WO2020207488A1 - 空间电场的发生装置 - Google Patents

空间电场的发生装置 Download PDF

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WO2020207488A1
WO2020207488A1 PCT/CN2020/084382 CN2020084382W WO2020207488A1 WO 2020207488 A1 WO2020207488 A1 WO 2020207488A1 CN 2020084382 W CN2020084382 W CN 2020084382W WO 2020207488 A1 WO2020207488 A1 WO 2020207488A1
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electric field
spatial
electrode
generating
space
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PCT/CN2020/084382
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French (fr)
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乐飚
王丽江
唐万福
王大祥
奚勇
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上海必修福企业管理有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36002—Cancer treatment, e.g. tumour
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/326—Applying electric currents by contact electrodes alternating or intermittent currents for promoting growth of cells, e.g. bone cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01T—SPARK GAPS; OVERVOLTAGE ARRESTERS USING SPARK GAPS; SPARKING PLUGS; CORONA DEVICES; GENERATING IONS TO BE INTRODUCED INTO NON-ENCLOSED GASES
    • H01T19/00—Devices providing for corona discharge
    • H01T19/04—Devices providing for corona discharge having pointed electrodes

Definitions

  • the invention relates to a medical field, in particular to a space electric field generator.
  • Electric field treatment of tumor technology is a comprehensive subject that applies electricity to the treatment of malignant tumors. It has been nearly a century since the effect of electric field on tumor cells was proposed. With the maturity of the theory, people have begun to apply electric field in clinical treatment of tumors. At present, the following three electric field technologies are mainly studied at home and abroad: space electric field (tumor treating fields, namely TT-Fields), DC electric field, and DC pulse electric field.
  • space electric field tumor treating fields, namely TT-Fields
  • DC electric field DC pulse electric field.
  • the AC electric field has been widely used in many fields such as disease diagnosis, scientific research and environmental protection. 10 3 z is less than the low frequency of the alternating electric field may be the cell action potential, it is thus used to stimulate the nerve or muscle electrical activity; high frequency alternating electric field is greater than 10 6 Hz thermal effect on some cell or tissue. Between 10 3 Hz and 10 6 Hz frequency AC electric field of the cause neither depolarization nor larger dielectric consumption, it has the effect interferes with cellular mitosis.
  • Some charged molecules (such as proteins, peptides, DNA, etc.) in the cell will oscillate with the change of the AC electric field.
  • the force will make the direction of movement of charged molecules parallel to the direction of the electric field.
  • a dipole is a molecule separated from the cathode and the anode, and its direction is consistent with the direction of the electric field. Almost all charged molecules move in the direction of the field strength in the constantly changing AC electric field.
  • the division groove is divided into two daughter cells connected by narrow cytoplasm. An uneven electric field is formed near the connection site. Polar molecules move toward the direction of field strength, thereby affecting mitosis.
  • the role of the spindle in mitosis is to divide the sister chromatids evenly into two daughter cells.
  • the microtubule subunits are arranged in parallel according to the direction of the electric field.
  • tubulin is subjected to an electric field with a suitable strength to cause polymerization barriers, which hinder the formation of microtubules. This leads to cell fragmentation.
  • TT-Fields is to place the insulated electrode sheet on the skin around the malignant tumor growth site to provide an electric field.
  • the electric field device works 18 hours a day on average to form two mutually perpendicular alternating electric fields with a strength of 1-2V/cm and a frequency of 200kHz. . It can cover almost all areas of the human body that may be affected, and has no effect on non-differentiated cells. According to reports, in TT-Fields treatment, apart from the contact of the electrode patch in the treatment device with the skin, which may cause local dermatitis, no other side effects have been found. In addition, some studies have pointed out that TT-Fields has low toxicity and good patient response, with high degree of co-treatment.
  • Direct current electric field therapy is electrochemical therapy, which uses electrodes as cathodes and anodes to insert into tumor tissues, and continuously energizes to produce electrolytic ionization, electrophoresis and electroosmosis and other electrochemical reactions to achieve the purpose of destroying or affecting tumor cells.
  • electrochemical therapy uses electrodes as cathodes and anodes to insert into tumor tissues, and continuously energizes to produce electrolytic ionization, electrophoresis and electroosmosis and other electrochemical reactions to achieve the purpose of destroying or affecting tumor cells.
  • the main principle may be that after electrochemical treatment, the pH value of the tissue around the electrode changes significantly, forcibly changing the tumor cells The pH value of surrounding tissues and surrounding tissues.
  • the pulsed electric field contains a variety of frequencies from low to high, and the action time is very short.
  • the biological effect caused is very different from the DC electric field or electrostatic field.
  • the cell can quickly reach the maximum membrane voltage under the action of this external electric field. Due to the electric field stress, it will become thinner and will be broken down to produce electroporation when it reaches a certain critical point.
  • the applied instantaneous pulsed electric field intensity is greater than 1kV/cm, the permeability of the molecules in the cell will be greatly increased; if the pulsed electric field intensity continues to increase, the cell membrane will undergo irreversible electroporation.
  • Pulsed electric fields can be divided into two categories, as follows:
  • Electric pulse chemotheraoy is a tumor treatment method that combines the action of pulsed electric field with chemical drugs.
  • Low-intensity pulsed electric fields can cause reversible electroporation of cell membranes, increase the permeability of cell membranes, and increase the exchange of internal and external molecules, which is conducive to local drug delivery.
  • the degree of cell membrane penetration depends on the intensity and duration of the pulsed electric field, the size of the target cell, and so on.
  • the therapeutic effect of EPCT on superficial tumors such as skin or subcutaneous area has been confirmed, but there is no relevant result on the therapeutic effect of deep tumors.
  • Nanosecond pulsed electric fields refers to a steep pulse electric field with a pulse width of the order of nanoseconds, which can cause irreversible electroporation of tumor cell membranes.
  • nsPEF nanosecond pulsed electric fields
  • NOVOTTF-100A intermediate frequency alternating electric field tumor treatment device developed by Novocure Co., Ltd. is used in clinical practice.
  • the device was approved by the US FDA in 2011 as a recurrent deformable glioblast Second-line treatment after failure of first-line treatment of tumor.
  • Novokule Co., Ltd. applied for a patent in China (CN1976738B, the name of the invention is the use of electric fields of different frequencies to treat tumors).
  • the device claimed in its patent includes an AC voltage source and an insulated electrode; the electric field frequency claimed by its exclusive right is medium and low Frequency, and the low frequency is the main (according to the frequency division standard, the low frequency is generally 30 ⁇ 300KHZ, and the intermediate frequency is generally 300 ⁇ 3000KHZ).
  • the existing electric field therapy equipment and technology have their drawbacks.
  • Second, the frequency of the electric field used is mainly low-frequency, which can generate low-frequency currents. Low-frequency currents will cause changes in the ion concentration in human cells, and electrolysis will occur in the tissue medium to form harmful substances.
  • the purpose of the present invention is to provide a space electric field generating device.
  • the present invention provides a space electric field generating device, which at least includes:
  • the space electric field outer electrode is used to form a space electric field between the space electric field contact electrode; it includes more than two different electrodes, and the position of each different electrode is adjustable to adjust the coverage area of the space electric field.
