CN103146750A - Electrotransfection method and device - Google Patents

Abstract

The invention relates to a method and equipment for electrotransfection. The method includes a positive high-voltage pulse discharge of 500-1500V, 20-60μs to the cells, a pause of 600-1200μs, followed by a negative high-voltage pulse discharge of 500-1500V, 20-60μs, and a pause of 600-1200μs , followed by a 500-1500V, 10-50μs forward high-voltage pulse discharge, followed by a 100-300V, 20-100ms low-voltage electrophoresis electric field discharge. The electrotransfection method and equipment of the present invention can greatly improve the success rate of electrotransfection, and the transfection efficiency has reached about 50% in the results of peripheral blood mononuclear cells transfected with green fluorescent protein cytology experiments, which is a good way for the development of tumors. Biotherapeutics provided key technical support.

Description

Electrotransfection method and equipment

technical field

The present invention relates to a method and equipment for gene transfection, especially suitable for but not limited to the method and equipment for electrotransfection in primary lymphocyte transgene.

Background technique

In the field of tumor immunotherapy, the use of transgenic technology to design effector T lymphocytes targeting tumor cells is a promising new method of immunotherapy. Successful allogeneic bone marrow stem cell transplantation and clinical cases of lymphocyte infusions in patients with high-grade hematological cancers have shown that the human immune system can control or even eliminate residual lesions. The main complication of transfusion of exogenous lymphocytes is the graft-versus-host reaction, which attacks normal cells in addition to tumor cells. Due to the tolerance mechanism of the autologous immune system, autologous lymphocytes do not contain high-affinity effector cells. It has become a new direction for tumor immunotherapy to make autologous lymphocytes target tumor antigens and kill tumor cells through transgenic technology.

Screen and clone specific T cell receptors targeting tumor antigens, transfer the T cell receptors into the patient's peripheral blood T lymphocytes through transgenic technology, then expand and activate them, and infuse them back into the patient's body. Lymphocytes can kill tumor cells without killing normal tissue cells. In particular, adoptive T lymphocyte therapy can achieve good results for tumor stem cells that have metastasized and are resistant to chemotherapy drugs, which makes up for the shortcomings of surgery and chemotherapy in the treatment of tumor stem cells.

So far, the use of adoptive transgenic T cells in tumor immunotherapy has only used retroviral vectors to produce effective and stable therapeutic effects, which also leads to some safety hazards. First: Due to the problem of random insertion and integration of transcriptional virus vectors, it will cause irreversible genetic manipulation to the patient's own cells, and there is a potential risk of canceration; Peripheral blood T lymphocytes transfected with lentivirus are screened and expanded in vitro to produce stable expression cells. How to eliminate these transgenic T cells when the treatment is completed or side effects occur becomes a problem; It is cumbersome and will lead to unfavorable factors in quality management and control. Safety concerns become an inherent flaw in viral vector transgenesis.

Among the non-viral vector transgenic methods, the electroporation technology is the most attractive one. According to the statistics of clinicaltrials.gov, there are 35 clinical trials of electroporation so far. There are 19 clinical trials of electroporation DNA vaccine, subcutaneous and intramuscular injection of vaccine supplemented by electroporation, including 1 case of colon cancer vaccine, 1 case of prostate cancer vaccine, 2 cases of H5N1 avian influenza virus DNA vaccine, and 2 cases of melanoma DNA vaccine Cases, 5 HIV vaccines, 2 papillomavirus DNA vaccines, 1 cervical intraepithelial neoplasia DNA vaccine, 2 chronic hepatitis C vaccines, 1 hematologic malignancy vaccine, 1 anti-Plasmodium falciparum DNA vaccine, advanced cancer One case of tumor vaccine.

Electrotransfection of cytokine plasmids, clinical trials of intratumoral injection of cytokine plasmids and electrotransfection in 3 cases, intratumoral injection and electrotransfer of IL2 plasmid in 2 cases, and intratumoral injection and electrotransfection of IL12 plasmid in 1 case example.

