Microsecond pulsed electric field parameter selection method and device for killing senescent cells

Abstract

The invention discloses a microsecond pulsed electric field parameter selection method and device for killing senescent cells, and the method comprises the steps: 1) obtaining biological tissue cells, and carrying out cell culture to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells; 2) setting n groups of pulse electric field parameters for killing senescent cells; 3) placing the ith group of cell reagents containing normal cells and the ith group of cell reagents containing senescent cells between the electrode pair, and performing electrical stimulation on the ith group of cell reagents containing normal cells and the ith group of cell reagents containing senescent cells by using the ith group of pulsed electric field parameters; and 4) performing cell activity detection, PI positive rate detection and electric sensitivity detection on each group of cell reagents subjected to electrical stimulation, and determining optimal pulse electric field parameters. The device comprises an electrode pair and a pulse generator. Compared with a traditional chronic wound surface treatment method, the senescence cells can be efficiently and accurately killed only through an electric field, and no heat effect and no side effect exist.

Description

Technical Field

[0001] The present invention relates to the technical field of wound treatment, and in particular to a method and device for selecting parameters of a microsecond pulse electric field for killing senescent cells. Background Art

[0002] Diabetes is a common chronic disease. According to statistics, as of 2022, the total number of diabetes patients in China has reached 140 million, ranking first in the world. It is a disease that is difficult to cure and has a low control rate, with a control rate of only 36.7%. Diabetic patients are often accompanied by many complications, the most common of which are chronic diabetic wounds or foot ulcers. Most chronic wounds of diabetic patients have a long treatment cycle, slow healing and high recurrence rate, and even cause amputation in severe cases. The typical characteristics of chronic wounds are poor blood circulation, abnormal inflammation and the inability to produce the outermost epidermis. Diabetic wounds are different from ordinary wounds. Their wound microenvironment is complex and has many influencing factors, such as hyperglycemia, excessive production of ROS, and persistent inflammatory state. Among them, wound cell senescence is one of the important factors in the pathophysiology of non-healing wounds in diabetic patients. Hyperglycemia, oxidative stress, and mitochondrial and DNA damage in the diabetic wound environment are potential factors that induce cell senescence. The number of senescent cells in diabetic wounds is much greater than that in normal wounds. Senescent cells are a type of cell that permanently proliferates and changes in morphology, secretes complex pro-inflammatory factors, increases autophagy, and resists apoptosis. Compared with normal cells, the cell membrane of senescent cells shrinks, making the cell volume larger, and the cell and nucleus become larger, while the distance between the nucleus and the cell membrane becomes smaller. The long-term presence of senescent cells keeps the wound in the inflammatory stage and is difficult to heal. Cell senescence hinders tissue regeneration and triggers persistent low-level inflammation, thereby accelerating the aging of other cells.

[0003] There are many types of cells in the wound, including fibroblasts, monocytes, endothelial cells, epidermal cells, etc. There is still no good solution to eliminate senescent cells without damaging normal cells. Due to the difference in morphology between senescent cells and normal cells in the wound, changes in their cell membranes and nuclear membranes will cause changes in the electroporation threshold, resulting in differences in their impedance values. At present, there are certain gaps in the electrical impedance testing of senescent cells and normal cells, and the measurement of cell morphology and nuclear membrane size. Similarly, research on how to achieve selective killing of senescent cells in the wound to promote wound healing is also slightly insufficient. Summary of the invention

[0004] The purpose of the present invention is to provide a method and device for selecting microsecond pulse electric field parameters for killing senescent cells.

[0005] The technical solution adopted to achieve the technical purpose of the present invention is as follows: a method for selecting parameters of a microsecond pulse electric field for killing senescent cells comprises the following steps:

[0006] 1) Obtain biological tissue cells and perform cell culture to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells, where n is a positive integer.

[0007] 2) Set n groups of pulse electric field parameters as follows:

[0008]

[0009] Where I is the pulse electric field parameter matrix. i is the i-th group of pulse electric field parameters, i=1,2,...,n, I ij is the jth parameter of the i-th group of pulse electric field parameters, j=1, 2, ..., m, and m is a positive integer.

[0010] 3) Place the i-th group of cell reagents containing normal cells between the electrode pairs, and set the parameters of the pulse generator to the i-th group of pulse electric field parameters I i .

[0011] The pulse generator transmits a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents including normal cells.

[0012] The i-th group of cell reagents containing senescent cells is placed between the electrode pairs, and the parameters of the pulse generator are set to the i-th group of pulsed electric field parameters I i .

