WO2018011989A1 - Graft material for treating nerve damage including dental pulp cells - Google Patents

Abstract

Provided is a graft material for treating nerve damage, the graft material including dental pulp cells, wherein properties required of dental pulp cells provided as a graft material for treating nerve damage are identified. The present invention pertains to a graft material for treating nerve damage, the graft material including dental pulp cells in which GABRB1 genes are expressed and which are resistant to active oxygen.

Description

Transplant for nerve injury treatment containing pulp cells

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.

Recently, it has been reported that the movement disorder is remarkably recovered in spinal cord injury model animals by transplantation of human dental pulp cells. Here, 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. As typical examples of autotransplantation, autologous tooth transplantation, autologous skin transplantation, and the like are actually performed in clinical practice. In the case of autotransplantation treatment, 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.

On the other hand, allogeneic transplantation is a method of transplanting another person's tissue or tissue-derived cells. When using cells from other people, immune rejection must be considered. The immune response provided as a self-defense function is an obstacle to transplantation treatment. In recent years, with the advance of immunosuppressive agents, it has become possible to suppress rejection reactions, and organ transplantation and hematopoietic stem cell transplantation have developed dramatically. However, even with the currently improved 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.

When considering other transplantation, 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. Here, 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.

According to a recent report, it is estimated that about 80% of Japanese people can be covered by preparing about 50 lines of HLA haplotype homo cells (Non-patent Document 2). In other words, if dental pulp cells with abundant sources can be used for treatment, HLA haplotype homodonor cells with low immune rejection can be collected, and a strategy using this as a transplantation source becomes realistic.
The present inventors have previously clarified that treatment of spinal cord injury by cell transplantation can be enhanced by treating and transplanting dental pulp cells isolated from dental pulp tissue with FGF2 (for example, Patent Documents). 1). 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.

WO2014 / 185470

So far, methods for isolating dental pulp cells from teeth and pulp tissues have been studied, and establishment of pulp cell culture methods suitable for regenerative medicine has been studied. Here, when subjecting dental pulp cells to regenerative medicine, an important issue different from isolation methods and culture methods is that it is necessary to grasp the difference in cell properties for each donor from which they are derived. . Not all dental pulp cells have a nerve damage recovery function because individual differences occur depending on gene expression patterns as well as age and sex. That is, depending on the donor from which it is derived, for example, the presence of dental pulp cells that do not have a therapeutic effect on nerve damage is assumed. In support of this, the present inventors have clarified that there is a significant difference in the effect of recovering motor function in the model animal after cell transplantation due to the difference in dental pulp cell donors (for example, Patent Document 1). . Thus, in order to realize the allograft treatment of dental pulp cells, the differences in the properties of dental pulp cells among donors that correlate with the clinical effects are elucidated, and the biomarkers that can be examined before transplantation are narrowed down. Is an important issue.

However, there have been no reports that have clarified the differences in cell properties due to differences between donors or the properties of dental pulp cells that are preferable for use in the treatment of nerve damage. In particular, changes in the properties of dental pulp cells due to treatment with FGF2 and differences in properties of dental pulp cells due to differences in donors were unknown. Therefore, 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.

The inventors of the present invention have found that 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. Here, 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.

However, as described above, it has been clarified that even dental pulp cells treated with FGF2 do not have a therapeutic effect on nerve damage due to differences in donors when transplanted into a spinal cord injury model animal. As a result of further intensive studies, the present inventors have found that the FGF2-treated dental pulp cells have properties that are particularly important for use in the treatment of nerve damage. The present invention has been completed based on this finding.
In addition, in the patent document 1, the present inventors have shown the result of comprehensive analysis and comparison of gene expression of dental pulp stem cells cultured in MSCGM without FGF2 and derived from different donors. However, 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.

That is, 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.
Moreover, the transplant material for treatment of nerve injury of the present invention is, in one embodiment,
[2] A transplant for treatment of nerve damage according to [1] above,
The dental pulp cells are those treated with FGF2.
Moreover, the transplant material for treatment of nerve injury of the present invention is, in one embodiment,
[3] The graft material for treatment of nerve injury according to [2] above,
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.

Moreover, 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.
Moreover, 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.
Moreover, 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.

Moreover, the transplant material for treatment of nerve injury of the present invention is, in one embodiment,
[7] The graft material for treatment of nerve injury according to [3] above,
The increased expression level of the GABRB1 gene is 10 times or more compared with the dental pulp cells not treated with FGF2.
Moreover, 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.
Moreover, 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.
Moreover, 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.

In another aspect, 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.
In one embodiment, the method for treating nerve injury of the present invention is as follows.
[12] 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.

In another aspect, 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.
In addition, in one embodiment, 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.
In addition, in one embodiment, 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.

In addition, in one embodiment, 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.
In addition, in one embodiment, 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.
In addition, in one embodiment, 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.

According to the present invention, 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.

