阅读说明：本技术 一种基于上转化发光技术的心脏节律与功能的光控制系统 (Optical control system for heart rhythm and function based on up-conversion luminescence technology ) 是由 王晞 程玥 王龙 饶盼盼 于 2018-08-10 设计创作，主要内容包括：本发明公开了一种基于上转换发光技术的心脏节律与功能的光控制系统。该系统包括光敏蛋白表达系统、光学控制系统、心脏功能监测系统、中央控制系统。光敏蛋白表达系统包括光敏蛋白、病毒载体或非病毒载体、启动子及示踪荧光蛋白；光学控制系统包括上转换发光材料混合高分子材料形成的膜形结构和光输出装置；心脏功能监测系统指监测心脏节律和舒缩功能的装置；中央控制系统指接受心脏功能监测系统信号并根据程序控制向光学控制系统发放指令的装置；该系统以心脏为靶器官,实现光敏蛋白的心肌特异性表达,利用上转换发光材料结合近红外光程控输出,实施对心脏节律与功能的光学控制,进行心血管疾病机制及防治策略的研究与应用。(The invention discloses a light control system for heart rhythm and function based on an up-conversion luminescence technology. The system comprises a photosensitive protein expression system, an optical control system, a heart function monitoring system and a central control system. The photosensitive protein expression system comprises photosensitive protein, viral vector or non-viral vector, promoter and tracing fluorescent protein; the optical control system comprises a film-shaped structure formed by mixing an up-conversion luminescent material and a high polymer material and an optical output device; the heart function monitoring system refers to a device for monitoring heart rhythm and relaxation function; the central control system is a device which receives the signals of the heart function monitoring system and sends instructions to the optical control system according to program control; the system takes the heart as a target organ to realize the myocardial specific expression of photosensitive protein, and utilizes the up-conversion luminescent material in combination with the near infrared light program control output to implement the optical control on the heart rhythm and function and carry out the research and application of cardiovascular disease mechanism and prevention strategy.)

1. A light control system for heart rhythm and function based on up-conversion luminescence technology is characterized in that the system comprises a photosensitive protein expression system expressed in the heart, an optical control system, a heart function monitoring system connected with the heart and a central control system; the light-sensitive protein expression system comprises light-sensitive protein, a vector, a promoter and tracing fluorescent protein; the optical control system comprises a membrane-shaped structure formed by mixing an up-conversion luminescent material and a high polymer material and a light output device which is embedded under the skin or arranged outside the body to be close to the heart; the cardiac function monitoring system is a device for monitoring the electrocardio-change, the cardiac rhythm and the cardiac contractility, and feeds back the monitored electrocardio-signals to the central control system; the input of the central control system is connected with the heart function monitoring system, the output of the central control system is connected with the light output device in the optical control system, an output control program is generated according to monitoring information, the light output device is started to adjust corresponding optical parameters, near infrared light (NIR) is emitted and directly or indirectly projected to a film-shaped structure formed by a conversion luminescent material and a mixed high polymer material, light waves with corresponding wavelengths are converted, and the light waves act on the heart targeted by the photosensitive protein expression system.

2. The light control system for cardiac rhythm and function based on upconversion luminescence technology according to claim 1, wherein the light-sensitive protein comprises any one of a light-sensitive cation channel, a light-sensitive anion channel, a light-sensitive chloride ion pump, a light-sensitive hydrogen ion pump, a light-sensitive metabotropic receptor, a light-sensitive adenylate cyclase, and a light-sensitive protein that generates Reactive Oxygen Species (ROS).

3. The system for controlling the heart rhythm and function based on upconversion luminescence technology according to claim 1, wherein the light sensitive protein is a mutant of the light sensitive protein according to claim 2.

