Application of ultrafine nanoparticles derived from human umbilical cord mesenchymal stem cells in preparation of medicine for preventing and / or treating diseases caused by nerve cell injury

Abstract

The invention provides application of ultrafine nanoparticles derived from human umbilical cord mesenchymal stem cells in preparation of a medicine for preventing and / or treating diseases caused by nerve cell injury, and belongs to the technical field of biological medicines. The invention provides an application of human umbilical cord mesenchymal stem cell (ucMSC-supermers) in preparation of a medicine for preventing and / or treating diseases caused by nerve cell injury, and particularly provides an application of the human umbilical cord mesenchymal stem cell (ucMSC-supermers) in preparation of the medicine for preventing and / or treating the diseases caused by the nerve cell injury. As novel non-membranous extracellular nanoparticles, the mesenchymal stem cell-derived ultramicro nanoparticles are small in particle size, are not wrapped by a membranous structure, have stronger tissue penetrating power and higher cellular uptake efficiency, enrich bioactive substances with higher abundance, can efficiently promote axonal regeneration, and can be used for preparing a novel non-membranous extracellular nanoparticle. The effect of promoting nerve regeneration and repair is obviously better than that of the HucMSC-sEV under the same condition, and the treatment effect on traumatic diseases of the central nervous system is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ultrafine nanoparticles derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for the prevention and / or treatment of diseases caused by nerve cell damage. Background Technology

[0002] Central nervous system traumatic diseases are a general term for diseases caused by sudden mechanical external forces acting on the central nervous system, resulting in damage to the integrity of the brain, spinal cord, and other central nervous system components, and subsequently causing related functional impairments such as sensory, motor, consciousness, and autonomic nervous system dysfunction. Spinal cord injury (SCI), as a typical example of a central nervous system traumatic disease, has a complex pathophysiological process, mainly including primary mechanical injury and subsequent secondary injury cascade reactions such as inflammatory responses and oxidative stress. This disease can lead to progressive impairment or even permanent loss of sensory, motor, and autonomic nervous system functions below the level of injury, and is one of the intractable diseases that urgently needs to be addressed in clinical practice.

[0003] Currently, clinical treatment strategies for spinal cord injury (SCI) mainly include hyperbaric oxygen therapy, corticosteroid pulse therapy, surgical decompression intervention, and rehabilitation electrical stimulation therapy. However, while these methods can alleviate secondary damage or improve the local microenvironment to some extent, due to the difficulty of central nervous system regeneration, there is still a lack of effective means to significantly promote neural structural reconstruction and radically cure SCI. In the repair process of spinal cord injury, axon regeneration is a key link in reconstructing neural circuits and restoring neural signal transmission, and it is also the core challenge in current treatment research. In recent years, stem cell therapy, represented by human umbilical cord-derived mesenchymal stem cells (hucMSCs), has attracted much attention in the field of neuroregenerative medicine. Small extracellular vesicles (sEVs) secreted by hucMSCs are vesicle-like bodies with a particle size of less than 200 nm and a lipid bilayer membrane structure, which can promote the repair of damaged neural tissue; however, the therapeutic effect of sEVs on SCI is limited. Summary of the Invention

[0004] In view of this, the present invention provides the application of human umbilical cord mesenchymal stem cell-derived ultrafine nanoparticles (hucMSC-supermeres) in the preparation of drugs for the prevention and / or treatment of nerve cell damage and / or traumatic diseases of the central nervous system. Compared with hucMSC-sEV, hucMSC-supermeres can efficiently promote axon regeneration, reconstruct neural circuits, and restore nerve signal transmission, thus significantly improving the therapeutic effect on SCI.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of ultrafine nanoparticles derived from human umbilical cord mesenchymal stem cells in the preparation of medicaments for the prevention and / or treatment of diseases caused by nerve cell damage.

[0006] Preferably, the nerve cell damage includes nerve cell mechanical damage and / or nerve cell oxidative stress damage.

[0007] Preferably, the diseases caused by nerve cell damage include traumatic diseases of the central nervous system.

[0008] Preferably, the traumatic disease of the central nervous system includes spinal cord injury.

[0009] Preferably, the symptoms of spinal cord injury include at least one of the following: motor dysfunction, impaired nerve conduction, syringomyelia, disordered spinal cord structure, Nissl body lesions, inflammatory reactions of the spinal cord, glial scars of the spinal cord, and axonal lesions.

[0010] Preferably, the treatment of spinal cord injury includes promoting the regeneration and repair of damaged spinal cord tissue and / or improving the symptoms of spinal cord injury; the promotion of the regeneration and repair of damaged spinal cord tissue includes at least one of the following: promoting axonal regeneration and repair, promoting nerve cell regeneration and repair, promoting nerve fiber regeneration and repair, and promoting angiogenesis.

[0011] Preferably, the nerve cells include PC12 cells and / or DRG cells.

[0012] Preferably, the method for preparing the mesenchymal stem cell-derived ultrafine nanoparticles includes the following steps: After removing cell debris and organelles from the culture supernatant of human umbilical cord mesenchymal stem cells, the supernatant was washed by ultrafiltration and the concentrate was collected. The concentrate is subjected to ultracentrifugation, which includes: centrifuging the concentrate at 80,000–120,000 g for 1–4 h to obtain a first precipitate and a first supernatant; centrifuging the first supernatant at 150,000–180,000 g for 14–18 h to obtain a second precipitate and a second supernatant; and centrifuging the second supernatant at 360,000–370,000 g for 14–18 h to obtain a third precipitate and a third supernatant. The third precipitate is ultrafine nanoparticles derived from the mesenchymal stem cells.

[0013] Preferably, the surface marker proteins of the mesenchymal stem cell-derived ultramicro nanoparticles include angiotensin-converting enzyme 2 and Argonaute protein 2.

[0014] Preferably, the dosage form of the drug includes an injection; In the injection, the concentration of mesenchymal stem cell-derived ultrafine nanoparticles is not less than 10. 7 Particle number / µl.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides the application of hucMSC-supermeres in the preparation of drugs for the prevention and / or treatment of diseases caused by neuronal injury. This invention is the first to propose that hucMSC-supermeres promote axonal regeneration and neurological function recovery after spinal cord injury. In this invention, hucMSC-supermeres, as novel non-vesicular extracellular nanoparticles (NVEPs), have a smaller particle size and are not encapsulated by a membrane structure, giving them stronger tissue penetration and higher cellular uptake efficiency in the microenvironment of spinal cord injury. Simultaneously, hucMSC-supermeres are enriched with higher abundances of bioactive substances, and their effect on promoting axonal regeneration is significantly superior to that of small extracellular vesicles hucMSC-sEVs with a lipid bilayer membrane structure under the same conditions, effectively improving the drug delivery efficiency of neuroregeneration therapy. Experimental verification shows that hucMSC-supermeres can significantly promote the survival of damaged neurons, induce neurite extension, and upregulate the expression of key axonal proteins, thereby further promoting the regeneration and remodeling of damaged axons. Meanwhile, in vivo experiments showed that hucMSC-supermeres were more effective than hucMSC-sEV in improving hindlimb motor function and reducing scar formation at the injury site in rats with spinal cord injury. Therefore, the axonal regeneration-promoting effect of hucMSC-supermeres provides a safer and more efficient new strategy for the clinical treatment of spinal cord injury. Attached Figure Description

