JPH07138183A - Therapeutic agent for disease in stomach and duodenum - Google Patents

Abstract

PURPOSE:To obtain a medicine, containing a hepatocyte growth factor (HGF) as an active ingredient and useful for preventing and treating chronic and acute diseases in the stomach and duodenum. CONSTITUTION:This therapeutic agent contains an HGF as an active ingredient. The HGF is capable of promoting the growth of an epithelial cell in the stomach and duodenum and regenerating the damaged stomach and duodenum. The HGF is prepared by extraction thereof from viscera such as the liver, spleen, the lungs, the bone marrow, a brain, the kidney or the placenta of a mammal, e.g. a rat, cattle or a horse, a hemocyte such as blood platelet or a leukocyte, blood plasma, blood serum, etc., and purification of the resultant extract. The HGF can be prepared into the form such as an oral agent or a parenteral injection and the daily dose thereof is 0.01-100mg expressed in terms of the HGF administered in one to several divided portions. The obtained therapeutic agent is useful for preventing and treating gastritis, gastric ulcer, duodenal ulcer, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a therapeutic agent for gastric and duodenal diseases. More specifically, HGF (Hepatocyte Growth Fact
or a hepatocyte growth factor) as an active ingredient.

[0002]

2. Description of the Related Art HGF is a protein found by the present inventors as a factor for proliferating mature hepatocytes from regenerated liver rat serum in vitro (Biochem Biophys Re.
S Commun, 122, 1450, 1984). We further
Succeeded in isolating HGF from rat platelets (Pro
c. Natl. Acad. Sci, 83 , 6489,1986, FFBS Letter, 2
2 , 311, 1987), and its amino acid sequence was partially determined. Furthermore, the present inventors have succeeded in cloning human and rat-derived HGF cDNA based on the elucidated HGF amino acid sequence, and recombining this cDNA into animal tissue to obtain hepatocyte growth factor as a protein ( Human HGF: Nature, 342 , 440, 1989; Rat HGF: Pro
c. Natl. Acad. Sci, 87 , 3200, 1990).

The molecular weight of HGF is 82-85 kD by SDS-polyacrylamide gel electrophoresis. Rat HG
The F molecule has a heterodimer structure in which an α chain consisting of 463 amino acid residues and a β chain consisting of 233 amino acid residues are crosslinked by one disulfide bond, and both α and β chains have 2 chains.
There are individual glucosamine-type sugar chain binding sites. Human HG
F also has almost the same physiological activity and consists of an α chain consisting of 463 amino acid residues and a β chain consisting of 234 amino acid residues. There are four kringle structures in the α chain, which are similar to the fibrinolytic enzyme plasmin, and the amino acid sequence of the β chain is about 3 times that of the B chain of plasmin that has serine protease activity.
It has a homology of 7%. Rat HGF and human HGF
The amino acid sequence homology is 91.6% in α chain, β
It has a very high homology of 88.9% in the chains and its activities are completely compatible.

HGF, which was discovered as a factor that specifically proliferates liver parenchymal cells, has been shown to exhibit various activities in vivo according to recent research results by the present inventors and other researchers. However, expectations are growing for its application not only as a research target but also as a therapeutic drug for humans and animals. The present inventors have clarified that HGF acts widely as a growth factor not only on hepatocytes but also on epithelial cells, and achieved several inventions. In Japanese Patent Application No. 2-158841, HGF promotes the proliferation of renal proximal tubule cells, so that the application development as a therapeutic agent for renal diseases is also described.
-419158, HGF is melanocyte,
By promoting the growth of normal epithelial cells such as keratinocytes, it can be used as an epithelial cell promoter to treat wounds and skin ulcers.
The application and development of hair root cells as a proliferative agent were accomplished and the details thereof were disclosed. In particular, HGF is more suitable for practical use because it does not have the canceration action and cancer cell growth activity found in many other growth factors such as EGF. Furthermore, the present inventors have disclosed that in Japanese Patent Application No. 3-140812, HGF human liver cancer-derived HepG2 cell line and lymphoblastic cancer-derived IM9.
It has been disclosed that it can be used as an antitumor agent by utilizing the cancer cell growth inhibitory activity of cell lines and the like.

