WO2014126175A1

WO2014126175A1 - Composition for preventing or treating diabetes

Info

Publication number: WO2014126175A1
Application number: PCT/JP2014/053382
Authority: WO
Inventors: 新恒川; 上田　実; 朗仁山本; 貴子泉本
Original assignee: 国立大学法人名古屋大学
Priority date: 2013-02-13
Filing date: 2014-02-13
Publication date: 2014-08-21
Also published as: JPWO2014126175A1

Abstract

[Problem] Provided is a composition for preventing or treating diabetes. [Solution] A culture supernatant obtained by culturing dental pulp stem cells is used as a composition for preventing or treating diabetes.

Description

Composition for preventing or treating diabetes

The present specification relates to a composition for preventing or treating diabetes.
This application claims priority to Japanese Patent Application No. 2013-25120 filed on Feb. 13, 2013, the contents of which are incorporated herein by reference.

Diabetes is a serious disease that continues to increase in many countries. Diabetes mellitus also develops various serious complications, and therefore an effective prevention / treatment method is expected. There are two types of diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes involves the death of pancreatic β cells by autoimmunity. Type 2 diabetes is accompanied by a decrease in pancreatic β cells and a decrease in insulin secretion and sensitivity. Accompanied by. Diabetes is thought to be caused by a genetic or environmental predisposition, but it is not always clear.

The treatment of diabetes is basically administration of insulin for type 1, administration of a hypoglycemic agent for type 2, and administration of insulin thereafter.

Recently, it has been reported that impaired glucose tolerance has been improved by transplanting bone marrow stem cells into streptozotocin (STZ) -induced diabetes model mice (Non-patent Document 1). In addition, islet transplantation was established by the University of Alberta in Edmonton, Canada. Islet transplantation in which islet tissue isolated from multiple brain death donors (tissue donors) is transplanted to a single type 1 diabetic patient in a relatively short period of time. 7 patients with type 1 diabetes mellitus have reported that all but seven have become insulin withdrawal (insulin injection is not necessary to maintain normal blood glucose levels) Patent Document 2).

It is described that a composition containing a culture supernatant of stem cells such as dental pulp stem cells is effective for the treatment of an injured part (Patent Document 1).

International Publication No. WO2011 / 118795

However, when aiming for long-term insulin withdrawal, the current technology requires multiple transplants, and the islet function gradually deteriorates, resulting in poor 5-year insulin withdrawal results. In the case of stem cell transplantation or islet transplantation, the possibility of becoming cancerous cannot be denied even if it is autologous. In addition, since immune rejection reaction cannot be avoided if it is of different types, the use of immunosuppressants cannot be avoided.

A composition containing a culture supernatant of stem cells such as dental pulp stem cells is effective for tissue damage and effective for periodontal disease, spinal cord injury, etc., but generally cannot be expected to be effective for diabetes.

This specification provides a composition for preventing or treating diabetes.

The present inventors were examining the treatment of diabetes and unexpectedly obtained the knowledge that the culture supernatant of dental pulp stem cells was effective. The present specification provides the following means based on this finding.

(1) A composition for preventing or treating diabetes, comprising a culture supernatant obtained by culturing dental pulp stem cells.
(2) The composition according to (1), which does not contain serum.
(3) The composition according to (1) or (2), which does not contain dental pulp stem cells.
(4) A method for producing the prophylactic or therapeutic composition according to any one of (1) to (3),
Select adherent cells from dental pulp cells,
Culturing the adherent cells;
A production method for collecting a culture supernatant.
(5) A method for preventing or treating diabetes,
A method comprising administering the composition according to any one of (1) to (3) to an individual at risk of diabetes or an individual suffering from diabetes in an amount effective for the prevention or treatment of diabetes.
(6) The composition is administered by an administration method selected from the group consisting of intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and intranasal administration. The method according to (5).

