A cell extract and cosmetics or pharmaceuticals containing the cell extract.

Abstract

This invention discloses a bioactive composition with synergistic effects. The composition comprises a highly active stem cell extract prepared by a sequential pretreatment of hypoxia combined with heat shock, and a high-affinity monoclonal antibody P3F6 targeting human PTEN protein. The stem cell extract is rich in heat shock proteins and various growth factors; the monoclonal antibody P3F6 possesses specific light and heavy chain variable region sequences. Experiments have demonstrated that this composition exhibits significant synergistic effects in promoting skin cell proliferation, inhibiting apoptosis, enhancing skin barrier function, and promoting collagen synthesis, with effects far exceeding those of single components. This invention also provides the application of the composition in the preparation of skin repair, anti-aging cosmetics, and medical wound healing dressings.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, specifically to a cell extract and cosmetics or pharmaceuticals containing the cell extract. Background Technology

[0002] With the continuous development of regenerative medicine and dermatology, cell-free extracts derived from mesenchymal stem cells (also known as cell secretomes or conditioned medium) have become a research hotspot in the fields of cosmetics and skin repair due to their excellent biocompatibility and multi-target regulatory properties. These extracts are rich in various growth factors, cytokines, exosomes, and signal peptides, and can mimic some of the paracrine functions of stem cells under cell-free conditions. They exert anti-aging and repair effects by promoting fibroblast proliferation, enhancing collagen synthesis, and improving the skin microenvironment.

[0003] However, existing stem cell extract preparation technologies have significant limitations. Under conventional culture conditions (20% O2, 37°C, standard serum system), mesenchymal stem cells (MSCs) are in a stable growth environment, and their secretory profile is biased towards basal maintenance, lacking high-level expression of repair-related factors. Studies have shown that stem cells can activate response pathways such as HIF-1α and Nrf2 under physiological stress conditions such as hypoxia, oxidative stress, or heat stimulation, thereby regulating the secretion of various repair-related factors. However, traditional single-stimulation strategies (such as hypoxia or heat shock treatment alone) often only upregulate the expression of some factors, and the effects are unstable, making it difficult to obtain highly active and balanced extracts.

[0004] At the molecular signaling level, skin aging and damage repair processes are closely related to imbalances in multiple signaling pathways. Among them, PTEN (phosphatase and tensin homolog), as an important negative regulator of the PI3K / Akt signaling pathway, is believed to inhibit cell survival and proliferation signals and limit tissue repair and regeneration when upregulated. While traditional stem cell extracts can provide various promoting factors, their promoting effects are often weakened in cellular environments with excessive PTEN activation, leading to inconsistent efficacy in clinical or cosmetic applications.

[0005] While recent studies have attempted to restore PI3K / Akt signaling pathway activity using PTEN inhibitors (including small molecule compounds or specific antibodies), these strategies are mostly used alone, lacking synergistic systems with complex biological extracts. In particular, the technical approach of combining functional antibodies with highly active stem cell extracts that have undergone multiple stress pretreatments to activate their secretory profile has not yet been systematically reported. Such combinations not only require balancing the bioactivity and compatibility of each component but also addressing their stability and synergistic mechanisms within the system.

[0006] Therefore, how to enhance the bioactivity of stem cell extracts through multidimensional stimulation strategies and synergize with signaling regulatory elements (such as PTEN pathway modulators) has become a key technical problem that urgently needs to be solved in this field. Innovation in this direction not only involves optimizing cell pretreatment processes, but also requires breakthroughs in composition design and molecular mechanisms of action. Summary of the Invention

[0007] The present invention aims to provide a highly active stem cell extract and its preparation method, and to provide a synergistic composition of the extract and a specific antibody, thereby solving the problems of insufficient activity and limited function of traditional stem cell extracts.

[0008] Therefore, this invention discloses a synergistic bioactive composition comprising a highly active stem cell extract and an anti-PTEN monoclonal antibody, P3F6. The highly active stem cell extract is prepared by concentrating the secretory supernatant of adipose-derived mesenchymal stem cells after hypoxia preconditioning and mild heat shock treatment. The hypoxia preconditioning treatment was conducted for 24 hours at an oxygen concentration of 1% and a temperature of 37°C. The mild heat shock treatment involved raising the temperature from 37°C to 39.5°C at a rate of 0.5°C / min and maintaining this temperature for two hours in a hypoxic environment. The concentrations of HSP70, VEGF, FGF-2, and TGF-β1 in the highly active stem cell extract are 52.8 ± 4.1 pg / μg total protein, 28.3 ± 2.2 pg / μg total protein, 18.9 ± 1.6 pg / μg total protein, and 35.6 ± 2.8 pg / μg total protein, respectively. The light chain variable region of the anti-PTEN monoclonal antibody P3F6 contains the amino acid sequence shown in SEQ ID NO: 1; the heavy chain variable region of the anti-PTEN monoclonal antibody P3F6 contains the amino acid sequence shown in SEQ ID NO: 2. The mass ratio of the highly active stem cell extract to the anti-PTEN monoclonal antibody P3F6 is 20:1.

[0009] In one aspect, the present invention also discloses a method for preparing the highly active stem cell extract, the method comprising the following steps: (1) Cell culture: ADSCs are cultured at a concentration of 1×10⁻⁶ cells / mL. 4 cells / cm 2The cells were seeded at a density of 10% in T175 culture flasks and cultured in DMEM / F12 medium containing 10% FBS at 20% O2, 5% CO2, and 37°C. When the cell confluence reached about 80%, the medium was discarded and the cells were washed twice with PBS buffer for later use. (2) Hypoxia preconditioning: The cells were replaced with serum-free DMEM / F12 medium and cultured in a hypoxia incubator at 1% O2, 5% CO2, and 37°C for 24 hours to activate the hypoxia stress response of the cells. During the culture period, the cell morphology changes were observed every 8 hours using an inverted microscope to confirm that the cells adhered well and had no obvious apoptosis characteristics; (3) Mild heat shock treatment: After 24 hours of hypoxic culture, the concentration of 1% O2 and 5% CO2 in the incubator was kept constant, and the temperature was gradually increased through the incubator temperature control system at a rate of 0.5℃ / min, from 37℃ to 39.5℃, and cultured for 2 hours; after the treatment, the temperature was quickly restored to 37°C; (4) Active factor induction secretion stage: after the treatment, the culture medium was replaced with fresh serum-free DMEM / F12 medium, and the cells were placed in 20% O2, 5% CO2, and 37°C for 24 hours; then the supernatant was collected, centrifuged at 4°C 3000×g for 15 minutes to remove cell debris, and then filtered through a 0.22 μm PES membrane for sterilization; (5) Preparation and preservation of extract: the filtered supernatant was concentrated to a total protein concentration of about 10 kDa using a 10 kDa ultrafiltration system. mg / mL, after aliquoting, is stored at −80°C to obtain a highly active stem cell extract.

[0010] In one aspect, the present invention also discloses an anti-PTEN monoclonal antibody P3F6, wherein the light chain variable region of the monoclonal antibody P3F6 has the amino acid sequence shown in SEQ ID NO: 1 and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2.

[0011] In one aspect, the present invention also discloses a cosmetic comprising the aforementioned synergistic bioactive composition and a cosmetically acceptable carrier.

[0012] In one aspect, the present invention also discloses a medical repair dressing comprising the aforementioned synergistic bioactive composition and a pharmaceutically acceptable dressing matrix.

[0013] In one aspect, the present invention also discloses the application of the aforementioned anti-PTEN monoclonal antibody P3F6 in the preparation of cosmetics or pharmaceuticals.

