KR102159766B1 - Medium Composition Containing Extract of Red Algae and the Method for Inducing of Immature Dendritic Cell - Google Patents

Abstract

The present invention relates to a technique for inducing differentiation of immature dendritic cells, and more specifically, to a medium composition and a method for inducing differentiation of immature dendritic cells from bone marrow cells using red algae extract.

Description

Medium Composition Containing Extract of Red Algae and the Method for Inducing of Immature Dendritic Cells Using Red Algae Extract

The present invention relates to a technique for inducing differentiation of immature dendritic cells, and more specifically, to a medium composition and a method for inducing differentiation of immature dendritic cells from bone marrow cells using a red algae extract.

Dendritic cells (DC) are the most powerful antigen presenting cells (APCs) among cells present in the immune system to deliver antigens to T lymphocytes. Dendritic cells can induce immunity by reacting with primitive T cells (naive T cells) that have never come into contact with an antigen, and unlike other antigen presenting cells, they effectively induce a primary immune response, thereby generating strong immune memory. It is the only immune cell with inducible properties. The reason dendritic cells can induce strong immune responses is the antigen presenting cell (APC), which expresses not only MHC molecules (I/II) but also co-stimulatory molecules at high concentration on the cell surface. It is known to be because it secretes cytokines (cytokine, IFN-alpha, IL-12, IL-18, etc.) necessary for T cell activation.

In addition, since type 1 dendritic cells secrete Th1 immune-inducing cytokines such as IFN-alpha and IL-12, they can induce proliferation of antigen-specific Th1 cells and activation of cytotoxic T lymphocytes (CTLs). It is usefully applicable.

However, in order to utilize dendritic cells for autoimmune diseases and anticancer immunotherapy using immune cells, technologies for differentiating dendritic cells in vitro and maturation technologies to effectively induce T cell immunity are essential.

In particular, in relation to the technology to differentiate and manufacture dendritic cells in vitro, differentiation of dendritic cells derived from bone marrow is indispensable. In the cultivation of dendritic cells, the cost of the inducing composition is very expensive. There is a need to develop a composition for cell differentiation culture medium.

Therefore, developing a medium composition for inducing the differentiation of undendritic cells with a new composition to manufacture undendritic cells at an economical price can be suggested as an important method for developing various immunological treatment methods such as autoimmune diseases and anticancer immunotherapy. .

Red algae (紅藻類) is a branch of protozoa classified as algae, and about 600 genera and 2,000 species are known around the world. All have pink or dark red pigments, and make reddish starch by photosynthesis. Most grow in the sea, but others grow in fresh water or on damp soil surfaces. On the other hand, except for the single cells of several genera, most are composed of multicellular, and the size ranges from several mm to tens of mm. Some bodies are in the shape of threads, leaves, branches, or mesh. Most of them are soft, but there are groups where calcium carbonate accumulates between the cell walls like coral horses and becomes hard like stones.

As previously reported, the antibacterial effect, antioxidant effect, protective effect against radioactivity, and anti-inflammatory effect of various red algae are known, but the efficacy of red algae on inducing the differentiation of microdendritic cells is not yet known.

Korean Patent Registration No. 10-1790647

The present inventors have completed the present invention by discovering a new use of red algae extract.

Therefore, the object of the present invention is to contain red algae extract and GM-CSF as active ingredients, so that the red algae extract can more effectively promote the differentiation ability from bone marrow cells to immature dendritic cells by GM-CSF. It is to provide a medium composition for inducing differentiation of captives and a method for inducing differentiation.

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

In order to achieve the object of the present invention described above, the present invention is a medium composition comprising red algae extract and GM-CSF as active ingredients, wherein the red algae extract promotes the differentiation ability of GM-CSF to differentiate bone marrow cells into immature dendritic cells. It provides a medium composition for inducing differentiation into immature dendritic cells, characterized in that to.

In a preferred embodiment, the extract is a water-soluble extract of red algae.

In a preferred embodiment, the red algae is one or more of the red crest leaves ( Callophyllis japonica ) and the leaf tail ( Gracilaria textorii ).

In addition, the present invention provides a method for inducing differentiation into immature dendritic cells comprising the step of culturing bone marrow cells isolated from a living body in any one of the above-described medium compositions.

