Application of Hsacirc0005379 in promoting healing of diabetic wound

Abstract

The invention provides an application of Hsacirc0005379 in promoting healing of diabetic wounds, a hair follicle dermal stem cell exosome derived from Hsacirc0005379 and an application of Hsacirc0005379 in promoting healing of chronic wounds and healing of diabetic wounds, which are beneficial to understanding a double-cell interaction mechanism delivered by HFDSC-Exos. The effects of mitochondria and polarization states of macrophages in the diabetic wound microenvironment are determined, and the method has important significance for developing a new strategy for treating the diabetic wound by stem cells.

Description

Technical Field

[0001] The present invention provides application of Hsa_circ_0005379 in promoting diabetic wound healing, and belongs to the technical field of biomedicine. Background Art

[0002] Diabetes is another major disease that endangers human physical and mental health after cancer and cardiovascular disease. According to data from the International Diabetes Federation, it is estimated that more than 700 million people worldwide will suffer from the disease by 2045. The probability of diabetic patients developing diabetic wounds in their lifetime is about 25%, and treatment is prone to recurrence and has high mortality and disability rates. The pathogenesis of diabetic wounds is complex and is closely related to the local hyperglycemia environment of the wound, accumulation of advanced glycation end products, oxidative damage, abnormal metabolism and persistent chronic inflammation. Stem cell therapy for diabetic wounds has been studied for a long time. In the past, it has basically become an industry consensus that adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, exosomes and platelet-rich plasma preparations promote wound healing. However, due to unstable effects and safety issues with stem cell preparations, the clinical application of "stem cell therapy for diabetic wounds" has progressed slowly. Therefore, there is an urgent need to explore new methods for the treatment of diabetic wounds.

[0003] Macrophages are central innate multifunctional immune cells that play different roles in different stages of wound healing. Macrophages can be induced to polarize into pro-inflammatory (M1) in the wound, producing important pro-inflammatory factors such as TNF-α and IL-6; or anti-inflammatory (M2), producing anti-inflammatory factors such as TGF-β and IL-4. In diabetic wounds, the macrophage phenotype is significantly characterized by an increase in the number and proportion of M1 polarization, accompanied by abnormal macrophage metabolism and persistent inflammation. Therefore, regulating the local abnormal metabolic level of diabetic wounds, breaking the persistent inflammatory state of macrophages, and targeted promotion of macrophage polarization to M2 are very promising and attractive methods for treating diabetic wounds.

[0004] Based on the above background, this study aims to provide a stable and biosafe molecule that promotes diabetic wound healing, which is of great significance for the development of new strategies for stem cell treatment of diabetic wounds. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a molecule that promotes diabetic wound healing with stable effects and biosafety, and its application in promoting diabetic wound healing.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a circular RNA for promoting wound healing. The circular RNA is encoded as Hsa_circ_0005379, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0007] A second aspect of the present invention provides a biomaterial comprising: 1) Primers, which can amplify the circular RNA described in the first aspect of the present invention; 2) A recombinant vector comprising the circular RNA described in the first aspect of the present invention; 3) a recombinant host cell comprising the circular RNA described in the first aspect of the present invention and / or the vector described in 2); or 4) A polypeptide or protein complex comprising the translation product of the circular RNA described in the first aspect of the present invention.

[0008] Furthermore, the primers include a forward primer and a reverse primer.

[0009] Furthermore, the nucleotide sequence of the forward primer is shown in SEQ ID NO:2.

[0010] Furthermore, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3.

[0011] Furthermore, the recombinant vector includes a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome and / or a phage vector.

[0012] Furthermore, the recombinant host cell is a prokaryotic cell or a eukaryotic cell.

[0013] Furthermore, the prokaryotic cells include Escherichia coli cells.

[0014] Furthermore, the eukaryotic cells include yeast cells, CHO cells, 293 cells, and plant cells.

[0015] The third aspect of the present invention provides an exosome that promotes wound healing. The exosome is secreted by hair follicle dermal stem cells and contains the circular RNA described in the first aspect of the present invention.

[0016] Furthermore, the exosome is a hemispherical tray-shaped structure with one side concave.

[0017] The exosomes express CD9, CD63, and TSG101.

[0018] The particle size of the exosomes is 50-300 nm.

