Application of mesenchymal stem cell apoptosis vesicles in preparation of medicine for treating dry eye

Abstract

The invention discloses application of mesenchymal stem cell apoptosis vesicles in preparation of a medicine for treating dry eyes. The human umbilical cord-derived mesenchymal stem cell apoptosis vesicles are applied to preparation of the medicine for treating the dry eye for the first time, the mesenchymal stem cell apoptosis vesicles have the effects of promoting repair and regeneration of ocular surface cells such as corneal epithelial cells and lacrimal gland cells, can inhibit expression of proinflammatory factors and increase expression of anti-inflammatory factors, and are relatively simple and convenient to prepare and low in cost. Side effects are small and curative effects are high. Under the condition that artificial tears and anti-inflammatory drugs for clinically treating the dry eye at present have certain limitations, the mesenchymal stem cell apoptosis vesicles are used as a therapy with immunoregulation and regeneration repair capabilities, and a new thought is provided for treatment of the dry eye.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of mesenchymal stem cell apoptotic vesicles in preparing medicine for treating dry eyes. Background Art

[0002] Dry eye disease (DED) is a chronic ocular surface disease caused by multiple factors. Epidemiological surveys show that 5% to 34% of people around the world are troubled by dry eyes, and the number of patients has been increasing year by year in recent years. The onset of DED is mainly due to the loss of tear film homeostasis or imbalance of the ocular surface microenvironment caused by abnormalities in the quality, quantity and dynamics of tears, which may be accompanied by ocular surface inflammatory reactions, tissue damage and neurological abnormalities, causing a variety of ocular discomfort symptoms and / or visual dysfunction. The main symptoms of DED are dry eyes, itching, fatigue, foreign body sensation, glare and decreased vision, which bring eye troubles to patients' lives and work. Severe DED can significantly reduce the quality of life of patients. Although the causes of DED are different, inflammatory response is a key factor in the development of its course. Currently, the treatment of DED mainly relies on artificial tears. At present, there are some drugs targeting inflammatory response such as cyclosporine eye drops in the market. On the one hand, the price of such drugs is high and the cost of long-term use is too high. On the other hand, the treatment effect is still unsatisfactory for some patients with severe dry eye caused by immune inflammatory diseases such as graft-versus-host disease (GVHD), Sjögren's syndrome, Stevens-Johnson syndrome, etc. In recent years, stem cell therapy, as a new therapy with immune regulation and regeneration and repair capabilities, has provided a new idea for the treatment of dry eye.

[0003] Mesenchymal stem cells (MSCs) are multipotent adult stem cells with multidirectional differentiation potential and self-replication biological characteristics. In 1976, Friedenstein et al. first isolated fibroblast-like colonies from the bone marrow. Later, Caplan named them "MSCs" based on the characteristics of these cells. However, the direct clinical application of MSCs itself has certain limitations for the topical treatment of ocular surface diseases. On the one hand, stem cells are expensive, and on the other hand, cell products still have the ability to self-replicate and may have potential tumorigenicity. Therefore, it is meaningful to further explore the active ingredients of MSCs.

[0004] The term exosome (Exos) was first proposed by Trams et al. in 1981. It is a bilayer lipid vesicle with a diameter of 40 to 100 nm. It is produced in multivesicular bodies and released into the extracellular environment through the fusion of multivesicular bodies with the plasma membrane. Recent studies have shown that mesenchymal stem cell-derived exosomes (MSC-Exos) play an important role in regulating immune responses and other aspects, providing a new direction for the treatment of dry eyes. By culturing MSCs in vitro and then inducing them to produce exosomes, which have the molecular characteristics and biological functions of parental cells, they can exert unique therapeutic effects. However, the application of exosomes has shortcomings such as relatively complex production steps and high requirements for cell status.

