Preparation method of engineered exosome with better treatment effect

Abstract

The invention belongs to the technical field of bioengineering, and discloses a preparation method of an engineered exosome with a better treatment effect, and the preparation method comprises the following steps: S1, stimulating mesenchymal stem cells by using 10 [mu] g / mL of polyinosinic acid cytidine monophosphate for 1 h, and removing the polyinosinic acid cytidine monophosphate; s2, introducing a DMEM / F12 culture medium containing 10% of fetal calf serum into the mesenchymal stem cells subjected to stimulation treatment; s3, putting the mesenchymal stem cells into a cell incubator, culturing for 24 hours, and separating and purifying the exosome by adopting a conventional method; according to the invention, stem cells are pretreated by polyinosinic acid cytidine monophosphate, the stem cells are induced to secrete exosomes rich in therapeutic factors, and advanced preparation and purification technologies are combined, so that the engineered exosomes which are stable in quality and have a better therapeutic effect on osteoarthritis are successfully prepared.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing engineered exosomes with better therapeutic effects. Background Art

[0002] Osteoarthritis (OA) is the most common joint disease in the elderly, with a high incidence, high disability rate, and heavy disease burden. The current treatment options are difficult to meet the needs, so patients with terminal OA have to undergo traumatic and expensive joint replacement surgery. In recent years, the development of cell biology therapy has provided promising hope for the early treatment of OA. Among them, exosomes containing various nutritional factors and regulatory factors released by mesenchymal stem cells (MSCs) have shown gratifying results in the treatment of OA.

[0003] However, existing methods for preparing stem cell exosomes have many problems: (1) Exosomes prepared from natural stem cells contain a large amount of RNA and protein that are not helpful for treatment; (2) The yield of exosomes prepared from natural stem cells is often low and unstable. Therefore, the present invention proposes a method for preparing engineered exosomes with better therapeutic effects to solve such problems. Summary of the invention

[0004] The present invention is intended to provide a method for preparing engineered exosomes with better therapeutic effects, using polyinosinic acid and polycytidylic acid to pre-treat and stimulate stem cells to induce them to secrete exosomes richer in therapeutic factors, and combining advanced preparation and purification technologies to successfully prepare engineered exosomes with stable quality and better therapeutic effects on osteoarthritis. The problems in the background technology are solved.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing engineered exosomes with better therapeutic effects comprises the following steps:

[0007] S1, stimulate mesenchymal stem cells with agonists, and remove the agonists after 1 hour of stimulation;

[0008] S2, introducing the cell culture medium into the stimulated mesenchymal stem cells;

[0009] S3. After culturing the mesenchymal stem cells in a cell incubator for 24 hours, the exosomes were isolated and purified using conventional methods.

[0010] Furthermore, in step S1, the agonist is polyinosinic-cytidylic acid.

[0011] Furthermore, the concentration of the polyinosinic-cytidylic acid is 10 μg / mL.

[0012] Furthermore, in step S1, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0013] Furthermore, in step S2, the cell culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum.

[0014] The beneficial effects of the technical solution are:

[0015] 1. The engineered stem cell exosomes prepared by Poly(I:C) stimulation are of higher quality and more significant therapeutic effect.

[0016] 2. Engineered stem cell exosomes after Poly(I:C) stimulation have unique proteomic results.

[0017] 3. This scheme can achieve early treatment of osteoarthritis, alleviate patient symptoms, and improve quality of life. It can more stably prepare engineered exosomes with better osteoarthritis treatment effects, thereby reducing patients' need for joint replacement surgery and reducing medical costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of separation and extraction of rat ADSCs in an embodiment of the present invention;

[0019] Figure 2 A flowchart of EVs acquisition in an embodiment of the present invention;

[0020] Figure 3 This is a schematic diagram of Transwell co-culture in an embodiment of the present invention;

[0021] Figure 4 This is a schematic diagram of EVs co-culture in an embodiment of the present invention;

[0022] Figure 5 This is a diagram for identifying the osteogenic (A), adipogenic (B), and chondrogenic (C) differentiation abilities of rat ADSCs in the examples of the present invention;

[0023] Figure 6 Schematic diagram of the expression of surface markers CD90, CD73, CD34, and CD45 of rat ADSCs detected by flow cytometry in an embodiment of the present invention;

[0024] Figure 7 This is a schematic diagram of detecting the mRNA levels of TLR3 and TLR4 in ADSCs by RT-PCR in an embodiment of the present invention;

