Application of M0 type microglial cell exosome in preparation of medicine for treating retinal vascular injury diseases

Abstract

The invention relates to application of an M0 type microglial cell exosome in preparation of a medicine for treating retinal vascular injury diseases. The preparation enters eyeballs of a DR mouse model in a vitreous body injection mode, can effectively cross a blood-retina barrier to diffuse into local retinas of a model mouse, promotes repair of fragile retina blood vessels, relieves retinal tissue inflammatory response and improves DR healing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of M0 type microglial cell exosomes in preparation of a drug for treating retinal vascular injury diseases. BACKGROUND

[0002] As a representative disease of retinal microvascular injury, diabetic retinopathy (DR) is a major factor leading to visual impairment and even blindness in patients. Long-term high blood sugar leads to injury of retinal microvascular endothelial cells, thickening of the basement membrane, pericyte shedding and capillary occlusion, followed by retinal hypoxia, blood-retinal barrier destruction, pathological neovascularization and macular edema. At present, anti-angiogenic therapy targeting vascular endothelial growth factor (VEGF) has become the main treatment for retinal neovascular diseases, although this therapy has achieved certain effect for some patients, but many patients still face problems such as drug resistance, repeated treatment and systemic adverse reactions, which need to be solved. In addition to simply inhibiting neovascularization (such as anti-VEGF alone), another intervention idea for pathological blood vessels is to develop therapeutic drugs that can play a dual role of promoting blood vessel maturation and anti-inflammatory repair in the retina, and restore blood vessel structure and function by promoting blood vessel "re-maturation", thereby improving DR from the root.

[0003] As a natural nanocarrier for intercellular communication, exosomes are rich in proteins, lipids and functional small RNAs, and can stably carry and deliver signal molecules to recipient cells, thereby regulating various biological processes such as cell communication and cell survival. Through exosomes, donor cells can transfer proteins, mRNAs, microRNAs (miRNAs) and lipids to recipient cells. Therefore, these naturally equipped nanocarriers can achieve targeted drug delivery, increase the local concentration of therapeutic drugs and minimize side effects. In addition, exosomes have high biocompatibility and high designability, not only can they cross the blood-brain barrier, but also can bypass the P-glycoprotein drug efflux system to reduce drug resistance. These advantages endow them with great potential in the field of intravitreal drug preparation. SUMMARY

[0004] The technical problem to be solved by the present application is to provide application of M0 type microglial cell exosomes in preparation of a drug for treating retinal vascular injury diseases. The exosomes enter the eyeball of a DR mouse model in the form of an intravitreal injection preparation, can effectively cross the blood-retinal barrier and diffuse into the local retina of the model mouse, promote repair of fragile retinal blood vessels, reduce inflammation of the retinal tissue, and improve the prognosis of DR.

[0005] The application provides application of M0 type microglial cell exosomes in preparation of a drug for treating retinal vascular injury diseases.

[0006] Preferably, the M0 type microglial cell exosomes are derived from microglial cells in a resting or un-polarized state, and the culture condition is a constant temperature and humidity environment of 37 DEG C, 21% O2 and 5% CO2.

[0007] Preferably, the microglial cells are mouse microglial cell line BV2 or human microglial cells HMC3.

[0008] Preferably, the preparation method of the M0 type microglial cell exosomes comprises the following steps:

[0009] (1) ultracentrifugation is used to collect supernatant of the microglial cells after normal culture treatment, 4 DEG C, 300g centrifugation for 10 minutes, taking supernatant, obtaining supernatant I; then supernatant I is placed at 4 DEG C and centrifuged at 2000g for 10 minutes, taking supernatant, obtaining supernatant II;

[0010] (2) supernatant II is placed at 4 DEG C and centrifuged at 10000g for 30 minutes, taking supernatant, obtaining supernatant III;

[0011] (3) supernatant III is placed at 4 DEG C and centrifuged at 100000g for 70 minutes, discarding supernatant, resuspending with filtered PBS buffer; again, the resuspended solution is placed at 4 DEG C and centrifuged at 100000g for 70 minutes, discarding supernatant, finally, the exosomes are resuspended with PBS buffer, obtaining the M0 type microglial cell exosomes.

