CN114736296A

CN114736296A - Medicine or cosmetic prepared from fibroblast outer vesicle

Info

Publication number: CN114736296A
Application number: CN202210235333.7A
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: 2022-07-12
Anticipated expiration: 2042-03-11
Also published as: CN114736296B

Abstract

The invention relates to a medicine or a cosmetic prepared from fibroblast outer vesicles. 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

Medicine or cosmetic prepared from fibroblast outer vesicle

Technical Field

The invention relates to the field of pharmacy, in particular to a medicine or cosmetic prepared from fibroblast outer vesicles.

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 are therefore actively investigating their role in diagnosis. The first evidence that demonstrates the interaction of extracellular vesicles with recipient cells is that prostasomes (prostasomes) are able to promote the motility 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.

The existing research shows that theoretically any kind of cells can secrete exosomes; however, exosomes currently under investigation in the field of skin care 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 aging mainly including photo-aging caused by 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 2F6 which specifically inhibits the activity of P53.

CDR name	CDR sequences
		LCDR1	PYKEVIHNYYMG
LCDR2	ALECQHE
		LCDR3	PKHIMLTVV
HCDR1	PDKTA
		HCDR2	NQGYMNLWVYAKFMLI
HCDR3	CRKQFKY

Light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCPYKEVIHNYYMGWYQQKSGISPKPWIYALECQHEGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPKHIMLTVVFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSPDKTAWIKQRPGQGLEWIGNQGYMNLWVYAKFMLIGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGCRKQFKYWGLGTTLAVSS。

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 contain 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 the entire murine variable region sequence to a human antibody constant region sequence. 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 mixed solution) culture solution, culturing in a 37 ℃ and 5% CO2 incubator, allowing the cells to grow to be more than 80%, washing with PBS for 2-3 times, adding 1 mL0.25% trypsin, digesting at normal temperature for 30-60 s, slightly beating the bottle wall to allow the cells to fall off, adding 2 times of fetal calf serum-containing culture medium to stop digestion, uniformly beating with a lance pipe, centrifuging at 1000r/min for 8min, removing supernatant, adding sediment into high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double antibody mixed solution and 2F6 monoclonal antibody with final concentration of 100 mu g/mL, and continuously culturing for 96h in a standard incubator.

Collecting supernatant, centrifuging at 300 Xg for 10min to remove dead cells and large cell fragments, centrifuging at 2000 Xg for 15min to remove dead cells and cell fragments, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell fragments, transferring the supernatant to an ultracentrifuge tube, centrifuging at 106 Xg for 70min to remove supernatant, and collecting precipitate to obtain outer vesicles, wherein the above operations are performed at 4 ℃ under aseptic conditions.

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 diagram 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 carried out according to 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) measuring the serum antibody titer by ELISA (enzyme-linked immuno sorbent assay) of tail vein blood collection 7-10 d after the 3 rd immunization. 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.

Taking the splenocytes of a mouse with enhanced immunity BALB/c aseptically, taking 50% polyethylene glycol as a fusion agent, fusing Sp2/0 myeloma cells and the splenocytes of the mouse according to a ratio of 1: 7, adding HAT selective culture solution, inoculating a 96-well plate, culturing in a 37 ℃ and 5% CO2 incubator, absorbing culture supernatant in growth clone wells after 8 days, respectively using recombinant protein as a coating antigen, performing primary screening by ELISA, screening out positive hybridoma cells only reacting with the recombinant protein, performing 3-4 rounds of subcloning by a limiting dilution method until all the single clones are positive, and performing expanded culture and strain establishment. The stable monoclonal antibody hybridoma cells 3E4 and 2F6 are obtained.

Injecting 3E4 and 2F6 hybridoma cells into the abdominal cavity of a BALB/c mouse for about 2 multiplied by 106 per mouse, sucking ascites after the abdomen of the mouse is obviously expanded after about 8 days, centrifuging for 20min at 2000r/min, and taking supernatant. 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 22F 6 monoclonal antibody affinity, subtype identification, sequence analysis, and specificity identification

Using AMC sensors, purified 2F6 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 1.69GLY solution and buffer 3, and data output are shown in Table 1. (KD represents the equilibrium dissociation constant, i.e.affinity)

TABLE 12F 6 monoclonal antibody affinity results

Name of antibody	KD(M)
		2F6 monoclonal antibody	9.86E-09

As can be seen from the results in Table 1, the 2F6 monoclonal antibody has strong affinity, which can reach 9.86E-09M. Meanwhile, the purified 2F6 monoclonal antibody which aims at the P53 protein is verified by an antibody subtype identification kit, the antibody subtype is analyzed, and the result shows that the antibody subtype is IgG2a subtype and Kappa chain.

