WO2020032186A1 - Topical composition containing extracellular vesicles produced by buccal epithelial cells - Google Patents
Topical composition containing extracellular vesicles produced by buccal epithelial cells Download PDFInfo
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- WO2020032186A1 WO2020032186A1 PCT/JP2019/031429 JP2019031429W WO2020032186A1 WO 2020032186 A1 WO2020032186 A1 WO 2020032186A1 JP 2019031429 W JP2019031429 W JP 2019031429W WO 2020032186 A1 WO2020032186 A1 WO 2020032186A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- the present invention relates to a composition for topical application to epithelium such as skin, which contains extracellular vesicles derived from oral epithelial cells, particularly exosomes.
- epithelial cells are known to promote wound healing by transplanting to epithelial defect sites, and products such as regenerative medicine using the same have been put on the market.
- cultures of oral epithelial cells have been used for applications such as corneal regeneration and improved esophageal mucosal healing.
- An object of the present invention is to provide a new topical composition which can be easily prepared and can improve the condition of body tissues, particularly epithelial tissues such as skin and mucous membranes.
- the present inventors have found that a composition containing extracellular vesicles secreted into a medium when culturing oral epithelial cells has an effect such as promoting wound healing of epidermis or epithelial tissue such as skin or mucous membrane. And completed the invention of the present application. That is, the present invention provides a topical composition comprising extracellular vesicles derived from oral epithelial cells. Further, the present invention provides a step of culturing oral epithelial cells in a medium, a step of separating oral epithelial cells from the culture to recover a conditioned medium, and a step of isolating extracellular vesicles of oral epithelial cells from the conditioned medium. And adding the isolated extracellular vesicles to a medium suitable for topical application.
- the topical composition of the present invention can be easily prepared by isolating extracellular vesicles from a culture solution of oral epithelial cells and the like, and can heal and fibrosis wounds and ulcers on the epidermis such as skin or mucous membrane. Can be used to treat or prevent scarring by alleviation. It also has a rejuvenating effect by improving the condition of the epidermis by promoting tissue repair. Further, as an effect of reducing fibrosis, fibrosis of the heart, lungs, liver, kidney and the like can be improved.
- FIG. 4 shows the results of Western blot of fractions containing extracellular vesicles.
- the electron micrograph of the isolated extracellular vesicle is shown (scale bar: 50 nm).
- 4 shows the results of a fibroblast proliferation assay.
- Fig. 3 shows the results of a cytotoxicity assay on fibroblasts.
- the results of gene expression analysis of various growth factors in fibroblasts (FIG. 5a), HGF-release assay by ELISA (FIG. 5b), and expression analysis of type IV collagen gene (FIG. 5c) are shown.
- 3 shows the results of a proliferation test using squamous cell carcinoma cells. The observation results of an in vivo test using a rat full-thickness wound healing model (FIG.
- FIG. 7a shows a micrograph after staining with hematoxylin / eosin and picrosirius red (FIG. 7b)
- FIG. 4 shows the results of wound area measurement in an in vivo test using a rat full thickness wound healing model.
- the micrograph after hematoxylin and eosin staining in an in vivo test using a porcine skin wound model is shown (scale bar: 500 nm).
- the topical composition of the present invention comprises extracellular vesicles derived from oral epithelial cells.
- Oral epithelial cells are cells that constitute the surface of the oral mucosa, and include any cells that constitute epithelial tissue.
- the sampling method and the instrument for the collection are well known to those skilled in the art.
- various cells have been developed or marketed for research, and these cells can also be used.
- oral epithelial cells derived from mammals are used.
- Oral epithelial cells may be derived from a species different from the species to which the final topical composition is applied, but from the viewpoint of safety and availability in consideration of application to humans,
- human oral epithelial cells are used.
- the human is not particularly limited and may be non-adult / adult, but preferably oral epithelial cells derived from an adult are used.
- the isolation of extracellular vesicles from oral epithelial cells can be performed by those skilled in the art by any method. For example, since extracellular vesicles are secreted into the medium by culturing oral epithelial cells in the medium, a medium (conditioned medium) obtained by removing cells and residues from the culture after culture is collected and The extracellular vesicles derived from oral epithelial cells can be isolated from the conditioned medium by the technique described in (1).
- the medium used for culturing the cells and the culture conditions are not particularly limited, but a liquid medium, for example, a high glucose Dulbecco's modified Eagle medium is preferably used. Other components necessary for the culture may be appropriately added to the medium, and may include, for example, a nutrient mixture such as F-12 @ Ham, a corticosteroid such as Sol Cortev, an antibiotic, an autologous serum, and the like.
- Culture conditions are not particularly limited, and can be appropriately set by those skilled in the art according to the cells to be used and the amount of extracellular vesicles required. For example, 33 to 40 ° C, preferably 35 to 38 ° C And more preferably at a temperature of 37 ° C.
- the medium may be changed during the culturing, and these mediums can be used collectively thereafter.
- the conditioned medium is recovered by separating the cells from the culture by centrifugation or filtration.
- the collected conditioned medium may be further subjected to centrifugation or filtration to further remove impurities such as cell residues.
- the conditions of centrifugation and filtration can be appropriately determined by those skilled in the art.For example, first, the culture is centrifuged at 200 ⁇ g to 400 ⁇ g, preferably at 250 ⁇ g to 350 ⁇ g for about 10 minutes, and the cells are separated by centrifugation. Fine impurities can be removed by centrifugation again at 2500 ⁇ g to 3500 ⁇ g or by filtration at, for example, 0.22 ⁇ m.
- the method for isolating extracellular vesicles from a conditioned medium is well known and is not particularly limited, and those skilled in the art can isolate them by any method.
- the medium is concentrated by ultrafiltration or the like, for example, size exclusion chromatography, ultracentrifugation, microfiltration, density gradient centrifugation, or the like can be performed alone or in an appropriate combination.
- extracellular vesicles are isolated by performing size exclusion chromatography on the concentrated medium after ultrafiltration.
- For ultrafiltration for example, Amicon (100 kDa, 1000 kDa, etc.) from Merck Millipore can be used according to the manufacturer's instructions.
- the isolated extracellular vesicle preferably contains exosomes.
- exosomes exosomes, microvesicles, etc.
- the modes and conditions for isolating extracellular vesicles are not limited to those that target the specific one by strictly distinguishing the type of extracellular vesicle. Absent.
- the isolation operation may be performed under conditions such that exosomes are concentrated among extracellular vesicles, and exosomes may be concentrated. May be additionally performed.
- the fraction containing exosomes having a size of 30 to 200 nm, preferably 40 to 150 nm, and more preferably 80 to 130 mm is collected to obtain a ratio of exosomes. To increase the concentration of exosomes.
- the topical composition of the present invention can be obtained by adding the extracellular vesicle to a medium suitable for topical application.
- the type of medium can be appropriately selected by those skilled in the art from liquids, solids, semi-solids, and the like depending on the mode of application to the body.For example, any medium for forming a solution, gel, lotion, cream, ointment, plaster Is mentioned.
- the topical composition of the present invention may further include any adjuvants, additives, and the like that are acceptable for the above embodiments.
- the concentration of extracellular vesicles in the final composition can be appropriately set based on the intended use of the composition, the desired effect, etc., so as to be an amount effective for producing a therapeutic effect or a desired effect.
- 0.2 ⁇ g ⁇ 2mg / cm 2 according to the area of the site preferably 2.0 ⁇ 200 ⁇ g / cm 2, more preferably 10 ⁇ 100 ⁇ g / cm 2, particularly preferably the composition at a concentration of 20 ⁇ 40 ⁇ g / cm 2 It is preferable to add it to the product.
- Topical application in the present invention means to apply in such a manner as to directly act on a site in the body where the effect of the composition of the present invention is intended, and preferably to use in such a mode as to contact the site.
- the compositions of the invention are applied by application to the epithelium on the body surface (including contacting the solid composition).
- Topical application includes so-called topical application, but the application site is not limited to application to the body surface (a site directly visible from the outside, for example, skin, cornea), but to mucous membranes such as oral cavity, nasal cavity, and digestive organs. Includes aspects of application.
- the present invention also includes a mode in which the method is directly applied to internal organs (heart, lung, liver, kidney, thyroid, parathyroid, pancreas, gallbladder, etc.) through a surgical procedure or the like.
- the composition of the present invention is applied to the skin or mucous membrane, and is applied by applying to these sites.
- a wound or disease state can be improved by promoting the healing of a wound of skin or mucous membrane.
- Wounds include those based on any aspect, including trauma, surgical scars, and even burns.
- composition of the present invention stimulates fibroblasts, as shown in the examples below, to give hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), And has been shown to enhance the release of growth factors such as connective tissue growth factor (CTGF), so that by applying the composition to a given site, any disease or disease in which the above growth factors have a therapeutic effect The effect of preventing or treating the condition is expected.
- HGF hepatocyte growth factor
- VEGF vascular endothelial growth factor
- FGF fibroblast growth factor
- CTGF connective tissue growth factor
- a drug having the above-mentioned therapeutic or preventive effect can be obtained.
- extracellular vesicles derived from oral epithelial cells do not promote tumor growth in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells. Therefore, it can be safely compounded with pharmaceuticals for various diseases or symptoms.
- composition of the present invention improves the condition of tissues such as the skin and has a rejuvenating effect on the epidermis by promoting tissue repair. Can bring.
- the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
- conditioned medium used to produce a clinical grade oral mucosal cell sheet from cells isolated by biopsy from the mouth of a healthy adult donor was obtained from CellSeed.
- the provided conditioned medium was a keratinocyte culture medium (75% high glucose Dulbecco's modified Eagle medium, 25% nutrient mixture F-12 Ham, 0.475 g / L L-glutamine, 5 ⁇ g / ml Humulin Regular (Humulin Regular).
- Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Dilute the extracellular vesicles 1: 1 with RIPA (Radioimmunoprecipitation assay) buffer, and ultrapure for 5 minutes (on / off: 15 seconds / 15 seconds, in an ice bath, high setting (Cosmo Bio / sonicator)) Sonicated. Sonicated extracellular vesicles and cell lysates were diluted in MilliQ water (Merck Millipore) and BCA (bicinchoninic acid) assay was performed according to the instructions.
- RIPA Radioimmunoprecipitation assay
- Cell lysates were obtained from fibroblasts (CC-2509, Lonza) and oral keratinocytes (# 2610, ScienCell). The cell suspension (about 4 ⁇ 10 6 cells) was centrifuged at 300 ⁇ g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in PBS and centrifuged again. The pellet is resuspended in RIPA (Radioimmunoprecipitation assay) buffer (Sigma) containing 1% protease / phosphatase inhibitor cocktail (Cell Signaling Technology) and placed in an ice bath for 5 minutes (15 second on, 15 second off interval) Sonicated.
- RIPA Radioimmunoprecipitation assay
- the sample was centrifuged at 8000 ⁇ g for 10 minutes at 4 ° C., and the supernatant was collected and divided into equal amounts for use in the subsequent analysis.
- extracellular vesicles and cell lysates prepared as described above, confirm and measure the presence and properties of extracellular vesicles by molecular biological techniques, electron microscopy, and nanoparticle tracking analysis. Function was analyzed in in vitro studies.
- Filters were blocked for 60 minutes using TBS-T (BioRad) containing 5% milk powder (CellSignaling) and exposed to primary antibody overnight at 4 ° C.
