JP2799455B2 - Method for producing hepatocyte growth factor - Google Patents
Method for producing hepatocyte growth factorInfo
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- JP2799455B2 JP2799455B2 JP63286844A JP28684488A JP2799455B2 JP 2799455 B2 JP2799455 B2 JP 2799455B2 JP 63286844 A JP63286844 A JP 63286844A JP 28684488 A JP28684488 A JP 28684488A JP 2799455 B2 JP2799455 B2 JP 2799455B2
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- growth factor
- hepatocyte growth
- activity
- sdgf
- hepatocyte
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、生体外において肝実質細胞の増殖を促進す
る活性を有する肝実質細胞増殖因子及びその製造法に関
する。Description: TECHNICAL FIELD The present invention relates to a hepatocyte growth factor having an activity of promoting proliferation of hepatocytes in vitro and a method for producing the same.
肝臓は、生体中で解毒、血清タンパクの生産、アンモ
ニアの処理、血糖調節、胆汁分泌等、多彩な機能をはた
し、肝実質細胞の機能障害は死をまねくことが多い。従
って、肝炎、肝硬変など肝実質細胞の傷害がある時に
は、生体内における健全な肝実質細胞の増殖を促すと
か、生体外から補充することが求められる。The liver performs various functions such as detoxification, production of serum proteins, processing of ammonia, regulation of blood sugar, and bile secretion in a living body, and dysfunction of hepatic parenchymal cells often leads to death. Therefore, when there is damage to liver parenchymal cells such as hepatitis or cirrhosis, it is required to promote healthy proliferation of liver parenchymal cells in a living body or to replenish it from outside the body.
肝細胞の生体外培養において、従来ホルモンとして、
上皮細胞成長因子（Epidermal Growth Factor:EGF,Carp
enter,G.et al.Ann.Rev.Biochem.48.193（1979））、イ
ンスリン（Insulin）、あるいは、肝細胞増殖因子（Hep
atocyte Growth Factor:HGF,Nakamura et al.Proc.Nat
l.Acad.Sci.USA.83,6489（1986））などが用いられ、こ
れらに肝細胞の増殖を促す活性があることが知られてい
る。しかし、これらによる肝実質細の分裂増殖は、従
来、１〜２回しか誘起できず、新しい増殖活性の高い増
殖因子の発見が望まれていた。In vitro culture of hepatocytes, as a conventional hormone,
Epidermal Growth Factor (EGF, Carp
enter, G.et al.Ann.Rev.Biochem. 48 .193 ( 1979)), insulin (Insulin), or hepatocyte growth factor (Hep
atocyte Growth Factor: HGF, Nakamura et al. Proc. Nat
l.Acad.Sci.USA. 83 , 6489 (1986)) and the like, which are known to have an activity of promoting hepatocyte proliferation. However, mitotic proliferation of the liver parenchyma by these methods has been conventionally induced only once or twice, and it has been desired to find a new growth factor having high growth activity.
ところで、最近、肝細胞を脾臓に注入すると、やがて
肝細胞は脾臓組織内で分裂をはじめ、旺盛に増殖するこ
とのあることが報告されている（水戸廸郎，医学のあゆ
み，125（４） p.573〜587,1983）。このような現象に
ついて、脾臓組織は肝細胞にとって、物理化学的に生存
しやすい場所なのか、何か特定の増殖因子が含まれてい
るのか、或いは、全く別の理由なのかは現在のところ全
く不明である。Recently, it has been reported that when hepatocytes are injected into the spleen, the hepatocytes may start to divide in the spleen tissue and proliferate vigorously (Dimio Mito, History of Medicine, 125 (4) p .573-587,1983). At this time, it is not at all clear whether the spleen tissue is a physicochemically viable place for hepatocytes, whether it contains any particular growth factor, or for a completely different reason. Unknown.
本発明者らは、肝細胞の増殖因子を探索する研究過程
において、上記肝細胞の脾臓移植に関する報告に注目
し、脾臓内に肝実質細胞増殖因子が含まれているか否か
の検討を注意深く行った結果、脾臓の細胞間物質のうち
生理食塩液に可溶性の分画から、肝実質細胞増殖因子が
得られることを見い出し、又その物理化学的性質を明ら
かにして本発明を完成するに至った。The present inventors have paid attention to the report on the spleen transplantation of hepatocytes during the research process of searching for hepatocyte growth factors, and carefully examined whether hepatocyte growth factors are contained in the spleen. As a result, he found that hepatocyte growth factor was obtained from the fraction soluble in physiological saline among the intercellular substances of the spleen, and clarified the physicochemical properties thereof, thereby completing the present invention. .
