Application of plasma exosome in treatment of optic nerve retinal degeneration diseases

Abstract

The invention discloses application of plasma exosomes in treatment of optic nerve retinal degeneration diseases. The invention further finds that the plasma exosome can obviously promote proliferation and function recovery of retinal ganglion cells for the first time, and can effectively improve optic nerve injury in optic nerve retinal degeneration diseases. Especially, the plasma exosome of the glaucoma patient has a more remarkable effect of improving optic nerve injury in neural retinal degeneration diseases. The plasma exosome provided by the invention is simple in preparation method, can be directly extracted from plasma, does not need a cell culture process, does not need additional components, and has the advantages of wide applicability, good safety, no animal-derived components and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the treatment of optic nerve and retinal degenerative diseases. Background Art

[0002] Extracellular vesicles (EVs) are expected to be used as liquid biopsy molecular markers, cell replacement therapy and drug delivery carriers, and have bright prospects in the development of precision medicine. Small extracellular vesicles (sEVs) are EVs with a diameter of less than 200 nm secreted by living cells. They carry biologically active molecules such as intracellular nucleic acids and are important mediators of intercellular information transmission. They are widely distributed in various body fluids and are closely related to a variety of pathological and physiological processes. They have great application potential in the diagnosis and treatment of diseases. Compared with the problems of immune rejection, survival, and tissue integration faced by cell therapy, sEVs have many advantages such as low immunogenicity and easy passage through tissue barriers (such as blood-brain barrier and blood-eye barrier). For example, sEVs derived from induced pluripotent stem cells (iPSCs) have significant therapeutic effects on osteoarthritis, while sEVs derived from mesenchymal stem cells (MSCs) have a neuroprotective effect in traumatic brain injury. sEVs derived from neural progenitor cells (NPCs) can significantly inhibit the apoptosis of photoreceptor cells in animal models of retinal degeneration. sEVs derived from bone marrow MSCs promoted the survival of RGC cells and axon regeneration in the rat optic nerve injury model through the miRNA they carried. Schwann cells differentiated from adipose stem cells can also transmit RNA information through sEVs to promote axon growth. sEVs derived from microglia can reduce photoreceptor cell damage in the ROP model and inhibit angiogenesis.

[0003] Optic nerve damage is a common feature of glaucoma, hereditary optic neuropathy, optic neuritis, and ischemic, toxic, traumatic and other optic neuropathies. So far, there is no effective treatment.

[0004] At present, there are some reports on the potential therapeutic effects of exosomes from different cell sources in corneal diseases, autoimmune uveitis, glaucoma and retinal diseases. For example, exosomes isolated from human corneal mesenchymal stem cells can be absorbed by corneal epithelial cells, promote corneal epithelial cell migration and proliferation, and inhibit corneal neovascularization; after bone mesenchymal stem cell exosomes (BMSCs-Exo), RGC neurological function was significantly improved; although UMSCs-Exo can significantly improve the survival rate of Brn3a+RGC in the retinal ganglion cell layer and increase the activation of glial fibrillary acid protein in glial cells, it cannot promote axon regeneration; MSCs-Exo contains many pro-survival related proteins from the cAMP response element binding protein pathway and the heat shock protein (HSP) pathway that supports survival, which can effectively reduce the severity of retinal ischemia in mice with oxygen-induced retinopathy (OIR); the above research results show that the differentiation of exosome miRNAs from different sources may be the main factor in their application in the treatment of different diseases.

[0005] Existing research focuses more on the mechanism of action of stem cell exosomes in eye diseases, but there is no corresponding research on the application of plasma exosomes in optic nerve and retinal diseases. Stem cell exosomes have problems such as time-consuming cultivation and collection, high cost, and inability to produce in large quantities, which makes them currently unable to be used clinically. Summary of the invention

[0006] The purpose of the present invention is to provide the first application of plasma exosomes in the treatment of optic nerve and retinal degenerative diseases.

[0007] The plasma exosomes are derived from the plasma of patients with optic nerve and retinal diseases or the plasma of normal people, and the eye diseases include optic nerve damage and retinal diseases.

[0008] Preferably, the exosomes are derived from the plasma of autologous neuroretinopathy patients or normal subjects.

[0009] Preferably, the exosomes derived from the plasma of patients with optic nerve damage diseases are specifically exosomes derived from the plasma of patients with primary glaucoma.

[0010] Preferably, the exosomes are derived from the plasma of patients with allogeneic neuroretinopathy or normal subjects.

[0011] Preferably, the exosomes derived from the plasma of patients with optic neuroretinopathy are specifically exosomes derived from the plasma of patients with primary glaucoma.

[0012] Preferably, the drug for treating optic nerve damage is one of the drugs for treating glaucoma, autoimmune, hereditary optic neuropathy, optic neuritis or ischemic, toxic or traumatic optic nerve damage.

