Preparation method and application of extracellular vesicles of gardenia jasminoides

Abstract

The invention discloses a preparation method and application of CWTEV (Chloranthus parasiticus extracellular vesicles), and belongs to the technical field of biomedicines.The CWTEV is prepared through polymer precipitation method extraction and chromatographic column purification, and the CWTEV is characterized from the aspects of morphology, particle size, Zeta potential and the like; in-vitro activity research is carried out on the CWTEV, and the influence of the CWTEV on proliferation and migration of skin HaCaT cells is detected through a CCK-8 method and a cell scratching method respectively; carrying out transcriptomics and small RNA detection on the CWTEV and the HaCaT cells subjected to interaction culture, constructing a high-throughput omics database, and screening out a target gene corresponding to the miRNA of the CWTEV; encapsulating candidate miRNAs by using a liposome microcapsule, and carrying out cell proliferation and cell migration experiments; the functions of the candidate target genes are verified through Q-PCR and WB experiments, and a molecular mechanism for promoting wound healing of the CWTEV is determined.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a method for preparing and applying extracellular vesicles of Citrus wilsonii Tanaka. Background Art

[0002] Traditional Chinese medicine Citrus wilsonii Tanaka is the dried ripe fruit of Citrus wilsonii Tanaka and Citrus medica L. (CML) of the Rutaceae family. Currently, Citrus wilsonii Tanaka is mainly used in the medicinal material market. The index component of Citrus wilsonii Tanaka is the flavonoid naringin, with a content of ≥2.5%. According to modern pharmacological research, the compounds contained in Citrus wilsonii Tanaka have various biological activities such as analgesic, anti-inflammatory, antiviral, antibacterial, and antioxidant effects. The naringin component contained in Citrus wilsonii Tanaka can block the signal transduction of NF-κB and alleviate lipopolysaccharide-induced acute lung injury in mice; the naringenin component can inhibit the pro-inflammatory signaling pathways of cells such as hepatocytes and macrophages and inhibit the production of inflammatory factors such as TNF-α and IL-6; the flavonoid active components are famous for their antioxidant and anti-inflammatory properties, can promote wound healing and maintain skin health. Therefore, Citrus wilsonii Tanaka can also be used as an external application drug and has a certain curative effect on skin diseases.

[0003] Plant extracellular vesicles are nanoscale vesicles secreted by plant cells. In 2007, nanoscale extracellular vesicles secreted by plant cells were discovered. Their morphology and structure are similar to those of animal cell exosomes. These vesicles also contain biomolecules such as proteins, RNAs, and lipids, and can be released outside plant cells to interact with surrounding cells. They are disc-shaped or cup-shaped, and their morphology can be observed by transmission electron microscopy (TEM). Plant extracellular vesicles contain 90% of the active ingredients in plants and are the essence of the concentration of plant active ingredients. The research background of plant extracellular vesicles involves their important roles in cell-to-cell communication, drug delivery, and interaction with microorganisms. These studies provide important theoretical and practical bases for exploring new biotechnological applications.

[0004] The skin accounts for about 70% of the human body surface area. It is the largest human tissue and organ for sweating and sensing environmental changes, and is also a natural barrier to protect the body from external environmental infringement. It is composed of the epidermis, dermis, and subcutaneous tissue. Keratinocytes are the main components of the human skin epidermis and play a key role in the maintenance and wound repair of the skin epidermis. They are not only important effector cells in skin regeneration but also the source cells of epidermal growth factor (EGF), and they affect the proliferation and differentiation of keratinocytes. A basic feature of wound healing is to repair the intact epidermal barrier through epithelial formation, and the proliferation and migration of the human keratinocyte cell line (HaCaT) can promote skin re-epithelialization and wound gap closure. Human keratinocytes are the most critical cell type for skin wound healing. Therefore, it is of great significance to study how to extract extracellular vesicles of Citrus wilsonii Tanaka (CWTEV) and apply them to skin wound healing. Summary of the Invention

[0005] To solve the above problems, the object of the present invention is to provide a method for preparing Eugenia uniflora extracellular vesicles and its application.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A method for preparing Eugenia uniflora extracellular vesicles includes four steps: preparation of supernatant, concentration, crude extraction, and purification.

[0008] The method for preparing Eugenia uniflora extracellular vesicles specifically includes the following steps:

[0009] 1) After chopping the washed Eugenia uniflora fruits, add them to a mixed solution of glycerol and PBS with a concentration of 10 mM to extract juice, filter, and centrifuge the obtained first filtrate at 4°C and 8000 rpm for 0.5 - 1 h using a high-speed refrigerated centrifuge to obtain a first supernatant. The obtained first supernatant is filtered through a 0.45 μm filter membrane, and the second filtrate is collected;

[0010] 2) Concentrate the second filtrate prepared in step 1) using a 300 KDa ultrafiltration membrane package to obtain a concentrated solution;

[0011] 3) Add the concentrated solution prepared in step 2) to polyethylene glycol 8000, stir at 4°C and 100 - 300 rpm for 12 - 24 h, then centrifuge at 4°C and 12000 rpm for 15 - 20 min. Dissolve the obtained precipitate in 10 mM PBS, and then centrifuge at 4°C and 12000 rpm for 15 - 20 min to obtain a second supernatant;

[0012] 4) Purify the second supernatant prepared in step 3) using a chromatographic column to obtain Eugenia uniflora extracellular vesicles.

