A method for preparing functional mesenchymal stem cell exosomes by co-culturing, exosomes and applications thereof

Abstract

The present invention belongs to the field of biotechnology, and discloses a functional mesenchymal stem cell exosome prepared by co-culture, wherein the nanoparticle size of the exosome is 126nm, the particle size distribution is more uniform, there is only a single peak, the nanoparticle size of 126nm accounts for 100%, and the morphology is saucer-shaped and uniform. The umbilical cord mesenchymal stem cell supernatant exosomes obtained by the present invention maintain high activity and a relatively complete morphology, while the function of the exosomes is improved, and the yield of the exosomes is effectively increased. The present invention proposes a principle and application of thrombin combined with cytokines to intervene in the secretion of mesenchymal stem cell exosomes.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a principle affecting the secretion of exosomes in the supernatant of mesenchymal stem cells, a preparation method and an application thereof, in particular to a method for preparing functional mesenchymal stem cell exosomes by co-culturing, exosomes and an application thereof. Background Art

[0002] Exosomes, also known as exosomes or exosomes, are small vesicles wrapped in lipid molecules with a diameter of about 30-150nm secreted by living cells, with a density of about 1.13-1.19g / mL. They have a typical lipid bilayer structure and have lipid and lipid envelope components of the source cells. They are a specific subcellular structure rather than a simple fragment after cell lysis. They are cup-shaped under an electron microscope and exist in cell culture supernatants, serum, urine and other biological fluids. They contain a variety of important information components such as proteins, lipids, DNA, mRNA and micro RNA. The surface of all exosomes is basically distributed with common and iconic proteins, such as heat shock family proteins (hsp70, hsp90, etc.), histocompatibility complex molecules (MHC), transmembrane proteins (CD63, CD9, CD81, etc.) and tumor susceptibility gene 101 protein (TSG101). These proteins play a key connecting role in the transmission of substances and information between cells, and are expected to become iconic substances for the early diagnosis of various diseases. Stem cell-derived exosomes play an important role in tissue reconstruction, especially the function of proteins and RNA components contained in exosomes to communicate with damaged cells, which can regulate the biological functions of target cells and achieve the purpose of repair. Therefore, exosomes have been widely studied in regenerative medicine.

[0003] In recent years, researchers have linked exosomes to skin diseases and found that exosomes are not only involved in the physiological and pathological processes of the skin, such as regulating the secretion of pro-inflammatory cytokines in the skin microenvironment, promoting angiogenesis and collagen deposition in skin defects, and regulating the proliferation and differentiation of skin fibroblasts, but also play a specific role in information transmission when the skin microenvironment is diseased. Mesenchymal stem cell exosomes have been proven to have broad application prospects in many fields. Further exploration is still needed in the future, including the establishment of effective and feasible preparation methods and application methods. Exosomes are different from cells. They are more flexible and easier to transform. Therefore, exosomes can be used as a new direction for disease treatment research. At present, exosomes are mainly obtained directly from the culture supernatant by ultracentrifugation, but the amount of cells used is large and the amount of exosomes obtained is limited, which to a certain extent limits the preparation and application of exosomes. Therefore, it is necessary to find other efficient ways to obtain exosomes to overcome these limitations and understand the underlying reasons.

[0004] Prothrombin (Human Plasma) is a + A multifunctional serine protease with ion-induced allosteric properties, molecular formula C 63 H 88 CON 14 O 14 P, contains two polypeptide chains, A and B, connected by an interchain disulfide bond. Chain B is a functional chain with a typical serine protease folding structure, containing an active center located between two β-folding barrels, two external binding sites (Exosite I, Exosite II), and a Na + From the ion binding site, there is an autolysis loop and a W60d loop, with Na + Ion-induced allosteric enzyme properties, in the presence of Na + There are two conformations, ion-bound and Na+ ion-dissociated, which can be converted into each other. Part of the mechanism of their conversion is that the effector or substrate binds to the specific site of thrombin, causing changes in the enzyme conformation and energy transfer, thereby changing the equilibrium state of Na+ ion binding and dissociation. It has anti-coagulation function and can activate receptors to produce cell biological effects, such as inducing inflammation, inducing cells to release cytokines, causing cell proliferation and other reactions.

[0005]

[0006] Skin is the largest organ of the human body and the outermost structure of the human body. The skin covers the whole body. The skin can protect various tissues and organs in the body from mechanical, physical, pathogenic microbial and chemical invasions, and has a protective effect on the body. The skin also has the function of wound healing. When the body is subjected to external forces, the skin and skin tissue of the human body can regenerate, which requires the cooperation of multiple cells and tissues, and includes the joint action of cell proliferation, migration, matrix synthesis, skin contraction and growth factor-related signal processes. Although wound healing is still roughly understood, with the continuous deepening of research, the research on which signal pathways play a leading role in the healing process has been gradually deepened. Most wound healing processes can be healed in 1-2 weeks, but for some difficult-to-heal chronic wounds, they may not heal for a long time. For example, people with diabetes and long-term bedridden patients experience the pain of chronic wound healing. Therefore, how to solve the problem of chronic wound healing has become an important issue in current research. In recent years, a large number of literature reports have pointed out that stem cells can treat the ability of chronic wound healing. Some literature also pointed out that stem cell exosomes play a decisive role in the treatment process, but the specific mechanism is still unclear. Therefore, it is urgent to study the mechanism that affects the secretion of mesenchymal stem cell exosomes and how to efficiently obtain mesenchymal stem cell exosomes and apply them to skin injury repair. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing functional mesenchymal stem cell exosomes by co-culturing, exosomes and applications thereof.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A functional mesenchymal stem cell exosome prepared by co-culture, wherein the nanoparticles of the exosomes have a particle size of 126 nm, a more uniform particle size distribution, only a single peak, particles with a nanoparticle size of 126 nm account for 100%, and the morphology is saucer-shaped and uniform.

