JPH114685A - Cell line producing hibernation specific protein and establishment thereof - Google Patents
Cell line producing hibernation specific protein and establishment thereofInfo
- Publication number
- JPH114685A JPH114685A JP9160258A JP16025897A JPH114685A JP H114685 A JPH114685 A JP H114685A JP 9160258 A JP9160258 A JP 9160258A JP 16025897 A JP16025897 A JP 16025897A JP H114685 A JPH114685 A JP H114685A
- Authority
- JP
- Japan
- Prior art keywords
- hibernation
- cells
- specific protein
- cell line
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、冬眠特異的タンパ
ク質産生樹立細胞株およびその創製方法に関する。[0001] The present invention relates to a cell line established for hibernation-specific protein production and a method for creating the same.
【０００２】[0002]
【従来の技術】冬眠特異的タンパク質はシマリスの血中
に存在し冬眠に同期して量が変化するタンパク質として
見出されたものである。冬眠特異的タンパク質は４種の
タンパク質(HP-20、HP-25、HP-27およびHP-55）で構成
される。HP-20、HP-25およびHP-27は、Ｎ−末端アミノ
酸の約４０残基にコラーゲン様配列（Gly-X-Yの繰り返
し）を含む互いに類似した構造を持つ。HP-55は、α1−
アンチトリプシン様の構造を有し、serpinスーパーファ
ミリーに属するものである。これらのいずれのタンパク
質も肝臓で特異的に発現されている。血中では、HP-2
0、HP-25およびHP-27は、コラーゲン様三重ラセン構造
と分子間ジスルフィド結合によりヘテロトリマー（HP-2
0C）を形成し、それにHP-55が会合し複合体を形成して
いる。2. Description of the Related Art Hibernation-specific proteins have been found as proteins that exist in the blood of chipmunk and change in amount in synchronization with hibernation. Hibernation-specific proteins are composed of four proteins (HP-20, HP-25, HP-27 and HP-55). HP-20, HP-25 and HP-27 have structures similar to each other with a collagen-like sequence (Gly-XY repeats) at about 40 residues of the N-terminal amino acid. HP-55 is α1-
It has an antitrypsin-like structure and belongs to the serpin superfamily. All of these proteins are specifically expressed in the liver. In the blood, HP-2
0, HP-25 and HP-27 form heterotrimers (HP-2) by a collagen-like triple helix structure and intermolecular disulfide bonds.
0C) to which HP-55 associates to form a complex.
【０００３】冬眠特異的タンパク質は齧歯目の冬眠動物
にのみ見出されており、冬眠に同期して年周性に血中の
量が変動し、非冬眠期に多く冬眠期に著しく減少する。
冬眠特異的タンパク質の減少したシマリスでは環境温度
を低下させることにより、冬眠を発現させることができ
ることから、冬眠特異的タンパク質が冬眠の発現に重要
な生理的役割を担っていると考えられている。[0003] Hibernation-specific proteins have been found only in rodent hibernation animals, and their amounts in the blood fluctuate annually in synchrony with hibernation, and are significantly reduced during non-hibernation and significantly during hibernation. .
Since a chipmunk with reduced hibernation-specific protein can express hibernation by lowering the environmental temperature, it is considered that the hibernation-specific protein plays an important physiological role in the expression of hibernation.
【０００４】[0004]
【発明が解決しようとする課題】上記のように冬眠特異
的タンパク質は冬眠の発現に重要であるため、その生理
学的機能の解明や期待されるその機能の種々の分野での
応用のために、冬眠特異的タンパク質を大量に得る手段
の確立が望まれる。従って、本発明は、高効率で冬眠特
異的タンパク質を産生する樹立細胞株を提供することを
課題とする。As described above, since the hibernation-specific protein is important for the expression of hibernation, it is necessary to clarify its physiological function and to apply the expected function to various fields. It is desired to establish means for obtaining hibernation-specific proteins in large quantities. Therefore, an object of the present invention is to provide an established cell line that produces hibernation-specific protein with high efficiency.
