Stem cell exosome and use thereof in preparation of anti-aging cosmetic or pharmaceutical

Abstract

The present application provides a kind of human umbilical cord mesenchymal stem cell secretory exosome and anti-aging essence based on the exosome and its preparation method and application.By activating Wnt / β-catenin signaling pathway, the yield of exosome and the secretion amount of miR-21 are significantly improved, and the purity and biological activity of exosome are effectively purified by using microfluidic technology. The essence formula contains exosome, HA-Lipo composite carrier, asiaticoside microspheres and dipropylene glycol, and has good skin repair and anti-aging effect. Animal experiments have verified that the essence of the present application can effectively improve skin wrinkles, epidermal thickness and skin barrier function in UVB-induced skin photoaging model, and its molecular mechanism is related to the regulation of collagen metabolism. The essence has significant application prospect in the field of skin beauty and anti-aging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical and cosmetic technology, and specifically relates to a stem cell exosome and application thereof in preparation of anti-aging cosmetic or medicine. BACKGROUND

[0002] With the growth of age, the aging process of skin gradually accelerates, mainly manifested as skin relaxation, increased wrinkles, decreased elasticity, and formation of color spots, etc. The main causes of skin aging include reduction of collagen and elastin, damage of skin barrier function, and decline of antioxidant capacity, etc. These changes not only affect appearance, but also can lead to decline of skin function, so the research and development of anti-aging cosmetic and skin care products has become an important direction in the modern cosmetic field.

[0003] At present, the anti-aging products on the market mainly rely on chemical ingredients such as vitamin C, retinol, peptides, etc. Although these ingredients can slow down the aging process to some extent, they often have problems such as limited effect and large side effects. In recent years, stem cell therapy as a new emerging biomedical technology has gradually emerged in the field of skin anti-aging. Stem cells can secrete various bioactive molecules such as growth factors, cytokines, exosomes, etc. These secretions can promote the repair, regeneration and delay of aging of skin cells.

[0004] In particular, exosomes, as a kind of natural nanoscale vesicles secreted by stem cells, carry various biological active substances such as proteins, nucleic acids and lipids, and play an important role in cell communication, tissue repair and immune regulation, etc. More and more studies have shown that stem cell exosomes have significant anti-aging effect, which can delay the aging process by promoting the proliferation of skin cells, the synthesis of collagen, and the reduction of inflammatory response. Therefore, the use of stem cell exosomes to prepare cosmetic or medicine has become a hot spot in the research of anti-aging in recent years.

[0005] However, traditional exosome preparation relies on ultracentrifugation, with low yield (<20 μg / 10^6 cells) and host protein residues >30%; the content of natural exosome active ingredients (such as miR-21, miR-146a) fluctuates greatly (CV>40%), affecting the stability of the product; the transdermal efficiency is insufficient (<10%), and invasive means such as microneedle is needed. Therefore, a new type of exosome needs to be developed to further improve its effect in anti-aging cosmetic or medicine. SUMMARY

[0006] The purpose of the present application is to provide a stem cell exosome and the use of the stem cell exosome in preparation of anti-aging cosmetic or medicine.

[0007] Therefore, one aspect of the present application discloses a preparation method of human umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:

[0008] (1) Activate the Wnt / β-catenin signaling pathway by simultaneously adding 3 μM CHIR99021 and 10 μg / mL anti-Gsk3β monoclonal antibody in the culture medium of human umbilical cord mesenchymal stem cells, and culturing the cells under 5% O2 conditions for 48 hours;

[0009] (2) Purify the cultured human umbilical cord mesenchymal stem cell exosomes using a microfluidic device, set the flow rate to 8 L / h, the acoustic power to 300 W, and the shear force to 6 dyn / cm 2 ; and finally obtain human umbilical cord mesenchymal stem cell exosomes.

[0010] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-Gsk3β monoclonal antibody of the present application are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0011] Preferably, the amino acid sequence of the Gsk3β protein polypeptide for preparing the anti-Gsk3β monoclonal antibody of the present application is Cys-Arg-Arg-Ala-pSer 9 -Val-Pro-Gly-Leu-Lys, wherein pSer 9 represents a phosphorylation modification.

