In-vitro extraction and amplification method of vascular development related dental pulp stem cell subpopulation

Abstract

The invention discloses an in-vitro extraction and amplification method of a vascular development related dental pulp stem cell subpopulation, which comprises the following steps: extracting primary DPSCs from a dental pulp tissue through trypsin and collagenase digestion, and preparing a single-cell suspension; sorting the cells using flow cytometry; and the PDGFR beta + DPSCs are sorted out through a flow cytometer. And finally, inoculating the separated PDGFR beta + DPSCs into a DMEM culture medium containing 10% of FBS and 1% of penicillin-streptomycin, and culturing under the conditions of 37 DEG C and 5% of CO2. The PDGFR beta + DPSCs purified and cultured through the method have high proliferation, migration and angiogenesis promoting performance. According to the method, autologous dental pulp stem cells which are from donors of different ages and under different conditions and have high angiogenesis promoting characteristics can be stably obtained in a clinical environment to a certain extent. The method provides a necessary research basis for exploring and maintaining a regulation mechanism of DPSCs self-renewal, stable amplification and promotion of vascular performance, further widening the clinical application prospect of DPSCs and promoting dental pulp regeneration treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology stem cell culture, and in particular to an in vitro extraction and amplification method of a dental pulp stem cell subpopulation associated with vascular development. Background Art

[0002] Dental pulp disease is often caused by caries and traumatic injury, and conventional treatments (such as root canal surgery) often lead to loss of pulp vitality and affect tooth development. Compared with traditional methods, regenerative medicine, including autologous stem cell homing and stem cell transplantation, has received attention in pulp regeneration. The dental pulp is rich in blood vessels, and vascular development must be promoted for successful regeneration and restoration of physiological function. Despite the progress made in these studies, the intricate regulatory mechanisms that control pulp vascular development remain unclear due to the complexity of the pulp development process and technical limitations.

[0003] Exploring the regulatory mechanisms of pulp vascular development and using this knowledge to guide pulp regeneration provides a novel research direction. Mesenchymal stem cells may play a crucial role in the vascularization process. As key players in pulp homeostasis and injury repair, dental pulp stem cells (DPSCs) have great potential in pulp regenerative therapy. Early studies revealed the presence of DPSCs in the "stem cell niche" around blood vessels and nerves, suggesting a potential direct or indirect regulatory relationship between them. Specifically, intercellular communication between various cells, including endothelial cells (ECs) and DPSCs, may be a key factor in inducing vascularization.

[0004] At present, related studies have determined that FN1-ITGA5-mediated intercellular communication between DPSCs and ECs is a crucial factor in promoting vascular development through the analysis of single-cell sequencing data and in vivo and in vitro experiments. This discovery has laid a theoretical foundation for subsequent research on the regulatory mechanism of DPSCs promoting angiogenesis and its clinical application.

[0005] As the pressure of an aging population increases, the prevention and treatment of aging-related diseases has become a scientific issue that urgently needs a breakthrough. DPSCs from different stages of dental pulp development show characteristics that adapt to their physiological functions. In the early stages of dental pulp development, dental pulp stem cells mainly contribute to tooth formation. These cells have high proliferation capacity and multidirectional differentiation potential, and can differentiate into odontoblasts and other cell types that form dental pulp tissue. As teeth mature, dental pulp stem cells undergo age-related changes, including a decrease in number and an enhanced ability to maintain internal homeostasis. With age, the regenerative potential of stem cells gradually decreases, which may be related to cell aging, changes in the microenvironment, and changes in the extracellular matrix. Similarly, existing studies have shown that DPSCs from mature permanent teeth show lower effectiveness in promoting angiogenesis compared with DPSCs from young deciduous teeth. In the clinical application of stem cells, how to obtain dental pulp stem cells of autologous origin with strong angiogenic properties has become a problem, and there is currently no effective method to solve this problem. Therefore, it is important to find an effective method to ensure that DPSCs from donors of different ages and conditions always show the ability to promote angiogenesis. Summary of the invention

[0006] The present invention provides an in vitro extraction and expansion method for dental pulp stem cell subpopulations related to vascular development, with the purpose of ensuring autologous dental pulp stem cells from donors of different ages and conditions with strong angiogenesis-promoting properties. This method explores the regulatory mechanism for maintaining DPSCs self-renewal, stable expansion, and promoting vascular performance, further broadens the clinical application prospects of DPSCs, and provides a necessary research basis for promoting dental pulp regeneration treatment.

