JP5309381B2 - Extraction method of substance released from beetle and its extraction substance - Google Patents
Extraction method of substance released from beetle and its extraction substance Download PDFInfo
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- JP5309381B2 JP5309381B2 JP2009136463A JP2009136463A JP5309381B2 JP 5309381 B2 JP5309381 B2 JP 5309381B2 JP 2009136463 A JP2009136463 A JP 2009136463A JP 2009136463 A JP2009136463 A JP 2009136463A JP 5309381 B2 JP5309381 B2 JP 5309381B2
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Description
本発明は、学名Trypoxylus dichotomusの甲虫（以下、カブト虫）から放出される物質の抽出方法およびその抽出物質に関する。より詳しくは、カブト虫の幼虫を高密度条件下で飼育する等、一定の条件下で飼育した場合に、カブト虫の幼虫がすみわけ行動を示すという知見に基づき、一定の条件下で飼育した場合に、カブト虫より放出される物質の抽出方法およびその抽出物質に関する。 The present invention relates to a method for extracting a substance released from a beetle (hereinafter referred to as a beetle) having a scientific name of Trypoxylus dichotomus, and the extracted substance . More specifically, when beetle larvae are raised under certain conditions, such as when reared under certain conditions, such as when the beetle larvae show their behavior, they are reared under certain conditions. The present invention relates to a method for extracting a substance released from a beetle and the extracted substance .
従来よりカブト虫は、幼虫から成虫に至る変態のそれぞれの段階での個体を商品として人工飼育によって育成され、販売されてきた。人工飼育は、広く既知のものではあるが、特定の飼育方法も存在する（特許文献１参照）。 Conventionally, beetles have been cultivated and sold by artificial breeding using individual individuals at each stage of transformation from larvae to adults. Although artificial breeding is widely known, there is also a specific breeding method (see Patent Document 1).
以下、カブト虫の一般的な人工飼育方法とその方法で育成されるカブト虫について説明する。通常、クヌギやコナラなどの広葉樹を細かく砕いて腐食した、所謂、腐葉土を平地や山地などに大量に準備する。家庭や小規模に飼育する場合には、プラスチック製の透明な飼育ケースを用いる。交尾後のカブト虫の雌は、夏季期間中に、その腐葉土中に直径約２ｍｍ程度の球形の卵を産む。卵が孵化した後、腐葉土が飼料となり、幼虫が成育する。尚、幼虫は、腐葉土中で越冬し、越冬した年の春から夏にかけての期間に、自己の大きさより１から２ｃｍ程度大きな空洞を腐葉土中に形成し、その空洞の中で蛹に変態する。そして、さらに３から４週間経過した後、蛹の皮を破って成虫が出てくる。地上へは、餌となる甘味を有する液体（自然界では、樹液であり、人工飼育では、砂糖水や蜂蜜水あるいはメロン・桃などの果実成分）を求めて這い出す。また、成体は、地上に這い出した際には、夜間に可視光領域の波長の光に対して集まる性質を有している。幼虫や蛹形成時期での可視光に対して集まる性質は、知られていない。尚、カブト虫は、卵から成虫に至るまでの成長の過程を経て飼育するために約１年間を必要とする。 Hereinafter, a general artificial breeding method for beetles and beetles grown by the method will be described. Usually, a large amount of so-called humus soil prepared by finely pulverizing and corroding hardwood such as Kunugi and Quercus is prepared on a flat ground or a mountainous area. When rearing at home or on a small scale, use plastic transparent rearing cases. The mating beetle female lays a spherical egg about 2 mm in diameter in the mulch during the summer. After the eggs hatch, the humus becomes feed and the larvae grow. The larvae overwinter in the humus, and form a cavity about 1 to 2 cm larger than their own size in the humus during the period from spring to summer in the year of the overwintering, and transform into a cocoon in the cavity. After another 3 to 4 weeks, adults emerge after breaking the wings. On the ground, it is sought out for sweet liquids (natural sap in nature and sugar water, honey water or fruit components such as melon and peach in artificial breeding). In addition, adults have the property of gathering with respect to light having a wavelength in the visible light region at night when they crawl on the ground. The nature of gathering against visible light during larvae and pupae formation is unknown. In addition, a beetle needs about one year in order to breed through the process of growth from an egg to an adult.
どのような生命体でも個体の密度が高まれば、相互に排除または捕食するなどの、異常が見られるが、本カブト虫の幼虫や蛹形成期では、個体間における上述の異常はない。このような個体間での異常が特定のヒトに存在するならば、そのヒトは、他者を傷つけることや自己を犠牲にするような精神疾患を伴う可能性がある。事実、ヒトの場合、ヒトどうしの空間的隔たりと鬱病との関わりを示すデータが存在する（非特許文献１参照）。 In any living organism, if the density of individuals increases, abnormalities such as mutual exclusion or predation are observed, but the above-mentioned abnormalities among individuals are not observed during the larvae or pupae formation period of this beetle. If such inter-individual abnormalities exist in a particular human, that human may be associated with a mental illness that hurts others or sacrifices herself. In fact, in the case of humans, there is data indicating the relationship between the spatial distance between humans and depression (see Non-Patent Document 1).
鬱病は、脳内の辺縁系でのセロトニン（アミノ酸の一つ）が減少することによって、発症すると考えられている。セロトニン自体は、血液脳関門を通過出来ないが、その分解産物である５ヒドロキシインドール酢酸は通過できる。従って、髄液中の５ヒドロキシインドール酢酸量の減少を測定することによって、セロトニンの減少と鬱病との関連が明らかになった（非特許文献２と非特許文献３参照）。 Depression is thought to develop as serotonin (an amino acid) decreases in the limbic system in the brain. Serotonin itself cannot pass through the blood brain barrier, but its degradation product, 5-hydroxyindoleacetic acid, can pass through. Therefore, by measuring the decrease in the amount of 5-hydroxyindoleacetic acid in the cerebrospinal fluid, the relationship between serotonin decrease and depression was clarified (see Non-Patent Document 2 and Non-Patent Document 3).
