Nerve activator for regulating and controlling opening of MSCs (mesenchymal stem cells) of nerve cell membrane and application of nerve activator

Abstract

The invention provides a nerve activator for regulating and controlling opening of MSCs of a nerve cell membrane and application, and belongs to the technical field of nerve activation. The nerve activator is composed of an optical tweezers system and microparticles. According to the invention, the optical tweezers system is utilized to convert dynamic light force into vibration mechanical force through a micron particle medium, then the vibration mechanical force accurately acts on the neuron cell membrane, opening of a mechanical sensitive ion channel on the surface of the neuron cell membrane is dynamically regulated and controlled, and equivalent internal flow of calcium ions is realized, so that accurate activation and function regulation and control of neurons in subcellular precision are completed. The method for regulating and controlling nerves by using the nerve activator has the characteristics of no contact, flexibility and accuracy, is expected to provide a non-genetic optical method for high-precision nerve regulation and control, and provides technical support for understanding neuron interaction and neurodegenerative disease pathogenesis and treatment from the subcellular level.

Description

Technical Field

[0001] The present invention belongs to the technical field of nerve activation, and particularly relates to a nerve activator that regulates MSCs on the nerve cell membrane and its application. Background Art

[0002] Mechanosensitive Ion Channels (MSCs) are common ion channels on the nerve cell membrane, which are pore proteins that respond to mechanical force stimuli. When the channels are opened under the stimulation of mechanical force (such as tensile force, pressure, shear force), substances (mainly ions) can be transported across the membrane, converting the mechanical force signal into an electrical signal or a chemical signal within milliseconds, and then triggering a series of cellular reactions.

[0003] Calcium signaling is an important mechanism for cells to regulate various life activities. Neurons regulate various biological processes, such as the regulation of action potentials, the release of neurotransmitters, axon growth, and nerve regeneration, etc., through the influx of extracellular calcium ions (Ca 2+ ) and the release of Ca 2+ from the endoplasmic reticulum. According to the calcium hypothesis of Alzheimer's disease, neurodegenerative diseases such as Alzheimer's disease can be treated by regulating calcium ion signaling. Neurons are the most basic structural and functional units in the nervous system. Abnormal calcium ion signaling in local neurons can also lead to abnormal nervous system function and cause various serious nervous system diseases. Therefore, achieving local nerve activation is of great significance for promoting the development of new nervous system treatment technologies.

[0004] Currently, the classic methods for treating nerve diseases clinically include transcranial magnetic stimulation (TMS), transcranial electrical stimulation (TES), drug treatment, etc. These technologies directly act on the patient's brain through electrodes or magnetic fields to generate current to stimulate brain cells, usually belonging to large-area stimulation and unable to achieve precise stimulation of single cells. For example, in the patent application No. CN118543036A, a transcranial magnetic stimulation system and its stimulation method were invented. By predicting the required stimulation intensity through three steps: pre-stimulation, predicting neuron action potentials, and formal stimulation, the appropriate magnetic stimulation for the brain can be accurately given to make the neuron action potential threshold reach the required value. However, due to the individual differences of the human body and the complexity of the brain structure, the positioning of the stimulation site by TMS is still not precise enough; and as the depth of the brain increases, the intensity of the magnetic field generated by TMS gradually attenuates when passing through the skull, resulting in limited stimulation depth of the brain. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a nerve activator that regulates MSCs on the nerve cell membrane and its application, which can achieve precise activation of single nerve cells.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a nerve activator for regulating the opening of mechanically sensitive ion channels in the nerve cell membrane, and the nerve activator is composed of an optical tweezers system and microparticles.

[0008] Preferably, the microparticles include silica particles or polystyrene microspheres.

[0009] Preferably, the diameter of the microparticles is 0.5 - 2 μm.

[0010] The present invention provides an application of an optical tweezers system or the above-mentioned nerve activator in the preparation of a product for regulating the opening of mechanically sensitive ion channels in the nerve cell membrane.

[0011] The present invention provides an application of an optical tweezers system or the above-mentioned nerve activator in the preparation of a product for regulating calcium ion signals.

