Extraction method and application of olfactory ensheathing cell exosome rich in phagocytic protein

Abstract

The invention discloses application of an olfactory ensheathing cell exosome rich in phagocytic protein in preparation of a medicine for preventing / treating retinitis pigmentosa. According to the method, the olfactory ensheathing cell exosome rich in phagocytic protein is successfully extracted; meanwhile, the OEC-Exo is transplanted into the subretinal space of the retinitis pigmentosa rat through the subretinal space; experimental techniques such as visual electrophysiology, WB (western blot), retina slice immunofluorescence histochemical, PCR (polymerase chain reaction) and the like verify that the position of OEC-Exo in the retina can be swallowed by RPE cells, expression of phagocytic protein in the RPE is improved, the phagocytic ability of the RPE cells to the retina ectonoma is improved, and the retina ectonoma can be effectively inhibited. It is proved that the olfactory ensheathing cell exosome rich in phagocytic protein has the effect of improving the retina visual function, the effect that RCS rats lose phagocytic exosome fragments can be improved, the phagocytic function of RPE cells in RCS is repaired, and the disease progress is delayed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a method for extracting and an application of olfactory ensheathing cell exosomes rich in phagocytic proteins. Background Art

[0002] Exosomes are extracellular lipid nanovesicles with a size of about 30 - 150 nm, which are actively secreted by most living cells through exocytosis and contain various biological substances, including various proteins, mRNAs, and microRNAs. Exosomes naturally exist in body fluids, including blood, saliva, urine, and breast milk. Exosomes from different specimens may have different proteins or lipids, lumen contents, and different deposition characteristics. They can be used for disease diagnosis, personalized prediction through signal molecules in exosomes, and can also be used in vaccine development, immunotherapy, gene therapy, targeted drug therapy, etc.

[0003] Retinitis pigmentosa (RP) is a hereditary retinal disease, the main causes of which are the degeneration of rod and cone photoreceptor cells and the degeneration of retinal pigment epithelial (RPE) cells. In RP patients, the abnormal function of RPE cells may lead to their inability to normally phagocytose and digest retinol, and there is currently no effective treatment for RP. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related technologies to some extent. For this purpose, the main object of the present invention is to provide a method for extracting and an application of olfactory ensheathing cell exosomes rich in phagocytosis-related proteins.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] Application of olfactory ensheathing cell exosomes rich in phagocytic proteins in the preparation of a drug for preventing / treating retinitis pigmentosa.

[0007] In some specific embodiments, the drug includes a pharmaceutically acceptable carrier.

[0008] In some specific embodiments, the dosage form of the drug is a pharmaceutically acceptable dosage form.

[0009] A drug for preventing / treating retinitis pigmentosa, comprising the aforementioned olfactory ensheathing cell exosomes rich in phagocytic proteins.

[0010] A method for preparing the aforementioned olfactory ensheathing cell exosomes rich in phagocytic proteins, comprising the following steps:

[0011] Balance the olfactory ensheathing cell buffer, centrifuge and take the supernatant. Balance the supernatant with the buffer and ultracentrifuge. After centrifugation, discard the supernatant, resuspend the precipitate, and ultracentrifuge again. After centrifugation, discard the supernatant to obtain a small amount of liquid at the bottom, which is the olfactory ensheathing cell exosomes rich in phagocytic proteins.

[0012] In certain specific embodiments, the olfactory ensheathing cells are obtained by the following culture method:

[0013] 1) Select Wistar rats within 8 weeks after birth, decapitate them after soaking with 75% ethanol, take the outermost two layers of the olfactory bulb, isolate the olfactory ensheathing cells by trypsin digestion to obtain a single-cell suspension;

[0014] 2) Place the single-cell suspension in an untreated culture flask for culture, collect the cell supernatant to remove the main fibroblasts, transfer it to a new culture dish and continue to culture, remove other glial cells, transfer it to a culture dish coated with collagen for normal culture to obtain olfactory ensheathing cells.

