CD24 positive nucleus pulposus progenitor cell for inhibiting ferroptosis of nucleus pulposus cell, pharmaceutical composition and application

Abstract

The invention discloses a CD24 positive nucleus pulposus progenitor cell for inhibiting nucleus pulposus cell ferroptosis, a pharmaceutical composition and application. The invention firstly protects an isolated CD24 + NPPCs population and a pharmaceutical composition thereof. The invention protects the application of the cell population and the pharmaceutical composition in preparation of drugs for inhibiting nucleus pulposus cell ferroptosis and / or improving intervertebral disc degeneration microenvironment. The nucleus pulposus progenitor cell subgroup is enriched and obtained by taking CD24 as a marker. Experiments prove that the cell population can play a role through a multiple synergistic mechanism: on one hand, the ferroptosis of nucleus pulposus cells is effectively inhibited by up-regulating proteins such as GPX4 and SLC7A11 and reducing lipid peroxidation; on the other hand, by reducing the level of proinflammatory factors such as TNF-alpha and promoting synthesis of extracellular matrixes such as Aggrecan and COL2A1, the healthy intervertebral disc microenvironment is remodeled, and tissue regeneration is promoted.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering, regenerative medicine, and molecular cell biology, specifically to a CD24-positive nucleus pulposus progenitor cell that inhibits ferroptosis in nucleus pulposus cells, a pharmaceutical composition, and its uses. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Intervertebral disc degeneration is one of the leading causes of lower back pain, radiculopathy, and disability, placing a huge burden on patients and society. Current clinical treatments (such as medication, physical therapy, and surgery) mainly focus on symptom relief (such as pain and nerve compression), lacking disease-modifying therapies that can effectively slow down or reverse the degenerative process and promote tissue repair.

[0004] The nucleus pulposus is the core functional unit of the intervertebral disc, and its homeostasis is highly dependent on healthy nucleus pulposus cells. These cells are responsible for synthesizing and maintaining the extracellular matrix (ECM), providing the disc with elasticity and hydration. A decrease in the number and function of nucleus pulposus cells is widely recognized as a key initiating and driving factor in the development of intervertebral disc degeneration. Recent studies have found that ferroptosis, an iron-dependent, lipid peroxidation-driven form of programmed cell death, plays a crucial role in intervertebral disc degeneration. Various degenerative stimuli (such as oxidative stress, inflammatory factors, nutritional deprivation, and mechanical overload) can induce ferroptosis in nucleus pulposus cells, leading to significant cell loss and accelerated ECM degradation.

[0005] Given the central role of ferroptosis in nucleus pulposus cell death and intervertebral disc degeneration, targeting and inhibiting the ferroptosis pathway in nucleus pulposus cells is considered a promising new therapeutic strategy that could protect nucleus pulposus cells at the source, maintain their number and function, and thus delay or even reverse intervertebral disc degeneration.

[0006] Nucleus pulposus progenitor cells (NPPCs) are stem cell-like cells within the intervertebral disc with self-renewal and multi-lineage differentiation potential, theoretically capable of replenishing lost nucleus pulposus cells. In degenerated intervertebral discs, the number of NPPCs is significantly reduced and their function is impaired; there is a lack of efficient methods for targeted enrichment, activation, and delivery of NPPCs. The degenerative microenvironment (such as inflammation and hypoxia) inhibits the survival and differentiation of NPPCs. CD24 + (Stem cell surface markers) can serve as screening markers for highly active NPPC subsets, enriching progenitor cells with stronger proliferation, anti-apoptosis, and ECM synthesis capabilities. Targeting CD24+ The strategy of specifically inhibiting the nucleus pulposus progenitor cell subset to improve intervertebral disc degeneration is unprecedented and represents a novel approach for treating intervertebral disc degeneration. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to overcome the deficiencies of the prior art in the treatment of intervertebral disc degeneration (IVDD). Specifically, this invention aims to solve the following technical problems: provide a functional cell population that can efficiently target and repair degenerated intervertebral discs; provide a method that can effectively inhibit ferroptosis, a key death pathway in nucleus pulposus cells; and provide a comprehensive solution that can simultaneously achieve the triple effects of cell protection (anti-ferroptosis), tissue regeneration (differentiation into functional cells), and microenvironment improvement (anti-inflammatory).

