CN109593753A - A method of it improving electric energy and induces living cells film electroporation efficiency - Google Patents
A method of it improving electric energy and induces living cells film electroporation efficiency Download PDFInfo
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- CN109593753A CN109593753A CN201811634056.7A CN201811634056A CN109593753A CN 109593753 A CN109593753 A CN 109593753A CN 201811634056 A CN201811634056 A CN 201811634056A CN 109593753 A CN109593753 A CN 109593753A
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Abstract
The electric energy that the present invention utilizes transmission line that will store in capacitor, is discharged into isotonic saline with exponential waveform nanosecond electric pulse, causes the cell membrane of wherein living cells that electroporation occurs.When isotonic saline impedance is higher than line characteristic impedance, because charge reflects in transmission line, the voltage for being discharged into the electric pulse of load is higher than the voltage of capacitor discharge starting point load, so that the same single pulse energy stored in capacitor is discharged into when being suspended in living cells in isotonic saline, the voltage peak-peak of pulse is higher, to improve the efficiency that function electroporation occurs for induction living cells.It exports less gross energy just and can induce higher proportion of living cells and electroporation of cell membrane occur.
Description
Technical field
The present invention relates to a kind of methods of raising electric energy induction living cells film electroporation efficiency.
Technical background
Living cells in biological tissue is isolated by the cell membrane that one layer of phospholipid bilayer forms with microenvironment, this is cell energy
Enough bases survived and be proliferated in the environment.Previous research is thought, square waveform or trapezoidal electric pulse are discharged into biological tissue,
When the pulsewidth of impulse electric field is more than 30 microseconds (us), field strength is more than 800 volt/cms (V/cm), and 8 or more electric pulses just lure
The perforation of guided cell theca cell film, this is to utilize high-intensitive pulse in field of biomedicine application high-voltage pulse electric field at present
Electric field can cause cell membrane that reversible or irreversible electroporation occurs.Reversible electroporation of cell membrane is commonly used to small molecule object
Matter imports in cell membrane.Irreversible electroporation is the basis of tumour non-thermal ablation techniques method.Using the pulse of this Microsecond grade
The single pulse energy of irreversible electroporation occurs for electric field inducing cell film usually more than the nanosecond pulsed electric field that uses in this patent
Height, when being applied to tumour ablation, because tumor tissues and body are in connected state, when electric pulse is discharged into tumor tissues,
Portion of electrical current can also be diffused into surrounding tissue, and caused side effect is to lead to contraction of muscle.
Report there is no to show that can result in cell membrane using the impulse electric field of nanosecond occurs irreversible electroporation at present.Edge
The method that microsecond pulse electric field biological effect is studied is attacked, the generation of the nanosecond pulse electric field in biological effect research is led to
Frequently with the square waveform or trapezoidal electric pulse that are generated by Blumlein transmission line, this design needs to guarantee that target organism imitates
This impedance is consistent with the characteristic impedance of Blumlein transmission line, past when biological sample impedance is less than line characteristic impedance
Line characteristic impedance is reduced toward using parallel connection Blumlein transmission line, but increases gross energy in this way；When biological sample impedance
When greater than line characteristic impedance, using the target biological sample both ends parallel resistance in output, electric energy will be exported again in this way
It has been diverted in parallel resistance.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of raising electric energy induction living cells film electroporation
The method of efficiency.
The present invention adopts the following technical scheme: a kind of method for improving electric energy induction living cells film electroporation efficiency of, the party
Method are as follows: disperse cell to be perforated in isotonic saline, a pair in isotonic saline will be located at transmission line
Electrode leads to storage capacitor, and the electric energy in storage capacitor is discharged into isotonic physiology salt the transmission line in the form of nanosecond pulse
In solution；Wherein, the resistance value of isotonic saline containing cell is higher than line characteristic impedance, to improve in solution
Electric pulse maximum voltage value is allowed to be higher than storage capacitor charging voltage, so that the electricity for improving cell in isotonic saline is worn
The efficiency in hole.
