WO2007141974A1

WO2007141974A1 - Nonhuman model animal of hypertension

Info

Publication number: WO2007141974A1
Application number: PCT/JP2007/059316
Authority: WO
Inventors: Kazutake Tsujikawa; Hiroshi Yamamoto; Masaru Okabe; Hiroshi Okamoto; Katsutoshi Yayama
Original assignee: Osaka University; Pca Intermed, Inc.
Priority date: 2006-06-02
Filing date: 2007-05-01
Publication date: 2007-12-13
Also published as: JP2009201355A

Abstract

It is intended to provide a nonhuman model animal of hypertension which is useful in treating a vascular disease, a method of efficiently screening a drug for preventing and/or treating a vascular disease with the use of the above animal, and a drug obtained by the above method. Namely, a nonhuman model animal of hypertension characterized by completely lacking the function of RAMP1 gene on the chromosome due to the partial or entire modification of the RAMP1 gene. This nonhuman model animal of hypertension can be constructed by, for example, either a method with the use of an ES cell obtained by treating an ES cell containing a region wherein at least a part of the exon of RAMP1 gene is sandwiched between LoxP sequences and another region wherein a selection marker gene is sandwiched between LoxP sequences on the chromosome with Cre enzyme, or a method with the use of mating an RAMP1-floxed mouse having been constructed by using an ES cell containing a region wherein at least a part of the exon of RAMP1 gene is sandwiched between LoxP sequences on the chromosome with a mouse capable of expressing a Cre enzyme.

Description

Specification

Hypertension model non-human animal

Technical field

[0001] A hypertensive model mouse useful as a model animal for hypertension-related diseases in which a specific gene is deleted and the function of the gene is completely lost, and prevention of hypertension-related diseases using the hypertension model mouse The present invention relates to a method for evaluating and Z or searching for a drug suitable for treatment.

Background art

[0002] RAMP1 belongs to the receptor activity-modifying protein (RAMP) family, and has a signal sequence at the N-terminus and a single transmembrane domain at the C-terminus. RAMP1 forms a heterodimer by co-expression with calcitonin receptor-like receptor (CRLR) and functions as a receptor for calcitonin gene-related protein (CGR P), a neuropeptide that exhibits potent vasorelaxation. Is known.

[0003] JP 2005-503809 discloses a mutant mouse capable of expressing the human 匕 CGRP receptor by a complex of endogenous CRLR and human 匕 RAMP1, which is knocked-in by RAMP1 force S that has been humanized by amino acid mutation. Is disclosed. This human 力 CGRP receptor has a function equivalent to that of the CGRP receptor antagonist shown in the wild type human CGRP receptor, and it is described that it can be used for screening assays for receptor-binding substances. The animals lacking RA MP 1 are shown! /.

[0004] Patent 3639555 discloses a genetically modified mouse homozygous for the RAMP1 gene disruption. According to the description of Japanese Patent No. 3639555, a fragmented RAMP1 gene is a cell activity of the RAMP1 polypeptide encoded by the fragmented gene in cells that normally express the wild type of the RAMP1 gene. It means a RAMP gene that has been genetically modified to decrease. In addition, the non-human mammal (mouse) genetically modified as described above expresses RAMP1 polypeptide, and the polypeptide activity is 50% or less of the wild type level. Decreased gene expression (ie, RAMP polypeptide levels due to decreased RAMP mRNA levels) And the Z or fragmented RAMP gene encodes a mutant polypeptide having a function lower than that of the wild-type RAMP polypeptide. Such mice cannot ignore the effects of the polypeptide of the RAMP1 gene expressed in individuals, even if it is less active than the wild type, and can be used as a disease model mouse. It is unsuitable.

[0005] On the other hand, adrenomedullin (AM) is a potent vasorelaxing peptide that has been strongly suggested to be associated with blood pressure and circulatory dynamics. Since the AM receptor is composed of a complex of CRLR and RAMP2 or RAMP3, it is similar to the CGRP receptor composed of a complex of CRLR and RAMP1, and thus CGRP receptor Forces suggesting functional similarity to the body The relationship between RAMP1 that characterizes the CGRP receptor and blood pressure is not well understood.

[0006] Patent Document 1: Japanese Translation of Special Publication 2005-503809

Patent Document 2: Patent No. 3639555

Non-Patent Document 1: Science. 1997 Feb 14; 275 (5302): 964-967

Non-Patent Document 2: Nat Med. 1999 Apr; 5 (4): 434-8

Non-Patent Document 3: J. Exp. Med. 2002 Jan 21; 195: 151-160

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to prevent a vascular disorder disease using a pathological condition (hypertension due to vasoconstrictive disorder) model non-human animal useful for the treatment of a vascular disorder disease, and the animal.

An object of the present invention is to provide a method for efficiently screening for Z or a therapeutic drug, and a drug obtained by the method.

Another object of the present invention is to provide a method for easily screening a functional food using a non-human animal model of hypertension, a method for evaluating the functional food, and a functional food obtained by the method.

Means for solving the problem

[0008] As a result of intensive investigations to achieve the above object, the present inventors have found that the function of the RAMP1 gene is completely deficient due to partial or complete modification of the RAMP1 gene on the chromosome. The present invention can be used as a high blood pressure model animal, and such a high blood pressure model animal has been found to be extremely useful for screening for drugs for the prevention and treatment of vascular disorder diseases and for evaluation of functional foods. completed.

[0009] That is, the present invention provides a hypertensive model non-human animal characterized in that the function of the RAMP1 gene is completely deficient by modification of part or all of the RAMP1 gene on the chromosome. The non-human animal of the present invention preferably has no selection marker gene inserted on the chromosome.

