WO2022011729A1 - 一种卵细胞多效能精准电刺激装置及方法 - Google Patents

一种卵细胞多效能精准电刺激装置及方法 Download PDF

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WO2022011729A1
WO2022011729A1 PCT/CN2020/103160 CN2020103160W WO2022011729A1 WO 2022011729 A1 WO2022011729 A1 WO 2022011729A1 CN 2020103160 W CN2020103160 W CN 2020103160W WO 2022011729 A1 WO2022011729 A1 WO 2022011729A1
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cell
electrical stimulation
egg
capillary
liquid metal
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French (fr)
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黄海波
马友文
戴辞海
朱易辰
陈立国
刘吉柱
王阳俊
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Suzhou University
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    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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

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  • the invention relates to the field of biotechnology related to cloning, in particular to a multi-effect precise electric stimulation device and method for egg cells oriented to the cloning process.
  • Cloning is the process of using biological technology to produce offspring with the same genetic organization as the original individual through asexual reproduction. Since its birth, cloning technology has always occupied the most important position in life science research and related medical technology. Egg cells contain important biological genetic information and are the main objects of operation in the cloning process. However, the success rate of cloning experiments is extremely low. One of the main reasons is that the activity of isolated egg cells will be greatly reduced, and they are very easy to die during the experiment. Even if they do not die during reconstruction, they are also prone to developmental arrest in subsequent cultivation. At this time, artificial stimulation to the egg cells is required to activate the cells.
  • electrical stimulation of egg cells has become the best solution due to its advantages of convenient operation, precise parameter control, high activation rate, high biocompatibility, no pollution, and little damage.
  • electrical activation is the increase in the permeability of the egg cell membrane under the excitation of DC pulse voltage, the influx of extracellular Ca 2+ into the cell makes the concentration of Ca 2+ in the cytoplasm increase, and the egg cell is activated. If the DC pulse voltage continues to rise, the cell membrane will be irreversibly perforated. At this time, if the two cells are close to each other, they will fuse into one cell, which is electrofusion.
  • the basic principle is that the stability of the phosphodiester bimolecular structure of the cell membrane changes under the stimulation of electrical pulses.
  • the traditional egg cell electrical stimulation device basically arranges a batch of egg cells between macroscopic parallel electrode plates, and generates a uniform electric field by passing the electrical stimulation signal. Low cell death rate.
  • the voltage required to apply electrical stimulation is also large, which is very easy to cause damage to the cells and is not safe; and the traditional electrical stimulator cannot complete the precise electrical stimulation of specific parts of the egg cells. It is also impossible to use a set of electrical stimulation devices to complete the various electrical stimulations (electrical activation, electrofusion, mitochondrial membrane potential enhancement, etc.) required for the cloning of egg cells at various stages.
  • the purpose of the present invention is to provide a multi-effect precise electrical stimulation device and method for egg cells oriented to the cloning process.
  • An oocyte multi-effect precise electrical stimulation device comprising:
  • the electrical stimulation mechanism includes two first electrical stimulation components and two second electrical stimulation components, each of the first electrical stimulation components includes a liquid metal capillary fine needle electrode, a liquid metal capillary fine needle electrode connected to the liquid metal capillary fine needle electrode a first needle holder, each of the second electrical stimulation components includes a metal parallel plate microelectrode and a second needle holder connected to the metal parallel plate microelectrode;
  • a cell pose adjustment chip mounted on the base, for adjusting the cell pose
  • the rotation mechanism is installed on the base and connected with the cell pose adjustment chip, and is used for driving the cell pose adjustment chip to rotate.
  • the liquid metal capillary electrode is made by the following steps: firstly drawing a glass tube to form capillary needles, then injecting liquid metal from the tail of the capillary needle, and then throwing the liquid metal towards the capillary needle Tip, and finally trim the tip of the capillary needle and lead out the wire from the tail of the capillary needle.
  • the tip diameter of the liquid metal capillary fine needle electrode is 10 ⁇ m.
  • the metal parallel-plate micro-electrode includes a rod-shaped electrode body and a parallel-plate electrode, and the rod-shaped electrode body and the parallel-plate electrode are integrally formed.
  • the diameter of the head of the rod-shaped electrode body is 0.5 mm, and the size of the parallel plate electrode is 2 mm ⁇ 1 mm.
  • the cell posture adjustment chip includes an upper substrate, a lower substrate, and an intermediate layer disposed between the upper substrate and the lower substrate, the upper substrate is provided with micropores, and the intermediate layer An air chamber is provided, and the lower substrate is provided with an air guide column.
  • the diameter of the micropores is 15 ⁇ m.
