Oyster-derived exosome-like vesicles, and preparation method and application thereof

Abstract

The application belongs to the technical field of nanobiomaterials, and discloses a kind of oyster source exosome-like vesicle and its preparation method and application, specifically discloses a kind of oyster source exosome-like vesicle, the oyster source exosome-like vesicle is separated from oyster meat;The exosome-like vesicle contains osteogenic mineral elements.The application first discloses the oyster source exosome-like vesicle, the oyster source exosome-like vesicle is nanoscale membrane vesicle, presents typical tea tray-like morphology, average particle size is less than 150nm, can carry calcium and phosphorus and other osteogenic mineral elements, can be effectively taken up by osteoblasts, has the effect of promoting osteoblast proliferation and differentiation, and has the effect of improving osteoporosis, and can be used as beneficial dietary supplements in functional foods related to delaying bone loss.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials technology, specifically relating to an oyster-derived exosome-like vesicle, its preparation method, and its application. Background Technology

[0002] Osteoporosis is a chronic metabolic disease characterized by decreased bone density and quality due to the degeneration and reduction in the number of trabecular bone microstructures, accompanied by an increased risk of fractures. According to WHO data, more than 200 million people worldwide suffer from osteoporosis, and with the increasing aging of society, it has become a significant public health problem. However, many existing synthetic drugs have certain side effects, making long-term use unsuitable. Therefore, the rational intake of dietary active ingredients with good bone-promoting effects is of great significance for the prevention, treatment, and adjuvant therapy of osteoporosis.

[0003] Exosomes are nanoscale vesicles with a phospholipid bilayer structure secreted by cells. They carry a variety of bioactive substances, including proteins, lipids, nucleic acids, and small molecules, and can mediate intercellular communication and signal transduction. Recent studies have found that plant and animal foods are rich in exosome-like nanovesicles, which can carry various bioactive substances such as functional proteins, flavonoids, and polyphenols. Notably, oral ingestion of food-derived exosome-like nanovesicles is unaffected by the digestive tract environment, such as enzymes and pH, allowing the bioactive components they carry to be directly absorbed into the bloodstream without degradation. These nanovesicles exhibit various biological effects, including improving enteritis, anti-cancer activity, promoting bone formation, protecting the liver, and improving memory.

[0004] Oysters are rich in protein, polyunsaturated fatty acids, and a large number of minerals and trace elements beneficial to bone growth, giving them significant nutritional and medicinal value. Both ancient Chinese herbal medicine texts and modern scientific research show that oysters promote bone health, reduce the rate of bone loss, and lower the risk of osteoporosis. Therefore, developing oyster-derived exosome-like vesicles related to bone protection has promising applications. Summary of the Invention

[0005] The first aspect of the present invention is to provide an oyster-derived exosome-like vesicle.

[0006] The second objective of this invention is to provide a method for preparing oyster-derived exosome-like vesicles according to the first aspect of this invention.

[0007] The third aspect of the present invention aims to provide the application of the oyster-derived exosome-like vesicles of the first aspect of the present invention and / or the preparation method of the second aspect.

[0008] The fourth aspect of this invention is to provide a product.

[0009] The fifth aspect of this invention is to provide a culture medium.

[0010] The sixth aspect of this invention aims to provide a method for improving the proliferative and / or differentiation capacity of osteoblasts.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] The first aspect of the present invention is to provide an oyster-derived exosome-like vesicle, the oyster-derived exosome-like vesicle being isolated from oyster meat, the exosome-like vesicle containing osteogenic mineral elements.

[0013] Preferably, the average particle size of the oyster-derived exosome-like vesicles is 80–150 nm.

[0014] More preferably, the average particle size of the oyster-derived exosome-like vesicles is 143.0 ± 5.5 nm.

[0015] Preferably, the oyster-derived exosome-like vesicles are nanoscale vesicles with a typical saucer-like structure.

[0016] Preferably, the osteogenic mineral elements include calcium and phosphorus.

