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CN-122012387-A - Chir 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer and preparation method thereof

CN122012387ACN 122012387 ACN122012387 ACN 122012387ACN-122012387-A

Abstract

The invention discloses a Chir 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer and a preparation method thereof, belonging to the technical field of biomedicine. The invention relates to a Chir 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer which consists of Chir99021, rapamycin, lithium chloride, ascorbic acid, beta-sodium glycerophosphate and a solvent. The invention realizes the cooperative regulation and control of autophagy and differentiation by constructing a composite induction system, can obviously improve the multidirectional differentiation efficiency of dental pulp stem cells by the cooperative action of all the components, improves the formation amount of osteogenic differentiated calcium nodules by more than 60 percent compared with the existing single inducer, has higher differentiation maturity and can better meet the requirements of dental pulp regeneration and bone tissue engineering.

Inventors

  • Xie Huilan
  • FANG FANG
  • LIN YI

Assignees

  • 福州大学附属省立医院

Dates

Publication Date
20260512
Application Date
20260306

Claims (10)

  1. The inducer for promoting dental pulp stem cell differentiation by regulating autophagy of Chir99021 is characterized by comprising Chir99021, rapamycin, lithium chloride, ascorbic acid, beta-sodium glycerophosphate and a solvent.
  2. 2. The agent for promoting dental pulp stem cell differentiation by regulating and controlling autophagy according to claim 1, wherein the concentration of the Chir99021 is 2-4 mu mol/L.
  3. 3. The agent for promoting differentiation of dental pulp stem cells by regulating autophagy according to claim 2, wherein the concentration of the Chir99021 is 3 μmol/L.
  4. 4. The agent for promoting dental pulp stem cell differentiation by regulating and controlling autophagy according to claim 1, wherein the concentration of rapamycin is 0.1-1 nmol/L.
  5. 5. The agent for promoting dental pulp stem cell differentiation by regulating autophagy according to claim 4, wherein the concentration of rapamycin is 0.5nmol/L.
  6. 6. The agent for promoting dental pulp stem cell differentiation by regulating and controlling autophagy according to claim 1, wherein the concentration of lithium chloride is 5-10 mmol/L, the concentration of ascorbic acid is 50 mug/mL, and the concentration of beta-sodium glycerophosphate is 10mmol/L.
  7. 7. The agent for promoting dental pulp stem cell differentiation by regulating and controlling autophagy according to claim 1, wherein the solvent is a serum-free culture medium, and the final concentration of DMSO in the system is less than or equal to 0.1%.
  8. 8. The method for preparing the chip 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer according to any one of claims 1 to 7, comprising the following steps: 1) Dissolving Chir99021 and rapamycin respectively with DMSO to obtain corresponding stock solutions, dissolving lithium chloride, ascorbic acid and beta-sodium glycerophosphate respectively with serum-free culture medium to obtain corresponding stock solutions; 2) And (3) sequentially adding the stock solutions prepared in the step (1) into a serum-free culture medium, uniformly mixing, diluting to a working concentration, ensuring that the final concentration of DMSO in a diluted system is less than or equal to 0.1%, and standing for 10-15 min at normal temperature to obtain the dental pulp stem cell differentiation inducer for promoting autophagy by Chir99021 regulation.
  9. 9. The method for preparing the agent for promoting dental pulp stem cell differentiation by regulating autophagy according to claim 8, wherein in the 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 a serum-free medium to prepare a lithium chloride stock solution with a concentration of 0.5-1 mol/L, ascorbic acid is dissolved in a serum-free medium to prepare an ascorbic acid stock solution with a concentration of 5mg/mL, and sodium beta-glycerophosphate is dissolved in a serum-free medium to prepare a beta-glycerophosphate stock solution with a concentration of 1 mol/L.
  10. 10. The method for preparing the agent for promoting dental pulp stem cell differentiation by regulating and controlling autophagy according to claim 8, wherein in the step 2), the adding sequence of each stock solution is that lithium chloride, ascorbic acid and beta-sodium glycerophosphate stock solution are added first, and then Chir99021 and rapamycin stock solution are added after uniform mixing.

