An autophagosome for promoting the healing of diabetic wounds, its preparation method and application

Abstract

The present invention discloses an autophagosome for promoting the healing of diabetic wounds, its preparation method and application, belonging to the technical field of biomedicine. The present invention provides a preparation method of the autophagosome, which extracts the autophagosome from starved vascular endothelial cells. The generation of the autophagosome does not require additional reagents for induction, and the extraction conditions are simple. It does not require an ultra-high speed centrifuge and can be experimented in an ordinary laboratory. Moreover, the yield is higher than that of exosomes, providing a non-toxic and side-effect-free, easy-to-generate and low-cost treatment method for patients with chronic refractory wounds. The autophagosome of the present invention can promote the healing of diabetic wounds and relieve the physiological and psychological burdens caused by diabetic wounds to patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an autophagosome for promoting diabetic wound healing, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes is a multi-faceted metabolic disorder, including biochemical disturbances and epigenetic factors, which ultimately translate into irreversible tissue changes in the process of glucose oxidation. Leg or foot ulcers are the most common complications in diabetic patients, accounting for about 19 - 34% of the diabetic population. The classical concept defines diabetic foot as deep tissue damage in the lower extremities, which Armstrong also called the "cancer analogy". This is mainly because the 5-year mortality rate associated with foot ulcers and amputations exceeds that of common cancers. And globally, about 50 - 70% of amputations are due to diabetic wounds. It is generally believed that the main characteristics of diabetic wounds are persistent infections, and the formation and remodeling of mature granulation tissue are impaired. Although there have been some advances in the understanding of impaired diabetic wound healing in recent years, its pathological and molecular mechanisms are still unclear, and the treatment effects of existing methods are not satisfactory.

[0003] Secretory autophagosomes (SAPs) are produced by secretory autophagy and also belong to the family of extracellular vesicles (EVs). It is a new mode of intercellular communication that transfers cargo to target cells. Exposure to stress, especially starvation, can enhance secretory autophagy and reduce degradative autophagy by inducing the loss of lysosomal integrity. In recent years, the research on SAPs has mainly focused on elucidating their roles in the occurrence and development of diseases such as cancer and acute respiratory distress syndrome, but their therapeutic roles in diseases have not been taken seriously. Summary of the Invention

[0004] The purpose of the present invention is to provide an autophagosome for promoting diabetic wound healing, a preparation method thereof, and an application thereof, so as to improve the healing effect of autophagosomes in diabetic wounds.

[0005] The present invention provides a preparation method of an autophagosome for promoting diabetic wound healing, which includes the following steps: culturing vascular endothelial cells in a serum-free medium, performing a first centrifugation on the cell culture solution, collecting the first supernatant, and performing a second centrifugation on the first supernatant, wherein the precipitate contains the autophagosome;

[0006] The centrifugal force of the first centrifugation is 2000g, and the time is 10 minutes;

[0007] The centrifugal force of the second centrifugation is 12000g, and the time is 15 minutes.

[0008] Preferably, the serum-free medium comprises Gibco high-glucose DMEM medium containing 0.1% penicillin-streptomycin (PS).

[0009] Preferably, the time for culturing vascular endothelial cells in the serum-free medium is 48 hours.

[0010] Preferably, the temperature for both the first centrifugation and the second centrifugation is 4°C.

[0011] Preferably, after collecting the precipitate, it further includes incubating the precipitate with LC3-labeled magnetic beads to capture autophagosomes.

[0012] The present invention also provides autophagosomes obtained according to the above preparation method.

[0013] The present invention also provides the application of the above autophagosomes in the preparation of drugs for promoting the healing of diabetic wounds.

[0014] Beneficial effects: The present invention provides a preparation method of autophagosomes for promoting the healing of diabetic wounds. The autophagosomes are extracted from starved vascular endothelial cells. The generation of the autophagosomes does not require additional reagents for induction, and the extraction conditions are simple. It does not require an ultra-high-speed centrifuge and can be experimented in an ordinary laboratory. Moreover, the yield is higher than that of exosomes, providing a non-toxic side-effect, easy-to-generate, and low-cost treatment method for patients with chronic refractory wounds. The autophagosomes of the present invention can promote the healing of diabetic wounds and relieve the physiological and psychological burdens caused by diabetic wounds to patients. Description of the Drawings