[0007] The technical solution provided by the present invention is as follows:

[0008] An in vitro method for extracting and amplifying a subpopulation of dental pulp stem cells related to vascular development comprises the following steps:

[0009] S1. Obtain pulp tissue;

[0010] S2, digestion of dental pulp tissue;

[0011] S3, filtering the digested dental pulp tissue through a cell strainer to obtain a first cell suspension, centrifuging the first cell suspension, discarding the supernatant, adding PBS to resuspend the cells, and obtaining a second cell suspension;

[0012] S4. Cell staining

[0013] Adding equal amounts of the second cell suspension into different test tubes, setting them as a blank control group, an isotype control group, and a PDGFRβ staining group respectively;

[0014] 1% BSA solution was added to each tube to block nonspecific binding, and the first incubation was performed in the dark;

[0015] Adding diluted PDGFRβ antibody to the PDGFRβ staining group, adding diluted isotype control antibody to the isotype control group, and performing a second incubation in the dark;

[0016] S5. Wash cells

[0017] Wash the cells by adding PBS to all tubes, centrifuge, discard the supernatant, and wash with PBS, repeating at least twice;

[0018] After the final wash, the cells were resuspended in PBS to obtain the third cell suspension;

[0019] S6. Flow cytometry sorting

[0020] Performing cell analysis and sorting on the third cell suspension using a flow cytometer, and collecting and sorting PDGFRβ+DPSCs;

[0021] S7. The collected and sorted PDGFRβ+DPSCs are inoculated into DMEM culture medium and cultured in an incubator to achieve the expansion of the PDGFRβ+DPSCs.

[0022] Furthermore, in step S1, the obtaining of dental pulp tissue is specifically:

[0023] Dental pulp tissues were obtained from healthy deciduous teeth extracted for tooth replacement or permanent premolars and third molars extracted for orthodontic reasons, and placed in a culture dish containing DMEM, 100 U / ml penicillin and 100 μg / ml streptomycin.

[0024] Furthermore, in step S2, the pulp tissue digestion is specifically performed as follows:

[0025] The dental pulp tissue was cut into small pieces, placed in a digestion solution containing 2 mg / ml collagenase and 0.25% trypsin, and digested in a 37° C. water bath for 1 hour.

[0026] Further, in step S3, the cell filter is a 70 μm cell filter;

[0027] The first cell suspension is centrifuged as follows: the first cell suspension is centrifuged at 1000 rpm for 5 minutes using a centrifuge.

[0028] Furthermore, in step S4, the first light-proof incubation is specifically: incubating at 4° C. in the dark for 10 minutes;

[0029] The dilution multiple of the PDGFRβ antibody is 100 times, and the dilution method is: add 2 μl of PDGFRβ antibody into 198 μl of PBS;

[0030] The dilution factor of the isotype control antibody is 100 times;

[0031] The second dark-proof incubation is specifically: incubating in the dark at 4° C. for 15 minutes.

[0032] Furthermore, in step S6, the parameters of the flow cytometer are set as follows: laser wavelength 488nm, detection channel FITC, and voltage setting 500V.

[0033] Further, in step S7, the DMEM culture medium contains 10% FBS and 1% penicillin-streptomycin;

[0034] The incubator is a 37° C., 5% CO 2 incubator.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides an in vitro extraction and amplification method for a subpopulation of dental pulp stem cells related to vascular development. The PDGFRβ+DPSCs purified and cultured by this method have strong proliferation, migration and angiogenesis-promoting properties. The present invention can, to a certain extent, achieve the stable acquisition of autologous dental pulp stem cells from donors of different ages and conditions with strong angiogenesis-promoting properties in a clinical environment. Compared with the existing scheme, the advantages of this scheme in stably obtaining autologous dental pulp stem cells from donors of different ages and conditions with strong angiogenesis-promoting properties are as follows:

[0037] ① Improve the stability and consistency of DPSCs: Accurately identify and sort out the PDGFRβ+DPSCs subpopulation through single-cell sequencing and flow cytometry, which can significantly improve the consistency of DPSCs between different donors and reduce the variability caused by differences in donor age and conditions. Methods such as gene editing and antioxidant treatment may have difficulty in dealing with the variability between different donors, but this solution solves this problem from the source by screening and enriching specific cell subpopulations;

