Preparation method and application of engineered exosome

Abstract

The invention discloses a preparation method and application of an engineered exosome, and relates to the technical field of biology. The invention provides a preparation method of an engineered exosome, which comprises the following steps: a carrier construction step: constructing CD63-VEGF into an FUGW carrier, and replacing a Ubc promoter in the FUGW carrier with EF1a to obtain FUGW (EF1a)-CD63-VEGF, and the sequence of the FUGW (EF1a)-CD63-VEGF is as shown in SEQ ID NO: 5. The engineering exosome capable of carrying the VEGF is successfully developed by utilizing a synthetic biological technology, and a thought is provided for development of exosome drugs and drugs taking the exosome as a delivery carrier. The engineered exosome carrying the VEGF has a remarkable effect on promoting hair growth.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of engineered exosomes. Background Art

[0002] Exosomes are extracellular vesicles with a diameter of 30-150nm and a phospholipid bilayer. They are membranous vesicles that are released outside the cell after the fusion of intracellular multivesicular bodies with the cell membrane. Exosomes contain many components, including proteins, lipids, mRNA, mitochondrial DNA, miRNA, and many other non-coding RNAs. According to proteomics studies, exosomes not only have specific proteins that depend on the secreting cell type, but also have specific subpopulations of cellular proteins found in exosomes that are independent of the cell type. Exosome components such as lipids, metabolites, functional proteins, and nucleic acids play an important role in intercellular and intracellular communication, and show great potential for using exosomes as drug delivery carriers. With the in-depth study of exosomes, it was found that in addition to being able to mediate the transmission of intercellular signals, exosomes play an important role in disease screening and diagnosis, disease treatment, and as drug delivery carriers.

[0003] All cells can produce exosomes, but different types of exosomes have different functions. For example, exosomes secreted by cancer cells can promote the spread and metastasis of cancer, while exosomes produced by stem cells can be used in medical cosmetology and immune regulation, can perform repair functions, and can be developed as drugs separately.

[0004] Exosomes are composed of a lipid membrane bilayer structure that expresses the surface ligands and receptors of donor cells. The exosome lipid membrane surrounds and contains a hydrophilic core. As a natural carrier, exosomes can target specific organs, protect drugs and prolong the circulation time of drugs, and mediate long-distance intercellular communication. Exosomes have the advantages of good biocompatibility, small size, and low immunogenicity. They can be used as natural carriers for small molecule drugs, transmembrane proteins, and nucleic acid drugs. There are two methods for loading drugs into exosomes: endogenous loading and exogenous loading. Endogenous loading is to load drug molecules into parent cells before isolating exosomes so that the exosomes secreted by them carry drug molecules. Exogenous loading is to first isolate exosomes and then load drug molecules directly onto the surface or cavity of the exosome membrane by passive or active means. Endogenous loading will not damage the exosome membrane. The most commonly used method is to incubate with engineered exosomes and donor cells. However, how to make exosomes carry specific gene expression products to further increase their functional effects and how to introduce drugs into exosomes are still problems that need to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of engineered exosomes.

[0006] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for preparing engineered exosomes, including a vector construction step, for constructing CD63-VEGF into a FUGW vector, and replacing the Ubc promoter in the FUGW vector with EF1a to obtain FUGW (EF1a)-CD63-VEGF, wherein the sequence of the FUGW (EF1a)-CD63-VEGF is shown in SEQ ID NO:5.

[0007] Specifically, the method for preparing engineered exosomes comprises the following steps: S1, vector construction; S2, lentiviral packaging; S3, lentiviral transfection of stem cells; S4. Exosome purification: collect the supernatant in the culture dish, perform the first centrifugation to collect the supernatant, perform the second centrifugation to collect the supernatant, filter, add purification reagent to the filtrate, mix well and centrifuge to collect the exosomes.

[0008] Specifically, the lentiviral packaging described in step S2 includes cell recovery, cell passaging, cell plasmid plasmid packaging steps.

[0009] Furthermore, the cells are selected from one or more of 293T cells, A549 cells, MCF-7 cells, HEL cells, HUVEC cells, and NIH3T3 cells; Furthermore, the cells are 293T cells.

[0010] According to some embodiments of the present invention, the cell recovery step is: placing the frozen cells in a 37°C water bath, and after thawing, aspirating the cells into a centrifuge tube containing cell culture medium, centrifuging at 800rpm for 5 minutes, discarding the supernatant, adding culture medium to resuspend the cells, inoculating the cells into a cell culture bottle containing culture medium, and placing it in a 37°C, 5% CO2 incubator for culturing.