[0015] In certain specific embodiments, the buffer is PBS buffer solution.

[0016] Compared with the prior art, the present invention has at least the following advantages:

[0017] 1) The olfactory ensheathing cell exosomes rich in phagocytic proteins provided by the present invention have a particle size less than or equal to about 200 nm and a round shape. A large number of vesicle-like enrichments with a bi-concave bilayer membrane structure can be observed, presenting a lipid bilayer and a cup shape; and the present application has successfully extracted the olfactory ensheathing cell exosomes rich in phagocytic proteins;

[0018] 2) The present invention uses the olfactory ensheathing cell exosomes rich in phagocytic proteins for the prevention / treatment of retinitis pigmentosa. The present invention transplanted OEC-Exo into the subretinal space of retinitis pigmentosa rats through the subretinal space, and verified the location of OEC-Exo in the retina, its ability to be phagocytosed by RPE cells, the increase in the expression of phagocytic proteins in RPE, and the improvement of the phagocytic ability of RPE cells for the outer segments of the retina through experimental techniques such as visual electrophysiology, WB (western blot), immunohistofluorescence histochemistry of retinal sections, and PCR. It is proved that the olfactory ensheathing cell exosomes rich in phagocytic proteins have the effect of improving the visual function of the retina, which can improve the function of RCS rats in losing the ability to phagocytose outer segment debris, repair the phagocytic function of RPE cells in RCS, and delay the progression of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention, the following will briefly introduce the drawings required for the specific embodiments or the description of the prior art.

[0020] Figure 1In Example 1 of the present invention, through electron microscopy detection of olfactory ensheathing cell exosomes rich in phagocytic proteins, a morphological structure diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins (Figure A1) was obtained, and primary rat olfactory ensheathing cells were cultured, and cell identification diagrams (Figure A2 and Figure A3) were obtained using olfactory ensheathing cell-specific markers S100 and P75;

[0021] Figure 2 This is a particle size test diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins detected by the present invention through nanoparticle tracking analysis (NTA);

[0022] Figure 3 This is a particle size distribution diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins detected by the present invention through nanoparticle tracking analysis (NTA);

[0023] Figure 4 This is a morphological electron microscopy diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins in the present invention;

[0024] Figure 5 This is a diagram of the proteomic analysis of olfactory ensheathing cell exosomes rich in phagocytic proteins in the present invention;

[0025] Figure 6 This is a protein IPP analysis diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins in the present invention;

[0026] Figure 7 This is a detection diagram of labeled proteins of olfactory ensheathing cell exosomes rich in phagocytic proteins in the present invention;

[0027] Figure 8 This is a diagram of the effect of olfactory ensheathing cell exosomes rich in phagocytic proteins transplanted into the subretinal space of RCS rats on RCS visual function and the protective effect on retinal tissue structure, and the effect on the expression of cone cells and rod cells;

[0028] Figure 9 This is a diagram of the expression of phagocytosis-related proteins detected by WB after transplantation of olfactory ensheathing cell exosomes rich in phagocytic proteins in the present invention;

[0029] Figure 10 This is a distribution diagram of olfactory ensheathing cell exosomes rich in phagocytic proteins in the RCS retina after transplantation through the subretinal space in the present invention;

[0030] Figure 11 This is a diagram of the detection of sections of the RCS retina at 2W and 4W after transplantation of olfactory ensheathing cell exosomes rich in phagocytic proteins through the subretinal space in the present invention;

[0031] Figure 12 This is a diagram of the retinal flat mounts and sections of the RCS retina at 2W and 4W after transplantation of olfactory ensheathing cell exosomes rich in phagocytic proteins through the subretinal space in the present invention;

[0032] Figure 13 Detection of the expression of RPE cells and genes related to RPE cell phagocytosis after transplantation of phagocytosis protein-rich olfactory ensheathing cell exosomes into RCS rats. Specific implementation manners

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments, but it should not be construed as a limitation to the protection scope of the present invention. Any formal equivalent transformation based on the concept of the present invention should be regarded as within the scope of the present invention.

