JPH05301824A - Liver parenchyma cell growing agent - Google Patents

Abstract

PURPOSE:To attain increase of activity of hHGF and stabilization of a hHGF molecule by combinedly using polysaccharide or its derivative and hHGF. CONSTITUTION:The objective liver parenchyma cell growing agent consists of a liver parenchyma cell growth factor (hHGF) obtained by purification from a plasma or a recombinant method, polysaccharide such as heparin, dextran sulfate, heparan sulfate and chondroitin sulfate or its derivative.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hepatocyte proliferation agent that promotes the proliferation of hepatocytes, and more specifically, it comprises a polysaccharide or a derivative thereof and hepatocyte growth factor. The present invention relates to a hepatocyte proliferative agent in which is stabilized and its proliferative activity is enhanced.

[0002]

BACKGROUND OF THE INVENTION In recent years, human-derived protein factors capable of promoting the proliferation of hepatocytes, namely human hepatocyte growth factor (hereinafter "hHGF").
May be abbreviated. ) Was found in plasma of patients with fulminant hepatitis (JP-A-63-22526), and hHGF
Amino acid sequence and gene encoding protein (cDN
A) Sequence (JP-A-3-72883), a method for producing hHGF protein using this cDNA, and a transformant (JP-A-3-285693) have been proposed. It has been recognized that such hHGF has a function of promoting proliferation of liver parenchymal cells in vitro. The present inventor studied the binding mode of these hHGF to hepatocytes in order to elucidate the growth mechanism of naturally occurring hHGF or recombinant hHGF to hepatocytes. Among them, h bound to hepatocytes
It was found that most of HGF was washed with a buffer containing a polysaccharide such as heparin or a derivative thereof. From these results, the polysaccharide or its derivative inhibits the binding of hHGF to hepatocytes, the uptake into hepatocytes, or the process of metabolism thereof, which is considered to be essential for inducing hepatocyte proliferative activity of hHGF. It was expected to act as an inhibitor.

[0003]

[Means for Solving the Problems]
The effect of the polysaccharides or their derivatives on the hepatocyte proliferation activity of was investigated further. That is, Segren (Se
Glen's method (Methods in cell)
biology, vol 13, p29, Academ
ic Press, New York (1976)), when (1) a polysaccharide or a derivative thereof and hHGF were mixed and added to hepatocytes, (2) a polysaccharide or a derivative thereof was added and then hHGF was added. The effects on the proliferative activity of hepatic parenchymal cells were measured for the case of addition of (3) hHGF and the case of addition of polysaccharide or its derivative, respectively.

As a result, interestingly, unlike the results expected in the prior art, polysaccharides or their derivatives and hH
HGGF was added when GF was mixed and added, and when hHGF was added after adding the polysaccharide or its derivative,
The ability to promote the proliferation of hepatocytes is greatly enhanced, and in addition, by adding a polysaccharide or a derivative thereof to the system for measuring the proliferation of hepatocytes of hHGF, the degradation of hHGF present in the culture supernatant is performed. It was found for the first time that the above was suppressed, that is, the hHGF molecule was stabilized, and the present invention was completed.

That is, the gist of the present invention resides in a hepatocyte proliferating agent comprising a polysaccharide or a derivative thereof and a hepatocyte growth factor. To explain the present invention below, hHGF used in the present invention is a proteinaceous factor of human origin and having an activity of promoting the growth of hepatocytes, which is obtained by purifying from plasma or the like and a recombinant method. Any of those obtained by expressing the cDNA by the method described above can be used. Specifically, for example, by protein-chemically separating and purifying plasma from a patient with fulminant hepatitis according to the method described in JP-A-63-22526, or in JP-A-3-28569.
Human placenta-derived cDNA according to the method described in Japanese Patent No.
CDNA encoding hHGF obtained from A library
It can also be obtained by constructing an expression vector containing the above and expressing it in a host such as CHO cells.

