Nerve growth factor functionalized exosome as well as preparation method and application thereof

Abstract

The invention provides a nerve growth factor functionalized exosome as well as a preparation method and application thereof. The functionalized exosome can activate a Wnt / beta-catenin signal channel to maintain dryness of corneal limbal stem cells (LSCs) so as to promote corneal epithelium repair, can activate a cAMP signal channel to relieve inflammatory response and promote corneal nerve regeneration, and can be used for treating or relieving corneal chemical burns, including relieving inflammatory response, reducing stromal scar formation and inhibiting corneal neovascularization. Corneal epithelium repair and nerve regeneration are promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a nerve growth factor functionalized exosome, a preparation method thereof and application thereof in treating corneal chemical burn. BACKGROUND

[0002] The cornea is the most important refractive medium of the eye and is of great significance to the formation of good vision. Due to its special location, the cornea is easily damaged by various injuries, such as chemical injury or thermal burn. For example, corneal alkali burn caused by alkaline chemicals, corneal injury usually lasts for a long time, which is the main cause of blindness.

[0003] The comprehensive recovery of corneal chemical burn not only needs to restore the corneal tissue structure, but also needs to rebuild the corneal homeostasis including nerves and limbus. The current clinical treatment methods for acute corneal chemical burn aim to reduce excessive inflammation or promote corneal epithelial repair. However, these methods often fail to achieve comprehensive effects. For example, corticosteroid eye drops play an important role in inhibiting inflammation, but long-term use may induce ocular complications, damage the integrity of the cornea, and even cause corneal melting. Amniotic membrane transplantation has been widely used in the clinical treatment of corneal chemical burn, but the rapid melting and shedding of amniotic membrane to some extent limit the repair effect. Therefore, the multi-dimensional and complex repair process of corneal chemical burn requires effective and comprehensive treatment.

[0004] With the continuous development of stem cell and regenerative medicine technology, a large number of studies have shown that mesenchymal stem cells (MSCs) have the ability of self-renewal and multi-directional differentiation. Exosomes are a kind of nanoscale bioactive vesicles with a diameter of 40-160 nm, which can transfer bioactive molecules such as DNA, mRNA, miRNA, protein and lipid from donor cells to recipient cells, realize cell-cell communication, and participate in the regulation of different physiological activities. Exosomes derived from stem cells retain the characteristics of stem cells, including promoting tissue repair and regeneration and immune regulation, and are increasingly used in tissue repair and reconstruction. Compared with stem cells, exosomes also have the following advantages: (1) low immunogenicity, avoiding the occurrence of immune rejection reaction; (2) high stability, easy to store; (3) the production of exosomes can be mass-produced by changing the culture conditions; (4) the function of exosomes can be changed by changing the cell environment.

[0005] At present, there are few reports about the effect of MSC-Exo on corneal nerves and limbal stem cells (LSCs), because simple MSC-Exo treatment is difficult to achieve this effect. SUMMARY

[0006] In order to solve the problems existing in the prior art, the present application provides an exosome, which is obtained by stimulating mesenchymal stem cells (MSCs) with growth factors or cytokines, thereby increasing the contents of miR-708-3p, miR-629-5p and / or miR-503-5p in the exosome, so that the exosome can activate the Wnt / β-catenin signaling pathway, maintain the stemness of the corneal limbal stem cells and promote the repair of the corneal epithelium.

[0007] Preferably, the contents of miR-99b-3p and miR-503-5p in the exosome are increased, so that the exosome can activate the cAMP signaling pathway and promote the regeneration of the corneal nerves.

[0008] Preferably, the contents of miR-99b-3p and miR-629-5p in the exosome are increased, so that the exosome can activate the cAMP signaling pathway, reduce the inflammatory response of the cornea, reduce the scar formation of the corneal stroma and inhibit the formation of the new blood vessels of the cornea.