[0010] The hypertensive model non-human animal of the present invention has, for example, a region in which at least a part of the RAMP1 gene exon is sandwiched between LoxP sequences and a selectable marker gene is sandwiched between LoxP sequences on a chromosome. Using ES cells obtained by allowing Cre enzyme to act on ES cells containing a region, or using ES cells containing a region where at least a part of the RAMP1 gene exon is sandwiched between LoxP sequences on the chromosome The RAMPl-floxed mouse prepared in this way and the method of mating with a mouse capable of expressing the Cre enzyme can be produced by a deviation. In the present invention, the expression of the RAMP1 gene may be conditionally suppressed. The non-human animal is, for example, a rat or a mouse.

[0011] The present invention provides a blood vessel characterized by administering a test substance to the above-mentioned hypertension model non-human animal of the present invention or bringing a tissue, organ or cell derived from the animal into contact with the test substance. Disclosed is a method for screening for drugs for the prevention and Z or treatment of disability diseases. The above method can be performed using Z or vasodilatory action as an index. The vascular disorder diseases in the present invention include, for example, hypertension, vascular endothelial disorder, arteriosclerotic disease, and blood flow disorder diseases including memory disorder, dementia, peripheral blood flow disorder disease.

[0012] The present invention also provides a preventive and Z or therapeutic agent for a vascular disorder disease obtained by the drug screening method of the present invention.

[0013] Furthermore, the present invention provides a blood flow improving drug containing, as an active ingredient, a substance that enhances the expression or function of the RAMP1 gene.

[0014] The present invention also provides a prophylactic and Z or therapeutic agent for vascular disorder diseases containing a substance that enhances the expression or function of the RAMP1 gene as an active ingredient.

[0015] In the present invention, a test substance is administered to the above-described hypertension model non-human animal of the present invention, or There is provided a screening method for functional food, which comprises contacting a tissue, organ or cell derived from the animal with a test substance. The method can be performed using blood pressure and Z or vasodilating action as indices.

[0016] The present invention provides a functional food obtained by the functional food screening method of the present invention.

[0017] Further, the present invention is a function characterized in that the test substance is administered to the non-human hypertensive model non-human animal of the present invention, or a tissue, organ or cell derived from the animal is brought into contact with the test substance. A method for evaluating sex foods is provided. The method can be performed using blood pressure and Z or vasodilating action as indices.

The invention's effect

[0018] According to the present invention, a model non-human animal that constantly shows hypertension due to vasodilatation failure is obtained by completely deleting the function of the RAMP1 gene by modifying the RAMP1 gene on the chromosome. Can do. By using the hypertensive model non-human animal of the present invention, a drug useful for the prevention and treatment of vascular disorder disease can be efficiently screened, and the efficacy of the test substance can be easily evaluated. Such non-hypertensive animals with hypertension can also be used for screening (selection) and evaluation of functional foods that exhibit a blood pressure lowering and blood vessel relaxation action to prevent or improve hypertension and blood flow disorders.

Brief Description of Drawings

FIG. 1 is a schematic sectional view of a targeting vector used in Example 1.

FIG. 2 is a photograph of agarose gel electrophoresis showing the expression pattern of each organ of 8-week-old mice of RAMP1 + / +, RAMP1 +/− and RAMP1 − / − obtained in Example 1.

[Fig. 3] (i) is a graph showing blood pressure at 10 weeks of age for each of the RAMP1 + / +, RAMP1 +/-, and RAMP1-/-mice obtained in Example 1, and (mouth) is the heart rate. It is a graph to show.

[Fig. 4] (i) is a graph showing blood pressure at 6-18 weeks of age for each of the RAMP1 + / +, RAMP1 +/-, and RAMP1-/-mice obtained in Example 1, and (mouth) is the same heart rate. It is a graph which shows a number.

[Fig. 5] (i) shows the vasoconstriction rate under the influence of three vasodilators on 12-week-old mice of RAMP1 + / +, RAMP1 +/-, and RAMP1-/-obtained in Example 1. The graph which shows is shown. BEST MODE FOR CARRYING OUT THE INVENTION [0020] RAMP1 belongs to the receptor activity-modifying protein (RAMP) family and has a signal sequence at the N-terminus and a single transmembrane domain at the C-terminus. The nucleotide sequence of RAMP1 gene and the amino acid sequence of RAMP1 are known. For example, the nucleotide sequence of cDNA of human and mouse RAMP1 includes the sequences represented by GenBank Accession No. AJ001014 and No. BAA76617.

[0021] The present inventors have produced a RAMP1 completely deficient mouse in which the function of the RAMP1 gene has been completely deleted by modifying a part or all of the RAMP1 gene on the chromosome, and the expression form is detailed. Analysis was performed. Figure 3 shows wild-type (RAMP1 + / +), RAMP1 heterozygous (RAMP1 +/-), and RAMP1 homozygous (RAMP1-/-) mice measured in 6-week-old mice. It is a graph of blood pressure and (mouth) heart rate. As shown in Fig. 3 (ii), the heart rate is comparable, while blood pressure is compared to RAMP1 + / + mice and RAMP1 +/- mice as shown in Fig. 3 (mouth). RAMP1-/-mice are already very high at 6 weeks of age. Fig. 4 is a graph of (i) blood pressure and (mouth) heart rate measured for each of the RAMPl + / +, RAMPl +/-, and RAMP1-/-mice every 3 weeks from 6 to 18 weeks of age. is there. As shown in Fig. 4 (i), the heart rate is about the same up to 6-week-old power up to 18-week-old, whereas blood pressure is about RAMP1-/-mice as shown in Fig. 4 (mouth). Was already very high at 6 weeks of age and tended to increase gradually with age. It was also revealed that this hypertension was caused by the complete suppression of the vasodilatory effect of CGRP. Thus, RAMP1-deficient mice maintained the state of hypertension due to vasoconstriction from an early age. Therefore, it can be used as a model for hypertension and vasodilatation disorder. In addition, the RAMP1 gene is known to have homologues such as human, rat, pig, and macaque monkeys, and these nucleotide sequences are disclosed in GenBank. Therefore, non-human animals other than mice can also be used as models for hypertension and vasodilatation disorders.

