CN114921404A - Method for promoting mineralization of dental pulp stem cells - Google Patents
Method for promoting mineralization of dental pulp stem cells Download PDFInfo
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- CN114921404A CN114921404A CN202210706020.5A CN202210706020A CN114921404A CN 114921404 A CN114921404 A CN 114921404A CN 202210706020 A CN202210706020 A CN 202210706020A CN 114921404 A CN114921404 A CN 114921404A
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- 210000005258 dental pulp stem cell Anatomy 0.000 title claims abstract description 43
- 230000001737 promoting effect Effects 0.000 title claims abstract description 14
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- 230000033558 biomineral tissue development Effects 0.000 title claims abstract description 9
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- 229920001184 polypeptide Polymers 0.000 claims abstract description 67
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 67
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 67
- 230000006698 induction Effects 0.000 claims abstract description 14
- 230000002308 calcification Effects 0.000 claims abstract description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 4
- 235000000069 L-ascorbic acid Nutrition 0.000 claims abstract description 4
- 239000002211 L-ascorbic acid Substances 0.000 claims abstract description 4
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 4
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims abstract description 4
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- 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
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1361—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from dental pulp or dental follicle stem cells
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Abstract
The invention provides a method for promoting dental pulp stem cell mineralization, and belongs to the technical field of dental pulp stem cells. The method provided by the invention is characterized in that Cr-ACP1 polypeptide is added into an osteogenic induction culture medium to promote the mineralization level of dental pulp stem cells, and the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV; the addition amount of the Cr-ACP1 polypeptide is more than 50 mug/ml, and the components of the osteogenesis induction culture medium are DMEM +10% FBS +10mmol/L, beta-sodium glycerophosphate, 50g/L ascorbic acid and 0.1 mug/L dexamethasone. The method provided by the invention can effectively promote the calcification level of the dental pulp stem cells.
Description
Technical Field
The invention belongs to the technical field of dental pulp stem cells, and particularly relates to a method for promoting mineralization of dental pulp stem cells.
Background
Dental pulp stem cells are odontogenic mesenchymal stem cells isolated from human dental pulp tissue, and have self-renewal ability and high proliferation ability. When it is stimulated by the apoptosis of odontoblasts, it proliferates and differentiates into odontoblasts, and further dentin can be formed. At the same time, when properly induced in vitro, it can differentiate in the osteogenic/odontoblastic, adipogenic and neurogenic directions. Thus, dental pulp stem cells provide a significant opportunity for the construction of tissue engineered teeth and for the modification of dental pulp lesions.
Caries is a common oral disease, and when caries progresses to proper dentin, toxic substances secreted by bacteria and cells invade dental pulp tissues through dentinal tubules in teeth, thereby causing damage to the teeth to various degrees. In this case, the reconstruction of the teeth is an ideal treatment. In the process of reconstructing teeth, osteogenic differentiation of dental pulp stem cells needs to be realized, so as to realize calcification and tooth reconstruction, and therefore how to realize rapid osteogenic differentiation and calcification of dental pulp stem cells is a problem to be solved at present.
The Cr-ACP1 is a polypeptide of 9 amino acids extracted from cycas revoluta, the amino acid sequence of the polypeptide is AWKLFDDGV, at present, Cr-ACP1 is mostly used for resisting tumors and bacteria, and no report is provided on research on dental pulp stem cells.
Disclosure of Invention
The invention aims to solve the problem of rapid osteogenic differentiation and calcification of dental pulp stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for promoting mineralization of dental pulp stem cells, which comprises the step of adding a Cr-ACP1 polypeptide to an osteogenic induction medium to promote the mineralization level of the dental pulp stem cells, wherein the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV; the addition amount of the Cr-ACP1 polypeptide is more than 50 mug/ml, and the components of the osteogenesis induction culture medium are DMEM +10% FBS +10mmol/L, beta-sodium glycerophosphate, 50g/L ascorbic acid and 0.1 mug/L dexamethasone.
The application of the Cr-ACP1 polypeptide as the only active component in preparing the dental pulp stem cell osteogenic differentiation promoting pharmaceutical preparation.
Preferably, the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV.
Preferably, the pharmaceutical formulation consists of a Cr-ACP1 polypeptide and a pharmaceutically acceptable carrier.
Preferably, the dosage form of the pharmaceutical formulation includes a solid dosage form and a liquid dosage form.
Preferably, the Cr-ACP1 polypeptide promotes protein expression of osteogenic differentiation related proteins RUNX2 and OCN in dental pulp stem cells, the Cr-ACP1 polypeptide promotes activity of ALP enzymes in dental pulp stem cells, and the Cr-ACP1 polypeptide promotes calcification of dental pulp stem cells.
Use of a Cr-ACP1 polypeptide as the sole active ingredient in the preparation of a medium for promoting osteogenic differentiation of dental pulp stem cells.
Preferably, the Cr-ACP1 polypeptide has a sequence of AWKLFDDGV.
