WO2017035767A1 - 声敏感离子通道遗传操作方法及系统 - Google Patents

声敏感离子通道遗传操作方法及系统 Download PDF

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WO2017035767A1
WO2017035767A1 PCT/CN2015/088706 CN2015088706W WO2017035767A1 WO 2017035767 A1 WO2017035767 A1 WO 2017035767A1 CN 2015088706 W CN2015088706 W CN 2015088706W WO 2017035767 A1 WO2017035767 A1 WO 2017035767A1
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ion channel
sensitive ion
exogenous
mechanically sensitive
nerve
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PCT/CN2015/088706
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English (en)
French (fr)
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郑海荣
牛丽丽
刘新
孟龙
邱维宝
钱明
蔡飞燕
肖杨
王丛知
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中国科学院深圳先进技术研究院
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Priority to CN201580000405.8A priority Critical patent/CN106687587A/zh
Priority to PCT/CN2015/088706 priority patent/WO2017035767A1/zh
Publication of WO2017035767A1 publication Critical patent/WO2017035767A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

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  • the invention relates to the technical field of brain nerve stimulation, and in particular to a method and system for genetic operation of an acoustic sensitive ion channel.
  • the nucleus stimulation and loop regulation in the deep brain is an important way to understand the pathogenesis of functional brain diseases and to intervene and treat them. It is also a major frontier problem in current brain science research. Studies have shown that the occurrence of functional brain disease is associated with a specific "cortical-basal ganglion brain loop" dysfunction in the brain, and the corresponding target can be stimulated by a corresponding circuit to cause changes in the cortex and descending fibers to alleviate or cure the symptoms. This provides a scientific basis for in-depth study of the mechanisms of functional brain disease and neurological regulation and interventions such as deep brain stimulation.
  • the nerve loop is mainly located in the deep (6 ⁇ 10cm) nerve nuclei of the brain. It is the close communication channel between the microscopic molecular cells in the brain and the macroscopic whole behavior. It is also the study between the function of molecular cells in the brain and the overall behavior of the brain. Key bridge. Different units of a single function loop may span multiple structures and spatially interact with other functional loops.
  • the feedback information of the high-level hub modulates the activity of the functional loop without interruption, and the resulting chemical transmitter changes may change the effective connection of the functional loop. Therefore, deep brain stimulation and neural loop regulation are one of the core contents of brain science and brain disease research.
  • Deep Brain Stimulation is a target of specific nucleus in the brain implanted by electrodes. It can suppress abnormal neurological function of target cells through controllable high-frequency current stimulation, and achieve effective intervention and treatment of diseases.
  • Purpose [Ref. Chinese Patent CN 102470247 A, CN 102762253 A]. Since it was first used for tremor control in 1987, more than 100,000 patients worldwide have implanted DBS devices (also known as "cerebral pacemakers") for many refractory brain diseases such as Parkinson's disease and depression. Symptomatic, refractory epilepsy, dystonia, refractory pain, obsessive-compulsive disorder, etc.
  • DBS Deep brain stimulation for epilepsy. Neurotherapeutics, 2008, 5 (1): 59-67].
  • DBS Deep brain stimulation for epilepsy. Neurotherapeutics, 2008, 5 (1): 59-67.
  • 1 or 2 electrodes are implanted into the deep brain tissue. Stimulation of the nucleus is a permanent trauma to the brain tissue and nerve circuit, and the target cannot be replaced. It is difficult to achieve stimulation of more parts of the nuclei, and the entire power supply equipment must be surgically implanted into the body.
  • the stimulating electrode applied to the brain of the individual will affect the normal function of the body.
  • a glial cell sheath will form around the electrode, which not only affects the efficiency of the electrode, but also affects the normal function of the body.
  • the applied electrical stimulation always causes an excitatory response, and only when the inhibitory nuclei are stimulated can the inhibitory response be caused.
  • Transcranial direct current stimulatioin and transcranial magnetic stimulation (TMS) are painless and non-invasive detection and treatment techniques.
  • the former changes the depolarization or hyperpolarization direction of the neuronal membrane potential on the surface of the brain through two saline-soaked scalp-attached electrodes to change the cortical excitability of spontaneous neural activity.
  • Chinese patent CN 202538169 U The TMS generates a magnetic field perpendicular to the plane of the coil by a momentary, high-voltage pulse generated by a magnetic coil placed on the scalp, passing through the scalp and skull almost without attenuation, reaching deep tissues of the brain and generating an induced current that causes the nerve cells to depolarize. And produce an evoked potential.
  • the function of the cortical layer is regulated by a mode such as single pulse, double pulse and repeated transcranial magnetic stimulation, thereby regulating the function of the cortex
  • a mode such as single pulse, double pulse and repeated transcranial magnetic stimulation, thereby regulating the function of the cortex
  • Kirton A Chen R, Friefeld S, Gunraj C, Pontigon AM, et al. (2008) Contralesional repetitive transcranial magnetic stimulation for chronic hemiparesis in subcortical paediatric stroke: a randomised trial.