The evaluation standard of a BBB score is shown. 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)) 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. 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. Significant difference test was performed by Tukey's multiple comparisons test after two-way ANOVA. Asterisk indicates statistical significance compared to all plots (** P <0.01). Error bars indicate standard error (± SEM) (n = 3). 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 μm. 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. 6d to 6f show images of the DP296-FGF2 (+) transplantation area. In the center of the image of FIG. 6a, GAP-43 positive growth cones were detected between GFAP positive cells. FIG. 6b 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. In 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. In FIG. 6f, myelinated axons have disappeared. The scale bar in FIG. 6 indicates 500 μm in FIGS. 6a and 6d, and 100 μm 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. The scale bar indicates 1.0 mm. FIG. 8 is a graph showing the expression level of GABRB1 in a group treated with FGF2 or a group not treated with FGF2 for human dental pulp cells derived from each donor. Error bars indicate standard deviation (± S.D.) (n = 3). An asterisk (*) indicates statistical significance as compared to the FGF2-untreated group in each cell line (* P <0.05, ** P <0.01). FIG. 9 shows a graph of the results of quantifying the expression of specific genes in a group treated with FGF2 or a group not treated with FGF2 by real-time PCR for human dental pulp cells derived from each donor. Error bars indicate standard deviation (± S.D.) (n = 3). An asterisk (*) indicates statistical significance compared to the FGF2-untreated group (* P <0.05 and *** P <0.001). 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 μm, 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 (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 μm. FIG. 15 shows a group in which spinal cord injury model rats were transplanted with dental pulp cells (DP31-S) together with administration of edaravone (n = 5) and a group in which dental pulp cells (DP31-S) were transplanted (n = 9). ) And the time course change of the BBB score in the group (n = 3) to which edaravone was administered daily. Values are expressed as mean ± standard error. Significance was determined by two-way ANOVA followed by Tukey's multiple comparisons test (asterisk (*) indicates comparison with control group: * p <0.05, ** p <0.01, ** * p <0.0001, **** p <0.0001. † indicates comparison with pulp cell transplantation: †† p <0.01, ††† p <0.001).

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.

In the present specification, 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. As a preferred embodiment, there can be mentioned a form in which all or most of the dental pulp cells used in the transplant material for treatment of nerve damage of the present invention are dental pulp stem cells.
Therefore, 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. In addition, in the preferred embodiment of the invention disclosed in the present specification, “dental pulp cell” can be read as “dental pulp stem cell” as described above.

In this specification, “dental pulp 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. In addition, it is also possible to identify dental pulp stem cells using neural crest cell markers such as Nestin, SOX10, and SOX11 as an index.

Here, "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. is there. 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. In addition, the method of taking out pulp tissue from a tooth is well-known, and those skilled in the art can implement suitably. In addition, when 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 α-MEM for transport, and the temperature is low (for example, 4 ℃). Preparation and storage of dental pulp cells is described in Takeda, T. et al .: J. Dent. Res., 87: 676-681, 2008; Tamaoki et al., J Dent Res. 2010 89: 773-778, etc. Can be done according to the method.

Specifically, 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. As described above, methods for collecting dental pulp cells from dental pulp tissues are known, and those skilled in the art can appropriately perform reagents and test conditions to be used.
In addition, dental pulp cells isolated from dental pulp tissues contain dental pulp stem cells in the cell population. In the present invention, when treated with FGF2, a dental pulp cell group containing dental pulp stem cells can be used as it is and cultured in a medium containing FGF2. Alternatively, 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. 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.
Here, the “GABRB1 gene” is a gene encoding gamma-aminobutyric acid type A receptor beta1 subunit.
In the present specification, when 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. Means that In addition, dental pulp cells isolated from dental pulp tissues are in a state where expression of GABRB1 gene is not enhanced unless specific conditions such as culturing in the presence of FGF2 are passed. In addition, the expression level of the GABRB1 gene is enhanced by culturing dental pulp cells in the presence of FGF2 for a certain period. In particular, the GABRB1 gene is the gene whose expression is most enhanced in the presence of FGF2. In addition, when the expression of GABRB1 gene is enhanced by a method other than FGF2 treatment, the expression level of GABRB1 gene is significantly increased compared to the expression level of GABRB1 gene in cells before applying this method. Means that.

In the present specification, when `` the dental pulp cell expresses the GABRB1 gene '', it is not limited to the following. For example, as a preferred embodiment in human dental pulp cells, 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.

Further, in one embodiment of dental pulp cells used in the transplant material for treatment of nerve damage of the present invention, 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). “DRD2 gene” is a gene encoding Dopamine receptor D2. The “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.

In addition to the GABRB1 gene, 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. In addition, 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.

In the present specification, 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. . Preferably, it means that the gene expression level is significantly increased compared to the expression level of the corresponding gene in dental pulp cells not treated with FGF2. Compared with 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. Thus, for example, 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.

