Application of Schwann cell exosome in preparation of medicine for treating osteoporosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly provides application of Schwann cell exosomes in preparation of medicines for treating osteoporosis. An exosome derived from Schwann cells is prepared, and the inhibition effect of SC-EXO on osteoclasts is proved through TRAP staining, immunofluorescence, bone resorption, RT-qPCR and other experiments on the cell molecule level; meanwhile, the effect of SC-EXO on reduction of bone loss of ovarian extirpation mice is determined through in-vivo experiments. According to the invention, osteoclast formation and bone resorption capability can be inhibited, bone loss of OVX mice can be reduced in vivo, and the compound can be used for treating osteoporosis and other related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of Schwann cell exosomes in the preparation of drugs for treating osteoporosis. Background Art

[0002] Osteoporosis is a common bone disease characterized by decreased bone density and deterioration of bone microarchitecture, resulting in fragile bones and susceptibility to fractures. This disease affects hundreds of millions of people worldwide, especially in the elderly population. As the global population ages, its incidence is on the rise. The main pathogenesis of osteoporosis is an imbalance in bone metabolism, that is, the balance between bone resorption (mainly performed by osteoclasts) and bone formation (mainly performed by osteoblasts) is broken. Normally, osteoclasts are responsible for dissolving bone matrix and releasing minerals such as calcium and phosphate, while osteoblasts are responsible for synthesizing new bone matrix to supplement. But in osteoporosis, the rate of bone resorption exceeds that of bone formation, resulting in a decrease in bone mass and weakened bone structure.

[0003] Schwann cells, as the main glial cells of the peripheral nervous system, play a central role in this process. They not only support and protect nerve fibers in the repair of nerve injury, but also studies have shown that Schwann cells can promote angiogenesis and osteoblast activity by secreting a variety of growth factors and cytokines, which has a positive effect on fracture healing and bone tissue engineering. The role of Schwann cells in promoting nerve repair, angiogenesis, and osteogenesis has been widely studied. Such cells show great potential in the field of bone tissue engineering and help improve the speed and quality of bone formation. However, the interaction between Schwann cells and osteoclasts, key cells in bone homeostasis, is not fully understood. Further research on how Schwann cells affect osteoclast function may provide new strategies and methods for the treatment of osteoporosis and other bone metabolic diseases.

[0004] Exosomes are nanoscale biological vesicles with a diameter of 30-200 nm secreted by cells. The contents contain proteins and various nucleic acids, especially miRNA, which can regulate the expression of multiple genes and play a huge role in cell differentiation, biological development and the occurrence and development of diseases. As a medium for intercellular communication, exosomes play a key role in the neuro-bone tandem. They are involved in regulating nerve repair and bone metabolism. Exosomes released by Schwann cells have been shown to play a positive role in nerve regeneration and bone tissue repair. They carry a variety of bioactive molecules and affect the behavior of receiving cells. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Schwann cell exosomes in the preparation of a drug for treating osteoporosis.

[0006] The technical solution adopted by the present invention is specifically as follows: Application of Schwann cell exosomes in the preparation of drugs for treating osteoporosis.

[0007] Furthermore, the Schwann cell exosomes have the property of targeting osteoclast precursors and are a type of vesicles secreted by Schwann cells.

[0008] Furthermore, the osteoporosis is primary osteoporosis or secondary osteoporosis.

[0009] Furthermore, the medicine is in the form of injection, tablet, capsule, powder or other medically acceptable dosage forms.

[0010] Furthermore, the injection was prepared by using phosphate buffered saline as solvent to extract fresh exosomes.

[0011] Furthermore, the exosomes can be freeze-dried and prepared into tablets, capsules, and powders.

[0012] Compared with the prior art, the present invention has the following effects: The present invention prepares exosomes derived from Schwann cells, and at the cellular molecular level, through TRAP staining, immunofluorescence, bone absorption, RT-qPCR and other experiments, it is confirmed that SC-EXO can effectively inhibit osteoclast formation and bone absorption ability. In vivo, it can be confirmed that SC-EXO can effectively inhibit bone loss in OVX mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 . Identification results of Schwann cell-derived exosomes, where A is a transmission electron microscopy image (scale bar is 50 nm); B is the result of nanoparticle tracking analysis; C is the WB result of exosome marker membrane proteins.

