CN114886538A

CN114886538A - Pulse ablation method for increasing tumor selective treatment by using low temperature

Info

Publication number: CN114886538A
Application number: CN202210309768.1A
Authority: CN
Inventors: 蓝闽波; 赵红莉; 潘巨敏; 高陈程; 陈开茶; 周芳芳
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-08-12

Abstract

The invention relates to a pulse ablation method and a cryogenic electrode for increasing ablation selectivity by using low temperature, wherein the cryogenic electrode comprises but is not limited to an electrode under single pulse electric field ablation, an electrode under the combined use of cryoablation and pulse ablation, and an electrode under the combined use of thermal ablation and pulse ablation. The method is to increase ablation selectivity by lowering the temperature of the treatment area at the time of pulse administration. Under the same pulse condition, the temperature of the treatment area is changed to ensure that cancer cells die normally, the survival rate of normal cells is greatly improved, and therefore, the method can be used for controlling the ablation boundary, ensuring that the ablation area is more accurate and reducing the damage of normal tissues. In cell model experiments, the survival rate of normal cells is greatly improved compared with that of normal cells at room temperature (20-26 ℃) when a certain pulse condition is given at low temperature (0-5 ℃), and the survival rate of cancer cells is not obviously changed under the same condition.

Description

Pulse ablation method for increasing tumor selective treatment by using low temperature

Technical Field

The present application relates to a pulse ablation method and a cryogenic electrode for increasing tumor ablation selectivity using low temperature (0-5 ℃). More particularly, the present application relates to an electrode for generating irreversible electroporation on cells of a biological tissue or inducing apoptosis of cells of the biological tissue, and a method of increasing ablation selectivity using cryogenics.

Background

The traditional tumor treatment means mainly comprise surgery, chemo-anti-cancer drug therapy (chemotherapy) and radiotherapy (radiotherapy), but the problems of tumor metastasis and recurrence, strong drug toxic and side effects and the like cannot be solved. Recently, physical thermotherapy for tumors attracts attention, mainly including radiofrequency ablation, microwave ablation, cryoablation at low temperature, focused ultrasound ablation and the like, but because blood, lymph and other circulation systems can take away heat, the temperature is difficult to rise to the temperature required by ablation, the ablation effect is greatly reduced, and in addition, because the thermal ablation has higher requirements on the temperature, the ablation for tumors close to sensitive parts such as important organs, large blood vessels, nerves and the like is difficult to realize, and the application range of the tumor is limited to a certain extent.

With the continuous research and development of the pulse bioelectricity mechanism and the related technology thereof, the pulse electric field attracts the wide attention of researchers in various countries around the world with the unique non-thermal biomedical effect, and is gradually applied to the treatment of tumors, and the satisfactory treatment effect is obtained in clinical application. Irreversible electroporation produces less thermal effects and leaves the surrounding tissue intact, and therefore can be applied to sites near major blood vessels, the intestinal wall, the pancreatic duct, or other important organ structures.

However, the pulsed electric field also has some problems to be solved, such as:

the pulsed electric field generated during the ablation treatment is generally ellipsoidal, so the injury area is also ellipsoidal, the tumor has various shapes, such as papillary, cauliflower, villous, mushroom, polyp, knot, lobular, infiltration block, diffuse pachynsis, ulcer, saccular and the like, so that excessive normal tissues are merged into the ablation area while the tumor area is completely covered and treated by the pulsed electric field, and the pulsed electric field kills cells in the normal area to cause excessive ablation, thereby generating a certain adverse effect on a patient.

Thus, precise control of the electric field distribution, control of the ablation boundary, and more precise ablation zone reduces irritation and damage to non-target tissue, which can be beneficial in reducing adverse effects of treatment on the patient.

CN113616315A discloses a method of controlling the distribution of a conductive liquid to control the direction of ablation to avoid over-ablation. CN106388933B discloses a method of suppressing electric field diffusion distribution in non-treatment region by using insulating material to reduce electric stimulation to non-target tissue. CN109481002A discloses a device combining cold ablation and electric ablation, in which the electric cold probe adopts a needle electrode structure, and a temperature reducing device and a temperature measuring device are provided therein. CN108024803B discloses a method for eliminating the generation of electric arc in the process of electroporation by using temperature-controlled perfusate, and the electric arc condition is discussed by changing the temperature of the perfusate.

Disclosure of Invention

The invention aims to provide a pulse ablation method for increasing ablation selectivity by using the ambient temperature of a treatment area to achieve low temperature (hereinafter referred to as low temperature) and a low-temperature electrode designed by using the method. The temperature control device is added on the original ablation electrode, so that the temperature of the pulse treatment area is reduced to improve the ablation selectivity. The method can be used for the reconstruction of all kinds of ablation electrodes. According to the method, a cryogenic electrode capable of increasing ablation selectivity is designed, and comprises but is not limited to an electrode under single pulse electric field ablation, an electrode under combined use of cryoablation and pulse ablation, and an electrode under combined use of thermal ablation and pulse ablation, before pulse parameter electric field ablation is given, the temperature of a treatment area is adjusted to be at a preset low temperature through a temperature control device, treatment under corresponding pulse conditions is carried out after temperature control is stable, and the temperature can be returned to be at a suitable temperature for a human body after pulse delivery is finished, so that more accurate and specific treatment is realized.

The technical scheme of the invention is as follows:

a pulse ablation method for increasing ablation selectivity by using low temperature (0-5 ℃) can enable an ablation area to be more accurate and reduce damage of normal tissues, and comprises the following steps:

by adding a temperature control device on the original pulsed electric field ablation electrode, a stable low-temperature field (0-5 ℃) can be formed before pulse voltage is applied. Before the pulse parameter electric field is given for ablation, the temperature of the treatment area is adjusted to a preset low temperature through a temperature control device, after the temperature is controlled stably, treatment is carried out under the corresponding pulse condition, and the temperature can be heated to a proper temperature of a human body after the pulse delivery is finished. Ablation selectivity is enhanced by lowering the temperature of the treatment area at the time of pulse administration. Under the same pulse condition, by changing the temperature of the treatment area, cancer cells can die as usual, and the survival rate of normal cells is greatly improved. In cell model experiments, the survival rate of normal cells is greatly improved compared with that of normal cells at room temperature (20-26 ℃) when a certain pulse condition is given at low temperature (0-5 ℃), and the survival rate of cancer cells is not obviously changed under the same condition.