【０００５】[0005]
【課題を解決するための手段】本発明者は上記課題を解
決すべく鋭意検討を重ねた結果、冬眠動物の肝細胞とヘ
パトーマ(hepatoma)の細胞とを融合させることによっ
て、冬眠特異的タンパク質を高効率で産生する樹立細胞
株が得られることを見出し、本発明を完成するに至っ
た。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor fused a hibernating animal hepatocyte with a hepatoma cell to thereby obtain a hibernation-specific protein. The present inventors have found that an established cell line that can be produced with high efficiency can be obtained, and have completed the present invention.
【０００６】すなわち、本発明は、齧歯目の冬眠動物の
肝細胞とヘパトーマ細胞とを融合させ、冬眠特異的タン
パク質を産生する融合細胞を選択することを含む、冬眠
特異的タンパク質を産生する樹立細胞株の創製方法（以
下、「本発明方法」ともいう）を提供する。That is, the present invention provides a method for producing a hibernation-specific protein, which comprises fusing hepatoma cells and hepatoma cells of a rodent hibernating animal and selecting a fusion cell producing a hibernation-specific protein. A method for creating a cell line (hereinafter, also referred to as “the method of the present invention”) is provided.
【０００７】本発明方法は、選択された融合細胞を無血
清無タンパク質培地へ順化させることをさらに含んでも
よい。肝細胞が由来する齧歯目の冬眠動物はシマリスで
あることが好ましい。また、ヘパトーマ細胞は、チオグ
アニン耐性ラットヘパトーマ細胞H4TGであることが好ま
しい。[0007] The method of the present invention may further comprise acclimating the selected fused cells to a serum-free and protein-free medium. Preferably, the rodent hibernating animal from which hepatocytes are derived is a chipmunk. Further, the hepatoma cells are preferably thioguanine-resistant rat hepatoma cells H4TG.
【０００８】また、本発明は、本発明方法によって得ら
れる、冬眠特異的タンパク質を産生する樹立細胞株（以
下、「本発明細胞株」ともいう）を提供する。本発明細
胞株は、冬眠特異的タンパク質を高効率に産生する樹立
された細胞株であるので、それを培養することによって
冬眠特異的タンパク質を容易に且つ大量に産生させるこ
とができる。[0008] The present invention also provides an established cell line that produces a hibernation-specific protein obtained by the method of the present invention (hereinafter, also referred to as “cell line of the present invention”). Since the cell line of the present invention is an established cell line that produces hibernation-specific proteins with high efficiency, it is possible to produce hibernation-specific proteins easily and in large quantities by culturing them.
【０００９】[0009]
【発明の実施の形態】以下、本発明の実施の形態につい
て、詳細に説明する。本発明方法は、齧歯目の冬眠動物
の肝細胞とヘパトーマ細胞とを融合させ、冬眠特異的タ
ンパク質を産生する融合細胞を選択することを特徴とす
る。Embodiments of the present invention will be described below in detail. The method of the present invention is characterized by fusing hepatoma cells and hepatoma cells of a rodent hibernating animal and selecting a fused cell that produces a hibernation-specific protein.
【００１０】齧歯目の冬眠動物とは、齧歯目に属する体
温低下に耐える動物を意味し、例えば、シマリス、ジリ
ス、ヤマネ、ハリネズミ、マーモット等が挙げられ、好
ましくはシマリスである。[0010] The rodent hibernating animal means an animal belonging to rodents that resists a decrease in body temperature, and examples thereof include chipmunk, ground squirrel, dormouse, hedgehog, marmot, and the like, and preferably chipmunk.
【００１１】齧歯目の冬眠動物からの肝細胞の分離は、
公知の細胞分離方法によって行うことができ、例として
は、動物を麻酔した後、開腹し、門脈を介して灌流し、
次いで、コラゲナーゼを含む灌流液でさらに灌流した
後、灌流液で満たしたペトリシャーレに移し、ハサミで
細切し、穏やかなピペッティングにより細胞を分散さ
せ、細胞懸濁液をナイロンメッシュで濾し、濾液から遠
心分離により細胞を得るという方法、および、麻酔下で
肝臓を摘出し、ハサミで細切した後、コラゲナーゼで処
理し細胞を分散させ、ナイロンメッシュで濾し、濾液か
ら遠心分離により細胞を得るという方法が挙げられる。[0011] Isolation of hepatocytes from rodent hibernating animals comprises:
It can be performed by a known cell separation method, for example, after anesthetizing an animal, laparotomy, perfusion through the portal vein,
Then, after further perfusion with a perfusion solution containing collagenase, the cells were transferred to a Petri dish filled with the perfusion solution, cut into small pieces with scissors, the cells were dispersed by gentle pipetting, and the cell suspension was filtered through a nylon mesh. The method of obtaining cells by centrifugation from, and removing the liver under anesthesia, shredding with scissors, treating with collagenase, dispersing the cells, filtering through a nylon mesh, and obtaining cells from the filtrate by centrifugation Method.
