Thymoma epithelial cell subpopulation with neuromuscular-like characteristics and applications

Abstract

The application relates to a thymoma epithelial cell subpopulation with neuromuscular characteristics and application, and a thymoma epithelial cell subpopulation with neuromuscular characteristics is obtained through single clone dilution culture screening from a thymoma cell line Thy0517 of a patient with myasthenia gravis (MG) in combination, and a thymoma epithelial cell with neuromuscular characteristics in the cell subpopulation is named as Thymus_NMi; the cell subpopulation has synapse-like structures and neuromuscular adhesion characteristics, and efficiently expresses genes participating in neural cell adhesion and synapse connection, highly integrates neuromuscular double characteristics, and simulates key pathological characteristics of abnormal thymus-induced immune tolerance of MG patients in a molecular phenotype and physiological function, so that a cell model closest to a real clinical state is provided for exploring a myasthenia gravis occurrence mechanism.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and in particular relates to a subpopulation of thymoma epithelial cells with neuromuscular characteristics and their applications. Background Technology

[0002] Current research on thymoma-associated myasthenia gravis (TAMG) mainly focuses on immune cell disorders caused by the thymic microenvironment and related antigens such as acetylcholine receptors. It is believed that thymomas promote autoimmune responses against muscle antigens, leading to impaired neuromuscular junction transmission. However, the neuromuscular junction is essentially a highly specialized structure composed of motor nerve endings and muscle cells, and its normal function depends on the precise synergy of neurogenic molecules, synaptic adhesion molecules, signal transduction proteins, and muscle-related factors. Explaining the pathogenesis of thymoma-associated MG solely from the perspective of muscle antigens is insufficient to fully reveal the immunological complexity of TAMG, and also fails to explain the neurogenic immune abnormalities observed in some patients with thymoma and MG.

[0003] Neuron-specific antigen PNMA2 is a neurological antigen known to be closely associated with paraneoplastic neurological syndromes. It is generally considered to be primarily expressed in the nervous system, with very low or no expression in peripheral immune organs. Previous studies have mainly focused on the role of PNMA2 in nervous system tumors and paraneoplastic neurological syndromes; its expression, regulatory characteristics, and immunological significance in the thymus and thymoma tissues have not been systematically reported. If neurological-specific antigens are abnormally or ectopically expressed in thymoma epithelial cells, they may be used as autoantigens in the thymus to participate in T cell selection, thereby directly affecting the establishment of central immune tolerance and playing a potential role in the development and progression of TAMG.

[0004] Recent studies have gradually revealed that thymic epithelial cells are not simply functionally singular structural support cells, but rather a population of cells with significant molecular and functional heterogeneity. Previous research has identified a small subset of epithelial cells expressing tissue-restricted antigens or myoid antigens in the normal thymus, but their origin, stability, and functional characteristics remain unclear. In the context of thymoma, this cellular heterogeneity may be further amplified. The lack of a stable, cultureable model of a thymoma epithelial cell subset with clearly defined molecular markers and functional neuromuscular characteristics severely limits in-depth research into the pathogenesis of TAMG.

[0005] In addition, existing TAMG-related research models mostly rely on whole thymoma tissue, mixed-origin cell populations, or animal models, which generally suffer from high cellular heterogeneity, functional instability, and difficulty in accurately identifying the source of key pathogenic cells, making it difficult to meet the actual needs of mechanism research and drug screening. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a thymoma epithelial cell subpopulation with neuromuscular characteristics and its applications.

[0007] The technical solution adopted in this invention is: a subpopulation of thymoma epithelial cells with neuromuscular characteristics, the cell line named Thy_NMi, with accession number CGMCC No. 46792, classified as a human thymoma epithelial cell line with neuromuscular characteristics, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 19, 2026, and tested as viable.

[0008] Preferably, the cell subset highly expresses one or more of the thymic epithelial cell markers KRT8, KRT18, and EPCAM.

[0009] Preferably, the expression of neurogenic antigen characteristic gene PNMA2, synaptic structure and nerve adhesion characteristic genes CNTN5 and PTPRD, and muscle cell characteristic gene SRPK3 is high.

[0010] Preferably, the characteristic genes are significantly enriched in neuromuscular junction-related functions and pathways.

[0011] Preferably, the cell subset highly expresses one or more of the following characteristic genes: QPRT, ZNF711, CNTN5, MMP20, CGA, DAPP1, PTPRD, C1QTNF3-AMACR, SRPK3, WDR72, LAMA1, CASC8, PNMA2, PCDHB5, MAGEB2, GOLGA6L7, ZNF208, C11orf86, MAP1LC3A, and SMARCA1.

[0012] A method for culturing a subset of thymoma epithelial cells with neuromuscular characteristics, using a special culture medium based on DMEM supplemented with nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), Y-27632, epidermal growth factor (EGF), and platelet-derived growth factor D (PDGFD recombinant protein).

[0013] Preferably, the concentration of nerve growth factor (NGF) is 5-30 ng / mL, the concentration of insulin-like growth factor-1 (IGF-1) is 10-50 ng / mL, the concentration of Y-27632 is 5-15 μM, the concentration of epidermal growth factor (EGF) is 10-25 ng / mL, and the concentration of platelet-derived growth factor D (PDGFD recombinant protein) is 10-30 ng / mL.

[0014] Application of thymoma epithelial cell subsets with neuromuscular features in cell models.

[0015] Preferably, it is used for research on immune abnormalities in myasthenia gravis, drug development, drug screening, or pharmaceutical development or screening.