[0013] The pulse generator is configured to transmit a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents containing senescent cells, where the initial value of i is 1.

[0014] 4) Let i=i+1, and repeat step 3) until all cell reagents are electrically stimulated.

[0015] 5) Perform cell activity detection, PI positivity detection, and electrical sensitivity detection on each group of cell reagents that have undergone electrical stimulation to determine the optimal pulse electric field parameters.

[0016] Furthermore, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse number.

[0017] Furthermore, the pulse amplitude ranges from 100V to 400V.

[0018] The electric field strength ranges from 500 V / cm to 2000 V / cm.

[0019] Furthermore, the pulse width ranges from 50us to 200us.

[0020] Further, the steps of the cell activity detection are as follows:

[0021] 5.1.1) Add the electrically stimulated cell reagent into a 96-well plate, and place the 96-well plate in a cell incubator for a period of time.

[0022] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0023] 5.1.3) Use an ELISA reader to measure the absorbance of the cultured cell reagent.

[0024] 5.1.4) Compare the difference in absorbance values ​​between normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that achieve the maximum difference in cell activity between normal cells and senescent cells.

[0025] Further, the steps of the PI positive rate detection are as follows:

[0026] 5.2.1) Add PI staining working solution to the cell reagent that has not been electrically stimulated and resuspend the cells to obtain a cell suspension containing normal cells and a cell suspension containing senescent cells.

[0027] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation is performed on the i-th group of cell suspensions containing normal cells and the i-th group of cell suspensions containing senescent cells.

[0028] 5.2.3) The cell suspension after the electrical stimulation treatment is incubated in the dark for a period of time, and the cell suspension after the incubation in the dark is detected by flow cytometry to obtain the PI positive rate of the cells.

[0029] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0030] Further, the steps of the electrical sensitivity detection are as follows:

[0031] 5.3.1) Add PBS to the cells that have not been electrically stimulated for washing.

[0032] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in dark for a period of time to obtain a detection solution containing normal cells and a detection solution containing senescent cells.

[0033] 5.3.3) Using the i-th group of pulse electric field parameters I i The i-th group of test fluids containing normal cells and the i-th group of test fluids containing senescent cells are electrically stimulated, and the fluorescence change process of the cells is recorded in real time using a fluorescence microscope.

[0034] 5.3.4) By comparing the fluorescence dissipation of normal cells and senescent cells and the PI content in the cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that make the fluorescence dissipation of senescent cells fast and the PI content in normal cells high are obtained.

[0035] A device for applying the method comprises an electrode pair and a pulse generator.

[0036] The electrode pair is used to electrically stimulate the cell reagent to be treated.

[0037] The pulse generator is used to generate a microsecond pulse electric field and transmit it to the electrode pair.

[0038] Furthermore, the electrode cup includes a positive electrode and a negative electrode.

[0039] The positive electrode and the negative electrode are placed opposite to each other.

[0040] The cell reagent to be treated is placed between the positive electrode and the negative electrode.

[0041] Furthermore, there is a gap between the positive electrode and the negative electrode.

[0042] The technical effect of the present invention is unquestionable. The present invention proposes a new method for selectively killing senescent cells with microsecond pulse electric fields. Compared with traditional treatment methods for chronic wounds, the present invention only requires electric fields to efficiently and accurately kill senescent cells, without thermal effects or side effects.

[0043] The present invention combines the volume difference and cell impedance characteristics between senescent cells and normal cells, and uses a pulsed electric field to achieve different degrees of electroporation of the two types of cells, thereby achieving the effect of causing senescent cell death but a higher survival rate of normal cells.

[0044] The present invention proposes to regulate the pulse electric field parameters (amplitude, pulse width, frequency, action time) acting on cells based on the difference in morphology and electrical impedance between senescent cells and normal cells, so as to efficiently and selectively remove senescent cells.

[0045] Compared with traditional chemotherapy or radiotherapy, electroporation technology is safer. Since electroporation only physically perforates the cell membrane, it does not involve the side effects of chemotherapy or radioactive damage, so it causes less damage to the wound surface and a shorter recovery period.