[0014] The technical solution provided by this invention has produced significant and unexpected beneficial effects. Most notably, the composition exhibits a powerful synergistic effect across multiple functional dimensions, rather than a simple additive effect. At the cellular level, the composition's effect on promoting the proliferation of human dermal fibroblasts far exceeds that of a single component, with a 72-hour proliferation rate as high as 368%, and it demonstrates excellent anti-apoptotic ability, with an apoptosis inhibition rate exceeding 84% in a hydrogen peroxide-induced stress model. In a three-dimensional model that more closely resembles the human skin environment, the composition also performs exceptionally well, significantly reducing transepidermal water loss by nearly 70% and simultaneously and substantially increasing collagen content and epidermal thickness. This demonstrates its ability to achieve comprehensive and synergistic skin structure reconstruction and functional recovery from deep repair to the surface barrier. The mechanism of this synergistic effect stems from the perfect complementarity of the two mechanisms of action: the stem cell extract provides cells with abundant nutrient signals and a stress-protective environment, while the P3F6 antibody precisely relieves the inhibition of PTEN on key pathways of cell proliferation and survival. Together, they form a powerful dual-engine of "signal activation and nutrient support." Furthermore, the stem cell extract prepared by this invention has a high content of active factors and strong antibody affinity, ensuring the high efficiency of the basic composition. Ultimately, these remarkable in vitro efficacy results have been successfully transformed into products with practical application value, providing novel and efficient solutions for skin anti-aging, barrier repair, and wound healing, with broad market prospects. Attached Figure Description

[0015] Figure 1 The SDS-PAGE results of monoclonal antibody P3F6, where 1 represents monoclonal antibody P3F6.

[0016] Figure 2 The results of Western Blot specificity detection of monoclonal antibody P3F6, where 1 is monoclonal antibody P3F6 and 2 is A12B. Detailed Implementation

[0017] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0018] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0019] Example 1: Preparation and activity detection of highly active stem cell extracts

[0020] This embodiment provides a method for activating adipose-derived mesenchymal stem cells (ADSCs) through sequential pretreatment of "hypoxia combined with heat shock" to prepare a highly bioactive stem cell extract, and to detect and analyze the content of key active factors in the extract.

[0021] 1. Materials and Reagents

[0022] (1) Cell source: 3rd-5th generation human adipose-derived mesenchymal stem cells (ADSCs, isolated from abdominal fat of healthy adults or commercially purchased, with a survival rate ≥98% after resuscitation);

[0023] (2) Culture flask treatment: T175 culture flasks were coated with 0.1% gelatin at 37℃ for 30 minutes in advance (10mL of gelatin per flask), the gelatin was discarded and the flasks were air-dried to avoid poor cell adhesion;

[0024] (3) Culture medium preparation: DMEM / F12 medium containing 10% fetal bovine serum, with 1% double antibiotics (penicillin 100U / mL + streptomycin 100μg / mL), stored at 4℃, and preheated in a water bath at 37℃ for 30 minutes before use.

[0025] (4) The ELISA kit used for detection includes HSP70, VEGF, FGF-2 and TGF-β1.

[0026] 2. Experimental Procedure

[0027] (1) Cell culture: Take the logarithmic growth phase ADSCs, digest them with 0.25% trypsin-EDTA at 37℃ for 2 minutes, add medium containing 10% FBS to stop the digestion, centrifuge at 1000×g for 5 minutes (room temperature), and discard the supernatant; resuspend the cells in the above complete medium, stain with trypan blue to count, and adjust the cell density to 1×10⁻⁶. 4 cells / cm 2 (The total number of cells seeded in the T175 culture flask was approximately 3.5 × 10⁻⁶) 6 Add 25 mL of preheated complete culture medium to the culture flask, gently shake to distribute the cells evenly, and avoid uneven density in some areas. Place in a Thermo incubator with parameters set to 20% O2, 5% CO2, 37°C, and 95% relative humidity. Observe the cell morphology (spindle-shaped fibrous appearance) and growth status daily using an inverted microscope. When the cells cover about 80% of the bottom of the culture flask under the microscope (without obvious gaps, cells are in contact but not overlapping), stop the culture. Discard all the culture medium in the culture flask, and gently wash the cells twice with PBS buffer (pH 7.4, calcium and magnesium-free) pre-cooled to 4°C, adding 10 mL of PBS each time. Soak for 5 minutes and then slowly pour out the PBS, avoiding strong washing that may cause cell detachment. After rinsing, invert the culture flask to drain the residual PBS for later use.

[0028] (2) Hypoxia preconditioning: Slowly add 20 mL of serum-free DMEM / F12 medium (same as step (1), without FBS and antibiotics) along the wall of the pretreated T175 culture flask to avoid cell detachment due to liquid impact; gently rotate the culture flask to ensure that the medium evenly covers the cell surface without any air bubbles. Start the hypoxia incubator (Thermo, hypoxia module) 1 hour in advance, introduce 1% O2 and 5% CO2, and balance until the O2 concentration in the incubator is stable, the temperature is 37℃, and the humidity is 95%; place the culture flask in the hypoxia incubator, record the start time, and continue incubation for 24 hours, during which the door of the incubator is closed to avoid gas concentration fluctuations. Every 8 hours (0h, 8h, 16h, 24h of culture), open the incubator and quickly remove the culture flask. Observe under an inverted microscope: the cells should maintain an adhesion rate of more than 95% and no large areas of floating; maintain the spindle shape and no obvious shrinkage, increase of cytoplasmic granules, or appearance of apoptotic bodies (circular bright areas); take 3 different field-of-view photos for each observation and mark the time points to ensure traceability. If the proportion of floating cells is found to be >5%, the experiment must be repeated.

[0029] (3) Mild heat shock treatment: After 24 hours of hypoxic culture, the concentration of 1% O2 and 5% CO2 in the incubator was kept constant. The temperature was gradually increased through the incubator temperature control system at a rate of 0.5℃ / min (to avoid sudden temperature rise that could damage the cells), from 37℃ to 39.5℃, over a period of about 5 minutes. After the temperature was increased, the temperature of the culture medium in the bottle was confirmed to be stable at 39.5℃±0.1℃ using the temperature probe built into the incubator. The start time of heat shock was recorded. The culture was continued for 2 hours, during which cell morphology was observed every 30 minutes, with a focus on confirming the absence of a large number of apoptotic cells (floating cells ≤3%). If any abnormality was observed, the treatment was terminated immediately. Five minutes before the end of the heat shock, the incubator temperature control system was adjusted back to 37℃ to prepare for temperature recovery. After 2 hours of heat shock, the incubator temperature was allowed to drop naturally to 37℃ (about 10 minutes, during which a hypoxic environment was maintained). After the temperature stabilized, the cell adhesion and morphology were observed again. After confirming that there was no obvious damage, subsequent operations were performed.

