Application of M2 type microglial cell exosome

Abstract

The invention discloses application of the M2 type microglial cell exosome. The invention discloses an application of an M2 type microglial cell exosome in preparation of a product for treating diabetic retina neuropathy. In combination with a specific embodiment, the M2 type microglial cell exosome (M2-Exos) is separated and proved to promote the survival and axon growth of primary RGCs in vitro. In an STZ-induced diabetic mouse model, in-vivo injection of M2-Exos prevents RGC loss by inhibiting a pro-inflammatory mediator and enhancing an anti-inflammatory factor, so that ganglion cell complex thickness and visual ability are retained. The axon regeneration promoting ability of the M2-Exos is further verified in an optic nerve crushing animal model. The discovery shows that the M2-Exos is a potential neuroprotection and axonal regeneration promotion tool for treating the diabetic retina neuropathy, and the M2-Exos can be used for preparing products for treating the diabetic retina neuropathy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology and molecular biology, in particular to the application of M2-type microglial cell exosomes. BACKGROUND

[0002] Diabetic retinopathy (DR) has long been considered a microvascular complication of diabetes, but increasing evidence shows that diabetes also causes retinal neurodegeneration, i.e. diabetic retinal neuropathy (Prog Retin Eye Res. 2024 Jul;101:101271). In clinical treatment, although there are various methods to reduce diabetic retinal neovascularization, there is currently no effective treatment for diabetic retinal neuropathy. Recent studies have shown that regulating the activation state of microglia cells may become a potential treatment strategy for managing diabetic retinopathy (Angiogenesis. 2024 Aug;27(3):311-331).

[0003] Apoptosis of retinal ganglion cells (RGCs) is an important event in the progression of diabetic retinal neuropathy. The pathogenesis of diabetic retinal neuropathy involves various pathological changes in the retinal microenvironment, among which the changes in inflammatory factors play an important role. It has been found that inflammatory factors are involved in the damage of diabetic retinal ganglion cells. In the process of central nervous system injury repair, M2-type microglia cells are activated, and their anti-inflammatory and neuroregenerative properties have been proven. However, their role and the mechanism behind diabetic retinal neuropathy are not yet clear.

[0004] The current main treatment for DR includes intravitreal injection of anti-vascular endothelial growth factor (VEGF) and steroid drugs, full retinal laser photocoagulation and vitrectomy surgery intervention, which only target the microvascular damage of patients with visual impairment in the late stage. In addition, intravitreal anti-vascular endothelial growth factor has been used as a first-line treatment for DR, especially DME, but many patients do not respond well to this treatment. In these patients with poor response, anti-vascular endothelial growth factor drugs seem to have a temporary effect on microvessels and are damaging to various neural cells in the retina. However, the loss of RGCs is often underestimated during the course of diabetic retinopathy, which can cause irreversible damage to the visual system. Studies have shown that RGC degeneration and loss have been observed in patients with early diabetic retinopathy. Some researchers have also found similar clinical findings in animal models of diabetic retinopathy. Once RGCs are lost, any existing therapy cannot reverse them.

[0005] Therefore, there is a need for new treatment methods to treat diabetic retinal neuropathy. SUMMARY

[0006] Based on this, it is necessary to provide an application of M2-type microglial cell exosomes which can solve the above problems.

[0007] An application of M2-type microglial cell exosomes in the preparation of a product for treating diabetic retinal neuropathy.

[0008] In one embodiment, the product is used to protect in diabetic retinal neuropathy.

[0009] In one embodiment, the product is used to reduce the loss of retinal ganglion cells and promote axon regeneration in diabetic retinal neuropathy.

[0010] In one embodiment, the M2-type microglial cell exosomes are prepared by the following operations: Providing cortical cells and dispersing them with an appropriate amount of culture medium to obtain a cell suspension, planting the cell suspension in a culture flask containing 7.5%-15% FBS and 1%-10% PS in the culture medium, replacing the culture medium every 5-7 days, waiting for the cells to cover the bottom of the culture flask, and then shaking the culture flask on a 37°C shaker for 120 minutes. The obtained cell suspension is separated and the precipitate is retained. The precipitate is further cultured in the culture medium containing IL-4. After the culture is completed, the supernatant is separated. The M2-type microglial cell exosomes are separated from the supernatant.