[0025] Figure 8This is a schematic diagram of RT-PCR detection of IL-6 and IL-10 mRNA levels in ADSCs in an embodiment of the present invention;

[0026] Fig. 9 Schematic diagram of Western Blot detection of IL-6 and IL-10 protein levels in ADSCs in an embodiment of the present invention;

[0027] Fig.10 This is a characterization diagram of EVs in an embodiment of the present invention;

[0028] Fig.11 Comparison of the relative proliferation rate of chondrocytes detected by CCK-8 method in the embodiment of the present invention Figure 1 ;

[0029] Fig.12 This is a graph showing the proportion of chondrocyte apoptosis detected by flow cytometry after Transwell co-culture in an embodiment of the present invention;

[0030] Fig.13 Comparison of the relative proliferation rate of chondrocytes detected by CCK-8 method in the embodiment of the present invention Figure 2 ;

[0031] Fig.14 This is a schematic diagram of the proportion of chondrocyte apoptosis detected by flow cytometry after EVs co-culture in an embodiment of the present invention;

[0032] Fig.15 Schematic diagram of immunofluorescence detection of chondrocyte MMP13, Aggrencan and CollagenⅡ expression after Transwell co-culture in the embodiment of the present invention;

[0033] Fig.16 Schematic diagram of WB detection of MMP13, Aggrencan and CollagenⅡ expression in chondrocytes after Transwell co-culture in the embodiment of the present invention;

[0034] Fig.17 Schematic diagram of immunofluorescence detection of chondrocyte MMP13, Aggrencan and CollagenⅡ expression after EVs co-culture in an embodiment of the present invention;

[0035] Fig.18 This is a schematic diagram of WB detection of chondrocyte MMP13, Aggrencan and CollagenⅡ expression after EVs co-culture in an example of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0037] A method for preparing engineered exosomes with better therapeutic effects comprises the following steps:

[0038] S1, stimulate mesenchymal stem cells with agonists, and remove the agonists after 1 hour of stimulation;

[0039] Preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells;

[0040] Preferably, the agonist is polyinosinic-cytidylic acid;

[0041] Preferably, the concentration of polyinosinic-cytidylic acid is 10 μg / mL.

[0042] S2, introducing the cell culture medium into the stimulated mesenchymal stem cells;

[0043] Preferably, the cell culture medium is DMEM / F12 medium containing 10% fetal bovine serum.

[0044] S3. After culturing the mesenchymal stem cells in a cell incubator for 24 hours, the exosomes were isolated and purified using conventional methods.

[0045] Animal experiments

[0046] 1. Main materials and reagents:

