Regulation analysis method and system for electrical stimulation induced tunnel nanotube mediated cell electrical coupling based on patch clamp

Abstract

The invention discloses a patch clamp-based regulation and control analysis method and a patch clamp-based regulation and control analysis system for electrical stimulation induced tunnel nanotube mediated cell electrical coupling, and relates to the technical field of cell biology. The patch clamp-based regulation and control analysis method for electrical stimulation induced tunnel nanotube mediated cell electrical coupling comprises the following steps: S1, sample preparation and drug intervention: adding at least one signal path inhibitor into a cell bath solution for intervention; s2, performing membrane potential imaging and data analysis; s3, applying electrical stimulation and coupling verification: applying electrical stimulation in a specific mode to one cell in the tunnel nanotube connection cell pair by using patch clamp equipment, and executing membrane potential imaging and data analysis; s4, measuring the traction force of the cells; s5, analyzing membrane potential change and traction force change; through accurate electrical stimulation and multi-parameter monitoring, the regulation and control mechanism of tunnel nanotube mediated cell electrical coupling is deeply analyzed, and an effective means is provided for research of intercellular signal transmission.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology technology, and particularly relates to a patch-clamp-based electrical stimulation-induced regulation analysis method and system for cell electrical coupling mediated by tunnel nanotubes. BACKGROUND

[0002] The diversity and complexity of bioelectric phenomena exhibited in different organisms have always been a key area of biological and medical research. Human and other higher animal nerve cells and muscle cells transmit information and control physiological functions through bioelectric activity. Plants also exhibit the propagation of bioelectric signals when responding to environmental stimuli such as light and mechanical touch.

[0003] The electrical coupling between cells is of great significance for understanding cell-to-cell signal transmission and physiological function coordination. Tunnel nanotubes (TNTs) provide a new way for cell-to-cell material exchange and signal transmission. Cell-to-cell communication is a core area in life sciences, especially in the nervous system and heart tissue. Ca 2+ As a key second messenger, it plays a crucial role in cell signal transmission. The response of cells to external environmental changes is usually accompanied by dynamic changes in Ca 2+ concentration, which plays a key role in various physiological and pathological states.

[0004] Electrical stimulation technology, as a method to simulate and influence cell activity, has received widespread attention in the scientific community in recent years. By applying an external electric field, researchers can simulate the natural intercellular electrical signal transmission and explore how these signals regulate cell function. This method not only provides a means to simulate natural electrical signals, but also provides an effective experimental strategy for analyzing cell-to-cell communication mechanisms.

[0005] In cell biology research, traditional analysis methods often have difficulty in precisely controlling the pattern and intensity of electrical stimulation, and cannot comprehensively monitor the changes in membrane potential and traction force of cells. Patch-clamp technology is not used to achieve electrical stimulation, which is not convenient for precise control of current and voltage at the single cell level, thereby accurately simulating and studying the response of cells to electrical stimulation. Therefore, a patch-clamp-based electrical stimulation-induced regulation analysis method and system for cell electrical coupling mediated by tunnel nanotubes is needed to accurately induce and analyze cell electrical coupling mediated by tunnel nanotubes. SUMMARY

[0006] In view of the above, the present application aims at the existing problems in the prior art, and the main purpose is to provide a patch clamp-based electrical stimulation-induced regulation analysis method and system for cell electrical coupling mediated by tunnel nanotubes, which can deeply analyze the regulation mechanism of cell electrical coupling mediated by tunnel nanotubes through precise electrical stimulation and multi-parameter monitoring, and provide an effective means for the research of intercellular signal transmission.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: A patch clamp-based electrical stimulation-induced regulation analysis method for cell electrical coupling mediated by tunnel nanotubes, comprising the following steps: S1, sample preparation and drug intervention: providing C2C12 cells mediated by tunnel nanotubes, and adding at least one signal pathway inhibitor to the cell bath solution for intervention; S2, membrane potential imaging and data analysis; S3, electrical stimulation application and coupling verification: using a patch clamp device to apply a specific mode of electrical stimulation to one cell in the tunnel nanotube-connected cell pair, performing membrane potential imaging and data analysis, and comparing the membrane potential change curves of the electrical stimulation cell and the tunnel nanotube-connected cell; S4, cell traction force measurement; S5, analysis of membrane potential change and traction force change, verification of tunnel nanotube-mediated cell electrical coupling effect.

[0008] As a preferred solution: the signal pathway inhibitor in step S1 is selected from at least one of the following: gap junction channel inhibitor, T-type calcium channel blocker, L-type calcium channel blocker, mechanically sensitive ion channel blocker, sarcoplasmic reticulum calcium pump inhibitor, IP3 receptor inhibitor or myosin II inhibitor, and the action time of the signal pathway inhibitor is 25 to 40 minutes.