It is a figure which shows the change of the blood glucose level by the difference in STZ dosage. It is a figure which shows the change of the blood glucose level at the time of administration of the human fallen deciduous dental pulp stem cell culture supernatant and SHED-CM with respect to the STZ low dose mouse | mouth. It is a figure which shows the change of the body weight at the time of SHED-CM administration with respect to a STZ low dose mouse | mouth. It is a figure which shows the IPGTT (day27) result (blood glucose level) at the time of SHED-CM administration with respect to a STZ low dose mouse | mouth. It is a figure which shows the IPGTT (day27) result (insulin) at the time of SHED-CM administration with respect to a STZ low dose mouse | mouth. It is a figure which shows the measurement result of the insulin content in the pancreas at the time of SHED-CM administration (day 34) with respect to a STZ low dose mouse | mouth. It is a figure which shows the HE dyeing | staining result (magnification x4) of the pancreatic tissue at the time of SHED-CM administration (day 34) with respect to a STZ low dose mouse | mouth. It is a figure which shows the HE dyeing | staining result (magnification x40) of the pancreatic tissue at the time of SHED-CM administration (day 34) with respect to a STZ low dose mouse | mouth. It is a figure which shows the immuno-staining result (magnification x40) of the pancreatic tissue at the time of SHED-CM administration (day 34) with respect to STZ low dose mouse | mouth. It is a figure which shows the change of the blood glucose level at the time of SHED-CM administration with respect to a STZ mass administration mouse | mouth. It is a figure which shows the change of the body weight at the time of SHED-CM administration with respect to a STZ mass administration mouse | mouth. It is a figure which shows the result (blood glucose level) of IPGGT (day31) at the time of SHED-CM administration with respect to a STZ mass administration mouse | mouth. It is a figure which shows the change of the blood glucose level at the time of SHED-CM administration with respect to a high fat diet mouse | mouth. It is a figure which shows the change of the body weight at the time of SHED-CM administration with respect to a high fat diet mouse | mouth. It is a figure which shows the result (blood glucose level) of IPGGT (day69) at the time of SHED-CM administration with respect to a high fat diet mouse | mouth. It is a figure which shows the result (insulin) of IPGGT (day69) at the time of SHED-CM administration with respect to a fat eating mouse | mouth. It is a figure which shows the result (blood glucose level and reduction rate) of ITT (day58) at the time of SHED-CM administration with respect to a fat eating mouse | mouth. It is a figure which shows necrosis (%) based on the DAP dyeing | staining result at the time of SHED-CM administration using mouse | mouth pancreatic beta cell line: MIN6. It is a figure which shows the mouse | mouth pancreatic beta cell strain | stump | stock: The insulin secretion experiment result (content, release / content) using MIN6. It is a figure which shows the effect with respect to Apoptosis of SHED-CM using mouse | mouth pancreatic beta cell line: MIN6. It is a figure which shows the change of the blood glucose level at the time of SHED-CM administration with respect to a dbdb mouse. It is a figure which shows the change of the body weight at the time of SHED-CM administration with respect to a dbdb mouse | mouth. It is a figure which shows the IPGGT (day36) result (blood glucose level) at the time of SHED-CM administration with respect to a dbdb mouse | mouth. It is a figure which shows the result (insulin) of IPGGT (day36) at the time of SHED-CM administration with respect to a dbdb mouse | mouth. It is a figure which shows the result (blood glucose level and reduction rate) of ITT (day51) at the time of SHED-CM administration with respect to a dbdb mouse | mouth. It is a figure which shows pancreatic insulin content (day61) at the time of SHED-CM administration with respect to a dbdb mouse | mouth.

This description relates to a composition for preventing or treating diabetes. The present composition can contain a culture supernatant obtained by culturing dental pulp stem cells. The main culture supernatant contains various cytokines. In Patent Literature 1, since the main culture supernatant contains such cytokines, cells in the damaged part are allowed to proliferate, and as a result, the tissue having the damaged part can be repaired. The present inventors have obtained the knowledge that these main culture supernatants can be treated by administering the main culture supernatant to an individual suffering from diabetes as an unknown action. Even though various cytokines are contained in the main culture supernatant, the present inventors, who are skilled in the art, could not have been expected to be effective for diabetes.

According to this composition, diabetes can be effectively prevented or treated while solving the problems of conventional diabetes treatment.

Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings as appropriate. This detailed description is intended merely to provide those skilled in the art with details for practicing the preferred embodiments of the present invention and is not intended to limit the scope of the present disclosure. In addition, the additional features and inventions disclosed below can be used separately or together with other features and inventions to provide further improved compositions for the prevention or treatment of diabetes.

Further, the combinations of features and steps disclosed in the following detailed description are not essential in carrying out the present disclosure in the broadest sense, and are particularly only for explaining representative specific examples of the present disclosure. It is described. Moreover, various features of the representative embodiments described above and below, as well as those described in the independent and dependent claims, are described herein in providing additional and useful embodiments of the present disclosure. They do not have to be combined in the specific examples given or in the order listed.

All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

In this specification, “diabetes” means a disease in which a blood glucose level is pathologically high regardless of the classification. More specifically, it refers to the case where the blood glucose level and hemoglobin A1c exceed a certain value. Furthermore, in this specification, “diabetes” includes diabetic acute or chronic disorders (complications) associated with diabetes. Acute disorders include diabetic coma (ketonic coma, non-ketonic hyperosmotic coma, lactic acidosis, hypoglycemic coma), and acute infections. Chronic disorders include diabetic neuropathy, diabetic retinopathy, microvascular disorders such as diabetic nephropathy, cerebrovascular disorders, macrovascular disorders such as diabetic gangrene, hyperlipidemia / chronic infection, cholelithiasis, Examples include cataracts.

