Use of phascolosoma esculenta exosome in preparation of preparation for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
By preparing an oral exosome preparation of Sipunculus nudus, the side effects and compliance issues of existing anti-osteoporosis drugs were resolved, achieving a highly efficient and safe effect in promoting bone differentiation and enhancing the osteogenic capacity of bone marrow mesenchymal stem cells.
Patent Information
- Application Number
- CN202610955279.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-30
- Publication Date
- 2026-07-28
AI Technical Summary
Existing osteoporosis drugs have significant side effects, poor compliance, and are expensive. There is a lack of highly effective and safe marine-derived osteopromoting agents.
Using Sipuncula salina exosomes as the active ingredient, an osteogenic differentiation formulation was prepared through specific centrifugation and filtration steps to promote the proliferation activity, alkaline phosphatase activity, and expression of osteogenic marker proteins of bone marrow mesenchymal stem cells.
It significantly enhances the proliferative activity of bone marrow mesenchymal stem cells, promotes calcium nodule deposition, and upregulates the expression of osteogenic marker proteins RUNX2, ALP, and OPN, achieving a natural, low-toxicity, and highly effective osteogenic differentiation effect.
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Figure CN122461350A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of marine biomedical materials technology, and in particular to the application of a type of *Sipunculus nudus* exosome in the preparation of a formulation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells. Background Technology
[0002] Osteoporosis is a systemic chronic metabolic bone disease characterized by persistent bone loss, trabecular bone fragmentation and breakage, bone microstructure damage, and increased bone fragility, making it highly susceptible to fragility fractures. This disease is prevalent in postmenopausal women and the elderly. With the increasing aging of my country's population, the incidence of osteoporosis is rising year by year, seriously affecting the quality of life of the elderly. Currently available anti-osteoporosis drugs have significant side effects, poor adherence, and high costs. Therefore, developing natural, low-toxicity, widely available, and highly safe marine bio-derived osteopromoting agents has significant clinical value. Summary of the Invention
[0003] The main purpose of this application is to propose the application of *Sipunculus nudus* exosomes in the preparation of a formulation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, aiming to solve or at least partially alleviate the problems of significant side effects, poor compliance, and high cost of existing anti-osteoporosis drugs.
[0004] To achieve the above objectives, in a first aspect, this application proposes the use of Sipunculus nudus exosomes in the preparation of a formulation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells.
[0005] In some embodiments, the effective concentration of the *Sipunculus nudus* exosomes for promoting osteogenic differentiation of bone marrow mesenchymal stem cells is 5–10 μg / mL.
[0006] In some embodiments, the preparation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells is used to achieve at least one of the following functions: (a) Enhances the proliferative activity of bone marrow mesenchymal stem cells; (b) Increase alkaline phosphatase activity; (c) Promotes calcium nodule deposition; (d) Upregulate the expression levels of osteogenic marker proteins RUNX2, ALP and OPN. In some embodiments, the oral sipuncula exosomes are prepared by a method comprising the following steps: Coelomic fluid was extracted from Sipunculus nudus; The body cavity fluid was mixed with sterile phosphate-buffered saline (PBS) and then filtered to obtain the first filtrate. The first filtrate is centrifuged, and the supernatant is collected to obtain the second filtrate; The second filtrate was centrifuged a second time, and the supernatant was collected to obtain the third filtrate; The third filtrate was filtered through micropores to obtain the fourth filtrate; The fourth filtrate was centrifuged a third time, the precipitate was collected, and resuspended in sterile buffer.
[0007] In some embodiments, the volume ratio of the body cavity fluid to sterile phosphate-buffered saline (PBS) is 1:2 to 1:5; The first centrifugation conditions are centrifugation at 2500-4000g speed at 4℃ for 20-30 minutes; The second centrifugation conditions were: centrifugation at 10000g speed and 4℃ for 30–40 min; The microporous filtration uses a 0.45μm sterile filter membrane; The conditions for the third centrifugation are centrifugation at 100,000g speed and 4℃ for 60-70 minutes.
