Preparation method and application of mesenchymal stem cell exosome

Abstract

The invention provides a preparation method and application of mesenchymal stem cell exosomes, and the method is characterized in that N-acetylcysteine and quercetin are added into a culture medium used for culturing MSC cells, so that the MSC cells are stimulated to generate exosomes with anti-aging and anti-apoptosis effects. In addition, the exosome generated by stimulating the MSC through a culture mode of gradually reducing oxygen partial pressure further has an anti-aging effect; the adverse effects of acute oxidative stress, decline of multiplication capacity, unstable quality and the like in a low-oxygen state can be inhibited, and the activity of related components of cells and exosomes is not damaged. The method shows good anti-aging and anti-apoptosis effects in vitro, and a new thought is provided for treating aging diseases by the exosome in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a preparation method of mesenchymal stem cell exosomes and an application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with self-replication and multidirectional differentiation potential. They are rich in sources and can be obtained from bone marrow, umbilical cord, cord blood, fat and other tissues, and have low immunogenicity. Cell therapy based on MSCs has been successfully applied to the treatment of cardiovascular diseases, bone and cartilage defects, diabetes and other diseases. MSCs release a variety of cytokines and growth factors through paracrine and autocrine secretion. These secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

[0003] Exosomes are nano-scale lipid inclusion structures with a diameter of 30 to 150 nm, which contain substances such as proteins, mRNA and microRNA. Exosomes exist naturally in body fluids, including blood, saliva, urine, breast milk, etc. Exosomes are membrane vesicles derived from late endosomes (also called multivesicular bodies) secreted by living cells. Almost all cells, including tumors, can produce and release exosomes. Exosomes are secreted and released by cells, spread in body fluids such as blood, and can finally be engulfed by other cells. They are important mediators of intercellular communication. Exosomes secreted by host cells or tumor cells are involved in cell growth, proliferation, metabolism and regulation.

[0004] N-acetyl Cysteine ​​(NAC) is produced by the acetylation of L-cysteine, and its precursor cysteine ​​(Cys) is a common polar α-amino acid containing sulfhydryl groups in organisms. It is a non-essential amino acid that can be converted into glucose through metabolism. It can be produced by methionine metabolism in the human body, and it can also be converted into cystine. The biological activity of N-acetyl cysteine ​​is mainly attributed to the sulfhydryl group (-SH) in its structure, while its acetyl group protects it from oxidation and metabolism, thereby maintaining its continuous biological activity. Its metabolic conversion product glutathione (GSH) in cells plays a great role in the protection of cells.

[0005] Studies have shown that reactive oxygen species (ROS) are important factors in cell aging and apoptosis, and can induce cell aging and apoptosis signal transduction. Seon-Heeoh et al. found that the glutathione content in cells with impaired oxidative function decreased sharply, causing the loss of intracellular redox function, and the cells began to age and undergo programmed cell death. If N-acetylcysteine ​​is added, it can remove reactive oxygen species in the cells to reduce damage, and metabolize to produce a large amount of glutathione, restore the redox function in the cells, and inhibit cell apoptosis. In addition, in the apoptosis experiment of endothelial cells, it was found that sodium arsenite (Ars) may induce the expression of cell adhesion molecules (ICAM-1) and cause cell apoptosis. After adding N-acetylcysteine ​​(NAC), the content of sodium arsenite (Ars) in the cells can be significantly reduced, the expression of cell adhesion molecules can be reduced, the intracellular oxidative environment can be changed, and the adhesion between cells can be reduced, thereby inhibiting cell apoptosis.

[0006] Quercetin, also known as quercetin and quercetin, is a natural flavonoid substance discovered by Hungarian physiologist Szent Gyorgyi in 1936. Quercetin has rich biological activities, the most notable of which is its strong antioxidant capacity: quercetin can effectively remove free radicals and protect cells from oxidative damage; quercetin can protect cell DNA from oxidative damage, thereby delaying cell aging; at the same time, quercetin has anti-inflammatory effects and can reduce inflammatory responses.

[0007] Oxygen concentration plays an important role in the proliferation, differentiation and self-renewal of mesenchymal stem cells. However, in vitro culture conditions, MSCs are usually exposed to normoxia (21% O 2 ), which is very different from the oxygen concentration in the body under natural physiological conditions. In fact, a large part of MSCs exist in the body under hypoxia (2%-8% O 2Even lower) environments, studies have shown that exosomes were isolated from MSCs cultured under hypoxic conditions. These exosomes grew in a culture medium similar to that of peripheral arterial disease (0% fetal bovine serum, 1% oxygen), and found that these exosomes contain many pro-angiogenic factors that may be beneficial to ischemic tissues. Other studies have found that after hypoxia induction, exosomes from bone marrow mesenchymal stem cells showed increased vascularization, reduced cardiomyocyte apoptosis rate, and increased recruitment of cardiac progenitor cells. Therefore, the biological function and activity of MSCs can be significantly enhanced under the premise of hypoxia induction, thereby improving the transplantation therapeutic effect of MSCs and their secreted exosomes in disease models. However, in current studies, the extraction of exosomes under hypoxic conditions usually takes the approach of directly reducing oxygen concentration, that is, directly reducing the oxygen partial pressure from 21% to below 5%. This will not only have an adverse effect on cell growth, such as: ① A sudden reduction in oxygen partial pressure will trigger an acute oxidative stress response in MSCs, resulting in an instantaneous increase in reactive oxygen species (ROS) levels; ② A sudden reduction in oxygen partial pressure may induce hypoxic apoptosis or necrosis of MSCs; ③ A rapid reduction in oxygen partial pressure may lead to changes in pH in the culture medium or accumulation of metabolites (such as lactic acid); thereby causing abnormal changes in the composition of miRNA, proteins, and lipids in exosomes, affecting their specific functions.