[0016] Figure 1 The images show the morphology of hucMSC cells in Example 1. The left image shows the morphology of P0 generation cells, the middle image shows the adipogenic differentiation of P3 generation cells, and the right image shows the osteogenic differentiation of P3 generation cells. Figure 2 The images are transmission electron microscope (TEM) images of hucMSC-sEV and hucMSC-supermeres in Example 1. The left image is hucMSC-sEV (sEV), and the right image is hucMSC-supermeres (Supermeres). Figure 3 This is a Western blotting image of the biomarkers hucMSC, hucMSC-sEV, and hucMSC-supermeres in Example 1. Figure 4 This is a graph showing the uptake of fluorescently labeled hucMSC-sEV and hucMSC-supermees at equal particle concentrations by PC12 cells in Example 2; where blue fluorescence represents cell nuclei (Hoechst staining), and red fluorescence represents hucMSC-sEV (CM-Dil staining) and hucMSC-supermees (Alexa Fluor). TM (647 staining); A shows the uptake maps of hucMSC-sEV and hucMSC-supermeres; B shows the statistical results of the relative fluorescence intensity of the uptake of the two by a single cell. This indicates that P < 0.05; Figure 5 This image shows the retention of hucMSC-supermeres in SD rats of the SCI model in Example 2 at different time points. A shows the fluorescence intensity in SD rats at different time points, and B shows the statistical results of fluorescence intensity over different days. This indicates that P < 0.001. P < 0.0001, ns indicates no significant difference; Figure 6 Gait analysis was performed on SD rats in the Sham group, PBS group, hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 3 to observe the recovery of motor function. Red ink was used to stain the hind limb phalanges, and black ink was used to stain the forelimb paw pads. A shows the SD rat ink footprint test results, and B shows a statistical graph of the relative dragging area of ​​the hind limbs in each experimental group. This indicates that P < 0.05. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 7 HE staining of spinal cord tissue sections from SD rats in the Sham group, PBS group, hucMSC-sEV treatment group and hucMSC-supermeres treatment group in Example 3; Figure 8 Nissell staining of spinal cord tissue sections from SD rats in the Sham group, PBS group, hucMSC-sEV treatment group and hucMSC-supermeres treatment group in Example 3; Figure 9 The images show the immunofluorescence staining results of spinal cord tissue sections from SD rats in the Sham group, PBS group, hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 3. Green fluorescence labels the axonal structure NF200 of nerve cells, red fluorescence labels the astrocytes GFAP that form glial scars, and blue fluorescence labels the cell nuclei (Hoechst staining). A shows the tissue fluorescence staining results, and B shows the statistical results of the relative fluorescence intensity percentages of green and red fluorescence. This indicates that P < 0.01. This indicates that P < 0.001. P < 0.0001, ns indicates no significant difference; Figure 10 This image shows the CCK8 assay results of PC12 cells from the NC group, H2O2 group, hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 4 after treatment with the same number of hucMSC-sEV and hucMSC-supermeres particles under H2O2 damage. This indicates that P < 0.0001; Figure 11The image shows the TUNEL assay results for PC12 cells in the normal group (NC group), damaged group (H2O2 group), hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 4, used to detect cell apoptosis. Blue fluorescence represents cell nuclei (Hoechst staining), and red fluorescence represents apoptotic cells (TUNEL staining). A shows the TUNEL staining results, and B is a statistical graph of the relative fluorescence intensity of apoptotic cells in each experimental group. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 12 The image shows the results of Western-Blot analysis of PC12 cell proteins extracted from the normal group (NC group), the damaged group (H2O2 group, hucMSC-sEV treatment group, and hucMSC-supermeres treatment group) in Example 4. Figure 13 The image shows the axonal regeneration of PC12 cells in the normal group (NC group), damaged group (H2O2 group), hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 4, detected by cell fluorescence. Red fluorescence represents the axonal protein Tuj-1 of PC12 cells (stained with Cyanine 3). A shows the cell fluorescence staining results, and B shows the statistical results of the relative axonal length of each experimental group. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.0001; Figure 14 This image shows the axon regeneration of primary rat DRG cells from the normal group (NC group), damaged group (H2O2 group), hucMSC-sEV treatment group, and hucMSC-supermeres treatment group in Example 4, detected by cell fluorescence. Red fluorescence represents the axon protein Tuj-1 of DRG cells (stained with Cyanine 3). A shows the cell fluorescence staining results, and B shows the statistical results of the relative axon lengths of cells in each experimental group. This indicates that P < 0.001. This means P < 0.0001. Detailed Implementation

[0017] This invention provides the application of human umbilical cord mesenchymal stem cell-derived ultramicro nanoparticles (hucMSC-supermeres) in the preparation of medicaments for the prevention and / or treatment of nerve cell damage and / or traumatic diseases of the central nervous system.

[0018] In this invention, the hucMSC-supermeres are non-vesicular extracellular particles (NVEPs) with a particle size of approximately 25 nm secreted by mesenchymal stem cells. As an optional embodiment, the preparation method of the hucMSC-supermeres includes the following steps: After removing cell debris and organelles from the culture supernatant of human umbilical cord mesenchymal stem cells, the supernatant was washed by ultrafiltration and the concentrate was collected. The concentrate is subjected to ultracentrifugation, which includes: centrifuging the concentrate at 80,000–120,000 g for 1–4 h to obtain a first precipitate and a first supernatant; centrifuging the first supernatant at 150,000–180,000 g for 14–18 h to obtain a second precipitate and a second supernatant; and centrifuging the second supernatant at 360,000–370,000 g for 14–18 h to obtain a third precipitate and a third supernatant. The third precipitate is the hucMSC-supermeres.

[0019] This invention involves removing cell debris and organelles from the culture supernatant of human umbilical cord mesenchymal stem cells (HMSCs), followed by ultrafiltration washing, and collecting the concentrated solution. In this invention, the use of HMSCs is beneficial for promoting axonal regeneration and neurological function recovery after spinal cord injury. The culture supernatant preferably includes the culture supernatant obtained from P3-P7 generation HMSC cultures. HMSCs are primary cultured cells; their proliferation capacity is poor after passage 8-9, and the number of cells after adhesion and migration from passage 1-2 is insufficient. Therefore, cells in good growth condition from passage 3-7 are selected, and the culture supernatant is collected for supermere extraction to obtain highly bioactive hucMSC-supermeres, which is beneficial for promoting axonal regeneration and neurological function recovery after spinal cord injury. The preferred method for removing cell debris and organelles includes differential centrifugation. This differential centrifugation involves sequentially centrifuging the human umbilical cord mesenchymal stem cell culture supernatant at 300g, 2000g, and 10000g. The centrifugation time at 2000g is preferably 8-12 minutes, more preferably 10 minutes; the centrifugation time at 300g is preferably 8-12 minutes, more preferably 10 minutes; and the centrifugation time at 10000g is preferably 25-35 minutes, more preferably 30 minutes. The preferred temperature for differential centrifugation is 0-4°C. This differential centrifugation method is beneficial for removing impurities such as cell debris and organelles. Centrifugation at 0-4°C reduces the loss of these bioactive substances and maintains the biological activity of the particles. After removing cell debris and organelles, ultrafiltration washing is performed, preferably using a 100 kDa MWCO ultrafiltration centrifuge tube. This invention does not specifically limit the source of the 100 kDa MWCO ultrafiltration centrifuge tubes; any source of 100 kDa MWCO ultrafiltration centrifuge tubes well known in the art may be used. After ultrafiltration and washing, a concentrated solution is obtained.

