JP2009201355A - Nonhuman model animal of hypertension - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nonhuman model animal of hypertension which is useful in treating a vascular disease, to provide a method of efficiently screening a medicine for preventing and/or treating a vascular disease with the use of the above animal, and to provide the medicine obtained by the above method. <P>SOLUTION: Provided is the 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. The 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. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  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 deleted, and suitable for prevention and treatment of hypertension-related diseases using the hypertension model mouse The present invention relates to a method for evaluating and / or searching for drugs.

  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 is known to function as a receptor for calcitonin gene-related protein (CGRP), a neuropeptide that forms a heterodimer by co-expression with calcitonin receptor-like receptor (CRLR) and exhibits a potent vasorelaxant action. It has been.

  JP 2005-503809 discloses a mutant mouse in which humanized RAMP1 is knocked in by amino acid mutation and humanized CGRP receptor can be expressed by a complex of endogenous CRLR and humanized RAMP1. Although this humanized CGRP receptor has a function equivalent to that of the CGRP receptor antagonist shown in the wild-type human CGRP receptor, it has been described that it can be used for screening assays for receptor-binding substances. Animals lacking are not shown.

  Japanese Patent No. 3639555 discloses genetically altered mice that are homozygous for the RAMP1 gene disruption. According to the description of Japanese Patent No. 3639555, the disrupted RAMP1 gene is a decrease in the cellular activity of the RAMP1 polypeptide encoded by the disrupted gene in cells that normally express the wild type of the RAMP1 gene. This means that the RAMP gene has been genetically modified. In addition, the non-human mammal (mouse) genetically altered as described above expresses RAMP1 polypeptide, and the polypeptide activity is 50% or less of the wild-type level, because the RAMP gene As a result of decreased expression (ie, decreased RAMP mRNA levels result in decreased RAMP polypeptide levels) and / or the fragmented RAMP gene encodes a mutant polypeptide that has a lower function than the wild-type RAMP polypeptide. It is described that it is because. Such a mouse cannot be ignored even if it is less active than the wild type due to the polypeptide of the RAMP1 gene expressed in the individual, and is not suitable for use as a disease model mouse. is there.

  On the other hand, adrenomedullin (AM) is a strong vasorelaxing peptide that is strongly suggested to be associated with blood pressure and circulatory dynamics. The AM receptor is similar to the CGRP receptor composed of a complex of CRLR and RAMP1 in that it is composed of a complex of CRLR and RAMP2 or RAMP3. Functional similarity is suggested, but the association between RAMP1 that characterizes the CGRP receptor and blood pressure is not well understood.

JP 2005-503809 A Japanese Patent No. 3639555 Science. 1997 Feb 14; 275 (5302): 964-967 Nat Med. 1999 Apr; 5 (4): 434-8 J. Exp. Med. 2002 Jan 21; 195: 151-160

An object of the present invention is to provide a disease state (hypertension caused by vasoconstrictive disorder) model non-human animal useful for treatment of vascular disorder disease, and a drug for preventing and / or treating vascular disorder disease using the animal. An object of the present invention is to provide an efficient screening method and a drug obtained by the method.
Another object of the present invention is to provide a method for simply screening and evaluating a functional food using a hypertensive model non-human animal, and a functional food obtained by the method.

  As a result of diligent studies to achieve the above object, the present inventors have used, as a hypertensive model animal, an animal in which the function of the RAMP1 gene is completely deficient due to partial or total modification of the RAMP1 gene on the chromosome. As a result, the present inventors have found that such a hypertension model animal is extremely useful for screening drugs for the prevention and treatment of vascular disorder diseases and for evaluating functional foods, thereby completing the present invention.

  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.

  The hypertensive model non-human animal of the present invention includes, for example, an ES cell comprising, on a chromosome, a region in which at least a part of an exon of the RAMP1 gene is sandwiched between LoxP sequences and a region in which a selection marker gene is sandwiched between LoxP sequences. In addition, a method using ES cells obtained by the action of Cre enzyme, or RAMP1-floxed produced using ES cells containing on the chromosome at least part of the exons of RAMP1 gene sandwiched by LoxP sequences It can be produced by any method by mating between a mouse and a mouse capable of expressing the Cre enzyme. 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.

  The present invention relates to a vascular disorder disease characterized by administering a test substance to the above-mentioned hypertension model non-human animal of the present invention or contacting a tissue, organ or cell derived from the animal with the test substance. A method for screening a prophylactic and / or therapeutic drug is provided. The method can be performed using and / 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.

