Living cell mitochondria H2O2 sensor based on gene coding fluorescent protein

Abstract

The invention discloses a living cell mitochondrial H2O2 sensor based on gene coding fluorescent protein. The invention provides a recombinant cell. A preparation method of the recombinant cell comprises the following steps: (1) preparing a recombinant lentivirus for expressing a mitochondrial targeting HyPer7 probe; the mitochondrial targeting HyPer7 probe is a protein, wherein a mitochondrial targeting section and a HyPer7 probe section are sequentially arranged from an N end to a C end; and (2) transfecting THP-1 cells with the recombinant lentivirus to obtain recombinant cells. The invention also provides a macrophage obtained by inducing the recombinant cell. The recombinant cell or the macrophage can be used for H2O2-related environmental toxicology research, and has important research value and application prospect.

Description

Technical Field

[0001] The invention belongs to the field of cell biology and biosensor technology, and relates to a living cell mitochondrial H2O2 sensor based on gene-encoded fluorescent protein. Background Art

[0002] Mitochondria are important energy metabolism and signal transduction centers in cells, and their dysfunction is closely related to a variety of diseases. Mitochondrial reactive oxygen species (ROS) refer to highly active oxidative intermediates produced by molecular oxygen (O2) during metabolism, including free radicals and non-free radical oxides, which play a key role in cell signal transduction, oxidative stress, metabolic regulation, and the occurrence and development of diseases. Among them, hydrogen peroxide (H2O2), as a relatively stable and important component of ROS, is one of the key intermediates in aerobic metabolism and has an important influence on stress response, cell apoptosis, and various pathological processes. Unlike O2· - Despite its extremely short lifespan, H2O2 can persist for several seconds in a liquid environment and regulate metabolism and signaling pathways by acting as a second messenger through the oxidation of specific protein sulfates by particles.

[0003] H2O2 mainly comes from the specific enzyme catalysis process, including NADPH oxidase, monoamine oxidase (MAO), α-ketoacid dehydrogenase (α-KGDH), flavoenzyme and other enzyme systems; electron leakage in the electron transport chain (ETC) during mitochondrial oxidative phosphorylation is also an important source, O2· - Superoxide dismutase (SOD) catalyzes the dismutation of H2O2 to produce H2O2. H2O2 levels are regulated by antioxidant systems such as catalase (CAT) and glutathione peroxidase (GPx). Among various cellular subcompartments, mitochondria are the primary source of H2O2 production. H2O2 acts as a cellular signaling molecule, regulating metabolism and combating oxidative stress. However, at high concentrations, H2O2 can also induce oxidative damage, promoting aging, neurodegenerative diseases, and cancer. Therefore, monitoring mitochondrial H2O2 metabolism and its dynamic changes is crucial for understanding cellular function, disease mechanisms, and pollutant toxicity.

[0004] Traditional methods for detecting H2O2 primarily include fluorescent dyes, chemical probes, electron spin resonance, colorimetry, and chromatography-mass spectrometry. However, these methods suffer from high cytotoxicity, low specificity, cumbersome sample pretreatment, or difficulty in achieving real-time dynamic monitoring. Fluorescent dye methods (such as DCFH-DA and Amplex Red) are limited in their specificity, high cytotoxicity, and susceptibility to photobleaching, making them difficult to achieve long-term dynamic monitoring and susceptible to interference from other reactive oxygen species. Chemical probe methods (such as boronate-based probes) offer improved specificity, but reaction byproducts may interfere with detection, and their poor permeability makes it difficult to accurately capture transient H2O2 changes. Electrochemical methods rely on external enzymes and are susceptible to environmental influences. Electrodes are prone to contamination, leading to signal drift and compromising long-term stability. Colorimetry, while simple, has limited sensitivity, requires cell lysis for detection, is incapable of real-time monitoring of live cells, and exhibits significant background interference. Electron spin resonance (EPR) can directly detect free radicals and their derivatives, but its sensitivity for direct detection of H2O2 is low, and the instrument is expensive and complex to operate, making it unsuitable for high-throughput analysis. Chromatography-mass spectrometry (such as HPLC-MS and GC-MS) can provide high-resolution and highly specific quantitative analysis of H2O2, but sample pretreatment is complex and often requires cell lysis, making it difficult to apply to real-time, live-cell environmental monitoring. These shortcomings make traditional methods unable to meet the needs of highly specific, dynamic, and non-invasive mitochondrial H2O2 detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a living cell mitochondrial H2O2 sensor based on gene-encoded fluorescent protein.

