WO2022113887A1 - Method for assessing inflammatory retinal disease, inflammatory retinal disease therapeutic, and screening method for inflammatory retinal disease therapeutics - Google Patents
Method for assessing inflammatory retinal disease, inflammatory retinal disease therapeutic, and screening method for inflammatory retinal disease therapeutics Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions
- the present invention relates to a method for determining an inflammatory retinal disease, a method for screening an inflammatory retinal disease therapeutic agent, and a method for screening an inflammatory retinal disease therapeutic agent.
- Extracellular fine particles such as exosomes secreted from cells contain lipids and proteins derived from cell membranes on the surface, and nucleic acids derived from intracellular substances (microRNA, messenger RNA, DNA, etc.), proteins, etc. inside. Includes.
- the microRNA (miRNA) contained in the extracellular fine particles is a non-coding RNA consisting of about 17 to 24 nucleotides that is not translated into protein.
- miRNA Currently, more than 2500 types of human miRNA have been discovered. This miRNA has the function of suppressing the translation of various genes having target sites complementary to itself, and controls basic biological functions such as cell development, differentiation, proliferation, and cell death. .. In this way, since microRNA regulates the expression of many genes in cells, changes in its expression are thought to be related to the development and progression of diseases. Research is also being conducted to use it for discovery.
- Patent Document 1 describes a method for detecting bladder cancer using 27 types of miRNA whose expression fluctuates in bladder cancer as an index.
- miRNA is closely related to diseases such as cancer, and it is considered that miRNA may be used for their diagnosis, prognosis prediction, and the like.
- age-related macular degeneration is one of the chronic inflammatory retinal diseases and is a major cause of blindness in the elderly in developed countries (see Non-Patent Document 3).
- various abnormalities occur in the macula in the center of the retina, causing a decrease in visual acuity.
- Dyschromia and drusen (deposits) in the macula are seen in the early stage of the disease, but the detection of the disease is often delayed because there are few subjective symptoms.
- abnormal angiogenesis occurs in the macula, and the disorder progresses, causing serious deterioration of visual acuity. Therefore, diagnosis and prognosis of age-related macular degeneration should be made easily and with high accuracy in the early stage of the disease. There is a need for a way to do this.
- the present invention provides a novel determination method capable of early detection of chronic inflammatory retinal disease such as age-related macular degeneration, determination of severity, determination of therapeutic effect, etc. under the above-mentioned circumstances.
- the purpose is. It is also an object of the present invention to provide a novel therapeutic agent for inflammatory retinal diseases such as age-related macular degeneration. Furthermore, it is also an object to provide a new screening method for a therapeutic agent for inflammatory retinal disease.
- the gist of the present invention is as follows.
- [1] A method for determining an inflammatory retinal disease accompanied by tissue inflammation, using extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index.
- RPE retinal pigment epithelial cells
- [2] The determination method according to [1], wherein the profile of miRNA contained in the extracellular fine particles (EV) is used as an index.
- [3] The determination method according to [2], wherein the miRNA contains miR494-3p and / or miR1246.
- the determination method according to any one of [1] to [4], which is an inflammatory disease.
- a therapeutic agent for an inflammatory retinal disease accompanied by tissue inflammation which contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
- RPE retinal pigment epithelial cells
- the inflammation according to [7] or [8], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miR494-3p and / or miR1246 contained in the extracellular fine particles (EV).
- the function-regulating molecule of the extracellular fine particle (EV) is any one of [7] to [9], which is at least one selected from the group consisting of nucleic acid oligos, small molecule compounds, peptides and antibodies.
- the described inflammatory retinal disease therapeutic agent is at least one selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, and diabetic retinopathy.
- the agent for treating an inflammatory retinal disease according to any one of [7] to [10], which is a disease.
- Inflammation associated with tissue inflammation characterized by variability in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages.
- RPE retinal pigment epithelial cells
- a screening method for therapeutic agents for sexual retinal diseases [13] The screening method according to [12], wherein the extracellular fine particles (EV) are secreted from retinal pigment epithelial cells (RPE).
- RPE retinal pigment epithelial cells
- the screening method according to [12] or [13], wherein the variation in the profile of the extracellular fine particles (EV) is a variation in the profile of miRNA contained in the extracellular fine particles (EV).
- the possibility, severity, therapeutic effect, etc. of an inflammatory retinal disease such as age-related macular degeneration can be easily determined with high accuracy. be able to.
- Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention.
- the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as.
- the screening method of the present invention it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.
- the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention uses extracellular fine particles (EV) produced by the retinal pigment epithelial cells (RPE) of a subject as an index.
- EV extracellular fine particles
- extracellular fine particles are fine particles released by cells and are vesicles that can be confirmed with an electron microscope.
- the extracellular fine particles (EV) contain nucleic acids (microRNA, messenger RNA, DNA, etc.) derived from intracellular substances, proteins, etc., and are covered with a lipid double layer composed of cell-derived lipids and proteins. ..
- Extracellular microparticles (EVs) are their diameter, intracellular origin, density of microparticles in sucrose, shape, sedimentation rate, lipid composition, protein markers and mode of secretion (ie, after signaling (inducible) or spontaneously (ie). It is classified into multiple types based on structural)) and so on.
- extracellular fine particles examples include membrane particles, membrane vesicles, microvesicles, exosome-like vesicles, exosomes, ectome-like vesicles, ectosomes, exovesicles and the like.
- exosomes are vesicles contained in blood, lymph, saliva, urine, breast milk, semen, etc., and released by substantially spherical cells distributed around a diameter of about 100 nm.
- Molecules such as proteins, mRNA, and miRNA are contained inside the vesicle.
- Exosomes are sometimes referred to as microvesicles or extracellular vesicles, and there are a plurality of names.
- the particles are fractionated into 1.13 to 1.19 g / mL, and the particle diameter (diameter) by the dynamic light scattering method or the like is contained in the range of about 40 nm to about 150 nm.
- CD9, CD63, CD81 and the like are expressed on the membrane of exosomes.
- the amount of extracellular fine particles (EV) such as exosomes released increases, and in particular, the amount of CD63-positive exosomes released increases and specific microRNAs derived from these. Content increases.
- the amount of extracellular fine particles (EV) released such as exosomes is significantly increased, and in particular, the amount of CD63-positive exosomes released is further increased and specific micros contained therein are further increased.
- the RNA content is also increased. Therefore, by using the extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index, the possibility, severity, therapeutic effect, etc. of inflammatory retinal diseases such as age-related macular degeneration can be determined. It becomes possible to do.
- the above-mentioned "using extracellular fine particles (EV) as an index” means the amount of extracellular fine particles (EV) released, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles (EV). It means that the profile of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly miRNA (microRNA), is used as an index.
- a substance contained in EV particularly a nucleic acid derived from an intracellular substance, particularly miRNA (microRNA)
- miRNA miRNA
- the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and /
- the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable.
- the amount of the specific miRNA (microRNA) contained in the secreted extracellular fine particles (EV) is increased. Therefore, these can be used as indicators.
- the biological sample of the subject in the determination method of the present invention is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), but for example, the subject's anterior chamber water.
- EV extracellular fine particles
- RPE retinal pigment epithelial cells
- Examples include blood (plasma, serum), tissue fluid and the like.
- examples of inflammatory retinal diseases associated with tissue inflammation include age-related macular degeneration, central serous chorioretinal disease, proliferative vitreous retinopathy, and diabetic retinopathy.
- the disease to which the determination method of the present invention is particularly suitable is age-related macular degeneration.
- the possibility of inflammatory retinal disease, the severity of symptoms, the risk of onset, the risk of aggravation, the therapeutic effect, etc. can be determined.
- the higher the RNA content than the reference value the higher the possibility, severity, risk of developing, and risk of aggravation of inflammatory retinal disease associated with tissue inflammation in the subject.
- a reference value for determining the possibility of morbidity an average value, a numerical range, or the like in a healthy person can be used.
- the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
- the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention can also be described as a method including the following steps.
- Step of preparing a sample from a subject (2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly.
- Step of preparing a sample from a subject In this step, a biological sample is collected and prepared from a subject who is judged to have an inflammatory retinal disease accompanied by tissue inflammation.
- the sample from the subject is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), and examples thereof include the subject's anterior chamber water and blood.
- EV extracellular fine particles
- RPE retinal pigment epithelial cells
- miRNA miRNA
- microRNA miRNA
- various exosome separation kits pellet down by centrifugation, immunoprecipitation, magnetic
- Purification with beads, fractionation by particle size, column adsorption, etc. is used to separate extracellular fine particles (EV), and the amount of obtained extracellular fine particles (EV) is measured.
- the method for separating and recovering extracellular fine particles includes, for example, ultracentrifuging a sample of a subject at about 50,000 to 150,000 G for 0.5 to 2 hours. Prior to ultracentrifugation, the sample can be centrifuged for 0.1-2 hours at about 100-20,000 G. Extracellular fine particles (EV) are freeze-dried at 4 ° C for about 1 week, -20 ° C for about 1 month, and -80 ° C for about 6 months if they are dissolved in a solution such as PBS. If there is, it can be stored at 4 ° C for about 3 years.
- the method for measuring the amount of extracellular fine particles (EV) is not particularly limited, but for example, a method for measuring the number of particles of extracellular fine particles (EV) in a liquid with a NanoSign LM10V-HS nanoparticle tracking system, Examples include a method for measuring the amount of protein.
- a method for measuring the amount of CD63-positive exosomes in the obtained extracellular fine particles (EV) for example, protein quantification is performed according to a conventional method, and the amount of protein in each sample is adjusted to be equal to Western blotting. , CD63 is detected, and the amount of CD63 in each sample is analyzed.
- the profile of the contained microRNA (expression intensity) can be analyzed using 3D-gene human microRNA chips (manufactured by Toray Industries, Inc.) or the like.
- miRNA As the profile of the microRNA (miRNA), the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and / Alternatively, the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable.
- the numerical value obtained in the step (2) above is compared with the reference value.
- a reference value for determining the possibility of morbidity an average value, a numerical range, or the like in a healthy person can be used.
- the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
- the determination method of the present invention it is possible to easily diagnose and predict the prognosis of age-related macular degeneration in the early stage of the disease with high accuracy.
- the diagnosis and therapeutic effect of the possibility / risk of developing inflammatory retinal disease and the risk of severe / severe symptoms are determined.
- the judgment item is not limited to this.
- Other determination items include determination of pre-illness, initial lesion, or pre-stage (state in which symptoms are not clear) in which a definitive diagnosis can be made for each disease.
- the condition in which the visual field is lacking on the Amsler examination and new blood vessels are observed on the fluorescence fundus angiography is already severe age-related macular degeneration.
- the therapeutic agent for inflammatory retinal disease of the present invention contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
- RPE retinal pigment epithelial cells
- the amount of extracellular fine particles (EV) released and the above-mentioned specific miRNA (microRNA) contained therein Due to the increased amount, molecules that regulate (suppress or promote) these functions are expected to have a therapeutic effect on inflammatory retinal diseases associated with tissue inflammation.
- the retinal pigment epithelial cells When the amount of the specific miRNA (microRNA) released in the retinal pigment epithelial cells (RPE) of a subject who develops inflammatory retinal disease accompanied by tissue inflammation is increased, the retinal pigment epithelial cells (RPE) are used. While it is possible that the specific amount of miRNA in the tissue is excessive, the amount of the specific miRNA in the retinal pigment epithelial cells (RPE) (including those containing mitochondria) decreases due to the increase in the release amount. It is possible that it is. Therefore, it is considered that regulating (suppressing or promoting) the expression of these miRNAs according to the state of the disease of the subject leads to the treatment of the disease.
- microRNA specific miRNA released in the retinal pigment epithelial cells
- the function-regulating molecule of the extracellular fine particles (EV) is not particularly limited as long as it is a molecule that regulates the function of the extracellular fine particles (EV), but the production (release) of the extracellular fine particles (EV) is limited. ) Is a broad concept including molecules that regulate. Further, as a function-regulating molecule of extracellular fine particles (EV), a function-regulating molecule of microRNA (miRNA) contained in extracellular fine particles (EV) is given as a preferable example, and among them, extracellular fine particles (EV) are contained.
- miRNA microRNA
- a function-regulating molecule of miR494-3p, miR1246, miR4741 and / or miR7110-5p is more preferably a function-regulating molecule of miR494-3p, miR1246, miR4741 and / or miR7110-5p, further preferably a function-regulating molecule of miR494-3p and / or miR1246, and a function-regulating molecule of miR494-3p.
- these function-regulating molecules include nucleic acid oligos, small molecule compounds, peptides, antibodies, and the like, and specific examples thereof include nucleic acid oligos that regulate the function of the specific microRNA (miRNA). ..
- nucleic acid oligo introduction of mimic
- nucleic acid oligo introduction of inhibitor
- the nucleic acid oligo is preferably modified for the purpose of suppressing decomposition when administered to a living body and / or for the purpose of specifically binding to a diseased site.
- siRNA means a double-stranded RNA consisting of short strands in a range that does not show toxicity in cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.
- shRNA is RNA in which a single-stranded RNA constitutes a double strand via a hairpin structure.
- nucleic acid oligo in the present invention is an oligo composed of natural and unnatural RNA or DNA, and means a nucleic acid oligomer that controls the functions of miR494-3p, miR1246, miR4741 and / or miR7110-5p.
- Nucleic acid oligos in the present invention include miRNA, siRNA, shRNA, antisense nucleic acid, decoy nucleic acid, and nucleic acid aptamer.
- SiRNA and shRNA do not have to be exactly the same as the target gene, but have at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.
- the portion of double-stranded RNA in which RNAs are paired with each other in siRNA and shRNA is not limited to a completely paired one, but is not limited to a completely paired one, but is a mismatch (corresponding bases are not complementary) and bulge (base corresponding to one strand). It may include a non-matching portion due to (there is no) or the like. In the present invention, both the bulge and the mismatch may be contained in the double-stranded RNA region in which the RNAs in the dsRNA are paired with each other.
- the therapeutic agent for inflammatory retinal disease in the present invention may contain a pharmaceutically acceptable material such as a preservative or a stabilizer.
- a pharmaceutically acceptable material such as a preservative or a stabilizer.
- Acceptable means that the material itself may have the above-mentioned therapeutic effect or may not have the above-mentioned therapeutic effect, and may be the material that can be administered together with the above-mentioned therapeutic agent. Means material. Further, a material having no therapeutic effect and having a synergistic effect or an additive stabilizing effect when used in combination with a therapeutic agent for inflammatory retinal disease may be used.
- Examples of the material permitted in the formulation include sterile water, physiological saline, preservatives, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders, and the like. Can be done.
- an isotonic solution containing, for example, physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol) , Sodium chloride), suitable solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbitol 80, HCO-50), etc. May be good.
- a diluent, a solubilizing agent, a pH adjuster, a pain-relieving agent, a sulfur-containing reducing agent, an antioxidant and the like may be further contained.
- the therapeutic agent for inflammatory retinal disease of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylate]), or a colloidal drug delivery system (liposomes, albumin microspheres). , Microemulsions, nanoparticles and nanocapsules, etc.). Further, a method of making a drug a sustained release drug is also known and can be applied to the present invention.
- the pharmaceutically acceptable carrier to be used is appropriately selected from the above or in combination depending on the dosage form, but is not limited thereto.
- the therapeutic agent for inflammatory retinal disease of the present invention is used as a human medicine, it is possible to formulate and administer these substances by a known pharmaceutical method, in addition to directly administering these substances to patients. be.
- the above-mentioned materials that are acceptable for the formulation may be added.
- the therapeutic agent for inflammatory retinal disease in the present invention can be administered in the form of a pharmaceutical product, and can be administered orally or parenterally systemically or topically.
- intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral intestinal solvent, etc. can be selected, and the administration method should be appropriately selected according to the age and symptoms of the patient.
- the effective dose is selected in the range of 0.000001 mg to 1 g per 1 kg of body weight at a time.
- a dose of 0.00001 to 100 mg, preferably 0.0001 to 50 mg per patient can be selected.
- Examples of the inflammatory retinal disease in the therapeutic agent for inflammatory retinal disease of the present invention include age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, diabetic retinopathy and the like.
- the disease for which the therapeutic agent for inflammatory retinal disease of the present invention is particularly effective is age-related macular degeneration.
- the method for screening a therapeutic agent for an inflammatory retinal disease associated with tissue inflammation of the present invention is a variation in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. Is a feature.
- RPE retinal pigment epithelial cells
- monocytes / macrophages constructed by the present inventors as a system for mimicking the state of inflammatory retinal disease accompanied by tissue inflammation, extracellular fine particles secreted.
- the amount of (EV) increases, and the amount of the specific miRNA (microRNA) contained therein increases.
- the screening method of the present invention the amount of extracellular fine particles (EV) secreted in the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, and the above-mentioned specific miRNA (micro) contained therein.
- EV extracellular fine particles
- RPE retinal pigment epithelial cells
- micro macrophages
- Substances that are effective in reducing the amount of RNA) can be screened.
- the substance selected by the screening method of the present invention can be expected to have an effect of improving and treating the symptoms of inflammatory retinal disease accompanied by tissue inflammation.
- the specific miRNA is preferably mitochondrial-derived miR494-3p, miR1246, miR4741 and / or miR7115-5p, more preferably miR494-3p and / or miR1246p, and miR494-3p. It is even more preferable to have.
- the screening method of the present invention can also be described as a method including the following steps.
- Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (2) Step of adding a test substance to the co-culture system prepared in (1) and culturing for a certain period of time.
- RPE retinal pigment epithelial cells
- a substance contained in EV particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, the content of the above-mentioned specific miRNA (microRNA) derived from mitochondria
- microRNA miRNA
- Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages As the retinal pigment epithelial cell (RPE), a primary cultured cell of retinal pigment epithelial cell (RPE) collected from human retinal tissue, a cell established from these, or the like may be used, or from iPS cells. Retinal pigment epithelial cells (RPE) (iPS-hRPE cells) produced by induction may be used.
- RPE retinal pigment epithelial cells
- iPS-hRPE cells Retinal pigment epithelial cells
- monocytes / macrophages in this step monocytes / macrophages isolated from human blood, tissues, etc. may be used, or THP-1 cells, which are established cells, may be treated with PMA or the like by a conventional method.
- THP-1 cells which are established cells, may be treated with PMA or the like by a conventional method.
- Cells differentiated into macrophages may be used.
- the retinal pigment epithelial cells for example, iPS-hRPE cells
- monocytes / macrophages for example, PMA-THP-1 cells
- co-culture both cells may be mixed and cultured in one incubator, or one may be adherently cultured and the other cell may be seeded after a certain period of time has passed.
- iPS-hRPE cells and iPS-hRPP cells using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.), Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) and the like. Co-culture may be carried out under the condition that the cells do not come into direct contact with each other.
- the medium used in the co-culture is not particularly limited as long as it is usually used for general cell culture or culture of the above-mentioned retinal pigment epithelial cells (RPE) and monocytes / macrophages.
- Serum such as fetal bovine serum may be added to the medium, but a serum-free medium is preferable in order to eliminate the influence of extracellular fine particles brought in from the serum.
- the conditions for inducing inflammation by adding the above LPS or the like include, for example, LPS of 1 ng to 500 ng / mL, preferably 10 ng to 200 ng / mL, more preferably 50 ng to 150 ng / mL, still more preferably 80 ng to 120 ng / mL.
- LPS LPS of 1 ng to 500 ng / mL, preferably 10 ng to 200 ng / mL, more preferably 50 ng to 150 ng / mL, still more preferably 80 ng to 120 ng / mL.
- the conditions added so as to have a concentration can be mentioned.
- the number of each cell in the co-culture can be appropriately adjusted according to the size, morphology and the like of the incubator.
- specific co-culture for example, when a 24-well plate is used, 1.0 ⁇ 10 4 cells to 1.0 ⁇ 10 6 cells / well, preferably 5.0 ⁇ 10 4 cells to 5.0 ⁇ .
- PMA-THP-1 cells of about 10 5 cells, more preferably about 2.5 ⁇ 10 5 cells are seeded, cultured for about 30 minutes to 12 hours, and then 5.0 ⁇ 10 3 cells to 5.0 ⁇ 10 5 IPS-hRPE cells of cells / well, preferably 2.0 ⁇ 10 4 cells to 2.0 ⁇ 10 5 cells, more preferably 1.0 ⁇ 10 5 cells, can be seeded and co-cultured.
- test substance A step of adding the test substance to the co-culture system prepared in (1) and culturing for a certain period of time: In this step, the test substance is added to the co-culture system prepared in (1), and the culture is carried out for a certain period of time.
- the test substance include small molecule compounds, natural products, nucleic acid oligos, peptides, antibodies and the like, and the amount of addition can be appropriately adjusted according to the substance.
- the culture supernatant after adding the test substance and culturing for a certain period in the above step (2) is collected, and the amount of extracellular fine particles (EV) in the culture supernatant and the extracellular fine particles (EV) are collected.
- the amount of CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically specific miRNA (microRNA) derived from mitochondria. ), Etc. are measured.
- the amount of extracellular fine particles (EV) in the sample of the subject prepared in step (2) "(1) of the determination method of the present invention described above, in the extracellular fine particles (EV)".
- the “sample of the subject” can be replaced with the "culture supernatant” and applied.
- Step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation In this step, the numerical values obtained in the above step (3) and the numerical values of the negative target are compared. At this time, as the negative target, the numerical value obtained in the test to which the test substance is not added can be adopted. Amount of extracellular fine particles (EV) in the culture supernatant and amount of CD63-positive exosomes in the extracellular fine particles (EV) due to the addition of the test substance, as compared with the values obtained in the test without the addition of the test substance.
- EV extracellular fine particles
- EV extracellular fine particles
- nucleic acids derived from intracellular substances especially the profile of miRNA (microRNA), specifically the content of specific miRNA (microRNA) derived from mitochondria is reduced. If so, it is judged to be a substance that can be expected to be effective as a therapeutic agent for inflammatory retinal diseases.
- the screening method of the present invention it is possible to easily screen substances that are effective in treating inflammatory retinal diseases associated with tissue inflammation such as age-related macular degeneration.
- RPE retinal pigment epithelial cells
- hRPE cells the paper by Sugita et al. (Sugita S. et. Al., Lack of T cell response to iPSC-derivated retinal pigment epithelium cells from epithelium cell6 cell16sor6sol6s. The cells prepared by inducing from iPS cells according to the method described in the above were used.
- HRPE cells produced by inducing from iPS cells (hereinafter, also referred to as "iPS-hRPE cells”) and PMA-treated THP-1 cells (hereinafter, also referred to as “PMA-THP-1 cells”) are serum-free medium.
- Co-culture was performed in DMEM / F12 in the presence or absence of 100 ng / mL LPS for 24 hours. Specifically, 2.5 ⁇ 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours. Then, 1.0 ⁇ 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R).
- the comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R).
- the culture supernatant of each well was collected, centrifuged at 2000 ⁇ g for 10 minutes, and the obtained supernatant was passed through a 0.22 ⁇ m filter (manufactured by Millipore) to prepare a sample for quantification of various inflammatory cytokines.
- the concentrations of IL-6, MCP-1, IL-8, VEGF, TNF- ⁇ , and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Four points were measured for each sample, and the average value was taken as the concentration of each sample and is shown in FIG.
- results of this test 1 show that when retinal pigment epithelial cells and macrophages coexist and inflammation is induced by LPS, the inflammatory cytokines IL-6, MCP-1, IL-8, and VEGF are produced. The amount is significantly increased, indicating that it moves in a direction that further exacerbates inflammation.
- Test 2 Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 ⁇ 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours.
- RPE retinal pigment epithelial cells
- monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 ⁇ 10 5 cells
- 1.0 ⁇ 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R).
- the comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R).
- the culture supernatant of each well was collected, centrifuged at 2,000 ⁇ g for 10 minutes, and the obtained supernatant was passed through a 0.22 ⁇ m filter (manufactured by Millipore). Then, the pellets were collected by ultracentrifugation (100,000 ⁇ g, 90 minutes) using an MLS50 swing rotor (manufactured by Beckman Coulter).
- the pellet was suspended in PBS and ultracentrifuged (100,000 ⁇ g, 90 minutes) was performed again.
- the obtained pellet was suspended in PBS and protein quantification was performed according to a conventional method.
- Western blotting was performed by adjusting the amount of protein in each sample to be equal, CD63 was detected, and the amount of CD63 in each sample was compared. The results are shown in FIG. 2 (a).
- co-culture was performed using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.) under the condition that iPS-hRPE cells and PMA-THP-1 cells do not come into direct contact with each other. Specifically, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells was seeded in the lower chamber, and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells was seeded in the upper chamber. , Was co-cultured for 24 hours. A test was also conducted in which the cells seeded in the upper and lower chambers were replaced.