【００１２】ヘパトーマ細胞は、肝細胞ガンの細胞であ
り、その由来や種類に特に制限はない。例えば、ATCC
（12301 Parklawn Drive, Rockville, MD 20852, U.S.
A.）から入手できるチオグアニン耐性ラットヘパトーマ
細胞H4TGが挙げられる。Hepatoma cells are hepatocellular carcinoma cells, and their origin and type are not particularly limited. For example, ATCC
(12301 Parklawn Drive, Rockville, MD 20852, US
A.), thioguanine-resistant rat hepatoma cells H4TG.
【００１３】肝細胞とヘパトーマ細胞との融合は、ポリ
エチレングリコール法、電気融合法、両方法の併用等の
公知の方法に従って行うことができる。融合条件は、肝
細胞やヘパトーマ細胞の由来、種類等により、適宜選択
できる。Fusion of hepatocytes and hepatoma cells can be performed according to a known method such as a polyethylene glycol method, an electrofusion method, or a combination of both methods. Fusion conditions can be appropriately selected depending on the origin and type of hepatocytes and hepatoma cells.
【００１４】例えば、ポリエチレングリコール法の場合
には、ウィリアムＥ培地に分散させたヘパトーマ細胞
を、10％牛胎児血清、 OPIサプリメント(Sigma), HATサ
プリメント, 10-7Mデキサメタゾン、10-6Ｍリノレン酸
を含むイスコDMEM培地(Iscow'sDMEM medium)（GIBCO BR
L）に分散し直し、肝細胞とヘパトーマ細胞とを4：1の
割合で混和し、1日間CO2インキュベーター内で培養し、
これを50%ポリエチレングリコール(PEG)（分子量3000-3
700）で45秒間処理し細胞を融合させるという条件が挙
げられる。For example, in the case of the polyethylene glycol method, hepatoma cells dispersed in a William E medium are mixed with 10% fetal bovine serum, OPI supplement (Sigma), HAT supplement, 10 -7 M dexamethasone, 10 -6 M linolene. Iscow's DMEM medium containing acid (GIBCO BR
L), hepatocytes and hepatoma cells are mixed at a ratio of 4: 1, and cultured in a CO 2 incubator for 1 day.
This is 50% polyethylene glycol (PEG) (molecular weight 3000-3
700) for 45 seconds to fuse the cells.
【００１５】また、例えば、電気融合法の場合には、肝
細胞とヘパトーマ細胞を1：2で混和し、遠心後（450rp
m, 2分）沈殿細胞を0.25M スクロース液に懸濁する操作
を繰り返した後、適量の0.25M スクロース液に懸濁し、
5 x 107 cells/mlの濃度に調整し、この細胞分散液を、
電極間隔が0.2mmのキュベットに入れ、2.5kV/cmの電圧
で65msecの間持続するパルスを10回与え細胞融合させる
という条件が挙げられる。For example, in the case of the electrofusion method, hepatocytes and hepatoma cells are mixed at a ratio of 1: 2, and centrifuged (450 rp).
m, 2 minutes) After repeating the operation of suspending the precipitated cells in 0.25M sucrose solution, suspending the cells in an appropriate amount of 0.25M sucrose solution,
Adjust the concentration to 5 x 10 7 cells / ml, and add this cell dispersion
The condition is that a cell is placed in a cuvette having an electrode spacing of 0.2 mm, and a pulse lasting 65 msec at a voltage of 2.5 kV / cm is given 10 times to cause cell fusion.
【００１６】融合細胞の選択は、例えば、細胞を２４穴
培養プレート等を用いて希釈培養し、培養上清について
冬眠特異的タンパク質を測定し、該冬眠特異的タンパク
質が検出されたクローンを選別することを数回例えば４
回繰り返すことによって行うことができる。For selection of the fused cells, for example, the cells are diluted and cultured using a 24-well culture plate or the like, the hibernation-specific protein is measured from the culture supernatant, and the clone in which the hibernation-specific protein is detected is selected. Do it a few times eg 4
This can be done by repeating the procedure several times.