[0006] The present invention provides a recombinant cell, the preparation method of which comprises the following steps:

[0007] (1) preparing a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe; the mitochondrial-targeted HyPer7 probe is a protein, which has a mitochondrial-targeting segment and a HyPer7 probe segment from the N-terminus to the C-terminus; the mitochondrial-targeting segment is as shown in positions 1-29 of SEQ ID NO: 2, and the HyPer7 probe segment is as shown in positions 42-520 of SEQ ID NO: 2;

[0008] (2) Transfect the recombinant lentivirus into THP-1 cells to obtain recombinant cells.

[0009] The present invention provides a method for preparing a recombinant cell, comprising the following steps:

[0010] (1) preparing a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe; the mitochondrial-targeted HyPer7 probe is a protein, which has a mitochondrial-targeting segment and a HyPer7 probe segment from the N-terminus to the C-terminus; the mitochondrial-targeting segment is as shown in positions 1-29 of SEQ ID NO: 2, and the HyPer7 probe segment is as shown in positions 42-520 of SEQ ID NO: 2;

[0011] (2) Transfect the recombinant lentivirus into THP-1 cells to obtain recombinant cells.

[0012] The recombinant cells obtained by transfecting the recombinant lentivirus into THP-1 cells are recombinant monocytes.

[0013] In the mitochondrial targeting HyPer7 probe, a connecting peptide is present between the mitochondrial targeting segment and the HyPer7 probe segment. Specifically, the connecting peptide is as shown at positions 30-41 in SEQ ID NO: 2.

[0014] Specifically, the mitochondrial-targeted HyPer7 probe is shown in positions 1 to 520 of SEQ ID NO: 2.

[0015] Specifically, the mitochondria-targeted HyPer7 probe is shown in SEQ ID NO: 2.

[0016] The method for preparing a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe comprises the following steps: co-transfecting a plasmid containing a DNA molecule encoding the mitochondrial-targeted HyPer7 probe and a lentiviral helper plasmid into lentiviral packaging cells, followed by culturing to produce the recombinant lentivirus. Specifically, the lentiviral helper plasmids are pLP1, pLP2, and pMD2.G. Specifically, the lentiviral packaging cells are 293T cells.

[0017] The preparation method of the recombinant lentivirus expressing the mitochondrial-targeted HyPer7 probe comprises the following steps: co-transfecting 293T cells with a plasmid having a DNA molecule encoding the mitochondrial-targeted HyPer7 probe, a pLP1 plasmid, a pLP2 plasmid and a pMD2.G plasmid, and culturing the cells to obtain the recombinant lentivirus.

[0018] Specifically, the preparation method of the recombinant lentivirus expressing the mitochondrial-targeted HyPer7 probe includes the following steps: using a transfection reagent to co-transfect 293T cells with a plasmid having a DNA molecule encoding a mitochondrial-targeted HyPer7 probe, a pLP1 plasmid, a pLP2 plasmid and a pMD2.G plasmid (the molar ratio of the plasmid having a DNA molecule encoding a mitochondrial-targeted HyPer7 probe, the pLP1 plasmid, the pLP2 plasmid and the pMD2.G plasmid is 3:1:1:1, respectively), and after culturing for 12 hours, the supernatant is discarded, and the culture is continued for 36 hours with DMEM culture medium containing 10% FBS, and then the supernatant is collected to obtain the recombinant lentivirus.

[0019] Specifically, the preparation method of the recombinant lentivirus expressing the mitochondrial-targeted HyPer7 probe includes the following steps: using a transfection reagent, a plasmid having a DNA molecule encoding the mitochondrial-targeted HyPer7 probe, a pLP1 plasmid, a pLP2 plasmid and a pMD2.G plasmid (the molar ratio of the plasmid having a DNA molecule encoding the mitochondrial-targeted HyPer7 probe, the pLP1 plasmid, the pLP2 plasmid and the pMD2.G plasmid is 3:1:1:1, respectively) are co-transfected into 293T cells, and after culturing for 12 hours, the supernatant is discarded, and the culture is continued for 36 hours with DMEM culture medium containing 10% FBS, and then the supernatant is collected and concentrated to obtain the recombinant lentivirus.

[0020] Specifically, the MOI value of the recombinant lentivirus transfected into THP-1 cells was 20.

[0021] The step (2) comprises the following steps: transfecting THP-1 cells with the recombinant lentivirus, discarding the supernatant after culturing for 24 hours, then culturing the cells in a culture medium containing puromycin for 5-7 days, and finally sorting to obtain the target recombinant cells.

[0022] The step (2) comprises the following steps: transfecting THP-1 cells with the recombinant lentivirus using a transfection reagent, discarding the supernatant after culturing for 24 hours, then culturing the cells in RPMI-1640 complete medium containing 2 μg / mL puromycin for 5-7 days, and finally sorting to obtain the target recombinant cells. Specifically, the transfection reagent can be Polybrene transfection reagent.