Electrotransfection chemotherapy drugs, 9 cases of clinical trials of injection of bleomycin combined with electrotransfection for refractory, skin squamous, and glial tumors, including 2 cases of injection of electrotransfected bleomycin for head and neck cancer, skin and One case of electrotransfer of bleomycin by subcutaneous tumor injection, one case of electrotransfer of bleomycin for pancreatic cancer, one case of electrotransfer of bleomycin for melanoma, one case of electrotransfer of bleomycin for rectal cancer, and one case of electrotransfer of bleomycin for brain metastases One case of electrotransfer of bleomycin was used to treat tumor skin ulcer metastasis, and one case of electrotransfer of bleomycin was used to treat recurrent breast cancer.

Other clinical trials of electroporation include 2 cases of electroporation safety and tolerance test, and 1 case of irreversible electroporation treatment of early primary liver cancer.

The above statistics show that electrotransfection technology has not been used for transgenic therapy of primary lymphocytes for the following four reasons:

First, the existing electrotransfection technology uses a single discharge of a capacitor to form a high-voltage electric field. As shown in FIG. 1 , the schematic diagram of the circuit principle of the existing electrotransfection apparatus, the circuit includes a charging circuit and a discharging circuit. When the switch K1 is turned on, the control circuit charges the capacitor C, and after the capacitor is charged, the switch K1 is turned off. When discharging, when K2 is turned on, the capacitor C discharges to the load. The discharge time of the entire circuit is determined by the value of the discharge time RC, R is the equivalent resistance of the discharge circuit, including the resistance of the load circuit, C is the capacity value of the capacitor, and this circuit can only generate a single, positive high-voltage discharge pulse , can not generate a low-voltage discharge pulse. In capacitive discharge, if the voltage is low, small pores cannot be effectively formed on the cell membrane. If the voltage is high, the energy will be too concentrated, and permanent perforation will be formed on the cell membrane, resulting in the death of the target cell. This shows that a single discharge of the capacitor can easily lead to an increase in the apoptosis rate of primary lymphocytes, and a decrease in the availability of successfully transgenic primary lymphocytes.

Secondly, the existing electrotransfection technology uses positive pulse discharge. Since the cells are negatively charged, the transfection efficiency of cells near the positive electrode is higher than that of cells near the negative electrode, and the cell membrane perforation is all facing the positive electrode. Direction, the reduction of the total number of electrotransfected cells and less membrane perforation lead to a decrease in the availability of transgenic primary lymphocytes.

Again, long-term low-voltage electrophoresis is necessary to improve the electrotransfection efficiency of primary lymphocytes immediately after high-voltage pulse discharge, but there is no such measure at present. Because in the short-term high-voltage electric field, the cell membrane will form a temporary small hole. At this time, a low-voltage current is applied, and the exogenous molecules are electrophoresed into the cytoplasm.

Finally, the existing electrotransfection technology is used for the transfection of plant cells, microorganisms and cultured cell lines, and has a certain transfection efficiency, but the transfection efficiency of primary lymphocytes is extremely low, because the existing gene electrotransfection All dyeing techniques rely on the mitosis of the nucleus. At this time, the nuclear membrane will temporarily depolymerize, and foreign DNA enters the nucleus by passive diffusion. Only a very small number of therapeutic genes can freely diffuse into the nucleus through the pores of the nuclear membrane. Therefore, for cells that are in resting phase or rarely divide, the transfection efficiency of the existing gene electrotransfection technology is extremely low.

Therefore, safe and efficient transgenic technology has become a key technical bottleneck for cellular immunotherapy.

Contents of the invention

Aiming at the technical problem of the extremely low transgene efficiency of primary lymphocytes by traditional electroporation apparatus, the present invention provides an electrotransfection method and equipment, which can increase the transfection rate in transgene and improve the safety of viral vector transgene.