[0009] As a preferred solution: the gap junction channel inhibitor is meclofenamate with a concentration of 100 μM, the T-type calcium channel blocker is mibefradil with a concentration of 1 μM, the L-type calcium channel blocker is nifedipine with a concentration of 10 μM, the mechanically sensitive ion channel blocker is gadolinium chloride with a concentration of 100 μM, the sarcoplasmic reticulum calcium pump inhibitor is thapsigargin with a concentration of 1 μM, the IP3 receptor inhibitor is 2-aminoethyl diphenyl borate with a concentration of 15 μM, and the myosin II inhibitor is butaclamol with a concentration of 3 μM.

[0010] As a preferred solution: the membrane potential imaging and data analysis in step S2 are specifically: loading the cells with a membrane potential sensitive fluorescent dye, using a fluorescence microscope for time series imaging, and using Image J and Matlab software to analyze the membrane potential fluorescence intensity change curves of the electrically stimulated cells, tunnel nanotubes, tunnel nanotube connected cells and reference cells.

[0011] As a preferred solution: the specific mode of electrical stimulation in step S3 is: clamping the voltage at -80 mV for 20 ms, then stepping to +80 mV and maintaining for 1000 ms, and finally returning to -80 mV for another 20 ms.

[0012] As a preferred solution: the cell traction force measurement in step S4 is specifically: measuring the change in traction force of the tunnel nanotube connected cells before and after the electrical stimulation is applied by Fourier transform traction microscopy; comparing the displacement images of the fluorescent microspheres produced when the cells are alive and after they are lysed by 10% SDS solution, and calculating the cell traction force by Matlab software.

[0013] As a preferred solution: the substrate for cell culture is a polyacrylamide gel coated with collagen, and the preparation method comprises: placing the prepared polyacrylamide gel at 4°C for 6 hours for crosslinking, then coating with collagen and placing at 4°C, and sterilizing by ultraviolet light irradiation for 60 minutes before use.

[0014] A system for implementing the membrane patch clamp-based electrical stimulation-induced regulation analysis method of cell electrical coupling mediated by tunnel nanotubes, comprising a membrane patch clamp electrical stimulation module for applying specific electrical stimulation to target cells, a membrane potential monitoring module for monitoring membrane potential, and a cell traction force measurement module for measuring cell traction force.

[0015] As a preferred solution: the membrane patch clamp electrical stimulation module comprises a membrane patch clamp device system configured to apply specific mode of electrical stimulation to C2C12 cells connected by tunnel nanotubes.

[0016] As a preferred solution: the membrane potential monitoring module acquires time series images, and delineates background regions, electrically stimulated cell regions, tunnel nanotube regions, tunnel nanotube connected cell regions and reference cell regions.

[0017] Compared with the prior art, the application has obvious advantages and beneficial effects. Specifically, according to the above technical scheme, through accurate electrical stimulation and multi-parameter monitoring, the regulation mechanism of cell electrical coupling mediated by tunnel nanotubes is analyzed in depth; through comprehensive analysis of membrane potential changes and traction force changes, the cell electrical coupling effect can be more comprehensively verified, and an effective method and system are provided for the research of intercellular signal transmission; the present application takes C2C12 cells as the research object to explore the mechanism of intercellular electrical signal coupling mediated by TNT caused by electrical stimulation. Combined with the cell traction force experiment, the potential influence of electrical signal transmission on the traction force of TNT connected cells is revealed. Electrical stimulation can induce the fluorescence intensity change of the membrane potential indicator of TNT connected cells, indicating that the depolarization process is directly mediated by TNT; not only provides new insights for the mechanism of intercellular electrical signal transmission, but also opens up a new way for future research and clinical application in the field of bioelectricity; patch clamp technology is the key to realize the electrical stimulation experiment. This technology enables researchers to accurately control current and voltage at the single cell level, thereby accurately simulating and studying the response of cells to electrical stimulation. Through the membrane breaking experiment operation, this technology can directly change the ion concentration in the cell, especially Ca 2+ , simulate the electrical signal under physiological conditions. This precise control and measurement make it possible to understand how cells respond to electrical stimulation, and also open up a new path for exploring the mechanism of intercellular communication through electrical signals.