  • the Lancet Neurology 7:507-513 Both techniques are used to evaluate neurophysiological pathways, as well as neurological rehabilitation of depression, epilepsy, stroke, schizophrenia, autism and other diseases.
  • Optogenetics which has been developed in the past 10 years, has enabled the selective regulation of a certain microcirculation at the cellular level, that is, by giving lasers of different wavelengths to achieve excitatory or inhibitory regulation of a certain loop. It has effectively promoted the development of neuroscience [refer to Chinese patent CN 103168236A].
  • optogenetics technology activates light-sensitive channels by giving lasers of different wavelengths. Since the strong absorption of light by biological tissues severely limits the distance of light travel (only a few millimeters), it is necessary for patients or test animals. Corresponding brain region Into the fiber and fiber optic catheter, which inevitably damage part of the brain area during operation, resulting in the loss of certain physiological functions of the nervous system.
  • the Drug Delivery Pump achieves neuromodulation by direct administration of the pump device at a precise location [refer to US Pat. No. 6,609,030 B1]. Since the drug acts directly on the part, the dosage is effectively reduced, and side effects are reduced. Long-term use of baclofen through the implanted pump sheath has become the basic method for the treatment of refractory spinal cord or sputum originating from the brain. Optogenetics are powerful tools for exploring neural circuits [refer to patent CN 102283145 A, US 20140142664 A1]. The basic principle is that the opsin gene plus a specific promoter is introduced into a specific neuron group by viral transfection, and the physiological activity of the neuron is changed by light stimulation of different parameters, thereby realizing the regulation of the adjacent neural pathway.
  • Transcranial ultrasound neuromodulation is a new non-invasive brain stimulation technique in recent years. It stimulates or inhibits the central nervous system of the stimulation site through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations. The reversible change of the adjustment [refer to the patent US 20130197401, US20110092800]. Recently, the Arizona State University team demonstrated low-frequency, low-pressure ultrasound-induced neuromodulation through mouse brain horse-horse test, and also proposed a possible regulatory mechanism, that is, ultrasound affects voltage-gated sodium and calcium channels [Tyler WJ, Tufail Y, Finsterwald M, Tauchmann ML, Olson EJ, et al.
  • DBS is an invasive technique.
  • the device and surgery are expensive. There are certain risks such as surgical complications, target tolerance, rejection, etc.
  • the life of the battery is 4-5 years. After the battery is exhausted, the battery must be replaced again.
  • Internal stimulators, causing greater pain and financial burden to users, have largely limited the popularity of DBS.
  • Optogenetic technology cannot be used for the treatment of clinical brain diseases due to the need to transfect viral proteins.
  • the non-invasive tDCS and TMS technologies require higher operator and experimental conditions. The results are affected by factors such as the frequency of treatment, the location of stimulation, the duration of stimulation, the severity of the disease, and the drug treatment. The evaluation of efficacy is still controversial.
  • photoreceptor gene there are limitations in the regulation of photoreceptor gene, such as the use of wide-field stimulation mode, which activates or inhibits neurons, but it activates the entire animal sample or the entire neural circuit, failing to achieve selective photo-stimulation activation of specific cells or a group of cells.
  • wide-field stimulation mode which activates or inhibits neurons
  • photoreceptor gene it activates the entire animal sample or the entire neural circuit, failing to achieve selective photo-stimulation activation of specific cells or a group of cells.
  • scanning mirrors, acousto-optic deflectors, LED arrays, spatial light modulators, liquid crystals or micro-mirror technology although high spatial and temporal resolution can be used to stimulate the activated cells, it is usually necessary to combine inverted or upright fluorescence microscopes.
  • it is only suitable for the activation of light stimulation of cultured cells and brain slices, and there is a small range of light stimulation, which is not suitable for studying large neural networks and regulating the behavioral activities of living animals.
  • transcranial ultrasound neuromodulation technology uses low-frequency and low-pressure ultrasound to achieve brain nerve stimulation, there is no specific mechanism for how to use ultrasound for neuromodulation to achieve non-invasive and accurate brain nerve stimulation.
  • Embodiments of the present invention provide a genetic operation method for acoustically sensitive ion channels to achieve non-invasive, precise, and selective specific neural operations and regulation.
  • the method includes: acquiring an acoustic wave; focusing the acoustic wave on an endogenous mechanically sensitive ion channel of a nerve cell to be stimulated, opening the endogenous mechanically sensitive ion channel; and/or focusing the acoustic wave
  • the exogenous mechanically sensitive ion channel is opened on an exogenous mechanically sensitive ion channel of the nerve cell to be stimulated.
  • focusing the sound wave on an exogenous mechanically sensitive ion channel of a nerve cell to be stimulated comprises: expressing the sound sensitive gene by transfecting it onto a nerve cell to be stimulated, An exogenous mechanically sensitive ion channel is transferred to the cell membrane of the neural cell; the acoustic wave is focused on the exogenous mechanically sensitive ion channel after the transfer.
  • the sound waves are ultrasonic waves.
  • the neural cells include nerve cells in the deep brain region and nerve cells in the shallow brain region.
  • the sound pressure of the sound wave is greater than or equal to 1 kPa.