[0008] Secondly, this application also proposes a method for preparing oral exosomes of Sipunculus nudus, comprising the following steps: Step S1: Extract coelomic fluid from Sipunculus nudus; Step S2: Mix the body cavity fluid with sterile phosphate-buffered saline (PBS) at a volume ratio of 1:2 to 1:5, filter through a 70-mesh sterile filter to obtain the first filtrate; Step S3: Centrifuge the first filtrate at 2500-4000g and 4℃ for 20-30min, collect the supernatant, and obtain the second filtrate; Step S4: Centrifuge the second filtrate at 10000g and 4℃ for 30-40 minutes, collect the supernatant, and obtain the third filtrate; Step S5: The third filtrate is filtered through a 0.45μm sterile filter membrane to remove bacteria, resulting in the fourth filtrate. Step S6: Centrifuge the fourth filtrate at 100,000g and 4℃ for 60-70 min, collect the precipitate, and resuspend it in sterile buffer to obtain the *Sipunculus nudus* exosomes.
[0009] In some embodiments, in step S2, the mixture is filtered sequentially using 70-mesh and 40-mesh sterile filters. In some embodiments, step S3 further includes centrifuging the second filtrate at 2500–4000 g for 10–20 min; and / or, Step S4 is followed by centrifuging the third filtrate at 2500–4000g for 10–20 min.
[0010] In some embodiments, the sterile buffer includes one of sterile phosphate-buffered saline (PBS), cell-based culture medium, and double-distilled water.
[0011] Thirdly, this application also proposes an agent that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, including *Sipunculus nudus* exosomes prepared by the method for preparing *Sipunculus nudus* exosomes proposed in the second aspect of this application.
[0012] The exosomes of Sipunculus nudus proposed in this application have good biocompatibility and no significant toxicity to human bone marrow mesenchymal stem cells. They can significantly enhance the proliferative activity of human bone marrow mesenchymal stem cells. In vitro experiments have confirmed that the exosomes of Sipunculus nudus can significantly increase alkaline phosphatase activity, promote calcium nodule deposition, upregulate the expression of osteogenic marker proteins RUNX2, ALP, and OPN, and accelerate the osteogenic differentiation process of bone marrow mesenchymal stem cells. They can be used to prepare agents that promote osteogenic differentiation of bone marrow mesenchymal stem cells. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0014] Figure 1 Transmission electron microscope image of Sipunculus nudus exosomes provided for this application; Figure 2 The particle size distribution diagram of *Sipunculus nudus* exosomes provided in this application; Figure 3 The image shows the results of the toxicity test of *Sipunculus nudus* exosomes on human bone marrow mesenchymal stem cells provided in this application. Figure 4 The following images show the results of alkaline phosphatase staining and alizarin red staining of human bone marrow mesenchymal stem cells after intervention with Sipunculus nudus exosomes provided in this application: A is an image of alkaline phosphatase staining, and B is an image of alizarin red staining. Figure 5 The following is a Western blot result showing the effect of exosomes from *Sipunculus nudus* on the expression of osteogenic differentiation markers RUNX2, ALP, and OPN, as provided in this application: A is the result of Western blot analysis; B is the RUNX2 expression analysis; C is the ALP expression analysis; D is the OPN expression analysis.
[0015] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0016] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.
[0017] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.
[0018] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.
[0019] The key term “Phascolosoma esculenta exosome” used in this application refers to nanoscale vesicles with a diameter in the range of 30-150 nm and a lipid bilayer membrane structure, which are isolated and purified from the coelomic fluid of Phascolosoma esculenta.
[0020] "Bone marrow mesenchymal stem cells" refer to adult stem cells that are capable of self-renewal and have the potential to differentiate into multiple lineages such as osteoblasts, chondrocytes, and adipocytes.
[0021] "Osteogenic differentiation" refers to the biological process by which bone marrow mesenchymal stem cells transform into osteoblasts under specific induction conditions, accompanied by increased alkaline phosphatase (ALP) activity, calcium nodule deposition, and upregulation of osteogenic marker proteins such as osteogenic-associated transcription factor 2 (RUNX2) and osteopontin (OPN).
[0022] Osteoporosis is a systemic chronic metabolic bone disease characterized by persistent bone loss, trabecular bone fragmentation and breakage, bone microstructure damage, and increased bone fragility, making it highly susceptible to fragility fractures. This disease is prevalent in postmenopausal women and the elderly. With the increasing aging of my country's population, the incidence of osteoporosis is rising year by year, seriously affecting the quality of life of the elderly. Currently available anti-osteoporosis drugs have significant side effects, poor adherence, and high costs. Therefore, developing natural, low-toxicity, widely available, and highly safe marine bio-derived osteopromoting agents has significant clinical value.
