancreatic duct differentiation, Notch / Rbp-j signaling may play a role in inhibiting early pancreatic duct differentiation.

Although the role of Notch signaling in terminally differentiated cells is not known, it is speculated that Notch provides some degree of plasticity in postmitotic neurons (Ahmad, I., et al., Mech. Dev. , 53, 73-85 (1995)). We detected Notch2 and Dll1 expression in endocrine cells of adult mice (data not shown). Furthermore, it has been suggested that neurogenin-3 positive endocrine precursor cells are within islets (Fernandes, A., et al., Endocrinology, 138, 1750-1762 (1997); Gu, G., et al. , Development, 129, 2447-2457 (2002)); however, we found no difference between Rbp-j ^{f / f} Rip.cre mice and control mice (FIG. 7). It has been reported that forced expression of Notch1 IC in adult pancreas does not disturb mature endocrine cells (Murtaugh, LC, et al., Proc. Natl. Acad. Sci., 100, 14920-14925 (2003)). Together with the results of Rbp-j ^{f / f} Pdx.cre mice, Notch signaling in the pancreas appears to be essential only in the early stages of development. In adult islets, β-cells are generated primarily by the self-proliferation of existing β-cells (Dor, Y., et al., Nature, 429, 41-46 (2004)). Although compensatory acinar cell proliferation is large, the absolute low beta cell mass seen in adult Rbp-j ^{f / f} Pdx.cre mice suggests that this is a limit to the ability of differentiated beta cells to proliferate However, some degree of self-growth or transdifferentiation may compensate for reduced beta cell production in mutant mice.

In the present invention, the present inventors showed the effect of Notch / Rbp-j signaling on pancreatic development by a time-specific conditional gene targeting method. In Rbp-j ^{f / f} Pdx.cre mice, in addition to early α-cell differentiation, promotion of pancreatic duct differentiation was observed, and the overall result was β-cell dysplasia and insulin-deficient diabetes. In Rbp-j ^{f / f} Pdx.cre mice, β cells were not affected. Based on our results, the role of Notch / Rbp-j signaling in the pancreas is thought to occur as in the model shown in FIG. 7F. Pdx1-positive progenitor cells around E9 are the source of all mature cells in the pancreas. Lineage follow-up studies have shown that Pdx1-positive exocrine and endocrine precursor cells are present throughout embryogenesis (Gu, G., et al., Development, 129, 2447-2457 (2002)). Around E10, active Notch / Rbp-j signaling helps maintain a pool of epithelial progenitor cells while inhibiting early alpha and PP cell differentiation. Then, during proliferation and branching morphogenesis, the common progenitor cells acquire the eligibility to gradually progress to pancreatic duct, beta cell or delta (somatostatin producing) cell fate. At these stages, Notch / Rbp-j signaling then maintains progenitor cells by inhibiting pancreatic duct differentiation. Depletion of early progenitor cells, PP cells, and common progenitor cells by pancreatic duct differentiation in Rbp-j ^{f / f} Pdx.cre mice is usually due to the late stage of β-cell, δ-cell, and most acinar cell engagement Significantly affects the production of cells. Notch / Rbp-j signaling basically has no role on differentiated pancreatic β cells.

  The data presented in the present invention clearly show that Rbp-j is a key molecule for the proliferation of pancreatic progenitor cells and their proper differentiation into mature pancreatic cells. A comprehensive understanding of Notch / Rbp-j signaling in the pancreas provides an opportunity for rational control of pancreatic stem / progenitor cells both in vivo and in vitro, enabling insulin producing cells to be treated for diabetes treatment There is hope for unlimited supply.

