CN103432007A - New use of tooth-related stem cells - Google Patents

New use of tooth-related stem cells Download PDF

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CN103432007A
CN103432007A CN2013102733426A CN201310273342A CN103432007A CN 103432007 A CN103432007 A CN 103432007A CN 2013102733426 A CN2013102733426 A CN 2013102733426A CN 201310273342 A CN201310273342 A CN 201310273342A CN 103432007 A CN103432007 A CN 103432007A
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王松灵
施松涛
丁刚
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Capital Medical University
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Abstract

The invention relates to a new use of tooth-related stem cells, provides a new use of tooth-related stem cells in preparation of products for prevention or treatment of tooth-related diseases, immunologic diseases, autoimmune diseases, diseases related to abnormal T lymphocyte activation or T lymphocyte increase, and lupus erythematosus or systemic lupus erythematosus, or in preparation of products for repairing tooth-related tissues, and further provides a composition containing tooth-related stem cells, and a method for preventing or treating diseases by using the tooth-related stem cells.

Description

The new purposes of tooth related stem cells
The application is dividing an application of application number is 201080005266.5, denomination of invention is " the new purposes of tooth related stem cells " patent application.
Invention field
The present invention relates to the new purposes of tooth related stem cells.Particularly, the present invention relates to the purposes of tooth related stem cells in the product for the preparation of prevention or the treatment disease relevant to tooth or the state of an illness, or the purposes in the product that forms or repair for the preparation of tooth linked groups; Also relate to the purposes of tooth related stem cells in the product for the preparation of prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, the disease relevant with the abnormal rising of T lymphocyte or the state of an illness, lupus erythematosus or systemic lupus erythematosus (sle); Also relate to the Therapeutic Method according to such use; Also relate to the compositions that comprises tooth related stem cells.
Background technology
Stem cell is the germinal cell that a class has self renewal and differentiation potential, can be divided into embryonic stem cell and adult stem cell.In adult tissue and organ, ubiquity special adult stem cell, the tooth related stem cells that for example derives from the people had been found that at present is all to derive from mesoblastic mescenchymal stem cell (mesenchymal stem cells, MSCs), comprise dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (stem cells from human exfoliated deciduous teeth comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP) (1-4, refer to document 1 to 4, lower same), they can derive from many animals (mammal for example, people for example).Research shows that tooth related stem cells has very high multiplication capacity and multi-lineage potential, can be to osteoblast, adipose cell, the differentiation of neural like cell.When the complex tooth related stem cells and three-dimensional stent material is implanted to nude mice by subcutaneous, can produce autologous pulpodentinal complex spline structure and cementum periodontal membrane complex spline structure (1,3).Applied at present PDLSCs and the SCAP biological root of the tooth (4) of successfully regenerating on autologous miniature pig model.One of stem-cell research focus in the world concentrates on and sets up the various soma cell banks that are organized at present, carries out the clinical experimental study of stem cell.
According to WHO (2003) statistics, show, crowd's scope that the tooth relevant disease involves is considerably beyond other any disease.At present, the annual medical expense for odontopathy in the whole world is over 20,000,000,000 dollars.In the tooth relevant disease, periodontal disease is the very high bacterial infection disease of sickness rate in a kind of worldwide, finally causes the tooth supporting tissue to be lost and absence of tooth, and can cause a series of whole body systemic disease (13); And absence of tooth can cause serious impact to digestion and whole body health, also can make the patient produce bad social mentality's impact simultaneously.But at present periodontal disease and the damaged medicine do not had of dental tissue or Therapeutic Method are treated or repaired; The treatment of relevant absence of tooth mainly is still the substitute that adopts artificial material to make tooth.
Systemic lupus erythematosus (sle) (systemic lupus erythematosus, SLE) be take many internal organs get involved and blood in have the autoimmune disease that Multiple Antibodies is feature, extend the life cycle that conventional immunosuppressant or immune modulating treatment can make most of patients, and quality of life improves.After at first Italian scholar Marmont in 1996 reports and achieves success by autologous bone marrow transplantation treatment serious symptom lupus erythematosus, carry out rapidly the multiple intractable immunological diseases such as autologous peripheral blood stem cell transplantation and autologous bone marrow transplantation treatment serious symptom SLE both at home and abroad, obtained curative effect preferably.Hematopoietic stem cell transplantation can make some patients were reach state of an illness control, but its expense is high, complication is many, and the disease relapse rate, up to 40%-50%, can not be used by routine; And, although after transplanting, some patients were likely reaches long-term remission, all can not thoroughly effect a radical cure autoimmune disease, there is the problem of recurrence in some patients were.
Therefore, still need effectively to treat the novel method of some tooth relevant disease and some immune disease at present.
Summary of the invention
The novel method that the purpose of this invention is to provide some tooth relevant disease of effective treatment and some immune disease.The inventor now have been surprisingly found that under study for action tooth related stem cells for example periodontal ligament stem cell (PDLSCs) not only in autologous tooth, make the regeneration of damaged dental tissue, and dental tissue that can be damaged at allosome makes dental tissue regeneration, PDLSCs also has inhibitory action to the T lymphocyte of abnormal rising simultaneously.The present invention is based on above-mentioned result of study is accomplished.
summary of the invention
At first the present invention relates to PDLSCs for the preparation of prevention or treatment periodontal disease, purposes in the product that tooth defect is repaired.
The invention still further relates to PDLSCs purposes in the product for the preparation of abnormal raise relevant disease or symptom of prevention or treatment and T lymphocyte.
The invention still further relates to a kind of prevention or treatment periodontal disease, the method for repair deficiency dental tissue, it comprises that the host that needs to prevent or treat periodontal disease or need the repair deficiency dental tissue is effectively to prevent or the PDLSCs of therapeutic dose or repair deficiency dental tissue amount.
The present invention relates to a kind of prevention or treatment and abnormal relevant disease or the method for symptom of raising of T lymphocyte, it comprises the PDLSCs that gives the abnormal host's prevention raise of T lymphocyte or treatment effective dose.
detailed Description Of The Invention
In more detail, the invention provides the concrete item of following various aspects and various aspects.
First aspect present invention provides the purposes of tooth related stem cells in the product for the preparation of prevention or the treatment disease relevant to tooth or the state of an illness, or the purposes in the product that forms or repair for the preparation of tooth linked groups.
According to the purposes of first aspect present invention any one, wherein said tooth related stem cells 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.
According to the purposes of first aspect present invention any one, wherein said tooth related stem cells is from mammal.In one embodiment, described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
Purposes according to the first aspect present invention any one, wherein said tooth related stem cells is from mammal, and be selected from: dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (the stem cells from human exfoliated deciduous teeth that comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP).
According to the purposes of first aspect present invention any one, the wherein said disease relevant to tooth or the state of an illness or tooth linked groups form or repair and be selected from: periodontal disease, periodontitis, tooth defect, dental tissue are damaged, the tooth defect reparation, tooth linked groups is damaged and reparation, the substitute regeneration of tooth linked groups, etc.
According to the purposes of first aspect present invention any one, wherein said tooth related stem cells is used for disease or the state of an illness autologous or that tooth allosome is relevant, or forms or repair for the tooth linked groups of autologous or allosome.
According to the purposes of first aspect present invention any one, wherein said tooth linked groups includes but not limited to tooth, root of the tooth, dental pulp, gingiva etc., and other tissue relevant to tooth inside, tooth top layer, dental surface or around teeth.
The feature and advantage of first aspect present invention and each subitem thereof are equally applicable to other either side of the present invention and each subitem thereof.
