An engineered extracellular vesicle enriched with circCDK13 and its preparation method and application

Abstract

The present invention discloses an engineered extracellular vesicle enriched with circCDK13, a preparation method and application thereof, and belongs to the field of biomedical technology. The present invention provides a preparation method of an engineered extracellular vesicle enriched with circCDK13, wherein a vector overexpressing circCDK13 is used to infect placental mesenchymal stem cells to collect cell supernatant to obtain extracellular vesicles overexpressing circCDK13 and use the supernatant for diabetic wounds, and the therapeutic effect of the extracellular vesicles on diabetic wounds is significantly better than that of natural extracellular vesicles secreted by placental mesenchymal stem cells, and the wounds are not only more advantageous in terms of healing speed, but also have greater application potential in terms of stimulating the regeneration of skin appendages and improving the quality of wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered extracellular vesicle enriched with circCDK13, and a preparation method and application thereof. Background Art

[0002] Circular RNAs (circRNAs) are a special type of non-coding RNA that does not have a 5' cap and a 3' poly (A) tail. It connects the 3' and 5' ends through exon cyclization or intron cyclization to form a complete circular structure, thereby avoiding degradation by nuclease exonucleases. Therefore, circRNAs have a longer half-life and stronger conservation than linear RNA. Therefore, circRNAs have unique advantages in the development of disease diagnosis and treatment strategies.

[0003] Delayed wound healing or non-healing is an important complication that leads to severe disability and death in diabetic patients, seriously affecting the quality of life of patients and bringing huge economic burden to families and society. In recent years, with the in-depth study of circRNAs, researchers have gradually realized that circRNAs are also important participants in the process of skin wound repair. Previous studies have confirmed that circCDK13 (circBase ID: hsa_circ_0079929) can improve the function of fibroblasts and keratinocytes in skin tissue, thereby promoting the healing of diabetic wounds. However, circCDK13 is composed of 660 bases, which is difficult to be directly synthesized in vitro like microRNAs. In addition, due to the large molecular diameter of circCDK13, it is difficult to be absorbed by cells through the cell membrane when directly applied. Therefore, it is particularly important to explore a safe, feasible and efficient circRNA delivery strategy.

[0004] Small extracellular vesicles (sEVs) are nanoscale particles wrapped in a lipid bilayer membrane with a diameter of 50 to 200 nm, which contain a variety of non-coding RNAs (such as microRNA, lncRNA, circRNA), mRNA, DNA, proteins, lipids and other substances. sEVs can transfer proteins and nucleic acids to adjacent or distant cells, so sEVs are considered to be important messengers for intercellular communication. Compared with cell therapy, sEVs as a cell-free treatment strategy have better stability, lower immunogenicity, fewer ethical issues, and lower risks of embolism and tumorigenesis. In addition, sEVs are easily taken up by cells and have good protection for the bioactive components they encapsulate. Therefore, sEVs are favored by researchers in the fields of immunotherapy, regenerative medicine and drug delivery. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered extracellular vesicle enriched with circCDK13, and a preparation method and application thereof. The engineered sEVs have stronger biological activity than natural sEVs (Nature sEVs, N-sEVs), and can accelerate wound healing and regeneration of its appendages.

[0006] The present invention provides a method for preparing engineered extracellular vesicles enriched in circCDK13, comprising the following steps: using a vector overexpressing circCDK13 to infect mesenchymal stem cells, and extracting the extracellular vesicles obtained from the infected positive cells, namely the engineered extracellular vesicles enriched in circCDK13.

[0007] Preferably, the vector overexpressing circCDK13 comprises a lentivirus overexpressing circCDK13.

[0008] Preferably, after obtaining the infection-positive cells, the method further includes culturing the infection-positive cells, collecting the cell supernatant, and extracting the engineered extracellular vesicles enriched with circCDK13 from the cell supernatant.

[0009] Preferably, the method comprises culturing the infection-positive cells using a mesenchymal stem cell serum-free culture medium.

[0010] Preferably, the extraction method comprises differential centrifugation.

[0011] Preferably, the differential centrifugation method comprises: performing a first centrifugation on the infection-positive cells, performing a second centrifugation on the first centrifugation supernatant, performing a third centrifugation on the second centrifugation supernatant, and collecting the precipitate;

[0012] The centrifugal force of the first centrifugation is 3000-4000g, the centrifugal force of the second centrifugation is 10000-12000g, and the centrifugal force of the third centrifugation is 100000-200000g.

