CN114349856A

CN114349856A - Fibroblast outer capsule preparation and application thereof in cosmetics and medicines

Info

Publication number: CN114349856A
Application number: CN202210234556.1A
Authority: CN
Inventors: 李少波; 亓爱杰; 黄昱; 李立华; 陈清轩
Original assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Current assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-04-15
Anticipated expiration: 2042-03-11
Also published as: CN114349856B

Abstract

The invention relates to a preparation method of fibroblast outer capsules and application of the fibroblast outer capsules in cosmetics and medicines. The effect of the invention is obtained by inhibiting the activity of P53, further improving the activity of fibroblast, preventing senescence and further producing outer vesicles with high yield. The monoclonal antibody capable of inhibiting the activity of P53 can inhibit the aging of fibroblasts and improve the content of outer vesicles secreted by the fibroblasts after being added into a culture medium of the fibroblasts, has good anti-aging activity, is suitable for preparing anti-aging beauty or pharmaceutical products, and has good market application value.

Description

Fibroblast outer capsule preparation and application thereof in cosmetics and medicines

Technical Field

The invention relates to the field of pharmacy or cosmetics, in particular to preparation of an extracellular fibroblast vesicle and application of the extracellular fibroblast vesicle in beauty treatment and medicines.

Background

The outer vesicle is also called as an exosome, is a double-layer phospholipid vesicle with the diameter of 30-150 nm secreted by cells, and contains multiple miRNAs, proteins and cell regulatory factors; research shows that the exosome can communicate among cells, transmit biological information and protein and play a role in regulating receptor cells.

Extracellular vesicles are widely present in the human body and play an important role in physiological and pathophysiological processes. Extracellular vesicles have in turn been identified in a variety of bodily fluids, which underlies their potential as disease markers, and scientists and clinicians therefore actively investigate their role in diagnosis. The first evidence that demonstrates the interaction of extracellular vesicles with recipient cells is that prostasomes (prostasomees) are able to promote the activity of sperm cells. Over the last few years, extracellular vesicles have proven to have a very broad range of biological functions. EVs can mediate important intercellular short-range or long-range communication. Tumor cells or other cells of the tumor microenvironment secrete exosomes that promote tumor development by promoting angiogenesis, tumor cell migration, or tumor metastasis. The vesicles produced by tumor cells also contain immunosuppressive molecules that inhibit T lymphocytes or NK cells, or promote differentiation of regulatory T cells to suppress immune responses. Epithelial cells or the nervous system also have the function of producing extracellular vesicles. Extracellular vesicles released by small intestine epithelial cells are involved in antigen presentation under validated conditions, and these extracellular vesicles can extend the range of action of epithelial cells. Extracellular vesicles in respiratory and bronchoalveolar lavage fluids can enhance the release of proinflammatory cytokines from airway epithelial cells in asthmatic patients.

Current studies demonstrate the association of membrane-bound morphogens with extracellular domains, including Wnt, Notch receptor DII 4. Through Wnt signaling, exosomes produced by fibroblasts can promote breast cancer cell dynamic development. Likewise, in drosophila, Wnt-related EVs may be associated with the formation of a gradient of Wnt in tissue. In the nervous system, neurons, oligodendrocytes, microglia secrete extracellular vesicles that can act as receptor cells for each other. Extracellular vesicles have been shown to be involved in neuronal synaptic growth and neuronal survival. These secretory vesicles are thought to spread the source of the disease by interacting with the receptor cells. Recently, studies to identify the association of extracellular vesicles as tumor markers with their molecular cellular biological mechanisms have been increasing. Exosomes can be engineered by bioengineering methods to be a tool for disease treatment due to the advantages they have with surface proteins. In addition, the different integrin combinations on the surface of the exosomes determine the specificity of the exosome receptor cells and thus the specificity of cancer tissue metastasis. The exosome produced by the adipose tissue is rich in multiple miRNAs, and can regulate the gene expression level of the far-end tissue, thereby regulating the metabolism of the far-end tissue or cells.

