Application of engineered dental epithelial cell exosome

Abstract

The invention discloses application of an engineered dental epithelial cell exosome, and belongs to the technical field of biomedicine. The invention discloses an application of an engineered tooth epithelial cell exosome. An engineered tooth epithelial cell comprises an engineered tooth epithelial cell line HERS-H1; the application comprises the application in preparation of medicines for preventing or / and treating osteoporosis. The medicine for preventing or / and treating osteoporosis disclosed by the invention comprises an exosome of an engineered tooth epithelial cell line HERS-H1. The dental epithelial cell exosome disclosed by the invention can be taken by the bone marrow mesenchymal stem cells, and the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells are promoted; in an ovariectomized induced osteoporosis model, the dental epithelial cell exosome is injected into the caudal vein, so that bone loss can be effectively reduced, the bone microstructure quality can be improved, and the progress of osteoporosis can be delayed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of engineered dental epithelial cell exosomes. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease, which is mainly manifested by decreased bone density, destruction of bone microstructure, and decreased bone strength, which greatly increases the risk of fractures in patients and is known as the "silent killer". The onset of osteoporosis is closely related to age, decreased hormone levels, lifestyle changes, and drug use. As my country gradually enters an aging society, the incidence of osteoporosis caused by aging and decreased sex hormone levels has greatly increased, especially osteoporosis in postmenopausal women. Osteoporosis and its related complications such as fractures, spinal deformities, and low back pain not only affect patients' daily living activities and reduce their quality of life, but also bring heavy medical and economic burdens to individuals and society. At present, the main clinical treatments for osteoporosis include drug therapy, physical therapy, and dietary conditioning, and osteoporosis cannot be completely cured. Therefore, the development and exploration of new osteoporosis treatment drugs has important clinical value and social significance.

[0003] Exosomes are a kind of membrane nano-scale vesicle structure secreted by cells, with a diameter of about 50-150nm. They are formed by budding from the limiting membrane of the intracellular multivesicular body (MVB) into the cavity, and are released outside the cell after direct fusion of MVB with the plasma membrane. Exosomes carry multiple functional proteins, coding and non-coding RNA, DNA and other active molecules of source cells. They are important structures for signal transmission between cells and play an important role in various biological processes such as organ development, immune regulation, and tumorigenesis. In the field of regenerative medicine, the important role of stem cell-derived exosomes in tissue regeneration has gradually become a research hotspot in recent years. Studies have shown that stem cell-derived exosomes have a stem cell-like function of promoting tissue regeneration. Stem cell-derived exosomes can activate endogenous stem cells in damaged tissues through the nucleic acid or protein components they contain, and achieve endogenous regeneration in tissues such as the heart, brain, and tendons. In the process of tooth development, dental epithelial cells have the function of inducing osteogenic / odontogenic differentiation of mesenchymal cells. However, the prior art does not disclose that dental epithelial exosomes have the effect of treating osteoporosis.

[0004] Therefore, providing an effect of exosomes from a dental epithelial cell line (HERS-H1) in preventing and / or treating osteoporosis has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide the use of engineered dental epithelial cell exosomes in the preparation of a drug for preventing and / or treating osteoporosis.

[0006] Another object of the present invention is to provide a drug for preventing and / or treating osteoporosis, comprising exosomes of the engineered dental epithelial cell line HERS-H1.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention discloses an application of engineered dental epithelial cell exosomes, wherein the engineered dental epithelial cells include an engineered dental epithelial cell line HERS-H1; the application includes application in the preparation of a drug for preventing and / or treating osteoporosis.

[0009] The engineered dental epithelial cell line HERS-H1 described in the present invention is deposited in the China Center for Type Culture Collection, and its name is: SD rat tooth germ Hertwig epithelial root sheath cell line HERS-H1 (Hertwig's epithelial root sheath, HERS-H1), the deposit number is CCTCC NO: C2017249, the deposit time is November 21, 2017, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] On the other hand, the present invention discloses a drug for preventing and / or treating osteoporosis, comprising exosomes of an engineered dental epithelial cell line HERS-H1.

