Tunnel nanotube mediated intercellular mechanical signal transduction regulation and control mechanism research method and system and application thereof

Abstract

The invention discloses a tunnel nanotube mediated intercellular mechanical signal transduction regulation and control mechanism research method and system and application, and relates to the technical field of biology. The regulation and control mechanism research method of tunnel nanotube mediated intercellular mechanical signal transduction comprises the following steps: S1, providing tunnel nanotube connection cells growing on a polyacrylamide hydrogel substrate; s2, performing drug treatment on the tunnel nanotube connected cells: applying an effective dose of at least one signal path inhibitor into a system in which the tunnel nanotube connected cells are cultured; s3, mechanical stimulation of tunnel nanotube connection cells; s4, measuring the change of the traction force of the cells and the change of the spreading area of the cells; by adopting the research method provided by the invention, the important influence of intercellular electric signal conduction on the cell connection function of the tunnel nanotube is revealed, and a precise tool is provided for researching intercellular mechanical communication.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for studying the regulation mechanism of intercellular mechanical signal transduction mediated by tunneling nanotubes, a system thereof and an application. BACKGROUND

[0002] Tunneling nanotubes (TNTs) as a physical channel between cells can achieve long-distance directional signal transmission. However, the specific mechanism of how TNT-mediated signal transduction affects cell traction force and cell spreading area is still unclear. In this study, C2C12 cells were used as a model to investigate the effects of mechanical stimulation on traction force and cell spreading area mediated by TNT-mediated electromechanical signal transduction. The results showed that mechanical stimulation depended on the Ca 2+ , which promoted electrical coupling between cells, and this process played a key role in regulating the traction force and spreading area of TNT-connected cells. In addition, mechanical stimulation could induce extracellular Ca 2+ influx through T-type calcium channels on the C2C12 cell membrane, thereby affecting traction force and spreading area. Further studies showed that disrupting gap junction proteins after mechanical stimulation could eliminate the changes in traction force between TNT-connected cells, while inhibiting cell contraction did not affect cell spreading area, suggesting that mechanical structural components play different roles in the regulation of cell behavior. This study revealed the intercellular electrical signal transduction and coupling mechanism mediated by TNTs, and expanded the understanding of electrical activity and cell dynamics in living organisms.

[0003] Although a large number of studies have focused on the role of TNTs in biochemical signal transduction, their function in mechanical signal transmission has not been fully revealed. Mechanical stimulation as a key regulatory factor in life processes such as cell migration, proliferation and differentiation, its propagation between cells may not only depend on chemical signals, but also involve TNT-mediated physical signal pathways. In this study, a series of advanced biophysical and cell biological techniques were used, including a patch clamp experimental device combined with a micro-manipulation system to apply precise mechanical stimulation to TNT-connected C2C12 cells. Through this method, the propagation of mechanical signals between cells and their effects on downstream functional responses were systematically analyzed, revealing the cellular mechanism of TNT-mediated mechanical signal transduction. Therefore, there is an urgent need in the art to establish a complete research method that can precisely manipulate the mechanical microenvironment of TNTs, systematically analyze their downstream signal pathways, and quantitatively read out the mechanical behavior changes of cells, thereby fully revealing the regulation mechanism of intercellular mechanical signal transduction mediated by TNTs. SUMMARY

[0004] Therefore, the present application aims at the defects of the prior art, and the main purpose is to provide a research method and system for the regulation mechanism of intercellular mechanical signal transduction mediated by tunnel nanotubes and application, which reveals the important influence of intercellular electrical signal transduction on the function of TNTs connected cells by using the research method provided by the present application, and provides a precise tool for studying intercellular mechanical communication.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: A research method for the regulation mechanism of intercellular mechanical signal transduction mediated by tunnel nanotubes, comprising the following steps: S1, providing tunnel nanotube connected cells grown on a polyacrylamide hydrogel substrate; S2, drug treatment of the tunnel nanotube connected cells: applying an effective dose of at least one signal pathway inhibitor to the system in which the tunnel nanotube connected cells are cultured, the signal pathway inhibitor being selected from one or more of a gap junction inhibitor, a calcium channel inhibitor, a mechanically sensitive ion channel inhibitor, an extracellular calcium remover, an intracellular calcium release inhibitor, or a cell contractility inhibitor; S3, mechanical stimulation of the tunnel nanotube connected cells: applying a directional mechanical stimulation to the target cells by a micromanipulator controlled by a patch clamp system; S4, measuring the biological response occurring in the tunnel nanotube connected cells triggered by the mechanical stimulation; the biological response includes changes in cell traction force and changes in cell spreading area.

[0006] As a preferred solution: the C2C12 cells in step S1 are cultured in a culture medium, and 10% fetal bovine serum and 100 U / mL penicillin-streptomycin complex antibiotic are added to the culture medium; the C2C12 cells are cultured in a 37°C, 5% CO2 humidified incubator.

[0007] As a preferred solution: the preparation of the polyacrylamide hydrogel substrate in step S1 comprises the following steps: S11, substrate pretreatment: plasma treating the bottom glass slide of the confocal culture dish, and then sequentially modifying with NaOH solution, APTES ethanol solution and glutaraldehyde solution to form an activated substrate surface; S12, gel solution preparation: preparing a polyacrylamide gel premix solution containing fluorescent microspheres; S13, gel polymerization: dropping the obtained gel premix solution onto the activated substrate, and covering with a silanized cover glass, avoiding light polymerization to form a gel layer; S14, post-treatment and protein cross-linking: after removing the coverslips and washing the gels, the gel surface is activated using a photo-activated cross-linking agent under UV light irradiation, followed by cross-linking of extracellular matrix proteins to the activated gel surface.

