CN113699103A

CN113699103A - Induced reinforcement preparation process and application of poultry mesenchymal stem cell exosomes

Info

Publication number: CN113699103A
Application number: CN202110794777.XA
Authority: CN
Inventors: 谢儒生; 金玉堂
Original assignee: Individual
Current assignee: Shengyi Biotechnology Co ltd
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2021-11-26
Anticipated expiration: 2041-07-14
Also published as: CN113699103B

Abstract

The invention discloses a process for preparing an induced aid of an avian mesenchymal stem cell exosome, which comprises the following steps: performing primary separation culture on the poultry mesenchymal stem cells, performing subculture, taking the poultry mesenchymal stem cells, culturing by using a cell growth medium, discarding the culture medium, washing the cells for three times by using a phosphate buffer solution, adding curcumin for stimulation, continuously culturing for 72 hours by using a serum-free culture medium, collecting the serum-free culture medium, and continuously performing the treatment on the cells which are not collected until a sufficient amount of supernatant is collected; centrifuging the collected curcumin-stimulated serum-free culture solution, extracting and concentrating the exosome filtrate, and sterilizing the obtained exosome concentrated filtrate. The invention stimulates and induces poultry mesenchymal stem cells by curcumin through improving the process formula, promotes the production of exosomes boosted by curcumin, and has more excellent effect compared with exosomes without the boosting effect of curcumin.

Description

Induced reinforcement preparation process and application of poultry mesenchymal stem cell exosomes

Technical Field

The invention relates to a preparation method of stem cell exosomes, in particular to a preparation process and application of induced aid of avian mesenchymal stem cell exosomes, and belongs to the technical field of stem cell biomedicines.

Background

The source of the poultry mesenchymal stem cells is wide, the materials are easy to obtain, the cell separation efficiency is high, the cell strain state is stable, and the medical ethical problem does not exist, so that the poultry mesenchymal stem cells are gradually paid attention to. Exosomes are nanoscale vesicles that are released outside of a cell upon environmental stimulation or activation of the cell. Vesicle structures are classified into 3 types according to their diameter: exosomes (diameter 40-150 nm), microvesicles (diameter 100-1000 nm) and apoptotic bodies (diameter 1-4 μm). The exosome is formed by gathering substances such as protein, DNA (deoxyribonucleic acid) fragments, miRNA (micro ribonucleic acid) and the like in cytoplasm into a multi-vesicle endosome through an endocytosis path, and then fusing and cracking the multi-vesicle and a plasma membrane to release the multi-vesicle endosome to the outside of cells to form the exosome. The internal structure of exosomes mainly depends on the cell from which they are derived, but most exosomes are rich in integrins, heat shock proteins, the tetraspanin superfamily (CD9, CD63, CD81 and CD82), the cell endogenous proteins alix and the Rab protein family, and these proteins can be used as reliable markers for identifying exosomes.

Exosomes have functions of helping to remove old and useless cells and substituting with new cells, and are regarded as important substances effective in combating the aging and repair of skin. In addition to the development of applications at the beauty end, it was also found that exosomes also have an improving effect on allergic rhinitis caused by air pollution or seasonal alternation. If the exosome is prepared into an inhalation spray type, and is inhaled through the nasal cavity and the oral cavity, the exosome can pass through the nasal mucosa, the inflammatory reaction at the nasal mucosa is relieved, and the symptoms of nasal obstruction, rhinorrhea, asthma and the like are improved.

At present, although studies have been made on the use of specific induction conditions for inducing stem cells to secrete supernatants in vitro for medical treatment, cosmetics and the like, these conditioned media achieve therapeutic effects by inducing stem cells to secrete certain specific factors using specific cytokines, and the cytokines themselves are expensive, which results in an abnormally high cost of application of stem cells.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide an induced reinforcement preparation process and application of the poultry mesenchymal stem cell exosomes, wherein curcumin is used for stimulating and inducing poultry mesenchymal stem cells to promote the production of exosomes reinforced by curcumin, and the effect of the exosomes is more excellent than that of exosomes not reinforced by curcumin.

In order to solve the technical problems, the invention provides a process for preparing an induced aid of an avian mesenchymal stem cell exosome, which comprises the following steps:

s1: carrying out primary isolation culture on the poultry mesenchymal stem cells, carrying out subculture, and taking subcultured P3-P30 generation human adipose mesenchymal stem cells;

s2: culturing with cell growth liquid until 75-85% of cells are fused, discarding the culture liquid, washing the cells with phosphate buffer solution for three times, adding curcumin, culturing with serum-free culture liquid for 72 hr, and collecting the serum-free culture liquid;

s3: continuing the curcumin stimulation treatment of the step until the serum-free culture solution is collected, wherein the cells which are not collected in the step S2 are not collected;

s4: collecting serum-free culture solution stimulated by curcumin in the steps S2 and S3, centrifuging, extracting and concentrating the exosome filtrate, and finally preparing the needed curcumin to induce the reinforcement poultry mesenchymal stem cell exosomes after degerming treatment is carried out on the obtained exosome concentrated filtrate;

s5: adding excipient and water into curcumin-induced aid-increasing poultry mesenchymal stem cell exosome, mixing, filtering for sterilization, and freeze-drying to obtain the stem cell extract freeze-dried powder.

