KR102129534B1 - Composition for Facilitating the Proliferation of Stem Cells Comprising the Extract of Bamboo - Google Patents

Abstract

The present invention relates to a composition for promoting the proliferation of stem cells and a method for promoting the proliferation of stem cells using the same.

Description

Composition for facilitating the Proliferation of Stem Cells Comprising the Extract of Bamboo, comprising bamboo extract as an active ingredient

The present invention relates to a composition for promoting the proliferation of stem cells and a method for promoting the proliferation of stem cells using the same.

Recently, in the field of regenerative medicine using tissue engineering, the use technology of stem cells has been proposed as a new field for the treatment of incurable diseases. As a result, interest in research on stem cells has increased, and it is recognized that the use of stem cells capable of forming tissues through proliferation and differentiation can solve various types of diseases as well as problems caused by tissue damage.

Stem cells have the ability to self-replicate in an undifferentiated state and differentiate into specific cells under suitable conditions. Stem cells can be divided into embryonic stem cells and adult stem cells according to their origin. Since human embryonic stem cells are obtained from embryos that can occur as human life forms, they have excellent cell proliferation and differentiation ability, but have bioethical problems. In contrast, adult stem cells have a limited differentiation ability compared to embryonic stem cells, but cells that already exist in various organs of the human body are collected from bone marrow, blood, brain, skin, etc., and developed into stem cells. . Adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neuroblasts, and epithelial stem cells. In the case of hematopoietic stem cells, the stem cells capable of producing blood cells and lymphocytes are helpful for the treatment of immune system diseases including blood cancer. Stem cells differentiate into bone, cartilage, fat, and fibrous tissues and are involved in their recovery when each tissue is damaged. However, these adult stem cells are present in a small amount in each tissue and may exist without division or proliferation for many years.

In other words, stem cells refer to'undifferentiated' cells that have such differentiation ability but have not yet differentiated. In this undifferentiated state, if appropriate conditions are met, stem cells can be differentiated into various tissue cells, and studies such as treatment of diseases using them have been actively conducted, but as ethical problems of embryonic stem cells emerge, relatively ethical Research to use adult stem cells with few problems is attracting attention.

The stem cells obtained from the tissue are in a small amount, and when using them, a large amount of cells are required, so a culture to proliferate the number of stem cells is performed. For example, in Korean Patent Application Publication No. Methods for propagating in vitro have been disclosed. However, when culturing for stem cell proliferation, research on the culture composition capable of promoting the proliferation of stem cells while maintaining the characteristics of the stem cells is somewhat insufficient.

Therefore, it has been shown that certain herbal extracts that have been used for a long time in traditional Asian medicine can be used as a source of new therapeutic agents (Jeong SH, Lee JE, Jin SH, Ko Y and Park JB: Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.Mol Med Rep 10: 3315-3319, 2014.). These extracts can be considered pre-validated to validate their effectiveness and are expected to have fewer safety issues than chemical synthetic drugs. Therefore, the researchers of the present application devised the present invention for safer and more effective proliferation of stem cells than chemical synthetic drugs.

An object of the present invention is to provide a composition for promoting stem cell proliferation, comprising a bamboo extract as an active ingredient, and a method for promoting the proliferation of stem cells using the composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The inventors of the present invention have come to the present invention by finding that the effect of promoting the proliferation of stem cells occurs when using an active ingredient derived from bamboo extract.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for the full understanding of the present invention, various specific details, such as specific forms, compositions and processes, have been described. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of “in one embodiment” or “an embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the present invention. Additionally, special features, shapes, compositions, or properties can be combined in any suitable way in one or more embodiments.