[0013] Preferably, the retinal degenerative diseases include hereditary retinal diseases such as ischemic retinopathy, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration or retinitis pigmentosa.

[0014] The present invention also provides a plasma-derived exosome composition, comprising: plasma-derived exosomes and a medically acceptable solvent, wherein the exosomes are derived from the patient's autologous plasma or the plasma of a normal person.

[0015] Through experimental verification in the examples, the present invention has found for the first time that plasma exosomes can significantly promote the proliferation and functional recovery of retinal ganglion cells, and can effectively improve optic nerve damage in optic nerve retinal degenerative diseases, especially plasma exosomes in patients with glaucoma are more effective in improving optic nerve damage in neuroretinal degenerative diseases. Plasma exosomes can maintain the thickness of ganglion cells, the inner plexiform layer and the entire retina, have the effect of protecting visual function, and have the effect of inhibiting retinal damage caused by glial cell activation.

[0016] The plasma exosome preparation method provided by the present invention is simple and can be directly extracted from plasma without the need for a cell culture process or the addition of additional components. It has the advantages of low cost, wide applicability, good safety, and no animal-derived components.

[0017] In addition, autologous plasma exosomes are used in the treatment and improvement of optic nerve and retinal degenerative diseases. They overcome the defects of exosomes cultured from extracellular cells, which are of allogeneic origin and require strict control during the preparation process, such as immunogens, contamination, etc. However, autologous plasma exosomes do not require additional culture and have excellent safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A is the TEM identification image of exosomes; 1B is the NTA particle size distribution image of exosomes; 1C is the characteristic protein marker of exosomes detected by WB;

[0019] Figure 2 Establish schematic diagrams for animal experimental models;

[0020] Figure 3 A is a representative optical coherence tomography (OCT) image of the in vivo RGCs complex cross section of the experimental and control groups; 3B is a statistical graph of the thickness of the RGCs complex cross section; 3C is the number of surviving RGCs in the experimental and control groups; 3D is Figure 3 Result statistics of C;

[0021] Figure 4 A is the fluorescence image and statistical result of the number of microglia in the retinal tissue of the experimental group and the control group; 4B is the result of microglia activation in the outer plexiform layer of the retinal tissue of the experimental group and the control group. DETAILED DESCRIPTION

[0022] As mentioned above, in view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be further disclosed and explained in conjunction with examples below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Example 1: Patient specimen collection

[0024] A total of 70 plasma samples from patients with primary glaucoma and 70 plasma samples from patients with cataract were collected from Wenzhou Medical University Eye and Optometry Hospital. This study was approved by the Ethics Committee of Wenzhou Medical University Eye and Optometry Hospital and was conducted in accordance with the principle of informed consent.

[0025] Cases were collected according to the following criteria: (1) minimum intraocular pressure (IOP) greater than 21 mmHg; (2) with or without abnormal angle structure; (3) characteristic optic disc damage of glaucoma (cup-to-disc ratio>0.6); (4) corresponding visual field loss; (5) no other fundus diseases, ocular trauma, and systemic diseases. The names, gender, age, eye disease history, and systemic clinical symptoms of all participants were recorded. Slit lamp examination, gonioscopy, intraocular pressure measurement, fundus photography, optical coherence tomography (OCT), and visual field (VF) examination were performed on both eyes or the affected eye. Patients with systemic hemorrhagic diseases, coagulation diseases, immune diseases, severe cardiovascular and cerebrovascular diseases, heart failure or renal failure, metabolic diseases such as diabetes, other intraocular vascular proliferative diseases, and those with abnormal refractive media who could not see the fundus clearly were excluded.

[0026] Example 2: Isolation and validation of plasma exosomes from patients with primary glaucoma

[0027] 2.1 Phenotypic identification of plasma exosomes

[0028] Take advantage of Ribo TM Exosome separation reagent combined with differential centrifugation separation method was used to separate total exosomes from plasma. Through transmission electron microscopy, it was found that the exosomes had a typical cup-shaped structure ( Figure 1A). NanoSight shows that the main peak particle size of the separated exosomes is 95nm, which is consistent with the size of plasma exosomes ( Figure 1 B). Immunoblotting experiments verified the exosome-specific positive and negative proteins: Alix, SDCBP, CD9 and Calnexin. Figure 1 As shown in C, Alix, SDCBP, and CD9 were significantly enriched in plasma exosomes, while Calnexin was not detected in the exosome samples, proving that the extracted samples were exosomes.