[0011] According to some embodiments of the present invention, the cell passage step is as follows: when the cell density is greater than 80%, trypsin is added for digestion, and then cell culture medium is added to terminate the digestion, centrifuged at room temperature and 800 rpm for 5 min, the supernatant is discarded, and cell culture medium is added to resuspend the cells, and 1×10 6 The cells were transferred into culture flasks containing culture medium and cultured in a cell culture incubator at 37°C and 5% CO2.

[0012] According to some embodiments of the present invention, the cell plating step is: when the cell density is greater than 80%, trypsin is added for digestion, and then cell culture medium is added to terminate the digestion, centrifugation is performed, the supernatant is discarded, and the cells are resuspended in cell culture medium, and 4×10 6 The cells were transferred into culture flasks containing culture medium and cultured in a cell culture incubator at 37°C and 5% CO2.

[0013] According to some embodiments of the present invention, the lentiviral plasmid packaging includes plasmid FUGW (EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid.

[0014] Furthermore, the mass ratio of the FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2 and envelope plasmid is (1-5):(1-3):(1-3):1.

[0015] Furthermore, the mass ratio of the FUGW (EF1a) -CD63-VEGF, packaging plasmid 1, packaging plasmid 2 and envelope plasmid is 2:1:1:1.

[0016] According to some embodiments of the present invention, the packaging plasmid 1 is pLP1-KANA, the packaging plasmid 2 is pLP2-KANA, and the envelope plasmid is PVSVG-KANA.

[0017] Specifically, the lentiviral packaging comprises the following steps: (1) Mix FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid according to the ratio and add them into the culture medium, marked as A; (2) Add transfection reagent to the culture medium, denoted as B; (3) Mix A and B and add them to a culture dish for culture.

[0018] Furthermore, in step (2), the mass ratio of the transfection reagent to the total plasmid is 1:(1-3); Furthermore, in step (2), the mass ratio of the transfection reagent to the total plasmid is 1:2.

[0019] Furthermore, the culture medium in step (2) is selected from any one of OptiMEM culture medium, DMEM culture medium and RPMI culture medium.

[0020] Furthermore, the culture medium in step (2) is OptiMEM culture medium.

[0021] Furthermore, the transfection reagent described in step (2) is PEI.

[0022] Furthermore, the culture conditions in step (3) are 35-38°C and 4-8% CO2; Furthermore, the culture conditions in step (3) are 37°C, 5% CO 2。

[0023] Specifically, in step (3), after culturing for 36-48 hours, the supernatant in the culture dish is collected.

[0024] Furthermore, after culturing for 48 hours in step (3), the supernatant was collected after centrifugation at 4°C and 2000g for 10 minutes, filtered through a 0.45 μm filter membrane, and then centrifuged at 4°C and 20,000g for 2 hours to concentrate the lentivirus.

[0025] Specifically, in step S3, the lentiviral transfection of stem cells comprises the following steps: 1) Infect stem cells with lentiviral concentrate, add infection-promoting reagents, and culture in an incubator; 2) Add puromycin to the culture medium, culture in an incubator, and then perform subculture.

[0026] Furthermore, 1 ml of the lentiviral concentrate in step 1) infected 6*10 6 Stem cells; Furthermore, in step 1), the final concentration of the infection-promoting reagent is 5-10 μg / ml; further, in step 1), the final concentration of the infection-promoting reagent is 8 μg / ml.

[0027] Furthermore, the culture conditions in step 1) are 37° C., 5% CO 2 .

[0028] Furthermore, the culture time in step 1) is 24-48 hours, and the cells are cultured until the confluence reaches 80%.

[0029] Furthermore, the stem cells in step 1) are selected from any one of human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells; Furthermore, the stem cells in step 1) are human umbilical cord mesenchymal stem cells.

[0030] Furthermore, in step 2), the final concentration of puromycin is 1-5 μg / ml; further, in step 2), the final concentration of puromycin is 2 μg / ml.

[0031] Furthermore, the culture conditions described in step 2) are 37° C. and 5% CO 2 until the cell confluence reaches 50%.

[0032] Further, the conditions of the first centrifugation in step S4 are 3-8°C, 1000-3000g for 10-30min; Furthermore, the conditions for the first centrifugation in step S4 are 4° C. and 2000 g for 20 min.

[0033] Further, the conditions of the second centrifugation in step S4 are 3-8°C, 8000-12000g for 50-70min; Furthermore, the conditions for the second centrifugation in step S4 are 4° C. and 10,000 g for 60 min.

[0034] Furthermore, the purification reagent described in step S4 is PEG8000.

[0035] Furthermore, the final concentration of the purification reagent is 8-12%; further, the final concentration of the purification reagent is 10%.

[0036] Specifically, the purification reagent has a final concentration of 8-12% by mass volume.

