WO2011118304A1 - Screening method for therapeutic drug for heart disease - Google Patents
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- WO2011118304A1 WO2011118304A1 PCT/JP2011/053608 JP2011053608W WO2011118304A1 WO 2011118304 A1 WO2011118304 A1 WO 2011118304A1 JP 2011053608 W JP2011053608 W JP 2011053608W WO 2011118304 A1 WO2011118304 A1 WO 2011118304A1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/20—Animal model comprising regulated expression system
- A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
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- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Definitions
- the present invention relates to a screening method for a therapeutic agent for heart disease.
- ischemic heart diseases such as myocardial infarction
- drugs for treating ischemic heart diseases have attracted much social interest. Since cardiomyocytes of mammals including humans are not regenerated after being damaged, the prognosis of ischemic heart disease is generally poor and the quality of life tends to be lowered. Therefore, so-called regenerative medical technology is expected to contribute to the solution of this problem.
- necrotic cardiomyocytes are replaced with other cells (for example, mesenchymal stem cells or skeletal muscle cells) to differentiate into myocardium.
- mesenchymal stem cells or skeletal muscle cells for example, mesenchymal stem cells or skeletal muscle cells
- model animals such as rodents are used.
- a model animal is used as a rat ligated with a coronary artery.
- bFGF basic fibroblast growth factor
- an inhibitor of p38 protein belonging to the MAPK family works effectively on myocardial infarction model rats in cooperation with FGF-1.
- proteins such as p38 and FGF-1 are signal molecules involved in various biological phenomena, and it cannot be denied that even if they are used as drug candidate compounds for heart disease, there is a high possibility of side effects. Therefore, development of a screening system with higher certainty is required for the development of an effective drug for heart disease.
- a screening system with higher certainty is required for the development of an effective drug for heart disease.
- myocardial regeneration evaluation is limited to indirect evaluation methods such as recovery of cardiac function after ligation and observation of the cell cycle and DNA replication in cardiomyocytes, and highly sensitive myocardial regeneration evaluation is extremely difficult.
- the Kaede protein can change the green band fluorescence, which is normally emitted by itself, into the red band fluorescence as energy for absorbing specific ultraviolet rays.
- Transgenic zebrafish in which the above Kaede protein is expressed under a promoter that specifically operates in cardiomyocytes described in Non-Patent Document 3 has been prepared (Non-Patent Document 4).
- the transgenic zebrafish is merely used for the study of heart morphogenesis in the early stages of zebrafish development, and there is no suggestion of screening candidate compounds that can be active ingredients of drugs for heart disease.
- the main object of the present invention is to provide a method for screening candidate compounds effective as therapeutic agents for heart diseases. That is, it is to provide a screening method for a therapeutic agent for heart disease.
- Item 1 A screening method for a therapeutic agent for heart disease using fish in which a protein whose emission wavelength is changed by stimulation is expressed specifically in cardiomyocytes Item 2.
- the screening method according to Item 1 or 2 wherein the protein is Kaede Item 3.
- Item 4. The heart disease according to any one of claims 1 to 3, wherein the heart disease is ischemic heart disease or cardiomyopathy.
- the screening method described A screening method for a therapeutic agent for heart disease using fish in which a protein whose emission wavelength is changed by stimulation is expressed specifically in cardiomyocytes Item 2.
- the screening method according to Item 1 or 2 wherein the protein is Kaede Item 3.
- the screening method according to claim 1 or 2 wherein the fish is zebrafish or medaka.
- Item 4. The heart disease according to any one of claims 1 to 3, wherein the heart disease is ischemic heart disease or cardiomyopathy.
- candidate compounds As an active ingredient of a drug for heart disease in a zebrafish individual of the present invention that has previously damaged the heart, the number of cells that express only green fluorescence at the damaged site is measured. Thus, the degree of myocardial regeneration can be quantified with good reproducibility.
- candidate drugs that can promote myocardial regeneration in mammals are also used in breeding water after red discoloration and damage treatment of zebrafish in the present invention.
- injecting into the thoracic cavity of an individual it becomes possible to screen as having a property in which cells having only green fluorescence after a certain period of time increase predominantly with respect to the control group.
- a schematic diagram of the transgene is shown.
- the photographic image of the mlc2a-kaede transgenic zebrafish fry with the transgene inserted is shown.
- Discoloration of mlc2a-kaede transgenic zebrafish larvae heart by UV irradiation The upper part shows before ultraviolet irradiation and the lower part shows after ultraviolet irradiation.
- Right column fluorescent image by Green long-pass filter (GFP2).
- the upper row shows a fluorescence microscope image of a cardiac injury region of 7 dpi
- the middle row is 14 dpi
- the lower row is 30 dpi.
- the left column shows a red fluorescent image using a red band-pass filter
- the center column shows a green fluorescent image using a green band-pass filter
- the right column shows a composite image of each.
- the bar indicates 50 ⁇ m.
- Quantification of the number of neonatal cardiomyocytes during myocardial regeneration (A) Micrograph image with image processing added for quantification; left is a red and black image of a regenerated myocardium of 14 dpi, taken as a black-and-white image after being taken with a band-pass filter. , The center is a green fluorescent image in the same area as the left, and is taken in black and white after being photographed with a bandpass filter, and the right is a composite image of both. The red dots indicate individual regenerative myocardium with significantly stronger green fluorescence in the damaged area. The area indicated by the arrow is a Low Photoconverted area.
- the left column shows a red fluorescent image using a red band-pass filter
- the center column shows a green fluorescent image using a green band-pass filter
- the right column shows a composite image of each.
- the bar indicates 50 ⁇ m.
- the left side is the result of quantifying the number of regenerative cardiomyocytes of zebrafish treated for 7 to 14 dpi with DMSO control, and the right side with FGFR inhibitor. Effect on myocardial regeneration by treatment with myocardial regeneration promoter:
- the upper row shows the control
- the lower row shows the damaged area of the heart derived from zebrafish injected with bFGF into the chest cavity.
- the left column shows a red fluorescent image using a red band-pass filter
- the center column shows a green fluorescent image using a green band-pass filter
- the right column shows a composite image of each.
- the bar indicates 50 ⁇ m.
- the left side is the result of quantifying the number of regenerative cardiomyocytes of zebrafish injected with control and the right side with bFGF.
- the fish of the present invention is not particularly limited as long as it has the ability to regenerate the heart, but is generally preferably a fish that is easy to handle as a model animal in biological experiments, such as zebrafish, medaka, goldfish, etc. Can be mentioned. Zebrafish is preferred.
- the protein of the present invention is a protein in which light emitted by itself is changed by stimulation. Stimulation is not particularly limited as long as it can give a stimulating effect to proteins expressed in fish, but for example, by light, heat, mechanical, ultrasonic, electrical, magnetic, chemical substances Stimulation by light is preferable in consideration of the fact that it does not affect the living body itself, such as the conventional action in the heart of fish.
- Kaede derived from the sea coral is preferable.
- Said Kaede is a protein containing the amino acid encoded by the gene sequence shown to (GenBank Accession NO.AB085641).
- Kaede When the above-mentioned Kaede is not processed, it usually emits excitation light around 518 nm, and emits excitation light around 582 nm by irradiating it with ultraviolet light of about 350 to 410 nm.
- a protein that changes the wavelength of excitation light by receiving an external stimulus such as ultraviolet light is a protein that changes the wavelength of excitation light by receiving an external stimulus such as ultraviolet light.
- a person skilled in the art can easily prepare the above-described fish as a method for expressing the above-mentioned protein specifically in cardiomyocytes according to a known method for producing a transgenic animal.
- a gene cassette prepared by linking a gene sequence encoding the above-described protein downstream of a promoter sequence that specifically works in cardiomyocytes, It can be produced by breeding a chimeric individual produced by microinjection into cells at the 1-2 cell stage and selecting fish individuals that stably express the gene cassette.
- Examples of the promoter that specifically works in the above-mentioned cardiomyocytes include mlc2a, CARP (Cardiac ankyrin repeat protein), cTnT (Cardiac troponin T), etc., and mlc2a is particularly preferable.
- the screening method of the present invention is performed using the fish described above.
- the fish of the present invention described above changes the wavelength of fluorescence emitted by a protein that is specifically expressed in cardiomyocytes at normal times by applying a stimulus. A portion of the heart tissue is lost by damaging the portion of the heart that contains proteins with altered fluorescence wavelengths.
- the heart tissue is deficient in the fish of the present invention, it is regenerated after a certain period of time, so that the deficient part of the heart is regenerated together with the above-mentioned protein. Since most of the regenerated heart tissue contains cardiomyocytes containing proteins in a non-stimulated state, the wavelength of fluorescence emitted differs from that of the heart tissue that is not damaged. Therefore, the fish heart is stimulated in advance, the fluorescence emitted by the protein expressed in the heart region is changed, and then a part of the heart is damaged, and it is a candidate as an active ingredient of a heart disease therapeutic drug.
- the heart disease in the present invention is not particularly limited, but is preferably a disease which shows a symptom of lack of heart tissue and requires regeneration of the heart tissue itself for treatment. Specific examples include ischemic heart disease, cardiomyopathy, myocardial trauma and the like, and ischemic heart disease or cardiomyopathy is particularly preferable.
- screening methods include (1) A step 1 for performing a treatment for changing the wavelength of fluorescence emitted from a protein specifically expressed in cardiomyocytes by stimulating the above-mentioned fish heart. (2) Step 2 of performing a process of damaging the above-mentioned fish heart, (3) Step 3 of administering a candidate compound to the fish described above, (4) Step 4 after breeding for a certain period of time after the treatment of Steps 1 to 3 above, and observing the above-mentioned damaged part of the fish with a fluorescence microscope, and (5) Microscopic image obtained by observation of Step 4 above And quantifying the regenerated heart tissue, Can be mentioned.
- Step 1 in the screening method of the present invention is a step of performing a treatment for changing the wavelength of fluorescence emitted from the protein specifically expressed in cardiomyocytes by stimulating the above-mentioned fish heart.
- the stimulus given in step 1 can be appropriately selected as long as it can change the wavelength of the fluorescence emitted by each of the above-mentioned proteins, and is not particularly limited, but is particularly stimulated by light such as ultraviolet rays. Is preferred.
- the time for applying the stimulus can be appropriately set within a range in which the wavelength of fluorescence emitted from the above-mentioned protein can be changed without adversely affecting the survival of fish.
- stimulation by light such as ultraviolet rays
- the above-mentioned stimulation can be given by an appropriate method depending on the kind of stimulation, and may be given to the fish individual from the outside, or may be given directly to the heart by performing thoracotomy after treatment such as anesthesia.
- a stimulus such as light from the outside.
- Step 2 in the screening method of the present invention is a step of performing a treatment for damaging the above-mentioned fish heart.
- the method of damaging the fish heart in step 2 may be such that the heart tissue is lost and needs to be regenerated. Further, a method in which almost the same damage is given with good reproducibility for quantitative evaluation is preferable.
- the damage is a damage that can be regenerated without causing lethal damage to fish, and a method in which the degree of regeneration is easily observed is preferable.
- a method that does not require a particularly skilled technique is preferable.
- a process of puncturing a portion of the heart that has been discolored in the process of step 1 above using a needle having the same diameter can be mentioned.
- the puncture site is preferably a site that does not adversely affect the life activity of the fish by the damage treatment, and for example, it is preferable to damage the distal portion of the ventricular ventral surface.
- Step 2 may be performed after breeding for an appropriate period after Step 1 described above, or may be performed continuously after breeding after Step 1 described above.
- step 2 may be performed after step 1 as described above, or may be performed after step 3 described in detail below.
- Step 3 according to the screening method of the present invention is a step of administering a candidate compound to the aforementioned fish.
- the candidate compound used in Step 3 is not particularly limited, and examples thereof include nucleic acids, peptides, proteins, organic compounds, inorganic compounds, and the like, which can be prepared by known methods.
- the method for administering the above-mentioned candidate compound is not particularly limited, but for example, a method of administering directly into the thoracic cavity, intraperitoneal cavity, etc. of a fish using a device such as a microtubule; Examples thereof include a method in which the above-described candidate compound is contained in food and the like, and is administered via an ella or digestive organ; an administration method by physical diffusion, or the like.
- Step 3 can be processed prior to step 1 or step 2 described above.
- the process of the process 1 and the process 2 may be performed continuously, and you may carry out after the raising of a fixed period.
- Step 4 according to the screening method of the present invention is a step of performing the above-mentioned steps 1 to 3 and rearing for a certain period of time, and observing the above-mentioned damaged sites of fish with a fluorescence microscope.
- Step 4 after the treatment in Steps 1 to 3, the fish is raised for a certain period of time, for example, the heart is removed by opening the thorax of the fish, the image obtained by using a fluorescence microscope is analyzed, and the heart of the above candidate compound is analyzed. Whether or not there is an effect related to reproduction can be determined.
- the above-mentioned breeding for a certain period is an appropriate period within a range in which the presence or absence of the regenerative effect of the heart becomes clearer and highly reliable quantitative data having a significant difference is acquired, and usually 10 days. Or more, preferably about 14 to 30 days.
- Step 5 As the fluorescence microscope used in Step 4, a fluorescence microscope usually used in the field of biological science can be used, as long as it can detect the fluorescence wavelength before and after the change emitted from the above-mentioned protein. Good.
- Step 5 according to the screening method of the present invention is a step of analyzing the micrograph image obtained by the observation in the above step 4 and quantifying the regenerated heart tissue.
- Step 5 according to the screening method of the present invention is a step of analyzing the fluorescence microscope image obtained by the observation in the above step 4 and quantifying the regenerated heart tissue. Specifically, the number of cardiomyocytes regenerated after administration of the candidate compound can be quantified by counting from the fluorescence microscope image obtained in the above step 4.
- transgenic zebrafish Coded fluorescent molecule Kaede (MBL, Japan) that changes color by ultraviolet irradiation downstream of the 1.6 kb upstream region of the zebrafish mlc2a protein (SEQ ID NO: 1).
- the gene cassette (FIG. 1) in which the gene to be placed was placed was microinjected into cells at the zebrafish 1-2 cell stage. Thereafter, offspring that stably incorporated the foreign gene into the genomic DNA were isolated from this chimeric zebrafish individual injected microscopically. This was mlc2a-kaede transgenic zebrafish that can visualize heart regeneration. In this transgenic zebrafish individual, only cardiomyocytes exhibit green fluorescence, and as shown in FIG.