[0006] Such hHGF is SD under non-reducing conditions.
Molecular weight of about 76,000-92.0 by S-PAGE
Band at 00 and SDS-P under reducing conditions
A large subunit having a molecular weight of about 56,000 to 65,000 and a small subunit having a molecular weight of about 32,000 to 35,000, which give major bands to the molecular weights of about 56,000 to 65,000 and about 32,000 to 35,000 by AGE. It is a heterodimeric proteinaceous substance in which subunits are linked by disulfide bonds. And this hHGF is 80
It has the property of losing the activity of promoting the growth of hepatocytes by heat treatment at 10 ° C for 10 minutes or by digestion with trypsin or chymotrypsin, and has a strong affinity for heparin. Generally, the hHGF used in the present invention is
It started to show proliferative activity of hepatocytes at a concentration of about 0.5 to 2 ng / ml, and exhibited good proliferative activity at a concentration of about 5 to 10 ng / ml.

In the present invention, the above-mentioned hHGF and a polysaccharide or a derivative thereof are used in combination. The term “polysaccharide or derivative thereof” as used in the present invention refers to a carbohydrate produced by dehydration condensation of two or more molecules of a monosaccharide through a glycoside bond, that is, all glycans or their derivatives. Such polysaccharides or derivatives thereof, like glycosaminoglycans,
It has a structure formed by repeating disaccharide units, and one of the disaccharides consists of glucosamine or galactosamine (Science of Life, 39 (4), p. 306-310 (19).
88)) and polysaccharides such as glucan, which are sulfated, are preferably used. Specifically, for example, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate,
Examples include heparin, dextran, dextran sulfate and the like. Particularly, sulfate derivatives of polysaccharides such as heparin, dextran sulfate, heparan sulfate and chondroitin sulfate can be preferably used.

The polysaccharide or its derivative is preferably used in an excess amount with respect to hHGF, and usually, the polysaccharide or its derivative is used in the range of 10 ² to 10 ⁵ mol with respect to 1 mol of hHGF. The hepatocyte proliferation agent of the present invention is mainly used as an injection. The injection can be prepared according to a conventional method. At that time, known additives such as human serum albumin and a surfactant may be used in combination. Specifically, for example, the composition shown below is dissolved in 10 mM phosphate buffer (PBS (-)) having a pH of 7.5 to make the total volume 5 ml, and then sterilized with a 0.22 μm filter, and then a vial. It can be prepared by dispensing in a bottle, or by further freeze-drying and storing it, and dissolving it in distilled water, physiological saline, or the like at the time of use.

(1) hHGF 100 μg, human serum albumin 50 mg, Triton X-100 0.5 mg (2) hHGF 100 μg, human serum albumin 50 m
g (3) hHGF 100 μg, Triton X-100
0.5 mg (4) hHGF 100 μg, Tween 80 0.5 mg (5) Heparin 5 mg (6) Heparin 5 mg, human serum albumin 50 mg,
Triton X-100 0.5 mg (7) hHGF 100 μg, heparin 5 mg, human serum albumin 50 mg, Triton X-100 0.5 m
g (8) hHGF 100 μg, heparin 5 mg, human serum albumin 50 mg (9) hHGF 100 μg, heparin 5 mg, Triton X-100 0.5 mg (10) hHGF 100 μg, heparin 5 mg, Tween 80 0.5 mg (11) hHGF 100 μg, heparin 5 mg

In the present invention, an injection containing both hHGF and a polysaccharide or a derivative thereof may be prepared and used, or separate injections may be prepared to prepare a polysaccharide or a derivative thereof. After using the injectable formulation containing hHGF, the injectable formulation containing hHGF may be used, or these injectable formulations may be used simultaneously. When the injection containing hHGF is used first, it is preferable to use the injection containing the polysaccharide or its derivative as soon as possible. The amount of the hepatocyte-proliferating agent of the present invention to be used varies depending on the activity of hHGF which is an active ingredient and the subject to be used, but is approximately 0.1 μg / kg.
It is selected from the range of up to 1,000 μg / kg.