[0030] (4) Active factor induction and secretion stage: Turn on the hypoxia incubator, quickly remove the culture flask, discard all serum-free culture medium in a sterile laminar flow hood (to avoid contamination), add 20 mL of fresh preheated (37℃) serum-free DMEM / F12 culture medium; transfer the culture flask to a normal oxygen incubator (20% O2, 5% CO2, 37℃, 95% humidity), and continue culturing for 24 hours to induce cell secretion of active factors. After 24 hours of culture, use a sterile pipette (10 mL) to gently aspirate the supernatant along the wall of the culture flask in a laminar flow hood, transfer it to a sterile 50 mL centrifuge tube (pre-cooled to 4℃), and collect about 18-19 mL of supernatant from each flask; if a small number of floating cells appear in the supernatant, filter it initially with a sterile filter (70 μm) before centrifugation. Place the centrifuge tube containing the supernatant into a high-speed centrifuge, set the parameters to 4℃ and 3000×g, and centrifuge for 15 minutes. After centrifugation, collect the supernatant and transfer it to a new sterile centrifuge tube. Filter the supernatant using a 0.22μm pore size PES filter bottle, collect the filtered supernatant into a sterile reagent bottle (pre-cooled to 4℃), and label it "coarse extraction supernatant". Take 100μL of the sample for later use (to measure protein concentration).

[0031] (5) Preparation and preservation of extract: Use ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa. Wash the membrane with sterile PBS three times in advance (add 10 mL of PBS each time, centrifuge at 4000×g for 10 minutes each time) to remove the protective agent on the membrane. Add the filtered crude supernatant (about 18 mL / bottle) to the ultrafiltration tube, centrifuge at 4℃ and 4000×g. Take out the ultrafiltration tube every 30 minutes and invert it once to mix (to avoid membrane blockage and uneven concentration). During this period, use the BCA protein quantitative kit to detect the protein concentration of the concentrate. When the protein concentration reaches 10±0.5 mg / mL, stop centrifugation and collect the concentrate (about 1.8 mL / bottle), which is the high-activity stem cell extract (High-activity Hypoxia-Heat-shock ADSC Extract, abbreviated as HH-ADSC-E). Inside the laminar flow hood, dispense 1 mL of HH-ADSC-E into sterile EP tubes (1.5 mL each), quickly cap each tube, and label it with the name, concentration, and preparation date. Immediately store the tubes in a -80°C ultra-low temperature freezer to avoid repeated freeze-thaw cycles.

[0032] (6) Preparation of control group (Normal ADSC Extract, N-ADSC-E): ADSCs were inoculated and cultured to 80% confluence according to step (1), without hypoxia preconditioning or heat shock treatment. The culture medium was directly replaced with serum-free DMEM / F12 medium and cultured under normal oxygen for 24 hours. The supernatant was collected, centrifuged, filtered, concentrated by ultrafiltration (to 10 mg / mL), and stored. The steps were completely consistent with HH-ADSC-E to ensure uniform control conditions.

[0033] (7) Detection and analysis of active ingredients: The contents of HSP70, VEGF, FGF-2 and TGF-β1 in the two groups of extracts were detected by commercial ELISA kits. Each group of samples was measured in triplicate, and the results were taken as mean ± standard deviation.

[0034] 3. The test results are shown in Table 1. Statistical analysis (t-test, n=3) showed that the contents of the four key factors in the HH-ADSC-E group were significantly higher than those in the N-ADSC-E group (p< 0.01). Among them, the contents of HSP70 increased by about 247%, VEGF by about 233%, FGF-2 by about 205%, and TGF-β1 by about 189%.

[0035] Table 1. Content of active factors in highly active stem cell extracts (pg / μg total protein)

[0036]

[0037] 3. Experimental Summary: Through sequential stimulation with hypoxia combined with heat shock, the stress protection mechanism of ADSCs was significantly activated, leading to a significant upregulation of the levels of heat shock proteins and various growth factors secreted by the cells, resulting in an extract with enhanced biological activity. Compared with the extract obtained under conventional culture conditions, HH-ADSC-E showed a significant increase in both the types and concentrations of cytokines and signal peptides, indicating that this method can effectively enhance the activity of stem cell extracts and provides a scientific basis for their further development in skin repair and anti-aging applications.

[0038] Example 2: Preparation, screening and performance verification of monoclonal antibody P3F6

[0039] I. Experimental Materials and Equipment

[0040] 1. Cell line: HEK293F suspension cells, initial viability ≥95%, passage number ≤20, culture density not exceeding 6×10⁻⁶. 6 cells / mL.

[0041] 2. Expression vector: pcDNA3.4, containing a strong CMV promoter and ampicillin resistance gene, with a light chain expression framework matching the human κ chain signal peptide and a heavy chain framework matching the human IgG1 signal peptide.

[0042] 3. Transfection reagent: PEI MAX, prepared with 1 mg / mL sterile water, dispensed at -20℃, avoid repeated freeze-thaw cycles.

[0043] 4. Culture medium: Expi293 Expression Medium, preheated at 37°C for 30 min before use, serum-free formulation.

[0044] 5. Purification medium: Protein A Sepharose Fast Flow, pre-packed with a 5 mL chromatography column.

[0045] 6. Analytical reagents: Human PTEN protein (ab157087); PTEN ELISA Kit; CCK-8 reagent, etc.

[0046] 7. Buffer: PBS (pH 7.4): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, sterilized by 0.22 μm filtration; 0.1 M glycine (pH 2.7): 7.51 g / L glycine, pH adjusted with HCl, sterilized by filtration; coating buffer (0.05 M carbonate buffer, pH 9.6).

[0047] II. Antibody Molecular Design and Preparation Method of Monoclonal Antibody P3F6

[0048] 1. Antibody molecule design

[0049] (1) Light chain molecular structure: It is formed by direct fusion of the variable region of the light chain of the anti-human PTEN monoclonal antibody (amino acid sequence as shown in SEQ ID NO:1, which is obtained after screening and optimization by the inventors, LC-VL) and the constant region of the human κ chain (Cκ) through peptide bonds, ensuring that the reading frame is continuous without frameshift and that there are no additional amino acid insertions in the fusion region, so as to maintain the natural folding and function of the antibody light chain.

[0050] (2) Heavy chain molecular structure: It is composed of the heavy chain variable region (amino acid sequence as shown in SEQ ID NO:2, which is obtained after screening and optimization by the inventors, HC-VH) of the anti-human PTEN monoclonal antibody and the human IgG1 constant region (Cγ1), which ensures that the reading frame is continuous without frameshift, and there is no extra amino acid insertion in the fusion region. The variable region and the constant region are connected by natural peptide bonds, ensuring the integrity of the effector function structure of the Fc segment.

[0051] 2. Preparation process of monoclonal antibody P3F6

[0052] (1) Construction of fusion gene synthesis and expression vector

[0053] Codon optimization: Based on the amino acid sequence of the light / heavy chain fusion molecule, a human codon preference algorithm is used for optimization to avoid rare codon clusters in order to adapt to the expression preferences of HEK293F cells.

[0054] Enzyme restriction site design and gene synthesis: The light chain fusion gene was introduced with a HindIII restriction endonuclease site and a protective base at the 5' end, and an XhoI restriction endonuclease site and a protective base at the 3' end; the heavy chain fusion gene was introduced with a HindIII restriction endonuclease site and a protective base at the 5' end, and a BamHI restriction endonuclease site and a protective base at the 3' end; the above fusion genes were synthesized by a commercial institution (GenScript), cloned into the pUC57 cloning vector, and the correctness of the gene sequence was verified by Sanger sequencing.

[0055] Vector digestion and recovery: The pcDNA3.4 expression vector was used. The light chain vector was double-digested with HindIII / XhoI, and the heavy chain vector was double-digested with HindIII / BamHI. The reaction system contained 10×CutSmart Buffer, restriction endonuclease and vector DNA, and was incubated at 37℃ for 2 h. The linearized vector fragments were separated by 1% agarose gel electrophoresis and recovered using a gel recovery kit.

