A nerve activator with MSCs opening of nerve cell membrane regulation and application

Abstract

The application provides a nerve activator for regulating opening of a MSC and application, and belongs to the technical field of nerve activation.The nerve activator is composed of an optical tweezer system and microparticles.The application utilizes the optical tweezer system to convert dynamic optical force into vibrating mechanical force through microparticle medium, and then precisely acts on a neuron cell membrane, dynamically regulates opening of a mechanically sensitive ion channel on the surface of the neuron cell membrane, realizes internal flow of calcium ions and the like, and thus completes precise activation and function regulation of the neuron at a subcellular precision.The nerve regulation mode of the application has the characteristics of non-contact and flexible precision, is expected to provide a non-genetic optical method for high-precision nerve regulation, and provides technical support for understanding neuron interaction and pathogenesis and treatment of neurodegenerative diseases from a subcellular level.

Description

Technical Field

[0001] This invention belongs to the field of neural activation technology, and in particular relates to a neural activator that regulates the activation of MSCs in the nerve cell membrane and its application. Background Technology

[0002] Mechanosensitive ion channels (MSCs) are common ion channels on the cell membranes of nerve cells. They are pore proteins that respond to mechanical stimuli. When stimulated by mechanical forces (such as tension, pressure, and shear), the channels open to allow the transmembrane transport of substances (mainly ions). Within milliseconds, they convert mechanical force signals into electrical or chemical signals, thereby triggering a series of cellular responses.

[0003] Calcium signaling is a crucial mechanism for regulating various cellular life activities. Neurons communicate via extracellular calcium ions (calcium ions, Ca2+). 2+ Influx, endoplasmic reticulum Ca 2+ Calcium release and other mechanisms regulate various biological processes, such as the regulation of action potentials, the release of neurotransmitters, axonal growth, and neurogenesis. According to the calcium hypothesis of Alzheimer's disease, regulating calcium ion signaling can be used to treat neurodegenerative diseases such as Alzheimer's. 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 various serious neurological diseases. Therefore, achieving local neural activation is of great significance for promoting the development of new neurological therapeutic technologies.

[0004] Currently, classic clinical treatments for neurological diseases include transcranial magnetic stimulation (TMS), transcranial electrical stimulation (TES), and drug therapy. These techniques directly apply electrodes or magnetic fields to the patient's brain to generate electrical currents that stimulate brain cells. They typically involve large-area stimulation and cannot achieve precise stimulation of individual cells. For example, patent application CN118543036A describes a TMS system and its stimulation method that predicts the required stimulation intensity through three steps: pre-stimulation, prediction of neuronal action potentials, and formal stimulation, accurately delivering appropriate magnetic stimulation to the brain to reach the required threshold for neuronal action potentials. However, due to individual differences and the complexity of brain structure, TMS still lacks precision in locating the stimulation site; furthermore, as the stimulation penetrates deeper into the brain, the magnetic field generated by TMS gradually weakens as it passes through the skull, resulting in limited stimulation depth. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a neural activator and its application for regulating the activation of MSCs in nerve cell membranes, which can achieve precise activation of individual nerve cells.

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

[0007] This invention provides a neural activator that regulates the opening of mechanosensitive ion channels in nerve cell membranes, the neural activator consisting 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 to 2 μm.

[0010] This invention provides an application of an optical tweezers system or the aforementioned neural activator in the preparation of products that regulate the opening of mechanosensitive ion channels in nerve cell membranes.

[0011] This invention provides an application of an optical tweezers system or the aforementioned neural activator in the preparation of products that regulate calcium ion signals.

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

[0013] This invention provides a method for regulating the opening of mechanosensitive ion channels in nerve cell membranes using the aforementioned neural activator, comprising the following steps:

[0014] Microparticle solution is added to nerve cells, and the first optical potential trap is established using the optical tweezers system. The optical potential trap captures the microparticles, causing them to attach to the nerve cell membrane. Then, the optical tweezers system is used to establish a second optical potential trap to stimulate the nerve cell membrane, thereby regulating the opening of mechanosensitive ion channels on the surface of the nerve cell membrane.

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

[0016] Preferably, the first optical potential trap is a single optical potential trap with a scanning frequency of 9500-10000Hz; the second optical potential trap is two or more optical potential traps with a scanning frequency of 50-500Hz.