[0008] As a preferred solution: the volume fraction of APTES ethanol solution in step S11 is 50%, and the mass-volume concentration of glutaraldehyde solution is 0.5%; in step S13, the coverslips are treated with a silane release agent to achieve smooth separation of the polymerized gel; in step S14, the photo-activated cross-linking agent is Sulfo-SANPAH, which is diluted in HEPES buffer at a ratio of 1:100, and the extracellular matrix protein is type I collagen, which is cross-linked at a concentration of 0.2 mg / mL.

[0009] As a preferred solution: the gap junction inhibitor in step S2 is meclofenamic acid, which is used at a concentration of 100 μM for 40 minutes; the calcium channel inhibitor includes a T-type calcium channel inhibitor and an L-type calcium channel inhibitor, the T-type calcium channel inhibitor is mibefradil, which is used at a concentration of 1 μM; the L-type calcium channel inhibitor is nifedipine, which is used at a concentration of 10 μM.

[0010] As a preferred solution: the mechanosensitive ion channel inhibitor in step S2 is gadolinium ions, which are used at a concentration of 100 μM; the extracellular calcium removal agent is calcium-free Hanks' balanced salt solution, and the treatment time is 30 minutes; the intracellular calcium release inhibitor uses 1 μM rapamycin combined with 15 μM 2-phenylboronic acid ethylamine, and the cells are treated at room temperature for 25 minutes; the cell contractility inhibitor is Blebbistatin, which is used at a concentration of 3 μM for 30 minutes.

[0011] As a preferred solution: the patch-clamp system in step S3 is the Axopatch 200B system, and the glass microelectrode mounted on the micromanipulator needs to be positioned above the target cell and ensure no bubble interference before applying mechanical pressure.

[0012] As a preferred solution: the measurement of cell traction force in step S4 is achieved by Fourier transform traction force microscopy; the measurement of cell spreading area includes the following steps: obtaining bright-field images of the same cell before and after mechanical stimulation is applied; using image analysis software, the cell boundaries before and after stimulation are marked respectively; the change in spreading area of the cell before and after mechanical stimulation is calculated and compared.

[0013] A system for implementing the regulation mechanism research method of the tunnel nanotube mediated intercellular mechanical signal transduction, comprising a mechanical stimulation module for applying directional mechanical pressure to target cells, a mechanical response measurement module for measuring cell traction force by Fourier transform traction force microscopy, and a morphological analysis module for quantitatively analyzing cell spreading area changes; the mechanical stimulation module comprises a patch clamp system, a micromanipulator and a glass microelectrode; the mechanical response measurement module comprises a confocal microscope and an image processing unit; and the morphological analysis module comprises image analysis software.

[0014] The application of a signal pathway inhibitor used in the regulation mechanism research method of the tunnel nanotube mediated intercellular mechanical signal transduction in the preparation of a kit for researching cell mechanical transduction, wherein the kit comprises one or more signal pathway inhibitors.

[0015] Compared with the prior art, the present application has obvious advantages and beneficial effects, specifically, as known from the above technical solution, by using the research method provided by the present application, the important influence of intercellular electrical signal transduction on the function of TNTs connected cells is revealed, and the key roles of calcium ions, specific types of calcium channels, gap junction proteins and cell contractility in TNTs mediated mechanical signal transduction are highlighted. Not only the understanding of the TNTs mediated cell communication mechanism is deepened, but also a theoretical basis is provided for further exploring the TNTs signal regulation mechanism in the future. In the future, in-depth research in this field is expected to provide new ideas for therapeutic strategies targeting abnormal cell communication and mechanical transduction; the present application constructs a complete research system integrating "directional mechanical stimulation-specific drug intervention-double channel mechanical response quantitative measurement", overcomes the limitations of traditional methods that cannot perform site-specific and quantitative mechanical intervention and synchronous multi-parameter detection on TNTs networks, and provides an unprecedented precise tool for studying intercellular mechanical communication; the present method systematically reveals the core role of calcium ion signals in TNTs mechanical transduction and the specific pathway; by quantifying the complex cell mechanical behavior into two clear and measurable parameters, "traction force change" and "spreading area change", and combining with a specific signal pathway inhibitor toolkit, the present method provides an efficient standardized platform for screening drugs regulating the function of TNTs.

[0016] In order to more clearly illustrate the structural features and effects of the present application, the following will be combined with the specific embodiments and detailed descriptions. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A schematic diagram of the influence of the mechanical stimulation of the present application on the traction force and spreading area of TNTs connected C2C12 cells (extracellular solution: DMEM); Figure 2This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connected to C2C12 cells (extracellular solution: HBSS); Figure 3 This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connecting C2C12 cells (extracellular solution: DMEM + TG + 2-APB); Figure 4 This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connecting C2C12 cells (extracellular solution: HBSS + TG + 2-APB); Figure 5 This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connected to C2C12 cells (extracellular solution: DMEM + Mibefradil); Figure 6 This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connected to C2C12 cells (extracellular solution: DMEM + Nifedipine); Figure 7 This is a schematic diagram illustrating the effect of mechanical stimulation of the present invention on the traction force and spreading area of ​​TNTs connecting C2C12 cells. Detailed Implementation

[0018] The present invention is as follows Figure 1 As shown in Figure 7, a method for studying the regulatory mechanism of intercellular mechanotransduction mediated by tunnel nanotubes includes the following steps: S1 provides tunnel nanotubes for cell connection grown on a polyacrylamide hydrogel substrate; S2. Drug treatment of tunnel nanotube-connected cells: In a system containing tunnel nanotube-connected cells, an effective dose of at least one signaling pathway inhibitor is administered, wherein the signaling pathway inhibitor is selected from one or more of the following: gap junction inhibitors, calcium channel inhibitors, mechanosensitive ion channel inhibitors, extracellular calcium scavengers, intracellular calcium release inhibitors, or cell contractility inhibitors. S3. Mechanical stimulation of cells connected by tunnel nanotubes: Targeted mechanical stimulation is applied to the target cells using a micromanipulator controlled by a patch-clamp system. S4. Measure the biological response induced by the mechanical stimulus in the tunnel nanotube-connected cells; the biological response includes changes in cell traction force and changes in cell spreading area.

