CN102292435A - New uses of tooth related stem cells - Google Patents

New uses of tooth related stem cells Download PDF

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CN102292435A
CN102292435A CN2010800052665A CN201080005266A CN102292435A CN 102292435 A CN102292435 A CN 102292435A CN 2010800052665 A CN2010800052665 A CN 2010800052665A CN 201080005266 A CN201080005266 A CN 201080005266A CN 102292435 A CN102292435 A CN 102292435A
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tooth
stem cells
illness
stem cell
disease
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CN102292435B (en
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王松灵
施松涛
丁刚
魏福兰
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CELGENIX BIOSCIENCE CORP
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

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Abstract

New uses of tooth related stem cells in preparing products for preventing or treating tooth related diseases, immune diseases, autoimmune diseases, diseases related to abnormal activation or enhancement of T lymphocytes, lupus erythematosus or systemic lupus erythematosus, or products for repairing the tooth related tissues are provided. Compositions containing the tooth related stem cells and methods for preventing or treating diseases by using the tooth related stem cells are also provided.

Description

New uses of tooth related stem cells
The new application of tooth related stem cells
Invention field
The present invention relates to the new application of tooth related stem cells.In particular it relates to purposes of the tooth related stem cells in the product of the preparation disease related to tooth for preventing or treating or the state of an illness, or the purposes in the product for the formation of tooth linked groups or reparation is prepared；Further relate to tooth related stem cells prepare be used to prevent treat immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, with the purposes in the abnormal product for raising relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus of T lymphocytes；Further relate to according to the above-mentioned treatment method for getting rid of way；Further relate to include the composition of tooth related stem cells.Background technology
Stem cell is the initial cell that a class has self-renewing and differentiation potential, can be divided into embryonic stem cell and adult stem cell.Generally existing special adult stem cell in adult tissue and organ, the tooth related stem cells for example from people having now been found that all are derived from mesoblastic mescenchymal stem cell (mesenchymal stem cells, MSCs), including dental pulp stem cell (dental pulp stem cells, DPSCs), come off deciduous teeth dental pulp stem cell (stem cells from human exfoliated deciduous teeth, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and just blunt canine tooth nipple stem cell (stem cells from apical papilla, SCAP) (1-4, refer to document 1 to 4, similarly hereinafter）, they can derive from many animals (such as mammal, such as people).Research shows that tooth related stem cells have blunt high multiplication capacity and multi-lineage potential, can break up to Gegenbaur's cell, fat cell, neural-like cells.When the compound of tooth related stem cells and three-dimensional stent material be implanted to a mouse it is subcutaneous when, autologous pulpodentinal complex's spline structure and cementum periodontal film composite spline structure (1,3) can be produced.Go out biological root of the tooth (4) using PDLSCs and SCAP successful regenerations on autologous Mini-pig model at present.One of stem-cell research focus concentrates on and sets up various tissue adult stem cell storehouses in the world at present, carries out the clinical experimental study of stem cell.
Shown according to WHO (2003) statistics, crowd's scope that tooth relevant disease is involved is considerably beyond other any diseases.At present, malignant boil expense is controlled in the whole world for odontopathy every year More than 20,000,000,000 dollars.In tooth relevant disease, periodontosis is the incidence of disease very high bacterial infection disease in a kind of worldwide, ultimately results in tooth supporting tissue and loses and absence of tooth, and can cause a series of whole body system disease (13);And absence of tooth can cause serious influence to digestion and whole body health, while can also make patient produce bad social mentality's influence.But to periodontosis and dental tissue defect, good medicine or treatment method are not treated or repaired at present；About absence of tooth treatment it is main be still the substitute that is made using artificial material tooth.
Systemic red yabbi epidemic disease (systemic lupus erythematosus, SLE it is) to be involved with multi viscera and there is the autoimmunity disease that Multiple Antibodies are characterized in blood, conventional immunosupress or immune modulating treatment can make the life span extension of most of patients, and quality of life is improved.The reports first of Italian scholar Marmont in 1996 with autologous bone marrow transplantation treat severe lupus erythematosus it is successful after, carry out a variety of intractable immunological diseases such as autologous peripheral blood stem cell transplantation and autologous bone marrow transplantation treatment severe SLE rapidly both at home and abroad, achieve preferable curative effect.HSCT can make some patientss reach Morbidity control, but its costly, complication is more, and disease relapse rate is up to 40%-50%, it is impossible to routinely used；Although also, some patientss are possible to reach long-term Slow solutions after transplanting, can not thoroughly effect a radical cure autoimmune disease, the problem of some patientss have recurrence.
Therefore, present need exist for effectively treating some tooth relevant diseases and the novel method of some immunity diseases.The content of the invention
It is an object of the invention to provide the novel method for effectively treating some tooth relevant diseases and some immunity diseases.The present inventor now have been surprisingly found that tooth related stem cells such as periodontal ligament stem cell (PDLSCs) not only regenerates in autologous tooth the dental tissue of defect under study for action, and can regenerate dental tissue in the dental tissue of allosome defect, while PDLSCs also has inhibitory action to abnormal elevated T lymphocytes.The present invention is accomplished based on the studies above result.
Summary of the invention
Prepared present invention firstly relates to PDLSCs for preventing or treating periodontosis, purposes in the product that tooth defect is repaired.
It is used to preventing or treat purposes in the product of the disease relevant with the abnormal rise of T lymphocytes or symptom preparing the invention further relates to PDLSCs.
The invention further relates to one kind prevention or treatment periodontosis, the method of repair deficiency dental tissue, it, which includes giving, need to prevent or treat periodontosis or need the host of repair deficiency dental tissue with effectively prevention or therapeutic dose or the PDLSCs. of repair deficiency dental tissue amount The present invention relates to a kind of prevention or the method for the treatment disease relevant with the abnormal rise of T lymphocytes or symptom, it includes the PDLSCso for giving the extremely elevated host's prevention of Τ lymphocytes or therapeutically effective amount
Invention is confused in detail
In more detail, the invention provides the specific item of following various aspects and various aspects.Purposes of the first aspect present invention there is provided tooth related stem cells in the product of the preparation disease related to tooth for preventing or treating or the state of an illness, or the purposes in the product for the formation of tooth linked groups or reparation is prepared.
Purposes any one of according to a first aspect of the present invention, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
Purposes any one of according to a first aspect of the present invention, wherein described tooth related stem cells come from mammal.In one embodiment, described tooth related stem cells are from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
The purposes of any one, wherein described tooth related stem cells come from mammal, and is selected from according to a first aspect of the present invention：Dental pulp stem cell (dental pulp stem cells, DPSCs), come off and inspire confidence in L sound of baby talk No. stem cells (stem cells from human exfoliated deciduous teeth, SHED) periodontal ligament stem cells（Periodontal ligament stem cells, PDLSCs) and the blunt canine tooth nipple stem cell (stem cells from apical papilla, SCAP) of.
The purposes of any one, is selected from wherein the disease related to tooth or the state of an illness or tooth linked groups form or repaired according to a first aspect of the present invention：Periodontosis, periodontitis, tooth defect, dental tissue defect, tooth defect reparation, tooth linked groups defect and reparation, the regeneration of tooth linked groups substitutes, etc..
Purposes any one of according to a first aspect of the present invention, wherein described tooth related stem cells are used for the related disease of tooth or the state of an illness of autologous either allosome or are formed or repaired for autologous or allosome tooth linked groups.
Purposes any one of according to a first aspect of the present invention, wherein described tooth linked groups include but is not limited to tooth, root of the tooth, dental pulp, gum etc., and other tissues related to around Its pulp, tooth top layer, dental surface or tooth.
First aspect present invention and its feature and advantage of each subitem are equally applicable to other either sides of the invention and its each subitem. Second aspect of the present invention provide tooth related stem cells prepare be used to prevent treat immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, with the purposes in the abnormal product for raising relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus of T lymphocytes.
Purposes any one of according to a second aspect of the present invention, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
Purposes any one of according to a second aspect of the present invention, wherein described tooth related stem cells come from mammal.In one embodiment, described tooth related stem cells are from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
The purposes of any one, wherein described tooth related stem cells come from mammal, and is selected from according to a second aspect of the present invention：Dental pulp stem cell (dental pulp stem cells, DPSCs), come off ^ L sound of baby talk No. stem cells (stem cells from human exfoliated deciduous teeth, SHED), periodontal ligament stem cell (periodontal ligament stem cells,) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP) PDLSCs.
