WO2021012718A1 - 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用 - Google Patents

用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用 Download PDF

Info

Publication number
WO2021012718A1
WO2021012718A1 PCT/CN2020/084109 CN2020084109W WO2021012718A1 WO 2021012718 A1 WO2021012718 A1 WO 2021012718A1 CN 2020084109 W CN2020084109 W CN 2020084109W WO 2021012718 A1 WO2021012718 A1 WO 2021012718A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdpscs
cells
culture
dental
pluripotent stem
Prior art date
Application number
PCT/CN2020/084109
Other languages
English (en)
French (fr)
Inventor
李中瀚
陈红
Original Assignee
四川大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川大学 filed Critical 四川大学
Publication of WO2021012718A1 publication Critical patent/WO2021012718A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2513/00—3D culture

Definitions

  • the invention belongs to the technical field of tissue engineering, in particular to a kind of pluripotent stem cell MDPSCs used for dental pulp regeneration and a method and application for separation and culture thereof.
  • Teeth are important organs responsible for human chewing, pronunciation and facial aesthetics, and the maintenance of their functions mainly depends on the pulp.
  • Dental pulp can induce dentin formation, maintain nutrient supply, and provide teeth with sensory nerve networks that respond to external stimuli. Inflammation and necrosis of dental pulp and periapical periodontitis are common and frequently-occurring diseases in oral clinic.
  • Root canal therapy is the most commonly used effective treatment method for such diseases in the world. The principle is to use mechanical and chemical methods. Remove the inflammatory and necrotic pulp tissue in the root canal, and prevent the recurrence of infection through drug disinfection and root canal filling. Root canal treatment requires removal of the dental pulp tissue of the affected tooth in order to preserve the entire tooth or root.
  • stem cells With the development of stem cells and regenerative medicine, cell therapy based on stem cells has attracted more and more attention. How to use stem cells to regenerate functional pulp instead of traditional root canal therapy has become a new trend in the treatment of pulpitis and pulp necrosis.
  • Current research shows that a variety of mesenchymal stem cell populations isolated from tooth tissues (such as SCAP, SHED, DPSCs and DFCs, etc.) have shown varying degrees of pulp-dentin regeneration potential, but there are batches of regeneration efficiency. Large differences, the lack of clear molecular markers to evaluate cell pluripotency, and the large heterogeneity of mesenchymal cells themselves that make quality control difficult and other defects have caused these cell products to be promoted in the market mainly in the form of storage services. It is widely used in clinical treatment.
  • Another defect of the existing dental stem cells is that their culture system relies on the traditional two-dimensional culture method, that is, the cell population is fed in a culture flask as a monolayer of cells, and is subcultured based on the growth rate and density of the cells. Amplification.
  • two-dimensional cell culture has played a very important role in the development of life science research.
  • the hardness of the culture bottle itself is far greater than the body tissue, its biomechanical environment and tissue physiological conditions
  • the maintenance of their pluripotency is highly dependent on the environment. It requires not only matrix materials that meet the physiological environment, but also supportive growth factors.
  • three-dimensional (3D) culture can simulate the natural microenvironment of stem cells in the body, maintain the pluripotent state of the cells, and enrich cells with strong self-renewal ability effect.
  • Previous studies on the in vitro 3D culture of adult stem cells mainly focused on stem cell populations derived from epithelial cells, such as breast epithelial stem cells, intestinal epithelial stem cells, etc., while there are few reports on mesenchymal stem cells.
  • epithelial cells such as breast epithelial stem cells, intestinal epithelial stem cells, etc.
  • mesenchymal stem cells The discovery of Muse cells is currently reported evidence A relatively sufficient three-dimensional culture system for mesenchymal cells.
  • the purpose of the present invention is to make up for the shortcomings of traditional root canal treatment technology, propose a pluripotent stem cell MDPSCs, clearly separate and culture methods, and define its characteristic surface markers, in order to obtain a large number of in vitro expansion applications to achieve regeneration of teeth Pulp, the purpose of preserving the vital pulp of the affected tooth.
  • a pluripotent stem cell MDPSCs used for dental pulp regeneration are Cd24a positive cells isolated from dental mesenchymal tissue.
  • the dental mesenchymal tissue is preferably dental papilla, dental pulp and the like.
  • the MDPSCs exhibit self-renewal ability under 3D culture conditions, forming cell pellets that positively express pluripotency-related marker genes Sox2, Nanog, Oct4 and Ki67.
  • a method for separating and cultivating pluripotent stem cells MDPSCs for pulp regeneration including the following steps:
  • step 1) is:
  • step 1) B the digestion is terminated using ⁇ -MEM medium containing chemical serum substitute or serum.
  • the culture medium is a medium containing 2% by volume of serum or serum substitute and 1% by volume of cell growth factor in a basic medium containing 100U/mL penicillin and 100U/mL streptomycin,
  • the cell growth factor is a mixture of factors such as TGF ⁇ , EGF, BMP-2, BMP-4 and FGF.
  • the digestion is digestion in a 37°C water bath for 20-30 minutes, shaking every 5 minutes; the incubator is 37°C, 5% CO 2 .
  • step 3 is:
  • the 3D culture solution is composed of a mixture of cell culture solution and extracellular matrix pre-preparation in a 3:2 volume ratio.
