Application of hypoxia mesenchymal stem cell exosome in preparation of medicine for promoting corneal epithelium injury healing

Abstract

The invention relates to the field of biological medicines, in particular to application of an anoxic mesenchymal stem cell exosome in preparation of a medicine for promoting corneal epithelium injury healing. In-vivo and in-vitro experiments prove that the hypoxia mesenchymal stem cell exosome can remarkably restore inhibition on corneal epithelium injury healing under a hypertonic condition in vivo and in vitro, can remarkably promote proliferation and migration of human corneal epithelium cells in vitro, can promote corneal epithelium injury healing in vivo, has a good treatment effect on corneal epithelium injury, and can be used for treating corneal epithelium injury. Good application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the fields of cell therapy and regenerative medicine, and specifically to the use of exosomes of hypoxic mesenchymal stem cells in the preparation of a drug for promoting the healing of corneal epithelial damage. Background Art

[0002] Corneal epithelial injury is a pathological condition characterized by the destruction of the corneal epithelial barrier function and integrity. It can be caused by a variety of reasons, including mechanical damage, infection, and dry eye, resulting in partial or complete loss of the corneal epithelial cell layer. The corneal epithelial wound healing process is crucial for maintaining corneal integrity and maintaining its barrier function (McKay TB, Hutcheon AEK, Zieske JD, et al. Extracellular Vesicles Secreted by Corneal Epithelial Cells Promote Myofibroblast Differentiation [J]. Cells, 2020, 9(5). DOI:10.3390 / cells9051080).

[0003] Exosomes are vesicles secreted by eukaryotic cells, ranging in diameter from 30 to 150 nm, and contain a variety of bioactive molecules. In recent years, exosomes have shown great potential in disease diagnosis, treatment, and drug delivery, attracting widespread attention (Kalluri R, LeBleu V S. The biology, function, and biomedical applications of exosomes [J]. Science, 2020, 367(6478). DOI:10.1126 / science.aau6977). In addition, there are reports that exosomes from different sources can mediate intercellular information transmission, thereby changing the biological functions of recipient cells (Colombo M, Raposo G, Thery C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles [J]. Annu Rev CellDev Biol, 2014, 30(1530-8995 (Electronic)): 255-289. DOI:10.1146 / annurev-cellbio-101512-122326).

[0004] In recent years, the research on stem cells in corneal damage repair has made remarkable progress. Mesenchymal stem cells (MSCs) are well-known for their regenerative ability and immunomodulatory properties and are considered to be the main candidates for stem cell therapy. Mesenchymal stem cells can promote the healing of damaged tissues by secreting cytokines, reducing inflammation, reducing fibrosis, and promoting the proliferation of endogenous stem cells. Mesenchymal stem cell-derived exosomes (MSC-Exos) are an important component of the paracrine regulatory pathway. These exosomes have been shown to regulate immune cell activity and promote tissue regeneration (Bhujel B, Oh SH, Kim CM, et al. Mesenchymal Stem Cells and Exosomes: A Novel Therapeutic Approach for Corneal Diseases [J]. Int J Mol Sci, 2023, 24(13). DOI:10.3390 / ijms241310917). In addition, exosomes released by MSCs cultured under hypoxic conditions showed stronger efficacy in promoting tissue repair (Bhujel B, Oh SH, Kim CM, et al. Mesenchymal Stem Cells and Exosomes: A Novel Therapeutic Approach for Corneal Diseases [J]. Int J MolSci, 2023, 24(13). DOI:10.3390 / ijms241310917). MSC-Exo may be a promising treatment for corneal damage.

[0005] However, there are currently no reports on the application of hypoxic mesenchymal stem cell exosomes in the preparation of drugs to promote the healing of corneal epithelial injuries. Summary of the Invention

[0006] The present invention aims to provide a novel application of exosomes derived from hypoxic mesenchymal stem cells in the preparation of a drug for promoting the healing of corneal epithelial lesions, and to provide a drug for promoting the healing of corneal epithelial lesions. This invention covers the biological properties of mesenchymal stem cells, the isolation and application of exosomes, corneal biology, and treatment methods for corneal lesions.