[0012] Preferably, the nerve cells include SD fetal rat neuron cells, mouse hippocampal neuron cells, rat primary sensory neuron cells, rat adrenal pheochromocytoma cells or mouse cerebral cortex cells.

[0013] The present invention provides a method for regulating the opening of mechanically sensitive ion channels in the nerve cell membrane by using the above-mentioned nerve activator, including the following steps:

[0014] Add a microparticle solution to nerve cells, establish a first optical potential well by using the optical tweezers system, capture the microparticles with the optical potential well, make the microparticles attach to the nerve cell membrane, and then establish a second optical potential well by using the optical tweezers system to stimulate the nerve cell membrane, thereby regulating the opening of the mechanically sensitive ion channels on the surface of the nerve cell membrane.

[0015] Preferably, the number of the nerve cells is 4 - 6×10 4 ; the microparticle solution is prepared by diluting a 2 - 3% silica microsphere ethanol suspension by volume with PBS to 90 - 110 times; the volume of the microparticle solution is 1 - 3 μL.

[0016] Preferably, the first optical potential well is a single optical potential well, and the scanning frequency is 9500 - 10000 Hz; the second optical potential well is two or more optical potential wells, and the scanning frequency is 50 - 500 Hz.

[0017] Preferably, the time for the optical potential well to capture the microparticles and make the microparticles attach to the nerve cell membrane is 0.5 - 1.5 min.

[0018] Preferably, the optical tweezers system is provided with a laser and an acousto-optic deflector that emit a laser beam of 1064 nm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a nerve activator for regulating the opening of mechanically sensitive ion channels on the nerve cell membrane and its application. The present invention uses an optical tweezers system to convert dynamic optical force into vibrational mechanical force through a micron particle medium and then precisely act on the neuron cell membrane, dynamically regulating the opening of mechanically sensitive ion channels on the surface of the neuron cell membrane, realizing the influx of calcium ions, etc., thereby completing the precise activation and functional regulation of neurons at the subcellular level. The method of regulating nerves using the above nerve activator has the characteristics of non-contact, flexibility and precision, and is expected to provide a new non-genetic optical method for high-precision nerve regulation, and provide new technical support for understanding neuron interaction and the pathogenesis and treatment of neurodegenerative diseases at the subcellular level. Description of the Drawings

[0021] Figure 1 Schematic diagram of the beam path structure of the optical tweezers system device;

[0022] Figure 2 Schematic diagram of the principle of activating and regulating neuron cells of the present invention;

[0023] Figure 3 Bright-field image of the cell membrane oscillation at different positions of neuron cells under a 60-fold microscope, where the red-marked area is the silica particles captured by the optical tweezers system;

[0024] Figure 4 Results of the change in calcium fluorescence signal in neuron cells monitored by calcium fluorescence imaging technology before and after stimulating the cell membrane to regulate the mechanically sensitive ion channels on the neuron cell membrane;

[0025] Figure 5 Local electrical signal changes at the stimulation site of neuron cells monitored by patch clamp technology before and after stimulating the cell membrane to regulate the mechanically sensitive ion channels on the neuron cell membrane. Detailed Embodiments

[0026] The present invention provides a nerve activator for regulating the opening of mechanically sensitive ion channels on the nerve cell membrane, and the nerve activator is composed of an optical tweezers system and microparticles.

[0027] In the present invention, the optical tweezers system is a well-known test platform, preferably Tweez250 high-speed multi-trap nano-optical tweezers and test platform, brand Aresis. The most important devices in the optical tweezers system are lasers, condensers, acousto-optic deflectors (AODs), and beam expanders, which are used to form focused light and light trap arrays for capture. Among them, the laser emits a 1064nm laser beam, which is modulated by the AOD to generate a light trap array to capture and manipulate particles for capturing biological cells. During the research process of the present invention, it was found that the capture under a 60x objective lens is the most stable, and the camera view is the clearest, so the optical tweezers system of the present invention is preferably provided with a 60x objective lens. However, the light trap formed by the light beams under the 40x and 20x objective lenses assembled in the optical tweezers system device is not ideal for capturing sample particles, which is not conducive to achieving precise control.

