Application of extracellular vesicles derived from Sipunculus truncatula in the preparation of anti-osteoporosis drugs

Abstract

The present invention relates to the field of biomedicine technology, and in particular to the use of exosomes derived from Siegesprung's worm in the preparation of anti-osteoporosis drugs. The present invention adopts an ultra-high-speed centrifugation method to obtain exosomes derived from Siegesprung's worm, and experiments prove that the exosomes derived from Siegesprung's worm have an excellent effect of promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce the up-regulation of marker proteins of osteogenic differentiation, can be used as an inducer of osteogenic differentiation of bone marrow mesenchymal stem cells, and can be used in the preparation of anti-osteoporosis drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of exosomes derived from Sipunculus quadrilaterals in the preparation of anti-osteoporosis drugs. Background Art

[0002] Osteoporosis is a common bone disease with clinical manifestations of decreased bone density, thinning of bone tissue and abnormal microstructure, which makes bones brittle and fragile; this disease makes bones prone to fractures, especially in the wrist, hip and spine. Common causes of osteoporosis include aging, malnutrition, lack of exercise, genetic factors, endocrine disorders (such as menopause), long-term use of certain drugs (such as hormones), smoking and excessive drinking. At present, there is no effective intervention and prevention method for this disease in clinical practice.

[0003] Extracellular vesicles are lipid membrane vesicles of different sizes that are secreted by cells outside the cell. These vesicles are able to transport various molecules from the producing cells to the target cells. The three main subtypes of extracellular vesicles are microvesicles, exosomes, and apoptotic bodies. Among the different types of extracellular vesicles, extracellular vesicles are nano-sized vesicles that exist in different organisms. More and more evidence shows that extracellular vesicles play an important role in information exchange between cells, affecting physiological and pathological processes, and have been widely studied as nanotherapeutics, drug delivery carriers, and biomarkers. At present, people mainly focus on the study of extracellular vesicles from mammals and plants, while the composition and function of extracellular vesicles from nematodes have not been reported.

[0004] Siegeworms, also known as sandworms, belong to the phylum Siegeworms and genus Siegeworms. They are non-segmented, worm-like marine coeloms and are warm-water animals. Many Chinese medical books have recorded their edible and medicinal values. In some areas, they are used to replace the traditional Chinese medicine Cordyceps sinensis to treat bone hot flashes, night sweats due to yin deficiency, chest tightness, cough due to tuberculosis, excessive sputum, and tooth swelling and pain. However, there are no reports on the use of Siegeworm-derived exocysts for the prevention and treatment of osteoporosis. Summary of the invention

[0005] The technical problem solved by the invention is to provide application of the exovesicles derived from Sipunculus quadripodius in the preparation of anti-osteoporosis drugs.

[0006] In order to solve the above technical problems, the present invention provides the use of extracellular vesicles derived from Sipunculus quadrilaterals in the preparation of anti-osteoporosis drugs; and the use of the anti-osteoporosis drug as an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0007] Optionally, the method for preparing the exovesicles derived from Sipuncula includes:

[0008] Step S1, obtaining the coelomic fluid of Silurus spp., mixing the coelomic fluid of Silurus spp. with a PBS buffer, and filtering the mixture to obtain a mixed solution;

[0009] Step S2, subjecting the mixed solution to a first centrifugation treatment, extracting a supernatant, and filtering the mixture to obtain a first supernatant;

[0010] Step S3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a bacterial sieve to obtain a second supernatant;

[0011] Step S4, subjecting the second supernatant to a third centrifugation treatment, and the resulting precipitate is the exovesicles derived from Sipunculus quadrilateral.

[0012] Optionally, in step S1, the volume ratio of the Siempuri coelomic fluid to the PBS buffer is 1:(2-5).

[0013] Optionally, in step S2, the first centrifugal treatment is performed at a rotation speed of 2000-3000 g and for a time of 20-30 min.

[0014] Optionally, in step S3, the rotation speed of the second centrifugation is 9500-10500g and the time is 30-40min.

[0015] Optionally, in step S4, the rotation speed of the third centrifugation is 95000-105000g, and the time is 60-70min.

[0016] Optionally, in step S1, the filtration process is performed using a 70-mesh filter.

[0017] Optionally, in step S3, the pore size of the bacteria sieve is 0.45 μm.

[0018] Optionally, in step S2, the filtration treatment includes: filtering the supernatant through sieves with pore sizes of 70 μm and 40 μm in sequence.

