Application of umbilical cord blood exosome in preparation of medicine for treating ophthalmic diseases caused by aging

Abstract

The invention discloses application of an umbilical cord blood exosome in preparation of a medicine for treating ophthalmic diseases caused by aging. It is found that the umbilical cord blood exosome can improve the biological characteristics of senescent trabecular meshwork cells, relieve the senescence characteristics of the trabecular meshwork cells, improve the DNA damage repair capacity of the cells, promote proliferation of the trabecular meshwork cells and effectively maintain the stability of cell nucleuses, and then growth and division of the cells are supported. The umbilical cord blood exosome shows a remarkable anti-aging effect in treatment of ophthalmic diseases caused by aging, can effectively improve the biological function of trabecular meshwork cells, slow down the aging process and promote cell proliferation and repair, and has wide application potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of umbilical cord blood exosomes in preparing drugs for treating ophthalmic diseases caused by aging. Background Art

[0002] Exosomes, also known as extracellular vesicles (EVs), are nanoscale, cell-derived membrane vesicles that carry bioactive substances, including mRNA, proteins, and lipids, which are crucial for intercellular communication. Umbilical cord blood, the residual blood within the umbilical cord connecting the mother and fetus, contains many active ingredients. Preserving cord blood clinically can treat a variety of diseases, such as malignant tumors, blood system diseases, and immune system diseases, making it a very important resource. The rich hematopoietic stem cells contained in cord blood make it a valuable source of hematopoietic stem cells. The hematopoietic function of cord blood is increasingly recognized by the medical community. Cord blood transplants can be used to treat a variety of difficult-to-cure diseases, such as leukemia, diabetes, and radiation sickness.

[0003] As we age, the effects of aging on eye health gradually become apparent. Aging-related eye diseases include, but are not limited to, cataracts, glaucoma, age-related macular degeneration (AMD), and dry eye. These conditions not only affect patients' visual quality but can also have a serious negative impact on their mental health and social activities. According to statistics, age-related vision impairment is a major health problem among the elderly worldwide, resulting in a high economic burden and medical demand.

[0004] While a variety of treatment options are available, existing treatments still have many limitations. For example, while cataract surgery can restore vision, it cannot reverse other degenerative changes in the eye caused by age. Medical and surgical treatments for glaucoma have limited effectiveness and may be accompanied by side effects. For age-related macular degeneration, anti-VEGF therapy has some efficacy, but the course of treatment is long and expensive, and it does not completely address the cause. In addition, treatments for dry eye, while somewhat relieving, have not yet effectively cured the condition.

[0005] Therefore, a new, more effective and safe treatment strategy is urgently needed to repair eye tissue damage caused by aging and slow down or reverse pathological changes in the aging process. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention proposes the use of umbilical cord blood exosomes in the preparation of drugs for treating ophthalmic diseases caused by aging.

[0007] The present invention provides the use of umbilical cord blood exosomes in the preparation of a medicament for treating ophthalmic diseases caused by aging.

[0008] In some embodiments, the dosage form of the drug is any one of drops, gels, injections, oral preparations, transdermal absorption preparations, and mucosal absorption preparations.

[0009] In some embodiments, the drug administration routes include, but are not limited to, oral administration, eye drops, gels, and intravitreal injections.

[0010] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient, wherein the excipient is selected from any one or more of a thickener, a stabilizer, a preservative, a buffer, and the like.

[0011] The present invention also provides the use of umbilical cord blood exosomes in the preparation of drugs for preventing ophthalmic diseases caused by aging.

[0012] The present invention also provides the use of umbilical cord blood exosomes in preparing products for preventing ocular cell aging.

[0013] The present invention also provides the use of umbilical cord blood exosomes in preparing a product for restoring the cell cycle of aging ocular cells.

[0014] In some embodiments, the ocular cells include any one or more of trabecular meshwork cells, retinal cells, corneal cells, lens cells, optic nerve cells, iris cells, ciliary body cells, intraocular smooth muscle cells, and corneal endothelial cells.