[0023] Extracellular vesicles (exosomes) are nanoscale lipid bilayer membrane vesicles secreted by cells, with a particle size ranging from 30 to 150 nm. They are rich in proteins, lipids, nucleic acids, and various bioactive small molecules, and can mediate intercellular signal transduction, regulate cell proliferation and differentiation, and improve the microenvironment. They have become novel nanobiomaterials in regenerative medicine, bone repair, and tissue engineering. Current research mainly focuses on mammalian and plant exosomes, while the exploration of marine invertebrate-derived exosomes is relatively low, especially the exosomes of marine organisms such as Sipunculus nudus, which have not yet been systematically developed.
[0024] Sipunculus esculenta, scientifically known as *Phascolosoma esculenta*, belongs to the phylum Sipunculidae and the genus *Phascolosoma*. It is widely inhabited in brackish water tidal flats along the southeastern coast of my country, and is abundant and easily accessible. Sipunculus esculenta is highly nutritious and delicious. Modern pharmacological studies have confirmed that it is rich in collagen, bioactive peptides, antioxidants, and trace elements, possessing anti-aging, antioxidant, anti-fatigue, hypoxia-resistant, heat-resistant, radiation-resistant, and thrombolytic effects. Currently, there are no reports on the use of *Phascolosoma esculenta* exosomes for regulating osteogenic differentiation of bone marrow mesenchymal stem cells and promoting bone formation.
[0025] Based on this, in a first aspect, embodiments of this application propose the application of *Sipunculus nudus* exosomes in the preparation of formulations that promote osteogenic differentiation of bone marrow mesenchymal stem cells. *Sipunculus nudus* exosomes can be taken up by bone marrow mesenchymal stem cells via endocytosis. The osteogenic-related active proteins, differentiation-promoting miRNAs, and functional small molecules carried within them can activate intracellular osteogenic-related signaling pathways, thereby synergistically solving the technical problem of the lack of efficient and safe marine-derived formulations for promoting osteogenic differentiation in the prior art, achieving the technical effect of naturally low-toxicity and highly efficient promotion of osteogenic differentiation.
[0026] In some embodiments, the effective concentration of *Sipunculus nudus* exosomes for promoting osteogenic differentiation of bone marrow mesenchymal stem cells is 5–10 μg / mL. For example, the effective concentrations of *Sipunculus nudus* exosomes for promoting osteogenic differentiation of bone marrow mesenchymal stem cells are 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL.
[0027] Limiting the effective concentration of exosomes to this range ensures that they provide sufficient bioactive molecules to effectively initiate and maintain osteogenic differentiation signals, while avoiding potential cytotoxicity from excessively high concentrations.
[0028] In some embodiments, the agent that promotes osteogenic differentiation of bone marrow mesenchymal stem cells is used to achieve at least one of the following functions: (a) Enhances the proliferative activity of bone marrow mesenchymal stem cells; (b) Increase alkaline phosphatase activity; (c) Promotes calcium nodule deposition; (d) Upregulate the expression levels of osteogenic marker proteins RUNX2, ALP and OPN.
[0029] Enhancing the proliferation activity of bone marrow mesenchymal stem cells can provide a sufficient cell quantity base for subsequent differentiation; alkaline phosphatase is a marker enzyme for early osteoblast differentiation, and its activity level directly reflects the degree of osteoblast differentiation initiation; calcium nodules are morphological evidence of late-stage osteoblast maturation and mineralization function; RUNX2 is the master-controlling transcription factor for osteoblast differentiation, while ALP and OPN are its downstream effector molecules. Upregulation of the protein expression levels of these three molecules confirms the complete activation of osteoblast differentiation at the molecular level. *Sipunculus nudus* exosomes, through their internally loaded active substances, can systematically initiate and promote this series of osteoblast differentiation events by synergistically regulating multiple targets of related signaling pathways.
[0030] In some embodiments, the exosomes of *Sipunculus nudus* are prepared by a method comprising the following steps: Coelomic fluid was extracted from Sipunculus nudus; The body cavity fluid was mixed with sterile phosphate-buffered saline (PBS) and then filtered to obtain the first filtrate. The first filtrate is centrifuged, and the supernatant is collected to obtain the second filtrate; The second filtrate was centrifuged a second time, and the supernatant was collected to obtain the third filtrate; The third filtrate was filtered through micropores to obtain the fourth filtrate; The fourth filtrate was centrifuged a third time, the precipitate was collected, and resuspended in sterile buffer.