- green fluorescence can be observed from the outside in the juvenile stage.
- the cardiomyocytes turn from green to red as shown in FIG.
- the respective fluorescence can be extracted by using an appropriate filter, and by using a so-called long-pass filter, it is possible to simultaneously observe, for example, green fluorescence and orange fluorescence.
- FIG. 1 shows that the respective fluorescence can be extracted by using an appropriate filter, and by using a so-called long-pass filter, it is possible to simultaneously observe, for example, green fluorescence and orange fluorescence.
- mlc2a-kaede transgenic zebrafish was able to change the irradiation surface from green to red with good reproducibility by irradiating ultraviolet rays after thoracotomy after anesthesia even in adult fish. . Therefore, quantification experiment of myocardial regeneration and evaluation of the effect of drugs were performed using adult fish of mlc2a-kaede transgenic zebrafish. Anesthetized adult fish of mlc2a-kaede transgenic zebrafish using 0.32 mg / ml Tricaine (Sigma, USA), fixed in a sponge with slits with the abdomen facing up, and thoracotomy was performed using tweezers .
- the average number of cells having only fluorescence was 6 (FGFR inhibitor treatment) and 21 (control), and the inhibitory effect of the drug on myocardial regeneration could be quantitatively evaluated.
- FGF especially bFGF or FGF-1
- FGF-1 which has been shown to be able to accelerate the myocardial regeneration process in mammalian myocardial infarction model rats, quantitatively analyzes myocardial regeneration using transgenic zebrafish. We examined whether it was possible. Using the above method, the mlc2a-kaede transgenic zebrafish heart was changed to red fluorescence and subsequently damaged with a tungsten needle.
- transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test.
- ⁇ About Wnt> The influence of the heart regeneration effect of mlc2a-kaede transgenic zebrafish was examined by using an inhibitor of Wnt signal, which plays an essential role in the zebrafish fin regeneration process. In fish cardiomyocytes, there is no information about what role Wnt signals play in myocardial regeneration.
- the number of cells having only green fluorescence in the damaged area was significantly smaller than the number of cells having only green fluorescence in the control individuals.
- the number of cells having only green fluorescence is 3 (Wnt signal inhibitor treatment) and 21 (control) on average as shown in FIG. 9 (B), and the myocardial regeneration is remarkably suppressed by suppressing the Wnt signal. It became clear to suppress. It has also become possible to quantitatively evaluate the inhibitory effect of drugs on the regeneration of heart cells. From the above results, it was examined whether or not the myocardial regeneration effect by the GSK-3 inhibitor acting as a promoter in the Wnt signal can be quantitatively analyzed.
- the above method caused the mlc2a-kaede transgenic zebrafish heart to turn red fluorescent, and subsequently damaged with a tungsten needle, after 1 and 8 days of 2.5 nM GSK-3 inhibitor (BIO, Calbiochem, USA) Breeding was carried out in the presence or absence of an aqueous phase. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG. In spite of the same damage, a significant increase in regenerative myocardium with only green fluorescence was observed in the individuals to which GSK-3 inhibitor acting as a promoter of Wnt signal was added, compared to the control individuals. It was.
- transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test. From the results of the above examples, by using the method of the present invention, it becomes possible to screen for candidate compounds that are active ingredients of drugs for heart disease. Also, confirm whether compounds already listed as candidate compounds have an effective effect on heart disease, study the optimal concentration of the compound, study the administration interval and duration, and examine the possibility of combination with other candidate compounds It is also possible to use for such as.
- the screening method of the present invention is suitable for searching for candidate compounds that are active ingredients of drugs for heart disease.
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Abstract
Disclosed is a method for screening candidate compounds effective as therapeutic drugs for heart disease. Using fish with specific cardiac muscle expression of a protein that changes the wavelength of emitted florescence with a stimulus, success was achieved in making regenerated damaged sites visible after the hearts of these fish were regenerably damaged without lethal consequences. Furthermore, success was achieved in quantifying the extent of the regeneration. The method for screening candidate compounds effective as therapeutic agents for heart disease can be achieved using this technique.
Description
本発明は、心疾患治療薬のスクリーニング方法に関する。
The present invention relates to a screening method for a therapeutic agent for heart disease.
心筋梗塞など虚血性の心疾患を治療するための薬剤は、社会的にも多くの関心を集めている。ヒトを初めとする哺乳動物の心筋細胞は、損傷を受けた後に再生しない為、一般に虚血性心疾患の予後は悪く、生活の質も低下しがちとなっている。
そこで、いわゆる再生医療的技術がこの問題の解決に寄与すると期待されている。主な技術としては、壊死した心筋細胞を他の細胞（例えば間葉系幹細胞や骨格筋細胞など）で置換し心筋に分化させようとするものがある。
心筋に関する再生医療には、今後心筋の再生過程を制御する上で心筋細胞の再生を促進したり抑制したりする薬剤の開発がなされると考えられており、実際いくつかの薬剤が候補として報告されている。これらの薬剤の効果を判定する方法として、げっ歯類などのモデル動物が用いられている。例えば非特許文献１に記載のように、冠動脈を結紮したラットがモデル動物が用いられている。また本文献では、当該ラットの心臓に対して、新たに培養心筋細胞を移植する際に、ｂＦＧＦ（塩基性繊維芽細胞増殖因子）を用いた処理を加えることによって心臓機能の改善や瘢痕（ｓｃａｒ）部への培養心筋細胞の生着を促すことに成功している。
また具体的な心臓再生に有効である分子に関する知見として、非特許文献５において、ＭＡＰＫファミリーに属するｐ３８蛋白質の阻害剤が、ＦＧＦ−１と協調的に心筋梗塞モデルラットに対して有効に働くことが示唆されている。しかしながら、ｐ３８やＦＧＦ−１といった蛋白質は、様々な生体現象に関与するシグナル分子であり、心疾患に対する薬剤の候補化合物として使用しても、副作用が生じる可能性も高いことは否めない。従って、心疾患に対して効果的な薬剤の開発のために、より確実性の高いスクリーニング系の開発が求められている。
哺乳類動物の場合、通常はほとんど、或いは全く心筋が再生せず、げっ歯類動物にてしばしば行われる冠動脈結紮実験などを採用したとしても、心筋再生の定量的解析は達成されていない。このような心筋再生の評価は、結紮後の心臓機能の回復や心筋細胞における細胞周期やＤＮＡ複製の観察といった間接的な評価法に留まり、高感度の心筋再生評価は著しく困難である。またげっ歯類の冠動脈結紮実験は熟練した技術を必要とし、実験者の技量によって実験結果が容易に異なる可能性が高いため、異なる実験者による実験結果の単純な比較が行ないにくく、信頼性や再現性に問題がある。加えて、半定量的な心筋再生実験も、統計上の有意差を出す為には多くの動物を必要とするなど、げっ歯類動物をモデルマウスとして用いた心筋再生の研究には様々な困難が伴っている。
生物学的実験におけるモデル動物として多用されるゼブラフィッシュは、哺乳動物とは異なり、損傷した心臓が再生することが知られている（非特許文献２）。また近年のバイオテクノロジー技術の進歩により、ある一定のエネルギーを吸収することで、自らが発光する光の波長を変化させることのできる分子の開発が進んでいる。例えばＫａｅｄｅ蛋白質は、特定の紫外線を吸収するエネルギーとして、通常自らが発光する緑色帯の蛍光を、赤色帯の蛍光に変化させることができる。このような蛋白質を用いることによって、様々な生体現象を解析することに成功している。
上記のＫａｅｄｅ蛋白質を非特許文献３に記載の心筋細胞において特異的に作動するプロモーターの下で発現させたトランスジェニックゼブラフィッシュが作製されているが（非特許文献４）、本文献では心筋細胞の当該トランスジェニックゼブラフィッシュを、単にゼブラフィッシュの発生初期の心臓の形態形成の研究に用いているのみであり、心疾患に対する薬剤の有効成分となりえる候補化合物のスクリーニングについては、何ら示唆されていない。 Drugs for treating ischemic heart diseases such as myocardial infarction have attracted much social interest. Since cardiomyocytes of mammals including humans are not regenerated after being damaged, the prognosis of ischemic heart disease is generally poor and the quality of life tends to be lowered.
Therefore, so-called regenerative medical technology is expected to contribute to the solution of this problem. As a main technique, there is a technique in which necrotic cardiomyocytes are replaced with other cells (for example, mesenchymal stem cells or skeletal muscle cells) to differentiate into myocardium.
In the regenerative medicine related to the myocardium, it is considered that the development of drugs that promote or inhibit the regeneration of cardiomyocytes in controlling the myocardial regeneration process will be made in the future. Has been. As a method for determining the effects of these drugs, model animals such as rodents are used. For example, as described in Non-PatentDocument 1, a model animal is used as a rat ligated with a coronary artery. In addition, in this document, when newly cultured cardiomyocytes are transplanted into the heart of the rat, treatment with bFGF (basic fibroblast growth factor) is added to improve cardiac function or scar. Succeeded in encouraging the engraftment of cultured cardiomyocytes in the
In addition, as a specific knowledge regarding a molecule effective for cardiac regeneration, in Non-PatentDocument 5, an inhibitor of p38 protein belonging to the MAPK family works effectively on myocardial infarction model rats in cooperation with FGF-1. Has been suggested. However, proteins such as p38 and FGF-1 are signal molecules involved in various biological phenomena, and it cannot be denied that even if they are used as drug candidate compounds for heart disease, there is a high possibility of side effects. Therefore, development of a screening system with higher certainty is required for the development of an effective drug for heart disease.
In the case of mammals, little or no myocardium is usually regenerated, and even if a coronary artery ligation experiment often performed in rodents is employed, quantitative analysis of myocardial regeneration has not been achieved. Such myocardial regeneration evaluation is limited to indirect evaluation methods such as recovery of cardiac function after ligation and observation of the cell cycle and DNA replication in cardiomyocytes, and highly sensitive myocardial regeneration evaluation is extremely difficult. In addition, coronary artery ligation experiments in rodents require skilled techniques, and it is highly likely that the experimental results will easily differ depending on the skill of the experimenter. There is a problem with reproducibility. In addition, semi-quantitative myocardial regeneration experiments also require many animals to produce statistically significant differences. For example, it is difficult to study myocardial regeneration using rodent animals as model mice. Is accompanied.
Zebrafish, which are frequently used as model animals in biological experiments, are known to regenerate damaged hearts, unlike mammals (Non-patent Document 2). Also, due to recent advances in biotechnology, the development of molecules that can change the wavelength of the light that they emit by absorbing a certain amount of energy is progressing. For example, the Kaede protein can change the green band fluorescence, which is normally emitted by itself, into the red band fluorescence as energy for absorbing specific ultraviolet rays. By using such proteins, we have succeeded in analyzing various biological phenomena.
Transgenic zebrafish in which the above Kaede protein is expressed under a promoter that specifically operates in cardiomyocytes described in Non-PatentDocument 3 has been prepared (Non-Patent Document 4). The transgenic zebrafish is merely used for the study of heart morphogenesis in the early stages of zebrafish development, and there is no suggestion of screening candidate compounds that can be active ingredients of drugs for heart disease.
そこで、いわゆる再生医療的技術がこの問題の解決に寄与すると期待されている。主な技術としては、壊死した心筋細胞を他の細胞（例えば間葉系幹細胞や骨格筋細胞など）で置換し心筋に分化させようとするものがある。
心筋に関する再生医療には、今後心筋の再生過程を制御する上で心筋細胞の再生を促進したり抑制したりする薬剤の開発がなされると考えられており、実際いくつかの薬剤が候補として報告されている。これらの薬剤の効果を判定する方法として、げっ歯類などのモデル動物が用いられている。例えば非特許文献１に記載のように、冠動脈を結紮したラットがモデル動物が用いられている。また本文献では、当該ラットの心臓に対して、新たに培養心筋細胞を移植する際に、ｂＦＧＦ（塩基性繊維芽細胞増殖因子）を用いた処理を加えることによって心臓機能の改善や瘢痕（ｓｃａｒ）部への培養心筋細胞の生着を促すことに成功している。
また具体的な心臓再生に有効である分子に関する知見として、非特許文献５において、ＭＡＰＫファミリーに属するｐ３８蛋白質の阻害剤が、ＦＧＦ−１と協調的に心筋梗塞モデルラットに対して有効に働くことが示唆されている。しかしながら、ｐ３８やＦＧＦ−１といった蛋白質は、様々な生体現象に関与するシグナル分子であり、心疾患に対する薬剤の候補化合物として使用しても、副作用が生じる可能性も高いことは否めない。従って、心疾患に対して効果的な薬剤の開発のために、より確実性の高いスクリーニング系の開発が求められている。
哺乳類動物の場合、通常はほとんど、或いは全く心筋が再生せず、げっ歯類動物にてしばしば行われる冠動脈結紮実験などを採用したとしても、心筋再生の定量的解析は達成されていない。このような心筋再生の評価は、結紮後の心臓機能の回復や心筋細胞における細胞周期やＤＮＡ複製の観察といった間接的な評価法に留まり、高感度の心筋再生評価は著しく困難である。またげっ歯類の冠動脈結紮実験は熟練した技術を必要とし、実験者の技量によって実験結果が容易に異なる可能性が高いため、異なる実験者による実験結果の単純な比較が行ないにくく、信頼性や再現性に問題がある。加えて、半定量的な心筋再生実験も、統計上の有意差を出す為には多くの動物を必要とするなど、げっ歯類動物をモデルマウスとして用いた心筋再生の研究には様々な困難が伴っている。
生物学的実験におけるモデル動物として多用されるゼブラフィッシュは、哺乳動物とは異なり、損傷した心臓が再生することが知られている（非特許文献２）。また近年のバイオテクノロジー技術の進歩により、ある一定のエネルギーを吸収することで、自らが発光する光の波長を変化させることのできる分子の開発が進んでいる。例えばＫａｅｄｅ蛋白質は、特定の紫外線を吸収するエネルギーとして、通常自らが発光する緑色帯の蛍光を、赤色帯の蛍光に変化させることができる。このような蛋白質を用いることによって、様々な生体現象を解析することに成功している。
上記のＫａｅｄｅ蛋白質を非特許文献３に記載の心筋細胞において特異的に作動するプロモーターの下で発現させたトランスジェニックゼブラフィッシュが作製されているが（非特許文献４）、本文献では心筋細胞の当該トランスジェニックゼブラフィッシュを、単にゼブラフィッシュの発生初期の心臓の形態形成の研究に用いているのみであり、心疾患に対する薬剤の有効成分となりえる候補化合物のスクリーニングについては、何ら示唆されていない。 Drugs for treating ischemic heart diseases such as myocardial infarction have attracted much social interest. Since cardiomyocytes of mammals including humans are not regenerated after being damaged, the prognosis of ischemic heart disease is generally poor and the quality of life tends to be lowered.
