WO2010083730A1 - 牙齿相关干细胞的新用途 - Google Patents
牙齿相关干细胞的新用途 Download PDFInfo
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- WO2010083730A1 WO2010083730A1 PCT/CN2010/000104 CN2010000104W WO2010083730A1 WO 2010083730 A1 WO2010083730 A1 WO 2010083730A1 CN 2010000104 W CN2010000104 W CN 2010000104W WO 2010083730 A1 WO2010083730 A1 WO 2010083730A1
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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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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
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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
Definitions
- the present invention relates to new uses of tooth-related stem cells.
- the present invention relates to the use of tooth-related stem cells in the preparation of a product for preventing or treating a disease or condition associated with teeth, or in the preparation of a product for the formation or repair of a tooth-related tissue;
- Stem cells are prepared for the prevention or treatment of an immune disease or condition, an autoimmune disease or condition, a disease or condition associated with abnormal activation of T lymphocytes, a disease or condition associated with abnormally elevated T lymphocytes, lupus erythematosus, or Use in products of systemic lupus erythematosus; also relates to a method of treatment according to the above-described phlegm; and to a composition comprising tooth-related stem cells.
- Stem cells are a class of primitive cells with self-renewal and differentiation potential, which can be divided into embryonic stem cells and adult stem cells. Specific adult stem cells are ubiquitous in adult tissues and organs.
- human-derived tooth-related stem cells are mesenchymal stem cells (MSCs) derived from mesoderm, including dental pulp stem cells ( Dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth ( SHED), periodontal stem cells (PDLSCs), and stem cells from apical papilla , SCAP) (1-4, refer to documents 1 to 4, the same below), which may be derived from a variety of animals (eg, mammals, such as humans).
- MSCs mesenchymal stem cells
- DPSCs dental pulp stem cells
- SHED human exfoliated deciduous teeth
- PDLSCs periodontal stem cells
- SCAP apical papilla
- tooth-related stem cells have high proliferative capacity and multi-directional differentiation potential, and can differentiate into osteoblasts, adipocytes, and neuron-like cells.
- the complex of the tooth-related stem cells and the three-dimensional scaffold material is implanted under the skin of the mouse, the autologous dental pulp dentin complex structure and the cementum periodontal ligament complex-like structure can be produced (1, 3).
- PDLSCs and SCAP have been successfully used to regenerate bio-roots on autologous miniature pig models (4).
- one of the focus of stem cell research in the world is to establish a variety of tissue adult stem cell banks for clinical trials of stem cells.
- Hematopoietic stem cell transplantation can enable some patients to achieve disease control, but its cost and complications are high, and the recurrence rate is as high as 40%-50%, which cannot be used routinely. Moreover, although some patients may achieve long-term remission after transplantation, they are all It is impossible to completely cure autoimmune diseases, and some patients have recurrence problems.
- tooth-related stem cells such as periodontal ligament stem cells (PDLSCs) not only regenerate defective tooth tissue in autologous teeth, but also regenerate tooth tissue in allogeneic defective tooth tissue, while PDLSCs also It has an inhibitory effect on abnormally elevated T lymphocytes.
- PDLSCs periodontal ligament stem cells
- the present invention first relates to the use of PDLSCs in the manufacture of a product for the prevention or treatment of periodontal disease, dental defect repair.
- the present invention also relates to a method for preventing or treating periodontal disease, repairing defective tooth tissue, comprising administering a host for preventing or treating periodontal disease or repairing a defective tooth tissue to effectively prevent or treat the amount or repair the amount of defective tooth tissue PDLSCs.
- the present invention relates to a method for preventing or treating a disease or a symptom associated with abnormally elevated T lymphocytes, comprising administering to a host having an abnormally elevated sputum lymphocyte a prophylactically or therapeutically effective amount of PDLSCso
- tooth-related stem cells are selected from the group consisting of: dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and root canine papilla stem cells.
- the disease or condition associated with the tooth or the formation or repair of the tooth related tissue is selected from the group consisting of: periodontal disease, periodontitis, tooth defect, tooth tissue defect, tooth defect Repair, tooth-related tissue defects and repairs, tooth-related tissue replacement, and more.
- a second aspect of the present invention provides a dental related stem cell in which a disease or condition associated with the prevention or treatment of an immune disease or condition, an autoimmune disease or condition, abnormal activation of T lymphocytes, and an abnormal increase in T lymphocytes are provided.
- a disease or condition associated with the prevention or treatment of an immune disease or condition, an autoimmune disease or condition, abnormal activation of T lymphocytes, and an abnormal increase in T lymphocytes are provided.
- tooth-related stem cells are selected from the group consisting of: dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and root canine papillary stem cells.
- the tooth-related stem cell is used for an autoimmune or allogeneic immune disease or condition, an autoimmune disease or condition, a disease or condition associated with abnormal activation of T lymphocytes, A disease or condition associated with an abnormal increase in T lymphocytes, lupus erythematosus, or systemic lupus erythematosus.
- a third aspect of the invention provides a method of preventing or treating a disease or condition associated with a tooth, or a method of forming or repairing a tooth-related tissue, the method comprising administering to a host in need of preventing or treating a disease or condition associated with the tooth
- the amount of tooth-related stem cells, or the method comprises administering an effective amount of tooth-related stem cells to a host in need of forming or repairing a tooth-related tissue.
- tooth-related disease or condition or tooth-related tissue formation or repair is selected from the group consisting of: periodontal disease, periodontitis, tooth defect, tooth tissue defect, tooth defect Repair, tooth-related tissue defects and repairs, tooth-related tissue replacement, and more.
- said tooth-related tissue includes, but is not limited to, teeth, roots, pulp, gums, etc., and other related to the interior of the teeth, the surface of the teeth, the surface of the teeth, or the periphery of the teeth. organization.
- the method comprises the following steps
- a fourth aspect of the present invention provides a disease or condition for preventing or treating an immune disease or condition, an autoimmune disease or condition, an abnormal activation of T lymphocytes, a disease or condition associated with abnormally elevated T lymphocytes, and lupus erythematosus Or a method of systemic lupus erythematosus, the method comprising administering to a host in need thereof an effective amount of a tooth-related stem cell.
- tooth-related stem cells are selected from the group consisting of: dental pulp cells, deciduous dental pulp stem cells, periodontal ligament stem cells, and root canine papilla stem cells.
- tooth-related stem cells are derived from a mammal.
- the tooth-related stem cells are from a mammal selected from the group consisting of humans, pigs (eg, Wuzhishan minipigs, Guizhou Xiang pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats.
- tooth-related stem cells are derived from a mammal, and are selected from the group consisting of: dental pulp stem cells (DPSCs), stem cells from human exfoliated Deciduous teeth, SHED). Periodontal stem cells (PDLSCs) and stem cells from apical papilla (SCAP).
- DPSCs dental pulp stem cells
- SHED stem cells from human exfoliated Deciduous teeth
- SHED human exfoliated Deciduous teeth
- PDLSCs Periodontal stem cells
- SCAP apical papilla
- the tooth-related stem cell is used for an autoimmune or allogeneic immune disease or condition, an autoimmune disease or condition, a disease or condition associated with abnormal activation of T lymphocytes, A disease or condition associated with an abnormal increase in T lymphocytes, lupus erythematosus, or systemic lupus erythematosus.
- a fifth aspect of the invention provides a composition comprising an effective amount of a tooth-related stem cell and, optionally, a pharmaceutically acceptable carrier.
- tooth-related stem cell selected from: dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and root canine papillary stem cells.
- the tooth-related stem cells are derived from a mammal.
- the tooth-related stem cells are from a mammal selected from the group consisting of: humans, pigs (eg, Wuzhishan minipigs, Guizhou Xiang pigs), cattle, horses, monkeys, rats, mice, mice, sheep , sheep, goats.
- tooth-related stem cells are derived from a mammal, and are selected from the group consisting of: dental pulp stem cells (DPSCs), stem cells from stem cells from stem cells from Human exfoliated deciduous teeth, SHED). Periodontal stem cells (PDLSCs) and stem cells from apical papilla (SCAP).
- DPSCs dental pulp stem cells
- SHED Human exfoliated deciduous teeth
- PDLSCs Periodontal stem cells
- SCAP apical papilla
- a composition according to any one of the fifth aspects of the present invention which is for use in preventing or treating a disease or condition associated with teeth, or for the formation or repair of a tooth-related tissue, or for preventing or treating an immune disease or condition, Autoimmune disease or condition, disease or condition associated with abnormal activation of T lymphocytes, disease or condition associated with abnormally elevated T lymphocytes, lupus erythematosus, or systemic lupus erythematosus.
- composition according to any one of the fifth aspects of the present invention wherein the effective amount is a dose effective for preventing or treating a disease or condition associated with teeth, or is effective for use in tooth-related tissue formation Or the dose to be repaired, or to be effective in the prevention or treatment of an immune disease or condition, an autoimmune disease or condition, a disease or condition associated with abnormal activation of T lymphocytes, or an abnormal increase in T lymphocytes.
- tooth-related stem cells such as PDLSCs regenerate defective tooth tissue in autologous teeth, and can regenerate tooth tissue in the body tissue of the allogeneic defect.
- the present invention has been completed based on the above findings.
- the present invention relates to the use of tooth-related stem cells, such as PDLSCs, in the manufacture of a product for preventing or treating periodontal disease, dental defect repair (for example, as shown in Figure 8); and to preventing or treating periodontal disease, repairing defective teeth
- a method of tissue comprising administering to a host in need of prevention or treatment of periodontal disease or a host in need of repairing a defective tooth tissue to effectively prevent or treat an amount or repair a defect in the amount of dental tissue, such as PDLSCs.
- the present invention proposes a new concept of biological root regeneration; using biological allogeneic SCAP/DPSCs and PDLSCs to regenerate biologically functioning biological roots, and then performing crown restoration on newly formed functional biological roots to restore patient chewing Function (for example, as shown in Fig. 10);
- the specific implementation method for providing biological root regeneration provides a basis for further research on the mechanism of biological root regeneration and commercialization of biological roots.
- the present invention relates to a new concept of biological root regeneration and relates to the use of dental-related stem cells such as SCAP, DPSCs, PDLSCs in the regeneration of biological roots.
- the present invention also relates to a specific embodiment of biological root regeneration, which comprises the preparation of a periodontal ligament stem cell membrane and the optimal number of cells and optimal growth time required.
- the present invention relates to the use of tooth-related stem cells, such as PDLSCs, in the manufacture of a product for preventing or treating a disease or condition associated with abnormal elevation of T lymphocytes.
- the present invention also relates to a method of preventing or treating a disease or condition associated with abnormally elevated T lymphocytes comprising administering to a host having an abnormally elevated T lymphocyte a prophylactically or therapeutically effective amount of a tooth-related stem cell such as PDLSCs.
- the invention further relates to the use of tooth-related stem cells, such as SHED, in the prevention or treatment of autoimmune diseases such as systemic lupus erythematosus (e. g., Figure 11).
- periodontal disease includes, but is not limited to, periodontitis.
- the term "defective tooth tissue” includes, but is not limited to, various defects of the host tooth, such as tooth loss caused by various causes.
- the term "host” generally refers to a mammal, including but not limited to humans, pigs, cows, horses, etc.
- the term “host” refers to humans, pigs (eg Wuzhishan minipigs, Guizhou) Xiang pig), cattle, horse, monkey, rat, mouse, guinea pig, sheep, sheep, goat.
- the term “product” refers to various forms suitable for PDLSCs applications. According to the invention, the term “product” also refers to various forms suitable for dental related stem cell applications, such as groups. Compounds, pharmaceutical compositions, and the like.
- the term "disease or condition associated with a tooth” means a disease, a condition, a body condition, a body condition, and the like which are afflicted or expressed by a host as described herein, and which are associated with teeth.
- tooth-related tissue formation or repair or the term “formation or repair of tooth-related tissue” or “formation or repair of tooth-related tissue”, etc., have the same or similar meaning and generally refer to the description herein.
- the tooth-related tissue of the host is processed or manipulated such as formation, repair, generation, regeneration, culture, or the like, or processing or operation of forming, repairing, generating, regenerating, culturing, or the like of a tooth-related tissue abnormality (for example, a defect).
- composition is used in the ordinary meaning as understood by those skilled in the art and generally refers to a form that can be used in conjunction with or indirectly (e.g., prior to dilution) for clinical use, such as dosage form, pharmaceutical dosage form, administration. Form, etc.
- composition also generally has the same meaning as “pharmaceutical composition”.
- the actual dosage level of the tooth-related stem cells in the composition or pharmaceutical composition of the invention can be varied so that the resulting amount of stem cells can be tailored to the particular host, the patient in the particular composition and its composition, and the corresponding mode of administration. Therapeutic or preventive response.
- the dosage level must be based on the activity of the particular stem cell, the route of administration, the severity of the condition being treated, the course of treatment of the disease or condition, the process of formation and repair (and production, regeneration, culture, etc.) or the course of treatment, and the patient to be treated.
- the condition and past medical history were chosen. However, it is the practice in the art that the dose of stem cells and the time of administration begin from a level lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is obtained.
- those skilled in the art may determine the specific dosages to be applied in the specific circumstances, without the need to specifically limited.
- the amount of use in any of the cases can be determined with reference to specific amounts used in the Examples section of the present invention.
- a specific dose of Wuzhishan miniature pig PDLSCs is used hereinafter, and those skilled in the art can combine and combine the above doses. It is well known in the art to convert this dosage to a dose under human conditions.
- the tooth-related stem cells of the present invention can be administered alone (i.e., in the form of the original) or in the form of a pharmaceutical composition.
- the pharmaceutical composition of the present invention can be formulated into various suitable dosage forms depending on the route of administration.
- the use of one or more physiologically acceptable carriers, including excipients and auxiliaries, facilitates processing of the tooth-related stem cells into a formulation that can be used pharmaceutically.
- the appropriate form of preparation will depend on the route of administration chosen and can be made according to common general knowledge in the art.
- the tooth-related stem cells are present in a cell compatible medium (eg, physiological saline such as 0.9% saline, etc.).