  • the cell culture medium is a medium in which 2% by volume of serum or serum substitutes and 1% by volume of cell growth factors are added to a basal medium containing 100U/mL penicillin and 100U/mL streptomycin, wherein the cells grow
  • the factor is a mixture of factors such as TGF ⁇ , EGF, BMP-2, BMP-4 and FGF;
  • the extracellular matrix pre-preparation is preferably collagen, methylcellulose.
  • step C the cells are seeded on a 6-well low-adhesion culture plate at a density of 1 ⁇ 10 4 cells/cm 2 ; the specific operation of supplementing fresh culture medium is: supplementing 1/10 every two days The initial volume of fresh culture medium.
  • MDPSCs of pluripotent stem cells for dental pulp regeneration which are used to construct complete tooth germs, enhance the ability of osteogenic/odontogenic differentiation in vivo and in vitro, and promote the application of dental pulp-dentin complex in vivo functional regeneration .
  • the present invention has the following beneficial effects:
  • the invention provides a pluripotent stem cell MDPSCs used for dental pulp regeneration, which is successfully separated and enriched by a 3D culture method, and the characteristic surface markers of MDPSCs are defined in combination with flow cytometry.
  • MDPSCs used for dental pulp regeneration
  • the pluripotent stem cell MDPSCs of the present invention have the ability to form a vascularized pulp-dentin complex, which provides a cellular basis for the study of functional pulp regeneration and clinical transformation.
  • Figure 1 is a schematic diagram of the primary DPCs isolation and culture three-dimensional cell pelleting experiment
  • Figure 2 shows the typical cell morphology of primary DPCs and DPC spheres under light and electron microscopes
  • Figure 3 shows the immunofluorescence and RT-PCR detection of pluripotency and proliferation-related marker genes Oct4, Sox2, Nanog, Ki67 expression; where a is immunofluorescence and b is RT-PCR detection;
  • Figure 4 shows the staining and quantitative analysis of DPC spheres Alizarin Red and alkaline phosphatase after 10 days and 7 days of osteogenic induction fluid;
  • Figure 5 shows the differentiation of DPC spheres into bone-like/dentin-like mineralized structures under the mouse renal capsule
  • Figure 6 is a flow chart of subcutaneous transplantation of DPC spheres composite TDM in nude mice.
  • Figure 7 shows that DPC spheres form a pulp-dentin complex-like structure in the subcutaneous TDM lumen of nude mice.
  • Figure 8 shows the expression of dentin-specific markers DMP1 and DSPP, blood vessel-specific markers VEGF and CD31, and neural tissue-specific markers GFAP, S100 and NF200 in the regenerated tissue of DPC spheres.
  • Figure 9 shows the enriched surface marker genes in DPC spheres.
  • Figure 10 shows the RT-qPCR to verify the accuracy of the transcriptome sequencing results.
  • Figure 11 shows the FACS scatter plot and histogram analysis results obtained by flow cytometry.
  • Figure 12 shows the immunofluorescence detection of CD24a expression in monolayer, spheres and DPC p2 cells.
  • Figure 13 shows FACS sorting Cd24a positive cells in primary dental papilla cells, and 3 subgroups are obtained according to the expression of Cd24a.
  • Figure 14 shows the analysis results of the spheroidization efficiency of each of the three subgroups obtained according to the expression of CD24a.
  • Figure 15 shows a tissue section of developing mouse tooth germs analyzed by immunofluorescence and immunohistochemistry.
  • Example 1 Isolation and culture of pluripotent stem cells from mouse dental papilla
  • the experimental model diagram of the three-dimensional ball formation of dental papilla cells in this embodiment is shown in FIG. 1.
  • the isolation and culture of pluripotent spheroid stem cells from mouse dental papilla specifically includes the following steps:
  • P2 generation of dental papilla cells discard the culture medium, wash once with 1 ⁇ PBS, digest with 1ml 0.25% trypsin for 1-2 minutes; stop the digestion with an equal volume of 10% FBS ⁇ -MEM, centrifuge, remove the supernatant, and regenerate an appropriate amount of culture medium Count after suspension; mix the cell culture medium and methyl cellulose in a volume ratio of 3:2 to prepare 3D cell culture medium; add the counted cells to the 3D cell culture medium by pipetting and mixing, to a concentration of 1 ⁇ 10 4 cells/cm 2 Density is inoculated to each well of the 6-well ultra-low adhesion culture plate; 1/10 of the initial volume of fresh culture medium is added to each well of the 6-well culture plate every two days; the cell formation is observed under the microscope during the culture, until DPC spheres were obtained in 8 days for subsequent experiments.
  • the cell culture medium is a medium containing 2% by volume of serum or serum substitute and 1% by volume of cell growth factor in a basic medium containing 100U/mL penicillin and 100U/mL streptomycin, wherein the cell growth factor is A mixture of factors such as TGF ⁇ , EGF, BMP-2, BMP-4 and FGF.
  • DPCs primary dental papilla cells
  • ii is the DPC spheres of Day 1
  • iii is the DPC spheres of Day 7
  • iv is the scanning electron microscope image of the DPC spheres.
  • primary DPCs show similar mineralization ability under the renal capsule as DPC spheres, primary DPCs cannot form a pulp-dentin complex structure under the induction of TDM, while DPC spheres can.
  • DPC spheres were inoculated in a confocal small dish, and after 30-60 minutes, the cell pellets settled and adhered to the wall, fixed with 4% paraformaldehyde, and immunofluorescence staining was used to identify the pluripotency of DPC spheres.
  • the immunofluorescence staining steps are:
  • the cell pellets were fixed with 4% paraformaldehyde for 1 hour and washed 3 times with PBS;
  • the primary antibodies used in immunofluorescence detection are: rabbit anti-Sox2 (ab97959, Abcam, USA), rabbit anti-Nanog (ab80892, Abcam, USA), rabbit anti-OCT4 (D121072, Sangon Biotech, China), rabbit anti -Ki67 (ab15580, Abcam, USA).
  • the results of immunofluorescence staining are shown in Figure 3. DPC spheres positively express pluripotency-related and proliferation marker genes Sox2, Nanog, Oct4 and Ki67.