Therefore, so-called regenerative medical technology is expected to contribute to the solution of this problem. As a main technique, there is a technique in which necrotic cardiomyocytes are replaced with other cells (for example, mesenchymal stem cells or skeletal muscle cells) to differentiate into myocardium.
In the regenerative medicine related to the myocardium, it is considered that the development of drugs that promote or inhibit the regeneration of cardiomyocytes in controlling the myocardial regeneration process will be made in the future. Has been. As a method for determining the effects of these drugs, model animals such as rodents are used. For example, as described in Non-Patent
In addition, as a specific knowledge regarding a molecule effective for cardiac regeneration, in Non-Patent
In the case of mammals, little or no myocardium is usually regenerated, and even if a coronary artery ligation experiment often performed in rodents is employed, quantitative analysis of myocardial regeneration has not been achieved. Such myocardial regeneration evaluation is limited to indirect evaluation methods such as recovery of cardiac function after ligation and observation of the cell cycle and DNA replication in cardiomyocytes, and highly sensitive myocardial regeneration evaluation is extremely difficult. In addition, coronary artery ligation experiments in rodents require skilled techniques, and it is highly likely that the experimental results will easily differ depending on the skill of the experimenter. There is a problem with reproducibility. In addition, semi-quantitative myocardial regeneration experiments also require many animals to produce statistically significant differences. For example, it is difficult to study myocardial regeneration using rodent animals as model mice. Is accompanied.
Zebrafish, which are frequently used as model animals in biological experiments, are known to regenerate damaged hearts, unlike mammals (Non-patent Document 2). Also, due to recent advances in biotechnology, the development of molecules that can change the wavelength of the light that they emit by absorbing a certain amount of energy is progressing. For example, the Kaede protein can change the green band fluorescence, which is normally emitted by itself, into the red band fluorescence as energy for absorbing specific ultraviolet rays. By using such proteins, we have succeeded in analyzing various biological phenomena.
Transgenic zebrafish in which the above Kaede protein is expressed under a promoter that specifically operates in cardiomyocytes described in Non-Patent
本発明の主な目的は、心疾患に対する治療剤として有効な候補化合物をスクリーニングする方法を提供することである。すなわち、心疾患治療薬のスクリーニング方法を提供することである。
The main object of the present invention is to provide a method for screening candidate compounds effective as therapeutic agents for heart diseases. That is, it is to provide a screening method for a therapeutic agent for heart disease.
本願発明者らは、上述の課題に鑑みて鋭意研究を重ねた結果、特定のエネルギーを吸収することによって、発する光の波長が変化する蛋白質を、心筋細胞特異的に発現させた魚類を用いることで、該魚類の心臓に対して致死に至らしめることのない、再生可能な損傷を加えた後に、再生した損傷部位を可視化することに成功し、さらに再生の程度を定量化することに成功した。
本発明は、係る知見に基づいて完成されたものであり、以下の態様の発明を包含する。
項１ 刺激によって発光波長が変化する蛋白質を心筋細胞特異的に発現させた魚類を用いた、心疾患治療薬のスクリーニング方法
項２ 前記蛋白質がＫａｅｄｅである、上記項１又は２に記載のスクリーニング方法
項３ 前記魚類が、ゼブラフィッシュ又はメダカである、請求項１または２に記載のスクリーニング方法
項４ 前記心疾患が虚血性心疾患又は心筋症である、請求項１~３のいずれか１項に記載のスクリーニング方法 As a result of intensive studies in view of the above-mentioned problems, the inventors of the present application use fish that specifically express cardiomyocytes, a protein that changes the wavelength of emitted light by absorbing specific energy. So, after adding reproducible damage that did not cause lethality to the heart of the fish, it succeeded in visualizing the regenerated damage site and succeeding in quantifying the degree of regeneration. .
This invention is completed based on the knowledge which concerns, and includes the invention of the following aspects.
Item 1. A screening method for a therapeutic agent for heart disease using fish in which a protein whose emission wavelength is changed by stimulation is expressed specifically in cardiomyocytes Item 2. The screening method according to Item 1 or 2, wherein the protein is Kaede Item 3. The screening method according to claim 1 or 2, wherein the fish is zebrafish or medaka. Item 4. The heart disease according to any one of claims 1 to 3, wherein the heart disease is ischemic heart disease or cardiomyopathy. The screening method described
本発明は、係る知見に基づいて完成されたものであり、以下の態様の発明を包含する。
項１ 刺激によって発光波長が変化する蛋白質を心筋細胞特異的に発現させた魚類を用いた、心疾患治療薬のスクリーニング方法
項２ 前記蛋白質がＫａｅｄｅである、上記項１又は２に記載のスクリーニング方法
項３ 前記魚類が、ゼブラフィッシュ又はメダカである、請求項１または２に記載のスクリーニング方法
項４ 前記心疾患が虚血性心疾患又は心筋症である、請求項１~３のいずれか１項に記載のスクリーニング方法 As a result of intensive studies in view of the above-mentioned problems, the inventors of the present application use fish that specifically express cardiomyocytes, a protein that changes the wavelength of emitted light by absorbing specific energy. So, after adding reproducible damage that did not cause lethality to the heart of the fish, it succeeded in visualizing the regenerated damage site and succeeding in quantifying the degree of regeneration. .
This invention is completed based on the knowledge which concerns, and includes the invention of the following aspects.
心疾患に対する薬剤の有効成分となる候補化合物を、予め心臓に損傷を与えた、本発明のゼブラフィッシュ個体に用いることによって、該損傷部位にあって緑色蛍光のみを発現する細胞数を計測することにより心筋の再生度合いを再現性良く定量化できる。これを利用することで、下記実施例のｂＦＧＦで例示したように、哺乳動物においても心筋再生を促進可能する候補薬剤は、本発明におけるゼブラフィッシュの赤色変色、損傷処理後に候補薬剤を飼育水に加えたり、個体の胸腔内に注射することで、一定時間後に緑色蛍光のみを有する細胞が対照群に対して優位に増加する性質をもつものとしてスクリーニング可能となる。
ヒトに対して有用な薬剤が、ゼブラフィッシュ、メダカ等の小型魚類においても機能する例は多く報告されており、既に小型魚類を用いてヒト薬剤の薬理特性や副作用を検討する製薬会社も存在している。従って本発明の方法を用いることにより、単に小型魚類に限らず、ヒトを初めとする哺乳動物の心筋再生制御に関わる薬剤のスクリーニングすることが可能となる。
さらに、既に候補化合物として挙げられる化合物の、様々な有用性を確認することも可能である。 By using a candidate compound as an active ingredient of a drug for heart disease in a zebrafish individual of the present invention that has previously damaged the heart, the number of cells that express only green fluorescence at the damaged site is measured. Thus, the degree of myocardial regeneration can be quantified with good reproducibility. By using this, as exemplified by bFGF in the following examples, candidate drugs that can promote myocardial regeneration in mammals are also used in breeding water after red discoloration and damage treatment of zebrafish in the present invention. In addition, by injecting into the thoracic cavity of an individual, it becomes possible to screen as having a property in which cells having only green fluorescence after a certain period of time increase predominantly with respect to the control group.
There have been many reports that useful drugs for humans function in small fish such as zebrafish and medaka, and there are already pharmaceutical companies that use small fish to study the pharmacological properties and side effects of human drugs. ing. Therefore, by using the method of the present invention, it is possible to screen for drugs involved in the control of myocardial regeneration in mammals including humans, not just small fish.
Furthermore, it is possible to confirm various usefulness of the compounds already listed as candidate compounds.
ヒトに対して有用な薬剤が、ゼブラフィッシュ、メダカ等の小型魚類においても機能する例は多く報告されており、既に小型魚類を用いてヒト薬剤の薬理特性や副作用を検討する製薬会社も存在している。従って本発明の方法を用いることにより、単に小型魚類に限らず、ヒトを初めとする哺乳動物の心筋再生制御に関わる薬剤のスクリーニングすることが可能となる。
さらに、既に候補化合物として挙げられる化合物の、様々な有用性を確認することも可能である。 By using a candidate compound as an active ingredient of a drug for heart disease in a zebrafish individual of the present invention that has previously damaged the heart, the number of cells that express only green fluorescence at the damaged site is measured. Thus, the degree of myocardial regeneration can be quantified with good reproducibility. By using this, as exemplified by bFGF in the following examples, candidate drugs that can promote myocardial regeneration in mammals are also used in breeding water after red discoloration and damage treatment of zebrafish in the present invention. In addition, by injecting into the thoracic cavity of an individual, it becomes possible to screen as having a property in which cells having only green fluorescence after a certain period of time increase predominantly with respect to the control group.
There have been many reports that useful drugs for humans function in small fish such as zebrafish and medaka, and there are already pharmaceutical companies that use small fish to study the pharmacological properties and side effects of human drugs. ing. Therefore, by using the method of the present invention, it is possible to screen for drugs involved in the control of myocardial regeneration in mammals including humans, not just small fish.
Furthermore, it is possible to confirm various usefulness of the compounds already listed as candidate compounds.
この発明におけるその他の用語や概念は、発明の実施形態の説明や実施例において詳しく規定する。また、この発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。例えば、遺伝子工学および分子生物学的技術はＳａｍｂｒｏｏｋ ａｎｄ Ｍａｎｉａｔｉｓ，”Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ”，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，２００１；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．ｅｔ ａｌ．”Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ”，Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ，Ｎｅｗ Ｙｏｒｋ，Ｎ．Ｙ，２００７等に記載されている。
〔本発明の魚類について〕
本発明の魚類は心臓の再生能を有する魚類であれば、特に限定されることは無いが、一般的に生物実験におけるモデル動物として扱い易い魚類が好ましく、例えば、ゼブラフィッシュ、メダカ、キンギョ等を挙げることができる。好ましくはゼブラフィッシュである。
本発明の蛋白質とは、刺激によって自らが発する光が変化するものである。刺激とは、魚類内部にて発現する蛋白質に対して、刺激効果を与えうるものであれば得に限定はされないが、例えば、光、熱、力学的、超音波、電気、磁気、化学物質による刺激などが挙げられ、魚類の心臓における従来の働きなど、生体そのものに影響を与えないことを勘案して、光による刺激が好ましい。特に、紫外光による刺激が好ましく、３５０~４１０ｎｍの波長を有する紫外光が最も好ましい。
本発明の蛋白質は、上記の刺激に応答して自らが発光する蛍光波長を変化させ得るものであれば特に限定はされない。具体的にはイシサンゴ（Ｔｒａｃｈｙｐｈｙｌｌｉａ ｇｅｏｆｆｒｏｙｉ）由来のＫａｅｄｅが好ましい。上記のＫａｅｄｅは、（ＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ ＮＯ．ＡＢ０８５６４１）に示す遺伝子配列によってコードされるアミノ酸を含む蛋白質である。
上述のＫａｅｄｅは、未処理の場合は、通常５１８ｎｍ付近の励起光を発し、ここで３５０~４１０ｎｍ程度の紫外光を照射することによって、５８２ｎｍ付近の励起光を発する。すなわち、紫外光という外部刺激を受けることによって、励起光の波長を変化させる蛋白質である。
上述の魚類に対して、上述の蛋白質を心筋細胞特異的に発現させる方法としては、公知のトランスジェニック動物の作製法に従えば、当業者であれば容易に作製することが可能である。具体的には、心筋細胞特異的にタンパク質を発現させるために、心筋細胞において特異的に働くプロモーター配列の下流に、上述のタンパク質をコードする遺伝子配列を繋いで作成した遺伝子カセットを、魚類個体の１−２細胞期の細胞に顕微注射することによって作成されるキメラ個体を育種し、安定的に前記の遺伝子カセットを発現する魚類個体を選抜することによって作製することができる。
上述の心筋細胞にて特異的に働くプロモーターの例としては、ｍｌｃ２ａ、ＣＡＲＰ（Ｃａｒｄｉａｃ ａｎｋｙｒｉｎ ｒｅｐｅａｔ ｐｒｏｔｅｉｎ）、ｃＴｎＴ（Ｃａｒｄｉａｃ ｔｒｏｐｏｎｉｎ Ｔ）等を挙げることができるが、特に、ｍｌｃ２ａが好ましい。
〔本発明のスクリーニング方法〕
本発明のスクリーニング方法は、上述した魚類を用いて行う。上述した本発明の魚類は、正常時に心筋細胞内にて特異的に発現している蛋白質が発する蛍光の波長を、刺激を与えることによって変化する。発する蛍光波長が変化した蛋白質を含む心臓の部位に損傷を与えることによって、心臓組織の一部分を欠損させる。本発明の魚類は、心臓組織が欠損したとしても、一定期間経過後には再生されるので、心臓の欠損部位は上述の蛋白質と共に再生される。
再生した心臓組織は、刺激を与えていない状態の蛋白質を含む心筋細胞が大部分に含まれるので、損傷を与えていない心臓組織の部位と比較して発する蛍光の波長が異なる。従って、上記魚類の心臓に予め刺激を与えて、心臓部位にて発現する蛋白質が発する蛍光を変化させておき、次に心臓の一部に損傷を与え、心疾患治療薬の有効成分となる候補化合物を投与すれば、一定期間経過後に再生する心臓組織の程度を、刺激を与える前の蛋白質が発する蛍光領域を再生して判断することができる。そしてその領域を定量化すれば、各候補化合物の効果を定量的に示すことが可能となる。
本発明における心疾患とは、特には限定されるものではないが、心臓組織が欠落する症状を示し、治療するにあたって心臓組織そのものの再生が求められる疾患が好ましい。具体的には、虚血性心疾患、心筋症、心筋外傷等を挙げることができるが、特に、虚血性心疾患又は心筋症が好ましい。
スクリーニング方法の具体例としては、
（１）上述の魚類の心臓に対して刺激を与え、心筋細胞にて特異的に発現している蛋白質が発する蛍光の波長を変化させる処理を行う工程１、
（２）上述の魚類の心臓に対して、損傷を与える処理を行う工程２、
（３）上述の魚類に対して、候補化合物を投与する工程３、
（４）上記工程１~３の処理を施した後、一定期間飼育し、上述の魚類の損傷部位を蛍光顕微鏡によって観察する工程４、及び
（５）上記工程４の観察によって得られる顕微鏡写真像を解析し、再生した心臓組織を定量化する工程５、
を含む方法を挙げることができる。
（工程１について）
本発明のスクリーニング方法における工程１とは、上述の魚類の心臓に対して刺激を与え、心筋細胞にて特異的に発現している蛋白質が発する蛍光の波長を変化させる処理を行う工程である。
工程１において与える刺激は、上述したそれぞれの蛋白質が発する蛍光の波長を変化させることができるものであれば、適宜選択することが可能であり、特には限定されないが、特に紫外線などの光による刺激が好ましい。また、刺激を与える時間についても、魚類の生存等に悪影響を与えず、上述の蛋白質が発する蛍光の波長を変化させ得る範囲において適宜設定することができる。例えば、紫外線などの光による刺激の場合、通常１０~１２０秒程度の刺激を与えればよく、好ましくは３０~６０秒である。
上記の刺激は、刺激の種類によってそれぞれ適当な方法によって与えることができ、魚類個体に対して外部から与えてもよく、麻酔等の処理の後に開胸して、直接心臓に与えても良い。例えば、透明な表皮を有する魚類の場合は、光等の刺激を外部から与えることが可能となる。
また工程１は、下記に詳述する工程２の後に行っても良い。その場合、工程２にて心臓に損傷を与えた部位に上述の刺激を与えることが好ましく、損傷を与えた後は一定期間の飼育をすることなく、連続的に工程１による処理を行うことが好ましい。
（工程２について）
本発明のスクリーニング方法における工程２とは、上述の魚類の心臓に対して、損傷を与える処理を行う工程である。
工程２において魚類の心臓に損傷を与える方法は、心臓の組織が欠損し、再生を要する状態にすればよい。また、定量的評価の為に再現性良くほぼ同一の損傷が与えられる方法が好ましい。さらに該損傷とは、魚類に対して致死的な損傷を与えることなく、再生可能な程度の損傷であり、再生の程度が容易に観察される方法が好ましい。これらの条件を満たすような損傷を与える方法については特には限定されないが、スクリーニング系の再現性、後に得られる定量データに統計学上の有意差を示せる程度に信頼度の高いデータを採取することに鑑みて、特段熟練した技術を要しない方法が好ましい。