The invention also provides a low-temperature electrode designed by the method, which has the functions of the existing pulse electrode, can output and generate a stable pulse electric field, is provided with a temperature control system and can form a stable low-temperature field, wherein the low-temperature treatment is carried out before the pulse electric field ablation, and can be returned to the temperature after the electric field ablation is finished.

The low-temperature electrode comprises an ablation electrode needle, a temperature control device and a temperature measuring device which are used in pairs;

the ablation electrode needle is used for puncturing a positioned ablation area in a body, and generates an inter-needle pulse electric field and changes the temperature outside the ablation electrode through heat conduction by utilizing the electric conduction and heat conduction effects of an exposed end material;

the temperature control device regulates the temperature controlled by the device after inputting the required temperature so as to complete low-temperature treatment before pulse ablation and timely temperature return after ablation;

the temperature measuring device is used for monitoring the temperature of the environment where the ablation electrode needle is located in real time, is matched with the temperature control device for real-time temperature control, and accurately judges whether the temperature required by treatment is reached.

The low-temperature electrode according to the invention further comprises, but is not limited to, an electrode under single pulse electric field ablation, an electrode under combined use of cryoablation and pulse ablation, and an electrode under combined use of thermal ablation and pulse ablation, so that the low-temperature treatment before pulse ablation can be satisfied, and the killing selectivity to tumors during ablation can be greatly improved under the combination of a stable pulse electric field and a low-temperature field.

According to the low-temperature electrode, the diameter of the ablation electrode needle is variable, and the inside of the ablation electrode needle is of a hollow structure and used for placing a temperature control device and a connecting circuit.

According to the low-temperature electrode, the material of the ablation electrode needle can be various conductive and heat-conductive materials such as gold, silver, copper, platinum, zinc, metal oxide, conductive carbon material and the like; the insulating material can be polyurethane, polytetrafluoroethylene, polyethylene, glass fiber and other materials.

When the low-temperature electrode is used, at least one electrode is electrified and at least one electrode is grounded, and when the electrode is electrified, the electrode can bear pulse voltage of 3000-4000V, the pulse width is 0.01-100 mu s, and the pulse voltage is 1 kHZ-1 MHZ.

According to the low-temperature electrode of the invention, furthermore, a self-made pulse generator can be used, and other existing pulse generators can also be used.

According to the low-temperature electrode, the ablation electrode needle can be replaced by a medical flexible catheter, and can be applied to minimally invasive ablation of human body lumen tissues.

According to the low-temperature electrode, further, the low-temperature electrode capable of increasing ablation selectivity comprises: the pair of ablation electrode needles are used, and the temperature control device and the temperature measuring device are used;

the ablation electrode needle comprises an ablation electrode needle front end, an ablation electrode needle middle section and an ablation electrode needle tail end;

the front end of the ablation electrode needle is an exposed end, the surface of the electrode needle at the front end is exposed, and an inter-needle pulse electric field is generated and the temperature outside the needle is changed through heat conduction by utilizing the electric conduction and heat conduction effects of the material of the exposed end; the front end of the ablation electrode needle is hollow, a semiconductor temperature control sheet is tightly attached to the front end of the ablation electrode needle, the semiconductor temperature control sheet is connected to a tail end temperature control terminal through an internal lead, and the expected temperature is regulated and controlled by the temperature control terminal; a temperature measuring couple is embedded on the semiconductor temperature control sheet and is tightly attached to the ablation electrode needle, and the temperature measuring couple is connected to the tail end temperature measuring terminal through an internal conducting wire to display the actual temperature in real time;

the middle section of the ablation electrode needle is wrapped by an insulating material and a heat insulating material, so that excessive damage to a non-treatment area in ablation is avoided, the ablation process is not involved, the puncture depth can be increased, an operator can conveniently puncture the ablation electrode needle by holding the ablation electrode needle in the hand, and a hollow wire for placing a temperature control and measurement device is arranged inside the ablation electrode needle;

the tail end of the ablation electrode needle is provided with a temperature control terminal, a temperature measurement terminal and a pulse generator connecting end, and the temperature control terminal, the temperature measurement terminal and the pulse generator connecting end are used for man-machine interaction so as to control temperature and a pulse electric field.

According to the low-temperature electrode, further, the low-temperature electrode capable of increasing ablation selectivity comprises: the ablation electrode needles, the gas delivery pipeline and the temperature measuring device are used in pairs;

the front end of the ablation electrode needle is an exposed end, the surface of the electrode needle at the front end is exposed, and an inter-needle pulse electric field is generated and the temperature outside the needle is changed through heat conduction by utilizing the electric conduction and heat conduction effects of the material of the exposed end; the front end of the ablation electrode needle is hollow and is provided with a gas delivery pipeline for delivering gas from the tail end to the front end, a porous plug is arranged at the opening of the pipeline positioned at the front end, a temperature measurement couple is tightly attached to the inside of the front end of the ablation electrode needle and is connected to a tail end temperature measurement terminal through an internal lead, and the actual temperature is displayed in real time;

the middle section of the ablation electrode needle is wrapped by an insulating material and a heat insulating material, so that excessive damage to a non-treatment area in ablation is avoided, the ablation process is not involved, the puncture depth can be increased, an operator can conveniently puncture the ablation electrode needle by holding the ablation electrode needle in the hand, and a hollow wire and a gas delivery pipeline for placing a temperature measuring device are arranged in the ablation electrode needle;

the tail end of the ablation electrode needle is provided with a temperature measuring terminal and a pulse generator connecting end, and the temperature measuring terminal and the pulse generator connecting end are used for monitoring temperature and a pulse electric field through man-machine interaction.