  • the embodiment of the invention also provides an acoustic-sensitive ion channel genetic operating system to achieve non-invasive and precise operation and regulation of selective specific nerves.
  • the system comprises: sound wave generating means for generating sound waves; sound wave propagation means for transmitting the sound waves to nerve cells to be stimulated, the sound waves focusing on endogenous mechanically sensitive ions of nerve cells to be stimulated Opening the endogenous mechanically sensitive ion channel on the channel, and/or the sound wave is focused on an exogenous mechanically sensitive ion channel of the nerve cell to be stimulated, opening the exogenous mechanically sensitive ion channel Changing the membrane potential of the nerve cells.
  • the exogenous mechanically sensitive ion channel is exogenous mechanical sensitivity transferred to the cell membrane of the neural cell after expression of the acoustically sensitive gene onto the neural cell for expression Ion channel.
  • the sound waves are ultrasonic waves.
  • the neural cells include nerve cells in the deep brain region and nerve cells in the shallow brain region.
  • the sound pressure of the sound wave is greater than or equal to 1 kPa.
  • the sound wave is focused on an endogenous mechanically sensitive ion channel of the nerve cell to open the endogenous mechanically sensitive ion channel, and/or to focus the sound wave on the exogenous machinery of the nerve cell
  • the sensitive ion channel is opened to open the exogenous mechanically sensitive ion channel, so that the membrane potential of the nerve cell is changed, thereby achieving the purpose of regulating the excitation or inhibition of the nerve cell. Since sound waves activate neurons by mechanical force, voltage-gated sodium ions and calcium ion channels are not classical mechanically sensitive proteins. Therefore, the present application proposes to focus sound waves on endogenous mechanically sensitive ion channels of nerve cells and/or Or exogenous mechanically sensitive ion channels to achieve non-invasive, precise, and selective specific neural manipulation and regulation in a sonic manner.
  • FIG. 1 is a flow chart of a method for genetically operating an acoustically sensitive ion channel according to an embodiment of the present invention
  • FIG. 2 is a schematic view showing the opening of a mechanically sensitive ion channel in an embodiment of the present invention
  • FIG. 3 is a schematic diagram of sound waves in an embodiment of the present invention.
  • FIG. 4 is a structural diagram of a genetically sensitive ion channel genetic operating system in accordance with an embodiment of the present invention.
  • the genetic operation method of the acoustic sensitive ion channel in the embodiment of the present invention may include:
  • Step 101 Acquire sound waves
  • Step 102 Acoustic wave cranial focusing, focusing the sound wave on an endogenous mechanically sensitive ion channel of the nerve cell to be stimulated, opening the endogenous mechanically sensitive ion channel; and/or, the sound wave
  • the exogenous mechanically sensitive ion channel is opened by focusing on an exogenous mechanically sensitive ion channel of the nerve cell to be stimulated.
  • the endogenous mechanically sensitive ion channel can be opened and/or the sound wave can be focused by focusing the sound wave on the endogenous mechanically sensitive ion channel of the nerve cell.
  • the mechanically sensitive ion channel in the middle portion ie, endogenous mechanical sensitivity
  • the ion channel or the exogenous mechanically sensitive ion channel is open under the action of sound waves, and the arrow indicates that the mechanically sensitive ion channel is stretched and opened, so that the membrane potential of the nerve cell is changed, thereby controlling the excitation or inhibition of the nerve cell.
  • the sound wave acts on the cell membrane.
  • the sound pressure is greater than or equal to 1 kPa.
  • the sound wave may be an ultrasonic wave, and the sound wave sequence of the ultrasonic wave is as shown in FIG. 3.
  • the acoustic wave is focused on the exogenous mechanically sensitive ion channel of the nerve cell to be stimulated. Including: transferring the exogenous mechanically sensitive ion channel to the cell membrane of the nerve cell by transfecting the acoustically sensitive gene onto the nerve cell to be stimulated; focusing the sound wave on the transferred Exogenous mechanically sensitive ion channels.
  • the exogenous mechanically sensitive ion channel can be opened under the action of small acoustic radiation, thereby causing the membrane potential of the nerve cell to change, reaching the nerve
  • the purpose of the cell is selectively excited or inhibited by regulation.
  • the sonic-focused nerve cells may be nerve cells in the deep brain region or nerve cells in the shallow brain region, so that brain nerve stimulation in different brain regions in the entire brain region can be achieved.
  • the exogenous mechanically sensitive ion channel (or receptor) described above may include genes and/or proteins extracted from all algae, bacteria, viruses, actinomycetes, fungi, protozoa, and various animals.
  • an acoustic-sensitive ion channel genetic operating system is further provided in the embodiment. As shown in FIG. 4, the acoustic-sensitive ion channel genetic operating system includes:
  • a sound wave propagation device 402 for transmitting the sound wave to a nerve cell to be stimulated, the sound wave focusing on an endogenous mechanically sensitive ion channel of the nerve cell to be stimulated, opening the endogenous mechanical sensitivity The ion channel, and/or focusing the sound wave on an exogenous mechanically sensitive ion channel of the nerve cell to be stimulated, opening the exogenous mechanically sensitive ion channel, altering the membrane potential of the nerve cell.
