JPH04120097A - Hepatic cell growth factor - Google Patents
Hepatic cell growth factorInfo
- Publication number
- JPH04120097A JPH04120097A JP2238040A JP23804090A JPH04120097A JP H04120097 A JPH04120097 A JP H04120097A JP 2238040 A JP2238040 A JP 2238040A JP 23804090 A JP23804090 A JP 23804090A JP H04120097 A JPH04120097 A JP H04120097A
- Authority
- JP
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- Prior art keywords
- growth factor
- eluted
- adsorbed
- cell growth
- substance
- 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
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- 210000003494 hepatocyte Anatomy 0.000 title claims abstract description 28
- 239000003102 growth factor Substances 0.000 title abstract description 7
- 230000010261 cell growth Effects 0.000 title abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 13
- 229920002684 Sepharose Polymers 0.000 claims abstract description 11
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960002897 heparin Drugs 0.000 claims abstract description 10
- 229920000669 heparin Polymers 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 230000001766 physiological effect Effects 0.000 claims abstract description 4
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 claims description 24
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 24
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 2
- 229920002401 polyacrylamide Polymers 0.000 claims description 2
- 210000000952 spleen Anatomy 0.000 abstract description 10
- 239000006228 supernatant Substances 0.000 abstract description 6
- 208000006454 hepatitis Diseases 0.000 abstract description 5
- 241000283690 Bos taurus Species 0.000 abstract description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 4
- 206010016654 Fibrosis Diseases 0.000 abstract description 3
- 230000007882 cirrhosis Effects 0.000 abstract description 3
- 208000019425 cirrhosis of liver Diseases 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 abstract description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 abstract description 3
- 206010008909 Chronic Hepatitis Diseases 0.000 abstract description 2
- 208000001940 Massive Hepatic Necrosis Diseases 0.000 abstract description 2
- 231100000354 acute hepatitis Toxicity 0.000 abstract description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 2
- 208000019423 liver disease Diseases 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 2
- 150000001408 amides Chemical class 0.000 abstract 1
- 238000001502 gel electrophoresis Methods 0.000 abstract 1
- 238000005342 ion exchange Methods 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000008057 potassium phosphate buffer Substances 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
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- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
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- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- QVLTXCYWHPZMCA-UHFFFAOYSA-N po4-po4 Chemical compound OP(O)(O)=O.OP(O)(O)=O QVLTXCYWHPZMCA-UHFFFAOYSA-N 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011012 sanitization Methods 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、新規な肝細胞増殖因子に関する。[Detailed description of the invention] [Industrial application field] The present invention relates to a novel hepatocyte growth factor.
従来より、肝細胞の生体外培養において上皮細胞成長因
子(Ellidermal Growth Facto
r:E G F Carpenter、 G、 et
at、 、 Ann、 Rev、 Biochem、
、 48.193(1979)) 、インスリン等が用
いられ、これらが肝細胞の増殖を促進することか知られ
ている。また哺乳動物血液中に肝細胞増殖促進因子(H
GF:Hepatocyte Growth Fact
or)か出現することか示唆され、該因子の単離・精製
に成功した例も報告されている( Biochem、
Biophys、 Res、 Comm、 、 I 6
3.967(1889);Proc、Natl、Aca
d、Sci、U、S、A、、 87,320(1990
)。Conventionally, epidermal growth factor has been used in in vitro culture of hepatocytes.
r: E G F Carpenter, G, et
at, Ann, Rev, Biochem,
, 48.193 (1979)), insulin, etc. are used, and it is known that these promote the proliferation of hepatocytes. In addition, hepatocyte proliferation promoting factor (H) is present in mammalian blood.
GF: Hepatocyte Growth Fact
It has been suggested that the factor or) appears, and there have been reports of successful isolation and purification of the factor (Biochem,
Biophys, Res, Comm, , I 6
3.967 (1889); Proc, Natl, Aca
d, Sci, U, S, A, 87, 320 (1990
).
