Exosome containing mitochondrial autophagy starting factor as well as preparation method and application of exosome
By preparing exosomes rich in mitochondrial autophagy initiation factors under hypoxic conditions, the problem of combining exosome preparation with the treatment of intervertebral disc degeneration was solved, achieving precise targeted treatment and improved safety for intervertebral disc degeneration.
Patent Information
- Application Number
- CN202511973826.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-02-24
AI Technical Summary
Existing methods for preparing exosomes cannot simulate the physiological state of stem cells in vivo, resulting in significant differences in their active ingredients and functional efficacy compared to those in vivo. Furthermore, they lack sufficient targeting for intervertebral disc degeneration, making it difficult to effectively reverse pathological progression.
Human bone marrow mesenchymal stem cells were pretreated at an oxygen concentration of 1-3% to prepare exosomes rich in mitochondrial autophagy initiation factors. By optimizing culture conditions and purification processes, the activity and targeting of the exosomes were ensured.
Exosomes can precisely target and improve mitochondrial function in nucleus pulposus cells, reverse intervertebral disc degeneration, have high safety, wide applicability, and good batch stability, which is significantly better than that of exosomes cultured under normoxic conditions.
Smart Images
Figure CN121555418A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an exosome containing a mitochondrial autophagy initiator, its preparation method, and its application. Background Technology
[0002] Lower back pain is a prevalent health problem worldwide, causing severe physical discomfort, substantial medical costs, and varying degrees of loss of working capacity, significantly impacting individual quality of life and social productivity. From a pathophysiological perspective, intervertebral disc degeneration (IVDD) is one of the core pathological factors causing lower back pain.
[0003] Currently, treatment options for intervertebral disc degeneration still have significant limitations. Traditional conservative treatments (such as analgesics and physical therapy) can only relieve symptoms and cannot stop the pathological progression of degeneration; surgical treatments (such as discectomy and fusion) can solve the problem of mechanical compression, but they carry risks such as significant trauma, numerous postoperative complications, and accelerated degeneration of adjacent segments. Therefore, identifying key regulatory targets for intervertebral disc degeneration at the molecular level and developing targeted therapies that are both safe and effective has become a pressing technical challenge in this field.
[0004] Bone marrow mesenchymal stem cells (BMSCs) have shown great promise in the treatment of orthopedic diseases due to their multi-lineage differentiation potential, low immunogenicity, and excellent tissue repair regulation capabilities. Stem cells can participate in the repair of damaged tissues through mechanisms such as direct differentiation, cell-cell interactions, and paracrine effects. However, stem cell transplantation therapy faces many challenges: the harsh local microenvironment of the intervertebral disc (such as ischemia, inflammation, and oxidative stress) leads to limited stem cell homing ability, poor targeted migration, and extremely low post-transplant survival rates, severely restricting its therapeutic efficacy.
[0005] Exosomes, as the core carriers of paracrine function in stem cells, can carry characteristic molecules of parental cells and transmit signals to recipient cells through non-cell-to-cell contact, circumventing the aforementioned drawbacks of stem cell transplantation and becoming an ideal alternative tool for stem cell therapy. However, current technologies mostly use normoxic conditions (21% O2) to culture BMSCs for exosome preparation, while the oxygen concentration in the human physiological microenvironment is only 2-8%. Normooxic culture cannot simulate the physiological survival state of stem cells, which may lead to significant differences in the composition and functional efficacy of exosomes compared to those under physiological conditions in vivo, thus affecting their therapeutic effects. In addition, existing exosome therapies lack sufficient targeting of core pathological aspects such as nucleus pulposus cell senescence and mitochondrial quality control imbalance, making it difficult to reverse the process of intervertebral disc degeneration.
[0006] Based on this, optimizing stem cell culture conditions to prepare exosomes rich in specific functional factors that can precisely target and improve the mitochondrial function and aging state of nucleus pulposus cells has become a key breakthrough in solving the treatment problem of intervertebral disc degeneration. Summary of the Invention
[0007] The purpose of this invention is to provide an exosome containing a mitochondrial autophagy initiator, its preparation method, and its application, in order to solve the problems mentioned in the background art.