[0009] The present application does not have special limitations on the stimulation method, specific steps, conditions, equipment and materials, and those skilled in the art can select them according to the actual situation. For example, one or a combination of the following stimulation methods can be used: growth factor or cytokine stimulation (such as NGF, BDNF (brain-derived neurotrophic factor), TGF-β (transforming growth factor-β), IFN-γ (interferon-γ) and the like), physicochemical condition stimulation (such as hypoxic culture, oxidative stress, heat shock, mechanical stress and the like), chemical small molecule or drug stimulation, co-culture with target cells or their conditioned medium, and pre-adaptation strategy and the like. After stimulation, the culture supernatant is collected under suitable conditions, and the desired exosomes are obtained through exosome separation and purification technology.

[0010] Preferably, the stimulation is to culture the mesenchymal stem cells with a nerve growth factor.

[0011] Preferably, the concentration of the nerve growth factor is 10-300 ng / mL; preferably, the concentration of the nerve growth factor is 50-200 ng / mL; more preferably, the concentration of the nerve growth factor is 100 ng / mL.

[0012] Preferably, the culture time is not less than 12 hours, for example, 36, 48, 96, 144 hours; preferably, 24-88 hours, more preferably, 24-72 hours.

[0013] The present application does not have limitations on the culture conditions. Preferably, the culture conditions are to use α-MEM (or DMEM-F12) medium containing 10% exosome-free fetal bovine serum, and the culture is carried out in a cell culture incubator at 37℃, 5% CO2 saturated humidity.

[0014] Preferably, the nerve growth factor is derived from a mammal, such as a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow; more preferably, it is derived from a human, such as recombinant human nerve growth factor (rhNGF).

[0015] Preferably, the MSCs are derived from the bone marrow of a mammal; more preferably, they are derived from the bone marrow of a human.

[0016] In a second aspect, the present application provides a method for preparing exosomes, comprising culturing mesenchymal stem cells with nerve growth factor.

[0017] Preferably, the concentration of the nerve growth factor is 10-300 ng / mL; preferably, the concentration of the nerve growth factor is 50-200 ng / mL; more preferably, the concentration of the nerve growth factor is 100 ng / mL.

[0018] Preferably, the culturing time is not less than 12 hours, such as 36, 48, 96, or 144 hours; preferably, the culturing time is 24-88 hours, more preferably, the culturing time is 24-72 hours.

[0019] The present application does not limit the culturing conditions. Preferably, the culturing conditions are culturing in a cell incubator at 37℃, 5% CO2 saturated humidity, using DMEM-F12 (or a-MEM) medium containing 10% exosome-free fetal bovine serum.

[0020] Preferably, the nerve growth factor is derived from a mammal, such as a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow; more preferably, it is derived from a human, such as recombinant human nerve growth factor (rhNGF).

[0021] Preferably, the MSCs are derived from the bone marrow of a mammal; more preferably, they are derived from the bone marrow of a human.

[0022] Optionally, the method further comprises a post-processing step of extracting exosomes by ultracentrifugation. The present application does not have special limitations on the method, specific steps, conditions, equipment, and materials of ultracentrifugation, which can be selected by a person skilled in the art according to the actual situation.

[0023] Preferably, the step of extracting exosomes by ultracentrifugation comprises: 2-8℃, 200-500g centrifugation for 5-20min to remove dead cells; 2-8℃, 1000-3000g centrifugation for 5-30min to remove cell debris; 2-8℃, 5000-20000g centrifugation for 20-60min to remove microvesicles; 2-8℃, 100000-200000g centrifugation for 60-120min, and the precipitate collected is exosomes.

[0024] More preferably, the step of extracting exosomes by ultracentrifugation includes: 300-400g centrifugation at 6-8℃ for 10-15min to remove dead cells; 1500-2500g centrifugation at 6-8℃ for 10-20min to remove cell debris; 10000-15000g centrifugation at 6-8℃ for 30-40min to remove microvesicles; 100000-150000g centrifugation at 6-8℃ for 80-90min, and the precipitate is collected as exosomes.