[0015] In some embodiments, the ocular cells are trabecular meshwork cells, and the application is specifically:

[0016] Reduce the proportion of Beam A cell subsets in trabecular meshwork cells, and increase the proportions of Beam B cell subsets and JCT cell subsets in trabecular meshwork cells.

[0017] In some embodiments, the aging includes one or more of individual aging, organ aging, and cell aging; individual aging refers to the decline process of the entire organism; organ aging refers to the decline of the function of a specific organ or system, usually accompanied by structural changes; cell aging is the process in which cells no longer divide but remain active. Senescent cells cannot continue to divide, affecting tissue repair and regeneration.

[0018] In some embodiments, the application is specifically: umbilical cord blood exosomes prevent, improve or restore the function of aging eye cells by regulating the cell growth cycle.

[0019] In some embodiments, the regulating cell growth cycle is specifically any one or more of the following:

[0020] (1) Restoring the function of aging cells in G2 / M; The G2 / M phase is a key phase in the cell cycle, responsible for converting cells from the G2 phase to the metaphase (M phase). This phase includes chromosome replication correction, the disintegration of the nuclear membrane, and the preparation of related proteins to ensure that the genetic material can be correctly distributed when the cell divides. This phase is crucial for cell proliferation and the maintenance of normal function.

[0021] (2) Upregulation of CDK1 and CDK2 expression; CDK1 upregulation: Exosomes may promote the transcription and translation of CDK1 by activating specific pathways, or increase its stability by inhibiting its degraders. Upregulation of CDK1 helps initiate the M phase, ensuring that cells can enter the division phase; CDK2 upregulation: Exosomes may promote the expression or stability of CDK2 through similar mechanisms. CDK2 plays an important role in the G2 / M transition and S phase, and its upregulation helps the smooth progress of the cell cycle.

[0022] (3) Down-regulation of P16 and P21 protein expression; Umbilical cord blood exosome treatment significantly reduced the mRNA and protein levels of P16 and P21, suggesting that they play a positive role in regulating cell cycle arrest.

[0023] (4) Upregulate the expression of Ki67 and promote cell cycle progression; after treatment with umbilical cord blood exosomes, the secretion of Ki67 increased significantly, indicating that it can push cells from the G0 / G1 phase to the S phase and M phase, thereby promoting cell proliferation.

[0024] (5) Downregulate the expression of H2A.X and improve the DNA damage response; umbilical cord blood exosome treatment reduced the phosphorylation of H2A.X, and flow cytometry showed that the cells' tolerance to DNA double-strand breaks was improved, indicating its role in the DNA repair mechanism.

[0025] (6) Upregulate the expression of LaminB1 protein and promote nuclear structural stability; after treatment with umbilical cord blood exosomes, the expression of LaminB1 increased significantly, accompanied by the improvement of cell nuclear morphology and the reduction of nuclear membrane rupture, suggesting its important role in maintaining nuclear structural stability.

[0026] (7) Inhibition of lysosomal function. Umbilical cord blood exosome treatment reduced the secretion of lysosomal active proteins, thereby reducing the risk of cell apoptosis, which suggests its potential mechanism in inhibiting programmed cell death.

[0027] Exosomes play an important role in anti-aging, mainly through the following pathways:

[0028] (1) Reduce oxidative stress: Umbilical cord blood exosomes contain antioxidant components, such as certain peptides and non-coding RNA, which can scavenge free radicals and reduce cell damage.

[0029] (2) Regulating key signaling pathways: Umbilical cord blood exosomes regulate growth factors, death receptor proteins, etc. in cells by transmitting specific information substances, thereby affecting cell lifespan.

[0030] (3) DNA repair and regeneration: Certain components in umbilical cord blood exosomes may promote DNA repair and tissue regeneration, delaying aging.

[0031] Umbilical cord blood exosomes regulate CDK1, CDK2, and LMNb1 through multiple pathways, thereby influencing the smooth progression of the cell cycle. Furthermore, cord blood exosomes play a vital role in anti-aging, primarily through free radical scavenging, signaling pathway regulation, and DNA repair. This regulatory network provides a dynamic and precise means of regulating cell proliferation and lifespan.