Further, it is assumed that the impedance Z of isotonic salineL, line characteristic impedance Z0, capacitor start to etc.
Initial voltage value is U0, the electric pulse ceiling voltage calculated value being discharged into isotonic saline when seeping saline electric discharge
UpeakCoincidence formula (1):
Further, when the metal needle or metal plate that the electrode is formed using aluminium, stainless steel, titanium alloy.
Further, the isotonic saline refers to: osmotic pressure value 280-330 milli osmol/liter (mOsm/
) and the physiological equilibrium salting liquid of pH value 7.0-7.4 L.
Further, concentration of the cell in isotonic saline is 0.5-4 × 106A every milliliter of living cells
(mL)。
Further, the resistance value of isotonic saline containing cell is higher than line characteristic impedance, according to formula
(1), the peak-peak of output voltage can be higher than voltage value when capacitor starts to discharge, and can improve the same energy stored in capacitor
Measure the efficiency of inducing cell electroporation.
Further, in capacitor same pulse capacitor be discharged into the electric pulse of higher peak-peak voltage it is isotonic
In saline, in the case where other parameters are constant, same overall pulse number can induce higher proportion of cell membrane and occur
Electroporation.
Further, using current research method, it is able to detect that cell membrane increases the transparency of small-molecule substance
There is microcellular structure with cell.
The beneficial effects of the present invention are: it is such the result is that improving the same electric energy inducing cell hair stored in capacitor
The efficiency of raw electroporation, i.e., gross energy as much are discharged into isotonic saline, can induce higher proportion of cell
Electroporation occurs for cell membrane；In other words if to reach a certain proportion of cell electroporation of cell membrane occurs, it is discharged into isotonic life
The gross energy requirement managed in salting liquid is lower.
Thermal energy can be finally changed by being discharged into the total electric energy of biological sample, and consuming lower heat in this way can be achieved with to mesh
The effective prevention of structure function of cell is marked, Apoptosis or necrosis are such as led to by electroporation.Electric energy inducing cell electricity is improved to wear
On the one hand the efficiency in hole can reduce caused temperature in treatment process and increase.Heating ablation has been widely applied to clinical tumor
Local ablation method, such as RF ablation, microwave ablation.But the pipeline configuration for often having blood vessel etc. important around tumor tissues,
The blood flowed in medium vessels can take away a portion heat, influence treatment and execute according to schedule.Meanwhile being transmitted to pipe
The heat of road system will affect that pipe-line system is fully functional so that close to the tissues such as larger blood vessel, bile duct, ureter tumour without
Method realizes ablation.The electric energy that raising is discharged into tissue, can be in ablation targets tissue to the functioning efficiency of target cell
While reduce influence other structures, such as close to tumor tissues pipe-line system, improve the scope of application of this kind of ablation techniques.Separately
On the one hand the electric energy that treatment site in therapeutic process is transmitted to surrounding tissue can be reduced, facilitates flesh caused by control therefore
The side effects such as meat contraction
Detailed description of the invention
Fig. 1: millimicrosecond pulse generator sketch (A)；Voltage and current waveform diagram (B) when impedance is 13 ohm；Impedance
Voltage and current waveform diagram (C) when being 25 ohm；Impedance is 50 ohmic value voltage and current waveform diagrams (D).
Fig. 2: SMMC7721 cell receives cell membrane after 200 nanosecond pulses in the isotonic saline of different impedances
Variation to PI transparency.Scheming A is the control group without Electric Pulse Treatment, the isotonic saline that figure B cell suspends
Electrode pair between impedance be 13 ohm, figure C cell suspend isotonic saline between electrode pair impedance be 25 ohm, scheme D
The impedance between isotonic saline electrode pair that cell suspends is 50 ohm.Scheming E is the thin of control group and three experimental groups
PI staining positive cells percentage changes in born of the same parents.
