CN111437244B - Preparation method and product of high-efficiency stem cell expression exosome for skin beauty - Google Patents

Abstract

The invention discloses a preparation method and a product of a stem cell high-efficiency expression exosome for skin beauty, wherein the preparation method comprises the following steps: separating stem cells from human tissues and culturing; constructing a recombinant expression vector, adding the recombinant expression vector into a culture suspension of stem cells, obtaining the stem cells capable of expressing target proteins by adopting an electrotransfection mode, extracting exosomes from a supernatant of the stem cells, adding auxiliary materials according to parts by weight, carrying out instant freeze-drying by utilizing liquid nitrogen, and drying the obtained tablets in a vacuum environment. Through the technical scheme of the invention, the recombinant expression vector can be transferred to stem cells to extract the target protein exosome, and the product for skin beauty is prepared by using a freeze-drying technology, so that the active ingredients of the exosome are effectively protected, and the exosome can be stored for a long time at room temperature and keep activity.

Description

Preparation method and product of stem cell high-efficiency expression exosome for skin beauty

Technical Field

The invention relates to the technical field of stem cell exosomes, in particular to a preparation method of a stem cell high-efficiency expression exosome for skin beautifying and a product of the stem cell high-efficiency expression exosome for skin beautifying.

Background

Stem cell exosomes have been studied abroad for many years, and are increasingly used in the market, while domestic application to stem cell exosomes is also increased, and the types of products for beauty skin care are also increased, but the expression efficiency of the stem cell exosomes is low, the protein expression is low, the effect of beauty skin care is general, and the stem cell exosomes can cause the defects of insufficient directional secretion, reduced activity and the like after long-term storage.

Disclosure of Invention

Aiming at least one of the problems, the invention provides a preparation method and a product for skin beauty by using a stem cell high-efficiency expression exosome, wherein a recombinant expression vector is transferred into a stem cell in an electrotransfection mode, so that a target protein exosome is extracted, and on the basis, a product for skin beauty is prepared by using a freeze-drying technology, so that active ingredients of the stem cell high-efficiency expression exosome are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored at room temperature for a long time, and overcomes the defects of directional secretion, low long-time storage activity and the like of the stem cell exosome. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that the required target proteins are directionally and efficiently expressed.

In order to achieve the above object, the present invention provides a method for preparing exosome efficiently expressed by stem cells for skin beauty, comprising: separating stem cells from human tissues and culturing; combining a PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed 1, a T2A sequence, a target protein sequence to be expressed 2, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; adding the recombinant expression vector into the culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode; aseptically extracting exosomes from the supernatant of the dry cells after electrotransfection, and adding 50 parts of the exosomes, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of Hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water according to parts by weight, mixing and stirring; and (3) carrying out three-second instant freeze-drying dormancy on the mixed materials by adopting liquid nitrogen, and drying the mixed materials in a vacuum environment to sublimate and discharge the ice crystals in the freeze-dried tablets.

In the above technical solution, preferably, the recombinant expression vector includes two sequences of target proteins, and the target protein sequence 1 to be expressed and the target protein sequence 2 to be expressed are respectively disposed on the front side and the rear side of the T2A sequence.

In the above technical solution, preferably, the target protein includes oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, regenerated protein, transforming factor and insulin-like growth factor.

In the above technical solution, preferably, the stem cells isolated from the human tissue include mesenchymal stem cells, adipose-derived stem cells and epithelial stem cells, and the human tissue includes human umbilical cord tissue and adipose tissue.

In the above technical solution, preferably, the step of performing the electrotransfection mode comprises: 1-2 days before the electrotransfection is carried out, transferring the stem cells to a cell culture flask, so that the cell confluence of the stem cells before the electrotransfection is 50% -70%; the stem cells were slowly rinsed with PBS and aspiratedPBS, digesting the stem cells by pancreatin protease, and then adding a culture medium containing serum to neutralize pancreatin; the stem cells were collected by centrifugation and resuspended in PBS, hepes and serum-free medium to a density of 1X 10⁶ ～5×10⁶ cell/ml; setting electrotransfection parameters to be 2.0kV and 25 uFD; adding the plasmid of the recombinant expression vector into an electric rotating cup, and setting the initial concentration of the plasmid to be 10-50 mug/ml according to the type of the stem cells; adding the resuspended cells into an electric shock cup, turning the electric shock cup for uniformly mixing; putting the electric shock cup into an electric shock groove, and carrying out pulse electric shock for one time; transferring the shocked cells to a well containing a culture medium; and (3) shaking the pore plate to uniformly mix the cells, and carrying out electrotransformation for 24-48 hours to realize transient expression of cell genes.