  The present invention also provides a prophylactic and / or therapeutic agent for vascular disorder disease obtained from the drug screening method of the present invention.

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

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

  The present invention provides a screening for a functional food comprising 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. Provide a method. The method can be performed using blood pressure and / or vasodilatory action as an index.

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

  Furthermore, the present invention provides a functional food 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. Provides an evaluation method. The method can be performed using blood pressure and / or vasodilatory action as an index.

  According to the present invention, a model non-human animal that constantly shows hypertension due to vasodilatation failure can be obtained by completely deleting the function of the RAMP1 gene by modifying the RAMP1 gene on the chromosome. By using the hypertensive model non-human animal of the present invention, it is possible to efficiently screen for drugs useful for the prevention and treatment of vascular disorder diseases, and to easily evaluate the efficacy of the test substance. Such a hypertension model non-human animal can also be used for screening (selection) and evaluation of functional foods that exhibit a reduction in blood pressure and blood vessel relaxation, and prevent or ameliorate hypertension and blood flow disorders.

  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 base sequence of the RAMP1 gene and the amino acid sequence of RAMP1 are known. Examples of the base sequences of human and mouse RAMP1 cDNA include sequences represented by GenBank Accession No. AJ001014 and No. BAA76617.

  The present inventors made a RAMP1 completely deficient mouse in which the function of the RAMP1 gene was completely deleted by modifying a part or all of the RAMP1 gene on the chromosome, and performed a detailed analysis of its phenotype . FIG. 3 shows (i) blood pressure and (B) blood pressure measured in 6-week-old mice for wild-type (RAMP1 + / +) mice, RAMP1 heterozygous (RAMP1 +/-) mice, and RAMP1 homozygous (RAMP1-/-) mice. B) A graph of heart rate. As shown in Fig. 3 (a), the heart rate is comparable, whereas blood pressure is compared to RAMP1 + / + mice and RAMP1 +/- mice, as shown in Fig. 3 (b). RAMP1-/-mice are already very high at 6 weeks of age. FIG. 4 is a graph of (b) blood pressure and (b) heart rate measured for each of the RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − mice every 3 weeks from 6 to 18 weeks of age. is there. As shown in FIG. 4 (a), the heart rate is about the same from 6 to 18 weeks of age, while the blood pressure is RAMP1 − / − mice as shown in FIG. 4 (b). Already showed a high value at an extremely early stage of 6 weeks of age and tended to gradually increase with age. It was also clarified that this hypertension was caused by the complete suppression of vasodilation by CGRP. Thus, RAMP1-deficient mice can be used as a model for hypertension and vasodilatation disorder because the state of hypertension due to vasoconstriction is maintained from an early stage after birth. In addition, the RAMP1 gene is known to have homologues such as humans, rats, pigs, rhesus monkeys, etc., 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.

  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. “The gene function is completely deficient” means that, for example, the function inherent to wild-type RAMP1 (for example, a function constituting a CGRP receptor) 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 RAMP1 dominant negative model or a dominant active model.

  In the present invention, a non-human animal in which the function of the RAMP1 gene on the chromosome is completely deficient can be produced according to a known method for producing a knockout non-human animal using ES cells.

  Examples of the structure of the model animal of the present invention include, for example, a structure in which the entire RAMP1 gene region (for example, exons 1 to 3) on the chromosome has been removed; a partial modification of the RAMP1 gene on the chromosome, for example, deletion in the nucleotide sequence , A configuration in which the function of the gene is completely lost by addition or substitution. 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 removed. 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 or polypeptide by partial transcription / translation. Fragments may be generated. In particular, 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 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 should be eliminated. Therefore, it is suitable for a wide range of uses such as screening (including evaluation) of preventive drugs and therapeutic drugs for vascular disorder diseases including blood flow disorder diseases (including evaluation) and research of the mechanism of the disease itself.

  A method for producing such a hypertension model non-human animal is not particularly limited as long as the function of the RAMP1 gene on the chromosome can be completely deleted. For example, a method using the Cre / LoxP system can be mentioned. The Cre / LoxP system is a system in which the Cre enzyme recognizes the LoxP sequence and causes specific recombination. The advantage of this system is, for example, that the region where the DNA region can be deleted is limited to about 20 kb for homologous recombination. In contrast, it is a large technique of about 200 kb, and in gene targeting, it is a technique that allows easy removal of the selection marker gene from the chromosome, can easily introduce point mutations, and conditional gene targeting can be achieved by the Cre enzyme expression method. This is possible.

