PRP stem cell exosome chitosan composition as well as preparation method and application thereof

Abstract

The invention discloses a PRP (platelet-rich plasma) stem cell exosome chitosan composition as well as a preparation method and application thereof. The PRP stem cell exosome chitosan composition comprises the following components: activated platelet-rich plasma, stem cell exosome and chitosan. The preparation method of the composite gel comprises the following steps: adding chitosan into a suspension containing activated platelet-rich plasma and stem cell exosome, uniformly mixing, adding a povidone suspending aid, oscillating to form a stable suspension state, adjusting the pH value to 7.5-7.8, mixing with thrombin and a calcium agent, stirring in a dark place, and solidifying to form a gelatinous substance, thereby obtaining the composite gel. The platelet-rich plasma-stem cell exosome-chitosan composite gel is obtained. The composition and the composite gel can promote microvascular endothelial cell regeneration and recover normal vascular endothelial morphology and functions; skin regeneration is promoted; broad-spectrum bacteriostasis and lipid reduction are realized, and local inflammation and immune response are inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a PRP stem cell exosome chitosan composition, a preparation method and an application thereof. Background Art

[0002] In recent years, with the aggravation of population aging and the change of people's diet structure, the incidence of diabetes has risen rapidly. The lower limb ischemic disease caused by diabetes (commonly known as "diabetic foot") can lead to amputation and even endanger life. Therefore, diabetic foot has attracted more and more attention. At present, the traditional method is debridement, and then it is bandaged with a vaseline sterile dressing and changed dressings regularly for observation. However, due to combined neurovascular lesions, local blood supply insufficiency, microcirculation disorders, an increase in local senescent cells, and a decrease in cell proliferation ability, traditional treatments often have poor effects and ultimately lead to amputation, seriously affecting the living standards and quality of patients.

[0003] The methods for treating diabetic foot in the prior art have limited repair ability. At the same time, they have no local antibacterial effect, resulting in infection inhibiting cell regeneration and being unable to promote cell regeneration and tissue repair.

[0004] Therefore, it has become an increasingly urgent need to find an effective new method for treating diabetic foot. Summary of the Invention

[0005] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a PRP stem cell exosome chitosan composition and an application thereof.

[0006] Another purpose of the present invention is to provide a PRP stem cell exosome chitosan composite gel, a preparation method and an application thereof.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A composition, comprising the following components: activated platelet-rich plasma, stem cell exosomes and chitosan; the ratio of the activated platelet-rich plasma, stem cell exosomes and chitosan is 0.1 - 0.6 mL: 0.4 - 0.6 mL: 10 7 - 7×10 10 pieces; preferably 0.6 mL: 0.6 mL: 7×10 10 pieces

[0009] Furthermore, the preparation method of the activated platelet-rich plasma comprises the following steps:

[0010] A1. Mix the platelet-rich plasma with a calcium chloride buffer solution containing thrombin, centrifuge to remove cell debris, and collect the supernatant for sterile microfiltration;

[0011] A2. Repeated freeze-thaw cycles, followed by centrifugation to collect the supernatant and sterile microfiltration.

[0012] Further, the thrombin is bovine thrombin.

[0013] Further, in the calcium chloride buffer solution containing thrombin, the active concentration of thrombin is 5 u / mL, and the concentration of calcium chloride is 40 mM.

[0014] Further, the platelet-rich plasma and the calcium chloride buffer solution containing thrombin are mixed at a volume ratio of 1:1; and further mixed at a volume ratio of 1:1 for 1 hour.

[0015] Further, the centrifugation is carried out at 2000×g for 10 minutes.

[0016] Further, the sterile microfiltration is carried out using a sterile filter with a pore size of 0.2 μm.

[0017] Further, the preparation of the stem cell exosomes includes the following steps:

[0018] After culturing mesenchymal stem cells for 48 hours, the supernatant is obtained by low-temperature centrifugation and then the exosomes are separated by ultracentrifugation.