[0008] Although MSC-derived exosomes have many advantages such as promoting angiogenesis, cell proliferation, growth, and migration, the vast majority of MSC-derived exosomes cultured using conventional methods currently do not have a significant effect on cell anti-aging regulation. At the same time, some related studies have shown that N-acetylcysteine ​​can inhibit T cell aging and apoptosis in the process of regulating immune cells, but it has not been applied to the anti-aging effect of exosomes produced by MSC cells, nor has there been any research on the combination of N-acetylcysteine ​​and quercetin in cell anti-aging. Summary of the invention

[0009] In view of this, the present invention aims to propose a method for preparing mesenchymal stem cell exosomes and its application, in which N-acetylcysteine ​​and quercetin are added to the culture medium used to culture MSC cells, thereby stimulating MSC to produce exosomes with anti-aging and anti-apoptotic effects, and the exosomes produced by MSC are further stimulated to have anti-aging effects by gradually reducing the oxygen partial pressure culture method, and after the cells are induced and cultured with N-acetylcysteine ​​and quercetin, the adverse effects such as acute oxidative stress, decreased proliferation ability and unstable quality under hypoxia can be inhibited, and the activity of cells and exosome-related components is not damaged. This method shows good anti-aging and anti-apoptotic effects in vitro, and provides a new idea for the future treatment of aging diseases with exosomes.

[0010] To achieve the above object, the technical solution of the present invention is achieved as follows: A method for preparing mesenchymal stem cell exosomes, the method comprising the following steps: S1. Primary culture: Umbilical cord mesenchymal stem cells were cultured in serum-free complete medium to obtain P0 cells; S2. Domestication and cultivation: N-acetylcysteine ​​and quercetin were added to serum-free complete medium to prepare an acclimation medium, and the P0 cells were subcultured to the P5 cells using the acclimation medium; S3. Stepwise low oxygen pressure separation and extraction of mesenchymal stem cell exosomes: Continue to acclimate and culture the P5 mesenchymal stem cells until the cell confluence is >80%, then replace with serum-free basal medium and place the culture flask in a 5Vt% CO 2 and 15Vt% O 2 The cells were cultured in an incubator at 37°C for 24 hours, and then the O2 was reduced by 4-9Vt% every 24 hours. 2 The content was adjusted 2-3 times, and the supernatant was aspirated for gradient centrifugation to extract exosomes.

[0011] The addition of N-acetylcysteine ​​during the MSC culture process can cause changes in the MSC microenvironment, thereby increasing the expression of certain microRNA (miRNA) levels that regulate cell aging in MSC, such as miR-21, miR-146a, etc. These miRNAs can be taken up by the exosomes produced by the cells to produce exosome particles with anti-aging levels. In addition, the added quercetin can change the composition and function of miRNA in the secreted exosomes by regulating the gene expression of MSCs, so that the exosomes may carry more functional proteins or miRNAs related to autophagy (such as miR-100), promote the autophagy activity in the receptor cells, and enhance their ability to cope with aging damage.

[0012] In some embodiments, S1 specifically includes the following steps: 1) Use 0.9% saline containing 1% by volume penicillin and 1% by volume streptomycin as tissue cleaning solution to clean the surface of the placenta to remove surface congestion, cut the umbilical cord and remove the internal blood vessels; 2) Remove the Wharton's jelly from the umbilical cord, clean it with tissue cleaning solution, and cut it into 2-3 mm pieces 3 Transfer the small pieces into T75 culture flasks, 1 mL per flask; 3) Slowly add 10 mL of serum-free complete medium (serum-free basal medium containing 2% human platelet lysate by volume) to the culture bottle, gently shake the culture bottle to spread the umbilical cord tissue pieces in the culture bottle, and incubate at 37°C and 5% CO 2The cells were cultured statically in an incubator, and the culture medium was replenished every 2-3 days (3-4 mL of serum-free complete medium was added each time). After replenishing the culture medium 2-3 times in total, the tissue blocks were removed and the serum-free complete medium was fully replaced to continue culturing until the cells grew to a confluency of 70%. The obtained cells were then digested; 4) Aspirate the culture medium in the culture flask, rinse the culture flask with 20 mL of PBS to remove the residual culture medium, add 3 mL of recombinant trypsin solution for digestion, tap the culture flask to make the cells fall off to the bottom of the culture flask, add 10 mL of MSC serum-free complete culture medium to neutralize trypsin to terminate the digestion process, blow off the cells with a pipette, collect the cell suspension in a centrifuge tube, centrifuge, discard the supernatant, and obtain the cell pellet, which is recorded as the P0 generation cells.