[0012] Preferably, the particle size of the human umbilical cord mesenchymal stem cell exosomes obtained in step (2) of the present application is 102±5 nm, the PDI is 0.08, and the residual amount of host protein of the human umbilical cord mesenchymal stem cell exosomes is 4.8 μg / mg.

[0013] In one aspect, the present application also discloses an anti-aging essence, which comprises the following components:

[0014] (1) Human umbilical cord mesenchymal stem cell exosomes, the concentration is 1×1011 particles / g, and the amount used is 2.5%;

[0015] (2) HA-Lipo complex carrier, the concentration is 4.0%;

[0016] (3) Asiaticoside microspheres, the concentration is 1.5%;

[0017] (4) Dipropylene glycol, the concentration is 5%.

[0018] Preferably, the HA-Lipo complex carrier of the present application is formed by compounding sodium hyaluronate and liposomes, the particle size of the liposomes is 100±10 nm, and the PDI value is <0.1.

[0019] Preferably, the gynostemma pentaphyllum glycosides microspheres are prepared by a spray drying method, and the loading capacity is more than 70%, and the particle size of the microspheres is in the range of 200-300 μm.

[0020] In one aspect, the application also discloses an application of the human umbilical cord mesenchymal stem cell exosome prepared by the method in the preparation of an anti-aging essence.

[0021] The beneficial effects of the application are summarized as follows:

[0022] (1) By activating the Wnt / β-catenin signaling pathway, the combined treatment of CHIR99021 and anti-Gsk3β monoclonal antibody significantly promotes the secretion of human umbilical cord mesenchymal stem cell (hUC-MSCs) exosomes, and the exosome yield is increased from 18.2 μg / 10 6 cells of the no-treatment control group to 76.5 μg / 10 6 cells, achieving a significant increase. At the same time, the content of miR-21 is also significantly increased, indicating that this combined activation scheme promotes the synthesis of functional components of exosomes.

[0023] (2) Compared with the traditional ultracentrifugation method, the microfluidic purification technology shows higher efficiency and consistency in terms of particle size, purity and recovery rate of exosomes. The microfluidic method can provide more accurate particle size control (particle size 102±5 nm, PDI=0.08) and lower host protein residues (4.8 μg / mg), and the yield is also better than that of the ultracentrifugation method (76.5 μg / 10 6 cells vs. 52.3 μg / 10 6 cells). Therefore, microfluidic purification not only improves the quality of exosomes, but also improves the recovery rate.

[0024] (3) The exosome-HA-Lipo complex preparation (test group 1) performs outstandingly in repairing skin barrier function, reduces epidermal water loss (TEWL=8.2±0.7 g / h·m 2 ), and improves the integrity of the stratum corneum (stratum corneum score 1.2±0.3), showing its good effect on skin hydration and barrier repair. In addition, the qPCR analysis results show that test group 1 effectively regulates the expression of collagen type I (COL1A1) and matrix metalloproteinase (MMP-1) in the dermis, further indicating that exosomes play a role in the process of skin anti-aging by regulating collagen metabolism and skin repair mechanisms.

[0025] (4)The anti-aging essence of the present application uses exosomes activated by Wnt / β-catenin signaling pathway, significantly improves the yield of exosomes and the secretion level of miR-21. The application of microfluidic technology effectively improves the purity of exosomes, retains the biological activity and characteristic size of exosomes. The essence can effectively improve the anti-aging effect of the skin, including reducing skin wrinkles, improving skin barrier function, increasing the proliferation activity of epidermal cells, etc. Animal experiments show that in the UVB-induced skin photoaging model, the test group 1 (exosome-HA-Lipo complex preparation) is significantly better than other groups in improving skin wrinkles, epidermal thickness and skin barrier function, and at the molecular mechanism level, it can effectively regulate collagen metabolism, enhance the expression of dermal layer type I collagen and inhibit the activity of matrix metalloproteinase. The essence formula of the present application has wide application prospect in the field of anti-aging, skin repair, etc., especially suitable for relieving skin aging caused by ultraviolet rays, environmental pollution, age growth, etc. By activating intracellular signaling pathways and using efficient purification technology, the essence provides a novel and effective solution for skin beauty products. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Western blot detection results of β-catenin protein.

[0027] Figure 2 SDS-PAGE detection results of anti-Gsk3β monoclonal antibody, wherein 1 is anti-Gsk3β monoclonal antibody. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] Unless specifically noted, the reagents, methods, and equipment employed by the present application are routine reagents, methods, and equipment in the art. Unless specifically noted, the reagents and materials used in the following examples are commercially available.