Unless otherwise specified in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

In the present invention, "stem cell (stem cell)" is a cell having the ability to differentiate into two or more cells while having self-replicating ability, totipotent stem cells, pluripotent stem cells (pluripotent) stem cells) and multipotent stem cells. In addition, stem cells refer to undifferentiated cells having multipotency derived from adult cells, including humans, and mammals, preferably human bone marrow, blood, brain, skin, fat (ie, adipose tissue). Or adipocytes), umbilical cord blood, and umbilical cord Barton Jelly (Wharton's jelly). The cells derived from the adult cells include mesenchymal stem cells derived from adult cells.

"Gingiva" according to the present invention may be a more favorable source of stem cells. Stem cells have autogenous regeneration and pluripotency with the ability to differentiate into various cell types, including bone, adipose tissue, and cartilage. Stem cells can be obtained from a variety of sources, including intraoral areas (Ballini A, De Frenza G, Cantore S, et al: In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry.Int J Immunopathol Pharmacol 20: 9-16, 2007). The dental pulp and periodontal ligament are considered preferred sources. However, since the amount of tissue is limited, an additional tooth extraction procedure is required. Therefore, the researchers of the present application have gingival-derived stem cells have the ability to form bones, plastic adhesion and multi-line differentiation ability to form bones, fats and cartilage, and stem cell markers including CD44, CD73, CD90 and CD105 It was found to express. In addition, harvesting stem cells from the gingiva has the advantage of being easier than the bone marrow of the maxilla and mandible due to invasiveness, pain and loss of sensation.

In addition, in the present invention, "extract" refers to a material extracted or separated by an arbitrary method from a raw material, and includes an extract extracted from a raw material, a concentrate obtainable therefrom, and a dry matter and powder of the concentrate without limitation. to be. Several methods can be applied to herbal extraction. For example, cold extrusion and expeller pressing are used in a thermal control environment and higher temperatures are applied to the compression extraction. Various solvents such as ethanol, methanol, butanol, hexane, methylene chloride, ethyl acetate and sterile water can be used for solvent extraction (Wang MH, Jeong SH, Guo H and Park JB: Anti-inflammatory and cytotoxic effects of methanol, ethanol , and water extracts of Angelicae Dahuricae Radix.J Oral Sci 58: 125-131, 2016). A new microwave assisted extraction method for extracting Bambusa tulda has also been developed (Soumya V, Muzib YI and Venkatesh P: A novel method of extraction of bamboo seed oil (Bambusa bambos Druce) and its promising effect on metabolic symptoms of experimentally induced polycystic ovarian disease.Indian J Pharmacol 48: 162-167, 2016). Different solubility and boiling points appear between different types of solvents. It should be noted that different effects can be achieved depending on the type and concentration of the solvent.

In addition, in the present invention, "proliferation" of stem cells is a concept distinguished from differentiation of stem cells, and stem cells are not differentiated into specific cells, and cells are divided while maintaining the characteristics of stem cells. It means that the total number of is increased.

In addition, in the present invention, "medium" refers to a composition containing nutrients necessary to maintain the growth and survival of cells in vitro (in vitro), generally known medium such as RPMI-1640, DMEM, MEM, Or it may include some components thereof, and may also include glutamine, fetal bovine serum, and the like, but is not limited if the cells contain components necessary for maintaining growth and survival.

In the present invention, "passage culture" means a method of continuously culturing cells continuously while transferring a part of the cells to a new culture vessel periodically and then changing the culture medium to continuously cultivate the cells in a healthy state for a long period of time. it means. As the number of cells increases in a culture vessel with a limited space, after a certain period of time, proliferated nutrients are consumed or contaminants accumulate and the cells naturally die, so it is used as a method to increase the number of healthy cells. Replacing (culture vessel) or culturing by dividing the cell population is called 1 passage.

In the present invention, "Bamboo" refers to an evergreen perennial flowering plant belonging to the subfamily Bambusoideae of the family Poaceae of thegrass family. In bamboo, the interstitial area of the stem is usually empty, and the vascular bundles in cross section are scattered all over the stem instead of a cylindrical arrangement, and most bamboo species inhabit warm, humid tropical and warm climates. Bamboo is of economic and cultural importance in South Asia, Southeast Asia, and East Asia, and is used as a building material, food ingredient, and multipurpose ingredient.