  • the first needle holding instrument includes a first screw cap, a second screw cap, a connecting seat, a third screw cap and a fixing rod, and the first screw cap and the second screw cap are threaded
  • the second screw cap and the third screw cap are both threadedly connected to the connection seat, and the fixing rod is screwed to the third screw cap.
  • the rotating mechanism includes a motor, a transmission gear connected with the motor, and a bearing seat connected with the transmission gear, and the bearing seat is connected with the cell posture adjustment chip.
  • a multi-effect precise electrical stimulation method for egg cells using the device, comprising the following steps:
  • Egg cell activation stage move the egg cell to the cell pose adjustment chip and fix it, control the two liquid metal capillary micro-needle electrodes to be close to the egg cell zona pellucida at an angle of 50° to 70°, and continuously supply high-voltage DC pulses , and then rotate the cell pose adjustment chip through the rotating mechanism, and drive the egg cells fixed on the cell pose adjustment chip to rotate horizontally until all cell membranes are fully stimulated;
  • Reconstituted embryo culture stage The electrofused reconstituted embryo is placed between two metal parallel plate microelectrodes, and the metal parallel plate microelectrodes are fed with low-frequency alternating current microcurrent signals.
  • the liquid metal capillary micro-needle electrode and the metal parallel plate micro-electrode provided by the present invention can perform precise electrical stimulation on a single egg cell and specific parts of the egg cell such as cell membrane and mitochondria, so as to reduce the electrical damage to other parts of the egg cell as much as possible, Thus, the success rate and survival rate of each stage of the somatic cell cloning process are improved.
  • Electrofusion completes the enhancement of the mitochondrial membrane potential of the recombinant embryo during the developmental stage of the recombinant embryo, and achieves multiple effects through targeted electrical stimulation at each stage of the somatic cell cloning process, thereby helping the operator to break through each stage and continue to develop.
  • the electrode spacing d is smaller, according to the formula When the stimulation of the same electric field is applied, the required voltage is smaller, which improves the safety of both the operator and the operating object.
  • FIG. 1 is a schematic structural diagram of an electrical stimulation device according to a preferred embodiment of the present invention.
  • Fig. 2 is the top view of the electrical stimulation device of the preferred embodiment of the present invention.
  • FIG. 3 is a partial enlarged view of the electrical stimulation device according to the preferred embodiment of the present invention.
  • FIG. 4 is a partial top view of an electrical stimulation device according to a preferred embodiment of the present invention.
  • FIG. 5 is a schematic structural diagram of a cell pose adjustment chip according to a preferred embodiment of the present invention.
  • FIG. 6 is a schematic diagram of the exploded structure of the cell pose adjustment chip according to the preferred embodiment of the present invention.
  • FIG. 7 is a schematic structural diagram of a first electrical stimulation component of a preferred embodiment of the present invention.
  • Fig. 8 is the enlarged schematic diagram of A in Fig. 7;
  • FIG. 9 is a schematic structural diagram of a second electrical stimulation component of a preferred embodiment of the present invention.
  • Fig. 10 is the enlarged schematic diagram of B in Fig. 9;
  • Fig. 11 is the preparation process of the liquid metal capillary fine needle electrode of the preferred embodiment of the present invention.
  • Figure 12 is a schematic diagram of applying electrical stimulation to cells at each stage of cloning according to a preferred embodiment of the present invention.
  • Fig. 13 is the flow chart of applying electrical stimulation during the somatic cell cloning process of the preferred embodiment of the present invention.
  • a multi-effect precise electrical stimulation device for egg cells includes:
  • the electrical stimulation mechanism includes two first electrical stimulation components 12 and two second electrical stimulation components 14, each first electrical stimulation component 12 includes a liquid metal capillary fine needle electrode 16 and is connected to the liquid metal capillary fine needle electrode 16
  • the first needle holding instrument 18, each of the second electrical stimulation components 14 includes a metal parallel plate microelectrode 20 and a second needle holding instrument 24 connected to the metal parallel plate microelectrode 20;
  • the cell pose adjustment chip 26 is installed on the base 10 and used to adjust the cell pose
  • the rotation mechanism is installed on the base 10 and connected with the cell pose adjustment chip 26 , and is used for driving the cell pose adjustment chip 26 to rotate.
  • the liquid metal capillary microneedle electrode 16 of the present invention is preferably prepared by the following process, as shown in FIG. 11 , the process steps are:
  • a glass tube is drawn to form capillary microneedles.
  • a capillary needle pulling instrument is used to heat and draw the glass tube to a set shape and size to form capillary needles, and the capillary needle pulling instrument adopts a structure commonly used in biological laboratories.
  • the liquid metal is injected from the tail of the capillary microneedle.