例えば、同一の径を有する針を用いて上記工程１の処理にて変色させた心臓の部位を穿刺する処理を挙げることができる。また穿刺する部位は、損傷処理によって魚類の生命活動に悪影響を与えない部位が好ましく、例えば心室腹面遠位部に損傷を与えることが好ましい。
工程２は、上記の工程１の後に適当な期間飼育した後に行ってもよく、上記の工程１の後、飼育することなく連続的に行ってもよい。また上述の蛋白質が、上述の工程１と同様の方法によって処理され、既に発する蛍光の波長が変化しているものを別途入手して用いることも可能である。
さらに工程２は、上述のように工程１の後に行ってもよく、下記に詳述する工程３の後に行ってもよい。
（工程３について）
本発明のスクリーニング方法にかかる工程３とは、上述の魚類に対して、候補化合物を投与する工程である。
工程３において用いる候補化合物は、特に限定されることは無く、核酸、ペプチド、タンパク質、有機化合物、無機化合物等を挙げることができ、それぞれ公知の方法によって作製することが出来る。
上述の候補化合物の投与方法は、特には限定されないが、例えば微小細管等の器具を用いて、直接魚類の胸腔、腹腔内等に投与する方法；魚類を飼育する飼育槽、飼育する際に与える餌等に上述の候補化合物を含有させて、エラや消化器官を介して投与する方法；物理的な拡散による投与方法等を挙げることができる。上述の候補化合物を使用するにあたり、経済的な点を勘案すると、胸腔、腹腔内等に直接投与する方法が好ましい。
工程３は、上記工程１又は工程２に先だって処理することも可能である。この場合、工程３による処理の後、連続的に工程１及び工程２の処理を行ってもよく、一定期間の飼育の後に行ってもよい。
（工程４について）
本発明のスクリーニング方法にかかる工程４とは、上記工程１~３の処理を施した後、一定期間飼育し、上述の魚類の損傷部位を蛍光顕微鏡によって観察する工程である。
工程４では、上記工程１~３の処理後に一定期間飼育し、例えば、魚類を開胸して心臓を摘出し、蛍光顕微鏡を用いることによって得られる像を解析して、上述の候補化合物の心臓再生に係る効果の有無を判断することができる。
上述の一定期間の飼育とは、心臓の再生効果の有無がより明確となり、且つ有意差を持った信頼度の高い定量的なデータが取得される範囲において、適当な期間であり、通常１０日程度以上であり、好ましくは１４~３０日程度である。
また、上述の顕微鏡観察については、一定期間の飼育後の魚類に対して開胸処理のみを施し、心臓を摘出することなく蛍光顕微鏡下にて像を取得することも可能である。さらに、上記の魚類が透明な表皮を有する魚類であれば、開胸処理すら行うことなく、心臓再生効果の有無を蛍光顕微鏡による像から判断することもできる。心臓を摘出しない方法によって蛍光顕微鏡像を取得した場合は、経時的に候補化合物による心臓再生効果を評価することができ、さらに好ましいスクリーニング方法となる。
工程４において用いる蛍光顕微鏡は、通常は生物科学分野にて用いられる蛍光顕微鏡が利用可能であり、上述の蛋白質が発する変化前の蛍光波長と変化後の蛍光波長をそれぞれ検出し得るものであればよい。
（工程５について）
本発明のスクリーニング方法にかかる工程５は、上記工程４における観察によって得られる顕微鏡写真像を解析し、再生した心臓組織を定量化する工程である。
本発明のスクリーニング方法にかかる工程５は、上記工程４における観察によって得られる蛍光顕微鏡像を解析し、再生した心臓組織を定量化する工程である。具体的には候補化合物の投与後に再生した心筋細胞の数を、上記工程４によって得られた蛍光顕微鏡像から計数することによって、定量化することができる。すなわち、再生した心筋細胞の数が多いほど、候補化合物による効果が高いと判断することができる。なお、上述した細胞数を計数するためには、公知の画像解析ソフトを用いることによって実施できる。
上述の工程１~５を含む方法を用いることで、心疾患に対する新規薬剤の有効成分となり得る候補化合物をスクリーニングすることが可能となる。また、既に候補化合物として挙げられている化合物の、有用性の検討、投与間隔・期間の検討、他の候補薬剤との併用による効果の検討等を行うことも可能である。
以下に本発明をより詳細に説明する。但し、本発明が以下に示す実施例に限定されないのは言うまでも無い。 Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly shows the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, F .; M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, N .; Y, 2007, and the like.
[Fish of the present invention]
The fish of the present invention is not particularly limited as long as it has the ability to regenerate the heart, but is generally preferably a fish that is easy to handle as a model animal in biological experiments, such as zebrafish, medaka, goldfish, etc. Can be mentioned. Zebrafish is preferred.
The protein of the present invention is a protein in which light emitted by itself is changed by stimulation. Stimulation is not particularly limited as long as it can give a stimulating effect to proteins expressed in fish, but for example, by light, heat, mechanical, ultrasonic, electrical, magnetic, chemical substances Stimulation by light is preferable in consideration of the fact that it does not affect the living body itself, such as the conventional action in the heart of fish. In particular, stimulation with ultraviolet light is preferable, and ultraviolet light having a wavelength of 350 to 410 nm is most preferable.
The protein of the present invention is not particularly limited as long as it can change the fluorescence wavelength emitted by itself in response to the above stimulus. Specifically, Kaede derived from the sea coral (Trachphyllia geoffroyi) is preferable. Said Kaede is a protein containing the amino acid encoded by the gene sequence shown to (GenBank Accession NO.AB085641).
When the above-mentioned Kaede is not processed, it usually emits excitation light around 518 nm, and emits excitation light around 582 nm by irradiating it with ultraviolet light of about 350 to 410 nm. That is, it is a protein that changes the wavelength of excitation light by receiving an external stimulus such as ultraviolet light.
A person skilled in the art can easily prepare the above-described fish as a method for expressing the above-mentioned protein specifically in cardiomyocytes according to a known method for producing a transgenic animal. Specifically, in order to express a protein specifically in cardiomyocytes, a gene cassette prepared by linking a gene sequence encoding the above-described protein downstream of a promoter sequence that specifically works in cardiomyocytes, It can be produced by breeding a chimeric individual produced by microinjection into cells at the 1-2 cell stage and selecting fish individuals that stably express the gene cassette.
Examples of the promoter that specifically works in the above-mentioned cardiomyocytes include mlc2a, CARP (Cardiac ankyrin repeat protein), cTnT (Cardiac troponin T), etc., and mlc2a is particularly preferable.
[Screening method of the present invention]
The screening method of the present invention is performed using the fish described above. The fish of the present invention described above changes the wavelength of fluorescence emitted by a protein that is specifically expressed in cardiomyocytes at normal times by applying a stimulus. A portion of the heart tissue is lost by damaging the portion of the heart that contains proteins with altered fluorescence wavelengths. Even if the heart tissue is deficient in the fish of the present invention, it is regenerated after a certain period of time, so that the deficient part of the heart is regenerated together with the above-mentioned protein.
Since most of the regenerated heart tissue contains cardiomyocytes containing proteins in a non-stimulated state, the wavelength of fluorescence emitted differs from that of the heart tissue that is not damaged. Therefore, the fish heart is stimulated in advance, the fluorescence emitted by the protein expressed in the heart region is changed, and then a part of the heart is damaged, and it is a candidate as an active ingredient of a heart disease therapeutic drug. When a compound is administered, the degree of heart tissue to be regenerated after a certain period of time can be determined by regenerating the fluorescent region emitted by the protein before giving the stimulus. And if the area | region is quantified, it will become possible to show the effect of each candidate compound quantitatively.
The heart disease in the present invention is not particularly limited, but is preferably a disease which shows a symptom of lack of heart tissue and requires regeneration of the heart tissue itself for treatment. Specific examples include ischemic heart disease, cardiomyopathy, myocardial trauma and the like, and ischemic heart disease or cardiomyopathy is particularly preferable.
Specific examples of screening methods include
(1) Astep 1 for performing a treatment for changing the wavelength of fluorescence emitted from a protein specifically expressed in cardiomyocytes by stimulating the above-mentioned fish heart.
(2)Step 2 of performing a process of damaging the above-mentioned fish heart,
(3)Step 3 of administering a candidate compound to the fish described above,
(4) Step 4 after breeding for a certain period of time after the treatment ofSteps 1 to 3 above, and observing the above-mentioned damaged part of the fish with a fluorescence microscope, and (5) Microscopic image obtained by observation of Step 4 above And quantifying the regenerated heart tissue,
Can be mentioned.
(About step 1)
Step 1 in the screening method of the present invention is a step of performing a treatment for changing the wavelength of fluorescence emitted from the protein specifically expressed in cardiomyocytes by stimulating the above-mentioned fish heart.
The stimulus given instep 1 can be appropriately selected as long as it can change the wavelength of the fluorescence emitted by each of the above-mentioned proteins, and is not particularly limited, but is particularly stimulated by light such as ultraviolet rays. Is preferred. Also, the time for applying the stimulus can be appropriately set within a range in which the wavelength of fluorescence emitted from the above-mentioned protein can be changed without adversely affecting the survival of fish. For example, in the case of stimulation by light such as ultraviolet rays, it is usually sufficient to apply stimulation for about 10 to 120 seconds, preferably 30 to 60 seconds.
The above-mentioned stimulation can be given by an appropriate method depending on the kind of stimulation, and may be given to the fish individual from the outside, or may be given directly to the heart by performing thoracotomy after treatment such as anesthesia. For example, in the case of fish having a transparent epidermis, it is possible to give a stimulus such as light from the outside.
Moreover, you may perform theprocess 1 after the process 2 explained in full detail below. In that case, it is preferable to give the above-mentioned stimulus to the site where the heart is damaged in Step 2, and after the injury, the processing according to Step 1 can be continuously performed without breeding for a certain period of time. preferable.
(About step 2)
Step 2 in the screening method of the present invention is a step of performing a treatment for damaging the above-mentioned fish heart.
The method of damaging the fish heart instep 2 may be such that the heart tissue is lost and needs to be regenerated. Further, a method in which almost the same damage is given with good reproducibility for quantitative evaluation is preferable. Furthermore, the damage is a damage that can be regenerated without causing lethal damage to fish, and a method in which the degree of regeneration is easily observed is preferable. Although there is no particular limitation on the method of damaging to satisfy these conditions, data should be collected with such a high degree of reliability that it can show a statistically significant difference in the reproducibility of the screening system and the quantitative data obtained later. In view of the above, a method that does not require a particularly skilled technique is preferable.
For example, a process of puncturing a portion of the heart that has been discolored in the process ofstep 1 above using a needle having the same diameter can be mentioned. Further, the puncture site is preferably a site that does not adversely affect the life activity of the fish by the damage treatment, and for example, it is preferable to damage the distal portion of the ventricular ventral surface.
Step 2 may be performed after breeding for an appropriate period after Step 1 described above, or may be performed continuously after breeding after Step 1 described above. In addition, it is possible to separately obtain and use a protein in which the above-mentioned protein is processed by the same method as in the above-mentioned step 1 and the wavelength of the emitted fluorescence is already changed.
Further,step 2 may be performed after step 1 as described above, or may be performed after step 3 described in detail below.
(About step 3)
Step 3 according to the screening method of the present invention is a step of administering a candidate compound to the aforementioned fish.
The candidate compound used inStep 3 is not particularly limited, and examples thereof include nucleic acids, peptides, proteins, organic compounds, inorganic compounds, and the like, which can be prepared by known methods.
The method for administering the above-mentioned candidate compound is not particularly limited, but for example, a method of administering directly into the thoracic cavity, intraperitoneal cavity, etc. of a fish using a device such as a microtubule; Examples thereof include a method in which the above-described candidate compound is contained in food and the like, and is administered via an ella or digestive organ; an administration method by physical diffusion, or the like. When using the above-mentioned candidate compounds, taking into account economical points, a method of direct administration to the thoracic cavity, intraperitoneal cavity or the like is preferable.