In the present invention, "Bambusa tulda (BBT)" is an Indian wood bamboo belonging to the bamboo subfamily of the bamboo subfamily as an active ingredient of the composition for promoting stem cell proliferation in the present invention. It is also from the Indian subcontinent, Indochina, Tibet and Yunnan, with origins in Iraq, Puerto Rico and South America. Bambusa tulda is widely used in the Indian paper industry. It can grow up to 15m in height and 8cm in thickness, and is commonly found in Southeast Asia.

The composition for promoting stem cell proliferation of the present invention includes an extract obtained from Bambusa tulda as an active ingredient. The extract may be obtained through any conventional extraction method such as a solvent extraction method, an ultrasonic extraction method, a filtration method, and a reflux extraction method, and preferably may be obtained using a solvent extraction method or a reflux extraction method.

In order to achieve the above object, the present invention provides a composition for promoting stem cell proliferation, comprising bamboo extract as an active ingredient.

In one embodiment of the invention, the bamboo extract is derived from Bambusa tulda. In another embodiment of the present invention, the bamboo extract is extracted using water, an organic solvent or a mixture thereof as an extraction solvent. In another embodiment of the present invention, the organic solvent is selected from the group consisting of C1-C4 alcohol, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate. In another embodiment of the invention, the extraction solvent is water. In another embodiment of the invention, the stem cells are adult stem cells. In another embodiment of the invention, adult stem cells are from gingiva.

In addition, to achieve the above object, the present invention provides a medium for culturing stem cells, comprising bamboo extract as an active ingredient.

In one embodiment of the invention, the bamboo extract is derived from Bambusa tulda. In another embodiment of the invention, the stem cells are adult stem cells. In another embodiment of the invention, adult stem cells are from gingiva.

In addition, to achieve the above object, the present invention provides a method of promoting stem cell proliferation, comprising the step of treating the isolated stem cells with bamboo extract.

In one embodiment of the invention, the bamboo extract is derived from Bambusa tulda.

The composition for promoting stem cell proliferation according to the present invention has an effect of promoting the proliferation of the stem cells while maintaining the characteristics of the stem cells, and prepares or supplies the stem cells in a sufficient amount within a short period of time before being differentiated into a specific tissue. It can be useful when there is a need.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

FIG. 1 shows a photograph in which BBT was added to a growth medium at various concentrations, and then cell morphology was evaluated on day 1 using an inverted microscope. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
FIG. 2 shows photographs in which BBT was added to the growth medium at various concentrations, and then the cell morphology was evaluated on the third day. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
FIG. 3 shows photographs in which BBT was added to the growth medium at various concentrations, and then the cell morphology was evaluated on the 5th day. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
FIG. 4 shows photographs in which BBT was added to the growth medium at various concentrations, and then cell morphology was evaluated on day 7. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
5 is a graph showing the results of cell counting kit-8 analysis for stem cells cultured at various concentrations of Bambusa tulda methanol extract for 7 days (A) 1, (B) 3, (C) 5, and (D), respectively. . *P<0.05 vs untreated control.
FIG. 6 is a photograph showing staining by collagen I after 1 day of culture with BBT by immunofluorescence. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
7 is a photograph showing staining by collagen I after 3 days of culture with BBT by immunofluorescence. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
8 is a photograph showing staining by immunofluorescence for collagen I after 5 days of culture with BBT. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.
9 is a photograph showing staining by immunofluorescence for collagen I after 7 days of culture with BBT. (A) control and (B) 0.001% by weight BBT group, (C) 0.01% by weight BBT group, (D) 0.1% by weight BBT group and (E) 1% by weight BBT group (initial magnification, 200 times, scale bar, 200 μm). BBT is Bambusa tulda methanol extract.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Materials and methods