[0022] The hypertensive model non-human animal of the present invention (hereinafter sometimes referred to as "model animal") is completely deficient in the function of the RAMP1 gene due to partial or complete modification of the RAMP1 gene on the chromosome. It is characterized by that. “The gene function is completely deficient” means, for example, the function that wild-type RAMP1 originally has (for example, it constitutes the CGRP receptor). Function etc.) is completely lost. That is, the model animal of the present invention has no influence (action) due to RAMP1 protein in the individual or can be ignored, and does not include a dominant negative model or dominant active model of RAMP1.

[0023] In the present invention, a non-human animal in which the function of the RAMP1 gene on the chromosome is completely deleted is prepared according to a method known as a method for producing a knockout non-human animal using ES cells. can do.

[0024] The model animal of the present invention includes, for example, a configuration in which the entire region of the RAMP1 gene on the chromosome (for example, exons 1 to 3) has been removed; a partial modification of the RAMP1 gene on the chromosome, for example, Examples include a configuration in which the gene function is completely deleted by deletion, addition, or substitution in the base sequence. According to the former configuration, no transcription'translation product is generated in the individual, and it is useful as a model animal in which the influence of the RAMP1 gene is completely eliminated. In addition, the latter structure is not particularly limited as long as the gene function is completely deleted, and an appropriate site and range of the RAMP1 gene can be modified, and mRNA by partial transcription and translation can be used. Or a polypeptide fragment may be produced. Among them, the model animal of the present invention preferably has a configuration in which at least exon 2 of the RAMP1 gene on the chromosome is removed. Exon 2 has a mature protein coding start site. Therefore, in the model animals from which exon 2 has been removed, the polypeptide fragment of RAMP1 that constitutes the CGRP receptor is not formed, and any influence on the CGRP receptor function associated with the production of the polypeptide fragment is completely eliminated. Therefore, it is suitable for a wide range of uses such as screening (including evaluation) of preventive and therapeutic agents for vascular disorder diseases including blood flow disorder diseases (including evaluation) and research of the mechanism of the disease itself.

[0025] The method for producing such a hypertensive model non-human animal is not particularly limited as long as it can completely lack the function of the RAMP1 gene on the chromosome, but for example, a method using the CreZLoxP system can be mentioned. . The CreZLoxP system is a system in which the Cre enzyme recognizes the LoxP sequence and causes specific recombination. The advantage of this system is that, for example, the region where the DNA region can be deleted is limited to about 20 kb for homologous recombination In contrast to gene targeting, which is about 200 kb This is an easy method, and point mutations can be easily introduced, and conditional gene targeting is possible by the expression method of Cre enzyme.

[0026] As a method of using the CreZLoxP system in the present invention, for example, a method in which Cre enzyme is transiently expressed in ES cells and used in a step of removing a drug selection marker can be used. Specifically, In an ES cell (RAMPl-floxed-marker ES cell) containing a region where at least a part of the exon of the RAMP1 gene is sandwiched between LoxP sequences and a region where a selectable marker gene is sandwiched between LoxP sequences on the chromosome, A method using ES cells obtained by the action of Cre enzyme can be mentioned. In particular, when the former LoxP sequence sandwiches a sequence containing at least the mature protein coding start site of the RAMP1 gene on the chromosome (eg, _eX0 n2), the RAMP1 protein is completely defective due to the action of the Cre enzyme. Can be easily configured.

[0027] FIG. 1 is a schematic diagram showing an example of a method for producing ES cells obtained by allowing a Cre enzyme to act on the RAMPl-floxed-marker ES cells. For example, as shown in FIG. 1, ES cells used for the production of a hypertensive model non-human animal of the present invention are (ii) against the RAMP1 gene consisting of exonl to 3 on the chromosome of the ES cell (ii) From the first selectable marker gene (thymidine kinase: TK) sandwiched between LoxP sequences, exon2 of the RAMP1 gene sandwiched between LoxP sequences, and the second selectable marker gene (neomycin: neo) sandwiched between LoxP sequences Using a targeting vector comprising the following structure, homologous recombination is caused, and TK activity is used as an index. (Iii) On the chromosome, the RAMP1 gene sandwiched between the exon2 and LoxP sequences sandwiched between the LoxP sequences and neo Select RAMPl-floxed-marker ES cells into which the gene sequence has been integrated, and then act on the Cre enzyme to cause specific recombination, and (iv) remove exon2 and neo genes on the chromosome Can be obtained. It has been confirmed that the one LoxP sequence remaining in (iv) does not affect the expression of the target gene. According to the method of FIG. 1, since exon2 of the RAMP1 gene is removed from the chromosome, the generation of a transcription Z translation product of part or all of the RAMP1 gene is completely suppressed. The ES cells obtained in this way are completely deficient in the function of the RAMP1 gene on the chromosome and have no insertion of a selectable marker gene. Subsequently, an ES cell-derived individual having the above-described configuration can be obtained according to a known method for producing a transgenic animal. [0028] As another example of the method of using the CreZLoxP system in the present invention, RAMPl- prepared using ES cells containing a region on the chromosome where at least a part of the exon of the RAMP1 gene is sandwiched between LoxP sequences. An example is a method of mating floxed mice with mice capable of expressing the Cre enzyme. The RAMPl-floxed mouse is not particularly limited as long as it contains at least part of the RAMP1 gene exon sandwiched between LoxP sequences on the chromosome. For example, two RAMP1 gene exons are included. By homologous recombination in a form sandwiched between ΙοχΡ sequences, the gene itself is damaged, and mice can be used. The mouse capable of expressing the Cre enzyme can be prepared by a known method such as a method of introducing a Cre enzyme expression vector. In particular, as a mouse capable of expressing the Cre enzyme, the RAMP1 gene can be obtained by using a mouse capable of expressing the Cre enzyme under the control of a yarn and tissue-specific or expression-inducible promoter (Cre enzyme conditional expression mouse). The conditional gene targeting can be easily performed.

