Glioma cell line GL261-EGFP-luc suitable for monitoring glioma growth in vitro and in vivo and construction method and application thereof

Abstract

This invention discloses a glioma cell line suitable for in vitro and in vivo monitoring of glioma growth, its construction method, and its applications. Based on GL261-luc, GL261-EGFP-luc is constructed to achieve in vitro and in vivo monitoring of glioma growth. By constructing an orthotopic glioma transplantation animal model using GL261-EGFP-luc, in vivo imaging techniques based on the interaction between luciferase and luciferin can be used to study the dynamic development and changes of glioma cells in the mouse brain, and in vitro, the distribution of glioma cells in tumor tissue sections can be studied using EGFP. The GL261-EGFP-luc constructed in this invention not only retains the ability of GL261-luc cells to monitor glioma growth in vivo, but also allows for precise cellular-level examination of tumor tissue sections in vitro, clearly distinguishing glioma boundaries. This provides a more precise and efficient glioma cell line for glioma research, enabling precise localization of each cancer cell and providing an efficient tool for precision treatment of gliomas.

Description

Technical Field

[0001] This invention belongs to the fields of biology and oncology, specifically relating to a method for constructing and applying a bioluminescent in vivo imaging glioma cell line. Background Technology

[0002] Gliomas are among the most common primary tumors of the central nervous system, characterized by high recurrence rates, poor prognosis, and high mortality, posing a serious threat to patients' survival time and quality of life. However, the medical community currently lacks highly effective treatments for gliomas. In recent years, the incidence of brain tumors has been rising continuously, and many brain tumor cases, especially astrocytomas, are difficult to cure surgically at the time of diagnosis, and postoperative prognosis is also poor. Therefore, early diagnosis and treatment of gliomas are crucial for prolonging patient survival.

[0003] Currently, clinical diagnostic techniques for gliomas primarily rely on large-scale instruments such as magnetic resonance imaging (MRI) and computed tomography (CT). These techniques can only locate and characterize the lesion area. In medical research, gliomas are mainly studied using tissue sections and conventional immunohistochemical staining for imaging, but these methods cannot dynamically monitor the development and changes of tumor tissue in living tissue. Furthermore, practical tools for simultaneously monitoring glioma development and changes in vivo and in vitro in living animals are lacking, making it difficult to accurately identify the invasive boundaries of gliomas. This poses significant challenges to glioma research, medical diagnosis, and even treatment.

[0004] In recent years, the application of bioluminescence imaging in in vivo animal imaging has enabled the direct observation of changes in tumor tissue within living animals. Furthermore, bioluminescence, through the interaction of luciferase and luciferin, produces fluorescence that is directly proportional to the size of the tumor tissue and exhibits a good linear relationship. Because bioluminescence imaging does not require excitation light and has high sensitivity (capable of monitoring 10⁻⁶ tumors), it is highly effective. 2 Bioluminescence imaging has advantages such as high specificity and large scale, and more importantly, it uses non-ionizing low-energy radiation, which is harmless to organisms and suitable for long-term, continuous, and real-time monitoring. However, after removing tumor tissue, it is impossible to accurately locate and efficiently distinguish tumor cells from tumor tissue slices, nor can it distinguish dormant tumor cells and drug efficacy.

[0005] Therefore, there is an urgent need in this field to further modify the existing luciferase-carrying mouse glioma (GL261-luc) to create a new glioma cell line that can monitor glioma growth in vitro and in vivo, and to use this new glioma cell line to construct corresponding animal models to achieve precise in vitro localization and observation of the distribution and density of tumor cells in tumor tissue sections. Summary of the Invention

[0006] The purpose of this invention is to construct a glioma cell line GL261-EGFP-luc that expresses enhanced green fluorescent protein and luciferase, and to use this cell line to construct an animal model of orthotopic transplantation of glioma cells, so as to monitor and study gliomas in vivo and in vitro with the help of bioluminescence in vivo imaging technology and fluorescence microscopy.

[0007] In a first aspect, the present invention provides a glioma cell line comprising a fluorescent reporter gene and a luciferase gene, thereby expressing a fluorescent reporter protein and a luciferase.

[0008] In another preferred embodiment, the glioma cell line comprises a mouse glioma cell line.

[0009] In another preferred embodiment, the mouse glioma cell line is GL261-EGFP-luc.

[0010] In another preferred embodiment, the mouse glioma cell line further comprises a promoter and an antibiotic resistance gene.

[0011] In another preferred embodiment, the promoter is the Ubi promoter.

[0012] In another preferred embodiment, the antibiotic resistance gene is the hygromycin B resistance gene.

[0013] In another preferred embodiment, the fluorescent reporter gene includes an enhanced green fluorescent protein gene.

[0014] In another preferred embodiment, the fluorescent reporter protein includes enhanced green fluorescent protein (EGFP).