Step 3 can be processed prior to step 1 or step 2 described above. In this case, after the process by the process 3, the process of the process 1 and the process 2 may be performed continuously, and you may carry out after the raising of a fixed period.
(About step 4)
Step 4 according to the screening method of the present invention is a step of performing the above-mentionedsteps 1 to 3 and rearing for a certain period of time, and observing the above-mentioned damaged sites of fish with a fluorescence microscope.
In Step 4, after the treatment inSteps 1 to 3, the fish is raised for a certain period of time, for example, the heart is removed by opening the thorax of the fish, the image obtained by using a fluorescence microscope is analyzed, and the heart of the above candidate compound is analyzed. Whether or not there is an effect related to reproduction can be determined.
The above-mentioned breeding for a certain period is an appropriate period within a range in which the presence or absence of the regenerative effect of the heart becomes clearer and highly reliable quantitative data having a significant difference is acquired, and usually 10 days. Or more, preferably about 14 to 30 days.
In addition, with respect to the above-described microscopic observation, it is also possible to perform only a thoracotomy process on fish after breeding for a certain period, and obtain an image under a fluorescence microscope without removing the heart. Furthermore, if the fish has a transparent epidermis, the presence or absence of a heart regeneration effect can be determined from an image obtained by a fluorescence microscope without performing a thoracotomy. When a fluorescence microscope image is obtained by a method that does not remove the heart, the cardiac regeneration effect of the candidate compound can be evaluated over time, which is a more preferable screening method.
As the fluorescence microscope used in Step 4, a fluorescence microscope usually used in the field of biological science can be used, as long as it can detect the fluorescence wavelength before and after the change emitted from the above-mentioned protein. Good.
(About step 5)
Step 5 according to the screening method of the present invention is a step of analyzing the micrograph image obtained by the observation in the above step 4 and quantifying the regenerated heart tissue.
Step 5 according to the screening method of the present invention is a step of analyzing the fluorescence microscope image obtained by the observation in the above step 4 and quantifying the regenerated heart tissue. Specifically, the number of cardiomyocytes regenerated after administration of the candidate compound can be quantified by counting from the fluorescence microscope image obtained in the above step 4. That is, it can be determined that the effect of the candidate compound is higher as the number of regenerated cardiomyocytes is larger. In addition, in order to count the cell number mentioned above, it can implement by using well-known image analysis software.
By using the method including the above-describedsteps 1 to 5, it becomes possible to screen for candidate compounds that can be active ingredients of novel drugs for heart disease. It is also possible to examine the usefulness of compounds already listed as candidate compounds, to examine the administration interval and period, and to examine the effects of combined use with other candidate drugs.
Hereinafter, the present invention will be described in more detail. However, it goes without saying that the present invention is not limited to the following examples.
〔本発明の魚類について〕
本発明の魚類は心臓の再生能を有する魚類であれば、特に限定されることは無いが、一般的に生物実験におけるモデル動物として扱い易い魚類が好ましく、例えば、ゼブラフィッシュ、メダカ、キンギョ等を挙げることができる。好ましくはゼブラフィッシュである。
本発明の蛋白質とは、刺激によって自らが発する光が変化するものである。刺激とは、魚類内部にて発現する蛋白質に対して、刺激効果を与えうるものであれば得に限定はされないが、例えば、光、熱、力学的、超音波、電気、磁気、化学物質による刺激などが挙げられ、魚類の心臓における従来の働きなど、生体そのものに影響を与えないことを勘案して、光による刺激が好ましい。特に、紫外光による刺激が好ましく、３５０~４１０ｎｍの波長を有する紫外光が最も好ましい。
本発明の蛋白質は、上記の刺激に応答して自らが発光する蛍光波長を変化させ得るものであれば特に限定はされない。具体的にはイシサンゴ（Ｔｒａｃｈｙｐｈｙｌｌｉａ ｇｅｏｆｆｒｏｙｉ）由来のＫａｅｄｅが好ましい。上記のＫａｅｄｅは、（ＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ ＮＯ．ＡＢ０８５６４１）に示す遺伝子配列によってコードされるアミノ酸を含む蛋白質である。
上述のＫａｅｄｅは、未処理の場合は、通常５１８ｎｍ付近の励起光を発し、ここで３５０~４１０ｎｍ程度の紫外光を照射することによって、５８２ｎｍ付近の励起光を発する。すなわち、紫外光という外部刺激を受けることによって、励起光の波長を変化させる蛋白質である。
上述の魚類に対して、上述の蛋白質を心筋細胞特異的に発現させる方法としては、公知のトランスジェニック動物の作製法に従えば、当業者であれば容易に作製することが可能である。具体的には、心筋細胞特異的にタンパク質を発現させるために、心筋細胞において特異的に働くプロモーター配列の下流に、上述のタンパク質をコードする遺伝子配列を繋いで作成した遺伝子カセットを、魚類個体の１−２細胞期の細胞に顕微注射することによって作成されるキメラ個体を育種し、安定的に前記の遺伝子カセットを発現する魚類個体を選抜することによって作製することができる。
上述の心筋細胞にて特異的に働くプロモーターの例としては、ｍｌｃ２ａ、ＣＡＲＰ（Ｃａｒｄｉａｃ ａｎｋｙｒｉｎ ｒｅｐｅａｔ ｐｒｏｔｅｉｎ）、ｃＴｎＴ（Ｃａｒｄｉａｃ ｔｒｏｐｏｎｉｎ Ｔ）等を挙げることができるが、特に、ｍｌｃ２ａが好ましい。
〔本発明のスクリーニング方法〕
本発明のスクリーニング方法は、上述した魚類を用いて行う。上述した本発明の魚類は、正常時に心筋細胞内にて特異的に発現している蛋白質が発する蛍光の波長を、刺激を与えることによって変化する。発する蛍光波長が変化した蛋白質を含む心臓の部位に損傷を与えることによって、心臓組織の一部分を欠損させる。本発明の魚類は、心臓組織が欠損したとしても、一定期間経過後には再生されるので、心臓の欠損部位は上述の蛋白質と共に再生される。
再生した心臓組織は、刺激を与えていない状態の蛋白質を含む心筋細胞が大部分に含まれるので、損傷を与えていない心臓組織の部位と比較して発する蛍光の波長が異なる。従って、上記魚類の心臓に予め刺激を与えて、心臓部位にて発現する蛋白質が発する蛍光を変化させておき、次に心臓の一部に損傷を与え、心疾患治療薬の有効成分となる候補化合物を投与すれば、一定期間経過後に再生する心臓組織の程度を、刺激を与える前の蛋白質が発する蛍光領域を再生して判断することができる。そしてその領域を定量化すれば、各候補化合物の効果を定量的に示すことが可能となる。
本発明における心疾患とは、特には限定されるものではないが、心臓組織が欠落する症状を示し、治療するにあたって心臓組織そのものの再生が求められる疾患が好ましい。具体的には、虚血性心疾患、心筋症、心筋外傷等を挙げることができるが、特に、虚血性心疾患又は心筋症が好ましい。
スクリーニング方法の具体例としては、
（１）上述の魚類の心臓に対して刺激を与え、心筋細胞にて特異的に発現している蛋白質が発する蛍光の波長を変化させる処理を行う工程１、
（２）上述の魚類の心臓に対して、損傷を与える処理を行う工程２、
（３）上述の魚類に対して、候補化合物を投与する工程３、
（４）上記工程１~３の処理を施した後、一定期間飼育し、上述の魚類の損傷部位を蛍光顕微鏡によって観察する工程４、及び
（５）上記工程４の観察によって得られる顕微鏡写真像を解析し、再生した心臓組織を定量化する工程５、
を含む方法を挙げることができる。
（工程１について）
本発明のスクリーニング方法における工程１とは、上述の魚類の心臓に対して刺激を与え、心筋細胞にて特異的に発現している蛋白質が発する蛍光の波長を変化させる処理を行う工程である。
工程１において与える刺激は、上述したそれぞれの蛋白質が発する蛍光の波長を変化させることができるものであれば、適宜選択することが可能であり、特には限定されないが、特に紫外線などの光による刺激が好ましい。また、刺激を与える時間についても、魚類の生存等に悪影響を与えず、上述の蛋白質が発する蛍光の波長を変化させ得る範囲において適宜設定することができる。例えば、紫外線などの光による刺激の場合、通常１０~１２０秒程度の刺激を与えればよく、好ましくは３０~６０秒である。
上記の刺激は、刺激の種類によってそれぞれ適当な方法によって与えることができ、魚類個体に対して外部から与えてもよく、麻酔等の処理の後に開胸して、直接心臓に与えても良い。例えば、透明な表皮を有する魚類の場合は、光等の刺激を外部から与えることが可能となる。
また工程１は、下記に詳述する工程２の後に行っても良い。その場合、工程２にて心臓に損傷を与えた部位に上述の刺激を与えることが好ましく、損傷を与えた後は一定期間の飼育をすることなく、連続的に工程１による処理を行うことが好ましい。
（工程２について）
本発明のスクリーニング方法における工程２とは、上述の魚類の心臓に対して、損傷を与える処理を行う工程である。
工程２において魚類の心臓に損傷を与える方法は、心臓の組織が欠損し、再生を要する状態にすればよい。また、定量的評価の為に再現性良くほぼ同一の損傷が与えられる方法が好ましい。さらに該損傷とは、魚類に対して致死的な損傷を与えることなく、再生可能な程度の損傷であり、再生の程度が容易に観察される方法が好ましい。これらの条件を満たすような損傷を与える方法については特には限定されないが、スクリーニング系の再現性、後に得られる定量データに統計学上の有意差を示せる程度に信頼度の高いデータを採取することに鑑みて、特段熟練した技術を要しない方法が好ましい。
例えば、同一の径を有する針を用いて上記工程１の処理にて変色させた心臓の部位を穿刺する処理を挙げることができる。また穿刺する部位は、損傷処理によって魚類の生命活動に悪影響を与えない部位が好ましく、例えば心室腹面遠位部に損傷を与えることが好ましい。
工程２は、上記の工程１の後に適当な期間飼育した後に行ってもよく、上記の工程１の後、飼育することなく連続的に行ってもよい。また上述の蛋白質が、上述の工程１と同様の方法によって処理され、既に発する蛍光の波長が変化しているものを別途入手して用いることも可能である。
さらに工程２は、上述のように工程１の後に行ってもよく、下記に詳述する工程３の後に行ってもよい。
（工程３について）
本発明のスクリーニング方法にかかる工程３とは、上述の魚類に対して、候補化合物を投与する工程である。
工程３において用いる候補化合物は、特に限定されることは無く、核酸、ペプチド、タンパク質、有機化合物、無機化合物等を挙げることができ、それぞれ公知の方法によって作製することが出来る。
上述の候補化合物の投与方法は、特には限定されないが、例えば微小細管等の器具を用いて、直接魚類の胸腔、腹腔内等に投与する方法；魚類を飼育する飼育槽、飼育する際に与える餌等に上述の候補化合物を含有させて、エラや消化器官を介して投与する方法；物理的な拡散による投与方法等を挙げることができる。上述の候補化合物を使用するにあたり、経済的な点を勘案すると、胸腔、腹腔内等に直接投与する方法が好ましい。
工程３は、上記工程１又は工程２に先だって処理することも可能である。この場合、工程３による処理の後、連続的に工程１及び工程２の処理を行ってもよく、一定期間の飼育の後に行ってもよい。
（工程４について）
本発明のスクリーニング方法にかかる工程４とは、上記工程１~３の処理を施した後、一定期間飼育し、上述の魚類の損傷部位を蛍光顕微鏡によって観察する工程である。
工程４では、上記工程１~３の処理後に一定期間飼育し、例えば、魚類を開胸して心臓を摘出し、蛍光顕微鏡を用いることによって得られる像を解析して、上述の候補化合物の心臓再生に係る効果の有無を判断することができる。
上述の一定期間の飼育とは、心臓の再生効果の有無がより明確となり、且つ有意差を持った信頼度の高い定量的なデータが取得される範囲において、適当な期間であり、通常１０日程度以上であり、好ましくは１４~３０日程度である。
また、上述の顕微鏡観察については、一定期間の飼育後の魚類に対して開胸処理のみを施し、心臓を摘出することなく蛍光顕微鏡下にて像を取得することも可能である。さらに、上記の魚類が透明な表皮を有する魚類であれば、開胸処理すら行うことなく、心臓再生効果の有無を蛍光顕微鏡による像から判断することもできる。心臓を摘出しない方法によって蛍光顕微鏡像を取得した場合は、経時的に候補化合物による心臓再生効果を評価することができ、さらに好ましいスクリーニング方法となる。
工程４において用いる蛍光顕微鏡は、通常は生物科学分野にて用いられる蛍光顕微鏡が利用可能であり、上述の蛋白質が発する変化前の蛍光波長と変化後の蛍光波長をそれぞれ検出し得るものであればよい。
（工程５について）
本発明のスクリーニング方法にかかる工程５は、上記工程４における観察によって得られる顕微鏡写真像を解析し、再生した心臓組織を定量化する工程である。
本発明のスクリーニング方法にかかる工程５は、上記工程４における観察によって得られる蛍光顕微鏡像を解析し、再生した心臓組織を定量化する工程である。具体的には候補化合物の投与後に再生した心筋細胞の数を、上記工程４によって得られた蛍光顕微鏡像から計数することによって、定量化することができる。すなわち、再生した心筋細胞の数が多いほど、候補化合物による効果が高いと判断することができる。なお、上述した細胞数を計数するためには、公知の画像解析ソフトを用いることによって実施できる。
上述の工程１~５を含む方法を用いることで、心疾患に対する新規薬剤の有効成分となり得る候補化合物をスクリーニングすることが可能となる。また、既に候補化合物として挙げられている化合物の、有用性の検討、投与間隔・期間の検討、他の候補薬剤との併用による効果の検討等を行うことも可能である。
以下に本発明をより詳細に説明する。但し、本発明が以下に示す実施例に限定されないのは言うまでも無い。 Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly shows the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, F .; M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, N .; Y, 2007, and the like.
[Fish of the present invention]
The fish of the present invention is not particularly limited as long as it has the ability to regenerate the heart, but is generally preferably a fish that is easy to handle as a model animal in biological experiments, such as zebrafish, medaka, goldfish, etc. Can be mentioned. Zebrafish is preferred.