[0011]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1 High activation of hHGF protein by adding heparin Method of Seglen (Methodes)
in cell biology, vol 13, p2
9, Academic Press, New York
(1976)), male Wistar rats (body weight 2
Hepatic parenchymal cells were isolated from 0.05 g) using 0.05% collagenase (type I, Sigma). Collagen-coated multi-well plastic dishes (Nun with 1.55 cm diameter wells)
c) was seeded at a concentration of 5 × 10 ⁴ cells / 0.2 ml / cm ² , and monolayer culture was carried out at 37 ° C. in an air vapor phase containing 5% carbon dioxide (Tanaka et al., J. Bioche.
m. 84 , 937-946 (1978)). As a culture medium, 5% fetal bovine serum (FBS, Filtron, Al
(Tona, Australia), 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin were used in Williams E medium (Flow Laboratories, abbreviated as “basic medium” hereinafter).

[0012] Three hours after the start of the culture, the medium was replaced with a new basic medium, and 20 hours later, the medium was replaced with a basic medium containing no fetal calf serum. After this medium exchange, the final concentration is 0, 50, 10
0, 200, 500 μg / ml heparin (molecular weight 40
00-6000, Sigma), and then recombinant hHGF at a final concentration of 100 ng / ml. After that, the culture was continued for 20 hours, and then the DNA synthesis was measured. DNA synthesis is ³ H- thymidine (Amers
ham) to a final concentration of 4 μCi / ml (2 Ci / mm
ol), and the culture was continued at 37 ° C. for 4 hours, and the incorporation of ³ H-thymidine into DNA was measured. As a control group, a group to which hHGF was not added was used. After labeling by the above culture, the cells were washed 3 times with cold PBS (−), 2% perchloric acid and 95% ethanol, respectively. The cells are then air dried and 1N
After solubilization with 500 μl of NaOH, a part thereof was taken for radioactivity measurement. The concentration of recombinant hHGF was measured by the enzyme immunoassay method. The activity value was determined as the difference in the counts of the control group from the count of the amount of ³ H-thymidine incorporated into liver parenchymal cell DNA by the test sample (hereinafter, all the activity values of hHGF were determined by this method). The result is shown in FIG. In FIG. 1, the horizontal axis represents the final concentration of added heparin and the vertical axis represents hHGF activity.

FIG. 2 shows a test sample having a final concentration of 100 μm.
Heparin (molecular weight 4000-6000, Sigma) of g / ml was added, and then recombinant hHGF was added to a final concentration of 0, 4.28, 8.75, 17.5, 35, 70, 14.
The results when added at a concentration of 0 ng / ml are shown. The horizontal axis represents recombinant hHGF when heparin was added.
And the vertical axis represents hHGF activity. In Fig. 2, the curve (1) shows a final concentration of 100 µg / ml.
Shows the hHGF activity with the addition of the heparin, and the curve (2) shows the hHGF without the addition of heparin.
GF activity is shown.

From the above results, the addition of heparin to hHGF
It can be seen that the protein is highly activated. Example 2 Inhibition of degradation of hHGF protein by addition of heparin The method of Seglen shown in Example 1 (M
etodes incell biology, vol
13 , p29, Academic Press, Ne
Hepatocytes are separated according to w Yorl (1976), and these hepatocytes are placed in a collagen-coated multiwell plastic dish (Nunc) having a well of 3.5 cm in diameter at 5 × 10 ⁴ cells / 0.2 ml / cm ^2.
At a concentration of 5% carbon dioxide gas in the air phase, 37
Monolayer culture was performed at ° C. (Tanaka et al.,
J. Biochem. 84 , 937-946 (197).
8)). As a culture medium, 5% fetal bovine serum (FBS, F
iltron, Altona, Australia),
Williams E medium supplemented with 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin (Flow Laboratories, hereinafter abbreviated as “basic medium”) was used.

