Preparation method and application of adipose tissue SVF vesicles

Abstract

The invention discloses a preparation method and application of adipose tissue SVF vesicles. The preparation method mainly comprises the following steps: extracting and culturing adipose tissue SVF cells; constructing a recombinant plasmid of an overexpression target protein; taking the recombinant plasmids and SVF cells of the overexpressed target protein, and transfecting the SVF cells by adopting a microchannel cell transfection technology to obtain the SVF cells of the overexpressed target protein; and culturing the SVF cells over-expressing the target protein, collecting the culture supernatant, and extracting the SVF vesicles. The preparation method provided by the invention is simple to operate, convenient, practical and high in efficiency, the adipose tissue SVF vesicles with high target protein expression can be obtained, the purity of the vesicles is high, the curative effect can be ensured, and a new strategy is provided for realizing precise medical treatment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation method of adipose tissue SVF vesicles and application thereof. Background Art

[0002] The stromal vascular fraction (SVF) is a cell population separated from adipose tissue, which mainly includes mesenchymal stem cells, endothelial cells, smooth muscle cells, immune cells and some vascular components. These cell populations not only have high proliferation capacity, but also have multiple functions such as tissue repair, promotion of angiogenesis and immune regulation, so they have great application potential in the field of regenerative medicine and tissue engineering.

[0003] In addition, SVF cell membrane components can be processed into vesicle-like tiny particles through specific means to form SVF vesicles. These vesicles can carry and transport bioactive substances such as proteins and nucleic acids. By regulating the bioactive substances in the vesicles, precise therapeutic effects can be achieved. Studies have shown that SVF vesicles play an important role in intercellular communication, tissue repair and regeneration, immune regulation, anti-oxidation and anti-apoptosis, and anti-tumor. For example, in the treatment of chronic wounds, the miRNA and growth factors carried by SVF vesicles can regulate cell proliferation, angiogenesis, and collagen deposition, significantly promoting the healing of diabetic ulcers, burns, and chronic wounds. SVF vesicles are also widely used in the treatment of bone tissue engineering, central nervous system diseases, autoimmune diseases, etc. Due to their natural targeting and biocompatibility, SVF vesicles are also used for the delivery of nucleic acid drugs, protein drugs, and small molecule drugs. By modifying SVF vesicles, their targeting and delivery efficiency can be enhanced, showing broad application prospects in tissue repair, anti-tumor, anti-inflammatory, and gene therapy.

[0004] Although SVF vesicles show great potential in basic research and clinical applications, they still face some challenges. At present, the isolation and purification methods of SVF vesicles have not been standardized, and the yield is low, which limits their widespread use in clinical applications. In addition, although SVF vesicles have shown therapeutic effects in a variety of diseases, their specific molecular mechanisms are not yet fully understood. Therefore, it is urgent to develop a method for preparing SVF vesicles to obtain engineered SVF vesicles with specific targeting and enhanced functions to achieve the goal of precision medicine. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a method for preparing adipose tissue SVF vesicles, which is simple to operate, can extract adipose tissue SVF vesicles containing highly expressed target proteins, and the SVF vesicles have high purity and can exert a good therapeutic effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One aspect of the present invention provides a method for preparing adipose tissue SVF vesicles, comprising the following steps:

[0008] Extract and culture adipose tissue SVF cells;

[0009] Constructing a recombinant plasmid that overexpresses the target protein;

[0010] Take the recombinant plasmid and SVF cells that overexpress the target protein, and transfect the SVF cells using microchannel cell transfection technology to obtain SVF cells that overexpress the target protein;

[0011] SVF cells overexpressing the target protein are cultured, and the culture supernatant is collected for SVF vesicle extraction.

[0012] The preparation method of the present invention is simple to operate and low in cost. The obtained SVF vesicles have high purity and efficient targeted delivery ability, and can protect the stability of the target protein and improve the therapeutic effect.

[0013] Another aspect of the present invention provides adipose tissue SVF vesicles prepared by the preparation method described above.

[0014] Another aspect of the present invention provides use of the adipose tissue SVF vesicles described above in the preparation of medicines or medical dressings.

[0015] Another aspect of the present invention provides a medical dressing comprising the adipose tissue SVF vesicles described above.

[0016] The adipose tissue SVF vesicles of the present invention are used to prepare drugs or medical dressings, which can be used to improve the treatment effect clinically and realize precision medicine.