[0015] A second aspect of the present invention provides a method for constructing the glioma cell line described in the first aspect of the present invention, comprising the steps of:

[0016] (a) Provide a glioma cell line;

[0017] (b) Upregulating the expression of fluorescent reporter gene and luciferase in the glioma cell line, wherein the upregulation includes delivering a vector containing a fluorescent reporter gene and a vector containing luciferase into the glioma cell line;

[0018] (c) The glioma cell lines are screened to obtain glioma cell lines expressing fluorescent reporter genes and luciferase.

[0019] In another preferred embodiment, the fluorescent reporter gene includes an enhanced green fluorescent protein gene.

[0020] In another preferred embodiment, the vector includes a plasmid vector or a viral vector.

[0021] In another preferred embodiment, the viral vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0022] In another preferred embodiment, the vector is a lentiviral vector.

[0023] In another preferred embodiment, the glioma cell line comprises a mouse glioma cell line.

[0024] In another preferred embodiment, the construction method includes the steps of:

[0025] (i) Provide a glioma cell line containing a luciferase carrier;

[0026] (ii) Delivering a vector containing a fluorescent reporter gene into the glioma cell line;

[0027] (iii) The glioma cell lines are screened to obtain glioma cell lines expressing fluorescent reporter genes and luciferase.

[0028] In another preferred embodiment, the glioma cell line containing the luciferase carrier is GL261-luc.

[0029] In another preferred embodiment, a vector containing a fluorescent reporter gene and a vector containing luciferase are delivered into the glioma cell line in the presence of a transfer aid.

[0030] In another preferred embodiment, a lentiviral vector containing a fluorescent reporter gene and a lentiviral vector containing luciferase are delivered into the glioma cell line in the presence of a transfer aid.

[0031] In another preferred embodiment, the conversion aid comprises polyacrylamide.

[0032] In another preferred embodiment, the conversion aid is polyacrylamide.

[0033] In another preferred embodiment, the concentration of the polyaluminum amine is 6-8 μg / mL, more preferably 7.5 μg / mL.

[0034] In another preferred embodiment, the glioma cell lines are screened in the presence of antibiotics to obtain glioma cell lines expressing fluorescent reporter genes and luciferase.

[0035] In another preferred embodiment, the antibiotic includes hygromycin B.

[0036] In another preferred embodiment, the concentration of hygromycin B is 700-800 μg / mL, more preferably 750 μg / mL.

[0037] A third aspect of the present invention provides a method for constructing an animal model of glioma cell transplantation, comprising the steps of:

[0038] (1) Provide an individual animal for transplantation;

[0039] (2) Transplanting the glioma cell line described in the first aspect of the present invention into the animal individual.

[0040] In another preferred embodiment, the animal individuals include mice, rats, rabbits, dogs, monkeys, and pigs.

[0041] In another preferred embodiment, the animal is a mouse.

[0042] In another preferred embodiment, the mouse is a C57BL / 6 mouse.

[0043] In another preferred embodiment, the C57BL / 6 mouse is a 6-8 week old mouse.

[0044] In another preferred embodiment, the C57BL / 6 mouse weighs 18-20g.

[0045] In another preferred embodiment, the transplantation is an in situ transplantation.

[0046] In another preferred embodiment, the glioma cell line is transplanted in situ into the hippocampus of the mouse brain.

[0047] In another preferred embodiment, the glioma cell line is transplanted in situ to a position 1.5±0.2 mm posterior to the anterior fontanelle, 1.8±0.2 mm lateral to it, and 1.7±0.2 mm deep in the mouse brain.

[0048] A fourth aspect of the present invention provides a method for monitoring the growth of gliomas in vitro and in vivo, comprising the steps of:

[0049] (A) Transplanting the glioma cell line described in the first aspect of the present invention into an animal individual, thereby causing the glioma cell line to proliferate in the animal individual;

[0050] (B) Inject the animal individual with luciferase substrate to allow the luciferase substrate to react with luciferase, and use an in vivo optical imaging system to acquire images of the animal individual to obtain information on the growth of glioma.

[0051] (C) Obtain tissue sections from the animal individual and observe the spatial distribution of the glioma cell line under the excitation wavelength of a fluorescence microscope to obtain information on glioma growth.

[0052] In another preferred embodiment, the animal individual includes a mouse.

[0053] In another preferred embodiment, the mouse is a C57BL / 6 mouse.

[0054] In another preferred embodiment, the glioma cell line is transplanted into the brain of the C57BL / 6 mouse.

[0055] In another preferred embodiment, the duration of the proliferation is 7-21 days, more preferably 14-21 days, and even more preferably 21 days.

[0056] In another preferred embodiment, the luciferase substrate comprises luciferin potassium salt.

[0057] In another preferred embodiment, the injection dose of the fluorescein potassium salt is 100-200 mg / kg, more preferably 150 mg / kg.

[0058] In another preferred embodiment, the reaction duration is 2 ± 0.5 min.

[0059] In another preferred embodiment, the excitation wavelength of the fluorescence microscope is 488 nm.

[0060] In another preferred embodiment, the fluorescent reporter gene includes an enhanced green fluorescent protein gene.

[0061] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0062] In a fifth aspect, the present invention provides a formulation or composition comprising the glioma cell line described in the first aspect of the present invention.