[0016] Preferably, it is used for neuromuscular junction function research, drug development, drug screening, pharmaceutical development, or pharmaceutical screening.

[0017] Preferably, it is used for drug development, drug screening, pharmacology development, or pharmacology screening for regulating neuromuscular thymoma epithelial cells.

[0018] Application of PTPRD or SRPK3 in predicting whether patients with thymoma will have myasthenia gravis.

[0019] Application of PTPRD or SRPK3 as biomarkers in the preparation of kits for predicting myasthenia gravis in patients with thymoma.

[0020] Preferably, when the pathological type of thymoma is AB, the expression level of the PTPRD gene is detected; when the pathological type of thymoma is B2 or B3, the expression level of the SRPK3 gene is detected.

[0021] The advantages and positive effects of this invention are as follows: It utilizes monoclonal isolation technology to identify functional subpopulations with specific neuromuscular characteristics from the maternal human thymoma cell line Thy0517; the selected cell subpopulations or cell lines, compared to the original heterogeneous maternal cells, have a clear origin, highly consistent genetic background, and stable gene expression characteristics; it eliminates interference from non-functional confounding cells, greatly improving the accuracy and reproducibility of the myasthenia gravis pathological model in in vitro studies, and enriching the existing thymic epithelial cell line cell bank.

[0022] The core targets of the neuromuscular-like thymic epithelial cell (NM-like TEC) subset include molecules closely related to the clinical pathogenesis of myasthenia gravis, such as PNMA2 and CNTN5. The specific high expression of these molecules in the NM-like TEC subset enables it to mimic the key pathological features of abnormal immune tolerance induced in the thymus of MG patients in terms of molecular phenotype and physiological function, providing the cell model that is currently closest to the real clinical state for exploring the pathogenesis of myasthenia gravis.

[0023] A dedicated culture medium is also provided for the NM-like TEC subpopulation, which can be continuously passaged for more than 30 generations, with the cell passage time maintained at 2-3 days / time, and the expression levels of core characteristic genes (PNMA2, SRPK3, CNTN5, PTPRD) are stable for a long time. This solves the technical bottleneck of long-term culture of this cell model and provides reliable technical conditions for subsequent applications. Attached Figure Description

[0024] Figure 1 GO and KEGG analysis of NM-like TEC subpopulations;

[0025] Figure 2 Bar chart showing the expression of thymic epithelial cell markers EPCAM, KRT8, and KRT18 in NM-like TEC and resting control group;

[0026] Figure 3 Screening and analysis of core characteristic genes in NM-like TEC subpopulations; A: Expression heatmap of core characteristic genes in Thy0517, resting subpopulation, and NM-like TEC subpopulation; B: Fold-change ratio and score of core characteristic genes in NM-like TEC subpopulations relative to resting subpopulation; C: Bar chart of expression of neuroantigens, muscle, and synapse-related genes in resting subpopulations and NM-like TEC subpopulations (P<0.001).

[0027] Figure 4 Growth, differentiation, and morphological observation of Thy_NMi cell line; A: Cell differentiation at 6h (200X, left is control group, right is Thy_NMi cell line, P<0.001); B: Cell differentiation at 24h (200X, left is control group, right is Thy_NMi cell line); C: Cell differentiation at 48h (200X, left is control group, right is Thy_NMi cell line); D: Neuron-like cell differentiation of Thy_NMi cell line;

[0028] Figure 5 The relative expression levels of core characteristic genes in cell lines;

[0029] Figure 6 Identification of co-expression of epithelial properties and neuromuscular-like characteristic proteins in Thy_NMi cell line;

[0030] Figure 7 Induced differentiation of Th17 and Treg cells by Thy_NMi cells

[0031] Figure 8 Flow cytometry plots of Thy_NMi cells at different passages; A: Thy_NMi cells cultured in conventional medium, passage 2; B: Thy_NMi cells cultured in conventional medium, passage 10; C: Thy_NMi cells cultured in special medium, passage 30; D: Control group cells cultured in conventional medium, passage 2; E: Control group cells cultured in conventional medium, passage 10; F: Control group cells cultured in conventional medium, passage 30; where the x-axis represents Annexin V and the y-axis represents 7-AAD;

[0032] Figure 9Core gene expression in Thy_NMi cells of different generations;

[0033] Biological material: Name: Thy_NMi, Classification: Human thymoma epithelial cell line with neuromuscular features, Accession number: CGMCC No.46792, Accession date: March 19, 2026, Accession address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing, tested as viable. Detailed Implementation

[0034] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0035] This invention relates to a subpopulation of thymoma epithelial cells with neuromuscular characteristics and its applications. The subpopulation was obtained from the Thy0517 thymoma cell line, derived from patients with myasthenia gravis (MG), through monoclonal dilution culture and screening. Monoclonal differentiation of the Thy0517 cell line yielded a resting subpopulation without significant functional enrichment and several functional subpopulations with different gene expression patterns. One of these functional subpopulations exhibited synaptic-like structures and neuron-like adhesion characteristics, and efficiently expressed genes involved in nerve cell adhesion and synaptic connections, demonstrating a high degree of integration of both neural and muscular characteristics at the molecular level. This subpopulation was defined as a thymoma epithelial cell subpopulation with neuromuscular characteristics. A thymoma epithelial cell line with neuromuscular characteristics, named Thy_NMi, was deposited from this subpopulation at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46792.