[0032] (4) Improve mitochondrial dysfunction; umbilical cord blood exosomes can reduce ROS accumulation and improve mitochondrial function by activating antioxidant pathways (such as PRDX6).

[0033] (5) Regulate the TGF-β signaling pathway; umbilical cord blood exosomes can reduce trabecular meshwork sclerosis by inhibiting TGF-β-mediated fibrosis.

[0034] (6) Promote autophagy and cell survival; Umbilical cord blood exosomes can eliminate senescent cells by enhancing autophagy. At the same time, the upregulation of Beam B C3 can activate lysosomal function and support cell metabolism.

[0035] (7) Restore cell mechanical sensitivity; Umbilical cord blood exosomes can improve the mechanical response of the trabecular meshwork to aqueous humor outflow by regulating cell contractility.

[0036] In some embodiments, the ophthalmic disease comprises any one of glaucoma, cataract, age-related maculopathy, corneal disease, and retinitis pigmentosa.

[0037] Since senescent cells in the trabecular meshwork impair the drainage of intraocular fluid and increase intraocular pressure, thereby damaging the optic nerve and causing glaucoma, umbilical cord blood exosomes can improve the function of the trabecular meshwork, improve the drainage function of the aqueous chamber, remodel the matrix to reduce intraocular pressure, and protect the optic nerve through antioxidant and anti-inflammatory effects, thereby preventing vision loss caused by glaucoma.

[0038] The occurrence of cataracts is related to the aging and opacity of lens epithelial cells. This application can repair aging lens epithelial cells through the growth factors and miRNAs in umbilical cord blood exosomes, thereby delaying the progression of lens opacity.

[0039] Age-related macular degeneration (AMD) is a retinal degenerative disease caused by aging, primarily characterized by degeneration of the macula. Umbilical cord blood exosomes can restore the growth and proliferation of optic nerve cells by promoting the G2 / M phase of the cell cycle. They also regulate oxidative stress and protect the retina from oxidative damage. They stimulate retinal cells to release protective factors, such as IL-6 and IL-8, promoting macular cell repair. The miRNAs and certain proteins in umbilical cord blood exosomes can reduce the inflammatory response of retinal cells and modulate related immune pathways. Furthermore, by influencing the expression of molecules such as VEGF and TNF-α, umbilical cord blood exosomes can regulate the pathological process, treat AMD, and improve macular function.

[0040] Corneal diseases, including corneal degeneration and keratitis, are typically caused by aging or damage to corneal cells. Umbilical cord blood exosomes can stimulate corneal cell proliferation and differentiation by regulating the G2 / M cycle, accelerating the repair process. Antioxidants in exosomes can scavenge free radicals and reduce retinal tissue damage. By regulating cell activity and reducing apoptosis, exosomes have the potential to accelerate corneal self-repair. By mitigating inflammatory responses, they can have a therapeutic effect on corneal damage caused by infection or inflammation, restoring corneal transparency.

[0041] Retinitis pigmentosa is primarily caused by the aging and degeneration of retinal pigment epithelial cells, which in turn affects vision. Umbilical cord blood exosomes can promote the proliferation and differentiation of photoreceptor cells. In retinitis pigmentosa (RP), diseased photoreceptor cells or RPE cells may release exosomes containing pro-apoptotic factors (such as caspases and inflammatory factors), accelerating photoreceptor cell degeneration. Umbilical cord blood-derived exosomes can carry anti-aging and anti-apoptotic miRNAs and neurotrophic factors to delay photoreceptor cell death.

[0042] In some embodiments, the drug is used in combination with other drugs for treating eye diseases associated with aging.

[0043] In some embodiments, the combined use comprises encapsulating other drugs for treating aging-induced ophthalmic diseases in the umbilical cord blood exosomes; further, the other drugs for treating aging-induced ophthalmic diseases comprise proteins or RNA that inhibit ocular cell aging, or inhibitors of proteins or RNA that promote ocular cell aging.