[0037] In another aspect, the present invention provides engineered exosomes prepared by the above preparation method.

[0038] In yet another aspect, the present invention provides use of the above-mentioned engineered exosomes in the preparation of a product for promoting hair growth.

[0039] Specifically, the products include but are not limited to medicines.

[0040] Furthermore, the engineered exosomes are used as active ingredients in the drug.

[0041] Furthermore, the medicine includes a pharmaceutically acceptable carrier.

[0042] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0043] Furthermore, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form and a parenteral dosage form according to the administration method.

[0044] Furthermore, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0045] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, skin dosage forms, mucosal dosage forms and cavity dosage forms.

[0046] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0047] Furthermore, the respiratory tract administration dosage forms include but are not limited to sprays, aerosols and powder sprays.

[0048] Furthermore, the skin administration dosage forms include but are not limited to lotions, ointments, external solutions, plasters, pastes, and patches.

[0049] Furthermore, the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, sublingual tablets, and patches.

[0050] Furthermore, the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops and pills.

[0051] In yet another aspect, the present invention provides a product for promoting hair growth, wherein the product comprises the above-mentioned engineered exosomes.

[0052] Specifically, the products include but are not limited to medicines.

[0053] Furthermore, the engineered exosomes are used as active ingredients in the drug.

[0054] Furthermore, the medicine includes a pharmaceutically acceptable carrier.

[0055] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0056] Furthermore, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form and a parenteral dosage form according to the administration method.

[0057] Furthermore, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0058] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, skin dosage forms, mucosal dosage forms and cavity dosage forms.

[0059] According to some embodiments of the present invention, the present invention provides a method for preparing engineered exosomes, comprising a vector construction step, for constructing CD63-GFP into a FUGW vector, and replacing the Ubc promoter in the FUGW vector with EF1a to obtain FUGW (EF1a)-CD63-GFP, wherein the sequence of the FUGW (EF1a)-CD63-GFP is shown in SEQ ID NO:4.

[0060] Specifically, the method for preparing engineered exosomes comprises the following steps: (1) Vector construction; (2) Transfecting cells: Add FUGW (EF1a)-CD63-GFP to the culture medium, denoted as A; add the transfection reagent to the culture medium, denoted as B; mix A and B and add them to the culture dish for culture; (3) Exosome purification: Collect the supernatant in the culture dish, perform the first centrifugation to collect the supernatant, perform the second centrifugation to collect the supernatant, filter, add purification reagent to the filtrate, mix well and centrifuge to collect the exosomes.

[0061] Furthermore, in step (2), the mass ratio of FUGW(EF1a)-CD63-GFP to the transfection reagent is 1:(1-3); Furthermore, in step (2), the mass ratio of FUGW(EF1a)-CD63-GFP to the transfection reagent is 1:2.5.

[0062] Furthermore, the culture medium in step (2) is selected from any one of OptiMEM culture medium, DMEM culture medium and RPMI culture medium.

[0063] Furthermore, the culture medium in step (2) is OptiMEM culture medium.

[0064] Furthermore, the transfection reagent described in step (2) is PEIpro.

[0065] Furthermore, the culture conditions in step (2) are 35-38°C and 4-8% CO2; Furthermore, the culture conditions in step (2) are 37°C, 5% CO 2。

[0066] Specifically, in step (3), after culturing for 36-48 hours, the supernatant in the culture dish is collected.

[0067] Furthermore, the conditions for the first centrifugation in step (3) are 3-8°C, 1000-3000g for 10-30 min; Furthermore, the conditions for the first centrifugation in step (3) are 4°C and 2000g for 20 min.

[0068] Furthermore, the conditions for the second centrifugation in step (3) are 3-8°C, 8000-12000g for 50-70 min; Furthermore, the conditions for the second centrifugation in step (3) are 4°C and 10,000 g for 60 min.

[0069] Furthermore, the purification reagent described in step (3) is PEG8000.

[0070] Furthermore, the final concentration of the purification reagent is 8-12%; further, the final concentration of the purification reagent is 10%.

[0071] The beneficial effects of the present invention are: The present invention uses synthetic biology technology to successfully develop engineered exosomes that can carry specific gene expression products, which will provide ideas for the development of exosome drugs and drugs using exosomes as delivery carriers. The engineered exosomes carrying VEGF in the present invention have a significant effect on promoting hair growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a graph showing the transcription results of the target gene mRNA level. The control group in the figure is 293T cells, Ubc-GFP is 293T cells introduced with FUGW (Ubc) -GFP plasmid, EF1a-GFP is 293T cells introduced with FUGW (EF1a) -GFP plasmid, Ubc-CD63-GFP is 293T cells introduced with FUGW (Ubc) -CD63-GFP plasmid, and EF1a-CD63-GFP is 293T cells introduced with FUGW (EF1a) -CD63-GFP plasmid.