ところで、最近、牌臓に肝細胞を注入すると、肝細胞は
、牌臓内て増殖することか報告された(水戸延部、医学
の歩み、 125(4)、573.(1983))。By the way, it has recently been reported that when hepatocytes are injected into the spleen, the hepatocytes proliferate within the spleen (Nobube Mito, Igaku no Hyaku, 125(4), 573. (1983)).
本発明者らは肝細胞の増殖因子を探索する研究の過程で
、上記肝細胞の牌臓移植に関する報告に注目し、牌臓内
に肝細胞増殖因子を探索し、ヘパリンに吸着しない分子
置駒40,000と約15o、ooo又はそれ以上の肝
細胞増殖因子(SDGF)を得ることに成功した(特開
平2−134323号)。In the process of research to search for hepatocyte growth factors, the present inventors focused on the above-mentioned report on spleen transplantation of hepatocytes, and searched for hepatocyte growth factors in the spleen. We succeeded in obtaining hepatocyte growth factor (SDGF) of 40,000 and about 15o, ooo or more (Japanese Patent Application Laid-Open No. 134323/1999).
今回、我々か哺乳動物牌臓より見出した物質は物理化学
的性質、特にヘパリンセファロースに吸着する点て、5
DGFとは異なる。This time, the substance we discovered from mammalian spleen has physicochemical properties, especially its ability to adsorb to heparin sepharose.
It is different from DGF.
また、HGFとは物理化学的性質、特に分子量の点で異
なる他、生理活性の面からも、明らかに異なる物質であ
る。Furthermore, it is a substance that is clearly different from HGF in terms of physicochemical properties, particularly molecular weight, and also in terms of physiological activity.
FGFは線維芽細胞、内皮細胞など広範な種類の細胞に
対して増殖因子として働くか、肝細胞に対しては増殖作
用は認められないとの報告、認められるとする報告の両
者かある。抗FGF抗体に対する反応性か違う点などか
ら、本発明の肝細胞増殖因子とFGFは異なる物質であ
る。There are reports that FGF acts as a growth factor for a wide variety of cells such as fibroblasts and endothelial cells, and that it does not have a proliferative effect on hepatocytes, while others say that it does. The hepatocyte growth factor of the present invention and FGF are different substances because of their different reactivity to anti-FGF antibodies.
肝臓は、生体内で必要とする蛋白の合成、各種栄養分の
代謝・貯蔵・分解・供給を行う他、不要物質の解毒・排
泄等、生体内の代謝の中心的役割を担っている高度に分
化した器官である。この肝臓の中ても肝細胞(肝実質細
胞)かこれらの機能を主に持っている。The liver is a highly differentiated organ that plays a central role in the body's metabolism, including synthesizing proteins needed by the body, metabolizing, storing, decomposing, and supplying various nutrients, as well as detoxifying and excreting unnecessary substances. It is an organ that Even within the liver, hepatocytes (hepatocytes) mainly have these functions.
肝炎・肝硬変等の疾患時には、肝細胞の障害を伴う。こ
のような状態においては肝機能を保持するために生体内
で肝細胞の増殖を促進することか求められる。Diseases such as hepatitis and cirrhosis are accompanied by damage to liver cells. In such conditions, it is required to promote the proliferation of hepatocytes in vivo in order to maintain liver function.
本発明は、新規な肝細胞増殖因子を提供することを目的
とする。The present invention aims to provide a novel hepatocyte growth factor.
本発明者らは、補乳動物牌臓内の肝細胞増殖因子を探索
し、先の発明とは別に肝細胞を極めて良好に増殖させる
新規な活性物質を見出したので、分離・精製してその物
理化学的性状を明らかにし、本発明を完成するに至った
。The present inventors searched for hepatocyte growth factor in the spleen of mammalian mammals, and, in addition to the previous invention, discovered a new active substance that allows hepatocytes to proliferate extremely well. The physicochemical properties were clarified and the present invention was completed.
ここでいう哺乳動物としては、例えば、ウシ、ウマ、ブ
タ、ウサギ、ラット等いずれも利用できる。Examples of mammals that can be used include cows, horses, pigs, rabbits, and rats.
また、ここでいう肝細胞とは肝実質細胞を指す。Moreover, hepatocytes here refer to hepatic parenchymal cells.