Fig. 3: SMMC7721 cell receives thin after 200 pulses are handled between electrode in 50 ohm of isotonic saline
After birth is able to detect that perforated phenomenon.Upper figure is cell in 50 ohm of isotonic salines, is not received at impulse electric field
The cell membrane surface ultra microstructure of the control group of reason, the upper figure upper right corner are cell overall picture.The following figure is cell in 50 ohm of isotonic lifes
It manages in salting liquid, receives the cell membrane surface ultra microstructure of 200 pulses processing, the following figure lower right corner is cell overall picture.
Fig. 4: SK-Hep1 cell receives cell membrane after 200 nanosecond pulses in the isotonic saline of different impedances
Variation to PI transparency.Nanosecond pulse is discharged by the 1.8nF capacitor for being charged to 10000 volts, passes through a pair of of parallel connection
50 ohm transmission line of characteristic impedance is discharged to the interelectrode isotonic saline of a pair of plates.Scheme A be without electric pulse at
The control group of reason, figure B cell suspend isotonic saline electrode pair between impedance be 13 ohm, figure C cell suspend etc.
Seeping saline impedance between electrode pair is 25 ohm, the resistance between isotonic saline electrode pair that figure D cell suspends
Resist is 50 ohm.A, respectively shown in B, C, D three times repeat test as a result, wherein PI signal value is determined as 103 or more
Cell PI stained positive, cell membrane are perforated.Scheming E is PI staining positive cells in control group and the cell of three experimental groups
Percentage variation.
Fig. 5: SK-Hep1 cell receives thin after 200 pulses are handled between electrode in 50 ohm of isotonic saline
After birth is able to detect that perforated phenomenon.Upper figure is pair for not receiving impulse electric field processing in 50 ohm of isotonic salines
According to the cell membrane surface ultra microstructure of group, it can be observed that cell surface has a large amount of micro- suede in upper figure upper left horn cells overall picture
Hair.Upper figure full figure is its upper left corner cell surface partial enlargement, it can be found that there is the diameter of a small amount of regular circle shapes on cell membrane
The micropore of 100nm or so.The following figure is to receive the cell membrane surface that 200 pulses are handled in 50 ohm of isotonic salines to surpass
Micro-structure, following figure upper right corner cell membrane surface microvillus disappear, can observe cell membrane and perforate.Following figure full figure is its upper right
Angle partial enlargement, it can be found that a large amount of irregular micropores occurs in cell membrane surface, partial pore minor axis is more than 100nm.
Fig. 6: Fig. 2 capacitor charging is released after reaching 10000 volts with cell of the frequency of 10HZ into isotonic saline
Put pulse.HCCLM3 cell receives 0,200 and 500 in the isotonic saline that output peak-peak voltage can reach 12kV
(field emission scanning electron microscope detects the variation of cell membrane surface pattern: each to scheme at once or after suspension incubation 2 hours after a pulse
The scale bar instruction distance of upper white is 1um, and image is sphaerocyst body structure surface partial enlargement on each figure corner).
Fig. 7 capacitor charging with cell of the frequency of 10HZ into isotonic saline discharges arteries and veins after reaching 10000 volts
Punching.SK-Hep1 cell receives 0,200 and 500 in the isotonic saline that output peak-peak voltage can reach 12kV
(field emission scanning electron microscope detects the variation of cell membrane surface pattern: on each figure at once or after suspension incubation 2 hours after pulse
The scale bar instruction distance of white is 1um, and image is sphaerocyst body structure surface partial enlargement on each figure corner).
Fig. 8 capacitor charging with cell of the frequency of 10HZ into isotonic saline discharges arteries and veins after reaching 10000 volts
Punching.SMMC7721 cell receives 0,200 and 500 in the isotonic saline that output peak-peak voltage can reach 12kV
(field emission scanning electron microscope detects the variation of cell membrane surface pattern: on each figure at once or after suspension incubation 2 hours after pulse
The scale bar instruction distance of white is 1um, and image is sphaerocyst body structure surface partial enlargement on each figure corner).