Existing studies indicate that in theory any kind of cell can secrete exosomes; however, exosomes currently under investigation in the skin care field are mainly derived from human pluripotent induced stem cells (IPSCs), bone marrow Mesenchymal Stem Cells (MSCs) and human fibroblasts. Several recent studies have demonstrated that exosomes have significant anti-aging effects on skin. The exosome has the effect of promoting fibroblast regeneration and collagen synthesis. The effect of exosomes on fibroblast proliferation and collagen synthesis was observed by cell scratch assay. The experimental results show that exosomes from bone marrow mesenchymal stem cells and fibroblasts can remarkably promote the proliferation of fibroblasts and increase the synthesis of collagen.

Skin aging is mainly caused by two major causes: one is endogenous natural aging caused by aging; and various exogenous ages mainly caused by photo-aging due to ultraviolet ray damage. The use of exovesicles or exosomes to treat skin aging is a major direction of current research. However, the problem existing in the prior art is that fibroblasts are easy to age in the culture process, so that the prepared exosome has low activity and low yield, and therefore, the improvement of the activity of the fibroblasts is the key direction of research.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a method for promoting fibroblast proliferation and high-yield outer vesicles.

The applicant is based on the research results of the prior art: by down-regulating the expression of P53, the senescence of human fibroblasts can be inhibited, and the cell density can be increased.

Further, the invention also provides a monoclonal antibody 3E4 which specifically inhibits the activity of P53.

Furthermore, the light chain variable region sequence of the monoclonal antibody is (SEQ ID NO: 2) DIVITQRPALMAASPGEKVTITCVLSFWSGCKACAWYQQKSGISPKPWIYDIKSGSVGVPARFSGSGSGTSYSLTITSMEAEDAATYYCMNSMGWEGCFGAGTKLELK, and the heavy chain variable region sequence is (SEQ ID NO: 3) EVQLEESATELARPGASVKLSCKASGYIFSKPYWGWIKQRPGQGLEWIGVRFDECWKHMEGHAKPGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGHQRPGVTWGLGTTLAVSS.

Further, the monoclonal antibodies in certain embodiments as is well known in the art, the antigen binding specificity of a murine monoclonal antibody is primarily determined by the hypervariable Complementarity Determining Region (CDR) sequences. Murine antibodies typically comprise 6 CDR sequences, 3 on the light chain and 3 on the heavy chain of the antibody. As described above, chimeric, humanized or humanized forms of murine antibodies can be constructed by techniques such as CDR grafting wherein murine CDR sequences are inserted into, for example, human antibody framework and constant region sequences, or by ligating entire murine variable region sequences to human antibody constant region sequences. In alternative embodiments, the variable region sequences of an antibody may be constructed, for example, by chemical synthesis and assembly of oligonucleotides encoding the entire light and heavy chain variable regions of the antibody.

The invention also provides a gene encoding the heavy chain variable region of the monoclonal antibody; a recombinant vector containing the above gene; and a host cell into which the above gene or recombinant vector is introduced.

The above gene includes not only a sequence encoding a heavy chain variable region but also a polynucleotide having substantially the same nucleotide sequence as the gene and a fragment thereof. A polynucleotide having substantially the same nucleotide sequence as the gene may have at least 80%, preferably 90%, more preferably at least 95% homology with the polypeptide of the present invention. As described above, the polynucleotide of the present invention may include a variant in which one or more nucleotide sequences are substituted, deleted or inserted, as long as the variant encodes a protein having equivalent activity.

Various host cells may be used to express the heavy chain variable region of the monoclonal antibody according to the invention. For example, the host cell may be a prokaryotic cell, such as an Escherichia, Bacillus, Streptomyces, Pseudomonas, Proteus or staphylococcal fungus, such as an Aspergillus yeast, e.g., Pichia, Saccharomyces, Schizosaccharomyces or Neurospora, other lower eukaryotic cell, or eukaryotic cell, such as animal and plant cells.