[0017] In this invention, "exosome-like vesicles" refers to nanoscale extracellular vesicles secreted by cells into the extracellular space. The term includes exosomes and is used in the broadest sense, encompassing vesicles similar to exosomes in nanoscale vesicular structures and compositions.

[0018] In this invention, "oyster-derived exosome-like vesicles" refers to nanoscale exosome-like vesicles secreted by oyster cells. These exosome-like vesicles can be isolated from the extracellular fluid of oysters, and can be physically separated completely or partially from existing tissues or cells.

[0019] The membrane components of the exosome-like vesicles can be chemically or physically modified to effectively perform the desired function in target cells. For example, the membrane components of the exosome-like vesicles can be chemically modified using thiol groups (-SH) or amino groups (-NH2), or by chemically binding an inducing substance, fusion agent, or polyethylene glycol to the exosome-like vesicles.

[0020] The second aspect of the present invention is to provide a method for preparing oyster-derived exosome-like vesicles according to the first aspect of the present invention, comprising the following steps: mixing oysters with an extract, homogenizing, and separating solids and liquids to obtain oyster-derived exosome-like vesicles.

[0021] Preferably, the mass-to-volume ratio of the oyster to the extract is 1:(1-20); further, it is 1:(1-10); and even further, it is 1:(5-10).

[0022] Preferably, the extraction solution comprises phosphate buffer.

[0023] Preferably, the extract further includes a protease.

[0024] Preferably, the extract further includes 10 to 30 units of protease.

[0025] Preferably, the protease includes at least one of papain, pepsin, trypsin, and neutral protease.

[0026] Preferably, before mixing the oysters with the extract, the oysters are shelled and their tissues are shredded on ice.

[0027] Preferably, the oysters are mixed with the extract and digested at 35–40°C for 0.5–24 hours; more preferably, they are digested at 37–40°C for 0.5–24 hours.

[0028] Preferably, the mixture of oysters and extract is stirred in an ice-water bath for 10–20 min before homogenization.

[0029] Preferably, the homogenization is performed intermittently.

[0030] Preferably, the homogenization is performed at 500-1000 rpm, with each homogenization session lasting 2-4 seconds and an interval of 30-40 seconds, for a total of 1-3 homogenization sessions.

[0031] Preferably, the solid-liquid separation includes at least one method selected from ultracentrifugation, differential centrifugation, equilibrium density centrifugation, density gradient centrifugation, filtration, dialysis, and free-flow electrophoresis.

[0032] Currently, the most common method for exosome isolation is differential centrifugation. On one hand, differential centrifugation can be used in conjunction with filtration, ultracentrifugation, etc. On the other hand, gel filtration or ultrafiltration can be used to separate exosome-like vesicles. Alternatively, dialysis can be used instead of filtration to remove small molecules. Free-flow electrophoresis can also be used. Furthermore, density gradient centrifugation can be used, where exosome-like vesicles are separated by density gradients due to their different densities. Examples of density gradient materials used include Ficoll, glycerol, sucrose, cesium chloride, iodixanol, etc., but are not limited to these.

[0033] Preferably, the solid-liquid centrifugation conditions are as follows: centrifuge at 300×g for 10–20 min at 1–4℃ and collect the supernatant; centrifuge at 3000×g at 1–4℃ for 10–20 min and collect the supernatant; centrifuge at 10000×g at 1–4℃ for 15–30 min and collect the supernatant; centrifuge at 100000×g at 1–4℃ for 70–120 min and collect the precipitate.

[0034] Preferably, the precipitate collected by solid-liquid separation is resuspended in a buffer solution, filtered through a filter membrane, and then centrifuged at 100,000 × g and 1–4 °C for 70–120 min.