- the filters were washed on an orbital shaker with TBS-T for 3 ⁇ 10 minutes, exposed to the secondary antibody for 60 minutes at room temperature, and then washed again with TBS-T for 3 ⁇ 10 minutes.
- the buffer was removed from the filter, and a detection agent (ECL Prime WB detection kit, Sigma-Aldrich) was added and left for 5 minutes. Thereafter, protein expression was confirmed using LAS 4000 mini (Fuji Film).
- Samples from the extracellular vesicle fraction were positive for the internationally recognized markers (CD9 and flotilin) and negative for the negative marker GRP94 (FIG. 1).
- the antibodies and concentrations used were as follows: CD9 (# 13174, Cell Signaling Technologies), Flotillin (# 18634, Cell Signaling Technologies), GRP94 (# 2104, Cell Signaling Technologies), anti-rabbit IgG, HRP-linked (# 7074, Cell Signaling Technologies, 1: 5000).
- Antibodies were diluted in Can Get Signal solution (Toyobo).
- Nanoparticle tracking analysis The size and number of extracellular vesicles were measured using the LM10 Nanoparticle Analyzer (NanoSight) according to the manufacturer's instructions. Vesicle size was approximately 125 nm (124.8 ⁇ 4.1 nm (standard deviation)).
- the primers used are as follows: ⁇ -actin (4326315E-1112022, Applied Biosystem), HGF (HS00300159_m1, ThermoFischer Scientific), VEGFA (HS00900055_m1, ThermoFischer Scientific), FGF2 (HS00266645_m1, ThermoFischer Scientific 269gFischer 010gFisher 010GFr) ).
- the type IV collagen analysis was similarly performed using NHDF cells at passages 5 and 17 and primer Col4a1 (HS00266237_m1, ThermoFischer Scientific).
- HGF hepatocyte growth factor
- VEGFA vascular endothelial growth factor A
- FGF2 fibroblast growth factor
- CTGF connective tissue growth factor
- the extracellular vesicles were labeled with a fluorescent membrane dye (PKH26-GL-1KT, Sigma Aldrich), the extracellular vesicles were diluted with 500 ⁇ L of diluent C, and immediately 500 ⁇ L of diluent C ( 2 ⁇ L of PKH26) was added again and an incubation step of 5 minutes was performed with gentle mixing. Thereafter, 2.5 mL of PBS was added, and the extracellular vesicles were re-concentrated using a 10 kDa filter (Amicon). This step was performed three times in total. As a control, all steps were performed without extracellular vesicles.
- Tissue sections (8 ⁇ m) were prepared using a cryostat (MicroEdge) and stained with hematoxylin and eosin and picrosirius red (Polysciences). Slides stained with picrosirius red were visualized under a polarizing microscope and the images were evaluated blind.
- FIG. 7 (a) signals from the fluorescently labeled extracellular vesicles were observed in the wound bed of the rat until 5 days after application.
- extracellular vesicles promoted wound healing in full-thickness skin wounds of rats, as indicated by hematoxylin-eosin and picrosirius red-staining.
- the treatment group with extracellular vesicles showed significantly smaller wound areas on days 6 and 17 compared to the control.
- PBS extracellular vesicle or control
- physiological saline was applied by directly applying a composition containing extracellular vesicles produced by human oral epithelial cells to a wound model from which rat skin (epidermal, dermis) was excised. Compared to the control group to which was applied, wound healing was promoted, scarring was reduced, and a rejuvenating effect by tissue repair was observed.
- extracellular vesicles derived from oral epithelial cells in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells, exhibited tumor growth. was not promoted. Therefore, the composition of the present invention has an advantage that it can be safely formulated into a drug for various diseases or conditions.
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Abstract
The present invention provides a novel topical composition that can be easily manufactured and is capable of improving the state of bodily tissue, especially epithelial tissue such as the skin or mucosae. In particular, the present invention provides a topical composition containing extracellular vesicles derived from buccal epithelial cells.
Description
本発明は、口腔上皮細胞由来の細胞外小胞、特にエクソソームを含む、皮膚等の上皮に局所適用するための組成物に関する。
The present invention relates to a composition for topical application to epithelium such as skin, which contains extracellular vesicles derived from oral epithelial cells, particularly exosomes.
近年、アンメットメディカルニーズを解決するために、細胞や組織を用いた再生医療等製品の研究や開発が進められている。例えば上皮細胞は上皮欠損部位に移植することにより、創傷治癒を促進することが知られており、これを用いた再生医療等製品などが上市されている。また、口腔上皮細胞の培養物が、角膜の再生や食道粘膜の治癒の改善などの用途に使用されている。
In recent years, research and development of products such as regenerative medicine using cells and tissues have been promoted to solve unmet medical needs. For example, epithelial cells are known to promote wound healing by transplanting to epithelial defect sites, and products such as regenerative medicine using the same have been put on the market. In addition, cultures of oral epithelial cells have been used for applications such as corneal regeneration and improved esophageal mucosal healing.
また、様々な細胞種から分泌されるエクソソーム等の細胞外小胞(Extracellular vesicles;EVs)についても、その再生促進効果についての研究が進められており、その研究の多くは骨髄間葉系間質細胞(BM-MSC)-由来のエクソソームについて行われている(Journal of Translational Medicine (2015), 13:49)。しかしながら、上皮細胞由来の細胞外小胞に関しての研究はほとんど報告されておらず、特に口腔上皮細胞由来の細胞外小胞については、ウイルス免疫学に関する報告(Journal of Virology, Vol.90 No.7 (2016), p. 3469-3479)が存在するに過ぎない。
Studies are also being conducted on the effect of promoting extracellular vesicles (EVs) such as exosomes secreted from various cell types, and most of the research is on bone marrow mesenchymal stromal cells. It is performed on exosomes derived from cells (BM-MSC)-(Journal of Translational Medicine (2015), 13:49). However, few studies have been reported on extracellular vesicles derived from epithelial cells. Particularly, extracellular vesicles derived from oral epithelial cells have been reported on viral immunology (Journal of Virology, Vol.90 No.7). (2016), p. 3469-3479) only exists.
本発明は、容易に作製可能であり、かつ、身体の組織、特に皮膚や粘膜などの上皮組織の状態を改善することができる新たな局所適用組成物を提供することを目的とする。
An object of the present invention is to provide a new topical composition which can be easily prepared and can improve the condition of body tissues, particularly epithelial tissues such as skin and mucous membranes.
本願発明者らは、口腔上皮細胞を培養する際などに培地中に分泌される細胞外小胞を含む組成物が、皮膚や粘膜等の表皮や上皮組織の創傷治癒促進等の効果を有することを見出し、本願発明を完成するに至った。
即ち、本発明は、口腔上皮細胞由来の細胞外小胞を含む局所適用組成物を提供する。さらに、本発明は、口腔上皮細胞を培地中で培養する工程、培養物から口腔上皮細胞を分離して馴化培地を回収する工程、馴化培地から口腔上皮細胞の細胞外小胞を単離する工程、及び、単離した細胞外小胞を局所適用に適した媒体中に添加する工程を含む、局所適用組成物の製造方法を提供する。 The present inventors have found that a composition containing extracellular vesicles secreted into a medium when culturing oral epithelial cells has an effect such as promoting wound healing of epidermis or epithelial tissue such as skin or mucous membrane. And completed the invention of the present application.
That is, the present invention provides a topical composition comprising extracellular vesicles derived from oral epithelial cells. Further, the present invention provides a step of culturing oral epithelial cells in a medium, a step of separating oral epithelial cells from the culture to recover a conditioned medium, and a step of isolating extracellular vesicles of oral epithelial cells from the conditioned medium. And adding the isolated extracellular vesicles to a medium suitable for topical application.
即ち、本発明は、口腔上皮細胞由来の細胞外小胞を含む局所適用組成物を提供する。さらに、本発明は、口腔上皮細胞を培地中で培養する工程、培養物から口腔上皮細胞を分離して馴化培地を回収する工程、馴化培地から口腔上皮細胞の細胞外小胞を単離する工程、及び、単離した細胞外小胞を局所適用に適した媒体中に添加する工程を含む、局所適用組成物の製造方法を提供する。 The present inventors have found that a composition containing extracellular vesicles secreted into a medium when culturing oral epithelial cells has an effect such as promoting wound healing of epidermis or epithelial tissue such as skin or mucous membrane. And completed the invention of the present application.
That is, the present invention provides a topical composition comprising extracellular vesicles derived from oral epithelial cells. Further, the present invention provides a step of culturing oral epithelial cells in a medium, a step of separating oral epithelial cells from the culture to recover a conditioned medium, and a step of isolating extracellular vesicles of oral epithelial cells from the conditioned medium. And adding the isolated extracellular vesicles to a medium suitable for topical application.
本発明の局所適用組成物は、口腔上皮細胞の培養液等から細胞外小胞を単離することにより容易に作製可能であり、皮膚又は粘膜などの表皮の熱傷や潰瘍における創傷治癒や線維化軽減による瘢痕の治療又は予防に使用できる。また、組織修復を促すことにより表皮の状態を改善することにより若返り効果を有する。さらに線維化軽減による効果として心臓・肺・肝臓・腎臓などの線維症を改善させることができる。
The topical composition of the present invention can be easily prepared by isolating extracellular vesicles from a culture solution of oral epithelial cells and the like, and can heal and fibrosis wounds and ulcers on the epidermis such as skin or mucous membrane. Can be used to treat or prevent scarring by alleviation. It also has a rejuvenating effect by improving the condition of the epidermis by promoting tissue repair. Further, as an effect of reducing fibrosis, fibrosis of the heart, lungs, liver, kidney and the like can be improved.
本発明の局所適用組成物は、口腔上皮細胞由来の細胞外小胞を含む。口腔上皮細胞は、口腔内粘膜の表面部を構成する細胞であり、上皮組織を構成する任意の細胞を含む。その採取方法及びそのための器具等は当業者に周知である。また、研究用に様々な細胞が開発又は市販されており、それらの細胞を使用することも可能である。
本発明では、好ましくは哺乳動物由来の口腔上皮細胞が使用される。口腔上皮細胞は、最終の局所適用組成物が適用される生物種とは異なる生物種由来のものであってもよいが、ヒトへの適用を考慮した安全性及び入手容易性などの観点から、好ましくはヒト由来の口腔上皮細胞が使用される。上記ヒトは特に限定されず非成人/成人を問わないが、好ましくは成人由来の口腔上皮細胞が使用される。 The topical composition of the present invention comprises extracellular vesicles derived from oral epithelial cells. Oral epithelial cells are cells that constitute the surface of the oral mucosa, and include any cells that constitute epithelial tissue. The sampling method and the instrument for the collection are well known to those skilled in the art. In addition, various cells have been developed or marketed for research, and these cells can also be used.
In the present invention, preferably, oral epithelial cells derived from mammals are used. Oral epithelial cells may be derived from a species different from the species to which the final topical composition is applied, but from the viewpoint of safety and availability in consideration of application to humans, Preferably, human oral epithelial cells are used. The human is not particularly limited and may be non-adult / adult, but preferably oral epithelial cells derived from an adult are used.
