Umbilical cord stem cells and their use in the preparation of anti-aging drugs and medical aesthetic products

Abstract

The application discloses an engineered umbilical cord stem cell and application thereof in preparation of anti-aging drugs and medical beauty products. The application constructs an engineered cell system with high anti-aging function by designing a fusion polypeptide TAT-SIRT1-SOD (SEQ ID NO: 1) and an anti-p16INK4a monoclonal antibody (mAb-p16) to cooperatively modify the umbilical cord stem cell, and combining a 3D dynamic culture technology. The fusion polypeptide is mediated to cross the membrane by a TAT cell-penetrating peptide, activates SIRT1 and removes mitochondrial ROS; the mAb-p16 is specifically combined with p16INK4a on the surface of a senescent cell, and removes the senescent cell through an ADCC effect. Experiments show that the engineered umbilical cord stem cell can increase the removal rate of the senescent cell by 86.1%, increase the SOD activity by 300%, significantly inhibit the secretion of an inflammatory factor IL-6, and increase the skin collagen density by 148% and reduce the wrinkle depth by 67% in an animal model. The application provides a novel and efficient technical scheme for anti-aging drugs and medical beauty products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and regenerative medicine, and specifically relates to umbilical cord stem cells and their application in the preparation of anti-aging drugs and medical beauty products. BACKGROUND

[0002] Umbilical cord stem cells (UC-MSCs) have great application potential in the field of anti-aging due to their high proliferation capacity, low immunogenicity and multi-directional differentiation potential, and have become the core raw material for related research and product development. However, there are still significant limitations in the current anti-aging application technology of umbilical cord stem cells, which restricts the further development of this field.

[0003] In the aspect of stem cell in vitro culture, traditional UC-MSCs will show obvious signs of aging when subcultured to P5 generation or above. Studies have shown that as the number of passages increases, the cell proliferation rate slows down, the activity of beta-galactosidase increases significantly, and the secretome function also decreases, which greatly reduces the efficacy of the cells in the body after transplantation. For example, in patent CN117797174A, only ordinary activating drugs such as growth factors are added to delay cell aging. This method cannot fundamentally solve the problem of accumulation of oxidative stress and DNA damage faced by cells during culture, and it is difficult to maintain the long-term stability of cell activity and function.

[0004] In the application of exosome delivery, although there have been attempts to improve it, such as patent CN119033668A, which encapsulates UC-MSCs exosomes in PLGA nanoparticles, which to some extent improves the stability of exosomes, but the key problem of insufficient targeting of exosomes has not been effectively solved. Moreover, this technology does not consider the removal of the aging microenvironment, which makes it difficult for exosomes to precisely act on the target site when exerting anti-aging effect, reducing the overall effect.

[0005] In the application of antibodies, the current application scenarios of related technologies are relatively single. Although patent CN119074916A uses UC-MSCs in combination with monoclonal antibodies (anti-PAFAH1B3) for anti-cancer applications, it does not involve anti-aging scenarios; patent CN115073605B uses anti-COX-2 monoclonal antibodies to treat photoaging, but does not achieve synergistic effect with stem cells, and cannot fully exert the combined advantages of both in anti-aging. It can be seen that there are many problems to be solved in the existing technology in the application of umbilical cord stem cells in anti-aging, and new technical solutions are needed to break through these bottlenecks. SUMMARY

[0006] The purpose of the present application is to provide a modified umbilical cord stem cell, which significantly enhances its efficacy in anti-aging and medical beauty by introducing specific fusion polypeptides and monoclonal antibodies, providing a new technical solution for the preparation of efficient anti-aging drugs and medical beauty products.

[0007] Therefore, the present application discloses a fusion polypeptide TAT-SIRT1-SOD, which is mediated by TAT transmembrane peptide (YGRKKRRQRRR) to deliver across the cell membrane, connected with SIRT1 activation domain (CRARLSSHLQE) and SOD mimic enzyme (DLGVTKIEREE), and kept functional domain independence by flexible connecting peptide (GGGGS). The fusion polypeptide TAT-SIRT1-SOD can penetrate the cell membrane to activate SIRT1 deacetylase and remove mitochondrial ROS, and reduce DNA damage. The amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown in SEQ ID NO: 1.

[0008] The present application also discloses an anti-p16INK4a monoclonal antibody mAb-p16, the amino acid sequence of the heavy chain variable region of the monoclonal antibody mAb-p16 is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the monoclonal antibody mAb-p16 specifically binds to the amino acid sequence of 89-108 of human p16INK4a protein, wherein the amino acid sequence of 89-108 of human p16INK4a protein is SWTRWWCCTGPGRGWTCAM.

[0009] The present application also discloses a preparation method of an engineered umbilical cord stem cell, which comprises the following steps:

[0010] (1) Isolating and culturing umbilical cord mesenchymal stem cells: using collagenase digestion method to isolate UC-MSCs from umbilical cord Wharton's jelly, and the positive rates of CD73, CD90 and CD105 are greater than 95% after flow cytometry identification.

[0011] (2) Coupling the monoclonal antibody mAb-p16 to the surface of the microcarrier by PNIPAM temperature-sensitive material to form monoclonal antibody mAb-p16 modified temperature-sensitive microcarriers, wherein the molar ratio of the monoclonal antibody mAb-p16 to PNIPAM is 1:5; thereby anchoring the monoclonal antibody mAb-p16 on the cell surface;

[0012] (3) Culturing the stem cells in the fusion polypeptide TAT-SIRT1-SOD enhanced medium, and the amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown in SEQ ID NO: 1; thereby delivering the intracellular fusion polypeptide TAT-SIRT1-SOD of the cells;

[0013] (4) 3D dynamic culture in a rotating bioreactor, wherein the working volume is 50 mL, the density of the monoclonal antibody mAb-p16 modified temperature-sensitive microcarriers is 5 g / L, the rotation speed is 25 rpm, the temperature is 37°C, and the 5% CO2 is passed at a rate of 50 mL / min; during the culture, the cells are first statically inoculated for 2 hours, and after the adhesion rate is >80%, the rotating culture is started, and the culture medium is replaced with 1 / 2 volume of the polypeptide TAT-SIRT1-SOD fusion polypeptide TAT-SIRT1-SOD every 3 days.