[0012] Preferably, the drug is an intravitreal administration preparation. The exosomes can enter the retinal area quickly through intravitreal injection, reduce inflammation caused by retinal vascular diseases, neovascularization hemorrhage, retinal fibrosis proliferation, and relieve the visual impairment process caused by retinal vascular diseases.

[0013] More preferably, the intravitreal administration preparation is one or more of exosome suspension, spray, powder, gel, microsphere, emulsion, and liposome.

[0014] More preferably, the total number of exosome particles in the intravitreal administration preparation for each injection into the vitreous cavity is of the order of 10 9 .

[0015] More preferably, the retinal vascular damage disease includes one or more of diabetic retinopathy, retinal vein occlusion (including central retinal vein occlusion and branch retinal vein occlusion), wet age-related macular degeneration (including polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV)), retinal artery occlusion (including central retinal artery occlusion and branch retinal artery occlusion), retinopathy of prematurity, hypertensive retinopathy, radiation retinopathy, drug-induced or inflammatory retinal microangiopathy.

[0016] Advantages

[0017] (1) The present application finds, through in vivo animal experiments, that M0 microglial cell exosomes in a resting state can significantly reduce the levels of inflammation and neovascularization in the retina, can significantly alleviate the inflammatory imbalance of the retinal microenvironment in the disease state, and has important clinical value. And the exosome, as a medium for carrying maternal cell information and intercellular communication signals, can be used for the development of therapeutic drugs for retinal diseases.

[0018] (2) When the M0 microglial cell exosomes used in the present application are used to prepare intravitreal preparations for treating mouse retinal neovascularization-related diseases, the thinning degree of the mouse retina is reduced, the number of neovascularization is reduced, and the expression level of inflammatory factors in the retina is significantly reduced. It is shown that M0 microglial cell exosomes can be used to effectively treat retinal neovascularization-related diseases, and provide a new treatment strategy for the disease.

[0019] (3) The present application uses M0 microglial cell exosomes to co-incubate with human retinal microvascular endothelial cells (HRVEC), and finds that human retinal microvascular endothelial cells will phagocytose M0 microglial cell exosomes, indicating that M0 microglial cell exosomes can be absorbed and taken up by HRVEC, and thus achieve the purpose of treating retinal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Identification of microglia-derived exosomes. A, Transmission Electron Microscope (TEM) imaging analysis of the morphology of exosomes secreted by HMC3 under normal culture conditions; B, Nanoparticle Tracking Analysis (NTA) particle size analysis distribution of exosomes secreted by HMC3 under normal culture conditions; C, Western blot analysis of exosome markers lysosome-associated membrane protein 3 (CD63) and Tumor Susceptibility Gene 101 (TSG101) in exosomes secreted by HMC3 under normal culture conditions (EXO-1, HMC3 microglial cell exosome lysate; EXO-2, BV2 microglial cell exosome lysate).

[0021] Figure 2 Confocal imaging of uptake of microglial cell exosomes by retinal microvascular endothelial cells. A, blank control; B, uptake of Dil-labeled exosomes by retinal microvascular endothelial cells. Red Dil-labeled exosomes, green F-actin-labeled retinal microvascular endothelial cell cytoskeleton, and blue DAPI-labeled nuclei.

[0022] Figure 3 Maturation of blood vessels formed by retinal microvascular endothelial cells in a high-glucose environment after addition of microglial cell exosomes. A, immunofluorescence images of retinal microvascular endothelial cells in each group after addition of microglial cell exosomes, with red representing platelet endothelial cell adhesion molecule-1 (CD31), an indicator of blood vessel formation, and blue representing 4',6-diamidino-2-phenylindole (DAPI), a nuclear stain; B, vascular network formation of retinal microvascular endothelial cells after addition of microglial cell exosomes (experiments were repeated at least 5 times independently). HG in the figure refers to a high-glucose environment.