[0013] In step 1), the mass concentration of glycerol in the mixed solution is 10%.

[0014] In step 1), the mass - to - volume ratio of Eugenia uniflora fruits to the mixed solution is 1 g: 2 - 4 mL.

[0015] During the concentration process in step 2), set the peristaltic pump pressure value not exceeding 0.2 Mpa.

[0016] In step 3), the volume ratio of the concentrated solution to polyethylene glycol 8000 is 1: 0.1 - 0.2; the mass ratio of the precipitate to PBS is 1: 5 - 7.

[0017] The chromatographic column purification conditions in step 4) are as follows: two C18 liquid chromatographic columns are used in series; the flow rate is 100 cm / h, the sample loading amount is 7.5% of the column volume, and the rinsing solution is a PB solution with a concentration of 20 mM.

[0018] The Eugenia uniflora extracellular vesicles described in step 4) are snap-frozen with liquid nitrogen and stored at -80°C.

[0019] The present invention also includes the application of Eugenia uniflora extracellular vesicles in the preparation of drugs for skin wound healing.

[0020] The present invention has the following advantages compared with the prior art:

[0021] The present invention uses the polymer precipitation method for extraction and chromatographic column purification to prepare CWTEV, and characterizes it in terms of morphology, particle size, Zeta potential, etc.; conducts in vitro activity research on CWTEV, and detects the effects of CWTEV on the proliferation and migration of skin HaCaT cells by CCK-8 method and cell scratch method respectively; conducts transcriptomics and small RNA detection on CWTEV and HaCaT cells after co-culture, constructs a high-throughput omics database, and screens out the target genes corresponding to CWTEV miRNA; uses liposome microcapsules to encapsulate candidate miRNAs and conducts cell proliferation and cell migration experiments; verifies the functions of candidate target genes by Q-PCR and WB experiments to determine the molecular mechanism of CWTEV promoting wound healing.

[0022] The miRNAs in the Eugenia uniflora extracellular vesicles of the present invention can regulate the PI3K / AKT signaling pathway to exert an anti-inflammatory effect, thereby showing the effect of promoting skin wound healing, fully confirming that Eugenia uniflora has a certain curative effect on skin diseases; the active substances contained in plant extracellular vesicles can play an important regulatory role in human cell signal transduction and physiological responses. Among them, the small RNAs (microRNA, miRNA) contained in extracellular vesicles can better play a regulatory role in skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a transmission electron micrograph of CWTEV prepared in Example 1 of the present invention at different scales;

[0024] Figure 2 It is a particle size detection diagram of CWTEV prepared in Example 1 of the present invention, where (a) is the particle size distribution diagram and (b) is the particle concentration diagram;

[0025] Figure 3 It is a potential analysis diagram of CWTEV prepared in Example 1 of the present invention;

[0026] Figure 4 It is a staining result diagram of CWTEV, scale bar: 100 μm;

[0027] Figure 5 It is a diagram of the uptake of CWTEV by HaCaT cells, scale bar: 100 μm;

[0028] Figure 6 This is a research figure on the promotion of HaCaT cell proliferation and migration by CWTEV. Among them, (a) is the survival rate graph of HaCaT cells treated with different concentrations of CWTEV, (b) is the bar graph of the cell migration results of HaCaT cells treated with 20 μg / mL CWTEV, and (c) is the result of the scratch assay of HaCaT cells. Scale bar: 100 μm;

[0029] Figure 7 This is a schematic diagram of transcript targeting;

[0030] Figure 8 This is the encapsulation efficiency result graph of LMC@miR156;

[0031] Figure 9 This is the result graph of HaCaT cells taking up LMC@miR156. Scale bar: 50 μm;

[0032] Figure 10 This is the survival rate graph of LMC@miR156 on HaCaT cells. Among them, (a) is the survival rate graph of HaCaT cells treated with different concentrations of LMC@miR156, and (b) is the survival rate graph of HaCaT cells treated with different concentrations of LMC;

[0033] Figure 11 This is the graph of the proliferation and migration effects of LMC@miR156 on HaCaT cells. Among them, (a) is the bar graph of the cell migration results at a sample concentration of 100 μmol / L, and (b) is the graph of the scratch assay results of each group of cells. Scale bar: 100 μm;

[0034] Figure 12 This is the gene expression result graph of CWTEV and csi-miR156 on HaCaT cells;

[0035] Figure 13 This is the graph of the gene and protein level expression results of CWTEV and csi-miR156 on HaCaT cells. Among them, (a) is the relative protein expression graph of the HSP90AA1 gene, (b) is the relative protein expression level graph of AKT, (c) is the detection result graph of regulating the PI3K / AKT signaling pathway, and (d) is the Western blotting experimental result graph. Detailed implementation method

[0036] To better understand the technical solution of the present invention, the above content of the present invention is further described in detail below through specific implementation methods in the form of examples. However, this should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

[0037] Example 1 Preparation of Xiangyuan extracellular vesicles

[0038] 1) After chopping 100 g of washed Xiangyuan fruits, add them to a mixed solution of 200 mL of glycerol and PBS with a concentration of 10 mM to extract juice. The mass concentration of glycerol in the mixed solution is 10%. Filter, and centrifuge the obtained first filtrate at 4 °C and 8000 rpm for 0.5 h using a high-speed refrigerated centrifuge to obtain a first supernatant. The obtained first supernatant is filtered through a 0.45 μm filter membrane, and the second filtrate is collected;