[0046] The selective killing of senescent cells in the present invention is superior to other traditional treatment methods in the process of wound repair. The electroporation technology can achieve precise killing of senescent cells. By precisely controlling the electric field intensity, pulse number and pulse width, etc., senescent cells can be selectively perforated, while causing less damage to normal cells. This selective killing can protect normal cells to the maximum extent and reduce the side effects of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A flow chart of a method for selecting parameters of a microsecond pulse electric field for killing senescent cells;

[0048] Figure 2 Schematic diagram of the morphology of 3T3 normal adherent cells and senescent adherent cells; Figure 2 (a) is a schematic diagram of the normal adherent morphology of 3T3 cells; Figure 2 (b) is a schematic diagram of the morphology of senescent adherent cells;

[0049] Figure 3 Schematic diagram of the morphology of 3T3 normal suspension cells and senescent suspension cells; Figure 3 (a) is a schematic diagram of the normal morphology of 3T3 suspension cells; Figure 3 (b) is a schematic diagram of the morphology of senescent suspension cells;

[0050] Figure 4 Schematic diagram of the real and imaginary parts of impedance of 3T3 normal cells and senescent cells;

[0051] Figure 5 Schematic diagram of cell activity of 3T3 normal cells and senescent cells; Figure 5 (a) Schematic diagram of cell activity with different electrical parameters under pulse width of 50us; Figure 5 (b) Schematic diagram of cell activity with different electrical parameters under pulse width of 100us; Figure 5 (c) Schematic diagram of cell activity with different electrical parameters at a pulse width of 200us;

[0052] Figure 6 Schematic diagram of flow cytometry PI single staining of 3T3 normal cells and senescent cells;

[0053] Figure 7 Schematic diagram of fluorescence dissipation of 3T3 normal cells and senescent cells; Figure 7 (a) is a fluorescence schematic diagram of 3T3 normal cells before electrical stimulation; Figure 7 (b) is a fluorescence schematic diagram of 3T3 normal cells after electrical stimulation; Figure 7 (c) is a schematic diagram of PI content in 3T3 normal cells after electrical stimulation; Figure 7 (d) is a fluorescence schematic diagram of senescent cells before electrical stimulation; Figure 7 (e) is a fluorescence schematic diagram of senescent cells after electrical stimulation; Figure 7 (f) Schematic diagram of PI content in senescent cells after electrical stimulation;

[0054] Figure 8 Schematic diagram of the measurement system. DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with the embodiments, but it should not be understood that the above subject matter of the present invention is limited to the following embodiments. Without departing from the above technical ideas of the present invention, various substitutions and changes are made according to the common technical knowledge and customary means in the art, which should all be included in the protection scope of the present invention.

[0056] Embodiment 1:

[0057] See also Figures 1 to 8 , a method for selecting parameters of a microsecond pulse electric field for killing senescent cells, comprising the following steps:

[0058] 1) Obtain biological tissue cells and perform cell culture to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells, where n is a positive integer.

[0059] 2) Set n groups of pulse electric field parameters as follows:

[0060]

[0061] Where I is the pulse electric field parameter matrix. i is the i-th group of pulse electric field parameters, i=1,2,...,n, I ij is the jth parameter of the i-th group of pulse electric field parameters, j=1, 2, ..., m, and m is a positive integer.

[0062] 3) Place the i-th group of cell reagents containing normal cells between the electrode pairs, and set the parameters of the pulse generator to the i-th group of pulse electric field parameters I i .

[0063] The pulse generator transmits a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents including normal cells.

[0064] The i-th group of cell reagents containing senescent cells is placed between the electrode pairs, and the parameters of the pulse generator are set to the i-th group of pulsed electric field parameters I i .

[0065] The pulse generator is configured to transmit a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents containing senescent cells, where the initial value of i is 1.

[0066] 4) Let i=i+1, and repeat step 3) until all cell reagents are electrically stimulated.

[0067] 5) Perform cell activity detection, PI positivity detection, and electrical sensitivity detection on each group of cell reagents that have undergone electrical stimulation to determine the optimal pulse electric field parameters.

[0068] Embodiment 2:

[0069] A method for selecting microsecond pulse electric field parameters for killing senescent cells. The main technical content is shown in Example 1. Furthermore, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse number.

[0070] Embodiment 3:

[0071] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical content of which is shown in any one of Examples 1 to 2, and further, the pulse amplitude ranges from 100V to 400V.

[0072] The electric field strength ranges from 500 V / cm to 2000 V / cm.

[0073] Embodiment 4:

[0074] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical content of which is shown in any one of Examples 1 to 3, and further, the pulse width ranges from 50us to 200us.

[0075] Embodiment 5:

[0076] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 1 to 4. Further, the steps of cell activity detection are as follows:

[0077] 5.1.1) Add the electrically stimulated cell reagent into a 96-well plate, and place the 96-well plate in a cell incubator for a period of time.

[0078] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0079] 5.1.3) Use an ELISA reader to measure the absorbance of the cultured cell reagent.