Bambusa tulda Preparation of ingredients

Bambusa tulda Roxburgh was taken from the village of Sonaimuri Upazilla Amki in Noakhali region of Bangladesh. Specimens have been deposited in the Korea Biotechnology Research Institute plant specimen storage (Accession No. PB022073). After drying and grinding the leaves of Bambusa tulda, 500 ml methanol was added to 63 g of powder. Extraction was performed using a method of refiltration at room temperature. The extract was filtered and concentrated under reduced pressure with a rotary evaporator to obtain 2.75 g BBT.

human From the gingiva Isolation and culture of derived stem cells

The gingiva tissue provided by a healthy patient who visited the Catholic University of Seoul, St. Mary's Hospital was used. After the approval of the Seoul St. Mary's Hospital Ethics Committee (no. KC11SISI0348), the study was conducted and the patient's written consent was obtained. All methods used in this study were conducted in accordance with the ethical standards of the Institutional Ethics Committee and the Helsinki Declaration of 1964 and subsequent amendments. The gingival tissue obtained was removed and the tissue was cut to 1-2 mm. The gingival tissue was treated with dispase (1 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and collagenase IV (2 mg/ml; Sigma-Aldrich; Merck KGaA). Digested. Cells that were not attached to the culture dish were removed. The medium was changed every 2-3 days, and the cells were cultured in an incubator at 37° C. in a humidified atmosphere of 5% CO 2 and 95% air.

Bambusa tulda After extract treatment, evaluation of stem cell cell morphology

Cells were seeded in 96-well plates at a density of 2.0x10 ³ cells/well. α-MEM medium (α-minimal essential medium; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used. The α-MEM medium was 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc), 100 U/mL penicillin, 100 μg/mL streptomycin, 200 mM L-glutamine (Sigma-Aldrich; Merck KGaA) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich; Merck KGaA). The concentration of BBT was set to 0 (control), 0.001, 0.01, 0.1 and 1% by weight. BBT was dissolved in dimethyl sulfoxide (DMSO), and the concentration of DMSO (0.1%) was set to be the same for each group. Stem cell morphology was evaluated using an inverted microscope (CKX41SF; Olympus Corp., Tokyo, Japan) on Days 1, 3, 5 and 7.

Bambusa tulda After treatment with the extract, confirm the cell proliferation effect

Using cell counting kit-8 (CCK-8; Dojindo, Tokyo, Japan), quantitative analysis of BBT on cell proliferation was performed on day 1, day 3, day 5, and day 7. After adding tetrazolium monosodium salt, it was measured after incubation at 37°C for 2 hours. By measuring the spectrophotometric absorbance at 450 nm using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA), the degree of stem cell proliferation was confirmed.

Immunofluorescence

Immunofluorescence analysis was performed on days 1, 3, 5 and 7 using collagen I antibody (cat. no. ab76956; Abcam, Cambridge, UK). After incubation with BBT, cells were fixed, made permeable, blocked, and cultured with primary antibody against collagen I. The mouse monoclonal collagen I antibody was diluted 1:67 and incubated overnight at 4°C. A secondary antibody (cat. no. F2761; Thermo Fisher Scientific, Inc.) bound with fluorescein isothiocyanate-conjugated (FITC) was diluted 1:100, left at room temperature for 2 hours, and stained with DAPI. It was observed with a fluorescence microscope (Axiovert 200; Zeiss, Jena, Germany).

Statistical analysis

The results were expressed as the mean±standard deviation of the experimental group. A regularity test was performed, and differences between groups were conducted through analysis of variance and post-analysis using a computer program (SPSS 12 for Windows; SPSS Inc., Chicago, IL, USA). The significance level was set to P <0.05.

result

Cell morphology evaluation results

The cell morphology for BBT 0, 0.001, 0.01, 0.1 and 1% by weight is shown in Figure 1. Stem cells of the control group (0% by weight BBT) had a primary form similar to fibroblasts. Even with BBT, there was no significant difference on the 1st day. The results of the 3, 5 and 7 days of the stem cells can be seen in FIGS. 2, 3 and 4. On days 5 and 7 of the 1% by weight BBT group, the cells showed more dense morphology.

