KR20200054722A - 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 particularly, to a medium composition for inducing differentiation of immature dendritic cells from bone marrow-derived cells using a red algae extract, and a method of inducing differentiation thereof.

Description

Medium composition containing extract of red algae and the method for inducing of immature dendritic cell

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

Dendritic Cells (DCs) are the most potent antigen-presenting cells (APCs) capable of delivering antigens to T lymphocytes among cells present in the immune system. Dendritic cells can induce immunity by reacting with naive T cells that have never encountered an antigen, so unlike other antigen-presenting cells, they effectively induce a primary immune response, resulting in strong immune memory. It is the only immune cell with inducible properties. The reason dendritic cells can strongly induce an immune response is as an antigen presenting cell (APC), which expresses not only MHC molecules (I / II) on the cell surface, but also co-stimulatory molecules at high concentrations. It is known to secrete cytokines (cytokine, IFN-alpha, IL-12, IL-18, etc.) necessary for T cell activation.

In addition, because 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 (CTL). It is useful.

However, in order to utilize dendritic cells for anti-immune treatment using autoimmune diseases and immune cells, it is necessary to develop a technique for differentiating dendritic cells in vitro and a maturation technique to effectively induce T cell immunity.

In particular, in relation to the technique of differentiating and producing dendritic cells in vitro, the differentiation of dendritic cells derived from bone marrow is essential, and since the price of the composition for inducing them in the culture of dendritic cells is very expensive, a stem that can replace them Development of a composition of a culture medium for cell differentiation is required.

Therefore, the development of a medium composition for inducing differentiation of non-dendritic cells of a new composition and manufacturing of non-dendritic cells at an economical price can be suggested as an important method for developing various immunological treatment methods such as autoimmune diseases and anti-cancer immunotherapy. .

Red algae (紅 藻類) is a branch of the protozoa classified as algae, about 2,000 species of about 600 genus are known around the world. They all have pink or dark pink pigments and make red starch by photosynthesis. Most of them grow in the sea, but some grow on fresh water or wet soil. On the other hand, most of them are multicellular except for a few single cells, and the size ranges from several mm to several tens of mm. The body may have a thread, leaf, twig shape, or mesh shape. Most of them are soft, but some are hardened like stones because calcium carbonate accumulates between the cell walls like coral fish.

As previously reported, the antimicrobial, anti-oxidant, and anti-inflammatory effects of various red algae are known, but the efficacy of red algae in inducing differentiation of microdendritic cells is not yet known.

Republic of Korea Patent No. 10-1790647

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

Accordingly, an object of the present invention is to provide a medium composition and a method for inducing differentiation of immature dendritic cell differentiation, which can effectively induce bone marrow-derived cells to differentiate into immature dendritic cells by including a red algae extract as an active ingredient.

The objects of the present invention are not limited to the objects mentioned above, 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 provides a medium composition for inducing differentiation of immature dendritic cells comprising red algae extract as an active ingredient.

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

In a preferred embodiment, the red algae is at least one of the crested red leaf ( Callophyllis japonica ) and the leaftail ( Gracilaria textorii ).

In a preferred embodiment, the extract differentiates bone marrow-derived cells into immature dendritic cells.

In addition, the present invention provides a method for inducing differentiation of immature dendritic cells comprising the step of treating a red algae extract in a culture medium of bone marrow-derived cells isolated from a living body.

In a preferred embodiment, the extract is a water-soluble extract of at least one of the crested leaf ( Callophyllis japonica ) and the leaftail ( Gracilaria textorii ).

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

Since the red algae extract of the present invention has the effect of effectively inducing bone marrow-derived cells to differentiate into immature dendritic cells at a concentration that does not have cytotoxicity, it is a pre-step of dendritic cells that can be used for various anticancer immunotherapy Immature dendritic cells can be effectively produced from bone marrow-derived cells in vitro.

These technical effects of the present invention are not limited to the above-mentioned ranges, and even if not explicitly mentioned, the effects of the invention that can be recognized by those skilled in the art from the description of specific contents for carrying out the invention described below are also Of course it is included.

1 is a graph showing the cytotoxicity measurement results of immature dendritic cells of mice 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 bone marrow-derived cells and immature dendritic cells, cultured by treating red algae extract according to an embodiment of the present invention.

The terminology used in the present invention is selected from the general terms that are currently widely used, but in certain cases, there are also terms that are arbitrarily selected by the applicant. In this case, the meanings described or used in the detailed description of the invention are not considered as simple names. Therefore, the 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 indicate the same components.

The technical feature of the present invention was devised in that the red algae extract has the effect of differentiating bone marrow-derived cells into immature dendritic cells, and immunity in various anticancer treatments using differentiation inducing activity to red immature dendritic cells, which is a new use It is to provide a medium composition for inducing immature dendritic cell differentiation and a method for inducing differentiation of immature dendritic cells that can easily prepare immature dendritic cells essential for producing dendritic cells having properties that enhance a reaction.