[0044] In some embodiments, the umbilical cord blood exosomes are obtained by mixing umbilical cord plasma with polyethylene glycol, incubating the mixture, and then separating the mixture by ultracentrifugation.

[0045] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient.

[0046] In some embodiments, the concentration of umbilical cord blood exosomes in the drug is 10 6~10 11 p / mL.

[0047] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0048] The present invention discovered that umbilical cord blood exosomes can improve the biological properties of aging trabecular meshwork cells, reduce the aging characteristics of trabecular meshwork cells, improve the cells' DNA damage repair capacity, promote cell proliferation, and effectively maintain nuclear stability, thereby supporting cell growth and division. Umbilical cord blood exosomes have demonstrated significant anti-aging effects in the treatment of aging-related ophthalmic diseases, effectively improving the biological functions of trabecular meshwork cells, slowing the aging process, and promoting cell proliferation and repair. These findings suggest that umbilical cord blood exosomes have broad application prospects and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0050] Figure 1 This is a transmission electron microscope image of exosomes in Example 1 of the present invention;

[0051] Figure 2 This is the nanoparticle tracking analysis result of Example 1 of the present invention;

[0052] Figure 3 This is the result of Western blot detection of exosome marker proteins in Example 1 of the present invention;

[0053] Figure 4 This is the change in the level of aging-associated β-galactosidase (SA-βgal) after treatment with umbilical cord blood exosomes in Example 2 of the present invention; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

[0054] Figure 5 The results of the changes in the rate of P21-positive cells, a cell cycle arrest marker, after treatment with umbilical cord blood exosomes in Example 2 of the present invention; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

[0055] Figure 6 The results of the change in the Ki67-positive cell rate, a cell proliferation marker, after treatment with umbilical cord blood exosomes in Example 2 of the present invention; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

[0056] Figure 7 The results of the changes in γ-H2A.X expression on senescent human trabecular meshwork cells after treatment with umbilical cord blood exosomes in Example 2 of the present invention are shown; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001;

[0057] Figure 8 The results of the changes in LMNB1 (Lamin B1) content after treatment with umbilical cord blood exosomes in Example 2 of the present invention; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

[0058] Figure 9 The mRNA expression of CDK1, CDK2, and LMNB1 in senescent human trabecular meshwork cells after treatment with umbilical cord blood exosomes in Example 3 of the present invention. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0059] Figure 10 This is the effect of inhibiting CDK1 expression on cell replication, hydrogen peroxide (H2O2) oxidative stress, and transforming growth factor-β2 (TGF-β2)-induced human trabecular meshwork cells in Example 3 of the present invention; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

[0060] Figure 11 The total cell counts of the trabecular meshwork and retinal tissues in different experimental groups in Example 4 of the present invention;

[0061] Figure 12 This is the single cell analysis result of Example 4 of the present invention;

[0062] Figure 13 The immunohistochemical staining results of MYOC in Example 4 of the present invention are shown in the right figure. The statistical results of the left figure are shown in the right figure. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0063] Figure 14 The percentage statistics of each cell cycle stage of Beam A cells in Example 4 of the present invention are as follows;

[0064] Figure 15 The statistical results of the number of γ-H2A.X positive cells in Example 4 of the present invention are shown in Figure 4; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0065] Figure 16 The expression statistical results of lamin B1 in Example 4 of the present invention are shown as follows; * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001;

[0066] Figure 17This is a heatmap (log2 EDG) of differentially expressed genes in the trabecular meshwork of Example 4 of the present invention. Young represents the young mouse group, Old represents the old mouse group, Old+PBS represents the control group in which the old mice were intravitreally injected with PBS, and Old+Exo represents the experimental group in which the old mice were intravitreally injected with 109 p / mL of umbilical cord blood exosomes. Red / blue colors indicate up-regulated / down-regulated expression of the corresponding marker genes, respectively. DETAILED DESCRIPTION