Purposes any one of according to a second aspect of the present invention, wherein described tooth related stem cells be used for autologous or allosome immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, with T lymphocytes are abnormal raises relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus.
Second aspect of the present invention and its feature and advantage of each subitem are equally applicable to other either sides of the invention and its each subitem.
The method that third aspect present invention provides prevention or the treatment disease related to tooth or the state of an illness, or the method for being formed or repairing tooth linked groups, this method applies the tooth related stem cells of effective dose including the host to prevention in need or the treatment disease related to tooth or the state of an illness, or this method includes applying the tooth related stem cells of effective dose to the host in need for being formed or repairing tooth linked groups.
Method any one of according to a third aspect of the present invention, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
Method any one of according to a third aspect of the present invention, wherein described tooth related stem cells come from mammal.In one embodiment, described tooth related stem cells are from selected from following Mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
The method of any one, wherein described tooth related stem cells come from mammal, and is selected from according to a third aspect of the present invention：Dental pulp stem cell (dental pulp stem cells, DPSCs), come off deciduous teeth tooth No. stem cells (stem cells from human exfoliated deciduous teeth, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and just blunt canine tooth Li stem cells (stem cells from apical papilla, SCAP).
The method of any one, is selected from wherein the disease related to tooth or the state of an illness or tooth linked groups form or repaired according to a third aspect of the present invention：Periodontosis, periodontitis, tooth defect, dental tissue defect, tooth defect reparation, tooth linked groups defect and reparation, the regeneration of tooth linked groups substitutes, etc..
Method any one of according to a third aspect of the present invention, wherein described tooth related stem cells are used for the related disease of tooth or the state of an illness of autologous either allosome or formed for autologous or allosome tooth linked groups or multiple.
Method any one of according to a third aspect of the present invention, wherein described tooth linked groups include but is not limited to tooth, root of the tooth, dental pulp, gum etc., and other tissues related to around Its pulp, tooth top layer, dental surface or tooth.
Method any one of according to a third aspect of the present invention, wherein described host is mammal.In one embodiment, described host is to be selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, Yang, Cotton sheep, goat.
In an embodiment of any one of third aspect present invention method, it includes following step
(a) people's parodontium is gathered；
(b) hPDLSCs is cultivated;
(c) optionally freezen protective hPDLSCs;
(d) optionally melt hPDLSCs, and mycoplasma, bacterium, Colony forming efficiency, interstital stem cell marking mode and karyotyping optionally are checked to hPDLSCs；
(e) hPDLSCs pieces are prepared；
(f) HA/TCP is placed in the device orphan for accommodating hPDLSCs pieces；
(j) after optional periodontal initial treatment, hPDLSCs pieces and HA/TCP are implanted to periodontal damage fault location； (k) optional progress clinic and radiography evaluation, hematology and immunological evaluation.Third aspect present invention and its feature and advantage of each subitem are equally applicable to other either sides of the invention and its each subitem.
Fourth aspect present invention provides the method for preventing or treating immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the thin abnormal activation of T lymphs or the state of an illness, the disease relevant with the exception rise of T lymphocytes or the state of an illness, lupus erythematosus or systemic loupus erythematosus, and this method is included to the tooth related stem cells for having the host that this needs using effective dose.
According to this;The method of any one of the bright fourth aspects of ^, wherein described tooth related stem cells are selected from：The cell of dental pulp thousand, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
Method any one of according to a fourth aspect of the present invention, wherein described tooth related stem cells come from mammal.In one embodiment, described tooth related stem cells are from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
The method of any one, wherein described tooth related stem cells come from mammal, and is selected from according to a fourth aspect of the present invention：Dental pulp stem cell (dental pulp stem cells, DPSCs), the hole sound of baby talk No. stem cells that come off (stem cells from human exfoliated deciduous teeth, SHED) periodontal ligament stem cells（Periodontal ligament stem cells, PDLSCs) and ability canine tooth nipple stem cell (stem cells from apical papilla, SCAP).
Method any one of according to a fourth aspect of the present invention, wherein described tooth related stem cells be used for autologous or allosome immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, with T lymphocytes are abnormal raises relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus.
Method any one of according to a fourth aspect of the present invention, wherein described host is mammal.In one embodiment, described host is to be selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
Fourth aspect present invention and its feature and advantage of each subitem are equally applicable to other either sides of the invention and its each subitem.
Fifth aspect present invention provides a kind of composition, and it includes the tooth related stem cells and optional pharmaceutically acceptable carrier of effective dose.
Composition any one of according to a fifth aspect of the present invention, wherein described tooth related stem cells It is selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
Composition any one of according to a fifth aspect of the present invention, wherein described tooth related stem cells come from mammal.In one embodiment, described tooth related stem cells are from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, arteries and veins mouse, sheep, sheep, goat.
The composition of any one, wherein described tooth related stem cells move ^ from lactation, and is selected from according to a fifth aspect of the present invention：Dental pulp stem cell (dental pulp stem cells, DPSCs), come off hole Ya Ya Body stem cells (stem cells from human exfoliated deciduous teeth, SHED) periodontal ligament stem cells (periodontal ligament stem cells,) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP) PDLSCs.
Composition any one of according to a fifth aspect of the present invention; it is for preventing or treating the disease or the state of an illness related to tooth; either formed or repaired or for preventing or treating immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, raise relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus extremely with T lymphocytes for tooth linked groups.
Composition any one of according to a fifth aspect of the present invention; wherein described effective dose is can be efficiently used for preventing or treat the dosage of the disease related to tooth or the state of an illness; either will be to be efficiently used for the dosage that tooth linked groups form or repaired, or will be to be efficiently used for preventing or treat immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, the dosage for raising with T lymphocytes exception relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus.
Fifth aspect present invention and its feature and advantage of each subitem are equally applicable to other either sides of the invention and its each subitem.
According to the detailed record of the context of the invention, the present invention satisfactorily realizes upper above-mentioned various aspects and its each subitem.
It is further described to various aspects of the present invention with feature below.
All documents recited in the present invention, their full content is incorporated herein by reference, and if implication expressed by these documents with it is of the invention inconsistent when, be defined by the statement of the present invention.In addition, the various terms and phrase that the present invention is used are with well known to a person skilled in the art general sense, nonetheless, the present invention remains desirable to that these terms and phrase are described in more detail and explained at this, the term and phrase referred to is if any implication inconsistent with common art-recognized meanings, being stated with the present invention It is defined.
The present inventor now have been surprisingly found that the related stem cell of the teeth such as PDLSCs not only regenerates in autologous tooth the dental tissue of defect under study for action, and can regenerate dental tissue in the dental tissue of allosome defect.The present invention is accomplished based on the studies above result.Therefore, prepared the present invention relates to tooth related stem cells such as PDLSCs for preventing or treating periodontosis, purposes (such as shown in Fig. 8) in the product that tooth defect is repaired；Further relate to a kind of prevention or treatment periodontosis, the method of repair deficiency dental tissue, it, which includes giving, need to prevent or treat periodontosis or need the host of repair deficiency dental tissue with effectively prevention or therapeutic dose or the tooth related stem cells such as PDLSCs. of repair deficiency dental tissue amount
Further it is proposed that the new concept of biological dental root regeneration；The biological root of the tooth with biological function is regenerated using autologous allosome SCAP/ DPSCs and PDLSCs, the reparation being preced with again on the functional living being root of the tooth newly formed recovers the masticatory function (such as shown in Figure 10) of patient；The specific implementation method of biological dental root regeneration is provided, is furtherd investigate for the mechanism further to biological dental root regeneration and the commercialization of biological root of the tooth provides foundation.Therefore, the present invention relates to the new concept of biological dental root regeneration, and it is related to SCAP, DPSCs, the purposes of the related stem cell of tooth in biological dental root regeneration such as PDLSCs.The invention further relates to the specific implementation method of biological dental root regeneration, preparation and its required optimum cell number and optimum growh time including periodontal ligament stem cell diaphragm.