  As a method of using the Cre / LoxP system in the present invention, for example, a method of transiently expressing Cre enzyme in ES cells and using it in a step of removing a drug selection marker can be used. Above, the Cre enzyme is added to an ES cell (RAMP1-floxed-marker ES cell) containing a region in which at least a part of the exon of the RAMP1 gene is sandwiched between LoxP sequences and a region in which the selectable marker gene is sandwiched between LoxP sequences. A method using ES cells obtained by acting is 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 exon2 etc.), the CreP enzyme facilitates the complete deletion of the RAMP1 protein. It is possible to configure.

  FIG. 1 is a schematic diagram showing an example of a method for producing ES cells obtained by allowing Cre enzyme to act on the RAMP1-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 (i) a RAMP1 gene consisting of exon1 to 3 on the ES cell chromosome, and (ii) a LoxP sequence. A composition comprising a first selectable marker gene (thymidine kinase: TK) sandwiched between exon 2 of RAMP1 gene sandwiched between LoxP sequences, and a second selectable marker gene (neomycin: neo) sandwiched between LoxP sequences. Using the target targeting vector, homologous recombination occurs, and using TK activity as an index, (iii) the sequence of exon2 of the RAMP1 gene sandwiched between the LoxP sequences and the sequence of the neo gene sandwiched between the LoxP sequences are incorporated on the chromosome In addition, RAMP1-floxed-marker ES cells are selected, and a Cre enzyme is allowed to act on the cells to cause specific recombination, and (iv) exon2 and neo genes on the chromosome are removed. 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, generation of a transcription / translation product of a part or all of the RAMP1 gene is completely suppressed. The ES cells thus obtained are completely deficient in the function of the RAMP1 gene on the chromosome and have no insertion of a selectable marker gene. Subsequently, according to a known method for producing a transgenic animal, an ES cell-derived individual having the above-described configuration can be obtained.

  As another example of the method of using the Cre / LoxP system in the present invention, a RAMP1-floxed mouse produced using an ES cell containing a region where at least a part of an exon of the RAMP1 gene is sandwiched between LoxP sequences on a chromosome And a method of mating with a mouse capable of expressing the Cre enzyme. The RAMP1-floxed mouse is not particularly limited as long as at least a part of the exon of the RAMP1 gene is sandwiched between LoxP sequences on the chromosome. For example, the exon of the RAMP1 gene is composed of two loxP sequences. Mice that are homologously recombined in a sandwiched manner and in which the gene itself is not impaired 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, a mouse that can express the Cre enzyme under the control of a tissue-specific or expression-inducible promoter (Cre enzyme-conditional expression mouse) or the like can be used to condition the RAMP1 gene. Gene targeting can be easily performed.

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

  The method for screening a prophylactic and / or therapeutic agent for a vascular disorder disease of the present invention comprises administering a test substance to the above-mentioned hypertensive model non-human animal of the present invention or subjecting a tissue, organ or cell derived from said animal to the subject. It is characterized by contacting with a test substance. Hereinafter, the former may be referred to as “screening method 1” and the latter as “screening method 2”.

  The test substance used in the screening method (1, 2) of the present invention may be any known or novel compound, such as a nucleic acid, a carbohydrate, a lipid, a protein, a peptide, or a low molecular weight organic compound. Synthetic or naturally derived organic compounds can be used.

  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, it is administered parenterally, more preferably administered locally 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.

  The evaluation in the screening method 1 can be performed using, for example, the blood pressure value of the model animal as an index. Specifically, for example, contraction after administering a specific amount of a test substance three times a day for two weeks (administration conditions such as the number of times, the number of days, etc.) to the hypertensive model non-human animal of the present invention. When the stage blood pressure value is 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 a drug for preventing and / or treating vascular disorder diseases It can be evaluated as useful. In the case where the model animal is a mouse, for example, the blood pressure after administration of the test substance three 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 to a wild-type mouse having an average blood pressure of about 100 mmHg. Is 130 mmHg or less (preferably 120 mmHg), it can be evaluated that the test substance is useful as a drug for preventing and / or treating vascular disorder diseases.

  In the screening method 2, the method of bringing the test substance into contact with the tissue, organ or cell derived from the model animal is not particularly limited, and a method or model in which the test substance is introduced into a tissue or organ sample collected from the model animal A known method such as a method of culturing cells collected from animals 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.