The protein of the present invention is a protein in which light emitted by itself is changed by stimulation. Stimulation is not particularly limited as long as it can give a stimulating effect to proteins expressed in fish, but for example, by light, heat, mechanical, ultrasonic, electrical, magnetic, chemical substances Stimulation by light is preferable in consideration of the fact that it does not affect the living body itself, such as the conventional action in the heart of fish. In particular, stimulation with ultraviolet light is preferable, and ultraviolet light having a wavelength of 350 to 410 nm is most preferable.
The protein of the present invention is not particularly limited as long as it can change the fluorescence wavelength emitted by itself in response to the above stimulus. Specifically, Kaede derived from the sea coral (Trachphyllia geoffroyi) is preferable. Said Kaede is a protein containing the amino acid encoded by the gene sequence shown to (GenBank Accession NO.AB085641).
When the above-mentioned Kaede is not processed, it usually emits excitation light around 518 nm, and emits excitation light around 582 nm by irradiating it with ultraviolet light of about 350 to 410 nm. That is, it is a protein that changes the wavelength of excitation light by receiving an external stimulus such as ultraviolet light.
A person skilled in the art can easily prepare the above-described fish as a method for expressing the above-mentioned protein specifically in cardiomyocytes according to a known method for producing a transgenic animal. Specifically, in order to express a protein specifically in cardiomyocytes, a gene cassette prepared by linking a gene sequence encoding the above-described protein downstream of a promoter sequence that specifically works in cardiomyocytes, It can be produced by breeding a chimeric individual produced by microinjection into cells at the 1-2 cell stage and selecting fish individuals that stably express the gene cassette.
Examples of the promoter that specifically works in the above-mentioned cardiomyocytes include mlc2a, CARP (Cardiac ankyrin repeat protein), cTnT (Cardiac troponin T), etc., and mlc2a is particularly preferable.
[Screening method of the present invention]
The screening method of the present invention is performed using the fish described above. The fish of the present invention described above changes the wavelength of fluorescence emitted by a protein that is specifically expressed in cardiomyocytes at normal times by applying a stimulus. A portion of the heart tissue is lost by damaging the portion of the heart that contains proteins with altered fluorescence wavelengths. Even if the heart tissue is deficient in the fish of the present invention, it is regenerated after a certain period of time, so that the deficient part of the heart is regenerated together with the above-mentioned protein.
Since most of the regenerated heart tissue contains cardiomyocytes containing proteins in a non-stimulated state, the wavelength of fluorescence emitted differs from that of the heart tissue that is not damaged. Therefore, the fish heart is stimulated in advance, the fluorescence emitted by the protein expressed in the heart region is changed, and then a part of the heart is damaged, and it is a candidate as an active ingredient of a heart disease therapeutic drug. When a compound is administered, the degree of heart tissue to be regenerated after a certain period of time can be determined by regenerating the fluorescent region emitted by the protein before giving the stimulus. And if the area | region is quantified, it will become possible to show the effect of each candidate compound quantitatively.
The heart disease in the present invention is not particularly limited, but is preferably a disease which shows a symptom of lack of heart tissue and requires regeneration of the heart tissue itself for treatment. Specific examples include ischemic heart disease, cardiomyopathy, myocardial trauma and the like, and ischemic heart disease or cardiomyopathy is particularly preferable.
Specific examples of screening methods include
(1) A
(2)
(3)
(4) Step 4 after breeding for a certain period of time after the treatment of
Can be mentioned.
(About step 1)
The stimulus given in
The above-mentioned stimulation can be given by an appropriate method depending on the kind of stimulation, and may be given to the fish individual from the outside, or may be given directly to the heart by performing thoracotomy after treatment such as anesthesia. For example, in the case of fish having a transparent epidermis, it is possible to give a stimulus such as light from the outside.
Moreover, you may perform the
(About step 2)
The method of damaging the fish heart in
For example, a process of puncturing a portion of the heart that has been discolored in the process of
Further,
(About step 3)
The candidate compound used in
The method for administering the above-mentioned candidate compound is not particularly limited, but for example, a method of administering directly into the thoracic cavity, intraperitoneal cavity, etc. of a fish using a device such as a microtubule; Examples thereof include a method in which the above-described candidate compound is contained in food and the like, and is administered via an ella or digestive organ; an administration method by physical diffusion, or the like. When using the above-mentioned candidate compounds, taking into account economical points, a method of direct administration to the thoracic cavity, intraperitoneal cavity or the like is preferable.
(About step 4)
Step 4 according to the screening method of the present invention is a step of performing the above-mentioned
In Step 4, after the treatment in
The above-mentioned breeding for a certain period is an appropriate period within a range in which the presence or absence of the regenerative effect of the heart becomes clearer and highly reliable quantitative data having a significant difference is acquired, and usually 10 days. Or more, preferably about 14 to 30 days.
In addition, with respect to the above-described microscopic observation, it is also possible to perform only a thoracotomy process on fish after breeding for a certain period, and obtain an image under a fluorescence microscope without removing the heart. Furthermore, if the fish has a transparent epidermis, the presence or absence of a heart regeneration effect can be determined from an image obtained by a fluorescence microscope without performing a thoracotomy. When a fluorescence microscope image is obtained by a method that does not remove the heart, the cardiac regeneration effect of the candidate compound can be evaluated over time, which is a more preferable screening method.
As the fluorescence microscope used in Step 4, a fluorescence microscope usually used in the field of biological science can be used, as long as it can detect the fluorescence wavelength before and after the change emitted from the above-mentioned protein. Good.
(About step 5)
By using the method including the above-described
Hereinafter, the present invention will be described in more detail. However, it goes without saying that the present invention is not limited to the following examples.
トランスジェニックゼブラフィッシュを用いた心筋細胞の再生
ゼブラフィッシュｍｌｃ２ａ蛋白質の上流領域１．６ｋｂ（配列番号１：非特許文献３）の下流に、紫外線照射で変色する蛍光分子Ｋａｅｄｅ（ＭＢＬ，日本）をコードする遺伝子を配置した遺伝子カセット（図１）を、ゼブラフィッシュ１−２細胞期の細胞に顕微注射した。その後、顕微注射したこのキメラゼブラフィッシュ個体から、ゲノムＤＮＡに外来遺伝子を安定的に取り込んだ子孫を単離した。これを、心臓再生を可視化できるｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュとした。このトランスジェニックゼブラフィッシュ個体は心筋細胞のみが緑色の蛍光を呈し、図２に示すように、幼魚期には外部から緑色蛍光を観察できる。
このｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの幼魚期の心臓に、紫外光（３３０−３８５ｎｍ）を照射すると、図３に示すように心筋細胞が緑から赤色に変色する。緑色と赤色の蛍光は適当なフィルターを使うことによりそれぞれの蛍光のみを抽出可能であると共に、いわゆるロングパスフィルターを使うことで例えば緑色の蛍光と橙色の蛍光として同時に観察することも可能である。
また、図４に示すようにｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュは成魚においても麻酔後に開胸した後に紫外線を照射することで、照射面を再現性良く緑から赤色に変色させることが可能であった。従って、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの成魚を用いて心筋再生の定量化実験ならびに、薬剤が及ぼす影響の評価を行なった。０．３２ｍｇ／ｍｌのＴｒｉｃａｉｎｅ（Ｓｉｇｍａ，ＵＳＡ）を用いてｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの成魚を麻酔後、腹部を上にしてスリットを入れたスポンジ内で固定し、ピンセットを用いて開胸した。落射蛍光顕微鏡（ＢＸ６０−ＢＨ２−ＲＦＬ−Ｔ：オリンパス，日本）を用いて心室腹側面の緑色蛍光を発する心筋細胞を赤色蛍光に変色後、蛍光実体顕微鏡下で０．２ｍｍ径のタングステン針（ニラコ，日本）で変色部分に穿刺により損傷を加えた。この方法により再現性良く同一の損傷を加えることができた。
損傷付与から７日後、１４日後、３０日後のｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ個体をそれぞれ解剖し、心臓を摘出して蛍光顕微鏡（ＢＩＯＲＥＶＯ ＢＺ−９０００：キーエンス，日本）により蛍光像を観察した。結果を図５に示す。損傷付与から７日目までは緑色蛍光のみを有する細胞は損傷部位に認められないが、損傷付与から１４、３０日目では緑色蛍光のみを有する細胞群が損傷部位において明瞭に認められた。これにより、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ個体を用いることによって、心筋再生を可視化することが可能となった。
図６（Ａ）に示すように、再生心筋の緑色蛍光像および赤色蛍光像をそれぞれバンドパスフィルターで別々に撮影し、ＢＺ ａｎａｌｙｚｅｒ ＩＩ（キーエンス日本）を用いて緑色蛍光像を白黒像とし、赤色蛍光像を白黒像にした後明暗を反転させ、両画像を重ね合わせることにより損傷領域において緑色蛍光を有意に強くを有する細胞を明瞭な白色像として判別することが出来る。この方法を用いて、図６（Ｂ）に示すように、再生した心筋細胞数を再現性良く計測することが可能となった。
トランスジェニックゼブラフィッシュを用いた心臓再生薬剤のスクリーニング
＜ＦＧＦについて＞
ゼブラフィッシュの心臓の再生プロセスにおいて必須の役割を担っているＦＧＦＲの阻害剤を用いることで、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓再生効果にどのような影響を与えるか検討した。上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓を赤色蛍光に変色させた後、タングステン針による損傷付与から７日目から１４日目の間、１７μＭのＦＧＦＲ阻害剤（ＳＵ５４０２，Ｃａｌｂｉｏｃｈｅｍ，米国）を存在下および非存在下でゼブラフィッシュ個体を飼育した。損傷後１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図７（Ａ）に示す。
ＦＧＦＲ阻害剤で処理した個体において、損傷領域における緑色蛍光のみを有する細胞群は、対照個体における緑色蛍光のみを有する細胞群に比べて有意に少なく、それぞれ３個体ずつを処理して認められた緑色蛍光のみを有する細胞数は平均で６個（ＦＧＦＲ阻害剤処理）と２１個（対照）であり、心筋再生に及ぼす薬剤の阻害効果を定量的に評価することが可能となった。
また、哺乳動物である心筋梗塞モデルラットで心筋再生課程を促進し得ることが示されているＦＧＦ（特にｂＦＧＦあるいはＦＧＦ−１）が、トランスジェニックゼブラフィッシュを用いることで心筋再生を定量的に解析できるかどうかについて検討した。
上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ心臓を赤色蛍光に変色させ、引き続いてタングステン針で損傷を与え、２日後及び４日後に３０μＭのｂＦＧＦ（Ｉｎｖｉｔｒｏｇｅｎ：米国）１μｌをゼブラフィッシュ胸腔内に先端を尖らせた微小ガラス管（ナリシゲ，日本）を用いて注入した。１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図８（Ａ）に示す。
同一の損傷を加えたにも関わらず、ｂＦＧＦを加えた個体においては対照の個体に比べて緑色蛍光のみを有する再生心筋の有意な増加が認められた。顕微鏡観察像から、再生した心筋細胞の数を解析した結果、図８（Ｂ）に示すように、対照群の平均２２個に対してｂＦＧＦ投与群の平均は３４個であることが判明した。従って、トランスジェニックゼブラフィッシュは、心疾患に対する薬剤の有効成分となる候補化合物を、再現性良く、さらに簡便な方法によってスクリーニングすることが可能となる。また、検定することも可能である。
＜Ｗｎｔについて＞
ゼブラフィッシュのヒレの再生プロセスにおいて必須の役割を担っているＷｎｔシグナルの阻害剤を用いることで、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓再生効果にどのような影響を与えるか検討した。
なお、魚類の心筋細胞において、Ｗｎｔシグナルが心筋再生にどのような役割を果たすかについて、何ら情報は無い。上述の方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓を赤色蛍光に変色させた後、タングステン針による損傷付与から７日目から１４日目の間、５μＭのＷｎｔシグナル阻害剤（ＸＡＶ９３９，Ｃａｙｍａｎ Ｃｈｅｍｉｃａｌ，米国）を存在下、と非存在下でゼブラフィッシュ個体を飼育した。２日おきに飼育水を交換し、新たに５μＭのＷｎｔシグナル阻害剤を含む飼育水を用いた。損傷付与から１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図９（Ａ）に示す。
Ｗｎｔシグナル阻害剤で処理した個体において、損傷領域における緑色蛍光のみを有する細胞群は、対照個体における緑色蛍光のみを有する細胞群に比べて有意に少なく、それぞれ３個体ずつを処理して認められた緑色蛍光のみを有する細胞数は、図９（Ｂ）に示すように平均で３個（Ｗｎｔシグナル阻害剤処理）と２１個（対照）であり、Ｗｎｔシグナルを抑制することで心筋再生を顕著に抑制することが明らかとなった。また、心臓細胞の再生に及ぼす薬剤の阻害効果を定量的に評価することが可能となった。
上記の結果から、Ｗｎｔシグナルにおいて促進剤として働くＧＳＫ−３阻害剤による心筋再生効果を定量的に解析できるかどうかについて検討した。上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ心臓を赤色蛍光に変色させ、引き続いてタングステン針で損傷を与え、１日後及び８日後に２．５ｎＭのＧＳＫ−３阻害剤（ＢＩＯ，Ｃａｌｂｉｏｃｈｅｍ，米国）の存在下又は非存在下の水相にて飼育を行った。損傷付与から１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図９（Ａ）に示す。
同一の損傷を与えたにも関わらず、Ｗｎｔシグナルの促進剤として働くＧＳＫ−３阻害剤を加えた個体においては、対照の個体に比べて緑色蛍光のみを有する再生心筋の有意な増加が認められた。顕微鏡観察像から、再生した心筋細胞の数を解析した結果、図９（Ｂ）に示すように。対照群の平均２１個に対し、ＧＳＫ−３阻害剤の投与群の平均は３３個であることが判明した。従って、トランスジェニックゼブラフィッシュは、心疾患に対する薬剤の有効成分となる候補化合物を、再現性良く、さらに簡便な方法によってスクリーニングすることが可能となる。また、検定することも可能である。
上記の実施例の結果から、本発明の方法を用いることにより、心疾患に対する薬剤の有効成分となる候補化合物のスクリーニングが可能となる。また、既に候補化合物として挙がる化合物が、心疾患に対して有効な効果を奏するかどうかの確認、化合物の至適濃度の検討、投与間隔・期間の検討、他の候補化合物と併用可能性の検討等に用いることも可能となる。
また上記実施例において、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュを用いることによって、ＦＧＦ−１もＧＳＫ−３阻害剤も心筋再生効果を導き出すことが確認された。ＦＧＦ−１はＦＧＦシグナルカスケード、ＧＳＫ−３はＷｎｔカスケードと、それぞれ心筋細胞においては異なるシグナルカスケードに関与する分子であると考えられるにもかかわらず、本発明のｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュを用いることによって、心疾患に対して有効な成分であるとの結果が得られた。従って、本発明のスクリーニング方法は、心疾患に対する薬剤の有効成分となる候補化合物の探索に好適である。
Regeneration of cardiomyocytes using transgenic zebrafish Coded fluorescent molecule Kaede (MBL, Japan) that changes color by ultraviolet irradiation downstream of the 1.6 kb upstream region of the zebrafish mlc2a protein (SEQ ID NO: 1). The gene cassette (FIG. 1) in which the gene to be placed was placed was microinjected into cells at the zebrafish 1-2 cell stage. Thereafter, offspring that stably incorporated the foreign gene into the genomic DNA were isolated from this chimeric zebrafish individual injected microscopically. This was mlc2a-kaede transgenic zebrafish that can visualize heart regeneration. In this transgenic zebrafish individual, only cardiomyocytes exhibit green fluorescence, and as shown in FIG. 2, green fluorescence can be observed from the outside in the juvenile stage.