FIG. 4 is a diagram and a photograph showing the generation of Rbp-j ^{f / f} Pdx.cre mice and the effect of Rbp-j deletion on downstream targets. A) Disruption of the Rbp-j gene by Cre recombinase. The figure above shows a partial genome map of the floxed Rbp-j allele. The figure below shows the target locus after excision of exons 6 and 7 adjacent to the loxP site. Exons, diagnostic restriction fragment lengths, and probe positions used for Southern blotting are shown. The SphI restriction site is also shown. B) Photographs showing the results of X-gal staining of whole embryos of E8.5 and E9.5 Rbp-j ^{f / f} Rosa26R ^{f / +} Pdx.cre mice. Pdx.cre has begun to recombine loxP sites before E9.5. C) Photograph showing the efficient and specific deletion of Rbp-j in Rbp-j ^{f / f} Pdx.cre mice. Genomic DNA from the brain, liver, tail and pancreas of Rbp-j ^{f / f} Pdx.cre mouse neonates was digested with SphI and hybridized with the probe shown in (A). It is a photograph showing the result of immunostaining two individual serial sections from different dorsal pancreatic buds of E10.5 Rbp-j ^{f / f} mice and Rbp-j ^{f / f} Pdx.cre mice. 2 shows reduced Hes1 expression and increased neurogenin-3-positive cells in E10.5 Rbp-j ^{f / f} Pdx.cre mice. For Hes1 immunostaining, counterstaining was omitted for clarity. FIG. 3 is a photograph showing early-onset endocrine cell differentiation and progenitor cell loss in E11.5 Rbp-j ^{f / f} Pdx.cre mice. Two each successive pancreatectomy piece from Rbp-j ^{f / f} mice and Rbp-j ^{f / f} Pdx.cre mouse E11.5, hematoxylin - eosin staining, is a photograph showing the results of immunohistochemical staining and TUNEL assay . Fig. 2 shows promotion of differentiation of α cells and PP cells in Rbp-j ^{f / f} Pdx.cre mice. It is a photograph which shows the result of the X-gal staining of the whole embryo from Rbp-j ^{f / f} Rosa26R ^{f / +} and Rbp-j ^{f / f} Rosa26R ^{f / +} Pdx.cre mouse | mouth of E11.5. Fig. 6 shows pancreas size reduction in Rbp-j ^{f / f} Pdx.cre mice. It is a photograph which shows the tubular structure expansion | extension accompanied by the branching reduction in the Rbp-j ^{f / f} Pdx.cre mouse | mouth of E15. It is a photograph showing the results of hematoxylin-eosin staining, immunostaining and TUNEL assay of serial pancreatic sections from Ebp Rbp-j ^{f / f} mice and Rbp-j ^{f / f} Pdx.cre mice. Shows expansion and elongation of tube-like structures in Rbp-j ^{f / f} Pdx.cre mice. B) Photographs showing immunostaining and Hes1 in situ hybridization of serial pancreatic sections from E15 Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice. The characteristics of pancreatic duct cells of Rbp-j ^{f / f} Pdx.cre mice are shown. C) Photographs showing the results of X-gal staining of a distal pancreatic section from Rbp-j ^{f / f} mice and Ebp Rbp-j ^{f / f} Pdx.cre mice and double staining of glucagon and insulin. Cre-mediated deletion in Rbp-j ^{f / f} Pdx.cre mice can be confirmed. It is a photograph showing pancreatic duct cell dominance and acini catch-up growth in the pancreas of E18.5 Rbp-j ^{f / f} Pdx.cre mice. A) Photographs showing the results of hematoxylin-eosin staining, BrdU labeling, and immunostaining of serial pancreatic sections from E18.5 Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice. In the photograph of the BrdU label, the outline of the acinar region is indicated by a black broken line. In Rbp-j ^{f / f} Pdx.cre mice, the number of Brdu-labeled cells in the acinar region was increasing, and pancreatic duct cells occupied a wider area. B) Photographs showing the results of hematoxylin-eosin staining of spaced abdominal sections from E18.5 Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice. The pancreatic tissue is surrounded by a black broken line. Figure 5 shows pancreas reduction in pre-born Rbp-j ^{f / f} Pdx.cre mice. It is the photograph and figure which show the character of postnatal Rbp-j ^{f / f} Pdx.cre mouse. FIG. 2 is a photograph and a diagram showing pancreas reduction in adult Rbp-j ^{f / f} Pdx.cre mice. At 12 weeks of age, the intestinal tract region was excised (A) and the pancreatic weight was measured (B). Bars represent mean ± SE. Indicates the level of significance (Student's t test) (* = P <0.05; ** = P <0.01). It is the photograph and figure which show the pancreatic