他の例では、性決定と社会性維持との関連として、ヒトにおける疾患である性同一性障害の存在を挙げることが出来る。外見上は、既に性別が決定しているにもかかわらず、精神的には、外見上と逆の性を有している場合がある。一生物の集団を考えた場合、特定の集団が引き起こす事例の法的解釈は別として、性同一性障害は、必ずしも周囲のヒトが形成する社会が許容しているわけではない。患者自身が不快感を有する場合には、治療が必要となることもあり得る。 In another example, the relationship between sex determination and social maintenance can include the presence of a gender identity disorder, a disease in humans. In terms of appearance, even if the gender has already been determined, there may be mentally opposite gender. When considering a single population, apart from the legal interpretation of the cases caused by a particular population, gender identity disorder is not necessarily tolerated by the society formed by the surrounding people. If the patient himself has discomfort, treatment may be necessary.
通常、雌雄が存在する生命体の中で、光のような物理的刺激によって性が決定されることは知られていない。唯一、物理的条件で雌雄を決定しうる生物の事例として、爬虫類に属するカメ（亀）の雌雄の決定では、卵の期間において、卵に接する物理適刺激として外部温度が重要である旨、約３０年前から知られている（非特許文献４参照）。 Normally, it is not known that sex is determined by physical stimuli such as light in living organisms where males and females exist. The only example of organisms that can determine their sex according to physical conditions is that the external temperature is important as the physical stimuli in contact with the eggs during the egg period in the determination of turtles (turtles) belonging to reptiles. It has been known for 30 years (see Non-Patent Document 4).
本発明は、カブト虫という個体を用いることによる、社会性に関与する生理活性物質の創出方法を提供することを延長線上の課題としつつ、カブト虫より放出される物質を抽出することを課題とする。より詳しくは、カブト虫の飼育条件を変えることによるカブト虫より放出される物質を抽出することを課題とする。 This invention makes it a subject to extract the substance discharge | released from a beetle, making it the subject on the extension line to provide the creation method of the bioactive substance which is concerned with sociality by using the individual called a beetle. More specifically, it is an object to extract substances released from beetles by changing the rearing conditions of beetles .
本発明者は、個体の密度を高く保って飼育したカブト虫の幼虫から蛹への変態の際に、個々の幼虫が距離をおき、飼育空間が秩序正しく保たれていることに着目し、鋭意研究を重ねた結果、蛹に変態する前のカブト虫の幼虫が、体外に社会性に関与する何らかの生理活性物質を放出することを見出し、本発明を完成した。さらに、上記のような飼育条件下で光照射した際に、光という物理的刺激を介して、カブト虫の性別が決定されることを見出した。このような知見から、上記個体の密度を高く保って飼育したカブト虫の幼虫から蛹への変態の際に、一定の物理学的条件下で、カブト虫の体内に社会性に関与する生理活性物質が放出されることを見出し、本発明の社会性に関与する生理活性物質の創出方法を完成した。 The present inventor has paid attention to the fact that individual larvae are separated from each other during transformation from larvae to pupae that were bred while keeping the density of the individual high, and the breeding space is maintained in an orderly manner. As a result, it was found that beetle larvae before transforming into pupae release some physiologically active substance involved in sociality outside the body, and the present invention was completed. Furthermore, when light irradiation was carried out under the above breeding conditions, it discovered that the sex of the beetle was determined through the physical stimulus of light. Based on these findings, during the transformation from the larvae of the beetles bred while maintaining the above-mentioned density to the cocoon, physiologically active substances involved in sociality are found in the beetles under certain physical conditions. As a result, the inventors have completed a method for creating a physiologically active substance involved in the social nature of the present invention.
すなわち、本発明は以下よりなる。
１．飼育媒体中に飼育された甲虫における幼虫が蛹形成し成虫へと変態した後、飼育媒体を収集し、その飼育媒体を生理食塩水またはＰＢＳの緩衝液に懸濁させ、その懸濁液をろ過することを特徴とする甲虫より放出された物質の抽出方法。
２．飼育条件として、飼育媒体中に複数の幼虫を飼育した前項１記載の甲虫より放出された物質の抽出方法。
３．飼育条件として、飼育面積１１１０ｃｍ ２ に対して少なくとも２匹以上の幼虫となる密度で飼育媒体中に飼育した前項２記載の甲虫より放出された物質の抽出方法。
４．飼育条件として、飼育媒体１５リットルに対して少なくとも２匹以上の幼虫を飼育し、形成した各々の蛹間の平面的な間隔が長くとも１０ｃｍの隔たりを保させる密度で飼育媒体中に飼育した前項２又は３記載の甲虫より放出された物質の抽出方法。
５．飼育条件として、飼育した幼虫から蛹への変態の際に、一定の光照射条件下で飼育した前項１から４のいずれかに記載の甲虫より放出された物質の抽出方法。
６．懸濁液をろ過した後、更に二次元電気泳動により分離する前項１から５のいずれかに記載の甲虫より放出された物質の抽出方法。
７．飼育媒体中に飼育された甲虫における幼虫が蛹形成し成虫へと変態した後、飼育媒体を収集し、その飼育媒体を生理食塩水またはＰＢＳの緩衝液に懸濁させ、その懸濁液をろ過することで得られることを特徴とする甲虫の幼虫より放出された抽出物質。
That is, this invention consists of the following.
1. After the larvae of the beetles bred in the breeding medium formed pupae and transformed into adults, the breeding medium was collected, suspended in saline or PBS buffer, and the suspension filtered. A method for extracting a substance released from a beetle, characterized in that:
2. 2. A method for extracting substances released from beetles according to item 1 above, wherein a plurality of larvae are reared in a rearing medium as rearing conditions.
