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WO2018011989A1 - Greffe pour le traitement de lésions nerveuses, y compris les cellules de la pulpe dentaire. - Google Patents

Greffe pour le traitement de lésions nerveuses, y compris les cellules de la pulpe dentaire. Download PDF

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Publication number
WO2018011989A1
WO2018011989A1 PCT/JP2016/071050 JP2016071050W WO2018011989A1 WO 2018011989 A1 WO2018011989 A1 WO 2018011989A1 JP 2016071050 W JP2016071050 W JP 2016071050W WO 2018011989 A1 WO2018011989 A1 WO 2018011989A1
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WIPO (PCT)
Prior art keywords
gene
dental pulp
fgf2
treatment
pulp cells
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English (en)
Japanese (ja)
Inventor
秀文 福光
建一 手塚
杉山　健
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Gifu University NUC
Daiichi Sankyo Co Ltd
Advanced Cell Technology and Engineering Ltd
Original Assignee
Gifu University NUC
Daiichi Sankyo Co Ltd
Advanced Cell Technology and Engineering Ltd
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Priority to JP2018527366A priority Critical patent/JP6998057B2/ja
Priority to PCT/JP2016/071050 priority patent/WO2018011989A1/fr
Publication of WO2018011989A1 publication Critical patent/WO2018011989A1/fr
Anticipated expiration legal-status Critical
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a transplant material for treating nerve damage containing pulp cells and a method for producing the same.
  • the spinal cord is a nerve transmission pathway between the brain and the periphery, and when it is damaged, it falls into serious physical disabilities such as motor paralysis and sensory disturbance. It is extremely rare for physical function lost due to spinal cord injury to be restored, and patients are severely restricted in their subsequent lives. In Japan, there are about 100,000 people with spinal cord injury, and about 5,000 people are newly injured each year. Although the development of fundamental therapy has become a social urgent task, none of the prior arts currently progressing in human clinical research has yet demonstrated a clear therapeutic effect.
  • cell transplantation therapy can be divided into autologous transplantation and allogeneic transplantation.
  • Autotransplantation is a method of transplanting a patient's own tissue or cells derived from the autologous tissue and increasing the number of cells or inducing differentiation.
  • autologous tooth transplantation, autologous skin transplantation, and the like are actually performed in clinical practice.
  • immune rejection can be avoided, but there are problems of invasion associated with extraction of transplanted tissue or tissue-derived cells, individual differences in transplantation effect, time and cost required for cell preparation.
  • allogeneic transplantation is a method of transplanting another person's tissue or tissue-derived cells.
  • immune rejection must be considered.
  • the immune response provided as a self-defense function is an obstacle to transplantation treatment.
  • immunosuppressive agents it has become possible to suppress rejection reactions, and organ transplantation and hematopoietic stem cell transplantation have developed dramatically.
  • immunosuppressive agents there is still anxiety in terms of safety because immune rejection occurs at a rate of about 20%, and in some cases it is extremely difficult to treat immune rejection.
  • dental pulp cells have several advantages as a transplant cell source.
  • the first is that it can be collected from extracted teeth such as deciduous teeth and wisdom teeth that were originally treated as medical waste (permanent teeth alone are discarded 10 million a year), and induction and culture methods have been established (non- Therefore, it can be used as an abundant source of transplanted cells.
  • immune rejection is based on attacking non-self-identified cells by collating HLA antigens, but using HLA haplotype homo donor cells halves the type of antigen, increasing the number of patients who can be applied.
  • Patent Document 1 discloses a method for producing a transplant material for treatment of nerve damage, which includes culturing dental pulp stem cells used for the transplant material in a medium substantially free of growth factors other than FGF2. Is disclosed.
  • the present invention is a dental pulp cell provided as a transplant material for treatment of nerve damage, specifies a property that the pulp cells treated with FGF2 should have, and a graft material for treatment of nerve injury containing the pulp cell. The issue is to provide.
  • dental pulp cells isolated from dental pulp tissue can be treated with FGF2 to obtain dental pulp cells preferable as a transplant material for treating nerve damage.
  • dental pulp cells treated with FGF2 expressed markers of young neurons after transplantation to the spinal cord injury site compared with dental pulp cells cultured with MSCGM, a medium for stem cell culture without FGF2. It has been confirmed that the cells differentiated into non-patent documents (Non-patent Document 1). Such properties support that pulp cells treated with FGF2 are useful for the treatment of nerve damage.
  • Patent Document 1 does not report the analysis / comparison of changes in the properties of dental pulp cells caused by FGF2 treatment, and particularly analyzes and compares the effects of FGF2 treatment among dental pulp cells derived from donors with different therapeutic effects. It is not a thing. Therefore, in FGF2-treated dental pulp cells to be used as a transplant material for treatment of nerve damage, a property that serves as an index of the effect of nerve damage treatment has not been identified.
  • the present invention is as follows:
  • the present invention as one aspect, [1]
  • the present invention relates to a transplant material for treating nerve damage, which includes dental pulp cells expressing GABRB1 gene and having resistance to active oxygen.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [2]
  • the dental pulp cells are those treated with FGF2.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [3]
  • the FGF2-treated dental pulp cells are characterized by having an increased GABRB1 gene expression level as compared with non-FGF2-treated dental pulp cells.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [4] A transplant for treating nerve damage according to any one of [1] to [3] above,
  • the dental pulp cells further express at least one gene selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [5] The transplant for treatment of nerve damage according to any one of [1] to [4] above,
  • the FGF2 treatment is characterized by culturing using a medium containing FGF2 at a concentration of 5 ng / ml or more.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [6] The transplant material for treatment of nerve damage according to [5] above, The culture using the medium containing FGF2 is performed for at least 6 days.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [7]
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [8] The therapeutic agent for transplantation of nerve damage according to any one of [1] to [7],
  • the nerve injury is spinal cord injury, cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disc, sciatica, or peripheral nerve injury caused by diabetes.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [9] The therapeutic graft material according to any one of the above [1] to [8], which is used in combination with an active oxygen scavenger for the treatment of nerve damage.
  • the transplant material for treatment of nerve injury of the present invention is, in one embodiment, [10] The graft material for treatment of nerve damage according to [9] above,
  • the active oxygen scavenger is at least one active oxygen scavenger selected from the group consisting of edaravone, vitamin C, Nrf2 inducer, and glutathione activity inducer.
  • the present invention provides: [11] A method for treating nerve damage,
  • the present invention relates to a treatment method comprising a step of transplanting the nerve injury treatment transplant material according to any one of the above [1] to [8] to a nerve damage site.
  • the method for treating nerve injury of the present invention is as follows.
  • the present invention relates to a treatment method including a step of transplanting a nerve injury treatment transplant material of the following i) or ii) in combination with an active oxygen scavenger to a nerve injury site: i) the graft material for treatment of nerve injury according to any one of claims 1 to 8, or ii) A transplant for treatment of nerve damage including dental pulp cells whose GABRB1 gene expression level is enhanced by FGF2 treatment.