[0031] Specifically, in some embodiments, the volume ratio of coelomic fluid to sterile phosphate-buffered saline (PBS) is 1:2 to 1:5. For example, the volume ratio of coelomic fluid to sterile phosphate-buffered saline (PBS) is 1:2, 1:3, 1:4, or 1:5. If the dilution ratio is lower than 1:2, the fluid is too viscous, and large particles do not settle completely during centrifugation; if the dilution ratio is higher than 1:5, the efficiency of subsequent ultracentrifugation for exosome enrichment decreases, the processing volume becomes too large, and the processing time is long.
[0032] The first centrifugation conditions are: centrifugation at 2500–4000 g at 4°C for 20–30 min. For example, the first centrifugation speed can be 2500 g, 3000 g, 3500 g, or 4000 g, and the first centrifugation time can be 20 min, 25 min, or 30 min, etc. The purpose of the first centrifugation is to remove cells, nuclear debris, and large particulate impurities. The above-mentioned speed and time are sufficient to completely settle these impurities while avoiding shear damage to exosomes.
[0033] The second centrifugation conditions are 10,000g centrifugation at 4°C for 30–40 minutes. For example, the second centrifugation time is 30 minutes, 35 minutes, or 40 minutes. The centrifugal force of 10,000g can effectively remove residual cell debris, as well as larger microvesicles and apoptotic bodies, precipitating interfering substances while retaining exosomes in the supernatant.
[0034] The microfiltration system uses a 0.45μm sterile filter membrane. The 0.45μm pore size of the filter membrane can trap residual bacteria and other microorganisms and particles larger than 450nm, while allowing all exosomes with a particle size smaller than 150nm to pass through freely, thus combining filtration and sterilization with particle sieving functions.
[0035] The third centrifugation conditions are 100,000g at 4°C for 60–70 minutes. For example, the third centrifugation time is 60 minutes, 65 minutes, or 70 minutes. 100,000g is the classic condition for ultracentrifugation to enrich exosomes. Under this centrifugal force, exosomes are effectively settled at the bottom of the tube to form a milky white precipitate, while most soluble proteins are retained in the supernatant and discarded.
[0036] This application also proposes a method for preparing oral exosomes of Sipunculus nudus, including the following steps: S1. Extracting coelomic fluid from fresh, palatable Sipunculus nudus; S2. Mix the body cavity fluid with sterile phosphate-buffered saline (PBS) at a volume ratio of 1:2 to 1:5, and filter through a 70-mesh sterile filter to obtain the first filtrate. S3. Centrifuge the first filtrate at 2500-4000g and 4℃ for 20-30min, collect the supernatant, and obtain the second filtrate; S4. Centrifuge the second filtrate at 10000g and 4℃ for 30-40 minutes, collect the supernatant, and obtain the third filtrate. S5. The third filtrate is filtered through a 0.45μm sterile filter membrane to remove bacteria, resulting in the fourth filtrate. S6. Centrifuge the fourth filtrate at 100,000g and 4℃ for 60-70 min, collect the precipitate, and resuspend it in sterile buffer to obtain the *Sipunculus nudus* exosomes.
[0037] The method for preparing exosomes from Sipunculus nudus provided in this application begins with selecting fresh, undamaged exosomes. Step S2 uses a 70-mesh sieve to initially remove large tissue fragments and sediment, creating conditions for subsequent high-precision centrifugation. Steps S3 and S4 constitute a differential centrifugation process, gradually removing impurities of different sizes. Step S5 achieves the dual purpose of sterilization and particle homogenization. Step S6 completes the high-purity enrichment of exosomes. In this process, the operating conditions at each step follow a gradual principle of impurities decreasing in size and centrifugal force increasing in intensity, ensuring not only the recovery rate and purity of exosomes but also giving the preparation method good operability and industrial scale-up potential.
[0038] In some embodiments, in step S2, the mixture is filtered sequentially through 70-mesh and 40-mesh sterile filters. The 70-mesh filter can trap coarse particles, while the 40-mesh filter can further trap particles larger than 380 μm. Introducing the 40-mesh filter creates a gradient filtration system. In practice, the coelomic fluid first flows through a 70-mesh sterile filter and then through a 40-mesh sterile filter. It can be observed that the 40-mesh filter traps more fine, transparent debris, resulting in a clearer filtrate. During subsequent centrifugation, the deposition of large impurities at the bottom is significantly reduced. By implementing graded filtration, the risk of clogging in subsequent operations is effectively reduced, and the final purification efficiency of exosomes is improved.
