CN114099412A

CN114099412A - Application of caspase inhibitor in preparation of stem cell freeze-dried powder and anti-aging anti-inflammatory drug

Info

Publication number: CN114099412A
Application number: CN202111430933.0A
Authority: CN
Inventors: 王京; 陈帅
Original assignee: Beijing Daiyu Biotechnology Co ltd
Current assignee: Beijing Daiyu Biotechnology Co ltd
Priority date: 2021-11-29
Filing date: 2021-11-29
Publication date: 2022-03-01

Abstract

The invention relates to application of a caspase inhibitor in preparation of stem cell freeze-dried powder and anti-aging anti-inflammatory drugs. The invention provides a specific adipose-derived mesenchymal stem cell freeze-dried powder, which is prepared by freeze-drying a freeze-drying protective agent containing a caspase-7 monoclonal antibody, has the technical effects of promoting fibroblast migration and inhibiting inflammation, and can be used in the fields of beauty treatment or medicines.

Description

Application of caspase inhibitor in preparation of stem cell freeze-dried powder and anti-aging anti-inflammatory drug

Technical Field

The invention relates to the field of cosmetics or medicines, and particularly relates to application of a caspase inhibitor in preparation of stem cell freeze-dried powder and anti-aging anti-inflammatory medicines.

Background

Stem Cells (SC) are the cells of origin of the body, and are the progenitor cells that form the tissues and organs of the human body, and they can differentiate all types of cells in the body, for example: cells such as tissues, organs, bones, cartilages, tendons, ligaments and the like, and pluripotent cells with self-replicating capacity, under a certain condition, the cells can be differentiated into various functional cells, which are called universal cells in the academic world; stem cell therapy, typically involving autologous adipose stem cells or stem cell culture media, is used for facial anti-aging, achieving a new rejuvenation of the skin.

The use of stem cells has become increasingly popular in the cosmetic industry. However, preservation of stem cells has been a technical problem. At present, most of MSCs clinically used are cultured in vitro and stored at low temperature. However, studies have shown that the treatment effect of MSCs is better in the early passages than in the late stages, which may be related to the fact that the differentiation capacity of MSCs decreases with passage of cells. Therefore, early cryopreservation of MSCs is a necessary condition to maintain their cellular status and effectiveness at the time of transplantation.

The purpose of cryopreservation is to slow down the metabolic activity of cells and sustain life in the low temperature environment of liquid nitrogen during the freezing process of cell suspension, ice crystal formation is one of the major causes of cell death. The cooling rate is kept slow to avoid ice crystal formation so that the cryoprotectant gradually enters the interior of the cell to form a concentration gradient. When the cooling is continuously carried out, the water in the cells gradually flows out of the cells under the osmotic pressure of the freezing agent, and the cells are completely dehydrated and shrunk to reach the optimal freezing state when the concentration of the freezing agent in the cells reaches the maximum. If the freezing rate is too fast, the water in the cells cannot be fully discharged, so that ice crystals in the cells are formed to damage the cells. However, there is a certain toxic effect on cells by the frozen stock solution, and the cell toxicity is caused by the exposure of the cells in the frozen stock solution for a long time due to the low freezing rate.

In addition to freezing rate, cryoprotectants are also one of the important factors to consider for reducing freezing damage. Cryoprotectants are classified as osmotic protectants and non-osmotic protectants based on their ability to cross cell membranes. The osmotic cryoprotectant is dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, methanol, ethanol, propanol, formamide, etc., and has the main functions of preventing the formation of ice crystals in cells, avoiding the toxicity of high-concentration cryopreservation agent and dehydrating cells within tolerance. The protective capacity of osmotic protectants is a highly soluble water-soluble complex composed of many small molecules that can depress the freezing point of water at high concentrations, thereby reducing the formation of large ice crystals during freezing. Non-osmotic cryoprotectants such as trehalose, sucrose, lactose, glucose, mannitol, sorbitol, polymeric hydroxyethyl starch and polyvinylpyrrolidone, which protect cells at lower concentrations but require higher freezing rates, for example, polymeric hydroxyethyl starch cannot penetrate to increase extracellular concentrations and form reverse osmosis at low temperatures to protect dehydration. In other words, the concentration of the non-penetrating cryoprotectant outside the cells results in dehydration of the cells primarily during the initial stages of freezing. The protective effect of trehalose is the interaction with lipid membranes, which maintains the stability of proteins during cryopreservation and recovery, and which is capable of forming a vitrified matrix to inhibit intracellular ice crystal production. Even non-permeable protective agents can cause cell damage, so the development of the most effective cryopreservation agent is one of the challenges of cell cryopreservation at present. There have been studies that have begun to focus on the mechanism of toxicity of the impermeable protectant to cells in an attempt to minimize its toxicity.

