Evaluation method for ferroptosis sensitivity of Schwann cells of NF2-related vestibular nerve sheath tumor

Abstract

The invention relates to a method for evaluating ferroptosis sensitivity of NF2-related vestibular nerve sheath tumor Schwann cells. The method comprises the following steps: constructing NF2-mutated immortalized Schwann cells; carrying out ferroptosis induction and intervention experiments, and establishing a plurality of experiment groups; detecting ROS level, Fe < 2 + > concentration, GSH content, MDA level and protein expression of GPX4 and the like of each group of cells; sample preparation is carried out through a TEM method, and mitochondrial volume and morphological characteristics of each group of cells in a ferroptosis state are observed; detecting the cell viability, cell membrane damage and apoptosis proportion of each group of cells; and evaluating the influence of NF2 deletion on ferroptosis sensitivity. According to the method, a ferroptosis cell modeling scheme in which Erastin (an inducer) and Fer-1 (an inhibitor) are jointly intervened is designed, and a multi-dimensional ferroptosis evaluation system is combined, so that the stability and the result reproducibility of NF2 related vestibular nerve sheath tumor Schwann cell ferroptosis state evaluation are remarkably improved.

Description

Technical Field

[0001] This application relates to the fields of cell biology and molecular medicine, and in particular to a method for assessing the ferroptosis sensitivity of Schwann cells associated with neurofibromatosis type 2 (NF2) vestibular schwannoma and a related ferroptosis sensitivity detection kit. Background Technology

[0002] Neurofibromatosis type 2 (NF2) is an autosomal dominant inherited disease caused by germline mutations in the NF2 gene. Clinically, it is characterized by bilateral acoustic neuromas (vestibular schwannomas), often accompanied by other neurofibromas, meningiomas, and other nervous system tumors. Its pathogenesis is closely related to the loss of function of the NF2 gene-encoded product—Merlin protein (also known as a neuromembrane protein). Merlin protein, as a key tumor suppressor, participates in the regulation of cell proliferation, differentiation, and apoptosis by modulating the Hippo signaling pathway and the mTOR signaling pathway. Abnormal function of Merlin protein can lead to uncontrolled cell growth and tumor formation.

[0003] In recent years, ferroptosis, as a novel iron-dependent programmed cell death mechanism, has become a research hotspot in life sciences and medicine due to its core characteristic of abnormal accumulation of lipid peroxidation products and its close association with the development and progression of various tumors, resistance to radiotherapy and chemotherapy, and neurodegenerative diseases. The regulatory network of ferroptosis involves multiple key factors, including glutathione peroxidase 4 (GPX4, a key inhibitor of lipid peroxidation) and systemic Xc... - The ferroptosis pathway, composed of SLC7A11 and SLC3A2, is responsible for cysteine ​​uptake to maintain glutathione synthesis. Other pathways include acyl-CoA synthase family member 4 (ACSL4, which promotes polyunsaturated fatty acid esterification and participates in lipid peroxidation) and transferrin receptor (TFRC, which mediates iron uptake). Previous studies have confirmed that regulating the ferroptosis pathway (Erastin-induced ferroptosis) can significantly affect the sensitivity of various tumor cells, such as glioma, liver cancer, and breast cancer, to chemotherapy and radiotherapy, providing a new strategy for tumor treatment. However, these studies have not systematically evaluated its responsiveness and sensitivity in NF2 cells. Furthermore, the specific mechanism of ferroptosis in NF2-related vestibular schwannomas, especially its regulatory relationship with Merlin protein deficiency, remains unclear.

