JP7637839B2 - Proliferation promoter for mesenchymal stem cells - Google Patents

Description

The present invention relates to a food composition and a drug that have the effect of promoting the proliferation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are cells that have the ability to differentiate into cells belonging to the mesenchymal system, such as bone cells, cardiomyocytes, chondrocytes, tendon cells, nerve cells, and adipocytes. Mesenchymal stem cells are expected to be applied in regenerative medicine, such as for the reconstruction of bone, blood vessels, and cardiomyocytes.

Mesenchymal stem cells have immune-regulating properties, so even if an organ made from mesenchymal stem cells derived from another person is transplanted, there will be no rejection reaction. In addition, treatment using mesenchymal stem cells has the advantage of having a lower risk of tumor formation compared to 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 mesenchymal stem cells obtained vary depending on the organ from which they are harvested.

There is a demand for a technique for proliferating mesenchymal stem cells for use in regenerative medicine. For example, Patent Document 1 discloses a method for proliferating mesenchymal stem cells, which comprises a step of generating a proliferated population of undifferentiated mesenchymal stem cells (MSCs) by culturing a population of MSCs containing undifferentiated MSCs in a medium containing nicotinamide and fibroblast growth factor 4 (FGF4), and the medium does not contain additionally added basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and endothelial growth factor (EGF).

Patent Document 2 also discloses a method for expanding mesenchymal stem cells (MSCs), which comprises: (a) isolating cells from a body tissue or organ; and (b) incubating the isolated cells obtained in (a) in a growth medium containing an apoptosis-inducing agent for at least three days to obtain a cell population enriched in MSCs.

JP 2018-64572 A Patent Publication No. 2018-528782

There is a demand for technology to efficiently proliferate mesenchymal stem cells for use in regenerative medicine.

The object of the present invention is to provide a new drug that can promote the proliferation of mesenchymal stem cells.

The inventors obtained tuna waste solubilized material (TWL) by subjecting waste tuna, which is the edible part removed, to high-temperature and high-pressure treatment at temperatures of 100-300°C and pressures of 0.1-8.6 MPa for 1-120 minutes. When this TWL was added to bone marrow-derived mesenchymal stem cells (BMSCs) and cultured, it was newly discovered that it had the effect of promoting the proliferation of BMSCs in a concentration-dependent manner.

The present invention provides a proliferation promoter for mesenchymal stem cells, which contains as an active ingredient a 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.

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

The present invention also provides a proliferation promoter for mesenchymal stem cells, in which the fish is a round fish.

The present invention also provides a food composition for promoting the proliferation of mesenchymal stem cells, which contains as an active ingredient 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.

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 fish tail.

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

An image of the equipment used to obtain high-temperature, high-pressure treated material from discarded tuna. A graph showing the molecular weight distribution of a product obtained from tuna waste bones after high-temperature, high-pressure treatment at 250°C. A graph showing the molecular weight distribution of a material obtained from tuna waste after high-temperature, high-pressure treatment at 250°C. A graph showing the molecular weight distribution of a material obtained from tuna waste bones after high-temperature, high-pressure treatment at 200°C. A graph showing the molecular weight distribution of a material obtained from tuna waste after high-temperature, high-pressure treatment at 200°C. A diagram showing the effect on cell proliferation when high-temperature, high-pressure treated material obtained from tuna waste bones is added to bone marrow-derived mesenchymal stem cells (BMSCs) and cultured. FIG. 1 shows the effect of peptones on cell proliferation when added to bone marrow-derived mesenchymal stem cells (BMSCs) and cultured.

The present invention provides a proliferation promoter for mesenchymal stem cells, which contains as an active ingredient a 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.

Mesenchymal stem cells are cells that have the ability to differentiate into cells belonging to the mesenchymal system. As used herein, "promoting the proliferation of mesenchymal stem cells" refers to promoting the proliferation of mesenchymal stem cells in vivo or in vitro, and increasing the number of mesenchymal stem cells.