[0013] In some embodiments, the concentration of N-acetylcysteine ​​is 3-7 mmol / L, and the concentration of quercetin is 1-3 μmol / L.

[0014] Preferably, the concentration of N-acetylcysteine ​​is 5 mmol / L, and the concentration of quercetin is 1 μmol / L.

[0015] In some embodiments, in step S3, after the last oxygen content adjustment, the oxygen content in the environment is controlled at 1-5Vt%.

[0016] In some embodiments, in step S3, the adjustment is performed twice. 2 Content: After the first 24 hours of cultivation, adjust the oxygen content to 7-8 Vt% and continue to cultivate for 24 hours; after 24 hours, adjust the oxygen content to 2-3 Vt% and continue to cultivate for 24 hours; the total cultivation time is 72 hours.

[0017] In some embodiments, the MSC cell seeding density in each subculture in step S2 is 1×10 4 Pieces / cm 2 , at 37°C, 5 Vt % CO 2 Culture in an incubator and replace the culture medium every 48 hours.

[0018] In some embodiments, the MSC cell seeding density in step S3 is 1×10 4 Pieces / cm 2 .

[0019] The present invention also provides an application of mesenchymal stem cell exosomes prepared by the above-mentioned preparation method in cell anti-aging.

[0020] In some embodiments, the amount of exosomes added is 2.5×10 8 Particles / mL-2.5×10 9 Particles / mL.

[0021] Compared with the prior art, the method for preparing mesenchymal stem cell exosomes of the present invention has the following advantages: The preparation method of mesenchymal stem cell exosomes of the present invention adds N-acetylcysteine ​​and quercetin to the culture medium used for culturing MSC cells, thereby stimulating MSC to produce exosomes with anti-aging and anti-apoptotic effects, and stimulating MSC to produce exosomes with further anti-aging effects by gradually reducing the oxygen partial pressure culture method, and after the cells are induced and cultured by N-acetylcysteine ​​and quercetin, they can inhibit the adverse effects such as acute oxidative stress, decreased proliferation ability and unstable quality under hypoxia and do not damage the activity of cells and exosome-related components. This method shows good anti-aging and anti-apoptotic effects in vitro, and provides a new idea for the future treatment of aging diseases with exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 This is an electron micrograph of exosomes; Figure 2 is the exosome particle size diagram; Figure 3 Flow cytometry was used to detect the surface membrane proteins CD81 and CD9 of exosomes. (a) is the surface membrane protein CD81, and (b) is the surface membrane protein CD9. Figure 4 Western Blot was used to detect intracellular proteins in exosomes; Figure 5 To establish the IMR-90 cell aging model using D-galactose, (a) is 0 mg / mL galactose, (b) is 20 mg / mL galactose, (c) is 40 mg / mL galactose, and (d) is 80 mg / mL galactose; Figure 6 The ROS staining results of the oxidative stress experiment, (a) is the negative group, (b) is the positive group, (c) is the normal group, (d) is the Min group, (e) is the Max group, (f) is the normoxic group, (g) is the single quercetin group, (h) is the single acetyl group, (i) is the factor group, (j) is the one-step hypoxia group, and (k) is this patent; Figure 7 This is the expression result of p16 cell cycle inhibitor protein; Figure 8 The results are for the expression of p53 cell cycle inhibitory protein. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 Preparation of exosomes The preparation method comprises the following steps: S1. Primary culture of umbilical cord mesenchymal stem cells S11. Place stainless steel trays, filter screens, stainless steel cups, lunch boxes, tissue cleaning solution (0.9% saline containing 1% penicillin and 1% streptomycin), pipettes and other items in appropriate locations in the biosafety cabinet. Use surgical tweezers to remove the placental tissue and place it in a stainless steel tray. Use tissue cleaning solution to clean the surface of the placenta and remove the blood on the surface of the placenta. Use surgical scissors and tweezers to cut the umbilical cord connected to the placenta, and cut the other end from behind the sterile clamp of the umbilical cord. Take out the umbilical cord and wash it 3 times with tissue cleaning solution to remove the blood and blood clots on the surface. Clamp one end of the umbilical cord with tweezers, clamp the umbilical cord with another pair of tweezers and slide along the umbilical cord tissue to remove the residual umbilical cord blood in the blood vessels. First cut the umbilical cord into several sections of 2-3 cm in length, then cut it open along the longitudinal section of the umbilical cord with scissors, flatten it, remove the 3 blood vessels inside (2 umbilical arteries and 1 umbilical vein), and transfer it to a new dish.