【００１７】冬眠特異的タンパク質の検出は、冬眠特異
的タンパク質に特異的な抗体を用いるELISA法等によっ
て行うことができる。冬眠特異的タンパク質に特異的な
抗体は、例えば、シマリス血漿からHP-20複合体（HP-2
0、HP-25およびHP-27の会合体）とHP-55を精製し（Kond
o, J. and Kondo, N., Structural aspects of complex
of hibernation-specific proteins. in Adaptation t
o the cold, eds. F. Geiser, A.J. Hulbert, S.C. Nic
ol, University of New England Press, 351-355 (199
6); Kondo, N. and Kondo, J., Indentification of no
vel blood proteins specific for mammalian hibernat
ion., J. Biol. Chem., 267, 473-478 (1992)）、ウサ
ギに免疫し、得られた抗血清からIgGを精製して抗HP-20
複合体ポリクローナル抗体および抗HP-55ポリクローナ
ル抗体を得ることができ、また、HP-20複合体を還元、
変性下で精製し（上記文献参照）得られた各HP-20、HP-
25、HP-27を、同様にウサギに免疫し、各HP-20、HP-2
5、HP-27に対するポリクローナル抗体を得ることができ
る。ELISA法としては、抗HP-20複合体抗体をELISAプレ
ートに固相化し、これに測定サンプルを反応させ、さら
に二次抗体としてビオチン化抗HP-25抗体を反応させ、
ＨＲＰ(horseradish peroxidase)発色法により定量する
ものを挙げることができる。The hibernation-specific protein can be detected by an ELISA method using an antibody specific to the hibernation-specific protein. Antibodies specific for hibernation-specific proteins include, for example, HP-20 complex (HP-2
0, an aggregate of HP-25 and HP-27) and HP-55 (Kond
o, J. and Kondo, N., Structural aspects of complex
of hibernation-specific proteins. in Adaptation t
o the cold, eds. F. Geiser, AJ Hulbert, SC Nic
ol, University of New England Press, 351-355 (199
6); Kondo, N. and Kondo, J., Indentification of no
vel blood proteins specific for mammalian hibernat
Ion., J. Biol. Chem., 267, 473-478 (1992)), rabbits were immunized, and IgG was purified from the obtained antiserum to obtain anti-HP-20.
A complex polyclonal antibody and an anti-HP-55 polyclonal antibody can be obtained, and the HP-20 complex is reduced,
Each HP-20, HP-
Rabbits were immunized with HP-27 and HP-27 in the same manner.
5. A polyclonal antibody against HP-27 can be obtained. As an ELISA method, an anti-HP-20 complex antibody is immobilized on an ELISA plate, a measurement sample is reacted with the immobilized antibody, and a biotinylated anti-HP-25 antibody is further reacted as a secondary antibody.
Examples include those determined by the HRP (horseradish peroxidase) color development method.
【００１８】また、融合細胞の選択は、融合後培養した
細胞を分散させ、これに冬眠特異的タンパク質抗体を反
応させ、細胞をセルソーターを用いて分取し、分取した
各画分の細胞を培養し、冬眠特異的タンパク質産生細胞
が分離された画分を選別することによって行うこともで
きる。The fusion cells are selected by dispersing the cells cultured after the fusion, reacting the cells with a hibernation-specific protein antibody, sorting the cells using a cell sorter, and sorting the cells in each fraction. It can also be carried out by culturing and selecting a fraction from which hibernation-specific protein-producing cells have been separated.
【００１９】上記のようにして融合および選択を行って
得られた細胞株は、樹立細胞株であり、冬眠特異的タン
パク質を培地中に分泌するという性質を有する。また、
チオグアニン耐性ラットヘパトーマ細胞H4TGに由来して
得られた細胞株は、ヌードマウス（胸腺欠如による免疫
不全実験動物）の皮下への注入により成長し、腫瘍を形
成することができる。しかし、腫瘍成長の間、動物は元
気に生存し、悪性腫瘍ではないと思われる。The cell line obtained by fusion and selection as described above is an established cell line and has the property of secreting a hibernation-specific protein into the medium. Also,
The cell line obtained from the thioguanine-resistant rat hepatoma cell H4TG can grow and form tumors by subcutaneous injection of nude mice (animal-deficient experimental animals due to lack of thymus). However, during tumor growth, the animals survived well and do not appear to be malignant.