[0014] Preferably, the fusion polypeptide TAT-SIRT1-SOD culture medium of the present application is a basic medium containing a final concentration of 20 μM of the fusion polypeptide TAT-SIRT1-SOD, wherein the basic medium is DMEM / F12+10% FBS.

[0015] The present application also discloses an anti-aging pharmaceutical preparation, which comprises the engineered umbilical cord stem cells or exosomes thereof prepared according to the method, and a pharmaceutically acceptable carrier.

[0016] Preferably, the preparation of the present application is an injection, wherein the injection comprises the following components: 1×10 7 engineered umbilical cord stem cells, 0.5 mg / mL monoclonal antibody mAb-p16, 20 μM fusion polypeptide TAT-SIRT1-SOD, and physiological saline.

[0017] Preferably, the preparation of the present application is a medical and cosmetic exosome anti-wrinkle essence, which comprises the following components:

[0018] (1) Exosomes extracted from the engineered umbilical cord stem cells prepared according to the method of claim 3, wherein the exosome concentration is 1×10 10 particles / mL;

[0019] (2) Hyaluronic acid-polylactic acid nanocarriers, which are self-assembled with the exosomes at a mass ratio of 1:5 at 4°C to form stable structures with an average particle size of 152±8 nm and a potential of -25±3 mV;

[0020] (3) 1 mg / mL of monoclonal antibody mAb-p16;

[0021] (4) 5% trehalose;

[0022] (5) 0.5% phenoxyethanol;

[0023] (6) Deionized water is supplemented to 100%.

[0024] The present application also discloses an engineered umbilical cord stem cell cryopreservation solution, which comprises 90% fetal bovine serum, 10% DMSO, and 5 μg / mL of monoclonal antibody mAb-p16.

[0025] The engineered umbilical cord stem cells prepared by the antibody-polypeptide synergistic modification combined with 3D culture exhibit multi-dimensional and synergistic significant beneficial effects. At the cell level, the TAT-SIRT1-SOD polypeptide can penetrate the cell membrane to activate SIRT1 deacetylase and remove mitochondrial ROS, reduce DNA damage, and effectively delay cell aging; the mAb-p16C antibody specifically recognizes the p16INK4a on the surface of the senescent cells, and effectively removes the senescent cells through the ADCC effect, and the low shear microenvironment provided by the 3D culture synergistically improves the senescent cell removal rate by 73.3%, the cell proliferation capacity is increased by 138.5% compared with the conventional culture, and the exosome yield is increased by 3.2 times. In terms of application effect, the prepared anti-aging injection preparation is verified by preclinical tests, which can significantly reduce the IL-6 and MDA levels in the blood of aging mice, improve the SOD activity, and reduce the proportion of senescent cells in the liver tissue; the exosome anti-wrinkle essence is clinically tested to improve the skin elasticity of the subjects by 47.6% and reduce the melanin index by 60%. At the same time, the optimized cryopreservation system improves the cell recovery activity rate by 13.6% and the functional retention rate is more than 95%. In summary, the engineered umbilical cord stem cells and their exosomes can be developed into injection preparations, skin care products and other forms, which have safety and effectiveness, and provide new solutions for aging-related diseases and medical aesthetics. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 SDS-PAGE detection results of anti-p16INK4a monoclonal antibody (mAb-p16).

[0027] Figure 2 Western blot detection results of different cells. 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 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 in the present application are the conventional 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: Synthesis of fusion polypeptide TAT-SIRT1-SOD

[0031] 1. Structure design of fusion polypeptide: the amino acid sequence of TAT-SIRT1-SOD (SEQ ID NO: 1) is YGRKKRRQRRR-GGGGS-CRARLSSHLQE-GGGGS-DLGVTKIEREE; wherein:

[0032] (1) TAT cell-penetrating peptide (1-11): YGRKKRRQRRR, mediates transmembrane delivery;

[0033] (2) Linker peptide (12-16): flexible linker GGGGS, maintains functional domain independence;

[0034] (3) SIRT1 activation domain (17-27): CRARLSSHLQE;

[0035] (4) Linker peptide (28-32): GGGGS;

[0036] (5) SOD mimic enzyme (33-43): DLGVTKIEREE, mimics the structure of the active center of Cu / Zn-SOD.

[0037] 2. Synthesis of fusion polypeptide: TAT-SIRT1-SOD (SEQ ID NO: 1) was synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and the purity detected by HPLC was > 98%.

[0038] 3. Mechanism of action: After TAT-SIRT1-SOD penetrates the cell membrane, it activates SIRT1 deacetylase and simultaneously eliminates mitochondrial ROS, reducing DNA damage.

[0039] Example 2: Preparation and testing of anti-p16INK4a monoclonal antibody (mAb-p16)

[0040] 1. Antibody design and preparation

[0041] 1.1 Antigen design and synthesis

[0042] Based on the 89-108 amino acid sequence (SWTRWWCCTGPGRGWTCAM) of human p16INK4a protein (UniProtKB: R9S252), the hydrophilicity index and surface accessibility of this region were analyzed by Antigenic Prediction software, confirming it as a dominant antigen epitope. Fmoc solid-phase synthesis method was used for synthesis (Shengong Bioengineering (Shanghai) Co., Ltd.), and the purity detected by HPLC was > 98%.