[0019] Further, the mesenchymal stem cells are obtained through the following steps:

[0020] Transfer the completely thawed cell suspension to a centrifuge tube containing cell culture medium, centrifuge and aspirate the supernatant, add medium to resuspend the cells, adjust the cell seeding density to 1.0×10 5 viable cells / cm 2 and inoculate into a culture vessel, shake to evenly distribute the cells, and culture in a CO2 incubator at 5% CO2, 37°C, and a saturated humidity of 95% CO2. Change the medium every other day until the cell confluence reaches 80 - 90%.

[0021] Further, the cell culture medium includes a serum-free mesenchymal stem cell medium.

[0022] Further, the low temperature is 4°C.

[0023] Further, the ultracentrifugation method is as follows: centrifuge at 1500 rpm for 5 minutes, 2000×g for 10 minutes, 10000×g for 20 minutes, and finally, ultracentrifuge at 100000×g for 120 minutes using a Beckman Coulter ultracentrifuge Type 70Ti rotor. Collect the precipitated exosomes and resuspend them in sterile PBS.

[0024] Use of the above composition in the preparation of a product for promoting cell proliferation and / or tissue repair.

[0025] Use of the above composition in the preparation of a product for wound and / or ulcer repair.

[0026] Use of the above composition in the preparation of a product for treating diabetic foot.

[0027] An activated platelet-rich plasma (PRP)-stem cell exosome-chitosan composite gel, comprising the above composition.

[0028] Preparation method of the above activated platelet-rich plasma (PRP)-stem cell exosome-chitosan composite gel, comprising the following steps:

[0029] Add chitosan to a suspension containing activated platelet-rich plasma and stem cell exosomes, mix evenly, add a povidone suspending agent, oscillate to form a stable suspension state, adjust the pH to between 7.5 and 7.8, mix with thrombin and calcium agent, stir in the dark, and form a gel-like substance after coagulation to obtain a platelet-rich plasma-stem cell exosome-chitosan composite gel.

[0030] Furthermore, the ratio of the activated platelet-rich plasma, stem cell exosomes and chitosan is 0.1 - 0.6 mL:0.4 - 0.6 mL:10 7 ~7×10 10 pieces.

[0031] Still further, in the suspension containing activated platelet-rich plasma, stem cell exosomes and chitosan, the volume percentage of the activated platelet-rich plasma is 6%, and the concentration of platelets is 800×10 9 ~1000×10 9 / L.

[0032] Still further, in the suspension containing activated platelet-rich plasma, stem cell exosomes and chitosan, the concentration of stem cell exosomes is 7×10 10 pieces / 10 mL.

[0033] Use of the above activated platelet-rich plasma (PRP)-stem cell exosome-chitosan composite gel in the preparation of a product for promoting cell proliferation and / or tissue repair.

[0034] Use of the above activated platelet-rich plasma (PRP)-stem cell exosome-chitosan composite gel in the preparation of a product for wound and / or ulcer repair.

[0035] Use of the above activated platelet-rich plasma (PRP)-stem cell exosome-chitosan composite gel in the preparation of a product for treating diabetic foot.

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] (1) The present invention first proposes a composition of activated PRP, stem cell exosomes and chitosan and forms a composite gel therefrom, which can promote the regeneration of microvascular endothelial cells, restore the normal morphology and function of vascular endothelium; promote skin regeneration; have broad-spectrum antibacterial and lipid-lowering effects, and inhibit local inflammation and immune responses.

[0038] (2) The present invention proposes a method for preparing a bio-gel by compounding activated PRP, combined with stem cell exosomes and chitosan. This method not only promotes the regeneration of vascular endothelial cells, improves microcirculation and promotes tissue repair, but also has antibacterial and anti-infection effects. Each component has good physical and chemical stability, good compatibility and good biological properties, and can act as a high-quality wound and ulcer dressing, which is beneficial to clinical application and is of great significance for the repair of various infectious, ischemic and difficult-to-heal wound ulcers.