[0029] The RAMP1 gene deficiency on the chromosome may be either constitutive or conditional [tissue-specific, time-specific (transient), etc.]. In particular, if the expression level of RAMP1 gene is suppressed S-conditionally, it is preferable in that a hypertension model can be constructed under a desired condition. For example, a mouse that expresses the above Cre enzyme in a conditional manner. It can construct | assemble by well-known methods, such as the method of using.

[0030] The method for screening a prophylactic and Z or therapeutic agent for a vascular disorder disease of the present invention comprises administering a test substance to the above-mentioned hypertension model non-human animal of the present invention, or a tissue, organ derived from the animal, Or it is characterized by making a cell contact a test substance. Hereinafter, the former may be referred to as “screening method 1” and the latter as “screening method 2”.

[0031] The test substance to be used in the screening method (1, 2) of the present invention may be any known or novel compound. For example, nucleic acids, carbohydrates, lipids, proteins, peptides, small organic molecules Synthetic or natural organic compounds such as compounds can be used.

[0032] In screening method 1, the method of administering the test substance to the model animal is not particularly limited, and may be either oral or parenteral administration. For parenteral administration, for example, systemic administration via the vein, artery, muscle, abdominal cavity, airway, etc., or local administration at or near the ischemic site can be used, but is not limited thereto. Preferably parenteral administration, more Preferably, it is locally administered to or near the blood vessel. The dose of the test substance can be appropriately set according to the type of test substance, the administration method, the type, size, etc. of the model animal.

[0033] The evaluation in screening method 1 can be performed using, for example, the blood pressure value of the model animal as an index. Specifically, for example, the contraction after administering a specific amount of the test substance to the non-human animal model of hypertension of the present invention 3 times a day for 2 weeks (administration conditions such as frequency and days). When the blood pressure value for the period shows about 130% or less (preferably about 120% or less) of the average blood pressure value of the corresponding wild-type animal, the test substance is used for vascular disorder disease prevention and Z or treatment. It can be evaluated that it is useful as a medicine. In the case where the model animal is a mouse, for example, after the test substance is administered 3 times a day for 2 weeks to the hypertensive model mouse of the present invention showing an average blood pressure of about 140 mmHg as compared with a wild-type mouse having an average blood pressure of about lOOmmHg. When the blood pressure is 130 mmHg or less (preferably 120 mmHg), it can be evaluated that the test substance is useful as a drug for the prevention and Z or treatment of vascular disorder diseases.

In screening method 2, the method of bringing a test substance into contact with a tissue, organ, or cell derived from a model animal is not particularly limited, and the test substance is applied to a sample of tissue or organ collected from model animal power. A known method such as a method of adding or culturing cells collected from a model animal in the presence of a test substance can be used. As a method of introducing the test substance into the tissue or organ specimen, the same method as the parenteral administration described above can be used.

[0035] The evaluation in the screening method 2 can be performed using, for example, a vasoconstriction rate as an index. Specifically, for example, a vasoconstrictor is administered to a blood vessel sample collected from a non-human animal model of hypertension of the present invention, and then a CGRP and a test substance are cumulatively added, and the ratio is compared by ED.

When a significant difference is observed when comparing 50 or by the area under the concentration curve (AUC), the test substance is considered to be useful as a drug for the prevention and Z or treatment of vascular disorders. Can be evaluated. In the example of an animal model mouse, for example, create a blood vessel specimens Hue - Refurin (10- ⁶ M) after the vessel was contracted by添Ka卩, a CGRP and test substance each final concentration lx 10- ^u ~ 3Xl0- ⁶ measures the tension variation when the cumulative added to a M, vasoconstriction refraction obtained by conversion is a ratio with ED optionally by cumulative added mosquito卩analyte

If there is a significant difference when comparing 50 or AUC, the test It can be evaluated that the substance is useful as a drug for prevention and Z or treatment of vascular disorder diseases. The change in tension of the blood vessel specimen can be measured using a recorder (model 8K21; NEC) isometrically via, for example, an FD pickup and a carrier amplifier.

[0036] By the screening method of the present invention, a drug useful for the prevention or treatment of vascular disorder disease can be obtained. The vascular disease in the present invention includes hypertension and diseases (complications) caused by hypertension, such as vascular endothelial disorder, arteriosclerotic disease, and blood flow disorder. Examples of atherosclerotic diseases include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, or cerebrovascular disorder diseases; coronary artery diseases such as myocardial infarction and angina; aortic diseases such as aortic aneurysm and aortic dissection; It includes renal arterial diseases such as sclerosis and resulting renal failure; and peripheral arterial diseases such as obstructive arteriosclerosis. Examples of blood flow disorders include memory disorders, dementia, and peripheral blood flow disorders.

[0037] The preventive and Z or therapeutic agent (hereinafter sometimes referred to as "drug") for vascular disorder disease of the present invention contains a test substance obtained by the screening method as an active ingredient. Such drugs are administered orally or parenterally, for example, dissolved or suspended in a suitable vehicle. For parenteral administration, for example, systemic administration via the vein, artery, muscle, abdominal cavity, airway, etc., or local administration to or near blood vessels can be used, but is not limited thereto. Depending on the symptoms of the disease, oral administration is preferably performed, and can also be administered parenterally to the blood vessel or the vicinity thereof. The drug can, for example, cause a substance that enhances the expression or function of the RAMP1 gene to stably reach the contracted blood vessel site or a cell in the vicinity thereof, permeate the cell membrane, and promote the release of the drug with lysosomal Z endosome force. It is also preferable that the delivery system is designed.