[0013] Preferably, the first centrifugation time is 15 min; the second centrifugation time is 60 min; and the third centrifugation time is 90 min.

[0014] Preferably, before the third centrifugation, the second centrifugation supernatant is filtered, and the pore size of the filtration membrane is 0.22 μm.

[0015] The present invention also provides engineered extracellular vesicles enriched in circCDK13 prepared by the above preparation method.

[0016] The present invention also provides the use of the engineered extracellular vesicles enriched with circCDK13 in the preparation of a drug for promoting diabetic wound healing.

[0017] Beneficial effects: The present invention provides a method for preparing engineered extracellular vesicles enriched with circCDK13, and collects cell supernatants from mesenchymal stem cells infected with circCDK13 vectors to obtain extracellular vesicles overexpressing circCDK13. The engineered sEVs have stronger biological activity than natural sEVs. The present invention uses the engineered extracellular vesicles enriched with circCDK13 for diabetic wounds, and the therapeutic effect on diabetic wounds is significantly better than that of natural extracellular vesicles secreted by placental mesenchymal stem cells. It not only has an advantage in healing speed, but also has greater application potential in stimulating the regeneration of skin appendages and improving the quality of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the particle size and particle concentration test results of the engineered placental mesenchymal stem cell small extracellular vesicles in Example 1 of the present invention;

[0019] Figure 2 This is a transmission electron microscopic image of the extracellular vesicles of engineered placental mesenchymal stem cells in Example 1 of the present invention;

[0020] Figure 3 This is a graph showing the expression of specific marker proteins on the surface of the extracellular vesicles of engineered placental mesenchymal stem cells in Example 2 of the present invention;

[0021] Figure 4 This is a graph showing the expression level of circCDK13 in the extracellular vesicles of engineered placental mesenchymal stem cells in Example 2 of the present invention;

[0022] Figure 5 This is a diagram showing the results of the effect evaluation analysis of using engineered placental mesenchymal stem cell extracellular vesicles to treat a diabetic mouse wound model in Example 3 of the present invention;

[0023] Figure 6 H&E staining of skin wounds in diabetic mice. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing engineered extracellular vesicles enriched in circCDK13, comprising the following steps: using a vector overexpressing circCDK13 to infect mesenchymal stem cells, and extracting the extracellular vesicles obtained from the infected positive cells, namely the engineered extracellular vesicles enriched in circCDK13.

[0025] The vector referred to in the present invention refers to a self-replicating DNA molecule that transfers DNA fragments (target genes) to recipient cells in genetic engineering recombinant DNA technology, including common bacterial plasmids, bacteriophages and animal and plant viruses. The Vector vector referred to in the embodiment refers to a lentivirus. The vector overexpressing circCDK13 in the present invention preferably includes a lentivirus overexpressing circCDK13. The present invention does not specifically limit the construction method of the lentivirus. The construction is entrusted to a company to meet the requirements of overexpressing circCDK13 (circBase ID: hsa_circ_0079929).

[0026] The present invention utilizes the vector overexpressing circCDK13 to infect mesenchymal stem cells, and the placental mesenchymal stem cells are preferably cells with a cell fusion degree of 40% to 50%. The present invention does not specifically limit the method of infection. In the embodiment, infection is carried out using an infection enhancement solution, and after 48 hours of infection, puromycin is added to obtain infection-positive cells that stably express circCDK3. The mesenchymal stem cells of the present invention preferably include placental mesenchymal stem cells (CP-MSCs), umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, and the like.

[0027] After obtaining the infection-positive cells, the present invention preferably further comprises culturing the infection-positive cells, collecting the cell supernatant, and extracting the engineered extracellular vesicles enriched in circCDK13 from the cell supernatant. The present invention preferably utilizes a mesenchymal stem cell serum-free culture medium to culture the infection-positive cells. The present invention preferably uses differential centrifugation to extract a large number of cultured infection-positive cells, and the extraction preferably comprises differential centrifugation, and more preferably comprises: performing a first centrifugation on the infection-positive cells, performing a second centrifugation on the first centrifugation supernatant, performing a third centrifugation on the second centrifugation supernatant, and collecting the precipitate;

[0028] The centrifugal force of the first centrifugation is 3000-4000g, the centrifugal force of the second centrifugation is 10000-12000g, and the centrifugal force of the third centrifugation is 100000-200000g.