[0039] 2) Concentrate the second filtrate prepared in step 1) using a 300 KDa ultrafiltration membrane package. During the concentration process, set the peristaltic pump pressure value not to exceed 0.2 Mpa to obtain a concentrated solution;

[0040] 3) Add the concentrated solution prepared in step 2) to polyethylene glycol 8000, where the volume ratio of the concentrated solution to polyethylene glycol 8000 is 1:0.1. Stir at 4 °C and 100 rpm for 12 h, then centrifuge at 4 °C and 12000 rpm for 15 min. The obtained precipitate is dissolved in 10 mM PBS, where the mass ratio of the precipitate to PBS is 1:5, and then centrifuge at 4 °C and 12000 rpm for 15 min to obtain a second supernatant;

[0041] 4) Purify the second supernatant prepared in step 3) using a chromatographic column. The chromatographic column purification conditions are as follows: The chromatographic column is composed of 2 C18 liquid chromatographic columns connected in series; the flow rate is 100 cm / h, the sample loading amount is 7.5% of the column volume, and the rinsing solution is a PB solution with a concentration of 20 mM to obtain Xiangyuan extracellular vesicles. The obtained Xiangyuan extracellular vesicles are quickly frozen in liquid nitrogen and then stored at -80 °C.

[0042] Perform transmission electron microscopy observation on the prepared CWTEV. The specific test method is as follows: Take 10 μL of the sample and drop it on a copper mesh to precipitate for 1 minute, and then suck off the floating liquid with filter paper; Drop 10 μL of uranyl acetate on the copper mesh to precipitate for 1 minute, and then suck off the floating liquid with filter paper; Dry at 25 °C and use a 100 kV electron microscope to detect and image to obtain electron microscope imaging pictures of different nano-sizes. The detection results are as Figure 1 shown. From Figure 1 the results, it can be seen that the prepared CWTEV presents a typical "cup and plate" morphology wrapped by a lipid bilayer membrane, indicating that the polymer precipitation method combined with the chromatographic column purification method can obtain qualified CWTEV, and the morphological structure of CWTEV will not be damaged during the operation process.

[0043] Perform particle size detection on the prepared CWTEV. The detection results are as Figure 2 (a) and (b) shown. From Figure 2 (a), it can be seen that the diameter of the CWTEV vesicles is between 30 and 150 nm, the average particle size is 75.2 nm, and the particle concentration is 1.54×1011 particles / mL (as shown in Figure 2 (b)), which is consistent with the particle size captured by transmission electron microscopy.

[0044] The potential analysis of cryopreserved CWTEV was carried out, and the test results are as shown in Figure 3 . It can be seen from Figure 3 that CWTEV is -43.94 ± 0.95 mV, showing a relatively stable negative potential, and the absolute value of the potential value is higher than 30 mV, indicating that the sample can avoid aggregation after dissolution or dispersion and has stable physical properties.

[0045] Example 2 Preparation of Xiangyuan extracellular vesicles

[0046] 1) After chopping 200 g of washed Xiangyuan fruits, add them to a mixed solution of 600 mL of glycerol and 10 mM PBS for juicing, where the mass concentration of glycerol in the mixed solution is 10%. Filter, and centrifuge the obtained first filtrate at 4 °C and 8000 rpm for 0.7 h using a high-speed refrigerated centrifuge to obtain a first supernatant. The obtained first supernatant is filtered through a 0.45 μm filter membrane, and the second filtrate is collected;

[0047] 2) Concentrate the second filtrate prepared in step 1) using a 300 KDa ultrafiltration membrane package. During the concentration process, set the peristaltic pump pressure value not exceeding 0.2 Mpa to obtain a concentrated solution;

[0048] 3) Add the concentrated solution prepared in step 2) to polyethylene glycol 8000, where the volume ratio of the concentrated solution to polyethylene glycol 8000 is 1:0.2. Stir at 4 °C and 200 rpm for 15 h, then centrifuge at 4 °C and 12000 rpm for 20 min. The obtained precipitate is dissolved in 10 mM PBS, where the mass ratio of the precipitate to PBS is 1:6, and then centrifuge at 4 °C and 12000 rpm for 20 min to obtain a second supernatant;

[0049] 4) Purify the second supernatant prepared in step 3) using a chromatographic column. The chromatographic column purification conditions are as follows: The chromatographic column is composed of 2 C18 liquid chromatographic columns in series; the flow rate is 100 cm / h, the sample loading amount is 7.5% of the column volume, and the washing solution is a 20 mM PB solution to obtain Xiangyuan extracellular vesicles. After quickly freezing the obtained Xiangyuan extracellular vesicles in liquid nitrogen, store them at -80 °C.

