Application of Schwann cell exosome in preparation of anti-osteoclast differentiation drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly provides application of Schwann cell exosomes in preparation of osteoclast differentiation resisting medicines. Firstly, a Schwann cell and osteoclast precursor cell co-incubation experiment proves that Schwann cells inhibit osteoclast differentiation through exosomes. Then miRNA sequencing analysis is conducted on Schwann cell exosomes, then a PTEN / AKT signal channel is screened out, and experiments such as TRAP dyeing, RT-qPCR and WB are conducted on the cell molecule level to prove that SC-EXO inhibits osteoclast differentiation through the PTEN / AKT signal channel; meanwhile, through in-vivo experiments, the inhibition effect of SC-EXO on the osteoclast activity of ovarian-removed mice through a PTEN / AKT signal channel is determined. Therefore, the Schwann cell exosome can be used as an osteoclast differentiation inhibitor drug, and can be used as an osteoclast differentiation inhibitor for treating related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of Schwann cell exosomes in the preparation of anti-osteoclast differentiation drugs. Background Art

[0002] Osteoclasts are the only multinuclear giant cells in the body responsible for bone resorption. They originate from mononuclear macrophage precursor cells in the bone marrow and differentiate and mature under the action of specific signaling factors such as RANKL and M-CSF. The main function of osteoclasts is to absorb bone to maintain the metabolic balance of bones. However, excessive activation of osteoclasts may lead to a variety of diseases, including osteoporosis, rheumatoid arthritis, bone tumors, and Paget's disease. These diseases are usually associated with bone resorption exceeding bone formation, leading to bone loss and destruction of bone structure. Therefore, the regulation of osteoclast activity is crucial to maintaining bone health, and therapeutic strategies targeting excessive osteoclast activation have also become the key to the treatment of related diseases.

[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 anti-osteoclast differentiation drugs.

[0006] The technical solution adopted by the present invention is specifically as follows: Application of Schwann cell exosomes in the preparation of anti-osteoclast differentiation drugs or medical materials.

[0007] Furthermore, the exosomes are secreted by Schwann cells.

[0008] Furthermore, the Schwann cell exosomes inhibit osteoclast differentiation through miRNA-mediated signaling pathways.

[0009] Furthermore, the signaling pathway mediated by miRNA in the Schwann cell exosomes is a PTEN / AKT signaling pathway.

[0010] Furthermore, the drug comprises Schwann cell exosomes and other pharmaceutically acceptable components.

[0011] Furthermore, the drug comprises Schwann cell exosomes and other pharmaceutically acceptable components, and the other components comprise one or a combination of pharmaceutically acceptable carriers, additives, and other acceptable pharmaceutically active ingredients.

[0012] Furthermore, the dosage form of the drug is a pharmaceutically acceptable oral dosage form, dressing dosage form, injection dosage form or other medically acceptable dosage form for administration.

[0013] Compared with the prior art, the present invention has the following effects: The present invention first proves that Schwann cells inhibit osteoclast differentiation through exosomes by co-incubation experiments of Schwann cells and osteoclast precursor cells. Subsequently, the PTEN / AKT signaling pathway was screened out after miRNA sequencing analysis of Schwann cell exosomes. At the cellular molecular level, TRAP staining, RT-qPCR and WB experiments proved that SC-EXO inhibited osteoclast differentiation through the PTEN / AKT signaling pathway; at the same time, in vivo experiments confirmed the inhibitory effect of SC-EXO on osteoclast activity in ovariectomized mice through the PTEN / AKT signaling pathway. Therefore, Schwann cell exosomes can be used as an osteoclast differentiation inhibitor drug for osteoclast differentiation inhibitors to treat related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 TRAP staining of Schwann cells, Schwann cell culture supernatant, Schwann cells treated with GW4869, and culture supernatant of Schwann cells treated with GW4869 co-cultured with BMMs (scale bar is 500um).

[0015] Figure 2. 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.

[0016] Figure 3 . miRNA sequencing analysis screened SC-EXOs to inhibit osteoclast differentiation through PTEN / AKT pathway, where A is PCA analysis; B is sample correlation analysis; C is differential miRNA heat map; D is differential miRNA volcano map; E is the top 10 miRNAs differentially upregulated in SC-EXOs; F is GO circleplot; G is BP heatmap cluster; H is KEGG barplot; I is String analysis.

[0017] Figure 4 . Expression levels of genes in the PTEN / AKT pathway after SC-EXO treatment of BMMs, where A is the expression level of related proteins in the PTEN / AKT pathway; B is the relative mRNA expression level of PTEN.

[0018] Figure 5 . Cell staining results of Schwann cells co-cultured with BMMs after intervention with PTEN inhibitors, where A is TRAP staining (scale bar is 400um); B and C are quantification of osteoclast number and semi-quantification of osteoclast-positive area based on Figure A, respectively.

[0019] Figure 6 . Gene expression results of PTEN inhibitor intervention in co-culture of Schwann cells and BMMs, where A is the relative mRNA expression level of PTEN; B is the relative mRNA expression level of osteoclast functional gene Nfatc1; C is the relative mRNA expression level of osteoclast functional gene Ctsk; D is the relative mRNA expression level of osteoclast functional gene ACP5.