本発明では、好ましくは哺乳動物由来の口腔上皮細胞が使用される。口腔上皮細胞は、最終の局所適用組成物が適用される生物種とは異なる生物種由来のものであってもよいが、ヒトへの適用を考慮した安全性及び入手容易性などの観点から、好ましくはヒト由来の口腔上皮細胞が使用される。上記ヒトは特に限定されず非成人/成人を問わないが、好ましくは成人由来の口腔上皮細胞が使用される。 The topical composition of the present invention comprises extracellular vesicles derived from oral epithelial cells. Oral epithelial cells are cells that constitute the surface of the oral mucosa, and include any cells that constitute epithelial tissue. The sampling method and the instrument for the collection are well known to those skilled in the art. In addition, various cells have been developed or marketed for research, and these cells can also be used.
In the present invention, preferably, oral epithelial cells derived from mammals are used. Oral epithelial cells may be derived from a species different from the species to which the final topical composition is applied, but from the viewpoint of safety and availability in consideration of application to humans, Preferably, human oral epithelial cells are used. The human is not particularly limited and may be non-adult / adult, but preferably oral epithelial cells derived from an adult are used.
口腔上皮細胞からの細胞外小胞の単離は、当業者が任意の方法により行うことが可能である。例えば、口腔上皮細胞を培地中で培養することにより細胞外小胞が培地中に分泌されるため、培養後の培養物から細胞及び残渣を除いた培地(馴化培地)を回収した上で、周知の手法によりその馴化培地から口腔上皮細胞由来の細胞外小胞を単離することができる。
外 The isolation of extracellular vesicles from oral epithelial cells can be performed by those skilled in the art by any method. For example, since extracellular vesicles are secreted into the medium by culturing oral epithelial cells in the medium, a medium (conditioned medium) obtained by removing cells and residues from the culture after culture is collected and The extracellular vesicles derived from oral epithelial cells can be isolated from the conditioned medium by the technique described in (1).
細胞の培養に使用される培地及び培養条件などは特に限定されないが、好ましくは液体培地、例えば高グルコースダルベッコ改変イーグル培地が使用される。培養に必要な他の成分を培地に適宜添加してもよく、例えばF-12 Hamなどの栄養混合物、ソル・コーテフ等のコルチコステロイド、抗生物質、自家血清等を含めてもよい。培養の条件は特に限定されず、使用する細胞や必要とする細胞外小胞の量に応じて当業者が適宜設定することが可能であるが、例えば33~40℃、好ましくは35~38℃、より好ましくは37℃の温度で、12時間~25日間、好ましくは1~20日間、より好ましくは3~18日間培養される。必要により培養の途中で培地交換を行ってもよく、それらの培地物はその後まとめて使用することができる。上記の培養を行った後、遠心分離又は濾過などにより培養物から細胞を分離することにより馴化培地が回収される。回収した馴化培地についてさらに遠心分離又は濾過を行って細胞残渣等の不純物をさらに取り除いてもよい。遠心分離及び濾過の条件は当業者が適宜決定することが可能であるが、例えば、培養物をまず200xg~400xg、好ましくは250xg~350xgで10分程度、遠心分離して細胞を分離し、その後2500xg~3500xgで再度遠心分離するか、例えば0.22μmの濾過を行うことにより細かい不純物を除去することができる。
培 地 The medium used for culturing the cells and the culture conditions are not particularly limited, but a liquid medium, for example, a high glucose Dulbecco's modified Eagle medium is preferably used. Other components necessary for the culture may be appropriately added to the medium, and may include, for example, a nutrient mixture such as F-12 @ Ham, a corticosteroid such as Sol Cortev, an antibiotic, an autologous serum, and the like. Culture conditions are not particularly limited, and can be appropriately set by those skilled in the art according to the cells to be used and the amount of extracellular vesicles required. For example, 33 to 40 ° C, preferably 35 to 38 ° C And more preferably at a temperature of 37 ° C. for 12 hours to 25 days, preferably 1 to 20 days, more preferably 3 to 18 days. If necessary, the medium may be changed during the culturing, and these mediums can be used collectively thereafter. After the above culture, the conditioned medium is recovered by separating the cells from the culture by centrifugation or filtration. The collected conditioned medium may be further subjected to centrifugation or filtration to further remove impurities such as cell residues. The conditions of centrifugation and filtration can be appropriately determined by those skilled in the art.For example, first, the culture is centrifuged at 200 × g to 400 × g, preferably at 250 × g to 350 × g for about 10 minutes, and the cells are separated by centrifugation. Fine impurities can be removed by centrifugation again at 2500 × g to 3500 × g or by filtration at, for example, 0.22 μm.
前述の通り、馴化培地から細胞外小胞を単離する方法は周知であって特に限定されず、当業者が任意の手法により単離することができる。必要により限外濾過等によって培地を濃縮した後、例えば、サイズ排除クロマトグラフィー、超遠心法、精密濾過法、密度勾配遠心法などを単独で、または適宜組み合わせて実施することができる。好ましくは、限外濾過を行って濃縮した培地についてサイズ排除クロマトグラフィーを行うことにより細胞外小胞が単離される。限外濾過には、例えばメルクミリポア社のアミコン(100kDa、1000kDa等)等を製造者の指示手順に従って用いることが可能である。
As described above, the method for isolating extracellular vesicles from a conditioned medium is well known and is not particularly limited, and those skilled in the art can isolate them by any method. If necessary, after the medium is concentrated by ultrafiltration or the like, for example, size exclusion chromatography, ultracentrifugation, microfiltration, density gradient centrifugation, or the like can be performed alone or in an appropriate combination. Preferably, extracellular vesicles are isolated by performing size exclusion chromatography on the concentrated medium after ultrafiltration. For ultrafiltration, for example, Amicon (100 kDa, 1000 kDa, etc.) from Merck Millipore can be used according to the manufacturer's instructions.
単離される細胞外小胞は、好ましくはエクソソームを含む。一般に細胞外小胞の種類の区別(エクソソーム、マイクロベシクル等)は必ずしもすべて学術的に明確にされておらず、さらに特定の種類の細胞外小胞を選択的に単離することは技術的に容易ではないため、いずれの手法を用いて細胞外小胞体を単離したとしても通常はエクソソームを含むこととなる。従って、本発明の実施において、細胞外小胞を単離するための態様や条件は、細胞外小胞の種類を厳密に区別して特定のものを標的とするようなものに限定されることはない。
外 The isolated extracellular vesicle preferably contains exosomes. In general, the distinction between types of extracellular vesicles (exosomes, microvesicles, etc.) is not always scientifically clarified, and it is technically necessary to selectively isolate specific types of extracellular vesicles. Because it is not easy, the extracellular endoplasmic reticulum will usually contain exosomes regardless of which technique is used to isolate it. Therefore, in the practice of the present invention, the modes and conditions for isolating extracellular vesicles are not limited to those that target the specific one by strictly distinguishing the type of extracellular vesicle. Absent.
しかしながら、実施する個別の単離手法について、その分離精度が許容するのであれば、細胞外小胞のうちエクソソームが濃縮されるような条件で単離操作を行ってもよく、また、エクソソームを濃縮する工程を別途追加で行ってもよい。例えば、サイズ排除クロマトグラフィーにより細胞外小胞を単離する場合、30~200nm、好ましくは40~150nm、さらに好ましくは80~130mmのサイズの物質を含む画分を回収することによって、エクソソームの割合を上昇させてエクソソームの濃縮することができる。
However, if the separation accuracy of the individual isolation technique to be performed is acceptable, the isolation operation may be performed under conditions such that exosomes are concentrated among extracellular vesicles, and exosomes may be concentrated. May be additionally performed. For example, in the case of isolating extracellular vesicles by size exclusion chromatography, the fraction containing exosomes having a size of 30 to 200 nm, preferably 40 to 150 nm, and more preferably 80 to 130 mm, is collected to obtain a ratio of exosomes. To increase the concentration of exosomes.
上記のように細胞外小胞を単離した後、その細胞外小胞を局所適用に適した媒体中に添加することにより本発明の局所適用組成物を得ることができる。媒体の種類は、身体に適用する態様に応じて液体、固体、半固体などから当業者が適宜選択することができ、例えば溶液、ゲル、ローション、クリーム、軟膏、硬膏とするための任意の媒体が挙げられる。また、本発明の局所適用組成物は、上記の態様について許容される任意のアジュバント、添加剤等をさらに含んでもよい。最終組成物中における細胞外小胞の濃度は、治療効果又は所望の効果を奏するのに有効な量となるように、組成物の用途や所望の効果等に基づいて適宜設定できるが、局所適用する部位の面積に応じて0.2μg~2mg/cm2、好ましくは2.0~200μg/cm2、さらに好ましくは10~100μg/cm2、特に好ましくは20~40μg/cm2の濃度となるように組成物中に添加することが好ましい。
After isolating the extracellular vesicle as described above, the topical composition of the present invention can be obtained by adding the extracellular vesicle to a medium suitable for topical application. The type of medium can be appropriately selected by those skilled in the art from liquids, solids, semi-solids, and the like depending on the mode of application to the body.For example, any medium for forming a solution, gel, lotion, cream, ointment, plaster Is mentioned. In addition, the topical composition of the present invention may further include any adjuvants, additives, and the like that are acceptable for the above embodiments. The concentration of extracellular vesicles in the final composition can be appropriately set based on the intended use of the composition, the desired effect, etc., so as to be an amount effective for producing a therapeutic effect or a desired effect. 0.2μg ~ 2mg / cm 2 according to the area of the site, preferably 2.0 ~ 200μg / cm 2, more preferably 10 ~ 100μg / cm 2, particularly preferably the composition at a concentration of 20 ~ 40μg / cm 2 It is preferable to add it to the product.
本発明における局所適用とは、本発明の組成物による効果を意図する身体の部位に直接作用する態様で適用することを意味し、好ましくは当該部位に接触する態様での使用を意味する。好ましくは、本発明の組成物は身体表面の上皮に塗布することにより適用される(固体の組成物を接触させることを含む)。局所適用はいわゆる外用を含むが、適用される部位は身体表面部(外部から直接視認可能な部位、例えば皮膚、角膜)への適用には限定されず、口腔、鼻腔、消化器官等の粘膜に適用する態様を含む。さらに、外科的処置等を通して内臓器官(心臓、肺、肝臓、腎臓、甲状腺、副甲状腺、膵臓、胆嚢など)に直接適用する態様も含む。好ましくは、本発明の組成物は皮膚又は粘膜に適用することが好ましく、これらの部位に塗布することにより適用される。
局 所 The topical application in the present invention means to apply in such a manner as to directly act on a site in the body where the effect of the composition of the present invention is intended, and preferably to use in such a mode as to contact the site. Preferably, the compositions of the invention are applied by application to the epithelium on the body surface (including contacting the solid composition). Topical application includes so-called topical application, but the application site is not limited to application to the body surface (a site directly visible from the outside, for example, skin, cornea), but to mucous membranes such as oral cavity, nasal cavity, and digestive organs. Includes aspects of application. Further, the present invention also includes a mode in which the method is directly applied to internal organs (heart, lung, liver, kidney, thyroid, parathyroid, pancreas, gallbladder, etc.) through a surgical procedure or the like. Preferably, the composition of the present invention is applied to the skin or mucous membrane, and is applied by applying to these sites.