[0047] Cell culture flasks (Wuxi Nice Biotechnology Co., Ltd.); Transwell cell chamber culture plates (Beijing Lanjieke Technology Co., Ltd.); cell culture plates (Beijing Lanjieke Technology Co., Ltd.); laser confocal dish (Wuxi Nice Biotechnology Co., Ltd.); 40μm filter (Wuxi Nice Biotechnology Co., Ltd.); centrifugal concentrator (Pall, USA); EPF purification column (Shanghai Yumeibo Biotechnology Co., Ltd.); collagenase type I (BioFroxx, Germany); ECS extraction reagent (Shanghai Yumeibo Biotechnology Co., Ltd.); 1× PBS buffer (Hefei White Shark Biotechnology Co., Ltd.); DMEM / F12 medium (Germany) ibco company); fetal bovine serum FBS (Gibco company, USA); penicillin-streptomycin (Hefei White Shark Biotechnology Co., Ltd.); trypsin (Gibco company, USA); rat adipose-derived mesenchymal stem cell osteogenic differentiation kit (Guangzhou Saiye Biotechnology Co., Ltd.); rat adipose-derived mesenchymal stem cell adipogenic differentiation kit (Guangzhou Saiye Biotechnology Co., Ltd.); rat adipose-derived mesenchymal stem cell chondrogenic differentiation kit (Guangzhou Saiye Biotechnology Co., Ltd.); mesenchymal stem cell (rat) surface marker detection kit (Guangzhou Saiye Biotechnology Co., Ltd.); 4% paraformaldehyde universal fixative (Hefei White Shark Biotechnology Co., Ltd.); Ploy I:C (Sigma company, USA); LPS (Sigma company, USA); 1× PBS buffer (Hefei White Shark Biotechnology Co., Ltd.); Trizol reagent (Ambion company, USA); ReverTra qPCR RT Master Mix (TOYOBO, Japan); TB Fast qPCR Mix (TaKaRa, Japan); RIPA lysis buffer (Shanghai Biotech Co., Ltd.); protease inhibitors (Shanghai Biotech Co., Ltd.); BCA detection kit (Shanghai Biotech Co., Ltd.); protein loading buffer (Shanghai Biotech Co., Ltd.); PVDF membrane (Millipore, USA); CCK-8 kit (Shanghai Biotech Co., Ltd.); RIPA lysis buffer (Shanghai Biotech Co., Ltd.); protease inhibitors (Shanghai Biotech Co., Ltd.); BCA detection kit (Shanghai Biotech Co., Ltd.); Annexin V-FITC / PI apoptosis detection kit (Shanghai Aibixin Biotechnology Co., Ltd.); 4% paraformaldehyde (Hefei White Shark Biotechnology Co., Ltd.); DAPI staining solution (Shanghai Biyuntian Biotechnology Co., Ltd.); immunostaining blocking solution (Shanghai Biyuntian Biotechnology Co., Ltd.); immunostaining permeabilization solution (Shanghai Biyuntian Biotechnology Co., Ltd.); recombinant rat IL-1β protein (PreproTech, USA); rabbit anti-rat CD81 antibody (Abcam, USA); rabbit anti-rat TSG101 antibody (Abcam, USA); rabbit anti-rat CollagenⅡ antibody (Affinity, USA); rabbit anti-rat Aggrecan antibody (Affinity, USA); rabbit anti-large MMP13 antibody (Affinity, USA); rabbit anti-rat IL-6 antibody (Chengdu Zhengneng Biotechnology Co., Ltd.); rabbit anti-rat IL-10 antibody (Chengdu Zhengneng Biotechnology Co., Ltd.); rabbit anti-rat β-actin antibody (Affinity, USA); Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Shanghai Biotime Biotechnology Co., Ltd.); goat anti-rabbit HRP secondary antibody (Shanghai Biotime Biotechnology Co., Ltd.).

[0048] 2. Main instruments:

[0049] Clean bench (Suzhou Antai Air Technology Co., Ltd.); optical microscope (Leica, Germany); cell culture incubator (Thermo, USA); pipette (Eppendorf, Germany); a set of small animal surgical instruments (Jiangsu Sihong Jinjing Medical Instrument Co., Ltd.); low-temperature centrifuge (Eppendorf, Germany); spectrophotometer (Thermo, USA); CFX96 fluorescence quantitative PCR instrument (Bio-Rad, USA); Western electrophoresis instrument (Bio-Rad, USA); Western transfer instrument (Bio-Rad, USA); multi-color fluorescence chemiluminescence imaging system (VILBER, France); laser confocal microscope (ZEISS, Germany); enzyme-linked immunosorbent assay (Thermo, USA); LSRFortessa TM Flow cytometer (BD, USA); NanoSight NS300 nanoparticle tracking analyzer (Malvern Panalytical, UK); transmission electron microscope (JEOL, Japan).

[0050] 3. Experimental methods

[0051] 3.1. Extraction and identification of ADSCs from rat abdominal adipose tissue

[0052] 1) The experimental animal procedures were approved by the Experimental Animal Welfare and Ethics Committee of the Army Medical University (AMUWEC20223809);

[0053] 2) Two-week-old male SD rats (n=2, provided by the Experimental Animal Center of Army Medical University) were euthanized by CO2, and adipose tissue was obtained from the abdomen;

[0054] 3) After the adipose tissue is fully minced, complete culture medium containing 0.1% type I collagenase is added for digestion overnight to isolate ADSCs;

[0055] 4) The cells were cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin;

[0056] 5) Replace the culture medium with fresh complete medium every other day. When the cells are 90% to 95% confluent, trypsinize them and subculture them. The fifth generation ADSCs were used in subsequent experiments. The process of isolating ADSCs from rat abdominal adipose tissue is as follows Figure 1 As shown;

[0057] 6) Induce osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation of ADSCs using a rat adipose-derived mesenchymal stem cell osteogenic differentiation kit, a rat adipose-derived mesenchymal stem cell adipogenic differentiation kit and a rat adipose-derived mesenchymal stem cell chondrogenic differentiation kit, respectively; after induction, fix the cells with 4% paraformaldehyde, stain the cells with Alizarin Red, Oil Red O and Alcian Blue, respectively, and observe the staining under a microscope and take photos for record.

