Human choroid plexus papilloma-derived cell line and use thereof

Abstract

Disclosed in the present application are a human choroid plexus papilloma-derived cell line and the use thereof. The cell line is derived from benign choroid plexus papilloma, is an immortalized infinite cell line, and has important scientific significance and application value for biological, medical, toxicological and pharmacological researches and the like using choroid plexus and blood-cerebrospinal fluid barriers as research objects.

Description

A cell line derived from human choroid plexus papilloma and its application Technical Field

[0001] The present application relates to the technical field of cell lines, and in particular to a cell line derived from human choroid plexus papilloma and its application. Background Art

[0002] The blood-cerebrospinal fluid barrier (BCB) is responsible for the exchange and transport of substances between peripheral blood and cerebrospinal fluid (CSF). The choroid plexus (CP) is the histological basis of the blood-cerebrospinal fluid barrier (BCB) in the central nervous system and plays an important role in brain development, maturation, aging, endocrine regulation, and the pathology of some neurodegenerative diseases. The BCB is a channel and one of the primary gateways for various exogenous pathogens (chemicals, pathogenic microorganisms, and toxins, etc.) and drugs to enter the central nervous system (CNS). Evidence has shown that pathogens enter the CNS through the BCB. The BCB may play a key role in inducing immune stress responses in the CNS and is involved in regulating the intensity and type of immune responses. The CP forms from the ependyma, which invaginate into the ventricles. It forms a reticular arrangement within the ventricles and can be divided into the choroid plexus of the lateral ventricle, the choroid plexus of the third ventricle, and the choroid plexus of the fourth ventricle, depending on its anatomical location. The choroid plexus is not only an important source of cerebrospinal fluid (CSF) but also forms the structural foundation of the blood-CSF barrier. It prevents harmful factors circulating in the peripheral blood from entering the brain tissue, while also selectively transporting blood-borne substances into the CSF or excreting metabolic waste into the peripheral circulation. It is crucial for maintaining central nervous system homeostasis.

[0003] From a physiological perspective, the choroid plexus has a protective effect on the central nervous system. First, it acts as a barrier, keeping harmful substances in the peripheral circulation out of the central nervous system and maintaining nervous system homeostasis. Second, the choroid plexus exhibits selective permeability. Its numerous surface microvilli and specialized protein receptors / transporters enable the selective and active absorption of nutrients or excretion of metabolic products. Third, the choroid plexus has a secretory function and is an important source of cerebrospinal fluid, which has a certain buffering effect on brain tissue. Furthermore, the growth factors and other active components secreted by the choroid plexus have neurotrophic functions. The choroid plexus also plays a crucial role in certain pathological conditions, such as neurodegenerative diseases, poisoning, and neuroinflammation. For example, in cases of heavy metal poisoning (such as lead, mercury, and cadmium), or when toxic symptoms progressively worsen, the choroid plexus serves as a primary target storage site for toxicants before they enter the brain parenchyma, thereby exhibiting an accumulation effect and acting as a buffer pool. Existing technologies have systematically reviewed the regulatory role of CP. CP / BCB is not only the "entrance" for exogenous pathogens to enter the CNS, but also the starting point for certain infections. It is an important participant in the pathogenesis of nervous system infections and a possible immune "hub," carrying the "mission" of coordinating the selective homing of immune cells to the brain parenchyma of the central nervous system. In recent years, in vivo and in vitro studies based on CP organoids, cells, and animals have shown that after hydrocephalus (PIH) and viral infections such as Zika virus (ZIKV), human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus (SARS-CoV-2), CP produces a specific cellular response to pathogens, which is closely related to the subsequent immune response of the CNS. Therefore, the choroid plexus is of great significance for maintaining the homeostasis of the internal environment of the nervous system. It is also the site of action and a key factor in the pathological processes of some diseases, and has important research value.

[0004] A cell line refers to a population of cells that is propagated after the first successful passage of a primary cell culture. It can be divided into finite and infinite cell lines; the difference between the two lies in whether the cell culture can proliferate and be passaged indefinitely. Cell lines are important tools for research in life sciences, biology, and medicine. In 1964, Wiktor et al. used WI-38 human lung fibroblasts to produce a rabies vaccine. In 1975, Kohler and Milstein invented hybridoma technology, which fuses myeloma cells with spleen cells from immunized animals to form a hybrid cell line that secretes homogeneous, highly specific antibodies against the antigen—monoclonal antibodies. This technology is not only of great value in basic biological and immunological research but also has widespread applications in medical diagnostics. In the 1980s, through studies of various tumor cells, gene expression regulation and oncogene transformation were deeply explored. In the 1990s, large-scale industrial cell culture was applied to the biopharmaceutical industry.

[0005] Cell lines are the foundation of in vitro research, especially immortalized cell lines, which are widely used and possess significant application value. To date, the American Type Culture Collection (ATCC), the world's largest biological resource center, has cataloged nearly 4,000 human cell lines and various cell lines related to 150 other species. Among these, the SCP cell line (derived from sheep choroid plexus tissue) can be used to study choroid plexus tissue function. Of the six other CP epithelial cell lines not included in this catalog, the most commonly used are the Z310 cell line (derived from rat choroid plexus tissue) and the HIBCPP cell line (derived from human choroid plexus papilloma). However, the species-specific nature of the Z310 cell line and the high malignancy of the HIBCPP cell line limit the translation of research findings into practical applications.