In the above technical scheme, preferably, when the target protein is oligopeptide-1 and basic fibroblast, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibroblast-CD 33-IRES-PURO, episome ORI-episome EBNA 1; when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO and episome ORI-episome EBNA 1.

In the above technical solution, preferably, the step of isolating the mesenchymal stem cells from the human umbilical cord tissue comprises: separating to obtain human umbilical cord tissue blocks, and digesting for 5-10 minutes by using pancreatin; collecting the tissue suspension and centrifuging, discarding the supernatant, and placing the tissue block in a plate; adding a DMEM cell culture medium, and periodically changing the liquid by half; removing the tissue blocks after the cells grow over the plate; after trypsinization, transferring the primary cells on the plate to a T75 culture flask; and (5) when the cells grow over the culture bottle and passage is carried out to 2-3 generations, and the mesenchymal stem cells are obtained.

In the above technical solution, preferably, the step of isolating adipose stem cells from human adipose tissue comprises: flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube; adding equal volume of 0.1-0.5% type I collagenase into adipose tissue, and placing into 37 deg.C steam bath oscillatorDigesting for 30-60 min; when digestion is completed, the undigested adipose tissues are discarded, and the cell suspension is centrifuged and the supernatant is discarded; the adipose-derived stem cell pellet was obtained at the bottom of the centrifuge tube and resuspended to a density of 1X 10⁵ mL^-1 (ii) a Inoculating the cell suspension into a cell culture bottle, and culturing by using a DMEM (DMEM) culture medium until the cell suspension grows to the fusion degree of more than 80%; collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating into cell culture bottle, and culturing to obtain adipose-derived stem cells.

In the above technical scheme, preferably, after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5-7 hours in a vacuum environment by using an ultra-vacuum freeze-drying oven, so that ice crystals in the freeze-dried tablet are sublimated and discharged to obtain a loose, porous and water-disintegrable tablet, and the loose, porous and water-disintegrable tablet is packaged by using a double aluminum structure and then is stored in a dark place.

The invention also provides a product for efficiently expressing the exosome by the stem cell for skin beauty, which comprises a tablet prepared by the preparation method for efficiently expressing the exosome by the stem cell for skin beauty according to any one of the technical schemes.

Compared with the prior art, the invention has the following beneficial effects: the recombinant expression vector is transferred to the stem cell in an electrotransfection mode, so that a target protein exosome is extracted, and on the basis, a product for skin beautifying is prepared by using a freeze-drying technology, so that the active ingredients of the exosome efficiently expressed by the stem cell are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored for a long time at room temperature, and overcomes the defects of directional secretion, low long-time storage activity and the like of the exosome of the stem cell. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that multiple required target proteins are directionally and efficiently expressed.

Drawings

Fig. 1 is a schematic flow chart of a preparation method for efficiently expressing exosomes by stem cells for skin beauty disclosed in an embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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 some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The invention is described in further detail below with reference to the attached drawing figures:

as shown in fig. 1, the preparation method of the high-efficiency stem cell expression exosome for skin beauty provided by the invention comprises the following steps: separating stem cells from human tissues and culturing; combining the PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed, a T2A sequence, a target protein sequence to be expressed, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; adding the recombinant expression vector into a culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode; the exosome is aseptically extracted from the supernatant of the stem cells after electrotransfection, and 50 parts of the exosome, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water are added according to the parts by weight for mixing and stirring; and (3) carrying out three-second instant freeze-drying dormancy on the mixed material by adopting liquid nitrogen, and drying in a vacuum environment to sublimate and discharge ice crystals in the freeze-dried tablets.