すなわち、本発明の肝細胞増殖因子は、以下の理化学的
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
ものである。That is, the hepatocyte growth factor of the present invention is characterized by being a substance having the following physicochemical properties and physiological activities.
(a) 5DS−ポリアクリルアミドゲルを用いた電
気泳動による分子量か約18,000である。(a) Molecular weight determined by electrophoresis using 5DS-polyacrylamide gel is approximately 18,000.
(b) ヘパリンセファロースに吸着し、塩化ナトリ
ウム濃度1.0〜2.0Mの間で溶出する。(b) Adsorbs to heparin sepharose and elutes at a sodium chloride concentration of 1.0 to 2.0M.
(c) 肝細胞を増殖させる活性を有する。(c) It has the activity of proliferating hepatocytes.
以下、本発明について、上記(a)、 (b)、(c)
を説明する。Hereinafter, regarding the present invention, the above (a), (b), (c)
Explain.
(a)分子量
本発明の肝細胞増殖因子を脱塩後、非還元状態て5DS
−ポリアクリルアミドゲル電気泳動を行う。分子量マー
カーとしては、リゾチーム、大豆トリプシン、インヒビ
ター、ウシ炭酸脱水酵素、オハルプミン、を使用した。(a) Molecular weight After desalting the hepatocyte growth factor of the present invention, 5DS in a non-reduced state
- Perform polyacrylamide gel electrophoresis. Lysozyme, soybean trypsin, inhibitor, bovine carbonic anhydrase, and ohalupin were used as molecular weight markers.
分子量マーカーによる計算値から本発明の肝細胞増殖因
子は分子置駒18.000のバンドとして検出される。The hepatocyte growth factor of the present invention is detected as a band at the molecular weight marker 18.000 from the calculated value using the molecular weight marker.
(b)カラム吸着性
本発明の肝細胞増殖因子は0.5MNaCIPBSとし
、予め、0.5MNaC1−PBSて平衡化しておいた
ヘパリンセファロースに吸着する。(b) Column adsorption property The hepatocyte growth factor of the present invention is adsorbed on heparin Sepharose which has been equilibrated with 0.5M NaC1-PBS in 0.5M NaCIPBS.
0.5MNaC]−PBSで洗浄後、0.5M〜2.5
MNaC] −PBSて溶出する。0.5M NaC] - After washing with PBS, 0.5M to 2.5
MNaC] - Elute with PBS.
(c)肝細胞増殖活性
本発明の肝細胞増殖因子は、ラット初代培養肝細胞の培
養液中に添加することにより、肝細胞を濃度依存的に増
殖する。(c) Hepatocyte proliferation activity When the hepatocyte growth factor of the present invention is added to the culture medium of primary cultured rat hepatocytes, the hepatocytes proliferate in a concentration-dependent manner.
本発明の肝細胞増殖因子は、例えば、次のようにし得る
ことかできる。The hepatocyte growth factor of the present invention can be used, for example, as follows.
すなわち、補乳動物を屠殺後、新鮮な牌臓を取り出し、
ステンレス等のメツシュ上にのせ、又はミンサーを用い
てこれを押しつぶして細胞集塊をほぐす。これに、適当
量のリン酸緩衛生理食塩液(P B S : Phos
phate Buffered 5aline)等の生
理的緩衛液(例えば、牌臓1kgに対して2〜2OAを
使用する。)を加えて洗い、溶液・洗浄を収集し、細胞
間物質溶液を得る。That is, after slaughtering the supplementary animal, remove the fresh spleen,
Loosen the cell aggregate by placing it on a mesh made of stainless steel or crushing it using a mincer. To this, add an appropriate amount of phosphoric acid saline solution (PBS: Phos
Wash by adding a physiological sanitizing solution such as phate Buffered 5aline (for example, use 2 to 2 OA per 1 kg of spleen), and collect the solution/wash to obtain an intercellular substance solution.