[0017] Preferably, the light potential trap captures microparticles, allowing the microparticles to attach to the nerve cell membrane for 0.5–1.5 min.

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

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

[0020] This invention provides a neural activator and its application for regulating the opening of MSCs (mesocellular cells) on the neuronal cell membrane. The invention utilizes an optical tweezers system to convert dynamic optical force into vibrational mechanical force through a micron-sized particle medium, which is then precisely applied to the neuronal cell membrane. This dynamically regulates the opening of mechanosensitive ion channels on the neuronal cell membrane surface, enabling the influx of calcium ions and other substances, thereby achieving precise activation and functional regulation of neurons at the subcellular level. This invention's method of regulating neurons using the aforementioned neural activator is non-contact, flexible, and precise, and is expected to provide a novel non-genetic optical method for high-precision neural regulation. It also promises to provide new technological support for understanding neuronal interactions and the pathogenesis and treatment of neurodegenerative diseases at the subcellular level. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the beam path structure of an optical tweezers system.

[0022] Figure 2 This is a schematic diagram illustrating the principle of activating and regulating neurons in this invention;

[0023] Figure 3 This is a bright-field image of cell membrane oscillations at different locations within a neuron under 60x magnification, where the red-marked areas are silica particles captured by an optical tweezers system.

[0024] Figure 4 The results of changes in intracellular calcium ion fluorescence signal monitored by calcium ion fluorescence imaging technology before and after stimulation of the cell membrane to regulate the mechanosensitive ion channels on the surface of the neuronal cell membrane;

[0025] Figure 5 To investigate the changes in local electrical signals at the site of neuronal stimulation before and after stimulation of the mechanosensitive ion channels on the surface of the neuronal cell membrane, patch-clamp technique was used to monitor these changes. Detailed Implementation

[0026] This invention provides a neural activator that regulates the opening of mechanosensitive ion channels in nerve cell membranes, the neural activator consisting of an optical tweezers system and microparticles.

[0027] In this invention, the optical tweezers system is a well-known testing platform, preferably the Tweez250 high-speed multi-optical-trap nano-optical tweezers and testing platform, brand Aresis. The most important components of this optical tweezers system are a laser, a condenser lens, an acousto-optic deflector (AOD), and a beam expander, used to form a focused beam and an optical trap array for particle capture. Specifically, the laser emits a 1064nm laser beam, which is modulated by the AOD to generate an optical trap array to capture and manipulate particles, specifically for capturing biological cells. During the research process, it was found that capture under a 60x objective lens was the most stable and produced the clearest image when captured by a camera; therefore, the optical tweezers system of this invention preferably includes a 60x objective lens. However, the optical traps formed by the beams under the 40x and 20x objective lenses of this optical tweezers system do not achieve ideal particle capture effects and are not conducive to precise control.

[0028] In this invention, the microparticles preferably include silica particles or polystyrene microspheres, more preferably silica particles. This invention does not have a particular limitation on the source of the silica particles or polystyrene microspheres; they can be prepared using methods known in the art or commercially available products. For example, silica particles can be purchased from Aladdin (catalog number: M120356-5mL, CAS number: 7631-86-9). The diameter of the microparticles is preferably 0.5–2 μm, more preferably 1–1.5 μm, and even more preferably 1 μm. This invention utilizes an optical tweezers system to precisely act on nerve cells through a microparticle medium such as silica particles, dynamically regulating the opening of mechanosensitive ion channels on the surface of the nerve cell membrane.

[0029] This invention provides an application of an optical tweezers system or the aforementioned neural activator in the preparation of products that regulate the opening of mechanosensitive ion channels in nerve cell membranes.

[0030] This invention provides an application of an optical tweezers system or the aforementioned neural activator in the preparation of products that regulate calcium ion signals.

[0031] In the above applications, the nerve cells preferably include SD fetal rat neurons, mouse hippocampal neurons, rat primary sensory neurons, rat adrenal pheochromocytoma cells, or mouse cerebral cortical cells, and more preferably SD fetal rat neurons. The nerve cells of the present invention exhibit a significant response to the oscillating stimulation signal of the optical tweezers system.