[0036] Consistent with the thymoma cell line Thy0517, both the NM-like TEC subset and the resting subset highly express thymic epithelial cell markers KRT8, KRT18, and EPCAM, exhibiting thymic epithelial cell characteristics. Studies on the NM-like TEC subset revealed that, in addition to maintaining stable epithelial properties during long-term passage, it also exhibits neuromuscular characteristics; such as high expression of the neurogenic antigen gene PNMA2, synaptic structure and nerve adhesion genes CNTN5 and PTPRD, and the muscle cell characteristic gene SRPK3. The upregulated genes of the NM-like TEC subset are significantly enriched in biological processes and signaling pathways related to neural development, muscle contraction, and the neuromuscular junction, demonstrating a significant enrichment in neuromuscular junction-related functions and pathways. The NM-like TEC subset promotes the differentiation of T cells from peripheral blood mononuclear cells (PBMCs) into the Th17 subset and inhibits differentiation into the Treg subset.

[0037] The NM-like TEC subset exhibits ectopic high expression of neuron-associated antigens at the mRNA level, with the core gene PNMA2 (paraneoplastic neuron antigen 2) being a clinically recognized neurological antigen that is typically expressed only in neurons. In this subset, PNMA2 expression was significantly higher than in the resting subset, with an FPKM value of 1.74±0.35 vs 0.34±0.24 (P<0.001), suggesting that it may serve as an "ectopic antigen" for myasthenia gravis, inducing the formation of cross-reactive antibodies and thus mimicking the abnormal immune microenvironment within the thymus of MG patients. This study of the NM-like TEC subset is the first to discover aberrant expression of the neuron-specific antigen PNMA in thymoma epithelial cells derived from patients with thymoma and myasthenia gravis. This aberrant expression could serve as a novel thymoma-associated autoimmune marker and could be used for research and diagnostic reagent development for myasthenia gravis and related paraneoplastic autoimmune diseases.

[0038] The NM-like TEC subpopulation also exhibits synaptic-like structures and cell adhesion characteristics. The hallmark genes CNTN5 (neural cell adhesion molecule 5) and PTPRD (protein tyrosine phosphatase receptor D) are involved in synaptic adhesion and axonal guidance in the nervous system. CNTN5, in particular, is primarily expressed in the nervous system, and the high abundance of these molecules in NM-like TEC provides molecular support for the formation of neuromuscular junction-like cell processes. The NM-like TEC subpopulation exhibits specific high expression of SRPK3 (SRSF protease 3), a skeletal muscle-specific regulator highly expressed in both skeletal and cardiac muscle, demonstrating strong muscle expression specificity. It participates in myofibril differentiation and structural maintenance. This characteristic indicates that this subpopulation possesses a muscle-like gene expression profile, providing strong evidence for its "muscle-like" characteristics. The NM-like TEC subset highly expresses LAMA1 (lamin alpha 1) and MMP20. LAMA1 is a major component of the basement membrane and plays a central role in the differentiation of the neuromuscular junction and postsynaptic membrane, while MMP20 is responsible for remodeling the extracellular matrix and influencing axonal growth. The NM-like TEC subset also highly expresses QPRT (quinolinate phosphoribosyltransferase), a gene involved in the NAD+ synthesis salvage pathway, suggesting that this subset possesses strong cell viability during active functional metabolism, ensuring the stability of cell growth.

[0039] Gene expression was compared between the NM-like TEC subset and the resting subset. Differentially expressed genes upregulated in the NM-like TEC subset compared to the resting subset were screened (log2FC > 1, adj. P < 0.05). The intersection of receptor genes and upregulated differentially expressed genes yielded nine receptor genes: NGFR (neurotrophic factor receptor), APLNR (apelin receptor), ACKR3 (atypical chemokine receptor), PDGFR (platelet-derived growth factor receptor), TNFRSF11A (RANK receptor), etc. Furthermore, IGFBP1 and IGFBP3 (insulin-like growth factor binding protein) were also significantly upregulated, suggesting that IGF signaling plays an important regulatory role in this subset. Differentially expressed genes downregulated in the NM-like TEC subset compared to the resting subset (log2FC < -1, adj. P < 0.05) were screened, and analysis revealed that the expression levels of EGF (epidermal growth factor, log2FC = -1.54) and PDGFD (platelet-derived growth factor D, log2FC = -5.27) were significantly downregulated in the NM-like TEC subset. Furthermore, the NM-like TEC subset exhibited gene expression characteristics of high cytoskeleton contractility (high expression of ACTN2 / ACTN3 / TNC genes), insufficient cell-matrix adhesion (low expression of ITGAX / LUM / PCOLCE), and weak anti-apoptotic ability (low expression of BIRC7).

[0040] Based on the above characteristics, the NM-like TEC subset (Thy_NMi cell line) can be used as a cell model to simulate the pathological features of neuromuscular-like cells in the thymus of MG patients, providing an ideal in vitro model for autoimmune mechanism research and drug screening. This solves the problems of high heterogeneity and lack of functionally specific subsets in existing cell lines. For example, it can be used to develop or screen candidate molecules, kits, or drugs related to neuromuscular junction function, or to study models related to neuromuscular junction function; or to screen candidate drugs that regulate the function of neuromuscular-like thymoma epithelial cells.