[0073] Figure 2 The fluorescence observation results of 293T cells expressing FUGW(EF1a)-GFP and FUGW(EF1a)-CD63-GFP plasmids are shown in Figure 2.

[0074] Figure 3 This is a WB detection of different exosome protein expression diagrams. In the figure, M is a marker. The control group is the exosomes produced by 293T cells, EF1a-GFP is the exosomes produced by 293T cells introduced with FUGW (EF1a)-GFP plasmid, Ubc-CD63-GFP is the exosomes produced by 293T cells introduced with FUGW (Ubc)-CD63-GFP plasmid, and EF1a-CD63-GFP is the exosomes produced by 293T cells introduced with FUGW (EF1a)-CD63-GFP plasmid.

[0075] Figure 4The expression of VEGF in engineered exosomes was detected by WB.

[0076] Figure 5 This is a comparison chart to verify the effects of engineered exosomes on growth at the animal level. DETAILED DESCRIPTION

[0077] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0078] Example 1 1. Vector construction GFP and CD63-GFP were constructed into FUGW vector using genetic engineering technology, and the Ubc promoter in the FUGW vector was replaced with EF1a. A total of four vectors were constructed, namely FUGW(Ubc)-GFP (SEQ ID NO: 1), FUGW(Ubc)-CD63-GFP (SEQ ID NO: 2), FUGW(EF1a)-GFP (SEQ ID NO: 3), and FUGW(EF1a)-CD63-GFP (SEQ ID NO: 4).

[0079] 2. Obtaining engineered exosomes carrying GFP (1) 293T cell recovery: One week in advance, take a tube of frozen 293T cells, quickly place it in a 37°C water bath, and shake it quickly until the cells melt. Disinfect the outer surface of the cryotube with 75% alcohol and transfer it to a biosafety cabinet. Use a 1 mL pipette to pipette the cells into a 15 mL centrifuge tube that has been pre-added with 5 mL of adherent 293T cell culture medium (DMEM basic containing 10% FBS). Centrifuge at room temperature, 800 rpm, for 5 min, discard the supernatant, add 1 mL of adherent 293T cell culture medium to resuspend the cells, take 300 μL of cells and inoculate them into a T25 cell culture flask that has been pre-added with 5 mL of adherent 293T cell culture medium, and transfer it to a 37°C, 5% CO2 cell culture incubator for culture.

[0080] (2) 293T cell culture: After 2 days of culture, when the cell density is greater than 80%, discard the culture medium, add 1 mL PBS to wash, repeat once, add 1 mL trypsin to digest at room temperature for 15 s, add 2 mL adherent 293T cell culture medium to terminate digestion, and transfer 3 mL of cell suspension to a 15 mL centrifuge tube. Centrifuge at room temperature, 800 rpm, for 5 min, discard the supernatant, add 1 mL adherent 293T cell culture medium to resuspend the cells, and take 100 μL of cell suspension for counting. According to the counting results, inoculate 1*106 cells into a T182 cell culture flask that has been added with 30 mL adherent 293T cell culture medium in advance, and transfer to a 37°C, 5% CO2 cell culture incubator for culture.

[0081] (3) 293T plating: After 2 days of culture, when the cell density is greater than 80%, discard the culture medium, add PBS for washing, add trypsin for digestion, add culture medium to terminate digestion, centrifuge and discard the supernatant, add culture medium to resuspend the cells, take 100 μL of cell suspension, and count. Based on the counting results, inoculate 2*10 6 The cells were transferred to a 10-cm dish pre-added with 10 mL of adherent 293T cell culture medium and cultured in a 37°C, 5% CO2 cell culture incubator.

[0082] (4) Transfection: After 1 day of culture, when the cell density is 80%, discard the supernatant and add 15 mL of adherent 293T cell culture medium. During transfection, take 8 μg of the constructed plasmid and add it to 500 μL OptiMEM, marked as tube A; 20 μg of PEIpro (with a mass ratio of 1:2.5 to the plasmid, that is, take 20 μL of 1 mg / mL stock solution) and add it to 500 μL OptiMEM, marked as tube B. After tubes A and B are allowed to stand at room temperature for 5 min, gently mix tubes A and B, let the mixed solution stand at room temperature for 15 min, then evenly add it dropwise to a 10 cm culture dish, and move the culture dish crosswise to mix the solution and culture medium evenly, and culture it in a 37°C, 5% CO2 cell culture incubator. From transfection to the recovery of the culture supernatant, the culture medium is not changed.