[0050] Example 3 Preparation of Xiangyuan extracellular vesicles

[0051] 1) After chopping 500 g of washed Xiangyuan fruits, add them to a mixed solution of 2000 mL of glycerol and PBS with a concentration of 10 mM for juicing. The mass concentration of glycerol in the mixed solution is 10%. Filter, and centrifuge the obtained first filtrate at 4 °C and 8000 rpm for 1 h using a high-speed refrigerated centrifuge to obtain a first supernatant. The obtained first supernatant is filtered through a 0.45 μm filter membrane, and the second filtrate is collected;

[0052] 2) Concentrate the second filtrate prepared in step 1) using a 300 KDa ultrafiltration membrane package. During the concentration process, set the peristaltic pump pressure value not to exceed 0.2 Mpa to obtain a concentrated solution;

[0053] 3) Add the concentrated solution prepared in step 2) to polyethylene glycol 8000. The volume ratio of the concentrated solution to polyethylene glycol 8000 is 1:0.2. Stir at 4 °C and 200 rpm for 24 h, then centrifuge at 4 °C and 12000 rpm for 20 min. The obtained precipitate is dissolved in 10 mM PBS. The mass ratio of the precipitate to PBS is 1:7, and then centrifuge at 4 °C and 12000 rpm for 15 min to obtain a second supernatant;

[0054] 4) Purify the second supernatant prepared in step 3) using a chromatographic column. The chromatographic column purification conditions are as follows: The chromatographic column is composed of 2 C18 liquid chromatographic columns in series; the flow rate is 100 cm / h, the sample loading amount is 7.5% of the column volume, and the rinsing solution is a PB solution with a concentration of 20 mM to obtain Xiangyuan extracellular vesicles. The obtained Xiangyuan extracellular vesicles are quickly frozen in liquid nitrogen and then stored at -80 °C.

[0055] Example 4 CWTEV Staining and HaCaT Cell Uptake

[0056] Label the CWTEV after extraction in Example 3 using a PKH 26 red fluorescent cell membrane staining kit to check whether CWTEV can penetrate the cell membrane and enter the interior of HaCaT cells to play a role. The final volume of the staining solution is 1 mL, and the concentration of PKH 26 is 2×10 -6 M. Operate in the dark. The specific steps are as follows:

[0057] (1) Make up the CWTEV to 500 μL with diluent C (20 μL CWTEV + 480 μL diluent C), and gently pipette and mix to make a 2× nanovesicle solution;

[0058] (2) Add 2 mL of PKH 26 dye to 500 μL of PB solution and mix well to obtain 2× dye (4×10 - 6 M);

[0059] (3) Quickly add 500 μL of 2× nanovesicle solution to 500 μL of 2× dye, mix immediately, and the final concentration of PKH 26 is 2×10 -6 M;

[0060] (4) Incubate for 1 - 5 minutes, mix regularly in the middle. Since the staining is very rapid, do not stain for a long time;

[0061] (5) Add an equal volume of 1% BSA solution to terminate the reaction, and incubate for 1 minute to bind the excess dye;

[0062] (6) Remove the excess dye again using a 10KDa ultrafiltration tube and a chromatographic column, and collect the stained nanovesicles using PBS as a suspension for standby;

[0063] (7) Seed 96 - well plates with HaCaT cells. After 24 hours, co - culture the stained nanovesicles with HaCaT cells for 48 hours, then stain the cell nuclei with DAPI staining solution, and observe the uptake of CWTEV by HaCaT cells under an inverted fluorescence microscope. The detection results are as Figure 4 and Figure 5 shown.

[0064] From Figure 4 the results, it can be seen that the Green picture is a blank background taken under green fluorescence without fluorescence imaging, indicating that there are no other interfering substances in the sample; the Red picture is the fluorescence spots of CWTEV that are red under the excitation wavelength of PKH 26, i.e., under red fluorescence; the merged picture of the Green picture and the Red picture is the Merge picture, and only red fluorescence spots are seen, indicating successful staining. From Figure 5 it can be seen that the cell nuclei are blue fluorescence, and the stained CWTEV is taken up by the cells and aggregates around the blue cell nuclei, emitting strong red fluorescence, indicating that CWTEV can be taken up by HaCaT cells.

[0065] Example 5 Study on the promotion of HaCaT cell proliferation and migration by CWTEV

[0066] The HaCaT cell proliferation experiment was carried out as follows:

[0067] (1) HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin. Add 0.25% trypsin to digest, collect by centrifugation, and seed 5×10 3 cells per well in 96 - well plates at a volume of 100 μL / well;

[0068] (2) After incubation in a 37 °C, 5% carbon dioxide incubator for 24 hours, discard the supernatant, and co-culture with cells at final sample concentrations of 6.25, 12.5, 25, 50, and 100 μg / mL; after 24 hours, discard the supernatant, add CCK-8 solution in the dark, taking care not to introduce air bubbles to prevent interference with the readings. After incubation in the carbon dioxide incubator for 2 hours, measure the absorbance at 37 °C and a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0069] (3) Conduct three independent experiments to generate the growth percentage relative to untreated control cells, and select the most appropriate sample concentration for subsequent experiments.

[0070] The HaCaT cell migration experiment was performed as follows:

[0071] According to the cytotoxicity experiment, the extracellular vesicles of Xiangyuan cells at a concentration of 20 - 40 μg / mL have a proliferative effect on cells (as shown in Figure 6 (a)), so 20 μg / mL was selected as the final drug concentration of the sample group. Three groups, namely the blank group, the positive control group (EGF 50 ng / mL), and the sample group (20 μg / mL), were set up for the experiment.

[0072] (1) Seed HaCaT cells onto a 6-well plate at a concentration of 7×10 5 cells / well. After the cells cover the bottom of the plate, use a 10 μL sterile pipette tip to scratch the bottom of the 6-well plate to form six cross lines, three horizontal and three vertical, with similar spacing.

[0073] (2) After washing three times with PBS, record the scratch condition under an inverted fluorescence microscope at hour (t = 0). Try to select pictures with small differences in scratch width for subsequent calculations to avoid large errors.