【００２０】本発明方法は、選択された融合細胞を無血
清無タンパク質培地へ順化させることをさらに含んでも
よい。融合細胞の無血清無タンパク質培地への順化は、
培養細胞を無血清無タンパク質培地へ順化させる公知の
方法に従って、無血清無タンパク質培地へ培養培地を交
換することによって行うことができる。[0020] The method of the present invention may further comprise acclimating the selected fused cells to a serum-free protein-free medium. Acclimation of the fused cells to serum-free protein-free medium
According to a known method for acclimating a cultured cell to a serum-free protein-free medium, the culture can be performed by replacing the culture medium with a serum-free protein-free medium.
【００２１】無血清無タンパク質培地へ順化した細胞株
は無血清無タンパク質培地で培養することができ、従っ
て、培地中に分泌された冬眠特異的タンパク質の精製が
容易になる。A cell line adapted to a serum-free protein-free medium can be cultured in a serum-free protein-free medium, thus facilitating purification of hibernation-specific proteins secreted into the medium.
【００２２】冬眠特異的タンパク質（以下、ＨＰともい
う）は齧歯目の動物において、冬眠動物だけに見出され
ている血中タンパク質で、冬眠が起こる時期に量が特異
的に変化する。ＨＰ濃度が冬眠様に変化したシマリス
は、環境温度を低下させることにより、直ちに冬眠を発
現させることができる。冬眠中は組織の低温に対する耐
性の増大や、細菌感染、肉腫、放射線などの有害因子に
対する生体の抵抗性の増大が観察されることから、ＨＰ
が生体防御に関わる因子であると考えられる。Hibernation-specific protein (hereinafter, also referred to as HP) is a blood protein found only in hibernating animals in rodents, and its amount is specifically changed when hibernation occurs. A chipmunk whose HP concentration has changed to hibernation can immediately cause hibernation by lowering the environmental temperature. During the hibernation period, increased resistance of tissues to low temperatures and increased resistance of living bodies to harmful factors such as bacterial infection, sarcoma, and radiation are observed.
Is considered to be a factor involved in host defense.
【００２３】ＨＰ構成成分の一つであるHP-55は、セリ
ンプロテアーゼ阻害物質であることが明らかにされてい
る。セリンプロテアーゼ阻害物質は、炎症、血液凝固、
補体系の活性化を仲介するプロテアーゼを制御すること
が知られており、組織をプロテアーゼによる損傷や崩壊
から保護する因子として注目される。HP-55, one of the HP components, has been shown to be a serine protease inhibitor. Serine protease inhibitors are used in inflammation, blood clotting,
It is known to regulate proteases that mediate the activation of the complement system, and is attracting attention as a factor that protects tissues from protease damage and disruption.
【００２４】さらに、HP-20Cは、セリンプロテアーゼ阻
害物質であるHP-55と特異的に会合することや、その構
造の類似性から、補体系を活性化するタンパク質である
Ｃ１ｑに類似している。補体系は、抗体を介して種々の
感染症の原因となるウィルスや細菌を排除する重要な役
割をすることから、ＨＰは補体系に関わる新たな生体防
御因子として期待できる。Furthermore, HP-20C is similar to C1q, a protein that activates the complement system, due to its specific association with HP-55, a serine protease inhibitor, and its structural similarity. . Since the complement system plays an important role in eliminating viruses and bacteria that cause various infectious diseases via antibodies, HP can be expected as a new biological defense factor related to the complement system.
【００２５】この様に、ＨＰは生体防御物質として、医
療の分野での応用も期待でき、ＨＰを容易に且つ大量に
産生することを可能とする本発明細胞株は産業上極めて
利用価値が高いと考えられる。As described above, HP can be expected to be applied in the medical field as a biological defense substance, and the cell line of the present invention, which enables easy and large-scale production of HP, has extremely high industrial value. it is conceivable that.