[0011] The exosomes of the present invention may be prepared by using existing techniques, such as the method described in the patent publication number CN 111388504A; or the method of the present invention may be used.

[0012] In some embodiments of the present invention, the method for preparing exosomes comprises the following steps:

[0013] S1. Cultivate engineered epithelial cells HERS-H1, collect the culture medium, centrifuge, collect the supernatant, add the supernatant to a 100KD centrifugal filter, centrifuge, and collect the concentrate;

[0014] S2. Add exosome extraction reagent to the concentrated solution, incubate, centrifuge, discard the supernatant, and the precipitate is the exosomes of the engineered dental epithelial cell line HERS-H1.

[0015] In some embodiments of the present invention, in step S1, the engineered epithelial cells HERS-H1 are cultured in an Epithelial cell medium containing 2% fetal bovine serum in an incubator at 37° C. and 5% CO 2 .

[0016] In some embodiments of the present invention, the culture time is 2 to 10 days, preferably 4 to 6 days.

[0017] In some embodiments of the present invention, in step S1, the culture fluid is collected and centrifuged at 2000-5000 rpm for 10-60 min; preferably at 3000 rpm for 30 min, and the supernatant is collected.

[0018] In some embodiments of the present invention, in step S1, the supernatant is added to a 100KD centrifugal filter, centrifuged under low temperature conditions, and the concentrate is collected;

[0019] Preferably, the centrifugation is carried out at 1 to 6°C, more preferably at 4°C;

[0020] The centrifugal speed is preferably 4000-6000 rpm, more preferably 5000 rpm;

[0021] The centrifugation time is preferably 20 to 60 min, more preferably 30 min.

[0022] In some embodiments of the present invention, in step S2, the volume ratio of the concentrate and the exosome extraction reagent is 2:1;

[0023] After adding the exosome reagent, incubate at 1-6°C for 10-40 hours, preferably at 4°C, and preferably for 20 hours.

[0024] In some embodiments of the present invention, after the incubation, the mixture is centrifuged at 8000-20000 rpm for 30-120 min; preferably, the mixture is centrifuged at 10000 rpm for 60 min.

[0025] Some embodiments of the present invention further include a cell recovery step: the frozen engineered epithelial cells HERS-H1 are recovered, and then resuspended with culture medium, centrifuged, the supernatant is discarded, and then the culture medium is added for a second resuspending for use.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention creatively utilizes dental epithelial cell exosomes from teeth to prevent and / or treat osteoporosis. Experimental tests show that the dental epithelial cell exosomes of the present invention can be taken up by bone marrow mesenchymal stem cells and promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells; in the osteoporosis model induced by ovariectomy, tail vein injection of dental epithelial cell exosomes can effectively reduce bone loss, improve the quality of bone microstructure, and delay the progression of osteoporosis. The present invention provides a new method for the treatment of osteoporosis, which has important clinical application value and socioeconomic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Attached Figure 1 Transmission electron microscopy images of exosomes derived from dental epithelial cells;

[0029] Attached Figure 2 The particle size and distribution of exosomes derived from dental epithelial cells;

[0030] Attached Figure 3 The WB detection results of exosome marker proteins derived from dental epithelial cells, where Exos represents exosomes and HERS represents dental epithelial cells;

[0031] Attached Figure 4 Figure 2 shows the uptake of dental epithelial cell-derived exosomes by bone marrow mesenchymal stem cells.

[0032] Attached Figure 5 This is the result of the experiment on exosomes derived from dental epithelial cells promoting the proliferation of bone marrow mesenchymal stem cells;

[0033] Attached Figure 6 This is the alizarin red staining result of Test Example 4;

[0034] Attached Figure 7 This is the quantitative analysis result of Alizarin Red staining in Experimental Example 4;

[0035] Attached Figure 8 This is the experimental flow chart of the in vivo treatment of osteoporosis in animals with exosomes derived from dental epithelial cells in Experimental Example 5;

[0036] Attached Fig. 9 This is a microCT scan of the distal femur of an ovariectomized rat after 6 weeks of treatment with dental epithelial cell exosomes in Experimental Example 5;

