Extracellular vesicle induction material

Abstract

While methods and means for inducing the release of extracellular vesicles from eukaryotic cells already exist, there is still room for development in induction materials and methods for more efficient and higher-level induction of extracellular vesicles. Therefore, this research aims to provide materials for inducing extracellular vesicles. [Solution] By fermenting and culturing eukaryotic cells using swiftlet nests as a substrate, extracellular vesicles can be induced more efficiently and at higher levels.

Description

Technical Field

[0001] The present invention relates to a material for inducing the release of extracellular vesicles from single - cell species of eukaryotes, using the nest of Anabathmis, which consists of one or a combination of two or more of the family Anabathmidae, the tribe Anabathmis, or the genus Anabathmis of the order Apodiformes.

[0002] Furthermore, the present invention relates to a method for inducing the release of extracellular vesicles from cells of single - cell and multi - cell species organisms, using the nest of Anabathmis, which consists of one or a combination of two or more of the family Anabathmidae, the tribe Anabathmis, or the genus Anabathmis of the order Apodiformes.

[0003] In addition, the present invention relates to a material for inducing the release of extracellular vesicles from single - cell species of eukaryotes, using N - acetylneuraminic acid, which is a characteristic component of the nest of Anabathmis and consists of one or a combination of two or more of natural, semi - synthetic, or synthetic substances.

[0004] Furthermore, the present invention relates to a method for inducing the release of extracellular vesicles from cells of single - cell and multi - cell species organisms, using N - acetylneuraminic acid, which is a characteristic component of the nest of Anabathmis and consists of one or a combination of two or more of natural, semi - synthetic, or synthetic substances. Technical Background

[0005] Conventionally, it is a known fact that extracellular vesicles, which are lipid - membrane vesicles, are released from cells of multi - cell and single - cell species of eukaryotes. Extracellular vesicles are classified into exosomes derived from endocytosis by which cells take up extracellular substances, microvesicles derived from the cell membrane, and apoptotic bodies. Among these extracellular vesicles, exosomes and microvesicles have functions related to cell - to - cell communication, and apoptotic bodies are known to have functions such as phagocytosis, which uses the cell membrane to take in large particles and induce the formation of an internal compartment called a phagosome. Also, it is known that exosomes and microvesicles are released from healthy cells, while apoptotic bodies are released from cells that have undergone apoptosis (programmed cell death).

[0006] Among the extracellular vesicles mentioned above, exosomes are formed when the membrane of an endosome, which is formed inside a multivesicular endosome, invaginates.

[0007] The aforementioned exosomes are rich in endosome-binding proteins such as Rab (Ras-related in brain) GTPase, SNARE (Ssoluble N-ethyimaleimide factor attachment protein receptor), and Annexin and Flotillin, which are protein families that bind to calcium and phospholipids.

[0008] Among the aforementioned, exosomes, in particular, are said to be involved in communication between nearby and distant cells, along with microvesicles. In the immune system, exosomes released from cells function as antigen-presenting vesicles and are known to induce antitumor immune responses and immune tolerance that suppresses inflammation. In the nervous system, they are also said to be involved in myelin sheath formation, neurite extension, and the maintenance of nerve cell survival. Furthermore, in the blood coagulation system, microvesicles derived from platelets and monocytes mediate the formation of complexes such as factor X involved in the blood coagulation cascade, causing cell fusion and inducing thrombus formation. In the process of inflammation, the function of microvesicles changes depending on the type of stimulus and the cells releasing them, sometimes being suppressive and sometimes enhanced. In either case, it is a known fact that when microvesicles bind to target cells, cytokines that regulate the inflammatory response are released.

[0009] Due to the properties and functions described above, extracellular vesicles are thought to be deeply involved in the exacerbation and disappearance of disease symptoms, and are beginning to be used in various forms for therapeutic purposes and disease prevention.

[0010] Furthermore, in recent years, as mentioned above, research has been conducted on methods and materials for inducing extracellular vesicles, particularly exosomes. However, a method for inducing extracellular vesicles using swiftlet nests and N-acetylneuraminic acid, a characteristic component of swiftlet nests, as induction materials has yet to be found. [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention makes it possible to induce the release of extracellular vesicles from single-celled eukaryotic species using the nest of a swiftlet, which consists of one or more combinations of members of the tribe Swiftle or the genus Swiftle, ​​family Apodiformes, family Apodidae.

[0012] Furthermore, N-acetylneuraminic acid, which consists of one or more combinations of naturally derived, semi-synthetic, or synthetic components characteristic of swiftlet nests, can be used to induce the release of extracellular vesicles from the cells of unicellular and multicellular organisms.