[0023] Figure 4Figure 6. The effect of HMC3-exosomes on reducing retinal tissue damage in a high glucose mouse model. A, HE staining of the treatment and model groups in a high glucose model; B, the thickness of the retina, the picture was taken 1 mm from the optic disc; C, the number of mature blood vessels in the retina. Data are expressed as mean ± SD, n = 5; data were analyzed by One Way ANOVA, followed by Tukey's post hoc analysis; n.s. = no significant, * p < 0.05, ** p < 0.01, *** p < 0.001; Ctrl, normal control group; DR, retinal vascular lesion damage group; DR + EXO, intravitreal injection of normal HMC3-exosomes.

[0024] Figure 5 Figure 7. The effect of HMC3-exosomes on inhibiting inflammation and promoting mature blood vessel formation in a high glucose mouse model. A, the expression levels of nuclear factor kappa-B (NF-κB) and Interleukin-1 beta (IL-1β) in the retinal tissue of the treatment and model groups in a high glucose model; B, the expression levels of zonula occludens-1 (ZO-1), vascular endothelial growth factor receptor-2 (VEGFR2), and vascular endothelial growth factor A (VEGFA) in the retinal tissue of the treatment and model groups in a high glucose model. Ctrl, normal control group; DR, retinal vascular lesion damage group; DR + EXO, intravitreal injection of normal HMC3-exosomes. DETAILED DESCRIPTION

[0025] The application will be further described below in connection with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims.

[0026] Example 1

[0027] 1. Experimental animals

[0028] Adult male db / db wild-type mice were used to construct an animal model of retinal vascular injury in this study. The mice were purchased from Jiangsu Jiquan Pharmaceutical Biotechnology Co., Ltd. After purchase, they were strictly fed in accordance with the SPF feeding standards and AAALAC international standards at the Shanghai Tongji Hospital Experimental Animal Center. The mice used in the experiment were 11-12 weeks old and had no abnormalities. The relevant experimental operations and feeding management met the requirements of the Shanghai Experimental Animal Management Regulations and other regulations. This study was reviewed and approved by the Shanghai Tongji Hospital Experimental Animal Ethics Committee (Ethical Number: SBKT-2024-237).

[0029] 2. Cells used in the experiment

[0030] The microglial cells used in this study, BV-2 microglial cell line (BV2, RRID: CVCL_0182) and human microglial cell line (HMC3 cells, RRID: CVCL_II76), were purchased from Servicebio Cell Center. Primary human retinal microvascular endothelial cells (HRVEC) were purchased from Fugan Cell Bank (Cat# FH1138).

[0031] 3. Cell culture and subculture

[0032] When the cells grow to 80%-90%, subculture begins. Subculture uses 0.25% trypsin solution, removes old culture medium, and rinses cells with DPBS. Add 0.125% trypsin solution, ensuring that the solution completely covers the cells. Place the culture dish in a 37°C incubator for 2 minutes or until the cells are completely rounded. At the end of the incubation, gently knock the side of the flask to detach the cells from the culture dish. Add AM complete medium to terminate the digestion, and transfer the cell suspension to a centrifuge tube. Centrifuge at 1000 r·min -1 for 5 minutes. Remove the supernatant, resuspend the cells with medium, and seed the cells at a density of 5000 / cm 2 .

[0033] 4. High glucose treatment of cells

[0034] High glucose solution preparation: weigh 0.225 mg of powder into 50 ml PBS to prepare a 50 mM solution of D-glucose (sigma). When human retinal microvascular endothelial cells are mature or when the cells grow to about 50%-60% confluence, add 30 mM D-glucose to the cell culture dish (i.e. 400 microliters of medium + 600 microliters of high glucose solution). The treatment conditions are: 21% (v / v) O2 concentration, 5% (v / v) CO2 concentration, temperature 37°C, critical relative humidity (CRH) 90%. Cells are subjected to subsequent fixation or protein extraction, RNA extraction, etc. The experiment is set up in triplicate.