[0080] 5.1.4) Compare the difference in absorbance values ​​between normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that achieve the maximum difference in cell activity between normal cells and senescent cells.

[0081] Embodiment 6:

[0082] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 1 to 5. Further, the steps of detecting the PI positive rate are as follows:

[0083] 5.2.1) Add PI staining working solution to the cell reagent that has not been electrically stimulated and resuspend the cells to obtain a cell suspension containing normal cells and a cell suspension containing senescent cells.

[0084] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation is performed on the i-th group of cell suspensions containing normal cells and the i-th group of cell suspensions containing senescent cells.

[0085] 5.2.3) The cell suspension after the electrical stimulation treatment is incubated in the dark for a period of time, and the cell suspension after the incubation in the dark is detected by flow cytometry to obtain the PI positive rate of the cells.

[0086] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0087] Embodiment 7:

[0088] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 1 to 6. Further, the steps of the electrical sensitivity detection are as follows:

[0089] 5.3.1) Add PBS to the cells that have not been electrically stimulated for washing.

[0090] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in dark for a period of time to obtain a detection solution containing normal cells and a detection solution containing senescent cells.

[0091] 5.3.3) Using the i-th group of pulse electric field parameters I i The i-th group of test fluids containing normal cells and the i-th group of test fluids containing senescent cells are electrically stimulated, and the fluorescence change process of the cells is recorded in real time using a fluorescence microscope.

[0092] 5.3.4) By comparing the fluorescence dissipation of normal cells and senescent cells and the PI content in the cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that make the fluorescence dissipation of senescent cells fast and the PI content in normal cells high are obtained.

[0093] Embodiment 8:

[0094] A device for applying the method described in any one of Examples 1 to 7, comprising an electrode pair and a pulse generator.

[0095] The electrode pair is used to electrically stimulate the cell reagent to be treated.

[0096] The pulse generator is used to generate a microsecond pulse electric field and transmit it to the electrode pair.

[0097] Embodiment 9:

[0098] A device for selecting parameters of a microsecond pulse electric field for killing senescent cells. The main technical content is shown in Example 8. Furthermore, the electrode cup includes a positive electrode and a negative electrode.

[0099] The positive electrode and the negative electrode are placed opposite to each other.

[0100] The cell reagent to be treated is placed between the positive electrode and the negative electrode.

[0101] Embodiment 10:

[0102] A device for selecting parameters of a microsecond pulse electric field for killing senescent cells. The main technical content is shown in any one of Examples 8 to 9. Furthermore, there is a gap between the positive electrode and the negative electrode.

[0103] Embodiment 11:

[0104] See also Figures 1 to 8 , a method for selecting parameters of a microsecond pulse electric field for killing senescent cells, comprising the following steps:

[0105] 1) Obtain biological tissue cells and perform cell culture to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells, where n is a positive integer.

[0106] The mouse fibroblast cell line 3T3-L1 used was routinely cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1% double antibody (100 μg / mL penicillin and streptomycin) and 10% inactivated fetal bovine serum (FBS) (placed in a constant temperature and saturated humidity incubator at 37°C and 5% CO2. The cell culture was performed by digestion and passage using 0.25% trypsin (0.25% Trypsin-EDTA) throughout the process to obtain a cell reagent containing normal cells.

[0107] 3T3-L1: Take cells with a density of 80%, discard the original culture medium, add culture medium supplemented with H2O2 (300uM / mL) to stimulate the cells for 4 hours, then replace with fresh culture medium and culture for 24 hours to obtain a cell reagent containing senescent cells.

[0108] 2) Set n groups of pulse electric field parameters as follows:

[0109]

[0110] Where I is the pulse electric field parameter matrix. i is the i-th group of pulse electric field parameters, i=1,2,...,n, I ij is the jth parameter of the i-th group of pulse electric field parameters, j=1, 2, ..., m, and m is a positive integer.

[0111] 3) Place the i-th group of cell reagents containing normal cells between the electrode pairs, and set the parameters of the pulse generator to the i-th group of pulse electric field parameters I i .

[0112] The pulse generator transmits a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents including normal cells.

[0113] The i-th group of cell reagents containing senescent cells is placed between the electrode pairs, and the parameters of the pulse generator are set to the i-th group of pulsed electric field parameters I i .

[0114] The pulse generator is configured to transmit a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents containing senescent cells, where the initial value of i is 1.

[0115] 4) Let i=i+1, and repeat step 3) until all cell reagents are electrically stimulated.