Hereinafter, the preferred form of the dental pulp cell in which the expression of each gene is enhanced will be specifically described taking human dental pulp cells as an example. However, the dental pulp cells of the present invention are not limited to the following as long as the expression of each gene is enhanced.
In a preferred embodiment, 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. Has an expression level.
In addition, in a preferred embodiment, 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. Has gene expression level.
In addition, in preferred embodiments, 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. Has gene expression level.
In addition, in a preferred embodiment, 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. Has gene expression level.
In addition, in preferred embodiments, 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.
In addition, in a preferred embodiment, 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.
In addition, in a preferred embodiment, 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).
Regarding the expression level of each gene, when comparing the gene expression level in FGF2-untreated dental pulp cells, the following Example 2-2. It is preferable to carry out according to the method described in Real-time PCR.
Specifically, 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.
Further, by using 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. In particular, 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.

That is, in one aspect, 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. By screening dental pulp cells treated with FGF2 using these genes as an index, for example, dental pulp cells having a therapeutic effect on nerve damage can be selected from dental pulp cells derived from a plurality of donors.

In the present specification, “having active oxygen resistance” means that dental pulp cells have resistance to the toxicity of active oxygen. In particular, 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.
Here, 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.

In the present specification, when referring to “dental pulp cells having resistance to active oxygen”, in one preferred embodiment, any one of the following conditions a) to f) is satisfied:
A) Living dental pulp cells cultured in 10% FCS-αMEM medium containing 0.5 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM 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.
B) Living dental pulp cells cultured in 10% FCS-αMEM medium containing 0.6 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM medium without addition of hydrogen peroxide for 24 hours. Alive at a rate of about 40% or more, more preferably about 50% or more, and still more preferably about 60% or more with respect to the number of living dental pulp cells.
C) Living dental pulp cells cultured in 10% FCS-αMEM medium containing 0.7 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM medium without addition of hydrogen peroxide for 24 hours. Alive at a rate of about 15% or more, more preferably about 20% or more, and even more preferably about 25% 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-αMEM 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. About 3 times or more, more preferably about 4 times or more, more preferably about 5 times or more of the number,
E) Living dental pulp cells when cultured in 10% FCS-αMEM 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. About 3 times or more, more preferably about 4 times or more, more preferably about 5 times or more of the number,
F) Living dental pulp cells when cultured in 10% FCS-αMEM 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. Although not limited to the following embodiments, specifically, for example, 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.

As used herein, 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.

Moreover, in this specification, 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. The 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.

In this specification, “basic medium” refers to a medium containing only known components of low molecular weight. Non-limiting examples of 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.

In the present specification, “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. For example, 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 (trademark) serum-free culture kit for human mesenchymal stem cells (BD bioscience). It is not limited.

In the present specification, “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. 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.

Also, as one embodiment in the pulp cell culture step using a medium containing FGF2, a “medium substantially free of growth factors other than FGF2” can be used. In the present specification, “medium substantially free of growth factors other than FGF2” means that the only growth factor that is intentionally added is FGF2.

As used herein, “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.

In addition, 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.

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. Therefore, 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. For example, 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. By imparting appropriate fluidity, the transplant can be fixed at the damaged site.
After mixing with a gel such as collagen gel, soft agar, or synthetic polymer, three-dimensional culture may be performed by culturing for a certain period of time.

As used herein, “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.
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. For example, 10 μl 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.

Motor function can be evaluated by 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. When dental pulp cells are transplanted to the nerve damage site, the regenerated axons of GAP43-positive cells extend through the damaged part, and the regenerated axons that are myelinated can be confirmed by surrounding the axons with MBP-positive cells. Alternatively, it is preferable that GFAP-positive endogenous astrocytes are preserved as a scaffold for the regenerating axon.
In addition, for example, when dental pulp cells are transplanted to the site of nerve injury, in a preferred form, one week after pulp cell transplantation, GAP-43-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.
Moreover, 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 according to the present invention includes the above-described dental pulp cells in which the GABRB1 gene is expressed by FGF2 treatment. As one embodiment of the method for producing a transplant material for treating nerve damage, 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. In addition, the transplanted material may be placed by cutting the damaged site. When the transplant material contains allogeneic cells, an immunosuppressant such as cyclosporine may be administered simultaneously. As long as 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).
In this specification, an example of a method for treating human spinal cord injury is shown below, but the method of treatment is not limited thereto.
Spinal cord injury of a human spinal cord injury patient under anesthesia using the therapeutic graft material manufactured by the above-described method for manufacturing a therapeutic implant for nerve injury according to the present invention or the transplant material for therapeutic nerve injury according to the present invention. An effective amount of dental pulp cells can be directly transplanted to the site using a syringe or the like.

In one embodiment, 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. .
As used herein, the term “reactive 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. 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.
For example, when 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. It has been confirmed that edaravone alone cannot be recovered by simply administering it to a spinal cord injury model.
Also, for example, as 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.