[0020] After obtaining the concentrate, the present invention subjectes the concentrate to ultracentrifugation. Preferably, the ultracentrifugation involves a first centrifugation at 80,000–120,000 g for 1–4 h to obtain a first precipitate and a first supernatant; the centrifugal force of the first centrifugation is preferably 90,000–110,000 g, more preferably 100,000 g. The first centrifugation time is preferably 2–4 h, more preferably 1–4 h, and most preferably 4 h. After obtaining the first supernatant, the first supernatant is subjected to a second centrifugation at 150,000–180,000 g for 14–18 h to obtain a second precipitate and a second supernatant; the centrifugal force of the second centrifugation is preferably 160,000–170,000 g, more preferably 167,000 g. The second centrifugation time is preferably 15–17 h, more preferably 16 h. After obtaining the second supernatant, the second supernatant is subjected to a third centrifugation at 360,000–370,000 g for 14–18 h to obtain a third precipitate and a third supernatant. The centrifugal force for the third centrifugation is preferably 365,000–368,000 g, more preferably 367,000 g. The centrifugation time for the third centrifugation is preferably 15–17 h, more preferably 16 h. This ultracentrifugation method does not require the introduction of exogenous reagents, avoiding chemical contamination and ensuring the natural properties of the extracted ultrafine nanoparticles, which is beneficial for subsequent research and application. The method described in this invention can effectively remove nanovesicles of other sizes and impurities, and the obtained ultrafine nanoparticles can effectively promote axonal regeneration and nerve function recovery after spinal cord injury.

[0021] The third precipitate obtained is the hucMSC-supermeres. Preferably, the hucMSC-supermeres are resuspended in PBS solution and then filtered sterile to obtain a hucMSC-supermeres suspension. The amount of PBS buffer added during resuscitation can be flexibly adjusted to control the volume and concentration of the final resuspension, without damaging the activity of hucMSC-supermeres, thus improving the effect on promoting axonal regeneration and neurological function recovery after spinal cord injury.

[0022] In this invention, it is preferable to identify the obtained hucMSC-supermeres. The identification includes basic morphological identification and identification of surface marker proteins. Basic morphological identification includes transmission electron microscopy (TEM) observation of the isolated and purified hucMSC-supermeres. TEM images show that the hucMSC-supermeres appear as elliptical particles without nanomembrane vesicles, with a particle size of approximately 25 nm. The identification of the surface marker proteins is preferably performed using Western blotting. The proteins detected by Western blotting preferably include angiotensin-converting enzyme 2 (ACE2) and Argonaute protein 2 (AGO2). Existing technology (Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets) indicates that ACE2 and Argonaute protein 2 can serve as marker proteins for supermeres. Western blotting results show that the surface marker proteins of the hucMSC-supermeres isolated and purified in this invention include angiotensin-converting enzyme 2 (ACE2) and Argonaute protein 2 (AGO2). The superior physicochemical properties of hucMSC-supermeres make them more easily taken up by damaged neurons and can penetrate deep into the core of the injury, improving bioavailability and achieving precise repair of damaged nerve tissue. HucMSC-supermeres are enriched with higher abundances of bioactive proteins (such as key metabolic enzymes) and nucleic acids (such as miRNAs), and due to the lack of a lipid bilayer membrane, their tissue penetration and cellular uptake rates in vivo are significantly superior to sEVs, making them a highly promising class of circulating biomarkers and novel therapeutic carriers. This invention demonstrates that, compared to sEVs (approximately 150 nm in diameter) with a lipid bilayer membrane structure, hucMSC-supermeres have a smaller particle size and are unmembrane-free, exhibiting higher tissue penetration and cellular uptake rates in the spinal cord injury microenvironment, thus optimizing the drug delivery efficiency for nerve regeneration therapy.

[0023] In this invention, the preferred dosage of the drug is 10. 9 The number of particles is more than 2.5 × 10⁻⁶, more preferably 2.5 × 1 9 Particle count. The dosage form of the drug preferably includes an injection. The solvent of the drug preferably includes a PBS solution. In the injection, the concentration of hucMSC-supermeres is preferably not less than 10. 7 Particle number / µl, more preferably not less than 5×10 7Particle number / µl, more preferably 10 8 ~10 9 Particle count / µl. This concentration of injection is effective in treating spinal cord injury.

[0024] In this invention, the nerve cells preferably include PC12 cells and / or DRG cells. The DRG cells preferably include primary rat DRG cells. The nerve cell damage includes mechanical nerve cell damage and / or oxidative stress damage. The oxidative stress damage preferably includes H2O2-induced oxidative damage. The concentration of H2O2 is preferably 100-300 μM, more preferably 200 μM. Embodiments of this invention demonstrate that the hucMSC-supermeres can effectively repair nerve cell damage caused by mechanical or oxidative stress.

[0025] In this invention, the disease caused by nerve cell damage preferably includes traumatic diseases of the central nervous system. The traumatic diseases of the central nervous system preferably include spinal cord injury. The symptoms of spinal cord injury preferably include at least one of the following: motor dysfunction, impaired nerve conduction, syringomyelia, disordered spinal cord structure, Nissl body lesions of the spinal cord, inflammatory response of the spinal cord, glial scarring of the spinal cord, and axonal lesions. The motor dysfunction preferably includes lower limb / hind limb motor dysfunction. The axonal lesions preferably include at least one of the following: axonal swelling, axonal breakage, axonal disintegration, and axonal misalignment. The Nissl body lesions of the spinal cord preferably include at least one of the following: reduced Nissl bodies, Nissl body dissolution, and Nissl body disappearance. Treatment of spinal cord injury preferably includes promoting the regeneration and repair of damaged spinal cord tissue and / or improving the symptoms of spinal cord injury. The promotion of the regeneration and repair of damaged spinal cord tissue preferably includes at least one of the following: promoting axonal regeneration and repair, promoting nerve cell regeneration and repair, promoting nerve fiber regeneration and repair, and promoting angiogenesis.

[0026] In this invention, a spinal cord injury (SCI) SD rat model was constructed, and hucMSC-sEVs and hucMSC-supermeres were used for SCI treatment. Experimental results showed that hucMSC-supermeres significantly promoted the survival of damaged neurons, induced neurite extension, and upregulated the expression of key axonal proteins, thereby further promoting the regeneration and remodeling of damaged axons. More importantly, because hucMSC-supermeres are enriched with higher abundances of bioactive components, their effect on promoting axonal regeneration is significantly superior to that produced by small extracellular vesicles (hucMSC-sEVs) under the same conditions. Simultaneously, in vivo experiments showed that hucMSC-supermeres more effectively improved hindlimb motor function in rats with spinal cord injury and reduced scar formation at the injury site compared to hucMSC-sEVs. Therefore, the axonal regeneration-promoting effect of hucMSC-supermeres provides a safer and more efficient new strategy for the clinical treatment of spinal cord injury.