[0015] Figure 1 It is a diagram showing the expression of LC3 protein in autophagosomes extracted from intermediate vascular endothelial cells in vascular endothelial cells in Example 2 of the present invention and the analysis results of nanoparticles;

[0016] Figure 2 It is the analysis result of nanoparticles of autophagosomes in Example 2 of the present invention;

[0017] Figure 3 It is a transmission electron micrograph of autophagosomes in Example 2 of the present invention;

[0018] Figure 4 It is a general wound diagram showing the treatment effect of autophagosomes on the wounds of diabetic mice in Example 2 of the present invention;

[0019] Figure 5 It is a healing trajectory diagram showing the treatment effect of autophagosomes on the wounds of diabetic mice in Example 2 of the present invention. Detailed Embodiments

[0020] The present invention provides a method for preparing autophagosomes that promote the healing of diabetic wounds, comprising the following steps: culturing vascular endothelial cells in a serum-free medium, performing a first centrifugation on the cell culture solution, collecting the first supernatant, performing a second centrifugation on the first supernatant, and the precipitate contains the autophagosomes;

[0021] The centrifugal force of the first centrifugation is 2000g and the time is 10 minutes;

[0022] The centrifugal force of the second centrifugation is 12000g and the time is 15 minutes.

[0023] The present invention uses a serum-free medium to culture vascular endothelial cells. The serum-free medium is preferably Gibco high-glucose DMEM medium containing 0.1% penicillin-streptomycin (PS). The present invention preferably cultures vascular endothelial cells in the serum-free medium for 48 hours. Using the serum-free medium of the present invention, without providing fetal bovine serum, the vascular endothelial cells are in a starvation state, thereby promoting the occurrence of secreted autophagosomes.

[0024] The present invention preferably performs a first centrifugation on the cell culture solution. The first centrifugation includes removing large particulate matter by a low-speed centrifugation method and retaining the supernatant. The first centrifugation of the present invention is carried out at a low temperature, such as 4°C. The present invention performs a second centrifugation on the first centrifugate, collects the precipitate, and also maintains a low temperature during the second centrifugation. The present invention preferably performs the second centrifugation twice, resuspending with PBS during this period, and crude-extracted autophagosomes can be obtained by high-speed centrifugation.

[0025] After collecting the precipitate, the present invention preferably further includes incubating the precipitate with LC3-labeled magnetic beads to capture autophagosomes. In the example, it is preferred to first co-incubate activated protein A magnetic beads with LC3 antibody, then perform magnetic separation, collect the magnetic beads, and apply them to the above-mentioned crude-extracted autophagosomes. After co-incubating at 37°C for 30 min - 2 h, elute with 0.1 M NaOH.

[0026] The present invention also provides autophagosomes obtained according to the above preparation method.

[0027] Using the above preparation method, the purified autophagosomes exhibit a classical double-membrane-like structure, presenting an overall cup-shaped morphology. The particle size of the autophagosomes is (415.8 ± 69 nm) and the concentration And the expression level of LC3II in the autophagosomes is significantly higher than that in the cell lysate, while the expression of LC3I is significantly reduced, confirming that the extracted extracellular vesicles are exosomes.

[0028] The present invention also provides the application of the above autophagosomes in the preparation of drugs for promoting the healing of diabetic wounds. The application of autophagosomes can significantly promote the healing and repair of wounds in diabetic mice, promote epithelialization and granulation tissue regeneration, and has greater application potential in stimulating the regeneration of skin appendages and improving the healing quality.

[0029] To further illustrate the present invention, the following describes in detail an autophagosome for promoting the healing of diabetic wounds, its preparation method and application provided by the present invention in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] Preparation of autophagosomes for promoting the healing of diabetic wounds

[0032] 1. Set the starvation environment as Gibco high-glucose DMEM medium containing only 5% PS.

[0033] 2. Culture vascular endothelial cells in a serum-free environment. After 48 hours, collect the culture medium, centrifuge at a low speed of 2000 rmp in a centrifuge for 10 minutes to remove large particulate matter (such as dead cells and debris), collect the supernatant and continue centrifugation (4°C, 12000 g, 15 min). After discarding the supernatant, resuspend with PBS and centrifuge (4°C, 12000 g, 15 min) to obtain crudely extracted autophagosomes.

[0034] 3. Subsequently, co-incubate protein A magnetic beads with LC3 antibody, then perform magnetic separation, collect the magnetic beads, and act on the above-mentioned crudely extracted autophagosomes to purify autophagosomes through the interaction of antigen and antibody.