[0035] 5. Exosome extraction and protein identification

[0036] Extracellular exosomes were extracted using size exclusion method, supernatant of normal cultured BV-2 microglial cell line and human microglial cell line HMC3 were collected, exosomes of cell supernatant were isolated using qEV size exclusion column (IZON, New Zealand), then purified using filter device (Millipore, USA) at 4,000 xg for 50 minutes. Western blotting was used to determine exosome protein markers CD63 (1:1000, Abeam) and TSG101 (1:1000, Abeam). Exosomes were stored at -80°C or immediately used for subsequent experiments.

[0037] 6. Measurement of diameter and number of exosomes

[0038] Take 50 μL of the exosome suspension of the isolated BV-2 microglial cell line and human microglial cell line HMC3, respectively, add filtered PBS solution, and make up to 1 mL. After dilution, filter using a 0.22 μm filter membrane. Use NanoSight NS3000 nanoparticle tracking analyzer to analyze the particle size and concentration of exosomes in different samples. Set parameters: number of shots is 4, and each shot lasts for 40 seconds. Set three replicates for the experiment.

[0039] 7. Transmission electron microscopy

[0040] According to the method in step 5 above, after the exosomes of the isolated BV-2 microglial cell line and human microglial cell line HMC3 are collected, they are sent to Sevyl (Wuhan) Co., Ltd. for transmission electron microscopy detection experiment. Set three replicates for the experiment.

[0041] 8. Cell immunofluorescence

[0042] (1) Fixation: Take the cell confocal dish to be subjected to cell immunofluorescence experiment from the cell room. Discard the culture medium, wash with PBS once, add 4% paraformaldehyde, and fix for 30 minutes.

[0043] (2) Membrane rupture: wash with PBS once, add 0.2% (v / v) Triton X-100, and rupture the membrane for 15 minutes.

[0044] (3) Primary antibody incubation: wash with PBS for 3 times, dilute the corresponding primary antibody (Anti-CD31 1:100 Abeam Cat# ab187377) with immunofluorescence antibody diluent, mix well, and then add to the dark wet box and incubate at 4°C overnight; after the primary antibody incubation, take the wet box out of the refrigerator, equilibrate at room temperature for 5 minutes, and then wash with PBS for 3 times.

[0045] (4) Secondary antibody incubation: Dilute the corresponding species fluorescent secondary antibody (Goat anti-Rabbit IgG antibody, Servicebio, Cat No. GB21404) with antibody diluent, mix well and add it to the sample. Incubate at room temperature in a dark and humidified box for 1 hour, and wash 3 times with PBS.

[0046] (5) Nucleus staining: Use DAPI solution (5 μl / slide), mix well and add, stain at room temperature for 15 minutes in a dark and humidified chamber; wash 3 times with PBS, then add 200 μL of PBS. (6) Imaging: Use laser confocal imaging, and analyze the image data using the accompanying software. The experiment was set up in triplicate.

[0047] 9. Exosome uptake

[0048] The preparation method of M0 type microglial cell exosomes includes the following steps:

[0049] (1) The supernatant of microglia after normal culture treatment was collected by ultracentrifugation. The supernatant was collected at 300g for 10 minutes at 4℃ to obtain supernatant I. Then, supernatant I was centrifuged at 2000g for 10 minutes at 4℃ to obtain supernatant II.

[0050] (2) Centrifuge the supernatant II at 4°C at 10000g for 30 minutes, and take the supernatant to obtain supernatant III;

[0051] (3) Centrifuge the supernatant III at 4°C for 70 minutes at 100,000 g, discard the supernatant, and resuspend it with filtered PBS buffer; then centrifuge the resuspended solution at 4°C for 70 minutes at 100,000 g, discard the supernatant, and finally resuspend the exosomes with PBS buffer to obtain the M0 type microglial cell exosomes.