The proliferation promoter of the present invention contains as an active ingredient fish or meat that has been treated at a temperature of 100 to 300°C and a pressure of 0.1 to 8.6 MPa for 1 to 120 minutes (i.e., high-temperature, high-pressure treatment).

The processed product of the present invention includes a polypeptide produced by hydrolyzing fish or meat proteins by high-temperature, high-pressure processing. In this specification, a polypeptide refers to a peptide formed by two or more amino acids linked by peptide bonds, and is a concept that also includes oligopeptides. The polypeptide of the present invention is a peptide produced by hydrolyzing proteins. The molecular weight of the polypeptide contained in the processed product of the present invention may be 10 or more, 50 or more, or 100 or more, for example, 10 to 10,000, preferably 50 to 5,000, and more preferably 100 to 1,000.

In this specification, when the molecular weight of a polypeptide is specified by a numerical range, it means that at least 10% or more of the polypeptide falls within that range, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, 90% or more of the polypeptide, and more preferably substantially all of the molecular weight of the polypeptide falls within that range.

The high-temperature, high-pressure processed product used in the present invention is produced 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, thereby hydrolyzing proteins and producing polypeptides with a molecular weight of 100 to 10,000. The high-temperature, high-pressure processed product used in the present invention is produced by treating fish or meat at high temperature and high pressure using the above-mentioned combination of temperature, pressure and time, thereby efficiently hydrolyzing proteins contained in the fish or meat and producing polypeptides with a molecular weight of 100 to 10,000.

In the present invention, the temperature when processing fish or meat is 100 to 300°C, preferably 130 to 300°C, and more preferably 150 to 250°C. The pressure when processing fish or meat is 0.1 to 8.6 MPa, and 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 longer when the processing temperature is low and shorter when the processing temperature is high. For example, when the processing temperature is 100-180°C, the processing time may be set to 60-120 minutes, when the processing temperature is 180-220°C, the processing time may be set to 20-60 minutes, and when the processing temperature is 220-300°C, the processing time may be set to 1-20 minutes. By setting the temperature and time in this way, the protein can be hydrolyzed appropriately and the solubilization rate of the protein can be increased. As a result, polypeptides with a molecular weight of 100-10,000 can be efficiently produced.

In addition, by setting the processing temperature at 130 to 300°C and the processing time at 5 to 60 minutes, the molecular weight of the resulting polypeptide can be controlled to 100 to 1000.

In the present invention, water may be added when processing fish or meat, but it is not necessary to add water. With the present invention, the moisture contained in the fish or meat can sufficiently hydrolyze proteins without adding water.

In addition, in the present invention, when processing fish or meat, ingredients 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, high-pressure processing. Examples of ingredients other than water include acids, bases, reducing agents, dispersants, surfactants, and complexing agents.

Fish in the present invention include tuna, bonito, salmon, horse mackerel, yellowtail, and red sea bream. In the present invention, the fish is preferably tuna or bonito. The fish in the present invention may be one type of fish or a mixture of two or more types of fish. For example, fish bones, waste, and meat can be used as the fish. Fish bones include heads, bones, fins, gills, and meat associated with these. Fish bones are often waste, but in the present invention, processed materials obtained from fish bones can be used as an active ingredient of the growth promoter. Therefore, the growth promoter of the present invention containing processed materials obtained by high-temperature and high-pressure processing of fish bones as an active ingredient is particularly useful.

Meat in the present invention includes beef, pork, chicken, wild boar, mutton, venison, horse, bear, crocodile, and whale meat. Meat in the present invention may be one type of meat or a mixture of two or more types of meat. Meat may also be any part of an animal. For example, chicken meat that can be used includes breast meat, chicken fillet, thigh meat, shank meat, and chicken wings.