[0011] In one embodiment, the concentration of IL-4 in the culture medium containing IL-4 is 20 ng / ml.

[0012] In one embodiment, the operation of further culturing the precipitate in the culture medium containing IL-4 is performed for 48 h.

[0013] In one embodiment, the culture medium is DMEM F12 complete medium.

[0014] In one embodiment, the M2-type microglial cells are murine cells.

[0015] In one embodiment, the operation of providing cortical cells is as follows: taking the head of a mammal, soaking it in antibiotics, and then moving it into the culture medium. The cortical area of the prehemisphere of the mammal's head is obliquely cut, the dura mater is removed, and the remaining cortex is moved into another culture medium. The obtained cortex is cut into fragments, 0.125% trypsin is added, and the mixture is placed in a 37°C incubator for digestion. After digestion is completed, fetal bovine serum is added to terminate digestion, and finally the precipitate is separated and taken, which is the cortical cells.

[0016] In one embodiment, the operation of isolating the M2-type microglial cell exosome from the supernatant is as follows: the supernatant is sequentially centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes and 10,000 g for 30 minutes at 4°C, and then filtered with a 0.22 μm sterile filter, the filtrate is ultracentrifuged at 140,000 g for 70 minutes at 4°C, resuspended in PBS and then centrifuged at 140,000 g for 70 minutes, the precipitate is reserved, and the precipitate is the M2-type microglial cell exosome.

[0017] In combination with specific embodiments, the present application isolates and demonstrates that this M2-type microglial cell exosome of the present application promotes the survival and axonal growth of primary RGCs in vitro. In vivo injection of M2-Exos prevents RGC loss by suppressing pro-inflammatory mediators and enhancing anti-inflammatory factors, thereby preserving the thickness of the ganglion cell complex and visual ability in a STZ-induced diabetic mouse model. The axonal regeneration promoting ability of M2-Exos is further verified in an optic nerve crush (ONC) animal model.

[0018] These findings indicate that this M2-type microglial cell exosome (M2-Exos) of the present application is a potential neuroprotective and axonal regeneration promoting tool for treating diabetic retinal neuropathy, and this M2-type microglial cell exosome of the present application can be used for preparing a product for treating diabetic retinal neuropathy.

[0019] The M2-type microglial cell exosome (M2-Exos) prepared by this M2-type microglial cell exosome of the present application plays a protective role in diabetic retinal neuropathy.

[0020] The M2-type microglial cell exosome (M2-Exos) prepared by this M2-type microglial cell exosome of the present application can significantly reduce the loss of retinal ganglion cells (RGCs) and promote axonal regeneration.

[0021] Furthermore, the M2 microglial exosomes (M2-Exos) of the present invention have the following advantages in repairing diabetic retinopathy: 1. Exosomes are naturally secreted nanoparticles. Compared to existing anti-VEGF and steroid drugs, they have higher biocompatibility and are less likely to trigger immune rejection or inflammatory reactions in the vitreous cavity. This avoids the potential genotoxicity of viral vectors or the side effects of chemically synthesized materials. 2. Exosomes can precisely reach the site of retinal or vitreous lesions; exosomes can encapsulate small RNAs, protecting them from degradation by vitreous enzymes and prolonging their duration of action. 3. Exosomes can cross the blood-retinal barrier, enabling systemic administration (e.g., intravenous injection) or local administration (intravitreal injection), reducing the need for invasive procedures. 4. Exosomes can modulate the retinal microenvironment and promote ganglion cell regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A The figure shows the results of identifying the polarization of primary microglia by immunofluorescence, where the scale bar is 50 μm.

[0023] Figure 1B The graph shows the results of flow cytometric analysis of primary microglial cells after induction with IL-4 for 0, 24, and 48 hours.

[0024] Figure 2 Figure 1 is the identification result of exosomes, where A is the morphology diagram, B is the diameter size, distribution and Zeta potential diagram, and C is the result diagram of Western blot determination of specific markers of exosomes.