[0028] In the present invention, the microparticles preferably include silica particles or polystyrene microspheres, and more preferably silica particles. The present invention does not specifically limit the source of silica particles or polystyrene microspheres, and they can be prepared by methods known in the art or commercially available products, such as silica particles purchased from aladdin / Aladdin, item number: M120356-5mL, CAS number: 7631-86-9. The diameter of the microparticles is preferably 0.5 to 2 μm, more preferably 1 to 1.5 μm, and more preferably 1 μm. The present invention utilizes an optical tweezers system to accurately act on nerve cells through microparticle media such as silica particles, and dynamically regulates the opening of mechanically sensitive ion channels on the surface of nerve cell membranes.

[0029] The present invention provides an application of an optical tweezers system or the above-mentioned neural activator in preparing a product for regulating the opening of mechanically sensitive ion channels of nerve cell membranes.

[0030] The present invention provides an optical tweezers system or application of the above-mentioned neural activator in preparing a product for regulating calcium ion signals.

[0031] In the above application, the neural cells preferably include SD fetal rat neural cells, mouse hippocampal neural cells, rat primary sensory neural cells, rat adrenal pheochromocytoma cells or mouse cerebral cortex cells, and are further preferably SD fetal rat neural cells. The neural cells of the present invention respond significantly to the oscillation stimulation signal of the optical tweezers system.

[0032] The present invention provides a method for regulating the opening of a mechanosensitive ion channel of a nerve cell membrane using the above-mentioned nerve activator, comprising the following steps:

[0033] Add a microparticle solution to nerve cells, use the optical tweezers system to establish a first optical potential well, capture the microparticles with the optical potential, attach the microparticles to the nerve cell membrane, and then use the optical tweezers system to establish a second optical potential well to stimulate the nerve cell membrane, thereby regulating the opening of the mechanically sensitive ion channels on the nerve cell membrane surface.

[0034] In the present invention, a microparticle solution is added to nerve cells. The number of the nerve cells is 4 - 6×10 4 cells, more preferably 4.5 - 5.5×10 4 cells, and even more preferably 5×10 4 cells; the microparticle solution is prepared by diluting a silica microsphere ethanol suspension with a mass - volume fraction of 2 - 3% to 90 - 110 times with PBS; the volume of the microparticle solution is preferably 1 - 3 μL, more preferably 1.5 - 2.5 μL, and even more preferably 2 μL. The nerve cells preferably include SD fetal rat neuron cells, mouse hippocampal neuron cells, rat primary sensory neuron cells, rat adrenal pheochromocytoma cells or mouse cerebral cortex cells, and more preferably SD fetal rat neuron cells.

[0035] In the present invention, use the optical tweezers system to establish a first optical potential well. The optical potential well captures the microparticles and attaches the microparticles to the nerve cell membrane. Then use the optical tweezers system to establish a second optical potential well to stimulate the nerve cell membrane, thereby regulating the opening of the mechanically sensitive ion channels on the nerve cell membrane surface. After adding the microparticle solution in the present invention, immediately use the optical tweezers system to establish the first optical potential well and the second optical potential well. First, capture the microparticles with the optical potential well, and then use the optical potential well to stimulate the nerve cell membrane, thereby regulating the opening of the mechanically sensitive ion channels on the nerve cell membrane surface. The first optical potential well is a single optical potential well, and the scanning frequency is 9500 - 10000 Hz, more preferably 9600 - 10000 Hz, and even more preferably 10000 Hz. When establishing the first optical potential well, the setting of the scanning frequency can stably and quickly capture the particles and make them adhere to the cell membrane; in the present invention, the time for the optical potential well to capture the microparticles and attach the microparticles to the nerve cell membrane is preferably 0.5 - 1.5 min, and more preferably 1 min. The second optical potential well is preferably more than 2 optical potential wells, more preferably 2 optical potential wells, and the scanning frequency is preferably 50 - 500 Hz, more preferably 200 - 400 Hz, and even more preferably 200 or 400 Hz. When establishing the second optical potential well, the setting of the scanning frequency can accurately activate the opening of the mechanically sensitive ion channels on the surface of a single nerve cell. The optical tweezers system is provided with a laser and an acousto - optic deflector that emit a laser beam of 1064 nm, thereby generating an optical trap array.

