JP2001103869A - Lipocalin-type prostaglandin d synthetase transgenic animal and examination using the animal - Google Patents
Lipocalin-type prostaglandin d synthetase transgenic animal and examination using the animalInfo
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- JP2001103869A JP2001103869A JP28599799A JP28599799A JP2001103869A JP 2001103869 A JP2001103869 A JP 2001103869A JP 28599799 A JP28599799 A JP 28599799A JP 28599799 A JP28599799 A JP 28599799A JP 2001103869 A JP2001103869 A JP 2001103869A
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】この出願の発明は、ヒト・リ
ポカリン型プロスタグランジンD合成酵素(L−PGD
S)遺伝子を導入したトランスジェニック動物と、この
動物を用いた各種試験方法に関するものである。さらに
詳しくは、この出願の発明は、L−PGDSの大量発現
によって中枢神経系機能、循環器系機能および生殖機能
等に、先天性または反応性の障害を呈するトランスジェ
ニック動物と、この動物を用いて上記機能障害に対する
予防または治療薬剤の有効成分を試験する方法に関する
ものである。The invention of this application relates to a human lipocalin-type prostaglandin D synthase (L-PGD).
S) The present invention relates to a transgenic animal into which a gene has been introduced and various test methods using the animal. More specifically, the invention of this application relates to a transgenic animal exhibiting a congenital or responsive disorder in the central nervous system function, circulatory system function, reproductive function, etc. due to the overexpression of L-PGDS, and the use of this animal. And a method for testing an active ingredient of a preventive or therapeutic agent for the above dysfunction.
【0002】[0002]
【従来の技術】L−PGDS(J.Biol.Chem. 260:12410
-12415, 1985; J.Biol.Chem. 264:1041-1045, 1989; Pr
oc.Natl.Acad.Sci.USA. 88:4020-4024, 1991; Proc.Nat
l.Acad.Sci.USA. 89:5376-5380,1992; J.Biol.Chem. 26
7:23202-23208, 1992; J.LipdMediators Cell Signalin
g 12:257-273, 1995; J.Biol.Chem. 270:1422-1428, 19
95)は、各種の生理活性の有する体内物質プロスタグラ
ンジンD2(PGD2 :Prostaglandins Leukotrienes E
ssent. Fatty Acids 37:219-234, 1989; FASEBJ. 5:257
5-2581, 1991; Adv.Neuroimmunol. 5:211-216, 1995;
J.Lipd Mediators Cell Signaling 14:71-82, 1996)の
産生機能と、細胞分化因子であるビタミンA群の輸送機
能(Proc.Natl.Acad.Sci.USA. 88:4020-4024, 1991; J.
Biol.Chem. 272:15789-15795, 1997)を併せ持つ酵素で
あり、中枢神経系、循環器系および生殖器官で発現し
(Arch.Biochem.Biophys. 260:521-531, 1988; Br.J.Op
hthalmology 72:461-464, 1988; Proc.Natl.Acad.Sci.U
SA. 90:9070-9074, 1993; Prostaglandins 51:298, 199
6; J.Neurosci. 16:6119-6124, 1996)、体液中に分泌
されている(Biochem.Biophys.Res.Commun. 203:1110-1
116, 1994; Proc.Japan.Acad. 72:108-111, 1996; Clin
ical Cheminsry 42:1984-1991, 1996)。そして、これ
らの各器官でのL−PGDSの発現量の増減が、種々の
身体障害や疾患に密接に関係していることも知られてい
る。2. Description of the Related Art L-PGDS (J. Biol. Chem. 260: 12410)
-12415, 1985; J. Biol. Chem. 264: 1041-1045, 1989; Pr.
oc.Natl.Acad.Sci.USA.88: 4020-4024, 1991; Proc.Nat
l.Acad.Sci.USA. 89: 5376-5380,1992; J.Biol.Chem. 26
7: 23202-23208, 1992; J. LipdMediators Cell Signalin
g 12: 257-273, 1995; J. Biol.Chem. 270: 1422-1428, 19
95) is a prostaglandin D 2 (PGD 2 : Prostaglandins Leukotrienes E) having various physiological activities.
ssent.Fatty Acids 37: 219-234, 1989; FASEBJ. 5: 257
5-2581, 1991; Adv.Neuroimmunol. 5: 211-216, 1995;
J. Lipd Mediators Cell Signaling 14: 71-82, 1996) and the transport function of vitamin A group which is a cell differentiation factor (Proc. Natl. Acad. Sci. USA. 88: 4020-4024, 1991; J; .
Biol. Chem. 272: 15789-15795, 1997) and is expressed in the central nervous system, circulatory system and reproductive organs (Arch. Biochem. Biophys. 260: 521-531, 1988; Br. J. Op
hthalmology 72: 461-464, 1988; Proc.Natl.Acad.Sci.U
SA. 90: 9070-9074, 1993; Prostaglandins 51: 298, 199
6; J. Neurosci. 16: 6119-6124, 1996), which is secreted into body fluids (Biochem. Biophys. Res. Commun. 203: 1110-1).
116, 1994; Proc. Japan.Acad. 72: 108-111, 1996; Clin
ical Cheminsry 42: 1984-1991, 1996). It is also known that the increase or decrease of the expression level of L-PGDS in each of these organs is closely related to various physical disorders and diseases.
【0003】例えば、PGD2は現在までに明らかにさ
れている内因性睡眠物質のうちで最も強い催眠作用を示
すが、その合成酵素であるL−PGDSの阻害物質(Ar
ch.Biochem.Biophys. 289:161-166, 1991)を動物の脳
室内に投与すると顕著な睡眠障害が生じる(Proc.Natl.
Acad.Sci.USA 88:9046-9050, 1991)ことから、脳内に
おけるL−PGDSの合成不全が不眠症の一因であるこ
とが示唆されている。また、中枢神経系でのPGD2の
増減は、侵害的な刺激(例えば痛覚刺激)に対する感受
性の変化(Brain Res. 510:26-32, 1990; J.Pharmacol.
Exp.Ther. 278:1146-1152, 1996; Proc. Natl. Acad.
Sci. USA. 96:726-730, 1999)および排卵誘発を促す黄
体ホルモンの分泌量の変化(Endocrinology 110:2207-2
209, 1982)にも関与することも知られている。For example, PGD 2 has the strongest hypnotic action among endogenous sleep substances that have been identified to date, but it is an inhibitor of the synthetic enzyme L-PGDS (Ar
Ch. Biochem. Biophys. 289: 161-166, 1991) results in significant sleep disorders when administered intraventricularly to animals (Proc. Natl.