[0014] Figure 2 . The results of SC-EXO inhibition on osteoclast differentiation, where A is TRAP staining of SC-EXO-intervention BMMs differentiation into osteoclasts (scale bar is 400um); B and C are quantification of osteoclast number and semi-quantification of osteoclast-positive area based on Figure A; D is immunofluorescence detection of actin ring after SC-EXO-intervention BMMs differentiation into osteoclasts for 5 days (scale bar is 100um); E and F are semi-quantification of actin ring area and quantification of osteoclast nucleus percentage based on Figure D, respectively.

[0015] Figure 3. The results of SC-EXO's inhibition of osteoclast bone resorption, where A is the toluidine blue staining result of SC-EXO intervening in osteoclast bone resorption on bone slices (scale bar is 200um); B is the semi-quantitative bone resorption positive area based on Figure A.

[0016] Figure 4 . SC-EXO inhibits the expression of osteoclast functional genes.

[0017] Figure 5 . Reconstructed images of micro CT scans of the distal femur in the sham-operated group (SHAM), OVX mice group (OVX), and SC-EXO-treated group (SC-EXO). DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0019] Example 1 Isolation and identification of Schwann cell exosomes S1: Culture of Schwann cells (SCs) Fetal bovine serum was first centrifuged to remove exosomes (4°C, 100,000 g, 70 minutes). Rat Schwann cells (RSC96) were cultured in high-glucose DMEM medium containing 10% exosome-free serum and 1% double antibody, and the medium was changed every 2 days. The incubator was kept at a constant temperature of 37°C, 5% CO2, and 70%-80% humidity. When the cell confluence reached 80%-90%, the culture supernatant was collected for exosome extraction.

[0020] S2: SC-EXO separation The supernatant collected above is used to extract exosomes. There are three specific methods: Gradient centrifugation method: centrifuge (4°C, 300×g, 10 minutes), take the supernatant and centrifuge again (4°C, 2000×g, 20 minutes), take the supernatant and centrifuge again (4°C, 10000×g, 30 minutes), discard the precipitate, centrifuge the supernatant at 100,000×g at 4°C for 70 minutes, collect the precipitate, resuspend it in PBS, and centrifuge it again (4°C, 100,000×g, 70 minutes), and take the precipitate to obtain Schwann cell exosomes.

[0021] 2. Kit extraction method: use a commercial kit such as (Total Exosome Isolation Reagent, No.4478359, Invitrogen) to extract according to the kit requirements.

[0022] 3. Extract exosomes using size exclusion chromatography.

[0023] S3: SC-EXO identification Transmission electron microscopy (TEM) was used to observe the morphology of the exosomes. The particle size and concentration were analyzed by NTA (Nanoparticle Tracking Analysis), and the exosome marker membrane proteins were detected by WB. Figure 1 As shown in A, TEM showed that the extracted exosomes had a typical exosome morphology, usually disc-shaped or hemispherical with concave sides. Figure 1 The NTA results in B show that the size of exosomes is mainly distributed at 114.3 nm, which is consistent with the size distribution range of exosomes reported in the literature, that is, cell vesicles with a diameter of 30 to 200 nm. Figure 1 Western blot analysis of SCs and SC-EXO in C showed that the extracted SC-EXO showed high levels of exosome marker proteins CD9, CD63, and TSG101. Taken together, these results indicate that the extracted and purified vesicles are exosomes (SC-EXO). Example 2 Inhibitory effect of SC-EXO on osteoclast differentiation and function S1: Isolation of mouse bone marrow-derived macrophages (BMMs) BMMs were extracted from the bone marrow of 4-week-old C57BL / 6 female mice. After euthanasia with 3% sodium pentobarbital, they were immersed in 75% ethanol for 10 minutes. The muscle tissue of the hind limbs was removed to obtain the femur and tibia. Under a sterile environment, the ends of the tibia and femur were cut off respectively, and then the complete culture medium was aspirated with a syringe to flush the bone marrow cells into the culture dish until the bone head turned white. The liquid in the culture dish was repeatedly blown and mixed, and the cells were filtered with a 200-mesh filter. The filtrate was collected in a centrifuge tube at a speed of 1000 rpm, centrifuged for 5 min, the supernatant was discarded, and the cells were resuspended in 5 mL of α-MEM complete culture medium (containing 10% FBS, 1% penicillin-streptomycin), and inoculated in a T-25 cm at 37°C. 2After 24 hours, the cells were collected and resuspended in 5 mL of α-MEM medium containing 30 ng / mL macrophage colony stimulating factor and cultured in the culture flask for another 3 days. Since only BMMs can adhere to the wall and survive under M-CSF stimulation among various cells, the adherent cells at the bottom of the culture flask were classified as such cells. Adherent cells grown to about 90% confluence were trypsinized for 15 minutes to obtain BMMs and used for subsequent in vitro experiments.