[0029] Example 3. Preparation of a chronic ocular hypertension mouse model

[0030] The mice used in the experiment were provided by Wenzhou Medical University Eye Hospital and approved by the Ethics Committee of Wenzhou Medical University Eye Hospital. We used 2-3 month old mice, and after 1 week of adaptive feeding, intraperitoneal injection anesthesia was performed. After general anesthesia, the eyeball was anesthetized and disinfected with iodine tincture. About 2 μl of silicone oil was pushed from the posterior chamber into the anterior chamber to cause aqueous humor block to create a chronic ocular hypertension (COH) model. The preparation process is as follows: Figure 2 shown.

[0031] Example 4. Therapeutic effects of plasma exosomes from different sources on a mouse model of chronic ocular hypertension

[0032] The chronic high intraocular pressure model mice were injected with 500ng plasma exosomes from primary glaucoma patients (denoted as Gla-PDEV) and plasma exosomes from cataract patients (denoted as Cont-PDEV) through the vitreous cavity per eye. After 7 days, the therapeutic effects of the above two exosomes on the chronic high intraocular pressure model mice were detected. A control group was set up, and 500ng PBS (denoted as Sham) was injected into each eye of the chronic high intraocular pressure model mice through the vitreous cavity.

[0033] The thickness of the retinal ganglion cell complex (GCC) of the retina was detected by OCT, and it was found that the thickness of the GCC layer in the plasma exosomes treated group of primary glaucoma patients and the plasma exosomes treated group of cataract patients was increased compared with that in the untreated group. Among them, the GCC layer in the plasma exosomes treated group of primary glaucoma patients was thicker. Figure 3 As shown in A and B.

[0034] By immunofluorescence, Figure 3 As shown in C and D, the number of retinal ganglion cells (RGC) in the plasma exosomes treated groups of primary glaucoma patients and cataract patients was significantly higher than that in the untreated group. At the same time, the number of plasma exosomes in the primary glaucoma patients was higher than that in the plasma exosomes treated group of cataract patients, indicating that the plasma sEVs in cataract patients also showed a certain neuroprotective effect, and the therapeutic effect of plasma sEVs in glaucoma patients was more significant.

[0035] The activation status of retinal microglial cells, including morphology, quantity, and migration, was observed by immunofluorescence, as Figure 4 shown in A. The quantity of retinal microglial cells in the Gla-PDEV group < the Cont-PDEV group < the Sham group. Meanwhile, as Figure 4 shown in B, it was found that the quantity of intersections between the synapses of retinal microglial cells and the grid in the Gla-PDEV group > the Cont-PDEV group > the Sham group. The fewer intersections between the synapses and the grid, the higher the activation status of the cells, and vice versa. Plasma exosomes from patients with primary glaucoma and the treatment group of plasma exosomes from patients with cataract could inhibit the activation state of microglial cells. The inhibitory effect on the activity of microglial cells in the plasma exosome group of patients with primary glaucoma was higher than that in the treatment group of plasma exosomes from patients with cataract.

[0036] Based on all the above results, we draw the conclusion that plasma-derived exosomes from patients with optic nerve injury diseases, especially plasma exosomes from patients with primary glaucoma, can improve the optic nerve injury in patients with optic nerve retinal degenerative diseases.

[0037] Finally, it is necessary to state here that the above embodiments are only used to further illustrate the technical solutions of the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention.

Claims

1. Use of plasma-derived exosomes in the preparation of a drug for treating eye diseases, characterized in that: The exosomes are derived from the plasma of patients with optic nerve and retinal diseases or the plasma of normal people, and the eye lesions include optic nerve damage lesions and retinal lesions.

2. Use of the plasma-derived exosomes according to claim 1 in the preparation of a drug for treating eye diseases, characterized in that: The exosomes are derived from the plasma of patients with autologous optic neuroretinopathy or normal subjects.

3. Use of the plasma-derived exosomes according to claim 1 in the preparation of a drug for treating eye diseases, characterized in that: The exosomes are derived from the plasma of patients with allogeneic neuroretinopathy or normal subjects.

4. The use of plasma-derived exosomes according to claim 1 in the preparation of a drug for treating eye diseases, characterized in that: The exosomes derived from the plasma of patients with optic neuroretinopathy are specifically exosomes derived from the plasma of patients with primary glaucoma.

5. Use of the plasma-derived exosomes according to claim 1, 2 or 3 in the preparation of a drug for treating eye diseases, characterized in that: The drug for treating optic nerve damage and lesions is one of the drugs for treating glaucoma, autoimmune, hereditary optic neuropathy, optic neuritis or optic nerve damage and lesions caused by ischemia, toxicity or trauma.

6. Use of plasma-derived exosomes according to claim 1 in the preparation of a drug for treating eye diseases, characterized in that: Retinal degenerative diseases include ischemic retinopathy, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration or hereditary retinal diseases such as retinitis pigmentosa.

7. A plasma-derived exosome composition, characterized in that: include: Plasma-derived exosomes and a medically acceptable solvent, wherein the exosomes are derived from the patient's autologous plasma or the plasma of a normal person.

Images