islet reduction and the endocrine cell amount reduction in a Rbp-j ^{f / f} Pdx.cre mouse | mouth. Pancreatic sections from 10-week-old Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice were double stained for insulin and glucagon (C) Number of islets (D), β cells and α Cell area (E) was determined and normalized to the total pancreatic area. Bars represent mean ± SE. Indicates the level of significance (Student's t test) (* = P <0.05; *** = P <0.001). It is a figure which shows the fall of pancreatic insulin content in a Rbp-j ^{f / f} Pdx.cre mouse | mouth. Pancreatic insulin content was measured with acid-ethanol extracts from 6-week-old Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice. Bars represent mean ± SE. Indicates the level of significance (Student's t test) (* = P <0.05; *** = P <0.001). It is a photograph showing the results of hematoxylin-eosin staining and immunostaining of serial pancreatic sections from 8-week-old Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice. Figure 5 shows pancreatic duct and duct-related endocrine cell expansion in Rbp-j ^{f / f} Pdx.cre mice. The number of β cells was significantly reduced. Endocrine cells are located near the pancreatic ductal structure. It is a photograph which shows the diabetes phenotype of Rbp-j ^{f / f} Pdx.cre mouse | mouth. FIG. 2 is a photograph and a diagram showing growth delay in Rbp-j ^{f / f} Pdx.cre mice. Shown are macroscopic findings of 6-day-old (left) and 3-week-old (right) Rbp-j ^{f / f} Pdx.cre mice (bottom) and control littermates (top). FIG. 3 shows the diabetes phenotype of Rbp-j ^{f / f} Pdx.cre mice. B) A littermate growth curve obtained by mating Rbp-j ^{f / f} mice and Rbp-j ^{f / f} Pdx.cre mice is shown. Each bar represents the mean ± SE. CE) Blood glucose levels (C) and plasma insulin levels (D) in 8-week-old male Rbp-j ^{f / f} and Rbp-j ^{f / f} Pdx.cre mice fed fast and randomly fed FIG. Each bar represents the mean ± SE. It is a figure which shows the result of having measured the food intake from the 16-week-old male Rbp-j ^{f / f} mouse ^| mouth and Rbp-j ^{f / f} Pdx.cre mouse | mouth for 24 hours (E). Each bar represents the mean ± SE of mice. F) Serum amylase activity measured from 10-week-old male Rbp-j ^{f / f} mice and Rbp-j ^{f / f} Pdx.cre mice. Rbp-j ^{f / f} Rip.cre mice show exocrine pancreatic dysfunction. Bars represent mean ± SE of mice. Indicates the level of significance (Student's t test) (** = P <0.01; *** = P <0.001). It is the photograph and figure which show the character in a beta cell specific Rbp-j knockout mouse. A) A photograph certifying the generation of Rbp-j ^{f / f} Rip.cre mice. Genomic DNA from tails or pooled isolated islets of 12 week old Rbp-j ^{f / f} Rip.cre mice was digested with SphI and hybridized with the probes shown in (FIG. 1A). (B to D) shows metabolic parameters in Rbp-j ^{f / f} Rip.cre mice. Body weight from 19-week-old male Rip.cre and Rbp-j ^{f / f} Rip.cre mice (B), blood glucose level randomly fed (C), and insulin concentration randomly fed (D) is shown. Bars represent mean ± SE of mice. Indicates the level of significance (Student's t test) (NS; not significant). It is a photograph which shows the result of the immunohistochemistry of the serial pancreas section from a 20-week-old Rip.cre mouse | mouth and a Rbp-j ^{f / f} Rip.cre mouse | mouth. No morphological changes are observed in pancreas from Rbp-j ^{f / f} Rip.cre mice. FIG. 6 shows a simplified model of the role of Notch / Rbp-j signaling in pancreatic development. PP and δ cell differentiation was omitted for simplicity.

Claims (21)

  A genetically modified non-human mammal, wherein the expression of the Rbp-j gene is artificially suppressed during the differentiation process of pancreatic cells.   The genetically modified non-human mammal according to claim 1, wherein a foreign gene is inserted into one or both of the Rbp-j gene pairs.   The genetically modified non-human mammal according to claim 1 or 2, wherein the expression of the Rbp-j gene is suppressed only in pancreatic cells.   The genetically modified non-human mammal according to any one of claims 1 to 3, which is a model animal for diabetes. The screening method of the pharmaceutical composition which suppresses pancreatic beta cell differentiation including the process of the following (a)-(c).