[0006] Summary of the Invention

[0007] To overcome the above-mentioned defects, the present invention constructs a human choroid plexus cell line derived from a benign choroid plexus papilloma to retain its epithelial cell-related biological characteristics to the greatest extent possible, meet relevant research and development needs, and provide a powerful tool for biological, medical, toxicological, pharmacological and other research with the choroid plexus and blood-cerebrospinal fluid barrier as research objects.

[0008] Specifically, in the first aspect, one of the objectives of the present invention is to provide a new human-derived choroid plexus papilloma-derived cell line, wherein the cell line expresses growth factors, and the growth factors include growth differentiation factor 15 (GDF15), transforming growth factor beta (TGFβ) and / or basic fibroblast growth factor (FGF2).

[0009] Preferably, the growth factor includes at least GDF15. More preferably, the growth factor also includes TGFβ and / or FGF2.

[0010] Preferably, the growth factor is expressed in the form of protein or mRNA.

[0011] Preferably, the cell line is derived from human choroid plexus papilloma, and the cell line has contact inhibition ability. Further preferably, the cell line is derived from benign human choroid plexus papilloma, such as benign human choroid plexus papilloma grade I according to the WHO.

[0012] Preferably, the cell line is an immortalized unlimited cell line.

[0013] Preferably, the cell line comprises the SV40 large T antigen fragment, and the cell line has integrated the SV40 large T antigen fragment into the cell nucleus of the cell line.

[0014] Preferably, the cell line expresses epithelial cell characteristic molecules.

[0015] Preferably, the epithelial cell characteristic molecules include KRT, and further preferably, the epithelial cell characteristic molecules include keratin-7 (KRT-7) and keratin-8 (KRT-8).

[0016] Preferably, the epithelial cell characteristic molecules are expressed in the form of proteins or mRNA.

[0017] Preferably, the cell line expresses adherens junction (AJ) proteins and tight junction (TJ) proteins. Further preferably, the adherens junction proteins include e-cadherin (CDH1), the tight junction proteins include claudins (CLDNs), tight junction proteins (TJPs), occludin (OCLN) and / or junctional adhesion molecules (JAMs). Even more preferably, the tight junction proteins include claudin-1 (CLDN1), zonula occludens-1 (ZO-1), occludin (OCLN), claudin-11 (CLDN11) and / or junctional adhesion molecules-A (JAM-A).

[0018] Preferably, the adherens junction protein or tight junction protein is expressed in the form of protein or mRNA.

[0019] In one embodiment of the present application, the cell line expresses CDH1, CLDN1, CLDN11, ZO1, OCLN and / or JAMA (JAM1) at the mRNA level.

[0020] In one embodiment of the present application, the cell line expresses CDH1, CLDN1, ZO-1 and / or OCLN at the protein level.

[0021] Preferably, the cell line expresses characteristic molecules of choroid plexus epithelial cells. Further preferably, the characteristic molecules of choroid plexus epithelial cells include transthyretin (TTR).

[0022] Preferably, the characteristic molecules of the choroid plexus epithelial cells are expressed in the form of protein or mRNA.

[0023] Preferably, the cell line expresses NADPH oxidase (NOX), and preferably, the NADPH oxidase includes NOX4, NOX5, DUOX1 and / or DUOX2.

[0024] Preferably, the characteristic molecules of the choroid plexus epithelial cells are expressed in the form of protein or mRNA.

[0025] Preferably, the cell line expresses the inflammatory factors interleukin-1α (IL1α), interleukin-1β (IL1β), interleukin-18 (IL18), interleukin-6 (IL6) and / or tumor necrosis factor-α (TNFα).

[0026] Preferably, the inflammatory factors are expressed in the form of protein or mRNA.

[0027] Preferably, the cell line expresses the anti-inflammatory factor leukemia inhibitory factor receptor (LIFR).

[0028] Preferably, the anti-inflammatory factor is expressed in the form of protein or mRNA.

[0029] Preferably, the cell morphology is polygonal, multi-angled or bamboo-shaped.

[0030] Preferably, the cell line comprises structures such as tight junctions and / or microvilli structures.

[0031] Preferably, the cell line comprises a loss of heterozygosity at the Amelogenin sex locus.

[0032] Preferably, the cell line is named hCPP (Human-derived Choroid Plexus Papilloma Cell Line), deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 45152 and a deposit date of May 17, 2022.

[0033] In a second aspect, a method for constructing the above-mentioned human choroid plexus papilloma-derived cell line is provided, wherein the method comprises:

[0034] (1) Primary culture: mince the choroid plexus papilloma tissue, separate the tissue cells, and then inoculate and culture the cells for passage;

[0035] (2) Immortality;

[0036] (3) Continuous subculture;

[0037] (4) Identification of biological characteristics and obtaining a cell line derived from human choroid plexus papilloma.

[0038] Preferably, the immortalization includes viral gene transfection method, telomerase activation method, oncogene method or reversion immortalization method.

[0039] Further preferably, the viral gene transfection method comprises using Epstein-Barr virus (EBV), Simian virus 40 (SV40) or human papillomavirus (HPV).

[0040] Further preferably, the oncogene method comprises gene transfection of a proto-oncogene, and the proto-oncogene comprises Myc, c-Jun, c-Ias, vsrc or Mdm2.

[0041] In one embodiment of the present application, the immortalization of the cell line is achieved by transfecting the expression plasmid of SV40 large T antigen using the liposome method.

[0042] In a third aspect, a cell culture or subcloned cells is provided, wherein the cell culture or subcloned cells are obtained by culturing or passage of the above-mentioned cell line derived from human choroid plexus papilloma.

[0043] In a fourth aspect, a tissue or organ or a culture thereof is provided, wherein the tissue or organ or a culture thereof is derived from the above-mentioned human choroid plexus papilloma-derived cell line or the above-mentioned cell culture or subclone cell.