4. The system for controlling heart rhythm and function based on upconversion luminescence technology according to claim 1, wherein the vector comprises a viral vector or a non-viral vector, and the viral vector comprises adenovirus and adeno-associated virus; wherein the adeno-associated virus comprises a plurality of serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV 9; the non-viral vector comprises liposome complex, cationic polymer, chitosan polymer and inorganic nanoparticles.

5. The light control system for cardiac rhythm and function based on upconversion luminescence technology as claimed in claim 1, wherein the promoter comprises specific promoter and broad-spectrum promoter, the tissue-specific promoter selected comprises myocardial-specific promoter α MHC, cTNT, CK1.3, CK0.4 and other myocardial-specific promoters, the broad-spectrum promoter selected comprises CAG, CMV, PGK1, EF1A, SV40, UBC, EFFS, c-FOS and other broad-spectrum promoters, and the promoter further comprises expression up-regulation element of the corresponding promoter.

6. The light control system for heart rhythm and function based on up-conversion luminescence technology according to claim 1, wherein the light-sensitive protein expression system is targeted to heart, i.e. the heart is used as a target organ, and the constructed light-sensitive protein expression system is used for realizing the expression of light-sensitive protein by injection and other ways; the heart target organ comprises cell and subcellular types including ventricular myocytes, atrial myocytes, sinoatrial node cells, atrioventricular node cells, fibroblasts, purkinje fibers and nerve fiber endings; the injection mode comprises intravenous injection, local myocardial injection, coronary intervention injection and other modes.

7. The system for controlling heart rhythm and function based on upconversion luminescence technology according to claim 1, wherein the structure of the upconversion luminescence material mixed polymer material is degradable or non-degradable through 3D printing, manual spin coating, screen printing, dip coating, inkjet printing, and spray pyrolysis; the up-conversion luminescent material comprises a main material NaYF₄、NaGdF₄A sensitizer Yb, activators Er, Tm and Ho, a main body material, the sensitizer and the activator are combined in any percentage; the structure of the up-conversion luminescent material comprises up-conversion nano-particles UCNPs, up-conversion fluorescent powder UCLPs, up-conversion fluorescent particles and up-conversion fluorescent powder; the polymer material comprises non-biodegradable material, polyethylene and polypropyleneAlkenes, polyacrylates, aromatic polyesters, silica gels, rubbers, polyurethanes, polyetheretherketones, and the like; degradable biological material, collagen, aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polylactic acid, polycaprolactone, polyphosphazene and silk fibroin.

8. The system of claim 1, wherein the light output device is a laser or LED emitting near-infrared light in a wavelength range of 780-1100 nm, and 1100-2526 nm; the light output device also comprises a power supply, an integrated chip, a pulse generator, an optical fiber and a joint; the integrated chip receives and processes a program instruction sent by the central control system, controls the pulse generator, starts the laser source laser or the LED, the laser source laser or the LED is connected with the optical fiber through one or more optical fiber connectors, and outputs near infrared light through the optical fiber; the illumination mode and optical parameters of the light output device are continuously adjustable within a certain range, and the parameters comprise illumination wavelength, illumination pulse width time, illumination frequency and illumination power.

9. A light control system for performing heart rhythm and function based on upconversion luminescence technology according to claim 1, wherein the optical control system is implemented in such a manner that the light output device emits near infrared light according to a predetermined program, and the near infrared light is directly or indirectly projected to a film structure formed by the upconversion luminescence material mixed polymer material, so that the upconversion luminescence material excites a light wave with a corresponding wavelength, and the light wave can activate the light-sensitive protein expressed by the heart; the choice of the wavelength of the near infrared light and the upconversion luminescent material depends on the properties of the light sensitive protein expressed in the heart.

Technical Field

The invention belongs to the field of medical instruments, and particularly relates to a light control system for carrying out heart rhythm and function based on an up-conversion luminescence technology.