[0023] The step (2) comprises the following steps: using Polybrene transfection reagent to transfect the recombinant lentivirus into THP-1 cells (the concentration of Polybrene transfection reagent in the culture system is 8 μg / mL), discarding the supernatant after culturing for 24 hours, and then culturing for 5-7 days using RPMI-1640 complete medium containing 2 μg / mL puromycin, and finally sorting to obtain the target recombinant cells.

[0024] Sorting criteria: showing fluorescence at an excitation wavelength of 405 nm and showing fluorescence at an excitation wavelength of 488 nm (the emission wavelength of the fluorescence is 525 / 50 nm or 530 / 30 nm).

[0025] The recombinant cells can be passaged, and the cells obtained through passage are also recombinant cells. The passage method includes culturing the cells in a cell culture vessel using RPMI-1640 complete medium containing 2 μg / mL puromycin for 2 days. The liquid phase from one cell culture vessel is then equally divided between two cell culture vessels, and each cell culture vessel is supplemented with an equal volume of RPMI-1640 complete medium containing 2 μg / mL puromycin as the liquid phase. Exemplarily, the number of passages is 20.

[0026] Specifically, the DNA molecule encoding the mitochondrial-targeted HyPer7 probe is shown in positions 3187-4755 of SEQ ID NO: 1.

[0027] Specifically, the plasmid containing the DNA molecule encoding the mitochondrial-targeted HyPer7 probe also contains a DNA molecule encoding a puromycin resistance protein. Specifically, the DNA molecule encoding the puromycin resistance protein is shown at positions 5283-5882 in SEQ ID NO: 1.

[0028] Specifically, the plasmid having the DNA molecule encoding the mitochondria-targeted HyPer7 probe is the pLVX-mito-HyPer7-Puro plasmid.

[0029] The recombinant cells obtained by the method also fall within the protection scope of the present invention.

[0030] The macrophages obtained by inducing the recombinant cells also fall within the scope of protection of the present invention. The macrophages obtained by inducing the recombinant cells are recombinant macrophages.

[0031] The induction was performed using PMA.

[0032] The induction step may specifically include: taking the recombinant cells and inducing them with 100 ng / mL PMA for 24 hours.

[0033] The induction step may specifically include: taking the recombinant cells and culturing them in RPMI-1640 complete medium containing 100 ng / mL PMA for 24 hours.

[0034] The present invention also protects a protein (mitochondrial targeting HyPer7 probe), which has a mitochondrial targeting segment and a HyPer7 probe segment from the N-terminus to the C-terminus; the mitochondrial targeting segment is shown as positions 1-29 in SEQ ID NO: 2, and the HyPer7 probe segment is shown as positions 42-520 in SEQ ID NO: 2.

[0035] In the protein, a connecting peptide is present between the mitochondrial targeting segment and the HyPer7 probe segment, specifically, the connecting peptide is as shown at positions 30-41 in SEQ ID NO: 2.

[0036] Specifically, the protein is shown in positions 1 to 520 of SEQ ID NO: 2.

[0037] Specifically, the protein is shown in SEQ ID NO: 2.

[0038] The present invention also protects nucleic acid molecules encoding the protein.

[0039] Specifically, the nucleic acid molecule encoding the protein is a DNA molecule, as shown in positions 3187-4755 of SEQ ID NO: 1.

[0040] The present invention also protects recombinant plasmids or recombinant viruses having nucleic acid molecules encoding the protein.

[0041] The recombinant plasmid containing the nucleic acid molecule encoding the protein also contains a DNA molecule encoding a puromycin resistance protein. Specifically, the DNA molecule encoding the puromycin resistance protein is shown at positions 5283-5882 in SEQ ID NO: 1.

[0042] Specifically, the recombinant plasmid is pLVX-mito-HyPer7-Puro plasmid.

[0043] The pLVX-mito-HyPer7-Puro plasmid is shown in SEQ ID NO: 1.

[0044] Specifically, the recombinant virus is a recombinant lentivirus.

[0045] Specifically, the recombinant lentivirus is a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe prepared by any of the above methods.

[0046] The present invention also protects the use of component 1 or component 2 in preparing recombinant cells;

[0047] The component 1 consists of the recombinant plasmid, lentiviral packaging helper plasmid, lentiviral packaging cells and THP-1 cells;

[0048] The component 2 consists of the recombinant virus and THP-1 cells.

[0049] Specifically, the lentiviral packaging helper plasmids are pLP1 plasmid, pLP2 plasmid and pMD2.G plasmid.

[0050] Specifically, the lentivirus packaging cells are 293T cells.

[0051] The present invention also protects the use of the recombinant cell (recombinant monocyte) or the macrophage (recombinant macrophage) as a cell sensor in detecting H2O2.

