Use of umbilical cord blood exosomes in the preparation of a medicament for treating an age-induced ophthalmic disease

Abstract

The application discloses application of umbilical cord blood exosomes in preparation of a medicine for treating eye diseases caused by aging. It is found that the umbilical cord blood exosomes can improve the biological characteristics of aged trabecular meshwork cells, reduce the aging characteristics of the trabecular meshwork cells, improve the DNA damage repair ability of the cells, promote the proliferation of the trabecular meshwork cells, and effectively maintain the stability of the cell nucleus, thereby supporting the growth and division of the cells. The umbilical cord blood exosomes show a remarkable anti-aging effect in the treatment of the eye diseases caused by aging, can effectively improve the biological functions of the trabecular meshwork cells, slow down the aging process, promote cell proliferation and repair, and have wide application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of umbilical cord blood exosomes in the preparation of drugs for treating age-related ophthalmic diseases. Background Technology

[0002] Exosomes, also known as extracellular vesicles (EVs), are nanoscale cell-derived membrane vesicles carrying 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 bioactive components. Clinically, preserved umbilical cord blood can be used to treat various diseases, such as malignant tumors, hematological disorders, and immune system diseases; umbilical cord blood is a vital resource. The abundant hematopoietic stem cells in umbilical cord blood make it a precious source of hematopoietic stem cells. The hematopoietic function of umbilical cord blood is increasingly recognized by the medical community. Umbilical cord blood transplantation can be used to treat leukemia, diabetes, radiation sickness, and many other intractable diseases.

[0003] As we age, the effects of aging on eye health become increasingly apparent. Age-related eye diseases include, but are not limited to, cataracts, glaucoma, age-related macular degeneration (AMD), and dry eye syndrome. These diseases not only affect patients' visual quality but can also have serious negative impacts on their mental health and social activities. Statistics show that age-related vision impairment is one of the leading health problems among the elderly population worldwide, leading to significant economic burdens and medical needs.

[0004] Despite the availability of various treatment options, existing methods still have many limitations. For example, while cataract surgery can restore vision, it cannot reverse other age-related degenerative changes in the eye. Drug 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 treatment course is long and expensive, and it does not completely address the underlying cause. Furthermore, while treatments for dry eye offer some relief, they have not yet provided a complete cure.

[0005] Therefore, there is an urgent need for a new, more effective, and safer treatment strategy to repair age-related eye tissue damage and slow down or reverse pathological changes during the aging process. Summary of the Invention

[0006] In view of the deficiencies in the prior art, this invention proposes the application of umbilical cord blood exosomes in the preparation of drugs for treating age-related ophthalmic diseases.

[0007] This invention provides the application of umbilical cord blood exosomes in the preparation of drugs for treating age-related ophthalmic diseases.

[0008] In some embodiments, the dosage form of the drug is any one of drops, gel, injection, oral dosage, transdermal absorption preparation, and mucosal absorption preparation.

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

[0010] In some embodiments, the drug further includes pharmaceutically acceptable excipients selected from any one or more of thickeners, stabilizers, preservatives, and buffers.

[0011] This invention also provides the application of umbilical cord blood exosomes in the preparation of drugs for preventing age-related ophthalmic diseases.

[0012] This invention also provides the application of umbilical cord blood exosomes in the preparation of products for preventing eye cell aging.

[0013] This invention also provides the application of umbilical cord blood exosomes in the preparation of products that restore the cell cycle of aging eye cells.

[0014] In some embodiments, the eye cells include any one or more of the following: 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 eye cells are trabecular meshwork cells, and the application specifically includes:

[0016] The proportion of Beam A cell subset in trabecular meshwork cells was reduced, while the proportions of Beam B cell subset and JCT cell subset in trabecular meshwork cells were increased.

[0017] In some embodiments, the aging includes one or more of individual aging, organ aging, and cellular 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; cellular aging is the process by which cells enter a state where they no longer divide but remain active, and senescent cells can no longer divide, affecting tissue repair and regeneration.

[0018] In some implementations, the application specifically involves umbilical cord blood exosomes preventing, improving, or restoring the function of aging eye cells by regulating the cell growth cycle.

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

[0020] (1) Restoring the role of aging cells in the G2 / M phase; the G2 / M phase is a critical stage in the cell cycle, responsible for transitioning cells from the G2 phase to metaphase (M phase). This phase includes chromosome replication correction, nuclear membrane disintegration, and the preparation of related proteins to ensure that cells can properly distribute genetic material during division. This phase is essential for cell proliferation and the maintenance of normal function.