[0019] Optionally, the particle size of the extracellular vesicles derived from Sipuncula quadrilateral is 129 nm.

[0020] Compared with the prior art, the present invention adopts ultrahigh-speed centrifugation to obtain extracellular vesicles derived from Siegesporium, and experiments have proved that the extracellular vesicles derived from Siegesporium have excellent effects in promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce the up-regulation of marker proteins of osteogenic differentiation. They can be used as inducers of osteogenic differentiation of bone marrow mesenchymal stem cells and can be used in the preparation of anti-osteoporosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a transmission electron microscope image of the exovesicles derived from Sipunculus in Example 1 of the present invention;

[0022] Figure 2 This is a particle size distribution diagram of the exovesicles derived from Sipuncula in Example 1 of the present invention;

[0023] Figure 3 This is a result diagram showing the effect of different concentrations of Sipunculus sphenotype-derived exosome solutions on the activity of hBMSCs in Example 3;

[0024] Figure 4 The alkaline phosphatase staining light microscopy images of hBMSCs osteogenic differentiation induced by the extracellular vesicle solutions derived from Sipunculus truncatula at different concentrations in Example 4;

[0025] Figure 5 These are light microscopic images of alizarin red staining of hBMSCs induced by exosome solutions derived from Sipunculus spp. at different concentrations in Example 4;

[0026] Figure 6 This is the gel electrophoresis diagram of the marker proteins of osteogenic differentiation of hBMSCs induced by extracellular vesicle solutions from Sipunculus truncatula at different concentrations in Example 5. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0028] It should be noted that, in the absence of conflict, the features in the embodiments of the present invention can be combined with each other. The meanings of the terms "comprising", "including", "containing", and "having" are non-restrictive, that is, other steps and other ingredients that do not affect the results can be added. The above terms cover the terms "consisting of" and "essentially consisting of". Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0029] The embodiment of the present invention provides the use of extracellular vesicles derived from Sipunculus quadratus in the preparation of an anti-osteoporosis drug; and the use of the anti-osteoporosis drug as an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0030] The embodiment of the present invention adopts ultrahigh-speed centrifugation to obtain extracellular vesicles derived from Siegesporium, and experiments have proved that the extracellular vesicles derived from Siegesporium have excellent effects on promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce the up-regulation of marker proteins of osteogenic differentiation. They can be used as inducers of osteogenic differentiation of bone marrow mesenchymal stem cells and can be used in the preparation of anti-osteoporosis drugs.

[0031] In some embodiments of the present invention, the method for preparing the exovesicles derived from Sipunculus truncatula comprises:

[0032] Step S1, obtaining the coelomic fluid of Silurus spp., mixing the coelomic fluid of Silurus spp. with a PBS buffer, and filtering the mixture to obtain a mixed solution;

[0033] Step S2, subjecting the mixed solution to a first centrifugation treatment, extracting a supernatant, and filtering the mixture to obtain a first supernatant;

[0034] Step S3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a bacterial sieve to obtain a second supernatant;

[0035] Step S4, subjecting the second supernatant to a third centrifugation treatment, and the resulting precipitate is the exovesicles derived from Sipunculus quadrilateral.

[0036] In some embodiments of the present invention, in step S1, the volume ratio of the Siempuri coelomic fluid to the PBS buffer is 1:(2-5).

[0037] In some embodiments of the present invention, in step S2, the first centrifugal treatment is performed at a speed of 2000-3000 g and for a time of 20-30 min.

[0038] In some embodiments of the present invention, in step S3, the second centrifugation is performed at a speed of 9500-10500 g and for a time of 30-40 min.

[0039] In some embodiments of the present invention, in step S4, the rotation speed of the third centrifugation is 95000-105000g, and the time is 60-70min.

[0040] In some embodiments of the present invention, in the step S1, the filtration process is performed using a 70-mesh filter.

[0041] In some embodiments of the present invention, in step S3, the pore size of the bacteria sieve is 0.45 μm.

[0042] In some embodiments of the present invention, in step S2, the filtration treatment includes: filtering the supernatant through sieves with pore sizes of 70 μm and 40 μm in sequence.

[0043] In some embodiments of the present invention, the particle size of the extracellular vesicles derived from Sipunculus quadrilateral is 129 nm.