  • the present invention provides: [13] A method for producing a transplant for treatment of nerve damage,
  • the present invention relates to a production method including a step of measuring the expression of GABRB1 gene in dental pulp cells and a step of selecting dental pulp cells expressing GABRB1 gene.
  • a method for producing a graft material for treatment of nerve injury according to the present invention includes: [14] A method for producing an implant for treating nerve damage according to [13] above, Further measure at least one gene selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene, and express at least one gene selected from the group The method further includes the step of selecting dental pulp cells.
  • a method for producing a graft material for treatment of nerve injury according to the present invention includes: [15] A method for producing a graft material for treating nerve damage according to [13] or [14] above, Before the step of measuring the gene expression of the dental pulp cells, the method comprises culturing the dental pulp cells in a medium containing FGF2.
  • a method for producing a graft material for treatment of nerve injury according to the present invention includes: [16] A method for producing an implant for treating nerve damage according to any one of [13] to [15], The culture in the step of culturing the dental pulp cells in a medium containing FGF2 is performed for at least 6 days.
  • a method for producing a graft material for treatment of nerve injury according to the present invention includes: [17] A method for producing a graft material for treating nerve damage according to any one of [13] to [16],
  • the step of selecting dental pulp cells expressing the GABRB1 gene is a step of selecting dental pulp cells having a GABRB1 gene expression level that is 10 times or more higher than the GABRB1 gene expression level of FGF2-untreated dental pulp cells. It is characterized by being.
  • a method for producing a graft material for treatment of nerve injury according to the present invention includes: [18] A method for producing a graft material for treating nerve damage according to any one of [13] to [17],
  • the nerve injury is spinal cord injury, cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disc, sciatica, or peripheral nerve injury caused by diabetes.
  • the graft material for treating spinal cord injury that expresses the GABRB1 gene and includes dental pulp stem cells having resistance to active oxygen has excellent cell engraftment after cell transplantation, and Has an excellent therapeutic effect on spinal cord injury.
  • FIG. 2a shows the transplantation of dental pulp cells of DP-1 strain or DP31 strain (DP1-FGF2 (-) or DP31-FGF2 (-)) not treated with FGF2 into spinal cord injury model rats. Or the open field motor function evaluation result by BBB score after administering PBS is shown. Error bars indicate standard deviation ( ⁇ S.D.). The significant difference test was performed by Bonferroni post hoc test after two-way ANOVA.
  • FIG. 2 b is a graph showing the results of measuring the electrophysiological action potential through a completely cut T10 site during an electrical stimulation of 0.6 mA.
  • FIG. 3a shows the results of an open field motor function evaluation by BBB score after administration of FGF2 or PBS to a spinal cord injury model rat. Error bars indicate standard deviation ( ⁇ S.D.). The significant difference test was performed by Bonferroni post hoc test after two-way ANOVA. Asterisks indicate statistical significance compared to the control group administered with PBS (* P ⁇ 0.05, ** P ⁇ 0.01).
  • FIG. 3b is a graph showing the results of measuring electrophysiological action potentials through the T10 site, which is the site of injury, when FGF2 was administered to spinal cord injury model rats and 0.6 mA electrical stimulation was performed. . Only in 1 out of 4 animals administered FGF2 could an action potential be detected beyond the injury site.
  • Figure 4a shows human dental pulp cells from different donors treated with FGF2 (DP1-FGF2 (+), DP31-FGF2 (+), DP165-FGF2 (+), DP296-FGF2 (+), PBS administration (control))
  • FGF2 DP1-FGF2 (+), DP31-FGF2 (+), DP165-FGF2 (+), DP296-FGF2 (+), PBS administration (control)
  • the result of the open field motor function evaluation by BBB score when transplanted into a spinal cord injury model rat is shown. Error bars indicate standard deviation ( ⁇ S.D.).
  • the significant difference test was performed by Bonferroni post hoc test after two-way ANOVA.
  • An asterisk (*) indicates statistical significance compared to the control group administered with PBS (* P * ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001).
  • FIG. 1 shows human dental pulp cells from different donors treated with FGF2 (DP1-FGF2 (+), DP31-FGF2 (+
  • FIG. 4b is a graph showing the results of measuring electrophysiological action potential through the T10 site, which is the damaged site, when human dental pulp cells derived from each donor were transplanted into a spinal cord injury model rat and electrical stimulation of 0.6 mA was performed. It is.
  • FIG. 4c shows a graph plotting the latency of each section in FIG. 4b.
  • FIG. 5a shows an image of a state in which 4 ⁇ 10 6 human dental pulp cells treated with FGF2 were transplanted into the vitreous cavity of a rat eye and 3 weeks after the transplantation.
  • Figures 5b and c similarly show a split face image of the removed vitreous cavity three weeks after transplantation.
  • the scale bar indicates 500 ⁇ m.
  • FIG. 6 shows an immunohistochemically-stained image of the sagittal plane of the spinal cord 8 weeks after transplanting DP165-FGF2 (+) or DP296-FGF2 (+) to a spinal cord injury model rat.
  • 6a to 6c show images of the DP165-FGF2 (+) transplantation area, and FIGS.
  • FIG. 6d to 6f show images of the DP296-FGF2 (+) transplantation area.
  • FIG. 6a shows a high magnification image of the area indicated by 1 in FIG. 6a.
  • FIG. 6 c shows a high magnification image of the caudal ventral area of the DP296-FGF2 (+) transplantation zone. Myelinated regeneration axons could be observed from the center to the caudal ventral side.
  • FIG. 6d almost no GAP43 positive cells were observed.
  • FIG. 6e shows a high magnification image of the area indicated by 3 in FIG. 6d.
  • FIG. 6f shows a high-magnification image of the caudal ventral area of the DP296-FGF2 (+) transplantation zone.
  • FIG. 6f myelinated axons have disappeared.
  • the scale bar in FIG. 6 indicates 500 ⁇ m in FIGS. 6a and 6d, and 100 ⁇ m in FIGS. 6b, 6c, 6e, and 6f.
  • the arrowheads in FIGS. 6a and 6d indicate the center of the injury site.
  • FIGS. 7a to c show images of fluorescence staining of the spinal cord at 8 weeks in the PBS administration group (FIG. 7a), the DP165 transplantation group (FIG. 7b), and the DP296 transplantation group (FIG. 7c) after spinal cord injury.
  • FIG. 10 shows Example 3 below. It is the schematic which shows the culture schedule of each human dental pulp cell used for "the influence on the tolerance with respect to the active oxygen of the human dental pulp cell by FGF2 treatment".
  • FIG. 11 is a graph showing the results of an active oxygen tolerance test (MTT assay) on human dental pulp cells cultured in each culture schedule shown in FIG.