すなわち本発明は、脾臓の細胞間物質から肝実質細胞
増殖因子を抽出分離することにより肝細胞増殖因子を製
造する方法と、下記の理化学的性質及び生理活性を有す
る物質であることを特徴とする肝実質細胞増殖因子（Sp
leen Drived Growth Factor,以下SDGFと呼ぶ）に係わ
る。That is, the present invention is a method for producing hepatocyte growth factor by extracting and separating hepatocyte growth factor from splenic intercellular substances, and a substance having the following physicochemical properties and physiological activities. Hepatocyte growth factor (Sp
leen Drived Growth Factor (hereinafter referred to as SDGF).
（ａ）分子量が約40,000と約150,000又はそれ以上。(A) The molecular weight is about 40,000 and about 150,000 or more.
（ｂ）ヘパリンカラムに吸着しない。(B) Does not adsorb to the heparin column.
（ｃ）100℃、２分間の加熱処理によって肝実質細胞増
殖促進活性が失活する。(C) Heat treatment at 100 ° C. for 2 minutes inactivates the hepatocyte proliferation promoting activity.
（ｄ）トリプシン添加（0.01％,2時間,37℃処理）によ
り肝実質細胞増殖促進活性が失活しない。(D) Addition of trypsin (0.01%, 2 hours, 37 ° C. treatment) does not inactivate the hepatocyte proliferation promoting activity.
（ｅ）肝実質細胞を生体外において増殖させる活性を有
する。(E) It has the activity of proliferating hepatocytes in vitro.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
これまで脾臓において肝実質細胞増殖因子（以下、HG
Fという）が存在することについては全く知られていな
い。又、後述するように、本発明のSDGFは凝集活性化物
質を作用させずに得られるものであり、物理化学的性
質、とくにヘパリンカラムに吸着しない点で、HGFとは
異なる。Hepatocyte growth factor (hereinafter HG)
F is not known at all. Further, as described later, the SDGF of the present invention is obtained without the action of an aggregation activating substance, and differs from HGF in physicochemical properties, particularly in that it does not adsorb to a heparin column.
又、脾臓組織には、線維芽細胞増殖因子（Fibrobrast
Growth Factor,FGF）が含まれており、抽出できること
が報告されている。In addition, spleen tissue contains fibroblast growth factor (Fibrobrast
Growth Factor (FGF) is reported to be extractable.
FGFは線維芽細胞、内皮細胞など広範な細胞に増殖因
子として働くが、肝細胞には増殖刺激効果が検出されな
いとの報告がある。加えて、FGFはヘパリンカラムに吸
着する性質をもち、本発明のSDGFとは物理化学的にも異
なった物質である。It has been reported that FGF acts as a growth factor on a wide range of cells such as fibroblasts and endothelial cells, but no growth stimulating effect is detected on hepatocytes. In addition, FGF has the property of adsorbing to a heparin column, and is a physicochemically different substance from the SDGF of the present invention.
SDGF活性分画を得るには次の方法による。 The SDGF active fraction is obtained by the following method.
すなわち、ラット、ウシ、ブタなどの動物を屠殺後、
新鮮な脾臓をとり出し、ステンレス等メッシュ上にの
せ、これを押しつぶして細胞集塊をほぐす。これを適当
量のリン酸緩衝生理食塩液（Phosphate Buffered Salin
e,PBS）等の生理的緩衝液にて洗い流し、メッシュ下の
容器中に細胞間物質を得る。なお、脾臓は如何なる動物
種のものでも使用できる。PBSの液量として例えば脾臓1
gに対し、20mlを使用する。That is, after slaughtering animals such as rats, cows and pigs,
Take out a fresh spleen, put it on a mesh such as stainless steel, crush it and loosen the cell clumps. This is added to an appropriate amount of Phosphate Buffered Salin
e, PBS) to wash out with a physiological buffer and obtain an intercellular substance in a container below the mesh. The spleen can be of any animal species. For example, spleen 1
Use 20 ml per g.
得られた細胞間物質溶液を遠心（例えば1300×g,10分
間）して、その上清液として可溶性細胞間物質分画を得
る。The obtained intercellular substance solution is centrifuged (for example, at 1300 xg for 10 minutes) to obtain a soluble intercellular substance fraction as a supernatant.