[0074] (3) Continue to culture the cells in the basal medium in a 37 °C incubator for 24 hours and 48 hours (t = n). At each time point, take pictures of the scratch migration under an inverted fluorescence microscope.

[0075] (4) Statistical method: Use Image J software to quantify the area of the cell-free region. Open the taken pictures, select 3 - 6 horizontal lines with similar spacing, and calculate the cell migration. The formula for the migration rate is:

[0076] The results of the ability of CWTEV to promote HaCaT migration are shown in Figure 6 (b) and (c). It can be seen from the figures that when co-cultured with HaCaT cells at a sample concentration of 20 μg / mL for 24 hours and 48 hours, the cell migration rate of the CWTEV group (CWTEV) is significantly higher than that of the blank control group (Control), indicating that CWTEV has a promoting effect on HaCaT cell migration.

[0077] Example 6: Study on the mechanism of CWTEV promoting skin wound healing

[0078] Step 1: Construction and analysis of the high-throughput omics database of CWTEV and HaCaT cells

[0079] (1) Perform small RNA detection on the CWTEV prepared in Examples 1 to 3 respectively, and the comparison gene bank is Citrus sinensis of the genus Citrus in the Rutaceae family. The final sequence reference comes from the non-coding RNA sequence information included in the miRBase and RNAcentral databases.

[0080] (2) Co-culture HaCaT cells with 20 μg / mL of CWTEV. After incubating for 24 hours, extract total RNA with RNA extraction reagent. Gene Denovo Biotechnology Co., Ltd. performs transcriptomics and small RNA sequencing of HaCaT cells, and three replicates are set for the blank control group (CK) and the drug-added group (CWTEV) respectively. The detection results are as Figure 7 shown.

[0081] As Figure 7 can be seen, according to the target gene prediction, csi-miR156 has a binding site with the target gene HSP90AA1 at the 3'UTR end. Cis-miR156 may target HSP90AA1 with the highest expression level, and it has more advantages in promoting the proliferation and migration of HaCaT cells. Therefore, verification experiments are performed using the predicted csi-miR156 and the target gene HSP90AA1.

[0082] Step 2: In vitro activity study of CWTEV candidate miRNAs

[0083] (1) Synthesize csi-miR156 mimics

[0084] According to the sequencing data results, the selected csi-miR156 sequence is UGACAGAAGAGAGUGAGCAC. The csi-miR156 mimic oligonucleotide is used for cell experiments composed of a sense strand and an antisense strand to create a double-stranded miRNA duplex: csi-miR156 sense 5'-UGACAGAAGAGAGUGAGCAC-3' and csi-miR156 antisense 5'-UGUGCUCACUCUUCUUCUUGUC-3'. In order to detect the encapsulation rate of liposome microcapsules for csi-miR156A and observe whether LMC@miR156 can be taken up by HaCaT cells, a csi-miR156 mimic with a green fluorescent label at the 5' end was synthesized, and the synthetic product was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0085] (2) Preparation of liposome microcapsules

[0086] ① Autoclave the liposome extruder and then place it in a drying oven to dry. Prepare a 0.1 μm filter membrane.

[0087] ② Prepare one unit of liposome solvent (44 mg egg yolk lecithin, 11 mg cholesterol dissolved in 2 mL absolute ethanol), and assist dissolution with ultrasound.

[0088] ③ Withdraw 2 mL of the liposome solution into a rotary evaporation flask, adjust the rotary evaporator, keep the water temperature at 45 - 50 °C, and rotary evaporate for about 5 minutes until a liposome film forms on the inner wall of the rotary evaporation flask.

[0089] ④ Dilute the synthesized csi - miR156 mimic with pure water to a solution with a final concentration of 625 μmol / L. Redissolve the liposome with 2 mL of the mimic and assist dissolution with ultrasound. Note that it should be completely redissolved within 5 minutes, otherwise it is prone to condensing into a frozen mass.

[0090] ⑤ Introduce nitrogen, and repeatedly extrude with the liposome extruder at least 10 times until the liposome solution appears transparent under light, that is, the liposome microcapsules are successfully prepared. This step is carried out on ice.

[0091] ⑥ Observe under a microscope that the liposome microcapsules are spherical vesicles. Using the same method, redissolve with PBS to prepare a 2 mL blank liposome microcapsule (Liposome microcapsule, LMC) for use as a control experiment. The detection results are as Figure 8 shown.

[0092] It can be seen from Figure 8 that the fluorescence positive rate of LMC@miR156 detected by a flow - through nano - analyzer is 26%, and the liposome microcapsules can successfully encapsulate csi - miR156.

[0093] (3) Proliferation experiment of HaCaT cells incubated with LMC@miR156

[0094] ① HaCaT cells are cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin, then digested with 0.25% trypsin, collected by centrifugation, and plated in a 96 - well plate at 5×10 3 , 100 μL per well.

[0095] ② After incubating in a carbon dioxide incubator for 24 hours, discard the supernatant, and co - culture with the cells according to the final concentrations of 50, 100, and 200 μmol / L of LMC@miR156; after 24 hours, discard the supernatant, add CCK - 8 solution in the dark, pay attention not to introduce air bubbles to prevent interference with the readings, and after incubating in a carbon dioxide incubator for 2 hours, detect the absorbance with an enzyme - linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm and a temperature of 37 °C.