[0007] The first aspect of the present invention provides the use of hypoxic mesenchymal stem cell exosomes in the preparation of a drug for promoting the healing of corneal epithelial damage.

[0008] The second aspect of the present invention provides the use of hypoxic mesenchymal stem cell exosomes in the preparation of a drug for treating corneal epithelial damage.

[0009] Furthermore, the hypoxic mesenchymal stem cell exosomes are exosomes isolated after bone marrow mesenchymal stem cells are cultured under hypoxic conditions in serum-free DMEM medium for 48 hours; the hypoxic conditions are 1% oxygen, 5% carbon dioxide, and the rest nitrogen.

[0010] Furthermore, the method for preparing the hypoxic mesenchymal stem cell exosomes comprises the following steps:

[0011] Bone marrow cells extracted from mouse tibias and femurs were resuspended in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin; the medium was changed after 24 hours and every 3 days thereafter; bone marrow mesenchymal stem cells (BM-MSCs) were cultured to 70-80% confluence; then cultured under hypoxic conditions in serum-free DMEM medium for 48 hours; separated by traditional differential centrifugation; the medium was collected and centrifuged at 300 g for 5 minutes to remove cells; the medium was then centrifuged at 2,000 g for 20 minutes and at 10,000 g for 30 minutes at 4°C to remove cell debris; the supernatant was then filtered through a 0.22 μm filter; the pretreated supernatant was ultracentrifuged at 100,000 g for 2 hours at 4°C; and the exosome pellet was resuspended in phosphate-buffered saline.

[0012] Furthermore, the bone marrow cells are from 2-4 week old C57BL / 6J mice.

[0013] In a third aspect, the present invention provides a drug for promoting the healing of corneal epithelial damage, wherein the active ingredient of the drug is hypoxic mesenchymal stem cell exosomes.

[0014] Furthermore, the medicine also includes pharmaceutically acceptable carriers or excipients.

[0015] Furthermore, the effective dose of hypoxic mesenchymal stem cell exosomes in the drug is 1.0 × 10 8 particles / mL.

[0016] The advantages of the present invention are:

[0017] Experiments have shown that exosomes from hypoxic mesenchymal stem cells can significantly restore the inhibition of corneal epithelial injury healing under hypertonic conditions both in vivo and in vitro, significantly promote the proliferation and migration of human corneal epithelial cells in vitro, and promote the healing of corneal epithelial injury in vivo. They have a good therapeutic effect on corneal epithelial injury and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Hypoxic mesenchymal stem cell exosomes promote human corneal epithelial cell (HCEC) proliferation in vitro.

[0019] Figure 2 Hypoxic mesenchymal stem cell exosomes promote human corneal epithelial cell (HCEC) migration in vitro.

[0020] Figure 3 . Exosomes derived from hypoxic mesenchymal stem cells promote healing of corneal epithelial injury in vivo in a mouse model. DETAILED DESCRIPTION

[0021] The specific implementation methods provided by the present invention are described in detail below with reference to the accompanying drawings and examples.

[0022] Example 1: Acquisition of Exosomes from Hypoxic Mesenchymal Stem Cells

[0023] First, bone marrow mesenchymal stem cells (BM-MSCs) were isolated and cultured. Briefly, 2- to 4-week-old C57BL / 6J mice were euthanized and soaked in 75% ethanol for 5 minutes. Tibias and femurs were then completely dissected using scissors and sterile forceps, passed through a 40μM cell strainer, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The resuspended cells were seeded into cell flasks and cultured at 37°C in 5% CO2. The medium was changed after 24 hours and every 3 days thereafter until passage. For exosome isolation, MSCs were cultured to 70-80% confluence and then cultured in serum-free DMEM under hypoxic conditions (1% oxygen, 5% carbon dioxide, and the remainder nitrogen) for 48 hours. Cells were isolated using conventional differential centrifugation. The culture medium was collected and centrifuged at 300 g for 5 minutes to remove the cells. The culture medium was then centrifuged at 2,000 g for 20 minutes and then at 10,000 g for 30 minutes at 4°C to remove cellular debris. The supernatant was then filtered through a 0.22 μm filter. The pretreated supernatant was ultracentrifuged at 100,000 g for 2 hours at 4°C. The exosome pellet was resuspended in phosphate-buffered saline and used immediately or stored at −80°C.