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

[0022] (3) Downregulation of P16 and P21 protein expression; treatment with umbilical cord blood exosomes significantly reduced the mRNA and protein levels of P16 and P21, suggesting that they play an active role in regulating cell cycle arrest.

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

[0024] (5) Downregulation of H2A.X expression improves DNA damage response; umbilical cord blood exosome treatment reduces H2A.X phosphorylation and flow cytometry shows that cells have increased tolerance to DNA double-strand breaks, indicating its role in DNA repair mechanisms.

[0025] (6) Upregulating the expression of LaminB1 protein and promoting 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 reduced 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 apoptosis, suggesting a 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 antioxidants, such as certain peptides and non-coding RNAs, which can scavenge free radicals and reduce cell damage.

[0029] (2) Regulation of key signaling pathways: Umbilical cord blood exosomes regulate intracellular growth factors, death receptor proteins, etc. 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, thus delaying aging.

[0031] Umbilical cord blood exosomes regulate CDK1, CDK2, and LMNb1 through multiple pathways, thereby influencing the smooth progression of the cell cycle. Simultaneously, umbilical cord blood exosomes play a crucial role in anti-aging, primarily through mechanisms such as free radical scavenging, signaling pathway regulation, and promoting DNA repair. This regulatory network provides 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) Regulation of TGF-β signaling pathway; umbilical cord blood exosomes can alleviate trabecular meshwork sclerosis by inhibiting TGF-β-mediated fibrosis.

[0034] (6) Promotes autophagy and cell survival; umbilical cord blood exosomes can clear senescent cells by enhancing autophagy. At the same time, 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 includes any one of glaucoma, cataracts, age-related macular degeneration, corneal disease, and retinitis pigmentosa.

[0037] Glaucoma is caused by senescent cells in the trabecular meshwork impairing the drainage of intraocular fluid and increasing intraocular pressure, which in turn damages the optic nerve. Umbilical cord blood exosomes can improve the function of the trabecular meshwork, improve aqueous humor drainage, remodel the matrix and reduce intraocular pressure, and protect the optic nerve with antioxidant and anti-inflammatory properties, thus preventing vision loss caused by glaucoma.

[0038] The development of cataracts is related to the aging and clouding of lens epithelial cells. This application can repair aging lens epithelial cells and slow down the process of lens clouding by using growth factors and miRNAs from umbilical cord blood exosomes.

[0039] Age-related macular degeneration (AMD) is a degenerative retinal disease caused by aging, primarily manifested as degeneration of the macular region. Umbilical cord blood exosomes can restore the growth and proliferation of retinal neurons by promoting the G2 / M phase of the cell cycle. They also regulate oxidative stress, protecting the retina from oxidative damage. Furthermore, they promote macular cell repair by stimulating retinal cells to release protective factors such as IL-6 and IL-8. miRNAs and certain proteins in umbilical cord blood exosomes can reduce inflammatory responses in retinal cells and regulate related immune pathways. In addition, umbilical cord blood exosomes can modulate the disease process, treat AMD, and improve macular function by affecting the expression of molecules such as VEGF and TNF-α.

[0040] Corneal diseases, including corneal degeneration and keratitis, are usually caused by corneal cell aging or damage. Umbilical cord blood exosomes can stimulate corneal cell proliferation and differentiation by regulating the G2 / M cell cycle, accelerating the repair process. The 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 the cornea's self-repair ability. Utilizing their anti-inflammatory properties, they have therapeutic effects on corneal damage caused by infection or inflammation, restoring corneal transparency.

[0041] Retinitis pigmentosa (RP) is primarily caused by the aging and degeneration of retinal pigment epithelial cells, thus affecting vision. Umbilical cord blood exosomes can promote the proliferation and differentiation of photoreceptor cells. During RP regeneration, 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, apoptosis-inducing miRNAs and neurotrophic factors to delay photoreceptor cell death.

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

[0043] In some embodiments, the combined use includes encapsulating other drugs for treating age-related ophthalmic diseases in the umbilical cord blood exosomes; further, the other drugs for treating age-related ophthalmic diseases include 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 incubating umbilical cord plasma with polyethylene glycol and then separating them by ultracentrifugation.

[0045] In some embodiments, the drug also includes pharmaceutically acceptable excipients.

[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] This invention discovers that umbilical cord blood exosomes can improve the biological characteristics of aging trabecular meshwork cells, alleviate aging features, 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 exhibit significant anti-aging effects in the treatment of age-related ophthalmic diseases, effectively improving the biological function of trabecular meshwork cells, slowing the aging process, and promoting cell proliferation and repair, demonstrating broad application prospects and clinical value. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is an image of exosomes under a transmission electron microscope according to Example 1 of the present invention;

[0051] Figure 2 The results of nanoparticle tracking analysis in Example 1 of this 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 The changes in aging-related β-galactosidase (SA-βgal) content after umbilical cord blood exosome treatment in Example 2 of this invention are shown; * indicates p<0.05, ** indicates p<0.01; *** indicates p<0.001.

