Method and device for monitoring dynamic response of cell nucleus to perceive extracellular mechanical stimulation
By designing a mechanical control system that includes single -cell compression, the device of the constant temperature hydraulic mechanics control experimental platform and the fluorescent micro imaging system, monitor the dynamic response of the nucleus perception of external mechanical stimuli, solve the difficulty of monitoring cell perception mechanical mechanical mechanical mechanical mechanical mechanical research The problem of stimulus mechanism realizes real -time monitoring and research on the cell response mechanism.
Patent Information
- Application Number
- CN202411455335.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to effectively monitor how cells perceive external mechanical stimulation and adaptively regulate their own behavior, especially in cell mechanics studies.
A device that monitors the dynamic response of an outerized mechanical stimulation of the cells, including the mechanical control system for single -cell compression, the constant temperature hydraulic mechanical control experimental platform, and the fluorescent microscopy imaging system. Through this device, a single live cells can be compressed at Norton's mechanical scale, and the cell morphological changes and the nuclear dynamic process of YAP protein can be monitored in real time.
Real -time monitoring of the mechanism of cell perception external mechanical stimulation and response, revealing how cells perceive and adapt to external mechanical changes through nucleus, thereby providing new insights for intervention of cancer cell metastasis and immune cell response.
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Figure CN119331722A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of in vivo cell research, and in particular to a method and device for monitoring the dynamic response of a cell nucleus to extracellular mechanical stimulation. Background Art
[0002] Cells in the human body are in a highly crowded microenvironment, and there are complex and difficult-to-measure forces between cells and neighboring cells and the extracellular microenvironment (ECM). Especially during tumor formation, the failure of cell growth and proliferation regulation makes the mechanical connection between cells and cells and cells and ECM closer. Cells have the ability to sense external mechanical signals and adaptively adjust their biochemical behavior, and the disorder of this mechanical response ability and the disorder of related signal transduction pathways often lead to the occurrence and development of various diseases (such as cancer). Studies have found that cells rely on the nucleus as a ruler to regulate power, and respond by sensing and generating signals used to change cell behavior through the nucleus to ensure that cells can adapt, survive and effectively pass through crowded and mechanically heterogeneous microenvironments. This process is related to cancer cell invasion, immune response and embryonic development. Therefore, exploring the mechanism of how single living cells perceive external mechanical constraints and adaptively adjust their own behavior has become one of the important topics in the study of cell mechanics.
[0003] To this end, we proposed a method and device to monitor the dynamic response of the cell nucleus to extracellular mechanical stimulation to solve the above problems. Summary of the invention
[0004] The purpose of the present invention is to provide a method and device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation, so as to solve the problems raised in the above-mentioned background technology.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] A device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation mainly includes three parts: a mechanical control system for single cell compression, a constant temperature liquid phase mechanical manipulation experimental platform, and a fluorescence microscopy imaging system.
[0007] A 37°C constant temperature controller is provided on one side of the constant temperature liquid phase mechanics manipulation experimental platform;
[0008] The mechanical manipulation system of single-cell compression is composed of a functional probe for single-cell mechanical manipulation and a mechanical feedback control loop. The mechanical manipulation system of single-cell compression uses a laser beam emitted by a 1# laser through a 1# converging lens and a 1# reflecting prism to hit the back of the functional probe to complete reflection. The reflected light enters the mechanical disturbance sensor and then enters the mechanical feedback controller. By comparing and feeding back the initial setting value and the actual measurement value, the feedback signal is input into the centralized control system.
[0009] The fluorescence microscopy imaging system emits wavelength-adjustable laser light through the 2# laser, which is focused by the 3# converging lens and reflected by the 2# reflecting prism, split by the dichroic filter, and then enters the fluorescence microscope eyepiece for fluorescence signal excitation; the obtained fluorescence signal is sequentially collected, enhanced and processed through the laser filter, the 2# converging lens and the fluorescence signal enhancement and processor, and the obtained signal is input into the centralized control system.
[0010] In a further embodiment, the bottom of the constant temperature liquid phase mechanical manipulation experimental platform is composed of a type I collagen coating spread on a PAA hydrogel substrate with a hardness of 3 KPa.
[0011] In a further embodiment, the functionalized probe is a flat tipless silicon microcantilever with a metal plated backside.
[0012] In a further embodiment, the PAA hydrogel substrate is obtained by mixing acrylamide and methylene bisacrylamide in a certain ratio, and then incubated with type I collagen for modification.
[0013] Another object of the present invention is to provide a method for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation, which is characterized by comprising the following steps:
[0014] S1, 24 h before, the actin cytoskeleton, nucleus and YAP protein of Hela cells were fluorescently labeled;
[0015] S2, 1 hour before, trypsinize the resuspended Hela cells, with a concentration of about 1*10 5 The cells were seeded at a density of 1.57 μg / cm2 on a 35 mm type I collagen-coated 3 kPa PAA hydrogel substrate and placed in a cell culture incubator to complete cell adhesion.