Second aspect present invention provides the purposes of tooth related stem cells in the product for the preparation of prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, the disease relevant with the abnormal rising of T lymphocyte or the state of an illness, lupus erythematosus or systemic lupus erythematosus (sle).
According to the purposes of second aspect present invention any one, wherein said tooth related stem cells 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.
According to the purposes of second aspect present invention any one, wherein said tooth related stem cells is from mammal.In one embodiment, described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
Purposes according to the second aspect present invention any one, wherein said tooth related stem cells is from mammal, and be selected from: dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (the stem cells from human exfoliated deciduous teeth that comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP).
According to the purposes of second aspect present invention any one, wherein said tooth related stem cells is for the immune disease of autologous or allosome or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, disease or the state of an illness, lupus erythematosus or the systemic lupus erythematosus (sle) relevant with the abnormal rising of T lymphocyte.
The feature and advantage of second aspect present invention and each subitem thereof are equally applicable to other either side of the present invention and each subitem thereof.
Third aspect present invention provides prevention or the treatment disease relevant to tooth or the method for the state of an illness, perhaps form or repair the method for tooth linked groups, the method comprises to there being the host who needs prevention or the treatment disease relevant to tooth or the state of an illness to use the tooth related stem cells of effective dose, or the method comprises the tooth related stem cells of using effective dose to the host who has needs to form or to repair tooth linked groups.
According to the method for third aspect present invention any one, wherein said tooth related stem cells 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.
According to the method for third aspect present invention any one, wherein said tooth related stem cells is from mammal.In one embodiment, described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
Method according to the third aspect present invention any one, wherein said tooth related stem cells is from mammal, and be selected from: dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (the stem cells from human exfoliated deciduous teeth that comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP).
According to the method for third aspect present invention any one, the wherein said disease relevant to tooth or the state of an illness or tooth linked groups form or repair and be selected from: periodontal disease, periodontitis, tooth defect, dental tissue are damaged, the tooth defect reparation, tooth linked groups is damaged and reparation, the substitute regeneration of tooth linked groups, etc.
According to the method for third aspect present invention any one, wherein said tooth related stem cells is used for disease or the state of an illness autologous or that tooth allosome is relevant, or forms or repair for the tooth linked groups of autologous or allosome.
According to the method for third aspect present invention any one, wherein said tooth linked groups includes but not limited to tooth, root of the tooth, dental pulp, gingiva etc., and other tissue relevant to tooth inside, tooth top layer, dental surface or around teeth.
According to the method for third aspect present invention any one, wherein said host is mammal.In one embodiment, described host is selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
In an embodiment of third aspect present invention any one method, it comprises the following steps:
(a) gather people's periodontal membrane;
(b) cultivate hPDLSCs;
(c) freezing preservation hPDLSCs optionally;
(d) optionally melt hPDLSCs, and optionally hPDLSCs is checked to mycoplasma, antibacterial, colony form efficiency, interstital stem cell marking mode and karyotyping;
(e) prepare the hPDLSCs sheet;
(f) HA/TCP is placed in to the vessel that accommodate the hPDLSCs sheet;
(j), after optional periodontal initial therapy, hPDLSCs sheet and HA/TCP are implanted to the periodontal damage fault location;
(k) optionally carry out clinical and radiography evaluation, hematology and immunological evaluation.
The feature and advantage of third aspect present invention and each subitem thereof are equally applicable to other either side of the present invention and each subitem thereof.
Fourth aspect present invention provides prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, the disease relevant with the abnormal rising of T lymphocyte or the method for the state of an illness, lupus erythematosus or systemic lupus erythematosus (sle), and the method comprises the tooth related stem cells of using effective dose to the host that these needs are arranged.
According to the method for fourth aspect present invention any one, wherein said tooth related stem cells 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.
According to the method for fourth aspect present invention any one, wherein said tooth related stem cells is from mammal.In one embodiment, described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
Method according to the fourth aspect present invention any one, wherein said tooth related stem cells is from mammal, and be selected from: dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (the stem cells from human exfoliated deciduous teeth that comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP).
According to the method for fourth aspect present invention any one, wherein said tooth related stem cells is for the immune disease of autologous or allosome or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, disease or the state of an illness, lupus erythematosus or the systemic lupus erythematosus (sle) relevant with the abnormal rising of T lymphocyte.
According to the method for fourth aspect present invention any one, wherein said host is mammal.In one embodiment, described host is selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
The feature and advantage of fourth aspect present invention and each subitem thereof are equally applicable to other either side of the present invention and each subitem thereof.
Fifth aspect present invention provides a kind of compositions, the tooth related stem cells that it comprises effective dose and optional pharmaceutically acceptable carrier.
According to the compositions of fifth aspect present invention any one, wherein said tooth related stem cells 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.
According to the compositions of fifth aspect present invention any one, wherein said tooth related stem cells is from mammal.In one embodiment, described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
Compositions according to the fifth aspect present invention any one, wherein said tooth related stem cells is from mammal, and be selected from: dental pulp stem cell (dental pulp stem cells, DPSCs), deciduous teeth dental pulp stem cell (the stem cells from human exfoliated deciduous teeth that comes off, SHED), periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) and tip of a root dental papilla stem cell (stem cells from apical papilla, SCAP).
Compositions according to the fifth aspect present invention any one; it is for prevention or treatment disease or the state of an illness relevant to tooth; perhaps for tooth linked groups, form or repair, or for prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, disease or the state of an illness, lupus erythematosus or the systemic lupus erythematosus (sle) relevant with the abnormal rising of T lymphocyte.
Compositions according to the fifth aspect present invention any one; wherein said effective dose be can be effectively for prevention or the treatment disease relevant to tooth or the dosage of the state of an illness; or will be with the dosage that effectively for tooth linked groups, forms or repair, or will be with effectively for prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, the disease relevant with the abnormal rising of T lymphocyte or the dosage of the state of an illness, lupus erythematosus or systemic lupus erythematosus (sle).
The feature and advantage of fifth aspect present invention and each subitem thereof are equally applicable to other either side of the present invention and each subitem thereof.
According to the detailed record of the context of the invention, the present invention realizes above-mentioned various aspects and each subitem thereof satisfactorily.
Below with characteristics, be further described to various aspects of the present invention.
All documents that the present invention quotes from, their full content is incorporated to this paper by reference, and if, when the expressed implication of these documents and the present invention are inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this, these terms and phrase to be described in more detail and to explain, the term of mentioning and phrase, if any inconsistent with known implication, are as the criterion with the implication that the present invention was explained.
The inventor now have been surprisingly found that the relevant stem cell of tooth such as PDLSCs not only makes damaged dental tissue regeneration in autologous tooth under study for action, and dental tissue that can be damaged at allosome makes dental tissue regeneration.The present invention is based on above-mentioned result of study is accomplished.Therefore, the present invention relates to tooth related stem cells for example PDLSCs for example, for the preparation of prevention or treatment periodontal disease, purposes (shown in Fig. 8) in the product that tooth defect is repaired; Also relate to a kind of prevention or treatment periodontal disease, the method of repair deficiency dental tissue, it comprises that the host that needs prevention or treat periodontal disease or need the repair deficiency dental tissue is effectively to prevent or the tooth related stem cells of therapeutic dose or repair deficiency dental tissue amount PDLSCs for example.
In addition, the present invention proposes the new concept of biological dental root regeneration; Utilize autologous allosome SCAP/DPSCs and PDLSCs to bear the biological root of the tooth with biological function, the reparation of being preced with again on the new functional living being root of the tooth formed, recover patient's masticatory function (for example, shown in Figure 10) again; Provide the specific implementation method of biological dental root regeneration, for further the mechanism of biological dental root regeneration being furtherd investigate 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 relate to SCAP, DPSCs, the purposes of stem cell in biological dental root regeneration that the teeth such as PDLSCs are relevant.The invention still further relates to the concrete implementing method of biological dental root regeneration, comprising preparation and required optimum cell number and the optimum growh time of periodontal ligament stem cell diaphragm.