[0116] 5) Perform cell activity detection, PI positivity detection, and electrical sensitivity detection on each group of cell reagents that have undergone electrical stimulation to determine the optimal pulse electric field parameters.

[0117] Embodiment 12:

[0118] A method for selecting microsecond pulse electric field parameters for killing senescent cells. The main technical content is shown in Example 11. Furthermore, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse number.

[0119] Embodiment 13:

[0120] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical content of which is shown in any one of Examples 11 to 12, and further, the pulse amplitude ranges from 100V to 400V.

[0121] The electric field strength ranges from 500 V / cm to 2000 V / cm.

[0122] Embodiment 14:

[0123] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells. The main technical content is shown in any one of Examples 11 to 13. Furthermore, the pulse width ranges from 50us to 200us.

[0124] Embodiment 15:

[0125] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 11 to 14. Further, the steps of cell activity detection are as follows:

[0126] 5.1.1) Add the electrically stimulated cell reagent into a 96-well plate, and place the 96-well plate in a cell incubator for a period of time.

[0127] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0128] 5.1.3) Use an ELISA reader to measure the absorbance of the cultured cell reagent.

[0129] 5.1.4) Compare the difference in absorbance values ​​between normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that achieve the maximum difference in cell activity between normal cells and senescent cells.

[0130] Take cells with good growth and a density of 70-80%, discard the original culture medium, add lysis protection reagent (Phosphate Buffer Solution, PBS) to rinse once, add trypsin and put into the cell incubator for digestion for 1 min to make the cells fall off, then add fresh culture medium to terminate the digestion, centrifuge the cell suspension at 800 rpm for 5 min, remove the supernatant, add culture medium to resuspend (3×10 6 cells / mL). The cell suspension was electrically stimulated by a self-developed pulse generator and an electrode cup (2mm between the positive and negative electrodes). Each time, 100uL of the cell suspension was added to the electrode cup and the whole was placed on the electrode base. The electric field strength and pulse width were adjusted according to different pulse electric field parameters. The cell suspension was treated with a pulse electric field at a frequency of 1Hz for 8s. After treatment, the cells were added to a 96-well plate and placed in a cell incubator for further culture for 3h. Then, 10uL of cell proliferation / toxicity detection reagent (Cell Counting Kit-8, CCK-8) was added to each well. After continuing to culture for 2h, the absorbance value at 450nm was measured with an enzyme reader. Data processing was obtained Figure 5The cell activity line graph shown in the figure shows that after experiments with different pulsed electric field parameters, it was found that when the electric field strength was 1000V / cm and the pulse width was 100μs for 8s, the 3T3 normal cells and the senescent cells could obtain the maximum cell activity difference.

[0131] Embodiment 16:

[0132] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 11 to 15. Further, the steps of detecting the PI positive rate are as follows:

[0133] 5.2.1) Add PI staining working solution to the cell reagent that has not been electrically stimulated and resuspend the cells to obtain a cell suspension containing normal cells and a cell suspension containing senescent cells.

[0134] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation is performed on the i-th group of cell suspensions containing normal cells and the i-th group of cell suspensions containing senescent cells.

[0135] 5.2.3) The cell suspension after the electrical stimulation treatment is incubated in the dark for a period of time, and the cell suspension after the incubation in the dark is detected by flow cytometry to obtain the PI positive rate of the cells.

[0136] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0137] Take cells with good growth status and density of 70-80%, digest and centrifuge, discard the supernatant, add propidium iodide (PI) staining working solution to resuspend the cells (1×10 7 Each time, 100uL of cell suspension was added to a 2mm electric shock cup and placed in the electrode tank. The pulsed electric field treatment was performed according to different electric field parameters. The treated cell suspension was transferred to a 1.5mL EP tube and incubated at 37℃ in the dark for 30min before flow cytometry detection. The PI positive rate of normal cells and senescent cells was counted, and the analysis results were as follows: Figure 6 As shown, when the electric field strength is 1000 V / cm and the pulse width is 100 μs and the electric treatment is performed for 8 s, the difference in PI positivity rate between 3T3 normal cells and senescent cells is maximized.

[0138] Embodiment 17:

[0139] A method for selecting parameters of a microsecond pulse electric field for killing senescent cells, the main technical content of which is shown in any one of Examples 11 to 16. Further, the steps of the electrical sensitivity detection are as follows:

[0140] 5.3.1) Add PBS to the cells that have not been electrically stimulated for washing.

[0141] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in dark for a period of time to obtain a detection solution containing normal cells and a detection solution containing senescent cells.