After 3 hours from the start of culture, the medium was replaced with a new basal medium, and 20 hours later, PB containing 0.25% gelatin was added.
After washing three times with S (-), Dulbecco's MEM containing 0.25% gelatin and 25 mM Hepes (hereinafter abbreviated as "Binding medium") was used for each well twice.
ml was added, and monolayer culture was carried out at 37 ° C. for 3 hours in an air-gas phase containing 5% carbon dioxide. After that, the binding medium was exchanged, 1 ml was added to each well, and heparin (molecular weight: 400 was added) so that the final concentration was 100 μg / ml.
0-6000, Sigma) was added. As a control group, a group without heparin added was prepared. On the other hand, the method of Hunter et al. (Nature 19
4 , 495-496 (1962), carrier-free Na ¹²⁵ I (3.7 GBq, NEN) was used to label the recombinant hHGF. The resulting labeled hHGF (hereinafter abbreviated as “labeled hHGF”) was added to each well so that the final concentration was 200 pM. After that, monolayer culture was performed at 37 ° C. in an air vapor phase containing 5% carbon dioxide. 0 minutes, 10 minutes, 30 minutes, 60 minutes,
The culture supernatant was collected over a period of 120 minutes and 180 minutes. The collected culture supernatant was sufficiently cooled on ice, after which transfer RNA was added so that the final concentration was 100 μg / ml, and then trichloroacetic acid (TCA) was added so that the final concentration was 10%. The test sample thus prepared was cooled under ice-cooling for 3 hours and then centrifuged at 4 ° C. at 10,000 G to collect the supernatant, which was used for measuring the amount of hHGF degraded. The supernatant thus obtained contains the labeled hHGF whose molecular weight has been reduced or decomposed by incubation with hepatocytes.

The results are shown in FIG. The horizontal axis represents the time from the addition of labeled hHGF to hepatocytes until the collection of the culture supernatant, and the vertical axis represents the amount of degraded labeled hHGF. In FIG. 3, curve (1) shows the amount of degraded labeled hHGF when heparin at a final concentration of 100 μg / ml was added, and curve (2) shows the degradation when heparin was not added. Shows the amount.

From the above results, the addition of heparin to hHGF
It can be seen that it is effective in inhibiting protein degradation. Example 3 High activation of hHGF by dextran sulfate and dextran Dextran sulfate having a similar molecular structure to dextran sulfate and dextran not having a sulfate group in the molecule showed the ability of hHGF to grow hepatocytes. The effect on it was investigated.

Hepatocytes were isolated and cultured in the same manner as in Example 1. After 3 hours from the start of the culture, the medium was exchanged with a basic medium and after 20 hours of culture, the final concentration was 200 ng.
/ Ml recombinant hHGF and 0, 1, 10, 50,
100, 500 μg / ml dextran sulfate (molecular weight 8000, Sigma), dextran (molecular weight 1000)
0, Sigma) or heparin (molecular weight 4000-6
000, Sigma), and the medium was replaced with a basic medium containing no fetal bovine serum. After culturing for another 20 hours, DN
A synthesis measurements were made. DNA synthesis, recombinant hHGF
Was measured in the same manner as in Example 1.

The results are shown in FIG. In FIG. 4, the horizontal axis represents the final concentration of added dextran sulfate, dextran or heparin, and the vertical axis represents the hHGF activity. As shown in FIG. 4, by adding dextran sulfate or dextran, hepatocyte proliferation activity of hHGF can be enhanced. In particular, dextran sulfate was found to remarkably enhance the activity of hHGF as well as heparin.

Example 4 High activation of hHGF by various polysaccharides and their derivatives As test samples, in addition to heparin, dextran sulfate and dextran used in the above examples, heparan sulfate,
Hyaluronic acid, chondroitin, chondroitin sulfate A
About -E, the effect of hHGF on the hepatocyte proliferation ability was examined.