[0025] Optionally, the method further comprises a pretreatment step of obtaining, culturing and subculturing mesenchymal stem cells. The method, specific steps, conditions, equipment and materials for obtaining, culturing and subculturing mesenchymal stem cells are not particularly limited in the present application, and can be selected by those skilled in the art according to the actual situation.

[0026] Preferably, the step of obtaining, culturing and subculturing mesenchymal stem cells includes: culturing bone marrow-derived MSCs in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody in a 37℃ constant temperature incubator, and changing the medium every 48h. When the cell density reaches 70-80%, subculture.

[0027] In a third aspect, the present application provides exosomes prepared by the method for preparing exosomes according to the second aspect.

[0028] The increased content of miR-708-3p, miR-629-5p and / or miR-503-5p in the exosomes enables the exosomes to activate the Wnt / β-catenin signaling pathway, maintain the stemness of the limbal stem cells, and promote the repair of the corneal epithelium.

[0029] Preferably, the increased content of miR-99b-3p and miR-503-5p in the exosomes enables the exosomes to activate the cAMP signaling pathway and promote the regeneration of corneal nerves.

[0030] Preferably, the increased content of miR-99b-3p and miR-629-5p in the exosomes enables the exosomes to activate the cAMP signaling pathway, reduce the inflammatory response of the cornea, reduce the formation of stromal scars in the cornea, and inhibit the formation of new blood vessels in the cornea.

[0031] In a fourth aspect, the present application provides the use of the exosomes according to the first aspect or prepared by the method for preparing exosomes according to the third aspect in the preparation of a medicament for treating or alleviating chemical burns of the cornea.

[0032] In a fifth aspect, the present application provides the use of the exosomes according to the first aspect or prepared by the method for preparing exosomes according to the third aspect in the preparation of a medicament for treating or alleviating the inflammatory response, stromal scars, new blood vessels in the cornea, damage to the corneal epithelium, or nerve damage caused by chemical burns of the cornea.

[0033] In the fourth and fifth aspects of the present application, preferably, the corneal chemical burn is an alkali burn.

[0034] Preferably, the corneal chemical burn is in the acute phase.

[0035] Preferably, the symptoms of the corneal chemical burn include corneal epithelial damage, corneal nerve damage, corneal stroma damage, or corneal inflammatory response, corneal stroma scarring, corneal neovascularization.

[0036] Preferably, the concentration of the exosomes in the drug is 10-200 ug / mL; more preferably, the concentration is 50-150 ug / mL.

[0037] The present application does not limit the dosage form of the drug, for example, hydrogel, eye drops, eye gel, patch, dressing.

[0038] The present application does not have a specific limitation on the excipients in the drug, and any pharmaceutically acceptable excipient can be used, and the preparation is adjusted according to the need.

[0039] Preferably, the drug is a hydrogel: NGF-MSC-Exo is dissolved and loaded in a temperature-sensitive hydrogel Pluronic F127; the pH value of the hydrogel is 7.2-7.8, for example 7.4.

[0040] Preferably, the drug is a nanoparticle eye drop: the exosomes are encapsulated in hyaluronic acid / chitosan nanoparticles to improve the retention time and reduce the frequency of administration. The eye drops can also be added with penetration enhancers to enhance the penetration (the sustained-release effect is close to that of hydrogel).

[0041] Preferably, the drug is a microneedle patch: a dissolvable microneedle array (PVA / PVP matrix loaded with exosomes) can painlessly penetrate the corneal barrier and has high delivery efficiency.

[0042] Preferably, the drug is a liposome spray: the exosomes are mixed with phospholipids to prepare liposomes, which are sprayed in the form of mist, have a large coverage area, and are suitable for large-area damage.