[0083] (5) Exosome purification (note that the operation should be performed on ice): After 36-48 h of culture, collect the supernatant from each dish into a 50 mL centrifuge tube (the supernatant from two dishes can be combined, i.e. 30 mL), place on ice and transfer to the cell compartment, centrifuge at 4°C, 2000 g for 20 min, and collect the supernatant into a new 50 mL centrifuge tube. Centrifuge at 4°C, 10,000 g for 60 min, collect the supernatant, and filter the supernatant into a new 50 mL centrifuge tube using a 0.22 μm filter membrane. Add 50% PEG 8000 to the filtrate to a final concentration of 10%, mix well, and let stand at 4°C overnight. The next day, centrifuge the treated solution at 4°C, 10,000 g for 60 min to collect exosomes. After centrifugation, a yellow-white precipitate can be seen on the wall of the centrifuge tube. Remove the supernatant, use a pipette to remove the residual supernatant, add 200 μL PBS to resuspend the precipitate, label the name and date, and store at -80°C. After resuspension, green fluorescence can be seen under a fluorescence microscope. Alternatively, 40 μL of sample can be taken and detected on a multifunctional microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The measured RFU value should be higher than that of the PBS control group.

[0084] 3 Verification of exosomes carrying GFP 3.1 Verification of target gene expression at the mRNA level and the effect of different promoters on target gene expression yield Different plasmids were transfected into 293T cells. After culturing for 36 h, the total RNA of the cells was extracted using the FastPure® Cell / TissueTotal RNA Isolation Kit V2 (Vazyme, Catalog No.: RC112-01). The steps for total RNA extraction were as follows: 293T cells were digested with trypsin, centrifuged at 1000 rpm for 5 min at room temperature, and the cells were collected; 3 x 10 6 Cells were added with 500 μl Buffer RL and vortexed until no obvious cell clusters were found. The subsequent steps were carried out according to the instructions for RNA extraction. Finally, 30-50 μl RNase-free ddH2O was added as needed. The RNA concentration was measured to be 777.7 - 1454.7 ng / μl; OD260 / OD280: 2.12 - 2.14, and the cells were stored at -80°C.

[0085] The transcription amount of different plasmids relative to the target gene was detected by qPCR using primers for detecting the CD63 gene. The experimental steps were as follows: Kit: 2× RealStar Fast SYBR qPCR Mix (UNG) (Genstar, Cat. No.: A302); Steps: Refer to the instructions, add the corresponding components according to the 20 μl system, and make 3 replicate wells for each sample; when adding Mix, be careful to avoid light; qPCR reaction procedure: Table 1 qPCR reaction procedure

[0086] Table 2 Related primers

[0087] Specific results such as Figure 1 As shown, from Figure 1 It can be seen that the introduced target gene was successfully transcribed and the EF1a promoter was stronger than the Ubc promoter.

[0088] 3.2 Observation of CD63-GFP fusion protein expression in 293T cells using fluorescence microscopy CD63 is a marker protein on the surface of exosomes, which belongs to the tetraspanin superfamily. If CD63-GFP is successfully expressed in 293T cells, fluorescence will be observed around the cell membrane under a fluorescence microscope, but no fluorescence will be found in the cytoplasm. By comparing the luminescence results of 293T cells containing FUGW(EF1a)-GFP and FUGW(EF1a)-CD63-GFP plasmids, it was found that the CD63-GFP fusion protein was successfully expressed on the surface of 293T cell membranes ( Figure 2 ).

[0089] 3.3 WB verification of engineered exosomes CD63 is a marker protein of exosomes. If the exosomes successfully contain the GFP target protein, the corresponding band can be observed by WB. The WB experimental results show that the obtained exosomes contain the GFP target protein ( Figure 3 ). As can be seen from the figure, exosomes containing GFP were successfully obtained, and the EF1a promoter was stronger than the Ubc promoter.

[0090] The specific process of WB detection is as follows: Preparation of glue: clamp two clean glass plates, fix them in a bracket, prepare 10% separation gel solution, add 5-7.5 ml of gel solution between the two glass plates, press the glue with anhydrous ethanol, pour out the ethanol after 30 minutes, and use filter paper to absorb the residual ethanol between the glass plates; then add 2-3 ml of concentrated gel solution, then insert a 10-hole comb between the glass plates, wait for 30-60 minutes until the protein gel solidifies, remove the glass plate, soak it in pure water, and store it at 4°C.