[0038] The drug dosage form may be either liquid or solid. For example, an effective amount of an inhibitor is dissolved, dispersed, or emulsified in a diluent or dispersion medium such as water or physiological saline. Liquid preparations; DDS preparations containing an effective amount of an inhibitor in the form of powder, granules, solid preparations such as sachets and tablets. These drugs can be added with known pharmaceutically acceptable additives.

[0039] The drug may be an encapsulated body in which an active ingredient is encapsulated in a ribosome, a sustained-release material, or the like, or a carrier supported on a carrier. By encapsulating in ribosomes, etc. This is advantageous in that the nuclease protects the decomposition ability of the protease and the ribosome membrane binds to the cell surface and easily reaches the cell by endocytosis. Encapsulation in sustained-release materials such as collagen provides long-term sustainability of the active ingredient. In addition to the above, known pharmaceutically acceptable additives can be added to the blood flow improving drug.

[0040] The dose of the drug varies depending on the type of active ingredient, administration method, symptom, type of administration target, size, drug characteristics, etc., but is usually preferably about 0.5 to 50 mg per day for an adult. It is about 1 to 20 mg, and can be administered once a day or divided into multiple doses.

[0041] The blood flow improving agent of the present invention contains, as an active ingredient, a substance that enhances the expression or function of the RAMP1 gene by alleviating vasoconstriction. Here, “expression” means that protein is produced, and “substance that enhances expression” means gene transcription, post-transcriptional regulation, translation into protein, and post-translational modification (tyrosine phosphorylation). It may act at any stage such as oxidation) or protein folding. “Substances that enhance function” include, for example, RAMP1 protein; it acts on functional sites such as protein-protein interaction sites with CRLR in RAMP1 and receptor active sites such as CGRP receptors to increase signal transduction. Or a substance that improves; RAMP1 or a dominant active mutant of a protein that interacts with RAMP1. As used herein, “dominant active mutant” refers to a substance whose physiological activity has been improved by introducing a mutation into a protein. These mutants can indirectly enhance their function by a synergistic effect with the wild type.

[0042] Substances that enhance the expression of RAMP1 include, for example, transcription promoting factors, protein synthesis promoters, protein stabilizing enzymes, splicing factors that can promote mRNA cytoplasmic transition, mRNA stabilizing enzymes, and mRNA binding. In particular, a substance capable of acting specifically on the target molecule is preferable in order to minimize side effects on other genes and proteins. These functional nucleic acids and proteins may be produced in the administration subject after administration, and can be prepared by known methods using expression vectors, cells, etc. as necessary. The dosage form, administration method, dosage and the like of these blood flow improving drugs are the same as those for the above drugs.

[0043] The preventive and Z or therapeutic agent for a vascular disorder disease of the present invention may also generate a RAMP1 gene. A substance that enhances the current or function may be contained as an active ingredient. That is, the above blood flow improving drug can be used as a prophylactic or therapeutic drug for vascular disorder diseases.

[0044] The non-human animal model of hypertension of the present invention can also be used for screening and evaluation of functional foods. Here, “functional food” means the nutritional function (primary function) inherent in food, sensory functions such as taste fragrance (secondary function), and ecological defense and disease prevention. • Recovery · Food that has been designed and processed so that biological regulation can be clarified scientifically by focusing on the presence of biological regulation functions (third function) such as adjustment of physical rhythm. In other words, foods with tertiary functions are defined as functional foods.

[0045] Examples of test substances used in the screening method and evaluation method for functional foods of the present invention include vitamins, minerals, oligosaccharides, sugar alcohols, amino acids, peptides, proteins, animal extracts, lactic acid bacteria, yeasts, and the like. And microorganisms and extracts thereof, plants such as herbs and medicinal herbs and extracts thereof, glycosides, oils and fats, and the like. In addition, the form of the test substance is not particularly limited, and liquids such as soft drinks, carbonated drinks, liquid seasonings such as dressings; solid seasonings such as table sugar; confectionery such as cookies; and instant foods A solid substance etc. are mentioned. In addition, these functional foods may be in a pharmaceutical form such as tablets and capsules.

[0046] The functional food screening method and evaluation method of the present invention include the ability to administer a test substance to the above-described high blood pressure model non-human animal of the present invention, or a tissue, organ, or animal derived from the animal. It is characterized by contacting cells with a test substance. Preferably, a method of administering a test substance to a model animal is preferred.

[0047] The screening method for functional foods of the present invention can be carried out in accordance with the above-described prevention method for vascular disorder diseases and screening method for Z or therapeutic agent. The evaluation method for functional food (including the evaluation in the screening method) can set a lower judgment criterion than the evaluation in the above-mentioned drug screening method (1, 2). More specifically, in the method for evaluating functional food, for example, in the aforementioned screening method 1 for drugs, the average blood pressure value of wild-type animals is about 135% or less (about 135 mmHg or less in the case of mouse blood pressure). A test substance can be evaluated as useful as a functional food. According to the above method, functional food that can alleviate hypertension and reduce the risk of vascular disorder disease can be easily obtained. Can be screened and evaluated.

[0048] The functional food evaluation method of the present invention can be used as a scientific basis for evaluating, for example, functional health foods including foods for specified health use and functional foods for nutrition. In addition, since the above method uses a decrease in blood pressure or a blood vessel relaxing action as an index, for example, the indication of “improvement of eating habits for people with higher blood pressure” in the food for specified health use is displayed on the obtained functional food. It may be possible to appeal the function directly to consumers.

[0049] The functional food of the present invention can be obtained by the screening method or the evaluation method described above. For this reason, it is extremely useful as a food that can promote the alleviation and improvement of hypertension and reduce or prevent the risk of diseases related to high blood pressure (eg, vascular disorder diseases).

Example

[0050] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples. “RAMP1 + / +” means wild type, “RAMP1 +/-” means RAMP1 heterozygote, and “RAMP1-/-” means RAMP1 homozygote.