3. 3. The method for extracting a substance released from a beetle as recited in the preceding item 2, which was reared in a rearing medium at a density of at least two larvae per rearing area of 1110 cm 2 as rearing conditions .
4). As a rearing condition, at least two larvae were reared with respect to 15 liters of the rearing medium, and reared in the rearing medium at a density that kept the distance of 10 cm at most even when the planar spacing between the formed pupae was at most A method for extracting a substance released from a beetle according to 2 or 3.
5. 5. The method for extracting a substance released from a beetle according to any one of the preceding items 1 to 4, which was reared under constant light irradiation conditions during transformation from a reared larva to a pupa as a rearing condition.
6). 6. The method for extracting a substance released from a beetle according to any one of items 1 to 5, wherein the suspension is filtered and further separated by two-dimensional electrophoresis.
7). After the larvae of the beetles bred in the breeding medium formed pupae and transformed into adults, the breeding medium was collected, suspended in saline or PBS buffer, and the suspension filtered. Extracted material released from beetle larvae, characterized in that it is obtained by
本発明の甲虫より放出された物質の抽出方法によれば、社会性に関与する関連遺伝子産物を含む生理活性物質の創出をなし得る。具体的には、カブト虫の幼虫を飼育容器の底面積１１１０ｃｍ２、高さ３１ｃｍに対して少なくとも２匹の密度条件で飼育した場合、幼虫から蛹への変体の際に、個々の幼虫が平面的な距離を長くとも１０ｃｍの間隔をおき、飼育空間が秩序正しく保たれていることから、各々の幼虫から放出された特定の物質は、社会秩序を一定に保つための社会性に関与する生理活性物質である可能性が考えられた。また、光照射条件下で幼虫を飼育した場合に、光を受容できない飼育容器内の部分からは、雌雄選別の実施段階で、１００％の割合で雄のカブト虫の蛹が見出された。そこで、光照射の条件下で、光受容の有無により、各々の幼虫から放出された特定の物質を分析することにより、雌雄決定に関与する関連遺伝子産物を含む生理活性物質をスクリーニングしうることが考えられた。関連遺伝子産物を含む生理活性物質の創出方法の延長上には、昆虫という種を超えて、ヒトの性同一性障害に関する知見がカブト虫を利用して得られる可能性がある。つまり、生理活性物質や類似の活性を有する分子は、社会秩序を逸脱する精神疾患の治療に応用することが出来る。さらに、性同一性障害の治療に応用することが出来る。また、光という比較的単純な刺激とその受容による性決定の関連性が明らかにされることによって、ヒトを含む哺乳類での性決定の機構究明の糧にもなり得る。 According to the method for extracting a substance released from a beetle of the present invention, a physiologically active substance containing a related gene product involved in sociality can be created . Specifically, when the beetle larvae are bred in a density condition of at least two animals with respect to a bottom area of 1110 cm 2 and a height of 31 cm, the individual larvae are planar when transformed from larvae to pupae. A specific substance released from each larva is a physiologically active substance involved in sociality to keep the social order constant because the rearing space is kept in order with a distance of 10 cm at the longest. The possibility that it is. In addition, when larvae were reared under light irradiation conditions, male beetle moths were found at a rate of 100% at the stage of sex selection from the portion in the rearing container that could not accept light. Therefore, by analyzing specific substances released from each larvae under the conditions of light irradiation based on the presence or absence of photoreception, it is possible to screen physiologically active substances including related gene products involved in sex determination. it was thought. Related to the extension of the creation process of physiologically active substance include the gene product is, across species of insects, may be obtained by utilizing Kabutochu findings regarding gender identity disorders in humans. That is, molecules with physiology active substances and similar activities can be applied to the treatment of mental disorders departing from the social order. Furthermore, it can be applied to the treatment of gender identity disorder. In addition, the elucidation of the relationship between light-relative simple stimuli and their determination of sex determination can serve as a basis for investigating the mechanisms of sex determination in mammals including humans.
さらに、上記の知見は、社会秩序を逸脱する疾患や性同一性障害の診断が、医師の主観ではなく、生化学的パラメータによって客観的に示される可能性があることから、診断薬としての応用をも望める。 In addition, the above findings suggest that diagnosis of diseases and gender identity disorders that deviate from social order may be objectively indicated by biochemical parameters rather than doctor's subjectivity. Can also be expected.
本発明は、一定の密度でカブト虫を飼育したとき、幼虫から蛹への変体の際に、個々の幼虫が距離をおき、飼育空間が秩序正しく保たれていることから、カブト虫が社会性に関与する生理活性物質を放出していると考えらる。個々の幼虫が距離をおき、飼育空間が秩序正しく保たれていることは、図１を参照されたい。 In the present invention, when beetles are reared at a constant density, when they are transformed from larvae to pupae, the individual larvae are separated from each other, and the rearing space is maintained in an orderly manner. Ru considered to be released a physiologically active substance to be. See FIG. 1 to see that the individual larvae are separated and the breeding space is kept in order.
社会性に関与する関連遺伝子産物を含む生理活性物質の創出方法は、野生に存在する遺伝的背景が雑多なカブト虫を用いた系では困難であり、前例や各種の媒体での報告・記述が存在しないため、遺伝的背景をほぼ同一にしたカブト虫を用いることが前提となる。 Creation method of a physiologically active substance containing the relevant gene products involved in social resistance is difficult in a system where genetic background were used miscellaneous Kabutochu present in the wild, the reporting and descriptions in the previous and various media due to the absence, it is assumed to use a Kabutochu which is substantially identical to heritage Den background.