[0067] To help those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0068] The present invention discloses the use of umbilical cord blood exosomes in the preparation of a drug for treating aging-related ophthalmic diseases. Exosomes derived from umbilical cord blood (UCB) plasma can reduce aging and restore the proliferation and function of human trabecular meshwork cells. In in vitro studies, treatment with UCB exosomes significantly reduced senescence in human trabecular meshwork cells. Furthermore, treatment with UCB exosomes downregulated pro-aging genes, upregulated LMNB1 expression, modulated the DNA damage response via γ-H2A.X, and promoted cell proliferation by upregulating Ki67 marker expression. In in vivo studies, treatment of aged mice with UCB exosomes increased the total number of trabecular meshwork cells and retinal cells. Single-cell analysis of trabecular meshwork tissue revealed an increase in non-senescent cells and a decrease in senescent cells within the trabecular meshwork after treatment with UCB exosomes; the proportion of trabecular meshwork cells in the G2 / M phase was significantly increased. UCB exosomes can reduce senescence in human trabecular meshwork cells, maintain cell activity, and promote cell growth, mitosis, and cell division. Umbilical cord blood exosomes also restored the composition of different cell types in the trabecular meshwork tissue of aged mice.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0070] Example 1 Isolation and Preparation of Umbilical Cord Blood Exosomes

[0071] Umbilical cord blood plasma was isolated from umbilical cord blood collected from healthy donors after neonatal delivery. Proteins and large particles were removed from the plasma by filtration through a 220 nm membrane. Subsequently, the cord blood plasma was incubated with polyethylene glycol (PEG 10000), allowing the exosomes to partition into the hydrophobic PEG moiety. After overnight incubation at 4°C, the exosomes were isolated by centrifugation at 3000 g for 30 minutes.

[0072] Nanoparticle tracking analysis (NTA) was used to measure the number and diameter of exosomes. Exosomes were characterized using markers such as CD63, CD81, and Flotillin. Finally, exosomes were prepared to an appropriate concentration using phosphate buffered saline.

[0073] like Figure 1 As shown in Figure 2, the size of exosomes under transmission electron microscopy is approximately 30 to 200 nm. The size and distribution of exosomes were quantified by nanoparticle tracking analysis, as shown in Figure 2. Figure 2 As shown, after sample dilution, the exosome detection concentration range is 10 per ml. 7 ~10 10 Exosome particles. Western blot detection results are as follows Figure 3 As shown, the specific band corresponding to the molecular weight of the exosome marker protein is clearly visible, indicating that umbilical cord blood exosomes were successfully prepared in this example.

[0074] Example 2 In vitro study of umbilical cord blood exosomes on aging human trabecular meshwork cells

[0075] Senescence of human trabecular meshwork (TM) cells can be induced by three pathways: natural cell replication, H2O2 oxidative stress, and TGF-β2 treatment.

[0076] In this example, human trabecular meshwork cells were replicated to passage 10 (P10) to construct a replicative aging model. To establish a hydrogen peroxide (H2O2)-induced aging model, human trabecular meshwork cells were treated with 200 μM H2O2 for 24 hours. To establish a TGF-β2-induced aging model, human trabecular meshwork cells were treated with 5 ng / ml TGF-β2 for 24 hours.

[0077] Adding 10 9 The researchers incubated umbilical cord blood exosomes at 100 p / mL for 24 hours to observe their effects on senescent cells. In oxidative stress-induced cellular senescence, H2O2, as a reactive oxygen species, can induce cellular senescence through oxidative stress. This process generates free radicals that damage DNA, proteins, and other cellular components, leading to oxidative stress. Following H2O2 exposure, cultured cells exhibit senescence-like growth arrest, characterized by decreased cell proliferation, cell cycle arrest, and an increase in senescence markers such as the enzyme β-galactosidase.

[0078] The effects of umbilical cord blood exosomes were analyzed by detecting the senescence marker β-galactosidase (SA-β-gal), the cell cycle arrest marker P21, the DNA damage response marker γ-H2A.X, the cell proliferation marker Ki67, and the nuclear stability marker LMNB1 (Lamin B1).