[0039] In some embodiments, step S3 is followed by centrifugation of the second filtrate at 2500–4000 g for 10–20 min; and / or, step S4 is followed by centrifugation of the third filtrate at 2500–4000 g for 10–20 min. Adding a low-speed centrifugation purification step after the conventional differential centrifugation step allows for secondary sedimentation and capture of a small number of denser particles still suspended in the supernatant after the initial centrifugation. These residual impurities may be particles that were stirred up and floated during the previous centrifugation operation or particles that were too small to settle sufficiently. This low-speed, high-speed secondary centrifugation removes stubborn impurities that are difficult to settle naturally at a relatively low time cost.
[0040] In some implementations, the sterile buffer includes one of sterile phosphate-buffered saline (PBS), cell basal medium, or double-distilled water. Sterile phosphate-buffered saline (PBS) maintains stable osmotic pressure and pH, making it suitable for routine in vivo animal injections and most cell experiments. Cell basal medium (such as α-MEM, DMEM, or RPMI-1640) is suitable for applications requiring direct addition to the cell culture system, avoiding alteration of the medium composition. Double-distilled water is suitable for analytical scenarios requiring subsequent lyophilization or strict control of ion concentration. The choice can be flexibly determined based on subsequent formulation and experimental objectives.
[0041] Thirdly, embodiments of this application also propose an agent that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, including *Sipunculus nudus* exosomes prepared by the method for preparing *Sipunculus nudus* exosomes proposed in the second aspect of this application.
[0042] The *Sipunculus nudus* exosomes in the formulation are the sole or main active ingredient, possessing natural osteogenic bioactivity that promotes bone differentiation, thus eliminating the need for additional complex chemical inducing factors. Because the preparation method proposed in this application ensures the high purity and high activity of the *Sipunculus nudus* exosomes, they can exert bone-repairing effects when formulated into different dosage forms (such as injections, cytokine supplements, and biomaterial coatings) through local or systemic application.
[0043] The following specific examples provide further details.
[0044] Unless otherwise specified, the reagents, materials and instruments used in the examples are known to those skilled in the art or are commercially available.
[0045] Example 1: Isolation and preparation of exosomes from Sipunculus nudus (Nepenthes spp.) Fresh, intact, and undamaged Sipuncula worms (collected from an artificial breeding base in the intertidal mudflats of coastal mangroves in Tangdong Village, Taiping Town, Mazhang District, Zhanjiang City, Guangdong Province) were selected. Coelomic fluid was extracted in a sterile operating table. The coelomic fluid was mixed with sterile phosphate-buffered saline (PBS) at a volume ratio of 1:3. The mixture was filtered through a 70-mesh sterile filter to remove tissue residue and impurities, yielding the first filtrate. The first filtrate was centrifuged at 4℃ and 2500g for 25 minutes, the bottom precipitate was discarded, and the supernatant was collected to obtain the second filtrate. The second filtrate was centrifuged at 4°C and 10,000g for 35 min to remove broken cells and macromolecular impurities, and the supernatant was collected as the third filtrate. The third filtrate was slowly passed through a 0.45 μm sterile filter membrane to obtain a clear fourth filtrate. The fourth filtrate was placed in an ultracentrifuge tube and centrifuged at 4°C and 100,000g for 65 min. The milky white precipitate at the bottom of the tube was collected and gently resuspended in sterile phosphate-buffered saline (PBS) to obtain the final exosome stock solution of *Sipunculus nudus*, which was stored at -80°C for later use. The obtained *Sipunculus nudus* exosomes were characterized by transmission electron microscopy, and the results are as follows: Figure 1 As shown. From Figure 1 As can be seen, the exosomes of Sipunculus nudus exhibit a typical cup-shaped vesicle structure with an intact membrane structure and uniform morphology, consistent with the microscopic morphological characteristics of exosomes.
[0046] Example 2: Detection of exosome size in *Sipunculus nudus* Nanoparticle tracking analysis (NTA) was used to detect the particle size distribution and concentration of *Sipunculus nudus* exosomes. The specific steps are as follows: 1 μL of the *Sipunculus nudus* exosome stock solution prepared in Example 1 was taken and serially diluted with sterile phosphate-buffered saline (PBS) to control the particle concentration at 1.0 × 10⁻⁶. 8 ~2.5×10 9 particles / mL; the data were analyzed using the instrument and 11 fields of view were collected for statistical analysis. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that the exosomes of Sipunculus nudus are concentrated at around 125 nm in size, with uniform particle size distribution and no obvious impurity particles, which meets the criteria for exosome particle size determination.