[0030] Example 1: Preparation of exosomes

[0031] I. Cell culture and activation

[0032] 1. Cell line and culture conditions

[0033] (1) Cell line: human umbilical cord mesenchymal stem cells (hUC-MSCs) from Beijing Bao Bo Wei Biotechnology Co., Ltd.

[0034] (2) Medium: a-MEM (containing 10% exosome-free fetal bovine serum, exosome-free FBS) supplemented with 1% antibiotics (penicillin-streptomycin solution).

[0035] (3) Culture conditions: Cells were cultured at 37°C in a 5% CO2 incubator, and the medium was replaced every 2-3 days. When the cells grew to 80-90% confluence, they were treated.

[0036] 2. Activation treatment

[0037] (1) Treatment group: CHIR99021 (3 μM) and anti-Gsk3β monoclonal antibody (10 μg / mL, as shown in Example 4) were added, and the cells were cultured under hypoxic conditions (5% O2) for 48 hours.

[0038] (2) Control group

[0039] ① CHIR99021 control group: CHIR99021 (3 μM) was added, and the cells were cultured for 48 hours.

[0040] ② Anti-Gsk3β monoclonal antibody control group: Only anti-Gsk3β monoclonal antibody (10 μg / mL) was added, and the cells were cultured for 48 hours.

[0041] ③ Untreated control group: No treatment was added, and the cells were cultured for 48 hours using the regular medium.

[0042] (3) All groups were cultured under the same culture conditions (5% O2) for 48 hours to ensure accurate comparison of the activation treatment conditions.

[0043] 3. Cell harvesting and exosome separation: After 48 hours of treatment, the cell culture supernatant was collected, and exosomes were separated using ultracentrifugation (100,000g, 2 hours, 4°C).

[0044] 4. Results analysis, as shown in Table 1

[0045] (1) Exosome yield: The untreated control group had the lowest exosome yield, which was 18.2 ± 2.1 μg / 10 6 cells. The CHIR99021 control group and the anti-Gsk3β monoclonal antibody control group had exosome yields of 26.5 ± 3.3 μg / 10 6 cells and 42.4 ± 5.6 μg / 10 6 cells, respectively, which were higher than the untreated control group. The treatment group had an exosome yield of 76.5 ± 4.3 μg / 10 6 cells, which was significantly higher than the other groups, indicating that the combined use of CHIR99021 and anti-Gsk3β monoclonal antibody more effectively promoted exosome secretion.

[0046] (2) miR-21 content: The miR-21 content in the untreated control group was 315±28 copies / μg. The miR-21 content in the CHIR99021 control group and the anti-Gsk3β monoclonal antibody control group was 480±32 copies / μg and 680±60 copies / μg, respectively, both significantly higher than that in the untreated control group. The miR-21 content in the treated group was 1200±85 copies / μg, significantly higher than that in the other groups, indicating that the combined activation of the Wnt / β-catenin signaling pathway promotes miR-21 synthesis.

[0047] (3) Western blot results (such as Figure 1 β-catenin protein expression was higher in the treated groups compared to the other groups, confirming effective activation of the Wnt / β-catenin signaling pathway. In the CHIR99021 or anti-Gsk3β mAb-treated groups, although exosome production and miR-21 levels increased, the upregulation of β-catenin expression was less pronounced than in the combined treatment group.

[0048] The above results showed that there were significant differences in exosome production and miR-21 content between the treatment group and the control group, indicating that the combined use of CHIR99021 and anti-Gsk3β monoclonal antibody showed a synergistic effect in activating the Wnt / β-catenin signaling pathway.

[0049] Table 1 Summary of test results

[0050]

[0051] All data were analyzed using SPSS 22.0 statistical software, and one-way analysis of variance (ANOVA) was used for comparison between groups, with the significance level set at p < 0.01.

[0052] 2. Microfluidic Purification

[0053] 1. Equipment and purification conditions

[0054] (1) Microfluidic purification equipment parameters: Exosomes were purified using a microfluidic device with a flow rate of 8 L / h, an acoustic power of 300 W, and a shear force of 6 dyn / cm 2 .