  • a high-precision syringe is used to inject liquid metal from the tail of the capillary needle, and the liquid metal is a gallium-based liquid metal.
  • the diameter of the tip 28 of the liquid metal capillary fine needle electrode 16 is preferably 10 ⁇ m.
  • the metal parallel plate microelectrode 20 is made of metal that does not chemically react with the cell culture medium when energized.
  • the metal parallel plate microelectrode 20 is made of platinum, but is not limited to platinum, and can also be gold, silver, or the like.
  • the metal parallel plate microelectrode 20 preferably includes a rod-shaped electrode body 30 and a parallel-plate electrode 31 , and the rod-shaped electrode body 30 and the parallel-plate electrode 31 are integrally formed. Further preferably, the diameter of the head 32 of the rod-shaped electrode body 30 is 0.5 mm, and the size of the parallel plate electrode 31 is 2 mm ⁇ 1 mm.
  • the cell posture adjustment chip 26 includes an upper substrate 33 , a lower substrate 34 and an intermediate layer 35 disposed between the upper substrate 33 and the lower substrate 34 .
  • the substrate 33 is provided with a micro-hole 36
  • the intermediate layer 35 is provided with an air chamber 38
  • the lower substrate 34 is provided with an air guide column 40.
  • the air chamber 38 is used to stabilize the air pressure and reduce fluctuations
  • the air guide column 40 is connected to an air pump (not shown in the figure) to generate negative pressure for fixing the egg cells.
  • the diameter of the micropores 36 is 15 ⁇ m.
  • the upper substrate 33 , the lower substrate 34 and the intermediate layer 35 are all rectangular, which is convenient for installation.
  • the center of the upper substrate 33 is provided with a groove 42 , and the micro-holes 36 are provided on the bottom wall of the groove 42 .
  • the groove 42 is circular, and the micro holes 6 are arranged on the central bottom wall of the groove 42 .
  • both the upper substrate 33 and the lower substrate 34 are made of plexiglass material, and the intermediate layer 35 is made of PDMS material.
  • the first needle holding instrument 18 of the present invention preferably includes a first screw cap 44 , a second screw cap 46 , a connecting base 48 , a third screw cap 50 and a fixing rod 52 , and the first screw cap 44
  • the second screw cap 46 is threadedly connected to the second screw cap 46 and the third screw cap 50 is screwed to the connection seat 48
  • the fixing rod 52 is screwed to the third screw cap 50 .
  • the second screw cap 46 is cushioned with a first annular gasket 54, and the connecting seat 48 A second annular gasket 56 is internally cushioned.
  • the first needle holder 18 and the second needle holder 24 have the same structure and size.
  • a channel 58 is preferably provided in the first screw cap 44 , the second screw cap 46 and the connecting base 48 , and the liquid metal capillary microneedle electrode 16 is partially clipped into the channel 58 , and the channel 58 can facilitate the liquid metal capillary microneedle electrode 16 . the lead out of the wire 59.
  • each first needle holder 18 and each second needle holder 24 are respectively fixed on the corresponding four-degree-of-freedom robotic arms (not shown in the figure), the four-degree-of-freedom manipulator can realize translation along the three directions of X, Y, Z and rotation around the X direction, and the Y direction refers to the direction parallel to the tip 28 or the head 32. direction.
  • the preferred rotating mechanism of the present invention includes a motor 60 , a transmission gear (not shown in the figure) connected to the motor 60 , and a carrier 62 connected to the transmission gear.
  • the cell posture adjustment chip 26 is mounted on the carrier 62 .
  • the bearing seat 62 is installed in the base 10, the bearing seat 62 is circular, the bearing seat 62 extends inwardly with at least one supporting plate 63, the cell posture adjustment chip 26 is fixed on the supporting plate 63, and the outer peripheral surface of the bearing seat 62 is A gear (not shown in the figure) is sleeved, the transmission gear is connected with the gear on the outer peripheral surface of the bearing seat 62, the motor 60 is activated, and the bearing seat 62 is driven to rotate through the transmission gear, thereby driving the cell posture adjustment chip 26 to rotate.
  • a multi-effect precise electrical stimulation method for egg cells of the present invention is introduced, using the above-mentioned device, including the following steps:
  • Egg cell activation stage move the egg cell 64 to the cell pose adjustment chip 26 and fix it, as shown in Figure 12-(a), control the two liquid metal capillary microneedle electrodes 16 to be close to each other at an angle of 50° to 70°
  • the zona pellucida of the egg cell 64 is continuously fed with high-voltage DC pulses, and then the cell posture adjustment chip 26 is rotated by the rotating mechanism, which drives the egg cell 64 fixed on the cell posture adjustment chip 26 to rotate horizontally until all cell membranes are fully stimulated;
  • Reconstituted embryo culture stage The electrofused reconstituted embryo 72 is placed between two parallel plate electrodes 31, as shown in Figure 12-(c).
