A method for detecting mitochondrial mass control
By preparing sample cell suspensions, labeling with fluorescent dyes, and detecting with the JC-1 probe, and combining this with doxorubicin to establish a cardiomyocyte damage model, the problem of not being able to perform rapid quantitative analysis and sorting of multiple parameters simultaneously in existing technologies has been solved, thus realizing rapid quantitative analysis and sorting of cardiomyocytes using multiple parameters.
Patent Information
- Application Number
- CN202310438032.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-23
- Publication Date
- 2026-01-27
- Estimated Expiration
- 2043-04-23
AI Technical Summary
Current technologies cannot perform multi-parameter, rapid quantitative analysis and sorting of cardiomyocytes simultaneously, and cannot effectively screen out specific cells for further culture and observation.
A cardiomyocyte damage model was established by preparing sample cell suspensions, labeling with fluorescent dyes, isolating cardiomyocytes, and detecting them with the JC-1 mitochondrial membrane potential fluorescent probe. By activating Bnip3-mediated mitochondrial autophagy with doxorubicin, rapid quantitative analysis and sorting of multiple parameters were achieved.
It enables rapid quantitative analysis and sorting of cardiomyocytes using multiple parameters, allowing for the selection of cells with specific characteristics for further culture and observation, and verification of mitochondrial membrane potential.
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Figure CN116754443B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of mitochondrial quality control detection technology, and more specifically, to a method for mitochondrial quality control detection. Background Technology
[0002] Mitochondria are among the most important organelles in the cell, and various mechanisms exist within the cell to control mitochondrial quality and maintain mitochondrial homeostasis. When mitochondrial damage is minor, mitochondrial condition can be improved through mitochondrial fusion and mitochondrial outer membrane vesicle (MDV). Mitophagy and mitochondrial protease degradation pathways primarily serve to clear severely damaged mitochondria from the cell. If severely damaged mitochondria are not cleared in time, they release large amounts of death factors, thereby affecting the homeostasis of surrounding healthy mitochondria and inducing apoptosis.
[0003] Studies have found that KIF5B, Myosin19, and Drp1 mediate mitochondrial secretion by regulating mitochondrial transport, localization, and division. Under external stimuli, most of the mitochondria entering the migratory body are damaged. The mechanism by which damaged mitochondria enter the migratory body is that the binding of damaged mitochondria to KIF5B is enhanced while the binding to Dynein is weakened. Damaged mitochondria are more easily transported to the cell edge by KIF5B and enter the migratory body.
[0004] Doxorubicin, an anthracycline, is a commonly used broad-spectrum antitumor drug in clinical practice, effective against various malignant tumors such as breast cancer, lymphoma, sarcoma, and certain types of leukemia. Its mechanism of action involves intercalation into human DNA, thereby blocking tumor cell proliferation and inducing apoptosis. However, doxorubicin was found to have dose-dependent cardiotoxicity upon its development, including various types of arrhythmias, ventricular dilation, and ultimately congestive heart failure. This cardiotoxicity significantly limits its clinical use. Despite the cardiotoxicity associated with doxorubicin, it remains a commonly used drug in clinical treatment due to the lack of suitable alternatives.
[0005] The mechanism of doxorubicin-induced cardiotoxicity mainly involves the combined involvement of DNA damage, mitochondrial dysfunction, oxidative stress, and various types of cell death. Myocardium requires a large number of normally functioning mitochondria to produce ATP to maintain the metabolism and function of cardiomyocytes. Therefore, mitochondrial homeostasis is mainly maintained through mitophagy, mitochondrial dynamics, and mitochondrial biosynthesis; its dysregulation can lead to severe damage to cardiac function. In the early stages of mitophagy, the mitochondrial membrane potential decreases or disappears. Therefore, we use JC-1 staining, an ideal fluorescent probe for detecting mitochondrial membrane potential. The detection principle is as follows: In normal cardiomyocytes, the mitochondrial membrane potential is high, and JC-1 accumulates in the mitochondrial matrix, forming a polymer that produces red fluorescence; after cardiomyocyte damage, the mitochondrial membrane potential is low, and JC-1 cannot accumulate in the mitochondrial matrix. At this time, JC-1 exists as a monomer, producing green fluorescence.