[0055] (2) Ultracentrifugation (traditional method): The cell culture supernatant was purified by ultracentrifugation at 100,000 g (2 h, 4°C) for exosome purification as a traditional control.

[0056] (3) Purification medium: For both methods, the same buffer / surfactant system was used for exosome isolation to ensure comparable particle size and purity.

[0057] 2. Results analysis, as shown in Table 2

[0058] (1) Particle size and PDI: The particle size of exosomes in the microfluidic purification group was 102 ± 5 nm, and the PDI was 0.08, showing good uniformity and stability, suitable for subsequent application. The particle size of exosomes in the ultracentrifugation purification group was 95 ± 4 nm, slightly smaller than that in the microfluidic purification group, but the PDI was 0.12, indicating that the particle size distribution was relatively wide, and there were more heterogeneous particles. Although the ultracentrifugation method can still separate exosomes, the particle size distribution is wide, and the purity may be slightly lower.

[0059] (2) Host protein residues: The amount of host protein residues in the microfluidic purification group was 4.8 μg / mg, which was significantly lower than that in the ultracentrifugation purification group (12.5 μg / mg). This shows that the microfluidic purification technology is more efficient in removing host proteins and can provide higher purity exosome products.

[0060] (3) Exosome recovery rate and yield: The yield of exosomes in the microfluidic purification group was 76.5 ± 4.3 μg / 10 6 cells, which was significantly higher than that of the traditional ultracentrifugation method (52.3 ± 6.8 μg / 10 6 cells), indicating that microfluidic technology can provide higher yield and recovery rate, especially in smaller sample size conditions.

[0061] Table 2 Detection results of products after purification

[0062]

[0063] All data were statistically analyzed using SPSS 22.0, and one-way ANOVA was used, with a significant level of p < 0.01. The microfluidic purification group performed better than the ultracentrifugation purification group in terms of particle size, PDI, host protein residues, and yield, with significant differences.

[0064] 3. Summary: Through comparison of microfluidic purification and traditional ultracentrifugation, it was found that microfluidic purification was significantly better than ultracentrifugation in terms of exosome particle size, uniformity, purity, and yield. Microfluidic technology not only improves the recovery rate of exosomes, but also has higher accuracy in host protein removal and particle size control, suitable for subsequent functional research and application. In contrast, the traditional ultracentrifugation method is widely used, but has certain limitations in yield and purity.

[0065] The above results show that the activation of the Wnt / β-catenin signaling pathway in human umbilical cord mesenchymal stem cells (hUC-MSCs) can significantly increase the production of exosomes and the secretion of miR-21. The microfluidic purification technology effectively improves the purity of exosomes and retains their characteristic size and biological activity. These results show that the activation scheme and purification method provide a reliable basis for further studying the potential of hUC-MSCs secreted exosomes in disease treatment.

[0066] Example 2: Anti-aging serum formula and preparation

[0067] I. Formula composition (w / w)

[0068] 1. Exosomes: concentration of 1 x 1011 particles / g, dosage of 2.5%.

[0069] Example 1: Exosomes obtained by microfluidic purification method, after standardized particle size analysis and protein residue analysis, ensure that the purity reaches ≥95%, stored in a -80°C refrigerator to avoid repeated freezing and thawing.

[0070] 2. HA-Lipo carrier: concentration of 4.0%.

[0071] HA-Lipo carrier is a composite carrier of sodium hyaluronate (HA) and liposome (Lipo). The particle size of liposome is controlled at 100 ± 10 nm, and the PDI value is <0.1 to ensure the effectiveness of skin absorption.

[0072] HA-Lipo carrier preparation method: HA-Lipo composite carrier is prepared by thin film hydration method. The specific operation is as follows: dissolve the liposome raw material (1,2-oleic acid phospholipid, cholesterol) in an appropriate amount of chloroform, and hydrate the film. Ultrasonic treatment is used to obtain uniform liposome particles, which are then filtered through a nanofiltration membrane to remove unencapsulated components. The particle size and distribution of the liposome are tested by dynamic light scattering (DLS) to ensure that they meet the expectations.

[0073] 3. Asiaticoside microspheres: concentration of 1.5%.