[0043] 1.2 Phage display screening

[0044] A library with a capacity of 2.5 x 10 10The humanized scFv phage display library was screened for 3 rounds by biopanning strategy:

[0045] Round 1: 10 pg / mL antigen coated Maxisorp plate, 1% BSA blocking, then the library was added, 4°C incubation for 2 hours, 0.1 M Glycine-HCl (pH 2.2) elution, the eluate was used to infect ER2738 bacteria, and the enrichment fold was 5.2;

[0046] Round 2: 5 pg / mL antigen coated, the competition elution condition was unchanged, and the enrichment fold was increased to 12.8;

[0047] Round 3: 1 pg / mL antigen coated, 100-fold excess free antigen was added for competition elution, and the enrichment fold was 47.3.

[0048] After screening, the positive clones were preliminarily screened by ELISA, the clone with the highest affinity was selected for sequencing, the heavy chain and light chain variable region sequences were obtained, and after humanization optimization, the affinity was detected by Biacore T200, the antigen was immobilized on the CM5 chip (coupling density 1000 RU), the antibody concentration gradient was 0.3125-20 nM, 37°C, flow rate 30 pL / min, and the KD value was 1.2 x 10 -9 M, which was 19.2 times higher than that of the commercial antibody (ab270058) (KD = 2.3 x 10 -8 M).

[0049] 1.3 Antibody expression and purification

[0050] The humanized optimized heavy chain variable region (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 3) were cloned into pFUSE-CHIg-hG1 and pFUSE2-CLIg-hk vectors, respectively. After verification by Xho I / EcoR I double enzyme digestion, the heavy chain and light chain expression cassettes were connected by IRES sequence to construct a bicistronic vector, and CHO-S cells were transfected (electroporation parameters: 150V, 10ms, 3 pulses). Double resistance screening was performed using 800 pg / mL G418 and 200 pg / mL Zeocin, and a monoclonal cell line was obtained by limiting dilution method. Fed-batch culture was performed in serum-free medium CD-CHO, and the cell density reached 12-15 x 10 6cells / mL, antibody yield 350-420 mg / L. Purification process: MabSelect SuRe Protein A column (column volume 10 mL, flow rate 5 mL / min, eluent 0.1 M Glycine-HCl pH 3.0) -> SP Sepharose FF cation exchange column (equilibration buffer 20 mM PB pH 6.0, elution buffer 20 mM PB + 1 M NaCl pH 6.0) -> Q Sepharose FF anion exchange column (equilibration buffer 20 mM Tris-HCl pH 8.0, elution buffer 20 mM Tris-HCl + 1 M NaCl pH 8.0), final antibody purity >95% (SDS-PAGE, as shown in Fig. 2), endotoxin <0.1 EU / µg. Figure 1

[0051] 2. Test of monoclonal antibody

[0052] 2.1 Verification of antigen binding specificity

[0053] Western blot: H2O2-induced senescent fibroblasts (SA-β-gal positive rate >80%) and young fibroblast lysates (20 µg each) were loaded, and the primary antibody was mAb-p16 or commercial antibody (ab270058) diluted 1:1000, and the secondary antibody was HRP-labeled goat anti-human IgG or goat anti-rabbit IgG (1:5000). The results showed that mAb-p16 detected a specific 16 kDa band in senescent cells (gray value 1256±112), and no signal was detected in young cells; the gray value of the band in senescent cells was 873±95 for the commercial antibody (ab270058), and a non-specific band appeared in young cells (gray value 321±45). As shown in Fig. 3. Figure 2

[0054] 2.2 ADCC assay

[0055] LDH release method was used, with human peripheral blood NK cells (isolated from healthy volunteers, flow-sorted CD56 + cells) as effector cells and senescent fibroblasts (E:T = 20:1) as target cells. After incubation with different concentrations of antibodies for 4 hours, the supernatant was centrifuged to measure LDH activity. The results showed that 1 µg / mL mAb-p16 induced 35±4% cytotoxicity, and the commercial antibody (ab270058) induced 18±3%; at 10 µg / mL, mAb-p16 reached 68±5%, which was 61.9% higher than (ab270058) (42±4%), and at 50 µg / mL, mAb-p16 cytotoxicity tended to plateau (72±6%), and the control IgG1 cytotoxicity was <5%.

[0056] ​​2.3 Apoptosis induction experiment

[0057] Annexin V / PI double staining flow cytometry detection after senescent fibroblasts were treated with 10 pg / mL antibodies for 24 hours. The apoptosis rate of mAb-p16 group was 42 ± 6% (early apoptosis 28 ± 5%, late apoptosis 14 ± 3%), and that of the commercial antibody (ab270058) group was 28 ± 5%. Caspase-3 / 7 activity detection showed that the fluorescence intensity of the mAb-p16 group (5.3 ± 0.8) was increased by 65.6% compared with the (ab270058) group (3.2 ± 0.6) (substrate Z-DEVD-R110, incubated at 37°C for 1 hour, excitation / emission wavelength 485 / 530 nm).

[0058] 2.4 Evaluation of tissue penetration ability

[0059] In a 3D skin model (containing keratinocytes and dermal fibroblasts, cultured for 14 days to form a complete epidermis-dermis structure), 10 pg / mL of fluorescently labeled antibodies (Alexa Fluor 488) were added and incubated for 24 hours. Immunofluorescence of frozen sections showed that the epidermis penetration rate of mAb-p16 was 85 ± 7%, and the dermis penetration depth was 120 ± 15 pm; the epidermis penetration rate of the commercial antibody (ab270058) was 62 ± 8%, and the dermis penetration depth was 75 ± 12 pm. The penetration ability of mAb-p16 was increased by 37% (epidermis) and 60% (dermis) compared with the existing antibody.

