KR20170135202A - A method for screening anti-acne meterials - Google Patents

Abstract

In the present specification, a method for screening an anti-acne substance through an exosome secretion amount derived from acne causing bacteria is disclosed. The method comprises a step for confirming an amount of exosome that is isolated from a causative agent of acne after treating a test substance with the causative agent of acne, wherein the causative agent of acne may be propionibacterium acnes. The method has the effect of screening anti-acne substances that reduce the secretion of exosomes from acne causing bacteria.

Description

METHOD FOR SCREENING ANTI-ACNE METERIALS

In this specification, a method for screening an anti-acne substance through an exosome secretion amount derived from acne causing bacteria is disclosed.

Most animal cells have the ability to secrete extracellular vesicles of intracellular origin with varying sizes and components and these extracellular vesicles are all biological (including blood, urine, saliva, and cell cultures) Are found in biological fluids (Loyer X, Vion AC, Tedgui A, Boulanger CM. Microvesicles as cell-cell messengers in cardiovascular diseases. Circ Res 2014; 114: 345-53) (Ohno S, Ishikawa A, Kuroda M Roles of exosomes and microvesicles in disease pathogenesis. Adv Drug Deliv Rev 2013; 65: 398-401).

The extracellular follicle is a membrane-structured oocyte having a diameter of about 20 nm to about 5 μm in diameter, and is heterogeneous in its size and composition. Exosome (about 30-100 nm), ectosome a number of different species such as ectosomes, microvesicles (about 100-1,000 nm), microparticles, apoptotic bodies (about 1-5 μm), and the like.

The different types of extracellular vesicles can be determined by their origin, diameter, density in sucrose, shape, sedimentation rate, lipid composition, protein marker or secretion mode (i.e. signal (inducible) or spontaneous ). For example, the micro-vesicle is a membrane vesicle with an irregular shape of about 100 to 1,000 nm, which emerges toward the outside of the plasma membrane (originating from the plasma membrane) and has a phospholipid containing an integrin, selectin, CD40 ligand, phosphatidylserine . On the other hand, exosomes are the smallest membrane vesicles having a cup shape of about 30 to 100 nm (<200 nm) and originate from endosomes (originating from the endosomes) of the late endosomes and contain CD63, CD9 tetraspanin, TSG101, It is known to have a lipid comprising a marker comprising ESCRT, cholesterol, sphingomyelin, ceramide, phosphatidylserine.

These extracellular follicles reflect the state of secretory origin cells (donor cells), exhibit various biological activities depending on the secretion from certain cells, transfer genetic material and proteins between cells, and play important roles in intercellular interactions .

Procaryotic or eukaryotic cells are also known to secrete extracellular vesicles (Camussi, G., Deregibus, MC, Bruno, S., Cantaluppi, V., & Biancone, L. (2010). Exosomes / microvesicles as a mechanism of the cell-to-cell communication. Kidney international, 78 (9), 838-848, Bang, Claudia, and Thomas Thum. 2012): 2060-2064. Kim, DK, Lee, J., Simpson, RJ, Lotvall, J., & Gho, YS (2015, April), EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research. Gram-negative and Gram (1), Gram-negative and Gram-negative, and Gram-negative and Gram- -positive bacterial extracellular vesicles, In Seminars in cell &amp; developmental biology (Vol.40, pp. 97-104), Academic Press).

On the other hand, acne is an inflammatory disease of the hair follicle that occurs mainly on the face, chest, neck and shoulders of the skin. The cause of acne is excessive secretion of sebum due to hormonal action, exfoliation of the keratinocytes of the sebaceous gland, It is known as a complex action by acne bacteria. As the inner wall of the sebaceous tube becomes thicker due to abnormal exaggeration of the follicular wall in the sebaceous gland, the sebaceous gland is blocked and the secretion of sebum is increased, so that sebum generated continuously accumulates in the sebaceous gland and promotes the occurrence of acne. As the acne bacterium grows vigorously in this area, it develops as a painful acne vulgaris.