[0004] Meanwhile, current research on NF2-related vestibular schwannomas also faces model and technical bottlenecks: On the one hand, no stable and reliable humanized NF2 model suitable for ferroptosis research has been established domestically or internationally. Existing commonly used cell models (such as HEI193 cells), although derived from NF2 patients, still retain some Merlin protein expression and have complex genetic backgrounds, making it difficult to accurately simulate the complete loss of Merlin function caused by germline mutations in the NF2 gene. On the other hand, research on the application of ferroptosis inducers (such as Erastin, RSL3, etc.) in vestibular schwannomas is extremely limited, and there is a lack of research on iron homeostasis (such as Fe²⁺) targeting the NF2 background. + A systematic quantitative assessment scheme for lipid peroxidation levels (such as malondialdehyde (MDA) and glutathione (GSH) content) and reactive oxygen species (ROS) levels is needed. Furthermore, existing research on ferroptosis often focuses on single-indicator detection (such as detecting ROS alone for oxidative stress studies, Fe...). 2+ The detection methods (used in neurodegenerative disease models, etc.) lack standardized system protocols for joint validation of multidimensional indicators in the same experimental system, and the existing indicator detections have not been biologically validated in the NF2 background; at the same time, research on the association between Merlin protein loss and the ferroptosis pathway is limited in exploring molecular mechanisms because a stable passaged human immortalized cell model has not yet been established.

[0005] In summary, current research primarily focuses on the relationship between NF2 gene mutations and cell proliferation and apoptosis, while insufficient research is conducted on the regulation of the ferroptosis pathway in the context of NF2 deficiency. There is a lack of stable NF2 cell models for in vitro functional assessment and ferroptosis sensitivity quantification protocols, hindering the effective evaluation of the mechanisms by which iron homeostasis, lipid peroxidation, and ROS levels affect NF2-related diseases. Furthermore, there is a lack of intervention pathways and quantitative detection techniques that can simulate actual pathological states. Therefore, given the gaps in research on the regulatory mechanisms of ferroptosis and its correlation with Merlin protein function in NF2-related vestibular schwannomas, it is urgent to establish stable and reliable NF2 cell models and develop a multi-indicator combined ferroptosis sensitivity assessment system to deeply elucidate the role of ferroptosis in the development and progression of NF2-related tumors, providing theoretical basis and technical support for targeted therapy and the screening and development of related drugs. Summary of the Invention

[0006] To address the aforementioned issues, this application proposes a novel method for assessing the sensitivity of Schwann cells in NF2-associated vestibular schwannomas to ferroptosis.

[0007] Specifically, this application provides the following technical solutions: In a first aspect, this application provides a method for assessing the sensitivity of Schwann cells in NF2-related vestibular schwannomas to ferroptosis, comprising the following steps: S1. Construct immortalized Schwann cells with NF2 mutations, the name of which is NF2-VS-03 (human NF2-associated acoustic neuroma immortalized cell line), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46597; S2. Ferrapoptosis induction and intervention experiment: Logarithmic growth phase NF2-VS-03 cells were seeded in 6-well plates, 1×10⁶ cells per well. 5 Cells were cultured for 24 hours until adherence was achieved; Erastin group, Fer-1 group, combined group and DMSO control group were established; S3. Detection of molecular markers of ferroptosis: The levels of ROS and Fe in each group of cells were measured. 2+ Concentration, GSH content, MDA level, and protein expression of GPX4, ACSL4, PCBP1, and E-cadherin; S4. Observation of mitochondrial ultrastructure: Samples were prepared using TEM to observe the volume and morphological characteristics of mitochondria in each group under ferroptosis. S5. Functional Response Experiment: Cell viability, cell membrane damage, and apoptosis rate were detected in each group of cells. S6. Statistical Analysis: Two-way ANOVA and t-tests were performed using GraphPad Prism to assess the impact of NF2 deficiency on ferroptosis sensitivity.

[0008] Furthermore, the Merlin protein expression in the NF2-mutant immortalized Schwann cells constructed in step S1 of the evaluation method of this application is completely absent.

[0009] Furthermore, the construction of NF2-mutant immortalized Schwann cells described in step S1 of the evaluation method of this application includes the following steps: Fresh tumor tissue from acoustic neuroma was selected; The obtained tumor tissue was cleaned and cut. Add a digestion solution containing 0.125% trypsin and 0.1% type II collagenase to the cut tumor tissue. The volume ratio of tumor tissue to digestion solution is 1:1. Digest at 37°C for 30 minutes, and then stop digestion. After digestion was terminated, the cells were centrifuged at 1000 rpm for 5 minutes, filtered, resuspended in complete culture medium, and seeded into T25 culture flasks pre-coated with Laminin for culture until an adherent cell population was formed. Lentiviral vectors containing SV40 T antigen and puromycin resistance gene were constructed and primary cells adhered to the cell wall for 24 hours were infected with MOI=20. The medium was changed after 12 hours, and 2 μg / ml puromycin was added after 48 hours for selection. The process lasted for 7 days. Clones with good activity were selected and passaged continuously. The absence of Merlin protein expression was verified by both qRT-PCR and Western blot.

