KR102066109B1 - A method for screening materials for suppressing immunocyte migration using exosomes - Google Patents

Abstract

The present disclosure discloses a method for screening a substance for inhibiting immune cell migration using exosomes. The method comprises the step of measuring the protein expression level of the vascular endothelial cells after treatment with vascular endothelial cells isolated from Staphylococcus sp . Strain, the method inhibits immune cell migration This has the effect of screening the material accurately and quickly.

Description

A method for screening a substance for inhibiting immune cell migration using exosomes {A METHOD FOR SCREENING MATERIALS FOR SUPPRESSING IMMUNOCYTE MIGRATION USING EXOSOMES}

The present disclosure discloses a method for screening a substance for inhibiting immune cell migration using exosomes.

A characteristic of inflammatory diseases is the accumulation of activated immune cells in affected tissues. The process by which immune cells migrate from blood vessels to sites of inflammation involves the interaction cascade of immune cells with vascular endothelial cells [Springer, T., Ann. Rev. Physiol., 57, 827, 1995]. Vascular cells include vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin (endothelial cells). By selectively expressing specific adhesion molecules, such as selectin), the type of leukocytes (monocytes, lymphocytes or neutrophils) is determined.

On the other hand, most animal cells have the ability to secrete extracellular vesicles of cellular origin of various sizes and components, which include extracellular vesicles, including blood, urine, saliva and cell culture. It is found in all biological fluids. Prokaryotic or eukaryotic cells are also known to secrete extracellular vesicles.

Extracellular vesicles are membrane structure vesicles ranging in size from about 20 nm in diameter to about 2 μm in diameter, and are heterogeneous in size and composition, and include exosomes, ectosomes, and microvesicles. and many different species such as microvesicles, microparticles, and the like. An exosome is a 20-400 nm membrane structure vesicle that is secreted from a cell, and binds to other cells and tissues to deliver membrane components, proteins (growth hormones, cytokines, etc.), RNA, etc. It plays a variety of roles.

Korean Patent Publication No. 10-1146821

In one aspect, the present disclosure finds that exosomes from the strain of Staphylococcus sp . Increase the expression of adhesive proteins in vascular endothelial cells to induce immune cell migration, thereby improving accuracy and rapidity. It is an object of the present invention to provide a method for screening the improved immune cell migration inhibitor.

In one aspect, the techniques disclosed herein comprise (1) culturing a Staphylococcus sp . Strain; (2) separating the exosomes from the culture medium of the strain; And (3) treating the isolated exosomes in vitro with vascular endothelial cells, and then measuring a protein expression level of the vascular endothelial cells.

In an exemplary embodiment, the Staphylococcus sp . Strain in step (1) may be Staphylococcus aureus .

In an exemplary embodiment, the exosome in the step (2) may be one having an average diameter of 20 to 100 nm.

In an exemplary embodiment, the time for treating the exosomes in vascular endothelial cells in step (3) may be 2 hours or more.

In an exemplary embodiment, the immune cell migration inhibitor may be to inhibit the migration of immune cells in vascular endothelial cells.

In an exemplary embodiment, the vascular endothelial cells may be human skin derived vascular endothelial cells.

In an exemplary embodiment, the substance for inhibiting immune cell migration may be to inhibit protein expression of vascular endothelial cells.

In an exemplary embodiment, the protein of the vascular endothelial cell may be one or more selected from the group consisting of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (IMC-1), and E-selectin. have.

In an exemplary embodiment, step (1) may include culturing the test substance to the strain.

In an exemplary embodiment, step (2) may include separating the exosomes after treating the test material in the culture medium of the strain.

In an exemplary embodiment, step (3) may comprise treating the isolated exosomes with vascular endothelial cells along with the test substance.

In an exemplary embodiment, the method selects a test substance that reduces the protein expression level of vascular endothelial cells as a substance for inhibiting immune cell migration, compared to the protein expression level of vascular endothelial cells of the control group not treated with the test substance. It may be to include the step.

In one aspect, the technique disclosed herein uses an exosome from a Staphylococcus sp . Strain to screen for substances that inhibit migration of immune cells that occur in the early stages of the immune response, thereby initiating an immune response. There is an effect that can screen the substances that can prevent or inhibit the hyperimmune reaction in the step.

In another aspect, the technology disclosed herein has the effect of screening the substance for inhibiting immune cell migration accurately and quickly.