[0074] Preparation method: spray drying method is used to prepare asiaticoside microspheres, and the loading capacity needs to reach more than 70%. Gelatin and polyvinyl alcohol (PVA) are used as polymer matrix, and asiaticoside is dissolved in solvent to prepare microspheres. The particle size range of the microspheres is 200-300 μm, which ensures slow drug release.

[0075] 4. Dipropylene glycol / water: the concentration of dipropylene glycol is 5%, and the rest is adjusted to 100% of the total formula volume with water. As a solvent and thickening agent, it helps to adjust the stability and use of the formula.

[0076] II. Formula preparation process

[0077] 1. Preparation of ingredients: Exosomes, HA-Lipo carrier, asiaticoside microspheres, and dipropylene glycol are added to a sterile stirring container in sequence. Use a precision electronic balance to weigh each ingredient to ensure accurate proportions.

[0078] 2. Mixing process: Under low-speed stirring, slowly add the HA-Lipo carrier to the solution containing the exosomes, continue stirring for 30 minutes to ensure uniform dispersion. Then add the asiaticoside microspheres and continue stirring for 1 hour to ensure complete uniformity of all ingredients.

[0079] 3. Final solution: Remove any undissolved impurities by filtration (0.22 μm) to obtain the final formulation. Check the pH value to ensure it is between 4.5-5.5 to maintain the skin's appropriate pH.

[0080] 4. Preservation and packaging: The prepared formulation is divided into sterile vials and sealed for storage. The product should be stored in a cool, dry, and dark place, and stored at room temperature, with a shelf life of 12 months.

[0081] Example 3: Application of Anti-aging Serum

[0082] I. Animal Model:

[0083] 1. Strain: BALB / c-nu nude mice (female, 8 weeks old, n=40), establish UVB-induced skin photoaging model;

[0084] 2. Induction method: UVB irradiation (30 mJ / cm 2 , 3 times a week for 4 weeks) to form typical photoaging characteristics (wrinkle proliferation, epidermal thickening).

[0085] II. Grouping and Treatment:

[0086] 1. Test group 1 (n=10): Exosome-HA-Lipo complex preparation (exosome dose 1×10^10 particles / cm 2 , applied topically daily;

[0087] 2. Test group 2 (n=10): Exosomes alone (exosome dose 1×10^10 particles / cm 2 , without HA-Lipo carrier, applied topically daily;

[0088] 3. Positive control group (n=10): 0.05% tretinoin cream;

[0089] 4. Blank control group (n=10): Base cream (without active ingredients).

[0090] III. Detection Indicators and Results

[0091] 1. Quantitative analysis of skin wrinkles: 3D skin imaging system (Antera ) captures the texture of the back skin, calculates the wrinkle density (number of wrinkles / cm 2 ). The results are shown in Table 3, and Test Group 1 has a very good effect, better than other groups.

[0092] Table 3 Results of quantitative analysis of skin wrinkles

[0093]

[0094] (**p <0.01 vs positive control group, n = 10)

[0095] 2. Epidermal thickness and proliferation activity: Epidermal thickness was measured by H&E staining; Ki67 positive cell rate (epidermal proliferation marker) was detected by immunohistochemistry. The results are shown in Table 4, and Test Group 1 has a very good effect, better than other groups.

[0096] Table 4 Results of epidermal thickness and proliferation activity

[0097]

[0098] (**p <0.001 vs positive control group, normal skin reference value: epidermal thickness 22 ± 3 μm, Ki67 positive rate 3-5%)

[0099] 3. Barrier function repair: Trans-epidermal water loss (TEWL) detection (Tewameter TM300); stratum corneum integrity score (0-5 points, the lower the score, the better the barrier). The results are shown in Table 5, and Test Group 1 has a very good effect, better than other groups.

[0100] Table 5 Results of barrier function repair

[0101]

[0102] (**p <0.01 vs positive control group, normal skin reference value: TEWL 5-7 g / h·m 2 , stratum corneum score 0.5-1.0)

[0103] Four, verification of molecular mechanism

[0104] 1. Regulation of collagen metabolism: qPCR detects the expression of dermal layer type I collagen (COL1A1) and matrix metalloproteinase (MMP-1). The results are shown in Table 6, and Test Group 1 has a very good effect, better than other groups.