[0052] Exosomes isolated from the BV-2 microglia and human microglia HMC3 cell lines were stained with a Dil kit for 30 minutes and then cultured with retinal microvascular endothelial cells for 2–4 hours. Cells were stained with 100 μL of phalloidin (MedChemExpress, China) for 10 minutes and mounted on medium containing DAPI (Servicebio). Exosome uptake by HRVECs was visualized under a microscope (ZEISS).

[0053] 10. Tube Formation Experiment

[0054] Free serum and endothelial cell culture medium (ECM) (Sigma-Aldrich, St. Louis, Missouri, USA) were added to pre-chilled 96-well plates at a 1:1 ratio (50 μL / well, BD, USA) and polymerized at 37°C for 30 min to form a thin gel layer. HRVEC (8 × 10⁻⁶) was then added. 4 Cells per well were suspended in conditioned medium supplemented with 5% FBS and injected into plates containing the above mixture. After incubation for 6–8 hours, capillary structures were observed under an optical microscope, and 3–5 fields of view per well were captured using a camera. The angiogenesis analyzer in ImageJ software was used to examine the total length of the tubular structures.

[0055] 11. Western blot analysis

[0056] (1) Sample preparation

[0057] A) Preparation of cell protein samples: After the cells (HRVEC) were treated accordingly, the culture medium was removed, and the cells were washed once with pre-cooled PBS (Gibco, Cat# .C20012500BT). The PBS was aspirated, and an appropriate amount of protein lysis buffer (ThermoFisher Scientific, Cat# .78503) was added. The cells were scraped off using a cell scraper.

[0058] B) Preparation of exosome protein samples: The exosomes obtained by ultracentrifugation were resuspended in protein lysis buffer.

[0059] C) Centrifugation: Incubate the sample on ice for 30 minutes, then centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new EP tube. Aliquot 10 μL of the tissue supernatant for relative protein quantification, and freeze the remainder at -80°C.

[0060] (2) Protein Quantification and Denaturation: Relative protein quantification was performed according to the BCA protein quantification kit (Thermo Fisher Scientific, Cat#.23225) instructions. After relative quantification, each group of samples was diluted to the lowest possible concentration as the final concentration. SDS-PAGE protein loading buffer (5X) (Beyotime, Cat#.P0015L) was added at a volume ratio of 4:1 and mixed thoroughly. The protein samples were placed at 100°C for 5 minutes. The denatured protein samples were stored at -80°C.

[0061] (3) Polyacrylamide gel electrophoresis: SDS-PAGE gel was prepared using the SDS-PAGE gel kit (Yazyme, Shanghai). After the gel was solidified, the prepared protein sample was added to the sample well, and the protein molecular electrophoresis was performed using the Bio-Rad electrophoresis system at a constant voltage of 60-80 V to make the protein electrophoresis to the top of the concentrated gel, and then the voltage was adjusted to 100-120 V until the electrophoresis was completed.

[0062] (4) Transferring: The PVDF membrane (Merck Millipore, Cat#.ISEQ00010) was soaked and activated in methanol, and then balanced with ultrapure water. The completed electrophoresis gel was cut, and the sponge, PVDF membrane, gel, and sponge were clamped into four layers in order, and the membrane was transferred at a constant current of 200 mA for 90-120 min.

[0063] (5) Blocking: After transferring, the PVDF membrane was washed with TBST (prepared by mixing TBS (Servicebio, Cat#.G0001) and Tween20 (Servicebio, Cat#.GC204002) at a volume ratio of 1000:1) 1-2 times, and the PVDF membrane was soaked with 5% (v / v) skimmed milk (FUJIFILM Wako, 190-12865) prepared with TBST, and incubated at room temperature for 1 hour on a shaker.