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In a preferred embodiment, the bone marrow cells are obtained by separating the isolated bone marrow into single cells and then dissolving red blood cells.

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Since the red algae extract of the present invention has the effect of effectively promoting the ability of bone marrow cells to differentiate into immature dendritic cells by GM-CSF at a concentration that does not have cytotoxicity, it can be used for various anticancer immunotherapy. Immature dendritic cells, the predecessor of cells, can be effectively produced from bone marrow cells in vitro.

These technical effects of the present invention are not limited only to the ranges mentioned above, and even if not explicitly mentioned, the effects of the invention that can be recognized by those of ordinary skill in the art from the description of specific details for the implementation of the invention described later are also Of course it is included.

1 is a graph showing the cytotoxicity measurement results of mouse immature dendritic cells treated with red algae extract according to an embodiment of the present invention.
FIG. 2 is a graph showing results obtained by staining CD11c, a marker of immature dendritic cells and bone marrow cells cultured by treatment with red algae extract according to an embodiment of the present invention.

As for terms used in the present invention, general terms that are currently widely used are selected, but in certain cases, some terms are arbitrarily selected by the applicant. In this case, the meanings described or used in the detailed description of the invention are considered rather than the names of simple terms. Therefore, its meaning should be grasped.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numerals used to describe the present invention throughout the specification denote the same elements.

The technical feature of the present invention was conceived in that red algae extract promotes the differentiation ability of GM-CSF to differentiate bone marrow cells into immature dendritic cells. The new use of red algae extract, GM-CSF, promotes differentiation into immature dendritic cells. A medium composition for inducing differentiation into immature dendritic cells and inducing differentiation into immature dendritic cells that can easily produce immature dendritic cells, which are essential for producing dendritic cells having the characteristics of enhancing immune response in various chemotherapy using To provide a way.

In other words, dendritic cells differentiate from progenitor cells derived from bone marrow and exist as immature dendritic cells with high antigenic phagocytosis. When external stimuli and antigens are recognized, the immature dendritic cells undergo maturation, resulting in cell surface Since the expression of the molecule is increased and the adaptive immune response is initiated by moving to the T-lymphocyte-dense area, a number of immature dendritic cells are essential to induce the adaptive immune response, but in the tumor microenvironment, various cellular responses of tumor cells Due to the lack of sufficient number of immature dendritic cells, it is difficult to induce an anti-tumor immune response even if inducing maturation. Therefore, developing a method for producing a large amount of immature dendritic cells is to develop various immunological treatment methods such as cancer immunotherapy. Because it is important.

Accordingly, the medium composition for inducing differentiation of immature dendritic cells of the present invention contains red algae extract and GM-CSF as active ingredients.

This is because, as described above, the red algae extract promotes the differentiation ability of GM-CSF in the bone marrow cell culture medium treated at a concentration that does not have cytotoxicity, thereby increasing the expression of CD11c, known as a marker of immature dendritic cells, in a concentration-dependent manner. As a result, if the medium composition of the present invention is treated with bone marrow cells to induce maturation after making immature dendritic cells, it can be used to enhance the immune response during various immunological treatments such as anticancer treatment in mammals including humans.

Red algae for obtaining red algae extract contained in the medium composition for inducing differentiation of immature dendritic cells of the present invention is not limited as long as red algae, but in the embodiment of the present invention, red algae extracts are used as red algae extracts ( Callophyllis japonica ) and leaf tail ( Gracaria textorii ) One or more extracts of were used.

The red crested leaf (Callophyllis japonica ) belongs to the Calimenia family, and has been widely distributed in the south coast of Korea and consumed as a food. According to previously reported, red crested leaves are known to have a protective effect against radiation irradiation in mice, and also have antioxidant and antibacterial effects. Gracilaria textorii belongs to the family Coliaceae, which is distributed all over Korea, and is also distributed in Japan, the East China Sea, and Indonesia. Leaf stalks have also been ingested as food for a long time in Korea, and the extracts from stalks of leaves are known to have anti-inflammatory effects on mouse macrophages. As described above, red crested leaves have antibacterial, antioxidant, and protective effects on radioactivity, and the leaves are known to have anti-inflammatory effects, but their efficacy in inducing the differentiation of microdendritic cells so far Was not known.