[0041] To maintain the proliferative capacity and optimal differentiation ability of the NM-like TEC subset during passage, a dedicated culture medium for the NM-like TEC subset was developed. The composition of this medium was designed based on the gene expression characteristics of the NM-like TEC subset. The NM-like TEC subset highly expresses NGFR; without exogenous NGF stimulation, NGFR will lead to specific atrophy and apoptosis due to the loss of characteristic survival signals. IGFBP1 and IGFBP3 are significantly upregulated in the NM-like TEC subset; exogenous IGF-1 supplementation helps ensure the continuous activation of this pathway, maintaining cell metabolism and proliferative activity. The upregulated genes associated with these subsets can be maintained by adding NGF and IGF-1. The expression of EGF and PGFDD in the NM-like TEC subset is downregulated. EGF is the most basic mitogen in epithelial cells, and its deficiency can directly lead to cell cycle arrest. PGFDD is crucial for the survival of mesenchyme and epithelial cells. This systemic deficiency of endogenous growth signals is the main reason for increased cell proliferation arrest and apoptosis. To overcome the deficiency of these two genes, exogenous supplementation of EGF and PGFDD can be used. Furthermore, the addition of the ROCK inhibitor Y-27632 can inhibit anoikis and promote cell adhesion and cytoskeleton homeostasis, thereby improving passage survival rate and colony formation efficiency. Based on the above receptor expression profile and signaling pathway activity characteristics, combined with the neuromuscular biological properties, receptor-ligand relationship and functional correlation of this cell subset, DMEM was used as the basal medium, supplemented with nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), Y-27632, epidermal growth factor (EGF), and platelet-derived growth factor D (PDGFD recombinant protein). Through passage stability experiments and core gene expression stability tests, the concentrations of various factors were optimized. The optimal formulation of the dedicated culture medium was finally determined to be: DMEM, 10% FBS, NGF (5-30 ng / mL), IGF-1 (10-50 ng / mL), Y-27632 (5-15 μM), EGF (10-25 ng / mL), and PDGFD (10-30 ng / mL). Experiments demonstrated that the above-mentioned dedicated culture medium allowed for continuous passage for more than 30 generations, maintaining high cell proliferation and differentiation capacity, efficiently expressing core characteristic genes, and maintaining the neuromuscular phenotype of the cell line for a long period.

[0042] Using monoclonal isolation technology, a functional subpopulation with specific neuromuscular characteristics was identified from the maternal human thymoma cell line Thy0517. Compared to the original heterogeneous maternal cells, the NM-like TEC subpopulation has a clear origin, highly consistent genetic background, and stable gene expression characteristics. This eliminates interference from non-functional confounding cells, significantly improving the accuracy and reproducibility of the myasthenia gravis pathological model in in vitro studies and enriching the existing thymic epithelial cell line bank. It overcomes the bottleneck of cell model heterogeneity, achieving high purity and high stability. The core targets of the NM-like TEC subpopulation include molecules closely related to the clinical pathogenesis of MG, such as PNMA2 and CNTN5. The specific high expression of these molecules in the NM-like TEC subpopulation highly mimics the key pathological features of abnormal immune tolerance induced in the thymus of MG patients in terms of molecular phenotype and physiological function, providing the most realistic clinical cell model for exploring the pathogenesis of myasthenia gravis. The NM-like TEC subset and its stable functional response characteristics make it an excellent in vitro platform for evaluating drugs related to neuromuscular junction function, screening molecules that regulate thymic epithelial phenotypic switching, and studying basic immunological mechanisms. It has good prospects for application in drug development, antibody screening, and medical research.

[0043] Transcriptomic data and clinical information from 105 thymoma samples were obtained from the TCGA database. The correlation between four core characteristic genes of the NM-likeTEC subset and different pathological types of thymoma and TAMG was analyzed. All four genes showed highly significant differences among different pathological types; PTPRD and SRPK3 showed significant differences in expression between the MG and non-MG groups. PTPRD expression was significantly increased in the AB type thymoma MG group, and SRPK3 expression was significantly increased in the B2 and B3 type thymoma MG groups. No significant differences were found between the MG and non-MG groups for CNTN5 and PNMA2 within each pathological type. PTPRD and SRPK3 can serve as molecular markers to differentiate whether different pathological types of thymoma are complicated with MG. Reagents detecting PTPRD and / or SRPK3 gene expression levels can be combined in kits to predict whether thymoma patients will have TAMG. For thymoma patients who have not yet developed MG symptoms, detecting PTPRD / SRPK3 expression can provide early warning of their future risk of developing MG. When the pathological type of thymoma is AB, the expression level of PTPRD gene is detected; when the pathological type of thymoma is B2 or B3, the expression level of SRPK3 gene is detected.

[0044] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0045] Example 1: Obtaining and dividing monoclonal cell subsets

[0046] 1.1 Maternal amplification of Thy0517 and establishment of a single-clone library

[0047] The human thymoma cell line Thy0517, cryopreserved in liquid nitrogen, was revived and seeded in complete growth medium (high glucose DMEM + 10% FBS) and cultured at 37°C in a 5% CO2 cell culture incubator until 80% confluence. Single-cell suspensions were prepared by digestion with 0.25% trypsin. After cell counting, 100 cells were diluted to 10 ml with complete growth medium and thoroughly mixed, then seeded into 96-well plates at 100 μL per well. Twenty-four hours after seeding, each well was labeled with a single cell under a microscope, and the medium was changed as needed. When the cell confluence in each well exceeded 80%, the cells were digested and transferred to 24-well plates. Cells were then progressively transferred to 12-well plates, 6-well plates, and T25 cell culture flasks according to cell growth density, ultimately yielding 14 stable passaged monoclonal isolates. All 14 cell lines were viable for in vitro growth and successfully revived after cryopreservation in liquid nitrogen.