[0043] The present application has the beneficial technical effects: the NGF functionalized exosomes provided by the present application can activate the Wnt / β-catenin signaling pathway to maintain the stemness of limbal stem cells (LSCs) to promote corneal epithelial repair, and can activate the cAMP signaling pathway to reduce the inflammatory response and promote corneal nerve regeneration. The nerve growth factor functionalized exosomes provided by the present application can promote the repair of corneal chemical burns, including but not limited to reducing the inflammatory response, reducing stroma scarring, inhibiting corneal neovascularization, promoting corneal epithelial repair and nerve regeneration. The present application provides a new idea and method for treating and relieving corneal chemical burns. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 : Morphology of MSC-Exo and rhNGF-MSC-Exo derived exosomes observed by transmission electron microscopy.

[0045] Figure 2 : Particle size analysis results of MSC-Exo and rhNGF-MSC-Exo.

[0046] Figure 3 : Expression of marker proteins CD9 and CD81 of MSC-Exo and rhNGF-MSC-Exo.

[0047] Figure 4 : Effect of rhNGF (50 ng / mL, 100 ng / mL, 150 ng / mL) at different concentrations on the proliferation of corneal limbal stem cells after different culture times (24 hours, 48 hours, 72 hours) (A, B).

[0048] Figure 5 : Effect of rhNGF (50 ng / mL, 100 ng / mL, 150 ng / mL) at different concentrations on the proliferation of corneal trigeminal neuron cells after different culture times (24 hours, 48 hours, 72 hours) (A, B).

[0049] Figure 6 : Shows the corneal epithelial wound repair area (A) and speed (B) of the control group, MSC-Exo group (100 ng / mL), and rhNGF-MSC-Exo group (100 ng / mL) eyedrops mice.

[0050] Figure 7 : Shows the corneal stroma transparency (A, B) and new blood vessel growth (A, C) of the control group, MSC-Exo group (100 ng / mL), and rhNGF-MSC-Exo group (100 ng / mL) eyedrops mice.

[0051] Figure 8 : Shows the corneal stroma edema (A) and corneal thickness (B) of the control group, MSC-Exo group (100 ng / mL), and rhNGF-MSC-Exo group (100 ng / mL) eyedrops mice.

[0052] Figure 9 : Corneal nerve regeneration graph of the control group, MSC-Exo group (100 ng / mL), and rhNGF-MSC-Exo group (100 ng / mL) eyedrops mice.

[0053] Figure 10 : MSC-Exo and rhNGF-MSC-Exo miRNA sequencing volcano plot.

[0054] Figure 11 Figure 2: MSC-Exo and rhNGF-MSC-Exo miRNA sequencing GO analysis plot.

[0055] Figure 12 Figure 3: MSC-Exo and rhNGF-MSC-Exo miRNA sequencing KEGG analysis plot. DETAILED DESCRIPTION

[0056] Corneal chemical burn refers to the damage to the cornea and ocular surface tissue caused by direct contact with chemical substances through corrosion, dissolution or chemical reaction, which is an ophthalmic emergency. Its severity is closely related to the type, concentration and contact time of the chemical substance. If not handled properly, it can lead to corneal scarring, permanent vision loss or even blindness. The core mechanism of corneal chemical burn is the chemical reaction between chemical substances and corneal tissue (such as protein denaturation, lipid dissolution, collagen destruction, etc.), which further damages the structure and function of the cornea. The symptoms of corneal chemical burn change over time after injury and can be divided into acute phase, repair phase and complication phase. Among them, the treatment focus in the acute phase is to control the injury, including: 1) neutralize residual chemicals; 2) prevent infection; 3) reduce inflammation and edema. Usually multiple drugs are used in combination therapy.

[0057] The present application is directed to corneal chemical burn, in particular acute phase chemical injury, by atopic modification of mesenchymal stem cell (MSC) exosomes (Exo) secreted after, for reducing inflammation, promoting corneal epithelial repair, nerve regeneration and preventing complications (inhibiting corneal scarring and neovascularization).