Acad. Sci. USA 88: 9046-9050, 1991) suggests that insufficiency of L-PGDS synthesis in the brain contributes to insomnia. Also, increasing or decreasing PGD 2 in the central nervous system results in altered susceptibility to noxious stimuli such as nociceptive stimuli (Brain Res. 510: 26-32, 1990; J. Pharmacol.
Exp. Ther. 278: 1146-1152, 1996; Proc. Natl. Acad.
Sci. USA. 96: 726-730, 1999) and changes in the secretion of progesterone that promotes ovulation induction (Endocrinology 110: 2207-2).
209, 1982).
【0004】一方、循環器系でのL−PGDS発現とし
ては、例えば、動脈硬化の病巣においてL−PGDSが
特異的に発現することが知られている(Proc.Natl.Aca
d.Sci.USA. 94:14689-14694, 1997)。これは、L−P
GDSにより合成されるPGD 2の血液凝固抑制作用(P
rostaglandins 16:373-388, 1978)によって狭窄した血
管を閉鎖させないためであると推測されている。従っ
て、血液中のL−PGDSは動脈硬化のマーカーとして
有望視されている。また、L−PGDSは腎臓の近尿細
管の特定部位に局在して発現しているが、その部位はヒ
トにおける塩分過多を原因とする腎機能低下に関係する
部位であるとされていることから、L−PGDSは腎機
能低下症の発症機構に密接に関係するものと考えられて
もいる。On the other hand, L-PGDS expression in the circulatory system
For example, L-PGDS may be used in atherosclerotic lesions.
It is known to be specifically expressed (Proc. Natl. Aca
d.Sci.USA. 94: 14689-14694, 1997). This is LP
PGD synthesized by GDS TwoOf blood coagulation (P
rostaglandins 16: 373-388, 1978)
It is speculated that this is not to close the tube. Follow
Thus, L-PGDS in blood is a marker of arteriosclerosis
Promising. In addition, L-PGDS is used in the near renal cells of the kidney.
It is expressed at a specific site in the duct, but that site is
Is associated with decreased renal function due to excessive salt in
L-PGDS is considered to be a renal
It is thought that it is closely related to the onset mechanism of hypoactivity
There are also.
【0005】さらに、生殖器官におけるL−PGDSの
発現は、不妊に関係していることも知られている。すな
わち、L−PGDSは精巣・副睾丸で産生され、精液中
に分泌されるが、男性不妊要因である精液減少症の患者
の精液中のL−PGDS濃度は健常人に比べて有意に低
い(Biol.Reprod. 58:600-607, 1998)。また、ウシ精
液の高受胎率関連蛋白質として報告されたSP26はL
−PGDSであることも判明している(Biol.Reprod. 5
8:826-833, 1998)。ただし、精液中にはPGD2がほと
んど検出されないことから(Biol.Reprod. 58:600-607,
1998)、L−PGDSはPGD2合成機能とは別の機能
によって生殖機能に関係していると考えられる。[0005] Furthermore, it is known that the expression of L-PGDS in the reproductive tract is related to infertility. That is, L-PGDS is produced in the testis and epididymis and secreted into the semen, but the L-PGDS concentration in the semen of a patient with sperm reduction, which is a factor of male infertility, is significantly lower than that in a healthy person ( Biol. Reprod. 58: 600-607, 1998). SP26 reported as a protein related to high fertility in bovine semen is L
-PGDS (Biol. Reprod. 5
8: 826-833, 1998). However, since PGD 2 is hardly detected in semen (Biol. Reprod. 58: 600-607,
1998), L-PGDS is thought to be related to reproductive function by another function and PGD 2 synthesis function.
【0006】またL−PGDは女性の妊娠にも関係して
いる。すなわち、L−PGDSは妊娠後期の胎児中枢神
経系で発現し、羊水中に分泌されるが、その量は胎児の
成長に伴って増加することがヒトおよび動物で確認され
ている。このため、L−PGDSは妊娠異常のマーカー
としての利用が期待されている。[0006] L-PGD is also involved in female pregnancy. That is, L-PGDS is expressed in the fetal central nervous system in late pregnancy and secreted into amniotic fluid, and it has been confirmed in humans and animals that the amount increases with the growth of the fetus. Therefore, L-PGDS is expected to be used as a marker for abnormal pregnancy.
【0007】[0007]
【発明が解決しようとする課題】以上のとおり、L−P
GDSが生物個体の様々な生理機能に密接に関係してお
り、その大量発現が種々のヒト疾患要因となりうること
が示唆されている。SUMMARY OF THE INVENTION As described above, LP
GDS is closely related to various physiological functions of living organisms, and it has been suggested that its overexpression may cause various human diseases.
【0008】しかしながら、L−PGDS活性の大量発
現が動物個体に対してどの様に作用するのかについて、
統制された条件下での研究を可能とするモデル動物系は
確立されていない。[0008] However, as to how the overexpression of L-PGDS activity acts on animal individuals,
No model animal system has been established that allows for research under controlled conditions.
【0009】また、そのようなモデル動物は、L−PG
DS活性の大量発現によって生じる各種疾患の予防もし
くは治療薬剤の開発等にも極めて有効であろうと期待さ
れる。Further, such a model animal is L-PG
It is expected to be extremely effective for the development of drugs for preventing or treating various diseases caused by the overexpression of DS activity.
【0010】この出願の発明は、以上のとおりの事情に
鑑みてなされたものであって、遺伝的にL−PGDS活
性を大量発現している動物個体を提供することを課題と
している。またこの出願は、この動物個体を用い、L−
PGDS活性の欠損によって生じる各種疾患の予防もし
くは治療物質の有効性を試験する方法を提供することを
課題としてもいる。The invention of this application has been made in view of the above circumstances, and has as its object to provide an animal individual that genetically expresses a large amount of L-PGDS activity. In addition, this application uses this animal individual,
It is another object of the present invention to provide a method for testing the efficacy of a substance for preventing or treating various diseases caused by deficiency of PGDS activity.