In the embodiment, the recombinant expression vector is transferred to the stem cell in an electrotransfection mode, so that the target protein exosome is extracted, and on the basis, a product for skin beautifying is prepared by using a freeze-drying technology, so that the active ingredients of the exosome efficiently expressed by the stem cell are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored at room temperature for a long time, and the defects of directional secretion, low long-time storage activity and the like of the stem cell exosome are overcome. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that the required target proteins are directionally and efficiently expressed.

In the above embodiment, preferably, the recombinant expression vector includes two sequences of the target protein, and the sequence 1 of the target protein to be expressed and the sequence 2 of the target protein to be expressed are respectively disposed on the front side and the back side of the T2A sequence.

In the above embodiments, preferably, the protein of interest includes oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, neogenin, transforming factor and insulin-like growth factor.

In the above examples, preferably, when the target protein is oligopeptide-1 and basic fibrin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibrin-CD 33-IRES-PURO, episome ORI-episome EBNA 1.

In the above examples, preferably, when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, episome ORI-episome EBNA 1.

In the above embodiment, preferably, the stem cells isolated from human tissue include mesenchymal stem cells, adipose stem cells and epithelial stem cells, and the human tissue includes human umbilical cord tissue and adipose tissue.

In the above embodiment, preferably, after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5 to 7 hours in a vacuum environment by using an ultra-vacuum freeze-drying oven, so that ice crystals in the freeze-dried tablet are sublimated and discharged, and the loose, porous and water-disintegrable tablet is obtained, and is packaged by using a double aluminum structure and then is stored in a dark place.

The product for skin beauty with the high-efficiency stem cell expression exosomes provided by the invention comprises the tablet prepared by the preparation method for skin beauty with the high-efficiency stem cell expression exosomes provided by any one of the embodiments.

The preparation method and the product of the high-efficiency expression exosome of the stem cell for skin beauty provided by the above embodiment are provided, and three embodiments are provided below to explain the above method in detail.

The first embodiment is as follows:

the invention provides a preparation method of high-efficiency expression oligopeptide-1 and basic fibroblast of human umbilical cord mesenchymal stem cells for skin beauty, which comprises the following steps:

1. isolating human umbilical cord mesenchymal stem cells from a human umbilical cord;

1) separating umbilical cord Wharton's jelly, and shearing the jelly into pieces;

2) digesting for 5-10 minutes by using 0.25% pancreatin;

3) collecting the tissue suspension, centrifuging for 2000 r and 10 min;

4) discarding the supernatant, and placing the tissue block in a 90mm plate;

5) adding 2-3 ml of DMEM cell culture medium, and changing the medium half every 2-3 days;

6) removing tissue blocks when the cells grow over the plate;

7) digesting with 0.25% pancreatin, and transferring the primary cells on the plate to a T75 culture flask;

8) after the cells grow over the culture bottle, passage is carried out to 2-3 generations;

2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-alkali fibrin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;

3. and (3) performing electrotransfection on the human umbilical cord mesenchymal stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain the human umbilical cord mesenchymal stem cells capable of efficiently expressing oligopeptide-1 and basic fibroblast:

suspension of cells:

1) 1-2 days before electroporation, cells were transferred to 75cm² Cell culture flasks with a cell confluency of about 50-70% before transformation, at which time the number of cells is about 2-10X 10⁶ And each electrical revolution is about 1-10X 10⁵ (ii) individual cells;

2) slowly washing cells with 12ml of PBS, sucking out the PBS, digesting the cells with 0.4ml of trypsin protease, adding 10ml of culture medium containing serum, and neutralizing the trypsin;

3) centrifuging to collect cells, resuspending the cells in PBS, Hepes, serum-free medium, etc. to density of 1-5 × 10⁶ cell/ml；

And (3) electric conversion:

1) setting a power conversion program: 2.0KV, 25 uFD;

2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is generally 10-50 ug/ml;

3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;

4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;

5) transferring the cells to wells already containing 0.5ml of medium;

6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;

7) detecting the activity of the cells after 24-48 h;

wherein, the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.

