Mitochondrial function evaluation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a mitochondrial function evaluation method and application thereof. The invention provides an innovative mitochondrial function evaluation method which comprises the following steps: reporting ATP concentration change and mitochondrial morphology in a mitochondrial matrix in real time, in situ and quantitatively through ATP response fluorescent protein Mito-AT1.03 with a mitochondrial positioning signal, and directly reflecting mitochondrial energy metabolism and mitochondrial fusion / division states; meanwhile, a mitochondrial autophagy key initiation factor Parkin protein is utilized to construct a fusion protein Parkin-mCherry, and the fusion protein Parkin-mCherry is used as an early-stage specific marker for autophagy initiation. The two fluorescent proteins are co-transfected into cells, and the signal levels and positioning conditions of the two fluorescent proteins are observed, so that the mitochondrial function can be accurately evaluated in real time, and a new thought is provided for methodology development of mitochondrial dysfunction mechanism research and mitochondrial function regulator screening.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for assessing mitochondrial function and its application. Background Technology

[0002] Mitochondria are vital organelles in cells, responsible for energy production and participating in numerous important physiological processes, including cell differentiation, proliferation, metabolism, inflammation, and apoptosis. Their proper function is crucial for maintaining cellular homeostasis. Oxidative stress and cellular senescence can lead to mitochondrial dysfunction, particularly impaired oxidative phosphorylation (decreased ATP levels) and compromised quality control (accumulation of damaged mitochondria), ultimately disrupting cellular metabolic homeostasis and causing cell death. Recent studies suggest that mitochondrial dysfunction may be associated with the development and progression of many major diseases, including neurodegenerative diseases (such as Parkinson's and Alzheimer's), metabolic diseases (such as diabetes and obesity), cardiovascular diseases, inflammatory bowel disease, cancer, and aging. Currently, there is a gap in the translation of research findings on mitochondrial dysfunction into effective targeted drugs. The mechanisms triggering mitochondrial dysfunction remain unclear, significantly limiting the further clinical application of mitochondrial function regulators. Therefore, developing mitochondrial function detection methods based on specific mechanisms will provide strong support for the future research and validation of various mitochondrial function regulatory chemicals.

[0003] Mitochondrial morphology is highly dynamic. To adapt to changes in intracellular and extracellular stress environments, it maintains the dynamic balance of the mitochondrial network through continuous division and fusion processes, thereby meeting energy metabolism and other physiological needs. Mitochondrial morphology is regulated by fusion proteins (such as MFN1 / 2 and OPA1) and splitting proteins (such as DRP1 and FIS1). Fusion proteins enhance intermitochondrial cooperation to facilitate energy transfer and substance exchange (such as maintaining membrane potential and sharing mitochondrial DNA); splitting proteins facilitate the transport of mitochondria within the cell or the isolation of damaged mitochondria, and selectively encapsulate and degrade damaged mitochondria through mitophagy. Mitophagy is a key mitochondrial quality control pathway in the cell, and the PINK1 / Parkin pathway is one of the most well-understood mitophagy pathways. PTEN-inducible kinase 1 (PINK1) is a highly conserved mitochondrial protein involved in the regulation of mitochondrial function. Parkin is an E3 ubiquitin ligase responsible for linking Ub molecules to substrate proteins; substrate proteins tagged with Ub are recognized and degraded by proteases. In healthy mitochondria, PINK1 targets mitochondria via its mitochondrial targeting sequence and enters the inner mitochondrial membrane via the TOM / TIM complex. It is then cleaved by proteases located on the mitochondrial matrix and inner membrane, and released into the cytoplasm for hydrolysis by ubiquitin-proteasomes. In damaged mitochondria, mitochondrial depolarization and decreased membrane potential obstruct the pathway of PINK1 into the inner mitochondrial membrane. This causes PINK1 to stably accumulate on the cytoplasmic surface of the outer mitochondrial membrane, recruiting Parkin and activating its E3 ubiquitin ligase activity. This ubiquitinates mitochondrial proteins, including p62, which accumulate on the outer mitochondrial membrane. The ubiquitinated products are then recruited to autophagosomes by binding to LC3. Mature autophagosomes fuse with lysosomes to form autolysosomes, which ultimately degrade the contained mitochondria.