[0010] The method for preparing functional mesenchymal stem cell exosomes by co-culturing the exosomes as described above comprises the following steps:

[0011] (1) Cell culture

[0012] The primary frozen mesenchymal stem cells were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2The cells were inoculated at a density of 1000 μg / mL in a cell culture flask, and co-cultured with DMEM / F12, 10% serum, and 1% double antibody for 24 h before transfection with miRNA. After 5 h of transfection, the culture medium was replaced with DMEM / F12 medium, 5% serum, and 1% double antibody in a volume of 22 ml / T175, and interleukin was added in an amount of IL-1 1000 U / 22 mL culture medium and IL-18 1000 U / 22 mL culture medium. The cells were co-cultured at 37°C and 5% CO2.

[0013] (6) After 24 hours of co-culture, when the cell growth density reached 70%, the culture medium was removed, the cells were washed twice with PBS, and serum-free DMEM-F1222 ml and thrombin 100 U / T175 / 22 ml were added for starvation culture;

[0014] (7) Starvation culture for 48 h, collecting co-cultured mesenchymal stem cells and cell culture supernatant;

[0015] (2) Exosome extraction

[0016] The cell culture supernatant was centrifuged at 400 g, 9 rpm, 9 rpm, for 10 min, and the supernatant was transferred; at 4°C, 2000 g, 9 rpm, 9 rpm, 20 min, and the supernatant was transferred; at 4°C, 10000 g, 9 rpm, 9 rpm, 30 min, and the supernatant was transferred. The cell debris in the supernatant was effectively removed by the above centrifugation process;

[0017] After centrifugation, filter once with a 0.45 μm filter membrane and once with a 0.22 μm filter membrane to remove residual cell debris and large particles in the liquid. Ultracentrifuge for 70 min at 4°C, 29800 r, increase speed by 100 r, decrease speed by 100 r, discard the supernatant, resuspend the precipitate with PBS, collect it, repeat the ultracentrifugation operation twice, resuspend the precipitate with PBS, and obtain exosomes.

[0018] Furthermore, the specific steps of transfection in step (1) are:

[0019] Add 500 μl of serum-free DMEM-F12 and 18 μl of transfection reagent to each 1.5 ml centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a transfection reagent-medium mixture;

[0020] At the same time, add 500 μl of serum-free basal medium to another 1.5 ml centrifuge tube, add 300 pmol of miR-1291 oligo, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a miR-1291 oligo-medium mixture;

[0021] Add the transfection reagent-medium mixture dropwise to the miR-1291 oligo-medium mixture, mix gently with a pipette, and let stand at room temperature for 18 min;

[0022] The culture medium in the culture flask was replaced with serum-free DMEM-F12, and the mixture was allowed to stand at room temperature for 18 min, and then immediately added to the cell culture medium for transfection.

[0023] The use of the exosomes as described above in the preparation of drugs that promote skin damage repair.

[0024] The use of exosomes as described above in the preparation of regenerative medicine and targeted therapy drugs.

[0025] The use of the exosomes as described above in the preparation of a drug for promoting full-thickness skin healing.

[0026] The use of the exosomes as described above in the preparation of drugs for promoting skin tissue regeneration and collagen formation.

[0027] The advantages and positive effects achieved by the present invention are:

[0028] 1. The differentially expressed miRNA obtained by gene sequencing in the present invention intervenes in the culture of mesenchymal stem cells, and stimulates mesenchymal stem cells under the combined action of thrombin and interleukin, which can significantly increase the yield of exosomes in the supernatant of mesenchymal stem cell culture, and maintain a high activity and a relatively complete morphology, while the function of the exosomes is improved. The present invention determines the action principle of the VEGF signaling pathway of miR-1291 affecting the secretion of exosomes by mesenchymal stem cells by WB and fluorescent quantitative PCR, and obtains more exosomes under the joint action of thrombin and interleukin, which is 5 times higher than the exosomes of normally cultured cells; and the exosomes derived from stem cells are applied in the process of skin damage repair, showing that the function of exosomes is improved, and the functional exosomes prepared by the present invention can further promote the wound healing speed. By clarifying the regulatory mechanism of exosomes in the supernatant of mesenchymal stem cells in tissue regeneration, the present invention can prepare customized exosomes with specific targets for efficient and precise medicine.

[0029] 2. The exosomes in the supernatant of umbilical cord mesenchymal stem cells obtained by the present invention maintain high activity and a relatively complete morphology. At the same time, the function of the exosomes is improved, and the output of the exosomes is effectively increased. The present invention proposes a principle and application of thrombin combined with cytokines to intervene in the secretion of mesenchymal stem cell exosomes.