Moreover, the present invention relates to for example PDLSCs purposes in the product for the preparation of abnormal raise relevant disease or symptom of prevention or treatment and T lymphocyte of tooth related stem cells.The invention still further relates to a kind of abnormal relevant disease or the method for symptom of raising of prevention or treatment and T lymphocyte, it comprises the tooth related stem cells PDLSCs for example that gives the abnormal host's prevention raise of T lymphocyte or treatment effective dose.The invention further relates to tooth related stem cells such as SHED purposes (for example, shown in Figure 11) in the autoimmune systemic diseases such as prevention or systemic lupus erythematosus disease.
According to the present invention, term " periodontal disease " includes but not limited to periodontitis.
According to the present invention, term " defective tooth tissue " includes but not limited to the various damaged situation of host's tooth, the loss of tooth caused as a variety of causes.
According to the present invention, term " host " is often referred to mammal, includes but not limited to the people, pig, cattle, horse etc.In one embodiment, term " host " refers to people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
According to the present invention, term " product " refers to the various forms that is suitable for the PDLSCs application.According to the present invention, term " product " also refers to be suitable for the various forms of tooth related stem cells application, such as compositions, pharmaceutical composition etc.
According to the present invention, term " disease relevant to tooth or the state of an illness " refers to disease that host described herein suffers from or show, the state of an illness, Signs, condition etc., and these diseases, the state of an illness, Signs, condition etc. are relevant to tooth.
According to the present invention, term " tooth linked groups forms or repairs " or term " formation of tooth linked groups or reparation " or " forming or repair tooth linked groups " etc., they have identical or approximate implication, and the tooth linked groups that typically refers to host described herein is formed, repairs, generates, regenerates, cultivation etc. processed or operation, or to tooth linked groups abnormal (such as damaged) formed, repaired, generated, regenerated, cultivation etc. processes or operation.
According to the present invention, term " compositions " is to have the implication that those skilled in the art usually understand, and typically refer to can connect or indirectly (for example dilution before use) for the form of clinical use, such as dosage form, pharmaceutical dosage form, form of medication, etc.At clinical practice field or drug world, term " compositions " also has equivalent meanings with " pharmaceutical composition " usually.
Can change the actual dose level of the present composition or pharmaceutical composition Tooth related stem cells, so as the stem cell amount of gained can be effectively for concrete host, patient in the situation that particular composition and composition thereof and corresponding administering mode obtain required treatment or prophylactic response.The dosage level fibrous root is selected according to treatment process, formation and the reparation of the order of severity, disease or the state of an illness of the activity of concrete stem cell, route of administration, the patient's condition of treat (and generation, regeneration, cultivation etc.) processing or the process operated and the patient's to be treated patient's condition and medical history.But the way of this area is, the dosage of stem cell and time of application are from lower than for obtaining, level that required therapeutic effect requires, increasing gradually dosage, until obtain required effect.Therefore, for the present invention, those skilled in the art, under the instruction of the detailed disclosed information of the present invention, can determine the concrete dosage be suitable under concrete condition according to the concrete condition such as but not limited to above-mentioned, and need not do concrete restriction.Particularly, can determine the use amount in arbitrary situation with reference to concrete amount used in embodiment of the present invention part, for example in the present invention, hereinafter used the WZSP PDLSCs of concrete dosage, those skilled in the art can convert as the people this dosage with the dosage under condition according to above-mentioned dosage and in conjunction with techniques well known instruction.
Tooth related stem cells of the present invention is (with the former state form) or with the form administration of pharmaceutical composition separately.Pharmaceutical composition of the present invention can be made into various suitable dosage forms according to route of administration.Use the upper acceptable carrier of one or more physiologys, comprise excipient and auxiliary agent, they are conducive to tooth related stem cells is processed into to the preparation that can pharmaceutically use.Suitable dosage form depends on selected route of administration, can be manufactured according to general knowledge well known in the art.In one embodiment of the invention, described tooth related stem cells for example is present in, in the medium (, normal saline is as 0.9% normal saline, etc.) of cytocompatibility.In one embodiment of the invention, described tooth related stem cells is present in the medium of cytocompatibility, and preserve at low temperatures, such as preserving, and can be optionally redissolve into before use applicable the and form used spiritual according to the present invention under cold preservation, the condition such as freezing.
The accompanying drawing explanation
Fig. 1. the expression of people PDLSCs immune molecule: applying flow cytometry detects the expression of fPDLSCs and cPDLSCs related immune molecule.HLA-I, HLA-Π DR, the expression of CD80 and CD86 as shown in the figure: fPDLSCs (76.2% ± 5.8%, n=5) and cPDLSCs (78.5% ± 6.4%, n=5) express HLA-I, but do not express HLA-Π DR and costimulatory molecules CD80, CD86.
Fig. 2 .PDLSCs suppresses the T lymphopoiesis: (a) fPDLSCs/cPDLSC can not cause the Allogeneic T lymphopoiesis.(b) fPDLSCs/cPDLSC dose dependent ground suppresses the T lymphopoiesis that mitogen PHA causes.(c) postpone to add fPDLSCs/cPDLSC also can suppress the T lymphopoiesis that PHA causes.(d) fPDLSCs/cPDLSC can suppress two-way mixed lymphocyte reaction.
Fig. 3 .PDLSCs suppresses the T lymphopoiesis by secretion PGE2: (a) in the Transwell culture experiment, PDLSCs also can suppress the T lymphopoiesis, and prompting PDLSCs secretion soluble factor is brought into play immunosuppressive action.(b) all there is TGF-β 1 in simple PDLSCs culture supernatant and MLR supernatant, but there is no significant difference between the two.(c) concentration of PGE2 significance in MLR raises.(d) the neutralization experiment shows that anti-TGF-beta 1 antibody does not recover the T lymphopoiesis, and the PGE2 inhibitor has been offset the immunosuppressive action of PDLSCs, and prompting PGE2 is the Main Factors of mediation PDLSCs immunosuppressive action.The immunosuppressive action of (e, f) PDLSCs does not cause the lymphocytic apoptosis of T (e), and merely with PHA, stimulates the apoptosis rate consistent (f) after the T lymphocyte.(g) the repressed T lymphocyte that PDLSCs causes can recover propagation again while stimulating again with PHA or IL-2.
The mensuration of Fig. 4 .HGF and IL-10: all do not measure HGF and IL-10 in simple PDLSCs culture supernatant and MLR supernatant, illustrate that these two kinds of factors do not participate in the immunosuppressive action of PDLSCs mediation.
The paradenlal tissue regeneration of Fig. 5 .PDLSCs mediation: (a)-4w and 0w, there is no the difference of significance between the clinical indices of four groups.But after treatment 12 weeks, the PD of autologous or allogeneic PDLSCs transplantation group, GR compares remarkable recovery with HA/TCP with the blank group with AL.(b, c, d, e) CT scan has obvious periodontal bone damaged before showing treatment, and defect degree is close.Latter 12 weeks for the treatment of, autologous PDLSCs group (h) and allogeneic PDLSCs group (i) have obtained periodontal Regeneration fully.The blank group does not almost have osteanagenesis (f), and the bone defect degree of HA/TCP group increases the weight of (g). (j, k, l, m) Histological research finds that autologous or allogeneic PDLSCs group has significantly new bone and paradenlal tissue regeneration at the bone defective region.But typical periodontitis performance is as still high-visible in HA/TCP group and blank group as dark periodontal pocket, shortage area of new bone and desmodontium.PD: visit and examine the degree of depth, GR: gingival recession, AL: attachment loss, D: dentin, C: cementum, PDL: periodontal membrane, B: bone.