- Transwell registered trademark
- the number of fine particles contained in) was measured using a NanoSigt LM10V-HS nanoparticular tracing system (manufactured by Quantum Design).
- Western blotting was performed on the sample (CS) of each culture supernatant and the sample (UC) obtained by ultracentrifuging the sample (CS) of each culture supernatant by the same method as in Test 2 above, and CD63 was detected. ..
- the results are shown in FIG.
- the number of fine particles secreted by co-culturing iPS-hRPE cells and PMA-THP-1 cells as compared with the case of culturing with only one of the cells. It turned out that it increased significantly.
- FIG. 3 (b) it was found that the proportion of CD63 + exosomes in the fine particles was significantly increased by co-culture. That is, the co-culture increased the amount of CD63 + exosomes secreted among the fine particles.
- Test 4 Analysis of microRNA in extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages.
- the profile of the microRNA contained in the cells was analyzed using 3D-gene human microRNA chips (manufactured by Toray Co., Ltd.).
- Table 1 below, for each microRNA, the expression intensity (THP-1 + RPE) in the co-cultured sample of iPS-hRPE cells and PMA-THP-1 cells is shown by the expression intensity (RPE) in the iPS-hRPE cell single culture sample. The divided value (ratio) is shown. # 0228, # 0259, and # 0619 are independent test numbers.
- FIG. 4 a Venn diagram shows a set of microRNAs whose expression was increased by co-culture in each test.
- miR-494-3p showed the largest increase in the first, second, and fourth place among the three measured values (Table 1). See).
- the expression of the specific microRNA in the secreted microparticles such as exosomes increases, especially miR-494-3p.
- miR-494-3p was considered to be usable as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease.
- miR1246 can also be used as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease.
- the Transwell (registered trademark) system has some limitations. Since the content of the upper chamber is smaller than that of the lower chamber, fine particles may preferentially settle at the bottom of the lower chamber. Also, a 0.40 ⁇ m filter is not sufficient to block cell penetration into other chambers. Therefore, a Nico-1 (registered trademark) system, which is a horizontally connected double chamber, was used, and a comparison was made between the case where a 0.03 ⁇ m filter was attached and the case where a 0.03 ⁇ m filter was attached between the two chambers. Both chambers each hold a final volume of 1.5 mL.
- iPS-hRPE using Transwell (registered trademark) cell culture cells (manufactured by Corning) and Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) with iPS-hRP. Co-culture was performed under the condition that one cell did not come into direct contact with the cell. Specifically, in Transwell®, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells. was seeded in the upper chamber and co-cultured for 24 hours.
- Transwell® 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells.
- Nico-1® 1.0 ⁇ 10 5 cells PMA-THP-1 cells were seeded in one chamber and 2.0 ⁇ 10 5 cells iPS-hRPE cells were seeded in the other chamber. Co-culture was performed for 24 hours.
- Nico-1® as described above, comparison was made with and without a 0.03 ⁇ m filter between the two chambers. Western blotting was performed on the sample obtained by ultracentrifuging each culture supernatant by the same method as in Test 2 above, and CD63 was detected. The results are shown in FIGS. 5 (a) and 5 (b).
- IPS-hRPE cells and PM cells directly with iPS-hRPE cells using Transwell® cell culture plants (Corning) and Nico-1® culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan). Co-culture was performed under the condition of no contact. Specifically, in Transwell®, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells. was seeded in the upper chamber and co-cultured for 24 hours.
- the concentrations of IL-6, MCP-1, IL-8, VEGF, TNF- ⁇ , and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). The ELISA measurement was performed in 3 divided doses and repeated as 3 independent experiments. The average value was calculated for each sample and the concentration of each sample is shown in FIGS. 6 (a) and 6 (b).
- TNF ⁇ Production of TNF ⁇ from macrophages by exosome stimulation derived from retinal pigment epithelial cells (RPE) and production of MCP-1 and IL-6 from RPE cells Cell-cell interaction considered to be involved in pathogenesis
- RPE retinal pigment epithelial cells
- MCP-1 and IL-6 production of MCP-1 and IL-6 from RPE cells
- the concentrations of TNF- ⁇ , MCP-1, and IL-6 in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Particle concentration was measured with the NanoSigt LM10V-HS nanoparticle tracking system. The results are shown in FIGS. 7 (a) and 7 (b).
- Exosomes derived from iPS-hRPE cells increased the production of TNF- ⁇ from PMA-THP-1 cells in a concentration-dependent manner.
- the exosomes derived from the co-culture of iPS-hRPE cells and PMA-THP-1 had a greater effect of promoting the production of TNF- ⁇ from PMA-THP-1 cells than the exosomes derived from iPS-hRPE cells alone.
- the number of particles in the culture supernatant was 3.5 times, and it was supercentrifuged. The concentrated one was 8.5 times, and the co-culture increased remarkably.
- TNF- ⁇ showed increased production of MCP-1 and IL-6 in parallel with increased production of CD63 + protein in microparticles from iPS-hRPE cells, but increased production of microparticles was confirmed. It was not done (Fig. 7 (b)).
- the inflammation exacerbation circuit shown in FIG. 7 (c) is involved in the pathogenesis. That is, when an inflammatory stimulus as symbolized by LPS is applied to the RPE, a signal stimulus is applied to the RPE via a receptor related to natural inflammation such as TLR4 existing on the cell. This is thought to lead to EV (fine particle) production in the presence of RPE alone. When this EV is produced, it acts on macrophages that have infiltrated the inflamed area in the vicinity and induces the production of TNF- ⁇ . TNF- ⁇ production is sufficiently induced even with fine particles concentrated twice by ultracentrifugation from the supernatant of the single culture, but the induction activity of the co-culture supernatant is higher.
- the amount of fine particles produced is considerably enhanced in the co-culture as described above. It is presumed that some Exosomes in the fine particles are responsible for the TNF- ⁇ production-inducing activity. There is no activity in the culture supernatant or the remaining supernatant after ultracentrifugation of the microparticles. This TNF- ⁇ then acts on the nearby RPE, inflammatory cytocan from the RPE, MCP-1 (has the effect of migrating macrophages to the inflammatory site), IL-6 (involved in choroidal tissue fibrosis). , VEGF (involved in angiogenesis) production is thought to be enhanced and EV production induced.
- EV production from RPE is not significantly enhanced, but CD63-positive exosomes contained in EV are increased. Similarly, it is considered that this circuit is rotated to enhance EV production, inflammatory cytokine production, and local macrophages migration by MCP-1. That is, it is considered that the coexistence of RPE and macrophages and the cell-cell interaction mediated by microparticles (exosomes) establish an inflammatory exacerbation circuit and are involved in the formation of pathological conditions.
- Exosome miRNAs in the interaction of retinal pigment epithelial cells (RPE) and macrophages The role of microparticles (exosomes) produced in co-culture of iPS-hRPE cells and PMA-THP-1 cells was analyzed in terms of low molecular weight RNA profile in secreted microparticles (exosomes). Fine particle concentration was measured by nanoparticle follow-up analysis. Table 2 summarizes the concentrations of microparticles released per 106 cells in the three cultures (iPS-hRPE cells, PMA-THP-1 cells, co-culture).
- the base sequence "UGAAACAUACACGGGAAACCUC” in the above miR494-3p mimic shows the sequence of miR-494-3p (SEQ ID NO: 1)
- the base sequence "AAUGGAUUUUUGGAGCAGG” in the miR1246 mimic shows the sequence of miR-1246 (SEQ ID NO: 2).
- FCCP Carbonyl cyanide-p-trifluoromethoxyphrydrazone
- ROT / AA Rotenone / Antimycin A
- OCR Oxygen Consumption Rate (pmole / min) (oxygen consumption rate), which is a value indicating the oxygen consumption of cells.
- ECAR Extracellular Acidification Rate (mpH / min) (extracellular acidification rate), which indicates the rate of extracellular pH production by metabolites excreted extracellularly. Both are widely used as indicators of energy metabolism at the cellular level. The results are shown in FIGS. 8 (a) to 8 (c).
- Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (primary cultured HRPE cells vs ARPE19 cell line) Human primary retinal pigment epithelial (HRPE) cells (P3) and ARPE19 cell line (P25) were seeded at 16 ⁇ 10 4 cells / well on a 6-well plate, respectively. The next day, the whole medium was replaced with DMEM (+ 10% FBS), and 1 hour later, miR494-3p mimic or miR1246 mimic was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 ⁇ 10 4 cells / 100 ⁇ L / well.
- FIG. 9 (a) and Table 3 below primary cultured human RPE cells
- FIG. 9 (b) and Table 4 below ARPE 19 cell line
- miR494-3 pinhibitor Anti-miR TM miRNA hsa-miR-494-3p, MH12409, Thermo Fisher Scientific, Waltham, MA, USA
- miR1246inhibitor Anti-miR TM miRNA hsa-miR-1246, MH13182, Thermo Fisher Scientific, Waltham, MA, USA
- FIG. 10 (a) and Table 5 below Nic-ARPE cell line
- FIG. 10 (b) and Table 6 below ARPE 19 cell line).
- Plasma 6 control samples 100 ⁇ L each, AMD patient 5 samples 100 ⁇ L each
- Aqueous humor control 5 samples 50 ⁇ L each, AMD patient 5 samples 50 ⁇ L each
- miRNA was extracted using miRNeasy Serum / Plasma kit (QIAGEN, Hilden, Germany) (extraction amount 10 ⁇ L). Reverse transcription reaction was performed using primers specific for each miRNA, and quantitative PCR was performed. The relative amount of miRNA was quantified by the ⁇ Ct method from each CT value and the CT value of the control using cel-miR-39 as a control (spire-in control) (the number of cycles of quantitative PCR was 40). The results are shown in FIG. 11 (miR-494-3P) and FIG. 12 (miR-1246).
- the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention it is possible to easily determine the susceptibility, severity, therapeutic effect, etc. of a chronic inflammatory retinal disease such as age-related macular degeneration.
- Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention.
- the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as.
- the screening method of the present invention it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.
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Abstract
A purpose of the present invention is to provide a novel assessment method that enables early detection, assessment of severity, and assessment of therapeutic effect, etc., of chronic inflammatory retinal diseases including age-related macular degeneration. Another purpose is to provide a novel therapeutic for inflammatory retinal diseases including age-related macular degeneration and to provide a novel screening method for inflammatory retinal disease therapeutics. The present invention is a method for assessing inflammatory retinal diseases associated with tissue inflammation, with extracellular vesicles (EV) produced by the retinal pigment epithelial cells (RPE) of a subject being employed as an indicator. This assessment method is characterized in that the profile of miRNA contained in the extracellular vesicles (EV) (especially miR494-3p and/or miR1246) is employed as an indicator.
Description
本発明は、炎症性網膜疾患の判定方法、炎症性網膜疾患治療剤、及び炎症性網膜疾患治療剤のスクリーニング方法に関する。
The present invention relates to a method for determining an inflammatory retinal disease, a method for screening an inflammatory retinal disease therapeutic agent, and a method for screening an inflammatory retinal disease therapeutic agent.
細胞から分泌されるエクソソーム等の細胞外微粒子（Ｅｘｔｒａｃｅｌｌｕｌａｒ ｖｅｓｉｃｌｅ）は、表面に細胞膜由来の脂質、タンパク質を含み、内部には細胞内物質由来の核酸（マイクロＲＮＡ、メッセンジャーＲＮＡ、ＤＮＡ等）、タンパク質等を含んでいる。上記細胞外微粒子が含むマイクロＲＮＡ（ｍｉｃｒｏ ＲＮＡ；ｍｉＲＮＡ）は、タンパク質に翻訳されない約１７～２４ヌクレオチドからなる非コードＲＮＡである。ヒトのｍｉＲＮＡは、現在２５００種類以上が発見されている。このｍｉＲＮＡは、自分自身と相補的な標的部位をもつ様々な遺伝子の翻訳を抑制する働きがあり、細胞の発生、分化、増殖、細胞死等の基本的な生物学的機能を制御している。このようにマイクロＲＮＡは細胞内の多くの遺伝子の発現制御を行っているため、その発現の変化は疾患の発生や進行等に関連すると考えられており、最近ではマイクロＲＮＡを疾患の診断、早期発見に利用しようという研究も行われている。
Extracellular fine particles such as exosomes secreted from cells contain lipids and proteins derived from cell membranes on the surface, and nucleic acids derived from intracellular substances (microRNA, messenger RNA, DNA, etc.), proteins, etc. inside. Includes. The microRNA (miRNA) contained in the extracellular fine particles is a non-coding RNA consisting of about 17 to 24 nucleotides that is not translated into protein. Currently, more than 2500 types of human miRNA have been discovered. This miRNA has the function of suppressing the translation of various genes having target sites complementary to itself, and controls basic biological functions such as cell development, differentiation, proliferation, and cell death. .. In this way, since microRNA regulates the expression of many genes in cells, changes in its expression are thought to be related to the development and progression of diseases. Research is also being conducted to use it for discovery.
例えば、癌とｍｉＲＮＡとの関連について、Ｃａｌｉｎらは、リンパ性白血病において、ｍｉＲ１５及びｍｉＲ１６が頻繁にダウンレギュレーションしていることを報告している（非特許文献１参照；Ｃａｌｉｎ ＧＡ，Ｄｕｍｉｔｒｕ ＣＤ，Ｓｈｉｍｉｚｕ Ｍら，Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ，２００２，ｖｏｌ．９９，ｐ．１５５２４－９）。また、Ｍｉｃｈａｅｌらは、ヒト大腸癌においてｍｉＲ－１４３及びｍｉＲ－１４５の発現が低下していることを報告している（非特許文献２参照；Ｍｉｃｈａｅｌ ＭＺ， ＳＭ ＯＣ， ｖａｎ Ｈｏｌｓｔ Ｐｅｌｌｅｋａａｎ ＮＧ，Ｙｏｕｎｇ ＧＰ，Ｊａｍｅｓ ＲＪ，Ｍｏｌ Ｃａｎｃｅｒ Ｒｅｓ，２００３，ｖｏｌ．１，ｐ．８８２－９１）。さらに特許文献１には、膀胱癌において発現変動している２７種類のｍｉＲＮＡを指標とする膀胱癌の検出方法が記載されている。このように、ｍｉＲＮＡは、癌等の疾病と深い関わりがあり、それらの診断、予後予測等に利用できる可能性があると考えられている。
For example, regarding the association between cancer and miRNA, Calin et al. Report that miR15 and miR16 are frequently downregulated in lymphocytic leukemia (see Non-Patent Document 1; Calin GA, Dumitru CD, Shimazu). M et al., Proc Natl Acad Sci USA, 2002, vol.99, p.15524-9). In addition, Michael et al. Reported that the expression of miR-143 and miR-145 was decreased in human colorectal cancer (see Non-Patent Document 2; Michael MZ, SM OC, van Holst Pellekaan NG, Young GP. , James RJ, Mol Cancer Res, 2003, vol. 1, p. 882-91). Further, Patent Document 1 describes a method for detecting bladder cancer using 27 types of miRNA whose expression fluctuates in bladder cancer as an index. As described above, miRNA is closely related to diseases such as cancer, and it is considered that miRNA may be used for their diagnosis, prognosis prediction, and the like.
一方、加齢黄斑変性は慢性の炎症性網膜疾患のひとつであり、先進国において高齢者の失明の主要原因となっている（非特許文献３参照）。加齢にともなって網膜中央の黄斑部に様々な異常が生じ、視力低下を引き起こす疾患である。疾患初期には黄斑部の色素異常、ドルーゼン（沈着物）がみられるが自覚症状が少ないため、疾患の発見が遅くなることが多い。疾患が進行すると黄斑部に異常な血管新生等が起こり、障害が進んで深刻な視力低下を引き起こすことから、疾患の初期に加齢黄斑変性の診断や予後予測等を高い確度で簡便に行うことができる方法が求められている。
On the other hand, age-related macular degeneration is one of the chronic inflammatory retinal diseases and is a major cause of blindness in the elderly in developed countries (see Non-Patent Document 3). With aging, various abnormalities occur in the macula in the center of the retina, causing a decrease in visual acuity. Dyschromia and drusen (deposits) in the macula are seen in the early stage of the disease, but the detection of the disease is often delayed because there are few subjective symptoms. As the disease progresses, abnormal angiogenesis occurs in the macula, and the disorder progresses, causing serious deterioration of visual acuity. Therefore, diagnosis and prognosis of age-related macular degeneration should be made easily and with high accuracy in the early stage of the disease. There is a need for a way to do this.
本発明は、上述のような状況の中、加齢黄斑変性を始めとする慢性の炎症性網膜疾患の早期発見、重症度の判定、治療効果等の判定が可能な新規の判定方法を提供することを目的とする。本発明はまた、加齢黄斑変性を始めとする炎症性網膜疾患の新規治療剤を提供することも目的とする。さらに、炎症性網膜疾患治療剤の新規スクリーニング方法を提供することも目的とする。
The present invention provides a novel determination method capable of early detection of chronic inflammatory retinal disease such as age-related macular degeneration, determination of severity, determination of therapeutic effect, etc. under the above-mentioned circumstances. The purpose is. It is also an object of the present invention to provide a novel therapeutic agent for inflammatory retinal diseases such as age-related macular degeneration. Furthermore, it is also an object to provide a new screening method for a therapeutic agent for inflammatory retinal disease.
前記課題を解決するために、本発明者が種々検討した結果、網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージの共培養系において、炎症を惹起する培養条件にすると、細胞外微粒子（Ｅｘｔｒａｃｅｌｌｕｌａｒ ｖｅｓｉｃｌｅｓ；ＥＶ）の分泌が促進されると共に、分泌されるＥＶ中の特定のマイクロＲＮＡの量が増大することを見出した。そして、これらのマイクロＲＮＡを指標として、慢性の炎症性網膜疾患の罹患可能性、重症度、治療効果等の判定を行う新規の判定方法を完成させた。また、これらの特定のマイクロＲＮＡの阻害剤は、慢性の炎症性網膜疾患の治療に有効であると考えられ、新規治療剤としての可能性が示唆された。さらに、網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において炎症を惹起する条件にすると、実際の炎症に近い状態を構築することができることから、その際に分泌される細胞外微粒子（ＥＶ）のプロファイルの変動、特に細胞外微粒子（ＥＶ）が含有する上記特定のマイクロＲＮＡのプロファイルの変動を指標として、炎症状態を改善する、即ち炎症性の網膜疾患を治療することが可能な薬剤（炎症性網膜疾患治療剤）をスクリーニングする方法も見出した。すなわち本発明の要旨は、以下の通りである。
As a result of various studies by the present inventor in order to solve the above-mentioned problems, in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, when the culture conditions are set to induce inflammation, extracellular fine particles (Extracellular secretions) It has been found that the secretion of EV) is promoted and the amount of specific microRNA in the secreted EV is increased. Then, using these microRNAs as an index, a new determination method for determining the susceptibility, severity, therapeutic effect, etc. of chronic inflammatory retinal disease has been completed. In addition, these specific microRNA inhibitors are considered to be effective in the treatment of chronic inflammatory retinal diseases, suggesting their potential as new therapeutic agents. Furthermore, if the conditions are such that inflammation is induced in the co-culture system of retinal pigment epithelial cells (RPE) and monospheres / macrophages, a state close to actual inflammation can be constructed, and thus the cells secreted at that time. It is possible to improve the inflammatory state, that is, to treat an inflammatory retinal disease, using the fluctuation of the profile of the external fine particles (EV), particularly the fluctuation of the profile of the specific microRNA contained in the extracellular fine particles (EV) as an index. We have also found a way to screen for possible drugs (therapeutic agents for inflammatory retinal diseases). That is, the gist of the present invention is as follows.
［１］被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を指標とする、組織炎症を伴う炎症性網膜疾患の判定方法。
［２］上記細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルを指標とすることを特徴とする、［１］に記載の判定方法。
［３］上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、［２］に記載の判定方法。
［４］上記ｍｉＲＮＡが、ミトコンドリア由来である、［２］又は［３］に記載の判定方法。
［５］上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症および、糖尿病性網膜症からなる群より選択される少なくとも１種の炎症性疾患である、［１］から［４］のいずれかに記載の判定方法。
［６］被験者における炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク及び治療効果から成る群より選択される少なくとも１種を判定する、［１］から［５］のいずれかに記載の判定方法。
［７］網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子を有効成分として含有する、組織炎症を伴う炎症性網膜疾患治療剤。
［８］上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡの機能調節分子である、［７］に記載の炎症性網膜疾患治療剤。
［９］上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の機能調節分子である、［７］又は［８］に記載の炎症性網膜疾患治療剤。
［１０］上記細胞外微粒子（ＥＶ）の機能調節分子が、核酸オリゴ、低分子化合物、ペプチド及び抗体から成る群より選択される少なくとも１種である、［７］から［９］のいずれかに記載の炎症性網膜疾患治療剤。
［１１］上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症、及び糖尿病性網膜症からなる群より選択される少なくとも１種の疾患である、［７］から［１０］のいずれかに記載の炎症性網膜疾患治療剤。
［１２］網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）のプロファイルの変動を指標とすることを特徴とする、組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法。
［１３］上記細胞外微粒子（ＥＶ）が、網膜色素上皮細胞（ＲＰＥ）から分泌される、［１２］に記載のスクリーニング方法。
［１４］上記細胞外微粒子（ＥＶ）のプロファイルの変動が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルの変動である、［１２］又は［１３］に記載のスクリーニング方法。
［１５］上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、［１４］に記載のスクリーニング方法。
［１６］網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において、被験物質の添加により分泌される細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６が抑制された場合に、上記被験物質を有効な組織炎症を伴う炎症性網膜疾患治療剤の候補として選択する、［１２］から［１５］のいずれかに記載のスクリーニング方法。 [1] A method for determining an inflammatory retinal disease accompanied by tissue inflammation, using extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index.
[2] The determination method according to [1], wherein the profile of miRNA contained in the extracellular fine particles (EV) is used as an index.
[3] The determination method according to [2], wherein the miRNA contains miR494-3p and / or miR1246.
[4] The determination method according to [2] or [3], wherein the miRNA is derived from mitochondria.
[5] At least one inflammatory retinal disease associated with tissue inflammation selected from the group consisting of age-related macular degeneration, central serous chorioretinal disease, proliferative vitreous retinopathy, and diabetic retinopathy. The determination method according to any one of [1] to [4], which is an inflammatory disease.
[6] Any one of [1] to [5], which determines at least one selected from the group consisting of the possibility of inflammatory retinal disease in the subject, the severity of the disease, the risk of developing the disease, the risk of aggravation, and the therapeutic effect. Judgment method described in.
[7] A therapeutic agent for an inflammatory retinal disease accompanied by tissue inflammation, which contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
[8] The therapeutic agent for inflammatory retinal disease according to [7], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miRNA contained in the extracellular fine particles (EV).
[9] The inflammation according to [7] or [8], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miR494-3p and / or miR1246 contained in the extracellular fine particles (EV). A therapeutic agent for retinal diseases.
[10] The function-regulating molecule of the extracellular fine particle (EV) is any one of [7] to [9], which is at least one selected from the group consisting of nucleic acid oligos, small molecule compounds, peptides and antibodies. The described inflammatory retinal disease therapeutic agent.
[11] The inflammatory retinal disease associated with the tissue inflammation is at least one selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, and diabetic retinopathy. The agent for treating an inflammatory retinal disease according to any one of [7] to [10], which is a disease.
[12] Inflammation associated with tissue inflammation, characterized by variability in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. A screening method for therapeutic agents for sexual retinal diseases.
[13] The screening method according to [12], wherein the extracellular fine particles (EV) are secreted from retinal pigment epithelial cells (RPE).
[14] The screening method according to [12] or [13], wherein the variation in the profile of the extracellular fine particles (EV) is a variation in the profile of miRNA contained in the extracellular fine particles (EV).
[15] The screening method according to [14], wherein the miRNA comprises miR494-3p and / or miR1246.
[16] In a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, miR494-3p and / or miR1246 contained in extracellular fine particles (EV) secreted by the addition of the test substance are suppressed. The screening method according to any one of [12] to [15], wherein the test substance is selected as a candidate for an effective therapeutic agent for inflammatory retinal disease associated with tissue inflammation.