得られた細胞間物質溶液を遠心(例えば7゜00xg、
10分間)して、その上清液として可溶性細胞間物質画
分を得る。本画分をガーゼ等で濾過して超遠心処理(例
えば100,000xg、30分間)し、ヘパリンセフ
ァロース・カラムに吸着させる。NaCl溶出画分に肝
細胞増殖因子か含まれる。更に、本溶出画分をヘパリン
セファロース・カラムに吸着させ、NaC1て溶出する
ことにより精製することかできる。The obtained intercellular substance solution is centrifuged (e.g. 7°00xg,
10 minutes) to obtain the soluble intercellular substance fraction as the supernatant. This fraction is filtered through gauze or the like, subjected to ultracentrifugation (eg, 100,000xg, 30 minutes), and adsorbed onto a heparin-Sepharose column. The NaCl elution fraction contains hepatocyte growth factor. Further, this elution fraction can be purified by adsorbing it on a heparin Sepharose column and eluting with NaCl.
以下に実施例及び試験例を示し、本発明をより具体的に
述−\る。しかしながら、本発明はこれらに限定される
ものではない。Examples and test examples are shown below to describe the present invention in more detail. However, the present invention is not limited thereto.
実施例
ウシ(ホルスタイン雌)の牌臓(1kg)を2anに切
り、水切り用のパッド上に載せ、PBS存在下てこれを
擦り潰した。パッドの下の容器中に収容された牌細胞、
細胞間物質の懸濁液を遠心分離(10,OOOxg)し
て上清を得た。上清をガーゼ、濾紙等で濾過し浮遊物等
を除去した。濾液を、更に、超遠心分離(100,00
0xg)して上清を得た。Example A spleen (1 kg) of a cow (Female Holstein) was cut into 2 ann pieces, placed on a draining pad, and ground in the presence of PBS. tile cells housed in a container under the pad;
The suspension of intercellular material was centrifuged (10,000 x g) to obtain a supernatant. The supernatant was filtered using gauze, filter paper, etc. to remove suspended matter. The filtrate was further subjected to ultracentrifugation (100,000
0xg) to obtain the supernatant.
本上清を3倍容の10mMリン酸カリウム緩衛液(pH
6,4)で3回、透析した。透析内液を以下の精製に供
した。Transfer this supernatant to 3 times the volume of 10mM potassium phosphate buffer solution (pH
6,4) and dialyzed three times. The dialysis fluid was subjected to the following purification.
本内液を予め10mMリン酸カウム緩衛液(pH6,4
)で平衡化したイオン交換カラム(SP−セルロース及
びDEAE−セルロース)に付したところ、非吸着画分
に肝細胞増殖因子(SDGF−1)か存在した。Prepare the internal solution in advance with 10mM phosphate phosphate buffer solution (pH 6.4).
), hepatocyte growth factor (SDGF-1) was present in the non-adsorbed fraction.
本非吸着画分を0.5MNaC1−PBSに対して透析
し、予め0.5MNaC1−PBSて平衡化しておいた
ヘパリンセファロース・カラムに吸着させた。This non-adsorbed fraction was dialyzed against 0.5M NaCl-PBS and adsorbed onto a heparin-Sepharose column that had been equilibrated with 0.5M NaCl-PBS.
0.5MNaCI−PBSて洗浄後、0.5M〜25M
NaC1(クラデイエンド) −PBSで溶出した。肝
細胞増殖因子は第1図のように10M 〜2.0MNa
C1−PBS画分に溶出された。After washing with 0.5M NaCI-PBS, 0.5M to 25M
NaCl (Cladiendo)-eluted with PBS. Hepatocyte growth factor is 10M to 2.0MNa as shown in Figure 1.
It was eluted in the C1-PBS fraction.
本画分を3倍容の20mMりン酸カリウム緩衛液(pH
7,0)に対して3回透析し、20mMリン酸カリウム
緩衛液(pH7,0)で平衡化したハイドロキシアパタ
イト・カラムに吸着させた。This fraction was added to 3 times the volume of 20mM potassium phosphate buffer solution (pH
7.0) three times and adsorbed onto a hydroxyapatite column equilibrated with 20mM potassium phosphate buffer solution (pH 7.0).