[0025] Figure 3A Microscopic images of primary RGC uptake of M2-Exos.

[0026] Figure 3B Microscopic view of an eyeball section from an STZ-induced diabetic mouse injected with 2 μg of M2-Exos (labeled with Dil).

[0027] Figure 4A This is a fluorescence image of RGC under a confocal microscope.

[0028] Figure 4B for Figure 4A Statistical chart of .

[0029] Figure 5A Microscopic view of a mouse eyeball section.

[0030] Figure 5B for Figure 5A Statistical chart of .

[0031] Figure 6 This is a fluorescence image of Tunel staining of mouse eyeball sections. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0033] The application discloses application of M2-type microglial cell exosomes in preparation of a product for treating diabetic retinal neuropathy.

[0034] In combination with specific embodiments, the present application separates and proves that the M2-type microglial cell exosomes of the present application promote the survival and axon growth of primary RGCs in vitro. In a STZ-induced diabetic mouse model, in vivo injection of M2-Exos prevents RGC loss by inhibiting pro-inflammatory mediators and enhancing anti-inflammatory factors, thereby preserving the thickness of the ganglion cell complex and visual ability. The axon regeneration promoting ability of M2-Exos is further verified in an optic nerve crush (ONC) animal model.

[0035] These findings show that the M2-type microglial cell exosomes (M2-Exos) of the present application are potential neuroprotective and axon regeneration promoting tools for treating diabetic retinal neuropathy, and the M2-type microglial cell exosomes of the present application can be used in the preparation of a product for treating diabetic retinal neuropathy.

[0036] In addition, the M2-type microglial cell exosomes (M2-Exos) of the present application have the following advantages in the repair of diabetic retinal neuropathy: 1. Exosomes are natural nanoparticles secreted by cells, and have higher biocompatibility than existing anti-VEGF and steroid drugs, and are less likely to cause immune rejection or inflammatory reactions in the vitreous cavity. Avoiding the gene toxicity caused by viral vectors or the side effects of chemically synthesized materials. 2. Exosomes can accurately reach the retinal or vitreous lesion site. Exosomes can encapsulate small RNAs and the like, protect them from being degraded by enzymes in the vitreous body, and prolong the action time. 3. Exosomes can cross the blood-retinal barrier, enabling systemic administration (such as intravenous injection) or local administration (intravitreal injection), reducing the need for invasive procedures. 4. Exosomes can regulate the retinal microenvironment and ganglion cell regeneration and repair.

[0037] Preferably, in the present embodiment, the M2-type microglial cell exosomes (M2-Exos) of the present application play a protective role in diabetic retinal neuropathy.

[0038] It can be seen from the above that the product is used to play a protective role in diabetic retinal neuropathy.

[0039] Preferably, in the present embodiment, the M2-type microglial cell exosome (M2-Exos) of the present application can significantly reduce the loss of retinal ganglion cells (RGCs) and promote axon regeneration.

[0040] It can be seen from the above that the product is used to reduce the loss of retinal ganglion cells and promote axon regeneration in diabetic retinal neuropathy.

[0041] Preferably, in the present embodiment, the M2-type microglial cell exosome is prepared by the following operation: S10, provide cortical cells and disperse them with an appropriate amount of culture medium to obtain a cell suspension, plant the cell suspension in a culture bottle containing 7.5%~15% FBS (preferably 10%) and 1%~10% PS (preferably 1%), replace the culture medium every 5~7 days, shake the culture bottle on a 37℃ shaker for 120 minutes when the cells cover the bottom of the culture bottle, separate the obtained cell suspension and reserve the precipitate, continue to culture the precipitate with IL-4-containing culture medium, and separate the supernatant after the culture is completed.

[0042] The culture medium containing 7.5%~15% FBS (preferably 10%) and 1%~10% PS (preferably 1%) can make the microglial cells adhere more, thereby providing more exosomes for subsequent experiments.

[0043] When the cortical mixed cells cover the bottom of the culture bottle, shake the culture bottle on a 37℃ shaker for 120 minutes to obtain relatively pure microglial cells, thereby providing purer target exosomes for subsequent experiments.