【0011】[0011]
【課題を解決するための手段】この出願は、上記の課題
を解決するための発明として、ヒト・リポカリン型プロ
スタグランジンD合成酵素遺伝子を導入した非ヒト動物
の全能性細胞を個体発生して得られる非ヒト動物および
その子孫動物であって、体細胞染色体中に上記遺伝子を
保有し、ヒト・リポカリン型プロスタグランジンD合成
酵素を大量発現することを特徴とするトランスジェニッ
ク動物を提供する。SUMMARY OF THE INVENTION The present invention is directed to an invention for solving the above-mentioned problems, in which a human lipocalin-type prostaglandin D synthase gene is introduced into a totipotent cell of a non-human animal. The present invention provides a transgenic animal, which is a non-human animal and its progeny, which has the gene in a somatic chromosome and expresses human lipocalin-type prostaglandin D synthase in large amounts.
【0012】また、このトランスジェニック動物におい
ては、非ヒト動物がマウスであることを好ましい態様と
してもいる。さらにこの出願は、以下の各種試験方法を
提供する。 (1) 睡眠調節物質の個体内活性を試験する方法であ
って、上記のトランスジェニック動物に候補物質を投与
し、この動物の睡眠状態を測定することを特徴とする試
験方法。 (2) 鎮痛物質の個体内活性を試験する方法であっ
て、上記のトランスジェニック動物に候補物質を投与
し、この動物の痛覚刺激に対する反応性を測定すること
を特徴とする試験方法。 (3) 血液凝固抑制物質の個体内活性を試験する方法
であって、上記のトランスジェニック動物に候補物質を
投与し、この動物の動脈硬化の程度を測定することを特
徴とする試験方法。 (4) 腎機能促進物質の個体内活性を試験する方法で
あって、上記のトランスジェニック動物に候補物質を投
与し、この動物の腎機能を測定することを特徴とする試
験方法。 (5) 不妊改善物質の個体内活性を試験する方法であ
って、上記のトランスジェニック動物の雄に候補物質を
投与し、この雄動物の精子の成熟の程度または繁殖率の
程度を測定することを特徴とする試験方法。 (6) 不妊改善物質の個体内活性を試験する方法であ
って、上記のトランスジェニック動物の雌に、妊娠前ま
たは妊娠後に候補物質を投与し、この雌動物の子宮にお
ける受精卵の着床状態または胎仔の発育状態を測定する
ことを特徴とする試験方法。 (7) 麻酔物質の個体内活性を試験する方法であっ
て、上記のトランスジェニック動物に候補物質を投与
し、この動物の麻酔状態および/または麻酔からの覚醒
状態を測定することを特徴とする試験方法。 (8) 生理不順改善物質の個体内活性を試験する方法
であって、上記のトランスジェニック動物の雌に候補物
質を投与し、この雌動物の性周期、排卵数および/また
は各種ホルモン量を測定することを特徴とする試験方法[0012] In a preferred embodiment of the transgenic animal, the non-human animal is a mouse. Further, this application provides the following various test methods. (1) A method for testing the activity of a sleep regulating substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the sleep state of the animal. (2) A method for testing the activity of an analgesic substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the responsiveness of the animal to a pain stimulus. (3) A method for testing the activity of an anticoagulant substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the degree of arteriosclerosis of the animal. (4) A method for testing the activity of a renal function promoting substance in an individual, comprising administering a candidate substance to the transgenic animal and measuring the renal function of the animal. (5) A method for testing the activity of a fertility-improving substance in an individual, which comprises administering a candidate substance to the male of the above-mentioned transgenic animal, and measuring the degree of maturation or reproductive rate of sperm of the male animal. A test method characterized by the following. (6) A method for testing the activity of an infertility-improving substance in an individual, comprising administering a candidate substance to a female transgenic animal before or after pregnancy, and implanting a fertilized egg in the uterus of the female animal. Alternatively, a test method comprising measuring the state of development of a fetus. (7) A method for testing the activity of an anesthetic substance in an individual, which comprises administering a candidate substance to the transgenic animal, and measuring the anesthesia state and / or arousal state from the anesthesia of the animal. Test method. (8) A method for testing the activity of a physiological disorder improving substance in an individual, which comprises administering a candidate substance to a female of the above-mentioned transgenic animal, and measuring the sexual cycle, ovulation count and / or various hormone levels of the female animal. Test method characterized by performing
【0013】[0013]
【発明の実施の形態】導入遺伝子であるヒトL−PGD
S遺伝子は、そのcDNAを用いることができる。この
L−PGDScDNAは公知の配列(J. Biol. Chem. 2
67:23202-23208, 1992)の任意部分の塩基配列に基づい
てオリゴヌクレオチドを合成し、これをプローブとして
用いてヒトcDNAライブラリーをスクリーニングする
方法や、目的とするcDNA断片の両末端にハイブリダ
イズするオリゴヌクレオチドを合成し、これをプライマ
ーとして用いてヒト細胞から単離したmRNAからRT
−PCR法により調製することもできる。DESCRIPTION OF THE PREFERRED EMBODIMENTS Human L-PGD as a transgene
As the S gene, its cDNA can be used. This L-PGDS cDNA has a known sequence (J. Biol. Chem. 2
67: 23202-23208, 1992), a method for screening a human cDNA library using the oligonucleotide as a probe based on an arbitrary nucleotide sequence, and hybridizing to both ends of a cDNA fragment of interest. And using the primers as primers to RT from mRNA isolated from human cells.
-It can also be prepared by the PCR method.
【0014】また、導入遺伝子には、その発現を制御す
るためのプロモーター配列やエンハンサー配列を連結す
る。この発明のトランスジェニック動物は、公知のトラ
ンスジェニック動物作製法(例えば、Proc. Natl. Aca
d. Scl. USA 77;7380-7384, 1980)に従って作成するこ
とができる。すなわち、前記導入遺伝子を非ヒト動物の
全能性細胞に導入し、この細胞を個体へと発生させ、体
細胞のゲノム中に導入遺伝子が組み込まれた個体を選別
することによって目的とするトランスジェニック動物を
作製することができる。非ヒト動物としては、技術的に
は全ての動物種を対象とすることが可能であるが、特に
近交系が多数作出されており、しかも受精卵の培養、体
外受精等の技術が整っているマウスが最適である。遺伝
子を導入する全能性細胞としては、マウスの場合、受精
卵や初期胚を用いることができる。また培養細胞への遺
伝子導入法としては、トランスジェニック動物個体の産
出高率や次代への導入遺伝子の伝達効率を考慮した場
合、DNAの物理的注入(マイクロインジェクション)
法が最適である。[0014] A promoter sequence and an enhancer sequence for controlling the expression of the transgene are linked to the transgene. The transgenic animal of the present invention can be prepared by a known transgenic animal production method (for example, Proc. Natl. Aca).