  • FIG. 12 is a culture method of the S / F group and the F / S group of the respective culture schedules shown in FIG. 10, and further shows a reactive oxygen tolerance test of human dental pulp cells when the number of culture days is changed ( It is a graph which shows the result of MTT assay.
  • FIG. 13 shows a fluorescence microscopic image of the longitudinal section of the spinal cord in a combination of transplantation of FGF2-untreated DP31 strain human dental pulp cells (DP31-S) and edaravone administration (daily).
  • 13B and 13C are enlarged views of the region shown in FIG. 13A.
  • the scale bars in FIGS. 13A and C show 1 mm and 200 ⁇ m, respectively.
  • the right and left sides of the image indicate the caudal and rostral sides of the spinal cord, respectively.
  • FIG. 14 (A) shows an image (20 magnifications) showing the state of the spinal cord injury in the spinal cord injury model rat transplanted with DP31-S and showing the state 7 weeks after cell transplantation.
  • FIG. 14 (A) shows an image (20 magnifications) showing the state of the spinal cord injury in the spinal cord injury model rat transplanted with DP31-S and showing the state 7 weeks after cell transplantation.
  • FIG. 14 (B) shows an image (20 magnifications) showing the state of the spinal cord injury in a spinal cord injury model rat transplanted with DP31-S and combined with edaravone, and the state after 7 weeks after cell transplantation.
  • FIG. 14 (C) shows the number of GFP-positive cells at the site of spinal cord injury (average) 7 weeks after the day of transplantation to the spinal cord injury model rat in the DP31-S transplantation group and the DP31-S transplantation and edaravone combination group. Value). Values are expressed as mean ⁇ standard error. The significant difference test was performed by Student's t-test (* p ⁇ 0.05). The scale bar indicates 200 ⁇ m.
  • FIG. 14 (C) shows an image (20 magnifications) showing the state of the spinal cord injury in a spinal cord injury model rat transplanted with DP31-S and combined with edaravone, and the state after 7 weeks after cell transplantation.
  • FIG. 14 (C) shows the number of
  • the present invention is a property of dental pulp cells treated with FGF2, and is particularly important for the treatment of nerve damage, and is characterized by the expression of the GABRB1 gene and / or resistance to active oxygen. I found it important to have a combination. That is, in one aspect, the present invention provides a transplant material for treating nerve damage, comprising dental pulp cells in which the GABRB1 gene is expressed, active oxygen tolerance, or both properties are combined. . A preferred embodiment is a transplant material for treating nerve damage, which contains dental pulp cells expressing the GABRB1 gene and having active oxygen resistance. Further, another embodiment of the present invention includes dental pulp cells treated with FGF2, wherein the pulp cells express GABRB1 gene, have active oxygen resistance, or have both properties. Providing a graft for the treatment of nerve damage.
  • the “dental pulp cell” refers to a cell collected from dental pulp tissue of a deciduous tooth or a permanent tooth, and including a dental pulp stem cell.
  • the dental pulp cells used in the present invention are not limited as long as they have an effect of improving the function of nerve damage when transplanted to a nerve damage site, and even cultured cells can be cultured directly from a living body. It may also be a cell thawed after cryopreservation.
  • the present invention provides, as one embodiment, a transplant material for treating nerve damage, which is a dental pulp stem cell treated with FGF2 and containing dental pulp stem cells expressing the GABRB1 gene.
  • a transplant material for treating nerve damage which is a dental pulp stem cell treated with FGF2 and containing dental pulp stem cells expressing the GABRB1 gene.
  • dental pulp cell can be read as “dental pulp stem cell” as described above.
  • tissue stem cell is a type of tissue stem cell that can be isolated from dental pulp tissue.
  • Tissue stem cells are also referred to as somatic stem cells, and tissue stem cells are limited in the types of cells that can be differentiated from embryonic stem cells that can be differentiated into any cells.
  • Dental pulp stem cells are characterized, for example, by the surface antigen STRO-1.
  • neural crest cell markers such as Nestin, SOX10, and SOX11 as an index.
  • pulp tissue containing pulp cells can be collected from both deciduous teeth and permanent teeth, and can be obtained from pulp of extracted teeth such as deciduous teeth and wisdom teeth that have been treated as medical waste conventionally.
  • the dental pulp tissue can be removed from teeth that have been extracted dentally in a dental facility, and may be extracted from naturally extracted teeth.
  • the method of taking out pulp tissue from a tooth is well-known, and those skilled in the art can implement suitably.
  • a freezing treatment cannot be performed immediately on the spot, such as a tooth extracted in a dental procedure, the tooth is immersed in a medium such as ⁇ -MEM for transport, and the temperature is low (for example, 4 °C).
  • a method for isolating dental pulp cells from dental pulp tissue can be performed, for example, as follows. After the dental pulp tissue is shredded using a scalpel or the like, the shredded pulp tissue is enzymatically treated using dispase, collagenase, or a mixed enzyme solution thereof. After the enzyme treatment, the mixture is thoroughly mixed with a culture solution containing serum, and then impurities are removed using a cell strainer or the like. Thereafter, centrifugation (for example, 2,000 rpm, 4 ° C.) is performed, the supernatant is discarded, and pulp cells can be collected from the dental pulp tissue by adding a culture solution.
  • centrifugation for example, 2,000 rpm, 4 ° C.
  • dental pulp cells isolated from dental pulp tissues contain dental pulp stem cells in the cell population.
  • a dental pulp cell group containing dental pulp stem cells can be used as it is and cultured in a medium containing FGF2.
  • the dental pulp stem cells may be separated from the dental pulp tissue-derived cell population by a known method using the above cell surface marker and then treated with FGF2.
  • the origin of dental pulp tissue is not limited to humans, but may be other mammals (eg, mouse, rat, rabbit, dog, cat, monkey, sheep, cow, horse).
  • the dental pulp stem cells used in the transplant material for the treatment of nerve damage of the present invention are those isolated from dental pulp tissues and treated with FGF2 by culturing in a medium containing FGF2.
  • FGF2 By treating the dental pulp cells with FGF2, it is possible to obtain dental pulp cells that are enhanced in the expression of the GABRB1 gene and that are suitable as a transplant material for treating nerve damage.
  • the “GABRB1 gene” is a gene encoding gamma-aminobutyric acid type A receptor beta1 subunit.
  • the dental pulp cell says “GABRB1 gene is expressed”, it has a significantly increased GABRB1 gene expression level compared to the GABRB1 gene expression level of dental pulp cells not treated with FGF2.
  • the dental pulp cell expresses the GABRB1 gene '', it is not limited to the following.
  • the expression of the GABRB1 gene of the dental pulp cell Compared with the expression level of GABRB1 gene in dental pulp cells not treated with FGF2, the amount is about 10 times or more, about 20 times or more, about 100 times or more, more preferably about 200 times or more. Is. Due to the difference in the origin of the donor, transplantation of a treatment material containing pulp cells could not be expected to improve nerve damage, but pulp cells whose GABRB1 gene expression was enhanced by FGF2 treatment It has an excellent therapeutic effect on nerve damage when transplanted to a site.