[0019] Furthermore, the average particle size of the exosomes is 150 nm.

[0020] The fourth aspect of the present invention provides the use of the circular RNA described in the first aspect of the present invention, the recombinant vector, recombinant host cell, polypeptide or protein complex in the biomaterial described in the second aspect of the present invention and / or the exosome described in the third aspect of the present invention, and the use includes the following: 1) Application in the preparation of drugs or products for promoting chronic wound healing; 2) Application in the preparation of drugs or products that promote diabetic wound healing; 3) Application in the preparation of products that promote macrophage M2 polarization in a high-glucose environment; 4) Application in the preparation of products for regulating the expression of SIRT2 and / or TFAM in macrophages.

[0021] Furthermore, the chronic wound includes one or more of chronic inflammatory wounds, surgical wounds, ulcer wounds, and burn wounds.

[0022] Furthermore, the chronic inflammatory wound surface includes one or more of a diabetic wound surface and a diabetic ulcer wound surface.

[0023] Furthermore, the drug may be used alone or in the form of a pharmaceutical composition.

[0024] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0025] Furthermore, the product includes a medical product, a skin care product or a cosmetic.

[0026] The fifth aspect of the present invention provides a method for promoting chronic wound healing or diabetic wound healing, the method comprising administering to a subject a drug or product comprising the circular RNA described in the first aspect of the present invention and / or the exosomes described in the third aspect of the present invention.

[0027] Furthermore, the chronic wound includes one or more of chronic inflammatory wounds, surgical wounds, ulcer wounds, and burn wounds.

[0028] Furthermore, the chronic inflammatory wound surface includes one or more of a diabetic wound surface and a diabetic ulcer wound surface.

[0029] Furthermore, the drug may be used alone or in the form of a pharmaceutical composition.

[0030] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0031] Furthermore, the product includes a medical product, a skin care product or a cosmetic.

[0032] The sixth aspect of the present invention provides a method for preparing the circular RNA described in the first aspect of the present invention, wherein the method comprises artificial synthesis or synthesis using genetic engineering technology.

[0033] Furthermore, the step of synthesizing by genetic engineering technology includes introducing 2) a recombinant vector in the biological material described in the second aspect of the present invention into 3) a recombinant host cell to express the circular RNA described in the first aspect of the present invention.