[0036] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] In the following examples, the composition of the complete cell culture medium is 98% cell basal culture medium (purchased from OriCell, BNRO-03011) + 2% rat neuronal cell culture supplement (purchased from OriCell, RAXFN-04011).

[0039] The monodisperse silica microspheres were purchased from aladdin, with the item number: M120356-5mL and the CAS number: 7631-86-9.

[0040] The monodisperse silica microsphere solution is prepared by mixing monodisperse silica microspheres with anhydrous ethanol at 2.5% (w / v, g / L) to prepare a silica microsphere ethanol suspension, which is then diluted to 100 times the volume with PBS to obtain a monodisperse silica microsphere solution.

[0041] Example 1

[0042] A method for regulating the opening of mechanically sensitive ion channels on the surface of neuronal cell membranes based on dynamic photomechanical forces, comprising the following steps:

[0043] (1) Cell culture was performed in an incubator at 37°C, 5% CO2, and saturated humidity. SD fetal rat hippocampal neurons (OriCell, SHCFN-00001) were cultured in complete cell culture medium to a density of 40% to 60%, and cell experiments were then performed;

[0044] (2) Technical equipment and experimental device: The device used is an optical tweezers system (Tweez250 high-speed multi-trap nano-optical tweezers and test platform, brand Aresis). The schematic diagram of the beam path structure of the optical tweezers system is shown in Figure 1 As shown, it mainly includes LED lighting source, focusing device, sample, objective lens, dichroic mirror, CMOS, beam expander, laser, AOD and computer.

[0045] Among them, the LED provides an illumination light source, which is focused by a condenser lens and irradiated on the sample on the stage. The generated image information is transmitted to a complementary metal oxide semiconductor (CMOS) through an objective lens and a dichroic mirror. The objective lens used in this embodiment is a 60x objective lens. The laser emits a laser beam of 1064 nm, which is modulated by an acousto-optic deflector (AOD) to generate an optical trap array for capturing and manipulating particles, and is used to capture biological cells; the laser beam is collimated and focused into a parallel beam through a beam expander; the optical trap can be programmed and adjusted by a computer (PC).

[0046] (3) Dynamically regulate the cells under the conditions of 37 °C, 5% CO2, and saturated humidity: directly add 2 μL of a monodisperse silica particle microsphere solution with a diameter of 1 μm to the culture dish containing 2.5×10 4 cells / mL of SD rat hippocampal neurons (the total volume of the culture medium in this culture dish is 2 mL).

[0047] (4) After adding the monodisperse silica particle microsphere solution, immediately observe the cells under bright field, use the above-mentioned optical tweezers system to establish a single optical potential well, set the scanning frequency to 10000 Hz (i.e., the maximum value), place the silica particles captured by the optical trap on a single cell membrane, and wait for 1 min, so that the silica particles adhere to the cell membrane.

[0048] (5) Stimulate the cell membrane to regulate the opening of the mechanosensitive ion channels on the surface of the neuron cell membrane: The laser power and scanning frequency are input by the computer, the laser power is 0.1 W, the scanning frequency is set to 200 Hz, and 2 optical potential wells are set, then the generated oscillation frequency is 100 Hz.

[0049] By stimulating the cell membrane to regulate the opening of the membrane mechanosensitive ion channels, triggering changes in intracellular calcium ion concentration and membrane potential, so the calcium ion fluorescence imaging technology and the patch clamp technology are used to monitor this process in real time respectively. Among them, the schematic diagram of the principle of activating and regulating neuron cells is shown in Figure 2 .

[0050] Figure 3 The results show that silica particles can be successfully captured at different positions on the neuron cell membrane using the optical tweezers system.

[0051] When stimulating the cell membrane to regulate the mechanically sensitive ion channels on the surface of the neuron cell membrane, while simultaneously photographing the sequence of calcium ion fluorescence images inside the selected cell for the selected cell oscillation stimulation, the experimental group (Experimental) curve is obtained. Meanwhile, before stimulating the cell membrane to regulate the mechanically sensitive ion channels on the surface of the neuron cell membrane, a cell is found around the selected cell, and the control group (Control) curve is obtained by photographing the sequence of natural quenching of calcium ion fluorescence inside this cell. Among them, for both the experimental group and the control group, the exposure time is 600 ms and the gain is 1.0.

