WO2017164659A2 - Method for examining ability of natural material to increase immunocompetence and method for providing customized diet using same - Google Patents

Abstract

The present invention relates to a method for examining individual subjects for immunocompetence wherein blood samples from individual subjects are added with antigen cells and assayed for the death of the antigen cells and the levels of cytokines including IFN-γ. The present invention relates to a method for examining a degree to which a natural material increases the immunocompetence of individual subjects, wherein blood samples from the individual subjects are added with antigen cells and the natural material and assayed for the death of the antigen cells and the level of cytokines including IFN-γ. The present invention relates to a method for providing customized diets for increasing the immunocompetence of individual subjects, comprising determining and selecting natural materials for immunopotentiating the individual subjects.

Description

Immunopotentiation Test of Natural Products and Method of Providing Customized Diet Using the Test Method

The present invention relates to a method for testing an individual's immune capacity by adding antigenic cells to blood of an individual and measuring the killing of antigenic cells and cytokines including IFN-γ.

The present invention, by adding antigen cells and natural material to the blood of the individual to measure the killing of antigen cells and cytokines including IFN-γ to a method that can examine the extent to which the natural material affects the individual's immune capacity enhancement It is about.

The present invention relates to a method for providing a customized diet for enhancing an individual's immunity after identifying and selecting a natural material for enhancing an individual's immunity.

The immune system consists of innate and acquired immunity. Congenital immune cells have evolved to take early defense by responding to pathogens with limited receptors, and acquired immune cells are driven by T cells, and have a wider variety of memory and specific responses to specific antigens of infectious organisms. The attack system has evolved to enable immediate and effective response to reinfection of the same pathogens.

Innate immune responses are important for inducing early immune responses as well as for determining the direction and intensity of responses. In addition, the acquired immune response itself acts to enhance the innate immune response, so that the two immune responses cooperatively form positive feedbacks to cope with infection effectively.

Innate immune responses and immunoregulatory cells regulate differentiation of acquired immune responses, that is, they are controlled to differentiate into Th1 responses that primarily represent cytotoxic responses or Th2 responses that mainly represent humoral responses, including antibody responses, depending on the nature of the infectious agent. Induces an immune response that is optimal for the removal of infectious organisms.

Natural killer cells (NK cells) , one of the innate immune cells, is known for its cytotoxicity (cytotoxicity) to cancer cells. In other words, NK cells play a key role in removing cancer cells and cells infected with viruses. It has also been found to play an important role in bone marrow transplantation, fetal implantation and fertility control.

In addition, NK cells play a key role in regulating immune responses through direct interactions with dendritic cells, macrophages, T cells, and indirect interactions with cytokines.

This process is outlined as a mechanism by which NK cells are activated by interferons (IFNs) or macrophages-derived cytokines. That is, when exposed to IFN-α, IFN-β or IL-12, one of the cytokines produced at the beginning of infection, its activity is amplified 20 to 100 times to produce IFN-γ. In other words, IFN- α and IFN-β synergize with IL-12, NK cells produce a large amount of IFN- γ, and this IFN- γ plays an essential role in controlling infection.

In addition, NK cells express various germ-line encoded immunoreceptors that induce or inhibit activity instead of lacking an antigen specific receptor. NK cells recognize target cells through these receptors and regulate their activity by a comprehensive signaling balance induced thereby.

Natural killer T cells ( NKT cells , Natual killer T cells) have less than 1% of the total lymphocytes, but have been known to play an important role in various immune responses and pathologies.

NKT is a major cell population that performs multifaceted immunomodulatory functions in a wide range of immune diseases. Many studies of NKT cells in the past have reported that natural or artificial activation of NKT suppresses these diseases in a variety of diseases, including autoimmune diseases, transplant rejection, infectious diseases, and cancer.

NKT cells, one of the immunoregulatory cells, are a heterogeneous group of T cells. They express NK cell markers, including NK cell receptors, on the cell surface. Unlike conventional T cell selection methods, CD 1d-glycolipid and Expresses the CD 1d-lipid complex. NKT cells are positively selected by CD4 ⁺ , CD8 ⁺ double positive (DP) thymic cells.

NKT cells  It has different properties from other cell populations Within a short time  With large amounts of IL-4 IFN secretes -γ, Th1 and On Th2 reaction  Central variety Cytokine  Secrete. As a result, T-cell Th1 Of Th2  Plays an important role in controlling differentiation.