[0047] Example 3: Determination of exosome granulocyte protein concentration in Sipuncula oryzae The protein concentration of exosome stock solution was detected using the BCA protein quantification kit. The specific steps are as follows: A gradient of protein standards (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) was prepared; 20 μL of the standards and the exosome sample to be tested were added to each well of a 96-well plate, with 200 μL of BCA working solution added to each well. The plates were incubated at 37 ℃ for 25 min; the absorbance at 562 nm was measured using a microplate reader, a standard curve was plotted, and the sample concentration was calculated. The results showed that the protein concentration of the *Sipunculus nudus* exosome stock solution prepared in Example 1 was 2.95 mg / mL, suitable for subsequent cell intervention experiments.
[0048] Example 4: Effects of *Sipunculus nudus* exosomes on the activity of human bone marrow mesenchymal stem cells (hBMSCs) Human bone marrow mesenchymal stem cells (P5 generation, purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd., product name OriCell® Adult Bone Marrow Mesenchymal Stem Cells, HUXMA-01001) were used for toxicity experiments. Cells were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells / well; blank control group, 5 μg / mL and 10 μg / mL Sipunculus nudus exosome intervention groups were set up, and the intervention was carried out for 24 h, 48 h and 72 h, respectively; after the intervention, 10% volume of CCK-8 detection solution was added to each well, and incubated at 37 ℃ for 2 h. The absorbance at 450 nm was measured, and the results are as follows: Figure 3 As shown. From Figure 3 It can be seen that at the three intervention time points of 24h, 48h and 72h, the exosomes of Sipunculus orientalis at 5μg / mL and 10μg / mL were not cytotoxic and could continuously enhance the proliferation activity of hBMSCs. As the intervention time was extended, the cell proliferation activity showed a slow upward trend, which confirmed that the exosomes of Sipunculus orientalis (0~10 μg / mL) have good biosafety.
[0049] Example 5: Analysis of the effect of *Sipunculus nudus* exosomes on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) Prepare osteogenic induction medium (α-MEM complete medium containing 100 nmol / L dexamethasone, 50 nmol / L ascorbic acid, and 10 nmol / L β-glycerophosphate); seed hBMSCs in 12-well plates at a density of 5 × 10⁻⁶. 5 Cells / well were divided into a blank control group, and 5 μg / mL and 10 μg / mL exosome intervention groups; the induction medium was changed fresh every 3 days. On day 3 of culture, alkaline phosphatase staining was performed. After fixing cells with 4% paraformaldehyde, staining was performed at room temperature in the dark for 60 min. After washing with sterile phosphate-buffered saline (PBS), the cells were photographed. On day 10 of culture, Alizarin Red S staining was performed. After fixing with ethanol, staining was performed for 20 min. After washing, the cells were photographed. The staining results are shown below. Figure 4 A and B in the middle. From Figure 4 As can be seen from Figures A and B, compared with the blank group, the staining depth of the exosome intervention group was significantly increased, the number of calcium nodules increased, and the staining effect of the high concentration group was better, showing a clear dose-dependent effect, which confirms that the exosomes of Sipunculus nudus can effectively promote osteogenic differentiation of hBMSCs.
[0050] Example 6: Effects of *Sipunculus nudus* exosomes on the expression of osteogenic marker proteins in human bone marrow mesenchymal stem cells (hBMSCs) hBMSCs were seeded in 60 mm cell culture dishes at a density of 5 × 10⁶ cells / mL. 5 At 80% density, different concentrations of *Sipunculus nudus* exosomes (5 μg / mL and 10 μg / mL) were added for intervention. Radioimmunoprecipitation lysis buffer (RIPA lysis buffer) was added, and cells were lysed on ice for 15 min, followed by centrifugation at 13000g for 15 min. The supernatant was collected, and protein concentration was determined using the BCA method and balanced. Samples were added to 5× Loading Buffer at a 1:4 ratio, boiled in 100℃ water for 15 min, loaded with 15 μg of total protein, and subjected to SDS-PAGE gel electrophoresis (80V constant voltage electrophoresis separation for 1.5 h). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane (transfer conditions: 100V, 100min), blocked with 5% skim milk for 2h, washed 5 times with TBST (Tris buffered saline containing Tween-20) for 10min each time, and then incubated overnight at 4℃ with primary antibodies RUNX2 (1:1000), ALP (1:1000), OPN (1:1000), and β-actin (1:5000). After thorough washing with TBST, the membrane was incubated with secondary antibody at room temperature for 1h, and the protein expression level was detected by chemiluminescence imaging. The results are as follows. Figure 5 As shown. From Figure 5As shown in A, B, C, and D, the exosomes of Sipunculus nudus can significantly upregulate the expression levels of RUNX2, ALP, and OPN proteins in a dose-dependent manner, indicating that the exosomes of Sipunculus nudus possess significant osteogenic activity.