[0044] In a fifth aspect, a use of the above-mentioned human choroid plexus papilloma-derived cell line, the above-mentioned cell culture or subcloned cell, or the above-mentioned tissue or organ or culture thereof is provided, wherein the use comprises:

[0045] 1) Construct a biomimetic model of choroid plexus epithelial cells or tissues;

[0046] 2) Construct an in vitro model of the blood-cerebrospinal fluid barrier;

[0047] 3) Extract various neurotrophic substances synthesized and secreted by the choroid plexus and develop related biological products.

[0048] Preferably, the bionic model or in vitro model is used for biological, medical, toxicological, pharmacological and other research with the choroid plexus and the blood-cerebrospinal fluid barrier as research objects. More preferably, the bionic model or in vitro model is used for drug development research on diseases related to the choroid plexus.

[0049] Preferably, the neurotrophic substances include but are not limited to various proteins, sugars, etc. required in cerebrospinal fluid.

[0050] Preferably, the choroid plexus-related diseases include but are not limited to inflammation, vascular disease, tumor, degeneration, deformity, genetic disease, immune disease, nutritional metabolic disease, poisoning, trauma, and parasitic disease related to the choroid plexus.

[0051] In a sixth aspect, a method for determining the toxicity of a test substance on the blood-cerebrospinal fluid barrier is provided, the method comprising incubating the test substance with the above-mentioned human choroid plexus papilloma-derived cell line or the above-mentioned cell culture or subclone cells and evaluating the viability of the cells.

[0052] Preferably, the test substances include but are not limited to various drugs, poisons or microorganisms.

[0053] Description of terms related to the present invention

[0054] Cell line: A population of cells expanded from one or a few common ancestral cells, such as, but not limited to, a clonal population of cells expanded from a single isolated cell.

[0055] Subcloning: In cell cloning, for cultured cells, screening out cells with certain characteristics from the original clones for cultivation is called subcloning.

[0056] Compared with the prior art, the present invention has the following advantages:

[0057] First, the cell line is an infinite cell line that can be continuously passaged;

[0058] Second, compared with Z310 and SCP cell lines, the cell line of the present application is a human cell line, which avoids the influence of species differences on experimental results;

[0059] Third, the cell line described in this application retains the biological characteristics of choroid plexus epithelial cells, with relatively intact tight junctions and secretory functions. Its malignancy is lower than that of existing human choroid plexus epithelial cells (HIBCPP). This cell line has high application value in basic medical research, biopharmaceutical development, toxicology, and pharmacology, focusing on choroid plexus epithelial cells or the blood-cerebrospinal fluid barrier.

[0060] Deposit information: The cell line was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on May 17, 2022, with the deposit number CGMCC No.45152. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1: Technical roadmap for establishing hCPP cell lines;

[0062] Figure 2: Patient's tumor histopathological diagnosis: choroid plexus papilloma (WHO grade I). Figures A and B show different observation fields, both at a magnification of 400x.

[0063] Figure 3: Morphological observation of hCPP under an optical microscope, 100×, where A1 and A2 are the low-density and high-density cell morphologies of hCPP passage64, respectively; B1 and B2 are the low-density and high-density cell morphologies of hCPP passage104, respectively; C1 and C2 are the low-density and high-density cell morphologies of hCPP passage134, respectively;

[0064] Figure 4: Ultrastructural observation of hCPP under transmission electron microscopy (A: 2500×; B: 2000×);

[0065] Figure 5: hCPP cell growth curve, n = 4;

[0066] Figure 6: STR profiling profiles of cell lines;

[0067] Figure 7: Representative images of karyotype analysis of cell lines;

[0068] Figure 8: mRNA expression of hCPP identification-related indicators, n = 3;

[0069] Figure 9: KRT protein expression levels, where “-”: TK-6 cells, used as negative control, “+”: Hepg2 cells, used as positive control, Pan-KRT represents all KRT antibodies;

[0070] Figure 10: Melting curve of TTR mRNA amplification;

[0071] Figure 11: Amplification curve of TTR mRNA;

[0072] Figure 12: TTR protein expression levels, where “-”: TK-6 cells, used as negative control, and “+”: HepG2 cells, used as positive control;

[0073] Figure 13: Expression of connexins, where "-": TK-6 cells, used as negative control, "+": Hepg2 cells, used as positive control;

[0074] Figure 14: ELISA method for detecting GDF15 secretion by hCPP;

[0075] Figure 15: Representative images of the soft agar colony formation assay to examine the degree of malignant transformation of hCPP cells;

[0076] Figure 16: Representative images of nude mouse tumor-bearing experiments examining the degree of malignant transformation of hCPP cells;

[0077] Figure 17: Mycoplasma contamination detection results in hCPP cell culture supernatant, where "M": DNA Marker, "-": human oral pharyngeal swab - negative control, "+": human oral pharyngeal swab - positive control, "empty": PBS - blank control group, p67, p107, and p137 represent different passage numbers;

[0078] Figure 18: Western Blot analysis of the reactivity of hCCP cells to the neurotoxin manganese chloride, wherein A is the expression level of GDF-15 protein, and B is the relative expression of GDF-15 protein at different manganese chloride concentrations;

[0079] Figure 19: Western Blot analysis of hCCP cell responsiveness to neuroprotective drug stimulation, where A represents the expression level of GDF-15 protein, and B represents the relative expression of GDF-15 protein under stimulation with different drugs. Memantine represents memantine, and amantadine represents amantadine.