Cell proliferation confirmation result

The effect of BBT on cell proliferation on day 1, 3, 5 and 7 was analyzed via CCK-8, and the results are shown in FIG. 5. After 1 day incubation, the relative proliferation rates of 0.001, 0.01, 0.1 and 1% by weight BBT groups were 112.5±17.5, 121.2±14.3, 126.9±10.5 and 157.0±13.1% of the control groups (P<0.05), respectively. Day 3 0.001, 0.01, 0.1 and 1% by weight BBT group results were 103.4±12.5, 116.7±15.1, 121.6±4.2 and 184.4±13.3% of the control group (P<0.05), respectively. The results of 0.001, 0.01, 0.1 and 1% by weight BBT group on Day 5 were 110.4±10.6, 113.4±14.8, 102.6±5.2 and 136.4±7.7% of the control group (P<0.05), respectively. The results of 0.001, 0.01, 0.1 and 1% by weight BBT group on Day 7 were 79.8±3.7, 75.0±2.4, 76.8±4.3 and 70.9±1.6% of the control group (P>0.05), respectively.

Immunofluorescence result

Representative immunofluorescence staining results for collagen I on days 1, 3, 5 and 7 are shown in FIGS. 6 to 9. On day 1, there was no clear change in collagen I. On day 3, there was an increase in collagen I expression, more clearly in the 1 wt% BBT group. However, day 5 results showed that the expression of collagen I gradually decreased in the high concentration BBT group, and showed a similar trend on the 7th day.

The present invention has provided for the effect of various concentrations of BBT on morphology and cell proliferation of human gingival tissue-derived stem cells. It was clearly demonstrated that short-term application of Bambusa tulda enhanced the proliferation of mesenchymal stem cells with increased collagen I expression.

In this study, application of BBT improved cell proliferation by 50% depending on concentration and incubation time. In this regard, BBT can be applied to overcome a limited number of cells or to improve the survival of transplanted stem cells. Herb extracts can be combined with growth factors for synergistic effects.

Based on these results, BBT showed a beneficial effect on the proliferation of mesenchymal stem cells with increased collagen I expression at an early time point.

Claims (13)

A composition for promoting stem cell proliferation, comprising bamboo extract as an active ingredient. According to claim 1,
The bamboo extract is derived from Bambusa tulda, a composition for promoting stem cell proliferation. According to claim 1,
The bamboo extract is extracted with water, an organic solvent or a mixture thereof as an extraction solvent, a composition for promoting stem cell proliferation. According to claim 3,
The organic solvent is one or more organic solvents selected from the group consisting of C1-C4 alcohol, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate, and methyl acetate, for promoting stem cell growth Composition. According to claim 3,
The extraction solvent is water, a composition for promoting stem cell proliferation. According to claim 1,
The stem cells are adult stem cells, stem cell growth promoting composition. The method of claim 6,
The adult stem cells are derived from gingiva (gingiva) tissue, a composition for promoting stem cell proliferation. Stem cell culture medium containing bamboo extract as an active ingredient. The method of claim 8,
The bamboo extract is derived from Bambusa tulda, a medium for culturing stem cells. The method of claim 8,
The stem cells are adult stem cells, the culture medium for stem cells. The method of claim 10,
The adult stem cells are derived from gingiva (gingiva) tissue, a medium for culturing stem cells. A method of promoting the proliferation of stem cells, comprising treating the isolated stem cells with bamboo extract. The method of claim 12,
The bamboo extract is derived from Bambusa tulda, a method for promoting the proliferation of stem cells.

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