[0037] Attached Fig.10 This is a graph showing the results of bone morphometry analysis of distal femurs in ovariectomized rats after 6 weeks of treatment with dental epithelial cell exosomes in Experimental Example 5;

[0038] Attached Fig.11 This is the HE staining image of distal femur bone tissue of ovariectomized rats after 6 weeks of treatment with dental epithelial cell exosomes in Experimental Example 5;

[0039] Attached Figure 9-10 In the figure, Sham represents the sham operation group, OVX represents the untreated ovariectomized group, OVX+Exo-100μg, OVX+Exo-300μg, and OVX+Exo-500μg represent the treatment groups with different doses of exosomes, respectively. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] The dental epithelial cells described in the embodiments of the present invention, unless otherwise specified, refer to the engineered dental epithelial cell line HERS-H1.

[0042] The exosome extraction reagents described in the present invention are prior art, including ES8500, The exosome extraction reagent used in the embodiment of the present invention is small extracellular vesicles purification kit.

[0043] Example 1

[0044] This Example 1 discloses a method for preparing exosomes of the present invention, which is as follows:

[0045] (1) Engineered dental epithelial cell culture:

[0046] 1.1 Take out the engineered dental epithelial cell line HERS-H1 stored in liquid nitrogen, and quickly revive it with warm water at 37℃-40℃. Transfer the cell suspension after cell recovery in each tube to a 15mL centrifuge tube, add 5mL Epithelial cell medium culture medium to resuspend, centrifuge at 1000rpm-1500rpm for 5min, discard the supernatant, add 2mL Epithelial cell medium culture medium containing 2% fetal bovine serum to resuspend for later use.

[0047] 1.2 After resuspending, count the HERS-H1 cells and take 1×10 5 The cells were inoculated into T175 cell culture flasks, and the cells were cultured in Epithelial cell medium containing 2% fetal bovine serum. The culture flasks were placed in an incubator at 37° C. and 5% CO 2 , and the medium was replaced every 2 days for 5 days.

[0048] (2) Acquisition of exosomes from dental epithelial cells:

[0049] 2.1 Collect the culture medium in the culture flask into a 50 mL centrifuge tube, centrifuge at 3000 rpm for 30 min to remove possible cell debris, and collect the supernatant.

[0050] 2.2 Take the supernatant after centrifugation and add it to a 100KD centrifugal filter. Centrifuge at 5000rpm for 30min at 4℃ to collect the concentrate.

[0051] 2.3 Add exosome extraction reagent to the concentrate (the concentrate and exosome extraction reagent are in a ratio of 2:1), incubate at 4°C for 20 hours, centrifuge at 10,000 rpm for 60 minutes, discard the supernatant, add an appropriate amount of 0.01 M PBS to the precipitate to dissolve the exosomes secreted by dental epithelial cells under 3D culture conditions (HERS-Exo), and determine the concentration of the exosome solution by BCA method and store it at -80°C for later use.

[0052] Test Example 1

[0053] In this test example, the exosomes prepared in Example 1 were detected.

[0054] 1. Take an appropriate amount of exosomes, dilute them appropriately, drop them on the transmission electron microscope sample copper grid, dry them, add phosphotungstic acid to stain for 3 minutes, and observe them under a transmission electron microscope. The results showed that the exosomes secreted by dental epithelial cells were round, double-layer membrane vesicle-like structures, such as attached Figure 1 shown.

[0055] 2. Take an appropriate amount of exosomes, dilute them appropriately, and use nanoparticle tracking analysis (NTA) to detect the size of exosomes. The particle size analysis shows that the peak value of the particle size distribution of exosomes secreted by dental epithelial cells is 100nm. Figure 2 shown.

[0056] 3. Western Blot Detection of Exosome Marker Proteins from Dental Epithelial Cells

[0057] An appropriate amount of dental epithelial cells HERS and their secreted exosomes were taken and added to pre-cooled RIPA Lysis Buffer, respectively, and lysed at 4°C for 30 minutes, followed by centrifugation at 12000 rpm at 4°C for 15 minutes to obtain cell protein supernatant and exosome protein supernatant. The concentration was determined by BCA method and then stored at -80°C for future use. The expression of exosome marker proteins CD63 and HSP70 was detected by Western Blot method.