The evaluation in the screening method 2 can be performed using, for example, the vasoconstriction rate as an index. Specifically, for example, when a vasoconstrictor is administered to a blood vessel sample collected from a hypertension model non-human animal of the present invention, αCGRP and a test substance are cumulatively added and compared by ED ₅₀ , or a concentration curve When a significant difference is observed when compared by the lower area (AUC), it can be evaluated that the test substance is useful as a drug for preventing and / or treating vascular disorder diseases. In the case where the model animal is a mouse, for example, a blood vessel specimen is prepared, and after the blood vessel is contracted by adding phenylephrine (10 ⁻⁶ M), αCGRP and the test substance are each set to a final concentration of 1 × 10 ^{−11 to} 3 × 10 ⁻⁶ M. Measure the change in tension when cumulatively added so that the vasoconstriction rate obtained by conversion is significantly different when compared with ED ₅₀ depending on the cumulative addition of the test substance or when compared with AUC. Can be evaluated as being useful as a drug for preventing and / or treating 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.

  By the screening method of the present invention, a drug useful for the prevention and treatment of vascular disorder diseases can be obtained. The vascular diseases in the present invention include hypertension and diseases (complications) caused by high blood pressure, such as vascular endothelial disorder, arteriosclerotic disease, and blood flow disorder. Arteriosclerotic diseases include, for example, 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; And renal arterial diseases such as renal failure; and peripheral arterial diseases such as obstructive arteriosclerosis. Examples of the blood flow disorder include memory disorders, dementia, and peripheral blood flow disorders.

  The preventive and / or therapeutic agent for vascular disorder disease of the present invention (hereinafter sometimes referred to as “drug”) contains a test substance obtained by the screening method as an active ingredient. Such a drug is dissolved or suspended in an appropriate vehicle and administered orally or parenterally. For parenteral administration, for example, systemic administration via routes such as veins, arteries, muscles, abdominal cavity, airways, etc., or local administration in or near blood vessels can be used, but not limited thereto. Depending on the symptom of the disease, oral administration is preferably performed, and can also be administered parenterally to or near blood vessels. The drug is, for example, a drug delivery that allows a substance that enhances the expression or function of the RAMP1 gene to stably reach a contracted blood vessel site or a cell in the vicinity thereof, permeates the cell membrane, and promotes the release of the drug from the lysosome / endosome. It is also preferable that the system is designed.

  The drug dosage form may be either a liquid or a solid, for example, a liquid obtained by dissolving, dispersing, and emulsifying an effective amount of an inhibitor in a diluent or dispersion medium such as water or physiological saline; Examples thereof include DDS preparations containing an effective amount of an inhibitor in the form of powder, granules, solid preparations such as sachets and tablets. To these drugs, known pharmaceutically acceptable additives can be added.

  The drug may also be an encapsulated body in which the active ingredient is encapsulated in liposomes, sustained-release materials, or the like, or a carrier supported on a carrier. Encapsulating in liposomes and the like is advantageous in that the active ingredient is protected from degradation by nucleases and proteases, and the liposome membrane binds to the cell surface and easily reaches inside the cell by endocytosis. By encapsulating in a sustained release material such as collagen, long-term persistence of the active ingredient can be obtained. In addition to the above, pharmaceutically acceptable known additives can be added to the blood flow improving agent.

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

  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 relaxing vasoconstriction. Here, “expression” means that a protein is produced, and “substance that enhances expression” means gene transcription, post-transcriptional regulation, translation into protein, post-translational modification (tyrosine phosphorylation) ), Which may act at any stage such as protein folding. “Substances that enhance functions” include, for example, RAMP1 protein; acting on functional sites such as a protein-protein interaction site with CRLR in RAMP1 and a receptor active site such as CGRP receptor to increase signal transmission Alternatively, it may be a substance that improves; RAMP1 or a dominant active mutant of a protein that interacts with RAMP1. Here, the “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.

  Substances that enhance the expression of RAMP1 include, for example, transcription promoting factors, protein synthesis promoters, protein stabilizing enzymes, factors that can promote splicing and cytoplasmic migration of mRNA, mRNA stabilizing enzymes, and binding to mRNA for activation. 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 in a form produced within the administration subject after administration, and can be prepared by known methods using expression vectors, cells, etc., if necessary. The dosage form, administration method, dosage and the like of these blood flow improving drugs are the same as those of the above drugs.