Figure 1 shows the results of analyzing the size of the exosomes isolated from Staphylococcus aureus in one test example of the present specification.
Figure 2a and 2b shows the results of confirming the effect of exosomes on the gene and protein expression of the adhesion protein by treating the exosomes in the skin vascular endothelial cells in one test example of the present specification.
Figure 3 compares the effect on the gene expression of the adhesion protein by treating the exosomes and Staphylococcus aureus extract to skin vascular endothelial cells for 4 to 8 hours in one test example of the present specification, respectively The results are shown.
Figure 4 compares the effect of the exosomes and Staphylococcus aureus extract on the skin vascular endothelial cells for 48 hours in one test example of the present disclosure on the gene expression of the adhesion protein It is shown.
Figure 5 shows the result of comparing the effect of the exosomes isolated from the various strains in the test example of the present invention on the gene expression of the adhesion protein of the skin vascular endothelial cells.
Figure 6 shows the results confirming the effect of the substances that inhibit the major signaling system of the immune response in the adhesion protein gene expression of skin vascular endothelial cells by exosomes in one test example of the present specification.

Hereinafter, the present invention will be described in detail.

In one aspect, the techniques disclosed herein comprise (1) culturing a Staphylococcus sp . Strain; (2) separating the exosomes from the culture medium of the strain; And (3) treating the isolated exosomes to vascular endothelial cells, and then measuring a protein expression level of the vascular endothelial cells.

The concentration of immune cells into an immune response occurs as the immune cells move through blood vessels. In order for this reaction to occur, the adhesion proteins of vascular endothelial cells must be overexpressed in the early stages of the reaction to move immune cells into the blood vessels. Therefore, reducing the expression of adhesion proteins in vascular endothelial cells may reduce or delay the immune response, and may result in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E- in blood vessels. Increased selectin protein expression indicates an immune response.

In an exemplary embodiment, the Staphylococcus sp . Strain in step (1) may be Staphylococcus aureus .

In an exemplary embodiment, the exosomes in the step (2) may have an average diameter of 20 to 100 nm, 20 to 90 nm, 30 to 80 nm or 40 to 70 nm.

In an exemplary embodiment, the time for treating the exosomes in vascular endothelial cells in step (3) may be 2 hours or more. In another exemplary embodiment, the time for treating the exosomes to vascular endothelial cells is 2 hours or more, 3 hours or more, or 4 hours or more and 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours. Or less than 5 hours or less than 4 hours. For example, it can be 2 to 4 hours, 4 to 8 hours, 4 to 6 hours or 6 to 8 hours.

In an exemplary embodiment, the immune cells are T lymphocytes (T cells), B lymphocytes (B cells), neutrophils (Neutrophil), eosinophils (Eosinophil), basophils (Basophil), monocytes (Macrophage) It may be one or more selected from the group consisting of natural killer cells (Natural Killer cells) and natural killer T cells (Natural Killer T cells).

In an exemplary embodiment, the immune cell migration inhibitor may be to inhibit the migration of immune cells in vascular endothelial cells.

In an exemplary embodiment, the vascular endothelial cells may be human skin derived vascular endothelial cells.

In an exemplary embodiment, the substance for inhibiting immune cell migration may be to inhibit protein expression of vascular endothelial cells.

In an exemplary embodiment, the protein of the vascular endothelial cell may be one or more selected from the group consisting of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (IMC-1), and E-selectin. have.

In an exemplary embodiment, the step of measuring the expression level of the protein is a process of confirming the presence or / and expression level of the protein or a gene encoding the same, and immunoprecipitation, radioimmunoassay (RIA) ELISA, immunohistochemistry, immunofluorescence staining, 2D electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS Liquid Chromatography-Mass Spectrometry / Mass Spectrometry (MS / MS), Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF), Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry (SELDI-TOF), RT -PCR, Western Blotting, Northern Blotting, Protein Chip Analysis and Flow Cytometry (FACS) may be appropriately selected by those skilled in the art. All.

In an exemplary embodiment, the immune cell migration inhibitor may be an active ingredient of a pharmaceutical composition for preventing or treating hyperimmune reactions.

In an exemplary embodiment, the immune cell migration inhibitor may be an active ingredient of the cosmetic composition for improving the immune response.

In an exemplary embodiment, step (1) may include culturing the test substance to the strain.

In an exemplary embodiment, step (2) may include separating the exosomes after treating the test material in the culture medium of the strain.

In an exemplary embodiment, step (3) may comprise treating the isolated exosomes with vascular endothelial cells along with the test substance.

In an exemplary embodiment, the method selects a test substance that reduces the protein expression level of vascular endothelial cells as a substance for inhibiting immune cell migration, compared to the protein expression level of vascular endothelial cells of the control group not treated with the test substance. It may be to include the step.

The method has an effect of improving the accuracy of the screening by determining the extent to which the movement of immune cells in the vascular endothelial cells by the exosome in the early stage of the immune response is controlled by the test substance.

In addition, the method has the effect of improving the accuracy of the screening by evaluating the activity of the test substance to regulate the production or secretion of the exosomes that induce an immune response by treating the test substance to the strain.