  • the second aspect of the present invention provides a method for generating a spatial electric field, which at least includes the following steps:
  • the spatial electric field outer electrode includes more than 2 different electrodes, and the position of each different electrode is adjustable to adjust the coverage area of the spatial electric field;
  • the third aspect of the present invention provides a method for using the aforementioned spatial electric field generating device, the method at least including the following steps:
  • the fourth aspect of the present invention provides a method for selectively destroying or inhibiting the growth of tumor cells in a test subject, which is to place the test subject in the aforementioned spatial electric field generating device.
  • the fifth aspect of the present invention provides the use of the aforementioned spatial electric field generating device or the aforementioned spatial electric field generating method including one or more of the following aspects: (1) selectively destroying or inhibiting the growth of tumor cells in the test subject;
  • the spatial electric field generating device of the present invention has the following beneficial effects:
  • Fig. 1 is a schematic diagram of the structure of a device for generating a spatial electric field of the present invention.
  • Fig. 2 is a schematic diagram showing the arrangement of different electrodes of the external electrodes of the spatial electric field of the present invention.
  • Figure 3 shows the imaging diagnosis results of human melanoma cells B16F10 before and after electric field treatment.
  • Figure 4 shows the imaging diagnosis results of human melanoma cell A431 before and after electric field treatment.
  • Figure 5 shows the imaging diagnosis results of the glioblastoma strain SNB-19 before and after electric field treatment.
  • Figure 6 shows the results of imaging diagnosis of human melanoma cells A172 before and after electric field treatment.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. Conventional technology.
  • the present invention provides a space electric field generating device, which at least includes:
  • the space electric field outer electrode is used to form a space electric field between the space electric field contact electrode; it includes more than two different electrodes, and the position of each different electrode is adjustable to adjust the coverage area of the space electric field.
  • the spatial electric field contact electrode is used to connect with the object to be measured, so that the object to be measured and the spatial electric field contact electrode are equipotential.
  • the object to be tested is a living animal. Further, it is a mammal.
  • the mammals are preferably rodents, artiodactyls, odd-hoofed animals, lagomorphs, primates and the like.
  • the primates are preferably monkeys, apes or humans.
  • the spatial electric field contact electrode is grounded.
  • the outer electrode of the spatial electric field does not contact the object to be detected.
  • the generating device further includes:
  • the power supply is used to provide electrical energy to the generating device
  • the regulating unit is used to regulate and control the performance of the space electric field, including one or more of electric field strength, electric field direction, electric field pulse frequency, and electric field lattice range.
  • control unit can adjust the frequency, waveform, and amplitude of the power supply, so that the output high voltage frequency, waveform, and voltage are changed. Changes the intensity, frequency and vibration of the energy acting on the cells. Distinguish the difference between normal tissues and controlled tissues.
  • control unit can adjust the frequency, size, and waveform of the voltage to generate the target electric field intensity. By adjusting the intensity of the target electric field, the effects on different types of tissues and cells can be achieved.
  • the control unit can be used to select different electrodes for power supply, so as to adjust the effect area and electric field intensity of the spatial electric field. By detecting the change of the electric field coupling current, it is possible to track the range, depth and position of the electric field.
  • the spatial electric field contacting the electrode does not affect the selection of the position of the different electrode or the power supply.
  • the position and/or spatial electric field performance of the different electrode is confirmed according to other diagnostic data; and/or, the position, number, and/or spatial electric field performance of the different electrode is diagnosed according to the electric field coupling current. Automatic tracking adjustment.
  • the method for confirming the electric field strength of the spatial electric field is:
  • the tumor cells are cultured under electric fields with different electric field strengths, and the lethality rate of the tumor cells is calculated.
  • the tumor cells are the same type of tumor as the subject to be tested;
  • water is used as a conductive contact between the spatial electric field contact electrode and the object to be detected.
  • the power supply is a high-voltage output power supply.
  • the voltage of the power supply is adjustable from 0.001 kv to 120 kv
  • the current output is adjustable from 0.001 ma to 10000 ma
  • the working distance of the electric field is adjustable from 0.1 cm to 100 cm.
  • the different electrode is a point probe.
  • the different electrodes may be arranged to form a matrix of different electrodes to form a spatial lattice electric field, and the output electric field of the spatial lattice can be precisely controlled according to the drug action range or tumor-related medical imaging data to adapt to the action range.
  • the arrangement of the different electrodes can be selected from a lattice arrangement, which can be a 2n lattice, where n is a non-zero natural number.
  • n is a non-zero natural number.
  • the density of the lattice depends on the accuracy of the electric field. The use of precise electric field to improve the efficiency of tumor cells and reduce the effect on normal tissues. However, limited by the size of the output electric field power supply, the lattice is dense and the power supply is bulky.
  • any one or more warp lines from a ⁇ b ⁇ c to xx and any one or more weft lines from 1 ⁇ 2 ⁇ 3 to nn activate one or more lattice different poles, which are composed of different poles and equipotential poles
  • the electric field is applied, and the electric field coverage is determined by the activated lattice pole.
  • This range can come from the coupling characteristics of tumor cells in the electric field or other digital signals of medical diagnostic imaging. It is confirmed that this electric field range can accurately act on tumor cells and reduce the impact on other tissues.
  • a 64 dot matrix can only distinguish 64 electric field positions
  • a 4096 dot matrix can distinguish 4096 electric field positions
  • a 16777216 dot matrix can distinguish 16777216 electric field positions. The effect of this dot matrix is relatively accurate, and the image of the surrounding normal tissues is small.
  • the present invention also provides a method for generating a spatial electric field, which at least includes the following steps:
  • the spatial electric field outer electrode includes more than 2 different electrodes, and the position of each different electrode is adjustable to adjust the coverage area of the spatial electric field;
  • the living organism refers to a living animal. Further, it is a mammal.
  • the mammals are preferably rodents, artiodactyls, odd-hoofed animals, lagomorphs, primates and the like.
  • the primates are preferably monkeys, apes or humans.
  • the method further includes adjusting and controlling the performance of the spatial electric field, including one or more of electric field strength, electric field direction, electric field pulse frequency, and electric field lattice range.
  • the spatial electric field contact electrode is grounded.
  • the different electrode is a point probe.
  • the arrangement of the different electrodes is selected from a lattice arrangement, which is a 2 n lattice, where n is a non-zero natural number.
  • n is a non-zero natural number.
  • 64 dot matrix 4096 dot matrix
  • 16777216 dot matrix etc.
  • the density of the lattice depends on the accuracy of the electric field.
  • water is used as a conductive contact between the spatial electric field contact electrode and the living organism.
  • the external electrode of the spatial electric field is not in contact with the living organism.
  • the voltage of the power supply is adjustable from 0.001 kv to 120 kv
  • the current output is adjustable from 0.001 ma to 10000 ma
  • the working distance of the electric field is adjustable from 0.1 cm to 100 cm.
  • the method for confirming the electric field strength of the spatial electric field includes the following steps:
  • the tumor cells are cultured under electric fields with different electric field strengths, and the lethality rate of the tumor cells is calculated.
  • the tumor cells are the same type of tumor as the subject to be tested;
  • the present invention also provides a method for using the aforementioned spatial electric field generating device, which is characterized in that the method at least includes the following steps:
  • the object to be tested is a living animal. Further, it is a mammal.
  • the mammals are preferably rodents, artiodactyls, odd-hoofed animals, lagomorphs, primates and the like.