(Composition for prevention or treatment of diabetes)
The present composition may contain somatic stem cells derived from dental pulp, that is, a culture supernatant obtained by culturing dental pulp stem cells.

(Dental pulp stem cells)
The dental pulp stem cells are not particularly limited as long as they are stem cells derived from dental pulp obtained from dental pulp. Although it may be a permanent dental pulp stem cell or a deciduous dental pulp stem cell, preferably a dental pulp stem cell derived from a dropped deciduous tooth is used from the viewpoint of cell proliferation ability. In relation to an individual to which the composition is applied, in order to suppress or avoid rejection, it is preferably a dental pulp stem cell of the same species (or human origin if human), more preferably an autologous dental pulp stem cell is used. .

Dental pulp stem cells can be selected as adherent cells in dental pulp cells. A culture supernatant obtained by culturing adherent cells or subcultured cells of dental pulp cells collected from deciduous deciduous teeth or permanent teeth can be used as “culture supernatant of dental pulp stem cells”. For example, it can be appropriately obtained by the method described in JP 2011-219432 A, for example.

In addition, immortalized cells of dental pulp stem cells can also be provided. Usually, in immortalization of dental pulp stem cells, 1 or 2 or more genes, preferably 3 or more genes, more preferably 4 or more genes are introduced. For this reason, immortalized cells no longer have the same properties as the original dental pulp stem cells. It is well known to those skilled in the art that the original cells and the immortalized cells differ in their products, secretions and amounts. Accordingly, the pulp stem cells, which are the original cells, and the immortalized cells derived from the dental pulp stem cells have different products, and the secretion status and the composition of the secretion are also different. Therefore, the culture supernatant of dental pulp stem cells and the culture supernatant of immortalized cells derived from dental pulp stem cells are greatly different in their compositions, that is, the types and proportions of the components. Therefore, the action and therapeutic effect of the culture supernatant on inflammatory diseases are also different between dental pulp stem cells and immortalized cells derived from the cells.

(1) Collection of dental pulp After naturally deciduous deciduous teeth (or extracted deciduous teeth or permanent teeth) are sterilized with a chlorohexidine or isodine solution, the crown portion is divided and the pulp tissue is collected with a dental reamer.
(2) Enzyme Treatment The collected dental pulp tissue is suspended in a basic medium (10% bovine serum / antibiotic Dulbecco's modified Eagle medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. The pulp cells after enzyme treatment are collected by centrifugation for 5 minutes (5000 rpm). In principle, cell sorting with a cell strainer is not used because it reduces the collection efficiency of neural stem cell fractions of SHED and DPSC.
(3) Cell culture (selection of adherent cells)
Cells are resuspended in 4 cc basal medium and seeded in 6 cm diameter adherent cell culture dishes. After culturing for 3 days in an incubator adjusted to 5% CO 2 and 37 ° C., the adherent cells that formed colonies are treated with 0.05% trypsin · EDTA for 5 minutes at 37 ° C. The dental pulp cells detached from the dish are seeded in an adherent cell culture dish having a diameter of 10 cm and expanded. For example, when observing with the naked eye, the cells are detached from the culture vessel and collected when the cells reach sub-confluence (a state in which the cells occupy about 70% of the surface of the culture vessel) or confluence, and the culture is filled again with the culture solution. Seed in containers. Subculturing may be repeated. For example, subculture is performed 1 to 8 times, and the cells are grown to the required number of cells (for example, about 1 × 10 ⁷ cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be recovered and stored (storage conditions are, for example, −198 ° C.).
(Alternative method)
Cells are resuspended in 4 cc basal medium and seeded in 6 cm diameter adherent cell culture dishes. After adding a culture solution (for example, 10% FCS-containing DMEM (Dulbecco's Modified Eagle's Medium)), the cells are cultured for about 2 weeks in an incubator adjusted to 5% CO ₂ and 37 ° C. After removing the culture solution, the cells are washed once or several times with PBS or the like. Instead of this operation (removal of culture solution and washing of cells), adhesive cells (dental pulp stem cells) that formed colonies may be collected. In this case, for example, the cells are treated with 0.05% trypsin / EDTA for 5 minutes at 37 ° C., and the detached cells are collected.
(4) Cell recovery Next, cells are recovered. The cells can be collected by centrifuging after detaching the cells from the culture vessel by trypsin treatment or the like. Using the recovered dental pulp stem cells, the composition of the present invention is prepared.