[0005] Apoptotic vesicles (apoVs) are bilayer lipid structures with unique biological characteristics and functional properties produced during apoptosis, and play an important role in maintaining tissue and organ homeostasis. AapoVs and exosomes (Exos) have unique advantages in biomedical applications. Compared with exosomes, apoVs have different functional protein carriers and surface markers. At least 13 specific proteins have been identified, which can play an important role in maintaining tissue and organ homeostasis. ApoVs are bilayer lipid structures produced by apoptosis, with large particle size, high density, simple and controllable generation process, and source specificity. Due to its large size and strong drug loading capacity, it can carry more drug molecules or therapeutic payloads. ApoVs can also inherit substances and information from parent cells, accurately transmit specific signals or molecules, and may play a more important role, especially under pathological conditions. In addition, apoVs have good biocompatibility and low immunogenicity in the body, which can effectively avoid immune rejection and ensure the safety and stability of treatment. In terms of tissue repair and regeneration, apoVs show higher efficiency and can promote cell proliferation and tissue repair. At the same time, apoVs also perform well in anti-inflammatory and antibacterial properties, effectively reducing infection and inflammatory responses, thereby promoting wound healing and tissue regeneration. However, the application of apoptotic vesicles in the treatment of dry eye has not been reported. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of apoptotic vesicles of mesenchymal stem cells in the preparation of a drug for treating dry eye, aiming to provide a new idea for the treatment of dry eye.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect, a use of mesenchymal stem cell apoptotic vesicles in the preparation of a drug for treating dry eyes is provided.

[0009] A preferred technical solution is a method for preparing mesenchymal stem cell apoptotic vesicles, comprising:

[0010] Adding staurosporine to mesenchymal stem cells for stimulation causes the mesenchymal stem cells to shrink;

[0011] The shrunken mesenchymal stem cells were washed by blowing, centrifuged at 600-1000 g for 5-10 min, and then at 1800-2200 g for 5-10 min, and the supernatant was collected;

[0012] The supernatant was centrifuged at 15000-20000 g for 30-40 min and the precipitate was collected;

[0013] The precipitate was resuspended in PBS and centrifuged at 20,000-22,000 g for 30-40 min, and the supernatant was discarded to obtain the apoptotic vesicles of mesenchymal stem cells.

[0014] In a preferred technical solution, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0015] In a preferred technical solution, the drug is used to promote the repair and regeneration of ocular surface cells to treat dry eyes.

[0016] In a preferred technical solution, the drug is used to inhibit pro-inflammatory factors and increase the expression of anti-inflammatory factors to treat dry eyes.

[0017] In a preferred technical solution, the drug is an ophthalmic preparation prepared with mesenchymal stem cell apoptotic vesicles as active ingredients and pharmaceutically acceptable excipients.

[0018] In a preferred technical solution, the medicine is eye drops.

[0019] In a further preferred technical solution, the concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 7 ~1×10 9 Pieces / mL.

[0020] In a further preferred technical solution, the concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 8 Pieces / mL.

[0021] According to a further preferred technical solution, the eye drops are administered 4 times a day, 0.05 to 0.1 mL each time.

[0022] Beneficial effects: The present invention provides the use of mesenchymal stem cell apoptotic vesicles in the preparation of drugs for treating dry eyes. Compared with exosomes, the advantages of apoptotic vesicles are: the generation process is relatively simple, and they can be obtained by inducing cell apoptosis; good biocompatibility and low immunogenicity, which are conducive to reducing immune rejection reactions; and higher efficiency in tissue repair and regeneration. In the application of treating dry eyes, mesenchymal stem cell apoptotic vesicles have the effect of promoting the repair and regeneration of ocular surface cells such as corneal epithelial cells and lacrimal gland cells, can inhibit the expression of pro-inflammatory factors and increase anti-inflammatory factors, with small side effects and high efficacy. In the case that the current clinical drugs for treating dry eyes have certain limitations and inevitably bring a series of adverse reactions, its application prospects are good. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of fluorescent staining of mouse cornea in Example 1.

[0024] Figure 2 This is a graph showing the test results of the tear secretion of mice in Example 1.