Fig. 6. allogeneic PDLSCs transplants and can not cause immunological rejection: (a) at each time point as shown in the figure, the CD3 of allogeneic PDLSCs transplantation group +, CD4 +, CD8 +the T lymphocyte does not have significant difference; (b) in transplanting latter 3 days, CD3 +, CD4 +, CD8 +the expression of the mark of T lymphocyte number and activated T lymphocytes---CD40L does not have notable difference between four treatment groups.
Fig. 7. the immune state of 12w after transplanting: 12w after treatment, CD3 +, CD4 +, CD8 +the expression of the mark of T lymphocyte number and activated T lymphocytes---CD40L does not have notable difference between four treatment groups yet.
Fig. 8. described the paradenlal tissue regeneration of PDLSCs mediation in one embodiment of the present of invention.
Fig. 9. described successful regeneration biological root of the tooth on miniature pig.
Figure 10. described to carry out biological dental root regeneration and succeeded on miniature pig.
Figure 11. described to come off deciduous teeth dental pulp stem cell systemic lupus erythematosus Mus serum and renal histology change.
Figure 12. illustrate that allogeneic Periodontal ligament stem cell (hPDLSCs) is used for the treatment of an example of the standard operation of periodontal disease.
The specific embodiment
By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.
The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although, for to realize that many materials and operational approach that the object of the invention is used are well known in the art, the present invention still does to describe in detail as far as possible at this.
embodiment 1:PDLSCs is to the lymphopoietic inhibitory action of T
materials and methods
1), people PDLSCs
Choose the normal third molar of the healthy patients in 18-28 that Beijing Stomatological Hospital Attached to Shoudu Medical Science Univ.'s Oral and Maxillofacial Surgery outpatient service pulls out year, according to the method for previous literature report, separate and cultivate PDLSCs (3).The utilization of tissue obtains the approval of Ethics Committee of the Capital University of Medical Sciences.For the frozen periodontal ligament stem cell of application (cryopreserved periodontal ligament stem cells in next step experiment, cPDLSCs), by frozen in liquid nitrogen 3 months of the periodontal ligament stem cell of part fresh separated (freshly isolated periodontal ligament stem cells, fPDLSCs).After 3 months, freeze-stored cell is quick-thawing, inoculation, cellar culture in 37 ℃ of water-baths.In this research, the cell of application is all in 2-4 generation.In same experiment, fPDLSCs and the cPDLSCs of application the same generation.
2), PERIPHERAL BLOOD MONONUCLEAR CELL
The PERIPHERAL BLOOD MONONUCLEAR CELL (peripheral blood mononuclear cells, PBMCs) of application Percoll gradient centrifugation separating health donor.
3), antibody
The anti-human human leucocyte antigen of application (human leukocyte antigen in this research, HLA)-I, HLA-Π DR, CD80, CD86 antibody (BD Biosciences), anti-CD3, CD4, CD8, CD40L, transforming growth factor β (transforming growth factor β, TGF-β), hepatocyte growth factor (hepatocyte growth factor, HGF) is anti- body (Abcam), the anti-mouse IgG antibody of anti-IL-10, FITC labelling (AbD Serotec).
4), flow cytometry
In order to study the expression of PDLSCs surface related immune molecule, by 1.0 * 10 6fPDLSCs or cPDLSCs are hatched 1 hour respectively with under anti-HLA-I, HLA-Π DR, CD80 or CD86 antibody room temperature.After rinsing through phosphate buffer (phosphate buffered saline, PBS), cell is hatched 30 minutes again with under the anti-room temperature of anti-mouse IgG two of FITC labelling.Hatch and finish rear up flow type cell instrument (BD Inmmunocytometry Systems) detection expression.
5), mixed lymphocyte reaction
As irritation cell, 5.0 * 10 4fPDLSCs or cPDLSCs first irradiate 20Gy under linear accelerator, the allogeneic PBMCs of cell concentration such as then add, and in 96 orifice plates, 0.2mlRPMI-1640, jointly hatch 5 days.Finishing first 18 hours of reaction, every hole adds 1 μ Ci's 3h-thymidine ( 3h-TdR).After 18 hours, collecting cell on glass fiber filter paper, using liquid scintiloscope (PerkinElmer) calculates 3the H-TdR incorporation efficiency. 3the result of H-TdR incorporation efficiency means with count per minute ± standard deviation (CPM ± SD).
In order to study PDLSCs, whether the propagation lymphocytic impact of T and this kind of impact are had to dose dependent, in the short proliferation experiment of mitogen, the PHA (Sigma-Aldrich) that the final concentration of take is 0.5 μ g/mL stimulates PBMCs (5.0 * 10 4) propagation, (amount of PDLSCs is respectively 1.0 * 10 to hatch altogether 5 days with the autologous fPDLSCs of various dose or cPDLSCs 4, 5.0 * 10 4, 2.5 * 10 5, 5.0 * 10 5).Finish to hatch first 18 hours every holes and add 1 μ Ci's 3h-TdR, collecting cell and calculating after 18 hours 3the H-TdR incorporation efficiency.
Further research postpones to add PDLSCs whether can affect the T lymphopoiesis.The PHA that is first 0.5 μ g/mL with final concentration stimulates PBMCs (5.0 * 10 4) breed 2 days, then add the fPDLSCs of Isodose or cPDLSCs jointly to hatch again 3 days.Within 3 days, measure afterwards 3the H-TdR incorporation efficiency.
After confirming that PDLSCs can suppress T lymphopoiesis that mitogen PHA causes, we observe the impact of PDLSCs to two-way mixed lymphocyte reaction (mixed lymphocyte reaction, MLR).PBMCs (5.0 * 10 from two Different Individual 4) with third party fPDLSCs or the cPDLSCs of equivalent, jointly hatch 5 days.Finish to hatch first 18 hours every holes and add 1 μ Ci's 3h-TdR, hatch and finish rear collecting cell, calculating 3the H-TdR incorporation efficiency.
6), with PDLSCs, the T after hatching altogether is lymphocytic stimulates again
In order to observe PDLSCs, the lymphocytic propagation of T is suppressed to whether reversible, we have carried out the reactivation experiment.PBMCs (5.0 * 10 4) first with PDLSCs and the PHA (0.5 μ g/mL) of equivalent, jointly hatch 5 days, then the T lymphocyte obtained by Percoll gradient centrifugation and PHA (0.5 μ g/mL) or rhIL-2 (interleukin2, IL-2,50U/mL; R& D systems) jointly react 2 days.After 2 days, application 3h-TdR mixes method and measures the T lymphocytic proliferation rate.
7), Transwell culture experiment
Mediate PDLSCs to the lymphocytic propagation inhibition of T in order to observe by the direct contact of cell-cell or PDLSCs secretion soluble factor, we have carried out the Transwell culture experiment.Transwell culture systems (Costar) has the mocromembrane that diameter is 0.4 μ m aperture, and this mocromembrane can be separated two kinds of cells artificially.By PBMCs (5.0 * 10 4) be placed in the epicoele of Transwell culture systems with PHA (0.5 μ g/mL), and 5.0 * 10 4pDLSCs (from this experiment, the following part of this research is all used fPDLSCs) is seeded in cavity of resorption.After 5 days, application 3h-TdR mixes method and measures the T lymphocytic proliferation rate.
