WO2023190267A1 - Fine particle, prophylactic drug or therapeutic drug for parkinson's disease, and method for improving parkinson's disease - Google Patents
Fine particle, prophylactic drug or therapeutic drug for parkinson's disease, and method for improving parkinson's disease Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
Definitions
- the present invention relates to microparticles, preventive or therapeutic agents for Parkinson's disease, and methods for improving Parkinson's disease.
- Parkinson's disease is a neurodegenerative disease that occurs frequently around the world, and further development of treatments and prevention methods for Parkinson's disease is desired. Research to date has made progress in elucidating the pathogenesis of Parkinson's disease.
- Non-Patent Document 1 states that among sirtuins (SIRTs), inhibition of SIRT2 exerts a neuroprotective effect in Parkinson's disease (PD), that SIRT2 protein level is highly increased in PD models, and that it is negatively affected by miR-212-5p. It is stated that it is controlled by Based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors have been described as a new therapeutic tool for PD. There is.
- Non-Patent Document 2 states that degeneration of dopaminergic (DA) neurons occurs in PD, and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of DA neurons in PD and increases the survival of primary mesencephalic neurons (PMNs). ) has been described to specifically increase the expression of miR-182-5p and miR-183-5p. It has also been described that miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
- DA dopaminergic
- GDNF glial cell line-derived neurotrophic factor
- Non-Patent Document 3 describes a review of miRNAs and new therapeutic targets in Parkinson's disease
- Figure 1 on p1957 lists 41 types of miRNAs as miRNAs that are down-regulated in the brain tissue of Parkinson's disease. Are listed.
- Non-Patent Documents 1 to 3 do not describe the use of microparticles such as exosomes as a means for introducing or transfecting the miRNA described in these documents into living organisms.
- the problem to be solved by the present invention is to provide microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.
- the microparticles contain hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group, [4 The microparticles described in ].
- Microparticles [8] The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells, The microparticle according to [1], wherein the microparticle does not contain components other than the culture supernatant of dental pulp-derived stem cells except for exosomes.
- a prophylactic or therapeutic agent for Parkinson's disease comprising the microparticle according to [1].
- Parkinson's disease comprising administering an effective amount of the microparticles according to [1] or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease according to [9] to a subject who has developed Parkinson's disease. How to improve.
- microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.
- FIG. 1 is a heat map showing the expression level of miRNA expressed in exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells.
- FIG. 2 is a heat map showing the expression level of each miRNA shown in FIG. 1 in the cerebral cortex.
- the microparticles of the present invention are microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
- the microparticles of the present invention contain specific miRNAs that are attracting attention as a means of treating Parkinson's disease.
- preferred embodiments of the microparticles of the present invention will be explained.
- ⁇ Mechanism of Parkinson's disease> An overview of the pathogenesis of Parkinson's disease is as follows. (1) Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta occurs. (2) Due to a decrease in dopaminergic neurons in the substantia nigra pars compacta, the amount of dopamine produced decreases. (3) Defects in dopaminergic neurons in the striatum disrupt basal function. (4) Bring about the manifestation of Parkinson's disease symptoms. In other words, under normal conditions, dopamine produced in the substantia nigra acts as a liaison with various parts of the brain in the striatum of the cerebrum and regulates body movements and functions, but when Parkinson's disease develops, dopamine becomes insufficient. The patient becomes unable to control body movements and functions, and motor and non-motor symptoms appear.
- Lewy bodies are known to be one of the causes of progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Then, ⁇ -synuclein protein aggregates and accumulates in dopaminergic neurons in the substantia nigra pars compacta, causing the formation of Lewy bodies.
- the disease in which Lewy bodies appear is called "Lewy body disease,” and in addition to Parkinson's disease, there is Lewy body dementia.
- the microparticles of the present invention can improve Parkinson's disease.
- the therapeutic agent for Parkinson's disease and its mechanism of action will be explained together with the mechanism of action of the microparticles of the present invention.
- SIRT2 inhibitors Front. Mol. Neurosci. vol. 11, 381 (2018) states that among sirtuins (SIRTs), inhibition of SIRT2 exerts neuroprotective effects in Parkinson's disease (PD), that SIRT2 protein levels are highly increased in PD models, and that miR-212-5p It has been described that it is negatively controlled by And based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors represent a new therapeutic tool for PD. Specifically, miR-212-5p transfection reduces SIRT2 expression, inhibits SIRT2 activity, and prevents dopaminergic neuron loss and DAT decline.
- miR-212-5p exhibits neuroprotective effects in PD.
- the mechanism of action is that nuclear acetylated p53 is upregulated by p53, which is the major deacetylation substrate of SIRT2.
- p53 which is the major deacetylation substrate of SIRT2.
- reduction of cytoplasmic p53 promotes autophagy in PD models.
- miR-212-5p attenuates apoptosis in PD models.
- the microparticles of the present invention contain miR-212-5p, they function as SIRT2 inhibitors.
- miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
- miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
- the microparticles of the present invention contain miR-182-5p or miR-183-5p, they function as an inhibitor of dopaminergic neuron degeneration or apoptosis.
- the microparticles of the present invention include hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p , hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa -If it contains at least one of miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543, Parkinson's It functions as a supplement for
- Amantadine Hydrochloride has the effect of promoting dopamine release in the striatum, and is also known to have the effect of suppressing L-DOPA-induced involuntary movements (dyskinesia) in which the body moves on its own. Note that amantadine hydrochloride has little relevance to the microparticles of the present invention.
- MAO-B inhibitor Selegiline hydrochloride, an MAO-B inhibitor, reduces the activity of MAO and suppresses the decomposition of dopamine, so taking it serves as a therapeutic means.
- MAO-B inhibitors also inhibit the breakdown of other neurotransmitters such as norepinephrine and serotonin. Note that MAO-B inhibitors have little relevance to the microparticles of the present invention.
- Droxidopa Donorepinephrine is a different neurotransmitter than dopamine.
- norepinephrine is synthesized from dopamine by ⁇ -hydroxylase, so it becomes insufficient when dopamine decreases.
- droxidopa which is a norepinephrine precursor
- norepinephrine can be supplemented and the stiffness of the legs caused by norepinephrine deficiency can be improved. Note that droxidopa has little relevance to the microparticles of the present invention.
- microparticles of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells such as dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium.
- the microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably are microparticles derived from the culture supernatant of dental pulp-derived stem cells.
- the microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily need to be obtained from the culture supernatant of dental pulp-derived stem cells.
- the microparticles inside dental pulp-derived stem cells are isolated by any method, if they are the same as the microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, then It can be said that they are microparticles derived from water.
- Microparticles derived from a culture supernatant such as dental pulp-derived stem cells may be used in a state contained in the culture supernatant, or may be used in a state purified from the culture supernatant.
- the microparticles are microparticles purified from culture supernatant.
- the origin of the microparticles can be determined by a known method. For example, it can be determined whether the microparticles are derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells using the method described in J Stem Cell Res Ther (2016) 8:2. I can do it. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticle.
- the microparticles contain miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
- miRNA miRNA
- miRNA is, for example, an RNA molecule of 21 to 25 bases (nucleotides). miRNAs can regulate gene expression by suppressing target gene (target) mRNA degradation or decoding steps.
- miRNA may be single-stranded (monomer) or double-stranded (dimer).
- the miRNA is preferably a mature miRNA that has been cleaved with a ribonuclease such as Dicer.
- miRNAs described herein are registered in known databases (e.g., miRBase database) in association with accession numbers, and those skilled in the art will be able to identify the sequences. It can be defined uniquely.
- the accession number of hsa-miR-127-5p is MI0000472, and the sequence is registered in miRBase database.
- the accession number of each miRNA will be omitted.
- miRNA in this specification also includes variants that differ by about 1 to 5 bases from mature miRNA such as hsa-miR-127-5p.
- each miRNA in this specification refers to a polynucleotide consisting of a base sequence having identity with the base sequence of each miRNA (for example, hsa-miR-127-5p), or a polynucleotide consisting of a complementary base sequence thereof. It includes a polynucleotide having the function of miRNA in the present invention.
- Identity refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the incidence (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST.
- Polynucleotides consisting of identical base sequences may have, for example, point mutations, deletions, and/or additions in the miRNA base sequence.
- the number of bases in the point mutation, etc. is, for example, 1 to 5, 1 to 3, 1 to 2, or 1.
- the polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence of miRNA, and includes a polynucleotide having the function of miRNA in the present invention.
- Stringent conditions are not particularly limited, and include, for example, the conditions described in [0028] of JP-A-2017-184642, the contents of which are incorporated herein by reference.
- the microparticles contain miRNA targeting a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
- miRNA targeting a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
- the microparticles contain at least one type of the following Parkinson's disease-related miRNA group; Parkinson's disease-related miRNA group: hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5
- the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
- the microparticles preferably contain hsa-miR-212-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 0.1 or more, more preferably 0.5 or more, and 1.0 It is particularly preferable to include the above.
- microparticles of the present invention express 1.1 times more hsa-miR-212-5p than exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. It is preferably at least 1.5 times, more preferably at least 1.5 times, particularly preferably at least 2 times.
- the expression of the SIRT2 gene in any cell can be suppressed to 0.8 times or less, and preferably 0.6 times or less compared to normal (in untreated cells). More preferably, it can be suppressed to 0.4 times or less, and particularly preferably to 0.4 times or less.
- the microparticles are inhibitors of degeneration or apoptosis of dopaminergic neurons, and hsa-miR-182 among Parkinson's disease-related miRNA group -5p or hsa-miR-183-5p, and more preferably both hsa-miR-182-5p and hsa-miR-183-5p. It is preferable that the microparticles independently contain hsa-miR-182-5p or hsa-miR-183-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and 10. It is more preferable to contain 0 or more, and it is particularly preferable to contain 15.0 or more.
- the expression level of hsa-miR-182-5p or hsa-miR-183-5p is compared with exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. is preferably 1.1 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more.
- microparticles function as a supplement for miRNAs whose expression is downregulated in Parkinson's disease brain tissue, hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330- 5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR- It is preferable that at least one of 485
- Parkinson's disease-related miRNA group may contain any one of hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p. More preferably, it is particularly preferred to contain at least hsa-miR-199a-3p.
- the microparticle preferably contains at least one type of miRNA that is down-regulated in Parkinson's disease brain tissue as a Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and preferably 10.0 or more. It is more preferable to include 15.0 or more, particularly preferably 15.0 or more.
- Microparticles are hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p as Log2 Ratio of read count number obtained by analysis using IMOTA. It is preferable that each of them contains 4.0 or more, and it is particularly preferable that hsa-miR-199a-3p contains 15.0 or more.
- the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Approximately 1800 of these are miRNAs. Among these miRNAs, there are 180 to 200 types of miRNAs that are abundant.
- the high content of miRNA in microparticles derived from dental pulp-derived stem cells is characterized by the fact that there are many microRNAs related to treatment of cranial nerve diseases and eye diseases. This is my knowledge. This feature is significantly different from the types of miRNA that are contained in large amounts in other mesenchymal stem cell microparticles. For example, miRNAs that are abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to treatment of cranial nerve diseases or eye diseases.
- the microparticles preferably contain 5 or more types of miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used for the treatment of Parkinson's disease, more preferably 10 or more types, and especially 15 or more types. Preferably, it is particularly preferable that 20 or more types are included. Furthermore, the microparticles preferably contain 5 or more types of the aforementioned Parkinson's disease-related miRNA group, more preferably 10 or more types, particularly preferably 15 or more types, and 20 or more types. is particularly preferred.
- the microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles. , more preferably exosomes.
- the diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, particularly preferably 50 to 150 nm.
- molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles, and it may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three thereof. good.
- exosomes are used as microparticles may be described, but the microparticles used in the present invention are not limited to exosomes.
- exosomes are extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane.
- the surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
- the interior of the exosome preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
- Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.
- the content of microparticles in the microparticle composition is not particularly limited.
- the microparticle composition preferably contains 0.5 x 10 8 or more microparticles, more preferably 1.0 x 10 8 or more, particularly preferably 2.0 x 10 8 or more, It is particularly preferable to contain 2.5 ⁇ 10 8 or more, and even more particularly preferably to contain 1.0 ⁇ 10 9 or more. Further, there is no particular restriction on the concentration of fine particles contained in the fine particle composition.
- the microparticle composition preferably contains 1.0 ⁇ 10 8 particles/mL or more, more preferably 2.0 ⁇ 10 8 particles/mL or more, and 4.0 ⁇ 10 8 particles/mL or more.
- a preferred embodiment of the microparticles of the present invention can maintain a high amount of miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease by containing the microparticles in such a large amount or at a high concentration.
- the microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, and the like.
- the purpose of the pharmaceutical composition is to facilitate administration of microparticles to the recipient.
- the pharmaceutically acceptable carrier is a carrier (including diluents) that does not cause significant irritation to the administered subject and does not inhibit the biological activity and properties of the administered compound.
- carriers are propylene glycol; (physiological) saline; emulsions; buffers; media, such as DMEM or RPMI; cryopreservation media containing components that scavenge free radicals.
- the microparticle composition may contain an active ingredient of a conventionally known therapeutic agent for Parkinson's disease. Those skilled in the art can make appropriate changes depending on the intended use, administration target, etc.
- the microparticle composition does not contain the predetermined substance.
- the microparticle composition preferably does not contain dental pulp-derived stem cells.
- the microparticle composition preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Other cytokines include those described in [0014] to [0020] of JP 2018-023343A.
- the microparticle composition does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.
- the microparticle composition preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.).
- the microparticle composition is substantially free of conventional serum replacements, such as Knockout serum replacement (KSR).
- KSR Knockout serum replacement
- the content (solid content) of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and 0.01% by mass or less. It is particularly preferable that
- the microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells, etc., and then purifying microparticles from the culture supernatant of dental pulp-derived stem cells.
- the microparticles of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells.
- the microparticles of the present invention are prepared by receiving a composition containing the culture supernatant of dental pulp-derived stem cells that had been disposed of (or purifying the composition as appropriate) and refining the microparticles therefrom. You may.
- the culture supernatant of dental pulp-derived stem cells there are no particular limitations on the culture supernatant of dental pulp-derived stem cells or the like. It is preferable that the culture supernatant of dental pulp-derived stem cells and the like is substantially free of serum.
- the serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and preferably 0.01% by mass or less. Particularly preferred.
- Dental pulp-derived stem cells may be derived from humans or animals other than humans.
- animals other than humans include those similar to the animals (species) to which the microparticles of the present invention are administered, which will be described later, and mammals are preferred.
- dental pulp-derived stem cells used in the culture supernatant.
- Stem cells from exfoliated deciduous teeth can be used.
- dental pulp stem cells from deciduous teeth obtained by other methods can be used.
- dental pulp-derived stem cells derived from animals other than humans such as pig deciduous tooth pulp stem cells, can be used.
- dental pulp-derived stem cells contain vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-).
- VEGF vascular endothelial growth factor
- HGF hepatocyte growth factor
- IGF insulin-like growth factor
- PDGF platelet-derived growth factor
- TGF- transforming growth factor-beta
- the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain many proteins, and it is preferable to use dental pulp stem cells of deciduous teeth. That is, in the present invention, it is preferable to use a culture supernatant of deciduous tooth pulp stem cells.
- the dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved.
- immortalized stem cells derived from dental pulp can be used.
- the immortalized stem cells are preferably immortalized stem cells that have not become cancerous.
- Immortalized stem cells of dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
- the TGF ⁇ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the transforming growth factor (TGF) ⁇ receptor, and for example, 2-(5-benzo[1,3 ] Dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1- Phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridine -2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine( Merck & Co.), SB431542 (S
- the ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-binding kinase.
- ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (Fujifilm Wako Pure Chemical Industries, Ltd.).
- Y-27632 Preferred is Y-27632.
- GSK3 inhibitors are not particularly limited as long as they inhibit GSK-3 (Glycogen Synthase Kinase 3), and include A 1070722, BIO, BIO-acetoxime (TOCRIS), etc. can be mentioned.
- MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), such as AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86 -9766), SL327, U0126-EtOH (all sold by Selleck), PD98059, U0124, and U0125 (all sold by Cosmo Bio Inc.).
- MEK MAP kinase-ERK kinase
- AZD6244 CI-1040
- PD0325901 PD0325901
- RDEA119 BAY86 -9766
- SL327 U0126-EtOH (all sold by Selleck)
- PD98059 U0124
- U0125 all sold by Cosmo Bio Inc.
- microparticles of the present invention When using the microparticles of the present invention in regenerative medicine, in accordance with the requirements of the Regenerative Medicine Safety Act, dental pulp-derived stem cells, culture supernatants of these immortalized stem cells, and compositions containing microparticles derived therefrom must be This embodiment does not contain any other somatic stem cells other than derived stem cells.
- the microparticle composition may contain mesenchymal stem cells and other somatic stem cells other than dental pulp-derived stem cells, it is preferable not to contain them. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc.
- Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like.
- somatic stem cells of the digestive system include pancreatic (general) stem cells, hepatic stem cells, and the like.
- Examples of myeloid somatic stem cells (other than mesenchymal stem cells) include hematopoietic stem cells and the like.
- Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, and the like.
- the microparticle composition may contain stem cells other than somatic stem cells, but preferably does not contain them.
- Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).
- a culture supernatant such as dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells.
- a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing dental pulp-derived stem cells.
- a culture supernatant that has been appropriately subjected to various treatments for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc. may be used.
- Dental pulp-derived stem cells for obtaining a culture supernatant of dental pulp-derived stem cells can be selected by conventional methods, and can be selected based on cell size and morphology, or as adherent cells. Dental pulp cells collected from fallen baby teeth or permanent teeth can be sorted as adherent cells or their successor cells. As the culture supernatant of dental pulp-derived stem cells, a culture supernatant obtained by culturing selected stem cells can be used.
- the "culture supernatant of dental pulp-derived stem cells, etc.” is preferably a culture solution that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc.
- the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain any cells (regardless of the type of cells) as a whole. Due to this feature, the composition of this embodiment is clearly distinguished from dental pulp-derived stem cells themselves as well as from various compositions containing dental pulp-derived stem cells.
- a typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of a culture supernatant of dental pulp-derived stem cells.
- the culture supernatant of dental pulp-derived stem cells used in the present invention may contain culture supernatants of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells.
- the culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of deciduous dental pulp-derived stem cells as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of dental pulp-derived stem cells of deciduous teeth.
- a basic medium or a basic medium to which serum or the like is added can be used.
- DMEM Dulbecco's modified Eagle's medium
- IMDM Iscove's modified Dulbecco's medium
- Ham F12 medium Ham F12, SIGMA, GIBCO, etc.
- RPMI1640 medium etc.
- a serum-free medium may be used throughout the entire process or during the last or several subcultures from the end. For example, by culturing dental pulp-derived stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant of dental pulp-derived stem cells can be prepared.
- the culture supernatant of dental pulp-derived stem cells used in the preparation of microparticles such as exosomes in the present invention may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it does not substantially contain other components. Preferably not. However, various types of additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.
- microparticles are preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles.
- Microparticles can be isolated by separating them from unassociated components based on the properties of the microparticles. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity.
- microparticles can be purified by separating a specific fraction (eg, precipitate) containing many microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the predetermined fractions may be removed.
- microparticles can be purified by filtering a culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration treatment. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed at the same time.
- the material, pore size, etc. of the filtration membrane used for the filtration treatment are not particularly limited. Filtration can be carried out using filtration membranes of appropriate molecular weight or size cut-off using known methods.
- light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track microparticles.
- specific enzyme activities, etc. can be used to track the activity in each fraction.
- As a method for purifying microparticles the method described in [0034] to [0064] of Japanese Patent Publication No. 2019-524824 may be used, and the contents of this publication are incorporated herein by reference.
- the final form of the microparticle composition is not particularly limited.
- the microparticle composition can be formed by filling a container with microparticles together with a solvent or dispersion medium; by gelling the microparticles together with a gel and filling the container; by freezing and/or drying the microparticles.
- it may be solidified into a formulation or filled into a container.
- containers include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation.
- the freezing temperature can be, for example, -20°C to -196°C.
- the microparticles of the present invention can be easily mass-produced compared to conventional compositions that can be used as therapeutic or preventive drugs for Parkinson's disease, and can be used in stem cell culture fluids that were previously discarded as industrial waste. It has advantages such as being able to be used for various purposes and reducing the cost of disposing of stem cell culture fluid.
- the culture supernatant of dental pulp-derived stem cells is the culture supernatant of human dental pulp-derived stem cells
- the microparticles of the present invention are highly safe from an immunological standpoint when applied to humans. Another advantage is that there are fewer ethical issues.
- the microparticles of the present invention will be safer and more ethical when applied to that patient. There will be fewer problems.
- the microparticles of the present invention are derived from the culture supernatant of dental pulp-derived stem cells, they can also be used in restorative medicine.
- compositions containing microparticles derived from culture supernatants such as dental pulp-derived stem cells are preferably used for restorative medical applications.
- stem cells are not the main actors in regeneration, but rather that the humoral components produced by stem cells repair organs together with one's own stem cells.
- Difficult issues associated with conventional stem cell transplantation such as canceration, standardization, administration methods, storage stability, and culture methods, have been resolved, and restorations can be achieved using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Medical care becomes possible.
- stem cell transplantation when using the microparticles of the present invention, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer.
- the microparticles of the present invention have the advantage of being of uniformly standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.
- the prophylactic or therapeutic agent for Parkinson's disease of the present invention contains the microparticles of the present invention.
- prevention refers to preventing the onset of a disease (neurodegenerative disease in this specification).
- treatment refers to alleviating, suppressing, or preventing the progression of symptoms of a disease that has developed, and improving symptoms.
- examples include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc.; intraarterial administration, intravenous administration, subcutaneous administration or Intraperitoneal administration is more preferred.
- various formulation techniques can be used to alter the in vivo distribution of microparticles. Many methods of altering in vivo distribution are known to those skilled in the art. Examples of such methods include protection of exosomes in vesicles composed of materials such as proteins, lipids (eg, liposomes), carbohydrates or synthetic polymers.
- the microparticles of the present invention administered to a subject who has developed Parkinson's disease may circulate within the subject's body and reach a predetermined tissue. There are no particular restrictions on the number of administrations or the interval between administrations.
- the frequency of administration can be one or more times per week, preferably five or more times, more preferably six or more times, particularly preferably seven or more times.
- the administration interval is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be adjusted as appropriate depending on the species to be administered and the symptoms of the subject.
- the microparticles of the present invention are preferably used to administer the microparticles to a subject who has developed dementia at least once a week over a therapeutically effective period.
- the amount is preferably 1 to 50 ⁇ g, more preferably 3 to 30 ⁇ g, and more preferably 5 to 50 ⁇ g per mouse (approximately 25 g). More particularly preferred is ⁇ 25 ⁇ g.
- the preferred range of dosage per body weight for other animals can be calculated from the dosage per body weight (about 25 g) for model mice using a proportional relationship. However, it can be adjusted as appropriate depending on the symptoms of the subject.
- the animals to which the microparticles of the present invention are administered are preferably mammals, birds (chickens, quail, ducks, etc.), and fish (salmon, trout, tuna, bonito, etc.).
- the mammal may be a human or a non-human mammal, but humans are particularly preferred. More preferably, the non-human mammal is a cow, pig, horse, goat, sheep, monkey, dog, cat, mouse, rat, guinea pig, or hamster.
- microparticles of the present invention may be used in combination with conventionally known therapeutic agents for Parkinson's disease. Specifically, it may be used in combination with, for example, L-DOPA (levodopa).
- L-DOPA levodopa
- Example 1 ⁇ Preparation of culture supernatant of dental pulp-derived stem cells> Using DMEM medium instead of the DMEM/HamF12 mixed medium and otherwise following the method described in Example 6 of Patent No. 6296622, a culture supernatant of human deciduous dental pulp stem cells was prepared, and the culture supernatant was fractionated. did. In primary culture, fetal bovine serum (FBS) is added to the culture, and in subculture, the supernatant of the subculture medium is cultured using the primary culture medium, and the supernatant of the subculture medium is separated so that it does not contain FBS. culture supernatant was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.