More important point in considering the practicality of HGF as a medicine is that HGF promotes the growth of only cells that have entered the G1 phase, that is, the growth phase, and acts on the cells in the G0 phase, that is, the quiescent phase. Do not do it. This is
It means that proliferation and regeneration of injured tissue is promoted, but it has no effect on undamaged tissue. Therefore, it is considered that even if HGF is excessively administered or HGF reaches the non-affected part via blood or the like, it does not induce canceration in normal tissues or cause excessive proliferation.

[0006]

As described above, HGF promotes the proliferation of not only hepatocytes but also epithelial cells in a wide range and has an activity of suppressing the proliferation of cancer cells.
It is expected that GF acts in healing tissue damage. H
GF-producing cells are not epithelial cells themselves, but Kup in the liver
It is known to be produced mainly by mesenchymal cells such as ffer cells and sinusoidal endothelial cells, capillary endothelial cells in the kidney, and alveolar macrophages and vascular endothelial cells in the lung. It has been clarified that a so-called paracrine mechanism in which HGF is supplied according to the above is established. However, when the liver or kidney is injured, HGF production is increased even in an uninjured organ, such as the lung, and therefore it is considered that HGF is also supplied by the so-called endocrine mechanism.

[0007] As described above, HGF is a growth factor that acts on wound healing in various organs / tissues. Whether HGF contributes to the repair of the stomach and duodenum when the stomach and duodenum are damaged. Not revealed. Since most of the target cells for HGF are epithelial cell lines, the present inventors have found that HGF contributes to the regeneration of the stomach and duodenum when the stomach and duodenum are damaged, and is effective for the prevention and treatment of diseases. Thinking about deafness, the effect of HGF on the stomach and duodenum was examined. As a result, it was found that HGF markedly promotes the proliferation of gastric mucosal cells. The present invention has been made based on such findings, and an object of the present invention is to provide a therapeutic agent for gastroduodenal disease useful for the prevention / treatment of gastric / duodenal injury.

[0008]

[Means for Solving the Problems] The gastric / duodenal disease therapeutic agent of the present invention made to solve the above problems is HG
Containing F as an active ingredient. The HGF having the above-mentioned constitution, which is the active ingredient of the present invention, can be prepared by various methods as long as it is purified to the extent that it can be used as a medicine. Various methods are known as methods for preparing HGF, and examples thereof include liver, spleen, lung, bone marrow, brain, kidney, placenta and other organs of mammals such as rats, cows, horses and sheep, platelets, leukocytes. It can be obtained by extraction and purification from blood cells such as blood plasma, plasma and serum. In addition, HGF can also be obtained by culturing primary culture cells or cell lines that produce HGF and separating and purifying from culture (culture supernatant, cultured cells, etc.). Alternatively, a gene encoding HGF can be incorporated into an appropriate vector by a genetic engineering method, and this can be inserted into an appropriate host for transformation, and the desired recombinant HGF can be obtained from the culture of this transformant. (For example, Nature, 34
2 , 440, 1989, JP-A-5-111383, Bioche
m. Biophys. Res. Commun., 163 , 967, 1989, etc.). The above-mentioned host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.

More specifically, as a method for extracting and purifying HGF from living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a rat in a hepatitis state is excised and crushed, and S- It can be purified by an ordinary protein purification method such as gel column chromatography using sepharose or heparin sepharose, and HPLC. In addition, an animal cell such as a Chinese hamster ovary (CHO) cell, a mouse C127 cell, etc., which is obtained by incorporating a gene encoding the amino acid sequence of human HGF into a vector such as bovine papilloma virus DNA using a gene recombination method. Transforming monkey COS cells,
It can be obtained from the culture supernatant.

The HGF thus obtained has a part of its amino acid sequence deleted or substituted with another amino acid, a part of another amino acid sequence inserted, or has 1 amino acid at the N-terminal and / or C-terminal. Alternatively, two or more amino acids may be linked, or the sugar chain may be similarly deleted or substituted.

The HGF has an action of promoting the proliferation of gastric mucosal cells, as shown in Examples described later, and in the living body suffering from gastric / duodenal injury, it is found in the stomach and duodenum (and liver and lungs). It became clear that HGF production is promoted. More specifically, the effect of HGF on cell growth was examined in primary culture of rabbit gastric mucosal cells (epithelial cells).
It was confirmed that A synthesis and proliferation showed a clear dose-dependent response. Since HGF promotes cell growth at physiological doses, it was revealed that HGF serves as a means for regulating the proliferation of gastric epithelial cells. The above-mentioned cell proliferation is EG
It was additive or synergistic to F and insulin and blocked by genistein, a tyrosine kinase-specific inhibitor. Therefore, it is considered that the pathway of tyrosine kinases, which mediates the actions of various growth factors, is involved in the HGF-induced gastric epithelial cell growth-promoting response.