本発明の組成物を身体の所定の部位に局所適用することにより、皮膚、粘膜、又は上皮を有する他の器官(心臓、肺、肝臓、腎臓、甲状腺、副甲状腺、膵臓、胆嚢など)の健康な組織構造又は機能を回復し、創傷治癒の促進や瘢痕の治療又は軽減等の効果を得ることができる。上記の態様として、例えば、皮膚又は粘膜の創傷の治癒を促進することより創傷又は疾患の状態を改善することができる。創傷としては任意の態様に基づくものが含まれ、外傷、手術痕、さらに熱傷を含む。この治癒の促進により、皮膚又は粘膜の痛みが緩和される共に、瘢痕を治療又は予防することによって適用部位の外観の見た目を向上させることができる。さらに、前述の通り繊維化が問題となる内臓器官(心臓、肺、肝臓、腎臓など)に適用することによって、該器官の線維化を治療又は予防することができる。
By topically applying the composition of the present invention to a predetermined part of the body, the health of skin, mucous membrane, or other organs having epithelium (heart, lung, liver, kidney, thyroid, parathyroid, pancreas, gallbladder, etc.) Recovery of various tissue structures or functions, and effects such as promotion of wound healing and treatment or reduction of scarring can be obtained. In the above embodiment, for example, a wound or disease state can be improved by promoting the healing of a wound of skin or mucous membrane. Wounds include those based on any aspect, including trauma, surgical scars, and even burns. By promoting this healing, the pain of the skin or mucous membrane is alleviated, and the appearance of the application site can be improved by treating or preventing scar. Further, as described above, by applying to internal organs where fibrosis is a problem (heart, lung, liver, kidney, etc.), fibrosis of the organ can be treated or prevented.
その他、本発明の組成物は、後述の実施例に示すように線維芽細胞を刺激して、肝細胞増殖因子(HGF)、血管内皮増殖因子(VEGF)、線維芽細胞増殖因子(FGF)、及び結合組織増殖因子(CTGF)等の増殖因子の放出を促進することが示されているため、組成物を所定の部位に適用することによって、上記の増殖因子が治療効果を有する任意の疾患又は状態を予防又は治療する効果が予想される。
In addition, the composition of the present invention stimulates fibroblasts, as shown in the examples below, to give hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), And has been shown to enhance the release of growth factors such as connective tissue growth factor (CTGF), so that by applying the composition to a given site, any disease or disease in which the above growth factors have a therapeutic effect The effect of preventing or treating the condition is expected.
本発明の組成物を医薬品に配合することにより、上記の治療又は予防効果を有する医薬品を得ることができる。特に、後述の実施例に示すように、口腔上皮細胞由来の細胞外小胞は、骨髄間葉系間質細胞由来の細胞外小胞とは対照的に腫瘍増殖を促進しないことが確認されているため、様々な疾患又は症状に対する医薬品に安全に配合することができる。
医 薬 品 By blending the composition of the present invention with a drug, a drug having the above-mentioned therapeutic or preventive effect can be obtained. In particular, as shown in the Examples below, it has been confirmed that extracellular vesicles derived from oral epithelial cells do not promote tumor growth in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells. Therefore, it can be safely compounded with pharmaceuticals for various diseases or symptoms.
さらに、本発明の組成物は、皮膚等の組織の状態を改善し、組織修復を促進することによる表皮の若返り効果を有するため、化粧品に配合することによって健康な皮膚に対しても好ましい効果をもたらすことができる。
以下、実施例により本発明をより詳細に説明するが、本発明はこれらの実施例の態様に限定されるものではない。 Furthermore, the composition of the present invention improves the condition of tissues such as the skin and has a rejuvenating effect on the epidermis by promoting tissue repair. Can bring.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
以下、実施例により本発明をより詳細に説明するが、本発明はこれらの実施例の態様に限定されるものではない。 Furthermore, the composition of the present invention improves the condition of tissues such as the skin and has a rejuvenating effect on the epidermis by promoting tissue repair. Can bring.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
1.細胞外小胞の単離
健常成人ドナーの口内から生検で単離した細胞から臨床グレードの口腔粘膜細胞シートを製造する際に使用した馴化培地をCellSeed社より得た。提供を受けた馴化培地は、ケラチノサイト培養培地(75%の高グルコースダルベッコ改変イーグル培地、25%の栄養混合物F-12 Ham、0.475g/LのL-グルタミン、5μg/mlのヒューマリンレギュラー(Humulin Regular)、0.4μg/mLのソル・コーテフ、1nMのコレラ毒素、2nMのT3、10ng/mLのEGF、40μg/mLのゲンスマイシン(Gensumycin)、0.14μg/mLのファンギゾンおよび5%の自家血清)で細胞を37℃で培養した際のものであり、培養の第5、8、10、12、13、14、15及び16日目における培地交換の際にそれぞれ8~12mLの馴化培地を回収したものを合わせて、細胞外小胞の単離に使用した。 1. Isolation of extracellular vesicles A conditioned medium used to produce a clinical grade oral mucosal cell sheet from cells isolated by biopsy from the mouth of a healthy adult donor was obtained from CellSeed. The provided conditioned medium was a keratinocyte culture medium (75% high glucose Dulbecco's modified Eagle medium, 25% nutrient mixture F-12 Ham, 0.475 g / L L-glutamine, 5 μg / ml Humulin Regular (Humulin Regular). Regular), 0.4 μg / mL sol Cortev, 1 nM cholera toxin, 2 nM T3, 10 ng / mL EGF, 40 μg / mL Gensumycin, 0.14 μg / mL fungizone and 5% autologous serum) When the cells were cultured at 37 ° C., 8 to 12 mL of the conditioned medium was collected when the medium was replaced on days 5, 8, 10, 12, 13, 14, 15, and 16 of the culture. The combination was used for the isolation of extracellular vesicles.
健常成人ドナーの口内から生検で単離した細胞から臨床グレードの口腔粘膜細胞シートを製造する際に使用した馴化培地をCellSeed社より得た。提供を受けた馴化培地は、ケラチノサイト培養培地(75%の高グルコースダルベッコ改変イーグル培地、25%の栄養混合物F-12 Ham、0.475g/LのL-グルタミン、5μg/mlのヒューマリンレギュラー(Humulin Regular)、0.4μg/mLのソル・コーテフ、1nMのコレラ毒素、2nMのT3、10ng/mLのEGF、40μg/mLのゲンスマイシン(Gensumycin)、0.14μg/mLのファンギゾンおよび5%の自家血清)で細胞を37℃で培養した際のものであり、培養の第5、8、10、12、13、14、15及び16日目における培地交換の際にそれぞれ8~12mLの馴化培地を回収したものを合わせて、細胞外小胞の単離に使用した。 1. Isolation of extracellular vesicles A conditioned medium used to produce a clinical grade oral mucosal cell sheet from cells isolated by biopsy from the mouth of a healthy adult donor was obtained from CellSeed. The provided conditioned medium was a keratinocyte culture medium (75% high glucose Dulbecco's modified Eagle medium, 25% nutrient mixture F-12 Ham, 0.475 g / L L-glutamine, 5 μg / ml Humulin Regular (Humulin Regular). Regular), 0.4 μg / mL sol Cortev, 1 nM cholera toxin, 2 nM T3, 10 ng / mL EGF, 40 μg / mL Gensumycin, 0.14 μg / mL fungizone and 5% autologous serum) When the cells were cultured at 37 ° C., 8 to 12 mL of the conditioned medium was collected when the medium was replaced on
回収した馴化培地を、300xg、10分間、4℃の遠心分離し、次いで、0.22μmの限外濾過によって細胞及び細胞残渣を取り除いた。限外濾過カラム(アミコン、メルクミリポア)100kDa(5,000xg、4℃)、次いで10kDa(7,500xg、4℃)により培地を濃縮し、細胞外小胞(エクソソームを含む)を遊離タンパク質から分離するためサイズ排除クロマトグラフィーカラム(qEVカラム、Izon)に加えた。カラム製造者の指示書に従い、画分7~9(各500μl)を細胞外小胞画分として回収した。
(4) The collected conditioned medium was centrifuged at 300 × g for 10 minutes at 4 ° C., and then cells and cell debris were removed by ultrafiltration at 0.22 μm. Concentrate the medium with an ultrafiltration column (Amicon, Merck Millipore) 100kDa (5,000xg, 4 ° C) and then 10kDa (7,500xg, 4 ° C) to separate extracellular vesicles (including exosomes) from free proteins It was applied to a size exclusion chromatography column (qEV column, Izon). Fractions 7-9 (500 μl each) were collected as extracellular vesicle fractions according to the column manufacturer's instructions.
2.総タンパク質濃度測定
Pierce BCAプロテインアッセイキット(ThermoFisher Scientific)を使用してタンパク質濃度を測定した。細胞外小胞をRIPA(Radioimmunoprecipitation assay)バッファーで1:1に希釈し、5分間(オン/オフ: 15秒/15秒、氷浴中、高セッティング(コスモバイオ・超音波破砕装置))で超音波処理した。超音波処理した細胞外小胞及び細胞溶解物をMilliQ水(メルクミリポア)で希釈し、BCA(ビシンコニン酸)アッセイを指示書に従って実施した。 2. Total protein concentration determination Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Dilute the extracellular vesicles 1: 1 with RIPA (Radioimmunoprecipitation assay) buffer, and ultrapure for 5 minutes (on / off: 15 seconds / 15 seconds, in an ice bath, high setting (Cosmo Bio / sonicator)) Sonicated. Sonicated extracellular vesicles and cell lysates were diluted in MilliQ water (Merck Millipore) and BCA (bicinchoninic acid) assay was performed according to the instructions.
Pierce BCAプロテインアッセイキット(ThermoFisher Scientific)を使用してタンパク質濃度を測定した。細胞外小胞をRIPA(Radioimmunoprecipitation assay)バッファーで1:1に希釈し、5分間(オン/オフ: 15秒/15秒、氷浴中、高セッティング(コスモバイオ・超音波破砕装置))で超音波処理した。超音波処理した細胞外小胞及び細胞溶解物をMilliQ水(メルクミリポア)で希釈し、BCA(ビシンコニン酸)アッセイを指示書に従って実施した。 2. Total protein concentration determination Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Dilute the extracellular vesicles 1: 1 with RIPA (Radioimmunoprecipitation assay) buffer, and ultrapure for 5 minutes (on / off: 15 seconds / 15 seconds, in an ice bath, high setting (Cosmo Bio / sonicator)) Sonicated. Sonicated extracellular vesicles and cell lysates were diluted in MilliQ water (Merck Millipore) and BCA (bicinchoninic acid) assay was performed according to the instructions.
3.細胞溶解物の調製
線維芽細胞(CC-2509、Lonza)および口腔ケラチノサイト(#2610、ScienCell)から細胞溶解物を得た。細胞懸濁液(細胞約 4x106個)を300xgで5分間遠心し、上清を捨て、ペレットをPBSに再懸濁して再度遠心した。ペレットを、1%のプロテアーゼ/ホスファターゼ阻害剤カクテル(Cell Signaling Technology)を含むRIPA(Radioimmunoprecipitation assay)バッファー(Sigma)に再懸濁し、氷浴中で5分間(15秒オン、15秒オフのインターバル)超音波処理した。次いで、サンプルを4℃、8000xgで10分間遠心し、上清を回収し、等量ずつに分けて以後の分析に使用した。
上記の通り調製した細胞外小胞及び細胞溶解物を使用して、分子生物学的手法、電子顕微鏡観察、ナノ粒子トラッキング解析により細胞外小胞の存在及び特性を確認・測定し、さらにそれらの機能をインビトロ研究で分析した。 3. Preparation of cell lysates Cell lysates were obtained from fibroblasts (CC-2509, Lonza) and oral keratinocytes (# 2610, ScienCell). The cell suspension (about 4 × 10 6 cells) was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in PBS and centrifuged again. The pellet is resuspended in RIPA (Radioimmunoprecipitation assay) buffer (Sigma) containing 1% protease / phosphatase inhibitor cocktail (Cell Signaling Technology) and placed in an ice bath for 5 minutes (15 second on, 15 second off interval) Sonicated. Next, the sample was centrifuged at 8000 × g for 10 minutes at 4 ° C., and the supernatant was collected and divided into equal amounts for use in the subsequent analysis.