  • the primates are preferably monkeys, apes or humans.
  • the spatial electric field contact electrode is grounded.
  • the outer electrode of the spatial electric field does not contact the object to be detected.
  • the generating device further includes:
  • the power supply is used to provide electrical energy to the generating device
  • the regulating unit is used to regulate and control the performance of the space electric field, including one or more of electric field strength, electric field direction, electric field pulse frequency, and electric field lattice range.
  • control unit can adjust the frequency, waveform, and amplitude of the power supply, so that the output high voltage frequency, waveform, and voltage are changed. Changes the intensity, frequency and vibration of the energy acting on the cells. Distinguish the difference in the role of normal organizations and controlled organizations.
  • control unit can adjust the frequency, size, and waveform of the voltage to generate the target electric field intensity. By adjusting the intensity of the target electric field, the effects on different types of tissues and cells can be achieved.
  • the control unit can be used to select different electrodes for power supply, so as to adjust the effect area and electric field intensity of the spatial electric field. By detecting the change of the electric field coupling current, it is possible to track the range, depth and position of the electric field.
  • the spatial electric field contacting the electrode does not affect the selection of the position of the different electrode or the electrification situation.
  • the position and/or spatial electric field performance of the different electrode is confirmed according to other diagnostic data; and/or, the position, number, and/or spatial electric field performance of the different electrode is diagnosed according to the electric field coupling current. Automatic tracking adjustment.
  • the method for confirming the electric field strength of the spatial electric field is:
  • the tumor cells are cultured under electric fields with different electric field strengths, and the lethality rate of the tumor cells is calculated.
  • the tumor cells are the same type of tumor as the subject to be tested;
  • water is used as a conductive contact between the spatial electric field contact electrode and the object to be detected.
  • the power supply is a high-voltage output power supply.
  • the voltage of the power supply is adjustable from 0.001 kv to 120 kv, and the current output is adjustable from 0.001 ma to 10000 ma.
  • the different electrode is a point probe.
  • the different electrodes may be arranged to form a matrix of different electrodes to form a spatial lattice electric field, and the output electric field of the spatial lattice can be precisely controlled according to the drug action range or tumor-related medical imaging data to adapt to the action range.
  • the arrangement of the different electrodes can be selected from a lattice arrangement, which can be a 2n lattice, where n is a non-zero natural number.
  • n is a non-zero natural number.
  • the density of the lattice depends on the accuracy of the electric field. The use of precise electric field to improve the efficiency of tumor cells and reduce the effect on normal tissues. However, limited by the size of the output electric field power supply, the lattice is dense and the power supply is bulky.
  • any one or more warp lines from a ⁇ b ⁇ c to xx and any one or more weft lines from 1 ⁇ 2 ⁇ 3 to nn activate one or more lattice different poles, which are composed of different poles and equipotential poles
  • the electric field is applied, and the electric field coverage is determined by the activated lattice pole.
  • This range can come from the coupling characteristics of tumor cells in the electric field or other digital signals of medical diagnostic imaging. It is confirmed that this electric field range can accurately act on tumor cells and reduce the impact on other tissues.
  • a 64 dot matrix can only distinguish 64 electric field positions
  • a 4096 dot matrix can distinguish 4096 electric field positions
  • a 16777216 dot matrix can distinguish 16777216 electric field positions. The effect of this dot matrix is relatively accurate, and the image of the surrounding normal tissues is small.
  • the present invention also provides a method for selectively destroying or inhibiting the growth of tumor cells in a subject to be tested, which is to place the subject to be tested in the aforementioned spatial electric field generating device.
  • the present invention also provides the use of the aforementioned spatial electric field generating device or the aforementioned spatial electric field generating method in one or more of the following aspects: (1) selectively destroying or inhibiting the growth of tumor cells in the test subject;
  • the electric field strength and direction of the spatial electric field generator of the present invention can be adjusted.
  • the electric field performance of the generator can also be adjusted as required. Make adjustments to achieve other functions.
  • Experiment 1 Find the optimal range of intensity variation between electrodes that can inhibit human melanoma.
  • Human melanoma has high proliferation and significant drug resistance.
  • Human melanoma cell lines B16-F10 and A431 (conventionally cultured in DMEM medium (containing 10% fetal calf serum, mass concentration of 100mg) /L streptomycin and 100mg/L penicillin), cultured at 37°C, 5% CO 2 , saturated humidity, and passaged once in 2 to 3 days.
  • the method for counting the number of cancer cells per 1 liter of cancer cell suspension Dilute the cancer cell suspension solution (dilute to different dilution levels if necessary), and measure the mixture of different dilution levels. Place 20 microliters of the suspension solution on a cell counting plate, select 3-5 fields at different positions, and count the changes in the number of cells.
  • 7 culture flask is used as a control without connecting electrodes. Add 2 ml of culture medium to each culture flask, and then add 1 ⁇ 10 5 cells respectively, and place them in 5% carbon dioxide; after incubating in a 37° C. incubator for 2-3 hours, implement the electric field treatments A1-A7 as described above. Take out the cell culture flask 24-48 hours after the start of treatment, and count the cells. According to the counting results, statistics of tumor cell changes.
  • the inhibition rate is maximum, which is marked as 100%.
  • the effect of electric field treatment has a tendency to increase significantly with the increase of electric field intensity. Only once, after 24 hours of treatment, when the electric field intensity is greater than 1KV/cm, the effect of killing human melanoma cell lines appears. When the electric field power reaches 10KV/cm, after 24 hours of treatment, almost all cells are killed (see Figure 3 for the effect of killing cells).
  • the electric field treatment effect has a tendency to increase significantly with the increase of electric field intensity.
  • the electric field intensity is greater than 1KV/cm
  • the effect of killing human melanoma cell lines appears.
  • the electric field power reached 3KV/cm, almost all the cells were killed after 48 hours of treatment (see Figure 4 for the effect of killing cells).
  • the same cell-killing effect requires nearly twice the electric field intensity in the 24hr treatment group. It can be predicted that there is a significant correlation between the electric field strength and the efficiency of killing cells.
  • Experiment 2 Find the optimal range of intensity variation between electrodes that can inhibit human glioblastoma.
  • the strongest electric field strength against glioblastoma strains is 120kV/cm.
  • the cells are routinely cultured in DMEM or RPMI1640 medium (containing 10% fetal bovine serum and mass concentration Both are 100mg/L streptomycin and penicillin), cultured under 5% CO 2 and saturated humidity, and passaged once in 2 to 3 days.
  • the method for counting the number of cancer cells per 1 liter of cancer cell suspension Dilute the cancer cell suspension to different dilution levels, and measure 20 ⁇ l of the suspension solution of different dilution levels Place it on the cell counting plate, select 3-5 fields at different positions, and count the changes in the number of cells.
  • 7 culture flask is used as a control without connecting electrodes. Add 2 ml of culture medium to each culture flask, and then add 1 ⁇ 10 5 cells respectively, and place them in 5% carbon dioxide; after incubating in a 37° C. incubator for 2-3 hours, implement the electric field treatments A1-A7 as described above.
  • the cell culture flask was taken out 24 hours and 48 hours after the treatment started, and the cell count was performed. According to the counting results, statistics of tumor cell changes.
  • the inhibition rate is maximum, which is marked as 100%.