In addition, 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. As described above, 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. Such a kit 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). Moreover, the active oxygen removal agent used at the time of cell transplantation may be contained. Examples of 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. In addition, 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. 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.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Moreover, the content of the literature referred in this specification is integrated in this specification as a reference.

The recovery and genome / gene analysis of human dental pulp tissue described in Examples 1 and 2 below were approved by the Gifu University Ethics Committee (approval number 26-116). All animal experiments strictly followed a protocol approved by the Animal Experiment Committee at Gifu University (approval numbers 25-20, 25-68, 27-83).

<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.

Specifically, the following method was used.

<1-1. Isolation and culture of human dental pulp cells and viral infection>
The inventors collected approximately 300 DPC strains at Gifu University Medical University Hospital. Human dental pulp cells derived from patients provided based on informed consent were isolated and cultured by a known method (Gronthos et al., 2000, Takeda et al., 2008).
Lentiviruses for gene transfer are classified into lentiviral vectors (EF-1α promoter, Venus) (Supplementary Table S.) and plasmids (pLP / VSV-G, pLP1, pLP2) (Miyoshi, H. et al., Development of a self-inactivating lentivirus vector. J. Virol. 1998) and produced in HEK293FT cell line. The lentiviral vector contains a sequence in which a Venus gene is bound downstream of the EF1α 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 α-MEM medium (Sigma). The efficiency of virus infection was confirmed using FACS.
In addition, FGF2 (R & D SYSTEMS) was added to α-MEM medium at a concentration of 10 ng / μl 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. In addition, as a control group for the addition of FGF2, DP1 or DP31 passages were produced in α-MEM medium not containing FGF2, and were similarly used for transplantation at passages 10 to 13 (DP1 respectively). -FGF (-) group or DP31-FGF (-) group).

<1-2. Animals and transplant surgery>
Female Wistar adult rats (148 to 168 g) were purchased from Nippon SLC. Rats were placed under appropriate anesthesia by inhalation of 1.5-2% isoflurane (TK-7, Biomachinery, Japan). After laminectomy, the tenth thoracic vertebra (Th10) of the spinal cord was replaced with a surgical scalpel (Surgical). The spinal cord was completely cut by cutting twice slowly and precisely using a blade stainless steel no. 11 mini, kai medical, Japan. Moreover, the cutting | disconnection was confirmed visually. After hemostasis treatment, medium containing cells at a concentration of about 1 × 10 ⁶ in 10 μl medium was carefully and slowly injected into the injury site using a Hamilton syringe (Sigma-Aldrich, Missouri, USA). After cell injection, the cells were left out of contact for 10 minutes. After leaving for 10 minutes, the wound was closed with sutures. In addition, instead of injecting the cells, the same amount of PBS was added to the spinal cord amputation site in the control group of rats, and after standing for 10 minutes, the wound was closed with sutures (PBS administration group). Rats were placed in a rewarming chamber until they woke up after surgery.

Thereafter, antibiotics (sulbactam / ampicillin, SANDOZ, 10 mg / kg weight) were administered daily for one week. In addition, 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.

<1-3. Transplantation of human dental pulp cells into rat vitreous cavity>
In order to examine the carcinogenicity of human dental pulp cells treated with FGF2, 1-1. Human dental pulp cells (DP31) cultured in a medium containing FGF2 by the method described in 1) were transplanted into the rat vitreous cavity.
Specifically, the rats were placed under anesthesia with 1.5-2% isoflurane and the rat cornea was further anesthetized with 4% oxybuprocaine (Benoxil, Santen). The cornea was punctured using a 27 gauge hypodermic needle (terumo, USA). The 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 μl of DMEM medium containing DP31 treated with FGF2 was injected into the vitreous cavity. Following cell injection, 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.

<1-4. 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.

<1-5. 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 (φ0.2 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.

<1-6. Immunohistochemistry>
For immunohistochemical staining, the rats were anesthetized 8 weeks after the surgery and fixed by transcardial perfusion using 1% PBS and 4% paraformaldehyde. Spinal cord tissue was removed and further postfixed with 4% paraformaldehyde for 24 hours and then placed in 30% sucrose solution overnight. Thereafter, the spinal cord was frozen in an OCT compound (Sakura Finetek), and a 25 mm sagittal section frozen section was prepared using a cryostat (CM3050S, Leica). 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).

<1-7. Result>
As a result, the group transplanted with DP1 (DP1-FGF2 (-) group) and DP31 (DP31-FGF2 (-) group) cultured without adding FGF2 had a significant motor function compared to the PBS-treated group. No recovery could be observed (Figure 2a). Similarly, among 6 animals transplanted with DP1-FGF2 (-) and DP31-FGF2 (-), an electrical action potential through a completely cut site could be detected at 8 weeks after spinal cord injury. There was only one (Figure 2b). Next, a similar test using α-MEM containing 10 μg / ml FGF2 (FGF2 alone administration group) was attempted, and a slight improvement in function was observed compared to the PBS administration group without using an immunosuppressant. Only shown (Figure 3a). In the FGF2 single administration group, action potential could be detected from only one of the 4 animals (FIG. 3b).
On the other hand, the group administered with DP1 or DP31 cultured in a medium containing FGF2 (DP1-FGF2 (+) group or DP31-FGF2 (+) group) showed significant recovery after spinal cord injury in any group, Improvement of action potential was observed (FIG. 4).