[0004] Fluorescent protein labeling technology uses fluorescent proteins (such as red fluorescent protein mCherry) to label and track specific proteins or biomolecules within cells. This is achieved by inserting the fluorescent protein-encoding gene into the target protein gene using transgenic technology, constructing a fusion expression vector, and then using transfection technology to express the fluorescently labeled target protein in cells. This technology can visually present the distribution and translocation of the target protein and subcellular structures such as mitochondria within the cell, maintaining cell viability and providing continuous imaging time-series data for precise observation of cellular dynamics. Furthermore, by combining multiple fluorescent proteins, multicolor labeling observation can be achieved, providing more comprehensive and detailed information on cell structure and function. Therefore, fluorescent protein labeling technology combined with cell imaging analysis is widely used in high-throughput screening, drug development, and toxicity assessment. Simultaneously monitoring intracellular ATP levels and the degree of mitophagy using fluorescent protein labeling technology can accurately reflect the energy production capacity and quality control process of mitochondria, providing a comprehensive and accurate description of mitochondrial function.

[0005] Currently, techniques for studying mitochondrial energy status mainly include Seahorse extracellular flux (XF) analysis, ATP content measurement, mitochondrial membrane potential (MMP) detection, and respiratory complex activity analysis. Among these, Seahorse extracellular flux (XF) analysis uses microsensors to monitor live-cell oxygen consumption rate (OCR, reflecting respiratory chain activity) and extracellular acidification rate (ECAR, reflecting glycolysis) in real time, analyzing the energy contribution of mitochondrial aerobic metabolism and glycolysis. However, it only provides population-wide average values ​​and cannot directly observe subcellular dynamics (e.g., individual mitochondria). Furthermore, it relies on exogenous inhibitors (e.g., oligomycin, FCCP), which may interfere with normal mitochondrial physiological states. ATP content measurement mainly includes chemiluminescence methods (e.g., luciferase reporter systems) and enzyme-coupled methods (hexokinase reactions). Both methods require cell lysis, cannot track real-time dynamic changes in mitochondrial ATP, and cannot distinguish between mitochondrial-derived and non-mitochondrial-derived ATP (e.g., glycolysis). Mitochondrial membrane potential (MMP) detection uses mitochondrial membrane potential-specific probes (e.g., JC-1, TMRM, etc.) to indicate MMP. However, the inherent instability and potential cytotoxicity of fluorescent probes not only limit their long-term observation of mitochondrial membrane potential but also lead to significant batch effects, making it difficult to guarantee the reproducibility and consistency of results across different experiments. Respiratory chain complex activity analysis, based on spectrophotometric detection of complex IV enzyme activity, also requires cell lysis, resulting in the loss of live cell dynamics information and failing to reflect the regulatory mechanisms of mitochondrial dysfunction.

[0006] There are many techniques for studying mitophagy, such as transmission electron microscopy (TEM), Western blotting, immunofluorescence, fluorescent probe labeling, and fluorescent protein labeling. Among these, TEM can observe substructural changes in mitochondria and the formation of mitophagosomes. It is mainly identified through the unique double membrane and cristae structures of mitochondria. However, it is easily affected by cell structure damage caused by human manipulation such as sectioning, and can also be misled by other double membrane structures or low electron density vesicles within the cell. Moreover, TEM sample preparation is complex and inefficient, which has significant limitations in quantitative analysis. Western blotting utilizes the principle of antigen-antibody reactions to indirectly reflect mitochondrial autophagy activity by detecting changes in the expression levels of autophagy-related proteins (such as mitochondrial membrane protein Tom20, autophagosome protein LC3, and lysosomal protein LAMP2). Immunofluorescence, similarly based on the principle of specific antigen-antibody binding, uses fluorescently labeled antibodies to achieve subcellular localization of autophagy-related proteins. Both techniques require expensive specific antibodies, are time-consuming, and involve cumbersome procedures. They cannot dynamically detect the autophagy process or perform high-throughput screening of the autophagy activity of various compounds. Fluorescent probe labeling utilizes mitochondrial and lysosomal specific fluorescent probes to achieve co-localization of mitochondria and lysosomes. Therefore, it can only observe the late stage of mitophagy (the stage of fusion between autophagic mitochondria and lysosomes), failing to observe the early stages of mitophagy and exhibiting the inherent limitations of fluorescent probes mentioned above. The conventional strategy for detecting mitophagy using fluorescent protein labeling technology involves fusing the genes of autophagosomes and mitochondria-specific proteins with different fluorescent protein genes, and then analyzing mitochondrial autophagy activity by observing the co-localization of the two fused fluorescent proteins (such as the technology disclosed in Chinese invention patent CN202010148310.3). This strategy determines whether mitophagy has occurred and the level of autophagy by observing the binding of mitochondria to autophagosomes. However, since it does not involve the early triggering mechanism of mitophagy, the mitophagy regulators obtained when applied to the screening of mitophagy compounds still require further mechanistic studies and verification, which greatly increases the workload.