In addition, the experimental result shows that intracellular free Ca ²⁺ is H
Ca ²⁺ did not appear to play an important role in the action of HGF on rabbit gastric epithelial cells, indicating that it was not increased by GF.

[0013] Furthermore, although there is a slight problem in distinguishability from myofibroblasts, the culture conditioned medium of fibroblast-like cells derived from the mesenchyme of the stomach is similar to HGF. It has been shown that a factor having a growth promoting effect is present, and the growth promoting effect of the culture supernatant of fibroblast-like cells is
It was synergistic with GF or insulin and was blocked by genistein. In other organs such as the liver, kidneys and lungs, fibroblasts secrete HGF, which promote the proliferation of epithelial cells and contribute to the healing process from injury. The above findings and findings and significant gastric tissue defects are first replaced by granulated tissue, followed by re-epithelialization (J. Clin.
Gastroenterol. 13, Suppl 2, S21-34, 1991), gastric mucosal repair and wound healing are regulated by the interaction between gastric epithelial cells and underlying connective tissue contributed by HGF. It is inferred that it will be done.

As described above, since HGF has an action of promoting the proliferation of gastric mucosal cells, the therapeutic agent of the present invention is effective for treating chronic or acute gastroduodenal diseases (for example, gastritis, gastric ulcer,
It can be used for the prevention and treatment of duodenal ulcers.

The therapeutic agent of the present invention can take various dosage forms (eg, liquid, solid, capsule, etc.), but generally, it is an injection with HGF as the active ingredient alone or with a conventional carrier. Or, it is made into an oral preparation together with a conventional carrier. The injection can be prepared by a conventional method. For example, HGF is dissolved in an appropriate solvent (eg, sterilized water, buffer solution, physiological saline, etc.), sterilized by filtration with a filter, and then sterilized. It can be prepared by filling a conventional container. The HGF content in the injection is usually about 0.0002 to 0.2 (W / V%), preferably 0.001 to 0.
It is adjusted to about 1 (W / V%). In addition, as an oral drug, for example, tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups and the like are formulated, and these formulations are prepared. It can be prepared according to a conventional method. The HGF content in the preparation can be appropriately adjusted depending on the dosage form, disease to be applied and the like.

Upon formulation, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Furthermore, the formulation of the present invention may contain additives necessary for formulation, for example, excipients, solubilizers, antioxidants, soothing agents, isotonic agents and the like. If it is a liquid formulation, freeze preservation,
Alternatively, it is desirable to remove water by freeze-drying or the like before storage. The lyophilized preparation is used by re-dissolving it by adding distilled water for injection or the like at the time of use.

The formulation of the present invention can be administered by an appropriate administration route depending on the form of the formulation. For example, it can be administered in the form of an injection such as vein, artery, subcutaneous or intramuscular. The dose is appropriately adjusted depending on the patient's symptoms, age, body weight, etc., but is usually 0.01 mg to 100 m as HGF.
g, and it is appropriate to administer this once or several times a day.

[0018]

INDUSTRIAL APPLICABILITY In the therapeutic agent of the present invention, HGF, which is an active ingredient, promotes the proliferation of gastric and duodenal cells and can regenerate and repair the damaged mucous membrane of the stomach and duodenum. Therefore, the therapeutic agent of the present invention is useful for the prevention and treatment of chronic and acute gastro-duodenal diseases (for example, gastritis, gastric ulcer, duodenal ulcer, etc.).

[0019]

The present invention will be described in more detail based on the following examples and formulation examples, but the present invention is not limited to these examples. The reagents used in Examples 1 to 3 below are as follows. Human EGF (Wakunaga), human insulin (Shionogi), and genistein (genistein, Sigma)
Used the purchased goods. HGF is the method of Nakamura et al. (Nature 342, 440-443,
1989), human recombinant HGF purified from the culture supernatant of CHO cells transfected with an expression vector containing human HGF full-length cDNA was used. The reagents for gastric epithelial cell isolation and culture were as follows. : Modified Coon's modified Ham's F-12 medium (KC biological), BME (Basal medium Eagle), MEM (minimal es)
essential medium, manufactured by Sigma), amino acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(N-2-hydroxyethylpiperadine-N-2-ethanesulonic aci
d, HEPES) buffer (Sigma), bovine serum albumin (B
SA) (Fraction V, Sigma), Hank's solution (HBS
S, manufactured by Gibco), crude type I collagenase (Sigma
), Ethylenediaminetetraacetic acid (EDTA, Sigma). Tritium-labeled thymidine ([ ³ H] -thymidine, New England Nuclear, Inc.), crystal violet (Crystal violet, Sigma) and Indo-1AM (fluorescent Ca ²⁺ probe, Wako) are purchased products. did.