[0006] The shortcoming of the existing technology is that it cannot perform multi-parameter, rapid quantitative analysis and sorting of test samples simultaneously, measure the multi-parameter characteristics of each cell, and separate cells with specified characteristics while performing cell characteristic analysis, so as to further culture, clone, observe or detect mitochondrial membrane potential of specific cells. Summary of the Invention
[0007] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a detection method for mitochondrial quality control. The technical problem to be solved by the present invention is: how to perform multi-parameter, rapid quantitative analysis and sorting of test samples simultaneously, so as to further culture, clone, observe or perform mitochondrial fragmentation tests on specific cells.
[0008] To achieve the above objectives, the present invention provides the following technical solution: a detection method for mitochondrial quality control, specifically including the following operational steps:
[0009] S1: Select myocardial tissue samples and immediately place them in pre-cooled tissue culture medium. Use medical scissors to cut the tissue blocks into small pieces of ±1 mm³. Add an appropriate amount of enzyme digestion solution and digest in a 37°C constant temperature water bath for 20-30 minutes, intermittently shaking or pipetting the cells during this period. Stop digestion with complete tissue block culture medium, transfer to a 300-mesh nylon mesh for filtration to remove tissue clumps, collect the filtered cells, centrifuge at 500g for 10 minutes and remove the supernatant. Wash the cell pellet twice with culture medium to prepare a sample cell suspension, place it in a culture dish, and incubate at 37°C in a constant temperature incubator containing 5% carbon dioxide for cell adhesion. After 12 hours, add an appropriate amount of culture medium and continue to culture under the aforementioned conditions. Change the culture medium once every 2 days and observe the adhesion of myocardial cells daily.
[0010] S2: When small, round, and bright cells are observed to divide on long spindle-shaped fibroblasts, a single-cell suspension is prepared from cardiomyocytes in the logarithmic growth phase using an enzyme digestion solution. c-Kit is selected as the surface marker to be tested on cardiomyocytes, and cardiomyocytes are labeled with anti-c-Kit specific fluorescent dye to obtain a stained sample cell suspension. The stained sample cell suspension is arranged in a single row under the coating of sheath fluid to form a sample liquid jet, which passes through the detection area in sequence.
[0011] S3: An ultra-high frequency piezoelectric crystal is placed above the nozzle of the flow chamber. The vibration generated causes the ejected liquid flow to form uniform droplets. The test cells in the sample cells are dispersed in the droplets. The droplets are charged with different positive and negative charges to complete the separation of cardiomyocytes and obtain purified cardiomyocytes with better viability.
[0012] S4: Doxorubicin can activate Bnip3-mediated mitophagy, causing the mitochondrial permeability transition pore to open and the mitochondria to depolarize, thereby impairing mitochondrial activity, losing membrane potential, and inducing cardiomyocyte apoptosis. Based on this cardiotoxic principle of doxorubicin, a cardiomyocyte damage model was established using doxorubicin.
[0013] S5: The mitochondrial membrane potential of normal cardiomyocytes obtained in S3 and damaged cardiomyocytes obtained in S4 was detected using the JC-1 mitochondrial membrane potential fluorescent probe.
[0014] Preferably, the myocardial sample tissue described in S1 above is washed to remove dead tissue components, fibers, fat and blood vessels from the tissue block.
[0015] Preferably, the tissue pieces described in S1 above are placed in pre-cooled tissue culture medium for rinsing to remove the shredded cell debris.
[0016] Preferably, the myocardial sample tissue in S1 is washed to remove dead tissue, fibers, fat and blood vessels from the tissue block.
