Chir99021 inducer for regulating autophagy and promoting differentiation of dental pulp stem cells and preparation method of Chir99021 inducer

Abstract

The invention discloses an inducer for promoting differentiation of dental pulp stem cells by regulating autophagy through Chir99021 and a preparation method of the inducer, and belongs to the technical field of biomedicine. The Chir99021 inducer for regulating and controlling autophagy and promoting dental pulp stem cell differentiation is prepared from Chir99021, rapamycin, lithium chloride, ascorbic acid, beta-sodium glycerophosphate and a solvent. According to the invention, by constructing a composite induction system, collaborative regulation of autophagy and differentiation is realized; through the synergistic effect of all the components, the multidirectional differentiation efficiency of the dental pulp stem cells can be remarkably improved, the osteogenic differentiation calcium nodule formation amount is improved by 60% or above compared with that of an existing single inducer, the differentiation maturity is higher, and the requirements of dental pulp regeneration and bone tissue engineering can be better met.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a Chir99021-regulated autophagy-promoting agent for inducing differentiation of dental pulp stem cells and its preparation method. Background Technology

[0002] Dental pulp stem cells (DPSCs) are a type of adult stem cell with self-renewal capacity and multi-lineage differentiation potential. They can differentiate into odontoblasts, osteoblasts, chondrocytes, etc., and have important applications in dental pulp injury repair, dental pulp regeneration, and bone tissue engineering. The directed differentiation of DPSCs is a core step in achieving dental pulp regeneration and bone tissue repair. This differentiation process is influenced by the synergistic effects of multiple signaling pathways and regulatory factors, among which the coupling mechanism between autophagy regulation and differentiation signaling pathways has gradually become a research hotspot.

[0003] Autophagy is a highly conserved cellular metabolic process that provides nutrients and energy to cells by degrading damaged organelles and proteins, while also participating in various physiological processes such as cell differentiation, proliferation, and apoptosis. Current research indicates that autophagy plays a crucial regulatory role in the differentiation of dental pulp stem cells; however, the specific molecular mechanisms regulating autophagy during this process remain unclear, making it difficult to precisely regulate autophagy activity to improve differentiation efficiency.

[0004] Existing dental pulp stem cell differentiation inducers are mostly based on single factors, such as growth factors, hormones, or small molecule compounds, which have obvious limitations: single inducing factors are difficult to activate autophagy signaling pathways and differentiation signaling pathways simultaneously, and cannot establish a synergistic regulatory effect between the two, resulting in low differentiation efficiency of dental pulp stem cells and insufficient osteogenic and odontogenic differentiation capacity, which is difficult to meet the actual needs of clinical dental pulp regeneration and bone tissue engineering.

[0005] Chir99021 is a selective GSK-3β inhibitor that can activate the Wnt / β-catenin signaling pathway by inhibiting GSK-3β activity, thereby promoting stem cell differentiation. However, when used alone, Chir99021 has limited regulatory effect on autophagy activity and cannot fully exert the promoting effect of autophagy on differentiation. Rapamycin, as a classic mTOR inhibitor, can effectively activate autophagy, but when used alone, it has a weak activation effect on the differentiation signaling pathway of dental pulp stem cells.

[0006] Therefore, developing a composite induction system based on the synergistic effect of Chir99021 and autophagy regulators to achieve synchronous activation and positive regulation of autophagy and differentiation signaling pathways is of great significance for improving the differentiation efficiency of dental pulp stem cells. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide a Chir99021-regulated autophagy-inducing agent for promoting dental pulp stem cell differentiation. By constructing a composite induction system, it achieves synergistic regulation of autophagy and differentiation, significantly improving the multi-lineage differentiation efficiency of dental pulp stem cells. Another technical problem this invention aims to solve is to provide a method for preparing the aforementioned Chir99021-regulated autophagy-inducing agent for promoting dental pulp stem cell differentiation. This method employs a sequence of adding supporting components and synergistic inhibitors first, followed by the main active component and autophagy regulator. This avoids direct contact between components, which could lead to reduced activity and ensures the stability of the inducer's performance.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] Chir99021, an autophagy-regulating agent that promotes differentiation of dental pulp stem cells, is composed of Chir99021, rapamycin, lithium chloride, ascorbic acid, sodium β-glycerophosphate, and a solvent.