[0105] Table 6 Results of COL1A1) and MMP-1 expression (relative expression)

[0106]

[0107] (**p<0.001 vs positive control group, blank control group as 1 times)

[0108] 2. Inflammation inhibition effect: ELISA was used to detect the content of IL-1β and TNF-α in skin tissue. As shown in Table 7, test group 1 had a very good effect, better than other groups.

[0109] Table 7: Results of IL-1β and TNF-α content detection (pg / mg)

[0110]

[0111] (**p<0.01 vs positive control group)

[0112] 3. Safety evaluation

[0113] (1) Skin irritation: erythema / edema was observed daily, and the cumulative irritation score of the test group was 0.15 (ISO 10993 standard, safety threshold <0.5);

[0114] (2) Systemic toxicity: after 4 weeks of treatment, there was no significant change in serum ALT, AST and Cr (p>0.05).

[0115] V. Experimental conclusion

[0116] 1. Efficacy advantage: Exosome-HA-Lipo complex preparation showed a significantly better anti-wrinkle effect than tretinoin cream in UVB-induced skin photoaging mouse model. The skin wrinkle improvement rate of the test group was 57.5%, which was significantly higher than that of tretinoin cream (36.9%). At the same time, the test group could effectively repair the skin barrier and improve TEWL and stratum corneum integrity. The exosome group without HA-Lipo improved the skin wrinkles and barrier function to some extent, but the effect was not as good as that of the exosome-HA-Lipo complex preparation.

[0117] 2. Clear mechanism: by regulating collagen metabolism, the test group significantly up-regulated the expression of type I collagen (COL1A1) (3.8 times) and inhibited the expression of MMP-1 (60%). In addition, the test group also showed a significant inflammation inhibition effect, with significantly reduced levels of IL-1β and TNF-α (47% and 46% respectively), thereby achieving a multi-target anti-aging effect. The exosome group without HA-Lipo showed moderate collagen metabolism regulation and inflammation inhibition, but the effect was limited.

[0118] Example 4: Preparation of anti-Gsk3β monoclonal antibody

[0119] I. Antibody preparation steps

[0120] 1. Antigen design and preparation

[0121] (1) Target selection: Select the key active site of Gsk3β protein (amino acid sequence: Ser 9 -Arg 215 ), design an antigen epitope with high affinity and high specificity. Synthesize the peptide segment (Gsk3β protein polypeptide): Cys-Arg-Arg-Ala-pSer 9 -Val-Pro-Gly-Leu-Lys, which contains a phosphorylation modification (i.e. pSer 9 expresses phosphorylation modification) to improve the selectivity and functionality of the antibody.

[0122] (2) Antigen construction: cross-link the synthesized peptide segment with KLH carrier protein through thio SMCC, the concentration of the cross-linked antigen is 2 mg / mL, and PBS (pH 7.4) buffer is used for dissolution to ensure immunogenicity and stability.

[0123] 2. Animal immunization and hybridoma screening

[0124] (1) Animal selection: 6-week-old BALB / c female mice (n = 5).

[0125] (2) Immunization program: for the first immunization, emulsify the antigen (50 μg per mouse) with Freund's complete adjuvant, and perform multiple subcutaneous injections; use Freund's incomplete adjuvant for booster immunization at weeks 2, 4, and 6. Collect serum at week 8, and detect the serum titer by ELISA to ensure that the titer is >1:10 5 (OD450≥2.0).

[0126] (3) Cell fusion: mix the spleen cells of the immunized mice with SP2 / 0 myeloma cells at a ratio of 5:1, and use 50% PEG-1500 for cell fusion.

[0127] (4) Screening medium: use a selection medium containing HAT (fluorouracil / thymidine), and add 20% FBS to the medium to promote the growth of fused cells.

[0128] 3. Monoclonal antibody production

[0129] (1) Positive clone screening: use ELISA to detect the binding of antibodies in the culture supernatant to Gsk3β antigen (positive threshold: OD450>1.0); use limited dilution method for 3 rounds of subcloning screening, and finally obtain a stable secreting monoclonal antibody (clone number: mAb-GSK3β-7D3).

[0130] (3) Antibody purification: Antibody purification was performed using Protein A affinity chromatography with glycine-HCl (pH 3.0) as elution buffer; ultrafiltration was used to concentrate to 5 mg / mL and stored in PBS (containing 0.02% NaN3) to ensure the stability and activity of the antibody.

[0131] II. Antibody performance test

[0132] 1. Basic characteristics: The results are shown in Table 8.