[0026] S12. Use tweezers to peel off the Wharton's jelly, put it into a culture dish filled with tissue cleaning solution, wash it with tissue cleaning solution and tear the Wharton's jelly into long strips. After processing, use tweezers to transfer the Wharton's jelly to a 50mL centrifuge tube and cut it into 2-3mm with scissors. 3 Use a 3 mL sterile dropper to transfer small pieces into T75 culture bottles, 1 mL per bottle. The tip of the dropper needs to be cut off with sterile scissors before use.

[0027] S13. Slowly add 10 mL of serum-free complete medium (serum-free basal medium containing 2% human platelet lysate, the serum-free basal medium is MSC NutriStem® XFBasal Medium from Biological Industries, catalog number 05-200-1A) to the culture bottle, and gently shake the culture bottle to spread the umbilical cord tissue pieces in the culture bottle. Place the T75 culture bottle at 37°C and 5% CO 2Culture in an incubator. On the third day of culture, remove the culture flask from the incubator and add 3.5 mL of serum-free complete medium. Replenish the medium every 3 days. After replenishing the medium 3 times, cells will crawl out from the bottom of the culture flask. Remove the tissue blocks and replace the serum-free complete medium to continue culturing. When the cells grow to a confluency of 70%, the obtained cells are then digested.

[0028] S14, before cell subculture, recombinant trypsin, serum-free complete medium, and PBS were placed at room temperature for equilibrium temperature. The culture flask was taken out of the incubator, the medium in the culture flask was discarded with a pipette, and 20 mL of PBS was drawn to rinse the culture flask to remove the residual medium. 3 mL of recombinant trypsin solution was added to the culture flask for digestion, the culture flask was gently shaken so that trypsin fully covered the cell surface, and digested for about 1 min. The cells were observed to shrink into a circle under a microscope, and the culture flask was gently tapped so that the cells fell off to the bottom of the culture flask in a quicksand-like manner. 10 mL of serum-free complete medium was added to neutralize trypsin to terminate the digestion process, and the cells were blown away with a pipette, and the cell suspension was collected in a 15 mL centrifuge tube. The centrifuge tube was placed in a centrifuge, centrifuged at 1400 rpm for 5 min, and the supernatant was removed. The precipitated cells were recorded as P0 generation cells.

[0029] S2. Acclimation of umbilical cord mesenchymal stem cells S21, adding 5 mmol / L N-acetylcysteine ​​and 1 μmol / L quercetin to serum-free complete medium to prepare an acclimation medium; S22, resuspend the P0 cells in 10 mL of acclimation medium, count the cells, and then inoculate at a density of 1×10 4 MSC cells / cm 2 Inoculate into a T75 culture flask and add the above acclimation medium to 30 mL. Incubate at 37°C and 5% CO 2 The cells were cultured statically in an incubator, and fresh acclimation medium was replaced every 48 hours until the cell confluence reached 80%. The medium in the culture flask was discarded, 20 mL of PBS was aspirated to rinse the culture flask to remove the residual medium, 3 mL of recombinant trypsin solution was added for digestion, the culture flask was tapped to make the cells fall off to the bottom of the culture flask, 10 mL of acclimation medium was added to neutralize the trypsin to terminate the digestion process, the cells were blown off with a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, the supernatant was discarded, and the cell pellet was recorded as P1 cells.

[0030] S23. In order to ensure that the mesenchymal stem cells can be fully and continuously nourished by the acclimated culture medium, the P1 generation cells are expanded, digested, and subcultured according to the above-mentioned method until the cell generation reaches P5 generation.

[0031] S3. Stepwise low oxygen pressure separation and extraction of mesenchymal stem cell exosomes S31. Resuspend the P5 cells in 10 mL of acclimation medium, count the cells, and then inoculate at a density of 1×10 4 MSC cells / cm 2 Inoculate into a T75 culture flask and add the above acclimation medium to 30 mL. Incubate at 37°C and 5% CO 2 The cells were cultured statically in an incubator and fresh acclimation medium was replaced every 48 hours until the cell confluence was >80%.

[0032] S32. Take out the culture flask, discard the culture medium, wash the culture flask three times with PBS, and then add 30 mL of serum-free basal medium (MSC NutriStem® XF Basal Medium from Biological Industries, Catalog No. 05-200-1A, without 2% human platelet lysate) to the flask. Then place the culture flask at 37°C, 15 Vt % O 2 、5 Vt % CO 2 After culturing in the incubator for 24 hours, the oxygen concentration was adjusted to 7.5 Vt % and culture was continued for 24 hours. After 24 hours, the oxygen concentration was adjusted to 2 Vt % and culture was continued for 24 hours. After a total of 72 hours of culture, the cell supernatant was collected into a 50 mL centrifuge tube.

[0033] S33. Centrifuge the centrifuge tube at 300g, 4°C, for 10 minutes to remove dead cells and larger cell fragments; then aspirate the supernatant into a new 50ml centrifuge tube.

[0034] S34. Centrifuge the centrifuge tube at 2000g, 4°C, for 20 minutes to further remove impurities such as cell debris; then aspirate the supernatant into a high-speed centrifuge tube (Beckman Coulter).