［２］上記細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルを指標とすることを特徴とする、［１］に記載の判定方法。
［３］上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、［２］に記載の判定方法。
［４］上記ｍｉＲＮＡが、ミトコンドリア由来である、［２］又は［３］に記載の判定方法。
［５］上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症および、糖尿病性網膜症からなる群より選択される少なくとも１種の炎症性疾患である、［１］から［４］のいずれかに記載の判定方法。
［６］被験者における炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク及び治療効果から成る群より選択される少なくとも１種を判定する、［１］から［５］のいずれかに記載の判定方法。
［７］網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子を有効成分として含有する、組織炎症を伴う炎症性網膜疾患治療剤。
［８］上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡの機能調節分子である、［７］に記載の炎症性網膜疾患治療剤。
［９］上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の機能調節分子である、［７］又は［８］に記載の炎症性網膜疾患治療剤。
［１０］上記細胞外微粒子（ＥＶ）の機能調節分子が、核酸オリゴ、低分子化合物、ペプチド及び抗体から成る群より選択される少なくとも１種である、［７］から［９］のいずれかに記載の炎症性網膜疾患治療剤。
［１１］上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症、及び糖尿病性網膜症からなる群より選択される少なくとも１種の疾患である、［７］から［１０］のいずれかに記載の炎症性網膜疾患治療剤。
［１２］網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）のプロファイルの変動を指標とすることを特徴とする、組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法。
［１３］上記細胞外微粒子（ＥＶ）が、網膜色素上皮細胞（ＲＰＥ）から分泌される、［１２］に記載のスクリーニング方法。
［１４］上記細胞外微粒子（ＥＶ）のプロファイルの変動が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルの変動である、［１２］又は［１３］に記載のスクリーニング方法。
［１５］上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、［１４］に記載のスクリーニング方法。
［１６］網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において、被験物質の添加により分泌される細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６が抑制された場合に、上記被験物質を有効な組織炎症を伴う炎症性網膜疾患治療剤の候補として選択する、［１２］から［１５］のいずれかに記載のスクリーニング方法。 [1] A method for determining an inflammatory retinal disease accompanied by tissue inflammation, using extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index.
[2] The determination method according to [1], wherein the profile of miRNA contained in the extracellular fine particles (EV) is used as an index.
[3] The determination method according to [2], wherein the miRNA contains miR494-3p and / or miR1246.
[4] The determination method according to [2] or [3], wherein the miRNA is derived from mitochondria.
[5] At least one inflammatory retinal disease associated with tissue inflammation selected from the group consisting of age-related macular degeneration, central serous chorioretinal disease, proliferative vitreous retinopathy, and diabetic retinopathy. The determination method according to any one of [1] to [4], which is an inflammatory disease.
[6] Any one of [1] to [5], which determines at least one selected from the group consisting of the possibility of inflammatory retinal disease in the subject, the severity of the disease, the risk of developing the disease, the risk of aggravation, and the therapeutic effect. Judgment method described in.
[7] A therapeutic agent for an inflammatory retinal disease accompanied by tissue inflammation, which contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
[8] The therapeutic agent for inflammatory retinal disease according to [7], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miRNA contained in the extracellular fine particles (EV).
[9] The inflammation according to [7] or [8], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miR494-3p and / or miR1246 contained in the extracellular fine particles (EV). A therapeutic agent for retinal diseases.
[10] The function-regulating molecule of the extracellular fine particle (EV) is any one of [7] to [9], which is at least one selected from the group consisting of nucleic acid oligos, small molecule compounds, peptides and antibodies. The described inflammatory retinal disease therapeutic agent.
[11] The inflammatory retinal disease associated with the tissue inflammation is at least one selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, and diabetic retinopathy. The agent for treating an inflammatory retinal disease according to any one of [7] to [10], which is a disease.
[12] Inflammation associated with tissue inflammation, characterized by variability in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. A screening method for therapeutic agents for sexual retinal diseases.
[13] The screening method according to [12], wherein the extracellular fine particles (EV) are secreted from retinal pigment epithelial cells (RPE).
[14] The screening method according to [12] or [13], wherein the variation in the profile of the extracellular fine particles (EV) is a variation in the profile of miRNA contained in the extracellular fine particles (EV).
[15] The screening method according to [14], wherein the miRNA comprises miR494-3p and / or miR1246.
[16] In a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, miR494-3p and / or miR1246 contained in extracellular fine particles (EV) secreted by the addition of the test substance are suppressed. The screening method according to any one of [12] to [15], wherein the test substance is selected as a candidate for an effective therapeutic agent for inflammatory retinal disease associated with tissue inflammation.
本発明の組織炎症を伴う炎症性網膜疾患の判定方法によると、加齢黄斑変性等の炎症性網膜疾患の罹患可能性、重症度、治療効果等の判定を、高い確度で、かつ容易に行うことができる。加齢黄斑変性は、患者において疾患初期の自覚症状が少ないため早期発見が困難であるという課題があったが、本発明の判定方法により早期発見が可能となる。また、網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子、特に特定のマイクロＲＮＡの機能調節分子は、加齢黄斑変性等の慢性の炎症性網膜疾患の新規治療剤として有効である。さらに、本発明のスクリーニング方法によると、炎症性網膜疾患に対して治療効果を有する物質を簡便に探索することができる。
According to the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention, the possibility, severity, therapeutic effect, etc. of an inflammatory retinal disease such as age-related macular degeneration can be easily determined with high accuracy. be able to. Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention. In addition, the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA, is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as. Furthermore, according to the screening method of the present invention, it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.
以下、本発明について詳細に説明する。なお、本明細書中で使用される用語は、特に言及しない限り、当該技術分野で通常用いられる意味で解釈される。
Hereinafter, the present invention will be described in detail. The terms used herein are to be construed as commonly used in the art, unless otherwise noted.
［組織炎症を伴う炎症性網膜疾患の判定方法］
本発明の組織炎症を伴う炎症性網膜疾患の判定方法は、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を指標とする。 [Method for determining inflammatory retinal disease with tissue inflammation]
The method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention uses extracellular fine particles (EV) produced by the retinal pigment epithelial cells (RPE) of a subject as an index.
本発明の組織炎症を伴う炎症性網膜疾患の判定方法は、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を指標とする。 [Method for determining inflammatory retinal disease with tissue inflammation]
The method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention uses extracellular fine particles (EV) produced by the retinal pigment epithelial cells (RPE) of a subject as an index.
本発明において細胞外微粒子（ＥＶ）とは、細胞が放出する微粒子であり、電子顕微鏡で確認することができる小胞である。細胞外微粒子（ＥＶ）は、内部に細胞内物質由来の核酸（マイクロＲＮＡ、メッセンジャーＲＮＡ、ＤＮＡ等）、タンパク質等を含み、細胞由来の脂質及びタンパク質から構成される脂質二重層で覆われている。細胞外微粒子（ＥＶ）は、その直径、細胞内起源、スクロース中での微粒子の密度、形状、沈降速度、脂質組成、タンパク質マーカー及び分泌の様式（即ち、シグナル（誘導性）後又は自発的（構成的））等に基づいて複数の種類に分類される。このような細胞外微粒子の種類としては、例えば、膜粒子、膜小胞、微小胞、エクソソーム様小胞、エクソソーム、エクトソーム様小胞、エクトソーム、エキソベシクル等が挙げられる。
In the present invention, extracellular fine particles (EV) are fine particles released by cells and are vesicles that can be confirmed with an electron microscope. The extracellular fine particles (EV) contain nucleic acids (microRNA, messenger RNA, DNA, etc.) derived from intracellular substances, proteins, etc., and are covered with a lipid double layer composed of cell-derived lipids and proteins. .. Extracellular microparticles (EVs) are their diameter, intracellular origin, density of microparticles in sucrose, shape, sedimentation rate, lipid composition, protein markers and mode of secretion (ie, after signaling (inducible) or spontaneously (ie). It is classified into multiple types based on structural)) and so on. Examples of such extracellular fine particles include membrane particles, membrane vesicles, microvesicles, exosome-like vesicles, exosomes, ectome-like vesicles, ectosomes, exovesicles and the like.
本発明においてエクソソームは、血液、リンパ液、唾液、尿、母乳、精液等に含まれている、直径１００ｎｍ程度を中心として分布している概略球形の細胞が放出する小胞である。小胞内部には、タンパク質、ｍＲＮＡ、ｍｉＲＮＡなどの分子を内包している。なお、エクソソームは、マイクロベシクル、細胞外小胞と称されることもあり、複数の呼称が存在する。例えば、密度勾配遠心法では１．１３～１．１９ｇ／ｍＬに分画され、動的光散乱法等による粒子径（直径）が約４０ｎｍ～約１５０ｎｍの範囲に含まれる微粒子である。また、エクソソームの膜上にはＣＤ９、ＣＤ６３、ＣＤ８１等が発現している。
In the present invention, exosomes are vesicles contained in blood, lymph, saliva, urine, breast milk, semen, etc., and released by substantially spherical cells distributed around a diameter of about 100 nm. Molecules such as proteins, mRNA, and miRNA are contained inside the vesicle. Exosomes are sometimes referred to as microvesicles or extracellular vesicles, and there are a plurality of names. For example, in the density gradient centrifugation method, the particles are fractionated into 1.13 to 1.19 g / mL, and the particle diameter (diameter) by the dynamic light scattering method or the like is contained in the range of about 40 nm to about 150 nm. In addition, CD9, CD63, CD81 and the like are expressed on the membrane of exosomes.
炎症条件下の網膜色素上皮細胞（ＲＰＥ）においては、エクソソーム等の細胞外微粒子（ＥＶ）の放出量が増大し、特にＣＤ６３陽性のエクソソームの放出量が増大すると共にこれらに由来する特定のマイクロＲＮＡの含有量が増大する。特にマクロファージが浸潤している炎症条件においては、エクソソーム等の細胞外微粒子（ＥＶ）の放出量が顕著に増大し、特にＣＤ６３陽性のエクソソームの放出量がより増大すると共にこれらに含まれる特定のマイクロＲＮＡの含有量もさらに増大する。そのため、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を指標とすることで、加齢黄斑変性等の炎症性網膜疾患の罹患可能性、重症度、治療効果等の判定をすることが可能となる。
In retinal pigment epithelial cells (RPE) under inflammatory conditions, the amount of extracellular fine particles (EV) such as exosomes released increases, and in particular, the amount of CD63-positive exosomes released increases and specific microRNAs derived from these. Content increases. Especially in inflammatory conditions infiltrated with macrophages, the amount of extracellular fine particles (EV) released such as exosomes is significantly increased, and in particular, the amount of CD63-positive exosomes released is further increased and specific micros contained therein are further increased. The RNA content is also increased. Therefore, by using the extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index, the possibility, severity, therapeutic effect, etc. of inflammatory retinal diseases such as age-related macular degeneration can be determined. It becomes possible to do.
本発明の判定方法において上記「細胞外微粒子（ＥＶ）を指標とする」とは、細胞外微粒子（ＥＶ）の放出量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイルを指標とすることをいう。上記マイクロＲＮＡ（ｍｉＲＮＡ）のプロファイルとしては、ミトコンドリア由来のｍｉＲ４９４－３ｐ、ｍｉＲ１２４６、ｍｉＲ４７４１及び／又はｍｉＲ７１１０－５ｐの含有量、発現量、放出量等が好ましいものとして挙げられ、中でもｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の含有量、発現量、放出量等がより好ましいものとして挙げられ、ｍｉＲ４９４－３ｐの含有量、発現量、放出量等がさらに好ましいものとして挙げられる。組織炎症を伴う炎症性網膜疾患を発症している被験者の網膜色素上皮細胞（ＲＰＥ）においては、分泌される細胞外微粒子（ＥＶ）が含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量が増加しているため、これらを指標とすることができる。
In the determination method of the present invention, the above-mentioned "using extracellular fine particles (EV) as an index" means the amount of extracellular fine particles (EV) released, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles (EV). It means that the profile of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly miRNA (microRNA), is used as an index. As the profile of the microRNA (miRNA), the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and / Alternatively, the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable. In the retinal pigment epithelial cells (RPE) of subjects developing inflammatory retinal disease with tissue inflammation, the amount of the specific miRNA (microRNA) contained in the secreted extracellular fine particles (EV) is increased. Therefore, these can be used as indicators.
本発明の判定方法における被験者の生体試料としては、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を含む試料であれば特に限定されないが、例えば、被験者の前房水、血液（血漿、血清）、組織液等が挙げられる。
The biological sample of the subject in the determination method of the present invention is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), but for example, the subject's anterior chamber water. Examples include blood (plasma, serum), tissue fluid and the like.
本発明において、組織炎症を伴う炎症性網膜疾患としては、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症、糖尿病性網膜症等が挙げられる。これらのうち、本発明の判定方法が特に適している疾患は加齢黄斑変性である。
In the present invention, examples of inflammatory retinal diseases associated with tissue inflammation include age-related macular degeneration, central serous chorioretinal disease, proliferative vitreous retinopathy, and diabetic retinopathy. Of these, the disease to which the determination method of the present invention is particularly suitable is age-related macular degeneration.
本発明の判定方法においては、炎症性網膜疾患の罹患可能性、症状の重軽度、発症リスク、重症化リスク、治療効果等を判定することができる。例えば、被験者の網膜色素上皮細胞（ＲＰＥ）が放出する細胞外微粒子（ＥＶ）の量が基準値より多い程、ＣＤ６３陽性のエクソソームの放出量が基準値より多い程、これらに含まれる特定のマイクロＲＮＡの含有量が基準値よりも多い程、被験者は組織炎症を伴う炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスクが高いと判断される。ここで、罹患可能性を判断する際の基準値としては、健常人における平均値、数値範囲等を用いることができる。また、組織炎症を伴う炎症性網膜疾患の治療効果を判定する場合には、治療前の値、治療後の値を比較し、その数値が低くなっていれば治療効果があったと判定され、その数値が変化しない場合は治療効果が見られなかったと判定され、その数値が高くなった場合には悪化したと判定される。
In the determination method of the present invention, the possibility of inflammatory retinal disease, the severity of symptoms, the risk of onset, the risk of aggravation, the therapeutic effect, etc. can be determined. For example, the greater the amount of extracellular fine particles (EV) released by the subject's retinal pigment epithelial cells (RPE), the greater the amount of CD63-positive exosomes released, the more specific micros contained in these. The higher the RNA content than the reference value, the higher the possibility, severity, risk of developing, and risk of aggravation of inflammatory retinal disease associated with tissue inflammation in the subject. Here, as a reference value for determining the possibility of morbidity, an average value, a numerical range, or the like in a healthy person can be used. In addition, when determining the therapeutic effect of inflammatory retinal disease accompanied by tissue inflammation, the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
本発明の組織炎症を伴う炎症性網膜疾患の判定方法は、以下の工程を含む方法として説明することもできる。
The method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention can also be described as a method including the following steps.
（１）被験者からの試料を準備する工程、
（２）（１）で準備された被験者の試料中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程、
（３）上記（２）で測定された数値と基準値を比較し、組織炎症を伴う炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク、治療効果等を判定する工程。 (1) Step of preparing a sample from a subject,
(2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly. A step of measuring a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, a content of a specific miRNA (microRNA) derived from mitochondria, etc.
(3) A step of comparing the numerical value measured in (2) above with a reference value to determine the possibility of inflammatory retinal disease accompanied by tissue inflammation, severe / mild, risk of onset, risk of aggravation, therapeutic effect, and the like.
（２）（１）で準備された被験者の試料中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程、
（３）上記（２）で測定された数値と基準値を比較し、組織炎症を伴う炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク、治療効果等を判定する工程。 (1) Step of preparing a sample from a subject,
(2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly. A step of measuring a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, a content of a specific miRNA (microRNA) derived from mitochondria, etc.
(3) A step of comparing the numerical value measured in (2) above with a reference value to determine the possibility of inflammatory retinal disease accompanied by tissue inflammation, severe / mild, risk of onset, risk of aggravation, therapeutic effect, and the like.
上記各工程について以下に説明する。
Each of the above processes will be described below.
（１）被験者からの試料を準備する工程：
本工程においては、組織炎症を伴う炎症性網膜疾患の判定を受ける被験者から、生体試料を採取し、準備する。被験者からの試料としては、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を含む試料であれば特に限定されないが、例えば、被験者の前房水、血液等が挙げられる。 (1) Step of preparing a sample from a subject:
In this step, a biological sample is collected and prepared from a subject who is judged to have an inflammatory retinal disease accompanied by tissue inflammation. The sample from the subject is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), and examples thereof include the subject's anterior chamber water and blood.
本工程においては、組織炎症を伴う炎症性網膜疾患の判定を受ける被験者から、生体試料を採取し、準備する。被験者からの試料としては、被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を含む試料であれば特に限定されないが、例えば、被験者の前房水、血液等が挙げられる。 (1) Step of preparing a sample from a subject:
In this step, a biological sample is collected and prepared from a subject who is judged to have an inflammatory retinal disease accompanied by tissue inflammation. The sample from the subject is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), and examples thereof include the subject's anterior chamber water and blood.
（２）（１）で準備された被験者の試料中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程：
本工程において細胞外微粒子（ＥＶ）の量を測定する場合には、試料に対して、例えば、超遠心分離法、密度勾配遠心法、及び各種エクソソーム分離キット（遠心によるペレットダウン、免疫沈降、磁気ビーズによる精製、粒子のサイズによる分画、カラム吸着等）を用いて、細胞外微粒子（ＥＶ）を分離し、得られた細胞外微粒子（ＥＶ）の量を測定する。 (2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly. Step of measuring the profile of nucleic acid derived from an intracellular substance, particularly miRNA (microRNA), specifically, the content of a specific miRNA (microRNA) derived from mitochondria:
When measuring the amount of extracellular fine particles (EV) in this step, for example, ultracentrifugation method, density gradient centrifugation method, and various exosome separation kits (pellet down by centrifugation, immunoprecipitation, magnetic) are used for the sample. Purification with beads, fractionation by particle size, column adsorption, etc.) is used to separate extracellular fine particles (EV), and the amount of obtained extracellular fine particles (EV) is measured.
本工程において細胞外微粒子（ＥＶ）の量を測定する場合には、試料に対して、例えば、超遠心分離法、密度勾配遠心法、及び各種エクソソーム分離キット（遠心によるペレットダウン、免疫沈降、磁気ビーズによる精製、粒子のサイズによる分画、カラム吸着等）を用いて、細胞外微粒子（ＥＶ）を分離し、得られた細胞外微粒子（ＥＶ）の量を測定する。 (2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly. Step of measuring the profile of nucleic acid derived from an intracellular substance, particularly miRNA (microRNA), specifically, the content of a specific miRNA (microRNA) derived from mitochondria:
When measuring the amount of extracellular fine particles (EV) in this step, for example, ultracentrifugation method, density gradient centrifugation method, and various exosome separation kits (pellet down by centrifugation, immunoprecipitation, magnetic) are used for the sample. Purification with beads, fractionation by particle size, column adsorption, etc.) is used to separate extracellular fine particles (EV), and the amount of obtained extracellular fine particles (EV) is measured.
細胞外微粒子（ＥＶ）の分離・回収方法は、例えば、被験者の試料を０．５～２時間、約５０，０００～１５０，０００Ｇで超遠心することを含む。超遠心を行う前に、上記試料を０．１～２時間、約１００～２０，０００Ｇで遠心することを含むことができる。細胞外微粒子（ＥＶ）は、ＰＢＳ等の溶液に溶解した状態であれば、４℃で１週間程度、－２０℃で１か月程度、－８０℃で６か月程度、凍結乾燥した状態であれば４℃で３年程度保存が可能である。
The method for separating and recovering extracellular fine particles (EV) includes, for example, ultracentrifuging a sample of a subject at about 50,000 to 150,000 G for 0.5 to 2 hours. Prior to ultracentrifugation, the sample can be centrifuged for 0.1-2 hours at about 100-20,000 G. Extracellular fine particles (EV) are freeze-dried at 4 ° C for about 1 week, -20 ° C for about 1 month, and -80 ° C for about 6 months if they are dissolved in a solution such as PBS. If there is, it can be stored at 4 ° C for about 3 years.
細胞外微粒子（ＥＶ）の量を測定する方法は、特に限定されるものではないが、例えばＮａｎｏＳｉｇｈｔ ＬＭ１０Ｖ‐ＨＳナノ粒子追跡システムで液体中の細胞外微粒子（ＥＶ）の粒子数を測定する方法、タンパク量を測定する方法等が挙げられる。得られた細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量を測定する方法としては、例えば、常法に従ってタンパク定量を行い、各サンプルのタンパク量が等量になるように調整してウェスタンブロッティングを行い、ＣＤ６３を検出し、各サンプルのＣＤ６３量を解析する方法等が挙げられる。また、各サンプルに含まれるマイクロＲＮＡについては、３Ｄ－ｇｅｎｅ ｈｕｍａｎ ｍｉｃｒｏＲＮＡ ｃｈｉｐｓ（東レ社製）等を用いて、含まれるマイクロＲＮＡ（発現強度）のプロファイルを解析することができる。
The method for measuring the amount of extracellular fine particles (EV) is not particularly limited, but for example, a method for measuring the number of particles of extracellular fine particles (EV) in a liquid with a NanoSign LM10V-HS nanoparticle tracking system, Examples include a method for measuring the amount of protein. As a method for measuring the amount of CD63-positive exosomes in the obtained extracellular fine particles (EV), for example, protein quantification is performed according to a conventional method, and the amount of protein in each sample is adjusted to be equal to Western blotting. , CD63 is detected, and the amount of CD63 in each sample is analyzed. Further, for the microRNA contained in each sample, the profile of the contained microRNA (expression intensity) can be analyzed using 3D-gene human microRNA chips (manufactured by Toray Industries, Inc.) or the like.
上記マイクロＲＮＡ（ｍｉＲＮＡ）のプロファイルとしては、ミトコンドリア由来のｍｉＲ４９４－３ｐ、ｍｉＲ１２４６、ｍｉＲ４７４１及び／又はｍｉＲ７１１０－５ｐの含有量、発現量、放出量等が好ましいものとして挙げられ、中でもｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の含有量、発現量、放出量等がより好ましいものとして挙げられ、ｍｉＲ４９４－３ｐの含有量、発現量、放出量等がさらに好ましいものとして挙げられる。
As the profile of the microRNA (miRNA), the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and / Alternatively, the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable.
（３）上記（２）で測定された数値と基準値を比較し、組織炎症を伴う炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク、治療効果等を判定する工程：
本工程においては、上記（２）の工程で得られた数値と基準値を比較する。このとき、罹患可能性を判断する際の基準値としては、健常人における平均値、数値範囲等を用いることができる。また、組織炎症を伴う炎症性網膜疾患の治療効果を判定する場合には、治療前の値、治療後の値を比較し、その数値が低くなっていれば治療効果があったと判定され、その数値が変化しない場合は治療効果が見られなかったと判定され、その数値が高くなった場合には悪化したと判定される。 (3) A step of comparing the numerical value measured in (2) above with a reference value to determine the possibility of inflammatory retinal disease accompanied by tissue inflammation, severe / mild, risk of onset, risk of aggravation, therapeutic effect, etc .:
In this step, the numerical value obtained in the step (2) above is compared with the reference value. At this time, as a reference value for determining the possibility of morbidity, an average value, a numerical range, or the like in a healthy person can be used. In addition, when determining the therapeutic effect of inflammatory retinal disease accompanied by tissue inflammation, the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
本工程においては、上記（２）の工程で得られた数値と基準値を比較する。このとき、罹患可能性を判断する際の基準値としては、健常人における平均値、数値範囲等を用いることができる。また、組織炎症を伴う炎症性網膜疾患の治療効果を判定する場合には、治療前の値、治療後の値を比較し、その数値が低くなっていれば治療効果があったと判定され、その数値が変化しない場合は治療効果が見られなかったと判定され、その数値が高くなった場合には悪化したと判定される。 (3) A step of comparing the numerical value measured in (2) above with a reference value to determine the possibility of inflammatory retinal disease accompanied by tissue inflammation, severe / mild, risk of onset, risk of aggravation, therapeutic effect, etc .:
In this step, the numerical value obtained in the step (2) above is compared with the reference value. At this time, as a reference value for determining the possibility of morbidity, an average value, a numerical range, or the like in a healthy person can be used. In addition, when determining the therapeutic effect of inflammatory retinal disease accompanied by tissue inflammation, the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
本発明の判定方法によると、疾患の初期の加齢黄斑変性の診断や予後予測等を高い確度で簡便に行うことができる。
According to the determination method of the present invention, it is possible to easily diagnose and predict the prognosis of age-related macular degeneration in the early stage of the disease with high accuracy.