[0008] To achieve the above objectives, the present invention provides the following technical solution: an exosome containing mitochondrial autophagy initiation factor, which is obtained by secretion from human bone marrow mesenchymal stem cells pretreated for an appropriate duration under 1-3% oxygen concentration conditions.
[0009] A method for preparing exosomes, specifically including the following steps:
[0010] S1. Culture and passage of human bone marrow mesenchymal stem cells: Human bone marrow mesenchymal stem cells are cultured under normoxic conditions until the confluence reaches 80-90%, and then passaged.
[0011] S2. Hypoxia pretreatment: Passaged human bone marrow mesenchymal stem cells were seeded into culture dishes, replaced with exosome serum-free culture medium, and placed in a culture environment of 37℃, 5%CO2, and 2%O2 for an appropriate period of time for pretreatment.
[0012] S3. Extraction and purification of exosomes: Collect the cell culture supernatant from step S2, centrifuge at differential speed, collect the precipitate and resuspend it to obtain exosomes.
[0013] Preferably, in step S1, the human bone marrow mesenchymal stem cells are passaged from P3 to P5, with a passage ratio of 1:3.
[0014] Preferably, in step S1, the culture medium used for normoxic culture is DMEM / F12 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin; in step S2, the serum-free exosome culture medium is DMEM / F12 basal medium containing 10% serum-free exosomes.
[0015] Preferably, in step S2, the seeding density of human bone marrow mesenchymal stem cells is 1×10⁻⁶. 6 Cells per 10cm culture dish, with a cell confluence of 70-80% before pretreatment.
[0016] Preferably, in step S3, the specific parameters of the continuous centrifugation process are as follows: under 4°C conditions, the mixture is centrifuged sequentially at 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min. The supernatant is collected and then subjected to ultracentrifugation at 100000×g for 70 min. The supernatant is discarded, and the precipitate is resuspended in sterile PBS. The mixture is then subjected to ultracentrifugation at 4°C at 100000×g for 70 min again. The precipitate is collected and resuspended in 200-500 μL of sterile PBS.
[0017] Preferably, in step S1, the human bone marrow mesenchymal stem cells are cryopreserved and thawed cells. The thaw operation includes: rapidly thawing the cryopreserved human bone marrow mesenchymal stem cells in a 37°C water bath, transferring them to a centrifuge tube containing the DMEM / F12 culture medium, centrifuging at 1000×g for 5 min, discarding the supernatant, and resuspending them in fresh culture medium for inoculation.
[0018] Preferably, in step S2, before replacing the exosome serum-free culture medium, the cell surface is rinsed 2-3 times with sterile PBS to remove residual serum.
[0019] Preferably, in step S3, the obtained exosomes are aliquoted and stored in a -80°C freezer, and repeated freeze-thaw cycles are avoided.
[0020] The use of exosomes in the preparation of drugs for the treatment or prevention of intervertebral disc degeneration.
[0021] The technical effects and advantages of this invention are as follows:
[0022] (1) In view of the shortcomings of traditional conservative treatment, which can only relieve symptoms and cannot stop the degeneration, the exosomes of the present invention are secreted by human bone marrow mesenchymal stem cells pretreated with 2% oxygen concentration. They are naturally rich in mitochondrial autophagy initiation factors, which can precisely target senescent nucleus pulposus cells of human intervertebral discs, promote the recovery of mitochondrial autophagy flux, improve mitochondrial functional damage and oxidative stress, and promote the synthesis of extracellular matrix. From the molecular mechanism level, they reverse the aging process of nucleus pulposus cells, delay or even block intervertebral disc degeneration, which is more targeted and radical than traditional treatment methods.
[0023] (2) This invention addresses the problems of limited homing ability, poor target migration, and extremely low survival rate after transplantation caused by the poor local microenvironment of the intervertebral disc in stem cell transplantation. This invention uses exosomes as the core carrier of the paracrine function of stem cells, which does not require cell transplantation and transmits biological signals through non-cell contact, thus avoiding the above-mentioned technical bottlenecks of stem cell transplantation. Moreover, exosomes have low immunogenicity and will not cause immune rejection. Compared with surgical treatment and stem cell transplantation, it has higher safety and wider applicability in clinical application.