Background

Optogenetics is a biotechnology that has attracted much attention in recent years, and is a biotechnology that integrates interdisciplinary multidisciplinary techniques such as optics, software control, and genetic manipulation techniques. The main principle is that firstly, a gene manipulation technology is adopted to transfer a light-sensitive protein gene (such as ChR2, eBR, NaHR3.0, Arch or OptoXR and the like) into a specific cell type to express a special ion channel or a G protein coupled receptor. The light sensitive ion channel can respectively generate selectivity to the passing of cations or anions under the stimulation of illumination with different wavelengths, thereby causing the change of membrane potential at two sides of the cell membrane and achieving the purpose of selectively exciting or inhibiting the cells. The optogenetic technology fully combines the advantages of optics and genetics, namely the cell accuracy of a gene editing technology, the micron-sized focusing property of specific monochromatic light, the microsecond-level transient property of current of a photosensitive channel and the medium-independent dependence of light propagation.

The key steps of applying the optogenetic technology in the heart research field mainly include selection of ① light-sensitive proteins, each light-sensitive protein has an activating light wave with a specific wavelength and can cause a cell state to generate corresponding functional change, so that the selection of a proper light-sensitive protein can realize regulation and control of excitation and contraction of cardiac muscle cells. ② expresses the light-sensitive protein in the heart, and the light-sensitive protein can be expressed in target cells by adopting genetic methods such as virus transfection, non-virus transfection, transgenic animals, cre-dependent expression systems and the like. ③ optical control technology is implemented to determine the optimal light control conditions for regulating and controlling the target cells by using parameters such as wavelength, power, pulse width, pulse interval, pulse repetition rate and the like. ④ biological effect evaluation utilizes the existing bioelectricity recording technology, cardiac contraction function evaluation technology and the like to evaluate the regulation and control effect of illumination on the cardiac muscle cells or the cardiac function.

Cardiovascular diseases are common diseases seriously threatening human health, and have the characteristics of high morbidity, high disability rate and high mortality rate. The current therapeutic means mainly include drug therapy, interventional therapy, surgical therapy, implantable electronic devices and the like. The implanted electronic device sends pulse current with certain frequency at fixed time through the pulse generator, the pulse current is transmitted to the myocardium contacted with the electrode through the lead and the electrode, local myocardial cells are excited by external electric stimulation, and the current is conducted to the peripheral myocardium through intercellular gap connection or intercalated disc connection, so that the whole atrium or ventricle is excited and further contraction activity is generated. However, this device requires a lead medium, causes pain when the lead medium is implanted, and also fails to achieve precise control of cardiac rhythm, synchronous contraction, and the like.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide an optical control system for carrying out heart rhythm and function by utilizing an upconversion luminescence technology, which can implement direct or tissue-penetrability heart optical control and apply the optogenetic technology to the research and application of the regulation and control of the electrophysiological and contractile function of the heart, the disease mechanism and the prevention and treatment strategy.

In order to realize the purpose, the invention adopts the following technical scheme: a light control system for heart rhythm and function based on up-conversion luminescence technology comprises a photosensitive protein expression system expressed in heart, an optical control system, a heart function monitoring system connected with heart, and a central control system; the light-sensitive protein expression system comprises light-sensitive protein, a vector, a promoter and tracing fluorescent protein; the optical control system comprises a membrane-shaped structure formed by mixing an up-conversion luminescent material and a high polymer material and a light output device which is embedded under the skin or arranged outside the body to be close to the heart; the central control system receives the electrocardio-blood flow signals measured by the heart function monitoring system, and sets a command to the light output device according to a program, and near infrared light (NIR) is directly or indirectly projected to a film-shaped structure formed by a conversion luminescent material and a mixed high polymer material, and light waves with corresponding wavelengths are converted to act on the heart targeted by the photosensitive protein expression system.