Using the extracellular vesicles and cell lysates prepared as described above, confirm and measure the presence and properties of extracellular vesicles by molecular biological techniques, electron microscopy, and nanoparticle tracking analysis. Function was analyzed in in vitro studies.
線維芽細胞(CC-2509、Lonza)および口腔ケラチノサイト(#2610、ScienCell)から細胞溶解物を得た。細胞懸濁液(細胞約 4x106個)を300xgで5分間遠心し、上清を捨て、ペレットをPBSに再懸濁して再度遠心した。ペレットを、1%のプロテアーゼ/ホスファターゼ阻害剤カクテル(Cell Signaling Technology)を含むRIPA(Radioimmunoprecipitation assay)バッファー(Sigma)に再懸濁し、氷浴中で5分間(15秒オン、15秒オフのインターバル)超音波処理した。次いで、サンプルを4℃、8000xgで10分間遠心し、上清を回収し、等量ずつに分けて以後の分析に使用した。
上記の通り調製した細胞外小胞及び細胞溶解物を使用して、分子生物学的手法、電子顕微鏡観察、ナノ粒子トラッキング解析により細胞外小胞の存在及び特性を確認・測定し、さらにそれらの機能をインビトロ研究で分析した。 3. Preparation of cell lysates Cell lysates were obtained from fibroblasts (CC-2509, Lonza) and oral keratinocytes (# 2610, ScienCell). The cell suspension (about 4 × 10 6 cells) was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in PBS and centrifuged again. The pellet is resuspended in RIPA (Radioimmunoprecipitation assay) buffer (Sigma) containing 1% protease / phosphatase inhibitor cocktail (Cell Signaling Technology) and placed in an ice bath for 5 minutes (15 second on, 15 second off interval) Sonicated. Next, the sample was centrifuged at 8000 × g for 10 minutes at 4 ° C., and the supernatant was collected and divided into equal amounts for use in the subsequent analysis.
Using the extracellular vesicles and cell lysates prepared as described above, confirm and measure the presence and properties of extracellular vesicles by molecular biological techniques, electron microscopy, and nanoparticle tracking analysis. Function was analyzed in in vitro studies.
4.ウエスタンブロット
細胞溶解物又は細胞外小胞単離物をLaemmliサンプルバッファー(BioRad)で1:1に希釈し、95℃で5分間加熱し、その後氷上で冷却した。サンプルをNuPage 4-12% 1.5x15 ウェルゲル(ThermoFisher)上にロードし、電気泳動(200V、125mA、25分;パワーステーション 1000XP;Atto)を行い、その後、iBlot(Invitrogen)の11分プログラムを使用してタンパク質をニトロセルロースメンブレン(タンパク質iBlotゲル転写スタック、Invitrogen)上に転写した。その後のブロッキング、抗体インキュベーション、及び洗浄のステップは傾斜式シェーカー上で実施した。5% 粉ミルク(CellSignaling)を含むTBS-T(BioRad)を使用して60分間フィルターのブロッキングを行い、4℃、オーバーナイトで一次抗体に曝露した。オービタルシェーカー上でフィルターをTBS-Tで3x10分間洗浄し、室温で二次抗体に60分間曝露し、その後再度3x10分間、TBS-Tで洗浄した。フィルターからバッファーを抜き、検出剤(ECL Prime WB 検出キット、Sigma-Aldrich)を加えて5分間置いた。その後、LAS 4000 mini(富士フィルム)を使用してタンパク質の発現を確認した。細胞外小胞の画分由来のサンプルは、国際的に認められたマーカー(CD9及びフロチリン)について陽性であり、陰性マーカーGRP94について陰性であった(図1)。
使用した抗体及び濃度は以下の通り:CD9(#13174、Cell Signaling Technologies)、Flotillin(#18634、Cell Signaling Technologies)、GRP94(#2104、Cell Signaling Technologies)、抗-ウサギIgG、HRP-linked(#7074、Cell Signaling Technologies、1:5000)。抗体はCan Get Signal solution(東洋紡)中に希釈した。各メンブレンをストリッピングして、異なる抗体で再使用した:30分、2% SDS、0.8%β-メルカプトエタノール、Tris-HCl(pH 6.6)、50℃、その後、TBS-Tで3x10分間洗浄した。 4. Western blot cell lysates or extracellular vesicle isolates were diluted 1: 1 with Laemmli sample buffer (BioRad), heated at 95 ° C. for 5 minutes, and then cooled on ice. Samples were loaded onto NuPage 4-12% 1.5x15 well gels (ThermoFisher), electrophoresed (200V, 125mA, 25 minutes; Power Station 1000XP; Atto), and then used with the 11 minute program of iBlot (Invitrogen). Proteins were transferred onto a nitrocellulose membrane (Protein iBlot gel transfer stack, Invitrogen). Subsequent blocking, antibody incubation, and washing steps were performed on a tilt shaker. Filters were blocked for 60 minutes using TBS-T (BioRad) containing 5% milk powder (CellSignaling) and exposed to primary antibody overnight at 4 ° C. The filters were washed on an orbital shaker with TBS-T for 3 × 10 minutes, exposed to the secondary antibody for 60 minutes at room temperature, and then washed again with TBS-T for 3 × 10 minutes. The buffer was removed from the filter, and a detection agent (ECL Prime WB detection kit, Sigma-Aldrich) was added and left for 5 minutes. Thereafter, protein expression was confirmed using LAS 4000 mini (Fuji Film). Samples from the extracellular vesicle fraction were positive for the internationally recognized markers (CD9 and flotilin) and negative for the negative marker GRP94 (FIG. 1).
The antibodies and concentrations used were as follows: CD9 (# 13174, Cell Signaling Technologies), Flotillin (# 18634, Cell Signaling Technologies), GRP94 (# 2104, Cell Signaling Technologies), anti-rabbit IgG, HRP-linked (# 7074, Cell Signaling Technologies, 1: 5000). Antibodies were diluted in Can Get Signal solution (Toyobo). Each membrane was stripped and reused with a different antibody: 30 minutes, 2% SDS, 0.8% β-mercaptoethanol, Tris-HCl (pH 6.6), 50 ° C., then washed with TBS-T for 3 × 10 minutes .
細胞溶解物又は細胞外小胞単離物をLaemmliサンプルバッファー(BioRad)で1:1に希釈し、95℃で5分間加熱し、その後氷上で冷却した。サンプルをNuPage 4-12% 1.5x15 ウェルゲル(ThermoFisher)上にロードし、電気泳動(200V、125mA、25分;パワーステーション 1000XP;Atto)を行い、その後、iBlot(Invitrogen)の11分プログラムを使用してタンパク質をニトロセルロースメンブレン(タンパク質iBlotゲル転写スタック、Invitrogen)上に転写した。その後のブロッキング、抗体インキュベーション、及び洗浄のステップは傾斜式シェーカー上で実施した。5% 粉ミルク(CellSignaling)を含むTBS-T(BioRad)を使用して60分間フィルターのブロッキングを行い、4℃、オーバーナイトで一次抗体に曝露した。オービタルシェーカー上でフィルターをTBS-Tで3x10分間洗浄し、室温で二次抗体に60分間曝露し、その後再度3x10分間、TBS-Tで洗浄した。フィルターからバッファーを抜き、検出剤(ECL Prime WB 検出キット、Sigma-Aldrich)を加えて5分間置いた。その後、LAS 4000 mini(富士フィルム)を使用してタンパク質の発現を確認した。細胞外小胞の画分由来のサンプルは、国際的に認められたマーカー(CD9及びフロチリン)について陽性であり、陰性マーカーGRP94について陰性であった(図1)。
使用した抗体及び濃度は以下の通り:CD9(#13174、Cell Signaling Technologies)、Flotillin(#18634、Cell Signaling Technologies)、GRP94(#2104、Cell Signaling Technologies)、抗-ウサギIgG、HRP-linked(#7074、Cell Signaling Technologies、1:5000)。抗体はCan Get Signal solution(東洋紡)中に希釈した。各メンブレンをストリッピングして、異なる抗体で再使用した:30分、2% SDS、0.8%β-メルカプトエタノール、Tris-HCl(pH 6.6)、50℃、その後、TBS-Tで3x10分間洗浄した。 4. Western blot cell lysates or extracellular vesicle isolates were diluted 1: 1 with Laemmli sample buffer (BioRad), heated at 95 ° C. for 5 minutes, and then cooled on ice. Samples were loaded onto NuPage 4-12% 1.5x15 well gels (ThermoFisher), electrophoresed (200V, 125mA, 25 minutes; Power Station 1000XP; Atto), and then used with the 11 minute program of iBlot (Invitrogen). Proteins were transferred onto a nitrocellulose membrane (Protein iBlot gel transfer stack, Invitrogen). Subsequent blocking, antibody incubation, and washing steps were performed on a tilt shaker. Filters were blocked for 60 minutes using TBS-T (BioRad) containing 5% milk powder (CellSignaling) and exposed to primary antibody overnight at 4 ° C. The filters were washed on an orbital shaker with TBS-T for 3 × 10 minutes, exposed to the secondary antibody for 60 minutes at room temperature, and then washed again with TBS-T for 3 × 10 minutes. The buffer was removed from the filter, and a detection agent (ECL Prime WB detection kit, Sigma-Aldrich) was added and left for 5 minutes. Thereafter, protein expression was confirmed using LAS 4000 mini (Fuji Film). Samples from the extracellular vesicle fraction were positive for the internationally recognized markers (CD9 and flotilin) and negative for the negative marker GRP94 (FIG. 1).
The antibodies and concentrations used were as follows: CD9 (# 13174, Cell Signaling Technologies), Flotillin (# 18634, Cell Signaling Technologies), GRP94 (# 2104, Cell Signaling Technologies), anti-rabbit IgG, HRP-linked (# 7074, Cell Signaling Technologies, 1: 5000). Antibodies were diluted in Can Get Signal solution (Toyobo). Each membrane was stripped and reused with a different antibody: 30 minutes, 2% SDS, 0.8% β-mercaptoethanol, Tris-HCl (pH 6.6), 50 ° C., then washed with TBS-T for 3 × 10 minutes .
5.透過型電子顕微鏡観察
10μLの細胞外小胞懸濁液を銅グリッド上に20分間置き、さらに2%酢酸ウラニルを加えて1分間置いた。酢酸ウラニルを取り除き、蒸留水を加えて1分間置き、その後取り除いた。グリッドをそのまま15分間空気乾燥させ、透過型電子顕微鏡(H7650、日立)を使用して細胞外小胞(エクソソーム)の形態を確認した(図2)。 5. Observation by transmission electron microscopy 10 μL of the extracellular vesicle suspension was placed on a copper grid for 20 minutes, and 2% uranyl acetate was added thereto for 1 minute. The uranyl acetate was removed, distilled water was added and left for 1 minute, then removed. The grid was air-dried for 15 minutes and the morphology of extracellular vesicles (exosomes) was confirmed using a transmission electron microscope (H7650, Hitachi) (FIG. 2).