[0063] In another preferred embodiment, the formulation or composition further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0064] In another preferred embodiment, the dosage form of the formulation or composition is a liquid formulation.

[0065] In another preferred embodiment, the dosage form of the preparation or composition is an injectable dosage form.

[0066] In another preferred embodiment, the carrier, diluent, or excipient includes a culture medium.

[0067] In another preferred embodiment, the culture medium is DMEM medium.

[0068] In another preferred embodiment, the density of the glioma cell line in the formulation or composition is 1 × 10⁻⁶. 6 per mL.

[0069] In a sixth aspect, the present invention provides a kit comprising one or more components selected from the following: the glioma cell line described in the first aspect of the present invention or the formulation or composition described in the fifth aspect of the present invention.

[0070] In another preferred embodiment, the kit also includes a label or instructions.

[0071] The seventh aspect of the present invention is the use of the glioma cell line described in the first aspect of the present invention, the formulation or composition described in the fifth aspect of the present invention, or the kit described in the sixth aspect of the present invention for the preparation of a medicament for the diagnosis or treatment of glioma.

[0072] In another preferred embodiment, the use also includes research on the mechanisms of glioma development.

[0073] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0074] Figure 1 The diagram shows the gene map of the lentiviral vector transfecting GL261 into GL261-luc, the gene map of the lentiviral vector Ubi-MCS-SV40-EGFP-IRES-Hygromycin used in this invention, and a schematic diagram of the procedure for transfecting GL261-luc (with the characteristic gene: CMV-luc-puro) cells using the lentiviral vector Ubi-MCS-SV40-EGFP-IRES-Hygromycin.

[0075] Figure 2 The results of microscopic observation of the inactivation of GL261-luc by hygromycin B at concentrations of 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, and 1000 μg / mL were shown to screen for the lowest concentration that could kill GL261-luc.

[0076] Figure 3 Images of GL261-EGFP-luc selected with hygromycin B, observed using a fluorescence microscope, are shown. Image A shows GL261-luc under a fluorescence microscope. Image B shows GL261-EGFP-luc selected with hygromycin B 72 hours after lentiviral transfection. Image C shows GL261-EGFP-luc on day 7 after hygromycin B selection 72 hours after lentiviral transfection.

[0077] Figure 4The signals of GL261-luc and GL261-EGFP-luc in the FITC channel were displayed during flow cytometry to verify the efficiency of lentiviral transfection and hygromycin B screening.

[0078] Figure 5 The results of signal acquisition by the PerkinElmer IVIS Lumina II small animal in vivo optical imaging system after GL261-luc and GL261-EGFP-luc were reacted with luciferase in vitro were shown. The imaging efficiency of the two was found to be the same, indicating that the introduction of the EGFP gene does not affect the luciferase function of GL261-EGFP-luc.

[0079] Figure 6 The display shows 5×10 3 GL261-EGFP-luc was injected into the brain of mice via stereotactic injection. Dynamic monitoring using bioluminescent in vivo imaging was performed on days 7, 14, and 21 to reflect the growth of the brain tumor. On day 21, the brain tumor was removed, weighed, and linear regression analysis was performed using the fluorescence signal.

[0080] Figure 7 The results of frozen tissue sections (40 μm) collected from tumor-bearing mice on day 21 are shown. The distribution of tumor cells was observed under a fluorescence microscope. Detailed Implementation

[0081] Through thorough and extensive experiments, the inventors successfully integrated the enhanced green fluorescent protein (EGFP) reporter gene into a glioma cell line originally used for bioluminescent in vivo imaging by transfecting it with a vector carrying EGFP. Using this cell line, a stable orthotopic glioma transplantation animal model was constructed. Bioluminescent in vivo imaging technology was then used to observe the dynamic development and changes of glioma cells in vivo in real time, as well as to precisely locate the distribution of tumor cells in vitro. This invention not only achieves high-resolution imaging of glioma cells in vivo and tracking the spread and invasion of tumor cells in vitro, but also provides a powerful tool for studying the mechanisms of glioma development and the development of glioma therapeutics. This invention was completed based on these findings.

[0082] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0083] the term

[0084] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value 20, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered by this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0085] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0086] As used herein, the terms "glioma cell line," "glioma cell line," and "glioma cell" are used interchangeably and all refer to tumor cells or tumor cell populations derived from glial cells. Glial cells are supporting cells widely distributed in the central and peripheral nervous systems, and gliomas are common primary tumors.

[0087] As used in this article, the term "orthotopic transplantation" is a method of tumor cell transplantation to prepare tumor models. It refers to the inoculation of tumor cell lines into specific sites or tissues. Compared to subcutaneous tumor models, it uses fewer cells and can better simulate the microenvironment in which tumor cells grow in the body.

[0088] As used herein, the term "screening" refers to the process of obtaining highly pure target cells. Target cells are transfected with a foreign gene to acquire antibiotic resistance; cells that are not successfully transfected with the foreign gene do not possess antibiotic resistance. By adding antibiotics to the cell culture system, the cells that are not successfully transfected with the foreign gene die, while the target cells survive. Through repeated screening, highly pure target cells can be obtained. The methods and procedures for screening are well known to those skilled in the art.