- FBS fetal bovine serum
- exosomes of dental pulp-derived stem cells were purified and collected by the following ultracentrifugation method. After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells through a filter with a pore size of 0.22 micrometers, the solution was centrifuged at 100,000 ⁇ g at 4° C. for 60 minutes. The supernatant was decanted and the exosome enriched pellet was resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 xg for 60 minutes.
- PBS phosphate buffered saline
- the pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 ⁇ l). Protein concentration was determined by microBSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C. A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the microparticle composition sample of Example 1.
- the average particle diameter and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated using a nanoparticle analysis system NanoSight (manufactured by Nippon Quantum Design Co., Ltd.).
- the average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm.
- the microparticle composition of Example 1 was a high concentration exosome solution of 1.0 ⁇ 10 9 particles/ml or more, specifically, a high concentration exosome solution of 2.0 ⁇ 10 9 particles/ml.
- the components of the obtained microparticle composition of Example 1 were analyzed by a known method.
- Example 1 did not contain stem cells of dental pulp-derived stem cells, did not contain MCP-1, and did not contain Siglec-9. Therefore, it was found that an active ingredient different from MCP-1 and Siglec-9, which are the active ingredients of the culture supernatant of mesenchymal stem cells, and their analogs, was the active ingredient of the microparticle composition of Example 1. .
- Example 1 MicroRNA expressed in exosomes Small RNA contained in the microparticle composition of Example 1 was analyzed by next generation sequencing (NGS) analysis. NGS analysis identified 1787 miRNAs contained in the microparticle composition (dental pulp-derived stem cell exosomes) of Example 1. The results obtained are shown in Table 1 below.
- IMOTA Interactive Multi-Omics-Tissue Atlas
- IMOTA is an interactive multi-omics atlas that allows you to investigate interactions and expression levels of miRNAs, mRNAs, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770- D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions").
- the Omic counts were 3071 proteins, 14182 mRNAs, and 1124 miRNAs.
- the counts of Omics with a high expression rate in the cerebral cortex were 1119 for proteins, 1110 for mRNA, and 145 for miRNA.
- Example 1 Exosomes of dental pulp-derived stem cells
- Example 2 highly expressed in the cerebral cortex as shown in Tables 2 and 3 below.
- Test Example 3 Search for microRNAs involved in diseases Based on the obtained analysis results, microRNAs involved in diseases were searched. Extraction of disease-related miRNAs was performed using IMOTA. Here, we investigated whether microRNAs that suppress proteins related to Parkinson's disease are contained, or microRNAs that control proteins that are targets of therapeutic drugs. In this example, microRNA, which is a gene related to Parkinson's disease, was searched. Non-patent document 1 (Front. Mol. Neurosci. vol. 11, 381(2017)) and non-patent document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-), which describe microRNAs that contribute to the treatment of Parkinson's disease.
- the expression levels of microRNAs expressed in the microparticle composition (SGF exosome) of Example 1 are shown in FIG. Furthermore, among the miRNAs that are down-regulated in the brain tissue of Parkinson's disease described in Non-Patent Document 3 (Neural Regen. Res. 12 (12) p1945-1959 (2017)), the microparticle composition of Example 1 (SGF The expression levels of microRNAs expressed in exosomes are also shown in Figure 1. Additionally, the expression level of miRNA expressed in the microparticle composition of Example 1 in the cerebral cortex is shown in FIG. 2. Blank rows in FIG.
- Example 2 indicate hsa-miR-182-5p and hsa-miR-183 among the miRNAs of the cerebral cortex registered in IMOTA that are expressed in the microparticle composition of Example 1. -5p, hsa-miR-330-5p, and hsa-miR-429 were absent.
- Example 1 contains hsa-, which is described in Non-Patent Document 1 (Front. Mol. Neurosci. vol. 11, 381 (2016)) as a microRNA that contributes to the treatment of Parkinson's disease. miR-212-5p, as well as hsa-miR-182-5p and hsa-miR-183-5p described in Non-Patent Document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018)) are expressed. I found out that there is. Moreover, from FIG.
- the microparticles of the present invention contain miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used in the treatment of Parkinson's disease.
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Abstract
Provided are, comprising miRNA downregulated in Parkinson's disease or miRNA used for the treatment of Parkinson's disease: a fine particle containing a prescribed miRNA of interest as a therapeutic means for Parkinson's disease; a prophylactic drug or therapeutic drug for Parkinson's disease; and a method for improving Parkinson's disease.
Description
本発明は、微小粒子、パーキンソン病の予防薬または治療薬およびパーキンソン病の改善方法に関する。
The present invention relates to microparticles, preventive or therapeutic agents for Parkinson's disease, and methods for improving Parkinson's disease.
パーキンソン病は、世界中で頻度の高い神経変性疾患であり、パーキンソン病の治療法や予防法のさらなる発展が望まれている。これまでの研究で、パーキンソン病の発生機序の解明が進んでいる。
Parkinson's disease is a neurodegenerative disease that occurs frequently around the world, and further development of treatments and prevention methods for Parkinson's disease is desired. Research to date has made progress in elucidating the pathogenesis of Parkinson's disease.
非特許文献1には、サーチュイン(SIRT)の中でもSIRT2の阻害はパーキンソン病(PD)で神経保護効果を発揮すること、SIRT2タンパク質レベルがPDモデルで高度に増加し、miR-212-5pによって負に制御されることが記載されている。そして、PDの病因におけるmiR-212-5pとSIRT2を介したp53依存性のプログラム細胞死との間の直接的な関連に基づき、SIRT2阻害剤がPDの新しい治療手段となることが記載されている。
非特許文献2には、PDではドパミン作動性(DA)ニューロンの変性が生じること、グリア細胞株由来神経栄養因子(GDNF)がPDでのDAニューロンの生存を増加させて一次中脳ニューロン(PMN)でmiR-182-5pおよびmiR-183-5pの発現を特異的に増加させることが記載されている。そして、miR-182-5pおよびmiR-183-5pは、PDへの新しい治療手段となることが記載されている。 Non-PatentDocument 1 states that among sirtuins (SIRTs), inhibition of SIRT2 exerts a neuroprotective effect in Parkinson's disease (PD), that SIRT2 protein level is highly increased in PD models, and that it is negatively affected by miR-212-5p. It is stated that it is controlled by Based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors have been described as a new therapeutic tool for PD. There is.
Non-Patent Document 2 states that degeneration of dopaminergic (DA) neurons occurs in PD, and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of DA neurons in PD and increases the survival of primary mesencephalic neurons (PMNs). ) has been described to specifically increase the expression of miR-182-5p and miR-183-5p. It has also been described that miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
非特許文献2には、PDではドパミン作動性(DA)ニューロンの変性が生じること、グリア細胞株由来神経栄養因子(GDNF)がPDでのDAニューロンの生存を増加させて一次中脳ニューロン(PMN)でmiR-182-5pおよびmiR-183-5pの発現を特異的に増加させることが記載されている。そして、miR-182-5pおよびmiR-183-5pは、PDへの新しい治療手段となることが記載されている。 Non-Patent
Non-Patent Document 2 states that degeneration of dopaminergic (DA) neurons occurs in PD, and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of DA neurons in PD and increases the survival of primary mesencephalic neurons (PMNs). ) has been described to specifically increase the expression of miR-182-5p and miR-183-5p. It has also been described that miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
一方、非特許文献3には、パーキンソン病におけるmiRNAと新たな治療ターゲットについての総説が記載されており、p1957のFigure 1にはパーキンソン病の脳組織においてダウンレギュレーションされるmiRNAとして41種類のmiRNAが記載されている。
On the other hand, Non-Patent Document 3 describes a review of miRNAs and new therapeutic targets in Parkinson's disease, and Figure 1 on p1957 lists 41 types of miRNAs as miRNAs that are down-regulated in the brain tissue of Parkinson's disease. Are listed.
しかし、非特許文献1~3には、これらの文献に記載のmiRNAを生体内に導入したりトランスフェクションしたりする手段として、エクソソーム等の微小粒子を用いることは記載されていなかった。
However, Non-Patent Documents 1 to 3 do not describe the use of microparticles such as exosomes as a means for introducing or transfecting the miRNA described in these documents into living organisms.
本発明が解決しようとする課題は、パーキンソン病の治療手段として注目される特定のmiRNAを含む微小粒子を提供することである。
The problem to be solved by the present invention is to provide microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.
本発明者らは、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む微小粒子により、上記課題を解決できることを見出した。
The present inventors have discovered that the above problems can be solved by microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
具体的に、本発明および本発明の好ましい構成は、以下のとおりである。
[1] パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む、微小粒子。
[2] 微小粒子がエクソソームである、[1]に記載の微小粒子。
[3] 微小粒子が、歯髄由来幹細胞の培養上清よりも高い濃度で、パーキンソン病に関する遺伝子を標的遺伝子とするmiRNAを含む、[1]に記載の微小粒子。
[4] 微小粒子が下記パーキンソン病関連miRNA群のうち少なくとも1種類を含む、[1]に記載の微小粒子;
パーキンソン病関連miRNA群:
hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-212-5p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543。
[5] 微小粒子がパーキンソン病関連miRNA群のうち、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pを含む、[4]に記載の微小粒子。
[6] 微小粒子が、SIRT2阻害剤であり、かつパーキンソン病関連miRNA群のうちhsa-miR-212-5pを含む、[4]に記載の微小粒子。
[7] ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤であり、かつ、パーキンソン病関連miRNA群のうちhsa-miR-182-5pまたはhsa-miR-183-5pを含む、[4]に記載の微小粒子。
[8] 微小粒子が歯髄由来幹細胞の培養上清から精製されて単離された微小粒子であり、
微小粒子が、歯髄由来幹細胞の培養上清からエクソソームを除いた成分を含まない、[1]に記載の微小粒子。
[9] [1]に記載の微小粒子を含む、パーキンソン病の予防薬または治療薬。
[10] 有効量の[1]に記載の微小粒子、あるいは有効量の[9]に記載のパーキンソン病の予防薬または治療薬を、パーキンソン病を発症した対象に投与することを含む、パーキンソン病の改善方法。 Specifically, the present invention and preferred configurations of the present invention are as follows.
[1] Microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
[2] The microparticle according to [1], wherein the microparticle is an exosome.
[3] The microparticle according to [1], wherein the microparticle contains miRNA that targets a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
[4] The microparticle according to [1], wherein the microparticle contains at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
[5] The microparticles contain hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group, [4 The microparticles described in ].
[6] The microparticle according to [4], wherein the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
[7] The agent according to [4], which is an inhibitor of dopaminergic neuron degeneration or apoptosis and includes hsa-miR-182-5p or hsa-miR-183-5p from the Parkinson's disease-related miRNA group. Microparticles.
[8] The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells,
The microparticle according to [1], wherein the microparticle does not contain components other than the culture supernatant of dental pulp-derived stem cells except for exosomes.
[9] A prophylactic or therapeutic agent for Parkinson's disease, comprising the microparticle according to [1].
[10] Parkinson's disease, comprising administering an effective amount of the microparticles according to [1] or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease according to [9] to a subject who has developed Parkinson's disease. How to improve.
[1] パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む、微小粒子。
[2] 微小粒子がエクソソームである、[1]に記載の微小粒子。
[3] 微小粒子が、歯髄由来幹細胞の培養上清よりも高い濃度で、パーキンソン病に関する遺伝子を標的遺伝子とするmiRNAを含む、[1]に記載の微小粒子。
[4] 微小粒子が下記パーキンソン病関連miRNA群のうち少なくとも1種類を含む、[1]に記載の微小粒子;
パーキンソン病関連miRNA群:
hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-212-5p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543。
[5] 微小粒子がパーキンソン病関連miRNA群のうち、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pを含む、[4]に記載の微小粒子。
[6] 微小粒子が、SIRT2阻害剤であり、かつパーキンソン病関連miRNA群のうちhsa-miR-212-5pを含む、[4]に記載の微小粒子。
[7] ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤であり、かつ、パーキンソン病関連miRNA群のうちhsa-miR-182-5pまたはhsa-miR-183-5pを含む、[4]に記載の微小粒子。
[8] 微小粒子が歯髄由来幹細胞の培養上清から精製されて単離された微小粒子であり、
微小粒子が、歯髄由来幹細胞の培養上清からエクソソームを除いた成分を含まない、[1]に記載の微小粒子。
[9] [1]に記載の微小粒子を含む、パーキンソン病の予防薬または治療薬。
[10] 有効量の[1]に記載の微小粒子、あるいは有効量の[9]に記載のパーキンソン病の予防薬または治療薬を、パーキンソン病を発症した対象に投与することを含む、パーキンソン病の改善方法。 Specifically, the present invention and preferred configurations of the present invention are as follows.
[1] Microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
[2] The microparticle according to [1], wherein the microparticle is an exosome.
[3] The microparticle according to [1], wherein the microparticle contains miRNA that targets a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
[4] The microparticle according to [1], wherein the microparticle contains at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
[5] The microparticles contain hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group, [4 The microparticles described in ].
[6] The microparticle according to [4], wherein the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
[7] The agent according to [4], which is an inhibitor of dopaminergic neuron degeneration or apoptosis and includes hsa-miR-182-5p or hsa-miR-183-5p from the Parkinson's disease-related miRNA group. Microparticles.
[8] The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells,
The microparticle according to [1], wherein the microparticle does not contain components other than the culture supernatant of dental pulp-derived stem cells except for exosomes.
[9] A prophylactic or therapeutic agent for Parkinson's disease, comprising the microparticle according to [1].
[10] Parkinson's disease, comprising administering an effective amount of the microparticles according to [1] or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease according to [9] to a subject who has developed Parkinson's disease. How to improve.
本発明によれば、パーキンソン病の治療手段として注目される特定のmiRNAを含む微小粒子を提供することができる。
According to the present invention, it is possible to provide microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.
以下において、本発明について詳細に説明する。以下に記載する構成要件の説明は、代表的な実施形態や具体例に基づいてなされることがあるが、本発明はそのような実施形態に限定されるものではない。なお、本明細書において「~」を用いて表される数値範囲は「~」前後に記載される数値を下限値および上限値として含む範囲を意味する。
The present invention will be explained in detail below. Although the constituent elements described below may be explained based on typical embodiments and specific examples, the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after the "~" as lower and upper limits.
[微小粒子]
本発明の微小粒子は、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む、微小粒子である。
本発明の微小粒子は、パーキンソン病の治療手段として注目される特定のmiRNAを含む。
以下、本発明の微小粒子の好ましい態様を説明する。 [Microparticles]
The microparticles of the present invention are microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
The microparticles of the present invention contain specific miRNAs that are attracting attention as a means of treating Parkinson's disease.
Hereinafter, preferred embodiments of the microparticles of the present invention will be explained.
本発明の微小粒子は、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む、微小粒子である。
本発明の微小粒子は、パーキンソン病の治療手段として注目される特定のmiRNAを含む。
以下、本発明の微小粒子の好ましい態様を説明する。 [Microparticles]
The microparticles of the present invention are microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
The microparticles of the present invention contain specific miRNAs that are attracting attention as a means of treating Parkinson's disease.
Hereinafter, preferred embodiments of the microparticles of the present invention will be explained.
<パーキンソン病の定義>
パーキンソン病(PD)は、中枢神経系の特定の神経細胞(ニューロン)内又はグリア細胞内に異常なタンパク質性の封入体が形成される結果、神経細胞が徐々に侵され、減退および消失していく進行性神経疾患(神経変性疾患)の一種である。
パーキンソン病は、解剖学および病理学的見地から、黒質緻密部(SNc;それら自体の投射を線状体に送る、メラニンを含有するドパミン作動性ニューロンにより構成されている神経核)の選択的変性により特徴付けられる。
パーキンソン病(PD)症状としては、四大症状(安静時振戦、動作緩慢・無動、筋強剛、姿勢反射障害)などの運動障害や、非運動症状(自律神経症状、感覚障害、精神症状、睡眠障害など)、衝動制御障害(病的賭博など)が含まれる。
病理的特徴として、黒質緻密部(SNc)におけるドパミン作動性ニューロン変性およびレビー小体の存在が主に挙げられ、さらに青班、視床下部、大脳皮質、基底核、自律神経系の中枢及び末梢構成部分を含む他の系統の変性も報告されている。黒質緻密部におけるドパミン作動性ニューロンの約80%の喪失があるとき、パーキンソン病症状が見られることが多い。 <Definition of Parkinson's disease>
Parkinson's disease (PD) is caused by the formation of abnormal proteinaceous inclusions within certain nerve cells (neurons) or glial cells in the central nervous system, which gradually attack the nerve cells, causing them to decline and disappear. It is a type of progressive neurological disease (neurodegenerative disease).
From an anatomical and pathological point of view, Parkinson's disease is a selective disease of the substantia nigra pars compacta (SNc; a neuronal nucleus composed of melanin-containing dopaminergic neurons that sends their own projections to the striatum). Characterized by degeneration.
Parkinson's disease (PD) symptoms include motor disorders such as the four major symptoms (resting tremor, bradykinesia/akinesia, muscle rigidity, postural reflex disorder), and non-motor symptoms (autonomic nervous symptoms, sensory disorders, mental disorders). symptoms, sleep disturbances, etc.), impulse control disorders (such as pathological gambling).
Pathological features mainly include dopaminergic neuron degeneration and the presence of Lewy bodies in the substantia nigra pars compacta (SNc), as well as the pallial cortex, hypothalamus, cerebral cortex, basal ganglia, and central and peripheral autonomic nervous system. Degeneration of other strains including component parts has also been reported. Parkinson's disease symptoms are often seen when there is approximately 80% loss of dopaminergic neurons in the substantia nigra pars compacta.
パーキンソン病(PD)は、中枢神経系の特定の神経細胞(ニューロン)内又はグリア細胞内に異常なタンパク質性の封入体が形成される結果、神経細胞が徐々に侵され、減退および消失していく進行性神経疾患(神経変性疾患)の一種である。
パーキンソン病は、解剖学および病理学的見地から、黒質緻密部(SNc;それら自体の投射を線状体に送る、メラニンを含有するドパミン作動性ニューロンにより構成されている神経核)の選択的変性により特徴付けられる。
パーキンソン病(PD)症状としては、四大症状(安静時振戦、動作緩慢・無動、筋強剛、姿勢反射障害)などの運動障害や、非運動症状(自律神経症状、感覚障害、精神症状、睡眠障害など)、衝動制御障害(病的賭博など)が含まれる。
病理的特徴として、黒質緻密部(SNc)におけるドパミン作動性ニューロン変性およびレビー小体の存在が主に挙げられ、さらに青班、視床下部、大脳皮質、基底核、自律神経系の中枢及び末梢構成部分を含む他の系統の変性も報告されている。黒質緻密部におけるドパミン作動性ニューロンの約80%の喪失があるとき、パーキンソン病症状が見られることが多い。 <Definition of Parkinson's disease>
Parkinson's disease (PD) is caused by the formation of abnormal proteinaceous inclusions within certain nerve cells (neurons) or glial cells in the central nervous system, which gradually attack the nerve cells, causing them to decline and disappear. It is a type of progressive neurological disease (neurodegenerative disease).
From an anatomical and pathological point of view, Parkinson's disease is a selective disease of the substantia nigra pars compacta (SNc; a neuronal nucleus composed of melanin-containing dopaminergic neurons that sends their own projections to the striatum). Characterized by degeneration.
Parkinson's disease (PD) symptoms include motor disorders such as the four major symptoms (resting tremor, bradykinesia/akinesia, muscle rigidity, postural reflex disorder), and non-motor symptoms (autonomic nervous symptoms, sensory disorders, mental disorders). symptoms, sleep disturbances, etc.), impulse control disorders (such as pathological gambling).
Pathological features mainly include dopaminergic neuron degeneration and the presence of Lewy bodies in the substantia nigra pars compacta (SNc), as well as the pallial cortex, hypothalamus, cerebral cortex, basal ganglia, and central and peripheral autonomic nervous system. Degeneration of other strains including component parts has also been reported. Parkinson's disease symptoms are often seen when there is approximately 80% loss of dopaminergic neurons in the substantia nigra pars compacta.
<パーキンソン病の発生機序>
パーキンソン病の発生機序の概要は以下のとおりである。
(1)黒質緻密部のドパミン作動性ニューロンの進行性変性が生じる。
(2)黒質緻密部のドパミン作動性ニューロンが減少することにより、ドパミンの作られる量が少なくなる。
(3)線状体のドパミン作動性神経の欠損が、基底の機能を破壊する。
(4)パーキンソン病症状の発現をもたらす。
すなわち、正常時には黒質で作られたドパミンは大脳の線条体で脳の各部分との連絡役として働き、体の動きや働きを調節するが、パーキンソン病を発症するとドパミンが不足して十分な体の動きや働きの調節が行えなくなり、運動症状や非運動症状が現れる。 <Mechanism of Parkinson's disease>
An overview of the pathogenesis of Parkinson's disease is as follows.
(1) Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta occurs.
(2) Due to a decrease in dopaminergic neurons in the substantia nigra pars compacta, the amount of dopamine produced decreases.
(3) Defects in dopaminergic neurons in the striatum disrupt basal function.
(4) Bring about the manifestation of Parkinson's disease symptoms.
In other words, under normal conditions, dopamine produced in the substantia nigra acts as a liaison with various parts of the brain in the striatum of the cerebrum and regulates body movements and functions, but when Parkinson's disease develops, dopamine becomes insufficient. The patient becomes unable to control body movements and functions, and motor and non-motor symptoms appear.
パーキンソン病の発生機序の概要は以下のとおりである。
(1)黒質緻密部のドパミン作動性ニューロンの進行性変性が生じる。
(2)黒質緻密部のドパミン作動性ニューロンが減少することにより、ドパミンの作られる量が少なくなる。
(3)線状体のドパミン作動性神経の欠損が、基底の機能を破壊する。
(4)パーキンソン病症状の発現をもたらす。
すなわち、正常時には黒質で作られたドパミンは大脳の線条体で脳の各部分との連絡役として働き、体の動きや働きを調節するが、パーキンソン病を発症するとドパミンが不足して十分な体の動きや働きの調節が行えなくなり、運動症状や非運動症状が現れる。 <Mechanism of Parkinson's disease>
An overview of the pathogenesis of Parkinson's disease is as follows.
(1) Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta occurs.
(2) Due to a decrease in dopaminergic neurons in the substantia nigra pars compacta, the amount of dopamine produced decreases.
(3) Defects in dopaminergic neurons in the striatum disrupt basal function.
(4) Bring about the manifestation of Parkinson's disease symptoms.
In other words, under normal conditions, dopamine produced in the substantia nigra acts as a liaison with various parts of the brain in the striatum of the cerebrum and regulates body movements and functions, but when Parkinson's disease develops, dopamine becomes insufficient. The patient becomes unable to control body movements and functions, and motor and non-motor symptoms appear.
黒質緻密部のドパミン作動性ニューロンの進行性変性が生じる原因の一つとして、レビー小体が知られている。そして、黒質緻密部のドパミン作動性ニューロンの中にαシヌクレインタンパク質が凝集して溜まることが、レビー小体を形成する原因となる。
なお、レビー小体が出現する疾患を「レビー小体病」といい、パーキンソン病のほかにはレビー小体型認知症などがある。 Lewy bodies are known to be one of the causes of progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Then, α-synuclein protein aggregates and accumulates in dopaminergic neurons in the substantia nigra pars compacta, causing the formation of Lewy bodies.
The disease in which Lewy bodies appear is called "Lewy body disease," and in addition to Parkinson's disease, there is Lewy body dementia.
なお、レビー小体が出現する疾患を「レビー小体病」といい、パーキンソン病のほかにはレビー小体型認知症などがある。 Lewy bodies are known to be one of the causes of progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Then, α-synuclein protein aggregates and accumulates in dopaminergic neurons in the substantia nigra pars compacta, causing the formation of Lewy bodies.