上述のとおり、前房水や血漿中の上記特定のｍｉＲＮＡを定量することにより、炎症性網膜疾患の罹患可能性・発症リスク、症状の重軽度・重症化リスクの診断及び治療効果が判定されるが、判定項目はこれに限定されない。その他の判定項目としては、未病、初期病変、あるいは各疾患にて確定診断を下せる前段階（症状が明確ではない状態）であることの判定等が挙げられる。特に、加齢黄斑変性においては、アムスラー検査、眼底検査及び蛍光眼底造影検査の診断方法があり得る。しかし、アムスラー検査で視野が欠けたり、蛍光眼底造影で新生血管が認められたりする状態はすでに重度の加齢黄斑変性である。そこで、加齢黄斑変性のより効果的な治療のためには、脈絡膜浸潤マクロファージが網膜色素上皮細胞と相互作用をすることなどにより惹起される組織炎症に係るより早期の、視野欠損や血管新生がまだない段階での診断方法、判定方法が望まれている。前房水や血漿、組織液中の上記特定のｍｉＲＮＡを定量することにより、未病、初期病変、あるいは確定診断をまだ下せない早期の段階での予測が可能であり、より効果的な薬剤治療が期待できるだけでなく、加齢黄斑変性の予防のための指標にもなり得る。
As described above, by quantifying the above-mentioned specific miRNA in the anterior aqueous humor or plasma, the diagnosis and therapeutic effect of the possibility / risk of developing inflammatory retinal disease and the risk of severe / severe symptoms are determined. However, the judgment item is not limited to this. Other determination items include determination of pre-illness, initial lesion, or pre-stage (state in which symptoms are not clear) in which a definitive diagnosis can be made for each disease. In particular, in age-related macular degeneration, there may be diagnostic methods of Amsler examination, fundus examination and fluorescence fundus contrast examination. However, the condition in which the visual field is lacking on the Amsler examination and new blood vessels are observed on the fluorescence fundus angiography is already severe age-related macular degeneration. Therefore, for more effective treatment of age-related macular degeneration, earlier-stage visual field defects and angiogenesis related to tissue inflammation caused by the interaction of choroidal infiltrating macrophages with retinal pigment epithelial cells are required. A diagnostic method and a judgment method at a stage that has not yet been achieved are desired. By quantifying the above-mentioned specific miRNAs in anterior aqueous humor, plasma, and tissue fluid, it is possible to predict pre-illness, early lesions, or early stages where a definitive diagnosis cannot be made, and more effective drug treatment. Not only can it be expected, but it can also be an index for the prevention of age-related macular degeneration.
また、前房水や血漿中の上記特定のｍｉＲＮＡを定量することは、加齢黄斑変性の罹患前の診断に有益であるだけでなく、疾患分類にも有効である。加齢黄斑変性は滲出型と委縮型に分別される。前駆病変として、脈絡膜新血管異常に基づく脈絡膜血管新生（ＰＮＶ）などとの関連が議論されているが、これら、多様な後眼部炎症性疾患の各々の表現型や治療方法は大きく異なる。マクロファージなどの炎症性免疫細胞の脈絡膜－網膜組織への浸潤を伴う組織炎症が、中心性漿液性脈絡網膜症（ＣＳＣ）の急性から慢性への進行を引き起こし得ることが示唆され始めている。しかし、早期に滲出型ＡＭＤか委縮型ＡＭＤかの診断は現実不可能である。前房水や血漿中、あるいはその他の組織液中の上記特定のｍｉＲＮＡを定量することにより、加齢黄斑変性の滲出型、委縮型を含む多様な後眼部炎症性疾患の疾患分類や、その診断にも寄与するものと考えられる。
In addition, quantifying the above-mentioned specific miRNA in anterior aqueous humor or plasma is not only useful for pre-diagnosis of age-related macular degeneration, but also for disease classification. Age-related macular degeneration is divided into exudative and atrophic types. As prodromal lesions, the relationship with choroidal neovascularization (PNV) based on choroidal neovascularization has been discussed, but the phenotypes and treatment methods of these various posterior ocular inflammatory diseases differ greatly. It is beginning to be suggested that tissue inflammation with infiltration of inflammatory immune cells such as macrophages into the choroid-retinal tissue can cause the acute to chronic progression of central serous chorioretinal disease (CSC). However, early diagnosis of wet AMD or atrophic AMD is not feasible. By quantifying the above-mentioned specific miRNA in anterior aqueous humor, plasma, or other tissue fluid, various posterior ocular inflammatory diseases including exudative and atrophic types of age-related macular degeneration can be classified and diagnosed. It is thought that it also contributes to.
［炎症性網膜疾患治療剤］
本発明の炎症性網膜疾患治療剤は、網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子を有効成分として含有する。組織炎症を伴う炎症性網膜疾患を発症している被験者の網膜色素上皮細胞（ＲＰＥ）においては、放出される細胞外微粒子（ＥＶ）の量やそれが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量が増加しているため、これらの機能を調節（抑制又は促進）する分子が、組織炎症を伴う炎症性網膜疾患の治療効果を奏することが期待される。なお、組織炎症を伴う炎症性網膜疾患を発症している被験者の網膜色素上皮細胞（ＲＰＥ）において上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の放出量が増加している場合、網膜色素上皮細胞（ＲＰＥ）中の上記特定のｍｉＲＮＡ量が過剰になっていることが考えられる一方で、放出量の増加によって網膜色素上皮細胞（ＲＰＥ）中の上記特定のｍｉＲＮＡ量（ミトコンドリア含有のものも含む）が減少していることも考え得る。そのため、被験者の疾患の状態に合わせて、これらのｍｉＲＮＡの発現を調節（抑制又は促進）することが疾患の治療につながると考えられる。 [Therapeutic agent for inflammatory retinal disease]
The therapeutic agent for inflammatory retinal disease of the present invention contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient. In the retinal pigment epithelial cells (RPE) of subjects developing inflammatory retinal disease accompanied by tissue inflammation, the amount of extracellular fine particles (EV) released and the above-mentioned specific miRNA (microRNA) contained therein. Due to the increased amount, molecules that regulate (suppress or promote) these functions are expected to have a therapeutic effect on inflammatory retinal diseases associated with tissue inflammation. When the amount of the specific miRNA (microRNA) released in the retinal pigment epithelial cells (RPE) of a subject who develops inflammatory retinal disease accompanied by tissue inflammation is increased, the retinal pigment epithelial cells (RPE) are used. While it is possible that the specific amount of miRNA in the tissue is excessive, the amount of the specific miRNA in the retinal pigment epithelial cells (RPE) (including those containing mitochondria) decreases due to the increase in the release amount. It is possible that it is. Therefore, it is considered that regulating (suppressing or promoting) the expression of these miRNAs according to the state of the disease of the subject leads to the treatment of the disease.
本発明の炎症性網膜疾患治療剤は、網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子を有効成分として含有する。組織炎症を伴う炎症性網膜疾患を発症している被験者の網膜色素上皮細胞（ＲＰＥ）においては、放出される細胞外微粒子（ＥＶ）の量やそれが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量が増加しているため、これらの機能を調節（抑制又は促進）する分子が、組織炎症を伴う炎症性網膜疾患の治療効果を奏することが期待される。なお、組織炎症を伴う炎症性網膜疾患を発症している被験者の網膜色素上皮細胞（ＲＰＥ）において上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の放出量が増加している場合、網膜色素上皮細胞（ＲＰＥ）中の上記特定のｍｉＲＮＡ量が過剰になっていることが考えられる一方で、放出量の増加によって網膜色素上皮細胞（ＲＰＥ）中の上記特定のｍｉＲＮＡ量（ミトコンドリア含有のものも含む）が減少していることも考え得る。そのため、被験者の疾患の状態に合わせて、これらのｍｉＲＮＡの発現を調節（抑制又は促進）することが疾患の治療につながると考えられる。 [Therapeutic agent for inflammatory retinal disease]
The therapeutic agent for inflammatory retinal disease of the present invention contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient. In the retinal pigment epithelial cells (RPE) of subjects developing inflammatory retinal disease accompanied by tissue inflammation, the amount of extracellular fine particles (EV) released and the above-mentioned specific miRNA (microRNA) contained therein. Due to the increased amount, molecules that regulate (suppress or promote) these functions are expected to have a therapeutic effect on inflammatory retinal diseases associated with tissue inflammation. When the amount of the specific miRNA (microRNA) released in the retinal pigment epithelial cells (RPE) of a subject who develops inflammatory retinal disease accompanied by tissue inflammation is increased, the retinal pigment epithelial cells (RPE) are used. While it is possible that the specific amount of miRNA in the tissue is excessive, the amount of the specific miRNA in the retinal pigment epithelial cells (RPE) (including those containing mitochondria) decreases due to the increase in the release amount. It is possible that it is. Therefore, it is considered that regulating (suppressing or promoting) the expression of these miRNAs according to the state of the disease of the subject leads to the treatment of the disease.
ここで、本発明において細胞外微粒子（ＥＶ）の機能調節分子とは、細胞外微粒子（ＥＶ）が有する機能を調節する分子であれば特に限定されないが、細胞外微粒子（ＥＶ）の産生（放出）を調節する分子も含む広い概念である。また、細胞外微粒子（ＥＶ）の機能調節分子としては、細胞外微粒子（ＥＶ）が含有するマイクロＲＮＡ（ｍｉＲＮＡ）の機能調節分子が好ましい例として挙げられ、中でも細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ、ｍｉＲ１２４６、ｍｉＲ４７４１及び／又はｍｉＲ７１１０－５ｐの機能調節分子であることがより好ましく、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の機能調節分子であることがさらに好ましく、ｍｉＲ４９４－３ｐの機能調節分子であることが特に好ましい。これらの機能調節分子としては、核酸オリゴ、低分子化合物、ペプチド、抗体等が挙げられ、具体的には、上記特定のマイクロＲＮＡ（ｍｉＲＮＡ）の機能を調節する核酸オリゴ等が好ましいものとして挙げられる。さらに詳細には、上記特定のマイクロＲＮＡ（ｍｉＲＮＡ）の機能を亢進する核酸オリゴ（ｍｉｍｉｃの導入）、上記特定のマイクロＲＮＡ（ｍｉＲＮＡ）の機能を抑制する核酸オリゴ（ｉｎｈｉｂｉｔｏｒの導入）である。なお、上記核酸オリゴは、生体に投与された際の分解を抑制する目的、及び／又は疾患部位に特異的に結合させる目的等により、修飾されていることが好ましい。
Here, in the present invention, the function-regulating molecule of the extracellular fine particles (EV) is not particularly limited as long as it is a molecule that regulates the function of the extracellular fine particles (EV), but the production (release) of the extracellular fine particles (EV) is limited. ) Is a broad concept including molecules that regulate. Further, as a function-regulating molecule of extracellular fine particles (EV), a function-regulating molecule of microRNA (miRNA) contained in extracellular fine particles (EV) is given as a preferable example, and among them, extracellular fine particles (EV) are contained. It is more preferably a function-regulating molecule of miR494-3p, miR1246, miR4741 and / or miR7110-5p, further preferably a function-regulating molecule of miR494-3p and / or miR1246, and a function-regulating molecule of miR494-3p. It is particularly preferable to have. Examples of these function-regulating molecules include nucleic acid oligos, small molecule compounds, peptides, antibodies, and the like, and specific examples thereof include nucleic acid oligos that regulate the function of the specific microRNA (miRNA). .. More specifically, it is a nucleic acid oligo (introduction of mimic) that enhances the function of the specific microRNA (miRNA), and a nucleic acid oligo (introduction of inhibitor) that suppresses the function of the specific microRNA (miRNA). The nucleic acid oligo is preferably modified for the purpose of suppressing decomposition when administered to a living body and / or for the purpose of specifically binding to a diseased site.
本発明における上記核酸オリゴとして、好ましくは、上記特定のマイクロＲＮＡ（ｍｉＲＮＡ）のｍｉｍｉｃ、ｓｉＲＮＡ又はｓｈＲＮＡを挙げることができ、より好ましくは、上記特定のマイクロＲＮＡ（ｍｉＲＮＡ）のｍｉｍｉｃ、ｓｉＲＮＡを挙げることができる。ｓｉＲＮＡは、細胞で毒性を示さない範囲の短鎖からなる二重鎖ＲＮＡを意味し、例えば１５～４９塩基対と、好適には１５～３５塩基対と、さらに好適には２１～３０塩基対とすることが出来る。また、ｓｈＲＮＡは、一本鎖のＲＮＡがヘアピン構造を介して二重鎖を構成しているＲＮＡである。
As the nucleic acid oligo in the present invention, preferably, mimic, siRNA or shRNA of the specific microRNA (miRNA) can be mentioned, and more preferably, mimic, siRNA of the specific microRNA (miRNA) can be mentioned. Can be done. SiRNA means a double-stranded RNA consisting of short strands in a range that does not show toxicity in cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Can be. Further, shRNA is RNA in which a single-stranded RNA constitutes a double strand via a hairpin structure.
本発明における「核酸オリゴ」は、天然及び非天然のＲＮＡまたはＤＮＡからなるオリゴであり、ｍｉＲ４９４－３ｐ、ｍｉＲ１２４６、ｍｉＲ４７４１及び／又はｍｉＲ７１１０－５ｐの機能を制御する核酸のオリゴマーを意味する。本発明における核酸オリゴとしては、ｍｉＲＮＡ、ｓｉＲＮＡ、ｓｈＲＮＡ、アンチセンス核酸、デコイ核酸、核酸アプタマーが含まれる。
The "nucleic acid oligo" in the present invention is an oligo composed of natural and unnatural RNA or DNA, and means a nucleic acid oligomer that controls the functions of miR494-3p, miR1246, miR4741 and / or miR7110-5p. Nucleic acid oligos in the present invention include miRNA, siRNA, shRNA, antisense nucleic acid, decoy nucleic acid, and nucleic acid aptamer.
ｓｉＲＮＡおよびｓｈＲＮＡは、標的遺伝子と完全に同一である必要はないが、少なくとも７０％以上、好ましくは８０％以上、さらに好ましくは９０％以上、最も好ましくは９５％以上の配列の相同性を有する。
SiRNA and shRNA do not have to be exactly the same as the target gene, but have at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.
ｓｉＲＮＡおよびｓｈＲＮＡにおけるＲＮＡ同士が対合した二重鎖ＲＮＡの部分は、完全に対合しているものに限らず、ミスマッチ（対応する塩基が相補的でない）、バルジ（一方の鎖に対応する塩基がない）等により不対合部分が含まれていてもよい。本発明においてはｄｓＲＮＡにおけるＲＮＡ同士が対合する二重鎖ＲＮＡ領域中に、バルジおよびミスマッチの両方が含まれていてもよい。
The portion of double-stranded RNA in which RNAs are paired with each other in siRNA and shRNA is not limited to a completely paired one, but is not limited to a completely paired one, but is a mismatch (corresponding bases are not complementary) and bulge (base corresponding to one strand). It may include a non-matching portion due to (there is no) or the like. In the present invention, both the bulge and the mismatch may be contained in the double-stranded RNA region in which the RNAs in the dsRNA are paired with each other.
本発明における炎症性網膜疾患治療剤には、保存剤や安定剤等の製剤上許容しうる材料が添加されていてもよい。製剤上許容しうるとは、それ自体は上記治療効果を有する材料であってもよいし、当該治療効果を有さない材料であってもよく、上記治療剤とともに投与可能な製剤上許容される材料を意味する。また、治療効果を有さない材料であって、炎症性網膜疾患治療剤と併用することによって相乗的効果もしくは相加的な安定化効果を有する材料であってもよい。
The therapeutic agent for inflammatory retinal disease in the present invention may contain a pharmaceutically acceptable material such as a preservative or a stabilizer. The term "acceptable" means that the material itself may have the above-mentioned therapeutic effect or may not have the above-mentioned therapeutic effect, and may be the material that can be administered together with the above-mentioned therapeutic agent. Means material. Further, a material having no therapeutic effect and having a synergistic effect or an additive stabilizing effect when used in combination with a therapeutic agent for inflammatory retinal disease may be used.
製剤上許容される材料としては、例えば滅菌水や生理食塩水、保存剤、安定剤、賦形剤、緩衝剤、防腐剤、界面活性剤、キレート剤（ＥＤＴＡ等）、結合剤等を挙げることができる。
Examples of the material permitted in the formulation include sterile water, physiological saline, preservatives, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders, and the like. Can be done.
本発明における炎症性網膜疾患治療剤を注射用の水溶液とする場合には、例えば生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ－ソルビトール、Ｄ－マンノース、Ｄ－マンニトール、塩化ナトリウムが挙げられる）、適当な溶解補助剤、例えばアルコール（エタノール等）、ポリアルコール（プロピレングリコール、ＰＥＧ等）、非イオン性界面活性剤（ポリソルベート８０、ＨＣＯ－５０）等と併用してもよい。所望によりさらに希釈剤、溶解補助剤、ｐＨ調整剤、無痛化剤、含硫還元剤、酸化防止剤等を含有してもよい。
When the therapeutic agent for inflammatory retinal disease in the present invention is used as an aqueous solution for injection, an isotonic solution containing, for example, physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol) , Sodium chloride), suitable solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbitol 80, HCO-50), etc. May be good. If desired, a diluent, a solubilizing agent, a pH adjuster, a pain-relieving agent, a sulfur-containing reducing agent, an antioxidant and the like may be further contained.
また、必要に応じ、本発明における炎症性網膜疾患治療剤をマイクロカプセル（ヒドロキシメチルセルロース、ゼラチン、ポリ［メチルメタクリル酸］等のマイクロカプセル）に封入したり、コロイドドラッグデリバリーシステム（リポソーム、アルブミンミクロスフェア、マイクロエマルジョン、ナノ粒子及びナノカプセル等）としたりすることもできる。さらに、薬剤を徐放性の薬剤とする方法も公知であり、本発明に適用し得る。使用される製剤上許容しうる担体は、剤型に応じて上記の中から適宜あるいは組合せて選択されるが、これらに限定されるものではない。
Further, if necessary, the therapeutic agent for inflammatory retinal disease of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylate]), or a colloidal drug delivery system (liposomes, albumin microspheres). , Microemulsions, nanoparticles and nanocapsules, etc.). Further, a method of making a drug a sustained release drug is also known and can be applied to the present invention. The pharmaceutically acceptable carrier to be used is appropriately selected from the above or in combination depending on the dosage form, but is not limited thereto.
本発明の炎症性網膜疾患治療剤をヒトの医薬として使用する場合には、これらの物質自体を直接患者に投与する以外に、公知の製剤学的方法により製剤化して投与を行うことも可能である。製剤化する場合には上記に記載の製剤上許容される材料を添加しても良い。
When the therapeutic agent for inflammatory retinal disease of the present invention is used as a human medicine, it is possible to formulate and administer these substances by a known pharmaceutical method, in addition to directly administering these substances to patients. be. In the case of formulation, the above-mentioned materials that are acceptable for the formulation may be added.
本発明における炎症性網膜疾患治療剤は、医薬品の形態で投与することが可能であり、経口的または非経口的に全身あるいは局所的に投与することができる。例えば、点滴等の静脈内注射、筋肉内注射、腹腔内注射、皮下注射、坐薬、注腸、経口性腸溶剤等を選択することができ、患者の年齢、症状により適宜投与方法を選択することができる。有効投与量は、一回につき体重１ｋｇあたり０．０００００１ｍｇから１ｇの範囲で選ばれる。あるいは、患者あたり０．００００１～１００ｍｇ、好ましくは０．０００１～５０ｍｇの投与量を選ぶことができる。
The therapeutic agent for inflammatory retinal disease in the present invention can be administered in the form of a pharmaceutical product, and can be administered orally or parenterally systemically or topically. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral intestinal solvent, etc. can be selected, and the administration method should be appropriately selected according to the age and symptoms of the patient. Can be done. The effective dose is selected in the range of 0.000001 mg to 1 g per 1 kg of body weight at a time. Alternatively, a dose of 0.00001 to 100 mg, preferably 0.0001 to 50 mg per patient can be selected.
本発明の炎症性網膜疾患治療剤における炎症性網膜疾患としては、例えば加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症、糖尿病性網膜症等が挙げられる。これらのうち、本発明の炎症性網膜疾患治療剤が特に有効である疾患は加齢黄斑変性である。
Examples of the inflammatory retinal disease in the therapeutic agent for inflammatory retinal disease of the present invention include age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, diabetic retinopathy and the like. Of these, the disease for which the therapeutic agent for inflammatory retinal disease of the present invention is particularly effective is age-related macular degeneration.
［組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法］
本発明の組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法は、網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）のプロファイルの変動を指標とすることを特徴とする。組織炎症を伴う炎症性網膜疾患の状態をミミックする系として、本発明者らが構築した網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系においては、分泌される細胞外微粒子（ＥＶ）の量が増加し、これらが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量が増加する。本発明のスクリーニング方法によると、網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）の量や、これらが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量を減少させる効果のある物質をスクリーニングすることができる。本発明のスクリーニング方法によって選定される物質は、組織炎症を伴う炎症性網膜疾患の症状を改善、治療する効果を奏することが期待できる。 [Screening method for therapeutic agents for inflammatory retinal diseases associated with tissue inflammation]
The method for screening a therapeutic agent for an inflammatory retinal disease associated with tissue inflammation of the present invention is a variation in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. Is a feature. In the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages constructed by the present inventors as a system for mimicking the state of inflammatory retinal disease accompanied by tissue inflammation, extracellular fine particles secreted. The amount of (EV) increases, and the amount of the specific miRNA (microRNA) contained therein increases. According to the screening method of the present invention, the amount of extracellular fine particles (EV) secreted in the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, and the above-mentioned specific miRNA (micro) contained therein. Substances that are effective in reducing the amount of RNA) can be screened. The substance selected by the screening method of the present invention can be expected to have an effect of improving and treating the symptoms of inflammatory retinal disease accompanied by tissue inflammation.
本発明の組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法は、網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）のプロファイルの変動を指標とすることを特徴とする。組織炎症を伴う炎症性網膜疾患の状態をミミックする系として、本発明者らが構築した網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系においては、分泌される細胞外微粒子（ＥＶ）の量が増加し、これらが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量が増加する。本発明のスクリーニング方法によると、網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）の量や、これらが含有する上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の量を減少させる効果のある物質をスクリーニングすることができる。本発明のスクリーニング方法によって選定される物質は、組織炎症を伴う炎症性網膜疾患の症状を改善、治療する効果を奏することが期待できる。 [Screening method for therapeutic agents for inflammatory retinal diseases associated with tissue inflammation]
The method for screening a therapeutic agent for an inflammatory retinal disease associated with tissue inflammation of the present invention is a variation in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. Is a feature. In the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages constructed by the present inventors as a system for mimicking the state of inflammatory retinal disease accompanied by tissue inflammation, extracellular fine particles secreted. The amount of (EV) increases, and the amount of the specific miRNA (microRNA) contained therein increases. According to the screening method of the present invention, the amount of extracellular fine particles (EV) secreted in the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, and the above-mentioned specific miRNA (micro) contained therein. Substances that are effective in reducing the amount of RNA) can be screened. The substance selected by the screening method of the present invention can be expected to have an effect of improving and treating the symptoms of inflammatory retinal disease accompanied by tissue inflammation.
上記特定のｍｉＲＮＡ（マイクロＲＮＡ）は、ミトコンドリア由来のｍｉＲ４９４－３ｐ、ｍｉＲ１２４６、ｍｉＲ４７４１及び／又はｍｉＲ７１１０－５ｐであることが好ましく、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６であることがより好ましく、ｍｉＲ４９４－３ｐであることがさらに好ましい。
The specific miRNA (microRNA) is preferably mitochondrial-derived miR494-3p, miR1246, miR4741 and / or miR7115-5p, more preferably miR494-3p and / or miR1246p, and miR494-3p. It is even more preferable to have.
本発明のスクリーニング方法は、以下の工程を含む方法として説明することもできる。
The screening method of the present invention can also be described as a method including the following steps.
（１）網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系を準備する工程
（２）（１）で準備された共培養系に被験物質を添加し、一定期間培養する工程、
（３）（２）の培養終了後、培養上清を回収し、培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程、及び
（４）被験物質が組織炎症を伴う炎症性網膜疾患治療剤として適しているか否かを判定する工程。 (1) Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (2) Step of adding a test substance to the co-culture system prepared in (1) and culturing for a certain period of time. ,
(3) After the culture of (2) is completed, the culture supernatant is collected, and the amount of extracellular fine particles (EV) in the culture supernatant, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles ( A step of measuring the content of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, the content of the above-mentioned specific miRNA (microRNA) derived from mitochondria, and ( 4) A step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation.
（２）（１）で準備された共培養系に被験物質を添加し、一定期間培養する工程、
（３）（２）の培養終了後、培養上清を回収し、培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の上記特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程、及び
（４）被験物質が組織炎症を伴う炎症性網膜疾患治療剤として適しているか否かを判定する工程。 (1) Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (2) Step of adding a test substance to the co-culture system prepared in (1) and culturing for a certain period of time. ,
(3) After the culture of (2) is completed, the culture supernatant is collected, and the amount of extracellular fine particles (EV) in the culture supernatant, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles ( A step of measuring the content of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, the content of the above-mentioned specific miRNA (microRNA) derived from mitochondria, and ( 4) A step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation.