[0096] ③Three independent experiments were conducted to generate the growth percentage relative to untreated control cells. The most suitable sample concentration was selected for subsequent experiments. The test results are shown as Figure 9 and Figure 10 (a) and 10(b).

[0097] As can be seen from Figure 9 , after co-culturing LMC@miR156 with green fluorescence at the 5' end of csi-miR156 and HaCaT cells for 24 hours, the cell membrane was stained with PKH 26 and the cell nucleus was stained with DAPI. The inverted fluorescence microscope could observe the uptake of csi-miR156 by HaCaT cells at their respective fluorescence wavelengths. As can be seen from Figure 10 (a), the CCK-8 experiment screened the survival rate of HaCaT cells with different concentrations (50 μmol / L - 200 μmol / L, quantified by the concentration of csi-miR156) of LMC@miR156. LMC@miR156 with a concentration as high as 100 μmol / L had no cytotoxic effect on HaCaT cells. As can be seen from Figure 10 (b), an equal dose of LMC also did not produce cytotoxicity. Therefore, 100 μmol / L was selected as the optimal concentration of LMC@miR156 for reference in subsequent experiments. The cell survival percentage was calculated by designating the absorbance value obtained from the negative control and cells treated with standard growth medium as 100%.

[0098] (4) Migration experiment of LMC@miR156 incubated with HaCaT cells

[0099] According to the cytotoxicity experiment, when the concentration of LMC@miR156 was 100 μmol / L, it had a promoting effect on cell proliferation, and the corresponding blank liposome (LMC) also did not produce toxicity to cells at a concentration of 100 μmol / L. Therefore, 100 μmol / L was selected as the final concentration of LMC@miR156 and LMC. Four groups were set up for the experiment: blank group (Control), CWTEV group (CWTEV, 20 μg / mL), LMC@miR156 group (LMC@miR156, 10 μmol / L), and LMC group (LMC, 100 μmol / L). The specific steps are as follows:

[0100] ①HaCaT cells were seeded into a 6-well plate at a concentration of 7×10 5 cells / well. After the cells grew to cover the bottom of the plate, a 10 μL sterile pipette tip was used to scratch the bottom of the 6-well plate to form six cross lines, three horizontal and three vertical, with similar spacing;

[0101] ② After washing three times with PBS, record the scratch condition at 0 hour (t = 0) under an inverted fluorescence microscope. Try to select pictures with small differences in scratch width for later calculation to avoid large errors.

[0102] ③ Continue to culture the cells in the basal medium for 2 hours and 48 hours (t = n) in a 37°C incubator. Take pictures of the scratch migration at each time point through an inverted fluorescence microscope.

[0103] ④ Statistical method: Use Image J software to quantify the area of the cell-free region. After opening the taken pictures, select 3 - 6 horizontal lines with similar spacing and calculate the cell migration. The formula for the migration rate is: The detection results are as Figure 11 (a) and 11(b) show.

[0104] As Figure 11 (a) and 11(b) show, when performing 24-hour and 48-hour cell migration experiments with a sample concentration of 100 μmol / L, the cell migration effect of the LMC@miR156 group is better than that of the LMC group and the Control group, effectively promoting cell migration.

[0105] Step 3: RNA extraction and Q-PCR

[0106] Through target gene data and enrichment analysis, it was found that Xiangyuan miRNA targets the HSP90AA1 gene and regulates the downregulation of this gene. Use Q-PCR and WB to verify the credibility of the omics. Design primer sequences on NCBI, and synthesize primers HSP90AA1 and GAPDH by Suzhou Hongxun Biotechnology Co., Ltd. Set up a blank group (Control), a CWTEV group (CWTEV, 20 μg / mL), an LMC@miR156 group (LMC@miR156, 100 μmol / L), and an LMC group (LMC, 100 μmol / L). Use RNA-easy Isolation Reagent to extract the total RNA of the four groups after co-culture with cells for 24 hours respectively, measure the RNA concentration with a ultra-micro spectrophotometer, and reverse transcribe the qualified total RNA into cDNA using a reverse transcription kit. Use Fast SYBR qPCR SuperMix kit for Q-PCR. The expression of HSP90AA1 is normalized to GAPDH. Q-PCR is repeated three times. Use the 2 -ΔΔCt method for normalization and quantification. The primer sequences are shown in Table 1.

[0107] Table 1 Primer sequences

[0108]

[0109]

[0110] (1) Total RNA extraction of co-cultured cells

[0111] ① Discard the cell culture medium and wash once with 1×PBS;

[0112] ② Add 500 μL of RNA-easy to each well of the six-well plate. Use a pipette to blow and beat the cells to detach them from the bottom of the plate, and transfer them to a 1.5 mL centrifuge tube. Use a pipette to repeatedly blow and beat until fully lysed;

[0113] ③ Add 2 / 5 volume of sterilized water (200 μL for every 500 μL of RNA-easy) to the above lysate, shake well, and let it stand at room temperature for about 5 minutes; Centrifuge at 12000×g at room temperature for 15 minutes;

[0114] ④ Take out the centrifuge tube. At this time, the solution is divided into an upper aqueous phase (containing RNA) and a lower precipitate. Carefully aspirate 600 μL of the upper aqueous phase into a new enzyme-free 1.5 mL EP tube; Add 600 μL of isopropanol, shake well, and let it stand at room temperature for 10 minutes; Centrifuge at 12000×g at room temperature for 10 minutes. See white precipitate, and carefully discard the supernatant;