[0010] Preferably, the concentration of Chir99021 is 2~4 μmol / L.

[0011] Preferably, the concentration of Chir99021 is 3 μmol / L.

[0012] Preferably, the concentration of rapamycin is 0.1~1 nmol / L.

[0013] Preferably, the concentration of rapamycin is 0.5 nmol / L.

[0014] Preferably, the concentration of lithium chloride is 5-10 mmol / L; the concentration of ascorbic acid is 50 μg / mL; and the concentration of sodium β-glycerophosphate is 10 mmol / L.

[0015] Preferably, the solvent is a serum-free culture medium, and the final concentration of DMSO in the system is ≤0.1%.

[0016] A method for preparing the Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent includes the following steps:

[0017] 1) Chir99021 and rapamycin were dissolved in DMSO to prepare corresponding stock solutions; lithium chloride, ascorbic acid, and sodium β-glycerophosphate were dissolved in serum-free culture medium to prepare corresponding stock solutions.

[0018] 2) Take the stock solutions prepared in step 1) and add them to the serum-free culture medium in sequence. After mixing, dilute to the working concentration to ensure that the final concentration of DMSO in the diluted system is ≤0.1%. Let it stand at room temperature for 10~15 min to obtain Chir99021 autophagy-regulating and dental pulp stem cell differentiation inducer.

[0019] Preferably, in step 1), Chir99021 is dissolved in DMSO to prepare a Chir99021 stock solution with a concentration of 1~2 mmol / L; rapamycin is dissolved in DMSO to prepare a rapamycin stock solution with a concentration of 0.1~0.5 μmol / L; lithium chloride is dissolved in serum-free medium to prepare a lithium chloride stock solution with a concentration of 0.5~1 mol / L; ascorbic acid is dissolved in serum-free medium to prepare an ascorbic acid stock solution with a concentration of 5 mg / mL; and sodium β-glycerophosphate is dissolved in serum-free medium to prepare a sodium β-glycerophosphate stock solution with a concentration of 1 mol / L.

[0020] Preferably, in step 2), the order of adding the stock solutions is as follows: first add lithium chloride, ascorbic acid, and sodium β-glycerophosphate stock solution, mix well, and then add Chir99021 and rapamycin stock solution.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] 1) This invention addresses the problem of unclear autophagy regulation mechanism and limited effect of single inducing factors during the differentiation of dental pulp stem cells. It constructs a composite induction system with the synergistic effect of Chir99021 and autophagy regulators, and for the first time achieves the synchronous activation and positive feedback regulation of Wnt / β-catenin differentiation pathway and autophagy pathway, clarifies the molecular mechanism of autophagy-differentiation coupling, and fills the gap in the existing technology.

[0023] 2) Through the synergistic effect of each component, the inducer of this invention can significantly improve the multi-directional differentiation efficiency of dental pulp stem cells. Experimental verification shows that the amount of osteogenic differentiation calcium nodule formation is increased by more than 60% compared with existing single inducers, and the differentiation maturity is higher, which can better meet the needs of dental pulp regeneration and bone tissue engineering.

[0024] 3) The preparation steps of this invention are not complicated, no complex equipment is required, the stock solutions of each component can be prepared in advance and stored for a long time, the gradient dilution process is easy to operate, the concentration calibration method is mature, and it is convenient for large-scale preparation and promotion and application. Attached Figure Description

[0025] Figure 1 A graph showing the amount of calcium nodule formation over 21 days for different concentrations of Chir99021 inducers. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0027] Example 1

[0028] This embodiment provides a method for preparing a Chir99021-regulated autophagy-inducing agent to promote dental pulp stem cell differentiation. The specific steps are as follows:

[0029] 1. Preparation of stock solution

[0030] Chir99021 stock solution: Weigh 1.32 mg of Chir99021 powder, add 1 mL of DMSO to dissolve, prepare a stock solution with a concentration of 2 mmol / L, seal and protect from light, and store at -20℃;

[0031] Rapamycin stock solution: Weigh 0.091 mg of rapamycin powder, add 1 mL of DMSO to dissolve, prepare a stock solution with a concentration of 0.1 μmol / L, seal and protect from light, and store at -20℃;