d. Scl. USA 77; 7380-7384, 1980). That is, the transgenic animal of interest is introduced by introducing the transgene into a totipotent cell of a non-human animal, generating the cell into an individual, and selecting an individual having the transgene integrated in the genome of a somatic cell. Can be produced. As a non-human animal, it is technically possible to target all animal species, but in particular many inbred strains have been created, and in addition, techniques such as fertilized egg culture and in vitro fertilization have been established. A mouse that works best. In the case of a mouse, a fertilized egg or an early embryo can be used as the totipotent cell into which the gene is introduced. As a method for introducing a gene into cultured cells, physical injection of DNA (microinjection) is considered in consideration of the yield rate of an individual transgenic animal and the efficiency of transgene transfer to the next generation.
The method is optimal.
【0015】遺伝子を注入した受精卵は、次に仮親の卵
管に移植され、個体まで発生し出生した動物を里親につ
けて飼育させたのち、体の一部(尾部先端)からDNA
を抽出し、サザン解析やPCR法により導入遺伝子の存
在を確認する。導入遺伝子の存在が確認された個体を初
代(Founder)とすれば、導入遺伝子はその子孫の50%に
伝達され、野性型または変異型の動物を効率よく作出す
ることが可能である。The fertilized egg into which the gene has been injected is then transplanted into the fallopian tube of the foster mother, and the animals that have developed and born to the individual are fostered and reared, and then DNA is obtained from a part of the body (tail tip).
And the presence of the transgene is confirmed by Southern analysis or PCR. If the individual in which the presence of the transgene is confirmed is defined as the first generation (Founder), the transgene is transmitted to 50% of its offspring, and a wild-type or mutant animal can be efficiently produced.
【0016】このようにして作出されたトランスジェニ
ック動物はL−PGDSを過剰産生するため、そのPG
D2の生理作用を検討する最適のモデルとなりうる。ま
た、L−PGDSを対象とした薬剤のスクリーニングに
も適したモデルとなるうる。Since the transgenic animal thus produced overproduces L-PGDS,
It can be a perfect model to study the physiological effects of D 2. In addition, it can be a model suitable for screening drugs for L-PGDS.
【0017】このようにして作成したトランスジェニッ
ク動物は、以下のとおりの試験方法に用いることができ
る。 (1)睡眠調節物質の試験方法 野生型マウス等にL−PGDS活性阻害剤を脳室内投与
すると、顕著な睡眠障害が認められるが、この発明のト
ランスジェニック動物は尾の切断による痛覚刺激により
一過性の過眠を呈する。これは、ストレス下および病的
条件における睡眠誘発にPGD2が貢献していることを
示している。そこで、催眠薬等の有効成分となる睡眠調
節物質の候補物質をトランスジェニック動物に投与し、
ストレス下または病的条件下での動物の睡眠状態を測定
することによって、例えば副作用の少ない覚醒物質を含
む睡眠調節物質の探索、そのような物質を有効成分とす
る催眠薬および覚醒薬の開発が可能となる。また、この
ような睡眠調節物質の探索等をとおして、哺乳動物にお
ける睡眠覚醒調節機構の解明も可能となる。なお、マウ
スの睡眠状態の測定は、例えば脳波、筋電、活動量、摂
食・摂水量、体温等を経時的に計測することによって行
うことができる。 (2)鎮痛物質の試験方法 ヒトは健康時には接触刺激によって痛みを感じることは
ないが、例えば帯状疱疹に罹患した場合のような病的状
況下では軽い触覚刺激でも激痛を起こす。これはアロデ
ィニアと呼ばれる現象であり、熱刺激や機械刺激による
痛覚過敏とは区別されている(Textbook of Pain, 3rd
Ed, pp165-200, 1994; Pain 68:13-23,1996)。また、
脊髄におけるPGD2はアロディニアの発現調節に関与
している(Proc. Natl. Acad. Sci. USA. 96:726-730,
1999)。The transgenic animal thus prepared can be used in the following test methods. (1) Test Method for Sleep Regulatory Substance When a L-PGDS activity inhibitor is intracerebroventricularly administered to a wild-type mouse or the like, a remarkable sleep disorder is observed. Presents with transient hypersomnia. This indicates that PGD 2 contributes to sleep induction under stress and in pathological conditions. Therefore, a candidate substance for a sleep regulating substance to be an active ingredient such as a hypnotic is administered to the transgenic animal,
By measuring the sleep state of animals under stress or pathological conditions, for example, the search for sleep regulating substances including stimulants with few side effects, the development of hypnotics and stimulants containing such substances as active ingredients have been promoted. It becomes possible. In addition, it is possible to elucidate the sleep-wake regulation mechanism in mammals through such a search for a sleep regulating substance. The sleep state of the mouse can be measured, for example, by measuring brain waves, myoelectricity, activity, eating / water consumption, body temperature, and the like over time. (2) Method of Testing Analgesic Substances Humans do not feel pain due to contact stimuli when healthy, but in severe illness such as when suffering from shingles, severe tactile stimuli cause severe pain. This is a phenomenon called allodynia, which is distinguished from hyperalgesia caused by thermal or mechanical stimulation (Textbook of Pain, 3rd
Ed, pp165-200, 1994; Pain 68: 13-23, 1996). Also,
PGD 2 in the spinal cord are involved in the regulation of expression of allodynia (Proc Natl Acad Sci USA 96: ..... 726-730,
1999).