  • the effect of electric field treatment tends to increase significantly with the increase of electric field intensity.
  • the electric field intensity is greater than 1KV/cm
  • the effect of killing human glioma cell lines appears.
  • the electric field power reached 3KV/cm, almost all cells were killed after 48 hours of treatment.
  • the same cell-killing effect requires nearly twice the electric field intensity in the 24hr treatment group. It can be predicted that there is a significant correlation between the electric field strength and the efficiency of killing cells.
  • Mouse melanoma cells were grown on coverslips and cultured in TT-fields for 24 and 48 hours. After treatment, remove the medium, wash the cells in a buffer solution [10mM 4-morpholineethanesulfonic acid, 150mM NaCl, 5mM EGTA, 5mM MgCl 2 and 5mM glucose (pH6.1)], infiltrate and use 0.5% Triton X-100 and Fix with 0.25% glutaraldehyde (sigma) for 5 minutes, then fix with 1% glutaraldehyde for 20 minutes and take pictures.
  • a buffer solution 10mM 4-morpholineethanesulfonic acid, 150mM NaCl, 5mM EGTA, 5mM MgCl 2 and 5mM glucose (pH6.1)
  • Triton X-100 Fix with 0.25% glutaraldehyde (sigma) for 5 minutes, then fix with 1% glutaraldehyde for 20 minutes and take pictures.
  • mice were cultured in separate cages under conditions without specific pathogens.
  • the tumor cells in the logarithmic growth phase were digested with 0.25% trypsin, resuspended in PBS at a cell density of 5 ⁇ 10 6 mL -1 each, and inoculated into the right forelimb of mice.
  • mice After 3 weeks, after forming a subcutaneous solid tumor with a size of 1cm ⁇ 1cm, they were randomly divided into 2 groups: control group and experimental group, each with 12 animals. Mice in the experimental group were treated with electric field, and mice in the control group were not treated with electric field.
  • HE staining to observe the infiltration of inflammatory cells in the tumor after treatment After 24 hours of electric field treatment, 4 mice were randomly selected from the experimental group and the control group, respectively, were sacrificed to take the tumor, and the volume fraction of 4% paraformaldehyde was used at room temperature. After fixation for 20 hours, routine dehydration, embedding, and paraffin sectioning were performed. 6 pieces of each specimen were taken. After HE staining, the inflammatory cell infiltration was observed under a light microscope.
  • the start date of electric field treatment was set to D0, and the experimental group was anesthetized every 2 days after the start of treatment, and the body weight and tumor volume were measured.
  • the weight of the experiment is in the interval D0 to D24, and there is almost no significant change. There were no obvious signs of scalding or depilation on the surface of the experimental animals.
  • 60KV/cm of the median electric field intensity is the first choice for verification.
  • D6-D12 is the stable phase. The tumor volume begins to decrease at D12 after continuous treatment, and until D24, the tumor volume shows a continuous downward trend.

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Abstract

本发明提供一种空间电场的发生装置，至少包括：空间电场接触电极；空间电场外电极，用于与所述空间电场接触电极之间形成空间电场；包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积。发明的空间电场的发生装置，生产成本低，适用性更强，治疗效果更好，安全性能更高，用途更广泛。

Description

空间电场的发生装置 技术领域
本发明涉及一种医疗领域，特别是涉及一种空间电场的发生装置。
背景技术
电场治疗肿瘤技术是将电学应用于治疗恶性肿瘤的综合性课题。电场对肿瘤细胞的作用从提出到现在已经经历了近一个世纪，随着理论的日趋成熟，人们已经开始将电场应用于临床治疗肿瘤。目前国内外主要研究以下3种电场技术：空间电场(tumor treating fields,即TT—Fields)、直流电场、直流脉冲电场。