Therefore, it has been reported that the influence of trehalose capable of stabilizing cell membranes on cells is investigated by the survival rate of cells, inhibitors of catalase (antioxidant enzyme) and caspase. Studies showed that freezing amniotic fluid MSCs for 3 weeks in liquid nitrogen using serum-free frozen stock solutions containing 2.5%, 5% and 10% DMSO, 60mmol/L trehalose, 100ug/mL catalase and 30ug/mL zVAD-fmk (caspase inhibitor) showed similar performance in population doubling, cell surface antigen expression and myoblast differentiation capacity using frozen stock solutions of 2.5% DMSO and 3 additives (trehalose, catalase and caspase inhibitor) and frozen stock solutions containing 10% DMSO and 30% FBS, demonstrating the feasibility of reducing DMSO and serum-free freezing to preserve MSCs survival and function.

Caspases are generally present in most metazoan cells as inactive zymogens, and family members are similar in amino acid sequence, structure and enzymatic properties. The zymogen is a single polypeptide with the molecular weight of 30-50ku, and consists of three structural domains, namely an N-terminal front domain, a large subunit of 20ku and a small subunit of 10 ku. Upon activation of the zymogen molecule, the size subunits dissociate and reassemble into an active complex in the form of a tetramer, with two small subunit in the middle and two large subunit in the periphery, each heterodimer containing one catalytic site. The most important of them is the executive type Caspase (executionercasepase), including Caspase-3, Caspase-6, Caspase-7, etc., which acts to specifically crack the substrate to make the cell undergo biochemical and morphological change, finally leading to the cell apoptosis. However, currently, there are few types of caspase inhibitors and there are not enough choices provided in the art, so there is an urgent need in the art to develop new caspase inhibitors for use in the preparation of lyophilized stem cell powder.

Disclosure of Invention

In one aspect of the invention, a monoclonal antibody specific for Caspase-7 is provided. The light and heavy chain variable region sequences of the antibody are respectively shown as

SEQ ID NO

2 and 3.

In another aspect, the invention provides adipose-derived mesenchymal stem cell lyophilized powder which is obtained by adopting a specific lyophilized agent to protect adipose-derived mesenchymal stem cells.

Correspondingly, the invention provides a cryoprotectant, which contains a Caspase-7 monoclonal antibody.

In another aspect, the cryopreservation protective agent of the invention is prepared by dissolving 2% of monoclonal antibody, 20% of polyvinylpyrrolidone, 25% of trehalose, 20% of glycerol, 5% of sucrose, 10% of mannitol and 0.5% of polyarginine in DMEM/F12 culture solution and mixing uniformly.

In another aspect, the present invention provides a method of lyophilizing adipose mesenchymal stem cells, the method comprising: collecting adipose-derived mesenchymal stem cells with 85% fusion degree in the 4 th generation, digesting the cells with trypsin/EDTA digestive juice, stopping digestion when the cells become round and just separate from the surface of the culture plate, washing for 2 times with PBS (PBS), 1000r/min, and centrifuging for 5 min. After centrifugation, taking adipose mesenchymal stem cells and a protective agent according to the volume ratio of 2: 1 and mixing uniformly. Firstly, the mixture is balanced for 15min at 4 ℃, and then the mixture is quickly placed into a low-temperature refrigerator of minus 80 ℃ for freezing for 2 h. Then transferred to the freeze dryer shelf pre-cooled to-60 ℃ for 2 h. After the freezing process is finished, a cold trap refrigerating valve is opened to reduce the temperature of the cold trap to be below minus 60 ℃, a vacuum pump is opened to enable the vacuum degree of a freeze-drying chamber to reach below 10Pa, a shelf refrigerating valve is closed to enable the temperature of a shelf to be raised to be below minus 35 ℃, and primary drying is maintained for 15 hours. Thereafter, the shelf temperature was raised to 20 ℃ at a heating rate of 0.2 ℃/min and maintained at this temperature until the end of the secondary drying process, which lasted for about 12 hours. After freeze-drying, the sample is in a dry cake shape without collapse, and is strictly sealed and stored in a refrigerator at 4 ℃.