[0079] The results are as follows Figures 4 to 8 As shown, after treatment of senescent trabecular meshwork cells with umbilical cord blood exosomes, the senescence markers SA-β-gal and P21 were significantly reduced, the DNA damage response marker γ-H2A.X was significantly decreased, and the cell proliferation marker Ki67 and the nuclear stability marker LMNB1 were significantly increased. This indicates that umbilical cord blood exosomes can significantly improve the biological characteristics of senescent trabecular meshwork cells, alleviate senescence characteristics, improve DNA damage repair, and promote cell proliferation and nuclear stability.

[0080] Example 3 RNA sequencing of exosome-treated human trabecular meshwork

[0081] By sequencing the mRNA of human trabecular meshwork cells, we examined whether umbilical cord blood exosomes participate in biological processes such as cell cycle and cell senescence. The expression of CDK1, CDK2, and LMNB1 genes in trabecular meshwork cells treated with umbilical cord blood exosomes was significantly upregulated compared with that in control trabecular meshwork cells ( Figure 9 shown).

[0082] The cyclin-dependent kinase 1 (CDK1) gene is a key factor in cell cycle regulation, especially in the G2-M phase. Upregulation of CDK1 helps initiate the M phase, allowing cells to enter the division phase. The cyclin-dependent kinase 2 (CDK2) gene is involved in the G1-S phase transition, such as Figure 10 As shown in the results, small interfering RNA (siRNA) was used to block the CDK1 and CDK2 genes in exosome-treated senescent human trabecular meshwork cells. Senescence-associated β-galactosidase (SA-β-gal) expression was found to rebound in exosome-treated senescent human trabecular meshwork cells. This suggests that umbilical cord blood exosomes promote the expression of multiple genes that inhibit aging, including CDK1 and CDK2.

[0083] Example 4 In vivo study and single cell analysis of umbilical cord blood exosomes on trabecular meshwork tissue in naturally aged mice

[0084] 15-16 month old C57BL / 6J mice (elderly exosome group) were injected intravitreally with umbilical cord blood exosomes once every two weeks with a single injection volume of 0.5 μL (concentration of 10 10p / mL) for a total of four injections, with PBS injected as a vehicle control. Treatment lasted for two months. After injection, fresh trabecular meshwork tissue was collected for single-cell RNA sequencing analysis, and paraffin-embedded eyeballs were subjected to HE staining and immunohistochemistry to evaluate the effects of umbilical cord blood exosomes on mouse ocular tissue. In addition, two non-therapeutic control groups included 2-month-old CBL / 6J mice (young group) and 15-16-month-old C57BL / 6J mice (old group).

[0085] The total cell counts of trabecular meshwork and retinal tissues in different experimental groups were as follows: Figure 11 As shown in the results, the total number of cells in old mice treated with umbilical cord blood exosomes increased and approached the number of cells in the young group. According to the expression of trabecular meshwork cell markers Aqp-1, Chi3I1, Co1a1, Ctgf and Myoc, Figure 12 Single-cell analysis of the trabecular meshwork cells divided them into three subpopulations, including Beam A cells, Beam B cells, and ciliary junction cells (JCT) cells. Beam A cell markers include MYOC, MGP, COL1A1, PDPN, TMEFF2, C3, and Fmo2; Beam B cell markers include MYOC, MGP, COL1A1, PDPN, TMEFF2, and AQP1; and JCT cell markers include MYOC, MGP, COL1A1, PDPN, FMOD, Tnmd, Nell2, Chad, Chil1, and Rgs5.

[0086] Umbilical cord blood exosome treatment restored the composition of different trabecular meshwork cell types in aging mouse trabecular meshwork tissue and partially reversed the decrease in the number of Beam B cells and JCT cells. Figure 13 Immunohistochemical staining of MYOC, a classic marker of trabecular meshwork cells, showed that trabecular meshwork cells were significantly reduced, and umbilical cord blood exosome treatment increased the number of trabecular meshwork cells in aged mice.