[0044] It should be noted that the culture medium used in the present application does not contain exosomes.

[0045] More preferably, in the present embodiment, the concentration of IL-4 in the IL-4-containing culture medium is 20 ng / ml.

[0046] Particularly preferably, in the present embodiment, the operation of continuing to culture the precipitate with IL-4-containing culture medium has a culture time of 48h.

[0047] More preferably, the operation of shaking the culture bottle on the shaker for 120 minutes is specifically: shaking the culture bottle on the shaker at a speed of 80 rpm for 30 min.

[0048] More preferably, in the present embodiment, the culture medium is DMEM F12 complete culture medium. In other embodiments, the culture medium can also be selected from other types.

[0049] More preferably, in the present embodiment, the M2-type microglial cells are murine cells.

[0050] More preferably, in the present embodiment, the operation of providing the cortical cells is as follows: the head of the mammal is taken, soaked with antibiotics, and then moved into a culture medium, the cortical area of the anterior half of the mammal's head is obliquely cut, the dura mater is removed, the remaining cortex is moved into another culture medium, the obtained cortex is cut into pieces (preferably cut into 1 mm x 1 mm x 1 mm pieces), 0.125% trypsin is added, and then placed in a 37°C incubator for digestion, and then observed under a microscope to determine when the digestion is completed, fetal bovine serum is added to stop the digestion, and finally separated and precipitated (preferably centrifuged at 1000 rpm for 10 minutes), and the precipitate is the cortical cells.

[0051] The antibiotics can be a 1% solution of double antibiotics (streptomycin + penicillin).

[0052] More preferably, in the present embodiment, the operation of separating the supernatant after the culture is completed is as follows: after the culture is completed, centrifuged at 1000 rpm for 5 minutes to obtain the supernatant.

[0053] S20, separating from the supernatant to obtain M2-type microglial cell exosomes.

[0054] More preferably, in the present embodiment, the operation of separating from the supernatant to obtain M2-type microglial cell exosomes is as follows: the supernatant is sequentially centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes at 4°C, and then filtered with a 0.22 μm sterile filter, the filtrate is ultracentrifuged at 140,000 g for 70 minutes at 4°C, resuspended in PBS and then centrifuged at 140,000 g for 70 minutes, the precipitate is retained, and the precipitate is the M2-type microglial cell exosomes.

[0055] The following is a specific embodiment.

[0056] Embodiment 1 1. Extraction and induction of M2-type microglial cells The head of a fresh SPF mouse (C57 mouse born 0-3 days, Nanjing Medical University Animal Center) was taken, soaked in a 1% double-antibiotic (streptomycin + penicillin) solution, and then moved into a 3-ml DMEM F12 medium (the medium mentioned below is this medium). The cortex area of the prefrontal hemisphere was obliquely cut using a microscissors, the dura mater was removed under a dissecting microscope, the remaining cortex was placed in another medium, the tissue was cut into pieces of about 1 mm x 1 mm x 1 mm, 0.125% trypsin was added, and the mixture was placed in a 37°C incubator for digestion. The digestion was observed under a microscope for 20 minutes, and then the digested tissue was sucked into a 50-ml centrifuge tube. Fetal bovine serum was added to stop the digestion, and then the mixture was centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was evenly blown and filtered three times with a filter. The cell suspension was seeded in a T75 culture bottle containing 10% FBS and 1% PS.

[0057] Part of the obtained microglial cells were induced to culture for 48 hours in a medium containing IL-4 (20 ng / ml), and then separated by centrifugation at 1000 rpm for 5 minutes to obtain the supernatant of the induced cells and the induced cells.

[0058] The above microglial cells and the above induced cells were taken respectively, and immunofluorescence staining was further confirmed to obtain Figure 1A .

[0059] Combining Figure 1A It can be seen that the above induced cells contain characteristic proteins in M2-type microglial cells.

