Culture method and application of stem cells for reversing replicative senescence-induced apical papilla

Abstract

The invention provides a culture method for reversing replicative aging root tip tooth papilla stem cells and application of the culture method, and belongs to the technical field of biological medicine. The method comprises the following steps: overexpressing an OCT4 gene in the replicative senescence tooth apex papilla stem cells, and culturing the cells by adopting a culture medium containing methyl cellulose to obtain the reversed replicative senescence tooth apex papilla stem cells. The method can be used for dental differentiation and angiogenic differentiation of stem cells. Through the synergistic effect of stable overexpression of the slow virus-mediated OCT4 gene and a methylcellulose 3D balling culture system, reversal of replicative senescence of the stem cells of the apical papilla is realized, the proliferation efficiency and differentiation potential of the stem cells are remarkably improved, the source of the stem cells is further expanded, and the yield of the stem cells is greatly increased.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, relates to dental pulp regeneration and anti-aging drug screening, and specifically relates to a culture method for reversing replicative aging apical dental papilla stem cells and its application. Background Art

[0002] The large number of cells required for clinical treatment (hundreds of thousands to millions) requires stem cells to undergo more than 25 doublings, which greatly consumes their replication potential. Once this potential is exhausted, cells enter a state of replicative senescence, characterized by enlarged and flattened cells, decreased proliferation, and reduced differentiation potential. Late-passage stem cells, such as replicatively aged stem cells from the apical papilla (SCAP), further lose their stemness, immune regulation, and homing abilities. Transplanting such senescent cells not only has poor repair effects but may even be harmful to the recipient, seriously affecting their application in regenerative medicine.

[0003] Replicative senescence involves a variety of factors, including the production of reactive oxygen free radicals, activation of oncogenes or inactivation of tumor suppressor genes, radiation, and various cytokines and cell signal transduction. These changes are believed to be caused by differences in the growth environment of stem cells in vitro and in vivo. Compared with traditional 2D culture, 3D culture can better simulate the cellular microenvironment of stem cells in vivo, promote interactions and signal transduction between cells and between cells and the matrix. This culture method optimizes the cell growth environment and reduces oxidative stress and DNA damage, thereby providing strong support for the long-term culture and application of stem cells. In the field of regenerative medicine, 3D cell culture technology can delay cell replicative senescence and maintain its high proliferation capacity and differentiation potential. However, the yield of stem cells obtained by 3D culture is low, which has become a bottleneck problem restricting the field of regenerative medicine. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a culture method and application of stem cells of the apical papilla for reversing replicative aging, so as to solve the technical problem of low yield of replicative aging apical papilla stem cell balls (SCAP) with good biological functions that can be used for clinical transplantation in the existing technology.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for culturing apical dental papilla stem cells for reversing replicative aging, wherein the OCT4 gene is overexpressed in the replicative aging apical dental papilla stem cells, and the cells are cultured using a culture medium containing methylcellulose to obtain reversed replicative aging apical dental papilla stem cells.

[0007] Specifically, the final concentration of methylcellulose in the culture medium is 0.1 to 1 g / mL, preferably 0.2 to 0.6 g / mL, and most preferably 0.4 g / mL.

[0008] Specifically, the number of passages of the replicatively aged apical dental papilla stem cells is greater than or equal to 15 times.

[0009] Specifically, the overexpression of the OCT4 gene is at least 8-fold, more specifically, 8-10-fold.

[0010] Specifically, the expression of pluripotency factors SOX2 and NANOG was upregulated in the reversed replicatively aged apical papilla stem cells, while the expression of senescence-related molecules P16, P21, and P53 was downregulated in the cell spheres.