[0029] The time of the first centrifugation of the present invention is preferably 15 minutes; the time of the second centrifugation is preferably 60 minutes; the time of the third centrifugation is preferably 90 minutes. Before the third centrifugation, the present invention preferably further comprises filtering the second centrifugation supernatant, and the pore size of the filtration membrane is preferably 0.22 μm. After the precipitation is obtained by the third centrifugation, the present invention preferably further comprises resuspending with PBS buffer (pH 7.2-7.4), and then performing the third centrifugation, and resuspending again to obtain engineered extracellular vesicles enriched with circCDK13.

[0030] The present invention also provides engineered extracellular vesicles enriched in circCDK13 prepared by the above preparation method.

[0031] The engineered extracellular vesicles enriched with circCDK13 described in the present invention have a typical cup-shaped structure with an average diameter of about 114 nm, and can overexpress circCDK13, and the overexpression of circCDK13 does not affect the morphology and diameter of sEVs; characteristic membrane proteins were detected by western blot, showing that CD63, CD9 and TSG101 were positive.

[0032] The present invention also provides the use of the engineered extracellular vesicles enriched with circCDK13 in the preparation of a drug for promoting diabetic wound healing.

[0033] In the examples of the present invention, through control experiments on diabetic mouse models, it was confirmed that natural sEVs (N-sEVs) secreted from CP-MSCs can promote wound healing in diabetic mice, and sEVs loaded with circCDK13 have significantly enhanced their ability to promote wound healing, and circCDK13-sEVs can accelerate wound healing and regeneration of its appendages, making wound repair more perfect.

[0034] In order to further illustrate the present invention, the engineered extracellular vesicles enriched in circCDK13 and the preparation method and application thereof provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Engineered extracellular vesicles enriched with circCDK13 OE Preparation of -sEVs

[0037] (1) CircCDK13 lentivirus and lentiviral vector were constructed by Guangzhou Gisai Biotechnology Co., Ltd.

[0038] (2) CP-MSCs were seeded into T25 culture flasks and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0039] (3) When the cell confluence reaches 40% to 50%, add circCDK13 lentivirus or Vector into the cultured cells and add the corresponding infection enhancement solution.

[0040] (4) 48 h after infection, puromycin (2 μg / mL) was added to the cell culture medium to obtain CP-MSCs stably expressing circCDK3.

[0041] (5) CircCDK13-overexpressing CP-MSCs (circCDK13-OE group) and their control CP-MSCs (Control group) were cultured on a large scale using MSC serum-free culture medium.

[0042] (6) When the cell confluence reaches 80% to 90%, the cell supernatant is collected for isolation of sEVs, while CP-MSCs continue to be subcultured.

[0043] (7) Place the cell supernatant in a centrifuge tube and centrifuge at 300 g for 10 min at 4°C.

[0044] (8) Collect the supernatant, discard the precipitate, and centrifuge at 3000 g for 15 min at 4°C.

[0045] (9) Collect the supernatant, discard the precipitate, and centrifuge at 10,000 g for 60 min at 4°C.

[0046] (10) Collect the supernatant, discard the precipitate, and filter the supernatant using a 0.22 μm filter.

[0047] (11) The collected filtrate was placed in an ultracentrifuge and centrifuged at 200,000 g for 90 min at 4°C.

[0048] (12) Discard the supernatant, add sterile PBS buffer to resuspend the pellet, and centrifuge at 200,000 g for 90 min at 4°C.

[0049] (13) Discard the supernatant and add sterile PBS buffer to resuspend the precipitate to obtain a sEV suspension. The collected sEVs are stored in separate devices at -80°C, and the separated sEVs are subsequently identified.

[0050] 2. Western blot detection of sEVs surface marker proteins

[0051] (1) The BCA protein quantification kit was used to determine the protein concentration of sEVs.

[0052] (2) Based on the protein quantification results, the amount of sEVs loaded (10–30 μg) was calculated, and 5× protein loading buffer was added in proportion. The mixture was vortexed and mixed, and the mixture was heated in a 100°C metal bath for 10 min. The denatured proteins were subjected to SDS-PAGE electrophoresis.

[0053] (3) Finally, the exosome positive protein markers CD9, TSG101, CD63 and the negative protein marker Calnexin were detected - "three positives and one negative". The primary antibody dilution ratio is as follows: CD63 (1:1000), CD9 (1:1000), TSG101 (1:2000), Calnexin (1:5000).