[0013] The swiftlet nests used in this invention belong to the Swiftle tribe or Swiftle genus of the family Apodidae, order Apodiformes, and include A. amelis (gray swiftlet), A. brevirostris (Himalayan swiftlet), A. elaphra (Seychelles swiftlet), A. francica (edible swiftlet), A. fuciphaga (Javan swiftlet), A. germani (Malayan swiftlet), and A. hirundinacea (Ya A. p A. alawanensis (Palawan Swift), A. papuensis (Papua Swift), A. pelewensis (Micronesian Swift), A. rogersi (Indochinese Swift), A. salangana (Cook's Swift), A. sawtelli (Cook's Swift), A. spodiopygius (White-rumped Swift), A. terraereginae (Australian Swift) One or a combination of two or more of the following species are preferred: A. unicolor (Indian Swiftlet), A. whiteheadi (Philippine Swiftlet), A. vanikorensis (Plain Swiftlet), A. vulcanorum (Mountain Swiftlet), C. esculenta (White-bellied Swiftlet), C. linchi (Cave Swiftlet), and C. troglodytes (Dwarf Swiftlet).

[0014] Furthermore, the swiftlet nests of the Swiftle tribe or Swiftle genus of the Apodiformes order, Apodidae family, used in this invention are those that have been washed to remove impurities and waste products and then dried.

[0015] Furthermore, this invention focuses on N-acetylneuraminic acid, a component characteristic of swiftlet nests, and its content is also considered to have a significant impact.

[0016] In this invention, the following species of swifts, belonging to the tribe A. amelis or genus A. infuscata, are used: A. amelis (gray swift), A. brevirostris (Himalayan swift), A. elaphra (Seychelles swift), A. francica (edible swift), A. fuciphaga (Javan swift), A. germani (Malayan swift), A. hirundinacea (mountain swift), A. infuscata ( Moluccan Swiftlet, A. inquieta (Caroline Swiftlet), A. leucophaeus (Tahitian Swiftlet), A. maxima (Giant Swiftlet), A. mearnsi (Philippine Swiftlet), A. nuditarsus (Papua Mountain Swiftlet), A. ocista (Marquesas Swiftlet), A. orientalis (Guadalcanal Swiftlet), A. palawanensis (Palawan Swiftlet), A. pap A. uensis (Papua Swift), A. pelewensis (Micronesian Swift), A. rogersi (Indochina Swift), A. salangana (Cook's Swift), A. sawtelli (Cook's Swift), A. spodiopygius (White-rumped Swift), A. terraereginae (Australian Swift), A. unicolor (Indian Swift), A. whitehea By using the nests of one or more of the following species—Di (Philippine Swift), A. vanikorensis (Plain Swift), A. vulcanorum (Mountain Swift), C. esculenta (White-bellied Swift), C. linchi (Cave Swift), and C. troglodytes (Dwarf Swift)—as a substrate for fermenting yeast, a single-celled eukaryotic species, extracellular vesicles are induced in the yeast.

[0017] The yeast described in the preceding section includes Saccharomyces cerecisiae, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces carioanus, Saccharomyces cariocus, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces florentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomycesuvarum, and Saccharomyces It consists of one or more members from the genera Zonatus, Saccharomycopsis, Candida kefyr, Candida bombicola, Candida utilis, and Candida saitoana, Zygosaccharomyces rouxii and Zygosaccharomyces microellipsoides, Kluyveromyces, Schizosaccharomyces pombe, Torulaspora delbrueckii, Pichia anomala and Pichia caribbica, Pseudoxyma tsukubaensis, and Endomyces.

[0018] In the present invention, the extracellular vesicles induced from the yeast described in the preceding paragraph consist of exosomes derived from endocytosis, in which cells take in extracellular substances, and microvesicles and apoptotic bodies derived from the cytoplasmic membrane.

[0019] Of the extracellular vesicles described in the previous section, exosomes and microvesicles are known to have functions related to intercellular communication, while apoptotic bodies are known to have functions such as phagocytosis, which involves taking in large particles using the cell membrane and inducing the formation of an internal compartment called a phagosome.

[0020] Among the extracellular vesicles mentioned above, exosomes have a particle size of 30-200 nanometers, and microvesicles have a particle size of 100-1000 nanometers, indicating a wide range of particle sizes. Furthermore, apoptotic bodies have a particle size of 800-5000 nanometers, representing an even wider range of particle sizes.

[0021] The difference in the release of these extracellular vesicles is that exosomes and microvesicles are released from healthy cells, while apoptotic bodies are released from cells that have undergone apoptosis (natural cell death).

[0022] Furthermore, exosomes are rich in endosome-binding proteins such as Rab (Ras-related in brain) GTPase, SNARE (Ssoluble N-ethyimaleimide factor attachment protein receptor), and Annexin and Flotillin, which are protein families that bind to calcium and phospholipids.

[0023] Moreover, exosomes, together with microvesicles, are said to be involved in cell communication between neighboring or distant cells. In the immune system, exosomes released from cells function as antigen-presenting vesicles, and are also known to induce immune tolerance that suppresses anti-tumor immune responses and inflammation. In the nervous system, it is also said to be involved in the formation of myelin sheaths, the extension of neurites, and the survival and maintenance of neurons. Furthermore, in the blood coagulation system, microvesicles derived from platelets and monocytes mediate the formation of complexes such as factor X involved in the blood coagulation cascade, induce cell fusion to induce thrombus formation, and in the process of inflammation, the function of microvesicles changes depending on the type of stimulus and the cells from which they are released, either suppressing or enhancing it. In this case, it is known that in any case, the binding of microvesicles to target cells releases cytokines that regulate the inflammatory response.