  • the sound wave generating device 401 includes a signal generator 4011 for generating a radio frequency signal.
  • the signal generator 4011 can be a single-element ultrasonic transducer or a needle ultrasonic transducer. , multi-array array, arc-array ultrasonic transducer, ultrasonic surface wave chip or micro-capacitive ultrasonic transducer CMUT; power amplifier 4012 for power amplification of the radio frequency signal; matching circuit 4013, Selecting a channel for outputting in the ultrasound probe for the amplified RF signal, setting a delay between the different channels, and using different channels for the amplified RF signal to be output in the ultrasound probe causes the ultrasound probe to generate The focus of the sound wave is different.
  • the adjustment of the position of the focus of the sound wave generated by the ultrasonic probe can be realized by adjusting the channel used for outputting the amplified RF signal in the ultrasonic probe; the ultrasonic probe 4014 is used for Converting the amplified RF signal into an acoustic wave according to a channel selected by the matching circuit for the amplified RF signal and a set delay, for example
  • the material of the ultrasonic probe 4014 may be a piezoelectric material such as a piezoelectric single crystal material, a piezoelectric ceramic material, or a thin film piezoelectric material.
  • the sound pressure of the sound wave is greater than or equal to 1 kPa.
  • the sound wave may be an ultrasonic wave, and the sound wave sequence of the ultrasonic wave is as shown in FIG. 3.
  • the sound wave propagation device 402 is an ultrasonic coupling agent, and the ultrasonic coupling agent isolates the air within a distance between the sound wave generating device 401 and the skull.
  • the ultrasound can be transmitted to the skull and then focused through the skull on the endogenous mechanically sensitive ion channels and/or exogenous mechanically sensitive ion channels of the nerve cells.
  • the exogenous mechanically sensitive ion channel in order to reduce the radiation force of the acoustic wave required to open the exogenous mechanically sensitive ion channel, in the present embodiment, the exogenous mechanically sensitive ion channel is transfected into the acoustic sensitive gene. After expression on nerve cells, it is transferred to an exogenous mechanically sensitive ion channel on the cell membrane of the nerve cell.
  • the sonic-focused nerve cells may be nerve cells in the deep brain region or nerve cells in the shallow brain region to achieve brain nerve stimulation in different brain regions in the entire brain region.
  • the sound waves are focused on endogenous mechanically sensitive ion channels of the neural cells to open the endogenous mechanically sensitive ion channels, and/or to focus the sound waves on the exogenous nature of the neural cells.
  • the mechanically sensitive ion channel is opened to open the exogenous mechanically sensitive ion channel, so that the membrane potential of the nerve cell is changed, thereby achieving the purpose of regulating the excitation or inhibition of the nerve cell. Since sound waves activate neurons by mechanical force, voltage-gated sodium ions and calcium ion channels are not classical mechanically sensitive proteins.
  • the present application proposes to focus sound waves on endogenous mechanically sensitive ion channels of nerve cells and/or Or exogenous mechanically sensitive ion channels, thereby achieving non-invasive, precise operation and regulation of selective specific nerves in a sonic manner.
  • embodiments of the present invention can be provided as a method, system, or computer program product. Accordingly, the present invention may take the form of an entirely hardware embodiment, an entirely software embodiment, or a combination of software and hardware. Moreover, the invention can take the form of a computer program product embodied on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) including computer usable program code.
  • computer-usable storage media including but not limited to disk storage, CD-ROM, optical storage, etc.
  • the computer program instructions can also be stored in a computer readable memory that can direct a computer or other programmable data processing device to operate in a particular manner, such that the instructions stored in the computer readable memory produce an article of manufacture comprising the instruction device.
  • the apparatus implements the functions specified in one or more blocks of a flow or a flow and/or block diagram of the flowchart.