The importance of acne causing bacteria in the development of acne has been acknowledged, and based on this, a method of screening anti-acne substances through the activity of inhibiting the growth of acne causative bacteria has been developed, but the activity of regulating the production of exosome secreted by acne causing bacteria A method for screening an anti-acne substance through the use of an anti-acne agent has not been disclosed.

Korean Patent Laid-Open No. 10-2012-0015021

In one aspect, the present disclosure aims to provide a method for screening anti-acne substances using exosomes derived from acne causing bacteria.

In one aspect, the technique disclosed herein includes the steps of treating a test agent with acne causing bacteria; Separating the exosome from the causative acne bacterium treated with the test substance; And confirming the amount of the exosome that has been separated. The present invention also provides a method for screening an anti-acne substance.

In one exemplary embodiment, the acne causing agent may be Propionibacterium acnes .

In an exemplary embodiment, the acne causing bacteria may be treated with the test substance and cultured for 72 to 96 hours.

In an exemplary embodiment, the step of isolating the exosomes comprises centrifuging the culture medium of acne causing bacteria treated with the test substance at 2,000 to 20,000 x g, collecting the supernatant, and centrifuging at 100,000 to 200,000 x g .

In an exemplary embodiment, the centrifugation may comprise centrifuging at 2,000 to 5,000 x g and then centrifuging at 8,000 to 20,000 x g.

In an exemplary embodiment, the step of separating the exosomes may include centrifuging the culture medium of acne causing bacteria, filtering the resultant with a filter of 0.2 to 0.5 mu m, and then ultracentrifuging.

In an exemplary embodiment, the isolated exosome may have a diameter of 20 to 500 nm.

In an exemplary embodiment, the isolated exosomes may be precipitated in ultracentrifugation of 100,000 to 200,000 x g of the acne-causing bacteria culture.

In an exemplary embodiment, the amount of the isolated exosomes may comprise comparing the amount of exosomes isolated from the untreated acne causative organism to the test substance.

In an exemplary embodiment, the test substance may be evaluated as an anti-acne substance when the amount of the separated exosome is less than the amount of exosome isolated from acne causal organisms not treated with the test substance.

In one aspect, the technique disclosed in this specification has an effect of providing a method of screening an anti-acne substance using a secretion amount of exosomes exocytically released from acne causing bacteria as a marker for screening of anti-acne substances.

FIG. 1 shows the size (FIG. 1A) and transmission electron microscope (FIG. 1B) of exosomes isolated from acne causing bacteria in one test example of the present specification.
Figure 2 shows the expression of the inflammatory cytokine gene (Fig. 2a) and protein (Fig. 2b) upon treatment of keratinocytes (NHEK) with exosomes (1-10 g / ml) in one test example of the present specification.
Figure 3 shows the increase in cell proliferation upon treatment of exosomes (1 to 20 占 퐂 / ml) on keratinocytes (HaCaT cells) in one test example of the present specification.
Figure 4 shows the expression of the genes of the cell differentiation markers (Figure 4a) and protein (Figure 4b) upon treatment with exogenous (1-10 mu g / ml) keratinocytes (NHEK) .

Hereinafter, the present invention will be described in detail.

In one aspect, the technique disclosed herein includes the steps of treating a test agent with acne causing bacteria; Separating the exosome from the causative acne bacterium treated with the test substance; And confirming the amount of the exosome that has been separated. The present invention also provides a method for screening an anti-acne substance.

In one exemplary embodiment, the acne causing agent may be Propionibacterium acnes .

In an exemplary embodiment, the acne causing bacteria may be treated with the test substance and cultured for 72 to 96 hours.

In one exemplary embodiment, the test material can be treated with diluted acne causing bacteria after inoculation into the medium by diluting the acne causative bacteria that have undergone pre-cultivation.

As used herein, the term "exosome" refers to nano-sized extracellular vesicles, that is, extracellular vesicles secreted from the cells and released into the extracellular space. The exosomes are internal and external to the membrane- And has bacterial plasma membrane lipids, plasma membrane proteins, nucleic acids and cytoplasmic components. In addition, exosomes bind to other cells and deliver membrane components, mRNAs, miRNAs, etc., and they are important extracellular transporters that mediate cell-cell communication by transferring these transporters to recipient cells.