[0048] 1.2 Expression profiling sequencing and data standardization

[0049] Total RNA was extracted from 14 monoclonal cell lines at passage 30 using the Trizol method (3 samples per cell line, 1 million cells per sample, cells cultured independently). After quality control, cDNA libraries were constructed, and transcriptome sequencing was performed using the Lumina platform. The raw sequencing data underwent Fastq quality control to remove adapters, and alignment to the human reference genome (GRCh38) was performed using HISAT2. The original gene counts were counted using the FeatureCounts tool. Simultaneously, FPKM values ​​were calculated using the alignment results to characterize the relative expression levels of genes in different clones, providing a quantitative basis for subsequent heterogeneity analysis.

[0050] 1.3 Core Clustering Algorithm and Subgroup Division

[0051] Based on the standardized expression matrix, the coefficient of variation for each gene among the 14 clones was calculated using analysis of dispersion (MAD), and the top 3000 highly variable genes were screened to capture heterogeneity among clones. The Pearson correlation coefficient matrix among each sample was calculated and converted into a distance matrix (1-Pearson), followed by clustering using the Ward.D2 discrete sum of squares method in hierarchical clustering. Iterative clustering was performed on the 14 clones, a method that minimizes intra-cluster variance to ensure compact clustering. The topological structure of the dendritic chart was plotted, precisely defining the 14 clones into 7 cell subpopulations with significant transcriptomic differences at the optimal shearing height.

[0052] 1.4 Establishment of the resting control group

[0053] A strategy of comparing one subgroup with all other subgroups was employed, and the characteristic genes of each subgroup were identified using the limma package in R software (screening criteria: log2FC>1, adj.P<0.05). The characteristic gene sets of each group were then subjected to GO biological processes and KEGG pathway enrichment analysis. The results showed that one subgroup's gene set failed to enrich any significant functional entries; therefore, this subgroup was established as the "resting subgroup" and used as a control subgroup for the other subgroups.

[0054] 1.5 Establishment of a subset of thymic epithelial cells with neuromuscular characteristics

[0055] One subpopulation underwent differential gene analysis with the resting subpopulation (screening criteria: log2FC>1, adj.P<0.05), further revealing the upregulated differentially expressed genes in this subpopulation. This differentially expressed gene set was then subjected to GO biological processes and KEGG pathway enrichment analysis, such as... Figure 1 As shown, this subset is enriched in neuromuscular-related biological processes and signaling pathways, and is named the neuromuscular-like thymic epithelial cell (NM-like TEC) subset.

[0056] Example 2: Characteristic gene analysis of thymic epithelial cell subsets with neuromuscular features

[0057] For the NM-like TEC subpopulation, a secondary gene screening was performed: First, the set of upregulated differentially expressed genes in the NM-like TEC subpopulation relative to the resting subpopulation was extracted, and its intersection with the characteristic gene set of the NM-like TEC subpopulation obtained from the strategy of comparing a certain group with all other groups was taken. Then, a weighted scoring algorithm was introduced (the calculation formula is: RankingScore = logFC × log 10 (MeanCounts+1) is used to rank the genes. MeanCounts is the average original expression value of the gene within the NM-like TEC subgroup to ensure that the core targets selected are not only statistically specific but also have sufficient gene abundance, and to identify the genes with the highest scores as the core characteristic genes of NM-like TEC.

[0058] To clarify the epithelial cell consistency of this subpopulation, the expression levels of the common markers of thymic epithelial cells EPCAM, KRT8, and KRT18 were detected in this subpopulation. The results are as follows: Figure 2 As shown, there was no statistically significant difference in the expression of the above markers between the NM-like TEC subset and the resting control group (P values ​​were 0.213, 0.495 and 0.0563, respectively), confirming that both subsets maintained stable basic biological properties of thymic epithelial cells.

[0059] Based on the core characteristic genes selected, expression heatmaps were drawn by comparing Thy0517, the resting subpopulation, and the NM-like TEC subpopulation. Results are as follows: Figure 3 A and Figure 3 As shown in Figure B, the screened core genes exhibited high abundance expression in the NM-like TEC subset, while almost all of them were expressed at low or very low levels in the maternal cell Thy0517 and resting subsets; this confirms the specificity of the core genes as the molecular fingerprint of the NM-like TEC subset. Further comparison of the FPKM values ​​of several core characteristic genes related to neuroantigens, muscle, and synapses, such as... Figure 3 As shown in Figure C, the mean expression of PNMA2 in the NM-like TEC subgroup was significantly higher than that in the resting control group (2.27±0.52 vs 0.6±0.2, P<0.001), suggesting that it may participate in autoimmune induction as an ectopic neural antigen; PTPRD expression was increased in the NM-like TEC subgroup (1.74±0.35 vs 0.34±0.24, P<0.001), providing a molecular basis for the formation of synapse-like structures; SRPK3 expression in the NM-like TEC subgroup was significantly higher than that in the resting control group (0.42±0.15 vs 0.03±0.02, P<0.001), showing that this subgroup has muscle-like gene expression characteristics; and the mean expression of CNTN5 in the NM-like TEC subgroup was significantly higher than that in the resting control group (0.51±0.04 vs 0.001). The difference (0.01±0.01, P<0.001) suggests that this subpopulation possesses synaptic-like structures and neuron-like adhesion characteristics, providing a molecular basis for the formation of cell processes resembling neuromuscular junctions. These results indicate that the NM-like TEC subpopulation possesses stable transcriptomic characteristics distinct from the maternal cell and other cell subpopulations, further validating its identifiability and stability as an independent thymic epithelial-like cell subpopulation.