遺伝的背景をほぼ同一にしたカブト虫の取得方法は、一対の雌雄からなる成体カブト虫を飼育し、産卵させ、翌年に成体を得ることによる。得られた成虫の外観が濃赤色の成体カブト虫のみを選別し、交配させ、再び成体を得るという操作を総じて３年間繰り返すことで、作出開始から３年（３年目）で遺伝的背景をほぼ同一にした純系の成体カブト虫（半クローン化カブト虫）を得ることができる。このような半クローン化カブト虫に関する既報の文献は存在しない。 The method for obtaining beetles with substantially the same genetic background is that an adult beetle consisting of a pair of males and females is bred and spawned, and adults are obtained in the following year. By selecting only adult red beetles whose appearance is dark red, mating, and re-establishing adults for 3 years in total, the genetic background is almost 3 years after the start of production (3rd year). The same pure adult beetle (semi-cloned beetle) can be obtained. There is no published literature on such semi-cloned beetles.
上記、得られた半クローン化カブト虫について、社会性に関与する生理活性物質の創出方法を実施するためには、飼育容器の底面積１１１０ｃｍ２、高さ３１ｃｍに対して少なくとも２匹の密度条件で飼育したカブト虫の幼虫を用いることを要する。当該幼虫は、比較的小規模でプラスチック製かガラス製の光を透過する容器を用いて飼育することが推奨され、少なくとも幼虫が蛹に変態する前の時点で、約１０匹以上１５匹未満の幼虫が存在していることが望ましい。その根拠（理由）は、通常、幼虫が蛹に変態するために必要な空間を飼育媒体（例えば、腐葉土）中に確保しなければならないが、その空間が広すぎても狭すぎても良好な結果が得られないことが本発明に関わる実験から明らかになったからである。上記高密度条件は、飼育媒体１５リットルに対して、少なくとも１０匹以上、好ましくは１０匹以上１５匹未満のカブト虫の幼虫が存在している条件であっても良い。 Above, the semi-cloned beetle obtained, to implement the creation method of a physiologically active substance involved in social resistance, rearing container bottom area 1110Cm2, at least two dogs density condition the height 31cm It is necessary to use reared beetle larvae. It is recommended that the larvae be kept in a relatively small plastic or glass light-transmitting container, and at least about 10 or more and less than 15 larvae before transformation into pupae. It is desirable that larvae exist. The reason (reason) is usually that the space necessary for the larvae to transform into pupae must be secured in the breeding medium (eg humus), but it is good if the space is too wide or too narrow It is because it became clear from the experiment regarding this invention that a result is not obtained. The high density condition may be a condition in which at least 10 or more, preferably 10 or more and less than 15 beetle larvae exist for 15 liters of the rearing medium.
本発明の明細書において、「社会性に関与する生理活性物質」とは、社会性を維持するのに必要な生理活性物質を意味する。「関連する遺伝子産物」とは、社会性の維持や雌雄決定に関与するペプチドやタンパク質を意味する。ここで、「社会性」とは、特別な定義はないが、例えばヒトの場合には、人間関係を維持し、他人と意思の疎通を図ることができ、平和に社会秩序を維持できること等を意味する。広辞苑には、集団をつくって生活しようとする人間の根本性質と記されている。どのような生物でも個体の密度が高まれば、相互に排除または捕食するなどの異常が見られる場合がある。上記のような異常が特定のヒトに存在するならば、そのヒトは、他者を傷つけることや自己を犠牲にするような精神疾患を伴う可能性がある。例えば、鬱病は、社会生活におけるストレスなどにより、脳内の辺縁系でのセロトニン（アミノ酸の一つ）が減少することによって発症すると考えられている。その結果、同疾患患者は、内向的になり、他者との接触も減少する。また、性決定と社会性維持との関連を示す一例として、ヒトにおける疾患である性同一性障害の存在を挙げることが出来る。外見上は、既に性別が決定しているにもかかわらず、精神的には、外見上と逆の性を有している場合がある。一生物の集団を考えた場合、特定の集団が引き起こす事例の法的解釈は別として、性同一性障害は、必ずしもヒトが形成する社会が許容しているわけではない。このような場合にも、「社会性」に関与する生理活性物質の生体内外の量的増減が関与していることも考えられる。患者自身が不快感を有する場合には、治療が必要となることもあり得る。 In the specification of the present invention, “a physiologically active substance involved in sociality” means a physiologically active substance necessary for maintaining sociality. “Related gene products” mean peptides and proteins involved in maintaining sociality and determining sex. Here, “social” has no special definition. For example, in the case of humans, it is possible to maintain human relations, communicate with others, and maintain social order peacefully. means. Kojien says that it is the fundamental nature of human beings who are trying to make a group and live. If the density of individuals in any organism increases, abnormalities such as mutual exclusion or predation may be observed. If such abnormalities are present in a particular person, that person may be associated with a mental illness that hurts others or sacrifices herself. For example, depression is thought to develop when serotonin (an amino acid) in the limbic system in the brain decreases due to stress in social life. As a result, patients with the disease are introverted and contact with others is reduced. An example of the relationship between sex determination and social maintenance is the presence of gender identity disorder, a disease in humans. In terms of appearance, even if the gender has already been determined, there may be mentally opposite gender. When considering a group of organisms, apart from legal interpretation of the cases caused by a particular group, gender identity disorder is not necessarily tolerated by the human society. Even in such a case, it is conceivable that the increase or decrease in quantity of the physiologically active substance involved in “sociality” inside or outside the living body is involved. If the patient himself has discomfort, treatment may be necessary.