- the tooth-related stem cells are present in a cell compatible medium and are stored at low temperatures, for example under refrigeration, freezing, etc., and may optionally be reconstituted prior to use.
- the form to be applied in accordance with the spirit of the present invention is applied.
- PDLSCs inhibit T lymphocyte proliferation: (a) fPDLSCs/cPDLSC does not cause proliferation of allogeneic T lymphocytes. (b) fPDLSCs/cPDLSC dose-dependently inhibits T lymphocyte proliferation induced by mitogen PHA. (c) Delayed addition of fPDLSCs/cPDLSC also inhibited PHA-induced T lymphocyte proliferation. (d) fPDLSCs/cPDLSC is able to inhibit bidirectional mixed lymphocyte responses.
- PDLSCs inhibit T lymphocyte proliferation by secreting PGE2:
- PDLSCs also inhibit T lymphocyte proliferation in Transwell culture experiments, suggesting that PDLSCs secrete soluble factors to exert immunosuppressive effects.
- TGF-pi was present in both the pure PDLSCs culture supernatant and the MLR supernatant, but there was no significant difference between the two.
- the concentration of PGE2 is significantly increased in MLR.
- Neutralization experiments showed that anti-TGF-pi antibodies did not restore T lymphocyte proliferation, and PGE2 inhibitors counteracted the immunosuppressive effects of PDLSCs, suggesting that PGE2 is a major factor mediating the immunosuppressive effects of PDLSCs.
- HGF and IL-10 were not detected in both PDLSCs culture supernatant and MLR supernatant, indicating that these two factors are not involved in PDLSCs-mediated immunosuppression.
- PDLSCs-mediated periodontal tissue regeneration (a) There were no significant differences between the four groups of clinical indicators at -4w and 0w. But 12 weeks after treatment, autologous or allogeneic The PD, GR and AL of the PDLSCs transplantation group were significantly restored compared with the blank control group and HA/TCP. (b, c, d, e) CT scans showed significant periodontal bone defects before treatment, and the degree of defect was similar. At 12 weeks after treatment, periodontal bone regeneration was completely achieved in the autologous PDLSCs group (h) and the allogeneic PDLSCs group (i).
- FIG. 1 Immunization status 12w after transplantation: At 12w after treatment, the number of CD3 + , CD4+, CD8+ T lymphocytes and the expression of CD40L, a marker of activated sputum lymphocytes, did not differ significantly between the four treatment groups. .
- Figure 8. depicts PDLSCs mediated periodontal tissue regeneration in one embodiment of the invention.
- Figure 9. depicts the successful regeneration of biological roots on small pigs.
- Figure 10 depicts the success of bioroot regeneration on small pigs.
- Figure 11 depicts the serum and renal histological changes in the systemic lupus erythematosus of the deciduous deciduous dental pulp stem cells.
- FIG. 12 illustrates an example of the standard procedure for the treatment of periodontal disease with allogeneic human periodontal ligament stem cells (hPDLSCs). detailed description
- Example 1 Inhibition of T lymphocyte proliferation by PDLSCs
- PB.MCs Peripheral blood mononuclear cells
- HLA human leukocyte antigen
- HLA- ⁇ DR. CD80 CD86 antibody
- CD40L CD40L
- transforming growth factor p transformation growth factor ⁇
- TGF- ⁇ TGF- ⁇
- HGF hepatocyte growth factor
- Anti-IL-10 FITC-labeled anti-mouse IgG antibody iAbD Serotec
- l.OxlO 6 fPDLSCs or cPDLSCs were incubated with anti-HLA-I, HLA- ⁇ DR, GD80 or CD86 antibodies for 1 hour at room temperature. After washing with phosphate buffered saline (PBS), the cells were incubated with FITC-labeled anti-mouse IgG secondary antibody for 30 minutes at room temperature. After the end of the incubation, the expression was detected by an upflow cytometer (BD Inmmunocytometry Systems).
- PBMCs 5.0 xlO 4
- PHA Sigma-Aldrich
- PBMCs 5.0 x lO 4
- PHA PHA
- cPDLSCs cPDLSCs
- PBMCs 5.0 x lO 4
- 3 Ci-TdR of l Ci was added to each well 18 hours before the end of the incubation. After the incubation, the cells were collected and the 3 H-TdR incorporation rate was calculated.
- T lymphocytes were incubated with equal amounts of PDLSCs and PHA (0.5 g/mL) for 5 days, then T lymphocytes obtained by gradient density centrifugation were combined with PHA (0.5 g/mL) or recombinant human interleukin 2 (intei eukin 2, IL-2, 50 U/mL; R&D systems) co-reacted for 2 days. After 2 days, the proliferation rate of T lymphocytes was determined by 3 H-TdR incorporation.
- the Transwell culture system (Costar) has a microcapsule with a diameter of 0.4 ⁇ , which allows the two cells to be artificially separated.
- PBMCs 5.0 xlO 4
- PHA 0.5 g/mL
- the real face begins, and the following parts of the study were inoculated in the inferior cavity with fPDLSCs.
- the proliferation rate of T lymphocytes was determined by 3 H-TdR incorporation.
- the 24 defects were randomly divided into the following 4 groups: [1] blank control group, no treatment; [2] simple material group, flap, cure, transplantation HA/TCP stent material (provided by Wuhan University of Technology), gelatin Sponge coverage defect, suture; [3] Autologous PDLSCs transplantation group, flap, curettage, transplantation of HA/TCP scaffold material +2.0 xlO 7 Wuzhishan miniature pig PDLSCs, gelatin sponge covering defect, suture; [4] Allogeneic PDLSCs transplantation Group, flap, curettage, transplantation HA/TCP scaffold material +2.0 X10 7 fragrant pig PDLSCs, gelatin sponge covering defect, suture.
- Clinical examinations were performed before the preparation of periodontitis bone defect model (-4w), before transplantation (0w) and 12 weeks after treatment, including probing depth (PD), gingival recession (GR) and Attachment loss (AL).
- the condition of bone regeneration was observed by Ow and CT (Siemens) 12 w after treatment.
- Blood routine examination, blood biochemical examination, immunoglobulin examination and T lymphocyte-related markers were examined at -4w, l-7d, 2w, 4w, 8w and 12w after treatment, including CD3+ cell count, CD4+ cell count, CD8+ Cell count and expression of CD40L, a marker of activated T lymphocytes.
- the animals were sacrificed, specimens were taken from the experimental site, fixed, decalcified, paraffin-embedded, HE stained, and tissue regeneration was observed.
- the stimulatory molecules CD80, CD86 were similar to the expression of BMSCs amplified in vitro (23).
- the effects of PDLSCs as antigen-presenting cells on the proliferation of T lymphocytes were further investigated.
- the experimental group was incubated with 5.0 x 10 4 PDLSCs previously irradiated with a linear accelerator 20 Gy with an equal amount of allogeneic PBMCs.
- the T lymphocyte proliferation group as a positive control was: 5.0 x 10 4 PBMCs were co-incubated with an equal amount of allogeneic PBMCs. Equal amounts of PBMCs were cultured alone as negative controls. The results showed that the experimental group PDLSCs did not cause proliferation of allogeneic PBMCs, while the positive control group could cause significant
- T lymphocyte proliferation suggests that PDLSCs have low immunogenicity.
- PDLSCs can inhibit T lymphocyte proliferation
- fPDLSCs and cPDLSCs were inoculated with a cell volume of 1.0 x 10 4 , 5.0 ⁇ 10 4 , 2.5 x 10 s and 5.0 x 10 5 , then autologous PBMCs (5.0 x 10 4 ) and a final concentration of 0.5 g/mL PHA.
- PHA stimulated the proliferation of PBMCs (5.0xl0 4 ) as a positive control. It was found that the proliferation of PHA-stimulated PBMCs was significantly inhibited by fPDLSCs or cPDLSCs, and this inhibition was dose-dependent.
- PDLSCs inhibit T lymphocyte proliferation by secreting PGE2
- the concentration of PGE2 was 15.19 ⁇ 1.26 ng/ml, which was significantly higher than that of pure PDLSCs.
- the concentration of TGF- ⁇ was 1459.79 ⁇ 109.49 pg/mL, no significant change.
- IL-10 is a major mediator of immunosuppressive effects of BMSCs.
- Di Nicola et al (7) found that TGF- ⁇ and HGF are the main factors that play a role in antibody neutralization experiments.
- Other studies have shown that inhibitors of PGE2 can counteract the immunosuppressive effects of BMSCs (25). These studies have shown that soluble factors are involved in the immunosuppressive effects of BMSCs.
- PGE2 is a major factor in the inhibition of T lymphocyte proliferation by PDLSCs.
- the immunosuppressive effect of PDLSCs is not related to T cell apoptosis, but induces T cell incompetence.
- FIG. 8 Another test result according to the present embodiment is shown in Fig. 8, in which PDLSCs-mediated periodontal tissue regeneration is described in detail, and the results shown in the figure show that it is consistent with the above test results in the present embodiment.
- the present invention shows that PDLSCs expressing HLA-I and not expressing HLA-Il DR CD80 and CD86 do not cause proliferation of allogeneic T lymphocytes, suggesting that PDLSCs have low immunogenicity; PDLSCs can inhibit mitogens and the same T lymphocyte proliferation induced by allogeneic antigens; PDLSCs do not cause apoptosis of T lymphocytes, and T lymphocytes can regain proliferative ability when stimulated again; PDLSCs exert their immunosuppressive function by secreting PGE2; allogeneic PDLSCs can be repaired Mini-pig periodontitis bone defect model does not cause immune rejection.
- Example 3 Tooth-related mesenchymal stem cell-mediated biological root regeneration
- Root tip nipple stem cells Under the anesthesia, the small pig fangs are aseptically removed, and the apical apex of the apex is removed. The D-Hank's solution is repeatedly washed, chopped, and placed in type I collagenase (3 g/L). Digestion with Dispase (4 g/L), digest at 37 °C for 1 h, collect the cells through a 70 ⁇ filter, centrifuge at 1000 r/min for 10 min, and resuspend into a single cell suspension with the culture solution. The apical papilla cells and periodontal ligament cells were seeded in a 6-well plate at 0.
- Dental pulp cells Under the anesthesia, the small pig canines are aseptically removed, the teeth are opened and the pulp tissue is removed, and the D-Hank's solution is repeatedly washed, chopped, digested and cultured, and the specific steps are the same as the apical papilla cells.
- Periodontal ligament stem cells Under the anesthesia, the small pig canines are aseptically removed, and the periodontal tissues around them are gently peeled off. The periodontal tissues of the middle segment are taken and washed repeatedly with D-Hank's solution, chopped, digested, and cultured. Tongue nipple cells.
- the vigorously growing 2xl0 5 second or third generation periodontal ligament stem cells were seeded in a 60 mm culture dish, and the culture medium was ⁇ - ⁇ medium (containing 15% fetal calf serum, 100 ⁇ /L of L-ascorbic acid 2-decanoic acid). , 2 mmol/L glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin). After 10 to 14 days of culture, the JUL border cells are creased, and the cell membrane is completely removed by a relatively blunt blade or a cell scraper, and the cell membrane is not dried during the process.
- the hydroxyapatite/trasalic tricalcium scaffold material is shaped like a root, and the dimensions are: a cone with a diameter of 5 mm and a length of 15 mm.
- Hydroxumsite/citrate tricalcium is a porous network structure with a pore diameter of 200 ⁇ 500 ⁇ .
- the dental pulp stem cells are compounded on a hydroxyapatite/tricalcium phosphate three-dimensional scaffold and grown on the fifth day in the bioreactor. The dental pulp stem cells, the cells are fully stretched, and the cell surface has many protrusions and secretory granules, which are connected into one piece and have a diameter of 20-50 ⁇ m.
- the lxlO 8 was cultured to the third generation of the apical papillae and suspended in the medium.
- the root-type scaffold material was placed in the cell-containing medium and mixed thoroughly on a shaker at 37 ° C for 2 h to carefully clamp the scaffold material. Placed in the culture, the remaining cell suspension is carefully added to the scaffold material, and after standing for 4 h, the culture solution is added, and cultured in a bioreactor for 5 days and then transplanted.
- the edentulous areas of small pigs were selected separately. Open the mucosa, open the periosteum flap, and expose the alveolar crest. Using a planter to drill a hole similar in shape to the hydroxylstone/tricalcium phosphate scaffold in the alveolar bone at a rate of 800 rpm, the above-mentioned apical papilla stem cells/dental stem cells and root-shaped hydroxyapatite The stone/stone tricalcium was mixed and cultured in a bioreactor for 5-7 days. The periodontal ligament stem cell membrane wrapped around the surface of hydroxyapatite/salt tricalcium and was implanted in the alveolar bone of small pigs.
- Periodontal ligament cell sheets have been used in periodontal tissue engineering for some time, but conventionally used Recell temperature response culture, the surface of the culture used a temperature sensitive hydrophobic material PIPA-Am. When the temperature is higher than 32. At C, the surface is hydrophobic, suitable for cell attachment and growth. When the temperature is lowered, the polymer becomes hydrophilic and swells, and the cells are naturally released. Harvesting the cells simply reduces the temperature to 20. C can, without enzyme digestion and treatment, can retain cell surface function and activity. However, the culture material requires a special material PIPA-Am. Therefore, we have explored a method for preparing a periodontal membrane cell membrane by using conventional JUL culture medium.
- Adding 100 ⁇ /L of L-ascorbic acid 2-phosphate to the medium component can promote the cells. It proliferates rapidly and secretes a large amount of extracellular matrix collagen. When all the cells are connected together and grow to a certain extent, we completely remove the entire cell membrane.
- the fine spore membrane does not destroy the scaffold formed by the extracellular matrix, and does not destroy the adhesion, proliferation and differentiation of cell surface proteins, and promotes the formation of periodontal tissues after returning to the body.
- the normal periodontal membrane is particularly thin about 0.2 mm. If the stent material is relatively thick, the void left after the material is degraded is a problem, and the periodontal membrane avoids this problem.
- the bioreactor can not simulate the material exchange mechanism of the internal circulation system, it can generate a hydrodynamic environment inside the scaffold material. If the mechanical strength is controlled, the damage to the cells can be reduced, and the cell growth can be provided. Favorable mechanical environment.