[0089] As used herein, the term "fluorescent reporter protein" refers to a class of proteins that emit fluorescence when excited by excitation light and are expressed by a "fluorescent reporter gene." After transfecting cells with a fluorescent reporter gene, the cells express the fluorescent reporter protein. In the presence of excitation light, the distribution of cells expressing the fluorescent reporter protein can be obtained through histological observation. Fluorescent reporter proteins include green fluorescent protein (GFP), yellow fluorescent protein (GFP), blue fluorescent protein (GFP), and red fluorescent protein (GFP). The fluorescent reporter protein used in this invention is enhanced green fluorescent protein (EGFP), which is modified from GFP and has a fluorescence intensity more than six times that of GFP. The glioma cell line in this invention contains a fluorescent reporter gene and can express the fluorescent reporter protein for in vitro monitoring of glioma growth.

[0090] As used herein, the term "luciferase" refers to a class of enzymes that fluoresce upon catalyzing a substrate, derived from the expression of a "luciferase gene." After transfecting cells with the luciferase gene, the cells express luciferase. Upon injection of a substrate (i.e., luciferin or its salt) into the body, luciferase interacts with the substrate and emits fluorescence, allowing for in vivo observation of the fluorescence phenomenon and thus determining the distribution of cells expressing luciferase. The glioma cell line in this invention contains the luciferase gene and is used for in vivo monitoring of glioma growth.

[0091] GL261-EGFP-luc

[0092] As used herein, the term "GL261-EGFP-luc" refers to a novel mouse glioma cell line created in this invention, which contains a fluorescent reporter gene and a luciferase gene, thereby expressing a fluorescent reporter protein and a luciferase.

[0093] Preferably, the fluorescent reporter gene includes an enhanced green fluorescent protein gene; and the fluorescent reporter protein includes enhanced green fluorescent protein.

[0094] Preferably, the GL261-EGFP-luc cell line is modified based on the GL261-luc cell line, specifically including transfecting the GL261-luc cell line with a vector containing the EGFP gene.

[0095] Preferably, the mouse glioma cell line further comprises a promoter and an antibiotic resistance gene, wherein the promoter is the Ubi promoter and the antibiotic resistance gene is the hygromycin B resistance gene.

[0096] Auxiliary agents

[0097] As used in this article, the term "transduction enhancer" also refers to a transfection agent that can improve the transfection efficiency of target cells and reduce the amount of viral vector required for transfection.

[0098] As used in this article, the term "polybrene" refers to a small, positively charged molecule that binds to anions on the cell surface, thereby effectively enhancing the infection efficiency of lentiviruses. It is commonly used as a lentiviral transfection aid. However, polybrene exhibits some cytotoxicity, and different cell types show varying sensitivities to it. Therefore, before using polybrene to assist cell transfection, it is necessary to screen for an appropriate concentration for different cell types. At this concentration, polybrene can effectively promote lentiviral transfection while ensuring that cells maintain their normal physiological state.

[0099] carrier

[0100] As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid molecule linked to it.

[0101] Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or those without free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and a wide variety of other polynucleotides known in the art. Vectors can be introduced into host cells through transformation, transduction, or transfection, enabling the expression of their carried genetic material elements in the host cells. A vector can be introduced into target cells, thereby producing transcripts, peptides, or proteins. A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain a replication initiation site.

[0102] Vectors include plasmid vectors and viral vectors. A plasmid vector is a circular double-stranded DNA loop in which another DNA fragment can be inserted, for example, using standard molecular cloning techniques. A viral vector contains a virus-derived DNA or RNA sequence within a vector used to package the virus. Viruses include, for example, lentiviruses, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses. Viral vectors also contain virally carried polynucleotides for transfection into a target cell. Some vectors (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) are capable of autonomous replication in the target cells into which they are introduced.

[0103] Other vectors (e.g., non-attachment mammalian vectors) integrate into the genome of the target cell after introduction and thereby replicate along with the target genome. Furthermore, some vectors can direct the expression of genes they are operatively linked to. Such vectors are called "expression vectors."

[0104] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired expression level.

[0105] Lentivirus vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type 1). They can effectively integrate exogenous genes into the genome of host cells, effectively infect dividing and non-dividing cells, greatly improve the transduction efficiency of target genes, and achieve long-term and stable expression of target genes relatively conveniently and quickly.

[0106] Method for constructing the glioma cell line GL261-EGFP-luc in this invention

[0107] This invention provides a method for constructing a glioma cell line GL261-EGFP-luc, which can be used to monitor glioma growth in vitro and in vivo. The construction method includes the following steps:

[0108] (A1) 12 hours in advance, lay 5×10⁶ cells in 96-well culture plates. 3 Once GL261-luc glioma cells reached approximately 60% confluence, 6-8 μg / mL of polybrene-assisted transfection agent was added to DMEM medium. The pre-coated GL261-luc glioma cells were then infected with Ubi-MCS-SV40-EGFP-IRES-Hygromycin lentivirus, which carries the Ubi promoter, an enhanced green fluorescent protein reporter gene, and a hygromycin B resistance gene.