[0054] Figure 5 The results of the change in the positive cell rate of cell cycle arrest marker P21 after umbilical cord blood exosome treatment in Example 2 of this 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 after umbilical cord blood exosome treatment in Example 2 of this invention; * indicates p<0.05, ** indicates p<0.01; *** indicates p<0.001;

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

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

[0058] Figure 9 This figure represents 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 this invention. * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001;

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

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

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

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

[0063] Figure 14 This is a statistical result of the percentage of each stage of the cell cycle in Beam A cells in Example 4 of the present invention;

[0064] Figure 15 This is the statistical result of the number of γ-H2A.X positive cells in Example 4 of the present invention; * indicates p<0.05, ** indicates p<0.01; *** indicates p<0.001;

[0065] Figure 16 The above are the statistical results of the expression level of laminin B1 in Example 4 of the present invention; * indicates p<0.05, ** indicates p<0.01; *** indicates p<0.001;

[0066] Figure 17This is a heatmap (log2 EDG) of differentially expressed genes in the trabecular meshwork of Embodiment 4 of this invention. Young represents the young mouse group, Old represents the aged mouse group, old+PBS represents the control group of aged mice receiving intravitreal injection of PBS, and old+Exo represents the experimental group of aged mice receiving intravitreal injection of 10⁹ p / mL umbilical cord blood exosomes. Red / blue indicates upregulation / downregulation of the corresponding marker genes, respectively. Detailed Implementation

[0067] To enable those skilled in the art to 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 some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0068] This invention discloses the application of umbilical cord blood exosomes in the preparation of drugs for treating age-related ophthalmic diseases. Umbilical cord blood (UCB) plasma exosomes can reduce aging and restore the proliferation and function of human ocular trabecular meshwork cells. In in vitro studies, treatment with UCB exosomes significantly reduced the aging of human ocular trabecular meshwork cells. Furthermore, UCB exosome treatment downregulated pro-aging genes, upregulated LMNB1 expression, regulated DNA damage response through γ-H2A.X, and promoted cell proliferation by upregulating Ki67 marker expression. In in vivo studies, treatment with UCB exosomes in aging mice increased the total number of trabecular meshwork cells and retinal cells. In single-cell analysis of trabecular meshwork tissue, UCB exosome treatment increased the number of non-senescent cells and decreased the number of senescent cells within the trabecular meshwork; the proportion of trabecular meshwork cells in the G2 / M phase significantly increased. UCB exosomes can reduce the aging of human ocular trabecular meshwork cells, maintain cellular activity, and also promote cell growth and drive mitosis and cell division. Umbilical cord blood exosomes also restored the composition of different cell types in the trabecular meshwork tissue of aging mice.

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

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

[0071] 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) to allow exosomes to partition into the hydrophobic PEG fraction. After overnight incubation at 4°C, exosomes were separated by centrifugation at 3000 g for 30 minutes.

[0072] The number and diameter of exosomes were determined using nanoparticle tracking analysis (NTA). Exosomes were characterized using markers such as CD63, CD81, and Flotillin. Finally, exosomes were prepared to appropriate concentrations using phosphate-buffered saline.

[0073] like Figure 1 As shown, exosomes appear to be approximately 30–200 nm in size under a transmission electron microscope. The size and distribution of exosomes were quantified using nanoparticle tracking analysis, such as... Figure 2 As shown, after sample dilution, the exosome detection concentration range is 10 mg / mL. 7 ~10 10 Exosome particles. Western blot results are as follows: Figure 3 As shown, the specific bands corresponding to the molecular weight of the exosome marker protein are clearly visible, indicating that umbilical cord blood exosomes were successfully prepared in this embodiment.

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

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

[0076] In this embodiment, to construct a replicative aging model, human trabecular meshwork cells were replicated to the 10th generation (P10). 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] Add 10 to senescent trabecular meshwork cells 9 Umbilical cord blood exosomes at a concentration of p / mL were incubated 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, causing damage to DNA, proteins, and other cellular components by generating free radicals, thereby inducing oxidative stress. After exposure to H2O2, cultured cells exhibit senescence-like growth arrest, characterized by decreased cell proliferation rate, cell cycle arrest, and an increase in senescence markers (such as senescence-associated β-galactosidase).