- the cells were cultured in the bioreactor for 5 days after being compounded into the material.
- the dynamic three-dimensional culture of the bioreactor promoted the exchange of nutrients inside the scaffold, promoted the uniform distribution of the seed cells in the scaffold material, and facilitated the cell retention. Bone phenotype and promotion of deposition of extracellular base shields in scaffold materials.
- the replanted bio-roots showed a high-density shadow that was not transmitted through the X-ray film, surrounded by low-density images.
- the morphology of the regenerated biological root tissue is completely different from that of the new bone tissue. It is composed of a number of spherical hard tissue masses at different growth stages, and the spherical masses are connected to each other to form a network.
- the new spherical hard tissue is small in size, surrounded by loosely arranged blue-violet cells, presumably odontoblasts or cementoblasts, with a uniform, powder-dyed matrix in the center.
- the more mature spherical hard tissue mass is larger, with no cells in the middle, or a very small amount of cell debris embedded therein.
- There is a dentin-like structure in the mass of which It can be seen that the irregular dentin tubule-like structure also has a cementum-like structure, and the microscopic appearance is a relatively uniform hard tissue.
- the newly formed hard tissue area is surrounded by fibrous connective tissue, and no inflammatory cell infiltration is observed. In some areas, a structure similar to Sharp's fiber-like structure is inserted into the cementoplast and dentin-like structure being formed.
- the present invention has important and far-reaching significance for the use of biological root regeneration to repair tooth loss:
- the present invention proposes a new concept of biological root regeneration based on tissue engineering technology.
- the present invention implements the new concept of biological root regeneration on large animal miniature pigs, and succeeds in demonstrating that the method of the invention is feasible, and is expected to be a new repair method for clinical application.
- the specific implementation method provided by the present invention is reliable and provides theoretical basis and technical support for in-depth research and commercialization of biological root regeneration.
- FIG. 9 An experimental result is shown in Fig. 9, in which the case of successfully regenerating the biological root on a small pig is described in detail.
- A experimental flow chart
- B, C formation of dentin cementous structure and periodontal membranous structure (HE staining);
- D periodontal membrane COL I staining positive;
- E dentin calculus The quality of the structure was positive for Dsp staining;
- F ⁇ I Scanning electron microscopy (SEM) of the regenerative roots:
- H newly formed periodontal membrane fibers are newly formed The dentinal cement structure and the alveolar bone (G arrow);
- I The newly formed dentin-like structure (referred to by the thick arrow in Figure G).
- BMMSCs Bone marrow mesenchymal stem cells
- PBMCs peripheral blood mononuclear cells
- SHED or BMMSCs (lxl0 5 cells/10 g body weight, cells suspended in 100 ml PBS) were injected into 16-week-old MRL/lpr mice through the tail vein, and the control group was injected with physiological saline.
- the mice were sacrificed at 20 weeks of age, and peripheral blood, kidney, long bone (femur and tibia) specimens were taken.
- Kidney specimens were fixed, paraffin-embedded, and HE, trichrome, periodic acid-schiff (pas) staining was performed to observe the recovery of glomerular basement membrane function and the recovery of excessive growth of mesangial cells.
- Serum dsDNA-IgG, dsDNA-IgM, and ANA levels were measured by ELISA to detect levels of complement 3 (C3) and creatinine urine protein in urine and serum.
- SLE is an autoimmune disease characterized by multiple organ involvement and multiple antibodies in the blood.
- T and ⁇ lymphocyte overactivation are the key link in the pathogenesis of SLE.
- T, ⁇ lymphocytes are derived from lymphoid stem/progenitor cells, and MSCs play an important role in the proliferation and differentiation of lymphoid stem/progenitor cells. Therefore, the role of MSCs in the pathogenesis of SLE has attracted more and more attention.
- the differentiated bone marrow stromal cells do not support lymphocyte growth well.
- the growth of lymphocytes depends on the various cytokines secreted by lymphocytes and bone marrow stromal cells.
- the abnormality of bone marrow stromal cells participates in lymphocytes. Abnormal activation.
- MSCs express a large number of cell adhesion molecules, which play an important role in cell adhesion, homing, supporting hematopoiesis, and regulating immune cell function.
- the tumor cells are implanted into human allogeneic mice, the tumor cells are cleared by the host immune system, and when the tumor cells and MSCs are simultaneously injected into the allogeneic mice, the tumor cells can survive in the receptor, indicating that the MSCs have immunosuppressive function in vivo. .
- MSCs retain their immunomodulatory effects when differentiated into other cell types, which means that transplanted MSCs can exert long-term immunomodulatory effects.
- SHED has the biological and immunological properties of MSCs and can interact with T lymphocytes. Compared with BMMSCs, SHED exerts an immunological effect by restoring the ratio of Tregs/Thl7 in peripheral blood and reducing the number of Th17 cells. After injection of SHED in 16-week-old MRL/lpr mice, trabecular bone regeneration was observed and osteoclast activity was inhibited. The SHED-injected mice, like the mice injected with BMMSCs, reconstituted the osteoblast microenvironment, thereby improving their dysfunction.
- hPDLSCs allogeneic human periodontal ligament stem cells
- hPDLSCs Melting hPDLSCs. After thawing, hPDLSCs were examined for mycoplasma, bacteria, colony formation efficiency, mesenchymal stem cell marker patterns, and karyotype analysis.
- Nitric oxide plays a critical role in suppression of T cell proliferation by mesenchymal stem cells, Blood; 109:228-234.
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Description
牙齿相关干细胞的新用途
发明领域
本发明涉及牙齿相关干细胞的新用途。 具体地, 本发明涉及牙齿相 关干细胞在制备用于预防或治疗与牙齿相关的疾病或病情的产品中的用 途,或者在制备用于牙齿相关组织形成或修复的产品中的用途; 还涉及牙 齿相关干细胞在制备用于预防或治疗免疫性疾病或病情、 自身免疫性疾 病或病情、 与 T淋巴细胞异常活化有关的疾病或病情、 与 T淋巴细胞异 常升高有关的疾病或病情、 红斑狼疮、 或者***性红斑狼疮的产品中的 用途; 还涉及根据上述甩途的治疗方法; 还涉及包含牙齿相关干细胞的 组合物。 背景技术
干细胞是一类具有自我更新和分化潜能的原始细胞, 可分为胚胎干 细胞和成体干细胞。 在成体组织及器官中普遍存在着特异的成体干细 胞,目前已经发现的例如来源于人的牙齿相关干细胞都是来源于中胚层的 间充质干细胞 (mesenchymal stem cells, MSCs), 包括牙髓干细胞 (dental pulp stem cells, DPSCs)、 脱落乳牙牙髓干细胞 (stem cells from human exfoliated deciduous teeth, SHED)、 牙周膜干细胞 (periodontal ligament stem cells, PDLSCs)和才艮尖牙***干细胞 (stem cells from apical papilla , SCAP) (1-4, 即指文献 1至 4, 下同), 它们可以来源于多种动物 (例如哺 乳动物, 例如人)。研究表明牙齿相关干细胞具有艮高的增殖能力和多向 分化潜能, 能够向成骨细胞、 脂肪细胞、 神经样细胞分化。 当把牙齿相 关干细胞和三维支架材料的复合物植入到棵鼠皮下时, 能够产生自体牙 髓牙本质复合体样结构和牙骨质牙周膜复合体样结构 (1,3)。 目前在自体 小型猪模型上已经应用 PDLSCs和 SCAP成功再生出生物牙根 (4)。目前 国际上干细胞研究焦点之一集中在建立各种组织成体干细胞库,进行干细 胞的临床试验研究。
根据 WHO(2003)统计数字表明, 牙齿相关疾病所波及的人群范围已 远远超过其它任何一种疾病。 目前, 全世界每年用于牙齿疾病的治疔费用
已超过 200亿美元。 在牙齿相关疾病中, 牙周病是一种全世界范围内发 病率很高的细菌感染性疾病, 最终导致牙齿支持组织丧失和牙齿缺失, 并能引起一系列的全身***性疾病 (13);而牙齿缺失可对人体消化***和 全身健康造成严重的影响, 同时也会使患者产生不良的社会心理影响。但 目前对牙周病及牙齿组织缺损没有好的药物或治疗方法进行治疗或修 复; 有关牙齿缺失的治疗主要仍是采用人工材料制作牙齿的替代物。
***性红斑狼疫 (systemic lupus erythematosus, SLE)是以多脏器受 累和血中存在多种抗体为特征的自身免疫病,常规的免疫抑制或免疫调节 治疗可使多数患者的生存期延长, 生活质量得到改善。 1996 年意大利学 者 Marmont首先报道用自体骨髓移植治疗重症红斑狼疮取得成功后, 国 内外迅速开展了自体外周血干细胞移植和自体骨髓移植治疗重症 SLE等 多种难治性免疫病,取得了较好的疗效。造血干细胞移植能使部分患者达 到病情控制, 但其费用高、 并发症多, 病情复发率高达 40%-50%, 不能 被常规使用; 并且, 移植后虽然部分患者有可能达到长期緩解, 但均不能 彻底根治自身免疫性疾病, 部分患者存在复发的问题。
因此,目前仍然需要可有效治疗某些牙齿相关疾病和某些免疫性疾病 的新颖方法。 发明内容
本发明的目的是提供有效治疗某些牙齿相关疾病和某些免疫性疾病 的新颖方法。 本发明人在研究中现出人意料地发现牙齿相关干细胞例如 牙周膜干细胞 (PDLSCs)不但在自体的牙齿中使缺损的牙齿组织再生,而 且能在异体缺损的牙齿组织使牙齿组织再生, 同时 PDLSCs还对异常升 高的 T淋巴细胞有抑制作用。 本发明基于上述研究结果得以完成。
发明概述
本发明首先涉及 PDLSCs在制备用于预防或治疗牙周病, 牙齿缺损 修复的产品中用途。
本发明还涉及 PDLSCs在制备用于预防或治疗与 T淋巴细胞异常升 高有关的疾病或症状的产品中用途。
本发明还涉及一种预防或治疗牙周病, 修复缺损牙齿组织的方法, 其包括给予需预防或治疗牙周病或需修复缺损牙齿组织的宿主以有效 预防或治疗量或修复缺损牙齿组织量的 PDLSCs.
