Use of a polypeptide in the preparation of a medium for promoting osteogenic differentiation of dental pulp stem cells

Abstract

The application provides a polypeptide in the preparation of a culture medium for promoting the osteogenic differentiation of dental pulp stem cells, and belongs to the technical field of dental pulp stem cells. The culture medium provided by the application contains an effective promoting component Cr-ACP1 polypeptide, and the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV. When the culture is used to culture dental pulp stem cells, the osteogenic differentiation of the dental pulp stem cells can be effectively accelerated.

Description

[0001] This case is a divisional application. The original application was entitled "A method for promoting the mineralization of dental pulp stem cells". The original application was filed on June 21, 2022, and the original application number was CN202210706020.5. Technical Field

[0002] This invention belongs to the field of dental pulp stem cell technology, and particularly relates to the application of a polypeptide in the preparation of a culture medium for promoting osteogenic differentiation of dental pulp stem cells. Background Technology

[0003] Dental pulp stem cells are dental mesenchymal stem cells isolated from human dental pulp tissue, possessing self-renewal and high proliferative capacity. When stimulated by odontoblast apoptosis, they proliferate and differentiate into odontoblasts, which can further form dentin. Simultaneously, under appropriate in vitro induction, they can differentiate into osteoblasts / odontoblasts, adipocytes, and neuroblasts. Therefore, dental pulp stem cells offer a significant opportunity for constructing tissue-engineered teeth and modifying dental pulp injuries.

[0004] Dental caries is a common oral disease. When caries progresses to the dentin, bacteria and toxic substances secreted by cells can invade the dental pulp tissue through the dentinal tubules, leading to varying degrees of tooth damage. In this case, tooth reconstruction is an ideal treatment. The reconstruction process requires the osteogenic differentiation of dental pulp stem cells to achieve calcification and tooth reconstruction. Therefore, how to achieve rapid osteogenic differentiation and calcification of dental pulp stem cells is a problem that needs to be solved.

[0005] Cr-ACP1 is a 9-amino acid polypeptide extracted from cycads, with the amino acid sequence AWKLFDDGV. Currently, Cr-ACP1 is mainly used for anti-tumor and antibacterial purposes, but there are no reports on its application in dental pulp stem cells. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of rapid osteogenic differentiation and calcification of dental pulp stem cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for promoting the mineralization of dental pulp stem cells, the method comprising adding Cr-ACP1 peptide to osteogenic induction medium to promote the mineralization level of dental pulp stem cells, wherein the amino acid sequence of Cr-ACP1 peptide is AWKLFDDGV; the amount of Cr-ACP1 peptide added is greater than 50 μg / ml; and the osteogenic induction medium consists of DMEM + 10% FBS + 10 mmol / L, sodium β-glycerophosphate, 50 g / L ascorbic acid and 0.1 μmol / L dexamethasone.

[0009] Application of Cr-ACP1 polypeptide as the sole active ingredient in the preparation of a drug formulation that promotes osteogenic differentiation of dental pulp stem cells.

[0010] Preferably, the amino acid sequence of the Cr-ACP1 polypeptide is AWKLFDDGV.

[0011] Preferably, the pharmaceutical preparation consists of a Cr-ACP1 peptide and a pharmaceutically acceptable carrier.

[0012] Preferably, the dosage form of the pharmaceutical preparation includes solid dosage form and liquid dosage form.

[0013] Preferably, the Cr-ACP1 polypeptide promotes the protein expression of osteogenic differentiation-related proteins RUNX2 and OCN in dental pulp stem cells, the Cr-ACP1 polypeptide promotes the activity of ALP enzyme in dental pulp stem cells, and the Cr-ACP1 polypeptide promotes calcification of dental pulp stem cells.

[0014] Application of Cr-ACP1 peptide as the sole active ingredient in the preparation of a culture medium for promoting osteogenic differentiation of dental pulp stem cells.

[0015] Preferably, the sequence of the Cr-ACP1 polypeptide is AWKLFDDGV.