[0039] (3) Chitosan has good biological properties and can form a gel-like substance by mixing and coagulating with PRP, stem cell exosomes, thrombin and calcium in a certain proportion. Description of the Drawings

[0040] Figure 1 It is a diagram showing the characterization results of exosomes. Among them, Fig. (A) is a diagram showing the size results of exosomes; Fig. (B) is an electron microscope result diagram of exosomes, scale = 200 nm; Fig. (C) is a diagram showing the detection results of exosome markers.

[0041] Figure 2 It is a diagram showing the research results of the effects of different concentrations of PRP on promoting cell proliferation.

[0042] Figure 3 It is a diagram showing the research results of the effects of different concentrations of activated PRP on promoting cell proliferation.

[0043] Figure 4 It is a diagram showing the research results of the effects of different amounts of stem cell exosomes on promoting cell proliferation.

[0044] Figure 5 It is a diagram showing the research results of the effects of different concentrations of chitosan on promoting cell proliferation.

[0045] Figure 6 It is a diagram showing the research results of the effects of PRP, stem cell exosomes, chitosan and the composition on promoting cell proliferation.

[0046] Figure 7 It is a three-dimensional network structure diagram after culturing human umbilical vein endothelial cells.

[0047] Figure 8It is a statistical result chart of the angioplasty and mesh distribution of the three-dimensional network structure diagram.

[0048] Figure 9 It is a result chart of the anti-inflammatory effects of PRP, stem cell exosomes, stem cell exosomes + chitosan, and the composition. Specific implementation manners

[0049] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0050] Example 1 Preparation of PRP-stem cell exosome-chitosan composite gel

[0051] Preparation method of PRP-stem cell exosome-chitosan composite gel:

[0052] (1) Mesenchymal stem cell culture:

[0053] Take out mesenchymal stem cells (MSCs) (provided by Guangdong Cord Blood Bank) from the cryogenic container, quickly place them in a 37°C water bath and shake continuously, and quickly thaw within 1 minute; under sterile conditions, transfer the completely thawed cell suspension to a centrifuge tube containing 10 mL of umbilical cord mesenchymal stem cell medium (Youkang Biotech mesenchymal stem cell serum-free medium); centrifuge at 1500 rpm for 5 minutes, aspirate and discard the supernatant after centrifugation; add an appropriate amount of fresh umbilical cord mesenchymal stem cell medium to resuspend the cells, and perform cell counting and viability detection; according to the cell number and viability, adjust the cell seeding density to 1.0×10 5 viable cells / cm 2 , add the cell suspension to the culture vessel, and gently shake to evenly distribute the cells; make marks on the culture vessel, and transfer it to a CO2 incubator with 5% CO2, 37°C, and a saturated humidity of 95% for culture. Observe the cell state and change the medium every other day; when the cell confluence reaches 80-90%, set aside.

[0054] (2) Stem cell exosome extraction

[0055] Perform exosome separation at 4°C: Take mesenchymal stem cells with a cell confluence of 80-90%, after culturing for 48 hours, extract the cell supernatant and centrifuge, and apply centrifugation at 1500 rpm for 5 minutes, 2000×g for 10 minutes, and 10000×g for 20 minutes respectively. Finally, separate the exosomes by ultracentrifugation at 100000×g for 120 minutes using a Beckman Coulter ultracentrifuge Type 70Ti rotor. Collect the precipitated exosomes and resuspend them in sterile PBS. Add sterile PBS and mix with the exosomes, and store the exosomes at -80°C at 1×10 10 / 1mL.

[0056] The characterization results of the extracted stem cell exosomes are as Figure 1 shown.

[0057] (3) Preparation of activated PRP

[0058] The platelet-rich plasma (Guangdong Cord Blood Bank) was centrifuged twice, the first time at 215×g for 10 min and the second time at 863×g for 10 min, and two methods were used to activate PRP:

[0059] ① Thrombin from bovine + CaCl2·2H2O: PRP was mixed with an equal volume of thrombin solution (containing 5 u / mL thrombin from bovine in 40 mM CaCl2·2H2O buffer) for about 1 hour. After activation, PRP was centrifuged at 2000×g for 10 minutes to remove cell debris and collected through a 0.2 μm filter pore.

