JPH0881378A - Human epidermal keratinized cell activator - Google Patents

Abstract

PURPOSE: To obtain the subject activator capable of shortening the culture time in a culture system of human epidermal keratinized cells, thus leading to increase in the passage number of cultured cells, or useful for treating burns, wounds etc., containing, as active ingredient, an oligosaccharide alginate (salt). CONSTITUTION: This activator contains, as active ingredient, an oligosaccharide alginate or a salt thereof. The active ingredient is obtained by subjecting an alginic acid lyase produced by Alteromonas microbes to alginic acid (salt) to carry out a treatment. It is preferable that the oligosaccharide alginate (salt) be <=1000 in molecular weight and the disaccharide to pentasaccharide constituent thereof account for 80-10wt.% of the whole active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human epidermal keratinocyte activating agent containing an alginic acid oligosaccharide or a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art A culture system for culturing human epidermal keratinocytes in a serum-free medium has been established, and recently, a cell culture kit for culturing human epidermal keratinocytes has been sold [eg,
Made by Morinaga Seigaku Kenkyusho Co., Ltd., Kurashiki Boshoku Co., Ltd., Far East Pharmaceutical Co., Ltd.], skin-related research technology has made great progress. Also, by using a serum-free medium, it is not necessary to consider unknown components contained in serum, which facilitates basic research. Moreover, it has become easier to reduce costs, eliminate lot differences, and maintain quality.

As a result, research and development using serum-free medium has been promoted in various fields, and particularly in the dermatological field, it is about to develop into clinical application for the purpose of treating skin such as wounds and burns. . With such technical progress, in the field of skin treatment, treatment using the skin obtained by culture has attracted attention. That is, in the treatment of patients who cannot regenerate the skin by themselves due to severe burns or the like, the skin fragments of the patients themselves are cultured to make skin having a large surface area, and the skin is transplanted.
Targets of this treatment include, for example, patients with burns covering 70% or more of the body surface, extensive bruising, and traces after burns. In foreign countries, transplantation of cultured epidermis was performed in patients with burns reaching 98% of the body surface, and treatment was successful in some cases.

However, the problem remains that the culture period of the patient's own skin used for treatment is as long as 2 to 3 weeks, or that it is difficult to carry out the number of times the culture is subcultured (passage number) 4 times or more. It remains as a problem.

[0005]

DISCLOSURE OF THE INVENTION The present invention can shorten the culture time required for culturing skin cells, and increase the passage number of the cultured cells to surely obtain a large number of target skin cells. It is an object of the present invention to provide a human epidermal keratinocyte activator that can be used.

[0006]

[Means for Solving the Problems] As a result of intensive studies based on the above-mentioned problems, the present inventors have cultivated human epidermal keratinocytes in a medium in which alginic acid oligosaccharide or a salt thereof is added to a serum-free culture system. By doing so, it is expected that the above problems will be solved and that a culture cell system will be constructed (a skin bank that can store and preserve human skin and make it available at any time). It was found that proliferative activity was obtained, and the present invention was completed.

That is, the present invention is a human epidermal keratinocyte activator containing an alginic acid oligosaccharide or a salt thereof as an active ingredient. Examples of the alginic acid oligosaccharide or a salt thereof include those obtained by treating alginic acid or a salt thereof with a microbial cell belonging to the genus Alteromonas or a treated product thereof. In addition, alginic acid oligosaccharides or salts thereof have a molecular weight
Although it is 1,000 or less, the ratio of disaccharide to pentasaccharide in the alginic acid oligosaccharide or its salt is 80 to 100% by weight.

Further, the present invention is for the addition of human serum epidermal keratinocyte serum-free medium to proliferate human epidermal keratinocytes, or for the treatment of burns, wounds or bruises.
It is the human epidermal keratinocyte activator described above. Hereinafter, the present invention will be described in detail. The alginic acid oligosaccharide or a salt thereof, which is the active ingredient of the present invention, can be obtained by acting on alginic acid or a salt thereof, a degrading enzyme of alginic acid or a salt thereof produced by a microorganism belonging to the genus Alteromonas, that is, alginate lyase. Alginic acid is one of the polysaccharides having D-mannuronic acid and L-guluronic acid as constituent sugars, and its salts include, for example, sodium alginate,
Examples include potassium alginate, or a mixture thereof. Examples of the alginic acid oligosaccharide salt include sodium alginate oligosaccharide, potassium alginate oligosaccharide, and a mixture thereof.