  • the DPC spheres in the low adhesion culture plate were collected by centrifugation, and the results of immunofluorescence staining were verified by RT-PCR at 2000 rpm for 5 min.
  • RNA dissolution add 10 ⁇ L RNase-free water with a pipette, and gently pipette to dissolve the precipitate;
  • RNA solution A260/A280 by UV spectrophotometer, and the ratio is between 1.8-2.2 as qualified RNA. Store at -80°C for later use.
  • RT reaction Reverse transcription is carried out in accordance with the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit manual. After mixing, it is centrifuged briefly and placed in a gradient PCR machine.
  • Reaction formula Using a 10ul reaction system, add premixed solutions into the PCR tube: Green mix (Cat#K1082, Thermofisher, USA), 5ul; Forward primer, 0.5ul; Reverse primer, 0.5ul; cDNA, 1ul; H 2 O, 3ul; to a total volume of 10 ⁇ l. The above reaction solution was shaken and mixed, and centrifuged briefly.
  • DPC spheres positively express pluripotency-related and proliferation marker genes Sox2, Nanog, Oct4 and Ki67.
  • Inoculate 2 ⁇ 10 5 cells into each well of a 6-well plate, culture in a 37°C, 5% CO 2 cell incubator; remove the cells when they grow to the logarithmic phase, discard the old culture medium, and rinse 3 times with PBS buffer , Add osteogenic induction liquid and culture for 7 days and 10 days respectively; discard the osteogenic induction liquid and rinse 3 times with PBS; for the 10 days of osteogenic induction group, fix with 4% paraformaldehyde for 30 minutes, wash with PBS to remove residual paraformaldehyde; Add the pre-configured Alizarin Red S (Cat#A5533, pH 4.3, Sigma-Aldrich, USA) to the well plate, stain for 5 min at room temperature, wash 3 times with PBS, observe under the microscope and take the picture, use 10% chlorine Cetylpyridine was dissolved mineralized nodules, and the absorbance was measured at 405nm.
  • ALP staining and ALP activity detection were performed in accordance with the instructions of the ALP coloring kit (Cat#C3206, Beyotime, China) and ALP activity kit (Cat#A059-2, Jiancheng, China).
  • the results are shown in Figure 4, where (a) and (c) are the staining results of Alizarin Red and ALP respectively, and (b) and (d) are the quantitative analysis of the staining results of Alizarin Red and ALP respectively. It can be seen that DPC spheres show stronger mineralization ability and ALP activity under the action of osteoinductive fluid.
  • DPC spheres-derived cells are mixed with Matrigel (Cat#354234, BD, USA) at a cell density of 1.0 ⁇ 10 6 /10 ⁇ l;
  • Samples were taken after 4 weeks, fixed with 4% paraformaldehyde, decalcified with 10% EDTA for 1 week, and paraffin-embedded sections for histological staining analysis.
  • Example 3 DPC spheres promote the functional regeneration of dental pulp-dentin complex in vivo
  • TDM preparation extract the central incisor from the mandible of the pig; carefully scrape the periodontal ligament tissue, grinding away the cementum, pulp tissue and dentin layer; cut the root canal into 2-3 lengths with a high-speed turbine bracelet Millimeter segments; the ultrasonic cleaning machine shakes and cleans the root canal 3 times, each time for 5-6 minutes; the root canal is transferred to a gradient EDTA solution (17% to 5%) for 5 minutes, during which it is rinsed with deionized water for 5 minutes (3 times); TDM is immersed in a mixture of penicillin (50U/ml) and streptomycin (50mg/ml) and stored at 4°C for later use.
  • penicillin 50U/ml
  • streptomycin 50mg/ml
  • DPC spheres-derived cells are mixed with Matrigel (Cat#354234, BD, USA) at a cell density of 1.0 ⁇ 10 6 /10 ⁇ l;
  • the temperature is 21°C, and the photoperiod is 12 hours;
  • Figure 7 shows that DPC spheres regenerated in the subcutaneous TDM lumen of nude mice to form a pulp-dentin complex-like structure similar to that of natural teeth.
  • Figure 8 shows that the dentin-specific markers DMP1 and DSPP, blood vessel-specific markers VEGF and CD31, and neural tissue-specific markers GFAP, S100 and NF200 in the regenerated tissue of DPC spheres are all positive, similar to the expression of natural dental pulp.
  • Reaction system 5 ⁇ l Premix Ex TaqTM II (2x), 0.5 ⁇ l, 10 ⁇ M upstream primer; 0.5 ⁇ l, 10 ⁇ M downstream primer; 1.0 ⁇ l cDNA template; 3 ⁇ l ddH2O to a total volume of 10 ⁇ l.
  • Reaction conditions Polymerase Activation: 95°C, 30s, PCR Cycling (40cycles): 95°C, 15s-60°C, 34s; MeltCurve: 95°C, 15s, 60°C, 15s, 95°C, 15s.
  • Figure 9 shows the enriched surface marker genes in DPC spheres. Fold change is shown in gray, and FPKM value is shown in black. Compare the differential gene expression profiles of DPC spheres and 2D cultured cells, and compare with The surface protein database overlapped (Fold change ⁇ 2), and 15 candidate surface markers were screened. Mark the candidate surface marke for further verification.
  • Figure 10 shows the accuracy of the transcriptome sequencing results verified by RT-qPCR, which confirms the accuracy of the sequencing results.
  • Example 5 CD24a-positive cells in the dental papilla have stronger ball-forming ability
  • the experimental process is as described in Example 2 for immunofluorescence detection.
  • rat anti-CD24 (ab64064, Abcam, USA). The results of immunofluorescence staining are shown in Figure 12: DPC spheres cells are rich in CD24a positive cell population.
  • Example 2 The experimental process was as described in step 3) in Example 1. The density of 5 ⁇ 10 3 cells/cm 2 was inoculated on a 24-well ultra-low adhesion culture plate.
  • tissue freezing medium embeds the dehydrated mandible and slices with a cryostat.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Transplantation (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Vascular Medicine (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供一种用于牙髓再生的多能干细胞MDPSCs及其制备方法。该MDPSCs为从牙间充质组织中分离的CD24a阳性细胞，在3D培养条件下展现自更新能力，形成阳性表达多能相关标志基因Sox2，Nanog，Oct4和Ki67的细胞小球。多能干细胞MDPSCs用3D培养的方法对其成功分离和富集，并结合流式细胞术定义了MDPSCs的特征性表面标记。MDPSCs裸鼠体内移植能够再生牙髓，可替代传统根管治疗，在消除牙髓炎症的前提下保存活髓，具有形成血管化牙髓-牙本质复合体的能力。