[0052] The present invention also protects the use of the recombinant cells (recombinant monocytes) or the macrophages (recombinant macrophages) in environmental toxicology research; the environmental toxicology research is an environmental toxicity research related to H2O2.

[0053] The present invention also protects the use of the recombinant cells (recombinant monocytes) or the macrophages (recombinant macrophages) as cell models.

[0054] The cell model is as follows (a) or (b) or (c):

[0055] (a) The cell model is used to screen substances that cause changes in H2O2 levels in cell mitochondria;

[0056] (b) The cell model is used to evaluate whether the test substance has toxicity that causes changes in mitochondrial H2O2 levels in cells;

[0057] (c) The cell model is used to observe the dynamic changes of H2O2 levels in cell mitochondria caused by the test substance.

[0058] Mitochondrial reactive oxygen species (ROS) play a key role in cellular oxidative stress, toxicological processes, and various pathological processes. Hydrogen peroxide (H2O2), a key component of ROS, is of great significance in the study of cellular damage and toxicity mechanisms induced by environmental pollutants. In particular, within the immune system, the rapid response of monocytes and macrophages to exogenous pollutants (such as heavy metals, nanoparticles, and environmental persistent organic pollutants), and their ROS production, directly influence inflammatory responses, phagocytic function, and cell survival. Therefore, accurate and real-time detection of mitochondrial H2O2 levels in monocytes and macrophages is of great value for assessing pollutant toxicity and studying its molecular mechanisms. However, existing H2O2 detection methods (such as fluorescent dyes, electrochemical sensors, electron spin resonance, and chromatography-mass spectrometry) have varying degrees of limitations, such as low specificity, high cytotoxicity, inability to achieve real-time dynamic monitoring, expensive detection equipment, and complex sample processing. These limitations make it difficult to meet the demand for highly sensitive, stable, and non-invasive detection of mitochondrial H2O2 in living cells.

[0059] The cell model provided by the present invention can monitor the dynamic changes of mitochondrial H2O2 in real time and with high sensitivity at the living cell level, providing a reliable detection tool for pollutant toxicity assessment, environmental health and pollution exposure-related diseases (such as chronic inflammation, oxidative damage and immune disorders).

[0060] Deficiencies of existing technologies: ① Unstable probe expression: Traditional methods using transient transfection to construct cell sensors have the limitation of unstable probe expression levels, making it difficult to maintain high expression and dynamic monitoring for a long time; ② Inaccurate mitochondrial localization: The localization efficiency of existing probes in mitochondria is low, making it difficult to achieve specific monitoring; ③ Insufficient detection sensitivity: Existing probes cannot meet the needs of high-precision research on the oxidation level of cells (reflected by H2O2 levels).

[0061] Compared with existing H2O2 detection technology, the present invention has the following significant advantages:

[0062] (1) Efficient and stable expression and dynamic monitoring capabilities:

[0063] At present, many H2O2 detection experiments use transient transfection or short-term viral infection methods, which makes the expression level of the probe vary greatly in the cell population, resulting in poor repeatability of the experimental results. Not only is it possible to cause cell damage during the transient transfection process, but the probe expression is short-lived (usually the expression level gradually decreases within 24-72 hours), making it difficult to perform long-term dynamic monitoring. The present invention successfully constructed a THP-1 cell sensor that stably expresses HyPer7 through a lentiviral-mediated stable transfection technology. The probe expression level is high and stable and continuous, avoiding the expression attenuation problem caused by transient transfection, and the probe expression is uniform in the cell population, improving the repeatability and stability of the experiment. Based on this stable characteristic, the present sensor can realize real-time dynamic monitoring of mitochondrial H2O2 in living cells, breaking through the limitation that traditional methods can only report the oxidation level of a single time point, and can dynamically track the generation, accumulation and clearance process of mitochondrial H2O2, which helps to deeply understand the oxidative stress process and the trend of mitochondrial function changes. It does not interfere with the physiological state of the cell and can achieve non-perturbation monitoring, which is particularly suitable for toxicity assessment, pollutant exposure and long-term ROS fluctuation analysis. Suitable for real-time imaging of living cells, it can observe the dynamic changes of H2O2 levels in the same cell, avoiding errors caused by cell-to-cell variation.

[0064] (2) Precise positioning of mitochondria improves spatial resolution and accuracy:

[0065] Traditional H2O2 detection methods are often based on the measurement of whole-cell or cytoplasmic H2O2 content, making it difficult to distinguish mitochondrial-derived H2O2 from other intracellular ROS. Extracting mitochondria separately and then detecting H2O2 levels is not only cumbersome, but also makes it difficult to determine the reaction process and the reliability of the results after the purified mitochondria lose their natural physiological environment. The present invention uses a mitochondrial localization signal sequence to precisely locate the HyPer7 probe, enabling it to specifically detect mitochondrial H2O2 content. This eliminates the need for mitochondrial purification, avoids interference from H2O2 in the cytoplasm or other organelles, improves the spatial resolution and accuracy of detection, and simplifies the experimental process.