1、空间电场(tumor treating fields，TT—Fields)概述
交流电场已在疾病诊断、科研和环保等多个领域应用广泛。低于10 3z的低频率交流电场可以使细胞产生动作电位，因而被用来刺激神经或肌肉的生物电活动；高于10 6Hz的高频交流电场对一些细胞或组织产生热效应。介于10 3Hz和10 6Hz的中频交流电场则既不会导致去极化也不会较大的介电消耗，具有干扰细胞有丝分裂的作用。
1.1 TT—Fields抑制癌细胞分裂的机制
细胞内的一些带电分子(如蛋白、多肽和DNA等)，它们会随所处交流电场的变化而发生振荡。在匀场电场中，作用力会使带电分子的运动方向平行于电场方向。偶极子是阴极和阳极分离的分子，它的方向与电场方向保持一致。几乎所有带电分子，在不断变化的交流电场中均会朝着场强强的方向运动。处在有丝分裂末期的细胞，分裂沟分割成两个由狭窄细胞质连接的子细胞，在连接部位附近形成不均匀电场，极性分子朝着场强强的方向运动，从而影响有丝分裂。
(1)有丝分裂纺锤体形成的停止
纺锤体在有丝分裂中的作用是将姐妹染色单体平均分到两个子细胞中。在没有进行分裂的细胞中，微管亚单位会根据电场方向而平行排列。细胞进行有丝分裂的间期时，微管蛋白因受到合适强度的电场力而引起聚合障碍，使微管形成受阻。进而导致细胞发生碎裂。
(2)有丝分裂沟的破坏
所有有丝分裂的细胞都会发生纺锤体形成受阻的情况。有丝分裂后期的细胞，两个子染色体将被拉到细胞达的两极。在有丝分裂末期刚进行时，分裂沟最终形成，并将形成两个完全一样的细胞。而在这个狭窄的细胞膜连接处，将产生一个沙漏样非均匀电场。场强强度最 大的位置是狭窄部位的中心位置，其中的场强将使带电粒子受到单向电场力的作用，使这些带电粒子向分裂沟方向发生移动。在TT—Fields也是利用此原理，使细胞内结构紊乱，产生细胞破坏作用。
1.2 TT—Fields的治疗特点
TT—Fields是将绝缘电极片放恶性肿瘤生长部位外周的皮肤来提供电场，电场装置平均每天工作18小时，形成两个相互垂直的强度在1-2V/cm之间、频率为200kHz的交流电场。可覆盖人体可能发病的几乎所有区域，且对非分化细胞无影响。据报道，TT—Fields治疗中，除了治疗设备中的电极贴片与皮肤接触可能导致局部皮炎外，尚未发现其他毒副作用。另外，有研究指出，TT—Fields毒性小且患者反应良好，配合治疗度高。
2直流电场
直流电场治疗即电化学治疗，是将电极分别用作阴极和阳极插入肿瘤组织中，持续地通电，产生电解电离、电泳电渗等电化学反应，达到破坏或影响肿瘤细胞的目的。近年来，虽然已有众多科学家研究电化学治疗肿瘤的机制，但机理仍不十分明确，其最主要的原理可能为电化学处理后，电极周围组织的pH值发生显著变化，强制改变了肿瘤细胞及周围组织的pH值，在pH≤4或pH≥10的环境中，大部分酶蛋白失活变性，细胞膜的通透性改变，使肿瘤发生诸如如细胞核凝固、细胞膜崩溃、线粒体消失、核蛋白凝固坏死产等多种病理效应，最终导致癌细胞死亡。
3、脉冲电场
脉冲电场含有从低至高的多种频率，作用时间极短，所引起的生物学效应与直流电场或静电场存在很大区别，细胞在这种外加电场的作用下可以快速达到最大膜电压，细胞膜由于受到电场应力的作用会变薄，达到某一临界点时会被击穿产生电穿孔。当施加的瞬时脉冲电场强度大于1kV/cm时，细胞中分子的渗透率会大大提高；如果脉冲电场强度继续增加，细胞膜将发生不可逆性电穿孔。脉冲电场可以分为两大类，具体如下：
3.1电脉冲化疗
电脉冲化疗(electric pulse chemotheraoy，EPCT)是一种将脉冲电场作用与化学药物相结合的肿瘤治疗方法。低强度的脉冲电场可以对细胞膜造成可逆性电穿孔，细胞膜通透性增大，内外分子交换增加，有利于局部给药发挥作用。细胞膜渗透程度取决于脉冲电场强度和持续时间、目标细胞的大小等。目前EPCT对皮肤或皮下等浅表部位肿瘤的治疗效果已得到肯定，而对深部肿瘤的治疗效果还没有相关的结果。
3.2纳秒级脉冲电场
纳秒级脉冲电场(nanosecond pulsed electric fields，nsPEF)指脉宽在纳秒数量级的陡脉冲电场，陡脉冲电场能使肿瘤细胞膜发生不可逆性电穿孔。目前国内外对陡脉冲电场治疗肿瘤的研究已经取得了很大的进展，nsPEF技术为恶性肿瘤治疗提供了一种高效、无能量依赖性的工具。其抗肿瘤机制比较复杂，除了直接破坏肿瘤细胞外还能导致其凋亡，但nsPEF诱导肿瘤细胞凋亡的具体机制尚未完全明确。
目前全球只有诺沃库勒(Novocure)有限公司开发的NOVOTTF-100A中频交变电场肿瘤治疗设备用于临床，所述设备于2011年通过美国FDA的审批，作为复发性变形性脑胶质母细胞瘤一线治疗失败后的二线治疗。诺沃库勒有限公司在中国申请的专利(CN1976738B，发明名称为使用不同频率的电场治疗肿瘤)，其专利权利要求的装置包括AC电压源、绝缘电极；其专权利要求的电场频率属于中低频率，并以低频为主(按频率划分标准低频一般为30～300KHZ，中频一般为300～3000KHZ)。
现有的电场治疗设备及技术有其缺陷性。第一，因为不同类型的肿瘤对场强的敏感性不同，所以固定强度的电场用于治疗肿瘤时，正常区的细胞、组织、器官会引起电场力线的改变，故难以确定肿瘤细胞所接触的真实电场强度，无法进行精准的电场治疗，可能会使电场强度达不到治疗的效果。第二，其使用的电场频率以低频为主，可产生低频电流，低频电流会导致人体细胞内离子浓度发生变化，并在组织的介质中发生电解形成有害物质。第三，如果绝缘电极绝缘层失效或击穿后则可造成机体的严重损害，故其临床应用存在安全隐患。第四，长时间佩戴与皮肤直接接触的电极会导致皮肤的过敏反应。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种空间电场的发生装置。
为实现上述目的及其他相关目的，本发明提供一种空间电场的发生装置，所述发生装置至少包括：
空间电场接触电极；
空间电场外电极，用于与所述空间电场接触电极之间形成空间电场；包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积。
本发明第二方面提供一种空间电场的发生方法，至少包括以下步骤：
1)提供空间电场接触电极，并使所述空间电场接触电极接触生物活体；
2)提供空间电场外电极，所述空间电场外电极包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积；
3)将所述空间电场接触电极及所述空间电场外电极接通电源，产生空间电场。
本发明第三方面提供前述空间电场的发生装置的使用方法，所述方法至少包括如下步骤：
1)将待检测对象空间电场接触电极连接，使待测对象与所述空间电场接触电极形成等电位；
2)调整各个异电极的位置，以调整空间电场的覆盖面积。
本发明第四方面提供一种选择性破坏或抑制待测对象体内肿瘤细胞生长的方法，为将待测对象置于前述空间电场的发生装置中。
本发明第五方面提供前述空间电场的发生装置或前述空间电场的发生方法在包括以下一个或多个方面的用途：(1)选择性破坏或抑制待测对象体内肿瘤细胞生长；
(2)选择性的调节或刺激如脑神经干细胞或神经元细胞的分裂/生长速率；
(3)降低血脂；(4)改善动脉硬化；(5)防止血栓堵塞血管；(6)清除血液中的毒素；
(7)调节自律神经；(8)通过调节/清除自由基，提高人体免疫力。
如上所述，本发明的空间电场的发生装置，具有以下有益效果：
(1)适用性更强，治疗效果更好。
(2)安全性能更高。
(3)生产成本低。
(4)用途广泛。
附图说明
图1显示为本发明空间电场的发生装置结构示意图。
图2显示为本发明空间电场外电极的异电极的排布方式示意图。
图3显示为人黑色素瘤细胞B16F10的电场治疗前后的影像学诊断结果。
图4显示为人黑色素瘤细胞A431的电场治疗前后的影像学诊断结果。
图5显示为胶质母细胞瘤株SNB-19的电场治疗前后的影像学诊断结果。
图6显示为人黑色素瘤细胞A172的电场治疗前后的影像学诊断结果。
