Application of outer vesicles derived from Rosella in preparation of inducing agent for osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

The invention provides application of outer vesicles derived from Rosella in preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells, and relates to the technical field of biomedicine.The outer vesicles derived from Rosella are obtained through an ultra-high-speed centrifugation method, and experiments prove that the outer vesicles derived from Rosella have the advantages that the inducer can be used for inducing osteogenic differentiation of bone marrow mesenchymal stem cells; the outer vesicles derived from Rosella have an excellent effect of promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), can significantly induce up-regulation expression of marker protein of osteogenic differentiation, can be used as an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells, and can be applied to 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 Roseburia in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells. Background Art

[0002] Osteoporosis (OP) is a systemic metabolic disease. Its clinical pathological characteristics include increased bone fragility and increased probability of fractures. Its microscopic manifestations include a decrease in trabeculae and destruction of bone microstructure. Currently, it is believed that the imbalance of bone metabolism is the main cause of the disease. Osteoporosis can occur in different genders and at any age, but it is more common in postmenopausal women and the elderly. Currently, there is no effective intervention and prevention method for the 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, exosomes are nano-sized vesicles (30nm to 150nm) 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 studying extracellular vesicles from mammals and plants, while bacterial extracellular vesicles and their composition and function are rarely reported.

[0004] Roseburia has received extensive attention in recent years. It is part of the commensal bacteria and belongs to the genus Roseburia of the family Lachnospiraceae, class Clostridiales, order Firmicutes. It is a Gram-positive anaerobic bacterium. Currently, Roseburia includes 5 species (Roseburia intestinalis, R. hominis, R. inulinivorans, R. faecis, and R. cecicola), accounting for 2-31% of the total number of healthy intestinal bacteria. It produces short-chain fatty acids, especially butyrate, which affects colon motility. It has been reported that Roseburia can improve intestinal biodiversity and improve glucose tolerance. The imbalance (too little) of Roseburia may affect multiple metabolic pathways, including irritable bowel syndrome, obesity, type 2 diabetes, and neurological diseases. However, there are no reports on the application of Roseburia-derived exosomes in the treatment of osteoporosis. Summary of the invention

[0005] The technical problem solved by the present invention is to provide application of exosomes derived from Roseburia in preparing anti-osteoporosis drugs.

[0006] In order to solve the above technical problems, the present invention provides the use of exosomes derived from Roseburia in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0007] Optionally, the method for preparing the exosomes derived from Roseburia comprises: Step S1, subculture and activate Roseburia and add it to the culture medium, culture it with shaking in a closed state, perform a first centrifugation, extract the supernatant, and filter it using a first bacterial sieve to obtain a first supernatant; Step S2, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a second bacterial sieve to obtain a second supernatant; Step S3, subjecting the second supernatant to a third centrifugation treatment, extracting the supernatant, and filtering it using a third bacterial sieve to obtain a third supernatant; Step S4, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant; Step S5, subjecting the fourth supernatant to a fifth centrifugation treatment, removing the supernatant, and obtaining a precipitate, which is the exosome derived from the genus Roseburia.

[0008] Optionally, the Roseburia genus is Roseburia intestinalis.

[0009] Optionally, in step S1, the rotation speed of the first centrifugation is 1500g to 2000g, and the time is 20min to 30min.

[0010] Optionally, in step S2, the rotation speed of the second centrifugation is 9500g to 10500g, and the time is 20min to 30min.

[0011] Optionally, in step S3, the rotation speed of the third centrifugation is 9500g to 10500g, and the time is 30min to 40min.

[0012] Optionally, in step S4, the rotation speed of the fourth centrifugation treatment is 9500g to 10500g, and the time is 30min to 40min.

[0013] Optionally, in step S5, the rotation speed of the fifth centrifugation is 145000 g to 155000 g, and the time is 90 min to 120 min.

[0014] Optionally, the pore size of the second bacteria sieve is 0.45 μm.

[0015] Optionally, the pore size of the third bacteria sieve is 0.22 μm.