RNA was extracted from 2F6 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 22F 6 monoclonal antibody CDR-philic sequences

Light chain variable region (SEQ ID NO: 2)

Heavy chain variable region (SEQ ID NO: 3)

The specificity of the 2F6 mAb is identified by a Westernblot method, the p53 recombinant protein, fibrin and BSA are subjected to SDS-PAGE electrophoresis, after membrane transfer, the 2F6 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 mixed solution), culturing in a 37 ℃ and 5% CO2 incubator, allowing the cells to grow to be more than 80%, washing with PBS for 2-3 times, adding 1 mL0.25% trypsin, digesting at normal temperature for 30-60 s, slightly beating the bottle wall to allow the cells to fall off, adding 2 times of fetal calf serum-containing culture medium to stop digestion, uniformly beating with a blowgun tube, centrifuging at 1000r/min for 8min, removing supernatant, precipitating, adding an appropriate amount of complete culture medium to prepare single-cell suspension, adjusting concentration, and using the HSF in log phase with good condition for experiment. HSF in logarithmic phase is inoculated into 96-well culture plates at the concentration of 1 × 105 cells/mL for culture, each well is inoculated with 90 μ L, the plates are placed into a cell culture box to be cultured at 37 ℃ and 5% CO2, and after 24 hours, the plates are taken out to establish an experimental group with 2F6 monoclonal antibody at different concentrations, a negative control group (containing equal cell resuspension) and a blank control group (containing equal DMEM culture solution). The experimental group was added with 10. mu.L/well of 2F6 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 compound wells are arranged for each concentration, after uniform oscillation, the mixture is placed in a standard incubator for continuous culture, after administration for 96 hours, the mixture is taken out and added with MTT20 mu L/well, after continuous culture for 4 hours, DMSO150 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 2F6 monoclonal antibody is administered for 96 hours, wherein OD after 100 mug/mL experimental group is 4.1, 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

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 fibroblast extracellular vesicles in 2F 6-containing antibody Medium

Culturing fibroblast HSF in a DMEM (containing 10% fetal calf serum and 1% double-antibody mixed solution) culture solution, placing in a 37 ℃ and 5% CO2 incubator for culture, growing the cells to be more than 80%, washing with PBS for 2-3 times, adding 1 mL0.25% trypsin for normal-temperature digestion for 30-60 s, slightly beating the bottle wall to make the cells fall off, adding 2 times of fetal calf serum-containing culture medium for stopping digestion, uniformly beating with a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding a high-sugar DMEM culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and a 2F6 monoclonal antibody with the final concentration of 100 mu g/mL, and placing in a standard incubator for continuous culture for 96 h.

The culture steps are operated in a large scale, 50mL of supernatant is collected, centrifugation is carried out for 10min at 300 Xg, dead cells and large cell fragments are removed, centrifugation is carried out for 15min at 2000 Xg, dead cells and cell fragments are removed, centrifugation is carried out for 30min at 10000 Xg/min, vesicles with large cell fragments are removed, the supernatant is transferred to an ultracentrifuge tube, centrifugation is carried out for 70min at 106 Xg, the supernatant is removed, 1.20g of precipitates in total are collected, the precipitates are dissolved and precipitated by PBS, and outer vesicles are obtained, and the operations are carried out at 4 ℃ under aseptic conditions.

Example 5 preparation of control fibroblast extracellular vesicles

Culturing fibroblast HSF in DMEM (containing 10% fetal calf serum and 1% double-antibody mixed liquid), culturing in an incubator at 37 ℃ and 5% CO2, allowing the cells to grow to be more than 80%, washing with PBS for 2-3 times, adding 1 mL0.25% trypsin, digesting at normal temperature for 30-60 s, slightly beating the bottle wall to allow the cells to fall off, adding 2 times of fetal calf serum-containing culture medium to stop digestion, uniformly beating with a lance pipe, centrifuging at 1000r/min for 8min, removing supernatant, precipitating, adding high-sugar DMEM culture medium containing 10% fetal calf serum and 1% double-antibody mixed liquid, and culturing in a standard incubator for 96 h.