[0060] Flow cytometry was performed on the above microglial cells to identify CD11b and CD45, and a flow cytometer was used to evaluate the purity of the obtained primary microglial cells and the percentage of polarization after induction. Part of the above microglial cells were seeded in a confocal dish and a large dish, and were stimulated with IL-4 (20 ng / ml) for 0 hours, 24 hours and 48 hours respectively. M2-type microglial cells were checked by flow cytometry, and the results were as follows Figure 1B .

[0061] Combining Figure 1B It can be seen that the CD11b+ / CD45 weakly positive microglial cells account for more than 85% of the total number of cells; after receiving IL-4 stimulation for 0, 24 and 48 hours, the expression of CD206 is the highest after 48 hours Figure 1B .

[0062] In combination Figure 1A and Figure 1B It can be determined that the induced cells are M2-type microglial cells, and the supernatant of the induced cells is M2-type microglial cell supernatant.

[0063] 2. Isolation and identification of exosomes The collected microglial cell supernatant and M2-type microglial cell supernatant were respectively treated as follows: The supernatant was centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes and 10,000 g for 30 minutes at 4°C, and then filtered with a 0.22 μm sterile filter. The filtrate was ultracentrifuged at 140,000 g for 70 minutes at 4°C, resuspended with PBS, and then centrifuged again at 140,000 g for 70 minutes, and the precipitate was retained, which was the exosome.

[0064] Through the above operation, microglial cell exosomes (M0-Exos) and M2-type microglial cell exosomes (M2-Exos) were obtained respectively.

[0065] The morphology of exosomes was observed by TEM, the diameter size, distribution and Zeta potential of exosomes were evaluated by NTA, and the specific markers of exosomes such as CD9, CD63, CD81 and TSG101 were determined by Western blot, and the following results were obtained Figure 2 .

[0066] In combination Figure 2 It can be seen that both M0-Exos and M2-Exos can be observed to have the typical cup-shaped vesicle morphology of exosomes, the diameters of M0-Exos and M2-Exos are both between 30-150 nm, and Western blot analysis shows that exosome markers such as CD63, CD81 and TSG101 are expressed in M0-Exos and M2-Exos, but not in cells as negative controls. In contrast, Calnexin was mainly detected in cells but not in exosomes.

[0067] 3. Uptake of exosomes by RGCs STZ-induced DR mouse model 90 male C57BL / 6 mice (Nanjing Medical University, weight 18-22 grams, 6 weeks) were fed adaptively for 2 weeks, and 30 of them were injected intraperitoneally with 55 mg / kg STZ (Med Chem Express, Cat. HY-13753) for 5 consecutive days; 30 mice were treated with normal saline as a control group. Serum glucose concentration was monitored 7 days after the start of treatment. Mice with blood glucose levels greater than 16.7 mmol / L were considered diabetic mice (DR mice).

[0068] After the DR mice were perfused with PBS from the heart, the eyeball was taken out, and the retinal tissue was peeled off under a body microscope and cut into about 1 mm x 1 mm x 1 mm pieces with an ophthalmic fiber scissors. A papain and collagenase type II mixture was added and digested for about 20 minutes. The digestion was then terminated, and then filtered through a 70um filter three times to obtain the primary RGCs. After incubation with the corresponding flow antibody for 2 hours, the excess antibody was washed off with PBS three times, and then the flow cytometer was obtained Figure 3A .

[0069] In combination Figure 3A It can be seen that the uptake of M2-Exos (labeled with Dil) by primary RGCs was captured under a confocal microscope.

[0070] The STZ-induced diabetic mice obtained above were injected intravitreally with 2ug of M2-Exos (labeled with Dil), and their eyeball sections were taken out 3 months later. Under a confocal microscope, it was observed that Figure 3B .

[0071] In combination Figure 3B It can be seen that the uptake of M2-exos by RGCs was captured under a confocal microscope.

[0072] 4. M2-Exos promotes the neuroprotection of RGCs PBS (NC), M2-type microglial cell conditioned medium (M2-CM), and M2-type microglial cell medium treated with exosome inhibitor GW4869 (Umibio, UR21021) (M2-CM-GW4869) were added to the primary RGC culture medium neurobasal / B27 (Gibco) to stimulate RGC cells for 24h. After the culture was completed, live and dead cells were distinguished by staining with calcein-AM and propidium iodide (PI) (Beyotime, Cat#C2015S) at 37°C for 15 minutes, and then observed under a confocal microscope to obtain Figure 4A The number of live and dead cells was counted to obtain Figure 4B .