[0016] Compared with the related art, the present invention adopts ultrahigh-speed centrifugation to obtain exosomes derived from Roseburia, and experiments have proved that the exosomes derived from Roseburia have excellent effects in promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce the up-regulation of the expression 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

[0017] Figure 1 This is a transmission electron microscopy image of the exosomes derived from Roseburia in Example 1 of the present invention; Figure 2 This is a particle size distribution diagram of the exosomes derived from Roseburia in Example 1 of the present invention; Figure 3 This is a result diagram showing the effect of different concentrations of Roseburia-derived exosome solutions on the activity of hBMSCs in Example 3; Figure 4 It is an alkaline phosphatase staining light microscopic picture of hBMSCs osteogenic differentiation induced by exosome solutions derived from Roseburia of different concentrations in Example 4; Figure 5 These are light microscopic images of alizarin red staining of hBMSCs induced by exosome solutions derived from Roseburia at different concentrations in Example 4; Figure 6 This is the gel electrophoresis diagram of the marker proteins of hBMSCs osteogenic differentiation induced by exosome solutions derived from Roseburia at different concentrations in Example 5. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] The embodiment of the present invention provides the use of exosomes derived from Roseburia in preparing an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0021] The embodiment of the present invention adopts ultrahigh-speed centrifugation to obtain exosomes derived from Roseburia, and experiments have proved that the exosomes derived from Roseburia have excellent effects on promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce up-regulation of osteogenic differentiation marker proteins, 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.

[0022] In some embodiments of the present invention, the method for preparing the exosomes derived from Roseburia comprises: Step S1, subculture and activate Roseburia and add it to the culture medium, culture it with shaking in a closed state, perform a first centrifugation, extract the supernatant, and filter it using a first bacterial sieve to obtain a first supernatant; Step S2, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a second bacterial sieve to obtain a second supernatant; Step S3, subjecting the second supernatant to a third centrifugation treatment, extracting the supernatant, and filtering it using a third bacterial sieve to obtain a third supernatant; Step S4, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant; Step S5, subjecting the fourth supernatant to a fifth centrifugation treatment, removing the supernatant, and obtaining a precipitate, which is the exosome derived from the genus Roseburia.

[0023] In some embodiments of the present invention, illustratively, the Roseburia species is Roseburia intestinalis.

[0024] In some embodiments of the present invention, in step S1, the rotation speed of the first centrifugation is 1500 g to 2000 g, and the time is 20 min to 30 min; and the pore size of the first bacterial sieve is 0.45 μm.

[0025] In some embodiments of the present invention, in step S2, the rotation speed of the second centrifugation is 9500 g to 10500 g, the time is 20 min to 30 min, and the pore size of the second bacteria sieve is 0.45 μm.

[0026] In some embodiments of the present invention, in step S3, the rotation speed of the third centrifugation is 9500 g to 10500 g, and the time is 30 min to 40 min; the pore size of the third bacteria sieve is 0.22 μm.

[0027] In some embodiments of the present invention, in step S4, the rotation speed of the fourth centrifugation is 9500 g to 10500 g, and the time is 30 min to 40 min.

[0028] In some embodiments of the present invention, in step S5, the rotation speed of the fifth centrifugation is 145000 g to 155000 g, and the time is 90 min to 120 min.

[0029] 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. Roseburia intestinalis used was purchased from Ningbo Testo Biotechnology Co., Ltd., product name Roseburia intestinalis-DSM 14610, item number TS253874. Figure 3 In the table, ns means no significant difference.

[0030] Example 1: Extraction of exosomes from Roseburia 1.1. After subculture and activation of Roseburia intestinalis, add it to the culture medium, shake and culture it in a closed state, perform a first centrifugation, extract the supernatant, and filter it using a first bacterial sieve to obtain a first supernatant; wherein the speed of the first centrifugation is 2000g, and the time is 25min; the pore size of the first bacterial sieve is 0.45μm; the components of the culture medium include: 10 parts of tryptic peptone, 10 parts of beef extract powder, 3 parts of yeast extract powder, 5 parts of glucose, 5 parts of sodium chloride, 1 part of soluble starch, 0.5 parts of L-cysteine ​​hydrochloride, 3 parts of sodium acetate, 0.001 parts of resazurin, and 1000 parts of deionized water.