[0058] 7) After completing labeling and staining according to the instructions of the Mesenchymal Stem Cell (Rat) Surface Marker Detection Kit, immediately detect ADSCs surface markers by flow cytometry.

[0059] 3.2. Induction of ADSCs in vitro using TLR3 or TLR4 specific agonists

[0060] 1) When the 5th generation ADSCs reached 90% confluence, TLR4 receptor agonist LPS, TLR3 receptor agonist Ploy I:C and complete culture medium were used to prepare induction medium, and the final concentration of the inducer was LPS 5 μg / mL, Ploy I:C 10 μg / mL according to the previous study;

[0061] 2) After removing the old culture medium of ADSCs, wash them three times with 1×PBS buffer, add the prepared induction medium, and then place them in a cell culture incubator at 37°C and 5% CO2 for induction for 1 hour;

[0062] 3) Remove the induction medium, rinse three times with 1× PBS buffer, add fresh complete serum medium, and continue culturing at 37° C. and 5% CO 2 for 24 hours.

[0063] 3.3. EVs acquisition and characterization

[0064] 1) Get EVs:

[0065] ① Centrifuge the collected cell supernatant at 4°C, 3000g for 10 minutes to remove cell debris;

[0066] ② Transfer to the upper chamber of the centrifugal concentrator tube and centrifuge at 4°C, 6000g for 1 hour to concentrate the supernatant;

[0067] ③ Add ECS extraction reagent to the concentrated supernatant at a volume ratio of 4:1, mix thoroughly and let stand at 4°C overnight (no more than 24 hours);

[0068] ④ Centrifuge at 4°C, 10,000 g for 60 min to obtain EVs precipitate, and add 1× PBS buffer to fully dissolve and resuspend;

[0069] ⑤ Transfer to the EPF purification column and centrifuge at 4°C, 3000g for 10 minutes to obtain filtered and purified EVs;

[0070] ⑥ The obtained EVs suspension was divided and stored at -80℃ for future use. Figure 2 shown.

[0071] 2) Characterization and identification:

[0072] The particle size of EVs was detected by nanoparticle tracking analyzer, and the morphology of EVs was observed by uranyl acetate negative staining technique and transmission electron microscopy. The protein concentration of EVs was determined by BCA method, and the surface markers of EVs CD81 and TSG101 were detected by Western Blot experiment.

[0073] 3.4. Total RNA acquisition, reverse transcription and RT-PCR detection of ADSCs after induction

[0074] 1) Add Trizol reagent to the induced AD-MSCs, homogenize at room temperature for 5 minutes, and then aspirate the lysate;

[0075] 2) Total RNA was extracted step by step using chloroform and isopropanol, and RNA concentration and purity were tested using a spectrophotometer;

[0076] 3) Use ReverTra qPCR RT Master Mix for reverse transcription reaction;

[0077] 4) Press TB The reaction system was constructed according to the instructions of Fast qPCR Mix reagent. The amplification conditions were as follows: a. Reverse transcription: 37°C for 15 minutes; b. Pre-denaturation: 95°C for 30 seconds; c. Denaturation: 95°C for 10 seconds; d. Annealing / extension: 60°C for 10 seconds; e. Repeat steps c and d for a total of 40 cycles;

[0078] 5) RT-qPCR reactions were performed using the Bio-Rad CFX96 Connect Real-Time System.

[0079] 6) The information of primers used is shown in Table 1. GAPDH was used as the reference gene, and the primers were designed and prepared by Shanghai Sangon Biotechnology Co., Ltd. The experiment was repeated three times for each sample. Statistical analysis was performed using 2 -ΔΔCt Law.