[0018] To make the structural features and effects of the application clearer, specific embodiments will be described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Ca 2+ Influence diagram of TNT-mediated electrical stimulation on cell membrane potential change; Figure 2 Extracellular Ca 2+ Influence diagram of TNT-mediated electrical stimulation on cell membrane potential change; Figure 3 Extracellular Ca 2+ Influence diagram of TNT-mediated electrical stimulation on cell membrane potential change; Figure 4 Influence diagram of GJ on TNT-mediated electrical stimulation on cell membrane potential change; Figure 5 Influence diagram of GJ on TNT-mediated electrical stimulation on cell membrane potential change; Figure 6 Influence diagram of T-type calcium channel on TNT-mediated electrical stimulation on cell membrane potential change; Figure 7This is a schematic diagram illustrating the effect of the T-type calcium channel of the present invention on the proportion of TNT-mediated electrical coupling between C2C12 cells; Figure 8 This is a schematic diagram illustrating the effect of the L-type calcium channel of the present invention on changes in cell membrane potential induced by TNT-mediated electrical stimulation; Figure 9 This is a schematic diagram illustrating the effect of the L-type calcium channel of the present invention on the proportion of TNT-mediated electrical coupling between C2C12 cells; Figure 10 This is a schematic diagram illustrating the effect of electrical stimulation of C2C12 cells on the change of traction force in TNT-connected cells according to the present invention. Figure 11 The extracellular fluid of this invention is free of Ca 2+ Schematic diagram of the effect of electrical stimulation on the changes in traction force of TNT-connected cells in C2C12 cells; Figure 12 This is a schematic diagram illustrating the effect of the GJ of the present invention on the changes in traction force of TNT-connected cells induced by electrical stimulation of C2C12 cells; Figure 13 This is a schematic diagram illustrating the effect of the T-type calcium channel of the present invention on the change in traction force of TNT-connected cells induced by electrical stimulation of C2C12 cells; Figure 14 This is a schematic diagram illustrating the effect of the L-type calcium channel of the present invention on the change in traction force of TNT-connected cells induced by electrical stimulation of C2C12 cells. Detailed Implementation

[0020] The present invention is as follows Figure 1 As shown in Figure 14, a patch-clamp-based method for analyzing the regulation of cell electrical coupling mediated by tunnel nanotubes induced by electrical stimulation includes the following steps: S1. Sample preparation and drug intervention: Provide tunnel nanotube-mediated C2C12 cells and add at least one signaling pathway inhibitor to the cell bath for intervention; the tunnel nanotube is also known as TNT. S2, membrane potential imaging and data analysis; S3. Electrical stimulation application and coupling verification: A specific pattern of electrical stimulation was applied to one cell in a tunnel nanotube-connected cell pair using a patch clamp device. Membrane potential imaging and data analysis were performed, and the membrane potential change curves of the electrically stimulated cell and the tunnel nanotube-connected cell were compared. S4: Cell traction force measurement; S5. Analyze the changes in membrane potential and traction force to verify the cell electrical coupling effect mediated by tunnel nanotubes.

[0021] The signaling pathway inhibitor in step S1 is selected from at least one of the following: gap junction channel inhibitor, T-type calcium channel blocker, L-type calcium channel blocker, mechanosensitive ion channel blocker, sarcoplasmic reticulum calcium pump inhibitor, IP3 receptor inhibitor, or myosin II inhibitor, and the duration of action of the signaling pathway inhibitor is 25 to 40 minutes.

[0022] The gap junction channel inhibitor is meclofenamic acid at a concentration of 100 μM, the T-type calcium channel blocker is mibediil at a concentration of 1 μM, the L-type calcium channel blocker is nifedipine at a concentration of 10 μM, the mechanosensitive ion channel blocker is gadolinium chloride at a concentration of 100 μM, the sarcoplasmic reticulum calcium pump inhibitor is carotenoid at a concentration of 1 μM, the IP3 receptor inhibitor is 2-aminoethyl diphenylboronic acid ester at a concentration of 15 μM, and the myosin II inhibitor is butadiene at a concentration of 3 μM.

[0023] The membrane potential imaging and data analysis in step S2 specifically involves: loading cells with a membrane potential-sensitive fluorescent dye, performing time-series imaging using a fluorescence microscope, and using ImageJ and Matlab software to analyze the changes in membrane potential fluorescence intensity of electrically stimulated cells, tunnel nanotubes, tunnel nanotube-connected cells, and reference cells.

[0024] The specific mode of electrical stimulation in step S3 is as follows: the clamp voltage is maintained at -80 mV for 20 ms, then stepped to +80 mV and maintained for 1000 ms, and finally returned to -80 mV for another 20 ms.

[0025] The cell traction force measurement in step S4 specifically involves: measuring the change in traction force between tunnel nanotubes and cells before and after electrical stimulation using Fourier transform traction force microscopy; comparing the displacement images of fluorescent microspheres generated when cells are alive with those generated after lysis and death in 10% SDS solution; and calculating the cell traction force using Matlab software.

[0026] The substrate used for cell culture is a polyacrylamide gel coated with collagen. The preparation method includes: placing the prepared polyacrylamide gel at 4°C for 6 hours for cross-linking, then coating it with collagen and placing it at 4°C, and sterilizing it with ultraviolet light for 60 minutes before use.

[0027] A system for implementing the patch-clamp-based electrical stimulation-induced modulation analysis method of cell electrical coupling mediated by tunnel nanotubes includes a patch-clamp electrical stimulation module for applying specific electrical stimulation to target cells, a membrane potential monitoring module for monitoring membrane potential, and a cell traction force measurement module for measuring cell traction force.