[0087] Senescent cells were identified as cells significantly enriched in the SenMayo gene set (PMID: 35974106). As shown in Table 1 below, in eyes treated with exosomes, the number of non-senescent cells in the Beam A subset of TM cells was significantly increased, while the number of senescent cells was decreased.

[0088] Table 1 Senescent cell count

[0089]

[0090] Figure 14The percentages of Beam A cells in each cell cycle phase are shown for young and aged animals, as well as for an aged control group (PBS) and a group treated with cord blood exosomes from aged animals (Old_Exo). Compared to the young group, aged animals showed a decreased percentage of TM cells in the G2 / M phase, which is responsible for cell growth and preparation for mitosis and cell division. In the aged group, exosome treatment increased the percentage of cells in the G2 / M phase, indicating that cord blood exosomes enhance and promote mitosis and cell division. Figures 15-16 The results showed that exosome treatment reduced the number of γ-H2A.X-positive cells and upregulated the expression of lamin B1. Lamin B1 is essential for maintaining the nuclear structure, regulating the cell cycle, participating in the chromosome condensation process in the interphase, and is indispensable in mitosis. This further confirmed the anti-aging effect of umbilical cord blood exosomes on aged trabecular meshwork (TM) tissue.

[0091] pass Figure 17 As shown in Table 2, the results of single-cell gene sequencing show that the analysis of the effects of exosomes on trabecular meshwork cell subsets partially reversed age-related gene expression abnormalities:

[0092] Table 2 Differentially expressed genes (log2 DEGs) in trabecular meshwork subtypes of the study groups

[0093]

[0094] In the Beam A and Beam B subpopulations, compared to the young group, the expression of extracellular matrix-related genes (such as Rgs5, Chil1, Tnmd, Fmod, and Chad) in the elderly control group (PBS+old) was significantly downregulated, while the expression of exosomes in the elderly group was partially restored (for example, Chil1 in Beam A recovered from -5.626 to -1.120, although still lower than the expression level in the young group), suggesting that exosomes may improve trabecular meshwork function by inhibiting matrix degradation or promoting matrix remodeling. In the JCT subpopulation, the expression of Tnmd and Fmod in the elderly group significantly recovered (Tnmd increased from -0.224 to 1.214, and Fmod increased from 0.923 to 1.038). Exosomes may enhance the elasticity of the extracellular matrix and alleviate age-related tissue sclerosis.

[0095] (1) Regulate inflammation and oxidative stress-related pathways. The complement component C3 of the Beam B subpopulation was significantly upregulated in the elderly group (from 0.54 to 2.923), which may alleviate chronic inflammation by regulating the complement system. The antioxidant-related gene Mgp of Beam A was slightly upregulated in the elderly group (from -0.224 to 0.125), which may inhibit calcification-related damage.

[0096] (2) Improvement of aqueous humor drainage function. Aqp1 (aquaporin 1) of Beam A was significantly upregulated in the elderly group (from 0.001 to 2.911), which may enhance the outflow of aqueous humor. Pdpn of JCT was upregulated in the elderly group (from -0.643 to 0.425), which may improve the permeability of the trabecular meshwork by regulating cell adhesion.

[0097] Umbilical cord blood exosomes can partially reverse trabecular meshwork aging through multiple targets, including:

[0098] 1) Matrix remodeling: inhibiting abnormal collagen deposition (decreased Col1a1) and enhancing elastin (increased Tnmd)

[0099] 2) Antioxidant and anti-inflammatory: Regulate genes such as Mgp and C3 to reduce oxidative damage and chronic inflammation.

[0100] 3) Improve aqueous humor dynamics: Enhance drainage function through Aqp1 and Pdpn.

[0101] 4) Cell function recovery: Regulating autophagy and mechanical sensitivity genes (Chil1, Rgs5) to delay aging.