[0017] Preferably, the stained sample cell suspension in S2 above enters the flow chamber under constant gas pressure, and the resulting sample liquid stream is cylindrical.
[0018] Preferably, the droplets in S3 flow through a deflector plate with several kilovolts, are deflected under the action of a high-voltage electric field, and fall into their respective collection containers. Droplets that are not charged fall into the waste liquid container in the middle.
[0019] Preferably, the cardiomyocytes obtained in S3 above are treated with doxorubicin 1 μmol / L for 24 h to obtain a cardiomyocyte damage model.
[0020] Preferably, the membrane potential of normal and damaged cardiomyocytes in S5 is measured to verify the detection quality of this mitochondrial quality control detection method.
[0021] The technical effects and advantages of this invention are as follows:
[0022] 1. This invention enables simultaneous multi-parameter, rapid quantitative analysis and sorting by preparing sample cell suspensions, labeling with fluorescent dyes, isolating cardiomyocytes, establishing a cardiomyocyte damage model, and detecting mitochondrial membrane potential. The measurement speed is fast, and multiple parameters of each cell can be measured at the same time. While performing cell characteristic analysis, cells with specific characteristics can be isolated for further culture, cloning, observation, or mitochondrial membrane potential detection of specific cells.
[0023] 2. Based on the cardiotoxicity principle of doxorubicin, this invention establishes a cardiomyocyte damage model using doxorubicin, and uses the JC-1 mitochondrial membrane potential fluorescent probe to detect the mitochondrial membrane potential of the obtained normal cardiomyocytes and damaged cardiomyocytes, thereby verifying the detection quality of this mitochondrial quality control detection method. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the detection process of the present invention. Implementation
[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0026] This invention provides a method for detecting mitochondrial quality control, specifically including the following steps:
[0027] S1: Select myocardial tissue samples and immediately place them in pre-cooled tissue culture medium. Use medical scissors to cut the tissue blocks into ±1mm³ pieces. Add an appropriate amount of enzyme digestion solution and digest in a 37℃ constant temperature water bath for 20-30 minutes, intermittently shaking or pipetting the cells during digestion. Terminate digestion with complete tissue block culture medium. Transfer to a 300-mesh nylon mesh for filtration to remove tissue clumps. Collect the filtered cells, centrifuge at 500g for 10 minutes, remove the supernatant, wash the cell pellet twice with culture medium to prepare a sample cell suspension, place it in a culture dish, and incubate at 37℃ in a constant temperature incubator containing 5% carbon dioxide for 12 hours. Add an appropriate amount of culture medium again and continue culturing under the aforementioned conditions. Change the culture medium every 2 days and observe the myocardial cell adhesion daily. S2: When small, round, and shiny cells are observed to divide from long spindle-shaped fibroblasts, prepare a single-cell suspension of myocardial cells in the logarithmic growth phase using enzyme digestion solution. Select c-Kit as the surface marker to be tested for myocardial cells and use... Anti-c-Kit specific fluorescent dye is used to label cardiomyocytes, resulting in a stained cell suspension. The stained cell suspension is arranged in a single row under the coating of sheath fluid to form a sample liquid jet, which passes through the detection area sequentially. S3: An ultra-high frequency piezoelectric crystal is placed above the nozzle of the flow chamber. The vibration generated causes the ejected liquid jet to form uniform droplets. The test cells in the sample cells are dispersed in the droplets. The droplets are charged with different positive and negative charges to complete the separation of cardiomyocytes and obtain purified cardiomyocytes with good viability. S4: Doxorubicin can activate Bnip3-mediated mitophagy, causing the opening of the mitochondrial permeability transition pore and mitochondrial depolarization, resulting in impaired mitochondrial activity, loss of membrane potential, and subsequent cardiomyocyte apoptosis. Based on this cardiotoxic principle of doxorubicin, a cardiomyocyte damage model is established using doxorubicin. S5: The mitochondrial membrane potential of the normal cardiomyocytes obtained in S3 and the damaged cardiomyocytes in S4 is detected using the JC-1 mitochondrial membrane potential fluorescent probe.