[0060] ② Repeated freezing and thawing method: Deep freezing at -80°C in a refrigerator for 24 h, water bath at 37°C for 1 h, repeated freezing and thawing 5 times, and centrifugation at 2000×g for 10 min. The supernatant was filtered using a 0.2 μm sterile filter.

[0061] (4) Preparation of the suspension containing activated PRP and stem cell exosomes

[0062] PRP was added to sterile PBS, and then stem cell exosomes were added. After vortex mixing evenly to form a stable suspension state, a suspension containing activated PRP and stem cell exosomes was prepared.

[0063] (5) Preparation of PRP-stem cell exosome-chitosan composite gel

[0064] Chitosan was added to the suspension containing activated PRP and stem cell exosomes. The volume percentages of PRP and chitosan were 6% respectively, and the concentration of exosomes was 7×10 10 / 10 mL. After vortex mixing evenly, 0.1 mL of povidone suspending agent was added, and the mixture was shaken in a water bath to form a stable suspension state. Then sodium bicarbonate solution was added to adjust the pH to between 7.5 and 7.8. Thrombin and calcium agent were added to make the final volume percentages of thrombin and calcium agent 1%. The mixture was stirred thoroughly in the dark on a magnetic stirrer and solidified to form a gel-like substance.

[0065] Example 2

[0066] (1) Study on the effect of PRP and activated PRP on promoting cell proliferation

[0067] 1) Study on the effect of PRP on promoting cell proliferation

[0068] PRP groups with different concentrations: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing in a 5% CO2 incubator at 37°C for 24 hours, the old medium was aspirated, different amounts of PRP were added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, so that the final concentrations of PRP in the medium were 0%, 1%, 2%, 4%, 6%, 8%, and 10% by volume percentage, with 3 replicates in each group.

[0069] 10% FBS group: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing in a 5% CO2 incubator at 37°C for 24 hours, the old medium was aspirated, and then 100 μL of 20% (v / v) fetal bovine serum was added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, with 3 replicates in each group.

[0070] After culturing for 24 hours respectively, CCK-8 solution (10 μL) was added to each well and incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a spectrophotometer. The results of promoting cell proliferation are as Figure 2 shown. The results showed that the ability of the unactivated PRP group to promote cell proliferation was weaker than that of the 10% FBS group.

[0071] 2) Study on the effect of activated PRP on promoting cell proliferation

[0072] PRP was activated through the following steps:

[0073] ① Thrombin + CaCl2·2H2O: PRP was mixed with an equal volume of thrombin solution (5 u / mL bovine thrombin in 40 mM CaCl2·2H2O buffer) for about 1 hour. After activation, PRP was centrifuged at 2000×g for 10 minutes to remove cell debris and collected through a 0.2 μm filter pore.

[0074] ② Repeated freeze-thaw method: Deep frozen at -80°C for 24 h, water bath at 37°C for 1 h, repeated freeze-thaw 5 times, centrifuged at 2000×g for 10 min. The supernatant was filtered through a 0.2 μm sterile filter. Activated PRP was obtained.

[0075] Activated PRP groups with different concentrations: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing for 24 hours in a 5% CO₂ incubator at 37°C, the old medium was aspirated, different amounts of activated PRP were added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, so that the final concentrations of activated PRP in the medium were 0%, 1%, 2%, 4%, 6%, 8%, and 10% (v / v) respectively, with 3 replicates in each group.

[0076] 10% FBS group: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing for 24 hours in a 5% CO₂ incubator at 37°C, the old medium was aspirated, 100 μL of 20% (v / v) fetal bovine serum was added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, with 3 replicates in each group.

[0077] After culturing for 24 hours respectively, CCK-8 solution (10 μL) was added to each well and incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a spectrophotometer. The results of promoting cell proliferation are as Figure 3 shown. The results showed that the activated PRP groups with concentrations of 1%, 2%, and 6% (v / v), especially the activated PRP group with a concentration of 6% (v / v), had significantly better cell proliferation-promoting ability than the 10% FBS group. Compared with the PRP group being weaker than the 10% FBS group in the experiment, it indicated that the activated PRP had better cell proliferation-promoting ability than PRP at specific concentrations, and the concentration of 6% (v / v) was the best.

