JP2021164407A - Growth stimulator for mesenchymal stem cell - Google Patents

Abstract

To provide a food composition and an agent having the action of stimulating the growth of a mesenchymal stem cell.SOLUTION: A growth stimulator for a mesenchymal stem cell has, as an active ingredient, a product from treating fish or meat at a temperature of 100-300°C and a pressure of 0.1-8.6 MPa for 1-120 minutes. There is also provided a food composition for stimulating the growth of a mesenchymal stem cell.SELECTED DRAWING: Figure 3

Description

The present invention relates to food compositions and agents having an action of promoting the proliferation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are cells capable of differentiating into cells belonging to the mesenchymal system such as bone cells, myocardial cells, cartilage cells, tendon cells, nerve cells and fat cells. Mesenchymal stem cells are expected to be applied to regenerative medicine such as reconstruction of bone, blood vessels and myocardium.

Since mesenchymal stem cells have an immunomodulatory ability, rejection does not occur even if an organ prepared from mesenchymal stem cells derived from another person is transplanted. In addition, treatment using mesenchymal stem cells has an advantage that the risk of tumorigenesis is lower than that of iPS cells.

Mesenchymal stem cells can be obtained from various tissues such as bone marrow and adipose tissue. It is known that the properties of the obtained mesenchymal stem cells differ depending on the organ to be collected.

There is a need for a technique for proliferating mesenchymal stem cells for use in regenerative medicine. For example, Patent Document 1 states that as a method for proliferating mesenchymal stem cells, MSCs containing undifferentiated mesenchymal stem cells (MSCs) in a medium containing nicotine amide and fibroblast growth factor 4 (FGF4). The medium comprises the step of producing a proliferated undifferentiated MSC population by culturing the population, wherein the medium is additionally added basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF),. And methods for growing undifferentiated MSCs that are free of endothelial growth factor (EGF) have been disclosed.

In addition, Patent Document 2 describes in a method for proliferating mesenchymal stem cells (MSC): (a) a step of isolating cells from a body tissue or organ; and (b) the single obtained in (a). A method is disclosed that comprises the step of obtaining an MSC-enriched cell population by incubating the detached cells in a growth medium containing an apoptosis-inducing agent for at least 3 days.

Japanese Unexamined Patent Publication No. 2018-64572 Special Table 2018-528782 Gazette

There is a need for a technique for efficiently proliferating mesenchymal stem cells for use in regenerative medicine.

Therefore, an object of the present invention is to provide a new drug capable of promoting the proliferation of mesenchymal stem cells.

The present inventors have solubilized the tuna waste portion by subjecting the waste portion from which the edible portion has been removed from the tuna to a high temperature and high pressure treatment at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes. TWL) was obtained. When this TWL was added to bone marrow-derived mesenchymal stem cells (BMSC) and cultured, it was newly found that it has a concentration-dependent effect of promoting the growth of BMSC.

The present invention provides a growth promoter for mesenchymal stem cells, which comprises a treated product of fish or meat treated at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient.

The present invention also provides a growth promoter for mesenchymal stem cells, wherein the fish is tuna.

The present invention also provides a growth promoter for mesenchymal stem cells, wherein the fish is ara.

The present invention also comprises a food product for promoting the proliferation of mesenchymal stem cells, which comprises a processed product obtained by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient. The composition is provided.

The present invention also provides a food composition in which the fish is tuna.

The present invention also provides a food composition in which the fish is ara.

The present invention can contribute to regenerative medicine because it provides a new drug that promotes the proliferation of mesenchymal stem cells.

Image of a device for obtaining high-temperature and high-pressure processed products from the tuna disposal section. The figure which shows the molecular weight distribution of the high-temperature and high-pressure processed product of 250 ° C obtained from the bone of tuna waste. The figure which shows the molecular weight distribution of the high-temperature and high-pressure processed product of 250 ° C obtained from the body of tuna waste. The figure which shows the molecular weight distribution of the high-temperature and high-pressure processed product of 200 ℃ obtained from the bone of tuna waste. The figure which shows the molecular weight distribution of the high-temperature and high-pressure processed material of 200 ℃ obtained from the body of tuna waste. The figure which shows the effect on cell proliferation when the high temperature and high pressure treated product obtained from the bone of tuna waste is added to bone marrow-derived mesenchymal stem cells (BMSC) and cultured. The figure which shows the effect on cell proliferation when peptones are added to bone marrow-derived mesenchymal stem cells (BMSC) and cultured.

The present invention provides a growth promoter for mesenchymal stem cells, which comprises a treated product of fish or meat treated at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient.