この可溶性細胞間物質分画（Soluble Matrix Fractio
n,SMF）にSDGFが含まれることが見い出され、それに基
づいて本発明を完成した。This soluble intercellular substance fraction (Soluble Matrix Fractio
n, SMF) was found to contain SDGF, based on which the present invention was completed.
参考例（SDGF活性の測定法） ウィスター系雌ラット（７〜８週令）を用い、コラー
ゲン潅流法（Seglen,P.O.,Exp.Cell Res.82,391−398
（1973））により肝実質細胞を分離した。この肝実質細
胞を、10％牛胎児血清,10-7Mインスリン及び10-7Mデキ
サメサゾンを含むウィリアムズＥ培地（フローラブラト
リー社製）に懸濁させ、12ウエル−マルチプレート（リ
ンブロー社製）に２×104個/cm2の密度でまき込み、５
％炭酸ガス、37℃下で培養した。12時間後に先のウィリ
アムズＥ培地を除去し、10％牛胎児血清を含む新鮮なウ
ィリアムズＥ培地を加え、更に12時間培養する。これに
所定量のSMF（本発明SDGFを含む）を添加し、２日後同
一培地及びSMFで培地交換し、更に２日間培養する。こ
の培養液における細胞数をコールターカウンター（コー
ルターエレクトロニクス社製）で測定することによりSD
GFの活性を調べる。Reference Example (Method for Measuring SDGF Activity) Using a Wistar female rat (7 to 8 weeks old), a collagen perfusion method (Seglen, PO, Exp. Cell Res. 82 , 391-398)
(1973)). The hepatic parenchymal cells are suspended in a Williams E medium (manufactured by Florabratry) containing 10% fetal calf serum, 10 -7 M insulin and 10 -7 M dexamethasone, and 12-well multiplate (manufactured by Limbro) 2 × 10 4 pieces / cm 2 at a density of 5
The cells were cultured at 37 ° C. in% carbon dioxide. After 12 hours, the previous Williams E medium is removed, fresh Williams E medium containing 10% fetal calf serum is added, and the cells are further cultured for 12 hours. A predetermined amount of SMF (including the SDGF of the present invention) is added thereto, and after 2 days, the medium is replaced with the same medium and SMF, and the cells are further cultured for 2 days. By measuring the number of cells in this culture using a Coulter counter (manufactured by Coulter Electronics), SD
Check the activity of GF.
以下に実施例を示し、本発明をより具体的に述べる
が、本発明はこれらに限定されるものではない。Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
実施例 （１） ２匹のラット（７〜８週令）から脾臓（0.96
g）を取り出し、ステンレスメッシュ上に載せ、シリン
ジの棒等で押しつぶすようにして細胞集塊をほぐし、PB
S適当量にて洗い流し、メッシュ下の容器中の脾細胞、
細胞間物質に全体量20mlになるようにPBSを加え遠心分
離（1,300×g,10分間）して上清（SMF）を得た。このSM
Fの添加量を変化させた場合のSDGF活性を第１図に示
す。Examples (1) Spleen (0.96 weeks) from two rats (7-8 weeks old)
g), place on a stainless steel mesh, crush the cell clumps by crushing with a stick of syringe, etc.
S Wash off with an appropriate amount, spleen cells in a container under the mesh,
PBS was added to the intercellular substance to a total volume of 20 ml, and the mixture was centrifuged (1,300 × g, 10 minutes) to obtain a supernatant (SMF). This SM
FIG. 1 shows the SDGF activity when the amount of F added was changed.
第１図において、縦軸はSDGF活性指数を横軸はSMF添
加量（１ウエル中）を示す。In FIG. 1, the vertical axis indicates the SDGF activity index, and the horizontal axis indicates the amount of SMF added (in one well).
（２） 牛（ハルスタイン雌）の脾臓（1kg）を2cm角に
切り、水切り用のバット上に載せ、PBS存在下でこれを
手でつぶす。バットの下の容器中に収容された脾細胞、
細胞間物質の懸濁液を遠心分離（10,000×g,30分間）し
て上清を得た。その上清にPBSを２になるまで加え、
更に遠心分離（100,000×g,30分間）して、上清（SMF）
を得た。そのSMFを、予め20mMリン酸ナトリウム緩衝液
（NaPPi,pH＝7.1）で平衡化したヒドロキシアパタイト
カラム（バイオラッド社製）に吸着させ、20mM,100mM,4
50mMと段階的にNaPPi（pH7.1）の濃度を変え溶出させて
精製した。その結果を第２図に示す。(2) Cut the spleen (1 kg) of a cow (Halstein female) into 2 cm squares, place it on a bat for draining, and crush it by hand in the presence of PBS. Splenocytes contained in a container under the vat,
The suspension of the intercellular substance was centrifuged (10,000 × g, 30 minutes) to obtain a supernatant. Add PBS to the supernatant until it becomes 2,
After further centrifugation (100,000 xg, 30 minutes), supernatant (SMF)
I got The SMF was adsorbed onto a hydroxyapatite column (manufactured by Bio-Rad) pre-equilibrated with 20 mM sodium phosphate buffer (NaPPi, pH = 7.1), and
Purification was carried out by changing the concentration of NaPPi (pH 7.1) stepwise to 50 mM and eluting. The result is shown in FIG.