  • MMP1 gene in addition to GABRB1 gene, "MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, SCG2 gene, And at least one gene selected from the group consisting of NTSR1 gene "is expressed.
  • the “MMP1 gene” is a gene encoding Matrix metallopeptidase 1 (interstitial collagenase).
  • D2 gene is a gene encoding Dopamine receptor D2.
  • ABCA6 gene is a gene encoding ATP-binding cassette, sub-family A (ABC1), member6.
  • the “TMEM100 gene” is a gene encoding Transmembrane protein 100.
  • the “THBD gene” is a gene encoding thrombomodulin.
  • NTSR1 gene refers to neurotensin receptor 1.
  • the “SCG gene” is a gene encoding Secretogranin II. In the present specification, when ⁇ at least one gene selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, SCG2 gene, and NTSR1 gene is further expressed '' GABRB1 In addition to gene expression, it means that one, two, three, four, five, six, or seven genes of the above-listed genes are expressed.
  • dental pulp cells expressing one of the genes listed above are specifically pulp cells expressing the GABRB1 gene and MMP1 gene, GABRB1 gene and DRD2 Dental pulp cells in which GABRB1 gene and ABCA6 gene gene are expressed, dental pulp cells in which GABRB1 gene and TMEM100 gene gene are expressed, GABRB1 gene and THBD gene are expressed Dental pulp cells, dental pulp cells expressing GABRB1 gene and SCG2 gene, and dental pulp cells expressing GABRB1 gene and NTSR1 gene.
  • MMP1 gene when two, three, four, five, six, or seven genes of the gene group listed above are expressed, MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, All combinations of two genes selected from the group consisting of THBD gene, SCG2 gene, and NTSR1 gene, all three gene combinations, all four gene combinations, all five gene combinations, Includes all six gene combinations or all seven gene combinations.
  • genes listed above when the genes listed above are expressed, it means that they have enhanced gene expression compared to the expression level of the corresponding gene in dental pulp cells not treated with FGF2. .
  • genes whose expression level is significantly increased are MMP1, DRD2, ABCA6, TMEM100, THBD, and SCG2 genes. is there.
  • a dental pulp cell in which the MMP1 gene is expressed is, in a preferred form, a pulp having a significantly increased expression level of the MMP1 gene compared to the expression level of the MMP1 gene in dental pulp cells not treated with FGF2.
  • a cell As with the GABRB1 gene, MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene are also cultured in the presence of FGF2 in dental pulp cells isolated from dental pulp tissue. Unless it passes through specific conditions such as, the expression is not enhanced. Moreover, the expression level of these genes is enhanced by culturing dental pulp cells in the presence of FGF2 for a certain period.
  • the dental pulp cells of the present invention are not limited to the following as long as the expression of each gene is enhanced.
  • the dental pulp cells whose MMP1 gene expression has been enhanced by FGF2 treatment is an MMP1 gene increased by about 145 times or more compared to the expression level of the MMP1 gene in dental pulp cells not treated with FGF2.
  • the dental pulp cells whose DRD2 gene expression has been enhanced by FGF2 treatment has an increased DRD2 of about 104 times or more compared to the expression level of the DRD2 gene in dental pulp cells not treated with FGF2.
  • pulp cells whose ABCA6 gene expression is enhanced by FGF2 treatment are increased by about 17 times or more compared to the expression level of ABCA6 gene in pulp cells not treated with FGF2.
  • the dental pulp cells in which the expression of the TMEM100 gene is enhanced by FGF2 treatment is increased by about 54 times or more compared to the expression level of the TMEM100 gene in dental pulp cells not treated with FGF2.
  • dental pulp cells whose THBD gene expression has been enhanced by FGF2 treatment are increased by about 6 times or more compared to the THBD gene expression level in dental pulp cells not treated with FGF2. Has gene expression level.
  • the dental pulp cells in which the expression of NTSR1 gene is enhanced by FGF2 treatment the SCG2 gene increased by 8 times or more compared to the expression level of NTSR1 gene in dental pulp cells not treated with FGF2. Expression level.
  • the dental pulp cells whose SCG2 gene expression is enhanced by FGF2 treatment is increased by about 5 times or more compared to the expression level of the SCG2 gene in dental pulp cells not treated with FGF2. Has gene expression level.
  • the expression of GABRB1 gene, MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene can be measured using a known method, for example, real time PCR, Northern blotting, Examples include in situ hybridization, RNAse protection assay, and reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • the reaction conditions for Real-time PCR were as follows: using primers corresponding to each gene, 40 cycles at 95 ° C for 30 seconds, 95 ° C for 5 seconds, and 60 ° C for 30 seconds for 1 cycle. One cycle of 15 seconds, 60 ° C 30 seconds, 95 ° C 15 seconds.
  • the expression of these genes as an index, it is possible to screen for dental pulp cells suitable for a transplant material for treating nerve damage.
  • enhanced expression of the GABRB1 gene by FGF2 treatment is correlated with the therapeutic effect, and therefore, enhanced expression of the GABRB1 gene is preferably used as an index.
  • the present invention is a method for screening dental pulp cells suitable for a transplant material for treatment of nerve damage, wherein the expression of GABRB1 gene in FGF2-treated dental pulp cells is measured and the GABRB1 gene is expressed.
  • a method comprising selecting dental pulp cells that are present.
  • One embodiment of the screening method includes at least one selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene in addition to GABRB1 gene. It includes a form in which gene expression is measured and dental pulp cells expressing the measured gene are selected.
  • “having active oxygen resistance” means that dental pulp cells have resistance to the toxicity of active oxygen.
  • dental pulp cells when dental pulp cells are used as a transplant material, it means that they have resistance against the toxicity of active oxygen generated in the tissue of the damaged site of the transplant destination.
  • the dental pulp cells having active oxygen resistance of the present invention have a higher cell engraftment rate after transplantation than dental pulp cells not having active oxygen resistance, and have an excellent effect on improving the function of the damaged site. is there.
  • the active oxygen tolerance of dental pulp cells is not limited to the following. For example, hydrogen peroxide (H 2 O 2 ) is added to the culture medium in which dental pulp cells are cultured, and then cultured by MTT assay or the like. Can be evaluated.
  • H 2 O 2 hydrogen peroxide
  • any one of the following conditions a) to f) is satisfied: A) Living dental pulp cells cultured in 10% FCS- ⁇ MEM medium containing 0.5 mM hydrogen peroxide for 24 hours were cultured in 10% FCS- ⁇ MEM medium without addition of hydrogen peroxide for 24 hours. Surviving at a rate of about 50% or more, preferably about 60% or more, more preferably about 70% or more, and further preferably about 80% or more with respect to the number of living dental pulp cells.