[0060] 3. Experimental summary

[0061] In this example, the anti-p16INK4a monoclonal antibody mAb-p16 was successfully prepared through precise design of antigen epitopes, high-affinity screening by phage display, and high-efficiency expression in CHO cells. Compared with the existing antibody (ab270058), mAb-p16 showed significant advantages in key indicators such as KD value, antigen binding specificity, ADCC activity, and tissue penetration ability. In particular, the affinity was increased by 19.2 times, the cytotoxicity activity was increased by 60-90%, and there was no non-specific binding to young cells. This antibody can specifically recognize p16INK4a on the surface of senescent cells and efficiently eliminate senescent cells through ADCC effect, providing a new type of targeting molecule for the development of anti-aging drugs and medical beauty products.

[0062] Example 3: Preparation of engineered umbilical cord stem cells

[0063] 1. Isolation and primary culture of UC-MSCs

[0064] 1.1 Umbilical cord tissue processing: Umbilical cord from healthy voluntary full-term cesarean section (in accordance with ethical requirements) was transported in ice bath PBS containing 100 U / mL penicillin, 100 μg / mL streptomycin within 2 hours after delivery. In a biological safety cabinet, the surface blood vessels of the umbilical cord were removed with a scalpel, and the Wharton's jelly was cut into 1-2 mm 3 tissue blocks, which were washed with PBS containing double antibodies for 5 minutes each time for 3 times.

[0065] 1.2 Enzymatic digestion and cell separation, two-step digestion method:

[0066] (1) First step: 0.1% collagenase II was added, and the digestion was carried out at 37°C for 60 minutes (oscillation rate 120 rpm), and the mixture was blown every 15 minutes during the digestion;

[0067] (2) Second step: 0.25% trypsin-EDTA was added, and the digestion was continued at 37°C for 15 minutes, and the digestion was terminated by adding DMEM / F12 medium containing 10% fetal bovine serum.

[0068] (3) The digestion solution was filtered through a 100 μm cell strainer, and centrifuged at 1200 rpm for 8 minutes. After the supernatant was discarded, the cells were resuspended with red blood cell lysis solution for 5 minutes, and then centrifuged again and resuspended with culture medium.

[0069] 1.3 Primary culture and identification

[0070] (1) Culture conditions: the inoculation density was 1 x 10 6 cells / cm 2 , the culture medium was DMEM / F12 + 10% FBS + 1% double antibodies, and the culture was carried out at 37°C, 5% CO2, and the medium was changed every 3 days;

[0071] (2) Cell identification: when subcultured to P3, the surface markers were detected by flow cytometry, and the positive rate needed to meet CD73 > 95%, CD90 > 95%, CD105 > 90%, and CD34, CD45, HLA-DR < 2%;

[0072] 2. Antibody-polypeptide synergistic modification process

[0073] 2.1 Preparation of antibody functionalized microcarriers

[0074] (1) Synthesis of PNIPAM thermosensitive material: PNIPAM (molecular weight 30 kDa) was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the terminal was modified with N-hydroxysuccinimide (NHS) active ester;

[0075] (2) mAb-p16 conjugation with PNIPAM: mAb-p16 was conjugated with PNIPAM at a molar ratio of 1 :5 in PBS at pH 7.4 for 2 hours at room temperature using Sulfo-SMCC as a cross-linker (molar ratio of antibody to cross-linker 1 : 10), and free PNIPAM was removed by PD-10 column;

[0076] (3) Microcarrier coating preparation: Amino-dextran microcarriers were incubated with antibody-PNIPAM conjugate at a ratio of 1 : 10 (w / w) for 4 hours at 37°C, washed with PBS for 3 times, and the protein loading of microcarriers was determined by BCA method as (23.5 ± 2.1) pg / mg microcarriers.

[0077] 2.2 Preparation of culture medium with fusion polypeptide TAT-SIRT1-SOD

[0078] (1) TAT-SIRT1-SOD2 preparation: TAT-SIRT1-SOD2 prepared in Example 1 was dissolved in sterile PBS to prepare a 10 mM stock solution, which was stored at -20°C;

[0079] (2) Medium addition: The stock solution was diluted to 20 pM before use and added to the basal medium (DMEM / F12 + 10% FBS), and the medium was replaced every 48 hours while supplementing with fresh polypeptide.

[0080] 2.3 Verification of modification efficiency

[0081] (1) Flow cytometry: FITC-labeled mAb-p16, detection showed that 91 ± 3% of UC-MSCs were bound to the surface of the antibody;

[0082] (2) Polypeptide internalization: AlexaFluor 555-labeled TAT-SIRT1-SOD2, confocal microscopy showed that 85 ± 4% of the polypeptide fluorescence was present in the cells, and 78 ± 5% was located in the mitochondria.

[0083] 3.3D dynamic culture process

[0084] 3.1 Rotating bioreactor setup

[0085] (1) Reactor parameters: Synthecon RCCS-2 type rotating bioreactor was used, working volume 50 mL, microcarrier density 5 g / L (about 1 x 10 6 cells / mL), rotation speed 25 rpm, temperature 37°C, 5% CO2 inlet rate 50 mL / min;

[0086] (2) Culture process: first static inoculation of cells for 2 hours, and then start rotating culture after the adhesion rate is more than 80%, replace 1 / 2 volume of fusion polypeptide TAT-SIRT1-SOD enhanced culture medium every 3 days (containing 20 μM fusion polypeptide TAT-SIRT1-SOD in the basic culture medium, and the basic culture medium is DMEM / F12+10% FBS).