[0010] Furthermore, the detection of ferroptosis molecular markers in step S3 of the evaluation method of this application includes: ROS level detection: The cells were stained with DCFH-DA fluorescent probe, incubated at 37°C for 30 minutes, washed with PBS, and the fluorescence intensity was detected by flow cytometry. Fe 2+ Concentration detection: Phen Green SK probe or FeRhoNox-1 probe, flow cytometry or colorimetric method for quantification; GSH content detection: The total glutathione content was detected by the DTNB method, and the colorimetric reading was performed. MDA level detection: MDA level is detected using the TBA method or the C11-BODIPY probe; Protein level detection: Total cell protein was extracted using RIPA lysis buffer, separated by SDS-PAGE, and the expression of GPX4, ACSL4, PCBP1, and E-cadherin proteins was detected by Western blot.

[0011] Furthermore, the sample preparation by TEM method described in step S4 of the evaluation method of this application includes: first fixing the cells in 2.5% glutaraldehyde for 2 hours, then replacing it with 1% osmium tetroxide for fixation, dehydrating with graded ethanol, embedding in epoxy resin, sectioning, and staining.

[0012] Furthermore, the functional responsiveness experiment described in step S5 of the evaluation method of this application includes: Cell viability assay: Cell viability was determined using a combination of CCK-8 assay and colorimetric method. Cell membrane damage: Cell membrane damage was detected using the LDH release assay; Apoptosis rate: The apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry.

[0013] Furthermore, the ferroptosis induction and intervention experiment described in step S2 of the evaluation method of this application also includes the use of the following ferroptosis intervention reagents: RSL3 (GPX4 irreversible inhibitor), FIN56 (GPX4 degradation promoter) or Sulfasalazine (SLC7A11 inhibitor), wherein RSL3 is suitable for high stress simulation.

[0014] Secondly, this application provides a detection kit for the sensitivity of Schwann cells in NF2-related vestibular schwannoma to ferroptosis, the detection kit comprising: Immortalized Schwann cells with NF2 mutations; ROS detection module: DCFH-DA fluorescent probe, PBS buffer, fluorescent calibration solution; Fe 2+ Concentration detection module: Phen Green SK probe or FeRhoNox-1 fluorescent probe, colorimetric detection standard curve solution (Fe... 2+ (Standard solution); MDA level detection module: TBA (thiobarbituric acid) reaction reagent, MDA standard (malondialdehyde solution); GSH content detection module: DTNB reaction solution, total GSH reduction buffer (containing NADPH and glutathione reductase); Positive drug intervention module: Erastin (inducer), Fer-1 (inhibitor), DMSO diluent.

[0015] Furthermore, the immortalized Schwann cells with the NF2 mutation included in the detection kit of this application are named NF2-VS-03, which are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46597 and deposit date of September 22, 2025.

[0016] Furthermore, the Merlin protein expression in the immortalized Schwann cells with NF2 mutations included in the detection kit of this application is completely absent.

[0017] In summary, the method for assessing the sensitivity of Schwann cells in NF2-related vestibular schwannomas according to this invention has the following advantages: (1) To assess and regulate the ferroptosis sensitivity of Schwann cells in NF2-related vestibular schwannomas, this invention successfully constructed an NF2-deficient immortalized cell model (NF2-VS-03 cell line). This cell model is the first domestic and international immortalized vestibular schwannomas cell model with a completely Merlin-deficient background, clear origin, and human specificity, completely solving the problems of residual Merlin expression and complex gene background in traditional HEI193 cells. This cell model can be used to simulate the abnormal iron homeostasis of vestibular schwannomas in neurofibromatosis (NF2), providing a standardized tool for the study of NF2 tumor mechanisms and is of great significance for studying the pathogenesis of NF2-related tumors. In addition, the cell platform and ferroptosis pathway intervention process constructed in this invention can be used to develop personalized intervention strategies and screen ferroptosis-targeting drugs suitable for NF2 patients, providing tools for translational medicine research.