Although the curcumin-induced reinforcement poultry mesenchymal stem cell exosome has various biological activities and is wide in application field, the biological activities of the curcumin-induced reinforcement poultry mesenchymal stem cell exosome are limited by various external conditions and are inconvenient to store, so that the invention also provides the cryolite powder of the curcumin-induced reinforcement poultry mesenchymal stem cell exosome, and the cryolite powder is prepared by the cryolite powder preparation method commonly used in the field; the frozen crystal powder is prepared by freezing liquid medicine into solid state in aseptic environment, and then carrying out vacuum pumping to sublimate and dry water.

In a further limited technical scheme of the present invention, in the induced rescue preparation process of the poultry mesenchymal stem cell exosome, the cell growth solution is a DMEM culture solution containing 5% fetal bovine serum, and the culture solution further contains an antibacterial antibiotic; the antibacterial antibiotic is penicillin and streptomycin, wherein the final concentration of penicillin in a cell growth solution is 200U/ml, the final concentration of streptomycin in the cell growth solution is 250U/ml, the DMEM culture solution containing 5% fetal calf serum is obtained by adding 5% of fetal calf serum into a commercially available basic DMEM culture solution, and the penicillin with the final concentration of 200U/ml and the streptomycin with the final concentration of 250U/ml are added to obtain the cell growth solution.

In the induced reinforcement preparation process of the poultry mesenchymal stem cell exosomes, the phosphate buffer solution is a PBS solution with a pH value of 7.0-7.2; the excipient is any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced reinforcement poultry mesenchymal stem cell exosomes.

In the induced reinforcement preparation process of the poultry mesenchymal stem cell exosomes, the water in the step S5 is ultrapure water, and the ultrapure water is added to a final volume of 100 ml; the freeze-drying treatment is that the freeze-drying temperature is-20 to-35 ℃, the vacuum degree is 50 to 200Pa, and the freeze-drying is carried out for 48 hours.

Further, in the aforementioned induced aid preparation process of the poultry mesenchymal stem cell exosomes, the centrifugation in step S4 is performed for 1,000xg10 minutes and 10,000xg10 minutes, after that, the supernatant is collected, the collected supernatant is subjected to tangential flow filtration through a hollow fiber membrane, the collected, filtered and concentrated exosome solution is then passed through an exosome extraction system to extract a high-purity exosome solution, and the sterilization treatment is performed by filtering and sterilizing through a sterile 0.22 μm filter membrane.

An application of fowl mesenchymal stem cell exosome comprises preparing wound healing medicine or cell repairing medicine from stem cell extract cryo-crystal powder; preparing a whitening product or a fine chemical product for resisting skin wrinkles.

The invention has the beneficial effects that: according to the invention, through improvement of a process formula, poultry mesenchymal stem cells are stimulated and induced by curcumin, and exosomes boosted by curcumin are promoted to be generated, so that the effect of the exosomes is more excellent than that of exosomes not boosted by curcumin; meanwhile, exosomes secreted by the isolated cells are collected and extracted by a special technology, and are called curcumin-induced reinforcement poultry mesenchymal stem cell exosomes; the curcumin induces the exosomes of the mesenchymal stem cells of the booster poultry to be frozen and dried to produce frozen crystal powder, thereby ensuring the biological activity and the efficacy. The process verified by the invention is mainly to discuss the effect of curcumin induced enhancement poultry mesenchymal stem cell exosome on skin repair.

Drawings

FIG. 1 is a transmission electron microscope image of the present invention before and after lyophilization.

FIG. 2 shows particle size analysis and particle number according to the present invention.

FIG. 3 shows the purity test of the frozen crystal powder of the present invention.

FIG. 4 shows the protein concentration contained in the frozen powder of the present invention.

FIG. 5 shows the RNA concentration of exosomes contained in the cryogel powder of the present invention.

FIG. 6 shows the cell proliferation effect of the cryo-crystal powder of the present invention on human skin fibroblasts.

FIG. 7 shows the effect of cryo-crystal powder of the present invention on cell proliferation of human hair follicle cells.

FIG. 8 is a graph showing the wound healing effect of the cryo-crystal powder of the present invention on human skin fibroblasts.