Further, the invention provides an application of the freeze-dried adipose tissue-derived mesenchymal stem cells prepared by the method in preparing a beauty product. It is achieved by promoting the secretion of collagen and the like of fibroblasts and inhibiting apoptosis.

In another aspect, an antioxidant is added to the cosmetic product of the present invention.

The antioxidant of the invention comprises: aspergillic acid and its salts and derivatives, such as: aspartame; asicolar ether; aspartame; aspergetin tryptophan and its salts; caffeic acid and its salts. These antioxidants may be used in an amount of 0.0001 to 3% by weight of the total weight of the article of manufacture.

In the present invention, the cosmetic product also contains a solvent.

Alcohols suitable for cosmetics such as ethanol and isopropyl alcohol can be used as volatile solvents. And, the content of the composition of the present invention to the volatile solvent to the total weight of the composition is in an amount of 1 to 10% by weight.

The cosmetic ingredient of the present invention includes, in addition to the active ingredient, ingredients generally used in cosmetic ingredients, such as stabilizers, emulsifiers, solubilizers, pigments, and perfumes, which are generally used as auxiliaries. The cosmetic composition of the present invention may be prepared in any conventionally manufactured form such as, but not limited to, a solution, a suspension, an emulsion, a face gel, an emulsion, etc. More particularly, the cream can be a skin-moistening emulsion essence cream eye cream mask or a spray type.

In another aspect, the invention provides an application of the freeze-dried adipose-derived mesenchymal stem cells prepared by the method in preparing anti-inflammatory drugs. Specifically, the anti-inflammation is realized by reducing the expression of an inflammation gene TNF-alpha.

The pharmaceutical composition of the present invention may be formulated into dosage forms in the form of tablets, reducing agents, powders, granules, capsules, suspensions, emulsions, syrups, aerosols, sterilizing agents, injections, etc. according to the usual methods for preventing and treating skin inflammation.

Solid preparations for oral administration include a purified epoxy preparation, a granular preparation, a capsule preparation and the like, and these solid preparations may be mixed to formulate at least one excipient such as a starch calcium carbonate hydrogel. Besides pure excipient, the magnesium alloy hard fat powder can also be used as lubricant such as magnesium alloy hard fat powder. The liquid preparation for oral administration includes suspension, liquid, emulsion syrup, etc., and can be used in various excipients such as wetting agent, sweetener, aromatic, preservative, etc. besides conventional pure diluent- -water cubic paraffin.

The preparation for non-oral administration can comprise sterile aqueous solution non-chiral solvent suspending agent latex freeze-dried preparation adjuvant and the like. Achiral and suspending solvents are useful for injectable esters such as propylene glycol polyethylene glycol olive oil and the like.

In addition, the pharmaceutical composition of the present invention may further comprise a carrier excipient or diluent. The carrier excipient or diluent is lactose, methylcellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose polyvinyl alcohol pyrrolidone; hydromethylhydroxybenzoylimine; a phthalimide; a phthalimide; magnesium removal; a stearylimine; and mineral oil such as silica.

The specific dosage of the pharmaceutical composition is determined according to the state of a patient in a preparation method and the degree of sex-age diseases of a patient; the route and period of administration of the drug form, the sensitivity of excretion rate response, and the like. The choice of the amount and number of bets may be varied by the practitioner and do not limit the scope of the invention in any way.

Advantageous effects

The invention provides a specific adipose-derived mesenchymal stem cell freeze-dried powder, which is prepared by freeze-drying a freeze-drying protective agent containing a caspase-7 monoclonal antibody, has the technical effects of promoting fibroblast migration and inhibiting inflammation, and can be used in the fields of beauty treatment or medicines.