[0030] 3. The functional exosomes prepared by the present invention are applied to the process of skin damage repair. After damage repair, the average fluorescence intensity of keratin CK14 is 2 times that of the model group and 1.45 times that of traditional exosomes, which not only increases the yield but also improves the function. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram for identifying overexpression of miR-1291 in the present invention;

[0032] Figure 2 This is the expression diagram of related genes in mesenchymal stem cells after co-culture of miR-1291 thrombin and interleukin in the present invention;

[0033] Figure 3 This is a graph showing changes in the expression of related proteins in mesenchymal stem cells after co-culture of miR-1291 thrombin and interleukin in the present invention;

[0034] Figure 4 The transmission electron microscopy and nanoparticle size identification results of the exosomes in the present invention; wherein, A: particle size of traditional exosomes, B: particle size of exosomes prepared in the present invention, C: electron microscopy of traditional exosomes, D: electron microscopy of exosomes prepared in the present invention, and E: Western Blot identification results;

[0035] Figure 5 This is a schematic diagram of the exosome production in the present invention;

[0036] Figure 6 Schematic diagram of the results of promoting cell migration in the present invention (comparison diagram of scratch test results);

[0037] Figure 7 This is a schematic diagram of the statistical results of the skin damage repair rate of each group of mice in the supernatant exosomes of mesenchymal stem cells of the present invention;

[0038] Figure 8 Schematic diagram of the skin damage repair of each group of exosomes in the supernatant of mesenchymal stem cells of the present invention (top) and schematic diagram of the final results of the skin damage repair of each group of mice (bottom);

[0039] Fig. 9 Schematic diagram of HE staining detection of wound pathology in each group of the present invention; wherein, A: normal mouse skin (10×), B: injured mouse skin (10×), C: injured mouse skin + exosomes (10×), D: injured mouse skin + thrombin-stimulated exosomes (10×), E: normal mouse skin (4×), F: injured mouse skin (4×), G: injured mouse skin + exosomes (4×), H: injured mouse skin + thrombin-stimulated exosomes (4×);

[0040] Fig.10 Schematic diagram of the Masoon staining test results of each group of wounds in the present invention; wherein, A: normal mouse skin (10×), B: injured mouse skin (10×), C: injured mouse skin + exosomes (10×), D: injured mouse skin + thrombin-stimulated exosomes (10×);

[0041] Fig.11 Schematic diagram of CK14 immunohistochemical detection results of wound surfaces in each group of the present invention; wherein the upper figure is an immunofluorescence imaging statistical diagram, and the lower figure is a data result diagram after Image statistics;

[0042] Fig.12 This is a schematic diagram of the results of the Collagen I immunohistofluorescence detection and chemical detection of wound surfaces in each group of the present invention.

[0043] Fig.13 This is a diagram for verifying the selection of thrombin dosage in the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0045] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0046] A functional mesenchymal stem cell exosome prepared by co-culture, wherein the nanoparticles of the exosomes have a particle size of 126 nm, a more uniform particle size distribution, only a single peak, particles with a nanoparticle size of 126 nm account for 100%, and the morphology is saucer-shaped and uniform.

[0047] The method for preparing functional mesenchymal stem cell exosomes by co-culturing the exosomes as described above comprises the following steps:

[0048] (1) Cell culture

[0049] The primary frozen mesenchymal stem cells were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2 The cells were inoculated at a density of 1000 μg / mL in a cell culture flask, and co-cultured with DMEM / F12, 10% serum, and 1% double antibody for 24 h before transfection with miRNA. After 5 h of transfection, the culture medium was replaced with DMEM / F12 medium, 5% serum, and 1% double antibody in a volume of 22 ml / T175, and interleukin was added in an amount of IL-1 1000 U / 22 mL culture medium and IL-18 1000 U / 22 mL culture medium. The cells were co-cultured at 37°C and 5% CO2.

[0050] (6) After 24 hours of co-culture, when the cell growth density reached 70%, the culture medium was removed, the cells were washed twice with PBS, and serum-free DMEM-F1222 ml and thrombin 100 U / T175 / 22 ml were added for starvation culture;

[0051] (7) Starvation culture for 48 h, collecting co-cultured mesenchymal stem cells and cell culture supernatant;

[0052] (2) Exosome extraction

[0053] The cell culture supernatant was centrifuged at 400 g, 9 rpm, 9 rpm, for 10 min, and the supernatant was transferred; at 4°C, 2000 g, 9 rpm, 9 rpm, 20 min, and the supernatant was transferred; at 4°C, 10000 g, 9 rpm, 9 rpm, 30 min, and the supernatant was transferred. The cell debris in the supernatant was effectively removed by the above centrifugation process;

[0054] After centrifugation, filter once with a 0.45 μm filter membrane and once with a 0.22 μm filter membrane to remove residual cell debris and large particles in the liquid. Ultracentrifuge for 70 min at 4°C, 29800 r, increase speed by 100 r, decrease speed by 100 r, discard the supernatant, resuspend the precipitate with PBS, collect it, repeat the ultracentrifugation operation twice, resuspend the precipitate with PBS, and obtain exosomes.

[0055] Preferably, the specific steps of transfection in step (1) are:

[0056] Add 500 μl of serum-free DMEM-F12 and 18 μl of transfection reagent to each 1.5 ml centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a transfection reagent-medium mixture;

[0057] At the same time, add 500 μl of serum-free basal medium to another 1.5 ml centrifuge tube, add 300 pmol of miR-1291 oligo, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a miR-1291 oligo-medium mixture;

[0058] Add the transfection reagent-medium mixture dropwise to the miR-1291 oligo-medium mixture, mix gently with a pipette, and let stand at room temperature for 18 min;

[0059] The culture medium in the culture flask was replaced with serum-free DMEM-F12, and the mixture was allowed to stand at room temperature for 18 min, and then immediately added to the cell culture medium for transfection.

[0060] The use of the exosomes as described above in the preparation of drugs that promote skin damage repair.

[0061] The use of exosomes as described above in the preparation of regenerative medicine and targeted therapy drugs.