[0034] Advantages and beneficial effects of the present invention: This study studied the mechanism of action of Hsa_circ_0005379 in mediating macrophage function and promoting diabetic wound healing at the in vitro and in vivo levels, which is helpful to understand the dual-cell interaction mechanism of HFDSC-Exos delivery, clarify the role of macrophage mitochondria and polarization state in the diabetic wound microenvironment, and is of great significance for the development of new strategies for stem cell treatment of diabetic wounds, and provides new ideas for the clinical treatment of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a picture of HFDSC-Exos promoting wound healing in diabetic mice. Figure 1 A in the figure is a diagram showing cell activity in vivo imaging of small animals. Figure 1 B in the figure is a picture of the wound healing of mice. Figure 1 C in the figure is the level of inflammatory factors in the wound of mice; Figure 2 The diagram of HFDSC-Exos regulating macrophage mitochondrial metabolism level. Figure 2 A in the figure shows the morphology of macrophage mitochondria under electron microscope and the expression of exosome markers detected by WB. Figure 2 B in the figure is the detection diagram of ROS level in macrophages. Figure 2 C in the figure is the flow cytometry detection of macrophage M2 levels. Figure 2 D in the figure is the level of advanced glycation end products and mitochondrial transcription factors. Figure 2 E in the figure is a statistical chart of macrophage M2 levels detected by flow cytometry; Figure 3 The result diagram of macrophages taking up HFDSC-Exos and highly expressing Hsa_circ_0005379. Figure 3 A in the figure is the RNA-SEQ result diagram. Figure 3 B is the expression level of Hsa_circ_0005379 in HFDSC-Exos detected by q-PCR. Figure 3 C in the figure is the fluorescence staining image of macrophages taking up exosomes observed by laser scanning confocal microscopy. Figure 3 D in the figure is the statistical graph of Hsa_circ_0005379 expression level after co-culture of macrophages and HFDSC-Exos; Figure 4 This is a diagram showing the state of Hsa_circ_0005379 in the cytoplasm of macrophages. Figure 4A in the figure is the cyclization site map of Hsa_circ_0005379. Figure 4 B in the figure is the expression level of Hsa_circ_0005379 in different intracellular locations detected by electrophoresis. Figure 4 C in the figure is the fluorescence localization map of Hsa_circ_0005379 in the cytoplasm of macrophages; Figure 5 This is the transcription factor SIRT2 protein binding Hsa_circ_0005379 map, Figure 5 A in the figure is the potential target map of Hsa_circ_0005379 predicted by bioinformatics software. Figure 5 B in the figure is the difference diagram of Hsa-miR-23a-3p verified by q-PCR. Figure 5 C in the figure is a detection diagram of the effect of high expression of Hsa-miR-23b-3p on macrophage phenotype under continuous high glucose environment. Figure 5 D in the figure is the result of the Hsa_circ_0005379 ligation probe and SIRT2 protein pull-down experiment. Figure 5 E in is the mass spectrum of peptide sequence, Figure 5 F in is the catRAPID predicted binding site map, Figure 5 G in the figure is the RIP method to detect SIRT2 pulling down Hsa_circ_0005379; Figure 6 This is a diagram showing that transcription factor SIRT2 regulates macrophage M2 polarization through TFAM. Figure 6 A in the figure is the flow cytometry detection of the polarization ratio of macrophages M2. Figure 6 B in the figure is the ROS staining of macrophages. Figure 6 C in the figure is the expression level of TFAM when overexpressing SIRT2 detected by WB. Figure 6 D in the figure is the AGE expression among the ELISA detection groups; Figure 7 Hsa_circ_0005379 regulates macrophage polarization by upregulating TFAM through SIRT2. Figure 7 A in the figure is the electron microscopic image of mitochondria in different groups. Figure 7 B in the figure is the ROS staining of macrophages. Figure 7 C in the figure is the M2 polarization ratio diagram detected by flow cytometry. Figure 7 D in the figure is the statistical diagram of ROS staining. Figure 7 E in the figure is the macrophage polarization statistics diagram. Figure 7 F in the figure is the statistical chart of TFAM expression level detected by ELISA. Figure 7 The G in the figure is the statistical chart of AGE expression level detected by ELISA. Figure 7 H in the figure is the result of RIP and PCR detection; Figure 8 This is a diagram showing how Hsa_circ_0005379 promotes wound healing in diabetic mice. Figure 8 A in the figure is a diagram of wound healing in a living animal model. Figure 8 B in the figure is the immunohistochemical CD26 detection image of the wound surface in each group. Figure 8 C in the figure is HE staining picture of the new epidermis in each group. Figure 8 D in the figure is the injection diagram of each group of mice. Figure 8 E in the figure is the statistical chart of wound healing rate in each group. Figure 8 F in the figure is the statistical diagram of CD206 expression level. Figure 8 G in the figure is the statistical graph of epidermal thickness stained with HE; Fig. 9 Schematic diagram of the mechanism by which Hsa_circ_0005379 promotes diabetic wound healing in mice. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] In the present invention, the term "circular RNA" refers to a special class of non-coding RNA molecules, which are closed RNA molecules formed by reverse splicing, unlike traditional linear RNA. Circular RNA does not have a 5' end cap and a 3' end poly (A) tail. Through exon cyclization or intron cyclization, the 3' and 5' ends are connected to form a complete circular structure to avoid degradation by nuclease exonucleases. It is more stable and more conservative than linear RNA. Hsa_circ_0005379 refers to a nucleic acid molecule named according to the standardized naming system of the circbank database, where "Hsa" refers to human origin and "circ" refers to circular. Those skilled in the art can clearly know the circular RNA it refers to through the number.

[0038] In the present invention, the term "wound" refers to the damage to normal skin (tissue) caused by external injury factors such as surgery, external force, heat, electric current, chemical substances, low temperature and internal factors of the body such as local blood supply disorder. It is often accompanied by the destruction of skin integrity and the loss of a certain amount of normal tissue. Wounds can usually be divided into acute wounds and chronic wounds according to the healing time, and can be divided into epidermal wounds, dermal wounds and full-thickness wounds according to the depth of the wound. And according to the degree of infection of the wound, it can be divided into clean wounds, contaminated wounds and infected wounds. According to the cause of injury, it can be divided into physical wounds, chemical wounds, electrical wounds, radiation wounds and pathological wounds. In some embodiments, the wound refers to a chronic wound, preferably a diabetic wound, including a diabetic wound wound and a diabetic ulcer wound, and a diabetic ulcer wound usually appears on the foot.