  • Egg cell activation stage move the egg cell 64 to the cell pose adjustment chip 26 and turn on the negative pressure pump to fix the egg cell 64, and control the two liquid metal capillary microneedle electrodes 16 to be close to the zona pellucida of the egg cell 64 at an included angle of 60°,
  • the needle tips of the two liquid metal capillary electrodes 16 are separated by 8-12 ⁇ m, so that the electric field between the two liquid metal capillary electrodes 16 is focused on the cell membrane of the egg cell 64, and a high-voltage DC pulse is continuously supplied, and the electric field strength is 0.5 ⁇ 1.0kv/ cm, the pulse width is 50-100 ⁇ s, then start the motor 60 to drive the rotating mechanism to rotate the cell pose adjustment chip 26, and drive the egg cell 64 fixed in the center of the cell pose adjustment chip 26 to rotate horizontally until all the cell membranes are fully stimulated.
  • the permeability of the egg cell membrane after electrical stimulation increases, which is beneficial for Ca 2+ in the culture medium to enter the egg cell 64 and complete the activation of the egg cell 64 .
  • the needle tips of the two liquid metal capillary fine needle electrodes 16 are separated by 10 ⁇ m.
  • the egg cells 64 are ovarian oocytes.
  • Reconstituted embryo culture stage place the electrofused reconstituted embryo 72 between the two parallel plate electrodes 31, and pass a low-frequency alternating current microcurrent signal to the metal parallel plate microelectrode 20, the voltage is 40-50V, and the pulse width is 20 ⁇ 30ms, the frequency is 30 ⁇ 50Hz, in order to increase the mitochondrial membrane potential of the recombinant embryo 72, and then increase the activity of the recombinant embryo 72 to promote the division of the recombinant embryo 72.

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Abstract

本发明公开了一种卵细胞多效能精准电刺激装置，包括：基座；电刺激机构，包括两个第一电刺激组件和两个第二电刺激组件，每个第一电刺激组件包括液态金属毛细微针电极、与液态金属毛细微针电极相连接的第一持针仪，每个第二电刺激组件包括金属平行板微电极、与金属平行板微电极相连接的第二持针仪；细胞位姿调整芯片，安装在基座上，用于调整细胞的位姿;旋转机构，安装在基座上且与细胞位姿调整芯片相连接，用于驱动细胞位姿调整芯片旋转。本发明还公开了一种卵细胞多效能精准电刺激方法。本发明能对整个克隆流程中不同阶段的卵细胞特定部位进行精准电刺激，对体细胞克隆流程各阶段的针对性电刺激实现多重效应，提升体细胞克隆全流程的成功率。