[0027] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments, unless otherwise described, can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0029] In the embodiments of the present invention, fetal bovine serum (purchased from Vazyme), α-MEM culture medium (purchased from Meilunbio), human umbilical cord mesenchymal stem cell adipogenic differentiation kit (purchased from OriCell), ACE2 antibody (purchased from Proteintech), AGO2 antibody (purchased from Proteintech), Alix antibody (purchased from CST), Calnexin antibody (purchased from CST), CD63 antibody (purchased from Proteintech), CD9 (purchased from CST), Hoechst dye (purchased from Sigma), Vybrant™ CM-DiI labeling solution (purchased from Invitrogen), and Alexa Fluor were used. TM647 protein labeling kit (purchased from Invitrogen), NF200 antibody (purchased from Proteintech), GFAP antibody (purchased from CST), PBS buffer (purchased from Shandong Qidu Biomedical Co., Ltd.), CCK8 assay kit (purchased from Vazyme), TUNEL BrightRed Apoptosis kit (purchased from Vazyme), Tuj-1 antibody (purchased from Proteintech), Case-3 antibody (purchased from CST), Bcl-2 antibody (purchased from CST), GAPDH antibody (purchased from Proteintech), DMEM medium (purchased from Meilunbio), Neurobasal™-A medium (purchased from Thermo Fisher Scientific), Cyanine 3 antibody (purchased from Abclonal).

[0030] Example 1: Isolation, acquisition and identification of hucMSC-sEV and hucMSC-supermeres.

[0031] (1) Isolation, culture and identification of hucMSCs: After obtaining approval from the ethics committee and informed consent from the parturients, umbilical cord tissues from full-term parturients were collected from the Fourth Affiliated Hospital of Jiangsu University (Fourth People's Hospital of Zhenjiang City) and transported to the laboratory aseptically. After removing the umbilical cord torsion vessels and veins using microsurgical instruments, the processed gelatinous umbilical cord tissue was trimmed into 1-3 mm pieces. 3 The fragments are evenly spread out at a depth of 3.5 cm. 2 Approximately 15 tissue fragments were placed on the bottom surface of each culture dish. The dishes were inverted and placed in a 37°C, 5% CO2 incubator for 2 hours. After the tissues adhered tightly to the bottom, 1 mL of complete culture medium (α-MEM basal medium containing 15% FBS and 1% penicillin-streptomycin solution) was added, ensuring the culture medium surface lightly covered the tissue fragments. The culture system underwent semi-quantitative medium replacement every 72 hours, and cell migration was continuously observed. P0 generation cells exhibited a fish-like aggregation pattern. A portion of cells cultured to P3 generation was used for adipogenic-osteogenic induction to identify the cells. Figure 1 The other part is cultured for subsequent separation and acquisition of hucMSC-sEV and hucMSC-supermeres.

[0032] Adipogenic induction assay of hucMSCs: 0.1% gelatin was evenly coated on the bottom of a 6-well plate and incubated at 37°C for 30 min. Excess solution was removed, and the cells were allowed to air dry at room temperature. Then, well-grown third-generation hucMSCs were seeded into the pretreated plates and cultured until 90% confluence was achieved. The original culture medium was discarded, and 2 mL of adipogenic differentiation inducer A was added to each well to begin induction culture. After 72 hours, the culture was replaced with differentiation promoter B, and after 24 hours, it was replaced back with inducer A. This alternating induction culture was repeated for 21 days until obvious lipid vacuolar structures appeared inside the cells. Finally, staining was performed. The induction solution was first discarded, and the cells were washed with PBS buffer. 4% paraformaldehyde solution was added for fixation for 30 min. After removing the fixative, the cells were washed three times with PBS to remove impurities. Freshly prepared Oil Red O working solution was added to completely cover the cell layer for staining in the dark for 20 min. After staining, the cells were thoroughly washed three times to remove excess dye, and then washed twice with PBS. The staining effect was observed under a microscope, and the results were recorded.

[0033] Osteogenic induction assay for hucMSCs: A 0.1% gelatin solution was evenly coated on the bottom of a 6-well plate and incubated at 37°C for 30 minutes. Excess solution was removed, and the cells were allowed to air dry at room temperature. Subsequently, healthy third-generation hucMSCs were seeded into the pretreated plates and cultured until confluence reached 80%-90%. The original culture medium was discarded, and the cells were washed with PBS and replaced with 2 mL of osteogenic induction medium. The medium was changed every 3 days. Approximately 15-30 days after adding the osteogenic induction medium, when a large number of calcium crystals were observed in the hucMSCs under a microscope, the original culture medium was aspirated, and Alizarin Red S staining solution was added. Staining was performed at room temperature for 10 minutes, followed by washing twice with PBS. The staining effect was observed under a microscope, and the results were recorded.

[0034] Figure 1 The left image shows primary hucMSCs emerging from the umbilical cord tissue, demonstrating the morphological characteristics of the cells; the middle image shows the adipogenic induction experiment of hucMSCs; and the right image shows the osteogenic induction experiment of hucMSCs. Figure 1 The results showed that the extracted cells were hucMSCs, which can be used for the subsequent isolation and acquisition of hucMSC-sEVs and hucMSC-supermeres.

[0035] (2) Isolation and acquisition of hucMSC-sEV and hucMSC-supermeres: The culture supernatant of P3-P7 generation hucMSCs that have not undergone adipogenic osteogenic induction was collected. At 4°C, the cells were removed by centrifugation at 300 g for 10 min, cell debris was removed by centrifugation at 2000 g for 10 min, and organelles were removed by centrifugation at 10000 g for 30 min. The supernatant was then collected and placed in an ultrafiltration concentrator with a molecular weight cutoff of 100 kDa. The concentrator was centrifuged at 2000 g for 10 min at 4°C to obtain a concentrate. The collected concentrate was then subjected to a first centrifugation at 100000 g for 2 h at 4°C to obtain a supernatant containing hucMSC-supermeres and a precipitate containing hucMSC-sEV.

[0036] After adding PBS to the precipitate containing hucMSC-sEV, the precipitate was washed by centrifugation at 100,000g for 2 h. The supernatant was then removed, and the obtained precipitate was hucMSC-sEV. The precipitate was resuspended in an appropriate amount of PBS and then collected through a 0.22μm sterile filter to obtain sterile hucMSC-sEV. The concentration was adjusted to 10 with PBS. 8 Particle number / μl, obtain hucMSC-sEV suspension, aliquot and store at -80℃; The supernatant containing hucMSC-supermeres was centrifuged a second time at 4°C at 167,000 g for 16 h to obtain cell exomeres and the supernatant after exomeres removal. The exomeres-free supernatant was then centrifuged a second time at 4°C at 367,000 g for 16 h to obtain the supernatant and hucMSC-supermeres precipitate. The hucMSC-supermeres precipitate appeared turbid. The hucMSC-supermeres precipitate was then passed through a 0.22 µm sterile filter to collect sterile hucMSC-supermeres. Particle count analysis showed that the concentration of collected sterile hucMSC-supermeres was close to 10. 8 Particle number / μl, adjust concentration to 10 with PBS 8 The number of particles per μl was used to obtain a hucMSC-supermeres suspension, which was then aliquoted and stored at -80°C.