The electrotransfection method of the present invention includes: discharging the cells four times, and performing two pauses between the discharges:

The first discharge: a positive pulse with a voltage of 500-1500V and a duration of 20-60μs (microseconds);

The first pause: pause duration 600 ~ 1200μs;

The second discharge: a negative pulse with a voltage of 500-1500V and a duration of 20-60μs;

The second pause: pause duration 600 ~ 1200μs;

The third discharge: a positive pulse with a voltage of 500-1500V and a duration of 10-50μs;

The fourth discharge: a forward electrophoretic electric field with a voltage of 100-300V and a duration of 20-100ms (milliseconds).

Through the combination of positive and negative high-voltage pulsed electric fields and low-voltage pulsed electric fields, the cell membrane can be perforated in two directions, and the long-term low-voltage pulse can cause foreign molecules to be electrophoresed into the cytoplasm, thereby increasing the total number of electrotransfected cells and The number of membrane perforations, thereby increasing the electrotransfection rate. After testing, the transfection efficiency of peripheral blood mononuclear cells transfected with pmaxGFP (green fluorescent protein) reached about 50%, which provided key technical support for the development of tumor biotherapeutic drugs.

Preferably, the voltage of the first discharge is a forward pulse of 800-1100V, and the duration is 35-45 μs.

Preferably, the pause duration of the first pause is 800-1000 μs.

Preferably, the voltage of the second discharge is a negative pulse of 800-1100V, and the duration is 35-45 μs.

Preferably, the pause duration of the second pause is 800-1000 μs.

Preferably, the voltage of the third discharge is a forward pulse of 800-1100V, and the duration is 15-25 μs.

Preferably, the voltage of the fourth discharge is a forward electrophoretic electric field of 150-200V, and the duration is 40-60ms.

Further, the components of the electrotransfection buffer used in conjunction with the above method can be: PBS (phosphate buffer) buffer, concentration 30-200mM; Tris-HCl, concentration 30-150mM; Ca(NO ₃ ) ₂ , Concentration 0.2-1mM; KCl, concentration 2-8mM; MgCl ₂ , concentration 10-20mM; NaCl, concentration 50-150mM; glucose, concentration 5-30mM; pH 6.5-pH7.2.

Another component of the electrotransfection buffer is: PBS buffer, with a concentration of 30-200 mM; Tris-HCl, with a concentration of 30-150 mM; Ca(NO ₃ ) ₂ , with a concentration of 0.2-1 mM; KCl, with a concentration of 2-1 mM. 8mM; MgCl ₂ , concentration 10-20mM; NaCl, concentration 50-150mM; pH 6.5-pH7.2.

The present invention also provides an electrotransfection device used in the method, comprising a sequentially connected display circuit, a control circuit, a logic circuit and a drive circuit, the output end of the drive circuit is connected to a low-voltage DC power supply through a conduction element, The output end of the drive circuit is also connected to the high-voltage DC power supply through the full bridge circuit, the output end of the full bridge circuit is connected to the load, and is connected to the control circuit through the current detection circuit. The conduction element may be a triode or a MOS transistor. in:

Control circuit: MCU control circuit for precise control of time, mainly to complete the generation of waveforms. It can be composed of common single-chip microcomputer or existing chips, modules and conventional peripheral circuits. The main function of the control circuit is to control, supervise and coordinate the normal operation of the entire equipment. Its main functions are to complete the generation of discharge waveforms, precisely control the discharge time, communicate with the operation display panel, send display information and record key operations, etc. Its core is a single-chip microcomputer, such as a PIC16F1937 single-chip microcomputer, or other single-chip microcomputers with similar functions.

Logic circuit: used to ensure the normal operation of the full bridge circuit, prevent the simultaneous conduction of the upper and lower bridges of the same arm, and quickly generate various waveforms and fast protection. The logic circuit uses the programmable device PLD (Programmable Logic Device) as the core of the circuit, and writes the corresponding embedded control program to ensure that the downstream bridge circuit is conducted according to the specified logic. The connection between the PLD and the peripheral circuit can be connected according to the specific PLD model.