First, the alginate lyase-producing microorganisms used for obtaining the alginate oligosaccharide or the salt thereof as the active ingredient of the present invention will be described. Examples of such microorganisms include microorganisms belonging to the genus Alteromonas, and examples thereof include Alteromonas sp. No. 1786. Alteromonas sp. No. 1786 was isolated from the intestine of horseshoe crab as a result of screening of the seafood intestine and its contents with sodium alginate as the sole carbon source, and its morphological and physiological properties. Is as shown in Table 1 below.

[0010]

[Table 1]

Based on the characteristics of the strains shown in Table 1 above, the method of Shimizu et al. [Marine Microbial Research Method, Academic Publishing Center, 228
239 (1985)], it was found that the above microorganism (this strain) belongs to the genus Alteromonas. This strain has been deposited as FERM P-11685 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

A strain having the properties shown in Table 1 above is cultivated, and alginate lyase is separated and purified from the resulting culture by a conventional purification method. As the medium, for example,
Sodium alginate (10 g), artificial seawater [sodium chloride 300 mM, potassium chloride 10 mM, magnesium sulfate (7
Hydrate) 50 mM, calcium chloride (dihydrate) 10 mM], ammonium iron citrate 10 g / 100 ml demineralized water, dipotassium hydrogen phosphate (trihydrate) 7.5 g / 100 ml demineralized water, ammonium chloride 20 g / A medium composition containing 100 ml demineralized water and 1 M Tris-HCl buffer is mentioned.

The culture is carried out, for example, by pre-culturing the freeze-dried bacterial strain Alteromonas sp. No. 1786 twice (20 ° C., 1 day).
After that, main culture (25 ° C, 1 day) is performed. By culturing the above-mentioned microorganism in this manner, a culture supernatant containing alginate lyase or a culture containing the microorganism can be obtained. Then, alginate lyase can be obtained using the culture as a material by the method used for protein separation and purification. For example, a molecular weight cutoff of 500,000 from the above culture solution
The crude alginate lyase solution is obtained by removing the cells with an ultrafiltration membrane (manufactured by Romicon), and the crude alginate lyase solution is appropriately subjected to salting out, centrifugation, various chromatography, electrophoresis, etc. You may combine and refine. Examples of various chromatographies include hydrophobicity, gel filtration, ion exchange, reverse phase, affinity chromatography and the like. In addition, SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis or the like can be used to confirm the purity of the purified product and its molecular weight.

The enzymatic properties of the alginate lyase thus obtained are as follows. Action: When alginic acid lyase produced by the above microorganisms is reacted with alginic acid or its salt as a substrate, increase in absorbance at 230 nm, which is the specific absorption wavelength derived from the double bond of the reaction product alginic acid oligosaccharide or its salt , And an increase in reducing power due to the resulting oligosaccharide was confirmed.

Optimum pH: The alginate lyase produced by the above microorganism has a high relative activity amount in the range of pH 7.0 to 7.5, and the optimum pH is pH 7.0 at which the relative activity amount is maximum. Optimum temperature and thermal stability: Alginate lyase produced by the above microorganisms increases in the range of 45 to 55 ° C. Enzymatic activity: Using a reaction solution having the following composition, sodium alginate and alginate lyase are reacted at 50 ° C for 10 minutes, and the amount of produced oligosaccharide is measured by the Nelson-Somogy method, whereby the enzymatic activity of alginate lyase can be measured. . This enzyme activity is shown as 1 unit of the amount of enzyme that produces sodium alginate oligosaccharide corresponding to 1 μmole of mannuronic acid. You may use potassium alginate for enzyme activity measurement. (Composition of reaction solution) ・ 0.05M sodium phosphate buffer (pH 7.0) containing 0.5% sodium alginate (manufactured by Wako Pure Chemical Industries) ··········································································· Alternatively, in order to produce a salt thereof, an alginate lyase obtained by culturing a strain having the ability to produce alginate lyase, which is a mutant strain obtained by applying the above-mentioned strain of the present invention to a normal mutation means, can also be used. .