One aspect of the present application relates to a method of pulse ablation with increased ablation selectivity using low temperature (0-5 ℃). The method is to increase ablation selectivity by lowering the temperature of the treatment area at the time of pulse administration. Under the same pulse condition, cancer cells can die as usual by changing the temperature of a treatment area, the survival rate of normal cells is greatly improved, and thus the treatment selectivity of an ablation area is improved, and the method specifically comprises the following steps: by adding the temperature control device on the original pulsed electric field ablation electrode, a stable low-temperature field can be formed before pulse voltage is applied. Before the pulse parameter electric field is given for ablation, the temperature of the treatment area is adjusted to a preset low temperature through a temperature control device, after the temperature is controlled stably, treatment is carried out under the corresponding pulse condition, and the temperature can be heated to a proper temperature of a human body after the pulse delivery is finished. The method can be used to control the ablation boundary, make the ablation zone more accurate and reduce damage to normal tissue. In cell model experiments, the survival rate of normal cells is greatly improved compared with that of normal cells at room temperature (20-26 ℃) when a certain pulse condition is given at low temperature (0-5 ℃), and the survival rate of cancer cells is not obviously changed under the same condition.

In some embodiments of the present application, the cryogenic electrode may be an electrode under single-pulse electric field ablation, comprising: the pair of ablation electrode needles are used, and the temperature control device and the temperature measuring device are used; the ablation electrode needle comprises an ablation electrode needle front end, an ablation electrode needle middle section and an ablation electrode needle tail end. The front end is an exposed end, namely a pulse voltage output end, the surface of the electrode needle at the front end is exposed, an inter-needle pulse electric field is generated and the external temperature of the needle is changed through heat conduction by utilizing the electric conduction and heat conduction effects of the material of the exposed end, and the interior of the electrode needle is hollow and is used for placing a temperature control device and a temperature measuring device; the middle section is externally wrapped by an insulating and heat-insulating material, so that excessive damage to a non-treatment area in ablation is avoided, the ablation process is not involved, the puncture depth can be increased, an operator can conveniently puncture by holding the puncture by hands, and a hollow wire for placing a temperature control and measurement device is arranged inside the middle section; the tail end is provided with a temperature control terminal, a temperature measurement terminal and a pulse generator connecting end for man-machine interaction to control the temperature and the pulse electric field. The temperature control device is connected with the temperature control terminal through a lead, the melting process comprises two processes of temperature reduction and temperature rise, and the temperature controlled by the temperature control device is adjusted by inputting the required temperature through the terminal. The temperature required by cooling needs to be close to 0 ℃, the input temperature of the terminal is set to 0 ℃, pulse ablation can be started when the temperature is stably controlled to 0-5 ℃, the input temperature of the terminal is adjusted to 37 ℃ after the pulse ablation, and the ablation electrode needle is taken out after the temperature is returned. The temperature measuring device is connected with the temperature measuring terminal through a lead, is used for monitoring the temperature of the environment where the front end of the ablation electrode needle is located in real time, displays the temperature through the temperature measuring terminal, is matched with the temperature control device to control the temperature in real time, and accurately judges whether the temperature required by treatment is reached.

In some embodiments of the present application, the cryogenic electrode may be an electrode for combined cryoablation and pulse ablation, comprising: the pair-used ablation electrode needle, the gas delivery pipeline and the temperature measuring device; the ablation electrode needle comprises an ablation electrode needle front end, an ablation electrode needle middle section and an ablation electrode needle tail end. The front end is an exposed end, namely a pulse voltage output end, the surface of the electrode needle at the front end is exposed, an inter-needle pulse electric field is generated and the temperature outside the needle is changed through heat conduction by utilizing the electric conduction and heat conduction effects of the exposed end material, and the interior is hollow and is used for placing a temperature measuring device; the middle section is externally wrapped by an insulating and heat-insulating material, so that excessive damage to a non-treatment area in ablation is avoided, the ablation process is not involved, the puncture depth can be increased, an operator can conveniently puncture by holding the puncture by hands, and a hollow wire for placing a temperature measuring device is arranged inside the middle section; the tail end is provided with a temperature measuring terminal and a pulse generator connecting end for man-machine interaction so as to monitor temperature and a pulse electric field. The gas delivery pipeline is arranged inside the ablation electrode needle and used for delivering gas to the front end from the tail end of the ablation electrode needle, the opening of the pipeline adopts a porous plug structure to generate Joule Thomson throttling reaction, different gases can reach different temperatures under throttling, the temperature can be reduced to-140 ℃ by introducing argon, the temperature can be increased to 40 ℃ by introducing helium, the helium can be controlled to be 0-5 ℃ by changing the internal and external pressure, and the purpose of controlling the temperature can be achieved by changing the types of the gases and the internal and external pressures. The temperature measuring device is connected with the temperature measuring terminal through a conducting wire and used for monitoring the temperature of the environment where the front end of the ablation electrode needle is located in real time, displaying the temperature of the gas after temperature control in real time through the temperature measuring terminal and judging whether the temperature is controlled to be required by treatment or not.

In some embodiments of this application, temperature regulating device can use semiconductor temperature control piece, just connecing the power before melting for press close to and melt electrode needle one side refrigeration, melt and accomplish the back and connect the power, make and press close to and melt electrode needle one side and become to heat from refrigerating to accomplish rewarming.

In some embodiments of the present application, the gas delivery pipeline adopts a porous plug structure, argon is introduced to reduce the temperature to-140 ℃ for cryoablation, helium is introduced to raise the temperature to 40 ℃ for rewarming, and the helium can be controlled at 0 ℃ for cryoablation by changing the internal and external pressure.

Description of the drawings:

FIG. 1 is a schematic illustration of tumor ablation with more accurate control of ablation boundaries according to the ablation method of the present application;

FIG. 2 is a schematic view of an electrode under single pulse electric field ablation according to an embodiment of the present application;

FIG. 3 is a schematic view of an electrode under a combination of freezing and pulsed electric field ablation according to an embodiment of the present application;

FIG. 4 is a schematic illustration of cell viability of normal cells pulsed ablation at low temperature;

FIG. 5 is a schematic illustration of cell viability of cancer cells pulsed ablation at low temperature;

FIG. 6 is a graph showing the comparison of the survival rate of normal cells after pulse ablation at pulse widths of 30 μ s, 10 μ s and 1 μ s;

FIG. 7 is a graph showing the pulse ablated cell viability of cancer cells at pulse widths of 30 μ s, 10 μ s, and 1 μ s.