The fish or meat may be boiled and pressed before being subjected to high temperature and high pressure treatment. For example, the fish or meat may be provided as a press cake. The press cake can be produced, for example, as follows: 1. The fish or meat is boiled in a kettle with an equal amount of water at 90-100°C for 1-2 hours. 2. The solids derived from the boiled fish or meat are then pressed in a press, and the solids remaining after solid-liquid separation are used as the press cake.

The growth promoter of the present invention may further contain any optional components. For example, the growth promoter of the present invention may contain a pharma- ceutically acceptable base, carrier, additive, excipient, binder, disintegrant, lubricant, stabilizer, colorant, pH regulator, buffer, solubilizer, etc.

The growth promoter of the present invention may be in any form, such as a solid, liquid, powder, granules, or paste. The growth promoter of the present invention may be in any form, such as a tablet, troche, pill, powder, capsule, granule, suspension, emulsion, syrup, or liquid, as an oral administration formulation. The growth promoter of the present invention may be in any form, such as a parenteral administration formulation, such as an intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, oral administration, or transmucosal administration.

The proliferation promoter of the present invention can promote the proliferation of mesenchymal stem cells by adding it to mesenchymal stem cells and culturing them. Therefore, it is particularly useful in regenerative medicine using mesenchymal stem cells. In addition, the proliferation promoter of the present invention can also promote the proliferation of mesenchymal stem cells in vivo by administering it to humans and non-human animals.

The present invention also provides a food composition for promoting the proliferation of mesenchymal stem cells, which contains as an active ingredient 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.

The food composition of the present invention can be provided as a nutritional supplement, a supplement, a health functional food, a food for specified health uses, a nutritional functional food, and the like. The food composition of the present invention may be a food that displays a function such as promoting the proliferation of mesenchymal stem cells. The food composition of the present invention may also be in the form of a general processed food, and may be in any form such as beverages, confectioneries, soups, dairy products, oil and fat processed products, and seasonings.

(Example 1) Molecular weight distribution of solubilized tuna waste (TWL) A solubilized tuna waste (TWL) was prepared by the following method. Tuna was provided by Sanki Feed Industry Co., Ltd. Waste from tuna with edible parts removed (head, bones, etc.) was used. 15 g of the flesh of the waste tuna (hereinafter referred to as "flesh") or the bone from which as much flesh as possible was removed (hereinafter referred to as "bone") was placed in a 50 ml pressure-resistant container, and high-temperature, high-pressure treatment was performed at 250°C or 200°C for 5 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes at 4.1 MPa to obtain a solubilized tuna waste (TWL). An image of high-temperature, high-pressure treatment is shown in Figure 1. The pressure-resistant container containing the waste tuna was placed in a molten salt furnace heated by a heater to perform high-temperature, high-pressure treatment. The temperature of the molten salt furnace was measured by a thermocouple. The treatment time was from the time the pressure-resistant container was placed in the molten salt furnace to the time it was removed. After a predetermined time, the pressure vessel was removed from the molten salt furnace and cooled by immersing it in a water bath at room temperature, thereby completing the treatment.

The molecular weight distribution of the centrifugal supernatant of the high-temperature, high-pressure treated product was measured by gel filtration chromatography using HPLC (column: TSK-GEL G2500PWXL 7.8 x 300 mm; eluent: 0.1% TFA: _CH3CN = 55:45; flow rate: 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 Figures 2 to 5. In the graphs of Figures 2 to 5, the horizontal axis represents the HPLC elution time, and the vertical axis represents absorbance (AU: Absorbance units).

Figure 2 shows the molecular weight distribution of the solubilized material obtained by treating discarded tuna bones at 250°C.

Figure 3 shows the molecular weight distribution of the solubilized material from discarded tuna meat treated at 250°C.

Figure 4 shows the molecular weight distribution of the solubilized material obtained by treating discarded tuna bones at 200°C.

Figure 5 shows the molecular weight distribution of the solubilized material from discarded tuna meat treated at 200°C.