The light-sensitive protein comprises a light-sensitive cation channel channelrhodopsin, a light-sensitive anion channel GtABCR, a light-sensitive chloride ion pump halorhodopsin, a light-sensitive hydrogen ion pump bacteriorhodopsin, a light-sensitive metabolic receptor optOXR, a light-sensitive adenylate cyclase photoactivated Adenylyl cyclases, a light-sensitive protein Killerred for generating Reactive Oxygen Species (ROS), and a mutant of the light-sensitive protein; for example, the light-sensitive cation channel ChR2 is selected to include all mutants of ChR2(H134R, C128X, D156A, ChETA, E123T/T159C, CatCh, XXL, XXM).

The vector comprises a viral vector or a non-viral vector, and the viral vector comprises adenovirus and adeno-associated virus; wherein the adeno-associated virus comprises a plurality of serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV 9; the non-viral vectors include liposome complexes, cationic polymers, chitosan polymers, inorganic nanoparticles, and the like.

The promoter comprises a specific promoter and a broad-spectrum promoter, the selected tissue-specific promoter comprises a myocardial-specific promoter α MHC, cTNT, CK1.3, CK0.4 and other myocardial-specific promoters, the selected broad-spectrum promoter comprises CAG, CMV, PGK1, EF1A, SV40, UBC, EFFS, c-FOS and other broad-spectrum promoters, and the promoter also comprises an expression up-regulation element of the promoter.

The tracing fluorescent protein comprises mCherry, EYFP, EGFP, dTomato and other tracing fluorescent proteins.

The heart target implementation mode of the light-sensitive protein expression system is that the heart is taken as a target organ, and the constructed light-sensitive protein expression system is used for realizing the expression of the light-sensitive protein in modes of injection and the like; the heart target organ comprises cell and subcellular types including ventricular myocytes, atrial myocytes, sinoatrial node cells, atrioventricular node cells, fibroblasts, purkinje fibers and nerve fiber endings; the injection mode comprises intravenous injection, local myocardial injection, coronary intervention injection and other modes; the successful expression of the light-sensitive protein can make the heart receive the regulation and control of the corresponding wavelength illumination, including regulating and controlling the heart rhythm, functions and the like, and activate the research of the heart optogenetics.

The up-conversion luminescent material is in a film-shaped structure formed by mixing a high polymer material with the up-conversion luminescent material, the up-conversion luminescent material has Anti-Stokes characteristics, long-wave radiation is converted into short-wave radiation through a multi-photon mechanism, and the up-conversion luminescent material can emit visible light under the excitation of near infrared light (NIR); the up-conversion materialComprises a main material NaYF₄、NaGdF₄A sensitizer Yb, activators Er, Tm and Ho, and the host material, the sensitizer and the activator are combined in any percentage; different combinations of upconverting materials can be excited to a plurality of different wavelength light waves; the up-conversion material comprises up-conversion nano particles (UCNPs), up-conversion fluorescent powder (UCLPs), up-conversion fluorescent particles, up-conversion fluorescent powder and up-conversion material with other structures;

the high polymer material comprises non-biodegradable materials, such as polyethylene, polypropylene, polyacrylate, aromatic polyester, silica gel, rubber, polyurethane, polyether ether ketone and the like; degradable biological materials, collagen, aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polylactic acid, polycaprolactone, polyphosphazene and silk fibroin;

the film-shaped structure is formed by mixing an up-conversion luminescent material and a high polymer material through modes of 3D printing, manual spin coating, a screen printing method, a dip coating method, an ink-jet printing method, a spray pyrolysis method and the like, and is degradable or non-degradable.

The light output device is a laser or an LED capable of emitting near-infrared wavelength, and the wavelength range comprises near-infrared short wave (780-1100 nm) and near-infrared long wave (1100-2526 nm); the light output device also comprises a power supply, an integrated chip, a pulse generator, an optical fiber and a joint; the integrated chip receives and processes a program instruction sent by the central control system, controls the pulse generator, starts the laser source laser or the LED, the laser source laser or the LED is connected with the optical fiber through one or more optical fiber connectors, and the near infrared light is output through the optical fiber.