[0091] Table 3 Composition of concentration gel and separation gel

[0092] Take 200 μl of exosomes, add RIPA (containing a final concentration of 1 mM phenylmethylsulfonyl fluoride (PMSF)) at a ratio of 1:1, lyse on ice for 20 min, mix three times during the period; centrifuge (13000 g, 5 min, 4°C), take the supernatant, and measure the total protein concentration by BCA; WB sample preparation: Take 80 μl of the supernatant after RIPA lysis, add 20 μl of 5x loading buffer (without 2-mercaptoethanol), and then incubate at 100 °C for 10 min; Sample loading: 15 μl of marker was loaded. After the sample was quantified by BCA, the total protein amount loaded was 200-300 μg and the volume was 30-35 μl. If multiple samples were run at the same time, equal amounts and volumes should be taken into consideration. For example, sample B is 5 mg / ml and sample C is 3 mg / ml. After the samples are mixed with 5× loading buffer and heated at 100°C for 10 min, take 3.75 μl of sample B and add 6.25 μl of 1× loading buffer to mix; take 6.25 μl of sample C and add 3.75 μl of 1× loading buffer to mix, which can meet the requirement of "the total protein amount of the sample is 15 μg and the volume is 10 μl"; Gel running: Place the glass plate containing the protein gel in the electrophoresis tank, pour in 1× running buffer, set a constant voltage of 100 V for 100-120 min. Stop electrophoresis when bromophenol blue migrates to the bottom of the gel; Transfer: Open the mold, separate the black and white sides, place filter paper on the negative electrode side (black) of the mold, and place it in the transfer solution; take out the protein gel, cut off the concentrated gel and excess separation gel, place it on the filter paper on the negative electrode side (black) of the mold, and expel the bubbles; soak the PVDF membrane in methanol, activate it for 15 seconds, take it out and place it on the gel; put the filter paper on after expelling the bubbles, and after expelling the bubbles again, clamp the mold, and keep the constant current at 300 mA for 120 minutes; Blocking and washing: Soak the transferred PVDF membrane in 5% blocking solution and shake at room temperature at 60 rpm for 60 min; then wash the membrane once with TBST and shake at room temperature at 100 rpm for 5 min; Primary antibody (CD63 Antibody (MX-49.129.5) (mouse)) incubation and washing: Place the PVDF membrane in a container, add 10 ml of diluted antibody, shake at 4°C, 60 rpm, and incubate overnight. The next day, recover the primary antibody and wash the membrane three times with TBST at room temperature, 100 rpm, for 10 min each time; Secondary antibody (HRP-labeled goat anti-mouse IgG (H+L)) incubation and washing: Place the PVDF membrane in a container, add 10 ml of diluted antibody, and shake at room temperature at 60 rpm for 60 min; recover the secondary antibody and wash the membrane 3 times with TBST at room temperature at 100 rpm for 10 min each time.

[0093] Development: Mix the two liquids in the developer kit at a ratio of 1:1 (500 μl: 500 μl), evenly drip them on the membrane, and place it in a multifunctional imager for development and photography.

[0094] 4 Obtaining engineered exosomes carrying VEGF According to the method described in step 1, the VEGF gene was constructed into the determined expression vector using genetic engineering technology, namely FUGW (EF1a) -CD63-VEGF (SEQ ID NO: 5). Lentivirus packaging was performed according to the following steps: 4.1 Lentivirus packaging 4.1.1 293T cell recovery: Take 1 frozen 293T cell, quickly place it in a 37℃ water bath, and shake it quickly until the cells melt. Disinfect the outer surface of the cryopreserved tube with 75% alcohol and transfer it to a biosafety cabinet. Use a 1 ml pipette to draw the cells into a 15 ml centrifuge tube that has been pre-added with 5 ml of adherent 293T cell culture medium (DMEM culture medium containing 10% FBS). Centrifuge at room temperature, 800rpm, for 5 min, discard the supernatant, add 1 ml of adherent 293T cell culture medium to resuspend the cells, take 500 μl of cells and inoculate them into a T75 cell culture flask that has been pre-added with 12 ml of adherent 293T cell culture medium, and transfer it to a 37℃, 5% CO2 cell culture incubator for culture.

[0095] 4.1.2 293T cell culture: When the cell confluence is greater than 80% (24-48 h), discard the culture medium, add 3 ml PBS to wash, repeat once, add 2 ml trypsin to digest at room temperature for 15 s, add 2 ml adherent 293T cell culture medium to terminate digestion, transfer 4 ml cell suspension to a 15 ml centrifuge tube. Centrifuge at room temperature, 800 rpm for 5 min, discard the supernatant, add 1 ml adherent 293T cell culture medium to resuspend the cells, and take 100 μl of cell suspension for counting. According to the counting results, inoculate 1*10 6 The cells were transferred to a T182 cell culture flask in which 25 ml of adherent 293T cell culture medium had been added in advance, and then cultured in a 37°C, 5% CO2 cell culture incubator.