The disease in which Lewy bodies appear is called "Lewy body disease," and in addition to Parkinson's disease, there is Lewy body dementia.
<パーキンソン病治療薬と作用機序>
本発明の微小粒子は、パーキンソン病の改善をできることが好ましい。以下、パーキンソン病治療薬と作用機序を、本発明の微小粒子の作用機序とあわせて説明する。
パーキンソン病治療薬と作用機序としては、以下のものが挙げられる。 <Parkinson's disease treatment drugs and mechanism of action>
Preferably, the microparticles of the present invention can improve Parkinson's disease. Hereinafter, the therapeutic agent for Parkinson's disease and its mechanism of action will be explained together with the mechanism of action of the microparticles of the present invention.
The following are examples of Parkinson's disease therapeutic drugs and their mechanisms of action:
本発明の微小粒子は、パーキンソン病の改善をできることが好ましい。以下、パーキンソン病治療薬と作用機序を、本発明の微小粒子の作用機序とあわせて説明する。
パーキンソン病治療薬と作用機序としては、以下のものが挙げられる。 <Parkinson's disease treatment drugs and mechanism of action>
Preferably, the microparticles of the present invention can improve Parkinson's disease. Hereinafter, the therapeutic agent for Parkinson's disease and its mechanism of action will be explained together with the mechanism of action of the microparticles of the present invention.
The following are examples of Parkinson's disease therapeutic drugs and their mechanisms of action:
(a)SIRT2阻害剤
Front. Mol. Neurosci. vol.11, 381(2018)には、サーチュイン(SIRT)の中でもSIRT2の阻害はパーキンソン病(PD)で神経保護効果を発揮すること、SIRT2タンパク質レベルがPDモデルで高度に増加し、miR-212-5pによって負に制御されることが記載されている。そして、PDの病因におけるmiR-212-5pとSIRT2を介したp53依存性のプログラム細胞死との間の直接的な関連に基づき、SIRT2阻害剤がPDの新しい治療手段となる。詳細には、miR-212-5pトランスフェクションはSIRT2の発現を低下させ、SIRT2の活性を阻害し、ドパミン作動性ニューロンの喪失とDATの低下を防ぐ。すなわち、miR-212-5pがPDで神経保護効果を示す。作用機序としては、核のアセチル化p53は、SIRT2の主要な脱アセチル化基質であるp53によりアップレギュレーションである。さらに、細胞質p53の減少は、PDモデルのオートファジーを促進する。これに対し、miR-212-5pは、PDモデルのアポトーシスを軽減する。
本発明の微小粒子がmiR-212-5pを含む場合、SIRT2阻害剤として機能する。 (a) SIRT2 inhibitor Front. Mol. Neurosci. vol. 11, 381 (2018) states that among sirtuins (SIRTs), inhibition of SIRT2 exerts neuroprotective effects in Parkinson's disease (PD), that SIRT2 protein levels are highly increased in PD models, and that miR-212-5p It has been described that it is negatively controlled by And based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors represent a new therapeutic tool for PD. Specifically, miR-212-5p transfection reduces SIRT2 expression, inhibits SIRT2 activity, and prevents dopaminergic neuron loss and DAT decline. That is, miR-212-5p exhibits neuroprotective effects in PD. The mechanism of action is that nuclear acetylated p53 is upregulated by p53, which is the major deacetylation substrate of SIRT2. Furthermore, reduction of cytoplasmic p53 promotes autophagy in PD models. In contrast, miR-212-5p attenuates apoptosis in PD models.
When the microparticles of the present invention contain miR-212-5p, they function as SIRT2 inhibitors.
Front. Mol. Neurosci. vol.11, 381(2018)には、サーチュイン(SIRT)の中でもSIRT2の阻害はパーキンソン病(PD)で神経保護効果を発揮すること、SIRT2タンパク質レベルがPDモデルで高度に増加し、miR-212-5pによって負に制御されることが記載されている。そして、PDの病因におけるmiR-212-5pとSIRT2を介したp53依存性のプログラム細胞死との間の直接的な関連に基づき、SIRT2阻害剤がPDの新しい治療手段となる。詳細には、miR-212-5pトランスフェクションはSIRT2の発現を低下させ、SIRT2の活性を阻害し、ドパミン作動性ニューロンの喪失とDATの低下を防ぐ。すなわち、miR-212-5pがPDで神経保護効果を示す。作用機序としては、核のアセチル化p53は、SIRT2の主要な脱アセチル化基質であるp53によりアップレギュレーションである。さらに、細胞質p53の減少は、PDモデルのオートファジーを促進する。これに対し、miR-212-5pは、PDモデルのアポトーシスを軽減する。
本発明の微小粒子がmiR-212-5pを含む場合、SIRT2阻害剤として機能する。 (a) SIRT2 inhibitor Front. Mol. Neurosci. vol. 11, 381 (2018) states that among sirtuins (SIRTs), inhibition of SIRT2 exerts neuroprotective effects in Parkinson's disease (PD), that SIRT2 protein levels are highly increased in PD models, and that miR-212-5p It has been described that it is negatively controlled by And based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors represent a new therapeutic tool for PD. Specifically, miR-212-5p transfection reduces SIRT2 expression, inhibits SIRT2 activity, and prevents dopaminergic neuron loss and DAT decline. That is, miR-212-5p exhibits neuroprotective effects in PD. The mechanism of action is that nuclear acetylated p53 is upregulated by p53, which is the major deacetylation substrate of SIRT2. Furthermore, reduction of cytoplasmic p53 promotes autophagy in PD models. In contrast, miR-212-5p attenuates apoptosis in PD models.
When the microparticles of the present invention contain miR-212-5p, they function as SIRT2 inhibitors.
(b)ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤
Molecular Therapy: Nucleic Acids, Vol.11 p9-22(2018)には、PDではドパミン作動性ニューロンの変性が生じること、グリア細胞株由来神経栄養因子(GDNF)がPDでのドパミン作動性ニューロンの生存を増加させて一次中脳ニューロン(PMN)でmiR-182-5pおよびmiR-183-5pの発現を特異的に増加させることが記載されている。そして、miR-182-5pおよびmiR-183-5pミミックのトランスフェクションは神経突起伸長の増加をもたらし、GDNF効果を模倣してドパミン作動性ニューロンの神経保護を仲介することが記載されている。さらにGDNFで処理されたPMNにおける内因性miR-182-5pまたはmiR-183-5pの阻害はGDNFのプロDA作動性効果を弱めたことが記載されている。これらのことから、miR-182-5pおよびmiR-183-5pは、PDへの新しい治療手段となる。
本発明の微小粒子がmiR-182-5pまたはmiR-183-5pを含む場合、ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤として機能する。 (b) Inhibitor of degeneration or apoptosis of dopaminergic neurons Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018) states that degeneration of dopaminergic neurons occurs in PD and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of dopaminergic neurons in PD and improves primary mesencephalic neurons. It has been described that the expression of miR-182-5p and miR-183-5p is specifically increased in (PMN). And it has been described that transfection of miR-182-5p and miR-183-5p mimics results in increased neurite outgrowth and mediates neuroprotection of dopaminergic neurons, mimicking the GDNF effect. Furthermore, it has been described that inhibition of endogenous miR-182-5p or miR-183-5p in PMNs treated with GDNF attenuated the pro-DAergic effects of GDNF. Based on these facts, miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
When the microparticles of the present invention contain miR-182-5p or miR-183-5p, they function as an inhibitor of dopaminergic neuron degeneration or apoptosis.
Molecular Therapy: Nucleic Acids, Vol.11 p9-22(2018)には、PDではドパミン作動性ニューロンの変性が生じること、グリア細胞株由来神経栄養因子(GDNF)がPDでのドパミン作動性ニューロンの生存を増加させて一次中脳ニューロン(PMN)でmiR-182-5pおよびmiR-183-5pの発現を特異的に増加させることが記載されている。そして、miR-182-5pおよびmiR-183-5pミミックのトランスフェクションは神経突起伸長の増加をもたらし、GDNF効果を模倣してドパミン作動性ニューロンの神経保護を仲介することが記載されている。さらにGDNFで処理されたPMNにおける内因性miR-182-5pまたはmiR-183-5pの阻害はGDNFのプロDA作動性効果を弱めたことが記載されている。これらのことから、miR-182-5pおよびmiR-183-5pは、PDへの新しい治療手段となる。
本発明の微小粒子がmiR-182-5pまたはmiR-183-5pを含む場合、ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤として機能する。 (b) Inhibitor of degeneration or apoptosis of dopaminergic neurons Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018) states that degeneration of dopaminergic neurons occurs in PD and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of dopaminergic neurons in PD and improves primary mesencephalic neurons. It has been described that the expression of miR-182-5p and miR-183-5p is specifically increased in (PMN). And it has been described that transfection of miR-182-5p and miR-183-5p mimics results in increased neurite outgrowth and mediates neuroprotection of dopaminergic neurons, mimicking the GDNF effect. Furthermore, it has been described that inhibition of endogenous miR-182-5p or miR-183-5p in PMNs treated with GDNF attenuated the pro-DAergic effects of GDNF. Based on these facts, miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
When the microparticles of the present invention contain miR-182-5p or miR-183-5p, they function as an inhibitor of dopaminergic neuron degeneration or apoptosis.
(c)パーキンソン病で発現が抑制されるmiRNAの補充
Neural Regen. Res., 12, (12), p1945-1959(2017)には、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAが記載されている。このようなmiRNAを補充することは、PDへの新しい治療手段となる。
本発明の微小粒子は、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する。特に、本発明の微小粒子が、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543のうち少なくとも1種類を含む場合、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する。 (c) Supplementation of miRNA whose expression is suppressed in Parkinson's disease Neural Regen. Res. , 12, (12), p1945-1959 (2017) describes miRNAs that are downregulated in brain tissue of Parkinson's disease. Supplementing such miRNAs provides a new means of treatment for PD.
The microparticles of the present invention function as a supplement for miRNAs that are downregulated in Parkinson's disease brain tissue. In particular, the microparticles of the present invention include hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p , hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa -If it contains at least one of miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543, Parkinson's It functions as a supplement for miRNAs that are down-regulated in diseased brain tissue.
Neural Regen. Res., 12, (12), p1945-1959(2017)には、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAが記載されている。このようなmiRNAを補充することは、PDへの新しい治療手段となる。
本発明の微小粒子は、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する。特に、本発明の微小粒子が、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543のうち少なくとも1種類を含む場合、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する。 (c) Supplementation of miRNA whose expression is suppressed in Parkinson's disease Neural Regen. Res. , 12, (12), p1945-1959 (2017) describes miRNAs that are downregulated in brain tissue of Parkinson's disease. Supplementing such miRNAs provides a new means of treatment for PD.
The microparticles of the present invention function as a supplement for miRNAs that are downregulated in Parkinson's disease brain tissue. In particular, the microparticles of the present invention include hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p , hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa -If it contains at least one of miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543, Parkinson's It functions as a supplement for miRNAs that are down-regulated in diseased brain tissue.
(d)L-DOPAやドパミンアゴニストの補充
ドパミンの前駆体であるL-DOPA(レボドパ)を服用することで、血液中から脳内に入り、パーキンソン病の脳内で不足するドパミンを補充する方法が治療手段となる(ドパミンは血液中から脳内に入らない)。また、L-DOPAよりも作用時間の長いドパミンアゴニスト(ドパミン受容体刺激薬)を服用することで同様の治療手段となる。
なお、L-DOPAやドパミンアゴニストの補充は、本発明の微小粒子とは関連性が低い。 (d) Replenishment of L-DOPA and dopamine agonists: By taking L-DOPA (levodopa), a precursor of dopamine, it enters the brain from the blood and replenishes the dopamine that is deficient in the brain in patients with Parkinson's disease. is the treatment (dopamine does not enter the brain from the blood). In addition, taking a dopamine agonist (dopamine receptor stimulant), which has a longer action time than L-DOPA, can be used as a similar treatment.
Note that supplementation with L-DOPA and dopamine agonists has little relevance to the microparticles of the present invention.
ドパミンの前駆体であるL-DOPA(レボドパ)を服用することで、血液中から脳内に入り、パーキンソン病の脳内で不足するドパミンを補充する方法が治療手段となる(ドパミンは血液中から脳内に入らない)。また、L-DOPAよりも作用時間の長いドパミンアゴニスト(ドパミン受容体刺激薬)を服用することで同様の治療手段となる。
なお、L-DOPAやドパミンアゴニストの補充は、本発明の微小粒子とは関連性が低い。 (d) Replenishment of L-DOPA and dopamine agonists: By taking L-DOPA (levodopa), a precursor of dopamine, it enters the brain from the blood and replenishes the dopamine that is deficient in the brain in patients with Parkinson's disease. is the treatment (dopamine does not enter the brain from the blood). In addition, taking a dopamine agonist (dopamine receptor stimulant), which has a longer action time than L-DOPA, can be used as a similar treatment.
Note that supplementation with L-DOPA and dopamine agonists has little relevance to the microparticles of the present invention.
(e)抗コリン薬
パーキンソン病ではドパミンの減少に伴って、別の神経伝達物質であるアセチルコリンが相対的に過剰になるため、アセチルコリンの作用を減らす方法が治療手段となる。
なお、抗コリン薬は、本発明の微小粒子とは関連性が低い。 (e) Anticholinergic Drugs In Parkinson's disease, as dopamine decreases, acetylcholine, another neurotransmitter, becomes relatively excessive, so methods to reduce the effects of acetylcholine are a means of treatment.
Note that anticholinergic drugs have little relevance to the microparticles of the present invention.
パーキンソン病ではドパミンの減少に伴って、別の神経伝達物質であるアセチルコリンが相対的に過剰になるため、アセチルコリンの作用を減らす方法が治療手段となる。
なお、抗コリン薬は、本発明の微小粒子とは関連性が低い。 (e) Anticholinergic Drugs In Parkinson's disease, as dopamine decreases, acetylcholine, another neurotransmitter, becomes relatively excessive, so methods to reduce the effects of acetylcholine are a means of treatment.
Note that anticholinergic drugs have little relevance to the microparticles of the present invention.
(f)塩酸アマンタジン
塩酸アマンタジンは線条体でのドパミン放出を促す働きがあるほか、身体が勝手に動くL-DOPA誘発性の不随意運動(ジスキネジア)を抑制する効果が知られている。
なお、塩酸アマンタジンは、本発明の微小粒子とは関連性が低い。 (f) Amantadine Hydrochloride Amantadine hydrochloride has the effect of promoting dopamine release in the striatum, and is also known to have the effect of suppressing L-DOPA-induced involuntary movements (dyskinesia) in which the body moves on its own.
Note that amantadine hydrochloride has little relevance to the microparticles of the present invention.
塩酸アマンタジンは線条体でのドパミン放出を促す働きがあるほか、身体が勝手に動くL-DOPA誘発性の不随意運動(ジスキネジア)を抑制する効果が知られている。
なお、塩酸アマンタジンは、本発明の微小粒子とは関連性が低い。 (f) Amantadine Hydrochloride Amantadine hydrochloride has the effect of promoting dopamine release in the striatum, and is also known to have the effect of suppressing L-DOPA-induced involuntary movements (dyskinesia) in which the body moves on its own.
Note that amantadine hydrochloride has little relevance to the microparticles of the present invention.
(g)L-DOPAとの併用薬
L-DOPAとの併用で、L-DOPAの効果が切れた際のウェアリングオフや振戦を改善する効果が知られているゾニサミド(パーキンソン症状を改善する作用機序は完全には解明されていない)、L-DOPAの効果が切れた際のウェアリングオフを改善する効果が知られているアデノシン受容体拮抗薬が知られている。L-DOPAを分解する血液中の酵素であるドパ脱炭酸酵素(DDC)阻害薬、L-DOPAを分解する血液中の酵素であるカテコール-O-メチル転移酵素(COMT)阻害薬なども知られている。
なお、以上に例示したL-DOPAとの併用薬は、本発明の微小粒子とは関連性が低い。 (g) Medications used in combination with L-DOPA Zonisamide (which improves Parkinson's symptoms) is known to be effective in improving wear-off and tremors when the effects of L-DOPA wear off when used in combination with L-DOPA. (The mechanism of action has not been completely elucidated), and adenosine receptor antagonists are known to be effective in improving wear-off when the effects of L-DOPA wear off. There are also drugs known to inhibit dopa decarboxylase (DDC), an enzyme in the blood that breaks down L-DOPA, and inhibitors of catechol-O-methyltransferase (COMT), an enzyme in the blood that breaks down L-DOPA. It is being
Note that the drugs used in combination with L-DOPA as exemplified above have little relevance to the microparticles of the present invention.
L-DOPAとの併用で、L-DOPAの効果が切れた際のウェアリングオフや振戦を改善する効果が知られているゾニサミド(パーキンソン症状を改善する作用機序は完全には解明されていない)、L-DOPAの効果が切れた際のウェアリングオフを改善する効果が知られているアデノシン受容体拮抗薬が知られている。L-DOPAを分解する血液中の酵素であるドパ脱炭酸酵素(DDC)阻害薬、L-DOPAを分解する血液中の酵素であるカテコール-O-メチル転移酵素(COMT)阻害薬なども知られている。
なお、以上に例示したL-DOPAとの併用薬は、本発明の微小粒子とは関連性が低い。 (g) Medications used in combination with L-DOPA Zonisamide (which improves Parkinson's symptoms) is known to be effective in improving wear-off and tremors when the effects of L-DOPA wear off when used in combination with L-DOPA. (The mechanism of action has not been completely elucidated), and adenosine receptor antagonists are known to be effective in improving wear-off when the effects of L-DOPA wear off. There are also drugs known to inhibit dopa decarboxylase (DDC), an enzyme in the blood that breaks down L-DOPA, and inhibitors of catechol-O-methyltransferase (COMT), an enzyme in the blood that breaks down L-DOPA. It is being
Note that the drugs used in combination with L-DOPA as exemplified above have little relevance to the microparticles of the present invention.
(h)MAO-B阻害薬
MAO-B阻害薬である塩酸セレギリンはMAOの活性を低下させてドパミンの分解を抑制するため、服薬することで治療手段となる。また、MAO-B阻害薬はノルエピネフリンやセロトニンなど他の神経伝達物資の分解も抑制する。
なお、MAO-B阻害薬は、本発明の微小粒子とは関連性が低い。 (h) MAO-B inhibitor Selegiline hydrochloride, an MAO-B inhibitor, reduces the activity of MAO and suppresses the decomposition of dopamine, so taking it serves as a therapeutic means. MAO-B inhibitors also inhibit the breakdown of other neurotransmitters such as norepinephrine and serotonin.
Note that MAO-B inhibitors have little relevance to the microparticles of the present invention.
MAO-B阻害薬である塩酸セレギリンはMAOの活性を低下させてドパミンの分解を抑制するため、服薬することで治療手段となる。また、MAO-B阻害薬はノルエピネフリンやセロトニンなど他の神経伝達物資の分解も抑制する。
なお、MAO-B阻害薬は、本発明の微小粒子とは関連性が低い。 (h) MAO-B inhibitor Selegiline hydrochloride, an MAO-B inhibitor, reduces the activity of MAO and suppresses the decomposition of dopamine, so taking it serves as a therapeutic means. MAO-B inhibitors also inhibit the breakdown of other neurotransmitters such as norepinephrine and serotonin.
Note that MAO-B inhibitors have little relevance to the microparticles of the present invention.
(i)ドロキシドパ
ドノルエピネフリンは、ドパミンとは別の神経伝達物質である。しかし、ノルエピネフリンは、β水酸化酵素によってドパミンから合成されるため、ドパミンが減ると不足する。ノルエピネフリン前駆体であるドロキシドパの服薬により、ノルエピネフリンを補うことができ、ノルエピネフリンの不足に伴う足のすくみを改善することができる。
なお、ドロキシドパは、本発明の微小粒子とは関連性が低い。 (i) Droxidopa Donorepinephrine is a different neurotransmitter than dopamine. However, norepinephrine is synthesized from dopamine by β-hydroxylase, so it becomes insufficient when dopamine decreases. By taking droxidopa, which is a norepinephrine precursor, norepinephrine can be supplemented and the stiffness of the legs caused by norepinephrine deficiency can be improved.
Note that droxidopa has little relevance to the microparticles of the present invention.
ドノルエピネフリンは、ドパミンとは別の神経伝達物質である。しかし、ノルエピネフリンは、β水酸化酵素によってドパミンから合成されるため、ドパミンが減ると不足する。ノルエピネフリン前駆体であるドロキシドパの服薬により、ノルエピネフリンを補うことができ、ノルエピネフリンの不足に伴う足のすくみを改善することができる。
なお、ドロキシドパは、本発明の微小粒子とは関連性が低い。 (i) Droxidopa Donorepinephrine is a different neurotransmitter than dopamine. However, norepinephrine is synthesized from dopamine by β-hydroxylase, so it becomes insufficient when dopamine decreases. By taking droxidopa, which is a norepinephrine precursor, norepinephrine can be supplemented and the stiffness of the legs caused by norepinephrine deficiency can be improved.
Note that droxidopa has little relevance to the microparticles of the present invention.
<微小粒子の詳細>
本発明の微小粒子の有効成分であるmiRNAは、微小粒子に含まれる。
本発明の微小粒子は、例えば、歯髄由来幹細胞等の間葉系幹細胞からの分泌、出芽または分散などにより、歯髄由来幹細胞等から導き出され、細胞培養培地に浸出、放出または脱落するものである。微小粒子は、歯髄由来幹細胞等の培養上清に含まれることが好ましく、歯髄由来幹細胞の培養上清に由来する微小粒子であることがより好ましい。ただし、歯髄由来幹細胞の培養上清に由来する微小粒子は、必ずしも歯髄由来幹細胞の培養上清から取得する必要がない。例えば、歯髄由来幹細胞の内部の微小粒子を任意の方法で単離したものであっても、歯髄由来幹細胞の培養上清から単離できる微小粒子と同じものであれば、歯髄由来幹細胞の培養上清に由来する微小粒子と言える。
歯髄由来幹細胞等の培養上清に由来する微小粒子は、培養上清に含まれた状態で用いてもよく、または培養上清から精製した状態で用いてもよい。微小粒子が培養上清から精製された微小粒子であることが好ましい。
微小粒子の由来は、公知の方法で判別することができる。例えば、微小粒子は、J Stem Cell Res Ther (2018) 8:2に記載の方法で、歯髄由来幹細胞、脂肪由来幹細胞、骨髄由来幹細胞、臍帯由来幹細胞などのいずれの幹細胞に由来するか判別することができる。具体的には、微小粒子のmiRNAパターンに基づいて、それぞれの微小粒子の由来を判別することができる。 <Details of microparticles>
miRNA, which is an active ingredient of the microparticles of the present invention, is contained in the microparticles.
The microparticles of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells such as dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium. The microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably are microparticles derived from the culture supernatant of dental pulp-derived stem cells. However, the microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily need to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if the microparticles inside dental pulp-derived stem cells are isolated by any method, if they are the same as the microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, then It can be said that they are microparticles derived from water.
Microparticles derived from a culture supernatant such as dental pulp-derived stem cells may be used in a state contained in the culture supernatant, or may be used in a state purified from the culture supernatant. Preferably, the microparticles are microparticles purified from culture supernatant.
The origin of the microparticles can be determined by a known method. For example, it can be determined whether the microparticles are derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells using the method described in J Stem Cell Res Ther (2018) 8:2. I can do it. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticle.