本发明涉及一种预防或治疗与 T淋巴细胞异常升高有关的疾病或症 状的方法,其包括给予 Τ淋巴细胞异常升高的宿主预防或治疗有效量的 PDLSCso
发明详迷
更详细地, 本发明提供了以下的各个方面以及各个方面的具体项。 本发明第一方面提供了牙齿相关干细胞在制备用于预防或治疗与牙 齿相关的疾病或病情的产品中的用途,或者在制备用于牙齿相关组织形成 或修复的产品中的用途。
根据本发明第一方面任一项的用途, 其中所述的牙齿相关干细胞选 自: 牙髓干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙*** 干细胞。
根据本发明第一方面任一项的用途, 其中所述的牙齿相关干细胞来 自哺乳动物。在一个实施方案中,所述的牙齿相关干细胞来自选自以下的 哺乳动物: 人、 猪 (例如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
根据本发明第一方面任一项的用途, 其中所述的牙齿相关干细胞来 自哺乳动物, 并且选自: 牙髓干细胞 (dental pulp stem cells, DPSCs)、 脱落孚 L牙牙號干细胞 (stem cells from human exfoliated deciduous teeth, SHED). 牙周膜干细胞(periodontal ligament stem cells, PDLSCs)和才艮 尖牙***干细胞 (stem cells from apical papilla , SCAP)。
根据本发明第一方面任一项的用途, 其中所述的与牙齿相关的疾病 或病情或者牙齿相关组织形成或修复选自: 牙周病、 牙周炎、 牙齿缺损、 牙齿组织缺损、 牙齿缺损修复、 牙齿相关组织缺损和修复、 牙齿相关组 织替代物再生、 等等。
根据本发明第一方面任一项的用途, 其中所述的牙齿相关干细胞用 于自体或者异体的牙齿相关的疾病或病情,或者用于自体或异体的牙齿相 关组织形成或修复。
根据本发明第一方面任一项的用途, 其中所述的牙齿相关组织包括 但不限于牙齿、 牙根、 牙髓、 牙龈等等, 以及其它与牙齿内部、 牙齿表层、 牙齿表面或者牙齿周围相关的组织。
本发明第一方面及其各子项的特征和优点同样适用于本发明其它 任一方面及其各子项。
本发明第二方面提供了牙齿相关干细胞在制备用于预防或治疗免疫 性疾病或病情、 自身免疫性疾病或病情、 与 T淋巴细胞异常活化有关的 疾病或病情、 与 T淋巴细胞异常升高有关的疾病或病情、 红斑狼疮、 或 者***性红斑狼疮的产品中的用途。
根据本发明第二方面任一项的用途, 其中所述的牙齿相关干细胞选 自: 牙髓干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙*** 干细胞。
根据本发明第二方面任一项的用途, 其中所述的牙齿相关干细胞来 自哺乳动物。在一个实施方案中,所述的牙齿相关干细胞来自选自以下的 哺乳动物: 人、 猪 (例如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
根据本发明第二方面任一项的用途, 其中所述的牙齿相关干细胞来 自哺乳动物, 并且选自: 牙髓干细胞 (dental pulp stem cells, DPSCs), 脱落^ L牙牙號干细胞 (stem cells from human exfoliated deciduous teeth, SHED), 牙周膜干细胞 (periodontal ligament stem cells, PDLSCs)和根 尖牙***干细胞 (stem cells from apical papilla, SCAP)。
根据本发明第二方面任一项的用途, 其中所述的牙齿相关干细胞用 于自体或者异体的免疫性疾病或病情、 自身免疫性疾病或病情、 与 T淋 巴细胞异常活化有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾病 或病情、 红斑狼疮、 或者***性红斑狼疮。
本发明第二方面及其各子项的特征和优点同样适用于本发明其它 任一方面及其各子项。
本发明第三方面提供了预防或治疗与牙齿相关的疾病或病情的方 法,或者形成或修复牙齿相关组织的方法,该方法包括给有需要预防或治 疗与牙齿相关的疾病或病情的宿主施用有效量的牙齿相关干细胞,或者该 方法包括给有需要形成或修复牙齿相关组织的宿主施用有效量的牙齿相 关干细胞。
根据本发明第三方面任一项的方法, 其中所述的牙齿相关干细胞选 自: 牙髓干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙*** 干细胞。
根据本发明第三方面任一项的方法, 其中所述的牙齿相关干细胞来 自哺乳动物。在一个实施方案中,所述的牙齿相关干细胞来自选自以下的
哺乳动物: 人、 猪 (例如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
根据本发明第三方面任一项的方法, 其中所述的牙齿相关干细胞来 自哺乳动物, 并且选自: 牙髓干细胞 (dental pulp stem cells , DPSCs)、 脱落乳牙牙號干细胞 (stem cells from human exfoliated deciduous teeth , SHED) , 牙周膜干细胞 (periodontal ligament stem cells , PDLSCs)和才艮 尖牙 Li头干细胞 (stem cells from apical papilla , SCAP)。
根据本发明第三方面任一项的方法, 其中所述的与牙齿相关的疾病 或病情或者牙齿相关组织形成或修复选自: 牙周病、 牙周炎、 牙齿缺损、 牙齿组织缺损、 牙齿缺损修复、 牙齿相关组织缺损和修复、 牙齿相关组 织替代物再生、 等等。
根据本发明第三方面任一项的方法, 其中所述的牙齿相关干细胞用 于自体或者异体的牙齿相关的疾病或病情,或者用于自体或异体的牙齿相 关组织形成或 复。
根据本发明第三方面任一项的方法, 其中所述的牙齿相关组织包括 但不限于牙齿、 牙根、 牙髓、 牙龈等等, 以及其它与牙齿内部、 牙齿表层、 牙齿表面或者牙齿周围相关的组织。
根据本发明第三方面任一项的方法, 其中所述的宿主是哺乳动物。 在一个实施方案中, 所述的宿主是选自以下的哺乳动物: 人、 猪 (例如五 指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 綿羊、 山羊。
在本发明第三方面任一项方法的一个实施方案中, 其包括以下步
(a)采集人牙周膜;
(b)培养 hPDLSCs;
(c)任选地冷冻保存 hPDLSCs;
(d)任选地融化 hPDLSCs ,以及任选地对 hPDLSCs检查支原体、 细菌、 集落形成效率、 间质干细胞标记模式和核型分析;
(e)制备 hPDLSCs片;
(f)将 HA/TCP置于容纳有 hPDLSCs片的器孤中;
(j)在任选的牙周初始治疗后, 将 hPDLSCs片与 HA/TCP植入 到牙周损伤缺陷处;
(k)任选的进行临床和放射照相评价、 血液学和免疫学评价。 本发明第三方面及其各子项的特征和优点同样适用于本发明其它 任一方面及其各子项。
本发明第四方面提供了预防或治疗免疫性疾病或病情、 自身免疫性 疾病或病情、 与 T淋巴细 异常活化有关的疾病或病情、 与 T淋巴细胞 异常升高有关的疾病或病情、 红斑狼疮、 或者***性红斑狼疮的方法, 该方法包括给有此需要的宿主施用有效量的牙齿相关干细胞。
根据本;^明第四方面任一项的方法, 其中所述的牙齿相关干细胞选 自: 牙髓千细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙*** 干细胞。
根据本发明第四方面任一项的方法, 其中所述的牙齿相关干细胞来 自哺乳动物。在一个实施方案中,所述的牙齿相关干细胞来自选自以下的 哺乳动物: 人、 猪 (例如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
根据本发明第四方面任一项的方法, 其中所述的牙齿相关干细胞来 自哺乳动物, 并且选自: 牙髓干细胞 (dental pulp stem cells, DPSCs)、 脱落孔牙牙號干细胞 (stem cells from human exfoliated deciduous teeth, SHED). 牙周膜干细胞(periodontal ligament stem cells, PDLSCs)和才 尖牙***干细胞 (stem cells from apical papilla, SCAP)。
根据本发明第四方面任一项的方法, 其中所述的牙齿相关干细胞用 于自体或者异体的免疫性疾病或病情、 自身免疫性疾病或病情、 与 T淋 巴细胞异常活化有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾病 或病情、 红斑狼疮、 或者***性红斑狼疮。
根据本发明第四方面任一项的方法, 其中所述的宿主是哺乳动物。 在一个实施方案中, 所述的宿主是选自以下的哺乳动物: 人、 猪 (例如五 指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
本发明第四方面及其各子项的特征和优点同样适用于本发明其它 任一方面及其各子项。
本发明第五方面提供了一种组合物, 其包含有效量的牙齿相关干细 胞和任选的药学可接受的载体。
根据本发明第五方面任一项的组合物, 其中所述的牙齿相关干细胞
选自: 牙髓干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙乳 头干细胞。
根据本发明第五方面任一项的组合物, 其中所述的牙齿相关干细胞 来自哺乳动物。在一个实施方案中,所述的牙齿相关干细胞来自选自以下 的哺乳动物: 人、 猪 (例如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 脉鼠、 羊、 绵羊、 山羊。
根据本发明第五方面任一项的组合物, 其中所述的牙齿相关干细胞 来自哺乳动^, 并且选自: 牙髓干细胞 (dental pulp stem cells , DPSCs)、 脱落孔牙牙體干细胞 (stem cells from human exfoliated deciduous teeth, SHED). 牙周膜干细胞 (periodontal ligament stem cells, PDLSCs)和根 尖牙***干细胞 (stem cells from apical papilla, SCAP)。
根据本发明第五方面任一项的组合物, 其是用于预防或治疗与牙齿 相关的疾病或病情,或者用于牙齿相关组织形成或修复,或者用于预防或 治疗免疫性疾病或病情、 自身免疫性疾病或病情、 与 T淋巴细胞异常活 化有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾病或病情、 红斑 狼疮、 或者***性红斑狼疮。
根据本发明第五方面任一项的组合物, 其中所述的有效量是可以有 效地用于预防或治疗与牙齿相关的疾病或病情的剂量, 或者是要以有效 地用于牙齿相关组织形成或修复的剂量,或者是要以有效地用于预防或治 疗免疫性疾病或病情、 自身免疫性疾病或病情、 与 T淋巴细胞异常活化 有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾病或病情、 红斑狼 疮、 或者***性红斑狼疮的剂量。
本发明第五方面及其各子项的特征和优点同样适用于本发明其它 任一方面及其各子项。
根据本发明上下文的详细记载, 本发明令人满意地实现上上述的各 个方面及其各子项。
下面对本发明的各个方面和特点作进一步的描述。
本发明所引述的所有文献, 它们的全部内容通过引用并入本文, 并且 如果这些文献所表达的含义与本发明不一致时, 以本发明的表述为准。此 外, 本发明使用的各种术语和短语具有本领域技术人员公知的一般含义, 即便如此, 本发明仍然希望在此对这些术语和短语作更详尽的说明和解 释,提及的术语和短语如有与公知含义不一致的, 以本发明所表述的含义
为准。
本发明人在研究中现出人意料地发现 PDLSCs等牙齿相关的干细胞 不但在自体的牙齿中使缺损的牙齿组织再生, 而且能在异体缺损的牙齿 组织使牙齿组织再生。 本发明基于上述研究结果得以完成。 因此, 本发 明涉及牙齿相关干细胞例如 PDLSCs在制备用于预防或治疗牙周病, 牙 齿缺损修复的产品中用途 (例如图 8所示); 还涉及一种预防或治疗牙周 病, 修复缺损牙齿组织的方法, 其包括给予需预防或治疗牙周病或需修 复缺损牙齿组织的宿主以有效预防或治疗量或修复缺损牙齿组织量的 牙齿相关干细胞例如 PDLSCs.
此外, 本发明提出生物牙根再生的新理念; 利用自体异体 SCAP/ DPSCs和 PDLSCs再生出了具有生物学功能的生物牙根,在新形成的功 能性生物牙根上再进行冠的修复,恢复患者的咀嚼功能 (例如图 10所示); 提供生物牙根再生的具体实施方法, 为进一步对生物牙根再生的机理进 行深入研究及生物牙根的商品化提供依据。 因此, 本发明涉及生物牙根 再生的新理念, 并涉及 SCAP, DPSCs, PDLSCs 等牙齿相关的干细胞 在生物牙根再生中的用途。 本发明还涉及生物牙根再生的具体实施办 法, 其中包括牙周膜干细胞膜片的制备及其所需最佳细胞数及最佳生长 时间。
再者, 本发明涉及牙齿相关干细胞例如 PDLSCs在制备用于预防或 治疗与 T淋巴细胞异常升高有关的疾病或症状的产品中用途。本发明还 涉及一种预防或治疗与 T淋巴细胞异常升高有关的疾病或症状的方法, 其包括给予 T淋巴细胞异常升高的宿主预防或治疗有效量的牙齿相关干 细胞例如 PDLSCs。 本发明进一步涉及牙齿相关干细胞例如 SHED在预 防或治疗***性红斑狼疮病等自身免疫***病中用途 (例如图 11所示)。
根据本发明, 术语"牙周病 "包括但不限于牙周炎。
根据本发明, 术语"缺损牙齿组织 "包括但不限于宿主牙齿的各种缺 损情况, 如各种原因引起的牙齿脱落。
根据本发明, 术语"宿主,,通常指哺乳动物, 包括但不限于人, 猪, 牛, 马等。 在一个实施方案中, 术语"宿主,,是指人、 猪 (例如五指山小型 猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵羊、 山羊。
根据本发明, 术语"产品"是指适于 PDLSCs应用的各种形式。 根据 本发明, 术语"产品"还指适于牙齿相关干细胞应用的各种形式, 例如组
合物、 药物组合物等。
根据本发明 , 术语"与牙齿相关的疾病或病情"是指本文所述宿主罹 患或者表现的疾病、 病情、 体症、 身体状态等, 这些疾病、 病情、 体症、 身体状态等与牙齿相关。