上記各工程について以下に説明する。
Each of the above processes will be described below.
（１）網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系を準備する工程：
本工程において、網膜色素上皮細胞（ＲＰＥ）としては、ヒトの網膜組織から採取した網膜色素上皮細胞（ＲＰＥ）の初代培養細胞、これらから株化した細胞等を用いてもよいし、ｉＰＳ細胞から誘導して作製された網膜色素上皮細胞（ＲＰＥ）（ｉＰＳ－ｈＲＰＥ細胞）を用いてもよい。ｉＰＳ細胞から網膜色素上皮細胞（ＲＰＥ）を誘導する方法については、杉田らの論文（Ｓｕｇｉｔａ Ｓ． ｅｔ． ａｌ．， Ｌａｃｋ ｏｆ Ｔ ｃｅｌｌ ｒｅｓｐｏｎｓｅ ｔｏ ｉＰＳＣ－ｄｅｒｉｖｅｄ ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ ｆｒｏｍ ＨＬＡ ｈｏｍｏｚｙｇｏｕｓ ｄｏｎｏｒｓ． Ｓｔｅｍ Ｃｅｌ Ｒｅｐｏｒｔｓ， ２０１６， ｖｏｌ．７， ｐａｇｅ６１９－６３４）に記載の方法を参考にすることができる。 (1) Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages:
In this step, as the retinal pigment epithelial cell (RPE), a primary cultured cell of retinal pigment epithelial cell (RPE) collected from human retinal tissue, a cell established from these, or the like may be used, or from iPS cells. Retinal pigment epithelial cells (RPE) (iPS-hRPE cells) produced by induction may be used. For a method of inducing retinal pigment epithelial cells (RPE) from iPS cells, see a paper by Sugita et al. (Sugita S. et. The method described in Reports, 2016, vol. 7, page 619-634) can be referred to.
本工程において、網膜色素上皮細胞（ＲＰＥ）としては、ヒトの網膜組織から採取した網膜色素上皮細胞（ＲＰＥ）の初代培養細胞、これらから株化した細胞等を用いてもよいし、ｉＰＳ細胞から誘導して作製された網膜色素上皮細胞（ＲＰＥ）（ｉＰＳ－ｈＲＰＥ細胞）を用いてもよい。ｉＰＳ細胞から網膜色素上皮細胞（ＲＰＥ）を誘導する方法については、杉田らの論文（Ｓｕｇｉｔａ Ｓ． ｅｔ． ａｌ．， Ｌａｃｋ ｏｆ Ｔ ｃｅｌｌ ｒｅｓｐｏｎｓｅ ｔｏ ｉＰＳＣ－ｄｅｒｉｖｅｄ ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ ｆｒｏｍ ＨＬＡ ｈｏｍｏｚｙｇｏｕｓ ｄｏｎｏｒｓ． Ｓｔｅｍ Ｃｅｌ Ｒｅｐｏｒｔｓ， ２０１６， ｖｏｌ．７， ｐａｇｅ６１９－６３４）に記載の方法を参考にすることができる。 (1) Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages:
In this step, as the retinal pigment epithelial cell (RPE), a primary cultured cell of retinal pigment epithelial cell (RPE) collected from human retinal tissue, a cell established from these, or the like may be used, or from iPS cells. Retinal pigment epithelial cells (RPE) (iPS-hRPE cells) produced by induction may be used. For a method of inducing retinal pigment epithelial cells (RPE) from iPS cells, see a paper by Sugita et al. (Sugita S. et. The method described in Reports, 2016, vol. 7, page 619-634) can be referred to.
本工程において単球／マクロファージとしては、ヒトの血液、組織等から分離した単球／マクロファージを用いてもよいし、株化細胞であるＴＨＰ－１細胞を常法によりＰＭＡ等で処理することでマクロファージに分化させた細胞（ＰＭＡ－ＴＨＰ－１細胞）を用いてもよい。
As the monocytes / macrophages in this step, monocytes / macrophages isolated from human blood, tissues, etc. may be used, or THP-1 cells, which are established cells, may be treated with PMA or the like by a conventional method. Cells differentiated into macrophages (PMA-THP-1 cells) may be used.
本工程においては、上記網膜色素上皮細胞（ＲＰＥ）（例えばｉＰＳ－ｈＲＰＥ細胞）と、単球／マクロファージ（例えばＰＭＡ－ＴＨＰ－１細胞）を、ＬＰＳ等を添加して炎症を惹起した条件下で共培養を行う。共培養としては、両方の細胞を１つの培養器に混合して培養してもよいし、片方を接着培養させ、一定時間が経過した後にもう片方の細胞を播種してもよい。また、Ｔｒａｎｓｗｅｌｌ（登録商標）ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔｓ（コーニング社製）、Ｎｉｃｏ‐１（登録商標）ｃｕｌｔｕｒｅ ｓｙｓｔｅｍ（ＧｉｎｒｅｉｌａｂＩｎｃ．， Ｕｃｈｉｎａｄａ， Ｉｓｈｉｋａｗａ， Ｊａｐａｎ）等を用いてｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とが直接接触しない条件で共培養を行ってもよい。
In this step, the retinal pigment epithelial cells (RPE) (for example, iPS-hRPE cells) and monocytes / macrophages (for example, PMA-THP-1 cells) are added under the condition of inducing inflammation by adding LPS or the like. Perform co-culture. As co-culture, both cells may be mixed and cultured in one incubator, or one may be adherently cultured and the other cell may be seeded after a certain period of time has passed. In addition, iPS-hRPE cells and iPS-hRPP cells using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.), Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) and the like. Co-culture may be carried out under the condition that the cells do not come into direct contact with each other.
共培養において用いられる培地は、一般的な細胞培養や、上記網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージの培養に通常用いられるものであれば、特に限定されない。培地に牛胎児血清等の血清を添加してもよいが、血清から持ち込まれる細胞外微粒子の影響をなくすためには、無血清培地が好ましい。
The medium used in the co-culture is not particularly limited as long as it is usually used for general cell culture or culture of the above-mentioned retinal pigment epithelial cells (RPE) and monocytes / macrophages. Serum such as fetal bovine serum may be added to the medium, but a serum-free medium is preferable in order to eliminate the influence of extracellular fine particles brought in from the serum.
上記ＬＰＳ等を添加して炎症を惹起した条件としては、例えばＬＰＳを１ｎｇ～５００ｎｇ／ｍＬ、好ましくは１０ｎｇ～２００ｎｇ／ｍＬ、より好ましくは５０ｎｇ～１５０ｎｇ／ｍＬ、さらに好ましくは８０ｎｇ～１２０ｎｇ／ｍＬの濃度になるように添加した条件が挙げられる。
The conditions for inducing inflammation by adding the above LPS or the like include, for example, LPS of 1 ng to 500 ng / mL, preferably 10 ng to 200 ng / mL, more preferably 50 ng to 150 ng / mL, still more preferably 80 ng to 120 ng / mL. The conditions added so as to have a concentration can be mentioned.
共培養におけるそれぞれの細胞の数については、培養器のサイズ、形態などに合わせて適宜調節することができる。具体的な共培養の一例としては、例えば２４ウェルプレートを用いる場合、１．０×１０４ｃｅｌｌｓ～１．０×１０６ｃｅｌｌｓ／ウェル、好ましくは５．０×１０４ｃｅｌｌｓ～５．０×１０５ｃｅｌｌｓ、より好ましくは２．５×１０５ｃｅｌｌｓ程度のＰＭＡ－ＴＨＰ－１細胞を播種し、３０分～１２時間程度培養した後、５．０×１０３ｃｅｌｌｓ～５．０×１０５ｃｅｌｌｓ／ウェル、好ましくは２．０×１０４ｃｅｌｌｓ～２．０×１０５ｃｅｌｌｓ、より好ましくは１．０×１０５ｃｅｌｌｓ程度のｉＰＳ－ｈＲＰＥ細胞を播種して共培養を行うことができる。
The number of each cell in the co-culture can be appropriately adjusted according to the size, morphology and the like of the incubator. As an example of specific co-culture, for example, when a 24-well plate is used, 1.0 × 10 4 cells to 1.0 × 10 6 cells / well, preferably 5.0 × 10 4 cells to 5.0 ×. PMA-THP-1 cells of about 10 5 cells, more preferably about 2.5 × 10 5 cells are seeded, cultured for about 30 minutes to 12 hours, and then 5.0 × 10 3 cells to 5.0 × 10 5 IPS-hRPE cells of cells / well, preferably 2.0 × 10 4 cells to 2.0 × 10 5 cells, more preferably 1.0 × 10 5 cells, can be seeded and co-cultured.
（２）（１）で準備された共培養系に被験物質を添加し、一定期間培養する工程：
本工程においては、（１）で準備された共培養系に被験物質を添加して一定期間の培養が行われる。被験物質としては、例えば低分子化合物、天然物、核酸オリゴ、ペプチド、抗体等が挙げられ、添加量は物質に合わせて適宜調整され得る。 (2) A step of adding the test substance to the co-culture system prepared in (1) and culturing for a certain period of time:
In this step, the test substance is added to the co-culture system prepared in (1), and the culture is carried out for a certain period of time. Examples of the test substance include small molecule compounds, natural products, nucleic acid oligos, peptides, antibodies and the like, and the amount of addition can be appropriately adjusted according to the substance.
本工程においては、（１）で準備された共培養系に被験物質を添加して一定期間の培養が行われる。被験物質としては、例えば低分子化合物、天然物、核酸オリゴ、ペプチド、抗体等が挙げられ、添加量は物質に合わせて適宜調整され得る。 (2) A step of adding the test substance to the co-culture system prepared in (1) and culturing for a certain period of time:
In this step, the test substance is added to the co-culture system prepared in (1), and the culture is carried out for a certain period of time. Examples of the test substance include small molecule compounds, natural products, nucleic acid oligos, peptides, antibodies and the like, and the amount of addition can be appropriately adjusted according to the substance.
（３）（２）の培養終了後、培養上清を回収し、培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程：
本工程においては、上記（２）の工程において被験物質を添加して一定期間培養した後の培養上清を回収し、培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する。なお、具体的な方法については、上述の本発明の判定方法の工程（２）「（１）で準備された被験者の試料中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程」における各物質の測定方法の説明において「被験者の試料」を「培養上清」に置き換えて適用できる。 (3) After the culture of (2) is completed, the culture supernatant is collected, and the amount of extracellular fine particles (EV) in the culture supernatant, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles ( Step of measuring the content of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, a specific miRNA (microRNA) derived from mitochondria:
In this step, the culture supernatant after adding the test substance and culturing for a certain period in the above step (2) is collected, and the amount of extracellular fine particles (EV) in the culture supernatant and the extracellular fine particles (EV) are collected. ), The amount of CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically specific miRNA (microRNA) derived from mitochondria. ), Etc. are measured. Regarding the specific method, the amount of extracellular fine particles (EV) in the sample of the subject prepared in step (2) "(1) of the determination method of the present invention described above, in the extracellular fine particles (EV)". CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically of specific miRNA (microRNA) derived from mitochondria. In the description of the method for measuring each substance in the "step of measuring the content and the like", the "sample of the subject" can be replaced with the "culture supernatant" and applied.
本工程においては、上記（２）の工程において被験物質を添加して一定期間培養した後の培養上清を回収し、培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する。なお、具体的な方法については、上述の本発明の判定方法の工程（２）「（１）で準備された被験者の試料中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量等を測定する工程」における各物質の測定方法の説明において「被験者の試料」を「培養上清」に置き換えて適用できる。 (3) After the culture of (2) is completed, the culture supernatant is collected, and the amount of extracellular fine particles (EV) in the culture supernatant, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles ( Step of measuring the content of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, a specific miRNA (microRNA) derived from mitochondria:
In this step, the culture supernatant after adding the test substance and culturing for a certain period in the above step (2) is collected, and the amount of extracellular fine particles (EV) in the culture supernatant and the extracellular fine particles (EV) are collected. ), The amount of CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically specific miRNA (microRNA) derived from mitochondria. ), Etc. are measured. Regarding the specific method, the amount of extracellular fine particles (EV) in the sample of the subject prepared in step (2) "(1) of the determination method of the present invention described above, in the extracellular fine particles (EV)". CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically of specific miRNA (microRNA) derived from mitochondria. In the description of the method for measuring each substance in the "step of measuring the content and the like", the "sample of the subject" can be replaced with the "culture supernatant" and applied.
（４）被験物質が組織炎症を伴う炎症性網膜疾患治療剤として適しているか否かを判定する工程：
本工程においては、上記（３）の工程で得られた数値と陰性対象との数値を比較する。このとき、陰性対象としては、被験物質を添加していない試験で得られた数値を採用することができる。被験物質を添加していない試験で得られた数値と比較して、被験物質の添加により培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量が低下している場合には、炎症性網膜疾患の治療剤としての効果が期待できる物質であると判断される。 (4) Step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation:
In this step, the numerical values obtained in the above step (3) and the numerical values of the negative target are compared. At this time, as the negative target, the numerical value obtained in the test to which the test substance is not added can be adopted. Amount of extracellular fine particles (EV) in the culture supernatant and amount of CD63-positive exosomes in the extracellular fine particles (EV) due to the addition of the test substance, as compared with the values obtained in the test without the addition of the test substance. , Substances contained in extracellular fine particles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically the content of specific miRNA (microRNA) derived from mitochondria is reduced. If so, it is judged to be a substance that can be expected to be effective as a therapeutic agent for inflammatory retinal diseases.
本工程においては、上記（３）の工程で得られた数値と陰性対象との数値を比較する。このとき、陰性対象としては、被験物質を添加していない試験で得られた数値を採用することができる。被験物質を添加していない試験で得られた数値と比較して、被験物質の添加により培養上清中の細胞外微粒子（ＥＶ）の量、細胞外微粒子（ＥＶ）中のＣＤ６３陽性エクソソームの量、細胞外微粒子（ＥＶ）が含有する物質、特に細胞内物質由来の核酸、中でもｍｉＲＮＡ（マイクロＲＮＡ）のプロファイル、具体的にはミトコンドリア由来の特定のｍｉＲＮＡ（マイクロＲＮＡ）の含有量が低下している場合には、炎症性網膜疾患の治療剤としての効果が期待できる物質であると判断される。 (4) Step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation:
In this step, the numerical values obtained in the above step (3) and the numerical values of the negative target are compared. At this time, as the negative target, the numerical value obtained in the test to which the test substance is not added can be adopted. Amount of extracellular fine particles (EV) in the culture supernatant and amount of CD63-positive exosomes in the extracellular fine particles (EV) due to the addition of the test substance, as compared with the values obtained in the test without the addition of the test substance. , Substances contained in extracellular fine particles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically the content of specific miRNA (microRNA) derived from mitochondria is reduced. If so, it is judged to be a substance that can be expected to be effective as a therapeutic agent for inflammatory retinal diseases.
本発明のスクリーニング方法によると、加齢黄斑変性等の組織炎症を伴う炎症性網膜疾患の治療に効果を奏する物質を簡便にスクリーニングすることが可能である。
According to the screening method of the present invention, it is possible to easily screen substances that are effective in treating inflammatory retinal diseases associated with tissue inflammation such as age-related macular degeneration.
以下に本発明を実施例に基づいて更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。
The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.
［試験１］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系における炎症性サイトカインの産生
ヒト単球様細胞株ＴＨＰ－１を、１００ｎｇ／ｍＬのＰＭＡ存在下で２４～４８時間培養してマクロファージ様に分化させた。ｈＲＰＥ細胞としては、杉田らの論文（Ｓｕｇｉｔａ Ｓ． ｅｔ． ａｌ．， Ｌａｃｋ ｏｆ Ｔ ｃｅｌｌ ｒｅｓｐｏｎｓｅ ｔｏ ｉＰＳＣ－ｄｅｒｉｖｅｄ ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ ｆｒｏｍ ＨＬＡ ｈｏｍｏｚｙｇｏｕｓ ｄｏｎｏｒｓ． Ｓｔｅｍ Ｃｅｌ Ｒｅｐｏｒｔｓ， ２０１６， ｖｏｌ．７， ｐａｇｅ６１９－６３４）に記載の方法に従ってｉＰＳ細胞から誘導して作製された細胞を用いた。 [Test 1] Production of inflammatory cytokines in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages Human monocyte-like cell line THP-1 was used for 24-48 hours in the presence of 100 ng / mL PMA. It was cultured and differentiated into macrophages. As hRPE cells, the paper by Sugita et al. (Sugita S. et. Al., Lack of T cell response to iPSC-derivated retinal pigment epithelium cells from epithelium cell6 cell16sor6sol6s. The cells prepared by inducing from iPS cells according to the method described in the above were used.
ヒト単球様細胞株ＴＨＰ－１を、１００ｎｇ／ｍＬのＰＭＡ存在下で２４～４８時間培養してマクロファージ様に分化させた。ｈＲＰＥ細胞としては、杉田らの論文（Ｓｕｇｉｔａ Ｓ． ｅｔ． ａｌ．， Ｌａｃｋ ｏｆ Ｔ ｃｅｌｌ ｒｅｓｐｏｎｓｅ ｔｏ ｉＰＳＣ－ｄｅｒｉｖｅｄ ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ ｆｒｏｍ ＨＬＡ ｈｏｍｏｚｙｇｏｕｓ ｄｏｎｏｒｓ． Ｓｔｅｍ Ｃｅｌ Ｒｅｐｏｒｔｓ， ２０１６， ｖｏｌ．７， ｐａｇｅ６１９－６３４）に記載の方法に従ってｉＰＳ細胞から誘導して作製された細胞を用いた。 [Test 1] Production of inflammatory cytokines in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages Human monocyte-like cell line THP-1 was used for 24-48 hours in the presence of 100 ng / mL PMA. It was cultured and differentiated into macrophages. As hRPE cells, the paper by Sugita et al. (Sugita S. et. Al., Lack of T cell response to iPSC-derivated retinal pigment epithelium cells from epithelium cell6 cell16sor6sol6s. The cells prepared by inducing from iPS cells according to the method described in the above were used.
ｉＰＳ細胞から誘導して作製されたｈＲＰＥ細胞（以下、「ｉＰＳ－ｈＲＰＥ細胞」ともいう）と、ＰＭＡ処理したＴＨＰ－１細胞（以下、「ＰＭＡ－ＴＨＰ－１細胞」ともいう）を無血清培地ＤＭＥＭ／Ｆ１２中で、１００ｎｇ／ｍＬのＬＰＳ存在下若しくは非存在下で２４時間、共培養を行った。具体的には、２４ウェルプレート（コーニング社製）に、２．５×１０５ｃｅｌｌｓ／ウェルのＰＭＡ－ＴＨＰ－１細胞を播種し、４時間培養した。その後、１．０×１０５ｃｅｌｌｓ／ウェルのｉＰＳ－ｈＲＰＥ細胞を播種し、２４時間共培養を行った（Ｔ／Ｒ）。比較対象は、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）、又はｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）とした。各ウェルの培養上清を回収し、２０００×ｇ、１０分遠心し、得られた上清を０．２２μｍのフィルター（ミリポア社製）を通し、各種炎症性サイトカインの定量用のサンプルとした。各サンプル中のＩＬ－６、ＭＣＰ－１、ＩＬ－８、ＶＥＧＦ、ＴＮＦ－α、ＰＥＤＦの濃度をＥＬＩＳＡキット（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて測定した。各サンプルにつき４点の測定を行い、平均値を各サンプルの濃度とし、図１に示した。
HRPE cells produced by inducing from iPS cells (hereinafter, also referred to as "iPS-hRPE cells") and PMA-treated THP-1 cells (hereinafter, also referred to as "PMA-THP-1 cells") are serum-free medium. Co-culture was performed in DMEM / F12 in the presence or absence of 100 ng / mL LPS for 24 hours. Specifically, 2.5 × 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours. Then, 1.0 × 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R). The comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R). The culture supernatant of each well was collected, centrifuged at 2000 × g for 10 minutes, and the obtained supernatant was passed through a 0.22 μm filter (manufactured by Millipore) to prepare a sample for quantification of various inflammatory cytokines. The concentrations of IL-6, MCP-1, IL-8, VEGF, TNF-α, and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Four points were measured for each sample, and the average value was taken as the concentration of each sample and is shown in FIG.
図１に示すとおり、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）、又はｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）と比較して、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを共培養すること（Ｔ／Ｒ）により、ＩＬ－６、ＭＣＰ－１、ＩＬ－８、ＶＥＧＦの産生量が顕著に増加した。一方、ＴＮＦ－αは、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）からの産生量が最も多いところ、ｉＰＳ－ｈＲＰＥ細胞（Ｒ）と共培養（Ｔ／Ｒ）することにより、その産生量が顕著に抑制された。なお、ｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）の培養上清中にはＴＮＦ－αは検出されなかった。また、ＰＥＤＦは、ｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）からの産生量が最も多いところ、ＰＭＡ－ＴＨＰ－１細胞（Ｔ）と共培養すること（Ｔ／Ｒ）により、その産生量が顕著に抑制された。なお、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）の培養上清中にはＰＥＤＦはほとんど検出されなかった。
As shown in FIG. 1, co-culture of iPS-hRPE cells and PMA-THP-1 cells as compared with PMA-THP-1 cells only (T) or iPS-hRPE cells only (R) (T). / R) markedly increased the production of IL-6, MCP-1, IL-8, and VEGF. On the other hand, the amount of TNF-α produced from only PMA-THP-1 cells (T) is the highest, but the amount produced by co-culturing (T / R) with iPS-hRPE cells (R) is remarkable. Was suppressed. In addition, TNF-α was not detected in the culture supernatant of only iPS-hRPE cells (R). In addition, the amount of PEDF produced from only iPS-hRPE cells (R) is the highest, but the amount produced is significantly suppressed by co-culturing with PMA-THP-1 cells (T) (T / R). Was done. In addition, PEDF was hardly detected in the culture supernatant of only PMA-THP-1 cells (T).
加齢黄斑変性等の網膜の炎症性疾患の初期には、網膜色素上皮細胞周辺にマクロファージが浸潤してくることが確認されている。この試験１の結果は、網膜色素上皮細胞とマクロファージが共存し、ＬＰＳによって炎症が惹起されている状態となると、炎症性のサイトカインであるＩＬ－６、ＭＣＰ－１、ＩＬ－８、ＶＥＧＦの産生量が顕著に増加し、炎症をさらに増悪させる方向に動くことを示している。
It has been confirmed that macrophages infiltrate around retinal pigment epithelial cells in the early stages of retinal inflammatory diseases such as age-related macular degeneration. The results of this test 1 show that when retinal pigment epithelial cells and macrophages coexist and inflammation is induced by LPS, the inflammatory cytokines IL-6, MCP-1, IL-8, and VEGF are produced. The amount is significantly increased, indicating that it moves in a direction that further exacerbates inflammation.
［試験２］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系におけるＣＤ６３＋エクソソームの産生
ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを、２４ウェルプレート中、無血清培地（ＬＰＳ存在下又は非存在下）にて共培養し、ＣＤ６３＋エクソソームの産生量を測定した。具体的には、２４ウェルプレート（コーニング社製）に、２．５×１０５ｃｅｌｌｓ／ウェルのＰＭＡ－ＴＨＰ－１細胞を播種し、４時間培養した。その後、１．０×１０５ｃｅｌｌｓ／ウェルのｉＰＳ－ｈＲＰＥ細胞を播種し、２４時間共培養を行った（Ｔ／Ｒ）。比較対象は、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）、又はｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）とした。各ウェルの培養上清を回収し、２，０００×ｇ、１０分遠心し、得られた上清を０．２２μｍのフィルター（ミリポア社製）を通した。その後、ＭＬＳ５０スウィングロータ（ベックマン・コールター社製）を用いて超遠心（１００，０００×ｇ、９０分）を行いペレットを回収した。ペレットをＰＢＳに懸濁し、再度超遠心（１００，０００×ｇ、９０分）を行った。得られたペレットをＰＢＳに懸濁し、常法に従ってタンパク定量を行った。各サンプルのタンパク量が等量になるように調整してウェスタンブロッティングを行い、ＣＤ６３を検出し、各サンプルのＣＤ６３量を比較した。結果を図２（ａ）に示す。 [Test 2] Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 × 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours. Then, 1.0 × 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R). The comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R). The culture supernatant of each well was collected, centrifuged at 2,000 × g for 10 minutes, and the obtained supernatant was passed through a 0.22 μm filter (manufactured by Millipore). Then, the pellets were collected by ultracentrifugation (100,000 × g, 90 minutes) using an MLS50 swing rotor (manufactured by Beckman Coulter). The pellet was suspended in PBS and ultracentrifuged (100,000 × g, 90 minutes) was performed again. The obtained pellet was suspended in PBS and protein quantification was performed according to a conventional method. Western blotting was performed by adjusting the amount of protein in each sample to be equal, CD63 was detected, and the amount of CD63 in each sample was compared. The results are shown in FIG. 2 (a).
ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを、２４ウェルプレート中、無血清培地（ＬＰＳ存在下又は非存在下）にて共培養し、ＣＤ６３＋エクソソームの産生量を測定した。具体的には、２４ウェルプレート（コーニング社製）に、２．５×１０５ｃｅｌｌｓ／ウェルのＰＭＡ－ＴＨＰ－１細胞を播種し、４時間培養した。その後、１．０×１０５ｃｅｌｌｓ／ウェルのｉＰＳ－ｈＲＰＥ細胞を播種し、２４時間共培養を行った（Ｔ／Ｒ）。比較対象は、ＰＭＡ－ＴＨＰ－１細胞のみ（Ｔ）、又はｉＰＳ－ｈＲＰＥ細胞のみ（Ｒ）とした。各ウェルの培養上清を回収し、２，０００×ｇ、１０分遠心し、得られた上清を０．２２μｍのフィルター（ミリポア社製）を通した。その後、ＭＬＳ５０スウィングロータ（ベックマン・コールター社製）を用いて超遠心（１００，０００×ｇ、９０分）を行いペレットを回収した。ペレットをＰＢＳに懸濁し、再度超遠心（１００，０００×ｇ、９０分）を行った。得られたペレットをＰＢＳに懸濁し、常法に従ってタンパク定量を行った。各サンプルのタンパク量が等量になるように調整してウェスタンブロッティングを行い、ＣＤ６３を検出し、各サンプルのＣＤ６３量を比較した。結果を図２（ａ）に示す。 [Test 2] Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 × 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours. Then, 1.0 × 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R). The comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R). The culture supernatant of each well was collected, centrifuged at 2,000 × g for 10 minutes, and the obtained supernatant was passed through a 0.22 μm filter (manufactured by Millipore). Then, the pellets were collected by ultracentrifugation (100,000 × g, 90 minutes) using an MLS50 swing rotor (manufactured by Beckman Coulter). The pellet was suspended in PBS and ultracentrifuged (100,000 × g, 90 minutes) was performed again. The obtained pellet was suspended in PBS and protein quantification was performed according to a conventional method. Western blotting was performed by adjusting the amount of protein in each sample to be equal, CD63 was detected, and the amount of CD63 in each sample was compared. The results are shown in FIG. 2 (a).
また、Ｔｒａｎｓｗｅｌｌ（登録商標）ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔｓ（コーニング社製）を用いてｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とが直接接触しない条件で共培養を行った。具体的には、５．０×１０５ｃｅｌｌｓ／ｍＬのＰＭＡ－ＴＨＰ－１細胞７００μＬを下のチャンバーに、２．０×１０５ｃｅｌｌｓ／ｍＬのｉＰＳ－ｈＲＰＥ細胞３００μＬを上のチャンバーに播種し、２４時間共培養を行った。上下の各チャンバーに播種する細胞を入れ替えた試験も行った。それぞれのチャンバーから培養上清を回収し、上記と同様に超遠心を行い、サンプルを調整してウェスタンブロッティングを行った。ＣＤ６３を検出し、各サンプルのＣＤ６３量を比較した。結果を図２（ｂ）に示す。
In addition, co-culture was performed using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.) under the condition that iPS-hRPE cells and PMA-THP-1 cells do not come into direct contact with each other. Specifically, 700 μL of 5.0 × 10 5 cells / mL PMA-THP-1 cells was seeded in the lower chamber, and 300 μL of 2.0 × 10 5 cells / mL iPS-hRPE cells was seeded in the upper chamber. , Was co-cultured for 24 hours. A test was also conducted in which the cells seeded in the upper and lower chambers were replaced. Culture supernatants were collected from each chamber, ultracentrifuged in the same manner as above, samples were prepared, and Western blotting was performed. CD63 was detected and the amount of CD63 in each sample was compared. The results are shown in FIG. 2 (b).
図２（ａ）に示すとおり、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを共培養することにより、いずれか一方の細胞のみでの培養の場合と比較して、ＣＤ６３エクソソームの産生が顕著に増加した。ＬＰＳ存在下、非存在下の両方で同様の傾向が見られたが、ＬＰＳ存在下の炎症状態をミミックした条件の方が、ＣＤ６３エクソソームの産生量が顕著に増大していた。
As shown in FIG. 2 (a), by co-culturing iPS-hRPE cells and PMA-THP-1 cells, the production of CD63 exosomes is remarkable as compared with the case of culturing with only one of the cells. Increased to. Similar tendencies were observed both in the presence and absence of LPS, but the production of CD63 exosomes was significantly increased in the mimic condition of the inflammatory state in the presence of LPS.
また、図２（ｂ）に示すとおり、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とが直接接触しない条件で共培養を行ってもＣＤ６３エクソソームの産生が顕著に増加した。
Further, as shown in FIG. 2 (b), the production of CD63 exosomes was remarkably increased even when co-culture was performed under the condition that iPS-hRPE cells and PMA-THP-1 cells did not come into direct contact with each other.
以上の結果から、網膜色素上皮細胞とマクロファージが共存し、ＬＰＳによって炎症が惹起された条件においては、ＣＤ６３＋エクソソームの産生量が増大することがわかった。また、Ｔｒａｎｓｗｅｌｌ（登録商標）ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔｓを用いた試験結果から、この共培養によるＣＤ６３＋エクソソームの産生量の増大には、両方の細胞の直接接触によるインターラクションは必要ないこともわかった。
From the above results, it was found that the production amount of CD63 + exosome increases under the condition that retinal pigment epithelial cells and macrophages coexist and inflammation is induced by LPS. In addition, from the test results using Transwell® cell culture inserts, it was found that the increase in the production of CD63 + exosomes by this co-culture does not require interaction by direct contact between both cells.
［試験３］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系において産生が増強される細胞外微粒子の解析
上記試験１で得られた各サンプル（ＬＰＳ（＋）の条件のもの）に含まれる微小粒子数を、ＮａｎｏＳｉｇｈｔ ＬＭ１０Ｖ－ＨＳ ｎａｎｏｐａｒｔｉｃｌｅ ｔｒａｃｋｉｎｇ ｓｙｓｔｅｍ（Ｑｕａｎｔｕｍ Ｄｅｓｉｇｎ社製）を用いて測定した。また各培養上清のサンプル（ＣＳ）と、各培養上清のサンプル（ＣＳ）を上記試験２と同様の方法で超遠心して得られたサンプル（ＵＣ）についてウェスタンブロッティングを行い、ＣＤ６３を検出した。結果を図３に示す。 [Test 3] Analysis of extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. The number of fine particles contained in) was measured using a NanoSigt LM10V-HS nanoparticular tracing system (manufactured by Quantum Design). In addition, Western blotting was performed on the sample (CS) of each culture supernatant and the sample (UC) obtained by ultracentrifuging the sample (CS) of each culture supernatant by the same method as inTest 2 above, and CD63 was detected. .. The results are shown in FIG.
上記試験１で得られた各サンプル（ＬＰＳ（＋）の条件のもの）に含まれる微小粒子数を、ＮａｎｏＳｉｇｈｔ ＬＭ１０Ｖ－ＨＳ ｎａｎｏｐａｒｔｉｃｌｅ ｔｒａｃｋｉｎｇ ｓｙｓｔｅｍ（Ｑｕａｎｔｕｍ Ｄｅｓｉｇｎ社製）を用いて測定した。また各培養上清のサンプル（ＣＳ）と、各培養上清のサンプル（ＣＳ）を上記試験２と同様の方法で超遠心して得られたサンプル（ＵＣ）についてウェスタンブロッティングを行い、ＣＤ６３を検出した。結果を図３に示す。 [Test 3] Analysis of extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. The number of fine particles contained in) was measured using a NanoSigt LM10V-HS nanoparticular tracing system (manufactured by Quantum Design). In addition, Western blotting was performed on the sample (CS) of each culture supernatant and the sample (UC) obtained by ultracentrifuging the sample (CS) of each culture supernatant by the same method as in
図３（ａ）に示すとおり、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを共培養することにより、いずれか一方の細胞のみでの培養の場合と比較して、分泌される微小粒子数自体が顕著に増加することがわかった。また、図３（ｂ）に示すとおり、共培養により微小粒子中にＣＤ６３＋エクソソームが占める割合も顕著に増加していることがわかった。即ち、共培養により、微小粒子のうち特にＣＤ６３＋エクソソームの分泌量が増加した。
As shown in FIG. 3 (a), the number of fine particles secreted by co-culturing iPS-hRPE cells and PMA-THP-1 cells as compared with the case of culturing with only one of the cells. It turned out that it increased significantly. In addition, as shown in FIG. 3 (b), it was found that the proportion of CD63 + exosomes in the fine particles was significantly increased by co-culture. That is, the co-culture increased the amount of CD63 + exosomes secreted among the fine particles.
［試験４］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系において産生が増強される細胞外微粒子中のマイクロＲＮＡの解析
上記試験１で得られた各サンプルに含まれる微小粒子について、３Ｄ－ｇｅｎｅ ｈｕｍａｎ ｍｉｃｒｏＲＮＡ ｃｈｉｐｓ（東レ社製）を用いて、含まれるマイクロＲＮＡのプロファイルを解析した。下記表１に、各マイクロＲＮＡについて、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞の共培養サンプル中の発現強度（ＴＨＰ－１＋ＲＰＥ）をｉＰＳ－ｈＲＰＥ細胞単独培養サンプル中の発現強度（ＲＰＥ）で除した値（ｒａｔｉｏ）を示した。＃０２２８、＃０５２９、＃０６１９は、独立して行った試験番号である。また、上記３回の各試験において、それぞれ８個、７個、６個のマイクロＲＮＡが共培養によって有意に発現増加していた。これらの結果を図４にまとめた。図４においては、各試験において共培養で発現増加していたマイクロＲＮＡの集合をベン図で示している。 [Test 4] Analysis of microRNA in extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. The profile of the microRNA contained in the cells was analyzed using 3D-gene human microRNA chips (manufactured by Toray Co., Ltd.). In Table 1 below, for each microRNA, the expression intensity (THP-1 + RPE) in the co-cultured sample of iPS-hRPE cells and PMA-THP-1 cells is shown by the expression intensity (RPE) in the iPS-hRPE cell single culture sample. The divided value (ratio) is shown. # 0228, # 0259, and # 0619 are independent test numbers. In addition, in each of the above three tests, the expression of 8, 7, and 6 microRNAs was significantly increased by co-culture. These results are summarized in FIG. In FIG. 4, a Venn diagram shows a set of microRNAs whose expression was increased by co-culture in each test.
上記試験１で得られた各サンプルに含まれる微小粒子について、３Ｄ－ｇｅｎｅ ｈｕｍａｎ ｍｉｃｒｏＲＮＡ ｃｈｉｐｓ（東レ社製）を用いて、含まれるマイクロＲＮＡのプロファイルを解析した。下記表１に、各マイクロＲＮＡについて、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞の共培養サンプル中の発現強度（ＴＨＰ－１＋ＲＰＥ）をｉＰＳ－ｈＲＰＥ細胞単独培養サンプル中の発現強度（ＲＰＥ）で除した値（ｒａｔｉｏ）を示した。＃０２２８、＃０５２９、＃０６１９は、独立して行った試験番号である。また、上記３回の各試験において、それぞれ８個、７個、６個のマイクロＲＮＡが共培養によって有意に発現増加していた。これらの結果を図４にまとめた。図４においては、各試験において共培養で発現増加していたマイクロＲＮＡの集合をベン図で示している。 [Test 4] Analysis of microRNA in extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. The profile of the microRNA contained in the cells was analyzed using 3D-gene human microRNA chips (manufactured by Toray Co., Ltd.). In Table 1 below, for each microRNA, the expression intensity (THP-1 + RPE) in the co-cultured sample of iPS-hRPE cells and PMA-THP-1 cells is shown by the expression intensity (RPE) in the iPS-hRPE cell single culture sample. The divided value (ratio) is shown. # 0228, # 0259, and # 0619 are independent test numbers. In addition, in each of the above three tests, the expression of 8, 7, and 6 microRNAs was significantly increased by co-culture. These results are summarized in FIG. In FIG. 4, a Venn diagram shows a set of microRNAs whose expression was increased by co-culture in each test.
表１に示すとおり、ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とを共培養することにより、分泌される微小粒子中のマイクロＲＮＡのうち、特にｍｉＲ－４９４－３ｐの量が顕著に増加してることが分かった。ｉＰＳ－ｈＲＰＥ細胞、ＰＭＡ－ＴＨＰ－１細胞、共培養における両細胞由来の微小粒子中のｍｉＲＮＡ（Ｅｘｏ ｍｉＲ）のプロファイルを検討し、ｉＰＳ‐ｈＲＰＥ培養におけるそれらの量に関して１００の上位ランクのｍｉＲＮＡ（Ｅｘｏ ｍｉＲ）の中から、共培養における上位ランクのｍｉＲを選択した。これらの中で、４つのｍｉＲは、少なくとも２回の反復実験で共培養で増加した（図４；上記３回の反復実験で各実験から選択した８、７、及び６つのｍｉＲを描いたベン図）。図４に示した４つのＥｘｏ ｍｉＲ候補のうち、ｍｉＲ－４９４－３ｐは３つの測定値の中で１位、２位、４位と最大の増加を示していることが立証された（表１を参照）。網膜色素上皮細胞とマクロファージが共存し、ＬＰＳによって炎症が惹起されている状態となると、分泌されるエクソソーム等の微小粒子中の上記特定のマイクロＲＮＡの発現が増加し、特にｍｉＲ－４９４－３ｐが顕著に増加していることから、ｍｉＲ－４９４－３ｐは、網膜の炎症性疾患のマーカーとして疾患の罹患可能性、重症度、治療効果の判定に使用できると考えられた。また、総合的に判断してｍｉＲ１２４６も網膜の炎症性疾患のマーカーとして疾患の罹患可能性、重症度、治療効果の判定に使用できると考えられた。
As shown in Table 1, co-culture of iPS-hRPE cells and PMA-THP-1 cells significantly increased the amount of miR-494-3p among the microRNAs in the secreted microparticles. I found out that it was. The profiles of miRNAs (Exo miRs) in microparticles derived from both iPS-hRPE cells, PMA-THP-1 cells, and co-cultures were examined and 100 top-ranked miRNAs (exo miR) in their amount in iPS-hRPE cultures ( From Exo miR), the top-ranked miR in co-culture was selected. Among these, 4 miRs increased in co-culture in at least 2 repeat experiments (Fig. 4; Venn diagram depicting 8, 7, and 6 miRs selected from each experiment in the above 3 repeat experiments. ). Of the four Exo miR candidates shown in FIG. 4, it was proved that miR-494-3p showed the largest increase in the first, second, and fourth place among the three measured values (Table 1). See). When retinal pigment epithelial cells and macrophages coexist and inflammation is induced by LPS, the expression of the specific microRNA in the secreted microparticles such as exosomes increases, especially miR-494-3p. Since there was a marked increase, miR-494-3p was considered to be usable as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease. In addition, it was considered that miR1246 can also be used as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease.
［試験５］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系におけるＣＤ６３＋エクソソームの産生（細胞間接触の必要性の検討）
Ｔｒａｎｓｗｅｌｌ（登録商標）系及びＮｉｃｏ‐１（登録商標）系におけるｈＲＰＥ細胞とマクロファージの共培養を比較し、ＣＤ６３＋エクソソームの産生における両細胞間の直接的な接触の必要性を検討した。 [Test 5] Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (examination of the need for cell-cell contact)
Co-cultures of hRPE cells and macrophages in Transwell® and Nico-1® systems were compared to examine the need for direct contact between the two cells in the production of CD63 + exosomes.
Ｔｒａｎｓｗｅｌｌ（登録商標）系及びＮｉｃｏ‐１（登録商標）系におけるｈＲＰＥ細胞とマクロファージの共培養を比較し、ＣＤ６３＋エクソソームの産生における両細胞間の直接的な接触の必要性を検討した。 [Test 5] Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (examination of the need for cell-cell contact)
Co-cultures of hRPE cells and macrophages in Transwell® and Nico-1® systems were compared to examine the need for direct contact between the two cells in the production of CD63 + exosomes.
Ｔｒａｎｓｗｅｌｌ（登録商標）系にはいくつかの限界がある。上部チャンバーの内容量が下部チャンバーより小さいため、微小粒子は下部チャンバーの底部に優先的に沈降する可能性がある。また、０．４０μｍフィルターでは、他のチャンバーへの細胞浸透を遮断するのに十分ではない。そこで、水平に連結した二重チャンバーであるＮｉｃｏ‐１（登録商標）系を用い、２つのチャンバー間に０．０３μｍのフィルターを装着した場合としなかった場合とを比較した。なお、両チャンバーはそれぞれ１．５ｍＬの最終容積を保持している。
The Transwell (registered trademark) system has some limitations. Since the content of the upper chamber is smaller than that of the lower chamber, fine particles may preferentially settle at the bottom of the lower chamber. Also, a 0.40 μm filter is not sufficient to block cell penetration into other chambers. Therefore, a Nico-1 (registered trademark) system, which is a horizontally connected double chamber, was used, and a comparison was made between the case where a 0.03 μm filter was attached and the case where a 0.03 μm filter was attached between the two chambers. Both chambers each hold a final volume of 1.5 mL.
具体的には、Ｔｒａｎｓｗｅｌｌ（登録商標）ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔｓ（コーニング社製）及びＮｉｃｏ‐１（登録商標）ｃｕｌｔｕｒｅ ｓｙｓｔｅｍ（ＧｉｎｒｅｉｌａｂＩｎｃ．， Ｕｃｈｉｎａｄａ， Ｉｓｈｉｋａｗａ， Ｊａｐａｎ）を用いてｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とが直接接触しない条件で共培養を行った。具体的には、Ｔｒａｎｓｗｅｌｌ（登録商標）では、５．０×１０５ｃｅｌｌｓ／ｍＬのＰＭＡ－ＴＨＰ－１細胞７００μＬを下のチャンバーに、２．０×１０５ｃｅｌｌｓ／ｍＬのｉＰＳ－ｈＲＰＥ細胞３００μＬを上のチャンバーに播種し、２４時間共培養を行った。Ｎｉｃｏ‐１（登録商標）では、１．０×１０５ｃｅｌｌｓのＰＭＡ－ＴＨＰ－１細胞を一方のチャンバーに、２．０×１０５ｃｅｌｌｓのｉＰＳ－ｈＲＰＥ細胞をもう一方のチャンバーに播種し、２４時間共培養を行った。Ｎｉｃｏ‐１（登録商標）では、上述のとおり、２つのチャンバー間に０．０３μｍのフィルターを装着した場合としなかった場合とを比較した。各培養上清を上記試験２と同様の方法で超遠心して得られたサンプルについてウェスタンブロッティングを行い、ＣＤ６３を検出した。結果を図５（ａ）及び図５（ｂ）に示す。
Specifically, iPS-hRPE using Transwell (registered trademark) cell culture cells (manufactured by Corning) and Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) with iPS-hRP. Co-culture was performed under the condition that one cell did not come into direct contact with the cell. Specifically, in Transwell®, 700 μL of 5.0 × 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 μL of 2.0 × 10 5 cells / mL iPS-hRPE cells. Was seeded in the upper chamber and co-cultured for 24 hours. In Nico-1®, 1.0 × 10 5 cells PMA-THP-1 cells were seeded in one chamber and 2.0 × 10 5 cells iPS-hRPE cells were seeded in the other chamber. Co-culture was performed for 24 hours. For Nico-1®, as described above, comparison was made with and without a 0.03 μm filter between the two chambers. Western blotting was performed on the sample obtained by ultracentrifuging each culture supernatant by the same method as in Test 2 above, and CD63 was detected. The results are shown in FIGS. 5 (a) and 5 (b).
Ｎｉｃｏ‐１（登録商標）の系において、０．０３μｍフィルターにより微小粒子の移動が遮断された場合には、ｉＰＳ‐ＲＰＥ細胞ではＥｘｏ関連ＣＤ６３＋蛋白質の産生増加が観察されたが、ＰＭＡ‐ＴＨＰ‐１細胞では観察されなかった。これは、ｈＲＰＥ細胞とＭｐｓの相互作用におけるＥＶナノ粒子の関与を支持した。なお、Ｔｒａｎｓｗｅｌｌ（登録商標）の系（図５（ａ））では、ｉＰＳ－ＲＰＥによるＥｘｏ関連ＣＤ６３＋産生の増加は影響を受けなかった。これらの結果は、０．０３μｍフィルターを備えたＮｉｃｏ‐１（登録商標）系において、ｉＰＳ‐ＲＰＥ細胞によるＥｘｏ関連ＣＤ６３＋蛋白質の産生抑制を支持したが、ＰＭＡ‐ＴＨＰ‐１による産生抑制は支持せず（図５（ｂ））、これら２つの細胞型におけるＣＤ６３＋Ｅｘｏは明確な分子的特徴を有することを示唆した。
In the Nico-1® system, increased production of Exo-related CD63 + protein was observed in iPS-RPE cells when the movement of fine particles was blocked by a 0.03 μm filter, but PMA-THP- Not observed in 1 cell. This supported the involvement of EV nanoparticles in the interaction of hRPE cells with Mps. In the Transwell® system (FIG. 5 (a)), the increase in Exo-related CD63 + production by iPS-RPE was not affected. These results supported the suppression of Exo-related CD63 + protein production by iPS-RPE cells in the Nico-1® system with a 0.03 μm filter, but the suppression of production by PMA-THP-1. (Fig. 5 (b)), it was suggested that CD63 + Exo in these two cell types had distinct molecular characteristics.
［試験６］網膜色素上皮細胞（ＲＰＥ）と単球／マクロファージとの共培養系における炎症性サイトカインの産生（細胞間接触の必要性の検討）
Ｔｒａｎｓｗｅｌｌ（登録商標）系及びＮｉｃｏ‐１（登録商標）系におけるｈＲＰＥ細胞とマクロファージの共培養を比較し、ＭＣＰ‐１、Ｉｌ‐６、ＩＬ‐８、ＴＮＦ‐α、ＶＥＧＦ及びＰＥＤＦの産生における両細胞間の直接的な接触の必要性を検討した。 [Test 6] Production of inflammatory cytokines in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (examination of the need for cell-cell contact)
Co-cultures of hRPE cells and macrophages in Transwell® and Nico-1® systems were compared and both in the production of MCP-1, Il-6, IL-8, TNF-α, VEGF and PEDF. The need for direct cell-cell contact was investigated.
Ｔｒａｎｓｗｅｌｌ（登録商標）系及びＮｉｃｏ‐１（登録商標）系におけるｈＲＰＥ細胞とマクロファージの共培養を比較し、ＭＣＰ‐１、Ｉｌ‐６、ＩＬ‐８、ＴＮＦ‐α、ＶＥＧＦ及びＰＥＤＦの産生における両細胞間の直接的な接触の必要性を検討した。 [Test 6] Production of inflammatory cytokines in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (examination of the need for cell-cell contact)
Co-cultures of hRPE cells and macrophages in Transwell® and Nico-1® systems were compared and both in the production of MCP-1, Il-6, IL-8, TNF-α, VEGF and PEDF. The need for direct cell-cell contact was investigated.