Since the raw material alginic acid or its salt contains a small amount of calcium, it is possible that the alginic acid oligosaccharide or its salt produced using them also contains calcium. Calcium is known as a substance that promotes keratinization (differentiation) of epidermal keratinocytes, and in the presence of calcium, it keratinizes before it proliferates sufficiently, so it is necessary to remove calcium from the culture medium at the growth stage. Therefore, it is preferable to subject the alginic acid oligosaccharide of the present invention or a salt thereof to desalting and calcium removal. For the treatment, it is preferable to use an electrodialysis device (a product of Asahi Kasei Kogyo Co., Ltd., trade name: Microacylizer) and the like.

Next, the alginic acid or its salt as a raw material is reacted with the alginate lyase to obtain an alginic acid oligosaccharide or its salt. In this case, the reaction temperature of alginic acid or a salt thereof with alginate lyase is preferably 45 to 55 ° C., and the pH during the reaction is preferably 7.0 to 7.5.
More preferably, it is carried out under conditions of ℃ and pH 7.0.

The alginic acid oligosaccharides or salts thereof of the present invention include those having a molecular weight of 1,000 or less centered on disaccharides to pentasaccharides, and the ratio of disaccharides to pentasaccharides in the alginic acid oligosaccharides or salts thereof is 80 to 50. 100% by weight. The active ingredient of the alginic acid oligosaccharide or a salt thereof obtained as described above can be used as it is as the human epidermal keratinocyte activating agent of the present invention, and dried by a method such as freeze-drying, spray-drying and hot-air drying. Can be set.

The human epidermal keratinocyte activator of the present invention can be used, for example, for the purpose of culturing and proliferating epidermal cells obtained from a patient. In addition, the human epidermal keratinocyte activating kit of the present invention may be added to the human epidermal keratinocyte activator of the present invention to prepare a culture solution, which can be used to promote cell growth. in this case,
The effective amount of human epidermal keratinocyte activator contained in the medium is 0.00
It is 1 to 100 mg / ml, preferably 0.01 to 0.1 mg / ml.

When the human epidermal keratinocyte activator of the present invention is applied as a medicine, the subject is particularly limited as long as the purpose is to treat or ameliorate various diseases or symptoms by the human epidermal keratinocyte activator action. do not do. For example, treatments for burns, wounds, bruises, etc. can be used for specific purposes. The method of administration is parenteral, and examples of the dosage form include creams, ointments, poultices and the like. In addition, the dose varies depending on whether it is an animal or a human, age, route of administration, and frequency of administration, and can be widely varied. In this case, the effective amount of the human epidermal keratinocyte activator of the present invention administered as a composition with an appropriate diluent and a pharmacologically usable carrier is 0.01
-100 mg / cm ² skin / day, which is administered once to several times a day for 2 days or more.

When the human epidermal keratinocyte activator of the present invention is parenterally administered, additives such as a stabilizer, a buffer, a preservative and an isotonicity agent may be added. In the present invention, a pharmacological test which proves that the alginic acid oligosaccharide or the salt thereof is effective as a human epidermal keratinocyte activating agent will be described below. [Human epidermal keratinocyte proliferation test] Using a normal human epidermal keratinocyte culture kit manufactured by Morinaga Institute of Scientific Research, the effect of the present invention on the proliferation of human epidermal keratinocytes is examined. Therefore, the following tests were performed.

[Test Example 1] Frozen normal human epidermal keratinocytes were thawed, cultured in a serum-free medium for epidermal keratinocytes, and further passaged twice. At the third passage, filter-sterilized potassium alginate oligosaccharide (concentration 1 mg / ml) was added to the above serum-free medium, the number of cells was counted over time, and the state of cell growth was morphologically examined. I observed. In addition, the thing which did not add potassium alginate oligosaccharide was compared as a control.

As a result, as shown in FIG. 1, in the culture medium containing potassium alginate oligosaccharide (“◯”), a remarkable increase in cells was observed as compared with the control culture medium (“●”).
On the other hand, the state of cell proliferation immediately after the addition of potassium alginate oligosaccharide, and 6 hours and 48 hours later, is shown as photographs in FIGS. 2, 3 and 4, respectively. It is also clear from the photographs that the culture medium containing potassium alginate oligosaccharide (lower photograph) has a larger number of cells than the control culture medium (upper photograph). From the above, it was possible to enhance the proliferative activity of human epidermal keratinocytes by adding potassium alginate oligosaccharide.