[0019] In step S1, C2C12 cells are cultured in a culture medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin compound antibiotics. The C2C12 cells are cultured in a 37°C, 5% CO2 humidified incubator.

[0020] The preparation of the polyacrylamide hydrogel substrate in step S1 includes the following steps: S11. Substrate pretreatment: The bottom slide of the confocal culture dish is subjected to plasma treatment, and then modified sequentially with NaOH solution, APTES ethanol solution and glutaraldehyde solution to form an activated substrate surface; S12, Gel solution preparation: Prepare a polyacrylamide gel premix containing fluorescent microspheres; S13, Gel polymerization: The obtained gel premix is ​​dropped onto the activated substrate and covered with a coverslip treated with silanization. The mixture is then polymerized in the dark to form a gel layer. S14. Post-treatment and protein crosslinking: After removing the coverslip and cleaning the gel, the gel surface is activated under ultraviolet light using a photo-activated crosslinking agent. Then, extracellular matrix proteins are crosslinked to the activated gel surface.

[0021] In step S11, the volume fraction of the APTES ethanol solution is 50%, and the mass-volume concentration of the glutaraldehyde solution is 0.5%. In step S13, the coverslip is treated with a silane stripper to facilitate the separation of the polymer from the gel. In step S14, the photoactivated crosslinking agent is Sulfo-SANPAH, which is diluted 1:100 in the HEPES buffer, and the extracellular matrix protein is type I collagen with a crosslinking concentration of 0.2 mg / mL.

[0022] In step S2, the gap junction inhibitor is meclofenac at a concentration of 100 μM and the treatment time is 40 minutes. The calcium channel inhibitors include T-type calcium channel inhibitors and L-type calcium channel inhibitors. The T-type calcium channel inhibitor is mibediil at a concentration of 1 μM, and the L-type calcium channel inhibitor is nifedipine at a concentration of 10 μM.

[0023] In step S2, the mechanosensitive ion channel inhibitor is gadolinium ion at a concentration of 100 μM; the extracellular calcium scavenger is a calcium-free Hanks balanced salt solution, and the treatment time is 30 minutes; the intracellular calcium release inhibitor is 1 μM rapamycin combined with 15 μM ethylamine 2-phenylboronic acid, and the cells are treated at room temperature for 25 minutes; the cell contractility inhibitor is Blebbistatin at a working concentration of 3 μM, and the treatment time is 30 minutes.

[0024] In step S3, the patch clamp system is the Axopatch 200B system. Before applying mechanical pressure, the glass microelectrode mounted on the micromanipulator must be positioned above the target cell and free from air bubbles.

[0025] In step S4, the measurement of cell traction force is achieved through Fourier transform traction force microscopy. The measurement of cell spreading area includes the following steps: acquiring bright-field images of the same cell before and after mechanical stimulation; using image analysis software to mark the cell boundaries before and after stimulation; and calculating and comparing the changes in cell spreading area before and after mechanical stimulation.

[0026] A system for studying the regulatory mechanisms of intercellular mechanotransduction mediated by tunnel nanotubes includes a mechanical stimulation module for applying directional mechanical pressure to target cells, a mechanical response measurement module for measuring cellular traction force using Fourier transform traction microscopy, and a morphological analysis module for quantitatively analyzing changes in cell spreading area. The mechanical stimulation module includes a patch-clamp system, a micromanipulator, and glass microelectrodes. The mechanical response measurement module includes a confocal microscope and an image processing unit. The morphological analysis module includes image analysis software.

[0027] Mechanical Stimulation Module: Utilizing a micromanipulator to precisely position glass microelectrodes, a patch-clamp system is used to apply quantitative, directional pressure stimulation to target cells, simulating physiological or pathological mechanical signals, and observing the response of tunnel nanotubes. Mechanical Response Measurement Module: Confocal microscopy is used to acquire images of cells placed on a fluorescent microsphere matrix. Fourier transform traction force microscopy analysis is performed using an image processing unit to quantitatively calculate the changes in traction force generated by cells before and after mechanical stimulation. Morphological Analysis Module: Image analysis software is used to automatically identify and segment cell images acquired by the microscope, quantitatively measuring and analyzing the dynamic changes in cell spreading area morphological parameters during signal transduction.

[0028] The application of signaling pathway inhibitors used in a method for studying the regulatory mechanism of intercellular mechanotransduction mediated by tunnel nanotubes in the preparation of a kit for studying cell mechanotransduction, the kit containing one or more signaling pathway inhibitors.

[0029] Example: Research methods, systems, and applications of the regulatory mechanism of tunnel nanotube-mediated intercellular mechanotransduction. Cell culture The C2C12 cells used in this study were purchased from the China Cell Resource Sharing Platform (National Cell Bank, Beijing). Cell culture was performed using Dulbecco modified Eagle medium (DMEM, Corning, USA), supplemented with 10% fetal bovine serum (Fetal Bovine Serum, FBS, Gibco, USA) and 100 U / mL penicillin-streptomycin combination antibiotics. Cells were cultured at 37°C in a humidified incubator with 5% CO2 to maintain optimal growth and cell viability.