[0028] The patch-clamp electrical stimulation module includes a patch-clamp device system configured to apply a specific pattern of electrical stimulation to C2C12 cells connected via tunnel nanotubes.

[0029] The membrane potential monitoring module acquires time-series images and delineates the background region, the electrically stimulated cell region, the tunnel nanotube region, the tunnel nanotube connected cell region, and the reference cell region.

[0030] Precise induction and monitoring: By applying specific patterns of electrical stimulation through patch-clamp devices, the parameters of the electrical stimulation can be precisely controlled. Combined with membrane potential imaging and cell traction force measurement, the electrophysiological and mechanical changes of cells can be comprehensively monitored, providing accurate data for the study of cell-cell electrical coupling.

[0031] Multi-signal pathway research: Intervention using inhibitors of multiple signaling pathways can help to explore the roles of different signaling pathways in tunnel nanotube-mediated cell electrical coupling, and help to reveal the regulatory mechanism of cell electrical coupling.

[0032] Comprehensive analysis method: By comprehensively analyzing changes in membrane potential and traction force, the cell electrical coupling effect mediated by tunnel nanotubes can be more fully verified, providing a new perspective and method for the study of intercellular signal transduction.

[0033] Example: A method and system for analyzing the regulation of cell-cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation. Materials used: DMEM cell culture medium (Thermo Fisher Scientific, USA), bis(1,3-dibarbituric acid)-trimethyloxenolol (DiBAC4(3)), (Invitrogen, USA), trypsin (Sigma-Aldrich, USA), penicillin / streptomycin (P / S) (Sigma-Aldrich, USA), poly-D-lysine (PDL), (Sigma-Aldrich, USA), fetal bovine serum (FBS) (Thermo Fisher Scientific, USA), acrylamide (Sigma-Aldrich, USA), bis-acrylamide (Sigma-Aldrich, USA), ammonium persulfate (Sigma-Aldrich, USA), tetramethylethylenediamine (TMEMD) (Sigma-Aldrich, USA), carboxylate fluorescent beads (200 nm in diameter) (Thermo Fisher Scientific, USA). Fisher Scientific (USA) L-type calcium channel blocker (Nifedipine) (Sigma-Aldrich, USA), T-type calcium channel blocker (Mibefradil) (Sigma-Aldrich, USA), Gadolinium chloride (Sigma-Aldrich, USA), Meclofenamate sodium salt (Sigma-Aldrich, USA), Thapsigargin (Sigma-Aldrich, USA), 2-aminoethyl diphenylborate (Sigma-Aldrich, USA), Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, USA), Carboxylate Fluorescent Microbeads (200 nm diameter) (Thermo Fisher Scientific, USA), Hydroxyethylpiperazine Ethiosulfonic Acid (HEPES) (Sigma-Aldrich, USA), Phosphate Buffered Salt Solution (PBS) (Beyotime, China).

[0034] Analysis method: Drug processing methods: Meclofenamate was dissolved in sterile water to prepare a 100 mM stock solution, and the final concentration in the bath solution was 100 μM, with an incubation time of 40 min. Mibefradil was dissolved in DMSO to prepare a 1 mM stock solution, and the Mibefradil stock solution was added to the bath solution to achieve an incubation concentration of 1 μM, with an incubation time of 30 min. Nifedipine was dissolved in anhydrous ethanol to prepare a 10 mM stock solution, and its final concentration in the bath solution was 10 μM, with an incubation time of 30 min. Gadolinium was dissolved in sterile water to prepare a 100 mM stock solution, and the Gadolinium was added to the bath solution to achieve an incubation concentration of 100 μM, with an incubation time of 30 min. Thapsigargin was dissolved in DMSO to prepare a 1 mM stock solution, and the Thapsigargin was added to the bath solution to achieve a final concentration of 1 μM, with an incubation time of 25 min. 2-Aminoethyl diphenylborate was dissolved in DMSO to prepare a 15 mM solution. 2-Aminoethyl diphenylborate was then added to the bath solution to achieve an effective concentration of 15 μM, with an action time of 25 min. Blebbistatin was dissolved in DMSO to prepare a 3 mM stock solution. The usage concentration was 3 μM, with an action time of 30 min. All prepared drugs were stored at -20°C.

[0035] Membrane potential imaging: (1) Prepare a 10 mM stock solution of DiBAC4(3) dye using DMSO (store the stock solution in a -20℃ refrigerator). (2) Take 1 μL of 10 mM membrane potential dye stock solution and dissolve it in 1 mL of LMEM medium to make membrane potential dye working solution; (3) Remove the prepared cells, discard the old culture medium, and wash the cells once with PBS; (4) Add the prepared membrane potential dye working solution to the culture dish and incubate in a 5% CO2, 37℃ incubator for 20-30 min; (5) Discard the membrane potential dye working solution and wash with calcium- and magnesium-free HBSS solution 2-3 times, 2 min each time; (6) Finally, add the appropriate bath solution and use a fluorescence microscope for imaging.