[0035] 4. Resuspend the precipitate with sterile PBS to obtain autophagosomes. It can be used immediately or stored frozen for later use.

[0036] Example 2

[0037] Based on the method described in Example 1, relevant identifications are carried out on the obtained exosomes, including the following steps:

[0038] 1. After extracting the total protein of autophagosomes, detect the protein concentration using a BCA protein detection kit. Use a Western blot experiment to detect the surface specific marker of autophagosomes: LC3 through electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and chemiluminescent imaging analysis. The specific operations are as follows:

[0039] (1) Protein concentration detection by BCA method: Take about 50 μL of autophagosome suspension and add 50 μL of RIPA lysis buffer (R0010, Solarbio). Lyse thoroughly at 4°C for 15 minutes, centrifuge (12,000 g, 4°C) for 20 minutes (cell lysate preparation uses 100 μL of RIPA lysis buffer from 1 well of a 6-well plate). Slowly aspirate the supernatant with a pipette and place it in a 1.5 mL EP tube. After measuring the protein concentration of the cell lysate and autophagosomes using a BCA protein concentration detection kit (PC0020, Solarbio), dilute the two groups of samples to the same concentration by calculation with PBS. Mix them thoroughly at a volume ratio of protein sample: loading buffer (5×) of 4:1 and boil in an iron bath at 105°C for 10 minutes.

[0040] (2) Electrophoresis: Operate according to the instructions of the gel preparation kit. The lower gel is the separating gel and the upper gel is the stacking gel. After adding the lower gel (separating gel), flatten it with anhydrous ethanol. Insert the comb as soon as possible after adding the upper gel (stacking gel) to avoid air bubbles in the slot (the presence of air bubbles affects the electrophoresis of samples). Take the samples out of the refrigerator to thaw, then vortex and centrifuge briefly (because a small amount of water vapor will condense on the tube cap, affecting the sample concentration). The loading volume is selected according to the protein concentration, with a loading volume of 20 μg / well. Add Rainbow Marker to the sample slots on both sides of the loading as a reference for the position of the target protein. The loading order is cell protein lysate and autophagosomes in sequence. Electrophoresis is carried out at room temperature, with a constant voltage of 70 mV for concentration, and a constant voltage of 130 mV after entering the separating gel, and electrophoresis until the required position for the experiment.

[0041] (3) Electrotransfer: Prepare the equipment for electrotransfer (electrotransfer tank, ice-making, etc.) and reagents (electrotransfer buffer preparation: each 1000 mL of electrotransfer buffer contains 100 mL of 10× electrotransfer buffer, 700 mL of pure water, and 200 mL of methanol). When transferring the membrane, soak the PVDF membrane in methanol for more than 10 seconds for activation. There should be no air bubbles between the gel and the membrane. The electrotransfer buffer can be pre-cooled in ice in advance. In an ice bath, transfer at a constant current of 300 mA for 1 hour (the time can be adjusted according to the size of the target protein);

[0042] (4) Blocking: Take out the PVDF membrane from the electrotransfer tank and soak it in skim milk (5%, 00 mL plus 5 g of milk powder) for more than 1 hour. Recycle the blocked milk and store it in a 4°C refrigerator for diluting the secondary antibody the next day;

[0043] (5) Primary antibody incubation: Dilute the LC3 antibody with a primary antibody diluent (1:1000, that is, 1 mL of primary antibody diluent plus 1 μL of primary antibody solution). Incubate the diluted antibody in the blocked PVDF membrane, and slowly shake on a shaker at 4°C overnight;

[0044] (6) Secondary antibody incubation: Recover the primary antibody, rinse the membrane with PBST (PBS + 0.1% Tween) or TBST (TBS + 0.1% Tween) on a shaker quickly for 3 times, 10 minutes each time. Then soak the membrane in the secondary antibody (the secondary antibody is diluted with 5% skim milk at a ratio of 1:10000) and incubate at room temperature for 1 hour;

[0045] (7) Exposure: Prepare the luminescent solution, put the strip into the exposure machine (GE company), drop the luminescent solution for exposure, and collect pictures. When necessary, the software Image J can be used to analyze the gray value of the protein strip.

[0046] The results are as Figure 1 shown. The expression level of LC3II in the purified autophagosomes is significantly higher than that in the cell lysate, and the expression of LC3II increases significantly, while the expression of LC3I decreases significantly. It is confirmed that the extracted extracellular vesicles are exosomes.

