JP3305355B2 - Hepatocyte proliferation agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hepatocyte proliferating agent for promoting the proliferation of hepatocytes, and more particularly to a hepatocyte growth factor comprising a polysaccharide or a derivative thereof and a hepatocyte growth factor. Is stabilized and further has an enhanced proliferative activity.

[0002]

2. Description of the Related Art In recent years, a human protein factor capable of promoting the proliferation of hepatocytes, that is, a human hepatocyte growth factor (hereinafter referred to as "hHGF").
Sometimes abbreviated. ) Was found in the plasma of a patient with fulminant hepatitis (JP-A-63-22526).
Amino acid sequences and genes encoding proteins (cDN
A) 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. And it has been recognized that such hHGF has a function of promoting the proliferation of hepatocytes in vitro. The present inventors have studied the binding mode of these hHGFs to hepatocytes in order to elucidate the growth mechanism of naturally occurring hHGF or recombinant hHGF to hepatocytes. Among them, h bound to hepatocytes
HGF was found to be largely 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 the induction of hepatocyte proliferation activity of hHGF. It was expected to act as an inhibitor.

[0003]

[Means for Solving the Problems] The present inventors have developed hHGF
The effect of polysaccharides or their derivatives on the hepatocyte proliferation activity of E. coli was further investigated. That is, Seglen (Se
Glen) method (Methods in cell)
biology, vol 13, p29, Academ
ic Press, New York (1976)), when (1) a polysaccharide or a derivative thereof and hHGF are mixed and added, (2) hHGF is added after adding the polysaccharide or a derivative thereof. The effect on the proliferative activity of hepatocytes was measured for the case where the addition was performed and (3) the case where the polysaccharide or a derivative thereof was added after the addition of hHGF.

As a result, interestingly, unlike the results expected in the prior art, polysaccharides or derivatives thereof and hH
When hHGF was added after the addition of the GF and the polysaccharide or its derivative, the hHGF
Greatly enhances the ability of hepatic parenchymal cells to promote the proliferation of hepatocytes, and in addition, by adding a polysaccharide or a derivative thereof to a system for measuring the proliferation of hepatocytes of hHGF, the degradation of hHGF present in the culture supernatant Was found for the first time, that is, hHGF molecules were stabilized, and the present invention was completed.

That is, the gist of the present invention resides in a hepatocyte proliferation agent comprising a polysaccharide or a derivative thereof and a hepatocyte growth factor. In the following, the present invention will be described. HHGF used in the present invention is a protein factor derived from human and having the activity of promoting the proliferation of hepatic parenchymal cells. Any of those obtained by expressing the cDNA can be used. Specifically, for example, the protein is separated and purified from plasma of a fulminant hepatitis patient according to the method described in JP-A-63-22526, or
CDN derived from human placenta in accordance with the method described in JP-A-3
CDNA encoding hHGF obtained from library A
Can also be obtained by constructing an expression vector containing and expressing in a host such as CHO cells.

[0006] Such hHGF can be used for SD under non-reducing conditions.
According to S-PAGE, the molecular weight is about 76,000-92.0.
00 to give a band, and SDS-P under reducing conditions.
A large subunit having a molecular weight of about 56,000 to 65,000 and a small molecule having a molecular weight of about 32,000 to 35,000 which give a main band at a molecular weight of about 56,000 to 65,000 and about 32,000 to 35,000 by AGE. A heterodimeric protein substance in which subunits are linked by disulfide bonds. And this hHGF is 80
It has the property that the activity of promoting the proliferation of hepatocytes is lost by heat treatment at 10 ° C. for 10 minutes or by digestion with trypsin or chymotrypsin, and has a strong affinity for heparin. Generally, hHGF used in the present invention comprises:
Hepatocyte proliferative activity starts to be exhibited at a concentration of about 0.5 to 2 ng / ml, and good proliferative activity is exhibited at a concentration of about 5 to 10 ng / ml.

[0007] In the present invention, the above hHGF and a polysaccharide or a derivative thereof are used in combination. The polysaccharide or a derivative thereof as referred to in the present invention refers to a carbohydrate generated by dehydration condensation of two or more monosaccharide molecules through glycosidic bonds, that is, all glycans or derivatives thereof. Such polysaccharides or derivatives thereof, like glycosaminoglycans,
The disaccharide unit has a repeating structure, and one of the disaccharides is composed of glucosamine or galactosamine (Science of Life, 39 (4), pp. 306-310 (19)
88)) Those in which polysaccharides such as glucans and the like are sulfated are preferably used. Specifically, for example, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate,
Heparin, dextran, dextran sulfate and the like. In particular, sulfate derivatives of polysaccharides such as heparin, dextran sulfate, heparan sulfate, and chondroitin sulfate can be suitably used.