In one aspect, the exosome may be an extracellular bezique with a diameter of 20 to 500 nm. In another aspect, the exosome is at least 20 nm, at least 30 nm, at least 40 nm or at least 50 nm, but at most 500 nm, at most 400 nm, at most 300 nm, at most 200 nm, at most 100 nm, at most 90 nm, lt; RTI ID = 0.0 &gt; nm, &lt; / RTI &gt; 70 nm or less, or 60 nm or less.

In an exemplary embodiment, the exosomes are prepared by centrifugation, ultracentrifugation, differential centrifugation, Equilibrium Density Centrifugation, density gradient, filtration, but it is not limited thereto by one or more methods selected from the group consisting of filtration, dialysis, and free-flow electrophoresis.

The density gradient is the most commonly used method for distinguishing substances having different densities. Specific examples of the method include ficoll, glycerol, sucrose, cesium chloride, (iodixanol), and the like, but the present invention is not limited thereto. In one aspect, the density gradient can be used with ultracentrifugation and the like. In another aspect, gel filtration or ultrafiltration can be used to screen exosomes. In another aspect, dialysis may be used instead of filtration to remove small molecules. In another aspect, free-flow electrophoresis can be used.

In one exemplary embodiment, the step of isolating the exosomes comprises culturing the culture medium of acne causative bacteria treated with the test substance at a concentration of 2,000 to 20,000 x g, or 2,000 to 15,000 x g, or 2,000 to 10,000 x g, X g and then the supernatant is collected and ultracentrifuged at 100,000 to 200,000 x g or 120,000 to 180,000 x g or 130,000 to 170,000 x g or 100,000 to 150,000 x g or 150,000 to 200,000 x g Step &lt; / RTI &gt;

In an exemplary embodiment, the centrifugation may be performed for 10 to 50 minutes, and the ultracentrifuge may be performed for 2 to 4 hours.

The centrifugal separation may be performed stepwise by changing the speed or time. For example, the centrifugation may be performed by centrifuging at 2,000 to 5,000 x g, or 2,000 to 4,000 x g, or 3,000 to 4,000 x g for 10 to 30 minutes, and then centrifuging at 8,000 to 20,000 x g or 8,000 to 15,000 x g, To 12,000 x g, or 9,000 to 12,000 x g for 10 to 30 minutes.

In an exemplary embodiment, the step of separating the exosomes comprises centrifuging the culture medium for acne causing bacteria and then filtering it with a filter of 0.2 to 0.5 mu m, or 0.3 to 0.5 mu m, or 0.4 to 0.5 mu m, . The purity of the culture solution centrifuged through the filtration step can be increased.

In an exemplary embodiment, the isolated exosomes may be precipitated in ultracentrifugation of 100,000 to 200,000 x g of the acne-causing bacteria culture. At this time. The acne causing bacteria culture solution may be a supernatant obtained by centrifuging the culture solution at 2,000 to 20,000 x g.

In an exemplary embodiment, the exosomal secretion amount can be determined, for example, by measuring the exosomes separated by the same separation method from the same cell number using nanoparticle tracking analysis (NTA) using a Q-Nano or NanoSight instrument, It can be confirmed by counting the number of proteins, lipids or proteins, peptides, RNA, miRNA (microRNA), lncRNA (long noncoding RNA) and metabolites in the exosome surface proteins, lipids or exosomes. PCR, RT-qPCR, GC-MS, LC-MS, NMR, radioimmunoassay, immunoprecipitation, immunoprecipitation, and immunoprecipitation were performed according to Western blotting, dot blot, ELISA, northern blot, Methods such as immunoprecipitation assay, radioimmunodiffusion, FACS and protein chip can be suitably selected by those skilled in the art.

In an exemplary embodiment, the method may include comparing the amount of isolated exosomes to the amount of exosomes isolated from the untreated acne causing bacteria.

In an exemplary embodiment, the method may include evaluating the test substance as an anti-acne substance when the amount of isolated exosome is less than the amount of exosome isolated from acne causing bacteria that have not been treated with the test substance .