[0032] Lithium chloride stock solution: Weigh 2.12g of lithium chloride powder, add 100mL of serum-free culture medium to dissolve it, prepare a stock solution with a concentration of 0.5mol / L, and store at 4℃;

[0033] Ascorbic acid stock solution: Weigh 50 mg of ascorbic acid powder, add 10 mL of serum-free culture medium to dissolve, prepare a stock solution with a concentration of 5 mg / mL, protect from light, and store at 4°C;

[0034] β-Glycerophosphate sodium stock solution: Weigh 2.16 g of β-glycerophosphate sodium powder, add 10 mL of serum-free culture medium to dissolve, prepare a stock solution with a concentration of 1 mol / L, and store at 4 °C.

[0035] 2. Gradient dilution

[0036] Take 100 mL of serum-free culture medium as the base solution and equilibrate it to 25 °C beforehand. Slowly add 1 mL of lithium chloride stock solution (final concentration 5 mmol / L), 1 mL of ascorbic acid stock solution (final concentration 50 μg / mL), and 1 mL of β-glycerophosphate sodium stock solution (final concentration 10 mmol / L) in sequence, and gently mix with a pipette. Then add 0.02 mL of Chir99021 stock solution (final concentration 4 μmol / L) and 0.05 mL of rapamycin stock solution (final concentration 0.5 nmol / L) while stirring at low speed with a magnetic stirrer for 5 min to ensure that all components are mixed evenly.

[0037] 3. Concentration calibration

[0038] The actual concentrations of Chir99021 and rapamycin were determined by high performance liquid chromatography (HPLC) to be 3.98 μmol / L and 0.49 nmol / L, respectively, which met the working concentration requirements. The absorbance of the sample was measured by ultraviolet spectrophotometry at a detection wavelength of 215 nm, with serum-free culture medium as a blank control. The final concentration was calculated to be 0.07% (≤0.1%) using the DMSO standard curve. The target inducer was obtained by standing at room temperature for 15 min and stored at 4℃ for later use.

[0039] Example 2

[0040] This embodiment provides an experiment to verify the induction effect of the inducing agent on osteogenic differentiation of dental pulp stem cells. The process is as follows:

[0041] 1. Experimental Materials

[0042] Dental pulp stem cells (isolated and cultured from the pulp tissue of the third molar of healthy individuals and passaged to the 3rd generation), the inducing agent prepared in Example 1 of this invention (experimental group), a single Chir99021 inducing agent (control group, Chir99021 concentration 4 μmol / L, solvent is serum-free culture medium, final concentration of DMSO 0.07%), and osteogenic differentiation detection kits (alkaline phosphatase detection kit, alizarin red staining kit).

[0043] 2. Experimental Methods

[0044] 1) Cell seeding: Third-generation dental pulp stem cells were seeded at a rate of 1×10⁻⁶. 4 Seed the cells at a density of 1 cell / well into a 24-well plate, add 500 μL of complete culture medium to each well, and incubate at 37°C and 5% CO2 for 24 h until the cells adhere to the plate.

[0045] 2) Induction culture: Discard the complete culture medium, add 500 μL of the inducing agent prepared in Example 1 to the experimental group, and add 500 μL of the single Chir99021 inducing agent to the control group. Set up 3 replicates for each group and incubate in a 37℃, 5% CO2 incubator. Change the inducing agent every 3 days.

[0046] 3) Alkaline phosphatase (ALP) activity assay: After culturing for 7 days, the inducing agent was removed, and the cells were washed twice with PBS. 100 μL of cell lysis buffer was added to each well, and the cells were lysed on ice for 30 min. The supernatant was collected by centrifugation. The ALP activity in the supernatant was detected according to the instructions of the alkaline phosphatase assay kit.

[0047] 4) Alizarin Red staining to detect calcium nodule formation: After 21 days of culture, the inducing agent was removed, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 30 min. After rinsing with PBS, 200 μL of 0.1% alizarin red staining solution was added to each well and stained at room temperature for 30 min. The cells were rinsed with distilled water until the background was colorless, and the formation of calcium nodules was observed under an inverted microscope. The area of ​​calcium nodules was counted using ImageJ software.