[0096] 4.1.3 293T plating: When the cell confluence is greater than 80% (24-48 h), discard the culture medium, refer to 4.1.2, add PBS for washing, add trypsin for digestion, add culture medium to terminate digestion, centrifuge and discard the supernatant, add culture medium to resuspend the cells, take 100μl of cell suspension, and count. Based on the counting results, inoculate 4*10 6 Place 100 cells into a 10 cm dish pre-added with 10 ml of adherent 293T cell culture medium. Repeat the procedure to prepare 4 dishes and transfer to a 37°C, 5% CO2 cell culture incubator for culture.

[0097] 4.1.4 Lentiviral plasmid packaging: 24 hours after plating, when the cell confluence is greater than 80%, perform the following operations on each dish.

[0098] The transfection operation was performed according to the plasmid ratio of FUGW-EF1a-CD63-VEGF: pLP1-KANA: pLP2-KANA: PVSVG-KANA = 2:1:1:1 (w / w / w / w). The total amount of plasmid required for each 10 cm culture dish during transfection was 25 μg (the specific volume of each plasmid added to each dish was calculated according to the ratio and concentration of each plasmid), added to 500 μl OptiMEM, marked as tube A; PEI 50 μg (the mass ratio of the required total plasmid was 1:2, that is, 50 μl of 1 mg / ml stock solution) was added to 500 μl OptiMEM, marked as tube B. After tubes A and B were allowed to stand at room temperature for 5 min, the solution in tube A was added to tube B and gently mixed. The mixed solution was allowed to stand at room temperature for 15 min, then evenly added dropwise to the 10 cm culture dish, and the culture dish was moved crosswise to mix the solution and culture medium evenly, and then cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium does not need to be changed from plating to recovery of culture supernatant, depending on the cell status (if medium change is required, it should be done 24 hours after plating and before transfection).

[0099] 4.1.5 Lentivirus collection: After 48 hours of culture, collect the supernatant from each dish into a 50 ml centrifuge tube, centrifuge at 4°C, 2000 g for 10 min, collect the supernatant, filter the supernatant into a 50 ml high-speed centrifuge tube using a 0.45 μm filter, balance it with an electronic balance, tighten the tube cap, centrifuge at 4°C, 20000 g for 2 hours to concentrate the lentivirus. After centrifugation, a white precipitate of virus can be seen on the centrifuge tube. Pour out the supernatant, and use a 200 ul pipette to remove the remaining supernatant. Add an appropriate amount of lentivirus preservation solution according to the amount of precipitated virus to resuspend the virus (4 10 cm culture dishes with a total of 40 ml culture supernatant, resuspend with 1 ml preservation solution; if used immediately, resuspend with 1 ml DMEM), gently blow evenly with a 1 ml pipette, transfer the concentrated virus solution to a 1.5 ml centrifuge tube and mark the name and date.

[0100] 4.1.5.1 Storage of lentivirus: Concentrated lentivirus solution can be stored at 4°C for 3 days or at -80°C.

[0101] 4.1.6 Human umbilical cord mesenchymal stem cells (hUC-MSC) lentiviral infection and culture supernatant collection (recovery on P3, infection on P4, screening and verification on P5, supernatant collection on P6-P10).

[0102] 4.1.6.1 Puromycin concentration: The final concentration of puromycin is 2 μg / ml.

[0103] 4.1.6.2 Lentivirus infection: 1 ml of virus concentrate was used to infect 6*10 6 hUC-MSC cells, as follows. Refer to 4.1.1, resuscitate and passage hUC-MSCs in advance, at 3*10 6 cells / 10 ml / 10 cm dish (2 dishes in total), subculture the cells into 10 cm dishes pre-added with 10 ml 3D FloTrix mesenchymal stem cell serum-free medium, and add 500 μl concentrated virus solution and 8 μg / ml final concentration of infection-promoting reagent Polybrene to each dish (8 μl of 10 mg / ml stock solution to each dish). After cross-movement and even mixing, culture in a 37°C, 5% CO2 cell culture incubator for 24-48 h until the cell confluence reaches 80%.

[0104] 4.1.6.3 Puromycin screening: Refer to 4.1.2, collect cells from two dishes, merge and subculture into a T182 culture flask. When subculture, add puromycin with a final concentration of 2 μg / ml to the culture medium (for example, 25 ml culture medium, add 50 μl 1 mg / ml puromycin), transfer to a 37℃, 5% CO2 cell culture incubator and culture for 48 h until the cell confluence is about 50%. At this time, hUC-MSC should be less than or equal to P6, and discard the supernatant of this round of culture.