[0034] The present invention generally and / or specifically describes the materials and test methods used in the experiments. For the test methods or testing methods involved, unless otherwise specified, they are all conventional methods; for the reagents or instruments used, unless the manufacturer is indicated, they are all commercially available conventional products and are prepared or used by conventional methods.

[0035] Example 1: Preparation of olfactory ensheathing cells

[0036] This example provides a method for culturing olfactory ensheathing cells. Specifically:

[0037] Select Wistar rats within 8 weeks after birth, soak them in 75% ethanol and then decapitate them. Take the outermost two layers of the olfactory bulbs and obtain a single-cell suspension by digestion with 1.25 g / L trypsin;

[0038] Culture the single-cell suspension in an untreated culture flask for 18 h, collect the cell supernatant to remove the main fibroblasts, transfer it to a new culture dish and continue to culture for 36 h to remove other glial cells, and transfer it to a culture dish coated with collagen for normal culture to obtain olfactory ensheathing cells.

[0039] This example identifies the purity of the prepared olfactory ensheathing cells. Specifically:

[0040] When the confluence of olfactory ensheathing cells reaches 80%, fix the olfactory ensheathing cells with 4% paraformaldehyde for 30 minutes, then permeabilize and block them with 3% Triton X100 - 3% BSA at 37°C for 20 minutes, add specific protein antibodies for olfactory ensheathing cells (S100, MA1-26621 Invitrogen) (P75, ab52987, Abcam) and incubate the antibodies overnight at 4°C. After 12 hours, remove the primary antibody, wash it 3 times with phosphate buffer, 3 minutes each time, and add the corresponding secondary antibody (Goat anti-Rabbit Alexa Fluor TM 488, Invitrogen, A-11008) (Goat anti-Mouse Alexa Fluor TM568, Invitrogen, A-11004), incubated at 37 °C for 1 hour, added nuclear staining solution and incubated at room temperature for 10 minutes, then washed 3 times with phosphate buffer for 3 minutes each time, and photographed with an anti-fluorescence quenching agent covering the surface. The results are as Figure 1 shown. It can be seen from the figure that the cell morphology of primary olfactory ensheathing cells of cultured rats is shown in Figure A1 under light microscopy, and the cells were identified with olfactory ensheathing cell-specific markers S100 and P75 (as shown in Figures A2 and A3),), for the purity detection of olfactory ensheathing cells. It can be known from the figure that the purity of the olfactory ensheathing cells is high.

[0041] Example 2: Preparation of olfactory ensheathing cell exosomes rich in phagocytic proteins

[0042] The extraction method of exosomes rich in phagocytic proteins provided by the present invention is specifically as follows: Take out olfactory ensheathing cells from the -80 °C refrigerator (the olfactory ensheathing cells obtained by the preparation method of Example 1 in this example), balance with PBS, centrifuge at 1500×g, 4 °C for 15 minutes; take the supernatant, centrifuge at 10000×g, 4 °C for 30 minutes; after centrifugation, take the supernatant, centrifuge at 14000×g, 4 °C for 30 minutes; after centrifugation, take the supernatant, transfer the supernatant to an ultra-high-speed centrifuge tube, balance with 1×PBS buffer on the balance with a pipette, carefully place it in an ultra-high-speed centrifuge, 4 °C, ultra-high-speed centrifuge at 110000×g for 120 minutes; after centrifugation, carefully discard the supernatant, resuspend the precipitate with 1×PBS buffer, 4 °C, ultra-high-speed centrifuge at 110000×g for 60 minutes; after centrifugation, carefully discard the supernatant, and the trace amount of liquid at the bottom of the centrifuge tube obtained is the exosomes. Carefully pipette the bottom of the centrifuge tube with PBS solution, aspirate it into a 0.5 - 1.5 ml centrifuge tube, and gently pipette with a pipette to completely dissolve it, that is, obtain olfactory ensheathing cell exosomes rich in phagocytic proteins (abbreviated as OEC-Exo).