[0030] Preparation of polyacrylamide (PA) hydrogels First, 18 mm diameter circular coverslips were cleaned and dried in an oven. Then, the bottom slides of the 20 mm confocal culture dishes used for culturing were plasma-treated (1 minute) to expose their surface hydroxyl groups. Next, 150 μL of 1 M NaOH solution was added to each culture dish, and the dishes were heated in an oven for 15 minutes to form a thin NaOH coating. Then, a 50% (v / v) APTES (3-aminopropyltriethoxysilane, Sigma) solution was prepared, mixed with anhydrous ethanol at a 1:1 (v / v) ratio, and added to each culture dish (200 μL). The dishes were incubated in a fume hood at room temperature for 30 minutes. After incubation, the dishes were washed three times with triple-distilled water (TDW) for 5 minutes each, with gentle shaking on a shaker to ensure thorough cleaning. Finally, 200 μL of 0.5% glutaraldehyde (Sigma) solution was added to each culture dish, and the dishes were incubated in a fume hood at room temperature for another 30 minutes. After treatment, remove the glutaraldehyde solution, rinse 1–2 times with pure water, and air dry. Simultaneously, apply a silane stripper evenly to the previously cleaned and dried 18 mm coverslip surface and wipe clean with lens paper. Prepare a PA gel premix containing 200 nm fluorescent microspheres (Invitrogen, added at a 1:250 ratio) and mix thoroughly. Add 20 μL of PA gel solution to each pretreated confocal culture dish, then gently cover it with an inverted coverslip, avoiding air bubbles to form a uniform gel layer. Place the culture dishes in the dark at room temperature for 30 minutes to complete the gel polymerization reaction. After gel solidification, soak in 2 mL of triple-distilled water for 10 minutes. Then carefully remove the coverslip with a scalpel, rinse twice with triple-distilled water, and refrigerate the culture dishes at 4°C overnight. Before use, wash the gel dishes twice with PBS. To cross-link the proteins, Sulfo-SANPAH (ProteoChem) was diluted 1:100 in HEPES buffer, and 400 μL of the solution was added to each culture dish. The dishes were then irradiated with UV light at a distance of approximately 10 cm for 20 minutes to activate the cross-linking reaction. After UV irradiation, the cross-linking agent was removed, and the dishes were washed three times with HEPES buffer, approximately 3 minutes each time. Finally, type I collagen was dissolved in 6 mM acetic acid to prepare a 0.2 mg / mL solution. After mixing, 400 μL of the solution was added to each dish and stored at 4°C for later use.

[0031] Drug treatment To inhibit gap junction communication, cells were treated with 100 μM meclofenamate (Sigma) at room temperature for 40 minutes. To block T-type calcium channels, cells were incubated with 1 μM Mibefradil (Sigma) for 30 minutes. Similarly, L-type calcium channels were inhibited with 10 μM nifedipine (Sigma) under the same conditions. For inhibition of mechanosensitive ion channels, cells were treated with 100 μM gadolinium (Sigma) for 30 minutes. To remove extracellular calcium, the original culture medium was replaced with calcium-free Hanks' balanced salt solution (Ca²⁺-free HBSS), and the cells were allowed to stand for 30 minutes to ensure adequate calcium removal. To inhibit intracellular calcium release, cells were treated with 1 μM rapamycin (Thapsigargin, Sigma) in combination with 15 μM 2-Aminoethyldiphenylborate (2-APB, Sigma) at room temperature for 25 minutes to block the endoplasmic reticulum calcium storage release pathway. These drug treatments aimed to systematically evaluate the functional roles of different calcium signaling pathways in cellular responses. Furthermore, to inhibit cell contraction, Blebbistatin was dissolved in DMSO at a concentration of 3 mM to prepare a stock solution, resulting in a final working concentration of 3 μM, and incubated at room temperature for 30 minutes.

[0032] Cellular traction force measurement After the PA hydrogel was prepared according to the aforementioned steps, it was first incubated at 4°C for 6 hours to promote the full reaction of the cross-linking agent and complete the initial cross-linking. Subsequently, type I collagen was coated onto the gel surface, and it was again incubated overnight at 4°C to stabilize the collagen coating. Once the gel was ready, it was placed in a biosafety cabinet and irradiated with ultraviolet light for 60 minutes to further enhance the cross-linking strength and ensure sterility. Cells were then seeded onto the PA hydrogel surface, and imaging was performed after adhesion. Imaging was conducted using a Zeiss LSM 710 confocal microscope (Carl Zeiss Microimaging, Japan) equipped with a 40× / 0.75 numerical aperture objective. Cell morphology and their interaction with the gel were acquired through bright-field and fluorescence channels, respectively. To obtain cell-free fluorescence images required for traction force analysis, 10% SDS solution was added to the culture dish to lyse the cells, and fluorescence images were then captured again for subsequent displacement field calculations. Quantitative analysis of cellular traction forces was performed using Fourier Transform Traction Microscopy (FTTC), with image processing and force field reconstruction conducted using the Matlab platform. This experimental procedure combines advanced imaging systems and computational analysis techniques to efficiently and accurately measure the traction forces of cells on a gel substrate, providing reliable experimental evidence for a deeper understanding of cellular mechanical behavior.

[0033] TNTs connect cells to force stimulation This experiment employed glass microelectrodes mounted on a micromanipulation system (MP-225) to apply mechanical stimulation to cells connected by tunnel nanotubes (TNTs). The operation was controlled by an Axopatch 200B patch-clamp system, enabling precise control of the stimulation intensity and location. The specific steps were as follows: pre-cultured cells were placed on the stage of an Olympus IX73 inverted microscope, and then the glass microelectrodes were quickly positioned above the target cells using the micromanipulation system. Ensuring the glass electrodes were free of air bubbles, the electrodes were slowly lowered while applying directional mechanical pressure to the target cells. Throughout the process, the confocal culture dish containing the target cells had to remain stable to avoid displacement interfering with measurement accuracy. This experimental system enables mechanical stimulation of TNT-connected cells, while simultaneously allowing for precise measurement of cellular responses using traction microscopy. This method provides a crucial technical means and experimental platform for studying the transmission of mechanical signals between TNT-connected cells and their regulatory mechanisms on cellular biomechanical behavior.