[0008] The method utilizes CD24 + NPPCs differentiate into functional nucleus pulposus cells, repairing and replenishing damaged nucleus pulposus cells in degenerated intervertebral discs; they activate CD24-mediated anti-ferroptosis pathways, such as upregulating GPX4 and inhibiting lipid peroxidation, protecting cells in situ. Simultaneously, CD24... + NPPCs inhibit the expression of inflammatory factors (such as IL-1β and TNF-α) in degenerated nucleus pulposus cells, alleviate hypoxic stress, remodel the degenerated microenvironment, and effectively improve nucleus pulposus cell function, providing a potential intervention mechanism and a transformable treatment option for intervertebral disc degenerative diseases.

[0009] The technical solution adopted in this invention is as follows: In a first aspect of the invention, an isolated CD24-positive nucleus pulposus progenitor cell (CD24) is provided. + A population of NPPCs, said population being prepared by a method comprising the following steps: a. Isolating primary nucleus pulposus cells from nucleus pulposus tissue; b. Using an antibody that specifically binds to CD24, CD24-positive cells are sorted from the primary nucleus pulposus cells.

[0010] Preferably, the sorting in step b is performed by immunomagnetic bead sorting.

[0011] In a second aspect of the invention, a pharmaceutical composition is provided comprising an effective amount of the aforementioned CD24. + NPPC populations and pharmaceutically acceptable carrier composition.

[0012] Preferably, the pharmaceutically acceptable carrier is phosphate-buffered saline.

[0013] Preferably, the CD24 + The effective dose for the NPPC population is 5 × 10⁻⁶ units per dose.4 Up to 5×10 5 Each cell.

[0014] In a third aspect of the invention, the CD24 is provided. + Use of NPPCs populations or the pharmaceutical composition thereof in the preparation of a reagent for inhibiting ferroptosis in nucleus pulposus cells.

[0015] Preferably, the inhibition of nucleus pulposus ferroptosis includes upregulating the expression of GPX4 protein and / or SLC7A11 protein in nucleus pulposus cells.

[0016] In a fourth aspect of the invention, the CD24 is provided. + Use of NPPCs populations or the pharmaceutical compositions thereof in the preparation of reagents for improving the microenvironment of intervertebral disc degeneration.

[0017] Preferably, the improvement of the intervertebral disc degenerative microenvironment includes reducing the level of pro-inflammatory factors, which are selected from one or more of TNF-α, IL-1β and IL-6.

[0018] In a fifth aspect of the invention, a non-therapeutic CD24 is provided. + The in vitro applications of NPPC populations, for studying the mechanisms of intervertebral disc degeneration in experimental models or screening therapeutic agents, include: CD24 + NPPCs populations were co-cultured with degenerated nucleus pulposus cells in vitro; Detect at least one indicator associated with intervertebral disc degeneration.

[0019] Preferably, the indicators related to intervertebral disc degeneration are selected from one or more of the following: cellular ferroptosis level, GPX4 / SLC7A11 protein expression level, Aggrecan / COL2A1 protein expression level, and inflammatory factor TNF-α / IL-1β / IL-6 level.

[0020] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) This invention is the first to use CD24 as a surface marker to screen and enrich progenitor cell subsets (CD24) with stronger proliferative, stress-resistance and differentiation potential in nucleus pulposus tissue. + NPPCs). This cell population exhibited significantly enhanced function in anti-ferroptosis and tissue regeneration compared to unsorted nucleus pulposus cells.

[0021] (2) This invention overcomes the limitations of a single mechanism by using CD24 + NPPCs achieve a triple synergistic therapeutic effect: potent anti-ferrode death: CD24 + NPPCs can significantly upregulate the expression of key anti-ferroptosis proteins such as GPX4 and SLC7A11 in degenerated nucleus pulposus cells, effectively inhibit lipid peroxidation, increase cell survival rate by more than 60%, and protect cells from programmed cell death pathways.