[0058] The brief steps of Western Blot are as follows:

[0059] (1) Take 1×10 5 1 mL of pre-cooled RIPA lysis buffer containing 1 mM PMSF was added to 0.5 mg of HERS-H1 cells or exosomes, mixed well, and lysed on ice for 10 min; centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant (i.e., protein sample) was taken for later use;

[0060] (2) The supernatant was denatured by heating at 100°C for 10 min after the protein concentration was determined by the BCA method, and then aliquoted and stored at -80°C for later use;

[0061] (3) Take the denatured protein samples from each group, mix them with 5× Loading buffer and perform protein PAGE electrophoresis; after the electrophoresis, transfer the separated protein samples to a PVDF membrane for later use;

[0062] (4) The transferred PVDF membrane was blocked with 5% skim milk (prepared in TBST) for 30 min, and CD63 (1:500 dilution), HSP70 (1:500 dilution), and β-actin (1:500 dilution) antibodies were added, respectively, and incubated at 4°C overnight;

[0063] (5) After the primary antibody incubation, the PVDF membrane was rinsed with TBST three times, each time for 10 min. After rinsing, HRP-labeled secondary antibody dilution was added and incubated at room temperature for 1 h;

[0064] (6) After the secondary antibody incubation, the PVDF membrane was rinsed three times with TBST, each time for 10 min. After the rinsing, the PVDF membrane was developed by chemiluminescence and the image was collected.

[0065] The results are attached Figure 3 The results showed that exosomes were significantly enriched in CD63 and HSP70, but β-actin was not detected; the expression of CD63 and HSP70 was not detected in the protein lysate of dental epithelial cells.

[0066] Test Example 2

[0067] This test example discloses an investigation of dental epithelial cell exosomes being taken up by bone marrow mesenchymal stem cells. The details are as follows:

[0068] 1. Take bone marrow mesenchymal stem cells with good growth status and use 2×10 4 The number of cells / dish was inoculated into a glass-bottomed culture dish and cultured in an incubator at 37°C and 5% CO2. The cells were cultured in α-MEM medium containing 5% fetal bovine serum.

[0069] 2. The HERS-Exo prepared according to the method of Example 1 was resuspended in PBS, DiO dye was added and mixed thoroughly, and then incubated at 37°C in the dark for 20 minutes, and DiO-labeled HERS-Exo was re-extracted;

[0070] 3. After the bone marrow mesenchymal stem cells adhered to the wall, the medium was replaced with α-MEM medium containing 5% fetal bovine serum and DiO-labeled HERS-Exo and cultured for 12 hours;

[0071] 4. Aspirate the culture medium, rinse with PBS, fix with fixative solution for 15 minutes, wash with PBS for 5 minutes × 3 times; add 0.5% tritonx-100 to break the membrane at room temperature for 10 minutes, wash with PBS; add 1% BSA solution to block at 37℃ for 30 minutes;

[0072] 5. Stain with Phalloidin dilution solution at room temperature in the dark for 30 minutes, then wash with PBS; dilute DAPI staining solution, stain at room temperature in the dark for 5 minutes, then wash with PBS and observe and collect images under a fluorescence confocal microscope.

[0073] The results are attached Figure 4 As shown: After being co-cultured with bone marrow mesenchymal stem cells for 12 hours, the exosomes secreted by dental epithelial cells can be taken up by bone marrow mesenchymal stem cells. Figure 4 DAPI staining in the middle shows the blue cell nucleus, red Phalloidin marks the cytoskeleton, and green fluorescence shows the ingested dental epithelial cell exosomes.

[0074] Test Example 3

[0075] This test example discloses a test on the effect of dental epithelial cell exosomes on the proliferation of bone marrow mesenchymal stem cells. The details are as follows:

[0076] 1. Take bone marrow mesenchymal stem cells with good growth status, prepare cell suspension, adjust cell concentration, and inoculate them into 96-well plates at a cell density of 1000 cells / well (100 μl cell suspension); culture in an incubator at 37°C and 5% CO2.