Example 1 Separation of gastric basal mucosal cells and cell culture method As gastric basal mucosal cells (hereinafter referred to as gastric mucosal cells), adult rabbits (body weight 2.5-3.0 kg of Japanese white rabbits were used regardless of sex). The method of Ota et al. (Gastroenterologia Ja
Ponica 25, 1-7, 1990). In general, the basal mucosa was rapidly separated from the rabbit stomach, the deposits were roughly scraped off and then cut into 2-3 mm ² pieces. The cut tissue was crude type I collagenase (0.35).
(mg / ml) in BME.
This was followed by an incubation in BME containing 1 mM EDTA, followed by an incubation in BME solution containing crude type I collagenase as described above, this incubation containing 5% CO ₂ and 9% CO _2.
It was carried out twice under the conditions of 37 ° C. and pH 7.4 in an environment of 5% O ₂ . Cells removed from the last incubation were washed with HBSS and 3% with 5% CO _2.
It was cultured in a humid environment at 7 ° C. The culture medium was F-12 medium, and 10% non-mobilized (56 ° C, 30 minutes) fetal bovine serum (FBS, manufactured by Gibco), 15 mM HE.
PES buffer, 100 units / ml penicillin, 10
0 unit / ml streptomycin and 5 μg / ml Fung
izone was added.

The cultured cells were prepared by the method of Terano et al. (Gastroe
According to Nterology 83, 1280-1291, 1982), it was examined 48 hours later by periodic acid Schiff (PAS) staining. The cultured cells were subsaturated at 48 hours after inoculation (Sub-c
onfluent) formed a single layer. 90 of these single layers
% Or more have PAS-positive substances in the cytoplasm,
It was shown that the cultured cells consisted mainly of mucosal cells. These cells died after one month, and fibroblast-like cells (distinguished from the cell morphology) dominated.

In addition, gastric basal fibroblast-like cells (hereinafter,
The term "fibroblast-like cells" was obtained when gastric mucosal cells were killed and dominated by fibroblasts when cultured for 1 month or longer as described above. 10% FBS during this period
The F-12 medium added with was changed every week. Fibroblast culture supernatant was obtained from the culture system after the predominant fibroblasts.

Example 2 Cell growth effect of various growth factors The growth effect of various growth factors on gastric mucosal cells was
It was measured by synthesis promotion and the crystal violet method. The details are as follows. A. Cell proliferation method and proliferation measurement method The effects of various proliferation (growth) factors on DNA synthesis were measured by the [ ³ H] -thymidine incorporation method. The separated gastric mucosal cells were inoculated on a 24-well culture plate (manufactured by Primaria) at a density of 1.4 × 10 ⁵ cells / cm ² , and 2 in F-12 medium supplemented with 10% FBS.
Cultured for 4 hours. Next, after culturing for 24 hours using serum-free F-12 medium, the medium was supplemented with a test substance (EGF, insulin, HGF, fibroblast culture supernatant or genistein) and 0.1% BSA without addition. Serum F-12
The medium was changed. At the same time, [ ³ H] -thymidine (final concentration:
1.0 mCi / ml) was added. Twenty-four hours after adding the test substance, the cells were washed and 5% trichloroacetic acid was added. The cells were left for 1 hour at 4 ° C, then 1N N
Solubilized in aOH and neutralized with HCl. The solution is a Readycap (Beckman ins
(manufactured by trument) and air dried overnight. Radioactivity was then counted using a liquid scintillation counter.

For the measurement of proliferation by the crystal violet method, gastric mucosal cells were seeded in 96-well culture plates (Primaria) and grown as described above. Twenty-four hours after the addition of the test substance, the cultured cells contained 25% of 0.1% crystal violet.
It was fixed and stained by immersion in 10% methanol for 10 minutes. The stained cell layer was then washed and dried. The absorbance at 600 nm was measured by a multi-well spectrophotometer (Immunoreader 2000, InterM).
ed).