(Culture supernatant of dental pulp stem cells)
The dental pulp stem cell culture supernatant is a supernatant of a culture solution obtained by culturing dental pulp stem cells. That is, it does not substantially contain cellular components (dental pulp stem cells or dental pulp cells). The present composition is typically a composition that does not contain dental pulp stem cells and dental pulp cells and is composed only of the culture supernatant of dental pulp stem cells. The cultured dental pulp stem cells are removed by separating and removing cell components after culturing. Separation of cell components from the culture medium is possible by methods well known to those skilled in the art. Furthermore, the culture supernatant obtained by appropriately performing various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) on the culture solution is used. Also good.

For the cultivation of dental pulp stem cells, a basic medium or a basic medium supplemented with serum or the like can be used. In addition to DMEM, Iskov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI 1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of components that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

The composition preferably does not contain serum. Its safety is enhanced by not containing serum. For example, a culture supernatant without serum can be prepared by culturing dental pulp stem cells in a medium without serum (serum-free medium). A serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.

(Acquisition of culture supernatant)
For the cultivation of dental pulp stem cells, the conditions usually used for stem cells can be applied as they are or as they are appropriately changed. Manufacture of dental pulp stem cell culture supernatant can be appropriately performed by those skilled in the art. For example, the culture supernatant may be obtained by the following operation.

First, as already described, the adherent cells (dental pulp stem cells) selected from the dental pulp are cultured in the medium described above. For example, the cells are seeded in an adherent cell culture dish and cultured in an incubator adjusted to 5% CO ₂ and 37 ° C. Subculture as necessary. For example, when observing with the naked eye, the cells are detached from the culture vessel and collected when the cells reach sub-confluence (a state in which cells occupy about 70% of the surface of the culture vessel) or confluence, and the culture is filled again with the culture solution. Seed in containers. Subculturing may be repeated. For example, subculture is performed 1 to 8 times, and the cells are grown to the required number of cells (for example, about 1 × 10 ⁷ cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be recovered and stored (storage conditions are, for example, −198 ° C.).

Next, the culture supernatant of the selected and cultured dental pulp stem cells is collected. For example, the culture solution can be collected by suction with a dropper or pipette. The collected culture supernatant is used as it is or after one or more treatments as an active ingredient of the composition of the present invention. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., −80 ° C.).

The main culture supernatant can be appropriately concentrated. That is, the main culture supernatant may be included as a concentrate. As a concentration method, those skilled in the art can appropriately select and use from known methods. For example, a culture supernatant concentrate can be obtained by spin column concentration method or ethanol precipitation concentration method. The main culture supernatant may be lyophilized. That is, the main culture supernatant may be a lyophilized product.

(Ingredients and forms of this composition)
The present composition is a culture supernatant of dental pulp stem cells, and may contain a low molecular organic compound in addition to a high molecular compound such as a protein secreted by dental pulp stem cells during culture. Furthermore, since this composition is a culture supernatant, it can also contain a component derived from a medium.

The present composition can take the form of liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.). Moreover, this composition can take various well-known formulation forms according to the kind of disease, the characteristic of the individual who has a disease, an administration method, and dosage. For example, tablets, powders, granules, granules, fine granules, capsules, solid injections that dissolve when used, solid agents such as suppositories, liquid injections (intravenous / intramuscular injection), injections And topical external preparations such as eye drops, sprays, lotions, creams, patches and the like. Moreover, the form supported by the indwelling medical instrument etc. can also be taken. In addition, the present composition can contain a known pharmaceutically acceptable salt. A person skilled in the art can make an appropriate formulation.

The present composition can contain other components that are acceptable for formulation depending on the type of disease and the formulation form. Contains other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) You can also. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used. Antibiotics, pH adjusting agents, growth factors (for example, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)) and the like may be included.

This composition can be used for the prevention or treatment of diabetes. Diabetes can include type 1 and type 2 diabetes, as well as acute and chronic diabetic disorders associated therewith, as already described.

The administration route of the composition is not particularly limited. Various known administration forms can be employed depending on the application site and the target disease. For example, parenteral administration may be systemic administration or local administration. More specifically, injection, application or spraying can be mentioned. In addition, intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, intraoral administration and the like can be mentioned.

The dosage of this composition is not particularly limited. It can be set in consideration of the age, weight, disease state, etc. of the test subject. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.

Examples of target individuals to which this composition is applied include mammals including humans (pets, domestic animals, laboratory animals, etc.). Examples thereof include dogs, cats, rabbits, mice, cows, pigs, goats, sheep, horses, monkeys, guinea pigs, rats and mice.