【0018】従って、この発明のトランスジェニック動
物は、アロディニアおよび痛覚過敏誘発機構の解明に利
用することができる。また、鎮痛物質の候補をマウスに
投与し、このマウスの痛覚刺激に対する反応性を測定す
ることによって、痛覚反応に選択的な鎮痛薬を開発する
ことが可能となる。さらに、痛覚誘発機構の解明と新規
の鎮痛薬の開発は、例えばモルヒネで抑えることのでき
ないガン末期の激痛メカニズムを解明し、その対処療法
を開発するにも有効である。 (3)血液凝固抑制物質の試験方法 従来、動脈硬化モデル動物としては、強制的に動脈硬化
を生じさせたラットやウサギが用いられていたが、マウ
スはヒトによく似た摂食による動脈硬化を発症させるこ
とができる(Atheroscleosis 57:65-73, 1985)。また
前述のとおり、動脈硬化の病巣には特異的にL−PGD
Sが発現し、それによって合成されるPGD2によって
凝固した血液を溶解して血管閉鎖を防止することが知ら
れている。この発明のトランスジェニック動物(特に、
トランスジェニックマウス)は、L−PGDSを大量に
発現するため、動脈硬化による心不全の発作を軽減でき
る可能性がある。Therefore, the transgenic animal of the present invention can be used for elucidating the mechanism of inducing allodynia and hyperalgesia. Further, by administering a candidate analgesic substance to a mouse and measuring the responsiveness of the mouse to a pain stimulus, it becomes possible to develop an analgesic which is selective for a pain sensation reaction. Furthermore, elucidation of the mechanism of pain induction and development of novel analgesics are effective in elucidating, for example, the mechanism of severe pain at the end of cancer that cannot be suppressed by morphine, and developing a treatment for the pain. (3) Test method for anticoagulant substances Conventionally, rats and rabbits that forcibly induced arteriosclerosis have been used as arteriosclerosis model animals, but mice have atherosclerosis caused by eating much like humans. (Atheroscleosis 57: 65-73, 1985). Also, as described above, L-PGD is specifically present in atherosclerotic lesions.
It is known that S expresses and lyses coagulated blood by PGD 2 synthesized thereby to prevent vascular closure. The transgenic animal of the present invention (particularly,
Transgenic mice) express L-PGDS in large amounts, and may be able to reduce attacks of heart failure due to arteriosclerosis.
【0019】従って、動脈硬化を生じさせたトランスジ
ェニック動物に血液凝固抑制物質の候補を投与し、この
マウスの動脈硬化の程度を測定することによって、ヒト
の動脈硬化発症機構の解明とともに、新しい血液凝固抑
制薬を開発することが可能である。 (4)腎機能促進物質の試験方法 前述のとおり、L−PGDSは腎機能の低下に関係する
部位に特異的に発現している。従って、このL−PGD
S遺伝子を導入したトランスジェニック動物は、例えば
塩分摂取量によっても腎機能低下症を生じにくい可能性
があり、そのようなモデルマウスに候補物質を投与して
マウスの腎機能を測定することにより、新規な腎機能促
進薬を開発することが可能となる。また、ヒトの腎機能
低下症の発症機構の解明にも有効である。 (5)男性要因による不妊改善物質の試験方法 前述のとおり、男性不妊要因である精子減少症患者の精
液中L−PGDS濃度は有意に低いが、高受胎率を示す
雄ウシの精液中のL−PGDS濃度は高い。従って、こ
の発明のトランスジェニック動物の雄に候補物質を投与
し、この雄動物の精子の成熟や繁殖率の程度を測定する
ことによって、不妊治療薬の開発が可能となる。また、
男性要因による不妊の解明にも有効である。 (6)女性要因による不妊改善物質の試験方法 野生型の雌マウスでは、妊娠中期の胎盤で特異的かつ一
過性にL−PGDSが発現する。一方、PGD2が減少
した場合には、分娩は正常ではあるが、妊娠期間の顕著
な延長が認められる。従って、このトランスジェニック
動物の妊娠前または妊娠後に候補物質を投与し、この雌
マウスの子宮における受精卵の着床状態または胎児の発
育状態を測定することによって、女性要因による不妊を
改善するための薬剤の開発が可能となる。 (7)麻酔物質の試験方法 外科手術等の際には吸入麻酔物質を用いて全身麻酔を行
う場合があるが、理想的な麻酔とは低濃度の吸入麻酔剤
による無痛・筋弛緩・意識消失をいう。しかし、麻酔状
態には、性別・年齢・体格・健康状態等に依存した個体
差があり、麻酔のかかりにくい患者では高濃度の吸入麻
酔剤の使用により死に至る場合もある。このため、吸入
麻酔剤の濃度は慎重に決定される必要があるが、吸入麻
酔物質の中枢神経系への作用機序がほとんど解明されて
いないために、医療現場では熟練医師の経験則に頼らざ
るを得ないのが現状である。ところで、脳内PGD2減
少は吸入麻酔物質による麻酔の深度が浅くなるため、L
−PGDSを大量発現するこの発明のトランスジェニッ
ク動物において麻酔下および/または覚醒後の状態を測
定することによって、痛覚消失・筋弛緩・睡眠に至るメ
カニズムを各々分離して解析することが可能である。こ
れにより、現在使用されている吸入麻酔物質のより詳細
な薬効評価が可能となるばかりか、新規の吸入麻酔物質
の開発も可能となる。なお、マウスの麻酔状態として
は、例えば正向反射の消失、無痛、筋弛緩、意識消失等
をそれぞれ常法に従って測定することができる。また、
覚醒状態は、例えば正向反射の回復、痛覚反応、筋力の
回復、覚醒を測定することができる。 (8)生理不順改善物質の試験方法 排卵周期と睡眠および活動量が同調することが知られて
いる(Gen. Comp. Endocrinol. 7:10-17, 1996; Physio
l. Behav. 49:1079-1084, 1991; Brain Res. 734:275-2
85, 1996; Brain Res. 811:96-104, 1998)。生活リズ
ムの変調により生理不順が起こるが、ホルモンの分泌異
常による生理不順は社会生活に支障をきたすような、例
えば過剰睡眠等を引き起こす。また、PGD2が減少し
た動物は、排卵期前後が延長された性周期を示し、その
結果、野生型動物に比べて性周期が長い。PGD2は排
卵誘発を促すホルモンの分泌量を変化させるため、この
発明のトランスジェニック動物は、排卵の誘発機構およ
び生理不順による過剰睡眠等の誘発機構を解明するため
に有効であり、新規の生理不順改善物質の開発が可能と
なる。Therefore, by administering a candidate for a blood coagulation inhibitor to a transgenic animal that has caused arteriosclerosis and measuring the degree of arteriosclerosis in the mouse, the mechanism of arteriosclerosis in humans can be elucidated and a new blood can be obtained. It is possible to develop anticoagulants. (4) Test method for renal function promoting substance As described above, L-PGDS is specifically expressed in a site related to a decrease in renal function. Therefore, this L-PGD
Transgenic animals into which the S gene has been introduced may be less likely to cause renal dysfunction due to, for example, salt intake, and by administering a candidate substance to such a model mouse to measure the renal function of the mouse, It becomes possible to develop new renal function promoters. It is also effective in elucidating the mechanism of onset of renal hypofunction in humans. (5) Test Method for Fertility-Improving Substances Due to Male Factors As described above, the L-PGDS concentration in semen of a spermatozoa patient who is a factor of male infertility is significantly low, but the L level in the semen of a bull showing a high conception rate is high. -PGDS concentration is high. Therefore, by administering a candidate substance to the male transgenic animal of the present invention and measuring the degree of sperm maturation and reproductive rate of the male animal, it is possible to develop a therapeutic agent for infertility. Also,