[0007] In summary, current methods for assessing mitochondrial function still have significant limitations: (1) Single evaluation indicators: Commonly used methods (such as JC-1 probe, MitoTracker probe, and Seahorse oxygen consumption rate assay) often only reflect one aspect of mitochondrial function (such as membrane potential, mitochondrial mass, and respiratory chain activity), lacking a comprehensive assessment of key functions (energy status and quality control). (2) Lack of dynamic correlation: Mitochondrial energy metabolism (ATP level) and quality control (especially autophagic clearance of damaged mitochondria) are closely coupled dynamic processes. Existing methods make it difficult to observe the changes of these two processes and their spatiotemporal relationship simultaneously, in the same living cell, or even on the same mitochondria at the same time point, in the same living cell, or even on the same mitochondria, which greatly limits the in-depth understanding of the physiological and pathological mechanisms of mitochondria. (3) Limitations in throughput and applicability: High-resolution techniques (such as transmission electron microscopy) cannot dynamically observe mitochondria; biochemical methods (such as ATP detection kits) are prone to damaging cells and losing spatial information; and there is a lack of highly specific tools suitable for long-term, low-toxicity observation of living cells. Therefore, it is necessary to develop new methods for assessing mitochondrial function to simultaneously assess mitochondrial energy status, kinetics, and the initiation of mitochondrial autophagy. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, this invention provides an innovative method for assessing mitochondrial function. By co-expressing the mitochondrial ATP-responsive fluorescent protein AT1.03 and the Parkin-mCherry fusion protein in living cells, it achieves both synchronous, dynamic, and visual tracking of mitochondrial energy metabolism (ATP levels) and mitochondrial quality control (fusion / fission balance and autophagy initiation-Parkin recruitment).

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of this invention provides a method for assessing mitochondrial function, specifically: co-expressing mitochondrial ATP-responsive fluorescent protein AT1.03 and Parkin-mCherry fusion protein in living cells, monitoring changes in mitochondrial matrix ATP concentration, fusion and division status, and Parkin protein recruitment dynamics through dual-channel fluorescence imaging; the sequence of the synthesized Parkin-mCherry fusion protein is shown in SEQ ID No. 1.

[0011] Preferably, the method for co-expressing the mitochondrial ATP-responsive fluorescent protein AT1.03 and the Parkin-mCherry fusion protein in living cells specifically includes the following steps:

[0012] S1. The sequence of synthesized Parkin-mCherry was cloned into the lentiviral vector LV6-EF1a-Puro to construct the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentiviral vector plasmid, and HEK293T cells were transfected to collect lentiviral particles.

[0013] S2. Live cells were infected with lentiviruses and selected with puromycin to obtain cell lines that stably express Parkin-mCherry fusion protein.

[0014] S3. Using liposomes, the plasmid of the ATP fluorescent probe Mito-AT1.03 expressing the mitochondrial localization signal was transfected into the cell line in S2 that stably expressed the Parkin-mCherry fusion protein. After G418 selection, a cell line stably expressing dual fluorescent proteins was obtained.

[0015] This invention utilizes the ATP-responsive fluorescent protein Mito-AT1.03, which carries a mitochondrial localization signal, to report changes in ATP concentration and mitochondrial morphology in the mitochondrial matrix in real time, in situ, and quantitatively, directly reflecting mitochondrial energy metabolism and mitochondrial fusion / division status. Simultaneously, a fusion protein, Parkin-mCherry, is constructed using Parkin protein, a key initiation factor for mitophagy, as an early specific marker for autophagy initiation. By co-transfecting cells with both proteins and observing the signal levels and localization of the two fluorescent proteins, mitochondrial function can be accurately assessed in real time, providing new insights for the development of methodologies for studying mitochondrial dysfunction mechanisms and screening mitochondrial function regulators.

[0016] More preferably, the living cells are human bronchial epithelioid cells 16HBE.

[0017] More preferably, in S2, the concentration of puromycin used for screening is not less than 1 μg / mL, and the screening time is more than 4 days.

[0018] Furthermore, the optimal concentration of puromycin used for screening was 1 μg / mL, and the screening time was 4 days.

[0019] More preferably, in S2, the live cells are revived and passaged 4-8 times before infection.