ILC (innate lymphoid cells) is a newly discovered cell population in 2010 that is characterized by the morphological characteristics of lymphoid cells without the presence of antigen receptors such as T cell receptors or B cell receptors. Cell group with

Unlike T cells, which are responsible for the acquired immune response, congenital lymphocytes do not possess antigen specificity, but they are anatomically similar to T cells and are congenital in that they play a role in T cells. 'S discovery caused a big wave in academia.

Congenital lymphocytes respond rapidly to innate cytokines (IL-1, IL-33, IL-25) and are responsible for maintaining tissue homeostasis, repairing damaged tissues, and protecting against pathogens. Although studies on the characteristics and functions of congenital lymphocytes are still underway, congenital lymphocytes can be classified into three groups.

Type 1 congenital lymphocytes (ILC1) have a phenotype that is quite similar to natural killer cells. Until recently, natural killer cells were thought to be primitives of congenital lymphocytes, and they were included in ILC1 in a broad sense, but in 2013, type 1 congenital lymphocytes (ILC1) were found in the colon of patients with Crohn's disease. The two are now being studied separately.

Both natural killer cells and type 1 congenital lymphocytes require T-bet, a transcription factor, and secrete IFN-γ in the differentiation process, but type 1 congenital lymphocytes are granzyme secreted by natural killer cells. The difference is that they do not secrete cytotoxic substances such as perforin. Instead, type 1 congenital lymphocytes can secrete large amounts of IFN- γ in response to IL-7 and IL-12 .

Therefore, by analyzing natural killer cells (NK cells), natural killer T cells (NKT cells) and congenital lymphocyte cells (ILC) present in the blood, the current health status can be confirmed by analyzing the individual's immune capacity and activity. I would say.

Applicants focus on the fact that natural killer cells (NK cells), natural killer T cells (NKT cells) and congenital lymphocyte cells (ILCs) secrete and interact with various cytokines, but specifically secrete IFN-γ to regulate immunity. The present invention has been devised.

(1) It was conceived that an individual's immune capacity can be tested by injecting antigen cells into the blood of an individual and measuring the death of antigen cells and cytokines including IFN-γ.

(2) It is possible to test whether natural material affects the immune ability measured in (1) by adding antigen cells and natural material to individual's blood and measuring the death of antigen cells and cytokines including IFN-γ. The point was focused.

(3) After identifying and screening natural materials for enhancing the individual's immunity through the process of (1) and (2), it was conceived that a customized diet for enhancing the individual's immunity can be provided.

Hereinafter, a brief look at the prior art related to the present invention. First, Korean Patent Publication No. 10-2011-0010030 discloses an 'immunity test system for food selection'.

Schematically, collecting the blood of the individual, separating and testing the immune cells from the collected blood to measure the activity of the immune cells; Injecting and culturing various foods into the immune cells; Inspecting the cultured cells to measure activity and confirm improvement effects for each food; It includes the step of selecting the best foods that have an effect on the immunity for each individual.

However, since the prior art does not substantially test the function on antigen cells, it is difficult to test immunity based on innate immunity and acquired immune mechanism. In addition, because the specific immune cells are isolated and cultured, efficiency problems may be raised in terms of test methods.

Secondly, Korean Patent Publication No. 10-2004-0047899 discloses 'a novel method for measuring immunological activity'.

In brief, methods for measuring NKT cells, NK cells, CD8, perforin producing cells, IL-12 and IFN-γ are shown in the detailed description.

Although the prior art has a similar aspect to the present invention in that it measures immune cells and cytokines, the overall function of antigen cells is not measured by measuring each factor separately, and thus immunity based on innate and acquired immune mechanisms. The ability test can be said to be difficult.

The problem to be solved by the present invention is to provide a method for testing an individual's immune capacity by adding antigenic cells to individual blood and measuring the killing of antigenic cells and cytokines including IFN-γ.

The problem of the present invention is to add the antigen cells and natural material to the blood of the individual to measure the killing of antigen cells and cytokines including IFN-γ to examine the extent to which the natural material affects the individual's immune capacity enhancement. To provide a way.

The problem of the present invention is to provide a method for providing a customized diet for enhancing the individual's immunity after identifying and selecting a natural material for enhancing the individual's immunity.