[0018] (2) Design a dynamic ferroptosis intervention modeling scheme and propose a cell modeling method that combines Erastin (inducer) and Fer-1 (inhibitor) to break through the limitations of traditional single induction, accurately simulate the dynamic evolution process of ferroptosis from initiation to termination, and better fit the pathophysiological characteristics of neurotumor.

[0019] (3) A multi-dimensional ferroptosis evaluation system is proposed, which includes ROS (reactive oxygen species), Fe... 2+ The quadruple quantitative evaluation index system of free ferrous iron, GSH (reduced glutathione), and MDA (malondialdehyde), combined with the dual pathways of flow cytometry (high-throughput) and colorimetry (routine detection), significantly improves the stability and reproducibility of ferroptosis status assessment.

[0020] (4) The morphology-molecular correlation mechanism was expanded. Typical morphological changes of ferroptosis, such as mitochondrial volume reduction and cristae destruction in NF2-deficient cells, were observed by transmission electron microscopy (TEM). For the first time, these changes were directly associated with GSH depletion and PCBP1 (polycytosine-binding protein 1) expression imbalance, which deepened the research dimension of ferroptosis mechanism.

[0021] (5) It provides theoretical support for targeted therapy. At the molecular level, it is confirmed that NF2 deficiency leads to the downregulation of PCBP1 expression by inhibiting PCBP1 transcription (NF2 protein directly regulates the PCBP1 promoter), which in turn causes iron metabolism disorder, increased lipid peroxidation and cell damage. This provides a theoretical basis and experimental foundation for individualized treatment targeting molecules such as GPX4 and PCBP1.

[0022] Other features and advantages of this application will be set forth in detail in the following description, or will become apparent through the implementation of the relevant technical solutions of this application. The objectives and other advantages of this application can be achieved through the technical features and means explicitly pointed out in the description, claims, and drawings, and will be obtained through the implementation of these technical contents. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings involved in the description of the embodiments will be briefly introduced below. It should be noted that the accompanying drawings only show some embodiments of this application. For those skilled in the art, other related drawings can be derived from these drawings without creative effort.

[0024] Figure 1 The image shows an immunofluorescence pattern of S100 staining on cells from the third passage of two groups of NF2-VS-03 cells in this embodiment. The cells are S100 positive, confirming that they are of schwannoma cell origin.

[0025] Figure 2The image shown is a TEM observation result from this embodiment, which shows that the mitochondria in the NF2 group exhibit a typical ferroptosis phenotype (reduced volume, membrane rupture, cristae collapse), while the mitochondria in the control group have intact structures.

[0026] Figure 3 This is a graph showing the fluorescence intensity changes detected by ROS in this embodiment.

[0027] Figure 4 In this embodiment, Fe 2+ Concentration detection results graph.

[0028] Figure 5 This is a graph showing the GSH / MDA level detection results in this embodiment.

[0029] Figure 6 This is the Western blot of the protein in this embodiment.

[0030] Figure 7 The figure shows the results of cell viability (CCK-8) and LDH release experiments in this embodiment.

[0031] Figure 8 This is a flow cytometry plot of Annexin V-FITC / PI apoptosis in this embodiment.

[0032] Figure 9 This is a flowchart illustrating the overall implementation process of the method of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the protection scope of this application.

[0034] In this document, the term "comprising" and any variations thereof (such as "including," "including," etc.) are open-ended expressions and should be understood as "including but not limited to," meaning that the listed content is not exhaustive and may include other content not explicitly mentioned. The term "based on" should be understood as "at least partially based on," meaning that the basis or condition referred to may not be the only factor and may involve other relevant factors. The term "one embodiment" should be understood as "at least one embodiment," meaning that the described embodiment is not the only possible implementation, and other similar embodiments may exist.