[0032] This invention provides a method for regulating the opening of mechanosensitive ion channels in nerve cell membranes using the aforementioned neural activator, comprising the following steps:

[0033] Microparticle solution is added to nerve cells, and the first optical potential trap is established using the optical tweezers system. The optical potential traps the microparticles, causing them to attach to the nerve cell membrane. Then, the optical tweezers system is used to establish a second optical potential trap to stimulate the nerve cell membrane, thereby regulating the opening of mechanosensitive ion channels on the surface of the nerve cell membrane.

[0034] In this invention, a microparticle solution is added to nerve cells. The number of nerve cells is 4–6 × 10⁶. 4 The number of units is further preferably 4.5–5.5 × 10⁻⁶. 4 More preferably 5 × 10 4 The microparticle solution is prepared by diluting a 2-3% (w / v) silica microsphere ethanol suspension with PBS to a volume ratio of 90-110 times. 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 neurons, mouse hippocampal neurons, rat primary sensory neurons, rat adrenal pheochromocytoma cells, or mouse cerebral cortical cells, more preferably SD fetal rat neurons.

[0035] In this invention, a first optical potential trap is established using the optical tweezers system. This trap captures microparticles, causing them to adhere to the nerve cell membrane. A second optical potential trap is then established using the same system to stimulate the nerve cell membrane, thereby regulating the opening of mechanosensitive ion channels on the nerve cell membrane surface. After adding the microparticle solution, the first and second optical potential traps are immediately established using the optical tweezers system. Microparticles are first captured using the optical potential traps, and then the nerve cell membrane is stimulated, thereby regulating the opening of mechanosensitive ion channels on the nerve cell membrane surface. The first optical potential trap is a single trap with a scanning frequency of 9500–10000 Hz, more preferably 9600–10000 Hz, and even more preferably 10000 Hz. By setting the scanning frequency during the establishment of the first optical potential trap, particles can be captured stably and rapidly and adhered to the cell membrane. In this invention, the time for the optical potential trap to capture microparticles and allow them to adhere to the nerve cell membrane is preferably 0.5–1.5 min, more preferably 1 min. The second optical potential trap preferably consists of two or more optical potential traps, more preferably two optical potential traps. The scanning frequency is preferably 50–500 Hz, further preferably 200–400 Hz, and more preferably 200 or 400 Hz. When establishing the second optical potential trap, the mechanosensitive ion channels on the surface of a single nerve cell can be precisely activated by setting the scanning frequency. The optical tweezers system is equipped with a laser that emits a 1064 nm laser beam and an acousto-optic deflector, thereby generating an optical trap array.

[0036] In this 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 will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0039] The monodisperse silica microspheres were purchased from Aladdin, catalog number: M120356-5mL, CAS number: 7631-86-9.

[0040] Preparation of the monodisperse silica microsphere solution: Monodisperse silica microspheres and anhydrous ethanol were mixed at a concentration of 2.5% (w / v, g / L) to prepare a silica microsphere ethanol suspension, which was then diluted with PBS to a volume of 100 times to obtain the monodisperse silica microsphere solution.

[0041] Example 1

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

[0043] (1) Cell culture was carried out in an incubator at 37℃, 5% CO2, and saturated humidity. SD fetal rat hippocampal neurons (OriCell, SHCFN-00001) were cultured in complete cell culture medium until the density reached 40%–60%, at which point cell experiments could be performed.

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

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

[0046] (3) Dynamic regulation of cells under conditions of 37℃, 5% CO2, and saturated humidity: The cells were prepared using a solution containing 2.5 × 10⁻⁶ ppm. 4 2 μL of monodisperse silica microspheres with a diameter of 1 μm were directly added to a culture dish of SD fetal rat hippocampal neurons (the total volume of the culture medium in the dish was 2 mL).

[0047] (4) After adding the monodisperse silica microsphere solution, observe the cells in bright field immediately. Use the above optical tweezers system to establish a single optical potential trap. Set the scanning frequency to 10000Hz (i.e., the maximum value). The optical trap captures silica particles and places them on a single cell membrane. Wait for 1 minute so that the silica particles are attached to the cell membrane.

[0048] (5) Stimulating the cell membrane to regulate the opening of mechanosensitive ion channels on the surface of neuronal cell membranes: The laser power and scanning frequency are input by the computer. The laser power is 0.1W, the scanning frequency is set to 200Hz, and two optical potential traps are set, so the oscillation frequency generated is 100Hz.