第２図において、縦軸（１）は細胞数（SDGF活性）
（２）はタンパク量、横軸はフラクションナンバーを示
す。（１〜７−20mM NaPPi,8〜16−100mM NaPPi,17〜La
st 450mM NaPPi）。In FIG. 2, the vertical axis (1) indicates the cell number (SDGF activity)
(2) shows the protein amount, and the horizontal axis shows the fraction number. (1-7-20 mM NaPPi, 8-16-100 mM NaPPi, 17-La
st 450 mM NaPPi).
この図からNaPPiの濃度が高い溶出液による溶出部分
から高純度のSDGFが得られることが判る。From this figure, it can be seen that high-purity SDGF can be obtained from the elution portion of the eluate having a high concentration of NaPPi.
（３） 分子量 SMFをセファクリルＳ−200によるゲル濾過カラム（フ
ァルマシア社製）に通すと活性は２つのピークに分か
れ、既知の分子量マーカーであるAldolase,BSA,RNase A
を用いてそれぞれの溶出位置と活性の溶出位置から分子
量は約40,000と約150,000又はそれ以上と決定した。(3) Molecular weight When SMF is passed through a gel filtration column (manufactured by Pharmacia) using Sephacryl S-200, the activity is divided into two peaks, and Aldolase, BSA, RNase A, which are known molecular weight markers.
The molecular weight was determined to be about 40,000 and about 150,000 or more from the respective elution positions and the elution positions of the activities.
（４） ヘパリンセファロースへの吸着性 このSMFをヘパリン・セファロースCL−6Bのカラム
（ファルマシア社製）を用いたヘパリン親和性カラムク
ロマトグラフィーを行い、SDGF活性を調べた。その結
果、未吸着画分にSDGF活性があらわれ、本発明のSDGFは
ヘパリン・セファロースにい吸着しないことが判る。し
たがって、本発明のSDGFは前記の血小板由来のHGFとは
異なるものである。(4) Adsorbability to Heparin Sepharose This SMF was subjected to heparin affinity column chromatography using a column of Heparin Sepharose CL-6B (manufactured by Pharmacia), and SDGF activity was examined. As a result, SDGF activity appeared in the unadsorbed fraction, indicating that the SDGF of the present invention did not adsorb to heparin-Sepharose. Therefore, the SDGF of the present invention is different from the aforementioned platelet-derived HGF.
（５） トリプシン耐性 SMF、ウィリアムズＥ培地にそれぞれトリプシンを0.0
1％加え、37℃,2時間で処理をした。これらをそれぞれ
アッセイ系にトリプシンの量が10μg/mlになるように加
え、SDGF活性をみた。その結果を第３図に示す。(5) Trypsin-resistant SMF and trypsin were added to Williams E medium for 0.0
1% was added, and the mixture was treated at 37 ° C. for 2 hours. These were each added to the assay system so that the amount of trypsin was 10 μg / ml, and the SDGF activity was observed. FIG. 3 shows the results.
この第３図において、１はSMF（−），トリプシン
（−）、２はSMF（−），トリプシン（＋）、３はSMF
（＋），トリプシン（＋）、４はSMF（＋），トリプシ
ン（−）である。この第３図において、SMF（＋）でト
リプシン（＋）とトリプシン（−）のもの、即ち、３及
び４が増殖活性指数において大差がないことから本発明
のSDGFは本条件下でSMFの中においてトリプシン耐性を
有することが判る。In FIG. 3, 1 is SMF (-), trypsin (-), 2 is SMF (-), trypsin (+), 3 is SMF
(+), Trypsin (+) and 4 are SMF (+) and trypsin (-). In FIG. 3, the SMF (+) of trypsin (+) and trypsin (−), that is, 3 and 4 have no significant difference in the growth activity index. It is found that the sample has trypsin resistance.