As a method for introducing the recombinant vector of the present invention into a host cell and transforming it, a conventional gene manipulation method can be used. For example, as physical methods, microinjection, a liposome-dependent method, a direct DNA uptake method, a receptor-mediated DNA transfer method, a cell purification method, and the like. Targeted DNA transfer or virus-mediated gene transfer may be used.

Further, the present invention provides a method for preventing senescence by increasing fibroblast activity by inhibiting P53 activity to produce high production of outer vesicles.

Further, the invention also provides a preparation method of the outer vesicle, which comprises the following specific steps:

culturing fibroblast HSF in DMEM (containing 10% fetal calf serum and 1% double antibody mixture), placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein the cells grow to more than 80%, washing with PBS for 2-3 times, adding 1mL0.25% trypsin, digesting at normal temperature for 30-60 s, tapping the bottle wall to make the cells fall off, adding 2 times of fetal calf serum-containing culture medium to stop digestion, blowing the mixture uniformly with a gun tube, centrifuging at 1000r/min for 8min, removing the supernatant, precipitating, adding high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and 3E4 monoclonal antibody with the final concentration of 100 mu g/mL, and placing the high-sugar DMEM culture medium in a standard incubatorThe culture was continued for 96 h.

Collecting supernatant, centrifuging at 300 × g for 10min to remove dead cells and large cell debris, centrifuging at 2000 × g for 15min to remove dead cells and cell debris, centrifuging at 10000 × g/min for 30min to remove vesicles with large cell debris, transferring supernatant into ultracentrifuge tube, and centrifuging at 10 × g/min⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to obtain outer vesicle, and performing above operations at 4 deg.C under aseptic condition.

Furthermore, the invention also provides application of the outer vesicle prepared by the method in preparing an anti-aging pharmaceutical composition.

The composition of the invention can be prepared for oral or parenteral administration by mixing with a commonly used diluent or excipient such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant and a surfactant. The oral solid preparation is tablet, pill, powder, granule, capsule and tablet. These solid preparations are prepared by mixing the composition of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Lubricating agents such as magnesium stearate and talc may also be included. Liquid preparations for oral administration are suspensions, solutions, emulsions and syrups, and the above-mentioned preparations may contain various excipients such as wetting agents, sweeteners, aromatics and preservatives.

The pharmaceutical compositions of the present invention may be formulated for parenteral administration as sterile aqueous solutions, water-insoluble excipients, suspensions and emulsions.

Water insoluble excipients and suspensions may contain propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethanol and the like.

The composition for preventing or treating MERS of the present invention may be administered orally or parenterally, including intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection.

Further, the invention also provides application of the outer vesicle prepared by the method in preparing a cosmetic product for resisting skin aging.

The active ingredients of the cosmetic products for the skin of the present invention are outer vesicles mixed in a base, and the content of these active ingredients is generally about 0.0001 to 10% by weight, preferably about 0.001 to 5% by weight. From the above-mentioned content range of the active ingredient, a satisfactory anti-aging effect can be obtained.

In the present invention, all kinds of cosmetic basic materials used so far, for example, various alcohols, animal or vegetable fats, surfactants, pectins, carboxymethylcellulose, alginates, etc., and other additives such as stabilizers, pigments, fragrances, etc. The mixture may be appropriately mixed and, if necessary, the blend may be melted by heating, or the blend may be melted and stirred. The compositions of the present invention can be used in these and all other considerable forms.

Advantageous effects

The invention provides a method for improving the activity of fibroblast by inhibiting the activity of P53, preventing senescence and further producing outer vesicles at high yield. The monoclonal antibody capable of inhibiting the activity of P53 can inhibit the aging of fibroblasts and improve the content of outer vesicles secreted by the fibroblasts after being added into a culture medium of the fibroblasts, has good anti-aging activity, is suitable for preparing anti-aging beauty or pharmaceutical products, and has good market application value.