[0016] S3. Move the fluorescently labeled and reattached cells to the constant temperature liquid phase mechanical manipulation experimental platform, and use a flat tipless microcantilever with precise control of disturbance to compress the single cell. Use an integrated fluorescence microscopy imaging system to record the morphology of the cells before compression and the localization information of YAP;
[0017] S4, then, a constant force of 1 nN was applied to the functionalized probe, the cell was compressed at a force loading rate of 0.5 μm / s, and a fluorescence image was recorded every 2 seconds;
[0018] S5. When the cell is compressed to 1 / 2 of the initial height of the nucleus (approximately half the height of the cell), stop force loading and record the fluorescence image for the last time. At the same time, pay attention to the morphology of the nucleus and cytoskeleton to prevent damage to the cell membrane and nuclear membrane caused by overload. If membrane damage occurs, the measurement results of the cell will not be included in the statistical queue.
[0019] In a further embodiment, the integrated fluorescence microscopy system needs to first fluorescently label the actin cytoskeleton, cell nucleus and YAP protein, and record a fluorescence image every 2 seconds from the beginning of cell compression. Assuming that the cell half-height is 10 microns and the force loading rate is about 0.5 microns / second, 10 fluorescence images will be recorded. According to the image information, the morphology and size changes of cells and cell nuclei during cell compression and the dynamic changes of YAP nuclear translocation can be monitored in real time.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention can compress a single living cell at a nanonewton (nN) mechanical scale, monitor the dynamic process of cell morphology changes and nuclear translocation of YAP (Yes-associated protein) protein in real time, and reveal the mechanism of cell perception of extracellular mechanical stimulation and response to mechanical stimulation by characterizing the dynamic process of YAP nuclear translocation (the ratio of fluorescence intensity of YAP protein in the nucleus and cytoplasm). The main methods adopted by the present invention are as follows: first, a functionalized probe that can be used to achieve nanonewton precision single-cell mechanical manipulation is artificially modified; and a 3KPa PAA hydrogel substrate coated with type I collagen is prepared to construct a constant temperature liquid phase mechanical manipulation experimental platform that simulates human physiological conditions; under the experimental platform, a fluorescence microscopy imaging system is integrated to monitor the dynamic response behavior of living cells under single-cell mechanical manipulation in real time. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 A partial structural schematic diagram of the device of the present invention is shown;
[0023] Figure 2 A schematic diagram of the process and steps for monitoring YAP nuclear translocation in the present invention is shown.
[0024] In the figure, 1. Mechanical disturbance sensor; 2. Mechanical feedback controller; 3. 1# reflecting prism; 4. 1# converging lens; 5. 1# laser; 6. Constant temperature liquid phase mechanical manipulation experimental platform; 7. Functionalized probe; 8. Type I collagen coating; 9. PAA hydrogel substrate; 10. Constant temperature controller; 11. Fluorescence microscope eyepiece; 12. Dichroic filter; 13. 2# reflecting prism; 14. Laser filter; 15. 2# converging lens; 16. 3# converging lens; 17. Fluorescence signal enhancement and processor; 18. 2# laser; 19. Centralized control system. DETAILED DESCRIPTION
[0025] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0026] The main experimental process of the present invention is as follows: First, the Hela cells resuspended in trypsin were digested with about 1*10 5 The cells were seeded at a density of 1.577 W / m2 on a 35 mm type I collagen coated with a 3 kPa PAA hydrogel substrate and placed in a cell culture incubator for 1 hour to complete cell adhesion. Then, the cells were moved to a constant temperature liquid phase mechanical manipulation experimental platform, and a flat tipless microcantilever with precisely controlled perturbations was used to compress the single cells, loading them with a constant force of about 1 nN until they were compressed to 1 / 2 of the initial height of the nucleus. Furthermore, by fluorescently labeling the nucleus, YAP protein, and actin cytoskeleton, an integrated fluorescence microscopy imaging system was used to monitor in real time the morphology and size changes of cells and nuclei during cell compression, as well as the dynamic process of YAP nuclear translocation. The experiment found that in the early stage of cell compression, the nucleus was round and the volume did not change significantly, and YAP was mainly distributed in the cytoplasm; with further compression, the actin microfilament skeleton was reconstructed, the nucleus began to deform, the height / width ratio decreased, and the volume increased, triggering YAP nuclear translocation; when the cell was compressed to 1 / 2 of the initial height of the nucleus, the nucleus further deformed, and the nuclear translocation reached the threshold, and further compression may cause damage to the cell membrane. It is confirmed that the cell nucleus can sense external mechanical stimuli through the remodeling of the actin microfilament skeleton and the coupling of related adaptor proteins, and adaptively adjust the volume of the cell nucleus, causing the nuclear membrane to wrinkle and stretch, triggering the opening of nuclear pores, thereby promoting the nuclear translocation of YAP protein. The present invention realizes real-time monitoring of the dynamic process of YAP protein nuclear translocation in a constant temperature liquid phase mechanical manipulation experimental platform that simulates physiological conditions by modifying a functional probe for single-cell mechanical manipulation with nanonewton accuracy and integrating a fluorescence microscopy imaging system. The present invention can reveal how cells sense and respond to external mechanical stimuli from the nanonewton mechanical scale, and through the dynamic monitoring of YAP nuclear translocation, it can provide new insights into extracellular microenvironmental targets that intervene in cancer cell metastasis and immune cell responses.