【００２６】[0026]
【実施例】以下に、実施例によってさらに具体的に説明
する。 １．シマリス由来肝細胞の分離 シマリスをネンブタール(1ml/kg)で麻酔した後、全身を
アルコール滅菌し、クリーンベンチ内で開腹し、門脈を
介して３７℃に加温したHanks液（GIBCO BRL）（Caイオ
ンおよびMgイオンを除き、10mM ＥＧＴＡと5mM HEPESを
加え、pH 7.5に調整）を10分間灌流した。次いで、0.05
％コラゲナーゼ（コラゲナーゼヤクルト、（株）三光純
薬）を含むMgイオン欠如Hanks液でさらに10分間灌流し
た後、10ml Hanks液で満たしたペトリシャーレに移し、
ハサミで細切し、穏やかなピペッティングにより細胞を
分散させた。細胞懸濁液をナイロンメッシュで濾し、濾
液から遠心分離により細胞を得た。The present invention will be described more specifically with reference to the following examples. 1. Separation of chipmunk-derived hepatocytes After the chipmunk was anesthetized with Nembutal (1 ml / kg), the whole body was sterilized with alcohol, laparotomy was performed in a clean bench, and the Hanks solution (GIBCO BRL) heated to 37 ° C. through the portal vein (GIBCO BRL) ( Excluding Ca ions and Mg ions, 10 mM EGTA and 5 mM HEPES were added, and the mixture was adjusted to pH 7.5) for 10 minutes. Then 0.05
% Collagenase (collagenase Yakult, Sanko Junyaku Co., Ltd.), perfused with Hanks solution lacking Mg ions for 10 minutes, and transferred to a Petri dish filled with 10 ml Hanks solution.
The cells were minced with scissors and dispersed by gentle pipetting. The cell suspension was filtered through a nylon mesh, and cells were obtained from the filtrate by centrifugation.
【００２７】本細胞を、７％牛胎児血清、10-7Ｍインス
リン、10-7Ｍデキサメタゾン、10-9Ｍ表皮成長因子、お
よび、10-8Ｍアプロチニン(aprotinin)を含むウィリア
ムＥ培地(Willium E medium)（GIBCO BRL)に懸濁し、ガ
ン細胞との細胞融合に使用した。The cells were cultured in a William E medium (Willium medium) containing 7% fetal calf serum, 10 -7 M insulin, 10 -7 M dexamethasone, 10 -9 M epidermal growth factor, and 10 -8 M aprotinin. E medium) (GIBCO BRL) and used for cell fusion with cancer cells.
【００２８】２．肝細胞とガン細胞の融合 (1) ポリエチレングリコール法 ガン細胞として、６−チオグアニン耐性ラットヘパトー
マ細胞（6-thioguanine-resistant rat hepatoma）H4TG
をATCCから購入した。ウィリアムＥ培地に分散させたH4
TG細胞を、再度、10％牛胎児血清、 OPIサプリメント(S
igma), HATサプリメント, 10-7Mデキサメタゾン、10-6
Ｍリノレン酸を含むイスコDMEM培地(Iscow's DMEM medi
um)（GIBCO BRL）に分散し直し、上記１で得られた肝細
胞とH4TG細胞とを4：1の割合で混和し、1日間CO2インキ
ュベーター内で培養した。これを50%ポリエチレングリ
コール(PEG)（分子量3000-3700）で45秒間処理し細胞を
融合させた。2. Fusion of hepatocytes and cancer cells (1) Polyethylene glycol method As cancer cells, 6-thioguanine-resistant rat hepatoma H4TG
Was purchased from ATCC. H4 dispersed in William E medium
TG cells, 10% fetal calf serum, OPI supplement (S
igma), HAT supplement, 10 -7 M dexamethasone, 10 -6
Iscow's DMEM media containing M linolenic acid
um) (GIBCO BRL), the hepatocytes obtained in 1 above and H4TG cells were mixed at a ratio of 4: 1, and cultured in a CO 2 incubator for 1 day. This was treated with 50% polyethylene glycol (PEG) (molecular weight 3000-3700) for 45 seconds to fuse the cells.