Fig. 9 capacitor charging voltage 10kV can reach to peak-peak voltage and discharge electricity in the isotonic saline of 12kV
Pulse.Hepatocellular carcinoma cells SMMC7721, HCCLM3 and SK-Hep1 receive 0,200,500,800 and 1000 nanosecond pulse electricity
Processing after at once or suspend be incubated for 2 hours after cell AnnexinV-FITC PI dyeing after FCM analysis results change.
Scheme the receiving 0 or 200 pulses that A is three kinds of different cell characteristics, flow cytometer detection Annexin at once or after being incubated for 2 hours after processing
V and PI signal scatter plot.Scheme B, C, D are respectively after SMMC7721, HCCLM3 and SK-Hep1 receive various dose pulse processing
Annexin V-PI- double negative cells ratio after treatment at once or be incubated for 2 hours after flow cytometer detection result histogram.It is three kinds thin
The cell that born of the same parents maintain and Annexin V not penetrating to PI that can not dye is reduced after 200 nanosecond pulsed electric fields act on, and right
The cytosis that PI is penetrating or Annexin V can be dyed.The cell number that cell membrane does not receive impulse electric field influence completely is reduced
It is further increased after being incubated for 2 hours.
Specific embodiment
The present invention forms the circuit RC by capacitor and load resistance, can be when release meets RC circuit discharging on load resistance
Between index of speciality waveform nanosecond pulse.And when exporting pulse to target biological sample by transmission line, it is exported from capacitor
The ENERGY E of single electric pulse can be calculated according to formula (2), and wherein C is capacitor capacitance value, and U is that pulse issues momentary capacitance
The voltage value of device load；Pulse is loaded into the tie point of biological sample, pulse voltage value U in transmission linepFormula can be passed through
(3) it calculates, wherein UiIt is transmission line top electrode to incidence wave voltage value between tie point, UrIt is transmission line top electrode to tie point
Between reflected wave voltage value, jk be transmission line propagation constant；Because the waveform for exporting pulse is the exponential wave for meeting equation (3)
Shape can choose the peak-peak for exporting the pulse voltage on biological sample after hardware system determines as measurement pulse
Electric field leads to the key parameter of living cells functional effect in biological sample, i.e., is loaded into biological sample by what formula (1) calculated
The attainable voltage peak-peak of impulse waveform institute in this.It can be able to achieve in this way to high electric field pulse generating device and biology
The dose-effect relationship of interaction between the two entirely different systems of sample, is reasonably analyzed and determined.This facilitates pair
The ablation of the main application scenarios of the technology --- entity tumor carries out more accurately preoperative therapy plan and designs.
Up=Uie-jk+Urejk (3)
Isotonic physiology is discharged into when the impedance of isotonic saline is greater than line characteristic impedance according to formula (1)
Pulse voltage peak-peak in salting liquid is higher than capacitance initial voltage value.It is such the result is that improve capacitor in store it is same
Electric energy inducing cell the efficiency of electroporation occurs, i.e., gross energy as much is discharged into isotonic saline, can induce
Electroporation occurs for the cell membrane of higher proportion of cell；It is worn in other words if to reach a certain proportion of cell and cell membrane electricity occur
Hole, the gross energy requirement being discharged into isotonic saline are lower.
On the one hand the efficiency for improving electric energy inducing cell electroporation can reduce caused temperature in treatment process and increase；
On the other hand the electric energy that treatment site in therapeutic process is transmitted to surrounding tissue can be reduced, caused by facilitating control therefore
The side effects such as contraction of muscle.
Case study on implementation 1: it is molten that 13 ohm, 25 ohm and 50 ohm isotonic physiology salts are discharged at 10000 volts of capacitor charging
To the perforation effect of SMMC7721 cell when in liquid
Hepatocellular carcinoma SMMC7721 from 1980 by The 2nd Army Medical College from 50 years old male's Patients with Primary sample
The hepatocellular carcinoma cells system for being is built in extraction, is widely used in liver cancer diagnosis and treatment research.