カブト虫の幼虫から蛹への変体の際に、個々の幼虫が距離をおき、飼育空間が秩序正しく保たれているので、各々の幼虫から放出される物質を分離して抽出することで、社会秩序を維持するための社会性に関与する生理活性物質を創出することができる。放出される生理活性物質は、幼虫を飼育した後の飼育媒体から検出し、分離することができる。ここで、飼育媒体とは、カブト虫の幼虫や蛹を飼育する際に必要な媒体であり、例えば腐葉土、飼育土壌、腐敗木材、おがくず、家畜糞便などが挙げられる。使用する飼育媒体は、カブト虫の蛹形成後、成虫へと変態し、蛹が存在しなくなった直後のものであり、蛹が存在していた飼育媒体を収集する。そして、その飼育媒体５００ミリリットルを約１０００ミリリットルの生理食塩水またはＰＢＳ等の緩衝液に懸濁させた後、その懸濁液をろ過することにより、カブト虫の幼虫より放出される生理活性物質を得ることができる。 During the transformation of beetle larvae to pupae, the individual larvae are separated from each other and the breeding space is maintained in an orderly manner. It is possible to create a physiologically active substance involved in sociality for maintaining the health. The released physiologically active substance can be detected and separated from the rearing medium after rearing the larvae. Here, the rearing medium is a medium necessary for rearing beetle larvae and pupae, and examples thereof include humus, rearing soil, spoiled wood, sawdust, and livestock feces. The breeding medium to be used is the one immediately after the beetle is transformed into an adult after forming the pupae, and the breeding medium in which the pupae are present is collected. Then, after suspending 500 ml of the rearing medium in a buffer solution such as about 1000 ml of physiological saline or PBS, the suspension is filtered to obtain a physiologically active substance released from the beetle larvae. be able to.
社会性に関与する生理活性物質は、カブト虫幼虫の飼育媒体を約１０００ミリリットルの生理食塩水またはＰＢＳ等の緩衝液に懸濁させ、その懸濁液をろ過後、液相クロマトグラフィー（ＬＣ）によって分離して抽出することにより、創出することができる。また、質量分析法（ＭＳ）による分子量の測定、ＮＭＲやＩＲなどの測定、アミノ酸配列情報等の結果を適宜取得し、解析することで創出された生理活性物質を同定することができる。 Physiologically active substances involved in sociality are obtained by suspending a beetle larvae rearing medium in a buffer solution such as about 1000 ml of physiological saline or PBS, and filtering the suspension, followed by liquid phase chromatography (LC). It can be created by separating and extracting. In addition, it is possible to identify a physiologically active substance created by appropriately obtaining and analyzing results such as measurement of molecular weight by mass spectrometry (MS), measurement of NMR, IR, and amino acid sequence information.
本発明の甲虫より放出された物質の抽出方法において、前記密度条件で飼育したカブト虫幼虫を、一定の物理学的条件下で飼育することで、より目的に応じた社会性に関与する生理活性物質を創出することができる。 In the method for extracting a substance released from a beetle of the present invention, a beetle larvae bred under the above-mentioned density conditions are bred under certain physical conditions, so that a physiologically active substance that is more involved in sociality according to the purpose. Can be created.
本発明のカブト虫の飼育では、幼虫から蛹に変態する時期に、各幼虫は、個体の密度を高い状態に保ちながら、自己の縄張り（場所）を確保している。ガラスやプラスチック製などの透明な飼育ケースを用いて飼育した場合、雌の蛹は、同ケースの周囲に位置し、雌の蛹が光を受容していることが観察できる。このことから、例えば、物理学的条件が光照射条件の場合に、雌雄の決定に係る関連遺伝子産物を含む物質を分離し、雌雄決定に係る社会性に関与する生理活性物質を創出することができる。この場合に、光照射は、一日あたり、８〜１２時間、好ましくは５月下旬の日照時間に合わせた連続した時間、照度２００〜８００ルクス、好ましくは４００ルクスを照射することが必要である。直射日光は、避けなければならないが、屋外で飼育する場合は、光源として日光を利用しても良い。このような条件を境意として、光を受容できた個体と受容できなかった個体について、カブト虫の雌雄が決定される（図１および図２参照）。 In the rearing of the beetle of the present invention, each larva secures its own territory (place) while maintaining the density of the individual at a time of transformation from the larva to the pupa. When bred using a transparent breeding case made of glass or plastic, the female pupae are located around the case and it can be observed that the female pupae receive light. From this, for example, when the physical condition is a light irradiation condition, it is possible to isolate a substance containing a related gene product related to sex determination and create a physiologically active substance involved in sociality related to sex determination. it can. In this case, the light irradiation needs to be performed for 8 to 12 hours per day, preferably for a continuous time according to the sunshine hours in late May, with an illuminance of 200 to 800 lux, preferably 400 lux. . Direct sunlight should be avoided, but when rearing outdoors, sunlight may be used as a light source. Based on such a condition, the sexes of beetles are determined for individuals that can and cannot receive light (see FIGS. 1 and 2).
性の決定および集団形成について分子生物学的に考察する。性を決定する遺伝子と集団形成を行う遺伝子は染色体上の近傍に位置し、相互が光という物理刺激を介して雌のカブト虫によって放出される生理活性物質によって遺伝子発現を制御され、それらが脳内の神経細胞の受容体（知覚、臭覚、視覚などの感覚を受容するタンパク質）に作用することによって自己と他者を認識し、その結果、個体として雌雄が分別されたと考えられる。 Consider molecular biology about sex determination and population formation. Genes that determine sex and genes that form populations are located close to each other on the chromosome, and their gene expression is controlled by physiologically active substances released by female beetles via a physical stimulus called light. It is thought that self and others are recognized by acting on the receptors (sensory, olfactory, visual and other sensations) of the nerve cells, and as a result, males and females are separated.