Abstract

一种声敏感离子通道遗传操作方法及系统，其中，该方法包括：获取声波（101）；将所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道，和/或将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道，改变所述神经细胞的膜电位（102）。该方案以声波的方式实现无创、精确、选择特异性的神经操作和调控。

Description

声敏感离子通道遗传操作方法及系统 技术领域
本发明涉及脑神经刺激技术领域，尤其涉及一种声敏感离子通道遗传操作方法及系统。
背景技术
帕金森病、癫痫和抑郁症等脑功能性疾病的有效干预和治疗是重大医学难题。由于我国人口基数庞大而且老龄化逐年提高，这些脑疾病患者的数量高达数千万人，已经成为我国沉重的经济负担和棘手的社会问题。功能性脑疾病主要病理改变导致的脑深部核团及神经环路的功能障碍。但是，目前对功能性脑疾病的确切机理仍不清楚，并缺乏有效的治疗措施，这仍然是全球共同面临的医学挑战。因此，认识这些脑疾病的发病机制和发展先进的干预与治疗技术是科学界的重大而紧迫的任务。
脑深部的神经核团刺激与环路调控是理解功能性脑疾病发病机制并对其进行干预和治疗的重要途径，也是目前脑科学研究的重大前沿问题。研究表明，功能性脑疾病的发生与大脑内特定的“皮质-基底神经节大脑环路”功能障碍有关，刺激相应的靶点可通过相应的回路引起皮质和下行纤维改变从而减轻或治愈症状，这为深入研究功能性脑疾病的发生机制和深部脑刺激等神经调控与干预手段提供了科学依据。
神经环路主要位于大脑深部的(6～10cm)神经核团之间，是大脑内微观分子细胞与宏观整体行为之间的密切联系通道，也是研究脑内分子细胞功能与整体行为功能之间的关键桥梁。单一功能环路的不同单元可能跨越多个结构，并在空间上与其它功能环路相交互。高级中枢的反馈信息无间断地调制功能环路的活动，所产生的化学递质变化则可能改变功能环路的有效连接方式。因此，深部脑刺激与神经环路调控是脑科学和脑疾病研究的核心内容之一。
神经刺激与环路调控的技术和工具是推动神经科学发展的重要动力。继1870年德国科学家报道了电刺激下犬的大脑皮层可引发特定的躯体反应之后的100多年里，电、磁、光等技术与神经科学相结合产生了深部脑电刺激、磁刺激、光基因调控等神经刺激与调控技术。
脑深部电刺激(Deep Brain Stimulation，DBS)是将电极植入的脑内特定神经核团靶点，通过可控的高频电流刺激抑制靶点细胞的异常神经功能，达到有效干预和治疗疾病 的目的【参考中国专利CN 102470247 A，CN 102762253 A】。自1987年首次被用于震颤的控制以来，全世界共有10多万名患者植入了DBS装置(亦称“脑起搏器”)，为众多难治性的脑疾病如帕金森病、抑郁症、难治性癫痫、肌张力失调、顽固性疼痛、强迫症等提供了一种有效的干预方法【Halpem CH,Samadani U,Litt B,et a1.Deep brain stimulation for epilepsy.Neurotherapeutics,2008,5(1)：59-67】。但是，DBS的应用也存在着重要的局限：临床通过开颅手术将1～2根电极植入深脑组织对于核团进行刺激是对脑组织和神经环路造成永久的创伤、靶点无法更换、难以实现更多部位核团的刺激，而且整个电源供给装备也要手术植入到身体中。在个体的脑部施加的刺激电极会影响机体的正常功能，DBS电极使用一段时间以后，在电极周围会形成胶质细胞鞘，不仅影响电极的效率，还会影响机体的正常功能，而且，在施加电刺激时，所施加的电刺激总是引起兴奋性反应，只有在刺激抑制性核团时，才能引起抑制性反应，这些缺点也限制了电刺激技术在调控神经环路方面的应用。
经颅直流电刺激(transcranial direct current stimulatioin，tDCS)和经颅磁刺激(transcranial magnetic stimulation，TMS)等技术是无痛无创的检测和治疗技术。前者经由两个盐水浸湿的头皮贴附电极片向颅内特定区域输入恒定电流，改变大脑表面神经元膜电位的去极化或超极化方向，从而改变自发神经活动的皮质兴奋性【参考中国专利CN 202538169 U】。TMS由放置于头皮上的磁性线圈产生的瞬时、高伏脉冲产生一个垂直于线圈平面的磁场域，几乎无衰减地通过头皮和颅骨，到达大脑的深部组织并产生感应电流，使神经细胞去极化并产生诱发电位。通过单脉冲、双脉冲和重复经颅磁刺激等模式，调控神经细胞的兴奋或抑制特性，从而调节皮层的功能【参考中国专利CN 102462892 A，参考美国专利US 6827681B2、Kirton A,Chen R,Friefeld S,Gunraj C,Pontigon A-M,et al.(2008)Contralesional repetitive transcranial magnetic stimulation for chronic hemiparesis in subcortical paediatric stroke:a randomised trial.The Lancet Neurology 7:507-513.】。两种技术均用于评价神经电生理传导通路，以及抑郁症、癫痫、中风、精神分裂症、自闭症等疾病的神经康复治疗。
近10多年来新兴发展起来的光遗传学技术(Optogenetics)，实现了在细胞水平选择性调控某一微环路，即通过给予不同波长的激光实现对某一环路的兴奋性或者抑制性调控，有力地推动了神经科学的发展【参考中国专利CN 103168236A】。但是，光遗传学技术是通过给与不同波长的激光来激活光敏感通道，由于生物组织对于光的强烈吸收严重限制了光的传播距离(仅有若干毫米)，因此需要在患者或被试动物的相应脑区插 入光纤和光纤导管，这在操作时不可避免的会损伤部分脑区，从而导致神经系统的某些生理功能丧失。同时，光遗传学技术的关键依赖于光敏感通道蛋白(转染病毒实现)的表达，目前仅用于神经科学小动物模型(老鼠)实验研究。但是对于非人灵长类高级大动物仍然未见报道，该技术面临复杂的挑战。
