JP4098372B2 - Method for producing heparin-binding growth factor - Google Patents

Description

【００２８】
以上の結果、ｈＨＧＦ産生細胞株の培養液中にヘパリンを添加することにより、回収されるｈＨＧＦ量が数倍に増大することが判明した。
【００２９】
実施例４ ｈＨＧＦ産生ＣＨＯ細胞株のｈＨＧＦ産生に及ぼす各種硫酸化多糖類の影響
次に、ヘパリン以外の硫酸化多糖類についても同様の検討を行った。
ＫＢＥ株を４×１０⁵ 個／ｍｌ／ウェルで１２ウェルマイクロプレ−トに播種し、培養５日後の上清を採取して、上述のＥＬＩＳＡ法にて培養上清中のｈＨＧＦ量を測定した。添加多糖類として、ヘパリン、ヘパラン硫酸、デキストラン（以上シグマ社）、デキストラン硫酸及びコンドロイチン硫酸（以上生化学工業社）を用いた。
【００３０】
表２に結果を示す。ヘパリン、ヘパラン硫酸、デキストラン硫酸、コンドロイチン硫酸のいずれにおいてもｈＨＧＦ含量の増大が認められた。ヘパリン、ヘパラン硫酸、デキストラン硫酸では１０−１００μｇ／ｍｌの添加範囲で最大効果が得られ、２倍ないしそれ以上の上昇が示された。一方、コンドロイチン硫酸では効果は認められるものの、その増大効果はやや低く、有効必要用量も高用量側にシフトしていた。硫酸基を持たないデキストランでは、全く効果が認められなかった。
【００３１】
【表２】

【００３２】
ＫＢＥ株は、マイクロキャリア−（ファルマシア社）を用いた浮遊培養法によって数カ月以上ＨＧＦ産生を維持させることが可能であるが、その場合も、硫酸化多糖類は同様の効果を数カ月間維持した。種々の培養形態・培養スケ−ルの検討においても、硫酸化多糖類の作用は付着培養（Ｔフラスコ、ディッシュ、ロ−ラ−ボトルなど）・浮遊培養（マイクロキャリア−を用いた、あるいは用いないスピナ−培養）に関わりなく、また９６ウェルプレ−トのマイクロウェル培養から数リットル以上の培養規模において、同様に認められた。
【００３３】
以上の結果から、ＨＧＦ生産細胞の培養系に種々の硫酸化多糖類を加えることにより、回収されるＨＧＦ量が２倍ないし数倍に増大することが判明した。この効果は低用量の硫酸化多糖類添加で生じ、培養の方法や硫酸化多糖類の種類に関わりなく、培養数時間から数カ月間の長期に渡って維持され、ＨＧＦ生産株で広く認められた。この効果の原理は、硫酸化多糖類がその硫酸基を介してＨＧＦを結合し、ＨＧＦ産生細胞上のレセプタ−（ｃ−Ｍｅｔ蛋白質及びヘパラン硫酸プロテオグリカンなど）へのＨＧＦ結合・消費・分解を著しく遅延させるためと理解された。
【００３４】
【発明の効果】
本発明の生産方法によれば、ヘパリン結合性増殖因子の生産及び回収量を数倍に高めることが可能であり、その結果、該増殖因子の生産におけるコストを著しく軽減できるようになった。本発明を基にヘパリン結合性増殖因子を生産することにより、医療分野での広範な利用が期待できる。
【図面の簡単な説明】
【図１】ＣＨＯ細胞におけるｈＨＧＦ／ｃ−Ｍｅｔ蛋白質複合体の形成についてＳＤＳ−ポリアクリルアミド電気泳動の結果を示した図面である。
【図２】ＣＨＯ細胞におけるｈＨＧＦの消費速度について、ヘパリン存在下及び非存在下での比較を示した図面である。
【図３】ｈＨＧＦ生産株における培養上清中のｈＨＧＦ量について、ヘパリン存在下及び非存在下での比較を経時的に示した図面である。
【図４】ｈＨＧＦ生産株における培養上清中のｈＨＧＦ量について、ヘパリン添加用量の影響を示した図面である。[0001]
[Industrial application fields]
The present invention relates to a method for producing a heparin-binding growth factor, and more specifically, by culturing a transformed cell that produces heparin-binding growth factor in the presence of a sulfated polysaccharide or an agonist thereof. The present invention relates to a method for efficiently obtaining the growth factor from Kiyo with high production.