Furthermore, it is used to preventing or treat purposes in the product of the disease relevant with the abnormal rise of T lymphocytes or symptom preparing the present invention relates to tooth related stem cells such as PDLSCs.The invention further relates to a kind of prevention or the method for the treatment disease relevant with the abnormal rise of T lymphocytes or symptom, it includes the tooth related stem cells such as PDLSCs for giving the extremely elevated host's prevention of T lymphocytes or therapeutically effective amount.The invention further relates to purposes in tooth related stem cells self immune system disease such as SHED is in prevention or systemic lupus erythematosus disease (such as shown in Figure 11）.
According to the present invention, term " periodontosis " includes but is not limited to periodontitis.
According to the present invention, term " defective tooth tissue " includes but is not limited to the various defect situations of host's tooth, the loss of tooth as caused by a variety of causes.
According to the present invention, term " host, it is often referred to mammal, including but not limited to people, pig, ox, horse etc..In one embodiment, term " host, refer to people, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
According to the present invention, term " product " refers to be suitable to the various forms that PDLSCs is applied.According to the present invention, term " product " also refers to the various forms applied suitable for tooth related stem cells, such as group Compound, pharmaceutical composition etc..
According to the present invention, term " disease or the state of an illness related to tooth " refers to disease, the state of an illness, Signs, condition that host described herein suffers from or showed etc., and these diseases, the state of an illness, Signs, condition etc. are related to tooth.
According to the present invention, term " tooth linked groups form or repaired " or term " formation or reparation of tooth linked groups " or " forming or repair tooth linked groups " etc., they have identical or approximate implication, and the tooth linked groups of host described herein are typically referred to the processing such as to be formed, repaired, generated, regenerated, cultivated or operating, or tooth linked groups abnormal (such as defect) are formed, repaired, are generated, are regenerated, cultivate handle or operation.
According to the present invention, term " composition " is the implication being generally understood that with those skilled in the art, and typically refer to connect or (such as before use dilute) be used for the form of Clinical practice indirectly, such as formulation, pharmaceutical dosage form, form of medication,.In clinical practice field or drug field, term " composition " also generally has equivalent meanings with " pharmaceutical composition ".
The actual dose level of the present composition or pharmaceutical composition Tooth related stem cells can be changed, so as to gained stem cell amount can by dare for specific host, patient particular composition and its constitute and corresponding administering mode in the case of obtain needed for treatment or prevention react.Dosage level must according to the treatment process of the activity of specific stem cell, method of administration, the order of severity for treating the patient's condition, disease or the state of an illness, formed and repair the processing such as (and generation, regeneration, culture) or the process of operation and the patient's condition and medical history of patient to be treated and selected.But, the way of this area is, the dosage and time of application of stem cell are since the level required less than therapeutic effect needed for obtaining, gradually incremental dose, until obtaining required effect.Therefore, for the present invention, those skilled in the art can determine specific dosage be applicable in particular situations under the teaching for the information that the present invention is disclosed in detail according to such as, but not limited to above-mentioned concrete condition, without to make specific restriction.Particularly, Specific amounts used in part of the embodiment of the present invention is may be referred to determine the usage amount under either case, the WZSP PDLSCs of specific dosage is for example used below in the present invention, those skilled in the art can instruct the dosage under the conditions of use that the dose lonvestion is behaved according to above-mentioned dosage and combination techniques well known.
Tooth related stem cells of the present invention can be independent（I.e. with intact form) or the administration in the form of pharmaceutical composition.Pharmaceutical composition of the present invention can be made into various suitable formulations according to method of administration.Using one or more physiologically acceptable carriers, comprising excipient and auxiliary agent, they are conducive to being processed into tooth related stem cells into the preparation that can pharmaceutically use.Appropriate dosage form depends on selected method of administration, can be manufactured according to general knowledge well known in the art.At one of the present invention In embodiment, the tooth related stem cells are present in the medium of cytocompatibility (for example, physiological saline such as 0.9% physiological saline, etc.).In one embodiment of the invention, the tooth related stem cells are present in the medium of cytocompatibility, and preserve, preserved such as under the conditions of refrigerating, freezing at low temperature, and optionally can redissolve into the form of administration according to of the invention spiritual that is applicable before use.Brief description of the drawings
The expression of Fig. 1 people PDLSCs immune molecules：Using the expression of Flow cytometry fPDLSCs and cPDLSCs related immune molecule.HLA-I, HLA- Π DR, CD80 and CD86 expression are as shown in the figure：(76.2% ± 5.8%, n=5) and cPDLSCs (78.5% ± 64%, η=5) express HLA-I to fPDLSCs, but do not express HLA-Π DR and costimulatory molecules CD80, CD860
Fig. 2 PDLSCs suppresses T lymphopoiesis：（A) fPDLSCs/cPDLSC will not cause Allogeneic T lymphopoiesis.（B) fPDLSCs/cPDLSC dose-dependently suppresses T lymphopoiesis caused by mitogen PHA.（C) delay, which adds fPDLSCs/cPDLSC, can also suppress T lymphopoiesis caused by PHA.（D) fPDLSCs/cPDLSC can suppress two-way mixed lymphocyte reaction (MLP).
Fig. 3 PDLSCs suppresses T lymphopoiesis by secreting PGE2：（A) in Transwell culture experiments, PDLSCs can also suppress T lymphopoiesis, point out PDLSCs secretion soluble factors to play immunosuppressive action.（B) all there is TGF-pi in simple PDLSCs culture supernatants and MLR supernatants, but between the two without significant difference.（C) PGE2 concentration conspicuousness in MLR is raised.（D) neutralize experiment and show that anti-TGF-pi antibody does not recover T lymphopoiesis, PGE2 inhibitor counteracts PDLSCs immunosuppressive action, and it is the Main Factors for mediating PDLSCs immunosuppressive actions to point out PGE2.（E, f) PDLSCs immunosuppressive action do not cause the apoptosis of T lymphocytes（E), with merely stimulate the apoptosis rate after T lymphocytes consistent (f) with PHA.（G) propagation can be recovered when repressed T lymphocytes are stimulated again with PHA or IL-2 caused by PDLSCs again.
Fig. 4 HGF and IL-10 measure：HGF and IL-10 are not all measured in simple PDLSCs culture supernatants and MLR supernatants, illustrates that both factors are not engaged in the immunosuppressive action of PDLSCs mediations.
The paradenlal tissue regeneration of Fig. 5 PDLSCs mediations：（A) there is no the difference of conspicuousness between -4w and 0 w, four groups of clinical indices.But treatment after 12 weeks, autologous or allogeneic PD, GR and the AL of PDLSCs transplantation groups significantly recover compared with blank control group and HA/TCP.（B, c, d, e) CT scan display has obvious periodontal bone defects before treating, and defect degree is close.12 weeks after treatment, autologous PDLSCs groups (h) and allogeneic PDLSCs groups (i) obtain periodontal Regeneration completely.Blank control group is almost without bone tissue regeneration（F), and the Cranial defect degree of HA/TCP groups aggravates (g) (j, k, l, m) Histological research and finds that autologous or allogeneic PDLSCs groups have obvious new bone and paradenlal tissue regeneration in Cranial defect area.But, typical periodontitis performance such as deep oral pocket, shortage area of new bone and periodontal fiber is still high-visible in HA/TCP groups and blank control group. PD:Depth, GR are examined in spy:Gingival recession, AL:Attachment loss, D:Dentine, C:Cementum, PDL:Parodontium, B:Bone.
Fig. 6 allogeneics PDLSCs transplanting will not cause immunological rejection：（A) in Each point in time as depicted, the CD3+, CD4 of allogeneic PDLSCs transplantation groups+, CD8+ T lymphocytes do not have significant difference；（B) after the transfer 3 days, CD3+, CD4+, CD8+ T lymphocyte numbers and activated T lymphocytes mark --- CD40L expression does not have notable difference between four treatment groups.
12w immune state after Fig. 7 transplanting：12w, CD3 after the treatment+, CD4+, the CD8+ T lymphocyte numbers and mark for activating Τ lymphocytes --- CD40L expression is between four treatment groups also without notable difference.
Fig. 8 depict the paradenlal tissue regeneration that PDLSCs is mediated in one embodiment of the present of invention.Fig. 9 depict the successful regeneration biology root of the tooth on miniature pig.