[0062] The use of the exosomes as described above in the preparation of a drug for promoting full-thickness skin healing.

[0063] The use of the exosomes as described above in the preparation of drugs for promoting skin tissue regeneration and collagen formation.

[0064] Specifically, the relevant preparation and detection are as follows:

[0065] 1. Cell Culture

[0066] (I) Co-culture of mesenchymal stem cells with miRNA, thrombin and interleukin

[0067] The definitions of some products involved in the present invention are as follows:

[0068] MSCs 1: Mesenchymal stem cells (MSCs 1) were collected from the control group

[0069] Stock solution 1: Cell culture supernatant collected from the control group (stock solution 1)

[0070] EV1: The supernatant exosomes extracted from the original solution 1 collected from the control group by ultracentrifugation are EV1

[0071] MSCs 2: Mesenchymal stem cells (MSCs 2) were collected from the co-culture group

[0072] Stock solution 2: Culture supernatant of mesenchymal stem cells collected from the combined culture group (stock solution 2)

[0073] EV2: The supernatant exosomes extracted from the stock solution 2 collected from the combined culture group by ultracentrifugation are EV2

[0074] 1. Collection of cells and supernatant from the control group

[0075] The primary frozen mesenchymal stem cells (cells purchased from the National Biomedical Experimental Cell Resource Bank, resource code 3101HUMSCSP403) were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a T175 cell culture flask and co-cultured with DMEM / F12, 10% serum and 1% double antibody for 24 h. The culture medium was replaced and co-cultured with DMEM / F12 and 1% double antibody for 72 h. Then, mesenchymal stem cells (MSCs 1) and cell culture supernatant (stock solution 1) were collected and compared with the cells and supernatant collected under the co-culture conditions.

[0076] 2. Cells and supernatants of the co-culture experimental group

[0077] The primary frozen mesenchymal stem cells (cells purchased from the National Biomedical Experimental Cell Resource Bank, resource code 3101HUMSCSP403) were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2 The cells were inoculated at a density of 1.50 μg / mL in a T175 cell culture flask and co-cultured with DMEM / F12, 10% serum, and 1% double antibody for 24 h before being transfected with miRNA. The miRNA was synthesized by a designated company, and the specific primer sequences are shown in Table 1.

[0078] Table 1 Primer sequences of miR-1291 constructed in the present invention

[0079]

[0080] The specific transfection steps are:

[0081] (1) Add 500 μl serum-free DMEM-F12 and 18 μl GP-transfect-mate transfection reagent to a 1.5 ml centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min;

[0082] (2) At the same time, add 500 μl of serum-free basal medium to another 1.5 ml centrifuge tube, add 300 pmol of miR-1291 oligo, mix gently with a pipette, and let stand at room temperature for 5 min;

[0083] (3) Add the GP-transfect-mate-medium mixture dropwise to the miR-1291 oligo-medium mixture, mix gently with a pipette, and let stand at room temperature for 18 min;

[0084] (4) The culture medium in the culture flask was replaced with serum-free DMEM-F12. After the mixture was allowed to stand at room temperature for 18 min, the mixture was immediately added to the cell culture medium for transfection;

[0085] (5) Five hours after transfection, the culture medium (DMEM / F12 medium, 5% serum, 1% double antibody) was replaced and interleukin (IL-11000U, IL-181000U) was added, and co-cultured at 37°C and 5% CO2.

[0086] (6) After 24 hours of co-culture, when the cell growth density reached about 70%, the culture medium was removed, the cells were washed twice with PBS, and serum-free DMEM-F12 and 100 U / T175 thrombin were added for starvation culture. The amount of thrombin was selected based on time-dependent and dose-dependent experiments.

[0087] (7) After starvation culture for 48 h, the co-cultured mesenchymal stem cells (MSCs 2) and the cell culture supernatant (stock solution 2) were collected.

[0088] The miR-1291 constructed by using the primer sequences in Table 1 in the present invention was qualified after identification. After transfection of mesenchymal stem cells with 18 μL of transfection reagent and 300 pmol of miR-1291 was overexpressed, Figure 1 In the experiment, the relative expression of miR-1291 mimics was 1635.26, and the relative expression of NC mimics was 1.15, indicating that the overexpression experiment was successful. Then, the effect of miR-1291 on the VEGF signaling pathway of exosomes secreted by mesenchymal stem cells was studied.

[0089] 3. Thrombin dosage time-dependent and dose-dependent experiments

[0090] The dosage of thrombin is selected through time-dependent and dose-dependent experiments. The specific experimental steps are as follows:

[0091] The primary frozen mesenchymal stem cells (cells purchased from the National Biomedical Experimental Cell Resource Bank, resource code 3101HUMSCSP403) were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2 The cells were inoculated at a density of 100 μg / mL in a T175 cell culture flask and co-cultured with DMEM / F12, 10% serum and 1% double antibody for 24 h. The culture medium was replaced and the dosage of thrombin was selected as 50 U / T175, 100 U / T175 and 200 U / T175, respectively. The cells were co-cultured with DMEM / F12 and 1% double antibody for 24 h, 48 h and 72 h. The cell culture supernatant was collected and exosomes were extracted from the supernatant by ultracentrifugation to compare the exosome secretion amount.