[0051] Example 1 (creation of a RAMP1 knockout mouse)

(1) Vector production

BAC clone: GSll- " ² 01 (Kurashiki Spinning Co., Ltd.) found a genome containing exon of RAMP1 in a 9 kbp fragment excised with Ba mHl. By subcloning, we confirmed exon2 of RA MP1. The LoxP sequence was inserted 200 bp upstream, and the LoxP-neomycin metagene (from pPNT) -LoxP cassette was inserted 2 kbp downstream 5. Upon of Exon2 5. The thymid ine kinase gene (from pPNT) was inserted 7 bp into the targeting vector did.

pPNT: Victor J. cell: 1991 (65) 1153—63

[0052] (2) ES cell production

20 g of the targeting vector linearized with Notl was transferred to D3 ES cells using Gene Puis er (Bio-Rad) with an electoral position (conditions 250≡V, 500≡F). From the following day, G418 was added as a selective medium at 150 μg / mL, and gancyclovir was added at 2 mol / L from the next day. One week after the electoral position, colonized ES cells were picked up and passaged for genome extraction and freezing. Genome is extracted from cells and synthesized oligonucleotides PCR using the primers Primer456 (SEQ ID NO: 1) and Primer298 (SEQ ID NO: 2) as primers and 94 ° C-30 sec + (94 ° C-30 sec 68 ° C- lmin) X 30cycle + 72 ° C-2 min By carrying out the reaction, clones that had homologous recombination on the short arm side were selected. For clones in which short-arm homologous recombination was confirmed, synthetic oligonucleotides Primer494 (SEQ ID NO: 3) and Primer495 (SEQ ID NO: 4) were used as primers, and 94 ° C-30sec + (94 ° C- 30sec 70 ° C -lmin) X 30cycle + 72 ° C-lmin By performing PCR reaction under the condition of 30sec, it is detected that Lo xP remains on the long arm side, and further, Southern blotting is used to detect short arm and long arm Homologous recombination on both sides was finally confirmed. Homologous recombination from 643 clones to 5 clones was confirmed, and 2 clones having normal karyotypes were designated as RAMPl-floxed ES cells.

SEQ ID NO: 1: 5,-TGTACCCTGT GCGGTTTGTC TGGGGGCGCG-3, (Primer456) SEQ ID NO: 2: 5,-AGGGGAGGAG TAGAAGGTGG CGCGAAGGGG-3 '(Primer298) SEQ ID NO: 3: 5,-GTCTTAACTG CACTGTGGGC-3' (Primer494)

SEQ ID NO: 4: 5,-GTGATAAAAG CAACTCCGGG-3 '(Primer495)

(3) Check Cre / LoxP system

The resulting RAMPl-floxed ES cells were transfected with 10 g of pCAGGS-Cre linearized with Notl using an erect mouth position using Gene Puller (Bio-Rad) (conditions were 250 ≡ V and 500 ≡ μ F). From the next day, G418 (150 μg / mL) was added as a selective medium. One week after electroporation, colonized ES cells were picked up and passaged for genome extraction and freezing. Using the extracted oligonucleotides Primer258 (SEQ ID NO: 5) and Primer259 (SEQ ID NO: 6) as primers, 94 ° C-30sec + (94 ° C-30sec 60 ° C-30sec 72 ° C-30sec ) Cre recombinase integration was confirmed by PCR reaction under conditions of X 40cycle + 72 ° C-2min. In addition, the clones with Cre were introduced using primers Primer494 (SEQ ID NO: 7) and Primer496 (SEQ ID NO: 8) as primers, and 94 ° C-30sec + (94 ° C-30sec 65 ° C- PCR was performed under the conditions of 30 sec 74 ° C-30 sec) X 40cycle + 72 ° C-2 min. Since the wild-type genome is more than 4kbp away, PCR does not run! / ヽ, but when Creombi has recombinasion! /, A 500bp band can be confirmed. This confirmed the introduction of Cre recombinase in both clones. Genomic recombinasion was confirmed in all clones.

SEQ ID NO: 5: 5,-GTTTCACTGG TTATGCGGCG G-3, (Primer258)

SEQ ID NO: 6: 5, TTCCAGGGCG CGAGTTGATA G-3, (Primer259)

SEQ ID NO: 7: 5,-GTCTTAACTG CACTGTGGGC-3 '(Primer494)

SEQ ID NO: 8: 5,-TTGGAACATC ACCACGCACG-3, (Primer496)

[0054] (4) Chimera mouse production, germ line transmission

Four to eight RAMP1-floxed ES cells were injected into blastocysts of unfertilized eggs collected from C57BL / 6 mice (Japan SLC) and returned to pseudopregnant ICR mice. The color of the litter obtained after 3 weeks was confirmed to be a chimeric mouse. Furthermore, it was crossed with chimeric mice and wild-type C57BL / 6 mice, and it was analyzed by hair color and dienotyping that the ESP-derived RAMP1 +/- mouse power ^, and the two clones were analyzed. Confirmed germ line transmission. In the dienotyping, the genome extracted from the tail was subjected to PCR reaction using Primer456 and Primer298 as described above (94 ° C-30 sec using primers of synthetic oligonucleotide Primer456 (SEQ ID NO: 1) and Primer298 (SEQ ID NO: 2)). + (94 ° C-30 sec, 68 ° C-lmin) X PCR at 30 cycles + 72 ° C-2 min) and Southern blotting.

[0055] (5) Homozygote and its phenotype

The obtained RAMP1 +/− mice were mated to obtain RAMP1 − / − mice, and the same lump RAMP1 + / + mice and RAMP1 +/− mice as controls. Dienotyping was performed on the genome extracted from the tail by a PCR reaction under the same conditions as in (3) above.