As shown in Figures 2 to 5, the molecular weight of the main components decreased from 1000 or more to 100 or less as the treatment time increased. In particular, the sample obtained under conditions of 250°C for 15 to 30 minutes showed a peak on the low molecular weight side from 1000 or more to 100, which suggests that there are many peptides with 10 amino acids or less.

(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 Sanki Feed Industry Co., Ltd. Waste from tuna (head, bones, etc.) was used after removing edible parts. The flesh of the tuna waste (hereinafter referred to as "flesh") or the bone from which as much flesh as possible was removed (hereinafter referred to as "bone") was placed in a 50 ml pressure-resistant container, and high-temperature and high-pressure treatment was performed at 200°C for 30 minutes at 1.6 MPa, 250°C for 15 minutes, or 250°C for 30 minutes at 4.1 MPa to obtain solubilized tuna waste (TWL). The high-temperature and high-pressure treatment was performed using the apparatus of FIG. 1 as in Example 1, and the pressure-resistant container containing the tuna waste was placed in a molten salt furnace heated by a heater to perform high-temperature and high-pressure treatment. The temperature of the molten salt furnace was measured by a thermocouple. The treatment time was from the time the pressure-resistant container was placed in the molten salt furnace to the time it was removed. After 15 or 30 minutes, the pressure vessel was removed from the molten salt furnace and cooled by immersing it in a room temperature water bath to complete the treatment.

The obtained TWL was subjected to amino acid analysis. The high-temperature, high-pressure treated product was centrifuged and the supernatant was collected. The amount of free amino acids and total amino acids in the centrifugal supernatant were measured. The amount of free amino acids was measured by HPLC amino acid automatic analysis. The amount of total amino acids was measured by decomposing the product with 6N hydrochloric acid at 105°C for 16 hours, removing the hydrochloric acid, and then adjusting the volume to a constant value and subjecting the product to HPLC amino acid automatic analysis.

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

These results indicate that the solubilized material (TWL) from tuna waste is rich in glutamic acid, alanine, glycine, proline, hydroxyproline, and leucine. The amino acid composition of the solubilized material (TWL) from tuna waste is different from the amino acid composition of tuna flesh listed in the Nutritional Composition Table. This suggests that the solubilized material (TWL) from tuna waste may contain amino acids and peptides specific to this part of the body.

(Example 3) Effect of tuna waste solubilized material (TWL) on stem cells (Primary culture of bone marrow-derived mesenchymal stem cells)
Male and female ddy mice (Tokyo Experimental Animals, Tokyo, Japan) aged 5 weeks or older were used. Two femurs and two tibias were removed from each mouse, and 2 mm of each bone end was cut off. The bone marrow was then centrifuged (Fisher Scientific) at 6000 rpm and 2000 × g to collect bone marrow fluid. The bone marrow fluid was suspended in 10 ml of Minimum Essential Alpha Medium (Life Technologies Japan, Tokyo, Japan) containing 5% FBS (Sigma-Aldrich Japan, LLC, Lot: 15K111) and antibiotics (48 unit/ml penicillin, 50 μg/ml streptomycin). The bone marrow was then removed using a cell strainer (FALCON, Kanagawa, Japan), and the cells were seeded on a ₁₀₀ mm dish (Corning Japan, Tokyo, Japan) and cultured at 37°C and 5% CO2 for 3 days. The floating cells were then removed by washing with Dulbecco's PBS (-) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and the resulting adherent cells were designated as mouse bone marrow-derived mesenchymal stem cells (BMSCs). The number of viable cells was counted using a Luna-FL (cell counter) (LMS).

(Cell proliferation evaluation (WST-1 method))
The sample used was tuna waste bone solubilized material (TWL: bone 250°C 30 min) obtained by high-temperature, high-pressure treatment at 250°C for 30 min. As a control, tryptone, which was made by depolymerizing bovine casein with an amino acid score of 100 using trypsin, was used.