[0024] (3) The present invention uses 2% hypoxia to pretreat human bone marrow mesenchymal stem cells to simulate the physiological survival microenvironment of stem cells in the body, so that the secreted exosomes are closer to the physiological state in the body in terms of active ingredients and functional characteristics. Its functional efficacy in promoting the repair of nucleus pulposus cells and improving intervertebral disc degeneration is better than that of exosomes cultured in normoxic conditions.
[0025] (4) In view of the shortcomings of existing exosome preparation processes, such as the lack of unified standards and large quality differences between different batches of products, this invention clearly defines the passage number (P3-P5 generation), hypoxia pretreatment conditions (1-3% O2, appropriate duration), and seeding density (1×10⁻⁶) of human bone marrow mesenchymal stem cells. 6 Key process parameters such as cell / 10cm culture dish, centrifugation purification parameters (multi-step gradient centrifugation and ultracentrifugation parameters at 4℃) were standardized. At the same time, the operation procedures for cell resuscitation, culture medium selection, residual serum removal, and exosome preservation were standardized, realizing the standardization and normalization of exosome preparation, effectively ensuring the batch stability of the product and the reproducibility of clinical applications, and solving the problem of fluctuation in therapeutic effect caused by process differences in the existing technology.
[0026] (5) The exosomes of the present invention can reduce the Pfirrmann grade of the intervertebral disc by 1-2 grades in rats with intervertebral disc degeneration (IVDD), increase the number of nucleus pulposus cells by more than 50%, and increase the proteoglycan staining intensity by 40%±5.3% (P<0.01). It can effectively improve the integrity of the intervertebral disc in aging rats and promote the synthesis of extracellular matrix. It is significantly superior to existing exosome therapy and traditional treatment methods, providing a safe, effective and targeted new treatment plan for intervertebral disc degeneration, reducing the medical costs associated with low back pain, and is of great significance for improving the quality of life of patients and social productivity. Attached Figure Description
[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings:
[0028] Figure 1 Characterization and identification of exosomes prepared from hypoxic and normoxic mesenchymal stem cells. A. Western blot image of hypoxia-inducible factor HIF-1 protein. B. Relative protein expression level. C. Content of exosome surface marker proteins. D. NTA particle size analysis. E. TEM microscopy results. F. Immunofluorescence detection of PKH26 endocytosis efficiency. G. Schematic diagram of PKH26 endocytosis;
[0029] Figure 2The images show the in vivo imaging recovery results of mesenchymal stem cell exosomes in rats under hypoxic and normoxic conditions. A. X-ray image of the rat caudal vertebrae. B. MRI image of the rat caudal vertebrae. C. DHI score of caudal intervertebral height and Pfirrmann classification of T2-weighted MRI images;
[0030] Figure 3 The histological recovery results of exosomes from hypoxic and normoxic mesenchymal stem cells in rats. A. Histological HE staining images of sections from rats at 4 and 8 weeks of treatment. B. Histological SO staining images of sections from rats at 4 and 8 weeks of treatment;
[0031] Figure 4 The results show the enrichment of BNIP3, a mitophagy factor, in exosomes of hypoxic and normoxic mesenchymal stem cells and the activation of mitophagy. A. Colocalization map of exosomes and mitochondria in nucleus pulposus cells. B. Quantitative colocalization map. C. Results of exosome-rescued mitochondrial morphology and function. D. Quantitative results of mitochondrial morphology and function. Detailed Implementation
[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0033] This invention provides, for example Figures 1-4 The image shows an exosome derived from hypoxic mesenchymal stem cells, which is secreted by human bone marrow mesenchymal stem cells pretreated with 2% oxygen for 24 hours.
[0034] The exosomes were secreted by human bone marrow mesenchymal stem cells (hBMSCs) pretreated with 2% oxygen for 24 h. Their core characteristics are as follows: rich in the mitochondrial autophagy initiator BNIP3, significantly higher than that of exosomes secreted by hBMSCs cultured in normoxic (21% O2); molecular markers: surface expression of exosome characteristic markers TSG101, HSP70, and CD63, but no expression of the endoplasmic reticulum marker Calnexin; morphology and particle size: under transmission electron microscopy (TEM), they exhibit a typical cup-shaped or saucer-shaped structure, and nanoparticle tracking analysis (NTA) shows that the particle size distribution is in the range of 50-120 nm, with an average particle size of 100 nm; targeting and endocytosis efficiency: after being fluorescently labeled with PKH26 / PKH67, after co-incubation with TBHP-induced senescent NPCs for 24 h, the endocytosis efficiency was significantly higher than that of exosomes cultured in normoxic conditions, and they could co-localize with the mitochondria of NPCs.