Figure 10 depict the biological dental root regeneration of the progress on miniature pig and succeeded.
Figure 11 depict the deciduous teeth dental pulp stem cell systemic lupus erythematosus mouse serum that comes off Ji the dirty Histological changes of Kidney.
Figure 12 illustrate that allogeneic Periodontal ligament stem cell (hPDLSCs) is used for an example for treating the standard operation of periodontosis.Embodiment
The present invention can be further described by the following examples, however, the scope of the present invention is not limited to following embodiments.One of skill in the art to the present invention it is understood that on the premise of without departing substantially from the spirit and scope of the present invention, can carry out various change and modification.
The present invention carries out general and/or specific description to the material and test method that are arrived used in experiment.Although to realize that many materials used in the object of the invention and operating method are it is known in the art that still the present invention is still described in detail as far as possible herein. Embodiment 1:PDLSCs is to the lymphopoietic inhibitory action of T
Material and method
1), people PDLSCs
The normal third molar for the healthy patients of 18-28 Sui that Beijing Stomatological Hospital Attached to Shoudu Medical Science Univ.'s Oral and Maxillofacial Surgery outpatient service is pulled out is chosen, the method reported according to previous literature is separated and culture PDLSCs (3).The utilization of tissue obtains the approval of Ethics Committee of the Capital University of Medical Sciences.Periodontal ligament stem cell is frozen in order to be applied in next step experiment（Cryopreserved periodontal ligament stem cells, cPDLSCs), the periodontal ligament stem cell (freshly isolated periodontal ligament stem cells, fPDLSCs) of part fresh separated is frozen in liquid nitrogen 3 months.After 3 months, freeze-stored cell quick-thawing, inoculation, cellar culture in 37 water-baths.In our current research, the cell of application is all in 2-4 generations.In same experiment, using the fPDLSCs and cPDLSCso of the same generation
2), PMNC
Using the PMNC (peripheral blood mononuclear cells, PB.MCs) of Percoll gradient centrifugation separating health donor.
3), antibody
Anti-human HLA (human leukocyte antigen are applied in this research, HLA)-I, HLA- Π DR. CD80, CD86 antibody (BD Biosciences), AntiCD3 McAb, CD4, CD8, CD40L, TGF p (transforming growth factor β, TGF- Jie）, HGF (hepatocyte growth factor, HGF) antibody（), Abeam the anti-mouse IgG antibody iAbD Serotec of anti-IL-10, FITC mark),
4), the thin bag art of streaming
In order to study the expression of PDLSCs surfaces related immune molecule, by l.OxlO6FPDLSCs or cPDLSCs under anti-HLA-I, HLA- Π DR, GD80 or CD86 antibody at room temperature respectively with being incubated 1 hour.After being rinsed through perphosphate Slow fliud flushings (phosphate buffered saline, PBS), cell is incubated 30 minutes at room temperature with the FITC anti-mouse IgG secondary antibodies marked again.Incubation terminates rear flow cytometer (BD Inmmunocytometry Systems) detection expression.
5), mixed lymphocyte reaction (MLP)
It is used as stimulation cell, 5.0 xlO4FPDLSCs or cPDLSCs first irradiates 20Gy under linear accelerator, then the allogeneic PBMCs of the cell concentration such as addition, common in 96 orifice plates, 0.2ml RPMI-1640 to be incubated 5 days.Terminating reaction preceding 18 hours, lpCi is added per hole 's3H-thymidine (3H-TdR).After 18 hours, cell is collected in glass fiber filter paper, is calculated using liquid glimmer instrument (PerkinElmer)3H-TdR incorporation efficiencies.3The result of H-TdR incorporation efficiencies is represented with count per minute scholar standard deviation (CPM ± SD).
In order to study whether PDLSCs has dose dependent to the influence and such a influence of breeding T lymphocytes, in mitogen promotees proliferation experiment, PBMCs (5.0 xlO are stimulated with final concentration of 0.5pg/mL PHA (Sigma-Aldrich)4) propagation, being incubated 5 days altogether with the autologous fPDLSCs or cPDLSCs of various dose, (PDLSCs amount is respectively 1.0xl04, 5.0 xl04, 2.5xl05, 5.0 xl05).Terminate to be incubated first 18 hours and add Ι μ Ο's per hole3H-TdR, collects cell and calculates after 18 hours3H-TdR incorporation efficiencies.
Further research prolongs whether addition PDLSCs can influence T lymphopoiesis.First PBMCs (5.0 xlO are stimulated with final concentration of 0.5pg/mL PHA4) breed 2 days, the fPDLSCs or cPDLSCs for then adding Isodose are incubated 3 days jointly again.Determined after 3 days3H-TdR incorporation efficiencies.
After confirming that PDLSCs can suppress T lymphopoiesis caused by mitogen PHA, we observe PDLSCs to two-way mixed lymphocyte reaction (MLP)（Mixed lymphocyte reaction, MLR) influence.PBMCs (5.0 xlO from two Different Individuals4) be incubated 5 days jointly with the third party fPDLSCs or cPDLSCs of equivalent.Terminate to be incubated first 18 hours and add l Ci's per hole3H-TdR, incubation is collected cell, calculated after terminating3H-TdR incorporation efficiencies.
6) stimulation again of the T lymphocytes after, being incubated altogether with PDLSCs
Whether reversible to the Proliferation Ability of T lymphocytes in order to observe PDLSCs, We conducted reactivation experiment. PBMCs (5.0 xlO4) be first incubated 5 days jointly with the PDLSCs and PHA (0.5 g/mL) of equivalent, then the T lymphocytes obtained by Percoll gradient centrifugation and PHA (0.5 g/mL) or rhIL-2 (intei eukin 2, IL-2,50U/mL;R＆D systems) react 2 days jointly.After 2 days, application3H-TdR incorporation methods determine T lymphocytic proliferation rates.
7), Transwell culture experiments
Directly contacted by cell-ECM or PDLSCs secretion soluble factors mediate the PDLSCs to the Proliferation Ability of T lymphocytes to observe, We conducted Transwell culture experiments.Transwell culture systems (Costar) have the mocromembrane in a diameter of 0.4 μ ι η apertures, and this mocromembrane can artificially separate two kinds of cells.By PBMCs (5.0 xlO4) it is placed in the epicoele of Transwell culture systems with PHA (0.5 g/mL), and 5.0 xlO4PDLSCs is (from this Real face starts, and the following part of this research is all with fPDLSCs) it is seeded in cavity of resorption.After 5 days, application3H-TdR incorporation methods determine T lymphocytic proliferation rates.
8), the measure of soluble factor and neutralization experiment
After Proliferation Abilities of the discovery soluble factor mediation PDLSCs to T lymphocytes, we determine simple PDLSCs culture supernatant (inoculation 1-5 days) and MLR culture supernatants (5.0X10 using liquor-saturated immunoadsorption assay (enzyme-linked immumosorbent assay, ELISA)4PDLSCs, equivalent PBMCs and 0.5 g/mL PHA) in TGF-β Ι (R＆D systems), HGF (R＆D systems), prostaglandin E2 (prostaglandin E2, PGE2;Assay Designs) and IL-10 (R＆D systems) concentration.OD value is read in 450nm (TGF-β Ι, HGF and IL-10) or 405nm (PGE2) by liquor-saturated mark instrument (Molecules Devices).
Then neutralization experiment is carried out, MLR reaction system, including PBMCs (5.0 xlO are set up in Transwell culture systems4), PDLSCs (5.0 xlO4) and PHA (0.5pg/mL), it is separately added into following antibody or reagent while reaction：Anti-TGF-beta antibodies (10ng/ mL), anti-HGF antibody (10ng/ mL) and anti-IL-10 antibody (10ng/mL) or PGE2 inhibitor Indomethacin (5 mol/L; Sigma-Aldrich)0After 5 days, pass through3H-TdR incorporation methods determine T lymphocytic proliferation rates.