[0036] (7) Import the collected images into Image J software, and use the ROI Manager plugin in the software to circle the five parts: background area (cell-free area), force-stimulated cell area, TNT area, TNT-connected cell area and reference cell area, and obtain the average gray value of each area in the time series. (8) Import the average gray value of each region into Matlab, and process it with software to obtain the curves of the fluorescence intensity of the membrane potential indicator in the four regions of the force-stimulated cell, TNT, TNT-connected cell, and reference cell as a function of time. The horizontal axis of the curve is time, and the vertical axis is ΔF / F0. Wherein, ΔF is the average fluorescence intensity of the cell at a certain moment - the average fluorescence intensity of the background region at the corresponding moment, and F0 is the average fluorescence intensity of the cell within 10 frames when the shooting begins.

[0037] Electrical stimulation application: Electrical stimulation was applied to TNT-bound cells using a patch-clamp device system (Axopatch 200B), as follows: (1) C2C12 cells were clamped at -80mV (for 20ms), the voltage was changed to +80mV (for 1000ms), and finally returned to -80mV (for 20ms). At the same time, fluorescence imaging was performed on electrically stimulated cells and TNT-connected cells for 5 minutes; (2) Import the collected images into Image J software, and use the ROI Manager plugin in the software to circle the five parts: background area (cell-free area), electrically stimulated cell area, TNT area, TNT-connected cell area and reference cell area, and obtain the average gray value of these five areas in the time series. (3) Import this average gray value into Matlab, and process it with software to obtain the fluorescence intensity curves of electrically stimulated cells, TNT, TNT-connected cells, and reference cell membrane potential indicators over time. The horizontal axis of the curve is time, and the vertical axis is ΔF / F0, where ΔF is the average fluorescence intensity of the cell at a certain moment - the average fluorescence intensity of the background area at the corresponding moment, and F0 is the average fluorescence intensity of the cell within 10 frames at the start of the shooting.

[0038] Cellular traction force measurement: First, PA gel was prepared and placed in a 4°C refrigerator for 6 hours to crosslink the gel (using a crosslinking agent) and coat collagen. Then, it was placed in a 4°C refrigerator overnight. The prepared PA gel was then irradiated with UV light in a clean bench for 60 minutes, and cells were seeded. Bright-field and fluorescence images were captured using a 40x fluorescence microscope. 10% SDS solution was added to the culture dish, and after cell death, fluorescence images were captured again. Finally, the traction force was calculated using the Fourier transform traction microscopy (FTTC) method in Matlab software.

[0039] Statistical Analysis: Experimental data were derived from at least three independent experiments, and are expressed as mean ± standard error (SEM). Student's t-test was used to analyze statistical significance when comparing two groups of data; analysis of variance (ANOVA) was used to compare three or more groups of data. All data analyses were performed using GraphPad Prism 9.5.1 software. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, and "ns" represents no statistical significance.

[0040] Experimental Results and Discussion: Electrical stimulation of C2C12 cells causes changes in the cell membrane potential across TNT cells; Ca 2+ Effects of TNT-mediated electrical stimulation on changes in membrane potential in C2C12 cells First, C2C12 cells were labeled with membrane potential indicators. Then, TNT-linked C2C12 cells were selected and electrically stimulated. The results showed that electrical stimulation induced changes in the fluorescence intensity of the membrane potential indicator in TNT-linked cells, but not in cells without TNT linkage. Figure 1 As shown in (b). This indicates that electrical stimulation can induce depolarization in C2C12 cells, and that the depolarization induced by electrical stimulation is mediated by TNT-mediated electrical signaling to TNT-connected cells, rather than by chemical diffusion induced by electrical stimulation.

[0041] Figure 1 (a) Transmission of electrical signals between TNT-connected C2C12 cells; (b) Effect of electrical stimulation on the fluorescence intensity of cell membrane potential indicators in TNT-connected C2C12 cells (external solution: DMEM); (c) Bright-field image of electrically stimulated C2C12 cells (external solution: DMEM).

[0042] Figure 2 (a) Effect of electrical stimulation on the fluorescence intensity of TNT-linked C2C12 cell membrane potential indicators (external solution: calcium-free HBSS); (b) Bright field image of electrically stimulated C2C12 cells (external solution: calcium-free HBSS).

[0043] Figure 3 In the control group: n=16, calcium-free HBSS: n=16.

[0044] This experiment investigated the effect of calcium-deficient HBSS on TNT-mediated intercellular electrical coupling induced by electrical stimulation. The results showed that in the absence of calcium in the extracellular fluid... 2+Under certain conditions, the fluorescence intensity of TNT-mediated cell membrane potential indicators showed little change after electrical stimulation of cells, such as... Figure 2 As shown in (a), the proportion of TNT-mediated electrical coupling between C2C12 cells decreased from 100% in the control group to 50%, as Figure 3 As shown. This significant change compared to the control group indicates that the extracellular fluid Ca... 2+ It plays an important regulatory role in the TNT-mediated intercellular electrical coupling between C2C12 cells induced by electrical stimulation.