10μLの細胞外小胞懸濁液を銅グリッド上に20分間置き、さらに2%酢酸ウラニルを加えて1分間置いた。酢酸ウラニルを取り除き、蒸留水を加えて1分間置き、その後取り除いた。グリッドをそのまま15分間空気乾燥させ、透過型電子顕微鏡(H7650、日立)を使用して細胞外小胞(エクソソーム)の形態を確認した(図2)。 5. Observation by transmission electron microscopy 10 μL of the extracellular vesicle suspension was placed on a copper grid for 20 minutes, and 2% uranyl acetate was added thereto for 1 minute. The uranyl acetate was removed, distilled water was added and left for 1 minute, then removed. The grid was air-dried for 15 minutes and the morphology of extracellular vesicles (exosomes) was confirmed using a transmission electron microscope (H7650, Hitachi) (FIG. 2).
6.ナノ粒子トラッキング解析
LM10 ナノ粒子解析装置(NanoSight)を用い、製造者の指示に従って細胞外小胞のサイズ及び数を測定した。小胞のサイズは約125nm(124.8±4.1nm(標準偏差))であった。 6. Nanoparticle tracking analysis The size and number of extracellular vesicles were measured using the LM10 Nanoparticle Analyzer (NanoSight) according to the manufacturer's instructions. Vesicle size was approximately 125 nm (124.8 ± 4.1 nm (standard deviation)).
LM10 ナノ粒子解析装置(NanoSight)を用い、製造者の指示に従って細胞外小胞のサイズ及び数を測定した。小胞のサイズは約125nm(124.8±4.1nm(標準偏差))であった。 6. Nanoparticle tracking analysis The size and number of extracellular vesicles were measured using the LM10 Nanoparticle Analyzer (NanoSight) according to the manufacturer's instructions. Vesicle size was approximately 125 nm (124.8 ± 4.1 nm (standard deviation)).
7.線維芽細胞増殖アッセイ
NHDFp13の細胞(CC-2509、Lonza)を、1,600個/ウェルの濃度で96-ウェルプレート中に播種した。翌日、培地を、細胞外小胞(2μg/mL、0.67μg/mL、0.2μg/mL)を含む血清非含有培地、細胞外小胞を含まないコントロール培地又はデキサメタゾン(10nm)含有培地に交換した。72時間後に、CCKキット(同仁化学研究所)を使用して細胞の代謝を測定した。細胞外小胞含有のサンプルは、抗瘢痕剤として一般に使用されるコルチコステロイドであるデキサメタゾンと同様に線維芽細胞の増殖を抑制することが確認された(図3)。 7. Fibroblast Proliferation Assay Cells of NHDFp13 (CC-2509, Lonza) were seeded in 96-well plates at a concentration of 1,600 cells / well. On the next day, the medium was replaced with a serum-free medium containing extracellular vesicles (2 μg / mL, 0.67 μg / mL, 0.2 μg / mL), a control medium containing no extracellular vesicles, or a medium containing dexamethasone (10 nm). . After 72 hours, cell metabolism was measured using a CCK kit (Dojindo Laboratories). It was confirmed that the sample containing extracellular vesicles suppressed the proliferation of fibroblasts in the same manner as dexamethasone, a corticosteroid generally used as an anti-scarring agent (FIG. 3).
NHDFp13の細胞(CC-2509、Lonza)を、1,600個/ウェルの濃度で96-ウェルプレート中に播種した。翌日、培地を、細胞外小胞(2μg/mL、0.67μg/mL、0.2μg/mL)を含む血清非含有培地、細胞外小胞を含まないコントロール培地又はデキサメタゾン(10nm)含有培地に交換した。72時間後に、CCKキット(同仁化学研究所)を使用して細胞の代謝を測定した。細胞外小胞含有のサンプルは、抗瘢痕剤として一般に使用されるコルチコステロイドであるデキサメタゾンと同様に線維芽細胞の増殖を抑制することが確認された(図3)。 7. Fibroblast Proliferation Assay Cells of NHDFp13 (CC-2509, Lonza) were seeded in 96-well plates at a concentration of 1,600 cells / well. On the next day, the medium was replaced with a serum-free medium containing extracellular vesicles (2 μg / mL, 0.67 μg / mL, 0.2 μg / mL), a control medium containing no extracellular vesicles, or a medium containing dexamethasone (10 nm). . After 72 hours, cell metabolism was measured using a CCK kit (Dojindo Laboratories). It was confirmed that the sample containing extracellular vesicles suppressed the proliferation of fibroblasts in the same manner as dexamethasone, a corticosteroid generally used as an anti-scarring agent (FIG. 3).
8.線維芽細胞の細胞毒性及びHGF-放出アッセイ
NHDFp15の細胞(CC-2509、Lonza)を、50,000個/ウェルの濃度で24-ウェルプレート中に播種した。翌日、培地を、2μg/mLの細胞外小胞を含む血清非含有培地又は細胞外小胞を含まない血清非含有培地に交換した。72時間後、培地を回収して-80℃で凍結し、CCKキット(同仁化学研究所)を使用して細胞の代謝を測定した結果、細胞外小胞は線維芽細胞に対して細胞毒性を示さないことが確認された(図4)。また、馴化培地中の肝細胞増殖因子(HGF)含有量をELISAキット(Abcam)により測定したところ、HGF含有量が上昇していることがタンパク質レベルで確認された(図5(b))。 8. Fibroblast cytotoxicity and HGF-release assay Cells of NHDFp15 (CC-2509, Lonza) were seeded at a concentration of 50,000 / well in 24-well plates. The next day, the medium was changed to a serum-free medium containing 2 μg / mL extracellular vesicles or a serum-free medium containing no extracellular vesicles. After 72 hours, the medium was collected, frozen at -80 ° C, and the cell metabolism was measured using the CCK kit (Dojindo Laboratories). As a result, extracellular vesicles showed cytotoxicity to fibroblasts. It was confirmed that it did not show (FIG. 4). Further, when the content of hepatocyte growth factor (HGF) in the conditioned medium was measured using an ELISA kit (Abcam), it was confirmed at the protein level that the HGF content was increased (FIG. 5 (b)).
NHDFp15の細胞(CC-2509、Lonza)を、50,000個/ウェルの濃度で24-ウェルプレート中に播種した。翌日、培地を、2μg/mLの細胞外小胞を含む血清非含有培地又は細胞外小胞を含まない血清非含有培地に交換した。72時間後、培地を回収して-80℃で凍結し、CCKキット(同仁化学研究所)を使用して細胞の代謝を測定した結果、細胞外小胞は線維芽細胞に対して細胞毒性を示さないことが確認された(図4)。また、馴化培地中の肝細胞増殖因子(HGF)含有量をELISAキット(Abcam)により測定したところ、HGF含有量が上昇していることがタンパク質レベルで確認された(図5(b))。 8. Fibroblast cytotoxicity and HGF-release assay Cells of NHDFp15 (CC-2509, Lonza) were seeded at a concentration of 50,000 / well in 24-well plates. The next day, the medium was changed to a serum-free medium containing 2 μg / mL extracellular vesicles or a serum-free medium containing no extracellular vesicles. After 72 hours, the medium was collected, frozen at -80 ° C, and the cell metabolism was measured using the CCK kit (Dojindo Laboratories). As a result, extracellular vesicles showed cytotoxicity to fibroblasts. It was confirmed that it did not show (FIG. 4). Further, when the content of hepatocyte growth factor (HGF) in the conditioned medium was measured using an ELISA kit (Abcam), it was confirmed at the protein level that the HGF content was increased (FIG. 5 (b)).
9.線維芽細胞における遺伝子発現解析
NHDFp14の細胞(CC-2509、Lonza)を、336,000個/ウェルの濃度で6-ウェルプレート中に播種した。翌日、培地を、2μg/mL細胞外小胞を含む血清非含有培地(コントロールのウェルについては細胞外小胞を含まない血清非含有培地)に交換した。72時間後、RNeasyキット(Qiagen)を使用してRNAを単離した。市販キット(OriGene)を使用してcDNAを合成し、TaqMan Fast Advanced Master Mix(ThermoFisher Scientific)を使用して、各種増殖因子について定量PCRを行った。
使用したプライマーは以下の通り:β-アクチン(4326315E-1112022、Applied Biosystem)、HGF(HS00300159_m1、ThermoFischer Scientific)、VEGFA(HS00900055_m1、ThermoFischer Scientific)、FGF2(HS00266645_m1、ThermoFischer Scientific)、CTGF(HS01026927_g1、ThermoFischer Scientific)。IV型コラーゲン解析については、継代5回、17回のNHDF細胞、及びプライマー Col4a1(HS00266237_m1、ThermoFischer Scientific)を使用して同様に行った。 9. Gene expression analysis in fibroblasts Cells of NHDFp14 (CC-2509, Lonza) were seeded at a concentration of 336,000 cells / well in a 6-well plate. The next day, the medium was replaced with serum-free medium containing 2 μg / mL extracellular vesicles (for control wells, serum-free medium without extracellular vesicles). After 72 hours, RNA was isolated using the RNeasy kit (Qiagen). CDNA was synthesized using a commercially available kit (OriGene), and quantitative PCR was performed for various growth factors using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific).
The primers used are as follows: β-actin (4326315E-1112022, Applied Biosystem), HGF (HS00300159_m1, ThermoFischer Scientific), VEGFA (HS00900055_m1, ThermoFischer Scientific), FGF2 (HS00266645_m1, ThermoFischer Scientific 269gFischer 010gFisher 010GFr) ). The type IV collagen analysis was similarly performed using NHDF cells at passages 5 and 17 and primer Col4a1 (HS00266237_m1, ThermoFischer Scientific).
NHDFp14の細胞(CC-2509、Lonza)を、336,000個/ウェルの濃度で6-ウェルプレート中に播種した。翌日、培地を、2μg/mL細胞外小胞を含む血清非含有培地(コントロールのウェルについては細胞外小胞を含まない血清非含有培地)に交換した。72時間後、RNeasyキット(Qiagen)を使用してRNAを単離した。市販キット(OriGene)を使用してcDNAを合成し、TaqMan Fast Advanced Master Mix(ThermoFisher Scientific)を使用して、各種増殖因子について定量PCRを行った。
使用したプライマーは以下の通り:β-アクチン(4326315E-1112022、Applied Biosystem)、HGF(HS00300159_m1、ThermoFischer Scientific)、VEGFA(HS00900055_m1、ThermoFischer Scientific)、FGF2(HS00266645_m1、ThermoFischer Scientific)、CTGF(HS01026927_g1、ThermoFischer Scientific)。IV型コラーゲン解析については、継代5回、17回のNHDF細胞、及びプライマー Col4a1(HS00266237_m1、ThermoFischer Scientific)を使用して同様に行った。 9. Gene expression analysis in fibroblasts Cells of NHDFp14 (CC-2509, Lonza) were seeded at a concentration of 336,000 cells / well in a 6-well plate. The next day, the medium was replaced with serum-free medium containing 2 μg / mL extracellular vesicles (for control wells, serum-free medium without extracellular vesicles). After 72 hours, RNA was isolated using the RNeasy kit (Qiagen). CDNA was synthesized using a commercially available kit (OriGene), and quantitative PCR was performed for various growth factors using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific).