[0066] (3) High sensitivity and specificity:

[0067] Existing H2O2 detection methods (such as DCFH-DA fluorescent probe and Amplex Red detection) usually rely on chemical probes, but these methods may produce nonspecific fluorescent signals after intracellular metabolism and are easily interfered with by other reactive oxygen species (such as superoxide anions and hydroxyl radicals). The present invention adopts the HyPer7 gene-encoded fluorescent probe, which specifically recognizes H2O2 through its chimeric OxyR sensing domain, avoiding the nonspecific interference of traditional chemical probes and making H2O2 detection more accurate. Compared with the transient transfection probe, the stably transfected HyPer7 probe has high sensitivity and rapid response to H2O2, avoids the errors caused by changes in transient transfection efficiency, can dynamically monitor the redox state in mitochondria in real time, improves signal sensitivity and data consistency, is suitable for quantitative analysis, and can provide a more accurate H2O2 concentration change curve.

[0068] (4) Suitable for large-scale high-throughput screening and broad application prospects:

[0069] Since the probe is stably integrated into the genome, there is no need for transfection before each experiment, and it is suitable for long-term follow-up experiments and high-throughput drug screening, saving experimental time and cost. It is suitable for cell model construction and can obtain more stable signal readings in long-term experiments such as pollutant exposure experiments and immune cell function studies. The cell sensor constructed by the present invention can be used for high-throughput and real-time dynamic research on the redox state of mitochondria in monocytes / macrophages, providing an efficient tool for high-throughput screening of compounds, research on the toxicity mechanism of pollutants, drug screening and disease mechanism research. In addition, the present invention is based on the THP-1 monocytic cell line, which can be induced to differentiate into macrophages by methods such as Phorbol 12-myristate 13-acetate (PMA), and the mitochondrial H2O2 level can be monitored under different cell states, thereby expanding the application of the sensor in the fields of immune cell research, inflammation models, pollutant toxicity assessment, etc.

[0070] In summary, the present invention is superior to existing technologies in terms of specificity, real-time performance, cell targeting, stability, scope of application, and environmental toxicology applications. It provides an efficient, stable, and non-invasive detection method for pollutant toxicity assessment and oxidative stress research, and has broad application value in the fields of environmental health and biomedical research.

[0071] The recombinant cells provided by the present invention can be used as cell sensors to detect H₂O₂ in the environment (with a sensitivity of up to 10 μM). The recombinant cells provided by the present invention, or the macrophages derived from their differentiation, can dynamically monitor H₂O₂ levels in the mitochondria of the cells in real time through fluorescence. Therefore, they can be used as cell models for environmental toxicology research, research on diseases related to mitochondrial reactive oxygen species, drug screening, and cell function studies. This invention overcomes the limitations of traditional detection methods and has significant research value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Schematic diagram of the components of pLVX-mito-HyPer7-Puro.

[0073] Figure 2 This is the result diagram of Example 4.

[0074] Figure 3 This is the result diagram of Example 5.

[0075] Figure 4 This is the result diagram of Example 6.

[0076] Figure 5 This is the result diagram of Example 7. DETAILED DESCRIPTION

[0077] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0078] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative experiments in the following examples were repeated three times, and the results were averaged. Unless otherwise specified, the cell culture conditions were: 37°C, 5% CO2. THP-1 cells (human monocytes): Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-567. 293T cells (human embryonic kidney cells): Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number GNHu17. PMA: Phorbol 12-myristate13-acetate.

[0079] Example 1: Construction of recombinant plasmid

[0080] The recombinant plasmid was constructed and named pLVX-mito-HyPer7-Puro plasmid. The pLVX-mito-HyPer7-Puro plasmid is a circular plasmid formed by a double-stranded DNA molecule. Its full sequence is shown in SEQ ID NO: 1 (9975 bp). The schematic diagram of the elements is shown in Figure 1In SEQ ID NO: 1, positions 3187-4755 encode the protein shown in SEQ ID NO: 2, and positions 5283-5882 encode the puromycin resistance protein. In SEQ ID NO: 2, the segment consisting of amino acid residues 1-29 functions to target mitochondria, and amino acid residues 42-520 constitute the HyPer7 probe.

[0081] The HyPer7 probe is a pH-stable, ultrafast, and ultrasensitive H2O2 probe. The HyPer7 protein has two maximum excitation wavelengths: one near 400nm and the other near 499nm. In the presence of H2O2, the fluorescence intensity of the HyPer7 probe decreases at excitation wavelengths near 400nm and increases at excitation wavelengths near 499nm.