本発明の微小粒子の有効成分であるmiRNAは、微小粒子に含まれる。
本発明の微小粒子は、例えば、歯髄由来幹細胞等の間葉系幹細胞からの分泌、出芽または分散などにより、歯髄由来幹細胞等から導き出され、細胞培養培地に浸出、放出または脱落するものである。微小粒子は、歯髄由来幹細胞等の培養上清に含まれることが好ましく、歯髄由来幹細胞の培養上清に由来する微小粒子であることがより好ましい。ただし、歯髄由来幹細胞の培養上清に由来する微小粒子は、必ずしも歯髄由来幹細胞の培養上清から取得する必要がない。例えば、歯髄由来幹細胞の内部の微小粒子を任意の方法で単離したものであっても、歯髄由来幹細胞の培養上清から単離できる微小粒子と同じものであれば、歯髄由来幹細胞の培養上清に由来する微小粒子と言える。
歯髄由来幹細胞等の培養上清に由来する微小粒子は、培養上清に含まれた状態で用いてもよく、または培養上清から精製した状態で用いてもよい。微小粒子が培養上清から精製された微小粒子であることが好ましい。
微小粒子の由来は、公知の方法で判別することができる。例えば、微小粒子は、J Stem Cell Res Ther (2018) 8:2に記載の方法で、歯髄由来幹細胞、脂肪由来幹細胞、骨髄由来幹細胞、臍帯由来幹細胞などのいずれの幹細胞に由来するか判別することができる。具体的には、微小粒子のmiRNAパターンに基づいて、それぞれの微小粒子の由来を判別することができる。 <Details of microparticles>
miRNA, which is an active ingredient of the microparticles of the present invention, is contained in the microparticles.
The microparticles of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells such as dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium. The microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably are microparticles derived from the culture supernatant of dental pulp-derived stem cells. However, the microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily need to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if the microparticles inside dental pulp-derived stem cells are isolated by any method, if they are the same as the microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, then It can be said that they are microparticles derived from water.
Microparticles derived from a culture supernatant such as dental pulp-derived stem cells may be used in a state contained in the culture supernatant, or may be used in a state purified from the culture supernatant. Preferably, the microparticles are microparticles purified from culture supernatant.
The origin of the microparticles can be determined by a known method. For example, it can be determined whether the microparticles are derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells using the method described in J Stem Cell Res Ther (2018) 8:2. I can do it. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticle.
(miRNA)
本発明では、微小粒子がパーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む。
本発明において、miRNA(MicroRNAs)は、例えば21~25塩基(ヌクレオチド)のRNA分子である。miRNAは、標的遺伝子(target;ターゲット)mRNAの分解または解読段階における抑制により遺伝子発現を調節できる。
本発明において、miRNAは、一本鎖(一量体)でもよいし、二本鎖(二量体)であってもよい。また、本発明において、miRNAは、Dicer等のリボヌクレアーゼにより切断された成熟型miRNAが好ましい。 (miRNA)
In the present invention, the microparticles contain miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
In the present invention, miRNA (MicroRNAs) is, for example, an RNA molecule of 21 to 25 bases (nucleotides). miRNAs can regulate gene expression by suppressing target gene (target) mRNA degradation or decoding steps.
In the present invention, miRNA may be single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, the miRNA is preferably a mature miRNA that has been cleaved with a ribonuclease such as Dicer.
本発明では、微小粒子がパーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む。
本発明において、miRNA(MicroRNAs)は、例えば21~25塩基(ヌクレオチド)のRNA分子である。miRNAは、標的遺伝子(target;ターゲット)mRNAの分解または解読段階における抑制により遺伝子発現を調節できる。
本発明において、miRNAは、一本鎖(一量体)でもよいし、二本鎖(二量体)であってもよい。また、本発明において、miRNAは、Dicer等のリボヌクレアーゼにより切断された成熟型miRNAが好ましい。 (miRNA)
In the present invention, the microparticles contain miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
In the present invention, miRNA (MicroRNAs) is, for example, an RNA molecule of 21 to 25 bases (nucleotides). miRNAs can regulate gene expression by suppressing target gene (target) mRNA degradation or decoding steps.
In the present invention, miRNA may be single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, the miRNA is preferably a mature miRNA that has been cleaved with a ribonuclease such as Dicer.
なお、hsa-miR-127-5pなどの本明細書に記載のmiRNAの配列は公知のデータベース(例えば、miRBase database)にアクセッション番号と関連づけられて登録されており、当業者であれば配列を一義的に定めることができる。例えば、hsa-miR-127-5pのアクセッション番号はMI0000472であり、miRBase databaseに配列が登録されている。以下、各miRNAのアクセッション番号は省略する。
ただし、本明細書におけるmiRNAは、hsa-miR-127-5pなどの成熟型miRNAに対して1~5個程度の塩基が異なるバリアントも含む。また、本明細書における各miRNAは、各miRNA(例えばhsa-miR-127-5p)の塩基配列と同一性を有する塩基配列からなるポリヌクレオチド、または、それらの相補的な塩基配列からなるポリヌクレオチドであって、本発明におけるmiRNAの機能を有するポリヌクレオチドを含む。「同一性」とは、比較する配列同士を適切にアライメントしたときの同一の程度であ
り、前記配列間のアミノ酸の正確な一致の出現率(%)を意味する。アライメントは、例えば、BLAST等の任意のアルゴリズムの利用により行うことができる。同一性は、例えば、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%または約99%である。同一性を有する塩基配列からなるポリヌクレオチドは、例えば、miRNAの塩基配列において、点変異、欠失および/または付加を有してもよい。前記点変異等の塩基数は、例えば、1~5個、1~3個、1~2個、または1個である。また、相補的な塩基配列からなるポリヌクレオチドは、例えば、miRNAの塩基配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、本発明におけるmiRNAの機能を有するポリヌクレオチドを含む。ストリンジェントな条件としては、特に限定されないが、例えば、特開2017-184642号公報の[0028]に記載の条件を挙げることができ、この公報の内容は参照して本明細書に組み込まれる。 Note that the sequences of miRNAs described herein, such as hsa-miR-127-5p, are registered in known databases (e.g., miRBase database) in association with accession numbers, and those skilled in the art will be able to identify the sequences. It can be defined uniquely. For example, the accession number of hsa-miR-127-5p is MI0000472, and the sequence is registered in miRBase database. Hereinafter, the accession number of each miRNA will be omitted.
However, miRNA in this specification also includes variants that differ by about 1 to 5 bases from mature miRNA such as hsa-miR-127-5p. In addition, each miRNA in this specification refers to a polynucleotide consisting of a base sequence having identity with the base sequence of each miRNA (for example, hsa-miR-127-5p), or a polynucleotide consisting of a complementary base sequence thereof. It includes a polynucleotide having the function of miRNA in the present invention. "Identity" refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the incidence (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST. Identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99%. Polynucleotides consisting of identical base sequences may have, for example, point mutations, deletions, and/or additions in the miRNA base sequence. The number of bases in the point mutation, etc. is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. In addition, the polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence of miRNA, and includes a polynucleotide having the function of miRNA in the present invention. . Stringent conditions are not particularly limited, and include, for example, the conditions described in [0028] of JP-A-2017-184642, the contents of which are incorporated herein by reference.
ただし、本明細書におけるmiRNAは、hsa-miR-127-5pなどの成熟型miRNAに対して1~5個程度の塩基が異なるバリアントも含む。また、本明細書における各miRNAは、各miRNA(例えばhsa-miR-127-5p)の塩基配列と同一性を有する塩基配列からなるポリヌクレオチド、または、それらの相補的な塩基配列からなるポリヌクレオチドであって、本発明におけるmiRNAの機能を有するポリヌクレオチドを含む。「同一性」とは、比較する配列同士を適切にアライメントしたときの同一の程度であ
り、前記配列間のアミノ酸の正確な一致の出現率(%)を意味する。アライメントは、例えば、BLAST等の任意のアルゴリズムの利用により行うことができる。同一性は、例えば、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%または約99%である。同一性を有する塩基配列からなるポリヌクレオチドは、例えば、miRNAの塩基配列において、点変異、欠失および/または付加を有してもよい。前記点変異等の塩基数は、例えば、1~5個、1~3個、1~2個、または1個である。また、相補的な塩基配列からなるポリヌクレオチドは、例えば、miRNAの塩基配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、本発明におけるmiRNAの機能を有するポリヌクレオチドを含む。ストリンジェントな条件としては、特に限定されないが、例えば、特開2017-184642号公報の[0028]に記載の条件を挙げることができ、この公報の内容は参照して本明細書に組み込まれる。 Note that the sequences of miRNAs described herein, such as hsa-miR-127-5p, are registered in known databases (e.g., miRBase database) in association with accession numbers, and those skilled in the art will be able to identify the sequences. It can be defined uniquely. For example, the accession number of hsa-miR-127-5p is MI0000472, and the sequence is registered in miRBase database. Hereinafter, the accession number of each miRNA will be omitted.
However, miRNA in this specification also includes variants that differ by about 1 to 5 bases from mature miRNA such as hsa-miR-127-5p. In addition, each miRNA in this specification refers to a polynucleotide consisting of a base sequence having identity with the base sequence of each miRNA (for example, hsa-miR-127-5p), or a polynucleotide consisting of a complementary base sequence thereof. It includes a polynucleotide having the function of miRNA in the present invention. "Identity" refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the incidence (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST. Identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99%. Polynucleotides consisting of identical base sequences may have, for example, point mutations, deletions, and/or additions in the miRNA base sequence. The number of bases in the point mutation, etc. is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. In addition, the polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence of miRNA, and includes a polynucleotide having the function of miRNA in the present invention. . Stringent conditions are not particularly limited, and include, for example, the conditions described in [0028] of JP-A-2017-184642, the contents of which are incorporated herein by reference.
本発明では、微小粒子が、歯髄由来幹細胞の培養上清よりも高い濃度で、パーキンソン病に関する遺伝子を標的遺伝子とするmiRNAを含むことが好ましい。
以下、微小粒子が含むmiRNAの好ましい態様を説明する。 In the present invention, it is preferable that the microparticles contain miRNA targeting a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
Hereinafter, preferred embodiments of miRNA contained in the microparticles will be described.
以下、微小粒子が含むmiRNAの好ましい態様を説明する。 In the present invention, it is preferable that the microparticles contain miRNA targeting a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
Hereinafter, preferred embodiments of miRNA contained in the microparticles will be described.
本発明では、微小粒子が下記パーキンソン病関連miRNA群のうち少なくとも1種類を含むことがより好ましい;
パーキンソン病関連miRNA群:
hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-212-5p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543。 In the present invention, it is more preferable that the microparticles contain at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
パーキンソン病関連miRNA群:
hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-212-5p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543。 In the present invention, it is more preferable that the microparticles contain at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
(1)SIRT2阻害剤
本発明では、微小粒子が、SIRT2阻害剤であり、かつパーキンソン病関連miRNA群のうちhsa-miR-212-5pを含むことが好ましい。
微小粒子は、hsa-miR-212-5pを、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、0.1以上含むことが好ましく、0.5以上含むことがより好ましく、1.0以上含むことが特に好ましい。 (1) SIRT2 inhibitor In the present invention, it is preferable that the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
The microparticles preferably contain hsa-miR-212-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 0.1 or more, more preferably 0.5 or more, and 1.0 It is particularly preferable to include the above.
本発明では、微小粒子が、SIRT2阻害剤であり、かつパーキンソン病関連miRNA群のうちhsa-miR-212-5pを含むことが好ましい。
微小粒子は、hsa-miR-212-5pを、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、0.1以上含むことが好ましく、0.5以上含むことがより好ましく、1.0以上含むことが特に好ましい。 (1) SIRT2 inhibitor In the present invention, it is preferable that the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
The microparticles preferably contain hsa-miR-212-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 0.1 or more, more preferably 0.5 or more, and 1.0 It is particularly preferable to include the above.
本発明の微小粒子は、脂肪由来幹細胞の培養上清から得られるエクソソームまたは臍帯由来幹細胞の培養上清から得られるエクソソームと比較して、hsa-miR-212-5pの発現量が1.1倍以上であることが好ましく、1.5倍以上であることがより好ましく、2倍以上であることが特に好ましい。
The microparticles of the present invention express 1.1 times more hsa-miR-212-5p than exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. It is preferably at least 1.5 times, more preferably at least 1.5 times, particularly preferably at least 2 times.
本発明の微小粒子がSIRT2阻害剤である場合、任意の細胞内におけるSIRT2遺伝子の発現を通常の(未処理の細胞の場合の)0.8倍以下に抑制できることが好ましく、0.6倍以下に抑制できることがより好ましく、0.4倍以下に抑制できることが特に好ましい。
When the microparticles of the present invention are SIRT2 inhibitors, it is preferable that the expression of the SIRT2 gene in any cell can be suppressed to 0.8 times or less, and preferably 0.6 times or less compared to normal (in untreated cells). More preferably, it can be suppressed to 0.4 times or less, and particularly preferably to 0.4 times or less.
(2)ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤
本発明では、微小粒子が、ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤であり、かつ、パーキンソン病関連miRNA群のうちhsa-miR-182-5pまたはhsa-miR-183-5pを含むことが好ましく、hsa-miR-182-5pおよびhsa-miR-183-5pを両方とも含むことがより好ましい。
微小粒子は、hsa-miR-182-5pまたはhsa-miR-183-5pを、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、それぞれ独立に4.0以上含むことが好ましく、10.0以上含むことがより好ましく、15.0以上含むことが特に好ましい。 (2) Inhibitor of degeneration or apoptosis of dopaminergic neurons In the present invention, the microparticles are inhibitors of degeneration or apoptosis of dopaminergic neurons, and hsa-miR-182 among Parkinson's disease-related miRNA group -5p or hsa-miR-183-5p, and more preferably both hsa-miR-182-5p and hsa-miR-183-5p.
It is preferable that the microparticles independently contain hsa-miR-182-5p or hsa-miR-183-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and 10. It is more preferable to contain 0 or more, and it is particularly preferable to contain 15.0 or more.
本発明では、微小粒子が、ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤であり、かつ、パーキンソン病関連miRNA群のうちhsa-miR-182-5pまたはhsa-miR-183-5pを含むことが好ましく、hsa-miR-182-5pおよびhsa-miR-183-5pを両方とも含むことがより好ましい。
微小粒子は、hsa-miR-182-5pまたはhsa-miR-183-5pを、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、それぞれ独立に4.0以上含むことが好ましく、10.0以上含むことがより好ましく、15.0以上含むことが特に好ましい。 (2) Inhibitor of degeneration or apoptosis of dopaminergic neurons In the present invention, the microparticles are inhibitors of degeneration or apoptosis of dopaminergic neurons, and hsa-miR-182 among Parkinson's disease-related miRNA group -5p or hsa-miR-183-5p, and more preferably both hsa-miR-182-5p and hsa-miR-183-5p.
It is preferable that the microparticles independently contain hsa-miR-182-5p or hsa-miR-183-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and 10. It is more preferable to contain 0 or more, and it is particularly preferable to contain 15.0 or more.
本発明では、脂肪由来幹細胞の培養上清から得られるエクソソームまたは臍帯由来幹細胞の培養上清から得られるエクソソームと比較して、hsa-miR-182-5pまたはhsa-miR-183-5pの発現量が1.1倍以上であることが好ましく、1.5倍以上であることがより好ましく、2倍以上であることが特に好ましい。
In the present invention, the expression level of hsa-miR-182-5p or hsa-miR-183-5p is compared with exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. is preferably 1.1 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more.
(3)パーキンソン病で発現が抑制されるmiRNAの補充
微小粒子が、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する場合、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543のうち少なくとも1種類を含むことが好ましい。さらにパーキンソン病関連miRNA群のうち、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pのうちいずれか1種類を含むことがより好ましく、hsa-miR-199a-3pを少なくとも含むことが特に好ましい。 (3) Supplementation of miRNAs whose expression is suppressed in Parkinson's disease If microparticles function as a supplement for miRNAs whose expression is downregulated in Parkinson's disease brain tissue, hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330- 5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR- It is preferable that at least one of 485-5p, hsa-miR-542-3p, and hsa-miR-543 is included. Furthermore, among the Parkinson's disease-related miRNA group, it may contain any one of hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p. More preferably, it is particularly preferred to contain at least hsa-miR-199a-3p.
微小粒子が、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAの補充として機能する場合、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543のうち少なくとも1種類を含むことが好ましい。さらにパーキンソン病関連miRNA群のうち、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pのうちいずれか1種類を含むことがより好ましく、hsa-miR-199a-3pを少なくとも含むことが特に好ましい。 (3) Supplementation of miRNAs whose expression is suppressed in Parkinson's disease If microparticles function as a supplement for miRNAs whose expression is downregulated in Parkinson's disease brain tissue, hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330- 5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR- It is preferable that at least one of 485-5p, hsa-miR-542-3p, and hsa-miR-543 is included. Furthermore, among the Parkinson's disease-related miRNA group, it may contain any one of hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p. More preferably, it is particularly preferred to contain at least hsa-miR-199a-3p.
微小粒子は、パーキンソン病の脳組織においてダウンレギュレーションされるmiRNAのうち少なくとも1種類を、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、4.0以上含むことが好ましく、10.0以上含むことがより好ましく、15.0以上含むことが特に好ましい。微小粒子は、IMOTAを用いた解析で得られるリードカウント数のLog2Ratioとして、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pをいずれも4.0以上含むことが好ましく、hsa-miR-199a-3pを15.0以上含むことが特に好ましい。
The microparticle preferably contains at least one type of miRNA that is down-regulated in Parkinson's disease brain tissue as a Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and preferably 10.0 or more. It is more preferable to include 15.0 or more, particularly preferably 15.0 or more. Microparticles are hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p as Log2 Ratio of read count number obtained by analysis using IMOTA. It is preferable that each of them contains 4.0 or more, and it is particularly preferable that hsa-miR-199a-3p contains 15.0 or more.
本発明の微小粒子は、脂肪由来幹細胞の培養上清から得られるエクソソームまたは臍帯由来幹細胞の培養上清から得られるエクソソームと比較して、hsa-miR-199a-3pの発現量が1.1倍以上であることが好ましく、1.5倍以上であることがより好ましく、2倍以上であることが特に好ましい。
The microparticles of the present invention express 1.1 times more hsa-miR-199a-3p than exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. It is preferably at least 1.5 times, more preferably at least 1.5 times, particularly preferably at least 2 times.
(miRNAの種類)
ここで、歯髄由来幹細胞の培養上清に由来する微小粒子には、約2600種類のsmall RNAが含まれる。このうちの約1800種類がmiRNAである。これらのmiRNAのうち、含有量が多いmiRNAは180~200種類である。歯髄由来幹細胞に由来する微小粒子における含有量が多いmiRNAは、脳神経疾患や眼疾患に治療に関連するマイクロRNAが多い点が特徴であり、従来知られておらず、本発明者が新たに見出した知見である。この特徴は、その他の間葉系幹細胞の微小粒子における含有量が多いmiRNAの種類とは大きく異なる。例えば、脂肪由来幹細胞の微小粒子や臍帯由来幹細胞の微小粒子における含有量が多いmiRNAには、脳神経疾患や眼疾患に治療に関連するマイクロRNAはほとんど含まれない。 (Type of miRNA)
Here, the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Approximately 1800 of these are miRNAs. Among these miRNAs, there are 180 to 200 types of miRNAs that are abundant. The high content of miRNA in microparticles derived from dental pulp-derived stem cells is characterized by the fact that there are many microRNAs related to treatment of cranial nerve diseases and eye diseases. This is my knowledge. This feature is significantly different from the types of miRNA that are contained in large amounts in other mesenchymal stem cell microparticles. For example, miRNAs that are abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to treatment of cranial nerve diseases or eye diseases.
ここで、歯髄由来幹細胞の培養上清に由来する微小粒子には、約2600種類のsmall RNAが含まれる。このうちの約1800種類がmiRNAである。これらのmiRNAのうち、含有量が多いmiRNAは180~200種類である。歯髄由来幹細胞に由来する微小粒子における含有量が多いmiRNAは、脳神経疾患や眼疾患に治療に関連するマイクロRNAが多い点が特徴であり、従来知られておらず、本発明者が新たに見出した知見である。この特徴は、その他の間葉系幹細胞の微小粒子における含有量が多いmiRNAの種類とは大きく異なる。例えば、脂肪由来幹細胞の微小粒子や臍帯由来幹細胞の微小粒子における含有量が多いmiRNAには、脳神経疾患や眼疾患に治療に関連するマイクロRNAはほとんど含まれない。 (Type of miRNA)
Here, the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Approximately 1800 of these are miRNAs. Among these miRNAs, there are 180 to 200 types of miRNAs that are abundant. The high content of miRNA in microparticles derived from dental pulp-derived stem cells is characterized by the fact that there are many microRNAs related to treatment of cranial nerve diseases and eye diseases. This is my knowledge. This feature is significantly different from the types of miRNA that are contained in large amounts in other mesenchymal stem cell microparticles. For example, miRNAs that are abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to treatment of cranial nerve diseases or eye diseases.
微小粒子は、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAとして5種類以上を含むことが好ましく、10種類以上を含むことがより好ましく、15種類以上を含むことが特に好ましく、20種類以上を含むことがより特に好ましい。また、微小粒子は、前述のパーキンソン病関連miRNA群のうち5種類以上を含むことが好ましく、10種類以上を含むことがより好ましく、15種類以上を含むことが特に好ましく、20種類以上を含むことがより特に好ましい。
The microparticles preferably contain 5 or more types of miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used for the treatment of Parkinson's disease, more preferably 10 or more types, and especially 15 or more types. Preferably, it is particularly preferable that 20 or more types are included. Furthermore, the microparticles preferably contain 5 or more types of the aforementioned Parkinson's disease-related miRNA group, more preferably 10 or more types, particularly preferably 15 or more types, and 20 or more types. is particularly preferred.
(微小粒子の種類)
微小粒子は、エクソソーム(exosome)、微小胞、膜粒子、膜小胞、エクトソーム(Ectosome)およびエキソベシクル(exovesicle)、またはマイクロベシクル(microvesicle)からなる群から選択される少なくとも1種類であることが好ましく、エクソソームであることがより好ましい。
微小粒子の直径は、10~1000nmであることが好ましく、30~500nmであることがより好ましく、50~150nmであることが特に好ましい。
また、微小粒子の表面には、CD9、CD63、CD81などのテトラスパニンという分子が存在することが望ましく、それはCD9単独、CD63単独、CD81単独でもよく、あるいはそれらの2つないしは3つのどの組み合わせでも良い。
以下、微小粒子として、エクソソームを用いる場合の好ましい態様を説明することがあるが、本発明に用いられる微小粒子はエクソソームに限定されない。 (Type of microparticles)
The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles. , more preferably exosomes.
The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, particularly preferably 50 to 150 nm.
Furthermore, it is desirable that molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles, and it may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three thereof. good.
Hereinafter, preferred embodiments of the case where exosomes are used as microparticles may be described, but the microparticles used in the present invention are not limited to exosomes.
微小粒子は、エクソソーム(exosome)、微小胞、膜粒子、膜小胞、エクトソーム(Ectosome)およびエキソベシクル(exovesicle)、またはマイクロベシクル(microvesicle)からなる群から選択される少なくとも1種類であることが好ましく、エクソソームであることがより好ましい。
微小粒子の直径は、10~1000nmであることが好ましく、30~500nmであることがより好ましく、50~150nmであることが特に好ましい。
また、微小粒子の表面には、CD9、CD63、CD81などのテトラスパニンという分子が存在することが望ましく、それはCD9単独、CD63単独、CD81単独でもよく、あるいはそれらの2つないしは3つのどの組み合わせでも良い。
以下、微小粒子として、エクソソームを用いる場合の好ましい態様を説明することがあるが、本発明に用いられる微小粒子はエクソソームに限定されない。 (Type of microparticles)
The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles. , more preferably exosomes.
The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, particularly preferably 50 to 150 nm.
Furthermore, it is desirable that molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles, and it may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three thereof. good.
Hereinafter, preferred embodiments of the case where exosomes are used as microparticles may be described, but the microparticles used in the present invention are not limited to exosomes.
エクソソームは、原形質膜との多胞体の融合時に細胞から放出される細胞外小胞であることが好ましい。
エクソソームの表面は、歯髄由来幹細胞の細胞膜由来の脂質およびタンパク質を含むことが好ましい。
エクソソームの内部には、核酸(マイクロRNA、メッセンジャーRNA、DNAなど)およびタンパク質など歯髄由来幹細胞の細胞内の物質を含むことが好ましい。
エクソソームは、ある細胞から別の細胞への遺伝情報の輸送による、細胞と細胞とのコミュニケーションのために使用されることが知られている。エクソソームは、容易に追跡可能であり、特異的な領域に標的化され得る。 Preferably, exosomes are extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane.
