Cell migration promoter and method

Abstract

This invention provides a composition that enhances cell migration activity. [Solution] A method for producing a cell migration activator, comprising the steps of: crushing lingonberry fruit and / or cranberry fruit; adding distilled water and extracting at room temperature for 24 to 48 hours; filtering the extract; sterilizing the filtrate by filtration; and adjusting the solid content to a constant concentration.

Description

Technical Field

[0001] The present invention relates to a cell migration promoter and a promoting method. More specifically, it relates to a cell migration promoter and a promoting method using exosomes and loquat extract and / or cranberry extract.

Background Art

[0002] Cell migration is the ability of cells to move and is considered to be related to wound healing and wrinkle formation. Exosomes are extracellular vesicles released by cells, and there are reports that they are effective in cell proliferation and inflammation suppression. One of the functions of exosomes is to activate cell migration, and it is considered that this promotes wound healing. There are also reports that cell migration is related to tumor metastasis and proliferation (Patent Document 1). However, the function has not necessarily been fully elucidated, and there has been a demand for a method to enhance the cell migration function.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] To provide a composition and a method for enhancing cell migration activity.

Means for Solving the Problems

[0005] (1) A method for enhancing cell migration activity by adding loquat extract and / or cranberry extract to a medium. (2) The method for enhancing cell migration activity according to (1), wherein the loquat extract or cranberry extract is a water extract. (3) A method for increasing the cell migration activity of (1), wherein the amount of lingonberry extract and / or cranberry extract added is 0.00001 to 0.001%. (4) A method for enhancing cell migration activity by adding exosomes and lingonberry extract and / or cranberry extract to a culture medium. (5) A method for enhancing the cell migration activity of (4), wherein the amount of exosomes added is 0.1% to 5%, and the amount of lingonberry extract and / or cranberry extract added is 0.00001% to 0.001%. (6) A cell migration activator comprising lingonberry extract and / or cranberry extract as its main components. (7) Furthermore, a cell migration activator of (6) that contains exosomes. (8) The process of grinding lingonberry or cranberry fruit, The process involves adding distilled water and extracting at room temperature for 24 to 48 hours, The process of filtering the extract, The process of sterilizing the filtered material, A process to adjust the solid content to a certain concentration and A method for producing a cell migration activator having the following characteristics. [Effects of the Invention]

[0006] According to the present invention, a novel cell migration activator and a method for activating cell migration are provided. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 shows a photograph investigating the effect of the cranberry extract of the present invention on cell migration activity. [Modes for carrying out the invention]

[0008] Cell migration plays a crucial role in wound healing and other processes, thus creating a need for methods and agents that enhance cell migration activity. Through diligent research, the inventors discovered that lingonberry extract and / or cranberry extract have the effect of enhancing cell migration activity, leading to the completion of the present invention. Specifically, the present invention provides a method for enhancing cell migration activity by adding lingonberry extract and / or cranberry extract. Furthermore, the present invention also provides a cell migration activator containing lingonberry extract and / or cranberry extract, and a method for producing the same. Additionally, the invention provides a cell migration activator obtained by adding exosomes to the aforementioned extract, and a method for enhancing cell migration activity.

[0009] In this specification, lingonberry (Vaccinium vitis-idaea) refers to an evergreen shrub belonging to the genus Vaccinium in the family Ericaceae, and varieties include cowberry and lingonberry. In this specification, cranberry refers to a general term for evergreen shrubs belonging to the subgenus Oxycoccos in the genus Vaccinium in the family Ericaceae. Cranberries are found in acidic swamps in the temperate regions of the Northern Hemisphere, and the main species include climbing cranberry, dwarf cranberry, large-fruited cranberry, and cranberry. Cranberry fruits are used for beverages, food, medicine, and healthcare, and contain benzoic acid.

[0010] As the lingonberry extract or cranberry extract, an aqueous extract of lingonberry or cranberry is preferably used, but is not limited to this. The extraction time may be preferably 3 hours or more, 5 hours or more, 8 hours or more, 10 hours or more, 15 hours or more, 18 hours or more, or 24 hours or more at room temperature. The upper limit of the extraction time is 48 hours. As long as the cell migration activation function is maintained, there are no particular restrictions on the extraction method, and other extraction methods include, but are not limited to, hot water extraction and organic solvent extraction.

[0011] In this specification, cell migration refers to the movement of cells within a culture medium. To investigate cell migration activity, a portion of the cells arranged in a petri dish is scratched to remove them, and then cultured in a culture medium. The extent to which cells migrate into the grooves created by the scratching can be used to determine the activity (scratch test). The more cells that migrate and cover the grooves, the higher the cell migration activity is considered to be. In the photograph in Figure 1, the area enclosed by horizontal lines at the top and bottom is the groove where cells were removed by scratching, and the presence of cells in this area indicates that cell migration is occurring.

[0012] When lingonberry extract was added to a 0.0001% (solids content, w / w) culture medium, an increase in cell migration activity was observed even with lingonberry extract alone. Specifically, in Figure 1, while almost no cells were present in the scratch area of ​​the control PBS, when 0.0001% lingonberry extract was added, numerous cells were observed in the scratch area (grooves) (see Figure 1B). From this, it was considered that lingonberry extract increased cell migration activity. The lower limit of the concentration of lingonberry extract to be added is 0.00001% or more, more preferably 0.00001% or more, and even more preferably 0.0001% or more, and the upper limit is 1% or less, more preferably 0.1% or less, even more preferably 0.01% or less, even more preferably 0.001% or less, and especially preferably 0.0001% or less.