Driving circuit: There are six driving conduction modules in total, and this circuit includes the power supply of the driving module. The drive module has an overcurrent protection function. The core conduction element in the drive conduction module is an IGBT (Insulated Gate Bipolar Transistor) device, and the IGBT is composed of a BJT (Bipolar Transistor) and a MOS (Insulated Gate Field Effect Transistor) tube. The composite fully controlled voltage-driven power semiconductor device has the advantages of high input impedance of MOSFET (metal-oxide-semiconductor-field-effect transistor) and low conduction voltage drop of GTR (power transistor). The saturation voltage of GTR is low, the carrying current density is large, but the driving current is large; the driving power of MOSFET is small, the switching speed is fast, but the conduction voltage drop is large, and the current carrying density is small. The IGBT combines the advantages of the above two devices, with low driving power and low saturation voltage. The IGBT is driven by a corresponding drive circuit. Each drive conduction module has an output terminal, and the output terminals of each module are connected and then output to the full bridge circuit.

Full bridge circuit: A full bridge circuit is composed of a single conduction element and four conduction elements of the same structure, assuming that the left is positive and the right is negative (that is, the left triode of the full bridge circuit part in Figure 3 conducts, and the right triode conduction, other transistors cut off), the high-voltage input or low-voltage input DC power supply can output positive and negative waves. The full bridge circuit is an oscillation composed of four triodes or MOS tubes. The advantage of the full bridge circuit is that it is not easy to produce effusion.

Low-voltage DC power supply: When used for overvoltage protection, when the voltage exceeds the low-voltage DC threshold, the input of this circuit is forcibly closed and an alarm is issued. Under normal circumstances, it will not exceed the low-voltage DC threshold; when used for self-discharge protection, under any circumstances , as long as the power plug is out of power, the self-discharge circuit will automatically discharge the electricity on the capacitor within a few minutes; when it is used in the charging circuit, it will charge the capacitor; when it is used in the power detection circuit, it will detect whether the voltage on the power supply meets the requirements. If the requirements are not met, the charging circuit continues to complete charging.

High-voltage DC power supply: When used for overvoltage protection, when the voltage exceeds the high-voltage DC, the input of this circuit is forced to close and an alarm is issued. Under normal circumstances, it will not exceed the high-voltage DC; when used for self-discharge protection, in any case, As long as the power plug is out of power, the self-discharging circuit will automatically discharge the electricity on the capacitor within a few minutes; when it is used in the charging circuit, it will charge the capacitor; when it is used in the power detection circuit, it will detect whether the voltage on the power supply meets the requirements. If the requirements are met, the charging circuit continues to complete charging.

Current detection circuit: the main function is to protect the discharge circuit and detect whether the charging voltage meets the requirements, so that the entire circuit of the device is protected. When there is no load in the equipment circuit, that is, when the cell electric shock cup is not connected (the cell electric shock cup is a small square cup-shaped metal box, and cells and cell transfection fluid are filled in the electric shock cup), in order to avoid the hazard of high-voltage electric shock , the entire discharge circuit of the device is open circuit, will not discharge, and is prompted by a signal light; when the device circuit is connected to the cell electric shock cup, the current detection circuit detects whether the voltage of the high-voltage DC power supply and the low-voltage DC power supply meet the requirements, if not , start the charging circuit to continue charging, when the voltage meets the requirements, the signal light will prompt.

The electrotransfection method and equipment of the present invention can greatly improve the success rate of electrotransfection, and the transfection efficiency in peripheral blood mononuclear cells transfected with pmaxGFP (green fluorescent protein) cytology experiment results reached about 50%. Provided key technical support for the development of tumor biotherapeutic drugs.