The red algae extract used in the present invention can be separated according to a conventional method known in the art for extracting an extract from a natural product, that is, using a conventional extraction solvent under conditions of a normal temperature and pressure. As the extraction solvent, a solvent that can be generally used in the extraction process can be used, including water, anhydrous or aqueous lower alcohol having 1-4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane and 1,3-butylene glycol. It can be extracted using a solvent selected from the group consisting of. In one embodiment, the red algae extract may be a water-soluble extract of red algae, particularly an ethanol extract, as described in Examples.

The medium composition of the present invention may be used alone as red algae extract, but may include red algae extract in a basic medium used for cell culture such as DMEM, and if necessary, various amino acids, vitamins, inorganic salts, etc. that are generally used for cell culture. Ascorbic acid 2-glucoside (AA2G), insulin-transferin-selenium, EGF, PDGF-BB, human serum, as well as various organic and inorganic ingredients that are acceptable in the culture medium according to the purpose of use. It may contain more.

The concentration of red algae extract, which is an active ingredient contained in the medium composition of the present invention, may be included in a concentration necessary to promote the differentiation ability of GM-CSF that differentiates bone marrow cells into immature dendritic cells, and is determined in consideration of cell status and required period. It may be, and is not limited to a concentration in a specific range, but may be included in an amount of 0.01 to 99% by weight in the medium composition.

Next, the method for inducing differentiation of immature dendritic cells of the present invention includes culturing bone marrow cells isolated from a living body in any one of the above-described medium compositions. As described above, when the red algae extract and GM-CSF are included in the bone marrow cell culture medium, the red algae extract promotes the differentiation ability of GM-CSF to induce the differentiation of bone marrow cells into immature dendritic cells. In one embodiment, the bone marrow cells are obtained by dissolving red blood cells after separating the isolated bone marrow into single cells, and the red algae extract may be a water-soluble extract of one or more of red crest leaves ( Callophyllis japonica ) and leaf tail ( Gracilaria textorii ).

Example 1

1. Preparation of red algae extract

Red crested leaves ( Callophyllis japonica ) were collected in winter from Wando, and washed three times with fresh water to remove seawater. The washed seaweed was dried in a dryer at 90° C. for 24 hours to remove moisture, and then made into a powder state using a blender. 100 g of seaweed powder was added to 900 ml of 70% ethanol, stirred, and filtered on a 3MM filter paper on the second day to collect the seaweed extract. The above method was repeated 3 times to extract for a total of 6 days. The obtained extract was concentrated using a vacuum concentrator, and all solvents were removed using a lyophilizer to obtain a powdery extract. The obtained extract was dissolved in sterilized tertiary distilled water and ethanol and used for cell experiments.

2. Preparation of bone marrow cells

C57BL/6 mice (8-10 weeks old) were purchased and bone marrow was isolated from the femur and tibia. The separated bone marrow was separated into single cells, red blood cells were lysed, and cultured in RPMI-1640 medium for cell culture for 4 hours.

3. Induction of differentiation of bone marrow cells into immature dendritic cells

After the prepared bone marrow cells were removed again, the cells were cultured in a medium containing GM-CSF for 6 days, and the cell culture solution was replaced every 2 days. During the cultivation period of 6 days, the red rice leaf extract was treated with GM-CSF, and the culture medium containing the extract was renewed every 2 days. At this time, the medium composition 1 containing GM-CSF (5ng/ml) and red rice red leaf extract (50 ng/ml) was used. Thereafter, on the 6th day, non-adherent dendritic cells and weakly attached dendritic cells were used as immature dendritic cells.

Example 2

The same method as in Example 1 was used, except that medium composition 2 containing GM-CSF (5 ng/ml) and leaf stalk extract (50 ng/ml) was used using not the red crested leaves, but Gracilaria textorii . To obtain immature dendritic cells.

Example 3

GM-CSF (5 ng/ml), red-crested leaf extract (25 ng/ml), and leaf stalk extract (25 ng) by using not only red-crested leaves but also Gracilaria textorii in the same amount. /ml) was used to obtain immature dendritic cells in the same manner as in Example 1, except that the medium composition 3 was used.