4. Sterile extraction of the culture supernatant obtained after electrotransfection:

1) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and discarding the precipitate;

2) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and removing the precipitate;

3) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;

4) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times;

5. adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 weight part of sodium chloride, 1-10 weight parts of pullulan, 1-10 weight parts of trehalose, 0.5-5 weight parts of mannitol, 1-10 weight parts of dextran, 1-5 weight parts of glucose, 0.5-10 weight parts of sodium lactate, 0.5-2 weight parts of potassium chloride and 5-10 weight parts of albumin, and deionized water is supplemented to the required amount.

6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.

Example two:

the invention provides a preparation method for efficiently expressing oligopeptide-1 and basic fibroblast by adipose-derived stem cells and used for skin beauty, which comprises the following specific steps:

1. separating and extracting adipose stem cells from adipose tissues;

1) flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube;

2) adding equal volume of type I collagenase of 0.1-0.5% into the tissue, and sterilizing in a 37 deg.C steam bath oscillator for 30-60 min;

3) when digestion is completed, discarding upper undigested adipose tissues, centrifuging the lower cell suspension for 10min at a speed of 300-400 g, and discarding the supernatant;

4) obtaining the adipose-derived stem cell mass at the bottom of the centrifuge tube, resuspending the adipose-derived stem cell mass, and adjusting the density to 1X 10⁵ mL^-1 ；

5) Inoculating to culture area of 25cm² Culturing the cells in the cell culture bottle by using a DMEM medium until the cells grow to the fusion degree of more than 80%;

6) collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating to 3 culture areas of 75cm² The cell culture flask of (1);

2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-alkali fibrin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;

3. and (3) performing electrotransfection on the adipose-derived stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain adipose-derived stem cells capable of efficiently expressing oligopeptide-1 and basic fibroblast:

suspension of cells:

1) 1-2 days before electroporation, cells were transferred to 75cm² Cell culture flasks, with a confluency of cells of about 50-70% before transformation. The number of cells in this case is about 2 to 10X 10⁶ And each electrical revolution is about 1-10X 10⁵ (ii) individual cells;

2) slowly washing cells with 12ml PBS, sucking out PBS, digesting cells with 0.4ml pancreatin protease, adding 10ml culture medium containing serum, and neutralizing pancreatin;

3) centrifuging to collect cells, resuspending the cells with PBS, Hepes, serum-free medium, etc. to density of 1-5 × 10⁶ cell/ml。

And (3) electric conversion:

1) setting a power conversion program: 2.0KV, 25 uFD;

2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is 10-50ug/ml generally;

3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;

4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;

5) transferring the cells to wells already containing 0.5ml of medium;

6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;

7) detecting the activity of the cells after 24-48 h;

wherein the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.

4. Sterile extraction of the culture supernatant obtained after electrotransfection:

5) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and removing the precipitate;

6) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and discarding the precipitate;

7) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;

8) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times;

5. adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 part by weight of sodium chloride, 1-10 parts by weight of pullulan, 1-10 parts by weight of trehalose, 0.5-5 parts by weight of mannitol, 1-10 parts by weight of dextran, 1-5 parts by weight of glucose, 0.5-10 parts by weight of sodium lactate, 0.5-2 parts by weight of potassium chloride and 5-10 parts by weight of albumin, and deionized water is supplemented to the required amount.

6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.

Example three:

the invention provides a preparation method for efficiently expressing oligopeptide-1 and keratin by human umbilical cord mesenchymal stem cells for skin beauty, which comprises the following specific steps:

1. isolating human umbilical cord mesenchymal stem cells from human umbilical cord;

1) separating umbilical cord Wharton's jelly, and shearing the jelly into pieces;

2) digesting for 5-10 minutes by using 0.25% pancreatin;

3) collecting the tissue suspension, centrifuging for 2000 r and 10 min;

4) discarding the supernatant, and placing the tissue block in a 90mm dish;

5) adding 2-3 ml of DMEM cell culture medium, and changing the culture medium half every 2-3 days;

6) removing tissue blocks when the cells grow over the plate;