Description

用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用 技术领域
本发明属于组织工程技术领域，具体为一种用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用。
背景技术
牙齿是承担人类咀嚼、发音以及维持颜面美学的重要器官，而其功能的维持则主要依靠牙髓。牙髓能够诱导牙本质形成，维持营养供应，并为牙齿提供对外界刺激作出反应的感觉神经网。牙髓炎症和坏死以及根尖周炎是口腔临床的常见病和多发病，根管治疗(root canal therapy)作为国际上针对此类疾病最常用的有效治疗手段，其原理是利用机械和化学方法去除根管内炎症、坏死的牙髓组织，并通过药物消毒和根管充填防止感染再次发生。根管治疗需要摘除患牙牙髓组织以达到保留整个牙齿或牙根的目的，这将导致牙髓的永久失活和功能丧失，同时患牙也会因此失去正常的生理弹性，牙体变脆易蹦碎。此外，根管治疗手术步骤繁琐，一般需要2-4次就诊，经过多个治疗步骤和拍摄多张X光片才能完成，治疗期间还可能出现疼痛等不适症状，病人要承受的痛苦也较大。若治疗失败，消毒不彻底导致牙髓腔再次感染，很容易导致牙齿拔除。
随着干细胞与再生医学的发展，基于干细胞的细胞治疗越来越受到人们的关注。如何利用干细胞再生功能性牙髓替代传统根管治疗术成为治疗牙髓炎和牙髓坏死的新趋势。目前的研究表明，牙齿组织中分离出的多种间充质干细胞群(如SCAP，SHED，DPSCs以及DFCs等)均显示有不同程度的牙髓-牙本质再生潜能，但是其存在再生效率批次差异大，缺乏明确的分子标记对细胞多能性进行评估以及间充质细胞本身存在较大异质性以致品控不易等缺陷，导致这些细胞产品主要以储存服务的形式在市场 上推行，尚未广泛用于临床治疗。
已有的牙源性干细胞的另一个缺陷在于，其培养体系依赖于传统的二维培养方式，即细胞群以单层细胞方式饲养于培养瓶中，并基于细胞的生长速度和密度进行传代和扩增。二维细胞培养作为非常经典的细胞培养方式，在生命科学研究发展过程中起到了非常大的促进作用，但是，由于培养瓶本身材质的硬度远远超过体内组织，其生物力学环境与组织生理条件存在显著差异。就干细胞而言，其多能性的维持对环境的依赖性较大，不仅需要符合生理环境的基质材料，还需要辅助以支持性的生长因子，而这些对生理微环境的模拟在二维培养环境中较难实现。
与传统的二维(2D)贴壁培养技术不同，三维(3D)培养可以模拟干细胞在体内的天然生存微环境，维持细胞的多能状态，并对有较强自更新能力的细胞有富集作用。以往对成体干细胞体外3D培养的研究，主要集中在上皮来源的干细胞群体，例如乳腺上皮干细胞、肠上皮干细胞等，而对间充质来源的干细胞报道较少，Muse细胞的发现是目前报道的证据较为充分的间充质细胞三维培养体系。因此，我们希望利用该系统研究异质性的牙乳头细胞群中是否存在尚未被定义的多能牙髓再生干细胞亚群。尽管3D培养可以对某些干细胞亚群进行富集和纯化，但纯化后的细胞群体仍可能是异质性的。鉴于流式细胞术的广泛应用，细胞表面蛋白在不影响细胞活力的前提下相对更易量化，使得它成为定义不同类型细胞的理想工具。对细胞表面标记物的识别和探索性研究使从异质性细胞群中分离出独特的亚群成为可能。因此，探索从牙源性异质细胞群中富集和分离多能干细胞的专门技术，以进一步应用于牙髓再生，对细胞治疗临床转化和生物医学研究具有广阔的应用前景。
发明内容
本发明的目的在于弥补传统根管治疗技术的缺陷，提出一种多能干细胞 MDPSCs，明确分离和培养方法，并对其特征性表面标志物进行定义，以期获得大量体外扩增应用，达到再生牙髓，保存患牙活髓的目的。
本发明目的通过以下技术方案来实现：
一种用于牙髓再生的多能干细胞MDPSCs，所述MDPSCs为从牙间充质组织中分离的Cd24a阳性细胞。其中，牙间充质组织优选为牙乳头，牙髓等。
进一步，所述MDPSCs在3D培养条件下展现自更新能力，形成阳性表达多能相关标志基因Sox2，Nanog，Oct4和Ki67的细胞小球。
一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，包括以下步骤：
1)采用酶与螯合剂复合消化法进行牙间充质细胞的原代分离培养：
2)当牙间充质细胞密度达到70-90％用酶消化进行传代培养得到P2代牙间充质细胞；
3)将P2代牙间充质细胞接种至培养板上进行3D培养获得呈细胞小球状态的MDPSCs细胞。
进一步，所述步骤1)的具体操作为：
A、分离牙齿完整牙胚，从牙冠钙化边缘下掏出牙间充质组织，冲洗后剪碎成乳糜状；
B、用酶和螯合剂消化液对剪碎后的组织进行消化，消化完成后终止消化；
C、经洗涤、离心、去上清后将培养基重悬细胞后置于原代培养瓶中，放入培养箱中培养；
D、补充培养液至稍稍浸没组织块，并视细胞生长情况适当换液或传代。
步骤1)B中，终止消化采用含有化学血清替代物或血清α-MEM培养基。
步骤1)D中，培养液是在含100U/mL青霉素、100U/mL链霉素的基础培养基中加入2％体积比血清或血清替代物、1％体积比的细胞生长因子的培养基，其中细胞生 长因子为TGFβ、EGF、BMP-2、BMP-4以及FGF等因子的混合物。
进一步，所述消化为置于37℃水浴锅中消化20-30分钟，每隔5分钟摇晃一次；所述培养箱为37℃，5％CO 2。
进一步，所述步骤3)的具体操作为：
A、将P2代牙间充质细胞弃培养液，洗涤后加入蛋白酶消化；
B、用α-MEM培养基终止消化，离心，去上清，适量培养液重悬后计数；
C、将计数后的所得细胞加入3D培养液中吹打混匀，并将细胞接种至培养板，培养期间补充新鲜培养液并观察细胞成球情况。
进一步，所述3D培养液由细胞培养液和胞外基质预制备物按3:2体积比混合组成。其中，细胞培养液是在含100U/mL青霉素、100U/mL链霉素的基础培养基中加入2％体积比血清或血清替代物、1％体积比的细胞生长因子的培养基，其中细胞生长因子为TGFβ、EGF、BMP-2、BMP-4以及FGF等因子的混合物；胞外基质预制备物优选胶原蛋白、甲基纤维素。
进一步，步骤C中，所述细胞以1×10 4cells/cm 2的密度接种至6-well低粘附培养板；所述补充新鲜培养液的具体操作为：每两日补充一次1/10初始体积的新鲜培养液。
一种用于牙髓再生的多能干细胞MDPSCs的应用，所述细胞在构建完整牙胚，增强体内外成骨/成牙向分化能力，促进牙髓牙本质复合体在体内功能再生中的应用。
与现有技术相比，本发明具有以下有益效果：
本发明提供一种用于牙髓再生多能干细胞MDPSCs，并用3D培养的方法对其成功分离和富集，同时结合流式细胞术定义了MDPSCs的特征性表面标记。MDPSCs裸鼠体内移植能够再生牙髓，或可在将来替代传统根管治疗，在消除牙髓炎症的前提下保存活髓。本发明多能干细胞MDPSCs具有形成血管化牙髓-牙本质复合体的能力， 为研究功能性牙髓再生以及临床转化提供了细胞基础。
附图说明
图1为原代DPCs分离培养三维细胞成球实验模式图；