[0043] This application conducts performance identification on the prepared olfactory ensheathing cell exosomes rich in phagocytic proteins, specifically as follows:

[0044] 1) Particle size distribution of olfactory ensheathing cell exosomes rich in phagocytic proteins

[0045] Wash the sample cell with deionized water, calibrate the instrument (ZetaView, S / N 20 - 577) with polystyrene microspheres (100 nm), then wash the sample cell with 1×PBS buffer. Take 1 ul of the olfactory ensheathing cell exosome sample rich in phagocytic proteins and dilute it 1000 times with 1×PBS buffer, then push it into the sample cell for injection detection; the results are as Figure 2 It can be seen that the size of the exosomes, Figure 3 as shown in the size distribution of the exosomes. It can be seen from the figure that the particle size range of the olfactory ensheathing cell exosomes is within 80 - 220 nm.

[0046] 2) Morphological structure of olfactory ensheathing cell exosomes rich in phagocytic proteins

[0047] The olfactory ensheathing cell exosome sample rich in phagocytic protein was shaken and mixed and centrifuged. Use a pipette to draw about 15ul of the olfactory ensheathing cell exosome sample rich in phagocytic protein onto the copper mesh and let it stand for 1min. The tweezers used should be gentle to clamp the copper mesh to prevent the copper mesh from being broken. Use filter paper to absorb the olfactory ensheathing cell exosome sample rich in phagocytic protein on the copper mesh, and then use a pipette to absorb about 15ul of 2% uranyl acetate staining solution and stain it at room temperature for 1min. If obvious adsorbents are visible on the copper mesh, pure water can be dripped onto the surface, quickly absorbed, and repeatedly washed several times. Use filter paper to absorb the olfactory ensheathing cell exosome sample rich in phagocytic protein on the copper mesh, air-dry the stained sample, observe and take pictures, and save the pictures. The results are as follows: Figure 4 As shown in the figure, it can be seen that the size of the exosome nanoparticles of the olfactory ensheathing cells is less than or equal to about 200nm and the shape is round. A large number of vesicle-like enrichments with a biconcave double-layer membrane structure can be observed, showing a lipid bilayer and a cup shape.

[0048] 3) Identification of phagocytic proteins in exosomes of olfactory ensheathing cells rich in phagocytic proteins

[0049] This application uses proteomics to analyze olfactory ensheathing cells and their secreted olfactory ensheathing cell exosomes rich in phagocytic proteins. The results are as follows Figure 5 As shown in the figure, it can be seen that there are 1609 significantly different proteins in the exosomes of the olfactory ensheathing cells rich in phagocytic proteins (such as Figure 5 A, 5B), of which 487 were up-regulated in the exosomes of olfactory ensheathing cells rich in phagocytic proteins, and 1122 were down-regulated in the exosomes of olfactory ensheathing cells rich in phagocytic proteins; the volcano plot (as shown in Figure 5 C), it can be seen that the components are highly reproducible; further analysis of the KEGG signaling pathway of the differentially expressed proteins showed that the endocytic signaling pathway of the proteins in the exosomes was highly expressed (such as Figure 5 D, 5E); further analysis using protein IPP showed that the differentially expressed proteins were divided into three clusters, and the first cluster with the highest content was mainly involved in the endocytic signaling pathway (as shown in Figure 6 The above results confirm that olfactory ensheathing cell exosomes are rich in endocytic proteins.