The polysaccharide or its derivative is preferably used in an excess amount relative to hHGF, and usually used in the range of 10 ² to 10 ⁵ mol of the polysaccharide or its derivative per 1 mol of hHGF. The hepatocyte proliferating agent of the present invention is mainly used as an injection. An 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 following composition is dissolved in a 10 mM phosphate buffer (PBS (-)) having a pH of 7.5, the total volume is adjusted to 5 ml, and then sterilized with a 0.22 μm filter. It can be prepared by dispensing in a bottle, or by further freeze-drying and preserving, and dissolving it in distilled water or physiological saline as needed before 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) 100 μg of hHGF, 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 and then the polysaccharide or a derivative thereof may be used. After using the injectable preparation containing hHGF, the injectable preparation containing hHGF may be used, or these injectable preparations may be used simultaneously. When an injection containing hHGF is used first, an injection containing a polysaccharide or a derivative thereof is preferably used as soon as possible. The amount of the hepatic parenchymal cell proliferating agent of the present invention varies depending on the activity of hHGF, which is an active ingredient, and the object to be used.
１，1,000 μg / kg.

[0011]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto without departing from the scope of the invention. Example 1 High Activation of hHGF Protein by Addition of Heparin Method of Seglen (Methods)
in cellbiology, vol 13, p2
9, Academic Press, New York
(1976)), according to Wistar male rats (body weight 2).
00g), liver parenchymal cells were isolated using 0.05% collagenase (Type I, Sigma). The hepatic parenchymal cells were placed in a collagen-coated multiwell plastic dish having 1.55 cm diameter wells (Nun
c), inoculated at a concentration of 5 × 10 ⁴ cells / 0.2 ml / cm ² , and cultured in a monolayer at 37 ° C. in an air gas 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), Williams E medium (Flow Laboratories, hereinafter abbreviated as "basal medium") supplemented with 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin was used.

Three hours after the start of the culture, the medium was replaced with a new basal medium, and 20 hours later, the medium was replaced with a basal medium not containing 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), followed by recombinant hHGF at a final concentration of 100 ng / ml. After the culturing was continued for 20 hours, DNA synthesis was measured. DNA synthesis was performed using ³ H-thymidine (Amers
ham) at a final concentration of 4 μCi / ml (2 Ci / mm
ol), 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 three times each with cold PBS (-), 2% perchloric acid and 95% ethanol. The cells are then air-dried and
After solubilization with 500 μl of NaOH, a portion was taken for radioactivity measurement. The concentration of the recombinant hHGF was measured by an enzyme immunoassay. The activity value was determined from the count of the amount of ³ H-thymidine incorporated into the DNA of the hepatic parenchyma cells by the test sample as the difference between the counts of the control group (hereinafter, all the hHGF activity values 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.
g / ml of heparin (molecular weight 4000-6000, Sigma) was added, and recombinant hHGF was added to final concentrations of 0, 4.28, 8.75, 17.5, 35, 70, 14
The results are shown for the case of adding at a concentration of 0 ng / ml. The horizontal axis shows the recombinant hHGF when heparin was added.
And the vertical axis indicates hHGF activity. In FIG. 2, the curve (1) shows that the final concentration is 100 μg / ml.
Shows the hHGF activity when heparin was added, and curve (2) shows the hHGF activity when heparin was not added.
Shows GF activity.

[0014] According to 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
methods incellbiology, vol
13 , p29, Academic Press, Ne
hepatocyte separated according w Yorl (1976)), collagen-coated multiwell plastic dishes (Nunc) to 5 × 10 ⁴ cells /0.2Ml/cm ² the hepatocytes having wells with a diameter of 3.5cm
Seeded at a concentration of 5% CO 2 in an air-gas phase containing 37%
The cells were cultured in a monolayer at ℃. (Tanaka et al.,
J. Biochem. 84 , 937-946 (197)
8)). As a culture medium, 5% fetal bovine serum (FBS, FBS
iltron, Altona, Australia),
A Williams E medium (Flow Laboratories, hereinafter abbreviated as “basal medium”) supplemented with 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin was used.