[0002]
[Prior art and problems to be solved by the invention]
In recent years, various cell growth factors have been cloned, and among them, a group of growth factors having a strong affinity for heparin has been found. This is generically called heparin-binding growth factor, which includes fibroblast growth factor (FGF), keratinocyte growth factor (KGF), pleiotrophin, granulocyte / macrophage colony formation stimulating factor (GM). -CSF), interleukins 3 and 7, and various known or unidentified growth factors such as vascular endothelial growth factor (VEGF) (Experimental Medicine, 9 (14), 1772 (1991)). These factors are known to bind to glycosaminoglycans represented by heparin and heparan sulfate. Among them, FGF is stored on the cell surface and extracellular matrix by binding to heparan sulfate proteoglycan, And it was confirmed that the biological activity of FGF can be regulated by heparin (Cell, 64 , 841 (1991)). Recently, a human-derived protein factor that can take out liver parenchymal cells from the living body and promote their growth in vitro, that is, human hepatocyte growth factor (hereinafter abbreviated as “hHGF”) has been found in the plasma of fulminant hepatitis patients. (Japanese Patent Laid-Open No. 63-22526), an amino acid sequence encoding hHGF protein, a sequence of a gene (cDNA) encoding the same (Japanese Patent Laid-Open No. 3-72883), and hHGF using this cDNA A protein production method and a transformant (JP-A-3-285893) have been reported. The recombinant hHGF protein produced by such a method has been found to promote the growth of hepatocytes in vitro. Moreover, it has been found that the HGF protein, not limited to humans, is a kind of the above heparin-binding growth factor having a strong affinity for heparin. The present inventors examined the influence of sulfated polysaccharides on HGF binding and consumption in target cells. By mixing HGF and various sulfated polysaccharides, binding of HGF to target cells and target It was found that the consumption of hHGF by cells was strongly suppressed, and as a result, the action of HGF was sustained and stabilized (European Patent Publication No. 517182).
[0003]
On the other hand, as a means for producing a growth factor, a method for introducing cDNA of the growth factor into microorganisms such as Escherichia coli or mammalian cells has been widely used (Molecular Cloning, 2nd ed., 16.3-17.40). , Cold Spring Harbor Laboratory Press (1989)). In the case of a protein having a complex structure having a sugar chain and having many disulfide bonds in the molecule, such as hHGF, animal cells are exclusively used for its production. Among them, the epithelial cell line CHO derived from Chinese hamster ovary is particularly widely used.
[0004]
On the other hand, a receptor molecule on the target cell of heparin-binding growth factor is also becoming clear. For example, the receptor molecule of HGF has been found to be a proto-oncogene c-met product (C-Met protein). (Science, 251 , 802 (1991)) and several FGF receptor families have already been clarified as receptors for acidic and alkaline FGF and KGF (Experimental Medicine, 10 , 25 (1992)). Further, as other receptors, heparan sulfate proteoglycan (HSPG) on the cell surface is also known to capture heparin-binding growth factor via intramolecular glycosaminoglycan (protein, nucleic acid, enzyme, 34 , 853 (1989)). HSPG is expressed in large amounts on a large number of cells. Furthermore, it is widely accepted that growth factors themselves are quickly taken up into cells and consumed when growth factors bind to specific receptors on the cells to induce their physiological activity. In the case of HGF, the present inventors have already analyzed from the analysis using an epithelial cell line, and hHGF is taken into the cell very rapidly after binding to the receptor (internalization) (t _1/2 : 5- 10 minutes), and it is revealed that it is decomposed and released into the culture supernatant (degradation) (t _1/2 : 30-60 minutes) (J. Cell. Biochem. Suppl 0 (16partB)) , 184 (1992)).