Drawings

FIG. 1 is a graph showing the detection result of Western blot

FIG. 2 is a graph showing the effect of fibroblast proliferation

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of P53 monoclonal antibody

Recombinant human p53 protein (abcam, cat ab84768, SEQ ID NO: 1) was used as immunogen. The recombinant protein was mixed well with an equal volume of Freund's complete adjuvant, and BALB/c mice were injected subcutaneously with 50. mu.g/mouse at multiple points. The primary immunization was performed at 4 weeks, and the postero-cutaneous multi-point injection was performed at the back, and the secondary immunization was performed at 4 weeks, with 50. mu.g of immunogen used per time. And (4) after 3 rd immunization, measuring the serum antibody titer by ELISA (enzyme-linked immunosorbent assay) in tail vein blood collection at 7-10 d. The 1 mouse with the highest antibody titer was selected 4d before the fusion and boosted by intraperitoneal injection of 150. mu.g of non-adjuvant antigen.

Aseptically taking splenocytes of the boosted BALB/c mice, fusing Sp2/0 myeloma cells and splenocytes of the mice according to a ratio of 1: 7 by using 50% polyethylene glycol as a fusion agent, adding HAT selective culture solution, inoculating a 96-well plate, placing at 37 ℃ and 5% CO₂Culturing in an incubator, sucking culture supernatants in growth cloning holes after 8 days, respectively using recombinant proteins as coating antigens, performing primary screening by ELISA, screening positive hybridoma cells only reacting with the recombinant proteins, performing 3-4 rounds of subcloning by a limiting dilution method until all monoclonals are positive, and performing expanded culture and strain building. The more stable monoclonal antibody hybridoma cells 3E4 and 2F6 were obtained.

Abdominal injection of 3E4 and 2F6 hybridoma cells into BALB/c mice approximately 2X 10⁶After about 8 days, the abdomen of the mouse is obviously expanded, ascites is sucked up, 2000r/min is carried out, centrifugation is carried out for 20min, and the supernatant is taken. After dilution with equilibration buffer, the mixture was filtered through a 0.45 μm filter. And (3) passing the filtered ascites through a Purify protein purifier at the flow rate of 1ml/min, washing the impure protein by a balanced buffer solution, eluting the antibody by an eluent (glycine-hydrochloric acid pH2.8100mmol/L), collecting the antibody with OD280 more than 0.5, dialyzing by PBS at 4 ℃, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification to obtain a relatively pure monoclonal antibody through identification.

Example 23E 4 monoclonal antibody affinity, subtype identification, sequence analysis and specificity identification

Using AMC sensors, purified 3E4 antibody was diluted to 10ug/ml with PBST and recombinant protein was diluted with PBST in a gradient: 444.4nmol/ml, 222.2nmol/ml, 111.1nmol/ml, 55.6nmol/ml, 27.8nmol/ml, 0 nmol/ml; the operation flow is as follows: equilibration for 60s in buffer 1(PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69 GLY solution and buffer 3, and data output are shown in Table 1. (KD represents the equilibrium dissociation constant, i.e.affinity)

TABLE 13E 4 monoclonal antibody affinity results

Name of antibody	KD（M）
		3E4 monoclonal antibody	3.42E-09

As can be seen from the results in Table 1, the 3E4 monoclonal antibody has strong affinity, which can reach 3.42E-09M. Meanwhile, the purified 3E4 monoclonal antibody aiming at the P53 protein is verified by an antibody subtype identification kit, the antibody subtype is analyzed, and the result shows that the result is IgG2a subtype and Kappa chain.

RNA was extracted from 3E4 hybridoma cell line, cDNA was synthesized by reverse transcription, the Light Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIg-kR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIg-HR primers. Sequencing the amplified product, and putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis to obtain light and heavy chain sequences of the antibody as shown in the following.