[0020] Figure 7 . In vivo verification that SC-EXO inhibits osteoclast differentiation through the PTEN / AKT signaling pathway, where A is TRAP staining of the distal femur pathological section (scale bar is 300um); B is PTEN immunohistochemistry of the distal femur pathological section (scale bar is 200um), C is p-AKT immunohistochemistry of the distal femur pathological section (scale bar is 200um), and D is Nfatc1 immunofluorescence of the distal femur pathological section (scale bar is 100um). DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Schwann cells inhibit osteoclast differentiation via exosomes S1: Co-culture of Schwann cells (SCs) and osteoclast precursor cells (BMMs) First, evenly spread the BMMs cells on a cell culture plate containing α-MEM complete culture medium, take a cell culture chamber that fits the well plate and place it in it, then inoculate the SCs cells, and finally place the well plate in a 37°C constant temperature 5% CO2 incubator for 24 hours to allow complete diffusion. Then replace the medium with complete culture medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL. At the same time, it can be treated differently, the culture medium is replaced every two days, and the samples are collected on the 5th day. After fixing the cells with paraformaldehyde, perform TRAP staining on the samples according to the instructions of the TRAP staining kit. Figure 1 As shown in the figure, co-culture of Schwann cells and BMMs inhibits the differentiation of BMMs into osteoclasts. The same inhibition of BMMs into osteoclasts occurred when BMMs were treated with Schwann cell culture supernatant. The addition of exosome inhibitor GW4869 offset the inhibitory effect of Schwann cells on BMMs into osteoclasts. Similarly, this inhibitory effect was also offset when BMMs were treated with Schwann cell culture medium treated with exosome inhibitor GW4869. These results indicate that Schwann cells inhibit osteoclast differentiation through exosomes.

[0023] S2: SC-EXO separation Rat Schwann cells (RSC96) were cultured in high-glucose DMEM medium containing 10% exosome-free serum and 1% double antibody, and the medium was replaced 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. There are 3 specific methods: 1. 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, centrifuge it again (4°C, 100,000×g, 70 minutes), and take the precipitate to obtain Schwann cell exosomes.

[0024] 2. Kit extraction method: use a commercial kit such as (Total Exosome Isolation Reagent, No.4478359, Invitrogen) to extract according to the kit requirements.

[0025] 3. Extract exosomes using size exclusion chromatography.

[0026] 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 2 As shown in A, TEM showed that the extracted exosomes had a typical exosome morphology, usually disc-shaped or hemispherical with concave sides. Figure 2 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 2 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).

[0027] Example 2 In vitro verification that SC-EXO inhibits osteoclast differentiation via the PTEN / AKT signaling pathway 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.

[0028] S2: Induction of osteoclasts The above cells were digested and seeded in 96-well plates, 8000 BMMs were plated in each well, and then the wells were cultured at 37°C and 5% CO2 for 24 hours to allow complete diffusion. Then, the medium was replaced with complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL, and the medium was replaced every two days. Mature osteoclasts were obtained on the 5th day of induction culture.

[0029] S3: SC-EXO inhibits osteoclast differentiation via PTEN / AKT signaling pathway We performed miRNA sequencing on SC-EXO, focusing on the miRNAs that were differentially expressed in exosomes compared with SCs cells, and sorted the 10 miRNAs that were differentially expressed in the SC-EXO group according to their expression levels. We then performed enrichment analysis on the differentially expressed target genes of miRNAs. Finally, we used the STRING database to perform interaction analysis on the target genes and predict key genes. Figure 3As shown in Figures AD, the two groups of samples of SCs and SC-EXOs were differentially analyzed, and it was found that the two groups had different miRNA expression patterns, and the reproducibility between samples was very good. Figure E sorted the 10 miRNAs that were differentially expressed in the SC-EXO group compared with the SC group from high to low according to the expression level. GO analysis in Figure FG showed that the target genes of these miRNAs were highly involved in "cell proliferation", "cell migration", "cytoskeleton organization" and "cell apoptosis regulation". KEGG enrichment analysis in Figure H showed that the target genes of these miRNAs were also directly involved in the relevant signaling pathways of bone metabolism, among which the phosphatidylinositol trikinase / protein kinase B (PI3K / Akt) pathway is worthy of attention. This signaling pathway can participate in the regulation of bone metabolism and is of great significance in regulating the growth, proliferation, differentiation, apoptosis and aging of cells such as bone marrow mesenchymal stem cells, osteoblasts and osteoclasts. In Figure I, the target gene interaction analysis was performed using the https: / / cn.string-db.org website, and it was found that the central hub gene was Pten. The Pten gene encodes the protein PTEN. When PTEN activity increases, it can inhibit the PI3K / AKT signaling pathway, thereby reducing the activity of osteoclasts and reducing bone resorption. Therefore, we speculate that when SC-EXO is internalized by osteoclast precursor cells BMMs, the released miRNA will promote the expression of PTEN, and the increase in PTEN will lead to the inhibition of the AKT pathway, thereby inhibiting the differentiation and maturation of osteoclasts. Together, these results show that the PTEN / AKT signaling pathway is a potential molecular mechanism for SC-EXO to inhibit osteoclast differentiation.