[0050] 4) Identification of phagocytic proteins in olfactory ensheathing cell exosomes enriched with phagocytic proteins

[0051] Protein detection further confirmed the exosomes (E). After vortexing and centrifugation, the exosome samples of olfactory ensheathing cells rich in phagocytic proteins were added with 50 μl of protein lysis buffer (Thermo Scientific TM, (89900) After thorough mixing, it was lysed on ice for 20 minutes. 1 μl was taken and mixed with the BCA protein concentration assay reagent (Sigma - Aldrich, BCA1 - 1KT), and then reacted at 37 °C for 30 minutes. The protein concentration was detected at a wavelength of 450 nm. According to the protein concentration, 30 μg of protein was loaded onto a 10% SDS - polyacrylamide gel and electrophoresed at 160 V for 40 minutes. After 40 minutes, it was transferred for 90 minutes. After blocking with 5% BSA at room temperature for 1 hour, primary antibodies CD63 (Invitrogen, MA5 - 35208), CD9 (Invitrogen, MA5 - 31980), TSG101 (Invitrogen, MA5 - 32463) were added and incubated overnight at 4 °C. After washing with the washing solution on a shaker for 30 minutes, secondary antibodies (Invitrogen 31430, 31460) were added and incubated at 37 °C for 1 hour. After washing with the washing solution on a shaker for another 30 minutes, it was developed and exposed. The results are as Figure 7 shown. The results suggest that the olfactory ensheathing cell exosomes rich in phagocytic proteins can express CD63, CD9, and TSG101 proteins.

[0052] Example 3: Application of olfactory ensheathing cell exosomes rich in phagocytic proteins in retinitis pigmentosa

[0053] This example provides the application of olfactory ensheathing cell exosomes rich in phagocytic proteins in retinitis pigmentosa. Specifically:

[0054] At 21 days of RCS rats, OEC - Exo was transplanted into the subretinal space of retinitis pigmentosa rats through the subretinal space. Through experimental techniques such as visual electrophysiology, WB (western blot), immunohistofluorescence histochemistry of retinal sections, PCR, etc., the location of OEC - Exo in the retina was verified, its ability to be phagocytosed by RPE cells was verified, the expression of phagocytic proteins in RPE was increased, and the phagocytic ability of RPE cells for the outer segments of the retina was increased.

[0055] 1) Visual electrophysiological detection

[0056] At 21 days of RCS rats, OEC - Exo was transplanted into the subretinal space of retinitis pigmentosa rats through the subretinal space. Visual electrophysiological detection was performed at 1W, 2W, and 4W after the operation. The results are as Figure 8 shown. It can be seen from the figure that after RCS rats were transplanted with olfactory ensheathing cell exosomes rich in phagocytic proteins, their visual function was improved (A - J); at the same time, it was observed through retinal sections that the number of apoptotic cells in the rats in the transplanted exosome group was significantly less than that in the PBS group (K - R), the outer nuclear layer thickness was also protected, and at the same time, the expression protein of cone cells was higher (S - V), indicating that OEC - Exo transplantation has the effect of improving the retinal visual function.

[0057] 2) Western Blot (WB) assay

[0058] At 21 days of age in RCS rats, OEC-Exo was transplanted into the subretinal space of rats with retinitis pigmentosa through the subretinal space. At 14 and 28 days after surgery, the eyeballs containing the RPE-Bruch membrane complex were gently picked up with forceps or carefully picked out with curved forceps and placed on the surface of a culture dish; washed 3 times with PBS for 5 minutes each time, and then 0.1% TritonX-100 permeabilization solution was added for 6 hours; 3% BSA-PBS blocking solution was added for 3 hours, the blocking solution was discarded, and then the primary antibodies (mertk, Invitrogen, PA5-102027) (Rab5a, Invitrogen, PA5-29022) (Rab7a, Invitrogen, PA5-52369) (β-actin, Abcam, ab8226) diluted with 3% BSA-PBS blocking solution were added, placed in a dark box and stored in a 4°C refrigerator for 24 hours; washed 3 times with PBS for 5 minutes each time, and then the secondary antibody diluted with 3% BSA-PBS blocking solution was added, placed in a dark box and incubated at room temperature for 6 hours; washed 3 times with PBS for 5 minutes each time, anti-fluorescence quenching mounting medium was added, and the slides were mounted and observed. Mertk is the main protein for the RPE cells of RCS rats to initiate the function of phagocytosing outer segment debris. The results are as Figure 9 shown. There is no Mertk protein in RCS rats. Therefore, RCS rats cannot phagocytose outer segment debris. As can be seen from the figure, after transplantation of olfactory ensheathing cell exosomes through the subretinal space, Mertk protein can be detected within 2 weeks, and the transplantation group highly expresses Rab5a and Rab7a proteins. Therefore, it can effectively protect the visual function of RCS rats, effectively protect retinal photoreceptor cells, reduce the apoptosis of photoreceptor cells, and protect the number of cone cells and rod cells; while the protein expression of Mertk cannot be detected 4 weeks after transplantation of RCS exosomes, and there is no significant difference in the protein expression levels of Rab5a and Rab7a compared with the RCS control group.