[0039] In the present invention, wound healing refers to a natural physiological process, which is essentially an inherent defensive adaptive response of the body to tissue cell damage caused by various harmful factors. The healing response includes: 1) regeneration of original tissue cells, and / or 2) non-specific connective tissue hyperplasia replacing original tissue cells. In some embodiments, wound healing refers to the reduction of wound area, and promoting wound healing means that the time for the wound area to be reduced is shorter than the original healing time required.

[0040] In the present invention, a recombinant vector comprising the circular RNA of the present invention is provided. The term "recombinant vector" refers to an artificial construct that is capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The recombinant vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) a transcription subunit of appropriate transcription and translation start and stop sequences. The recombinant vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentiviruses, retroviruses, vaccinia, adenoviruses, fowlpox, baculoviruses, SV40, and pseudorabies. In the present invention, the term "recombinant host cell" refers to introducing the vector of the present invention into a host cell, so that the recombinant host cell can transcribe the target nucleic acid sequence and / or translate the target protein.

[0041] In the present invention, the term "polypeptide or protein complex" refers to a single polypeptide or multiple associated polypeptides. In some embodiments, it refers to the translation product of the circular RNA described in the first aspect of the present invention, which includes a polypeptide chain translated from any start site of the circular RNA, or a polypeptide chain encoded by any open reading frame in the circular RNA.

[0042] In the present invention, the term "exosome" is an extracellular vesicle, generally having a hydrodynamic diameter of nanometer scale, i.e., a particle size of less than 1000 nm. Exosomes can be derived from any cell type in vivo, in vitro and in vitro, including but not limited to primary cells, cell lines, cells present in multicellular organisms, or substantially any other type of cell source. The term should also be understood to refer to exosome mimetics, cell membrane-based vesicles obtained by, for example, membrane extrusion, ultrasonic treatment or other techniques, and their contents. It is obvious to those skilled in the art that when describing the medical and scientific uses and applications of exosomes, the present invention generally relates to a plurality of exosomes, i.e., exosome populations that may contain thousands, millions, billions or even trillions of exosomes. In some embodiments, the exosomes are exosomes derived from human hair follicle dermal stem cells.

[0043] In the present invention, the term "medical product", "skin care product" or "cosmetics" refers to medical, cosmetic, and cosmetic products whose efficacy includes damaged skin repair and skin barrier repair, and can be used alone or in combination with other products that promote damaged skin repair, reduce skin inflammation, and promote wound healing. As long as the target tissue can be reached, the medical product, skin care product or cosmetic of the present invention can be administered to the receptor by any route. Its preparation type is essence, eye cream, cream, freeze-dried powder, solution, emulsion, cream, gel, mask or dressing.

[0044] In the present invention, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implanted drug storage devices. The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant, or excipient. In some cases, a pharmaceutical acid, base, or buffer may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention may be administered to the receptor by any route. In some embodiments, administration by injection is preferred. The pharmaceutical composition of the present invention can also be used in combination with other drugs that promote chronic wound healing or diabetic wound healing. Other compounds that promote chronic wound healing or diabetic wound healing can be administered simultaneously with the main active ingredient (e.g., the circular RNA described in the first aspect of the present invention), or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient.