[0044] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The human bone marrow mesenchymal stem cells (hBMSCs) used in the examples of the present invention were purchased from Saiye (Guangzhou) Biotechnology Co., Ltd., and the product name is OriCell ®Adult bone marrow mesenchymal stem cells, model number HUXMA-01001, and the PBS buffer used in the embodiments of the present invention were purchased from Wuhan Pronocell Life Science Co., Ltd., model number PB180327. Figure 3 In the table, * is used to indicate the significant difference between different treatments, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0045] Example 1: Extraction of extracellular vesicles from Sipunculus

[0046] 1.1. Take fresh Sipuncula spp. to obtain the Sipuncula spp. coelomic fluid, mix the Sipuncula spp. coelomic fluid with PBS buffer, and filter the mixture with a 70-mesh filter to obtain a mixed solution; wherein the volume ratio of the Sipuncula spp. coelomic fluid to the PBS buffer is 1:2.5.

[0047] 1.2. The mixed solution is centrifuged for the first time, the supernatant is extracted, and filtered through sieves with pore sizes of 70 μm and 40 μm in turn to obtain a first supernatant; wherein the first centrifugation is performed at a speed of 2500 g and for 25 min.

[0048] 1.3. The first supernatant is centrifuged for a second time, the supernatant is extracted, and filtered using a bacterial sieve with a pore size of 0.45 μm to obtain a second supernatant; the second centrifugation is performed at a speed of 10,000 g and a time of 35 min.

[0049] 1.4. The second supernatant is centrifuged for a third time to obtain a precipitate, which is the exovesicles derived from Silurus truncatus. The precipitate is resuspended in PBS buffer to obtain an exovesicle suspension derived from Silurus truncatus. The third centrifugation is performed at 100,000 g for 70 min.

[0050] The extracellular vesicles derived from Sipunculus were identified by transmission electron microscopy. Figure 1 Transmission electron microscopy image of an extracellular vesicle from Sipunculus spp. Figure 1 It can be seen that the morphology of the exovesicles from Siegesporium is a typical membrane "cup-and-disc" structure. The particle size of the exovesicles from Siegesporium was measured using a nanoparticle tracking analyzer. Figure 2 This is the particle size distribution diagram of the extracellular vesicles from Sipunculus. Figure 2 It can be seen that the particle size of the extracellular vesicles derived from Sipuncula is concentrated around 129nm.

[0051] Example 2: Experimental study on the concentration of extracellular vesicle proteins from Sipunculus truncatula

[0052] The obtained extracellular vesicles from Sipunculus were tested using a BCA protein assay kit. The specific steps are as follows:

[0053] (1) Dilute the protein standard with sterile PBS buffer to prepare protein standards with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively; take 20 μL of each concentration of protein standard and add it to the standard wells of a 96-well plate to prepare a standard curve;

[0054] (2) Dissolving the exovesicles derived from Silurus punctatus obtained in Example 1 in PBS buffer to obtain an exovesicle solution derived from Silurus punctatus; taking 1 μL of the exovesicle solution derived from Silurus punctatus, diluting it to 20 μL with PBS buffer, and adding it to the sample wells of a 96-well plate;

[0055] (3) Add 200 μL of BCA working solution to the standard wells and sample wells of the 96-well plate and place at 37°C for 30 min;

[0056] (4) Determine the A562 value using an ELISA reader;

[0057] (5) Calculate the protein concentration of the extracellular vesicle solution derived from Silurian worms based on the standard curve.

[0058] After measurement, the protein concentration in the extracellular vesicle solution derived from Sipunculus obtained in Example 2 was 3 mg / mL.

[0059] Example 3: Experiment on the effect of extracellular vesicles from Sipunculus truncatula on the activity of human bone marrow mesenchymal stem cells (hBMSCs)

[0060] The specific contents are as follows:

[0061] (1) adding the exovesicles derived from Siegespermum prepared in Example 1 to an α-MEM complete medium to obtain a first exovesicle solution and a second exovesicle solution, wherein the concentration of the exovesicles derived from Siegespermum in the first exovesicle solution is 30 μg / ml, and the concentration of the exovesicles derived from Siegespermum in the second exovesicle solution is 60 μg / ml; and the α-MEM complete medium is used as a blank control solution;

[0062] (2) P5 human bone marrow mesenchymal stem cells (hBMSCs) were inoculated and cultured in 96-well plates at a density of 5×10 3 / well; after the cells adhered to the wall, the first exosome solution, the second exosome solution and the blank control solution obtained in step (1) were incubated for 24 hours and 48 hours respectively; CCK-8 reagent was added to each well at a ratio of 10:1, incubated at 37°C for 2 hours, and the absorbance at 452nm was detected using a UV spectrophotometer. The results are shown in Figure 3 .