It is also effective in elucidating infertility due to male factors. (6) Test method for fertility-improving substance due to female factors In wild-type female mice, L-PGDS is specifically and transiently expressed in the placenta in the second trimester. On the other hand, when PGD 2 is decreased, the delivery is normal, but the gestation period is markedly prolonged. Therefore, by administering a candidate substance before or after pregnancy of the transgenic animal and measuring the implantation state of the fertilized egg in the uterus of the female mouse or the developmental state of the fetus, it is possible to improve infertility due to female factors. Drug development becomes possible. (7) Test method of anesthetic substance In general, general anesthesia is performed using an inhaled anesthetic substance during surgery, etc., but ideal anesthesia is painlessness, muscle relaxation and loss of consciousness due to a low concentration of inhaled anesthetic. Say. However, there are individual differences in the state of anesthesia depending on gender, age, physique, health status, and the like. In patients who are hardly anesthetized, death may be caused by use of a high-concentration inhalation anesthetic. For this reason, the concentration of inhaled anesthetics must be carefully determined.However, since the mechanism of action of inhaled anesthetics on the central nervous system has not been elucidated, medical practice relies on the rules of thumb of experienced physicians. At present it is inevitable. By the way, the decrease in PGD 2 in the brain is due to the fact that the depth of anesthesia with an inhaled anesthetic substance becomes shallow,
-The mechanism leading to analgesia, muscle relaxation, and sleep can be separately analyzed by measuring the state under anesthesia and / or after arousal in the transgenic animal of the present invention that overexpresses PGDS. . This not only allows more detailed evaluation of the efficacy of currently used inhalational anesthetics, but also allows the development of new inhalational anesthetics. In addition, as an anesthesia state of a mouse, for example, loss of righting reflex, painlessness, muscle relaxation, loss of consciousness, etc. can be measured according to a conventional method. Also,
The arousal state can be measured by, for example, recovery of righting reflex, pain response, recovery of muscle strength, and arousal. (8) Test method of physiological disorder improving substance It is known that ovulation cycle and sleep and activity are synchronized (Gen. Comp. Endocrinol. 7: 10-17, 1996; Physio
l. Behav. 49: 1079-1084, 1991; Brain Res. 734: 275-2
85, 1996; Brain Res. 811: 96-104, 1998). Physiological irregularities occur due to the modulation of life rhythm, but physiological irregularities due to abnormal secretion of hormones cause social life, such as excessive sleep. In addition, animals in which PGD 2 has been reduced show an estrous cycle in which the period before and after the ovulation period is prolonged. Since PGD 2 changes the secretion of hormones that promote ovulation induction, the transgenic animal of the present invention is effective for elucidating the ovulation induction mechanism and the induction mechanism such as excessive sleep due to irregular menstruation, and a novel physiological It becomes possible to develop irregular improvement substances.
【0020】なお、以上のとおりの各試験方法において
は、L−PGDS発現量の異なるトランスジェニック動
物(例えば、2倍体染色体の両方に導入遺伝子を有する
ホモ接合型や一方に導入遺伝子を有するヘテロ接合型)
を適宜に使い分けることによって、L−PGDS産生量
に依存した各種症状と、それに対する試験物質の効果を
詳細に分析することが可能である。In each of the test methods described above, transgenic animals having different L-PGDS expression levels (for example, homozygous animals having a transgene in both diploid chromosomes and heterozygous animals having a transgene in one of them) are used. Junction type)
It is possible to analyze in detail the various symptoms depending on the amount of L-PGDS production and the effects of the test substances on them by properly using.
【0021】[0021]
【実施例】以下、実施例を示してこの発明をさらに詳細
に説明するが、この発明は以下の例に限定されるもので
はない。 実施例1 (1)トランスジェニックマウスの作製 ヒト細胞のmRNAから調製したcDNAライブラリー
から、ラットL−PGDS遺伝子のcDNAをプローブ
としてヒトL−PGDSのcDNAをクローニングし
た。EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Example 1 (1) Preparation of Transgenic Mouse Human L-PGDS cDNA was cloned from a cDNA library prepared from human cell mRNA using rat L-PGDS gene cDNA as a probe.
【0022】次にベクター(pCAGGS)のクローニ
ング部位(Sal I/Not I)にヒトL−PGDSのc
DNAを挿入結合し、導入ベクターを構築した。図1
は、この導入ベクターにおける導入遺伝子の構成であ
る。Next, the human L-PGDS c was inserted into the cloning site (Sal I / Not I) of the vector (pCAGGS).
The DNA was inserted and ligated to construct a transfer vector. FIG.
Is the structure of the transgene in this transfection vector.
【0023】この導入ベクターをマイクロインジェクシ
ョン法によってFVBマウスの受精卵に注入した。遺伝
子導入受精卵は定法に従って仮親の卵管に移植し、個体
へと発生させ出生させた。This transfection vector was injected into fertilized eggs of FVB mice by the microinjection method. The transgenic fertilized egg was transplanted into the fallopian tube according to a standard method, developed into an individual, and born.