[0073] In combination Figure 4A andFigure 4B It can be seen that M2-CM (containing M2-Exos) reduced RGC apoptosis, while M2-CM-GW4869 (containing exosome inhibitor) had no such effect.

[0074] 5. M2-exo promotes RGC axon regeneration Optic nerve injury (ONC) mouse model Mice (6-week-old male, Nanjing Medical University) were anesthetized with sodium pentobarbital (20 mg / kg) for 2 weeks before the experiment. Lateral keratotomy was then performed on the left eye using a surgical microscope. The conjunctiva was incised lateral to the cornea, and the muscles were separated to expose the optic nerve while avoiding damage to the small blood vessels surrounding the nerve. Fiber forceps were used to clamp the optic nerve 1 mm behind the eyeball for 10 seconds. After surgery, the eyelids were closed, and the integrity of the retinal blood supply was verified. Using a 33G microsyringe needle, 2 mm behind the limbus, 1.5 μL of the various treatment reagents was slowly injected into the vitreous. Aureomycin ointment was applied to the wound to prevent bacterial infection. Two days before the mice were harvested for experimental purposes, CTB-FITC (2 μL; 1 mg / mL) was injected into the vitreous to label regenerating axons.

[0075] After anesthesia, the eyeballs of mice in each experimental group were removed and sliced ​​on ice. Figure 5A , and the experimental results were statistically analyzed to obtain Figure 5B .

[0076] Combine Figure 5A and Figure 5B As can be seen, PBS, M0-Exos, and M2-Exos were injected intravitreally before optic nerve injury. Compared to the PBS group, the regenerated axons in the M2-Exos group were longer. This further confirms that M2-Exos can promote optic nerve axon regeneration.

[0077] 6. M2-exo reduces RGC apoptosis in DR To clarify the neuroprotective effect of M2-exos on RGCs in DR, PBS, M0-Exos, or M2-Exos were injected into the vitreous cavity before DR. Three months after DR, the eyeballs of each experimental group were harvested and paraffin sections were made. Apoptosis was analyzed by Tunel staining using an apoptosis detection kit (Beyotime, Cat#C0006S). Figure 6 .

[0078] Combine Figure 6It can be seen that the eyeball sections of the mice in the above experimental groups were subjected to Tunel staining, and under the confocal microscope, it can be observed that compared with the PBS and M0-Exos groups, the RGCs apoptosis of the M2-Exos group is reduced.

[0079] In the above operations, part of the operations involved are as follows: 1) Dil labeling According to the working solution configuration of Dil (ThermoFisher, D282), incubate M2-exos with Dil working solution at room temperature for 5-30 minutes, and then wash away the excess Dil working solution according to the extraction method of exosomes.

[0080] 2) Transmission electron microscopy observation of ultrastructure First, fix in 4% glutaraldehyde in 0.1 mmol / L cacodylate buffer (pH 7.4) for 1 hour, then fix in 1% osmium tetroxide in 0.1 mmol / L cacodylate buffer for 2 hours. The treated sections are gradually dehydrated in ethanol, then dehydrated with dry acetone, and then embedded in Durcupan ACM. Cut ultra-thin sections (0.1 μm), mount on Formvar-coated grooves, and stain with 3% lead citrate. Observe and take pictures of the ultrastructure by transmission electron microscopy.

[0081] 3) Immunofluorescence staining Fix the cells or tissue sections with 4% paraformaldehyde, permeabilize with 0.3% Triton X-100, block with 5% bovine serum albumin (BSA), then incubate with the corresponding primary antibody at 4°C overnight. Incubate the sections and cells with the corresponding secondary antibody and DAPI reagent for 2 hours, then wash with PBS three times, then mount with mounting medium, cover the coverslips, and finally observe and take pictures of the immunofluorescence images with a fluorescence microscope.

