NF2-related auditory neuroma immortalized cell strain as well as construction method and application thereof

Abstract

The invention relates to an NF2-related auditory neuroma immortalized cell strain as well as a preparation method and application thereof. The name of the cell strain is NF2-VS-03, the cell strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46597. As a standardized human-derived Schwann cell model which is clear in source, complete in Merlin protein deletion, stable in passage and sensitive to ferroptosis inducers, the cell strain can be used for mechanism research and drug screening of NF2-related auditory neuroma, and is particularly suitable for ferroptosis pathway target screening, action mechanism analysis and precise drug screening. The cell strain provided by the invention overcomes the defects of insufficient traceability and phenotype accuracy of the existing model cell source, residual Merlin expression, unstable drug response, non-passage or serious drifting, lack of clinical treatment conversion docking capability and the like, and has wide development and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell engineering and neuro-oncology model construction, and particularly relates to a neurofibromatosis type 2 (NF2) related acoustic neuroma immortalized cell line and a construction method and application thereof. BACKGROUND

[0002] In the field of neuro-oncology research, especially in the mechanism exploration and drug screening of vestibular schwannoma (VS), cell model construction is the core basis. In particular, in the research of neurofibromatosis type 2 (NF2) related acoustic neuroma, since the disease has a clear genetic background (NF2 gene mutation leads to loss of function of Merlin protein), therefore, constructing a cell model with high accuracy, stable passage and high similarity to clinical pathology is of great significance for in-depth elucidation of pathogenesis and screening of targeted drugs.

[0003] At present, the cell model technology system related to NF2 mainly includes the following types, but all have significant defects, which are difficult to meet the deep needs of NF2 related acoustic neuroma research: (1) Public cell line HEI-193: This cell line is derived from an NF2 patient and obtained by SV40 T antigen transformation. Although it carries the NF2 mutation background, the expression of Merlin protein is not completely lost; long-term in vitro culture leads to significant phenotype drift, and the response to ferroptosis inducers is unstable. In addition, its source is single, and lacks traceable histological verification, which limits the clinical comparability and reproducibility.

[0004] (2) CRISPR / Cas9 knockout model: NF2 or Merlin gene is knocked out in the established cell line by gene editing technology, which can theoretically achieve precise knockout. However, this technology is complex and costly, and the gene introduction efficiency and growth stability in Schwann cells are poor; most models are artificially modified backgrounds, and there are significant differences in transcriptome characteristics and actual pathological tissues, which are difficult to reflect the real pathological state.

[0005] (3) Cross-species animal cell line model: mouse or rat-derived Schwann cell lines (such as RT4-D6P2T, RSC96, etc.) are commonly used to simulate NF2 function. However, the differences in genetic background, translation regulation mechanism and drug targets between species greatly limit the translational value of preclinical research.

[0006] (4) Selection of immortalization system: although SV40 T antigen is widely used for cell immortalization, it lacks a supporting phenotype verification system and is prone to cause abnormal phenotypes; E6 / E7 and hTERT systems have the risk of dedifferentiation, especially in Schwann cells, which can easily activate unintended signaling pathways and affect the reliability of the model.

[0007] In summary, the current NF2-related acoustic neuroma research, the core bottleneck of cell model is reflected in the following aspects: Lack of traceability and accurate phenotype of cell source: lack of fresh surgical tissue from sporadic or genetic patients, and fully identified NF2 knockout cell lines; existing models are mostly animal-derived or artificially modified, which is difficult to truly reflect the cell behavior under the NF2 mutation background.

[0008] Merlin expression remains and protein function is not completely inactivated: some cell lines still have residual Merlin protein, which leads to confusion in experimental results.

[0009] Drug response is unstable: the existing model has inconsistent response to ferroptosis inducers (such as Erastin, RSL3), which cannot effectively support ferroptosis mechanism research and precise drug screening.

[0010] Poor passage stability: most cell lines are difficult to be stably passaged or have serious phenotype drift, which limits long-term experimental application.

[0011] Lack of clinical translation docking ability: lack of in vitro and in vivo evaluation platform compatible with clinical treatment strategies, which is difficult to verify the actual effect of drug intervention.

[0012] It is particularly pointed out that the inventors found in the research process that although some NF2-related cell models have been developed, for example, Chinese patent CN109486765B discloses a method for establishing an NF2 vestibular schwannoma Schwann cell line and its cell line; Chinese patent CN110904051B discloses a human acoustic neuroma immortalized cell line, a preparation method and its application; Chinese patent application CN117660356A discloses an NF2 type neurofibromatosis-related meningioma immortalized cell line and its construction method and application, but the above-mentioned cell lines have not been tested for sensitivity to ferroptosis inducers during construction, so they cannot be directly used for ferroptosis pathway target screening, mechanism analysis, and precise drug screening and development.