Drawings

FIG. 1 graph showing the results of the number of migrating cells

FIG. 2 is a graph showing the results of expression levels of individual genes in fibroblasts

FIG. 3 is a graph showing the results of the expression level of TNF-. alpha.as a cytokine

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of Caspase-7 monoclonal antibody

According to the amino acid sequence of Caspase-7, through analyzing the situation of corresponding main activity binding epitope, the high-activity antigen epitope is obtained by screening and is shown as SEQ ID NO: 1, as an immunogen for the preparation of corresponding monoclonal antibodies.

rgtelddgiqadsgpindtdanprykipveadflfaystvpgyyswrspgrgsw(SEQ ID NO：1)。

The coding sequence corresponding to the amino acid sequence shown in the sequence 1 is used for expressing a fusion protein GST-Caspase-7 with glutathione-S-transferase (GST) in escherichia coli BL21 through a prokaryotic expression plasmid PGEX-4T-1. The fusion protein is purified by glutathione agarose gel affinity chromatography, then is subjected to enzyme digestion by thrombin, and Caspase-7 truncated peptide is collected. SDS-PAGE electrophoresis detection and Western blot identification confirm that the target protein Caspase-7 truncated peptide is correctly expressed, and the protein concentration is adjusted to 1mg/mL for later use.

The 1 st immunization was performed by injecting the immunogen into Balb/c mice at a dose of 50. mu.g recombinant protein per mouse. After 15d, 2 nd immunization was performed at a dose of 25. mu.g recombinant protein/mouse. Day 25 later, the 3 rd immunization (same as the 2 nd immunization) was performed. After 35 days, the fourth immunization (the same as the 3 rd immunization) is carried out, 7d after the immunization is finished, the indirect ELISA method is used for detecting the serum antibody titer of the mice, and the result shows that the mice can generate the antibody against Caspase-7, and the antibody titer reaches 4 multiplied by 10⁶And compounding fusion requirements. The mice were boosted by direct mouse tail intravenous injection at a dose of 120. mu.g recombinant protein per mouse 3d prior to fusion, without adjuvant, in preparation for fusion.

Cell fusion experiments were performed using PEG-induced semi-solid medium culture methods commonly used in the art, and spleen cells with an immune titer meeting the fusion requirement were combined with SP2/0 cells at a ratio of 10: mixed cell culture was performed at a ratio of 1. And screening out cell strains positive to the recombinant protein by adopting an indirect ELISA method. And (3) carrying out single cell isolation culture on the screened positive hybridoma cells by a limiting dilution method, detecting by an indirect ELISA method, selecting the clones 2D5 and 4F3 which have the strongest positive result on the detection result of the recombinant protein, and carrying out continuous yaking until the cell positive rate is 100%, thus determining the strains. Healthy adult F1 mice were selected and injected intraperitoneally with 0.5 mL/mouse of liquid paraffin. One week later, the well-grown hybridoma cells were collected at a cell concentration of 0.5X 10⁶-1×10⁶Each mouse was injected intraperitoneally with 0.5 mL. Ascites from mice were collected for about 12 days. Ascites was purified by the n-octanoic acid-saturated ammonium sulfate method. Protein concentration after purification was determined by the Bradford method. Adjusting the ascites concentration to 1mg/ml, diluting the purified antibody with diluent 10 respectively³、10⁴、10⁵、10⁶And 10⁷The titer of ascites was determined as the value at which the OD value was greater than the negative control value by a dilution factor of 2.1 or more. The serum of the fusion mouse is diluted 1000 times to be used as a positive control, the serum of the non-immunized mouse is diluted 1000 times to be used as a negative control, and the diluent is used as a blank control. The results are shownIt is shown that the ascites titers of 2D5 and 4F3 are both at 10⁶The concentrations of both purified proteins were 6.52mg/mL and 5.14mg/mL, respectively.

EXAMPLE 22D 5 monoclonal antibody identification

Identification of monoclonal antibody subclasses: the unlabeled SEQ ID NO: the recombinant protein of 1 is diluted to 0.25ug/mL and added into a 96-well enzyme label plate for coating antigen. The monoclonal antibody subclasses were determined using an ELISA kit according to the protocol. The results show that 2D5 is of the IgG2b type.