[0027] The details are as follows:
[0028] A device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation, such as Figure 1 As shown, it mainly includes three parts: a mechanical control system for single-cell compression, a constant-temperature liquid-phase mechanical manipulation experimental platform, and a fluorescence microscopy imaging system.
[0029] The details are as follows:
[0030] The bottom of the constant temperature liquid phase mechanical manipulation experimental platform 6 is composed of a type I collagen coating 8 spread on a PAA hydrogel 9 with a hardness of 3 KPa, and is equipped with a 37° C. constant temperature controller 10, allowing the measurement of the cell samples to be tested under physiological conditions.
[0031] The mechanical manipulation system for completing single-cell compression is composed of a functional probe 7 for single-cell mechanical manipulation and a mechanical feedback control loop. In order to achieve mechanical control with nanonewton accuracy, the laser beam emitted from the 1# laser 5 through the 1# converging lens and the 1# reflecting prism 3 hits the back of the functional probe 7 to complete reflection. The reflected light enters the mechanical disturbance sensor 1 and then enters the mechanical feedback controller 2. Through the comparative feedback of the initial setting value and the actual measured value, the feedback signal is input into the centralized control system 19, thereby completing the cell mechanical manipulation with nanonewton accuracy.
[0032] A fluorescence microscopy imaging system for real-time monitoring of cell dynamic responses is integrated under the platform. The system emits wavelength-adjustable laser light from the 2# laser 18, which is focused by the 3# converging lens 16 and reflected by the 2# reflecting prism 13, split by the dichroic filter 12, and then enters the fluorescence microscope eyepiece 11 for fluorescence signal excitation; the obtained fluorescence signal is collected, enhanced and processed by the laser filter 14, the 2# converging lens 15 and the fluorescence signal enhancement and processor 17 in turn, and the obtained signal is input into the centralized control system 19.
[0033] The functionalized probe 7 is a flat silicon microcantilever without a tip, and the back of the cantilever is plated with metal to achieve laser emission. The mechanical disturbance sensor can accurately measure the position disturbance information of the emitted laser, and then compare the initial input value with the actual measured value in the mechanical feedback controller 2, thereby completing the cell mechanical manipulation with nanonewton accuracy.
[0034] Among them, the constant temperature liquid phase mechanical manipulation experimental platform 6, its 3KPa PAA hydrogel substrate 9 is obtained by adjusting the mixing ratio of acrylamide and methylene bisacrylamide, and then incubating type I collagen for modification. In order to simulate the physiological environment suitable for cell growth, the platform is equipped with a constant temperature controller 10 with a temperature of 37°C, and the temperature control signal is also adjusted by the centralized control system 19.
[0035] Among them, the integrated fluorescence microscopy system, according to the experimental objectives, needs to first fluorescently label the actin cytoskeleton, cell nucleus and YAP protein, and record a fluorescence image every 2 seconds from the beginning of cell compression. Assuming that the cell half-height is 10 microns and the force loading rate is about 0.5 microns / second, 10 fluorescence images will be recorded. Based on the image information, the morphology and size changes of cells and nuclei during cell compression and the dynamic changes of YAP nuclear translocation can be monitored in real time.
[0036] Based on the device, the specific process and steps of using single cell mechanical manipulation technology to monitor the dynamic response of living cells are as follows ( Figure 2 shown):
[0037] (1) 24 h before the experiment, the actin cytoskeleton, nucleus and YAP protein of Hela cells were fluorescently labeled.
[0038] (2) One hour before the experiment, trypsin was used to digest the resuspended Hela cells at a concentration of approximately 1 × 10 5 The cells were seeded at a density of 35 mm type I collagen coated with 3 kPa PAA hydrogel substrate and placed in a cell culture incubator to complete cell adhesion.
[0039] (3) The experiment was started. The fluorescently labeled and reattached cells were moved to the constant temperature liquid phase mechanical manipulation experimental platform, and single cell compression was performed using a flat tipless microcantilever with precisely controlled perturbations. An integrated fluorescence microscopy system was used to record the morphology of the cells before compression and the localization information of YAP (e.g. Figure 2 as shown in step 1).