Description

一种卵细胞多效能精准电刺激装置及方法 技术领域
本发明涉及与克隆相关的生物技术领域，特别是涉及一种面向克隆过程的卵细胞多效能精准电刺激装置及方法。
背景技术
克隆是利用生物技术由无性繁殖产生与原个体有完全相同基因组织后代的过程。自诞生以来，克隆技术在生命科学研究与相关医学技术中一直占据着最重要的位置。卵细胞包含着重要的生物遗传信息，是克隆过程的主要操作对象。然而克隆实验的成功率极低，其一主要原因是离体的卵细胞活性会大幅下降，在实验过程中极易死亡，即使重构时未死亡，在后续的培育中也极易发生发育阻滞，这时就需要人工对卵细胞施加刺激，来激活细胞。在众多刺激方法中，电刺激卵细胞由于具有操作方便、参数控制精确、激活率高、生物相容性高、无污染、损伤小等优点成为最佳方案。针对卵细胞的电刺激主要分为电激活和电融合两种。电激活是卵胞膜在直流脉冲电压激励下细胞膜通透性增加，细胞外Ca 2+流入细胞使得胞质内Ca 2+浓度升高，卵细胞被激活。而如果直流脉冲电压继续升高，细胞膜将发生不可逆的穿孔，此时如果两个细胞互相紧贴将会融合成一个细胞，这就是电融合。其基本原理都是由于细胞膜的磷酸二酯双分子结构在电脉冲刺激下稳定性发生变化。
传统的卵细胞电刺激装置基本上都是在宏观平行电极板间排列一批卵细胞，通入电刺激信号产生匀强电场，通过精确调整寻找最佳电刺激参数得到较高的电刺激成功率和较低的细胞死亡率。但由于宏观平行板电极的间距较大，施加电刺激所需的电压也较大，极易对细胞造成损伤也不安全；且传统的电刺激仪无法完成对卵细胞特定部位的实施精准电刺激，也无法用一套电 刺激装置完成克隆各阶段卵细胞所需的多种电刺激(电激活、电融合、线粒体膜电位提升等)。
发明内容
针对现有技术不足，本发明的目的在于提供一种面向克隆过程的卵细胞多效能精准电刺激装置及方法。
为了实现上述目的，本发明一实施例提供的技术方案如下：
一种卵母细胞多效能精准电刺激装置，包括：
基座；
电刺激机构，包括两个第一电刺激组件和两个第二电刺激组件，每个所述第一电刺激组件包括液态金属毛细微针电极、与所述液态金属毛细微针电极相连接的第一持针仪，每个所述第二电刺激组件包括金属平行板微电极、与所述金属平行板微电极相连接的第二持针仪；
细胞位姿调整芯片，安装在所述基座上，用于调整细胞的位姿；
旋转机构，安装在所述基座上且与所述细胞位姿调整芯片相连接，用于驱动所述细胞位姿调整芯片旋转。
作为本发明的进一步改进，所述液态金属毛细微针电极采用以下步骤制成：先拉制玻璃管形成毛细微针，接着从毛细微针尾部注入液态金属，然后将液态金属甩向毛细微针尖端，最后修剪毛细微针尖端，从毛细微针尾部引出导线。
作为本发明的进一步改进，所述液态金属毛细微针电极的尖端直径为10μm。
作为本发明的进一步改进，所述金属平行板微电极包括杆状电极本体和平行板电极，所述杆状电极本体与所述平行板电极一体成型。
作为本发明的进一步改进，所述杆状电极本体的头部直径为0.5mm，所 述平行板电极的尺寸为2mm×1mm。
作为本发明的进一步改进，所述细胞位姿调整芯片包括上基板、下基板以及设于所述上基板与下基板之间的中间层，所述上基板内设置有微孔，所述中间层设置有气室，所述下基板设置有导气柱。
作为本发明的进一步改进，所述微孔的直径为15μm。
作为本发明的进一步改进，所述第一持针仪包括第一旋盖、第二旋盖、连接座、第三旋盖和固定杆，所述第一旋盖与所述第二旋盖螺纹连接，所述第二旋盖、第三旋盖均与所述连接座螺纹连接，所述固定杆与所述第三旋盖螺纹连接。
作为本发明的进一步改进，所述旋转机构包括电机、与所述电机相连接的传动齿轮以及与所述传动齿轮相连接的承载座，所述承载座与所述细胞位姿调整芯片相连接。
一种卵细胞多效能精准电刺激方法，使用所述的装置，包括以下步骤：
(1)卵细胞激活阶段：将卵细胞移动至细胞位姿调整芯片上并固定，控制两个液态金属毛细微针电极呈50°～70°夹角紧贴卵细胞透明带，持续通入较高压直流脉冲，然后通过旋转机构使细胞位姿调整芯片旋转，带动固定于细胞位姿调整芯片上的卵细胞水平旋转直至所有的细胞膜都被充分刺激；
(2)卵细胞去核阶段：对激活后的卵细胞进行去核；
(3)重构胚电融合阶段：在将核供体注入去核卵细胞构成重构胚后，将重构胚固定在细胞位姿调整芯片上，移动两个液态金属毛细微针电极呈180°夹角紧贴重构胚，并使核供体与去核卵细胞的接触面与电场方向垂直，通入高压直流脉冲，使核供体和去核卵细胞发生电融合，形成重组胚胎；
(4)重组胚胎培养阶段：将电融合后的重组胚胎置于两个金属平行板微电极间，给金属平行板微电极通入低频交流微电流信号。
本发明的有益效果是：