[0035] A third aspect of the present invention is the application of the oyster-derived exosome-like vesicles of the first aspect of the present invention and / or the preparation method of the second aspect in any one of (1) to (9):

[0036] (1) Prepare drugs for the treatment and / or adjuvant treatment of osteoporosis;

[0037] (2) To prepare products that delay bone loss;

[0038] (3) To develop products that reduce the risk of osteoporosis;

[0039] (4) Prepare products that improve bone density;

[0040] (5) Prepare products that supplement osteogenic mineral elements;

[0041] (6) Enhances osteoblast proliferation capacity;

[0042] (7) Prepare products that enhance the proliferation capacity of osteoblasts;

[0043] (8) Enhance osteoblast differentiation capacity;

[0044] (9) Prepare products that enhance osteoblast differentiation ability.

[0045] Preferably, the product includes any one of food, medicine, and reagent.

[0046] A fourth aspect of the present invention is to provide an oyster-derived exosome-like vesicle product comprising the first aspect of the present invention.

[0047] Preferably, the product further includes a carrier, excipients, or diluents, which can be obtained by those skilled in the art from professional books on the subject and are pharmaceutically acceptable.

[0048] Preferably, the product includes any one of food, medicine, and reagent.

[0049] Preferably, the concentration of oyster-derived exosome-like vesicles in the product is 5–500 μg / mL; more preferably 10–200 μg / mL; and even more preferably 20–80 μg / mL.

[0050] A fifth aspect of the present invention is to provide an oyster-derived exosome-like vesicle culture medium comprising the first aspect of the present invention.

[0051] Preferably, the concentration of oyster-derived exosome-like vesicles in the culture medium is 5–500 μg / mL; more preferably 10–200 μg / mL; and even more preferably 20–80 μg / mL.

[0052] Preferably, the culture medium also contains the nutrients required by the cells, which can be obtained by those skilled in the art from professional books in the field.

[0053] A sixth aspect of the present invention provides a method for improving the proliferation, differentiation and / or uptake of exogenous calcium ions by osteoblasts, comprising the step of treating osteoblasts with oyster-derived exosome-like vesicles according to the first aspect of the present invention; or culturing osteoblasts with the culture medium according to the fifth aspect of the present invention.

[0054] The beneficial effects of this invention are:

[0055] This invention discloses for the first time an oyster-derived exosome-like vesicle. This oyster-derived exosome-like vesicle is a nanoscale membrane vesicle with a typical saucer-like morphology and an average particle size of less than 150 nm. It can carry osteogenic mineral elements such as calcium and phosphorus, and can be effectively taken up by osteoblasts. It has the effect of promoting osteoblast proliferation and differentiation, and can improve osteoporosis. It can be used as a beneficial dietary supplement in functional foods related to delaying bone loss.

[0056] This invention requires no chemical reagents during preparation and the preparation method is simple. Oysters are a common aquatic product, and oysters are readily available as raw materials, meeting the needs of large-scale production. Attached Figure Description

[0057] Figure 1 A flowchart of a method for preparing oyster-derived exosome-like vesicles.

[0058] Figure 2 The above is a statistical chart showing the extraction rate of oyster-derived exosome-like vesicles in Examples 1-3.

[0059] Figure 3 Figure showing the particle size analysis results of oyster-derived exosome-like vesicles.

[0060] Figure 4 Transmission electron microscopy image of oyster-derived exosome-like vesicles.

[0061] Figure 5 Figure showing the results of trace mineral element analysis of oyster-derived exosome-like vesicles.

[0062] Figure 6 The graph shows the statistical results of the effect of oyster-derived exosome-like vesicles on osteoblast proliferation. In the graph, * indicates p < 0.05, and ** indicates p < 0.01.

[0063] Figure 7 The figure shows the statistical results of the effect of oyster-derived exosome-like vesicles on osteoblast differentiation ability. In the figure, * indicates p < 0.05 and ** indicates p < 0.01.

