JP7339101B2 - Composition for Proliferating Intestinal Epithelial Stem Cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to novel uses of proteoglycan (foods, etc.) based on the enterocyte activating action of proteoglycan.

Maintaining the intestinal barrier function is considered important for maintaining the intestinal environment and maintaining health (Patent Documents 1 and 2).

JP 2019-43879 JP 2019-11315

However, in animals such as humans, effective means for improving intestinal function have not yet been found, and more effective means are desired.

Accordingly, an object of the present invention is to provide means for easily improving intestinal function in animals such as humans.

The present invention was made to solve such problems, and when a composition (agent) containing proteoglycan was injected into the lumen of the mouse intestine (intestinal organoid), the possibility of proliferating intestinal epithelial stem cells was demonstrated. newly found.

Preferably, the proteoglycan is a chondroitin sulfate-type proteoglycan, preferably a proteoglycan derived from salmon nasal cartilage.

By using this composition, the intestinal function of animals such as humans can be improved more easily.

The present invention will be described below.

((intestinal) epithelial stem cells)
Epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissue. The term "epithelial stem cell" refers to a cell that has a long-term self-renewal function and the ability to differentiate into an epithelial differentiated cell, and refers to a stem cell derived from an epithelial tissue. Epithelial tissues include, for example, cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver , pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair root, trachea, lung and the like. The term “organoid” refers to a three-dimensional cell organization that is self-organized by accumulating cells in a controlled space at high density.

(proteoglycan)
Proteoglycans are glycoproteins in which sugar chains called glycosaminoglycans (hereinafter referred to as GAGs) such as chondroitin sulfate and dermatan sulfate are covalently bound to core proteins. Proteoglycans are widely distributed in the body, such as skin and cartilage, as one of the major components of extracellular matrix. GAG chains have a long linear structure with no branches. Since it has many sulfate groups and carboxyl groups, it is negatively charged, and the GAG chain assumes an elongated shape due to its electrical repulsion. In addition, proteoglycans can retain a large amount of water due to the water affinity of sugars. A large number of GAG chain groups contained in proteoglycans retain water flexibly like a sponge, and perform functions unique to cartilage such as elasticity and resistance to impact.

The proteoglycan core protein has the property of binding to various molecules in the matrix. Cartilage proteoglycan has a binding region with hyaluronic acid or link protein on the N-terminal side, and may bind to these substances or associate between the same molecules. It has a lectin-like domain, an EGF-like domain, etc. at its C-terminus and binds to various other molecules. Due to this property, proteoglycan builds a structure suitable for each tissue.

Among proteoglycans, chondroitin sulfate-type proteoglycans are proteoglycans in which chondroitin sulfate is covalently bound to a core protein.
Salmon nasal cartilage-derived proteoglycan is proteoglycan obtained by extraction from salmon nasal cartilage. Here, salmon is, for example, a fish belonging to the genus Oncorhynchus, and salmon with the scientific name "Oncorhynchus keta" is preferably selected.

In addition, the lower limit of the content of proteoglycan contained in the composition according to the present invention is preferably 5 μg/mL or more, more preferably 50 μg/mL or more, and still more preferably 500 μg/mL, from the viewpoint of proliferation of intestinal epithelial stem cells, for example. (0.5 mg/mL) or more, for example, the upper limit is preferably 5000 μg/mL or less, more preferably 2500 μg/mL or less, from the viewpoint of proteoglycan solubility when preparing a composition (solution) containing proteoglycan, It is more preferably 1000 μg/mL (1 mg/mL) or less.
The proteoglycan contained in the composition according to the present invention is produced, for example, by the method described in the publication (Japanese Patent No. 6317053).

(Improvement of intestinal function, etc.)
“Improve intestinal function” includes not only treatment of deteriorated intestinal function, but also prevention of intestinal deterioration (disease, etc.). It also includes preventing the onset of the disease by using it, and allowing the diseased lesion to acquire functions that exceed those in normal or remission in advance, thereby reducing damage to the lesion caused after the onset of the disease.