[0017] Beneficial effects of the present invention:

[0018] The preparation method of adipose tissue SVF vesicles provided in the present invention is simple to operate, convenient, practical and efficient, and can obtain adipose tissue SVF vesicles that highly express the target protein. The vesicles have high purity and can prevent adverse reactions (such as immune reactions, etc.) caused by direct administration of proteins to subjects, thereby ensuring therapeutic efficacy and providing a new strategy for achieving precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a flow chart of the preparation of adipose tissue SVF vesicles in a preferred embodiment of the present invention.

[0020] Figure 2 This is the morphology of the SVF-P0 primary cells extracted in Example 1.

[0021] Figure 3 This is a simplified diagram of the GFP-LBP plasmid map constructed in Example 1.

[0022] Figure 4 The statistical results of qPCR verification of transfection efficiency after microchannel cell transfection in Example 1.

[0023] Figure 5 This is the nanoflow cytometry result of the SVF vesicles extracted in Example 1.

[0024] Figure 6 These are the results of the efficacy evaluation of the SVF vesicles that highly express LBP in Example 2. DETAILED DESCRIPTION

[0025] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] The first aspect of the present invention discloses a method for preparing adipose tissue SVF vesicles. The main process of the preparation method is as follows: Figure 1 As shown in , the preparation method of the present invention can be used to prepare SVF vesicles with high purity and efficient targeted delivery ability, and the SVF vesicles contain overexpressed target proteins, which can achieve good therapeutic effects.

[0027] Combine the following Figure 1 The preparation method of adipose tissue SVF vesicles in the present invention is described in detail.

[0028] Extraction and culture of adipose tissue SVF cells

[0029] This step mainly includes two steps: extraction and cultivation of adipose tissue SVF cells.

[0030] The extraction of SVF cells from adipose tissue includes the following steps: obtaining adipose tissue, washing and chopping it into a minced meat state; digesting the minced meat-like adipose tissue in sequence, terminating the digestion, and centrifuging it for the first time, discarding the supernatant and retaining the precipitate; resuspending the precipitate and collecting the filtrate by sieving, and after the filtrate is centrifuged for the second time, discarding the supernatant and retaining the precipitate.

[0031] In the present invention, the biological source of the adipose tissue is a mammal, such as a mouse, a rabbit, a sheep or a human. In some embodiments of the present invention, female or male mice aged 3 to 4 weeks are used.

[0032] Among them, the acquisition of adipose tissue can be carried out by means or methods that are relatively well known or conventional in the art, without special restrictions. For example, in some specific embodiments of the present invention, the experimental animals used are mice. First, the mice need to be weighed and anesthetized and killed as needed (for example, by injection of tribromoethanol), and a circle of hair around the chest at the base of the mouse forelimbs is removed, and then immersed in 75% alcohol for 5 to 10 minutes to prevent mycoplasma and chlamydia contamination caused by hair loss in subsequent experiments, which may lead to inaccurate or unstable experimental results. Then, the mouse is taken out of the alcohol, and the liquid is absorbed with sterile gauze. The mouse skin is cut along the hair shaved part, and the skin incision is fixed symmetrically with two tissue forceps. The skin is pulled horizontally backward along the direction of the mouse trunk to the base of the tail, and the symmetrical inguinal white adipose tissue (iWAT) can be exposed (note that the tissue forceps should be embedded in the outside of the skin to avoid contaminating the inside of the skin and iWAT); and the mouse iWAT is suspended and separated using sterile elbow forceps, and immersed in a small dish filled with phosphate buffer to complete the sampling of adipose tissue.

[0033] Furthermore, the washing of adipose tissue is a conventional washing method in the art, usually by gently rinsing in a buffer to avoid damaging the tissue. The specific buffer can be selected according to the needs of the experiment or tissue, and the technicians in this field have such ability. In the present invention, the buffer used is a phosphate buffer, that is, a commercial PBS buffer, and the buffer is sterilized by 0.1μm filtration. Preferably, when rinsing the tissue, a penicillin-streptomycin-amphotericin B additive can be added in advance at a volume ratio of 1:100, in which the penicillin is 10000U / mL, the streptomycin is 10mg / mL, and the amphotericin B is 25μg / mL.

[0034] Furthermore, the washed fat tissue needs to be chopped for subsequent experiments. In the specific operation, the washed tissue is slightly drained of liquid, transferred to the surface of a clean container (such as a dish lid), and the tissue is chopped with sterile scissors until there are no large pieces of tissue and it is in a minced meat state. The specific chopping time can be adjusted according to needs, but it should not be too long. The best control is about 5 minutes.