(Methods for preventing or treating diabetes)
The prophylactic or therapeutic methods disclosed herein can comprise administering the composition to an individual at risk for diabetes or an individual suffering from diabetes in an amount effective to prevent or treat diabetes. According to this treatment method, it is possible to prevent or treat diabetes by solving conventional disadvantages all at once. About this composition, an administration method, etc., the aspect already demonstrated can be applied to this method.

(Method for screening factors or combinations effective for prevention or treatment of diabetes)
According to the disclosure of the present specification, the step of supplying one or more components contained in the culture supernatant obtained by culturing dental pulp stem cells to an evaluation system for diabetes and evaluating the action on inflammatory diseases A method is provided comprising:

According to this screening method, it is possible to screen which components are effective against diabetes among the components contained in the dental pulp stem cell culture supernatant, and the identified dental pulp stem cell culture supernatant components are contained as active ingredients That is, it is possible to obtain a preventive or therapeutic composition mainly containing only the specified culture supernatant component as an active ingredient. In addition, such a composition is not derived from dental pulp stem cell culture supernatant, and an effective prophylactic or therapeutic composition can be obtained by combining commercially available and / or purified specific components.

Note that the evaluation system for diabetes used in this screening method is known for diabetes. For example, streptozotocin-induced diabetes model mouse litter, NOD mouse (NOD.Cg-Prkdcscid Il2rgtm1Sug / Jic), dbdb mouse (C57BLKS / JIar- + Lepr db / + Lepr db), obob mouse (C57BL / 6JHamSlc ob / ob), Diabetes model animals such as Goto-Kakizaki rat (GK rat), Zucker fatty rat (ZDF-Leprfa / CrlCrlj) = ZDF rat (Zucker Diabetic fatty), Wistar fatty rat (Wistar- Leprfa / Leprfa), high-fat diet model animal, etc. In addition, an evaluation system using a pancreatic β cell line such as MIN6 cells, INS-1 cells, βTC cells, HIT cells, etc. can be appropriately selected and used. Such various evaluation systems can be appropriately selected by those skilled in the art, and can also be appropriately selected by referring to Examples and the like in the present specification.

The dental pulp stem cell culture supernatant contains the following components (each component is described in terms of protein, gene or substance name, common name, etc.). One, two or more selected from these components, or three or more can be appropriately combined and applied to screening of factors effective for diabetes.

Hereinafter, the present invention will be specifically described with reference to examples. However, the following examples do not limit the present invention. In the following examples,% means mass%.

The experimental methods used in the following examples are described below.

(Preparation of human fallen deciduous dental pulp stem cell culture supernatant (SHED-CM))
Using 10 cm dish, DMEM (SIGMA ALORICH Co USA) + 10% FBS (SIGMA ALORICH Co USA) + 1% Penicillin Streptomycin (Life Technologies Japan Ltd.) is used to culture deciduous dental pulp cells to 90%. After washing twice with PBS, the medium was changed to a serum-free medium (DMEM) and cultured for 48 hours. The supernatant was collected and centrifuged at 1500 rpm for 4-5 minutes and 3000 rpm for 5 minutes, and the supernatant was used as SHED-CM in the following examples.

(Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT))
Mice fasted for 16 hours were given IPGTT intraperitoneally at 2 g / kg of glucose and ITT at 2 U / kg of humanin R. Orbital blood sampling was performed before administration, 15 minutes, 30 minutes, 60 minutes, and 120 minutes after administration, and blood glucose level measurement and blood insulin level were measured.
For blood glucose level measurement, whole blood was measured using Antsense (HORIBA). The blood insulin level was measured by measuring the serum separated by EDTA by ELISA (Morinaga Insulin Measurement Kit: trade name (Morinaga Seika)).

(Isolated pancreatic insulin content)
The isolated pancreas was agitated using a homogenizer in 10 ml of KRB, administered with 25% asidethanol, and allowed to react overnight. The insulin content of the centrifuged supernatant was measured using the HTRF method (Homogenous Time Resolved Fluorescence. Cibi Bioassays).

(Histopathological examination)
The isolated islets were immersed in 4% PFA for 4 hours to fix the tissue. The histopathological examination was compared by immunostaining and HE staining.

(Immunostaining)
After fixing with PFA, it was immersed in 10% sucrose for 8 hours and in 20% sucrose overnight, and embedded in OCT compound. Sections were prepared to a thickness of 10 μm, and the expression of insulin and glucagon was compared using a fluorescent immunostaining method.