Mesenchymal stem cells are cells capable of differentiating into cells belonging to the mesenchymal system. As used herein, the term "proliferation promotion for mesenchymal stem cells" means promoting the proliferation of mesenchymal stem cells in vivo or in vitro to increase the number of mesenchymal stem cells.

The growth promoter of the present invention contains a treated product obtained by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes (that is, high temperature and high pressure treatment) as an active ingredient.

The processed product in the present invention includes a polypeptide produced by hydrolyzing a protein of fish or meat by high-temperature and high-pressure treatment. As used herein, a polypeptide refers to a peptide in which two or more amino acids are linked by peptide bonds, and is a concept including oligopeptides. The polypeptide in the present invention is a peptide produced by hydrolyzing a protein. Even if the molecular weight of the polypeptide contained in the processed product in the present invention is 10 or more, 50 or more, 100 or more, for example, 10 to 10000. It may be good, preferably 50 to 5000, and more preferably 100 to 1000.

In the present specification, when the molecular weight of a polypeptide is defined by a numerical range, it means that at least 10% or more of the polypeptide is contained in the range, preferably 20%, 30%, 40% of the polypeptide. , 50%, 60%, 70%, 80% or more, 90% or more, and more preferably the molecular weights of substantially all polypeptides are included in the range.

The high-temperature and high-pressure treated product used in the present invention hydrolyzes proteins by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes to have a molecular weight of 100 to 10000. Produces a polypeptide. The high-temperature and high-pressure processed product used in the present invention efficiently hydrolyzes proteins contained in fish or meat by subjecting fish or meat to high-temperature and high-pressure treatment at the above-mentioned combination of temperature, pressure and time, and has a molecular weight of 100 to 100. Produced as 10000 polypeptides.

In the present invention, the temperature at which fish or meat is processed is 100 to 300 ° C, preferably 130 to 300 ° C, and more preferably 150 to 250 ° C. The pressure for processing fish or meat is 0.1 to 8.6 MPa, preferably 0.2 to 4.0 MPa. The time for processing fish or meat is 1 to 120 minutes.

The time for processing the fish or meat can be appropriately set according to the temperature at which the fish or meat is processed. For example, the processing time may be set long when the processing temperature is low and short when the processing temperature is high. For example, when the processing temperature is 100 to 180 ° C, the processing time may be set to 60 to 120 minutes, and when the processing temperature is 180 to 220 ° C, the processing time may be set to 20 to 60 minutes. When the processing temperature is 220 to 300 ° C., the processing time may be set to 1 to 20 minutes. By setting the temperature and time in this way, the protein can be appropriately hydrolyzed and the solubilization rate of the protein can be increased. As a result, a polypeptide having a molecular weight of 100 to 10000 can be efficiently produced.

Further, by setting the treatment temperature to 130 to 300 ° C. and the treatment time to 5 to 60 minutes, the molecular weight of the produced polypeptide can be controlled to 100 to 1000.

In the present invention, water may or may not be added when treating fish or meat. According to the present invention, the protein can be sufficiently hydrolyzed by the water contained in fish or meat without adding water.

Further, in the present invention, when treating fish or meat, components other than water may or may not be added. That is, in the present invention, only fish or meat may be subjected to high temperature and high pressure treatment. Ingredients other than water include, for example, acids, bases, reducing agents, dispersants, surfactants, complexing agents and the like.

Fish in the present invention include tuna, bonito, salmon, horse mackerel, yellowtail, red sea bream and the like. In the present invention, the fish is preferably tuna or bonito. The fish in the present invention may be one kind of fish or a mixture of two or more kinds of fish. As the fish, for example, fish ara, waste and flesh can be used. Fish gills include heads, bones, fins, gills and their associated bodies. Fish ara is often a waste product, but in the present invention, a processed product obtained from ara can be used as an active ingredient of a growth promoter. Therefore, in the present invention, the growth promoter containing a treated product obtained by treating fish ara at high temperature and high pressure as an active ingredient is particularly useful.

Meat in the present invention includes beef, pork, chicken, wild boar meat, sheep meat, venison, horse meat, bear meat, eel meat, whale meat and the like. The meat in the present invention may be one kind of meat or a mixture of two or more kinds of meat. Also, the meat may be any part of the animal. For example, as chicken, breast meat, chicken breast, thigh meat, shin meat, chicken wings and the like can be used.