The affinity constant was determined using a non-competitive ELISA. The unlabeled SEQ ID NO: 1 to 1ug/mL, diluting in a multiple ratio, diluting in a serial way by 11 gradients, reading OD450 values by an enzyme-labeling instrument, selecting 4 most appropriate coating concentrations, coating each concentration by 2 inches, repeating the experiment, and showing that the affinity constant of 2D5 is 5.21 multiplied by 10¹¹And has better combination effect.

And (3) specific identification: caspase-3, Caspase-7, GST and BSA are used as specific antigens, and western blot detection shows that the 2D5 monoclonal antibody only forms specific bands with Caspase-7 and no bands are generated with other monoclonal antibodies, which shows that the 2D5 has better specificity.

The light chain variable region sequence of the antibody is identified and obtained by adopting the conventional monoclonal antibody light chain variable region sequence identification method in the field and is shown as SEQ ID NO 2, and the heavy chain variable region sequence is shown as SEQ ID NO 3.

Light chain variable region (SEQ ID NO:2)

DIVITQRPALMAASPGEKVTITCFRYMWWDWIYVDWYQQKSGISPKPWIYILMSNTVGVPARFSGSGSGTSYSLTITSMEAEDAATYYCVPASDDYESFGAGTKLELK

Heavy chain variable region (SEQ ID NO:3)

EVQLEESATELARPGASVKLSCKASGYIFSKCNCVWIKQRPGQGLEWIGLWMILHLEHVKTYWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGIHRPRKWWGLGTTLAVSS

Example 3 isolation and preparation of adipose-derived mesenchymal Stem cells

15ml of fat particles are centrifuged for 5min at 1000 r/min. Taking the upper adipose tissue and the lower cells. Trypsin and collagenase were added to the adipose tissue at a final concentration of 0.1%. Digesting in a water bath shaker at 37 deg.C for 30 min. Centrifuging at 700g/min for 5 min. And (4) reserving the lower layer cells, mixing the two cells uniformly, adding a PBS buffer solution to resuspend the cells, and centrifuging again for 700g/min and 5 min. The primary culture solution is inoculated to a culture flask after the cells are resuspended, and cultured in a 5% carbon dioxide incubator at 37 ℃. Is marked as P0 generation. The growth of the cells was observed. Half of the liquid is changed on the first 3 days, and then every 3 days, the cells are subcultured when the cells reach 90% fusion, digested by 0.05% trypsin and subcultured according to the proportion of 1: 4. Passage 3 for immunophenotypic identification, expression of surface markers CD13, CD29, CD31, CD34, CD45, CD44, CD73, CD90, CD105 and HLA-DR was determined by flow cytometry. The result shows that the expressions of CD90 and CD105 of the adipose-derived mesenchymal stem cells are positive, and the positive rate is more than 96%; CD34, CD45 and HLA-DR expression are negative, and the positive rate is less than 2 percent.

Example 4 freezing experiment of adipose mesenchymal stem cells

Freezing and storing protective agent: dissolving 2% monoclonal antibody, 20% polyvinylpyrrolidone, 25% trehalose, 20% glycerol, 5% sucrose, 10% mannitol and 0.5% polyarginine in DMEM/F12 culture solution, and mixing.

Control cryopreservation protectant: 20% of polyvinylpyrrolidone, 25% of trehalose, 20% of glycerol, 5% of sucrose, 10% of mannitol and 0.5% of polyarginine, and dissolving and uniformly mixing in a DMEM/F12 culture solution.

Collecting adipose-derived mesenchymal stem cells with 85% fusion degree in the 4 th generation, digesting the cells with trypsin/EDTA digestive juice, stopping digestion when the cells become round and just separate from the surface of the culture plate, washing for 2 times with PBS (PBS), 1000r/min, and centrifuging for 5 min. After centrifugation, taking adipose mesenchymal stem cells and a protective agent according to the volume ratio of 2: 1 and mixing uniformly. Firstly, the mixture is balanced for 15min at 4 ℃, and then the mixture is quickly placed into a low-temperature refrigerator of minus 80 ℃ for freezing for 2 h. Then transferred to the freeze dryer shelf pre-cooled to-60 ℃ for 2 h. After the freezing process is finished, a cold trap refrigerating valve is opened to reduce the temperature of the cold trap to be below minus 60 ℃, a vacuum pump is opened to enable the vacuum degree of a freeze-drying chamber to reach below 10Pa, a shelf refrigerating valve is closed to enable the temperature of a shelf to be raised to be below minus 35 ℃, and primary drying is maintained for 15 hours. Thereafter, the shelf temperature was raised to 20 ℃ at a heating rate of 0.2 ℃/min and maintained at this temperature until the end of the secondary drying process, which lasted for about 12 hours. After freeze-drying, the sample is in a dry cake shape without collapse, and is strictly sealed and stored in a refrigerator at 4 ℃.