[0035] S35. Place the centrifuge tube in a high-speed centrifuge and centrifuge at 10,000 g, 4° C., for 30 minutes to further remove smaller cell fragments and impurities; then pipette the supernatant into a 50 ml centrifuge tube.

[0036] S36. Filter the supernatant with a 0.22 μm PES filter membrane (Millipore). Place the filtered supernatant in an ultracentrifuge tube (Beckman Coulter), balance it, and place it in an ultracentrifuge.

[0037] S37. Centrifuge the filtered supernatant at 100,000 g, 4°C, and 90 min. After centrifugation, discard the supernatant, resuspend with 20 ml of sterile PBS, and repeatedly blow the bottom of the ultracentrifuge tube to resuspend the exosome precipitate at the bottom; after balancing, place it in an ultracentrifuge and centrifuge it again at 100,000 g, 4°C, and 90 min. After centrifugation, discard the supernatant, resuspend with 1 mL of sterile PBS, repeatedly blow the bottom of the centrifuge tube, and then store the extracted exosome suspension at -80°C.

[0038] Experiment 1 Transmission electron microscopy observation and particle size detection of exosome particles The exosomes obtained in Example 1 were observed in the following steps: 1. Take 50 μl of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and place it in a 4°C refrigerator for fixation for 1 h; 2. Add 20 μl of the fixed exosome suspension onto the front of the copper mesh and let it stand for 20 minutes; 3. Carefully absorb the excess solution with absorbent filter paper; then wash the copper mesh with ultrapure water 5 times, 30 seconds each time, and dry it with filter paper; 4. Add 1 drop of 2% uranyl acetate dye solution to the front of the copper mesh, dye for 1 minute, and then use filter paper to absorb the excess dye along the edge of the copper mesh; 5. Place the copper mesh in air at room temperature to dry naturally, and then observe it on the machine after it is dry.

[0039] 6. Dilute 50 μl of the separated and purified exosome precipitate with PBS 100-500 times, and take 50 μl of the sample for nanoparticle size analyzer to detect the particle size and peak value of the exosomes.

[0040] like Figure 1 The morphology of exosomes can be observed. Under the electron microscope, the exosomes have a complete envelope and a double-layer lipid membrane structure. The exosome morphology presents a typical "tray-like" structure. At the same time, the nanoparticle size analysis of the exosomes was performed, and the results are as follows Figure 2 As shown in the figure, it can be seen that the exosome particle size peaks at 81nm and 110nm, and the number of exosome particles with a size less than 200nm accounts for about 99.9% of the total number of particles.

[0041] Experiment 2 Detection of exosome surface protein expression by flow cytometry The surface protein expression of the exosomes obtained in Example 1 was detected in the following steps: 1. Mix the components of Thermofisher CD63 magnetic beads (Cat. No. 10606D) by inversion for 10 minutes; pipette 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube; 2. Add 200 μl of magnetic bead cleaning solution to the EP tube and mix thoroughly with a pipette tip; 3. Place the EP tube on the magnetic rack for 1 minute, then discard the supernatant; 4. Take 50 μl of the extracted exosome suspension, add 50 μl of washing solution to a final volume of 100 μl, and mix thoroughly; 5. Place the exosome-washing solution mixture on a rotary mixer, set the speed to 10 rpm, and incubate overnight at 2-8°C; 6. The next day, centrifuge the sample for 3 to 5 seconds to collect the precipitate; 7. Add 300 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds; 8. Place the sample on the magnetic rack for about 1 minute and discard the supernatant; 9. Add 400 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds; 10. Place the sample on the magnetic rack for about 1 minute, discard the supernatant, and resuspend with 300 μl of washing solution; 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow cytometry antibodies to the samples, and incubate at 4°C in the dark for 30 minutes. 12. Place the incubated sample on the magnetic rack for about 1 minute, discard the supernatant, and add 300 μl of cleaning solution for washing; 13. Repeat step 12 once, resuspend the sample with 300 μl PB and then test on the instrument.

[0042] Test results such as Figure 3 As shown in the figure, it can be seen that when the exosome particles are labeled with CD81-PE antibody, the number of exosome particles with positive CD81 expression is detected to account for 97.53%; when the exosome particles are labeled with CD9-PE antibody, the number of exosome particles with positive CD9 expression is detected to account for 95.51%.

[0043] Experiment 3: Detection of intracellular proteins in exosomes The exosomes obtained in Example 1 were subjected to exosome intracellular protein detection, and the steps were as follows: 1. Protein quantification: Quantitative detection of exosome protein was performed according to the instructions of QubitTM Protein Assay Kit.

[0044] 2. Sample preparation: Mix the exosome sample with the loading buffer and place in a 100℃ water bath for 10 minutes to denature the protein. After cooling, centrifuge.

[0045] 3. Prepare the precast gel: Place the ExoressPAGE Gels precast gel in the electrophoresis tank, add pre-cooled Running Buffer to the electrophoresis tank, and gently pull out the comb with both hands.