[0035] In addition, it should be noted that during the steps of cleaning and chopping adipose tissue, it is generally recommended to change into new gloves for subsequent operations to reduce the occurrence of contamination.

[0036] Furthermore, the minced fat tissue needs to be digested, digested, centrifuged for the first time, and the supernatant is discarded to keep the precipitate, which are all conventional methods in the art without special limitations.

[0037] Among them, digestion is generally achieved by adding cell digestion solution to the tissue. According to the difficulty of tissue digestion obtained, technicians in this field can select the corresponding cell digestion solution or combine multiple digestion solutions. In some specific embodiments of the present invention, the cell digestion solution used is type I collagenase solution. The specific concentration can be selected as needed. Preferably, the concentration of type I collagenase solution is 2 mg / mL, and it is dissolved in phosphate buffer and sterilized by 0.22 μm filtration. The amount is added according to the specific tissue situation. Taking mice as an example, 1 mL is used for each mouse tissue. In addition, the specific parameters of digestion can be determined by technicians in this field based on known methods or experiments. Preferably, the shredded iWAT is poured into a container containing type I collagenase solution, sealed with a sealing film, placed in a 37°C shaker, and digested at a speed of 100 to 300 rpm. It should be noted that the digestion time should not exceed 30 minutes to prevent cell damage. Preferably, the digestion time is 20 to 30 minutes.

[0038] The termination of digestion herein is carried out in a conventional manner in the art, that is, adding an equal volume of fresh complete medium to the digested cell container to achieve the termination effect. There is no particular limitation on the specific fresh complete medium, and the corresponding medium can generally be selected according to the needs of the tissue. In some specific embodiments of the present invention, the complete medium used is DMEM / F12+10% special grade fetal bovine serum+1% penicillin-streptomycin-amphotericin B additive.

[0039] The first centrifugation is performed after the digestion is terminated. The specific centrifugation conditions can be designed according to the actual situation of the tissue. Preferably, the centrifugation temperature is recommended to be 4°C to maintain cell activity. The centrifugation method is recommended to be horizontal centrifugation (which can be achieved by using a horizontal centrifuge) to prevent cell loss. At the same time, the centrifugation speed is recommended not to exceed 3000rpm to avoid damaging the tissue. The preferred centrifugation speed is 1500-3000rpm. In addition, the centrifugation time is not particularly limited and can be adjusted according to the specific tissue situation (such as the number of tissues or the volume of tissues, etc.).

[0040] Further, the cell precipitate collected after termination of digestion and centrifugation is resuspended in fresh complete medium and sieved to remove impurities such as large tissue blocks. In the specific operation, the resuspended solution is passed through a 40 μm sieve, and the filtered solution (containing SVF cells) is collected and then centrifuged for the second time. The conditions or parameters of the second centrifugation are the same as those of the first centrifugation and will not be described in detail here.

[0041] In the present invention, the culture of adipose tissue SVF cells adopts the conventional primary cell culture steps in the art. Specifically, the precipitate after the second centrifugation is resuspended, inoculated into a cell culture container, and observed and replaced 24 hours after inoculation. Among them, the cell culture container used is a conventional T25 cell culture flask or a 6cm small dish in the art. The liquid replacement operation is first repeatedly rinsed with phosphate buffer until the liquid is clear (preferably washed 3 times with PBS buffer), and then fresh complete culture medium is added; thereafter, the liquid is replaced every 3 days, and 3 to 5 generations can be passed, and the passage ratio is 1:2 or 1:3, and cells with fewer passages are used as much as possible for subsequent experiments.

[0042] Construction of recombinant plasmids overexpressing target proteins

[0043] In the present invention, the corresponding target protein is selected according to the needs of disease treatment, and a recombinant plasmid overexpressing the target protein is constructed. The target protein here is not particularly limited and can be a therapeutic target for any known disease in the art. By overexpressing these target proteins and using SVF vesicles for treatment, excellent therapeutic effects can be exerted. In some specific embodiments of the present invention, the target protein used is lipopolysaccharide binding protein (LBP), which is an acute phase response protein synthesized by the liver and secreted into the blood. It belongs to the lipid transport protein family, and its main function is to recognize and bind lipopolysaccharide (LPS), which plays a key role in the host immune defense response; in addition, it also has a lipid transport region that can bind triglycerides (TG) and participate in the formation of fat droplets in adipose tissue; the fat cell niche located in the dermis is generally considered to be related to the hair cycle and skin regeneration, and therefore, LBP is a potential clinical treatment target in wound repair.