The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
The interior of the exosome preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.
エクソソームの表面は、歯髄由来幹細胞の細胞膜由来の脂質およびタンパク質を含むことが好ましい。
エクソソームの内部には、核酸(マイクロRNA、メッセンジャーRNA、DNAなど)およびタンパク質など歯髄由来幹細胞の細胞内の物質を含むことが好ましい。
エクソソームは、ある細胞から別の細胞への遺伝情報の輸送による、細胞と細胞とのコミュニケーションのために使用されることが知られている。エクソソームは、容易に追跡可能であり、特異的な領域に標的化され得る。 Preferably, exosomes are extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane.
The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
The interior of the exosome preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.
(微小粒子の含有量)
微小粒子組成物における、微小粒子の含有量は特に制限はない。微小粒子組成物は、微小粒子を0.5×108個以上含むことが好ましく、1.0×108個以上含むことがより好ましく、2.0×108個以上含むことが特に好ましく、2.5×108個以上含むことがより特に好ましく、1.0×109個以上含むことがさらにより特に好ましい。
また、微小粒子組成物における、微小粒子の含有濃度は特に制限はない。微小粒子組成物は、微小粒子を1.0×108個/mL以上含むことが好ましく、2.0×108個/mL以上含むことがより好ましく、4.0×108個/mL以上含むことが特に好ましく、5.0×108個/mL以上含むことがより特に好ましく、2.0×109個/mL以上含むことがさらにより特に好ましい。
本発明の微小粒子の好ましい態様は、微小粒子をこのように多量または高濃度で含むことにより、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAの量を高く維持できる。 (Content of fine particles)
The content of microparticles in the microparticle composition is not particularly limited. The microparticle composition preferably contains 0.5 x 10 8 or more microparticles, more preferably 1.0 x 10 8 or more, particularly preferably 2.0 x 10 8 or more, It is particularly preferable to contain 2.5×10 8 or more, and even more particularly preferably to contain 1.0×10 9 or more.
Further, there is no particular restriction on the concentration of fine particles contained in the fine particle composition. The microparticle composition preferably contains 1.0×10 8 particles/mL or more, more preferably 2.0×10 8 particles/mL or more, and 4.0×10 8 particles/mL or more. It is particularly preferable to contain 5.0×10 8 pieces/mL or more, and it is even more particularly preferable to contain 2.0×10 9 pieces/mL or more.
A preferred embodiment of the microparticles of the present invention can maintain a high amount of miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease by containing the microparticles in such a large amount or at a high concentration.
微小粒子組成物における、微小粒子の含有量は特に制限はない。微小粒子組成物は、微小粒子を0.5×108個以上含むことが好ましく、1.0×108個以上含むことがより好ましく、2.0×108個以上含むことが特に好ましく、2.5×108個以上含むことがより特に好ましく、1.0×109個以上含むことがさらにより特に好ましい。
また、微小粒子組成物における、微小粒子の含有濃度は特に制限はない。微小粒子組成物は、微小粒子を1.0×108個/mL以上含むことが好ましく、2.0×108個/mL以上含むことがより好ましく、4.0×108個/mL以上含むことが特に好ましく、5.0×108個/mL以上含むことがより特に好ましく、2.0×109個/mL以上含むことがさらにより特に好ましい。
本発明の微小粒子の好ましい態様は、微小粒子をこのように多量または高濃度で含むことにより、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAの量を高く維持できる。 (Content of fine particles)
The content of microparticles in the microparticle composition is not particularly limited. The microparticle composition preferably contains 0.5 x 10 8 or more microparticles, more preferably 1.0 x 10 8 or more, particularly preferably 2.0 x 10 8 or more, It is particularly preferable to contain 2.5×10 8 or more, and even more particularly preferably to contain 1.0×10 9 or more.
Further, there is no particular restriction on the concentration of fine particles contained in the fine particle composition. The microparticle composition preferably contains 1.0×10 8 particles/mL or more, more preferably 2.0×10 8 particles/mL or more, and 4.0×10 8 particles/mL or more. It is particularly preferable to contain 5.0×10 8 pieces/mL or more, and it is even more particularly preferable to contain 2.0×10 9 pieces/mL or more.
A preferred embodiment of the microparticles of the present invention can maintain a high amount of miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease by containing the microparticles in such a large amount or at a high concentration.
<その他の成分>
微小粒子組成物は、微小粒子の他に、投与する対象の動物の種類や目的に応じて、本発明の効果を損なわない範囲でその他の成分を含有していてもよい。その他の成分としては、栄養成分、抗生物質、サイトカイン、保護剤、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤などを挙げられる。
栄養成分としては、例えば、脂肪酸等、ビタミン等を挙げることができる。
抗生物質としては、例えば、ペニシリン、ストレプトマイシン、ゲンタマイシン等が挙げられる。
担体としては、薬学的に許容可能な担体として公知の材料を挙げることができる。
微小粒子組成物は、歯髄由来幹細胞の培養上清それ自体または微小粒子それ自体であってもよく、薬学的に許容可能な担体や賦形剤などをさらに含む医薬組成物であってもよい。医薬組成物の目的は、投与対象への微小粒子の投与を促進することである。 <Other ingredients>
The microparticle composition may contain other components in addition to the microparticles, depending on the type of animal to be administered and the purpose, to the extent that the effects of the present invention are not impaired. Other ingredients include nutritional ingredients, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, etc. It will be done.
Examples of nutritional components include fatty acids, vitamins, and the like.
Examples of antibiotics include penicillin, streptomycin, and gentamicin.
The carrier may include materials known as pharmaceutically acceptable carriers.
The microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, and the like. The purpose of the pharmaceutical composition is to facilitate administration of microparticles to the recipient.
微小粒子組成物は、微小粒子の他に、投与する対象の動物の種類や目的に応じて、本発明の効果を損なわない範囲でその他の成分を含有していてもよい。その他の成分としては、栄養成分、抗生物質、サイトカイン、保護剤、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤などを挙げられる。
栄養成分としては、例えば、脂肪酸等、ビタミン等を挙げることができる。
抗生物質としては、例えば、ペニシリン、ストレプトマイシン、ゲンタマイシン等が挙げられる。
担体としては、薬学的に許容可能な担体として公知の材料を挙げることができる。
微小粒子組成物は、歯髄由来幹細胞の培養上清それ自体または微小粒子それ自体であってもよく、薬学的に許容可能な担体や賦形剤などをさらに含む医薬組成物であってもよい。医薬組成物の目的は、投与対象への微小粒子の投与を促進することである。 <Other ingredients>
The microparticle composition may contain other components in addition to the microparticles, depending on the type of animal to be administered and the purpose, to the extent that the effects of the present invention are not impaired. Other ingredients include nutritional ingredients, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, etc. It will be done.
Examples of nutritional components include fatty acids, vitamins, and the like.
Examples of antibiotics include penicillin, streptomycin, and gentamicin.
The carrier may include materials known as pharmaceutically acceptable carriers.
The microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, and the like. The purpose of the pharmaceutical composition is to facilitate administration of microparticles to the recipient.
薬学的に許容可能な担体は、投与対象に対して顕著な刺激性を引き起こさず、投与される化合物の生物学的活性および特性を抑止しない担体(希釈剤を含む)であることが好ましい。担体の例は、プロピレングリコール;(生理)食塩水;エマルション;緩衝液;培地、例えばDMEMまたはRPMIなど;フリーラジカルを除去する成分を含有する低温保存培地である。
Preferably, the pharmaceutically acceptable carrier is a carrier (including diluents) that does not cause significant irritation to the administered subject and does not inhibit the biological activity and properties of the administered compound. Examples of carriers are propylene glycol; (physiological) saline; emulsions; buffers; media, such as DMEM or RPMI; cryopreservation media containing components that scavenge free radicals.
微小粒子組成物は、従来公知のパーキンソン病の治療薬の有効成分を含んでいてもよい。当業者であれば用途や投与対象などにあわせて適切に変更することができる。
The microparticle composition may contain an active ingredient of a conventionally known therapeutic agent for Parkinson's disease. Those skilled in the art can make appropriate changes depending on the intended use, administration target, etc.
一方、微小粒子組成物は、所定の物質を含まないことが好ましい。
例えば、微小粒子組成物は、歯髄由来幹細胞を含まないことが好ましい。
また、微小粒子組成物は、MCP-1を含まないことが好ましい。ただし、MCP-1以外のサイトカインを含んでいてもよい。その他のサイトカインとしては、特開2018-023343号公報の[0014]~[0020]に記載のもの等が挙げられる。
また、微小粒子組成物は、シグレック9を含まないことが好ましい。ただし、シグレック9以外のその他のシアル酸結合免疫グロブリン様レクチンを含んでいてもよい。
なお、微小粒子組成物は、血清(ウシ胎仔血清、ヒト血清、羊血清等)を実質的に含まないことが好ましい。また、微小粒子組成物は、Knockout serum replacement(KSR)などの従来の血清代替物を実質的に含まないことが好ましい。
微小粒子組成物は、上記したその他の成分の含有量(固形分量)がいずれも1質量%以下であることが好ましく、0.1質量%以下であることがより好ましく、0.01質量%以下であることが特に好ましい。 On the other hand, it is preferable that the microparticle composition does not contain the predetermined substance.
For example, the microparticle composition preferably does not contain dental pulp-derived stem cells.
Furthermore, the microparticle composition preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Other cytokines include those described in [0014] to [0020] of JP 2018-023343A.
Moreover, it is preferable that the microparticle composition does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.
Note that the microparticle composition preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.). It is also preferred that the microparticle composition is substantially free of conventional serum replacements, such as Knockout serum replacement (KSR).
In the microparticle composition, the content (solid content) of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and 0.01% by mass or less. It is particularly preferable that
例えば、微小粒子組成物は、歯髄由来幹細胞を含まないことが好ましい。
また、微小粒子組成物は、MCP-1を含まないことが好ましい。ただし、MCP-1以外のサイトカインを含んでいてもよい。その他のサイトカインとしては、特開2018-023343号公報の[0014]~[0020]に記載のもの等が挙げられる。
また、微小粒子組成物は、シグレック9を含まないことが好ましい。ただし、シグレック9以外のその他のシアル酸結合免疫グロブリン様レクチンを含んでいてもよい。
なお、微小粒子組成物は、血清(ウシ胎仔血清、ヒト血清、羊血清等)を実質的に含まないことが好ましい。また、微小粒子組成物は、Knockout serum replacement(KSR)などの従来の血清代替物を実質的に含まないことが好ましい。
微小粒子組成物は、上記したその他の成分の含有量(固形分量)がいずれも1質量%以下であることが好ましく、0.1質量%以下であることがより好ましく、0.01質量%以下であることが特に好ましい。 On the other hand, it is preferable that the microparticle composition does not contain the predetermined substance.
For example, the microparticle composition preferably does not contain dental pulp-derived stem cells.
Furthermore, the microparticle composition preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Other cytokines include those described in [0014] to [0020] of JP 2018-023343A.
Moreover, it is preferable that the microparticle composition does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.
Note that the microparticle composition preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.). It is also preferred that the microparticle composition is substantially free of conventional serum replacements, such as Knockout serum replacement (KSR).
In the microparticle composition, the content (solid content) of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and 0.01% by mass or less. It is particularly preferable that
<微小粒子の製造方法>
微小粒子の製造方法は、特に制限はない。
歯髄由来幹細胞等の培養上清を調製し、続けて歯髄由来幹細胞の培養上清から微小粒子を精製して、本発明の微小粒子を調製してもよい。あるいは、商業的に購入して入手した歯髄由来幹細胞の培養上清から微小粒子を精製して、本発明の微小粒子を調製してもよい。さらには、廃棄処理されていた歯髄由来幹細胞の培養上清を含む組成物を譲り受けて(またはその組成物を適宜精製して)、そこから微小粒子を精製して、本発明の微小粒子を調製してもよい。 <Method for manufacturing microparticles>
There are no particular limitations on the method for producing microparticles.
The microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells, etc., and then purifying microparticles from the culture supernatant of dental pulp-derived stem cells. Alternatively, the microparticles of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells. Furthermore, the microparticles of the present invention are prepared by receiving a composition containing the culture supernatant of dental pulp-derived stem cells that had been disposed of (or purifying the composition as appropriate) and refining the microparticles therefrom. You may.
微小粒子の製造方法は、特に制限はない。
歯髄由来幹細胞等の培養上清を調製し、続けて歯髄由来幹細胞の培養上清から微小粒子を精製して、本発明の微小粒子を調製してもよい。あるいは、商業的に購入して入手した歯髄由来幹細胞の培養上清から微小粒子を精製して、本発明の微小粒子を調製してもよい。さらには、廃棄処理されていた歯髄由来幹細胞の培養上清を含む組成物を譲り受けて(またはその組成物を適宜精製して)、そこから微小粒子を精製して、本発明の微小粒子を調製してもよい。 <Method for manufacturing microparticles>
There are no particular limitations on the method for producing microparticles.
The microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells, etc., and then purifying microparticles from the culture supernatant of dental pulp-derived stem cells. Alternatively, the microparticles of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells. Furthermore, the microparticles of the present invention are prepared by receiving a composition containing the culture supernatant of dental pulp-derived stem cells that had been disposed of (or purifying the composition as appropriate) and refining the microparticles therefrom. You may.
(歯髄由来幹細胞等の培養上清の調製方法)
歯髄由来幹細胞等の培養上清は、特に制限はない。
歯髄由来幹細胞等の培養上清は、血清を実質的に含まないことが好ましい。例えば、歯髄由来幹細胞等の培養上清は、血清の含有量が1質量%以下であることが好ましく、0.1質量%以下であることがより好ましく、0.01質量%以下であることが特に好ましい。 (Method for preparing culture supernatant of dental pulp-derived stem cells, etc.)
There are no particular limitations on the culture supernatant of dental pulp-derived stem cells or the like.
It is preferable that the culture supernatant of dental pulp-derived stem cells and the like is substantially free of serum. For example, the serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and preferably 0.01% by mass or less. Particularly preferred.
歯髄由来幹細胞等の培養上清は、特に制限はない。
歯髄由来幹細胞等の培養上清は、血清を実質的に含まないことが好ましい。例えば、歯髄由来幹細胞等の培養上清は、血清の含有量が1質量%以下であることが好ましく、0.1質量%以下であることがより好ましく、0.01質量%以下であることが特に好ましい。 (Method for preparing culture supernatant of dental pulp-derived stem cells, etc.)
There are no particular limitations on the culture supernatant of dental pulp-derived stem cells or the like.
It is preferable that the culture supernatant of dental pulp-derived stem cells and the like is substantially free of serum. For example, the serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and preferably 0.01% by mass or less. Particularly preferred.
歯髄由来幹細胞は、ヒト由来であっても、ヒト以外の動物由来であってもよい。ヒト以外の動物としては、後述する本発明の微小粒子を投与する対象の動物(生物種)と同様のものを挙げることができ、哺乳動物が好ましい。
Dental pulp-derived stem cells may be derived from humans or animals other than humans. Examples of animals other than humans include those similar to the animals (species) to which the microparticles of the present invention are administered, which will be described later, and mammals are preferred.
培養上清に用いられる歯髄由来幹細胞としては、特に制限はない。脱落乳歯歯髄幹細胞(stem cells from exfoliated deciduous teeth)や、その他の方法で入手される乳歯歯髄幹細胞や、永久歯歯髄幹細胞(dental pulp stem cells;DPSC)を用いることができる。ヒト乳歯歯髄幹細胞やヒト永久歯歯髄幹細胞の他、ブタ乳歯歯髄幹細胞などのヒト以外の動物由来の歯髄由来幹細胞を用いることができる。
歯髄由来幹細胞は、エクソソームに加え、血管内皮増殖因子(VEGF)、肝細胞増殖因子(HGF)、インシュリン様成長因子(IGF)、血小板由来成長因子(PDGF)、形質転換成長因子-ベータ(TGF-β)-1および-3、TGF-α、KGF、HBEGF、SPARC、その他の成長因子、ケモカイン等の種々のサイトカインを産生し得る。また、その他の多くの生理活性物質を産生し得る。
本発明では、歯髄由来幹細胞の培養上清に用いられる歯髄由来幹細胞が、多くのタンパク質が含まれる歯髄由来幹細胞であることが特に好ましく、乳歯歯髄幹細胞を用いることが好ましい。すなわち、本発明では、乳歯歯髄幹細胞の培養上清を用いることが好ましい。 There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, dental pulp stem cells from deciduous teeth obtained by other methods, and dental pulp stem cells (DPSCs) from permanent teeth can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, dental pulp-derived stem cells derived from animals other than humans, such as pig deciduous tooth pulp stem cells, can be used.
In addition to exosomes, dental pulp-derived stem cells contain vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-). β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines. It can also produce many other physiologically active substances.
In the present invention, it is particularly preferable that the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain many proteins, and it is preferable to use dental pulp stem cells of deciduous teeth. That is, in the present invention, it is preferable to use a culture supernatant of deciduous tooth pulp stem cells.
歯髄由来幹細胞は、エクソソームに加え、血管内皮増殖因子(VEGF)、肝細胞増殖因子(HGF)、インシュリン様成長因子(IGF)、血小板由来成長因子(PDGF)、形質転換成長因子-ベータ(TGF-β)-1および-3、TGF-α、KGF、HBEGF、SPARC、その他の成長因子、ケモカイン等の種々のサイトカインを産生し得る。また、その他の多くの生理活性物質を産生し得る。
本発明では、歯髄由来幹細胞の培養上清に用いられる歯髄由来幹細胞が、多くのタンパク質が含まれる歯髄由来幹細胞であることが特に好ましく、乳歯歯髄幹細胞を用いることが好ましい。すなわち、本発明では、乳歯歯髄幹細胞の培養上清を用いることが好ましい。 There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, dental pulp stem cells from deciduous teeth obtained by other methods, and dental pulp stem cells (DPSCs) from permanent teeth can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, dental pulp-derived stem cells derived from animals other than humans, such as pig deciduous tooth pulp stem cells, can be used.
In addition to exosomes, dental pulp-derived stem cells contain vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-). β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines. It can also produce many other physiologically active substances.
In the present invention, it is particularly preferable that the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain many proteins, and it is preferable to use dental pulp stem cells of deciduous teeth. That is, in the present invention, it is preferable to use a culture supernatant of deciduous tooth pulp stem cells.
本発明に用いられる歯髄由来幹細胞は、目的の処置を達成することができれば、天然のものであってもよく、遺伝子改変したものであってもよい。
特に本発明では、歯髄由来幹細胞の不死化幹細胞を用いることができる。実質的に無限増殖が可能な不死化幹細胞を用いることで、幹細胞の培養上清中に含まれる生体因子の量と組成を、長期間にわたって安定させることができる。歯髄由来幹細胞の不死化幹細胞としては、特に制限はない。不死化幹細胞は、癌化していない不死化幹細胞であることが好ましい。歯髄由来幹細胞の不死化幹細胞は、歯髄由来幹細胞に、以下の低分子化合物(阻害剤)を単独または組み合わせて添加して培養することにより、調製することができる。
TGFβ受容体阻害薬としては、トランスフォーミング増殖因子(TGF)β受容体の機能を阻害する作用を有するものであれば特に限定されることはなく、例えば、2-(5-ベンゾ[1,3]ジオキソール-4-イル-2-tert-ブチル-1H-イミダゾール-4-イル)-6-メチルピリジン、3-(6-メチルピリジン-2-イル)-4-(4-キノリル)-1-フェニルチオカルバモイル-1H-ピラゾール(A-83-01)、2-[(5-クロロ-2-フルオロフェニル)プテリジン-4-イル]ピリジン-4-イルアミン(SD-208)、3-[(ピリジン-2-イル)-4-(4-キノニル)]-1H-ピラゾール、2-(3-(6-メチルピリジン-2-イル)-1H-ピラゾール-4-イル)-1,5-ナフチリジン(以上、メルク社)、SB431542(シグマアルドリッチ社)などが挙げられる。好ましくはA-83-01が挙げられる。
ROCK阻害薬としては、Rho結合キナーゼの機能を阻害する作用を有するものであれば特に限定されない。ROCK阻害薬としては、例えば、GSK269962A(Axonmedchem社)、Fasudil hydrochloride(Tocris Bioscience社)、Y-27632、H-1152(以上、富士フイルム和光純薬株式会社)などが挙げられる。好ましくはY-27632が挙げられる。
GSK3阻害薬としては、GSK-3(Glycogen synthase kinase 3,グリコーゲン合成酵素3)を阻害するものであれば特に限定されることはなく、A 1070722、BIO、BIO-acetoxime(以上、TOCRIS社)などが挙げられる。
MEK阻害薬としては、MEK(MAP kinase-ERK kinase)の機能を阻害する作用を有するものであれば特に限定されることはなく、例えば、AZD6244、CI-1040(PD184352)、PD0325901、RDEA119(BAY86-9766)、SL327、U0126-EtOH(以上、Selleck社)、PD98059、U0124、U0125(以上、コスモ・バイオ株式会社)などが挙げられる。 The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved.
In particular, in the present invention, immortalized stem cells derived from dental pulp can be used. By using immortalized stem cells that can proliferate virtually indefinitely, the amount and composition of biological factors contained in the culture supernatant of stem cells can be stabilized over a long period of time. There are no particular restrictions on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably immortalized stem cells that have not become cancerous. Immortalized stem cells of dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
The TGFβ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the transforming growth factor (TGF)β receptor, and for example, 2-(5-benzo[1,3 ] Dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1- Phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridine -2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine( Merck & Co.), SB431542 (Sigma-Aldrich), and the like. Preferred is A-83-01.
The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-binding kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (Fujifilm Wako Pure Chemical Industries, Ltd.). Preferred is Y-27632.
GSK3 inhibitors are not particularly limited as long as they inhibit GSK-3 (Glycogen Synthase Kinase 3), and include A 1070722, BIO, BIO-acetoxime (TOCRIS), etc. can be mentioned.
MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), such as AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86 -9766), SL327, U0126-EtOH (all sold by Selleck), PD98059, U0124, and U0125 (all sold by Cosmo Bio Inc.).
特に本発明では、歯髄由来幹細胞の不死化幹細胞を用いることができる。実質的に無限増殖が可能な不死化幹細胞を用いることで、幹細胞の培養上清中に含まれる生体因子の量と組成を、長期間にわたって安定させることができる。歯髄由来幹細胞の不死化幹細胞としては、特に制限はない。不死化幹細胞は、癌化していない不死化幹細胞であることが好ましい。歯髄由来幹細胞の不死化幹細胞は、歯髄由来幹細胞に、以下の低分子化合物(阻害剤)を単独または組み合わせて添加して培養することにより、調製することができる。
TGFβ受容体阻害薬としては、トランスフォーミング増殖因子(TGF)β受容体の機能を阻害する作用を有するものであれば特に限定されることはなく、例えば、2-(5-ベンゾ[1,3]ジオキソール-4-イル-2-tert-ブチル-1H-イミダゾール-4-イル)-6-メチルピリジン、3-(6-メチルピリジン-2-イル)-4-(4-キノリル)-1-フェニルチオカルバモイル-1H-ピラゾール(A-83-01)、2-[(5-クロロ-2-フルオロフェニル)プテリジン-4-イル]ピリジン-4-イルアミン(SD-208)、3-[(ピリジン-2-イル)-4-(4-キノニル)]-1H-ピラゾール、2-(3-(6-メチルピリジン-2-イル)-1H-ピラゾール-4-イル)-1,5-ナフチリジン(以上、メルク社)、SB431542(シグマアルドリッチ社)などが挙げられる。好ましくはA-83-01が挙げられる。
ROCK阻害薬としては、Rho結合キナーゼの機能を阻害する作用を有するものであれば特に限定されない。ROCK阻害薬としては、例えば、GSK269962A(Axonmedchem社)、Fasudil hydrochloride(Tocris Bioscience社)、Y-27632、H-1152(以上、富士フイルム和光純薬株式会社)などが挙げられる。好ましくはY-27632が挙げられる。
GSK3阻害薬としては、GSK-3(Glycogen synthase kinase 3,グリコーゲン合成酵素3)を阻害するものであれば特に限定されることはなく、A 1070722、BIO、BIO-acetoxime(以上、TOCRIS社)などが挙げられる。
MEK阻害薬としては、MEK(MAP kinase-ERK kinase)の機能を阻害する作用を有するものであれば特に限定されることはなく、例えば、AZD6244、CI-1040(PD184352)、PD0325901、RDEA119(BAY86-9766)、SL327、U0126-EtOH(以上、Selleck社)、PD98059、U0124、U0125(以上、コスモ・バイオ株式会社)などが挙げられる。 The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved.
