Method for evaluating mitochondrial function and use thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a mitochondrion function evaluation method and application thereof. The application provides an innovative mitochondrion function evaluation method, which reports the ATP concentration change in the mitochondrion matrix and the mitochondrion morphology in real time, in situ and quantitatively through ATP response fluorescent protein Mito-AT1.03 with a mitochondrion positioning signal, directly reflects the mitochondrion energy metabolism and the mitochondrion fusion / fission state; meanwhile, a fusion protein Parkin-mCherry is constructed by using Parkin protein, a key starting factor of mitochondrion autophagy, as an early specific mark of autophagy initiation. The two are co-transfected into cells, and the signal level and positioning condition of the two fluorescent proteins are observed, so that the mitochondrion function can be accurately and timely evaluated, and a new idea is provided for the mechanism research of mitochondrion dysfunction and the methodological development of the screening of mitochondrion function regulators.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for evaluating mitochondrial function and application thereof. BACKGROUND

[0002] Mitochondria are important organelles for energy production in cells, and are involved in multiple important physiological processes such as cell differentiation, proliferation, metabolism, inflammation and apoptosis. Whether the function of mitochondria is normal or not is a key to maintaining cell homeostasis. Under stress conditions such as oxidative stress and cell aging, mitochondrial dysfunction occurs, especially oxidative phosphorylation is blocked (ATP level is decreased) and quality control is failed (accumulation of damaged mitochondria), which eventually destroys the metabolic homeostasis of cells and leads to cell death. Recent studies have shown that mitochondrial dysfunction may be related to the occurrence and development of many major diseases, including neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), metabolic diseases (such as diabetes and obesity), cardiovascular diseases, inflammatory bowel disease, cancer and aging, etc. At present, the research results of mitochondrial dysfunction still have a blank in the conversion to effective targeted drugs. The mechanism triggering mitochondrial dysfunction is not clear, which greatly limits the further application of mitochondrial function regulators in clinic. Therefore, the development of a mitochondrial function detection method based on a specific mechanism will provide strong support for the research and verification of various types of mitochondrial function regulation chemicals in the future.

[0003] Mitochondrial morphology is highly dynamic, in order to adapt to the changes of the stress environment inside and outside the cell, it will maintain the dynamic balance of mitochondrial network through continuous division and fusion process, so as to meet the energy metabolism and other physiological needs. Mitochondrial morphology is regulated by fusion proteins (such as MFN1 / 2, OPA1) and division proteins (such as DRP1, FIS1), among which fusion proteins enhance inter-mitochondrial cooperation to facilitate energy transfer and material exchange (such as maintaining membrane potential, sharing mitochondrial DNA); division proteins facilitate mitochondrial transport within the cell or isolate damaged parts, and selectively wrap and degrade damaged mitochondria through the mitochondrial autophagy mechanism. Mitophagy is a key mitochondrial quality control pathway in cells, and the PINK1 / Parkin pathway is one of the most clearly studied mitochondrial autophagy pathways. PTEN-induced kinase 1 (PINK1) is a highly conserved mitochondrial protein involved in the regulation of mitochondrial function. Parkin is an E3 ubiquitin ligase responsible for connecting Ub molecules and substrate proteins, and Ub-tagged substrate proteins are recognized and degraded by proteases. In healthy mitochondria, PINK1 is targeted to mitochondria by mitochondrial targeting sequences and enters the mitochondrial inner membrane through the TOM / TIM complex, then is cleaved by proteases located in the mitochondrial matrix and inner membrane, and is released into the cytoplasm to be ubiquitinated-proteasome hydrolysis. In damaged mitochondria, mitochondrial depolarization and reduced membrane potential block the entry of PINK1 into the mitochondrial inner membrane, causing PINK1 to accumulate stably on the cytoplasmic side of the outer mitochondrial membrane, recruit Parkin and activate its E3 ubiquitin ligase activity, and then ubiquitinate proteins on the mitochondria. After ubiquitination, receptor proteins including p62 accumulate on the outer membrane of mitochondria, leading to the recruitment of ubiquitinated products into autophagosomes through binding with LC3, and mature autophagosomes fuse with lysosomes to form autolysosomes, and finally degrade the contained mitochondria.