Fish or meat may be boiled and squeezed before high temperature and high pressure treatment. For example, fish or meat may be served as a press cake. Press cakes can be produced, for example, as follows: 1. Boil fish or meat in a kettle with about the same amount of water for 1-2 hours at 90-100 ° C. 2. Next, the solid content derived from fish or meat after simmering is squeezed with a squeezing machine, and the solid content remaining after solid-liquid separation is used as a press cake.

The growth promoter of the present invention can further contain any component. For example, the growth promoters of the present invention are pharmaceutically acceptable bases, carriers, additives, excipients, binders, disintegrants, lubricants, stabilizers, colorants, pH regulators, buffers. Agents, solubilizers and the like can be included.

The growth promoter of the present invention can be in any form such as solid, liquid, powder, granules and paste. In addition, the growth promoter of the present invention may be any preparation such as tablets, lozenges, pills, powders, capsules, granules, suspensions, emulsions, syrups and liquids as orally administered preparations. can. The growth promoter of the present invention may be a parenteral administration preparation, for example, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, enteral administration, enteral administration. It can be any formulation for administration, such as oral administration and transmucosal administration.

The growth promoter of the present invention can promote the growth of mesenchymal stem cells by adding it to the mesenchymal stem cells and culturing the cells. Therefore, it is particularly useful for regenerative medicine using mesenchymal stem cells. In addition, the growth promoter of the present invention can promote the growth of mesenchymal stem cells in vivo by administering to humans and animals other than humans.

The present invention also comprises a food product for promoting the proliferation of mesenchymal stem cells, which comprises a processed product obtained by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient. The composition is provided.

The food composition of the present invention can be provided as a dietary supplement, a supplement, a food with a health function, a food for specified health use, a food with a nutritional function, and the like. The food composition of the present invention may be a food exhibiting a function such as promoting the proliferation of mesenchymal stem cells. In addition, the food composition of the present invention may be in the form of a general processed food, for example, in any form such as beverages, confectionery, soups, dairy products, processed fats and oils and seasonings. There can be.

(Example 1) Molecular weight distribution of solubilized tuna (TWL) in the tuna waste part A solubilized product (TWL) in the tuna waste part was prepared by the following method. Tuna was provided by Saniku Feed Industry Co., Ltd. The waste part (head and bone, etc.) from which the edible part was removed from the tuna was used. In a 50 ml pressure-resistant container, put 15 g of the body of the tuna disposal part (hereinafter referred to as "body") or the bone from which the body of the waste part has been removed as much as possible (hereinafter referred to as "bone"), and at 250 ° C or 200 ° C. High-temperature and high-pressure treatment was carried out at 4.1 MPa for 5 minutes, 15 minutes, 20 minutes, 30 minutes or 60 minutes to obtain a solubilized tuna waste portion (TWL). Figure 1 shows an image of high-temperature and high-pressure processing. The pressure-resistant container containing the tuna disposal part was placed in a molten salt reactor heated by a heater to perform high-temperature and high-pressure treatment. The temperature of the molten salt reactor was measured with a thermocouple. The treatment time was from the time when the pressure-resistant container was put into the molten salt reactor to the time when it was taken out. After a predetermined time, the pressure-resistant container was taken out from the molten salt reactor and cooled by immersing it in a water bath at room temperature to complete the treatment.

The molecular weight distribution of the obtained centrifugal supernatant of the high-temperature and high-pressure treated product was measured. The molecular weight distribution was measured by gel filtration chromatography using HPLC (column: TSK-GEL G2500PWXL 7.8 x 300 mm; eluent: 0.1% TFA: CH ₃ CN = 55: 45; flow velocity: 0.5 ml / min; temperature: 40 ° C; detection: UV at 210 nm).

The results of measuring the molecular weight distribution of these samples (TWL) are shown in FIGS. 2 to 5. In the graphs of FIGS. 2 to 5, the horizontal axis represents the elution time of HPLC, and the vertical axis represents the absorbance (AU).

FIG. 2 shows the molecular weight distribution of the solubilized product obtained by treating the bone of the tuna waste portion at 250 ° C.

FIG. 3 shows the molecular weight distribution of the solubilized tuna waste treated at 250 ° C.

FIG. 4 shows the molecular weight distribution of the solubilized product obtained by treating the bone of the tuna waste portion at 200 ° C.

FIG. 5 shows the molecular weight distribution of the solubilized tuna waste treated at 200 ° C.

As shown in FIGS. 2 to 5, the molecular weight of the main component decreased from 1000 or more to 100 or less as the treatment time became longer. In particular, the samples obtained under the conditions of 250 ° C. for 15 to 30 minutes showed peaks from 1000 or more to 100 on the small molecule side, so it is considered that there are many peptides with 10 or less amino acids.