Description

Chir 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer and preparation method thereof Technical Field The invention belongs to the technical field of biomedicine, and particularly relates to a Chir 99021-regulated autophagy-promoting dental pulp stem cell differentiation inducer and a preparation method thereof. Background Dental Pulp stem cells (Dental Pulp STEM CELLS, DPSCS) are adult stem cells with self-renewal capacity and multidirectional differentiation potential, can be differentiated into odontoblasts, osteoblasts, chondroblasts and the like, and have important application values in the fields of Dental Pulp injury repair, dental Pulp regeneration and bone tissue engineering. The directional differentiation of dental pulp stem cells is a core link for realizing dental pulp regeneration and bone tissue repair, and the differentiation process is subjected to the synergistic effect of various signal paths and regulatory factors, wherein the coupling mechanism of autophagy regulation and differentiation signal paths gradually becomes a research hot spot. Autophagy is a highly conserved cellular metabolic process that provides nutrients and energy to cells by degrading damaged organelles and proteins, while participating in various physiological processes such as cell differentiation, proliferation, apoptosis, etc. The prior researches show that autophagy plays a key regulation role in the differentiation process of dental pulp stem cells, but the specific molecular mechanism of autophagy regulation in the differentiation process of dental pulp stem cells is still unknown, so that the differentiation efficiency is difficult to improve by accurately regulating autophagy activity. The existing dental pulp stem cell differentiation inducer mainly uses single factors, such as growth factors, hormones or small molecular compounds, and the like, has obvious limitations that the single induction factors are difficult to synchronously activate autophagy signal paths and differentiation signal paths, and cannot establish a synergistic regulation effect between the autophagy signal paths and the differentiation signal paths, so that the dental pulp stem cells have low differentiation efficiency, the osteoblast odontoblast differentiation capability is insufficient, and the actual requirements of clinical dental pulp regeneration and bone tissue engineering are difficult to meet. Chir99021 is a selective GSK-3 beta inhibitor, and can activate Wnt/beta-catenin signal channels by inhibiting GSK-3 beta activity so as to promote stem cell differentiation, but the regulation and control effect of the independent use of Chir99021 on autophagy activity is limited, and the promotion effect of autophagy on differentiation cannot be fully exerted. Rapamycin acts as a classical mTOR inhibitor and is effective in activating autophagy, but has a weak effect on activation of the differentiation signaling pathway of dental pulp stem cells when used alone. Therefore, a composite induction system based on the synergistic effect of Chir99021 and autophagy regulator is developed, synchronous activation and forward regulation of autophagy and differentiation signal channels are realized, and the method has important significance for improving the differentiation efficiency of dental pulp stem cells. Disclosure of Invention Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide the inducer for promoting dental pulp stem cell differentiation by regulating autophagy through Chir99021, and the synergistic regulation of autophagy and differentiation is realized by constructing a composite induction system, so that the multidirectional differentiation efficiency of dental pulp stem cells is remarkably improved. The invention aims to solve the other technical problem of providing the preparation method of the inducer for promoting dental pulp stem cell differentiation by regulating and controlling autophagy by Chir99021, which adopts the sequence of adding supporting components and synergistic inhibitor firstly and adding main active components and autophagy regulator secondly, so that the reduction of activity caused by direct contact of the components can be avoided, and the stability of the inducer performance is ensured. In order to solve the technical problems, the technical scheme adopted by the invention is as follows: chir99021 controls autophagy and promotes differentiation inducer of dental pulp stem cells, which consists of Chir99021, rapamycin, lithium chloride, ascorbic acid, beta-sodium glycerophosphate and solvent. Preferably, the concentration of the Chir99021 is 2-4 mu mol/L. Preferably, the concentration of Chir99021 is 3. Mu. Mol/L. Preferably, the concentration of the rapamycin is 0.1-1 nmol/L. Preferably, the rapamycin concentration is 0.5nmol/L. Preferably, the concentration of the lithium chloride is 5-10 mmol/L, the concentration of t