[0115] ⑤ Add 500 μL of 75% ethanol, flick the bottom of the tube to suspend the precipitate, try not to invert up and down to avoid the white precipitate sticking to the tube cap or the tube wall;

[0116] ⑥ Centrifuge at 8000×g at room temperature for 3 minutes and discard the supernatant;

[0117] ⑦ Repeat steps ⑤ and ⑥ once to discard all the supernatant;

[0118] ⑧ Let it dry at room temperature (2 - 3 minutes), add 20 - 30 μL of sterilized water to dissolve the precipitate, and use a pipette to gently blow and beat to fully dissolve the RNA precipitate;

[0119] ⑨ Measure the RNA content with a ultra-micro spectrophotometer, and evaluate the RNA quality by looking at the A260 / A280 and A260 / A230 values. After aliquoting, store at -80 °C.

[0120] (2) Reverse transcription of RNA into cDNA

[0121] ① Calculate the amount of RNA required for the reverse transcription reaction system, and configure a 20 μL reverse transcription reaction system (see Table 2. This operation needs to be done on ice);

[0122] Table 2 Reverse transcription reaction solution

[0123]

[0124]

[0125] ② Set the reverse transcription reaction conditions: 50°C for 15 min; 75°C for 5 min; end the reaction when the temperature drops to 4°C, and place the reaction system in a reverse transcriptase instrument.

[0126] ③ Obtain the reverse transcription product cDNA. After diluting it 10-fold with sterile water, store the cDNA sample at -20°C for short-term or -80°C for long-term.

[0127] (3) Q-PCR reaction

[0128] Use the specific primers for HSP90AA1 and GAPDH in Table 1, diluted 10-fold before use. Prepare the PCR reaction system on ice and perform amplification using Q-PCR. The conditions are shown in Tables 3 and 4.

[0129] Table 3 Composition of the reaction solution

[0130]

[0131] Table 4 Reaction conditions

[0132] Loop Reaction temperature Reaction time 1 95℃ 30s 39 95℃ 5s 39 60℃ 10s 39 70℃ 30s 1 65℃ 5s 1 95℃ 5s

[0133] After the reaction on the machine is completed, export the data and use the 2 -ΔΔCt method to analyze the relative expression level of the target gene. This analysis method can intuitively reflect the relative content of the target gene among different experimental groups. The detection results are as Figure 12 shown.

[0134] As Figure 12 can be seen, CWTEV is consistent with the transcriptomics detection and downregulates the HSP90AA1 gene. LMC@miR156 also downregulates the HSP90AA1 gene.

[0135] (4) Preparation of cell proteins

[0136] ① The culture method of HaCaT cells is the same as in step 2. Set four groups: blank group (Control), CWTEV group (CWTEV, 20 μg / mL), LMC@miR156 group (LMC@miR156, 100 μmol / L), and LMC group (LMC, 100 μmol / L).

[0137] ② Discard the old culture medium, place the cells on ice, and carefully wash the cells 2 times with pre-cooled PBS, then discard the PBS solution.

[0138] ③ Add Western and IP cell lysis buffer and lyse the cells on ice for 30 minutes. Pipette the cells to collect the lysed cells into a 1.5 mL centrifuge tube.

[0139] ④ Perform ultrasonic lysis for 5 s / 3 times / 50% power, and operate on ice.

[0140] ⑤ Incubate at -80 °C in a refrigerator for 30 minutes. After lysis, place it in a pre-cooled low-temperature centrifuge at 4 °C, centrifuge at 12,000 rpm for 10 minutes;

[0141] ⑥ Collect the supernatant. Conduct the entire process on ice, aliquot and store at -80 °C in a refrigerator for later use;

[0142] ⑦ Quantify the protein concentration of the sample using a BCA protein quantification detection kit, and normalize the protein concentrations of the four samples;

[0143] ⑧ Take 90 μL of the protein extraction solution, add 30 μL of 4× Loading buffer, and heat in a metal bath at 100 °C for 5 minutes to denature it.

[0144] (5) Loading protein samples onto the gel

[0145] Take out the denatured protein sample, fix the SDS-PAGE gel plate, vertically pull out the 10-well comb upwards, fill the glass plate up to the upper edge with 1× electrophoresis buffer. According to the experimental requirements, use a 200 μL pipette to gently add 20 μL of the protein sample to the upper edge of the bottom of the well, avoiding the protein sample from overflowing from the well and causing cross-contamination of adjacent loading wells.

[0146] (6) Running the gel

[0147] Turn on the power switch, set the voltage to 80 V. After 30 minutes, when each group of protein samples has dropped below the plane of the stacking gel, increase the voltage to about 180 V for electrophoresis. Adjust the electrophoresis duration according to the protein marker, and pay attention that the bands do not run out of the gel.

[0148] (7) Transferring the membrane

[0149] ① Carefully pry open the glass plate, take out the gel and cut off the stacking gel, and immediately place it in 1× electrotransfer buffer;

[0150] ② Cut a PVDF membrane slightly larger than the gel, activate it with methanol for 5 - 10 minutes, and place it in 1× electrotransfer buffer;

[0151] ③ Between the positive and negative electrodes of the transfer device, place an asbestos mesh, filter paper, PVDF membrane, gel, filter paper, and asbestos mesh in sequence;

[0152] ④ Clamp the "sandwich" structure and place it in the transfer instrument. After filling it with 1× electrotransfer buffer, apply a constant voltage of 100 V and a limited current of 330 mA, and transfer the membrane for about 1 hour and 15 minutes;

[0153] ⑤ Note that no bubbles should be generated during the entire operation process. Do not directly touch the PVDF membrane with your hands, and fill the outer periphery of the transfer tank with ice cubes for cooling.