After 3 hours from the start of the culture, the culture is cultivated in a new basic medium.
PB containing 0.25% gelatin after 20 hours
After washing three times with S (-), 0.25% gelati
Dulbecco MEM containing 25 mM Hepes (hereinafter
Abbreviated as "Binding medium". ) For each well
Add 3 ml of air containing 5% carbon dioxide at 37 ° C for 3 hours.
Monolayer culture was performed under the phase. After this, change the binding medium
And add 1 ml for each well,
100 μg / ml of heparin (molecular weight 400
0-6000, Sigma). Control group
As a group, heparin was not added. Hand
Hunter et al. (Nature19
4, 495-496 (1962)).
Lee Na¹²⁵I (3.7 GBq, NEN)
Labeling of the recombinant hHGF was performed. The resulting labeling
hHGF (hereinafter abbreviated as “label hHGF”) in each well
To a final concentration of 200 pM. This
After that, a single layer culture was performed at 37 ° C in an air gas phase containing 5% carbon dioxide.
Nourished. 0 minutes, 10 minutes, 30 minutes, 60 minutes,
The culture supernatant was collected over time at 120 minutes and 180 minutes. Times
The collected culture supernatant is sufficiently cooled under ice-cooling and the final concentration is
Transfer RNA to 100 μg / ml
After addition, add trichloro vinegar to a final concentration of 10%.
Acid (TCA) was added. The test sample prepared in this way
After cooling the sample under ice cooling for 3 hours, centrifugation at 10,000 G at 4 ° C
Perform the operation and collect the supernatant to measure the amount of hHGF degradation.
Using. The supernatant thus obtained contains hepatocytes and
Incubation reduces the molecular weight, that is, decomposes
Labeled hHGF.

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

[0017] According to the above results, the addition of heparin to hHGF
It turns out that it is effective in inhibiting protein degradation. Example 3 High Activation of hHGF by Dextran Sulfate and Dextran Regarding dextran sulfate having a similar molecular structure as compared with heparin and dextran having no sulfate group in the molecule, the hepatic parenchymal cell proliferating ability of hHGF was determined. The effects were examined.

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 culturing for 20 hours, 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)
(Sigma, Sigma) and a basal medium without fetal calf serum. After further culturing for another 20 hours, DN
A synthesis was measured. DNA synthesis, recombinant hHGF
Was measured in the same manner as in Example 1.

FIG. 4 shows the result. In FIG. 4, the horizontal axis indicates the final concentration of added dextran sulfate, dextran or heparin, and the vertical axis indicates hHGF activity. As shown in FIG. 4, the hepatic parenchymal cell proliferation activity of hHGF can be enhanced by adding dextran sulfate or dextran. In particular, dextran sulfate was found to significantly enhance the activity of hHGF, like heparin.

Example 4 High Activation of hHGF by Various Polysaccharides and Derivatives As test samples, heparin, dextran sulfate and dextran used in the above-mentioned examples, heparan sulfate,
Hyaluronic acid, chondroitin, chondroitin sulfate A
The effect of hHGF on the hepatic parenchymal cell proliferation ability was examined for E.

First, hepatic parenchymal cells were isolated according to Example 1.
Was wound at a concentration of 5 × 10 ⁴ /0.2 ml / cm ² into a collagen-coated multi-well plastic dish having the above-mentioned wells and cultured in a monolayer at 37 ° C. in an air gas phase containing 5% carbon dioxide gas. As a culture medium, 5% fetal bovine serum (FB
S) A Williams E medium (hereinafter abbreviated as "basal medium") supplemented with 1 μM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin was used. Three hours after the start of the culture, the medium was replaced with a basal medium. After culturing for 20 hours, the medium was replaced with a basal medium containing no fetal calf serum.

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

In FIG. 5, the vertical axis represents the various polysaccharides used,
Glycosaminoglycans and derivatives thereof are shown.
The horizontal axis indicates HGF activity. In particular, it was found that heparin, dextran sulfate, heparan sulfate, and chondroitin sulfate significantly induced the enhancement of HGF activity.

Example 5 Preparation of Polysaccharide or Its Derivative-Binding Type hHGF Protein and Enhancement of Its Hepatocyte Proliferation Activity Capability hHGF has a strong affinity for polysaccharides such as heparin and dextran sulfate or derivatives thereof. So hHG
After removing the polysaccharide or its derivative from F and 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, it is known that heparin, dextran sulfate and the like exhibit an anticoagulant effect of blood in a living body. However, by using this method, it is expected that the effect of a simple polysaccharide or a derivative thereof, eg, heparin or dextran sulfate, which did not bind to hHGF on the living body can be extremely suppressed.
Further, as in the case shown in Example 1, hHGF bound with such a molecule was found to induce hepatic parenchymal cell proliferation ability extremely strongly as compared with the case where only hHGF was added.