[0035] This invention also provides the application of the above-mentioned hypoxic bone marrow mesenchymal stem cell exosomes in the preparation of treatments for intervertebral disc degeneration.
[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be interpreted as...
[0037] This is to limit the scope of protection of the present invention.
[0038] Example 1: The method for preparing exosomes includes three core steps: hBMSCs culture and passage, hypoxia pretreatment, and exosome extraction and purification. The specific operations are as follows:
[0039] Culture and passage of hBMSCs
[0040] Frozen hBMSCs were rapidly thawed in a 37°C water bath. The cell suspension was transferred to centrifuge tubes containing 5 mL of DMEM / F12 medium (supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). The cells were centrifuged at 1000 × g for 5 min, and the supernatant was discarded. Subsequently, the cell pellet was resuspended in 2 mL of fresh DMEM / F12 medium and seeded into 6 cm cell culture dishes. The cells were then incubated in a normoxic incubator at 37°C, 5% CO2, and 21% O2. Change the culture medium every 2-3 days. When the cell confluence reaches 80-90%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin-EDTA digestion solution, incubate at 37°C for 2 min, and add 2 mL of culture medium to stop digestion after the cells become rounded and detached from the cell wall. Centrifuge at 1000×g for 5 min and discard the supernatant. Resuspend the cells in fresh culture medium and passage them to new culture dishes at a ratio of 1:3. Use hBMSCs in the logarithmic growth phase from passages 3-5 for subsequent hypoxia pretreatment.
[0041] Hypoxia pretreatment of hBMSCs
[0042] The 3rd to 5th generation hBMSCs were used at a rate of 1×10 6 Seed cells at a density of 10 cm cells / dish in 10 cm culture dishes and cultured in a normal oxygen incubator until the cell confluence reaches 70-80%. Discard the original culture medium and gently rinse the cell surface three times with sterile PBS to remove residual serum. Replace with DMEM / F12 basal medium containing 10% exosomes and serum-free. Transfer the culture dishes to a tri-gas incubator and set the culture conditions to 37°C, 5% CO2, 2% O2, and perform hypoxia pretreatment for 24 h.
[0043] Extraction and purification of exosomes
[0044] Collect the supernatant of hBMSCs culture after 24 h of hypoxia pretreatment and transfer it to a 50 mL sterile centrifuge tube. Centrifuge at 300 × g for 10 min at 4 °C to remove intact cells, centrifuge at 2000 × g for 10 min to remove dead cells and cell debris, and centrifuge at 10000 × g for 30 min to remove larger extracellular vesicles and residual debris. After each centrifugation, carefully aspirate the supernatant into a new centrifuge tube. Transfer the supernatant to an ultracentrifuge tube and ultracentrifuge at 100000 × g for 70 min at 4 °C. Discard the supernatant; the milky white precipitate at the bottom is the crude exosome extract. Resuspend the crude exosome extract in 1 mL of sterile PBS and ultracentrifuge again at 100000 × g for 70 min at 4 °C. Discard the supernatant. Resuspend the final precipitate in 200-500 μL of sterile PBS; this is the exosome containing BNIP3. Aliquot and store at -80 °C to avoid repeated freeze-thaw cycles.
[0045] Characterization of exosomes
[0046] To ensure that exosomes meet the requirements of this invention, the following characteristic verifications are required. The verification methods and judgment criteria are as follows:
[0047] Protein marker detection (Western Blot): Take 50 μL of exosome suspension, add 12.5 μL of 5×SDS loading buffer, and boil in water for 10 min to denature the protein; perform 12% SDS-PAGE gel electrophoresis (stacking gel 80V, separating gel 120V), and transfer the protein to a PVDF membrane using wet transfer method (300mA, 90 min); block with 5% skim milk for 2 h, add primary antibody (rabbit anti-human TSG101, HSP70, CD63, Calnexin antibody, 1:1000 dilution) and incubate overnight at 4℃, wash 3 times with TBST, add HRP-labeled secondary antibody (1:5000 dilution) and incubate at room temperature for 1 h, and develop with ECL chemiluminescence; TSG101, HSP70, and CD63 expression were positive, and Calnexin expression was negative.