The illumination mode and optical parameters of the light output device are continuously adjustable within a certain range, and the parameters comprise illumination wavelength, illumination pulse width time, illumination frequency, illumination power and the like.

The optical control system has an implementation mode that the light output device emits near infrared light according to a set program, and the near infrared light directly or indirectly (penetrates through biological tissues) projects to a film-shaped structure formed by the upconversion luminescent material and the high polymer material, so that the upconversion luminescent material excites light waves with corresponding wavelengths, and the light waves can activate photosensitive protein expressed by the heart; the choice of the wavelength of the near infrared light and the upconversion luminescent material depends on the properties of the above-mentioned light-sensitive proteins expressed in the heart.

The cardiac function monitoring system is a device for monitoring the electrocardio-change, the cardiac rhythm and the cardiac contractility, and feeds back the monitored electrocardio-signals to the central control system.

The input of the central control system is connected with the heart function monitoring system, the output of the central control system is connected with the optical output device in the optical control system, an output control program is generated according to the monitoring information, and the optical output device is started to adjust corresponding optical parameters.

The invention has the beneficial effects that: the light control system for cardiac rhythm and function by using the up-conversion luminescence technology has the advantages of tissue penetrability, flexibility, accuracy, wireless property, no pain and the like, can flexibly control cardiac activity, realizes accurate regulation and control on cardiac rhythm, synchronous contraction and the like, and completes research and application on cardiac electrophysiological function regulation and control, disease mechanism and prevention and treatment strategies.

Drawings

FIG. 1 is a schematic diagram of an optical control system for performing heart rhythm and function using upconversion luminescence.

FIG. 2 is a schematic diagram of an optical control system for performing heart rhythm and function by the laboratory animal of FIG. 1 using an upconversion luminescence technique.

FIG. 3 is a schematic diagram of an implementation of the light sensitive protein expression system.

Fig. 4 is a schematic diagram of an implementation of an optical control system comprising a film-shaped structure based on an up-converting material and a light output device.

FIG. 5 is a spectrum of the visible light spectrum of 980nm near infrared light converted by the UCNP as the up-conversion material in example 1.

FIG. 6 is an electrocardiogram of the heart rate of rat in example 1 successfully captured by the visible light after the programmed 980nm near infrared light is converted by the up-conversion material UCNP.

The system comprises a central control system 101, a photosensitive protein expression system 102, an optical control system 103, a cardiac function monitoring system 104, a viral vector schematic diagram for constructing and expressing photosensitive protein 201, a heart for expressing photosensitive protein 202, a mechanism mode diagram for photosensitive protein action 203, a schematic diagram for membrane-shaped structure action formed by mixing up-conversion luminescent materials and high polymer materials 310, an up-conversion material 311, a high polymer material 312, a schematic diagram for composite membrane-shaped structure action 313, an optical output device 320, a power supply 321, an integrated chip 322, a laser light source 323, a pulse generator 324, an optical fiber connector 325 and an optical fiber, and an experimental animal 500.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

As shown in fig. 1 and fig. 2, the light-controlled cardiac rhythm and contractile function test is performed on a test animal 500, which comprises a central control system 101, a light-sensitive protein expression system 102, an optical control system 103 and a cardiac function monitoring system 104.

The central control system 101 of the example is connected with the cardiac function monitoring system 104 at its input and with the optical control system 103 at its output, and the central control system 101 generates an output control program based on the monitoring information to control the optical control system 103.

The example light-sensitive protein expression system 102, including vectors, promoters, regulatory elements, light-sensitive proteins and fluorescent proteins, transfects light-sensitive proteins into the heart of an experimental animal 500 by intravenous injection, local injection into the myocardium, coronary intervention, and other means.

The example optical control system 103 includes a film-shaped structure 310 formed by applying an up-conversion luminescent material mixed with a polymer material and a light output device 320, the light output device 320 receiving a signal from the central control system 101 and emitting a light stimulus; the cardiac function monitoring system 104 is configured to monitor changes in the electrophysiology and function of the experimental animal 500, and feed back the detected information to the central control system 101.