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

Next, the heparin-binding hHGF thus obtained was measured for its hepatocyte proliferation activity. Liver parenchymal cells were isolated and cultured in the same manner as in Example 1. Three hours after the start of the culture, the medium was replaced with a basal medium, and after culturing for 20 hours, the medium was replaced with a basal medium (Flow Laboratories) containing no fetal calf serum. After this medium exchange, the final concentration of the test sample is 0, 0.5, 2, 10, 50, 100, 300
Heparin-bound hHGF at μg / ml or hHGF not bound to heparin was added. After culturing for 24 hours or 48 hours, DNA synthesis was measured. DNA synthesis was performed using ³ H-thymidine (Ame
rsham) at a final concentration of 4 μCi / ml (2 Ci / m
mol), the culture was continued at 37 ° C. for 5 hours, and the incorporation of ³ H-thymidine into DNA was measured. HHG as control group
The group to which F was not added was used. After labeling by the above culture,
The cells were washed three 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 portion thereof was taken for radioactivity measurement. The concentrations of the recombinant hHGF and heparin-bound hHGF used here were measured by the enzyme immunoassay in the same manner as in Example 1. The activity value was determined as the difference between the count of the control group and the count of the amount of ³ H-thymidine incorporated into the DNA of the liver parenchyma cells by the test sample.

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

Example 6 Preparation of hHGF proteins conjugated with various polysaccharides and derivatives thereof and its inhibitory activity on hHGF protein degradation In the same manner as in Example 2, the recombinant hHGF with Na ¹²⁵ I was prepared according to the method of Hunter et al. Labeling was performed. A final concentration of 50 μl was obtained with respect to 200 μl of a PBS (−) solution containing 3 μg of the obtained labeled recombinant hHGF protein.
mg / ml of heparin (Sigma molecular weight, 400
0-6000) or dextran sulfate (Sigma,
200 μl of a PBS (−) solution containing 8000) was added. After standing at 4 ° C all day and night, Sephad
Gel filtration using an ex G-50 column (Pharmacia) was performed to remove heparin or dextran sulfate that did not bind to hHGF.
That is, PB containing Tween 80 with a final concentration of 0.01%
Sephadex G-50 column (1.5 ×) equilibrated with S (−) solution (hereinafter abbreviated as “elution buffer”)
After adding a mixture of heparin or dextran sulfate and hHGF to 12 cm), the elution buffer was added to the same column. Labeled hHGF protein bound to heparin or dextran sulfate molecule (hereinafter referred to as heparin-bound
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 a method of preparing the liver parenchymal cells using a collagen-coated multi-well plastic dish having a 3.5 cm diameter well (Nun)
C) was rolled in at a concentration of 5 × 10 ⁴ cells / 0.2 ml / cm ² , and cultured in a monolayer at 37 ° C. in an air gas phase containing 5% carbon dioxide gas. 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 “basal medium”) was used. After 3 hours from the start of the culture, the medium was replaced with a basal medium.
After washing three times with PBS (-) containing 25% gelatin, the above-mentioned binding medium was added to each well for 2 times.
The mixture was added, and monolayer culture was performed at 37 ° C. for 3 hours in an air gas phase containing 5% carbon dioxide. Thereafter, the binding medium was exchanged, and 1 ml of the binding medium was added to each well.

The prepared hepatocytes were tested with heparin-bound ¹²⁵ I-hHGF or dextran sulfate-bound ¹²⁵ I-hHGF at a final concentration of 200 pM as a test sample.
Was added so that ¹²⁵ I for the control group
-Only hHGF was added. Thereafter, monolayer culture was performed at 37 ° C. in an air gas phase containing 5% carbon dioxide. After the start of culture, 0
Minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 3
The culture supernatant was collected over a period of 60 minutes. The collected culture supernatant was sufficiently cooled under ice cooling, and then the final concentration was 100 μg /
After adding the transfer RNA to a volume of 0.1 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 10,000 G at 4 ° C. to collect a supernatant and a precipitated fraction. The supernatant thus obtained (hereinafter referred to as TCA-solubilized fraction) contains the labeled hHGF which has been reduced in molecular weight, ie, degraded by incubation with hepatocytes. On the other hand, the precipitate fraction (hereinafter TC
A precipitate fraction) contains undegraded hHGF. Radioactivity contained in the obtained TCA solubilized fraction and TCA precipitated fraction was measured, and the ratio was calculated.