[0080] Figure 20: Real-time fluorescence quantitative PCR was used to measure the expression of inflammatory cytokine mRNA in hCCP cells after 24 hours of stimulation with 100 ng / ml LPS, n=3;

[0081] Figure 21: Real-time fluorescence quantitative PCR was used to measure the expression of inflammatory cytokines mRNA in hCCP cells after stimulation with 25, 50, and 100 μM manganese for 24 hours, n=4; Note: *: p < 0.05 compared with the control group or the 25 μM dose group; **: p < 0.01 compared with the control group, the 25 or 50 μM dose group; ***: p < 0.001 compared with the control group, the 25 or 50 μM dose group; NS: p > 0.05 compared with the control group;

[0082] Figure 22: Real-time fluorescence quantitative PCR was used to measure the basal expression of NADPH oxidase (NOX) isoform gene transcription levels in hCCP cells under physiological conditions, n = 3;

[0083] Figure 23: Real-time fluorescence quantitative PCR was used to measure the expression changes of NOX(s) gene transcription levels in hCCP cells after 24 hours of stimulation with 100 μM manganese (n=3); *: p<0.05 compared with the control group. DETAILED DESCRIPTION

[0084] The following describes the implementation of the present application through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present application from the contents disclosed in this specification.

[0085] Before further describing the specific embodiments of this application, it should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of this application are intended to describe specific embodiments and are not intended to limit the scope of protection of this application. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions or the conditions recommended by the respective manufacturers.

[0086] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials in the prior art that are similar or equivalent to those described in the examples may be used to implement this application, based on the prior art knowledge of those skilled in the art and the description of this application.

[0087] Example 1 Preparation of hCPP cell line

[0088] The technical route of this application is shown in Figure 1. First, the patient's choroid plexus papilloma tissue was subjected to primary culture, immortalization, and continuous subculture to obtain a new human choroid plexus papilloma cell line. The biological characteristics of this cell were systematically identified, including the expression levels of characteristic molecules of choroid plexus epithelial cells, identification of tight junction-related molecules, identification of secretory function, as well as growth curves, genetics, morphology, and other indicators. This experiment was conducted with the approval of the Medical Ethics Review Committee and informed consent.

[0089] (1) Organizational Source

[0090] Tissue was obtained from a pathological space-occupying lesion in the third ventricle of a 2-year-old male patient at Beijing Tiantan Hospital, affiliated with Capital Medical University, who was diagnosed with a choroid plexus papilloma (WHO grade I). Choroid plexus tissue, stored at 4°C postoperatively, was retrieved for tissue culture on the day of surgery (March 21, 2016). The pathological diagnosis of the patient's tumor tissue is shown in Figure 2. The intraventricular tumor tissue exhibited an exophytically growing papillary structure, with a small amount of loose connective tissue and mature capillaries forming the core of the papilla. The surface was lined with a single layer of well-differentiated cuboidal epithelial-like tumor cells, with minimal cellular heterogeneity, relatively uniform nuclear alignment, and no infiltration. The lesion was consistent with a choroid plexus papilloma (WHO grade I). The pathological classification was choroid plexus papilloma (WHO grade I).

[0091] (2) Primary culture

[0092] After the tissue is minced, trypsin digestion is used to separate the tissue cells. The steps are as follows:

[0093] (1) Cut the tissue into pieces of about 1 to 3 mm 3 The tissue was divided into small pieces and transferred into a 15 mL centrifuge tube. DMEM was added to wash the tissue pieces to remove blood. After natural sedimentation, the supernatant was removed.

[0094] (2) Add 2 mL of trypsin digestion solution (0.25%) to the tissue block and incubate on a 37°C constant temperature shaker for 5 minutes. After natural precipitation, collect the supernatant.

[0095] (3) The supernatant was transferred to a 50 mL centrifuge tube and 4 mL of fetal bovine serum was added to neutralize the digestion effect of trypsin; 2 mL of trypsin digestion solution was added to the precipitate for further digestion.

[0096] (4) Repeat the above steps 5 to 10 times until the tissue block is completely digested.

[0097] (5) Collect the supernatant from each time, centrifuge at 300g for 10 min, remove the supernatant, add an appropriate amount of complete culture medium (DMEM medium containing 10% FBS, 10 ng / mL human epidermal growth factor, 100 U / mL penicillin, and 100 μg / mL streptomycin) to resuspend the cells.

[0098] (6) The cell suspension was passed through a 70 μm cell sieve to remove tissue debris, and then the cells were counted and resuspended to 1×10 6 pieces / mL.

[0099] (7) The cells were seeded in a 10 cm dish and cultured at 37°C and 5% CO2.

[0100] (8) The complete culture medium was replaced every 3 to 4 days, and the cells were passaged when they proliferated to about 90% confluence.

[0101] (3) Cell immortalization

[0102] The cultured cells were immortalized by transfection with the SV40 large T antigen expression plasmid (pLenti-EF1a-SV40 Large T) using the lipofectamine 3000 method. The steps are as follows:

[0103] (1) The primary culture was subcultured and seeded into 6-well plates.

[0104] (2) Transfection was performed after the cells grew to a confluence of 70% to 80%.

[0105] (3) Take a 0.6 mL centrifuge tube, mark it as "A", add 125 μL Opti-MEM medium and 7.5 μL Lipofectamine 3000 reagent, and mix thoroughly.

[0106] (4) Take a 0.6 mL centrifuge tube, mark it as "B", and add 125 μL Opti-MEM medium, 2.5 μg pLenti-EF1a-SV40 Large T plasmid and 5 μL P3000 reagent, and mix thoroughly.