[0080] Table 1 Target gene printing sequence

[0081]

[0082] 3.5. Total protein acquisition of induced ADSCs and Western Blot experiment

[0083] 1) Add RIPA lysis buffer and protease inhibitors to the induced ADSCs and lyse on ice for 10 minutes;

[0084] 2) After fully scraping the cells, aspirate the lysate and centrifuge at 12000g for 15 minutes at 4°C, and aspirate the supernatant to obtain the total protein lysate;

[0085] 3) Detect protein concentration using BCA kit;

[0086] 4) Add protein loading buffer at a volume ratio of 4:1, boil in a 100°C metal bath for 10 minutes, and place on ice for electrophoresis;

[0087] 5) Use 20 μg of protein sample for electrophoresis and transfer to a PVDF membrane with a pore size of 0.22 μm;

[0088] 6) Block with Western blocking solution for 2 hours at room temperature and incubate with primary antibodies against IL-6, IL-10, and β-actin at 4°C overnight;

[0089] 7) After washing, the membrane was incubated with the secondary antibody at room temperature for 1 hour and developed using a multi-color fluorescent chemiluminescent imaging system;

[0090] 8) β-actin was used as a reference protein and the gray value of the bands was measured using Image J software.

[0091] 3.6. Obtaining rat knee chondrocytes

[0092] 1) Two-week-old male SD rats (n=2, provided by the Experimental Animal Center of Army Medical University) were euthanized by CO2, the knee joint cavity was opened, and the cartilage tissue was carefully removed and fully minced;

[0093] 2) Add complete culture medium containing 0.2% type II collagenase and place in an incubator for digestion overnight to isolate chondrocytes;

[0094] 3) Use DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and culture in a 37°C incubator with 5% CO2;

[0095] 4) Replace the culture medium with fresh complete medium every other day. When the cells reach 90% confluence, digest them with trypsin and subculture. The first generation chondrocytes were used for subsequent experiments.

[0096] 3.7 Transwell co-culture

[0097] 1) Experimental design: Control group (blank control group), IL-1β group (10 ng / mL IL-1β was added to the lower chamber only), IL-1β+ADSC group (10 ng / mL active IL-1β was added to the lower chamber and co-cultured with uninduced ADSCs), IL-1β+ADSC TLR4 Group (10 ng / mL active IL-1β was added to the lower chamber and co-cultured with ADSCs activated with TLR4), IL-1β+ADSC TLR3 Group (10 ng / mL active IL-1β was added to the lower chamber and co-cultured with ADSCs that activated TLR3); for proliferation detection and immunofluorescence experiments, 24-well plates and 24-well confocal dishes were used, 1×104 chondrocytes were seeded in each well, and 2×104 ADSCs were planted in the upper chamber of each 24-well Transwell culture plate. For apoptosis and Western Blot detection, 6-well plates were used, 1×106 chondrocytes were planted in each well, and 2×106 ADSCs were planted in the upper chamber of each 6-well Transwell culture plate;

[0098] 2) The same number of ADSCs were seeded into the upper chamber of the Transwell culture plate, and after the ADSCs adhered to the plate, they were pretreated according to the induction scheme of this study.

[0099] 3) The same number of chondrocytes were seeded into 6-well plates or 24-well plates. After the cells adhered to the wall, the upper chamber of the Transwell seeded with ADSCs was transferred to the 6-well plate or 24-well plate.

[0100] 4) At the same time, IL-1β (10 ng / mL) was added to the lower chamber of the co-culture system and co-cultured with chondrocytes in an incubator at 37°C and 5% CO2 for 24 hours. Figure 3 shown.

[0101] 3.8. EVs co-culture

[0102] The groups were divided into: Control group (blank control group), IL-1β group (only 10 ng / mL IL-1β active protein was added), IL-1β+ADSC EVs Group (addition of 10 ng / mL active IL-1β and uninduced ADSCs-derived EVs), IL-1β+ADSC TLR4-EVs Group (addition of 10 ng / mL active IL-1β and EVs derived from ADSCs that activated TLR4), IL-1β+ADSC TLR3-EVsGroup (addition of 10 ng / mL active IL-1β and EVs derived from ADSCs that activated TLR3). For proliferation detection and immunofluorescence experiments, 24-well plates and 24-well confocal dishes were used, 1×104 chondrocytes were seeded in each well, and 20 μg / well EVs were added for co-culture for 24 hours. For cell apoptosis and Western Blot detection, 6-well plates were used, 1×106 chondrocytes were seeded in each well, and 40 μg / well EVs were added for co-culture for 24 hours. Figure 4 shown.

[0103] 3.9. CCK-8 detection

[0104] 1) After the co-culture is completed, the old culture medium in the chondrocytes is removed and fresh complete culture medium containing 10% CCK-8 reagent is added;

[0105] 2) Place in a cell culture incubator at 37°C and 5% CO2 and incubate for 2 hours;

[0106] 3) The absorbance value was then measured at 450 nm using an ELISA reader and statistical analysis was performed.