图2为原代DPCs及DPC spheres光镜和电镜下的典型细胞形态；
图3为免疫荧光及RT-PCR检测多能性和增殖相关标志基因Oct4,Sox2,Nanog,Ki67的表达情况；其中a为免疫荧光，b为RT-PCR检测；
图4为成骨诱导液分别作用10天和7天后DPC spheres茜素红和碱性磷酸酶染色情况及定量分析；
图5为DPC spheres在小鼠肾被膜下分化为骨样/牙本质样矿化结构；
图6为DPC spheres复合TDM裸鼠皮下移植流程图。
图7为DPC spheres在裸鼠皮下TDM管腔内形成牙髓-牙本质复合体样结构。
图8为DPC spheres再生组织中牙本质特异性标志物DMP1和DSPP，血管特异性标志物VEGF和CD31，神经组织特异性标志物GFAP、S100和NF200的表达情况。
图9为DPC spheres中富集的表面标志基因。
图10为RT-qPCR验证转录组测序结果的准确性。
图11为采用流式细胞术得到的FACS散点图和直方图分析结果。
图12为免疫荧光检测monolayer、spheres和DPC p2细胞各自CD24a表达情况。
图13为FACS对原代牙乳头细胞中的Cd24a阳性细胞进行分选，根据Cd24a表达情况得到3个亚群。
图14为根据CD24a表达情况得到的3个亚群各自成球效率的分析结果。
图15为免疫荧光和免疫组化分析小鼠发育中的牙胚组织切片。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1 小鼠牙乳头中多能干细胞的分离培养
本实施例牙乳头细胞分离培养三维成球实验模式图如图1所示。小鼠牙乳头中多能成球干细胞的分离培养具体包括以下步骤：
1)原代牙乳头细胞的分离培养
选择10只出生后1-3天的C57BL/6小鼠乳鼠，脱颈处死后75％乙醇浸泡5-10min消毒，分离上下颌骨，用显微镊在体式显微镜下剥离第一二磨牙完整牙胚，从牙冠钙化边缘下方掏出牙乳头组织，用含抗生素的PBS冲洗后眼科剪剪碎成乳糜状；
用胶原酶(625CDU/ml)和螯合剂对组织进行消化，37℃水浴锅中消化20-30分钟，每隔5分钟摇晃一次；
用含10％FBS的α-MEM培养基终止消化，PBS洗涤，1000rpm离心3-5min，去上清，1ml新鲜EpiCM培养基重悬细胞后转移至T25培养瓶中，于37℃，5％CO 2的培养箱中培养过夜；
次日补充2-3ml新鲜培养液继续培养，视细胞生长情况每2-3天换液一次。
2)当DPCs密度达到约70-90％的时候用0.25％胰蛋白酶消化1-2min，10％FBS的α-MEM培养基终止消化，1000rpm离心3-5min，去上清，牙间充质培养基重悬后将1×10 6个细胞接种至T25进行传代培养；后述实验所用细胞均为P2代牙乳头细胞。
3)3D培养细胞成球
P2代牙乳头细胞，弃培养液，用1×PBS洗一次，0.25％胰蛋白酶1ml消化1-2分钟；等体积10％FBS的α-MEM终止消化，离心，去上清，适量培养液重悬后计数； 细胞培养液和甲基纤维素按3:2体积比混合配制成3D细胞培养液；将计数所得细胞加入3D细胞培养液中吹打混匀，以1×10 4cells/cm 2的密度接种至6-well超低粘附培养板每个孔；每两日补充1/10初始体积的新鲜培养液至6-well培养板每孔；培养期间显微镜下观察细胞成球情况，至第8天获得DPC spheres用于后续实验。
细胞培养液是在含100U/mL青霉素、100U/mL链霉素的基础培养基中加入2％体积比血清或血清替代物、1％体积比的细胞生长因子的培养基，其中细胞生长因子为TGFβ、EGF、BMP-2、BMP-4以及FGF等因子的混合物。
本实施例原代牙乳头细胞(DPCs)及DPC spheres光镜和电镜下的典型细胞形态如图2所示。其中i是原代DPCs，ii是Day 0的DPC spheres，iii是Day 7的DPC spheres，iv是DPC spheres的扫描电镜图。尽管原代DPCs在肾被膜下显示同DPC spheres相似的矿化能力，但原代DPCs在TDM诱导下无法形成牙髓牙本质复合体结构，而DPC spheres可以。
实施例2 多能干细胞的检测
1、免疫荧光检测
将DPC spheres接种于共聚焦小皿中，待30-60min细胞小球沉降贴壁，4％多聚甲醛固定，免疫荧光染色对DPC spheres的多能性进行鉴定。
免疫荧光染色步骤为：
a.细胞小球经4％多聚甲醛固定1h，用PBS洗涤3次；
b.加入0.5％TritionX-100室温打孔30min，PBS洗3次；
c.5％BSA室温封闭30min；
d.弃封闭液，加一抗孵育，4℃过夜；
e.复温10min，PBS洗5min/次，共3次，加相应荧光二抗，37℃孵育1h；
f.PBS洗3次，DAPI染细胞核5min；
g.PBS洗3次，荧光显微镜下观察。
免疫荧光检测所使用的一抗主要有：rabbit anti-Sox2(ab97959,Abcam,USA),rabbit anti-Nanog(ab80892,Abcam,USA),rabbit anti-OCT4(D121072,Sangon Biotech,China),rabbit anti-Ki67(ab15580,Abcam,USA)。免疫荧光染色结果如图3所示，DPC spheres阳性表达多能相关及增殖标志基因Sox2，Nanog，Oct4和Ki67。
2、RT-PCR
离心收集低粘附培养板中的DPC spheres，2000rpm，5min，RT-PCR对免疫荧光染色结果进行验证。
a.提取细胞小球总RNA
1)PBS清洗离心获得的DPC spheres，加入1ml RNAiso Plus裂解液，混匀后室温静置5min；
2)向裂解液中加入0.2ml氯仿，振荡器剧烈振荡15s，待其充分乳化后，静置5min；
3)12000g，4℃，离心15min；
4)离心后分为三层：无色上清层、中间蛋白层及下层有机相。转移上清液至新的RNase-free的EP管中；
5)吸取等体积异丙醇加入上清液中，混匀，室温静置10min；
6)12000g，4℃，离,10min；
7)离心后管底可见白色沉淀物，即总RNA；
8)沿EP管壁缓慢地加入RNase-free水配制的75％乙醇1ml，轻轻上下颠倒洗涤；
9)12000g，4℃，离心5min；
10)弃去乙醇，室温干燥5min；
11)RNA溶解：移液枪加入10μL RNase-free水，轻轻吹打溶解沉淀；
12)紫外分光光度计检测RNA溶液A260/A280，比值在1.8-2.2之间为合格RNA，-80℃保存备用。
b.逆转录(Reverse transcription，RT)
1)RT反应：反转录按照Thermo Scientific RevertAid First Strand cDNA Synthesis Kit说明书进行，混匀后瞬时离心，放入梯度PCR仪。
2)反应条件：42℃，60分钟；70℃，5分钟。