Ｔｒａｎｓｗｅｌｌ（登録商標）ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔｓ（コーニング社製）及びＮｉｃｏ‐１（登録商標）ｃｕｌｔｕｒｅ ｓｙｓｔｅｍ（ＧｉｎｒｅｉｌａｂＩｎｃ．， Ｕｃｈｉｎａｄａ， Ｉｓｈｉｋａｗａ， Ｊａｐａｎ）を用いてｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞とが直接接触しない条件で共培養を行った。具体的には、Ｔｒａｎｓｗｅｌｌ（登録商標）では、５．０×１０５ｃｅｌｌｓ／ｍＬのＰＭＡ－ＴＨＰ－１細胞７００μＬを下のチャンバーに、２．０×１０５ｃｅｌｌｓ／ｍＬのｉＰＳ－ｈＲＰＥ細胞３００μＬを上のチャンバーに播種し、２４時間共培養を行った。Ｎｉｃｏ‐１（登録商標）では、１．０×１０５ｃｅｌｌｓのＰＭＡ－ＴＨＰ－１細胞を一方のチャンバーに、２．０×１０５ｃｅｌｌｓのｉＰＳ－ｈＲＰＥ細胞をもう一方のチャンバーに播種し、２４時間共培養を行った。上下の各チャンバーに播種する細胞を入れ替えた試験も行った。各ウェルの培養上清を回収し、２０００×ｇ、１０分遠心し、得られた上清を０．２２μｍのフィルター（ミリポア社製）を通し、各種炎症性サイトカインの定量用のサンプルとした。各サンプル中のＩＬ－６、ＭＣＰ－１、ＩＬ－８、ＶＥＧＦ、ＴＮＦ－α、ＰＥＤＦの濃度をＥＬＩＳＡキット（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて測定した。ＥＬＩＳＡ測定を３回に分けて行い、３回の独立した実験として繰り返した。各サンプルにつき平均値を算定し各サンプルの濃度として図６（ａ）及び図６（ｂ）に示した。
IPS-hRPE cells and PM cells directly with iPS-hRPE cells using Transwell® cell culture plants (Corning) and Nico-1® culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan). Co-culture was performed under the condition of no contact. Specifically, in Transwell®, 700 μL of 5.0 × 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 μL of 2.0 × 10 5 cells / mL iPS-hRPE cells. Was seeded in the upper chamber and co-cultured for 24 hours. In Nico-1®, 1.0 × 10 5 cells PMA-THP-1 cells were seeded in one chamber and 2.0 × 10 5 cells iPS-hRPE cells were seeded in the other chamber. Co-culture was performed for 24 hours. A test was also conducted in which the cells seeded in the upper and lower chambers were replaced. The culture supernatant of each well was collected, centrifuged at 2000 × g for 10 minutes, and the obtained supernatant was passed through a 0.22 μm filter (manufactured by Millipore) to prepare a sample for quantification of various inflammatory cytokines. The concentrations of IL-6, MCP-1, IL-8, VEGF, TNF-α, and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). The ELISA measurement was performed in 3 divided doses and repeated as 3 independent experiments. The average value was calculated for each sample and the concentration of each sample is shown in FIGS. 6 (a) and 6 (b).
０．０３μｍフィルターを用いないＮｉｃｏ‐１（登録商標）系での共培養では、Ｔｒａｎｓｗｅｌｌ（登録商標）培養でみられた上昇に匹敵するＭＣＰ－１、ＩＬ－６、ＩＬ－８の産生の著明なアップレギュレーションが認められた（図６（ａ））が、０．０３μｍフィルターを設置すると、このアップレギュレーションは完全に消失した（図６（ａ））。対照的に、ＶＥＧＦの産生増加は０．０３μｍフィルターによって部分的に抑制され（図６（ｂ））、ｉＰＳ－ＲＰＥ細胞によるＰＥＤＦ又はＰＭＡ－ＴＨＰ－１によるＴＮＦαの産生は、フィルター非存在下のレベルと比較すると抑制されなかった（図６（ｂ））。
Co-cultures in Nico-1® systems without a 0.03 μm filter resulted in significant production of MCP-1, IL-6, IL-8 comparable to the elevation seen in Transwell® cultures. Clear upregulation was observed (FIG. 6 (a)), but this upregulation disappeared completely when the 0.03 μm filter was installed (FIG. 6 (a)). In contrast, increased VEGF production was partially suppressed by a 0.03 μm filter (FIG. 6 (b)), and production of PEDF or TNFα by PMA-THP-1 by iPS-RPE cells was in the absence of the filter. It was not suppressed when compared to the level (Fig. 6 (b)).
［試験７］網膜色素上皮細胞（ＲＰＥ）由来のエクソソーム刺激によるマクロファージからのＴＮＦαの産生及びＲＰＥ細胞からのＭＣＰ－１、ＩＬ－６の産生
病態形成に関与していると考えられる細胞間相互作用による炎症増悪回路を明らかにするために、ｉＰＳ－ｈＲＰＥ細胞単独由来の、又はｉＰＳ－ｈＲＰＥ細胞及びＰＭＡ‐ＴＨＰ‐１の共培養由来のエクソソームの、マクロファージからのＴＮＦ－α産生促進効果を検討した。また、そのＴＮＦ－α処理によってｉＰＳ－ｈＲＰＥ細胞から産生される各種炎症性サイトカイン、微小粒子を定量した。また、ＣＤ６３＋微小粒子（ＥＸＯ）量を確認した。各サンプル中のＴＮＦ－α、ＭＣＰ－１、ＩＬ－６の濃度は、ＥＬＩＳＡキット（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて測定した。粒子濃度はＮａｎｏＳｉｇｈｔ ＬＭ１０Ｖ‐ＨＳナノ粒子追跡システムで測定した。結果を図７（ａ）及び図７（ｂ）に示す。 [Test 7] Production of TNFα from macrophages by exosome stimulation derived from retinal pigment epithelial cells (RPE) and production of MCP-1 and IL-6 from RPE cells Cell-cell interaction considered to be involved in pathogenesis In order to clarify the inflammation exacerbation circuit, the effect of promoting TNF-α production from macrophages of exosomes derived from iPS-hRPE cells alone or from co-culture of iPS-hRPE cells and PMA-THP-1 was investigated. .. In addition, various inflammatory cytokines and fine particles produced from iPS-hRPE cells by the TNF-α treatment were quantified. In addition, the amount of CD63 + fine particles (EXO) was confirmed. The concentrations of TNF-α, MCP-1, and IL-6 in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Particle concentration was measured with the NanoSigt LM10V-HS nanoparticle tracking system. The results are shown in FIGS. 7 (a) and 7 (b).
病態形成に関与していると考えられる細胞間相互作用による炎症増悪回路を明らかにするために、ｉＰＳ－ｈＲＰＥ細胞単独由来の、又はｉＰＳ－ｈＲＰＥ細胞及びＰＭＡ‐ＴＨＰ‐１の共培養由来のエクソソームの、マクロファージからのＴＮＦ－α産生促進効果を検討した。また、そのＴＮＦ－α処理によってｉＰＳ－ｈＲＰＥ細胞から産生される各種炎症性サイトカイン、微小粒子を定量した。また、ＣＤ６３＋微小粒子（ＥＸＯ）量を確認した。各サンプル中のＴＮＦ－α、ＭＣＰ－１、ＩＬ－６の濃度は、ＥＬＩＳＡキット（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて測定した。粒子濃度はＮａｎｏＳｉｇｈｔ ＬＭ１０Ｖ‐ＨＳナノ粒子追跡システムで測定した。結果を図７（ａ）及び図７（ｂ）に示す。 [Test 7] Production of TNFα from macrophages by exosome stimulation derived from retinal pigment epithelial cells (RPE) and production of MCP-1 and IL-6 from RPE cells Cell-cell interaction considered to be involved in pathogenesis In order to clarify the inflammation exacerbation circuit, the effect of promoting TNF-α production from macrophages of exosomes derived from iPS-hRPE cells alone or from co-culture of iPS-hRPE cells and PMA-THP-1 was investigated. .. In addition, various inflammatory cytokines and fine particles produced from iPS-hRPE cells by the TNF-α treatment were quantified. In addition, the amount of CD63 + fine particles (EXO) was confirmed. The concentrations of TNF-α, MCP-1, and IL-6 in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Particle concentration was measured with the NanoSigt LM10V-HS nanoparticle tracking system. The results are shown in FIGS. 7 (a) and 7 (b).
ｉＰＳ－ｈＲＰＥ細胞由来のエクソソームは、濃度依存的にＰＭＡ－ＴＨＰ－１細胞からのＴＮＦ－αの生産を増加させた。ｉＰＳ－ｈＲＰＥ細胞及びＰＭＡ‐ＴＨＰ‐１の共培養由来のエクソソームの方が、ｉＰＳ－ｈＲＰＥ細胞単独由来のエクソソームより、ＰＭＡ－ＴＨＰ－１細胞からのＴＮＦ－αの生産促進効果は大きかった。培養上清中のｉＰＳ－ｈＲＰＥ細胞由来のエクソソーム、ｉＰＳ－ｈＲＰＥ細胞及びＰＭＡ‐ＴＨＰ‐１の共培養由来のエクソソームの粒子数を比較すると、培養上清で３．５倍、それを超遠心して濃縮したもので８．５倍と、共培養の方が顕著に増加していた。
Exosomes derived from iPS-hRPE cells increased the production of TNF-α from PMA-THP-1 cells in a concentration-dependent manner. The exosomes derived from the co-culture of iPS-hRPE cells and PMA-THP-1 had a greater effect of promoting the production of TNF-α from PMA-THP-1 cells than the exosomes derived from iPS-hRPE cells alone. Comparing the number of particles of iPS-hRPE cell-derived exosomes, iPS-hRPE cells, and PMA-THP-1 co-culture-derived exosomes in the culture supernatant, the number of particles in the culture supernatant was 3.5 times, and it was supercentrifuged. The concentrated one was 8.5 times, and the co-culture increased remarkably.
ＴＮＦ‐αは、ｉＰＳ‐ｈＲＰＥ細胞からの微小粒子中のＣＤ６３＋蛋白質の産生増加と並行して、ＭＣＰ‐１とＩＬ‐６の産生増加を示したが、微小粒子の産生量については増大は確認されなかった（図７（ｂ））。
TNF-α showed increased production of MCP-1 and IL-6 in parallel with increased production of CD63 + protein in microparticles from iPS-hRPE cells, but increased production of microparticles was confirmed. It was not done (Fig. 7 (b)).
これらの結果から、図７（ｃ）に示す炎症増悪回路が病態形成に関与していると考えられる。即ち、ＲＰＥに対してＬＰＳで象徴されるような炎症刺激が加わると、その細胞上に存在するＴＬＲ４等の自然炎症に係る受容体を介してＲＰＥ内に信号刺激が加わる。そのことにより、まず、ＲＰＥ単独存在下でのＥＶ（微小粒子）産生に繋がると考えられる。このＥＶが産生されると、それが近傍に存在する炎症局所に浸潤してきたマクロファージに作用し、ＴＮＦ－αを産生誘導する。単独培養の上清から超遠心２回で濃縮した微小粒子でも十分ＴＮＦ－α産生は誘導されているが、共培養培養上清のものの方が誘導活性が高い。但し微小粒子の産生量は、上述のとおり共培養においてかなり増強される。おそらく微小粒子の中の一部のＥｘｏｓｏｍｅにＴＮＦ－α産生誘導活性が担われていると想定される。培養上清や超遠心で微小粒子を遠沈させた残りの上清には活性は全くない。このＴＮＦ－αは次に傍にいるＲＰＥに作用し、ＲＰＥからの炎症性サイトカン、ＭＣＰ－１（マクロファージを炎症局所に遊走させる作用を有する）、ＩＬ－６（脈絡膜組織線維化に関与）、ＶＥＧＦ（血管新生に関与）の産生を増強するとともにＥＶの産生を誘導すると考えられる。ＲＰＥからのＥＶ産生は有意には増強していないが、ＥＶ中に含まれるＣＤ６３陽性エクソソームは増加している。以下同様に、この回路が周り、ＥＶ産生増強と炎症性サイトカイン産生、ＭＣＰ－１による局所へのマクロフアージ遊走が増強されると考えられる。即ち、ＲＰＥとマクロファージの共存と、微小粒子（エクソソーム）を介する細胞間相互作用により炎症増悪回路が成立し病態形成に関与していると考えられる。
From these results, it is considered that the inflammation exacerbation circuit shown in FIG. 7 (c) is involved in the pathogenesis. That is, when an inflammatory stimulus as symbolized by LPS is applied to the RPE, a signal stimulus is applied to the RPE via a receptor related to natural inflammation such as TLR4 existing on the cell. This is thought to lead to EV (fine particle) production in the presence of RPE alone. When this EV is produced, it acts on macrophages that have infiltrated the inflamed area in the vicinity and induces the production of TNF-α. TNF-α production is sufficiently induced even with fine particles concentrated twice by ultracentrifugation from the supernatant of the single culture, but the induction activity of the co-culture supernatant is higher. However, the amount of fine particles produced is considerably enhanced in the co-culture as described above. It is presumed that some Exosomes in the fine particles are responsible for the TNF-α production-inducing activity. There is no activity in the culture supernatant or the remaining supernatant after ultracentrifugation of the microparticles. This TNF-α then acts on the nearby RPE, inflammatory cytocan from the RPE, MCP-1 (has the effect of migrating macrophages to the inflammatory site), IL-6 (involved in choroidal tissue fibrosis). , VEGF (involved in angiogenesis) production is thought to be enhanced and EV production induced. EV production from RPE is not significantly enhanced, but CD63-positive exosomes contained in EV are increased. Similarly, it is considered that this circuit is rotated to enhance EV production, inflammatory cytokine production, and local macrophages migration by MCP-1. That is, it is considered that the coexistence of RPE and macrophages and the cell-cell interaction mediated by microparticles (exosomes) establish an inflammatory exacerbation circuit and are involved in the formation of pathological conditions.
［試験８］網膜色素上皮細胞（ＲＰＥ）とマクロファージの相互作用におけるエクソソームｍｉＲＮＡ
ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞との共培養で産生された微小粒子（エクソソーム）の役割を、分泌された微小粒子（エクソソーム）における低分子ＲＮＡプロファイルの観点から解析した。微小粒子濃度はナノ粒子追跡分析により測定した。３つの培養（ｉＰＳ－ｈＲＰＥ細胞、ＰＭＡ－ＴＨＰ－１細胞、共培養）において、細胞１０６個あたりに放出される微小粒子の濃度を表２にまとめた。 [Test 8] Exosome miRNAs in the interaction of retinal pigment epithelial cells (RPE) and macrophages
The role of microparticles (exosomes) produced in co-culture of iPS-hRPE cells and PMA-THP-1 cells was analyzed in terms of low molecular weight RNA profile in secreted microparticles (exosomes). Fine particle concentration was measured by nanoparticle follow-up analysis. Table 2 summarizes the concentrations of microparticles released per 106 cells in the three cultures (iPS-hRPE cells, PMA-THP-1 cells, co-culture).
ｉＰＳ－ｈＲＰＥ細胞とＰＭＡ－ＴＨＰ－１細胞との共培養で産生された微小粒子（エクソソーム）の役割を、分泌された微小粒子（エクソソーム）における低分子ＲＮＡプロファイルの観点から解析した。微小粒子濃度はナノ粒子追跡分析により測定した。３つの培養（ｉＰＳ－ｈＲＰＥ細胞、ＰＭＡ－ＴＨＰ－１細胞、共培養）において、細胞１０６個あたりに放出される微小粒子の濃度を表２にまとめた。 [Test 8] Exosome miRNAs in the interaction of retinal pigment epithelial cells (RPE) and macrophages
The role of microparticles (exosomes) produced in co-culture of iPS-hRPE cells and PMA-THP-1 cells was analyzed in terms of low molecular weight RNA profile in secreted microparticles (exosomes). Fine particle concentration was measured by nanoparticle follow-up analysis. Table 2 summarizes the concentrations of microparticles released per 106 cells in the three cultures (iPS-hRPE cells, PMA-THP-1 cells, co-culture).
表２に示すとおり、共培養では有意な微小粒子濃度の増加は認められなかったが、３回の測定すべてにおいて２種類の細胞に対して相加効果は確認された。１０６の微小粒子中のｍｉＲＮＡは、３つの実験間でわずかに変化したが、共培養についての結果は、ｉＰＳ‐ｈＲＰＥ細胞培養由来の微小粒子中のわずかに低い量を除いて、単一の細胞培養のものと大きな違いは見られなかった。しかし、１０６細胞あたりのｍｉＲの量は３つの培養間で大きな差が認められ、ｉＰＳ－ＲＰＥ細胞単独の培養と比較して共培養では１０６細胞あたりのｍｉＲの量が約５～９倍と有意に増加していた。
As shown in Table 2, no significant increase in the concentration of fine particles was observed in the co-culture, but the additive effect was confirmed for all two types of cells in all three measurements. The miRNAs in 106 microparticles changed slightly between the three experiments, but the results for co-culture were single except for slightly lower amounts in the microparticles from iPS-hRPE cell culture. No significant difference was seen from that of cell culture. However, the amount of miR per 106 cells was significantly different among the three cultures, and the amount of miR per 106 cells was about 5 to 9 times higher in the co-culture than in the culture of iPS-RPE cells alone. Was significantly increased.
［試験９］ｍｉＲ４９４－３ｐ及びｍｉＲ１２４６のｍｉｍｉｃ導入によるミトコンドリア機能修復（ＡＲＰＥ１９細胞株）
ＡＲＰＥ１９細胞株（Ｐ２５）、２．５×１０４ｃｅｌｌｓ／ｗｅｌｌを６ｗｅｌｌ ｐｌａｔｅ（２ ｐｌａｔｅ）に播種した。３日後、ＤＭＥＭ（＋１０％ＦＢＳ）に培地を全量交換し、１時間後にｍｉＲ４９４－３ｐ ｍｉｍｉｃ（mirVana（商標） miRNA hsa-miR-494-3p UGAAACAUACACGGGAAACCUC、MC12409、Thermo Fisher Scientific, Waltham, MA,USA）またはｍｉＲ１２４６ ｍｉｍｉｃ（mirVana（商標） miRNA hsa-miR-1246 AAUGGAUUUUUGGAGCAGG、MC13182、Thermo Fisher Scientific, Waltham, MA,USA）をＴｒａｎｓｆｅｃｔｉｏｎした。上記のｍｉＲ４９４－３ｐ ｍｉｍｉｃ中の塩基配列「UGAAACAUACACGGGAAACCUC」はｍｉＲ－４９４－３ｐの配列（配列番号１）を、ｍｉＲ１２４６ ｍｉｍｉｃ中の塩基配列「AAUGGAUUUUUGGAGCAGG」はｍｉＲ－１２４６の配列（配列番号２）を示している。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。１時間後にＤＭＥＭ（＋１０％ＦＢＳ）を１５０μＬ／ｗｅｌｌ添加した。播種２４時間後に、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒ(Agilent Technologies, Santa Clara, CA, USA) にて解析した。また、解析後の細胞をＤＡＰＩ染色し、Ｃｅｌｌ ｉｎｓｉｇｈｔにて細胞数を測定した。 [Test 9] Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (ARPE19 cell line)
ARPE 19 cell line (P25), 2.5 × 10 4 cells / well was seeded on 6 well plates (2 plates). After 3 days, the whole medium was replaced with DMEM (+ 10% FBS), and after 1 hour, miR494-3p mimic (mirVana ™ miRNA hsa-miR-494-3p UGAAACAUACACGGGAAACCUC, MC12409, Thermo Fisher Scientific, Waltham, MA, USA) Alternatively, miR1246 mimic (mirVana ™ miRNA hsa-miR-1246 AAUGGAUUUUUGGAGCAGG, MC13182, Thermo Fisher Scientific, Waltham, MA, USA) was transfected. The base sequence "UGAAACAUACACGGGAAACCUC" in the above miR494-3p mimic shows the sequence of miR-494-3p (SEQ ID NO: 1), and the base sequence "AAUGGAUUUUUGGAGCAGG" in the miR1246 mimic shows the sequence of miR-1246 (SEQ ID NO: 2). ing. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. After 1 hour, DMEM (+ 10% FBS) was added at 150 μL / well. Twenty-four hours after seeding, cell mitochondrial function was analyzed by Flux analyzer (Agilent Technologies, Santa Clara, CA, USA). In addition, the cells after analysis were stained with DAPI, and the number of cells was measured by Cell insight.
ＡＲＰＥ１９細胞株（Ｐ２５）、２．５×１０４ｃｅｌｌｓ／ｗｅｌｌを６ｗｅｌｌ ｐｌａｔｅ（２ ｐｌａｔｅ）に播種した。３日後、ＤＭＥＭ（＋１０％ＦＢＳ）に培地を全量交換し、１時間後にｍｉＲ４９４－３ｐ ｍｉｍｉｃ（mirVana（商標） miRNA hsa-miR-494-3p UGAAACAUACACGGGAAACCUC、MC12409、Thermo Fisher Scientific, Waltham, MA,USA）またはｍｉＲ１２４６ ｍｉｍｉｃ（mirVana（商標） miRNA hsa-miR-1246 AAUGGAUUUUUGGAGCAGG、MC13182、Thermo Fisher Scientific, Waltham, MA,USA）をＴｒａｎｓｆｅｃｔｉｏｎした。上記のｍｉＲ４９４－３ｐ ｍｉｍｉｃ中の塩基配列「UGAAACAUACACGGGAAACCUC」はｍｉＲ－４９４－３ｐの配列（配列番号１）を、ｍｉＲ１２４６ ｍｉｍｉｃ中の塩基配列「AAUGGAUUUUUGGAGCAGG」はｍｉＲ－１２４６の配列（配列番号２）を示している。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。１時間後にＤＭＥＭ（＋１０％ＦＢＳ）を１５０μＬ／ｗｅｌｌ添加した。播種２４時間後に、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒ(Agilent Technologies, Santa Clara, CA, USA) にて解析した。また、解析後の細胞をＤＡＰＩ染色し、Ｃｅｌｌ ｉｎｓｉｇｈｔにて細胞数を測定した。 [Test 9] Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (ARPE19 cell line)
ARPE 19 cell line (P25), 2.5 × 10 4 cells / well was seeded on 6 well plates (2 plates). After 3 days, the whole medium was replaced with DMEM (+ 10% FBS), and after 1 hour, miR494-3p mimic (mirVana ™ miRNA hsa-miR-494-3p UGAAACAUACACGGGAAACCUC, MC12409, Thermo Fisher Scientific, Waltham, MA, USA) Alternatively, miR1246 mimic (mirVana ™ miRNA hsa-miR-1246 AAUGGAUUUUUGGAGCAGG, MC13182, Thermo Fisher Scientific, Waltham, MA, USA) was transfected. The base sequence "UGAAACAUACACGGGAAACCUC" in the above miR494-3p mimic shows the sequence of miR-494-3p (SEQ ID NO: 1), and the base sequence "AAUGGAUUUUUGGAGCAGG" in the miR1246 mimic shows the sequence of miR-1246 (SEQ ID NO: 2). ing. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. After 1 hour, DMEM (+ 10% FBS) was added at 150 μL / well. Twenty-four hours after seeding, cell mitochondrial function was analyzed by Flux analyzer (Agilent Technologies, Santa Clara, CA, USA). In addition, the cells after analysis were stained with DAPI, and the number of cells was measured by Cell insight.
図中、「ＦＣＣＰ」は、Ｃａｒｂｏｎｙｌ ｃｙａｎｉｄｅ－ｐ－ｔｒｉｆｌｕｏｒｏｍｅｔｈｏｘｙｐｈｅｎｙｌｈｙｄｒａｚｏｎｅ、「ＲＯＴ／ＡＡ」は、Ｒｏｔｅｎｏｎ／ＡｎｔｉｍｙｃｉｎＡである。また、「ＯＣＲ」はＯｘｙｇｅｎ Ｃｏｎｓｕｍｐｔｉｏｎ Ｒａｔｅ（ｐｍｏｌｅ／ｍｉｎ）（酸素消費速度）であり、細胞の酸素消費を示す値である。「ＥＣＡＲ」はＥｘｔｒａｃｅｌｌｕｌａｒ Ａｃｉｄｉｆｉｃａｔｉｏｎ Ｒａｔｅ（ｍｐＨ／ｍｉｎ）（細胞外酸性化速度）であり、細胞外に排出される代謝物による細胞外のｐＨの産生化速度を示す。いずれも、細胞レベルでのエネルギー代謝状態の指標として広く用いられている。結果を図８（ａ）～（ｃ）に示す。
In the figure, "FCCP" is Carbonyl cyanide-p-trifluoromethoxyphrydrazone, and "ROT / AA" is Rotenone / Antimycin A. Further, "OCR" is an Oxygen Consumption Rate (pmole / min) (oxygen consumption rate), which is a value indicating the oxygen consumption of cells. "ECAR" is an Extracellular Acidification Rate (mpH / min) (extracellular acidification rate), which indicates the rate of extracellular pH production by metabolites excreted extracellularly. Both are widely used as indicators of energy metabolism at the cellular level. The results are shown in FIGS. 8 (a) to 8 (c).
ｍｉＲ１２４６ ｍｉｍｉｃ、ｍｉＲ４９４－３ｐ ｍｉｍｉｃ導入時において、Ｍａｘｉｍｕｍ ｒｅｓｐｉｒａｔｉｏｎ（図８（ａ））、ＥＣＡＲが有意に増加した（図８（ｂ））。また、ｍｉＲ１２４６導入時においては、ＯＣＲ／ＥＣＡＲも有意に増加した（図８（ｃ））。ｍｉＲ１２４６ ｍｉｍｉｃ、ｍｉＲ４９４－３ｐ ｍｉｍｉｃの導入により、ミトコンドリア機能の低下しているＡＲＰＥ１９細胞株におけるミトコンドリア機能の回復効果が得られた。
At the time of introduction of miR1246 mimic and miR494-3p mimic, Maximum rejection (FIG. 8 (a)) and EPAR increased significantly (FIG. 8 (b)). In addition, OCR / EPAR also increased significantly when miR1246 was introduced (FIG. 8 (c)). By introducing miR1246 mimic and miR494-3p mimic, the effect of restoring mitochondrial function in the ARPE19 cell line with reduced mitochondrial function was obtained.