[0013] In summary, it is urgent to construct a standardized human Schwann cell model with clear source, complete loss of Merlin expression, stable passage, especially suitable for ferroptosis research and precise drug screening, to break through the bottleneck of the prior art and promote the mechanism research and clinical translation of NF2-related acoustic neuroma. SUMMARY

[0014] In order to solve the above problems, the present application provides a new human-derived acoustic neuroma immortalized cell line with the characteristics of NF2 gene deletion and sensitivity to ferroptosis inducers, as well as a construction and verification method thereof. The cell line can be applied to NF2-related mechanism research and drug screening.

[0015] In view of the above problems existing in the existing NF2-related cell model, the present application proposes the following solutions: 1. In view of the problems of insufficient traceability and phenotype accuracy of the source of the existing model cells, the present application separates cells from fresh surgical tissues of NF2 patients, completely retains the original tumor characteristics, and realizes accurate confirmation of the source and identity through STR comparison and immunophenotypic identification.

[0016] 2. In view of the problem of residual Merlin expression and incomplete inactivation of protein function in the existing model, the cell line constructed by the present application clearly identifies NF2 gene mutations (such as c.499delA) through Sanger sequencing and verifies the complete deletion of Merlin protein, eliminating the confusing effects of residual Merlin.

[0017] 3. In view of the problem that the drug response of the existing model is unstable and cannot be used for ferroptosis mechanism research, through functional verification during the construction of the cell line, the cell line of the present application is sensitive to typical ferroptosis inducers such as Erastin and RSL3, and ROS and lipid peroxidation are significantly increased, which can be used for ferroptosis pathway target screening, mechanism analysis and precise drug screening.

[0018] 4. In view of the problem that the existing model cells cannot be passaged or have serious drift, the present application realizes cell immortalization through the SV40T lentivirus system, selects the growth stable, GFP positive clone NF2-VS-03, which has consistent proliferation rate and gene expression profile, and is suitable for long-term passage and standardized experiment.

[0019] 5. In view of the problem that the existing model lacks the ability to interface with clinical treatment, the cell line of the present application can construct a subcutaneous tumor model, and the growth curve significantly changes after responding to ferroptosis intervention, providing an in vitro and in vivo evaluation platform that matches the precise treatment strategy.

[0020] In order to achieve the above purpose, the present application provides the following technical solutions: The present application provides an NF2-related acoustic neuroma immortalized cell line, the name of the cell line is NF2-VS-03 (human NF2-related acoustic neuroma immortalized cell line), which is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC No. 46597, and the preservation date is September 22, 2025.

[0021] Further, the Merlin protein of the cell strain of the application is completely deleted.

[0022] Further, the cell strain of the application is sensitive to the iron death inducer Erastin, and can be used for screening of iron death pathway targets, mechanism analysis and precise drug screening.

[0023] Further, the application also relates to the use of the cell strain in the preparation of an acoustic neuroma cell model.

[0024] Further, the application also relates to the use of the cell strain in the preparation of an acoustic neuroma cell model.

[0025] In another aspect, the application also provides a construction method of the cell strain, comprising the following steps: (1) Isolation and culture of primary cells: Fresh acoustic neuroma tissue is taken; The obtained acoustic neuroma tissue is sequentially subjected to washing, shearing, enzyme digestion, termination reaction and filtration to obtain a cell suspension; The obtained cell suspension is subjected to primary culture to form a group of adherent cells; (2) Immortalization treatment: The primary cells are transfected with a lentivirus system carrying an immortalization gene; The transfected cells are sequentially subjected to resistance screening and clone screening to obtain an immortalized NF2-related acoustic neuroma cell strain.

[0026] Further, in the construction method of the cell strain of the application, in the isolation and culture step of the primary cells, 0.125% trypsin + 0.1% collagenase type II mixed enzyme is used, mixed at a ratio of 1 mL mixed enzyme: 50 mg acoustic neuroma tissue, incubated in a 37°C water bath for 30 minutes, and gently blown every 10 minutes to accelerate digestion.

[0027] Further, in the construction method of the cell strain of the application, in the immortalization treatment step, the lentivirus system carrying the immortalization gene is a third-generation lentivirus system, carrying SV40 T antigen and GFP / puromycin double selection markers, with a virus titer of ≥1×10 8 TU / mL; the transfection operation is performed when the primary cell density reaches 40%~50%, and the MOI is set to 20.