In particular, in the present invention, immortalized stem cells derived from dental pulp can be used. By using immortalized stem cells that can proliferate virtually indefinitely, the amount and composition of biological factors contained in the culture supernatant of stem cells can be stabilized over a long period of time. There are no particular restrictions on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably immortalized stem cells that have not become cancerous. Immortalized stem cells of dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
The TGFβ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the transforming growth factor (TGF)β receptor, and for example, 2-(5-benzo[1,3 ] Dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1- Phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridine -2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine( Merck & Co.), SB431542 (Sigma-Aldrich), and the like. Preferred is A-83-01.
The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-binding kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (Fujifilm Wako Pure Chemical Industries, Ltd.). Preferred is Y-27632.
GSK3 inhibitors are not particularly limited as long as they inhibit GSK-3 (Glycogen Synthase Kinase 3), and include A 1070722, BIO, BIO-acetoxime (TOCRIS), etc. can be mentioned.
MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), such as AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86 -9766), SL327, U0126-EtOH (all sold by Selleck), PD98059, U0124, and U0125 (all sold by Cosmo Bio Inc.).
本発明の微小粒子を再生医療に用いる場合、再生医療等安全性確保法の要請から、歯髄由来幹細胞またはこれらの不死化幹細胞の培養上清や、それに由来する微小粒子を含む組成物は、歯髄由来幹細胞等以外のその他の体性幹細胞を含有しない態様とする。微小粒子組成物は、歯髄由来幹細胞等以外の間葉系幹細胞やその他の体性幹細胞を含有していてもよいが、含有しないことが好ましい。
間葉系幹細胞以外のその他の体性幹細胞の例としては、真皮系、消化系、骨髄系、神経系等に由来する幹細胞が含まれるが、これらに限定されるものではない。真皮系の体性幹細胞の例としては、上皮幹細胞、毛包幹細胞等が含まれる。消化系の体性幹細胞の例としては膵臓(全般の)幹細胞、肝幹細胞等が含まれる。(間葉系幹細胞以外の)骨髄系の体性幹細胞の例としては、造血幹細胞等が含まれる。神経系の体性幹細胞の例としては、神経幹細胞、網膜幹細胞等が含まれる。
微小粒子組成物は、体性幹細胞以外の幹細胞を含有していてもよいが、含有しないことが好ましい。体性幹細胞以外の幹細胞としては、胚性幹細胞(ES細胞)、誘導多能性幹細胞(iPS細胞)、胚性癌腫細胞(EC細胞)が含まれる。 When using the microparticles of the present invention in regenerative medicine, in accordance with the requirements of the Regenerative Medicine Safety Act, dental pulp-derived stem cells, culture supernatants of these immortalized stem cells, and compositions containing microparticles derived therefrom must be This embodiment does not contain any other somatic stem cells other than derived stem cells. Although the microparticle composition may contain mesenchymal stem cells and other somatic stem cells other than dental pulp-derived stem cells, it is preferable not to contain them.
Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like. Examples of somatic stem cells of the digestive system include pancreatic (general) stem cells, hepatic stem cells, and the like. Examples of myeloid somatic stem cells (other than mesenchymal stem cells) include hematopoietic stem cells and the like. Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, and the like.
The microparticle composition may contain stem cells other than somatic stem cells, but preferably does not contain them. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).
間葉系幹細胞以外のその他の体性幹細胞の例としては、真皮系、消化系、骨髄系、神経系等に由来する幹細胞が含まれるが、これらに限定されるものではない。真皮系の体性幹細胞の例としては、上皮幹細胞、毛包幹細胞等が含まれる。消化系の体性幹細胞の例としては膵臓(全般の)幹細胞、肝幹細胞等が含まれる。(間葉系幹細胞以外の)骨髄系の体性幹細胞の例としては、造血幹細胞等が含まれる。神経系の体性幹細胞の例としては、神経幹細胞、網膜幹細胞等が含まれる。
微小粒子組成物は、体性幹細胞以外の幹細胞を含有していてもよいが、含有しないことが好ましい。体性幹細胞以外の幹細胞としては、胚性幹細胞(ES細胞)、誘導多能性幹細胞(iPS細胞)、胚性癌腫細胞(EC細胞)が含まれる。 When using the microparticles of the present invention in regenerative medicine, in accordance with the requirements of the Regenerative Medicine Safety Act, dental pulp-derived stem cells, culture supernatants of these immortalized stem cells, and compositions containing microparticles derived therefrom must be This embodiment does not contain any other somatic stem cells other than derived stem cells. Although the microparticle composition may contain mesenchymal stem cells and other somatic stem cells other than dental pulp-derived stem cells, it is preferable not to contain them.
Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like. Examples of somatic stem cells of the digestive system include pancreatic (general) stem cells, hepatic stem cells, and the like. Examples of myeloid somatic stem cells (other than mesenchymal stem cells) include hematopoietic stem cells and the like. Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, and the like.
The microparticle composition may contain stem cells other than somatic stem cells, but preferably does not contain them. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).
歯髄由来幹細胞またはこの不死化幹細胞の培養上清の調製方法としては特に制限はなく、従来の方法を用いることができる。
歯髄由来幹細胞等の培養上清は、歯髄由来幹細胞を培養して得られる培養液である。例えば歯髄由来幹細胞の培養後に細胞成分を分離除去することによって、本発明に使用可能な培養上清を得ることができる。各種処理(例えば、遠心処理、濃縮、溶媒の置換、透析、凍結、乾燥、凍結乾燥、希釈、脱塩、保存等)を適宜施した培養上清を用いることにしてもよい。 There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used.
A culture supernatant such as dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing dental pulp-derived stem cells. A culture supernatant that has been appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may be used.
歯髄由来幹細胞等の培養上清は、歯髄由来幹細胞を培養して得られる培養液である。例えば歯髄由来幹細胞の培養後に細胞成分を分離除去することによって、本発明に使用可能な培養上清を得ることができる。各種処理(例えば、遠心処理、濃縮、溶媒の置換、透析、凍結、乾燥、凍結乾燥、希釈、脱塩、保存等)を適宜施した培養上清を用いることにしてもよい。 There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used.
A culture supernatant such as dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing dental pulp-derived stem cells. A culture supernatant that has been appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may be used.
歯髄由来幹細胞の培養上清を得るための歯髄由来幹細胞は、常法により選別可能であり、細胞の大きさや形態に基づいて、または接着性細胞として選別可能である。脱落した乳歯や永久歯から採取した歯髄細胞から、接着性細胞またはその継代細胞として選別することができる。歯髄由来幹細胞の培養上清には、選別された幹細胞を培養して得られた培養上清を用いることができる。
Dental pulp-derived stem cells for obtaining a culture supernatant of dental pulp-derived stem cells can be selected by conventional methods, and can be selected based on cell size and morphology, or as adherent cells. Dental pulp cells collected from fallen baby teeth or permanent teeth can be sorted as adherent cells or their successor cells. As the culture supernatant of dental pulp-derived stem cells, a culture supernatant obtained by culturing selected stem cells can be used.
なお、「歯髄由来幹細胞等の培養上清」は、歯髄由来幹細胞等を培養して得られる細胞そのものを含まない培養液であることが好ましい。本発明で用いる歯髄由来幹細胞の培養上清は、その一態様では全体としても細胞(細胞の種類は問わない)を含まないことが好ましい。当該態様の組成物はこの特徴によって、歯髄由来幹細胞自体は当然のこと、歯髄由来幹細胞を含む各種組成物と明確に区別される。この態様の典型例は、歯髄由来幹細胞を含まず、歯髄由来幹細胞の培養上清のみで構成された組成物である。
本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞および大人歯髄由来幹細胞の両方の培養上清を含んでいてもよい。本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞の培養上清を有効成分として含むことが好ましく、50質量%以上含むことがより好ましく、90質量%以上含むことが好ましい。本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞の培養上清のみで構成された組成物であることがより特に好ましい。 The "culture supernatant of dental pulp-derived stem cells, etc." is preferably a culture solution that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc. In one embodiment, the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain any cells (regardless of the type of cells) as a whole. Due to this feature, the composition of this embodiment is clearly distinguished from dental pulp-derived stem cells themselves as well as from various compositions containing dental pulp-derived stem cells. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of a culture supernatant of dental pulp-derived stem cells.
The culture supernatant of dental pulp-derived stem cells used in the present invention may contain culture supernatants of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of deciduous dental pulp-derived stem cells as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of dental pulp-derived stem cells of deciduous teeth.
本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞および大人歯髄由来幹細胞の両方の培養上清を含んでいてもよい。本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞の培養上清を有効成分として含むことが好ましく、50質量%以上含むことがより好ましく、90質量%以上含むことが好ましい。本発明で用いる歯髄由来幹細胞の培養上清は、乳歯歯髄由来幹細胞の培養上清のみで構成された組成物であることがより特に好ましい。 The "culture supernatant of dental pulp-derived stem cells, etc." is preferably a culture solution that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc. In one embodiment, the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain any cells (regardless of the type of cells) as a whole. Due to this feature, the composition of this embodiment is clearly distinguished from dental pulp-derived stem cells themselves as well as from various compositions containing dental pulp-derived stem cells. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of a culture supernatant of dental pulp-derived stem cells.
The culture supernatant of dental pulp-derived stem cells used in the present invention may contain culture supernatants of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of deciduous dental pulp-derived stem cells as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of dental pulp-derived stem cells of deciduous teeth.
培養上清を得るための歯髄由来幹細胞の培養液には基本培地、或いは基本培地に血清等を添加したもの等を使用可能である。なお、基本培地としてはダルベッコ改変イーグル培地(DMEM)の他、イスコフ改変ダルベッコ培地(IMDM)(GIBCO社等)、ハムF12培地(HamF12)(SIGMA社、GIBCO社等)、RPMI1640培地等を用いることができる。また、培地に添加可能な成分の例として、血清(ウシ胎仔血清、ヒト血清、羊血清等)、血清代替物(Knockout serum replacement(KSR)など)、ウシ血清アルブミン(BSA)、抗生物質、各種ビタミン、各種ミネラルを挙げることができる。
但し、血清を含まない「歯髄由来幹細胞の培養上清」を調製するためには、全過程を通して或いは最後または最後から数回の継代培養についは無血清培地を使用するとよい。例えば、血清を含まない培地(無血清培地)で歯髄由来幹細胞を培養することによって、血清を含まない歯髄由来幹細胞の培養上清を調製することができる。1回または複数回の継代培養を行うことにし、最後または最後から数回の継代培養を無血清培地で培養することによっても、血清を含まない歯髄由来幹細胞等の培養上清を得ることができる。一方、回収した培養上清から、透析やカラムによる溶媒置換などを利用して血清を除去することによっても、血清を含まない歯髄由来幹細胞の培養上清を得ることができる。 As a culture solution for dental pulp-derived stem cells to obtain a culture supernatant, a basic medium or a basic medium to which serum or the like is added can be used. In addition to Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12, SIGMA, GIBCO, etc.), RPMI1640 medium, etc. may be used as the basic medium. I can do it. In addition, examples of components that can be added to the culture medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.
However, in order to prepare a "culture supernatant of dental pulp-derived stem cells" that does not contain serum, a serum-free medium may be used throughout the entire process or during the last or several subcultures from the end. For example, by culturing dental pulp-derived stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant of dental pulp-derived stem cells can be prepared. It is also possible to obtain serum-free culture supernatant of dental pulp-derived stem cells, etc. by performing one or more subcultures and culturing the last or several subcultures in a serum-free medium. I can do it. On the other hand, a serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.
但し、血清を含まない「歯髄由来幹細胞の培養上清」を調製するためには、全過程を通して或いは最後または最後から数回の継代培養についは無血清培地を使用するとよい。例えば、血清を含まない培地(無血清培地)で歯髄由来幹細胞を培養することによって、血清を含まない歯髄由来幹細胞の培養上清を調製することができる。1回または複数回の継代培養を行うことにし、最後または最後から数回の継代培養を無血清培地で培養することによっても、血清を含まない歯髄由来幹細胞等の培養上清を得ることができる。一方、回収した培養上清から、透析やカラムによる溶媒置換などを利用して血清を除去することによっても、血清を含まない歯髄由来幹細胞の培養上清を得ることができる。 As a culture solution for dental pulp-derived stem cells to obtain a culture supernatant, a basic medium or a basic medium to which serum or the like is added can be used. In addition to Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12, SIGMA, GIBCO, etc.), RPMI1640 medium, etc. may be used as the basic medium. I can do it. In addition, examples of components that can be added to the culture medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.
However, in order to prepare a "culture supernatant of dental pulp-derived stem cells" that does not contain serum, a serum-free medium may be used throughout the entire process or during the last or several subcultures from the end. For example, by culturing dental pulp-derived stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant of dental pulp-derived stem cells can be prepared. It is also possible to obtain serum-free culture supernatant of dental pulp-derived stem cells, etc. by performing one or more subcultures and culturing the last or several subcultures in a serum-free medium. I can do it. On the other hand, a serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.
培養上清を得るための歯髄由来幹細胞の培養には、通常用いられる条件をそのまま適用することができる。歯髄由来幹細胞の培養上清の調製方法については、幹細胞の種類に応じて幹細胞の単離および選抜工程を適宜調整する以外は、後述する細胞培養方法と同様とすればよい。歯髄由来幹細胞の種類に応じた歯髄由来幹細胞の単離および選抜は、当業者であれば適宜行うことができる。
また、歯髄由来幹細胞の培養には、エクソソームなどの微小粒子を多量に生産させるために、特別な条件を適用してもよい。特別な条件として、例えば、低温条件、低酸素条件、微重力条件など、何らかの刺激物と共培養する条件などを挙げることができる。 For culturing dental pulp-derived stem cells to obtain a culture supernatant, commonly used conditions can be applied as they are. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells according to the type of the dental pulp-derived stem cells.
Furthermore, special conditions may be applied to culturing dental pulp-derived stem cells in order to produce a large amount of microparticles such as exosomes. Examples of special conditions include conditions for co-cultivation with some stimulus, such as low temperature conditions, hypoxic conditions, microgravity conditions, and the like.
また、歯髄由来幹細胞の培養には、エクソソームなどの微小粒子を多量に生産させるために、特別な条件を適用してもよい。特別な条件として、例えば、低温条件、低酸素条件、微重力条件など、何らかの刺激物と共培養する条件などを挙げることができる。 For culturing dental pulp-derived stem cells to obtain a culture supernatant, commonly used conditions can be applied as they are. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells according to the type of the dental pulp-derived stem cells.
Furthermore, special conditions may be applied to culturing dental pulp-derived stem cells in order to produce a large amount of microparticles such as exosomes. Examples of special conditions include conditions for co-cultivation with some stimulus, such as low temperature conditions, hypoxic conditions, microgravity conditions, and the like.
本発明でエクソソームなどの微小粒子の調製に用いる歯髄由来幹細胞の培養上清は、歯髄由来幹細胞の培養上清の他にその他の成分を含んでいてもよいが、その他の成分を実質的に含まないことが好ましい。
ただし、エクソソームの調製に使用する各種類の添加剤を、歯髄由来幹細胞の培養上清に添加してから保存しておいてもよい。 The culture supernatant of dental pulp-derived stem cells used in the preparation of microparticles such as exosomes in the present invention may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it does not substantially contain other components. Preferably not.
However, various types of additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.
ただし、エクソソームの調製に使用する各種類の添加剤を、歯髄由来幹細胞の培養上清に添加してから保存しておいてもよい。 The culture supernatant of dental pulp-derived stem cells used in the preparation of microparticles such as exosomes in the present invention may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it does not substantially contain other components. Preferably not.
However, various types of additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.
(微小粒子の調製)
歯髄由来幹細胞等の培養上清から、微小粒子を精製して、微小粒子を調製することができる。 (Preparation of microparticles)
Microparticles can be prepared by purifying microparticles from culture supernatants such as dental pulp-derived stem cells.
歯髄由来幹細胞等の培養上清から、微小粒子を精製して、微小粒子を調製することができる。 (Preparation of microparticles)
Microparticles can be prepared by purifying microparticles from culture supernatants such as dental pulp-derived stem cells.
微小粒子の精製は、歯髄由来幹細胞の培養上清から微小粒子を含む画分の分離であることが好ましく、微小粒子の単離であることがより好ましい。
微小粒子は、微小粒子の特性に基づいて非会合成分から分離されることにより、単離され得る。例えば、微小粒子は、分子量、サイズ、形態、組成または生物学的活性に基づいて単離され得る。
本発明では、歯髄由来幹細胞の培養上清を遠心処理して得られた、微小粒子を多く含む特定の画分(例えば沈殿物)を分取することにより、微小粒子を精製することができる。所定の画分以外の画分の不要成分(不溶成分)は除去してもよい。微小粒子組成物からの、溶媒および分散媒、ならびに不要成分の除去は完全な除去でなくてもよい。遠心処理の条件を例示すると、100~20000gで、1~30分間である。
本発明では、歯髄由来幹細胞の培養上清またはその遠心処理物を、ろ過処理することにより、微小粒子を精製することができる。ろ過処理によって不要成分を除去することができる。また、適切な孔径のろ過膜を使用すれば、不要成分の除去と滅菌処理を同時に行うことができる。ろ過処理に使用するろ過膜の材質、孔径などは特に限定されない。公知の方法で、適切な分子量またはサイズカットオフのろ過膜でろ過をすることができる。ろ過膜の孔径はエクソソームを分取しやすい観点から、10~1000nmであることが好ましく、30~500nmであることがより好ましく、50~150nmであることが特に好ましい。
本発明では、歯髄由来幹細胞の培養上清またはその遠心処理物あるいはそれらのろ過処理物を、ラムクロマトグラフィーなど、さらなる分離手段を用いて分離することができる。例えば様々なカラムを用いた高速液体クロマトグラフィー(HPLC)を使用できる。カラムは、サイズ排除カラムまたは結合カラムを使用できる。
各処理段階におけるそれぞれの画分中で、微小粒子(またはその活性)を追跡するために、微小粒子の1つ以上の特性または生物学的活性を使用できる。例えば、微小粒子を追跡するために、光散乱、屈折率、動的光散乱またはUV-可視光検出器を使用できる。または、それぞれの画分中の活性を追跡するために、特定の酵素活性などを使用できる。
微小粒子の精製方法として、特表2019-524824号公報の[0034]~[0064]に記載の方法を用いてもよく、この公報の内容は参照して本明細書に組み込まれる。 Purification of microparticles is preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles.
Microparticles can be isolated by separating them from unassociated components based on the properties of the microparticles. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity.
In the present invention, microparticles can be purified by separating a specific fraction (eg, precipitate) containing many microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the predetermined fractions may be removed. Removal of solvents and dispersion media and unnecessary components from the microparticle composition may not be complete. Examples of conditions for centrifugation are 100 to 20,000 g and 1 to 30 minutes.
In the present invention, microparticles can be purified by filtering a culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration treatment. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed at the same time. The material, pore size, etc. of the filtration membrane used for the filtration treatment are not particularly limited. Filtration can be carried out using filtration membranes of appropriate molecular weight or size cut-off using known methods. The pore size of the filtration membrane is preferably from 10 to 1000 nm, more preferably from 30 to 500 nm, and particularly preferably from 50 to 150 nm, from the viewpoint of easy separation of exosomes.
In the present invention, a culture supernatant of dental pulp-derived stem cells, a centrifuged product thereof, or a filtered product thereof can be separated using an additional separation means such as lamb chromatography. For example, high performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column.
One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing step. For example, light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track microparticles. Alternatively, specific enzyme activities, etc. can be used to track the activity in each fraction.
As a method for purifying microparticles, the method described in [0034] to [0064] of Japanese Patent Publication No. 2019-524824 may be used, and the contents of this publication are incorporated herein by reference.
微小粒子は、微小粒子の特性に基づいて非会合成分から分離されることにより、単離され得る。例えば、微小粒子は、分子量、サイズ、形態、組成または生物学的活性に基づいて単離され得る。
本発明では、歯髄由来幹細胞の培養上清を遠心処理して得られた、微小粒子を多く含む特定の画分(例えば沈殿物)を分取することにより、微小粒子を精製することができる。所定の画分以外の画分の不要成分(不溶成分)は除去してもよい。微小粒子組成物からの、溶媒および分散媒、ならびに不要成分の除去は完全な除去でなくてもよい。遠心処理の条件を例示すると、100~20000gで、1~30分間である。
本発明では、歯髄由来幹細胞の培養上清またはその遠心処理物を、ろ過処理することにより、微小粒子を精製することができる。ろ過処理によって不要成分を除去することができる。また、適切な孔径のろ過膜を使用すれば、不要成分の除去と滅菌処理を同時に行うことができる。ろ過処理に使用するろ過膜の材質、孔径などは特に限定されない。公知の方法で、適切な分子量またはサイズカットオフのろ過膜でろ過をすることができる。ろ過膜の孔径はエクソソームを分取しやすい観点から、10~1000nmであることが好ましく、30~500nmであることがより好ましく、50~150nmであることが特に好ましい。
本発明では、歯髄由来幹細胞の培養上清またはその遠心処理物あるいはそれらのろ過処理物を、ラムクロマトグラフィーなど、さらなる分離手段を用いて分離することができる。例えば様々なカラムを用いた高速液体クロマトグラフィー(HPLC)を使用できる。カラムは、サイズ排除カラムまたは結合カラムを使用できる。
各処理段階におけるそれぞれの画分中で、微小粒子(またはその活性)を追跡するために、微小粒子の1つ以上の特性または生物学的活性を使用できる。例えば、微小粒子を追跡するために、光散乱、屈折率、動的光散乱またはUV-可視光検出器を使用できる。または、それぞれの画分中の活性を追跡するために、特定の酵素活性などを使用できる。
微小粒子の精製方法として、特表2019-524824号公報の[0034]~[0064]に記載の方法を用いてもよく、この公報の内容は参照して本明細書に組み込まれる。 Purification of microparticles is preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles.
Microparticles can be isolated by separating them from unassociated components based on the properties of the microparticles. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity.
In the present invention, microparticles can be purified by separating a specific fraction (eg, precipitate) containing many microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the predetermined fractions may be removed. Removal of solvents and dispersion media and unnecessary components from the microparticle composition may not be complete. Examples of conditions for centrifugation are 100 to 20,000 g and 1 to 30 minutes.
In the present invention, microparticles can be purified by filtering a culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration treatment. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed at the same time. The material, pore size, etc. of the filtration membrane used for the filtration treatment are not particularly limited. Filtration can be carried out using filtration membranes of appropriate molecular weight or size cut-off using known methods. The pore size of the filtration membrane is preferably from 10 to 1000 nm, more preferably from 30 to 500 nm, and particularly preferably from 50 to 150 nm, from the viewpoint of easy separation of exosomes.
In the present invention, a culture supernatant of dental pulp-derived stem cells, a centrifuged product thereof, or a filtered product thereof can be separated using an additional separation means such as lamb chromatography. For example, high performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column.
One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing step. For example, light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track microparticles. Alternatively, specific enzyme activities, etc. can be used to track the activity in each fraction.