[0078] (2) Study on the effect of stem cell exosomes on promoting cell proliferation

[0079] Groups with different amounts of stem cell exosomes: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing for 24 hours in a 5% CO₂ incubator at 37°C, the old medium was aspirated, different amounts of stem cell exosomes were added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, so that the exosome concentration in the well plate was 10 5 / 10 mL, 10 6 / 10 mL, 10 7 / 10 mL), with 3 replicates in each group.

[0080] 10% FBS group: Human peritoneal mesothelial cells (HMrSV5 cells) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing in a 5% CO₂ incubator at 37 °C for 24 hours, the old medium was aspirated, and 100 μL of 20% (v / v) fetal bovine serum was added. DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, with 3 replicate wells in each group.

[0081] After culturing for 24 hours respectively, CCK-8 solution (10 μL) was added to each well and incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a spectrophotometer. The results of promoting cell proliferation are as Figure 4 shown. The results showed that when the amount of stem cell exosomes was 10 7 , the effect of promoting cell proliferation was significantly better than that of other groups. Therefore, 10 7 / 10 mL or more of stem cell exosomes was added to the composition.

[0082] (3) Chitosan promotes cell repair and proliferation

[0083] Human peritoneal mesothelial cells (HMrSV5 cells) (Guangdong Cord Blood Bank) in the logarithmic growth phase were seeded at 10,000 cells / well in a 96-well plate with 100 μL of 10% DMEM complete medium. Then, after culturing in a 5% CO₂ incubator at 37 °C for 24 hours, the old medium was aspirated, different amounts of chitosan were added, and DMEM complete medium containing 1% 100 U / mL penicillin / streptomycin was added to make up the total volume to 200 μL, so that the final concentrations of chitosan in the medium were 0%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, and 10% by volume percentage, with 3 replicate wells in each group.

[0084] After culturing for 24 hours respectively, CCK-8 solution (10 μL) was added to each well and incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a spectrophotometer. The results of culturing for 24 hours are as Figure 5 shown. The results of the 24-hour culture showed that the groups with 2%, 4%, and 6% by volume percentage were significantly better than the 0% group. It can be seen that the promotion of cell viability by chitosan at different concentrations showed a trend of first increasing and then decreasing, and chitosan at 4% - 6% could significantly improve cell activity.

[0085] Example 3

[0086] Study on promoting the proliferation of vascular endothelial cells

[0087] Umbilical vein endothelial cells (Guangdong Cord Blood Bank) (1×10 6) Inoculate into a 12-well plate for culture, and add 2 mL of DMEM / F12 medium to each well.

[0088] Control group: Culture umbilical vein endothelial cells alone;

[0089] Stem cell exosome group (10 9 EXO group): Add stem cell exosomes to the well plate where endothelial cells have been cultured for co-culture, so that the concentration of exosomes in the well plate is 1×10 9 / 10 mL;

[0090] 6% PRP group: Add activated PRP to the well plate where endothelial cells have been cultured for co-culture, so that the volume percentage of activated PRP in the well plate is 6%

[0091] 6% PRP + stem cell exosome + chitosan group (6% PRP & 10 9 EXO & 6% Chitosan group): Add stem cell exosomes, activated PRP and chitosan to the well plate where endothelial cells have been cultured for co-culture, so that the volume percentages of activated PRP and chitosan in the well plate are 6% respectively, and the concentration of exosomes is 1×10 9 / 10 mL.

[0092] The results are as Figure 6 shown. The 6% PRP + stem cell exosome + chitosan group significantly promoted the proliferation of vascular endothelial cells.

[0093] (2) Promotion of endothelial cell tube formation experiment (tube formation)

[0094] In this experiment, Matrigel (354263, BD, USA) with low concentration growth factors was used as the matrix gel. About 50 μL of basement gel was spread in each well of the 96-well plate, and human umbilical vein endothelial cells (HUVEC, 4×10 4 ) were combined with DMEM / F12 medium and inoculated on the basement.