根据本发明, 术语"牙齿相关组织形成或修复"或者术语"牙齿相关组 织的形成或修复"或者"形成或修复牙齿相关组织"等, 它们具有相同或近 似的含义, 并且通常是指本文所述宿主的牙齿相关组织进行形成、 修复、 生成、 再生、 培养等处理或操作, 或者对牙齿相关组织异常 (例如缺损)进 行形成、 修复、 生成、 再生、 培养等处理或操作。
根据本发明, 术语"组合物 "是具有本领域技术人员通常理解的含 义, 并且通常是指可接或间接 (例如临用前稀释)用于临床使用的形式, 例如剂型、 药物剂型、 给药形式、 等。 在临床应用领域或者药物领域, 术语"组合物"还通常与"药物组合物"具有同等含义。
可改变本发明组合物或药物组合物中牙齿相关干细胞的实际剂量水 平, 以便所得的干细胞量能有敢针对具体宿主、 患者在特定组合物及其 组成以及相应给药方式的情况下得到所需的治疗或预防反应。 剂量水平 须根据具体干细胞的活性、 给药途径、 所治疗病况的严重程度、 疾病或 病情的治疗进程、 形成和修复 (以及生成、 再生、 培养等)处理或操作的进 程、 以及待治疗患者的病况和既往病史来选定。但是, 本领域的做法是, 干细胞的剂量以及施用时间从低于为得到所需治疗效果而要求的水平 开始, 逐渐增加剂量, 直到得到所需的效果。 因此, 就本发明而言, 本 领域技术人员在本发明详细公开的信息的教导下, 可以根据例如但不限 于上述的具体情况来确定在具体情况下所适用的具体剂量, 而无需要作 具体限定。 特别是, 可以参考本发明实施例部分中所用的具体量来确定 任一情况下的使用量, 例如在本发明下文使用了具体剂量的五指山小型 猪 PDLSCs, 本领域技术人员可根据上述剂量并结合本领域公知技术教 导将该剂量换算为人用条件下的剂量。
本发明牙齿相关干细胞可以单独(即以原样形式)或以药物组合物的 形式给药。本发明药物组合物可根据给药途径配成各种适宜的剂型。使用 一种或多种生理学上可接受的载体, 包含赋形剂和助剂, 它们有利于将牙 齿相关干细胞加工成可以在药学上使用的制剂。适当的制剂形式取决于所 选择的给药途径,可以按照本领域熟知的常识进行制造。在本发明的一个
实施方案中, 所述牙齿相关干细胞存在于细胞相容的介质 (例如, 生理盐 水如 0.9%生理盐水, 等等)中。 在本发明的一个实施方案中, 所述牙齿相 关干细胞存在于细胞相容的介质中, 并且在低温下保存, 例如在冷藏、 冷 冻等条件下保存,并且可任选在在临用前复溶成适用根据本发明精神而施 用的形式。 附图说明
图 1. 人 PDLSCs免疫分子的表达: 应用流式细胞术检测 fPDLSCs 和 cPDLSCs相关免疫分子的表达。 HLA-I, HLA-Π DR, CD80和 CD86 的表达情况如图所示: fPDLSCs (76.2%±5.8%, n= 5)和 cPDLSCs (78.5%±6·4%, η= 5) 表达 HLA-I, 但是不表达 HLA -Π DR 和共刺激 分子 CD80、 CD860
图 2. PDLSCs抑制 T淋巴细胞增殖:(a) fPDLSCs/cPDLSC 不会引 起同种异体 T 淋巴细胞增殖。(b) fPDLSCs/cPDLSC 剂量依赖性地抑制 丝裂原 PHA 引起的 T 淋巴细胞增殖。 (c) 延迟加入 fPDLSCs/cPDLSC 也能够抑制 PHA 引起的 T 淋巴细胞增殖。 (d) fPDLSCs/cPDLSC能够 抑制双向混合淋巴细胞反应。
图 3. PDLSCs 通过分泌 PGE2 抑制 T 淋巴细胞增殖: (a) 在 Transwell培养实验中, PDLSCs也能抑制 T淋巴细胞增殖,提示 PDLSCs 分泌可溶性因子来发挥免疫抑制作用。 (b)在单纯 PDLSCs 培养上清和 MLR上清中都存在 TGF-pi ,但是两者之间没有显著性差异。(c) PGE2 的浓度在 MLR中显著性升高。(d) 中和实验表明抗 TGF-pi抗体没有恢 复 T淋巴细胞增殖, PGE2抑制剂抵消了 PDLSCs的免疫抑制作用, 提 示 PGE2 是介导 PDLSCs 免疫抑制作用的主要因子。 (e, f) PDLSCs 的 免疫抑制作用并没有引起 T淋巴细胞的凋亡 (e), 与单纯用 PHA 刺激 T 淋巴细胞后的凋亡率一致 (f)。 (g) PDLSCs引起的被抑制的 T淋巴细胞 用 PHA或者 IL-2再刺激时可以重新恢复增殖。
图 4. HGF 和 IL-10的测定:在单纯 PDLSCs 培养上清和 MLR 上 清中都没有测到 HGF和 IL-10,说明这两种因子没有参与 PDLSCs介导 的免疫抑制作用。
图 5. PDLSCs介导的牙周组织再生: (a)在 -4w和 0 w, 四组的临床 指标之间没有显著性的差异。 但是治疗后 12 周, 自体或者同种异体
PDLSCs移植组的 PD, GR和 AL与空白对照组和 HA/TCP相比显著恢 复。 (b,c,d,e) CT扫描显示治疗前都有明显的牙周骨缺损, 而且缺损程 度相近。治疗后 12 周, 自体 PDLSCs 组 (h) 和同种异体 PDLSCs 组 (i) 完全获得了牙周骨再生。 空白对照组几乎没有骨组织再生(f), 而 HA/TCP 组的骨缺损程度加重 (g). (j,k,l,m)组织学研究发现自体或者同 种异体 PDLSCs组在骨缺损区有明显的新骨和牙周组织再生 。但是, 典 型的牙周炎表现如深牙周袋、 缺乏新生骨和牙周纤维在 HA/TCP 组和 空白对照组仍然清晰可见。 PD: 探诊深度, GR: 牙龈退缩, AL: 附着丧 失, D: 牙本质, C: 牙骨质, PDL: 牙周膜, B: 骨。
图 6. 同种异体 PDLSCs 移植不会引起免疫排斥反应: (a) 在如图 所示的各个时间点, 同种异体 PDLSCs 移植组的 CD3+, CD4+, CD8+ T 淋巴细胞没有显著性差异; (b)在移植后 3天, CD3+, CD4+, CD8+ T淋巴 细胞数目以及活化 T淋巴细胞的标志物—— CD40L 的表达情况在四个 治疗组之间没有明显差异。
图 7. 移植后 12w的免疫状况:在治疗后 12w, CD3+, CD4+, CD8+ T 淋巴细胞数目以及活化 Τ淋巴细胞的标志物—— CD40L 的表达情况在 四个治疗组之间也没有明显差异。
图 8. 描绘了本发明的一个实施例中 PDLSCs介导的牙周组织再生。 图 9. 描绘了在小型猪上成功再生生物牙根。
图 10. 描绘了在小型猪上进行生物牙根再生获得成功。
图 11. 描绘了脱落乳牙牙髓干细胞治疗***性红斑狼疮鼠血清及腎 脏组织学改变。
图 12. 说明同种异体人牙周膜干细胞 (hPDLSCs)用于治疗牙周病的 标准操作的一个实例。 具体实施方式
通过下面的实施例可以对本发明进行进一步的描述, 然而, 本发明的 范围并不限于下述实施例。 本领域的专业人员能够理解, 在不背离本发明 的精神和范围的前提下, 可以对本发明进行各种变化和修饰。
本发明对试验中所使用到的材料以及试验方法进行一般性和 /或具 体的描述。 虽然为实现本发明目的所使用的许多材料和操作方法是本领 域公知的, 但是本发明仍然在此作尽可能详细描述。
实施例 1 : PDLSCs对 T淋巴细胞增殖的抑制作用
材料和方法
1)、 人 PDLSCs
选取首都医科大学附属北京口腔医院口腔颌面外科门诊拔除的 18-28 岁的健康患者的正常第三磨牙, 按照以往文献报道的方法分离和 培养 PDLSCs(3)。人体组织的利用得到首都医科大学伦理委员会的批准。 为了下一步实验中应用冻存牙周膜干细胞(cryopreserved periodontal ligament stem cells , cPDLSCs) , 将部分新鲜分离的牙周膜干细胞 (freshly isolated periodontal ligament stem cells, fPDLSCs)冻存在液氮 中 3个月。 3个月后, 冻存细胞在 37Ό水浴中快速解冻、 接种、 常规培 养。 在本研究中, 应用的细胞都在第 2-4代。 在同一实验中, 应用同一 代的 fPDLSCs 和 cPDLSCso
2)、 外周血单个核细胞
应用梯度密度离心法分离健康供者的外周血单个核细胞 (peripheral blood mononuclear cells, PB.MCs)。
3)、 抗体
本研究中应用抗人白细胞抗原 (human leukocyte antigen , HLA)-I、 HLA-Π DR. CD80、 CD86抗体 (BD Biosciences) , 抗 CD3、 CD4、 CD8、 CD40L、 转化生长因子 p(transforming growth factor β, TGF-卩)、 肝 细胞生长因子 (hepatocyte growth factor, HGF) 抗体(Abeam), 抗 IL-10、 FITC标记的抗小鼠 IgG抗体 iAbD Serotec),
4)、 流式细包术
为了研究 PDLSCs表面相关免疫分子的表达, 将 l.OxlO6 fPDLSCs 或者 cPDLSCs分别与抗 HLA-I、 HLA-Π DR、 GD80或者 CD86抗体 室温下孵育 1小时。经过磷酸盐緩冲液 (phosphate buffered saline, PBS) 沖洗后, 细胞再与 FITC标记的抗小鼠 IgG二抗室温下孵育 30分钟。 孵育结束后上流式细胞仪 (BD Inmmunocytometry Systems)检测表达情 况。
5)、 混合淋巴细胞反应
作为刺激细胞, 5.0 xlO4 fPDLSCs 或者 cPDLSCs 先在直线加速器 下照射 20Gy,然后加入等细胞量的同种异体 PBMCs,在 96孔板、 0.2ml RPMI-1640中共同孵育 5天。 在结束反应前 18个小时, 每孔加入 lpCi
的 3H-thymidine (3H-TdR)。 18个小时后, 在玻璃纤维滤纸上收集细胞, 应用液体闪烁仪 (PerkinElmer)计算 3H-TdR掺入率。 3H-TdR掺入率的 结果以每分钟计数士标准差 (CPM±SD)来表示。
为了研究 PDLSCs对增殖 T淋巴细胞的影响以及此种影响是否具有 剂量依赖性, 在丝裂原促增殖实验中, 以终浓度为 0.5pg/mL 的 PHA(Sigma-Aldrich)刺激 PBMCs (5.0 xlO4)增殖, 与不同剂量的自体 fPDLSCs 或者 cPDLSCs共孵育 5天 (PDLSCs的量分别为 1.0xl04, 5.0 xl04,2.5xl05,5.0 xl05 )。结束孵育前 18小时每孔加入 ΙμΟ的 3H-TdR , 18小时后收集细胞并且计算 3H-TdR掺入率。
进一步研究延 加入 PDLSCs是否会影响 T淋巴细胞增殖。先用终 浓度为 0.5pg/mL的 PHA刺激 PBMCs (5.0 xlO4)增殖 2天, 然后加入同 等剂量的 fPDLSCs 或者 cPDLSCs再共同孵育 3天。 3天后测定 3H-TdR 掺入率。
在证实 PDLSCs能够抑制丝裂原 PHA引起的 T淋巴细胞增殖后, 我们观察 PDLSCs 对双向混合淋巴细胞反应(mixed lymphocyte reaction, MLR)的影响。 来自两个不同个体的 PBMCs (5.0 xlO4 )与等量 的第三方 fPDLSCs 或者 cPDLSCs共同孵育 5天。 结束孵育前 18小时 每孔加入 l Ci的 3H-TdR , 孵育结束后收集细胞、 计算 3H-TdR掺入 率。
6)、 与 PDLSCs共孵育后的 T淋巴细胞的再次刺激
为了观察 PDLSCs对 T淋巴细胞的增殖抑制是否可逆,我们进行了 再激活实验。 PBMCs (5.0 xlO4) 先与等量的 PDLSCs 和 PHA (0.5 g/mL) 共同孵育 5天,然后把通过梯度密度离心法获得的 T淋巴细 胞与 PHA (0.5 g/mL) 或者重组人白介素 2(intei eukin 2, IL-2, 50U/mL; R&D systems)共同反应 2天。 2天后, 应用 3H-TdR掺入法测定 T淋巴 细胞增殖率。
7)、 Transwell培养实验
为了观察通过细胞 -细胞直接接触还是 PDLSCs 分泌可溶性因子来 介导 PDLSCs对 T淋巴细胞的增殖抑制, 我们进行了 Transwell培养实 验。 Transwell 培养*** (Costar)具有直径为 0.4μιη 孔径的微膜, 此微 膜可以把两种细胞人为地分隔开来。 将 PBMCs (5.0 xlO4) 与 PHA (0.5 g/mL)置于 Transwell培养***的上腔, 而 5.0 xlO4 PDLSCs (从本
实脸开始, 本研究的以下部分都用 fPDLSCs)接种在下腔。 5天后, 应用 3H-TdR掺入法测定 T淋巴细胞增殖率。
8)、 可溶性因子的测定及中和实验
当发现可溶性因子介导 PDLSCs对 T淋巴细胞的增殖抑制后,我们 应用醉联免疫吸附实验 (enzyme-linked immumosorbent assay, ELISA) 来测定单纯 PDLSCs的培养上清 (接种 1-5天)和 MLR培养上清 (5.0X104 PDLSCs, 等量 PBMCs 和 0.5 g/mL 的 PHA)中 TGF-βΙ (R&D systems), HGF (R&D systems), ***素 E2 (prostaglandin E2, PGE2; Assay Designs)和 IL-10 (R&D systems)的浓度。通过醉标仪 (Molecules Devices) 在 450nm (TGF-βΙ, HGF和 IL-10) 或者 405nm (PGE2)读取光密度值。
然后进行中和实验, 在 Transwell培养***中建立 MLR 的反应体 系 , 包括 PBMCs (5.0 xlO4), PDLSCs (5.0 xlO4) 和 PHA (0.5pg/mL), 在反应的同时分别加入以下抗体或者试剂: 抗 TGF-β 抗体 (10ng/ mL), 抗 HGF抗体 (10ng/ mL)和抗 IL-10 抗体 (10ng/mL)或者 PGE2的抑制 剂吲哚美辛 (5 mol/L; Sigma-Aldrich)0 5天后, 通过 3H-TdR掺入法测 定 T淋巴细胞增殖率。
9)、 测定凋亡 T淋巴细胞的百分率
建立 MLR 的反应体系,包括 PBMCs (5.0 xlO4), PDLSCs (5.0 xlO4) 和 PHA (0.5 g/mL), 5天后应用 Annexin V-Fluos 凋亡试剂盒 (Roche Diagnostics)来测定凋亡 T淋巴细胞的百分率。 实施例 2: PDLSCs介导的牙周骨缺损修复
1)、 材料和方法