[0034] Measurement of cell spreading area before and after force stimulation Bright-field images of cells were imported into ImageJ image analysis software to quantitatively measure the spreading area of ​​TNT-connected cells. In the images, the cell boundaries before mechanical stimulation were marked with magenta outlines, while yellow outlines corresponded to the boundary morphology of the same cell after mechanical stimulation. By employing a uniform color coding method, the contour changes of the same cell at different time points were clearly distinguished, thus ensuring the accuracy of comparative analysis. This method effectively reflects the changes in the spreading area of ​​TNT-connected cells before and after mechanical stimulation, providing a quantitative basis for assessing mechanosensitive cell morphological remodeling.

[0035] Discussion of Results: Ca 2+ Changes in traction force and spreading area of ​​TNT-connected cells under mechanical stimulation First, C2C12 cells with TNT-linked structures were screened, and then... 2+ Mechanical stimulation was performed on cells in DMEM medium. The results showed that the average traction force between TNT-connected cells was 12.0165 Pa before mechanical stimulation, but significantly increased to 14.234 Pa after stimulation. Figure 1 As shown in (a) to (d), this significant change in traction force indicates that the electrical signals induced by mechanical stimulation can effectively propagate between TNTs and enhance their traction force. Further analysis showed that, under the same conditions, the average spreading area of ​​TNT-connected cells before mechanical stimulation was 684.257 μm. 2 After stimulation, it significantly decreased to 585.87 μm. 2 ;like Figure 1 (e) Figure 1 As shown in (f). This change suggests that the electrical signals induced by mechanical stimulation, after being transmitted to the connective cells via TNTs, cause a significant reduction in cell spreading area. In summary, the experimental results indicate that in the presence of Ca... 2+ Under the culture conditions, mechanical stimulation can regulate the traction and spreading behavior of connected cells through TNT-mediated electromechanical signal transmission, indicating that calcium ions play a key regulatory role in TNT-mediated electromechanical signal transmission.

[0036] Figure 1 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0037] Extracellular Ca 2+ Regulation of traction force and spreading area in TNT-connected cell mechanostimulation This experiment was conducted in the absence of Ca. 2+ Hanks equilibrium salt solution (Ca 2+ C2C12 cells were mechanically stimulated in a TNT-free HBSS (HBSS-free) medium. The results showed that before stimulation, the mean traction force of TNT-connected cells was 11.0638 Pa, which slightly decreased to 11.03 Pa after stimulation. Figure 2 As shown in (a) to (d), the differences were not significant. This result indicates that when the extracellular environment lacks Ca2+, the differences are not statistically significant. 2+ At that time, the mechanically induced electrical signals could not be effectively transmitted to the connective cells via TNT, and therefore the traction force did not change significantly. Under the same conditions without Ca2+... 2+ Under these conditions, the average spreading area of ​​TNT-connected cells increased from 459.13 μm before stimulation. 2 The slight change was 464.296 μm after stimulation. 2 ,like Figure 2 (e) and Figure 2 As shown in (f), no significant differences were observed. This result further indicates that in the absence of extracellular Ca2+, no significant differences were observed. 2+ In this environment, mechanically stimulated TNT-mediated signals cannot effectively propagate between connecting cells, resulting in a relatively constant spreading area. In summary, extracellular Ca2+... 2+ The presence of TNTs is crucial for TNT-mediated mechanotransduction, and their absence will significantly weaken the transcellular transmission of signals, thereby blocking the regulatory effects of traction and spreading behavior.

[0038] Figure 2 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0039] Intracellular Ca 2+ Regulatory Role of TNTs in Trapural Force and Cell Spreading in Mechanostimulation Response Deletion of extracellular Ca 2+This weakens the regulatory effect of mechanical stimulation on the traction force between TNTs and cells. To further investigate the role of intracellular calcium storage (mainly derived from the endoplasmic reticulum) in this process, this experiment added 1 μM rapamycin (Thapsigargin, TG, endoplasmic reticulum calcium) to the culture medium of C2C12 cells. 2+ TG (a type of ATPase inhibitor) and 15 μM 2-APB (an IP3 receptor blocker) were used to effectively inhibit the release of intracellular calcium ions. Mechanical stimulation experiments were conducted in DMEM culture medium containing TG and 2-APB. The results showed that the average traction force between TNTs and cells was 13.3615 Pa before stimulation and 13.4045 Pa after stimulation. Figure 3 As shown in (a) to (d), there is no significant difference between the two. This indicates that when intracellular Ca... 2+ When release is blocked, the electrical signals induced by mechanical stimulation cannot effectively propagate to the connective cells via TNTs, resulting in no significant change in traction force. Under the same treatment conditions, the average spreading area of ​​TNT-connected cells increased from 969.106 μm before stimulation. 2 The size changed slightly to 968.89 μm after stimulation. 2 , Figure 3 As shown in (e) and (f), there were also no significant differences. This result further supports the finding that intracellular Ca2+... 2+ The crucial role of intracellular Ca2+ in the transduction of mechanostimulation signals means that impaired release will significantly limit the efficiency of TNT-mediated signal propagation between cells. In summary, intracellular Ca2+ plays a vital role in this process. 2+ Normal dynamic release is crucial for TNT-mediated mechanotransduction, and interference with it will significantly weaken the mechanoresponsiveness of traction and cell spreading behavior.