[0049] By stimulating the cell membrane to regulate the opening of mechanosensitive ion channels, changes in intracellular calcium ion concentration and membrane potential are triggered. Therefore, calcium ion fluorescence imaging and patch-clamp techniques are used to monitor this process in real time. A schematic diagram illustrating the principle of activating and regulating neurons is shown below. Figure 2 .

[0050] Figure 3 The results showed that silica particles could be successfully captured at different locations on the neuronal cell membrane using an optical tweezers system.

[0051] When stimulating the mechanosensitive ion channels on the surface of neuronal cell membranes, a sequence of intracellular calcium ion fluorescence images was simultaneously captured while oscillating the selected cells, resulting in the experimental curve. Simultaneously, before stimulating the mechanosensitive ion channels on the surface of neuronal cell membranes, a cell was located near the selected cells, and a sequence of images showing the natural quenching of intracellular calcium ion fluorescence was captured, resulting in the control curve. In both the experimental and control groups, the exposure time was 600 ms and the gain was 1.0.

[0052] Figure 4 The results showed that the fluorescence signal in the experimental group was enhanced compared with that in the control group, indicating the opening of the calcium ion mechanosensitive channel.

[0053] After stimulating the mechanosensitive ion channels on the surface of neuronal cell membranes to regulate their activity, patch-clamp techniques were used to monitor changes in neuronal cell membrane current signals. Normally, there is no current signal when the calcium ion mechanosensitive channels are not open, but a change in current signal occurs once they are opened.

[0054] like Figure 5 As shown, this invention utilizes an optical tweezers system to stimulate the cell membrane and regulate the mechanosensitive ion channels on the surface of neuronal cell membranes, resulting in a significant change in current signal, indicating the opening of calcium ion mechanosensitive channels.

[0055] Figures 3-5 The results showed that by using the optical force generated by the optical tweezers system to attach silica particles to the neuronal cell membrane, the opening of mechanosensitive ion channels on the surface of the neuronal cell membrane could be dynamically regulated at different cell membrane oscillation positions, thereby achieving precise activation and functional regulation at the subcellular level.

[0056] Example 2

[0057] The difference between this embodiment and Embodiment 1 is that: the stimulation of the cell membrane regulates the opening of the mechanosensitive ion channels on the surface of the neuronal cell membrane: the laser power and scanning frequency are input by the computer, the laser power is 0.1mW, the scanning frequency is set to 400Hz, and two optical potential traps are set, so the oscillation frequency generated is 200Hz. Other steps are the same as in Embodiment 1.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for regulating the opening of mechanosensitive ion channels in nerve cell membranes using a neural activator, characterized in that, The neural activator consists of an optical tweezers system and microparticles; The method includes the following steps: Microparticle solution is added to nerve cells. The optical tweezers system is used to establish a first optical potential trap. The optical potential trap captures the microparticles and causes them to attach to the nerve cell membrane. The optical tweezers system is then used to establish a second optical potential trap to generate an oscillation frequency, which stimulates the nerve cell membrane and thereby regulates the opening of mechanosensitive ion channels on the surface of the nerve cell membrane. The first optical potential trap is a single optical potential trap with a scanning frequency of 9500-10000Hz; the second optical potential trap is two or more optical potential traps with a scanning frequency of 50-500Hz; the optical potential traps capture microparticles, and the time for the microparticles to attach to the nerve cell membrane is 0.5-1.5min. The optical tweezers system includes a laser that emits a 1064nm laser beam, a focusing mirror, an acousto-optic deflector, and a beam expander. By using an optical tweezers system to convert dynamic optical force into vibrational mechanical force through a micron-sized particle medium, the force is precisely applied to the neuronal cell membrane. This dynamically regulates the opening of mechanosensitive ion channels on the surface of the neuronal cell membrane, enabling calcium ion influx and thus achieving precise activation and functional regulation of neurons at the subcellular level.

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

3. The method according to claim 2, characterized in that, The diameter of the microparticles is 0.5 to 2 μm.

4. The method according to claim 1, characterized in that, The nerve cells include SD fetal rat neurons, mouse hippocampal neurons, rat primary sensory neurons, rat adrenal pheochromocytoma cells, or mouse cerebral cortex cells.

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

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