In order to confirm that human dental pulp cells treated with FGF2 are not maintained for a long time at the site after transplantation and disappear immediately from the site of spinal cord injury, human dental pulp cells expressing Venus protein containing a nuclear translocation signal were transplanted . As a result, almost no fluorescence was detected at the injured site at 8 weeks after the spinal cord injury. To support this result, when the same human dental pulp cells were transplanted into the vitreous cavity, the cleavage plane of the vitreous was observed with a fluorescence microscope (SZX-RFL2, OLYMPUS) three weeks after the transplantation. The fluorescence could not be detected (FIG. 5). This indicates that the cells were not maintained long enough for the transplanted cells to be detected in the spinal cord injury model system of the present study, and no neoplasia of the transplanted cells was observed (note that the Examples The results of 4 showed that FGF2-treated dental pulp cells remained slightly after 7 weeks of transplantation and were not detected, although there were differences in detection power due to differences in the structure of lentiviral vectors, but two results. All indicate that most of the transplanted cells are not maintained in the transplanted spinal cord tissue.)

In addition, when comparing the therapeutic effect for each donor derived, DP1, DP31, or DP165 cultured in a medium containing FGF2 (DP1-FGF (+) group, DP31-FGF2 (+) group, or In the DP165-FGF2 (+) group), the motor function was significantly improved in the BBB score as compared with the control group (PBS administration group) (FIG. 4a). However, interestingly, the same effect could not be confirmed in the group transplanted with DP296 cultured in a medium containing FGF2 (DP296-FGF2 (+) group) (FIG. 4a).

To support this, in the DP296-FGF2 (+) group, when an electrical stimulus of 0.6 mA was applied, the DP1-FGF (+) group, the DP31-FGF2 (+) group, or the DP165-FGF2 group Compared with the latencies (4.24 ± 0.38ms, 3.67 ± 0.34ms, 3.72 ± 0.32ms) of the three wards of (+) ward, the latency was significantly extended (6.14 ± 1.4ms) (Fig. 4b) And c).
In the absence of spinal cord injury, stimulation at Th8 causes a short latency evoked potential at Th13. No action potential occurred in the PBS administration group. On the other hand, in the group transplanted with DP1, DP31, and DP165, recovery of the evoked response with an extended latency was observed. In DP296, the action potential could be observed as described above, but the latency response was considerably prolonged when compared with the section without spinal cord injury.

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. In the DP165-FGF (+) group, GAP43-positive axons increased along the GFAP-positive astrocytes (Figs. 6a and b).
In addition, the regenerated axon was also involved in myelination, and it was possible to confirm a myelin that was MBP positive (FIG. 6c). On the other hand, only a very limited regeneration was observed in the DP296-FGF (+) group (FIGS. 6d, e, and f). In the PBS-treated group and the DP296-FGF (+) group, most of the GFAP-positive cells disappeared in the caudal spinal cord. On the other hand, in the DP165-FGF (+) group, it was maintained even when 8 weeks after the spinal cord was completely cut (FIG. 7).

<Example 2. Regulation of GABRB1 expression in human dental pulp cells by FGF2>
In order to confirm that gene expression of dental pulp cells is controlled by FGF2 treatment, comprehensive gene expression analysis was performed using DP1, DP31, DP165, and DP296. As shown in Example 1, DP296 is a dental pulp cell 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. Then, FGF2 (R & D SYSTEMS) was added to α-MEM medium at a final concentration of 10 ng / μl every day to prepare human dental pulp cells treated with FGF2 (FGF2 treatment group). In addition, as an FGF2 non-added group, a similarly isolated human dental pulp cell cultured in an α-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). After 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).

<2-2. Real-time PCR>
Total RNA from cultured human dental pulp cells was isolated using Rneasy (registered trademark) Plus Mini Kit (Qiagen, Valencia, CA, USA). For real-time PCR, 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.

<2-3. Result>
As a result of the cDNA microarray analysis, there were 131 genes whose expression was significantly increased 5 times or more by the FGF2 treatment compared with the FGF-treated group and the non-FGF-treated group. In particular, GABRB1 showed a 357-fold increase in expression due to FGF2 treatment. The following table is a list of genes whose expression was significantly increased by 5 times or more by the FGF2 treatment compared with the FGF treatment group and the non-FGF treatment group.