[0035] In this application, the terms "a" and "a plurality of" are used to modify related elements or features, and their expression is illustrative rather than restrictive. Unless otherwise expressly stated in the context, "a" should be understood as "at least one," and "a plurality of" should be understood as "at least two." Those skilled in the art should reasonably interpret these terms based on the semantic and logical relationships of the context to ensure that they cover the possibility of "one or more."

[0036] Example: A method for assessing the sensitivity of Schwann cells in NF2-associated vestibular schwannomas to ferroptosis ( Figure 9 ) This method includes the following steps: (a) Construction of immortalized Schwann cells with NF2 mutation (NF2-VS-03) Source tissue: Fresh tumor tissue from patients who underwent surgery for sporadic acoustic neuroma with ethical approval; Primary culture: Wash the tumor tissue, then cut the tissue into 1 mm pieces. 3 To determine the size, digest the sample using a digestive solution containing 0.125% trypsin and 0.1% type II collagenase (volume ratio 1:1) at 37°C for 30 minutes, gently mixing by blowing and cooling during digestion. Cell adhesion: After digestion was terminated, the cells were centrifuged at 1000 rpm for 5 minutes, resuspended in complete culture medium, and seeded into T25 culture flasks pre-coated with Laminin. Lentiviral infection: A lentiviral vector containing the SV40 T antigen (containing the puromycin resistance gene) was constructed and used to infect primary cells adherent to the culture medium for 24 hours at an MOI of 20; the medium was changed after 12 hours, and 2 μg / ml puromycin was added after 48 hours for selection, which lasted for 7 days. Clone screening and validation: Clones with good activity were selected for continuous passage, and the absence of Merlin protein expression was verified by both qRT-PCR and Western blot. Nomenclature and Preservation: This cell line was named NF2-VS-03. It can be stably passaged for more than 20 generations, exhibits a stable cell growth curve, and demonstrates good morphological uniformity and biological reproducibility. Figure 1 ).

[0037] (II) Experiments on Ferric Death Induction and Intervention Cell seeding: Logarithmic growth phase NF2-VS-03 cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. 5 One cell was cultured for 24 hours until it adhered to the culture vessel. Drug intervention groups: Erastin group (5 μM, ferroptosis inducer), Fer-1 group (32 μM, ferroptosis inhibitor), combination group (Erastin + Fer-1) and DMSO control group were set up, with duplicate wells in each group; Intervention time: uniform treatment for 24 hours to ensure that the drug acts completely on the iron homeostasis pathway and lipid peroxidation chain; Drug solution preparation: Both Erastin and Fer-1 are prepared using DMSO as the stock solution, and the DMSO content in the final concentration does not exceed 0.1% to avoid solvent interference.

[0038] In addition, other ferroptosis inducers can be selected in this scheme, such as RSL3 (an irreversible inhibitor of GPX4), FIN56 (which promotes GPX4 degradation), and Sulfasalazine (which inhibits SLC7A11). Among them, RSL3 has the strongest effect and is suitable for high stress simulation.

[0039] (III) Detection of molecular indicators of ferroptosis ROS detection: The cells were stained with DCFH-DA fluorescent probe (10 μM), incubated at 37°C for 30 minutes, washed three times with PBS, and the fluorescence intensity was detected by flow cytometry. Fe 2+ Concentration detection: Quantification was performed using a Phen Green SK probe (5 μM) by flow cytometry or colorimetric method (OD 510 nm); In addition to Phen Green SK, Fe... 2+ Concentration detection can also be performed using the iron ion fluorescent dye FeRhoNox-1 in conjunction with a laser confocal microscope to enhance subcellular localization capabilities; GSH level detection: Total glutathione was detected using a GSH detection kit (DTNB method), with colorimetric readings at 450nm; MDA assay: MDA was detected using the thiobarbituric acid (TBA) method, and the extract was analyzed colorimetrically at 532 nm. In addition to the TBA method, the C11-BODIPY (581 / 591) probe can be introduced for flow cytometry detection. Its advantages are high specificity, dynamic and real-time detection, and suitability for live cell labeling. Protein level detection: Total cell protein was extracted using RIPA lysis buffer, separated by SDS-PAGE, and the expression changes of GPX4, E-cadherin, PCBP1, and ACSL4 were detected by Western blotting.