[0005]
In the conventional technique, when an expression vector having a heparin-binding growth factor cDNA is introduced into an appropriate host cell to produce the growth factor, a part of the expressed growth factor is a growth factor receptor of the producing cell itself. The amount of the growth factor recovered from the production cell culture supernatant was relatively low because it could not be prevented from being decomposed and consumed via -and HSPG molecules.
[0006]
[Means for Solving the Problems]
The inventors added various sulfated polysaccharides to the culture system of the growth factor-producing cells (transformants), and compared the growth factor production amounts. For example, a sulfated polysaccharide is added at a concentration of 1-1000 μg / ml in the culture solution of HGF-producing CHO cells into which hHGF cDNA has been introduced (JP-A-3-285893), and the culture supernatant is recovered over time. HGF content was quantified. As a result, interestingly, about 1.5 to 5-fold enhancement of HGF production was recognized by the addition of sulfated polysaccharide.
[0007]
On the other hand, the present inventors examined the HGF responsiveness of the host CHO cells used for HGF expression, and found that the host cells have C-Met protein and that DNA synthesis is actually promoted by the addition of HGF. It has been found that cells consume and degrade HGF. Furthermore, the present inventors investigated the effect of sulfated polysaccharides on HGF binding / degradation of host cells. By mixing HGF and sulfated polysaccharides, binding of HGF to host cells and host It has been found that consumption of HGF by cells is strongly suppressed.
[0008]
From the above results, by adding sulfated polysaccharides to the culture system in the HGF-producing cell culture system, HGF binding / consumption / degradation by the production cells themselves is avoided, and the amount of HGF recovered from the culture supernatant is greatly enhanced. As a result, the present invention has been completed. That is, the gist of the present invention is that a cell line transformed by introducing a gene encoding a heparin-binding growth factor is cultured in the presence of a sulfated polysaccharide or an agonist thereof, and the heparin-binding growth factor is obtained from the culture solution. And a method for producing a heparin-binding growth factor characterized in that a sulfated polysaccharide or an agonist thereof is introduced into a culture solution of a cell line transformed by introducing a gene encoding a heparin-binding growth factor. The present invention resides in a method for inhibiting degradation of a heparin-binding growth factor characterized by coexistence.
[0009]
Hereinafter, the present invention will be described in detail.
The heparin-binding growth factor defined in the present invention corresponds to all heparin-binding growth factors having affinity for sulfated polysaccharides. Specifically, proteins such as acidic FGF, alkaline FGF (Methods in Enzymology, 147 , 120 (1987)), KGF (Science, 245 , 752 (1989)), HGF, etc. whose entire amino acid sequence has been clarified Sex factor etc. are mentioned.
[0010]
The growth factor-producing cell used in the present invention is a cell having a sulfated polysaccharide structure typified by HSPG on its surface, and into which the above growth factor cDNA has been introduced by a recombinant method. Specifically, a production strain in which an expression vector containing cDNA encoding hHGF is constructed according to the method described in JP-A-3-285893, and the expression vector is introduced into a host such as CHO cells. Is mentioned. In such HGF-producing cell lines, an increase in HGF recovery of about 1.5 to 5 times or more is observed by addition of sulfated polysaccharides. Such a cell line can be cultured by suspension culture or adherent culture by a conventional method. As the medium, MEM, RPMI-1640 or the like is used, and the medium is cultured in the presence of 5-10% serum, or in the presence of an appropriate amount of insulin, dexamethasone, transferrin or the like.
[0011]
In the present invention, the sulfated polysaccharide added to the transformed cell culture system producing the heparin-binding growth factor includes proteoglycans including natural glycosaminoglycans, glycosaminoglycans, glucans, and derivatives thereof. . A sulfated polysaccharide is a carbohydrate produced by dehydration condensation of a sugar by a glycosidic bond, that is, a glycan having a sulfate group added, and such a polysaccharide is composed of repeating disaccharide units such as glycosaminoglycan. One of which disaccharides are composed of glucosamine or galactosamine (Science of Life, 39 (4), 306 (1988)), polysaccharides sulfated like glucan, These derivatives are included. Specific examples include chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, heparin, dextran sulfate and the like. In addition, these sulfated polysaccharide agonists can also be added to the culture system of the present invention.