Here, in order to evaluate the expression level of GABRB1 in DP1, DP31, DP165, and DP296, a real-time PCR assay was performed. As a result, as expected, in DP1, DP31, and DP165, the expression of GABRB1 was significantly increased by FGF2 treatment, and the expression level was the highest among the genes whose expression was enhanced. However, the expression of GABRB1 in DP296 did not show a significant change (FIG. 8). This result indicates that GABRB1 gene expression by FGF2 treatment can be used as an index of the therapeutic effect of nerve damage. 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.

<Example 3. Effect of FGF2 treatment on resistance of human dental pulp cells to active oxygen>
Transplantation of FGF2-untreated dental pulp cells did not show an effective effect on improving nerve damage. Here, it is known that in the spinal cord injury part, the concentration of active oxygen increases in the acute phase. Reactive oxygen is thought to damage not only the nervous system cells in the spinal cord but also the transplanted cells. In order to confirm whether FGF2 treatment is resistant to the cytotoxicity of dental pulp cells to active oxygen, the following test was performed.

<3-1. Experimental Materials>
Human dental pulp tissue was collected after obtaining written consent from the patient at the Gifu University School of Medicine, Department of Oral Pathology. In addition, induced and cultured dental pulp cells are cryopreserved and managed in an anonymized form that cannot be connected, and personal information is strictly protected. A research plan using the same cells was submitted to the bioethics committees of Gifu University and Gifu Pharmaceutical University, and after receiving approval, some cells were donated from the stock and used for experiments. Mesenchymal stem cell basal medium (MSCBM) was purchased from Lonza Japan Co., Ltd., and α-MEM medium was purchased from Sigma Aldrich Japan Co., Ltd. Fetal calf serum (FCS) was purchased from Hyclone, and FGF-2 was purchased from R & D System.

<3-2. Pulp Cell Culture>
From the state in which human dental pulp cells DP31 isolated from dental pulp tissue (ie, dental pulp cells derived from the same donor as DP31 used in Examples 1 and 2) became semi-confluent in one 10 cm dish, 2-3 days 1 passage (1 passage to 3 passages) and cultured up to passage 12 in MSCBM. Then, in order to evaluate the tissue kinetics, the EGFP gene linked to the CAG promoter and the puromycin resistance gene were introduced into dental pulp cells using a lentiviral vector, and puromycin was added and cultured to select only the transgenic cells. . At this time, almost no cells that caused cell death were observed. After passage of the gene-introduced dental pulp cells, dental pulp cells were cultured according to the following culture schedule.
First, in order to examine the relationship between the timing of addition of FGF2 and the effect on resistance to active oxygen, 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 α-MEM medium containing 10% FCS (10% FCS-αMEM) are further cultured in 10% FCS-αMEM medium for 49 hours (S / S: DPC-S Ward).
ii) Dental pulp cells cultured for 6 passages in 10% FCS-containing αMEM medium (10% FCS-αMEM) were further cultured in 10% FCS-αMEM medium for 48 hours, and then FGF-2 (final concentration: 10 Incubate for 1 hour in 10% FCS-αMEM containing (ng / ml) (S / F: DPC-S).
iii) Pulp cells cultured for 6 passages in 10% FCS-αMEM medium containing FGF-2 (final concentration: 10 ng / ml) were treated with 10% FCS containing FGF-2 (final concentration: 10 ng / ml). -Incubate for another 49 hours in αMEM medium (F / F: DPC-FS).
iv) Pulp cells cultured for 6 passages in 10% FCS-αMEM medium containing FGF-2 (final concentration: 10 ng / ml) were treated with 10% FCS containing FGF-2 (final concentration: 10 ng / ml). -After further culturing in αMEM medium for 24 hours, culture in 10% FCS-αMEM medium for 25 hours (F / S: DPC-FS).
In addition, the relationship between the culture period and the effect on active oxygen tolerance when dental pulp cells cultured as S / F: DPC-S section were cultured in FGF2-containing medium, or cultured as F / S: DPC-FS section In order to examine the relationship between the culture period when the dental pulp cells were cultured in a medium not containing FGF2 and the effect on the resistance to active oxygen, the dental pulp cells were cultured under the following conditions v) and vi) with the culture period changed.
v) S / F: Pulp cells obtained by DPC-S culture were further cultured in 10% FCS-αMEM medium containing FGF-2 (final concentration: 10 ng / ml) for 1 or 2 days. Culture for 5 days or 11 days (S / F1, S / F2, S / F5, or S / F11).
vi) F / S: Pulp cells obtained by culturing in DPC-FS section are further cultured in 10% FCS-αMEM 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.

<3-3. Resistance test against active oxygen>
The resistance test against active oxygen is conducted according to the above 3-1. Culturing for 24 hours in 10% FCS-αMEM 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.