[0040] Figure 3 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0041] No Ca was found in either the extracellular or intracellular space. 2+ Effects of traction force and spreading area on the mechanical stimulation response of TNT-connected cells To completely block the role of calcium signaling in TNT-mediated mechanostimulation, this experiment also inhibited extracellular calcium. 2+The influx of calcium and the release of calcium from intracellular stores. Specifically, C2C12 cells were cultured in a calcium-free environment... 2+ The HBSS solution was added with 1 μM rapamycin (Thapsigargin, TG) and 15 μM 2-APB to jointly inhibit endoplasmic reticulum calcium deficiency. 2+ -ATPase and IP3 receptor channels, thereby achieving dual deprivation of calcium signaling. After mechanical stimulation under HBSS+TG+2-APB conditions, the mean traction force between TNTs and cells was 13.3027 Pa before stimulation and 13.2868 Pa after stimulation. Figure 4 As shown in (a) to (d), there is no significant difference between the two. This indicates that when extracellular Ca... 2+ Deletion and intracellular Ca 2+ When release is simultaneously inhibited, the electrical signals induced by mechanical stimulation cannot effectively propagate through the TNT structure to the connective cells, thus the traction force does not change significantly. Under the same treatment conditions, the average spreading area of ​​TNT-connected cells increased from 687.979 μm before stimulation. 2 The size changed slightly to 687.541 μm after stimulation. 2 , Figure 4 As shown in (e) and (f), the differences were not statistically significant. This result further illustrates that the integrity of calcium signaling is crucial for TNT-mediated electro-signal transduction. Lack of extracellular calcium and blockage of intracellular calcium release significantly weaken the propagation efficiency of mechanical stimuli between cells, resulting in no significant response in cell spreading behavior. In summary, calcium signaling plays a decisive role in TNT-mediated mechano-electro-signal coupling transduction; both extracellular uptake and intracellular mobilization are indispensable, making it a core factor regulating traction force and changes in cell spreading area.

[0042] Figure 4 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0043] The regulatory role of calcium channels in the mechanical load response of TNT-connected cells, controlling traction force and spreading area. To investigate the function of calcium channels in the response to mechanical stimulation, this study added 1 μM Mibefradil (a T-type calcium channel blocker) to the culture medium of C2C12 cells to specifically inhibit the activity of T-type calcium channels on the cell membrane. The results showed that before mechanical stimulation, the average traction force between TNTs and cells was 10.3277 Pa, which slightly increased to 10.3912 Pa after stimulation. Figure 5 As shown in (a) to (d), the differences were not significant. This result indicates that inhibition of T-type calcium channels weakens the cells' ability to respond to mechanical stimuli, possibly because the electrical signals induced by mechanical stimulation cannot be effectively transmitted to neighboring cells connected to TNTs, resulting in limited changes in their traction force. Under the same Mibefradil treatment conditions, the average spreading area of ​​TNT-connected cells increased from 1241.98 μm before stimulation. 2 A slight decrease to 1241.65 μm after stimulation. 2 ,like Figure 5 As shown in (e) and (f), the differences are also not significant. This further illustrates that T-type calcium channels are sensitive to ions (such as Ca2+). 2+ TNTs play a crucial role in transmembrane entry; their blockage hinders the propagation of mechanically induced electrical signals between cells, thus preventing significant changes in the spreading behavior of TNT-connected cells. In summary, T-type calcium channels play a vital role in the response of the C2C12 cell membrane to mechanical signals and in signal transduction. Their normal permeability is indispensable for the transcellular propagation of TNT-mediated electrical signals and the regulation of traction force and spreading area.

[0044] To investigate the function of L-type calcium channels in mechanostimulation-induced signal transduction, 10 μM nifedipine was added to the culture medium of C2C12 cells to specifically block L-type calcium channels on the cell membrane. The results showed that before mechanical stimulation, the average traction force of TNT-connected cells was 13.3087 Pa, which significantly increased to 14.717 Pa after stimulation. Figure 6 As shown in (a) to (d), significant changes were observed. This indicates that although L-type calcium channels were inhibited, mechanical stimulation could still induce effective electrical signals, which were successfully transmitted to the connective cells through the TNT structure, thereby enhancing their traction. Under the same conditions, the average spreading area of ​​TNT-connective cells increased from 1252.27 μm before stimulation. 2 Significantly decreased to 1182.66 μm 2 ,like Figure 6As shown in (e) and (f), significant changes were also observed. This result indicates that even when L-type calcium channels on the C2C12 cell membrane are blocked, signals generated by mechanical stimulation can still propagate to TNT-connected cells through other mechanisms or channel pathways, thereby affecting their morphological changes. In summary, although L-type calcium channels play a role in calcium signaling regulation, their inhibition does not block TNT-mediated electrical signal transduction. This suggests that other types of calcium channels (such as T-type calcium channels) or mechanisms may play a more crucial role in this process, thereby maintaining the sensitivity and responsiveness of TNT-connected cells to mechanical stimulation.

[0045] Figure 5 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0046] Figure 6 shows: (a) the distribution of traction force in TNT-connected cells before mechanical stimulation; (b) the distribution of traction force in TNT-connected cells after mechanical stimulation; (c) a bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) a statistical comparison of the traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) a bright-field image showing the morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); and (f) a statistical comparison of the spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0047] The role of gap connexins in traction regulation and the effect of cell contractility on TNT-connected cell spreading behavior To investigate the role of connexins in TNT-mediated mechanostimulation, this experiment added 100 μM meclofenamic acid (MFA) to the culture medium of C2C12 cells to block gap junctions (GJs), and measured the traction force of TNT-connected cells. The results showed that under GJ blocking conditions, the average traction force of TNT-connected cells was 11.4378 Pa before stimulation and 11.287 Pa after stimulation. Figure 7As shown in (a) to (d), the differences were not significant. This indicates that GJ blockade hindered the propagation of electro-inducible electrical signals induced by mechanical stimulation between TNT-connected cells, thus limiting the change in traction force, suggesting that gap junctions play a crucial role in signal transduction. On the other hand, to assess the effect of cell contractility on the spreading behavior of TNT-connected cells, this experiment used DMEM culture medium containing the myosin II inhibitor Blebbistatin to reduce cell contractility. Under these conditions, the average spreading area of ​​TNT-connected cells before stimulation was 491.44 μm. 2 The value after stimulation was 491.07 μm. 2 ,like Figure 7 As shown in (e) and (f), no significant differences were observed. These results suggest that when cell contractility is inhibited, mechanical stimulation no longer significantly affects the spreading behavior of TNT-connected cells. In summary, gap connexins play an important role in the transcellular propagation of TNT-mediated electrical signals, and their blockade will inhibit changes in traction force; while cell contractility may be involved in regulating the spreading response of TNT-connected cells to mechanical stimulation, providing important clues for further elucidating the coupling mechanism between cellular mechanical behavior and signal transduction.