[0038] ② Stem cells have stable angiogenesis ability: PDGFRβ+DPSCs subpopulation showed strong angiogenesis ability in in vitro experiments, which is of great significance in dental pulp regeneration and helps to improve the regeneration effect. Although environmental factor regulation and drug intervention can improve cell function to a certain extent, their effects may not be as significant and stable as directly using cell subpopulations with high angiogenesis ability;

[0039] ③ Overcoming the problem of stem cell aging: Through the regulation of the PDGFR / PI3K / AKT signaling pathway, the aging of PDGFRβ+DPSCs can be effectively delayed, and their high proliferation and migration abilities can be maintained, thereby ensuring their long-term effects in dental pulp regeneration. Methods such as gene editing and antioxidant treatment may have long-term safety issues, while this approach can more safely delay stem cell aging by naturally selecting highly functional cell subpopulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the flow cell sorting and purification culture process of PDGFRβ+DPSCs in an embodiment of the present invention and a laser scanning confocal microscope image;

[0041] Figure 2 This is a diagram showing the effects of proliferation, migration and angiogenesis promotion of PDGFRβ+DPSCs in an embodiment of the present invention;

[0042] Figure 3 This is the application of the role of PDGFRβ+ cells in the development of dental pulp blood vessels in an example of the present invention, wherein A is a schematic diagram of the construction of PDGFRβ-CreERT2 / ROSA-DTA double transgenic mice, and B is a schematic diagram of the immunofluorescence results. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0044] Therefore, the detailed description of the embodiments of the present application provided below in conjunction with the accompanying drawings is intended to represent only selected embodiments of the present application, and is not intended to limit the scope of protection claimed in the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

[0045] See also Figure 1 The present invention provides an in vitro method for extracting and amplifying a subpopulation of dental pulp stem cells related to vascular development, comprising the following steps:

[0046] S1. Obtain dental pulp tissue.

[0047] S2. Digest the dental pulp tissue.

[0048] S3. Filter the digested dental pulp tissue through a cell strainer to obtain a first cell suspension, centrifuge the first cell suspension, discard the supernatant, add PBS to resuspend the cells, and obtain a second cell suspension.

[0049] S4. Cell staining

[0050] The same amount of the second cell suspension was added to different test tubes, which were set as a blank control group, an isotype control group and a PDGFRβ staining group, respectively.

[0051] 1% BSA solution was added to each tube to block nonspecific binding, and the first incubation was performed in the dark;

[0052] The diluted PDGFRβ antibody was added to the PDGFRβ staining group, and the diluted isotype control antibody was added to the isotype control group, and then incubated in the dark for a second time.

[0053] S5. Wash cells

[0054] Wash the cells by adding PBS to all tubes, centrifuge, discard the supernatant, and wash with PBS, repeating at least twice.

[0055] After the final wash, the cells were resuspended in PBS to obtain a third cell suspension.

[0056] S6. Flow cytometry sorting

[0057] The third cell suspension is subjected to cell analysis and sorting using a flow cytometer, and PDGFRβ+DPSCs are collected and sorted.

[0058] S7. The collected and sorted PDGFRβ+DPSCs are inoculated into DMEM culture medium and cultured in an incubator to achieve the expansion of the PDGFRβ+DPSCs.

[0059] The above method is further described below in conjunction with embodiments:

[0060] Example 1 In vitro extraction and culture of primary DPSCs

[0061] 1.1 Materials and reagents

[0062] Pulp tissue (teeth replacing extracted healthy deciduous teeth or permanent premolars and third molars extracted for orthodontic reasons)

[0063] Dulbecco's Modified Eagle Medium(DMEM)

[0064] Fetal bovine serum (FBS)

[0065] Penicillin-Streptomycin Solution

[0066] Trypsin-EDTA solution (0.25%)

[0067] Phosphate buffered saline (PBS)

[0068] Collagenase (2 mg / ml)

[0069] Antibiotics and antifungal medications

[0070] 6-well culture plate

[0071] 37℃, 5% CO2 incubator

[0072] 1.2 Experimental procedures

[0073] 1. Obtaining dental pulp tissue:

[0074] Dental pulp tissues were obtained from healthy deciduous teeth extracted for tooth replacement or permanent premolars and third molars extracted for orthodontic reasons, and placed in a culture dish containing DMEM and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin).