8), the mensuration of soluble factor and neutralization experiment
After finding soluble factor mediation PDLSCs lymphocytic propagation suppress to T, we apply enzyme-linked immunosorbent assay (enzyme-linked immumosorbent assay, ELISA) and measure the culture supernatant of simple PDLSCs (inoculation 1-5 days) and MLR culture supernatant (5.0 * 10 4pDLSCs, the PHA of equivalent PBMCs and 0.5 μ g/mL) middle TGF-β 1 (R& D systems), HGF (R& D systems), PGE2 (prostaglandin E2, PGE2; Assay Designs) and IL-10 (R& D systems) concentration.Read optical density value by microplate reader (Molecules Devices) at 450nm (TGF-β 1, HGF and IL-10) or 405nm (PGE2).
Then neutralize experiment, set up the reaction system of MLR in the Transwell culture systems, comprise PBMCs (5.0 * 10 4), PDLSCs (5.0 * 10 4) and PHA (0.5 μ g/mL); add respectively following antibody or reagent in reaction: anti-TGF-beta antibodies (10ng/mL), inhibitor indomethacin (the 5 μ mol/L of anti-HGF antibody (10ng/mL) and anti-IL-10 antibody (10ng/mL) or PGE2; Sigma-Aldrich).After 5 days, by 3h-TdR mixes method and measures the T lymphocytic proliferation rate.
9), measure the lymphocytic percentage rate of apoptosis T
Set up the reaction system of MLR, comprise PBMCs (5.0 * 10 4), PDLSCs (5.0 * 10 4) and PHA (0.5 μ g/mL), apply Annexin V-Fluos apoptosis test kit (Roche Diagnostics) after 5 days and measure the lymphocytic percentage rate of apoptosis T.
the periodontal bone defect repair of embodiment 2:PDLSCs mediation
1), materials and methods
WZSP and Guizhou Xiang pig (6-8 monthly age, body weight 30-40 kilogram) are provided by China Agricultural University's Experimental Animal Center.This experiment is by the approval of Ethics Committee of the Capital University of Medical Sciences.The separation of miniature pig canine tooth PDLSCs and cultural method are with people PDLSCs.According to the method (13) of bibliographical information, prepare the periodontitis bone defect model of 12 female WZSPs, the periodontitis bone that altogether prepares 24 place's lower jaw sixth-year molars is damaged.24 places are damaged is divided into following 4 groups at random: [1] blank group, do not do any treatment; [2] simple material group, turn over lobe, scrape control, transplant HA/TCP timbering material (Wuhan University of Technology provides), gelfoam cover damaged, sew up; [3] autologous PDLSCs transplantation group, turn over lobe, scrape and control, transplant HA/TCP timbering material+2.0 * 10 7wZSP PDLSCs, gelfoam cover damaged, stitching; [4] allosome PDLSCs transplantation group, turn over lobe, scrape and control, transplant HA/TCP timbering material+2.0 * 10 7fragrant pig PDLSCs, gelfoam cover damaged, stitching.Before preparing periodontitis bone defect model before (4w), transplantation treatment (0w) and the treatment after 12w carry out clinical examination, comprise visiting and examine the degree of depth (probing depth, PD), gingival recession (gingival recession, GR) and attachment loss (attachment loss, AL).The situation that 12w application CT (Siemens) observes osteanagenesis after 0w and treatment.After-4w, treatment, 1-7d, 2w, 4w, 8w and 12w carry out the inspection of blood routine examination, blood biochemical analysis, immunoglobulin inspection and T lymphocyte Research of predicting markers, comprise CD3 +cell counting, CD4 +cell counting, CD8 +the expression of the mark of cell counting and activated T lymphocytes---CD40L.12w after treatment, put to death animal, from the experiment position, obtains specimen, fixing, decalcification, paraffin embedding, row HE dyeing, tissues observed regeneration situation.
Pass through Student ' t check and variance analysis and carry out statistical analysis, p<0.05 thinks that significant difference is arranged.
2), result and explanation
2-1), PDLSCs has reduced immunogenicity
At first observe the immunophenotype of people PDLSCs, find fPDLSCs (76.2% ± 5.8%, n=5) and cPDLSCs (78.5% ± 6.4%, n=5) express HLA-I, but do not express HLA-Π DR and costimulatory molecules CD80, CD86 (Fig. 1), to the BMSCs of cultured and amplified in vitro, express situation similar (23).
Further research PDLSCs as antigen presenting cell on the lymphopoietic impact of T.Experimental group is in advance through 5.0 * 10 of linear accelerator 20Gy irradiation 4the allogeneic PBMCs of PDLSCs and equivalent is hatched altogether.T lymphopoiesis group as positive control is: 5.0 * 10 4pBMCs with the allogeneic PBMCs of equivalent, hatch altogether.The negative contrast of the PBMCs of single culture equivalent.Result shows, experimental group PDLSCs does not cause allogeneic PBMCs propagation, and positive controls can cause significantly
(a), prompting PDLSCs has reduced immunogenicity to Fig. 2 to the T lymphopoiesis.
2-2), PDLSCs can suppress the T lymphopoiesis
Further observe the lymphopoietic impact of T that PDLSCs causes mitogen and alloantigen.By fPDLSCs and cPDLSCs inoculation, cell concentration is respectively: 1.0 * 10 4, 5.0 * 10 4, 2.5 * 10 5with 5.0 * 10 5, then add autologous PBMCs (5.0 * 10 4) and the final concentration PHA that is 0.5 μ g/mL.PHA stimulates PBMCs (5.0 * 10 4) breed positive contrast.Found that, the PBMCs propagation that PHA stimulates is suppressed by fPDLSCs or cPDLSCs significantly, and this inhibition is dose dependent.This kind of immunosuppressive action necessarily requires the existence of PDLSCs, rather than caused by bulk effect (bulk effect), because add the autologous PBMCs of equivalent can not cause inhibitory action (Fig. 2 b) in the breeder reaction system.In addition, even stimulate PBMCs (5.0 * 10 at PHA 4) breed and within latter two days, add again PDLSCs, still can obviously suppress the lymphocytic propagation of T (Fig. 2 c).We further observe the impact of PDLSCs on MLR.PBMCs and third-party PDLSCs from two Different Individual are hatched jointly.Result shows, fPDLSCs and cPDLSCs can both suppress two-way MLR (Fig. 2 d).This part experimental data shows, PDLSCs can dose dependent ground and the antigen non-specific ground T lymphopoiesis caused with mitogen that suppresses that the Allogeneic T cell receptor causes.
2-3), PDLSCs suppresses the T lymphopoiesis by secretion PGE2
We further study PDLSCs and suppress the lymphopoietic mechanism problem of T.Whether in order to illustrate, directly to contact and cause with PBMCs cell-cell by PDLSCs, at first we carried out the Transwell culture experiment, and PDLSCs and PBMCs are separated artificially.Result of study shows, no matter be that Transwell culture experiment or cell-cell directly contact experiment, can both obtain close T lymphopoiesis inhibition, point out this immunosuppressive action and cell-cell directly to contact irrelevant, but rely on soluble factor have that (Fig. 3 a).
By ELISA, we measure in simple PDLSCs culture supernatant and the MLR supernatant in the soluble factor of several probabilities.No matter PDLSCs culture supernatant or MLR supernatant, all do not find HGF and IL-10 (Fig. 4).Then we continue to observe TGF-β 1 and PGE2, because the two is considered to the Main Factors (7,25) of BMSCs performance immunoloregulation function.As shown in Fig. 3 b and Fig. 3 c, after inoculation, in the PDLSCs culture supernatant of 1-5d, the relative concentration of TGF-β 1 and PGE2 is stable, not significantly fluctuation.But, in the MLR supernatant, the concentration of PGE2 reaches 15.19 ± 1.26ng/ml, compare obvious rising with the concentration that simple PDLSCs cultivates, the concentration of TGF-β 1 is 1459.79 ± 109.49pg/mL, there is no significant change.