[0056] Ligation and transformation: The linearized pcDNA3.4 vector and the light chain fusion gene were mixed at a ratio of 2:1 (mass ratio), and T4 DNA ligase was added. The mixture was ligated at 16°C for 16 h. The linearized pcDNA3.4 vector and the heavy chain fusion gene were also mixed at a ratio of 2:1 (mass ratio) and ligated at 16°C for 16 h using T4 DNA ligase. 5 μL of each ligation product was used to transform DH5α competent cells, which were then plated on LB solid medium containing ampicillin (100 μg / mL) and cultured at 37°C for 16 h.

[0057] Positive clone identification: Three single colonies were picked and inoculated into LB liquid medium containing ampicillin for shaking culture. Plasmids were extracted using a plasmid mini-prep kit. The size of the plasmid insert fragment was verified by double enzyme digestion. Sanger sequencing was performed on the enzyme-positive plasmids to cover the variable-constant region fusion region and the full length of the constant region, confirming the absence of frameshift mutations and point mutations. Finally, the light chain expression plasmid (pcDNA3.4-LC) and the heavy chain expression plasmid (pcDNA3.4-HC) were obtained.

[0058] (2) Transient cotransfection expression

[0059] Cell pretreatment: 24 h before transfection, HEK293F cells in logarithmic growth phase (viability ≥96%) were harvested and the cell density was adjusted to 2 × 10⁻⁶ cells using fresh Expi293 Expression Medium. 6 Cells / mL were inoculated into 125 mL shake flasks at a working volume of 30 mL and cultured in a shaking incubator at 37℃, 8% CO2, and 120 rpm (amplitude 25 mm).

[0060] Preparation of transfection complex: On the day of transfection, take 15 μg each of pcDNA3.4-LC and pcDNA3.4-HC plasmids (mass ratio 1:1) and dilute them in 1.5 mL of Opti-MEM medium; separately take 135 μL of PEI MAX transfection reagent (1 mg / mL) (DNA to PEI mass ratio 1:3) and dilute it in 1.5 mL of Opti-MEM medium; slowly mix the two dilutions, vortex for 10 seconds, and then let stand at room temperature in the dark for 10 minutes to form DNA-PEI complex.

[0061] Transfection and Feeding: The DNA-PEI complex was slowly added dropwise to the HEK293F cell suspension, and the flask was gently shaken to disperse the complex evenly. The cells were then returned to the incubator for further culture. 24 h after transfection, 50% glucose solution (final concentration 5 g / L) and 100 mM sodium pyruvate solution (final concentration 1 mmol / L) were added to the culture system. 48 h after transfection, 500 μL of the sample was taken, centrifuged at 1000×g for 5 min, and the supernatant was used to screen antibody expression using Protein A test strips (positive color development indicates successful expression). On day 7 of culture, cell viability was measured (≥60%) and the culture supernatant was collected.

[0062] (3) Collection of supernatant and purification of antibody

[0063] Supernatant pretreatment: Aliquot 30 mL of culture supernatant into 50 mL centrifuge tubes, centrifuge at 4 °C and 3000 × g for 15 min to remove cell debris; take the centrifuged supernatant and vacuum filter it through a 0.22 μm PES filter membrane to further remove minute impurities; store the filtered supernatant on ice.

[0064] Protein A affinity chromatography purification: Connect a pre-packed Protein A Sepharose Fast Flow column (5 mL) to the chromatography system. Equilibrate the column with PBS (pH 7.4) at a flow rate of 5 mL / min until the UV 280 nm absorbance baseline stabilizes (approximately 10 column volumes). Load the pretreated supernatant onto the column at a flow rate of 2 mL / min and collect the breakthrough buffer (for detecting unbound antibodies). After loading, continue washing the column with PBS (pH 7.4) at a flow rate of 5 mL / min until the UV 280 nm absorbance returns to baseline (approximately 5 column volumes). Elute with 0.1 M glycine buffer (pH 2.7) at a flow rate of 1 mL / min, collecting 1 mL of eluent from each tube. Immediately after collection, add 1 M Tris-HCl buffer (pH 8.0) to neutralize to pH 7.2-7.4 to prevent antibody denaturation.

[0065] Antibody concentration and buffer replacement: Combine the UV280 nm positive elution peaks (approximately 3-5 mL in volume), transfer to a 10 kDa molecular weight cutoff ultrafiltration tube, and centrifuge at 4000×g at 4°C, mixing by inverting the ultrafiltration tube every 30 min; when the volume is concentrated to approximately 500 μL, add 5 mL of PBS (pH 7.4) and continue centrifugation, repeating 3 times to completely remove glycine from the elution buffer; finally concentrate to 1 mL, and use NanoDrop 2000 to detect protein concentration, with the target concentration controlled at 2.0±0.2 mg / mL.

[0066] SDS-PAGE detection ( Figure 1 ): Under reducing conditions (with the addition of β-mercaptoethanol), 1 μg of antibody was loaded onto a 12% separating gel, and electrophoresis was performed at a constant voltage of 120 V for 90 min. After staining with Coomassie Brilliant Blue R-250, a single band was clearly visible for the heavy chain (approximately 50 kDa) and the light chain (approximately 25 kDa), with no obvious extraneous bands or degradation products.

[0067] III. Screening and Performance Validation of P3F6 Antibodies

[0068] (1) ELISA detection of PTEN binding affinity: human PTEN protein was diluted to 1 μg / mL with coating buffer, 100 μL was added to each well of a 96-well plate, and incubated at 4℃ for 16 h; after discarding the solution, the plate was washed 3 times with PBST, 200 μL of 1% BSA was added, and the plate was incubated at 37℃ for 1 h.

[0069] P3F6 and control antibody A12B (ab32199) were diluted with 1% BSA to 0.1, 0.3, 1, 3, 10, 30, and 100 nM (7 gradients), with 100 μL added to each well (3 replicates), and incubated at 37°C for 1 h; washed 5 times with PBST. HRP-labeled anti-human IgG or HRP-labeled anti-rabbit secondary antibody (both 1:5000) were added, and incubated at 37°C for 30 min; washed 5 times with PBST, TMB was added for color development for 15 min, and the reaction was stopped with 2 M H2SO4; absorbance was measured at 450 nm using a microplate reader, and Kd values ​​were fitted using GraphPad Prism. The results showed that P3F6 Kd = 0.46 ± 0.05 nM, A12B Kd = 2.68 ± 0.32 nM, and the affinity of P3F6 was increased by 5.8 times.

[0070] (2) Western Blot validation of specificity: Human dermal fibroblasts were lysed on ice for 30 min with RIPA lysis buffer (containing protease inhibitor), centrifuged at 12000×g for 15 min at 4℃, and the protein concentration was adjusted to 2 mg / mL using the BCA method. 20 μg of protein was denatured and loaded onto a 10% gel electrophoresis plate (80 V stacking gel for 30 min, 120 V separating gel for 90 min); transferred to a PVDF membrane (300 mA for 90 min), and blocked with 5% skim milk powder for 1 h. Primary antibody (P3F6 / A12B, 1 μg / mL) was incubated overnight at 4℃; after washing 3 times with TBST, secondary antibody (1:10000) was added and incubated at room temperature for 1 h; ECL staining was performed, and the bands were analyzed using ImageJ. The results showed that ( Figure 2 P3F6 showed only a single band at approximately 55 kDa (PTEN molecular weight); A12B showed a nonspecific band.