First, hepatocytes were isolated according to Example 1 and, in the same manner as in Example 1, the hepatocytes had a diameter of 1.55 cm.
The collagen-coated multi-well plastic dish having the wells described above was rolled up at a concentration of 5 × 10 ⁴ cells / 0.2 ml / cm ² and subjected to monolayer culture at 37 ° C. in an air-gas phase containing 5% carbon dioxide. As a culture medium, 5% fetal bovine serum (FB
S) Williams E medium (hereinafter abbreviated as "basal medium") supplemented with 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin was used. The medium was exchanged with a basal medium 3 hours after the start of culturing, and after culturing for 20 hours, the medium was exchanged with a basal medium containing no fetal bovine serum.

Next, each test sample having a final concentration of 100 μg / ml and 200 ng / ml of recombinant hHGF were added.
After culturing for 20 hours, DNA synthesis was measured. DNA synthesis was performed in the same manner as in the method described in Example 1. As a control group, a group to which hHGF was not added was used. After labeling with the above culture, the cells are placed in cold PBS.
(−) Washed 3 times each with 2% perchloric acid and 95% ethanol. The cells are then air dried and 1N NaOH 50
After solubilization with 0 μl, a part of the solution was taken for radioactivity measurement. The concentration of recombinant hHGF was measured by the enzyme immunoassay method. The activity value was determined as the difference in the counts of the control group from the counts of the amount of ³ H-thymidine incorporated into the liver parenchymal cell DNA by the test sample. The result is shown in FIG.

In FIG. 5, the vertical axis represents the various polysaccharides used,
1 illustrates glycosaminoglycans and their derivatives.
The horizontal axis shows HGF activity. In particular, it was found that heparin, dextran sulfate, heparan sulfate, and chondroitin sulfate markedly enhance the HGF activity.

Example 5 Preparation of Polysaccharide or Derivative-Binding hHGF Protein and Enhancement of Hepatocyte Proliferation Activity The hHGF has a strong affinity for polysaccharides such as heparin and dextran sulfate, or their derivatives. So hHG
After removing the polysaccharide or its derivative from F, mixing the polysaccharide or its derivative, the polysaccharide or its derivative that did not bind to hHGF was removed, and only the polysaccharide or its derivative-bound hHGF was prepared. In particular, heparin, dextran sulfate and the like are known to exhibit blood anticoagulant activity in vivo. However, by using this method, it is expected that the effect of a single polysaccharide or a derivative thereof, which did not bind to hHGF, such as heparin or dextran sulfate, on the body can be extremely suppressed.
Further, it was found that hHGF bound with such a molecule induces hepatocyte proliferation ability extremely strongly as compared with the case where only hHGF is added, as in the case shown in Example 1.

The following is a polysaccharide or its derivative-bonded hH
The method for preparing the GF protein and its ability to enhance hepatocyte proliferation activity will be described. Recombinant hHGF protein 1m
The final concentration is 5 for 1 ml of PBS (-) solution containing g.
0 mg / ml heparin (molecular weight 4000-6000,
100 μl of PBS (-) solution containing Sigma) was added. After standing at 4 ℃ for 24 hours, Sephadex
Gel filtration using a G-50 column (Pharmacia) was performed to remove heparin that did not bind to hHGF. That is, T with a final concentration of 0.01%
Sephadex G equilibrated with PBS (-) solution containing ween80 (hereinafter abbreviated as "elution buffer")
A mixture of heparin and hHGF was added to a −50 column (1.5 × 12 cm), and then an elution buffer was added to the same column. The hHGF protein bound to the heparin molecule (hereinafter referred to as heparin-bound hHGF) is collected in the excluded volume fraction of the same column. On the other hand, heparin (Sigma) used in this operation has a molecular weight of 4000-6000.
Since the low molecular weight heparin is used, heparin-bound hHGF is not eluted in the excluded volume fraction from which it is recovered.