Comparative Examples 1 to 3

Bone marrow cells were differentiated into immature dendritic cells using Comparative Prediction Compositions 1 to 3 consisting of 5, 10, and 20 ng/ml of GM-CSF without red algae extract.

Experimental Example 1

After inducing immature dendritic cells by treating bone marrow-derived precursor cells with medium compositions 1 to 3 containing GM-CSF and red algae extract, respectively, as in Examples 1 to 3, immature dendritic stained through Annexin/PI staining on day 6 Cells were measured for cytotoxicity through flow cytometry and the results are shown in FIG. 1. Data are representative of the results of 4 individual replicates.

As shown in Figure 1, it was confirmed that all of the medium compositions 1 to 3 in which the concentration of the red algae extract was 50 ng/ml did not exhibit cytotoxicity to dendritic cells.

Experimental Example 2

In order to confirm whether the cells finally obtained in Examples 1 to 3 and Comparative Examples 1 to 3 exhibit a phenotype of dendritic cells, that is, to confirm whether bone marrow cells have differentiated into immature dendritic cells, present in the culture medium obtained on the 6th day. The phenotype of cells was analyzed as follows and the results are shown in FIG. 2. The phenotype of immature dendritic cells was analyzed using a flow cytometry method for cell surface molecules.

The cells obtained on the 6th day of culture were washed twice with cold PBS. The washed cells were treated with an anti-CD11c antibody (a marker of dendritic cells) with FITC attached, and then reacted in a refrigerator at 4° C. for 30 minutes. The stained cells were washed twice with cold PBS and then fixed with 4% paraformaldehyde. The fixed cells were measured using an FC500 (Beckman coulter) instrument, and then analyzed using kaluza software (Beckman coulter). Data are representative of the results of 4 individual replicates.

As shown in FIG. 2, it was confirmed that the differentiation of immature dendritic cells was increased in a concentration-dependent manner when GM-CSF, a cytokine for dendritic cell differentiation, was treated.

In particular, in the case of medium composition 3 containing both red rice crest and leaf extract in precursor cells treated with 5 ng/ml of GM-CSF, comparative plant composition 3 consisting of GM-CSF 20 ng/ml was treated. It was confirmed that CD11c was expressed at a level similar to that of when.

From these experimental results, when the red crested leaf extract and the leaf extract were treated at the same time. It can be seen that the differentiation rate of dendritic cells is further increased than when the red crested red leaf extract and the leaf stalk extract were added to GM-CSF as a single unit.

Therefore, the present invention shows that the red algae extract, in particular, the red crest of a red leaf and the leaf tail, promotes the differentiation-inducing activity by GM-CSF, which differentiates bone marrow cells into immature dendritic cells, thereby enhancing immune response during immunological treatment such as anticancer treatment. It can be seen that, by using the present invention, dendritic cells essential for a new pharmaceutical composition to be enhanced can be easily prepared from bone marrow cells.

Although the present invention has been illustrated and described with a preferred embodiment as described above, it is not limited to the above-described embodiment, and within the scope not departing from the spirit of the present invention, to those of ordinary skill in the technical field to which the present invention belongs. Various changes and modifications will be possible.

Claims (7)

As a medium composition containing red algae extract and GM-CSF as active ingredients,
The red algae extract is a medium composition for inducing differentiation into immature dendritic cells, characterized in that promoting the differentiation ability of GM-CSF to differentiate bone marrow cells into immature dendritic cells.
The method of claim 1,
The red algae extract is a medium composition for inducing differentiation into immature dendritic cells, characterized in that the water-soluble extract of red algae.
The method of claim 1,
The red algae is a medium composition for inducing differentiation into immature dendritic cells, characterized in that it is one or more of one of red crested leaves ( Callophyllis japonica ) and leaf tail ( Gracilaria textorii ).
delete A method for inducing differentiation into immature dendritic cells comprising; culturing bone marrow cells isolated from a living body in the medium composition of any one of claims 1 to 3.
delete The method of claim 5,
The bone marrow cells are obtained by separating the isolated bone marrow into single cells and then dissolving red blood cells.

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