[0064] Figure 8 The figures show the effects of oyster-derived exosome-like vesicles on micro-CT regulation of the femur in ovariectomized mice. Figure a shows the effect of oyster-derived exosome-like vesicles on bone mineral density (BMD) in osteoporotic mice; figure b shows the effect of oyster-derived exosome-like vesicles on the bone volume to bone tissue volume ratio (BV / TV) in osteoporotic mice; figure c shows the effect of oyster-derived exosome-like vesicles on bone surface area density (BS / TV) in osteoporotic mice; and figure d shows the effect of oyster-derived exosome-like vesicles on the number of trabeculae (Tb.N) in osteoporotic mice. In the figures, ## indicates p < 0.01 between the normal control group and the osteoporosis model group, and ** indicates p < 0.01 between the osteoporosis model group and the oyster-derived exosome-like vesicle intervention group. Detailed Implementation

[0065] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0066] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0067] Example 1

[0068] A method for preparing oyster-derived exosome-like vesicles includes the following steps:

[0069] (1) Take fresh oysters, transport them to the laboratory on ice, open the shells and take out the oyster meat, cut it into small pieces (2mm×2mm) with scissors, add pre-cooled PBS at a material-to-liquid ratio of 1:8, stir magnetically in an ice-water bath for 10 minutes, and then homogenize at 1000 rpm. Each homogenization time is 3s, with an interval of 30s. Homogenize a total of 3 times to obtain oyster homogenate.

[0070] (2) The oyster homogenate was transferred to a centrifuge tube and subjected to gradient centrifugation. The steps of gradient centrifugation are as follows: the supernatant collected after filtration through a 60-mesh sieve was centrifuged at 300×g and 4℃ for 10 min (first centrifugation), and the supernatant was collected; the supernatant collected after the first centrifugation was centrifuged at 3000×g and 4℃ for 10 min (second centrifugation), and the supernatant was collected; the supernatant collected after the second centrifugation was centrifuged at 10000×g and 4℃ for 20 min (third centrifugation), and the supernatant was collected; the supernatant collected after the third centrifugation was centrifuged at 100000×g and 4℃ for 70 min (fourth centrifugation), and the precipitate was collected; the precipitate collected after the fourth centrifugation was resuspended in PBS, filtered through a 0.45μm filter membrane, and then centrifuged at 100000×g and 4℃ for 70 min to obtain oyster-derived exosome-like vesicles.

[0071] Example 2

[0072] A method for preparing oyster-derived exosome-like vesicles includes the following steps:

[0073] (1) Take fresh oysters, transport them to the laboratory on ice, open the shells and take out the oyster meat, cut it into small pieces (2mm×2mm) with scissors, add PBS containing 20 units of papain at a material-to-liquid ratio of 1:8, digest at 37°C for 30 min; then place it in an ice-water bath and stir magnetically for 10 min, then homogenize at 1000 rpm, each homogenization time is 3 s, the interval time is 30 s, and a total of 3 homogenizations are performed to obtain oyster homogenate;

[0074] (2) The oyster homogenate was transferred to a centrifuge tube and subjected to gradient centrifugation. The steps of gradient centrifugation are as follows: the supernatant collected after filtration through a 60-mesh sieve was centrifuged at 300×g and 4℃ for 10 min (first centrifugation), and the supernatant was collected; the supernatant collected after the first centrifugation was centrifuged at 3000×g and 4℃ for 10 min (second centrifugation), and the supernatant was collected; the supernatant collected after the second centrifugation was centrifuged at 10000×g and 4℃ for 20 min (third centrifugation), and the supernatant was collected; the supernatant collected after the third centrifugation was centrifuged at 100000×g and 4℃ for 70 min (fourth centrifugation), and the precipitate was collected; the precipitate collected after the fourth centrifugation was resuspended in PBS, filtered through a 0.45μm filter membrane, and then centrifuged at 100000×g and 4℃ for 70 min to obtain oyster-derived exosome-like vesicles.