[0048] Figure 7 (a) Tether force distribution of TNT-connected cells before mechanical stimulation (extracellular solution: DMEM + MFA); (b) Tether force distribution of TNT-connected cells after mechanical stimulation; (c) Bright-field image of cells receiving mechanical stimulation during traction force measurement; (d) Statistical comparison of traction force of TNT-connected cells before and after mechanical stimulation (n = 20); (e) Bright-field image (extracellular solution: DMEM + Blebbistatin) showing morphological changes of TNT-connected cells before mechanical stimulation (magenta outline) and after stimulation (yellow outline); (f) Statistical comparison of spread area of ​​TNT-connected cells before and after mechanical stimulation (n = 20).

[0049] Tunnel nanotubes (TNTs) are unique long-distance intercellular connection structures, providing a distinctive pathway for directional and long-range intercellular communication. Through TNT structures, cells can transmit various signals, including electrical and biochemical signals, enabling coordinated responses across spatial distances. This study aims to investigate how TNT-mediated electrical signal transduction affects the traction force and spreading area of ​​connected cells, thereby deepening our understanding of TNT-mediated cell communication mechanisms.

[0050] The research results indicate that Ca 2+TNTs play a crucial regulatory role in cellular responses to mechanical stimuli. Specifically, blocking extracellular calcium influx or intracellular calcium release inhibits mechanically stimulated TNT-mediated electrotransduction, thus preventing the expected changes in traction force and spreading area between connected cells. This result highlights the indispensable role of calcium ions in TNT-mediated electrotransduction, participating in signal transmission and regulating cellular responses to mechanical stimulation. Furthermore, when both extracellular calcium influx and intracellular calcium release are simultaneously inhibited, the electrocoupling between C2C12 cells via TNTs is almost completely disrupted, further emphasizing the crucial role of calcium ions. 2+ Its central role in TNT signal transmission.

[0051] Furthermore, even with sufficient extracellular calcium concentration, blocking intracellular calcium release significantly weakens cellular mechanoresponsiveness. This suggests that effective cellular regulation of mechanosignals depends on the dynamic balance between intracellular and extracellular calcium sources. Intracellular calcium storage, particularly the calcium reservoir from the endoplasmic reticulum, may serve as a crucial buffer system for regulating intracellular calcium concentration in response to changes in external mechanical stimuli. This calcium homeostasis mechanism enables cells to precisely regulate their mechanical behavior, further highlighting the critical role of calcium homeostasis in TNT-mediated mechanotransduction.

[0052] The findings highlight the distinct roles of T-type and L-type calcium channels in TNT-mediated cellular responses. Activation of T-type calcium channels is crucial for TNT signaling, and extracellular calcium... 2+ Entering cells through these channels significantly altered traction and cell spreading area. Conversely, when T-type calcium channels were inactive, electrical signal transduction was blocked, and the changes in traction and spreading area disappeared. Blocking L-type calcium channels had no significant effect on the response of TNT-connected cells to mechanical stimulation, further highlighting the specific mechanoregulatory role of T-type calcium channels in TNT signaling. These results suggest that T-type calcium channels may have evolved specific functions in specific cellular environments to promote calcium-dependent mechanotransduction, thereby enhancing the responsiveness of TNT-connected cells to mechanical stimulation.

[0053] The gap junction protein Connexin 43 (Cx43) plays a crucial role in TNT-mediated electro-signal transduction. Impaired Cx43 function not only weakens the communication capabilities of gap junctions but may also indirectly reduce the efficiency of TNT-mediated electro-signal transduction, thereby affecting mechanical responses such as traction and cell spreading. This study further confirms that gap junctions play a key role in regulating cellular traction during TNT signaling, and inhibiting their function weakens cellular responses to mechanical stimuli. This suggests that Cx43 not only supports TNT signaling through direct electrocoupling but may also enhance the sensitivity of TNT-connected cells to mechanical signals by regulating downstream signaling pathways.

[0054] Furthermore, under conditions inhibiting cell contractility, the spreading response of TNT-connected cells to mechanical stimuli almost ceased. Although TNT-mediated signals could be transmitted to connected cells, the cell spreading area did not change significantly. These results suggest that cell contractility may provide necessary structural support for TNT-mediated morphological changes. In cells lacking sufficient contractility, the cytoskeleton may be insufficient to support effective mechanotransduction, thereby reducing the responsiveness to mechanical stimuli.

[0055] The key design focus of this invention is to reveal the significant impact of intercellular electrical signal transduction on the function of TNTs in connecting cells, using the research methods provided in this application. It highlights the crucial roles of calcium ions, specific types of calcium channels, gap junction proteins, and cell contractility in TNT-mediated mechanotransduction. This not only deepens our understanding of the mechanisms of TNT-mediated cell communication but also provides a theoretical foundation for further exploration of TNT signaling regulation mechanisms. In the future, in-depth research in this field is expected to provide new ideas for therapeutic strategies targeting abnormal cell communication and mechanotransduction. This application constructs a complete research system integrating "directional mechanical stimulation - specific drug intervention - dual-channel mechanical response quantitative measurement". By combining precise patch-clamp manipulation with traction force microscopy and morphological analysis, it overcomes the limitations of traditional methods in performing targeted and quantitative mechanical intervention and simultaneous multi-parameter detection on TNT networks, providing an unprecedentedly precise tool for studying intercellular mechanical communication. This method systematically reveals the core role and specific pathway of calcium ion signaling in TNT mechanotransduction. By quantifying complex cell mechanical behavior into two clear and measurable parameters, namely "traction force change" and "spreading area change", and combining them with a specific signaling pathway inhibitor toolkit, this method provides an efficient and standardized platform for screening drugs that regulate TNT function.