本発明のカブト虫より放出される物質を抽出することにより解決されるべき社会的課題は、３つ存在する。性同一性障害によるいじめなどによるトラウマ、社会性の欠如などの症状を呈する患者数が急増している。疾患は、解剖学的に見た所見としての性を受け入れることが出来ず、多岐にわたる疾患を併発する場合が多い。医師と患者間での問診などによって、病であるか否かが診断される。その結果が病と判断された場合は、患者が希望すれば、主としてホルモン療法や外科的治療が行われる。しかし、副作用も多くみられることから新たな医薬品の開発が求められている。従って、発明によって創出された生理活性物質または作用点が類似の化合物を投与することにより、新規の治療薬として応用することが本発明の延長線上の１つ目の課題である。 There are three social problems to be solved by extracting substances released from the beetles of the present invention. The number of patients presenting with symptoms such as trauma due to bullying due to gender identity disorder and lack of sociality is increasing rapidly. Diseases cannot accept sexuality as an anatomical finding and often accompany a wide variety of diseases. Whether or not the patient is ill is diagnosed by an inquiry between the doctor and the patient. If the result is determined to disease, if desired patients, primarily Hol mode emission therapy or surgical treatment is performed. However, since there are many side effects, the development of new drugs is required. Therefore, it is the first problem on the extension of the present invention to apply as a novel therapeutic agent by administering a compound having a similar physiologically active substance or action point created by the invention.
また、上述のような疾患時に、血液中、尿中、髄液中あるいは、体内の特定の部位にカブト虫幼虫の社会性を維持する遺伝子産物のヒトのホモログが減少、もしくは、増加している場合、本発明を応用し、関連遺伝子産物の変動を生化学的に検査することにより明らかにされる定量的または定性的な結果から、性同一性障害であることを客観的に診断することが可能になる。つまり、診断薬として応用することが本発明の延長線上の２つ目の課題である。 In addition, the human homologue of the gene product that maintains the sociality of beetle larvae in the blood, urine, cerebrospinal fluid, or a specific part of the body during the above-mentioned diseases is reduced or increased Applying the present invention, it is possible to objectively diagnose gender identity disorder based on quantitative or qualitative results revealed by biochemical examination of related gene product variation become. In other words, application as a diagnostic agent is the second problem on the extension of the present invention.
前述の方法により近親交配を行い、作製した半クローン化カブト虫の成虫は、外観が濃赤色であるのみならず、攻撃性や集光性も同一世代間で類似している。以上から、半クローン化カブト虫は、ほぼ遺伝的背景が同一なカブト虫であると考えられる。この幼虫が蛹になる際に、雌となる蛹は、透明な飼育容器の周囲に位置し、光に曝されることを本願発明者は見出した。さらに、そのカブト虫は、幼虫から蛹に変態する際に、関連遺伝子産物を含む生理活性物質を飼育媒体中に放出していることも確認した。つまり、この生理活性物質によってカブト虫の雌雄は決定されていると考えられる。この生理活性物質は、ヒトの性ホルモンではないことから、性同一性障害の治療に使用されている現存の医薬品とは異なる新薬を開発することが可能となり、１つ目の課題を解決させることが出来る。 The semi-cloned beetle adults produced by inbreeding by the above-mentioned method are not only dark red in appearance, but also have similar aggression and light collecting properties among the same generation. From the above, the semi-cloned beetle is considered to be a beetle having almost the same genetic background. The inventor of the present application has found that when this larva becomes a pupa, a female pupa is located around a transparent breeding container and is exposed to light. Furthermore, when the beetle transformed from a larva to a pupa, it was also confirmed that a physiologically active substance including a related gene product was released into the breeding medium. That is, it is considered that the male and female beetles are determined by this physiologically active substance. Since this bioactive substance is not a human sex hormone, it is possible to develop a new drug that is different from existing drugs used to treat gender identity disorder, and to solve the first problem. I can do it.
以下、本発明の理解を深めるために、実施例を示して具体的に本発明を説明するが、本発明は以下に限定されるものではないことはいうまでもない。 Hereinafter, in order to deepen an understanding of the present invention, the present invention will be specifically described with reference to examples, but it is needless to say that the present invention is not limited to the following.
（実施例１）光照射条件下でのカブト虫の飼育
本発明で取得したカブト虫は、初発の段階で、一対の親となる野生のカブト虫から数十個の卵を得た後、孵化させた幼虫を飼育媒体（市販の飼育用腐葉土）中で越冬飼育して得たものである。得られた幼虫は、５月頃に蛹に変態した後、成虫となった。成虫の中から、濃赤色の外観を有する個体を選別し、近親交配を行った。同様の近親交配の操作を繰り返し、全工程後の３年目に遺伝的背景をほぼ同一にしたカブト虫を取得した。尚、上記の発明操作は、全て室内で実施した。
(Example 1) Breeding of beetles under light irradiation conditions The beetles obtained in the present invention were larvae hatched after obtaining dozens of eggs from a pair of parental wild beetles at the initial stage. Was obtained by rearing in winter in a breeding medium (commercially available humus for breeding). The obtained larvae became adults after transforming into moths around May. Individuals having a dark red appearance were selected from adults and inbred. Similar inbreeding operations were repeated, and beetle insects having substantially the same genetic background were obtained in the third year after all the steps. All the above-described invention operations were performed indoors.