[0077] 2. Prepare medium containing HERS-Exo at different concentrations, namely 80 μg / mL, 160 μg / mL, and 240 μg / mL, and use α-MEM medium containing 5% fetal bovine serum without HERS-Exo as control; after the cells adhere to the wall, replace the medium containing HERS-Exo at different concentrations for further culture in each group of cells;

[0078] 3. At the same time every day, mix α-MEM with CCK-8 reagent in a volume ratio of 10:1 to prepare CCK-8 working solution. Add 100 μl of working solution to the corresponding wells of each group. Incubate in an incubator away from light for 90 minutes, measure the OD value at a wavelength of 450 nm using an enzyme reader, and continue to detect for 5 days, recording the data for analysis.

[0079] The CCK8 results are attached. Figure 5 The results showed that after 3 days of HERS-Exo intervention, the proliferation of bone marrow mesenchymal stem cells in each group was significantly higher than that in the control group (Con), indicating that HERS-Exo can significantly promote the proliferation of bone marrow mesenchymal stem cells.

[0080] Test Example 4

[0081] This test example discloses an experiment in which dental epithelial cell exosomes promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0082] The details are as follows:

[0083] 1. Prepare osteoinduction fluid: Prepare osteoinduction medium using α-MEM containing 5% fetal bovine serum as the basic medium (the ingredients and final concentrations are as follows: 100nM dexamethasone, 50μg / mL vitamin C, 10mM β-sodium glycerophosphate); add different concentrations of HERS-Exo to the osteogenic induction medium, which are 80μg / mL, 160μg / mL, and 240μg / mL, respectively, and use the osteogenic induction medium without adding HERS-Exo as the control;

[0084] 2. Take bone marrow mesenchymal stem cells with good growth status and inoculate them into 12-well plates. After the cell growth density reaches 70%, replace it with osteogenic induction medium containing different concentrations of HERS-Exo. Use osteogenic induction medium without HERS-Exo as control and continue to culture in a cell culture incubator. Change the medium every 2 days for 21 days.

[0085] 3. After the cells were cultured in osteogenic induction medium for 21 days, the induction medium was discarded, and the cells were rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. The fixative was discarded, and the cells were rinsed with PBS for 3 times. The cells were gently stained with Alizarin Red working solution for 15 minutes, and the staining solution was discarded. After rinsing with PBS, the staining effect was observed under a microscope and pictures were taken;

[0086] 4. Quantitative analysis of Alizarin Red staining: add 10% cetylpyridinium chloride solution to the stained and rinsed well plate, incubate in a 37°C incubator for 15 minutes to release the Alizarin Red stain, dilute the resulting solution by an appropriate multiple, measure the absorbance at a wavelength of 562nm in a 96-well plate using an enzyme reader, and record the data and draw graphs using software.

[0087] The results of Alizarin Red staining are shown in the attached Figure 6 As shown: After the control group (Con) cells were cultured in osteogenic induction medium for 21 days, Alizarin red-positive stained mineralized nodules were deposited in the cell matrix; after adding different concentrations of HERS-Exo to the osteogenic induction medium, the number of Alizarin red-positive stained mineralized nodules increased significantly.

[0088] The results of quantitative analysis of Alizarin Red are shown in the attached Figure 7 As shown: As the concentration of HERS-Exo increases, the positive staining of Alizarin Red of BMSCs increases significantly, indicating that HERS-Exo can significantly promote the osteogenic differentiation of BMSCs.

[0089] Test Example 5

[0090] This test example discloses an animal experiment on the use of dental epithelial cell exosomes of the present invention for the treatment of osteoporosis. In this test example, HERS-Exo was prepared according to the method of Example 1, and PBS was added to prepare PBS solutions of HERS-Exo of different concentrations. Figure 8 The process shown is to conduct an in vivo osteoporosis treatment experiment in ovariectomized animals. The details are as follows:

[0091] Eight-week-old female SD rats (weight 150-200 g) were selected to prepare an ovariectomized (OVX) osteoporosis animal model. 10% chloral hydrate solution was injected intraperitoneally, and the animals were fixed on the experimental table in a supine position after anesthesia.