[0046] 4. Sample loading: Load 20-70 μL into each well and record the loading order.

[0047] 5. Electrophoresis: upper gel at 80 V for 30 min; lower gel at 110 V for 120 min. After electrophoresis, rinse the gel in 1X PBS for 2 min, then soak in transfer buffer for equilibrium.

[0048] 6. Soak the PVDF membrane in 100% ethanol for 10 min, then in ddH 2 O for 5 min, and finally equilibrated in Transfer buffer for 20 min.

[0049] 7. Transfer: Perform semi-dry transfer in Trans-Blot® Turbo™ Transfer System for 30 min at 25 V / 1.3 mA / cm 2 After transfer, soak the membrane in PBS for 3-5 min.

[0050] 8. Blocking: Place the transferred membrane in 20 ml of blocking solution and place on a shaker at room temperature for 0.5-1 h.

[0051] 9. Wash membrane: 1X PBST (1000ml 1×PBS+1ml Tween-20) for 5-10 min, wash four times.

[0052] 10. Incubate with primary antibody: dilute the primary antibody (1:1000) according to the antibody manual, incubate overnight at 4°C on a shaker. Recover the primary antibody solution and rinse the membrane 5 times with 1X PBST, 5-10 min each time. The primary antibody used is rabbit anti-human antibody, and the antibodies are: CD63, CD81, CD9, Alix, Calnexin, Flot1.

[0053] 11. Incubate with secondary antibody: Add secondary antibody (1:3000), incubate at 4°C, shaker, incubate for 0.5-1h. Recover the secondary antibody solution and use 1×PBST. The secondary antibody is mouse anti-rabbit antibody.

[0054] 12. Rinse the membrane 5 times, 5-10 min each time.

[0055] 13. Chemiluminescent gel imaging: Add ECL luminescent solution and allow to react for 2 minutes, then place in a chemiluminescent imaging system for imaging.

[0056] Figure 4Western Blot test results showed that the intracellular proteins Alix and Flot1 in the extracted exosome particles had bands, and the protein size was in line with the expected level. Exosomes express CD63, CD9, and CD81 transmembrane proteins, so these proteins all have bands, and the size is in line with the expected expression. Exosomes do not express Calnexin protein, so there is no band for this protein.

[0057] Experiment 4 Comparison of different preparation methods In order to verify the effect of culture method on the anti-aging function of exosomes, the following experiment was designed: 1. Exosomes in the control group (hereinafter referred to as the normal group): On the basis of Example 1, the acclimation medium used in step S2 was completely changed to a serum-free complete medium, and the rest was the same as in Example 1, and finally exosomes were obtained.

[0058] 2. Exosomes in the low-dose group (hereinafter referred to as the Min group): On the basis of Example 1, the N-acetylcysteine ​​added to the acclimation medium used in step S2 was 1 mmol / L, the quercetin added was 0.5 μmol / L, and the rest was the same as Example 1, and finally exosomes were obtained.

[0059] 3. Exosomes in the high-dose group (hereinafter referred to as the Max group): On the basis of Example 1, the N-acetylcysteine ​​added to the acclimation medium used in step S2 was 10 mmol / L, the quercetin added was 5 μmol / L, and the rest was the same as Example 1, and finally exosomes were obtained.

[0060] 4. N-acetylcysteine ​​exosomes (hereinafter referred to as monoacetyl group): On the basis of Example 1, quercetin was not added to the acclimation medium used in step S2, and the rest was the same as Example 1, and finally exosomes were obtained.

[0061] 5. Quercetin exosomes (hereinafter referred to as single quercetin group): On the basis of Example 1, N-acetylcysteine ​​was not added to the acclimation medium used in step S2, and the rest was the same as in Example 1, and finally exosomes were obtained.

[0062] 6. Normoxia group: Based on Example 1, in step S3, mesenchymal stem cell exosomes are separated and extracted using normoxic partial pressure: The P5 mesenchymal stem cells were cultured according to step S31 of Example 1 until the cell confluence was >80%, and the medium was replaced with serum-free basal medium. The culture flask was placed at 37°C and 5% CO 2 , 21%O 2The cells were cultured in an incubator for 72 hours, and exosomes were obtained after 72 hours.

[0063] 7. One-step hypoxia group: Based on Example 1, in step S3, mesenchymal stem cell exosomes are separated and extracted using a one-step low oxygen partial pressure separation method: The P5 mesenchymal stem cells were cultured according to step S31 of Example 1 until the cell confluence was >80%, and the medium was replaced with serum-free basal medium. The culture flask was placed at 37°C and 5% CO 2 , 2%O 2 The cells were cultured in an incubator for 72 hours, and exosomes were obtained after 72 hours.

[0064] Experiment 5: Study on the anti-aging effects of exosomes at the in vitro cellular level 1. Establishment of IMR-90 cell senescence model using D-galactose 1) Take 2.5g of D-galactose powder, dissolve it in 34.5mL of sterile PBS, and dispense it into 1mL EP tubes. The D-galactose concentration in each tube is 80mg / mL.