第１図はラットのSMFのSDGF活性を示す図、第２図はウ
シのSMFのヒドロキシアパタイトカラムによる精製を示
す図、第３図はSDGFのトリプシン耐性を示す図である。FIG. 1 is a diagram showing SDGF activity of rat SMF, FIG. 2 is a diagram showing purification of bovine SMF by a hydroxyapatite column, and FIG. 3 is a diagram showing trypsin resistance of SDGF.
───────────────────────────────────────────────────── フロントページの続き 特許法第30条第１項適用申請有り 昭和63年11月16日銀 座ガスホールにおいて開催された通商産業省工業技術院 微生物工業技術研究所の研究発表会の要旨集に発表 (72)発明者 鈴木 徹 茨城県つくば市東１丁目１番３号 工業 技術院微生物工業技術研究所内 (72)発明者 古賀 信光 東京都江東区牡丹２―13―１ 株式会社 前川製作所内 審査官 内田 淳子 (56)参考文献 特開 昭63−188697（ＪＰ，Ａ) (58)調査した分野(Int.Cl.6，ＤＢ名) A61K 35/28 C07K 14/46 - 14/47────────────────────────────────────────────────── ─── Continuing from the front page There is an application for the application of Article 30, Paragraph 1 of the Patent Act. Abstracts of research presentations held by the Research Institute of Microorganisms and Technology of the Ministry of International Trade and Industry held at the Ginza Gas Hall on November 16, 1988. (72) Inventor Tohru Suzuki 1-3-1 Higashi, Tsukuba, Ibaraki Pref.Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology Government Atsuko Uchida (56) References JP-A-63-188697 (JP, A) (58) Fields investigated (Int. Cl. 6 , DB name) A61K 35/28 C07K 14/46-14/47
Claims (3)
殖因子を抽出分離することを特徴とする肝実質細胞増殖
因子の製造法。1. A method for producing hepatocyte growth factor, comprising extracting and separating hepatocyte growth factor from intercellular substances of spleen tissue.
として分離することを特徴とする、特許請求の範囲第１
項に記載の肝実質細胞増殖因子の製造法。2. The method according to claim 1, wherein the extract is extracted with a buffer, centrifuged, and separated as a soluble fraction.
6. The method for producing hepatocyte growth factor according to item 6.
因子であることを特徴とする肝実質細胞増殖因子。 （ａ）脾臓組織の細胞間物質から緩衝液で抽出され、遠
心によって得られる可溶性画分に存在する。 （ｂ）分子量が約40,000と約150,000又はそれ以上。 （ｃ）ヘパリンカラムに吸着しない。 （ｄ）100℃、２分間の加熱処理によって肝実質細胞増
殖促進活性が失活する。 （ｅ）トリプシン添加（0.01％、２時間、37℃処理）に
より肝実質細胞増殖促進活性が失活しない。 （ｆ）肝実質細胞を生体外において増殖させる活性を有
する。3. A hepatocyte growth factor characterized by having the following physicochemical properties and physiological activities. (A) Extracted with a buffer solution from intercellular substances of spleen tissue and present in a soluble fraction obtained by centrifugation. (B) molecular weight of about 40,000 and about 150,000 or more. (C) Does not adsorb to the heparin column. (D) Hepatocyte proliferation promoting activity is inactivated by heat treatment at 100 ° C. for 2 minutes. (E) Addition of trypsin (0.01%, 2 hours, 37 ° C. treatment) does not inactivate the hepatocyte proliferation promoting activity. (F) It has the activity of proliferating hepatocytes in vitro.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63286844A JP2799455B2 (en) | 1988-11-15 | 1988-11-15 | Method for producing hepatocyte growth factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63286844A JP2799455B2 (en) | 1988-11-15 | 1988-11-15 | Method for producing hepatocyte growth factor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02134323A JPH02134323A (en) | 1990-05-23 |
| JP2799455B2 true JP2799455B2 (en) | 1998-09-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH083195A (en) * | 1991-10-04 | 1996-01-09 | Toshiichi Nakamura | Tissue injury healing factor |
| JP3394982B2 (en) * | 1991-11-07 | 2003-04-07 | 敏一 中村 | Side effects inhibitor for cancer therapy |
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| JPH0720995B2 (en) * | 1987-01-29 | 1995-03-08 | 一丸フアルコス株式会社 | Cell growth promoter derived from bovine spleen |
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| JPH02134323A (en) | 1990-05-23 |
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