[Test Example 2] After thawing the frozen normal human epidermal keratinocytes, a medium (0.01, in which serum alginate oligosaccharides sterilized by filtration were added to a serum-free medium for epidermal keratinocytes
The cells were cultured at 0.10 and 1.00 mg / ml), and when the cells reached semi-confluence (state in which adherent cells occupy about 80% of the area of the incubator), passage was performed. The time to reach semi-confluence and the number of passages were examined, and the state of cells was observed morphologically. In addition, the one to which sodium alginate oligosaccharide was not added was compared as a control.

Table 2 shows the semi-confluent achievement time at each passage number.

[0026]

[Table 2]

In the sodium alginate-oligosaccharide-containing culture medium, the time required to become semi-confluent, which is a state suitable for passage, gradually increased after 4 passages, but 10
The cells were able to grow even after passage. On the other hand, in the control culture medium, no growth was observed at the 6th passage and the passage could not be performed. FIG. 5 shows the time-dependent changes in the number of cells at 6 passages. In the culture medium containing sodium alginate oligosaccharide, 0.01 (“●”), 0.10 (“▲”), 1.00
(“■”) At any concentration of mg / ml, it reached semi-confluent in 4 days and could be passaged.
In the control culture medium (“◯”), no growth was observed even after culturing for 7 days, and the cells could not be passaged. Usually 4 passages
After 10 times, the proliferation activity of the cells declined, a normal state could not be expected, and the cells could not be passaged more than 6 times. However, the addition of sodium alginate oligosaccharide increased the proliferation activity of the cells, and they were passaged 10 times. However, it was confirmed that the cells could grow. Fig. 6 shows the morphological appearance of the cells on the 7th day of the culture in the control culture medium at the 6th passage, and the morphology of the cells which reached the semi-confluent state of the culture medium containing the sodium alginate oligosaccharide at the 6th to 10th passages. The state is shown as a photograph in FIGS. As is clear from the photographs, the control culture medium (Fig. 6) stopped growing at the 6th passage, while the culture medium containing sodium alginate oligosaccharide (Figs. 7 to 11) showed normal regardless of the number of passages. It has been found that the growth can be maintained.

From the above, the human epidermal keratinocyte activator of the present invention not only enhances the proliferative activity of cells with a small number of passages and shortens the culturing period, but also the cells whose proliferative activity is attenuated by four or more passages. It was also found to have an effect on the activation of, and increase the number of passages.

[0029]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. [Example 1] Production of alginate oligosaccharide (1) Preparation of alginate lyase (medium composition) ・ Sodium alginate ・ ・ ・ ・ ・ ・ ・ ・ 10 g ・ Artificial Seawater ^{* 1}・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 1000 ml ・ Fe stock ^{* 2}・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 1 ml ・ Pi stock ^{* 3}・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 2 ml ・ NH ₄ stock ^{* 4}・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 5 ml ・ 1M Tris-HCl buffer (pH 7.8) ・ ・・ ・ ・ ・ ・ ・ ・ ・ 50 ml ──────────────────────────────────── * 1: Artificial Seawater: Sodium chloride 300 mM, potassium chloride 10 mM, magnesium sulfate (heptahydrate) 50 mM, calcium chloride (dihydrate) 10 mM * 2: F e stock: Ammonium iron citrate 10g / 100ml Demineralized water * 3: Pi stock: Dipotassium hydrogen phosphate (trihydrate) 7.5g / 100ml Demineralized water * 4: NH ₄ stock: Ammonium chloride 20g / 100ml Demineralized water Above Freeze-dried bacterial cells Alteromonas
The SP No. 1786 strain was precultured twice (20 ° C., 1 day) and then main culture (25 ° C., 1 day). As a result, an alginate lyase culture solution having an enzyme activity of 0.90 unit per ml of the culture solution was produced.

The cells were removed from the above culture broth with an ultrafiltration membrane having a molecular weight cut off of 500,000 (manufactured by Romicon) to obtain a crude alginate lyase solution. (2) Production of potassium alginate oligosaccharides After dissolving potassium alginate (10.0 kg) in 90 L of demineralized water, the crude alginate lyase solution (50,
000 U) was added and reacted at 40 ° C. for 20 hours with stirring. The reaction solution was deproteinized, desalted, and then freeze-dried to obtain 4.2 kg of potassium alginate oligosaccharide powder.