Intestinal deterioration (disease, etc.) is a disease that develops in the intestine. Intestinal diseases accompanied by inflammation involving the entire intestine such as infectious enteritis induced by allergies, food poisoning, drugs, alcohol, etc.; irritable bowel syndrome and the like. An intestinal disease in which the site of inflammation is from the duodenum to the rectum is preferable, and an intestinal disease in which the small intestine or the large intestine is more preferable.

(others)
The composition of the present invention can be used in external preparations such as pharmaceuticals, foods (including beauty and health-conscious food and drink), beverages, and cosmetics. Specific examples of foods and beverages include nutritional supplements, nourishing tonics, recovery from fatigue, constitution improvement, whitening, skin beautifying, hair beautifying, hair growth/growth, slimming, mental stability, and other beauty/health-oriented processed foods, supplements, and nutritional supplements. In addition to foods and drinks, general favorite foods and beverages such as soft drinks can be mentioned. In addition, the composition of the present invention can be produced by arbitrarily selecting and combining other components, if necessary, within a range that does not impair the effects of the present invention.

[Preparation of sample used in test (composition containing proteoglycan, etc.)]
As a control solution, a liquid composition (a composition containing no proteoglycan) having the following composition was prepared, and as Example 1, a liquid composition containing proteoglycan having the following composition was prepared.

(control solution)
・PBS

(Example 1)
• Proteoglycan: solution with PBS as solvent to a final concentration of 1 mg/mL.

The components contained in the control and the solution of Example 1 were as follows.
PBS: phosphate buffered saline (without calcium and magnesium)
・Proteoglycan: proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133))

Next, mouse intestinal organoids used in the test were produced. Cultrex (registered trademark) HA-R-Spondin1-Fc 293T cells (TREVIGEN, Gaithersburg, MD, USA) were cultured in advance, and the solution in which R-Spondin1 was secreted into the medium was collected to condition R-spondin-1. used as a medium. Mouse intestinal organoids were constructed as follows. Specifically, 15 cm of the proximal small intestine (including the duodenum and jejunum) of the mouse was dissected, removed, opened vertically, and washed several times with ice-cold PBS. After cutting into small pieces, tissue pieces were incubated in ice-cold PBS containing 5 mM EDTA for 40 minutes. After removing the EDTA solution, the tissue pieces were triturated vigorously with ice-cold PBS by pipetting and the supernatant was discarded. After further trituration with ice-cold PBS, the crypt-rich supernatant was collected by filtration through a 70 μm cell strainer. The filtrate was centrifuged at 100×g for 1 min, and the precipitated crypts were added to organoid basal medium (OBM: Advanced DMEM/F12 containing 2 mM L-glutamine, 1 mM N-acetyl-L-cysteine, 100 units/mL penicillin, 100 μg /mL streptomycin, 10 mM HEPES, pH 7.0). Matrigel was mixed 1:1 with the crypt suspension and 20 μL of the mixture was plated in a 48-well culture plate. After solidification of Matrigel at 37° C., 0.25 mL organoid growth medium (OGM: OBM supplemented with 12.5 ng/mL EGF, 25 ng/mL Noggin, 5% FBS, 20% R-spondin-1 conditioned medium). was added to each well and cultured at 37° C. with 5% CO ₂ concentration to generate mouse intestinal organoids.

About 20 organoids were suspended in Matrigel so that one dome would grow, and one dome was dispensed into a 35 mm dish and placed in a CO ₂ incubator for about 5 minutes to solidify the Matrigel. 2 mL of 40% R-spondin-1 conditioned medium containing 5% FBS growth medium was added per dish and cultured for 24 hours before microinjection into the lumen of intestinal organoids. A micropipette (needle) having an outer diameter of 10 μm or less was used for microinjection. The amount injected into the intestinal organoids in microinjection was 5 nL, and the injection rate was 1 nL/sec.