B. Test Results The results of the above tests were as follows. Proliferative effect of HGF on gastric mucosal cells The amount of DNA synthesis (based on [ ³ H] -thymidine uptake, based on [ ³ H] -thymidine uptake, hereinafter the same) of cells cultured for 24 hours using HGF as a test substance was measured by the crystal violet staining method. The growth response is shown in Figure 2 [mean + standard error,
*: Comparison result with control p <0.0
1]. The test results are shown as a ratio (%) to the control (hereinafter the same). As shown in FIG. 1, HGF
Significantly promotes DNA synthesis in a dose-dependent manner, and FIG.
This proliferation was confirmed by the crystal violet staining method as shown in FIG.

Proliferative effect of HGF, EGF and insulin on gastric mucosal cells HGF only, EGF only and HGF + E as test substances
The amount of DNA synthesized in cells cultured for 24 hours using GF is shown in FIG. 3, and the amount of DNA synthesized in cells cultured for 24 hours using HGF alone, insulin (INS) alone and HGF + INS as test substances is shown in FIG. [Average value + standard error, *: Comparison result with control p <0.01, **: H
Comparison results with GF only and EGF only (or INS only) p <0.01]. As shown in FIGS. 3 and 4, EG
F and INS also promoted DNA synthesis and acted additively or synergistically with HGF.

Proliferative effect of culture supernatant of fibroblast-like cells on gastric mucosal cells DN of cells cultured for 24 hours using culture supernatant of cultured fibroblast-like cells as a test substance
A synthetic amount is shown in FIG. 5 (mean value + standard error, *: comparison result with control p <0.01). Moreover, the amount of DNA synthesis of cells cultured for 24 hours using the culture supernatant that coexisted with the cultured fibroblast-like cells for 10 minutes or 120 minutes is shown in FIG. 6 (average value + standard error, *: Comparison result with control p <0.01). As shown in FIGS. 5 and 6, the culture supernatant of fibroblast-like cells promoted DNA synthesis in a dose-dependent and coexistence time-dependent manner. A separate test showed that the culture supernatant of fibroblast-like cells act additively or synergistically with EGF and insulin.

Inhibitory Effect of Genistein on Cell Proliferation As mentioned above, is DNA synthesis, which was found to be promoted by HGF and fibroblast-like cell culture supernatant, inhibited by genistein which is a tyrosine kinase-specific inhibitor? I examined whether or not. FIG. 7 shows the result of suppression of HGF-stimulated DNA synthesis by genistein. Judgment was based on [ ³ H] -thymidine incorporation (average value +
Standard error, *: Comparison result with HGF + DMSO p <0.
01). Further, FIG. 8 shows the results of inhibition of DNA synthesis promoted by the culture supernatant of fibroblast-like cells by genistein. The determination was based on the amount of [ ³ H] -thymidine incorporation (mean value + standard error, *: comparison result with culture supernatant of fibroblast-like cells + DMSO, p <0.01). In addition,
In the figure, DMSO is dimethyl sulfoxide, GS is genistein, and FIB is the culture supernatant of fibroblast-like cells.
As shown in FIG. 7, it was revealed that the promotion of DNA synthesis by HGF was suppressed in a dose-dependent manner by genistein, which is a tyrosine kinase-specific inhibitor.
Further, as shown in FIG. 8, the promotion of DNA synthesis by the culture supernatant of fibroblast-like cells was also suppressed by genistein.