  The prophylactic and / or therapeutic agent for vascular disorder disease of the present invention may also contain a substance that enhances the expression or function of RAMP1 gene as an active ingredient. That is, the blood flow improving agent can be used as a prophylactic or therapeutic agent for vascular disorder diseases.

  The hypertensive model non-human animal of the present invention can also be used for screening and evaluation of functional foods. Here, “functional food” refers to nutritional functions (primary functions) inherent in foods, sensory functions (secondary functions) such as taste and aroma, as well as ecological defense and disease prevention / Focusing on the fact that there are biological regulation functions (tertiary functions) such as recovery and adjustment of physical rhythm, this food is designed and processed so that biological regulation can be scientifically elucidated and functions can be demonstrated. That is, a food having a tertiary function is defined as a functional food.

  Examples of the test substance used in the screening method and evaluation method of the functional food of the present invention include, for example, vitamins, minerals, oligosaccharides, sugar alcohols, amino acids, peptides, proteins, animal extracts, microorganisms such as lactic acid bacteria and yeast, and the like Examples include substances containing constituents such as extracts, herbs and herbs, and extracts thereof, glycosides, 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. These functional foods may be in a pharmaceutical form such as tablets and capsules.

  The functional food screening method and the evaluation method of the present invention include administering a test substance to the above-described hypertensive 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. It is characterized by that. A method of administering a test substance to a model animal is preferable.

  The screening method for functional foods of the present invention can be performed according to the screening method for preventive and / or therapeutic agents for vascular disorder diseases exemplified above. 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-described drug screening methods (1, 2). More specifically, in the method for evaluating a functional food, for example, in the drug screening method 1, the average blood pressure value of a wild-type animal is about 135% or less (about 135 mmHg or less in the case of mouse blood pressure). It can be evaluated that the test substance is useful as a functional food. According to the above method, functional foods that can alleviate hypertension and reduce the risk of vascular disorder diseases can be easily screened (selected) and evaluated.

  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 specific health use and functional foods for nutrition. In addition, since the above method uses a decrease in blood pressure or blood vessel relaxation as an index, for example, an indication such as “improvement of eating habits for people with higher blood pressure” in the food for specified health use is obtained. It may be possible to appeal the function directly to consumers.

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

  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.

Example 1 (creation of a RAMP1 knockout mouse)
(1) Vector production
BAC clone: GS11-99201 (Kurashiki Spinning Co., Ltd.) found a genome containing exon of RAMP1 in a 9kbp fragment excised by BamH1. RAMP1 exon2 was confirmed by subcloning. A LoxP sequence was inserted 200 bp upstream of exon2, and a LoxP-neomycin resistance gene (from pPNT) -LoxP cassette was inserted 2 bp downstream. The thymidine kinase gene (from pPNT) was inserted at 5 kbp upstream of Exon2 to obtain a targeting vector.
pPNT: Victor LJ cell: 1991 (65) 1153-63

(2) ES cell production
20 μg of targeting vector linearized with NotI was transfected into D3 ES cells by electroporation using Gene Pulser (Bio-Rad) (conditions 250≡V, 500≡μF). From the next day, G418 was added as a selective medium at 150 μg / mL, and gancyclovir was added at 2 μmol / L from the next day. One week after electroporation, colonized ES cells were picked up and passaged for genome extraction and freezing. Genome is extracted from the cells, and the synthetic oligonucleotides Primer456 (SEQ ID NO: 1) and Primer298 (SEQ ID NO: 2) are used as primers, 94 ° C-30sec + (94 ° C-30sec 68 ° C-1min) x 30cycle + 72 ° C- By performing a PCR reaction under the condition of 2 min, clones having homologous recombination on the short arm side were selected. For clones in which short arm homologous recombination was confirmed, 94 ° C-30 sec + (94 ° C-30 sec 70 ° C-1 min) using synthetic oligonucleotides Primer494 (SEQ ID NO: 3) and Primer495 (SEQ ID NO: 4) as primers ) × 30cycle + 72 ° C-1min 30sec to detect the presence of LoxP on the long arm side, and finally perform homologous recombination on both the short arm and long arm by Southern blotting Confirmed. Homologous recombination was confirmed from 643 clones to 5 clones, and 2 clones having normal karyotype were designated as RAMP1-floxed ES cells.
Sequence number 1: 5'-TGTACCCTGT GCGGTTTGTC TGGGGGCGCG-3 '(Primer456)
Sequence number 2: 5'-AGGGGAGGAG TAGAAGGTGG CGCGAAGGGG-3 '(Primer298)
Sequence number 3: 5'-GTCTTAACTG CACTGTGGGC-3 '(Primer494)
Sequence number 4: 5'-GTGATAAAAG CAACTCCGGG-3 '(Primer495)