[0107] 3.10 Annexin V-FITC / PI apoptosis detection

[0108] 1) After co-culture, remove the old culture medium in the 6-well plate and wash the chondrocytes three times with 1× PBS;

[0109] 2) Add 0.25% trypsin to digest the cells, centrifuge at 2000 rpm for 5 minutes to collect the chondrocytes, discard the supernatant, add pre-cooled 1× PBS to resuspend the cells, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant to obtain the washed chondrocytes;

[0110] 3) Then, 300 μL of 1× Binding Buffer was added to suspend the chondrocytes, and 5 μL of Annexin V-FITC was added to mix well, protected from light, and incubated at room temperature for 15 minutes.

[0111] 5) Before testing, add 5 μL of PI staining and 200 μL of 1× Binding Buffer

[0112] 6) In BD LSRFortessa TM The detection was performed on a flow cytometer and analyzed using Flowjo 10.8.1 software.

[0113] 3.11. Obtaining total protein from chondrocytes and Western Blot

[0114] 1) After co-culture, chondrocytes were lysed using RIPA lysis buffer;

[0115] 2) Measure protein concentration using the BCA method;

[0116] 3) Add loading buffer to the lysate and boil at 100°C for 10 minutes;

[0117] 4) 20 μg of protein sample was used for electrophoresis, and the lysate was separated by electrophoresis on SDS-PAGE (10% gel). Then, the membrane was transferred at a constant current of 200 mA for 1.5 hours;

[0118] 5) PVDF membrane was incubated with CollagenⅡ, Aggrecan, and MMP13 overnight;

[0119] 6) After washing, incubate with secondary antibody at room temperature for 1 hour. After washing again, detect the bands using a multi-color fluorescent chemiluminescent imaging system.

[0120] 3.12 Immunofluorescence staining

[0121] 1) After co-culture, chondrocytes were fixed with 4% paraformaldehyde for 30 min at room temperature;

[0122] 2) Wash with pre-cooled 1× PBS three times, then add immune permeabilization solution and permeabilize at room temperature for 30 minutes;

[0123] 3) Discard the permeabilization solution, wash with immunostaining washing solution, add blocking solution and block at room temperature for 60 minutes;

[0124] 4) After discarding the blocking solution, add primary antibodies against CollagenⅡ, Aggrecan, and MMP13 and incubate overnight in a 4°C refrigerator;

[0125] 5) After thorough washing, cells were incubated with Alexa Fluor 488-labeled secondary antibodies for 60 min at room temperature;

[0126] 6) Use DAPI staining solution to stain nuclei for 10 minutes at room temperature;

[0127] 7) Observe cells and take pictures using a laser confocal microscope;

[0128] 8) The relative fluorescence intensity (total fluorescence intensity / number of cell nuclei) was analyzed using ZEN software (2012 version). Three different images were taken from each group for statistical analysis.

[0129] 4. Experimental results

[0130] 4.1 Obtaining and identifying rat ADSCs Microscopic observation showed that as the passage progressed, the obtained rat ADSCs gradually showed a typical spindle-shaped fibroblast-like morphology and grew closely attached to the wall. The results of Alizarin red, Oil Red O, and Alcian blue staining showed that the obtained rat ADSCs had good osteogenic, adipogenic, and chondrogenic differentiation potentials. Figure 5 shown.

[0131] The results of flow cytometry showed that the obtained ADSCs highly expressed positive markers CD90 (94.60%) and CD73 (93.70%), and almost did not express negative markers CD34 (2.72%) and CD45 (0.81%). Figure 6 shown.

[0132] 4.2 Activation of TLR3 and TLR4

[0133] RT-PCR results showed that under our induction scheme, ADSCs pretreated with LPS upregulated the mRNA expression of TLR4 compared with the Control group (P < 0.0001) and the Poly I:C group (P < 0.0001); and ADSCs induced by Poly I:C upregulated the mRNA expression of TLR3 compared with the Control group (P < 0.0001) and the LPS group (P < 0.001). Figure 7 As shown (*: P < 0.05; ****: P < 0.0001).