9) percentage of apoptosis T lymphocytes, is determined
Set up MLR reaction system, including PBMCs (5.0 xlO4), PDLSCs (5.0 xlO4) and PHA (0.5 g/mL), determine the percentage of apoptosis T lymphocytes using Annexin V-Fluos apoptosis kit (Roche Diagnostics) after 5 days.Embodiment 2:The periodontal bone defects reparation of PDLSCs mediations
1), material and method
WZSP and Guizhou Xiang pig (6-8 monthly ages, 30-40 kilograms of body weight）By China Agricultural University, Experimental Animal Center is provided.This experiment is ratified by Ethics Committee of the Capital University of Medical Sciences.Miniature pig canine tooth PDLSCs separation and cultural method are with people PDLSCs.According to the method (13) of document report, the periodontitis Cranial defect model of 12 female WZSPs is prepared, the periodontitis Cranial defect of first permanent mandibular molar at 24 is prepared altogether.Defect is randomly divided into following 4 groups at 24：【1】Blank control group, any treatment is not done；【2】Simple material group, turns over valve, scrapes and control, transplant HA/TCP timbering materials (Wuhan University of Technology's offer), gelfoam covering defect, suture；【3】Autologous PDLSCs transplantation groups, turn over valve, scrape and control, transplant the xlO of HA/TCP timbering materials+2.07WZSP PDLSCs, gelfoam covering defect, suture；【4】Allosome PDLSCs is transplanted Group, turns over valve, scrapes and control, transplant the X10 of HA/TCP timbering materials+2.07Fragrant pig PDLSCs, gelfoam covering defect, suture.In 12 w progress clinical examinations after (0w) and treatment before (- 4w), transplantation treatment before preparing periodontitis Cranial defect model, depth (probing depth are examined including visiting, PD), tooth silver is shunk back (gingival recession, GR) and attachment loss (attachment loss, AL) Ow and treatment after 12 w application CT (Siemens) observation osteanagenesis situation.L-7d, 2w, 4w, 8w and 12w carry out blood routine examination, blood bio-chemistry checking, immunoglobulin inspection and the inspection of T lymphocyte Research of predicting markers after -4w, treatment, including CD3+ cell counts, CD4+ cell counts, mark --- the CD40L expression of CD8+ cell counts and activated T lymphocytes.12w, puts to death animal after the treatment, and sample, fixation, decalcification, FFPE, row HE dyeing, tissues observed regeneration situation are obtained from Test sites.
Examined by Student't and variance analysis carries out statistical analysis, p<0.05 is considered there is significant difference.
2), result and explanation
2-1), PDLSCs has low immunogenicity
It is initially observed people PDLSCs immunophenotype, it was found that fPDLSCs (76.2% ± 5.8%, n=5) and cPDLSCs (78.5% ± 6.4%, n=5) express HLA-I, but HLA-Π DR and costimulatory molecules CD80, CD86 (Fig. 1) are not expressed, it is similar (23) to the BMSCs expression situation of cultured and amplified in vitro.
PDLSCs is further studied as antigen presenting cell on the lymphopoietic influences of T.Experimental group is 5.0 xlO for first passing through linear accelerator 20Gy irradiations in advance4PDLSCs and the allogeneic PBMCs of equivalent are incubated altogether.It is as the T lymphopoiesis groups of positive control： 5.0 xlO4PBMCs is incubated altogether with the allogeneic PBMCs with equivalent.Individually the PBMCs of culture equivalent is negative control.As a result show, experimental group PDLSCs does not cause allogeneic PBMCs to breed, and positive controls can cause significantly
T lymphopoiesis（Fig. 2 a), point out PDLSCs that there is low immunogenicity.
2-2), PDLSCs can suppress T lymphopoiesis
PDLSCs is further looked on the lymphopoietic influences of T caused by mitogen and alloantigen.FPDLSCs and cPDLSCs is inoculated with, cell concentration is respectively： 1 .0 xlO4, 5.0χ104, 2.5x10sAnd 5.0xl05, then add autologous PBMCs (5.0xl04) and final concentration of 0.5 g/mL PHA.PHA stimulates PBMCs (5.0xl04) breed as positive control.As a result find, the PBMCs propagation that PHA is stimulated significantly is suppressed by fPDLSCs or cPDLSCs, and this suppression is dose dependent.Such a immunosuppressive action is necessarily required PDLSCs presence, rather than by bulk effect（Bulk effect) cause, it will not cause inhibitory action because adding the autologous PBMCs of equivalent in breeder reaction system（Fig. 2 b).In addition, stimulating PBMCs (5.0X10 even in PHA4) PDLSCs is added within two days after propagation, remain able to substantially suppress the propagation of T lymphocytes（Fig. 2 c).We further look at influences of the PDLSCs to MLR.PBMCs and third-party PDLSCs from two Different Individuals are incubated jointly.As a result show, fPDLSCs and cPDLSCs can suppress two-way MLR (Fig. 2 d).This part experimental data shows that PDLSCs dose-dependently can suppress caused by allogeneic T cells acceptor and T lymphopoiesis caused by mitogen with antigen-non-specific.
2-3), PDLSCs suppresses T lymphopoiesis by secreting PGE2
We further study PDLSCs and suppress the lymphopoietic mechanism problems of T.Whether caused to illustrate by the directly contact of PDLSCs and PBMCs cell-ECMs, we have carried out Transwell culture experiments first, and PDLSCs and PBMCs is artificially separated.Result of study shows, either Transwell culture experiments or cell-ECM directly contact experiment, close T lymphproliferative diseases effects can be obtained, point out this immunosuppressive action directly to contact unrelated with cell-ECM, but rely on the presence of soluble factor（Fig. 3 a).
By ELISA, we determine the soluble factor with several possibilities in MLR supernatants in simple PDLSCs culture supernatants.No matter PDLSCs culture supernatants or MLR supernatants, all do not find HGF and IL-10 (Fig. 4).Then we continue to observe TGF-β Ι and PGE2, because both is considered as the Main Factors (7,25) that BMSCs plays immunoloregulation function.As shown in Fig. 3 b and Fig. 3 c, after inoculation in 1-5 d PDLSCs culture supernatants, TGF-pi and PGE2 concentration are stablized relatively, do not fluctuate significantly.But, in MLR supernatants, PGE2 concentration is significantly raised compared with the concentration that simple PDLSCs is cultivated up to 15.19 ± 1.26ng/ml, and TGF-β Ι concentration is 1459.79 ± 109.49 pg/mL, without significant change.
We recover the lymphopoietic abilities of T by adding specific anti-TGF-beta, IL-10, HGF antibody and PGE2 inhibitor in MLR systems to observe it, further determine that PDLSCs suppresses the lymphopoietic Main Factors of T.As a result find, the propagation of T lymphocytes can significantly be recovered by adding PGE2 inhibitor, and proliferative ability now is close with the simple proliferative ability with PHA stimulations PBMCs（Fig. 3 d).And anti-TGF-p is added in experimental system, IL-10, HGF neutralizing antibody does not recover the propagation of T lymphocytes（Fig. 3 d).These as shown by data PGE2 is that mediation PDLSCs suppresses the lymphopoietic Main Factors of T.
For BMSCs immunosuppression mechanism, although it is not immediately clear that any factor plays the effect and some researchs of dominance and also exists inconsistent, but most research understanding is also It is that antiproliferative soluble factor such as IL-10 is secreted by BMSCs, HGF, TGF-β Ι, PGE2, indoles amine -2,3- dioxygenase and nitric oxide（5,6,17,19,20,24-28) cause.
Beyth etc. (28) reports IL-10 is the main mediated factor of BMSCs immunosuppressive actions.Di Nicola etc. (7) are by being the principal element played a role with experiment discovery TGF-β Ι and HGF in antibody.It is another that there are some researches show PGE2 inhibitor can offset BMSCs immunosuppressive action (25).These researchs all indicate the immunosuppressive action that soluble factor take part in BMSCs.Our research prompting PGE2 is that PDLSCs suppresses the lymphopoietic Main Factors of T.
2-4), PDLSCs immunosuppressive action is unrelated with t cell proliferation, but inducing T cell is incompetent
Further whether research PDLSCs immunosuppressive action causes the apoptosis of T lymphocytes.It is now discovered that, in PDLSCs, PBMCs and PHA reaction system, the T percentage of lymphocyte of apoptosis is similar to simple PBMC, PHA reaction system, and it is to cause immunosuppressive possibility to eliminate T Lymphocyte Apoptosis（Fig. 3 e, f).Then, being inhibited the T lymphocytes of 5 days to extract by PDLSCs, stimulated again with PHA or IL-2 two days.As a result find, the T lymphocytes being inhibited by had significant proliferation again, this propagation degree stimulates PBMCs degree close with simple PHA（Fig. 3 g).It is therefore contemplated that PDLSCs T lymphproliferative diseases are reversible, caused by inducer T lymphocyte incapability.