[0154] (8) Blocking

[0155] ① After taking out the membrane, it was observed that the marker had completely transferred from the gel to the PVDF membrane;

[0156] ② Wash the electrotransfer solution on the membrane 3 times with 1×TBST on a shaker, 10 minutes each time;

[0157] ③ Immerse the PDVF membrane in 5% blocking milk, slowly shake on a shaker, and incubate at room temperature for 1 hour.

[0158] (9) Antibody Incubation and Chemiluminescence Imaging

[0159] ① After blocking, wash the excess milk 3 times with 1×TBST on a shaker, 10 minutes each time;

[0160] ② Dilute the primary antibody according to the ratio, cut the membrane into four parts according to the protein molecular weight size, and immerse them in the corresponding primary antibodies respectively, and incubate with shaking overnight at 4°C;

[0161] ③ Recover the primary antibody, store it at -4°C, soak the PVDF membrane in 1×TBST and shake it quickly at room temperature on a shaker 3 times, 5 minutes each time;

[0162] ④ Dilute the secondary antibody with 5% blocking solution at 1:5000, incubate the PVDF membrane, and shake it slowly at room temperature for 1 hour;

[0163] ⑤ After incubation, recover the blocking solution and store it at -20°C, wash it 3 times with 1xTBST;

[0164] ⑥ After washing the membrane, prepare the luminescent solution and developer solution at a ratio of 1:1;

[0165] ⑦ After evenly wetting the membrane, take a picture with a BIO-RAD instrument;

[0166] ⑧ Use Image J software to digitize the gray value of the scanned picture strip, and perform statistical analysis on the obtained values. The detection results are as Figure 13 (a), (b), (c) and (d) shown.

[0167] As Figure 13 (a), (b), (c) and (d) show, compared with the Control group, the protein expression level of HSP90AA1 decreased. AKT is a downstream protein of HSP90AA1, and its protein expression level was also reduced, but AKT was phosphorylated, indicating that the PI3K / AKT signaling pathway can be activated, and the phosphorylation ratio of AKT increased, promoting the migration of HaCaT cells.

[0168] In summary, the miRNAs of CWTEV can regulate the PI3K / AKT signaling pathway to exert anti-inflammatory effects, thus showing the role of promoting skin wound healing.

[0169] Although the specific implementation manners of the present invention are described above, they are not limitations on the protection scope of the present invention. Based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A method for preparing extracellular vesicles of Xiangyuan, characterized in that: The method comprises four steps: preparation, concentration, crude extraction and purification of supernatant.

2. The method for preparing the extracellular vesicles of Xiangyuan according to claim 1, characterized in that: The specific steps include: 1) After the washed Xiangyuan fruit is chopped, the juice is squeezed into a mixed solution of glycerol and 10 mM PBS, and filtered. The obtained first filtrate is centrifuged at 4°C and 8000 rpm for 0.5 to 1 h using a high-speed refrigerated centrifuge to obtain a first supernatant. The obtained first supernatant is filtered through a 0.45 μm filter membrane to collect a second filtrate; 2) using a 300 KDa ultrafiltration membrane to concentrate the second filtrate obtained in step 1) to obtain a concentrated solution; 3) adding the concentrated solution obtained in step 2) to polyethylene glycol 8000, stirring at 4°C, 100-300 rpm for 12-24 hours, and then centrifuging at 4°C, 12000 rpm for 15-20 minutes, adding 10 mM PBS to the obtained precipitate to dissolve, and then centrifuging at 4°C, 12000 rpm for 15-20 minutes to obtain a second supernatant; 4) Purifying the second supernatant obtained in step 3) using a chromatographic column to obtain Xiangyuan extracellular vesicles.

3. The method for preparing the Xiangyuan extracellular vesicles according to claim 2, characterized in that: The mass concentration of glycerol in the mixed solution in step 1) is 10%.

4. The method for preparing the Xiangyuan extracellular vesicles according to claim 2, characterized in that: The mass volume ratio of the Xiangyuan fruit and the mixed solution in step 1) is 1g:2-4mL.

5. The method for preparing the Xiangyuan extracellular vesicles according to claim 2, characterized in that: During the concentration process of step 2), the peristaltic pump pressure is set to a value not exceeding 0.2 MPa.

6. The method for preparing the Xiangyuan extracellular vesicles according to claim 2, characterized in that: In step 3), the volume ratio of the concentrate to polyethylene glycol 8000 is 1:0.1-0.2; the mass ratio of the precipitate to PBS is 1:5-7.

7. The method for preparing Xiangyuan extracellular vesicles according to claim 2, characterized in that: The purification conditions of the chromatographic column in step 4) are as follows: the chromatographic column is two C18 liquid chromatography columns connected in series; the flow rate is 100 cm / h, and the sample loading amount is 7.5% of the column volume.

8. The method for preparing Xiangyuan extracellular vesicles according to claim 2, characterized in that: The Xiangyuan extracellular vesicles described in step 4) are quickly frozen with liquid nitrogen and then stored at -80°C.

9. Use of the Xiangyuan extracellular vesicles according to claim 1 in preparing a skin wound healing drug.

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