[0011] The present invention also has the following technical features:

[0012] Specifically, the culture method comprises the following steps:

[0013] Step 1: Obtain replicative senescent apical papilla stem cells:

[0014] Step 1.1, isolate and obtain primary apical dental papilla cells from teeth: store clinically collected teeth in basal culture medium, rinse until there is no obvious blood clot on the tooth surface, carefully separate the apical dental papilla tissue with scissors, then mince, add type I collagenase, and digest in a constant temperature environment; add basal culture medium to terminate digestion, centrifuge and discard the supernatant; add basal culture medium containing fetal bovine serum to resuspend the tissue block, inoculate it into a cell culture flask, and culture it in a constant temperature environment. During the culture period, replace fresh culture medium and observe under a microscope until the primary cells crawl out of the tissue block.

[0015] Specifically, in step 1.1, the centrifugal speed is 200-1000 rpm / min, the centrifugation time is 2-10 min, and the culture medium is replaced with fresh culture medium every 1-2 days during the culture period.

[0016] Step 1.2, subculture the primary apical dental papilla cells obtained in step 1.1 to obtain replicatively aged apical dental papilla stem cells: when the primary cells grow to 80% to 90% confluence, add trypsin containing EDTA to digest the cells, then subculture them into new culture dishes, and continuously subculture them to P15 to obtain replicatively aged apical dental papilla stem cells.

[0017] Specifically, in step 1.2, the concentration of trypsin is 0.25%.

[0018] Step 2: Overexpress the OCT4 gene in the replicatively aged apical papilla stem cells obtained in step 1:

[0019] Step 2.1, transfect the OCT4 gene recombinant lentiviral vector into the replicatively aged apical dental papilla stem cells: After the replicatively aged apical dental papilla stem cells were digested with trypsin containing EDTA, the cells were transfected with 1×10 5 Cells were seeded in a 6-well plate at a density of 1:100 / well. After 24 hours, the cell confluence reached 40%. Fresh basal medium was replaced. The infection volume of each well was 1 mL. Lentiviral transfection reagent was added at a concentration of 1:25 and mixed evenly with the cell culture supernatant. Subsequently, OCT4 gene recombinant lentiviral particles were added at a concentration of 5 MOI.

[0020] Step 2.2: Package and express the recombinant lentiviral vector encoding the OCT4 gene in replicatively aged apical papilla stem cells to obtain replicatively aged apical papilla stem cells harboring the OCT4 gene. Each time the cell culture medium is changed, puromycin is added to a final working concentration of 2.5 μg / mL to screen the cells. After 7 days, green fluorescence is detected in the nuclei of the replicatively aged apical papilla stem cells, demonstrating successful overexpression of the OCT4 gene.

[0021] Step 3: Establishment of 3D spheroid culture system:

[0022] Step 3.1, prepare a culture medium containing methylcellulose: add 50 mL of FBS, 5 mL of epithelial cell growth factor, and 5 mL of penicillin / streptomycin solution to 500 mL of endothelial cell culture medium and mix well to prepare an epithelial cell culture medium; mix the prepared epithelial cell culture medium with methylcellulose culture medium in a volume ratio (v / v) of 3:2, and mix well to obtain a culture medium containing methylcellulose, wherein the final concentration of methylcellulose is 0.4 g / mL.

[0023] Step 3.2, 3D sphere culture: Digest the replicatively aged apical papilla stem cells with integrated OCT4 gene cultured in step 2.2 into single cells, add 1 mL of basal medium to resuspend the cell pellet and count the cells. 4 The cells were mixed with the culture medium containing methylcellulose prepared in step 3.1 and plated into an ultra-low adsorption 24-well plate with an initial volume of 500 μL per well. 100 μL of fresh 3D culture medium was added every 2 days for 7 days.

[0024] Specifically, the content of fetal bovine serum in the cell culture medium is 10%.

[0025] Specifically, the basal culture medium is α-modified MEM medium.

[0026] Specifically, the constant temperature environment is an environment of 37° C. and a carbon dioxide concentration of 5%.