(3) Confirmation of Cre / LoxP system The obtained RAMP1-floxed ES cells were transfected with 10 μg of pCAGGS-Cre linearized with NotI by electroporation using Gene Pulser (Bio-Rad). ≡V and 500≡μF). 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. For the extracted genome, synthetic oligonucleotides Primer258 (SEQ ID NO: 5) and Primer259 (SEQ ID NO: 6) were used as primers, and 94 ° C-30sec + (94 ° C-30sec 60 ° C-30sec 72 ° C-30sec) x 40cycle + The integration of Cre recombinase was confirmed by performing a PCR reaction at 72 ° C. for 2 min. Furthermore, for the clone into which Cre was introduced, synthetic oligonucleotides Primer494 (SEQ ID NO: 7) and Primer496 (SEQ ID NO: 8) were used as primers, and 94 ° C-30 sec + (94 ° C-30 sec 65 ° C-30 sec 74 ° C- PCR reaction was performed under the conditions of 30 sec) × 40 cycle + 72 ° C.-2 min. Since the wild-type genome is more than 4 kbp apart, PCR does not run, but if recombinasion is occurring due to Cre, a 500 bp band can be confirmed. As a result, genomic recombinasion was confirmed in all clones in which introduction of Cre recombinase was confirmed in both of the two clones.
Sequence number 5: 5'-GTTTCACTGG TTATGCGGCG G-3 '(Primer258)
Sequence number 6: 5'-TTCCAGGGCG CGAGTTGATA G-3 '(Primer259)
Sequence number 7: 5'-GTCTTAACTG CACTGTGGGC-3 '(Primer494)
Sequence number 8: 5'-TTGGAACATC ACCACGCACG-3 '(Primer496)

(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. It was confirmed that it was a chimeric mouse with the color of the litter obtained after 3 weeks. Furthermore, we crossed chimera mice with wild-type C57BL / 6 mice, analyzed the presence of ESP-derived RAMP1 +/- mice in their offspring by coat color and genotyping, and gave germ line transmission to two clones. confirmed. Genotyping was performed by using a PCR reaction using Primer 456 and Primer 298 as described above (94 ° C-30 sec + using synthetic oligonucleotide Primer 456 (SEQ ID NO: 1) and Primer 298 (SEQ ID NO: 2)) as primers. (PCR reaction under the conditions of 94 ° C.-30 sec 68 ° C.-1 min) × 30 cycles + 72 ° C.-2 min) and Southern blotting.

(5) Homozygote and its phenotype The obtained RAMP1 +/− mice were mated with each other to obtain RAMP1 − / − mice, and the same lump RAMP1 + / + mice and RAMP1 +/− mice as controls. Genotyping 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 each organ of brain, thymus, spleen, and lung was confirmed. The result is shown in FIG.

Example 2 (Constant RAMP1 KO Mouse Production)
By mating pCAGGS-Cre transgenic mice and RAMP1-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 yield RAMP1 homo-deficient mice. Genotyping uses the genome extracted from the tail as a template and synthetic oligonucleotides Primer494 (SEQ ID NO: 7) and Primer496 (SEQ ID NO: 8) as primers, 94 ° C-30sec + (94 ° C-30sec 65 ° C-30sec PCR reaction under conditions of 74 ° C.-30 sec) × 40 cycle + 72 ° C.-2 min, and 94 ° C.-30 sec + (94 ° C.) using synthetic oligonucleotides Primer494 (SEQ ID NO: 3) and Primer495 (SEQ ID NO: 4) as primers -30 sec 70 ° C.-1 min) × 30 cycle + 72 ° C.-1 min 30 seconds was determined as a hetero-deficient mouse or a homo-deficient mouse in combination with a PCR reaction.

Example 3
The RAMP1 − / − mouse obtained in Example 1 is intravenously injected with a 15 mg / 0.5 ml injection prepared by dissolving the test substance in physiological saline, and the mean blood pressure is measured over time according to the method described later. To do. 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 the RAMP1 − / − mouse injected with the test substance is 120 mmHg or less, the test substance is useful as a prophylactic and / or therapeutic drug for vascular disorder diseases.