The sections were washed 3 times with PBS, then treated with 0.3% Triton-Xin PBS for 2 minutes, and blocked with 1% BSA + 0.1% Triton-Xin PBS for 30 minutes. Thereafter, the primary antibody diluted 1/500 with the blocking solution was reacted at 4 ° C. overnight (insulin: abcam; ab7842, glucagon: abcam; ab8055). After the primary antibody reaction, it was washed 3 times with PBS, and the fluorescently labeled secondary antibody Alexa Fluor (invitrogen) was diluted 1/1000 with a blocking solution and reacted at room temperature for 2 hours. After the secondary antibody reaction, it was washed 3 times with PBS, mounted, and then observed with a fluorescence microscope.

5 random non-consecutive sections were randomly selected and observed for 5 or more fields per section (25 fields or more per animal). ImageJ was used to analyze the evaluation of the area where fluorescence was expressed.

(HE staining)
The tissue fixed with PFA was replaced with PBS (PBS was exchanged in the order of 2 hours, 8 hours, and 2 hours), then embedded in paraffin using a paraffin embedding device (Sakura Finetex Japan), and sliced to a thickness of 4 μm. It was created. Sections were deparaffinized (xylene 3 min x 3 times, 100% ethanol 3 min x 3 times, 95% ethanol 1 min, 90% ethanol 1 min, 70% ethanol 1 min, 95% ethanol 1 min, milliQ water ( MQ) and washed with x5 hematoxylin solution (Meyer: diluted with MQ) for 5 minutes and washed with MQ for 10 minutes. Then, it was reacted with × 5 1% eosin Y solution (WAKO: diluted with 70% ethanol) for 2 minutes, washed once with MQ, and dehydrated (70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1). And 100% ethanol 3 minutes x 3 times, xylene 3 minutes x 3 times in this order), and after mounting, observation was performed with a fluorescence microscope.

(Nuclear staining)
After loading with STZ, the slide glass was washed 3 times with PBS, and the cells on the slide glass were fixed with 4% PFA for 30 minutes and washed 3 times with PBS. After washing, DAPI (Dojindo; Japan) diluted to 1/2000 in PBS was reacted for 5 minutes, washed 3 times with PBS, mounted, and then observed with a fluorescence microscope. Using imageJ, the number of necrosis cells / total number of cells in one visual field was counted for comparison. (6 views from 1 slide glass n: 3)

(Western blotting)
After loading with STZ, the plate was washed 3 times with PBS, administered with 50 × protease inhibitor + Cdc42 lysis buffer 400 μl / well, and allowed to react for 15 minutes. The entire contents of the well were collected and centrifuged at 15000 rpm for 20 minutes at 4 ° C. A sample obtained by mixing 30 μl of the centrifuged supernatant + 1 × 30 μl of SDS and reacting at 100 ° C. for 3 minutes was used as a sample. (The amount of protein in all samples: about 2000 μl / ml is confirmed using BCAassay; SIGMA ALORICH.)

10 μl of the sample was electrophoresed on SDS polyamide gel (20 mA / 1.5 hours) and then transferred to the membrane (250 mA / 1 hour). Blocking the membrane with Blockingone (Nacalai Tesque) for 30 minutes, and after blocking, react the primary antibody (caspase3: cellsignaling, cleaved: caspase: cellsignaling) diluted 1/3000 with canget signal (Toyobo) at 4 ℃ overnight. And washed 3 times with TBS-T. Thereafter, a secondary antibody (rabit: SIGMA ADLICH) diluted 1/10000 with canｃget signal (Toyobo) was reacted at room temperature for 2 hours, washed 3 times with TBS-T, and then fluorescently developed using ECL-prime. , Developed.

(Preparation of model mouse for diabetes by destruction of pancreatic β cells by administration of STZ and administration of SHED-CM)
Streptozotocin (hereinafter STZ, SIGMA ALORICH Co. USA) was dissolved in physiological saline to prepare a 5 mg / ml solution, and this solution was 50 mg / kg once a day for 9 to 10-week-old C57B16 / J mice. Was intraperitoneally administered for 5 days to prepare a diabetes model mouse.

The start date of STZ administration was day 1, and SHED-CM was administered by 2 ml / day tail vein for 14 days from day 1. As a control group, DMEM (serum-free medium) was administered into the tail vein at 2 ml / day, and as an Exendin-4 (hereinafter referred to as Ex-4) group, Ex-4 was administered intraperitoneally at 48 nmol / day for 14 days. Went. Body weight and blood glucose were measured every 24 hours, and blood glucose level and blood insulin concentration were measured at days 15 and 27 by an intraperitoneal glucose tolerance test (hereinafter IPGTT). The pancreas was isolated on day 34, and the histopathological examination and the insulin content of the pancreas were measured. The results are shown in FIGS.