[0071] (3) Cellular level functional verification: Human skin fibroblasts were used at a concentration of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates and adhered for 24 h. After 12 h of serum-free DMEM synchronization, cells were added to each well and cultured at 37°C for 24 h with PBS (control), A12B (10 μg / mL), and P3F6 (10 μg / mL). Proteins were extracted and Western blots were used to detect p-Akt and total Akt. The p-Akt / total Akt ratio was calculated using ImageJ. Results showed that the ratio was 3.2 in the P3F6 group, 1.5 in the A12B group, and 1.0 in the control group, indicating that P3F6 significantly activated the pathway.

[0072] (4) Cell proliferation promotion experiment: Human skin fibroblasts were used at 3×10 3 Cells were seeded into 96-well plates and, after 24 h of adhesion, were divided into groups and treated with PBS, A12B (10 μg / mL), and P3F6 (10 μg / mL), with 5 replicates per group. The cells were then incubated at 37°C for 48 h. 10 μL of CCK-8 was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was measured. The proliferation rate was calculated as (experimental group OD - blank OD) / (control OD - blank OD) × 100%. The results showed that the proliferation rate of the P3F6 group was 162.5 ± 8.4%, the A12B group was 124.8 ± 7.1%, and the control group was 100% (p < 0.01).

[0073] Example 3: Preparation of a synergistic composition of HH-ADSC-E and monoclonal antibody P3F6

[0074] I. Materials and Reagents

[0075] 1. Highly active human adipose-derived stem cell extract (HH-ADSC-E), with a concentration adjusted to 10.0 mg / mL (prepared in Example 1).

[0076] 2. Monoclonal antibody P3F6 (prepared in Example 2), with the concentration adjusted to 1.0 mg / mL before use.

[0077] 3. Buffer system: PBS (pH 7.4) + 5% trehalose (m / v).

[0078] 4. 0.22 μm polyethersulfone filter membrane (sterile), rotary mixer (4 °C), sterile cryovials.

[0079] II. Preparation method of the composition

[0080] 1. Ratio determination: Based on the results of previous optimization experiments, the optimal ratio was determined to be extract:antibody = 20:1 (m / m).

[0081] 2. Mixing procedure: At 4 °C, take 2 mL of HH-ADSC-E extract (10 mg / mL), slowly add 1 mL of P3F6 antibody solution (1 mg / mL), and then add buffer to bring the total volume to 4 mL. Gently mix at 15 rpm for 30 min on a rotary mixer to ensure the system is homogeneous and free of precipitation.

[0082] 3. Sterilization and preservation: The mixture was sterilized by filtration through a 0.22 μm PES membrane with a recovery rate of 98.4%. After filtration, it was aliquoted into sterile cryovials (1 mL per tube) and stored at −80°C.

[0083] Example 4: Cell proliferation experiment (CCK-8 assay)

[0084] I. Materials and Reagents

[0085] 1. Cells: Human dermal fibroblasts (HDFs), passages 5-8, passage ratio 1:3, viability ≥95%.

[0086] 2. Culture medium: DMEM high glucose medium + 10% FBS + 1% penicillin 100 U / mL + streptomycin 100 μg / mL.

[0087] 3. Detection reagent: CCK-8 kit, stored at 4°C protected from light, preheated at 37°C for 10 min before use.

[0088] 4. Test samples: HH-ADSC-E (prepared in Example 1, 10 mg / mL, diluted to 50 μg / mL with culture medium); P3F6 antibody (prepared in Example 2, 2 mg / mL, diluted to 2.5 μg / mL with culture medium); composition (HH-ADSC-E 50 μg / mL + P3F6 2.5 μg / mL, freshly prepared).

[0089] II. Experimental Procedure

[0090] 1. Cell pretreatment and seeding: Take frozen HDFs, thaw them rapidly in a 37°C water bath, add 9 mL of complete culture medium, centrifuge at 1000×g for 5 min, and discard the supernatant; resuspend the cells in complete culture medium, seed them in T25 culture flasks, and incubate at 37°C in a 5% CO2 incubator. When the confluence reaches 80%-90%, passage (digest with 0.25% trypsin for 2 min, then centrifuge after termination); take cells in the logarithmic growth phase, stain with trypan blue for counting, and adjust the density to 5×10⁶ cells / year. 4 cells / mL; add 100 μL of cell suspension (i.e., 5 × 10⁶ cells / mL) to each well of a 96-well plate. 3 Cells / well), add 100 μL PBS to the edge wells (to avoid edge effect); incubate at 37℃ and 5% CO2 for 24 h to ensure cell adhesion ≥90% (observe under an inverted microscope).

[0091] 2. Group treatment: Discard the original culture medium in the 96-well plate, add 100 μL / well of treatment solution according to the following groups (5 replicates per group), gently shake the plate to mix the liquid, and return it to the incubator to continue incubation:

[0092] (1) G1 (PBS control group): DMEM medium containing 1% FBS (to maintain cell survival and reduce serum interference) + 10 μL PBS;

[0093] (2) G2 (HH-ADSC-E group): DMEM medium containing 1% FBS + HH-ADSC-E (final concentration 50 μg / mL);

[0094] (3) G3 (P3F6 group): DMEM medium containing 1% FBS + P3F6 antibody (final concentration 2.5 μg / mL);

[0095] (4) G4 (combination group): DMEM medium containing 1% FBS + HH-ADSC-E (50 μg / mL) + P3F6 (2.5 μg / mL);

[0096] 3. CCK-8 assay (24 h, 48 h, 72 h time points): Two hours before each time point, add 10 μL CCK-8 reagent to each well (avoiding air bubbles); incubate at 37℃ in a 5% CO2 incubator in the dark for 2 hours (to ensure sufficient color development); measure the absorbance at 450 nm using a microplate reader (reference wavelength 630 nm), and record the OD value of each replicate well. Proliferation rate = (OD of experimental group - OD of blank well) / (OD of G1 group - OD of blank well) × 100% (blank wells are those containing only culture medium + CCK-8, OD...) 450 =0.05±0.01).

[0097] III. The experimental results are shown in Table 2.

[0098] 1. Proliferation Trend: The cell proliferation rate of each group gradually increased over time, reaching a peak at 72 h. Group G1 (control) showed slow proliferation, with a proliferation rate of only 100% at 72 h; Group G2 (HH-ADSC-E) showed significant proliferation, reaching 252.6% at 72 h, indicating that stem cell extract alone can effectively promote HDF proliferation; Group G3 (P3F6) showed weak proliferation, with only 118.4% at 72 h, suggesting that antibody alone has limited effect on promoting proliferation at this concentration.

[0099] 2. Synergistic effect: Group G4 (combination) showed the strongest proliferation ability, with proliferation rates of 290.0%, 351.6%, and 368.4% at 24 h, 48 h, and 72 h, respectively, which were significantly higher than those of Group G2 (p<0.01) and Group G3 (p<0.001). The synergistic index (CI) was <1.5 for all groups (CI=1.38 at 72 h), indicating that the combined use of HH-ADSC-E and P3F6 produced a synergistic proliferation effect, rather than a simple additive effect.

[0100] 3. Concentration dependence: The OD values ​​of group G4 were the highest among all groups at each time point, and the difference between group G4 and group G2 increased with time (difference of 0.22 at 24 h and 0.37 at 72 h), suggesting that the synergistic effect increased with culture time.

[0101] Table 2 OD at various time points 450 Calculation results of values ​​and cell proliferation rate

[0102]

[0103] Example 5: Apoptosis detection (Annexin V / PI double staining flow cytometry)

[0104] I. Materials and Reagents

[0105] 1. Cells: Human dermal fibroblasts (HDFs), passages 5-8, viability ≥95%.

[0106] 2. Apoptosis inducer: 30% H2O2, diluted to 500 μM with PBS (prepare fresh and protect from light).