[0025] Figure 3 This is a graph showing the test results of the tear film breakup time of mice in Example 1. DETAILED DESCRIPTION

[0026] The present invention provides use of apoptotic vesicles of mesenchymal stem cells in the preparation of a drug for treating dry eye. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0027] Studies in recent years have shown that mesenchymal stem cells (MSCs) and exosomes (exos) have great potential and practicability in inflammatory response, drug delivery, nerves and tissues. However, most of the current research is based on animal models. Exosomes are relatively difficult to prepare and lack large-sample clinical studies. Compared with exosomes (exos), apoptotic vesicles (apovs) have a relatively simple production process and can be obtained by inducing cell apoptosis; good biocompatibility and low immunogenicity, which are conducive to reducing immune rejection reactions; and show higher efficiency in tissue repair and regeneration.

[0028] Based on this, an embodiment of the present invention provides the use of mesenchymal stem cell apoptotic vesicles in the preparation of a drug for treating dry eyes.

[0029] In the comparison of the effects of treating dry eyes, studies have shown that adipose mesenchymal stem cell-derived exosomes (ADSC-Exos) may reduce cytokines by inhibiting the TLR4 signaling pathway / NLRP3-IL-1β signaling pathway, and have a significant therapeutic effect on benzalkonium chloride-induced dry eyes in mice (Wang Guifang, Long Hongmei, Gong Xileyuan, et al. The therapeutic effect and mechanism of adipose mesenchymal stem cell-derived exosomes on benzalkonium chloride-induced dry eyes in mice [J]. New Progress in Ophthalmology, 2021, 41(10): 930-934.). In this example, human umbilical cord mesenchymalstem cells apoptotic vesicles (hUCMSCs-apoVs) are used to prepare eye drops for the treatment of dry eyes. Existing results show that hUCMSCs-apoVs eye drops can reduce ocular surface damage caused by dry eyes, and the mechanism-related research needs further research and confirmation. Since apoVs can promote cell proliferation and tissue repair, and also have excellent anti-inflammatory and antibacterial properties, apoVs can alleviate dry eye symptoms and reduce corneal and conjunctival damage in DED patients through immune regulation.

[0030] In one embodiment, the method for preparing the mesenchymal stem cell apoptotic vesicles comprises:

[0031] Adding staurosporine to mesenchymal stem cells for stimulation causes the mesenchymal stem cells to shrink;

[0032] The shrunken mesenchymal stem cells were washed by blowing, centrifuged at 600-1000 g for 5-10 min, and then at 1800-2200 g for 5-10 min, and the supernatant was collected;

[0033] The supernatant was centrifuged at 15000-20000 g for 30-40 min and the precipitate was collected;

[0034] The precipitate was resuspended in PBS and centrifuged at 20,000-22,000 g for 30-40 min, and the supernatant was discarded to obtain the apoptotic vesicles of mesenchymal stem cells.

[0035] In one embodiment, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0036] In one embodiment, the drug is a drug for promoting the repair and regeneration of ocular surface cells to treat dry eyes.

[0037] In one embodiment, the drug is a drug for inhibiting pro-inflammatory factors and increasing the expression of anti-inflammatory factors to treat dry eyes.

[0038] In one embodiment, the concentration of staurosporine (STS) is 400-600 nM, and the stimulation time is 5-7 hours. Preferably, the concentration of staurosporine is 500 nM, and the stimulation time is 6 hours. Specifically, 4 mL STS is added to a 10 cm culture dish, and 10 mL STS is added to a 15 cm culture dish.

[0039] In one embodiment, the drug is an ophthalmic preparation prepared with mesenchymal stem cell apoptotic vesicles as active ingredients and pharmaceutically acceptable excipients.

[0040] In one embodiment, the drug is eye drops.