Rehydration and revival of freeze-dried cells: the lyophilized stem cells were reconstituted by rehydration in a rehydration solution (DMEM/F12 medium containing 20% fetal bovine serum at 37 ℃). The proportion of viable cells was calculated by trypan blue exclusion (ScienCeH, USA), and the cell recovery rate R was calculated as follows: r ═ N1/N2XE1 × 100%, where N1 is the number of cells after lyophilization and rehydration, N2 is the number of cells before lyophilization, and E1 is the trypan blue exclusion of the cells after lyophilization and rehydration. The results of 12h incubation were defined as the resuscitation rate. The results are shown in Table 1.

TABLE 1 cell recovery

Group of	Cell recovery (%)
		Control cryopreservative protectant lyophilized stem cells	59.3±5.2
Freeze-dried stem cells of protective agent	92.4±8.6*

The stem cells frozen after preservation are revived for 12 hours, the average reviving rate of the stem cells frozen by the freeze-drying protective agent of the contrast is (59.3 +/-5.2)%, while the reviving rate of the stem cells frozen by the freeze-drying protective agent of the invention reaches (92.4 +/-8.6)%, which has significant improving effect, thus fully showing that the Caspase monoclonal antibody of the invention can effectively inhibit the apoptosis stimulation of the cells in the freeze-drying process caused by environmental change, realizing the effect of improving the cell survival rate, and the spindle-shaped form of the cells observed under an optical microscope is better maintained, indicating that P is less than 0.05, and the difference is significant.

Example 5 Effect of adipose-derived Stem cells on fibroblast migration experiment before and after lyophilization

Fibroblast migration experiment Radius was first applied before the cells were added^TMTreating the migration plate with pre-prepared glue solution for 20min, adding washing solution to wash, and washing with HFF-1 at 2 × 10⁵And/ml, adding 500 mu l of the mixture into each well, culturing for 24h, removing gel after the cells grow full, sucking the culture medium, adding the complete culture medium into a control group, adding DMEM/F12 culture medium for recovering 20% of lyophilized dry cell powder rehydration solution of the frozen protective agent in the example 4 into the experimental group 1, adding DMEM/F12 culture medium for recovering 20% of lyophilized dry cell powder rehydration solution of the frozen protective agent in the example 4 into the experimental group 2, placing the mixture into a living cell workstation for culturing for 24h, observing the cell migration condition and counting the cell migration area. Cell migration experiments are detailed in Radius^TM24-WellCellMigrationAssay kit instructions. Cell migration was observed by Transwell: the 24-well plate was filled with 600. mu.l (lower chamber) of DMEM/F12 medium containing 20% of the rehydrated lyophilized powder of example 4, and HFF-1 cells were cultured at 3X 10⁴200 μ l serum-free DMEM medium was added to the upper chamber of the transwell and placed at 37 ℃ in 5% CO₂After 24 hours of incubation in an incubator, the transwell chamber was removed, the non-migrated cells on the inner membrane of the chamber were wiped off with a cotton swab, the membrane on the lower side was inverted, placed in a new 24-well plate, fixed for 20min with 600. mu.l of 4% paraformaldehyde, and stained with 0.1% crystal violet staining solution for 3 min. PBS rinse 3 times, remove unbound crystal violet, cotton swab gently wipe off cell side wall crystal violet dye, appropriate air drying. Each chamber was placed in a new 24-well plate under an inverted microscope (10 ×), 6 fields were randomly photographed, and the number of migrated cells was statistically analyzed. The results are shown in FIG. 1.