Propidium iodide (Propidium Iodide, PI) cannot penetrate complete cell membrane, and PI enters in cell cytosol, energy
Red fluorescent is issued in conjunction with DNA or RNA in cell membrane, whether a kind of commonly detect perforates on cell membrane
Fluorescent dye.
Field emission scanning electron microscope (field emission scanning electron microscope,
FESEM) there is ultrahigh resolution, biological sample such as tissue, cell, microorganism and large biological molecule can be observed on nanoscale
Deng the extremely strong sample surfaces nanotopographical structural information of three-dimensional sense of the loyal original appearance of acquisition.
In Fig. 1 figure, " millimicrosecond pulse generator " capacitor C charging voltage is set in 10000 volts, passes through a pair of spy in parallel
Property impedance be 50 ohm transmission line (in parallel after line characteristic impedance be 25 ohm) be discharged into " cell suspension reaction system "
In isotonic saline in.When the impedance difference of isotonic saline, it is discharged into the top of the electric pulse in solution
Threshold voltage is different, and voltage and current waveform such as schemes B when impedance is 13 ohm, and the peak of measurement voltage peak value is 9 kilovolts, low
The voltage loaded before capacitor discharge；Voltage and current waveform such as schemes C, measurement voltage peak value peak when impedance is 25 ohm
For 10 kilovolts, equal to the voltage loaded before capacitor discharge；Impedance is that 50 ohmic value voltage and current waveform diagrams such as scheme D, real
The peak for surveying voltage peak is 12 kilovolts, higher than the voltage loaded before capacitor discharge.
In Fig. 2,2 × 106A SMMC7721 cell receives nanosecond pulsed electric field processing in 1ml physiological equilibrium salting liquid,
As the result is shown as the impedance of isotonic saline increases, the SMMC7721 cell PI handled by 200 impulse electric fields contaminates
The percentage of cells of the color positive dramatically increases.The control group of electric pulse is not received, PI positive cell percentage is 3.06 ± 0.65；
The processing group PI positive cell percentage 4.63 ± 0.18 in 13 ohm of isotonic salines；It is molten in 25 ohm of isotonic physiology salts
Processing group PI positive cell percentage is 14.6 ± 3.72 in liquid；5 in 50 ohm of isotonic salines processing group PI it is positive
Percentage of cells is 60.0 ± 2.33.After experiment, compared with the control group, three isotonic salines of experimental group are averaged
Temperature increases 3.7 ± 0.3 DEG C, 3.8 ± 0.2 DEG C, 3.8 ± 0.3 DEG C respectively.
Fig. 3 SMMC7721 cell receives thin after 200 pulses are handled between electrode in 50 ohm of isotonic saline
After birth is able to detect that perforated phenomenon.Upper figure is cell in 50 ohm of isotonic salines, is not received at impulse electric field
The cell membrane surface ultra microstructure of the control group of reason, it can be observed that cell surface has largely in upper figure upper right horn cells overall picture
Microvillus.Upper figure full figure is upper right corner cell surface partial enlargement, it is possible to find has the diameter of a small amount of regular circle shapes small on cell membrane
In the micropore of 100nm.The following figure is cell in 50 ohm of isotonic salines, receives the cell membrane table of 200 pulses processing
Face ultra microstructure, it can be observed that cell membrane surface microvillus disappears in the horn cells overall picture of following figure bottom right, cell membrane is perforated.
Following figure full figure is its lower right corner cell surface partial enlargement, it can be found that a large amount of irregular micropores, partial pore occurs in cell membrane
Minor axis is more than 100nm.
Result above index support waveform nanosecond pulsed electric field from function and structure can result in SMMC7721 cell membrane
Electroporation occurs.And the impedance of isotonic saline is higher than the characteristic impedance of transmission line between electrode, impulse electric field induces wherein
The efficiency that electroporation occurs for the cell membrane of cell improves.