[0029] The cell proliferation by culture supernatant of Example 3 intracellular free Ca ²⁺ measurement HGF and fibroblast-like cells was determined whether the free intracellular Ca ²⁺ increases. The measuring method is as follows. Gastric mucosal cells were cultured on coverslips using F-12 medium. Cultured cells contained 5 mM indo-1AM (fluorescent Ca ²⁺ probe)
Was incubated for 15 minutes at 37 ° C. The coverslips were washed in indo-1AM free solution for 30 minutes and then transferred to the flow-through cell chamber. On the above cover glass, 137 mM NaCl, 3.7 mM KCl, 0.5 mM MgCl ₂ , 1.8 mM C
A HEPES buffer containing aCl ₂ , 5.6 mmol glucose and 4.0 mmol HEPES was run continuously.
The intracellular free calcium concentration was determined by the method of Peeters et al. (Am. J.
Physiol. 253, H1400-1408, 1987).
The fluorescence was collected by an objective lens and split by a beam splitter so that measurements at both 410 nm and 480 nm wavelengths could be made simultaneously using two separate photomultiplier tubes. [Ca ²⁺ ] i is 410 in both fluorescence emission.
/ 480 fluorescence ratio to [Ca ²⁺ ] i = 250 nm (R-Rm
in) / (Rmax-R) b. Rmax and Rmin correspond to the values when Ca ²⁺ is bound to indo-1AM and when Ca ²⁺ is not present, respectively. Rmi
To find n, load the cultured gastric cells with indo-1AM,
After washing, 140 mmol KCl, 4 mmol HEPES (pH 7.05) and 50 mmol with "0" Ca ²⁺ (0.1 mmol EGTA added) for 2 minutes at 37 ° C.
Soaked in digitonin. Rmax is [Ca ²⁺ ]
It was determined by adding sufficient Ca ²⁺ to raise o to 0.1 mmol (“High Ca ²⁺ ”). The result is shown in FIG. As shown in FIG. 9, HGF and EGF did not increase intracellular free Ca ²⁺ .

Preparation Example 1 A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to give a lyophilized formulation. Got

Formulation Example 2 0.02M phosphate buffer (containing 0.15M NaCl and 0.01% polysorbate 80, pH 7.4) 10
HGF 1mg and human serum albumin 100m in 0ml
An aqueous solution containing g was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to obtain a lyophilized preparation.

Formulation Example 3 HGF 1 mg, sorbitol 2 g, glycine 2 g and polysorbate 80 10 m in 100 ml of distilled water for injection.
A solution containing g was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to obtain a lyophilized preparation.

[Brief description of drawings]

FIG. 1 is a diagram showing the amount of DNA synthesis of cells cultured for 24 hours using HGF as a test substance (average value + standard error, *: comparison result with control p <0.01).

FIG. 2 is a view showing a growth reaction (crystal violet staining method) of cells cultured for 24 hours using HGF as a test substance (mean value + standard error, *: comparison result with control p <0.01). .

FIG. 3: HGF alone, EGF alone or H as a test substance
It is a figure which shows the amount of DNA synthesis of the cell culture | cultivated for 24 hours using GF + EGF (mean value + standard error, *: Comparison result with control p <0.01, **: Comparison result with HGF only or EGF only) p <0.01).

FIG. 4 shows only HGF as a test substance, insulin (IN
(S) alone or a figure showing the amount of DNA synthesis of cells cultured for 24 hours using HGF + INS (mean value + standard error, *: comparison result with control p <0.01, **: HGF)
Comparison result with only INS or only INS p <0.01).

FIG. 5 is a diagram showing the amount of DNA synthesis of cells cultured for 24 hours using culture supernatants of cultured fibroblast-like cells as test substances (average value + standard error,
*: Comparison result with control p <0.01).

[Fig. 6] Fig. 6 shows the use of a culture supernatant in which cultured fibroblast-like cells were allowed to coexist for 10 minutes or 120 minutes as a test substance.
It is a figure which shows the amount of DNA synthesis of the cell culture | cultivated for 4 hours (mean value + standard error, *: comparison result with control p <0.0.
1).

FIG. 7 is a graph showing the results of suppression of DNA synthesis promoted by HGF by genistein (judgment depends on [ ³ H] -thymidine incorporation amount) (mean value + standard error, *:
Results of comparison with HGF + DMSO p <0.01). In addition,
In the figure, DMSO represents dimethyl sulfoxide and GS represents genistein.

FIG. 8 shows the result of suppression of DNA synthesis promoted by the culture supernatant of fibroblast-like cells by genistein (judgment: [ ³
FIG. 7 is a diagram showing (H] -thymidine incorporation amount) (mean value + standard error, *: culture supernatant of fibroblast-like cells + D).
Results of comparison with MSO p <0.01). In the figure, DM
SO is dimethyl sulfoxide, GS is genistein, F
IB indicates the culture supernatant of fibroblast-like cells.

FIG. 9 is a graph showing changes in intracellular free Ca ²⁺ concentration in cells cultured with HGF and EGF. Deoxycholic acid was used as a positive control.

Claims (1)

[Claims] 1. A therapeutic agent for gastroduodenal diseases, which comprises HGF as an active ingredient.