[0022] Promotes tissue regeneration: CD24 + NPPCs can differentiate into functional nucleus pulposus cells, directly replenishing cell numbers and efficiently synthesizing and secreting key components of the extracellular matrix (such as Aggrecan and COL2A1), restoring them to 70%-85% of normal levels, thus fundamentally repairing the intervertebral disc structure.

[0023] Improving the pathological microenvironment: CD24 + NPPCs can significantly inhibit the expression of pro-inflammatory factors (such as IL-1β and TNF-α), alleviate hypoxic stress, and create a favorable environment for cell survival and tissue regeneration.

[0024] (3) Through rigorous in vitro co-culture models and in vivo animal models, this invention has confirmed that CD24 + NPPC transplantation can effectively restore the disc height index (DHI) by 40%-60% and significantly improve the histological morphology score, demonstrating a definite therapeutic effect.

[0025] (4) This invention not only provides a novel cell therapy product, but also clarifies its mechanism of action and pharmaceutical use, laying a solid foundation for the development of innovative biological agents to delay, prevent or even reverse the process of intervertebral disc degeneration, and has a clear clinical translation path and huge market potential. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 This is a Western blot result of the protein expression levels of inflammatory factors (TNF-α, IL-1β, IL-6) and key matrix components (COL2A1, Aggrecan) in the nucleus pulposus tissue.

[0028] Figure 2 This is a Western blot result of the detection of key proteins (GPX4 and SLC7A11) in the ferroptosis signaling pathway in nucleus pulposus cells; Figure 3 This is a graph showing the fluorescence intensity results of iron ions; Figure 4This is a diagram showing the morphological structure of intervertebral disc tissue in an animal model; Figure 5 This is an image showing the immunohistochemical results of key extracellular matrix components (COL2A1, Aggrecan) in an animal model. Figure 6 This is a comparison of intervertebral disc height in animal models. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0031] This invention provides a method using CD24 + A method for treating intervertebral disc degeneration using NPPC subgroups. This subgroup can repair degenerated intervertebral disc tissue, improve its microenvironment, and simultaneously achieve the dual effects of tissue regeneration and anti-cell death by inhibiting the ferroptosis signaling pathway in nucleus pulposus cells, providing a new strategy for the clinical treatment of intervertebral disc degenerative diseases.

[0032] To verify the effectiveness and mechanism of action of the method, the patented technical solution includes the following experimental steps: ① In vitro co-culture experiment: Establishing CD24 + Co-culture system of NPPCs and lipopolysaccharide (LPS)-induced degenerated nucleus pulposus cells; ② In vivo injection therapy experiment: CD24 + NPPCs were injected into the caudal intervertebral disc of a rat model of intervertebral disc degeneration.

[0033] The above experiments verified CD24 + NPPCs can effectively improve intervertebral disc degeneration. Their mechanism of action involves inhibiting the ferroptosis pathway in nucleus pulposus cells, thereby achieving the dual effects of promoting tissue regeneration and resisting cell death.

[0034] This invention innovatively targets CD24 +Nucleus pulposus progenitor cells (NPPCs) intervene in intervertebral disc degeneration through a dual mechanism: on the one hand, they inhibit the ferroptosis signaling pathway and activate the GPX4 pathway, significantly enhancing the cells' antioxidant stress capacity (experiments show a cell survival rate increase of over 60%), effectively curbing nucleus pulposus cell degeneration; on the other hand, they inhibit key inflammatory factors (such as IL-1β and TNF-α) and alleviate hypoxic stress, reshaping the microenvironment of the degenerated intervertebral disc. This technology also drives CD24... + NPPCs differentiate into functional cells, repairing and replenishing damaged nucleus pulposus tissue, promoting the restoration of extracellular matrix synthesis to 70-85% of normal levels, achieving a 40-60% recovery rate in intervertebral disc height, and effectively improving nucleus pulposus cell function. This invention achieves this through "synergistic targeting of ferroptosis, inflammation, and hypoxic stress" and "promoting CD24." + The dual strategy of "NPPC differentiation and extracellular matrix repair" provides an integrated and innovative treatment option for intervertebral disc degenerative diseases with potential curative value and clear clinical translation prospects.