[0020] More preferably, in S3, the plasmid expressing the ATP fluorescent probe Mito-AT1.03, which expresses the mitochondrial localization signal, is the pCMV-Mito-AT1.03 plasmid.

[0021] More preferably, in S3, the concentration of G418 used for G418 screening is not less than 400 μg / mL, and the screening time is more than 14 days.

[0022] Furthermore, the optimal G418 concentration used for G418 screening was 400 μg / mL, and the screening time was 14 days.

[0023] The second aspect of this invention provides the application of the mitochondrial function assessment method described in the first aspect in screening compounds that regulate mitochondrial function.

[0024] Preferably, the mitochondrial function regulating compound includes rotenone and doxorubicin.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) Real-time synchronous monitoring of dual functions: For the first time, the correlation between mitochondrial energy metabolism (ATP level) and quality control (fusion / fission balance and autophagy initiation-Parkin recruitment) can be tracked synchronously, dynamically, and visually at live cell and single-cell / subcellular organelle resolution. This provides an unprecedentedly powerful tool for revealing the dynamic regulation of mitochondrial function and understanding the causal and temporal relationships between the energy crisis and mitochondrial fusion / fission balance and autophagy clearance.

[0027] (2) Solving key scientific problems: greatly promotes the understanding of the mitochondrial dysfunction mechanism in neurodegenerative diseases (such as Parkinson's disease-Parkin gene-related diseases, Alzheimer's disease), cardiovascular diseases, metabolic syndromes (such as diabetes, obesity), cancer, aging and related diseases, because these diseases all have the core problem of energy imbalance and / or autophagy defects.

[0028] (3) Facilitates drug development and toxicity evaluation:

[0029] 1) High-throughput drug screening: Imaging-based high-content screening platforms can be developed to efficiently screen novel therapeutic compounds (such as neuroprotective agents, cardioprotective agents, anti-aging drugs, and anticancer drugs) that can synergistically improve mitochondrial energy production (increase ATP) and promote the clearance of damaged mitochondria (induce Parkin recruitment).

[0030] 2) Drug mechanism research: to accurately elucidate the real-time effects of candidate drugs on the dynamic processes of mitochondrial dual function and their interactions.

[0031] 3) Toxicity assessment: Real-time monitoring of the multiple toxicities of drugs or environmental pollutants to mitochondrial energy, mitochondrial fusion / fission balance and mitophagy function provides a more comprehensive safety evaluation.

[0032] (4) Technical advantages: It has the advantages of high spatiotemporal resolution, suitability for long-term live cell imaging, high genetic coding specificity, and relatively low phototoxicity. Attached Figure Description

[0033] Figure 1This is a schematic diagram illustrating the principle of mitochondrial function evaluation.

[0034] Figure 2 A schematic diagram of the LV6-EF1a-Parkin-mCherry-Puro fusion expression vector;

[0035] Figure 3 The fluorescence expression pattern of the LV6-EF1a-Parkin-mCherry-Puro fusion expression vector;

[0036] Figure 4 The sequencing sequence alignment results for the LV6-EF1a-Parkin-mCherry-Puro fusion expression vector.

[0037] Figure 5 The fluorescence image of the 16HBE control group stably expressing the Parkin-mCherry fusion protein; the right-hand composite image: the top left image is the superimposed effect of the fluorescence of the blue, green and red channels, the top right image is the DAPI-stained cell nucleus, the bottom left image is the immunofluorescence image of the mitochondrial protein Tom20, and the bottom right image is the intracellular localization of the Parkin-mCherry fusion protein; the left-hand image is a magnified view of the superimposed image.

[0038] Figure 6 Fluorescence image of 16HBE after 12 h exposure to rotenone to stably express the Parkin-mCherry fusion protein; in the right composite image: the top left image is the superimposed effect of the blue, green and red channels fluorescence, the top right image is the DAPI-stained cell nucleus, the bottom left image is the immunofluorescence image of the mitochondrial protein Tom20, and the bottom right image is the intracellular localization of the Parkin-mCherry fusion protein; the left image is a magnified view of the superimposed image.

[0039] Figure 7 The first image shows the fluorescence of the mitochondrial functional dual fluorescent reporter gene 16HBE cells in the control group; the top left image shows the superimposed effect of the green and red channels fluorescence; the top middle image shows the intracellular localization of the ATP-responsive fluorescent protein Mito-AT1.03 containing mitochondrial localization signals; the top right image shows the intracellular localization of the Parkin-mCherry fusion protein; and the bottom image is a magnified view of the superimposed image.