The above-mentioned content of the present invention will be further described in detail below in conjunction with the specific implementation manners of the examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. Without departing from the above-mentioned technical idea of the present invention, various replacements or changes made according to common technical knowledge and customary means in this field shall be included in the scope of the present invention.

Description of drawings

Figure 1 is a circuit block diagram of a traditional electroporation instrument.

Fig. 2 is a schematic diagram of discharge pulses in the electrotransfection method of the present invention.

Fig. 3 is a schematic circuit diagram of the electrotransfection device of the present invention.

FIG. 4 is a schematic circuit diagram of the driving circuit module in FIG. 3 .

Fig. 5 is a microscope comparison diagram under the white light field of view and the fluorescence field of view of electrotransfection with buffer ML16 in the method of the present invention.

Fig. 6 is a microscope comparison diagram under white light field of view and fluorescence field of view of electrotransfection with buffer GD20 in the method of the present invention.

Fig. 7 is a graphical comparison of the transfection rate of peripheral blood mononuclear cells detected by flow cytometry after the traditional method and the method of the present invention.

Fig. 8 is a graph showing the comparison of the apoptotic rate and availability rate of peripheral blood lymphocytes after the traditional method and the method of the present invention.

Detailed ways

Cell electrotransfection was performed using the device of the present invention.

experiment method:

The electrotransfection method of the present invention is shown in Figure 2. In the experiment of this example, human peripheral blood mononuclear cells were used to transfect the pmaxGFP (green fluorescent protein) plasmid of AMAXA Company with the article number VPA1001 through the method of the present invention, and the cell flow cytometry Analyze transfection efficiency. The method includes performing a positive high-voltage pulse on the cells, pausing for a period of time, followed by a negative high-voltage pulse, pausing for a period of time, followed by a positive high-voltage pulse, and then a long-term low-voltage electrophoresis electric field.

Test equipment:

The equipment circuit for electrotransfection is shown in Figure 3, including a display circuit, a control circuit, a logic circuit, and a drive circuit connected in sequence. The output end of the drive circuit is connected to a low-voltage DC power supply through a conduction element of a triode structure, and the output of the drive circuit The terminal is also connected to the high-voltage DC power supply through a full bridge circuit, the output terminal of the full bridge circuit is connected to a load, and is connected to the control circuit through a current detection circuit. in:

Control circuit: MCU control circuit for precise control of time, mainly to complete the generation of waveforms.

Logic circuit: used to ensure the normal operation of the full bridge circuit and prevent the simultaneous conduction of the upper and lower bridges of the same arm.

Driving circuit: a total of six driving triode modules as shown in Figure 4, and the driving module has an over-current protection function. Each drive conduction module has an output terminal OUTPUT1, and the output terminal OUTPUT1 of each module is connected to output to the full bridge circuit. The E201 in Figure 4 is an integrated IGBT (Insulated Gate Bipolar Transistor, insulated gate bipolar transistor) drive module, the model is VLA517-01R, and the port OutA in the circuit is controlled by a PLD (programmable logic device). Control the input terminal, the E201 module controls the base G of the triode M201, the current flows from the collector C to the transmitter E, and finally from the output terminal OUTPUT1.

Full-bridge circuit: A full-bridge circuit is composed of a single conduction element and four conduction elements of the same structure. Assuming that the left is positive and the right is negative, the high-voltage input DC can output positive and negative waves, and the low-voltage input DC can only output positive waves. . The full bridge circuit is an oscillation composed of four triodes or MOS tubes. The advantage of the full bridge circuit is that it is not easy to produce effusion.

Low-voltage DC power supply: When used for overvoltage protection, when the voltage exceeds the low-voltage DC threshold, the input of this circuit is forced to close and an alarm is issued. Under normal circumstances, it will not exceed the low-voltage DC threshold; when used for self-discharge protection, in any case, As long as the power plug is out of power, the self-discharging circuit will automatically discharge the electricity on the capacitor within a few minutes; when it is used in the charging circuit, it will charge the capacitor; when it is used in the power detection circuit, it will detect whether the voltage on the power supply meets the requirements. If the requirements are met, the charging circuit continues to complete charging.