Detailed Description

The induced reinforcement preparation process of the poultry mesenchymal stem cell exosome provided by the embodiment comprises the following steps:

s4: collecting serum-free culture solution stimulated by curcumin in the steps S2 and S3, centrifuging, extracting and concentrating the exosome filtrate, and finally preparing the needed curcumin to induce the reinforcement poultry mesenchymal stem cell exosomes after degerming treatment is carried out on the obtained exosome concentrated filtrate; the centrifugation operation adopts the centrifugation process of 1,000Xg10 minutes and 10,000Xg10 minutes, the supernatant is collected after the centrifugation is finished, the collected supernatant flows through a hollow fiber membrane to be subjected to tangential flow filtration, the collected, filtered and concentrated exosome solution is then passed through an exosome extraction system to extract high-purity exosome solution, and the sterilization treatment adopts a sterile 0.22 mu m filter membrane to carry out filtration sterilization;

s5: adding excipient and ultrapure water into curcumin-induced aid-increasing poultry mesenchymal stem cell exosomes, mixing, filtering for sterilization, and then performing freeze-drying treatment to obtain stem cell extract freeze-dried powder; adding ultrapure water to a final volume of 100 ml; the freeze-drying treatment is that the freeze-drying temperature is-20 to-35 ℃, the vacuum degree is 50 to 200Pa, and the freeze-drying is carried out for 48 hours.

The cell growth medium in this example is DMEM medium containing 5% fetal bovine serum, and the culture medium further contains an antibacterial antibiotic; the antibacterial antibiotic is penicillin and streptomycin, wherein the final concentration of penicillin in the cell growth solution is 200U/ml, the final concentration of streptomycin in the cell growth solution is 250U/ml, and the DMEM culture solution containing 5% fetal calf serum is obtained by adding 5% fetal calf serum to a commercially available basic DMEM culture solution, and adding penicillin with the final concentration of 200U/ml and streptomycin with the final concentration of 250U/ml to obtain the cell growth solution. The phosphate buffer solution is a PBS solution with the pH value of 7.0-7.2; the excipient is any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced reinforcement poultry mesenchymal stem cell exosomes.

Reagent instruments are used in this example:

BCA protein detection kit: # 23225; thermo Fisher Scientific, Waltham, MA, USA;

RIPA buffer: 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 1% Na-deoxyholate, 0.1% SDS, 0.1 mM CaCl₂， and 0.01 mM MgCl₂；

qEV RNA extraction kit: IZON Science LTD, Christchurch, New Zealand;

protease inhibitor cocktail: thermo Fisher Scientific;

full wavelength microplate reader: molecular Devices, San Jose, Calif., USA;

HitachiHT7700 electron microscope: hitachi, Tokyo, Japan;

a spectral luminance meter: NanoDrop ™ 2000c, Thermo Fisher Scientific;

silica gel insert: culture inserts, 2-well plates, ibiTreat; ibidi, Martinsried, Germany.

Transmission electron microscope using procedure

Curcumin-induced enhancement poultry mesenchymal stem cell exosome frozen crystal powder samples are prepared for transmission electron microscopy. Briefly, exosomes were immobilized on copper mesh, fixed with 1% glutaraldehyde in cold PBS for 5 min to stabilize the immune response, washed with sterile distilled water, compared with a uranium oxalate solution at pH7 for 5 min, and then embedded with methylcellulose on ice for 10 min. Excess cellulose was removed and the samples were dried for permanent storage. The exosome samples were imaged using a HitachiHT7700 electron microscope at 80kV voltage.

Detection of contaminating heteroprotein

The pierce tmbca protein assay kit was used for purity testing. A standard curve (range 0-2000. mu.g/mL) was obtained from nine serial dilutions of Bovine Serum Albumin (BSA) and working reagent. All samples and standard spots were repeated 3 times. Dissolving samples (10 μ l each) (100 mg curcumin-induced rescue poultry mesenchymal stem cell exosome freeze-dried powder or freeze-dried powder of control exosome in 500 μ lQH₂O) was mixed with 200 μ l of the working reagent and incubated at 65 ℃ for 30 minutes. After cooling to room temperature, each difference in absorbance (minus the average absorbance of the 562nm blank standard replicate) was measured by a full wavelength microplate reader and converted to μ g/ml through standard curve. If the protein concentration exceeds the upper limit of the standard curve, 2000. mu.g/mL, the sample is diluted to a level that can be determined within the standard range and the final concentrate is calibrated to take into account the dilution factor.

Exosome protein concentration detection step curcumin induces the frozen crystal powder of the exosomes of the rescue poultry interstitial stem cells of 100mg and the frozen crystal powder of the control exosomes to be resuspended in RIPA buffer solution and supplemented with protease inhibitor mixture. The BCA protein assay kit was used for purity testing. Nine serial dilutions were made using Bovine Serum Albumin (BSA) and working reagent to generate a standard curve (range 0-250. mu.g/mL). All samples and standard spots were repeated 3 times. Samples (10. mu.l each) were mixed with 200. mu.l of working reagent and incubated at 65 ℃ for 30 minutes. After cooling to room temperature, each difference in absorbance was measured by a full wavelength microplate reader, the average absorbance at 562nm of the blank standard replicate was subtracted, and the difference in absorbance was converted to μ g/ml by the standard curve.