The specific implementation mode is as follows:

various exemplary embodiments of the present application are described in detail below with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps set forth in these embodiments, numerical values do not limit the scope of the present application unless specifically stated otherwise.

The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the application, its application, or uses.

FIG. 1 is a schematic view of tumor ablation with more accurate control of ablation boundaries according to the ablation method of the present application

The schematic diagram is a radial sectional diagram, wherein a gray area is a tumor 11, the tumor is in an irregular spherical shape, a blank area is a normal tissue 12, the tumor 11 is wrapped in the blank area, a positive electrode 13 and a negative electrode 14 of an ablation electrode needle are inserted into a preset position for pulse ablation, a formed pulse electric field is in a regular ellipsoid shape, an ablation and killing area 15 of the pulse electric field is far larger than the volume range of the tumor 11, and excessive normal tissues are killed by a pulse electric field; under the low temperature environment, the survival rate of normal cells is greatly improved, but the survival rate of cancer cells is not changed, so that after the treatment area is subjected to low temperature treatment, the killing area 16 approaches to the tumor boundary to form a more accurate ablation area, thereby protecting normal tissues and avoiding excessive damage.

FIG. 2 is a schematic view of an electrode under single pulse electric field ablation according to an embodiment of the present application

The surface of the front end 21 of the ablation electrode needle is exposed, the insulating and heat-insulating materials 24 are wrapped outside the middle section 22 and the tail end 23 needle, a semiconductor temperature control sheet 25 is tightly attached to the front end 21 and is connected to a tail end temperature control terminal 27 through an internal lead 26, and the expected temperature is regulated and controlled by the temperature control terminal 27. The temperature control sheet 25 is embedded with a temperature measuring couple 28 and is tightly attached to the ablation electrode needle, and is connected to the tail end temperature measuring terminal 210 through an internal lead 29, so as to display the actual temperature in real time. The tail end 23 is further provided with a pulse generator connection end 211 for externally connecting a pulse electric field generator.

During actual treatment, the front end 21 of the ablation electrode needle is inserted into a preset ablation area, the temperature is reduced to be 0-5 ℃ through the semiconductor temperature control sheet 25, the temperature of the terminal 210 to be measured stops being reduced after the temperature is displayed stably, then the pulse generator is turned on to generate pulse voltage for the conductive material after being electrified, so that a pulse electric field is formed, after pulse ablation is finished, the temperature is returned to 37 ℃ through the semiconductor temperature control sheet 25, and finally the ablation electrode needle is taken out.

FIG. 3 is a schematic view of an electrode under a combination of freezing and pulsed electric field ablation according to an embodiment of the present application

The surface of the front end 31 of the ablation electrode needle is exposed, the insulating and heat insulating materials 34 are wrapped outside the middle section 32 and the tail end 33 needles, a gas conveying pipeline 35 is arranged inside the electrode needle and conveys gas to the front end 31 from the tail end 33, a porous plug 36 is arranged at the opening of the pipeline so as to generate Joule Thomson reaction, different gases are input through the gas conveying pipeline 35 to reach different temperatures, a temperature measuring couple 37 is tightly attached to the ablation electrode needle and is connected to a tail end temperature measuring terminal 39 through an internal lead 38, and the actual temperature is displayed in real time. The tail end 33 is provided with a pulse generator connection end 310 for externally connecting a pulse electric field generator.

During actual treatment, the front end 31 of the ablation electrode needle is inserted into a preset ablation area, then argon is introduced to reduce the temperature to-140 ℃ for cryoablation, after cryoablation is finished, helium is introduced to enable the temperature to return to 0-5 ℃, the helium introduction is stopped after the temperature of the terminal 39 to be measured is stably displayed, then the pulse generator is started to be powered on to enable the conductive material to generate pulse voltage, so that a pulse electric field is formed, after pulse ablation is finished, helium is introduced again to enable the temperature to return to 37 ℃, and finally the ablation electrode needle is taken out.

The cell suspension model ablation embodiment of the invention.

The electroporation experiment of cells is carried out by using a self-made pulse generator to carry a 4mm electrode cup of American BTX, the increasing effect of low temperature (0-5 ℃) on the pulse ablation selectivity, the safety and the stability of the pulse ablation are verified, and the subsequent experiment in a mouse body can be further verified. In the experiment, firstly, normal cells and cancer cells cooled at normal temperature (22-26 ℃) and ice-water bath low temperature (0-5 ℃) are respectively subjected to pulsed electric field ablation with the same parameters, and the survival rate of the normal cells is greatly changed under different temperature conditions, but the cancer cells are not obviously changed; then, by changing the parameter setting of the pulse electric field, the influence of different pulse widths on the survival rate of normal cells is found.

Example 1: normal cells were subjected to pulsed electric field ablation experiments at room temperature (22-26 ℃) and cooling in an ice-water bath at low temperature (0-5 ℃).

Three normal cells of L929, 293T and HC11 are selected as experimental objects in the experiment, an electrode cup is used for placing cell suspension blown by a culture medium in the experiment, the cell suspension is placed in a safe electrode chamber, and then the cell suspension is connected with a self-made pulse generator for pulse ablation. The experiment is divided into a room temperature group and a low temperature group, wherein the room temperature group adopts a centrifuge tube to be placed in a 26 ℃ temperature control environment in a naked mode, and the low temperature group adopts the centrifuge tube to be placed in an ice water bath for temperature reduction control. The temperature of the room temperature group is stable at 22-26 ℃ and the temperature of the low temperature group is stable at 0-5 ℃ through measurement. The pulse parameters used for both the room temperature group and the low temperature group were: the pulse voltage was 0 to 800V, and one set of experiments (where 0V was a control group to which no pulse intensity voltage was applied) was set at intervals of 100V, the pulse width was 30 μ s, the number of pulses was 60, and the pulse frequency was 1Hz, wherein each set of experiments was provided with multiple wells, and 3 sets were measured in parallel, for a total of 2 x 9 x 2 x 3 to 108 sets.