High-voltage DC power supply: When used for overvoltage protection, when the voltage exceeds the high-voltage DC, the input of this circuit is forced to close and an alarm is issued. Under normal circumstances, it will not exceed the high-voltage DC; when used for self-discharge protection, in any case, as long as When the power plug is out of power, the self-discharging circuit will automatically discharge the electricity on the capacitor within a few minutes; when used in a charging circuit, it will charge the capacitor; Requirements, the charging circuit continues to complete charging.

In this embodiment, a comparative test is carried out with the discharge method of combining the positive and negative direction high voltage pulse electric field and low voltage pulse electric field of the present invention through traditional three positive high voltage pulse discharges and one positive low voltage pulse discharge.

The traditional method is: 3 forward high voltages are 800V, 1 forward low voltage is 200V, the pulse width is 50μs+T+50μs+T+50μs+20ms, and the pause time T=1000μs.

The method of the present invention is as follows: the positive high voltage is 800V, the negative high voltage is -800V, the low voltage is 200V, the pulse width is 50μs+T+50μs+T+50μs+20ms, and the pause time T=1000μs, specifically:

The first discharge: a positive pulse with a voltage of 800V and a duration of 50μs (microsecond);

The first pause: pause duration 1000μs;

The second discharge: a negative pulse with a voltage of -800V and a duration of 50μs;

Second pause: pause duration 1000μs;

The third discharge: a positive pulse with a voltage of 800V and a duration of 50μs;

The fourth discharge: a forward electrophoretic electric field with a voltage of 200V and a duration of 20ms (milliseconds).

Adopt two kinds of damping solutions to carry out matching test in the present embodiment:

Buffer ML16 composition: PBS buffer (phosphate buffer), concentration 30-200mM; Tris-HCl, concentration 30-150mM; Ca(NO ₃ ) ₂ , concentration 0.2-1mM; KCl, concentration 2-8mM; MgCl ₂ , Concentration 10-20mM; NaCl, 50-150mM; Glucose, concentration 5-30mM; pH6.5-pH7.2.

Buffer GD20 composition: PBS buffer, concentration 30-200mM; Tris-HCl, concentration 30-150mM; Ca(NO ₃ ) ₂ , concentration 0.2-1mM; KCl, concentration 2-8mM; MgCl ₂ , concentration 10-20mM; NaCl, 50~150mM; pH6.5~pH7.2.

Test data and results:

As shown in Figure 5, Figure 5A is a photomicrograph of electrotransfection with buffer ML16 under white light field of view, and Figure 5B is a photomicrograph of electrotransfection with buffer ML16 under fluorescent field of view. The ratio of transfected cells under visual fluorescence and white light field was 40% to 50%.

As shown in Figure 6, Figure 6A is a photomicrograph of electrotransfection with buffer GD20 under a white light field of view, and Figure 6B is a photomicrograph of electrotransfection with buffer GD20 under a fluorescent field of view. The ratio of cells under visual fluorescence and white light field of view was 30% to 40%.

Buffer ML16 was used to conduct a comparative test of the accurate data of the transfection rate of the traditional method and the method of the present invention.

As shown in Figure 7, Figure 7A is the result of the transfection rate test of the traditional method, and Figure 7B is the result of the transfection rate test of the method of the present invention. As can be seen from the comparison diagram in Figure 7, the transfection rate of the traditional method in Figure 7A is 39.6%, and the transfection rate of the inventive method in Figure 7B is 51.3%, and the transfection rate of the inventive method is 11.7% higher than that of the traditional method.