[0028] The myocardial tissue sample described in S1 was washed to remove dead tissue, fibers, fat, and blood vessels. The tissue pieces from S1 were then rinsed in pre-cooled tissue culture medium to remove any shredded cell debris. The myocardial tissue sample from S1 was washed again to remove dead tissue, fibers, fat, and blood vessels. The stained cell suspension from S2 was introduced into a flow chamber under constant gas pressure, forming a cylindrical sample stream. The droplets from S3 flowed through a deflector plate with several kilovolts, were deflected under the influence of a high-voltage electric field, and fell into their respective collection containers. Uncharged droplets fell into the central waste container. The myocardial cells obtained in S3 were treated with 1 μmol / L doxorubicin for 24 hours to obtain a damaged myocardial cell model. Membrane potential was measured in both normal and damaged myocardial cells from S5 to verify the detection quality of this mitochondrial quality control method.
[0029] See attached document Figure 1 Preparation of sample cell suspension: Cardiac tissue samples were selected and washed to remove dead tissue, fibers, fat, and blood vessels. The myocardial tissue samples were immediately placed in pre-cooled tissue culture medium. Medical scissors were used to cut the tissue blocks into ±1 mm³ pieces. These pieces were then rinsed in pre-cooled tissue culture medium to remove any remaining cell fragments. An appropriate amount of enzyme digestion solution was added, and the mixture was digested in a 37°C water bath for 20–30 minutes, with intermittent shaking or pipetting during digestion. Digestion was terminated using complete tissue block culture medium. The cells were then transferred to a 300-mesh nylon mesh for filtration to remove tissue clumps. The filtered cells were collected and centrifuged at 500g for 10 minutes before the supernatant was removed. Cell suspension was prepared by washing the cell pellet twice with culture medium and placing it in a culture dish. The cells were then incubated at 37°C in a 5% carbon dioxide incubator for 12 hours. After 12 hours, an appropriate amount of culture medium was added, and the incubation was continued under the same conditions. The culture medium was changed every two days, and the adhesion of cardiomyocytes was observed daily. Fluorescent dye labeling: When small, round, and bright cells were observed to divide from the elongated spindle-shaped fibroblasts, cardiomyocytes in the logarithmic growth phase were digested with enzyme solution to prepare a single-cell suspension. c-Kit was selected as the surface marker to be tested on the cardiomyocytes, and the cardiomyocytes were labeled with an anti-c-Kit specific fluorescent dye to obtain a stained sample cell suspension. Cell suspensions, coated with sheath fluid, are arranged in a single row to form a sample stream. This stream passes sequentially through the detection area. The stained cell suspensions enter the flow chamber under constant gas pressure, forming a cylindrical sample stream. Cardiac cell separation: An ultra-high frequency piezoelectric crystal is placed above the nozzle of the flow chamber. The vibration generated causes the ejected stream to form uniform droplets. The test cells are dispersed within these droplets, which are charged with different positive and negative charges. The droplets flow through a deflector plate with several kilovolts, where they are deflected under the influence of a high-voltage electric field and fall into their respective collection containers. Uncharged droplets fall into the waste container in the middle, completing the separation of cardiomyocytes and yielding purified cells with good viability. Cardiomyocytes were used to establish a cardiomyocyte damage model. Cardiomyocytes were treated with doxorubicin (1 μmol / L) for 24 h to obtain a cardiomyocyte damage model. Doxorubicin can activate Bnip3-mediated mitophagy, causing the opening of the mitochondrial permeability transition pore and mitochondrial depolarization, resulting in impaired mitochondrial activity, loss of membrane potential, and subsequent cardiomyocyte apoptosis. Based on this cardiotoxic principle of doxorubicin, a cardiomyocyte damage model was established using doxorubicin. Mitochondrial membrane potential was detected using the JC-1 mitochondrial membrane potential fluorescent probe to detect the mitochondrial membrane potential of normal and damaged cardiomyocytes, verifying the detection quality of this mitochondrial quality control detection method.