TABLE 23E 4 monoclonal antibody CDR-philic sequences

CDR name	CDR sequences
		LCDR1	VLSFWSGCKACA
LCDR2	DIKSGSV
		LCDR3	MNSMGWEGC
HCDR1	KPYWG
		HCDR2	VRFDECWKHMEGHAKP
HCDR3	HQRPGVT

Light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCVLSFWSGCKACAWYQQKSGISPKPWIYDIKSGSVGVPARFSGSGSGTSYSLTITSMEAEDAATYYC MNSMGWEGCFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSKPYWGWIKQRPGQGLEWIGVRFDECWKHMEGHAKPGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGHQRPGVTWGLGTTLAVSS。

The specificity of the 3E4 mAb is identified by a Westernblot method, the p53 recombinant protein, fibrin and BSA are subjected to SDS-PAGE electrophoresis, after membrane transfer, the 3E4 mAb is diluted by 1: 500 and incubated, and goat anti-mouse IgG labeled by HRP (diluted by 1: 7500) is used as a secondary antibody, and the operation is carried out according to the Westernblot step. The results show that the mAb can react specifically with the P53 recombinant protein (with a specific reaction band), and that neither BSA nor fibrin react (FIG. 1), which indicates that the antibody of the invention has better specificity.

EXAMPLE 3 culture of fibroblasts

Culturing fibroblast HSF in DMEM (containing 10% fetal calf serum and 1% double antibody mixture), placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein cells grow to be more than 80%, washing for 2-3 times by PBS, adding 1mL0.25% trypsin, digesting for 30-60 s at normal temperature, tapping the bottle wall slightly to enable the cells to fall off, adding 2 times of volume of culture medium containing fetal calf serum to stop digestion, blowing and beating uniformly by a gun barrel, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding a proper amount of complete culture medium to prepare single cell suspension, adjusting concentration, and using the HSF in logarithmic phase with good condition for experiment. Taking HSF of logarithmic growth phase at 1 × 10⁵Inoculating each cell/mL into 96-well culture plate, culturing, inoculating 90 μ L per well, placing in cell culture box at 37 deg.C and 5% CO₂Culturing, and taking out after 24h to set up an experimental group with different concentrations of 3E4 monoclonal antibody, a negative control group (containing an equal amount of cell resuspension) and a blank control group (containing an equal amount of DMEM culture solution). The experimental group was added with 10. mu.L/well of 3E4 monoclonal antibody to final concentrations of 100, 50, 10 and 1. mu.g/mL, and the negative control group and the blank control group were added with the same amount of DMEM culture solution per well. At least 3 multiple wells are arranged for each concentration, after uniform oscillation, the mixture is placed in a standard incubator for continuous culture, after administration is carried out for 96 hours, the mixture is taken out and added with MTT20 mu L/well, after continuous culture is carried out for 4 hours, DMSO 150 mu L/well is added, after uniform oscillation, the absorbance value (OD) of each well is measured at the position of 490nm wavelength of an enzyme-labeling instrument, and the result is shown in figure 2.

As can be seen from the results in FIG. 2, the fibroblast cells have better effects of reducing apoptosis and promoting cell proliferation after the 3E4 monoclonal antibody is administered for 96 hours, wherein OD after 100 mug/mL experimental group is 4.2, the fibroblast cells have better proliferation effect, and the difference between the administration and non-administration is significant (P < 0.05).

The expression level of p53 protein in fibroblasts after 96h was identified by the Westernblot method, and the results are shown in Table 3.

TABLE 3 expression level of p53 protein in fibroblasts

Experiment of each group	Expression level of p53 protein
		100μg/mL	0.29±0.03*
50μg/mL	0.43±0.04*
		10μg/mL	0.58±0.04*
1μg/mL	0.62±0.03*
		Negative control group	1.00
Blank control group	1.00

As can be seen from the results in table 3, the P53 protein expression level in the fibroblasts after 96h culture was significantly inhibited in the experimental group relative to the negative control group and the blank group (P < 0.05). This also indicates that inhibition of the activity of P53 protein effectively inhibits apoptosis of fibroblasts, thereby promoting proliferation of fibroblasts.