As can be seen from the results in FIG. 1, after HFF-1 overgrows, the migration capacity of the fibroblasts was found to be higher than that of the control group in all of the 2 experimental compositions, and the number of dead cells was small, and most of the scratches were repaired. Particularly, the stem cells freeze-dried by the cryopreservation protective agent disclosed by the invention in the experimental group 1 have the best effect of promoting the migration of the fibroblasts, the migration cells of the stem cells achieve the effect of (235 +/-7), and compared with the PP (propene Polymer) of a control group, the experimental group 1 and the experimental group 2 have obvious difference and have statistical significance.

Example 6 Effect of different lyophilizates treatment on fibroblast Gene expression

And detecting the mRNA expression condition of the cell gene by adopting qRT-PCR. Fibroblast cells were arranged at 5X 10⁴Per cm²Six well plates were inoculated and 2ml of complete medium was added to each well of the blank control. Adding 2ml complete culture medium containing 15% lyophilized adipose-derived mesenchymal stem cell lyophilized powder into each well of experimental group, setting 3 groups in parallel, placing at 37 deg.C and 5% CO₂Culturing in an incubator for 48 h. Extracting total RNA from cells, reverse transcribing to cDNA, and storing at-20 deg.C. qRT-PCR measures mRNA expression of extracellular matrix-associated protein type I collagen (collagen I), MMP-1, elastin (elastin), fibronectin (fibronectin), Caspase-7. PCR amplification conditions: keeping at 50 deg.C for 2min, pre-denaturing at 95 deg.C for 10min, denaturing at 95 deg.C for 15s, annealing at 60 deg.C for 1min, and circulating 40 times.

Specific primer sequences are as follows (5 '-3'):

beta-actin internal reference:

upstream Forward AGAAAATCTGGCACCACACC

Downstream Reverse AGAGAGGGTACAGGGATAGCA

Collagen I：

Upstream Forward CACACAGAGGTTTCAGTGGTTTGG

Downstream Reverse GCACCAGTAGCACCATCATCTTC

Elastin：

Upstream Forward GGGTTGTGTCACCAGAAGCA

Downstream Reverse CAACCCCCGTAAGGAGAATGC

Fibronectin：

Upstream Forward AAGATTGGAGAGAAGTGGGACC

Downstream Reverse GAGCAAATGGCACCGAGATA

MMP-1：

Upstream Forward TTGAGAAAGCCTTCCAACTCTG

Downstream Reverse CCGCAACACGATGTTAAGTTGTA

Caspase-7：

Upstream Forward AGTGACAGGTATGGGCGTTCG

Downstream Reverse GCATCTATATCCCCCCCTAAAGTGG

The results are shown in FIG. 2.

As can be seen from the results in FIG. 2, the fibronectin, elastin and type I collagen of the fibroblasts treated by the freeze-dried cell conditioned medium are obviously increased, and the mRNA level expression of MMP-1 and caspase-7 is obviously reduced. Particularly, the adipose-derived mesenchymal stem cells treated by the caspase monoclonal antibody freeze-drying agent have higher effect of promoting the activity of fibroblasts. Wherein the type I collagen is relatively improved by 3.4 times, and the difference is obvious and has statistical significance.

Example 7 verification of anti-inflammatory effect of adipose mesenchymal stem cell lyophilized powder

Evaluation of anti-inflammatory efficacy based on LPS-stimulated macrophages. Raw264.7 macrophages of a mouse are used as a model, and are induced to generate inflammatory reaction by LPS, so that the anti-inflammatory effect of the human adipose mesenchymal stem cell freeze-dried powder is researched. Raw264.7 macrophage inoculation amount is 1 multiplied by 10⁵One cell/well was inoculated in 12-well culture plates, 1ml of complete medium was added per well, divided into five groups: control, Lipopolysaccharide (LPS), LPS + Dex (dexamethasone positive control), LPS + 50% stem cells and LPS + 50% control stem cells. Adding LPS (100ng/ml), Dex (5 μ g/ml), 50% dry cells (lyophilized powder diluted with 1ml distilled water and mixed with complete culture medium 1: 1), standing at 37 deg.C and 5% CO₂Culturing for 24h in an incubator; collecting cell supernatant, detecting the level of cell inflammatory factor TNF-alpha in the cell culture supernatant by using a TNF-alpha ELISA kit, and detecting the absorbance value of 450nm by using an enzyme-labeling instrument. The results are shown in FIG. 3.