[0075] 2. Tissue Digestion:

[0076] The pulp tissue was cut into small pieces and placed in a digestion solution containing 2 mg / ml collagenase and 0.25% trypsin, and digested in a 37°C water bath for 1 hour. The test tube was gently shaken every 15 minutes to promote digestion.

[0077] 3. Preparation of single cell suspension:

[0078] After digestion, the cells were filtered through a 70 μm cell strainer to obtain a single-cell suspension.

[0079] Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add PBS to resuspend the cells, and repeat twice.

[0080] 4. Cell culture:

[0081] The cells were resuspended in DMEM medium containing 10% FBS and 1% penicillin-streptomycin and seeded in 6-well culture plates with 2 ml per well.

[0082] The culture plate was placed in a 37°C, 5% CO2 incubator and the culture medium was replaced regularly (every 2-3 days).

[0083] Example 2 Flow cytometric purification and in vitro culture of PDGFRβ+ DPSCs

[0084] 2.1 Materials and reagents

[0085] PDGFRβ antibody (Abcam, ab69506)

[0086] Isotype control antibody

[0087] Flow cytometer (Beckman Coulter, USA)

[0088] Flow Cytometry Analysis Software

[0089] Sterile PBS

[0090] BSA (1%)

[0091] 4℃ refrigerator

[0092] Centrifuge

[0093] 2.2 Experimental operation

[0094] 1. Cell collection and preparation:

[0095] DPSCs were collected from the culture dish and digested with 0.25% trypsin to obtain a single-cell suspension.

[0096] Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with PBS, and repeat twice.

[0097] 2. Cell staining:

[0098] Equal amounts of cell suspension were added to three test tubes: blank control group, isotype control group and PDGFRβ staining group.

[0099] 1% BSA solution was added to each tube to block nonspecific binding and incubated at 4°C in the dark for 10 minutes.

[0100] PDGFRβ antibody (diluted 1:100, about 2 μl antibody added to 198 μl PBS) was added to the PDGFRβ staining group, and the corresponding isotype control antibody (diluted 1:100) was added to the isotype control group, and incubated at 4°C in the dark for 15 min.

[0101] 3. Cell washing:

[0102] Add 1 ml of PBS to wash the cells, centrifuge (1000 rpm, 5 minutes), discard the supernatant, wash again with PBS, and repeat twice.

[0103] After the final wash, cells were resuspended in 500 μl PBS and analyzed by flow cytometry within 2 h.

[0104] 4. Flow cytometry analysis and sorting:

[0105] Flow cytometer (Beckman Coulter, USA) was used for cell analysis and sorting, and PDGFRβ+DPSCs were selected for subsequent experiments.

[0106] Flow cytometer parameter settings: laser wavelength 488nm, detection channel FITC, voltage set to 500V.

[0107] The sorted PDGFRβ+ DPSCs were collected and inoculated into DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured in a 37°C, 5% CO2 incubator.

[0108] Example 3 Identification of the proliferation, migration and angiogenesis-promoting abilities of PDGFRβ+ DPSCs

[0109] 3.1 Materials and reagents

[0110] Proliferation kit (such as CCK-8, Dojindo)

[0111] DMEM medium, FBS, penicillin-streptomycin solution

[0112] 6-well culture plate

[0113] HUVEC cells (human umbilical vein endothelial cells)

[0114] Matrigel (Corning)

[0115] Scratch test equipment (such as pipette tips)

[0116] Fluorescence microscope (such as Leica DMi8)

[0117] 96-well plate

[0118] 3.2 Experimental operation

[0119] 1. Proliferation assay:

[0120] Use the CCK-8 kit and follow the kit instructions.

[0121] PDGFRβ+ DPSCs were seeded into 96-well plates at 5000 cells per well and 100 μl of culture medium was added.

[0122] At 0, 24, 48, and 72 hours, 10 μl of CCK-8 solution was added to each well and incubated at 37°C for 2 hours.

[0123] The absorbance (450 nm) was measured using a microplate reader (such as BioTek ELx808) to analyze the cell proliferation ability.

[0124] 2. Migration test (scratch test):

[0125] PDGFRβ+ DPSCs were cultured in 6-well culture plates until confluence.