When the larvae heart of the mlc2a-kaede transgenic zebrafish is irradiated with ultraviolet light (330-385 nm), the cardiomyocytes turn from green to red as shown in FIG. For the green and red fluorescence, only the respective fluorescence can be extracted by using an appropriate filter, and by using a so-called long-pass filter, it is possible to simultaneously observe, for example, green fluorescence and orange fluorescence.
In addition, as shown in FIG. 4, mlc2a-kaede transgenic zebrafish was able to change the irradiation surface from green to red with good reproducibility by irradiating ultraviolet rays after thoracotomy after anesthesia even in adult fish. . Therefore, quantification experiment of myocardial regeneration and evaluation of the effect of drugs were performed using adult fish of mlc2a-kaede transgenic zebrafish. Anesthetized adult fish of mlc2a-kaede transgenic zebrafish using 0.32 mg / ml Tricaine (Sigma, USA), fixed in a sponge with slits with the abdomen facing up, and thoracotomy was performed using tweezers . Using epi-illumination fluorescence microscope (BX60-BH2-RFL-T: Olympus, Japan) after changing the color of myocardial cells emitting green fluorescence on ventricular ventricle to red fluorescence, 0.2mm diameter tungsten needle (Niraco) under fluorescent stereomicroscope In Japan), the discolored part was damaged by puncture. By this method, the same damage could be added with good reproducibility.
7 days, 14 days, and 30 days after the damage was imparted, each of the mlc2a-kaede transgenic zebrafish individuals was dissected, the heart was removed, and the fluorescence image was observed with a fluorescence microscope (BIOREVO BZ-9000: Keyence, Japan). The results are shown in FIG. Cells having only green fluorescence were not observed at the damaged site from the 7th day after the damage was applied, but a group of cells having only green fluorescence was clearly observed at the damaged site on the 14th and 30th days after the damage was given. Thereby, it became possible to visualize myocardial regeneration by using mlc2a-kaede transgenic zebrafish individuals.
As shown in FIG. 6 (A), a green fluorescence image and a red fluorescence image of the regenerated myocardium were separately photographed with a bandpass filter, and the green fluorescence image was converted into a black and white image using BZ analyzer II (Keyence Japan). After the fluorescence image is converted into a black and white image, the brightness and darkness are reversed, and by superimposing both images, it is possible to discriminate cells having significantly strong green fluorescence in the damaged area as a clear white image. Using this method, as shown in FIG. 6B, the number of regenerated cardiomyocytes can be measured with good reproducibility.
Screening of cardiac regenerative drugs using transgenic zebrafish
<About FGF>
The influence of the FGFR inhibitor, which plays an essential role in the zebrafish heart regeneration process, on the heart regeneration effect of mlc2a-kaede transgenic zebrafish was examined. After the heart of mlc2a-kaede transgenic zebrafish was changed to red fluorescence by the above method, 17 μM FGFR inhibitor (SU5402, Calbiochem, USA) was added during the 7th to 14th days after the injury by the tungsten needle. Zebrafish individuals were bred in the presence and absence. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In the individual treated with the FGFR inhibitor, the number of cells having only green fluorescence in the damaged area was significantly smaller than the group of cells having only green fluorescence in the control individual. The average number of cells having only fluorescence was 6 (FGFR inhibitor treatment) and 21 (control), and the inhibitory effect of the drug on myocardial regeneration could be quantitatively evaluated.
In addition, FGF (especially bFGF or FGF-1), which has been shown to be able to accelerate the myocardial regeneration process in mammalian myocardial infarction model rats, quantitatively analyzes myocardial regeneration using transgenic zebrafish. We examined whether it was possible.
Using the above method, the mlc2a-kaede transgenic zebrafish heart was changed to red fluorescence and subsequently damaged with a tungsten needle. After 2 and 4 days, 1 μl of 30 μM bFGF (Invitrogen: USA) was placed in the zebrafish chest cavity. Injection was performed using a sharpened micro glass tube (Narishige, Japan). After 14 days, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
Despite the same damage, a significant increase in regenerated myocardium with only green fluorescence was observed in the individuals with bFGF compared to the control individuals. As a result of analyzing the number of regenerated cardiomyocytes from a microscopic observation image, as shown in FIG. 8B, it was found that the average of the bFGF administration group was 34 compared to the average of 22 of the control group. Therefore, transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test.
<About Wnt>
The influence of the heart regeneration effect of mlc2a-kaede transgenic zebrafish was examined by using an inhibitor of Wnt signal, which plays an essential role in the zebrafish fin regeneration process.
In fish cardiomyocytes, there is no information about what role Wnt signals play in myocardial regeneration. After the heart of mlc2a-kaede transgenic zebrafish was changed to red fluorescence by the above-described method, 5 μM Wnt signal inhibitor (XAV939, Cayman Chemical, Zebrafish individuals were bred in the presence and absence of the United States). The breeding water was changed every two days, and the breeding water newly containing 5 μM Wnt signal inhibitor was used. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In the individuals treated with the Wnt signal inhibitor, the number of cells having only green fluorescence in the damaged area was significantly smaller than the number of cells having only green fluorescence in the control individuals. The number of cells having only green fluorescence is 3 (Wnt signal inhibitor treatment) and 21 (control) on average as shown in FIG. 9 (B), and the myocardial regeneration is remarkably suppressed by suppressing the Wnt signal. It became clear to suppress. It has also become possible to quantitatively evaluate the inhibitory effect of drugs on the regeneration of heart cells.
From the above results, it was examined whether or not the myocardial regeneration effect by the GSK-3 inhibitor acting as a promoter in the Wnt signal can be quantitatively analyzed. The above method caused the mlc2a-kaede transgenic zebrafish heart to turn red fluorescent, and subsequently damaged with a tungsten needle, after 1 and 8 days of 2.5 nM GSK-3 inhibitor (BIO, Calbiochem, USA) Breeding was carried out in the presence or absence of an aqueous phase. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In spite of the same damage, a significant increase in regenerative myocardium with only green fluorescence was observed in the individuals to which GSK-3 inhibitor acting as a promoter of Wnt signal was added, compared to the control individuals. It was. As a result of analyzing the number of regenerated cardiomyocytes from a microscopic observation image, as shown in FIG. It was found that the average of the GSK-3 inhibitor administration group was 33 compared to the average of 21 in the control group. Therefore, transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test.
From the results of the above examples, by using the method of the present invention, it becomes possible to screen for candidate compounds that are active ingredients of drugs for heart disease. Also, confirm whether compounds already listed as candidate compounds have an effective effect on heart disease, study the optimal concentration of the compound, study the administration interval and duration, and examine the possibility of combination with other candidate compounds It is also possible to use for such as.
Moreover, in the said Example, it was confirmed by using mlc2a-kaede transgenic zebrafish that both FGF-1 and a GSK-3 inhibitor lead to a myocardial regeneration effect. The mlc2a-kaede transgenic zebrafish of the present invention is used even though FGF-1 is considered to be a molecule involved in the FGF signal cascade and GSK-3 is a Wnt cascade and a signal cascade different in cardiomyocytes. As a result, a result that it is an effective ingredient for heart disease was obtained. Therefore, the screening method of the present invention is suitable for searching for candidate compounds that are active ingredients of drugs for heart disease.
ゼブラフィッシュｍｌｃ２ａ蛋白質の上流領域１．６ｋｂ（配列番号１：非特許文献３）の下流に、紫外線照射で変色する蛍光分子Ｋａｅｄｅ（ＭＢＬ，日本）をコードする遺伝子を配置した遺伝子カセット（図１）を、ゼブラフィッシュ１−２細胞期の細胞に顕微注射した。その後、顕微注射したこのキメラゼブラフィッシュ個体から、ゲノムＤＮＡに外来遺伝子を安定的に取り込んだ子孫を単離した。これを、心臓再生を可視化できるｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュとした。このトランスジェニックゼブラフィッシュ個体は心筋細胞のみが緑色の蛍光を呈し、図２に示すように、幼魚期には外部から緑色蛍光を観察できる。
このｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの幼魚期の心臓に、紫外光（３３０−３８５ｎｍ）を照射すると、図３に示すように心筋細胞が緑から赤色に変色する。緑色と赤色の蛍光は適当なフィルターを使うことによりそれぞれの蛍光のみを抽出可能であると共に、いわゆるロングパスフィルターを使うことで例えば緑色の蛍光と橙色の蛍光として同時に観察することも可能である。
また、図４に示すようにｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュは成魚においても麻酔後に開胸した後に紫外線を照射することで、照射面を再現性良く緑から赤色に変色させることが可能であった。従って、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの成魚を用いて心筋再生の定量化実験ならびに、薬剤が及ぼす影響の評価を行なった。０．３２ｍｇ／ｍｌのＴｒｉｃａｉｎｅ（Ｓｉｇｍａ，ＵＳＡ）を用いてｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの成魚を麻酔後、腹部を上にしてスリットを入れたスポンジ内で固定し、ピンセットを用いて開胸した。落射蛍光顕微鏡（ＢＸ６０−ＢＨ２−ＲＦＬ−Ｔ：オリンパス，日本）を用いて心室腹側面の緑色蛍光を発する心筋細胞を赤色蛍光に変色後、蛍光実体顕微鏡下で０．２ｍｍ径のタングステン針（ニラコ，日本）で変色部分に穿刺により損傷を加えた。この方法により再現性良く同一の損傷を加えることができた。
損傷付与から７日後、１４日後、３０日後のｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ個体をそれぞれ解剖し、心臓を摘出して蛍光顕微鏡（ＢＩＯＲＥＶＯ ＢＺ−９０００：キーエンス，日本）により蛍光像を観察した。結果を図５に示す。損傷付与から７日目までは緑色蛍光のみを有する細胞は損傷部位に認められないが、損傷付与から１４、３０日目では緑色蛍光のみを有する細胞群が損傷部位において明瞭に認められた。これにより、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ個体を用いることによって、心筋再生を可視化することが可能となった。
図６（Ａ）に示すように、再生心筋の緑色蛍光像および赤色蛍光像をそれぞれバンドパスフィルターで別々に撮影し、ＢＺ ａｎａｌｙｚｅｒ ＩＩ（キーエンス日本）を用いて緑色蛍光像を白黒像とし、赤色蛍光像を白黒像にした後明暗を反転させ、両画像を重ね合わせることにより損傷領域において緑色蛍光を有意に強くを有する細胞を明瞭な白色像として判別することが出来る。この方法を用いて、図６（Ｂ）に示すように、再生した心筋細胞数を再現性良く計測することが可能となった。
トランスジェニックゼブラフィッシュを用いた心臓再生薬剤のスクリーニング
＜ＦＧＦについて＞
ゼブラフィッシュの心臓の再生プロセスにおいて必須の役割を担っているＦＧＦＲの阻害剤を用いることで、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓再生効果にどのような影響を与えるか検討した。上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓を赤色蛍光に変色させた後、タングステン針による損傷付与から７日目から１４日目の間、１７μＭのＦＧＦＲ阻害剤（ＳＵ５４０２，Ｃａｌｂｉｏｃｈｅｍ，米国）を存在下および非存在下でゼブラフィッシュ個体を飼育した。損傷後１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図７（Ａ）に示す。
ＦＧＦＲ阻害剤で処理した個体において、損傷領域における緑色蛍光のみを有する細胞群は、対照個体における緑色蛍光のみを有する細胞群に比べて有意に少なく、それぞれ３個体ずつを処理して認められた緑色蛍光のみを有する細胞数は平均で６個（ＦＧＦＲ阻害剤処理）と２１個（対照）であり、心筋再生に及ぼす薬剤の阻害効果を定量的に評価することが可能となった。
また、哺乳動物である心筋梗塞モデルラットで心筋再生課程を促進し得ることが示されているＦＧＦ（特にｂＦＧＦあるいはＦＧＦ−１）が、トランスジェニックゼブラフィッシュを用いることで心筋再生を定量的に解析できるかどうかについて検討した。
上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ心臓を赤色蛍光に変色させ、引き続いてタングステン針で損傷を与え、２日後及び４日後に３０μＭのｂＦＧＦ（Ｉｎｖｉｔｒｏｇｅｎ：米国）１μｌをゼブラフィッシュ胸腔内に先端を尖らせた微小ガラス管（ナリシゲ，日本）を用いて注入した。１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図８（Ａ）に示す。
同一の損傷を加えたにも関わらず、ｂＦＧＦを加えた個体においては対照の個体に比べて緑色蛍光のみを有する再生心筋の有意な増加が認められた。顕微鏡観察像から、再生した心筋細胞の数を解析した結果、図８（Ｂ）に示すように、対照群の平均２２個に対してｂＦＧＦ投与群の平均は３４個であることが判明した。従って、トランスジェニックゼブラフィッシュは、心疾患に対する薬剤の有効成分となる候補化合物を、再現性良く、さらに簡便な方法によってスクリーニングすることが可能となる。また、検定することも可能である。
＜Ｗｎｔについて＞
ゼブラフィッシュのヒレの再生プロセスにおいて必須の役割を担っているＷｎｔシグナルの阻害剤を用いることで、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓再生効果にどのような影響を与えるか検討した。
なお、魚類の心筋細胞において、Ｗｎｔシグナルが心筋再生にどのような役割を果たすかについて、何ら情報は無い。上述の方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュの心臓を赤色蛍光に変色させた後、タングステン針による損傷付与から７日目から１４日目の間、５μＭのＷｎｔシグナル阻害剤（ＸＡＶ９３９，Ｃａｙｍａｎ Ｃｈｅｍｉｃａｌ，米国）を存在下、と非存在下でゼブラフィッシュ個体を飼育した。２日おきに飼育水を交換し、新たに５μＭのＷｎｔシグナル阻害剤を含む飼育水を用いた。損傷付与から１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図９（Ａ）に示す。
Ｗｎｔシグナル阻害剤で処理した個体において、損傷領域における緑色蛍光のみを有する細胞群は、対照個体における緑色蛍光のみを有する細胞群に比べて有意に少なく、それぞれ３個体ずつを処理して認められた緑色蛍光のみを有する細胞数は、図９（Ｂ）に示すように平均で３個（Ｗｎｔシグナル阻害剤処理）と２１個（対照）であり、Ｗｎｔシグナルを抑制することで心筋再生を顕著に抑制することが明らかとなった。また、心臓細胞の再生に及ぼす薬剤の阻害効果を定量的に評価することが可能となった。
上記の結果から、Ｗｎｔシグナルにおいて促進剤として働くＧＳＫ−３阻害剤による心筋再生効果を定量的に解析できるかどうかについて検討した。上記方法によりｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュ心臓を赤色蛍光に変色させ、引き続いてタングステン針で損傷を与え、１日後及び８日後に２．５ｎＭのＧＳＫ−３阻害剤（ＢＩＯ，Ｃａｌｂｉｏｃｈｅｍ，米国）の存在下又は非存在下の水相にて飼育を行った。損傷付与から１４日後、ゼブラフィッシュ個体より心臓を切除、回収し、蛍光顕微鏡下で再生心筋細胞の観察を行なった。結果を図９（Ａ）に示す。