[0051] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.
Claims
1. Application of Sipunculus nudus exosomes in the preparation of formulations that promote osteogenic differentiation of bone marrow mesenchymal stem cells.
2. The application as described in claim 1, characterized in that, The effective concentration of the *Sipunculus nudus* exosomes for promoting osteogenic differentiation of bone marrow mesenchymal stem cells is 5–10 μg / mL.
3. The application as described in claim 1, characterized in that, The preparation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells is used to achieve at least one of the following functions: (a) Enhances the proliferative activity of bone marrow mesenchymal stem cells; (b) Increase alkaline phosphatase activity; (c) Promotes calcium nodule deposition; (d) Upregulate the expression levels of osteogenic marker proteins RUNX2, ALP and OPN.
4. The application as described in claim 1, characterized in that, The delicious Sipunculus exosomes were prepared by a method comprising the following steps: Coelomic fluid was extracted from Sipunculus nudus; The body cavity fluid was mixed with sterile phosphate-buffered saline (PBS) and then filtered to obtain the first filtrate. The first filtrate is centrifuged, and the supernatant is collected to obtain the second filtrate; The second filtrate was centrifuged a second time, and the supernatant was collected to obtain the third filtrate; The third filtrate was filtered through micropores to obtain the fourth filtrate; The fourth filtrate was centrifuged a third time, the precipitate was collected, and resuspended in sterile buffer.
5. The application as described in claim 4, characterized in that, The volume ratio of the body cavity fluid to sterile phosphate-buffered saline (PBS) is 1:2 to 1:
5. The first centrifugation conditions are centrifugation at 2500-4000g speed at 4℃ for 20-30 minutes; The second centrifugation conditions were: centrifugation at 10000g speed and 4℃ for 30–40 min; The microporous filtration uses a 0.45μm sterile filter membrane; The conditions for the third centrifugation are centrifugation at 100,000g speed and 4℃ for 60-70 minutes.
6. A method for preparing palatable exosomes of Sipunculus nudus, characterized in that, Includes the following steps: Step S1: Extract coelomic fluid from Sipunculus nudus; Step S2: Mix the body cavity fluid with sterile phosphate-buffered saline (PBS) at a volume ratio of 1:2 to 1:5, filter through a 70-mesh sterile filter to obtain the first filtrate; Step S3: Centrifuge the first filtrate at 2500-4000g and 4℃ for 20-30min, collect the supernatant, and obtain the second filtrate; Step S4: Centrifuge the second filtrate at 10000g and 4℃ for 30-40 minutes, collect the supernatant, and obtain the third filtrate; Step S5: The third filtrate is filtered through a 0.45μm sterile filter membrane to remove bacteria, resulting in the fourth filtrate. Step S6: Centrifuge the fourth filtrate at 100,000g and 4℃ for 60-70 min, collect the precipitate, and resuspend it in sterile buffer to obtain the *Sipunculus nudus* exosomes.
7. The preparation method according to claim 6, characterized in that, In step S2, the mixture is filtered sequentially using 70-mesh and 40-mesh sterile filters.
8. The preparation method according to claim 6, characterized in that, Following step S3, the method further includes centrifuging the second filtrate at 2500–4000 g for 10–20 min; and / or, Step S4 is followed by centrifuging the third filtrate at 2500–4000g for 10–20 min.
9. The preparation method according to claim 6, characterized in that, The sterile buffer solution includes one of sterile phosphate-buffered saline (PBS), cell basal culture medium, and double-distilled water.
10. A preparation for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, The exosomes of *Sipunculus nudus* prepared by the method for preparing *Sipunculus nudus* exosomes according to any one of claims 6 to 9.