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0036] Terminology Explanation:

[0037] Primary cells: These are cells that are directly isolated from animal or human tissues and cultured immediately or after a short period of adaptation without undergoing immortalization treatment. Generally, the cells initially cultured and their early passaged cells (usually referring to passages 1 to 10, P1-P10) are collectively referred to as primary cells.

[0038] 10% complete medium: refers to a nutrient solution for nucleus pulposus cell culture prepared by adding 10% fetal bovine serum (FBS) and necessary antibiotics (such as 1% penicillin / streptomycin) to DMEM / F12 basal medium.

[0039] 0.25% trypsin solution: This refers to a cell digestion solution prepared by dissolving trypsin in a buffer solution (such as PBS or HBSS) at a concentration of 0.25%. That is, each 100 mL of solution contains 0.25 grams of trypsin.

[0040] 0.1% Type II Collagenase: This refers to a tissue digestion solution prepared by dissolving Type II collagenase at a concentration of 0.1% in a buffer solution (such as PBS, physiological saline, or serum-free basal medium). That is, each 100 mL solution contains 0.1 g of lyophilized Type II collagenase powder.

[0041] Example 1: Isolation and culture of nucleus pulposus progenitor cells (NPPCs)

[0042] 1. Isolation of primary nucleus pulposus cells (NPCs) After anesthetizing rats with intraperitoneal injection of tribromoethanol (250 mg / kg), the intervertebral discs of the caudal vertebrae were isolated under aseptic conditions, and the nucleus pulposus tissue was collected. The nucleus pulposus tissue was minced and digested with 0.1% type II collagenase (dissolved in PBS or serum-free DMEM / F12) at 37°C for 3-4 hours. The digestion product was filtered through a 40 μm cell filter, centrifuged (300-400 g, 5-10 min), and the cell pellet, i.e., primary NPCs, was collected. The cells were resuspended in DMEM / F12 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and incubated at 37°C with 5%... Cultured in an incubator. Cells were grown to passage 2 (P2) for subsequent experiments.

[0043] 2. CD24 + NPPCs magnetic bead sorting Discard the NPCs culture supernatant and wash the cells twice with PBS buffer. Digest the cells with 0.25% trypsin solution and centrifuge (4... Collect the cell pellet at 1200 rpm for 5 min. Add an appropriate amount of PE-conjugated CD24 antibody and incubate on ice in the dark for 15-20 minutes. Resuspend the cells in sufficient MojoSort™ Buffer and centrifuge (4... Wash the cells at 1200 rpm for 5 min. Discard the supernatant. Add an appropriate amount of PE nanobeads and incubate on ice in the dark for 15 minutes. Then, wash the cells with an appropriate amount of MojoSort™ Buffer. Place the cell suspension on a magnetic rack and let it stand for 5 minutes. After 5 minutes, carefully aspirate and collect the CD24-negative cells that have not bound to the magnetic rack (flow-through solution). The cells remaining on the magnetic rack are CD24-positive cells. Elute the CD24-positive cells from the magnetic rack with an appropriate amount of buffer (such as serum-containing medium or MojoSort™ Buffer) and seed them into culture flasks for subsequent experiments.

[0044] Example 2: CD24 + Functional validation of NPPCs' anti-ferroptosis signaling pathway 1. Western Blot detection of relevant protein expression: An indirect co-culture system was used. Experimental group: NPPCs were co-cultured with LPS-induced degenerated NPCs for 3-4 days; control group: LPS-induced degenerated NPCs were cultured alone in complete medium for the same time. After co-culture, NPCs from each group were collected and washed with PBS. Cells were lysed using RIPA lysis buffer containing 1 mM PMSF. The lysis buffer was centrifuged at 4°C, 12,000 rpm for 30 minutes, and the supernatant (total protein) was collected. Protein concentration was determined using a BCA protein quantification kit. Absorbance was read at 562 nm using a microplate reader, a standard curve was plotted, and the protein concentration of each sample was calculated.