Similarly, if sodium alginate or a mixture of sodium alginate and potassium alginate is used, a sodium alginate oligosaccharide or a mixture of sodium alginate oligosaccharide and potassium alginate oligosaccharide is obtained. (3) Preparation of calcium-free alginate oligosaccharides The potassium alginate oligosaccharides prepared in (2) above (40.0
g) was subjected to an electrodialysis device (trade name: Microacylizer, manufactured by Asahi Kasei Kogyo Co., Ltd.) to remove calcium ions, and purified potassium alginate oligosaccharide (39.5 g) was obtained. The oligosaccharides were compared at the elemental level before and after purification.
Table 3 shows the results. From the result, it was confirmed that calcium could be removed.

[0032]

[Table 3]

Incidentally, calcium-free sodium alginate oligosaccharides can be prepared in the same manner. [Example 2] Preparation of culture solution containing human epidermal keratinocyte activator (1) Medium for normal human epidermal keratinocyte (I) Morinaga Institute of Scientific Research Basic liquid of serum-free basic liquid medium for keratinocytes To 250 ml of medium, 2.0 ml of bovine pituitary gland extract and 2.5 μg of epidermal growth factor are added and mixed to prepare a complete liquid medium. The sodium alginate oligosaccharide prepared in Example 1 (3) was sterilized by filtration and then added to a complete liquid medium to a concentration of 1 mg.
/ Medium was added to give a culture solution containing the human epidermal keratinocyte activator of the present invention.

(2) Normal human epidermal keratinocyte medium (II) 250 ml of basal liquid medium of serum-free basal liquid medium for keratinocytes manufactured by Morinaga Institute of Scientific Research Co., Ltd. 2.0
ml and epidermal growth factor 2.5 μg are added and mixed to prepare a complete liquid medium. The potassium alginate oligosaccharide produced in Example 1 (3) was sterilized by filtration, and then added to a complete liquid medium at a concentration of 1 mg / ml of medium, and a culture solution containing the human epidermal keratinocyte activator of the present invention. Got

Reference Example 1 This Reference Example shows an analysis example of the degree of polymerization of the potassium alginate oligosaccharide of the present invention. The degree of polymerization is determined by thin layer chromatography (TL
C). First, it was prepared by hydrolyzing sodium alginate as a standard substance with 1N tolyloroacetic acid at 100 ° C. for 24 hours. Incidentally, potassium alginate may be used as the standard substance.

TLC was performed under the following conditions. Thin layer plate: Silica gel 60 HPTLC plate (manufactured by Merck) Developing solvent: 1-butanol / acetic acid / water (5/2/3) Color reagent: diphenylamine-aniline-phosphate reagent The above sodium alginate hydrolyzate (standard substance) was used. T
When subjected to LC, 6 spots were obtained. Of the 6 spots, the spot with the highest mobility is D
-Mannuronic acid (D-mannuronic acid lactone manufactured by Sigma Co., which has been treated with an alkali to remove the lactone ring, R
It was in agreement with f value of 0.38 and DP = 1). Further, the other spots were subjected to gel filtration with Bio-Gel P-2 (1.5 × 46 cm, eluted with 0.1 M acetate buffer (pH 4.0)) to obtain a purified oligosaccharide. About this purified oligosaccharide, the amount of uronic acid (orcinol iron-hydrochloric acid method, Brown; Arch. Biochem., 11 , 269-27
8 (1946)) and reducing sugars (Somogie Nelson method, Somogyi;
J. Biol. Chem., 195 , 19-23 (1952), Nelson; J. Biol.
Chem., 153 , 375-380 (1944)) was measured and the amount of uronic acid /
When the degree of polymerization (DP) was calculated from the amount of reducing sugar, it was 2, 3, 4, 5, and 6, respectively.

Then, these 6 kinds of purified oligosaccharides (D
-Including mannuronic acid), the relationship between the logarithmic value of relative mobility (Rf / 1-Rf) and the degree of polymerization (DP) in thin layer chromatography was examined, and a linear calibration curve was obtained. (Fig. 12). In FIG. 12, “●” indicates sodium alginate hydrolyzate (standard substance), and “◯” indicates alginic acid oligosaccharides (a, b, c, d).