[0126] Use a 200 μl pipette tip to make a scratch on the cell monolayer and wash away floating cells with PBS.

[0127] Add 2 ml of fresh culture medium, culture for 24 hours, and record cell migration.

[0128] Scratch pictures were taken at 0 h and 24 h using a fluorescence microscope, and the scratch healing rate was analyzed using ImageJ software.

[0129] 3. Promote angiogenesis experiment (tube formation experiment):

[0130] Matrigel was spread in a 24-well plate, 100 μl was added to each well, and incubated at 37°C for 30 minutes to solidify.

[0131] HUVEC cells were mixed with PDGFRβ+DPSCs at a ratio of 1:1, seeded on Matrigel, and 200 μl of cell suspension (10,000 cells of each type) was added to each well.

[0132] The tube formation of cells was observed and recorded after 4 and 8 hours, and images were taken using a fluorescence microscope.

[0133] The number of formed tubes and branch points were counted and analyzed using ImageJ software to evaluate the angiogenic ability of PDGFRβ+DPSCs.

[0134] 4. Colony formation assay

[0135] After trypsinization of cells in the logarithmic growth phase, resuspend them in complete medium (basal medium + 10% fetal bovine serum) to form a cell suspension and count them;

[0136] Inoculate 400-1,000 cells / well (depending on cell growth, generally 700 cells / well) in each experimental group in a 6-well culture plate;

[0137] Continue culturing until 14 days or until the number of cells in most single clones is greater than 50, changing the medium every 3 days and observing the cell status;

[0138] After cloning was completed, the cells were photographed under a microscope, then washed once with PBS, 1 mL of 4% paraformaldehyde was added to each well for fixation for 30-60 min, and washed once with PBS;

[0139] 1 ml of crystal violet stain was added to each well, and the cells were stained for 10-20 min. The cells were washed with PBS several times, dried, and photographed with a digital camera (the entire six-well plate and each well were photographed separately).

[0140] Through the above detailed steps and conditions, we can systematically extract and culture primary DPSCs, sort and culture PDGFRβ+DPSCs, and identify their ability to proliferate, migrate and promote angiogenesis, providing an experimental basis for in-depth research on their application in dental pulp regeneration.

[0141] Example 4 Animal experiment on the role of PDGFRβ+DPSCs in the development of dental pulp blood vessels

[0142] 4.1 Materials and reagents

[0143] Transgenic mice: PDGFRβ-CreERT2 and ROSA-DTA

[0144] SPF animal breeding facilities

[0145] 12-hour light / dark cycle

[0146] Tamoxifen corn oil solution

[0147] 1% sodium pentobarbital

[0148] Normal saline

[0149] Tissue fixatives, dehydrators, permeabilizers, and paraffin

[0150] Rotary microtome

[0151] Antigen retrieval reagents

[0152] Serum blocking solution

[0153] PDGFRβ, CD31 Antibodies

[0154] Fluorescence microscopy

[0155] 4.2 Experimental operation

[0156] 1. Mouse Model Establishment

[0157] PDGFRβ-CreERT2 and ROSA-DTA transgenic mice were housed under SPF conditions with a 12-h light / dark cycle.

[0158] Conditional knockout of PDGFRβ+ cells was achieved by breeding double transgenic mice (PDGFRβ-CreERT2 and ROSA-DTA).

[0159] In 4-week-old mice, Cre activity was induced using tamoxifen in corn oil (100 μg / g body weight, intraperitoneal injection, for three consecutive days).

[0160] 2. Experimental Grouping

[0161] The mice were divided into three groups: a developing permanent teeth control group (4 weeks old, 10 mice), an adult permanent teeth control group (8 weeks old, 10 mice), and a tamoxifen-treated group (8 weeks old, 10 mice).

[0162] 3. Specimen Collection and Processing

[0163] Mandibular molar samples were collected at different developmental stages for histological analysis. Mice were anesthetized with 1% sodium pentobarbital (50 mg / kg body weight) and the oral cavity was rinsed with normal saline. Mandibular molars were carefully dissected using a scalpel, and the collected mandibular molar samples were fixed, dehydrated, infiltrated, and embedded in paraffin.

[0164] 4. Tissue Sectioning and Staining

[0165] Tissue sections were prepared using a rotary microtome, followed by heat-induced antigen retrieval and antibody incubation.