[0012]
At least one kind of the sulfated polysaccharide or its agonist is added to a cell culture medium that produces heparin-binding growth factor. The amount to be added is not particularly limited as long as it does not have a strong influence on the growth of production cells, but it is usually suitably used in the range of 1-1000 μg / ml, more preferably 10-100 μg / ml. The addition is usually performed from the beginning of the culture, and a method of adding a new medium every time the medium is exchanged. However, it may be added during the culture.
[0013]
By such culture, heparin-binding growth factor is produced in the culture supernatant of the transformed cell culture system, so that the desired heparin-binding growth factor can be separated and purified from this culture supernatant according to a conventional method. . Specifically, the culture supernatant can be easily isolated and purified by using various types of chromatography, for example, S-Sepharose, sulfated cellulofine, and the like.
[0014]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples as long as the gist thereof is not exceeded.
Example 1 Detection of HGF receptor on CHO cells A purified recombinant hHGF preparation (prepared according to the method described in JP-A-3-72883) was prepared using the chloramine T method (Nature, 194 , 495 (1962)). It was labeled with iodine. That is, the reaction was allowed to proceed by adding 100 μl of 1 mg / ml chloramine T to 20 μg of hHGF dissolved in 100 μl of 0.5 M sodium chloride and 0.5 M phosphate buffer (pH 7.4), and then 2.5 mg / ml pyrol. The reaction was stopped by adding 100 μl of sodium sulfite. The reaction solution was added to a Sephadex G-25 (Pharmacia) column equilibrated with PBS (-) containing 0.25% gelatin and 5 mM potassium iodide to remove unlabeled iodine. ¹²⁵ I-labeled hHGF (hereinafter abbreviated as “labeled hHGF”) was sterilized by passing through a 0.2 μm filter, dispensed, and stored at −20 ° C. until use.
[0015]
CHO cells were cultured in a plastic dish (Corning) having a bottom area of 78.5 cm ^{2, and} the one that reached a nearly saturated density (semi-confined) was used for the experiment. With the cells attached to the dish, the cells were washed 3 times with 0.25% gelatin and 25 mM hepes-containing DMEM medium (Gibco) pH 7.4 (hereinafter abbreviated as “binding medium”), and then labeled with 0-200 pM. The culture was gently shaken for 4 hours at 4 ° C. with 10 ml of a binding medium containing hHGF. As a control, a dish in which 2 nM unlabeled hHGF was further added to 200 pM labeled hHGF was also prepared. The supernatant was removed, washed 4 times each with ice-cold binding medium and then with ice-cold PBS (-), and then 5 ml of cross-linking buffer (140 mM sodium chloride, 1 mM magnesium chloride, 10 mM phosphoric acid containing 100 μg / ml bis-suberate) The reaction was carried out in sodium (pH 8.3) at 4 ° C. for 30 minutes to chemically crosslink the labeled hHGF and the hHGF binding protein on the cell surface. After stopping the reaction by adding 5 ml of 25 mM Tris-HCl buffer (pH 7.4), 140 mM sodium chloride, and 1 mM EDTA, the cells were detached with a scraper, and 400 μl of lysate (50 mM Tris -Hydrochloric acid (pH 7.4), 140 mM sodium chloride, 1% nonidet P-40, 1 mM EDTA, 2 mM phenylmethylsulfonic acid fluoride) were added, and the mixture was reacted on ice for 1 hour to solubilize the cells. A supernatant was obtained from the cell lysate by centrifugation at 12,000 × g, 4 ° C. for 30 minutes, and a part thereof was subjected to immunoprecipitation, electrophoresis and autoradiography.