As shown in FIG. 3, the example light-sensitive protein expression system 102 includes the expression of light-sensitive protein genes in experimental animals 500 by intravenous injection, local injection into myocardium, coronary intervention, and other means.

The example light-sensitive protein expression system 102, light-sensitive protein selected for use includes light-sensitive cation channel channelrhodopsin, light-sensitive anion channel GtACR, light-sensitive chloride pump halorhodopsin, light-sensitive hydrogen ion pump bacteriorhodopsin, light-sensitive metabotropic receptor optoXR, light-sensitive adenylate cyclase photoactivatydnylyl cyclases and corresponding light-sensitive protein mutants; if the photosensitive cation channel ChR2 is selected, all mutants of ChR2(H134R, XXM, XXL, C128X, D156A, ChETA, E123T/T159C, CatCh) and the like are included.

The example photoprotein expression system 102, viral vectors used include adenovirus and adeno-associated virus; the adeno-associated virus comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, AAV9 is selected as an implementation example, and a target tissue is a heart; the non-viral vectors include liposome complexes, cationic polymers, chitosan polymers, inorganic nanoparticles, and the like.

In the example light-sensitive protein expression system 102, the selected promoters are tissue-specific promoters, myocardial-specific promoters α MHC, cTNT, CK1.3, CK0.4 and other myocardial-specific promoters, and broad-spectrum expression promoters including CAG, CMV, PGK1, EF1A, SV40, UBC, EFFS, c-FOS and other broad-spectrum promoters may also be selected.

The example light sensitive protein expression system 102, selected for use with regulatory elements to increase promoter expression, is selected from-CS-CRM 4-.

The example photoprotein expression system 102, which constructs a gene (viral vector-promoter-regulatory element-photoprotein-fluorescent protein) 201 expressing photoprotein, was injected intravenously and after 8 weeks the heart was able to stably express photoprotein.

For example, 203 said light-sensitive protein ChR2 is a light-controlled nonselective cation channel, and experimental animal 500 cardiomyocytes express ChR2, and the protein can be activated by blue light at about 470nm to promote the change of ChR2 molecular conformation, form cation channels, flow in monovalent and divalent cations, depolarize and excite cells, change the membrane potential of cardiomyocytes to generate induced current, thereby playing a role in light-controlled myocardial electrical activity.

If the photosensitive protein is ChR2, the light output device of the example light output device adopts a near-infrared light laser light source with the wavelength of about 980nm to irradiate the film-shaped structure 310 formed by 3D printing of the up-conversion luminescent material mixed with the silica gel, so as to excite ChR2 expressed on the whole heart of the experimental animal, and the film-shaped structure is attached to the surface of the heart; the light-sensitive protein can be selected from mutants of ChR2, including ChR2(H134R, XXM, XXL, C128X, D156A, ChETA, E123T/T159C, CatCh), and the like, the example light output device can also be selected from a film-shaped structure 310 formed by irradiating the upconversion luminescent material and silica gel mixed 3D printing by a near-infrared laser light source with the wavelength of about 980nm, so as to excite the ChR2 mutant expressed in the whole center of an experimental animal 500, and the excitation wavelength of the upconversion luminescent material is about 980nm with the luminescence wavelength of about 470 nm.

As shown in fig. 4, the example optical control system includes a film-shaped structure formed by 3D printing of an upconversion luminescent material mixed with silicone and a light output device; the up-conversion luminescent material is mixed with a film-shaped structure formed by 3D printing of silica gel, and is used for receiving 980nm near infrared light (NIR) emitted by a light output device, converting the NIR into blue light of about 480nm, and further exciting light-sensitive protein ChR2 expressed on the heart.