本実施例では、まず、幅４３ｃｍ、奥行き２６ｃｍ、高さ３１ｃｍの透明なプラスチック製の飼育容器を準備した。その中に、飼育媒体を当該容器の底面から約１５ｃｍまで万遍なく入れた。成虫になるまでの変態過程のいずれの個体も、上記で準備した容器の中に１５匹程度を育成させた。特に、蛹に変態する直前の幼虫の段階では、当該容器中の幼虫の数が本発明を遂行する上で重要な要素となる。蛹に変態する直前の幼虫を含む当該容器は、雌雄選別するために、光を照射する必要がある。ここで述べる光とは、日光、電力を用いた蛍光灯や白熱灯を指す。光は、常時、照射する必要は無く、太陽光の日周期を基本とし、１日当たり連続して約１２時間、４００ルクス（lx）で照射する必要がある。直射日光は、避けなければならないが、屋外で飼育する場合は、光源として日光を利用しても良い。 In this example, first, a transparent plastic breeding container having a width of 43 cm, a depth of 26 cm, and a height of 31 cm was prepared. Inside, the breeding medium was evenly placed from the bottom of the container to about 15 cm. About 15 individuals were grown in the container prepared above in any of the transformation processes until adulthood. In particular, the number of larvae in the container is an important factor in carrying out the present invention at the stage of the larvae immediately before transformation into a pupa. The container containing the larvae just before being transformed into a pupa needs to be irradiated with light in order to select the sex. The light described here refers to a fluorescent lamp and an incandescent lamp using sunlight and electric power. It is not necessary to irradiate light constantly, and it is necessary to irradiate at 400 lux (lx) continuously for about 12 hours per day based on the daily cycle of sunlight. Direct sunlight should be avoided, but when rearing outdoors, sunlight may be used as a light source.
上記操作後の雄の蛹は、容器の中央若しくは、光を避けるように容器の中に配置される。よって、容器の中央のみを掘り出すことによって、雄の蛹を効率よく回収することが可能である（図１参照）。 The male cage after the above operation is arranged in the middle of the container or in the container so as to avoid light. Therefore, it is possible to efficiently recover male pupae by digging out only the center of the container (see FIG. 1).
また、雄の成虫のみを選別する際には、容器の周囲の飼育媒体を別の容器に移すことによって、雌のカブト虫を排除する。この操作によって、成虫の雌雄の選別が可能となる。 When selecting only male adults, female beetles are eliminated by transferring the breeding medium around the container to another container. This operation makes it possible to select adult males and females.
雌雄選別における実施例を数値化すると、２つの容器に分けて飼育した合計３０匹のカブト虫のうち、雄の蛹は１９匹（光が照射される容器の外側に０匹、光が照射されない内側に１９匹が集団化）であり、雌の蛹は１１匹（光が照射される容器の外側に６匹、光が照射されない内側に５匹が集団化）であった。従って、カブト虫総数に対する雄の選別効率は、６３．３％であるが、光が照射されない容器の内側に限定すれば、雄の選別効率は１００％に達した。 Quantifying the examples in male and female selection, out of a total of 30 beetles reared in two containers, there were 19 male wings (0 outside the container irradiated with light, inside not irradiated with light) 19 were grouped), and 11 female pupae (6 on the outside of the container irradiated with light and 5 on the inside not irradiated with light). Therefore, the male selection efficiency relative to the total number of beetles was 63.3%, but the male selection efficiency reached 100% when limited to the inside of the container not irradiated with light.
（実施例２）選別されたカブト虫からの関連遺伝子産物を含む生理活性物質の創出
カブト虫を飼育していない媒体（培養土）対照とし、実施例１の方法で培養した飼育媒体（飼育容器内の全培養土）について、以下の処理を行い、二次元電気泳動およびＬＣ−ＭＳによりカブト虫が放出したと考えられる生理活性物質について分析した。
(Example 2) Creation of a physiologically active substance containing a related gene product from a selected beetle A culture medium (cultured soil) cultivated by the method of Example 1 as a medium (cultured soil) control that does not breed beetles All culture soils were subjected to the following treatment, and analyzed for physiologically active substances considered to be released by beetles by two- dimensional electrophoresis and LC-MS.
（１）二次元電気泳動による分離
５００ｍｌの飼育媒体をＰＢＳ約１０００ｍｌに混合し、懸濁液を調製した。同様に、カブト虫を飼育していない媒体をＰＢＳに混合し、懸濁液を調製したものを対照とした。上記調製した各懸濁液を限外濾過し、その後得られた溶液をＴＣＡ（トリクロル酢酸）沈澱させ、得られた沈澱物を可溶化させたものをサンプルとして用いた。可溶化に用いる溶液は、尿素（０．５１ｇ）、１０％（ｗ／ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）（０．０２ｍｌ）、２０％（ｖ／ｖ）トリトンＸ（polyoxyethylene-p -isooctylphenol）-100 （０．１０ｍｌ）、ジチオスレイトール（ＤＴＴ） （０．０１ｇ）、PharmalyteTM(GE ヘルスケア)（０．０１ｍｌ）をMilli-Q水で１．００ｍｌにメスアップして調製した。
(1) Separation by two- dimensional electrophoresis 500 ml of breeding medium was mixed with about 1000 ml of PBS to prepare a suspension. Similarly, a medium in which beetles were not raised was mixed with PBS and a suspension was prepared as a control. Each of the prepared suspensions was ultrafiltered, and then the obtained solution was precipitated with TCA (trichloroacetic acid), and the resulting precipitate was solubilized and used as a sample. The solution used for solubilization was urea (0.51 g), 10% (w / v) sodium dodecyl sulfate (SDS) (0.02 ml), 20% (v / v) Triton X (polyoxyethylene-p-isooctylphenol) − 100 (0.10 ml), dithiothreitol (DTT) (0.01 g) and Pharmalyte ™ (GE Healthcare) (0.01 ml) were prepared by making up to 1.00 ml with Milli-Q water.
上記調製した各サンプル８０μｌを、サンプル塗布用ろ紙５枚に染み込ませ、アプライした。 二次元電気泳動は、 一次元目はドライストリップ（ｐＨ４−７，１８ｃｍ）、 二次元目は１５％アクリルアミドゲルを用いて行なった。泳動により得られたスポットは、高感度タンパク質染色試薬SYPRO(R) Ruby（プロメガ社）を用いて染色した。 80 μl of each of the prepared samples was soaked in 5 sample application filter papers and applied. Two-dimensional electrophoresis was performed using a dry strip (pH 4-7, 18 cm) for the first dimension and a 15% acrylamide gel for the second dimension. The spots obtained by electrophoresis were stained using a highly sensitive protein staining reagent SYPRO (R) Ruby (Promega).