The apoptosis rate and availability rate are shown in Fig. 8, wherein Fig. 8A is the test result of the traditional method, and Fig. 8B is the test result of the method of the present invention. As can be seen from the comparison diagram of Figure 8, the apoptosis rate of the traditional method in Figure 8A is 8.8%, and the yield rate is 91.2%, and the apoptosis rate of the method of the present invention in Figure 8B is 11%, and the yield rate is 89%. Compared with the traditional method, the apoptosis rate of the method is only increased by 2.2% (that is, the yield is reduced by 2.2%), and the reduction of the yield is completely within the acceptable range.

By comparing the data, it can be seen that through the method of combining the high-voltage pulsed electric field and the low-voltage pulsed electric field in the positive and negative directions of the present invention, after the cell membrane is perforated in two directions and the low-voltage pulse is used for a long time, the exogenous molecules can be electrophoresed into the cytoplasm, In this way, the total number of electrotransfected cells and the number of membrane perforations are increased, and the electrotransfection rate is greatly improved, so that the transfection efficiency of peripheral blood mononuclear cells transfected with pmaxGFP (green fluorescent protein) cytology experiment results reaches about 50%. Provided key technical support for the development of tumor biotherapeutic drugs.

Claims (10)

1. The method for electrotransfection, which is characterized in that: the cells are discharged four times, with two pauses between the discharges: The first discharge: a positive pulse with a voltage of 500-1500V and a duration of 20-60μs; The first pause: pause duration 600 ~ 1200μs; The second discharge: a negative pulse with a voltage of 500-1500V and a duration of 20-60μs; The second pause: pause duration 600 ~ 1200μs; The third discharge: a positive pulse with a voltage of 500-1500V and a duration of 10-50μs; The fourth discharge: a forward electrophoretic electric field with a voltage of 100-300V and a duration of 20-100ms. 2. The electrotransfection method according to claim 1, characterized in that: the voltage of the first discharge is a forward pulse of 800-1100V, and the duration is 35-45 μs. 3. The electrotransfection method according to claim 1, characterized in that: the pause duration of the first pause is 800-1000 μs. 4. The electrotransfection method according to claim 1, characterized in that: the voltage of the second discharge is a negative pulse of 800-1100V, and the duration is 35-45 μs. 5. The electrotransfection method according to claim 1, characterized in that: the pause duration of the second pause is 800-1000 μs. 6. The electrotransfection method according to claim 1, characterized in that: the voltage of the third discharge is a forward pulse of 800-1100V, and the duration is 15-25 μs. 7. The electrotransfection method according to claim 1, characterized in that: the voltage of the fourth discharge is a forward electrophoresis electric field of 150-200V, and the duration is 40-60ms. 8. The electrotransfection method according to any one of claims 1 to 7, characterized in that: the electrotransfection buffer components used together are: PBS buffer, concentration 30-200mM; Tris-HCl, concentration 30 ～150mM; Ca(NO ₃ ) ₂ , concentration 0.2～1mM; KCl, concentration 2～8mM; MgCl ₂ , concentration 10～20mM; NaCl, concentration 50～150mM; glucose, concentration 5～30mM; pH 6.5～pH7. 2. 9. The electrotransfection method according to any one of claims 1 to 7, characterized in that: the electrotransfection buffer components used together are: PBS buffer, concentration 30-200mM; Tris-HCl, concentration 30mM ～150mM; Ca(NO ₃ ) ₂ , concentration 0.2～1mM; KCl, concentration 2～8mM; MgCl ₂ , concentration 10～20mM; NaCl, concentration 50～150mM; pH 6.5～pH7.2. 10. The equipment for the electrotransfection of the method according to claim 1 is characterized in that: it comprises a display circuit, a control circuit, a logic circuit and a driving circuit connected in sequence, and in the driving circuit, the driving circuit is connected to the low voltage by a conduction element. The DC power supply is connected, and the output end of the drive circuit is also connected to the high-voltage DC power supply through the full bridge circuit. The output end of the full bridge circuit is connected to the load, and is connected to the control circuit through the current detection circuit.

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