[0040] Figure 8 The image shows the fluorescence of the doxorubicin-exposed group of 16HBE cells containing the mitochondrial functional dual fluorescent reporter gene; the top left image shows the superimposed effect of the green and red channels fluorescence; the top middle image shows the intracellular localization of the ATP-responsive fluorescent protein Mito-AT1.03 containing mitochondrial localization signals; the top right image shows the intracellular localization of the Parkin-mCherry fusion protein; and the bottom image is a magnified view of the superimposed image (i.e., the mitophagy that occurs in the cell).

[0041] Figure 9 Statistical graphs were created to quantitatively represent changes in mitochondrial matrix ATP concentration, fusion and division states, or Parkin protein recruitment dynamics using green fluorescence intensity, mitochondrial fragmentation factor, and number of mitophagy particles, respectively. Green fluorescence intensity results were statistically analyzed from fluorescence images of 5 random fields of view, and mitochondrial fragmentation factor and number of mitophagy particles were randomly analyzed from 50 cells. **p < 0.01, ***p < 0.001. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0044] This invention provides a method for evaluating mitochondrial function and its application:

[0045] (I) Overall Framework

[0046] By constructing a dual fluorescent probe co-expression system ( Figure 1 In living cells, it performs the following functions:

[0047] (1) Real-time quantification of mitochondrial matrix ATP (green channel);

[0048] (2) Real-time observation of mitochondrial morphology (green channel);

[0049] (3) Dynamic tracking of Parkin protein recruitment on the mitochondrial surface (red channel).

[0050] (II) Implementation Steps

[0051] 1. Constructing the Parkin-mCherry fusion expression lentiviral vector plasmid

[0052] The LV6-EF1a-Parkin-mCherry-Puro lentiviral vector plasmid was synthesized by Gemma Genetics. Following the Homosapiens parkin RBR E3 ubiquitin protein ligase (NCBI Reference Sequence: NM_004562.3) and the synthetic construct mCherry gene (GenBank: MZ027319.1), the Parkin-mCherry sequence was synthesized using gene synthesis methods. The synthesized sequence was cloned into the lentiviral vector LV6-EF1a-Puro, constructing the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentiviral vector plasmid. This plasmid was then transfected into HEK293T cells to collect lentiviral particles.

[0053] 2. Construction of a cell line for mitochondrial autophagy reporter gene therapy

[0054] 16HBE cells were resuscitated and passaged 5 times. 16HBE cells were then infected with lentivirus, and cell lines stably expressing the Parkin-mCherry fusion protein were selected using puromycin (1 μg / mL, 4 days). Cells were treated with a mitophagy inducer (rotenone, 100 μM) for 6 h, fixed, and permeabilized. Mitochondrial protein Tom20 was labeled using immunofluorescence, and the nuclei were labeled with the nuclear dye DAPI. The expression of the Parkin-mCherry fusion protein was observed and verified under a laser confocal microscope.

[0055] 3. Construction of a mitochondrial functional dual-fluorescent reporter gene cell line

[0056] The plasmid (pCMV-Mito-AT1.03) containing the ATP fluorescent probe Mito-AT1.03, which expresses mitochondrial localization signals, was transfected into 16HBE cells stably expressing the Parkin-mCherry fusion protein using liposomes. Cell lines stably expressing dual fluorescent proteins were selected using G418 (400 μg / mL, 14 days). Cells were treated with a mitochondrial toxic compound (doxorubicin, 10 μM) for 6 h, and the expression of dual fluorescent reporter genes in live cells was observed and verified under a laser confocal microscope.

[0057] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0058] Example 1: Construction of Parkin-mCherry fusion expression lentiviral vector plasmid

[0059] The LV6-EF1a-Parkin-mCherry-Puro lentiviral vector plasmid was synthesized by Gemma Genetics Co., Ltd. Specifically, referencing the Homo sapiens parkin RBR E3 ubiquitin protein ligase (NCBI Reference Sequence: NM_004562.3) and the synthetic construct mCherry gene (GenBank: MZ027319.1), the Parkin-mCherry sequence (SEQ ID No. 1) was synthesized using gene synthesis methods, as follows:

[0060]