As a method for purifying microparticles, the method described in [0034] to [0064] of Japanese Patent Publication No. 2019-524824 may be used, and the contents of this publication are incorporated herein by reference.
微小粒子組成物の最終的な形態は、特に制限はない。例えば、微小粒子組成物は、微小粒子を溶媒または分散媒とともに容器に充填してなる形態;微小粒子をゲルとともにゲル化して容器に充填してなる形態;微小粒子を凍結および/または乾燥して固形化して製剤化または容器に充填してなる形態などが挙げられる。容器としては、例えば凍結保存に適したチューブ、遠沈管、バッグなどが挙げられる。凍結温度は、例えば-20℃~-196℃とすることができる。
The final form of the microparticle composition is not particularly limited. For example, the microparticle composition can be formed by filling a container with microparticles together with a solvent or dispersion medium; by gelling the microparticles together with a gel and filling the container; by freezing and/or drying the microparticles. For example, it may be solidified into a formulation or filled into a container. Examples of containers include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.
本発明の微小粒子は、従来のパーキンソン病の治療薬または予防薬として用いることができる組成物と比較して、大量生産しやすい、従来は産業廃棄物等として廃棄されていた幹細胞の培養液を利活用できる、幹細胞の培養液の廃棄コストを減らせる等の利点がある。特に歯髄由来幹細胞の培養上清が、ヒト歯髄由来幹細胞の培養上清である場合は、本発明の微小粒子をヒトに対して適用する場合に、免疫学上などの観点での安全性が高く、倫理性の問題も少ないという利点もある。歯髄由来幹細胞の培養上清が、認知症の患者からの歯髄由来幹細胞の培養上清である場合は、本発明の微小粒子をその患者に対して適用する際により安全性が高まり、倫理性の問題も少なくなるであろう。
本発明の微小粒子が、歯髄由来幹細胞の培養上清に由来する場合、修復医療の用途にも用いられる。特に歯髄由来幹細胞等の培養上清に由来する微小粒子を含む組成物は、修復医療の用途に好ましく用いられる。ここで、幹細胞移植を前提とした再生医療において、幹細胞は再生の主役ではなく、幹細胞の産生する液性成分が自己の幹細胞とともに臓器を修復させる、ということが知られている。従来の幹細胞移植に伴うがん化、規格化、投与方法、保存性、培養方法などの困難な問題が解決され、歯髄由来幹細胞の培養上清またはそれに由来する微小粒子を用いた組成物により修復医療が可能となる。幹細胞移植と比較すると、本発明の微小粒子を用いた場合は細胞を移植しないために腫瘍化などが起こりにくく、より安全と言えるだろう。また、本発明の微小粒子は一定に規格化した品質のものを使用できる利点がある。大量生産や効率的な投与方法を選択することができるので、低コストで利用ができる。 The microparticles of the present invention can be easily mass-produced compared to conventional compositions that can be used as therapeutic or preventive drugs for Parkinson's disease, and can be used in stem cell culture fluids that were previously discarded as industrial waste. It has advantages such as being able to be used for various purposes and reducing the cost of disposing of stem cell culture fluid. In particular, when the culture supernatant of dental pulp-derived stem cells is the culture supernatant of human dental pulp-derived stem cells, the microparticles of the present invention are highly safe from an immunological standpoint when applied to humans. Another advantage is that there are fewer ethical issues. If the culture supernatant of dental pulp-derived stem cells is a culture supernatant of dental pulp-derived stem cells from a patient with dementia, the microparticles of the present invention will be safer and more ethical when applied to that patient. There will be fewer problems.
When the microparticles of the present invention are derived from the culture supernatant of dental pulp-derived stem cells, they can also be used in restorative medicine. In particular, compositions containing microparticles derived from culture supernatants such as dental pulp-derived stem cells are preferably used for restorative medical applications. In regenerative medicine based on stem cell transplantation, it is known that stem cells are not the main actors in regeneration, but rather that the humoral components produced by stem cells repair organs together with one's own stem cells. Difficult issues associated with conventional stem cell transplantation, such as canceration, standardization, administration methods, storage stability, and culture methods, have been resolved, and restorations can be achieved using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Medical care becomes possible. Compared to stem cell transplantation, when using the microparticles of the present invention, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer. Furthermore, the microparticles of the present invention have the advantage of being of uniformly standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.
本発明の微小粒子が、歯髄由来幹細胞の培養上清に由来する場合、修復医療の用途にも用いられる。特に歯髄由来幹細胞等の培養上清に由来する微小粒子を含む組成物は、修復医療の用途に好ましく用いられる。ここで、幹細胞移植を前提とした再生医療において、幹細胞は再生の主役ではなく、幹細胞の産生する液性成分が自己の幹細胞とともに臓器を修復させる、ということが知られている。従来の幹細胞移植に伴うがん化、規格化、投与方法、保存性、培養方法などの困難な問題が解決され、歯髄由来幹細胞の培養上清またはそれに由来する微小粒子を用いた組成物により修復医療が可能となる。幹細胞移植と比較すると、本発明の微小粒子を用いた場合は細胞を移植しないために腫瘍化などが起こりにくく、より安全と言えるだろう。また、本発明の微小粒子は一定に規格化した品質のものを使用できる利点がある。大量生産や効率的な投与方法を選択することができるので、低コストで利用ができる。 The microparticles of the present invention can be easily mass-produced compared to conventional compositions that can be used as therapeutic or preventive drugs for Parkinson's disease, and can be used in stem cell culture fluids that were previously discarded as industrial waste. It has advantages such as being able to be used for various purposes and reducing the cost of disposing of stem cell culture fluid. In particular, when the culture supernatant of dental pulp-derived stem cells is the culture supernatant of human dental pulp-derived stem cells, the microparticles of the present invention are highly safe from an immunological standpoint when applied to humans. Another advantage is that there are fewer ethical issues. If the culture supernatant of dental pulp-derived stem cells is a culture supernatant of dental pulp-derived stem cells from a patient with dementia, the microparticles of the present invention will be safer and more ethical when applied to that patient. There will be fewer problems.
When the microparticles of the present invention are derived from the culture supernatant of dental pulp-derived stem cells, they can also be used in restorative medicine. In particular, compositions containing microparticles derived from culture supernatants such as dental pulp-derived stem cells are preferably used for restorative medical applications. In regenerative medicine based on stem cell transplantation, it is known that stem cells are not the main actors in regeneration, but rather that the humoral components produced by stem cells repair organs together with one's own stem cells. Difficult issues associated with conventional stem cell transplantation, such as canceration, standardization, administration methods, storage stability, and culture methods, have been resolved, and restorations can be achieved using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Medical care becomes possible. Compared to stem cell transplantation, when using the microparticles of the present invention, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer. Furthermore, the microparticles of the present invention have the advantage of being of uniformly standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.
[パーキンソン病の予防薬または治療薬]
本発明のパーキンソン病の予防薬または治療薬は、本発明の微小粒子を含む。
本明細書において「予防」とは、疾患(本明細書では神経変性疾患)の発症を未然に防ぐことをいう。また、本明細書において「治療」とは、発症した疾患における症状の進行を緩和し、抑制し、又は阻止すること、及び症状を改善することをいう。 [Prophylactic or therapeutic drug for Parkinson's disease]
The prophylactic or therapeutic agent for Parkinson's disease of the present invention contains the microparticles of the present invention.
As used herein, "prevention" refers to preventing the onset of a disease (neurodegenerative disease in this specification). In addition, as used herein, "treatment" refers to alleviating, suppressing, or preventing the progression of symptoms of a disease that has developed, and improving symptoms.
本発明のパーキンソン病の予防薬または治療薬は、本発明の微小粒子を含む。
本明細書において「予防」とは、疾患(本明細書では神経変性疾患)の発症を未然に防ぐことをいう。また、本明細書において「治療」とは、発症した疾患における症状の進行を緩和し、抑制し、又は阻止すること、及び症状を改善することをいう。 [Prophylactic or therapeutic drug for Parkinson's disease]
The prophylactic or therapeutic agent for Parkinson's disease of the present invention contains the microparticles of the present invention.
As used herein, "prevention" refers to preventing the onset of a disease (neurodegenerative disease in this specification). In addition, as used herein, "treatment" refers to alleviating, suppressing, or preventing the progression of symptoms of a disease that has developed, and improving symptoms.
[パーキンソン病の改善方法]
本発明のパーキンソン病の改善方法は、有効量の本発明の微小粒子あるいは有効量の本発明のパーキンソン病の予防薬または治療薬を、パーキンソン病を発症した対象に投与することを含む。 [How to improve Parkinson's disease]
The method for improving Parkinson's disease of the present invention includes administering an effective amount of the microparticles of the present invention or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease of the present invention to a subject who has developed Parkinson's disease.
本発明のパーキンソン病の改善方法は、有効量の本発明の微小粒子あるいは有効量の本発明のパーキンソン病の予防薬または治療薬を、パーキンソン病を発症した対象に投与することを含む。 [How to improve Parkinson's disease]
The method for improving Parkinson's disease of the present invention includes administering an effective amount of the microparticles of the present invention or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease of the present invention to a subject who has developed Parkinson's disease.
本発明の微小粒子を、パーキンソン病を発症した対象に投与する工程は特に制限はない。
投与方法は、口腔、鼻腔または気道への噴霧または吸引、点滴、局所投与、点鼻薬などを挙げることができ、侵襲が少ないことが好ましい。局所投与の方法としては、注射が好ましい。また、皮膚表面に電圧(電気パルス)をかけることにより細胞膜に一時的に微細な穴をあけ、通常のケアでは届かない真皮層まで有効成分を浸透させられるエレクトロポレーションも好ましい。局所投与する場合、静脈内投与、動脈内投与、門脈内投与、皮内投与、皮下投与、筋肉内投与、腹腔内投与などを挙げることができ、動脈内投与、静脈内投与、皮下投与または腹腔内投与であることがより好ましい。
また、種々の製剤化技法を用い、微小粒子のインビボ分布を変えることができる。インビボ分布を変える多数の方法が当業者に既知である。そのような方法の例には、たとえば、タンパク質、脂質(たとえば、リポソーム)、炭水化物または合成ポリマーのような物質で構成される小胞におけるエクソソームの保護が挙げられる。
パーキンソン病を発症した対象に投与された本発明の微小粒子は対象の体内を循環し、所定の組織に到達してもよい。
投与回数および投与間隔は、特に制限はない。投与回数は1週間当たり1回以上とすることができ、5回以上であることが好ましく、6回以上であることがより好ましく、7回以上であることが特に好ましい。投与間隔は、1時間~1週間であることが好ましく、半日間~1週間であることがより好ましく、1日(毎日1回)であることが特に好ましい。ただし、投与対象の生物種や投与対象の症状に応じて、適宜調整することができる。
本発明の微小粒子は、微小粒子を治療有効期間にわたって1週間に1回以上、認知症を発症した対象に投与する用途であることが好ましい。投与対象がヒトである場合は、1週間当たりの投与回数は多い方が好ましく、治療有効期間にわたって1週間に5回以上投与することが好ましく、毎日投与することが好ましい。
2.0×109個/mlの濃度の歯髄由来幹細胞の培養上清を用いる場合、マウスモデルでは、マウス1匹(約25g)当たり0.1~5mlであることが好ましく、0.3~3mlであることがより好ましく、0.5~1mlであることがより特に好ましい。
0.1×108個/μgの濃度の微小粒子を用いる場合、マウスモデルでは、マウス1匹(約25g)当たり1~50μgであることが好ましく、3~30μgであることがより好ましく、5~25μgであることがより特に好ましい。
その他の動物への体重当たりの投与量の好ましい範囲は、モデルマウスへの体重(約25g)当たりの投与量から比例関係を用いて計算することができる。ただし、投与対象の症状に応じて、適宜調整することができる。 There are no particular restrictions on the process of administering the microparticles of the present invention to a subject who has developed Parkinson's disease.
Administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, dripping, local administration, nasal spray, etc., and minimally invasive methods are preferred. Injection is a preferred method of local administration. Also preferred is electroporation, which temporarily creates minute holes in cell membranes by applying voltage (electrical pulses) to the skin surface, allowing active ingredients to penetrate into the dermal layer, which cannot be reached with normal skin care. In the case of local administration, examples include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc.; intraarterial administration, intravenous administration, subcutaneous administration or Intraperitoneal administration is more preferred.
Also, various formulation techniques can be used to alter the in vivo distribution of microparticles. Many methods of altering in vivo distribution are known to those skilled in the art. Examples of such methods include protection of exosomes in vesicles composed of materials such as proteins, lipids (eg, liposomes), carbohydrates or synthetic polymers.
The microparticles of the present invention administered to a subject who has developed Parkinson's disease may circulate within the subject's body and reach a predetermined tissue.
There are no particular restrictions on the number of administrations or the interval between administrations. The frequency of administration can be one or more times per week, preferably five or more times, more preferably six or more times, particularly preferably seven or more times. The administration interval is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be adjusted as appropriate depending on the species to be administered and the symptoms of the subject.
The microparticles of the present invention are preferably used to administer the microparticles to a subject who has developed dementia at least once a week over a therapeutically effective period. When the subject to be administered is a human, the number of administrations per week is preferably large, preferably 5 or more times per week over the effective therapeutic period, and preferably daily.
When using a culture supernatant of dental pulp-derived stem cells at a concentration of 2.0 × 10 9 cells/ml, in a mouse model, the amount is preferably 0.1 to 5 ml per mouse (about 25 g), and 0.3 to 5 ml per mouse (approximately 25 g). The amount is more preferably 3 ml, and particularly preferably 0.5 to 1 ml.
When using microparticles at a concentration of 0.1×10 8 particles/μg, in mouse models, the amount is preferably 1 to 50 μg, more preferably 3 to 30 μg, and more preferably 5 to 50 μg per mouse (approximately 25 g). More particularly preferred is ~25 μg.
The preferred range of dosage per body weight for other animals can be calculated from the dosage per body weight (about 25 g) for model mice using a proportional relationship. However, it can be adjusted as appropriate depending on the symptoms of the subject.
投与方法は、口腔、鼻腔または気道への噴霧または吸引、点滴、局所投与、点鼻薬などを挙げることができ、侵襲が少ないことが好ましい。局所投与の方法としては、注射が好ましい。また、皮膚表面に電圧(電気パルス)をかけることにより細胞膜に一時的に微細な穴をあけ、通常のケアでは届かない真皮層まで有効成分を浸透させられるエレクトロポレーションも好ましい。局所投与する場合、静脈内投与、動脈内投与、門脈内投与、皮内投与、皮下投与、筋肉内投与、腹腔内投与などを挙げることができ、動脈内投与、静脈内投与、皮下投与または腹腔内投与であることがより好ましい。
また、種々の製剤化技法を用い、微小粒子のインビボ分布を変えることができる。インビボ分布を変える多数の方法が当業者に既知である。そのような方法の例には、たとえば、タンパク質、脂質(たとえば、リポソーム)、炭水化物または合成ポリマーのような物質で構成される小胞におけるエクソソームの保護が挙げられる。
パーキンソン病を発症した対象に投与された本発明の微小粒子は対象の体内を循環し、所定の組織に到達してもよい。
投与回数および投与間隔は、特に制限はない。投与回数は1週間当たり1回以上とすることができ、5回以上であることが好ましく、6回以上であることがより好ましく、7回以上であることが特に好ましい。投与間隔は、1時間~1週間であることが好ましく、半日間~1週間であることがより好ましく、1日(毎日1回)であることが特に好ましい。ただし、投与対象の生物種や投与対象の症状に応じて、適宜調整することができる。
本発明の微小粒子は、微小粒子を治療有効期間にわたって1週間に1回以上、認知症を発症した対象に投与する用途であることが好ましい。投与対象がヒトである場合は、1週間当たりの投与回数は多い方が好ましく、治療有効期間にわたって1週間に5回以上投与することが好ましく、毎日投与することが好ましい。
2.0×109個/mlの濃度の歯髄由来幹細胞の培養上清を用いる場合、マウスモデルでは、マウス1匹(約25g)当たり0.1~5mlであることが好ましく、0.3~3mlであることがより好ましく、0.5~1mlであることがより特に好ましい。
0.1×108個/μgの濃度の微小粒子を用いる場合、マウスモデルでは、マウス1匹(約25g)当たり1~50μgであることが好ましく、3~30μgであることがより好ましく、5~25μgであることがより特に好ましい。
その他の動物への体重当たりの投与量の好ましい範囲は、モデルマウスへの体重(約25g)当たりの投与量から比例関係を用いて計算することができる。ただし、投与対象の症状に応じて、適宜調整することができる。 There are no particular restrictions on the process of administering the microparticles of the present invention to a subject who has developed Parkinson's disease.
Administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, dripping, local administration, nasal spray, etc., and minimally invasive methods are preferred. Injection is a preferred method of local administration. Also preferred is electroporation, which temporarily creates minute holes in cell membranes by applying voltage (electrical pulses) to the skin surface, allowing active ingredients to penetrate into the dermal layer, which cannot be reached with normal skin care. In the case of local administration, examples include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc.; intraarterial administration, intravenous administration, subcutaneous administration or Intraperitoneal administration is more preferred.
Also, various formulation techniques can be used to alter the in vivo distribution of microparticles. Many methods of altering in vivo distribution are known to those skilled in the art. Examples of such methods include protection of exosomes in vesicles composed of materials such as proteins, lipids (eg, liposomes), carbohydrates or synthetic polymers.
The microparticles of the present invention administered to a subject who has developed Parkinson's disease may circulate within the subject's body and reach a predetermined tissue.
There are no particular restrictions on the number of administrations or the interval between administrations. The frequency of administration can be one or more times per week, preferably five or more times, more preferably six or more times, particularly preferably seven or more times. The administration interval is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be adjusted as appropriate depending on the species to be administered and the symptoms of the subject.
The microparticles of the present invention are preferably used to administer the microparticles to a subject who has developed dementia at least once a week over a therapeutically effective period. When the subject to be administered is a human, the number of administrations per week is preferably large, preferably 5 or more times per week over the effective therapeutic period, and preferably daily.
When using a culture supernatant of dental pulp-derived stem cells at a concentration of 2.0 × 10 9 cells/ml, in a mouse model, the amount is preferably 0.1 to 5 ml per mouse (about 25 g), and 0.3 to 5 ml per mouse (approximately 25 g). The amount is more preferably 3 ml, and particularly preferably 0.5 to 1 ml.
When using microparticles at a concentration of 0.1×10 8 particles/μg, in mouse models, the amount is preferably 1 to 50 μg, more preferably 3 to 30 μg, and more preferably 5 to 50 μg per mouse (approximately 25 g). More particularly preferred is ~25 μg.
The preferred range of dosage per body weight for other animals can be calculated from the dosage per body weight (about 25 g) for model mice using a proportional relationship. However, it can be adjusted as appropriate depending on the symptoms of the subject.
本発明の微小粒子を投与する対象の動物(生物種)は、特に制限はない。本発明の微小粒子を投与する対象の動物は、哺乳動物、鳥類(ニワトリ、ウズラ、カモなど)、魚類(サケ、マス、マグロ、カツオなど)であることが好ましい。哺乳動物としては、ヒトであっても、非ヒト哺乳動物であってもよいが、ヒトであることが特に好ましい。非ヒト哺乳動物としては、ウシ、ブタ、ウマ、ヤギ、ヒツジ、サル、イヌ、ネコ、マウス、ラット、モルモット、ハムスターであることがより好ましい。
There are no particular restrictions on the animal (species) to which the microparticles of the present invention are administered. The animals to which the microparticles of the present invention are administered are preferably mammals, birds (chickens, quail, ducks, etc.), and fish (salmon, trout, tuna, bonito, etc.). The mammal may be a human or a non-human mammal, but humans are particularly preferred. More preferably, the non-human mammal is a cow, pig, horse, goat, sheep, monkey, dog, cat, mouse, rat, guinea pig, or hamster.
本発明の微小粒子は、従来公知のパーキンソン病の治療薬と併用してもよい。具体的には、例えばL-DOPA(レボドパ)と併用してもよい。
The microparticles of the present invention may be used in combination with conventionally known therapeutic agents for Parkinson's disease. Specifically, it may be used in combination with, for example, L-DOPA (levodopa).
以下に実施例と比較例または参考例とを挙げて本発明の特徴をさらに具体的に説明する。以下の実施例に示す材料、使用量、割合、処理内容、処理手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。
The features of the present invention will be explained in more detail below with reference to Examples and Comparative Examples or Reference Examples. The materials, usage amounts, proportions, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.
[実施例1]
<歯髄由来幹細胞の培養上清の調製>
DMEM/HamF12混合培地の代わりにDMEM培地を用い、その他は特許第6296622号の実施例6に記載の方法に準じて、ヒト乳歯歯髄幹細胞の培養上清を調製して、培養上清を分取した。初代培養ではウシ胎仔血清(FBS)を添加して培養し、継代培養では初代培養液を用いて培養した継代培養液の上清をFBSが含まれないように分取し、乳歯歯髄幹細胞の培養上清を調製した。なお、DMEMはダルベッコ改変イーグル培地であり、F12はハムF12培地である。 [Example 1]
<Preparation of culture supernatant of dental pulp-derived stem cells>
Using DMEM medium instead of the DMEM/HamF12 mixed medium and otherwise following the method described in Example 6 of Patent No. 6296622, a culture supernatant of human deciduous dental pulp stem cells was prepared, and the culture supernatant was fractionated. did. In primary culture, fetal bovine serum (FBS) is added to the culture, and in subculture, the supernatant of the subculture medium is cultured using the primary culture medium, and the supernatant of the subculture medium is separated so that it does not contain FBS. culture supernatant was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.
<歯髄由来幹細胞の培養上清の調製>
DMEM/HamF12混合培地の代わりにDMEM培地を用い、その他は特許第6296622号の実施例6に記載の方法に準じて、ヒト乳歯歯髄幹細胞の培養上清を調製して、培養上清を分取した。初代培養ではウシ胎仔血清(FBS)を添加して培養し、継代培養では初代培養液を用いて培養した継代培養液の上清をFBSが含まれないように分取し、乳歯歯髄幹細胞の培養上清を調製した。なお、DMEMはダルベッコ改変イーグル培地であり、F12はハムF12培地である。 [Example 1]
<Preparation of culture supernatant of dental pulp-derived stem cells>
Using DMEM medium instead of the DMEM/HamF12 mixed medium and otherwise following the method described in Example 6 of Patent No. 6296622, a culture supernatant of human deciduous dental pulp stem cells was prepared, and the culture supernatant was fractionated. did. In primary culture, fetal bovine serum (FBS) is added to the culture, and in subculture, the supernatant of the subculture medium is cultured using the primary culture medium, and the supernatant of the subculture medium is separated so that it does not contain FBS. culture supernatant was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.
<エクソソームの調製>
得られた歯髄由来幹細胞の培養上清から、歯髄由来幹細胞のエクソソームを以下の超遠心法により、精製および回収した。
乳歯歯髄幹細胞の培養上清(100mL)を0.22マイクロメーターのポアサイズのフィルターで濾過したのち、その溶液を、60分間、4℃で100000×gで遠心分離した。上清をデカントし、エクソソーム濃縮ペレットをリン酸緩衝食塩水(PBS)中に再懸濁した。再懸濁サンプルを、60分間100000×gで遠心分離した。再度ペレットを濃縮サンプルとして遠心チューブの底から回収した(およそ100μl)。タンパク濃度は、マイクロBSAタンパク質アッセイキット(Pierce、Rockford、IL)によって決定した。エクソソームを含む組成物(濃縮溶液)は、-80℃で保管した。
歯髄由来幹細胞の培養上清から精製したエクソソームを含む組成物を、実施例1の微小粒子組成物サンプルとした。 <Preparation of exosomes>
From the obtained culture supernatant of dental pulp-derived stem cells, exosomes of dental pulp-derived stem cells were purified and collected by the following ultracentrifugation method.
After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells through a filter with a pore size of 0.22 micrometers, the solution was centrifuged at 100,000×g at 4° C. for 60 minutes. The supernatant was decanted and the exosome enriched pellet was resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 xg for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 μl). Protein concentration was determined by microBSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C.