上記の結果を図３に示した。対照媒体から調製したサンプルを二次元泳動後に出現したスポットとカブト虫飼育媒体から調製したサンプルを二次元泳動後に出現したスポットを比較したところ、塩基性部分に異なるスポットを確認することができた。カブト虫が飼育媒体中に放出している因子が、ペプチドおよびポリペプチドであるならば、生理活性物質は、当該スポットに現れるものであると思慮される。 The above results are shown in FIG. When spots prepared after two-dimensional migration of the sample prepared from the control medium were compared with spots appeared after two-dimensional migration of the sample prepared from the beetle rearing medium, different spots could be confirmed in the basic portion. If the factors released by the beetle into the breeding medium are peptides and polypeptides, the bioactive substance is considered to appear at the spot.
（２）ＬＣ−ＭＳによる分離
上記調製した各サンプルは、それぞれ直径０．２２μｍのフィルターでろ過し、ろ過後の各１００μｌをＬＣ−ＭＳ解析装置に注入した。ＬＣ−ＭＳは、解析時間：６０分、０．１％蟻酸アセトニトリル溶液の濃度勾配：２０−９５％、０．１％蟻酸水溶液の濃度勾配：８０−５％の条件で解析を行い、注入後９分後辺りに認められたピークの分子量を測定した（図４および図５参照）。
(2) Separation by LC-MS Each of the prepared samples was filtered through a 0.22 μm diameter filter, and 100 μl of each filtered sample was injected into an LC-MS analyzer. LC-MS performs analysis under the conditions of analysis time: 60 minutes, concentration gradient of 0.1% formic acid acetonitrile solution: 20-95%, concentration gradient of 0.1% formic acid aqueous solution: 80-5%, after injection The molecular weight of the peak observed around 9 minutes was measured (see FIGS. 4 and 5).
上記の結果、ＬＣ分析においてカブト虫を飼育した飼育媒体から得たサンプルでは、９．３２分においてピークを認めたのに対し、対照から得たサンプルではそのようなピークは認めなかった（図４）。この９．３２分において認められたピークに係る物質は、カブト虫から放出された生理活性であると考えられる。 As a result of the above, a peak obtained at 9.32 minutes was observed in the sample obtained from the culture medium in which the beetle was reared in LC analysis, whereas no such peak was observed in the sample obtained from the control (FIG. 4). . The substance related to the peak observed at 9.32 minutes is considered to be the physiological activity released from the beetle.
さらに、ＭＳ分析を行った結果、カブト虫を飼育した飼育媒体から得たサンプルでは、対照から得たサンプルに比べ、４４８．４ｍ／ｚ，ａｍｕの部分でピークの大きさに顕著な違いが認められた（図５）。この４４８．４ｍ／ｚ，ａｍｕの部分において認められたピークに係る物質は、カブト虫から放出された生理活性であると考えられる。 Furthermore, as a result of MS analysis, in the sample obtained from the rearing medium where the beetle was reared, a significant difference was observed in the peak size in the portion of 448.4 m / z, amu compared to the sample obtained from the control. (FIG. 5). The substance related to the peak observed in this 448.4 m / z, amu portion is considered to be a physiological activity released from a beetle.
以上詳述したように、本発明の生理活性物質の創出方法により、飼育面積１１１０ｍｍ２に対して少なくとも２匹以上の密度条件で飼育したカブト虫において、幼虫から蛹への変体の際に、個々の幼虫が距離をおき、飼育空間が秩序正しく保たれていることから、各々の幼虫から放出され、分離された特定の生理活性物質は、社会秩序を一定に保つための社会性に関与する生理活性物質可能性が考えられた。 As described above in detail, in the beetle bred under a density condition of at least two animals with respect to a breeding area of 1110 mm 2 by the method for creating a physiologically active substance of the present invention, individual larvae are transformed during the transformation from larvae to pupae. Because the rearing space is kept in order and the breeding space is kept in order, the specific physiologically active substances released and separated from each larva are physiologically active substances involved in sociality to keep the social order constant. The possibility was considered.
２００７年末に集計された性同一性障害と診断された人は、約７０００人である。現状に不快感を持つ人には、治療が施される。医薬品としては、男性・女性ホルモンが使用されているが、副作用も多く、副作用の少ない医薬品の開発が求められている。 About 7000 people were diagnosed with gender identity disorder as of the end of 2007. People who are uncomfortable with the current situation are treated. Male and female hormones are used as pharmaceuticals, but there are demands for the development of pharmaceuticals with many side effects and few side effects.
本発明の結果、カブト虫から得られた関連遺伝子産物を含む生理活性物質をさらに分析して同定し、ヒトに対して重篤な副作用や毒性を示さず、薬効を示す物質を得ることができれば、上述のような治療薬として利用が可能である。 As a result of the present invention, if a physiologically active substance containing a related gene product obtained from a beetle is further analyzed and identified, and a substance exhibiting a medicinal effect can be obtained without showing serious side effects and toxicity to humans, It can be used as a therapeutic agent as described above.
さらには、性同一性障害の疾患患者の体内（血液、髄液、唾液など）から、本発明で創出された生理活性物質と同様の物質の増減が観察されれば、生化学的パラメータによる客観性を有した診断薬として利用が可能である。
Furthermore, if an increase or decrease in a substance similar to the physiologically active substance created in the present invention is observed in the body (blood, cerebrospinal fluid, saliva, etc.) of a patient with a gender identity disorder, an objective by biochemical parameters is observed. It can be used as a diagnostic agent with sex.
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