20mMリン酸カリウム緩衛液(pH7,0)で洗浄後
、20mM〜700mMリン酸カリウム緩衛液(pH7
,0)で溶出させた。200mM〜300mMリン酸カ
リウム緩衛液(pH7,0)溶出画分に肝細胞増殖因子
(SDGF−1)を得た。その結果を第2図に示す。After washing with 20mM potassium phosphate washing solution (pH 7.0), wash with 20mM to 700mM potassium phosphate washing solution (pH 7.0).
, 0). Hepatocyte growth factor (SDGF-1) was obtained in the eluted fraction of 200mM to 300mM potassium phosphate buffer solution (pH 7,0). The results are shown in FIG.
更に、本溶出画分をOy 5MNaCI−PBSに対し
て透析し、予め0.5MNaC1−PBSて平衡化して
おいたヘパリンセファロース・カラムに吸着させた。0
.5MNaC1−PBSて洗浄後、0.5M 〜2.5
MNaC1(グラデイエンド’)−PBSて溶出したと
ころ、第3図に示すように1.0M 〜2.0MNaC
1−PBS付近に肝細胞増殖因子(SDGF−1)か溶
出された。Furthermore, this elution fraction was dialyzed against Oy 5M NaCI-PBS and adsorbed onto a heparin Sepharose column that had been equilibrated with 0.5M NaCl-PBS. 0
.. After washing with 5M NaCl-PBS, 0.5M ~ 2.5
When eluted with MNaC1 (Gradiend')-PBS, 1.0M to 2.0M NaC was eluted as shown in Figure 3.
Hepatocyte growth factor (SDGF-1) was eluted near 1-PBS.
試験例
〈検体〉
実験例で得られた検体(第3図に示すように1゜0M
〜2.0MNaC1−PBS付近に溶出された肝細胞増
殖因子)を以下に記載したような肝細胞増殖因子活性の
測定法を用いて調整したラット肝細胞培養液中に添加し
、本発明の肝細胞増殖因子の肝細胞増殖活性を検討した
。その結果を第4図に示す。Test example <Sample> The sample obtained in the experiment example (1°0M as shown in Figure 3)
Hepatocyte growth factor (eluted around ~2.0 M NaCl-PBS) was added to a rat hepatocyte culture medium prepared using the method for measuring hepatocyte growth factor activity as described below. The hepatocyte proliferation activity of cell growth factors was investigated. The results are shown in FIG.
〈肝細胞増殖因子活性の測定法〉
ウィスター系雄ラット(180〜220 g)を用い、
コラーゲン潅流法(Seglen、 P、 O9,Ex
p、 Ce1Res、、 82,391−398(19
73))により肝細胞を分離・調整した。<Method for measuring hepatocyte growth factor activity> Using Wistar male rats (180-220 g),
Collagen perfusion method (Seglen, P, O9, Ex
p, Ce1Res, 82, 391-398 (19
Hepatocytes were isolated and prepared according to 73).
この細胞をlO%ウシ胎児血清、10−7Mインスリン
及び10−’Mデキサメサゾンを含むウィリアムスE培
地(フローラボラトリー社製)に懸濁させ、24ウェル
−マルチプレート(ファルコン社製)にlXl0’個/
alの密度になるようまき込み、5%炭酸ガス存在下で
37°Cにて培養した。The cells were suspended in Williams E medium (manufactured by Flow Laboratories) containing 10% fetal bovine serum, 10-7M insulin, and 10-'M dexamethasone, and placed in a 24-well multiplate (manufactured by Falcon) at 1X10'/cells.
The cells were sown at a density of 5% carbon dioxide and cultured at 37°C in the presence of 5% carbon dioxide.
5時間後に培地を除去し、まき込み時と同じ組成に更に
所定量のヘパリンセファロース溶出画分(肝細胞増殖因
子を含む)を添加した培地を加え培養した。25時間後
に3H−チミジン(終濃度0゜251t Ci/ mβ
、1.5μM)を添加し、更に24時間培養した。この
間にDNAに取り込まれた放射活性を測定することによ
り肝細胞増殖因子の活性を調へた。After 5 hours, the medium was removed, and a medium with the same composition as at the time of inoculation but further supplemented with a predetermined amount of heparin sepharose elution fraction (containing hepatocyte growth factor) was added and cultured. After 25 hours, 3H-thymidine (final concentration 0°251t Ci/mβ
, 1.5 μM) and further cultured for 24 hours. During this time, the activity of hepatocyte growth factor was determined by measuring the radioactivity incorporated into the DNA.