微量药物泵技术(Drug Delivery Pump)通过在精确位置植入泵装置直接给药的手段来实现神经调控【参考美国专利US 6609030 B1】。由于药物直接作用到局部，有效减少了用药剂量，减少了副作用。长期通过植入泵鞘内使用巴氯芬已经成为治疗难治性脊髓或者大脑起源的痉挛的基本方法。光感基因神经调控(Optogenetics)是探索神经环路的有力工具【参考专利CN 102283145 A、US 20140142664 A1】。其基本原理是将视蛋白基因加上特异启动子通过病毒转染导入特定的神经元类群，并通过不同参数的光刺激，来改变该神经元的生理活动，从而实现所属神经通路的调控。
经颅超声神经调控是近年来出现的无创性脑刺激新技术，通过不同的强度、频率、脉冲重复频率、脉冲宽度、持续时间使刺激部位的中枢神经产生刺激或抑制效应，对神经功能产生双向调节的可逆性变化【参考专利US 20130197401，US20110092800】。最近，亚利桑那州立大学小组通过小鼠脑海马片实验证明了低频低压超声波诱发神经调控，而且也提出可能的调控机制，即超声波影响电压门控的钠离子和钙离子通道【Tyler WJ,Tufail Y,Finsterwald M,Tauchmann ML,Olson EJ,et al.(2008)Remote excitation of neuronal circuits using low-intensity,low-frequency ultrasound.PLoS One 3:e3511.】。后来，该小组首次通过活体动物实验证明了利用低频低压超声波实现神经调控【Tufail Y,Matyushov A,Baldwin N,Tauchmann ML,Georges J,et al.(2010)Transcranial pulsed ultrasound stimulates intact brain circuits.Neuron 66:681-694.】。弗吉尼亚理工大学Carilion研究所的Legon Wynn等将低频低压超声波直接作用于脑部特定区域，能增强人们对触觉的分辨能力。这项发现第一次证明了低强度、经颅聚焦超声波能调节人类脑活动，提高觉察能力【Legon W,Sato TF,Opitz A,Mueller J,Barbour A,et al.(2014)Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans.Nature neuroscience 17:322-329.】。
尽管上述脑刺激和神经调控技术的临床或科学实验效果已得到肯定，但其作用机制尚不清楚。目前认为脑深部电刺激改变了电压门控通道的活性，神经递质耗竭，从而阻碍了突触信息的传递，阻抑了电极周围的神经信号输出，对所刺激的核团产生了功能性损毁效应；刺激作用于与电极周围神经元有突触联系的轴突终末，间接调节神经信号的输出，进而改变了病理性神经网络功能。
上述各种脑刺激装置仍存在多方面的局限性和技术挑战。例如，DBS是有创技术，装置和手术费用昂贵，存在一定的手术并发症、靶点耐受、排斥反应等风险，供电电池寿命为4-5年，电池耗尽后必须再次手术更换电池或者内部刺激器，给使用者造成更大的痛苦和经济负担，在很大程度上限制了DBS的普及。光遗传学技术由于需要转染病毒蛋白，所以无法用于临床脑疾病治疗。无创的tDCS和TMS技术外对操作者和实验条件要求较高，结果受治疗频率、刺激部位、刺激持续的时间、病情严重程度、药物治疗情况等因素影响，疗效评价尚有争议。光感基因神经调控存在局限性，如采用宽场刺激模式，虽可激活或抑制神经元，但它会把整个动物样品或整个神经环路激活，无法实现特定细胞或一群细胞选择性光刺激激活；如采用基于扫描镜、声光偏转器、发光二极管阵列、空间光调制器、液晶或微反射镜技术，虽可实现高时空分辨率的刺激激活细胞，但通常必须结合倒置或正置荧光显微镜上，只适用于培养细胞、脑切片的光刺激激活，存在光刺激范围小，不适合研究大的神经网络和调控活体动物的行为活动。这些限制了光感基因神经调控技术在神经环路研究中的应用。此外，虽然经颅超声神经调控技术利用低频低压超声波实现了脑神经刺激作用，但目前并没有具体的如何利用超声波进行神经调控的机制，以实现无创、精准的脑神经刺激。
发明内容
本发明实施例提供一种声敏感离子通道遗传操作方法，以实现无创、精准、选择特异性的神经操作和调控。该方法包括：获取声波；将所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道；和/或，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道。
在一个实施例中，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，包括：通过将声敏感基因转染到待刺激的神经细胞上进行表达，使所述外源性机械敏感性离子通道转移到所述神经细胞的细胞膜上；将所述声波聚焦在转移后的外源性机械敏感性离子通道上。
在一个实施例中，所述声波为超声波。
在一个实施例中，所述神经细胞包括深脑区域的神经细胞和浅脑区域的神经细胞。
在一个实施例中，所述声波的声压大于等于1千帕。