FIG. 4 is a graph showing the comparison of the cell viability in pulsed electric field ablation experiments of normal cells L929, 293T and HC11 at room temperature (22-26 ℃) and at low temperature (0-5 ℃) in an ice-water bath, wherein the abscissa is a pulse voltage of 0-800V, the step size is 100V, the ordinate is the calculated cell viability, and 0V (no pulse voltage control group) is taken as 100% viability. The room temperature curves 41, 43, 45 and the ice-water bath curves 42, 44, 46 all show a tendency of first falling smoothly and then falling sharply and then falling smoothly, that is, with the increasing pulse voltage, the survival rate of the cells changes slowly, then suddenly falls rapidly and finally returns to slow change, wherein the sudden change area is the pulse intensity threshold of the cells.

FIG. 4(A) is a graph showing the cell viability of L929, which is indicated by the room temperature curve 41 and the ice-water bath curve 42, wherein the cell viability of L929 at 100V, 200V and 300V is 94.51%, 93.84% and 78.67%, and the cell viability of L929 at 100V, 200V and 300V is 95.18%, 92.43% and 85.07%, and the cell viability in both environments is maintained at a relatively high level; the survival rate of the L929 at the room temperature at 400V is 20.50%, the survival rate of the ice-water bath at 400V is 50.44%, the survival rate at the room temperature at 500V is 14.60%, the survival rate of the ice-water bath at 500V is 26.08%, the survival rates under the two environments are greatly different, and the cell survival rate of the ice-water bath is obviously higher than that under the room temperature; the survival rates of L929 at room temperature at 600V, 700V and 800V are 13.51%, 8.64% and 8.18%, the survival rates of ice water bath at 600V, 700V and 800V are 14.36%, 11.66% and 9.54%, and the survival rates under the two environments are nearly consistent and are maintained at a relatively low state.

FIG. 4(B) is a graph showing the cell viability of 293T, which is indicated by the room temperature curve 43 and the ice-water bath curve 44, wherein the cell viability of 293T is 90.61% and 83.76% at 100V and 200V, and the cell viability of 293T is 92.72% and 87.13% at 100V and 200V, and the cell viability in both environments is maintained at a relatively high level; the room temperature survival rate of 293T at 300V is 39.18%, the survival rate of ice-water bath at 300V is 72.41%, the survival rates under the two environments are different greatly, and the cell survival rate of the ice-water bath is obviously higher than that under the room temperature; the survival rates of 293T at room temperature of 400V, 500V, 600V, 700V and 800V are 9.55%, 5.62%, 3.39% and 1.92%, the survival rates of ice-water bath at 400V, 500V, 600V, 700V and 800V are 12.64%, 7.2%, 5.13%, 2.99% and 2.68%, and the survival rates under the two environments are nearly consistent and are maintained in a relatively low state.

FIG. 4(C) is a graph showing the cell viability of HC11, and it can be seen from the room temperature curve 45 and the ice-water bath curve 46 that the room temperature viability of HC11 at 100V and 200V is 93.11% and 89.83%, and the ice-water bath viability at 100V and 200V is 96.48% and 94.50%, respectively, and the viability in both environments is maintained at a relatively high level; the survival rate of HC11 at room temperature at 300V is 41.36%, the survival rate of ice-water bath at 300V is 63.92%, the survival rates under the two environments are greatly different, and the cell survival rate of the ice-water bath is obviously higher than that under room temperature; the survival rates of HC11 at room temperature at 400V, 500V, 600V, 700V and 800V are 15.59%, 5.99%, 5.08%, 4.58% and 3.56%, and the survival rates of HC11 at 400V, 500V, 600V, 700V and 800V in ice-water bath are 20.57%, 6.49%, 5.17%, 4.40% and 3.91%, and the survival rates under the two environments are nearly consistent and are maintained at a relatively low state.

Example 2: pulsed electric field ablation experiments of cancer cells at normal temperature (22-26 ℃) and under cooling of ice-water bath at low temperature (0-5 ℃).

Three cancer cells B16, Hela and 4T1 are selected as experimental objects in the experiment, an electrode cup is used for placing cell suspension blown by a culture medium in the experiment, the cell suspension is placed in a safe electrode chamber, and then the safe electrode chamber is connected with a self-made pulse generator for pulse ablation. The experiment is divided into a room temperature group and a low temperature group, wherein the room temperature group adopts a centrifuge tube to be placed in a 26 ℃ temperature control environment in a naked mode, and the low temperature group adopts the centrifuge tube to be placed in an ice water bath for temperature reduction control. The temperature of the room temperature group is stable at 22-26 ℃ and the temperature of the low temperature group is stable at 0-5 ℃ through measurement. The pulse parameters used for both the room temperature group and the low temperature group were: the pulse voltage was 0 to 800V, and one set of experiments (where 0V was a control group to which no pulse intensity voltage was applied) was set at intervals of 100V, the pulse width was 30 μ s, the number of pulses was 60, and the pulse frequency was 1Hz, wherein each set of experiments was provided with multiple wells, and 3 sets were measured in parallel, for a total of 2 x 9 x 2 x 3 to 108 sets.

FIG. 5 is a graph of the comparison of cell viability for pulsed electric field ablation experiments of cancer cells B16, Hela and 4T1 at room temperature (22-26 ℃) and cooling in an ice water bath at low temperature (0-5 ℃), wherein the abscissa is a pulse voltage of 0-800V, the step size is 100V, the ordinate is the calculated cell viability, and 0V (no pulse voltage control group) is taken as 100% viability. The room temperature curves 51, 53, 55 and the ice-water bath curves 52, 54, 56 all show a tendency of first decreasing steadily and then decreasing sharply in a steady decrease, i.e. with a gradual increase of the applied pulse voltage, the survival rate of the cells changes slowly, then suddenly decreases and finally returns to a slow change, wherein the abrupt change region is the pulse intensity threshold of the cells.