[0045] In the present invention, the term "pharmaceutically acceptable excipient" refers to any pharmaceutical excipient that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be used without excessive toxicity. Suitable excipients can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such excipients are well known to those of ordinary skill in the art. The pharmaceutically acceptable excipient in the pharmaceutical composition can include fluids, such as water, saline, glycerol and ethanol. Adjuvants, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0046] In the present invention, the term "subject" includes any individual of interest, preferably a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0047] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are commercially available unless otherwise specified. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0048] Example 1 Study on the mechanism of action of HFDSCs-Exos and Hsa_circ_0005379 in vitro 1. Acquisition and characterization of HFDSC-Exos: Human hair follicle dermal stem cells (HFDSCs) and human macrophages (purchased from Pricella Biotechnology, Wuhan, China, and ATCC, Manassas, USA). Exosomes derived from HFDSCs (HFDSC-Exos) were obtained using a differential ultracentrifugation protocol. The particle size distribution of exosomes was characterized using nanoparticle tracking analysis (NTA) technology. The ultrastructure of exosomes was observed using transmission electron microscopy. Western blot analysis was used to evaluate the expression of HFDSC exosome-positive markers, including CD9, CD63, and TSG101 (Proteintech, China). The characterization results of human hair follicle dermal stem cell exosomes HFDSC-Exos are shown in Figure 1. Figure 2 As shown in A, HFDSC-Exos were observed to be a hemispherical saucer-shaped structure with one side concave under electron microscopy. WB detected positive expression of exosome markers CD9, CD63 and TSG101. NTA detected that the particle size range was 50-300 nm, with an average particle size of about 150 nm.

[0049] 2. Study on the mechanism of action of HFDSCs-Exos and Hsa_circ_0005379 1) Flow cytometry analysis: The density of macrophages was adjusted to 1×10 6 / ml, gently suspend the cells in PBS and transfer to a flow cytometer tube. After centrifugation, discard the supernatant and resuspend the cells in binding buffer. Then, add CD11b and CD206 (Proteintech, China) and mix thoroughly in the dark. After 15 minutes, analyze the cells by flow cytometry. Cell apoptosis was analyzed using FlowJo_V10 software. The results are shown in Figure 2 As shown in C and E, the M2 polarization of macrophages increased in the HFDSC-Exos group, and after the addition of exosome inhibitors, the M2 polarization of macrophages decreased.

[0050] 2) ROS fluorescence staining: Macrophages were evenly plated on a confocal culture dish, and the working solution of MitoSOX Red (Beyotime Biotechnology, China) was diluted 1:1000 to a final concentration of 5 μM to cover the cell surface. The cells were incubated at 37°C in the dark for 30 minutes. After incubation, the cells were gently washed three times with preheated buffer and imaged using a laser scanning confocal microscope. The results are shown in Figure 2. Figure 2 As shown in B and D, ROS reactive oxygen species in macrophages in the HFDSC-Exos group decreased, and ROS increased after the addition of exosome inhibitors.

[0051] 3) Enzyme-linked immunosorbent assay (ELISA): Select an appropriate ELISA kit based on the target protein and add samples and standards according to the kit instructions. After adding antibodies and colorimetric substrates, use a microplate reader to measure the optical density (OD value) of each well. Calculate the concentration of the analyte in the sample based on the standard curve. The results are as follows: Figure 2 As shown in D, the advanced glycation end products (AGE) in macrophages in the HFDSC-Exos group decreased and the expression of mitochondrial transcription factor A (TFAM) increased, indicating that HFDSC-Exos can regulate macrophage mitochondrial metabolism and cellular oxidation levels.

[0052] 4) Staining of exosome uptake by macrophages: The cells were fixed in 4% formaldehyde solution for 30 min at room temperature. The cells were washed three times with PBS to remove the fixative. The working solution of phalloidin (Biosharp, China) was added to the cells and incubated for 60 min at room temperature. The nuclei were stained with DAPI to facilitate the observation of the overall cell structure recorded using confocal microscopy. The results are shown in Figure 2. Figure 3 As shown in C, PKH26 labeling exosomes shows red fluorescence, Phalloidin labeling actin shows green fluorescence, and DAPI labeling cell nuclei shows blue fluorescence, showing that macrophages take up HFDSC-Exos.

[0053] 5) RNA library preparation and high-throughput sequencing: In a continuous high-glucose environment (25 mM), HFDSC Exos (1×10 9 ) and macrophages (1×10 5 / mL) for 72 hours. Whole transcriptome sequencing (YUNXU, China) of macrophage RNA before and after culture was performed to detect differentially expressed circular RNAs (circRNAs), which were then further validated and screened using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Figure 3 As shown in A.