[0134] 4.3 Cytokine secretion characteristics of ADSCs after induction

[0135] RT-PCR and Western Blot results showed that compared with the Control group and Poly I:C group, ADSCs after LPS pretreatment stably expressed the pro-inflammatory cytokine IL-6 at a high level (P < 0.05); compared with the Control group and LPS group, ADSCs after Poly I:C induction stably expressed a higher level of anti-inflammatory cytokine IL-10 (P < 0.05). RT-qPCR results also showed the same trend (P < 0.05). Figure 8 , Fig. 9 As shown (in Figure 8 *: P < 0.05; ****: P < 0.0001; Fig. 9 **: P<0.05; **: P<0.01).

[0136] EVs characterization

[0137] Transmission electron microscopy showed that most of the EVs obtained from each group showed a typical spherical double-layer membrane structure; the results of particle size analysis showed that the diameter distribution range of the vesicles in each group was not completely consistent: ADSC EVs Distributed at 222±6.2nm, ADSC TLR4-EVs Mainly distributed at 330.8±33.5nm, ADSC TLR3-EVs The main distribution is 236.9±8.2nm. WB results show that all groups of vesicles highly express CD81 and TSG101. Fig.10 As shown (A is a schematic diagram of EVs particle size distribution; B is a schematic diagram of EVs morphology under transmission electron microscopy; C is a schematic diagram of EVs marker protein detection).

[0138] 4.5 ADSC TLR3 ADSC TLR4 Regulatory effects on chondrocyte proliferation and apoptosis

[0139] CCK-8 experimental data showed that IL-1β had a significant inhibitory effect on chondrocyte proliferation (P < 0.0001). TLR3 Both induced and uninduced ADSCs could significantly increase the proliferation rate of chondrocytes (P < 0.0001), while ADSCs TLR4 It is worth noting that compared with uninduced ADSCs, ADSCs TLR3 It has a more obvious effect on promoting chondrocyte proliferation (P < 0.01). Fig.11 As shown (the statistical graphs from left to right represent the Control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 group) (****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; ##: P < 0.01, compared with the IL-1β+ADSC group).

[0140] The results of apoptosis detection showed that IL-1β significantly increased the proportion of apoptotic chondrocytes (P<0.0001). TLR3 Both induced and uninduced ADSCs could effectively reduce the proportion of apoptotic chondrocytes (P < 0.0001), while ADSCs TLR4 Compared with uninduced ADSCs, ADSCs TLR3 It showed a stronger ability to inhibit chondrocyte apoptosis (P<0.05). Fig.12As shown (****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; #: P < 0.05, compared with the IL-1β+ADSC group).

[0141] 4.6 ADSC TLR3-EVs ADSC TLR4-EVs Regulatory effects on chondrocyte proliferation and apoptosis

[0142] The results of CCK-8 assay showed that IL-1β significantly inhibited the proliferation of chondrocytes (P<0.0001). EVs (P<0.01) and ADSC TLR3-EVs (P<0.05) can effectively improve the inhibition of IL-1β on chondrocyte proliferation, but there is no statistical difference between the two groups (P>0.05). TLR4-EVs It could not improve the inhibitory effect of IL-1β on chondrocyte proliferation (P>0.05). Fig.13 As shown (the statistical graphs from left to right represent the Control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 group) (*: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; ns: P > 0.05, compared with the IL-1β+ADSC group).

[0143] The results of apoptosis detection showed that IL-1β significantly increased the proportion of apoptotic chondrocytes (P < 0.0001). EVs ADSC TLR3-EVs ADSC TLR4-EVs Both showed a trend of decreasing the proportion of apoptotic chondrocytes, but there was no statistical difference between them (all P>0.05). Fig.14 As shown (****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; #: P < 0.05, compared with the IL-1β+ADSC group).

[0144] 4.7 ADSC TLR3 and ADSCs TLR4Different regulatory effects on the OA-like phenotype of chondrocytes induced by IL-1β MMP13, Aggrecan and CollagenⅡ are key markers of the catabolism and anabolism processes during cartilage degradation, and can evaluate the osteoarthritis phenotype of chondrocytes. Immunofluorescence results showed that IL-1β treatment significantly increased the expression of matrix degrading enzyme MMP13 in chondrocytes, while reducing the expression of Aggrecan and CollagenⅡ (both P < 0.0001). Compared with the IL-1β group, co-culture with uninduced ADSCs effectively inhibited the expression of MMP13 in chondrocytes (P < 0.001), and upregulated the expression of Aggrecan (P < 0.01) and CollagenⅡ (P < 0.01). Compared with uninduced ADSCs, ADSC TLR3 The expression of MMP13 was significantly downregulated (P < 0.05), while the expression of Aggrecan (P < 0.01) and CollagenⅡ (P < 0.01) was upregulated. TLR4 It only showed a down-regulation effect on the expression of MMP13 (P < 0.05), but had no up-regulation effect on the expression levels of Aggrecan (P > 0.05) and CollagenⅡ (P > 0.05). Fig.15 As shown (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; #: P < 0.05, ##: P < 0.01, compared with the IL-1β+ADSC group), (bar graphs B / C / D are all according to the Control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 groups in order).