FIG. 5(A) is a graph of cell viability for B16, and from the room temperature curve 51 and the ice water bath curve 52, the viability in both environments was nearly identical between 0 and 800V, with no significant difference. Wherein the survival rate is maintained at a relatively high level at 100V and 200V, and is 98.33% and 89.21% at room temperature and 97.45% and 93.78% in ice-water bath; when the temperature is 300V, mutation occurs, the survival rate drops to 9.82% of room temperature, and the ice-water bath is 13.32%; the survival rate has been reduced to a very low level at 400V, 500V, 600V, 700V, 800V, 5.00%, 3.25%, 2.76%, 2.15%, 1.49% at room temperature, and 5.41%, 3.92%, 3.24%, 2.68%, 2.60% under ice-water bath.

FIG. 5(B) is a graph showing the cell viability of Hela, and it can be seen from the room temperature curve 53 and the ice-water bath curve 54 that the viability in both environments is nearly consistent between 0 and 800V, with no significant difference. Wherein the survival rate is maintained at a relatively high level at 100V and 200V, wherein the survival rate is 93.07% and 91.77% at room temperature, and the survival rate is 96.06% and 95.05% under ice-water bath; when the temperature is 300V, mutation occurs, the survival rate drops to 26.15 percent of room temperature, and the ice-water bath is 29.95 percent; the survival rate slowly decreases and is stabilized at a lower level at 400V, 500V, 600V, 700V and 800V, and the survival rate is 20.09%, 14.89%, 14.37%, 7.71% and 4.76% at room temperature and 25.09%, 16.21%, 14.01%, 7.23% and 7.05% under an ice-water bath.

FIG. 5(C) is a graph of cell viability for 4T1, and from the room temperature curve 55 and the ice water bath curve 56, the viability in both environments was nearly identical between 0 and 800V, with no significant difference. Wherein the survival rate is kept at a higher level at 100V, 96.44 percent at room temperature and 95.54 percent under ice-water bath; the survival rate is gradually reduced when the mutant is generated at 200V, 300V and 400V, wherein the survival rate is 74.81%, 49.75% and 16.92% at room temperature, and the survival rate is 80.79%, 50.56% and 21.31% in ice-water bath; the survival rate has been reduced to a very low level at 500V, 600V, 700V, 800V, 8.91%, 5.34%, 3.18%, 3.82% at room temperature, and 8.43%, 5.20%, 2.60%, 4.21% in ice-water bath.

As can be seen from examples 1 and 2, the cell selectivity of normal cells and cancer cells under different temperature environments is obviously different from the results of pulsed electric field ablation under cooling at room temperature (22-26 ℃) and ice-water bath at low temperature (0-5 ℃). The cell survival rates of the cancer cells at room temperature and low temperature are always consistent, which shows that the reduction of the temperature has no influence on the ablation capacity of the pulsed electric field of the cancer cells; the cell survival rate curve of normal cells at low temperature is always above the cell survival rate curve at room temperature, which shows that the cell survival rate can be improved to a certain extent by reducing the temperature, and particularly, the reduction speed of the cell survival rate at low temperature is greatly reduced compared with that at room temperature along with the increase of the pulse voltage near the cell threshold value, so that the cell survival rate curve has obvious ablation selectivity near the cell threshold value. Therefore, when the normal cells and the cancer cells are subjected to cooling treatment and pulse treatment at the same time, the survival rate of the cancer cells is kept unchanged, and the survival rate of the normal cells is greatly improved, so that the survival of the normal cells is protected to the greatest extent when the cancer cells are ablated and killed, and the operation of the normal cells is maintained.

Then, in order to find out the condition parameters with larger difference of the cell survival rates under two temperature environments, the parameter setting of the pulse electric field is tried to be changed, the fact that different pulse widths have certain influence on the survival rate of normal cells is found, and the difference of the cell survival rates at low temperature and room temperature is larger and larger along with the reduction of the pulse widths.

Example 3: pulsed electric field ablation experiments of normal cells at different pulse widths.

Three normal cells of L929, 293T and HC11 are selected as experimental objects in the experiment, an electrode cup is used for placing cell suspension blown by a culture medium in the experiment, the cell suspension is placed in a safe electrode chamber, and then the cell suspension is connected with a self-made pulse generator for pulse ablation. The experiment is divided into a room temperature group and a low temperature group, wherein the room temperature group adopts a centrifuge tube to be placed in a 26 ℃ temperature control environment in a naked mode, and the low temperature group adopts the centrifuge tube to be placed in an ice water bath for temperature reduction control. The temperature of the room temperature group is stable at 22-26 ℃ and the temperature of the low temperature group is stable at 0-5 ℃ through measurement. Wherein aiming at different pulse widths, two groups of experiments are additionally arranged, pulse ablation is respectively carried out under the pulse widths of 10 mus and 1 mus, and the pulse parameters used by the room temperature group and the low temperature group are as follows: at 10. mu.s: the pulse voltage is 0-800V, and every 100V set up a series of experiments (wherein 0V is the control group, do not give any pulse intensity voltage), the pulse quantity is 60, the pulse frequency is 1Hz, wherein each series of experiments have multiple holes, and 3 groups of parallel determination, total 2 x 9 x 2 x 3 is 108 groups; at 1. mu.s: the pulse voltage was 0-1400V, and a set of experiments (where 0V is a control group to which no pulse intensity voltage was applied) was set at intervals of 200V, the number of pulses was 60, and the pulse frequency was 1Hz, wherein each set of experiments was provided with multiple wells, and 3 sets were measured in parallel, for a total of 2 x 8 x 2 x 3 to 96 sets.

FIG. 6 is a graph showing the comparison of cell viability in pulsed electric field ablation experiments of normal cells L929, 293T and HC11 at pulse widths of 30 μ s, 10 μ s and 1 μ s, wherein the abscissa is a pulse voltage of 0-800V, the step size is 100V, the ordinate is the calculated cell viability, and 0V (non-pulse voltage control group) is taken as 100% viability. The

room temperature curve

61, 63, 65, 67, 69, 611, 613, 615, 617 and the ice-

water bath curve

62, 64, 66, 68, 610, 612, 614, 616, 618 all show a tendency of first falling smoothly and then falling sharply on a smooth falling curve, i.e. with the gradual increase of the applied pulse voltage, the survival rate of the cells changes slowly at first, then suddenly falls, and finally returns to a slow change, wherein the sudden change area is the pulse intensity threshold of the cells.