13 ohm, 25 ohm and 50 ohm isotonic salines are discharged at 10000 volts of 2 capacitor charging of case study on implementation
To the perforation effect of SK-Hep1 cell when middle
SK-Hep1 cell is U.S.'s Memorial Sloan-Kettering Cancer center 1970s at one 52
Year the postoperative sample of male adenoma patients in separate the tumour cell identified, cell type is endothelium in type, but is showed on morphology
For epitheliated type, can be formed in model animal body and the consistent lump of liver cancer cells characteristic.Be in a kind of medical basic research often
Tumour cell.
In Fig. 4,2 × 106A SK-Hep1 cell receives nanosecond pulsed electric field processing, knot in 1ml physiological equilibrium salting liquid
Fruit shows the impedance raising with isotonic saline, and the SK-Hep1 cell PI handled by 200 impulse electric fields dyes sun
The percentage of cells of property dramatically increases.The control group of electric pulse is not received, PI positive cell percentage is 3.29 ± 0.19；13
Processing group PI positive cell percentage 24.37 ± 5.25 in the isotonic saline of ohm；In 25 ohm of isotonic salines
Middle processing group PI positive cell percentage is 31.13 ± 2.95；5 in 50 ohm of isotonic salines processing group PI it is positive thin
Born of the same parents' percentage is 50.53 ± 5.19.After experiment, compared with the control group, three isotonic salines of experimental group are averaged
Temperature increases 3.9 ± 0.2 DEG C, 4.0 ± 0.1 DEG C, 3.8 ± 0.3 DEG C respectively.
Fig. 5 SK-Hep1 cell receives cell after 200 pulse processing between electrode in 50 ohm of isotonic saline
Film is able to detect that perforated phenomenon.Upper figure is the control for not receiving impulse electric field processing in 50 ohm of isotonic salines
The cell membrane surface ultra microstructure of group, it can be observed that cell surface has a large amount of microvillus in upper figure upper left horn cells overall picture.
Upper figure full figure is its upper left corner cell surface partial enlargement, it can be found that there is the diameter 100nm of a small amount of regular circle shapes on cell membrane
The micropore of left and right.The following figure is the cell membrane surface ultra micro knot for receiving 200 pulses processing in 50 ohm of isotonic salines
Structure, following figure upper right corner cell membrane surface microvillus disappear, can observe cell membrane and perforate.Following figure full figure is its upper right corner office
Portion's amplification, it can be found that a large amount of irregular micropores occurs in cell membrane surface, partial pore minor axis is more than 100nm.
Result above index support waveform nanosecond pulsed electric field from function and structure can result in SK-Hep1 cell membrane hair
Raw electroporation.And the impedance of isotonic saline is higher than the characteristic impedance of transmission line between electrode, impulse electric field induction is wherein thin
The efficiency that electroporation occurs for the cell membrane of born of the same parents improves.
At 10000 volts of 3 capacitor charging of case study on implementation discharge 200 and 500 pulses induction SMMC7721, HCCLM3 and
Irreversible electroporation occurs for SK-Hep1 cell
Hepatocellular carcinoma HCCLM3 be where Zhongshan Hospital Attached to Fudan Univ liver cancer research in human liver cancer sample separation and
The hepatocellular carcinoma cells for the plant height metastatic potential established are widely used in liver cancer diagnosis and treatment research.
Annexin V-FITC is the luciferase assay reagent that can be specifically bound with phosphatidyl serine.Phosphatidyl silk ammonia
Acid typically occurs in the inside of living cells film, if can detect that Annexin V-FITC shows that cell membrane is perforated in cell
Or the phosphatidyl serine of intercellular membrane appears in outside cell membrane.If PI can enter in cell membrane, Annexin V-FITC energy
It is enough also then further to prove that cell membrane is perforated with cell combination.