[0052] Figure 4 The results show that compared with the control group, the fluorescence signal in the experimental group is enhanced, indicating the opening of the calcium ion mechanically sensitive channel.

[0053] After stimulating the cell membrane to regulate the mechanically sensitive ion channels on the surface of the neuron cell membrane, the patch clamp technique is used to monitor the changes in the neuron cell membrane current signal. Under normal circumstances, when the calcium ion mechanically sensitive channel is not open, there is no current signal, and once it opens, there will be a change in the current signal.

[0054] As Figure 5 shown, the present invention uses an optical tweezer system to stimulate the cell membrane to regulate the mechanically sensitive ion channels on the surface of the neuron cell membrane, and obvious changes in the current signal occur, indicating the opening of the calcium ion mechanically sensitive channel.

[0055] Figures 3 to 5 The results show that by using the optical force generated by the optical tweezer system to attach silica particles to the neuron cell membrane, the opening of the mechanically sensitive ion channels on the surface of the neuron cell membrane can be dynamically regulated at different cell membrane oscillation positions, realizing precise activation and functional regulation at the subcellular precision.

[0056] Example 2

[0057] The difference between this example and Example 1 is as follows: To stimulate the cell membrane to regulate the opening of the mechanically sensitive ion channels on the surface of the neuron cell membrane: The laser power and scanning frequency are input by a computer. The laser power is 0.1 mW, the scanning frequency is set to 400 Hz, and 2 optical traps are set, then the generated oscillation frequency is 200 Hz. Other steps are the same as those in Example 1.

[0058] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A neural activator for regulating the opening of mechanically sensitive ion channels of nerve cell membranes, characterized in that: The neural activator is composed of an optical tweezers system and microparticles.

2. The neural activator according to claim 1, characterized in that: The microparticles include silica particles or polystyrene microspheres.

3. The neural activator according to claim 2, characterized in that: The diameter of the microparticles is 0.5-2 μm.

4. Use of an optical tweezers system or a neural activator as described in any one of claims 1 to 3 in the preparation of a product for regulating the opening of mechanically sensitive ion channels of nerve cell membranes.

5. Use of an optical tweezers system or a neural activator as described in any one of claims 1 to 3 in the preparation of a product for regulating calcium ion signals.

6. The use according to claim 4 or 5, characterized in that: The neural cells include SD fetal rat neuron cells, mouse hippocampal neuron cells, rat primary sensory neuron cells, rat adrenal pheochromocytoma cells or mouse cerebral cortex cells.

7. A method for regulating the opening of a mechanosensitive ion channel of a nerve cell membrane using the nerve activator according to any one of claims 1 to 3, characterized in that: The following steps are involved: A microparticle solution is added to the nerve cells, and the optical tweezers system is used to establish a first optical potential trap, which captures the microparticles so that the microparticles adhere to the nerve cell membrane. The optical tweezers system is then used to establish a second optical potential trap to stimulate the nerve cell membrane, thereby regulating the opening of the mechanically sensitive ion channels on the surface of the nerve cell membrane.

8. The method according to claim 7, characterized in that The number of the nerve cells is 4 to 6×10 4 The microparticle solution is prepared by diluting a silica microsphere ethanol suspension having a mass volume fraction of 2 to 3% with PBS to a volume of 90 to 110 times; the volume of the microparticle solution is 1 to 3 μL.

9. The method according to claim 7, characterized in that: The first optical potential well is a single optical potential well with a scanning frequency of 9500-10000 Hz; the second optical potential well is more than two optical potential wells with a scanning frequency of 50-500 Hz; the optical potential well captures microparticles, and the time for the microparticles to adhere to the nerve cell membrane is 0.5-1.5 minutes.

10. The method according to claim 7, characterized in that The optical tweezers system is provided with a laser emitting a 1064 nm laser beam and an acousto-optic deflector.

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