7) digesting with 0.25% trypsin, and transferring the primary cells on the plate to a T75 culture flask;

8) after the cells grow over the culture bottle, passage is carried out to 2-3 generations;

2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;

3. and (3) performing electrotransfection on the human umbilical cord mesenchymal stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain the human umbilical cord mesenchymal stem cells capable of efficiently expressing oligopeptide-1 and keratin:

suspension of cells:

1) 1-2 days before electroporation, cells were transferred to 75cm² Cell culture flasks with a cell confluency of about 50-70% before transformation, at which time the number of cells is about 2-10X 10⁶ And each electrical revolution is about 1-10X 10⁵ (ii) individual cells;

2) slowly washing cells with 12ml PBS, sucking out PBS, digesting cells with 0.4ml pancreatin protease, adding 10ml culture medium containing serum, and neutralizing pancreatin;

3) centrifuging to collect cells, resuspending the cells with PBS, Hepes, serum-free medium, etcSo that it has a density of 1-5 x 10⁶ cell/ml。

And (3) electric conversion:

1) setting a power conversion program: 2.0KV, 25 uFD;

2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is generally 10-50 ug/ml;

3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;

4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;

5) transferring the cells to wells already containing 0.5ml of medium;

6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;

7) detecting the activity of the cells after 24-48 h;

wherein, the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.

4. And (3) performing sterile extraction on the culture supernatant obtained after electrotransfection:

1) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and discarding the precipitate;

2) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and discarding the precipitate;

3) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;

4) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times.

5. Adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 part by weight of sodium chloride, 1-10 parts by weight of pullulan, 1-10 parts by weight of trehalose, 0.5-5 parts by weight of mannitol, 1-10 parts by weight of dextran, 1-5 parts by weight of glucose, 0.5-10 parts by weight of sodium lactate, 0.5-2 parts by weight of potassium chloride and 5-10 parts by weight of albumin, and deionized water is supplemented to the required amount.

6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.

According to the extraction and preparation method of the stem cell high-efficiency expression exosome for skin beauty provided by the embodiment, the extract is also suitable for liquid skin care products.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that the present invention can be applied to simultaneously express one target protein and a plurality of target proteins, and the constructed vector can be applied to expression of a plurality of stem cells. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing Benzhen workshop Biotechnology Co., Ltd

<120> preparation method and product of high-efficiency expression exosome of stem cell for skin beauty

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cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 780

ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 840

aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 900

ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 960

gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1020

atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1080

taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1140

acagtggttc aaagtttttt tcttccattt caggtgtcgt ga 1182

<210>2

<211>48

<212>DNA

<213> unknown

<400>2

atgcccctgc tgctgctgct gcccctgctg tgggccggcg ccctggcc 48

<210> 3

<211>6

<212>DNA

<213> unknown

<400> 3

gccacc 6

<210> 4

<211>465

<212>DNA

<213> Artificial sequence

<400> 4

atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60

ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120

ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180

aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240

cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300

tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360

accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420

cctgggcaga aagctatact ttttcttcca atgtctgcta agagc 465

<210>5

<211>54

<212>DNA

<213> unknown

<400>5

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210>6

<211>574

<212>DNA

<213> unknown

<400>6

cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60

tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120

gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180

aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240

aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300

ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360

cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420

ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480

cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540

ggacgtggtt ttcctttgaa aaacacgatg ataa 574

<210>7

<211>600

<212>DNA

<213> unknown

<400>7

atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60

cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120

cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180

atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240

agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300

tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360

cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 420

agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480

gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540

gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600

<210>8

<211>1790

<212>DNA

<213> unknown

<400>8

aacgggtagc atatgcttcc cgggtagtag tatatactat ccagactaac cctaattcaa 60

tagcatatgt tacccaacgg gaagcatatg ctatcgaatt agggttagta aaagggtcct 120

aaggaacagc gatatctccc accccatgag ctgtcacggt tttatttaca tggggtcagg 180

attccacgag ggtagtgaac cattttagtc acaagggcag tggctgaaga tcaaggagcg 240

ggcagtgaac tctcctgaat cttcgcctgc ttcttcattc tccttcgttt agctaataga 300

ataactgctg agttgtgaac agtaaggtgt atgtgaggtg ctcgaaaaca aggtttcagg 360

tgacgccccc agaataaaat ttggacgggg ggttcagtgg tggcattgtg ctatgacacc 420

aatataaccc tcacaaaccc cttgggcaat aaatactagt gtaggaatga aacattctga 480

atatctttaa caatagaaat ccatggggtg gggacaagcc gtaaagactg gatgtccatc 540

tcacacgaat ttatggctat gggcaacaca taatcctagt gcaatatgat actggggtta 600

ttaagatgtg tcccaggcag ggaccaagac aggtgaacca tgttgttaca ctctatttgt 660

aacaagggga aagagagtgg acgccgacag cagcggactc cactggttgt ctctaacacc 720

cccgaaaatt aaacggggct ccacgccaat ggggcccata aacaaagaca agtggccact 780

cttttttttg aaattgtgga gtgggggcac gcgtcagccc ccacacgccg ccctgcggtt 840

ttggactgta aaataagggt gtaataactt ggctgattgt aaccccgcta accactgcgg 900

tcaaaccact tgcccacaaa accactaatg gcaccccggg gaatacctgc ataagtaggt 960

gggcgggcca agataggggc gcgattgctg cgatctggag gacaaattac acacacttgc 1020

gcctgagcgc caagcacagg gttgttggtc ctcatattca cgaggtcgct gagagcacgg 1080

tgggctaatg ttgccatggg tagcatatac tacccaaata tctggatagc atatgctatc 1140

ctaatctata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 1200

ctaatctata tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc 1260

ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 1320

ctaatctgta tccgggtagc atatgctatc ctaatagaga ttagggtagt atatgctatc 1380

ctaatttata tctgggtagc atatactacc caaatatctg gatagcatat gctatcctaa 1440

tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagcatag gctatcctaa 1500

tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 1560

tttatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 1620

tctatatctg ggtagtatat gctatcctaa tctgtatccg ggtagcatat gctatcctca 1680

tgcatataca gtcagcatat gatacccagt agtagagtgg gagtgctatc ctttgcatat 1740

gccgccacct cccaaggggg cgtgaatttt cgctgcttgt ccttttcctg 1790

<210>9

<211>1926

<212>DNA

<213> unknown

<400>9

tcactcctgc ccttcctcac cctcatctcc atcacctcct tcatctccgt catctccgtc 60

atcaccctcc gcggcagccc cttccaccat aggtggaaac cagggaggca aatctactcc 120