[0054] 6) Quantitative real-time PCR: The top 10 circRNAs that were significantly highly expressed in macrophages after co-culture were selected from the RNA-SEQ results, and total RNA was extracted and then converted to cDNA using the PrimeScript RT reagent kit (Takara, Tokyo, Japan). qRT-PCR analysis was then performed using the Sybr green system, with GAPDH as an internal standard. -ΔΔCT Methods: The relative expression levels of RNA were calculated. The primer sequences are shown in Table 1. The results are shown in Figure 3 As shown in B, the expression level of Hsa_circ_0005379 increased most significantly.

[0055] Table 1. Circular RNA amplification primer sequence list

[0056] 7) Western blotting (WB): Establish a co-culture system of macrophages and HFDSC-Exos, extract proteins from each group of cells and quantify them. Separate proteins with SDS-polyacrylamide gel and transfer them to nitrocellulose membranes. Incubate the membrane with primary antibodies CD9, CD63, TSG101 and anti-β-actin (1:1000, Cell Signaling Technology, USA) antibodies at room temperature for 2 hours, and then incubate with secondary antibodies. Use Image Pro Plus 6.0 to analyze the grayscale value of the target protein. Calculate the expression level of the target protein relative to β-actin. The results are shown in Figure 2. Figure 3 As shown in D, the expression level of Hsa_circ_0005379 was significantly increased after macrophages were co-cultured with HFDSC-Exos.

[0057] 8) Fluorescence in situ hybridization (FISH): The Cy3-labeled Hsa_circ_0005379 probe was designed and synthesized by GenePharma. According to the manufacturer's instructions, the probe signal in macrophages was detected using a fluorescence in situ hybridization (FISH) kit (GenePharma, China), and the cell nuclei were stained with DAPI. All images were acquired using a Leica TCS SP8 laser scanning confocal microscope. Macrophages were treated with exoribonuclease, and it was found that the expression of Hsa_circ_0005379 was not affected; the cyclization site of Hsa_circ_0005379 was detected by Sanger sequencing to further confirm its circularization. Agarose gel electrophoresis and immunofluorescence labeling showed that the circular RNA molecule Hsa_circ_0005379 was present in the cytoplasm of macrophages, and the results are shown in Figure 2. Figure 4 shown.

[0058] 9) Bioinformatics software predicts targets: The potential targets of Hsa_circ_0005379 were predicted by bioinformatics software, and Hsa-miR-23a-3p was verified by q-PCR to be significantly different; however, it was verified that high expression of Hsa-miR-23b-3p had no significant effect on the phenotype of macrophages under a continuous high glucose environment. The results are as follows Figure 5 As shown in AC, Hsa_circ_0005379 does not regulate macrophage function through the ceRNA mechanism.

[0059] 10) Biotin RNA pull-down analysis: Macrophages were lysed by sonication in RIP buffer containing protease inhibitors and RNase inhibitors, and the cell lysate was pre-washed with streptavidin magnetic beads (Invitrogen, USA). In vitro transcribed biotin-labeled RNA or DNA probes adsorbed on streptavidin magnetic beads were incubated with cell lysates at 4°C for 6 hours and then washed three times with RIP buffer. Potential interacting proteins were evaluated by Western blotting or mass spectrometry analysis. The results are shown in Figure 5 As shown in DG in Figure , it can be seen that Hsa_circ_0005379 may play a role by binding to SIRT2 protein.

[0060] 3. Study on the mechanism of Hsa_circ_0005379 regulating SIRT2 1) Effect of transcription factor SIRT2 on macrophages: Through flow cytometry, WB and ELISA detection, it was found that after overexpression of SIRT2, the M2 polarization ratio of macrophages increased, ROS decreased, and TFAM expression increased; when SIRT2 and TFAM were overexpressed at the same time, the M2 polarization ratio increased further and ROS decreased further, showing the synergistic effect of SIRT2 and TFAM on macrophage polarization and anti-oxidation. The results are as follows Figure 6 shown.