[0082] Example 2: Preparation of recombinant cells

[0083] RPMI-1640 complete medium: RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0084] 1. Culture 293T cells in a 10 cm diameter culture dish using DMEM medium containing 10% FBS until the cell confluence reaches 50-60%, and then discard the supernatant.

[0085] 2. After completing step 1, remove the culture dish and co-transfect with the pLVX-mito-HyPer7-Puro plasmid, pLP1 plasmid, pLP2 plasmid, and pMD2.G plasmid using jetPRIME transfection reagent (the molar ratio of pLVX-mito-HyPer7-Puro plasmid, pLP1 plasmid, pLP2 plasmid, and pMD2.G plasmid is 3:1:1:1, respectively). Incubate for 12 hours. jetPRIME transfection reagent: Polyplus, catalog number 101000046. pLP1: Addgene, catalog number #209988. pLP2: Addgene, catalog number #209989. pMD2.G: Addgene, catalog number #12259.

[0086] 3. After completing step 2, remove the culture dish, discard the supernatant, add new DMEM medium containing 10% FBS, and culture for 36 hours. Then collect the supernatant and concentrate it using a virus concentration kit (Biyuntian Company, product number C2901S) to obtain a concentrated virus solution (containing the recombinant lentivirus).

[0087] 4. Take a 12-well plate and inoculate THP-1 cells (1×10 5 -3×10 5cells) and cultured in RPMI-1640 complete medium for 12 hours.

[0088] 5. After completing step 4, add Polybrene transfection reagent to a concentration of 8 μg / mL in the culture system. Then add the concentrated virus solution prepared in step 3 (MOI of 20) and incubate for 24 hours. Polybrene Infection / Transfection Reagent: Sigma-Aldrich, catalog number TR-1003-G.

[0089] 6. After completing step 5, centrifuge at 300g for 5 minutes, discard the supernatant, and wash twice with PBS buffer to remove uninfected virus particles.

[0090] 7. After completing step 6, add RPMI-1640 complete medium containing 2 μg / mL puromycin and culture for 5-7 days (replace half the amount of RPMI-1640 complete medium containing 2 μg / mL puromycin every 2 days).

[0091] 8. After completing step 7, obtain the P0 generation target recombinant cells by flow cytometry sorting.

[0092] Sorting criteria: showing fluorescence at an excitation wavelength of 405 nm and at an excitation wavelength of 488 nm, and the fluorescence emission wavelengths are 525 / 50 nm.

[0093] Example 3: Passaging of recombinant cells

[0094] RPMI-1640 complete medium: RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0095] The P0 generation target recombinant cells obtained in Example 2 were serially passaged. The cell culture and passaging methods for each generation were as follows: cells were cultured in a cell culture flask using RPMI-1640 complete medium containing 2 μg / mL puromycin for 2 days. The liquid phase from one cell culture flask was then divided equally between two cell culture flasks, and each cell culture flask was supplemented with an equal volume of RPMI-1640 complete medium containing 2 μg / mL puromycin as the liquid phase.

[0096] Cells from passages P1 to P20 were obtained. Each passage was analyzed using flow cytometry, and the percentage of target cells relative to total cells was consistently greater than 95%. Target cells were defined as cells that exhibited fluorescence at both 405 nm and 488 nm excitation wavelengths, with emission wavelengths of 525 / 50 nm.

[0097] Example 4: Subcellular localization

[0098] 1. The target recombinant cells prepared in Example 2 were collected and resuspended in RPMI-1640 medium to obtain a cell suspension of 800,000 cells / mL.

[0099] 2. Take 500 μL of the cell suspension prepared in step 1, add the mitochondrial probe and make the concentration in the system 200 nM, mix well, and incubate at 37°C in the dark for 20 minutes. TM Dyes for Mitochondria Labeling): Invitrogen, catalog number M7512.

[0100] 3. After completing step 2, centrifuge at 1000 rpm, discard the supernatant, resuspend the cell pellet with 100 μL PBS buffer, then slowly add 1 mL of methanol (pre-cooled at -20°C), mix gently, and incubate for 10 minutes.

[0101] 4. After completing step 3, centrifuge at 2000 rpm to collect the cell pellet.

[0102] 5. Resuspend the cell pellet obtained in step 4 in 1 mL of PBS buffer (pre-cooled at -20°C), centrifuge at 2000 rpm, and collect the cell pellet.

[0103] 6. Resuspend the cell pellet obtained in step 5 in 1 mL of PBS buffer (pre-cooled at -20°C), centrifuge at 2000 rpm, and collect the cell pellet.