图7显示为电场治疗效果示意图(n＝6，电场60KV/cm)。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1至图7。须知，本说明书所附图式所绘示的结构、比例、大小等，均仅用以配合说明书所揭示的内容，以供熟悉此技术的人士了解与阅读，并非用以限定本发明可实施的限定条件，故不具技术上的实质意义，任何结构的修饰、比例关系的改变或大小的调整， 在不影响本发明所能产生的功效及所能达成的目的下，均应仍落在本发明所揭示的技术内容能涵盖的范围内。同时，本说明书中所引用的如“上”、“下”、“左”、“右”、“中间”及“一”等的用语，亦仅为便于叙述的明了，而非用以限定本发明可实施的范围，其相对关系的改变或调整，在无实质变更技术内容下，当亦视为本发明可实施的范畴。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
如图1所示，本发明提供一种空间电场的发生装置，所述发生装置至少包括：
空间电场接触电极；
空间电场外电极，用于与所述空间电场接触电极之间形成空间电场；包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积。
进一步的，所述空间电场接触电极用于与待测对象相连接，使待测对象与所述空间电场接触电极形成等电位。
所述待测对象为动物活体。进一步的，为哺乳动物。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
在一种实施方式中，所述空间电场接触电极接地。
进一步的，所述空间电场外电极不与待检测对象接触。
在一种实施方式中，所述发生装置还包括：
供电电源，用于给所述发生装置提供电能；
调控单元，用于调节控制空间电场的性能，包括电场强度、电场方向、电场脉冲频率以及电场点阵范围中的一种或多种。
在一种实施方式中，所述调控单元可以对电源的频率、波形、幅度调整，从而输出高压频率、波形和电压发生改变。使作用在细胞上的能量强度、频次、震动发生变化。区别正常组织以及被控组织的作用差异。
在一种实施方式中，所述调控单元可以对电压的频率、大小以及波形进行调整，从而产生目标电场强度。通过对目标电场强度的调整，实现分别对不同类型组织以及细胞的作用。
可以利用所述调控单元选择给不同的异电极进行供电，以便调节空间电场的作用区域及电场强度。通过检测电场耦合电流变化，可以跟踪电场作用范围、电场的作用深度与位置。
空间电场接触电极不影响异电极位置或通电情况的选择。
在一种实施方式中，所述异电极的位置和/或空间电场的性能根据其他诊断数据确认；和 /或，所述异电极的位置、数量和/或空间电场性能根据电场耦合电流诊断后自动跟踪调整。
在一种实施方式中，所述空间电场的电场强度的确认方法为：
1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
在一种实施方式中，所述空间电场接触电极与待检测对象之间通过水作为导电接触。
在一种实施方式中，所述供电电源为高压输出电源。
在一种实施方式中，所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000ma，电场的工作距离为可调的0.1cm-100cm。
在一种实施方式中，所述异电极为点状探头。
在一种实施方式中，所述异电极可以排布形成矩阵异电极，来组成空间点阵电场，根据药物作用范围或肿瘤相关医学影像学数据，精确控制空间点阵输出电场，适应作用范围。
在一种实施方式中，所述异电极的排布方式可选自点阵排布，可以为2 n的点阵，其中n为非0自然数。例如64点阵、4096点阵、16777216点阵等，如图2所示。点阵密集程度取决于电场作用精确度。利用精确作用电场提高对肿瘤细胞作用效率、减少对正常组织作用影响。但受输出电场电源体积限制，点阵密集而电源体积庞大。其中a\b\c一直到xx的任意一或多条经线和1\2\3一直到nn任意一或多条纬线激活一个或多个点阵异极，这些异极和等电位位极组成作用电场，电场覆盖范围是激活的点阵极决定的。这个范围可以来自肿瘤细胞在电场中的耦合特性或其他医学诊断影像数字信号。确认了这个电场范围，可以精确作用肿瘤细胞，减少对其他组织影响。一般64点阵只能区分64个电场位置，4096点阵可以区分4096个电场位置，16777216点阵可以分辨16777216个电场位置，这个点阵作用就相对精确，对周围正常组织影像就较小。
本发明还提供空间电场的发生方法，至少包括以下步骤：
1)提供空间电场接触电极，并使所述空间电场接触电极接触生物活体；
2)提供空间电场外电极，所述空间电场外电极包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积；
3)将所述空间电场接触电极及所述空间电场外电极接通电源，产生空间电场。
所述生物活体是指动物活体。进一步的，为哺乳动物。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
进一步的，所述方法还包括调节控制空间电场的性能，包括电场强度、电场方向、电场 脉冲频率以及电场点阵范围中的一种或多种。
在一种实施方式中，所述空间电场接触电极接地。
在一种实施方式中，所述异电极为点状探头。
在一种实施方式中，所述异电极的排布方式选自点阵排布，为2 n点阵，其中n为非0自然数。例如64点阵、4096点阵、16777216点阵等，如图2所示。点阵密集程度取决于电场作用精确度。
在一种实施方式中，所述空间电场接触电极与生物活体之间通过水作为导电接触。
在一种实施方式中，所述空间电场外电极不与生物活体接触。
在一种实施方式中，所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000ma，电场的工作距离为可调的0.1cm-100cm。
在一种实施方式中，所述空间电场的电场强度的确认方法包括以下步骤：
1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
本发明还提供前述空间电场的发生装置的使用方法，其特征在于，所述方法至少包括如下步骤：
1)将待检测对象空间电场接触电极连接，使待测对象与所述空间电场接触电极形成等电位；
2)调整各个异电极的位置，以调整空间电场的覆盖面积。
进一步的，所述待测对象为动物活体。进一步的，为哺乳动物。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
在一种实施方式中，所述空间电场接触电极接地。
进一步的，所述空间电场外电极不与待检测对象接触。
在一种实施方式中，所述发生装置还包括：
供电电源，用于给所述发生装置提供电能；
调控单元，用于调节控制空间电场的性能，包括电场强度、电场方向、电场脉冲频率以及电场点阵范围中的一种或多种。
在一种实施方式中，所述调控单元可以对电源的频率、波形、幅度调整，从而输出高压频率、波形和电压发生改变。使作用在细胞上的能量强度、频次、震动发生变化。区别正常 组织以及被控组织的作用差异。
在一种实施方式中，所述调控单元可以对电压的频率、大小以及波形进行调整，从而产生目标电场强度。通过对目标电场强度的调整，实现分别对不同类型组织以及细胞的作用。
可以利用所述调控单元选择给不同的异电极进行供电，以便调节空间电场的作用区域及电场强度。通过检测电场耦合电流变化，可以跟踪电场作用范围、电场的作用深度与位置。
空间电场接触电极不影响异电极位置或通电情况的选择。
在一种实施方式中，所述异电极的位置和/或空间电场的性能根据其他诊断数据确认；和/或，所述异电极的位置、数量和/或空间电场性能根据电场耦合电流诊断后自动跟踪调整。
在一种实施方式中，所述空间电场的电场强度的确认方法为：
1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
在一种实施方式中，所述空间电场接触电极与待检测对象之间通过水作为导电接触。
在一种实施方式中，所述供电电源为高压输出电源。
在一种实施方式中，所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000ma。