[0027] The present invention also protects the application of the above-mentioned method for culturing apical dental papilla stem cells for reversing replicative aging in stem cell odontogenic differentiation; specifically, by upregulating the expression levels of odontogenic-related genes RUNX2, ALP, OCN, DSP and DMP-1, the odontogenic differentiation of apical dental papilla stem cells is promoted.

[0028] The present invention also protects the use of the above-mentioned method for culturing apical papilla stem cells for reversing replicative aging in stem cell angiogenic differentiation; specifically, by upregulating the expression of angiogenic differentiation-related genes CD31 and VEGF, the angiogenic differentiation of apical papilla stem cells is promoted.

[0029] Compared with the prior art, the present invention has the following technical effects:

[0030] (I) The present invention reverses the replicative aging of apical papilla stem cells through the synergistic effect of lentivirus-mediated stable overexpression of the OCT4 gene and a methylcellulose 3D sphere culture system, significantly improving their proliferation efficiency and differentiation potential, further expanding the source of stem cells and greatly increasing the yield of stem cells.

[0031] (II) The present invention can be applied to the fields of dental pulp regeneration, anti-aging drug screening, etc., and has industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the fluorescence image of cells infected with OCT4 lentivirus;

[0033] Figure 2 Overexpression of OCT4 resulted in upregulation of SCAP stemness genes in 3D cultured replicative senescence.

[0034] Figure 3 Overexpression of OCT4 upregulated the protein expression of the aging SCAP pluripotency factor;

[0035] Figure 4 Overexpression of OCT4 downregulated the expression of senescence-associated β-galactosidase in senescent SCAPs;

[0036] Figure 5 Overexpression of OCT4 reduces the expression of senescence-associated genes in replicative senescence SCAP

[0037] Figure 6 Overexpression of OCT4 reduced the expression of senescence-associated proteins in senescent SCAPs;

[0038] Figure 7 Overexpression of OCT4 enhanced the proliferation capacity of replicatively aged SCAPs;

[0039] Figure 8Overexpression of OCT4 upregulated the expression of replicatively aged SCAP odontogenic differentiation proteins;

[0040] Figure 9 Overexpression of OCT4 enhanced the mineralization capacity of replicatively senescent SCAPs;

[0041] Figure 10 for Figure 11 HE staining of regenerated dental pulp-like tissue in nude mice;

[0042] Figure 11 Masson staining of regenerated dental pulp-like tissue in nude mice;

[0043] Figure 12 To investigate the expression of odontoblast differentiation proteins in dental pulp regeneration tissue in nude mice;

[0044] Figure 13 Expression of angiogenic differentiation proteins in dental pulp regeneration tissue in nude mice.

[0045] Figure 14 represents the expression levels of odontogenic differentiation and angiogenic differentiation genes in replicatively aged SCAP under different expression systems.

[0046] The specific contents of the present invention are further explained in detail below with reference to the embodiments. DETAILED DESCRIPTION

[0047]

[0048] It should be noted that all culture media and carriers used in the present invention, unless otherwise specified, are culture media known in the art. For example:

[0049] Endothelial cell culture medium is a commercially available product, brand ScienCell, product number 29679.

[0050] Methylcellulose culture medium is a commercially available product, brand name Stemcell, product number MethoCult TM H4100.

[0051] CMV-MCS-GFP-SV40-Puromycin is a commercially available product purchased from Shandong Weizhen Biotechnology Co., Ltd.

[0052] Specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made on the basis of the technical solution of this application fall within the protection scope of the present invention.