The results are shown in figure 3, the stem cell lyophilized powder has obvious anti-inflammatory effect, can obviously inhibit the secretion of TNF-alpha, the inhibition effect can reach the positive control dexamethasone level, and the difference has statistical significance. The adipose-derived stem cell lyophilized powder has good anti-inflammatory effect. Particularly, the adipose mesenchymal stem cells treated by the freeze-dried preservative of the monoclonal antibody have higher activity and only 5417pg/ml of TNF-alpha level, and the effect of inhibiting anti-inflammatory action is obviously improved compared with the effect of a control freeze-dried preservative without the monoclonal antibody.

Those skilled in the art to which the present invention pertains from the above description will understand that the present invention may be embodied in other specific forms without changing its technical idea or essential features. In this connection, the embodiments described above are prophetic in all respects and must be understood as non-limiting. The scope of the present invention should be construed that the meaning and scope of the claims to be described later and any modification or variation derived from the equivalent concept thereof are included in the scope of the present invention as compared with the above detailed description.

Sequence listing

<110> Beijing Biotechnology Ltd

Application of <120> caspase inhibitor in preparation of stem cell freeze-dried powder and anti-aging anti-inflammatory drug

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Arg Gly Thr Glu Leu Asp Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile

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Asn Asp Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp

20 25 30

Phe Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser

35 40 45

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50

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Tyr Val Asp Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp

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Ile Tyr Ile Leu Met Ser Asn Thr Val 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 Val Pro Ala Ser Asp Asp Tyr

85 90 95

Glu Ser Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 3

<211> 116

<212> PRT

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

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

20 25 30

Asn Cys Val Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Leu Trp Met Ile Leu His Leu Glu His Val Lys Thr Tyr Trp Gln

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Ala 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 Ile His Arg Pro Arg Lys Trp Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

Claims

1. The application of the adipose-derived mesenchymal stem cell freeze-dried powder in the preparation of cosmetics is provided, and the freeze-dried powder can promote the activity of fibroblasts and delay cell aging; the adipose-derived mesenchymal stem cell freeze-dried powder is prepared by mixing adipose-derived mesenchymal stem cells with a freeze-drying protective agent and then freeze-drying the mixture; the freeze-drying protective agent is prepared by dissolving and uniformly mixing 2% of monoclonal antibody, 20% of polyvinylpyrrolidone, 25% of trehalose, 20% of glycerol, 5% of sucrose, 10% of mannitol and 0.5% of polyarginine in a DMEM/F12 culture solution; the monoclonal antibody is Caspase-7 monoclonal antibody 2D5, and its light chain variable region sequence is shown in SEQ ID No. 2 and its heavy chain variable region sequence is shown in SEQ ID No. 3.

2. The use according to claim 1, wherein an antioxidant is further added to the cosmetic.

3. Use according to claim 2, characterized in that the cosmetic composition further comprises usual auxiliaries such as stabilizers, emulsifiers, solubilizers, pigments and fragrances.

4. The application of the adipose-derived mesenchymal stem cell lyophilized powder in the preparation of medicines is characterized in that the lyophilized powder can resist inflammation, and the anti-inflammation is realized by reducing the expression of an inflammation gene TNF-alpha; the adipose-derived mesenchymal stem cell freeze-dried powder is prepared by mixing adipose-derived mesenchymal stem cells with a freeze-drying protective agent and then freeze-drying the mixture; the freeze-drying protective agent is prepared by dissolving and uniformly mixing 2% of monoclonal antibody, 20% of polyvinylpyrrolidone, 25% of trehalose, 20% of glycerol, 5% of sucrose, 10% of mannitol and 0.5% of polyarginine in a DMEM/F12 culture solution; the monoclonal antibody is Caspase-7 monoclonal antibody 2D5, and its light chain variable region sequence is shown in SEQ ID No. 2 and its heavy chain variable region sequence is shown in SEQ ID No. 3.

5. The use according to claim 4, wherein the medicament is formulated in the form of tablets, reducing agents, powders, granules, capsules, suspensions, emulsions, syrups, aerosols, injections, etc.

6. The use according to claim 5, wherein the medicament comprises an excipient which is starch.