[0105] 4.1.6.4 Passaging and collection of culture supernatant: Passage the cells after puro screening in the previous step to a new T182 culture flask without adding puromycin. Passage at a ratio of one to two, culture each generation for 48 - 72 h, collect the culture supernatant of P6 (20 ml), P7 (40 ml), P8 (80 ml), P9 (160 ml), and P10 (320 ml), centrifuge at 4°C, 3000 g, 20 min, retain the supernatant, and store temporarily at -20°C.

[0106] 4.1.7 Exosome purification (note that it is operated on ice): After 36-48 hours of culture, collect the supernatant of each dish into a 50 mL centrifuge tube, place it on ice and transfer it to the outside of the cell, centrifuge it at 4°C, 2000 g for 20 min, and collect the supernatant into a new 50 mL centrifuge tube. Collect the supernatant after centrifugation at 4°C, 10,000 g for 60 min, and filter the supernatant into a new 50 mL centrifuge tube using a 0.22 μm filter membrane. Add 50% PEG 8000 to the filtrate to a final concentration of 10%, mix well, and let it stand at 4°C overnight. The next day, centrifuge the treated solution at 4°C, 10,000 g for 60 min to collect exosomes. After centrifugation, a yellow-white precipitate can be seen on the wall of the centrifuge tube. Remove the supernatant, and use a pipette to remove the residual supernatant. Add 200 μL PBS to resuspend the precipitate, mark the name and date, and store it at -80°C.

[0107] The engineered exosomes carrying VEGF were verified as described in step 3. Figure 4 shown.

[0108] 5 Functional verification of engineered exosomes carrying VEGF After obtaining engineered exosomes carrying VEGF, animal experiments were conducted to verify whether they have a better hair growth effect. The verification was carried out according to the following experimental steps: 5.1 Experimental Grouping After 3 days of adaptation, C57BL6 mice (6 weeks old) were randomly divided into groups and photographed (before hair removal). The specific grouping is shown in Table 4 below: Table 4 Experimental groups

[0109] 5.2 Hair removal: After anesthesia, shave the back, use depilatory cream for 4 minutes, remove the hair, and then use depilatory cream for 2 minutes to remove all the hair on the back; the hair removal area is 0.5 cm behind the ear to 1 cm in front of the tail, about 4 cm long × 3 cm wide, and take pictures (after hair removal); 5.3 Administration: 1 ml syringe subcutaneously inject exosomes 50 μl × 4 points on the back, a total of 200 μl, 1.2 × 10 9 Number of particles; 5.4 Observation: Exosomes were observed regularly before and after administration. In each round of photography, the background was white, and the photography height, focus light source, light source direction and brightness were the same. The experimental results are shown in the figure below. Figure 5 As shown in the figure, it can be seen that engineered exosomes carrying VEGF can significantly promote hair growth.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing engineered exosomes, characterized in that: The method comprises a vector construction step, wherein CD63-VEGF is constructed into a FUGW vector, and the Ubc promoter in the FUGW vector is replaced with EF1a to obtain FUGW (EF1a)-CD63-VEGF, wherein the sequence of the FUGW (EF1a)-CD63-VEGF is shown in SEQ ID NO:

5.

2. The preparation method according to claim 1, characterized in that: The method for preparing engineered exosomes comprises the following steps: S1, vector construction; S2, lentiviral packaging; S3, lentiviral transfection of stem cells; S4. Exosome purification: collect the supernatant in the culture dish, perform the first centrifugation to collect the supernatant, perform the second centrifugation to collect the supernatant, filter, add purification reagent to the filtrate, mix well and centrifuge to collect the exosomes.

3. The preparation method according to claim 2, characterized in that: The lentiviral packaging described in step S2 includes cell recovery, cell passaging, cell plating and lentiviral plasmid packaging steps; The lentiviral packaging comprises the following steps: (1) Mix FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid according to the ratio and add them into the culture medium, marked as A; (2) Add transfection reagent to the culture medium, denoted as B; (3) Mix A and B and add them to a culture dish for culture.

4. The preparation method according to claim 3, characterized in that: The mass ratio of FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2 and envelope plasmid described in step (1) is (1-5):(1-3):(1-3):

1.

5. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of transfection reagent to total plasmid is 1:(1-3).

6. The preparation method according to claim 2, characterized in that: The purification reagent described in step S4 is PEG8000; the final concentration of the purification reagent is 8-12%.

7. The engineered exosomes prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the engineered exosomes according to claim 7 in the preparation of a product for promoting hair growth.

9. A product for promoting hair growth, characterized in that: The product includes the engineered exosomes described in claim 7.

10. The product according to claim 9, characterized in that The product is a medicine, and the medicine includes a pharmaceutically acceptable carrier.

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