同一の損傷を与えたにも関わらず、Ｗｎｔシグナルの促進剤として働くＧＳＫ−３阻害剤を加えた個体においては、対照の個体に比べて緑色蛍光のみを有する再生心筋の有意な増加が認められた。顕微鏡観察像から、再生した心筋細胞の数を解析した結果、図９（Ｂ）に示すように。対照群の平均２１個に対し、ＧＳＫ−３阻害剤の投与群の平均は３３個であることが判明した。従って、トランスジェニックゼブラフィッシュは、心疾患に対する薬剤の有効成分となる候補化合物を、再現性良く、さらに簡便な方法によってスクリーニングすることが可能となる。また、検定することも可能である。
上記の実施例の結果から、本発明の方法を用いることにより、心疾患に対する薬剤の有効成分となる候補化合物のスクリーニングが可能となる。また、既に候補化合物として挙がる化合物が、心疾患に対して有効な効果を奏するかどうかの確認、化合物の至適濃度の検討、投与間隔・期間の検討、他の候補化合物と併用可能性の検討等に用いることも可能となる。
また上記実施例において、ｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュを用いることによって、ＦＧＦ−１もＧＳＫ−３阻害剤も心筋再生効果を導き出すことが確認された。ＦＧＦ−１はＦＧＦシグナルカスケード、ＧＳＫ−３はＷｎｔカスケードと、それぞれ心筋細胞においては異なるシグナルカスケードに関与する分子であると考えられるにもかかわらず、本発明のｍｌｃ２ａ−ｋａｅｄｅトランスジェニックゼブラフィッシュを用いることによって、心疾患に対して有効な成分であるとの結果が得られた。従って、本発明のスクリーニング方法は、心疾患に対する薬剤の有効成分となる候補化合物の探索に好適である。
Regeneration of cardiomyocytes using transgenic zebrafish Coded fluorescent molecule Kaede (MBL, Japan) that changes color by ultraviolet irradiation downstream of the 1.6 kb upstream region of the zebrafish mlc2a protein (SEQ ID NO: 1). The gene cassette (FIG. 1) in which the gene to be placed was placed was microinjected into cells at the zebrafish 1-2 cell stage. Thereafter, offspring that stably incorporated the foreign gene into the genomic DNA were isolated from this chimeric zebrafish individual injected microscopically. This was mlc2a-kaede transgenic zebrafish that can visualize heart regeneration. In this transgenic zebrafish individual, only cardiomyocytes exhibit green fluorescence, and as shown in FIG. 2, green fluorescence can be observed from the outside in the juvenile stage.
When the larvae heart of the mlc2a-kaede transgenic zebrafish is irradiated with ultraviolet light (330-385 nm), the cardiomyocytes turn from green to red as shown in FIG. For the green and red fluorescence, only the respective fluorescence can be extracted by using an appropriate filter, and by using a so-called long-pass filter, it is possible to simultaneously observe, for example, green fluorescence and orange fluorescence.
In addition, as shown in FIG. 4, mlc2a-kaede transgenic zebrafish was able to change the irradiation surface from green to red with good reproducibility by irradiating ultraviolet rays after thoracotomy after anesthesia even in adult fish. . Therefore, quantification experiment of myocardial regeneration and evaluation of the effect of drugs were performed using adult fish of mlc2a-kaede transgenic zebrafish. Anesthetized adult fish of mlc2a-kaede transgenic zebrafish using 0.32 mg / ml Tricaine (Sigma, USA), fixed in a sponge with slits with the abdomen facing up, and thoracotomy was performed using tweezers . Using epi-illumination fluorescence microscope (BX60-BH2-RFL-T: Olympus, Japan) after changing the color of myocardial cells emitting green fluorescence on ventricular ventricle to red fluorescence, 0.2mm diameter tungsten needle (Niraco) under fluorescent stereomicroscope In Japan), the discolored part was damaged by puncture. By this method, the same damage could be added with good reproducibility.
7 days, 14 days, and 30 days after the damage was imparted, each of the mlc2a-kaede transgenic zebrafish individuals was dissected, the heart was removed, and the fluorescence image was observed with a fluorescence microscope (BIOREVO BZ-9000: Keyence, Japan). The results are shown in FIG. Cells having only green fluorescence were not observed at the damaged site from the 7th day after the damage was applied, but a group of cells having only green fluorescence was clearly observed at the damaged site on the 14th and 30th days after the damage was given. Thereby, it became possible to visualize myocardial regeneration by using mlc2a-kaede transgenic zebrafish individuals.
As shown in FIG. 6 (A), a green fluorescence image and a red fluorescence image of the regenerated myocardium were separately photographed with a bandpass filter, and the green fluorescence image was converted into a black and white image using BZ analyzer II (Keyence Japan). After the fluorescence image is converted into a black and white image, the brightness and darkness are reversed, and by superimposing both images, it is possible to discriminate cells having significantly strong green fluorescence in the damaged area as a clear white image. Using this method, as shown in FIG. 6B, the number of regenerated cardiomyocytes can be measured with good reproducibility.
Screening of cardiac regenerative drugs using transgenic zebrafish
<About FGF>
The influence of the FGFR inhibitor, which plays an essential role in the zebrafish heart regeneration process, on the heart regeneration effect of mlc2a-kaede transgenic zebrafish was examined. After the heart of mlc2a-kaede transgenic zebrafish was changed to red fluorescence by the above method, 17 μM FGFR inhibitor (SU5402, Calbiochem, USA) was added during the 7th to 14th days after the injury by the tungsten needle. Zebrafish individuals were bred in the presence and absence. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In the individual treated with the FGFR inhibitor, the number of cells having only green fluorescence in the damaged area was significantly smaller than the group of cells having only green fluorescence in the control individual. The average number of cells having only fluorescence was 6 (FGFR inhibitor treatment) and 21 (control), and the inhibitory effect of the drug on myocardial regeneration could be quantitatively evaluated.
In addition, FGF (especially bFGF or FGF-1), which has been shown to be able to accelerate the myocardial regeneration process in mammalian myocardial infarction model rats, quantitatively analyzes myocardial regeneration using transgenic zebrafish. We examined whether it was possible.
Using the above method, the mlc2a-kaede transgenic zebrafish heart was changed to red fluorescence and subsequently damaged with a tungsten needle. After 2 and 4 days, 1 μl of 30 μM bFGF (Invitrogen: USA) was placed in the zebrafish chest cavity. Injection was performed using a sharpened micro glass tube (Narishige, Japan). After 14 days, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
Despite the same damage, a significant increase in regenerated myocardium with only green fluorescence was observed in the individuals with bFGF compared to the control individuals. As a result of analyzing the number of regenerated cardiomyocytes from a microscopic observation image, as shown in FIG. 8B, it was found that the average of the bFGF administration group was 34 compared to the average of 22 of the control group. Therefore, transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test.
<About Wnt>
The influence of the heart regeneration effect of mlc2a-kaede transgenic zebrafish was examined by using an inhibitor of Wnt signal, which plays an essential role in the zebrafish fin regeneration process.
In fish cardiomyocytes, there is no information about what role Wnt signals play in myocardial regeneration. After the heart of mlc2a-kaede transgenic zebrafish was changed to red fluorescence by the above-described method, 5 μM Wnt signal inhibitor (XAV939, Cayman Chemical, Zebrafish individuals were bred in the presence and absence of the United States). The breeding water was changed every two days, and the breeding water newly containing 5 μM Wnt signal inhibitor was used. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In the individuals treated with the Wnt signal inhibitor, the number of cells having only green fluorescence in the damaged area was significantly smaller than the number of cells having only green fluorescence in the control individuals. The number of cells having only green fluorescence is 3 (Wnt signal inhibitor treatment) and 21 (control) on average as shown in FIG. 9 (B), and the myocardial regeneration is remarkably suppressed by suppressing the Wnt signal. It became clear to suppress. It has also become possible to quantitatively evaluate the inhibitory effect of drugs on the regeneration of heart cells.
From the above results, it was examined whether or not the myocardial regeneration effect by the GSK-3 inhibitor acting as a promoter in the Wnt signal can be quantitatively analyzed. The above method caused the mlc2a-kaede transgenic zebrafish heart to turn red fluorescent, and subsequently damaged with a tungsten needle, after 1 and 8 days of 2.5 nM GSK-3 inhibitor (BIO, Calbiochem, USA) Breeding was carried out in the presence or absence of an aqueous phase. Fourteen days after the injury, the heart was excised and collected from the zebrafish individuals, and the regenerated cardiomyocytes were observed under a fluorescence microscope. The results are shown in FIG.
In spite of the same damage, a significant increase in regenerative myocardium with only green fluorescence was observed in the individuals to which GSK-3 inhibitor acting as a promoter of Wnt signal was added, compared to the control individuals. It was. As a result of analyzing the number of regenerated cardiomyocytes from a microscopic observation image, as shown in FIG. It was found that the average of the GSK-3 inhibitor administration group was 33 compared to the average of 21 in the control group. Therefore, transgenic zebrafish can be screened for candidate compounds that are active ingredients of drugs for heart disease by a simpler method with good reproducibility. It is also possible to test.
From the results of the above examples, by using the method of the present invention, it becomes possible to screen for candidate compounds that are active ingredients of drugs for heart disease. Also, confirm whether compounds already listed as candidate compounds have an effective effect on heart disease, study the optimal concentration of the compound, study the administration interval and duration, and examine the possibility of combination with other candidate compounds It is also possible to use for such as.
Moreover, in the said Example, it was confirmed by using mlc2a-kaede transgenic zebrafish that both FGF-1 and a GSK-3 inhibitor lead to a myocardial regeneration effect. The mlc2a-kaede transgenic zebrafish of the present invention is used even though FGF-1 is considered to be a molecule involved in the FGF signal cascade and GSK-3 is a Wnt cascade and a signal cascade different in cardiomyocytes. As a result, a result that it is an effective ingredient for heart disease was obtained. Therefore, the screening method of the present invention is suitable for searching for candidate compounds that are active ingredients of drugs for heart disease.
Claims (4)
- 刺激によって発光波長が変化する蛋白質を心筋細胞特異的に発現させた魚類を用いた、心疾患治療薬のスクリーニング方法。 A screening method for a therapeutic agent for heart disease, using fish in which a protein whose emission wavelength is changed by stimulation is expressed specifically in cardiomyocytes.
- 前記蛋白質がＫａｅｄｅである、請求項１又は２に記載のスクリーニング方法。 The screening method according to claim 1 or 2, wherein the protein is Kaede.
- 前記魚類が、ゼブラフィッシュ又はメダカである、請求項１または２に記載のスクリーニング方法。 The screening method according to claim 1 or 2, wherein the fish is zebrafish or medaka.
- 前記心疾患が虚血性心疾患又は心筋症である、請求項１~３のいずれか１項に記載のスクリーニング方法。 The screening method according to any one of claims 1 to 3, wherein the heart disease is ischemic heart disease or cardiomyopathy.
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| CN102520130A (en) * | 2011-12-07 | 2012-06-27 | 复旦大学 | Screening method of medicines for inducing formation of myocardial cells by using model organism zebrafish |
| CN103540611A (en) * | 2013-09-26 | 2014-01-29 | 马明 | Method for producing color-changeable light-induced fluorescent spectacular fish |
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Non-Patent Citations (2)
| Title |
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| DE PATER E ET AL.: "Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart", DEVELOPMENT, vol. 136, no. 10, May 2009 (2009-05-01), pages 1633 - 41 * |
| KAWASAKI, T. ET AL.: "Investigation of isoproterenol-effective elements for enhanced genes in medaka cardiac hypertrophy model", J PHARMACOL SCI, vol. 112, no. SUPPLE, 15 February 2010 (2010-02-15), pages 246P * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520130A (en) * | 2011-12-07 | 2012-06-27 | 复旦大学 | Screening method of medicines for inducing formation of myocardial cells by using model organism zebrafish |
| CN103540611A (en) * | 2013-09-26 | 2014-01-29 | 马明 | Method for producing color-changeable light-induced fluorescent spectacular fish |
| CN103540611B (en) * | 2013-09-26 | 2015-09-30 | 马明 | A kind of method of producing changeable colour light induced fluorescence aquarium fish |
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