[0065] 2) Resuscitate IMR-90 cells in a 37°C water bath and inoculate at a density of 2×10 4 / cm 2 , inoculated into several wells of 6-well plates, added 2 mL of DMEM medium containing 10% FBS, and replaced with fresh medium every 2-3 days until the confluence of IMR-90 cells was ≥80%. 0 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL of D-galactose were added to the medium and cultured for 24 hours.

[0066] 3) SA-β-galactosidase labeling method was used to detect the phenotypic changes of cell aging under different concentrations of D-galactose to screen the appropriate D-galactose concentration.

[0067] SA-β-gal is a typical marker of senescent cells, and the state of cell senescence can be detected by changes in β-galactosidase activity.

[0068] Here are the steps: I. After discarding the culture medium supernatant in each well after modeling, wash the cells with PBS 2-3 times, and then fix the cells with 4% paraformaldehyde for 15-30 minutes.

[0069] II. Stain the cells with the staining solution in the SA-β-galactosidase kit (usually containing X-gal substrate). The staining process needs to be carried out at 37°C for 12 hours. During the staining process, β-galactosidase will convert X-gal into a blue product, and blue cells are senescent cells. After staining, wash away the unbound dye with PBS. Observe the cells under a microscope and record the number and morphology of blue-stained cells.

[0070] III. Calculate the number of senescent cells produced by IMR-90 cells induced by different concentrations of D-galactose. The well with the largest number of senescent cells is the optimal D-galactose induction concentration.

[0071] like Figure 5 As shown in the figure, the cell senescence at different concentrations, it can be seen from the figure that the cell senescence model obtained using 80 mg / mL galactose is the best.

[0072] 2. Oxidative stress assay to detect the inhibitory effect of exosomes on IMR-90 cell senescence Oxidative stress refers to the excessive production of reactive oxygen species (ROS) in cells, which exceeds the cells' antioxidant defense capacity, thereby triggering a series of cell damage and aging.

[0073] The aging model of cells was established using D-galactose, and the exosomes obtained by the patented method and the exosomes obtained from each group of Experiment 4 were added to the cells in the modeling. At the same time, a control group was set up to observe the antioxidant stress effect of each group of exosomes in the process of cell aging.

[0074] Here’s how: 1) 80 mg / mL D-galactose was selected to induce senescence of IMR-90 cells for 24 hours. The experimental group and the control group were designed as follows: The positive control group (hereinafter referred to as the positive group) consisted of IMR-90 cells induced by D-galactose for 24 hours; The negative control group (hereinafter referred to as negative group) consisted of IMR-90 cells that were not induced by senescence; The experimental group consisted of IMR-90 cells induced with D-galactose for 24 hours and supplemented with 1×10 9 particles / mL of exosomes in each group of Experiment 4 (including normal group, Min group, Max group, monoacetyl group, monoquercetin group, normoxic group, and one-step hypoxic group); The positive control group with only added factors (hereinafter referred to as factor group) was composed of IMR-90 cells induced by D-galactose for 24 hours, to which only N-acetylcysteine ​​and quercetin at the concentrations described in Example 1 were added, without the addition of exosomes.

[0075] Add 2 mL of DEME medium containing 10% FBS to each well. Place the plate in a 37°C, 5% CO 2 Incubate in an incubator for 24 hours.

[0076] 2) After 24 hours, the well plate was removed, the supernatant culture medium was discarded, and the cells of each group were stained using a ROS detection kit (Shanghai Zhuocai Biotechnology Co., Ltd., Cat. No.: ZC-A4108) to observe the changes in the reactive oxygen species in the cells. If the cells appear green under fluorescence, it means that the reactive oxygen species in the cells is high and the cells are in a state of aging.

[0077] The results are as follows Figure 6 As shown in the figure, it can be seen that in the ROS oxidative stress experiment, the IMR-90 cells in each group were observed under a fluorescence microscope, and the degree of cell aging was evaluated by comparing the fluorescence intensity. It can be seen that the exosomes prepared by the method of this patent group have the strongest antioxidant and anti-aging effects on cells, which is almost the same as the negative group, that is, no fluorescence is visible to the naked eye, and the other groups all have fluorescence of varying intensities.

[0078] 3. Cycle inhibitory protein assay to detect the inhibitory effect of exosomes on IMR-90 cell senescence Aging can cause an increase in cell cycle inhibitory proteins P16 and P21, leading to cell cycle arrest. The expression level of cell cycle inhibitory proteins was detected by Elisa method to illustrate the anti-aging effect of exosomes.

[0079] Here’s how to do it: 80 mg / mL D-galactose was selected to induce senescence of IMR-90 cells for 24 hours to create a model. The experimental group and the control group were designed as follows: The positive group was IMR-90 cells after 24 h of D-galactose induction; The negative group consisted of IMR-90 cells that were not induced by senescence; The experimental group consisted of IMR-90 cells induced with D-galactose for 24 hours and supplemented with 1×10 9 particles / mL of exosomes in each group of Experiment 4 (including normal group, Min group, Max group, monoacetyl group, monoquercetin group, normoxic group, and one-step hypoxic group); The factor group was that only N-acetylcysteine ​​and quercetin at the concentrations described in Example 1 were added to IMR-90 cells after D-galactose induction for 24 hours, and no exosomes were added.