atcgtcaaag ctgcacacag tcaccctgat attgcaggta ggagcgggct ttgtcataac 180

aaggtcctta atcgcatcct tcaaaacctc agcaaatata tgagtttgta aaaagaccat 240

gaaataacag acaatggact cccttagcgg gccaggttgt gggccgggtc caggggccat 300

tccaaagggg agacgactca atggtgtaag acgacattgt ggaatagcaa gggcagttcc 360

tcgccttagg ttgtaaaggg aggtcttact acctccatat acgaacacac cggcgaccca 420

agttccttcg tcggtagtcc tttctacgtg actcctagcc aggagagctc ttaaaccttc 480

tgcaatgttc tcaaatttcg ggttggaacc tccttgacca cgatgctttc caaaccaccc 540

tccttttttg cgcctgcctc catcaccctg accccggggt ccagtgcttg ggccttctcc 600

tgggtcatct gcggggccct gctctatcgc tcccgggggc acgtcaggct caccatctgg 660

gccaccttct tggtggtatt caaaataatc ggcttcccct acagggtgga aaaatggcct 720

tctacctgga gggggcctgc gcggtggaga cccggatgat gatgactgac tactgggact 780

cctgggcctc ttttctccac gtccacgacc tctccccctg gctctttcac gacttccccc 840

cctggctctt tcacgtcctc taccccggcg gcctccacta cctcctcgac cccggcctcc 900

actacctcct cgaccccggc ctccactgcc tcctcgaccc cggcctccac ctcctgctcc 960

tgcccctcct gctcctgccc ctcctcctgc tcctgcccct cctgcccctc ctgctcctgc 1020

ccctcctgcc cctcctgctc ctgcccctcc tgcccctcct gctcctgccc ctcctgcccc 1080

tcctcctgct cctgcccctc ctgcccctcc tcctgctcct gcccctcctg cccctcctgc 1140

tcctgcccct cctgcccctc ctgctcctgc ccctcctgcc cctcctgctc ctgcccctcc 1200

tgctcctgcc cctcctgctc ctgcccctcc tgctcctgcc cctcctgccc ctcctgcccc 1260

tcctcctgct cctgcccctc ctgctcctgc ccctcctgcc cctcctgccc ctcctgctcc 1320

tgcccctcct cctgctcctg cccctcctgc ccctcctgcc cctcctcctg ctcctgcccc 1380

tcctgcccct cctcctgctc ctgcccctcc tcctgctcct gcccctcctg cccctcctgc 1440

ccctcctcct gctcctgccc ctcctgcccc tcctcctgct cctgcccctc ctcctgctcc 1500

tgcccctcct gcccctcctg cccctcctcc tgctcctgcc cctcctcctg ctcctgcccc 1560

tcctgcccct cctgcccctc ctgcccctcc tcctgctcct gcccctcctc ctgctcctgc 1620

ccctcctgct cctgcccctc ccgctcctgc tcctgctcct gttccaccgt gggtcccttt 1680

gcagccaatg caacttggac gtttttgggg tctccggaca ccatctctat gtcttggccc 1740

tgatcctgag ccgcccgggg ctcctggtct tccgcctcct cgtcctcgtc ctcttccccg 1800

tcctcgtcca tggttatcac cccctcttct ttgaggtcca ctgccgccgg agccttctgg 1860

tccagatgtg tctcccttct ctcctaggcc atttccaggt cctgtacctg gcccctcgtc 1920

agacat 1926

<210>10

<211>468

<212>DNA

<213> Artificial sequence

<400>10

atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60

ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120

ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180

aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240

cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300

tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360

accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420

cctgggcaga aagctatact ttttcttcca atgtctgcta agagctga 468

<210>11

<211>546

<212>DNA

<213> Artificial sequence

<400> 11

atggggaaaa tcagcagtct tccaactcaa ttatttaaga tctgcctctg tgacttcttg 60

aagataaaga tacacatcat gtcgtcttca catctcttct acctggcact ctgcttgctc 120

acctttacca gctcggccac agccggacca gagacccttt gcggggctga gctggtggac 180

gctcttcagt tcgtgtgtgg accaaggggc ttttacttca acaagcccac aggctatggc 240

tccagcattc ggagggcacc acagacgggc attgtggatg agtgttgctt ccggagctgt 300

gatctgagga ggctggagat gtactgtgct ccgctgaagc ctacaaagtc agctcgttcc 360

atccgggccc agcgccacac tgacatgccc aagactcaga agtcccagcc cctatcgaca 420

cacaagaaaa ggaagctgca aaggagaagg aaaggtgagt caaaggcaca cccaggaggg 480

gaacaggagg agggagcaga ggcaactcag aaaatcagag gtgacagaga aaggaggccg 540

agctag 546

Claims (8)

1. A preparation method of a stem cell high-efficiency expression exosome for skin beauty is characterized by comprising the following steps:

separating stem cells from human tissues and culturing;

combining the PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed, a T2A sequence, a target protein sequence to be expressed, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; the target protein sequence 1 to be expressed and the target protein sequence 2 to be expressed are respectively arranged on the front side and the rear side of the T2A sequence, and the target proteins comprise oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, regenerated protein, transforming factor and insulin-like growth factor;

adding the recombinant expression vector into the culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode;

aseptically extracting exosomes from the supernatant of the dry cells after electrotransfection, adding 50 parts of the exosomes, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of Hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water according to parts by weight, mixing and stirring;

and (3) carrying out three-second instant freeze-drying dormancy on the mixed materials by adopting liquid nitrogen, and drying the mixed materials in a vacuum environment to sublimate and discharge the ice crystals in the freeze-dried tablets.