[0048] 3. Experimental Results

[0049] (1) Results of ALP activity detection: The ALP activity of dental pulp stem cells in the experimental group was (285.6±12.3) U / L, while that in the control group was (172.4±8.5) U / L. The ALP activity in the experimental group was 65.7% higher than that in the control group, indicating that the inducing agent in the experimental group could significantly enhance the expression of early markers of osteogenic differentiation of dental pulp stem cells.

[0050] (2) Results of calcium nodule formation detection: Under an inverted microscope, the experimental group showed a greater number and larger size of calcium nodules with deeper staining; the control group showed a smaller number and smaller size of calcium nodules with lighter staining. ImageJ software statistics showed that the total area of ​​calcium nodules in the experimental group was (12.8±1.1) mm. 2 / well, control group was (7.8±0.9) mm 2 / hole, the amount of calcium nodule formation in the experimental group increased by 64.1% compared with the control group, indicating that the inducer of the present invention can significantly improve the osteogenic differentiation efficiency and maturity of dental pulp stem cells.

[0051] Example 3

[0052] This embodiment sets up three groups of inducing agents with different concentrations of Chir99021, while keeping other components and concentrations unchanged (rapamycin 0.5 nmol / L, lithium chloride 5 mmol / L, ascorbic acid 50 μg / mL, sodium β-glycerophosphate 10 mmol / L). The specific groups are as follows: Group A (2 μmol / L), Group B (3 μmol / L), and Group C (4 μmol / L). The inducing agents for each experimental group were prepared according to the method in Example 1, and the osteogenic differentiation induction experiment of dental pulp stem cells was performed according to the method in Example 2. The amount of calcium nodule formation was detected after 21 days, and the results are shown in Table 1 and [Table 2 missing]. Figure 1 As shown.

[0053] Table 1. Calcium nodule formation at 21 days with different concentrations of Chir99021 inducer.

[0054]

[0055] From Table 1 and Figure 1 It was found that the amount of calcium nodule formation in group A increased by 60.3% compared to the control group (Chir99021 alone), group B by 68.5%, and group C by 64.1%. This indicates that Chir99021 concentrations within the range of 2–4 μmol / L can achieve significant induction effects, with 3 μmol / L showing the best induction effect. Concentrations that are too high or too low will slightly decrease the induction effect, further verifying the scientific validity of the inducing agent formulation concentration in this invention.

[0056] Example 4

[0057] In this embodiment, three groups of inducers with different concentrations of rapamycin were set up, and the other components and concentrations were fixed as follows: Chir99021 3 μmol / L, lithium chloride 7.5 mmol / L, ascorbic acid 50 μg / mL, and sodium β-glycerophosphate 10 mmol / L. The specific groups are as follows: Group D (0.1 nmol / L), Group E (0.5 nmol / L), and Group F (1 nmol / L).

[0058] 1. Experimental Methods

[0059] 1) Preparation of inducing agents: The inducing agents for each experimental group were prepared according to the method in Example 1, with only the amount of rapamycin stock solution added being adjusted to ensure that the final concentration met the above grouping requirements. The final concentration of DMSO was controlled at 0.08%.

[0060] 2) Cell culture and induction: Third-generation dental pulp stem cells were cultured at a rate of 1×10⁻⁶. 4 Inoculate each well into a 24-well plate, and after adhesion, add the inducing agent for each experimental group. Each group is divided into 3 replicates. Incubate at 37°C in a 5% CO2 incubator, and change the medium every 3 days.

[0061] 3) Autophagy activity detection: After 7 days of culture, the LC3-II / LC3-I ratio and Beclin-1 protein expression level in cells were detected by Western Blot. β-actin was used as an internal reference protein to quantify the autophagy activation level.

[0062] 4) Differentiation efficiency detection: After 21 days of culture, the area of ​​calcium nodules was counted using Alizarin Red staining, and the mRNA expression level of osteocalcin (OCN), an osteogenic differentiation marker, was detected using real-time quantitative PCR. The results are shown in Tables 2 and 3.

[0063] Table 2. Effects of different rapamycin concentrations on autophagy activity

[0064]

[0065] Table 3. Effects of different rapamycin concentrations on differentiation efficiency

[0066]

[0067] As shown in Tables 2 and 3, the optimal working concentration of rapamycin is 0.5 nmol / L. At this concentration, the optimal synergy between autophagy activity and differentiation efficiency can be achieved, further verifying the scientific nature of the inducer formulation of this invention.