<3-4. Result>
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. As shown in FIG. 11, compared with S / S: DPC-S and S / F: DPC-S, F / F: DPC-FS and F / S: DPC-FS Viable cells could be confirmed. In particular, when 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. In contrast, more viable cells could be confirmed in the F / F: DPC-FS group and the F / S: DPC-FS group.
Moreover, the result of the tolerance test with respect to the active oxygen of the pulp cell cultured by said v) and vi) culture schedule is shown in FIG. As shown in FIG. 12, even in the pulp cells in the S / F: DPC-S group, resistance to active oxygen increased in the group cultured for 11 days in a medium containing FGF2. Although not shown, the pulp is subcultured 2 to 4 times (approximately 6 to 12 days) in a medium containing FGF2 regardless of the culture period (passage 1 to 7) in a medium not containing FGF2. It was confirmed that the resistance of the cells to active oxygen increased. On the other hand, even 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.

<Example 4. Dental pulp cell transplantation with active oxygen scavenger>
<4-1. Spinal Cord Injury Model Rat>
Wistar rats (7 weeks old, female) were purchased from Japan SLC, Inc. after approval by the Animal Care and Animal Experiment Committee of Gifu Pharmaceutical University. 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.

<4-2. Preparation of spinal cord injury model rat and combination with edaravone in dental pulp cell transplantation into spinal cord injury model rat>
Medetomidine hydrochloride, midazolam, and butorphanol tartrate were mixed and prepared as a three-type mixed anesthetic, and intraperitoneally administered to 7-week-old female Wistar rats at a dose of 5 ml / kg. After confirming deep anesthesia by the presence or absence of forward reflexes, stimulation of the paws, etc., the back was incised 2 cm along the midline around the 10th thoracic vertebra, and the spine was exposed by exfoliating fat and muscle tissue. Thereafter, 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). After confirming hemostasis, 1.0 x 10 ⁶ pulp cells suspended in 10 μl 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. After surgery, atipamezole hydrochloride, a medetomidine hydrochloride antagonist, was administered intraperitoneally in a volume of 0.75 mg / kg. Rats that had their entire spinal cord disconnected lost their ability to urinate completely, so the bladder was stimulated and urinated twice a day during the breeding period. In addition, because of the xenotransplantation, cyclosporin A, an immunosuppressant, was administered intraperitoneally once a day at a dose of 10 mg / kg body weight.
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.

<4-3. Preparation of frozen section>
Seven weeks after cell transplantation, spinal cord injury model rats were perfused and fixed transcardially with 4% (w / v) paraformaldehyde (PFA) solution under ether anesthesia. Subsequently, the tissue was excised and postfixed with the same solution at 4 ° C. overnight and then immersed in PBS containing 20% (w / v) sucrose to replace the fixative (overnight). After that, it was frozen and embedded in an embedding agent OCT compound and stored at −80 ° C. A cryostat (Leika, M1800) was used to prepare a 25 μm-thick sagittal slice from the prepared frozen block and affixed to a MAS-coated fluorescent slide glass for histological analysis. Using.

<4-4. Immunohistochemical staining>
Reagents used for immunohistochemical staining were prepared as follows. Block Ace is Dainippon Sumitomo Pharma Co., Ltd., Vectastain ABC elite kit is Vector, MAS (Matsunami adhesive silane) coated slide glass is Matsunami Glass Co., Ltd. Embedding agent OCT Compound is Sakura Finetech, Inc. PermaFluor Aqueous Mounting Medium was purchased from Thermo Fisher Scientific. Anti-growth-associated protein (GAP) 43 mouse antibody, anti-glial fibrillary acidic protein (GFAP) rabbit antibody purchased from Chemicon, Alexa Fluor 488-labeled anti-mouse IgG antibody, Alexa Fluor 546-labeled anti-rabbit IgG antibody purchased from Molecular Probes, respectively did.

The specific method is described below. 4-3. After washing the thin sections prepared in PBS with PBS, in order to increase the antibody permeability, 0.1 M sTris-HCl buffer (pH.7.4) containing 0.3% (v / v) Triton X-100 at 37 C for 30 minutes Immersion treatment. Tissue sections were washed with PBS and then blocked with 2% Block Ace. Thereafter, a primary antibody specifically recognizing GFAP, GAP-43 or GFP was added and reacted overnight at 4 ° C. After washing 3 times with PBS, add secondary antibody (Alexa 488 labeled anti-mouse IgG antibody or Alexa 546 labeled anti-rabbit IgG antibody) し た diluted 500-fold with PBS containing 2% block ace, and add 3 at room temperature. Reacted for hours. After washing 3 times with PBS, it was encapsulated using PermaFluor Aqueous Mounting Medium. After encapsulating, the sample was sufficiently dried, and the stained image was observed and photographed using an all-in-one fluorescence microscope (KEYENCE, BZ-9000).