The primers used are as follows: β-actin (4326315E-1112022, Applied Biosystem), HGF (HS00300159_m1, ThermoFischer Scientific), VEGFA (HS00900055_m1, ThermoFischer Scientific), FGF2 (HS00266645_m1, ThermoFischer Scientific 269gFischer 010gFisher 010GFr) ). The type IV collagen analysis was similarly performed using NHDF cells at
図5(a)に示すように、細胞外小胞は線維芽細胞を刺激して、肝細胞増殖因子(HGF)、血管内皮増殖因子A(VEGFA)、線維芽細胞増殖因子(FGF2)、及び結合組織増殖因子(CTGF)などの重要な増殖因子の遺伝子発現を有意に増加させた。なお、肝細胞増殖因子(HGF)の結果については、前述の図5(b)において肝細胞増殖因子放出の増加がタンパク質レベルで確認されたELISAの結果とも対応した。また、図5(c)に示すように、古い(高継代)線維芽細胞は、若い(継代5回)線維芽細胞と比較して、IV型コラーゲン-遺伝子を低いレベルで発現した。しかしながら、「古い」線維芽細胞が一旦細胞外小胞に曝露されると「若い」細胞と同様の発現を示した。
As shown in FIG. 5 (a), extracellular vesicles stimulate fibroblasts, and hepatocyte growth factor (HGF), vascular endothelial growth factor A (VEGFA), fibroblast growth factor (FGF2), and Gene expression of important growth factors such as connective tissue growth factor (CTGF) was significantly increased. The results of hepatocyte growth factor (HGF) corresponded to the results of ELISA in which an increase in hepatocyte growth factor release was confirmed at the protein level in FIG. 5 (b) described above. In addition, as shown in FIG. 5 (c), the old (high passage) fibroblasts expressed the type IV collagen-gene at a lower level than the young (5 passages) fibroblasts. However, once "old" fibroblasts were exposed to extracellular vesicles, they showed similar expression as "young" cells.
10.癌細胞増殖試験
市販の扁平上皮癌細胞(TR146)を、10% FBS、1%抗生物質を含むDMEM中で培養した。細胞を、10,000個/cm2の密度で96-ウェルプレート中に播種し、翌日、培地を様々な濃度の細胞外小胞を含む血清非含有培地、又は、細胞外小胞を含まないコントロール培地と交換した。48時間後、Cell Counting Kit-8(CCK、同仁化学研究所)を用いて細胞数を測定した。その結果、口腔上皮細胞由来の細胞外小胞は、扁平上皮癌細胞(TR146)の増殖を濃度依存的に低減することが確認された(図6)。 10. Cancer cell proliferation test Commercially available squamous cell carcinoma cells (TR146) were cultured in DMEM containing 10% FBS and 1% antibiotics. Cells were seeded in 96-well plates at a density of 10,000 / cm 2, the next day, serum-free medium containing the extracellular vesicles of varying concentrations of the medium, or a control medium without extracellular vesicles Was replaced. After 48 hours, the number of cells was measured using Cell Counting Kit-8 (CCK, Dojindo Laboratories). As a result, it was confirmed that extracellular vesicles derived from oral epithelial cells reduced the proliferation of squamous cell carcinoma cells (TR146) in a concentration-dependent manner (FIG. 6).
市販の扁平上皮癌細胞(TR146)を、10% FBS、1%抗生物質を含むDMEM中で培養した。細胞を、10,000個/cm2の密度で96-ウェルプレート中に播種し、翌日、培地を様々な濃度の細胞外小胞を含む血清非含有培地、又は、細胞外小胞を含まないコントロール培地と交換した。48時間後、Cell Counting Kit-8(CCK、同仁化学研究所)を用いて細胞数を測定した。その結果、口腔上皮細胞由来の細胞外小胞は、扁平上皮癌細胞(TR146)の増殖を濃度依存的に低減することが確認された(図6)。 10. Cancer cell proliferation test Commercially available squamous cell carcinoma cells (TR146) were cultured in DMEM containing 10% FBS and 1% antibiotics. Cells were seeded in 96-well plates at a density of 10,000 / cm 2, the next day, serum-free medium containing the extracellular vesicles of varying concentrations of the medium, or a control medium without extracellular vesicles Was replaced. After 48 hours, the number of cells was measured using Cell Counting Kit-8 (CCK, Dojindo Laboratories). As a result, it was confirmed that extracellular vesicles derived from oral epithelial cells reduced the proliferation of squamous cell carcinoma cells (TR146) in a concentration-dependent manner (FIG. 6).
11.細胞外小胞のラベリング
蛍光メンブレン色素(PKH26-GL-1KT、Sigma Aldrich)で細胞外小胞をラベリングし、細胞外小胞を500μLの希釈液Cで希釈し、すぐに500μLの希釈液C(2μLのPKH26を含む)を再度加え、穏やかに混合しながら5分間のインキュベーション工程を行った。その後、2.5mLのPBSを加え、10kDaフィルター(アミコン)を使用して細胞外小胞を再濃縮した。この工程を合計3回行った。コントロールとして、細胞外小胞無しで全ての工程を同様に行った。 11. Labeling of extracellular vesicles The extracellular vesicles were labeled with a fluorescent membrane dye (PKH26-GL-1KT, Sigma Aldrich), the extracellular vesicles were diluted with 500 μL of diluent C, and immediately 500 μL of diluent C ( 2 μL of PKH26) was added again and an incubation step of 5 minutes was performed with gentle mixing. Thereafter, 2.5 mL of PBS was added, and the extracellular vesicles were re-concentrated using a 10 kDa filter (Amicon). This step was performed three times in total. As a control, all steps were performed without extracellular vesicles.
蛍光メンブレン色素(PKH26-GL-1KT、Sigma Aldrich)で細胞外小胞をラベリングし、細胞外小胞を500μLの希釈液Cで希釈し、すぐに500μLの希釈液C(2μLのPKH26を含む)を再度加え、穏やかに混合しながら5分間のインキュベーション工程を行った。その後、2.5mLのPBSを加え、10kDaフィルター(アミコン)を使用して細胞外小胞を再濃縮した。この工程を合計3回行った。コントロールとして、細胞外小胞無しで全ての工程を同様に行った。 11. Labeling of extracellular vesicles The extracellular vesicles were labeled with a fluorescent membrane dye (PKH26-GL-1KT, Sigma Aldrich), the extracellular vesicles were diluted with 500 μL of diluent C, and immediately 500 μL of diluent C ( 2 μL of PKH26) was added again and an incubation step of 5 minutes was performed with gentle mixing. Thereafter, 2.5 mL of PBS was added, and the extracellular vesicles were re-concentrated using a 10 kDa filter (Amicon). This step was performed three times in total. As a control, all steps were performed without extracellular vesicles.
12.ラット全層創傷治癒モデル
6週齢のオスのSprague Dawleyラット(n=3)を使用した。イソフルランを使用して知覚麻酔を起こさせ、ドミトール(0.375mg/kg)、ミダゾラム(2mg/kg)、酒石酸ブトルファノール(2.5mg/kg)の混合物を用いてその状態を維持した。背部の被毛を取り除き、5mm 生検トレパン(Kai メディカル)を使用して全層創傷(一匹あたりn=4)を作成した。20μl(4.44μg)の細胞外小胞懸濁液又はコントロール懸濁液を創傷に加え、45分間保持させ、デジタルカメラ及び実体顕微鏡(オリンパス社製:MVX10)を用いてイメージを取得し、その後、テガダーム創傷包帯を使用して創傷を被覆した。翌日、ラットを麻酔して写真撮影した後、1日目と同様に14μL(3.1μg)の細胞外小胞懸濁液を追加して創傷に加えた。
第3、4、5日目に動物を麻酔してイメージを取得し、第6日にと殺した。8mm生検トレパン(Kai メディカル)を用いて創傷を摘出して組織学分析に使用した。また、別途実施した同様の実験において、ラット(n=3)を第17日にと殺した。クリオスタット(MicroEdge)を用いて組織切片(8μm)を作製し、ヘマトキシリン・エオシン及びピクロシリウスレッド(Polysciences)で染色した。ピクロシリウスレッドで染色したスライドを偏光顕微鏡下で可視化し、盲検でイメージを評価した。
図7(a)に示すように、蛍光標識した細胞外小胞からのシグナルは、適用後5日までラットの創傷ベッドで確認された。また、図7(b)に示すように、ヘマトキシリン・エオシン及びピクロシリウスレッド-染色により示されるように、細胞外小胞は、ラットの全層皮膚創傷における創傷治癒を促進した。さらに、図8に示すように、盲検評価に基づいて、細胞外小胞による治療群は、第6日及び第17日において、コントロールと比較して有意に小さい創傷面積を示した。 12. Rat full-thickness wound healing model Six-week-old male Sprague Dawley rats (n = 3) were used. Perceptual anesthesia was induced using isoflurane and maintained with a mixture of domitol (0.375 mg / kg), midazolam (2 mg / kg), and butorphanol tartrate (2.5 mg / kg). The hair on the back was removed, and a full-thickness wound (n = 4 per animal) was made using a 5 mm biopsy trepan (Kai Medical). 20 μl (4.44 μg) of extracellular vesicle suspension or control suspension was added to the wound, kept for 45 minutes, and an image was obtained using a digital camera and a stereomicroscope (Olympus: MVX10). The wound was covered using a Tegaderm wound dressing. The next day, the rats were anesthetized and photographed, and then 14 μL (3.1 μg) of extracellular vesicle suspension was added to the wound as in Day 1.
Animals were anesthetized ondays 3, 4 and 5 and images were acquired and sacrificed on day 6. The wound was excised using an 8 mm biopsy trepan (Kai Medical) and used for histological analysis. In a similar experiment performed separately, rats (n = 3) were killed on day 17. Tissue sections (8 μm) were prepared using a cryostat (MicroEdge) and stained with hematoxylin and eosin and picrosirius red (Polysciences). Slides stained with picrosirius red were visualized under a polarizing microscope and the images were evaluated blind.
As shown in FIG. 7 (a), signals from the fluorescently labeled extracellular vesicles were observed in the wound bed of the rat until 5 days after application. Also, as shown in FIG. 7 (b), extracellular vesicles promoted wound healing in full-thickness skin wounds of rats, as indicated by hematoxylin-eosin and picrosirius red-staining. Furthermore, as shown in FIG. 8, based on the blinded evaluation, the treatment group with extracellular vesicles showed significantly smaller wound areas on days 6 and 17 compared to the control.