[0048] Morphological observation (TEM): 10 μL of exosome suspension was dropped onto a copper grid, allowed to stand at room temperature for 5 min, excess liquid was absorbed with filter paper, 2% phosphotungstic acid solution (pH 7.0) was added for negative staining for 5 min, and the mixture was allowed to air dry. Observe under a Hitachi H-7650 transmission electron microscope (100,000× magnification); qualified exosomes have typical cup-shaped or saucer-shaped structures and regular morphology.
[0049] Particle size distribution assay (NTA): The exosome suspension was diluted to 1×10⁸ particles / mL with sterile PBS; assay: Malvern NanoSight NS300 nanoparticle tracking analyzer, assayed at 25°C for 60 s; judgment criteria: particle size distribution in the range of 30-150 nm.
[0050] Verification of endocytosis efficiency (fluorescence microscopy)
[0051] Exosomes were labeled with PKH26 dye, incubated at 37°C for 30 min, and centrifuged at 100,000 × g for 70 min to remove free dye. The labeled exosomes were co-incubated with TBHP-induced senescent NPCs for 24 h, and the nuclei were stained with DAPI. The cells were observed under a laser confocal microscope. Red fluorescence was visible in the NPCs cells, and the fluorescence intensity was significantly higher than that of the normoxic exosome group.
[0052] The results showed that hypoxic exosomes did not differ significantly from normoxic exosomes in physical properties, but had significant advantages in endocytosis efficiency and secretion quantity.
[0053] Example 2: Construction of an IVDD rat model of intervertebral disc degeneration, evaluation of intervertebral disc imaging, and evaluation of intervertebral disc tissue recovery.
[0054] IVDD rat intervertebral disc degeneration model construction
[0055] SPF-grade Sprague-Dawley (SD) rats were selected as experimental animals, with a weight of 250-300g.
[0056] SD rats were subjected to general anesthesia (e.g., intraperitoneal injection of 10% chloral hydrate at a dose of 3.5 ml / kg). After the rats lost consciousness and pain reflexes, they were fixed prone on the X-ray imaging equipment operating table, exposing their tails.
[0057] Radiographic guidance: The intervertebral disc of the 7th / 8th caudal vertebra (Co7 / 8) of the rat is located using X-ray imaging equipment to identify the puncture target and ensure puncture accuracy.
[0058] Using a 22G puncture needle, under radiographic guidance, the needle was vertically inserted into the Co7 / 8 intervertebral disc space to a fixed depth of 3mm. After insertion, the needle was rotated 360° clockwise and held for 30 seconds to disrupt the annulus fibrosus and nucleus pulposus of the intervertebral disc and induce degeneration.
[0059] After the puncture, the rats were placed in a warm environment to awaken, and then fed and watered freely. The temperature of the rearing environment was controlled at 22-25℃, the humidity at 50-60%, and the light cycle was 12h light / 12h dark.
[0060] The results confirmed that the exosomes of the present invention can effectively delay the progression of IVDD and promote intervertebral disc repair in vivo.
[0061] Intervertebral disc imaging evaluation
[0062] Radiographic imaging (X-ray):
[0063] Lateral radiographs of the Co7 / 8 intervertebral discs in rats were taken to observe changes in disc height.
[0064] The disc height loss rate was calculated using the Disc Height Index (DHI) formula: DHI = (anterior disc height + posterior disc height) / 2 / average height of adjacent vertebral bodies × 100%.
[0065] Magnetic resonance imaging (MRI):
[0066] The tail of rats was scanned using a 3.0T MRI device to obtain T2-weighted images. The degree of intervertebral disc degeneration was graded according to the Pfirrmann grading system (Pfirrmann grade I is normal, and grade V is severe degeneration).