【００２９】(2) 電気融合(electrofusion)法 また、ガン細胞として、アザグアニン耐性マウスミエロ
ーマ細胞(azaguanin-resistant mouse myeloma)P3U1を
大日本製薬から購入した。上記１で得られた肝細胞とP3
U1細胞を1：2で混和し、遠心後（450rpm, 2分）沈殿細
胞を0.25M スクロース液に懸濁する。この操作を繰り返
し適量の0.25M スクロース液に懸濁し、5 x 107 cells/
mlの濃度に調整する。この細胞分散液を、電極間隔が0.
2mmのキュベットに入れ、2.5kV/cmの電圧で65msecの間
持続するパルスを10回与え細胞融合させた。(2) Electrofusion Method As a cancer cell, azaguanin-resistant mouse myeloma P3U1 was purchased from Dainippon Pharmaceutical. Hepatocytes obtained in 1 above and P3
U1 cells are mixed 1: 2, and after centrifugation (450 rpm, 2 minutes), the precipitated cells are suspended in 0.25 M sucrose solution. Repeat this operation repeatedly in an appropriate amount of 0.25M sucrose solution, and suspend at 5 x 10 7 cells /
Adjust to a concentration of ml. This cell dispersion was used with an electrode spacing of 0.
The cells were placed in a 2 mm cuvette and subjected to 10 pulses at a voltage of 2.5 kV / cm for 65 msec to cause cell fusion.
【００３０】３．冬眠特異的タンパク質（ＨＰ）産生細
胞の選択と試験 細胞融合後、24穴培養プレート5枚に細胞を分注し、10
％牛胎児血清、 OPIサプリメント(Sigma), HATサプリメ
ント, 10-7Mデキサメタゾンを含むイスコDMEM培地（GIB
CO BRL）を用いて、10日間培養を行い、ELISA法によっ
て各細胞上清をテストした。この結果５つのHP産生クロ
ーンが得られた。これに対し、さらに４回の希釈分散、
培養を繰り返し、当初の５つのクローンに比べ2500倍の
効率でHPを産生する４クローンを得た。これら４つのク
ローンは、肝細胞とヘパトーマ細胞との融合細胞に由来
するものであった。3. Selection and testing of hibernation-specific protein (HP) -producing cells After cell fusion, cells were dispensed into five 24-well culture plates and
% Fetal bovine serum, OPI supplement (Sigma), HAT supplement, Isco DMEM medium containing 10 -7 M dexamethasone (GIB
(CO BRL) for 10 days, and each cell supernatant was tested by ELISA. As a result, five HP-producing clones were obtained. On the other hand, four more dilution dispersions,
The culture was repeated to obtain 4 clones producing HP with 2500 times the efficiency of the initial 5 clones. These four clones were derived from fusion cells of hepatocytes and hepatoma cells.
【００３１】なお、上記ELISA法に使用した抗体は以下
のようにして得た。シマリス血漿からHP-20複合体（HP-
20、HP-25およびHP-27の会合体）とHP-55を精製し（Kon
do,J. and Kondo, N., Structural aspects of complex
of hibernation-specificproteins. in Adaptation to
the cold, eds. F. Geiser, A.J. Hulbert, S.C.Nico
l, University of New England Press, 351-355 (199
6); Kondo, N. and Kondo, J., Indentification of no
vel blood proteins specific for mammalianhibernati
on., J. Biol. Chem., 267, 473-478 (1992)）、ウサギ
に免疫し、得られた抗血清からIgGを精製して抗HP-20複
合体ポリクローナル抗体および抗HP-55ポリクローナル
抗体を得た。また、HP-20複合体を還元、変性下で精製
し（上記文献参照）得られた各HP-20、HP-25、HP-27
を、同様にウサギに免疫し、各HP-20、HP-25、HP-27に
対するポリクローナル抗体を得た。ELISA法は、抗HP-20
複合体抗体をELISAプレートに固相化し、これに測定サ
ンプルを反応させ、さらに二次抗体としてビオチン化抗
HP-25抗体を反応させ、ＨＲＰ(horseradish peroxidas
e)発色法により定量するものであった。The antibodies used in the above ELISA were obtained as follows. Chipmunk plasma from HP-20 complex (HP-
20, HP-25 and HP-27) and HP-55 (Kon
do, J. and Kondo, N., Structural aspects of complex
of hibernation-specific proteins. in Adaptation to
the cold, eds. F. Geiser, AJ Hulbert, SCNico
l, University of New England Press, 351-355 (199
6); Kondo, N. and Kondo, J., Indentification of no
vel blood proteins specific for mammalianhibernati
on., J. Biol. Chem., 267, 473-478 (1992)), rabbits were immunized, and IgG was purified from the obtained antiserum to obtain an anti-HP-20 complex polyclonal antibody and an anti-HP-55 polyclonal. Antibodies were obtained. In addition, the HP-20 complex was purified under reduction and denaturation (see the above literature), and the obtained HP-20, HP-25, and HP-27 were obtained.