[0095] ① On the first day, matrigel (BD, growth-factor-reduced, 356234), 96-well plate, pipette tips (200 μL), and EP tubes (1.5 mL) were pre-cooled in advance and placed in the 4 °C refrigerator overnight.

[0096] ② On the second day, after matrigel (10 mL) was thawed, it was ready for aliquoting. Operating on ice, matrigel was mixed with pre-cooled pipette tips, and EP tubes were pre-cooled in advance, and aliquoted at 500 μL each.

[0097] ③Pre-cool the 96-well plates in advance. Add 50 μL of matrigel to each well, avoiding the generation of bubbles. Leave it on ice for 5 minutes and place it in an incubator at 37 °C for about 30 minutes.

[0098] ④Control group: Human umbilical vein endothelial cells (HUVEC, 4×10 4 ) are further mixed with 100 μL of the culture medium, and 100 μL of the resuspension is added to each well.

[0099] Stem cell exosome group (EXO group): Add stem cell exosomes to the well plates where endothelial cells have been cultured and co-culture them so that the concentration of exosomes in the well plates is 7×10 10 / 10 mL.

[0100] 6% PRP group (PRP group): Add activated PRP to the well plates where endothelial cells have been cultured and co-culture them so that the volume percentage of activated PRP in the well plates is 6%.

[0101] 6% PRP + stem cell exosome + chitosan group (EXO&PRP&CHITOSAN group): Add stem cell exosomes, activated PRP and chitosan to the well plates where endothelial cells have been cultured and co-culture them so that the volume percentages of activated PRP and chitosan in the well plates are 6% respectively, and the concentration of exosomes is 7×10 10 / 10 mL.

[0102] ⑤Incubate in an incubator at 37 °C and observe after 4 hours.

[0103] Observe the three-dimensional reticular structure, and quantitatively analyze the tubule results through the software image J (National Institutes of Health, USA), and compare the angiogenesis and mesh distribution of the three groups of cells. The results are as Figure 7 and Figure 8 shown. The angiogenesis and the number of meshes in the 6% PRP + stem cell exosome + chitosan group are significantly increased.

[0104] (3) Anti-inflammatory effect of the PRP combined with the stem cell exosome chitosan mixture

[0105] Seed HMrSV5 cells in the logarithmic growth phase at 1.5×10 6 cells / well in 6-well plates (add DMEM / F12 medium to each well). When the HMrSV5 cells reach 70% confluence, randomly divide them into 3 groups according to the CCK-8 detection results:

[0106] ①Control group: Add 1 mL of high-glucose solution with a volume percentage of 4.5%;

[0107] ②Exosome group: Add 1 mL of high-glucose solution with a volume percentage of 4.5%, and then add exosomes to make the concentration of exosomes in the well plate 7×10 10 / 10 mL;

[0108] ③6% PRP + exosome + chitosan group: Add 1 mL of high-glucose solution with a volume percentage of 4.5%, activated PRP, chitosan, and exosomes to make the volume percentages of activated PRP and chitosan in the well plate 6% respectively, and the concentration of exosomes 1×10 9 / 10 mL.

[0109] ④Chitosan group: Add 1 mL of high-glucose solution with a volume percentage of 4.5% and chitosan to make the volume percentage of chitosan in the well plate 6%. After grouping treatment, continue to culture for 48 h.

[0110] ⑤Exosome + chitosan group: Add 1 mL of high-glucose solution with a volume percentage of 4.5%, chitosan, and exosomes to make the volume percentage of chitosan in the well plate 6%, and the concentration of exosomes 1×10 9 / 10 mL.