FIG. 6(A) is a graph of cell viability for L929, with the upper panel being a graph at a pulse width of 30. mu.s, the lower left being a graph at a pulse width of 10. mu.s, and the lower right being a graph at a pulse width of 1. mu.s. From a comparison of the room temperature curve 61 and the ice-water bath curve 62, the room temperature curve 63 and the ice-water bath curve 64, the room temperature curve 65 and the ice-water bath curve 66, it can be concluded that: the cell survival rate of the L929 in the ice-water bath low-temperature environment is greatly improved; the survival rate at room temperature is obviously different from that of the ice-water bath when the pulse width is 30 mu s and the survival rate at room temperature is 20.50% at 400V and 50.44% at 500V, and the survival rate at room temperature is 14.60% at 500V and 26.08% at 500V; the survival rate at room temperature and the survival rate in an ice water bath are obviously different when the pulse width is 10 mu s and the pulse voltage is 500V, 600V and 700V, the survival rate at room temperature is 34.53% at 500V, the survival rate in the ice water bath is 62.41%, the survival rate at room temperature is 9.59% at 600V, the survival rate in the ice water bath is 26.48%, the survival rate at room temperature is 4.15% at 700V, and the survival rate in the ice water bath is 17.13%; the survival rate at room temperature was clearly different between the room temperature survival rate at 800V and 1000V at a pulse width of 1 μ s and the survival rate in the ice-water bath at 800V, 32.18% at room temperature, 82.19% at ice-water bath, 16.28% at 1000V and 45.93% at ice-water bath.

FIG. 6(B) is a graph of cell viability at 293T, top 30 μ s pulse width, bottom left 10 μ s pulse width, and bottom right 1 μ s pulse width. From a comparison of the room temperature curve 67 and the ice-water bath curve 68, the room temperature curve 69 and the ice-water bath curve 610, the room temperature curve 611 and the ice-water bath curve 612, it can be derived: the cell survival rate of 293T in the ice-water bath low-temperature environment is greatly improved; when the pulse width is 30 mus, the survival rate at room temperature and the survival rate in ice-water bath are obviously different when the pulse voltage is 300V, and at 300V, the survival rate at room temperature is 39.18 percent and the survival rate in ice-water bath is 72.41 percent; when the pulse width is 10 mu s, the survival rate at room temperature is obviously different from that of the ice-water bath when the pulse voltage is 400V and 500V, the survival rate at room temperature is 30.12 percent when the pulse width is 400V, the survival rate of the ice-water bath is 58.07 percent, the survival rate at room temperature is 13.73 percent when the pulse width is 500V, and the survival rate of the ice-water bath is 30.61 percent; the survival rates at room temperature and ice water bath were clearly different at pulse voltages of 800V, 1000V and 1200V at a pulse width of 1 μ s, 38.12% at room temperature and 62.58% at 800V, 9.66% at 1000V, 35.38% at ice water bath, 8.31% at 1200V and 24.77% at 1000V.

FIG. 6(C) is a graph of HC11 cell viability, top 30 μ s pulse width, bottom left 10 μ s pulse width, and bottom right 1 μ s pulse width. From a comparison of the room temperature curve 613 with the ice-water bath curve 614, the room temperature curve 615 with the ice-water bath curve 616, the room temperature curve 617 with the ice-water bath curve 618, it can be concluded that: the cell survival rate of HC11 in the low-temperature environment of ice-water bath is greatly improved; when the pulse width is 30 mus, the survival rate at room temperature and the survival rate in the ice-water bath are obviously different when the pulse voltage is 300V, and the survival rate at room temperature is 41.36% and the survival rate in the ice-water bath is 63.92% when the pulse voltage is 300V; the survival rate at room temperature and the survival rate of the ice-water bath are obviously different when the pulse width is 10 mu s and the pulse voltage is 300V, 400V, 500V and 600V, the survival rate at room temperature is 72.99% at 300V, the survival rate of the ice-water bath is 89.55%, the survival rate at room temperature is 66.07% at 400V, the survival rate of the ice-water bath is 79.91%, the survival rate at room temperature is 39.65% at 500V, the survival rate of the ice-water bath is 52.41%, the survival rate at room temperature is 12.23% at 600V, and the survival rate of the ice-water bath is 26.69%; the survival rates at room temperature and ice water bath were clearly different at pulse voltages of 600V, 800V, 1000V, and 1200V at 1 μ s, 32.54% at room temperature and 54.86% at 600V, 16.35% at room temperature and 42.47% at 800V, 8.70% at 1000V, 28.26% at ice water bath, 6.00% at 1200V, and 16.56% at 1000V.

Example 4: pulsed electric field ablation experiments of cancer cells at different pulse widths.

Three cancer cells B16, Hela and 4T1 are selected as experimental objects in the experiment, an electrode cup is used for placing cell suspension blown by a culture medium in the experiment, the cell suspension is placed in a safe electrode chamber, and then the cell suspension is connected with a self-made pulse generator to carry out pulse ablation. The experiment is divided into a room temperature group and a low temperature group, wherein the room temperature group adopts a centrifuge tube to be placed in a 26 ℃ temperature control environment in a naked mode, and the low temperature group adopts the centrifuge tube to be placed in an ice water bath for temperature reduction control. The temperature of the room temperature group is stable at 22-26 ℃ and the temperature of the low temperature group is stable at 0-5 ℃ through measurement. Wherein aiming at different pulse widths, two groups of experiments are additionally arranged, pulse ablation is respectively carried out under the pulse widths of 10 mus and 1 mus, and the pulse parameters used by the room temperature group and the low temperature group are as follows: at 10. mu.s: the pulse voltage is 0-800V, and every 100V set up a series of experiments (wherein 0V is the control group, do not give any pulse intensity voltage), the pulse quantity is 60, the pulse frequency is 1Hz, wherein each series of experiments have multiple holes, and 3 groups of parallel determination, total 2 x 9 x 2 x 3 is 108 groups; at 1 μ s: the pulse voltage was 0 to 1400V, and a set of experiments (where 0V was a control group to which no pulse intensity voltage was given) was set at intervals of 200V, the number of pulses was 60, and the pulse frequency was 1Hz, wherein each set of experiments was provided with multiple wells, and 3 sets were measured in parallel, for a total of 2 × 8 × 2 × 3 and 96 sets.