[0067] 3. Intervertebral disc sampling and evaluation of intervertebral disc tissue recovery
[0068] Sacrifice of rats: In accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare", rats were euthanized by professionals using an overdose of isoflurane combined with cervical dislocation. Anesthesia was induced for 5-8 minutes with 5% isoflurane + oxygen (1.5L / min). After slow and irregular breathing, loss of corneal reflex, and no limb response, the head was stabilized with the left hand, and the torso was dislocated by lifting with the right hand. The procedure was completed once breathing and heartbeat had stopped.
[0069] Caudal vertebrae sample collection: Rats were placed supine on a sterile operating table. The skin of the caudal vertebrae was cut and the bones were exposed. The Co7 / 8 intervertebral disc (Co7 is slightly larger than Co8, and the intervertebral disc is an indentation between the vertebral bodies) was located using a 10× dissecting microscope and preoperative X-ray. A 4-5 mm tissue block containing "lower Co7 + Co7 / 8 intervertebral disc + upper Co8" was cut with microsurgical scissors. After washing away blood contamination with PBS at 4℃, the tissue was fixed with 10 times the volume of 4% paraformaldehyde (pH 7.4) at 4℃ for 24 h (the centrifuge tube was inverted once every 6 h).
[0070] Tissue dehydration and paraffin embedding: After fixation, wash three times with PBS (5 min each time), dehydrate at room temperature gradient (70% - anhydrous ethanol), clear twice with xylene (15 min each time), and immerse three times in molten paraffin (melting point 56-58℃) at 60℃ (1 h each time). In the embedding cassette, adjust the tissue so that the intervertebral disc is perpendicular to the bottom surface. After paraffin injection and cooling, label the group, number, and date.
[0071] HE staining: ① 5μm paraffin sections were spread in 40℃ warm water, and poly-L-lysine slides were taken out and baked at 60℃ for 2 hours; ② Xylene-gradient ethanol dewaxing to water; ③ Harris hematoxylin staining for 5 min, washing with tap water for 10 min, differentiation with 1% hydrochloric acid ethanol for 30 s, soaking in 0.5% ammonia water for 30 s, and rinsing with distilled water; ④ 0.5% eosin staining for 2 min, rinsing with distilled water; ⑤ Gradient ethanol-xylene dehydration and clearing, and mounting with neutral resin; ⑥ Microscopic observation (100×, 200×, 400×) showed that the model group had broken annulus fibrosus and fewer nucleus pulposus cells, while the treatment group showed significant improvement.
[0072] SO-FG staining: ① Same section preparation and dewaxing as HE; ② Staining with 0.1% FastGreen solution (prepared with 0.2% glacial acetic acid, pH 3.0) for 5 min, followed by rinsing 3 times with 0.2% glacial acetic acid; ③ Staining with 0.1% Safranin O for 10 min; ④ Rapid dehydration and clearing (95% ethanol-xylene), mounting with neutral resin; ⑤ Microscopic observation, ImageJ quantitative IOD value, the treatment group showed stronger Safranin O staining (red) in the nucleus pulposus than the model group, indicating higher proteoglycan content.
[0073] The results showed that the exosomes of the present invention can effectively improve the integrity of the intervertebral discs in aging rats and promote the synthesis of extracellular matrix.
[0074] Example 3: Verification of Mitochondrial Function
[0075] Double fluorescent staining of exosomes and mitochondria: To visually observe and verify the endocytosis of exosomes by nucleus pulposus cells (NPCs) and their interaction with mitochondria, a double staining method combining exosome membrane fluorescent labeling and mitochondrial-specific fluorescent labeling was used. The specific procedure is as follows:
[0076] Take the exosome suspension containing the mitochondrial autophagy initiation factor of this invention (concentration 1×10¹). 0 According to the instructions for the PKH67 fluorescent dye, the dye and exosomes were incubated at 37°C for 15 min, and 10% fetal bovine serum was added to terminate the reaction. The free dye was then removed by ultracentrifugation at 100000×g for 30 min (4°C) to obtain PKH67-labeled fluorescent exosomes, which were then resuspended in sterile PBS for later use.
[0077] Senescent nucleus pulposus cells induced by TBHP (treated with 200 μM TBHP for 24 h) were seeded in confocal culture dishes. When the cell adhesion density reached 60%-70%, PKH67-labeled fluorescent exosomes were added to bring the final exosome concentration to 1 × 10⁻⁶. 9 The particles / mL were incubated together at 37℃ and 5% CO2 for 6 hours.