[0104] 7. Resuspend the cell pellet obtained in step 6 in 1 mL of PBS buffer (pre-cooled at -20°C), then take a 200 μL sample and spin it using a Thermo Scientific Cytospin 4 to allow the cells to adhere to the slide.

[0105] 8. Seal the slides with antifade mounting medium containing DAPI (Beyotime, P0131) and then cover with a coverslip.

[0106] 9. Observe and photograph using a confocal microscope (Leica TCS SP8 STED, Leica). DAPI (blue): Ex / Em = 350 / 450 nm. HyPer7 probe (green): Ex / Em = 488 / 525 nm. Mitochondrial probe (red): Ex / Em = 577 / 602 nm.

[0107] See the sample photos Figure 2 It can be observed that the HyPer7 probe co-localizes with the mitochondrial probe, that is, the HyPer7 probe specifically binds to the mitochondria.

[0108] Example 5: Verification of Recombinant Cell Response to H2O2

[0109] RPMI-1640 complete medium: RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0110] Experimental group (expressed as H2O2): The target recombinant cells prepared in Example 2 were collected and resuspended in RPMI-1640 complete medium to a cell content of 500,000 to 1,000,000 cells / mL. A 100 mM H2O2 aqueous solution was then added to bring the H2O2 concentration in the system to 1 mM. The cells were then quickly added to a confocal microplate and incubated for 10 min. The cells were then excited under a confocal fluorescence microscope using 405 nm and 488 nm excitation light, respectively, and fluorescence was detected (fluorescence emission wavelength was 525 / 50 nm) and imaged.

[0111] Control group (denoted as Control): The target recombinant cells prepared in Example 2 were collected and resuspended in RPMI-1640 complete medium to a cell content of 500,000 to 1,000,000 cells / mL. The cells were then added to a confocal microplate and incubated for 10 minutes. The cells were then excited under a confocal fluorescence microscope using 405 nm and 488 nm excitation light, respectively, and fluorescence was detected (fluorescence emission wavelength was 525 / 50 nm) and imaged.

[0112] See the sample photos Figure 3 a.

[0113] The image data obtained by confocal microscopy were quantitatively analyzed by ImageJ software. The fluorescence intensity was quantified by calculating the average gray value of the pixels. The ratio of the fluorescence intensity under 488 nm excitation light to the fluorescence intensity under 405 nm excitation light was used as the response signal. Figure 3 The b signal can reach about 15 times, indicating that the sensor has good response performance.

[0114] Example 6: Sensitivity of recombinant cells in response to H2O2

[0115] RPMI-1640 complete medium: RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0116] 1. Collect 200,000 target recombinant cells prepared in Example 2, resuspend them in 500 μL of RPMI-1640 complete medium containing H2O2 (set multiple H2O2 concentrations: 1 μM-400 μM), incubate at room temperature for 2 minutes, and then detect them using a flow cytometer, using 405nm and 488nm excitation wavelengths to collect fluorescence signals (fluorescence emission wavelength is 525 / 50nm). 10,000 cell events were collected for each culture medium for statistics, and the average value of the response signal (the ratio of the fluorescence intensity under 488nm excitation light to the fluorescence intensity under 405nm excitation light) was taken. The results are shown in Figure 4 (The horizontal axis represents the H2O2 concentration in the culture medium). The recombinant cells used as a cellular sensor to detect ambient H2O2 concentrations showed a sensitivity of approximately 10 μM, a linear range of 10-100 μM, and a maximum change factor of approximately 10. This demonstrates that the cellular sensor can effectively reflect H2O2 levels and is highly sensitive to low H2O2 concentrations, with a detection limit of 10 μM.

[0117] 2. Collect the target recombinant cells prepared in Example 2, resuspend them in RPMI-1640 complete medium containing H2O2 (the H2O2 concentration is set to 10μM, 40μM or 100μM respectively), and then use flow cytometry to continuously detect for 12 minutes (time interval is 30s), using 405nm and 488nm excitation wavelengths to collect fluorescence signals (fluorescence emission wavelength is 525 / 50nm). 10,000 cell events were collected for each culture medium for statistics, and the average value of the response signal (the ratio of the fluorescence intensity under 488nm excitation light to the fluorescence intensity under 405nm excitation light) was taken. The results are shown in Figure 4 b. The mitochondrial H2O2 production rate increases with increasing ambient H2O2 concentration. At an ambient H2O2 concentration of 100 μM, the response reaches over five times within 30 seconds and stabilizes after approximately 2 minutes. At an ambient H2O2 concentration of 40 μM, the response begins at 1.5 minutes and continues to increase after 12 minutes. This demonstrates that the sensor's response time sensitivity is related to H2O2 concentration, with extremely rapid responses occurring at higher concentrations, demonstrating excellent performance.