FIG. 7 is a graph of the comparison of cell viability in pulsed electric field ablation experiments for cancer cells B16, Hela and 4T1 at pulse widths of 30 μ s, 10 μ s and 1 μ s, with a pulse voltage of 0-800V on the abscissa, with a step size of 100V, and a calculated cell viability on the ordinate, with 0V (no pulse voltage control group) as 100% viability. The room temperature curves 71, 73, 75, 77, 79, 711, 713, 715, 717 and the ice-water bath curves 72, 74, 76, 78, 710, 712, 714, 716, 718 all show a tendency of first decreasing smoothly and then decreasing sharply in a smooth manner, i.e., with a gradual increase in the applied pulse voltage, the cell survival rate changes slowly at first, then suddenly decreases suddenly, and finally returns to a slow change, wherein the sudden change region is the pulse intensity threshold of the cell, and under the ablation of different pulse widths of 30 mus, 10 mus and 1 mus, the cell survival rate at room temperature is consistent with that under the ice-water bath and shows no obvious difference.

As can be seen from examples 3 and 4, the ablation of normal cells under the pulse widths of 30 μ s, 10 μ s and 1 μ s shows a certain cell ablation selectivity under different temperature environments, while the cell ablation survival rate of cancer cells after the pulse widths are changed is not changed, and the cell survival rates at room temperature and low temperature are consistent. And the normal cells have larger increase amplitude of the cell survival rate at low temperature along with the reduction of the pulse width, and the crossed voltage interval is also larger, so that the ablation contrast at room temperature is more obvious, and the cell threshold value is more conveniently controlled to selectively kill the cells.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and amendments can be made without departing from the spirit of the present invention, and these modifications and amendments should also be considered as within the scope of the present invention.

Claims

1. A pulse ablation method for increasing ablation selectivity by using low temperature is characterized in that under the same pulse condition, cancer cells can die by changing the temperature of a treatment area, the survival rate of normal cells is greatly improved, namely the treatment selectivity of the ablation area can be improved, and the method specifically comprises the following steps:

by adding a temperature control device on the original pulsed electric field ablation electrode, a stable low-temperature field (0-5 ℃) can be formed before pulse voltage is applied; before the pulse parameter electric field is given for ablation, the temperature of the treatment area is adjusted to a preset low temperature through a temperature control device, after the temperature is controlled stably, treatment is carried out under the corresponding pulse condition, and after the pulse delivery is finished, the temperature is returned to the proper temperature of the human body.

2. A cryogenic electrode capable of increasing ablation selectivity is characterized in that the cryogenic electrode has the functions of the existing pulse electrode, can output and generate a stable pulse electric field, is provided with a temperature control system, and can form a stable cryogenic field, wherein cryogenic treatment is performed before the pulse electric field is ablated, and can be returned to the temperature after the electric field is ablated;

the temperature measuring device is used for monitoring the temperature of the environment where the ablation electrode is located in real time, is matched with the temperature control device for real-time temperature control, and accurately judges whether the temperature required by treatment is reached.

3. The cryogenic electrode capable of increasing ablation selectivity according to claim 2, wherein the cryogenic electrode comprises, but is not limited to, an electrode under single-pulse electric field ablation, an electrode under combined use of cryoablation and pulse ablation, and an electrode under combined use of thermal ablation and pulse ablation, and is capable of satisfying cryotreatment before pulse ablation and greatly improving killing selectivity of tumors during ablation under the combination of a stable pulsed electric field and a cryogenic field.

4. The cryogenic electrode capable of increasing ablation selectivity according to claim 2, wherein the diameter of the ablation electrode needle is variable, and the inside of the ablation electrode needle is of a hollow structure and is used for placing the temperature control device, the temperature measuring device and the connecting circuit.

5. The cryogenic electrode capable of increasing ablation selectivity according to claim 2, wherein the material of the ablation electrode needle can be various conductive and heat conductive materials such as gold, silver, copper, platinum, zinc, metal oxides, conductive carbon materials and the like; the insulating material can be polyurethane, polytetrafluoroethylene, polyethylene, glass fiber and other materials.

6. The cryogenic electrode capable of increasing ablation selectivity according to claim 2, wherein the ablation electrode needle can be replaced by a medical flexible catheter and can be applied to minimally invasive ablation of human body lumen tissue.

7. A cryogenic electrode for increasing ablation selectivity according to claim 2 comprising, in use, at least one electrode which is energised and at least one electrode which is earthed, the electrode being capable of withstanding a pulse voltage of 3000 to 4000V, a pulse width of 0.01 to 100 μ s, and a pulse voltage of 1kHz to 1 MHz.

8. The cryogenic electrode of claim 2 which increases ablation selectivity, comprising: the pair of ablation electrode needles are used, and the temperature control device and the temperature measuring device are used;

9. The cryogenic electrode of claim 2 with increased ablation selectivity comprising: the pair-used ablation electrode needle, the gas delivery pipeline and the temperature measuring device;

the tail end of the ablation electrode needle is provided with a temperature measuring terminal and a pulse generator connecting end, and the temperature measuring terminal and the pulse generator connecting end are used for man-machine interaction to monitor temperature and a pulse electric field.

10. A method of pulse ablation with increased ablation selectivity according to claim 1, wherein the method can make the ablation zone more accurate and reduce the damage of normal tissues, and comprises the following steps:

the cryogenic electrode capable of increasing ablation selectivity according to any one of claims 2-9 is applied, and a temperature control device is added on the existing pulsed electric field ablation electrode, so that a stable cryogenic field can be formed before pulse voltage is applied; before the pulse parameter electric field is given for ablation, the temperature of the treatment area is adjusted to a preset low temperature through a temperature control device, after the temperature is controlled stably, treatment is carried out under the corresponding pulse condition, and the temperature can be heated to a proper temperature of a human body after the pulse delivery is finished. Ablation selectivity is enhanced by lowering the temperature of the treatment area at the time of pulse administration. Under the same pulse condition, by changing the temperature of the treatment area, cancer cells can die as usual, and the survival rate of normal cells is greatly improved.