[0044] There is no particular limitation on the construction method of the recombinant plasmid, and the gene engineering methods familiar in the art, such as PCR amplification, cloning or artificial synthesis, can be used. Preferably, the target gene is inserted into the vector backbone by PCR amplification. There is no particular limitation on the selection of the vector backbone, and a suitable vector backbone can be selected according to the different target genes, as long as the purpose of overexpressing the target gene can be achieved.

[0045] Preferably, a fluorescent gene is also inserted into the recombinant plasmid, so as to facilitate the screening and identification of the target gene expression vector by fluorescent staining. The fluorescent gene can be a common type in the art, including but not limited to green fluorescent protein (GFP), red fluorescent protein (RFP), fluorescent dye (FAM or Cy3), etc. In some specific embodiments of the present invention, the fluorescent gene used is GFP.

[0046] After the recombinant plasmid is constructed, the plasmid is extracted using an endotoxin-free extraction kit conventionally used in the art. The mass concentration of the extraction is preferably 500-1000 ng / μL, but is not limited thereto and can generally be adjusted according to the subsequent transfection concentration.

[0047] SVF cell transfection to obtain SVF cells overexpressing the target protein

[0048] In this step, the present invention preferably uses microchannel cell transfection technology, which has high transfection efficiency, simple operation, no interference from exogenous chemicals, and is applicable to a variety of cell types, especially difficult-to-transfect cells. In particular, the absence of exogenous chemicals and viral vector residues makes subsequent clinical applications less controversial.

[0049] During the specific transfection, the extracted recombinant plasmid and the SVF cells in the adherent state are taken, and the instrument manual is used to operate, and the corresponding parameters are set for transfection through a microchannel cell transfection instrument to obtain SVF cells overexpressing the target protein. In some specific embodiments of the present invention, the cell density during the experiment is 80%, and the electrotransfection parameters are LEVEL=4, CYCLE=5.

[0050] Extraction of SVF vesicles

[0051] In this step, the transfected SVF cells are cultured, and when the desired conditions are reached, the cell culture supernatant is collected and SVF vesicles are extracted.

[0052] In some specific embodiments of the present invention, when the cell density is 80%, the complete medium is replaced with DMEM, and the cell culture supernatant is collected once every 24 hours, and once again after 24 hours. The cell culture supernatant is placed in a horizontal centrifuge and centrifuged for 10 minutes at 4°C and 1000 rpm to remove suspended cells. The supernatant is filtered through a 0.22 micron filter membrane and then SVF vesicles are extracted.

[0053] Among them, the extraction method of SVF vesicles can adopt the conventional method in this field. Preferably, a fully automatic exocrine extraction system is used, which is a sine wave negative pressure oscillation system (NPO) combined with a double coupling resonance system (HO) acting on a nano-ultrafiltration chip. Free nucleic acids and protein and other impurities in the sample are quickly separated through the nano-chip, thereby purifying and enriching exosomes. Compared with traditional differential centrifugation, density gradient centrifugation, membrane ultrafiltration, polymer-based precipitation and immunoseparation, it has the advantages of fast purification speed, high product purity, small sample volume, no introduction of exogenous impurities, and simple operation.

[0054] The second aspect of the present invention discloses an adipose tissue SVF vesicle, which is prepared by the preparation method described above.

[0055] The third aspect of the present invention discloses the use of the adipose tissue SVF vesicles as described above in the preparation of medicines or medical dressings.

[0056] The fourth aspect of the present invention discloses a medical dressing comprising the adipose tissue SVF vesicles described above.

[0057] It is understandable that the SVF vesicles prepared in the present invention can be used as an active ingredient of a drug or medical dressing, and thus be administered to a subject to exert a corresponding therapeutic effect.

[0058] It is understandable that the drug not only includes SVF vesicles, but also includes any pharmaceutically acceptable excipients and / or carriers. These excipients and / or carriers are selected or designed in order to prepare corresponding pharmaceutical preparations, or to improve the delivery effect of active ingredients, or to improve the efficacy of drugs, etc. Those skilled in the art have such capabilities, so they will not be elaborated here.