Bone marrow-derived mesenchymal stem cells (BMSCs) attached to a 100 mm dish were washed with Dulbecco's PBS (-), detached using a CELL LIFTER, and seeded at 8.0 × 10 ³ cells/well in a 96-well plate. One day later, tuna waste solubilized (TWL) or tryptone (control) was added, and six days later, cell proliferation test reagent WST-1 (Roche Diagnostics, Tokyo, Japan) was dissolved in the medium to a final concentration of 10%, and the entire medium was replaced. The medium was incubated at 37°C and 5% CO ₂ , and the absorbance at 440 nm was measured after 1 hour using Spectra Max M2e (Molecular Devices Japan, Tokyo, Japan). The results are shown relative to the proliferation rate after 6 days, with the rate at the time of addition of each sample taken as 1. Table 2 shows the mean and standard error of the proliferation rates in four independent wells.

The results are also shown graphically in Figure 6. Figure 6 shows the mean ± standard error of 4 wells. ** indicates p<0.01.

In Figure 6, "normal" indicates the control with nothing added. Figure 6 shows the relative cell proliferation rate when TWL and tryptone were added at various concentrations, with the control with nothing added being set at 1. As shown in Figure 6, cell proliferation was significantly promoted in a concentration-dependent manner in the group to which TWL was added to mesenchymal stem cells, compared to cells cultured in normal medium alone (Normal group) and the group to which tryptone was added. In particular, when TWL was added at 100 μg/mL, approximately 1.8-fold cell proliferation was observed compared to the Normal group.

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

Peptones were purchased from the market. The peptones used in the experiment are as follows. Peptone is a general term for substances made by enzymatically treating proteins to reduce their molecular weight (oligopeptides).
1. Tryptone (tryptic digest of casein)
2. Lactalbumin hydrolysate (LAH)
3. Casein peptone (Cpe) (pepsin digest of casein)
4. Beef peptone (Mpe)
5. Soybean-derived peptone (Spe)

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

After washing with Dulbecco's PBS (-), bone marrow-derived mesenchymal stem cells (BMSCs) attached to a 100 mm dish were detached using a CELL LIFTER and seeded at 8.0 × 10&sup3; cells/well in a 96-well plate. One day later, peptones were added, and six days later, cell proliferation test reagent WST-1 (Roche Diagnostics K.K., Tokyo, Japan) was dissolved in the medium to a final concentration of 100 μg/ml, and the entire medium was replaced. The medium was incubated at 37°C and 5% CO&#8322;, and the absorbance at 440 nm was measured one hour later using a Spectra Max M2e (Molecular Devices Japan, Tokyo, Japan). The results are shown in Table 3 and Figure 7.

In Table 3 and Figure 7, N indicates the control where nothing was added. The results are shown as the average value ± standard error for four independent wells. The results are shown relative to the proliferation rate 6 days after addition of each sample, with the rate at the time of addition being set at 1. As shown in Figure 7, in the group to which peptones were added, no cell proliferation activity was observed in bone marrow-derived mesenchymal stem cells. It was revealed that peptones containing various amino acids and oligopeptides do not affect the proliferation of bone marrow-derived mesenchymal stem cells.

The results of Examples 2 and 3 revealed that the peptides contained in the high-temperature, high-pressure treated tuna algae are likely to contain characteristic components that differ from the hydrolyzates derived from casein, beef, and soybeans.

The present invention promotes the proliferation of mesenchymal stem cells and can therefore be suitably used in the field of regenerative medicine using mesenchymal stem cells.

Claims (2)

A food composition for promoting the proliferation of mesenchymal stem cells, comprising as an active ingredient a processed product of tuna bones treated at a temperature of 100-300°C and a pressure of 0.1-8.6 MPa for 1-120 minutes. A proliferation promoter for mesenchymal stem cells, comprising as an active ingredient a product obtained by treating tuna bones at a temperature of 100-300°C and a pressure of 0.1-8.6 MPa for 1-120 minutes.

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