[0037] (3) Identification of hucMSC-sEV and hucMSC-supermeres: Morphology of hucMSC-sEV and hucMSC-supermeres observed by transmission electron microscopy: 20 µl of hucMSC-sEV suspension or hucMSC-supermeres suspension was evenly spread on a copper mesh supporting the membrane and allowed to adsorb at room temperature for 10 min. After adsorption equilibrium, residual droplets were removed by gently touching the edge of the copper mesh with sterile filter paper (avoiding contact with the central sample), and the copper mesh was quickly inverted and stained in 3% phosphotungstic acid at room temperature in the dark for 5 min. After staining, the copper mesh was placed vertically with the aid of filter paper and dried in a 37℃ incubator for 15 min. The dried samples were then examined by transmission electron microscopy.

[0038] Transmission electron microscopy observations revealed its microscopic morphological characteristics ( Figure 2 ): hucMSC-sEV is typically disc-shaped, while hucMSC-supermeres are elliptical particles without nanomembrane vesicles. Compared with the scale bar, hucMSC-sEV has a diameter of about 150 nm, while hucMSC-supermeres has a diameter of about 25 nm. Western blotting detection of protein biomarkers in hucMSC-sEV and hucMSC-supermeres: HucMSC-sEV and hucMSC-supermeres suspensions were taken, and an appropriate amount of pre-chilled RIPA lysis buffer was added. Indirect vortexing was performed (high-speed vortexing for 2 min, followed by an ice bath for 5 min), repeated 10 times to ensure complete lysis. After lysis, the mixture was centrifuged at 12000g for 15 min at 4°C. The supernatant was collected and transferred to a new EP tube. 3×SDS-PAGE loading buffer was added at a volume ratio of 3:1, and the mixture was thoroughly mixed. The tube was then placed in a water bath and heated at 100°C for 10 min to fully denature the proteins. The tube was then allowed to cool naturally to room temperature before being stored at -20°C for subsequent experiments. Select the appropriate concentration of precast gel kit according to the molecular weight of the target protein, prepare the gel according to the ratio, and install the prepared gel into the electrophoresis clamp according to the corresponding positive and negative electrodes. Add the protein sample to the corresponding lane, and add the pre-stained protein marker to both lanes for electrophoresis (60V for 30 min first, then 100V for 100 min) until the tracer is 0.5 cm from the bottom of the gel. After electrophoresis, use the wet transfer method to assemble the "sponge, filter paper, PVDF membrane (activated by methanol), gel, filter paper, sponge" into a "sandwich" structure, place it in the black and white clamp, and transfer the membrane under low temperature conditions (ice-water bath) (350mA for 120 min). After the transfer, block with 5% skim milk-TBST solution at room temperature for 2 h. Then, incubate with primary antibody (diluted, 4℃ overnight) and corresponding species secondary antibody (1:2000 dilution, room temperature for 1.5 h). After each antibody incubation, wash with TBST 3 times, and place on a shaker for 10 min after each wash. Finally, a freshly prepared, highly sensitive ECL chemiluminescent substrate was used for development and detection.

[0039] The final imaging and exposure detected hucMSC positive markers Calnexin, hucMSC-sEV positive markers Alix, CD9, CD63, and hucMSC-supermeres positive markers ACE2 and AGO2. Figure 3 This indicates that hucMSC-sEV and hucMSC-supermeres were obtained.

[0040] Example 2: Comparison of uptake efficiency between hucMSC-sEV and hucMSC-supermeres.

[0041] (1) Detection of hucMSC-sEV uptake: Take an appropriate amount of the hucMSC-sEV suspension prepared in Example 1, add the lipophilic fluorescent dye CM-Dil at a volume ratio of 1:300, mix thoroughly, and incubate in a 37℃ constant temperature shaker for 30 min in the dark for labeling. Transfer the labeled mixture to a 100 kDa ultrafiltration centrifuge tube, add pre-cooled PBS to 2 mL, and centrifuge at 1500 g for 30 min at 4℃. Then wash and centrifuge twice with PBS, and collect the concentrated fluorescently labeled hucMSC-sEV.

[0042] Rat adrenal medullary pheochromocytoma cells PC12 (purchased from Shanghai Enzyme Research Biotechnology Co., Ltd.) were seeded into poly-L-lysine-coated culture plates (culture plate preparation method: in a clean bench, 1 ml of poly-L-lysine was added to each well, allowed to stand for 30 min, then the poly-L-lysine was discarded, the cells were washed three times with sterile ddH2O, and irradiated with ultraviolet light for at least 15 min until the bottom of the plate was dry). The cells were expanded using RPIM 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Experiments were performed when the cells reached approximately 70% confluence. The old medium was discarded, and the cells were washed with PBS. Fresh medium containing 200 µg / mL of fluorescently labeled hucMSC-sEV was added, and the cells were incubated at 37°C in a 5% CO2 incubator for 12 h.

[0043] After incubation, cells were washed three times with PBS to remove untaken vesicles, and then fixed with 4% paraformaldehyde at room temperature for 20 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min, followed by nucleus staining with 1:300 Hoechst working solution in the dark for 10 min, and then washed three times with PBS. Finally, anti-fluorescence quenching mounting medium was added, and images of each channel were acquired using a laser confocal microscope. The results are shown in the figure. Figure 4 .

[0044] (2) Detection of hucMSC-supermeres uptake: The hucMSC-supermeres suspension prepared in Example 1 was centrifuged at 12000 g for 10 min at 4°C to remove impurities and precipitates. The concentration of hucMSC-supermeres was adjusted to 1 mg / mL using PBS, and then Alexa Fluor™ 647 dye was added at a molar ratio of 1:10 according to the instructions. The bio-dye was only used for labeling and would not change other properties of the supermeres. The mixture was placed at room temperature in the dark for 60 min, and gently shaken every 15 min to ensure sufficient binding. After the reaction was completed, a stop buffer containing glycine was added, and the mixture was allowed to stand at room temperature for 10 min to quench the free dye. The reaction system was transferred to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and centrifuged at 4000 g for 20 min at 4°C. The lower filtrate was discarded. The filtrate was washed repeatedly with PBS and centrifuged twice to completely remove unbound dye. Finally, the filtrate was resuspended in 200 µl of PBS to obtain fluorescently labeled hucMSC-supermeres and stored in the dark for later use. Following the above-described uptake assay procedure for hucMSC-sEV, fluorescently labeled hucMSC-supermeres were co-incubated with PC12 cells. Finally, the uptake was observed using a laser confocal microscope under 647 nm excitation light. The results are shown in [Figure number missing]. Figure 4 .

[0045] In situ retention detection of hucMSC-supermeres in spinal cord injury in SD rats with SCI model: Method for constructing the SCI model in SD rats: Female SD rats weighing approximately 200g were anesthetized with 40mg / Kg sodium pentobarbital. Using the spinous process of T10 vertebra as a landmark, the skin was incised along the midline of the back, the fascia and muscle layer were separated, and the laminae of T9-T11 were removed to expose the spinal dura mater. The spinal cord was exposed by vertically striking it with a 5g metal rod from a height of 15cm for 5 seconds. Tail spasm and hind limb retraction were used as markers of successful modeling.