[0045] Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The transferred membrane was blocked with 5% skim milk (dissolved in TBST) at room temperature for 1-2 hours. Primary antibody working solution was prepared using antibody dilution buffer according to the manufacturer's instructions and incubated overnight at 4°C with shaking. The next day, the membrane was washed three times with TBST (5-10 minutes each time). Secondary antibody labeled with HRP of the corresponding species (diluted according to manufacturer's instructions) was added and incubated at room temperature for 1-2 hours. The membrane was washed thoroughly three times with TBST (5-10 minutes each time). The membrane was immersed in ECL chemiluminescence imaging solution for 1-2 minutes, and exposure and image acquisition were performed using a chemiluminescence imaging system. The expression levels of target proteins were analyzed: ferroptosis key proteins (GPX4, SLC7A11), extracellular matrix proteins (Aggrecan, COL2A1), and pro-inflammatory factors (such as IL-1β, IL-6, TNF-α).

[0046] Result: As Figure 1 As shown, compared with the control group, the experimental group (CD24) + In degenerated NPCs (co-cultured with NPPCs), the protein expression levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 were significantly reduced; while the protein expression levels of key extracellular matrix components Aggrecan and COL2A1 were significantly upregulated.

[0047] like Figure 2 As shown, compared with the control group, the experimental group (CD24) + The protein expression levels of GPX4 and SLC7A11 were significantly upregulated in degenerated NPCs (co-cultured with NPPCs), indicating that CD24 + NPPCs effectively activate the antiferroptosis pathway in degenerated cells.

[0048] 2. Measurement of intracellular iron concentration (FerroOrange staining): The above-described indirect co-culture model (experimental group and control group) was used. After co-culture, the culture medium was discarded, and the cells were gently washed three times with pre-warmed HBSS. HBSS working solution containing 1-2 μmol / L FerroOrange probe was added, and the cells were incubated at 37℃ with 5% [presumably a specific concentration]. Incubate in the dark for 25-30 minutes. After incubation, discard the probe solution and wash the cells 2-3 times with HBSS. Immediately observe and quantitatively compare the intracellular iron ion concentrations in the two groups using a confocal microscope. The fluorescence intensity of ).

[0049] Result: As Figure 3 As shown, in the control group cells, LPS-induced intracellular Fe... 2+ The fluorescence intensity was significantly enhanced. And compared to CD24... + The experimental group co-cultured with NPPCs showed intracellular Fe 2+ The fluorescence intensity was significantly reduced, indicating that the accumulation of intracellular ferrous ions, a key indicator of ferroptosis, was effectively inhibited.

[0050] Example 3: CD24 + Animal model validation of NPPCs for treating intervertebral disc degeneration (IVDD) 1. Establishment of a rat IVDD model and cell transplantation: Six- to eight-week-old SPF-grade SD rats were selected and acclimatized for one week. Rats were anesthetized by intraperitoneal injection of tribromoethanol (20 mg / ml, dose 250 mg / kg or 10-15 ml / kg). After tail disinfection, a 21G needle was used to vertically puncture the target intervertebral disc (e.g., Co3 / 4, Co4 / 5, Co5 / 6) to a depth of approximately 3 mm (or penetrate the annulus fibrosus to enter the nucleus pulposus). The needle was rotated 360° and held for 30 seconds before being withdrawn, and hemostasis was achieved by compression. Preliminary experiments confirmed that this method could induce significant disc degeneration two weeks post-surgery.

[0051] Two weeks after the puncture, patients were randomly assigned to two groups: control group (PBS): 10 μL of PBS was injected into the degenerated intervertebral disc; experimental group (CD24...). + NPPCs): Injection of 5× into the degenerated intervertebral disc CD24 + NPPCs were suspended in 10 μL PBS (cell viability >95%). Further analysis was performed 4 weeks after cell transplantation.

[0052] 2. Disc Height Index (DHI) Measurement Four weeks after intervention, rats were anesthetized by intraperitoneal injection of tribromoethanol (20 mg / ml, 250 mg / kg). The anesthetized rats were then fixed onto a Micro-CT scanning table. The Micro-CT scanner was preheated for 15 minutes. Scanning parameters were set as follows: voltage 90 kV, current 88 μA, field of view (FOV) 72 mm, and scan resolution (e.g., 18 μm). The target intervertebral disc was located at the center of the scanning field of view using anatomical landmarks. Scanning was performed and data acquired. Images were reconstructed using the accompanying software, and the disc height index (DHI) of the target intervertebral disc was calculated.