As shown in FIG. 1, the mouse prepared in this example gradually increased in blood glucose level, and reached the maximum blood glucose level (400 mg / ml) about 2 weeks later. As shown in FIG. 2, a remarkable blood glucose level-lowering effect was observed in the group to which SHED-CM supernatant was added. In contrast, the bone marrow stem cell culture supernatant administration group and the Ex-4 administration group both showed blood glucose levels equivalent to those of the Control group. In addition, as shown in FIG. 3, the weight of each group was substantially equal throughout the observation period.

As shown in FIG. 4 and FIG. 5, the results of IPGTT showed that the blood glucose level was the lowest in the SHED-CM administration group, the insulin concentration in the blood was high, and insulin secretion was promoted. Further, as shown in FIG. 6, the insulin content of day 34 in the pancreas was highest in the SHED-CM administration group.

As shown in FIGS. 7 and 8, the results of HE staining of the pancreas collected on day 34 confirmed the presence of the best β cells in the SHED-CM administration group. As shown in FIG. 9, from the results of immunostaining after 34 days, in the SHED-CM administration group, it was possible to observe a tissue in which β cells producing and secreting insulin were enriched and surrounded by α cells. On the other hand, in the bone marrow stem cell culture supernatant administration group and the Ex-4 administration group, a good tissue morphology as in the SHED-CM administration group could not be observed.

(Preparation of model mouse for diabetes by destruction of pancreatic β cells by administration of STZ and administration of SHED-CM)
A diabetic model mouse was prepared by intraperitoneally administering 150 mg / kg of STZ physiological saline solution 15 mg / ml to 9 to 10-week-old C57B16 / J mice once. The start date of STZ administration was day 1, and after confirming that blood glucose level was 400 mg / dL or more in day 3, SHED-CM and DMEM were administered from day 3 to day 16 at 2 ml / day in each group, and Ex-4 was administered. Intraperitoneal administration was performed at 48 nmol / day. Body weight and blood glucose level were measured every 24 hours, and blood glucose level and blood insulin concentration were measured at days 17 and 31 by IPGTT. The pancreas was isolated on day 37, and the insulin content of the pancreas was measured. The results are shown in FIG. 1 and FIGS.

As shown in FIG. 1, the mouse prepared in this example rapidly increased in blood glucose level and reached a maximum blood glucose level (about 500 to 600 mg / ml). As shown in FIG. 10, in the SHED-CM administration group, the blood glucose level tended to decrease early, and the effect was maintained. As shown in FIG. 11, regarding the body weight, the SHED-CM administration group and the Ex-4 administration group showed the same tendency of weight loss. As shown in FIG. 12, the result of the day 17 IPGTT showed that the SHED-CM administration group had the highest blood glucose level suppressing effect.

(Preparation of diabetes model mice with high fat diet and administration of SHED-CM)
A 4-week-old C57B16 / J was fed a high fat diet (High Fat Diet 32: HFD) for 36 days to increase the body weight to 39.5 g blood glucose level of 200 mg / dL. Thereafter, SHED-CM and DMEM were intraperitoneally administered with 2 ml / day while continuing to feed HFD. Body weight and occasional blood glucose measurements were taken every 7 days after the start of the high fat diet. The day of SHED-CM administration start was day 1, and insulin resistance was evaluated by day 58 with an insulin tolerance test (ITT), and blood glucose level and blood insulin concentration were measured with day 69 by IPGTT. The results are shown in FIGS.

As shown in FIG. 13, the SHED-CM administration group maintained a significantly lower blood glucose level than Control. As shown in FIG. 14, the increase in body weight between the SHED-CM administration group and the Control group was comparable. As shown in FIGS. 15 and 16, the IPGTT result of the SHED-CM administration group was better than that of the Control group. As shown in FIG. 17, according to the results of ITT, insulin resistance was almost the same in the SHED-CM group and the Control group.

(Evaluation of SHED-CM using mouse pancreatic β cells)
Mouse pancreatic β-cell line: MIN6 (1 × 10 ⁶ / well) is cultured until it becomes 90% confluent, washed with DMEM (serum-free medium) three times, and then the cultures are respectively SHED-CM, DMEM, Exchanged to DMEM + Ex-4 (10 nM). Note that DMEM (SIGMA ADLICH Co USA) + 10% FBS (SIGMA ALORICH Co USA) + 1% Penicillin Streptomycin (invitrogen USA) was used as the MIN6 medium. Simultaneously with the exchange of the culture solution, STZ (0 mM, 1 mM, 5 mM) was added so as to have each concentration, and the culture was performed for 6 hours. After fixing the cells with PFA, MIN6 on the slide glass was stained with DAPI and observed with a fluorescence microscope. The number of necrosis cells / total number of cells (100 to 200) was counted using imageJ, and the ratio of necrosis (cell death) in each group was evaluated. The results are shown in FIG.