The invention has the beneficial effects that:
the invention finds a new application of the Cr-ACP1 polypeptide in promoting calcification and osteogenic differentiation of dental pulp stem cells, and invents a new method for promoting calcification of dental pulp stem cells.
Drawings
FIG. 1 shows the effect of Cr-ACP1 polypeptides at different concentrations on the osteogenic differentiation-associated proteins RUNX2 and OCN in dental pulp stem cells;
FIG. 2 effect of various concentrations of Cr-ACP1 polypeptide on dental pulp stem cell ALP enzyme;
figure 3 effect of different concentrations of Cr-ACP1 polypeptide on the level of calcification of dental pulp stem cells.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
(1) Inoculating the dental pulp stem cells into a 6-hole cell culture plate, and grouping the experiment into a blank control group, a 0 mug/ml Cr-ACP1 polypeptide group, a 50 mug/ml Cr-ACP1 polypeptide group, a 100 mug/ml Cr-ACP1 polypeptide group and a 200 mug/ml Cr-ACP1 polypeptide group, wherein each group is provided with 3 repetitions;
(2) blank control group: culturing with complete culture medium; 0 μ g/ml Cr-ACP1 polypeptide group was cultured using osteogenic induction medium containing 0 μ g/ml Cr-ACP1 polypeptide (DMEM +10% FBS +10mmol/L, beta-sodium glycerophosphate, 50g/L ascorbic acid and 0.1 μmol/L dexamethasone); 50 microgram/ml Cr-ACP1 polypeptide group: culturing in an osteogenic induction medium containing 50 microgram/ml Cr-ACP1 polypeptide; 100 mug/ml Cr-ACP1 polypeptide group: culturing in an osteogenic induction medium containing 100 mug/ml Cr-ACP1 polypeptide; 200 mug/ml Cr-ACP1 polypeptide group: culturing in an osteogenic induction medium containing 200 microgram/ml Cr-ACP1 polypeptide;
(3) after osteogenic differentiation culture for 8 days, the medium was removed and the cells were washed with PBS;
(4) adding 100 ul of protein lysate into each hole, scraping the cells by using a cell scraper, collecting the cells into an EP (EP) tube, and cracking the cells for 30min on ice;
(5) after cracking, placing the EP tube in a centrifuge, adjusting the temperature to 4 ℃, the rotating speed to 12000rpm, and the centrifuging time to 20min for centrifuging;
(6) after centrifugation is finished, collecting supernatant, detecting the protein concentration by using a Byunnan BCA kit, adding a protein loading buffer solution, and boiling for 5min to obtain a required protein sample;
(7) preparing separation gel and concentrated gel, installing an electrophoresis frame, carrying out loading on a protein sample and a protein Marker, carrying out electrophoresis, firstly carrying out 90V electrophoresis until the protein Marker is layered, and then increasing the voltage to 120V until bromophenol blue approaches the bottom of the separation gel;
(8) after electrophoresis is finished, assembling an electric rotating clamp according to a classic sandwich model, assembling an electric rotating box, and performing 250mA electric rotation for 1.5 h;
(9) placing the membrane after the electrotransformation in a sealing solution, and sealing for 1h at room temperature;
(10) after the blocking is finished, incubating RUNX2 (1: 5000), OCN (1: 3000) and beta-actin, and incubating overnight at 4 ℃;
(11) after the incubation is finished, recovering the primary antibody, washing the membrane with TBST, incubating the corresponding secondary antibody, and incubating for 1h at room temperature;
(12) after the incubation, the secondary antibody was discarded, and after washing the membrane, development was performed in a dark room.
Example 2
(1) Dental pulp stem cells were seeded in 12-well plates, and the cells were grouped as: blank control group, 3 replicates, 0 mug/ml Cr-ACP1 polypeptide group, 6 replicates; 100 mug/ml Cr-ACP1 polypeptide group, 6 replicates;
(2) blank control group: complete medium was used for the culture, 0 μ g/ml Cr-ACP1 polypeptide group: using an osteogenic induction medium containing 0 mug/ml of Cr-ACP1 polypeptide, 200 mug/ml of Cr-ACP1 polypeptide group: using an osteogenic induction medium containing 200 mug/ml of Cr-ACP1 polypeptide;
(3) removing the culture medium after osteogenic culture for 14 days, washing with PBS, fixing cells with formaldehyde, adding alkaline phosphatase staining solution for staining, removing the staining solution after 30min, and taking a picture of the small hole of the culture plate;
(4) meanwhile, the Cr-ACP1 polypeptide group with the concentration of 0 mug/ml and the Cr-ACP1 polypeptide group with the concentration of 200 mug/ml are quantitatively detected by referring to an alkaline phosphatase detection kit, and the absorbance is measured at the wavelength of 405 nm.