[0004] Fluorescent protein labeling technology realizes the labeling and tracking of specific proteins or biological macromolecules in cells by means of fluorescent proteins (such as red fluorescent protein mCherry): the fluorescent protein coding gene is inserted into the target protein gene by transgenic technology, a fusion expression vector is constructed, and the transfection technology is used to make the cells express the target protein carrying fluorescent label. This technology can directly present the distribution and translocation of target proteins and subcellular structures such as mitochondria in cells, can maintain cell activity, can provide continuous imaging time sequence, and can facilitate fine observation of cell dynamic process. In addition, through the combination of various fluorescent proteins, multi-color labeling observation can be realized, which can provide 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 research and development, toxicity evaluation and other fields. By simultaneously monitoring the ATP level and the degree of mitochondrial autophagy in cells through fluorescent protein labeling technology, the energy generation capacity of mitochondria and the mitochondrial quality control process can be accurately reflected, and the mitochondrial function can be comprehensively and accurately described.

[0005] Currently, the techniques for studying mitochondrial energy status mainly include Seahorse extracellular flux (XF) analysis, ATP content determination, mitochondrial membrane potential (MMP) detection and respiratory complex activity analysis. Among them, Seahorse extracellular flux (XF) analysis can monitor the oxygen consumption rate (OCR, reflecting the activity of respiratory chain) and extracellular acidification rate (ECAR, reflecting glycolysis) of living cells in real time through microsensors, and analyze the energy contribution of mitochondrial aerobic metabolism and glycolysis. However, it can only provide the average value of the population cells, cannot directly observe the dynamics at the subcellular level (such as single mitochondria), and needs to rely on exogenous inhibitors (such as oligomycin, FCCP), which may interfere with the normal physiological state of mitochondria. ATP content determination mainly includes chemiluminescence method (such as luciferase reporter system) and enzyme coupling method (hexokinase reaction). Both methods need to lyse cells, cannot track the dynamic changes of ATP in mitochondria in real time, and cannot distinguish ATP from mitochondria and non-mitochondrial sources (such as glycolysis). Mitochondrial membrane potential (MMP) detection uses mitochondrial membrane potential specific probes (such as 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 cause obvious batch effects in experiments, making it difficult to ensure the repeatability and consistency of results in different experiments. Respiratory chain complex activity analysis is based on spectrophotometric method to detect complex I-V enzyme activity. This method also needs to lyse cells, which not only loses the dynamic information of living cells, but also cannot reflect the regulation mechanism of mitochondrial dysfunction.

[0006] There are many techniques for studying mitochondrial autophagy, such as biological transmission electron microscopy, protein immunoblotting, immunofluorescence, fluorescent probe labeling, and fluorescent protein labeling. Among them, biological transmission electron microscopy can observe the substructure changes of mitochondria and the formation of mitochondrial autophagosomes. It mainly identifies the structures specific to mitochondria, such as double membranes and cristae. However, it is easily disturbed by cell structure damage caused by human operation such as sectioning, and it is also easily misled by other double-membrane structures or low-electron-density vesicles in the cell. Moreover, the electron microscopy sample preparation operation is complex, inefficient, and has great deficiencies in quantitative analysis. Protein immunoblotting uses the principle of antigen-antibody reaction to indirectly reflect the autophagic activity of mitochondria by detecting the expression changes of mitochondrial autophagy-related proteins (such as mitochondrial membrane protein Tom20, autophagosome protein LC3, lysosome protein LAMP2, etc.). Immunofluorescence also uses the principle of specific binding of antigens and antibodies to realize the subcellular localization of mitochondrial autophagy-related proteins using fluorescently labeled antibodies. Both of them require expensive specific antibodies, and the experimental period is long and the operation steps are complicated. They cannot dynamically detect the process of mitochondrial autophagy, nor can they high-throughput screen the mitochondrial autophagy activity of various compounds. Fluorescent probe labeling technology uses mitochondrial and lysosomal specific fluorescent probes to realize the colocalization of mitochondria and lysosomes, so it can only observe the late stage of mitochondrial autophagy (the stage of autophagic mitochondria and lysosome fusion), and cannot observe the early process of mitochondrial autophagy. It also has the inherent defects of the above fluorescent probes. The conventional strategy for detecting mitochondrial autophagy using fluorescent protein labeling technology is to fuse the genes of autophagosome and mitochondria specific proteins with different fluorescent protein genes, respectively, and analyze the autophagic activity of mitochondria by observing the colocalization of the two fusion fluorescent proteins (such as the technology disclosed in Chinese invention patent CN202010148310.3). This strategy determines whether mitochondrial autophagy occurs and the level of autophagy by observing the combination of mitochondria and autophagosomes. Since it does not involve the triggering mechanism of the early stage of mitochondrial autophagy, when applied to mitochondrial autophagy compound screening, the obtained mitochondrial autophagy modulators still need further mechanism research and verification, greatly increasing the workload.