[0040] (4) Then, a constant force of 1 nN was applied to the functionalized probe, and the cell was compressed at a force loading rate of 0.5 μm / s, and the fluorescence image was recorded every 2 seconds (e.g. Figure 2 as shown in step 2).
[0041] (5) Finally, when the cell is compressed to 1 / 2 of the initial height of the nucleus (approximately half the height of the cell), the force loading is stopped and the fluorescence image is recorded for the last time. Figure 2 (as shown in step 3) At the same time, pay attention to the morphology of the cell nucleus and cytoskeleton to prevent damage to the cell membrane and nuclear membrane due to overload. If membrane damage occurs, the measurement results of the cell will not be included in the statistical queue.
[0042] (6) Through the above steps, the dynamic response of living cells (YAP nuclear translocation) can be monitored based on single-cell mechanical manipulation technology.
[0043] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
[0044] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation, characterized in that: It mainly consists of three parts: a mechanical control system for single-cell compression, a constant-temperature liquid-phase mechanical manipulation experimental platform (6), and a fluorescence microscopy imaging system. A 37°C constant temperature controller (10) is provided on one side of the constant temperature liquid phase mechanics manipulation experimental platform (6); The mechanical manipulation system for single-cell compression is composed of a functional probe (7) for single-cell mechanical manipulation and a mechanical feedback control loop. The mechanical manipulation system for single-cell compression uses a laser beam emitted by a 1# laser (5) through a 1# converging lens (4) and a 1# reflecting prism (3) to hit the back of the functional probe (7) for reflection. The reflected light enters a mechanical disturbance sensor (1) and then enters a mechanical feedback controller (2). By comparing and feeding back the initial setting value and the actual measured value, the feedback signal is input into a centralized control system (19). The fluorescence microscopy imaging system emits wavelength-adjustable laser light through the 2# laser (18), which is focused by the 3# converging lens (16) and reflected by the 2# reflecting prism (13), split by the dichroic filter (12), and then enters the fluorescence microscope eyepiece (11) for fluorescence signal excitation; the obtained fluorescence signal is sequentially passed through the laser filter (14), the 2# converging lens (15) and the fluorescence signal enhancement and processor (17) to complete the collection, enhancement and processing of the fluorescence signal, and the obtained signal is input into the centralized control system (19).
2. The device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation according to claim 1, characterized in that: The bottom of the constant temperature liquid phase mechanical manipulation experimental platform (6) is composed of a type I collagen coating (8) spread on a PAA hydrogel substrate (9) with a hardness of 3 KPa.
3. The device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation according to claim 1, characterized in that: The functionalized probe (7) is a flat silicon microcantilever without a tip, and the back side of the cantilever is plated with metal.
4. The device for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation according to claim 2, characterized in that: The PAA hydrogel substrate (9) is obtained by mixing acrylamide and methylene bisacrylamide in a certain ratio, and then incubated with type I collagen for modification.
5. A response method based on the dynamic response device for monitoring cell nuclei sensing extracellular mechanical stimulation according to any one of claims 1 to 4, characterized in that: The steps include: S1, 24 h before, the actin cytoskeleton, nucleus and YAP protein of Hela cells were fluorescently labeled; S2, 1 hour before, trypsinize the resuspended Hela cells, with a concentration of about 1*10 5 The cells were seeded at a density of 1.57 μg / cm2 on a 35 mm type I collagen-coated 3 kPa PAA hydrogel substrate and placed in a cell culture incubator to complete cell adhesion. S3. Move the fluorescently labeled and reattached cells to the constant temperature liquid phase mechanical manipulation experimental platform, and use a flat tipless microcantilever with precise control of disturbance to compress the single cell. Use an integrated fluorescence microscopy imaging system to record the morphology of the cells before compression and the localization information of YAP; S4, then, a constant force of 1 nN was applied to the functionalized probe, the cell was compressed at a force loading rate of 0.5 μm / s, and a fluorescence image was recorded every 2 seconds; S5. When the cell is compressed to 1 / 2 of the initial height of the nucleus (approximately half the height of the cell), stop force loading and record the fluorescence image for the last time. At the same time, pay attention to the morphology of the nucleus and cytoskeleton to prevent damage to the cell membrane and nuclear membrane caused by overload. If membrane damage occurs, the measurement results of the cell will not be included in the statistical queue.
6. The method for monitoring the dynamic response of cell nuclei to extracellular mechanical stimulation according to claim 5, characterized in that: The integrated fluorescence microscopy imaging system needs to first fluorescently label the actin cytoskeleton, cell nucleus and YAP protein, and record a fluorescence image every 2 seconds starting from cell compression. Based on the image information, the morphology and size changes of cells and cell nuclei as well as the dynamic changes of YAP nuclear translocation during cell compression can be monitored in real time.