[0016]
For the purpose of separating c-Met protein known as one of functional receptor molecules of HGF from the cell solubilized fraction, immunoprecipitation was performed. That is, 5 μl of anti-mouse c-Met antiserum (Eur. J. Biochem., 204 , 857 (1992)) and 20 μl of Protein-A Sepharose (Pharmacia) were added to the cell lysate, and c- Met protein / HGF complex was immunoprecipitated. The precipitate was boiled in SDS-sample buffer (0.1 M Tris-HCl (pH 6.8), 10% glycerol, 1% sodium dodecyl sulfate (SDS)) and then non-reduced to a concentration of 6% polyacrylamide. The sample was subjected to SDS-polyacrylamide electrophoresis under conditions, the gel was fixed and dried, and the protein to which labeled hHGF was bound was detected by autoradiography.
[0017]
The results are shown in FIG. In FIG. 1, lanes 1, 2, 3, and 4 represent the results of samples with labeled hHGF addition amounts of 50 pM, 100 pM, 200 pM, 200 pM + unlabeled hHGF 2 nM, respectively. The numbers on the left indicate the mobility of the molecular weight marker in kilodaltons (kDa). Under non-reduction, a band of labeled hHGF alone was observed in the vicinity of 70 kDa, and a specific band was detected in the vicinity of 250 kDa. It is concluded that this band is a complex of hHGF and c-Met protein because it is specifically precipitated with an anti-c-Met antibody and is about 180 kDa when the molecular weight of hHGF is subtracted.
[0018]
From the above results, C-Met protein already known as a constituent molecule of HGF functional receptor already exists on CHO cells which are parent strains of hHGF-producing cell lines. It was found to bind specifically to the Met protein.
[0019]
Example 2 Measurement of remaining amount of added hHGF molecule in CHO cell culture system Next, the following experiment was conducted for the purpose of examining the degradation rate of hHGF in CHO cells. That is, a certain amount of hHGF was added to the culture supernatant, and the amount of HGF remaining in the supernatant was measured over time by an enzyme immunoassay (ELISA) method. The difference in the rate of HGF degradation depending on the presence or absence of sulfated polysaccharides A comparative study was conducted.
[0020]
The CHO cells were detached with 10 mM phosphate buffer-physiological saline pH 7.4 (PBS (-)) containing 0.02% trypsin-EDTA (ethylenediaminetetraacetic acid), washed three times by low speed centrifugation, and then the cells. The suspension was suspended in an eRDF medium (Kyokuto Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (FBS) at a concentration of 5 × 10 ⁵ cells / ml. 1 ml of this cell solution was added to a 12-well microplate (Costa) and cultured at 37 ° C. for one day in an air gas phase containing 5% carbon dioxide. After 16 hours, the culture solution in the culture well was removed, and after washing twice with PBS (−), 1 ml of fresh medium containing 2 μg / ml human recombinant HGF was added. At this time, heparin (molecular weight: 4000-6000, Sigma) was added to some wells to a final concentration of 100 μg / ml. Cultivation was continued at 37 ° C. in an air gas phase containing 5% carbon dioxide, and 50 μl of each supernatant was collected after 1, 2, 5, 24, and 68 hours, and the amount of residual HGF in the supernatant was measured by ELISA. did. The supernatant was stored frozen at −80 ° C. until just before measurement. The amount of residual HGF in the test culture supernatant was measured by the hHGF-specific sandwich ELISA method. Specifically, the test supernatant was 0.1% CHAPS, 0.4 M sodium chloride, 0.1% bovine serum albumin (BSA) (Sigma, RIA grade), 0.05% Tween20-containing 10 mM phosphate buffer. (PH 7.4) was diluted 50, 100, 200 times. As an ELISA plate, an anti-hHGF monoclonal antibody was previously adsorbed on a 96-well multiplate (NUNK, for ELISA) (0.5 μg / 50 μl-50 mM carbonate buffer (pH 9.6) / well), Subsequently, PBS (-) containing 1% BSA and 0.05% sodium azide was allowed to react for one day or more to block the well wall.
[0021]
50 μl of the diluted test solution was added to the ELISA plate well and allowed to stand overnight at 4 ° C. The next day, the wells were washed 4 times with PBS (-) (PBST) containing 0.05% Tween 20, and then a peroxidase-conjugated anti-hHGF polyclonal antibody was added as a secondary antibody and reacted at room temperature for 2 hours or more. I let you. The secondary antibody was affinity-purified with a protein A column from rabbit serum immunized with human recombinant HGF and conjugated with peroxidase by the periodate method (J. Histochem. Cytochem., 22 , 1084 (1974). )) Made.