[0016] The beneficial effects of this invention are:

[0017] This invention discovers a new use of Cr-ACP1 peptide in promoting dental pulp stem cell calcification and osteogenic differentiation, and invents a new method for promoting dental pulp stem cell calcification. Attached Figure Description

[0018] Figure 1 Effects of different concentrations of Cr-ACP1 peptide on osteogenic differentiation-related proteins RUNX2 and OCN in dental pulp stem cells;

[0019] Figure 2 Effects of different concentrations of Cr-ACP1 peptide on ALP enzyme in dental pulp stem cells;

[0020] Figure 3 The effect of different concentrations of Cr-ACP1 peptide on the calcification level of dental pulp stem cells. Detailed Implementation

[0021] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0022] Example 1

[0023] (1) Dental pulp stem cells were seeded in 6-well cell culture plates. The experimental groups were blank control group, 0 μg / ml Cr-ACP1 peptide group, 50 μg / ml Cr-ACP1 peptide group, 100 μg / ml Cr-ACP1 peptide group, and 200 μg / ml Cr-ACP1 peptide group, with 3 replicates in each group.

[0024] (2) Blank control group: cultured in complete culture medium; 0 μg / ml Cr-ACP1 peptide group: cultured in osteogenic induction medium containing 0 μg / ml Cr-ACP1 peptide (DMEM + 10% FBS + 10 mmol / L, β-glycerophosphate sodium, 50 g / L ascorbic acid and 0.1 μmol / L dexamethasone); 50 μg / ml Cr-ACP1 peptide group: cultured in osteogenic induction medium containing 50 μg / ml Cr-ACP1 peptide; 100 μg / ml Cr-ACP1 peptide group: cultured in osteogenic induction medium containing 100 μg / ml Cr-ACP1 peptide; 200 μg / ml Cr-ACP1 peptide group: cultured in osteogenic induction medium containing 200 μg / ml Cr-ACP1 peptide.

[0025] (3) After 8 days of osteogenic differentiation culture, the culture medium was removed and the cells were washed with PBS;

[0026] (4) Add 100 μl of protein lysis buffer to each well, scrape the cells off with a cell scraper, collect the cells into an EP tube, and lyse on ice for 30 min;

[0027] (5) After the pyrolysis is completed, place the EP tube in a centrifuge, adjust the temperature to 4℃, the rotation speed to 12000rpm, and the centrifugation time to 20min.

[0028] (6) After centrifugation, collect the supernatant, use the Beyotime BCA kit to detect the protein concentration, add protein loading buffer, boil for 5 minutes, and obtain the required protein sample.

[0029] (7) Prepare separating gel and stacking gel, install electrophoresis rack, load protein samples and protein markers, and perform electrophoresis. First, electrophore at 90V until the protein markers separate, then increase the voltage to 120V until the bromophenol blue is close to the bottom of the separating gel.

[0030] (8) After electrophoresis, assemble the electrophoresis clamp and electrophoresis box according to the classic "sandwich" model, and electrophoresis at 250mA for 1.5h;

[0031] (9) Place the electrospun membrane in a blocking solution and seal it at room temperature for 1 hour;

[0032] (10) After the sealing is completed, RUNX2 (1:5000), OCN (1:3000) and β-actin are incubated at 4°C overnight;

[0033] (11) After incubation, the primary antibody was recovered, the membrane was washed with TBST, and the corresponding secondary antibody was incubated at room temperature for 1 hour.

[0034] (12) After incubation, discard the secondary antibody, wash the membrane, and develop it in a dark room.

[0035] Example 2

[0036] (1) Dental pulp stem cells were seeded in 12-well plates and the cells were divided into the following groups: blank control group, 3 replicates; 0 μg / ml Cr-ACP1 peptide group, 6 replicates; 100 μg / ml Cr-ACP1 peptide group, 6 replicates;

[0037] (2) Blank control group: cultured in complete culture medium; 0 μg / ml Cr-ACP1 peptide group: osteogenic induction culture medium containing 0 μg / ml Cr-ACP1 peptide; 200 μg / ml Cr-ACP1 peptide group: osteogenic induction culture medium containing 200 μg / ml Cr-ACP1 peptide.