[0058] We found through research that Wnt / β-catenin and cAMP signaling pathway activators can have a repair and / or therapeutic effect on corneal chemical burn. Currently known Wnt / β-catenin signaling pathway activators include HLY78, CHIR99021 and R-spondin protein. cAMP signaling pathway activators include Forskolin, PGE2 and Isoproterenol. However, the potential problem of Wnt / β-catenin signaling pathway activators is that over-activation of the Wnt / β-catenin pathway can induce corneal scarring, leading to decreased transparency; or cause corneal neovascularization, affecting vision; or continuous activation can lead to hyperkeratosis, disrupting the smoothness of the corneal surface. The potential problem of cAMP signaling pathway activators is that they can exacerbate the inflammatory response and delay corneal epithelial repair; can over-stimulate nerve growth, leading to abnormal innervation; can affect aqueous humor dynamics, leading to elevated intraocular pressure.

[0059] The present application selects exosomes (NGF-MSC-Exo) secreted by mesenchymal stem cells (MSC) after being stimulated by growth factors or cytokines, such as nerve growth factor (NGF), to regulate through multiple targets, more effectively maintain the stemness of limbal stem cells (LSCs) and promote the regeneration of corneal nerves, meet the needs of comprehensive and efficient repair and / or treatment of corneal chemical burns, including but not limited to neurotrophic, anti-inflammatory, and anti-scarring.

[0060] Specifically, nerve growth factor (NGF), for example, recombinant human nerve growth factor (rhNGF), can indirectly activate the Wnt / β-catenin signaling pathway and / or the cAMP signaling pathway through downstream signaling. NGF-MSC-Exo treated with NGF not only carries a large amount of bioactive substances (proteins, mRNAs, miRNAs, lncRNAs), but also optimizes its content spectrum, containing more factors for promoting repair, anti-inflammatory, anti-fibrosis, and promoting vascular normalization. These factors and NGF signals produce multi-level, network-like synergistic and amplification effects, forming a complex molecular network regulation beyond single molecular mechanisms. This systematic and self-organizing repair ability is an "emergent" effect that single drugs or traditional combinations cannot achieve, with high unpredictability. For example, NGF-MSC-Exo carries a large amount of non-coding RNAs such as miRNAs and lncRNAs. A single miRNA can target and regulate dozens or even hundreds of mRNA molecules to remodel the entire transcriptome and signaling network in the recipient cells, and its comprehensive effect is a highly complex and unpredictable "systems biology" effect. For another example, the molecules carried by Exo (such as certain lncRNAs and histone modification enzymes) can affect the epigenetic state (DNA methylation, histone modification) of the recipient cells, thereby persistently changing the behavior of the recipient cells (such as maintaining the resting state of corneal cells and inhibiting the transformation of fibroblasts), and this long-acting regulation mechanism is difficult to achieve by traditional drugs.

[0061] More specifically, the NGF-MSC-Exo provided by the present application contains the following non-coding RNAs: miR-493-3p, miR-708-3p, miR-99b-3p, miR-589-5p, miR-629-5p, miR-522-3p, miR-503-5p, miR-214-3p, miR-27a-5p, miR-23b-5p and miR-451a. This specific group of miRNAs in NGF-MSC-Exo constitutes a precise regulatory network that maintains the stemness of limbal stem cells, promotes the expression of nerve regeneration factors, and inhibits excessive inflammatory response and fibroblast activation by positively regulating the Wnt / β-catenin and cAMP signaling pathways. Multi-target, multi-pathway synergy achieves the triple therapeutic goals of anti-inflammation, promotion of epithelial and nerve repair, and anti-scarring, providing a treatment strategy with repair potential for chemical burns, especially those accompanied by nerve damage.

[0062] In summary, unlike the single effect of the current conventional treatment methods, the NGF-MSC-Exo provided by the present application has the characteristics of comprehensive and efficient repair in the treatment and relief of corneal chemical burns, including but not limited to achieving the most extensive recovery of corneal morphology and function, which is manifested in reducing inflammatory response, promoting the healing of corneal epithelium and limbus, inhibiting corneal stroma scarring and neovascularization, and promoting the repair of corneal nerves.