Make there it can be seen that HCCLM3, SK-Hep1 and SMMC7721 cellule receive 200 and 500 nanosecond electric pulse
With rear, cell membrane microvillus is reduced, and a large amount of micropores occurs in cell membrane surface, and partial pore minor axis is more than 100 nanometers.200 arteries and veins
There is a degree of recovery in microvillus structure behind cell 2 hours of punching processing, but micropore still largely exists.500 pulses
2 hours after processing, microvillus structure increases without recovery, micro-pore diameter.And the cell without nanosecond pulsed electric field processing compareed
The phenomenon that above-mentioned microvillus structure change and micropore increase is not observed.And PI and Annexin V is utilized to detect cell membrane function
Can the result of the property completed also the irreversible electroporation of cell membrane caused by the nanosecond pulsed electric field observed is verified.
The above result shows that three kinds of different tumour cells receive 200 or 500 times repetition rate exponential waveform nanosecond arteries and veins
After rushing electric field treatment, there is the electroporation of cell membrane that current research method is capable of detecting when in a large amount of cell membranes.These three cells connect
2 hours after by the processing of 200 or 500 subnanosecond impulse electric fields, the complete cell of cell membrane is persistently reduced, and shows three kinds of equal energy of cell
Irreversible electroporation is occurred by impulse electric field induction.
Detection method in case study on implementation is as follows:
1. exporting electric current as shown in Fig. 1-A after transformer boosts to 10000 volts, being passed through in the structure shown in nsPulsor
It charges after diode (D) rectification to the capacitor (C) of 1.8nF, capacitive energy storage is under air gap switch (S) control that signal triggers
The resistive load being discharged into processor.Processor is 50 a pair of in parallel ohm transmission lines to BTX4mm pole cup (Bio-
Rad Laboratories, Califolia, USA) in be loaded in shock treatment solution cell suspension release high-voltage electricity arteries and veins
Punching.
2. electroporation buffer be the isotonic mannitol of 290mmol/L (Sinopharm Chemical Reagent Co.Ltd,
Shanghang, PRC) solution and MEM be mixed in a certain ratio the PH7.4 to be formed (measured by PB-10meter,
Sartorius Lab Instruments, Gottingen, Germany) in isotonic balanced salt solution, suspension resistance is by digital electricity
Bridge (TH2830, Tonghui Electronics Inc.Changzhou, PRC) calibration.In 1ml cell suspension in 4mm BTX electricity
It hits in cup, the resistance value of the electroporation buffer of different formulations is respectively 13.0 ± 0.24,25.1 ± 0.36,50.6 ± 0.8 (N=
5,P<0.01)。
3. the voltage signal being discharged into isotonic physiological equilibrium salting liquid utilizes PVM-4 high voltage probe (North Star
High Voltage, Washington, USA) it acquires, current signal utilizes current transformer (7805, Pearson
Electronics Inc, California, USA) acquisition, voltage and current signal with oscillograph (DPO 4054B, Tektronix,
Pennsylvania, USA) it records and analyzes.
4. cell maintains growth in adhere-wall culture ware (Corning, the U.S.), culture medium is containing 10% fetal calf serum
(Gibco Life Technologies, the U.S.) and 100 units/ml penicillin (Sigma, the U.S.) and 0.1mg/mL streptomysin
(Gibco Life Technologies, the U.S.) minimum essential medium (minimum essential medium, MEM,
Biological Industries Israel Beit Haemek Ltd, Shanghai, China).5% carbon dioxide 95% of cell
Air wetting incubator (II Water Jacket CO of Froma Series2Incubator, Thermo Scientific, beauty
State) 37 DEG C be incubated for culture.