(1)本发明所提供的液态金属毛细微针电极和金属平行板微电极可以对单个卵细胞以及卵细胞的特定部位如细胞膜、线粒体进行精准电刺激，尽可能的减少对卵细胞其他部位的电损伤，从而提高体细胞克隆过程各阶段成功率和存活率。
(2)可以完成体细胞克隆各阶段卵细胞所需的电刺激，包括在去核前对卵细胞的细胞膜进行电刺激以激活卵细胞，在重构胚电融合阶段完成对核供体和去核卵细胞的电融合，在重组胚胎发育阶段完成对重组胚胎线粒体膜电位的提升，通过对体细胞克隆流程各阶段的针对性电刺激实现多重效应，从而帮助操作对象突破各个阶段继续发育。
(3)由于采用了液态金属毛细微针电极和金属平行板微电极，电极间距d更小，根据公式
Figure PCTCN2020103160-appb-000001
在施加同样大小电场的刺激时，所需的电压更小，不论对操作者还是操作对象安全性都有提高。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术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中记载的一些实施例，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。
图1为本发明的优选实施例的电刺激装置的结构示意图；
图2为本发明的优选实施例的电刺激装置的俯视图；
图3为本发明的优选实施例的电刺激装置的局部放大图；
图4为本发明的优选实施例的电刺激装置的局部俯视图；
图5为本发明的优选实施例的细胞位姿调整芯片的结构示意图；
图6为本发明的优选实施例的细胞位姿调整芯片的分解结构示意图；
图7为本发明的优选实施例的第一电刺激组件的结构示意图；
图8为图7中A的放大示意图；
图9为本发明的优选实施例的第二电刺激组件的结构示意图；
图10为图9中B的放大示意图；
图11为本发明的优选实施例的液态金属毛细微针电极的制备流程；
图12为本发明的优选实施例的克隆各阶段对细胞施加电刺激的示意图；
图13为本发明的优选实施例的体细胞克隆过程中施加电刺激的流程图；
图中：10、基座，12、第一电刺激组件，14、第二电刺激组件，16、液态金属毛细微针电极，18、第一持针仪，20、金属平行板微电极，24、第二持针仪，26、细胞位姿调整芯片，28、尖端，30、杆状电极本体，31、平行板电极，32、头部，33、上基板，34、下基板，35、中间层，36、微孔，38、气室，40、导气柱，42、凹槽，44、第一旋盖，46、第二旋盖，48、连接座，50、第三旋盖，52、固定杆，54、第一环形垫圈，56、第二环形垫圈，58、通道，59、导线，60、电机，62、承载座，63、支撑板，64、卵细胞，66、核供体，68、去核卵细胞，70、重构胚，72、重组胚胎。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
如图1-图4所示，一种卵细胞多效能精准电刺激装置，包括：
基座10；
电刺激机构，包括两个第一电刺激组件12和两个第二电刺激组件14，每个第一电刺激组件12包括液态金属毛细微针电极16、与液态金属毛细微针电 极16相连接的第一持针仪18，每个第二电刺激组件14包括金属平行板微电极20、与金属平行板微电极20相连接的第二持针仪24；
细胞位姿调整芯片26，安装在基座10上，用于调整细胞的位姿；
旋转机构，安装在基座10上且与细胞位姿调整芯片26相连接，用于驱动细胞位姿调整芯片26旋转。
为了更精准的实现对单个细胞以及细胞的特定部位如细胞膜、线粒体进行电刺激，本发明优选液态金属毛细微针电极16采用以下工艺制备而成，如图11所示，工艺步骤为：
(1)拉制玻璃管形成毛细微针。作为优选方案，用毛细微针拉针仪加热并拉制玻璃管至设定形状和尺寸，形成毛细微针，毛细微针拉针仪采用生物实验室中常用结构。
(2)从毛细微针尾部注入液态金属。作为优选方案，用高精度注射器从毛细微针尾部注入液态金属，液态金属为镓基液态金属。
(3)将液态金属甩向毛细微针尖端。作为优选方案，利用离心机产生的离心力将液态金属甩向毛细微针尖端，直至在显微镜下观察液态金属填满针尖同时没有气泡，在离心机工作前，设置离心机的转速为6500r/min，持续时间1-2分钟，也可以根据毛细微针的直径进行转速和时间的调整。
(4)修剪毛细微针尖端，从毛细微针玻璃管尾部引出导线，形成液态金属毛细微针电极。作为优选方案，利用锻针仪修剪毛细微针尖端，使得修剪后的尖端直径满足要求，在液态金属氧化凝固前，从毛细微针玻璃管尾部引出导线以用于连接电源。液态金属暴露于空气中表面很快被氧化形成一层薄薄的约1nm的固态氧化膜，这有助于稳定液态金属毛细微针电极的形状，避免液态金属从毛细微针尖端处流出，避免影响毛细微针电极的性能。
如图8所示，本发明优选液态金属毛细微针电极16的尖端28直径为10μm。