[0064] (6) Antibody incubation: The membrane was washed with TBST 3 times for 5 minutes each, and the target protein band was cut and placed in an incubation box containing the corresponding primary antibody (VEGFA 1:1000, ServiceBio Cat# GB15165; ZO-1 1:1000, Affinity Biosciences Cat# AF5145; VEGFR2 1:1000, Affinity Biosciences Cat# AF4726; β-actin 1:1000, Abeam Cat# 1854-1; NF-ΚB 1:100, Abeam Cat# ab32536; IL-1β 1:100, Abeam Cat# ab216995), and incubated at 4°C on a shaker overnight. After incubation, the primary antibody was recovered (4-6 times), and the membrane was washed with TBST 3 times for 5 minutes each, and then the membrane was placed in a corresponding secondary antibody (Goat anti-Rabbit IgG antibody, Arigobio, Cat No.ARG65351) containing anti-primary antibody species, and incubated at room temperature for 1 hour on a shaker.

[0065] (7) Expose and develop: Discard the secondary antibody, wash the membrane with TBST for 3 times, 5 minutes each time, prepare HRP chemiluminescence developing solution (Merck Millipore, Cat# WBKLS0500) by mixing A and B at 1:1, immerse the PVDF membrane in the developing solution, and develop and image using the Bio-Rad chemiluminescence system. Set up three replicates for the experiment.

[0066] 12. Construction of mouse retinal vascular injury model and intravitreal injection of drugs

[0067] The purchased mice were raised to 10-12 weeks to test whether the model establishment was successful, and intravitreal injection was performed after the above modeling was completed under a dissecting microscope: a puncture was made about 1 mm behind the corneoscleral limbus of the mouse eyeball using a 30G needle, a microsyringe was used to enter the vitreous cavity obliquely behind the puncture, and the exosome was slowly injected. After keeping the posture for 3 minutes, it was slowly pulled out. The puncture site was lightly pressed with a cotton swab, and tobramycin eye ointment was applied to the wound to prevent infection. The intravitreal injection drugs were 1 ul of normal control group and microglial cell exosome (Exo) resuspension under normal culture conditions with a concentration of 1 x 1010 / mL, and no treatment as the model blank control group (DR), blank control (Ctrl).

[0068] 13. Eye HE staining

[0069] (1) After taking the material, wash the eyeball with PBS, and then place the eyeball in 4% paraformaldehyde for fixation for at least 10 minutes.

[0070] (2) The eyeball section is fixed in a wax block, and the paraffin section is placed in an oven for 30 minutes to make the tissue adhere firmly to the glass slide. The section is sequentially placed in xylene I (8 minutes), xylene II (8 minutes), xylene III (8 minutes), and then sequentially passes through anhydrous ethanol I (5 minutes), anhydrous ethanol II (5 minutes), 85% alcohol (5 minutes), 75% alcohol (5 minutes), and finally rinsed with tap water for 2 minutes.

[0071] (3) The section is placed in hematoxylin staining solution for 6 minutes to make the cell nucleus blue-purple. After staining, differentiate with hydrochloric acid alcohol (1%) for 2 seconds to remove non-specific staining, and then rinse with tap water. Then, return blue with ammonia solution for 15-30 seconds to restore the blue color of the cell nucleus, and finally rinse with tap water.

[0072] (4) The sections are placed in 95% alcohol for 1 minute to dehydrate, and then in eosin staining solution for 10-30 seconds to stain the cytoplasm and collagen fibers pink. After staining, rinse with tap water. (5) The sections are then subjected to dehydration and clearing treatment in the following order: anhydrous ethanol I (30 seconds), anhydrous ethanol II (2.5 minutes), anhydrous ethanol III (2.5 minutes), xylene I (2.5 minutes), and xylene II (2.5 minutes). Finally, the sections are mounted with neutral resin and covered with a coverslip. (6) The sections are examined and analyzed under a microscope.