[0030] Finally, it should be noted that although the present invention has been described in detail above with general descriptions and specific embodiments, the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for detecting mitochondrial quality control, characterized in that, The specific steps include the following: S1: Prepare sample cell suspension. Select myocardial tissue samples and immediately place them in pre-cooled tissue culture medium. Use medical scissors to cut the tissue blocks into small pieces of ±1 mm³. Add an appropriate amount of enzyme digestion solution and digest in a 37°C constant temperature water bath for 20-30 minutes. During this period, intermittently shake or pipette the cells. Stop digestion with complete tissue block culture medium. Transfer to a 300-mesh nylon mesh for filtration to remove tissue clumps. Collect the filtered cells and centrifuge at 500g for 10 minutes to remove the supernatant. Wash the cell pellet twice with culture medium to prepare sample cell suspension. Place it in a culture dish and incubate at 37°C in a constant temperature incubator containing 5% carbon dioxide for cell adhesion. After 12 hours, add an appropriate amount of culture medium and continue to culture under the aforementioned conditions. Change the culture medium once every 2 days and observe the adhesion of myocardial cells daily. S2: Fluorescent dye labeling. When small, round, and bright cells are observed to divide on long spindle-shaped fibroblasts, a single-cell suspension is prepared from cardiomyocytes in the logarithmic growth phase using an enzyme digestion solution. c-Kit is selected as the surface marker to be tested on cardiomyocytes. The cardiomyocytes are labeled with anti-c-Kit specific fluorescent dye to obtain a stained sample cell suspension. The stained sample cell suspension is arranged in a single row under the coating of sheath fluid to form a sample liquid jet, which passes through the detection area in sequence. S3: Cardiac cell separation. An ultra-high frequency piezoelectric crystal is placed above the nozzle of the flow chamber. The vibration generated causes the ejected liquid flow to form uniform droplets. The test cells in the sample cells are dispersed in the droplets. The droplets are charged with different positive and negative charges to complete the separation of cardiac cells and obtain purified cardiac cells with good viability. S4: Establish a cardiomyocyte damage model. Based on the cardiotoxicity principle of doxorubicin, a cardiomyocyte damage model is established using doxorubicin. S5: Detection of mitochondrial membrane potential. The JC-1 mitochondrial membrane potential fluorescent probe was used to detect the mitochondrial membrane potential of normal cardiomyocytes obtained in S3 and damaged cardiomyocytes obtained in S4.
2. The detection method for mitochondrial quality control according to claim 1, characterized in that: The myocardial sample tissue described in S1 above is washed to remove dead tissue, fibers, fat and blood vessels from the tissue block.
3. The detection method for mitochondrial quality control according to claim 1, characterized in that: The tissue pieces described in S1 above are placed in pre-cooled tissue culture medium for rinsing to remove the shredded cell debris.
4. The detection method for mitochondrial quality control according to claim 1, characterized in that: The stained sample cell suspension in S2 above enters the flow chamber under constant gas pressure, and the resulting sample liquid stream is cylindrical.
5. The detection method for mitochondrial quality control according to claim 1, characterized in that: The droplets in S3 flow through a deflector plate with several kilovolts and are deflected under the action of a high-voltage electric field, falling into their respective collection containers. Droplets that are not charged fall into the waste liquid container in the middle.
6. The method for detecting mitochondrial quality control according to claim 1, characterized in that: the cardiomyocytes obtained in S3 above are treated with doxorubicin 1 μmol / L for 24 h to obtain a cardiomyocyte damage model.
Citation Information
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