金属平行板微电极20由在通电时不会和细胞培养液发生化学反应的金属制成。本发明实施例中，金属平行板微电极20由铂制成，但并不局限于铂，也可以为金、银等。本发明优选金属平行板微电极20包括杆状电极本体30和平行板电极31，杆状电极本体30与平行板电极31一体成型。进一步优选杆状电极本体30的头部32直径为0.5mm，平行板电极31的大小为2mm×1mm。
如图5、图6所示，为了便于固定住细胞，本发明优选细胞位姿调整芯片26包括上基板33、下基板34以及设于上基板33与下基板34之间的中间层35，上基板33内设置有微孔36，中间层35设置有气室38，下基板34设置有导气柱40，微孔36、气室38、导气柱40依次相连通，微孔36用于吸持细胞，气室38用于稳定气压，减小波动，导气柱40与气泵(图中未示出)相连接，用于产生固定卵细胞的负压。
进一步优选微孔36的直径为15μm。进一步优选上基板33、下基板34以及中间层35均呈矩形，便于安装。优选上基板33的中心设置有凹槽42，微孔36设置在凹槽42的底壁上。进一步优选凹槽42呈圆形，微孔6设置在凹槽42的中心底壁上。进一步优选上基板33、下基板34均采用有机玻璃材质制成，中间层35采用PDMS材质制成。
如图7、图8所示，本发明优选第一持针仪18包括第一旋盖44、第二旋盖46、连接座48、第三旋盖50和固定杆52，第一旋盖44与第二旋盖46螺纹连接，第二旋盖46、第三旋盖50均与连接座48螺纹连接，固定杆52与第三旋盖50螺纹连接。为了提高第一旋盖44与第二旋盖46、第二旋盖46与连接座48之间连接的稳固性，本发明优选第二旋盖46内垫有第一环形垫圈54，连接座48内垫有第二环形垫圈56。如图9、图10所示，本发明优选第一持针仪18与第二持针仪24的结构和大小相同。本发明优选第一旋盖44、第二旋盖46和连接座48内设置有通道58，液态金属毛细微针电极16部分卡入通 道58内，同时通道58能够便于液态金属毛细微针电极16的导线59的引出。
为了便于液态金属毛细微针电极16和金属平行板微电极20的位置调整，本发明优选每个第一持针仪18、每个第二持针仪24分别固定在对应的四自由度机械臂(图中未示出)上，四自由度机械臂能够实现沿X、Y、Z三个方向的平移以及绕X方向的旋转，沿Y方向指的是沿与尖端28或头部32平行的方向。
本发明优选旋转机构包括电机60、与电机60相连接的传动齿轮(图中未示出)以及与传动齿轮相连接的承载座62，细胞位姿调整芯片26安装在承载座62上。进一步优选承载座62安装在基座10内，承载座62呈圆形，承载座62向内延伸有至少一个支撑板63，细胞位姿调整芯片26固定在支撑板63上，承载座62外周面套设有齿轮(图中未示出)，传动齿轮与承载座62外周面的齿轮相连接，电机60启动，通过传动齿轮带动承载座62旋转，从而带动细胞位姿调整芯片26旋转。
如图12所示，介绍本发明的一种卵细胞多效能精准电刺激方法，使用上述装置，包括以下步骤：
(1)卵细胞激活阶段：将卵细胞64移至细胞位姿调整芯片26上并固定，如图12-(a)，控制两个液态金属毛细微针电极16呈50°～70°夹角紧贴卵细胞64透明带，持续通入较高压直流脉冲，然后通过旋转机构使细胞位姿调整芯片26旋转，带动固定于细胞位姿调节芯片26上的卵细胞64水平旋转直至所有的细胞膜都被充分刺激；
(2)卵细胞去核阶段：对激活后的卵细胞64进行去核；
(3)重构胚电融合阶段：将核供体66注入去核卵细胞68构成重构胚70，如图12-(b)，将重构胚70固定在细胞位姿调节芯片26上，移动两个液态金属毛细微针电极16呈180°夹角紧贴重构胚70，并使核供体66与去核卵细胞68的接触面与电场方向垂直，图中的箭头方向代表电场方向，通入高压直 流脉冲，使核供体66和去核卵细胞68发生电融合，形成重组胚胎72；
(4)重组胚胎培养阶段：将电融合后的重组胚胎72置于两个平行板电极31间，如图12-(c)，给金属平行板微电极20通入低频交流微电流信号。
如图13所示，为了更进一步的说明本发明的电刺激方法，作为优选方案，包括以下步骤：
(1)卵细胞激活阶段：将卵细胞64移动至细胞位姿调整芯片26上并开启负压泵固定卵细胞64，控制两个液态金属毛细微针电极16呈60°夹角紧贴卵细胞64透明带，两个液态金属毛细微针电极16的针尖相距8-12μm，实现两液态金属毛细微针电极16间电场聚焦于卵细胞64细胞膜部位，持续通入较高压直流脉冲，电场强度为0.5～1.0kv/cm，脉宽为50～100μs，然后启动电机60驱动旋转机构使细胞位姿调整芯片26旋转，带动固定于细胞位姿调节芯片26中心的卵细胞64水平旋转直至所有的细胞膜都被充分刺激，施加电刺激后的卵细胞膜通透性增加，有利于培养液中的Ca 2+进入卵细胞64，完成对卵细胞64的激活。进一步优选两个液态金属毛细微针电极16的针尖相距10μm。进一步优选卵细胞64为卵巢卵母细胞。
(2)卵细胞去核阶段：在显微镜的辅助下，采用盲吸法对激活后的卵细胞64进行去核。