[0024] Example 2: Cell proliferation assay (EDU assay)

[0025] 1. Experimental methods

[0026] (1) Cell seeding: Digest HCEC cells with a density of about 80% to 90% and count the cells. Take about 2×10 cells per well. 3 Cells were seeded into 96-well plates and incubated in a 37°C 5% CO2 incubator. After the cells attached to the wall, the original culture medium was discarded and the cells were treated with serum-free medium containing PBS or hypMSC-Exo for 24 hours. Then, isotonic medium, hypertonic medium (500mOsm) containing PBS or hypMSC-Exo (1.0 × 10 6The cells were treated with 5% CO2 (particles / mL) and incubated in a 37°C 5% CO2 incubator for 24 to 48 hours.

[0027] EdU labeling: dilute the EdU stock solution of the EdU proliferation (555) kit with culture medium at a ratio of 1:8000; discard the HUVEC primary cell culture medium in the 96-well plate and replace it with the above dilution solution, 100 μL per well, and place it in a 37°C 5% CO2 incubator for about 2 hours.

[0028] (2) Cell fixation: Remove the culture medium from the 96-well plate and wash the cells with PBS solution for 3 times, 5-10 minutes each time. After washing, add 100µL of 4% paraformaldehyde solution to each well and fix at room temperature for 30 minutes. Discard the cell fixative. After cell fixation, add 100µL of 2mg / ml glycine solution to each well and decolorize on a shaker at room temperature for 5-10 minutes. Discard the glycine solution. Add 100µL of PBS solution to each well and wash the cells for 5 minutes. Add 100µL of 0.5% TritonX-100 permeabilization agent to each well and wash on a shaker at room temperature for 5 minutes. Add 100µL of PBS to each well and wash the cells. Decolorize on a shaker at room temperature for 5 minutes. Discard the PBS.

[0029] (3) Apollo staining: Before staining, first prepare the EdU reaction solution according to the ratio of PB: CU: AC: 488 chromogen = 860: 40: 100: 5. After the cells are fixed and washed, add 100 μL of the above dye solution to each well, incubate on a shaker at room temperature in the dark for 30 minutes, and discard the dye solution; add 100 μL of 0.5% TritonX-100 permeabilization agent to each well, and wash on a shaker at room temperature 2 to 3 times, each time for 10 minutes.

[0030] (4) DAPI staining: dilute the DAPI stock solution with PBS solution at a ratio of 1:10000, add 100µL of the diluted DAPI staining solution to each well, stain at room temperature in the dark, shake on a plate for 15 minutes, and discard the staining solution; add 100µL of PBS solution to each well, shake on a plate at room temperature for three times, each time for 5 to 10 minutes, and do not discard the PBS for the last wash.

[0031] (5) Observation: Use a fluorescence microscope to observe and take pictures, and calculate the number of stained nuclei in the proliferative phase and the number of all nuclei and their ratio.

[0032] 2. Experimental results

[0033] Hypertonicity significantly inhibited the proliferation of HCEC cells, and hypMSC-Exo could restore the inhibition of hypertonicity on HCEC migration (see Figure 1 ).

[0034] Example 3: Cell migration assay

[0035] 1. Experimental methods

[0036] (1) Cell seeding: Place the ibidi Culture-insert on the surface of a dry cell culture dish. Digest HCEC cells with a cell density of about 80% to 90%. 4 100 μL of cell suspension was seeded into the Culture-insert wells and placed in a 37°C 5% CO2 incubator for incubation. After the cells attached to the wall, the original culture medium was discarded and replaced with PBS or hypoxic mesenchymal stem cells (hypMSC-Exo, 1.0 × 10 6 particles / mL) in serum-free medium for 24 h.