[0166] 5. Fluorescence Microscope Observation

[0167] The samples were observed and images were captured using a fluorescence microscope to analyze vascularization.

[0168] like Figure 1 As shown, the present invention provides an in vitro extraction and amplification method for vascular development-related dental pulp stem cell subpopulations. First, primary DPSCs are extracted from dental pulp tissue by trypsin and collagenase digestion to prepare a single cell suspension. Then, these cells are sorted using flow cytometry. During the sorting process, the cell suspension is divided into a blank control group, an isotype control group, and a PDGFRβ staining group, and a PDGFRβ antibody is added to the PDGFRβ staining group. After incubation for 15 minutes, PDGFRβ+DPSCs are sorted by flow cytometry. Finally, the sorted PDGFRβ+DPSCs are inoculated in a DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2 for subsequent research. The control group is primary DPSCs (see Appendix Figure 1 A).

[0169] The cells were incubated with primary antibodies (anti-FN1 antibody, anti-PDGFRβ antibody) at 4°C overnight, and then treated with FITC-conjugated IgG secondary antibody for 2 h at room temperature. The cells were washed with PBS and fixed with 4% paraformaldehyde and DAPI reagent. Images were collected using a laser scanning confocal microscope. The results showed that this extraction protocol effectively sorted PDGFRβ+DPSCs, among which FN1 expression was strong (see Appendix Figure 1 BE).

[0170] like Figure 2As shown in the figure, the in vitro experiment studied the effect of PDGFRβ+DPSCs on the biological function of co-cultured HUVECs, and the conditioned medium was used to simulate the co-culture culture (see Appendix Figure 2 A), and compared the tube formation, colony formation and wound healing abilities of HUVECs. The proportion of G0 / G1 phase cells in HUVECs co-cultured with PDGFRβ+DPSCs was significantly reduced, suggesting that PDGFRβ+DPSCs enhanced their proliferation activity (see Appendix Figure 2 BC). The scratch, clone formation and tube formation experiments of HUVECs showed that PDGFRβ+DPSCs exhibited strong wound healing and tube formation abilities (see Appendix Figure 2 DJ). This indicates that PDGFRβ+DPSCs promote the proliferation and tube-forming ability of HUVECs and have a significant potential to promote vascularization.

[0171] like Figure 3 To further elucidate the role of PDGFRβ+ cells in the development of dental pulp blood vessels, PDGFRβ-CreERT2 / ROSA-DTA double transgenic mice were successfully constructed in vivo to conditionally knock out PDGFRβ+ cells (see Appendix Figure 3 A). The experimental mice were divided into three groups: a control group with developing permanent teeth (4 weeks old), a control group with adult permanent teeth (8 weeks old), and a tamoxifen-treated group (8 weeks old). Immunofluorescence results showed that the elimination of PDGFRβ+ cells led to a decrease in the number of blood vessels in the pulp tissue, as well as a decrease in the number of PDGFRβ+ cells surrounding the blood vessels (see Figure 2). Figure 3 B). This in vivo experiment validates the key role of PDGFRβ+ cells in the development of dental pulp blood vessels.

[0172] The PDGFRβ+DPSCs purified and cultured by the present invention have strong proliferation, migration and angiogenesis-promoting properties. In view of the above results, the present invention can, to a certain extent, achieve the stable acquisition of autologous dental pulp stem cells from donors of different ages and conditions with strong angiogenesis-promoting properties in a clinical environment. Compared with the existing scheme, the advantages of this scheme in stably obtaining autologous dental pulp stem cells from donors of different ages and conditions with strong angiogenesis-promoting properties are as follows:

[0173] ① Improve the stability and consistency of DPSCs: Accurately identifying and sorting the PDGFRβ+DPSCs subpopulation through single-cell sequencing and flow cytometry can significantly improve the consistency of DPSCs between different donors and reduce the variability caused by differences in donor age and conditions. Methods such as gene editing and antioxidant treatment may have difficulty dealing with the variability between different donors, but this solution solves this problem from the source by screening and enriching specific cell subpopulations.

[0174] ② Stem cells have stable angiogenesis ability: PDGFRβ+DPSCs subpopulation showed strong angiogenesis ability in in vitro experiments, which is of great significance in pulp regeneration and helps to improve the regeneration effect. Although environmental factor regulation and drug intervention can improve cell function to a certain extent, their effects may not be as significant and stable as directly using cell subpopulations with high angiogenesis ability.