Exosome RNA concentration detection

Curcumin induces the frozen crystal powder of the exosomes of the enhancement poultry mesenchymal stem cells of 100mg and the frozen crystal powder of the exosomes of 100mg are resuspended in the lysis buffer A of the qEVRNA extraction kit. The detailed extraction method follows the user specification. And detecting the RNA concentrations of the extracted exosomes of the curcumin-induced enhancement poultry interstitial stem cell exosome frozen crystal powder and the control exosomes by using a spectrophotometer.

Cell proliferation assay procedure

To determine cell viability, human skin fibroblasts and human hair follicle cells were cultured in their growth media until they reached 80% density. Cells were treated at 5X 10³The total volume of individual cells per well was seeded in 96-well plates at a density of 100 μ l. After inoculation, cells were incubated at 37 ℃ and 5% CO₂Medium for 24 hours to allow cell attachment. Cells were then washed once with PBS and the medium was replaced with medium containing 0.25% stripped FBS, starved for 24 hours. Supplemented with curcumin-induced rescue-enhancing avian mesenchymal stem cell exosome cryogels at different concentrations and medium containing 0.25% stripped FBS and treated for 72 hours (human dermal fibroblasts) or 96 hours (human hair follicle cells). Curcumin induces the supplement poultry interstitial stem cell exosome frozen crystal powder culture medium mixture to be renewed every other day. After 72 hours or 96 hours of treatment, the medium containing 20% MTS solution was changed to growth medium and incubated for 2 hours. The absorbance at 490nm was measured for each well using a full wavelength microplate reader.

Procedure for wound healing test

To examine the wound healing effect of curcumin-induced enhancement of avian mesenchymal stem cell exosome cryogel powder on the treatment of human dermal fibroblasts, a wound healing experiment was performed using a silica gel insert, and cells were plated in 24-well plates. Specifically, the cells were incubated at 2.5X 105 cells/cm²Is seeded in two compartments of a silica gel insert. The cells were grown for 24 hours, then the medium was changed to starvation medium (no serum) and cultured for another 24 hours. The culture inserts were removed using sterile forceps, creating a 500 μm wide gap. Media supplemented with curcumin-induced rescue avian mesenchymal stem cell exosome cryogels at different concentrations and 0.25% stripped FBS were added and incubated for 24 hours.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Claims

1. An induced reinforcement preparation process of an avian mesenchymal stem cell exosome is characterized by comprising the following steps:

2. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein: the cell growth solution is DMEM culture solution containing 5% fetal calf serum, and the culture solution also contains antibacterial antibiotics; the antibacterial antibiotic is penicillin and streptomycin, wherein the final concentration of penicillin in a cell growth solution is 200U/ml, the final concentration of streptomycin in the cell growth solution is 250U/ml, the DMEM culture solution containing 5% fetal calf serum is obtained by adding 5% of fetal calf serum into a commercially available basic DMEM culture solution, and the penicillin with the final concentration of 200U/ml and the streptomycin with the final concentration of 250U/ml are added to obtain the cell growth solution.

3. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein: the phosphate buffer solution is a PBS solution with the pH value of 7.0-7.2.

4. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein: the excipient is any one of mannitol, trehalose or sucrose; the dosage of the excipient accounts for 5-10% of the total weight of the curcumin-induced reinforcement poultry mesenchymal stem cell exosomes.

5. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein: the water in the step S5 is ultrapure water, and ultrapure water is added to a final volume of 100 ml.

6. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein: the freeze-drying treatment is that the freeze-drying temperature is-20 to-35 ℃, the vacuum degree is 50 to 200Pa, and the freeze-drying is carried out for 48 hours.

7. The process for preparing exosomes of avian mesenchymal stem cells according to claim 1, wherein:

the centrifugation operation of the step S4 is to adopt the centrifugation process of 1,000Xg10 minutes and 10,000Xg10 minutes, collect the supernatant after the centrifugation is finished, flow the collected supernatant through the hollow fiber membrane to carry out tangential flow filtration, collect the filtered and concentrated exosome solution and then pass through an exosome extraction system to extract the high-purity exosome solution, and the sterilization treatment adopts a sterile 0.22 mu m filter membrane to carry out filtration sterilization.

8. The application of the poultry mesenchymal stem cell exosome is characterized in that: preparing a wound healing medicament or a cell repairing medicament by using the stem cell extract cryolite powder; preparing a whitening product or a fine chemical product for resisting skin wrinkles.