在一种实施方式中，所述异电极为点状探头。
在一种实施方式中，所述异电极可以排布形成矩阵异电极，来组成空间点阵电场，根据药物作用范围或肿瘤相关医学影像学数据，精确控制空间点阵输出电场，适应作用范围。
在一种实施方式中，所述异电极的排布方式可选自点阵排布，可以为2 n的点阵，其中n为非0自然数。例如64点阵、4096点阵、16777216点阵等，如图2所示。点阵密集程度取决于电场作用精确度。利用精确作用电场提高对肿瘤细胞作用效率、减少对正常组织作用影响。但受输出电场电源体积限制，点阵密集而电源体积庞大。其中a\b\c一直到xx的任意一或多条经线和1\2\3一直到nn任意一或多条纬线激活一个或多个点阵异极，这些异极和等电位位极组成作用电场，电场覆盖范围是激活的点阵极决定的。这个范围可以来自肿瘤细胞在电场中的耦合特性或其他医学诊断影像数字信号。确认了这个电场范围，可以精确作用肿瘤细胞，减少对其他组织影响。一般64点阵只能区分64个电场位置，4096点阵可以区分4096个电场位置，16777216点阵可以分辨16777216个电场位置，这个点阵作用就相对精确，对周围正常组织影像就较小。
本发明还提供一种选择性破坏或抑制待测对象体内肿瘤细胞生长的方法，为将待测对象 置于前述空间电场的发生装置中。
本发明还提供前述空间电场的发生装置或前述空间电场的发生方法在包括以下一个或多个方面的用途：(1)选择性破坏或抑制待测对象体内肿瘤细胞生长；
(2)选择性的调节或刺激如脑神经干细胞或神经元细胞的分裂/生长速率；
(3)降低血脂；(4)改善动脉硬化；(5)防止血栓堵塞血管；(6)清除血液中的毒素；
(7)调节自律神经；(8)通过调节/清除自由基，提高人体免疫力。
本发明所述的空间电场的发生装置的电场强度及方向等性能均能调节，除了可以选择性破坏或抑制待测对象体内肿瘤细胞生长外，也可根据需要，对所述发生装置的电场性能进行调节，以实现其他功能。
实验方案
实验一：寻找对人类黑色素瘤产生抑制作用的最优的电极间强度变化范围。
人类黑色素瘤具有较高的增殖性，显著的耐药性，将人类黑色素瘤细胞株B16-F10与A431(常规培养于DMEM培养液(含体积分数为10％的胎牛血清、质量浓度为100mg/L的链霉素和质量浓度为100mg/L的青霉素)中，在37℃温度、体积分数为5％的CO 2、饱和湿度下培养，2～3d时间传代1次。
接上所述，癌细胞混悬溶液中每1升中含癌细胞个数的计数方法：将癌细胞混悬溶液(如有需要稀释到不同稀释级数)，量取不同稀释级数的混悬溶液20微升置细胞计数盘上，在不同位置选取3-5个视野，计数细胞数量的变化情况。
取编号为1号-7号细胞培养瓶，1号培养瓶安装电极对A1，电场强度为1V/cm；2号培养瓶安装电极对A2，电极间的电场强度为10V/cm；3号培养瓶安装电极对A3，电极间的电场强度为100V/cm；4号培养瓶安装电极对A4，电极间的电场强度为1KV/cm；5号培养瓶安装电极对A5，电极间的电场强度为10KV/cm；6号培养瓶安装电极对A6，电极间的电场强度为120KV/cm；7号培养瓶作为对照不接入电极。各培养瓶各加入培养液2毫升，再分别添加1x10 5个细胞后，静置于5％二氧化碳；37℃培养箱孵育2-3小时后实施如上所述A1-A7的电场治疗。治疗开始24-48小时后取出细胞培养瓶，进行细胞计数。根据计数结果，统计肿瘤细胞的变动情况。
实验结果如表1与表2所示。
Figure PCTCN2020084382-appb-000001
None：治疗组与对照组的细胞数量无差异
细胞抑制效率＝(对照组的活细胞数量-治疗组存活的细胞数量)/对照组的活细胞数量
当治疗组的存活细胞数量为零或与对照组的活细胞数量相比较数量少于千分之一时，抑制率最大，标记为100％。
如表1所示，电场治疗效果伴随电场强度的增加，有明显增强的趋势。仅仅1次，24hr的治疗后，当电场强度大于1KV/cm时，出现杀死人类黑色素瘤细胞株的效果。当电场功率达到10KV/cm时，治疗24hr，几乎全部的细胞都被杀伤(杀伤细胞效果图见图3)。
Figure PCTCN2020084382-appb-000002
如表2所示，电场治疗效果伴随电场强度的增加，有明显增强的趋势。1次，48hr的治疗后，当电场强度大于1KV/cm时，出现杀死人类黑色素瘤细胞株的效果。当电场功率达到3KV/cm时，治疗48hr后，几乎全部的细胞都被杀伤(杀伤细胞效果图见图4)。相同的杀伤细胞效果，在24hr治疗组需要近乎2倍的电场强度。可以预见，电场强度与杀伤细胞效率中存在显著的关联。
实验二：寻找对人类胶质母细胞瘤产生抑制作用的最优的电极间强度变化范围。
对神经胶质母细胞瘤株(SNB-19与A172)抑制最强的电场强度为120kV/cm，细胞常规培养于DMEM或RPMI1640培养基中(含有体积分数为10％的胎牛血清以及质量浓度均为100mg/L的链霉素和青霉素)，在体积分数为5％的CO 2、饱和湿度下培养，2～3d时间传代1次。
接上所述，癌细胞混悬溶液中每1升中含癌细胞个数的计数方法：将癌细胞混悬溶液稀释到不同稀释级数，量取不同稀释级数的混悬溶液20微升置于细胞计数盘上，在不同位置选取3-5个视野，计数细胞数量的变化情况。
取编号为1号-7号细胞培养瓶，1号培养瓶安装电极对A1，电场强度为1V/cm；2号培养瓶安装电极对A2，电极间的电场强度为10V/cm；3号培养瓶安装电极对A3，电极间的电场强度为100V/cm；4号培养瓶安装电极对A4，电极间的电场强度为1KV/cm；5号培养瓶安装电极对A5，电极间的电场强度为10KV/cm；6号培养瓶安装电极对A6，电极间的电场强度为120KV/cm；7号培养瓶作为对照不接入电极。各培养瓶各加入培养液2毫升，再分别添加1x10 5个细胞后，静置于5％二氧化碳；37℃培养箱孵育2-3小时后实施如上所述A1-A7的电场治疗。治疗开始24小时及48小时后取出细胞培养瓶，进行细胞计数。根据计数结果，统计肿瘤细胞的变动情况。
None：治疗组与对照组的细胞数量无差异
细胞抑制效率＝(对照组的活细胞数量-治疗组存活的细胞数量)/对照组的活细胞数量
当治疗组的存活细胞数量为零或与对照组的活细胞数量相比较数量少于千分之一时，抑制率最大，标记为100％。
如表3所示，电场治疗效果伴随电场强度的增加，有明显增强的趋势。仅仅1次，24hr的治疗后，当电场强度大于1KV/cm时，出现杀死人类脑胶质瘤细胞株的效果。当电场功率达到10KV/cm时，治疗24hr，几乎全部的细胞都被杀伤。杀伤细胞效果图见图5-6。
Figure PCTCN2020084382-appb-000003
如表4所示，电场治疗效果伴随电场强度的增加，有明显增强的趋势。1次，48hr的治疗后，当电场强度大于1KV/cm时，出现杀死人类胶质瘤细胞株的效果。当电场功率达到3KV/cm时，治疗48hr后，几乎全部的细胞都被杀伤。相同的杀伤细胞效果，在24hr治疗组需要近乎2倍的电场强度。可以预见，电场强度与杀伤细胞效率中存在显著的关联。
Figure PCTCN2020084382-appb-000004
在表3与表4所述实验中有A72在1KV/cm(24hr治疗组)与100v/cm及1KV/cm(48hr)治疗组中首次发现治疗后，肿瘤细胞的数量出现增长的现象。以100v/cm到3KV/cm为ROI(核心管擦区域)展开了进一步的探索(date not shown)。我们发现了一个有趣的现象。类似与脑胶质瘤细胞A172这样的电场敏感型细胞在不同的电场应激状态下，出现细胞增长速率两极明显分化的现象。该现象的发现，意味着电场敏感型的细胞或生体组织甚至是器官，软组织等都将具备以下多方面的应用。如：选择性破坏或抑制待测对象体内肿瘤细胞生长；选择性的调节或刺激如脑神经干细胞或神经元细胞的分裂/生长速率；降低血脂；改善动脉硬化；防止血栓堵塞血管；清除血液中的毒素；调节自律神经或通过调节/清除自由基，提高人体免疫力。
实验三：治疗前后肿瘤细胞增长情况的影像学观察
小鼠黑色素瘤细胞在盖玻片上生长，并在TT-fields中培养24与48小时。处理后，去除培养基，在缓冲溶液[10mM 4-吗啉乙磺酸、150mMNaCl、5mM EGTA、5mM MgCl 2和5mM葡萄糖(pH6.1)]中清洗细胞，渗透并用0.5％Triton X-100和0.25％戊二醛(sigma)固定5分钟，然后用1％戊二醛固定20分钟并拍照。