[0041] In one embodiment, the method for preparing the eye drops comprises:

[0042] (1) Preparing freeze-dried powder of mesenchymal stem cell apoptotic vesicles: mixing the mesenchymal stem cell apoptotic vesicles with the freeze-dried solution, centrifuging, placing at -80°C overnight and then freeze-drying to obtain freeze-dried powder of mesenchymal stem cell apoptotic vesicles;

[0043] Wherein, the lyophilized liquid includes 100mM trehalose and PVP-40 with a mass concentration of 5%;

[0044] (2) Preparation of mesenchymal stem cell apoptotic vesicle eye drops: The freeze-dried powder of the mesenchymal stem cell apoptotic vesicle is dissolved in double distilled water, diluted with PBS filtered through a 0.22 μm filter membrane, and then the diluted solution is filtered through a 0.22 μm filter membrane to obtain the mesenchymal stem cell apoptotic vesicle eye drops.

[0045] In one embodiment, the concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 7 ~1×10 9 Pieces / mL.

[0046] In one embodiment, the concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 8 Pieces / mL.

[0047] In one embodiment, the eye drops are administered 4 times a day, 0.05 to 0.1 mL each time.

[0048] The present invention will be further described below by means of specific examples.

[0049] Example 1

[0050] 1. Preparation process of mesenchymal stem cell apoptotic vesicles:

[0051] 1. Staurosporine (STS) stimulates cell shrinkage: 4 mL of STS was added to each 10 cm cell culture dish of human umbilical cord mesenchymal stem cells (hucMSCs) (10 mL of STS was added to 15 cm cell culture dish) for about 5 hours to cause cell shrinkage;

[0052] 2. Centrifugation after washing: Wash the shrunken cells into a 1.5 mL EP tube and centrifuge at 4°C (800g for 10 min → 2000g for 10 min);

[0053] 3. Take the supernatant: take the supernatant after centrifugation and dispense it into EP tubes (840 μL per EP tube × 2);

[0054] 4. Centrifugation: 17500g for 30 min at 4°C;

[0055] 5. Resuspend: discard the supernatant and resuspend in PBS filtered through a 0.22 μm filter membrane;

[0056] 6. Centrifugation: 21000g for 30 min at 4°C.

[0057] 2. Preparation process of freeze-dried powder of mesenchymal stem cell apoptotic vesicles:

[0058] 1. Prepare lyophilized solution: 100mM trehalose + 5% PVP-40 (Polyvinylpyrrolidone, average MW40000, polyvinylpyrrolidone), the configuration method refers to Baradie et al. (Youssef BK BE, Mohamed N, Frederick IO, et al. Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury. [J]. Frontiers in cell and developmental biology, 2020, 8181.): 0.191g trehalose + 0.25g PVP40 + 4mL 0.65×PBS;

[0059] 2. Mix the mesenchymal stem cell apoptotic vesicles with the lyophilized solution: 100 μL of mesenchymal stem cell apoptotic vesicles + 400 μL of lyophilized solution, mix well, centrifuge, and place at -80°C overnight;

[0060] 3. Preparation of freeze-dried powder of apoptotic vesicles of mesenchymal stem cells: freeze-drying in a freeze dryer for about 12 hours.

[0061] 3. Preparation process of eye drops containing mesenchymal stem cell apoptotic vesicles:

[0062] 1. Filter PBS: Filter PBS through a 0.22 μm filter membrane;

[0063] 2. Dissolve the freeze-dried powder of mesenchymal stem cell apoptotic vesicles in double distilled water: Dissolve the freeze-dried powder of mesenchymal stem cell apoptotic vesicles in each EP tube with 500 μl of double distilled water;

[0064] 3. PBS dilution: Use filtered PBS to dilute the dissolved mesenchymal stem cell apoptotic vesicle solution;

[0065] 4. Filtration: Filter the diluted mesenchymal stem cell apoptotic vesicle solution through a 0.22 μm filter membrane to obtain eye drops containing mesenchymal stem cell apoptotic vesicles.

[0066] Fourth, C57BL / 6 mice were used to establish a dry eye model and the therapeutic effect of mesenchymal stem cell apoptotic vesicles was confirmed by corneal fluorescence staining and phenol red cotton thread tear analysis.

[0067] 1. The experiment was divided into four groups: blank control group, dry eye disease (DED) model group, mesenchymal stem cell apoptosis vesicle treatment group, and solvent group, with 5 mice in each group.