[0092] The results are as follows Fig.13 As shown, from Fig.13 It can be seen that after 72 hours of starvation, the extracted exosomes of the normal control group were 134.22±36.784μg, 50U / T175 were 292.33±43.34, 100U / T175 were 646.96±42.96, 200U / T175 were 740.11±14.01, and thrombin can be selected as 100U / T175 and 200U / T175; after 72 hours of starvation, the extracted exosomes of the normal control group were 137.52±15.54μg, 50U / T175 were 348.81±25.32, 100U / T175 were 769.52±13.59, 200U / T175 were 774.19±31.81, and thrombin can be selected as 100U / T175 and 200U / T175. Based on the above experimental results and experimental costs, through the time-dependent and dose-dependent experiments of thrombin dosage, it can be determined that the optimal dosage of thrombin is 100U / T175, and the action time can be selected as 24h or 48h.

[0093] (II) Effect of co-culture on exosome secretion in cells

[0094] WB and fluorescence quantitative PCR were used to investigate the expression of genes and proteins that promote cell secretion of exosomes, and exosomes were obtained by low-temperature ultrahigh-speed centrifugation.

[0095] 1. Specific experimental steps of WB

[0096] (1) Sample preparation: The collected mesenchymal stem cells MSCs1 and MSCs2 were washed three times with PBS, mixed at a ratio of 1 ml of cell lysis buffer plus 1 μl of a universal 100X protease inhibitor cocktail (P1003), and shaken at 0-4°C for 30 min. After sufficient lysis, the upper suspension was obtained by centrifugation at 4°C and 12,000 rpm for 5 min. The protein content was determined by BCA protein quantification method. The protein concentration of the suspension was diluted to 1 μg / μl, and 100 μl of the suspension was mixed with 20 μl of 5X loading buffer, boiled in boiling water for 15 min, and immediately placed on ice or stored at -20°C.

[0097] (2) Electrophoresis: The electrophoresis time is generally 2 to 3 hours, the voltage is 80V, 30 minutes; 120V, 1 hour, run until the DNA marker is completely separated, then start transfer to the membrane;

[0098] (3) Transfer: Place the clips correctly into the transfer tank and transfer at 200 mA for 1.5 h;

[0099] (4) Blocking: After transfer, stain with Ponceau red, cut the membrane according to molecular weight, wash twice with ddH2O, and block with 5% skim milk powder at 25°C for 60 min;

[0100] (5) Antibody incubation: The primary antibody was diluted 1:1000, incubated at 37°C for 120 min or at 4°C overnight, and washed with TBST 5 times for 10 min. The secondary antibody was incubated at room temperature for 60 min, and washed with TBST 5 times for 10 min.

[0101] (6) Development and analysis of results: After fully mixing ECL colorimetric solution A and B, drop them on the membrane, place it in a multifunctional imaging device, and take a picture. Use image processing software to systematically analyze the protein molecule expression level of each band of Western Blot.

[0102] 2. Fluorescence quantitative PCR method steps (1) Extraction of RNA

[0103] The collected mesenchymal stem cells MSCs1 and MSCs2 were washed 3 times with PBS, added with Triol, lysed, transferred into EP tubes, and chloroform (1 / 5 of the total volume of Trizol) was added, mixed by oscillation for 30 seconds, and allowed to stand at room temperature for 3 minutes;

[0104] Centrifuge at 4°C, 12000 g for 15 min, and transfer the supernatant to a new centrifuge tube;

[0105] An equal volume of isopropanol was added to the supernatant, mixed by oscillation for 30 seconds, and allowed to stand at room temperature for 10 minutes; centrifuged at 12000 g for 10 minutes at 4°C;

[0106] Carefully discard the supernatant, add 1 mL of pre-cooled 75% alcohol to the centrifuge tube, vortex to mix, and centrifuge at 7500 g at 4°C for 5 min;

[0107] Dissolve RNA precipitate with appropriate amount of ddH2O;

[0108] The RNA concentration was measured by microplate reader.

[0109] (2) Preparation of gene-specific reverse transcription reaction system

[0110] Mix the following reagents in an RNase-free centrifuge tube for pre-denaturation:

[0111] Reagents Final concentration Dosage hole RTprimer(1uM) 60nM 1.2uL RNASample 1ug 2uL

[0112] After heating at 70℃ for 5 minutes, place on ice immediately.

[0113] Mix other reagents according to the 20uL reverse transcription reaction system provided in the table below

[0114]

[0115]

[0116] Mix slowly, incubate at 26°C for 40 min, 42°C for 40 min, 85°C for 10 min, incubate at 4°C until reverse transcription is complete, and store at 4°C.

[0117] (3) Establishment of 20uL system of dye-based quantitative PCR

[0118] Reagents Final concentration Dosage hole 2×Real-timePCRMasterMix(SYBR) 1× 10uL PCR primer set (10uM) 0.2uM 0.4uL TaqDNApolymerase (5U / uL) 1U 0.2uL RTproduct 2uL <![CDATA[RNase-freeH2O]]> To20ul

[0119] Real-time quantitative PCR one-step amplification:

[0120] ①95℃3min 1× cycle

[0121] ②95℃12s

[0122] ③62℃40s

[0123] ②-③ were cycled 40 times, the fluorescence signals were collected, and photos were taken after each cycle.