[0053] Example 1:

[0054] This example provides a method for culturing apical papilla stem cells to reverse replicative aging, which specifically includes the following steps:

[0055] Step 1: Obtain replicative senescent apical papilla stem cells:

[0056] Step 1.1, primary cell culture: clinically collected teeth were stored in α-MEM and rinsed with PBS until there was no obvious blood clot on the tooth surface. The root apex dental papilla tissue was carefully separated with scissors and cut into pieces of 1 mm in size. 3 About 1 mL of type I collagenase was added and digested in a 37°C, 5% CO2 incubator for 30 min. An equal volume of cell culture medium (α-MEM containing 20% ​​FBS) was added to terminate the digestion. The mixture was centrifuged at 800 rpm / min for 5 min and the supernatant was discarded. Cell culture medium (α-MEM containing 20% ​​FBS) was added to resuspend the tissue blocks and inoculated into T25 cell culture flasks. The cells were cultured in a constant temperature 37°C, 5% CO2 incubator. Fresh culture medium was replaced every 2 days and the primary cells were observed under a microscope to crawl out of the tissue blocks.

[0057] Step 1.2, establish a replicative aging model of apical papilla stem cells: when the screened primary cells grow to about 80% to 90% confluence, add 0.25% trypsin (containing EDTA) for digestion, subculture into 10 cm culture dishes, and continuously subculture to P15.

[0058] Step 2: Overexpression of the OCT4 gene in replicatively aged apical dental papilla stem cells:

[0059] Step 2.1, expression vector transfection: P5 generation SCAP was used to transfect CMV-MCS-GFP-SV40-Puromycin vector as a positive control (denoted as P5 GFP+ ), and P15 SCAP was transfected with CMV-MCS-GFP-SV40-Puromycin vector as negative control (denoted as P15 GFP+ ), P15 SCAP was transfected with CMV-MCS-GFP-Pou5f1-SV40-Puromycin vector as the experimental group (denoted as P15 OCT4+ ). P5 and P15 SCAP were digested with 0.25% trypsin-EDTA and the number of cells was 1×10 5 The cells were seeded in a 6-well plate at a density of 1:100 / well. After 24 hours, the cell confluence reached 40%. Fresh α-MEM culture medium was replaced with 1 mL of culture medium per well for infection. Lentiviral transfection reagent was added at a concentration of 1:25 and mixed evenly with the cell culture supernatant. Subsequently, CMV-MCS-GFP-SV40-Puromycin and CMV-MCS-GFP-Pou5f1-SV40-Puromycin viruses were added at an MOI of 5.

[0060] Step 2.2, intracellular expression of lentivirus: After 48 hours of cell incubation, the cells were observed under a fluorescence microscope, and more than 85% of the cells expressed green fluorescence. Subsequently, puromycin was added to the cells at a final working concentration of 2.5 μg / mL at each medium change to screen the cells. One week later, the positive control group SCAP P5 GFP+ and negative control group SCAP P15 GFP+ Empty vector was expressed in the cytoplasm, and green fluorescent marker protein GFP was expressed in the cytoplasm. OCT4+ After infection with lentivirus, only the nucleus expressed green fluorescence (e.g. Figure 1 shown).

[0061] Step 3: Establishment of 3D spheroid culture system:

[0062] Step 3.1, Preparation of 3D culture medium: Add 50 mL of FBS, 5 mL of epithelial cell growth factor, and 5 mL of penicillin / streptomycin solution to 500 mL of endothelial cell medium (ScienCell) and mix well to prepare epithelial cell culture medium. Mix the prepared epithelial cell culture medium with methylcellulose culture medium (containing 2.6% methylcellulose, when the volume is adjusted to 100 mL, the concentration of methylcellulose is 1.0%) in a volume ratio (v / v) of 3:2. Place on a vortex mixer and mix gently and thoroughly to avoid bubbles. After mixing, obtain 3D culture medium (the final concentration of methylcellulose in the culture medium is 0.4 g / mL).

[0063] Step 3.2, 3D sphere culture: Digest the SCAP cultured on the 2D plate into single cells, add 1 mL of α-MEM to resuspend the cell pellet and count the cells, using a 1×10 4 The cells were mixed with the 3D culture medium prepared in step 3.1 and plated into a 24-well ultra-low adsorption plate with an initial volume of 500 μL per well. 100 μL of fresh 3D culture medium was added every 2 days for 7 days.