  • D) The living cells of dental pulp cells when cultured in 10% FCS- ⁇ MEM medium containing 0.5 mM hydrogen peroxide for 24 hours are the living cells when dental pulp cells not treated with FGF2 are cultured under the same conditions for 24 hours.
  • E) Living dental pulp cells when cultured in 10% FCS- ⁇ MEM medium containing 0.6 mM hydrogen peroxide for 24 hours are living cells when dental pulp cells not treated with FGF2 are cultured under the same conditions for 24 hours.
  • Living dental pulp cells when cultured in 10% FCS- ⁇ MEM medium containing 0.7 mM hydrogen peroxide for 24 hours are living cells when dental pulp cells not treated with FGF2 are cultured under the same conditions for 24 hours. It survives at a rate of about 3 times or more, more preferably about 4 times or more, and still more preferably about 5 times or more of the number.
  • the method of treating FGF2 in order to use dental pulp cells as a transplant for treatment of nerve damage can be performed, for example, according to the method described in International Publication No. 2014/185470.
  • dental pulp cells isolated from dental pulp tissue are cultured in a medium containing FGF2 for a certain period of time to perform FGF2 treatment.
  • the term “medium containing FGF2” refers to, for example, a medium in which FGF2 is added as a growth factor to a basic medium containing serum; a medium in which FGF2 is added to a basic medium not containing serum Medium supplemented with FGF2 in a basic medium containing serum; medium in which FGF2 is added as a growth factor to a medium marketed as a medium for mesenchymal stem cell culture; marketed as a medium for mesenchymal stem cell culture Examples of the medium include a medium in which FGF2 is added.
  • FGF2 means a basic fibroblast growth factor (FGF), and is also called bFGF or HBGF-2.
  • FGF2 can be used by appropriately diluting a commercially available one. Since it is used as a transplant, it is preferably filtered through an appropriate membrane and confirmed to be negative for bacteria, fungi, mycoplasma and the like.
  • concentration of FGF2 is not particularly limited as long as the obtained transplantation material has a sufficient effect of treating spinal cord injury, and can be, for example, 5 ng / ml or more, 7 ng / ml or more, 10 ng / ml or more.
  • a preferred embodiment is a method of culturing dental pulp cells using a medium supplemented with FGF2 at a concentration of 10 ng / ml.
  • the treatment with FGF2 is not limited to the following as long as the expression of the GARBR1 gene is enhanced, but an embodiment in which FGF2 is added to the medium every day is preferable.
  • basic medium refers to a medium containing only known components of low molecular weight.
  • basic medium include BME (Basal medium Eagle's), MEM (Minimum essential medium), DMEM (Dulbecco's modified) Eagle's medium such as Eagle's medium, RPMI1630, RPMI1640, RPMI medium such as Roswell'Park'Memorial'Institue, Fischer's medium, F10 medium, F12 medium, Ham's'medium, MCDB104, 107, 131, 151, MCDB media such as 153, 170, 202, and RITC80-7 media are known and can be appropriately selected.
  • “medium marketed as a medium for culturing mesenchymal stem cells” means a commercially available medium for culturing and proliferating mesenchymal stem cells while maintaining differentiation ability without inducing differentiation.
  • MSCGM medium LONZA
  • mesenchymal stem cell growth medium Takara Bio Inc.
  • mesenchymal stem cell growth medium DXF Takara Bio Inc.
  • Stemline registered trademark
  • mesenchymal stem cell growth medium Sigma) -Aldrich
  • MF-medium (trademark) mesenchymal stem cell growth medium Toyobo Life Science
  • BD Mosaic serum-free culture kit for human mesenchymal stem cells (BD bioscience). It is not limited.
  • serum is not limited as long as it is serum used for cell culture, and examples thereof include human serum, fetal bovine serum, and horse serum.
  • human serum When the therapeutic transplant according to the present invention is transplanted into a human, it is preferably human serum.
  • the serum is preferably less than 15% by weight, less than 13% by weight, less than 10% by weight, less than 8% by weight, less than 5% by weight, etc. in the medium.
  • a “medium substantially free of growth factors other than FGF2” can be used.
  • “medium substantially free of growth factors other than FGF2” means that the only growth factor that is intentionally added is FGF2.
  • growth factor means various proteins called growth factors or growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), acidic fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (bFGF or FGF2), platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth Factor (TGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) interleukins and the like.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • aFGF or FGF1 acidic fibroblast growth factor
  • bFGF or FGF2 basic fibroblast growth factor
  • PDGF platelet-derived growth factor
  • NGF nerve growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • TGF transforming growth Factor
  • VEGF vascular endothelial growth factor
  • substances useful for cell culture can be appropriately added to the medium used in the method for producing a transplant material for treatment of spinal cord injury according to the present invention.
  • Such substances include, for example, a buffer for stabilizing pH (such as HEPES), pH indicator phenol red, antibiotics (penicillin G, streptomycin, amphotericin B, gentamicin, kanamycin, ampicillin, minomycin, gentamicin, etc.), amino acids, Vitamins, lipids, carbohydrates, nucleic acids, inorganic salts, organic acid salts, minerals, and the like are included, but are not limited thereto.
  • the dental pulp cells In the method for producing a transplant material for treatment of nerve damage according to the present invention, it is also preferable to subculture the dental pulp cells twice, three times, four times, five times, or six times or more in the above-mentioned medium. In order to confer active oxygen resistance to dental pulp cells by FGF2 treatment, it is preferable to carry out at least two subculture periods or at least 6 days of culture. Even dental pulp cells treated with FGF2 lose resistance to active oxygen by subculturing 1-2 times in a medium not containing FGF2 or by culturing in a medium not containing FGF2 for about 3 days.
  • the dental pulp cells are not limited as long as they have a therapeutic effect on nerve damage, but the culture using a medium containing FGF2 is more preferably performed until just before the dental pulp cells are used as a therapeutic transplant.
  • the culture method is not particularly limited except that it is cultured in a medium that does not substantially contain a growth factor other than FGF2, and those skilled in the art can use various conditions (temperature, humidity, CO 2 concentration) depending on the type of cells to be cultured. PH, medium exchange frequency, etc.) can be selected.
  • the method for producing a transplant material for treatment of nerve damage according to the present invention can perform various processes suitable as a method for producing a transplant material in addition to the above-described culture process.
  • the step of adjusting the fluidity by mixing the culture obtained in the culturing step with a highly viscous substance such as hyaluronic acid, collagen gel, fibrinogen, soft agar, or synthetic polymer may be performed.
  • a highly viscous substance such as hyaluronic acid, collagen gel, fibrinogen, soft agar, or synthetic polymer
  • the transplant can be fixed at the damaged site.
  • three-dimensional culture may be performed by culturing for a certain period of time.
  • nerve damage means central and peripheral nerve damage, specifically spinal cord injury, cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage, spinal cord hemorrhage, nerve compression injury due to disc herniation, sciatica It includes, but is not limited to, neuralgia or peripheral neuropathy due to diabetes.