[0059] The medical dressing described in the present invention refers to a medical material used to cover sores, wounds or other lesions. Any common type in the art can be used. A typical example is a medical patch. In some specific embodiments of the present invention, the medical patch is a microneedle patch.

[0060] It is understandable that the adipose tissue SVF vesicles contained in the medicine or medical dressing are in an effective amount, and the effective amount here means that when applied in such an amount, the disease or wound can achieve the expected effect, and the expected effect includes but is not limited to the relief or improvement of the symptoms.

[0061] The present invention is described below by specific examples, it should be noted that the following specific examples are only for illustrative purposes, and do not limit the scope of the present invention in any way, unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The term used in the specification of the present invention herein is only for the purpose of describing specific embodiments, and is not intended to limit the present invention. In addition, if not otherwise specified, the method for not specifically recording conditions or steps is a conventional method, and the reagents and materials adopted can be obtained from commercial sources.

[0062] Example 1

[0063] This example discloses a method for preparing SVF vesicles that highly express LBP, and the specific steps are as follows:

[0064] 1. Extraction and culture of mouse adipose stromal cells

[0065] (1) Three female C57BL / 6J mice aged 3 to 4 weeks were injected with a 1.25% (mass concentration) tribromoethanol solution to a lethal dose. The hair around the chest at the base of the forelimbs of the mice was shaved and then immersed in 75% alcohol for 5 to 10 minutes.

[0066] (2) Remove the mouse from the alcohol, dry the liquid with sterile gauze, and cut the mouse skin along the shaved hair area. Use two tissue forceps to symmetrically fix the skin incision, and pull the skin horizontally backward along the mouse trunk to the base of the tail to expose the symmetrical inguinal white adipose tissue (iWAT).

[0067] (3) Use sterile elbow forceps to suspend and isolate mouse iWAT, and immerse it in a small dish filled with phosphate buffer, and gently rinse the tissue in the phosphate buffer; the phosphate buffer is a commercial PBS buffer that has been sterilized by 0.1 μm filtration. When scrubbing the tissue, add penicillin-streptomycin-amphotericin B additive in advance at a ratio of 1:100 (v / v), and the additive contains 10,000 U / mL penicillin, 10 mg / mL streptomycin, and 25 μg / mL amphotericin B.

[0068] (4) After rinsing the tissue, drain the liquid slightly, transfer the iWAT to the lid of the dish, and use sterile scissors to chop the tissue until there are no large pieces of tissue and it is in a minced meat state.

[0069] (5) Pour the shredded iWAT into a 15 mL centrifuge tube containing type I collagenase solution, seal it with a sealing film, and place it in a shaker at 37°C at 200 rpm / min for 30 min. During the digestion process, take out the centrifuge tube and shake it vigorously several times every 10 minutes to make the digestion more uniform. The concentration of type I collagenase solution is 2 mg / mL. After dissolving it in phosphate buffer, filter it through 0.22 μm for sterilization. Use 1 mL for each mouse tissue. Excess type I collagenase needs to be aliquoted and frozen at -20°C in the dark.

[0070] (6) After digestion is complete, add fresh complete medium to a 15 mL centrifuge tube to terminate digestion. Place the centrifuge tube in a horizontal centrifuge and centrifuge for 10 minutes at 4°C and 2000 rpm. The complete medium is DMEM / F12 (PM150312B, Pronocell) + 10% premium fetal bovine serum + 1% penicillin-streptomycin-amphotericin B additive.

[0071] (7) Discard the supernatant of the liquid after centrifugation in step (6), resuspend the precipitate at the bottom of the centrifuge tube with 5 mL of fresh complete culture medium, pass the resuspended solution through a 40-μm sieve, use a new 15 mL centrifuge tube to collect the filtered solution, and place it in a horizontal centrifuge again, and centrifuge it at 4°C and 2000 rpm for 10 minutes.

[0072] (8) Discard the supernatant of the liquid after centrifugation in step (7), resuspend the sediment at the bottom of the centrifuge tube with 2 mL of fresh complete medium, and inoculate the cell suspension evenly into two T25 cell culture flasks. Use a 1 mL pipette tip to carefully discard the supernatant to avoid losing the cells at the bottom; the inoculation volume is 1.5 mice per T25 cell culture flask.