[0092] 1. Establishment of the ovariectomy-induced osteoporosis animal model: 8-week-old female SD rats (weight 150-200g) were selected to prepare the ovariectomy-induced osteoporosis animal model. 10% chloral hydrate solution was injected intraperitoneally, and the animals were anesthetized and fixed on the experimental table in a supine position. A longitudinal incision of about 15 mm was made along the midline skin of the lower abdomen of the rats to expose the subcutaneous tissue and abdominal wall muscles, the abdominal wall was cut open, the fallopian tubes were ligated, and the bilateral ovaries were removed. Suture layer by layer, and penicillin was injected intramuscularly every day for 1 to 3 days after surgery to prevent postoperative infection.

[0093] 2. Starting from one week after surgery, HERS-Exo was injected into the tail vein every week, and three dose groups were set up, with doses of 100μg / Kg body weight, 300μg / Kg body weight, and 500μg / Kg body weight, respectively; HERS-Exo was dissolved in 500μL 0.01M sterile PBS. Rats in the sham group (Sham) were not injected with exosomes, and rats in the ovariectomized control group (OVX) were only injected with the same volume of PBS. The other groups of ovariectomized rats were injected with exosomes at the corresponding doses; treatment was twice a week, with an interval of 2-3 days each time, and the treatment lasted for a total of 6 weeks.

[0094] 3 At 7 weeks after surgery (6 weeks after HERS-Exo treatment), the animals were killed by overdose of anesthesia, and the femur specimens were collected and fixed with 4% paraformaldehyde overnight. The femur specimens were washed with PBS and then subjected to microCT scanning of the distal femur.

[0095] The microCT scan results are shown in the attached Fig. 9 Compared with the sham operation group ( Fig. 9 Compared with the Sham group in the untreated ovariectomized group ( Fig. 9 The trabeculae at the distal end of the femur of the OVX group (in the study), became thinner and sparser, indicating bone degeneration and severe trabecular loss. After HERS-Exo treatment, different dose groups showed varying degrees of delayed trabecular loss and degeneration, among which the 300 μg / Kg body weight dose group had the most significant therapeutic effect.

[0096] The results of bone tissue morphometric analysis are shown in the attached Fig.10 As shown: Compared with the sham operation group, the parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the OVX group were significantly downregulated, and the trabecular dispersion (Tb.Sp) was significantly increased; after HERS-Exo treatment, the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular dispersion (Tb.Sp) were significantly improved, among which the treatment effect of the 300μg / Kg body weight dose group was the most significant, while there was no significant change in trabecular thickness (Tb.Th).

[0097] The above-mentioned microCT scanning and trabecular morphometric analysis results showed that intervention in ovariectomized rats with a dose of 300 μg / Kg body weight for 6 weeks can significantly inhibit bone loss caused by ovariectomy, and there are significant improvements in bone morphometric parameters such as bone density, bone volume fraction, trabecular number, and trabecular discreteness, indicating that dental epithelial cell-derived exosomes treatment can effectively prevent the occurrence and progression of osteoporosis caused by ovariectomy.

[0098] 4. After the femur samples were scanned by microCT, the bone samples were decalcified with 10% EDTA decalcification solution for 8 weeks (the decalcification solution was prepared with 4% paraformaldehyde and the decalcification solution was changed once a week); after decalcification, the bone tissue was rinsed with running water overnight. After rinsing, the samples of each group were dehydrated with 75%, 85%, 95%, and 100% gradient alcohol (30-60 minutes for each concentration of alcohol dehydration). After dehydration, each sample was transparentized with xylene (15 minutes, 2 times) and then immersed in wax overnight. After paraffin embedding, 8μm paraffin sections were made for subsequent staining.