[0107] 3. Detection reagents: Annexin V-FITC / PI apoptosis detection kit, containing Annexin V binding buffer (10×), Annexin V-FITC, and PI staining solution.

[0108] 4. Test samples: Same as in Example 4 (HH-ADSC-E 50 μg / mL, P3F6 2.5 μg / mL, composition as above). II. Experimental Procedure

[0109] 1. Cell seeding and apoptosis induction: HDFs were seeded at a rate of 1×10⁻⁶ cells / year. 5 Cells were seeded into 6-well plates, with 2 mL of complete culture medium added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h until adherence (60%-70% confluence). The culture medium was discarded, and the cells were washed twice with PBS. 2 mL of serum-free DMEM containing 500 μM H2O2 was added to each well, and the plates were incubated at 37°C for 4 h (some cells were observed to be wrinkled and floating under an inverted microscope, confirming successful apoptosis induction).

[0110] 2. Grouping: Discard the H2O2 induction solution, wash twice with PBS, and add 2 mL / well of treatment solution according to the following groups (3 replicates per group). Incubate at 37℃ with 5% CO2. 22 After culturing for 24 hours, observe cell morphology:

[0111] (1) G1 (normal): No apoptosis was induced, only complete culture medium was added (negative control);

[0112] (2) G1 (induction): After inducing apoptosis, add DMEM containing 1% FBS (positive control).

[0113] (3) G2: After inducing apoptosis, add DMEM+HH-ADSC-E (50 μg / mL) containing 1% FBS.

[0114] (4) G3: After inducing apoptosis, add DMEM+P3F6 (2.5 μg / mL) containing 1% FBS.

[0115] (5) G4: After inducing apoptosis, add DMEM+ composition containing 1% FBS (HH-ADSC-E 50 μg / mL + P3F6 2.5 μg / mL).

[0116] 3. Cell Collection and Staining: Collect the supernatant (containing floating apoptotic cells) from each well into a 15 mL centrifuge tube. Add 0.5 mL of 0.25% trypsin (without EDTA) to each well, digest at 37°C for 1 min, add 1 mL of complete culture medium to stop digestion, pipette to detach cells, and transfer to the centrifuge tubes. Centrifuge at 1000×g for 5 min, discard the supernatant, resuspend cells in PBS, and wash twice (to remove residual trypsin and culture medium). Adjust the cell density to 1×10⁶ cells / well using 1×Annexin V binding buffer. 6 For each cell / mL sample, transfer 100 μL of cell suspension to a flow cytometer, add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, mix gently, and incubate at room temperature in the dark for 15 min. Before loading the sample, add 400 μL of 1×Annexin V binding buffer to each tube and mix gently.

[0117] 4. Flow cytometry assay: excitation wavelength 488 nm, FITC detection channel (FL1), PI detection channel (FL2); blank control (buffer only), Annexin V-FITC single staining control, and PI single staining control were used for compensation adjustment; 1×10⁻⁶ samples were collected from each group. 4 Cells, recording Annexin V + / PI - (Early apoptosis), Annexin V + / PI + (Late apoptosis) cell proportion. Apoptosis inhibition rate = (G1 (induced) total apoptosis rate - experimental group total apoptosis rate) / G1 (induced) total apoptosis rate × 100%.

[0118] III. The experimental results are shown in Table 3.

[0119] 1. Apoptosis induction effect: After treatment with 500 μM H2O2, the total apoptosis rate of the G1 (induction) group reached 36.5±2.4%, which was significantly higher than that of the normal group (3.9±0.5%), indicating that the apoptosis model was successfully constructed.

[0120] 2. Anti-apoptotic effect of single treatment: The total apoptosis rate of the G2 group (HH-ADSC-E) was 12.1±1.0%, and the apoptosis inhibition rate was 66.8%; the total apoptosis rate of the G3 group (P3F6) was 20.5±1.6%, and the apoptosis inhibition rate was 43.8%, indicating that the anti-apoptotic effect of stem cell extract was better than that of antibody alone.

[0121] 3. Synergistic anti-apoptotic effect: The total apoptosis rate of the G4 group (combination) was only 5.8±0.6%, which was not significantly different from the normal group (3.9±0.5%) (p>0.05); the apoptosis inhibition rate reached 84.1%, which was significantly higher than that of the G2 group (p<0.01) and the G3 group (p<0.001), suggesting that the combined use of HH-ADSC-E and P3F6 can synergistically inhibit H2O2-induced apoptosis of HDFs, and the effect is close to that of normal cells.

[0122] 4. Distribution of apoptosis stages: The proportions of early apoptosis (2.5±0.3%) and late apoptosis (3.3±0.3%) in the G4 group were significantly lower than those in other treatment groups, indicating that the composition can simultaneously reduce apoptosis initiation and apoptosis progression.

[0123] Table 3. Distribution of apoptosis rate and calculation results of apoptosis inhibition rate.

[0124]

[0125] Example 6: Barrier Function Assessment of a 3D Skin Model

[0126] I. Materials and Reagents

[0127] 1. 3D Skin Model: Reconstruction of Human Epidermal Model (MatTek, EpiDerm) TM EPI-200 is constructed from normal adult epidermal keratinocytes, containing a complete stratum corneum, and is cultured for 7 days upon receipt (in the early stage of barrier function maturation).

[0128] 2. Model culture medium: EpiDerm TM Maintenance Medium, antibiotic-free, store at 4°C, preheat at 37°C for 30 minutes before use.

[0129] 3. Test samples: Same as in Examples 4-5: HH-ADSC-E (50 μg / mL, diluted with culture medium), P3F6 antibody (2.5 μg / mL, diluted with culture medium), and composition (HH-ADSC-E 50 μg / mL + P3F6 2.5 μg / mL, freshly prepared).

[0130] 4. Detection reagents: Hydroxyproline detection kit; HE staining kit; Masson's trichrome staining kit.

[0131] II. Experimental Procedure (Step-by-Step Operation)

[0132] 1. 3D Skin Model Preprocessing and Grouping:

[0133] Model reception and adaptation: Upon arrival of the model, immediately transfer it to a 37℃, 5% CO2 incubator. Use sterile forceps to remove the model inserts and place them in a 6-well plate. Add 2 mL of preheated Maintenance Medium to each well (the culture medium level should be 1 mm below the surface of the insert membrane to avoid submerging the model surface and to simulate the skin exposure environment). Adapt to culture for 24 hours (to ensure model viability and observe the epidermal cells under an inverted microscope to ensure they are neatly arranged and not detached).

[0134] Group design: The adapted models were divided into 4 groups (3 replicates per group, i.e., 3 independent models). The treatment solution was diluted with Maintenance Medium. Specific grouping details:

[0135] G1 (blank control): Maintenance Medium only;

[0136] G2 (HH-ADSC-E group): Maintenance Medium + HH-ADSC-E (final concentration 50 μg / mL);

[0137] G3 (P3F6 group): Maintenance Medium + P3F6 (final concentration 2.5 μg / mL);

[0138] G4 (composition group): Maintenance Medium + HH-ADSC-E (50 μg / mL) + P3F6 (2.5 μg / mL).

[0139] Continuous treatment: Change the treatment solution once a day (discard the old culture medium before changing the solution, and gently rinse the bottom of the insert membrane once with PBS to avoid residue). Add 2 mL of fresh treatment solution to each well each time, and continue treatment for 14 days. During this period, observe the appearance of the model every day (no shrinkage or browning is considered as normal activity).