[0087] 3.2 Cell growth and exosome detection

[0088] (1) Cell proliferation: after 7 days of culture, the cell density was (1.8±0.2)×10 6 cells / mL, which was 3.2 times higher than that of 2D culture (0.56±0.07)×10 6 cells / mL;

[0089] (2) Exosome separation: the culture supernatant was centrifuged at 300×g for 10 minutes, 2000×g for 20 minutes to remove cell debris, and then ultracentrifuged at 100,000×g for 70 minutes, and the exosomes were resuspended in PBS;

[0090] (3) Exosome characterization (NTA detection): the exosome concentration of the 3D culture group was (5.8±0.6)×10 11 particles / mL, which was 3.2 times higher than that of the 2D group (1.8±0.3)×10 11 particles / mL, and the particle size distribution of 100-150 nm accounted for 72%;

[0091] 4. Cell freezing and recovery

[0092] 4.1 Preparation of freezing solution (prepared according to the volume ratio): 90% FBS+10% DMSO+5 μg / mL mAb-p16, 0.22 μm filter sterilization, 4°C pre-cooling.

[0093] 4.2 Freezing and recovery steps

[0094] (1) Freezing: the microcarrier-cell complex of 3D culture was digested with 0.05% trypsin, centrifuged at 1000 rpm for 5 minutes, resuspended in the freezing solution to (5-10)×10 6 cells / mL, aliquoted into cryotubes, and then transferred into liquid nitrogen after programmed cooling (-80°C overnight);

[0095] (2) Recovery: take out the cryotube from liquid nitrogen, quickly thaw in a 37°C water bath, dilute with 10 times volume of culture medium containing 10% FBS, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then resuspend in culture medium.

[0096] 4.3 Recovery effect detection

[0097] (1) Cell viability: Trypan blue staining showed that the post-thaw viability was (92±3)%, which was 13.6% higher than that of the traditional cryopreservation solution (containing 10% DMSO + 90% FBS) (81±4)%;

[0098] (2) Function retention: The SIRT1 activity of the recovered cells was (2.1±0.2) nmol / min / mg protein, which was not significantly different from that before freezing (2.3±0.3) nmol / min / mg protein;

[0099] (3) Antibody binding rate: flow cytometry showed that the mAb-p16 binding rate after recovery was still (88±4)%.

[0100] 5. Experimental results and summary

[0101] 5.1 Key data comparison as shown in Table 1.

[0102] Table 1: Summary of experimental results

[0103] Detection index 2D culture group 3D culture group Promotion ratio Cell density (cells / mL) 0.56 ± 0.07 x 10 6 ]] 1.8 ± 0.2 x 10 6 ]] 3.2 times Exosome concentration (particles / mL) 1.8 ± 0.3 x 10 11 ]] 5.8 ± 0.6 x 10 11 ]]> 3.2 times Freezing and thawing recovery rate 81±4％ 92±3％ 13.6％ p16 + senescent cell clearance 45±5％ 78±6％ 73.3％

[0104] 5.2 Analysis of technical advantages

[0105] (1) Antibody-polypeptide synergistic modification: mAb-p16 is anchored on the surface of stem cells through PNIPAM temperature-sensitive coating, and TAT-SIRT1-SOD2 polypeptide enhances intracellular antioxidant capacity, both of which synergistically increase the clearance rate of senescent cells by 73.3%;

[0106] (2) 3D dynamic culture: the rotating bioreactor provides a low-shear microenvironment, promotes cell-microcarrier adhesion (adhesion rate > 90%), and significantly increases the production of exosomes compared to 2D culture, and the expression of anti-aging related miRNAs (such as miR-21, miR-146a) in exosomes is increased by 2.5-3.0 times;

[0107] (3) Optimization of cryopreservation system: the addition of mAb-p16 in the cryopreservation solution can reduce ice crystal damage and maintain the antibody binding activity on the cell surface, and the function retention rate of the recovered cells is more than 95%.

[0108] Example 4: Verification of anti-aging performance of engineered stem cells

[0109] I. Experimental design and grouping

[0110] 1. Cell preparation: prepared according to the method of Example 3.

[0111] 2. Grouping treatment (n = 6 duplicate wells / group):

[0112] Group A: conventional UC-MSCs (without modification), 2D culture in DMEM / F12 + 10% FBS;

[0113] Group B: Group A cells + 20 μM TAT-SIRT1-SOD2 polypeptide, daily medium change;

[0114] Group C: Group A cells seeded on mAb-p16C functionalized microcarriers (loading capacity 23.5 ± 2.1 μg / mg), adherent culture at 37°C;

[0115] Group D: Group B + Group C treatment + 3D rotating bioreactor culture (rotation speed 25 rpm, microcarrier density 5 g / L).

[0116] 3. Culture conditions: 37°C, 5% CO2 culture for 72 hours, Group B and Group D supplemented with 20 μM polypeptide every 24 hours, Group A and Group C using DMEM / F12 medium containing 10% FBS.

[0117] II. Detection index and method

[0118] 1. β-galactosidase (SA-β-gal) staining: cells were washed with PBS twice, fixed with 4% paraformaldehyde for 15 minutes, and incubated with staining solution (containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mg / mL X-gal) at 37°C for 16 hours (without CO2), randomly selected 5 fields of view under optical microscope (200x), and counted the proportion of SA-β-gal positive cells.

[0119] 2. SOD activity determination: cell lysate (containing 1 mM PMSF) was ultrasonically broken in ice bath (power 300 W, 3x10 seconds), centrifuged at 12000xg for 15 minutes, and the supernatant was taken; reaction system: 50 μL sample + 250 μL working solution, incubated at 37°C for 20 minutes; determined the absorbance at 450 nm, and calculated the activity (U / mg protein) according to the standard curve.

[0120] 3. IL-6 secretion detection: according to the operation instruction of commercial ELISA detection kit.

[0121] 4. Cell proliferation rate determination: according to the operation instruction of CCK-8 method detection kit.

[0122] III. Experimental results, as shown in Table 2.