[0107] (5) Mix the reagents in tubes A and B and incubate at room temperature for 10 to 15 minutes.

[0108] (6) Replace the cell culture medium by adding 2 mL of complete culture medium. Then add the mixture to the culture medium and mix thoroughly.

[0109] (7) The cells were cultured in a 37°C, 5% CO2 incubator for subsequent serial passage.

[0110] (IV) Continuous subculture

[0111] The transfected primary culture was continuously cultured for six years using conventional subculture methods, reaching over 130 generations. During this process, the cell culture maintained stable morphology and good growth, confirming that it is a novel, immortalized cell line derived from human choroid plexus papilloma tissue and designated the hCPP (Human-derived Choroid Plexus Papilloma Cell Line).

[0112] Example 2 Biological Characterization of hCPP Cell Line

[0113] Tight junctions between human choroid plexus epithelial cells are a core component of the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells also have the important function of secreting cerebrospinal fluid. Therefore, the biological characterization of this cell line will focus closely on these two functions. Three passages of hCPP cells were systematically characterized using morphological, molecular, and cell biological indicators to clarify their biological characteristics and to assess the stability of these characteristics through comparison of cells across the three passages.

[0114] The present invention detects cell morphology, growth dynamics, genetics, malignancy, etc. respectively to determine the biological characteristics of the cell line.

[0115] 1. Morphological characteristics

[0116] Cell morphology was observed using an optical microscope, and ultrastructure, including microvilli, was observed using an electron microscope. Under a light microscope, hCPP cells appeared polygonal (edge-shaped) or bamboo-shaped; at medium to high confluence, the cells interconnected to form a "netting" pattern. As shown in Figure 3, cells from all three passages were round or oval in shape, exhibiting a uniform polygonal epithelial morphology and a cobblestone-like mosaic arrangement. Furthermore, contact inhibition was observed when hCPP cells reached high cell density, suggesting a low degree of malignancy.

[0117] hCPP cells were observed under a transmission electron microscope, as shown in Figure 4. The black arrow in Figure 4A points to an obvious junction-like structure, which is a structure that only exists between epithelial and endothelial cells. Figure 4B shows clearly visible microvilli on the cell surface, which are involved in the secretory function of the cells. In addition, basic cell structures such as the cell nucleus and abundant mitochondria were also observed under the electron microscope.

[0118] 2. Cell Growth Kinetics

[0119] A real-time cell assay (RTCA) system was used to monitor cell replication and proliferation. The software calculated the population doubling time of the cells based on the growth curve, as shown in Figure 5. The population doubling time was 16.0 ± 1.3 h.

[0120] 3. Genetic characterization

[0121] (1) STR genotype identification

[0122] Using short tandem repeats (STRs) as genetic markers for cell typing is one of the most effective and accurate methods for detecting cell cross-contamination and identifying cell properties. STR genotyping has been strongly recommended for cell identification by organizations such as the ATCC. The ATCC Cell Bank in the United States, the DSMZ Cell Bank in Germany, and the JCRB Cell Bank in Japan have provided data on various cell lines for comparison using STR typing.

[0123] STR loci consist of short, tandemly repeated sequences of 3 to 7 base pairs in length. These repeats are ubiquitous in the human genome and serve as highly polymorphic markers, known as the DNA fingerprint of cells. They can be detected using PCR (polymerase chain reaction). Alleles at STR loci can be distinguished by the number of copies of the repeat sequence within the amplified region. After capillary electrophoresis separation, they can be identified using fluorescence detection. STR typing results can then be compared with specialized cell STR databases using specific calculation methods to infer the cell line to which the sample belongs, or the name of any possible cross-contamination.

[0124] The STR identification results of the hCPP cells constructed in Example 1 and their tissue sources are shown in FIG6 and Table 1. The results show that:

[0125] Table 1 Genotyping results of hCPP (p64) (STR analysis, 9 loci recognized by ATCC)

[0126] 1) No cross-contamination of other human cells was found in hCPP cells, indicating that they are a single-source human cell line;

[0127] 2) Comparison of the peak results of 21 STR loci in hCPP with those in the patient's tumor tissue revealed complete consistency across all 20 STR loci, with the exception of the sex locus, indicating that the cell line was derived from the patient's tumor tissue and that hCPP cells exhibited loss of heterozygosity (LOH) at the sex locus, Amelogenin.

[0128] 3) The STR typing results of hCPP were compared with cells in the STR database of ATCC in the United States and DSMZ in Germany. No cell line with exactly the same loci as this cell line was found, indicating that the hCPP cell line is a completely new cell line.

[0129] (2) Chromosome karyotype analysis

[0130] Karyotype analysis focuses on metaphase chromosomes. Using banding techniques, the chromosomes are analyzed, compared, sorted, and numbered based on characteristics such as chromosome length, centromere location, long-to-short arm ratio, and the presence of satellites. Diagnosis is based on variations in chromosome structure and number. Karyotype analysis provides an important basis for studying cytogenetic classification, chromosome number, and structural variation, and can determine a cell's genomic ploidy and chromosomal aberrations.

[0131] The results of karyotype analysis of the hCPP cells constructed in Example 1 are shown in Figure 7, Table 2 and Table 3. The results show that the 20 mitotic phases randomly selected for analysis were all diploid karyotypes, and the cell line had certain numerical and structural abnormalities, as follows: 39-46, X, -Y, add(5)(q35), -6, -7, add(8)(q24), +11, add(12)(q24), der(14; 15)(q10; q10), add(15)(p11), add(15)(p11), der(15), -19, -21, i(21)(q10), -22, +mar1.