[0078] After co-incubation, discard the culture medium in the culture dish, gently rinse the cells twice with sterile PBS, add serum-free culture medium containing MitoTracker Red mitochondrial-specific dye, and incubate at 37°C for 30 min.
[0079] Discard the MitoTrackerRed staining solution, wash twice with PBS, add fixative containing 4′,6-diamidinyl-2-phenylindole, fix at room temperature for 15 min and stain the cell nuclei; after fixation, wash three times with PBS, and observe the imaging using a laser confocal microscope with excitation wavelengths of PKH67 (488 nm), MitoTrackerRed (579 nm), and DAPI (358 nm).
[0080] Fluorescence images showed that PKH67-labeled exosomes (green fluorescence) and MitoTrackerRed-labeled mitochondria (red fluorescence) exhibited significant colocalization signals (yellow overlapping area) in the cytoplasm of nucleus pulposus cells, confirming that exosomes can be effectively endocytosed by senescent nucleus pulposus cells and interact directly with mitochondria, providing a basis for subsequent experiments on mitochondrial function regulation.
[0081] JC-1 fluorescent staining
[0082] The JC-1 fluorescent probe (which detects mitochondrial membrane potential and reflects mitochondrial functional status) was used to perform quantitative and qualitative analysis of mitochondrial membrane potential in nucleus pulposus cells. The specific procedures are as follows:
[0083] Dissolve JC-1 fluorescent dye in sterile deionized water according to the instructions to prepare a 1 mg / mL stock solution, and store it at -20°C protected from light. Before use, dilute the stock solution with serum-free DMEM / F12 medium to obtain a JC-1 working solution with a final concentration of 10 μg / mL.
[0084] Nucleus pulposus cells (sham-operated group, TBHP-induced senescence group, normoxic exosome treatment group, hypoxic exosome treatment group, and hypoxic exosome treatment + HIF-1α silencing group) were seeded into 12-well plates (5 × 10⁴ cells per well) and cultured to adherence according to standard procedures. The culture medium of each well was discarded, and the cells were gently washed twice with PBS. 500 μL of JC-1 working solution was added to each well, and the cells were incubated at 37°C in a 5% CO₂ incubator in the dark for 30 min.
[0085] After incubation, discard the JC-1 working solution and rinse the cells twice with pre-cooled JC-1 staining buffer to remove free dye. Add fresh serum-free culture medium and immediately observe the images using a laser confocal microscope. The excitation wavelength for JC-1 polymers (high membrane potential, normal mitochondrial function) is 585 nm (red fluorescence), and the excitation wavelength for JC-1 monomers (low membrane potential, impaired mitochondrial function) is 529 nm (green fluorescence).
[0086] The fluorescence images of each group were quantified using ImageJ software, and the ratio of "red fluorescence intensity / green fluorescence intensity" was calculated to reflect the mitochondrial membrane potential level. The results showed that the JC-1 ratio in the TBHP aging group was significantly lower than that in the sham-operated group, while the JC-1 ratio in the hypoxic exosome treatment group was higher than that in the aging group and the normoxic exosome group (P<0.01). However, its effect disappeared after HIF-1α silencing, confirming that the hypoxic exosomes of the present invention can effectively restore the mitochondrial membrane potential of aging nucleus pulposus cells and improve mitochondrial functional damage.
[0087] MitoSOX fluorescent staining of mitochondria
[0088] The MitoSOXRed fluorescent probe (which specifically detects superoxide anions in mitochondria and reflects the level of mitochondrial oxidative stress) was used to detect reactive oxygen species (ROS) in mitochondria of nucleus pulposus cells. The specific procedure is as follows:
[0089] Dissolve MitoSOXRed powder in dimethyl sulfoxide to prepare a 5 mM stock solution, aliquot it, and store it at -80°C protected from light. Before use, dilute the stock solution to a 5 μM working solution with serum-free DMEM / F12 medium.
[0090] Five groups of nucleus pulposus cells (sham-operated group, TBHP-induced senescence group, normoxic exosome treatment group, hypoxic exosome treatment group, and hypoxic exosome treatment + HIF-1α silencing group) were seeded into confocal culture dishes. After adhesion, the culture medium was discarded, and the cells were washed twice with PBS. 500 μL LitoSOXRed working solution was added to each dish, and the cells were incubated in a 37°C, 5% CO2 incubator in the dark for 15 min.