[0007] In summary, current methods for assessing mitochondrial function still have significant limitations: (1) single evaluation index: common methods (such as JC-1 probe, MitoTracker probe, Seahorse oxygen consumption rate determination) can only reflect one aspect of mitochondrial function (such as membrane potential, mitochondrial mass, respiratory chain activity), and lack comprehensive evaluation of key functions (energy state and quality control).(2) Lack of dynamic correlation: mitochondrial energy metabolism (ATP level) and quality control (especially autophagic removal of damaged mitochondria) are closely coupled dynamic processes. Existing methods cannot simultaneously, real-time, and visualize the changes of these two aspects and their spatiotemporal relationship in the same living cell, even in the same mitochondria, which greatly limits the understanding of the physiological and pathological mechanisms of mitochondria.(3) Flux and applicability limitations: high-resolution techniques (such as transmission electron microscopy) cannot dynamically observe mitochondria; biochemical methods (such as ATP detection kits) easily damage cells and lose spatial information; and there is a lack of high-specificity 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 state, dynamics, and mitochondrial autophagy initiation. SUMMARY

[0008] In order to overcome the deficiencies of the prior art, the present application provides an innovative method for assessing mitochondrial function, which co-expresses mitochondrial ATP-responsive fluorescent protein AT1.03 and Parkin-mCherry fusion protein in living cells, simultaneously and dynamically visualizes tracking mitochondrial energy metabolism (ATP level) and mitochondrial quality control (fusion / fission balance and autophagy initiation-Parkin recruitment).

[0009] In order to achieve the above object, the technical scheme adopted by the present application is:

[0010] The first aspect of the present application 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 mitochondrial matrix ATP concentration changes, fusion and fission state, and Parkin protein recruitment dynamics by dual-channel fluorescence imaging; the sequence of the Parkin-mCherry fusion protein is shown in SEQ ID No. 1.

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

[0012] S1, clone the sequence of synthesizing Parkin-mCherry into a lentivirus vector LV6-EF1a-Puro, construct a LV6-EF1a-Parkin-mCherry-Puro fusion expression lentivirus vector plasmid, and transfect HEK293T cells to collect lentivirus particles;

[0013] S2, infect living cells with the lentivirus, and obtain a cell strain stably expressing the Parkin-mCherry fusion protein after screening by puromycin;

[0014] S3, transfect the cell strain stably expressing the Parkin-mCherry fusion protein in S2 with a plasmid expressing the mitochondrial localization signal ATP fluorescent probe Mito-AT1.03 by using a liposome method, and obtain a cell strain stably expressing double fluorescent proteins after screening by G418.

[0015] The application directly reflects mitochondrial energy metabolism and mitochondrial fusion / fission state by using the ATP response fluorescent protein Mito-AT1.03 with a mitochondrial localization signal to report the ATP concentration change in the mitochondrial matrix and the mitochondrial morphology in real time, in situ and quantitatively; meanwhile, the application constructs a fusion protein Parkin-mCherry by using the mitochondrial autophagy key initiator Parkin protein as an early specific marker of autophagy initiation. The two are co-transfected into cells, and the signal level and localization of the two fluorescent proteins are observed, so that the mitochondrial function can be accurately and timely evaluated, and a new idea is provided for the methodology development of the mechanism research of mitochondrial dysfunction and the screening of mitochondrial function regulators.

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

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

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

[0019] More preferably, in S2, the living cells are resuscitated and subcultured 4-8 times before being infected.

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

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

[0022] Further, the optimal G418 concentration used in the G418 screening is 400 μg / mL, and the screening time is 14 days.

[0023] The second aspect of the present application provides the use of the mitochondrial function evaluation method of the first aspect in screening a mitochondrial function regulating compound.

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

[0025] Compared with the prior art, the present application has the following beneficial effects:

[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) is synchronously, dynamically and visualized tracked at the resolution of living cells, single cells / subcellular organelles. This provides an unprecedented powerful tool for revealing the dynamic regulation of mitochondrial function and understanding the causal and temporal relationship between energy crisis and mitochondrial fusion / fission balance and autophagy clearance.