本発明の肝細胞増殖因子は、急性肝炎、劇症肝炎、慢性
肝炎、肝硬変等の肝疾患の治療薬として有用である。更
には、肝細胞を生体外で増殖させるための研究用試薬と
して有用である。The hepatocyte growth factor of the present invention is useful as a therapeutic agent for liver diseases such as acute hepatitis, fulminant hepatitis, chronic hepatitis, and cirrhosis. Furthermore, it is useful as a research reagent for growing hepatocytes in vitro.
第1図及び第3図は、ヘパリンセファロース・カラムク
ロマトグラフィーの結果を示す図である。
第2図はハイドロキシアパタイト・カラムクロマトグラ
フィーの結果を示す図である。第4図は本発明の肝細胞
増殖因子の量とDNA合成量との関係を示す図である。
第
図
フラクションNo。
第3図
O
菊
8゜
フラクションN。
第4図
O
1,5
因子投与量
(μp/培讐液)FIG. 1 and FIG. 3 are diagrams showing the results of heparin Sepharose column chromatography. FIG. 2 is a diagram showing the results of hydroxyapatite column chromatography. FIG. 4 is a diagram showing the relationship between the amount of hepatocyte growth factor of the present invention and the amount of DNA synthesis. Figure Fraction No. Figure 3 O chrysanthemum 8° fraction N. Figure 4 O 1,5 Factor dose (μp/culture solution)
Claims (1)
とを特徴とする肝細胞増殖因子。 (a)SDS−ポリアクリルアミドゲルを用いた電気泳
動による分子量が約18,000である。 (b)ヘパリンセファロースに吸着し、塩化ナトリウム
濃度1.0〜2.0Mの間で溶出する。 (c)肝細胞を増殖させる活性を有する。[Scope of Claims] A hepatocyte growth factor characterized by being a substance having the following physicochemical properties and physiological activities. (a) Molecular weight determined by electrophoresis using SDS-polyacrylamide gel is approximately 18,000. (b) Adsorbs to heparin sepharose and elutes at a sodium chloride concentration of 1.0 to 2.0M. (c) It has the activity of proliferating hepatocytes.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2238040A JPH04120097A (en) | 1990-09-07 | 1990-09-07 | Hepatic cell growth factor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2238040A JPH04120097A (en) | 1990-09-07 | 1990-09-07 | Hepatic cell growth factor |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH04120097A true JPH04120097A (en) | 1992-04-21 |
Family
ID=17024279
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2238040A Pending JPH04120097A (en) | 1990-09-07 | 1990-09-07 | Hepatic cell growth factor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH04120097A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5547856A (en) * | 1992-05-18 | 1996-08-20 | Genentech, Inc. | Hepatocyte growth factor variants |
US5580963A (en) * | 1992-05-18 | 1996-12-03 | Genentech, Inc. | Single-chain hepatocyte growth factor variants |
WO1998024467A1 (en) * | 1996-12-05 | 1998-06-11 | Toshikazu Nakamura | Remedies for fulminant hepatitis |
US5879910A (en) * | 1992-05-18 | 1999-03-09 | Genetech, Inc. | Hepatocyte growth factor protease domain variants |
-
1990
- 1990-09-07 JP JP2238040A patent/JPH04120097A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5547856A (en) * | 1992-05-18 | 1996-08-20 | Genentech, Inc. | Hepatocyte growth factor variants |
US5580963A (en) * | 1992-05-18 | 1996-12-03 | Genentech, Inc. | Single-chain hepatocyte growth factor variants |
US5879910A (en) * | 1992-05-18 | 1999-03-09 | Genetech, Inc. | Hepatocyte growth factor protease domain variants |
WO1998024467A1 (en) * | 1996-12-05 | 1998-06-11 | Toshikazu Nakamura | Remedies for fulminant hepatitis |
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