[0061] 2) Transmission electron microscopy: Macrophages were pre-fixed with a 1:1 ratio of growth medium to 2.5% glutaraldehyde for 5 min at room temperature. After washing and serial dehydration in ethanol, the samples were embedded in SPON12 resin, polymerized at 60°C, and ultrathin 70 nm sections were cut. The sections were double stained with uranyl acetate and lead citrate. After air drying, mitochondrial morphology was examined by transmission electron microscopy at an accelerating voltage of 80 kV. The results are shown in Figure 2. Figure 7 As shown in the figure, after overexpression of Hsa_circ_0005379 in macrophages, the expression of SIRT2 and TFAM increased, AGE decreased, and ROS decreased; the mitochondrial ultrastructure was normal, mainly oval or elliptical; after overexpression of Hsa_circ_0005379 and SIRT2, the expression of ROS and AGE decreased, and abundant cristae structures were visible in mitochondria; at the same time, under this condition, the M2 polarization ratio increased. In further SIRT2 recovery experiments, downregulation of SIRT2 can effectively reverse the translation regulation of TFAM by Hsa_circ_0005379, increase ROS; increase mitochondrial vacuoles, and irregular distribution; and after upregulation of Hsa_circ_0005379, RIP and PCR results show that SIRT2 has an enhanced ability to upregulate TFAM.

[0062] The above results indicate that HFDSC-Exos-derived Hsa_circ_0005379 can bind to Silent Information Regulator 2 (SIRT2) and mediate abnormal metabolism and polarization function of macrophages through mitochondrial transcription factor A (TFAM).

[0063] Example 2 Study on the mechanism of action of HFDSCs-Exos and Hsa_circ_0005379 at the in vivo level 1. Establishment of diabetic wound model: 46-week-old BALB / c nude mice (sex is not limited) were purchased from the Animal Center of Xuzhou Medical University for experiments approved by the Animal Ethics Committee (approval number: 202309T011). Diabetes was induced with streptozotocin (50 mg / kg) within two weeks. Mice with blood glucose of 16.7 to 28.0 mmol / L and diabetic symptoms were selected. A 15 mm full-thickness skin wound was created and monitored daily until healing.

[0064] 1) Grouping: The mice were randomly divided into 5 groups, 4 in each group, and subcutaneously injected with PBS and MSCs (mesenchymal stem cells), ADSCs (adipose stem cells), HFDSCs (hair follicle dermal stem cells), and HFDSCs-Exos (hair follicle dermal stem cell exosomes).

[0065] 2) Calculation of healing rate: On days 0, 3, 7, and 14, the area was measured using ImageJ software (National Institutes of Health), and the wound healing rate was determined by applying the following formula: (initial wound area - area on day X) / initial wound area.

[0066] 3) Survival of wound cells: After collecting cells or exosomes, they were labeled with CM-Dil (1 μmol / L) and incubated for 30 minutes. The labeled samples were then injected into the wounds of mice (1×10 9 / 0.2 mL). Fluorescence images were taken using the IVIS Lumina S5 system at 24 and 36 hours to evaluate cell survival and exosome retention in the wound.

[0067] 2. Detection of the effect of Hsa_circ_0005379 on wound healing and M2 polarization: Hsa_circ_0005379 overexpression plasmid, empty overexpression plasmid (VEC1), knockout plasmid and empty knockout plasmid (VEC2) were co-transfected with auxiliary virus packaging plasmids pMD2.G and pSPAX2 (Obio, China) for virus packaging. After 48 hours of transfection, the supernatant (containing virus) was collected, concentrated and purified to infect macrophages.

[0068] 1) Grouping: The mice were randomly divided into 5 groups, 4 mice in each group, and subcutaneously injected with HFDSC-Exos, HFDSCcirc+-Exos, VEC1, HFDSCirc-Exos, and VEC2.

[0069] 2) HE and immunohistochemical staining: Skin samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​at 5 μm. The sections were dewaxed, rehydrated, and stained with H&E for histological examination. After blocking with 1% goat serum albumin, they were incubated with anti-CD206 and anti-F4 / 80 primary antibodies at 4°C overnight, and then stained with rabbit anti-mouse secondary antibodies for 1 hour at room temperature. The slides were washed and mounted with medium containing DAPI.

[0070] 3) Statistical analysis: SPSS 22.0 was used for statistical analysis, and data are presented as mean ± standard deviation. One-way ANOVA and t-test found significant differences at P < 0.05. GraphPad software (San Diego, USA) was used to manage and analyze data.

[0071] 3. Experimental results 1) Compared with PBS, MSCs and ADSCs, the wound surface of HFDSCs and HFDSC-Exos groups was ruddy in color, had less exudation, less secretion of inflammatory factors, and accelerated the healing of diabetic wounds. Figure 1 shown.