[0082] 4) Western blotting experiment Proteins in cells or tissues are lysed and extracted with RIPA solution. The protein concentration is detected with a BCA kit. After denaturation, the proteins are separated on a SDS-PAGE gel and transferred to a PVDF membrane. Incubate with blocking solution at room temperature for 1 hour, then incubate with primary antibody at 4°C overnight. Wash the PVDF membrane with TBST, incubate with the corresponding secondary antibody for 2 hours, then wash the membrane with TBST, dry the PVDF membrane with filter paper, drop the exposure solution (prepared according to the instructions), then develop with a gel image detection system to observe the protein bands and take pictures.

[0083] 5) Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., USA). Student's t-test was used for comparison of two groups of data. Multivariate analysis used one-factor or two-factor analysis of variance. Data were expressed as mean ± standard deviation. P value less than 0.05 was considered statistically significant.

[0084] In the description of the embodiments of the present application, it should be noted that the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the embodiments of the present application. In addition, the terms "first", "second", "third" are only for the purpose of description and cannot be understood as indicating or implying relative importance.

[0085] In the description of the embodiments of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0086] In the embodiments of the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "above" and "above" the second feature can be that the first feature is directly above or obliquely above the second feature, or it only means that the horizontal height of the first feature is higher than that of the second feature. The first feature "below", "below" and "below" the second feature can be that the first feature is directly below or obliquely below the second feature, or it only means that the horizontal height of the first feature is less than that of the second feature.

[0087] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0088] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of M2 microglial exosomes in the preparation of a product for the treatment of diabetic retinopathy.

2. The use according to claim 1, characterized in that The product is used to play a protective role in diabetic retinopathy.

3. The use according to claim 2, characterized in that The product is used to reduce the loss of retinal ganglion cells and promote axonal regeneration in diabetic retinopathy.

4. The use according to any one of claims 1 to 3, characterized in that The M2 microglial exosomes were prepared by the following operation: Providing cortical cells and dispersing them with an appropriate amount of culture medium to obtain a cell suspension, seeding the cell suspension in a culture flask containing the culture medium containing 7.5% to 15% FBS and 1% to 10% PS, replacing the culture medium every 5 to 7 days, and shaking the culture flask on a shaker at 37° C. for 120 minutes after the cells have covered the bottom of the culture flask. After the obtained cell suspension is separated, the precipitate is retained, and the precipitate is further cultured with the culture medium containing IL-4. After the cells adhere, the culture medium is replaced with a complete DMEM F12 medium without exosomes, and after culturing for 48 hours, the supernatant is separated and obtained; The M2 microglial exosomes are separated from the supernatant to obtain the M2 microglial exosomes.

5. The use according to claim 4, characterized in that The IL-4 concentration in the culture medium containing IL-4 was 20 ng / ml.

6. The use according to claim 5, characterized in that The precipitate was further cultured with the culture medium containing IL-4 for 48 hours.

7. The use according to claim 4, characterized in that The culture medium is DMEM F12 complete culture medium.

8. The use according to claim 4, characterized in that The M2 microglial cells are mouse cells.

9. The use according to claim 8, characterized in that The operation of providing cortical cells is as follows: taking the head of a mammal, soaking it in antibiotics, and then transferring it into the culture medium; cutting the cortical area of ​​the anterior hemisphere of the head of the mammal obliquely, removing the dura mater, and transferring the remaining cortex into another culture medium; cutting the obtained cortex into pieces, adding 0.125% trypsin, and placing it in a 37°C incubator for digestion; observing under a microscope after the digestion is complete, adding fetal bovine serum to terminate the digestion, and finally separating and obtaining the precipitate, wherein the precipitate is the cortical cells.

10. The use according to claim 9, characterized in that The M2 microglial exosomes were isolated from the supernatant by centrifuging the supernatant at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes at 4°C, filtering the supernatant through a 0.22 μm sterile filter, ultracentrifuging the filtrate at 140,000 g for 70 minutes at 4°C, resuspending the filtrate in PBS, and centrifuging it again at 140,000 g for 70 minutes. The precipitate was retained, which was the M2 microglial exosomes.

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