[0099] 5. After being baked at 60℃ overnight, the sample slices of each group were treated with xylene, 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol for 10 minutes each, rinsed with PBS twice, 5 minutes each time, and stained with hematoxylin staining solution for 5 minutes. The hematoxylin was absorbed, rinsed with PBS twice, 5 minutes each time, and stained with eosin staining solution for 5 minutes. The slices were then dehydrated with gradient alcohol, transparentized with xylene, sealed with neutral resin, observed under a microscope, and photographed.

[0100] The results of HE staining are shown in the attached Fig.11 As shown: in the distal femur, the trabeculae of the sham group (Sham group) were dense and neatly arranged; in the ovariectomy group (OVX group), obvious trabeculae were lost and broken, the trabeculae were sparse under the field of vision, and a large number of multinucleated cells could be formed in the bone marrow cavity; in the exosome treatment group, sparse and broken trabeculae were observed in the 100μg / Kg body weight group, while relatively complete and continuously arranged trabeculae were observed in the 300μg / Kg body weight treatment group, and relatively complete trabeculae were observed in the 500μg / Kg body weight treatment group, and the trabeculae density was lower than that in the 300μg / Kg body weight treatment group.

[0101] The above histological observation results show that after 6 weeks of intervention with a dose of 300μg / Kg body weight or 500μg / Kg body weight in ovariectomized rats, bone loss can be significantly improved and the progression of osteoporosis can be delayed.

[0102] The present invention extracts exosomes secreted by dental epithelial cells and applies them to the treatment of osteoporosis caused by ovariectomy. The exosomes from dental epithelial cells promote the osteogenic differentiation of bone marrow mesenchymal stem cells, thereby achieving the treatment of osteoporosis. This invention provides a new method for the treatment of osteoporosis and has important clinical application value and socioeconomic significance.

[0103] The above-mentioned embodiments only express the specific implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An application of engineered dental epithelial cell exosomes, characterized in that: The engineered dental epithelial cells include the engineered dental epithelial cell line HERS-H1; and the application includes application in preparing a drug for preventing and / or treating osteoporosis.

2. A drug for preventing and / or treating osteoporosis, characterized in that: Includes exosomes from the engineered dental epithelial cell line HERS-H1.

3. The drug according to claim 2, characterized in that The method for preparing exosomes comprises the following steps: S1. Cultivate engineered epithelial cells HERS-H1, collect the culture medium, centrifuge, collect the supernatant, add the supernatant to a 100KD centrifugal filter, centrifuge, and collect the concentrate; S2. Add exosome extraction reagent to the concentrated solution, incubate, centrifuge, discard the supernatant, and the precipitate is the exosomes of the engineered dental epithelial cell line HERS-H1.

4. The drug according to claim 3, characterized in that In step S1, the engineered epithelial cells HERS-H1 are cultured in an Epithelial cell medium containing 2% fetal bovine serum in an incubator at 37° C. and 5% CO 2 .

5. The drug according to claim 3, characterized in that In step S1, the culture solution is collected and centrifuged at 2000-5000 rpm for 10-60 min; preferably at 3000 rpm for 30 min, and the supernatant is collected.

6. The drug according to claim 3, characterized in that In step S1, the supernatant is added to a 100KD centrifugal filter, centrifuged under low temperature conditions, and the concentrate is collected; Preferably, the centrifugation is carried out at 1 to 6°C, more preferably at 4°C; The centrifugal speed is preferably 4000-6000 rpm, more preferably 5000 rpm; The centrifugation time is preferably 20 to 60 min, more preferably 30 min.

7. The drug according to claim 3, characterized in that In step S2, the volume ratio of the concentrate and the exosome extraction reagent is 2:1; After adding the exosome reagent, incubate at 1-6°C for 10-40 hours, preferably at 4°C, and preferably for 20 hours.

8. The drug according to claim 3, characterized in that After the incubation, centrifuge at 8000-20000 rpm for 30-120 min; preferably, centrifuge at 10000 rpm for 60 min.

9. The drug according to claim 3, characterized in that The method also includes a cell recovery step: recovering the frozen engineered epithelial cells HERS-H1, and then resuspending them with culture medium, centrifuging, discarding the supernatant, and then adding culture medium for a second resuspending for use.

10. The drug according to claim 3, characterized in that The medicine is an injection.

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