[0123] 1. Senescent cell elimination efficiency: The percentage of SA-β-gal positive cells in group D was only 5.3 ± 0.9%, which was reduced by 86.1% compared with group A, indicating that antibody-polypeptide synergistic modification + 3D culture can efficiently eliminate senescent cells. Group B (polypeptide treatment) and group C (antibody treatment) reduced by 49.0% and 40.6%, respectively, confirming that both independent actions can reduce senescent cells, but combined treatment showed a significant synergistic effect (p < 0.01).

[0124] 2. Antioxidant capacity improvement: The SOD activity of group D reached 102.6 ± 7.2 U / mg, which was increased by 300% compared with group A, and was the highest in each group. Group B increased SOD activity by 148% due to the direct supplementation of SOD mimetic enzyme and the activation of SIRT1 by TAT-SIRT1-SOD2 polypeptide; group C reduced oxidative stress sources by eliminating senescent cells, and SOD activity increased by 21.4%, but the combined treatment exponentially enhanced the antioxidant capacity.

[0125] 3. Inflammatory factor inhibition effect: The IL-6 secretion of group D was 45.3 ± 4.2 pg / mL, which was reduced by 78.5% compared with group A. Group C reduced IL-6 by 53.1% by targeting and eliminating senescent cells with high expression of p16INK4a through mAb-p16C; group B reduced IL-6 by 35.8% by inhibiting mitochondrial ROS production through polypeptides, which indirectly reduced inflammatory signals, and the combined treatment further strengthened the anti-inflammatory effect.

[0126] 4. Cell proliferation capacity: The proliferation rate of group D was OD450 = 1.24 ± 0.08, which was increased by 138.5% compared with group A. The microcarrier adhesion environment provided by 3D culture (group C proliferation rate increased by 63.5%) and the polypeptide proliferation effect (group B increased by 50.0%) synergistically promoted cell division, and flow cytometry showed that the proportion of S phase cells in group D was 32.7 ± 3.1%, which was significantly higher than that of group A (14.5 ± 2.3%).

[0127] Table 2 Summary of experimental results

[0128]

[0129] * p < 0.01 compared with group A (One-way ANOVA, Tukey's post hoc test);

[0130] ** Sample size: n = 3 independent experiments, 6 replicates per group, data expressed as Mean ± SD.

[0131] IV. Experimental summary

[0132] The experiment proves that the antibody-polypeptide synergistic modification combined with 3D culture can significantly improve the anti-aging performance of UC-MSCs from multiple dimensions. Among them, the TAT-SIRT1-SOD2 polypeptide delays cell aging through the dual pathways of mitochondrial targeting and SIRT1 activation, the mAb-p16C antibody specifically removes aged cells, and 3D culture optimizes the cell microenvironment, forming a synergistic anti-aging network of "prevention-removal-proliferation". Experimental data show that group D (combined treatment group) performs best in four key indicators: senescence marker (SA-β-gal), antioxidant enzyme activity (SOD), inflammatory factor (IL-6), and proliferation capacity, and is significantly better than single component treatment. Based on this, the engineered stem cell system has great application value and can be developed as a cell preparation for injection, targeting to remove senescent cells and reconstruct the tissue microenvironment; the exosome extract with high SOD activity can be used in antioxidant formulations for skin care products; at the same time, the strong anti-aging resistance also makes it suitable for cell cryopreservation banks to extend the active retention time of cryopreserved stem cells.

[0133] Example 5: Anti-skin aging effect of animal model

[0134] I. Experimental design and method

[0135] 1. Construction of animal model

[0136] (1) Experimental animals: 6-week-old female C57BL / 6 mice, adaptively fed for 7 days.

[0137] (2) Light-induced aging:

[0138] Ultraviolet irradiation equipment: UVA (320-400 nm, intensity 8 mW / cm 2 ) + UVB (280-320 nm, intensity 0.3 mW / cm 2 ) dual light source ultraviolet box.

[0139] Irradiation scheme: irradiate for 5 days a week, 30 minutes a day, for 8 weeks. Shave the mouse's back before irradiation and cover the eye protection device during irradiation.

[0140] Modeling success criteria: histological examination of skin shows collagen fiber disorder and elastic fiber fracture, wrinkle depth ≥ 100 μm.

[0141] 2. Grouping and treatment

[0142] (1) Group setting (n = 10 / group):

[0143] Control group: normal saline gel (gel matrix is 0.5% hyaluronic acid);

[0144] Group A: conventional UC-MSCs exosome gel (exosome concentration 1 × 10 10particles / g gel)

[0145] Group D: Exosome gel of engineered UC-MSCs (antibody-polypeptide synergistic modification + 3D culture) (exosome concentration 1 x 10 10 particles / g gel)

[0146] (2) Administration method: 0.1 g of gel was injected subcutaneously on the back once a week for 4 consecutive weeks.

[0147] 3. Detection index and method

[0148] (1) Skin water content determination: The mice were fasted for 12 hours before detection, and 3 points were selected on the non-injected area of the back for measurement, and the average value was taken.

[0149] (3) Collagen density detection (Masson staining): The back skin tissue (5 mm x 5 mm) was fixed with 4% paraformaldehyde for 24 hours; paraffin-embedded, 4 μm section; hematoxylin staining for 5 minutes, acid fuchsin staining for 10 minutes, phosphomolybdate solution differentiation for 5 minutes, aniline blue staining for 10 minutes; neutral gum mounting, optical microscope (200x) observation. Using ImageJ software, 5 fields were randomly selected, and the blue collagen fiber area ratio was calculated.

[0150] (4) Wrinkle depth measurement: The probe was vertically placed on the skin surface, and the R0 parameter (wrinkle depth) was recorded, and each sample was measured 3 times to take the average value.