The present invention comprises the steps of: a) collecting blood;

b) culturing by adding antigen cells to the blood;

c) analyzing the cultured blood and measuring the concentration of cytokines including killing of antigen cells and IFN-γ;

d) culturing by adding antigen cells and natural material to the blood of step a);

e) analyzing the cultured blood in step d) to determine the death of antigen cells and the concentration of cytokines including IFN-γ; And

Comprising: f) comparing the measurement results of step c) and the measurement results of step e); to provide a method for testing the immune augmentation of natural materials, to solve the technical problem.

The present invention comprises the steps of performing the steps d) to f) on a plurality of natural products, respectively, analyzing the immune enhancing effect of each natural material; Providing a personalized diet providing method for immunological enhancement, comprising: providing a personalized diet based on the analysis result, to solve the technical problem.

The immunoassay method of the natural product material according to the present invention has a remarkable effect of providing an immunocompetence test method based on innate immunity and acquired immune mechanisms by examining the action on antigen cells.

Immunoassay of natural products according to the present invention, the remarkable effects that can be examined including the enhanced activity of natural killer cells (NK cells), natural killer T cells (NKT cells) and innate lymphoid cells (Innate lymphoid cells) Holds.

The method of providing a customized diet according to the present invention has a remarkable effect of providing an optimal immune enhancing diet for each individual after examining the immune enhancement of each natural material.

1 is a diagram schematically illustrating a method for testing an individual's immune capacity.

Figure 2 is a schematic diagram illustrating a method for testing the immune capacity enhancement of natural materials.

Example  1. Immunopotentiation Test of Natural Products

1 is a view schematically showing a method for testing an individual's immune capacity, Figure 2 is a diagram schematically showing a method for testing the immune capacity enhancement of a natural material.

1) testing the individual's immune capacity

1-1) Blood Collection Step

Collect blood from a healthy person or sick person. Here, the sick includes a cancer patient or a person infected with a virus or the like.

1-2) culturing by adding antigenic cells to blood

Antigen cells are added and cultured to normal human blood, and the blood of the sick is cultured according to the characteristics of the antigen cells. The antigen cells to be injected and the antigen cells of the sick are subject to floating cells. This is because adherent cells cannot be used because NK cells are floating cells.

For example, the antigen cells to be added may be human cancer cells, preferably K562 cells. K562 cells are a cell line derived from a patient with chronic myelogenous leukemia, and are originally Philaderphia chromosome positive (Ph ⁺ ), bcr-Abl positive cells. It shows pluripotent stem cell-like properties that differentiate into erythrocytes, granulocytes, and monocytes upon stimulation.In addition to its use in stem cell research, it has high susceptibility to cytotoxicity by NK cells. Can be.

In addition, DMEM and RPMI medium containing 10% FBS, 1% penicilin and streptomycin are used for culturing human cancer cells. Measure the number of cells at 1 × 10 ⁸ cells / mL using a hemocytometer, and incubate in T25 cell culture flasks for 24 hours at 37 ° C., 5%, CO ₂ incubator. Subcultures should be done once every two to three days. However, the culture conditions or the duration of the culture may be changed depending on the characteristics of the antigen cells.

1-3) Cell death ability and cytokine measurement step

The cultured blood is analyzed to determine the death of antigen cells and cytokines including IFN-γ.

The killing assay of antigen cells can be made by measuring the cell proliferation rate using MTT assay.

MTT (3- [4,5-dimethyl thiazol-2-yl] -2,5-yl tetrazolium bromide) assay is a measure of the toxicity and viability of cells. The yellow water-soluble substance MTT is responsible for the dehydrogenase activity in mitochondria. It uses the principle of producing purple formazan that is insoluble in water. To this end, each cell line is added 200μL to 96well at the concentration of 1 × 10 ⁴ cells / well and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours to stabilize. Prepared extracts are treated in medium at an appropriate concentration and incubated for 48 hours. MTT (3- [4,5-dimethyl thiazol-2-yl] -2,5-yl tetrazolium bromide) assay reagent was added to the medium and reacted at 37 ° C., 5% CO ₂ incubator for 4 hours, followed by MTT reaction solution. Remove it. In order to dissolve the generated formazan, 200 μL of DMSO was treated and the absorbance was measured at 540 nm with an ELISA spectrometer.