[0145] Western Blot results also showed that ADSC TLR4 It was not able to effectively down-regulate the expression of MMP13 and increase the expression of Aggrecan and CollagenⅡ in chondrocytes induced by IL-1β (all P>0.05). Compared with the IL-1β group, the uninduced ADSCs could reduce the expression of MMP13 (P<0.05) and increase the expression of Aggrecan (P<0.01), but could not up-regulate the expression of CollagenⅡ (P>0.05). TLR3 It can more stably inhibit the expression of MMP13 (P<0.05), and significantly upregulate the expression of chondrocyte Aggrecan (P<0.01) and CollagenⅡ (P<0.001). Fig.16As shown (*: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: P > 0.05, compared with IL-1β group; #: P < 0.05, ###: P < 0.001, compared with IL-1β+ADSC group), (bar graphs B / C / D are all according to Control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 groups in order).

[0146] 4.8 ADSC TLR3-EVs and ADSCs TLR4-EVs The results of immunofluorescence staining showed that IL-1β treatment significantly increased the expression of metabolic marker MMP13 in chondrocytes, while reducing the expression of synthetic markers Aggrecan and CollagenⅡ (all P < 0.0001). EVs It effectively inhibited the expression of MMP13 in chondrocytes (P<0.0001) and upregulated the expression of Aggrecan (P<0.05) and CollagenⅡ (P<0.0001). EVs Compared with ADSC TLR3-EVs The expression of MMP13 was significantly downregulated (P < 0.01), while the expression of Aggrecan (P < 0.05) and CollagenⅡ (P < 0.01) was upregulated. TLR4-EVs It neither down-regulated the expression of MMP13 (P>0.05) nor up-regulated the expression levels of Aggrecan (P>0.05) and CollagenⅡ (P>0.05). Fig.17 As shown (*: P < 0.05, ***: P < 0.001, ****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; #: P < 0.05, compared with the IL-1β+ADSC group), (the bar graphs B / C / D are all according to the Control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 groups in order).

[0147] Western Blot results showed that ADSC TLR4-EVsThe expression of MMP13 in chondrocytes induced by IL-1β was upregulated, but the expression of Aggrecan and CollagenⅡ was not increased (all P>0.05). EVs The expression of MMP13 was significantly reduced (P < 0.05), while the expression of Aggrecan (P < 0.01) and Collagen Ⅱ (P < 0.05) were increased. EVs Compared with ADSC TLR4-EVs It can more significantly inhibit the expression of MMP13 (P<0.05), and significantly upregulate the expression of chondrocyte Aggrecan (P<0.05) and CollagenⅡ (P<0.05). Fig.18 (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: P > 0.05, compared with the IL-1β group; #: P < 0.05, compared with the IL-1β+ADSC group) as shown in (bar graphs B / C / D are all according to the control group; IL-1β group; IL-1β+ADSC group; IL-1β+ADSC TLR4 Group; IL-1β+ADSC TLR3 groups in order).

[0148] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing engineered exosomes with better therapeutic effects, characterized in that: The following steps are involved: S1, stimulate mesenchymal stem cells with agonists, and remove the agonists after 1 hour of stimulation; S2, introducing the cell culture medium into the stimulated mesenchymal stem cells; S3. After culturing the mesenchymal stem cells in a cell incubator for 24 hours, the exosomes were isolated and purified using conventional methods.

2. The method for preparing engineered exosomes with better therapeutic effects according to claim 1, characterized in that: In step S1, the agonist is polyinosinic-cytidylic acid.

3. The method for preparing engineered exosomes with better therapeutic effects according to claim 2, characterized in that: The concentration of polyinosinic-polycytidylic acid is 10 μg / mL.

4. The method for preparing engineered exosomes with better therapeutic effects according to claim 1, characterized in that: In step S1, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

5. The method for preparing engineered exosomes with better therapeutic effects according to claim 1, characterized in that: In step S2, the cell culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum.

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