实验四：小鼠皮下肿瘤模型的建立以及电场处理实验
小鼠在无特定病原条件下分笼培养。取对数生长期的肿瘤细胞，用体积分数为0.25％的胰酶消化后，按每只5×10 6mL -1的细胞数密度重悬于PBS后，接种于小鼠右前肢。经过3周时间，在形成尺寸为1cm×1cm的皮下实体瘤后，随机分为2组：对照组和实验组，每组12只。实验组小鼠进行电场处理，对照组小鼠不进行电场处理。
记录肿瘤生长抑制情况：电场治疗当天作为起始第1d，每隔1d，拍照记录裸鼠肿瘤和体质量变化，每隔2d用游标卡尺测量实验组和对照组肿瘤的最大横径a和最大纵径b，按计算式V＝ab 2/2计算肿瘤体积V。
HE染色观察治疗后的肿瘤内炎性细胞浸润情况：在电场治疗24h时间后，从实验组和对照组分别随机抽取4只小鼠处死取瘤，用体积分数为4％的多聚甲醛在室温下固定20h时间后，常规脱水、包埋、石蜡切片，每个标本取6片，HE染色后在光镜下观察炎性细胞浸润情况。
如图7所示，电场治疗开始日设定为D0，治疗开始后每隔2天将实验组麻醉后检测体重与肿瘤体积。肿瘤体积按椭圆体积计算公式V＝长径*短径*短径/2计算得到。首先实验的体重在D0到D24区间，几乎无明显变动。实验动物体表无明显烫伤，脱毛等迹象。治疗电场强度首选中位电场强度区间的60KV/cm实施验证。治疗开始一周左右，实验动物表面的肿瘤体积增长趋势趋于稳定。D6-D12为稳定期，连续治疗D12开始肿瘤体积开始下降，到D24为止，肿瘤体积呈现持续下降趋势。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (14)

  1. 一种空间电场的发生装置，其特征在于，所述发生装置至少包括：
    空间电场接触电极；
    空间电场外电极，用于与所述空间电场接触电极之间形成空间电场；包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积。
  2. 根据权利要求1所述的空间电场的发生装置，其特征在于：还包括以下特征中的一项或多项：
    1)所述空间电场接触电极用于与待测对象相连接，使待测对象与所述空间电场接触电极形成等电位；
    2)所述空间电场接触电极接地；
    3)所述发生装置还包括：
    供电电源，用于给所述发生装置提供电能；
    调控单元，用于调节控制空间电场的性能，包括电场强度、电场方向、电场脉冲频率以及电场点阵范围中的一种或多种；
    4)所述异电极为点状探头；
    5)所述异电极的排布方式选自点阵排布，为2 n点阵，其中n为非0自然数。
  3. 根据权利要求2所述的空间电场的发生装置，其特征在于：还包括以下特征中的一项或多项：
    1)特征1)中，所述空间电场接触电极与待检测对象之间通过水作为导电接触；
    2)特征1)中，所述空间电场外电极不与待检测对象接触；
    3)特征3)中，所述异电极的位置和/或空间电场的性能根据其他诊断数据确认；和/或，所述异电极的位置、数量和/或空间电场性能根据电场耦合电流诊断后自动跟踪调整；
    4)特征3)中，所述供电电源为高压输出电源；
    5)特征3)中，所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000ma，电场的工作距离为可调的0.1cm-100cm。
  4. 根据权利要求1所述的空间电场的发生装置，其特征在于：所述空间电场的电场强度的确认方法包括以下步骤：
    1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
    2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
  5. 一种空间电场的发生方法，至少包括以下步骤：
    1)提供空间电场接触电极，并使所述空间电场接触电极接触生物活体；
    2)提供空间电场外电极，所述空间电场外电极包括2个以上异电极，各个异电极的位置可调，用于调整空间电场的覆盖面积；
    3)将所述空间电场接触电极及所述空间电场外电极接通电源，产生空间电场。
  6. 根据权利要求5所述的空间电场的发生方法，其特征在于，还包括调节控制空间电场的性能，包括电场强度、电场方向、电场脉冲频率以及电场点阵范围中的一种或多种。
  7. 根据权利要求5所述的空间电场的发生方法，其特征在于，所述发生方法还包括以下特征中的一个或多个：
    1)所述空间电场接触电极接地；
    2)所述异电极为点状探头；
    3)所述异电极的排布方式选自点阵排布，为2 n点阵，其中n为非0自然数；
    4)所述空间电场接触电极与生物活体之间通过水作为导电接触；
    5)所述空间电场外电极不与生物活体接触；
    6)所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000ma，电场的工作距离为可调的0.1cm-100cm。
  8. 根据权利要求6所述的空间电场的发生方法，其特征在于，所述空间电场的电场强度的确认方法包括以下步骤：
    1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
    2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
  9. 根据权利要求1-4任一所述的空间电场的发生装置的使用方法，其特征在于，所述方法至少包括如下步骤：
    1)将待检测对象空间电场接触电极连接，使待测对象与所述空间电场接触电极形成等电位；
    2)调整各个异电极的位置，以调整空间电场的覆盖面积。
  10. 根据权利要求9所述的空间电场的发生装置的使用方法，其特征在于，所述使用方法还包括以下特征中的一个或多个：
    1)所述空间电场接触电极接地；
    2)所述异电极为点状探头；
    3)所述异电极的排布方式选自点阵排布，为2 n点阵，其中n为非0自然数；
    4)所述空间电场接触电极与待检测对象之间通过水作为导电接触；
    5)所述空间电场外电极不与待检测对象接触；
    6)所述供电电源的电压为可调的0.001kv-120kv，电流输出为可调的0.001ma-10000m，电场的工作距离为可调的0.1cm-100cm；
    7)所述使用方法还包括以下步骤：
    调节控制空间电场的性能，包括电场强度、电场方向、电场脉冲频率以及电场点阵范围中的一种或多种。
  11. 根据权利要求10所述的空间电场的发生装置的使用方法，其特征在于，特征7)中，所述异电极的位置和/或空间电场的性能根据其他诊断数据确认；和/或，所述异电极的位置、数量和/或空间电场性能根据电场耦合电流诊断后自动跟踪调整。
  12. 根据权利要求10所述的空间电场的发生装置的使用方法，其特征在于，特征7)中，所述空间电场的电场强度的确认方法包括以下步骤：
    1)将肿瘤细胞在不同电场强度的电场下进行培养，统计肿瘤细胞致死率，所述肿瘤细胞与待测对象所患的肿瘤种类相同；
    2)选择肿瘤细胞致死率最高的一组的电场强度范围作为所述空间电场的电场强度范围。
  13. 一种选择性破坏或抑制待测对象体内肿瘤细胞生长的方法，为将待测对象置于如权利要求1-4任一所述的空间电场的发生装置中。
  14. 如权利要求1-4任一所述的空间电场的发生装置或权利要求5-8任一所述的空间电场的发生方法在包括以下一个或多个方面的用途：
    (1)选择性破坏或抑制待测对象体内肿瘤细胞生长；
    (2)选择性的调节或刺激如脑神经干细胞或神经元细胞的分裂/生长速率；
    (3)降低血脂；(4)改善动脉硬化；(5)防止血栓堵塞血管；(6)清除血液中的毒素；
    (7)调节自律神经；(8)通过调节/清除自由基，提高人体免疫力。
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