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 + / + 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 administered with physiological saline was 140 mmHg, while the blood pressure of RAMP1 − / − mice injected with the test substance was 120 mmHg or less, The test substance is useful as a preventive and / or therapeutic agent for vascular disorder diseases.

Example 5
Based on the description in JP-T-2005-503809, a RAMP1 mutant protein with enhanced RAMP1 function was prepared by converting lysine of amino acid sequence 74 of RAMP1 into tryptophan, and dissolved in physiological saline to give 15 mg A RAMP1 − / − mouse obtained in Example 1 is intravenously injected as an /0.5 ml injection solution, and systolic blood pressure is measured over time according to the method described later. As a result, since the blood pressure of RAMP1 − / − mice after injection shows 120 mmHg or less, the RAMP1 mutant protein is useful as a blood flow improving agent and as a prophylactic and / or therapeutic agent for vascular disorder diseases. .

Example 6
A hypertensive model with a mean blood pressure of 140 mmHg using a solid food for mice crushed, mixed with the RAMP1 mutant protein prepared in Example 6 at a concentration of 15 mg / g and molded into a solid form again. The mice are ingested for 1 week instead of normal food, and then the systolic blood pressure is measured according to the method described later. As a result, the blood pressure of RAMP1 − / − mice ingested with the test substance decreases to 120 mmHg or less, and therefore the test substance is useful as a functional food useful for the prevention of vascular disorder diseases.

(Evaluation methods)
Measurement of Heart Rate and Systolic Blood Pressure For each of the RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − mice prepared in Example 1, the heart rate and systolic blood pressure were measured by the following method.
The mouse was held under anesthesia, and the heart rate and systolic blood pressure were measured by the tail-cuff method using MK2000 (Muromachi Kikai Co., Ltd.). In addition, in order to acclimatize to the environment at the time of measurement, after measuring 30 times or more for each individual, 10 measurements were performed as the main measurement, and the average value was taken as the value of the individual. The result is shown in FIG. As shown in FIG. 3, the mean blood pressure of wild type mice and heterozygotes was about 100 mmHg, whereas the mean blood pressure of RAMP1 KO mice obtained above was about 140 mmHg, indicating clear hypertension. Further, changes in heart rate and blood pressure were measured for mice aged 6 to 18 weeks by the same method. The result is shown in FIG. As shown in FIG. 4, the heart rate was not changed in any mouse. The blood pressure of RAMP1 KO mice was already high at the age of 6 weeks, and a tendency to gradually increase with age was observed.

Vasoconstriction experiment For each of the RAMP1 + / +, RAMP1 +/-, and RAMP1-/-mice prepared in Example 1, the tension change in the blood vessel specimen prepared by the following method is measured and converted into the vasoconstriction rate. Was used to evaluate the vasoconstriction characteristics.
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 KCl, 2.5 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ .7H ₂ O, 25 mM NaHCO ₃ , After removing the connective tissue of the blood vessel in 11.1 mM glucose), the blood vessel was cut to a width of 3 mm to prepare a ring specimen. A tungsten wire attached with a support rod made of acrylic resin was inserted into the blood vessel specimen and fixed in 10 mL of Klebs-Henseleit solution kept at 37 ° C. by aeration of 95% O ₂ /5% CO ₂ mixed gas. Another tungsten wire was inserted into the specimen and connected to the FD pickup. The specimen tension change is recorded on the recorder (model 8K21; NEC) isometrically via the FD pickup and carrier amplifier (1.0 g = 5 cm), and the simple manipulator (model t-7; NEC) is operated. A 0.7 g basic tension was then applied to the specimen. After 30 minutes, a vasoconstriction reaction with phenylephrine (final concentration 3 × 10 ⁻⁷ M) and a vasorelaxant reaction with acetylcholine (final concentration 10 ⁻⁷ M) were confirmed. After confirmation of the blood vessel specimen, equilibrate again in Klebs-Henseleit solution for 60 minutes, and the vessel is contracted by adding phenylephrine (10 ^-6 M), and αCGRP, βCGRP, or adrenomedullin (AM) is added to a final concentration of 1x 10 ^-11 The vascular relaxation response was recorded by cumulative addition of -3x10 ^-6 M. After equilibrating again for 30 minutes, after contracting with phenylephrine (10 ⁻⁶ M), papaverine (10 ⁻⁴ M) was added to relax the blood vessels, and the maximum relaxation ability was determined. These results are shown in FIG.