2. The method for preparing the exosome with high stem cell expression efficiency for skin beauty according to claim 1, wherein the stem cells separated from the human tissue comprise mesenchymal stem cells, adipose-derived stem cells and epithelial stem cells, and the human tissue comprises human umbilical cord tissue and adipose tissue.

3. The method for preparing the high-expression exosome for skin beauty according to claim 1, wherein the step of implementing the electrotransfection mode comprises the following steps:

1-2 days before the electrotransfection is carried out, transferring the stem cells to a cell culture flask, so that the cell confluence of the stem cells before the electrotransfection is 50% -70%;

slowly flushing the stem cells by using PBS, sucking out the PBS, digesting the stem cells by using pancreatin protease, and adding a culture medium containing serum to neutralize pancreatin;

the stem cells were harvested by centrifugation and resuspended at a density of 1X 10 using PBS, hepes and serum-free medium⁶ ～5×10⁶ cell/ml；

Setting electrotransfection parameters to be 2.0kV and 25 uFD;

adding the linearized plasmid of the recombinant expression vector into an electric rotor, and setting the initial concentration of the plasmid to be 10-50 mu g/ml according to the type of the stem cells;

adding the resuspended cells into an electric shock cup, turning the electric shock cup upside down and mixing uniformly;

putting the electric shock cup into an electric shock groove, and carrying out primary pulse electric shock;

transferring the shocked cells to a well containing a culture medium;

and (4) shaking the pore plate to uniformly mix the cells, and performing electrotransformation for 24-48 hours to realize instantaneous expression of cell genes.

4. The method for preparing the exosome for skin beauty according to claim 1, wherein when the target protein is oligopeptide-1 and basic fibroblast, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibroblast-CD 33-IRES-PURO, episome ORI-episome EBNA 1;

when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, and episome ORI-episome EBNA 1.

5. The method for preparing the exosome with high expression stem cell for skin beauty according to claim 2, wherein the step of separating the mesenchymal stem cell from the human umbilical cord tissue comprises the following steps:

separating to obtain human umbilical cord tissue blocks, and digesting for 5-10 minutes by using pancreatin;

collecting the tissue suspension, centrifuging, removing supernatant, and placing the tissue block in a plate;

adding a DMEM cell culture medium, and periodically changing the liquid by half;

removing the tissue mass after the cells grow over the plate;

after trypsinization, transferring the primary cells on the plate to a T75 culture flask;

and (5) when the cells grow over the culture bottle and passage is carried out to 2-3 generations, and the mesenchymal stem cells are obtained.

6. The method for preparing exosome efficiently expressed by stem cells for skin beauty according to claim 2, wherein the step of separating adipose stem cells from human adipose tissue comprises:

flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube;

adding equal volume of type I collagenase of 0.1-0.5% into adipose tissue, and digesting in 37 deg.C steam bath oscillator for 30-60 min;

when digestion is completed, the undigested adipose tissues are discarded, and the cell suspension is centrifuged and the supernatant is discarded;

the adipose-derived stem cell pellet was obtained at the bottom of the centrifuge tube and resuspended to a density of 1X 10⁵ mL^-1 ；

Inoculating the cell suspension into a cell culture bottle, and culturing by using a DMEM (DMEM) culture medium until the cell suspension grows to the fusion degree of more than 80%;

collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating into cell culture bottle, and culturing to obtain adipose-derived stem cells.

7. The method for preparing the exosome with the high expression stem cell for skin beauty according to claim 1, is characterized in that after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5-7 hours in a vacuum environment by using an ultra-vacuum freeze-drying box, so that ice crystals in the freeze-dried tablet are sublimated and discharged to obtain a loose and porous tablet disintegrating in water, and the loose and porous tablet is packaged by using a double-aluminum structure and then is stored in a dark place.

8. A product for beautifying skin by using the stem cell high-efficiency expression exosome, which comprises a tablet prepared by the preparation method for beautifying skin by using the stem cell high-efficiency expression exosome according to any one of claims 1 to 7.

Images