[0045] The effect of GJ on cell membrane potential changes induced by TNT-mediated electrical stimulation 100 μM MFA was added to the extracellular fluid of C2C12 cells, and TNT-connected cells were electrically stimulated. The results showed that both the electrically stimulated cells and the fluorescence intensity of membrane potential indicators related to their TNT-connected cells increased. Figure 4 As shown in (a). With the addition of MFA, the proportion of TNT-mediated intercellular electrical coupling was observed to decrease slightly from 100% under DMEM conditions to 85.71% under DMEM plus MFA conditions, as shown in (a). Figure 5 As shown in the figure. However, this change was not statistically significant. This indicates that gap junction proteins are not involved in TNT-mediated intercellular electrical coupling induced by electrical stimulation of C2C12 cells.

[0046] Figure 4 (a) Effect of electrical stimulation on the fluorescence intensity of TNT-linked cell membrane potential indicators in C2C12 cells (external solution: DMEM+MFA); (b) Bright field image of electrically stimulated C2C12 cells (external solution: DMEM+MFA). Figure 5 Control group: n=16, MFA: n=16.

[0047] Effects of calcium channels on changes in cell membrane potential induced by TNT-mediated electrical stimulation Adding Mibefradil to the extracellular fluid increased the fluorescence intensity of membrane potential indicators in cells when T-type calcium channels were blocked, but did not increase the fluorescence intensity of membrane potential indicators that connect to cells via TNT. Figure 6 As shown in (a), the proportion of TNT-mediated intercellular electrical coupling in C2C12 cells was significantly reduced. When the extracellular fluid was DMEM + Mibefradil, the proportion of TNT-mediated intercellular electrical coupling induced by electrical stimulation decreased from 100% in the control to 55.55%, as shown in (a). Figure 7 As shown in the figure. This result was statistically significant compared to the control group. This indicates that T-type calcium channels on the C2C12 cell membrane play an important role in TNT-mediated intercellular electrical coupling induced by electrical stimulation, especially in maintaining calcium... 2+During the transmission of charged ions across the cell membrane, T-type calcium channels play a crucial role in the TNT-mediated intercellular electrical signaling induced by electrical stimulation.

[0048] Figure 6 (a) Effect of electrical stimulation on the fluorescence intensity of a cell membrane potential indicator for TNT-linked C2C12 cells (external solution: DMEM + Mibefradil); (b) Bright-field image of electrically stimulated C2C12 cells (external solution: DMEM + Mibefradil). Figure 7 In China: control group: n=16, Mibefradil: n=16.

[0049] Figure 8 (a) Effect of electrical stimulation on the fluorescence intensity of TNT-linked C2C12 cell membrane potential indicator (external solution: DMEM+Nifedipine); (b) Bright field image of electrically stimulated C2C12 cells (external solution: DMEM+Nifedipine). Figure 9 In the control group: n=16, Nifedipine: n=16.

[0050] In this experiment, we investigated the potential impact of L-type calcium channels on electrocoupling between C2C12 cells. Adding the L-type calcium channel blocker Nifedipine (10 μM) to the extracellular fluid increased the fluorescence intensity of both the electrically stimulated cells and the membrane potential indicator that connects them via TNTs. Figure 8 As shown in (a), electrical stimulation slightly decreased the proportion of TNT-mediated intercellular electrical coupling, from 100% in the control to 87.5%, as shown in (a). Figure 9 As shown in the figure, this change did not show a statistically significant difference compared to the control group. This indicates that L-type calcium channels do not play a key role in the TNT-mediated electrical coupling between C2C12 cells induced by electrical stimulation.

[0051] Electrical stimulation of C2C12 cells causes changes in the traction force of TNT-connected cells. Ca 2+ Effects of electrical stimulation on changes in traction force in TNT-connected cells Experimental results showed that when the extracellular fluid was DMEM, the average traction force of TNT-connected cells before electrical stimulation was 14.8831 Pa, and after electrical stimulation, the traction force of TNT-connected cells increased to 16.3001 Pa. Figure 10 As shown, the changes in the traction force of TNT-connected cells before and after electrical stimulation are significant. This result indicates that the electrical signals induced by electrical stimulation can be successfully transmitted to TNT-connected cells, leading to an increase in the traction force of TNT-connected cells.

[0052] Figure 10 (a) Traction force of TNT-connected cells before electrical stimulation; (b) Traction force of TNT-connected cells after electrical stimulation; (c) Bright field image; (d) Comparison of traction force of TNT-connected cells before and after electrical stimulation (n=15); Figure 11 In the case of n=15.