In other words, dendritic cells differentiate from precursor cells derived from bone marrow and exist as immature dendritic cells with high antigen phagocytosis, and when external stimuli and antigens are recognized, maturation of immature dendritic cells progresses to the cell surface. Since the expression of the molecule increases and the T lymphocytes migrate to a dense region and initiate an adaptive immune response, a large number of immature dendritic cells are essential to cause the adaptive immune response, but in a tumor microenvironment, various cell responses of tumor cells Because immature dendritic cells do not exist in sufficient numbers, it is difficult to produce an anti-tumor immune response even when inducing maturation. Developing a method for producing a large number of immature dendritic cells develops various immunological treatment methods such as cancer immunotherapy. Because it is important.

Therefore, the medium composition for inducing differentiation of immature dendritic cells of the present invention includes a red algae extract as an active ingredient.

This is because the red algae extract, as described above, increases the expression of CD11c, known as a marker of immature dendritic cells, in a concentration-dependent manner in a bone marrow-derived cell culture medium treated with a concentration that does not have cytotoxicity. As a result, when the medium composition of the present invention is processed into bone marrow-derived cells to induce immature dendritic cells and then induce maturation, it can be used to enhance the immune response in various immunological treatments such as anticancer treatment of mammals including humans.

The red algae for obtaining the red algae extract contained in the medium composition for inducing immature dendritic cell differentiation of the present invention is not limited to red algae, but in the embodiment of the present invention, the red algae extract is the red leaf ( Callophyllis japonica ) and the leaf tailer ( Gracilaria textorii ). One or more extracts were used.

The crested red leaf (Callophyllis japonica ) belongs to the family of the Californian family, and has been widely distributed in the south coast of Korea and consumed as food. According to the previous reports, the crested red leaf is known to have a protective effect against radiation irradiation to mice, and is also known to have an antioxidant effect and an antibacterial effect. The leaf cactus ( Gracilaria textorii ) belongs to the family Cacti, and the leaf cactus is distributed all over Korea and is also distributed in Japan, the East China Sea, and Indonesia. Leaf clover has also been consumed as a food product for a long time in Korea, and extracts of cactus are known to have anti-inflammatory effects on mouse macrophages. As such, the crested red leaves have antimicrobial, anti-oxidant, and protective effects against radioactivity, and leaf tailings are known to have anti-inflammatory effects, and so far, their efficacy in inducing differentiation of microcage cells is completely absent. It was not known.

The red algae extract used in the present invention can be separated using a conventional extraction solvent according to a conventional method known in the art for extracting an extract from natural products, that is, under conditions of conventional temperature and pressure. As an extraction solvent, a solvent that can be generally used in the extraction process can be used, such as water, anhydrous or hydrous lower alcohols having 1 to 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. As an embodiment, the red algae extract may be a water soluble extract of red algae, particularly an ethanol extract as described in the Examples.

The medium composition of the present invention may be used alone as a red algae extract, but may include a red algae extract in a basic medium used for cell culture such as DMEM, and various amino acids, vitamins, and inorganic salts commonly used in cell culture, if necessary. A mixture of nutrient mixtures (Nutrient Mixture), ascorbic acid 2-glucoside (AA2G), insulin-transferrin-selenium, EGF, PDGF-BB, human serum, etc. It may further include.

The concentration of the red algae extract, which is an active ingredient included in the medium composition of the present invention, may be included in a concentration necessary to differentiate bone marrow-derived cells into immature dendritic cells, and may be determined in consideration of cell conditions, required periods, etc. Although not limited, the medium composition may be included in an amount of 0.01 to 99% by weight.

Next, the method for inducing differentiation of immature dendritic cells of the present invention includes treating a red algae extract in a culture medium of bone marrow-derived cells isolated from a living body. This is because, when the red algae extract is included in the bone marrow-derived cell culture medium, bone marrow-derived cells are induced to differentiate into immature dendritic cells by the red algae extract. As an embodiment, the bone marrow-derived cells are obtained by lysing the separated bone marrow into single cells and lysing the red blood cells, and the red algae extract may be one or more water-soluble extracts of crested leaf ( Callophyllis japonica ) and leaftail ( Gracilaria textorii ). .

Example 1

1. Preparation of red algae extract

Crested red leaves ( Callophyllis japonica ) were collected in Wando in winter 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 powdered using a blender. 100 g of seaweed powder was added to 900 ml of 70% ethanol, stirred, and filtered on a 3 mm filter paper on the second day to collect 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 then all solvents were removed using a freeze dryer to obtain a powdery extract. The obtained extract was dissolved in sterilized tertiary distilled water and ethanol and used in cell experiments.