[0075] Example 3

[0076] A method for preparing oyster-derived exosome-like vesicles includes the following steps:

[0077] (1) Take fresh oysters, transport them to the laboratory on ice, open the shells and take out the oyster meat, cut it into small pieces (2mm×2mm) with scissors, add PBS containing 20 units of papain at a material-to-liquid ratio of 1:8, and digest at 37°C for 24 hours; then place it in an ice-water bath and stir magnetically for 10 minutes, and homogenize at 1000 rpm for 3 seconds each time with an interval of 30 seconds, for a total of 3 homogenizations to obtain oyster homogenate;

[0078] (2) The oyster homogenate was transferred to a centrifuge tube and subjected to gradient centrifugation. The steps of gradient centrifugation are as follows: the supernatant collected after filtration through a 60-mesh sieve was centrifuged at 300×g and 4℃ for 10 min (first centrifugation), and the supernatant was collected; the supernatant collected after the first centrifugation was centrifuged at 3000×g and 4℃ for 10 min (second centrifugation), and the supernatant was collected; the supernatant collected after the second centrifugation was centrifuged at 10000×g and 4℃ for 20 min (third centrifugation), and the supernatant was collected; the supernatant collected after the third centrifugation was centrifuged at 100000×g and 4℃ for 70 min (fourth centrifugation), and the precipitate was collected; the precipitate collected after the fourth centrifugation was resuspended in PBS, filtered through a 0.45μm filter membrane, and then centrifuged at 100000×g and 4℃ for 70 min to obtain oyster-derived exosome-like vesicles.

[0079] Example 4

[0080] This embodiment describes the various characterizations of the oyster-derived exosome-like vesicles prepared.

[0081] 1. Extraction rate of oyster-derived exosome-like vesicles

[0082] The concentration of oyster-derived exosome-like vesicles prepared in Examples 1-3 was determined using a BCA reagent kit (BCA Protein Assay, beyotime, P0009). The extraction rate of oyster-derived exosome-like vesicles prepared in Examples 1-3 was calculated according to the following formula: Extraction rate = Oyster exosome-like vesicle protein content (mg) / Oyster meat weight (g).

[0083] The results are as follows Figure 2 As shown, the extraction rates of oyster-derived exosome-like vesicles prepared in Examples 2 and 3 are not significantly different, and both are higher than those in Example 1, indicating that papain digestion can increase the extraction rate of oyster-derived exosome-like vesicles.

[0084] 2. Size of oyster-derived exosome-like vesicles

[0085] The particle size of the oyster-derived exosome-like vesicles prepared in Examples 1-3 was detected by nanoparticle tracking analysis, as follows: The particle concentration of the oyster-derived exosome-like vesicles was diluted to an appropriate concentration, filtered through a 0.22 μm filter membrane, and then injected into the nanoparticle tracking analyzer for detection.

[0086] The results are as follows Figure 3 As shown, there was no significant difference in the average particle size of the oyster-derived exosome-like vesicles prepared in Examples 1-3, indicating that enzymatic digestion does not affect the particle size of the oyster-derived exosome-like vesicles. Therefore, Example 2 was selected as the preferred scheme for subsequent analysis.

[0087] 3. Morphology of oyster-derived exosome-like vesicles

[0088] The morphology of the oyster-derived exosome-like vesicles prepared in Example 2 was observed using transmission electron microscopy. The specific steps included: after fixing the oyster-derived exosome-like vesicles, a copper mesh was placed over the sample drop, and excess liquid was absorbed with filter paper. 10 μL of uranium water was dropped into a round droplet, and a copper mesh was placed over the droplet. After drying at room temperature, the droplet was observed and photographed under a transmission electron microscope.

[0089] The results are as follows Figure 4 As shown, the morphology of oyster-derived exosome-like vesicles is a typical saucer-like shape.

[0090] 4. Determination of trace mineral elements in oyster-derived exosome-like vesicles

[0091] Trace mineral elements in the oyster-derived exosome-like vesicles prepared in Example 2 were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results are as follows: Figure 5 As shown, the oyster-derived exosome-like vesicles prepared in Example 2 carry trace mineral elements such as calcium, phosphorus, copper, and iron, especially calcium and phosphorus.