[0140] 2. Operational details of testing indicators

[0141] (1) Barrier function: TEWL value detection (day 0, day 7, day 14)

[0142] Environmental preparation: 1 hour in advance, move the TEWL instrument and 3D model to a constant temperature and humidity room (22±1℃, humidity 50±5%) to avoid airflow interference (close the fan and doors and windows).

[0143] Detection steps: Gently wipe the model surface with a sterile cotton swab (to remove residual culture medium and avoid affecting the detection); place the TEWL probe vertically in the center of the model surface (1 mm away from the surface to avoid contact), detect 3 different regions for each model (center + two sides, 2 mm apart), detect each region 3 times, and take the average value; record the TEWL values ​​on day 0 (baseline before treatment), day 7, and day 14, and calculate the TEWL reduction rate within 14 days (reduction rate = (TEWL on day 0 - TEWL on day 14) / TEWL on day 0 × 100%).

[0144] (2) Collagen content: Hydroxyproline method (day 14)

[0145] Sample preparation: After processing, scrape the model off the insert membrane with sterile scissors, weigh it (approximately 20 mg per model), cut it into small pieces, add 1 mL of 6M HCl, and hydrolyze at 110℃ for 12 h (to ensure complete hydrolysis of collagen into hydroxyproline); after cooling the hydrolysate, adjust the pH to 6.0-6.8 with 10M NaOH, add distilled water to a final volume of 10 mL, centrifuge at 4℃ and 12000×g for 10 min, and collect the supernatant (to remove residue).

[0146] Color development and detection: Follow the kit instructions. Take 100 μL of supernatant, add 50 μL of chloramine T reagent, and incubate at 37℃ for 20 min; add 50 μL of Ehrlich reagent, and incubate at 60℃ for 20 min (protected from light); measure the absorbance at 550 nm using a microplate reader, and determine the absorbance based on the hydroxyproline standard curve (0-10 μg / mL, R...). 2 The hydroxyproline content in the sample was calculated using the formula (=0.998), and then converted to collagen content using the formula "collagen content = hydroxyproline content × 7.46". The final result is expressed as "μg collagen / mg model wet weight".

[0147] (3) Histological examination: HE staining and Masson staining (day 14)

[0148] Sample fixation and embedding: The model was fixed with 4% paraformaldehyde for 24 h (4℃), dehydrated with a gradient of ethanol (70%→80%→90%→100%, 30 min each), and cleared with xylene (twice, 15 min each); embedded in paraffin (60℃ paraffin, ensuring the epidermis is perpendicular to the section surface), cut 5 μm thick continuous sections with a microtome, mounted on glass slides (coated with poly-L-lysine to prevent detachment), and baked at 60℃ for 2 h.

[0149] HE staining (observation of epidermal structure and thickness): xylene twice (10 min each) → graded ethanol rehydration (100% → 90% → 80% → 70%, 5 min each) → rinse with distilled water for 5 min; hematoxylin staining for 5 min → rinse with tap water for 10 min (until cell nuclei are clear) → eosin staining for 2 min → rinse gently with distilled water; graded ethanol dehydration → xylene clearing → mounting with neutral resin; under a 200× field of view microscope, select 5 complete epidermal regions for each section, and measure the epidermal thickness (vertical distance from the top of the stratum corneum to the bottom of the basal layer) using ImageJ software, and take the average value.

[0150] Masson staining (quantitative collagen distribution): Dewaxing and rehydration as with HE staining; Weigert iron hematoxylin staining for 10 min → rinsing with tap water → Ponceau S and acid fuchsin staining for 5 min → differentiation with 1% phosphomolybdic acid solution for 3 min → aniline blue staining for 5 min → rinsing with 1% glacial acetic acid for 1 min; dehydration, clearing, and mounting as with HE staining; at 200× field of view, select 3 dermal regions (collagen-rich areas at the bottom of the model) for each section, and calculate the "collagen area percentage" (area of ​​blue collagen region / total field of view area × 100%) using ImageJ software.

[0151] III. Experimental results are shown in Tables 4 to 6.

[0152] 1. Barrier function repair (TEWL value): There was no significant difference in TEWL values ​​among the groups at baseline (day 0) (18.4-18.7 g / (h·m)). 2 The results indicated that the initial barrier state of the model was consistent; after 7 days of treatment, the TEWL values ​​of groups G2 and G4 decreased significantly (G2: 12.5±0.9, G4: 9.8±0.6), while the changes in groups G1 and G3 were minimal; at the 14-day endpoint, the TEWL value of group G4 was the lowest (5.5±0.4 g / (h·m)). 2 The TEWL value was close to that of normal human skin (4-6 g / (h·m²)), with a reduction rate of 69.52%, which was significantly better than that of group G2 (55.38%, p<0.01) and group G3 (13.04%, p<0.001), indicating that the composition can effectively repair the barrier function of the 3D skin model and reduce moisture loss.

[0153] 2. Collagen Synthesis and Deposition: Hydroxyproline method showed that the collagen content in group G4 (118.5±9.2μg / mg) was 32.8% higher than that in group G2 (89.2±6.8μg / mg) and 114.3% higher than that in group G3 (55.3±4.2μg / mg) (p<0.01). Masson staining further verified that the collagen area ratio in the dermis of group G4 (45.8±3.1%) was 2.5 times that of group G1 (18.2±1.5%), and the collagen fibers were more densely arranged (blue collagen bundles were uniformly distributed in a network under the microscope), while group G3 only slightly promoted collagen deposition, suggesting that HH-ADSC-E and P3F6 synergistically activate the collagen synthesis pathway.

[0154] 3. Epidermal structural integrity: The epidermal thickness of group G4 (82.3±6.5μm) was significantly higher than that of other groups, increasing by 20.1% compared to group G2. The stratum corneum thickness (15.6±1.2μm) was 1.8 times that of group G1 (8.5±0.7μm). HE staining showed that the epidermal cell layers in group G4 reached 15-17 layers, with tightly packed cells (basal cells were columnar and the intercellular spaces in the spinous layer were small), and the stratum corneum was continuous and intact. In contrast, the epidermis in group G1 was thinner and the stratum corneum was discontinuous, suggesting that the composition can promote the proliferation and differentiation of epidermal cells and build a more mature epidermal structure (consistent with the cell proliferation results in Example 4).

[0155] 4. Indicator correlation: The decrease in TEWL value was negatively correlated with epidermal thickness and stratum corneum thickness (r=-0.89, p<0.001), while collagen content was positively correlated with epidermal thickness (r=0.92, p<0.001). This indicates that the composition repairs the skin barrier through a dual mechanism of "promoting epidermal structure maturation + increasing collagen deposition", and the various indicators are synergistically improved, verifying the consistency of the mechanism of action.

[0156] Table 4 Dynamic changes in TEWL values ​​(g / (h·m)) 2 ))

[0157]

[0158] Table 5. Results of Collagen-Related Detections

[0159]

[0160] Table 6. Epidermal structural parameters

[0161]

[0162] IV. Experiment Summary

[0163] Synergistic effect verification: In a 3D skin model (which is closer to the in vivo environment), the combination of HH-ADSC-E and P3F6 showed significant synergistic effects in three core dimensions: barrier repair (TEWL reduction), collagen synthesis (content + distribution), and epidermal remodeling (thickness + number of cell layers). The effect was better than using either component alone (p<0.01), and no model toxicity was observed after 14 days of continuous treatment (normal appearance, no cell atrophy), proving its safety and efficacy.