[0064] Effect verification:

[0065] (A) Biological function assay of replicatively senescent SCAP cell spheroids overexpressing OCT4. The specific experimental steps are as follows:

[0066] Step 1: SCAP cell sphere stemness detection: qRT-PCR and immunofluorescence staining were used to detect P5 GFP+ 、P15 GFP+ 、P15 OCT4+ The expression of stemness factors OCT4, SOX2, and NANOG in cell spheres; Figure 2 As shown, qRT-PCR results showed that compared with senescent SCAP cell spheres (P15 GFP+ ) compared with OCT4 overexpression cell spheres (P15 OCT4+ ) in the OCT4 gene expression level is 8-10 times that of the senescent SCAP cell spheres, and the pluripotency factors SOX2 and NANOG are expressed. Figure 3 As shown, the results of immunofluorescence staining were consistent with those of qRT-PCR.

[0067] Real-time quantitative primer information is as follows: For OCT4, the upstream primer is 5'-gccgtatgagttctgtgggg-3', and the downstream primer is 5'-ctccttctccagcttcacgg-3'. For SOX2, the upstream primer is 5'-ACACC AATCCCATCCACACT-3', and the downstream primer is 5'-gcaaacttcctgcaaagctc-3'. For NANOG, the upstream primer is 5'-tgaacctcagctacaaacag-3', and the downstream primer is 5'-ctggatgttctgggtctggt-3'.

[0068] Step 2: SCAP cell spheroid senescence phenotype detection:

[0069] Step 2.1, galactosidase staining: P5 after 7 days of 3D culture GFP+ 、P15 GFP+ 、P15 OCT4+ The SCAP cell spheres were digested into single cells with 0.25% trypsin (containing EDTA), and cell culture medium (α-MEM containing 10% FBS) was added and seeded in 6-well plates, with 2×10 cells per well. 4 , culture overnight until the cells are stable. Remove the culture medium, wash twice with PBS, add 1mL SA-β-Gal galactosidase staining fixative, and fix at room temperature for 15 minutes. Remove the staining fixative, wash the 6-well plate 3 times with PBS, 5 minutes each time, remove PBS, and add 1mL of the staining working solution prepared according to the instructions to each well. Seal the 6-well plate with a film, incubate at 37°C overnight, and observe under an ordinary optical microscope. Figure 4 As shown, OCT4 overexpression cell spheres (P15 OCT4+ ) was significantly higher than that of the senescent SCAP cell spheres (P15 GFP+ ) dropped by 60%.

[0070] Step 2.2, senescence-related molecular detection: qRT-PCR and immunofluorescence staining detection of P5 GFP+ 、P15 GFP+ 、P15 OCT4+ The expression of senescence-related molecules P16, P21 and P53 in cell spheroids. Figure 5 and Figure 6 As shown, qRT-PCR results showed that compared with senescent SCAP cell spheres (P15 GFP+ ) compared with OCT4 overexpression cell spheres (P15 OCT4+ )'s P16, P21 and P53 expressions were significantly downregulated.