(Example 2) Amino acid analysis of solubilized tuna waste (TWL) A solubilized tuna waste (TWL) was prepared by the following method. Tuna was provided by Saniku Feed Industry Co., Ltd. The waste part (head and bone, etc.) from which the edible part was removed from the tuna was used. In a 50 ml pressure-resistant container, put the body of the tuna disposal part (hereinafter referred to as "body") or the bone from which the body of the disposal part has been removed as much as possible (hereinafter referred to as "bone"), and put it at 200 ° C for 30 minutes at 1.6 MPa. High temperature and high pressure treatment was carried out at 250 ° C. for 15 minutes or 250 ° C. for 30 minutes at 4.1 MPa to obtain a solubilized tuna waste portion (TWL). The high-temperature and high-pressure treatment was carried out by using the apparatus shown in FIG. 1 and placing a pressure-resistant container containing a tuna disposal part in a molten salt reactor heated by a heater, as in Example 1. The temperature of the molten salt reactor was measured with a thermocouple. The treatment time was from the time when the pressure-resistant container was put into the molten salt reactor to the time when it was taken out. After 15 or 30 minutes, the pressure-resistant container was taken out of the molten salt reactor and cooled by immersing it in a water bath at room temperature to complete the treatment.

Amino acid analysis of the obtained TWL was performed. The processed product treated at high temperature and high pressure was centrifuged, and the supernatant was collected. The amount of free amino acids and the total amount of amino acids were measured in the obtained centrifugal supernatant. Free amino acids were measured by automatic HPLC amino acid analysis. The amount of total amino acids was measured by decomposing at 105 ° C. for 16 hours with 6N hydrochloric acid, removing the hydrochloric acid, and subjecting this to automatic HPLC amino acid analysis.

Table 1 shows the amino acid analysis results of TWL obtained from "bone" or "body" at 250 ° C for 15 or 30 minutes. As shown in Table 1, the amino acids contained in these TWLs are aspartic acid (Asp), glutamate (Glu), proline (Pro), glycine (Gly), alanine (Ala), valine (Val), and methionine (Val). They were Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys), arginine (Arg) and hydroxyproline (Hyp). In addition, the TWL obtained under the condition of 200 ° C. for 30 minutes contained threonine (Thr) and serine (Ser).

From these results, it was found that the solubilized product (TWL) of the tuna waste part contains a large amount of glutamic acid, alanine, glycine, proline, hydroxyproline and leucine. The amino acid composition of the solubilized product (TWL) of the tuna waste part is different from the amino acid composition of the tuna meat described in the food nutrition ingredient table. Therefore, it is suggested that the solubilized product (TWL) of the tuna waste part may contain amino acids and peptides peculiar to this site.

(Example 3) Effect of solubilized tuna (TWL) on stem cells (primary culture of bone marrow-derived mesenchymal stem cells)
Ddy male and female mice aged 5 weeks or older (Tokyo Experimental Animal Co., Ltd., Tokyo, Japan) were used. Two femurs and two tibias of mice were removed, and 2 mm of both epiphysis were cut, and then centrifuged at a rotation speed of 6000 rpm and a centrifugal force of 2000 × g (Fisher Scientific) to collect bone marrow fluid. Minimum Essential Alpha Medium (Life Technologies Japan Co., Ltd., Tokyo, Japan) containing 5% FBS (Sigma-Aldrich Japan LLC Lot: 15K111) and antibiotics (48unit / ml Penicillin, 50μg / ml Streptomycin) Suspended. Next, remove meat pieces using a cell strainer (FALCON, Kanagawa, Japan), sow in a 100 mm dish (Corning Japan Co., Ltd., Tokyo, Japan), and incubate for 3 days under _{37 ° C, 5% CO 2 conditions.} bottom. Then, the floating cells were removed by washing with Dulbecco PBS (-) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and the obtained adherent cells were used as mouse bone marrow-derived mesenchymal stem cells (BMSC). The number of viable cells was measured using a Luna-FL (cell measuring device) (LMS).

(Cell proliferation evaluation (WST-1 method))
As a sample, a solubilized bone of tuna waste obtained by high-temperature and high-pressure treatment at 250 ° C. for 30 minutes (TWL: bone 250 ° C. for 30 minutes) was used. As a control, trypton, which is a small molecule of bovine casein having an amino acid score of 100 and reduced in molecular weight with trypsin, was used.