[0124] Since thrombin stimulates cells to produce many different biological effects, such as chemotaxis, cell proliferation, extracellular matrix synthesis and cytokine release, it can stimulate platelets to release many cytokines, such as PDGFB, TGF-β, etc. The protein encoded by the SPHK1 gene catalyzes the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), a lipid mediator with intracellular and extracellular functions. This protein and its product S1P play a key role in VEGF signal transduction and in inflammation, anti-apoptosis and immune processes. Figure 2 Compared with the NC transfection group, the expression of three genes, PLA2G4C, PDGFB, and SPHK1, in the miR-1291mimics transfected cells was significantly increased. Compared with the miR-1291inhibitor transfection group, the expression of the four genes in the miR-1291inhibitor transfection group did not change significantly. Figure 3 In the experiment, transfection of miR-1291 significantly increased the expression of PLA2G4C and PDGFB proteins and inhibited the expression of SPHK1 protein. It can be concluded that miR-1291 can significantly promote the expression of the three genes and may be able to change the regulatory mechanism of the three genes.

[0125] 2. Exosome Extraction and Identification

[0126] (I) Extraction of exosomes

[0127] The cell culture supernatant solution 1 and solution 2 were centrifuged at 400 g, 9 rpm, 9 rpm, and centrifuged for 10 min, and the supernatant was transferred; at 4°C, 2000 g, 9 rpm, 9 rpm, and centrifuged for 20 min, and the supernatant was transferred; at 4°C, 10000 g, 9 rpm, 9 rpm, and centrifuged for 30 min, and the supernatant was transferred. The cell debris in the supernatant was effectively removed through the above centrifugation process.

[0128] After centrifugation, filter once with a 0.45μm filter membrane and once with a 0.22μm filter membrane to remove residual cell debris and large particles in the liquid. Ultracentrifuge for 70min at 4°C, 29800r, increase speed by 100r, decrease speed by 100r, and discard the supernatant. Resuspend the precipitate in the ultracentrifuge tube with 10mL PBS, collect it in an ultracentrifuge tube, repeat the operation twice, resuspend the precipitate with PBS, take a small amount of exosomes and determine the protein content by the BCA method, extract the morphology and particle size of the exosomes by transmission electron microscopy and nanoparticle size detection, and obtain exosomes EV1 and EV2.

[0129] (II) Identification of exosomes

[0130] 1. Western Blot Identification of the Expression of Exosome Surface Markers

[0131] (1) Sample preparation: The protein content of the obtained exosomes EV1 and EV2 was determined by BCA protein quantification method. The protein concentration of the exosomes was diluted to 1 μg / μl with cell lysis buffer, and 100 μl of the suspension was mixed with 20 μl of 5X loading buffer, boiled in boiling water for 15 min, and immediately placed on ice or stored at -20°C;

[0132] (2) Electrophoresis: The electrophoresis time is generally 2 to 3 hours, the voltage is 80V, 30 minutes; 120V, 1 hour, run until the DNA marker is completely separated, then start transfer to the membrane;

[0133] (3) Transfer: Place the clips correctly into the transfer tank and transfer at 200 mA for 1 hour;

[0134] (4) Blocking: After transfer, stain with Ponceau red, cut the membrane according to molecular weight, wash twice with ddH2O, and block with 5% skim milk powder at 25°C for 60 min;

[0135] (5) Antibody incubation: The primary antibody was diluted 1:1000, incubated at 37°C for 120 min or at 4°C overnight, and washed with TBST 5 times for 10 min. The secondary antibody was incubated at room temperature for 60 min, and washed with TBST 5 times for 10 min.

[0136] (6) Development and analysis of results: After fully mixing ECL colorimetric solution A and B, drop them on the membrane, place it in a multifunctional imaging device, and take a picture. Use image processing software to systematically analyze the protein molecule expression level of each band of Western Blot.

[0137] 2. Exosomes Transmission Electron Microscopy Identification Steps

[0138] (1) Dilute the extracted exosomes to 0.5 μg / μL, add 5 μL onto a 200-mesh carbon film copper grid, and allow the carbon film to absorb in a dry environment. Repeat this process twice;

[0139] (2) Fix the sample with 100 μL of 2.5% glutaraldehyde at room temperature for 12 min, and wash the copper mesh with distilled water five times, 30 s each time;

[0140] (3) Use filter paper to absorb excess water around the copper mesh;

[0141] (4) Add 50 μL of acetic acid hydrogen peroxide glaze staining solution and stain at room temperature for 17 min;

[0142] (5) Use paper to absorb the moisture around and on the copper mesh, dry it in air for 1 min, observe and collect images under a transmission electron microscope, obtain exosome morphology images, and analyze the results.

[0143] 3. Exosome Nanoparticle Size Identification Steps

[0144] (1) Turn on the power of the nanoparticle size analyzer and wait for 30 minutes to stabilize the light source and calibrate;

[0145] (2) dilute the extracted exosome sample to 0.5 μg / μL;

[0146] (2) Take 2.5 μl of the diluted sample and dilute it 4000 times to 1 mL;

[0147] (3) Use a 1 mL syringe to draw the diluted sample, add it to the nanoparticle size loading tank, and measure the sample according to the set conditions;

[0148] (4) After the measurement is completed, the light intensity particle size distribution diagram and nanoparticle size are obtained, the collected images are observed, and the results are analyzed.

[0149] Figure 4 The functional exosomes extracted by the method of the present invention are detected by nanoparticle size. It can be observed in the figure that the particle size of the nanoparticles is 126nm, the particle size distribution is more uniform, there is only a single peak, and the particles with a nanoparticle size of 126nm account for 100%, while the particle size distribution in traditional exosomes is uneven, there are multiple peaks, and particles of different sizes have a certain proportion, particles with a particle size of 109.2nm account for 70.9%, and particles with a particle size of 149.4nm account for 29.1%. The exosomes prepared by the present invention have a more uniform nanoparticle size distribution than traditional exosomes; transmission electron microscopy shows that the thrombin exosomes prepared by the present invention have a saucer-shaped morphology and a relatively uniform morphology. At the same preparation concentration, the number of exosomes EV1 prepared in the control group is larger and the morphology is more uniform under the transmission electron microscope field of view; through Western Blot detection, multiple surface markers of exosomes are clearly expressed, which is consistent with the phenotypic characteristics of exosomes. It is proved that the thrombin-induced functional exosomes extracted by the present invention have more advantages than traditional exosomes in terms of apparent morphology, nanoparticle size distribution and surface marker protein expression.