For 8-week-old RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − mice, the expression of RAMP1 and CRLR at the mRNA level in the brain, thymus, spleen, and lung organs was confirmed. The results are shown in Fig. 1.

Example 2 (Constant RAMP1 KO mouse production)

By mating pCAGGS- Cre transgenic mice and RAMPl-floxed mice that express Cre recombinase systemically, pCAGGS- Cre positive and RAMP1- floxed mice were obtained as offspring. By mating the mouse with a wild type mouse, a RAMP1 hetero-deficient mouse was born. The heterozygous mice obtained were mated to produce RAMP1-homozygous mice. In dienotyping, the genome extracted from the tail is made into a saddle shape, and synthetic oligonucleotides are used. 94 ° C-3 Osec + (94 ° C-30sec 65 ° C-30sec 74 ° C-30sec) X 40cycle + 72 ° using Primer494 (SEQ ID NO: 7) and Primer496 (SEQ ID NO: 8) as primers 94 ° C-30 sec + (94 ° C-30 sec 70 ° C- using the PCR reaction under the condition of C-2 min, and the synthetic oligonucleotides Primer494 (SEQ ID NO: 3) and Primer495 (SEQ ID NO: 4) as primers lmin) X 30cycle + 72 ° C-1 min 30 sec was used in combination with the PCR reaction to determine whether it was a hetero-deficient mouse or a homo-deficient mouse.

[0057] Example 3

The RAMP1 − / − mice obtained in Example 1 were intravenously injected with a 15 mg / 0.5 ml injection prepared by dissolving the test substance in physiological saline, and the mean blood pressure was measured over time according to the method described later. taking measurement. As a control, RAMP1 + / + mice and RAMP1 − / − mice at 6 weeks of age are injected with 0.5 ml of physiological saline, and blood pressure is similarly measured over time. As a result, when the blood pressure of RAMP1 − / − mice injected with the test substance is 120 mmHg or less, the test substance is useful as a prophylactic and Z or therapeutic agent for vascular disorder diseases.

[0058] Example 4

A 15 mg / 0.5 ml injection prepared by dissolving the test substance in physiological saline is intravenously injected, and the systolic blood pressure is measured over time according to the method described later. As a control, RAMP1 + / + and RAMP1-/-mice at 6 weeks of age are injected with 0.5 ml of physiological saline and blood pressure is similarly measured over time. As a result, if the blood pressure of RAMP1-/-mouse administered with physiological saline is 140 mmHg, while the blood pressure of RAMP1-/-mice injected with the test substance is 120 mmHg or less, The test substance is useful as a preventive and Z or therapeutic agent for vascular disorder diseases.

[0059] Example 5

Based on the description in JP 2005-503809, a RAMP1 mutant protein with enhanced RAMP1 function was prepared by converting the 74th amino acid sequence of RAMP1 into tributophan, and dissolved in physiological saline. Then, it is intravenously injected into the RAMP1 − / − mouse obtained in Example 1 using it as an injection solution of 15 mg / 0.5 ml, and the systolic blood pressure is measured over time according to the method described later. As a result, the blood pressure of RAMP1 − / − mice after injection showed 120 mmHg or less. Therefore, the RAMP1 mutein was used as a blood flow improving agent and as a vascular disorder disease. It is useful as a preventive and z or therapeutic agent for patients.

[0060] Example 6

The solid food for mice was crushed, and the RAMP1 mutant protein prepared in Example 6 was mixed at a concentration of 15 mg / g and molded again into a solid form as a test substance, with an average blood pressure of 140 mmHg. After taking hypertension model mice for 1 week instead of normal food, measure systolic blood pressure according to the method described below. As a result, the blood pressure of RAMP1-/-mice fed with the test substance decreases to 120 mmHg or less, so that the test substance is useful as a functional food useful for the prevention of vascular disorder diseases.

[0061] (Evaluation method)

Measurement of heart rate and systolic blood pressure

For the RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − mice prepared in Example 1, the heart rate and systolic blood pressure were measured by the following methods.

Mice were held under anesthesia, and heart rate and systolic blood pressure were measured by tan- cuff removal using MK2000 (Muromachi Kikai Co., Ltd.). In order to acclimatize to the environment at the time of measurement, each individual was measured 30 times or more, then 10 measurements were performed as the main measurement, and the average value was taken as the value of that individual. The results are shown in Fig. 3. As shown in Figure 3, the mean blood pressure of wild-type mice and heterozygotes is about lOOmmHg, while the mean blood pressure of RAMP1 KO mice obtained above is about 140 mmHg, indicating clear hypertension. It was. In the same manner, changes in heart rate and blood pressure were measured for 6-18 week old mice. The results are shown in Fig. 4. As shown in Figure 4, heart rate did not change in any mouse. The blood pressure of the RAMP1 KO mice tended to gradually increase with the age of 6 weeks, which was already high.

[0062] Vasoconstriction experiment

For each of the RAMP1 + / +, RAMP1 +/-, and RAMP1-/-mice prepared in Example 1, the change in tension in the blood vessel specimen prepared by the following method was measured, and converted into a vasoconstriction rate. Shrinkage properties were evaluated.