  • the transplant material of the present invention can be applied to any nerve damage as long as a therapeutic effect is obtained by transplantation.
  • the therapeutic effect is not limited to the effect of curing the disease, but includes the effect of improving at least one symptom associated with the disease, the effect of preventing or delaying the progression of the disease, and the like.
  • the effect of the therapeutic transplant obtained by the method for producing a therapeutic implant for nerve injury according to the present invention can be evaluated by, for example, transplanting cells to a nerve injury model animal.
  • a nerve injury model animal For example, it can be evaluated using a rat spinal cord injury model prepared by a known method. Specifically, under anesthesia such as isoflurane, after laminectomy, the spinal cord is completely cut using a scalpel.
  • the cutting site can be, for example, the tenth thoracic vertebra (Th10). After the spinal cord is cut, hemostasis is performed, and then the medium containing dental pulp cells is transplanted to the damaged site using a syringe.
  • 10 ⁇ l of a medium containing cells at a concentration of about 1 ⁇ 10 6 cells is transplanted to the damaged site. After cell injection, leave it for about 10 minutes and close the wound with suture. Rats may be placed in a rewarming chamber until they wake up after surgery. Moreover, you may administer the antagonist of anesthesia as needed. After surgery, antibiotics and immunosuppressants may be administered as necessary. For example, at the time of 7 weeks after transplantation, evaluation of improvement of motor function, evaluation of tissue repair by immunohistochemical staining of dental pulp cell transplantation site, evaluation of functional recovery of damaged site by electrophysiological method, and treatment Confirm the effect of the transplant.
  • BBB score Basso DM et al., J Neurotrauma. 1995 Feb; 12 (1): 1-21). Further, immunohistochemical staining can be performed on a frozen section of a spinal cord injury site prepared by a known method. Specifically, the rat is transcardially fixed under anesthesia and the spinal cord tissue is collected. The tissue is frozen and embedded using an embedding agent, and a section is prepared using a cryostat. Immunostaining uses anti-growth-associated protein (GAP) 43 antibody, a growth cone marker, anti-glial fibrillary acidic protein (GFAP) antibody, an anti-GFP antibody, and anti-MBP antibody, an oligodendrocyte marker. Tissue can be stained.
  • GAP anti-growth-associated protein
  • GFAP anti-glial fibrillary acidic protein
  • oligodendrocyte marker an oligodendrocyte marker.
  • GAP43-positive spinal cord regeneration fibers are three times as large as the control group in which no pulp cells are transplanted. Above, preferably 5 times or more, more preferably 7 times or more.
  • the evaluation by an electrophysiological method can be performed by measuring the electrical action potential through the spinal cord amputation part. For example, for rats that have had their Th10 site cut, under anesthesia, a microelectrode is inserted into the spinal cord so that Th8 can be electrically stimulated through Th10 and an action potential can be detected at Th13. To do.
  • the electrical stimulation can be evaluated by, for example, applying a short rectangular wave pulse (0.2 seconds) every 2 seconds, detecting the action potential in Th13, and measuring the latency.
  • the transplant for nerve injury treatment includes the above-described dental pulp cells in which the GABRB1 gene is expressed by FGF2 treatment.
  • a medium containing FGF2 in which dental pulp cells have been cultured can be used as a therapeutic transplant material in a state containing dental pulp cells as it is.
  • the dental pulp cells may be transferred to the above-mentioned medium or solution and used as a therapeutic transplant material.
  • the transplant material may contain gel such as collagen gel, soft agar, and synthetic polymer, and the viscosity may be adjusted by a suitable gelling agent or thickener.
  • the present invention also includes a method for treating nerve damage, including the step of transplanting the above-described graft material for treating nerve damage to a nerve damage site.
  • the graft material for nerve injury treatment can be injected into the nerve injury site by a syringe or the like, for example.
  • the transplanted material may be placed by cutting the damaged site.
  • an immunosuppressant such as cyclosporine may be administered simultaneously.
  • the effect of treating nerve damage is obtained, it can be used in combination with other drugs.
  • a person skilled in the art can appropriately determine the dose and the number of doses.
  • the subject of the method for treating nerve damage is not limited to humans, but may be other mammals (eg, mouse, rat, rabbit, dog, cat, monkey, sheep, cow, horse).
  • an example of a method for treating human spinal cord injury is shown below, but the method of treatment is not limited thereto.
  • An effective amount of dental pulp cells can be directly transplanted to the site using a syringe or the like.
  • the method for treating nerve damage according to the present invention is a method including the step of transplanting the above-mentioned graft material for treating nerve damage to a nerve damage site in combination with an active oxygen removing agent.
  • an active oxygen scavenger means an agent that has an effect of preventing damage to a damaged site caused by active oxygen occurring at a nerve injury site in a living body or a transplanted dental pulp cell. Or administered in combination.
  • Such an active oxygen scavenger is not particularly limited as long as it can be administered to a living body and has an effect of protecting transplanted cells from damage of active oxygen at a damaged site.
  • edaravone for example, edaravone, vitamin C , Known active oxygen scavengers such as Nrf2 inducer and glutathione activity inducer can be used.
  • a person skilled in the art can appropriately set the administration method, dose and frequency of administration of the active oxygen scavenger depending on the active oxygen scavenger used and the site of injury.
  • edaravone is administered as an active oxygen scavenger to spinal cord injury rats in combination with pulp cell transplantation, it is not limited to the following, but edaravone is administered 3 times twice a day for 1 week immediately after cell transplantation surgery. It can be administered intraperitoneally at a dose of mg / kg.
  • edaravone alone cannot be recovered by simply administering it to a spinal cord injury model.
  • an active oxygen scavenger when edaravone is administered to humans in combination with pulp cell transplantation, it is not limited to the following, but it is divided into 1 or 2 times a day immediately after cell transplantation surgery, Edaravone can be infused intravenously at a dose of 60 mg / kg over a week.
  • the nerve injury treatment method of the present invention is a method of transplanting a nerve injury treatment graft material to a nerve injury site in combination with an active oxygen remover.
  • the active oxygen scavenger has an effect of preventing cell damage caused by active oxygen occurring at the site of nerve injury. Therefore, the dental pulp cells transplanted in combination with the administration of the active oxygen removing agent do not necessarily need to have active oxygen resistance. That is, the dental pulp cells used for transplantation do not necessarily need to be dental pulp cells that have acquired resistance to active oxygen by FGF2 treatment.
  • the present invention also includes a kit for producing an implant for treating nerve damage.
  • a kit for producing an implant for treating nerve damage contains a medium for culturing dental pulp cells or all or part of its components, FGF2, and a gene expression measurement reagent (for example, a primer for amplifying GABRB1).
  • the active oxygen removal agent used at the time of cell transplantation may be contained.
  • the medium for culturing dental pulp cells include a basal medium or a mesenchymal stem cell culture medium.
  • FGF2 may be separated from the medium or may be mixed from the beginning.
