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CN-121975728-A - Exosome for promoting hair follicle cell growth and preparation method and application thereof

CN121975728ACN 121975728 ACN121975728 ACN 121975728ACN-121975728-A

Abstract

The invention provides an exosome for promoting hair follicle cell growth, a preparation method and application thereof, wherein the method firstly adopts extremely low concentration Triton X-100, and reversible micropores are formed on the surface of a membrane on the premise of ensuring the stability of the whole structure of the exosome; and then the astragaloside IV and oleanolic acid are effectively introduced into the exosome by utilizing the ultrasonic action. Compared with the traditional electrotransformation method, the method provided by the invention has the advantages that the damage of the exosome membrane is obviously reduced, and the integrity of the membrane protein and the natural function is maintained. Meanwhile, the anti-apoptosis of astragaloside IV and the Wnt/beta-catenin activation of oleanolic acid are mutually synergistic, so that the proliferation of hair follicle cells and the growth period of hair follicles can be remarkably promoted, and a brand-new technical path is provided for alopecia treatment and hair follicle regeneration.

Inventors

  • WEI QING
  • XIAO HAIRONG
  • LIU QINGXI
  • HAN HAO
  • WANG XINYAN

Assignees

  • 博雅干细胞科技有限公司

Dates

Publication Date
20260505
Application Date
20251216

Claims (10)

  1. 1. A preparation method of exosomes for promoting hair follicle cell growth is characterized by comprising the following steps: (1) Adding Triton X-100 into the mesenchymal stem cell exosome solution for light-shielding incubation, wherein the final concentration of Triton X-100 is 0.1wt% to 0.5wt%; (2) Adding an astragaloside IV solution and an oleanolic acid solution into the solution after the incubation in the step (1) for continuous incubation; (3) After the incubation in the step (2), performing ultrasonic treatment on the mixture by using an ultrasonic crusher, and incubating again after the treatment; (4) And (3) filtering the solution after incubation in the step (3) to obtain a precipitate, namely the exosome.
  2. 2. The method of claim 1, wherein in the step (1), the concentration of the exosome solution is 1-11X 10 10 particles/mL, and the incubation condition is 15-45 min.
  3. 3. The method of claim 1, wherein in the step (2), the concentration of the astragaloside IV solution is 100-150. Mu.g/mL, and the concentration of the oleanolic acid solution is 200-450. Mu.g/mL.
  4. 4. The method of claim 1, wherein in the step (2), the incubation is performed in an incubator containing 37℃and 5% CO 2 for 1 hour.
  5. 5. The method of claim 1, wherein in the step (3), the intensity of the ultrasonic wave is 5-20%.
  6. 6. The method for preparing an exosome for promoting hair follicle cell growth according to claim 1, wherein in the step (3), the ultrasonic treatment is performed for 6 to 8 cycles of ultrasonic treatment for 1 to 3 minutes each, ultrasonic treatment is performed for 30 seconds in ultrasonic treatment per minute, ultrasonic treatment is performed for 30 seconds, and ultrasonic treatment is performed for off, and cooling is performed for 1 to 2 minutes after ultrasonic treatment.
  7. 7. The method of claim 1, wherein in step (3), the incubation is performed in an incubator containing 37℃and 5% CO 2 for 0.5-1 hr.
  8. 8. The method of claim 1, wherein in the step (4), the filtration is performed by centrifugation through an ultrafiltration tube of 100kd at 4000g for 30min for 2 times.
  9. 9. An exosome produced by the method for producing an exosome that promotes hair follicle cell growth of any one of claims 1 to 8.
  10. 10. Use of an exosome according to claim 9 for promoting hair follicle cell growth.

Description

Exosome for promoting hair follicle cell growth and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to an exosome for promoting hair follicle cell growth, and a preparation method and application thereof. Background Umbilical Cord Mesenchymal Stem Cells (UCMSCs) are a type of multipotent stem cells derived from umbilical cord tissue and have self-renewal and multipotent differentiation potential. Compared with stem cells derived from bone marrow or fat, UCMSCs are convenient to obtain materials, rich in sources, low in ethical risk, weak in immunogenicity and high in safety. Studies have shown that UCMSCs secrete a variety of bioactive factors and exosomes through paracrine effects, regulating cell proliferation, differentiation and apoptosis. In the field of hair follicle regeneration, UCMSCs are considered to be capable of improving the microenvironment of hair follicles and promoting the activation of hair follicle stem cells, so that the growing period of the hair follicles is prolonged, and a new idea is provided for treating alopecia and hair rarefaction. The exosomes are nanoscale vesicles secreted by cells (about 30-150nm in diameter), and have lipid bilayer membranes on the surfaces and are internally rich in active ingredients such as miRNAs, mRNAs, proteins, lipids and the like. Exosomes are important mediators of the biological effects of stem cells, capable of crossing intercellular barriers and precisely delivering signaling molecules. In hair follicle research, stem cell derived exosomes are proved to activate Wnt/beta-catenin and other key signal pathways, promote proliferation and migration of hair papilla cells and hair follicle stem cells, and thereby induce hair follicles to enter the anagen phase. In addition, the exosomes also have the effects of anti-inflammation, antioxidation and the like, can improve the microenvironment around the hair follicle, inhibit the injury of inflammatory factors to the hair follicle, and indirectly delay the decay of the hair follicle. The prior researches show that the exosomes derived from the mesenchymal stem cells play a certain role in promoting the proliferation of hair follicle cells, activating the hair follicle stem cells and improving the microenvironment of hair follicles. However, there is a limit to the pure use of exosomes in that ① exosomes have a limited endogenous active molecule content, which does not significantly promote the growth phase of hair follicle cells, especially in pathological conditions of hair loss or hair follicle dysfunction, and have insufficient repairability. ② Exosomes are easily cleared by metabolism in the body and their duration of action to stimulate hair follicle cells is relatively short. ③ Exosomes naturally load signal molecules of limited species, it is difficult to fully activate multiple signal pathways associated with hair follicle regeneration. In addition, in the prior art, a common method for exosome loading of small molecule compounds is mainly an electrotransport method. Although the method can improve the loading efficiency of the exosomes to a certain extent, the high-voltage electric field is easy to cause irreversible damage to the exosome membrane structure in the electrotransformation process, so that the membrane protein is lost, vesicle aggregation and even the functionality is reduced, thereby limiting the effect of the exosomes in drug delivery and hair follicle cell promotion application. Disclosure of Invention In view of the above, the present invention aims to provide an exosome for promoting hair follicle cell growth, a preparation method and application thereof, wherein Astragaloside IV and oleanolic acid are efficiently introduced into the exosome by combining ultra-low concentration Triton X-100 perforation with ultrasonic transduction. Compared with the traditional electrotransformation method, the method provided by the invention has the advantages that the damage of the exosome membrane is obviously reduced, and the integrity of the membrane protein and the natural function is maintained. Meanwhile, the anti-apoptosis of astragaloside IV and the Wnt/beta-catenin activation of oleanolic acid are mutually synergistic, so that the proliferation of hair follicle cells and the growth period of hair follicles can be remarkably promoted, and a brand-new technical path is provided for alopecia treatment and hair follicle regeneration. In order to achieve the above purpose, the technical scheme of the invention is realized as follows: a method of preparing an exosome that promotes hair follicle cell growth, the method comprising the steps of: (1) Adding Triton X-100 into the mesenchymal stem cell exosome solution for light-shielding incubation, wherein the final concentration of Triton X-100 is 0.1wt% to 0.5wt%; (2) Adding an astragaloside IV solution and an oleanolic acid solution i