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

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

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

[0081] mRNA sequencing of human trabecular meshwork cells was used to investigate whether umbilical cord blood exosomes participate in biological processes such as cell cycle and cellular senescence. Compared with control trabecular meshwork cells, umbilical cord blood exosome-treated trabecular meshwork cells showed significant upregulation of CDK1, CDK2, and LMNB1 gene expression. Figure 9 (As shown).

[0082] Cyclin-dependent kinase 1 (CDK1) is a key factor in cell cycle regulation, especially during the G2-M phase. Upregulation of CDK1 facilitates the initiation of M phase, enabling cells to enter the division phase. Cyclin-dependent kinase 2 (CDK2) is involved in the G1-S phase transition, such as... Figure 10 As shown, blocking the CDK1 and CDK2 genes in exosome-treated aging human trabecular meshwork cells with small interfering RNA (siRNA) resulted in a rebound in the expression of aging-associated β-galactosidase (SA-β-gal) in exosome-treated aging human trabecular meshwork cells. This indicates that umbilical cord blood exosomes promote the expression of multiple aging-inhibiting genes, including CDK1 and CDK2.

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

[0084] C57BL / 6J mice aged 15–16 months (aged exosome group) were used for intravitreal injection of umbilical cord blood exosomes every two weeks, with a single injection volume of 0.5 μL (concentration of 10). 10The mice were injected four times (p / mL), with PBS used as a control. Treatment lasted for two months. After injection, fresh trabecular meshwork tissue was collected for single-cell RNA sequencing analysis. Simultaneously, paraffin-embedded eyeballs were stained with hematoxylin and eosin (HE) and immunohistochemically to assess the effects of umbilical cord blood exosomes on mouse ocular tissue. Two non-treatment control groups included 2-month-old CBL / 6J mice (young group) and 15-16-month-old C57BL / 6J mice (aged group).

[0085] Total cell counts of trabecular meshwork and retinal tissue in different experimental groups, as shown below Figure 11 As shown, the total cell count in aged mice treated with umbilical cord blood exosomes increased and approached that of the younger group. Based on trabecular meshwork cell markers Aqp-1, Chi3I1, Co1a1, Ctgf, and Myoc, Figure 12 Single-cell analysis divided trabecular meshwork cells into three subpopulations: Beam A cells, Beam B cells, and ciliary body junctional cells (JCT) cells. Beam A cells were identified by markers including MYOC, MGP, COL1A1, PDPN, TMEFF2, C3, and Fmo2; Beam B cells by markers including MYOC, MGP, COL1A1, PDPN, TMEFF2, and AQP1; and JCT cells by markers including MYOC, MGP, COL1A1, PDPN, FMOD, Tnmd, Nell2, Chad, Chil1, and Rgs5.

[0086] Umbilical cord blood exosome therapy restored the composition of different trabecular meshwork cell types in the trabecular meshwork tissue of aging mice, and partially reversed the reduction in the number of Beam B cells and JCT cells. Furthermore, Figure 13 Immunohistochemical staining of MYOC, a classic marker of trabecular meshwork cells, showed a significant reduction in trabecular meshwork cells, while 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 exosome-treated eyes, the number of non-senescent cells and the number of senescent cells in the Beam A subgroup of TM cells were significantly increased.

[0088] Table 1. Count of senescent cells

[0089]

[0090] Figure 14This study presents the percentage of Beam A cells in each cell cycle phase in young animals, aged animals, the aged animal control group (PBS), and the aged animal cord blood exosome treatment group (Old_Exo). Compared to the young animal group, the percentage of TM cells in the G2 / M phase was decreased in the aged animals; the G2 / M phase is responsible for cell growth and preparation for mitosis and cell division. In the aged group, the exosome treatment group showed an increased percentage of cells in the G2 / M phase, indicating that cord blood exosomes enhance and promote mitosis and cell division. Figures 15 to 16 The results showed that exosome treatment reduced the number of γ-H2A.X positive cells and upregulated the expression of lamin B1, which is essential for maintaining the structure of the cell nucleus and can regulate the cell cycle, participate in the chromosome condensation process during interphase, and is indispensable in mitosis. This further confirmed the anti-cellular senescence effect of umbilical cord blood exosomes on senescent trabecular meshwork (TM) tissue.

[0091] pass Figure 17 The analysis of the effects of exosomes on trabecular meshwork cell subsets, as shown in Table 2 (single-cell gene sequencing results), reveals that some age-related gene expression abnormalities are reversed.