[0037] (2) Scratch formation: After gently removing the Culture-insert, use isotonic medium, hypertonic medium (500mOsm) containing PBS or hypMSC-Exo (1.0 × 10 6 The cells were treated with 5% CO2 (particles / mL) and incubated in a 37°C 5% CO2 incubator for 12 to 24 hours.

[0038] (3) Observation: Observe cell migration under a microscope and take photos.

[0039] 2. Experimental results

[0040] Hypertonicity significantly inhibited the migration of HCEC cells, and hypMSC-Exo could restore the inhibition of hypertonicity on HCEC migration (see Figure 2 ).

[0041] Example 4: Study on mouse corneal epithelial injury model

[0042] 1. Experimental methods

[0043] (1) Preparation of mouse corneal epithelial injury model: Male C57BL / 6J mice (6 weeks old) were randomly divided into a control group, a hypertonic group, and a hypertonic group containing hypMSC-Exo. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). After topical anesthesia with 0.5% proparacaine, a 2.0 mm central epithelial section was marked and then gently scraped with a blunt corneal scraper.

[0044] (2) Exosome treatment: The control group was given normal saline, and the other two groups were given hypertonic solution (500mOsm) to soak their eyes for 30 minutes. At the same time, 5 μL of exosome suspension (1.0 × 10 8The hypMSC-Exo group was topically treated with PBS to injure the cornea at time intervals of 0, 10, 20, and 30 minutes.

[0045] (3) Corneal wound healing observation: Wound healing was assessed using fluorescein staining at 0, 24, and 48 hours, and photographs were taken using a camera attached to a slit lamp biomicroscope. The wound healing percentage of each mouse was compared with the baseline using ImageJ software.

[0046] 2. Experimental results

[0047] Hypertonicity significantly inhibited the healing of corneal epithelial lesions in mice, and hypMSC-Exo could restore the inhibition of hypertonicity on the healing of corneal epithelial lesions (see Figure 3 ).

[0048] Experiments have shown that exosomes derived from hypoxic mesenchymal stem cells can significantly restore the inhibition of corneal epithelial injury healing under hypertonic conditions both in vivo and in vitro, have a good therapeutic effect on corneal epithelial injury under hypertonic conditions, and have good application prospects.

[0049] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of exosomes derived from hypoxic mesenchymal stem cells in the preparation of drugs to promote the healing of corneal epithelial damage.

2. Application of hypoxic mesenchymal stem cell exosomes in the preparation of drugs for the treatment of corneal epithelial damage.

3. The use according to claim 1 or 2, characterized in that The hypoxic mesenchymal stem cell exosomes are exosomes isolated after bone marrow mesenchymal stem cells are cultured under hypoxic conditions in serum-free DMEM medium for 48 hours; the hypoxic conditions are 1% oxygen, 5% carbon dioxide, and the rest nitrogen.

4. The use according to claim 3, characterized in that The method for preparing hypoxic mesenchymal stem cell exosomes comprises the following steps: Bone marrow cells extracted from mouse tibias and femurs were resuspended in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin; the medium was changed after 24 hours and every 3 days thereafter; bone marrow mesenchymal stem cells (BM-MSCs) were cultured to 70-80% confluence; then cultured under hypoxic conditions in serum-free DMEM medium for 48 hours; separated by traditional differential centrifugation; the medium was collected and centrifuged at 300 g for 5 minutes to remove cells; the medium was then centrifuged at 2,000 g for 20 minutes and at 10,000 g for 30 minutes at 4°C to remove cell debris; the supernatant was then filtered through a 0.22 μm filter; the pretreated supernatant was ultracentrifuged at 100,000 g for 2 hours at 4°C; and the exosome pellet was resuspended in phosphate-buffered saline.

5. A drug for promoting the healing of corneal epithelial damage, wherein the active ingredient of the drug is hypoxic mesenchymal stem cell exosomes.

6. The drug according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable carriers or excipients.

7. The drug according to claim 5, characterized in that The effective dose of hypoxic mesenchymal stem cell exosomes in the drug is 1.0 × 10 8 particles / mL.

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