[0109] (A2) After the GL261-luc glioma cells that have been infected with lentivirus have grown for 72 hours, replace them with fresh DMEM medium containing hygromycin B at a concentration of 700-800 μg / mL. Select the GL261-luc glioma cells by replacing the medium with fresh hygromycin B every 2-3 days for 7-10 days.

[0110] (A3) The glioma cells (GL261-EGFP-luc) with green fluorescent tags obtained in (A2) were amplified by single cloning and cryopreserved for propagation.

[0111] Preferably, the concentration of the polyaluminum glycoside transfer aid is 7.5 μg / ml.

[0112] Preferably, the concentration of hygromycin B is 750 μg / ml.

[0113] Method for constructing an animal model of orthotopic transplantation of glioma cells in this invention

[0114] This invention provides a method for constructing an animal model of orthotopic transplantation of glioma cells, comprising the following steps:

[0115] (B1) The GL261-EGFP-luc glioma cells, which were in good growth condition and stably expressed green fluorescence as obtained through steps A1-A3, were digested from the culture flasks with trypsin, and the cell density was adjusted to 1×10⁶ cells / year using PBS medium. 6 A cell density of cells / mL was prepared for use;

[0116] (B2) Select C57BL / 6 mice aged 6-8 weeks and weighing 18-20g, and use a stereotaxic apparatus and a microinjector to administer the 5×10⁻⁶ mice obtained in step one. 3 One GL261-EGFP-luc cell expressing green fluorescence was transplanted into the mouse brain at a position 1.5 mm posterior to Bregma point, 1.8 mm to the right, and 1.7 mm deep.

[0117] (B3) C57BL / 6 mice that had undergone orthotopic transplantation in (B2) were provided with sufficient water and food and kept individually in a clean environment at room temperature (22±2℃) with a 12h / 12h light-dark cycle to allow GL261-EGFP-luc glioma cells with green fluorescent tags to proliferate in the mouse brain.

[0118] The method for monitoring glioma growth in vitro and in vivo in this invention

[0119] This invention provides a method for studying the growth of gliomas in mice in vivo and in vitro using bioluminescence in vivo imaging and fluorescence microscopy, comprising the following steps:

[0120] (C1) The tumor-bearing C57BL / 6 mice were anesthetized with isoflurane, and the hair on the heads of the tumor-bearing mice was removed with animal depilatory cream to avoid the influence of hair on the collection of fluorescence signals.

[0121] (C2) Inject 150 mg / kg of fluorescein potassium salt, wait 2 min, and then use the PerkinElmer IVIS Lumina II small animal in vivo optical imaging system to acquire images of glioma cells in the mouse brain, and obtain fluorescence signals of GL261-EGFP-luc glioma cells in the mouse brain.

[0122] (C3) Mice were anesthetized with isoflurane and their hearts were perfused with PBS and 4% PFA. After removing the mouse brain tissue, it was placed in 4% PFA for 24 hours, followed by dehydration in 30% sucrose solution for 72 hours. After dehydration, the brain tissue was placed in OCT and stored overnight at -80°C. 40 μm sections of tumor-bearing mouse brain tissue were sectioned using a cryostat, mounted directly with DAPI, and observed under a fluorescence microscope at an excitation wavelength of 488 nm to determine the distribution and infiltration of GL261-EGFP-luc in the brain tissue.

[0123] This invention utilizes bioluminescence in vivo imaging technology and fluorescence microscopy technology to monitor the dynamic development of glioma cells. It can also locate and study individual cancer cells that have spread and metastasized, enabling precise examination and tracking of gliomas at the cellular level.

[0124] The main advantages of this invention include:

[0125] (1) This invention uses lentiviral genetic engineering technology to specifically modify tumor cells GL261-luc with enhanced green fluorescent protein EGFP. In vivo bioluminescence in vivo imaging technology can be used to monitor the dynamic development and changes of tumor tissue in real time, and in vitro fluorescence microscopy technology can be used to observe the distribution of tumor cells GL261-EGFP-luc in the brain tissue of tumor-bearing mice.

[0126] (2) The GL261-EGFP-luc glioma cells modified in this invention can be successfully used to construct an in vivo animal model of orthotopic glioma transplantation. The fluorescence response value of in vivo imaging can reflect the size of the tumor tissue, and the glioma boundary can be clearly distinguished by in vitro tissue sections (without HE staining or immunofluorescence staining), which effectively improves the accuracy of tumor localization and targeted treatment.

[0127] (3) Each GL261-EGFP-luc cancer cell proliferated in this invention is labeled with green fluorescence, which enables precise localization and study of individual cancer cells that have spread, providing a possible solution for further precision treatment.

[0128] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0129] method:

[0130] 1. Culture of GL261-luc and GL261-EGFP-luc glioma cells

[0131] GL261-luc and GL261-EGFP-luc cells were cultured in DMEM medium supplemented with 1% penicillin-streptomycin (10000 U / mL) and 10% fetal bovine serum (Biological Industries). The cell culture environment was maintained at 37°C, 95% humidity, and 5% CO2. When the cell confluence reached approximately 90%, the cells were passaged at a ratio of 1:4.