[0028] Further, in the construction method of the cell strain of the application, the resistance screening and clone screening steps are specifically as follows: 2 μg / mL puromycin is added 48 hours after transfection, and continuous treatment is performed for 7 days; under a microscope, a GFP-positive clone with uniform morphology and vigorous proliferation is selected, and is expanded and passaged one by one to obtain an immortalized NF2-related acoustic neuroma cell strain.

[0029] Further, the method for constructing the cell strain of the application further comprises the step of identifying the immortalized NF2-related acoustic neuroma cell strain, specifically comprising: S100 typical pericyte marker is detected by immunofluorescence, and MBP negative cells are excluded; STR analysis is used to compare with the original tissue to confirm the complete consistency of the source; Sanger sequencing or qPCR is used to verify the NF2 mutation site.

[0030] In summary, compared with the existing NF2-related cell model and construction system, the NF2-related acoustic neuroma immortalized cell strain and the construction method of the application have the following advantages: (1) The first human acoustic neuroma cell strain derived from NF2 background with Merlin deletion is constructed, which breaks through the limitation of traditional model.

[0031] (2) The cell strain of the application is sensitive to typical ferroptosis inducers such as Erastin and RSL3, and ROS and lipid peroxidation are significantly increased, which can be used for ferroptosis pathway target screening, mechanism analysis and precise drug screening.

[0032] (3) It can be used as a supporting platform for Merlin pathway and NF2 targeted pathway research.

[0033] (4) It overcomes the defects of short lifespan and heterogeneity of primary cells, and establishes a unified standard model.

[0034] (5) It maintains the genotype consistent with the clinical sample, has repeatability and standardization of drug verification function.

[0035] Other features and advantages of the application will be described in detail in the subsequent description, or can be understood by implementing the related technical solutions of the application. The purpose and other advantages of the application can be achieved by the technical features and technical means explicitly indicated in the specification, claims and drawings, and obtained by the implementation process of these technical contents. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly set forth the technical solutions of the embodiments of the application, the drawings involved in the embodiment description will be briefly introduced as follows. It should be pointed out that the drawings only show part of the embodiments of the application. For those skilled in the art, other related drawings can be deduced from these drawings without creative labor.

[0037] Figure 1 The whole process schematic diagram of the method for constructing the NF2-related acoustic neuroma immortalized cell strain of the application.

[0038] Figure 2 This is a flowchart of the lentivirus infection and screening process in the construction method of this embodiment.

[0039] Figure 3 The immunofluorescence image from the construction method in this embodiment shows that S100 is positive.

[0040] Figure 4 This is a comparison diagram of the STR map and NF2 mutation sequencing in the construction method of this embodiment. Sanger sequencing shows that the NF2 exon mutation leads to the complete loss of Merlin expression.

[0041] Figure 5 This is a cell growth curve diagram from the construction method of this embodiment.

[0042] Figure 6 This is a drug sensitivity response curve (Erastin, Fer-1) constructed using the method described in this embodiment.

[0043] Figure 7 This is a flowchart of the method for constructing immortalized cell lines for NF2-related acoustic neuroma according to the present invention, showing the complete design process of cell construction → characterization and verification → application platform. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] In the present application, the terms "one" and "multiple" used to modify the relevant elements or features are illustrative rather than restrictive. Unless explicitly stated otherwise in the context, "one" should be understood as "at least one" and "multiple" should be understood as "at least two". Those skilled in the art should reasonably interpret these terms according to the semantics and logical relationship of the context, ensuring that they cover the possibility of "one or more".

[0047] Embodiment: A method for constructing an NF2-related acoustic neuroma immortalized cell line Figure 1 、 Figure 7 ) (I) Acquisition and culture of primary cells: Surgical tissue processing: During the operation, a sterile centrifuge tube is used to take about 0.5-1 cm 3 Fresh acoustic neuroma tissue is immediately placed in an ice bath containing high-sugar DMEM + double antibodies, and is transported to the laboratory within 30 minutes; Washing step: Place the acoustic neuroma tissue in a sterile Petri dish and gently rinse it with 1x PBS for 3 times to remove blood clots and impurities; Tissue cutting: Use ophthalmic scissors and toothless forceps to cut the tissue into 1 mm 3 Size; Enzymatic digestion: Mix 1 mL of mixed enzymes (0.125% trypsin + 0.1% collagenase type II) with 50 mg of acoustic neuroma tissue, incubate in a 37°C water bath for 30 minutes, and gently blow every 10 minutes to accelerate digestion; Termination and filtration: Add an equal volume of FBS to terminate the reaction, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and filter the cell suspension using a 70 μm cell screen; Primary culture: Resuspend the cells in preheated complete medium (DMEM high sugar + 10% FBS + Insulin + 1x P / S), inoculate in a Laminin-coated T25 bottle, and incubate at 37°C, 5% CO2; Frequency of medium change: No medium change for the first 48 hours, medium change every 2 days from the 3rd day, and about 5-7 days to form a clear adherent cell population.