The culture steps are operated in a large scale, 50mL of supernatant is collected, centrifugation is carried out for 10min at 300 Xg, dead cells and large cell fragments are removed, centrifugation is carried out for 15min at 2000 Xg, dead cells and cell fragments are removed, centrifugation is carried out for 30min at 10000 Xg/min, vesicles with large cell fragments are removed, the supernatant is transferred to an ultracentrifuge tube, centrifugation is carried out for 70min at 106 Xg, the supernatant is removed, the total amount of precipitate is 0.95g, the precipitate is dissolved and precipitated by PBS, and outer vesicles are obtained, and the operations are carried out at 4 ℃ under aseptic conditions.

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 (WNP4), 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

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 reached 149.47 +/-3.22, the MDA activity of the mouse sera of the WNP4 group and the WNP5 group is particularly and remarkably reached 11.24 +/-0.46, and the improvement effect of the mouse sera of the WNP4 group is particularly and remarkably reached, which fully indicates that the outer vesicles obtained by treating the 2F6 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 these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

<110> Nosai Union (Beijing) biomedical science and technology Co., Ltd

<120> drugs or cosmetics prepared from fibroblast extracellular vesicles

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 393

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln

1 5 10 15

Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu

20 25 30

Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp

35 40 45

Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro

50 55 60

Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro

65 70 75 80

Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser

85 90 95

Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly

100 105 110

Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro

115 120 125

Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln

130 135 140

Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met

145 150 155 160

Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys

165 170 175

Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln

180 185 190

His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp

195 200 205

Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu

210 215 220

Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser

225 230 235 240

Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr

245 250 255

Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val

260 265 270

Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn

275 280 285

Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr

290 295 300

Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys

305 310 315 320

Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu

325 330 335

Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp

340 345 350

Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His

355 360 365

Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met

370 375 380

Phe Lys Thr Glu Gly Pro Asp Ser Asp

385 390

<210> 2

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Val Ile Thr Gln Arg Pro Ala Leu Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Pro Tyr Lys Glu Val Ile His Asn Tyr

20 25 30

Tyr Met Gly Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp

35 40 45

Ile Tyr Ala Leu Glu Cys Gln His Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Thr Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Pro Lys His Ile Met Leu Thr

85 90 95

Val Val Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 3

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Glu Glu Ser Ala Thr Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Pro Asp

20 25 30

Lys Thr Ala Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Gln Gly Tyr Met Asn Leu Trp Val Tyr Ala Lys Phe Met Leu

50 55 60

Ile Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Cys Arg Lys Gln Phe Lys Tyr Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

Claims

1. A monoclonal antibody 2F6 that specifically binds P53 activity, characterized in that 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. Monoclonal antibody 2F6 according to claim 1, characterized in that the CDR sequences are shown below

。

3. 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 1 mL0.25% 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 2F6 monoclonal antibody of claim 1 with the final concentration of 100 mu g/mL, and placing the DMEM culture medium in a standard incubator for continuous culture for 96 h; collecting supernatant, centrifuging at 300 Xg for 10min, and removing dead cellsAnd 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 ×⁶Centrifuging for 70min at x g, removing supernatant, and collecting precipitate to obtain outer vesicle.

4. The application of the outer vesicle in preparing the anti-aging cosmetic is characterized in that: the outer vesicle is prepared by the following method: culturing fibroblast HSF in DMEM culture solution containing 10% fetal calf serum and 1% double-antibody mixed solution, culturing in a culture box containing 37 ℃ and 5% CO2, growing the cells to be more than 80%, washing for 2-3 times by PBS, adding 1 mL0.25% trypsin, digesting for 30-60 s at normal temperature, slightly beating the bottle wall to make the cells fall off, adding 2 times of fetal calf serum-containing culture medium to stop digestion, uniformly blowing by using a gun tube, centrifuging for 8min at 1000r/min, removing supernatant, precipitating, adding high-sugar DMEM (DMEM) culture medium containing 10% fetal calf serum, 1% double-antibody mixed solution and the 2F6 monoclonal antibody of claim 1 with the final concentration of 100 mu g/mL, and continuously culturing for 96h in a standard culture box; 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 for 70min at x g, removing supernatant, and collecting precipitate to obtain outer vesicle.