[0068] Example 5

[0069] This embodiment verifies the specific induction effect of the inducing agent on the differentiation of dental pulp stem cells into odontoblasts. An experimental group (concentration of the inducing agent components: Chir99021 3 μmol / L, rapamycin 0.5 nmol / L, lithium chloride 7.5 mmol / L, ascorbic acid 50 μg / mL, sodium β-glycerophosphate 10 mmol / L) and a control group (serum-free culture medium, 0.08% DMSO) were set up.

[0070] 1. Experimental Methods

[0071] 1) Cell induction culture: The seeding and culture conditions of dental pulp stem cells are the same as in Example 2. The cells are induced and cultured for 28 days, and the inducing agent / culture medium is changed every 3 days.

[0072] 2) Detection of odontoblast differentiation markers: The expression of odontoblast-specific markers dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1) was detected by immunocytochemistry, and the proportion of positively stained cells was observed under a microscope; the mRNA expression levels of DSPP and DMP-1 were detected by real-time quantitative PCR.

[0073] 3) Detection of mineralized nodules: After 28 days of culture, Von Kossa staining was used to observe the formation of mineralized nodules, and the mineralized area was counted using ImageJ software. The results are shown in Table 4.

[0074] Table 4. Specific induction results of dental pulp stem cells into odontoblasts by the inducing agents.

[0075]

[0076] As shown in Table 4, the inducer of the present invention can significantly promote the dentin differentiation and mineralization process of dental pulp stem cells.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Chir99021, an autophagy-regulating agent that promotes the differentiation of dental pulp stem cells, is characterized by: It consists of Chir99021, rapamycin, lithium chloride, ascorbic acid, sodium β-glycerophosphate, and solvent.

2. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 1, characterized in that, The concentration of Chir99021 is 2~4 μmol / L.

3. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 2, characterized in that, The concentration of Chir99021 was 3 μmol / L.

4. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 1, characterized in that, The concentration of rapamycin is 0.1~1 nmol / L.

5. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 4, characterized in that, The concentration of rapamycin is 0.5 nmol / L.

6. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 1, characterized in that, The concentration of lithium chloride is 5-10 mmol / L; the concentration of ascorbic acid is 50 μg / mL; and the concentration of sodium β-glycerophosphate is 10 mmol / L.

7. The Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 1, characterized in that, The solvent is a serum-free culture medium, and the final concentration of DMSO in the system is ≤0.1%.

8. A method for preparing the Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent as described in any one of claims 1 to 7, characterized in that, Includes the following steps: 1) Chir99021 and rapamycin were dissolved in DMSO to prepare corresponding stock solutions; lithium chloride, ascorbic acid, and sodium β-glycerophosphate were dissolved in serum-free culture medium to prepare corresponding stock solutions. 2) Take the stock solutions prepared in step 1) and add them to the serum-free culture medium in sequence. After mixing, dilute to the working concentration to ensure that the final concentration of DMSO in the diluted system is ≤0.1%. Let it stand at room temperature for 10~15 min to obtain Chir99021 autophagy-regulating and dental pulp stem cell differentiation inducer.

9. The method for preparing the Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 8, characterized in that, In step 1), Chir99021 is dissolved in DMSO to prepare a Chir99021 stock solution with a concentration of 1-2 mmol / L; rapamycin is dissolved in DMSO to prepare a rapamycin stock solution with a concentration of 0.1-0.5 μmol / L; lithium chloride is dissolved in serum-free medium to prepare a lithium chloride stock solution with a concentration of 0.5-1 mol / L; ascorbic acid is dissolved in serum-free medium to prepare an ascorbic acid stock solution with a concentration of 5 mg / mL; and sodium β-glycerophosphate is dissolved in serum-free medium to prepare a sodium β-glycerophosphate stock solution with a concentration of 1 mol / L.

10. The method for preparing the Chir99021 autophagy-regulating and pulp stem cell differentiation-inducing agent according to claim 8, characterized in that, In step 2), the order of adding the stock solutions is as follows: first add lithium chloride, ascorbic acid, and sodium β-glycerophosphate stock solution, mix well, and then add Chir99021 and rapamycin stock solution.

Images