[0150] Figure 5Thrombin can induce inflammation, induce cells to release cytokines, cause cell proliferation and other reactions, interleukin can also induce inflammatory reactions. In the process of mesenchymal stem cell culture, adding thrombin and interleukin to promote stem cells to release cytokines into the culture supernatant will promote the content of exosomes in the supernatant. Through the culture method of the present invention, it can be found that the total amount of exosomes secreted by the culture supernatant of mesenchymal stem cells under the combined action of thrombin and interleukin in multiple batches of cells is 790.40±23.98μg, and the exosomes secreted by the supernatant of normal starved cells are 2.377±2.17μg. The total amount of thrombin functional exosomes extracted from multiple batches of cells is 159.22±26.34. The thrombin functional exosomes extracted by the present invention are 7 times that of the exosomes secreted by the supernatant of normal starved cells, and the difference between different batches is very small, indicating that thrombin and interleukin can significantly stimulate mesenchymal stem cells to secrete exosomes under the joint action. The present invention can provide a culture method that is better than various current culture methods and has a higher yield, which greatly improves the yield of exosomes, reduces the cost of experimental consumables, and saves experimental time, and is urgently needed to be applied to scientific research experiments and large-scale production.

[0151] 3. Verification of exosome function

[0152] 1. Verification of the ability of exosomes to promote migration

[0153] (1) Use a marker pen and a ruler to evenly draw horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the holes. Make at least 5 lines across each hole;

[0154] (2) Hacat (human immortalized epidermal cells) in the logarithmic growth phase were inoculated into a 6-well plate at 25,000 cells / well;

[0155] (3) After 24 h of cell culture, when the cells have covered the bottom of the flask, use the tip of the gun to measure against a ruler and make a scratch perpendicular to the horizontal line on the back;

[0156] (4) Wash the cells three times with PBS, remove the scratched cells, add basal clear culture medium, add EV1 20 μg / well, EV2 20 μg / well, and design the wells without added substances as the control group;

[0157] (6) Place the cells in a 37°C 5% CO2 incubator and take samples at 0, 6, 12, and 24 hours and take photos.

[0158] Figure 6 The exosomes obtained by the combined culture method provided in the present invention and the exosomes extracted from the supernatant of mesenchymal stem cells collected according to the combined culture stimulation provided in the present invention have better migration-promoting performance than the exosomes provided by the culture supernatant of normal starved mesenchymal stem cells, and the effect becomes more obvious as time goes by.

[0159] 2. Effects of exosomes on skin damage repair

[0160] Babl / c mice aged 6 to 8 weeks were selected, and after weighing the animals, 18g / 130μL 1% pentobarbital was injected into the abdominal cavity of mice. After the mice fell into a coma, the hair on the back of the mice was removed, and the hair was removed with a depilatory cream until the skin was exposed. After cleaning the depilatory cream, 75% alcohol was disinfected, and a wound was made with an 80mm puncher. The mice were divided into a control group (mass concentration 0.9% normal saline), EV1 (20μg / mouse / day), and EV2 (20μg / mouse / day). Each group used 100μl each time, and the drug was administered for 9 consecutive days and photographed every other day. On the 10th day, the skin at the skin damage repair site was taken for pathological examination. Pathological examination mainly includes HE staining, immunohistochemistry and immunofluorescence detection, and the skin damage repair, collagen, angiogenic factors, etc. of the control group and exosome group are detected.

[0161] Figure 7 and Figure 8 Thrombin is a multifunctional serine protease. During the coagulation process, thrombin can regulate platelet aggregation, cleave fibrinogen and convert it into insoluble fibrin. It is an important initiating molecule in the coagulation process and promotes VEGF production, which is of great significance for wound healing and angiogenesis under other pathological conditions. The therapeutic effect of exosomes secreted from the culture supernatant of mesenchymal stem cells under the combined action of thrombin and interleukin is better than that of exosomes extracted from the normal supernatant. From the analysis of the results, after 10 days of administration, the average wound repair rate of the exosome treatment group under the combined action of thrombin and interleukin reached 97.24%, and the control group was 88.75%, which was 9.49% higher than the control group, and the repair speed was fast, indicating that thrombin and interleukin can promote the formation of skin epidermis faster, change the functional characteristics of exosomes, and indicate that exosomes can be used in regenerative medicine and targeted therapy.

[0162] Fig. 9The wound healing process is a dynamic process, including hemostasis, inflammation, proliferation and remodeling stages. The H&E results showed that the skin tissue of the control group had no obvious dermal-epidermal boundary, while the wounds of the thrombin exosome group and the exosome group were observed to have obvious undulating dermal-epidermal junctions and smaller immature granulation tissues in the epidermis. Hair follicles were formed in the wound healing sites treated with the thrombin group, indicating that the skin wound was effectively repaired, and the effect of thrombin exosomes was better than that of the exosome group. In the quantitative analysis of the length of the wound that was not completely healed, the unhealed length of the control group was 7.066mm, and the healing length of the thrombin exosomes was 2.405mm, which was 65.96 lower than that of the control group. The unhealed length of the exosome group was 4.772mm, which was 32.47% lower than that of the control group, effectively shortening the wound length, indicating that the exosomes obtained by co-culture are more effective in promoting full-thickness skin healing.