[0013] Furthermore, while the addition of 1% to 3% mouse fibroblast-derived exosomes increased cell migration activity, the addition of 0.3% resulted in almost no cell migration to the scratched area.

[0014] In this specification, "exosome" refers to a membrane vesicle made of a lipid bilayer, approximately 50-200 nm in diameter, secreted by cells. The surface of an exosome contains lipids and proteins derived from the cell membrane, while the interior contains intracellular substances such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.

[0015] The origin of exosomes in the present invention may be experimental animals such as mice, rats, pigs, etc., cows, and humans, and is not limited thereto. In particular, experimental animals such as mice are used as models before human tests, so it can be expected that they will have the same effects as humans.

[0016] Furthermore, when both exosomes and loquat extract were added to the medium, a further increase in cell migration activity was observed. In the group added with 0.0001% loquat extract and 1% or 3% exosomes, significantly more cells migrated to the scratched area compared to the group with only exosomes (C - H in Fig. 1). From the visual observation results, a synergistic effect was observed between loquat extract and exosomes regarding cell migration activation. From these results, it is conceivable that loquat extract activates cell migration by a mechanism different from that of exosomes.

Example

[0017] (Example 1) Method for preparing exosomes Mouse fibroblasts were seeded at a density of 2x10 6 cells / 10 cm dish. They were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2 for 2 days. After the cells reached confluence, they were washed twice with 3 mL of PBS(-) and replaced with 10 mL of DMEM FBS(-). After culturing at 37°C and 5% CO2 for 24 hours, the medium was collected. To remove dead cells in the medium, it was centrifuged at 2,000 xg for 5 min, and the supernatant was collected. Concentration of exosomes was performed according to a conventional method. The supernatant was concentrated approximately 70-fold using a molecular weight 100 kDa ultrafiltration filter (Millipore Amicon Ultra-15) to obtain exosomes.

[0018] Method for preparing loquat extract Cranberry (Vaccinium vitis-idaea), also known as mossberry, is a small evergreen shrub in the genus Vaccinium of the Ericaceae family. Its fruit is edible, but it is a plant that is difficult to cultivate. Cranberries grow wild on Mount Fuji, and local residents have been consuming them as a nourishing and strengthening food and using them as a wound medicine since ancient times. Since the activity of treating wounds is considered to be the activity of promoting cell migration, cell experiments were conducted in the next section. The cranberry fruit was mashed with a mixer and distilled water was added to make it 10% (w / w). After standing at room temperature for 24 hours, it was roughly filtered through a cloth, filtered through a 2.7 μm filter, and then filtered through a 0.45 μm filter. The solid content was measured with a halogen moisture meter (Ohaus halogen moisture meter MB45), and distilled water was added to make it 1% (w / w) solid content to obtain cranberry extract. Cranberry extract can be prepared in the same way.

[0019] Analysis of cell migration by scratch assay The cell migration ability was analyzed by scratch assay. Scratch assay is a simple and widely used method. Human epidermal cells were seeded at a density of 1X10 6 cells / 6 cm dish, cultured in MCDB153 medium with 1% FBS at 37 °C and 5% CO2 for 6 hours. After the cells adhered to the dish, the medium was changed. When treated with cranberry extract, it was replaced with a medium containing 0.0001% solid content. The next day, it was replaced with MCDB153 medium containing the sample and 0.5% FBS, and a scratch was made on the dish with a pipette tip to create a groove without cells. After 24 hours, microscopic observation was carried out, and photos were taken with an iPhone (registered trademark) equipped with an i-NTER LENS (Micro Net Co., Ltd.).

[0020] In the control (PBS--), almost no cells were observed in the cell-free groove (within the black frame), indicating that cells had not migrated into the black frame (Figure 1A). With 0.0001% lingonberry extract (solid content), 3% exosomes, and 1% exosomes (v / v concentration), cells were observed within the black frame (Figures 1B, C, D), demonstrating activity to promote cell migration. Furthermore, combining lingonberry extract and exosomes resulted in cells filling almost the entire black frame (Figures 1F, G). Therefore, it was shown that combining lingonberry extract and exosomes can further promote cell migration. Since cell migration was not sufficiently promoted at an exosome concentration of 0.3% (Figures 1E, H), an exosome concentration of approximately 1% is preferable. [Industrial applicability]

[0021] This invention can be used in the medical and cosmetics industries.

Claims

1. A method for enhancing cell migration activity by adding lingonberry extract and / or cranberry extract to a culture medium.

2. The method for enhancing cell migration activity according to claim 1, wherein the lingonberry extract or cranberry extract is an aqueous extract.

3. The method for enhancing cell migration activity according to claim 1, wherein the amount of the lingonberry extract and / or cranberry extract added is 0.00001 to 0.001%.

4. A method for enhancing cell migration activity by adding exosomes and lingonberry extract and / or cranberry extract to a culture medium.

5. A method for enhancing cell migration activity according to claim 4, wherein the amount of exosomes added is 0.1% to 10%, and the amount of lingonberry extract and / or cranberry extract added is 0.00001% to 0.001%.

6. A cell migration activator primarily composed of lingonberry extract and / or cranberry extract.

7. Furthermore, the cell migration activator according to claim 6 contains exosomes.

8. The process involves grinding lingonberry or cranberry fruits, The process involves adding distilled water and extracting at room temperature for 24 to 48 hours, The process of filtering the extract, The process of sterilizing the filtered material, A process to adjust the solid content to a certain concentration and A method for producing a cell migration activator having the following characteristics.

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