In another aspect, the present disclosure provides a substance selection kit for inhibiting immune cell migration using the screening method.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Test Example 1. Staphylococcus aureus ( Staphylococcus aureus Isolation of Exosomes from

Staphylococcus aureus (Staphylococcus aureus; ATCC 6538) was inoculated in BHI medium, incubated at 37 ° C. for 72-96 hours and the culture was harvested. The collected culture solution was centrifuged at 4 ° C. and 3,000 × g for 10 minutes to precipitate bacteria, and the supernatant was collected and centrifuged at 4 ° C. and 10,000 × g for 20 minutes. The supernatant was collected and the remaining bacteria were removed using a 0.45 μm bottle-top filter, and the solution passed through the filter was collected and ultracentrifuged at 4 ° C. and 150,000 × g for 3 hours. Thereafter, the precipitated exosomes were dissolved in HEPES-buffered saline, and the size of the exosomes was measured as follows.

The size distribution of exosomes was measured by dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, U.K.). Size distribution based on relative frequency was measured by infrared light (wavelength = 633 nm) through the sample at scattering intensity for 10 × 30 s. As shown in Figure 1, the exosomes were confirmed to have a size distribution of 20 to 400 nm and an average diameter of 20 to 100 nm.

Test Example 2 Effect of Exosomes on Gene and Protein Expression of Adhesion Proteins in Skin Vascular Endothelial Cells

Human dermal microvascular Endothelial cells (HDMEC) were cultured in a 37 ° C. incubator in a medium containing SupplementMix (PromoCell # 39226) in Endothelial Cell Growth Medium MV2. After 24 hours, 100 ng / ml of the exosome isolated in Test Example 1 was treated, and the cells were harvested at 90 minutes to 6 hours later, RNA was isolated, and cDNA was synthesized through RT-PCR (reverse transcriptional polymerase chain reaction). . Taqman real-time PCR was performed using the synthesized cDNA to confirm gene expression levels of adhesion proteins ICAM-1, VCAM-1, and E-SEL. In addition, 6 hours after the exosome treatment, the protein of the skin vascular endothelial cells was extracted and the protein expression levels of the adhesion proteins ICAM-1, VCAM-1, and E-SEL were confirmed by Western blotting and are shown in FIG. 2B.

As a result, it was confirmed that exosomes isolated from Staphylococcus aureus increase the expression of genes and proteins of adhesion proteins ICAM-1, VCAM-1, and E-SEL in cutaneous vascular endothelial cells.

Test Example 3 Exosomes and Staphylococcus aureus ( Staphylococcus aureus Comparison of Effects of Extracts on Gene Expression of Adhesion Proteins in Skin Vascular Endothelial Cells

Staphylococcus aureus extract was prepared and used as follows.

Specifically, Staphylococcus aureus ( Staphylococcus aureus ) was inoculated in BHI medium, incubated at 37 ℃ for 72-96 hours and the collected culture solution was sonicated, and then 15 ℃ at 4 ℃, 13,000 × g 15 The cells were precipitated by centrifugation for minutes, and the precipitated bacteria were dissolved in water to measure the amount of protein.

After culturing the skin vascular endothelial cells in the same manner as in Test Example 2, after 24 hours, 100 ng / ml of the exosomes isolated from Test Example 1 and 100 ng / ml of the prepared strain extract were treated for 4 to 8 hours, respectively. RNA was isolated by harvesting cells over time and cDNA was synthesized through reverse transcriptional polymerase chain reaction (RT-PCR). Taqman real-time PCR was performed using the synthesized cDNA to confirm gene expression levels of adhesion proteins ICAM-1, VCAM-1, and E-SEL. In addition, after treating the exosomes and strain extracts to the skin vascular endothelial cells for 48 hours, the cells were harvested to compare gene expression levels of the adhesion proteins ICAM-1, VCAM-1, and E-SEL. .

As a result, the exosomes significantly increased the gene expression levels of the adhesion proteins ICAM-1, VCAM-1, and E-SEL in a faster time than the same amount of the extract of the strain, thereby speeding and accuracy of the screening method using the exosomes. It was confirmed that can be improved. That is, the exosomes derived from Staphylococcus aureus strains have a very fast and responsive effect on vascular endothelial cells and have been confirmed that the screening method according to the present specification provides further improved accuracy and speed. .

Test Example 4 Comparison of the Effect of Exosomes Isolated from Various Strains on Gene Expression of Adhesion Proteins of Skin Vascular Endothelial Cells

Exosomes were isolated from the culture medium of Propionibacterium acnes , Staphylococcus aureus and Lactobacillus plantarum in the same manner as in Test Example 1, and the effects of these on the expression of adhesion proteins of skin vascular endothelial cells were compared.