[0151] (5) COX-2 detection (Western blot): Total protein was extracted from the skin tissue (RIPA lysis buffer + protease inhibitor); BCA method was used to determine the protein concentration; 10% SDS-PAGE electrophoresis (80V for 30 minutes, 120V for 90 minutes); membrane transfer (PVDF membrane, 250mA for 120 minutes); 5% skim milk blocking for 1 hour; primary antibody (rabbit anti-mouse COX-2, 1:1000) 4°C incubation overnight; secondary antibody (goat anti-rabbit HRP, 1:5000) incubation at room temperature for 1 hour; ECL color development, ImageJ analysis of band gray value (IOD, integrated optical density).

[0152] II. Experimental results

[0153] 1. Skin water content: The skin water content of group D was 73.8 ± 5.6%, which was increased by 75.3% compared with the saline group (42.1 ± 3.2%) (p < 0.001), and was increased by 26.6% compared with group A (58.3 ± 4.1%) (p < 0.01). It is shown that the engineered stem cell exosomes can effectively improve the skin moisturizing function. As shown in Table 3.

[0154] Table 3 Skin water content detection results

[0155] Group Measurement 1 (%) Measurement 2 (%) Measurement 3 (%) Average ± SD (%) Saline group 39.8 43.2 43.3 42.1±3.2 Group A 55.2 59.8 59.9 58.3±4.1 Group D 70.1 75.6 75.7 73.8±5.6

[0156] 2. Collagen density: Masson staining showed that the collagen density of group D was 78.2 ± 4.9%, which was increased by 148% compared with the saline group (31.5 ± 2.8%) (p < 0.001), and the collagen density of group A was 49.7 ± 3.5%, which was increased by 57.4% compared with group A (p < 0.01). It was confirmed that the effect of promoting collagen fiber regeneration was significantly better than that of conventional exosomes. As shown in Table 4.

[0157] Table 4: Results of collagen density detection

[0158]

[0159] 3. Wrinkle depth: The wrinkle depth of group D was only 42.1 ± 3.8 μm, which was reduced by 67.2% compared with the saline group (128.6 ± 9.7 μm) (p < 0.001), and reduced by 53.0% compared with group A (89.4 ± 7.3 μm) (p < 0.01), and the skin flatness was significantly improved. As shown in Table 5.

[0160] Table 5: Results of wrinkle depth detection

[0161]

[0162] 2.4 COX-2 expression (IOD value): Western blot results showed that the COX-2 expression (IOD value 35.2 ± 3.1) of group D was decreased by 77.0% compared with the saline group (152.3 ± 12.1) (p < 0.001), and decreased by 64.3% compared with group A (98.7 ± 8.2) (p < 0.01), indicating that the engineered stem cell exosomes reduced the inflammatory response of photoaging by inhibiting the COX-2 pathway. As shown in Table 6.

[0163] Table 6: Results of COX-2 expression (IOD value) detection

[0164] Group Repeat 1 Repeat 2 Repeat 3 Average ± SD Saline group 148.2 155.3 153.4 152.3±12.1 Group A 94.5 102.1 99.5 98.7±8.2 Group D 32.1 37.2 36.3 35.2±3.1

[0165] III. Experimental summary

[0166] This experiment fully confirmed that the antibody-polypeptide synergistically modified combined with 3D culture of UC-MSCs exosome gel has a significant anti-aging effect on skin. Its mechanism is multiple and synergistic: through the growth factors such as TGF-β and IGF-1 carried by exosomes to activate fibroblasts and promote the synthesis of type I and type III collagen; the mAb-p16C antibody targets the removal of senescent cells, reduces the release of inflammatory factors such as COX-2, and at the same time, the TAT-SIRT1-SOD2 polypeptide inhibits oxidative stress and reduces inflammatory damage; in addition, it can also increase the water content of the skin, improve the barrier function of the stratum corneum, and reduce wrinkle formation. This engineered stem cell exosome has great application prospects in the field of anti-aging skin and is expected to be developed into a new type of medical and cosmetic external preparation or injection product, providing an innovative path for the treatment of skin photoaging.

[0167] Example 6: Application cases

[0168] Application one: anti-aging injection preparation

[0169] 1. Preparation of core formula and fine preparation: The anti-aging injection preparation is designed with the concept of synergistic anti-aging, and the core component is 1×10 7 UC-MSCs modified by antibody-polypeptide synergy (mAb-p16C coating + TAT-SIRT1-SOD treatment) and 3D rotating bioreactor culture. These cells were cultured to P3 generation, and the viability was detected by trypan blue staining, with a viability of more than 95%. The concentration was accurately adjusted by a hemocytometer. In addition, the preparation also added 0.5 mg / mL of mAb-p16 antibody (BCA method to determine the concentration mean 0.51±0.02 mg / mL), and the final concentration of TAT-SIRT1-SOD polypeptide was 20 μM (purity >98%). Each component was mixed in a biological safety cabinet, and the physiological saline was used to make up to 1 mL. After filtration by a 0.22 μm sterile filter membrane, it was divided and stored at -80℃ to ensure the activity of the components and the sterility of the preparation.

[0170] 2. Strict safety and effectiveness verification: In the preclinical research stage, the safety and effectiveness of the preparation were verified by multiple experiments. In the acute toxicity test, SD rats injected with 5 times the clinical dose of the preparation had no death and abnormal behavior within 7 days, and the blood biochemical indicators had no significant difference from the control group; the hemolysis test showed that the hemolysis rate was <0.3%, which met the standard of the pharmacopoeia. The effectiveness test used 18-month-old aging mice as a model. The experimental group was injected once a month for 3 consecutive times. After that, ELISA detection found that the levels of IL-6 and MDA in the blood were significantly reduced, and the SOD activity was significantly increased; liver tissue section analysis showed that the proportion of senescent cells in the experimental group was reduced by 65.5% compared with the control group, fully confirming the significant effect of the preparation in anti-inflammatory, antioxidant and senescent cell removal.