[0132] 2. Constructing glioma cells stably expressing enhanced green fluorescent protein (EGFP).

[0133] 2.1 Screening for the optimal concentration of polybrene, a lentiviral transgenic aid

[0134] GL261-luc cells in good growth condition were placed at a pre-adjusted cell concentration of 5 × 10⁻⁶. 4 Cells were seeded at a rate of 100 μL / mL in one well of a 96-well plate 12 h in advance. Cells were then seeded in the remaining 15 wells of the 96-well plate using the same method (3 replicates per group). After 12 h, five different concentration gradients of polybrene (0 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, and 10 μg / mL) were added to each well. Observations were performed at 12 h, 24 h, 36 h, and 48 h after the addition of polybrene. No significant toxicity was observed in the cells at concentrations of 0 μg / mL, 2.5 μg / mL, 5 μg / mL, and 7.5 μg / mL. Therefore, the appropriate working concentration of polybrene was determined to be 5–7.5 μg / mL; 7.5 μg / mL was preferred as the working concentration of polybrene for lentiviral cell infection.

[0135] 2.2 Determination of the optimal screening concentration of hygromycin B

[0136] GL261-luc cells in good growth condition were placed at a pre-adjusted cell concentration of 5 × 10⁻⁶. 4 Cells were seeded at a concentration of 100 μL / mL in one well of a 96-well plate 12 hours in advance. Cells were then seeded in the remaining 18 wells of the 96-well plate using the same method (3 replicates per group). 12 hours after seeding, six different concentration gradients of hygromycin B (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, and 1000 μg / mL) were added to each well. The number and density of surviving cells were observed and counted on day 7 after the addition of hygromycin B. The study found that the minimum concentration of hygromycin B that could completely kill GL261-luc cells on day 7 was 750 μg / mL.

[0137] 2.3 Lentiviral infection of GL261-luc glioma cells

[0138] The lentivirus used in this invention is Ubi-MCS-SV40-EGFP-IRES-Hygromycin, which has a hygromycin B resistance gene and an EGFP fluorescent reporter gene.

[0139] GL261-luc cells in good growth condition were placed at a pre-adjusted cell concentration of 5 × 10⁻⁶. 4 100 μL of the lentivirus Ubi-MCS-SV40-EGFP-IRES-Hygromycin was seeded into one well of a 96-well plate 12 h in advance and incubated overnight at 37 °C. The frozen lentivirus Ubi-MCS-SV40-EGFP-IRES-Hygromycin was then thawed on ice. The medium in the well was removed, and 50 μL of DMEM medium containing a multiplicity of infection (MOI) of 100 and 7.5 μg / ml polybrene was added. The plate was incubated at 37 °C for 12 h, and the medium was brought to a final volume of 100 μL. 24 h after infection, the virus-containing medium was aspirated, replaced with fresh DMEM medium, and the plate was incubated at 37 °C. 72 h post-infection, the expression of the EGFP fluorescent reporter gene was detected using a fluorescence microscope. Figure 3 As shown, some cells exhibit green fluorescence, indicating that GL261-luc cells were successfully infected with Ubi-MCS-SV40-EGFP-IRES-Hygromycin lentivirus and that the EGFP gene was successfully expressed.

[0140] 2.4 Hygromycin B screening of GL261-EGFP-luc cells expressing green fluorescent protein

[0141] The lentivirus used in this invention contains a hygromycin B resistance gene. GL261-luc cells successfully infected with the lentivirus will possess hygromycin B resistance and can survive in culture medium supplemented with hygromycin B. Cells not infected with the lentivirus do not possess hygromycin B resistance and cannot survive in culture medium containing hygromycin B. After 72 hours of viral infection, GL261-luc cells are cultured in fresh DEME medium containing 750 μg / mL hygromycin B. The medium is replaced every two days with fresh DEME medium containing 750 μg / mL hygromycin B until GL261-EGFP-luc cells stably expressing green fluorescence are selected. After 7 days of selection with 750 μg / mL hygromycin B, all cells exhibit green fluorescence. After continuous selection and passage, the GL261-EGFP-luc cells stably expressing green fluorescence are cryopreserved for seed culture.

[0142] 3. Bioluminescent in vivo imaging of mouse brain glioma cells

[0143] 3.1 Construction of an animal model of orthotopic transplantation of GL261-EGFP-luc cells

[0144] 3.1.1 Preparation of GL261-EGFP-luc cells for transplantation

[0145] GL261-EGFP-luc cells in good growth condition were digested from the cell culture flask using trypsin. After cell counting, the density of GL261-EGFP-luc cells was adjusted to 1×10⁻⁶ cells using PBS. 6 1 / mL, placed on ice.

[0146] 3.1.2 Anesthesia and skull fixation in mice

[0147] Six- to eight-week-old C57BL / 6 mice, weighing 18-20g, in good condition and with glossy fur, were selected as recipients for glioma cell transplantation. After anesthetizing the mice with isoflurane, their heads were fixed to a stereotaxic apparatus. The hair on their heads was shaved, and the shaved skin was disinfected with iodine. The skin on the top of the head was then cut open to expose the mouse skull.