3)cDNA产物置于-20℃保存备用。
c.PCR
1)反应配方：采用10ul反应体系，在PCR管内各加入预混溶液：Green mix(Cat#K1082,Thermofisher,USA)，5ul；Forward primer，0.5ul；Reverse primer，0.5ul；cDNA，1ul；H 2O，3ul；至总体积为10μl。将上述反应液震荡混匀，瞬时离心。
2)反应条件：95℃，2min→95℃，30s→55℃，30s→72℃，36s→72℃，5min→4℃，循环数35cycles。
d.琼脂糖核酸电泳
RT-PCR结果如图3所示：DPC spheres阳性表达多能相关及增殖标志基因Sox2，Nanog，Oct4和Ki67。
本实施例所涉及的引物序列如下表1所示：
表1
Figure PCTCN2020084109-appb-000001
3、成骨诱导液诱导体外成骨分化
将2×10 5个细胞接种至6孔板每个孔中，37℃、5％CO 2细胞培养箱中培养；细胞生长至对数期时取出，弃旧培养液，PBS缓冲液漂洗3遍，加入成骨诱导液分别培养7天和10天；弃成骨诱导液，使用PBS漂洗3遍；成骨诱导10天组，4％多聚甲醛固定30分钟，PBS洗去除残留多聚甲醛；将预先配置好的茜素红S(Cat#A5533，pH＝4.3，Sigma-Aldrich，USA)加入孔板中，室温下染色5min，PBS清洗3次，显微镜下观察并采图，用10％氯化十六烷基吡啶溶解矿化结节，于405nm处测吸光度。成骨诱导7天组，按照ALP显色试剂盒(Cat#C3206，Beyotime，China)和ALP活性试剂盒(Cat#A059-2，Jiancheng，China)说明书指导，ALP染色和ALP活性检测。结果如图4所示，其中(a)和(c)分别为茜素红和ALP染色结果，(b)和(d)分别为对茜素红和ALP染色结果的定量分析。由此可见，成骨诱导液作用下DPC spheres显示更强的矿化能力和ALP活性。
4、小鼠肾被膜移植
a.DPC spheres来源的细胞按1.0×10 6/10μl的细胞密度与Matrigel(Cat#354234,BD,USA)混合；
b.将上述混合液制成cell-matrigel小球(规格3.3×10 5/3μl)；
c.移植cell-matrigel小球到8周龄的雄性C57BL/6小鼠肾被膜下；
d.标准条件下饲养，温度21℃，光照周期12小时；
e.4周后取材，4％多聚甲醛固定，10％EDTA脱钙1周，石蜡包埋切片，以备组织学染色分析。
结果如图5所示，monolayer cells在肾被膜下移植4周后仅可见胶原纤维样结构形成，且DMP1和DSPP表达相对较弱，而DPC spheres可形成明显阳性表达DMP1和DSPP的矿化结构，这说明DPC spheres在体内显示更强的成骨/成牙分化能力。
实施例3 DPC spheres促进牙髓-牙本质复合体在体内的功能再生
1、裸鼠皮下移植，流程图如图6所示，具体操作过程如下：
a.TDM制备：猪下颌骨中拔出中切牙；小心地刮除牙周韧带组织，磨去牙骨质、牙髓组织和牙本质层；高速涡轮手环将根管切成长2-3毫米的小段；超声波清洗机震荡清洗根管3次，每次5～6min；根管转移至梯度EDTA溶液(17％到5％)5min，期间去离子水冲洗5min(3次)；将制备的TDM浸泡于青霉素(50U/ml)和链霉素(50mg/ml)混合液中4℃保存备用。
b.DPC spheres来源的细胞按照1.0×10 6/10μl的细胞密度与Matrigel(Cat#354234,BD,USA)混合；
c.将上述混合液用移液枪打入一端用盖随剂(Dycal,Cat#10800,DENSPLY,USA)封口的TDM管腔中，37℃培养箱培养至凝固；
d.移植TDM-cell-matrigel样品到8周龄的雄性裸鼠皮下；
e.标准条件下饲养，温度21℃，光照周期12小时；
f.4周后取材，4％多聚甲醛固定，10％EDTA脱钙6周，石蜡包埋切片，以备组织学染色分析。
结果如图7和图8所示，图7表明DPC spheres在裸鼠皮下的TDM管腔内再生形成与天然牙相似的牙髓-牙本质复合体样结构。图8表明DPC spheres再生组织中牙本质特异性标志物DMP1和DSPP，血管特异性标志物VEGF和CD31，神经组织特异性标志物GFAP、S100和NF200均呈阳性，与天然牙髓表达相似。
实施例4 转录组测序分析DPC spheres中富集的表面蛋白标志基因
1、RNA-seqencing
提取总RNA，NanoDrop检测纯度；Agilent Bioanalyer 2100 system(Agilent  Technologies,USA)验证RNA样品的完整性；RNA-seq分析各组样品mRNA整体表达谱，产生150bp reads数；fastq文件经HISAT2v2.1.0比对到小鼠基因组(Mus musculus,GRCm38)；FeatureCounts v1.6.0统计比对到每个基因上的reads数；以log FoldChange>2，p-adjusted value<0.01为标准，用R and DESeq2 package分析差异表达的基因。CD标记物的收集：细胞表面蛋白数据库(http://wlab.ethz.ch/cspa/)和Human/Mouse CD标记手册(BD Biosciences)。
2、RT-qPCR验证RNA-seqencing结果
a.提取总RNA；
b.反转录为cDNA；
c.实时荧光定量PCR。
反应体系:5μl
Figure PCTCN2020084109-appb-000002
Premix Ex TaqTMⅡ(2x)，0.5μl，10μM上游引物；0.5μl，10μM下游引物；1.0μl cDNA模板；3μl ddH2O至总体积为10μl。
反应条件：Polymerase Activation:95℃，30s，PCRCycling(40cycles)：95℃，15s-60℃，34s；MeltCurve:95℃，15s，60℃，15s，95℃，15s。
结果如图9和图10所示，图9为DPC spheres中富集的表面标志基因，Fold change以灰色显示，FPKM值以黑色显示，比较DPC spheres与2D培养细胞的差异基因表达谱，并与表面蛋白数据库重叠(Fold change≥2)，筛选出15个候选表面标志物。标记准备进一步验证的候选表面marke。图10为RT-qPCR验证转录组测序结果的准确性，证实了测序结果的准确性。
本实施例所涉及的引物序列如下表2所示：
表2
Figure PCTCN2020084109-appb-000003
Figure PCTCN2020084109-appb-000004
实施例5 牙乳头中CD24a阳性细胞成球能力更强
1、流式细胞术