Example 4 preparation of extracellular vesicles of fibroblasts in antibody-containing Medium

Fibroblast HSF was cultured in DMEM (containing 10% fetal bovine serum, 1% double antibody mixture) mediumIn the reaction solution, the mixture is placed at 37 ℃ and 5% CO₂Culturing in an incubator, wherein cells grow to more than 80%, washing for 2-3 times by PBS, adding 1mL0.25% trypsin, digesting at normal temperature for 30-60 s, slightly tapping the bottle wall to make the cells fall off, adding 2 times of volume of culture medium containing fetal calf serum to stop digestion, uniformly blowing by a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and 3E4 monoclonal antibody with final concentration of 100 mu g/mL, and placing in a standard incubator for further culturing for 96 h.

Operating the above culture steps in a large scale, collecting 50mL of supernatant, centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 15min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, transferring the supernatant to an ultracentrifuge tube, and centrifuging at 10 Xg/min to remove vesicles⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to total 1.23g, dissolving the precipitate with PBS to obtain outer vesicle, and performing above operations at 4 deg.C under aseptic condition.

Example 5 preparation of control fibroblast extracellular vesicles

Culturing fibroblast HSF in DMEM (containing 10% fetal calf serum and 1% double antibody mixture), placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein cells grow to more than 80%, washing for 2-3 times by PBS, adding 1mL0.25% trypsin, digesting at normal temperature for 30-60 s, slightly tapping the bottle wall to make the cells fall off, adding 2 times of culture medium containing fetal calf serum to stop digestion, uniformly blowing by a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding a high-sugar DMEM culture medium containing 10% fetal calf serum and 1% double-antibody mixed solution, and placing in a standard incubator for continuous culture for 96 h.

Operating the above culture steps in a large scale, collecting 50mL of supernatant, centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 15min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, transferring the supernatant to an ultracentrifuge tube, and centrifuging at 10 Xg/min to remove vesicles⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to total 0.96g, dissolving the precipitate with PBS to obtain outer vesicle, and performing above operations at 4 deg.C under aseptic condition.

As can be seen from the results of example 5, without treatment with mabs, the decrease in cell activity resulted in a relative decrease in the overall production of outer vesicles.

Example 6 verification of the Activity of fibroblast extracellular vesicles

The outer vesicles prepared in example 4 and example 5 were adjusted to the same concentration.

Grouping experimental animals: the 50 mice were randomly divided into 5 groups of 10 mice per group by weight, each group being: normal control group (NC), model control group (MC), positive control group (PC), example 4 outer vesicle stem-control group (WNP 4), example 5 outer vesicle stem-control group (WNP 5). Except for NC group mice injected with equal volume of normal saline subcutaneously, other groups injected with 150mg/kg of D-galactose subcutaneously; and (3) performing molding while performing intragastric intervention, performing intragastric administration on the NC group and the MC group with equal volume of distilled water, performing intragastric administration on the PC group with 100mg/kg/d and equal volume of Vc, and performing intragastric administration on the outer vesicle of the other mice of the group shown in the

embodiment

4 or 5 for 56 d. After the experiment is finished, all mice are fasted for 12h, the eyeballs are picked up to take blood, and the mice are killed quickly by a neck-breaking method. Centrifuging the obtained blood sample at 3500r/min for 15min, and separating serum sample; SOD is important antioxidant enzyme in vivo, and the improvement of activity can indirectly reflect the enhancement of the ability of organism to remove free radicals, weaken the damage to lipid peroxidation, and protect the structural and functional integrity of cell membrane. MDA is lipid peroxide formed by oxygen free radicals attacking unsaturated fatty acid in biological membrane, and the change of the concentration of MDA can indirectly reflect the change of the concentration of oxygen free radicals in tissues, and is a commonly used membrane lipid peroxidation index. Therefore, detecting the contents of SOD and MDA is an important index for detecting the activity of the outer vesicle. The SOD and MDA levels were determined according to the kit instructions. The results are shown in Table 4.