[0148] 3.1.3 Orthotopic transplantation of GL261-EGFP-luc cells

[0149] After fixing the mouse head onto a stereotaxic instrument, using Bregma as a reference, a fenestration was created 1.5 mm posterior and 1.8 mm to the right using a skull drill to avoid damaging brain tissue. 5 μL of GL261-EGFP-luc cell suspension (1 × 10⁻⁶ cells) was aspirated using a 5 μL microsyringe. 6 (Number of mice / mL). Place the microsyringe at the cranial window of the mouse and slowly insert the needle into the mouse brain tissue to a depth of 2.0 mm. After insertion, wait 2 minutes and then withdraw the needle 0.3 mm, resulting in an injection depth of 1.7 mm. Control the injection rate at 1 μL / min, leave the needle in place for 5 minutes after injection, and then slowly withdraw the needle. Suture the mouse's head skin. After the mouse regains consciousness and normal activity, place it in a clean environment, provide ample water and food, and house it at room temperature (22±2℃) with a 12h / 12h light / dark cycle.

[0150] 3.2 In vivo imaging and in vitro fluorescence microscopy observation of tissue sections after orthotopic transplantation of GL261-EGFP-luc cells

[0151] C57BL / 6 mice bearing gliomas were anesthetized with isoflurane. Hair on the heads of the mice was removed with animal depilatory cream to avoid interference with fluorescence signal collection. 150 mg / kg of fluorescein potassium was injected, and after 2 minutes, images of the mouse brain gliomas were acquired using the PerkinElmer IVIS Lumina II small animal in vivo optical imaging system. Figure 6 Fluorescence signals of GL261-EGFP-luc glioma cells in mouse brains were obtained. On day 21 of tumor bearing, after bioluminescence in vivo imaging, tumor-bearing mice were anesthetized with isoflurane and subjected to cardiac perfusion with PBS and 4% PFA. After removing the mouse brain tissue, it was placed in 4% PFA for 24 hours, followed by dehydration in 30% sucrose solution for 72 hours. After dehydration, the brain tissue was placed in an OCT chamber and incubated overnight at -80°C. 40μm sections of tumor-bearing mouse brain tissue were obtained using a cryostat, mounted directly with DAPI, and observed under a fluorescence microscope at an excitation wavelength of 488nm. Figure 7 The image shows the distribution and infiltration of GL261-EGFP-luc in brain tissue.

[0152] Example 1: Construction of a glioma cell line expressing green fluorescence (GL261-EGFP-luc)

[0153] This embodiment involves using lentivirus to infect GL261-luc glioma cells to construct a glioma cell line stably expressing green fluorescence. The specific steps are as follows:

[0154] (W1) 12 hours in advance, lay 5×10⁶ cells in 96-well culture plates. 3 Once GL261-luc glioma cells reached approximately 60% confluence, 7.5 μg / mL of polybrene-assisted transfection agent was added to DMEM medium. The pre-coated GL261-luc glioma cells were then infected with Ubi-MCS-SV40-EGFP-IRES-Hygromycin lentivirus, which carries the Ubi promoter, an EGFP fluorescent reporter gene with an excitation band of 488 nm, and a hygromycin B resistance gene.

[0155] (W2) After the GL261-luc glioma cells that have been infected with lentivirus have grown for 72 hours, replace them with fresh DMEM medium containing 750 μg / mL of hygromycin B to screen the GL261-luc glioma cells. Replace the medium with fresh hygromycin B every 2 days.

[0156] (W3) The glioma cells (GL261-EGFP-luc) with green fluorescent tags obtained from W2 were amplified by single cloning and cryopreserved for propagation.

[0157] (W4) Flow cytometry was used to verify the difference between GL261-EGFP-luc and GL261-luc cells after lentiviral transfection and hygromycin B selection: GL261-EGFP-luc and GL261-luc were diluted to 1×10⁻⁶. 6 Samples / mL, 100 μL sample, and the signal difference in the FITC channel was observed.

[0158] Example 2: In vitro luminescence verification of GL261-EGFP-luc and GL261-luc

[0159] This embodiment involves the use of PerkinElmer IVIS Lumina II to perform in vitro luminescence imaging verification of GL261-EGFP-luc and GL261-luc.

[0160] (X1) Digest and count the cells, then resuspend them to a concentration of 1×10⁻⁶. 5 Cells / mL, take 100μL and add it to a 96-well chemiluminescent plate, and perform serial dilutions to make the number of cells per well 10,000, 5,000 and 2,500;

[0161] (X2) Add 100 μL of 300 μg / mL fluorescein potassium salt to each well;

[0162] (X3) After 2 minutes, the plate was placed in the PerkinElmer IVIS Lumina II for imaging.

[0163] Example 3: Construction of an animal model of orthotopic transplantation of GL261-EGFP-luc

[0164] This embodiment involves constructing an animal model by implanting GL261-EGFP-luc proto-transfer to an animal individual.