In an exemplary embodiment, the test substance may be evaluated as an anti-acne substance when the amount of the separated exosome is decreased by 5% or more based on the amount of exosome isolated from acne causative organisms not treated with the test substance. Specifically, when the amount of the exosome isolated is reduced from 5% or more to 100% or less based on the amount of exosome isolated from untreated acne causing bacteria, more specifically, 10% or more, 20% or more, , 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more, the test substance may be evaluated as an anti-acne substance.

Extracellular vesicles, more specifically exosomes, are a complex physiologically active substance that secretes acne causing bacteria to the outside of the cell. It exacerbates the expression of inflammatory cytokines and proteins in keratinocytes and promotes cell proliferation And the expression of genes and proteins of cell differentiation markers is reduced, thereby damaging the skin barrier and inhibiting the differentiation of keratin, thereby directly causing acne. Accordingly, a substance that reduces the exocrine secretion amount of the causative agent of acne by treating the test substance with the causative agent of acne can be screened with an anti-acne substance.

The techniques disclosed herein provide a method of screening anti-acne substances by comparing the increase or decrease in the amount of exosomes from an acne candidate substance, i.e., the presence or absence of the test substance. Specifically, a substance that indirectly or directly reduces the secretion of exosomes can be selected as an anti-acne substance. That is, after comparing the exosome secretion amount in the absence of the test substance with the exosome secretion amount in the presence of the test substance, the substance that reduces the exosome secretion amount in the presence of the test substance to the exosome secretion amount in the absence of the test substance, .

Anti-acne substances through the inhibition of the growth of acne causative microorganisms are releasing a large amount of adverse effect substances including exosomes in the process of killing microbes, so that they have side effects other than anti-acne effect. On the other hand, the anti-acne substance that reduces the secretion amount of exosomes disclosed in the present invention has an effect of suppressing the secretion of exosome itself that causes acne and thereby reducing side effects compared with the conventional antimicrobial agent.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Test Example  1. From acne causing bacteria Exosomatic  detach

Propionibacterium acnes (ATCC 6919), a causative agent of acne, was inoculated into Brain Heart Infusion (BHI) medium and incubated at 35 ~ 37 ℃ in anaerobic condition for 72 hours and collected by centrifugation. The collected bacteria were inoculated in a BHI medium to be diluted 1/100, and cultured at 35 to 37 ° C in an anaerobic state for 72 to 96 hours to collect the culture. The collected cultures were centrifuged at 3,000 × g for 10 minutes to precipitate bacteria, and the supernatant was collected and centrifuged at 10,000 × g for 20 minutes at 4 ° C. to collect the supernatant. Then, 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 150,000 × g for 3 hours at 4 ° C. The precipitated exosomes were dissolved in HEPES-buffered saline and the sizes of the exosomes were measured using dynamic light scattering analysis and the morphology was observed with a transmission electron microscope.

Test Example 2. Expression of Inflammatory Cytokine Gene and Protein in Exoose Treatment of Keratinocytes

Human keratinocyte (NHEK) was cultured in a 6-well plate incubator, and after 24 hours, the culture medium containing exosomes 1, 5 and 10 μg / ml derived from the acne causing bacteria isolated in Test Example 1 . Twenty-four hours later, the cells were harvested, RNA was isolated, and cDNA was synthesized by RT-PCR (reverse transcriptional polymerase chain reaction). Then, Taqman real-time PCR was performed using the synthesized cDNA to detect the inflammatory cytokines IL-8 and IL -6, and TNF-alpha gene expression levels (Fig. 2a). In addition, the culture medium of exogenous keratinocyte-treated cells from the acne-producing bacteria was collected after 24 hours, and the cytokine was quantified using IL-8 and IL-6 quantitative ELISA kits (see FIG. As a result, it was confirmed that the exosome derived from acne causing bacteria increased the expression of genes and proteins of inflammatory cytokines in keratinocytes.

Test Example 3. Promoting cell proliferation upon treatment of exosome in keratinocytes

Human keratinocytes (HaCaT cells) were cultured in a 6-well plate incubator, and after 24 hours, they were replaced with medium containing exosomes 1, 5, 10, and 20 μg / ml derived from the acne causative bacteria isolated in Test Example 1 Respectively. After 24 hours of incubation, the CCK-8 solution was added, incubated for 2 hours, and absorbance was measured at 540 nm (see FIG. 3). As a result, it was confirmed that exosome derived from acne causing bacteria increased cell proliferation in keratinocytes.