[0053] Result: As Figure 6 As shown, compared with the control group, CD24 + The DHI value was significantly higher in the NPPCs treatment group, indicating that the loss of intervertebral disc height was effectively restored.

[0054] 3. Hematoxylin-eosin (H&E) staining: After micro-CT scanning or at another time point, rats were sacrificed, and target intervertebral disc tissue was collected. The tissue was fixed in 4% paraformaldehyde (PFA) for 48-72 hours. After fixation, the sample was placed in EDTA decalcification solution (or a suitable decalcification solution depending on the tissue), with the solution changed daily until the tissue softened (needle penetration was possible). The decalcification solution was removed by rinsing with running water for 24 hours. Dehydration was performed sequentially with a gradient of ethanol (70% → 80% → 90% → 95% → 100% → 100%, 1-2 hours each). After dehydration, the tissue was cleared with xylene (or an alternative clearing agent) (I → II, 30-60 minutes each) until the tissue was translucent. After clearing, the tissue was immersed in paraffin at 60°C (I → II → III, 1-2 hours each). The tissue was embedded in paraffin and allowed to cool and solidify. Serial sections were prepared using a microtome to a thickness of 4-5 μm. The slides were floated in 40°C warm water, then scooped out and attached to poly-L-lysine-coated glass slides. They were then baked in a 60°C oven for 2 hours or overnight.

[0055] Dewaxing and rehydration are performed sequentially through xylene I and II (10 min each) → anhydrous ethanol I and II (5 min each) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → distilled water (5 min). Hematoxylin staining is applied to the nucleus for 3-5 minutes, followed by rinsing with running water. Differentiation is performed with 1% hydrochloric acid ethanol for a few seconds, followed by rinsing with running water to achieve blue reversion (or using a blue reversion solution). Staining is performed with 0.5-1% eosin aqueous solution for 1-5 minutes, followed by rinsing with running water for 30 seconds. Dehydration and clearing are performed sequentially through 70% ethanol (5 min) → 80% ethanol (5 min) → 90% ethanol (5 min) → 95% ethanol I and II (5 min each) → anhydrous ethanol I and II (5 min each) → xylene I and II (5 min each). The slides are air-dried in a fume hood and mounted with neutral resin. After the slides were left to stand for 24 hours, images of the entire slides were acquired using a digital pathology slide scanner.

[0056] Result: As Figure 4 As shown, the control group (Co4 / 5) exhibited severe damage to the nucleus pulposus structure, a significant reduction in the number of nucleus pulposus cells, and disordered annulus fibrosus. Meanwhile, CD24... + The NPPCs treatment group (Co5 / 6) showed significant improvement in nucleus pulposus structure, increased cell number, and tissue structure closer to normal.

[0057] 4. Immunohistochemical (IHC) staining: Prepare paraffin sections using the same H&E staining steps as described above (until baking is complete). Dewaxing and rehydration are the same as for H&E staining (until distilled water). Immerse sections in preheated sodium citrate antigen retrieval solution (pH 6.0) or EDTA retrieval solution (pH 8.0-9.0), microwave to boiling, and maintain a gentle boil for 15-20 minutes (or autoclave or water bath retrieval). Allow to cool naturally to room temperature. Rinse three times with PBS (pH 7.4), 5 minutes each time. Incubate with 3% H2O2 (prepared with methanol or PBS) at room temperature for 10-15 minutes. Rinse three times with PBS, 5 minutes each time. Add 5-10% normal serum from animals homologous to the secondary antibody (e.g., goat serum) or BSA, and block in a humidified chamber at 37°C for 30 minutes. Aspirate the blocking solution; do not wash.