[0142] 5.3.3) Using the i-th group of pulse electric field parameters I i The i-th group of test fluids containing normal cells and the i-th group of test fluids containing senescent cells are electrically stimulated, and the fluorescence change process of the cells is recorded in real time using a fluorescence microscope.

[0143] 5.3.4) By comparing the fluorescence dissipation of normal cells and senescent cells and the PI content in the cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that make the fluorescence dissipation of senescent cells fast and the PI content in normal cells high are obtained.

[0144] Take cells with good growth status and density of 70-80% and prepare them into cell suspension (5×10 6 cells / mL), 5×10 5 cells / well were inoculated into a laser confocal dish with a diameter of 35 mm, cultured for 24 h, the original culture medium was removed, PBS was added to wash slowly twice, 1 mL of Calcein AM / PI detection working solution was added to each dish, and incubated at 37 ° C in the dark for 30 min. The laser confocal dish was placed on the stage of the laser confocal microscope. Based on the optimal parameters of the above experiment, the electric field intensity of 1000 V / cm, pulse width of 100 μs, and pulse number of 8 were selected for pulsed electrical treatment of cells, and the fluorescence changes of the cells were observed and recorded in real time under a fluorescence microscope during the stimulation process. Figure 7 As shown, it was observed that senescent cells were more sensitive to pulsed electric fields, and the fluorescence dissipated too quickly, while the fluorescence of normal cells was concentrated inside the cells. After reversible electroporation, normal cells were more active, and a large amount of PI entered the cells.

[0145] Embodiment 18:

[0146] A device for applying the method described in any one of Examples 11 to 17, comprising an electrode pair and a pulse generator.

[0147] The electrode pair is used to electrically stimulate the cell reagent to be treated.

[0148] The pulse generator is used to generate a microsecond pulse electric field and transmit it to the electrode pair.

[0149] Embodiment 19:

[0150] A device for selecting parameters of a microsecond pulse electric field for killing senescent cells. The main technical content is shown in Example 18. Furthermore, the electrode cup includes a positive electrode and a negative electrode.

[0151] The positive electrode and the negative electrode are placed opposite to each other.

[0152] The cell reagent to be treated is placed between the positive electrode and the negative electrode.

[0153] The electrode used in this shooting has the dimensions of 35 mm long × 5.4 mm wide × 1.6 mm high.

[0154] Embodiment 20:

[0155] A device for selecting parameters of a microsecond pulse electric field for killing senescent cells. The main technical content is shown in any one of Examples 18 to 19. Furthermore, there is a gap between the positive electrode and the negative electrode.

[0156] Embodiment 21:

[0157] See also Figures 1 to 8 , a method for selecting parameters of microsecond pulse electric field for killing senescent cells, the main technical contents are as follows:

[0158] Cell culture: The mouse fibroblast cell line 3T3-L1 was used and routinely cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1% double antibody (100 μg / mL penicillin and streptomycin) and 10% inactivated fetal bovine serum (FBS) in a constant temperature and saturated humidity incubator at 37°C and 5% CO2. The cells were digested and passaged using 0.25% trypsin (0.25% Trypsin-EDTA) throughout the cell culture process.

[0159] Construction of cell senescence model: 3T3-L1: Take cells with a density of 80%, discard the original culture medium, add culture medium supplemented with H2O2 (300uM / mL) to stimulate the cells for 4 hours, and then replace with fresh culture medium for 24 hours.

[0160] Experiment on selective killing of senescent cells by pulsed electric field:

[0161] 1. Size measurement of 3T3 normal cells and senescent cells

[0162] Take cells in good growth state, centrifuge and resuspend them, and then add 1×10 6cells / well were inoculated into 6-well plates. After culturing in an incubator for 24 hours, the cells in each well were digested and collected into 1.5mL EP tubes. The supernatant was discarded after centrifugation. 500uL / tube of cell membrane red fluorescent dye DiI (Cell Plasma Membrane Staining Kit with DiI) and cell nucleus blue fluorescent dye Hoechst (Hoechst 33258) were added to stain the cell membrane and cell nucleus respectively. The cells were incubated at 37℃ in the dark for 20 minutes, centrifuged at 500rpm for 5 minutes, and the staining solution was discarded. The cells were washed with PBS for 3 times and then dropped onto a glass slide. The slide was covered with a coverslip and observed by laser confocal microscopy. The cell and nuclear sizes of aging and normal cells were statistically analyzed by Image processing software. Figure 2 and Figure 3 The test results showed that the morphology and nuclear membrane size of 3T3 normal cells were smaller than those of aging cells.