[0092] Example 5

[0093] This embodiment uses in vitro cell experiments to investigate the effect of oyster-derived exosome-like vesicles on osteoblast proliferation using oyster-derived exosome-like vesicles obtained in Example 2.

[0094] The effect of oyster-derived exosome-like vesicles on osteoblast proliferation was detected using the CCK-8 assay. Specifically, MC3T3-E1 cells at a density of 10,000 cells / well were seeded in 96-well plates and cultured in α-MEM medium containing 10 v / v% FBS at 37°C for 24 h at 5% CO2. The culture medium was then replaced with different concentrations of oyster-derived exosome-like vesicles (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL), with three replicates for each treatment, and cultured at 37°C at 5% CO2 for 48 h. The viability of MC3T3-E1 cells was detected using the CCK-8 assay kit (CCK-8assay, beyotime, C0038) to observe the effect of oyster-derived exosome-like vesicles on osteoblast proliferation.

[0095] The results are as follows Figure 6 As shown, within the concentration range of 5–160 μg / mL of oyster-derived exosome-like vesicles, the viability of MC3T3-E1 cells increased with the increase of oyster-derived exosome-like vesicle concentration, indicating that oyster-derived exosome-like vesicles have a significant promoting effect on cell proliferation. However, when the concentration of oyster-derived exosome-like vesicles reaches 320 μg / mL, the promoting effect on cell proliferation weakens.

[0096] Example 6

[0097] This embodiment uses in vitro cell experiments to investigate the effect of oyster-derived exosome-like vesicles on osteoblast differentiation capacity.

[0098] Osteoblasts (MC3T3-E1 cells) were arranged at an appropriate density (2×10⁻⁶). 4 Oyster-derived exosome-like vesicles (20 μg / mL, 40 μg / mL, and 80 μg / mL) were seeded in 12-well plates and divided into a control group and groups with different concentrations of oyster-derived exosome-like vesicles (20 μg / mL, 40 μg / mL, and 80 μg / mL). Induction was performed using α-MEM medium containing 10 v / v% FBS, 1 w / v% penicillin-streptomycin, 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate sodium. After 7 days of culture, ALP activity was analyzed using an alkaline phosphatase (ALP) kit (ALP Assay, beyotime, P0321S) to evaluate the effect of oyster-derived exosome-like vesicles on osteoblast differentiation capacity.

[0099] The results are as follows Figure 7 As shown, ALP analysis results indicated that the addition of oyster-derived exosome-like vesicles significantly increased ALP activity compared to the control group, and this increase was dose-dependent, suggesting that oyster-derived exosome-like vesicles have a role in promoting osteogenic differentiation.

[0100] Example 7

[0101] This embodiment was used to investigate the regulatory effect of oyster-derived exosome-like vesicles on bone loss in osteoporotic mice.

[0102] Twelve-week-old female C57BL6 mice were selected to establish an osteoporosis model through bilateral ovariectomy. Three different groups were established: a sham group (with periovarian fat removal), an osteoporosis model group (OVX) with ovariectomy, and an intervention group (OVX-OEV) where mice underwent ovariectomy and were administered oyster-derived exosome-like vesicles prepared in Example 2 via gavage. All mice rested for one week after surgery before intervention. The OVX-OEV group received 50 mg / kg body weight of oyster-derived exosome-like vesicles daily via gavage, while the OVX and Sham groups received the same volume of physiological saline. After a total of 6 weeks of gavage, changes in bone three-dimensional structure and bone histomorphometric parameters were analyzed using Micro-CT scanning.