[0040] Figures 3-5 The results of multi-index detection are shown, including changes in ROS fluorescence intensity and Fe. 2+ Concentration fluctuations and GSH / MDA contrastive expression levels.

[0041] As shown in the figure, compared with the DMSO control, Erastin treatment significantly increased MDA levels in both the VS and NF2 groups, with a greater increase in the NF2 group (p<0.01). Furthermore, the MDA level in the NF2 group was significantly higher than that in the VS group (p<0.05), indicating more severe lipid peroxidation damage. The VS group showed some increase in MDA levels, but this difference was not statistically significant compared to the control group. After adding the ferroptosis inhibitor Fer-1, the MDA levels in both groups decreased.

[0042] GSH assay results showed that Erastin treatment significantly reduced GSH levels in both the VS and NF2 groups, with a more pronounced decrease in the NF2 group (p<0.01), indicating that their antioxidant capacity was more easily impaired. The addition of Fer-1 partially reduced MDA and increased GSH levels, suggesting that the ferroptosis pathway plays an important role in this process. In summary, the NF2 group was more prone to enhanced lipid peroxidation and GSH depletion under Erastin stimulation, leading to greater cellular damage, while Fer-1 intervention could alleviate this process to some extent.

[0043] Intracellular ROS levels were significantly elevated in both the VS and NF2 groups (p<0.01), but there was no statistically significant difference between the two groups. After the addition of the ferroptosis inhibitor Fer-1, ROS levels decreased in both groups, but failed to return to normal levels.

[0044] Fe 2+ Fluorescent probe detection results showed that, compared with the DMSO control, Erastin treatment significantly increased intracellular Fe in the NF2 group. 2+ The content (p<0.01) of Fe in the two groups after the addition of the ferroptosis inhibitor Fer-1 was significantly reduced. 2+ While the levels of iron ions decreased, the NF2 group still showed higher levels than the VS group. This result suggests that under Erastin-induced oxidative stress, the NF2 group is more prone to iron ion accumulation, thus exacerbating ferroptosis; while Fer-1 can partially alleviate this iron overload. Combined with previous measurements of GSH, MDA, and other indicators, this further demonstrates that NF2 is closely related to the ferroptosis pathway, and iron ion accumulation is a key characteristic.

[0045] Figure 6This is a Western blot analysis of proteins, including the relative expression levels and semi-quantitative statistical results of GPX4, PCBP1, and E-cadherin in different treatment groups. Western blot results showed significant changes in each protein under different treatment conditions. Comparing Erastin treatment and the DMSO control in each group, in the VS group, Lats1 protein was significantly upregulated, GPX4 was significantly downregulated, while E-cadherin and PCBP1 showed no significant changes. In the NF2 group, Lats1 was also significantly upregulated, while GPX4 and PCBP1 were significantly downregulated. Notably, after Erastin treatment, the comparison between the two groups revealed that the expression levels of Lats1 and ACSL4 in the NF2 group were significantly higher than in the VS group, and the downregulation of E-cadherin, GPX4, and PCBP1 was also more significant. This indicates that under Erastin-induced conditions, cells in the NF2 group are more sensitive to the regulation of related proteins, with a stronger response in the expression of Lats1, ACSL4, and PCBP1 compared to the VS group, while E-cadherin and GPX4 were further reduced. After the addition of the ferroptosis inhibitor Fer-1, the expression levels of each protein tended to partially recover, but the overall trend of change still existed, indicating that the regulation of protein expression after Erastin treatment differed significantly among different groups.

[0046] (iv) Observation of mitochondrial ultrastructure TEM sample preparation: Cells were fixed in 2.5% glutaraldehyde for 2 hours, then replaced with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, sectioned, and stained. Observation content: Under typical ferroptosis, mitochondria exhibit characteristics such as increased membrane density, membrane rupture, cristae collapse, and reduced volume. The differences between the NF2 group and the control group were compared. Figure 2 ).