[0038] (3) After osteogenic culture for 14 days, the culture medium was removed, the cells were washed with PBS, fixed with formaldehyde, and then alkaline phosphatase staining solution was added for staining. After 30 minutes, the staining solution was removed and the wells of the culture plate were photographed.

[0039] (4) At the same time, the 0 μg / ml Cr-ACP1 polypeptide group and the 200 μg / ml Cr-ACP1 polypeptide group were quantitatively detected according to the alkaline phosphatase detection kit, and the absorbance was measured at a wavelength of 405 nm.

[0040] Example 3

[0041] (1) Dental pulp stem cells were seeded in 12-well plates and the cells were divided into the following groups: blank control group (3 replicates), 0 μg / ml Cr-ACP1 peptide group (3 replicates), and 100 μg / ml Cr-ACP1 peptide group (3 replicates).

[0042] (2) Blank control group: cultured in complete culture medium; 0 μg / ml Cr-ACP1 peptide group: osteogenic induction culture medium containing 0 μg / ml Cr-ACP1 peptide; 200 μg / ml Cr-ACP1 peptide group: osteogenic induction culture medium containing 200 μg / ml Cr-ACP1 peptide.

[0043] (3) After culturing for 14 days, remove the culture medium and add paraformaldehyde to fix the cells;

[0044] (4) After fixation, wash the cells with PBS and incubate them with Alizarin Red S staining solution in the dark for 30 min.

[0045] (5) After removing the staining solution, wash the cells with PBS and take pictures;

[0046] (6) Then, 0 μg / ml Cr-ACP1 peptide group and 200 μg / ml Cr-ACP1 peptide group were added with 10% CPC, and the absorbance at 570 nm was detected with reference to the Alizarin Red S quantitative kit.

[0047] Experimental results:

[0048] (1) The experimental results of Example 1 are as follows Figure 1 As shown in the figure, treatment of dental pulp stem cells with Cr-ACP1 peptide can effectively promote the expression of osteogenic differentiation-related proteins RUNX2 and OCN, and the promoting effect is more obvious with increasing concentration.

[0049] (2) The experimental results of Example 2 are as follows Figure 2 As shown in the figure, the staining intensity and quantitative data of ALP enzyme in the 200 μg / ml Cr-ACP1 peptide group were 2.11 ± 0.16, which were significantly higher than those in the 0 μg / ml Cr-ACP1 peptide group. Therefore, the addition of Cr-ACP1 peptide can effectively promote the activity of ALP enzyme in dental pulp stem cells.

[0050] (3) The experimental results of Example 3 are as follows Figure 3 As shown, the calcium nodules in the 200 μg / ml Cr-ACP1 peptide group are more tightly packed and more numerous, and the quantitative result is 1.93±0.07, which is also significantly higher than that in the 0 μg / ml Cr-ACP1 peptide group. Therefore, the addition of Cr-ACP1 peptide can effectively promote the calcification of dental pulp stem cells, thereby helping to further generate teeth.

[0051] Based on the above results, we can conclude that Cr-ACP1 peptide can effectively promote the expression of osteogenic transformation-related proteins RUNX2 and OCN in dental pulp stem cells, and can effectively promote the activity of ALP enzyme. At the same time, it can effectively promote the calcification of dental pulp stem cells. Therefore, it can be used to prepare drugs that promote the calcification and osteogenic differentiation of dental pulp stem cells.

Claims

1. The application of Cr-ACP1 polypeptide as the sole active ingredient in the preparation of a culture medium for promoting osteogenic differentiation of dental pulp stem cells, characterized in that, The sequence of the Cr-ACP1 polypeptide is AWKLFDDGV. The amount of Cr-ACP1 peptide added to the culture medium is greater than 50 µg / ml; The culture medium consisted of DMEM, 10% FBS, 10 mmol / L sodium β-glycerophosphate, 50 g / L ascorbic acid, and 0.1 µmol / L dexamethasone.

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