[0061] NotI / BamHI restriction sites were added to both ends of the sequence, and the synthesized sequence was cloned into the lentiviral vector LV6-EF1a-Puro to construct the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentiviral vector plasmid. The constructed plasmid was transformed into Top10 competent E. coli, and the bacteria were then plated onto ampicillin-resistant LB agar plates and cultured overnight at 37°C. Single clones were picked and identified by colony PCR. Positive single clones were selected for amplification culture, and endotoxin-free plasmids were extracted using a high-purity plasmid extraction kit (Genepharma, P4170). After verification by sequencing, the plasmids were co-transfected into HEK293T cells using the transfection reagent RNAi-Mate (Genepharma, G04001) along with three helper packaging plasmids (pGag / Pol, pRev, pVSV-G). The specific steps are as follows: One day before transfection, HEK293T cells were seeded in 15cm culture dishes. Before transfection, 30μg of plasmid (7.5μg each of the four plasmids) was added to a centrifuge tube containing 1.5mL of serum-free DMEM medium and mixed well. Separately, 300μL of RNAi-Mate was added to another centrifuge tube containing 1.5mL of serum-free DMEM and mixed well. After incubation at room temperature for 5 minutes, the two were mixed and incubated at room temperature for another 20 minutes. The old medium in the 15cm culture dish was discarded, and 8mL of serum-free DMEM was added. The transfection mixture was then added dropwise to the culture dish, and the cells were incubated at 37℃ and 5% CO2 for 6 hours. The transfection solution was discarded, and fresh complete DMEM medium containing 10% FBS was added, and the cells were incubated for another 72 hours. The cell supernatant rich in lentiviral particles was collected and concentrated by ultracentrifugation at 4℃ and 20,000rpm for 2 hours to obtain a high-titer lentiviral concentrate. HEK293T cells were then cultured at a rate of 5×10⁻⁶ cells / year. 4 Cells were seeded in 24-well plates. The lentivirus stock solution was diluted 10-fold with DMEM medium containing 10% FBS. The medium in the 24-well plates was aspirated, and 500 μL of the diluted virus solution was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 72 h. HEK293T cell expression was verified by observation under a fluorescence microscope. The experimental results are as follows: Figure 2 As shown, fluorescence microscopy (100x) 72 h after lentiviral infection revealed that red fluorescent protein was diffusely distributed in HEK293T cells, with some cells containing several small particles, demonstrating the successful construction and expression of the Parkin-mCherry fusion protein expression vector. Figure 3 Furthermore, sequencing alignment of the LV6-EF1a-Parkin-mCherry-Puro fusion expression vector showed that the sequence was completely correct and contained no mutated bases. Figure 4 ).

[0062] Example 2: Construction of a cell line using a mitochondrial autophagy reporter gene

[0063] (1) 16HBE cell resuscitation and culture: The frozen 16HBE cells were rapidly thawed in a 37°C water bath. After thawing, the cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and MEM complete medium containing 10% FBS, 1% penicillin / streptomycin and 1% GlutaMAX was added. The cells were then cultured in a 37°C, 5% CO2 cell culture incubator.

[0064] (2) Cell infection: After 5 passages of cells, inoculate with 5×10 4 16HBE cells were seeded at a concentration of 500 μL / mL into 24-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. The virus concentrate was diluted 10-fold with fresh complete culture medium, and Polybrene was added to a final concentration of 5 μg / mL to prepare a total virus solution of 500 μL. The old culture medium in the 24-well plates was discarded, and the diluted virus solution was added to the cells. The cells were cultured for another 24 h, the virus solution was removed, and fresh culture medium was added. The cells were then cultured overnight. The cells were washed once with PBS, digested with trypsin for 2 min, and then digested with complete culture medium to terminate the digestion. Cells were collected by pipetting and counting, and the cell density was adjusted to 2 × 10⁶ cells / mL. 4 Cells per mL were used for passage to obtain transiently transformed cells for selection.

[0065] (3) Screening of stable transfected cells: The above transiently stained cells were seeded into 24-well plates at 500 μL per well and cultured for 24 h. Then, complete medium containing 1 μg / mL puromycin was added. The selection medium was changed every 2 days. After 4 days of pressure selection, the cells were digested with trypsin for 2 min, centrifuged to collect the cells, resuspended in 2 mL of maintenance medium containing 0.5 μg / mL hygromycin B, and cultured in 6-well plates for 3 days to obtain stable transfected cell lines (16HBE cells stably expressing Parkin-mCherry fusion protein).