FIG. 8 shows the result. FIG. 8A shows ¹²⁵
FIG. 8B shows the case where only I-hHGF was added, and FIG. 8C shows the case where heparin-bound ¹²⁵ I-hHGF was added.
Shows the case where dextran sulfate-bound ¹²⁵ I-hHGF was added. The horizontal axis indicates the time from the addition of the test sample to the liver parenchymal cells to the collection of the culture supernatant, and the vertical axis indicates 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 hHGF molecule was extremely suppressed by using GF. Example 7 Promotion of Rat Liver Regeneration by Heparin-Adding hHGF Rats (body weight: about 200 g) were subjected to surgery to remove 2/3 of the liver under anesthesia, filled with 200 μl of a test sample solution, and kept at 37 ° C. overnight in advance. Osmotic pump (A
lzet 2001) into the jugular vein,
The test sample solution was continuously supplied to the venous blood at a rate of 1 μl per hour. Five days later, the animals were sacrificed and the liver was weighed.

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

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

[0035]

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

[0036]

[Table 1]

[0037]

In the past, there has been no known industrial use of hHGF with respect to the existence of a factor which highly activates or stabilizes such a protein and how to utilize it. As shown by the present invention, by using a polysaccharide or a derivative thereof in combination with hHGF, the enhancement of hHGF activity and hHGF
The stability of the HGF molecule is improved, and as a result, the effect of hHGF on liver regeneration and the like at the time of liver injury in a living body can be further improved. Furthermore, 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 the drawings]

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

FIG. 2 is a graph showing the relationship between hHGF concentration and hHGF activity for high activation of hHGF upon addition of a fixed amount of heparin.

FIG. 3. Labeled hHG when a fixed amount of heparin is added
It is a figure showing inhibition of decomposition of F.

FIG. 4 is a graph showing the hepatic parenchymal cell proliferation activity of hHGF when various concentrations of dextran sulfate, dextran, and heparin are added to a constant concentration of hHGF.

FIG. 5 is a graph showing the hepatic parenchymal cell proliferation activity of hHGF when various polysaccharides and derivatives thereof are added to a certain concentration of hHGF.

FIG. 6 is a graph showing the relationship between the concentration of heparin-bound hHGF and hHGF not bound to heparin and the hepatic parenchymal cell proliferative ability obtained by culturing for 24 hours after the addition of each test sample.

FIG. 7 is a graph showing the relationship between the concentration of heparin-bound hHGF and hHGF not bound to heparin and the hepatic parenchymal cell proliferation ability, which was obtained by culturing for 48 hours after the addition of each test sample.

FIG. 8 is a diagram showing inhibition of degradation of heparin-bound hHGF and dextran sulfate-bound hHGF.

──────────────────────────────────────────────────の Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/22 A61K 47/36 A61P 1/16 CA (STN) BIOSIS / WPI (DIALOG) (STN)

Claims (10)

(57) [Claims] 1. A hepatocyte proliferation agent comprising glycosaminoglycan or dextran sulfate and HGF (hepatocyte growth factor). 2. The hepatocyte proliferating agent according to claim 1, wherein the glycosaminoglycan is heparin, heparan sulfate or chondroitin sulfate. 3. The hepatocyte proliferating agent according to claim 1, wherein glycosaminoglycan or dextran sulfate is bound to HGF (hepatocyte growth factor). 4. The hepatocyte proliferating agent according to claim 1, which is an injection. 5. A method for increasing the activity of HGF (hepatic parenchymal cell growth factor) by adding glycosaminoglycan or dextran sulfate to a preparation. 6. The method according to claim 5, wherein the glycosaminoglycan is heparin, heparan sulfate or chondroitin sulfate. 7. A method for stabilizing HGF (hepatocyte growth factor) using glycosaminoglycan or dextran sulfate. 8. The method according to claim 7, wherein the glycosaminoglycan is heparin, heparan sulfate or chondroitin sulfate. 9. HGF (hepatocyte growth factor) and HG
A preparation comprising glycosaminoglycan or dextran sulfate for enhancing the activity of F (hepatocyte growth factor). 10. The preparation according to claim 9, wherein the glycosaminoglycan is heparin, heparan sulfate or chondroitin sulfate.