[0063] The present application is further exemplified by the following examples, which are only used to illustrate the present application and should not be considered as limiting the scope of the present application. Unless otherwise specified, the technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present application belongs. Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0064] In the examples of the present application, part of the materials are as follows:

[0065] Mesenchymal stem cells (MSCs): purchased from Suzhou Saiye Biotechnology Co., Ltd., item number HUXMA-01001.

[0066] Recombinant human nerve growth factor (rhNGF): purchased from Wuhan Sanyi Biotechnology Co., Ltd., item number HZ-1222.

[0067] Example 1

[0068] The present application does not limit the method, specific steps, conditions, reagents, materials, equipment, etc. of cell culture and exosome extraction, and those skilled in the art can prepare them according to actual needs and conventional means.

[0069] The present embodiment provides a preferred method for preparing exosomes, and the specific steps are as follows:

[0070] (1) Culture of MSCs: Bone marrow-derived MSCs were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody in a 37°C constant temperature incubator, and the medium was changed every 48h. When the cell density reached 70-80%, the cells were passaged. MSCs after the 3rd passage (P3) were used in subsequent steps.

[0071] (2) Collection of MSC-Exo: When MSCs were passaged to 4-5 generations and grew to cover 70-80% of the bottle bottom, the medium was replaced with exosome-free serum and cultured for 24-72h, and the cell supernatant was collected. The obtained medium was separated by a conventional ultracentrifuge to separate exosomes, specifically 10min at 300g at 4°C to remove dead cells; 10min at 2000g at 4°C to remove cell debris; 30min at 10000g at 4°C to remove microvesicles; 90min at 100000g at 4°C to collect the precipitate, which was exosomes. The exosomes were resuspended in PBS twice and filtered through a 0.22μm filter. The obtained exosome suspension was stored in a -80°C refrigerator for standby use.

[0072] (3) Collection of rhNGF-MSC-Exo: When MSCs were passaged to 4-5 generations and grew to cover 70-80% of the bottle bottom, the medium was replaced with exosome-free serum and rhNGF and cultured for 24-72h, such as 30, 36, 42, 48, 54, 60, 66. The concentration of rhNGF was 50-150ng / mL, for example, 100ng / mL. The cell supernatant was collected by ultracentrifugation to obtain rhNGF-MSC-Exo, and the specific steps refer to step (2).

[0073] Morphology, particle size analysis and identification of marker proteins of Exo: Transmission electron microscopy detection results showed that MSC-Exo and rhNGF-MSC-Exo presented a double-sided concave disc-shaped vesicle with a double-layer vesicle structure Figure 1 ); Particle size analysis showed that the diameter of MSC-Exo was 132.4nm, and the diameter of rhNGF-MSC-Exo was 134.5nm, both of which met the standard diameter of Exo (40-160nm) Figure 2 ); Conventional Western blotting detection results showed that both kinds of exosomes expressed exosome marker proteins CD9 and CD81 Figure 3 ).

[0074] Evaluation of Exo function: rhNGF-MSC-Exo obtained by culturing MSCs with different concentrations of rhNGF (50 ng / mL, 100 ng / mL, 150 ng / mL) for 24 hours, 48 hours, and 72 hours, respectively, were used to co-culture with corneal limbal stem cells and corneal trigeminal neurons. Cell counting kit (CCK-8) and 5-ethynyl-2-deoxyuridine (EDU) proliferation test results showed that rhNGF-MSC-Exo obtained by culturing MSCs with 50-150 ng / mL of rhNGF for different times (24 hours, 48 hours, and 72 hours) could improve the proliferation ability of corneal limbal stem cells and corneal trigeminal neurons. Figure 4 , Figure 5 ) rhNGF-MSC-Exo obtained by culturing MSCs with 100 ng / mL of rhNGF had the most significant effect on cell proliferation.

[0075] Example 2

[0076] Animal experiment: rhNGF-MSC-Exo can promote corneal repair.