预冷PBS冲洗monolayer cells，DPCs P2和DPC spheres的单细胞悬液两次；Anti-CD24a抗体(ab64064,Abcam,USA)冰上孵育约5×10 5个细胞30min；预冷PBS洗涤2-3次；anti-rat IgG-FITC(Cat#A21208，Thermofisher，USA)孵育30min；预冷PBS洗涤3次，70μm细胞筛过滤；Rat normal IgG(Cat#10700,Thermofisher,USA)为同型对照。采用Becton-Dickinson Accuri C6(BD biosciences,USA)进行分析，BD Aria III(BD biosciences,USA)进行分选，FlowJo(版本7.6.1)处理数据。
得到的FACS散点图和直方图分析结果如图11所示，从上到下分别为散点图和直方图：以Rat normal IgG为阴性对照，DPC spheres分别为CD24a++(72.9％)、CD24a+(19.7％)、CD24a-(2.91％)；monolayer cells分别为CD24a++(0.179％)、CD24a+(4.99％)、CD24a-(93.8％)；DPCs P2分别为CD24a++(9.32％)、CD24a+(50.8％)、CD24a-(33％)。DPC spheres细胞中CD24a++细胞比例较高。
2、免疫荧光检测
实验过程如实施例2中免疫荧光检测所述。
免疫荧光检测所使用的一抗：rat anti-CD24(ab64064,Abcam,USA)。免疫荧光染色结果如图12所示：DPC spheres细胞中含有丰富的CD24a阳性细胞群。
3、流式分选后成球实验
实验过程如实施例1中的步骤3)所述，5×10 3cells/cm 2的密度接种至24-well超低粘附培养板。
结果如图13和图14所示，根据CD24a的表达情况，用流式细胞仪(FACS)对牙乳头细胞3个亚群进行分选。RT-qPCR进一步证实FACS分选获得的各细胞亚群 CD24a的表达情况。将分选得到的细胞在MC培养基中3D培养7天后拍照并进行统计分析，结果表明只有CD24a++表现出较强的成球能力，CD24a++细胞成球效率(17.04±3.87‰)明显高于CD24-细胞(1.76±0.24‰)**P<0.01(student t-test)。
4、牙胚组织切片染色
a.选择1只出生后1-3天的C57BL/6小鼠乳鼠，脱颈处死后75％乙醇浸泡5-10min消毒，分离完整上下颌骨。
b.将分离得到的颌骨分别于10％，20％，30％蔗糖梯度脱水至沉底；
c.tissue freezing medium包埋脱水后的上下颌骨，冷冻切片机切片。
d.后续实验过程如实施例2中免疫荧光检测所述。
采用免疫荧光和免疫组化分析小鼠发育中的牙胚组织切片，结果如图15所示，小鼠发育中的牙胚组织中存在Cd24a阳性细胞。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种用于牙髓再生的多能干细胞MDPSCs，其特征在于，所述MDPSCs为从牙间充质组织中分离的CD24a阳性细胞。
  2. 如权利要求1所述一种用于牙髓再生的多能干细胞MDPSCs，其特征在于，所述MDPSCs在3D培养条件下展现自更新能力，形成阳性表达多能相关标志基因Sox2，Nanog，Oct4和Ki67的细胞小球。
  3. 一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，包括以下步骤：
    1)采用酶与螯合剂复合消化法进行牙间充质细胞的原代分离培养：
    2)当牙间充质细胞密度达到70-90％用酶消化进行传代培养得到P2代牙间充质细胞；
    3)将P2代牙间充质细胞接种至培养板上进行3D培养获得呈细胞小球状态的MDPSCs细胞。
  4. 如权利要求3所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，所述步骤1)的具体操作为：
    A、分离牙齿完整牙胚，从牙冠钙化边缘下掏出牙间充质组织，冲洗后剪碎成乳糜状；
    B、用酶和螯合剂消化液对剪碎后的组织进行消化，消化完成后终止消化；
    C、经洗涤、离心、去上清后将培养基重悬细胞后置于原代培养瓶中，放入培养箱中培养；
    D、补充培养液至稍稍浸没组织块，并视细胞生长情况适当换液或传代。
  5. 如权利要求4所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，所述消化为置于37℃水浴锅中消化20-30分钟，每隔5分钟摇晃一次；所述培养箱为37℃，5％CO 2。
  6. 如权利要求3所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，所述步骤3)的具体操作为：
    A、将P2代牙间充质细胞弃培养液，洗涤后加入蛋白酶消化；
    B、用α-MEM培养基终止消化，离心，去上清，适量培养液重悬后计数；
    C、将计数后的所得细胞加入3D培养液中吹打混匀，并将细胞接种至培养板，培养期间补充新鲜培养液并观察细胞成球情况。
  7. 如权利要求6所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，所述3D培养液由细胞培养液和胞外基质预制备物按3:2体积比混合组成。
  8. 如权利要求7所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，所述细胞培养液是在含100U/mL青霉素、100U/mL链霉素的基础培养基中加入2％体积比血清或血清替代物、1％体积比的细胞生长因子的培养基，其中细胞生长因子为TGFβ、EGF、BMP-2、BMP-4以及FGF因子的混合物。
  9. 如权利要求6所述一种用于牙髓再生的多能干细胞MDPSCs的分离培养方法，其特征在于，步骤C中，所述细胞以1×10 4cells/cm 2的密度接种至6-well低粘附培养板；所述补充新鲜培养液的具体操作为：每两日补充一次1/10初始体积的新鲜培养液。
  10. 如权利要求1所述一种用于牙髓再生的多能干细胞MDPSCs的应用，其特征在于，所述细胞在构建完整牙胚，增强体内外成骨/成牙向分化能力，促进牙髓牙本质复合体在体内功能再生中的应用。
PCT/CN2020/084109 2019-07-25 2020-04-10 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用 WO2021012718A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910674796.1A CN110408592B (zh) 2019-07-25 2019-07-25 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用
CN201910674796.1 2019-07-25