[0063] Figure 3 The results of the effects of different concentrations of extracellular vesicle solutions from Sipuncula on the activity of hBMSCs are shown in Figure 2. Figure 3 0 μg / ml, 30 μg / ml and 60 μg / ml correspond to the experimental results of the blank control solution, the first exosome solution and the second exosome solution, respectively, and 24h and 48h correspond to the experimental results of the incubation treatment for 24h and 48h, respectively. Figure 3 It can be seen that after incubation with extracellular vesicles derived from Siempre crusoeii for 24h and 48h, the proliferation activity of human bone marrow mesenchymal stem cells (hBMSCs) was upregulated, indicating that extracellular vesicles derived from Siempre crusoeii are not toxic to human bone marrow mesenchymal stem cells (hBMSCs) and can promote the proliferation activity of human bone marrow mesenchymal stem cells (hBMSCs).

[0064] Example 4: Experiment on promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) by extracellular vesicles derived from Sipunculus

[0065] The specific contents are as follows:

[0066] (1) The osteogenic medium is an α-MEN complete medium containing dexamethasone, ascorbic acid and β-glycerophosphate, wherein the concentration of dexamethasone is 100 nmol / L, the concentration of ascorbic acid is 50 nmol / L, and the concentration of β-glycerophosphate is 10 nmol / L. The exovesicles derived from Silurus prepared in Example 1 are added to the osteogenic medium to obtain a first exovesicle solution derived from Silurus and a second exovesicle solution derived from Silurus, wherein the concentration of the exovesicles derived from Silurus in the first exovesicle solution derived from Silurus is 30 μg / ml, and the concentration of the exovesicles derived from Silurus in the second exovesicle solution derived from Silurus is 60 μg / ml. The osteogenic medium is used as a blank control solution.

[0067] (2) P5 human bone marrow mesenchymal stem cells (hBMSCs) were inoculated and cultured in 12-well plates at a density of 5×10 5 / well; after the cells adhered to the wall, the first sichuan worm-derived extracellular vesicle solution, the second sichuan worm-derived extracellular vesicle solution and the blank control solution obtained in step (1) were used as culture medium, and the culture medium was replaced every 3 days. After 3 days of osteogenic differentiation, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 15 minutes. Then, alkaline phosphatase staining solution was added to each well for staining, and incubated at room temperature for 60 minutes. The non-specifically bound alkaline phosphatase staining solution was washed away with PBS buffer. The image after staining is shown in FIG. Figure 4 .

[0068] (3) P5 human bone marrow mesenchymal stem cells (hBMSCs) were inoculated and cultured in 12-well plates at a density of 5×10 5 / well; after the cells adhered to the wall, the first Siegesporium-derived extracellular vesicle solution, the second Siegesporium-derived extracellular vesicle solution and the blank control solution obtained in step (1) were used as culture medium for culture, and the culture medium was replaced every 3 days. After 10 days of osteogenic differentiation culture, the culture medium was removed, and the cells were fixed with 75% ethanol for 15 minutes. Then, Alizarin Red S staining solution was added to each well for staining, and incubated at room temperature for 20 minutes. PBS buffer was used to remove non-specifically bound Alizarin Red S staining solution. The image after staining is shown in FIG. Figure 5 .

[0069] Figure 4 The alkaline phosphatase staining light microscopy images of hBMSCs osteogenic differentiation induced by extracellular vesicle solutions from Sipunculus truncatula at different concentrations. Figure 5 Light microscopic images of alizarin red staining showing osteogenic differentiation of hBMSCs induced by extracellular vesicle solutions from Siegesporium at different concentrations. Figure 4 and Figure 5 0 μg / ml, 30 μg / ml and 60 μg / ml correspond to the experimental results of the blank control solution, the first Siegesporium-derived exosome solution and the second Siegesporium-derived exosome solution, respectively. Figure 4 and Figure 5 It can be seen that the degree of staining of cells treated with the blank control solution, the first Siegesporium-derived exosome solution, and the second Siegesporium-derived exosome solution deepened in turn, indicating that the exosomes derived from Siegesporium can significantly promote the osteogenic differentiation of human bone marrow mesenchymal stem cells, and human bone marrow mesenchymal stem cells have been successfully differentiated into osteoblasts.