【0024】得られたマウス個体の尾部からDNAを抽
出し、導入遺伝子の配列にもとづき合成されたプローブ
を用い、サザン解析法によりトランスジェニックマウス
を選別した。L−PGDS遺伝子の発現量の異なる独立
した3系統のトランスジェニックマウスを確立した。結
果は図2に示したとおりである。 (2)トランスジェニックマウスの遺伝子発現の検討 トランスジェニックマウスの全身における導入遺伝子の
発現をサザン解析法により調べた。その結果B20マウス
において、L−PGDS遺伝子は骨格筋、心臓、肺、大
腸、肝臓に高レベルで発現していることが確認された。
結果は図3に示したとおりである。 (3)トランスジェニックマウスのPGD酵素活性の検
討 トランスジェニックマウスの各種臓器におけるPGD酵
素活性を、基質であるPGH2を用いて調べた。各種臓
器においてトランスジェニックマウスでは酵素活性に著
しい増加がみられた。また3系統間で比較したところ、
B20>B25>B7の順で酵素活性の増加が観察された。
結果は図4に示したとおりである。 実施例2 実施例1で得たトランスジェニックマウスを用いて、痛
覚刺激による睡眠誘発の解析を行った。DNA was extracted from the tail of the obtained mouse individual, and transgenic mice were selected by Southern analysis using a probe synthesized based on the sequence of the transgene. Three independent transgenic mice with different L-PGDS gene expression levels were established. The results are as shown in FIG. (2) Examination of gene expression in transgenic mice The expression of transgenes in whole body of transgenic mice was examined by Southern analysis. As a result, in B20 mice, it was confirmed that the L-PGDS gene was expressed at high levels in skeletal muscle, heart, lung, large intestine, and liver.
The results are as shown in FIG. (3) Examination of PGD enzyme activity in transgenic mice PGD enzyme activities in various organs of transgenic mice were examined using PGH 2 as a substrate. In various organs, transgenic mice showed a marked increase in enzyme activity. Also, when comparing the three systems,
An increase in enzyme activity was observed in the order of B20>B25> B7.
The results are as shown in FIG. Example 2 Using the transgenic mouse obtained in Example 1, analysis of sleep induction by pain stimulus was performed.
【0025】トランスジェニックマウスでは、御の先端
を切断する刺激により、野生型マウスに比較して、1〜
2時間後の徐波睡眠量が有意に増加していることが観察
された。結果は図5に示したとおりである。In the transgenic mouse, the stimulus for cutting off the tip of the transgenic mouse resulted in 1 to 1 compared to the wild type mouse.
It was observed that the amount of slow wave sleep after 2 hours was significantly increased. The results are as shown in FIG.
【0026】脳を摘出してPGD2量を定量したとこ
ろ、睡眠量の増加に伴い、その濃度が上昇していること
が明らかになった。結果は図6に示したとおりである。
以上の結果から、この発明のトランスジェニック動物
は、病的あるいはストレスによる睡眠誘発の機構を解明
するためのモデル動物として有用であり、新規睡眠覚醒
調節物質をスクリーニングする系としても有効であるこ
とが確認された。When the brain was excised and the amount of PGD 2 was quantified, it was found that the concentration increased with an increase in the amount of sleep. The results are as shown in FIG.
From the above results, the transgenic animal of the present invention is useful as a model animal for elucidating the mechanism of sleep induction caused by pathology or stress, and is also effective as a system for screening a novel sleep-wake modulator. confirmed.
【0027】[0027]
【発明の効果】以上詳しく説明したとおり、この発明に
よって、ヒトL−PGDS遺伝子が形質導入され、この
合成酵素の大量発現によて中枢神経系機能、循環器系機
能および生殖機能等に、先天性または反応性の障害を呈
するトランスジェニック動物が提供される。このトラン
スジェニック動物を用いることによって、上記の機能障
害に対する予防または治療薬剤の有効成分を動物個体レ
ベルで試験することが可能となる。As described above in detail, according to the present invention, the human L-PGDS gene is transduced, and a large amount of this synthetic enzyme is exerted on the central nervous system function, circulatory system function, reproductive function, etc. Transgenic animals exhibiting sexual or responsive disorders are provided. By using this transgenic animal, it becomes possible to test the active ingredient of the drug for preventing or treating the above-mentioned dysfunction at the animal individual level.
【図1】この発明のトランスジェニックマウスの作成に
用いた導入ベクターの構造を示す模式図である。FIG. 1 is a schematic diagram showing the structure of an introduction vector used for producing the transgenic mouse of the present invention.
【図2】3系統のトランスジェニックおよび野生型マウ
スの各種臓器より抽出したmRNAのノーザンブロット
解析の結果である。FIG. 2 shows the results of Northern blot analysis of mRNAs extracted from various organs of three lines of transgenic and wild-type mice.
【図3】トランスジェニックマウスの全身臓器から抽出
したmRNAのノーザンブロット分析の結果である。FIG. 3 shows the results of Northern blot analysis of mRNA extracted from systemic organs of transgenic mice.
【図4】3系統のトランスジェニックおよび野生型マウ
スの各種臓器より抽出したタンパク質分画を用いたL−
PGDS酵素活性の分析結果である。FIG. 4 shows L-proteins using protein fractions extracted from various organs of three lines of transgenic and wild-type mice.
It is an analysis result of PGDS enzyme activity.
【図5】トランスジェニックマウスおよび野生型マウス
における尾先端切断後の徐波睡眠量の変化を示すグラフ
である。●は尾切断、○はコントロールである。FIG. 5 is a graph showing changes in the amount of slow-wave sleep after transection of the tail tip in transgenic mice and wild-type mice. ● is tail cutting, ○ is control.