[0031] 1.2. The first supernatant is subjected to a second centrifugation treatment, the supernatant is extracted, and a second bacterial sieve is used for filtering treatment to obtain a second supernatant; wherein the second centrifugation treatment is performed at a speed of 10000 g for 25 min; and the pore size of the second bacterial sieve is 0.45 μm; 1.3. The second supernatant is subjected to a third centrifugation treatment, the supernatant is extracted, and a third bacterial sieve is used for filtering treatment to obtain a third supernatant; the third centrifugation treatment is carried out at a speed of 10000 g and a time of 35 min; the pore size of the third bacterial sieve is 0.22 μm; 1.4. The third supernatant is subjected to a fourth centrifugal treatment, and the supernatant is extracted to obtain a fourth supernatant; the fourth centrifugal treatment is performed at a speed of 10000 g and a time of 35 min; 1.5. The fourth supernatant is subjected to a fifth centrifugation treatment, and the supernatant is removed. The resulting precipitate is the exosomes derived from the genus Roseburia. The fifth centrifugation treatment is performed at a speed of 150,000 g for 105 min.

[0032] The exosomes derived from Roseburia were identified by transmission electron microscopy. Figure 1 Transmission electron microscopy images of exosomes from Roseburia spp. Figure 1 It can be seen that the exosomes derived from Roseburia have a typical membrane-like "cup-and-disc" structure. The particle size of the exosomes derived from Roseburia was measured using a nanoparticle tracking analyzer. Figure 2 Figure 2 shows the particle size distribution of exosomes derived from Roseburia spp. Figure 2 It can be seen that the particle size of the exosomes derived from Roseburia is concentrated around 110 nm.

[0033] Example 2: Experimental study on the determination of exosome protein concentration from Roseburia The obtained Roseburia exosomes were tested using a BCA protein assay kit. The specific steps are as follows: (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; (2) Dissolving the exosomes derived from the genus Roseburia prepared in Example 1 in a sterile PBS buffer to obtain an exosome solution derived from the genus Roseburia; taking 1 μL of the exosome solution derived from the genus Roseburia, diluting it to 20 μL with PBS buffer, and adding it to the sample wells of a 96-well plate; (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; (4) Determine the A562 value using an ELISA reader; (5) Calculate the protein concentration of the extracellular vesicle solution derived from Roseburia based on the standard curve.

[0034] After measurement, the protein concentration of the Roseburia-derived exosome solution obtained in Example 2 was 1 mg / mL.

[0035] Example 3: Experiment on the effect of exosomes derived from Roseburia on the activity of human bone marrow mesenchymal stem cells (hBMSCs) The specific contents are as follows: (1) adding the exovesicles derived from the genus Roseburia 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 the genus Roseburia in the first exovesicle solution is 1 μg / ml, and the concentration of the exovesicles derived from the genus Roseburia in the second exovesicle solution is 10 μg / ml; and the α-MEM complete medium is used as a blank control solution; (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 72 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 450nm was detected using a UV spectrophotometer. The results are shown in Figure 3 .

[0036] Figure 3 The results of the effects of different concentrations of Roseburia exosome solutions on the activity of hBMSCs are shown in FIG. Figure 3 0 μg / ml, 1 μg / ml and 10 μg / ml correspond to the experimental results of the blank control solution, the first extracellular vesicle solution and the second extracellular vesicle solution, respectively. Figure 3 It can be seen that after incubation with the exosomes derived from Roseburia for 72 hours, the proliferation activity of human bone marrow mesenchymal stem cells (hBMSCs) was not significantly affected, indicating that the exosomes derived from Roseburia of the present invention have no toxicity to human bone marrow mesenchymal stem cells (hBMSCs).

[0037] Example 4: Experiment on the promotion of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) by exosomes derived from Roseburia The specific contents are as follows: (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 the genus Roseburia prepared in Example 1 are added to the osteogenic medium to obtain a first exovesicle solution derived from the genus Roseburia and a second exovesicle solution derived from the genus Roseburia, wherein the concentration of the exovesicles derived from the genus Roseburia in the first exovesicle solution derived from the genus Roseburia is 1 μg / ml, and the concentration of the exovesicles derived from the genus Roseburia in the second exovesicle solution derived from the genus Roseburia is 10 μg / ml. The osteogenic medium is used as a blank control solution.

[0038] (2) P5 human bone marrow mesenchymal stem cells (hBMSCs) were inoculated and cultured in 12-well plates at a density of 5×105 / well; after the cells adhered to the wall, the first Roseburia exosome solution, the second Roseburia exosome solution and the blank control solution obtained in step (1) were used as culture medium, and the culture medium was changed 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 .