[0102] Through a series of in vitro and in vivo experiments, the present invention reveals the significant effects of umbilical cord blood exosomes in improving the characteristics of senescent cells, promoting cell proliferation, and maintaining nuclear stability. In in vitro experiments, by constructing aging models induced by replicative senescence, HO oxidative stress, and TGF-β, it was found that umbilical cord blood exosomes can significantly reduce the expression of senescence markers (such as SA-β-gal and P21), reduce the level of DNA damage marker γ-H2A.X, and upregulate the expression of cell proliferation marker Ki67 and nuclear stability marker LMNB1. In addition, exosomes inhibit cell senescence and promote the normal progression of the cell cycle by regulating the expression of cell cycle-related genes (such as CDK1 and CDK2). In in vivo experiments, by intravitreally injecting umbilical cord blood exosomes into an aged mouse model, it was found that exosome treatment can increase the total number of cells in the trabecular meshwork and retinal tissue, restore the composition of trabecular meshwork cell subpopulations, and reduce the number of senescent cells. Single-cell RNA sequencing analysis and immunohistochemical staining results further confirmed the protective effects of umbilical cord blood exosomes on aging trabecular meshwork cells, including promoting cell cycle progression and maintaining the integrity of cell nuclear structure, and improving aqueous humor dynamics and restoring trabecular meshwork function through matrix remodeling, anti-oxidation and anti-inflammation.

[0103] In summary, this study provides a novel therapeutic strategy based on umbilical cord blood exosomes that can effectively ameliorate age-related ocular pathologies and demonstrates significant anti-aging and cell repair potential. This discovery provides important scientific evidence and promising application prospects for the development of innovative therapies for age-related ophthalmic diseases.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of umbilical cord blood exosomes in the preparation of drugs for the treatment of ophthalmic diseases caused by aging.

2. Application of umbilical cord blood exosomes in the preparation of drugs for preventing ophthalmic diseases caused by aging.

3. Application of umbilical cord blood exosomes in the preparation of products to prevent ocular cell aging.

4. Application of umbilical cord blood exosomes in the preparation of products for restoring or slowing down the cell cycle of aging ocular cells.

5. The use according to claim 4, characterized in that The eye cells include any one or more of trabecular meshwork cells, retinal cells, corneal cells, lens cells, optic nerve cells, iris cells, ciliary body cells, intraocular smooth muscle cells and corneal endothelial cells.

6. The use according to claim 5, characterized in that The eye cells are trabecular meshwork cells, and the application is specifically: Reduce the proportion of Beam A cell subsets in trabecular meshwork cells, and increase the proportions of Beam B cell subsets and JCT cell subsets in trabecular meshwork cells.

7. The use according to any one of claims 1 to 5, characterized in that: The aging includes one or more of individual aging, organ aging and cell aging.

8. The use according to any one of claims 1 to 5, characterized in that The specific application is: umbilical cord blood exosomes prevent, improve or restore the function of aging eye cells by regulating the cell growth cycle.

9. The use according to claim 8, characterized in that The cell growth cycle regulation is specifically any one or more of the following: (1) Restoring cells in G2 / M; (2) upregulating the expression of CDK1 and CDK2; (3) downregulating the expression of P16 and P21 proteins; (4) Upregulate the expression of Ki67 and promote cell cycle progression; (5) downregulating H2A.X expression and improving DNA damage response; (6) Upregulate the expression of LaminB1 protein and promote nuclear structural stability; (7) Inhibit lysosomal function.

10. The use according to any one of claims 1 to 2, characterized in that: The ophthalmic disease includes any one of glaucoma, cataract, age-related maculopathy, corneal disease and retinitis pigmentosa.

11. The use according to claim 1, characterized in that The drug is used in combination with other drugs for treating eye diseases caused by aging.

12. The use according to claim 11, characterized in that The combined use method includes encapsulating other drugs for treating aging-induced ophthalmic diseases in the umbilical cord blood exosomes.

13. The use according to any one of claims 1 to 6, characterized in that: The umbilical cord blood exosomes are obtained by mixing umbilical cord plasma with polyethylene glycol and incubating the mixture, followed by ultracentrifugation and separation.

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