After culturing the skin vascular endothelial cells in the same manner as in Test Example 2, after 24 hours, each treated with 100ng / ml of exosomes, and after 4 hours, the cells were harvested to separate RNA and subjected to reverse transcriptional polymerase chain reaction (RT-PCR). CDNA was synthesized. Taqman real-time PCR was performed using the synthesized cDNA to confirm the gene expression level of the adhesion protein ICAM-1 and is shown in FIG. 5.

As a result, only some Staphylococcus aureus derived exo shown to increase the expression of adhesion proteins was confirmed that the specific bit Staphylococcus aureus derived exo typically leads to migration of immune cells. The same amount of exosomes isolated from other strains did not appear to increase the expression of adhesion proteins in vascular endothelial cells.

Test Example  5. Exosomes  Substances that inhibit the major signaling system of immune response activated by treatment On exosomes  Effect on Gene Expression of Adhesion Proteins in Skin Vascular Endothelial Cells

Staphylococcus aureus ( Staphylococcus aureus ) was used to isolate exosomes from the culture medium of the strain in the same manner as in Test Example 1. In addition, after culturing the skin vascular endothelial cells in the same manner as in Test Example 2, BAY 11-7082 (NF-kB inhibitor) and SP600125 [c-Jun N, which are substances that inhibit the main signaling system of the immune response after 24 hours -terminal kinase (JNK) inhibitor] were pretreated for 1 hour each, and 100ng / ml of the isolated exosomes were treated for 6 hours, followed by harvesting cells to separate RNA and reverse transcriptional polymerase chain reaction (RT-PCR). CDNA was synthesized. Taqman real-time PCR was performed using the synthesized cDNA to confirm gene expression levels of adhesion proteins ICAM-1, VCAM-1, and E-SEL.

As a result, it was confirmed that the expression of the adhesion protein was significantly reduced in skin vascular endothelial cells treated with exosomes and a substance that suppresses the immune response, compared with only exosomes.

Test Example 6. Statistical analysis

Statistical analysis herein was analyzed using GraphPad Prism software (GraphPad Software). Data are shown as mean ± SD. P-values were calculated using unpaired two-tailed Student's t-test, and P <0.05 was considered significant (*: P <0.05 , **: P <0.01 , ***: P <0.001 ).

As described above, specific portions of the present disclosure have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

(1) culturing the strain of Staphylococcus sp .
(2) separating the exosomes from the culture medium of the strain; And
(3) treating the isolated exosomes to vascular endothelial cells and then measuring the protein expression level of the vascular endothelial cells;
The vascular endothelial protein is at least one of VCAM-1 (vascular cell adhesion molecule-1) and E-selectin,
A method for screening a substance for inhibiting immune cell migration, which screens a substance for inhibiting immune cell migration in vascular endothelial cells.
The method of claim 1,
Staphylococcus sp . Strain in step (1) is Staphylococcus aureus ( Staphylococcus aureus ), a method for screening a substance for inhibiting immune cell migration.
The method of claim 1,
Exosome in the step (2) has an average diameter of 20 to 100 nm in size, the method for screening immune cell migration inhibitory material.
The method of claim 1,
In the step (3), the exosomes are treated in vascular endothelial cells for more than 2 hours, the method for screening a substance for inhibiting immune cell migration.
delete The method of claim 1,
The vascular endothelial cells are human skin-derived vascular endothelial cells, a method for screening a substance for inhibiting immune cell migration.
The method of claim 1,
The immune cells include T lymphocytes (T cells), B lymphocytes (B cells), neutrophils (Neutrophil), eosinophils, Basophils, monocytes, macrophages, macrophages, and natural killer cells. cell) and natural killer T cells (Natural Killer T cell) is a method for screening a substance for inhibiting immune cell migration that is at least one selected from the group consisting of.
The method of claim 1,
The immune cell migration inhibitory material is to inhibit the expression of vascular endothelial cells, a method for screening immune cell migration inhibitory material.
delete The method of claim 1,
Step (1) comprises screening a substance for inhibiting immune cell migration, comprising culturing the test substance to the strain.
The method of claim 1,
Step (2) is a method for screening a substance for inhibiting immune cell migration, comprising separating the exosomes after treating the test substance in the culture medium of the strain.
The method of claim 1,
Step (3) is a method for screening a substance for inhibiting immune cell migration, comprising treating the isolated exosomes with vascular endothelial cells along with the test substance.
The method according to any one of claims 10 to 12,
The method comprises the step of selecting a test substance for reducing the level of protein expression of vascular endothelial cells as a substance for inhibiting immune cell migration, compared to the degree of protein expression of vascular endothelial cells of the control group not treated with the test substance, A method for screening a substance for inhibiting cell migration.

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