[0073] 14. Statistical Analysis

[0074] In this study, multiple groups of quantitative data that conformed to a normal distribution and homogeneity of variance were compared pairwise using one-way ANOVA and Tukey post-hoc analysis; Student's t-test was used for comparisons between two groups of quantitative data. Data were analyzed and plotted using Graphpad Prism 9.0 software. All quantitative data are presented as mean ± standard deviation. p-values ​​were used to indicate statistical significance (asterisk: *p < 0.05; **p < 0.01; ***p < 0.001).

[0075] Experimental results:

[0076] 1. Extraction and identification of exosomes

[0077] The morphology of exosomes secreted by microglia was analyzed using transmission electron microscopy (TEM). The exosomes exhibited a clearly defined saucer-like membrane structure under TEM, and their diameter was approximately 100 nm. Figure 1 A). Nanoparticle tracking analysis (NTA) was used to analyze the number and size distribution of exosomes secreted by microglia. The vast majority of exosomes had a diameter less than 200 nm. Figure 1 B) Western blot analysis of exosome markers in normal BV2 and HMC3 exosomes showed that the exosomes contained abundant expression of exosome marker proteins TSG101 and CD63. Figure 1 C).

[0078] 2. Microglial cell exosomes can be absorbed and taken up by retinal microvascular endothelial cells.

[0079] Exosomes from the supernatant of microglial cells HMC3 and BV2 cells were isolated by size exclusion method, Dil staining was performed, and after filtration with a 0.22 μm filter membrane, the exosomes were added to human retinal microvascular endothelial cells for 2 hours of co-incubation, then the cells were fixed and imaged using confocal imaging technology. Dil is a major component of cell membranes and can label exosomes. In this experiment, the HRVEC skeleton was labeled with phalloidin, and Dil-labeled exosomes were taken up by HRVEC into the cell. It is shown that the exosomes secreted by microglial cells can be taken up by HRVEC, thereby serving as a tool for intercellular communication and playing a certain function. Figure 2

[0080] 3. M0 microglial cell exosomes can promote the formation of retinal microvascular endothelial cells

[0081] Using high glucose as an in vitro model of retinal microvascular endothelial cell damage, the results show that the expression level of CD31 in HRVEC cells co-cultured with M0 microglial cell exosomes is significantly increased compared with the high glucose group, and there is no significant difference compared with the control group Figure 3 A) Tube formation experiment shows that M0 microglial cell exosomes can significantly improve the damage of HRVEC lumen caused by high glucose, and can form a more stable and tight vascular network Figure 3 B) It is shown that M0 microglial cell exosomes can alleviate the damage of high glucose to the tube formation ability of retinal microvascular endothelial cells.

[0082] 4. Hypoxia-induced astrocyte exosomes can reduce retinal tissue damage

[0083] The db / db mouse model was used to simulate in vivo retinal vascular damage. BV2 exosomes cultured under normal conditions were injected into the vitreous cavity, and the model blank group and the control blank group were used. The results show that the retinal thickness of the model group mice is significantly thinned, and the injection of M0 microglial cell exosomes into the vitreous cavity can significantly reduce such damage, and there is no statistical difference compared with the normal group. In the detection of the number of mature blood vessels, the number of blood vessels of the model group mice is significantly reduced, and the injection of M0 microglial cell exosomes into the vitreous cavity increases the number of mature blood vessels in the retina. It is shown that M0 microglial cell exosomes can effectively reduce the damage degree of retinal blood vessels Figure 4 ).

[0084] 5. M0 microglial cell exosomes can promote the formation of mature blood vessels and reduce inflammatory response

[0085] In the db / db mouse model, the retinal tissue of the normal group injected with microglial cell exosomes under normal conditions was taken for protein detection, and the results showed that the expression level of inflammatory factors (NF-κB, IL-1β) in the retinal tissue of the retinal vascular damage mice was significantly increased​Figure 5 A), accompanied by the decrease of mature vascular markers (VEGFR2 and ZO-1) and the increase of neovascular markers (VEGFA) Figure 5 B), while intravitreal injection of M0 microglial cell exosomes can reverse this change, and after reversal, each detection index has no statistical difference compared with the normal control group.