[0047] (v) Functional Response Experiment: Cell viability: The absorbance was measured at 450 nm using the CCK-8 reagent to reflect changes in cell metabolic activity. LDH release: Detecting the content of lactate dehydrogenase in culture supernatant reflects the degree of damage to cell membrane integrity; Apoptosis analysis: Annexin V-FITC / PI double staining was used to detect the proportion of early and late apoptosis by flow cytometry.

[0048] (vi) Data statistical analysis: We used GraphPad Prism for two-way ANOVA and between-group t-tests to assess the effect of NF2 deficiency on ferroptosis sensitivity.

[0049] Figure 7Cell viability (CCK-8) and LDH release curves were displayed, showing that Erastin-induced cell damage was more significant in the NF2 group. CCK-8 assays showed that cell viability decreased in both the VS and NF2 groups with increasing Erastin concentration (5 μM, 10 μM), but at the same concentration, the viability of the NF2 group was significantly lower than that of the VS group (p<0.05). A significant difference was observed at the 5 μM concentration compared to the control group, and this concentration was used in subsequent experiments. It should be noted that a large number of NF2 cells induced by 5 μM Erastin died in subsequent experiments, suggesting that the NF2 group is more sensitive to Erastin-induced cell damage. LDH assays also confirmed this: after Erastin treatment, LDH release in the NF2 group was significantly higher than that in the VS group (p<0.01), indicating more severe disruption of cell membrane integrity. In the control group, there was no significant difference in cell viability and LDH release between the two groups. In conclusion, the NF2 group is more prone to decreased viability and increased cytotoxicity under Erastin stimulation. In addition, in the group that added the ferroptosis inhibitor Fer-1, tumor cells were cultured by co-incubating Fer-1 and Erastin, and Fer-1 was added 1 hour before Erastin induction. When comparing within the group, it can be seen that although the ferroptosis process was improved by adding Fer-1 (p<0.05), it was not completely offset.

[0050] Figure 8 The Annexin V-FITC / PI apoptosis flow cytometry plot visually shows the increased apoptosis rate in the NF2 group after Erastin treatment. Flow cytometry apoptosis detection results showed that, compared with the DMSO control, Erastin treatment significantly increased the proportion of early and late apoptotic cells in the NF2 group (p<0.01). After adding the ferroptosis inhibitor Fer-1, the apoptosis rate decreased in both groups, but the NF2 group was still higher than the VS group, suggesting that the NF2 group was more prone to apoptosis in Erastin-induced cell damage.

[0051] While the foregoing discussion contains several specific implementation details, these details should not be construed as limiting the scope of this application. The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of this application is not limited to technical solutions formed by specific combinations of the above-described technical features. Furthermore, this application should also cover other technical solutions formed by any combination of the above-described technical features or their equivalents without departing from the foregoing disclosed concept.

[0052] Those skilled in the art should also understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features, without departing from the spirit and scope of the technical solutions of the embodiments of this application. These modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the core spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for assessing the sensitivity of Schwann cells in NF2-associated vestibular schwannoma to ferroptosis, characterized in that, The method includes the following steps: S1. Construct immortalized Schwann cells with NF2 mutation, the name of which is NF2-VS-03, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.46597; S2. Ferrapoptosis induction and intervention experiment: Logarithmic growth phase NF2-VS-03 cells were seeded in 6-well plates, 1×10⁶ cells per well. 5 Cells were cultured for 24 hours until adherence was achieved; Erastin group, Fer-1 group, combined group and DMSO control group were established; S3. Detection of molecular markers of ferroptosis: The levels of ROS and Fe in each group of cells were measured. 2+ Concentration, GSH content, MDA level, and protein expression of GPX4, ACSL4, PCBP1, and E-cadherin; S4. Observation of mitochondrial ultrastructure: Samples were prepared using TEM to observe the volume and morphological characteristics of mitochondria in each group under ferroptosis. S5. Functional Response Experiment: Cell viability, cell membrane damage, and apoptosis rate were detected in each group of cells. S6. Statistical Analysis: Two-way ANOVA and t-tests were performed using GraphPad Prism to assess the impact of NF2 deficiency on ferroptosis sensitivity.