[0059] 3) Immunofluorescence immunohistochemistry detection of retinal sections

[0060] At 21 days in RCS rats, OEC-Exo was transplanted into the subretinal space of retinitis pigmentosa rats through the subretinal space. At 14 days and 28 days after surgery, fresh eyeballs were taken as soon as possible, placed in 4% paraformaldehyde for 30 minutes, and after removing the excess tissue for 60 minutes, placed in 30% sucrose overnight. After 12 hours, the excess moisture was removed by air drying. After placing the eye cup in the embedding tube, OCT embedding agent was added to immerse the tissue, and then the special small box was slowly placed flat in a small cup containing liquid nitrogen. When the bottom of the box touched the liquid nitrogen, it began to vaporize and boil. At this time, the small box was kept in place and not immersed in the liquid nitrogen. After about 10 - 20 s, the tissue quickly froze into a block. After making the frozen block, it could be placed in a cryostat for frozen sectioning. A layer of OCT embedding glue was applied on the sample holder, and the frozen tissue was placed on it. The tissue was pre-cooled in a 4℃ refrigerator for 5 - 10 min to allow the OCT glue to penetrate the tissue. The thin slices were 5 - 10 μm. After cutting, they were placed at room temperature for 30 min, then fixed in 4℃ acetone for 5 - 10 min, and dried in an oven for 20 min. Washed with PBS for 5 min × 3 times. Blocked the sections with PBS containing 10% normal goat serum at room temperature for 1 hour, dropped the appropriately diluted primary antibody or primary antibody working solution, placed the sections in an immunohistochemistry wet box with PBS added, and incubated at room temperature for 2 hours or overnight at 4℃. The next day, first place the wet box at 37℃ to warm up for 1 hour, then aspirate the primary antibody on the slides for recovery, and insert the sections into a small staining cylinder for PBS rinsing. Drop the secondary antibody diluted with PBS (protected from light) and incubate in a molecular hybridization box at 37℃ for 1 hour. Recover the secondary antibody, place the sections in the staining cylinder, and wash with PBS for 5 min × 3 times. Drop the DAPI working solution to stain the nuclei, at room temperature for 10 - 20 min (working concentration 0.1% for 15 min). Recover the DAPI, drop 5 - 10 μl of anti-fluorescence quenching mounting medium, cover the slides with a clean coverslip, and then observe and take pictures under a fluorescence microscope or a confocal microscope. The results are as Figure 10 shown. It can be seen from the figure that after transplantation of olfactory ensheathing cell exosomes through the subretinal space, they are distributed in the subretinal space in the RCS retina; as Figure 11 shown, after transplantation of olfactory ensheathing cell exosomes through the subretinal space, they are phagocytosed by retinal pigment epithelial cells in the 2W and 4W RCS retinas, and the expression of the RPE marker protein can be effectively protected and significantly increased; as Figure 12 shown, after transplantation of olfactory ensheathing cell exosomes through the subretinal space, the expression of the photoreceptor cell rhodopsin marker Opsin can be observed in the retinal pigment epithelial cells in the RCS retina 2W and 4W after transplantation, while there is no expression of Opsin in the control group protein, suggesting that olfactory ensheathing cell exosomes can improve the function of RCS rats in losing the phagocytosis of outer segment debris and restore the phagocytic function of RPE in RCS.