[0046] 25 µl of fluorescently labeled hucMSC-supermeres (containing a total of 2.5 × 10⁻⁶) was added. 9 The location of the spinal cord injury center in SD rats with the SCI model was determined by in situ injection of hucMSC-supermeres. The fluorescence intensity of hucMSC-supermeres was detected by in vivo imaging before injection (blank group), immediately after injection (0h), 1 day after injection (24h), 3 days after injection (72h), 5 days after injection (120h), and 5 days after injection (spinal cord group). The results are shown in […]. Figure 5 .

[0047] Based on nuclear localization analysis of the uptake efficiency of hucMSC-sEV and hucMSC-supermeres by individual PC12 cells, it was found that the uptake efficiency of hucMSC-supermeres by PC12 cells was superior to that of hucMSC-sEV. Figure 4 (See Table 1). Furthermore, hucMSC-supermeres can persist in situ for approximately 5 days in the SCI model of SD rats with spinal cord injury. During the acute axonal protection period (24-72 hours post-injury), they can exert a good therapeutic and protective effect against spinal cord injury during the critical period. Figure 5 (and Table 2).

[0048] Table 1. Uptake efficiency (au) of hucMSC-sEV and hucMSC-supermeres by PC12 cells.

[0049] Table 2. Fluorescence intensity results of hucMSC-supermeres at different time points (p / sec / cm) 2 / sr] / [uw / cm 2 ])

[0050] Example 3: hucMSC-supermeres alleviate spinal cord injury in SD rats.

[0051] (1) Footprint ink test of SD rats with SCI model: SD rats were modeled for SCI as described in Example 2 above. Among them, the control group (referred to as Sham group) was formed by only exposing the spinal cord and not performing the impact injury model (female SD rats weighing about 200g were anesthetized with 40mg / Kg sodium pentobarbital, the skin was cut along the midline of the back with the spinous process of T10 vertebra as the landmark, the fascia and muscle layer were separated, the T9-T11 lamina were removed, and the spinal dura mater was exposed) of the modeled SD rats. After the other SD rats were successfully modeled, they were slowly injected with a micro-syringe at 5 sites above, below, left and right of the injury center. The total volume of the drug injected at the 5 sites was 25 µl. According to the different injected drugs, the SD rats with SCI model were divided into three groups: sterile PBS buffer (referred to as PBS group), PBS prepared in Example 1 with a concentration of 10 8 The hucMSC-sEV suspension with particle number / μl (denoted as hucMSC-sEV group) and the 10 μl concentration prepared for Example 1. 8The hucMSC-supermeres suspension (particle count / μl) was designated as the hucMSC-supermeres group. Rats were fasted for 24 hours post-surgery, and bladder compression was performed daily to assist urination. On post-surgery day 28, a footprint ink test was performed. Red ink was applied to the hind paws of the experimental rats, and black ink to the fore paws. The rats were immediately placed at one end of a white sheet of paper and made to walk in a straight line forward. The footprint ink marks of each group of rats were scanned into images.

[0052] The rat exercise inkblot experiment showed that ( Figure 6 (See Table 3). Compared to the Sham group, the PBS group, which received no treatment, had the largest hindlimb dragging area, indicating extremely poor hindlimb motor function. In contrast, the dragging area was reduced in both the hucMSC-sEV and hucMSC-supermeres groups, indicating that both treatments promoted recovery from spinal cord injury in rats. Compared to the hucMSC-sEV group, the dragging area was significantly reduced in the hucMSC-supermeres group, indicating that its therapeutic effect was superior to that of the hucMSC-sEV group.

[0053] Table 3. Results of the rat exercise ink stain test (au)

[0054] (2) Spinal cord tissue of SD rats with HE staining for SCI model: 28 days after SCI modeling, rats from the above experimental groups (Sham group, PBS group, hucMSC-sEV group, and hucMSC-supermeres group) were selected, anesthetized by intraperitoneal injection of pentobarbital, fixed on the operating table, and the body cavity was opened to expose the heart. Physiological saline buffer (approximately 200 mL / rat) was slowly perfused into the rats through the left ventricle. Simultaneously, the blood vessels (veins or arteries) below the thoracic cavity were cut as drainage outlets. The perfusion fluid was used to flush the circulatory system until the outflow was clear, thus draining all blood from the body. Subsequently, the syringe was changed to infuse 4% paraformaldehyde solution (approximately 250 mL / rat) until the rat's body became rigid, completing the systemic fixation. Spinal cord tissue was harvested 2 cm from both ends of the original spinal cord injury point. The obtained samples were placed in 4% paraformaldehyde and fixed at 4°C. Then, they were paraffin-embedded and sectioned according to standard procedures. The prepared paraffin sections were baked in a 60℃ oven for 6 hours. After removal, they were sequentially immersed in xylene I and xylene II for 10 minutes each to completely dewax. Then, a gradient ethanol rehydration treatment was performed: the sections were successively immersed in 100%, 95%, 80%, and 70% ethanol for 2 minutes each. Finally, the sections were washed three times with PBS buffer for 5 minutes each time. The rehydrated sections were then immersed in hematoxylin staining solution for 5 minutes. After removal, they were placed in 1% hydrochloric acid ethanol solution for differentiation for 10 seconds, followed by a blueing treatment in 0.5% ammonia water for 10 seconds. The sections were rinsed with running water for 15 minutes to remove residual reagents, and then counterstained in eosin staining solution for 3 minutes. After staining, the sections were dehydrated with a gradient ethanol treatment, cleared with xylene, mounted with neutral resin, observed under a microscope, and images were acquired.

[0055] HE staining revealed findings in rat spinal cord tissue ( Figure 7 In the PBS group, significant cavitation and structural disorder were observed at the site of spinal cord injury in rats, while the hucMSC-sEV and hucMSC-supermeres groups promoted spinal cord tissue recovery. Furthermore, compared to hucMSC-sEV, hucMSC-supermeres more effectively reduced the area of ​​spinal cord cavity, further promoting tissue structural recovery.

[0056] (3) Spinal cord tissue of SD rats in the Nissl staining SCI model: Following the same HE staining procedure as described above, paraffin sections were baked overnight in a 60°C incubator, then routinely dewaxed and rehydrated using xylene and gradient ethanol solutions (100%, 95%, 80%, 70%), and finally washed in distilled water for later use. The rehydrated sections were then immersed in Nissl staining solution (1% toluidine blue) and incubated at 37°C (or room temperature) in the dark for 15 min until the neuronal cell bodies turned deep blue. The sections were removed and gently rinsed twice with double-distilled water to remove excess stain. The sections were then placed in 95% ethanol for differentiation for 30 s. When the background color faded, the Nissl bodies turned a clear deep blue, and the nucleoli were clearly visible, the sections were quickly placed in distilled water to terminate differentiation. After differentiation, the sections were rapidly dehydrated in 95% ethanol and 100% ethanol, approximately 2 min each time. They were then cleared in xylene (I, II) for 5 min each. Remove the slide, let the surrounding liquid dry, add neutral resin to mount the slide, and observe and acquire images under a microscope.

[0057] Nissler staining revealed the following in rat spinal cord tissue: Figure 8 In the PBS group, normal Nissl bodies at the site of spinal cord injury were significantly dissolved or disappeared, while this condition was alleviated in the hucMSC-sEV and hucMSC-supermeres groups. Furthermore, compared to hucMSC-sEV, hucMSC-supermeres more effectively supported Nissl body survival, resulting in superior neuronal health.