6週齢のオスのSprague Dawleyラット(n=3)を使用した。イソフルランを使用して知覚麻酔を起こさせ、ドミトール(0.375mg/kg)、ミダゾラム(2mg/kg)、酒石酸ブトルファノール(2.5mg/kg)の混合物を用いてその状態を維持した。背部の被毛を取り除き、5mm 生検トレパン(Kai メディカル)を使用して全層創傷(一匹あたりn=4)を作成した。20μl(4.44μg)の細胞外小胞懸濁液又はコントロール懸濁液を創傷に加え、45分間保持させ、デジタルカメラ及び実体顕微鏡(オリンパス社製:MVX10)を用いてイメージを取得し、その後、テガダーム創傷包帯を使用して創傷を被覆した。翌日、ラットを麻酔して写真撮影した後、1日目と同様に14μL(3.1μg)の細胞外小胞懸濁液を追加して創傷に加えた。
第3、4、5日目に動物を麻酔してイメージを取得し、第6日にと殺した。8mm生検トレパン(Kai メディカル)を用いて創傷を摘出して組織学分析に使用した。また、別途実施した同様の実験において、ラット(n=3)を第17日にと殺した。クリオスタット(MicroEdge)を用いて組織切片(8μm)を作製し、ヘマトキシリン・エオシン及びピクロシリウスレッド(Polysciences)で染色した。ピクロシリウスレッドで染色したスライドを偏光顕微鏡下で可視化し、盲検でイメージを評価した。
図7(a)に示すように、蛍光標識した細胞外小胞からのシグナルは、適用後5日までラットの創傷ベッドで確認された。また、図7(b)に示すように、ヘマトキシリン・エオシン及びピクロシリウスレッド-染色により示されるように、細胞外小胞は、ラットの全層皮膚創傷における創傷治癒を促進した。さらに、図8に示すように、盲検評価に基づいて、細胞外小胞による治療群は、第6日及び第17日において、コントロールと比較して有意に小さい創傷面積を示した。 12. Rat full-thickness wound healing model Six-week-old male Sprague Dawley rats (n = 3) were used. Perceptual anesthesia was induced using isoflurane and maintained with a mixture of domitol (0.375 mg / kg), midazolam (2 mg / kg), and butorphanol tartrate (2.5 mg / kg). The hair on the back was removed, and a full-thickness wound (n = 4 per animal) was made using a 5 mm biopsy trepan (Kai Medical). 20 μl (4.44 μg) of extracellular vesicle suspension or control suspension was added to the wound, kept for 45 minutes, and an image was obtained using a digital camera and a stereomicroscope (Olympus: MVX10). The wound was covered using a Tegaderm wound dressing. The next day, the rats were anesthetized and photographed, and then 14 μL (3.1 μg) of extracellular vesicle suspension was added to the wound as in Day 1.
Animals were anesthetized on
As shown in FIG. 7 (a), signals from the fluorescently labeled extracellular vesicles were observed in the wound bed of the rat until 5 days after application. Also, as shown in FIG. 7 (b), extracellular vesicles promoted wound healing in full-thickness skin wounds of rats, as indicated by hematoxylin-eosin and picrosirius red-staining. Furthermore, as shown in FIG. 8, based on the blinded evaluation, the treatment group with extracellular vesicles showed significantly smaller wound areas on
13.ブタ皮膚創傷治癒試験
ブタ(Japanese farm pig)(n=1)を、ドミトール、ミダゾラム及び酒石酸ブトルファノールで鎮静化し、セボフルランを用いて麻酔を維持した。背部の皮膚を剃り、ヨウ素で滅菌した。生検トレパン(Kai メディカル)を使用して5mmの皮膚創傷を作り、20μL(4.75μg)の細胞外小胞又はコントロール(PBS)懸濁液を創傷に加え、45分間保持させ、その後創傷をテガダームで覆った。7日後に電気メスを用いて創傷を解剖した。解剖したサンプルをクリオスタット(MicroEdge)により切片化し、ヘマトキシリン・エオシンで染色した。その結果、コントロール群について観察された再上皮化は限定的であったのに対して、細胞外小胞処置群については完全な再上皮化が確認された(図9の矢印)。また、細胞外小胞処置群について副作用の兆候はみられなかった。 13. Pig Skin Wound Healing Test Pigs (Japanese farm pig) (n = 1) were sedated with domitol, midazolam and butorphanol tartrate, and anesthesia was maintained using sevoflurane. The skin on the back was shaved and sterilized with iodine. A 5 mm skin wound is made using a biopsy trepan (Kai Medical) and 20 μL (4.75 μg) of extracellular vesicle or control (PBS) suspension is added to the wound and allowed to hold for 45 minutes, after which the wound is tegaderm Covered. Seven days later, the wound was dissected using an electric scalpel. Dissected samples were sectioned with a cryostat (MicroEdge) and stained with hematoxylin and eosin. As a result, re-epithelialization observed in the control group was limited, whereas complete re-epithelialization was confirmed in the extracellular vesicle-treated group (arrow in FIG. 9). No signs of side effects were observed in the extracellular vesicle treatment group.
ブタ(Japanese farm pig)(n=1)を、ドミトール、ミダゾラム及び酒石酸ブトルファノールで鎮静化し、セボフルランを用いて麻酔を維持した。背部の皮膚を剃り、ヨウ素で滅菌した。生検トレパン(Kai メディカル)を使用して5mmの皮膚創傷を作り、20μL(4.75μg)の細胞外小胞又はコントロール(PBS)懸濁液を創傷に加え、45分間保持させ、その後創傷をテガダームで覆った。7日後に電気メスを用いて創傷を解剖した。解剖したサンプルをクリオスタット(MicroEdge)により切片化し、ヘマトキシリン・エオシンで染色した。その結果、コントロール群について観察された再上皮化は限定的であったのに対して、細胞外小胞処置群については完全な再上皮化が確認された(図9の矢印)。また、細胞外小胞処置群について副作用の兆候はみられなかった。 13. Pig Skin Wound Healing Test Pigs (Japanese farm pig) (n = 1) were sedated with domitol, midazolam and butorphanol tartrate, and anesthesia was maintained using sevoflurane. The skin on the back was shaved and sterilized with iodine. A 5 mm skin wound is made using a biopsy trepan (Kai Medical) and 20 μL (4.75 μg) of extracellular vesicle or control (PBS) suspension is added to the wound and allowed to hold for 45 minutes, after which the wound is tegaderm Covered. Seven days later, the wound was dissected using an electric scalpel. Dissected samples were sectioned with a cryostat (MicroEdge) and stained with hematoxylin and eosin. As a result, re-epithelialization observed in the control group was limited, whereas complete re-epithelialization was confirmed in the extracellular vesicle-treated group (arrow in FIG. 9). No signs of side effects were observed in the extracellular vesicle treatment group.
上記の各実施例に示すように、ヒト口腔上皮細胞の産生する細胞外小胞を含む組成物を、ラットの皮膚(表皮、真皮)を切除した創傷モデルに直接塗布することにより、生理食塩水を塗布したコントロール群に比べて、創傷治癒が促進、瘢痕が軽減、さらに組織修復による若返り効果が認められた。
また、特に図6の癌細胞増殖試験の結果に示されるように、口腔上皮細胞由来の細胞外小胞は、骨髄間葉系間質細胞由来の細胞外小胞とは対照的に、腫瘍増殖を促進しないことが確認された。従って、本発明の組成物は、様々な疾患又は症状に対する医薬品に安全に配合することができる利点を有する。 As shown in each of the above Examples, physiological saline was applied by directly applying a composition containing extracellular vesicles produced by human oral epithelial cells to a wound model from which rat skin (epidermal, dermis) was excised. Compared to the control group to which was applied, wound healing was promoted, scarring was reduced, and a rejuvenating effect by tissue repair was observed.
In addition, as shown particularly in the results of the cancer cell proliferation test in FIG. 6, extracellular vesicles derived from oral epithelial cells, in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells, exhibited tumor growth. Was not promoted. Therefore, the composition of the present invention has an advantage that it can be safely formulated into a drug for various diseases or conditions.
また、特に図6の癌細胞増殖試験の結果に示されるように、口腔上皮細胞由来の細胞外小胞は、骨髄間葉系間質細胞由来の細胞外小胞とは対照的に、腫瘍増殖を促進しないことが確認された。従って、本発明の組成物は、様々な疾患又は症状に対する医薬品に安全に配合することができる利点を有する。 As shown in each of the above Examples, physiological saline was applied by directly applying a composition containing extracellular vesicles produced by human oral epithelial cells to a wound model from which rat skin (epidermal, dermis) was excised. Compared to the control group to which was applied, wound healing was promoted, scarring was reduced, and a rejuvenating effect by tissue repair was observed.
In addition, as shown particularly in the results of the cancer cell proliferation test in FIG. 6, extracellular vesicles derived from oral epithelial cells, in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells, exhibited tumor growth. Was not promoted. Therefore, the composition of the present invention has an advantage that it can be safely formulated into a drug for various diseases or conditions.
Claims (15)
- 口腔上皮細胞由来の細胞外小胞を含む、局所適用組成物。 (4) A composition for topical application comprising extracellular vesicles derived from oral epithelial cells.
- 細胞外小胞がエクソソームを含む、請求項1に記載の組成物。 The composition of claim 1, wherein the extracellular vesicle comprises an exosome.
- 細胞外小胞がエクソソームについて濃縮されている、請求項1又は2に記載の組成物。 The composition of claim 1 or 2, wherein the extracellular vesicles are enriched for exosomes.
- 口腔上皮細胞がヒト由来である、請求項1-3のいずれか1項に記載の組成物。 (4) The composition according to any one of (1) to (3), wherein the oral epithelial cells are derived from a human.
- 上皮への塗布により適用される、請求項1-4のいずれか1項に記載の組成物。 (5) The composition according to any one of (1) to (4), which is applied by application to an epithelium.
- 皮膚、粘膜、又は上皮を有する他の器官の健康な組織構造又は機能を回復するための、請求項1-5のいずれか1項に記載の組成物。 The composition according to any one of claims 1 to 5, for restoring healthy tissue structure or function of skin, mucous membrane, or another organ having epithelium.
- 肝細胞増殖因子、血管内皮増殖因子、線維芽細胞増殖因子、及び結合組織増殖因子からなる群から選択される1以上の増殖因子が治療効果を有する疾患又は状態を治療するための、請求項1-6のいずれか1項に記載の組成物。 2. For treating a disease or condition in which one or more growth factors selected from the group consisting of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, and connective tissue growth factor have a therapeutic effect. The composition according to any one of -6.
- 皮膚又は粘膜の創傷を治療するための、請求項1-7のいずれか1項に記載の組成物。 The composition according to any one of claims 1 to 7, for treating skin or mucous membrane wounds.
- 皮膚又は粘膜の瘢痕を治療又は予防するための、請求項1-7のいずれか1項に記載の組成物。 (8) The composition according to any one of (1) to (7) for treating or preventing scarring of skin or mucous membranes.
- 内臓器官の線維化を治療又は予防するための、請求項1-7のいずれか1項に記載の組成物。 The composition according to any one of claims 1 to 7, for treating or preventing fibrosis of internal organs.
- 請求項1-10のいずれか1項に記載の組成物を含む医薬品。 医 薬 品 A pharmaceutical comprising the composition according to any one of claims 1 to 10.
- 請求項1-10のいずれか1項に記載の組成物を含む化粧品。 化粧 Cosmetics containing the composition according to any one of claims 1 to 10.
- 口腔上皮細胞を培地中で培養する工程、
培養物から口腔上皮細胞を分離して馴化培地を回収する工程、
馴化培地から口腔上皮細胞の細胞外小胞を単離する工程、及び、
単離した細胞外小胞を局所適用に適した媒体中に添加する工程、
を含む、局所適用組成物の製造方法。 Culturing the oral epithelial cells in a medium,
Recovering the conditioned medium by separating the oral epithelial cells from the culture,
Isolating the extracellular vesicles of the oral epithelial cells from the conditioned medium, and
Adding the isolated extracellular vesicles to a medium suitable for topical application,
A method for producing a topical composition, comprising: - 細胞外小胞中のエクソソームを濃縮する工程をさらに含む、請求項13に記載の方法。 14. The method of claim 13, further comprising the step of enriching exosomes in extracellular vesicles.
- 上皮細胞がヒト由来である、請求項13又は14に記載の方法。 方法 The method according to claim 13 or 14, wherein the epithelial cells are of human origin.
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