[0060] The differential expression of core characteristic genes reveals that this subpopulation highly integrates both neural and muscular features at the molecular level. Specifically, CNTN5, PTPRD, and LAMA1 are involved in neuronal adhesion and synaptic connections, forming the molecular basis for the "neuron-like" branching processes of this subpopulation. High expression of PNMA2, a paraneoplastic antigen associated with myasthenia gravis (MG), reveals a potential link between this subpopulation and autoimmune pathogenesis. Furthermore, genes such as SRPK3 and QPRT are involved in muscle-specific kinase regulation and metabolic pathways. Because normal thymic epithelial cells are limited by their epithelial lineage, their gene transcription is mainly concentrated on structural keratins and conventional immune-presenting molecules, while the aforementioned neuromuscular specific genes are usually suppressed or at low transcriptional levels under normal physiological conditions. The specific activation of these genes by the NM-like TEC subpopulation constitutes its exclusive molecular fingerprint, distinguishing it from conventional epithelial cells.

[0061] Example 3: Growth, differentiation, and morphological characteristics identification of NM-like TEC subpopulations

[0062] A single clonal cell line from the NM-like TEC subgroup was selected, named Thy_NMi, and deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 46792.

[0063] A single clone cell line from the resting subpopulation was selected to represent the resting subpopulation and used as the control group.

[0064] Thy_NMi cell lines and control cell lines with the same logarithmic growth phase and number of passages (30 passages) were taken separately. Both cell lines were digested and diluted into suspension cells. After trypan blue staining, the cell viability of both the Thy_NMi and control cell lines at inoculation was greater than 95%. The suspension cells were then divided into 1×10⁻⁶ cells / cells. 5 2 ml of the solution was seeded into each well of a 6-well plate. The plates were incubated at 37°C and 5% CO2. Cell growth and morphological characteristics were observed and recorded at 6 h, 24 h, and 48 h post-inoculation.

[0065] like Figure 4 As shown in Figure A, at 6 hours after inoculation, 54.5 ± 5.2% of the cells in the control group had completed adherent differentiation, mostly exhibiting typical polygonal epithelial-like differentiation; while 8.7 ± 4.7% of the cells in the Thy_NMi cell line had begun adherent differentiation, with most still showing round or oval shapes, indicating a significant delay in adherent differentiation. Figure 4 B and Figure 4 As shown in Figure C, at 24 and 48 hours after inoculation, the resting subpopulation exhibited high homogeneity, with uniform polygonal epithelial cells arranged in a cobblestone-like compact pattern. The NM-like TEC subpopulation, however, showed significant heterogeneity and delayed differentiation. At 24 hours, a small number of cells in the NM-like TEC subpopulation remained in a round or semi-spreading state, exhibiting a unique pattern of mixed growth of polygonal and spindle-shaped epithelial cells. At 48 hours, the cell line still stably maintained the characteristic of mixed growth of polygonal and spindle-shaped cells, with a significantly higher proportion of spindle-shaped cells than the control group, indicating that this subpopulation possesses stronger cell plasticity and the potential to construct a neuromuscular-like microenvironment.

[0066] Against a background of abundant adherent epithelial cells, the NM-like TEC subset contains 1%-3% highly neuron-like cells, such as... Figure 4 As shown in D, its cell body is contracted and rounded, and extends outward with multiple slender filamentous processes similar to neuronal axons or dendrites. These processes exhibit strong polarity and spatial orientation, with an extension length that can reach more than 3 times the diameter of the cell body, and obvious swelling or branching points can be seen at the ends. Morphologically, this directly confirms the differentiation potential of this subgroup to specialize in a neuron-like phenotype.

[0067] The delayed adherent differentiation of the NM-like TEC subpopulation is highly correlated with core characteristic genes. High expression of SRPK3 and LAMA1 suggests that NM-like TEC cells preferentially undergo complex cytoskeleton rearrangement and neuromuscular junction-like basement membrane synthesis, rather than performing a simple epithelial adhesion procedure. This physical phenotype provides a structural basis for the neuromuscular-like function of the NM-like TEC subpopulation.

[0068] Example 4: Validation of core characteristic genes of NM-like TEC subpopulation

[0069] 4.1 Detection of expression levels of PNMA2, SRPK3, CNTN5 and PTPRD by real-time quantitative PCR

[0070] To verify the accuracy of transcriptome sequencing data analysis and further confirm the characteristic expression profile of NM-like TEC, this embodiment uses real-time quantitative PCR (RT-qPCR) to quantitatively detect the mRNA levels of four core characteristic genes of the Thy_NMi cell line: PNMA2, SRPK3, CNTN5, and PTPRD.

[0071] Control cell lines and Thy_NMi cell lines in the 30th generation logarithmic growth phase were collected, with a cell count of 2 × 10⁶ cells. 6 Cells were centrifuged at 2000 RPM / min to obtain cell pellets, and washed twice with PBS. Total RNA was extracted using the Trizol method. After the concentration and OD value were verified to be within acceptable limits, 1 μg of total RNA was reverse transcribed into cDNA. GAPDH was used as an internal control gene, and amplification was performed using the SYBR Green fluorescent dye method. Each sample was tested in triplicate for quantitative PCR. The relative expression levels of each gene were calculated using the 2-ΔΔCt method.