(3)重构胚电融合阶段：在将核供体66注入去核卵细胞68构成重构胚70后，将重构胚70固定在细胞位姿调节芯片26上，移动两个液态金属毛细微针电极16呈180°夹角紧贴重构胚70，并使核供体66与去核卵细胞68的接触面与电场方向垂直，通入高压直流脉冲，电场强度为1～2kv/cm，脉宽为50～100μs，使核供体66和去核卵细胞68发生电融合，形成重组胚胎72。进一步优选将核供体66注入去核卵细胞68的卵周隙内。
(4)重组胚胎培养阶段：将电融合后的重组胚胎72置于两个平行板电极31间，给金属平行板微电极20通入低频交流微电流信号，电压为40～50V， 脉宽为20～30ms，频率为30～50Hz，以提升重组胚胎72线粒体膜电位，进而提高重组胚胎72活性以促进重组胚胎72分裂。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (10)

  1. 一种卵细胞多效能精准电刺激装置，其特征在于，包括：
    基座；
    电刺激机构，包括两个第一电刺激组件和两个第二电刺激组件，每个所述第一电刺激组件包括液态金属毛细微针电极、与所述液态金属毛细微针电极相连接的第一持针仪，每个所述第二电刺激组件包括金属平行板微电极、与所述金属平行板微电极相连接的第二持针仪；
    细胞位姿调整芯片，安装在所述基座上，用于调整细胞的位姿；
    旋转机构，安装在所述基座上且与所述细胞位姿调整芯片相连接，用于驱动所述细胞位姿调整芯片旋转。
  2. 根据权利要求1所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述液态金属毛细微针电极采用以下步骤制成：先拉制玻璃管形成毛细微针，接着从毛细微针尾部注入液态金属，然后将液态金属甩向毛细微针尖端，最后修剪毛细微针尖端，从毛细微针尾部引出导线。
  3. 根据权利要求2所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述液态金属毛细微针电极的尖端直径为10μm。
  4. 根据权利要求1所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述金属平行板微电极包括杆状电极本体和平行板电极，所述杆状电极本体与所述平行板电极一体成型。
  5. 根据权利要求4所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述杆状电极本体的头部直径为0.5mm，所述平行板电极的尺寸为2mm×1mm。
  6. 根据权利要求1所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述细胞位姿调整芯片包括上基板、下基板以及设于所述上基板与下基板之间的中间层，所述上基板内设置有微孔，所述中间层设置有气室，所述下基板设置有导气柱。
  7. 根据权利要求6所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述微孔的直径为15μm。
  8. 根据权利要求1所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述第一持针仪包括第一旋盖、第二旋盖、连接座、第三旋盖和固定杆，所述第一旋盖与所述第二旋盖螺纹连接，所述第二旋盖、第三旋盖均与所述连接座螺纹连接，所述固定杆与所述第三旋盖螺纹连接。
  9. 根据权利要求1所述的一种卵细胞多效能精准电刺激装置，其特征在于，所述旋转机构包括电机、与所述电机相连接的传动齿轮以及与所述传动齿轮相连接的承载座，所述承载座与所述细胞位姿调整芯片相连接。
  10. 一种卵细胞多效能精准电刺激方法，其特征在于，使用如权利要求1-9中任一项所述的装置，包括以下步骤：
    (1)卵细胞激活阶段：将卵细胞移动至细胞位姿调整芯片上并固定，控制两个液态金属毛细微针电极呈50°～70°夹角紧贴卵细胞透明带，持续通入较高压直流脉冲，然后通过旋转机构使细胞位姿调整芯片旋转，带动固定于细胞位姿调整芯片上的卵细胞水平旋转直至所有的细胞膜都被充分刺激；
    (2)卵细胞去核阶段：对激活后的卵细胞进行去核；
    (3)重构胚电融合阶段：在将核供体注入去核卵细胞构成重构胚后，将重构胚固定在细胞位姿调整芯片上，移动两个液态金属毛细微针电极呈180°夹角紧贴重构胚，并使核供体与去核卵细胞的接触面与电场方向垂直，通入高压直流脉冲，使核供体和去核卵细胞发生电融合，形成重组胚胎；
    (4)重组胚胎培养阶段：将电融合后的重组胚胎置于两个金属平行板微电极间，给金属平行板微电极通入低频交流微电流信号。
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