Example 3
(1) The dental pulp stem cells were seeded in 12-well plates and grouped as: 3 replicates in a blank control group, and 3 replicates in a 0 mug/ml Cr-ACP1 polypeptide group; 100 mug/ml Cr-ACP1 polypeptide group, 3 replicates;
(2) blank control group: complete culture medium was used for culture, 0 μ g/ml Cr-ACP1 polypeptide group: using an osteogenic induction medium containing 0 mug/ml of Cr-ACP1 polypeptide, 200 mug/ml of Cr-ACP1 polypeptide group: using an osteogenic induction medium containing 200 mug/ml of Cr-ACP1 polypeptide;
(3) after 14 days of culture, removing the culture medium, and adding paraformaldehyde to fix the cells;
(4) after fixation is finished, washing cells by using PBS, and incubating for 30min in a dark place by using alizarin red S staining solution;
(5) after removing the staining solution, washing the cells with PBS, and taking a picture;
(6) and then adding 10% CPC into the 0 mug/ml Cr-ACP1 polypeptide group and the 200 mug/ml Cr-ACP1 polypeptide group, and detecting the light absorption value at 570nm by referring to an alizarin red S quantitative kit.
The experimental results are as follows:
(1) as shown in fig. 1, the experimental results of example 1 show that, after the dental pulp stem cells are treated with the Cr-ACP1 polypeptide, the expression of osteogenic differentiation-associated proteins RUNX2 and OCN in the dental pulp stem cells can be effectively promoted, and the promoting effect is more obvious as the concentration is increased.
(2) The experimental result of example 2 is shown in fig. 2, and it can be seen from the figure that the staining intensity and quantitative data of the ALP enzyme in the Cr-ACP1 polypeptide group of 200 mug/ml are 2.11 ± 0.16, which are significantly higher than those in the Cr-ACP1 polypeptide group, so that the addition of the Cr-ACP1 polypeptide can effectively promote the activity of the ALP enzyme in the dental pulp stem cells.
(3) The experimental result of example 3 is shown in fig. 3, and it can be seen that calcium nodules of the 200 mug/ml Cr-ACP1 polypeptide group are more closely arranged and more increased, and the quantitative result is 1.93 ± 0.07, which is also significantly higher than that of the 0 mug/ml Cr-ACP1 polypeptide group, so that the addition of the Cr-ACP1 polypeptide can effectively promote calcification of dental pulp stem cells, thereby facilitating further tooth generation.
In combination with the above results, we have found that Cr-ACP1 polypeptide can effectively promote the expression of proteins RUNX2, and OCN associated with osteogenic transformation in dental pulp stem cells, and can effectively promote the activity of ALP enzyme, and at the same time, can effectively promote calcification of dental pulp stem cells, and thus can be used for the preparation of a medicament for promoting calcification and osteogenic differentiation of dental pulp stem cells.
Claims (8)
1. A method for promoting mineralization of dental pulp stem cells, which is characterized in that Cr-ACP1 polypeptide is added into an osteogenesis induction medium to promote the mineralization level of the dental pulp stem cells, wherein the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV; the addition amount of the Cr-ACP1 polypeptide is more than 50 mug/ml, and the components of the osteogenesis induction culture medium are DMEM +10% FBS +10mmol/L, beta-sodium glycerophosphate, 50g/L ascorbic acid and 0.1 mug/L dexamethasone.
The application of the Cr-ACP1 polypeptide as the only active component in preparing the dental pulp stem cell osteogenic differentiation promoting pharmaceutical preparation.
3. The use of claim 2, wherein the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV.
4. The use of claim 2, wherein said pharmaceutical formulation consists of a Cr-ACP1 polypeptide and a pharmaceutically acceptable carrier.
5. The use according to claim 2, wherein the dosage form of the pharmaceutical formulation comprises a solid dosage form and a liquid dosage form.
6. The use of claim 2, wherein said Cr-ACP1 polypeptide promotes protein expression of osteogenic differentiation-associated proteins RUNX2 and OCN in dental pulp stem cells, said Cr-ACP1 polypeptide promotes activity of dental pulp stem cell ALP enzyme, and said Cr-ACP1 polypeptide promotes calcification of dental pulp stem cells.
Use of a Cr-ACP1 polypeptide as the sole active ingredient in the preparation of a medium for promoting osteogenic differentiation of dental pulp stem cells.
8. The use of claim 7, wherein the Cr-ACP1 polypeptide has a sequence of AWKLFDDGV.
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| CN202210706020.5A CN114921404B (en) | 2022-06-21 | 2022-06-21 | Method for promoting mineralization of dental pulp stem cells |
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| CA2875418A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| US20200306338A1 (en) * | 2019-03-26 | 2020-10-01 | National Guard Health Affairs | Antiviral peptides for treatment of the middle east respiratory syndrome |
| WO2022036010A1 (en) * | 2020-08-11 | 2022-02-17 | Cellacure Llc | Green closed loop bio-waste refining process for producing smart active extracts and delivery systems for their application |
| CN114948985A (en) * | 2022-06-06 | 2022-08-30 | 东枫堂(山东)生物科技发展有限公司 | Biological preparation for inhibiting adipose tissue-derived stem cells from generating adipose cells |
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| CN114921404B (en) | 2023-07-21 |
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