[0066] The main steps in determining the optimal screening concentration of puromycin are as follows: 16HBE cells are charged at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 24-well plates. After 24 hours of culture, the old culture medium was removed, and a gradient concentration of puromycin was added: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 4, and 8 μg / mL. The culture medium was changed every 2 days. It was observed that the minimum puromycin concentration that killed all cells on day 4 was 1 μg / mL. Therefore, 1 μg / mL was selected as the optimal screening concentration of puromycin.

[0067] (4) Phenotypic verification: Stable cells were divided into groups of 1×10⁻⁶ cells. 5Two cells were seeded per mL in glass-bottom confocal dishes, and cultured at 37°C in a 5% CO2 incubator for 24 h. The old culture medium was removed, and fresh culture medium containing 100 μM rotenone (Sigma, R8875), a mitophagy inducer, was added. The cells were incubated at 37°C in a 5% CO2 incubator for 12 h. The old culture medium was discarded, and the cells were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS, permeabilized with 0.3% Triton X-100 permeabilization buffer at room temperature for 15 min, washed three times with PBS, blocked with 5% BSA at room temperature for 1 h, and incubated overnight at 4°C with a 1:200 dilution of anti-mitochondrial protein Tom20 primary antibody (Cell Signaling Technology, 42406). The cells were then washed three times with PBST (0.1% Tween 20) and incubated with a 1:500 dilution of Alexa... The cells were incubated with 488-labeled fluorescent secondary antibody (Cell Signaling Technology, 4412) at room temperature for 1 hour, then washed three times with PBST. The nuclei were then labeled with ready-to-use nuclear dye DAPI (Invitrogen, R37606) (two drops per mL), stained for 30 minutes, washed twice with PBS, and finally the fluorescence image of Parkin-mCherry was captured by laser confocal microscopy.

[0068] The results are as follows Figure 5 As shown, in the control group without rotenone, Parkin-mCherry red fluorescent protein was diffuse, with few particles and no co-localization with the mitochondrial protein Tom20, indicating a low level of mitophagy. In contrast, the experimental group with rotenone showed a significant increase in Parkin-mCherry red fluorescent protein particles and extensive co-localization with the mitochondrial protein Tom20, indicating significant mitophagy. Figure 6 ).

[0069] Example 3: Construction of a mitochondrial functional dual-fluorescent reporter gene cell line

[0070] (1) According to 1.1×10 5 16HBE cells stably expressing the Parkin-mCherry fusion protein were seeded at a concentration of 500 μL / mL into 24-well plates and cultured at 37°C in a 5% CO2 cell incubator for 24 h, as per reference. The transfection system for the ATP fluorescent probe plasmid containing mitochondrial localization signals (pCMV-Mito-AT1.03, Beyotime, D2606) prepared according to the methods of the 3000 kit (Thermo Fisher Scientific, L3000008) is as follows: 0.5 μg plasmid, 1.5 μL Lipofectamine 3000 reagent, 1 μL LP3000 TM Reagents and 450 μL Opti-MEMI medium (Gibco, A3635101). The transfection solution was added to the cells, and the cells were incubated at 37°C in a 5% CO2 incubator for 6 hours. The transfection solution was discarded, and fresh medium was added for overnight incubation. The cells were washed once with PBS, digested with trypsin for 2 minutes, and then the digestion was stopped by adding complete medium. Cells were collected by pipetting and counting, and the cell density was adjusted to 2 × 10⁶ cells / year. 4 Cells per mL were used for passage to obtain transiently transformed cells for selection.

[0071] (2) Screening of stable transfected cells: The above transiently stained cells were seeded into 24-well plates at 500 μL per well and cultured for 24 h. Then, complete medium containing 400 μg / mL G418 was added. The screening medium was changed every 2-3 days. After 14 days of pressure screening, positive clones appeared. After being marked under a microscope, positive clones were picked in a biosafety cabinet and cultured in 12-well plates with maintenance medium containing 200 μg / mL G418 for 7 days. The medium was changed once during the period to obtain stable transfected cell lines.

[0072] The main steps in determining the optimal screening concentration of G418 are as follows: 16HBE cells are prepared at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 24-well plates. After culturing for 24 hours, the old culture medium was removed, and a gradient concentration of G418 was added: 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg / mL. The culture medium was changed every 2-3 days. It was observed that the minimum G418 concentration that killed all cells on day 10 was 400 μg / mL. Therefore, 400 μg / mL was selected as the optimal screening concentration of G418.