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

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

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

[0030] 2) Drug mechanism research: accurately elucidating the real-time effects of candidate drugs on the dynamic process of mitochondrial dual functions and their interaction.

[0031] 3) Toxicity evaluation: real-time monitoring of the multiple toxicities of drugs or environmental pollutants on mitochondrial energy, mitochondrial fusion / fission balance and mitochondrial autophagy function, providing more comprehensive safety evaluation.

[0032] (4) Technical advantages: high spatiotemporal resolution, suitable for long-term live cell imaging, high genetic coding specificity, relatively low phototoxicity, etc. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Schematic diagram of the principle for evaluation of mitochondrial function;

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

[0035] Figure 3 Fluorescence expression diagram of LV6-EF1a-Parkin-mCherry-Puro fusion expression vector;

[0036] Figure 4 Sequencing sequence alignment results of LV6-EF1a-Parkin-mCherry-Puro fusion expression vector.

[0037] Figure 5 Fluorescence diagram of 16HBE control group stably expressing Parkin-mCherry fusion protein; In the right combination diagram: the upper left diagram is a fluorescence superposition effect diagram of blue, green and red channels, the upper right diagram is a DAPI stained nucleus, the lower left diagram is an immunofluorescence diagram of mitochondrial protein Tom20, and the lower right diagram is the intracellular localization of Parkin-mCherry fusion protein; The left diagram is a partial enlarged view of the superposition diagram.

[0038] Figure 6 Fluorescence diagram of 16HBE stably expressing Parkin-mCherry fusion protein after exposure to rotenone for 12h; In the right combination diagram: the upper left diagram is a fluorescence superposition effect diagram of blue, green and red channels, the upper right diagram is a DAPI stained nucleus, the lower left diagram is an immunofluorescence diagram of mitochondrial protein Tom20, and the lower right diagram is the intracellular localization of Parkin-mCherry fusion protein; The left diagram is a partial enlarged view of the superposition diagram.

[0039] Figure 7 Fluorescence diagram of mitochondrial function double fluorescence reporter gene 16HBE cell control group; The upper left diagram is a fluorescence superposition effect diagram of green and red channels, the upper middle diagram is the localization of ATP responsive fluorescent protein Mito-AT1.03 containing mitochondrial localization signal in cells, and the upper right diagram is the localization of Parkin-mCherry fusion protein in cells; The lowermost diagram is a partial enlarged view of the superposition diagram.

[0040] Figure 8 Fluorescence diagram of mitochondrial function double fluorescence reporter gene 16HBE cell doxorubicin exposure group; The upper left diagram is a fluorescence superposition effect diagram of green and red channels, the upper middle diagram is the localization of ATP responsive fluorescent protein Mito-AT1.03 containing mitochondrial localization signal in cells, and the upper right diagram is the localization of Parkin-mCherry fusion protein in cells; The lowermost diagram is a partial enlarged view of the superposition diagram (i.e. mitochondrial autophagy occurring in cells).

[0041] Figure 9 Statistical graphs were used to quantitatively represent the changes in mitochondrial matrix ATP concentration, fusion and fission state or Parkin protein recruitment dynamics using green fluorescence intensity, mitochondrial fragmentation factor and mitochondrial autophagic particle number, respectively; green fluorescence intensity results were statistically analyzed from random fluorescence images of 5 fields, mitochondrial fragmentation factor and mitochondrial autophagic particle number were randomly analyzed from 50 cells; **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0042] The specific embodiments of the present application are further described below. It should be noted that the description of these embodiments is intended for purposes of illustration, and is not intended to limit the present application. Furthermore, the various features of the present application described in the various embodiments below can be combined with each other, as long as there is no conflict.

[0043] The experimental methods in the following examples are all conventional methods, and the experimental materials used in the following examples are all commercially available, unless otherwise specified.