The heparin-binding hHGF thus obtained was then assayed for hepatocyte proliferation activity. Hepatocytes were isolated and cultured in the same manner as in Example 1. After 3 hours from the start of the culture, the medium was replaced with a basic medium, and after 20 hours of culture, the medium was replaced with a basic medium containing no fetal bovine serum (Flow Laboratory). After this medium exchange, the final concentration of the test sample was 0, 0.5, 2, 10, 50, 100, 300.
μg / ml heparin-bound hHGF or heparin-unbound hHGF was added. After this, after culturing for 24 hours or culturing for 48 hours, DNA synthesis was measured. DNA synthesis uses ³ H-thymidine (Ame
Rsham) at a final concentration of 4 μCi / ml (2 Ci / m
mol), and then the culture was continued at 37 ° C. for 5 hours, and the incorporation of ³ H-thymidine into DNA was measured. HHG as a control group
A group to which F was not added was used. After labeling by the above culture,
The cells were washed 3 times each with cold PBS (−), 2% perchloric acid and 95% ethanol. Then air dry the cells,
After solubilization with 500 μl of 1N NaOH, a part of the solution was taken and measured for radioactivity. The concentrations of the recombinant hHGF and heparin-bound hHGF used here were measured by the enzyme immunoassay method in the same manner as in Example 1. The activity value was determined as the difference in the counts of the control group from the counts of the amount of ³ H-thymidine incorporated into the liver parenchymal cell DNA by the test sample.

The results of culturing for 24 hours after addition of the test sample are shown in FIG. 6, and the results of culturing for 48 hours are shown in FIG.
6 and 7, the horizontal axis represents the concentration of the added test sample and the vertical axis represents the hHGF activity. 6 and 7
As shown in, the heparin-binding hHGF was observed to enhance and maintain the hepatic parenchymal cell proliferation ability at a high addition amount from a low concentration to a high concentration, as compared with hHGF not binding heparin. Especially H that does not bind heparin
It was found that heparin-bound HGF stably maintained a high activity, as compared with the case where GF was added at a concentration of 50-300 ng / ml, which markedly reduced the proliferation ability of hepatocytes.

Example 6 Preparation of Various Polysaccharides and Derivative-Binding hHGF Proteins and Degradation Inhibitory Activity of the hHGF Proteins Similar to Example 2, according to the method of Hunter et al., Recombinant hHGF with Na ¹²⁵ I was prepared. Labeling was performed. The final concentration was 50 with respect to 200 µl of the PBS (-) solution containing 3 µg of the obtained labeled recombinant hHGF protein.
Heparin (molecular weight of Sigma, 400
0-6000) or dextran sulfate (Sigma,
200 μl of PBS (−) solution containing a molecular weight of 8000) was added. After leaving it overnight at 4 ℃, Sephad
Gel filtration was performed using an ex G-50 column (Pharmacia) to remove heparin or dextran sulfate that did not bind to hHGF.
That is, PB containing Tween 80 at a final concentration of 0.01%
Sephadex G-50 column (1.5 x) equilibrated with S (-) solution (hereinafter abbreviated as "elution buffer")
12 cm), a mixture of heparin or dextran sulfate and hHGF was added, and then an elution buffer was added to the same column. Labeled hHGF protein bound to heparin or dextran sulfate molecule (hereinafter heparin-bound type)
¹²⁵ I-hHGF or dextran sulfate-bound ¹²⁵
I-hHGF) is collected in the excluded volume fraction of the same column.

Next, hepatocytes were prepared according to Example 2. That is, the method of Seglen and this hepatocyte were used to prepare a collagen-coated multi-well plastic dish (Nun) having a well with a diameter of 3.5 cm.
C) was rolled up at a concentration of 5 × 10 ⁴ cells / 0.2 ml / cm ² , and monolayer culture was carried out at 37 ° C. in an air vapor phase containing 5% carbon dioxide. Culture medium is 5% fetal bovine serum (FBS), 1μ
M dexamethasine, 100 U / ml penicillin and 100
Williams E supplemented with μg / ml streptomycin
A medium (hereinafter abbreviated as "basic medium") was used. After 3 hours from the start of the culture, the medium was exchanged with a basic medium, and 20 hours later, the medium was exchanged.
After washing three times with PBS (-) containing 25% gelatin, the above-mentioned Binding medium was added to each well by 2 times.
ml was added, and monolayer culture was carried out at 37 ° C. for 3 hours in the air-gas phase containing 5% carbon dioxide. After that, the binding medium was exchanged, and 1 ml of the binding medium was added to each well.