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

[0093]

[0094] In the Beam A and Beam B subgroups, compared with the young group, extracellular matrix-related genes (such as Rgs5, Chil1, Tnmd, Fmod, and Chad) were significantly downregulated in the elderly control group (PBS+old), while exosomes partially recovered in the elderly group (e.g., 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 subgroup, the expression of Tnmd and Fmod was significantly restored in the elderly exosome group (Tnmd increased from -0.224 to 1.214, and Fmod increased from 0.923 to 1.038), suggesting that exosomes may enhance the elasticity of the extracellular matrix and alleviate age-related tissue sclerosis.

[0095] (1) Regulation of inflammation and oxidative stress-related pathways. The complement component C3 of Beam B subset was significantly upregulated in the aged exosome 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 aged exosome group (from -0.224 to 0.125), which may inhibit calcification-related damage.

[0096] (2) Improved aqueous humor drainage. Beam A's Aqp1 (aquaporin 1) was significantly upregulated in the aged exosome group (from 0.001 to 2.911), which may enhance aqueous humor outflow. JCT's Pdpn expression was increased in the aged exosome group (from -0.643 to 0.425), which may improve trabecular meshwork permeability by regulating cell adhesion.

[0097] Umbilical cord blood exosomes can partially reverse trabecular meshwork aging through multi-target action, including:

[0098] 1) Matrix remodeling: Inhibits abnormal collagen deposition (decreased Col1a1) and enhances elastin (increased Tnmd).

[0099] 2) Antioxidant and anti-inflammatory: Regulates 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 mechanosensitive genes (Chil1, Rgs5) delays aging.

[0102] This invention, through a series of in vitro and in vivo experiments, 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 replicative senescence, HO oxidative stress, and TGF-β-induced senescence models, it was found that umbilical cord blood exosomes significantly reduced the expression of senescence markers (such as SA-β-gal and P21), decreased the level of the DNA damage marker γ-H2A.X, and upregulated the expression of the cell proliferation marker Ki67 and the nuclear stability marker LMNB1. Furthermore, exosomes inhibited cellular senescence and promoted normal cell cycle progression by regulating the expression of cell cycle-related genes (such as CDK1 and CDK2). In in vivo experiments, by intravitreal injection of umbilical cord blood exosomes into an aged mouse model, it was found that exosome treatment increased the total number of cells in the trabecular meshwork and retinal tissue, restored the composition of trabecular meshwork cell subsets, and reduced the number of senescent cells. Single-cell RNA sequencing analysis and immunohistochemical staining results further confirmed the protective effect of umbilical cord blood exosomes on senescent trabecular meshwork cells, including promoting cell cycle progression and maintaining the integrity of the cell nuclear structure, and improving and restoring the function of trabecular meshwork through matrix remodeling, anti-oxidation and anti-inflammation, and improving aqueous humor dynamics.

[0103] In summary, this invention provides a novel therapeutic strategy based on umbilical cord blood exosomes that can effectively improve age-related ocular pathological changes and has significant anti-aging and cell repair potential. This discovery provides important scientific evidence and application prospects for developing innovative therapies for age-related ocular 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 within the protection scope of the present invention.

Claims

1. Application of umbilical cord blood exosomes in the preparation of products that restore or slow down the cell cycle of aging eye cells; The eye cells are derived from the trabecular meshwork tissue; The aging referred to is replicative aging, hydrogen peroxide-induced aging, or TGF-β2-induced aging; The specific application is to reduce the proportion of Beam A cell subsets in trabecular meshwork tissue and increase the proportion of Beam B cell subsets and JCT cell subsets in trabecular meshwork tissue. The specific application is that umbilical cord blood exosomes can prevent, improve, or restore the function of aging eye cells by regulating the cell growth cycle. The regulation of the cell growth cycle specifically includes any one or more of the following: (1) Restore the cell's function in G2 / M; (2) Upregulate the expression of CDK1 and CDK2; (3) Downregulate the expression of P16 and P21 proteins; (4) Upregulates Ki67 expression and promotes cell cycle progression; (5) Downregulate H2A.X expression and improve DNA damage response; (6) Upregulates the expression of LaminB1 protein, promoting nuclear structural stability; (7) Inhibits lysosomal function.

2. The application according to claim 1, characterized in that, The product is a drug, which is used in combination with other drugs for treating age-related eye diseases.

3. The application according to claim 2, characterized in that, The ophthalmic diseases mentioned include any one of glaucoma, cataracts, age-related macular degeneration, corneal diseases, and retinitis pigmentosa.

4. The application according to claim 2, characterized in that, The combined use includes encapsulating other drugs for treating age-related ophthalmic diseases in the umbilical cord blood exosomes.

5. The application according to claim 1, characterized in that, The umbilical cord blood exosomes were obtained by incubating umbilical cord plasma with polyethylene glycol and then separating them by ultracentrifugation.

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