Bone marrow-derived mesenchymal stem cells (BMSC) adhering to a 100 mm dish were washed with Dulbecco PBS (-), exfoliated using CELL LIFTER, and seeded on a ^{96-well plate at 8.0 × 10 3 cells / well.} One day later, solubilized tuna (TWL) or tryptone (control) was added, and six days later, the cell proliferation test reagent WST-1 (Roche Diagnostics Co., Ltd., Tokyo, Japan) was added. The solution was dissolved in the medium to a final concentration of 10%, and the whole amount of the medium was replaced. Incubation was carried out under 37 ° C. and 5% CO ₂ conditions, and after 1 hour, the absorbance at 440 nm was measured with Spectra Max M2e (Molecular Device Japan Co., Ltd., Tokyo, Japan). The results showed the relative growth rate after 6 days, with the addition of each sample as 1. Table 2 shows the mean and standard errors of growth rates in the four independent wells.

The results are shown in a graph in Fig. 6. Figure 6 shows the 4-well mean ± standard error. ** indicates p <0.01.

In FIG. 6, noramal indicates a control to which nothing was added. FIG. 6 shows the relative cell proliferation rate when TWL and tryptone at each concentration were added, with the control normal to which nothing was added as 1. As shown in FIG. 6, compared with the cells cultured only in the normal medium (Normal group) and the group to which tryptone was added, the group to which TWL was added to the mesenchymal stem cells showed significant cell proliferation in a concentration-dependent manner. Promoted. In particular, when TWL was added at 100 μg / mL, cell proliferation was observed about 1.8 times that of the Normal group.

(Example 3) Effect of peptones on stem cells (primary culture of bone marrow-derived mesenchymal stem cells)
A primary culture of bone marrow-derived mesenchymal stem cells was prepared in the same procedure as in Example 3.

Peptones were purchased commercially. The peptones used in the experiment are as follows. Peptone is a general term for substances in which proteins are enzymatically treated to reduce their molecular weight (oligopeptides).
1. 1. Tryptone (trypsin digest of casein)
2. Lactalbumin hydrolyzate (LAH)
3. 3. Casein Peptone (Cpe) (Pepsin digest of casein)
4. Beef-derived peptone (Mpe)
5. Soybean-derived peptone (Spe)

(Cell proliferation evaluation (WST-1 method))
The above peptones, namely tryptone (Try), lactalbumin hydrolyzate (LAH), casein peptone (Cpe), beef-derived peptone (Mpe) and soybean-derived peptone (Spe) were used as samples. again,

Bone marrow-derived mesenchymal stem cells (BMSC) adhering to a 100 mm dish were washed with Dulbecco PBS (-), exfoliated using CELL LIFTER, and seeded on a 96-well plate at 8.0 × 10 &sup3; cells / well. One day later, peptones were added, and six days later, the cell proliferation test reagent WST-1 (Roche Diagnostics Co., Ltd., Tokyo, Japan) was dissolved in the medium to a final concentration of 100 μg / ml, and the total amount was medium. Exchanged. Incubation was carried out under 37 ° C. and 5% CO &#8322; conditions, and after 1 hour, the absorbance at 440 nm was measured with Spectra Max M2e (Molecular Device Japan Co., Ltd., Tokyo, Japan). The results are shown in Table 3 and FIG.

In Table 3 and FIG. 7, N indicates a control with no addition. The results show mean ± standard error in 4 independent wells. The results showed the relative growth rate after 6 days, with the addition of each sample as 1. As shown in FIG. 7, no cell proliferation activity against bone marrow-derived mesenchymal stem cells was observed in the group to which peptones were added. It was revealed that peptones containing various amino acids and oligopeptides do not affect the proliferation of bone marrow-derived mesenchymal stem cells.

From the results of Examples 2 and 3, it was clarified that the peptides contained in the tuna high-temperature and high-pressure treated products are likely to contain characteristic components different from those of casein, beef, and soybean-derived decomposition products. rice field.

Since the present invention promotes the proliferation of mesenchymal stem cells, it can be suitably used in the field of regenerative medicine using mesenchymal stem cells.

Claims (6)

A food composition for promoting the proliferation of mesenchymal stem cells, which comprises a processed product obtained by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient. The food composition according to claim 1, wherein the fish is tuna. The food composition according to claim 1, wherein the fish is ara. A growth promoter for mesenchymal stem cells containing a treated product obtained by treating fish or meat at a temperature of 100 to 300 ° C. and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes as an active ingredient. The growth promoter for mesenchymal stem cells according to claim 4, wherein the fish is tuna. The growth promoter for mesenchymal stem cells according to claim 4, wherein the fish is ara.

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