[0133] Table 8 Summary of basic characteristics test results of anti-Gsk3β monoclonal antibody

[0134]

[0135] 2. Inhibition of Gsk3β activity: In vitro kinase reaction was performed using recombinant Gsk3β protein (50 nM) and β-catenin (25 nM), and the inhibition of β-catenin phosphorylation was observed under the action of different concentrations of antibody (12 nM to 50 nM). The results showed that the IC 50 value was 12 nM, indicating that the anti-Gsk3β monoclonal antibody prepared in the present application could effectively inhibit the kinase activity of Gsk3β at a low concentration.

[0136] III. Performance comparison with commercially available antibodies

[0137] 1. Cross-reactivity: ELISA was used to detect the binding signal of the antibody to Gsk3α recombinant protein (1 μg / mL), and OD450 value was used to determine whether cross-reaction occurred;

[0138] 2. Inhibition efficiency: In vitro kinase experiment was performed to detect the inhibition efficiency of the antibody at different concentrations on the phosphorylation of β-catenin by Gsk3β;

[0139] 3. Thermal stability: The antibody was stored at 37°C for 4 weeks, and the antibody titer decay rate was detected by ELISA to evaluate its thermal stability.

[0140] The specific test results are shown in Table 9, and the anti-Gsk3β monoclonal antibody (mAb-GSK3β-7D3) prepared in the present application has better effect than the commercially available antibody.

[0141] Table 9 Summary of performance comparison results with commercially available antibodies

[0142]

[0143] IV. Summary of technical advantages

[0144] 1. High specificity: By precisely designing the antigen epitope, the monoclonal antibody completely eliminates the cross-reaction with Gsk3a in the recognition of Gsk3b, ensuring extremely high specificity (the cross-reaction rate of traditional antibodies is >15%).

[0145] 2. Strong functionality: The monoclonal antibody shows excellent effect in activating the Wnt pathway and inhibiting the activity of Gsk3b, and the IC 50 value is 67% lower than that of the commercially available antibodies, significantly improving the activity of the Wnt signaling pathway.

[0146] 3. Process stability: After 30 generations of continuous passage, the antibody yield is stable at 25±3mg / L, and there is no activity attenuation, which proves the superior production stability and industrial production potential of the antibody.

[0147] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for preparing human umbilical cord mesenchymal stem cell exosomes, characterized in that: The method comprises the following steps: (1) Activate the Wnt / β-catenin signaling pathway by adding 3 μM CHIR99021 and 10 μg / mL anti-Gsk3β monoclonal antibody to the culture medium of human umbilical cord mesenchymal stem cells and culturing the cells under 5% O2 conditions for 48 hours; (2) Purifying the cultured human umbilical cord mesenchymal stem cell exosomes using a microfluidic device with a flow rate of 8 L / h, an acoustic power of 300 W, and a shear force of 6 dyn / cm²; ultimately obtaining human umbilical cord mesenchymal stem cell exosomes; The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-Gsk3β monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the amino acid sequence of the Gsk3β protein polypeptide used to prepare the anti-Gsk3β monoclonal antibody is Cys-Arg-Arg-Ala-pSer 9 -Val-Pro-Gly-Leu-Lys, where pSer 9 Describe phosphorylation modification.

2. The method according to claim 1, characterized in that The particle size of the human umbilical cord mesenchymal stem cell exosomes obtained in step (2) is 102±5 nm, the PDI is 0.08; and the residual host protein content of the human umbilical cord mesenchymal stem cell exosomes is 4.8 μg / mg.

3. An anti-aging essence, characterized in that: The anti-aging essence contains the following components: (1) The human umbilical cord mesenchymal stem cell exosomes prepared in claim 1, with a concentration of 1×10¹¹ particles / g and an amount of 2.5%; (2) HA-Lipo composite carrier, concentration of 4.0%; (3) Centella asiatica microspheres, concentration of 1.5%; (4) Dipropylene glycol, concentration is 5%; The HA-Lipo composite carrier is composed of sodium hyaluronate and liposomes, the particle size of the liposomes is 100±10nm, and the PDI value is less than 0.1; the asiaticoside microspheres are prepared by spray drying, the loading amount reaches more than 70%, and the particle size range of the microspheres is 200-300μm.

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