[0058] (4) Immunofluorescence staining of spinal cord tissue from SCI model SD rats: Prepared paraffin sections were baked in a 60℃ oven for 6 hours. After removal, they were immersed sequentially in xylene I and xylene II (xylene was used in two staining tanks; the first immersion was called xylene I, and the second immersion was called xylene II), each for 10 minutes to completely dewax. Then, a gradient ethanol rehydration treatment was performed: the sections were successively immersed in 100%, 95%, 80%, and 70% ethanol for 2 minutes each. Finally, the sections were washed three times with histochemical PBS buffer for 5 minutes each time. They were placed in sodium citrate buffer for heat antigen retrieval and boiled in water for 30 minutes. After removal, they were washed three times with histochemical PBS for 5 minutes each time. 5% BSA was added for blocking at room temperature for 30 minutes. The BSA was discarded, and the primary antibody of the target protein (NF200) was added, and incubated overnight at 4℃. The next day, the primary antibody was recovered, and the sections were washed three times with histochemical PBS for 5 minutes each time. The corresponding species of fluorescently labeled secondary antibody was added, and incubated at room temperature for 90 minutes. Wash the tissue section three times with PBS for 5 minutes each time. Add another target antibody (GFAP) and incubate overnight at 4°C. Repeat the above steps until the secondary antibody is eluted. Stain the cell nuclei with Hochest staining and incubate at room temperature for 10 minutes. Wash the tissue section three times with PBS for 5 minutes each time. Add an anti-fluorescence quenching mounting medium and seal the tissue section with a coverslip of appropriate size using clear nail polish. After drying in the dark, observe the sample under a fluorescence microscope and acquire fluorescence signal images of each channel.

[0059] Tissue fluorescence staining of rat spinal cord tissue revealed ( Figure 9 (See Table 4). The higher intensity of red fluorescence in the PBS group indicates that astrocytes are activated after spinal cord injury, forming glial scars and releasing various inflammatory factors. Persistent inflammation is not conducive to tissue recovery. In contrast, the lower expression of red fluorescence in the hucMSC-supermeres group indicates that hucMSC-supermeres have the function of reducing inflammatory response, which can improve the inflammatory microenvironment and thus protect nerve cells. On the other hand, the higher expression of green fluorescence in the hucMSC-supermeres group indicates good axonal regeneration.

[0060] Table 4. Results of tissue fluorescence staining of rat spinal cord tissue (au)

[0061] Example 4: hucMSC-supermeres promote PC12 cell survival and axonal regeneration.

[0062] (1) CCK8 assay to detect the effect of hucMSC-supermeres on the proliferation activity of PC12 cells after H2O2 damage: PC12 cells were cultured to the logarithmic growth phase to obtain cell suspension. The culture medium was RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. The cell suspension was seeded into 96-well plates, with 100 µl of cell suspension seeded into each well, and the cell suspension concentration was 1×10⁻⁶. 4 Cells / 100 μl, different treatment groups were set up according to different added components: normal group (NC group: no H2O2 and drug added), damaged group (H2O2 group: added with working concentration of 200 μM H2O2), and hucMSC-sEV treatment group (added with working concentration of 200 μM H2O2 and 25 μl of the concentration prepared in Example 1 of 10 8 The hucMSC-sEV group (particle number / μl), the hucMSC-supermeres treatment group (added with 200 μM H2O2 and 25 μl of the 10-particle-number-per-μl preparation from Example 1) 8 The number of particles per μl of hucMSC-supermeres was calculated, with 6 replicate wells per group. The wells were incubated at 37°C with 5% CO2 for 24 h. The culture medium in each well was discarded, and 100 µl of serum-free medium containing 10% CCK8 reagent was added. The wells were then incubated at 37°C for 2–4 h. Immediately after the reaction was terminated, the OD value of each well was measured at 450 nm using a multi-mode microplate reader.

[0063] The test results show that ( Figure 10 (As shown in Table 5), when PC12 cells were treated with H2O2-damaged cells by hucMSC-sEV and hucMSC-supermeres with the same number of particles, the cell proliferation activity of the hucMSC-supermeres group was significantly improved.

[0064] Table 5. Results of tissue fluorescence staining of rat spinal cord tissue (au)

[0065] (2) TUNEL assay to detect apoptosis in PC12 cells after H2O2 damage by hucMSC-supermeres: PC12 cells in logarithmic growth phase were seeded in 6-well plates (2 mL cell suspension per well) and cultured at 37°C with 5% CO2 until the cell density reached 50%. Following the same steps, normal, damaged, hucMSC-sEV, and hucMSC-supermeres treatment groups were established based on the different added components. The cells were cultured at 37°C for 24 h. The supernatant was discarded, and the cells were washed three times with PBS. 4% paraformaldehyde was added for fixation at room temperature for 15 min, followed by two PBS washes for 3 min each. Liquid around the slide was absorbed using filter paper, and a proteinase K working solution with a final concentration of 20 μg / mL was prepared and added to the tissue section. The solution was incubated at room temperature for 5 min, followed by two PBS washes for 3 min each. Add 100 μl of 1× Equilibration Buffer and incubate at room temperature for 15 min, then discard. Add 50 μl of TdT buffer and incubate at 37°C for 1 h, then discard. Wash twice with PBS, 3 min each time. Add Hoechst dye for nuclear staining and incubate at room temperature for 10 min. Add anti-fluorescence quenching mounting medium and invert the cell-side down onto a glass slide. Finally, seal the edges of the coverslip with clear nail polish. After the mounting medium has dried in the dark, observe the sample under a fluorescence microscope and acquire fluorescence signal images of each channel.

[0066] Tunel detection shows ( Figure 11 (As shown in Table 6) The number of apoptotic cells marked with red fluorescent dye in the hucMSC-supermeres group was significantly reduced, indicating that the cells had good anti-apoptotic properties.

[0067] Table 6 Tunel detection results (au)

[0068] (3) Western-Blot detection of protein expression in PC12 cells: PC12 cells in logarithmic growth phase were seeded into 6-well plates (2 mL of cell suspension per well) and cultured in a 5% CO2, 37°C incubator until the cell density reached 50%. Following step (1), different groups were set up according to the different components added to the PC12 cells: normal group, damaged group, hucMSC-sEV treatment group, and hucMSC-supermeres treatment group. After 24 h of culture, the old culture medium in the wells was discarded, and the culture plate was quickly placed in an ice bath to maintain low temperature. The cells were washed three times with PBS buffer, and 100 μl of RIPA lysis buffer was added to each well. Adherent cells were scraped off in a single direction using a sterile cell scraper. The collected cell lysis suspension was transferred into EP tubes and subjected to indirect vortexing (high-speed shaking for 2 min, ice bath for 5 min), repeated several times to ensure complete lysis. After lysis, the samples were centrifuged at 12000 g for 15 min at 4°C. Aspirate the supernatant and add 3×SDS-PAGE loading buffer at a volume ratio of 3:1. Mix well and heat in boiling water at 100℃ for 10 min to induce protein denaturation. Remove and allow to cool naturally to room temperature, then store at -20℃ for subsequent experiments. The same Western blotting procedure as in Example 1 was used to detect cell proliferation marker Bcl-2, apoptosis marker Case-3, and axonal protein markers NF200 and Tuj-1.