[0175] ③ Overcoming the problem of stem cell aging: Through the regulation of the PDGFR / PI3K / AKT signaling pathway, the aging of PDGFRβ+DPSCs can be effectively delayed, and their high proliferation and migration abilities can be maintained, thereby ensuring their long-term effects in dental pulp regeneration. Methods such as gene editing and antioxidant treatment may have long-term safety issues, while this approach can more safely delay stem cell aging by naturally selecting highly functional cell subpopulations.

[0176] The above is only the best specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions within the technical scope disclosed in the present application shall be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for in vitro extraction and amplification of dental pulp stem cell subpopulations related to vascular development, characterized in that: The following steps are involved: S1. Obtain pulp tissue; S2, digestion of dental pulp tissue; S3, filtering the digested dental pulp tissue through a cell strainer to obtain a first cell suspension, centrifuging the first cell suspension, discarding the supernatant, adding PBS to resuspend the cells, and obtaining a second cell suspension; S4. Cell staining Adding equal amounts of the second cell suspension into different test tubes, setting them as a blank control group, an isotype control group, and a PDGFRβ staining group respectively; 1% BSA solution was added to each tube to block nonspecific binding, and the first incubation was performed in the dark; Adding diluted PDGFRβ antibody to the PDGFRβ staining group, adding diluted isotype control antibody to the isotype control group, and performing a second incubation in the dark; S5. Wash cells Wash the cells by adding PBS to all tubes, centrifuge, discard the supernatant, and wash with PBS, repeating at least twice; After the final wash, the cells were resuspended in PBS to obtain the third cell suspension; S6. Flow cytometry sorting Performing cell analysis and sorting on the third cell suspension using a flow cytometer, and collecting and sorting PDGFRβ+DPSCs; S7. The collected and sorted PDGFRβ+DPSCs are inoculated into DMEM culture medium and cultured in an incubator to achieve the expansion of the PDGFRβ+DPSCs.

2. The in vitro extraction and amplification method of vascular development-related dental pulp stem cell subpopulations according to claim 1, wherein in step S1, the dental pulp tissue is obtained, characterized in that: Specifically: Dental pulp tissues were obtained from healthy deciduous teeth extracted for tooth replacement or permanent premolars and third molars extracted for orthodontic reasons, and placed in a culture dish containing DMEM, 100 U / ml penicillin and 100 μg / ml streptomycin.

3. The in vitro extraction and amplification method of vascular development-related dental pulp stem cell subpopulations according to claim 1, wherein in step S2, the dental pulp tissue digestion is performed, characterized in that: Specifically: The dental pulp tissue was cut into small pieces, placed in a digestion solution containing 2 mg / ml collagenase and 0.25% trypsin, and digested in a 37° C. water bath for 1 hour.

4. The in vitro extraction and amplification method of vascular development-related dental pulp stem cell subpopulations according to claim 1, characterized in that: In step S3, the cell filter is a 70 μm cell filter; The first cell suspension is centrifuged as follows: the first cell suspension is centrifuged at 1000 rpm for 5 minutes using a centrifuge.

5. The in vitro extraction and amplification method of a vascular development-related dental pulp stem cell subpopulation according to any one of claims 1 to 4, characterized in that: In step S4, the first dark-proof incubation is specifically: incubating at 4° C. in dark for 10 minutes; The dilution multiple of the PDGFRβ antibody is 100 times, and the dilution method is: add 2 μl of PDGFRβ antibody into 198 μl of PBS; The dilution factor of the isotype control antibody is 100 times; The second dark-proof incubation is specifically: incubating in the dark at 4° C. for 15 minutes.

6. The in vitro extraction and amplification method of vascular development-related dental pulp stem cell subpopulations according to claim 5, characterized in that: In step S6, the parameters of the flow cytometer are set as follows: laser wavelength 488nm, detection channel FITC, and voltage setting 500V.

7. The in vitro extraction and amplification method of vascular development-related dental pulp stem cell subpopulations according to claim 5, characterized in that: In step S7, the DMEM medium contains 10% FBS and 1% penicillin-streptomycin; The incubator is a 37° C., 5% CO 2 incubator.

Images