We pass through to add specific anti-TGF-beta in the MLR system, IL-10, and HGF antibody and PGE2 inhibitor are observed it and are recovered the lymphopoietic ability of T, further determine that PDLSCs suppresses the lymphopoietic Main Factors of T.Found that, add the PGE2 inhibitor can significantly recover the lymphocytic propagation of T, and proliferative ability now is with stimulate merely the proliferative ability close (Fig. 3 d) of PBMCs with PHA.And add anti-TGF-beta in experimental system, and IL-10, the neutralizing antibody of HGF does not recover the lymphocytic propagation of T (Fig. 3 d).These data show that PGE2 is that mediation PDLSCs suppresses the lymphopoietic Main Factors of T.
With regard to the immunosuppression mechanism of BMSCs, although it is inconsistent to it be not immediately clear that the effect of any factor performance dominance and some research also exist, most research understanding or secrete antiproliferative soluble factor such as IL-10 by BMSCs, HGF, TGF-β 1, PGE2, indole amine-2,3-dioxygenase and nitric oxide (5,6,17,19,20,24-28) cause.Beyth etc. (28) report IL-10 is the main mediated factor of BMSCs immunosuppressive action.DiNicola etc. (7) are by finding that with experiment TGF-β 1 and HGF are the principal elements played a role in antibody.Separately there are some researches show that the inhibitor of PGE2 can offset the immunosuppressive action of BMSCs (25).These researchs have all shown that soluble factor has participated in the immunosuppressive action of BMSCs.Our research prompting PGE2 is that PDLSCs suppresses the lymphopoietic Main Factors of T.
2-4), the immunosuppressive action of PDLSCs and t cell proliferation irrelevant, but inducing T cell incapability
Further whether the immunosuppressive action of research PDLSCs has caused the lymphocytic apoptosis of T.Now find, in the reaction system of PDLSCs, PBMCs and PHA, the T percentage of lymphocyte of apoptosis and PBMC merely, PHA reaction system are similar, and having got rid of the T Lymphocyte Apoptosis is to cause immunosuppressant probability (Fig. 3 e, f).Then, the T lymphocyte suppressed by PDLSCs 5 days is extracted, stimulate again two days with PHA or IL-2.Found that, suppressed T lymphocyte is had significant proliferation again, the degree close (Fig. 3 g) that this propagation degree and simple PHA stimulate PBMCs.Therefore, can think that the T lymphopoiesis inhibition of PDLSCs is reversible, cause by the inducer T lymphocyte incapability.
2-5), the paradenlal tissue regeneration of PDLSCs mediation
In view of the immunomodulatory properties of PDLSCs, can research allogeneic PDLSCs repair miniature pig periodontitis bone defect model.After transplanting 12 weeks, the PD of allograft PDLSCs group was 3.5 ± 0.6mm, and the PD of autotransplantation group is 3.3 ± 0.4mm, and the HA/TCP group is 13.1 ± 1.1mm, and the blank group is 10.6 ± 1.3mm.Statistical analysis shows that autologous or allogeneic PDLSCs transplantation group compares periodontal tissue with the HA/TCP group with the blank group and obtained obvious regeneration, and do not have significant difference between autologous or allogeneic PDLSCs group, (Fig. 5 a).CT scan demonstration alveolar bone autologous and allogeneic PDLSCs group is obviously regenerated, and substantially returned to normal level, and HA/TCP group and blank group is only regenerated on a small quantity or there is no regeneration (Fig. 5 a-h).Histological observation shows, in autologous and allogeneic PDLSCs group, bone, cementum and the desmodontium of making new advances of obviously regenerating, in HA/TCP group and blank group, still visible significantly periodontitis performance, comprise dark periodontal pocket, lack area of new bone and desmodontium (Fig. 5 j-m).In addition, with 4w before transplantation treatment, compare, blood routine examination (table 1), blood biochemical analysis (table 2), immunoglobulin inspection (table 3) and immunology index of correlation (Fig. 6 of each time point after treatment, Fig. 7) all there is no significant change, show that allograft PDLSCs repairs the damaged generation that there is no rejection of periodontitis bone.
Table 1
Table 2
Table 3
In addition, according to another result of the test of the present embodiment, see Fig. 8, wherein depicted in greater detail the paradenlal tissue regeneration of PDLSCs mediation, the result shown from figure shows, consistent with the result of the test above in the present embodiment.
In a word, the present invention shows, PDLSCs expresses the HLA-I, does not express HLA-II DR, CD80 and CD86, also can not cause the Allogeneic T lymphopoiesis, and prompting PDLSCs has reduced immunogenicity; PDLSCs can suppress the T lymphopoiesis that mitogen and alloantigen cause; PDLSCs can not cause the lymphocytic apoptosis of T, when the T lymphocyte is upset again, can recover multiplication capacity; PDLSCs brings into play its immune suppression function by secretion PGE2; Allogeneic PDLSCs can repair miniature pig periodontitis bone defect model, and can not cause immunological rejection.
embodiment 3: the biological dental root regeneration of the relevant mescenchymal stem cell mediation of tooth
1) materials and methods
1-1) separation of seed cell, cultivation
tip of a root dental papilla stem cell:the lower aseptic miniature pig canine tooth of pulling out of anesthesia, cut root tip root division canine tooth nipple, with D-Hank's liquid, repeatedly clean respectively, shred, be placed in the Digestive system containing NTx enzyme (3g/L) and Dispase (4g/L), digest 1h, mistake 70 μ m filter screen collecting cells under 37 ℃, the centrifugal 10min of 1000r/min, become single cell suspension with the culture fluid Eddy diffusion.By 0.01～1 * 10 5/ hole is inoculated in tip of a root dental papilla cells and periodontal ligament cell in 6 orifice plates respectively, at 37 ℃ of α-MEM culture medium (containing 15% hyclone, 2mmol/L glutamine, 100U/ml penicillin, 100 μ g/ml streptomycins), 5%CO 2cultivate, within every 2～3 days, change liquid 1 time.The cultivation of going down to posterity while reaching 80% fusion.
dental pulp stem cell:the lower aseptic miniature pig canine tooth of pulling out of anesthesia, the tooth of riving is got pulp tissue, with D-Hank's liquid, repeatedly cleans respectively, shreds, and digestion, cultivate, and concrete steps are with tip of a root dental papilla cells.
periodontal ligament stem cell:the lower aseptic miniature pig canine tooth of pulling out of anesthesia, peel off its periodontal tissue on every side gently, gets the periodontal tissue in stage casing, with D-Hank's liquid, repeatedly cleans respectively, shred, and digestion, cultivation, concrete steps are with tip of a root dental papilla cells.
1-2) periodontal ligament cell diaphragm preparation
By eugonic 2 * 10 5second or third generation periodontal ligament stem cell be seeded in the 60mm culture dish, cultivating composition is that α-MEM culture medium (contains 15% hyclone, the L-AA 2-phosphoric acid of 100 μ mol/L, 2mmol/L glutamine, the 100U/ml penicillin, 100 μ g/ml streptomycins).Cultivate 10-14 days, gauffer appears in the culture dish border cell, uses than blunt blade or cell sleaker cell patch integral body is peeled, and in process, cell patch is not dry.