[0163] 2.3T3 normal cell and senescent cell impedance test

[0164] The impedance spectrum of the cell suspension is measured by the cooperation of a precision impedance analyzer and a test fixture. Before the measurement, the impedance analyzer is calibrated for open circuit, short circuit and load compensation. During the measurement, 20 μL of cell suspension is aspirated and placed in a homemade electrode trough, the dimensions of which are 35 mm long × 5.4 mm wide × 1.6 mm high. The gold electrode at the center is 5 mm × 1.6 mm high, with a spacing of 2 mm. It is fixed to the bottom of a standard 35 mm culture dish, and its impedance spectrum in the frequency range of 20 Hz to 20 MHz is measured by an impedance analyzer. The schematic diagram of the measurement system is shown in the figure. Figure 8 As shown, each sample was read three times, and then the measured impedance spectrum data was fitted using the software EIS Spectrum Analyzer 1.0, and the cell membrane electrical parameters were calculated using the equivalent circuit inversion. The fitting results are shown in Figure 4 As shown, there is a certain difference in the real part of the impedance value between 3T3 normal cells and senescent cells.

[0165] 3. CCK8 cell activity detection

[0166] Take cells with good growth and density of 70-80%, discard the original culture medium, add PBS to rinse once, add trypsin and put into the cell incubator to digest for 1 min to make the cells fall off, then add fresh culture medium to stop digestion, centrifuge the cell suspension at 800 rpm for 5 min, remove the supernatant, add culture medium to resuspend (3×10 6cells / mL). The cell suspension was electrically stimulated by a self-developed pulse generator and an electrode cup (2mm between positive and negative electrodes). Each time, 100uL of cell suspension was added to the electrode cup and the whole was placed on the electrode base. The electric field strength and pulse width were adjusted according to the pulse electric field parameters in Table 1. The cell suspension was treated with a pulse electric field at a frequency of 1Hz for 8s. After treatment, the cells were added to a 96-well plate and placed in a cell incubator for further culture for 3h. Then, 10uCCK-8 (Cell Counting Kit-8) reagent was added to each well. After further culture for 2h, the absorbance value at 450nm was measured with an enzyme reader. Data processing was obtained Figure 5 The cell activity line graph shown in the figure shows that after experiments with different pulsed electric field parameters, it was found that when the electric field strength was 1000V / cm and the pulse width was 100μs for 8s, the 3T3 normal cells and the senescent cells could obtain the maximum cell activity difference.

[0167] Table 1 Experimental electrical parameters for selective killing of 3T3 senescent cells

[0168]

[0169] 4. Flow cytometry PI single staining after pulse electric field treatment

[0170] Take cells with good growth status and density of 70-80%, digest and centrifuge, discard the supernatant, add propidium iodide (PI) staining working solution to resuspend the cells (1×10 7 Each time, 100uL of cell suspension was added to a 2mm electric shock cup and the whole was placed in the electrode tank. The pulsed electric field treatment was performed according to the electric field parameters in Table 2. The treated cell suspension was transferred to a 1.5mL EP tube and incubated at 37℃ in the dark for 30min before flow cytometry detection. The PI positive rates of normal cells and senescent cells were counted and the analysis results were as follows: Figure 6 As shown, when the electric field strength is 1000 V / cm and the pulse width is 100 μs and the electric treatment is performed for 8 s, the difference in PI positivity rate between 3T3 normal cells and senescent cells is maximized.

[0171] Table 2 Flow cytometry parameters of 3T3 normal cells and senescent cells

[0172]

[0173] 5. Real-time photography of PI fluorescence staining (to explore the electrical sensitivity of 3T3 senescent cells and normal cells)

[0174] Take cells with good growth status and density of 70-80% and prepare them into cell suspension (5×10 6 cells / mL), 5×10 5cells / well were inoculated into a laser confocal dish with a diameter of 35 mm, cultured for 24 h, the original culture medium was removed, PBS was added to wash slowly twice, 1 mL of Calcein AM / PI detection working solution was added to each dish, and incubated at 37 ° C in the dark for 30 min. The laser confocal dish was placed on the stage of the laser confocal microscope. Based on the optimal parameters of the above experiment, an electric field strength of 1000 V / cm, a pulse width of 100 μs, and a pulse number of 8 were selected to perform pulsed electrical treatment on the cells and the fluorescence changes of the cells were recorded in real time under a fluorescence microscope during the stimulation process. The electrode used in this shooting is 35 mm long × 5.4 mm wide × 1.6 mm high. The gold electrode at the center is 5 × 1.6 mm in size and 2 mm in spacing. Figure 7 As shown, it was observed that senescent cells were more sensitive to pulsed electric fields, and the fluorescence dissipated too quickly, while the fluorescence of normal cells was concentrated inside the cells. After reversible electroporation, normal cells were more active, and a large amount of PI entered the cells.