[0044] The present application provides a mitochondrial function evaluation method and its application:

[0045] (I) Overall framework

[0046] By constructing a dual-fluorescent probe co-expression system, Figure 1 the following functions can be realized in living cells:

[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. Construction of Parkin-mCherry fusion expression lentivirus vector plasmid

[0052] LV6-EF1a-Parkin-mCherry-Puro lentivirus vector plasmid was synthesized by GenScript, referring to Homosapiens parkin RBR E3 ubiquitin protein ligase (NCBI Reference Sequence: NM_004562.3) and Synthetic construct mCherry gene (GenBank: MZ027319.1), using the method of gene synthesis, synthesizing the sequence of Parkin-mCherry, cloning the synthesized sequence into the lentivirus vector LV6-EF1a-Puro, constructing the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentivirus vector plasmid and transfecting HEK293T cells to collect lentivirus particles.

[0053] 2, Construction of mitochondrial autophagy reporter gene cell strain

[0054] Resuscitate 16HBE cells and pass 5 times, infect 16HBE cells with lentivirus, and screen stable expression of Parkin-mCherry fusion protein cell strain with puromycin (1 μg / mL, 4 days). After 6h treatment with mitochondrial autophagy inducer (rotenone, 100 μM), fixation and permeabilization of cells, mitochondrial protein Tom20 was labeled using immunofluorescence method, and cell nucleus was labeled with DAPI, and expression of Parkin-mCherry fusion protein was observed and verified under laser confocal microscope.

[0055] 3, Construction of mitochondrial function dual fluorescence reporter gene cell strain

[0056] The plasmid (pCMV-Mito-AT1.03) expressing mitochondrial localization signal ATP fluorescent probe Mito-AT1.03 was transfected into the above stable expression of Parkin-mCherry fusion protein 16HBE cells using liposome method, and G418 (400 μg / mL, 14 days) was used to screen stable expression of dual fluorescence protein cell strain. After 6h treatment with mitochondrial toxicity compound (doxorubicin, 10 μM), the expression of dual fluorescence reporter gene in living cells was observed and verified under laser confocal microscope.

[0057] In order to comprehensively and clearly present the technical solutions of the present application and its significant advantages, the present application will be described in detail below combined with specific examples.

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

[0059] LV6-EF1a-Parkin-mCherry-Puro lentivirus vector plasmid was synthesized by Genscript, the specific method was as follows: referring to Homo sapiens parkin RBR E3 ubiquitin protein ligase (NCBI Reference Sequence: NM_004562.3) and Synthetic construct mCherry gene (GenBank: MZ027319.1), the sequence of Parkin-mCherry (SEQ ID No. 1) was synthesized by gene synthesis method, as follows:

[0060]

[0061] The NotI / BamHI enzyme cutting sites were added at both ends of the sequence, and the synthetic sequence was cloned into the lentivirus vector LV6-EF1a-Puro to construct the LV6-EF1a-Parkin-mCherry-Puro fusion expression lentivirus vector plasmid. The constructed plasmid was transformed into Top10 competent E. coli, which was then spread on LB plates containing ampicillin and incubated at 37°C overnight. After single colony PCR identification, a positive single colony was selected for expansion and endotoxin-free plasmid extraction using a high-purity plasmid extraction kit (Genepharma, P4170). After sequencing alignment verification, the plasmid was co-transfected with three auxiliary packaging plasmids (pGag / Pol, pRev, and pVSV-G) into HEK293T cells using transfection reagent RNAi-Mate (Genepharma, G04001). The specific steps were as follows: one day before transfection, HEK293T cells were seeded in a 15 cm culture dish. Before transfection, 30 μg of plasmid (7.5 μg of each of the four plasmids) was added to a centrifuge tube containing 1.5 mL of serum-free DMEM medium and mixed well. Another centrifuge tube containing 1.5 mL of serum-free DMEM was added with 300 μL of RNAi-Mate and mixed well. After 5 min of room temperature standing, the two mixtures were mixed and continued to stand at room temperature for 20 min. The old medium in the 15 cm culture dish was aspirated and replaced with 8 mL of serum-free DMEM. The transfection mixture was added dropwise to the culture dish, which was then incubated at 37°C in a 5% CO2 incubator for 6 h. The transfection solution was aspirated, and fresh complete DMEM medium containing 10% FBS was added for continued incubation for 72 h. The cell supernatant rich in lentivirus particles was collected and concentrated by ultracentrifugation at 20000 rpm for 2 h at 4°C to obtain high-titer lentivirus concentrate. HEK293T cells were seeded at 5×10 4 The cells were seeded in a 24-well plate at 5×10 Figure 2 As shown in the experimental results, after 72 h of lentivirus infection, red fluorescent protein was observed in the HEK293T cells under a fluorescence microscope (100x), with some cells containing small particles, indicating that the Parkin-mCherry fusion protein expression vector was successfully constructed and expressed. Figure 3 ), and sequencing alignment of the LV6-EF1a-Parkin-mCherry-Puro fusion expression vector showed that the sequence was completely correct without mutation bases. Figure 4