1-3) timbering material
The hydroxyapatite/tricalcium phosphate timbering material is made to the profile of similar root of the tooth, be of a size of: diameter 5mm, the cone of length 15mm.Hydroxyapatite/tricalcium phosphate is porous network structure, aperture mostly is 200～500 μ m, dental pulp stem cell is compounded on the hydroxyapatite/tricalcium phosphate three-dimensional rack, the growth regulation dental pulp stem cell of 5 days in bioreactor, cell fully stretches, and projection and the secretory granule of cell surface are a lot, connect into a slice, diameter is many at 20-50 μ m.
1-4) before Hui Zhi, cell is compound
By 1 * 10 8the tip of a root dental papilla stem cell that was cultured to for the 3rd generation is suspended from culture medium, root of the tooth type timbering material is placed in to celliferous culture medium, fully mix 2h on 37 ° of C shaking tables, timbering material is carefully pressed from both sides out, be positioned in culture dish, remaining cell suspension is carefully dripped on timbering material, after standing 4h, add culture fluid, Hui Zhi after cultivation 5d in bioreactor.
1-5) return method for planting
Choose respectively the edentulous region of miniature pig.Cut mucosa, open glutinous periosteum lobe, expose alveolar ridge crest.Bore a hole similar to hydroxyapatite/tricalcium phosphate timbering material shape with planting machine with the speed of 800 rev/mins in alveolar bone, by above-mentioned tip of a root dental papilla stem cell/dental pulp stem cell with in bioreactor, cultivate 5-7 days after the dental root shaped hydroxyapatite/tricalcium phosphate mixes, the periodontal ligament stem cell diaphragm holds the hydroxyapatite/tricalcium phosphate surface, and Hui Zhi is in the miniature pig alveolar bone.
2) results and discussions
The periodontal ligament cell sheet is applied to history for some time of periodontal tissue engineering, but the conventional Recell temperature-responsive culture dish that adopts, a kind of temperature sensitive hydrophobic material PIPA-Am has been used on the surface of culture dish.When temperature, during higher than 32 ℃, surface has hydrophobicity, is applicable to adhering to and grow of cell, when temperature reduces, and the polymer hydrophilic and expansion that becomes, Spontaneous release cell.Harvesting only need reduce the temperature to 20 ℃ and get final product, and without enzymic digestion and processing, can retain cell surface function and activity.But culture dish needs special material PIPA-Am, therefore we have found out the method that common culture dish prepares the periodontal ligament cell diaphragm of applying, the L-AA 2-phosphoric acid that adds 100 μ mol/L in medium component, can promote the cell fast breeding, and can secrete a large amount of extracellular matrix collagen compositions, all cells is linked together, and after growing into to a certain degree, we peel whole cell patch is complete.Cell patch does not destroy the support that extracellular matrix is connected to form, and does not destroy the functions such as the adhesion propagation of cell surface protein and differentiation, forms periodontal tissue after can promoting back to plant in body.The normal thin especially about 0.2mm of periodontal membrane, if adopt timbering material, relatively thick, the space of leaving over after material degradation is a problem, the periodontal diaphragm has been avoided this problem.
Due to the factor of gravity and nutrition supply, the cytoskeleton complex is carried out in the process of inactive dimensional culture, find that cell gathers bottom and the surface of timbering material more, the cell quantity that material internal sticks is seldom.In incubation, static cultivation has also limited the degree of depth of Growth of Cells, and dental pulp stem cell is uniformly distributed in timbering material inside, could form the tissue of uniformity.Want to allow cell also can well grow in timbering material inside, nutrient substance can be effectively in the internal stent transmission with to discharge timely the cellular metabolism refuse very important.Although the mass exchange mechanism that bioreactor can't the analogue body internal circulation system, but can be at the inner hydrodynamics environment that produces of timbering material, if control the size of mechanical strength well, both can reduce the damaging action to cell, the growth that can be again cell provides favourable mechanical environment.In this experiment, cell is cultivated 5 days after being compound to material in bioreactor, bioreactor dynamic property dimensional culture has promoted the exchange of internal stent nutrient substance, promoted seed cell being uniformly distributed in timbering material, be conducive to cell and be held in bone phenotype and promote the deposition of extracellular matrix in timbering material.
After returning and planting 6～JIUYUE, the biological root of the tooth of Hui Zhi shows as the high density shadow of not transmission at X-ray film, have on every side the low-density shadow picture around.The histology is upper, and the biological root of the tooth tissue morphology of regeneration is fully different from newborn osseous tissue, many spherical sclerous tissueses in different growth stages agglomerate, consists of, and spherical agglomerate interconnects and reticulates.Newborn spherical sclerous tissues small volume, outer surrounding loose arrangement and is hepatic cell, and supposition may be odontoblast or cementoblast, the substrate that central authorities dye for even, powder.More ripe spherical sclerous tissues agglomerate volume is larger, and centre is not substantially containing cell, or visible minute quantity is embedded in cell debris wherein.The dentinoid structure of class is arranged in agglomerate, and wherein dentinal tubule's spline structure of visible irregular arrangement, also have similar cementoid structure, shows as the sclerous tissues than uniformity under mirror.The new hard tissue area formed is overseas has no cell infiltration for fibrous connective tissue holds, and is also shown in the fibroid structure of similar Sharp's in subregion and inserts in the cementoid and dentin spline structure formed.
From above-mentioned disclosed technical scheme, the present invention has very important and profound significance for utilizing biological dental root regeneration to repair absence of tooth: at first, the present invention proposes the new concept of the biological dental root regeneration based on tissue engineering technique.Secondly, the present invention implements the new concept of biological dental root regeneration on the larger animal miniature pig, succeeds, and proves that this inventive method is feasible, is expected to become a kind of new repair mode and is applied to clinical.The 3rd, specific implementation method system provided by the invention, reliable, for further investigation and the commercialization of carrying out biological dental root regeneration provides theoretical foundation and technical support.
In the present embodiment method, the experimental result provided as shown in Figure 9, wherein depicted in greater detail on miniature pig the situation of successful regeneration biological root of the tooth.Wherein, A: experiment flow figure; (B, C): formed dentin cementum spline structure and periodontal membrane spline structure (HE dyeing); D: periodontal membrane COL I stained positive; E: dentin cementum spline structure Dsp stained positive; F～I: regeneration biological root of the tooth scanning electron microscope (SEM) is observed: the F:HA/TCP material is multi-pore structure (100-400um); H: the new periodontal membrane fiber formed inserts in the new dentin cementum structure formed and alveolar bone (G figure arrow indication); I: the new dentin spline structure (G schemes thick arrow indication) formed.
In the present embodiment method, another experimental result provided is as shown in Figure 10, similar with Fig. 9, in Figure 10 depicted in greater detail carry out the situation that biological dental root regeneration succeeds on miniature pig.
embodiment 4: deciduous teeth dental pulp stem cell transplantation treatment systemic lupus erythematosus (sle) (SLE) comes off
1) materials and methods
1-1) people SHED
Choose the normal deciduous teeth of the healthy patients in 6-8 year of pulling out, according to the method for previous literature report, separate and cultivate 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 PERIPHERAL BLOOD MONONUCLEAR CELL
Bone marrow stroma stem cell (the bone marrow mesenchymal stem cells of application Percoll gradient centrifugation separating health donor, BMMSCs) and PERIPHERAL BLOOD MONONUCLEAR CELL (peripheral blood mononuclear cells, PBMCs).