[0022]
After incubation, the wells were washed 6 times with PBST, and a coloring solution (0.04% orthophenylenediamine, 0.02% hydrogen peroxide-containing phosphate citrate buffer (pH 5.0)) was added at 50 μl / The well was added and left at room temperature for several minutes to several tens of minutes. When moderate color development was obtained, 4.5 N sulfuric acid was added (50 μl / well) to stop the reaction, and the absorbance at 490 nm was measured with an immunoleader (Nihon Intermed). In order to prepare a calibration curve, a standard hHGF whose concentration was calculated in advance from the extinction coefficient was used by serial dilution in the range of 0-40 ng / ml. In addition, it confirmed from the correspondence with the band of the electrophoresis of hHGF after enzyme digestion that the ELISA method used here did not detect an HGF degradation product at all.
[0023]
The results are shown in FIG. The horizontal axis represents the time after the start of culture, and the vertical axis represents hHGF (μg / ml) in the culture supernatant. In the culture supernatant, the amount of hHGF rapidly decreased in the group without addition of heparin (●), whereas it was confirmed that the decrease rate of hHGF was extremely delayed in the addition group (■). From the slope of the graph, the decrease rate t _{1/2 of} hHGF in the supernatant was calculated to be about 12 hours in the heparin-free group and about 135 hours in the added group. From the above results, it was found that the degradation of hHGF in CHO cells was remarkably suppressed by adding heparin, which is a kind of oxidized polysaccharide, to the culture medium.
[0024]
Example 3 Effect of heparin on hHGF production of hHGF-producing CHO cell line In the actual hHGF-producing strain, sulfated polysaccharide was added to the culture solution, and the amount of hHGF recovered was examined.
According to the method described in JP-A-3-285893, transformed hHGF production strains KBE, KT4-3, and GE43-19, which introduce an expression vector containing hHGF cDNA into CHO cells and produce hHGF constitutively and stably. Got.
The hHGF-producing strain was suspended in 5% FBS-containing eRDF medium at a cell concentration of 4 × 10 ⁴ cells / ml, and 1 ml each was seeded on a 12-well microplate. Heparin (Sigma) was added thereto so that the final concentration was 0 to 1000 μg / ml, and the culture was started. A part of the supernatant was collected after a certain period of time by the hHGF specific ELISA method (see Example 2). The hHGF content was measured.
[0025]
The results are shown in FIGS. FIG. 3 shows a case in which the medium was changed every 2-3 days after the start of KBE cell culture, the supernatant was collected, and the amount of hHGF was traced over time until the 11th day of culture. The horizontal axis indicates the number of days of culture, and the vertical axis indicates the accumulated amount of hHGF in the culture supernatant as an ELISA unit (U; OD490 × dilution rate). Under the present conditions, the cells almost reached saturation density on the 4th day of culture, and thereafter the number of cells was gradually decreased, and the cell viability was 30% or less on the 13th day of culture. In the heparin-added group, a significant increase in the amount of hHGF was observed from the addition of a low concentration of 1 μg / ml, and a 2-3-fold increase in the amount of HGF was evident in any culture period in the presence of 10-100 μg / ml heparin. became. FIG. 4 shows the result of measuring the hHGF content by exchanging the medium immediately after the KBE cells reach the saturation density and collecting the supernatant after 2 days. The horizontal axis represents the heparin concentration, and the vertical axis represents the relative value when the hHGF content is 1.0 in the heparin-free group. In the heparin-added group, an increase in the hHGF amount of 2 times or more was already observed with the addition of 10 μg / ml compared to the non-addition group, and a 2-2.5-fold increase in the hHGF amount was shown with the addition of 1000 μg / ml. It was.