[0056] 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 method for studying the regulation mechanism of intercellular mechanical signal transduction mediated by a tunnel nanotube, characterized in that: The method comprises the following steps: S1, providing tunnel nanotube-connected cells grown on a polyacrylamide hydrogel substrate; S2, drug treatment of the tunnel nanotube-connected cells: applying an effective dose of at least one signal pathway inhibitor selected from one or more of a gap junction inhibitor, a calcium channel inhibitor, a mechanically sensitive ion channel inhibitor, an extracellular calcium remover, an intracellular calcium release inhibitor, or a cell contractility inhibitor to a system in which the tunnel nanotube-connected cells are cultured; S3, mechanical stimulation of the tunnel nanotube-connected cells: applying a directional mechanical stimulation to the target cells by a micromanipulator controlled by a patch clamp system; S4, measuring a biological response induced by the mechanical stimulation and occurring in the tunnel nanotube-connected cells; the biological response includes a change in cell traction force and a change in cell spreading area.

2. The method according to claim 1, wherein the method is characterized by: In the step S1, the C2C12 cells are cultured in a culture medium added with 10% fetal bovine serum and 100 U / mL penicillin-streptomycin complex antibiotics; the C2C12 cells are cultured in a 37°C, 5% CO2 humidified incubator.

3. The method according to claim 1, wherein the method is characterized by: The preparation of the polyacrylamide hydrogel substrate in the step S1 comprises the following steps: S11, substrate pretreatment: subjecting the bottom glass sheet of a confocal culture dish to plasma treatment, and then sequentially modifying the bottom glass sheet with a NaOH solution, an APTES ethanol solution, and a glutaraldehyde solution to form an activated substrate surface; S12, gel solution preparation: preparing a polyacrylamide gel premix solution containing fluorescent microspheres; S13, gel polymerization: dropping the obtained gel premix solution onto the activated substrate, and covering the gel with a silanized cover glass, polymerizing in the dark to form a gel layer; S14, post-treatment and protein crosslinking: removing the cover glass, washing the gel, and then activating the gel surface under ultraviolet light irradiation using a photoactivatable crosslinking agent, and subsequently crosslinking extracellular matrix proteins to the activated gel surface.

4. The method according to claim 3, wherein the method is for studying the mechanism of the regulation of the intercellular mechanical signal transduction mediated by the tunnel nanotube. In the step S11, the volume fraction of the APTES ethanol solution is 50%, and the mass-volume concentration of the glutaraldehyde solution is 0.5%; in the step S13, the cover glass is treated with a silane release agent to facilitate the separation of the cover glass from the gel after polymerization; in the step S14, the photoactivatable crosslinking agent is Sulfo-SANPAH, which is diluted in a HEPES buffer at a dilution ratio of 1:100, and the extracellular matrix protein is type I collagen, which is crosslinked at a concentration of 0.2 mg / mL.

5. The method according to claim 1, wherein the method is characterized by: In the step S2, the gap junction inhibitor is meclofenamic acid, which is used at a concentration of 100 μM and for a treatment time of 40 minutes; the calcium channel inhibitor includes a T-type calcium channel inhibitor and an L-type calcium channel inhibitor; the T-type calcium channel inhibitor is mibefradil, which is used at a concentration of 1 μM; the L-type calcium channel inhibitor is nifedipine, which is used at a concentration of 10 μM.

6. The method according to claim 1, wherein the method is characterized by: The mechanical sensitive ion channel inhibitor in step S2 is gadolinium ion, and the using concentration is 100 μM; the extracellular calcium removal agent is calcium-free Hanks balanced salt solution, and the processing time is 30 minutes; the intracellular calcium release inhibitor is 1 μM rapamycin combined with 15 μM 2-phenylboronic acid ethylamine, and the cell is treated at room temperature for 25 minutes; the cell contraction inhibitor is Blebbistatin, and the working concentration is 3 μM, and the processing time is 30 minutes.

7. The method for studying the regulatory mechanism of intercellular mechanotransduction mediated by tunnel nanotubes according to claim 1, characterized in that: The patch clamp system in step S3 is Axopatch 200B system, and the glass microelectrode installed on the micromanipulator needs to be positioned above the target cell and ensure no bubble interference before mechanical pressure is applied.

8. The method according to claim 1, wherein the method is characterized by: The measurement of cell traction force in step S4 is realized by Fourier transform traction force microscopy; the measurement of cell spreading area includes the following steps: obtaining the bright field images of the same cell before and after mechanical stimulation is applied; using image analysis software, the cell boundaries before and after stimulation are respectively marked; the change of cell spreading area before and after mechanical stimulation is calculated and compared.

9. A system for performing the method of investigating the regulatory mechanism of the tunneling nanotube-mediated intercellular mechanical signal transduction according to any one of claims 1 to 8, characterized in that, The mechanical stimulation module for applying directional mechanical pressure to the target cell, the mechanical response measurement module for measuring cell traction force by Fourier transform traction force microscopy, and the morphological analysis module for quantitatively analyzing the change of cell spreading area; the mechanical stimulation module includes a patch clamp system, a micromanipulator and a glass microelectrode; the mechanical response measurement module includes a confocal microscope and an image processing unit; the morphological analysis module includes image analysis software.

10. Use of a signal pathway inhibitor used in the method of studying the mechanism of regulation of intercellular mechanical signal transduction mediated by nanotubules according to any one of claims 1 to 8 in the preparation of a kit for studying cell mechanical transduction, characterized in that: The kit contains one or more signal pathway inhibitors.

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