[0053] The mean traction force of TNT-connected C2C12 cells was measured when the extracellular fluid was calcium-free HBSS. Before electrical stimulation, the mean traction force of TNT-connected cells was 12.6098 Pa, and after electrical stimulation, the mean traction force was 12.7873 Pa. Figure 11 As shown, there was no significant change in the traction force of TNT-connected cells before and after electrical stimulation. This result indicates that, under conditions of calcium ion absence in the extracellular fluid, the electrical signals generated by electrical stimulation of cells were not effectively transmitted to TNT-connected cells. Therefore, electrical stimulation did not induce any change in the traction force of TNT-connected cells.

[0054] Effects of GJ on changes in traction force in TNT-connecting cells induced by electrical stimulation When the extracellular fluid was DMEM+MFA, the average traction force of TNT-connected cells before electrical stimulation was 12.7498 Pa, and after electrical stimulation, the average traction force of TNT-connected cells increased to 14.1006 Pa. Figure 12 As shown, the traction force of TNT-connecting cells changed significantly before and after electrical stimulation. This result indicates that even with gap junction protein failure, the electrical signal induced by electrical stimulation can still be transmitted to TNT-connecting cells, leading to an increase in the traction force of these cells. Figure 12 In the case of n=15.

[0055] Effects of calcium channels on changes in traction force in TNT-connected cells induced by electrical stimulation In this experiment, extracellular fluid of DMEM + Mibefradil was used to investigate the effect of T-type calcium channels on the C2C12 cell membrane on the traction force of TNT-connected cells after electrical stimulation of C2C12 cells. Before electrical stimulation, the mean traction force of TNT-connected cells was 11.4165 Pa; after electrical stimulation, the mean traction force increased to 11.7673 Pa. Figure 13 As shown, this change was not statistically significant. This indicates that even with the blockade of T-type calcium channels, the electrical stimulation-induced intercellular electrical coupling between C2C12 cells has little effect on the traction force of TNT-connected cells. Figure 13 and Figure 14 In the case of n=15.

[0056] In this experiment, extracellular fluid of DMEM + Nifedipine was used to investigate the effect of electrical stimulation on the traction force of TNT-connected cells after L-type calcium channel blockade. The results showed that the average traction force of TNT-connected cells before and after electrical stimulation was 16.0831 Pa and 17.3606 Pa, respectively. Figure 14 As shown, the traction force of TNT-connecting cells changed significantly before and after electrical stimulation. This indicates that even with the blockage of L-type calcium channels, the electrical signals induced by electrical stimulation of C2C12 cells can still be transmitted to TNT-connecting cells, leading to an increase in the traction force of TNT-connecting cells.

[0057] Table 1: Proportion of TNT-mediated intercellular electrical coupling induced by electrical stimulation of C2C12 cells Bath Force stimulated cell number Cell number with electrical coupling Electrical coupling ratio (%) DMEM 16 16 100% (n=8) HBSS 16 8 50% (n=8) DMEM + MFA 16 14 85.71% (n=7) DMEM + Mibefradil 16 9 55.55% (n=9) DMEM + Nifedipine 16 14 87.5% (n=8) When C2C12 cells were electrically stimulated, the fluorescence intensity of an indicator of TNT-connected cell membrane potential changed significantly, while this phenomenon was not observed in cells without TNT connections. This result indicates that electrical stimulation of C2C12 cells can induce depolarization in TNT-connected cells. 2+ In the extracellular fluid, the proportion of TNT-mediated intercellular electrical coupling induced by electrical stimulation reached 100%. In the absence of Ca2+, 2+ Under extracellular fluid conditions, the proportion of intercellular electrical coupling decreased significantly to 50%, as shown in Table 1. This indicates that extracellular fluid Ca 2+ It is a key factor in maintaining intercellular electrical coupling. Furthermore, when T-type calcium channels in C2C12 cells were inhibited, the proportion of intercellular electrical coupling also decreased to 55.55%. These results indicate that extracellular calcium... 2+ TNTs can enter C2C12 cells via T-type calcium channels. The electrical signals transmitted through TNTs not only induce changes in the fluorescence intensity of membrane potential indicators in TNT-connected cells, but also affect the traction force between TNT-connected cells.

[0058] The key design focus of this invention is to deeply analyze the regulatory mechanism of TNT-mediated cell-cell electrical coupling through precise electrical stimulation and multi-parameter monitoring. By comprehensively analyzing changes in membrane potential and traction force, the cell-cell electrical coupling effect can be more fully verified, providing an effective method and system for the study of intercellular signal transduction. This application uses C2C12 cells as the research object to explore the mechanism of TNT-mediated intercellular electrical signal coupling induced by electrical stimulation. Combined with cell traction force experiments, the potential influence of electrical signal transmission on the traction force of TNT-connected cells is revealed. Electrical stimulation can induce changes in the fluorescence intensity of membrane potential indicators in TNT-connected cells, indicating that the depolarization process is directly mediated by TNT. This not only provides new insights into the mechanism of intercellular electrical signal transduction but also opens up new avenues for future research and clinical applications in the field of bioelectricity. Patch-clamp technique is key to realizing electrical stimulation experiments. This technique allows researchers to precisely control current and voltage at the single-cell level, thereby accurately simulating and studying the cell response to electrical stimulation. Through membrane perforation experiments, this technique can directly change the intracellular ion concentration, especially Ca2+. 2+ This simulates electrical signals under physiological conditions. This precise control and measurement makes it possible to gain a deeper understanding of how cells respond to electrical stimulation, and also opens new avenues for exploring the mechanisms of intercellular communication via electrical signals.