[0111] After 48 hours, extract the cell supernatant, centrifuge at 5000 g for 1 minute for ELISA detection of IL2. The detection reagent is purchased from Boao Yijie Technology Co., Ltd. Set standard wells, blank wells, and sample wells to be tested respectively. Add 50 μL to the standard wells on the enzyme-coated plate, add 40 μL of sample diluent first to the sample wells to be tested, and then add 10 μL of the sample to be tested. Add the sample to the bottom of the enzyme-coated plate wells, try not to touch the well walls, and gently shake to mix evenly. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells. Seal the plate with a sealing film and incubate at 37°C for 60 minutes. Then discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat 5 times, and pat dry. Add 50 μL of chromogenic reagent A to each well first, then add 50 μL of chromogenic reagent B, gently shake to mix evenly, and develop color at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well. Zero with the blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be carried out within 15 minutes after adding the stop solution. Repeat the biological samples three times. After calculation, the results of the expression levels of cytokines IL2, IL6, and IL8 are as Figure 9 shown. Under the action of the 6% PRP + exosome + chitosan group, the expression levels of IL2, IL6, and IL8 decreased significantly, showing an obvious anti-inflammatory effect.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composition, characterized in that, It comprises the following components: activated platelet-rich plasma, stem cell exosomes, and chitosan.

2. The composition according to claim 1, characterized in that, The ratio of the activated platelet-rich plasma, stem cell exosomes and chitosan is 0.1 - 0.6 mL: 0.4 - 0.6 mL: 10 7 - 7×10 10 cells.

3. The composition according to claim 1, wherein the preparation method of the activated platelet-rich plasma comprises the following steps: A1. Mix the platelet-rich plasma with a calcium chloride buffer solution containing thrombin, centrifuge to remove cell debris, collect the supernatant and filter it through a sterile microporous filter; A2. Perform repeated freezing and thawing, centrifuge to collect the supernatant and filter it through a sterile microporous filter.

4. The composition according to claim 2, wherein the thrombin is bovine thrombin; in the calcium chloride buffer solution containing thrombin, the active concentration of thrombin is 5 u / mL, and the concentration of calcium chloride is 40 mM; the platelet-rich plasma is mixed with the calcium chloride buffer solution containing thrombin in a volume ratio of 1:

1.

5. The composition according to claim 1, wherein the preparation of the stem cell exosomes comprises the following steps: After culturing mesenchymal stem cells for 48 hours, centrifuge at low temperature to separate the supernatant, and then separate the exosomes by ultracentrifugation; the mesenchymal stem cells are obtained through the following steps: Transfer the completely thawed cell suspension into a centrifuge tube containing cell culture medium. After centrifugation, aspirate and discard the supernatant, add medium to resuspend the cells, and adjust the cell seeding density to 1.0×10 5 viable cells / cm 2 and inoculate into a culture vessel. Shake to evenly distribute the cells and culture them in a CO2 incubator at 5% CO2, 37 °C, and a saturated humidity of 95%. Change the medium every other day until the cell confluence reaches 80-90%.

6. An activated platelet-rich plasma-stem cell exosome-chitosan composite gel, characterized in that, It includes the composition described in claims 1 to 5.

7. The preparation method of the activated platelet-rich plasma-stem cell exosome-chitosan composite gel according to claim 6, characterized in that, It includes the following steps: Add chitosan to the suspension containing activated platelet-rich plasma and stem cell exosomes and mix evenly, then add polyvinylpyrrolidone suspending agent, oscillate to form a stable suspension state, adjust the pH to between 7.5 and 7.8, mix with thrombin and calcium agent, stir in the dark, and form a gel-like substance after solidification to obtain a platelet-rich plasma-stem cell exosome-chitosan composite gel.

8. Use of the composition described in claims 1 to 5 or the platelet-rich plasma-stem cell exosome-chitosan composite gel described in claim 6 in the preparation of a product for promoting cell proliferation and / or tissue repair.

9. Use of the composition described in claims 1 to 5 or the platelet-rich plasma-stem cell exosome-chitosan composite gel described in claim 6 in the preparation of a product for wound and / or ulcer repair.

10. Use of the composition described in claims 1 to 5 or the platelet-rich plasma-stem cell exosome-chitosan composite gel described in claim 6 in the preparation of a product for treating diabetic foot.

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