2-5), the paradenlal tissue regeneration of PDLSCs mediations
In view of can PDLSCs immunomodulatory properties, research allogeneic PDLSCs repair miniature pig periodontitis Cranial defect model.12 weeks after the transfer, the PD of allograft PDLSCs groups was the mm of 3.5 scholar 0.6, and the PD of autotransplantation group is 3.3 ± 0.4 mm, and HA/TCP groups are 13.1 ± 1.1 mm, and blank control group is 10.6 ± 1.3 mm.Statistical analysis shows that autologous or allogeneic PDLSCs moves plant Group periodontium compared with HA/TCP groups and blank control group and obtained obvious regeneration, and there were significant differences between autologous or allogeneic PDLSCs groups（Fig. 5 a)0CT scan shows that autologous and allogeneic PDLSCs groups alveolar bone substantially regenerates, and has substantially returned to normal level, and HA/TCP groups and blank control group only regenerate or do not regenerated on a small quantity（Fig. 5 a-h).Histological observation shows, in autologous and allogeneic PDLSCs groups, hence it is evident that new bone, cementum and periodontal fiber are regenerated, in HA/TCP groups and blank control group, still visible obvious periodontitis performance, including deep oral pocket, shortage area of new bone and periodontal fiber（Fig. 5 j-m).In addition, compared with 4w before transplantation treatment, the blood routine examination (table 1) at each time point, blood bio-chemistry checking (table 2), immunoglobulin inspection (table 3) and immunology index of correlation (Fig. 6 after treatment, Fig. 7) all without significant change, show that allograft PDLSCs repairs periodontitis Cranial defect and do not had There is the generation of rejection.
Table 1
Project； ―
I 2i 3d 4d 5d 6d 7d 2w 8w 12w C leg 11.9＆1.2 12.M321Z6feO. the Surface of 2 Ul, 132 56 12 Μ, 21 12 Μ, 091 Body 1 turn over the Chant of .1811 1
RBC M U the flat Knitting .47 of ± 0. 6 0., 5I Let Sa Meetings Hidden of M 6 35 6.30 6
HGB g 121±62 117±5.1 119±6.1 121±3.9 " leakages of Μ 7 ＆ 5.1 of .6 12 double 126 ± 5.3
HCT % draw ± (the tender 93 δ .1 .7 37.7 ± 3.5 of 3 shoulders of 36M3 35.W.1 Chant 34.7 ± 3.6 353
PLT 136 ± 10.1 turn over delete 137 10.6 Body the δ 137 ± 10.2) 39 ± 9.5 of 2 i I S mi 141 ± 10.
The Entries mesh of table 2 is turned round：
•4w Id 2d 3d 4d 5d li 2w 4w 12w
ALT L turn over ■ 42.9+4.1 .1±4.4 ■ .6 Medical Chant
The 0M1 ■ Medical Should .1 mi of 41 ± 32 Na .1 of AST ML 38.5+3.1 ± 3.8
TP m are flat to delete the .1 74.fe6.6 76M.3 .1 5.9 74.4 ± 62 of 71. .3 Legs 73
The ■ 42 ± 055 4.1 ± 0. of 1. 0. Honor of WG Honor Honor, 1. 1 19 1.M.26 gangsters TO* of Honor 1M29 l. .36 That Nursing 3.8 ± 0.39 delete the .s of 3 Continued im, 4 t0.47,3 Gang 42 0.56 3.
The Chant Hidden ■ of 1 Meetings ■ ■ of CERA Hidden 65.3 72 62
Mi i turn over the ＆ 3.1 of ± 3.3 1 l of lake 142i2.1 139 1 ± 2.1 13
K+335 3. Meetings 3 22 3. .57 ■ .87 41 ± 076 " ± 021 3.7 Ο Let " θ .92 3
CI rat console the brains 101 of 3 105 ± 32 101 ± 28 104+2.6 l l, 10 δ ± 1.9 106 ± 22±3.6 1W±2J 10¾3.9
PA 2 6 m 2 236±11.5 ¾1±7.9 252±6.3 2M.1 23«±1Z7 i 253±122 The i of table 3
Xiang Mu Miscellaneous
Id 2d 3d 6d ½ 12w
The i of 19. 3.1 legs of IgE lliil leg .3 fat .2 legs 2,1M9 7
M. the mi mm 10.3i1.5 m Brain of 8.13 ± 1.9 9.2i2.3 legs 9
1. the Medical 1 of Wrapping Meetings Jane 1M7 131 ± 06
U2 i.i .3 the Read 1.M.3 1.1fe0.3 Ι Ι Ι Ο .1 of M2 Fan Meetings 11 turn over 2 121 ± 02
In addition, seeing Fig. 8 according to another result of the test of the present embodiment, wherein illustrate in detail the paradenlal tissue regeneration of PDLSCs mediations, the result shown from figure shows, consistent with experiment above result in the present embodiment.
In a word, present invention demonstrates that, PDLSCs expression HLA- I do not express HLA- Il DR CD80 and CD86, will not cause Allogeneic T lymphopoiesis yet, point out PDLSCs to have low immunogenicity；PDLSCs can suppress T lymphopoiesis caused by mitogen and alloantigen；PDLSCs will not cause the apoptosis of T lymphocytes, can recover multiplication capacity when T lymphocytes are upset again；PDLSCs plays its immune suppression function by secreting PGE2；Allogeneic PDLSCs can repair miniature pig periodontitis Cranial defect model, and will not cause immunological rejection.Embodiment 3:The biological dental root regeneration of tooth related mesenchymal stem cell mediation
1) material and method
1-1) separation, the culture of seed cell
Tip of a root dental papilla stem cell：It is sterile under anesthesia to pull out miniature pig canine tooth, cut tip of a root part tip of a root dental papilla, cleaned repeatedly respectively with D-Hank's liquid, shred, it is placed under enzyme containing type i collagen (3 g/L) and Dispase (4 g/L) digestive juice, 37 °C and digests 1 h, crosses 70 μ η ι filter screens and collect cell, 1000 r/min centrifuge 10 min, and single cell suspension is suspended into again with nutrient solution.By 0. 01-lxlO5Tip of a root dental papilla cells and periodontal ligament cell are inoculated in 6 orifice plates by/hole respectively, (contain 15% hyclone, 2 mmol/L paddy atmosphere acid amides, 100 U/ ml penicillin, 100 pg/ ml in α-Μ Ε Μ culture mediums Streptomysin) 37 °C, 5% C02Culture, changes liquid 1 time in every 2 ~ 3 days.Secondary Culture when being merged up to 80 %.
The cell of dental pulp thousand：Sterile under anesthesia to pull out miniature pig canine tooth, tooth of riving takes pulp tissue, is cleaned, shredded repeatedly respectively with D-Hank's liquid, digests, culture, and specific steps are with tip of a root dental papilla cells.
Periodontal ligament stem cell：The sterile periodontium pulled out miniature pig canine tooth, gently peel off around it, takes the periodontium in stage casing under anesthesia, is cleaned, shredded repeatedly respectively with D-Hank's liquid, digests, culture, and specific steps are with tip of a root dental papilla cells.
1-2) prepared by periodontal ligament cell diaphragm
By eugonic 2xl05Second or third generation periodontal ligament stem cell be seeded in 60mm culture dishes, it is trained and is divided into α-Μ Ε Μ culture mediums (containing 15% hyclone, 100 μ ι η ο Ι/L L-AA 2- Wagtail acid, 2 mmol/L glutamine, 100 U/ ml penicillin, 100 pg/ ml streptomysins）.Culture10One14My god, culture JUL border cell there is gauffer, cell patch is integrally peeled with compared with blunt blade or cell sleaker, during cell patch should not dry.