[0077] (1) Construction of mouse corneal chemical burn model: After anesthesia, C57BL / 6 mice (8-10 weeks old) were used to immerse a 2 mm diameter filter paper in a 1 mol / L sodium hydroxide solution, and the excess liquid was absorbed after the filter paper was placed in the center of the right cornea of the mouse for 30 seconds. Then, the filter paper was removed immediately, and the eye surface and conjunctival sac were rinsed with 20 ml of normal saline to establish a mouse corneal burn model. This method of construction is a conventional method in the art and is suitable for simulating the condition and symptoms of acute phase of corneal chemical burn, especially alkali burn.

[0078] (2) Experimental design: In this study, mice were randomly divided into three groups: control group (no drug given, only hydrogel eyedrops), Exo group (given 100 ug / ml MSC-Exo hydrogel eyedrops), and N-Exo group (given 100 ug / ml rhNGF-MSC-Exo hydrogel eyedrops). At different time points after surgery, slit lamp microscopy, fluorescein sodium staining, and corneal optical coherence tomography (OCT) examination were used to verify the therapeutic effect of rhNGF-MSC-Exo.

[0079] Hydrogel preparation process: The purified MSC-Exo and rhNGF-MSC-Exo were mixed with Pluronic F127 solution on ice (to avoid inactivation of exosomes caused by high temperature). The gelling mixture was placed at 37°C for 10 minutes to form a temperature-sensitive hydrogel.

[0080] (3) Postoperative follow-up: At 0, 3, 7, 10 days after modeling, the slit lamp microscope, fluorescein sodium staining and OCT examination were used to evaluate the corneal epithelial damage repair, stromal edema, stromal transparency and neovascularization of each group of mice. The results showed that compared with the control group and Exo group, the rhNGF-MSC-Exo group could significantly promote corneal epithelial regeneration( Figure 6 ), maintain corneal stromal transparency (no corneal scar) and inhibit corneal neovascularization( Figure 7 ), reduce stromal edema and reduce inflammatory response( Figure 8 ).

[0081] (4) Immunofluorescence detection: One month after operation, the specific marker of neurons (β3-tubulin) was detected by routine immunofluorescence. The results showed that compared with the control group and Exo group, the rhNGF-MSC-Exo group could significantly promote corneal nerve regeneration( Figure 9 ).

[0082] Example 3

[0083] Mechanism of rhNGF-MSC-Exo promoting corneal chemical burn repair

[0084] (1) Exo miRNA sequencing analysis: Total RNA of exosomes was extracted using TRlzol reagent according to the method provided in the instructions. The total amount and integrity of RNA were evaluated using Nanodrop2000 (Thermo Fisher Scientific Inc., USA) and Agilent2100 bioanalyzer (Agilent Technology, USA), respectively. 1 μg of total RNA was taken from each sample, and NEB Next Small RNA Library Prep Set for Illumina kit (Cat. No. NEB#E7330S, NEB, USA) was used to construct Small RNA library. Subsequently, agarose gel electrophoresis was performed, and the 140-160 bp PCR product was separated and purified as a Small RNA library. After the library quality was qualified by Agilent2100 bioanalyzer, Illumina Novaseq6000 platform was used for sequencing to generate 150 bp double-end sequences, and software program was used to analyze the data.

[0085] (2) Volcano plot results show that the contents of 11 miRNAs (the names of RNAs are based on the international unified naming rules) in rhNGF-MSC-Exo, miR-493-3p, miR-708-3p, miR-99b-3p, miR-589-5p, miR-629-5p, miR-522-3p, miR-503-5p, miR-214-3p, miR-27a-5p, miR-23b-5p, miR-451a, have changed, of which 7 are up-regulated (miR-493-3p, miR-708-3p, miR-99b-3p, miR-589-5p, miR-629-5p, miR-522-3p, miR-503-5p) and 4 are down-regulated (miR-214-3p, miR-27a-5p, miR-23b-5p, miR-451a) Figure 10 ) Among them, miR-708-3p, miR-629-5p and miR-503-5p can activate the Wnt / β-catenin signaling pathway, maintain the stemness of corneal limbal stem cells, and promote the repair of corneal epithelium; miR-99b-3p and miR-503-5p can activate the cAMP signaling pathway, promote corneal nerve regeneration; miR-99b-3p and miR-629-5p can activate the cAMP signaling pathway, reduce corneal inflammatory response, and thus reduce corneal stroma scarring and inhibit corneal neovascularization.