As shown in FIG. 18, it was found that necrosis was most significantly suppressed in the SHED-CM administration group.

(Evaluation of insulin secretion promotion by SHED-CM using mouse pancreatic β cells)
MIN6 (1 × 10 ⁶ / well) is cultured until it becomes 90% confluent, washed 3 times with DMEM (serum-free medium), and then the culture is SHED-CM, DMEM, DMEM + Ex-4 (10 nM), respectively. Was replaced. Simultaneously with the exchange of the culture solution, STZ (0 mM, 1 mM, 5 mM) was added so as to have each concentration, and the culture was performed for 6 hours. Then, after a 30-minute starvation with KRB buffer of 2.5 mM glucose, stimulation was performed with a low glucose KRB buffer (2.5 mM) and a high glucose KRB buffer (16.7 mM) for 30 minutes. The supernatant after stimulation with low glucose and high glucose was collected, and after collecting the supernatant, the insulin content was finally collected by the asidethanol method. Using the HTRF method, the amount of insulin in the supernatant (release) and the insulin content (content) after recovery of the supernatant were measured, and the insulin secretion ability was compared with release / content (%). The results are shown in FIG.

As shown in FIG. 19, it was found that insulin secretion was promoted in the SHED-CM administration group.

(Evaluation of apoptosis of SHED-CM using mouse pancreatic β cells)
MIN6 (1 × 10 ⁶ / well) is cultured until it becomes 90% confluent, washed 3 times with DMEM (serum-free medium), and then the culture is SHED-CM, DMEM, DMEM + Ex-4 (10 nM), respectively. Was replaced. Simultaneously with the exchange of the culture solution, STZ (0 mM, 1 mM, 5 mM) was added and cultured for 6 hours. Thereafter, the MIN6 protein was recovered, and the expression levels of caspase 3 and cleaved caspase 3 were compared by Western blotting. The results are shown in FIG.

As shown in FIG. 20, the SHED-CM administration group exhibited a lower level of cleaved caspase 3 expression than the Control group and bone marrow stem cell culture supernatant administration group.

From the above results, it was considered that SHED-CM suppresses β cell necrosis and apoptosis and promotes β cell insulin secretion ability.

(Preparation of type 2 diabetes model mouse and administration of SHED-CM)
1 ml of SHED-CM prepared in Example 1 was administered intravenously and intraperitoneally every 13 hours for 13 days (female) of leptin receptor-deficient mice (spontaneously developed) (dbdb mice). As a control, DMEM was intravenously administered in the same manner. Body weight and occasional blood glucose were measured every 5 days from the start of administration. The start date of SHED-CM administration was day 1, and blood glucose level and blood insulin concentration were measured at day 36 by a glucose tolerance test (IPGTT). In addition, insulin resistance was evaluated on day 51 by an insulin tolerance test (ITT). The results are shown in FIGS.

As shown in FIG. 21, the SHED-CM administration group maintained a generally low blood glucose level with respect to Control. As shown in FIG. 22, the body weight gain was similar between the SHED-CM administration group and the Control group. As shown in FIGS. 23 and 24, the results of IPGTT in the SHED-CM administration group were better than those in the Control group. As shown in FIG. 25, according to the results of ITT, insulin resistance was almost the same in the SHED-CM group and the Control group. In addition, as shown in FIG. 26, the pancreatic insulin content of day 61 was high in the SHED-CM administration group, supporting the insulin secretion promoting effect of SHED-CM.

From the above results, it was found that this composition is useful for the treatment of diabetes.

Claims

A composition for preventing or treating diabetes, comprising a culture supernatant obtained by culturing dental pulp stem cells.
The composition according to claim 1, which does not contain serum.
The composition according to claim 1 or 2, which does not contain dental pulp stem cells.
A method for producing the prophylactic or therapeutic composition according to any one of claims 1 to 3,
Select adherent cells from dental pulp cells,
Culturing the adherent cells;
A production method for collecting a culture supernatant.
A method for preventing or treating diabetes,
A method comprising administering the composition according to any one of claims 1 to 3 to an individual at risk of diabetes or an individual suffering from diabetes in an amount effective for the prevention or treatment of diabetes.
The composition is administered by an administration method selected from the group consisting of intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and intranasal administration. Item 6. The method according to Item 5.
Method for screening factor or combination effective for prevention or treatment of diabetes One or more components contained in culture supernatant obtained by culturing dental pulp stem cells are supplied to an evaluation system related to diabetes, and action on diabetes Evaluating the method.