The example light output device 320 comprises a power source 321, an integrated chip 322, a laser light source 323, a pulse generator 324, an optical fiber connector and an optical fiber 325 in sequence; the integrated chip 322 receives and processes the optical signal sent by the controller, controls the laser light source 323 and the pulse generator 324, and is connected with the optical fiber 325 through the optical fiber joint; and performing light control on the myocardium expressing the photosensitive protein through the optical fiber.

A light control system for carrying out heart rhythm and light function based on up-conversion luminescence technology is implemented in the process that a central control system 101 receives signals of a heart function monitoring system 104, sends instructions to an optical control system 103 according to program control, outputs light to project to a membrane-shaped structure 310 formed by mixing polymer materials with up-conversion luminescence materials, so that the up-conversion luminescence materials excite visible light with corresponding wavelengths, a light output device 320 acts on a heart targeted by a photosensitive protein expression system 102, the molecular conformation of ChR2 changes to form a cation channel, monovalent and divalent cations flow, cells depolarize and excite, and the membrane potential of cardiac muscle cells is changed to generate induced current, so that the light control heart muscle electrical activity effect is achieved.

Example 1 study of rare earth upconversion nanoparticle-mediated optogenetic pacing of rat hearts

Selecting excitatory light sensitive protein Channelrhodopsin-2(ChR2) mediating cation inflow as a tool protein, constructing a myocardial specific vector (viral vector-promoter-regulatory element-light sensitive protein-fluorescent protein), selecting ChR2 mutant H134R with strong photocurrent as a transfection fragment, α -myosin heavy chain α -MHC combined expression up-regulation element CS-CRM4 as a promoter, red fluorescent protein mCherry as a marker and a transcription stability regulatory element WPRE-pA to be packaged in AAV/2-9, and constructing a ChR2 heart-specific tool virus (rAAV-CS-CRM4- α MHC-hCHR2(H134R) -mChery-WPRE-Pa) (SEQ ID NO: 1). SD rats are injected with a virus vector coding ChR2 through 1ml syringe jugular veins to realize the full-heart expression of ChR 2.

After 8 weeks, SD rats were thoracically opened under anesthesia and different concentrations (2.5, 5, 10, 20mg/ml) of rare earth upconversion nanoparticles (UCNP, NaYF)₄: yb, Er/Tm, diameter 30nm) in a composite silica gel thin film, attaching UCNP membrane to the right ventricle, the 980nm near infrared light source is provided by the optical fiber coupling semiconductor laser, the pulse stimulation of the light source is realized in a TTL modulation mode, the divergence angle of the light beam is compressed by a collimating mirror, the power density is measured and calculated by laser power output display and laser power meter, the light mode is accurately adjusted (the light spot area is selected to have gradient diameters of 0.4mm, 1mm, 2mm and 5mm, the light power gradient is increased from 2mW to 50mW, the light pulse width gradient is increased from 5ms to 50ms, and the light frequency gradient is increased from 4Hz to 8Hz), different parts of the heart are stimulated (atrioventricular sequence, left ventricular synchronization, left ventricular sequence, right ventricular sequence and left ventricular synchronization), and the electrophysiological changes of the heart (ECG, MAP, MEA and hemodynamics) are recorded to obtain the effectiveness evaluation of the upconversion fluorescent material in cardiac light pacing.

A980 nm near infrared light (power 1w) is projected to a UCNP film packaged by 20mg/ml to emit blue visible light, a spectrogram of the emitted visible light measured by an Acton general spectrometer SP2750 is shown in figure 5, most of wave peak values are 450-500nm, ChR2(134R) can be activated under the irradiation of blue light with the wavelength range of 400-500 nm, and the visible light emitted by 980nm projected to the UCNP film can activate ChR2 (134R).