[0164] Mechanism correlation: Based on the results of Examples 4 (cell proliferation) and 5 (anti-apoptosis), the mechanism of action of the composition is speculated to be: P3F6 promotes epidermal cell proliferation and differentiation by inhibiting PTEN activation of the PI3K / Akt pathway; HH-ADSC-E provides active factors such as TGF-β1 and FGF-2, which synergistically enhance collagen synthesis and barrier-related protein (such as filaggrin) expression, ultimately achieving the linkage of "structural repair-functional recovery".

[0165] Application value: The barrier repair ability (TEWL is close to normal skin) and collagen enhancement effect exhibited by this composition in 3D models make it suitable for sensitive skin repair, wound healing, and improvement of photo-aged skin. It provides key in vitro data support for subsequent animal experiments (such as mouse skin barrier damage model) and preclinical studies. At the same time, the detailed experimental steps (such as TEWL environmental control and collagen hydrolysis conditions) ensure the reproducibility and reliability of the results.

[0166] Example 7: Cosmetic Formulation and Preparation of Highly Active Stem Cell Extracts and Antibodies

[0167] 1. Formulation composition (percentage by mass)

[0168] (1) HH-ADSC-E extract: 3%;

[0169] (2) Monoclonal antibody P3F6: 0.15%;

[0170] (3) Trehalose: 5%;

[0171] (4) Hyaluronic acid (HA, MW 1.2 MDa): 0.5%;

[0172] (5) Preservative (phenoxyethanol / potassium sorbate compound): 0.5%;

[0173] (6) Deionized water: balance to 100%.

[0174] 2. Preparation Method: Heat deionized water to 40 °C, add trehalose and hyaluronic acid, and stir with a magnetic stirrer until completely dissolved. Slowly add HH-ADSC-E extract, stirring at low speed for 15 min to avoid foaming. Add monoclonal antibody P3F6 at 4 °C, and gently swirl to mix for 30 min. Add preservative and mix thoroughly. Sterilize through a 0.22 μm sterile filter membrane, dispense into sterile emulsion bottles, and store sealed at 4 °C.

[0175] 3. This cosmetic formula has both cell proliferation promotion and antioxidant functions, making it suitable for the development of skin repair and anti-aging products.

[0176] Example 8: Formulation and preparation of medical repair dressing

[0177] 1. Formulation composition (percentage by mass)

[0178] (1) HH-ADSC-E extract: 5%;

[0179] (2) Monoclonal antibody P3F6: 0.25%;

[0180] (3) Cellulose gel matrix (microcellulose / hydroxypropyl methylcellulose): 20%;

[0181] (4) Glycerin: 10%;

[0182] (5) Preservative (phenoxyethanol / potassium sorbate): 0.3%;

[0183] (6) Purified water: balance to 100%.

[0184] 2. Preparation method: Add the cellulose gel matrix to purified water and stir in a 50 °C water bath for 30 min to form a homogeneous gel matrix. Add glycerol and stir until completely dissolved to form a flexible gel. Slowly add HH-ADSC-E extract and monoclonal antibody P3F6, and stir at low speed for 20 min to avoid foaming and protein denaturation. Add preservative, mix well, and sterilize with a 0.22 μm sterile filter membrane. Aliquot into medical dressing molds, allow to solidify at room temperature, and then seal and store at 4 °C.

[0185] This medical dressing combines highly active stem cell extracts and antibodies, and has the functions of promoting cell proliferation and accelerating wound healing, making it suitable for wound repair and tissue regeneration scenarios.

[0186] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A synergistic bioactive composition, characterized in that, The composition comprises a highly active stem cell extract and an anti-PTEN monoclonal antibody P3F6.

2. The synergistic bioactive composition according to claim 1, characterized in that, The highly active stem cell extract was prepared by concentrating the secretory supernatant of adipose-derived mesenchymal stem cells after hypoxia preconditioning and mild heat shock treatment. The hypoxia preconditioning treatment was carried out for 24 hours in an environment with an oxygen concentration of 1% and a temperature of 37°C. The mild heat shock treatment was carried out in a hypoxia environment by raising the temperature from 37°C to 39.5°C at a rate of 0.5°C / min and maintaining it for two hours.

3. The synergistic bioactive composition according to claim 1, characterized in that, The concentrations of HSP70, VEGF, FGF-2, and TGF-β1 in the highly active stem cell extract were 52.8 ± 4.1 pg / μg total protein, 28.3 ± 2.2 pg / μg total protein, 18.9 ± 1.6 pg / μg total protein, and 35.6 ± 2.8 pg / μg total protein, respectively.

4. The synergistic bioactive composition according to claim 1, characterized in that, The light chain variable region of the anti-PTEN monoclonal antibody P3F6 contains the amino acid sequence shown in SEQ ID NO: 1; the heavy chain variable region of the anti-PTEN monoclonal antibody P3F6 contains the amino acid sequence shown in SEQ ID NO:

2.

5. The synergistic bioactive composition according to claim 1, characterized in that, The mass ratio of the highly active stem cell extract to the anti-PTEN monoclonal antibody P3F6 is 20:

1.

6. A method for preparing the highly active stem cell extract as described in claim 1, characterized in that, The method includes the following steps: (1) Cell culture: ADSCs were cultured at a concentration of 1×10⁻⁶. 4 cells / cm 2 The cells were seeded at a density in T175 culture flasks, and DMEM / F12 medium containing 10% FBS was added. The flasks were then cultured at 20% O2, 5% CO2, and 37°C. When the cells reached approximately 80% confluence, the medium was discarded, and the cells were washed twice with PBS buffer for later use. (2) Hypoxia preconditioning: The cells were replaced with serum-free DMEM / F12 medium and cultured in a hypoxia incubator at 1% O2, 5% CO2, and 37°C for 24 hours to activate the hypoxia stress response of the cells. During the culture period, the cell morphology was observed every 8 hours using an inverted microscope to confirm that the cells adhered well and showed no obvious apoptotic characteristics; (3) Mild heat shock treatment: After 24 hours of hypoxic culture, the concentration of 1% O2 and 5% CO2 in the incubator was kept constant. The temperature was gradually increased through the incubator temperature control system at a rate of 0.5℃ / min from 37℃ to 39.5℃ and cultured for 2 hours. After the treatment, the temperature was quickly restored to 37°C. (4) Active factor induction and secretion stage: After the treatment, the medium was replaced with fresh serum-free DMEM / F12 medium and the cells were cultured at 20% O2, 5% CO2, and 37°C for 24 hours. Then the supernatant was collected, centrifuged at 4°C and 3000×g for 15 minutes to remove cell debris, and then filtered through a 0.22 μm PES membrane for sterilization. (5) Preparation and preservation of extract: The supernatant after filtration is concentrated to a total protein concentration of about 10 mg / mL by a 10 kDa ultrafiltration system, and then aliquoted and stored at −80°C to obtain a highly active stem cell extract.

7. An anti-PTEN monoclonal antibody P3F6, characterized in that, The monoclonal antibody P3F6 has the amino acid sequence shown in SEQ ID NO: 1 in its light chain variable region and the amino acid sequence shown in SEQ ID NO: 2 in its heavy chain variable region.

8. A cosmetic product, characterized in that, The cosmetic comprises the synergistic bioactive composition as described in claim 1 and a cosmetically acceptable carrier.

9. A medical repair dressing, characterized in that, The medical repair dressing comprises the synergistic bioactive composition as described in claim 1 and a pharmaceutically acceptable dressing matrix.

10. The use of the anti-PTEN monoclonal antibody P3F6 as described in claim 7 in the preparation of cosmetics or pharmaceuticals.

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