1-3) timbering material
The sour DFP timbering material of hydroxyapatite/upright stone tablet is made to the profile of similar root of the tooth, size is：The mm of diameter 5, the mm Round cones of length 15.The sour DFP of hydroxyl upright stone tablet lime stone/crack is porous network structure, aperture is generally 200 ~ 500 μ ι η, dental pulp stem cell is compounded on hydroxyapatite/tricalcium phosphate three-dimensional rack, the growth regulation dental pulp stem cell of 5 days in bioreactor, cell is fully extended, and the projection and secretory granules of cell surface are a lot, connect into a piece of, diameter is more in 20-50 μ ι η.
1-4) cell is combined before Hui Zhi
By lxlO8Cultivate to the tip of a root dental papilla in the 3rd generation to do and be carefully suspended from culture medium, root of the tooth type timbering material is placed in celliferous culture medium, 2 h are sufficiently mixed on 37 °C of shaking tables, timbering material is carefully pressed from both sides out, it is positioned in culture, remaining cell suspension is carefully added dropwise on timbering material, stands and adds nutrient solution after 4 h, Hui Zhi after 5d is cultivated in bioreactor.
1-5) return plant method
The edentulous region of miniature pig is chosen respectively.Mucous membrane is cut, glutinous periosteum valve, exposure crest of alveolar ridge is opened.A hole similar to hydroxyl upright stone tablet lime stone/tricalcium phosphate timbering material shape is bored in alveolar bone with 800 revs/min of speed with planting machine, cultivated 5 ~ 7 days in bioreactor after the sour DFP of above-mentioned tip of a root dental papilla stem cell/dental pulp stem cell and dental root shaped hydroxyapatite/upright stone tablet is mixed, the periodontal ligament stem cell diaphragm wrapping hydroxyl base phosphorus lime stone/sour DFP surfaces of Scales, Hui Zhi is in miniature pig alveolar bone.
2) result and discussion Periodontal ligament cell piece is applied to the history that periodontal tissue engineering has been for some time, but conventional sub- using Recell temperature-responsives culture, and the surface of culture has used a kind of temperature sensitive hydrophobic material PIPA-Am.When temperature is higher than 32.During C, surface has hydrophobicity, is adapted to the attachment and growth of cell, when temperature reduction, and condensate becomes hydrophilic and expansion, Spontaneous release cell.Harvesting need to only reduce the temperature to 20.C, without enzymic digestion and processing, can retain cell surface function and activity.But culture is sub- to need special material PIPA-Am, therefore we have found out the method for preparing periodontal ligament cell diaphragm using common culture JUL, 100 μ π ι ο Ι/L L-AA 2- phosphoric acid is added in medium component, cell fast breeding can be promoted, and a large amount of extracellular matrix collagen compositions can be secreted, all cells are linked together, after growth to a certain extent, we completely peel whole cell patch.Thin spore diaphragm does not destroy extracellular matrix and connects the support to be formed, and the functions such as adhesion propagation and the differentiation of cell surface protein are not destroyed, periodontium is formed after can promoting back in implant.The extremely thin about 0.2mm of normal parodontium, if using timbering material, relatively thick, the space left after material degradation is a problem, and periodontal diaphragm avoids this problem.
Due to the factor of gravity and nutrition supply, during carrying out inactive dimensional culture to cytoskeleton compound, the bottom and surface of timbering material being gathered discovery cell, the cell quantity that material internal sticks is seldom more.In incubation, static gas wave refrigerator also limit the depth of cell growth, and dental pulp stem cell is distributed in timbering material inner homogeneous, could form the tissue of uniformity.Want cell also can well be grown inside timbering material, nutriment effectively can be transmitted in internal stent and timely discharge cell metabolism waste is critically important.Although bioreactor can't analogue body internal circulation system mass exchange mechanism, but hydrodynamics environment can be produced inside timbering material, if controlling the size of mechanical strength, the damaging action to cell can be both reduced, favourable mechanical environment can be provided for the growth of cell again.Cell is compound to after material in this experiment cultivates 5 days in bioreactor, bioreactor dynamic property dimensional culture promotes the exchange of internal stent nutriment, seed cell being uniformly distributed in timbering material is promoted, is conducive to cell to keep Osteogenesis phenotype and promotes deposition of the extracellular base shield in timbering material.
Return and plant after 6 ~ September, the high density shadow that Hui Zhi biological root of the tooth shows as not transmiting in X-ray film around has low-density shadow picture to surround.In histology, the biological root of the tooth tissue morphology of regeneration and newborn bone tissue are entirely different, are made up of many spherical sclerous tissues's agglomerates in different growth phases, spherical agglomerates are connected with each other and reticulated.Newborn spherical sclerous tissues's small volume, outside is around cell of the loose arrangement in bluish violet, thus it is speculated that be probably odontoblast or cementoblast, and center is the matrix of uniform powder dye.More ripe spherical sclerous tissues's bolus volume is larger, and centre is substantially free of cell, or visible very small amount is embedded in cell fragment therein.There is the dentinoid structure of class in agglomerate, wherein It can be seen that the dentinal tubule spline structure of irregular arrangement, also has and more uniform consistent sclerous tissues is shown as under similar cementoid structure, mirror.The overseas hard tissue area newly formed is that fiber knot Parties organizes wrapping, has no and is also shown in cell infiltration, subregion in cementoid and dentine spline structure that the similar fibroid structure insertions of Sharp's are being formed.
From technical scheme disclosed above, the present invention has particularly significant and profound significance for repairing absence of tooth using biological dental root regeneration：First, the present invention proposes the new concept that base knits the biological dental root regeneration of engineering technology in Group.Secondly, the present invention implements the new concept of biological dental root regeneration on larger animal miniature pig, succeeds, it was demonstrated that the inventive method is feasible, is expected to turn into a kind of new repair mode applied to clinic.3rd, it is specific implementation method system that the present invention is provided, reliable, provide theoretical foundation and technical support for the further investigation and commercialization that carry out biological dental root regeneration.
In the present embodiment method there is provided an experimental result as shown in Figure 9, wherein illustrate in detail the situation of on miniature pig successful regeneration biology root of the tooth.Wherein, A:Experiment flow figure；（B, C):Form dentine cementum spline structure and parodontium spline structure（HE is dyed）; D:Parodontium COL I stained positives； E:Dentine cementum spline structure Dsp stained positives； F ~ I:Regeneration biological root of the tooth ESEM（SEM) observe： F:HA/TCP material porous gap structures（ 100-400um ); H:In the dentine cementum structure and alveolar bone that the periodontal membrane fiber insertion newly formed is newly formed（G figures arrow is signified）； I：The dentine spline structure newly formed（G figures block arrow is signified）.
In the present embodiment method there is provided another experimental result as shown in Figure 10, it is similar with Fig. 9, the situation that biological dental root regeneration succeeds that carried out on miniature pig is illustrate in detail in Figure 10.Embodiment 4:Come off deciduous teeth dental pulp stem cell transplantation treatment systemic loupus erythematosus (SLE) 1) material and method
1-1) people SHED
The normal deciduous teeth for the healthy patients of 6-8 Sui pulled out are chosen, the method reported according to previous literature is separated and culture SHED (3).The utilization of tissue obtains the approval of Ethics Committee of the Capital University of Medical Sciences.
1-2) human bone marrow stroma stem cell and PMNC
Using the bone marrow stroma stem cell of Percoll gradient centrifugation separating health donor（Bone marrow mesenchymal stem cells, BMMSCs) and all blood mononuclear cell (the peripheral blood mononuclear cells of sunset fore-telling PBMCs).
1-3) SHED systemic lupus erythematosus (SLE)
C57BL/6J and C3MRL-Fas,pr/ J (MRL/lpr) mouse（Female, 6-7 week old), Beige nude/nude Xid (III) mouse (female, 8-12 week old）By providing.This experiment is ratified by Ethics Committee of the Capital University of Medical Sciences.Experiment is divided into following 3 groups：1st, blank control group (n=3), any treatment is not done.2nd, transplanting BMMSCs groups (n=3).3rd, transplanting SHED groups (n=3).Under general anesthesia, by SHED or BMMSCs (lxl05Cells/10g body weight, cell is suspended in 100 ml PBS) 16 week old MRL/lpr mouse, control group injecting normal saline are entered by tail vein injection.Put to death in 20 week old of mouse, take peripheral blood, kidney, long bone (femur and shin bone) sample.Kidney sample is fixed, FFPE, row HE, trichrome, periodic acid-schiff (pas) dyeing, and observation glomerular basement membrane dysfunction recovers and mesangial cell excessively increases recovery situation.Serum dsDNA-IgG is detected by ELISA, dsDNA-IgM, ANA levels, detection urine neutralizes complement component 3 (complement 3, C3), kreatinin (creatinine) Urine proteins (urine protein) level in serum.