2. The method according to claim 1, characterized in that, Merlin protein expression was completely absent in the NF2-mutant immortalized Schwann cells constructed in step S1.

3. The method according to claim 2, characterized in that, The construction of immortalized Schwann cells with NF2 mutations described in step S1 includes the following steps: Fresh tumor tissue from acoustic neuroma was selected; The obtained tumor tissue was cleaned and cut. Add a digestion solution containing 0.125% trypsin and 0.1% type II collagenase to the cut tumor tissue. The volume ratio of tumor tissue to digestion solution is 1:

1. Digest at 37°C for 30 minutes, and then stop digestion. After digestion was terminated, the cells were centrifuged at 1000 rpm for 5 minutes, filtered, resuspended in complete culture medium, and seeded into T25 culture flasks pre-coated with Laminin for culture until an adherent cell population was formed. Lentiviral vectors containing SV40 T antigen and puromycin resistance gene were constructed and primary cells adhered to the cell wall for 24 hours were infected with MOI=20. The medium was changed after 12 hours, and 2 μg / ml puromycin was added after 48 hours for selection. The process lasted for 7 days. Clones with good activity were selected and passaged continuously. The absence of Merlin protein expression was verified by both qRT-PCR and Western blot.

4. The method according to claim 1, characterized in that, The detection of ferroptosis molecular indicators in step S3 includes: ROS level detection: The cells were stained with DCFH-DA fluorescent probe, incubated at 37°C for 30 minutes, washed with PBS, and the fluorescence intensity was detected by flow cytometry. Fe 2+ Concentration detection: Phen Green SK probe or FeRhoNox-1 probe, flow cytometry or colorimetric method for quantification; GSH content detection: The total glutathione content was detected by the DTNB method, and the colorimetric reading was performed. MDA level detection: MDA level is detected using the TBA method or the C11-BODIPY probe; Protein level detection: Total cell protein was extracted using RIPA lysis buffer, separated by SDS-PAGE, and the expression of GPX4, ACSL4, PCBP1, and E-cadherin proteins was detected by Western blot.

5. The method according to claim 1, characterized in that, The sample preparation by TEM method described in step S4 includes: first fixing the cells in 2.5% glutaraldehyde for 2 hours, then replacing it with 1% osmium tetroxide for fixation, dehydrating with graded ethanol, embedding in epoxy resin, sectioning, and staining.

6. The method according to claim 1, characterized in that, The functional responsiveness experiment described in step S5 includes: Cell viability assay: Cell viability was determined using a combination of CCK-8 assay and colorimetric method. Cell membrane damage: Cell membrane damage was detected using the LDH release assay; Apoptosis rate: The apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry.

7. The method according to claim 1, characterized in that, The ferroptosis induction and intervention experiment described in step S2 also includes the use of the following ferroptosis intervention reagents: RSL3, FIN56 or Sulfasalazine, wherein RSL3 is suitable for high stress simulation.

8. A detection kit for the sensitivity of Schwann cells in NF2-associated vestibular schwannoma to ferroptosis, characterized in that, The test kit includes: Immortalized Schwann cells with NF2 mutations; ROS detection module: DCFH-DA fluorescent probe, PBS buffer, fluorescent calibration solution; Fe 2+ Concentration detection module: Phen Green SK probe or FeRhoNox-1 fluorescent probe, colorimetric detection standard curve solution; MDA level detection module: TBA reaction reagent, MDA standard; GSH content detection module: DTNB reaction solution, total GSH reduction buffer; Positive drug intervention module: Erastin, Fer-1, DMSO dilution.

9. The detection kit according to claim 8, characterized in that, The immortalized Schwann cell with the NF2 mutation is named NF2-VS-03 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46597.

10. The detection kit according to claim 8, characterized in that, The Merlin protein expression in the immortalized Schwann cells with the NF2 mutation was completely absent.

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