[0091] After incubation, discard the MitoSOX working solution, wash three times with PBS (5 min each time), add fixative containing DAPI (4% paraformaldehyde), fix at room temperature for 10 min, and wash twice more with PBS.
[0092] Observation was performed using laser confocal microscopy. The excitation wavelength of MitoSOXRed was 510 nm (red fluorescence, reflecting the level of mitochondrial superoxide anion). The average fluorescence intensity of MitoSOXRed in nucleus pulposus cells of each group was quantitatively analyzed using ImageJ software. The results showed that the fluorescence intensity of the hypoxic exosome treatment group was lower than that of the TBHP group and the normoxic exosome group (P<0.01), indicating that the exosomes of the present invention can significantly inhibit the generation of superoxide anion in mitochondria of senescent nucleus pulposus cells and reduce mitochondrial oxidative stress damage.
[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. An exosome containing a mitophagy initiator, characterized in that, It is obtained by secretion from human bone marrow mesenchymal stem cells that have been pretreated for an appropriate period of time under conditions of 1-3% oxygen concentration.
2. A method for preparing exosomes, characterized in that, The preparation method includes the exosomes as described in claim 1, and specifically includes the following steps: S1. Culture and passage of human bone marrow mesenchymal stem cells: Human bone marrow mesenchymal stem cells are cultured under normoxic conditions until the confluence reaches 80-90%, and then passaged. S2. Hypoxia pretreatment: Passaged human bone marrow mesenchymal stem cells were seeded into culture dishes, replaced with exosome serum-free culture medium, and placed in a culture environment of 37℃, 5%CO2, and 2%O2 for an appropriate period of time for pretreatment. S3. Extraction and purification of exosomes: Collect the cell culture supernatant from step S2, centrifuge at differential speed, collect the precipitate and resuspend it to obtain exosomes.
3. The method for preparing exosomes according to claim 2, characterized in that, In step S1, the human bone marrow mesenchymal stem cells are passaged from P3 to P5, with a passage ratio of 1:
3.
4. The method for preparing exosomes according to claim 2, characterized in that, In step S1, the culture medium used for normoxic culture is DMEM / F12 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin; in step S2, the serum-free exosome culture medium is DMEM / F12 basal medium containing 10% serum-free exosomes.
5. The method for preparing exosomes according to claim 2, characterized in that, In step S2, the seeding density of human bone marrow mesenchymal stem cells is 1×10⁻⁶. 6 Cells per 10cm culture dish, with a cell confluence of 70-80% before pretreatment.
6. The method for preparing exosomes according to claim 2, characterized in that, In step S3, the specific parameters of the continuous centrifugation process are as follows: under 4°C conditions, the mixture is centrifuged sequentially at 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min. The supernatant is collected and then ultracentrifuged at 100000×g for 70 min. The supernatant is discarded, and the precipitate is resuspended in sterile PBS. The mixture is then ultracentrifuged again at 4°C at 100000×g for 70 min. The precipitate is collected and resuspended in 200-500 μL of sterile PBS.
7. The method for preparing exosomes according to claim 2, characterized in that, In step S1, the human bone marrow mesenchymal stem cells are cryopreserved and thawed cells. The thaw operation includes: placing the cryopreserved human bone marrow mesenchymal stem cells in a 37°C water bath for rapid thawing, transferring them to a centrifuge tube containing the DMEM / F12 culture medium, centrifuging at 1000×g for 5 min, discarding the supernatant, and resuspending them in fresh culture medium for inoculation.
8. The method for preparing exosomes according to claim 2, characterized in that, In step S2, before changing the exosome serum-free culture medium, rinse the cell surface 2-3 times with sterile PBS to remove residual serum.
9. The method for preparing exosomes according to claim 2, characterized in that, In step S3, the obtained exosomes are aliquoted and stored in a -80°C freezer, and repeated freeze-thaw cycles are avoided.
10. The use of the exosomes of claim 1 in the preparation of a medicament for treating or preventing intervertebral disc degeneration.