Under pentobarbital sodium anesthesia, the mouse carotid artery was cut and blood was removed. Next, the thoracic aorta was removed and 37 ° C Klebs-Henseleit solution (118.4 mM NaCl, 4.7 mM KC1, 2.5 mM) CaCl, 1.2 mM KH PO, 1.2 mM MgSO-7H 0, 25 mM NaHCO, 11.1 mM Darco

2 2 4 4 2 3

After removing the connective tissue of the blood vessel in 3), the blood vessel was cut to a width of 3 mm to prepare a ring specimen. Insert a tungsten wire with a support rod made of acrylic resin into the blood vessel specimen, 95% 0

2 I 5%

The mixture was fixed in 10 mL of Klebs-Henseleit solution kept at 37 ° C by ventilating CO gas mixture. Tan

2

Another dust wire was inserted into the specimen and connected to the FD pickup. The change in tension of the specimen is recorded on the recorder (model 8K21; NEC) isometrically via an FD pickup and carrier amplifier (1.0 g = 5 cm), and a simple manipulator (model t-7; NEC). ) To apply 0.7 g basic tension. After 30 minutes, Hue - Refurin and vasoconstriction that by the (final concentration of 3 X 10- ⁷ M), confirmed the blood vessel relaxation reaction by acetylcholine (final concentration of 10- ⁷ M). After confirmation of the blood vessel specimens finished, again equilibrated for 60 min at Klebs-Henseleit solution, the vessel is contracted by off two Refuri emissions (10- ⁶ M)添Ka卩, a CGRP, j8 CGRP or address drink Jiyu phosphorus (AM ) was recorded relaxation response of the cumulative added vessels to a final concentration of lx 10- ^u -3xl0- ⁶ M. After equilibration spoon again 30 minutes, Hue - after contracted with Refurin (10- ⁶ M), Papabe phosphorus (10 - ⁴ M) added to relax blood vessels to determine the maximum relaxation ability. These results are shown in FIG.

[0063] As shown in FIG. 5, the RAMP1 + / + mouse thoracic aorta contracted with furephrine was remarkably relaxed by a CGRP supplementation. On the other hand, the thoracic aorta of RAMP1-/-mice showed no relaxation reaction by α-CGRP. This indicates that RAMP1 constitutes a CGRP receptor. On the other hand, i8 CGRP did not show significant vasorelaxing activity in wild-type mouse thoracic aorta. In addition, the activity of AM, which is known to have a strong vasodilatory effect, was clearly attenuated in the thoracic aorta of RAMP1 − / − mice contracted by fe-rephrin compared to RAMP1 + / + mice. This indicates that the vasodilatory effect of adrenomedullin is involved, at least in part, in the receptor composed of RAMP1 as in CGRP.

[0064] Immunohistochemical staining

Human pathological tissue was fixed with 10% neutral buffered formalin, embedded in norafine, and cut into 4 / zm. After deparaffinization, the sections were incubated with anti-RAMP1 antibody (SantaCruz) for 1 hour at room temperature. Subsequent to the reaction, Histofine SAB-PO kit (manufactured by Chirei Co., Ltd.) and diaminobenzidine as a dye were visualized and counterstained with hematoxylin. as a result In addition, expression of RAMP1 was observed in colon cancer cells as well as brain and colon mucosa cells. This indicates that CGRP signals are involved in the growth and metastasis of cancer cells as well as vasodilatory effects.

Claims

The scope of the claims

[1] A hypertensive model non-human animal, wherein the function of the RAMP1 gene is completely deficient due to partial or complete modification of the RAMP1 gene on the chromosome.

[2] The hypertension model non-human animal according to claim 1, wherein a selection marker gene is not inserted on the chromosome.

[3] Cre enzyme is allowed to act on ES cells containing a region where at least part of the exon of the RAMP1 gene is sandwiched between LoxP sequences and a region where a selectable marker gene is sandwiched between LoxP sequences on the chromosome. A method using ES cells obtained by the above, or a RAMPl-floxed mouse prepared using ES cells containing a region in which at least part of the exon of the RAMP1 gene is sandwiched between LoxP sequences on the chromosome, and Cre enzyme The hypertensive model non-human animal according to claim 1 or 2, which is prepared by crossing the method with an expressible mouse!

[4] Claims 1 to 4 in which the function of the RAMP1 gene is conditionally defective

4. The hypertensive model non-human animal according to any one of items 3.

[5] The hypertensive model non-human animal according to any one of claims 1 to 4, wherein the non-human animal is a rat or a mouse.

[6] The test substance is administered to the hypertensive model non-human animal according to any one of claims 1 to 5, or a tissue, organ or cell derived from the animal is used as the test substance. A method for screening a drug for prevention and Z or treatment of a vascular disorder disease, which comprises contacting with a blood vessel.

7. The drug screening method according to claim 6, wherein blood pressure and Z or vasodilatory action are used as indices.

[8] The vascular disorder is at least selected from the group consisting of hypertension, vascular endothelial disorder, arteriosclerotic disorder, and blood flow disorder including memory disorder, dementia and peripheral blood flow disorder. The method for screening a drug according to any one of claims 6 and 7, which is a disease.

[9] A preventive and Z or therapeutic agent for a vascular disorder disease obtained by the method for screening a drug according to any one of claims 6 to 8.

[10] Blood flow modification containing a substance that enhances the expression or function of the RAMP1 gene as an active ingredient Good medicine.

[11] A prophylactic and Z or therapeutic agent for a vascular disorder disease comprising a substance that enhances the expression or function of the RAMP1 gene as an active ingredient.

[12] The vascular disorder is at least selected from the group consisting of hypertension, vascular endothelial disorder, arteriosclerotic disorder, and blood flow disorder including memory disorder, dementia and peripheral blood flow disorder. 12. The prophylactic and Z or therapeutic agent for vascular disorder according to claim 11, which is a disease.

[13] The test substance is administered to the hypertensive model non-human animal according to any one of claims 1 to 5, or a tissue, organ, or cell derived from the animal is used as the test substance. A method for screening functional foods, characterized in that the functional foods are brought into contact with each other.

[14] The method for screening a functional food according to claim 13, wherein blood pressure and Z or vasodilatory action are used as indices.

[15] A functional food obtained by the method for screening a functional food according to claim 13 or 14.

[16] The test substance is administered to the hypertensive model non-human animal according to any one of claims 1 to 5, or a tissue, organ or cell derived from the animal is used as the test substance. A method for evaluating functional foods, characterized in that it is brought into contact with a food product.

[17] The method for evaluating a functional food according to claim 16, wherein blood pressure and Z or vasodilatory action are used as indices.