A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the microparticle composition sample of Example 1.
得られた歯髄由来幹細胞の培養上清から、歯髄由来幹細胞のエクソソームを以下の超遠心法により、精製および回収した。
乳歯歯髄幹細胞の培養上清(100mL)を0.22マイクロメーターのポアサイズのフィルターで濾過したのち、その溶液を、60分間、4℃で100000×gで遠心分離した。上清をデカントし、エクソソーム濃縮ペレットをリン酸緩衝食塩水(PBS)中に再懸濁した。再懸濁サンプルを、60分間100000×gで遠心分離した。再度ペレットを濃縮サンプルとして遠心チューブの底から回収した(およそ100μl)。タンパク濃度は、マイクロBSAタンパク質アッセイキット(Pierce、Rockford、IL)によって決定した。エクソソームを含む組成物(濃縮溶液)は、-80℃で保管した。
歯髄由来幹細胞の培養上清から精製したエクソソームを含む組成物を、実施例1の微小粒子組成物サンプルとした。 <Preparation of exosomes>
From the obtained culture supernatant of dental pulp-derived stem cells, exosomes of dental pulp-derived stem cells were purified and collected by the following ultracentrifugation method.
After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells through a filter with a pore size of 0.22 micrometers, the solution was centrifuged at 100,000×g at 4° C. for 60 minutes. The supernatant was decanted and the exosome enriched pellet was resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 xg for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 μl). Protein concentration was determined by microBSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C.
A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the microparticle composition sample of Example 1.
実施例1の微小粒子組成物に含まれる微小粒子の平均粒径、濃度をナノ粒子解析システム NanoSight(ナノサイト)(日本カンタム・デザイン株式会社製)を用いて評価した。
実施例1の微小粒子組成物に含まれる微小粒子の平均粒径は50~150nmであった。
実施例1の微小粒子組成物は1.0×109個/ml以上の高濃度エクソソーム溶液であり、具体的には2.0×109個/mlの高濃度エクソソーム溶液であった。
また、得られた実施例1の微小粒子組成物の成分を公知の方法で分析した。その結果、実施例1の微小粒子は、歯髄由来幹細胞の幹細胞を含まず、MCP-1を含まず、シグレック9も含まないことがわかった。そのため、間葉系幹細胞の培養上清の有効成分であるMCP-1およびシグレック9ならびにこれらの類縁体とは異なる有効成分が、実施例1の微小粒子組成物の有効成分であることがわかった。 The average particle diameter and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated using a nanoparticle analysis system NanoSight (manufactured by Nippon Quantum Design Co., Ltd.).
The average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm.
The microparticle composition of Example 1 was a high concentration exosome solution of 1.0×10 9 particles/ml or more, specifically, a high concentration exosome solution of 2.0×10 9 particles/ml.
In addition, the components of the obtained microparticle composition of Example 1 were analyzed by a known method. As a result, it was found that the microparticles of Example 1 did not contain stem cells of dental pulp-derived stem cells, did not contain MCP-1, and did not contain Siglec-9. Therefore, it was found that an active ingredient different from MCP-1 and Siglec-9, which are the active ingredients of the culture supernatant of mesenchymal stem cells, and their analogs, was the active ingredient of the microparticle composition of Example 1. .
実施例1の微小粒子組成物に含まれる微小粒子の平均粒径は50~150nmであった。
実施例1の微小粒子組成物は1.0×109個/ml以上の高濃度エクソソーム溶液であり、具体的には2.0×109個/mlの高濃度エクソソーム溶液であった。
また、得られた実施例1の微小粒子組成物の成分を公知の方法で分析した。その結果、実施例1の微小粒子は、歯髄由来幹細胞の幹細胞を含まず、MCP-1を含まず、シグレック9も含まないことがわかった。そのため、間葉系幹細胞の培養上清の有効成分であるMCP-1およびシグレック9ならびにこれらの類縁体とは異なる有効成分が、実施例1の微小粒子組成物の有効成分であることがわかった。 The average particle diameter and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated using a nanoparticle analysis system NanoSight (manufactured by Nippon Quantum Design Co., Ltd.).
The average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm.
The microparticle composition of Example 1 was a high concentration exosome solution of 1.0×10 9 particles/ml or more, specifically, a high concentration exosome solution of 2.0×10 9 particles/ml.
In addition, the components of the obtained microparticle composition of Example 1 were analyzed by a known method. As a result, it was found that the microparticles of Example 1 did not contain stem cells of dental pulp-derived stem cells, did not contain MCP-1, and did not contain Siglec-9. Therefore, it was found that an active ingredient different from MCP-1 and Siglec-9, which are the active ingredients of the culture supernatant of mesenchymal stem cells, and their analogs, was the active ingredient of the microparticle composition of Example 1. .
[試験例1]:エクソソームで発現するマイクロRNA
実施例1の微小粒子組成物に含まれるsmall RNAを次世代シーケンシング(NGS)解析により解析した。NGS解析により、実施例1の微小粒子組成物(歯髄由来幹細胞のエクソソーム)に含まれるmiRNAが1787個同定された。得られた結果を下記表1に示す。 [Test Example 1]: MicroRNA expressed in exosomes
Small RNA contained in the microparticle composition of Example 1 was analyzed by next generation sequencing (NGS) analysis. NGS analysis identified 1787 miRNAs contained in the microparticle composition (dental pulp-derived stem cell exosomes) of Example 1. The results obtained are shown in Table 1 below.
実施例1の微小粒子組成物に含まれるsmall RNAを次世代シーケンシング(NGS)解析により解析した。NGS解析により、実施例1の微小粒子組成物(歯髄由来幹細胞のエクソソーム)に含まれるmiRNAが1787個同定された。得られた結果を下記表1に示す。 [Test Example 1]: MicroRNA expressed in exosomes
Small RNA contained in the microparticle composition of Example 1 was analyzed by next generation sequencing (NGS) analysis. NGS analysis identified 1787 miRNAs contained in the microparticle composition (dental pulp-derived stem cell exosomes) of Example 1. The results obtained are shown in Table 1 below.
[試験例2]:大脳皮質のmiRNAとの対比
マイクロRNAの解析にはIMOTA(Interactive Multi-Omics-Tissue Atlas)を用いた。
タンパクを制御するマイクロRNAをIMOTAで大脳皮質(Cerebral Cortex)を中心に検索した。IMOTAは、各組織や細胞におけるmiRNAやmRNA、タンパク質の相互作用や発現量について調べることができる対話型のマルチオミクスアトラスである(Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770-D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions")。大脳皮質では、Omicのカウント数は、タンパク質が3071個、mRNAが14182個、miRNAが1124個であった。また、大脳皮質で発現率(Expression Rate)がHighのOmicのカウント数は、タンパク質が1119個、mRNAが1110個、miRNAが145個であった。 [Test Example 2]: Comparison with miRNA in cerebral cortex IMOTA (Interactive Multi-Omics-Tissue Atlas) was used for microRNA analysis.
MicroRNAs that control proteins were searched for using IMOTA, focusing on the cerebral cortex. IMOTA is an interactive multi-omics atlas that allows you to investigate interactions and expression levels of miRNAs, mRNAs, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770- D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions"). In the cerebral cortex, the Omic counts were 3071 proteins, 14182 mRNAs, and 1124 miRNAs. Furthermore, the counts of Omics with a high expression rate in the cerebral cortex were 1119 for proteins, 1110 for mRNA, and 145 for miRNA.
マイクロRNAの解析にはIMOTA(Interactive Multi-Omics-Tissue Atlas)を用いた。
タンパクを制御するマイクロRNAをIMOTAで大脳皮質(Cerebral Cortex)を中心に検索した。IMOTAは、各組織や細胞におけるmiRNAやmRNA、タンパク質の相互作用や発現量について調べることができる対話型のマルチオミクスアトラスである(Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770-D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions")。大脳皮質では、Omicのカウント数は、タンパク質が3071個、mRNAが14182個、miRNAが1124個であった。また、大脳皮質で発現率(Expression Rate)がHighのOmicのカウント数は、タンパク質が1119個、mRNAが1110個、miRNAが145個であった。 [Test Example 2]: Comparison with miRNA in cerebral cortex IMOTA (Interactive Multi-Omics-Tissue Atlas) was used for microRNA analysis.
MicroRNAs that control proteins were searched for using IMOTA, focusing on the cerebral cortex. IMOTA is an interactive multi-omics atlas that allows you to investigate interactions and expression levels of miRNAs, mRNAs, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770- D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions"). In the cerebral cortex, the Omic counts were 3071 proteins, 14182 mRNAs, and 1124 miRNAs. Furthermore, the counts of Omics with a high expression rate in the cerebral cortex were 1119 for proteins, 1110 for mRNA, and 145 for miRNA.
大脳皮質で発現している1124個のmiRNAと、歯髄由来幹細胞の培養上清から精製したエクソソーム(実施例1の微小粒子組成物;SGF exosome)で発現しているmiRNA1787個の間で、共通に発現しているmiRNAは859個であった。すなわち、実施例1の微小粒子組成物(歯髄由来幹細胞のエクソソーム)で発現しているmiRNAのうち、大脳皮質でも発現しているmiRNAの割合は48.1%であり、非常に高かった。
A common difference between 1124 miRNAs expressed in the cerebral cortex and 1787 miRNAs expressed in exosomes (microparticle composition of Example 1; SGF exosome) purified from the culture supernatant of dental pulp-derived stem cells. There were 859 miRNAs expressed. That is, among the miRNAs expressed in the microparticle composition of Example 1 (exosomes of dental pulp-derived stem cells), the proportion of miRNAs also expressed in the cerebral cortex was 48.1%, which was very high.
また、実施例1の微小粒子組成物(歯髄由来幹細胞のエクソソーム)で発現があり、大脳皮質での発現がHighであるmiRNAは、下記表2および表3に記載の122個であった。
Additionally, there were 122 miRNAs expressed in the microparticle composition of Example 1 (exosomes of dental pulp-derived stem cells) and highly expressed in the cerebral cortex as shown in Tables 2 and 3 below.
[試験例3]:疾患に関わるマイクロRNAの探索
得られた解析結果をもとに疾患に関わるマイクロRNAを探索した。疾患に関連するmiRNAの抽出を、IMOTAを用いて行った。ここでは、パーキンソン病に関連するタンパクを抑制するマイクロRNAが含まれているか、または、治療薬のターゲットとなるタンパクを制御しているマイクロRNAが含まれているかを探索した。本実施例では、パーキンソン病に関連する遺伝子であるマイクロRNAを探索した。パーキンソン病の治療に寄与するマイクロRNAが記載された非特許文献1(Front. Mol. Neurosci. vol.11, 381(2018))、非特許文献2(Molecular Therapy: Nucleic Acids, Vol.11 p9-22(2018))に記載のマイクロRNAのうち、実施例1の微小粒子組成物(SGF exosome)で発現しているマイクロRNAの発現量を図1に示した。
また、非特許文献3(Neural Regen. Res. 12 (12) p1945-1959(2017))に記載のパーキンソン病の脳組織においてダウンレギュレーションされるmiRNAのうち、実施例1の微小粒子組成物(SGF exosome)で発現しているマイクロRNAの発現量もあわせて図1に示した。
あわせて、実施例1の微小粒子組成物で発現しているmiRNAの大脳皮質(Cerebral Cortex)における発現量を図2に示した。図2の空欄行は、IMOTAに登録されている大脳皮質のmiRNAの中に、実施例1の微小粒子組成物で発現しているmiRNAのうちhsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-330-5p、hsa-miR-429がなかったことを示す。 [Test Example 3]: Search for microRNAs involved in diseases Based on the obtained analysis results, microRNAs involved in diseases were searched. Extraction of disease-related miRNAs was performed using IMOTA. Here, we investigated whether microRNAs that suppress proteins related to Parkinson's disease are contained, or microRNAs that control proteins that are targets of therapeutic drugs. In this example, microRNA, which is a gene related to Parkinson's disease, was searched. Non-patent document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) and non-patent document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-), which describe microRNAs that contribute to the treatment of Parkinson's disease. 22 (2018)), the expression levels of microRNAs expressed in the microparticle composition (SGF exosome) of Example 1 are shown in FIG.
Furthermore, among the miRNAs that are down-regulated in the brain tissue of Parkinson's disease described in Non-Patent Document 3 (Neural Regen. Res. 12 (12) p1945-1959 (2017)), the microparticle composition of Example 1 (SGF The expression levels of microRNAs expressed in exosomes are also shown in Figure 1.
Additionally, the expression level of miRNA expressed in the microparticle composition of Example 1 in the cerebral cortex is shown in FIG. 2. Blank rows in FIG. 2 indicate hsa-miR-182-5p and hsa-miR-183 among the miRNAs of the cerebral cortex registered in IMOTA that are expressed in the microparticle composition of Example 1. -5p, hsa-miR-330-5p, and hsa-miR-429 were absent.
得られた解析結果をもとに疾患に関わるマイクロRNAを探索した。疾患に関連するmiRNAの抽出を、IMOTAを用いて行った。ここでは、パーキンソン病に関連するタンパクを抑制するマイクロRNAが含まれているか、または、治療薬のターゲットとなるタンパクを制御しているマイクロRNAが含まれているかを探索した。本実施例では、パーキンソン病に関連する遺伝子であるマイクロRNAを探索した。パーキンソン病の治療に寄与するマイクロRNAが記載された非特許文献1(Front. Mol. Neurosci. vol.11, 381(2018))、非特許文献2(Molecular Therapy: Nucleic Acids, Vol.11 p9-22(2018))に記載のマイクロRNAのうち、実施例1の微小粒子組成物(SGF exosome)で発現しているマイクロRNAの発現量を図1に示した。
また、非特許文献3(Neural Regen. Res. 12 (12) p1945-1959(2017))に記載のパーキンソン病の脳組織においてダウンレギュレーションされるmiRNAのうち、実施例1の微小粒子組成物(SGF exosome)で発現しているマイクロRNAの発現量もあわせて図1に示した。
あわせて、実施例1の微小粒子組成物で発現しているmiRNAの大脳皮質(Cerebral Cortex)における発現量を図2に示した。図2の空欄行は、IMOTAに登録されている大脳皮質のmiRNAの中に、実施例1の微小粒子組成物で発現しているmiRNAのうちhsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-330-5p、hsa-miR-429がなかったことを示す。 [Test Example 3]: Search for microRNAs involved in diseases Based on the obtained analysis results, microRNAs involved in diseases were searched. Extraction of disease-related miRNAs was performed using IMOTA. Here, we investigated whether microRNAs that suppress proteins related to Parkinson's disease are contained, or microRNAs that control proteins that are targets of therapeutic drugs. In this example, microRNA, which is a gene related to Parkinson's disease, was searched. Non-patent document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) and non-patent document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-), which describe microRNAs that contribute to the treatment of Parkinson's disease. 22 (2018)), the expression levels of microRNAs expressed in the microparticle composition (SGF exosome) of Example 1 are shown in FIG.
Furthermore, among the miRNAs that are down-regulated in the brain tissue of Parkinson's disease described in Non-Patent Document 3 (Neural Regen. Res. 12 (12) p1945-1959 (2017)), the microparticle composition of Example 1 (SGF The expression levels of microRNAs expressed in exosomes are also shown in Figure 1.
Additionally, the expression level of miRNA expressed in the microparticle composition of Example 1 in the cerebral cortex is shown in FIG. 2. Blank rows in FIG. 2 indicate hsa-miR-182-5p and hsa-miR-183 among the miRNAs of the cerebral cortex registered in IMOTA that are expressed in the microparticle composition of Example 1. -5p, hsa-miR-330-5p, and hsa-miR-429 were absent.
図1より、実施例1の微小粒子組成物には、パーキンソン病の治療に寄与するマイクロRNAとして非特許文献1(Front. Mol. Neurosci. vol.11, 381(2018))に記載のhsa-miR-212-5p、ならびに非特許文献2(Molecular Therapy: Nucleic Acids, Vol.11 p9-22(2018))に記載のhsa-miR-182-5pおよびhsa-miR-183-5pが発現していることがわかった。
また、図1より、実施例1の微小粒子組成物には、非特許文献3(Neural Regen. Res., 12, (12), p1945-1959(2017))にパーキンソン病の脳細胞で抑制(ダウンレギュレート)されているマイクロRNA、すなわちパーキンソン病の新たな治療標的として挙げられた41種類のマイクロRNAのうち、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543の18種類が発現していることがわかった。 From FIG. 1, the microparticle composition of Example 1 contains hsa-, which is described in Non-Patent Document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) as a microRNA that contributes to the treatment of Parkinson's disease. miR-212-5p, as well as hsa-miR-182-5p and hsa-miR-183-5p described in Non-Patent Document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018)) are expressed. I found out that there is.
Moreover, from FIG. 1, the microparticle composition of Example 1 has the effect of inhibiting Parkinson's disease brain cells ( hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- among the 41 types of microRNAs that are down-regulated (down-regulated), i.e., hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- 5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR- 337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa- It was found that 18 types of miR-542-3p and hsa-miR-543 were expressed.
また、図1より、実施例1の微小粒子組成物には、非特許文献3(Neural Regen. Res., 12, (12), p1945-1959(2017))にパーキンソン病の脳細胞で抑制(ダウンレギュレート)されているマイクロRNA、すなわちパーキンソン病の新たな治療標的として挙げられた41種類のマイクロRNAのうち、hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543の18種類が発現していることがわかった。 From FIG. 1, the microparticle composition of Example 1 contains hsa-, which is described in Non-Patent Document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) as a microRNA that contributes to the treatment of Parkinson's disease. miR-212-5p, as well as hsa-miR-182-5p and hsa-miR-183-5p described in Non-Patent Document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018)) are expressed. I found out that there is.
Moreover, from FIG. 1, the microparticle composition of Example 1 has the effect of inhibiting Parkinson's disease brain cells ( hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- among the 41 types of microRNAs that are down-regulated (down-regulated), i.e., hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- 5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR- 337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa- It was found that 18 types of miR-542-3p and hsa-miR-543 were expressed.
以上より、本発明の微小粒子は、パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含むことがわかった。
From the above, it was found that the microparticles of the present invention contain miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used in the treatment of Parkinson's disease.
Claims (10)
- パーキンソン病で発現が抑制されるmiRNAまたはパーキンソン病の治療に用いられるmiRNAを含む、微小粒子。 Microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
- 前記微小粒子がエクソソームである、請求項1に記載の微小粒子。 The microparticle according to claim 1, wherein the microparticle is an exosome.
- 前記微小粒子が、歯髄由来幹細胞の培養上清よりも高い濃度で、前記パーキンソン病に関する遺伝子を標的遺伝子とするmiRNAを含む、請求項1に記載の微小粒子。 The microparticle according to claim 1, wherein the microparticle contains miRNA that targets the gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
- 前記微小粒子が下記パーキンソン病関連miRNA群のうち少なくとも1種類を含む、請求項1に記載の微小粒子;
パーキンソン病関連miRNA群:
hsa-miR-127-5p、hsa-miR-133b、hsa-miR-155-5p、hsa-miR-182-5p、hsa-miR-183-5p、hsa-miR-184、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-212-5p、hsa-miR-299-5p、hsa-miR-3200-3p、hsa-miR-330-5p、hsa-miR-337-5p、hsa-miR-339-5p、hsa-miR-382-5p、hsa-miR-421、hsa-miR-423-5p、hsa-miR-429、hsa-miR-485-5p、hsa-miR-542-3p、hsa-miR-543。 The microparticle according to claim 1, wherein the microparticle includes at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543. - 前記微小粒子が前記パーキンソン病関連miRNA群のうち、hsa-miR-155-5p、hsa-miR-199a-3p、hsa-miR-382-5pおよびhsa-miR-423-5pを含む、請求項4に記載の微小粒子。 4. The microparticles include hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group. Microparticles described in .
- 前記微小粒子が、SIRT2阻害剤であり、かつ前記パーキンソン病関連miRNA群のうちhsa-miR-212-5pを含む、請求項4に記載の微小粒子。 The microparticle according to claim 4, wherein the microparticle is a SIRT2 inhibitor and includes hsa-miR-212-5p among the Parkinson's disease-related miRNA group.
- ドパミン作動性ニューロンの変性またはアポトーシスの抑制剤であり、かつ、前記パーキンソン病関連miRNA群のうちhsa-miR-182-5pまたはhsa-miR-183-5pを含む、請求項4に記載の微小粒子。 The microparticle according to claim 4, which is an inhibitor of dopaminergic neuron degeneration or apoptosis and contains hsa-miR-182-5p or hsa-miR-183-5p from the Parkinson's disease-related miRNA group. .
- 前記微小粒子が歯髄由来幹細胞の培養上清から精製されて単離された微小粒子であり、
前記微小粒子が、前記歯髄由来幹細胞の培養上清から前記エクソソームを除いた成分を含まない、請求項1に記載の微小粒子。 The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells,
The microparticle according to claim 1, wherein the microparticle does not contain any components other than the exosomes from the culture supernatant of the dental pulp-derived stem cells. - 請求項1に記載の微小粒子を含む、パーキンソン病の予防薬または治療薬。 A prophylactic or therapeutic agent for Parkinson's disease, comprising the microparticles according to claim 1.
- 有効量の請求項1に記載の微小粒子、あるいは有効量の請求項9に記載のパーキンソン病の予防薬または治療薬を、パーキンソン病を発症した対象に投与することを含む、パーキンソン病の改善方法。
A method for improving Parkinson's disease, which comprises administering an effective amount of the microparticles according to claim 1 or an effective amount of the preventive or therapeutic agent for Parkinson's disease according to claim 9 to a subject who has developed Parkinson's disease. .
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Non-Patent Citations (4)
Title |
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AKVILE JARMALAVICIUTE VIRGINIJUS TUNAITIS, UGNE PIVORAITE, ALGIRDAS VENALIS, AUGUSTAS PIVORIUNAS: "Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine–induced apoptosis", CYTOTHERAPY, ISIS MEDICAL MEDIA, OXFORD,, GB, vol. 17, no. 7, 1 July 2015 (2015-07-01), GB , pages 932 - 939, XP055434854, ISSN: 1465-3249, DOI: 10.1016/j.jcyt.2014.07.013 * |
NARBUTE KARĪNA, PIĻIPENKO VLADIMIRS, PUPURE JOLANTA, DZIRKALE ZANE, JONAVIČĖ UGNĖ, TUNAITIS VIRGINIJUS, KRIAUČIŪNAITĖ KAROLINA, JA: "Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats", STEM CELLS TRANSLATIONAL MEDICINE, ALPHAMED PRESS, INC., US, vol. 8, no. 5, 1 May 2019 (2019-05-01), US , pages 490 - 499, XP093094866, ISSN: 2157-6564, DOI: 10.1002/sctm.18-0162 * |
SHARMA YOGITA, SHOBHA K, SUNDEEP MATA, PINNELLI VENKATA BHARATKUMAR, PARVEEN SHAGUFTA, DHANUSHKODI ANANDH: "Neural Basis of Dental Pulp Stem Cells and its Potential Application in Parkinson’s Disease", CNS & NEUROLOGICAL DISORDERS, BENTHAM SCIENCE PUBLISHERS LTD.,, NL, vol. 21, no. 1, 1 January 2022 (2022-01-01), NL , pages 62 - 76, XP093094873, ISSN: 1871-5273, DOI: 10.2174/1871527320666210311122921 * |
VENUGOPAL CHAITRA, K SHOBHA, RAI KIRANMAI S., PINNELLI VENKATA BHARATKUMAR, KUTTY BINDU M., DHANUSHKODI ANANDH: "Neuroprotection by Human Dental Pulp Mesenchymal Stem Cells: From Billions to Nano", CURRENT GENE THERAPY, BENTHAM SCIENCE PUBLISHERS LTD., NL, vol. 18, no. 5, 12 November 2018 (2018-11-12), NL , pages 307 - 323, XP093094869, ISSN: 1566-5232, DOI: 10.2174/1566523218666180913152615 * |
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