TABLE 4 Effect of the Components on SOD and MDA content

Group of	SOD（U/mL）	MDA（nmol/mL）
			NC	136.42±3.45	13.51±0.73
MC	122.42±5.32	16.87±0.82
			PC	136.04±2.31	15.14±0.58
WNP4	150.36±6.03	11.08±0.67
			WNP5	141.29±2.10	12.66±1.03

As shown in table 4, the levels of SOD in serum of mice in MC group were significantly lower than those in NC group (P <0.05) and the MDA concentration was significantly higher than that in NC group (P <0.05), so that it was found that the model of aging of mice by D-galactose was successfully prepared. The SOD activity of the mouse sera of the WNP4 group and the WNP5 group is higher than that of the mouse sera of the MC group, particularly the improvement effect of the WNP4 group is particularly and remarkably achieved 150.36 +/-6.03, the MDA activity of the mouse sera of the WNP4 group and the WNP5 group is particularly and remarkably achieved 11.08 +/-0.67, and the improvement effect of the mouse sera of the WNP4 group is particularly and remarkably achieved 11.08 +/-0.67, so that the outer vesicles obtained by treating the 3E4 monoclonal antibody have higher activity. The result also shows that the external vesicle can remove free radicals in vivo and relieve the oxidative stress level of organisms by improving the activity of the antioxidant enzyme of the mice.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims

1. A monoclonal antibody 3E4 specific for P53 activity, characterized by: the light chain variable region sequence of the monoclonal antibody is SEQ ID NO: 2, the heavy chain variable region sequence is SEQ ID NO: 3, respectively.

2. An application of an external vesicle in preparing an anti-aging medicine is characterized in that: the outer vesicle is prepared by the following method: culturing fibroblast HSF in DMEM containing 10% fetal calf serum and 1% double antibody mixture, placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein cells grow to more than 80%, washing for 2-3 times by PBS, adding 1mL0.25% trypsin, digesting at normal temperature for 30-60 s, tapping the bottle wall to make the cells fall off, adding 2 times of volume of culture medium containing fetal calf serum to stop digestion, blowing the mixture uniformly by a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and the 3E4 monoclonal antibody of claim 1 with the final concentration of 100 mu g/mL, and placing the DMEM culture medium in a standard incubator to continue culturing for 96 h; collecting supernatant, centrifuging at 300 × g for 10min to remove dead cells and large cell debris, centrifuging at 2000 × g for 15min to remove dead cells and cell debris, centrifuging at 10000 × g/min for 30min to remove vesicles with large cell debris, transferring supernatant into ultracentrifuge tube, and centrifuging at 10 × g/min⁶Centrifuging at x g for 70min, removing supernatant, and collecting precipitate to obtain outer vesicle.

3. External vesicle in preparation of anti-aging cosmeticThe application in the product is characterized in that: the outer vesicle is prepared by the following method: culturing fibroblast HSF in DMEM containing 10% fetal calf serum and 1% double antibody mixture, placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein cells grow to more than 80%, washing for 2-3 times by PBS, adding 1mL0.25% trypsin, digesting at normal temperature for 30-60 s, tapping the bottle wall to make the cells fall off, adding 2 times of volume of culture medium containing fetal calf serum to stop digestion, blowing the mixture uniformly by a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and the 3E4 monoclonal antibody of claim 1 with the final concentration of 100 mu g/mL, and placing the DMEM culture medium in a standard incubator to continue culturing for 96 h; collecting supernatant, centrifuging at 300 × g for 10min to remove dead cells and large cell debris, centrifuging at 2000 × g for 15min to remove dead cells and cell debris, centrifuging at 10000 × g/min for 30min to remove vesicles with large cell debris, transferring supernatant into ultracentrifuge tube, and centrifuging at 10 × g/min⁶Centrifuging at x g for 70min, removing supernatant, and collecting precipitate to obtain outer vesicle.