[0054] 3. Electron microscopy detection of sEVs

[0055] (1) Take 10 μL of sEVs solution and drop it on the copper grid. After incubation at room temperature for 10 min, rinse it with sterile distilled water and finally absorb the excess liquid with absorbent paper.

[0056] (2) Pipette 10 μL of 2% uranyl acetate and drop it onto the copper grid for negative staining for 1 min. Use filter paper to absorb the floating liquid and dry it under an incandescent lamp for 2 min.

[0057] (3) The copper mesh was placed under a transmission electron microscope and imaged at 80 kV.

[0058] 4. Nanoparticle size analysis (NTA) of sEVs

[0059] (1) Clean the sample pool with deionized water.

[0060] (2) The instrument was calibrated with polystyrene microspheres (100 nm).

[0061] (3) Wash the sample pool with 1× PBS buffer.

[0062] (4) Take an appropriate amount of sample, dilute it with 1× PBS buffer, and then add it to the ParticleMetrix nanosystem sample pool for detection.

[0063] (5) After the detection, the software ZetaView 8.04.02SP2 was used for data processing to record the sEVs particle concentration and particle size distribution.

[0064] The results are as follows Figures 1 to 3 As shown, TEM observation of sEVs showed a typical cup-shaped structure ( Figure 2 ), NTA particle size analysis showed that its average diameter was about 114nm ( Figure 1 ), overexpression of circCDK13 did not affect the morphology and diameter of sEVs. In addition, western blot experiments of sEVs characteristic membrane proteins (CD63, CD9 and TSG101) further confirmed the identity of sEVs ( Figure 3 ).

[0065] 5. qRT-PCR detection

[0066] (1) Extraction of total RNA

[0067] Steady Pure rapid RNA extraction kit was used to extract total RNA from cells and sEVs.

[0068] (2) Reverse transcription of RNA into cDNA

[0069] RNA was reverse transcribed into cDNA using the Evo M-MLV reverse transcription reagent premix kit.

[0070] (3) RT-qPCR

[0071] a. Primer synthesis: Primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0072] b. Primer dilution: Before primer dilution, centrifuge at 4000 rpm for 1 min, then add an appropriate amount of RNase free water according to the instructions to dilute the primers shown in Table 1 to 10 μM.

[0073] Table 1 qRT-qPCR primer sequences

[0074]

[0075] c. Preparation of reaction solution: It is best to prepare the reaction solution on ice. The specific preparation method of the reaction solution is shown in Table 2.

[0076] (Note: This is the amount for one reaction)

[0077] Table 2 qRT-PCR reaction system

[0078]

[0079]

[0080] *1 Generally, a final primer concentration of 0.4 μM gives good results. If the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1 to 1.0 μM.

[0081] *2 In a 20μL system, the amount of DNA template added is usually less than 100ng. If necessary, gradient dilution can be performed to determine the appropriate amount of template added; in addition, the volume of cDNA stock solution used should not exceed 10% of the total volume of the qPCR reaction.

[0082] d. PCR reaction procedure

[0083] Pre-denaturation stage: 95℃30sec 1 cycle

[0084] PCR stage: 95°C for 5 sec, 60°C for 30 sec, 40 cycles;

[0085] Melting curve stage: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds for 1 cycle;

[0086] e. Transfer the reaction solution to the PCR amplification microplate and use Micro Amp TMThe plate was sealed with an optical adhesive sealing film, centrifuged at 1000 rpm for 1 min, and finally detected using Quant Studio 5 fluorescent quantitative PCR instrument.

[0087] The results are as follows Figure 4 As shown, the abundance of circCDK13 in circCDK13-sEVs was significantly increased compared with that in N-sEVs, indicating that circCDK13 overexpressed in CP-MSCs was successfully loaded into sEVs.

[0088] 6. CircCDK13 protection experiment in sEVs

[0089] circCDK13-sEVs (1.02×10 12 The number of particles) was measured with 20 μL RNaseA / T1 mixture with or without 1% Triton X-100 and incubated at 37°C for 30 min. Then, the RNase was inactivated at 75°C for 5 min, and total RNA was extracted using the SteadyPure Rapid RNA Extraction Kit. Finally, the copy number of circCDK13 was estimated by absolute quantitative PCR.

[0090] (1) The 220 bp characteristic fragment containing the reverse splicing site of circCDK13 was cloned and inserted into the pUC57 vector, which was designed and constructed by Haichuang Keye Biotechnology Co., Ltd.

[0091] (2) Steady Pure rapid RNA extraction kit was used to extract RNA from cells and sEVs.