五指山小型猪和贵州香猪 (6-8月龄, 体重 30-40千克)由中国农业大 学实验动物中心提供。 本实验通过首都医科大学伦理委员会批准。 小型 猪尖牙 PDLSCs的分离和培养方法同人 PDLSCs。 按照文献报道的方法 (13), 制备 12 只雌性五指山小型猪的牙周炎骨缺损模型, 总共制备 24 处下颌第一恒磨牙的牙周炎骨缺损。24处缺损随机分为以下 4个组:【1】 空白对照组,不做任何治疗;【2】单纯材料组,翻瓣、刮治、移植 HA/TCP 支架材料 (武汉理工大学提供)、 明胶海绵覆盖缺损、 缝合; 【3】 自体 PDLSCs移植组, 翻瓣、 刮治、 移植 HA/TCP支架材料 +2.0 xlO7五指山 小型猪 PDLSCs、 明胶海绵覆盖缺损、 缝合; 【4】 异体 PDLSCs移植
组, 翻瓣、 刮治、 移植 HA/TCP支架材料 +2.0 X107香猪 PDLSCs、 明胶 海绵覆盖缺损、缝合。在制备牙周炎骨缺损模型前 (-4w)、移植治疗前 (0w) 和治疗后 12 w进行临床检查, 包括探诊深度 (probing depth, PD)、 牙 银退缩 (gingival recession, GR)和附着丧失 (attachment loss, AL). 在 Ow和治疗后 12 w应用 CT (Siemens)观察骨再生的情况。 在 -4w、 治疗 后 l-7d、 2w、 4w、 8w和 12w进行血常规检查、 血生化检查、 免疫球蛋 白检查和 T淋巴细胞相关标志物的检查, 包括 CD3+细胞计数、 CD4+ 细 胞计数、 CD8+ 细胞计数以及活化 T淋巴细胞的标志物—— CD40L的表 达情况。 在治疗后 12w, 处死动物, 从实验部位获取标本, 固定、 脱钙、 石蜡包埋, 行 HE染色, 观察组织再生情况。
通过 Student' t检验和方差分析进行统计学分析, p<0.05认为是有 统计学差异。
2)、 结果和说明
2-1)、 PDLSCs具有低免疫原性
首先观察到人 PDLSCs的免疫表型, 发现 fPDLSCs (76.2%±5.8%, n=5)和 cPDLSCs (78.5%±6.4%, n=5) 表达 HLA-I, 但是不表达 HLA -Π DR 和共刺激分子 CD80、 CD86 (图 1), 与体外培养扩增的 BMSCs 表达状况相似 (23)。
进一步研究 PDLSCs作为抗原提呈细胞对 T淋巴细胞增殖的影响。 实验组为预先经过直线加速器 20Gy照射的 5.0 xlO4 PDLSCs与等量的 同种异体 PBMCs共孵育。作为阳性对照的 T淋巴细胞增殖组为: 5.0 xlO4 PBMCs与与等量的同种异体 PBMCs共孵育。 单独培养等量的 PBMCs 为阴性对照。 结果表明, 实验组 PDLSCs没有引起同种异体 PBMCs增 殖, 而阳性对照组可以引起显著的
T淋巴细胞增殖(图 2a), 提示 PDLSCs具有低免疫原性。
2-2)、 PDLSCs能够抑制 T淋巴细胞增殖
进一步观察 PDLSCs对丝裂原和同种异体抗原引起的 T淋巴细胞增 殖的影响。 将 fPDLSCs和 cPDLSCs接种, 细胞量分别为: 1 .0 xlO4, 5.0χ104, 2.5x10s 和 5.0xl05,然后加入 自体 PBMCs (5.0xl04) 和终浓度 为 0.5 g/mL的 PHA。 PHA刺激 PBMCs (5.0xl04)增殖为阳性对照。 结 果发现, PHA刺激的 PBMCs增殖明显地被 fPDLSCs或者 cPDLSCs所 抑制, 而且这种抑制是剂量依赖性的。 此种免疫抑制作用必须要求
PDLSCs的存在, 而不是由体积效应(bulk effect)引起, 因为在增殖反应 体系中加入等量的自体 PBMCs不会引起抑制作用(图 2b)。 此外, 即使 在 PHA刺激 PBMCs (5.0X104)增殖后两天再加入 PDLSCs, 仍然能够明 显抑制 T淋巴细胞的增殖(图 2c)。 我们进一步观察 PDLSCs对 MLR的 影响。 来自两个不同个体的 PBMCs和第三方的 PDLSCs共同孵育。 结 果表明, fPDLSCs和 cPDLSCs都能够抑制双向 MLR (图 2d)。 本部分 实验数据表明, PDLSCs能够剂量依赖性地和抗原非特异性地抑制同种 异体 T细胞受体引起的和丝裂原引起的 T淋巴细胞增殖。
2-3)、 PDLSCs通过分泌 PGE2抑制 T淋巴细胞增殖
我们进一步研究 PDLSCs抑制 T淋巴细胞增殖的机制问题。为了阐 明是否通过 PDLSCs和 PBMCs细胞-细胞直接接触引起,我们首先进行 了 Transwell培养实验, 将 PDLSCs和 PBMCs人为地分隔开来。 研究 结果表明, 无论是 Transwell培养实验还是细胞-细胞直接接触实验, 都 能够取得相近的 T淋巴细胞增殖抑制效果,提示这种免疫抑制作用与细 胞-细胞直接接触无关, 而是依靠可溶性因子的存在(图 3a)。
通过 ELISA,我们测定单纯 PDLSCs培养上清中和 MLR上清中几 种可能性的可溶性因子。 无论 PDLSCs培养上清还是 MLR上清, 都没 有发现 HGF和 IL-10 (图 4)。 然后我们继续观察 TGF-βΙ和 PGE2, 因 为这两者被认为是 BMSCs发挥免疫调节功能的主要因子 (7, 25)。 如图 3b和图 3c所示,在接种后 1-5 d的 PDLSCs培养上清中, TGF-pi和 PGE2 的浓度相对稳定, 没有明显的波动。 但是, 在 MLR上清中, PGE2的 浓度达 15.19±1.26ng/ml, 与单纯 PDLSCs培养的浓度相比明显升高, TGF-βΙ的浓度为 1459.79±109.49 pg/mL, 没有明显变化。
我们通过在 MLR体系中添加特异性的抗 TGF-β, IL-10, HGF抗体 和 PGE2 抑制剂来观察其恢复 T 淋巴细胞增殖的能力, 进一步确定 PDLSCs抑制 T淋巴细胞增殖的主要因子。 结果发现,加入 PGE2 抑制 剂能够显著恢复 T淋巴细胞的增殖, 而且此时的增殖力同单纯用 PHA 刺激 PBMCs的增殖力相近(图 3d)。 而实验体系中加入抗 TGF-p, IL-10, HGF的中和抗体并没有恢复 T淋巴细胞的增殖(图 3d)。 这些数据表明 PGE2是介导 PDLSCs抑制 T淋巴细胞增殖的主要因子。
就 BMSCs的免疫抑制机制而言, 虽然目前尚不清楚哪一种因子发 挥主导性的作用以及某些研究还存在不一致, 但是大多数的研究认识还
是通过 BMSCs 分泌抗增殖的可溶性因子比如 IL-10, HGF, TGF-βΙ, PGE2, 吲哚胺 -2, 3-二氧化酶和一氧化氮 (5, 6, 17, 19, 20, 24-28)引起。
Beyth 等 (28) 报道 IL-10是 BMSCs免疫抑制作用的主要介导因子。 Di Nicola等 (7)通过抗体中和实验发现 TGF-βΙ和 HGF是发挥作用的主要 因素。 另有研究表明 PGE2的抑制剂能够抵消 BMSCs的免疫抑制作用 (25)。这些研究都表明了可溶性因子参与了 BMSCs的免疫抑制作用。我 们的研究提示 PGE2是 PDLSCs抑制 T淋巴细胞增殖的主要因子。
2-4)、 PDLSCs的免疫抑制作用与 T细胞凋亡无关, 而是诱导 T细 胞无能
进一步研究 PDLSCs 的免疫抑制作用是否引起了 T淋巴细胞的凋 亡。 现发现, 在 PDLSCs、 PBMCs和 PHA的反应体系中, 凋亡的 T淋 巴细胞比例与单纯 PBMC、 PHA反应体系相似, 排除了 T淋巴细胞凋 亡是引起免疫抑制的可能性(图 3e,f)。 然后, 把被 PDLSCs抑制了 5天 的 T淋巴细胞提取, 用 PHA或者 IL-2再刺激两天。 结果发现, 已经被 抑制了的 T 淋巴细胞再次明显增殖, 这种增殖程度与单纯 PHA 刺激 PBMCs的程度相近似(图 3g)。 因此, 可以认为 PDLSCs的 T淋巴细胞 增殖抑制是可逆的, 通过诱导 T淋巴细胞无能引起。
2-5)、 PDLSCs介导的牙周组织再生
鉴于 PDLSCs的免疫调节特性,研究同种异体 PDLSCs能否修复小 型猪牙周炎骨缺损模型。 在移植后 12周, 同种异体移植 PDLSCs组的 PD 为 3.5士0.6 mm, 自体移植组的 PD 为 3.3±0.4 mm, HA/TCP组为 13.1±1.1 mm, 空白对照组为 10.6±1.3 mm。 统计学分析表明自体或者同 种异体 PDLSCs 移植組与 HA/TCP 组和空白对照组相比牙周组织得到 了明显再生, 而自体或者同种异体 PDLSCs 组之间并没有显著差异(图 5a)0 CT扫描显示自体和同种异体 PDLSCs组的牙槽骨明显再生, 基本 恢复到了正常水平, 而 HA/TCP 组和空白对照组仅仅少量再生或者没 有再生(图 5a-h)。 组织学观察表明, 在自体和同种异体 PDLSCs组, 明 显再生出新骨、 牙骨质和牙周纤维, 在 HA/TCP 组和空白对照组, 仍 可见明显的牙周炎表现, 包括深牙周袋、 缺乏新生骨和牙周纤维(图 5j-m)。 此外, 与移植治疗前 4w相比, 治疗后各时间点的血常规检查 (表 1)、 血生化检查 (表 2)、 免疫球蛋白检查 (表 3)和免疫学相关指标 (图 6, 图 7)都没有明显变化,表明同种异体移植 PDLSCs修复牙周炎骨缺损没
有排斥反应的发生。
表 1
项目 ; ―
·½ \i 2i 3d 4d 5d 6d 7d 2w ¼ 8w 12w C 腿 11.9&1.2 12.M321Z6feO.¾ \2 Ul 132 56 12Μ21 12Μ091體 1麵 翻.1811 1誦
RBC M U\M 6 35 6.30±0.« 6¾0.¾ . 5I 讓 薩 議 隱 6扁 編.47
HGB g 121±62 117±5.1 119±6.1 121±3.9 "Μ7 漏.6 12 & 5.1 翻 翻 126±5.3
HCT % 画 36M3 35.W.1 誦 34.7±3.6 353±(3 肩 嫩 9 3δ.1 .7 37.7±3.5
PLT 136±10.1« 翻 删 137ί10.6 體 \ 2 i I S mi 141±10.δ 137±10.2 )39±9.5
表 2 項目 扭 :
•4w Id 2d 3d 4d 5d li 2w 4w 12w
ALT L 翻 ■ 42.9+4.1 «.1±4.4 ■ .6 醫 .\ 誦
AST ML 38.5+3.1 4ί1±32 娜 «.1±3.8 0M1 ■ 醫 應 .1 mi
TP m 扁 删 71. .3 腳 73 .1 74.fe6.6 76M.3 .1 5.9 74.4±62
WG 謹 謹 1.¾0.¾ 謹 1. 謹 1M29 l. .36 謂 護 1 19 1.M.26 匪 TO* 3.8±0.39 ■ 42±055 4.1±0. 删 3 續 im 4¾t0.47 3綱 42ί0.56 3. .s
CERA 隱 1 議 ■ ■ 65.3ί72 62 誦 隱 ■
Mi i 翻 湖 142i2.1 139±3.3 1 l 1«±2.1 13 & 3.1
K+ 3 3 5 3.議 3 22 3. .57 ■.87 41±076 "±021 3.7ίΟ.¾ 讓 "ίθ.92 3
CI rat 慰3 105±32 101±28 104+2.6 l l 10δ±1.9 106±22 脑 101±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
表 3 i
项目 雜
Id 2d 3d 6d ½ 12w
IgE lliil 腿.3 脂.2 腿 21M9 19.¾3.1 腿 7 i\
m.\ 8.13±1.9 9.2i2.3 腿 9 mi mm 10.3i1.5 m 腦
1.纏 議 簡 1M7 1·31±0·6 醫 1
\.\U2 i.i .3 \M2 翻 議 1 1 讀 1.M.3 1.1fe0.3 Ι.ΙΙίΟ.1 翻 2 121±02
此外, 根据本实施例的另一次试验结果见图 8 , 其中详细描绘了 PDLSCs介导的牙周组织再生, 从图中显示的结果表明, 与本实施例中 上面的试验结果一致。
总之, 本发明表明, PDLSCs 表达 HLA- I , 不表达 HLA- Il DR CD80 和 CD86, 也不会引起同种异体 T淋巴细胞增殖, 提示 PDLSCs 具有低免疫原性; PDLSCs能够抑制丝裂原和同种异体抗原引起的 T淋巴 细胞增殖; PDLSCs不会引起 T淋巴细胞的凋亡, T淋巴细胞再次受到刺 激时可以恢复增殖能力; PDLSCs通过分泌 PGE2来发挥其免疫抑制功能; 同种异体 PDLSCs 可以修复小型猪牙周炎骨缺损模型, 且不会引起免疫 排斥反应。 实施例 3: 牙齿相关间充质干细胞介导的生物牙根再生
1) 材料和方法
1-1) 种子细胞的分离、 培养
根尖牙***干细胞: 麻醉下无菌拔除小型猪尖牙, 切取根尖部分根尖 牙***, 用 D-Hank's液分别反复清洗, 剪碎, 置于含 I型胶原酶 (3 g/L) 和 Dispase (4 g/L)的消化液, 37 °C下消化 1 h, 过 70 μηι滤网收集细胞, 1000 r/min离心 10 min,用培养液重新悬浮成单细胞悬液。按 0. 01-lxlO5/ 孔分别将根尖牙***细胞及牙周膜细胞接种于 6孔板中 ,在 α-ΜΕΜ培养 基 (含 15%胎牛血清, 2 mmol/L谷氛酰胺, 100 U/ ml青霉素, 100 pg/ ml
链霉素 )37 °C、 5% C02培养, 每 2 ~ 3天换液 1次。 达 80 %融合时传代培 养。
牙髓千细胞: 麻醉下无菌拔除小型猪尖牙, 劈开牙齿取牙髓组织, 用 D-Hank's 液分别反复清洗, 剪碎, 消化, 培养, 具体步骤同根尖牙*** 细胞。
牙周膜干细胞:麻醉下无菌拔除小型猪尖牙,轻轻剥离其周围的牙周 组织, 取中段的牙周组织, 用 D-Hank's液分别反复清洗, 剪碎, 消化, 培养, 具体步骤同根尖牙***细胞。
1-2) 牙周膜细胞膜片制备
将生长旺盛的 2xl05第二或第三代牙周膜干细胞接种在 60mm培养 皿, 培养成分为 α-ΜΕΜ培养基 (含 15%胎牛血清, 100 μιηοΙ/L的 L-抗坏 血酸 2-鴒酸, 2 mmol/L谷氨酰胺, 100 U/ ml青霉素, 100 pg/ ml链霉素)。 培养 10一14天, 培养 JUL边缘细胞出现皱褶, 用较钝刀片或细胞刮子将细 胞膜片整体揭下来, 过程中细胞膜片不要干燥。
1-3) 支架材料
将羟基磷灰石 /碑酸三钙支架材料做成类似牙根的外形, 尺寸为: 直 径 5 mm, 长度 15 mm的圓锥体。 羟基碑灰石 /罅酸三钙为多孔网状结构, 孔径多为 200 ~ 500 μιη, 牙髓干细胞复合在羟基磷灰石 /磷酸三钙三维支 架上, 在生物反应器内生长第 5天的牙髓干细胞, 细胞充分伸展, 细胞表 面的突起和分泌颗粒很多,, 连接成一片, 直径多在 20-50 μιη。
1-4) 回植前细胞复合