[0068] 2. Establishment of dry eye model: Mice were placed in cages with fans on both sides and raised in an environmental control room with a humidity of 30%, and scopolamine hydrobromide (0.5 mg / 0.2 mL) was injected subcutaneously into the mice 4 times a day for 7 consecutive days to reduce tear production. Each injection should be performed subcutaneously at different sites to avoid leakage of fluid caused by multiple injections.

[0069] 3. The blank control group did not receive any treatment. The dry eye model group did not receive any treatment after modeling. The mesenchymal stem cell apoptotic vesicle treatment group was treated with eye drops containing mesenchymal stem cell apoptotic vesicles (the concentration of mesenchymal stem cell apoptotic vesicles in the eye drops was about 1×10 8 The solvent group was modeled with PBS, which was applied 4 times a day (9:00, 11:00 in the morning, 15:00, 18:00 in the afternoon) for 7 days. The dry eye condition in each group was evaluated by corneal fluorescence staining and phenol red cotton thread tear analysis.

[0070] 4. Sodium fluorescein staining and scoring: Sodium fluorescein staining was used to evaluate the degree of corneal epithelial damage. Five mice were selected from each group 7 days after the administration of eye drops containing apoptotic vesicles of mesenchymal stem cells. First, the conjunctival sac of the mouse was rinsed 1 to 2 times with 0.9% (w / v) NaCl to ensure the cleanliness of the ocular surface, and then excess moisture around the eye was wiped off with a cotton swab; 10 μL of 0.1% (w / v) sodium fluorescein solution was carefully dropped into 1 / 3 of the conjunctival sac of the outer canthus of the mouse, and the upper and lower eyelids were artificially closed to make it blink three times. After the sodium fluorescein solution covered the entire cornea, the mouse was kept in a normal open-eye state and carefully observed and photographed under a slit lamp microscope with a blue cobalt lamp. The results are as follows: Figure 1 As shown, A is the blank control group, B is the dry eye model group, C is the mesenchymal stem cell apoptotic vesicle treatment group, and D is the solvent group. The damaged part of the cornea is the part that is stained with fluorescent yellow-green. The specific degree is judged according to the quadrant scoring method: the cornea is divided into 4 quadrants (superior, inferior, temporal, and nasal); the staining of each quadrant is scored separately, and then the score of each quadrant is added up. The scoring standard is (Liu Zuguo, Xiao Qiguo. Diagnosis and treatment of allergic conjunctivitis [J]. Chinese Journal of Ophthalmology, 2004, (07): 72-74.): 0 points: no staining; 1 point: slight dot-like staining and less than 30 points; 2 points: staining of not less than 30 points, but no diffuse phenomenon; 3 points: diffuse staining but no plaques; 4 points: plaque-like staining. The total score is 16 points. Each eye of the mouse was measured three times, and the average value was used as the score of corneal fluorescein sodium staining of the eye. Figure 1 It can be seen that after treatment, compared with the dry eye model group, the fluorescence staining of mice in the mesenchymal stem cell apoptotic vesicle treatment group was improved, and the fluorescence staining score was significantly reduced (n=5).

[0071] 5. Tear secretion experiment: The tear secretion of each group of mice was detected by the phenol red cotton thread experiment. Five mice were taken from each group at 3d and 7d after the administration of eye drops containing apoptotic vesicles of mesenchymal stem cells. The phenol red cotton thread was clamped with ophthalmic microtweezers and placed in the middle and outer 1 / 3 of the mouse conjunctival sac. It was taken out after staying for 60s. The wet length of the phenol red cotton thread was measured with a vernier caliper under a microscope, that is, the length of the cotton thread that changed from yellow to red. Each eye was tested 3 times, and the average length of the red part was considered to be the final length. The experimental results are shown in Tables 1 and Figure 2 As shown in Table 1 and Figure 2It can be seen that compared with the normal control group, the tear secretion of the dry eye model group was greatly reduced, and the difference was significant, indicating that the dry eye model was successfully established; compared with the dry eye model group, the tear secretion of the mesenchymal stem cell apoptotic vesicle treatment group was significantly increased; compared with the solvent group, the tear secretion of the mesenchymal stem cell apoptotic vesicle treatment group was also increased. Among them, the normal control group versus the dry eye model group, ****P<0.0001; the mesenchymal stem cell apoptotic vesicle treatment group versus the dry eye model group, ****P<0.0001; the mesenchymal stem cell apoptotic vesicle treatment group versus the solvent group, ***P<0.001; all were statistically significant.