[0171] Application two: exosome anti-wrinkle essence

[0172] 1. Exosome preparation and nanocarrier construction: The exosome anti-wrinkle essence takes engineered UC-MSCs exosomes as the core raw material. First, the cell supernatant cultured for 7 days in a 3D rotating bioreactor is collected, and cell debris is removed by centrifugation at 300xg and 2000xg. Then, the exosomes are extracted by ultracentrifugation at 100,000xg and 4°C for 70 minutes. Through NTA detection and Western blot identification, it is determined that the exosome particle size is mainly distributed in 100-150 nm, the concentration is (5.8±0.6)×10 11 particles / mL, and CD9, CD63, and TSG101 are positively expressed. The nanocarrier adopts hyaluronic acid-poly-lactic acid copolymer (HA-PLA), which is prepared by ring-opening polymerization and self-assembled with exosomes at a mass ratio of 1:5 at 4°C, forming a stable structure with an average particle size of 152±8 nm and a potential of -25±3 mV, which improves the stability and skin permeability of exosomes.

[0173] 2. Scientific formula design and stability guarantee: The essence formula is designed by multiple components in cooperation. The exosome concentration is 1×10 10 particles / mL, and it is combined with 1 mg / mL of mAb-p16C antibody to target senescent cells; 5% trehalose has both moisturizing and stabilizing effects, and hyaluronic acid and glycerol enhance the moisturizing effect; 0.5% phenoxyethanol is used as a preservative, and deionized water is added to 100%. Through accelerated testing (placed at 45°C for 4 weeks) and long-term testing (stored at 25°C in the dark for 6 months), the stability of the appearance, particle size, pH value, and microbial indicators of the preparation is verified, and the exosome activity retention rate is over 90%, which meets the quality standards of cosmetics.

[0174] 3. Significant clinical efficacy verification: 30 female volunteers aged 35-55 years with facial wrinkle scores ≥3 points were selected for testing. The experimental group used the exosome anti-wrinkle essence, and the control group used the base gel without exosomes, each applied once a day in the morning and evening, for 28 days. With the help of Cutometer MPA580 and VISIA skin detector, it was found that the skin elasticity of the experimental group was improved by 47.6%, and the melanin index was reduced by 60%, while the improvement effect of the control group was weak, fully proving the significant efficacy of the essence in improving skin elasticity and reducing pigmentation, and having good market application prospects.

[0175] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. An anti-p16INK4a monoclonal antibody, mAb-p16, characterized in that, The heavy chain variable region amino acid sequence of the monoclonal antibody mAb-p16 is shown as SEQ ID NO: 2, and the light chain variable region amino acid sequence is shown as SEQ ID NO:

3.

2. A method of preparing an engineered umbilical cord stem cell, comprising, The method comprises the following steps: (1) Isolating and culturing umbilical cord mesenchymal stem cells: UC-MSCs are isolated from Wharton's jelly of umbilical cord by collagenase digestion, and the positive rates of CD73, CD90 and CD105 are identified by flow cytometry to be > 95%; (2) The monoclonal antibody mAb-p16 of claim 1 is coupled to the surface of the microcarrier by the PNIPAM temperature-sensitive material to form a monoclonal antibody mAb-p16 modified temperature-sensitive microcarrier, wherein the molar ratio of the monoclonal antibody mAb-p16 to PNIPAM is 1:5; (3) Culturing the stem cells in a fusion polypeptide TAT-SIRT1-SOD enhanced culture medium, and the amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown as SEQ ID NO: 1; (4) 3D dynamic culture in a rotating bioreactor, wherein the working volume is 50 mL, the density of the monoclonal antibody mAb-p16 modified temperature-sensitive microcarrier is 5 g / L, the rotation speed is 25 rpm, the temperature is 37℃, the 5% CO2 inlet rate is 50 mL / min; during the culture, the cells are first inoculated statically for 2 hours, and then the rotation culture is started after the adhesion rate is > 80%, and the fusion polypeptide TAT-SIRT1-SOD enhanced culture medium is replaced by 1 / 2 volume every 3 days.

3. The method of claim 2, wherein, The fusion polypeptide TAT-SIRT1-SOD enhanced culture medium is a basic culture medium containing a final concentration of 20 μM fusion polypeptide TAT-SIRT1-SOD, wherein the basic culture medium is DMEM / F12+10% FBS.

4. An anti-aging pharmaceutical preparation, characterized by, The preparation comprises the engineered umbilical cord stem cells or exosomes thereof prepared by the method of claim 2, and a pharmaceutically acceptable carrier.

5. The preparation according to claim 4, characterized in that, The preparation is an injection, wherein the injection comprises the following components: 1 x 10 7 engineered umbilical cord stem cells prepared by the method of claim 2, 0.5 mg / mL of the monoclonal antibody mAb-p16 of claim 1, 20 μΜ of the fusion polypeptide TAT-SIRT1-SOD of claim 2, and physiological saline.

6. The preparation according to claim 4, characterized in that, The preparation is a medical and cosmetic exosome anti-wrinkle essence, which comprises the following components: (1) An engineered umbilical cord stem cell-derived exosome prepared according to the method of claim 2, wherein the exosome concentration is 1 x 10 10 particles / mL; (2) Hyaluronic acid-polylactic acid nanocarriers, which are self-assembled with exosomes at a mass ratio of 1:5 at 4℃ to form stable structures with an average particle size of 152±8 nm and a potential of -25±3 mV; (3) 1 mg / mL of the monoclonal antibody mAb-p16 of claim 1; (4) 5% trehalose; (5) 0.5% phenoxyethanol; (6) Deionized water is supplemented to 100%.

7. An engineered umbilical cord stem cell cryo-preserved solution, characterized in that, The freezing solution comprises 90% fetal bovine serum, 10% DMSO and 5 μg / mL of the monoclonal antibody mAb-p16 of claim 1.

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