[0163] Fig.10 Thrombin exosomes promote fibrosis and increase epithelial thickness to the greatest extent, which is related to the parallel transfer of a large number of growth factors to skin target cells, which is beneficial to the healing of previous wounds. Thrombin exosomes maximize the enhancement of skin tissue regeneration and collagen formation in full-thickness skin injuries, and promote the transformation of collagen fibers into muscle fibers.

[0164] Fig.11 The expression of CK14 at the base of the wound showed active epidermal cell proliferation at the wound edge, and the stratum corneum at the wound edge was significantly thickened. CK14-positive cells were mainly distributed in the basal layer and subbasal layer of the wound. In the treatment group, CK14-positive epidermal cell layer was visible under the blood scab on the wound, and as time went on, CK14-positive cells gradually migrated to the center of the wound. The thickness of CK14-positive epidermal cells in the model control group was significantly lower than that in the blank control group. The thrombin-induced exosome group was significantly better than the exosome group. In the mean fluorescence intensity statistics, the treatment group was significantly different from the model group, p < 0.05.

[0165] Fig.12 Compared with the control group, after skin injury treatment, the expression level of Collagen I protein in the skin tissue of mice in the thrombin exosomes group was significantly increased, while the expression of Collagen I protein in the skin tissue of mice in the exosomes group decreased to a certain extent compared with the thrombin exosomes group, but the effect was also better than that of the model group.

[0166] It can be seen that compared with the model control group, the expression level of related proteins in the mouse skin tissue after thrombin treatment with exosomes was significantly increased, and the healing speed of mouse epidermal wounds was significantly accelerated, indicating that the exosomes prepared by the present invention can effectively promote the healing of skin wounds.

[0167] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A functional mesenchymal stem cell exosome prepared by co-culture, characterized in that: The particle size of the nanoparticles of the exosomes is 126 nm, and the particle size distribution is more uniform, with only a single peak. The particles with a nanoparticle size of 126 nm account for 100%, and the shape is saucer-shaped and uniform.

2. The method for preparing functional mesenchymal stem cell exosomes by co-culturing exosomes according to claim 1, characterized in that: The steps include: (1) Cell culture The primary frozen mesenchymal stem cells were revived and cultured. After the third generation, the cells were cultured at 25,000 cells / cm 2 The cells were inoculated at a density of 1000 μg / mL in a cell culture flask, and co-cultured with DMEM / F12, 10% serum, and 1% double antibody for 24 h before transfection with miRNA. After 5 h of transfection, the culture medium was replaced with DMEM / F12 medium, 5% serum, and 1% double antibody in a volume of 22 ml / T175, and interleukin was added in an amount of IL-1 1000 U / 22 mL culture medium and IL-18 1000 U / 22 mL culture medium. The cells were co-cultured at 37°C and 5% CO2. (6) After 24 hours of co-culture, when the cell growth density reached 70%, the culture medium was removed, the cells were washed twice with PBS, and serum-free DMEM-F1222 ml and thrombin 100 U / T175 / 22 ml were added for starvation culture; (7) Starvation culture for 48 h, collecting co-cultured mesenchymal stem cells and cell culture supernatant; (2) Exosome extraction The cell culture supernatant was centrifuged at 400 g, 9 rpm, 9 rpm, for 10 min, and the supernatant was transferred; at 4°C, 2000 g, 9 rpm, 9 rpm, 20 min, and the supernatant was transferred; at 4°C, 10000 g, 9 rpm, 9 rpm, 30 min, and the supernatant was transferred. The cell debris in the supernatant was effectively removed by the above centrifugation process; After centrifugation, filter once with a 0.45 μm filter membrane and once with a 0.22 μm filter membrane to remove residual cell debris and large particles in the liquid. Ultracentrifuge for 70 min at 4°C, 29800 r, increase speed by 100 r, decrease speed by 100 r, discard the supernatant, resuspend the precipitate with PBS, collect it, repeat the ultracentrifugation operation twice, resuspend the precipitate with PBS, and obtain exosomes.

3. The method according to claim 2, characterized in that: The specific steps of transfection in step (1) are: Add 500 μl of serum-free DMEM-F12 and 18 μl of transfection reagent to each 1.5 ml centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a transfection reagent-medium mixture; At the same time, add 500 μl of serum-free basal medium to another 1.5 ml centrifuge tube, add 300 pmol of miR-1291 oligo, mix gently with a pipette, and let stand at room temperature for 5 min to obtain a miR-1291 oligo-medium mixture; Add the transfection reagent-medium mixture dropwise to the miR-1291 oligo-medium mixture, mix gently with a pipette, and let stand at room temperature for 18 min; The culture medium in the culture flask was replaced with serum-free DMEM-F12, and the mixture was allowed to stand at room temperature for 18 min, and then immediately added to the cell culture medium for transfection.

4. Use of the exosomes as claimed in claim 1 in the preparation of a drug for promoting skin damage repair.

5. Use of the exosomes according to claim 1 in the preparation of regenerative medicine and targeted therapy drugs.

6. Use of the exosomes according to claim 1 in the preparation of a drug for promoting full-thickness skin healing.

7. Use of the exosomes according to claim 1 in the preparation of a drug for promoting skin tissue regeneration and collagen formation.

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