[0080] Add 2 mL of DEME medium containing 10% FBS to each well, and set up 2 parallel wells for each group. Place the plate at 37°C and 5% CO 2The cells were cultured in an incubator for 24 hours. After 24 hours, the cell supernatant was collected and the expression levels of p21 and p16 proteins, inflammatory factors associated with aging, were detected using Elisa kits (Shanghai Zhuocai Biotechnology Co., Ltd., catalog numbers: ZC-32275, ZC-32019).

[0081] The results are as follows Figure 7-8 As shown, this patent group is Example 1. Compared with other methods, the method of the present invention (ie, Example 1) releases the least amount of cycle inhibitory proteins p16 and p21 from cells, indicating that the method of the present invention has the best anti-aging effect on cells.

[0082] At the same time, the anti-aging effect of adding only two factors without adding exosomes (factor group) and exosomes without adding factor domestication (ordinary group) on cells is inferior to that of the patent group, indicating that it is not just the two factors of N-acetylcysteine ​​and quercetin that play a role in the anti-aging level of cells, nor is it the exosomes of untamed MSCs, but the exosomes domesticated by the two factors. However, adding only one factor, that is, adding only N-acetylcysteine ​​(monoacetyl group) or adding only quercetin (monoquercetin group) is not better than the present invention in terms of the anti-aging level of cells, indicating that the two factors described in this patent must exist at the same time and the exosomes produced after domestication of MSCs can achieve the best cell anti-aging effect. After reducing the addition concentration of the two factors (such as the Min group), the anti-aging effect of exosomes on cells is inferior to the method described in this patent, and after increasing the addition concentration of the two factors (Max group), there is no significant improvement in the anti-aging effect of cells. Therefore, it is believed that the concentrations of the two factors used in the present invention are the optimal concentration range for MSC domestication.

[0083] In addition, it can be seen from the attached figure that when the two factors coexist and are used at the same concentration, the exosomes obtained by the stepwise hypoxia method are more advantageous than the exosomes obtained by the normoxic group and the one-step hypoxia group in terms of cellular anti-aging level, indicating that the exosomes obtained by the stepwise hypoxia method are also more conducive to the cellular anti-aging effect.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The method comprises the following steps: S1. Primary culture: Umbilical cord mesenchymal stem cells were cultured in serum-free complete medium to obtain P0 cells; S2. Domestication and cultivation: N-acetylcysteine ​​and quercetin were added to serum-free complete medium to prepare an acclimation medium, and the P0 cells were subcultured to the P5 cells using the acclimation medium; S3. Stepwise low oxygen partial pressure separation and extraction of mesenchymal stem cell exosomes: Continue to acclimate and culture the P5 generation mesenchymal stem cells until the cell confluence is >80%, replace with serum-free basal medium, place the culture flask in an incubator containing 5Vt% CO2 and 15Vt% O2 at 37°C for 24 hours, then reduce the O2 content by 4-9Vt% every 24 hours, adjust 2-3 times, and aspirate the supernatant for gradient centrifugation to extract exosomes.

2. The method for preparing mesenchymal stem cell exosomes according to claim 1, characterized in that: The concentration of N-acetylcysteine ​​was 3-7mmol / L, and the concentration of quercetin was 1-3μmol / L.

3. The method for preparing mesenchymal stem cell exosomes according to claim 1, characterized in that: In step S3, after the last oxygen content adjustment, the oxygen content in the environment is controlled at 1-5Vt%.

4. The method for preparing mesenchymal stem cell exosomes according to claim 3, characterized in that: In step S3, the O2 content is adjusted twice: after the first 24 hours of cultivation, the oxygen content is adjusted to 7-8 Vt%, and the cultivation is continued for 24 hours; after 24 hours, the oxygen content is adjusted to 2-3 Vt%, and the cultivation is continued for 24 hours; the total cultivation time is 72 hours.

5. The method for preparing mesenchymal stem cell exosomes according to claim 1, characterized in that: In step S2, the seeding density of MSC cells in each subculture was 1×10 4 Pieces / cm 2 , cultured in a 37°C, 5 Vt% CO2 incubator, and the culture medium was replaced every 48 hours.

6. The method for preparing mesenchymal stem cell exosomes according to claim 1, characterized in that: The MSC cell seeding density in step S3 was 1×10 4 Pieces / cm 2 .

7. Use of mesenchymal stem cell exosomes prepared by the preparation method according to any one of claims 1 to 6 in cell anti-aging.

8. The use of mesenchymal stem cell exosomes in anti-aging of cells according to claim 7, characterized in that: The amount of exosomes added was 2.5×10 8 Particles / mL -2.5×10 9 Particles / mL.

Images