[0030] For hepatocytes prepared, heparin-bound ¹²⁵ I-hHGF or dextran sulfate-bound ¹²⁵ I-hHGF a final concentration as the test sample 200pM
Was added. ¹²⁵ I as control group
-Only hHGF was added. After that, monolayer culture was performed at 37 ° C. in an air vapor phase containing 5% carbon dioxide. 0 after the start of culture
Minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 3
The culture supernatant was collected over 60 minutes. The collected culture supernatant was sufficiently cooled under ice-cooling, and then the final concentration was 100 μg /
After addition of transfer RNA to a final concentration of 10 ml, trichloroacetic acid (TCA) was further added to a final concentration of 10%. The test sample thus prepared was cooled under ice-cooling for 3 hours and then centrifuged at 4 ° C. at 10,000 G to collect a supernatant and a precipitate fraction. The supernatant thus obtained (hereinafter referred to as the TCA-solubilized fraction) contains the labeled hHGF which has been depolymerized or decomposed by incubation with hepatocytes. On the other hand, the precipitate fraction (hereinafter TC
The A-precipitated fraction) includes hHGF that has not undergone degradation. The radioactivity contained in the obtained TCA-solubilized fraction and TCA-precipitated fraction was measured and the ratio thereof was calculated.

The results are shown in FIG. ^{125 in} FIG. 8 (A)
When only I-hHGF was added, FIG. 8 (B) shows when heparin-bound ¹²⁵ I-hHGF was added, FIG. 8 (C).
Shows the case where dextran sulfate-bound ¹²⁵ I-hHGF was added. The horizontal axis represents the time from the addition of the test sample to hepatocytes until the collection of the culture supernatant, and the vertical axis represents the ratio of the TCA-solubilized fraction and the TCA-precipitated fraction to the total radioactivity. ..

As shown in FIGS. 8B and 8C,
Heparin-bound or dextran sulfate-bound hH
It was found that the degradation of the hHGF molecule was extremely suppressed by using GF. Example 7 Acceleration of rat liver regeneration by heparin-added hHGF After performing an operation to excise 2/3 of the liver of a rat (body weight: about 200 g) under anesthesia, 200 μl of a test sample solution was filled and preliminarily kept at 37 ° C. overnight. The osmotic pump (A
lzet 2001) is cannulated into the jugular vein,
The test sample solution was continuously supplied into the venous blood at a rate of 1 μl per hour. After 5 days, the animals were sacrificed and the weight of the liver was measured.

The test sample solution was a buffer solution containing 10 mM sodium phosphate and 0.7 M sodium chloride and having a pH of 7.5, and the hHGF-administered group (6 animals) had a recombinant hHGF concentration of 0.8 μg / ml. In the hHGF / heparin administration group (5 animals), a solution containing
Heparin at a concentration of ml (Sigma, molecular weight 4,000-
A liquid containing 6,000) was used as a test sample liquid. In addition,
The buffer administration group (6 animals) served as a control.

As shown in Table 1, the liver regeneration rate was significantly (P <0.05) in the hHGF administration group as compared with the buffer administration group.
Although it was high, hHGF / heparin administration group showed hHG
Even when compared with the F-administered group, a significantly high value (P <0.05) was shown, and heparin also increased in hepatocyte proliferation in animal individuals.
It was found to promote the action of hHGF. The liver regeneration rate was calculated according to the following formula.