1-3) SHED systemic lupus erythematosus (SLE)
C57BL/6J and C3MRL-Fas lpr/ J (MRL/lpr) mice (female, 6-7 week age), Beige nude/nude Xid (III) mice (female, 8-12 age in week) is by providing.This experiment is by the approval of Ethics Committee of the Capital University of Medical Sciences.Experiment is divided into following 3 groups: 1, blank group (n=3), do not do any treatment.2, transplant BMMSCs group (n=3).3, transplant SHED group (n=3).Under general anesthesia, by SHED or BMMSCs (1x10 5the cells/10g body weight, cell suspension is in 100ml PBS) enter MRL/lpr mice in 16 week age, matched group injecting normal saline by tail vein injection.It is put to death during 20 week age mice, get peripheral blood, kidney, long bone (femur and tibia) specimen.The kidney specimen is fixed, paraffin embedding, and row HE, trichrome, periodic acid-schiff (pas) dyeing, observe the glomerular basement membrane dysfunction and recover and mesangial cell excessive increase recovery situation.Detect serum dsDNA-IgG by ELISA, dsDNA-IgM, the ANA level, detect in urine and complement component 3 (complement3, C3), kreatinin (creatinine), urine protein (urine protein) level in serum.
2) result and explanation
SLE be take that many internal organs are got involved and blood in have the autoimmune disease that Multiple Antibodies is feature, many T of studies confirm that, bone-marrow-derived lymphocyte overactivity are the key links in SLE morbidity.T, bone-marrow-derived lymphocyte derive from the lymph ancestral cells, and MSCs has important effect in lymphatic trunks/progenitor cell proliferation differentiation, so the effect of MSCs in SLE causes a disease more and more receives publicity.SLE patient MSCs exists abnormal; the marrow stromal cell of its differentiation can not be supported lymphocyte growth preferably; lymphocytic growth depends on the various cytokines of lymphocyte itself and marrow stromal cell secretion, and marrow stromal cell abnormal participated in lymphocytic abnormal activation.The researchs such as Majumdar show that MSCs expresses a large amount of cell adhesion molecules, stick at iuntercellular, go back to the nest, play an important role in hematopoiesis support, adjusting immune cell function.When tumor cell plantation people allosome mice, tumor cell can be removed by host immune system, and, after tumor cell and MSCs inject the allosome mice simultaneously, tumor cell can be survived in receptor, shows that MSCs has immune suppression function in vivo.MSCs still retains its immunoregulation effect when being divided into other cell types, and this MSCs that just means transplanting can bring into play long-term immunoregulation effect.
Experiment in vitro confirms that SHED has MSCs biology and immunological characteristic, can influence each other with the T lymphocyte.With BMMSCs, compare, SHED, by recovering peripheral blood Tregs/Th17 ratio, reduces Th17 cell number performance immunology effect.After the MRL/lpr injected in mice SHED in 16 week age, can find trabecular bone regeneration, osteoclast activity has been subject to inhibition.The mice of injection SHED is the same with the mice of injection BMMSCs, has rebuild the osteoblast microenvironment, thereby has improved its dysfunction.After MRL/lpr injected in mice SHED, though the Treg level does not improve, the Treg/Th17 cell proportion obviously increases, and this shows that the immunoloregulation function of SHED may come from Treg inhibition autoimmune Th17 and can promote autoimmune and inflammation.
In the present embodiment method, the experimental result provided as shown in Figure 11, wherein depicted in greater detail come off deciduous teeth dental pulp stem cell systemic lupus erythematosus Mus serum and renal histology situation about changing.From figure, the visible the present embodiment of result has been realized purpose of the present invention.
In a word, the present invention shows, from the deciduous teeth dental pulp come off, can extract SHED, and it is to derive from mesoblastic mescenchymal stem cell, has biology and immunological characteristic and the function of MSCs.Although the immunoregulation effect mechanism of SHED is not yet clear and definite, due to its immunoregulation effect, potential therapeutical effect is arranged aspect autoimmune disease, SHED transplants the new trial that can be used as the treatment autoimmune disease.
embodiment 5: allogeneic Periodontal ligament stem cell (hPDLSCs) is used for the treatment of the standard operation example of periodontal disease
The present embodiment illustrates that with reference to Figure 12 allogeneic Periodontal ligament stem cell (hPDLSCs) is used for the treatment of an example of the standard operation of periodontal disease:
(a) gather people's periodontal membrane (periodontal ligament, PDL).Gather normal impacted third molar (impacted third molar) from 18-28 year patient.PDL is separated from tooth surface lightly.
(b) cultivate hPDLSCs.Separate hPDLSCs.Original cultivation is after 15 days, from the quantity of the hPDLSCs of an impacted third molar, is about 4.90 ± 0.34 * 10 5(P0; N=10).After cultivating other 15 days, the quantity of hPDLSCs (P3) is increased to 8.86 ± 0.46 * 10 again 6(n=10).HPDLSCs is aobvious positive to CD146 and CD90.
(c) freezing preservation hPDLSCs.By 10%DMSO and the freezing preservation of 90%FBS for third generation hPDLSCs, and be kept in liquid nitrogen.
(d) melt hPDLSCs.After thawing, hPDLSCs is checked to mycoplasma, antibacterial, colony form efficiency, interstital stem cell marking mode and karyotyping.
(e) prepare the hPDLSCs sheet.In the 100mm culture dish to three kinds of different cell number (1 * 10 5, 1 * 10 6with 2 * 10 6; N=3) cultivate 12-15 days, 1 * 10 6with 2 * 10 6form cell sheet in group, but 1 * 10 5do not form cell sheet in group.For this reason, by 1 * 10 6hPDLSCs be inoculated in the 100mm culture dish, reach 15 days.
(f) then, 40mg HA/TCP is placed in to these culture dishs.
(g) full figure of hPDLSCs sheet and HA/TCP.
(h) oral cavity periodontal damage figure.
In periodontal initial therapy (i) afterwards, two allogeneic hPDLSCs sheets and HA/TCP are implanted to the periodontal damage fault location (j) of 3mm * 5mm * 7mm.
(k) ensuing project comprises clinical and radiography evaluation, hematology and immunological evaluation.
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Claims (9)

1. the purposes of tooth related stem cells in the product for the preparation of prevention or the treatment disease relevant to tooth or the state of an illness, or the purposes in the product that forms or repair for the preparation of tooth linked groups.
2. according to the purposes of claim 1, wherein said tooth related stem cells 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.
3. according to the purposes of claim 1 or 2, wherein said tooth related stem cells is from mammal, and for example described tooth related stem cells is from being selected from following mammal: people, pig (for example WZSP, Guizhou Xiang pig), cattle, horse, monkey, rat, mice, Cavia porcellus, sheep, sheep, goat.
4. according to the purposes of claims 1 to 3 any one, the wherein said disease relevant to tooth or the state of an illness or tooth linked groups form or repair and be selected from: periodontal disease, periodontitis, tooth defect, dental tissue are damaged, the tooth defect reparation, tooth linked groups is damaged and reparation, the substitute regeneration of tooth linked groups, etc.
5. the purposes of tooth related stem cells in the product for the preparation of prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, the disease relevant with the abnormal rising of T lymphocyte or the state of an illness, lupus erythematosus or systemic lupus erythematosus (sle).
6. according to the purposes of claim 5, wherein said tooth related stem cells 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.
7. a compositions, the tooth related stem cells that it comprises effective dose and optional pharmaceutically acceptable carrier.
8. according to the compositions of claim 7, wherein said tooth related stem cells 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.
9. according to the compositions of claim 7 or 8; it is for prevention or treatment disease or the state of an illness relevant to tooth; perhaps for tooth linked groups, form or repair, or for prevention or treatment immune disease or the state of an illness, autoimmune disease or the state of an illness, the disease relevant with the T Abnormal lymphocyte activation or the state of an illness, disease or the state of an illness, lupus erythematosus or the systemic lupus erythematosus (sle) relevant with the abnormal rising of T lymphocyte.
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