[0071] Step 2.3, SCAP cell sphere proliferation capacity detection: P5 after 7 days of 3D culture GFP+ 、P15 GFP+ 、P15 OCT4+ The SCAP cell spheres were digested into single cells with 0.25% trypsin (containing EDTA) and seeded into a small glass dish at a cell number of 1.2×10 5 , culture overnight until the cells are stable; dilute the 10mM EdU (5-Ethynyl-2'-deoxyuridine) stock solution to a working concentration of 20μM at a ratio of 1:500 (culture medium), add it to the small dish at a volume ratio (v / v, culture medium: working solution) of 1:1 and continue culturing to ensure that its final concentration is 10μM; after EdU labeling is completed, remove the culture medium, fix it with 4% PFA at room temperature for 15 minutes, wash it three times with washing solution, each time for 3 minutes; then add PBS containing 3% Triton X-100 to break the membrane for 10 minutes, wash it three times with PBS, each time for 5 minutes; according to the Click reaction table in the instruction manual, prepare an appropriate volume of Click reaction solution and add it to the well plate, incubate it at room temperature in the dark for 30 minutes. Finally, perform cell nucleus staining, incubate it at room temperature in the dark for 10 minutes, wash and seal the slides, and observe the positive staining results under a confocal microscope. Figure 7 As shown, compared with senescent SCAP cell spheres (P15 GFP+ ) compared with OCT4 overexpression cell spheres (P15 OCT4+ ), indicating that the cell proliferation activity of OCT4-overexpressing SCAP cell spheres was significantly increased.

[0072] Step 3: Detection of odontogenic differentiation ability of SCAP cell spheres: OCT4 cell spheres cultured for 7 days in 3D were taken, digested, and inoculated into 6-well culture plates, with 4×10 cells per well. 5 When the cells reached 80-90% confluence, the odontogenesis induction medium (α-MEM culture medium containing 10% FBS, dexamethasone 10%) was replaced. -8 The odontogenesis-related proteins RUNX2 / ALP / OCN / DSP / DMP-1 were analyzed by Western blot.

[0073] The final experimental results are as follows Figure 8 As shown in the figure, it can be seen that the expression of SCAP odontoblastic differentiation protein in replicative aging is decreased, and after overexpression of OCT4, the expression of SCAP odontoblastic differentiation protein in replicative aging is upregulated. Figure 9 As shown in Figure 3, the results of Alizarin red staining further confirmed the above results.

[0074] (B) Subcutaneous transplantation of SCAP cells into nude mice to verify the regenerative capacity of dental pulp in vivo. The specific experimental steps are as follows:

[0075] Step 1: Subcutaneous transplantation in nude mice: Extracted orthodontic premolars were collected, and the root section was removed to prepare dentin septa 3-5 mm long. After decalcification and disinfection, one end was sealed with MTA and placed in double-antibody α-MEM medium and stored at 4°C. Matrigel hydrogel containing the three SCAP cell spheres was injected into the prepared dentin cannula and incubated at 37°C for 30 minutes. After the Matrigel hydrogel solidified, α-MEM medium was added and stored. Eighteen 6-week-old nude mice of either sex, weighing 60-80 g, were randomly divided into three groups. Each nude mouse was anesthetized with 1% pentobarbital (0.005 mL / g) intraperitoneally. The dorsal skin was disinfected with 1% iodine. A 5-6 mm midline incision was made along the dorsal surface with a No. 11 scalpel. The skin on both sides was separated with a vascular clamp to form a 6-8 mm pouch with the subcutaneous space. The prepared dentin cannula and cell mixture were implanted. The suture was sutured with No. 4 sterile silk suture, and the dorsal skin was disinfected again with 75% alcohol. Provide good postoperative care and raise the animal under normal conditions after the anesthetic wears off and the animal wakes up.

[0076] Step 2: Histological examination of the regenerated pulp-dentin complex: Samples were collected 12 weeks after surgery, and each nude mouse was anesthetized with an overdose of 1 mL of pentobarbital, followed by spinal cord amputation. The dorsal skin was incised, and the dentin cannula was removed with a vascular clamp. The connective tissue on the surface of the cannula was carefully separated and fixed in 4% paraformaldehyde at 4°C for 24 hours. The samples were decalcified with rapid decalcification solution for 5 weeks, during which the decalcification solution was changed every 3 days. The samples were dehydrated with 30% sucrose at 4°C for 24 hours, embedded in OCT, and frozen into sections with a thickness of 7 mm. The samples were stained with HE and Masson staining to observe the newly formed calcified tissue, collagen matrix, and vascular tissue in the root canal cavity; immunofluorescence staining was used to detect the expression of proteins related to odontogenic differentiation (DSPP, DMP-1) and blood vessels (CD31, VEGF).