[0035]

[Equation 1] Estimated liver weight = body weight x liver weight to body weight ratio ^* * Ratio of liver weight to body weight 5 days after sham surgery (only laparotomy without liver resection) (0.0431 in this example)

[0036]

[Table 1]

[0037]

EFFECTS OF THE INVENTION In the past, the existence of a factor that highly activates or highly stabilizes such a protein in the use of hHGF in industry and the method of using it have not been known. As shown in the present invention, by using a polysaccharide or a derivative thereof in combination with hHGF, enhancement of hHGF activity and h
The stability of the HGF molecule is improved, and as a result, the effect of hHGF can be further improved on liver regeneration and the like during liver injury in vivo. Further, when a polysaccharide or a derivative thereof is used in combination according to the present invention, the storage stability of the hHGF preparation can be expected.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between heparin concentration and hHGF activity for high activation of hHGF by addition of heparin.

FIG. 2 is a diagram showing the relationship between hHGF concentration and hHGF activity for high activation of hHGF when a certain amount of heparin is added.

FIG. 3 Labeled hHG after addition of a fixed amount of heparin
It is the figure which showed the inhibition of the decomposition of F.

FIG. 4 is a diagram showing the hepatocyte proliferation activity of hHGF when various concentrations of dextran sulfate, dextran, and heparin were added to a constant concentration of hHGF.

FIG. 5 is a diagram showing the hepatocyte proliferation activity of hHGF when various polysaccharides and their derivatives were added to a constant concentration of hHGF.

FIG. 6 is a diagram showing the relationship between the concentration of heparin-binding hHGF and heparin-unbound hHGF and the hepatocyte proliferation ability thereof, after the culture for 24 hours after the addition of each test sample.

FIG. 7 is a diagram showing the results of culturing for 48 hours after addition of each test sample, regarding the relationship between the concentration of heparin-binding hHGF and hHGF that does not bind heparin and its hepatocyte proliferation ability.

FIG. 8 shows inhibition of heparin-bound hHGF and dextran sulfate-bound hHGF degradation.

Claims (17)

[Claims] 1. A hepatocyte proliferation agent comprising a polysaccharide or a derivative thereof and a hepatocyte growth factor. 2. The hepatocyte proliferation agent according to claim 1, wherein the polysaccharide is glycosaminoglycan. 3. The hepatocyte proliferation agent according to claim 1, wherein the polysaccharide derivative is a sulfuric acid derivative. 4. The hepatocyte proliferation agent according to claim 3, wherein the polysaccharide is glycosaminoglycan. 5. A hepatocyte proliferation agent comprising at least one compound selected from heparin, dextran sulfate, heparan sulfate or chondroitin sulfate and a hepatocyte growth factor. 6. The hepatocyte proliferation agent according to claim 1, wherein the polysaccharide or derivative thereof is bound to hepatocyte growth factor. 7. The hepatocyte proliferation agent according to claim 1, which is an injection. 8. A method for enhancing the activity of hepatocyte growth factor using a polysaccharide or a derivative thereof. 9. The method according to claim 8, wherein the polysaccharide is glycosaminoglycan. 10. The method according to claim 8, wherein the polysaccharide derivative is a sulfuric acid derivative. 11. The method according to claim 10, wherein the polysaccharide is glycosaminoglycan. 12. A method for enhancing the activity of hepatocyte growth factor using at least one compound selected from heparin, dextran sulfate, heparan sulfate or chondroitin sulfate. 13. A method for stabilizing hepatocyte growth factor using a polysaccharide or a derivative thereof. 14. The method according to claim 13, wherein the polysaccharide is glycosaminoglycan. 15. The method according to claim 13, wherein the polysaccharide derivative is a sulfuric acid derivative. 16. The method according to claim 15, wherein the polysaccharide is glycosaminoglycan. 17. A method for increasing the activity of hepatocyte growth factor using at least one compound selected from heparin, dextran sulfate, heparan sulfate or chondroitin sulfate.