[0086] Summary:

[0087] The present application provides a new strategy for treating retinal vascular injury diseases by using M0 microglial cell exosomes. Compared with the traditional therapies such as anti-VEGF drugs, laser or steroids, the breakthrough of the present application lies in using the exosomes produced by M0 microglial cells (such as BV2 or HMC3) cultured under standard physiological conditions as the treatment core.

[0088] In terms of application scenarios, the present application breaks through the limitation of existing therapies which usually target a single or a few diseases, and covers a wide range of retinal vascular injury diseases such as diabetic retinopathy, retinal vein / artery obstruction, wet age-related macular degeneration, and retinopathy of prematurity.

[0089] Compared with the prior art, the present application has multiple outstanding advantages: first, the exosomes of M0 microglial cells, as endogenous substances, have excellent biocompatibility and low immunogenicity, and the safety is much higher than the side effect risks brought by drug-derived or plant-derived (such as patent CN202510998276.1) or induced exosomes (such as patent CN202311635652.8). Second, it realizes precise and efficient drug delivery through intravitreal injection (which can be made into suspension, gel and other dosage forms), not only can quickly target the retinal lesion area, but also through the characteristics of its natural carrier, realizes multi-target point synergistic therapy, at the same time relieves inflammation, inhibits pathological neovascularization, reduces bleeding and fibrosis proliferation, so as to block the visual impairment process from multiple links, and overcome the bottleneck of single target drug (such as anti-VEGF drug) with single efficacy and easy drug resistance.

[0090] In summary, the standardized M0 microglial cell exosomes not only represent a new category of "drugs", but also construct a highly potential natural drug delivery platform, which provides a safer and more comprehensive innovative treatment strategy beyond the traditional thinking for the clinical treatment of a series of refractory retinal vascular diseases.

Claims

1. Use of M0 type microglial cell exosomes in the preparation of a medicament for treating retinal vascular injury diseases.

2. Use according to claim 1, characterized in that: The M0 type microglial cell exosomes are derived from microglial cells in a resting or un-polarized state, and the culture conditions are a constant temperature and humidity environment of 37℃, 21% O2 and 5% CO2.

3. Use according to claim 2, characterized in that: The microglial cells are mouse microglial cell line BV2 or human microglial cells HMC3.

4. Use according to claim 1, characterized in that: The preparation method of the M0 type microglial cell exosomes comprises the following steps: (1) The supernatant of the microglial cells after normal culture treatment is collected by ultracentrifugation, centrifuged at 300g for 10 minutes at 4℃, and the supernatant is taken to obtain supernatant I; then supernatant I is centrifuged at 2000g for 10 minutes at 4℃, and the supernatant is taken to obtain supernatant II; (2) Supernatant II is centrifuged at 10000g for 30 minutes at 4℃, and the supernatant is taken to obtain supernatant III; (3) Supernatant III is centrifuged at 100000g for 70 minutes at 4℃, the supernatant is discarded, and the filtered PBS buffer is used for resuspension; then the resuspended solution is centrifuged at 100000g for 70 minutes at 4℃, the supernatant is discarded, and finally the exosomes are resuspended with PBS buffer to obtain the M0 type microglial cell exosomes.

5. The use according to claim 1, characterized in that: The medicament is a preparation for intravitreal administration.

6. Use according to claim 5, characterized in that: The preparation for intravitreal administration is one or more of exosome suspension, spray, powder, gel, microsphere, emulsion, and liposome.

7. Use according to claim 5, characterized in that: The intravitreal administration preparation has a total particle number of the exosomes in the order of 10 9 .

8. The use according to claim 1, characterized in that: The retinal vascular injury diseases include one or more of diabetic retinopathy, retinal vein occlusion, wet age-related macular degeneration, retinal artery occlusion, retinopathy of prematurity, hypertensive retinopathy, radiation retinopathy, and drug-induced or inflammatory retinal microangiopathy.

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