[0086] (3) Gene ontology (GO) analysis plot results show that the miRNAs in rhNGF-MSC-Exo are mainly enriched in biological processes (BP) such as positive regulation of synapse assembly, regulation of cell-cell adhesion, and regulation of transforming growth factor beta receptor signaling pathway Figure 11 ).

[0087] (4) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis results show: Figure 11 , 12The GO and KEGG analysis chart in the figure is the differential miRNA screened out by comparing MSC-Exo and rhNGF-MSC-Exo with a P value greater than 0.05. Compared with MSC-Exo, rhNGF-MSC-Exo significantly activated the Wnt / β-catenin and cAMP signaling pathways, which are important pathways for maintaining the stemness of corneal limbal stem cells, reducing inflammatory response, and promoting corneal nerve regeneration. Figure 12

[0088] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to the details and dosages in accordance with all the teachings disclosed, and such changes are within the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.​

Claims

1. An exosome, characterized in that, The miR-708-3p, miR-629-5p and / or miR-503-5p content in the exosomes secreted by the mesenchymal stem cells stimulated by the growth factors or cytokines is increased, so that the exosomes can activate the Wnt / β-catenin signaling pathway, maintain the stemness of the corneal limbal stem cells, and promote the repair of the corneal epithelium.

2. The exosome of claim 1, wherein, The increased miR-99b-3p and miR-503-5p content in the exosomes can activate the cAMP signaling pathway, promote the regeneration of the corneal nerves, and reduce the inflammation of the cornea. The increased miR-99b-3p and miR-629-5p content in the exosomes can activate the cAMP signaling pathway, reduce the inflammation of the cornea, reduce the formation of the stromal scar of the cornea, and inhibit the formation of the neovascularization of the cornea.

3. The exosome of claim 1, wherein, The mesenchymal stem cells are cultured with the nerve growth factor.

4. The exosome of claim 3, wherein, The concentration of the nerve growth factor is 10-300 ng / mL, and the culture time is not less than 12 hours.

5. A method for preparing an exosome, characterized by, The mesenchymal stem cells are cultured with the nerve growth factor. The concentration of the nerve growth factor is 10-300 ng / mL, and the culture time is not less than 12 hours.

6. The exosome prepared according to the method of claim 5, wherein The nerve growth factor is a recombinant human nerve growth factor. The increased miR-708-3p, miR-629-5p and / or miR-503-5p content in the exosomes can activate the Wnt / β-catenin signaling pathway, maintain the stemness of the corneal limbal stem cells, and promote the repair of the corneal epithelium. The increased miR-99b-3p and miR-503-5p content in the exosomes can activate the cAMP signaling pathway, promote the regeneration of the corneal nerves, and reduce the inflammation of the cornea. The increased miR-99b-3p and miR-629-5p content in the exosomes can activate the cAMP signaling pathway, reduce the inflammation of the cornea, reduce the formation of the stromal scar of the cornea, and inhibit the formation of the neovascularization of the cornea.

7. The exosomes of any one of claims 1 or 4, or the exosomes of claim 6 for use in the preparation of a medicament for treating or alleviating a chemical burn of the cornea.

9. Use according to claim 7 or 8, wherein the compound is ###0002### 8. The exosomes of any one of claims 1 or 4, or the exosomes of claim 6 for use in the preparation of a medicament for treating or alleviating an inflammation, a stromal scar, a neovascularization, a corneal epithelial damage, or a nerve damage caused by a chemical burn of the cornea.

10. Use according to claim 7 or 8, wherein the compound is ###00002### The chemical burn of the cornea is an alkali burn. The chemical burn of the cornea is in an acute phase.

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