[0047] 2. Analyze and detect the concentration and particle size of autophagosomes with nanoparticles. The detection method is as follows: Take out 20 μL of the autophagosome suspension obtained by centrifugation, dilute it with an appropriate amount of PBS, put it into the nano-system sample chamber (ParticleMetrix) for detection, and then use the software ZetaView8.04.02 to process and analyze the result data, and record the particle size and particle concentration of each group of autophagosomes. The results are as Figure 2 shown. The particle size of autophagosomes is (415.8 ± 69 nm) and the concentration is similar, indicating that there is no significant difference between the two.

[0048] 2. Detect and observe the morphology of exosomes by transmission electron microscopy, take pictures and record. The detection method is as follows:

[0049] (1) Osmium acid fixation: Add 20 μL of the suspension containing autophagosomes to 500 μL of osmium acid solution (2%), and place it in a refrigerator at 4 °C for 2 hours for fixation;

[0050] (2) Dehydration: After fixation, wash it 3 times with PBS, let it stand for about 15 minutes each time, and then perform dehydration treatment in ethanol solutions with concentration gradients of 50%, 70%, 80% and 90% in sequence. After dehydration is completed, let it stand for 15 minutes, and finally soak it in absolute ethanol for dehydration for 20 minutes. Perform dehydration again according to the above process;

[0051] (3) Replacement: Immerse the sample obtained after the above dehydration in acetone solution for 15 minutes to replace the dehydration solution, and then repeat this replacement process once;

[0052] (4) Impregnation: Impregnation solution I is prepared from acetone and embedding agent in a ratio of 2:1. Impregnation solution II is prepared from acetone and embedding agent in a ratio of 1:1. Impregnation solution III is prepared from acetone and embedding agent in a ratio of 1:2. Impregnation solution IV is prepared from 100% embedding agent. Immerse successively in impregnation solution I, impregnation solution II and impregnation solution III for 2 hours, and finally immerse in impregnation solution IV for 24 hours, and repeat the immersion in impregnation solution IV once;

[0053] (5) Embedding: After impregnation, put the sample into an embedding plate filled with pure embedding agent for embedding treatment;

[0054] (6) Polymerization and sectioning: After embedding, polymerize the sample at 65 °C. After 48 hours, cut the sample into thin slices with a thickness of 60 - 80 nm;

[0055] (7) Staining: Use uranyl acetate to stain the sectioned sample, soak for 10 minutes, wash, then stain with lead acetate for 10 minutes, wash again, and observe it with an electron microscope;

[0056] (8) Photographing: Conduct electron microscope (Hitachi) detection and imaging under the condition of 100 kv, and set the acceleration voltage to 40 - 120 kV (increments of 100 V). The experimental results are as Figure 3 shown. The autophagosomes present a classic double - membrane - like structure and an overall cup - shaped morphology.

[0057] Example 3

[0058] Based on the method described in Example 1, after the completion and qualified identification of Example 2, a therapeutic control trial of autophagosome preparation on diabetic mice was carried out. The specific steps are as follows:

[0059] 1. Establishment of diabetic wound model

[0060] Select 20 male diabetic gene mice (db / db mice) at 12 weeks old with similar gender, age, weight and growth status, and randomly divide them into 2 groups, with 10 mice in each group. After anesthesia, depilate and disinfect the back and cover it with a sterile cloth. Under sterile conditions, create a wound with a diameter of 1 cm on the back of the mice.

[0061] 2. Treatment of diabetic wound healing with autophagosomes

[0062] The experiment was divided into a PBS control group and an autophagosome group. After the establishment of the wound model in each group, in the autophagosome group, injections were performed at 3, 6, 9, and 12 o'clock around the wound margin every other day, and a suspension of 3.75 mM autophagosomes was injected at each site. The control group of mice was injected with sterile PBS, and the injection method and volume were the same as those in the autophagosome group.