(A) contacting Rbp-j with a test compound (b) detecting a binding between Rbp-j and the test compound (c) selecting a test compound that binds to Rbp-j. The screening method of the pharmaceutical composition which suppresses pancreatic beta cell differentiation including the process of the following (a)-(c).
(A) A step of bringing a test compound into contact with a cell expressing the Rbp-j gene (b) A step of measuring the expression level of Rbp-j (c) Rbp as compared with the case where the test compound is not contacted a step of selecting a test compound having a decreased expression level of -j. The screening method of the pharmaceutical composition which suppresses pancreatic (beta) cell differentiation including the process of the following (a)-(d).
(A) a step of providing a cell or cell extract having DNA to which a reporter gene is operably linked downstream of a promoter region of DNA encoding Rbp-j (b) a test compound in the cell or cell extract (C) measuring the expression level of the reporter gene in the cell or cell extract (d) reducing the expression level of the reporter gene compared to the case where the test compound is not contacted A step of selecting the test compound that has been allowed to enter. The screening method of the pharmaceutical composition which suppresses pancreatic beta cell differentiation including the process of the following (a)-(c).
(A) a step of contacting a test compound with a cell expressing the Rbp-j gene (b) a step of measuring the activity of Rbp-j in the cell (c) as compared with the case where the test compound is not contacted A step of selecting a test compound having reduced activity.   9. The screening method according to claim 5, wherein the pharmaceutical composition that suppresses pancreatic β-cell differentiation is a pharmaceutical composition for treating or preventing a disease associated with excessive insulin secretion. .   The method according to claim 9, wherein the disease associated with excessive insulin secretion is fatty liver, arteriosclerosis, hyperinsulinemia, or metabolic syndrome.   A pharmaceutical composition for treating or preventing a disease associated with excessive secretion of insulin, selected by the screening method according to claim 5. A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) contacting Rbp-j with a test compound (b) detecting a binding between Rbp-j and the test compound (c) selecting a test compound that binds to Rbp-j. A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) A step of bringing a test compound into contact with a cell expressing the Rbp-j gene (b) A step of measuring the expression level of Rbp-j (c) Rbp as compared with the case where the test compound is not contacted a step of selecting a test compound having an increased expression level of -j. A method for screening a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (d):
(A) a step of providing a cell or cell extract having DNA to which a reporter gene is operably linked downstream of a promoter region of DNA encoding Rbp-j (b) a test compound in the cell or cell extract (C) measuring the expression level of the reporter gene in the cell or the cell extract (d) increasing the expression level of the reporter gene compared to the case where the test compound is not contacted A step of selecting the test compound that has been allowed to enter. A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell expressing the Rbp-j gene (b) a step of measuring the activity of Rbp-j in the cell (c) as compared with the case where the test compound is not contacted A step of selecting a test compound having increased activity. A screening method for a pharmaceutical composition that promotes pancreatic β-cell differentiation, comprising the following steps (a) to (c).
(A) a step of administering a test compound to a genetically modified non-human mammal characterized in that the expression of the Rbp-j gene is artificially suppressed (b) pancreatic β cells in the genetically modified non-human mammal A step of measuring the amount (c) a step of selecting a compound that increases the amount of pancreatic β cells in the genetically modified non-human mammal as compared to a case where the test compound is not administered.   The method for screening according to any one of claims 12 to 16, wherein the pharmaceutical composition for promoting pancreatic β-cell differentiation is a pharmaceutical composition for treating or preventing a disease associated with insufficient insulin secretion. .   The pharmaceutical composition for treating or preventing diabetes selected by the screening method in any one of Claims 12-17.   A method of promoting pancreatic β-cell differentiation, comprising a step of promoting Rbp-j gene expression or activity in a cell expressing an Rbp-j gene at an early stage of pancreatic cell differentiation.   A method for treating or preventing a disease associated with insufficient secretion of insulin, comprising a step of promoting Rbp-j gene expression or activity in a cell that expresses an Rbp-j gene at an early stage of pancreatic cell differentiation.   21. The method according to claim 20, wherein the disease associated with insufficient insulin secretion is diabetic insulin secretion type diabetes.