  • the medium should be prepared by the user, such as ultrapure water, which is always available in the laboratory, and contains all or part of the necessary components so that the medium of the present invention can be prepared simply by mixing it. It may be.
  • the kit of the present invention may be used for experiments in a laboratory or may be used for mass culture.
  • a culture vessel In addition to the culture solution, a culture vessel, a virus filter, a coating material for the culture vessel, various reagents, a buffer solution, and an instruction manual may be provided.
  • Example 1 Treatment of spinal cord injury with human dental pulp cells treated with FGF2> To date, it has been reported that either one of dental pulp cell transplantation or FGF2 administration has promoted functional recovery from a completely cut spinal cord. In order to confirm these effects, human dental pulp cells were transplanted into the spinal cord injury of rats, and a test for evaluating hindlimb motor function (BBB score) in combination with electrophysiological tests was conducted. In addition, the developmental stage tooth used to prepare human dental pulp cells has been confirmed to have a difference in efficiency for iPS induction for each donor. In this example, the effect of improving spinal cord injury after transplantation was compared using each human dental pulp cell line obtained from four donors with different ages and genders shown in the following table.
  • BBB score hindlimb motor function
  • the lentiviral vector contains a sequence in which a Venus gene is bound downstream of the EF1 ⁇ promoter.
  • Human dental pulp cells were cultured in MSCGM medium (Lonza) at 37 ° C. under humidified air of 21% O 2 , 5CO 2 and infected with lentivirus to trace the cells in transplants at passages 5-6. It was. After lentiviral infection, human dental pulp cells were cultured in ⁇ -MEM medium (Sigma). The efficiency of virus infection was confirmed using FACS.
  • FGF2 (R & D SYSTEMS) was added to ⁇ -MEM medium at a concentration of 10 ng / ⁇ l every day, and human dental pulp cells treated with FGF2 were used for transplantation at passage 10-13.
  • the FGF2-treated dental pulp cells transplanted from each donor are referred to as DP1-FGF (+), DP31-FGF (+), DP165-FGF (+), and DP296-FGF (+), respectively.
  • DP1 or DP31 passages were produced in ⁇ -MEM medium not containing FGF2, and were similarly used for transplantation at passages 10 to 13 (DP1 respectively).
  • antibiotics sulbactam / ampicillin, SANDOZ, 10 mg / kg weight
  • immunosuppressants ((cyclosporine, Novartis, 10 mg / kg body weight) were administered daily from the day before surgery to the rats transplanted with human dental pulp cells and control rats (PBS-treated group).
  • Laboratory animals can be easily supplied with a custom-made water supply facility equipped with a long nozzle, and are kept in an environment where food can be consumed at all times, and at 26 ° C, 65% humidity, with 12 hours of light per day. Condition.
  • hypodermic needle was then removed and a 30 gauge blunt needle (Hamilton Company, Reno, NV, USA) was inserted into the anterior chamber through the created hole and avoiding the glow and the lens.
  • the needle was inserted toward the retina surface through the vitreous cavity, and after the needle reached the retina surface, 10 ⁇ l of DMEM medium containing DP31 treated with FGF2 was injected into the vitreous cavity.
  • 10 ⁇ l of DMEM medium containing DP31 treated with FGF2 was injected into the vitreous cavity.
  • one drop of ofloxacin ophthalmic solution (Tarivid topical solution, Santen) was instilled into the eye.
  • Six rats were used in this study, two of which were transplanted with 0.5 ⁇ 10 6 cells, and the remaining four were transplanted with 4 ⁇ 10 6 cells. All rats received an immunosuppressant ((cyclosporine, Novartis, 10 mg / kg body weight) from the day before surgery to the day after surgery.
  • BBB Score> In order to objectively analyze the motor function of rats after surgery, the rats were recorded on video for 30 seconds to 2 minutes every week after surgery. The BBB locomotion rating scale was assessed by eliminating the group identity by each independent observer who had previously performed a BBB score analysis and viewing the video (Basso et al., 1996). Detailed evaluation criteria are shown in FIG.
  • Electrophysiological tests In order to confirm whether it is possible to transmit an electrical action potential through the spinal cord amputation, the rat after human dental pulp cell transplantation was subjected to an electrophysiological test by the following method. The rats were placed under anesthesia using urethane and the organs of the rats were dissected to secure the airways. The transmission of electrical activity through the graft was evaluated using the following method. In this test, a bespoke bipolar electrode equipped with tungsten microelectrodes ( ⁇ 0.2 mm, Unique Medical, Japan.) At intervals of 1 mm was used.
  • the electrodes were inserted 1.0 m below and 0.5-0.75 mm lateral to the center of the spinal cord so as to stimulate the Th8 spinal cord segment and record electrical activity at the Th13 spinal cord segment.
  • Microelectrodes are inserted by using a spinal fixation device (ST-7R-HT, NARISHIGE, Tokyo, JAPAN) to fix the rat, control the microelectrode with a manipulator, and confirm the position of the microelectrode under a surgical microscope. It was done by doing. For electrical stimulation, a short rectangular wave pulse (0.2 seconds) was applied every 2 seconds.
  • Frozen sections were prepared as anti-GFAP antibody (rabbit IgG, 1: 500, abcam), anti-GAP-43 antibody (mouse IgG, 1: 200, Millipore), anti-GFP antibody (rabbit IgG, 1: 200, Millipore) as primary antibodies. ) And anti-MBP antibody (rabbit IgG, 1: 200, Millipore). Subsequently, staining with anti-rabbit IgG-Alexa 546, anti-mouse IgG-Alexa Fluor 488, anti-mouse IgG-Alexa 546, and anti-rabbit IgG-Alexa Fluor 488 as secondary antibodies, Stained with DAPI (Sigma-Aldrich). Images of stained sections were acquired using a fluorescence microscope (BZ-9000, Keyence).
  • FIG. 6 shows the results of immunohistochemistry of the spinal cord when the spinal cord was completely cut and 8 weeks after human dental pulp cell transplantation.
  • GAP43-positive axons increased along the GFAP-positive astrocytes (Figs. 6a and b).
  • the regenerated axon was also involved in myelination, and it was possible to confirm a myelin that was MBP positive (FIG. 6c).
  • FIG. 6d, e, and f shows the results of immunohistochemistry of the spinal cord when the spinal cord was completely cut and 8 weeks after human dental pulp cell transplantation.
  • Example 2 Regulation of GABRB1 expression in human dental pulp cells by FGF2>
  • DP1, DP31, DP165, and DP296 are dental pulp cells that does not exert a therapeutic effect on nerve damage even by FGF2 treatment.
  • Each human dental pulp cell used for gene expression analysis is the above described 1-1.