［試験１０］ｍｉＲ４９４－３ｐ及びｍｉＲ１２４６のｍｉｍｉｃ導入によるミトコンドリア機能修復（初代培養ＨＲＰＥ細胞 ｖｓ ＡＲＰＥ１９細胞株）
ヒト初代網膜色素上皮（ＨＲＰＥ）細胞（Ｐ３）、ＡＲＰＥ１９細胞株（Ｐ２５）をそれぞれ１６×１０４ｃｅｌｌｓ／ｗｅｌｌを６ｗｅｌｌ ｐｌａｔｅに播種した。翌日、ＤＭＥＭ（＋１０％ＦＢＳ）に培地を全量交換し、１時間後にｍｉＲ４９４－３ｐ ｍｉｍｉｃまたはｍｉＲ１２４６ ｍｉｍｉｃをＴｒａｎｓｆｅｃｔｉｏｎした。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。２時間後にＤＭＥＭ（＋１０％ＦＢＳ）を１５０μＬ／ｗｅｌｌ添加した。播種２４時間後に、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒにて解析した。結果を図９（ａ）及び下記表３（初代培養ヒトＲＰＥ細胞）、並びに図９（ｂ）及び下記表４（ＡＲＰＥ１９細胞株）に示す。 [Test 10] Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (primary cultured HRPE cells vs ARPE19 cell line)
Human primary retinal pigment epithelial (HRPE) cells (P3) and ARPE19 cell line (P25) were seeded at 16 × 10 4 cells / well on a 6-well plate, respectively. The next day, the whole medium was replaced with DMEM (+ 10% FBS), and 1 hour later, miR494-3p mimic or miR1246 mimic was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. After 2 hours, DMEM (+ 10% FBS) was added at 150 μL / well. Twenty-four hours after sowing, the mitochondrial function of the cells was analyzed by a Lux analyzer. The results are shown in FIG. 9 (a) and Table 3 below (primary cultured human RPE cells), and FIG. 9 (b) and Table 4 below (ARPE 19 cell line).
ヒト初代網膜色素上皮（ＨＲＰＥ）細胞（Ｐ３）、ＡＲＰＥ１９細胞株（Ｐ２５）をそれぞれ１６×１０４ｃｅｌｌｓ／ｗｅｌｌを６ｗｅｌｌ ｐｌａｔｅに播種した。翌日、ＤＭＥＭ（＋１０％ＦＢＳ）に培地を全量交換し、１時間後にｍｉＲ４９４－３ｐ ｍｉｍｉｃまたはｍｉＲ１２４６ ｍｉｍｉｃをＴｒａｎｓｆｅｃｔｉｏｎした。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。２時間後にＤＭＥＭ（＋１０％ＦＢＳ）を１５０μＬ／ｗｅｌｌ添加した。播種２４時間後に、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒにて解析した。結果を図９（ａ）及び下記表３（初代培養ヒトＲＰＥ細胞）、並びに図９（ｂ）及び下記表４（ＡＲＰＥ１９細胞株）に示す。 [Test 10] Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (primary cultured HRPE cells vs ARPE19 cell line)
Human primary retinal pigment epithelial (HRPE) cells (P3) and ARPE19 cell line (P25) were seeded at 16 × 10 4 cells / well on a 6-well plate, respectively. The next day, the whole medium was replaced with DMEM (+ 10% FBS), and 1 hour later, miR494-3p mimic or miR1246 mimic was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. After 2 hours, DMEM (+ 10% FBS) was added at 150 μL / well. Twenty-four hours after sowing, the mitochondrial function of the cells was analyzed by a Lux analyzer. The results are shown in FIG. 9 (a) and Table 3 below (primary cultured human RPE cells), and FIG. 9 (b) and Table 4 below (ARPE 19 cell line).
ｍｉＲ１２４６ ｍｉｍｉｃ、ｍｉＲ４９４－３ｐ ｍｉｍｉｃ導入によって、初代培養ＨＲＰＥ細胞においては、ＯＣＲ（酸素消費速度）の変化は認められなかった（図９（ａ））。一方、ＡＲＰＥ１９細胞株では、ＯＣＲ（酸素消費速度）の顕著な回復が認められた（図９（ｂ））。初代培養ＨＲＰＥ細胞と比較するとＡＲＰＥ１９細胞株ではＯＣＲ（酸素消費速度）の値が、１／４程度まで低下しており、ミトコンドリア機能が低下していると考えられる。ｍｉＲ１２４６ ｍｉｍｉｃ、ｍｉＲ４９４－３ｐ ｍｉｍｉｃの導入により、初代培養ＨＲＰＥ細胞における正常ミトコンドリア機能の亢進は殆ど認められないが、ＡＲＰＥ１９細胞株のようにミトコンドリア機能が低下した細胞に対しては顕著な修復効果が得られた。
No change in OCR (oxygen consumption rate) was observed in the primary cultured HRPE cells due to the introduction of miR1246 mimic and miR494-3p mimic (FIG. 9 (a)). On the other hand, in the ARPE19 cell line, a remarkable recovery of OCR (oxygen consumption rate) was observed (FIG. 9 (b)). Compared with the primary cultured HRPE cells, the OCR (oxygen consumption rate) value was reduced to about 1/4 in the ARPE19 cell line, and it is considered that the mitochondrial function was reduced. The introduction of miR1246 mimic and miR494-3p mimic hardly enhances normal mitochondrial function in primary cultured HRPE cells, but a remarkable repair effect is obtained for cells with reduced mitochondrial function such as ARPE19 cell line. Was done.
［試験１１］ｍｉＲ４９４－３ｐのｉｎｈｉｂｉｔｏｒ導入によるミトコンドリア機能修復（Ｎｉｃ－ＡＲＰＥ細胞株 ｖｓ ＡＲＰＥ１９細胞株）
ＡＲＰＥ－１９細胞株をニコチンアミド（Ｎｉｃ）添加ＭＥＭ培地で２ケ月培養することで細胞分化を進めたＮｉｃ－ＡＲＰＥ細胞株（ＭＥＭ－Ｎｉｃで２カ月培養）、ＡＲＰＥ１９細胞株（Ｐ２５；ＤＭＥＭ＋１０％ＦＢＳ＋１％Ｐ．Ｓ．で培養）を用いた。ＡＲＰＥ１９細胞株（ＤＭＥＭ＋１０％ＦＢＳ）及びＮｉｃ－ＡＲＰＥ細胞株（Ｐ．Ｓ．なしの培地に交換）をそれぞれ１６×１０４ｃｅｌｌｓ／ｗｅｌｌで６ｗｅｌｌ ｐｌａｔｅに播種した。翌日、ｍｉＲ４９４－３ｐｉｎｈｉｂｉｔｏｒ（Anti-miR（商標） miRNA hsa-miR-494-3p、MH12409、Thermo Fisher Scientific, Waltham, MA,USA）、またはｍｉＲ１２４６ｉｎｈｉｂｉｔｏｒ（Anti-miR（商標） miRNA hsa-miR-1246、 MH13182、Thermo Fisher Scientific, Waltham, MA,USA）をＴｒａｎｓｆｅｃｔｉｏｎした。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。ｏｖｅｒｎｉｇｈｔ静置し、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒにて解析した。結果を図１０（ａ）及び下記表５（Ｎｉｃ－ＡＲＰＥ細胞株）、並びに図１０（ｂ）及び下記表６（ＡＲＰＥ１９細胞株）に示す。 [Test 11] Mitochondrial function repair by introduction of miR494-3p inhibitor (Nic-ARPE cell line vs ARPE19 cell line)
Nic-ARPE cell line (cultured in MEM-Nic for 2 months), ARPE19 cell line (P25; DMEM + 10% FBS + 1), which promoted cell differentiation by culturing ARPE-19 cell line in nicotine amide (Nic) -added MEM medium for 2 months. % PS) was used. ARPE 19 cell lines (DMEM + 10% FBS) and Nic-ARPE cell lines (replaced in medium without PS) were seeded on 6 well plates at 16 × 10 4 cells / well, respectively. The next day, miR494-3 pinhibitor (Anti-miR ™ miRNA hsa-miR-494-3p, MH12409, Thermo Fisher Scientific, Waltham, MA, USA), or miR1246inhibitor (Anti-miR ™ miRNA hsa-miR-1246, MH13182, Thermo Fisher Scientific, Waltham, MA, USA) was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. Overnight was allowed to stand and the mitochondrial function of the cells was analyzed by the Lux analyzer. The results are shown in FIG. 10 (a) and Table 5 below (Nic-ARPE cell line), and FIG. 10 (b) and Table 6 below (ARPE 19 cell line).
ＡＲＰＥ－１９細胞株をニコチンアミド（Ｎｉｃ）添加ＭＥＭ培地で２ケ月培養することで細胞分化を進めたＮｉｃ－ＡＲＰＥ細胞株（ＭＥＭ－Ｎｉｃで２カ月培養）、ＡＲＰＥ１９細胞株（Ｐ２５；ＤＭＥＭ＋１０％ＦＢＳ＋１％Ｐ．Ｓ．で培養）を用いた。ＡＲＰＥ１９細胞株（ＤＭＥＭ＋１０％ＦＢＳ）及びＮｉｃ－ＡＲＰＥ細胞株（Ｐ．Ｓ．なしの培地に交換）をそれぞれ１６×１０４ｃｅｌｌｓ／ｗｅｌｌで６ｗｅｌｌ ｐｌａｔｅに播種した。翌日、ｍｉＲ４９４－３ｐｉｎｈｉｂｉｔｏｒ（Anti-miR（商標） miRNA hsa-miR-494-3p、MH12409、Thermo Fisher Scientific, Waltham, MA,USA）、またはｍｉＲ１２４６ｉｎｈｉｂｉｔｏｒ（Anti-miR（商標） miRNA hsa-miR-1246、 MH13182、Thermo Fisher Scientific, Waltham, MA,USA）をＴｒａｎｓｆｅｃｔｉｏｎした。Ｔｒａｎｓｆｅｃｔｉｏｎから２４時間後、細胞をＸＦｅ２４ ｐｌａｔｅへ、６×１０４ｃｅｌｌｓ／１００μＬ／ｗｅｌｌで播種した。ｏｖｅｒｎｉｇｈｔ静置し、細胞のミトコンドリア機能をＦｌｕｘ ａｎａｌｙｚｅｒにて解析した。結果を図１０（ａ）及び下記表５（Ｎｉｃ－ＡＲＰＥ細胞株）、並びに図１０（ｂ）及び下記表６（ＡＲＰＥ１９細胞株）に示す。 [Test 11] Mitochondrial function repair by introduction of miR494-3p inhibitor (Nic-ARPE cell line vs ARPE19 cell line)
Nic-ARPE cell line (cultured in MEM-Nic for 2 months), ARPE19 cell line (P25; DMEM + 10% FBS + 1), which promoted cell differentiation by culturing ARPE-19 cell line in nicotine amide (Nic) -added MEM medium for 2 months. % PS) was used. ARPE 19 cell lines (DMEM + 10% FBS) and Nic-ARPE cell lines (replaced in medium without PS) were seeded on 6 well plates at 16 × 10 4 cells / well, respectively. The next day, miR494-3 pinhibitor (Anti-miR ™ miRNA hsa-miR-494-3p, MH12409, Thermo Fisher Scientific, Waltham, MA, USA), or miR1246inhibitor (Anti-miR ™ miRNA hsa-miR-1246, MH13182, Thermo Fisher Scientific, Waltham, MA, USA) was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 × 10 4 cells / 100 μL / well. Overnight was allowed to stand and the mitochondrial function of the cells was analyzed by the Lux analyzer. The results are shown in FIG. 10 (a) and Table 5 below (Nic-ARPE cell line), and FIG. 10 (b) and Table 6 below (ARPE 19 cell line).
ｍｉＲ４９４－３ｐ ｉｎｈｉｂｉｔｏｒ導入によって、Ｎｉｃ－ＡＲＰＥ細胞株においては、ＯＣＲ（酸素消費速度）の低下が見られ、亢進ミトコンドリア機能の抑制効果が確認できた（図１０（ａ））。一方、ＡＲＰＥ１９細胞株では、ＯＣＲ（酸素消費速度）の変化は認められなかった（図１０（ｂ））。Ｎｉｃ－ＡＲＰＥ細胞株と比較するとＡＲＰＥ１９細胞株ではＯＣＲ（酸素消費速度）の値が１／４程度まで低下しており、ミトコンドリア機能が低下していると考えられる。ｍｉＲ４９４－３ｐのｉｎｈｉｂｉｔｏｒ導入により、Ｎｉｃ－ＡＲＰＥ細胞株における正常ミトコンドリア機能の抑制は認められたが、ＡＲＰＥ１９細胞株のようにミトコンドリア機能が低下した細胞に対して更なる抑制効果は認めがたい。
With the introduction of miR494-3p inhibitor, a decrease in OCR (oxygen consumption rate) was observed in the Nick-ARPE cell line, and the effect of suppressing enhanced mitochondrial function was confirmed (FIG. 10 (a)). On the other hand, in the ARPE19 cell line, no change in OCR (oxygen consumption rate) was observed (FIG. 10 (b)). Compared with the Nic-ARPE cell line, the OCR (oxygen consumption rate) value was reduced to about 1/4 in the ARPE19 cell line, and it is considered that the mitochondrial function was reduced. Inhibitor introduction of miR494-3p was observed to suppress normal mitochondrial function in Nic-ARPE cell line, but further inhibitory effect was not observed for cells with reduced mitochondrial function such as ARPE19 cell line.
［試験１２］ＡＭＤ（加齢黄斑変性症）患者の前房水及び血漿中のｍｉＲ－４９４－３ＰおよびｍｉＲ－１２４６の発現
血管新生を伴う加齢黄斑変性症患者から、前房水及び血漿を採取した。血管新生を伴う加齢黄斑変性症患者としては、ドルーゼンが関与している未治療症例を選択した。また正常コントロールとしては、角膜、ぶどう膜炎、緑内障、網膜疾患などを有さない白内障症例を選択し、白内障手術時に前房水及び血漿を採取した。前房水は採取後早急に－８０℃で保管した。血液サンプルは一時的に冷蔵保存し、遠心分離により血漿を分離し－８０℃で保管した。 [Test 12] Expression of miR-494-3P and miR-1246 in anterior aqueous humor and plasma of AMD (age-related macular degeneration) patients. Collected. As a patient with age-related macular degeneration with angiogenesis, an untreated case involving drusen was selected. As normal control, cataract cases without cornea, uveitis, glaucoma, retinal disease, etc. were selected, and anterior chamber water and plasma were collected at the time of cataract surgery. The anterior aqueous humor was stored at -80 ° C immediately after collection. Blood samples were temporarily refrigerated, plasma separated by centrifugation and stored at -80 ° C.
血管新生を伴う加齢黄斑変性症患者から、前房水及び血漿を採取した。血管新生を伴う加齢黄斑変性症患者としては、ドルーゼンが関与している未治療症例を選択した。また正常コントロールとしては、角膜、ぶどう膜炎、緑内障、網膜疾患などを有さない白内障症例を選択し、白内障手術時に前房水及び血漿を採取した。前房水は採取後早急に－８０℃で保管した。血液サンプルは一時的に冷蔵保存し、遠心分離により血漿を分離し－８０℃で保管した。 [Test 12] Expression of miR-494-3P and miR-1246 in anterior aqueous humor and plasma of AMD (age-related macular degeneration) patients. Collected. As a patient with age-related macular degeneration with angiogenesis, an untreated case involving drusen was selected. As normal control, cataract cases without cornea, uveitis, glaucoma, retinal disease, etc. were selected, and anterior chamber water and plasma were collected at the time of cataract surgery. The anterior aqueous humor was stored at -80 ° C immediately after collection. Blood samples were temporarily refrigerated, plasma separated by centrifugation and stored at -80 ° C.
使用した検体は以下のとおりである。
血漿：コントロール ６検体 各１００μＬ、ＡＭＤ患者５検体 各１００μＬ
前房水：コントロール ５検体 各５０μＬ、ＡＭＤ患者５検体 各５０μＬ The samples used are as follows.
Plasma: 6control samples 100 μL each, AMD patient 5 samples 100 μL each
Aqueous humor: control 5samples 50 μL each, AMD patient 5 samples 50 μL each
血漿：コントロール ６検体 各１００μＬ、ＡＭＤ患者５検体 各１００μＬ
前房水：コントロール ５検体 各５０μＬ、ＡＭＤ患者５検体 各５０μＬ The samples used are as follows.
Plasma: 6
Aqueous humor: control 5
各検体から、ｍｉＲＮｅａｓｙ Ｓｅｒｕｍ／Ｐｌａｓｍａ ｋｉｔ（QIAGEN, Hilden, Germany）を用いてｍｉＲＮＡを抽出した（抽出量１０μＬ）。それぞれｍｉＲＮＡに特異的なプライマーを用いて逆転写反応し、定量ＰＣＲを行った。ｍｉＲＮＡ相対量は、ｃｅｌ－ｍｉＲ－３９をコントロール（ｓｐｉｋｅ－ｉｎ ｃｏｎｔｒｏｌ）として、各ＣＴ値及びコントロールのＣＴ値からΔＣｔ法にて数値化した（定量ＰＣＲのＣｙｃｌｅ数は４０とした）。結果を図１１（ｍｉＲ－４９４－３Ｐ）、図１２（ｍｉＲ－１２４６）に示す。
From each sample, miRNA was extracted using miRNeasy Serum / Plasma kit (QIAGEN, Hilden, Germany) (extraction amount 10 μL). Reverse transcription reaction was performed using primers specific for each miRNA, and quantitative PCR was performed. The relative amount of miRNA was quantified by the ΔCt method from each CT value and the CT value of the control using cel-miR-39 as a control (spire-in control) (the number of cycles of quantitative PCR was 40). The results are shown in FIG. 11 (miR-494-3P) and FIG. 12 (miR-1246).
図１１及び１２に示すとおり、加齢黄斑変性症患者の前房水において、コントロールと比較して、ｍｉＲ－４９４－３Ｐ及びｍｉＲ－１２４６の増加が確認できた。なお、血漿検体での明確な傾向は確認されなかった。
As shown in FIGS. 11 and 12, in the anterior chamber water of patients with age-related macular degeneration, an increase in miR-494-3P and miR-1246 was confirmed as compared with the control. No clear tendency was confirmed in plasma samples.
本発明の組織炎症を伴う炎症性網膜疾患の判定方法によると、加齢黄斑変性等の慢性の炎症性網膜疾患の罹患可能性、重症度、治療効果等の判定を容易に行うことができる。加齢黄斑変性は、患者において疾患初期の自覚症状が少ないため早期発見が困難であるという課題があったが、本発明の判定方法により早期発見が可能となる。また、網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子、特に特定のマイクロＲＮＡの機能調節分子は、加齢黄斑変性等の慢性の炎症性網膜疾患の新規治療剤として有効である。さらに、本発明のスクリーニング方法によると、炎症性網膜疾患に対して治療効果を有する物質を簡便に探索することができる。
According to the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention, it is possible to easily determine the susceptibility, severity, therapeutic effect, etc. of a chronic inflammatory retinal disease such as age-related macular degeneration. Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention. In addition, the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA, is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as. Furthermore, according to the screening method of the present invention, it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.
Claims (16)
- 被験者の網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）を指標とする、組織炎症を伴う炎症性網膜疾患の判定方法。 A method for determining inflammatory retinal disease accompanied by tissue inflammation, using extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index.
- 上記細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルを指標とすることを特徴とする、請求項１に記載の判定方法。 The determination method according to claim 1, wherein the profile of miRNA contained in the extracellular fine particles (EV) is used as an index.
- 上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、請求項２に記載の判定方法。 The determination method according to claim 2, wherein the miRNA comprises miR494-3p and / or miR1246.
- 上記ｍｉＲＮＡが、ミトコンドリア由来である、請求項２又は３に記載の判定方法。 The determination method according to claim 2 or 3, wherein the miRNA is derived from mitochondria.
- 上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症および、糖尿病性網膜症からなる群より選択される少なくとも１種の炎症性疾患である、請求項１から４のいずれか１項に記載の判定方法。 The inflammatory retinal disease associated with the above-mentioned tissue inflammation is at least one inflammatory disease selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinosis, proliferative vitreous retinopathy, and diabetic retinopathy. The determination method according to any one of claims 1 to 4.
- 被験者における炎症性網膜疾患の罹患可能性、重軽度、発症リスク、重症化リスク及び治療効果から成る群より選択される少なくとも１種を判定する、請求項１から５のいずれか１項に記載の判定方法。 The invention according to any one of claims 1 to 5, wherein at least one selected from the group consisting of susceptibility, severity, risk of onset, risk of aggravation and therapeutic effect of inflammatory retinal disease in a subject is determined. Judgment method.
- 網膜色素上皮細胞（ＲＰＥ）が産生する細胞外微粒子（ＥＶ）の機能調節分子を有効成分として含有する、組織炎症を伴う炎症性網膜疾患治療剤。 A therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation, which contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
- 上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡの機能調節分子である、請求項７に記載の炎症性網膜疾患治療剤。 The therapeutic agent for inflammatory retinal disease according to claim 7, wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miRNA contained in the extracellular fine particles (EV).
- 上記細胞外微粒子（ＥＶ）の機能調節分子が、細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６の機能調節分子である、請求項７又は８に記載の炎症性網膜疾患治療剤。 The therapeutic agent for inflammatory retinal disease according to claim 7 or 8, wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miR494-3p and / or miR1246 contained in the extracellular fine particles (EV). ..
- 上記細胞外微粒子（ＥＶ）の機能調節分子が、核酸オリゴ、低分子化合物、ペプチド及び抗体から成る群より選択される少なくとも１種である、請求項７から９のいずれか１項に記載の炎症性網膜疾患治療剤。 The inflammation according to any one of claims 7 to 9, wherein the function-regulating molecule of the extracellular fine particle (EV) is at least one selected from the group consisting of nucleic acid oligos, small molecule compounds, peptides and antibodies. A therapeutic agent for sexual retinal diseases.
- 上記組織炎症を伴う炎症性網膜疾患が、加齢黄斑変性、中心性漿液性脈絡網膜症、増殖性硝子体網膜症、および、糖尿病性網膜症 からなる群より選択される少なくとも１種の疾患である、請求項７から１０のいずれか１項に記載の炎症性網膜疾患治療剤。 The inflammatory retinal disease associated with the tissue inflammation is at least one disease selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, and diabetic retinopathy. The inflammatory retinal disease therapeutic agent according to any one of claims 7 to 10.
- 網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において分泌される細胞外微粒子（ＥＶ）のプロファイルの変動を指標とすることを特徴とする、組織炎症を伴う炎症性網膜疾患治療剤のスクリーニング方法。 Inflammatory retinal disease with tissue inflammation, characterized by variability in the profile of extracellular microparticles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. A method for screening therapeutic agents.
- 上記細胞外微粒子（ＥＶ）が、網膜色素上皮細胞（ＲＰＥ）から分泌される、請求項１２に記載のスクリーニング方法。 The screening method according to claim 12, wherein the extracellular fine particles (EV) are secreted from retinal pigment epithelial cells (RPE).
- 上記細胞外微粒子（ＥＶ）のプロファイルの変動が、細胞外微粒子（ＥＶ）が含有するｍｉＲＮＡのプロファイルの変動である、請求項１２又は１３に記載のスクリーニング方法。 The screening method according to claim 12 or 13, wherein the variation in the profile of the extracellular fine particles (EV) is a variation in the profile of miRNA contained in the extracellular fine particles (EV).
- 上記ｍｉＲＮＡが、ｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６を含む、請求項１４に記載のスクリーニング方法。 The screening method according to claim 14, wherein the miRNA comprises miR494-3p and / or miR1246.
- 網膜色素上皮細胞（ＲＰＥ）と、単球／マクロファージとの共培養系において、被験物質の添加により分泌される細胞外微粒子（ＥＶ）が含有するｍｉＲ４９４－３ｐ及び／又はｍｉＲ１２４６が抑制された場合に、上記被験物質を有効な組織炎症を伴う炎症性網膜疾患治療剤の候補として選択する、請求項１２から１５のいずれか１項に記載のスクリーニング方法。 When miR494-3p and / or miR1246 contained in extracellular fine particles (EV) secreted by the addition of the test substance are suppressed in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. The screening method according to any one of claims 12 to 15, wherein the test substance is selected as a candidate for an effective therapeutic agent for inflammatory retinal disease associated with tissue inflammation.
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