本发明实施例还提供一种声敏感离子通道遗传操作系统，以实现无创、精准地对选择性特异神经的操作和调控。该系统包括：声波产生装置，用于产生声波；声波传播装置，用于将所述声波传输到待刺激的神经细胞处，所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道，和/或所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道，改变所述神经细胞的膜电位。
在一个实施例中，所述外源性机械敏感性离子通道是在将声敏感基因转染到所述神经细胞上进行表达后，转移到所述神经细胞的细胞膜上的外源性机械敏感性离子通道。
在一个实施例中，所述声波为超声波。
在一个实施例中，所述神经细胞包括深脑区域的神经细胞和浅脑区域的神经细胞。
在一个实施例中，所述声波的声压大于等于1千帕。
本发明实施例中，通过将声波聚焦在神经细胞的内源性机械敏感性离子通道上，以开放该内源性机械敏感性离子通道，和/或将声波聚焦在神经细胞的外源性机械敏感性离子通道上，以开放该外源性机械敏感性离子通道，使得改变该神经细胞的膜电位，进而达到对神经细胞兴奋或抑制进行调控的目的。由于声波是通过机械力激活神经元，但是电压门控钠离子和钙离子通道都不是经典的机械敏感蛋白，因此，本申请提出将声波聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上，从而以声波的方式实现无创、精确、选择特异性的神经操作和调控。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。在附图中：
图1为本发明实施例中声敏感离子通道遗传操作方法的流程图；
图2为本发明实施例中机械敏感性离子通道开放的示意图；
图3为本发明实施例中声波的示意图；
图4为本发明实施例中声敏感离子通道遗传操作系统的结构图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚明白，下面结合附图对本发明实施例做进一步详细说明。在此，本发明的示意性实施例及其说明用于解释本发明，但并不作为对本发明的限定。
图1为本发明实施例中声敏感离子通道遗传操作方法的流程图。如图1所示，本发明实施例中声敏感离子通道遗传操作方法可以包括：
步骤101：获取声波；
步骤102：声波穿颅聚焦，将所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道；和/或，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道。
由图1可以得知，在本发明实施例中，通过将声波聚焦在神经细胞的内源性机械敏感性离子通道上，可以开放该内源性机械敏感性离子通道，和/或将声波聚焦在神经细胞的外源性机械敏感性离子通道上，以开放该外源性机械敏感性离子通道，例如，如图2所示，中间部分的机械敏感性离子通道(即内源性机械敏感性离子通道或外源性机械敏感性离子通道)在声波的作用下开放，箭头表示机械敏感性离子通道拉伸进而开放，使得改变神经细胞的膜电位，进而达到对神经细胞兴奋或抑制进行调控的目的，由于声波是通过机械力激活神经元，但是电压门控的钠离子和钙离子通道都不是经典的机械敏感蛋白，因此，本申请提出将声波聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上，从而以声波的方式实现无创、精确地对选择性特异神经的操作和调控，为脑疾病的研究和治疗以及神经科学研究提供了一种革新的工具。
具体实施时，为了实现声波可以穿过颅骨聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上，在本实施例中，上述声波作用在细胞膜上的声压大于等于1千帕。具体的，上述声波可以是超声波，超声波的声波序列如图3所示。
具体实施时，为了减小开放外源性机械敏感性离子通道所需声波的辐射力，在本实施例中，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，包括：通过将声敏感基因转染到待刺激的神经细胞上进行表达，使所述外源性机械敏感性离子通道转移到所述神经细胞的细胞膜上；将所述声波聚焦在转移后的外源性机械敏感性离子通道上。即在神经细胞转染声敏感基因进行表达后，在较小的声波辐射力作用下即可开放转移后的外源性机械敏感性离子通道，从而造成神经细胞的膜电位发生变化，达到对神经细胞选择性地兴奋或抑制的调控目的。
具体实施时，上述声波聚焦的神经细胞可以是深脑区域的神经细胞，也可以是浅脑区域的神经细胞，以可以实现整个大脑区域中不同脑区域的脑神经刺激。
具体的，上述外源性机械敏感性离子通道(或受体)可以包括从所有藻类、细菌、病毒、放线菌、真菌、原生动物以及各种动物中提取的基因和/或蛋白。
具体实施时，在本实施例中还提供了一种声敏感离子通道遗传操作系统，如图4所示，该声敏感离子通道遗传操作系统包括：
声波产生装置401，用于产生声波；
声波传播装置402，用于将所述声波传输到待刺激的神经细胞处，所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道，和/或将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道，改变所述神经细胞的膜电位。
具体的，如图4所示，该声波产生装置401包括：信号发生器4011，用于产生射频信号，例如，该信号发生器4011可以是单阵元超声换能器、针式超声换能器、多阵元面阵、弧面阵超声换能器、超声表面波芯片或微型电容式超音波换能器CMUT；功率放大器4012，用于对所述射频信号进行功率放大；匹配电路4013，用于为放大后的射频信号选择在超声探头中输出时所用的通道，在不同通道之间设置延时，放大后的射频信号在所述超声探头中输出时所用的通道不同使得所述超声探头生成的声波的聚焦点不同，例如，可以通过调整放大后的射频信号在所述超声探头中输出时所用的通道，来实现对超声探头生成声波聚焦点位置的调整；所述超声探头4014，用于根据所述匹配电路为所述放大后的射频信号选择的通道和设置的延时，将所述放大后的射频信号转换成声波，例如，该超声探头4014的材料可以是压电单晶材料、压电陶瓷材料或薄膜压电材料等压电材料。