2. Bone-derived cell preparation

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

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

After removing the prepared bone marrow-derived cells again, the cells were cultured for 6 days in a medium containing GM-CSF and the cell culture solution was newly replaced every 2 days. During the 6-day culture period, the crested red leaf extract was treated with GM-CSF, and the culture medium containing the extract was newly replaced every 2 days. At this time, a medium composition 1 containing GM-CSF (5 ng / ml) and crest red leaf extract (50 ng / ml) was used. 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 the medium composition 2 containing GM-CSF (5 ng / ml) and leaf tailings extract (50 ng / ml) was used by using leaf custard ( Gracilaria textorii ) rather than crest red leaf. To obtain immature dendritic cells.

Example 3

GM-CSF (5 ng / ml), Crested Red Leaf Extract (25 ng / ml), and Leaf Cranberry Extract (25 ng) using not only the Crest red leaf but also the Crest red leaf and Gracilaria textorii at the same content. / ml) except for using the medium composition 3 was carried out in the same manner as in Example 1 to obtain immature dendritic cells.

Comparative Examples 1 to 3

Bone marrow-derived cells were differentiated into immature dendritic cells using a comparative preservation composition 1 to 3 composed of GM-CSF 5, 10, 20 ng / ml without red algae extract.

Experimental Example 1

Immature dendritic cells stained through Annexin / PI staining on the 6th day after inducing immature dendritic cells by treating medium compositions 1 to 3 each containing GM-CSF and red algae extract to bone marrow-derived precursor cells as in Examples 1 to 3 Cells were measured for cytotoxicity through a flow cytometer and the results are shown in FIG. 1. Data are representative of the results of four individual repeat experiments.

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

Experimental Example 2

Cells finally obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were present in the culture medium obtained on the 6th day to confirm that the phenotype of dendritic cells was displayed, that is, to determine whether the bone marrow-derived cells were differentiated into immature dendritic cells. The phenotype of the cells to be analyzed was analyzed as follows and the results are shown in FIG. 2. For the phenotype of immature dendritic cells, cell surface molecules were analyzed using flow cytometry.

Cells obtained on the 6th day of culture are washed twice with cold PBS. After treating the washed cells with FITC-attached anti-CD11c antibody (a marker of dendritic cells), they were 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. Fixed cells were measured using an FC500 (Beckman coulter) device, and then analyzed using kaluza software (Beckman coulter). Data are representative of the results of four individual repeat experiments.

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

Particularly, in the case of the medium composition 3 in which the precursor cells treated with 5 ng / ml of GM-CSF contain both the crest red leaf extract and the leaftail extract, the comparative culture medium composition consisting of 20 ng / ml of GM-CSF is treated. It was confirmed that CD11c was expressed at a level similar to that of the test.

From the results of these experiments, when the crested red leaf extract and the leaf tailings extract were treated simultaneously. It can be seen that the differentiation rate of dendritic cells is higher than when the crest red leaf extract and the leaf tailings extract are added to GM-CSF.

Therefore, the present invention shows that the red algae extract, particularly the crest red leaf and leaf tailings extract, has a differentiation-inducing activity that differentiates bone marrow-derived cells into immature dendritic cells, thereby enhancing the immune response during immunological treatment such as anti-cancer treatment. It can be seen that dendritic cells necessary for the use of the present invention can be easily prepared from bone marrow-derived cells.

The present invention has been illustrated and described with reference to preferred embodiments as described above, but is not limited to the above-described embodiments and is within the scope of the present invention without departing from the spirit of the present invention. By doing so, various changes and modifications will be possible.

Claims (7)

A medium composition for inducing differentiation of immature dendritic cells comprising red algae extract as an active ingredient.
According to claim 1,
The extract is a medium composition for inducing differentiation of immature dendritic cells, characterized in that it is a water-soluble extract of red algae.
According to claim 1,
The red algae is a medium composition for inducing differentiation of immature dendritic cells, characterized in that at least one of a crested red leaf ( Callophyllis japonica ) and a leaftail ( Gracilaria textorii ).
The method according to any one of claims 1 to 3,
The extract is a medium composition for inducing differentiation of immature dendritic cells, characterized in that the bone marrow-derived cells are differentiated into immature dendritic cells. A method for inducing differentiation of immature dendritic cells comprising the step of treating a red algae extract in a culture medium derived from bone marrow derived cells isolated from a living body.
The method of claim 5,
The extract is a method of inducing differentiation of immature dendritic cells, characterized in that it is a water-soluble extract of at least one of a crested red leaf ( Callophyllis japonica ) and a leaftail ( Gracilaria textorii ).
The method according to claim 5 or 6,
The bone marrow-derived cell is a method for inducing differentiation of immature dendritic cells, characterized by being obtained by lysing red blood cells after separating the separated bone marrow into single cells.

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