Test Example 4. Reduction of gene and protein expression of cell differentiation markers upon exosome treatment in keratinocytes

Human keratinocytes (NHEK) were cultured in a 6-well plate incubator and replaced with medium containing 1, 5, and 10 μg / ml of exosome derived from acne causing bacteria, which was isolated in Test Example 1 after 24 hours. After 4 days of culture, the cells were harvested, RNA was isolated, and cDNA was synthesized by RT-PCR (reverse transcriptional polymerase chain reaction). Then, Taqman real-time PCR was performed using the synthesized cDNA to obtain keratinocyte differentiation markers , KRT10) was measured (see Fig. 4A). In addition, proteins of keratinocytes were extracted and protein expression of keratinocyte differentiation markers (KRT1, KRT10) was confirmed by Western blotting (see FIG. 4B). As a result, it was confirmed that the exosome derived from acne causing agent decreased the expression of gene and protein of cell differentiation marker in keratinocyte.

Test Example  5. Anti acne  Screening of substances

Propionibacterium acnes (ATCC 6919), a causative agent of acne, was inoculated into Brain Heart Infusion (BHI) medium and incubated at 35 ~ 37 ℃ in anaerobic condition for 72 hours and collected by centrifugation. The collected bacteria were inoculated into BHI medium to be diluted 1/100, treated with the test substance, incubated at 35 to 37 ° C in anaerobic condition for 72 to 96 hours, and the culture solution was collected and the number of bacteria was counted. The collected cultures were centrifuged at 3,000 × g for 10 minutes to precipitate bacteria, and the supernatant was collected and centrifuged at 10,000 × g for 20 minutes at 4 ° C. to collect the supernatant. Then, 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 150,000 × g for 3 hours at 4 ° C. The amount of exosomes secreted from the same number of germs was measured by dissolving the precipitated exosomes in HEPES-buffered saline and confirming the amount of exosomes secreted from Propionibacterium acnes (ATCC 6919) cultured in the same manner without treating the test substance The amount of exosome) was evaluated to determine whether the test substance was an anti-acne substance. Exosomal secretion was determined by protein quantification of exosomes using the Bradford protein assay.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Treating the test substance with acne causing bacteria;
Separating the exosome from the causative acne bacterium treated with the test substance; And
And confirming the amount of the exosome that has been separated.
The method according to claim 1,
Wherein said acne causing bacteria is Propionibacterium acnes .
The method according to claim 1,
A method for screening an anti-acne substance, comprising culturing a causative agent of acne for 72 to 96 hours after treating the acne causing bacteria with the test substance.
The method according to claim 1,
The step of isolating the exosome comprises centrifuging the culture medium of acne causing bacteria treated with the test substance at 2,000 to 20,000 x g and then collecting the supernatant and ultracentrifuging at 100,000 to 200,000 x g. How to screen for acne substances.
5. The method of claim 4,
Wherein said centrifugation comprises centrifuging at 2,000 to 5,000 x g and then centrifuging at 8,000 to 20,000 x g.
5. The method of claim 4,
The method of screening an anti-acne substance according to claim 1, wherein the step of separating the exosomes comprises centrifuging the culture medium of acne causing bacteria and filtering the resultant with a filter of 0.2 to 0.5 mu m followed by ultracentrifugation.
The method according to claim 1,
Wherein said isolated exosome has a diameter of 20 to 500 nm.
The method according to claim 1,
Wherein the separated exosomes are sedimented by ultracentrifugation at 100,000 to 200,000 x g of the culture medium for causative acne causing acne.
The method according to claim 1,
And comparing the amount of the exosome thus isolated with the amount of exosome isolated from the acne causal organism not treated with the test substance.
10. The method of claim 9,
And evaluating the test substance as an anti-acne substance when the amount of the separated exosome is less than the amount of exosome isolated from acne causing bacteria that have not been treated with the test substance.

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