[0132] Table 2 Abnormal distribution of hCPP (p72) chromosome numbers Note: All 20 cell division phases are diploid karyotypes

[0133] Table 3 Distribution of major structural abnormalities of hCPP (p72) chromosomes Note: “-”: missing chromosome; “add”: chromosome fragment of unknown origin attached to the chromosome band; “i”: isochromosome; “+mar”: marker chromosome, a chromosome with structural abnormality that cannot be identified by banding method.

[0134] 4. Detection of Characteristic Molecules in Choroid Plexus Epithelial Cells

[0135] First, real-time PCR and Western blot experiments showed that hCPP expressed high levels of KERATIN-7 and 8. KERATIN is recognized as an intermediate filament protein expressed by epithelial cells, as shown in Figures 8-9 and Table 5, suggesting that the cell line is of epithelial tissue origin. The primers used in the PCR process are shown in Table 4.

[0136] Table 4 Real-time fluorescence quantitative PCR primer sequences

[0137] Table 5 KRT mRNA expression, n=3

[0138] In addition, hCPP also expresses TTR, a characteristic molecule of choroid plexus epithelial cells, as shown in Figures 8, 10-12, and Table 6, demonstrating that this cell line is derived from choroid plexus epithelial tissue and retains certain characteristics of choroid plexus epithelial cells. The TTR primers used in the PCR process are shown in Table 4.

[0139] Table 6 TTR mRNA expression, n=3

[0140] 5. Cell tight junctions

[0141] Tight junctions between cells are impermeable junctions, ubiquitous in various epithelial and endothelial cells of vertebrates. In the region of the tight junction between two adjacent cells, the plasma membrane forms cords. These cylindrical cords, held together by specialized proteins and divalent cations, seal the intercellular space, preventing the passage of macromolecules while allowing only water molecules and ions to pass through the small pores where the cords meet. The cords form a network of interwoven plasma membrane bands, increasing the area and density of tight junctions and more effectively preventing the passage of macromolecules between cells. Furthermore, the cords are connected to filaments within the cytoplasm, enhancing cell toughness and providing mechanical support for the tight junctions. Each cord is composed of a series of transmembrane protein complexes embedded in two plasma membranes. The extracellular domains of these proteins directly connect them to each other.

[0142] The protein components that make up the tight junction transmembrane protein complex include CLAUDINs, ZOs, OCCLUDINs, and / or JAMs. Real-time PCR and Western blot techniques were used to detect the expression levels of tight junction-related genes in hCPP cell lines. The results are shown in Figure 8 and Table 7. At the mRNA level, hCPP cells express CDH1, CLDN1, CLDN11, ZO1, OCLN, and JAMA. The primer sequences are shown in Table 4. At the protein level, as shown in Figure 13, hCPP cells express junction-related proteins such as ZO-1, OCLN, CDH1, and CLDN1 at certain levels.

[0143] Table 7 Expression of some connexin mRNA, n=3

[0144] 6. Cerebrospinal fluid secretion function

[0145] Choroid plexus epithelial tissue is the primary source of cerebrospinal fluid, and the various protein molecules it secretes have the function of nourishing neurons. We selected four secreted proteins characteristic of choroid plexus epithelial cells for testing, including three nutritional factors: TGFβ, GDF15, and FGF2. At the mRNA level, hCPP cells expressed GDF15, TGFβ, and FGF2, as shown in Figure 8 and Table 8. The primer sequences are shown in Table 4. At the protein level, this application also used ELISA to detect the expression of GDF15 protein, as shown in Figure 14.

[0146] Table 8 Secretory protein mRNA expression, n=3

[0147] 7. Other features

[0148] (1) Soft agar colony formation assay and nude mouse tumor bearing assay were used to evaluate the malignancy of the cell lines.

[0149] Normal cells rely on contact with the extracellular matrix to grow and divide. In contrast, cells with a high malignancy have the ability to grow and differentiate regardless of their surrounding environment. Therefore, cells that can form colonies in an anchorage-independent manner are considered to be more malignant to a certain extent. The overall goal of the soft agar colony formation assay is to semi-quantitatively determine the malignancy of cells by combining the results of other experiments. The colony formation rate = (number of colonies / number of cells seeded) × 100%. The results showed that the colony formation rate of hCPP (passage64+) cells was 0.79% ± 0.47% (n = 3). The colony formation images are shown in Figure 15.

[0150] Nude mouse tumor-bearing experiments involve transplanting cells or tissues orally or subcutaneously into nude mice, allowing them to form tumors. Balb / c nude mice are currently a commonly used nude mouse tumor-bearing model for in vivo evaluation of cell malignancy. Due to their congenital thymic defect and lack of T lymphocyte function, they hold great research value in oncology, immunology, safety evaluation of pharmaceuticals and biologics, and screening for effective drugs. Results showed that compared to the positive control group (100% tumor formation rate), the hCPP nude mouse tumor formation rate was 0%. Typical results from nude mouse tumor-bearing experiments are shown in Figure 16.

[0151] The results of soft agar colony formation assay and nude mouse tumor bearing assay indicated that the cells had a weak ability to grow in an anchored manner and were not tumorigenic. The malignancy of hCPP was low.

[0152] (3) PCR method was used for nucleic acid detection of mycoplasma.

[0153] The supernatants of hCPP cell cultures at passages 67, 107, and 137 were tested using primers shown in Table 4. The test results are shown in FIG17 , which showed that the nucleic acid of mycoplasma was negative, indicating that the hCPP cells were free of mycoplasma contamination.