[0077] The final experimental results are as follows Figures 10 to 13 As shown. Figure 10 As shown, HE staining results showed that P5 GFP+ and P15 OCT4+ Vascular-like tissues can be seen in the regenerated tissue (indicated by black arrows). Figure 11 As shown in Figure 2, Masson staining showed a more compact collagen formation. Figure 12 As shown in the figure, the results of immunofluorescence staining showed that the expression of odontogenic differentiation-related proteins DSPP and DMP-1 was GFP+ and P15 OCT4+ The SCAP cell spheres were higher than those in the negative control group P15 GFP+ .like Figure 13As shown in Figure 2, the expression of angiogenic differentiation-related proteins CD31 and VEGF was significantly increased in P5 GFP+ and P15 OCT4+ The SCAP cell spheres were higher than those in the negative control group P15 GFP+ .

[0078] (C) Detection of gene expression related to replicative senescence SCAP cell spheres:

[0079] In order to further confirm the synergistic effect of overexpression vector and 3D spheroid culture, the present invention set up the following experimental groups and control groups: 2D culture + P15 generation SCAP transfected with CMV-MCS-GFP-SV40-Puromycin vector (blank group, marked as 2DP15), 2D culture + P15 generation SCAP transfected with CMV-MCS-GFP-Pou5f1-Puromycin vector (control group 1, marked as 2DP15 OCT4+ ), 3D culture + P15 generation SCAP transfected with CMV-MCS-GFP-SV40-Puromycin vector (control group 2, marked as 3DP15), 3D culture + P15 generation SCAP transfected with CMV-MCS-GFP-Pou5f1-Puromycin vector (experimental group, marked as 3DP15 OCT4+ ).

[0080] After the above cells were cultured and collected, the expression levels of RUNX2, ALP, OCN, CD31 and VEGF genes in the cells were detected by real-time quantitative PCR. The results were as follows: Figure 14 As shown. Figure 14 It can be seen that 3D P15 OCT4+ In the 3D spheroid culture, the expression levels of vascular-associated proteins CD31 and VEGF, as well as odontogenesis-related proteins RUNX2, ALP, and OCN, were much higher than those in the other three groups. This suggests that the overexpression vector and 3D spheroid culture have a synergistic effect in reversing replicatively senescent SCAP cells and can significantly promote the redifferentiation of reversed replicatively senescent SCAP cells.

[0081] Primer information for real-time quantification is as follows:

[0082] The upstream primer for qRT-PCR of RUNX2 gene was: 5′-tgcggtgcaaactttctcca-3′, and the downstream primer was: 5′-gcttgc agccttaaatgactctg-3′.

[0083] The upstream primer for qRT-PCR of the ALP gene was: 5′-ccttgtagccaggcccattg-3′, and the downstream primer was: 5′-ggaccattc ccacgtcttcac-3′.

[0084] The upstream primer for qRT-PCR of the OCN gene was: 5′-cccaggcgctacctgtatcaa-3′, and the downstream primer was: 5′-ggtcagc caactcgtcacagtc-3′.

[0085] The upstream primer for qRT-PCR of CD31 gene was: 5′-tggccagtcacttgaagaca-3′, and the downstream primer was: 5′-cagttgtt gtagccagccatt-3′.

[0086] The upstream primer of qRT-PCR for VEGF gene was: 5′-agcacagcagatgtgaatgc-3′, and the downstream primer was: 5′-aatgctt tctccgctctgaa-3′.

[0087] Table 1. Expression levels of vascular differentiation and odontogenic differentiation genes

[0088]

[0089] Note: The data in Table 1 are the data after the original data are normalized with 2D P15 (blank group) as 1.