[0073] (9) Observe and change the medium 24 hours after cell inoculation (the morphology of SVF-P0 primary cells is shown in Figure 2 ). Repeat the washing with phosphate buffer until the liquid is clear, then add fresh complete medium, and change the medium every 3 days. The cells can be subcultured for 3 to 5 generations for subsequent experiments. When changing the medium, wash with PBS 3 times, using 1 mL each time; the subculture ratio is 1:3, and try to use cells with fewer subcultures for experiments.

[0074] 2. Overexpression of LBP using a microchannel cell transfection instrument

[0075] (1) A plasmid for overexpressing the GFP-LBP gene was constructed (the control group was set up as a GFP plasmid), and an endotoxin-free extraction kit (DP117, TIANGEN) was used to extract the plasmid at a concentration of 0.5 μg / μL.

[0076] Among them, the GFP plasmid comes from Aibimeng Biotechnology Co., Ltd. (Cat. No: LV590-Custom, Lot No: K23P14BA); the GFP-LBP plasmid was commissioned to Aibimeng Biotechnology Co., Ltd. for synthesis, and the LBP sequence (nucleotide sequence shown in SEQID NO.1) and 3×Flag tag were inserted into the GFP plasmid for recombination. The schematic diagram of the plasmid is shown in the following figure. Figure 3 As shown in , the specific construction methods are well known in the art and will not be elaborated here.

[0077] (2) The P1 cells collected from the culture above and the plasmid extracted in step (1) were transfected using a microchannel cell transfection instrument (MDMP E-1, Shanghai Mengde Biotechnology). The specific operations were carried out according to the instrument manual. The additional consumables that needed to be prepared before transfection were disposable cell embedding dishes (Guangzhou Jiete Biofiltration Co., Ltd., TCS016006 01.TC.012.0156). The remaining operations were carried out according to the operating instructions of the electroporator. The cell density during the experiment was 80%, and the electroporation parameters were LEVEL=4, CYCLE=5.

[0078] (4) 48 h after P1 cell transfection, cell mRNA was extracted and RT-qPCR was performed to verify the efficiency. The results are as follows Figure 4 As shown in , it shows that SVF cells with high expression of LBP were successfully constructed.

[0079] 3. Collect cell culture supernatant and use fully automated exosome extraction system for vesicle extraction

[0080] (1) After mouse adipose stromal cells successfully expressed GFP and GFP-LBP, when the cell density reached 80%, the complete culture medium was replaced with DMEM medium (PM150312, Pronocell), and the cell culture supernatant was collected every 24 hours and once again after another 24 hours.

[0081] (2) The cell culture supernatant was placed in a horizontal centrifuge and centrifuged at 4°C, 1000 rpm for 10 minutes to remove suspended cells.

[0082] (3) The supernatant (cell culture medium without serum) in step (2) was filtered through a 0.22 μm filter membrane and then vesicles were extracted using the fully automatic exosome extraction system (H-300, Huixin Biotechnology) with the program "CM.Feeble-MA.A03" selected. During the sample processing, bubbles should be avoided. The M chip is recommended to be resuspended in 350-400 μl PBS. The front and back sides of the chip, 5 points on each side, were rinsed with PBS 3-5 times for each point, and the chip was tilted and rinsed quickly.

[0083] Among them, the storage conditions of cell culture supernatant and vesicles are: placed at 4°C for use within 1 week and at -80°C for long-term storage.

[0084] The extracted SVF vesicles were detected by nanoflow cytometry, and the results were as follows: Figure 5 As shown in , it can be seen that the diameter of the SVF vesicles extracted in this example is about 56.2±22.1 nm, and the number of particles after 10-fold dilution is 1.83E+9 / mL; the particle distribution is concentrated, indicating that the vesicle purity is high.

[0085] Example 2

[0086] In this example, an acute wound model of the back skin of mice was constructed and wound treatment was performed to verify the therapeutic effect of the SVF vesicles with high expression of LBP prepared in Example 1.

[0087] 1. Construction of acute wound model on mouse back skin

[0088] Female mice (C57BL / 6J Lbp-KO mice) aged 7 to 8 weeks were fasted and deprived of water for 6 hours before bearing weight and anesthetized with 1.25% tribromoethanol at a dose of 0.2 mL / 10 g; after confirming the depth of anesthesia, the mice were fixed on the dissecting table and the hair on the back was shaved; after disinfecting the skin with alcohol cotton balls, two symmetrical wounds with a diameter of 6 mm were made on the back skin of the mice with a 6 mm skin punch.