[0072] like Figure 5As shown, RT-qPCR results revealed that the relative expression of the four core characteristic genes in the Thy_NMi cell line was significantly higher than that in the control cell line, and the expression trend was completely consistent with the RNA-seq data. The mRNA level of PNMA2 in the Thy_NMi cell line was approximately 1.87 times that of the control cell line (P<0.05), confirming its expression characteristic as an ectopic neural antigen. The expression of PTPRD in the Thy_NMi cell line was significantly higher than that in the control cell line by 4.73 times (P<0.01), suggesting that this subpopulation has a molecular basis in mimicking neural synaptic signal transduction. The expression of SRPK3 in the Thy_NMi cell line was significantly higher than that in the control cell line by 23.52 times (P<0.01), supporting the muscle-like characteristics of this subpopulation. The expression of CNTN5 in the Thy_NMi cell line was significantly higher than that in the control cell line by 4.73 times (P<0.01), providing molecular evidence for its neuron-like adhesion behavior during early adhesion.

[0073] 4.2 Multiplex fluorescent immunocytochemical staining (TSA method) to locate the expression of PNMA2, SRPK3, CNTN5 and PTPRD proteins

[0074] Thy_NMi cells in the 40th generation logarithmic growth phase were passaged and seeded into 24-well plates. When the cell density reached 60%, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 20 min. Inactivation and blocking were then performed using endogenous peroxidase blocking solution and 5% normal sheep serum, respectively. The first round of labeling followed: incubation with primary antibody KRT18, HRP secondary antibody, and TSA-570 working solution, covalently binding to KRT to produce red fluorescence. After washing, peroxidase blocking solution was added again and the cells were incubated at room temperature for 30 min. The second round of labeling was then performed: incubation with secondary antibodies (PNMA2, SRPK3, CNTN5, and PTPRD), HRP secondary antibody, and TSA-520 working solution, producing green fluorescence for protein B. Finally, the nuclei were stained with DAPI for 5-10 min and thoroughly washed with TBST before observation and imaging under a fluorescence microscope.

[0075] The results are as follows Figure 6 As shown, in Thy_NMi cells, KRT18 (red) and PNMA2, SRPK3, CNTN5 and PTPRD proteins (green) exhibit different levels of co-localization expression, confirming that the cells stably maintain epithelial properties and neuromuscular characteristics during long-term passage, and demonstrating that they can serve as ectopic antigen carriers and pathological structural models for myasthenia gravis.

[0076] Example 5: Effects of NM-like TEC subsets on immune cell differentiation

[0077] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers using lymphocyte separation medium, and the cell concentration was adjusted to 10⁻⁶ cells / mL using DMEM medium. 6 per ml.

[0078] A co-culture system of PBMCs and Thy_NMi cells was established. Control cell lines or Thy_NMi cells in the 30th generation logarithmic growth phase were passaged and seeded into 6-well plates at 2 × 10⁶ cells per well. 5 Add 10g of complete culture medium to each well of a six-well plate and incubate for 24 hours until the cells adhere well, thus establishing a co-culture system. 5 PBMCs were divided into an experimental group (Thy_NMi cells + PBMCs) and a control group (control cell line + PBMCs), with three replicates in each group. After co-culturing for 72 h, PBMCs were harvested, centrifuged, resuspended in 500 μL RMPI 1640 medium, and stimulated at 37°C for 4 h with 1 μL of cocktail. Flow cytometry staining was performed 4 h later to detect the ratio of Th17 (CD4+IL17+) and Treg (CD4+CD25+FOXP3+).

[0079] The results are as follows Figure 7 As shown, the proportion of the Th17 subset in the experimental group was significantly higher than that in the control group (3.67±0.56 vs 1.32±0.15), P<0.01, indicating a significant promotion of inflammation. The proportion of the Treg subset in the experimental group was significantly lower than that in the control group (3.13±0.68 vs 5.38±1.00), P<0.05, indicating a reduction in T cell immune tolerance. These results indicate that Thy_NMi cells can significantly induce T cell immune homeostasis imbalance by reshaping the Th17 / Treg cell balance, thus successfully simulating the pathogenic immune dysregulation characteristics in the thymic microenvironment of myasthenia gravis patients in vitro.

[0080] Example 6: Optimization of Culture Medium Conditions for Thy_NMi Cells

[0081] Studies have found that the proliferation capacity of Thy_NMi cells decreases after 10 passages. To maintain the long-term stable passage and optimal proliferation capacity of Thy_NMi cells, a special culture medium for Thy_NMi cells was designed based on the characteristic genes of this cell line. Different concentrations of recombinant proteins NGF, IGF-1, Y-27632, EGF, and PGFDD were added to the basal DMEM medium to form a special culture medium suitable for Thy_NMi cells. The concentrations of each factor were optimized through passage stability experiments and core gene expression stability tests. The optimal formulation of the special culture medium is: based on DMEM, supplemented with 10% FBS, NGF (5-30 ng / mL), IGF-1 (10-50 ng / mL), Y-27632 (5-15 μM), EGF (10-25 ng / mL), and PGFDD (10-30 ng / mL).

[0082] Thy_NMi cells were cultured using both conventional and specialized culture media, while a control group was cultured using conventional culture media. Apoptosis in both cell lines was detected by flow cytometry at passages 2, 10, and 30. RNA was extracted from Thy_NMi cells at passages 2, 10, and 30 using the Trizol method, and the expression levels of core characteristic genes PNMA2, SRPK3, CNTN5, and PTPRD were detected by RT-PCR.

[0083] The results are as follows Figure 8 As shown, when Thy_NMi cells were cultured in conventional medium (DMEM, 10% FBS), proliferation arrested and apoptosis increased by the 10th passage. The cells failed to adhere to the culture vessel after passage and could not be cultured further. However, when Thy_NMi cells were cultured in a specialized medium, they could be passaged for more than 30 consecutive passages at a frequency of 2-3 days per passage, and apoptosis was significantly reduced. In continuous culture of resting subsets, there was no statistically significant difference in apoptosis between the 2nd and 10th passages at the 30th passage.