[0063] 3. Evaluation of wound healing and efficacy evaluation of autophagosomes

[0064] (1) Wound healing evaluation

[0065] Photograph the wounds of the two groups of mice, record the daily wound healing situation, and replace and update the damaged rubber rings. The wound area healing rate is: (A0 - A n ) / A0 × 100%, where A0 is the wound area on the day of modeling, and A n is the wound area on the nth day after modeling. The area is calculated using ImageJ software. After the experiment, statistical analysis and graphing of the wound healing rate are performed. On the 3rd, 7th, and 14th days after constructing the wound model, randomly select 6 mice from each of the 3 groups, euthanize them, and use tissue scissors to cut the wound skin completely (reaching the muscle layer) along the periphery of the wound edge (about 1 cm away from the wound edge), and place it in 4% paraformaldehyde fixative for fixation for subsequent HE staining analysis. The results are as Figure 4 shown. The addition of exosomes can significantly shorten the wound healing cycle and improve the wound repair process.

[0066] (2) HE staining evaluation

[0067] ① Tissue dehydration: Take the wound tissue block out of the fixative, rinse it in running water for 30 minutes, and sequentially place the tissue block in 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol for dehydration, and finally place it in xylene solution to remove the alcohol in the tissue.

[0068] ② Paraffin embedding and sectioning: Place the tissue blocks in the melted paraffin in the order of the wound facing up for embedding. After the paraffin cools and solidifies, use a microtome to section the tissue blocks with a thickness of 5 μm. Gently lay the cut thin slices on warm water, insert a glass slide under the water surface where the thin slices are located, gently lift the glass slide, and let the thin slices lie flat on the glass slide. Mark the grouping on the slide with a pencil;

[0069] ③ Dewaxing: Bake the paraffin sections in an oven at 60°C for 2 hours to soften the paraffin. Subsequently, immerse the paraffin sections in xylene (I) and xylene (II) successively, and let them stand at room temperature for 10 minutes. Then place them in absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol in turn, let them stand for 2 minutes each time, and finally soak them in clear water for 2 minutes;

[0070] ④ Staining: Immerse the dewaxed sections in hematoxylin solution for 20 minutes, then wash them 3 times with pure water, differentiate them in 1% hydrochloric acid ethanol for 10 seconds, after differentiation, dip them in 5% ammonia solution for 3 seconds for blue return, and then wash them 3 times with pure water. Finally, soak them in eosin staining solution and let them stand at room temperature for 5 minutes, then rinse them 3 times with pure water;

[0071] ⑤ Dehydration, clearing, and mounting: The sections were dehydrated using ethanol with a low to high concentration gradient, then immersed in xylene for 2 minutes to clear the sections. Finally, an appropriate amount of neutral gum was dropped on the tissue section on the glass slide, and a coverslip was pressed on for mounting. Observation, photography, and analysis were performed using a microscope (Olympus).

[0072] The results showed that: As Figure 4 shown, after applying autophagosomes to diabetic wounds, differences in wound size could be observed on the 7th day. On the 7th day, the wound area of the autophagosome group was smaller than that of the control group. Among them, the wound proportion of the control group was 73.5 ± 4.1%, and the wound proportion of the autophagosome group was 45.3 ± 2.5%. By the 14th day, the wound healing proportion of the control group was 49.3 ± 1.7%, while the wound healing rate of the autophagosome group was as high as 90.4 ± 1.4%. The above results indicate that the autophagosome group can significantly shorten the wound healing cycle. HE staining was performed on the collected wound tissues. The experimental results showed that compared with the control group, the autophagosome group could significantly promote the epithelialization degree of diabetic wounds and the regeneration of granulation tissue, and no obvious granulation tissue filling was seen. In contrast, no obvious epithelialization and granulation tissue were seen in the control group, and a large number of adipocytes were filled at the lower edge of the wound ( Figure 5 ).

[0073] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A preparation method of autophagosomes for promoting the healing of diabetic wounds, characterized in that, It includes the following steps: culturing vascular endothelial cells in a serum-free medium, performing a first centrifugation on the cell culture medium, collecting the first supernatant, performing a second centrifugation on the first supernatant, and the autophagosomes are contained in the precipitate; The centrifugal force of the first centrifugation is 2000g and the time is 10 minutes; The centrifugal force of the second centrifugation is 12000g and the time is 15 minutes; The serum-free medium includes Gibco high-glucose DMEM medium containing 0.1% penicillin-streptomycin, and the time for culturing vascular endothelial cells in the serum-free medium is 48 hours.

2. The preparation method according to claim 1, wherein The temperatures of the first centrifugation and the second centrifugation are both 4°C.

3. The preparation method according to claim 1, characterized in that, After collecting the precipitate, it further includes incubating the precipitate with LC3-labeled magnetic beads to capture autophagosomes.

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