As shown in figure 6, the rat basal heart rate is 333 times/minute, near infrared light of 980nm (power 2w, frequency 7Hz) is projected and packaged on a UCNP silica gel membrane of 20mg/ml, the emitted blue light is projected on the heart of the SD rat, the ChR2(134R) molecular conformation expressed on the heart of the rat is changed, a cation channel, monovalent and divalent cation inflow are formed, the cell depolarizes and excites, membrane potential of cardiac muscle cells is changed to generate induction current, the rat heart rate is captured by a program control light source, and the electrocardiogram shows that the rat heart rate is controlled by light stimulation, so that the function of optically pacing the heart is achieved.

Optogenetics is a multi-disciplinary crossed innovative technology, and by combining the fields of materials science and optics, the optogenetics completes an optical control system for carrying out heart rhythm and function by utilizing an up-conversion luminescence technology, and is beneficial to research and application of heart electrophysiological function regulation, disease mechanism and prevention and treatment strategies of organisms.

The above examples are merely illustrative of the embodiments of the present invention and are not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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agagatttct ccaacccgtc gacgccacca tggactatgg cggcgctttg tctgccgtcg 960

gacgcgaact tttgttcgtt actaatcctg tggtggtgaa cgggtccgtc ctggtccctg 1020

aggatcaatg ttactgtgcc ggatggattg aatctcgcgg cacgaacggc gctcagaccg 1080

cgtcaaatgt cctgcagtgg cttgcagcag gattcagcat tttgctgctg atgttctatg 1140

cctaccaaac ctggaaatct acatgcggct gggaggagat ctatgtgtgcgccattgaaa 1200

tggttaaggt gattctcgag ttcttttttg agtttaagaa tccctctatg ctctaccttg 1260

ccacaggaca ccgggtgcag tggctgcgct atgcagagtg gctgctcact tgtcctgtca 1320

tccttatccg cctgagcaac ctcaccggcc tgagcaacga ctacagcagg agaaccatgg 1380

gactccttgt ctcagacatc gggactatcg tgtggggggc taccagcgcc atggcaaccg 1440

gctatgttaa agtcatcttc ttttgtcttg gattgtgcta tggcgcgaac acattttttc 1500

acgccgccaa agcatatatc gagggttatc atactgtgcc aaagggtcgg tgccgccagg 1560

tcgtgaccgg catggcatgg ctgtttttcg tgagctgggg tatgttccca attctcttca 1620

ttttggggcc cgaaggtttt ggcgtcctga gcgtctatgg ctccaccgta ggtcacacga 1680

ttattgatct gatgagtaaa aattgttggg ggttgttggg acactacctg cgcgtcctga 1740

tccacgagca catattgatt cacggagata tccgcaaaac caccaaactg aacatcggcg 1800

gaacggagat cgaggtcgag actctcgtcg aagacgaagc cgaggccgga gccgtgccag 1860

cggccgccgt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct 1920

tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 1980

agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc 2040

tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga 2100

agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg 2160

agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc 2220

aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc 2280

ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg 2340

acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg 2400

acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca 2460

acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt 2520

acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagaat 2580

tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg 2640

gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 2700

atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 2760

tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 2820

ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 2880

ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 2940

gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat 3000

cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 3060

gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 3120

tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 3180

cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc 3240

ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct 3300

aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg 3360

gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt 3420

ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc 3480

ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat 3540

gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag 3600

gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg 3660

attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg 3720

cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct 3780

cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 3840

gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc 3900

cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct 3960

gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 4020

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 4080

gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 4140

ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct 4200

gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 4260

tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt 4320

tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 4380

ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg 4440

ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 4500

gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 4560

agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 4620

ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 4680

gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 4740

tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 4800

ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 4860

ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 4920

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 4980

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 5040

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 5100

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 5160

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 5220

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 5280

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 5340

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 5400

aacaattaatagactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 5460

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 5520

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 5580

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 5640

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 5700

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 5760

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 5820

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 5880

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 5940

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 6000

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 6060

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 6120

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 6180

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 6240

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 6300

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 6360

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 6420

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt 6474