[0072] 2) Overexpression and knockdown of Hsa_circ_0005379 in HFDSCs, enrichment of exosomes in each group and injection, found that the wound healing in the Hsa_circ_0005379 overexpression group was the fastest, the CD26 positive rate (M2 polarization ratio) was higher, and HE staining showed that the new epidermis was thicker. The results are as follows Figure 8 shown.

[0073] The above results indicate that HFDSC-Exos and Hsa_circ_0005379 can promote the healing of chronic wounds, especially the healing of diabetic wounds.

[0074] In summary, Hsa_circ_0005379 is delivered from HFDSCs to recipient macrophages via exosomes. Under continuous high-glucose environment, Hsa_circ_0005379 upregulates TFAM by binding to SIRT2 to increase the metabolic level of macrophages and promote their polarization to M2, thereby promoting the healing of diabetic wounds. Fig. 9 The schematic diagram shows the mechanism by which Hsa_circ_0005379 promotes diabetic wound healing in the present invention.

[0075] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A circular RNA for promoting wound healing, characterized in that: The circular RNA is encoded as Hsa_circ_0005379, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. A biomaterial comprising: 1) A primer capable of amplifying the circular RNA according to claim 1; 2) A recombinant vector comprising the circular RNA according to claim 1; 3) A recombinant host cell comprising the circular RNA of claim 1 and / or the vector of claim 2); or 4) A polypeptide or protein complex comprising the translation product of the circular RNA according to claim 1.

3. The biomaterial according to claim 2, characterized in that The primers include a forward primer and a reverse primer; Preferably, the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 2; Preferably, the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 3; Preferably, the recombinant vector comprises a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome and / or a phage vector; Preferably, the recombinant host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cells include Escherichia coli cells; Preferably, the eukaryotic cells include yeast cells, CHO cells, 293 cells, and plant cells.

4. An exosome that promotes wound healing, characterized in that: The exosomes are secreted by hair follicle dermal stem cells, and contain the circular RNA according to claim 1.

5. The exosome according to claim 4, characterized in that The exosome is a hemispherical saucer-shaped structure with one side concave; The exosomes express CD9, CD63, and TSG101; The particle size of the exosomes is 50-300 nm; Preferably, the average particle size of the exosomes is 150 nm.

6. Use of the circular RNA according to claim 1, the recombinant vector, recombinant host cell, polypeptide or protein complex in the biomaterial according to claim 2 or 3, and / or the exosome according to claim 4 or 5, the use comprising the following: 1) Application in the preparation of drugs or products for promoting chronic wound healing; 2) Application in the preparation of drugs or products that promote diabetic wound healing; 3) Application in the preparation of products that promote macrophage M2 polarization in a high-glucose environment; 4) Application in the preparation of products for regulating the expression of SIRT2 and / or TFAM in macrophages.

7. The use according to claim 6, characterized in that: The chronic wound surface includes one or more of chronic inflammatory wound surface, surgical wound surface, ulcer wound surface, and burn wound surface; Preferably, the chronic inflammatory wound surface includes one or more of a diabetic wound surface and a diabetic ulcer wound surface; Preferably, the drug can be used alone or in the form of a pharmaceutical composition; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the product comprises a medical product, a skin care product or a cosmetic.

8. A method for promoting the healing of chronic wounds or diabetic wounds, characterized in that: The method comprises administering to a subject a drug or product comprising the circular RNA of claim 1 and / or the exosomes of any one of claims 4-5.

9. The method according to claim 8, characterized in that The chronic wound surface includes one or more of chronic inflammatory wound surface, surgical wound surface, ulcer wound surface, and burn wound surface; Preferably, the chronic inflammatory wound surface includes one or more of a diabetic wound surface and a diabetic ulcer wound surface; Preferably, the drug can be used alone or in the form of a pharmaceutical composition; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the product comprises a medical product, a skin care product or a cosmetic.

10. A method for preparing the circular RNA according to claim 1, characterized in that: The method includes artificial synthesis or synthesis by genetic engineering technology; Preferably, the step of synthesizing by genetic engineering technology comprises introducing 2) a recombinant vector in the biological material of claim 2 into 3) a recombinant host cell to make it express the circular RNA of claim 1.

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