5. the hepatocellular carcinoma cells of the logarithmic growth phase of adhere-wall culture, using containing 0.25% (W/V) trypsase and
0.02%EDTA (W/V) solution (the silent winged generation that science and technology of match, Shanghai, China) is at single cell suspension, after supernatant is abandoned in 500g centrifugation, carefully
Born of the same parents are with 2 × 106The concentration of a/mL is resuspended in shock treatment liquid.10000 volts of charging voltage, pulse frequency 10HZ are maintained, cell
It is placed in 4 DEG C of pulse buffer liquid, the pulse for discharging setting under room temperature is counted in cell suspension.
6. shock treatment or cellular control unit with 0.01mmol/L phosphate buffer (PBS) with after being washed, 500g is centrifuged 5 points
Clock abandons supernatant, and cell is resuspended in 200ul combination buffer, after 10ul PI is added, is protected from light incubation at room temperature 15 minutes, is added
After 800ul combination buffer in 60 minutes with flow cytometer (II type of FCM, Canto, Becton Dickinson company, CA,
The U.S.) it is detected.Under 488nm laser excitation, 575/30nm signal is collected as PI signal.Data FlowJo 10
(FlowJo, LLC, Oregon, the U.S.) is analyzed.
7. being prepared referring to field emission scanning electron microscope sample preparation methods, 106Cell be used to detect.Simply, sample
2.5% glutaraldehyde through PH7.40.01%PBS buffer is 1-2 hours fixed, after PBS washing three times, using containing 1% osmium
2-3 hours are fixed after the PBS room temperature of acid.Washed with PBS and remove osmic acid three times, sample through 30%, 50%, 70%, 80%,
100% Gradient elution using ethanol of 90%, 95%and.After cell count, sample critical point drying instrument (EM CPD 300, comes card,
Germany) in it is dry, after injection plated film instrument (EM ACE 200 comes card, German) gold-plated film, utilize Nova Nano 450
Emit scanning electron microscope (FEI, Holland) observation.
Claims (8)
1. a kind of method for improving electric energy induction living cells film electroporation efficiency, which is characterized in that this method are as follows: will be to be perforated thin
Born of the same parents are scattered in isotonic saline, and a pair of electrodes being located in isotonic saline is led to energy storage electricity with transmission line
Hold, the electric energy in storage capacitor is discharged into isotonic saline the transmission line in the form of nanosecond pulse；Wherein, contain
There is the resistance value of the isotonic saline of cell to be higher than line characteristic impedance, to improve the electric pulse ceiling voltage in solution
Value is allowed to be higher than storage capacitor charging voltage, to improve the efficiency of the electroporation of cell in isotonic saline.
2. electroporation method according to claim 1, which is characterized in that it is assumed that the impedance Z of isotonic salineL, transmission
Line characteristic impedance is Z0, initial voltage value is U0 when capacitor starts to discharge to isotonic saline, is discharged into isotonic physiology
Electric pulse ceiling voltage calculated value U in salting liquidpeakCoincidence formula (1):
3. the method according to claim 1, wherein when the electrode is formed using aluminium, stainless steel, titanium alloy
Metal needle or metal plate.
4. the method according to claim 1, wherein the isotonic saline refers to: osmotic pressure value 280-
The physiological equilibrium salting liquid of 330 milli osmols/liter (mOsm/L) and pH value 7.0-7.4.
5. the method according to claim 1, wherein concentration of the cell in isotonic saline is
0.5-4×106A every milliliter of living cells (mL).
6. the method according to claim 1, wherein the resistance value of isotonic saline containing cell is higher than
Line characteristic impedance, according to formula (1), the peak-peak of output voltage can be higher than capacitor and start voltage value when electric discharge,
The efficiency of the same energy inducing cell electroporation stored in capacitor can be improved.
7. the method according to claim 1, wherein same pulse capacitor is in capacitor with higher top
The electric pulse of threshold voltage is discharged into isotonic saline, in the case where other parameters are constant, same overall pulse number energy
Induce higher proportion of cell membrane that electroporation occurs.
8. the method according to claim 1, wherein being able to detect that cell membrane pair using current research method
The transparency of small-molecule substance increases and microcellular structure occurs in cell.
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