[0070] Example 5: Effect of extracellular vesicles from Sipunculus truncatula on osteogenic marker proteins of human bone marrow mesenchymal stem cells (hBMSCs)

[0071] The induction effect was detected by Western blot. The specific contents are as follows:

[0072] (1) adding the exovesicles derived from Silurus punctatus prepared in Example 1 to an α-MEM complete medium to obtain a third exovesicle solution derived from Silurus punctatus and a fourth exovesicle solution derived from Silurus punctatus, wherein the concentration of the exovesicles derived from Silurus punctatus in the third exovesicle solution derived from Silurus punctatus is 30 μg / ml, and the concentration of the exovesicles derived from Silurus punctatus in the fourth exovesicle solution derived from Silurus punctatus is 60 μg / ml; and the α-MEM complete medium is used as a blank control solution;

[0073] (2) P5 human bone marrow mesenchymal stem cells (hBMSCs) were seeded in a 60 mm cell culture dish at a seeding density of 5 × 10 5 / well; after the cell density grows to 80%, the three solutions obtained in step (1) (the third siphonophore-derived extracellular vesicle solution, the fourth siphonophore-derived extracellular vesicle solution and the blank control solution) are used as culture medium for culture respectively, the cells are lysed in RIPA lysis buffer for 30 minutes, centrifuged at 12000g for 10 minutes, the total cell protein concentration of the obtained supernatant is determined by BCA protein assay kit, the protein loading buffer is added for balancing, and then boiled in 100℃ boiling water for 10 minutes; 15μg protein is extracted from each group corresponding to the above three solutions, added to 10% SDS-PAGE gel, 60 V constant voltage electrophoresis separation for 2h; the separated proteins in each group were transferred to the PVDF membrane, and the PVDF membrane in each group was divided into 4 test membranes, and the test membranes were blocked in 5% skim milk for 1h, and then the 4 test membranes in each group were placed in specific primary antibodies such as RUNX2 (1:1000), ALP (1:1000), OPN (1:1000) and β-actin (1:1000) and incubated overnight at 4°C; after recovering the primary antibody, the test membrane was washed with TBST for 30min, and the test membrane was incubated with goat anti-rabbit secondary antibody (1:5000) at room temperature for 1h, and then rinsed with TBST again for 1h. Use fast chemiluminescent solution to evenly cover the test membrane, leave it for 2min, then take out the test membrane and place it in the chemiluminescent imager for detection, the results are shown in Figure 6 .

[0074] Figure 6 Gel electrophoresis of marker proteins of osteogenic differentiation of hBMSCs induced by extracellular vesicle solutions derived from Siegesporium at different concentrations. Figure 6 0 μg / ml, 30 μg / ml and 60 μg / ml correspond to the experimental results of the blank control solution, the third siphonophore-derived exosome solution and the fourth siphonophore-derived exosome solution, respectively. Figure 6 It can be seen that the expression of osteogenic marker proteins such as ALP, RUNX2, and OPN increased significantly, indicating that the extracellular vesicles derived from Siemprenidae can significantly induce the up-regulated expression of marker proteins of osteogenic differentiation, and can significantly promote the osteogenic differentiation of human bone marrow mesenchymal stem cells in a dose-dependent manner.

[0075] In addition, it should be noted that, although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. Application of extracellular vesicles derived from Sipunculus truncatula in the preparation of inducers for osteogenic differentiation of bone marrow mesenchymal stem cells; in, The method for preparing the exovesicles derived from Sipuncula includes: Step S1, obtaining the coelomic fluid of the Siegesner worm, mixing the coelomic fluid of the Siegesner worm with a PBS buffer, and filtering to obtain a mixed solution; the volume ratio of the coelomic fluid of the Siegesner worm to the PBS buffer is 1:2.5; Step S2, subjecting the mixed solution to a first centrifugal treatment, extracting the supernatant, and filtering to obtain a first supernatant; the first centrifugal treatment is performed at a speed of 2500 g and a time of 25 min; Step S3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a bacterial sieve to obtain a second supernatant; the second centrifugation treatment is performed at a speed of 10000g and a time of 35min; Step S4, subjecting the second supernatant to a third centrifugation treatment, and the resulting precipitate is the exovesicles derived from Sipunculus quadrilateral; the third centrifugation treatment is performed at a speed of 100000 g for 70 min; In the step S1, the filtering process is performed using a 70-mesh filter; In the step S2, the filtration treatment includes: filtering the supernatant through sieves with pore sizes of 70 μm and 40 μm in sequence; In step S3, the pore size of the bacteria sieve is 0.45 μm; The particle size of the extracellular vesicles derived from Sipunculus quadrilaterals is 129 nm.

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