[0024] From the above content, among extracellular vesicles, exosomes are also used in regenerative medicine such as for the skin, and in recent years, because they are contained in body fluids such as blood and urine, they are expected to be used as diagnostic markers for diseases, particularly as tumor markers.

[0025] From the above viewpoints, it is considered that the induction of extracellular vesicles including exosomes is useful for maintaining a healthy human body and alleviating various diseases.

Effects of the Invention

[0026] The extracellular vesicle-inducing material obtained by the present invention is the nest of the Japanese pond turtle of the family Emydidae, order Testudines, tribe Mauremys or genus Mauremys. By fermenting yeast, which is a eukaryotic single-cell species, using this as a substrate, the particle size distribution of the induced extracellular vesicles showed a higher value than when fermented with only water.

[0027] Specifically show the results of the previous paragraph.

[0028] The particle size distribution of extracellular vesicles released into the fermentation broth of both cases was measured when yeast, a eukaryotic single-cell species, was fermented with only water and when fermented using the Japanese pond turtle nest as a substrate.

[0029] The measurement of the particle size distribution was carried out by wet measurement using the laser diffraction / scattering method.

[0030] The temperature during measurement was 25 degrees Celsius. Sixty measurements were taken within a measurement time of 10 seconds, and the particle size distribution was confirmed. This operation was repeated three times, and finally, it was described based on the scattering intensity criterion, which is the ratio of the scattering intensity to the total intensity of scattering generated from the particles. In this scattering intensity criterion, it contributes to the ratio by the sixth power of the particle size.

[0031] The table of the measurement results is shown below.

[0032] Graph 1. Measurement results of extracellular vesicles in the fermentation broth when yeast was fermented with only water. TIFF2026136035000001.tif59158

[0033] Graph 2. Measurement results of extracellular vesicles in the fermentation broth when the nest of the Japanese swiftlet was used as a substrate. TIFF2026136035000002.tif58155

[0034] Graph 3. Comparison when the vertical axis scales of Graph 1 and Graph 2 are made the same. TIFF2026136035000003.tif64107

[0035] Table 1. The comparison numerical values in the previous paragraph. TIFF2026136035000004.tif243152

[0036] From the results up to the previous paragraph, when yeast was fermented using the nest of the Japanese swiftlet as a substrate, the particle size distribution showed higher values than when yeast was fermented with only water, and it was confirmed that the nest of the Japanese swiftlet induces extracellular vesicles.

[0037] As an effect of the invention, although the test results using yeast, a single-celled eukaryotic species, suggest that culturing cells using swiftlet nests as a substrate can induce the formation of extracellular vesicles, making it possible to efficiently collect these extracellular vesicles. [Modes for carrying out the invention]

[0038] The implementation of the present invention will be described below. [Examples]

[0039] After removing impurities and waste products, the dried swiftlet nests are disinfected with a 6% sodium hypochlorite solution diluted with water to 1 / 100th of its original volume.

[0040] The swiftlet nests, which were disinfected in the previous step, are soaked in water at a rate of 10% by weight.

[0041] The swiftlet nests, which have been soaked in water, are fermented with the water extract along with yeast (Saccharomyces cerecisiae). The fermentation conditions are 35 to 45 degrees Celsius for 48 hours.

[0042] After fermentation, sterilize by heating at 85 degrees Celsius for 30 to 60 minutes.

[0043] After heat sterilization, the sterile filtered fermentation supernatant is used as the extracellular vesicle-containing solution.

[0044] The extracellular vesicle-containing solution obtained in the previous section can be incorporated into cosmetics, foods, processed foods, soft drinks, and quasi-drugs in a stable state.

[0045] Furthermore, it can selectively collect only extracellular vesicles for use as active pharmaceutical ingredients. [Industrial applicability]

[0046] This invention is used in industries that manufacture cosmetics, foods, processed foods, soft drinks, and quasi-drugs.

[0047] The present invention can also be used in industries that manufacture active pharmaceutical ingredients for drugs that selectively utilize extracellular vesicles.

Claims

1. Material for inducing extracellular vesicles containing exosomes using swiftlet nests

2. A method for inducing extracellular vesicles containing exosomes using swiftlet nests.

3. An exosome-containing extracellular vesicle induction material using N-acetylneuraminic acid, which consists of one or more combinations of naturally derived, semi-synthetic, or synthetic components characteristic of swiftlet nests.

4. A method for inducing extracellular vesicles containing exosomes using N-acetylneuraminic acid, which consists of one or more combinations of naturally derived, semi-synthetic, or synthetic components characteristic of swiftlet nests.

5. Cosmetic composition containing claim 1

6. Food, processed food, or soft drink containing the contents of claim 1

7. Quasi-drug containing Claim 1

8. Active pharmaceutical ingredient using claim 1 as the material

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