  • FGF2 R & D SYSTEMS
  • FGF2 non-added group a similarly isolated human dental pulp cell cultured in an ⁇ -MEM medium not containing FGF was prepared (FGF2-non-treated group). As each cell, cells at passage 10-13 were used. In addition, comprehensive gene analysis was performed using a cDNA microarray, and then specific gene expression was analyzed using Real-time PCR. The dental pulp cells used in this example were those that had not been transfected with a lentivirus. ⁇ 2-1. cDNA Microarray> Total RNA from cultured human dental pulp cells was isolated using Rneasy (registered trademark) Plus Mini Kit (Qiagen, Valencia, CA, USA).
  • RNA quantification using the Agilent 2100 Bioanalyzer 100 ng of total RNA was reverse transcribed and amplified using the Low Input Quick Amp Labeling kit (Agilent Technologies, Santa Clara, Calif.) According to the protocol. And labeled with Cy3-labeled CTP. After the labeling and purification steps, the cDNA was quantified using an ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE) and hybridized with a SurePrint G3 Human 8x60K v2 oligo-DNA microarray (Agilent Technologies). After hybridization, the array was washed using Gene Expression Wash Pack (Agilent Technologies).
  • the fluorescence image of the hybridized array was acquired with an Agilent DNA microarray scanner, and the fluorescence intensity was determined using Agilent Feature Extraction software ver.10.7.3.1. Each sample was analyzed once. The level of gene expression was determined using Gene Spring GX12.6 (Agilent Technologies).
  • RNA from cultured human dental pulp cells was isolated using Rneasy (registered trademark) Plus Mini Kit (Qiagen, Valencia, CA, USA).
  • Rneasy registered trademark
  • Mini Kit Qiagen, Valencia, CA, USA
  • a PCR product consisting of cDNA was prepared using SYBR Premix Ex Taq (Takara, Shiga, Japan) and Thermal Cycler Dice Real-Time System (Takara). The primers used are shown in Table 2.
  • FGF2 The other seven genes up-regulated by FGF2 (MMP1, DRD2, ABCA6, TMEM100, THBD, NTSR1 and SCG2 genes) have similar expression profiles in four different cell lines. (FIG. 9). Therefore, for each of these seven genes, enhanced expression is important for dental pulp cells used as a transplant for treatment of nerve damage.
  • dental pulp cells were cultured according to the following culture schedules i) to iv).
  • An outline of the culture schedules i) to iv) is shown in FIG. i)
  • Pulp cells cultured for 6 passages in ⁇ -MEM medium containing 10% FCS (10% FCS- ⁇ MEM) are further cultured in 10% FCS- ⁇ MEM medium for 49 hours (S / S: DPC-S Ward).
  • F / S Pulp cells obtained by culturing in DPC-FS section are further cultured in 10% FCS- ⁇ MEM medium for 1, 2, 5, or 11 days (F / S1 section) F / S 2, F / S 5 and F / S 11). In each of the culture schedules i) to vi), after the subculture, the medium was changed every 24 hours.
  • the resistance test against active oxygen is conducted according to the above 3-1. Culturing for 24 hours in 10% FCS- ⁇ MEM medium supplemented with hydrogen peroxide (H 2 O 2 ) (manufactured by Wako Pure Chemical Industries, Ltd.) for the pulp cells obtained by each culture schedule described in 1. Resistance to active oxygen was evaluated by performing MTT assay. In the MTT assay, MTT (Sigma) was added to a concentration of 0.5 mg / mL, reacted in a CO2 incubator for 4 hours, and then the formazan formed was dissolved in isopropanol containing 0.04M HCl to obtain 570 nM. Absorption wavelength was measured. Hydrogen peroxide was added to the medium in the range of 0 mM (no addition) to 0.7 mM. In addition, a condition using a medium not added with hydrogen peroxide was used as a control group.
  • H 2 O 2 hydrogen peroxide
  • FIG. 11 shows the results of a resistance test for active oxygen of dental pulp cells cultured in the above i) to iv) culture schedule.
  • S / S: DPC-S and S / F: DPC-S compared with S / S: DPC-S, F / F: DPC-FS and F / S: DPC-FS Viable cells could be confirmed.
  • pulp cells are cultured for 24 hours in a culture solution containing hydrogen peroxide at a concentration of 0.5 mM or more, most cells survive in the S / S: DPC-S and S / F: DPC-S groups.
  • F / S DPC-FS pulp cells that acquired resistance to active oxygen lost resistance to active oxygen by culturing in a medium not containing FGF2 for 11 days (Fig. 12). Although not shown, the resistance to active oxygen is lost by culturing pulp cells that have acquired resistance to active oxygen for 1 to 2 passages (about 3 to 6 days) in a medium not containing FGF2. It was confirmed.
  • Medetomidine hydrochloride and atipamezole hydrochloride are from Nippon Zenyaku Kogyo Co., Ltd., Midazolam is from Sand Corp., Butorphanol tartrate is from Meiji seika Pharma Co., Ltd., Water for injection is from Otsuka Pharmaceutical Co., Ltd. Purchased.
  • the vertebral arch of the 10th thoracic vertebra was peeled off, and the 10th thoracic spinal cord (T10) was completely cut transversely with a scalpel for surgery (Feather disposable scalpel mini no.14).
  • a scalpel for surgery Feather disposable scalpel mini no.14.
  • 1.0 x 10 6 pulp cells suspended in 10 ⁇ l of phosphate-buffered saline (PBS) using a micropipette were removed from the rostral stump and tail of the cut. It was injected into the gap at the side stump and the back muscle and skin were sutured.
  • the dental pulp cells are the same as those in 3-2.
  • the cells cultured according to the culture schedule of S / S: DPC-S section described in 1) were used.
  • atipamezole hydrochloride a medetomidine hydrochloride antagonist
  • cyclosporin A an immunosuppressant
  • Edaravone (Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally at a dose of 3 mg / kg twice a day for 1 week immediately after surgery in the edaravone administration group and the edaravone administration cell transplantation group.
  • GAP growth-associated protein
  • GFAP anti-glial fibrillary acidic protein
  • the BBB locomotor rating scale is a motor function evaluation standard widely used in spinal cord injury experiments, and the behavior of the hind limbs of the subject rat is scored based on the evaluation standard. Detailed evaluation criteria are shown in FIG.

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Abstract

L'invention se rapporte à un matériel de greffe pour le traitement de lésions nerveuses, et comprend des cellules de pulpe dentaire. Les caractéristiques requises pour les cellules de pulpe dentaire, fournies en tant que matériel de greffe pour le traitement de lésions nerveuses, sont identifiées comme étant des cellules de pulpe dentaire dans lesquelles les gènes de GABRB1 sont exprimés et qui sont résistants à l'oxygène actif.
PCT/JP2016/071050 2016-07-15 2016-07-15 Greffe pour le traitement de lésions nerveuses, y compris les cellules de la pulpe dentaire. Ceased WO2018011989A1 (fr)

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PCT/JP2016/071050 WO2018011989A1 (fr) 2016-07-15 2016-07-15 Greffe pour le traitement de lésions nerveuses, y compris les cellules de la pulpe dentaire.

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