<4-5. Quantitative analysis of engrafted dental pulp cells in spinal cord tissue after transplantation of dental pulp cells into spinal cord injury model rats>
4-3. Among the serial sagittal slices prepared in step 1, 5 slices were prepared, one each at a position of 0.2 mm and 0.4 mm respectively on the left and right sides from the midline of the spinal cord and the midline. Then, the above 4-4. According to the method described in 1., immunostaining using an anti-GFP antibody was performed. The number of GFP positive cells within a range of 2 mm from the center of injury to the rostral and caudal sides was measured, and the number of GFP positive cells per section was quantified.

<4-6. Evaluation of hindlimb motor function>
Using the BBB score, the motor function of the hindlimbs of spinal cord injury model rats was evaluated every week for 7 weeks immediately after the injury. 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.

<4-7. Result>
The results of immunohistochemical staining are shown in FIG. As shown in FIG. 13, most of the regenerated axons showing GAP-43 positivity infiltrated into GFAP-positive cells by the combined use of cell transplantation in the S / S: DPC-S section and daily administration of edaravone.
In addition, GFP positive cells at the site of spinal cord injury when cell transplantation of S / S: DPC-S group and edaravone administration were used in combination, compared with the group of cell transplantation only of S / S: DPC-S group Was significantly increased (FIGS. 14a-c). This shows that engraftment of dental pulp cells can be significantly increased by using an active oxygen scavenger in combination even when transplanting dental pulp cells not treated with FGF2 at the site of nerve injury.
Furthermore, as shown in the results of the BBB score in FIG. 15, in the case where cell transplantation in the S / S: DPC-S section and edaravone administration were performed in combination, only edaravone administration and S / S: DPC-S Compared with the BBB score of the cell transplantation alone (no edaravone administration), the motor activity was significantly improved. This result shows that when pulp cells are transplanted into nerve damage sites, the use of an active oxygen remover can improve the effect of improving motor activity in addition to improving the engraftment rate of dental pulp cells. .

Claims (18)

1. A transplant for treatment of nerve damage, which contains dental pulp cells expressing GABRB1 gene and having resistance to active oxygen.
2. The graft material for treatment of nerve injury according to claim 1,
   A therapeutic transplant, wherein the dental pulp cells are treated with FGF2.
3. The graft material for treatment of nerve injury according to claim 2,
   A therapeutic transplant material, wherein the FGF2-treated dental pulp cells have an increased expression level of GABRB1 gene compared to dental pulp cells not treated with FGF2.
4. The graft material for treatment of nerve damage according to any one of claims 1 to 3,
   A therapeutic transplant material, wherein 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.
5. The graft material for treatment of nerve damage according to any one of claims 1 to 4,
   A therapeutic transplant, wherein the FGF2 treatment is a culture using a medium containing FGF2 at a concentration of 5 ng / ml or more.
6. The graft material for treatment of nerve injury according to claim 5,
   A therapeutic transplant, wherein the culture using the medium containing FGF2 is performed for at least 6 days.
7. The graft material for treatment of nerve injury according to claim 3,
   Compared with the dental pulp cells not treated with FGF2, the increased expression level of GABRB1 gene is 10 times or more,
   Therapeutic implants.
8. A therapeutic agent for transplantation of nerve damage according to any one of claims 1 to 7,
   A therapeutic transplant, wherein the nerve damage 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.
9. The therapeutic transplant according to any one of claims 1 to 8, which is used in combination with an active oxygen scavenger for the treatment of nerve damage.
10. The graft material for treatment of nerve injury according to claim 9,
    A therapeutic transplant, wherein 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.
11. A method of treating nerve damage,
    A therapeutic method comprising the step of transplanting the nerve injury treatment graft material according to any one of claims 1 to 8 to a nerve injury site.
12. A method of treating nerve damage,
    In combination with an active oxygen scavenger, a method of treatment comprising the step of transplanting the following i) or ii) nerve injury treatment graft material into 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.
13. A method of manufacturing an implant for the treatment of nerve injury comprising:
    A production method comprising a step of measuring the expression of a GABRB1 gene in a dental pulp stem cell and a step of selecting a dental pulp cell expressing the GABRB1 gene.
14. A method for producing an implant for the treatment of nerve injury according to claim 13,
    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 manufacturing method which further includes the process of selecting the dental pulp cell which is carrying out.
15. A method for producing an implant for treating nerve damage according to claim 13 or 14,
    A production method comprising culturing the dental pulp cell in a medium containing FGF2 before the step of measuring gene expression of the dental pulp cell.
16. A method for producing a graft for treatment of nerve damage according to any one of claims 13 to 15, comprising
    The production method, wherein the culturing in the step of culturing the dental pulp cells in a medium containing FGF2 is performed for at least 6 days.
17. A method for producing an implant for the treatment of nerve damage according to any one of claims 13 to 16, comprising
    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. A manufacturing method.
18. A method for producing an implant for treating nerve damage according to any one of claims 13 to 17, comprising
    The production method, wherein the nerve damage is spinal cord injury, cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage, spinal cord hemorrhage, nerve compression injury due to disc herniation, sciatica, or peripheral nerve injury due to diabetes.

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