[0154] Example 3 Responsiveness of hCPP Cells to Toxicological and Pharmacological Exogenous Factors

[0155] GDF15 has important physiological functions, including neurotrophic and neuroprotective effects on dopamine neurons. It is closely associated with the development of brain dysfunction and neurodegenerative diseases. Furthermore, choroid plexus epithelial cells (CPEs) are the primary source of GDF15 in the central nervous system. Therefore, CPEs are frequently studied in neurodegenerative diseases, stroke / cerebral hemorrhage, and traumatic brain injury. In this study, we measured GDF15 expression in hCPP cells and observed changes in GDF15 expression in response to various toxicants (such as heavy metal manganese) and neuroprotective drugs (memantine and amantadine). Using GDF15 as a marker, we assessed the responsiveness of hCPP cells to various toxicological and pharmacological exogenous factors. The results are shown below.

[0156] hCPP cells were seeded in six-well plates and treated when they were in the logarithmic growth phase and reached approximately 80% confluence. Experiment 1: The toxic effects of heavy metal manganese (Mn) were investigated using hCPP. The treatment groups were as follows: 0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM. Experiment 2: The effects of neuroprotective drugs (memantine and amantadine) were investigated using hCPP. The treatment groups were as follows: control group (no substance added), memantine 20 μM, and amantadine 30 μM. After 24 hours of treatment with exogenous factors, cell samples were lysed, sonicated, centrifuged, and denatured before Western blotting to determine the level of GDF-15 in the cell lysates.

[0157] In this experiment, hCPP cells were stimulated with 25μM, 50μM, 75μM, 100μM, 150μM, and 200μM manganese to assess changes in GDF15 expression in hCPP cells. The results, shown in Figure 18, show that GDF-15 concentrations in hCPP whole-cell lysates increased with increasing Mn exposure, with statistically significant differences observed at doses of 50μM and 75μM (P < 0.05), indicating that Mn, at certain doses, can upregulate GDF-15 expression in hCPP cells. Because the blood-cerebrospinal fluid barrier (BCB / CP) serves as a crucial pathway for Mn transport into the brain following excessive Mn exposure, and GDF-15 plays important physiological roles in vivo, the establishment of human choroid plexus epithelial cell-derived hCPPs can better investigate the pathogenesis of neurodegenerative diseases and can be applied to investigate the mechanisms of action of Mn-induced changes in GDF15 expression.

[0158] hCPP cells were stimulated with 20 μM memantine and 30 μM amantadine, respectively, to examine changes in GDF15 expression in hCPP cells under the effects of these neuroprotective drugs. The results, shown in Figure 19, show that GDF-15 expression in hCPP cells was slightly upregulated relative to the control group under the effects of 20 μM memantine, but was downregulated under the effects of 30 μM amantadine. These results suggest that the AD drug memantine may exert its protective effect by upregulating GDF-15 expression in CP cells, while the PD drug amantadine may not be involved in the regulatory mechanisms related to GDF-15 in CP cells, or that these results are due to the short duration of drug action (24 hours). However, due to the structure and function of the blood-cerebrospinal fluid barrier (BCB) / CP, human choroid plexus cells (hCPP) can still be used to investigate the mechanisms of drug entry into the brain parenchyma.

[0159] Example 4 Inflammatory responsiveness of hCPP cells to exogenous neurostimulatory factors (eg, LPS and manganese)

[0160] After hCPP cells were treated with 100 ng / ml lipopolysaccharide (LPS) for 24 hours, real-time quantitative PCR was used to measure transcriptional expression of the inflammatory factors IL1α, IL1β, IL18, IL6, and TNFα in hCPP cells. This trend was shown in Figure 20. Furthermore, heavy metal manganese (Mn) compounds have demonstrated neurotoxicity and can contribute to neurodegenerative diseases. Their toxic effects involve complex regulatory mechanisms. Although Mn has been reported to induce structural and functional changes in CP cells, inducing the secretion of inflammatory cytokines by microglia in the brain parenchyma, and further leading to inflammatory neuronal damage, the role of CP in this inflammatory process remains unknown. Whether excessive exposure to heavy metal Mn can induce an inflammatory response in CP has not been reported. Real-time quantitative PCR was also used to measure transcriptional changes in the expression of related inflammatory cytokine genes in hCPP cells after 24 hours of stimulation with 25, 50, and 100 μM Mn. The results are shown in Figure 21. In the 100 μM Mn dose group, the gene transcription levels of pro-inflammatory factors (IL-1α, IL-1β, IL-18, IL-6, TNF-α) in the cells were significantly increased; while the gene expression of the anti-inflammatory factor leukemia inhibitory factor receptor (LIFR) was significantly reduced.

[0161] The above results found for the first time that LPS and Mn can induce hCPP cells to secrete inflammatory cytokines. These important experimental results suggest that CP / BCB has inflammatory reactivity and is likely to serve as a special immune site in the CNS, participating in the immune regulation of the CNS, and even serving as the initial occurrence point of immune inflammatory response. After being stimulated by exogenous factors, it secretes inflammatory factors and transmits inflammatory factors into the brain parenchyma through CSF, further having the immune regulatory function of inducing, initiating and promoting inflammatory responses in the microglia and neurons of the CNS.

[0162] In the study of the molecular mechanisms of chronic inflammatory responses in the CNS, among the seven isoforms of NADPH oxidase (NOX), NOX2 is a key regulator of chronic neuroinflammation and progressive neurodegeneration caused by microglial activation. Increased NOX2 expression in neurons also promotes inflammatory neurodegeneration. However, there are no reports on the NOX isoforms in CP / BCB and their effects on immune regulation.