【図6】トランスジェニックマウスおよび野生型マウス
における尾先端切断後の脳内PGD2量の変化を示すグ
ラフである。FIG. 6 is a graph showing changes in the amount of PGD 2 in the brain after transection of the tail tip in transgenic mice and wild-type mice.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) G01N 33/50 C12N 5/00 B 33/88 15/00 A (72)発明者 裏出 良博 京都府京都市中京区西洞院通蛸薬師下ル古 西町440 藤和シティーコープ706号 (72)発明者 江口 直美 大阪府大阪市東淀川区瑞光1−8−7 (72)発明者 北山 博章 京都府京都市西京区御陵峰ヶ堂町3丁目5 番地の12 (72)発明者 林 直木 神奈川県横浜市泉区上飯田町2838−4 Fターム(参考) 2G045 AA01 AA08 AA29 CB17 4B024 AA01 AA11 BA07 BA80 CA04 CA09 DA02 EA04 GA12 HA13 HA14 4B065 AA91X AA93Y AB01 AC14 AC16 BA04 BD50 CA44 CA46 CA60 ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 C12N 5/00 B 33/88 15/00 A (72) Inventor Yoshihiro Urade Kyoto, Kyoto, Japan 440 Towa City Corp. 706 (72) Inventor Naomi Eguchi 1-8-7, Mizumitsu, Higashiyodogawa-ku, Osaka-shi, Osaka (72) Inventor Hiroaki Kitayama Goryomine, Saikyo-ku, Kyoto-shi, Kyoto 3-5-5 Kadomachi 12-72 (72) Inventor Naoki Hayashi 2838-4 Kamiida-cho, Izumi-ku, Yokohama-shi, Kanagawa F-term (reference) 2G045 AA01 AA08 AA29 CB17 4B024 AA01 AA11 BA07 BA80 CA04 CA09 DA02 EA04 GA12 HA13 HA14 4B065 AA91X AA93Y AB01 AC14 AC16 BA04 BD50 CA44 CA46 CA60
Claims (10)
D合成酵素遺伝子を導入した非ヒト動物の全能性細胞を
個体発生して得られる非ヒト動物およびその子孫動物で
あって、体細胞染色体中に上記遺伝子を保有し、ヒト・
リポカリン型プロスタグランジンD合成酵素を大量発現
することを特徴とするトランスジェニック動物。1. A non-human animal and its progeny obtained by ontogenizing a totipotent cell of a non-human animal into which a human lipocalin-type prostaglandin D synthase gene is introduced, and a progeny animal thereof, wherein Gene
A transgenic animal characterized by expressing a large amount of lipocalin-type prostaglandin D synthase.
ランスジェニック動物。2. The transgenic animal according to claim 1, wherein the non-human animal is a mouse.
法であって、請求項1または2のトランスジェニック動
物に候補物質を投与し、この動物の睡眠状態を測定する
ことを特徴とする試験方法。3. A method for testing the activity of a sleep regulating substance in an individual, the method comprising administering a candidate substance to the transgenic animal of claim 1 or 2, and measuring the sleep state of the animal. Method.
あって、請求項1または2のトランスジェニック動物に
候補物質を投与し、この動物の痛覚刺激に対する反応性
を測定することを特徴とする試験方法。4. A method for testing the activity of an analgesic substance in an individual, which comprises administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the responsiveness of the animal to a pain stimulus. Test method.
る方法であって、請求項1または2のトランスジェニッ
ク動物に候補物質を投与し、この動物の動脈硬化の程度
を測定することを特徴とする試験方法。5. A method for testing the activity of a blood coagulation inhibitor in an individual, the method comprising administering a candidate substance to the transgenic animal of claim 1 or 2, and measuring the degree of arteriosclerosis of the animal. Test method.
方法であって、請求項1または2のトランスジェニック
動物に候補物質を投与し、この動物の腎機能を測定する
ことを特徴とする試験方法。6. A method for testing the activity of a renal function promoting substance in an individual, comprising administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the renal function of the animal. Test method.
法であって、請求項1または2のトランスジェニック動
物の雄に候補物質を投与し、この雄動物の精子の成熟の
程度および/またはこの雄動物の繁殖率の程度を測定す
ることを特徴とする試験方法。7. A method for testing the activity of an infertility-improving substance in an individual, the method comprising administering a candidate substance to a male transgenic animal according to claim 1 or 2, and subjecting the male animal to sperm maturation and / or A test method characterized by measuring the degree of reproduction rate of the male animal.
法であって、請求項1または2のトランスジェニック動
物の雌に、妊娠前または妊娠後に候補物質を投与し、こ
の雌動物の子宮における受精卵の着床状態および/また
は胎仔の発育状態を測定することを特徴とする試験方
法。8. A method for testing the activity of an infertility-improving substance in an individual, which comprises administering a candidate substance to a female of the transgenic animal according to claim 1 before or after pregnancy, A test method comprising measuring the implantation state of a fertilized egg and / or the development state of a fetus.
あって、請求項1または2のトランスジェニック動物に
候補物質を投与し、このマウスの麻酔状態および/また
は麻酔からの覚醒状態を測定することを特徴とする試験
方法。9. A method for testing the activity of an anesthetic substance in an individual, comprising administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the anesthesia state and / or arousal state from the anesthesia of the mouse. A test method characterized by:
する方法であって、請求項1または2のトランスジェニ
ック動物の雌に候補物質を投与し、この雌動物の性周
期、排卵数および/または各種ホルモン量を測定するこ
とを特徴とする試験方法10. A method for testing the in vivo individual activity of a physiological disorder improving substance, which comprises administering a candidate substance to a female transgenic animal according to claim 1 or 2, wherein the female animal has a sexual cycle, ovulation number and / or ovulation rate. Or a test method characterized by measuring the amount of various hormones
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Cited By (3)
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---|---|---|---|---|
WO2004006956A1 (en) * | 2002-07-12 | 2004-01-22 | Japan Science And Technology Agency | Drugs for improving the prognosis of brain injury and a method of screening the same |
WO2004056992A1 (en) * | 2002-12-19 | 2004-07-08 | Riken | Three-dimensional structure of lipocalin-type prostaglandin d synthase and utilization of the same |
WO2019131879A1 (en) * | 2017-12-28 | 2019-07-04 | 学校法人兵庫医科大学 | Lipocalin-type prostaglandin d2 synthase production accelerating agent |
-
1999
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004006956A1 (en) * | 2002-07-12 | 2004-01-22 | Japan Science And Technology Agency | Drugs for improving the prognosis of brain injury and a method of screening the same |
WO2004056992A1 (en) * | 2002-12-19 | 2004-07-08 | Riken | Three-dimensional structure of lipocalin-type prostaglandin d synthase and utilization of the same |
WO2019131879A1 (en) * | 2017-12-28 | 2019-07-04 | 学校法人兵庫医科大学 | Lipocalin-type prostaglandin d2 synthase production accelerating agent |
JP6607655B1 (en) * | 2017-12-28 | 2019-11-20 | 学校法人兵庫医科大学 | Lipocalin-type prostaglandin D2 synthase production promoter |
JP2020007376A (en) * | 2017-12-28 | 2020-01-16 | 学校法人兵庫医科大学 | Lipocalin-type prostaglandin D2 synthase production promoter |
CN111511403A (en) * | 2017-12-28 | 2020-08-07 | 学校法人兵库医科大学 | Lipocalin-type prostaglandin D2 synthase production promoter |
JP7411175B2 (en) | 2017-12-28 | 2024-01-11 | 学校法人兵庫医科大学 | Lipocalin-type prostaglandin D2 synthase production promoter |
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