Claims

1. A method for culturing apical papilla stem cells for reversing replicative aging, characterized in that: The OCT4 gene was overexpressed in replicatively aged apical dental papilla stem cells, and the cells were cultured in a medium containing methylcellulose to obtain reversed replicatively aged apical dental papilla stem cells.

2. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 1, wherein: The final concentration of methylcellulose in the culture medium is 0.1-1 g / mL.

3. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 1, wherein: The number of passages of replicatively aged apical dental papilla stem cells is greater than or equal to 15 times.

4. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 1, wherein: The overexpression of OCT4 gene was at least 8-fold.

5. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 1, wherein: The method comprises the following steps: Step 1: Obtaining replicatively aged apical dental papilla stem cells: isolating and obtaining primary apical dental papilla cells from teeth; then subculturing the obtained primary apical dental papilla cells to obtain replicatively aged apical dental papilla stem cells; Step 2: Overexpressing the OCT4 gene in the replicatively aged apical dental papilla stem cells obtained in step 1: transfecting the OCT4 gene recombinant lentiviral vector into the replicatively aged apical dental papilla stem cells; packaging and expressing the OCT4 gene recombinant lentiviral vector in the replicatively aged apical dental papilla stem cells to obtain replicatively aged apical dental papilla stem cells integrated with the OCT4 gene; Step 3: Establishment of a 3D sphere culture system: Prepare a culture medium containing methylcellulose; Use the prepared culture medium containing methylcellulose to subculture the replicatively aged apical papilla stem cells integrated with the OCT4 gene.

6. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 5, wherein: Step one includes: storing the collected teeth in a basal culture medium, rinsing until there is no obvious blood clot on the surface of the teeth, carefully separating the dental papilla tissue of the root apex with scissors, then chopping it into pieces, adding type I collagenase and digesting it in a constant temperature environment; adding a basal culture medium to terminate the digestion, discarding the supernatant after centrifugation; adding a basal culture medium containing fetal bovine serum to resuspend the tissue blocks, inoculating them into a cell culture flask, and culturing them in a constant temperature environment. During the culture period, fresh culture medium is replaced, and primary cells are observed under a microscope to crawl out of the tissue blocks; when the primary cells grow to 80% to 90% fusion, the cells are digested and then continuously passaged.

7. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 5, wherein: Step 2 includes: digesting the replicative senescent apical papilla stem cells and 5 The cells were seeded at a density of 100 μg / well in a cell culture multi-well plate. When the cell confluence reached 40-50%, fresh basal medium was replaced, and lentiviral transfection reagent was added at a concentration of 1:

25. The mixture was evenly mixed with the cell culture supernatant. Then, OCT4 gene recombinant lentiviral particles were added at a concentration of 5 MOI for cell culture. Each time the cell medium was changed, puromycin was added to screen the cells until the OCT4 gene was successfully overexpressed in the replicatively aged apical papilla stem cells.

8. The method for culturing apical papilla stem cells for reversing replicative aging according to claim 5, wherein: Step three includes: adding fetal bovine serum, epithelial cell growth factor and penicillin-streptomycin double antibody solution to the endothelial cell culture medium, mixing and preparing the epithelial cell culture medium; mixing the prepared epithelial cell culture medium with methylcellulose culture medium in a volume ratio of 3:2, and obtaining a culture medium containing methylcellulose; digesting the replicative senescent root apical papilla stem cells integrated with the OCT4 gene into single cells, adding basal culture medium to resuspend the cell pellet and counting, and 1×10 per well. 4 The cells were mixed with a culture medium containing methylcellulose, plated into a cell culture multi-well plate, and cultured for at least 7 days.

9. Use of the culture method for reversing replicative aging of apical dental papilla stem cells according to any one of claims 1 to 8 in odontogenic differentiation of stem cells.

10. Use of the culture method for reversing replicative aging of apical papilla stem cells according to any one of claims 1 to 8 in angiogenic differentiation of stem cells.

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