Claims

1. A method for selecting parameters of microsecond pulse electric field for killing senescent cells. It is characterized in that The following steps are involved: 1) obtaining biological tissue cells and performing cell culture to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells, where n is a positive integer; 2) Set n groups of pulse electric field parameters as follows: Where, I is the pulse electric field parameter matrix; I i is the i-th group of pulse electric field parameters, i=1,2,...,n, I ij is the jth parameter of the i-th group of pulse electric field parameters, j=1, 2, ..., m, and m is a positive integer. 3) The i-th group of cell reagents containing normal cells is placed between the electrode pairs, and the parameters of the pulse generator are set to the i-th group of pulse electric field parameters I i ; The pulse generator transmits a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the cell reagent of the i-th group including normal cells; The i-th group of cell reagents containing senescent cells is placed between the electrode pairs, and the parameters of the pulse generator are set to the i-th group of pulsed electric field parameters I i ; The pulse generator is configured to transmit a pulse signal to the electrode pair, so that the electrode pair generates electrical stimulation acting on the i-th group of cell reagents containing senescent cells, where the initial value of i is 1. 4) Let i=i+1, and repeat step 3) until all cell reagents are electrically stimulated; 5) Perform cell activity detection, PI positivity detection, and electrical sensitivity detection on each group of cell reagents that have undergone electrical stimulation to determine the optimal pulse electric field parameters.

2. A method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 1, It is characterized in that The pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse number.

3. A method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 2, It is characterized in that The pulse amplitude ranges from 100V to 400V; The electric field strength ranges from 500 V / cm to 2000 V / cm.

4. A method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 2, It is characterized in that The pulse width ranges from 50us to 200us.

5. The method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 1, wherein the steps of detecting cell activity are as follows: 5.1.1) Add the electrically stimulated cell reagent into a 96-well plate, and place the 96-well plate in a cell incubator for a period of time; 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time; 5.1.3) Measure the absorbance value of the cultured cell reagent using an ELISA reader; 5.1.4) Compare the difference in absorbance values ​​between normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that achieve the maximum difference in cell activity between normal cells and senescent cells.

6. A method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 1, It is characterized in that The steps of the PI positive rate detection are as follows: 5.2.1) Add PI staining working solution to the cell reagent that has not been electrically stimulated and resuspend the cells to obtain a cell suspension containing normal cells and a cell suspension containing senescent cells; 5.2.2) Using the i-th group of pulse electric field parameters I i Electrically stimulating the i-th group of cell suspensions containing normal cells and the i-th group of cell suspensions containing senescent cells; 5.2.3) Incubating the cell suspension after the electrical stimulation treatment in the dark for a period of time, and detecting the cell suspension after the incubation in the dark by flow cytometry to obtain the PI positive rate of the cells; 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

7. A method for selecting parameters of a microsecond pulse electric field for killing senescent cells according to claim 1, It is characterized in that The steps of the electrosensitivity detection are as follows: 5.3.1) Add PBS to the cell reagent that has not been electrically stimulated for washing; 5.3.2) Adding Calcein AM / PI detection working solution to the washed cell reagent and incubating in the dark for a period of time to obtain a detection solution containing normal cells and a detection solution containing senescent cells; 5.3.3) Using the i-th group of pulse electric field parameters I i Electrically stimulating the i-th group of test fluids containing normal cells and the i-th group of test fluids containing senescent cells, and recording the fluorescence change process of the cells in real time through a fluorescence microscope; 5.3.4) By comparing the fluorescence dissipation of normal cells and senescent cells and the PI content in the cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that make the fluorescence dissipation of senescent cells fast and the PI content in normal cells high are obtained.

8. A device for applying the method according to any one of claims 1 to 7, It is characterized in that It includes an electrode pair and a pulse generator; The electrode pair is used to electrically stimulate the cell reagent to be treated; The pulse generator is used to generate a microsecond pulse electric field and transmit it to the electrode pair.

9. The device according to claim 8, It is characterized in that The electrode cup includes a positive electrode and a negative electrode; The positive electrode and the negative electrode are placed opposite to each other; The cell reagent to be treated is placed between the positive electrode and the negative electrode.

10. The device according to claim 9, It is characterized in that The positive electrode and the negative electrode have a gap therebetween.

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