[0024] S2: Induction of osteoclasts The above cells were digested and seeded in 96-well plates, 8,000 BMMs were plated in each well, and the plates were then cultured at 37°C and 5% CO2 for 24 hours to allow for complete diffusion. The medium was then replaced with complete culture medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL, and the culture medium was replaced every two days. Mature osteoclasts were obtained on the fifth day of induction culture.

[0025] S3: Inhibitory effects of SC-EXO on osteoclast differentiation and function Set BMMs+RANKL as the positive control group (Con): 8000 BMMs were inoculated in a 96-well plate. To conduct bone resorption experiments, bovine bone slices were placed on the bottom of the well plate and replaced with complete culture medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL. The culture medium was replaced every two days and samples were collected on the 5th day. BMMs+RANKL+SC-EXO was set as the experimental group (SC-EXO): 8000 BMMs were inoculated in a 96-well plate. To carry out the bone absorption experiment, bovine bone slices were laid on the bottom of the well plate in advance, and the medium was replaced with complete culture medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL. The culture medium was replaced every two days, and SC-EXO was added to a final concentration of 100ug / ml until the samples were collected on the 5th day; all groups were observed by TRAP staining to observe the formation of osteoclasts, and the effects on osteoclast function were observed by actin ring immunofluorescence and bone absorption experiments, and the expression of osteoclast functional genes was detected by RT-qPCR. Figure 2 TRAP staining and quantitative results of AC showed that a large number of mature osteoclasts were formed in the positive control group. After the addition of SC-EXO, osteoclast fusion was significantly inhibited, and the number of mature osteoclasts and the proportion of positive area were significantly reduced, which indicates that SC-EXO can effectively inhibit the formation of osteoclasts. Figure 2The immunofluorescence staining and quantitative results of actin ring of DF showed that the positive control group had a clear and complete actin ring. After the addition of SC-EXO, the formation of actin ring was significantly inhibited, which also shows that SC-EXO can effectively inhibit the formation of osteoclasts. Figure 3 The results of toluidine blue staining and quantitative analysis of the AB bone absorption experiment showed that the positive control group had obvious bone absorption areas. After the addition of SC-EXO, the positive area of ​​bone absorption was significantly reduced, which indicates that SC-EXO can effectively inhibit the function of osteoclasts. Figure 4 AC showed that after adding SC-EXO, the expression of osteoclast functional genes Nfatc1, Ctsk, and ACP5 was significantly reduced compared with the positive control group, which further demonstrated that SC-EXO can effectively inhibit the formation and function of osteoclasts.

[0026] Example 3 SC-EXO reduces bone loss in osteoporotic mice Ovariectomized (OVX) mice were obtained by routine ovarian removal surgery on 6-week-old female mice. Four weeks later, 100uL of 0.1ug / uL SC-EXO (solvent: PBS buffer) was injected through the tail vein three times a week. Eight weeks after the injection, micro CT was used to analyze the bone formation in the distal femoral tissue of OVX mice regularly injected with SC-EXO and the control group injected with solvent (phosphate buffered saline). Figure 5 As shown in the reconstructed images of micro-CT scans, compared with the sham operation group, the OVX group had a large amount of bone loss and a significant decrease in the number of trabeculae, which confirmed the successful establishment of the osteoporosis mouse model. After treatment with SC-EXO, compared with the OVX group, the SC-EXO group had significantly reduced bone loss and a significant increase in the number of trabeculae, indicating that SC-EXO can effectively improve the bone loss of OVX mice.

Claims

1. A use of Schwann cell exosomes in the preparation of a drug for treating a disease, characterized in that: The disease is osteoporosis.

2. The use according to claim 1, characterized in that: The Schwann cell exosomes have the property of targeting osteoclast precursors and are a type of vesicles secreted by Schwann cells.

3. The use according to claim 1, characterized in that: The osteoporosis is primary osteoporosis or secondary osteoporosis.

4. The use according to claim 1, characterized in that: The medicine is in the form of injection, tablet, capsule, powder and other medically acceptable dosage forms.

5. The use according to claim 1, characterized in that: The injection is prepared by using phosphate buffered saline as a solvent to extract fresh exosomes.

6. The use according to claim 1, characterized in that: The exosomes can be prepared into tablets, capsules, and powders after freeze-drying.

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