In addition, the concentration change of cytokines including IFN-γ may be performed by ELISA technique. Dilute purified anti-human cytokines (IFN-γ) monoclonal antibodies in a 96 well plate at 2 μg / mL in binding buffer (0.1 M sodium phosphate buffer, pH 9.0), 50 μL / well, and leave at 4 ° C overnight. do. After washing with PBS / Tween, blocking buffer (1% BSA / PBS) was added to 200 μL / well and left at 37 ° C. for 2 hours. After washing, 100 μL of the supernatant collected by treating with standard cytokine and extracts was left at 4 ° C. overnight. After washing with PBS / Tween, the biotinylated anti-cytokine detection antibody was diluted in blocking buffer / Tween and treated with 100 μL / well and left at 37 ° C for 1 hour. After washing with PBS / Tween again, streptavidin-HRP diluted to 1/1000 was diluted in blocking buffer / Tween at 100 μg / well and left for 1 hour before washing. 15 minutes after the TMB solution was added at 100 μg / well, 1 M HCl was added at 50 μL / well as a stop reagent, and the absorbance was measured at 450 nm using an ELISA spectrometer.

Depending on design conditions, other methods may be used for techniques for measuring the killing of antigen cells and cytokines including IFN-γ.

1-4) Immunity test step

The killing of antigen cells is analyzed and the difference between the concentration of cytokines analyzed in the blood of normal humans and the concentration of cytokines after injection of antigen cells is analyzed.

Subsequently, the values presented in the medical database may be utilized or a separate reference condition may be prepared to score immunity according to the death of antigen cells and changes in cytokine concentration.

2) step of examining the immune enhancement of natural products

2-1) Blood Collection Step

The same procedure as in 1-1) above is performed.

2-2) incubating antigen cells and natural material into blood

Perform the same as in step 1-2) above, but additional natural material is added to the blood.

Here, the ingredient of the natural product material may be a herbal preparation or a food preparation or a main ingredient of the food. The natural product material may be an extract or concentrate extracted using hot water, ethanol, fermentation or fractions in the case of herbal preparations, and may be a sample obtained by pulverizing, powdering and dissolving foods in the case of food preparations or main ingredients of foods.

2-3) Apoptosis and Cytokine Measurement

Perform the same procedure as in step 1-3).

2-4) Examination of Immune Enhancement

Perform the same as in step 1-4), but compare the results of step 1-4) to examine the immune enhancing ability of the natural material. In other words, if the killing of antigen cells and the secretion of cytokines including IFN-γ were increased than the results analyzed in step 1-4), it can be said that the immune enhancing ability of the natural material to be tested was confirmed.

After this, the optimal concentration may be analyzed by repeating steps 2-1) to 2-4) according to the concentration of the same natural material.

In addition, by performing steps 2-1) to 2-4) under the same conditions on the plurality of natural materials, the optimal natural materials may be analyzed.

Example  2. How to provide a customized diet using natural immunoassay

According to Example 1, steps 1-1) to 1-4) are performed to measure the individual's immune capacity. By repeating steps 2-1) to 2-4) for various natural materials, an optimal natural material for each individual can be selected. Thereafter, using the selected natural product material to provide a personalized diet.

Claims (5)

1. a) collecting blood;

   b) injecting the antigen cells into the blood and culturing;

   c) analyzing the cultured blood and measuring the concentration of cytokines including killing of antigen cells and IFN-γ;

   d) culturing by adding antigen cells and natural material to the blood of step a);

   e) analyzing the cultured blood in step d) to determine the death of antigen cells and the concentration of cytokines including IFN-γ; And

   and f) comparing the measurement result of step c) with the measurement result of step e).
2. The method according to claim 1,

   When the blood is the blood of the sick, steps of b) and d) characterized in that the antigen cells are not added, the method for testing the immune enhancement of the natural material.
3. The method according to claim 1,

   The antigen cell is characterized in that the floating cells, a method for examining the immune enhancement of the natural material.
4. The method according to claim 1,

   Immunity enhancement of the natural material, characterized in that to enhance the activity of natural killer cells (NK cells), natural killer T cells (NKT cells) and innate lymphoid cells (Innate lymphoid cells), How to check.
5. Analyzing the immuno-enhancing effect of each natural material by performing steps d) to f) of each of the natural material; And providing a personalized diet based on the analysis result.

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