2) result and explanation
SLE is that, there is the autoimmunity disease that Multiple Antibodies are characterized in multi viscera involvement and blood, many researchs confirm that T, Β lymphocyte overactivity are the key links in SLE morbidities.T, Β lymphocyte derive from lymph ancestral cells, and MSCs has important effect in lymph trunk/progenitor cell proliferation differentiation, so effects of the MSCs in SLE causes a disease increasingly attracts attention.
SLE patient MSCs exists abnormal; the marrow stromal cell that it breaks up can not preferably support lymphocyte growth; the various cell factors that the growth of lymphocyte is secreted with marrow stromal cell dependent on lymphocyte, the exception of marrow stromal cell in itself take part in the abnormal activation of lymphocyte.
The research such as Majumdar shows that MSCs expresses substantial amounts of cell adhesion molecule, in cell-cell adhesion, goes back to the nest, plays an important role in hematopoiesis support, regulation immune cell function.When tumour cell is planted people's Allogeneic mouse, tumour cell can be removed by host immune system, and after tumour cell and MSCs inject Allogeneic mouse simultaneously, tumour cell can be survived in acceptor, show that MSCs has immune suppression function in vivo.MSCs still retains its immunoregulation effect when being divided into other cell types, and this means that the MSCs of transplanting can play long-term immunoregulation effect.
Experiment in vitro confirms that SHED has MSCs biology and immunological characteristic, can be influenced each other with T lymphocytes.Compared with BMMSCs, SHED reduces Thl7 cell numbers and plays immunology effect by recovering peripheral blood Tregs/Thl7 ratios.After the MRL/lpr mouse injection SHED of 16 week old, it can be found that girder osteanagenesis, osteoclast activity is inhibited.SHED mouse is injected as injecting BMMSCs mouse, Gegenbaur's cell microenvironment has been rebuild, so as to improve its dysfunction.After MRL/lpr mouse injection SHED, though Treg levels are not improved, Treg/Thl7 cell proportions substantially increase, and this shows SHED Immunoloregulation function may come from TregSuppress autoimmunity and Thl7 can then promote autoimmunity and inflammation.
In the present embodiment method there is provided an experimental result as shown in Figure 11, wherein illustrate in detail the situation that come off deciduous teeth dental pulp stem cell systemic lupus erythematosus mouse serum and renal histology change.The visible the present embodiment of result realizes the purpose of the present invention from figure.
In a word, present invention demonstrates that, can extract out SHED from the deciduous teeth dental pulp come off, it is derived from mesoblastic mescenchymal stem cell, biology and immunological characteristic and function with MSCs.Although SHED immunoregulation effect mechanism is not yet clear and definite, due to its immunoregulation effect, there is potential therapeutic action in terms of autoimmunity disease, SHED transplanting can be used as the new try for treating autoimmunity disease.Embodiment 5:Allogeneic Periodontal ligament stem cell OiPDLSCs) it is used to treat the standard operation example of periodontosis
The present embodiment illustrates that allogeneic Periodontal ligament stem cell (hPDLSCs) is used for an example for treating the standard operation of periodontosis with reference to Figure 12：
(a) collection people's parodontium (periodontal ligament, PDL).The normal impaction mat woven of fine bamboo strips three is gathered from 18-28 Sui patient to grind one's teeth in sleep（impacted third molar).PDL is lightly separated from tooth surface.
(b) hPDLSCs is cultivated, hPDLSCs is separated.After original culture 15 days, the quantity of the hPDLSCs from an impacted third molar is about 4.90 ± 0.34 xl05(P0;The of n=10) is further cultured for after other 15 days, and hPDLSCs quantity (P3) increases to 8.86 ± 0.46xl06(n=10), hPDLSCs is to the aobvious positives of CD146 and CD90.
(c) freezen protective hPDLSCs, by third generation hPDLSCs 10% DMSO and 90% FBS freezen protectives, and is stored in liquid nitrogen.
(d) hPDLSCs is melted.After thawing, mycoplasma, bacterium, Colony forming efficiency, interstital stem cell marking mode and karyotyping are checked to hPDLSCs.
(e) hPDLSCs pieces are prepared.To three kinds of different cell number (lxlO in 100 mm culture dishes5, lxlO6And 2xl06;N=3) cultivate 12-15 days, in lxlO6And 2xl06Cell sheet is formed in group, but in lxlO5Cell sheet is not formed in group.Therefore, by lxlO6HPDLSCs be inoculated into 100 mm culture jni in, up to 15 days.
(f) then, 40 mg HA/TCP are placed in these culture Asias.
(g) hPDLSCs pieces and HA/TCP full figure. (h) oral cavity periodontal damage figure.
In periodontal initial treatment（I) after, two allogeneic hPDLSCs pieces and HA/TCP are implanted to 3mmx5mmx7mm periodontal damage fault location (j).
(k) ensuing project includes clinical and radiography evaluation, hematology and immunological evaluation.Bibliography：
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Claims (15)

  1. Claim
    1st, purposes of the tooth related stem cells in the product of the preparation disease related to tooth for preventing or treating or the state of an illness, or the purposes in the product for the formation of tooth linked groups or reparation is prepared.
    2nd, purposes according to claim 1, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
    3rd, according to the purposes of claim 1 or 2, wherein described tooth related stem cells come from mammal, example as mentioned tooth related stem cells from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, sheep, goat.
    4th, according to the purposes of any one of claims 1 to 3, it is selected from wherein the disease related to tooth or the state of an illness or tooth linked groups form or repaired：Periodontosis, periodontitis, tooth defect, dental tissue defect, tooth defect reparation, tooth linked groups defect and reparation, the regeneration of tooth linked groups substitutes, etc..
    5th, tooth related stem cells prepare be used to prevent treat immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, with the purposes in the abnormal product for raising relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus of T lymphocytes.
    6th, purposes according to claim 5, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
    7th, the method for prevention or the treatment disease related to tooth or the state of an illness, or the method for being formed or repairing tooth linked groups, this method applies the tooth related stem cells of effective dose including the host to prevention in need or the treatment disease related to tooth or the state of an illness, or this method includes applying the tooth related stem cells of effective dose to the host in need for being formed or repairing tooth linked groups.
    8th, method according to claim 7, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
    9th, according to the method for claim 7 or 8, wherein described tooth related stem cells come from mammal, example as mentioned tooth related stem cells from selected from following mammal：People, pig (such as WZSP, Guizhou Xiang pig), ox, horse, monkey, rat, mouse, cavy, sheep, silk floss Sheep, goat.
    10th, according to the method for any one of claim 7 to 9, it is selected from wherein the disease related to tooth or the state of an illness be confused or tooth linked groups form or repaired：Periodontosis, periodontitis, tooth defect, dental tissue defect, tooth defect reparation, tooth linked groups defect and reparation, the regeneration of tooth linked groups substitutes, etc..
    11st, prevent or treat immunity disease or the method for the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, the disease relevant with the exception rise of T lymphocytes or the state of an illness, lupus erythematosus or systemic loupus erythematosus, this method is included to the tooth related stem cells for having the host that this needs using effective dose.
    12nd, method according to claim 12, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
    13rd, a kind of composition, it includes the tooth related stem cells and optional pharmaceutically acceptable carrier of effective dose.
    14th, composition according to claim 13, wherein described tooth related stem cells are selected from：Dental pulp stem cell, the deciduous teeth dental pulp stem cell that comes off, periodontal ligament stem cell and tip of a root dental papilla stem cell.
    15th, according to the Group compounds of claim 13 or 14; it is for preventing or treating the disease or the state of an illness related to tooth; either formed or repaired or for preventing or treating immunity disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with T Abnormal lymphocyte activations or the state of an illness, raise relevant disease or the state of an illness, lupus erythematosus or systemic loupus erythematosus extremely with T lymphocytes for tooth linked groups.
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