[0030] BMMs + RANKL was set as the positive control group (Con): 8000 BMMs were inoculated in a 96-well plate and the medium was replaced with complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL. The 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 and the medium was replaced with complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL. The 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; Set up BMMs+RANKL+SC-EXO+PTEN inhibitor as the experimental group (SC-EXO+ PTEN Inhibitor): 8000 BMMs were inoculated in complete culture medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL, and the culture medium was changed every two days. At the same time, SC-EXO was added to a final concentration of 100ug / ml, and the concentration of PTEN inhibitor was 4uM until the samples were collected on the 5th day; The formation of osteoclasts in all groups was determined by TRAP staining, RT-qPCR and WB. Figure 4 In A, the relative protein expression levels of PTEN and its downstream signaling pathway genes AKT and m-Tor were analyzed by Western blotting. The relative protein expression level of PTEN was significantly increased, while the relative protein expression levels of AKT and m-Tor did not change significantly. However, the phosphorylation activation of AKT and mTOR was significantly inhibited, indicating that SC-EXO activated the expression of PTEN gene through miRNA, thereby negatively regulating the AKT / mTOR signaling pathway, and further inhibiting the differentiation of BMMs into osteoclasts. Figure 4 In B, qRT-PCR was used to detect the relative mRNA levels of PTEN in the osteoclast control group and the SC-EXO treatment group. The results showed that compared with the osteoclast control group, the expression level of the PTEN gene was significantly increased after SC-EXO treatment. Figure 5 In AC, the PTEN inhibitor SF1670 was added to the SC-EXO treatment group, and the results of TRAP staining and quantitative analysis showed that the PTEN inhibitor SF1670 could effectively offset the inhibitory effect of SC-EXO on osteoclast differentiation. Figure 6 qRT-PCR was used to detect the expression of PTEN and osteoclast functional genes Nfatc1, Ctsk, and ACP5 in AD. The results showed that compared with the SC-EXO treatment group, the intracellular PTEN level decreased after the addition of PTEN inhibitor SF1670, and the relative mRNA expression levels of osteoclast functional genes Nfatc1, Ctsk, and ACP5 increased significantly. The above results indicate that SC-EXO inhibits osteoclast differentiation through the PTEN / AKT signaling pathway.

[0031] Example 3 In vivo verification that SC-EXO inhibits osteoclast differentiation via the PTEN / AKT signaling pathway 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, the femurs of mice in each group were collected, and the activity of osteoclasts in the distal femur tissue and markers related to the PTEN / AKT signaling pathway were analyzed by TRAP staining, immunohistochemistry, and immunofluorescence. Figure 7As shown in Figure A, TRAP staining was used to detect the differentiation of osteoclasts in bone tissue. The results of TRAP staining showed that SC-EXO effectively reduced the presence of TRAP-positive osteoclasts in trabecular bone, which means that SC-EXO can significantly prevent the differentiation and maturation of osteoclasts in vivo. Figure BC used immunohistochemistry to detect the protein levels of PTEN and p-AKT. The results showed that compared with the sham operation group, the expression level of PTEN was significantly increased, while the protein level of AKT phosphorylation was significantly reduced, indicating that SC-EXO inhibited the differentiation of BMMs into osteoclasts by activating the expression of the PTEN gene and negatively regulating the AKT / mTOR signaling pathway. Figure D used immunofluorescence to detect the protein expression level of NFATc1, a functional gene of osteoclasts. The results showed that SC-EXO effectively inhibited the expression of NFATc1. In summary, these results show that the addition of SC-EXO can effectively inhibit the osteoclast activity of OVX mice, and the activation of the PTEN / AKT signaling pathway is inhibited, which further indicates that SC-EXO inhibits osteoclast differentiation through the PTEN / AKT signaling pathway.

Claims

1. A use of Schwann cell exosomes in the preparation of drugs or medical materials, characterized in that: The medicine or medical material is used for resisting osteoclast differentiation.

2. The use according to claim 1, characterized in that: The exosomes are secreted by Schwann cells.

3. The use according to claim 1, characterized in that: The Schwann cell exosomes inhibit osteoclast differentiation through miRNA-mediated signaling pathways.

4. The use according to claim 1, characterized in that: The signaling pathway mediated by miRNA in Schwann cell exosomes is the PTEN / AKT signaling pathway.

5. The use according to claim 1, characterized in that: The medicine comprises Schwann cell exosomes and other pharmaceutically acceptable components.

6. The use according to claim 1, characterized in that: The drug comprises Schwann cell exosomes and other pharmaceutically acceptable components, and the other components comprise one or a combination of pharmaceutically acceptable carriers, additives, and other acceptable active pharmaceutical ingredients.

7. The use according to claim 1, characterized in that: The dosage form of the drug is a pharmaceutically acceptable oral dosage form, dressing dosage form, injection dosage form or other medically acceptable dosage forms for achieving administration.

Images