[0026]
Next, Table 1 shows the results of measuring changes in the amount of hHGF in the supernatant due to the addition of heparin in three types of hHGF-producing strains. The test supernatant was measured 2 days after the medium was changed immediately after cell saturation. The hHGF content of the heparin-free group was taken as 1.00, and the hHGF content of the heparin-added 10 μg / ml group was shown as a relative value. In all strains, 2-5-fold increase in the amount of recovered hHGF was observed.
[0027]
[Table 1]

[0028]
As a result, it was found that the amount of hHGF recovered increases several times by adding heparin to the culture solution of the hHGF-producing cell line.
[0029]
Example 4 Effect of various sulfated polysaccharides on hHGF production of hHGF-producing CHO cell line Next, the same investigation was performed on sulfated polysaccharides other than heparin.
The KBE strain was seeded at 4 × 10 ⁵ cells / ml / well in a 12-well microplate, the supernatant after 5 days of culture was collected, and the amount of hHGF in the culture supernatant was measured by the ELISA method described above. . As added polysaccharides, heparin, heparan sulfate, dextran (Sigma Co.), dextran sulfate and chondroitin sulfate (Seikagaku Corporation) were used.
[0030]
Table 2 shows the results. Increased hHGF content was observed in any of heparin, heparan sulfate, dextran sulfate, and chondroitin sulfate. In the case of heparin, heparan sulfate, and dextran sulfate, the maximum effect was obtained in the addition range of 10-100 μg / ml, and an increase of 2 times or more was shown. On the other hand, although chondroitin sulfate had an effect, its increase effect was somewhat low, and the effective necessary dose was shifted to the higher dose side. With dextran having no sulfate group, no effect was observed.
[0031]
[Table 2]

[0032]
The KBE strain can maintain HGF production for several months or more by a suspension culture method using microcarrier (Pharmacia), but in this case, the sulfated polysaccharide maintained the same effect for several months. Even in the examination of various culture forms and culture scales, the action of sulfated polysaccharides is due to adherent culture (T flasks, dishes, roller bottles, etc.) and suspension culture (with or without microcarriers). Regardless of the spinner culture), the same was observed in microwell cultures of 96-well plates at culture scales of several liters or more.
[0033]
From the above results, it was found that the amount of HGF recovered increases two to several times by adding various sulfated polysaccharides to the culture system of HGF-producing cells. This effect is caused by the addition of low-dose sulfated polysaccharides, and is maintained for a long period of several hours to several months regardless of the culture method and the type of sulfated polysaccharides. . The principle of this effect is that sulfated polysaccharide binds HGF via its sulfate group and significantly binds, consumes and degrades HGF to receptors (such as c-Met protein and heparan sulfate proteoglycan) on HGF-producing cells. It was understood to delay.
[0034]
【The invention's effect】
According to the production method of the present invention, the production and recovery amount of heparin-binding growth factor can be increased several times, and as a result, the cost in production of the growth factor can be significantly reduced. By producing a heparin-binding growth factor based on the present invention, widespread use in the medical field can be expected.
[Brief description of the drawings]
FIG. 1 is a drawing showing the results of SDS-polyacrylamide electrophoresis for the formation of hHGF / c-Met protein complex in CHO cells.
FIG. 2 shows a comparison of hHGF consumption rate in CHO cells in the presence and absence of heparin.
FIG. 3 is a drawing showing a comparison of the amount of hHGF in the culture supernatant of a hHGF-producing strain in the presence and absence of heparin over time.
FIG. 4 is a graph showing the influence of the heparin addition dose on the amount of hHGF in the culture supernatant of the hHGF-producing strain.

Claims (3)

A liver characterized by culturing a CHO cell line transformed by introducing a gene encoding hepatocyte growth factor in the presence of a sulfated polysaccharide and collecting hepatocyte growth factor from the culture medium. Method for producing parenchymal cell growth factor.   The method according to claim 1, wherein the sulfated polysaccharide is selected from proteoglycans, glycosaminoglycans, glucans and derivatives thereof containing sulfate groups. A method for inhibiting degradation of hepatocyte growth factor, characterized in that a sulfated polysaccharide is allowed to coexist in a culture solution of a CHO cell line transformed by introducing a gene encoding hepatocyte growth factor.

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