[0092] (3) The RNA concentration of the samples was determined using a micro-UV spectrophotometer.

[0093] (4) Reverse transcribe RNA into cDNA using the Evo M-MLV reverse transcription reagent premix kit.

[0094] (5) The plasmid was diluted 10-fold in sequence and a standard curve was drawn by real-time PCR.

[0095] (6) The calculation formula is as follows:

[0096] Copy number (copy / μL) = 6.02 × 10 23 ×Plasmid concentration (ng / μL)×10 -9 / [(number of bases in the vector + number of bases in the inserted fragment) × 660](g / mol).

[0097] The equations for the amplification curve and the standard curve are:

[0098] Y=aX+b [the log 10 value of the initial amplification copy number is the abscissa (X), and the corresponding cycle number is the ordinate (Y)].

[0099] The plasmid standard linear equation was used to calculate the copy number of circCDK13 according to the circCDK13 cycle threshold (CT). Figure 4 As shown, treating circCDK13-sEVs with RNaseA / T1 mixture alone did not reduce the abundance of circCDK13; when circCDK13-sEVs were treated with 1% TritonX-100 (which can break the double-layer membrane structure of sEVs) combined with RNaseA / T1 mixture, the abundance of circCDK13 was significantly reduced, indicating that circCDK13 was protected in intact sEVs.

[0100] Table 3. Copy number statistics of circCDK13

[0101]

[0102] Example 2

[0103] Control experiment on the treatment of diabetic mice with the circCDK13-sEVs preparation prepared in Example 1

[0104] 1. Establishment of diabetic wound model

[0105] (1) Anesthesia: Anesthesia was performed with sodium pentobarbital (mice: concentration 1%, dose 0.1 mL / 20 g; rats: concentration 3%, dose 0.1-0.2 mL / 100 g).

[0106] (2) Surgery: Shave the hair on the surgical area on the back of mice and rats, disinfect the surgical area with iodine, and then create a 10mm diameter full-thickness skin excision wound on both sides of the back spine. (Note: All mice and rats must be kept in separate cages after surgery to prevent them from biting each other's wounds.)

[0107] (3) Grouping: The wounds were randomly divided into three groups: PBS group, N-sEVs group, and circCDK13-sEVs group, with 24 wounds in each group.

[0108] (4) Administration: PBS (100 μL), N-sEVs (100 μL, 2×10 11 / mL), circCDK13-sEVs (100μL, 2×10 11 / mL) were injected subcutaneously at 4 points on the wound edge using a microsyringe (25 μL at each site) on the day after surgery and on the 3rd, 7th, 10th, 14th, and 17th days after surgery.

[0109] (5) Photographic records: On the day after surgery and on days 3, 7, 10, 14, 17, and 21 after surgery, wound healing was observed and photographed. Image J software was used to calculate the wound area, and then the wound healing curves of each group of mice were drawn. Wound size = At / A0×100%, A0 is the initial wound area, and At is the wound area on day t.

[0110] (6) Sampling: The experimental animals were euthanized by an overdose of sodium pentobarbital on the 3rd, 7th, 14th and 21st days after surgery, and the wound surface and the surrounding normal skin were cut. The wound surface was divided into two parts, one half was fixed in 4% paraformaldehyde for histological examination, and the other half was quickly frozen in liquid nitrogen and then stored at -80℃ for future use.

[0111] The general view of the wound surface is as follows Figure 5 As shown in the figure, on days 7, 14, and 21 after wound surgery, the wounds treated with N-sEVs had smaller wound areas compared with the PBS group; and compared with the N-sEVs group, the wounds treated with circCDK13-sEVs had significantly smaller wound areas, and significant hair regeneration was observed on day 21 ( Figure 5 In addition, through quantitative analysis of wound images at each time point, we found that the wound area of ​​the PBS group and the N-sEVs group slightly increased on the third day, and the wounds of each group gradually healed over time. The wound healing rate of the PBS group was the slowest, followed by the N-sEVs group, and the wound healing rate of the circCDK13-sEVs group was the fastest ( Figure 5 The above experimental results show that natural sEVs (N-sEVs) secreted from CP-MSCs can promote wound healing in diabetic mice, and the wound healing ability of sEVs loaded with circCDK13 is significantly enhanced.

[0112] 2. H&E staining of skin wounds

[0113] (1) Dehydration: Use a fully automatic dehydrator to dehydrate the skin samples.