将 lxlO8培养至第 3代的根尖牙***干细 悬于培养基, 将牙根型支 架材料置于含细胞的培养基,在 37°C摇床上充分混合 2 h,将支架材料小 心夹出, 放置于培养 中, 将剩余的细胞悬液小心滴加于支架材料上, 静 置 4 h后加培养液, 在生物反应器内培养 5d后回植。
1-5) 回植方法
分别选取小型猪的缺牙区。 切开黏膜, 翻开黏骨膜用瓣, 暴露牙槽嵴 顶。 用种植机以 800转 /分钟的速度在牙槽骨内钻一个与羟基碑灰石 /磷酸 三钙支架材料形状相似的孔, 将上述根尖牙***干细胞 /牙髓干细胞与牙 根形羟基磷灰石 /碑酸三钙混合后在生物反应器内培养 5~7天, 牙周膜干 细胞膜片包绕羟基磷灰石 /鱗酸三钙表面, 回植于小型猪牙槽骨内。
2 ) 结果和讨论
牙周膜细胞片应用于牙周组织工程已经有一段时间的历史,但常规采 用 Recell 温度响应培养亚, 培养 的表面使用了一种温度敏感的疏水材 料 PIPA-Am。 当温度高于 32。C时, 表面具有疏水性, 适合细胞的附着和 生长, 当温度降低, 聚合体变得亲水及膨胀, 自然释放细胞。 收获细胞只 需将温度降到 20。C即可, 无需酶消化和处理, 可以保留细胞表面功能和 活性。 但培养亚需要特殊的材料 PIPA-Am, 因此我们摸索出了应用普通 的培养 JUL制备牙周膜细胞膜片的方法, 培养基成分中加入 100 μπιοΙ/L的 L-抗坏血酸 2-磷酸,可以促进细胞快速增殖,并能分泌大量细胞外基质胶 原成分, 将所有细胞连接在一起, 生长到一定程度后, 我们将整个细胞膜 片完整揭下来。 细孢膜片不破坏细胞外基质连接形成的支架, 不破坏细胞 表面蛋白的粘附增殖及分化等功能,可促进回植体内后形成牙周组织。正 常牙周膜特别薄约 0.2mm, 如果釆用支架材料, 相对较厚, 材料降解后 遗留的空隙是个问题, 牙周膜片避免了此问题。
由于重力及营养供应的因素,对细胞支架复合物进行静止性三维培养 的过程中,发现细胞多聚集到支架材料的底部和表面,材料内部黏附的细 胞数量很少。 培养过程中, 静止培养还限制了细胞生长的深度, 而牙髓干 细胞在支架材料内部均匀分布, 才能形成均匀一致的组织。 想要让细胞能 在支架材料内部也能很好的生长,营养物质能够有效的在支架内部传输和 及时的排出细胞代谢废物很重要。生物反应器虽然还不能模拟体内循环系 统的物质交换机制,但可在支架材料内部产生流体力学环境, 如果控制好 力学强度的大小, 既可减少对细胞的损伤作用, 又可为细胞的生长提供有 利的力学环境。本实验中细胞复合到材料后在生物反应器内培养 5天, 生 物反应器动力性三维培养促进了支架内部营养物质的交换,促进了种子细 胞在支架材料中的均匀分布,有利于细胞保持成骨表型及促进细胞外基盾 在支架材料中的沉积。
回植 6 ~ 9月后,回植的生物牙根在 X线片表现为不透射的高密度影, 周围有低密度影像环绕。组织学上,再生的生物牙根组织形态与新生的骨 组织完全不同,是由许多处于不同生长阶段的球形硬组织团块组成,球形 团块相互连接成网状。新生的球形硬组织体积较小, 外面围绕着疏松排列 呈蓝紫色的细胞,推测可能是成牙本质细胞或成牙骨质细胞,中央为均匀、 粉染的基质。 较成熟的球形硬组织团块体积较大, 中间基本不含细胞, 或 可见极少量被包埋于其中的细胞碎片。 团块中有类牙本质样的结构, 其中
可见排列不规则的牙本质小管样结构,也有类似牙骨质样的结构,镜下表 现为较均匀一致的硬组织。 新形成的硬组织区域外为纤维结締组织包绕, 未见炎细胞浸润, 部分区域内还可见类似 Sharp's纤维样的结构***正在 形成的类牙骨质和牙本质样结构中。
由上述公开的技术方案可见,本发明对于利用生物牙根再生修复牙齿 缺失有十分重要和深远的意义: 首先, 本发明提出了基于組织工程技术的 生物牙根再生的新理念。其次,本发明将生物牙根再生的新理念在大型动 物小型猪上实施, 获得成功, 证明该发明方法可行, 有望成为一种新的修 复方式应用于临床。 第三, 本发明提供的具体实施方法***、 可靠, 为进 行生物牙根再生的深入研究及商品化提供理论依据和技术支持。
在本实施例方法中,提供的一个实验结果见图 9所示,其中详细描绘 了在小型猪上成功再生生物牙根的情况。 其中, A: 实验流程图; (B,C ): 形成了牙本质牙骨质样结构及牙周膜样结构( HE染色); D:牙周膜 COL I 染色阳性; E: 牙本质牙骨质样结构 Dsp染色阳性; F ~ I: 再生生物牙根 扫描电镜(SEM )观察: F: HA/TCP材料呈多孔隙结构 ( 100-400um ); H: 新形成的牙周膜纤维***新形成的牙本质牙骨质结构及牙槽骨内 (G 图箭头所指); I: 新形成的牙本质样结构 (G图粗箭头所指)。
在本实施例方法中, 提供的另一个实验结果见图 10所示, 与图 9类 似, 图 10中详细描绘了在小型猪上进行生物牙根再生获得成功的情况。 实施例 4: 脱落乳牙牙髓干细胞移植治疗***性红斑狼疮 (SLE) 1) 材料和方法
1-1) 人 SHED
选取拔除的 6-8岁的健康患者的正常乳牙, 按照以往文献报道的方 法分离和培养 SHED (3)。人体组织的利用得到首都医科大学伦理委员会 的批准。
1-2) 人骨髓基质干细胞和外周血单个核细胞
应用梯度密度离心法分离健康供者的骨髓基质干细胞(bone marrow mesenchymal stem cells , BMMSCs)和夕卜周血单个核细胞 (peripheral blood mononuclear cells? PBMCs)。
1-3) SHED治疗***性红斑狼疮 (SLE)
C57BL/6J and C3MRL-Fas,pr/J (MRL/lpr)小鼠(雌性, 6-7 周龄),
Beige nude/nude Xid (III) 小鼠 (雌性,8-12 周龄)由提供。 本实验通过首 都医科大学伦理委员会批准。实验分为以下 3个组: 1、空白对照组 (n=3), 不做任何治疗。 2、 移植 BMMSCs 组 (n=3)。 3、 移植 SHED 组 (n=3)。 在全麻下,将 SHED或 BMMSCs (lxl05cells/10g 体重, 细胞悬浮于 100 ml PBS)通过尾静脉注射入 16周龄 MRL/lpr小鼠,对照组注射生理盐水。 在小鼠 20周龄时将其处死, 取外周血、 肾脏、 长骨 (股骨和胫骨)标本。 肾脏标本固定、 石蜡包埋, 行 HE、 trichrome, periodic acid-schiff(pas) 染色, 观察肾小球基底膜功能异常恢复及肾小球膜细胞过度增长恢复情 况。 通过 ELISA检测血清 dsDNA-IgG, dsDNA-IgM, ANA水平, 检测 尿中和血清中补体 3 (complement 3, C3)、 肌酸酐 (creatinine) 尿蛋白 (urine protein)水平。
2)结果和说明
SLE是以多脏器受累和血中存在多种抗体为特征的自身免疫病, 许 多研究证实 T、 Β 淋巴细胞过度活化是 SLE发病中的关键环节。 T、 Β 淋巴细胞来源于淋巴干 /祖细胞, 而 MSCs 在淋巴干 /祖细胞增殖分化中 具有重要的作用, 所以 MSCs 在 SLE 致病中的作用越来越受到关注。
SLE患者 MSCs存在异常, 它分化的骨髓基质细胞不能较好地支持淋巴 细胞生长, 淋巴细胞的生长依赖于淋巴细胞本身和骨髓基质细胞分泌的 各种细胞因子, 骨髓基质细胞的异常参与了淋巴细胞的异常活化。
Majumdar等研究表明 MSCs表达大量的细胞黏附分子,在细胞间黏附、 归巢、 支持造血、 调节免疫细胞功能中起重要作用。 当把肿瘤细胞种植 人异体小鼠时,肿瘤细胞会被宿主免疫***清除,而当肿瘤细胞和 MSCs 同时注入异体小鼠后, 肿瘤细胞可在受体内存活, 表明 MSCs在体内具 有免疫抑制功能。 MSCs在分化成其他细胞类型时仍保留其免疫调节作 用, 这就意味着移植的 MSCs可发挥长期的免疫调节作用。
体外实验证实 SHED具有 MSCs生物学及免疫学特性, 可以与 T 淋巴细胞相互影响。 与 BMMSCs 相比, SHED 通过恢复外周血 Tregs/Thl7 比例, 减少 Thl7 细胞数目发挥免疫学效应。 16 周龄的 MRL/lpr小鼠注射 SHED后, 可以发现小梁骨再生, 破骨细胞活性受到 了抑制。 注射 SHED的小鼠与注射 BMMSCs的小鼠一样, 重建了成骨 细胞微环境, 从而改善了其功能紊乱。 MRL/lpr小鼠注射 SHED后, 虽 说 Treg水平未提高, 但 Treg/Thl7细胞比例明显增高, 这表明 SHED
的免疫调节功能可能源于 Treg抑制自身免疫而 Thl7则可促进自身免疫 和炎症。
在本实施例方法中, 提供的一个实验结果见图 11所示, 其中详细描 绘了脱落乳牙牙髓干细胞治疗***性红斑狼疮鼠血清及肾脏组织学改变 的情况。 从图中结果可见本实施例实现了本发明的目的。
总之, 本发明表明, 从脱落的乳牙牙髓可提取出 SHED, 它是来源 于中胚层的间充质干细胞, 具有 MSCs的生物学和免疫学特性及功能。 尽管 SHED的免疫调节作用机制尚未明确, 但由于它的免疫调节作用, 在自身免疫病方面有潜在的治疗作用, SHED移植可作为治疗自身免疫 病的新尝试。 实施例 5: 同种异体人牙周膜干细胞 OiPDLSCs)用于治疗牙周病的 标准操作实例
本实施例参考图 12来说明同种异体人牙周膜干细胞 (hPDLSCs)用于 治疗牙周病的标准操作的一个实例:
(a)采集人牙周膜 (periodontal ligament, PDL)。 从 18-28岁患者采集 正常的阻生笫三磨牙(impacted third molar)。 将 PDL轻轻地从牙表面分 离。
(b)培养 hPDLSCs, 分离 hPDLSCs。 原始培养 15天后, 来自一个阻 生第三磨牙的 hPDLSCs的数量约为 4.90±0.34 xl05 (P0; n=10). 再培养另 外 15天后, hPDLSCs的数量 (P3)增加到 8.86±0.46xl06 (n=10), hPDLSCs 对 CD146和 CD90显阳性。
(c) 冷冻保存 hPDLSCs, 将第三代 hPDLSCs用 10% DMSO和 90% FBS冷冻保存, 并保存在液氮中。
(d)融化 hPDLSCs。 融化之后, 对 hPDLSCs检查支原体、 细菌、 集 落形成效率、 间质干细胞标记模式和核型分析。
(e)制备 hPDLSCs片。 在 100 mm培养皿中对三种不同细胞数 (lxlO5, lxlO6和 2xl06; n=3)培养 12-15天, 在 lxlO6和 2xl06组中形成细胞片, 但在 lxlO5组中未形成细胞片。为此,将 lxlO6的 hPDLSCs接种到 100 mm 培养 jni中, 达 15天。
(f)然后, 将 40 mg HA/TCP置于这些培养亚中。
(g) hPDLSCs片与 HA/TCP的全图。
(h)口腔牙周损伤图。
在牙周初始治疗(i)之后, 将两个同种异体 hPDLSCs 片与 HA/TCP 植入到 3mmx5mmx7mm的牙周损伤缺陷处 (j)。
(k)接下来的项目包括临床和放射照相评价、 血液学和免疫学评价。 参考文献:
1. Gronthps, S., M.Mankani, J.Brahim, P*G,,Robey, and S.Shi.2000. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 97: 13625-13630.
2. Miura, Μ·, S.Gronthos, M, Zhao, B.Lu, L.W.Fisher, P.G.Robey, S.Shi. 2003. SHED:
Stem cells from human exfoliated deciduous teeth. Proc. Natl. Acad. Sci. UiSA. 100 :5807-5812, 3 . Seo, Β·Μ,, M.Miura, S.Gronthos, P.M.Bartold, S.Batouli, J.Brahim, M.Young,
P.C.Robey, C.Y. Wang, and S.Shi. 2004, Multipotent postnatal stem cells from human periodontal ligament. Lancet.364: 149-155.
4. Sonoyama, W,, Y.Liu, D.Fang., T.Yamaza, B.M.Seo, C.Zhang, H.Liu, S.Gronthos,
C.Y.Wang, S.Wang and S.Shi. 2006. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE.l: 79-92.
5. Le Blanc, Κ·, C.Tammik, K.Rosendahl, E.Zetterberg, and O.Ringden.2003. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp. Hematol,31:890-896.
6 . Di Nicola, Μ·, C.Carlo-Stella, M.Magni, M.Milanesi, P.D.Longoni, P.Matteucci, S.Grisanti, and A.M.GiannL2002. Human bone marrow stromal cells suppress T-Iymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood .99:3838-3843.
7 . Djouad, F., P.Plence, C.Bony, P.Tropel, F.Apparailly, J.Sany, D.Noel, and C.Jorgensen.2003. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood .102:3837-3844.
8. Tse, W.T., J.D.Pendleton, W.M.Beyer, M.C.Egalka, and E.C.Guinan. 2003. Suppression of allogeneic T-cell proliferation by human marrow stromal ceIIs:implications in transplantation. Transplantation .75:389-397.
9. Lazarus H, Curtin P, Devine S, et al. Role of mesenchymal stem cells(MSC) in allogeneic transplantation: early phase 1 clinical results[abstract]. Blood 2000;392:1691.