[0118] Example 7: Application example of evaluating the oxidative stress effect of pollutants based on sensor cells

[0119] RPMI-1640 complete medium: RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin. Test substances: bisphenol A (BPA), bisphenol B (BPB), bisphenol C (BPC), bisphenol F (BPF), bisphenol AF (BPAF), or bisphenol S (BPS).

[0120] 1. Functional Validation of the Monocyte Model

[0121] 1. Take the target recombinant cells prepared in Example 2, resuspend them in RPMI-1640 complete medium containing 100 μM of the test substance, then seed them into a 24-well plate and culture for 24 hours. Set up a control, that is, replace the RPMI-1640 complete medium containing 100 μM of the test substance with RPMI-1640 complete medium.

[0122] 2. After completing step 1, use flow cytometry to detect the fluorescence signal using 405nm and 488nm excitation wavelengths (fluorescence emission wavelength is 525 / 50nm). Collect 10,000 cell events for each culture medium and calculate the average value of the response signal (the ratio of the fluorescence intensity under 488nm excitation light to the fluorescence intensity under 405nm excitation light).

[0123] See the results Figure 5 a. Five compounds induced a significant increase in mitochondrial H2O2 levels in the monocyte model.

[0124] 2. Functional Verification of Macrophage Model

[0125] 1. Take the target recombinant cells prepared in Example 2 and resuspend them in RPMI-1640 complete medium containing 100 ng / mL PMA. Then, seed them into a 24-well plate. After culturing for 24 hours, discard the supernatant containing non-adherent cells. Then, add RPMI-1640 complete medium containing 100 μM of the test substance and continue culturing for 24 hours. Set up a control, that is, replace the RPMI-1640 complete medium containing 100 μM of the test substance with RPMI-1640 complete medium.

[0126] 2. After completing step 1, use flow cytometry to detect the fluorescence signal using 405nm and 488nm excitation wavelengths (fluorescence emission wavelength is 525 / 50nm). Collect 10,000 cell events for each culture medium and calculate the average value of the response signal (the ratio of the fluorescence intensity under 488nm excitation light to the fluorescence intensity under 405nm excitation light).

[0127] See the results Figure 5 b. In the macrophage model, all six compounds led to a significant increase in mitochondrial H2O2 levels, among which the signal of the most toxic BPB-exposed sample increased by more than 50%.

[0128] Figure 5 The results showed that both cell models can be used in the evaluation of the oxidative stress effects of pollutants.

[0129] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A recombinant cell, the preparation method of which comprises the following steps: (1) preparing a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe; the mitochondrial-targeted HyPer7 probe is a protein, which has a mitochondrial-targeting segment and a HyPer7 probe segment from the N-terminus to the C-terminus; the mitochondrial-targeting segment is as shown in positions 1-29 of SEQ ID NO: 2, and the HyPer7 probe segment is as shown in positions 42-520 of SEQ ID NO: 2; (2) Transfect the recombinant lentivirus into THP-1 cells to obtain recombinant cells.

2. A method for preparing a recombinant cell, comprising the following steps: (1) preparing a recombinant lentivirus expressing a mitochondrial-targeted HyPer7 probe; the mitochondrial-targeted HyPer7 probe is a protein, which has a mitochondrial-targeting segment and a HyPer7 probe segment from the N-terminus to the C-terminus; the mitochondrial-targeting segment is as shown in positions 1-29 of SEQ ID NO: 2, and the HyPer7 probe segment is as shown in positions 42-520 of SEQ ID NO: 2; (2) Transfect the recombinant lentivirus into THP-1 cells to obtain recombinant cells.

3. Macrophages obtained by inducing the recombinant cells according to claim 1.

4. A protein comprising a mitochondrial targeting segment and a HyPer7 probe segment from N-terminus to C-terminus; the mitochondrial targeting segment is as shown at positions 1-29 in SEQ ID NO: 2, and the HyPer7 probe segment is as shown at positions 42-520 in SEQ ID NO:

2.

5. A nucleic acid molecule encoding the protein of claim 4.

6. A recombinant plasmid or recombinant virus comprising the nucleic acid molecule according to claim 5.

7. Use of component 1 or component 2 in preparing recombinant cells; The component 1 is composed of the recombinant plasmid described in claim 6, the lentiviral packaging helper plasmid, the lentiviral packaging cells and THP-1 cells; The component 2 consists of the recombinant virus described in claim 6 and THP-1 cells.

8. Use of the recombinant cell according to claim 1 or the macrophage according to claim 3 as a cell sensor for detecting H2O2.

9. Use of the recombinant cell of claim 1 or the macrophage of claim 3 in environmental toxicology research; the environmental toxicology research is an environmental toxicology research related to H2O2.

10. Use of the recombinant cell according to claim 1 or the macrophage according to claim 3 as a cell model.

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