Publications (1)

Publication Number Publication Date
WO2021012718A1 true WO2021012718A1 (zh) 2021-01-28

Family

ID=68363041

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/084109 WO2021012718A1 (zh) 2019-07-25 2020-04-10 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用

Country Status (2)

Country Link
CN (1) CN110408592B (zh)
WO (1) WO2021012718A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110408592B (zh) * 2019-07-25 2021-07-20 四川大学 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101970022A (zh) * 2008-03-12 2011-02-09 财团法人日本健康科学振兴财团 根管填充材料以及牙组织再生方法
CN103402558A (zh) * 2011-02-28 2013-11-20 独立行政法人国立长寿医疗研究中心 含有间充质干细胞的根管充填材料及使用该材料的牙组织再生方法
CN110408592A (zh) * 2019-07-25 2019-11-05 四川大学 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2015037945A1 (ko) * 2013-09-13 2015-03-19 서울대학교산학협력단 프롤릴 히드록실라아제 저해제 또는 히스톤 탈아세틸라아제 저해제, 및 항생제를 포함하는 치수 재생 치료 또는 치근첨 형성 유도 치료용 약학적 조성물, 및 이를 포함하는 합성고분자 나노섬유메시
CN109251889A (zh) * 2018-09-18 2019-01-22 中晶生物技术股份有限公司 一种移植用牙髓间充质干细胞微球的制备体系

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101970022A (zh) * 2008-03-12 2011-02-09 财团法人日本健康科学振兴财团 根管填充材料以及牙组织再生方法
CN105582575A (zh) * 2008-03-12 2016-05-18 财团法人日本健康科学振兴财团 根管填充材料以及牙组织再生方法
CN103402558A (zh) * 2011-02-28 2013-11-20 独立行政法人国立长寿医疗研究中心 含有间充质干细胞的根管充填材料及使用该材料的牙组织再生方法
CN110408592A (zh) * 2019-07-25 2019-11-05 四川大学 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WATARU SONOYAMA ET AL.: "Mesenchymal Stem Cell-Mediated Functional Tooth Regeneration in Swine", PLOS ONE., vol. 1, no. 1, 20 December 2006 (2006-12-20), XP055116639, ISSN: 1932-6203 *

Also Published As

Publication number Publication date
CN110408592A (zh) 2019-11-05
CN110408592B (zh) 2021-07-20

Similar Documents

Publication Publication Date Title
Chen et al. Regeneration of pulpo-dentinal–like complex by a group of unique multipotent CD24a+ stem cells
JP4365406B2 (ja) 骨髄細胞から歯を作製する方法
JP2008200033A (ja) 間葉系細胞の製造方法、歯の製造方法及び歯形成用間葉系細胞
CN103442724A (zh) 含有可从机体组织中分离的ssea-3阳性的多能干细胞的同种异体移植用细胞治疗用组合物
Zhang et al. Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro
CN103459590A (zh) 可由机体的脐带或脂肪组织分离的多能干细胞
JP2008029756A (ja) 歯の製造方法
WO2021012718A1 (zh) 用于牙髓再生的多能干细胞MDPSCs及其分离培养方法和应用
CN102755667A (zh) 一种组织工程化人牙根植入材料的制备方法及其用途
JP4748222B2 (ja) 軟骨細胞調製方法
JP4991204B2 (ja) 歯の製造方法
CN1884494A (zh) 诱导人胚胎干细胞向肝脏细胞分化的方法及其专用培养基
Tominaga et al. Isolation and characterization of epithelial and myogenic cells by “fishing” for the morphologically distinct cell types in rat primary periodontal ligament cultures
CN109294994A (zh) 有效修复地中海贫血Westmead突变的方法及应用
Popuri Concerns of a pediatric dentist in dental stem cells: an overview
Honda et al. Mesenchymal Dental Stem Cells for Tissue Regeneration.
CN104306958B (zh) 胰岛素样生长因子结合蛋白5在促进牙周组织再生中的应用
Bi et al. Hertwig's epithelial root sheath cells show potential for periodontal complex regeneration
CN102250841A (zh) 可回复性永生化大鼠骨髓间充质干细胞及其制备方法和应用
CN104928319B (zh) hTERT慢病毒重组体永生化人牙周膜干细胞系的方法
Dannan Dental-derived stem cells and whole tooth regeneration: an overview
CN107460166A (zh) 一种鸡ghr缺失型突变体成肌细胞的分离培养方法
WO2013123607A1 (zh) 用于成体干细胞的体外无血清培养的方法和培养基
KR101738715B1 (ko) 식물 줄기세포 또는 식물 역분화 줄기 세포의 추출물을 이용한 맞춤형 만능줄기세포의 유도 방법 및 상기 방법에 의해 제조된 만능줄기세포
CN111454892A (zh) 牙间充质干细胞培养基及在牙髓干细胞内的活性验证方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20844089

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20844089

Country of ref document: EP

Kind code of ref document: A1