  As shown in FIG. 5, the RAMP1 + / + mouse thoracic aorta contracted with phenylephrine was significantly relaxed by the addition of αCGRP. On the other hand, the thoracic aorta of RAMP1 − / − mice showed no relaxation reaction by αCGRP. This indicates that RAMP1 constitutes a receptor for αCGRP. On the other hand, βCGRP did not show significant vasorelaxing activity even 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 with phenylephrine 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.

Immunohistochemical staining Human pathological tissue was fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 μm. After deparaffinization, the sections were incubated with anti-RAMP1 antibody (SantaCruz) for 1 hour at room temperature. Following the reaction, Histofine SAB-PO kit (manufactured by Nichirei Co., Ltd.) and diaminobenzidine as a dye were visualized and counterstained with hematoxylin. As a result, expression of RAMP1 was observed in colon cancer cells as well as brain and colon mucosa cells. This indicates that the CGRP signal is involved in the proliferation and metastasis of cancer cells as well as the vasodilatory effect.

1 is a schematic sectional view of a targeting vector used in Example 1. FIG. FIG. 2 is an agarose gel electrophoresis photograph showing the expression pattern of each organ of 8-week-old mice of RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − obtained in Example 1. (A) is a graph showing blood pressure at 10 weeks of age for each mouse of RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − obtained in Example 1, and (b) is a graph showing the same heart rate. . (A) is a graph showing blood pressure at 6-18 weeks of age of each of RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − obtained in Example 1, and (B) is a graph showing the same heart rate. It is. (A) is a graph showing the vasoconstriction rate under the influence of three kinds of vasodilators for 12-week-old mice of RAMP1 + / +, RAMP1 +/−, and RAMP1 − / − obtained in Example 1. Show.

Claims (17)

  A hypertensive model non-human animal characterized in that the function of the RAMP1 gene is completely deficient due to partial or complete modification of the RAMP1 gene on the chromosome.   The hypertensive model non-human animal according to claim 1, wherein a selection marker gene is not inserted on the chromosome.   ES cell obtained by allowing Cre enzyme to act on an ES cell containing a region where at least a part of exon of RAMP1 gene is sandwiched between LoxP sequences and a region where a selectable marker gene is sandwiched between LoxP sequences on a chromosome Or a RAMP1-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 a chromosome, and a mouse capable of expressing a Cre enzyme The hypertensive model non-human animal according to claim 1 or 2, produced by any of the methods by crossing.   The hypertensive model non-human animal according to any one of claims 1 to 3, wherein the function of the RAMP1 gene is conditionally deleted.   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.   A test substance is administered to the hypertension model non-human animal according to any one of claims 1 to 5, or a tissue, organ, or cell derived from the animal is brought into contact with the test substance. A method for screening a drug for preventing and / or treating a vascular disorder disease.   The method for screening a drug according to claim 6, wherein blood pressure and / or vasodilatory action is used as an index.   The vascular disorder disease is at least one disease selected from the group consisting of hypertension, vascular endothelial disorder, arteriosclerotic disease, and blood flow disorder diseases including memory disorders, dementia, peripheral blood flow disorder diseases The method for screening a drug according to any one of claims 6 and 7.   A prophylactic and / or therapeutic drug for a vascular disorder disease obtained by the method for screening a drug according to any one of claims 6 to 8.   A blood flow improving agent comprising a substance that enhances the expression or function of the RAMP1 gene as an active ingredient.   A preventive and / 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.   The vascular disorder disease is at least one disease selected from the group consisting of hypertension, vascular endothelial disorder, arteriosclerotic disease, and blood flow disorder diseases including memory disorders, dementia, peripheral blood flow disorder diseases The prophylactic and / or therapeutic agent for a vascular disorder disease according to claim 11.   A test substance is administered to the hypertension model non-human animal according to any one of claims 1 to 5, or a tissue, organ, or cell derived from the animal is brought into contact with the test substance. Functional food screening method.   The method for screening a functional food according to claim 13, wherein blood pressure and / or vasodilatory action is used as an index.   The functional food obtained by the screening method of the functional food of Claim 13 or 14.   A test substance is administered to the hypertension model non-human animal according to any one of claims 1 to 5, or a tissue, organ, or cell derived from the animal is brought into contact with the test substance. Evaluation method for functional foods.   The method for evaluating a functional food according to claim 16, wherein blood pressure and / or vasodilatory action is used as an index.