[0062] ​Example 2: Construction of Mitophagy Reporter Cell Line

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

[0064] (2) Cell infection: After the cells were passaged for 5 times, 16HBE cells were inoculated in a 24-well plate at a concentration of 5x10 4 individuals / mL, 37°C, 5% CO2 cell incubator for 24h. The virus concentrate was diluted 10 times with fresh complete medium and added to the virus solution with a final concentration of 5μg / mL Polybrene to prepare a total volume of 500μL. The old culture solution in the 24-well plate was aspirated, and the diluted virus solution was added to the cells. The cells were cultured for another 24h, and then the virus solution was removed. After the cells were washed with PBS once and trypsinized for 2min, the digestion was terminated by adding complete medium. After the cells were collected by pipetting, the cell density was adjusted to 2x10 4 individuals / mL for subculture to obtain the transient cells for screening.

[0065] (3) Screening of stably transfected cells: The above-mentioned transient cells were inoculated in a 24-well plate at 500μL per well and cultured for another 24h. Then, complete medium containing 1μg / mL puromycin was added, and the selection medium was changed every 2 days. After 4 days of pressure selection, the cells were collected by trypsinization for 2min and centrifugation. The cells were resuspended in 2mL of maintenance medium containing 0.5μg / mL of hygromycin B and cultured in a 6-well plate for another 3 days to obtain the stable cell line (16HBE cells stably expressing Parkin-mCherry fusion protein).

[0066] The main steps for determining the optimal selection concentration of puromycin are as follows: 16HBE cells were inoculated in a 24-well plate at a density of 1x10 4 individuals / well, and cultured for 24h. The old culture medium was then removed and replaced with puromycin at different concentrations: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 4, and 8μg / mL. The medium was changed every 2 days. The minimum puromycin concentration that could kill all cells at the 4th day was observed to be 1μg / mL. Therefore, 1μg / mL was chosen as the optimal selection concentration of puromycin.

[0067] (4) Phenotype verification: The stable cells were inoculated in a 6-well plate at a density of 1x10 5Seeds were planted at a density of 2 mL / 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. 3000 kit (Thermo Fisher Scientific, L3000008) related method to prepare transfection system containing ATP fluorescent probe plasmid with mitochondrial localization signal (pCMV-Mito-AT1.03, Beyotime, D2606): 0.5 μg plasmid, 1.5 μL Lipofectamine 3000 reagent, 1 μL P3000 TM Reagent and 450 μL Opti-MEM I medium (Gibco, A3635101). The transfection solution was added to the cells and incubated at 37°C in a 5% CO2 cell incubator for 6 h. The transfection solution was then discarded and fresh medium was added for overnight incubation. The cells were then washed once with PBS, trypsinized for 2 min, and then stopped by adding complete medium. After pipetting and blowing, the cells were collected and counted. The cell density was adjusted to 2 x 10 4 cells / mL for subculture, and the transiently transfected cells were obtained.

[0071] (2) Screening of stably transfected cells: The transiently transfected cells were seeded in a 24-well plate at 500 μL per well and incubated for 24 h. Then, complete medium containing 400 μg / mL G418 was added, and the selection medium was replaced every 2-3 days. After 14 days of pressure selection, positive clones appeared. The positive clone cells were marked under a microscope and then picked in a biological safety cabinet. The cells were cultured in a 12-well plate using maintenance medium containing 200 μg / mL G418, and the medium was replaced once during the 7-day culture to obtain a stably transfected cell line.

[0072] The main steps for determining the optimal selection concentration of G418 are as follows: 16HBE cells were seeded in a 24-well plate at a density of 1 x 10 4 cells per well and incubated for 24 h. The old medium was then removed and replaced with G418 at gradient concentrations: 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg / mL. The medium was replaced every 2-3 days. The minimum G418 concentration that could kill all cells by the 10th day was 400 μg / mL, so 400 μg / mL was selected as the optimal selection concentration of G418.

[0073] (3) Phenotype verification: The stably transfected cells were seeded in a 24-well plate at a density of 1 x 10 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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