[0059] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A patch-clamp-based method for analyzing the regulation of cell electrical coupling induced by tunneling nanotubes through electrical stimulation, characterized in that: Includes the following steps: S1. Sample preparation and drug intervention: Provide tunnel nanotube-mediated C2C12 cells and add at least one signaling pathway inhibitor to the cell bath for intervention; S2, membrane potential imaging and data analysis; S3. Electrical stimulation application and coupling verification: A specific pattern of electrical stimulation was applied to one cell in a tunnel nanotube-connected cell pair using a patch clamp device. Membrane potential imaging and data analysis were performed, and the membrane potential change curves of the electrically stimulated cell and the tunnel nanotube-connected cell were compared. S4: Cell traction force measurement; S5. Analyze the changes in membrane potential and traction force to verify the cell electrical coupling effect mediated by tunnel nanotubes.

2. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes based on patch-clamp electrical stimulation according to claim 1, characterized in that: The signaling pathway inhibitor in step S1 is selected from at least one of the following: gap junction channel inhibitor, T-type calcium channel blocker, L-type calcium channel blocker, mechanosensitive ion channel blocker, sarcoplasmic reticulum calcium pump inhibitor, IP3 receptor inhibitor, or myosin II inhibitor, and the duration of action of the signaling pathway inhibitor is 25 to 40 minutes.

3. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation according to claim 2, characterized in that: The gap junction channel inhibitor is meclofenamic acid at a concentration of 100 μM, the T-type calcium channel blocker is mibediil at a concentration of 1 μM, the L-type calcium channel blocker is nifedipine at a concentration of 10 μM, the mechanosensitive ion channel blocker is gadolinium chloride at a concentration of 100 μM, the sarcoplasmic reticulum calcium pump inhibitor is carotenoid at a concentration of 1 μM, the IP3 receptor inhibitor is 2-aminoethyl diphenylboronic acid ester at a concentration of 15 μM, and the myosin II inhibitor is butadiene at a concentration of 3 μM.

4. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation according to claim 1, characterized in that: The membrane potential imaging and data analysis in step S2 specifically involves: loading cells with a membrane potential-sensitive fluorescent dye, performing time-series imaging using a fluorescence microscope, and using ImageJ and Matlab software to analyze the changes in membrane potential fluorescence intensity of electrically stimulated cells, tunnel nanotubes, tunnel nanotube-connected cells, and reference cells.

5. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation according to claim 1, characterized in that: The specific mode of electrical stimulation in step S3 is as follows: the clamping voltage is maintained at -80 mV for 20 ms, then stepped to +80 mV and maintained for 1000 ms, and finally returned to -80 mV and maintained for another 20 ms.

6. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation according to claim 1, characterized in that: The cell traction force measurement in step S4 specifically involves: measuring the change in traction force connecting cells to tunnel nanotubes using Fourier transform traction force microscopy before and after the application of electrical stimulation; comparing the displacement images of fluorescent microspheres generated when cells are alive with those generated after lysis and death in 10% SDS solution; and calculating the cell traction force using Matlab software.

7. The method for analyzing the regulation of cell electrical coupling mediated by tunneling nanotubes induced by patch-clamp electrical stimulation according to claim 1, characterized in that: The cell culture substrate is a polyacrylamide gel coated with collagen. The preparation method includes: placing the prepared polyacrylamide gel at 4°C for cross-linking for 6 hours, then coating it with collagen and placing it at 4°C, and sterilizing it by irradiation with ultraviolet light for 60 minutes before use.

8. A system for implementing the patch-clamp-based electrical stimulation-induced cell electrical coupling mediated by tunnel nanotubes as described in any one of claims 1-7, characterized in that: It includes a patch-clamp electrical stimulation module for applying specific electrical stimulation to target cells, a membrane potential monitoring module for monitoring membrane potential, and a cell traction force measurement module for measuring cell traction force.

9. The system according to claim 8, characterized in that: The patch-clamp electrical stimulation module includes a patch-clamp device system configured to apply a specific pattern of electrical stimulation to C2C12 cells connected via tunnel nanotubes.

10. The system according to claim 8, characterized in that: The membrane potential monitoring module acquires time-series images and delineates the background region, the electrically stimulated cell region, the tunnel nanotube region, the tunnel nanotube connected cell region, and the reference cell region.

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