Was similarly immunized to rabbits to obtain polyclonal antibodies against HP-20, HP-25 and HP-27. ELISA method is anti-HP-20
The conjugate antibody is immobilized on an ELISA plate, and a measurement sample is reacted with the immobilized antibody.
HP-25 antibody was reacted and HRP (horseradish peroxidas
e) It was determined by the color development method.
【００３２】得られたクローンで産生されたHPを簡便に
精製できるようにするため、無血清、無タンパク質培地
PFHM-II(Gibco BRL)にOPIサプリメント(Sigma), 10-7M
デキサメタゾンを加えた培養液へ培養培地を交換するこ
とにより、無血清無タンパク質培地への細胞の順化を行
った。４クローン中２クローンで順化に成功した。な
お、HP-20、HP-25、HP-27およびHP-55に対するポリクロ
ーナル抗体を用いたウェスタンブロット(western blot)
法で各HP-20ファミリーとHP-55を測定した結果、２つの
クローン間でHP産生効率は200倍の差があった。In order to be able to easily purify the HP produced by the obtained clone, a serum-free and protein-free medium
OPI supplement (Sigma) with PFHM-II (Gibco BRL), 10 -7 M
The cells were adapted to a serum-free protein-free medium by replacing the culture medium with a culture solution containing dexamethasone. Acclimation was successful in 2 out of 4 clones. Incidentally, Western blot using a polyclonal antibody against HP-20, HP-25, HP-27 and HP-55 (western blot)
As a result of measuring each HP-20 family and HP-55 by the method, there was a 200-fold difference in HP production efficiency between the two clones.
【００３３】[0033]
【発明の効果】以上説明したように、本発明によって、
冬眠特異的タンパク質を高効率で産生する樹立細胞株が
提供される。本樹立細胞株の提供によって、希少な冬眠
特異的タンパク質を培養により容易に且つ大量に入手す
ることが可能になる。As described above, according to the present invention,
An established cell line that produces hibernation-specific proteins with high efficiency is provided. By providing the established cell line, rare hibernation-specific proteins can be obtained easily and in large amounts by culturing.
Claims (5)
細胞とを融合させ、冬眠特異的タンパク質を産生する融
合細胞を選択することを含む、冬眠特異的タンパク質を
産生する樹立細胞株の創製方法。1. Creation of an established cell line producing a hibernation-specific protein, comprising fusing hepatoma cells and hepatoma cells of a rodent hibernating animal and selecting a fusion cell producing a hibernation-specific protein. Method.
質培地へ順化させることをさらに含む請求項１に記載の
創製方法。2. The method according to claim 1, further comprising acclimating the selected fused cells to a serum-free and protein-free medium.
請求項１または２に記載の創製方法。3. The method according to claim 1, wherein the rodent hibernating animal is a chipmunk.
性ラットヘパトーマ細胞H4TGである請求項１〜３のいず
れか１項に記載の創製方法。4. The method according to claim 1, wherein the hepatoma cell is a thioguanine-resistant rat hepatoma cell H4TG.
製方法によって得られる、冬眠特異的タンパク質を産生
する樹立細胞株。5. An established cell line that produces a hibernation-specific protein, obtained by the creation method according to any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9160258A JPH114685A (en) | 1997-06-17 | 1997-06-17 | Cell line producing hibernation specific protein and establishment thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9160258A JPH114685A (en) | 1997-06-17 | 1997-06-17 | Cell line producing hibernation specific protein and establishment thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH114685A true JPH114685A (en) | 1999-01-12 |
Family
ID=15711126
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9160258A Pending JPH114685A (en) | 1997-06-17 | 1997-06-17 | Cell line producing hibernation specific protein and establishment thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH114685A (en) |
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