[0061] 4) RT-PCR detection in OEC-Exo retinal tissue

[0062] At 21 days in RCS rats, OEC-Exo was transplanted into the subretinal space of retinitis pigmentosa rats through the subretinal space. At 14 days and 28 days after surgery, fresh retinal tissues were taken, 1 ml of TRIzol reagent was added, and the tissues were minced with sterile scissors. After sonication at 50 Hz for 1 minute using a tissue sonicator, 200 μl of chloroform was added to each tube, and after thorough mixing, the mixture was left standing on ice for 10 minutes. Then, it was centrifuged at 12,000 g for 15 minutes at 4°C. Take a new EP tube, add 400 μl of isopropanol, pre-cool on ice. After centrifugation, transfer the upper aqueous phase (about 300 μl) to this new EP tube, let it stand on ice, precipitate with alcohol for 10 min, centrifuge at 13,000 rpm for 10 minutes, discard the supernatant, dry the RNA, add 10 μl of EEPC water, measure the concentration, reverse transcribe the RAN into cDNA, and then perform RT-PCR detection according to the reagent instructions and instrument requirements. The results are as Figure 13 shown. It can be seen from the figure that after transplanting olfactory ensheathing cell exosomes into RCS rats, the expression of the RPE marker gene RPE65 in the retina can be increased. At the same time, within 2 weeks after transplantation, the expression levels of the RPE cell phagocytosis genes Mertk, Mertk ligand Gas6, ProS1, the phagocytosis-related gene FAK, the early phagosome marker gene Rab5a, and the late phagosome marker gene Rab7a show significant differences; while at 4 weeks, there are significant differences in the expression of the RPE cell phagocytosis-related genes only for Mertk and Rab5a, and there are no significant differences in other related genes.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered within the scope of the claims and the description of the present invention.

Claims

1. Application of olfactory ensheathing cell exosomes rich in phagocytic proteins in the preparation of drugs for the prevention / treatment of retinitis pigmentosa.

2. The use according to claim 1, characterized in that: The drug includes a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form.

4. A drug for preventing / treating retinitis pigmentosa, characterized in that: The invention relates to an olfactory ensheathing cell exosome rich in phagocytic proteins according to any one of claims 1 to 3.

5. A method for preparing olfactory ensheathing cell exosomes rich in phagocytic proteins according to any one of claims 1 to 3, characterized in that: The steps include: The olfactory ensheathing cell buffer was balanced, and the supernatant was obtained by centrifugation. The supernatant was balanced with buffer and ultra-high-speed centrifugation was performed. After centrifugation, the supernatant was discarded, and the precipitate was resuspended and ultra-high-speed centrifugation was performed. After centrifugation, the supernatant was discarded to obtain a small amount of liquid at the bottom, which was the olfactory ensheathing cell exosomes rich in phagocytic proteins.

6. The method for preparing olfactory ensheathing cell exosomes rich in phagocytic proteins according to claim 5, characterized in that: The olfactory ensheathing cells are obtained by the following culture method: 1) Wistar rats within 8 weeks of birth were selected, soaked in 75% ethanol and then decapitated. The outermost two layers of the olfactory bulb were removed, and the olfactory ensheathing cells were isolated by trypsin digestion to obtain a single cell suspension; 2) Place the single cell suspension in an untreated culture flask, culture it, collect the cell supernatant to remove the main fibroblasts, transfer it to a new culture dish to continue culturing, remove other glial cells, and transfer it to a collagen-coated culture dish for normal culture to obtain olfactory ensheathing cells.

7. The method for preparing olfactory ensheathing cell exosomes rich in phagocytic proteins according to claim 5, characterized in that: The buffer solution is PBS buffer solution.

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