[0103] Figure 8 In Figure 'a', the effect of oyster-derived exosome-like vesicles on bone mineral density (BMD) in osteoporotic mice was shown. The results indicated that the BMD in the OVX-OEV group recovered to the level of the Sham group, with an average of 0.078 g / cm³. 3 The concentration was significantly higher than that in the OVX group (0.036 g / cm³). 3 ; Figure 8 In Figure b, the effect of oyster-derived exosome-like vesicles on the bone volume to bone tissue volume ratio (BV / TV) in osteoporotic mice was shown. The results showed that the BV / TV of the OVX-OEV group recovered to the level of the Sham group, with an average of 12.23%, which was significantly higher than the 2.17% of the OVX group. Figure 8 In the middle, c represents the effect of oyster-derived exosome-like vesicles on bone surface area density (BS / TV) in osteoporotic mice. The results showed that the BS / TV of the OVX-OEV group recovered to the level of the Sham group, with an average of 8.21 1 / mm, which was significantly higher than that of the OVX group (1.82 1 / mm). Figure 8 The middle d represents the effect of oyster-derived exosome-like vesicles on the number of trabeculae (Tb.N) in osteoporotic mice. The results showed that the Tb.N in the OVX-OEV group recovered to the level of the Sham group, with an average of 1.73 1 / mm, which was significantly higher than that in the OVX group (0.36 1 / mm).

[0104] The above results indicate that the oyster-derived exosome-like vesicles prepared using the present invention have a significant preventive and therapeutic effect on osteoporosis in mice.

[0105] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An oyster-derived exosome-like vesicle, wherein the oyster-derived exosome-like vesicle is isolated from oyster meat, the exosome-like vesicle contains osteogenic mineral elements, and the oyster-derived exosome-like vesicle is a nanoscale vesicle with a typical saucer-like structure. The osteogenic mineral elements include calcium and phosphorus; The method for preparing oyster-derived exosome-like vesicles includes the following steps: Oysters were mixed with the extract, homogenized, and then separated into solid and liquid components to obtain oyster-derived exosome-like vesicles. The extraction solution includes phosphate buffer and papain; The mass-to-volume ratio of the oyster to the extract is 1:(1~20). The oysters were mixed with the extract and digested at 35-40°C for 0.5-24 hours. The solid-liquid separation conditions are as follows: centrifuge at 300×g for 10–20 min at 1–4℃ and collect the supernatant; centrifuge at 3000×g at 1–4℃ for 10–20 min and collect the supernatant; centrifuge at 10000×g at 1–4℃ for 15–30 min and collect the supernatant; centrifuge at 100000×g at 1–4℃ for 70–120 min and collect the precipitate. The precipitate collected by solid-liquid separation was resuspended in a buffer solution, filtered through a filter membrane, and then centrifuged at 100,000 × g and 1–4 °C for 70–120 min.

2. The use of the oyster-derived exosome-like vesicles according to claim 1 in any one of (1) to (9): (1) Preparation of drugs for the treatment and / or adjuvant treatment of osteoporosis; (2) To prepare products that delay bone loss; (3) To develop products that reduce the risk of osteoporosis; (4) Prepare products that improve bone density; (5) Prepare products that supplement osteogenic mineral elements; (6) Enhancing osteoblast proliferation capacity in vitro for non-disease treatment purposes; (7) Prepare products that enhance the proliferation capacity of osteoblasts; (8) Enhancing osteoblast differentiation capacity for non-disease treatment purposes in vitro; (9) Prepare products that enhance osteoblast differentiation ability.

3. A product comprising the oyster-derived exosome-like vesicles of claim 1.

4. The product according to claim 3, characterized in that, The concentration of oyster-derived exosome-like vesicles in the product is 5~500 μg / mL.

5. A culture medium comprising the oyster-derived exosome-like vesicles of claim 1.

6. The culture medium according to claim 5, characterized in that, The concentration of the oyster-derived exosome-like vesicles in the culture medium is 5~500 μg / mL.

7. A method for enhancing the proliferation and / or differentiation capacity of osteoblasts for non-disease treatment purposes in vitro, comprising the step of treating osteoblasts with oyster-derived exosome-like vesicles as described in claim 1; or culturing osteoblasts with the culture medium as described in claim 5 or 6.

Images