[0089] 2. Wound treatment

[0090] (1) Two days before the wound treatment of mice, the SVF vesicles containing GFP and GFP-LBP extracted in Example 1 were thawed at 4°C.

[0091] (2) After dissolution, the vesicles were diluted in HAMA solution at a volume ratio of 1:10. The preparation of HAMA solution was carried out according to the instruction manual (EFL-HAMA-150K methacryloyl hyaluronic acid).

[0092] (3) According to the product instructions, HAMA microneedle patches loaded with vesicles derived from mouse adipose stromal cells with high LBP expression were prepared, and the control group was a HAMA microneedle patch loaded with vesicles derived from mouse adipose stromal cells containing GFP. After preparation, the patches were placed in a sterile culture dish, the dish was wrapped with sealing film, and stored at room temperature for 1 day or at 4°C for 3 days.

[0093] (4) The microneedle patch was sutured and fixed on the wound surface of the mouse. The wound on the left side was the control group, where a HAMA microneedle patch containing GFP vesicles was placed. The wound on the right side was the experimental group, where a HAMA microneedle patch containing GFP-LBP vesicles was placed. Subsequently, 0.5 mL of normal saline and 0.1 mL of acetaminophen solution were injected intraperitoneally for fluid resuscitation and analgesia. The mouse was placed on a warming board and returned to the cage after recovery. The mouse was covered with an Elizabethan collar to prevent it from chewing the wound surface and the patch. The patch was replaced every three days for a total of 9 days.

[0094] The results are as follows Figure 6 As shown in , it can be seen that compared with the control group, the wound closure speed of the experimental group was accelerated in the first 9 days, indicating that the SVF vesicles of GFP-LBP extracted in the present invention have excellent therapeutic effects and can achieve precision medicine.

[0095] It should be noted that this article takes SVF vesicles with high expression of LBP as an example. The preparation method of adipose tissue SVF vesicles in the present invention is not only applicable to the preparation of SVF vesicles with high expression of LBP. Disease targets known in the art can all be used as target proteins for the preparation of SVF vesicles and improve the therapeutic effect, which will not be elaborated one by one here.

[0096] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing adipose tissue SVF vesicles, characterized in that: The following steps are involved: Extract and culture adipose tissue SVF cells; Constructing a recombinant plasmid that overexpresses the target protein; Take the recombinant plasmid and SVF cells that overexpress the target protein, and transfect the SVF cells using microchannel cell transfection technology to obtain SVF cells that overexpress the target protein; SVF cells overexpressing the target protein are cultured, and the culture supernatant is collected for SVF vesicle extraction.

2. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The extraction of adipose tissue SVF cells comprises the following steps: Obtain adipose tissue, wash and mince into minced meat; Digest the minced fat tissue in sequence, terminate the digestion, centrifuge for the first time, discard the supernatant and keep the precipitate; The precipitate is resuspended and then sieved to collect the filtrate, and the filtrate is centrifuged for the second time, and the supernatant is discarded and the precipitate is retained; Preferably, the digestion time does not exceed 30 min; Preferably, the tissue digestion solution used in the digestion is type I collagenase solution; Preferably, the first centrifugation and the second centrifugation are both performed by horizontal centrifugation, the centrifugation temperature is 4° C., and the centrifugation speed does not exceed 3000 rpm.

3. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The culturing of adipose tissue SVF cells comprises the following steps: The extracted cell pellet was resuspended and inoculated into the cell culture container, and the culture medium was changed after 24 hours of inoculation. Preferably, the cell culture container is a T25 cell culture flask or a 6 cm small dish; Preferably, the medium is changed every 3 days, and the subculture ratio is 1:2 or 1:

3.

4. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The target protein is lipopolysaccharide binding protein.

5. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The recombinant plasmid also includes a fluorescent group; Preferably, the fluorescent group is GFP.

6. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The concentration of the recombinant plasmid is 500-1000 ng / μL.

7. The method for preparing adipose tissue SVF vesicles according to claim 1, characterized in that: The SVF vesicle extraction adopts a fully automatic exosome extraction system.

8. A fat tissue SVF vesicle, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the adipose tissue SVF vesicles according to any one of claims 1 to 8 in the preparation of medicines or medical dressings.

10. A medical dressing, characterized in that: Containing the adipose tissue SVF vesicles according to any one of claims 1 to 8; Preferably, the medical dressing is a microneedle patch.

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