[0048] (II) Immortalization operation Figure 2 ): Virus preparation: Use a third-generation lentivirus system carrying SV40 T antigen and GFP / puromycin double selection markers; virus titer requirement ≥1x10 8 TU / mL; (Note: In addition to SV40 T antigen, immortalization systems such as hTERT, E6 / E7, etc. can also be flexibly selected according to research needs); Transfection procedure: Transfection was performed when the primary cell density reached 40-50%, with an MOI of 20. After adding the virus, the cells were gently shaken for 30 seconds to mix thoroughly. The cells were then incubated at 37°C for 12 hours, after which the medium was changed. Resistance screening: Add 2 μg / mL puromycin 48 hours after transfection and treat for 7 consecutive days; Clone screening: GFP-positive clones with uniform morphology and vigorous proliferation were selected under a microscope, amplified and passaged one by one, and then identified. Naming: Clones with consistent proliferation rates and stable adherent growth were selected and named NF2-VS-03; Biological markers: S100 typical sheath cell markers were detected by immunofluorescence, excluding MBP-negative cells (non-neuronal contamination). Figure 3 ); Genetic identification: STR analysis was used to compare with the original tissue, confirming 100% consistency and origin; Sanger sequencing or qPCR was used to verify the NF2 mutation site. Figure 4 ).

[0049] (III) Cell function and application verification: Proliferation and Apoptosis: The proliferation advantage and apoptosis resistance of NF2-VS-03 cells compared to primary cells were detected by CCK-8 and Annexin V-FITC / PI flow cytometry. Figure 5 ); Drug sensitivity: Intervention experiments were conducted using drugs such as Erastin and Ferrostatin-1 to establish standard drug-efficacy response curves. Figure 6 ).

[0050] 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.

[0051] 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. An NF2-associated acoustic neuroma immortalized cell line, characterized in that, The cell strain is named NF2-VS-03, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 46597.

2. The cell line of claim 1, wherein, The cell strain is completely deficient in Merlin protein.

3. The cell line of claim 1, wherein, The cell strain is sensitive to the iron death inducer Erastin, and can be used for screening of iron death pathway targets, analysis of action mechanism, and screening of precise drugs.

4. Use of the cell strain of claim 1 in preparation of a schwannoma cell model.

5. Use of the cell strain of claim 1 in screening of schwannoma treatment drugs.

6. The method of constructing the cell strain according to claim 1, characterized in that, The construction method comprises the following steps: (1) Isolation and culture of primary cells: fresh schwannoma tissue is taken; the obtained schwannoma tissue is sequentially subjected to washing, shearing, enzyme digestion, termination reaction and filtration operations to obtain a cell suspension; the obtained cell suspension is subjected to primary culture to form an adherent cell group; (2) Immortalization treatment: the primary cells are transfected using a lentivirus system carrying an immortalization gene; the transfected cells are sequentially subjected to resistance screening and cloning screening to obtain an immortalized NF2-related schwannoma cell strain.

7. The method of claim 6, wherein the cell line is constructed by, In the step of isolation and culture of primary cells, 0.125% trypsin + 0.1% type II collagenase mixed enzyme is used, mixed at a ratio of 1 mL mixed enzyme: 50 mg schwannoma tissue, incubated in a 37°C water bath for 30 minutes, and gently blown every 10 minutes to accelerate digestion.

8. The method of claim 6, wherein the cell line is constructed by, In the immortalization step, the lentivirus system carrying the immortalization gene is a third-generation lentivirus system, carrying SV40 T antigen and GFP / puromycin double selection markers, and the virus titer is ≥1×10 8 TU / mL; the transfection operation is performed when the primary cell density reaches 40%~50%, and the MOI is set to 20.

9. The method of claim 6, wherein the cell line is constructed by, The resistance screening and cloning screening steps specifically comprise: 2 μg / mL puromycin is added 48 hours after transfection, and continuous treatment is performed for 7 days; under a microscope, a GFP positive clone with uniform morphology and vigorous proliferation is selected, and is expanded and passed one by one to obtain an immortalized NF2-related schwannoma cell strain.

10. The method of claim 6, wherein the cell line is constructed by, The construction method further comprises a step of identifying the immortalized NF2-related schwannoma cell strain, specifically comprising: S100 typical pericyte marker positive is detected by immunofluorescence, and MBP negative cells are excluded; STR analysis and original tissue comparison are used to confirm complete consistency of the source; NF2 mutation sites are verified by Sanger sequencing or qPCR.

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