[0114] (2) Embedding: Use pathological grade paraffin with a melting point of 56-58°C for embedding.

[0115] (3) Sectioning: Section the wound from the surface with the largest transverse diameter.

[0116] (4) Baking: Before staining, place the paraffin sections in a 60°C constant temperature drying oven for baking.

[0117] (5) Dewaxing and hydration of paraffin sections: dewaxing with xylene (I) for 10 min, dewaxing with xylene (II) for 10 min, anhydrous ethanol (I) for 2 min, anhydrous ethanol (II) for 2 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 75% ethanol for 2 min, and distilled water for 2 min.

[0118] (6) Stain with hematoxylin solution for 3 to 10 minutes and rinse with tap water for 5 to 10 seconds.

[0119] (7) Use differentiation solution (1% hydrochloric acid alcohol) for 1 to 5 seconds and rinse with tap water for 20 to 30 seconds.

[0120] (8) Ammonia water (1% ammonia water) turns blue for 10 to 30 seconds, and then rinse with tap water for 20 to 30 seconds.

[0121] (9) Stain with eosin for 30 to 2 minutes and rinse with tap water for 1 to 5 seconds.

[0122] (10) Dehydration and transparency: 75% ethanol for 2 to 3 seconds, 85% ethanol for 2 to 3 seconds, 95% ethanol for 2 to 3 seconds, anhydrous ethanol (I) for 2 to 3 seconds, anhydrous ethanol (II) for 1 minute, xylene (I) for 1 minute, xylene (II) for 1 minute.

[0123] (11) Sealing and observation: Neutral gum was used for sealing, and finally the H&E sections were scanned panoramically using a fully automatic digital pathology scanner.

[0124] The results are as follows Figure 6 As shown in the figure, compared with the N-sEVs group, the epithelial tongue in the circCDK13-sEVs group was longer and thicker, indicating that circCDK13-sEVs has a stronger ability to promote epidermal cell proliferation and migration ( Figure 6 Middle a). On days 3, 7, 14, and 21 after wound surgery, the skin wounds were stained with H&E and quantified. The results showed that the wound healing rate in the PBS group was the slowest, followed by the N-sEVs group, and the wound healing rate in the circCDK13-sEVs group was the fastest ( Figure 6 (a). On the 21st day, no new hair follicles were observed in the wound of the PBS group, while a large number of regenerated skin appendages (hair follicles and sebaceous glands) were observed in the wound of the circCDK13-sEVs group. The above experimental results show that circCDK13-sEVs can accelerate wound healing and the regeneration of its appendages, making wound repair more perfect.

[0125] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing engineered extracellular vesicles enriched with circCDK13, characterized in that: The following steps are involved: Placental mesenchymal stem cells are infected with a vector that overexpresses circCDK13, and the extracellular vesicles extracted from the infected positive cells are the engineered extracellular vesicles enriched in circCDK13.

2. The preparation method according to claim 1, characterized in that: The vector overexpressing circCDK13 includes a lentivirus overexpressing circCDK13.

3. The preparation method according to claim 1, characterized in that: After obtaining the infection-positive cells, the method further includes culturing the infection-positive cells, collecting the cell supernatant, and extracting the engineered extracellular vesicles enriched with circCDK13 from the cell supernatant.

4. The preparation method according to claim 3, characterized in that: The method comprises culturing the infection-positive cells using a mesenchymal stem cell serum-free culture medium.

5. The preparation method according to claim 1 or 3, characterized in that: The extraction method includes differential centrifugation.

6. The preparation method according to claim 5, characterized in that: The differential centrifugation method comprises: performing a first centrifugation on the infection-positive cells, performing a second centrifugation on the first centrifugation supernatant, performing a third centrifugation on the second centrifugation supernatant, and collecting a precipitate; The centrifugal force of the first centrifugation is 3000-4000g, the centrifugal force of the second centrifugation is 10000-12000g, and the centrifugal force of the third centrifugation is 100000-200000g.

7. The preparation method according to claim 6, characterized in that: The first centrifugation time is 15 minutes; the second centrifugation time is 60 minutes; and the third centrifugation time is 90 minutes.

8. The preparation method according to claim 6, characterized in that: Before the third centrifugation, the second centrifugation supernatant is filtered, and the pore size of the filter membrane is 0.22 μm.

9. Engineered extracellular vesicles enriched in circCDK13 prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the engineered extracellular vesicles enriched with circCDK13 according to claim 9 in the preparation of a drug for promoting diabetic wound healing.

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