[0039] (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 Roseburia exosome solution, the second Roseburia exosome 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, 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 .

[0040] Figure 4 The alkaline phosphatase staining light microscopy images of hBMSCs osteogenic differentiation induced by exosome solutions derived from Roseburia at different concentrations. Figure 5 Light microscopic images of alizarin red staining of hBMSCs induced by exosome solutions from Roseburia at different concentrations. Figure 4 and Figure 5 0 μg / ml, 1 μg / ml and 10 μg / ml correspond to the experimental results of the blank control solution, the first Roseburia-derived exosome solution and the second Roseburia-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 cells treated with the first Roseburia-derived exosome solution, and the cells treated with the second Roseburia-derived exosome solution deepened in turn, indicating that Roseburia-derived exosomes 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.

[0041] Example 5: Effect of Roseburia-derived exosomes on osteogenic marker proteins of human bone marrow mesenchymal stem cells (hBMSCs) The induction effect was detected by Western blot. The specific contents are as follows: (1) adding the exovesicles derived from the genus Roseburia prepared in Example 1 to an α-MEM complete medium to obtain a third exovesicle solution derived from the genus Roseburia and a fourth exovesicle solution derived from the genus Roseburia, wherein the concentration of the exovesicles derived from the genus Roseburia in the third exovesicle solution derived from the genus Roseburia is 1 μg / ml, and the concentration of the exovesicles derived from the genus Roseburia in the fourth exovesicle solution derived from the genus Roseburia is 10 μg / ml; and the α-MEM complete medium is used as a blank control solution; (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 Roseburia exosome solution, the fourth Roseburia exosome solution and the blank control solution) are used as culture medium 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; 15g of protein is extracted from each group corresponding to the above three solutions and 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 membrane was washed with TBST for 30min, and the 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 membrane, leave it for 2min, then take out the membrane and place it in the chemiluminescent imager for detection, the results are shown in Figure 6 .

[0042] Figure 6 Gel electrophoresis of marker proteins of hBMSCs osteogenic differentiation induced by extracellular vesicle solutions from Roseburia at different concentrations. Figure 6 0 μg / ml, 1 μg / ml and 10 μg / ml correspond to the experimental results of the blank control solution, the exosome solution derived from the third Roseburia genus and the exosome solution derived from the fourth Roseburia genus, respectively. Figure 6It 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 Roseburia 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.

[0043] 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 Roseburia in the preparation of inducers for osteogenic differentiation of bone marrow mesenchymal stem cells.

2. The use according to claim 1, characterized in that: The method for preparing the exosomes derived from the genus Roseburia comprises: Step S1, subculture and activate Roseburia and add it to the culture medium, culture it with shaking in a closed state, perform a first centrifugation, extract the supernatant, and filter it using a first bacterial sieve to obtain a first supernatant; Step S2, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and filtering it using a second bacterial sieve to obtain a second supernatant; Step S3, subjecting the second supernatant to a third centrifugation treatment, extracting the supernatant, and filtering it using a third bacterial sieve to obtain a third supernatant; Step S4, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant; Step S5, subjecting the fourth supernatant to a fifth centrifugation treatment, removing the supernatant, and obtaining a precipitate, which is the exosome derived from the genus Roseburia.

3. The use according to claim 2, characterized in that: In the step S1, the Roseburia genus is Roseburia intestinalis.

4. The use according to claim 2, characterized in that: In the step S1, the rotation speed of the first centrifugation is 1500g to 2000g, and the time is 20min to 30min.

5. The use according to claim 2, characterized in that: In step S2, the rotation speed of the second centrifugation is 9500g to 10500g, and the time is 20min to 30min.

6. The use according to claim 2, characterized in that: In step S3, the rotation speed of the third centrifugation is 9500g to 10500g, and the time is 30min to 40min.

7. The use according to claim 2, characterized in that: In step S4, the rotation speed of the fourth centrifugation is 9500g to 10500g, and the time is 30min to 40min.

8. The use according to claim 2, characterized in that: In step S5, the rotation speed of the fifth centrifugal treatment is 145000g to 155000g, and the time is 90min to 120min.

9. The use according to claim 2, characterized in that: In the step S2, the pore size of the second bacteria sieve is 0.45 μm.

10. The use according to claim 2, characterized in that: In the step S3, the pore size of the third bacteria sieve is 0.22 μm.

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