[0058] Add the primary antibody working solution diluted according to the instructions (diluted with antibody diluent or blocking solution), and incubate overnight in a humidified chamber at 4°C. The next day, allow the slides to warm to room temperature for 30 minutes. Rinse three times with PBS, 5 minutes each time. Add the HRP-labeled secondary antibody (corresponding to the primary antibody species, diluted according to the instructions), and incubate at room temperature or in a humidified chamber at 37°C for 30-60 minutes. Rinse three times with PBS, 5 minutes each time. Add freshly prepared DAB chromogenic working solution, monitor the color development under a microscope (when a brownish-yellow positive signal appears), and rinse with tap water as needed (usually a few seconds to a few minutes) to stop the reaction. Lightly stain the nuclei with hematoxylin for 1-2 minutes, rinse with running water to return to blue (same as H&E). Dehydrate, clear, and mount the slides as per the H&E staining procedure (starting with 70% ethanol). Air dry in a fume hood, mount with neutral resin, let stand for 24 hours, and image using a digital pathology slide scanner.

[0059] Result: As Figure 5 As shown, compared with the control group, CD24 + In the NPPCs treatment group, the positive staining area and intensity of COL2A1 and Aggrecan in the nucleus pulposus tissue were significantly increased, indicating that the synthesis and deposition of extracellular matrix were effectively promoted.

[0060] Conclusion: The above embodiments demonstrate that the present invention successfully isolated and identified functional CD24. + NPPC population. This cell population effectively inhibited LPS-induced ferroptosis in nucleus pulposus cells in vitro, upregulated key anti-ferroptosis proteins, and reduced ferrous ion accumulation; in an in vivo rat intervertebral disc degeneration model, local injection of CD24... + NPPCs can effectively restore intervertebral disc height, improve tissue morphology, and promote the synthesis of extracellular matrix. This indicates that CD24 + NPPCs have great potential for use in the treatment of intervertebral disc degeneration.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An isolated population of CD24-positive notochordal progenitor cells (NPPCs) characterized by, + CD24 The population is prepared by a method comprising the steps of: a. isolating primary nucleus pulposus cells from nucleus pulposus tissue; b. sorting CD24 positive cells from the primary nucleus pulposus cells using an antibody that specifically binds to CD24.

2. The isolated population of CD24-positive notochordal progenitor cells (NPPCs) of claim 1, characterized in that, + NPPCs) of claim 1, wherein the population of NPPCs is characterized in that, The sorting in step b is performed by immunomagnetic bead sorting.

3. A pharmaceutical composition, characterized by, CD24 according to claim 1 or 2 + a population of NPPCs and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein The pharmaceutically acceptable carrier is phosphate buffered saline; Preferably, the CD24 + An effective amount of the population of NPPCs is 5 x 10 4 to 5 x 10 5 cells per administration unit.

5. The CD24 of claim 1 or 2 + Use of the population of NPPCs or the pharmaceutical composition of claim 3 or 4 in the preparation of an agent for inhibiting ferroptosis of nucleus pulposus cells.

6. Use according to claim 5, characterized in that, The inhibiting of ferroptosis in nucleus pulposus cells comprises upregulating GPX4 protein expression and / or SLC7A11 protein expression in nucleus pulposus cells.

7. The CD24 of claim 1 or 2 + Use of the population of NPPCs or the pharmaceutical composition of claim 3 or 4 in the preparation of an agent for ameliorating the degenerative microenvironment of the intervertebral disc.

8. The use of claim 7, wherein the ameliorating of intervertebral disc degeneration microenvironment comprises reducing the level of pro-inflammatory factors selected from one or more of TNF-a, IL-1b, and IL-6.

9. A CD24 for non-therapeutic purposes + In vitro use of a population of NPPCs, characterized in that it is for The use of claim 7, wherein the intervertebral disc degeneration is studied in an experimental model or a therapeutic drug is screened, the use comprising: The CD24 + NPPC populations were co-cultured with degenerated nucleus pulposus cells in vitro; detecting at least one indicator associated with intervertebral disc degeneration.

10. The CD24 for non-therapeutic purposes according to claim 9 + In vitro use of a population of NPPCs, characterized in that, The indicator associated with intervertebral disc degeneration is selected from one or more of: the level of cellular ferroptosis, the level of GPX4 / SLC7A11 protein expression, the level of Aggrecan / COL2A1 protein expression, the level of inflammatory factors TNF-a / IL-1b / IL-6.

Images