具体实施时，为了实现声波可以穿过颅骨聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上，在本实施例中，上述声波的声压大于等于1千帕。具体的，上述声波可以是超声波，超声波的声波序列如图3所示，此时，声波传播装置402为超声耦合剂，该超声耦合剂在声波产生装置401和颅骨之间的距离内隔离空气，使得超声波可以传播到颅骨处，进而穿过颅骨聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上。
具体实施时，为了减小开放外源性机械敏感性离子通道所需声波的辐射力，在本实施例中，所述外源性机械敏感性离子通道是在将声敏感基因转染到所述神经细胞上进行表达后，转移到所述神经细胞的细胞膜上的外源性机械敏感性离子通道。
具体实施时，上述声波聚焦的神经细胞可以是深脑区域的神经细胞，也可以是浅脑区域的神经细胞，以实现整个大脑区域中不同脑区域的脑神经刺激。
在本发明实施例中，通过将声波聚焦在神经细胞的内源性机械敏感性离子通道上，以开放该内源性机械敏感性离子通道，和/或将声波聚焦在神经细胞的外源性机械敏感性离子通道上，以开放该外源性机械敏感性离子通道，使得改变该神经细胞的膜电位，进而达到对神经细胞兴奋或抑制进行调控的目的。由于声波是通过机械力激活神经元，但是电压门控钠离子和钙离子通道都不是经典的机械敏感蛋白，因此，本申请提出将声波聚焦在神经细胞的内源性机械敏感性离子通道和/或外源性机械敏感性离子通道上，从而以声波的方式实现无创、精确地对选择性特异神经的操作和调控。
本领域内的技术人员应明白，本发明的实施例可提供为方法、系统、或计算机程序产品。因此，本发明可采用完全硬件实施例、完全软件实施例、或结合软件和硬件方面的实施例的形式。而且，本发明可采用在一个或多个其中包含有计算机可用程序代码的计算机可用存储介质(包括但不限于磁盘存储器、CD-ROM、光学存储器等)上实施的计算机程序产品的形式。
本发明是参照根据本发明实施例的方法、设备(系统)、和计算机程序产品的流程图和/或方框图来描述的。应理解可由计算机程序指令实现流程图和/或方框图中的每一流程和/或方框、以及流程图和/或方框图中的流程和/或方框的结合。可提供这些计算机程序指令到通用计算机、专用计算机、嵌入式处理机或其他可编程数据处理设备的处理器以产生一个机器，使得通过计算机或其他可编程数据处理设备的处理器执行的指令产生用于实现在流程图一个流程或多个流程和/或方框图一个方框或多个方框中指定的功能的装置。
这些计算机程序指令也可存储在能引导计算机或其他可编程数据处理设备以特定方式工作的计算机可读存储器中，使得存储在该计算机可读存储器中的指令产生包括指令装置的制造品，该指令装置实现在流程图一个流程或多个流程和/或方框图一个方框或多个方框中指定的功能。
这些计算机程序指令也可装载到计算机或其他可编程数据处理设备上，使得在计算机或其他可编程设备上执行一系列操作步骤以产生计算机实现的处理，从而在计算机或 其他可编程设备上执行的指令提供用于实现在流程图一个流程或多个流程和/或方框图一个方框或多个方框中指定的功能的步骤。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种声敏感离子通道遗传操作方法，其特征在于，包括：
    获取声波；
    将所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道；和/或，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道。
  2. 如权利要求1所述的方法，其特征在于，将所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，包括：
    通过将声敏感基因转染到待刺激的神经细胞上进行表达，使所述外源性机械敏感性离子通道转移到所述神经细胞的细胞膜上；
    将所述声波聚焦在转移后的外源性机械敏感性离子通道上。
  3. 如权利要求1或2所述的方法，其特征在于，所述声波为超声波。
  4. 如权利要求1或2所述的方法，其特征在于，所述神经细胞包括深脑区域的神经细胞和浅脑区域的神经细胞。
  5. 如权利要求1或2所述的方法，其特征在于，所述声波的声压大于等于1千帕。
  6. 一种声敏感离子通道遗传操作系统，其特征在于，包括：
    声波产生装置，用于产生声波；
    声波传播装置，用于将所述声波传输到待刺激的神经细胞处，所述声波聚焦在待刺激的神经细胞的内源性机械敏感性离子通道上，开放所述内源性机械敏感性离子通道，和/或所述声波聚焦在待刺激的神经细胞的外源性机械敏感性离子通道上，开放所述外源性机械敏感性离子通道，改变所述神经细胞的膜电位。
  7. 如权利要求6所述的系统，其特征在于，所述外源性机械敏感性离子通道是在将声敏感基因转染到所述神经细胞上进行表达后，转移到所述神经细胞的细胞膜上的外源性机械敏感性离子通道。
  8. 如权利要求6或7所述的系统，其特征在于，所述声波为超声波。
  9. 如权利要求6或7所述的系统，其特征在于，所述神经细胞包括深脑区域的神经细胞和浅脑区域的神经细胞。
  10. 如权利要求6或7所述的系统，其特征在于，所述声波的声压大于等于1千帕。
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