[0073] (3) Phenotypic verification: Stable cells were divided into groups of 1×10⁻⁶ cells. 5Two cells were seeded per glass-bottom confocal dish at a density of 2 mL / mL and cultured at 37°C in a 5% CO2 incubator for 24 h. The old culture medium was removed, and fresh culture medium containing 10 μM doxorubicin (Selleck, E2516), a mitochondrial toxic compound, was added. The cells were then incubated at 37°C in a 5% CO2 incubator for 6 h. After washing twice with PBS, fluorescence images of the mitochondrial fluorescent proteins Mito-AT1.03 and Parkin-mCherry in live cells were captured using a laser confocal microscope. Results are shown below. Figure 7 As shown, in the control group without doxorubicin, mitochondria in the cells were branched and exhibited bright green fluorescence, while Parkin-mCherry red fluorescent protein was diffuse with fewer particles, indicating normal mitochondrial ATP synthesis function and low levels of mitophagy. In contrast, in the experimental group with doxorubicin, mitochondria in the cells were fragmented and spherical, with relatively dim green fluorescence, but a significant increase in Parkin-mCherry red fluorescent protein particles, which showed obvious co-localization with mitochondria (indicated by arrows), indicating significant mitochondrial damage. Figure 8 To quantitatively detect changes in mitochondrial matrix ATP concentration, fusion and fission states, and Parkin protein recruitment dynamics, we statistically analyzed the green fluorescence intensity of five random fields to represent the relative ATP levels within mitochondria before and after doxorubicin treatment. Figure 9 A) Meanwhile, the mitochondrial fusion and division status was determined by statistically analyzing the mitochondrial fragmentation factors (mitochondrial perimeter) of 50 cells using ImageJ software. 2 The value of 4π / (4π × mitochondrial area) indicates the degree of fragmentation; a larger value indicates a higher degree of fragmentation. Figure 9 B), In addition, the level of mitophagy was represented by counting intracellular red fluorescent particles (out of 50 cells) Figure 9 C). The results showed that after 6 hours of treatment with doxorubicin, the relative ATP level in the mitochondria of cells decreased significantly, the degree of mitochondrial fragmentation increased significantly, and the number of mitochondrial autophagy particles increased significantly.

[0074] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for assessing mitochondrial function, characterized in that, The mitochondrial ATP-responsive fluorescent protein AT1.03 and the Parkin-mCherry fusion protein were co-expressed in living cells. The changes in mitochondrial matrix ATP concentration, fusion and division status and Parkin protein recruitment dynamics were monitored by dual-channel fluorescence imaging. The sequence of the synthesized Parkin-mCherry fusion protein is shown in SEQ ID No.

1.

2. The method for assessing mitochondrial function according to claim 1, characterized in that, The method for co-expressing the mitochondrial ATP-responsive fluorescent protein AT1.03 and the Parkin-mCherry fusion protein in living cells specifically includes the following steps: S1. The sequence of synthesized Parkin-mCherry was cloned into the lentiviral vector LV6-EF1a-Puro to construct the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentiviral vector plasmid, and HEK293T cells were transfected to collect lentiviral particles. S2. Live cells were infected with lentiviruses and selected with puromycin to obtain cell lines that stably express Parkin-mCherry fusion protein. S3. Using liposomes, the plasmid of the ATP fluorescent probe Mito-AT1.03 expressing the mitochondrial localization signal was transfected into the cell line in S2 that stably expressed the Parkin-mCherry fusion protein. After G418 selection, a cell line stably expressing dual fluorescent proteins was obtained.

3. A method for assessing mitochondrial function according to claim 1 or 2, characterized in that, The live cells are human bronchial epithelioid cells 16HBE.

4. The method for assessing mitochondrial function according to claim 2, characterized in that, In S2, the concentration of puromycin used for screening is not less than 1 μg / mL, and the screening time is more than 4 days.

5. The method for assessing mitochondrial function according to claim 2, characterized in that, In S2, live cells are revived and passaged 4-8 times before infection.

6. The method for assessing mitochondrial function according to claim 2, characterized in that, In S3, the plasmid for the ATP fluorescent probe Mito-AT1.03 expressing the mitochondrial localization signal is pCMV-Mito-AT1.03 plasmid.

7. The method for assessing mitochondrial function according to claim 2, characterized in that, In S3, the concentration of G418 used for screening is not less than 400 μg / mL, and the screening time is more than 14 days.

8. The application of the mitochondrial function assessment method according to any one of claims 1-7 in screening compounds that regulate mitochondrial function.

9. The application according to claim 8, characterized in that, The mitochondrial function-regulating compounds include rotenone and doxorubicin.

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