[0069] Western-Blot results showed that ( Figure 12 In the hucMSC-supermeres group, the expression levels of axonal markers (NF200, Tuj-1) and anti-apoptotic markers (Bcl-2) of cellular proteins were significantly increased, while the expression level of pro-apoptotic markers (Caspase-3) was significantly decreased.

[0070] (4) Cell fluorescence staining was used to detect the axonal morphology expression of hucMSC-supermeres in PC12 cells and primary rat DRG cells after H2O2 damage.

[0071] PC12 cell fluorescence staining: Logarithmic-phase PC12 cells were seeded into 6-well plates (2 mL cell suspension per well) and cultured at 37°C with 5% CO2 until the cell density reached 50%. Following step (1), normal, damaged, hucMSC-sEV, and hucMSC-supermeres treatment groups were established based on the different components added to the PC12 cells. These groups were seeded into 12-well plates with sterile coverslips (climbing slides) and cultured in an incubator. After the cells had fully adhered, the old culture medium was aspirated from the wells, and the cells were washed twice with PBS buffer. 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 30 min. The cells were washed twice with PBS, and 0.1% Triton X-100 was added for membrane permeation treatment for 15 min. The cells were washed twice with PBS, and the cell climbing slides were removed and placed on a glass slide (with the cell side facing up). 5% BSA blocking solution was added to cover the tissue for 1 h. Remove excess blocking solution and immediately add primary antibody (Tuj-1 antibody) diluted 1:50, incubating overnight at 4°C. The next day, recover or discard the primary antibody and transfer the slide back into the 12-well plate. Wash three times with PBS for 5 min each time. After washing, transfer the slide back onto a glass slide and add diluted fluorescently labeled secondary antibody, incubating at room temperature in the dark for 2 h. After secondary antibody incubation, place the slide back into the 12-well plate and wash three times with PBS for 10 min each time. Then add 1:300 diluted Hoechst dye and stain at room temperature in the dark for 10 min to label cell nuclei. After staining, wash three times with PBS for 10 min each time. Aspirate any remaining moisture from the edges of the slide, add anti-fluorescence quenching mounting medium, and invert the slide cell-side down onto a glass slide. Finally, seal the edges of the coverslip with clear nail polish. After the mounting medium has dried in the dark, observe the sample under a fluorescence microscope and acquire fluorescence signal images for each channel.

[0072] Extraction and fluorescence staining of primary rat DRG neurons: In adult SD rats, the back skin was quickly peeled off to expose and completely remove the spine. The spinal canal was cut along the midline of the dorsal side of the spine, and the lamina was carefully removed to expose the DRG. The nerve roots connected to the DRG were grasped with microforceps, and the axons were gently pulled apart. The DRG was completely removed from the vertebral foramen and placed in prepared DMED culture medium (DMEM basal medium, 10% FBS, and 1% penicillin-dextrose antibody). This process was repeated to collect a sufficient number of DRG cells. 1 mL of 3 mg / mL collagenase I was added, and after dispersing, the cells were digested at 37°C for 90 min (with gentle pipetting every 30 min). 0.25% trypsin was added, and digestion continued for 5 min, followed by pipetting. Digestion was stopped by adding twice the volume of DMEM medium. The cells were then collected in a 15 mL centrifuge tube through a 70 μm filter and centrifuged at 800 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 6 mL of 15% BSA solution. The cells were centrifuged at 720 rpm for 5 min and the supernatant was discarded. The cell pellet was then resuspended in DMEM medium. The cell suspension was seeded onto cell culture slides pre-coated with poly-lysine. After cell attachment, the medium was replaced with Neurobasal™-A medium (NB-A + 2% B27 + 1% Gln + 1% penicillin-streptomycin solution) and cultured until the cells were fully extended and the axons had fully grown. Subsequently, primary rat DRG cells were subjected to the same fluorescent staining method as PC12 cells.

[0073] Cell fluorescence showed that PC12 cells in the hucMSC-supermeres group had significantly increased axon length and were in good growth condition. Figure 13 (See Table 7) The results of primary rat DRG cells showed that the cell growth status of the hucMSC-supermeres group was well restored, and the axon length was significantly improved. Figure 14 (See Table 8). The above experiments demonstrate that the relative axon length of nerve cells increased significantly under hucMSC-Supermeres treatment intervention, indicating that its efficacy in promoting axon extension is significantly better than that of hucMSC-sEV with the same number of particles.

[0074] Table 7. Fluorescence staining results of PC12 cells (au)

[0075] Table 8. Fluorescence staining results of primary rat DRG cells (au)

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative intent, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of ultrafine nanoparticles derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for the prevention and / or treatment of diseases caused by nerve cell damage.

2. The application according to claim 1, characterized in that, The neuronal injury includes neuronal mechanical injury and / or neuronal oxidative stress injury.

3. The application according to claim 1, characterized in that, The diseases caused by nerve cell damage include traumatic diseases of the central nervous system.

4. The application according to claim 3, characterized in that, The traumatic diseases of the central nervous system include spinal cord injury.

5. The application according to claim 4, characterized in that, The symptoms of spinal cord injury include at least one of the following: motor dysfunction, impaired nerve conduction, syringomyelia, disordered spinal cord structure, Nissl body lesions, inflammatory reactions of the spinal cord, glial scars of the spinal cord, and axonal lesions.

6. The application according to claim 4, characterized in that, Treatment of spinal cord injury includes promoting the regeneration and repair of damaged spinal cord tissue and / or improving the symptoms of spinal cord injury; the promotion of the regeneration and repair of damaged spinal cord tissue includes at least one of the following: promoting axonal regeneration and repair, promoting nerve cell regeneration and repair, promoting nerve fiber regeneration and repair, and promoting angiogenesis.

7. The application according to claim 1, characterized in that, The nerve cells include PC12 cells and / or DRG cells.

8. The application according to claim 1, characterized in that, The method for preparing the mesenchymal stem cell-derived ultrafine nanoparticles includes the following steps: After removing cell debris and organelles from the culture supernatant of human umbilical cord mesenchymal stem cells, the supernatant was washed by ultrafiltration and the concentrate was collected. The concentrate is subjected to ultracentrifugation, which includes: centrifuging the concentrate at 80,000–120,000 g for 1–4 h to obtain a first precipitate and a first supernatant; centrifuging the first supernatant at 150,000–180,000 g for 14–18 h to obtain a second precipitate and a second supernatant; and centrifuging the second supernatant at 360,000–370,000 g for 14–18 h to obtain a third precipitate and a third supernatant. The third precipitate is ultrafine nanoparticles derived from the mesenchymal stem cells.

9. The application according to claim 1, characterized in that, The surface-labeled proteins of the mesenchymal stem cell-derived ultramicro nanoparticles include angiotensin-converting enzyme 2 and Argonaute protein 2.

10. The application according to any one of claims 1 to 9, characterized in that, The dosage form of the drug includes injections; In the injection, the concentration of mesenchymal stem cell-derived ultrafine nanoparticles is not less than 10. 7 Particle number / µl.

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