Next, the potassium alginate oligosaccharide prepared by the method of Example 1 (2) was subjected to high performance liquid chromatography (HPLC) by a 0 to 2M gradient elution with NaCl using DEAE GLASS (manufactured by Nakanai Tesque). , Fractionation with a fraction collector (JP-A-4-2)
No. 66896). This elution pattern is shown in FIG. Of the fractions 1 to 8 having absorption at 230 nm (hereinafter referred to as "P-1", "P-2", ... "P-8"), the main fraction P- 2, P-3, P-5,
P-8 was recovered, and each fraction was desalted using a column packed with Bio-Gel P-2 (manufactured by Bio-Rad) (see JP-A-4-169188), lyophilized, and TL.
Subjected to C. As a result, a thin layer chromatogram as shown in FIG. 14 was obtained. In FIG. 14, O indicates the origin. M
Is D-mannuronic acid, S is an enzymatically-degraded oligosaccharide of sodium alginate, 2 is a P-2 fraction, 3 is a P-3 fraction,
5 shows a P-5 fraction and 8 shows a P-8 fraction. Also,
Symbols a, b, c and d indicate spots for which the Rf value was calculated.

As can be seen from the thin layer chromatogram in FIG. 14, one spot was not formed in each sample. Therefore, the Rf values of the main spots were obtained and plotted on the calibration curve shown in FIG. 12 to examine the degree of polymerization. As a result, it was revealed that the degree of polymerization of the fractionated oligosaccharides (P-2, P-3, P-5, P-8) was mainly 2 to 5. That is, the potassium alginate oligosaccharide was a mixture having a degree of polymerization of 2 to 5.

An example of the structure of the potassium alginate oligosaccharide of the present invention is shown in FIG. (a) shows a degree of polymerization of 2, (b) shows a degree of polymerization of 3, (c) shows a degree of polymerization of 4, and (d) shows an oligosaccharide having a degree of polymerization of 5, respectively. The constituent sugars are D-mannuronic acid and L-
Although it is guluronic acid, it may be either a homo type or a hetero type, and R of the “—COOR” group may be H, Na or K.

[0041]

INDUSTRIAL APPLICABILITY The present invention provides a human epidermal keratinocyte activator that further stabilizes the culture system of human epidermal keratinocytes and enables practically four or more passages. .
The human epidermal keratinocyte activator of the present invention can not only increase the proliferative activity of cells with a small number of passages and shorten the culture period, but also have the effect of inactivating cells whose proliferative ability has significantly decreased by repeated passages. Yes, it was possible to grow even after 10 passages.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of a human epidermal keratinocyte proliferation test.

FIG. 2 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 3 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 4 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 5 is a diagram showing the results of a proliferation test on human epidermal keratinocytes that were repeated 6 times in subculture.

FIG. 6 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 7 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 8 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 9 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 10 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 11 is a photograph showing the morphology of organisms in human epidermal keratinocytes.

FIG. 12 is a diagram showing the relationship between the degree of polymerization of sodium alginate oligosaccharides and the logarithmic value of relative mobility.

FIG. 13 is a diagram showing a result of HPLC of potassium alginate oligosaccharide.

FIG. 14 is a diagram showing a thin-layer chromatogram of potassium alginate oligosaccharide.

FIG. 15 is a schematic diagram showing an example of the structure of an alginate oligosaccharide.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07H 3/06 C12N 5/06 9/88 C12P 19/04 C 7432-4B // A61L 27/00 C (C12N 9/88 C12R 1:01) (C12P 19/04 C12R 1:01) (72) Inventor Eiji Yamamoto 16-2 Maruha Co., Ltd. Central Research Institute, Wadai, Tsukuba, Ibaraki

Claims (6)

[Claims] 1. A human epidermal keratinocyte activator containing an alginic acid oligosaccharide or a salt thereof as an active ingredient. 2. The human epidermal keratinocyte activator according to claim 1, wherein the alginic acid oligosaccharide or a salt thereof is obtained by treating alginic acid or a salt thereof with cells of a microorganism belonging to the genus Alteromonas or a treated product thereof. 3. The human epidermal keratinocyte activator according to claim 1, wherein the alginic acid oligosaccharide or a salt thereof has a molecular weight of 1,000 or less. 4. The human epidermal keratinocyte activator according to claim 3, wherein the proportion of disaccharide to pentasaccharide in the alginic acid oligosaccharide or a salt thereof is 80 to 100% by weight. 5. The human epidermal keratinocyte activator according to any one of claims 1 to 4, which is added to a serum-free medium for human epidermal keratinocytes to proliferate human epidermal keratinocytes. 6. For the treatment of burns, wounds or bruises,
The human epidermal keratinocyte activator according to any one of claims 1 to 4.