[0072] Table 1 Test results of tear secretion in mice

[0073]

[0074] 6. Tear film breakup time: The BUT detection method was used to detect the tear film breakup time of each group of mice. Five mice were selected from each group at 3d and 7d after the administration of eye drops containing apoptotic vesicles of mesenchymal stem cells, and 10μL of 0.1% (w / v) sodium fluorescein solution was carefully dropped into the 1 / 3 of the conjunctival sac of the outer canthus of the mouse. The upper and lower eyelids were artificially closed to make them blink three times, and then the mice were kept in a normal open-eye state. They were immediately observed under a slit lamp microscope with a blue cobalt lamp, and the time from this time to the first appearance of black spots (breakup points) in the staining range was recorded. The operation was repeated 3 times in a row and the average value was recorded. The screening criterion for tear film breakup time is: ≤4s. The experimental results are shown in Tables 2 and Figure 3 As shown in Table 2 and Figure 3 It can be seen that the tear film breakup time of the dry eye model group was significantly shortened compared with the normal control group; the tear film breakup time of the mesenchymal stem cell apoptotic vesicle treatment group was significantly prolonged compared with the dry eye model group; the tear film breakup time of the mesenchymal stem cell apoptotic vesicle treatment group was also increased compared with the solvent group. Among them, the normal control group versus the dry eye model group, ****P<0.0001; the mesenchymal stem cell apoptotic vesicle treatment group versus the dry eye model group, ****P<0.0001; the mesenchymal stem cell apoptotic vesicle treatment group versus the solvent group, ***P<0.001; all were statistically significant.

[0075] Table 2 Mouse tear film breakup time test results

[0076]

[0077] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Application of mesenchymal stem cell apoptotic vesicles in the preparation of drugs for the treatment of dry eyes.

2. The use according to claim 1, characterized in that: The method for preparing mesenchymal stem cell apoptotic vesicles comprises: Adding staurosporine to mesenchymal stem cells for stimulation causes the mesenchymal stem cells to shrink; The shrunken mesenchymal stem cells were washed by blowing, centrifuged at 600-1000 g for 5-10 min, and then at 1800-2200 g for 5-10 min, and the supernatant was collected; The supernatant was centrifuged at 15000-20000 g for 30-40 min and the precipitate was collected; The precipitate was resuspended in PBS and centrifuged at 20,000-22,000 g for 30-40 min, and the supernatant was discarded to obtain the apoptotic vesicles of mesenchymal stem cells.

3. The use according to claim 2, characterized in that: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

4. The use according to claim 1, characterized in that: The drug is used for promoting the repair and regeneration of ocular surface cells to treat dry eyes.

5. The use according to claim 1, characterized in that: The drug is used for inhibiting pro-inflammatory factors and increasing the expression of anti-inflammatory factors to treat dry eyes.

6. The use according to claim 1, characterized in that: The medicine is an ophthalmic preparation prepared by taking mesenchymal stem cell apoptotic vesicles as active ingredients and adding pharmaceutically acceptable excipients.

7. The use according to claim 1, characterized in that: The medicine is eye drops.

8. The use according to claim 7, characterized in that: The concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 7 ~1×10 9 Pieces / mL.

9. The use according to claim 7, characterized in that: The concentration of mesenchymal stem cell apoptotic vesicles in the eye drops is 1×10 8 Pieces / mL.

10. The use according to claim 9, characterized in that: The eye drops are administered 4 times a day, 0.05 to 0.1 mL each time.

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