[0084] Further RT-qPCR analysis was performed on Thy_NMi cells at passages 2, 10, and 30 to detect the expression of core characteristic genes PNMA2, SRPK3, CNTN5, and PTPRD. The results are as follows: Figure 9 As shown, there was no significant difference in their relative expression levels (P > 0.05), indicating that the special culture medium can maintain the neuromuscular phenotype of this cell line for a long time.

[0085] Example 7: Correlation between core characteristic genes and pathological types of thymoma and TAMG.

[0086] Thymoma mRNA sequencing datasets (120 cases in total) and corresponding patient clinical data were downloaded from the TCGA database. After rigorous data cleaning to remove samples with mismatched information and missing key variables, 105 thymoma samples with complete expression profiles and clinical information were finally included. Clinical data included WHO pathological classifications (A, AB, B1, B2, B3) and thymoma muscular dystrophy (TAMG) status. R software was used to analyze four core genes (PNMA2, SRPK3, CNTN5, PTPRD) to assess the correlation between each gene and thymoma pathological type and TAMG. The results are shown in Table 1.

[0087] Table 1. Correlation analysis between core genes and pathological types of thymoma and MG.

[0088]

[0089] The data in the table show that gene expression is correlated with pathological type, and the expression of the four core characteristic genes differed significantly among different thymoma pathological types (P < 0.001). Analysis of the correlation between characteristic genes and TAMG revealed significant differences in the expression of PTPRD (P < 0.001) and SRPK3 (P < 0.001) between the MG and non-MG groups in the comparison of groups with and without MG.

[0090] The differences in gene expression between those with and without MG were compared within the same pathological type. The differences in characteristic gene expression between the MG and no_MG groups were compared within the same pathological type. The results are shown in Table 2.

[0091] Table 2. Core gene expression analysis between MG and non-MG groups in each pathological type.

[0092]

[0093] In type AB thymoma, the expression level of PTPRD in the MG group was significantly higher than that in the non-MG group (log2FC=1.79, P<0.01). In type B2 thymoma, the expression level of SRPK3 in the MG group was significantly higher than that in the non-MG group (log2FC =2.14, P<0.01); in type B3 thymoma, the expression of SRPK3 in the MG group was also significantly increased (log2FC=2.97, P<0.01).

[0094] The data above show that all four core genes are significantly associated with thymoma pathological types. Specifically, PTPRD and SRPK3 are significantly associated with TAMG: PTPRD is associated with MG in AB type thymoma, and SRPK3 is associated with MG in B2 / B3 type thymoma. These findings provide new clues to elucidating the molecular mechanisms of MG associated with different pathological types of thymoma, and PTPRD and SRPK3 may serve as potential biomarkers or therapeutic targets for TAMG.

[0095] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A subset of thymoma epithelial cells with neuromuscular characteristics, characterized in that: The cell line was named Thy_NMi and its accession number is CGMCC No. 46792.

2. The thymoma epithelial cell subset with neuromuscular characteristics according to claim 1, characterized in that: Cell subsets highly express one or more of the thymic epithelial cell markers KRT8, KRT18, and EPCAM.

3. The thymoma epithelial cell subset with neuromuscular characteristics according to claim 2, characterized in that: High expression of neurogenic antigen characteristic gene PNMA2, synaptic structure and nerve adhesion characteristic genes CNTN5 and PTPRD, and muscle cell characteristic gene SRPK3.

4. The thymoma epithelial cell subset with neuromuscular characteristics according to claim 1, characterized in that: The characteristic genes are significantly enriched in neuromuscular junction-related functions and pathways.

5. A method for culturing a subset of thymoma epithelial cells with neuromuscular characteristics as described in any one of claims 1-4, characterized in that: Cultured using a special culture medium, which is based on DMEM and supplemented with nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), Y-27632, epidermal growth factor (EGF) and platelet-derived growth factor D (PDGFD recombinant protein).

6. The method for culturing a subset of thymoma epithelial cells with neuromuscular characteristics according to claim 5, characterized in that: The concentrations of nerve growth factor (NGF) were 5-30 ng / mL, insulin-like growth factor-1 (IGF-1) were 10-50 ng / mL, Y-27632 were 5-15 μM, epidermal growth factor (EGF) were 10-25 ng / mL, and platelet-derived growth factor D (PDGFD recombinant protein) were 10-30 ng / mL.

7. The use of the thymoma epithelial cell subset with neuromuscular characteristics as described in any one of claims 1-4 in a cell model for non-disease diagnosis and treatment purposes.

8. The application according to claim 7, characterized in that: Used for research on immune abnormalities in myasthenia gravis, drug development, drug screening, pharmaceutical development, or pharmaceutical screening; Alternatively, it can be used for neuromuscular junction function research, drug development, drug screening, pharmaceutical development, or pharmaceutical screening. Alternatively, it can be used for drug development, drug screening, or formulation development or formulation screening for regulating neuromuscular thymoma epithelial cells.

9. Application of PTPRD or SRPK3 as biomarkers in the preparation of kits for predicting myasthenia gravis in patients with thymoma.

10. The application according to claim 9, characterized in that: When the pathological type of thymoma is AB, the expression level of PTPRD gene is detected; when the pathological type of thymoma is B2 or B3, the expression level of SRPK3 gene is detected.

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