10. Le Blanc, K" I.Rasmusson, B.Sundberg, C.G. others trom, M.Hassan, M.Uzunel, and O.Ringden. 2004. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet .363: 1439-1441.
11 . Pierdomenico, L., L.Bonsi, M.Calvitti, D.Rondelli, M.Arpinati, G.Chirumbolo,
E.Becchetti, C.Marchionni, F.AIviano, V.Fossati, N.Staffolam, M.Franchina, A.Grossi, and G.P.Bagnara.2005. Multipotent mesenchymal stem cells with immunosuppressive activity can
be easily isolated from dental pulp. Transplantation.80:836-842
12. Kinane, D,F,, G.J.Marshall. 2001. Periodontal manifestations of systemic disease. Aust Dent J. 46:2-12.
13. Liu, Υ·, Y.Zheng, G.Ding, D.Fang, C.Zhang, P.M.Bartold, S.Gronthos, S.Shi, and S.Wang. 2008. Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem cells. 26: 1065-1073.
14. Seo, Β·Μ·, M,Miura, W.Sonoyama, C.Coppe, R.Stanyan, and S.Shi. 2005. Recovery of stem cells from cryopreserved periodontal ligament. J. Dent. Res. 84:907-912.
15. Muller, L, S.Kordowich, C.Holzwarth, G.Isensee, P.Lang, F.Neunhoeffer, M.Dominici, J.Greil, and R.Handgretinger. 2008. Application of multipotent mesenchymal stromal cells in pediatric patients following allogeneic stem cell transplantation. Blood. Cells. MoL Dis.40:25-32.
16 . Ringden, 0·, M.Uzunel, LRasmusson, M.Remberger, B.Sundberg, H.Lonnies, H.U.Marschall, A.Dlugosz, A.Szakos, Z.Hassan, B.Omazic, J.Aschan, L.Barkholt, and K.Le Blanc. 2006. Mesenchymal stem cell for treatment of therapy-resistant graft-versus-host disease. Transplantation. 81: 1390-1397.
17. Uccelli,A,, V.Pistoia, L.Moretta.2007. Mesenchymal stem cells:a new strategy for immunosuppression? Trends. Immunol 28:219-226.
18. Giordano, A., U.Galderisi, I.R.Marino. 2007. From the laboratory bench to the patient,bedside:an update on clinical trials with mesenchymal stem cells. J. Cell.
PhysioI.211:27-35.
19. Krampera, Μ·, S.GIennie, J.Dyson, D.Scott, R.Laylor, E.Simpson, and F.Dazzi. 2003..Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood.101 :3722-3729.
20. Maccario, R., M.Podesta, A.Moretta, A.Cometa, P.Comoli, D.Montagna, L.Daudt,
A.Ibaticia, G.Piaggio, S.Pozzi, F.Frassoni, and F.Locatelli. 2005. Interaction of human mesenchymal stem cells with cells involved in alloantigen-speciHc immune response favors the differentiation of CD4+ T- cell subsets expressing a regulatory/suppressive phenotype. Haematologica .90:516-525.
21 , Corcione,A., F.Benvenuto, E.Ferretti, D.Giunti, V.Cappiello, F.Cazzanti, M.Risso,
F.Gualandi, G.L.Mancardi, V.Pistoia, and A.Uccelli.2006. Human mesenchymal stem cells modulate B-cell functions. Blood .107:367-372.
22. Sotiropoulou, Ρ·Α·, S.A.Perez, A.D.Gritzapis, C.N.Baxevanis, and M.Papamichail. 2006. Interactions between human mesenchymal stem cells and natural killer cells. Stem. Cells,24:74-85,
23. Devine, S,M., and R.Hoffman.2000.Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr. Opin, Hematol.7:358-363.
24. Aggarwal, S., and M.F.Pittinger.2005. Human mesenchymal stem cells modulate
allogeneic immune cell responses.Blood.105: 1815-1822.
25. Le Blanc, K" and O.Ringden.2005. Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation. Biol. Blood. Marrow. Transplant.ll:321-334.
26. Le Blanc, K" and O.Ringden.2006. Mesenchymal stem cells:properties and role in clinical bone marrow transplantation. Curr. Opin. Immunol.l8:586-591.
27. Sato K, Ozaki K, Oh I, et al. 2007, Nitric oxide plays a critical role in suppression of T cell proliferation by mesenchymal stem cells, Blood;109:228-234.
28. Beyth S, Borovsky Z, Mevorach D, et al. 2005, Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T- cell unresponsiveness.Blood;105: 2214-2219.
Claims
1、 牙齿相关干细胞在制备用于预防或治疗与牙齿相关的疾病或病情 的产品中的用途,或者在制备用于牙齿相关组织形成或修复的产品中的用 途。
2、 根据权利要求 1的用途, 其中所述的牙齿相关干细胞选自: 牙髓 干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙***干细胞。
3、根据权利要求 1或 2的用途, 其中所述的牙齿相关干细胞来自哺 乳动物,例如所述的牙齿相关干细胞来自选自以下的哺乳动物:人、猪 (例 如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵 羊、 山羊。
4、根据权利要求 1至 3任一项的用途,其中所述的与牙齿相关的疾 病或病情或者牙齿相关组织形成或修复选自: 牙周病、 牙周炎、 牙齿缺 损、 牙齿组织缺损、 牙齿缺损修复、 牙齿相关组织缺损和修复、 牙齿相 关组织替代物再生、 等等。
5、 牙齿相关干细胞在制备用于预防或治疗免疫性疾病或病情、 自身 免疫性疾病或病情、 与 T淋巴细胞异常活化有关的疾病或病情、 与 T淋 巴细胞异常升高有关的疾病或病情、 红斑狼疮、 或者***性红斑狼疮的 产品中的用途。
6、 根据权利要求 5的用途, 其中所述的牙齿相关干细胞选自: 牙髓 干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙***干细胞。
7、 预防或治疗与牙齿相关的疾病或病情的方法, 或者形成或修复牙 齿相关组织的方法, 该方法包括给有需要预防或治疗与牙齿相关的疾病 或病情的宿主施用有效量的牙齿相关干细胞,或者该方法包括给有需要形 成或修复牙齿相关组织的宿主施用有效量的牙齿相关干细胞。
8、 根据权利要求 7的方法, 其中所述的牙齿相关干细胞选自: 牙髓 干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙***干细胞。
9、根据权利要求 7或 8的方法, 其中所述的牙齿相关干细胞来自哺 乳动物,例如所述的牙齿相关干细胞来自选自以下的哺乳动物:人、猪 (例 如五指山小型猪、 贵州香猪)、 牛、 马、 猴、 大鼠、 小鼠、 豚鼠、 羊、 绵
羊、 山羊。
10、 根据权利要求 7至 9任一项的方法, 其中所迷的与牙齿相关的 疾病或病情或者牙齿相关组织形成或修复选自: 牙周病、 牙周炎、 牙齿 缺损、 牙齿组织缺损、 牙齿缺损修复、 牙齿相关组织缺损和修复、 牙齿 相关组织替代物再生、 等等。
11、 预防或治疗免疫性疾病或病情、 自身免疫性疾病或病情、 与 T 淋巴细胞异常活化有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾 病或病情、 红斑狼疮、 或者***性红斑狼疮的方法, 该方法包括给有此 需要的宿主施用有效量的牙齿相关干细胞。
12、 根据权利要求 12的方法, 其中所述的牙齿相关干细胞选自: 牙 髓干细胞、脱落乳牙牙髓干细胞、 牙周膜干细胞、和根尖牙***干细胞。
13、 一种组合物, 其包含有效量的牙齿相关干细胞和任选的药学可 接受的载体。
14、 根据权利要求 13的组合物, 其中所述的牙齿相关干细胞选自: 牙髓干细胞、 脱落乳牙牙髓干细胞、 牙周膜干细胞、 和根尖牙***干细 胞。
15、 根据权利要求 13或 14的組合物, 其是用于预防或治疗与牙齿 相关的疾病或病情,或者用于牙齿相关组织形成或修复,或者用于预防或 治疗免疫性疾病或病情、 自身免疫性疾病或病情、 与 T淋巴细胞异常活 化有关的疾病或病情、 与 T淋巴细胞异常升高有关的疾病或病情、 红斑 狼疮、 或者***性红斑狼疮。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102755667A (zh) * | 2012-07-05 | 2012-10-31 | 中国人民解放军第四军医大学 | 一种组织工程化人牙根植入材料的制备方法及其用途 |
CN102807967A (zh) * | 2012-08-31 | 2012-12-05 | 沙船(天津)生物科技发展有限公司 | 乳牙牙髓在制备间充质干细胞中的应用和乳牙牙髓间充质干细胞的培养方法 |
WO2015110082A1 (zh) * | 2014-01-27 | 2015-07-30 | 首都医科大学附属北京口腔医院 | 牙源性干细胞和基因修饰的牙源性干细胞的用途 |
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Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104232568B (zh) * | 2014-08-29 | 2016-07-20 | 丁刚 | 一种牙周膜干细胞的骨向诱导方法 |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004094588A2 (en) * | 2003-04-19 | 2004-11-04 | Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health | Postnatal stem cells and uses thereof |
CN1587393A (zh) * | 2004-07-15 | 2005-03-02 | 福建师范大学 | 利用角朊细胞干细胞和牙髓干细胞进行牙齿再生的技术 |
WO2005052140A2 (en) * | 2003-11-20 | 2005-06-09 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Multipotent postnatal stem cells from human periodontal ligament and uses thereof |
CN1896228A (zh) * | 2005-07-12 | 2007-01-17 | 叶庆良 | 一种牙齿干细胞及其制备方法 |
WO2008067488A2 (en) * | 2006-11-29 | 2008-06-05 | University Of Southern California | Mesenchymal stem cell-mediated functional tooth regeneration |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101144069A (zh) * | 2007-08-22 | 2008-03-19 | 黄显成 | 从口腔组织中培养间质干细胞的方法 |
-
2010
- 2010-01-22 CN CN2010800052665A patent/CN102292435B/zh not_active Expired - Fee Related
- 2010-01-22 WO PCT/CN2010/000104 patent/WO2010083730A1/zh active Application Filing
- 2010-01-22 CN CN2013102733426A patent/CN103432007A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004094588A2 (en) * | 2003-04-19 | 2004-11-04 | Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health | Postnatal stem cells and uses thereof |
WO2005052140A2 (en) * | 2003-11-20 | 2005-06-09 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Multipotent postnatal stem cells from human periodontal ligament and uses thereof |
CN1587393A (zh) * | 2004-07-15 | 2005-03-02 | 福建师范大学 | 利用角朊细胞干细胞和牙髓干细胞进行牙齿再生的技术 |
CN1896228A (zh) * | 2005-07-12 | 2007-01-17 | 叶庆良 | 一种牙齿干细胞及其制备方法 |
WO2008067488A2 (en) * | 2006-11-29 | 2008-06-05 | University Of Southern California | Mesenchymal stem cell-mediated functional tooth regeneration |
Non-Patent Citations (4)
Title |
---|
ANTONIO UCCELLI ET AL.: "Mesenchymal stem cells: a new strategy for immunosuppression?", TRENDS IN IMMUNOLOGY, vol. 28, no. 5, 31 May 2007 (2007-05-31), pages 219 - 226 * |
GAO, QING ET AL.: "Stem cells in periodontal ligament", JOURNAL OF CLINICAL STOMATOLOGY, vol. 24, no. 2, 29 February 2008 (2008-02-29), pages 121 - 122 * |
JIANG, MING ET AL.: "Comparison of odontogenic ability between dental pulp stem cells and dental papillae mesenchymal cells", JOURNAL OF PRACTICAL STOMATOLOGY, vol. 23, no. 3, 31 May 2007 (2007-05-31), pages 418 - 422 * |
MASAKO MIURA ET AL.: "SHED: stem cells from human exfoliated deciduous teeth", PNAS, vol. 100, no. 10, 13 May 2003 (2003-05-13), pages 5807 - 5812 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102755667A (zh) * | 2012-07-05 | 2012-10-31 | 中国人民解放军第四军医大学 | 一种组织工程化人牙根植入材料的制备方法及其用途 |
CN102755667B (zh) * | 2012-07-05 | 2014-06-25 | 中国人民解放军第四军医大学 | 一种组织工程化人牙根植入材料的制备方法及其用途 |
CN102807967A (zh) * | 2012-08-31 | 2012-12-05 | 沙船(天津)生物科技发展有限公司 | 乳牙牙髓在制备间充质干细胞中的应用和乳牙牙髓间充质干细胞的培养方法 |
WO2015110082A1 (zh) * | 2014-01-27 | 2015-07-30 | 首都医科大学附属北京口腔医院 | 牙源性干细胞和基因修饰的牙源性干细胞的用途 |
CN106062184A (zh) * | 2014-01-27 | 2016-10-26 | 首都医科大学附属北京口腔医院 | 牙源性干细胞和基因修饰的牙源性干细胞的用途 |
US20160348076A1 (en) * | 2014-01-27 | 2016-12-01 | Beijing Stomatology Hospital, Capital Medical University | The Usage of Odontogenic Stem Cells and Genetically Modified Odontogenic Stem Cells |
US10000738B2 (en) * | 2014-01-27 | 2018-06-19 | Beijing Stomatology Hospital, Capital Medical University | Usage of odontogenic stem cells and genetically modified odontogenic stem cells |
CN106062184B (zh) * | 2014-01-27 | 2019-10-25 | 首都医科大学附属北京口腔医院 | 牙源性干细胞和基因修饰的牙源性干细胞的用途 |
CN112481364A (zh) * | 2020-12-07 | 2021-03-12 | 上海交通大学医学院附属第九人民医院 | 基于单细胞测序筛选牙周膜特异性干细胞新亚群的方法及其应用 |
CN112481364B (zh) * | 2020-12-07 | 2023-03-24 | 上海交通大学医学院附属第九人民医院 | 基于单细胞测序筛选牙周膜特异性干细胞新亚群的方法及其应用 |
CN113755481A (zh) * | 2021-08-31 | 2021-12-07 | 上海市口腔医院(上海市口腔健康中心) | 一种无支架牙周膜干细胞球及其制备方法与应用 |
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