[0163] Next, we examined the basal expression of NOX(s) gene transcripts, a regulatory factor associated with the inflammatory response in hCPP. The results are shown in Figure 22. Of the seven NOX(s) primers amplified, hCPP only expressed four isoforms: NOX4, NOX5, DUOX1, and DUOX2. The primer sequences are shown in Table 9. Next, we examined changes in the transcriptional expression of NOX(s) genes, a regulatory factor associated with the inflammatory response, after 24 hours of stimulation with 100 μM manganese. As shown in Figure 23, NOX4 gene transcript expression increased significantly after 24 hours of stimulation with 100 μM manganese, and the other isoforms showed an increasing trend in expression.

[0164] Table 9 Real-time fluorescence quantitative PCR primer sequences for seven NADPH oxidase (NOX) isoforms

[0165] In summary, CP cells have the dominant expression of four subtypes: NOX4, NOX5, DUOX1, and DUOX2, and can induce the production of proinflammatory cytokines under exogenous stimulation. These characteristics make CP / BCB the "starting site and initiator" in the CNS inflammatory response and immune response chain, with important physiological, pathological, and toxicological significance and research value. They are also important targets for various drug development studies targeting the CNS for therapeutic effects and regulation of toxic side effects.

[0166] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A cell line derived from human choroid plexus papilloma, characterized in that: The cell line expresses growth factors, and the growth factors include GDF15, FGF2 and / or TGFβ.

2. The cell line according to claim 1, characterized in that The growth factors include at least GDF15. Preferably, the growth factors also include FGF2 and / or TGFβ.

3. The cell line according to claim 1 or 2, characterized in that The cell line has contact inhibition ability.

4. The cell line according to any one of claims 1 to 3, characterized in that The cell line is an immortalized unlimited cell line. Preferably, the cell line comprises the large T antigen fragment of SV40.

5. The cell line according to any one of claims 1 to 4, characterized in that The cell line expresses epithelial cell characteristic molecules. Preferably, the epithelial cell characteristic molecules include KRT. More preferably, the epithelial cell characteristic molecules include KRT-7 and KRT-8.

6. The cell line according to any one of claims 1 to 5, characterized in that The cell line expresses adhesion junction proteins and tight junction proteins. Preferably, the adhesion junction proteins include CDH1, and the tight junction proteins include CLDNs, TJPs, OCLN and / or JAMs. Further preferably, the tight junction proteins include CLDN1, ZO-1, OCLN, CLDN11 and / or JAMA.

7. The cell line according to any one of claims 1 to 6, characterized in that The cell line expresses characteristic molecules of choroid plexus epithelial cells. Preferably, the characteristic molecules of choroid plexus epithelial cells include TTR.

8. The cell line according to any one of claims 1 to 7, characterized in that The cell line expresses NADPH oxidase, and preferably, the NADPH oxidase includes NOX4, NOX5, DUOX1 and / or DUOX2.

9. The cell line according to any one of claims 1 to 8, characterized in that The cell line expresses inflammatory factors IL1α, IL1β, IL18, IL6 and / or TNFα.

10. The cell line according to any one of claims 1 to 9, characterized in that The cell line expresses the anti-inflammatory factor LIFR.

11. The cell line according to any one of claims 1 to 10, characterized in that The cells include structures such as tight junctions and / or microvilli.

12. The cell line according to any one of claims 1 to 11, characterized in that The growth factors, epithelial cell characteristic molecules, adhesion junction proteins, tight junction proteins, choroid plexus epithelial cell characteristic molecules, NADPH oxidase, inflammatory factors or anti-inflammatory factors are expressed in the form of proteins or mRNA.

13. The cell line according to any one of claims 1 to 12, characterized in that The cell line is named hCPP, and its deposit number is CGMCC No.45152.

14. The method for constructing a cell line according to any one of claims 1 to 13, characterized in that: The construction method comprises: (1) Primary culture: mince the choroid plexus papilloma tissue, separate the tissue cells, and then inoculate and culture the cells for passage; (2) Immortality; (3) Continuous subculture; (4) Identification of biological characteristics and obtaining a cell line derived from human choroid plexus papilloma.

15. The construction method according to claim 14, characterized in that: The immortalization methods include viral gene transfection, telomerase activation, oncogene or reversion immortalization.

16. A cell culture or subclone cell, characterized in that The cell culture or subclone cells are obtained by culturing or subculturing the cell line derived from the human choroid plexus papilloma according to any one of claims 1-13.

17. A tissue or organ or a culture thereof, characterized in that: The tissue or organ or its culture is derived from the human choroid plexus papilloma-derived cell line according to any one of claims 1 to 13 or the cell culture or subclone cell according to claim 16.

18. A use of the human choroid plexus papilloma-derived cell line according to any one of claims 1 to 13, the cell culture or subclone cell according to claim 16, or the tissue or organ or culture thereof according to claim 17, the use comprising: 1) Construct a bionic model of choroid plexus epithelial cells or tissues; 2) construct an in vitro model of the blood-cerebrospinal fluid barrier; 3) Extract various neurotrophic substances synthesized and secreted by the choroid plexus and develop related biological products; Preferably, the bionic model or in vitro model is used for biological, medical, toxicological, pharmacological and other research with the choroid plexus and the blood-cerebrospinal fluid barrier as research objects. More preferably, the bionic model or in vitro model is used for drug development research on diseases related to the choroid plexus.

19. A method for determining the toxicity of a test substance on the blood-cerebrospinal fluid barrier, characterized in that The method comprises incubating the test substance with the human choroid plexus papilloma-derived cell line according to any one of claims 1 to 13 or the cell culture or subclone cell according to claim 16 and evaluating the viability of the cells.

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