Of the 1:1 mixture of the solution of Example 1 above and 0.7 mg/mL of phenol red solution in PBS, 5 nL was injected into the lumen of the intestinal organoid (the final proteoglycan concentration was 500 μg/mL). mL, 2.5 ng proteoglycan injected per intestinal organoid).

In addition, as a control (composition containing no proteoglycan), 5 nL of a 1:1 mixture of the above control solution and 0.7 mg/mL phenol red solution dissolved in PBS was added to the lumen of the intestinal organoid. Injected groups were also generated.

After injection, the medium was changed to 5% R-spondin-1 conditioned medium containing 5% FBS growth medium and cultured at 37° C. with 5% CO ₂ concentration for 24, 48 and 72 hours. Organoids were observed under a microscope and digital images were taken. The obtained images were analyzed with Image J (NIH, Bethesda, MD, USA). The increase in the number of crypts was determined by continuously observing specific organoids over time and subtracting the number of crypts/organoid at the start of culture from the number of crypts/organoid at each measurement point to obtain the increase in the number of crypts/organoid. The amount of increase in cross-sectional area was obtained by continuously observing a specific organoid over time and subtracting the cross-sectional area at the start of culture from the cross-sectional area of the organoid at each measurement point to obtain the amount of increase in cross-sectional area/organoid. For the cross-sectional area increase rate, continue to observe specific organoids over time, express the organoid cross-sectional area at each measurement time point as a ratio to the cross-sectional area at the start of culture, and obtain the cross-sectional area increase rate/organoid (%). .

[Results of measuring the increase in the number of crypto]
Twenty-four hours after injecting the sample into the lumen of the intestinal organoid and culturing, the average crypt number increase/organoid in the control was 1.89 ± 1.45, whereas the solution of Example 1 (proteoglycan-containing composition A significant increase of 4.67±3.45 was observed in the case of injecting the substance). In addition, even 72 hours after injection and culture, the average crypt number increase/organoid in the control was 4.11±2.62, whereas the solution of Example 1 (proteoglycan-containing composition) was injected. case, a significant increase of 8.83±5.94 was observed.

[Results of measurement of cross-sectional area of organoids]
Twenty-four hours after injecting the sample into the lumen of the intestinal organoid and culturing, the average cross-sectional area increase/organoid in the control was 2379.3 ± 854.1 pixels, whereas Example 1 (proteoglycan-containing composition) was injected, a significant increase of 4597.7±2583.3 was observed. In addition, even 72 hours after injection and culture, the average cross-sectional area increase/organoid in the control was 5487.6 ± 1794.2, whereas when Example 1 (proteoglycan-containing composition) was injected, , 10252.7±5023.5. In addition, the cross-sectional area increase rate was 158.5 ± 21.1 pixels in the control (PBS) 24 hours after injecting the sample into the lumen of the intestinal organoid and culturing it, while Example 1 (proteoglycan-containing composition), a significant increase of 192.7±41.4 was observed. In addition, even 72 hours after injection and culture, the average cross-sectional area increase rate/organoid of the control was 234.3 ± 36.6, whereas when Example 1 (proteoglycan-containing composition) was injected, , 302.6±54.8, a significant increase was seen.

Therefore, it is considered that the inclusion of proteoglycan acts on intestinal stem cells to increase the proliferative activity of intestinal epithelial stem cells. Proliferation of intestinal epithelial stem cells is thought to contribute to intestinal metabolism.

Although the embodiments (including examples) of the present invention have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and is within the scope of the present invention. , even if there is a design change, etc., it is included in the present invention.

Claims (3)

A composition for intestinal organoid formation containing proteoglycans. 2. The composition according to claim 1, wherein the proteoglycan is a chondroitin sulfate-type proteoglycan. 3. The composition according to claim 1 or 2, wherein the proteoglycan is salmon nasal cartilage-derived proteoglycan.