[0165] (Y1) The GL261-EGFP-luc glioma cells, which were in good growth condition and stably expressed green fluorescence as obtained in the above steps, were digested from the culture flasks with trypsin, and the cell density was adjusted to 1×10⁻⁶ cells / year using DMEM medium. 6 A cell density of cells / mL was prepared for use;

[0166] (Y2) Select C57BL / 6 mice aged 6-8 weeks and weighing 18-20g. Use a stereotaxic apparatus and a microinjector to administer the 5×10⁻⁶ mice obtained in step one. 3One green fluorescent cell was transplanted into the mouse brain at a position 1.5 mm posterior to Bregma point, 1.8 mm to the right, and 1.7 mm deep.

[0167] (Y3) C57BL / 6 mice orthotopically transplanted in Y2 were provided with sufficient water and food and kept individually in a clean environment at room temperature (22±2℃) with a 12h / 12h light-dark cycle. This allowed GL261-EGFP-luc glioma cells with green fluorescent tags to proliferate in the mouse brain for use in subsequent studies.

[0168] Example 4: Studying the growth of gliomas in mice in vitro and in vivo using bioluminescence in vivo imaging technology and fluorescence microscopy.

[0169] This embodiment involves using bioluminescence in vivo imaging technology and fluorescence microscopy to study the in vivo and in vitro monitoring of glioma growth in mice.

[0170] (Z1) The tumor-bearing C57BL / 6 mice were anesthetized with isoflurane, and the hair on the heads of the tumor-bearing mice was removed with animal hair removal cream to avoid the influence of hair on fluorescence signal collection.

[0171] (Z2) After injecting 150 mg / kg of fluorescein potassium salt, wait for 2 min, and use the PerkinElmer IVIS Lumina II small animal in vivo optical imaging system to acquire images of gliomas in the mouse brain and obtain fluorescence signals of GL261-EGFP-luc glioma cells in the mouse brain.

[0172] (Z3) Mice were anesthetized with isoflurane and their hearts were perfused with PBS and 4% PFA. After removing the mouse brain tissue, it was placed in 4% PFA for 24 h and then in 30% sucrose solution for 72 h for dehydration. After dehydration, the brain tissue was placed in OCT and stored at -80°C overnight. The brain tissue of tumor-bearing mice was sectioned using a cryostat to obtain 40 μm sections, which were then mounted directly with DAPI and observed under a fluorescence microscope at an excitation wavelength of 488 nm to observe the distribution and infiltration of GL261-EGFP-luc in the brain tissue.

[0173] This technology utilizes bioluminescence in vivo imaging to monitor the dynamic development of glioma cells in vivo. In vitro, tumor cells are observed directly under a fluorescence microscope through tissue sections for precise localization and study, allowing for more accurate examination and tracking of gliomas at the cellular level.

[0174] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A glioma cell line, characterized in that, The cell line contains a fluorescent reporter gene and a luciferase gene, thereby expressing a fluorescent reporter protein and a luciferase.

2. A method of constructing a glioma cell line according to claim 1, wherein, Including the following steps: (a) Provide a glioma cell line; (b) Upregulating the expression of fluorescent reporter gene and luciferase in the glioma cell line, wherein the upregulation includes delivering a vector containing a fluorescent reporter gene and a vector containing luciferase into the glioma cell line; (c) The glioma cell lines are screened to obtain glioma cell lines expressing fluorescent reporter genes and luciferase.

3. The method of claim 2, wherein, In the presence of a transduction aid, a vector containing a fluorescent reporter gene and a vector containing luciferase are delivered into the glioma cell line.

4. The method of claim 3, wherein, The conversion aid is polybrene, and the concentration of polybrene is 6-8 μg / mL.

5. A method of constructing a glioma cell transplantation animal model, characterized by, Including the following steps: (1) Provide an individual animal for transplantation; (2) Transplanting the glioma cell line of claim 1 into the animal individual.

6. A method of monitoring the growth of a glioma in vivo and in vitro, characterized by, Including the following steps: (A) Transplanting the glioma cell line of claim 1 into an animal individual, thereby causing the glioma cell line to proliferate in the animal individual; (B) Inject the animal individual with luciferase substrate to allow the luciferase substrate to react with luciferase, and use an in vivo optical imaging system to acquire images of the animal individual to obtain information on the growth of glioma. (C) Obtain tissue sections from the animal individual and observe the spatial distribution of the glioma cell line under the excitation wavelength of a fluorescence microscope to obtain information on glioma growth.

7. A formulation or composition, characterized in that, The formulation or composition comprises the glioma cell line of claim 1.

8. A reagent kit, characterized in that, The kit comprises one or more components selected from the following: The glioma cell line of claim 1 or the formulation or composition of claim 7.

9. Use of the glioma cell line of claim 1, the formulation or composition of claim 7, or the kit of claim 8, characterized in that, Drugs used for the diagnosis or treatment of gliomas.

10. The use as described in claim 9, characterized in that, The applications also include research on the mechanisms of glioma development.

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