Microspectroscopic studies on effects of nanosecond pulsed electric fields (nsPEFs) on cells

概要

博⼠論⽂

Microspectroscopic studies on effects of
nanosecond pulsed electric fields (nsPEFs) on cells
（細胞に対するナノ秒パルス電場の影響の顕微分光研究）

令和四年度
東北⼤学⼤学院薬学研究科
分⼦薬科学専攻
楊

啓

1

General background ............................................................................................................... 6
1.1

NsPEFs and Electroporation ........................................................................................... 6

1.2

Biophysical Principles of nsPEFs Processing................................................................. 7

1.3

Membrane Permeabilization Induced by nsPEFs ......................................................... 13

1.4

Cellular Responses Induced by nsPEFs Treatment ...................................................... 16

1.4.1

Ca2+........................................................................................................................ 16

1.4.2

Reactive Oxygen Species ...................................................................................... 18

1.4.3

Genes and Metabolism .......................................................................................... 19

1.4.4

Apoptosis .............................................................................................................. 20

1.5

1.5.1

Application in Human Heath ................................................................................ 21

1.5.2

Application in Industry ......................................................................................... 23

1.6
2

Applications of nsPEFs................................................................................................. 21

Purpose of this Study .................................................................................................... 24

Effects of nsPEFs on cell volume ........................................................................................ 26
2.1

Introduction................................................................................................................... 26

2.2

Material and methods ................................................................................................... 27

2.2.1

Electrode fabrication ............................................................................................. 27

2.2.1.1 Fabrication of the conductive part .......................................................................... 29
2.2.1.2 Fabrication of the control part ................................................................................ 30

2.2.2

Experiments of Raman imaging ............................................................................ 33

2.2.2.1 Cell preparation ...................................................................................................... 33
2.2.2.2 Raman imaging system ........................................................................................... 34
2.2.2.3 Treatment of nsPEFs ............................................................................................... 36
2.2.2.4 Measurement on multi-confocal Raman microscope ............................................. 36
2.2.2.5 Data analysis of Raman images .............................................................................. 37

2.2.3

Experiments of fluorescence imaging ................................................................... 38

2.2.3.1 Preparation of dye solution ..................................................................................... 38
2.2.3.2 Cell preparation and stain ....................................................................................... 39
2.2.3.3 Fluorescence imaging system and measurement .................................................... 40
2.2.3.4 Treatment of nsPEFs ............................................................................................... 41
2.2.3.5 Data analysis ........................................................................................................... 41

2.3

Results........................................................................................................................... 43

2.3.1

Cell volume changes observed in Raman spectra of cells. ................................... 45
1

2.3.2

Cell volume changes observed in fluorescence imaging of cells.......................... 53

2.3.3

Effects of nsPEFs on the intracellular Ca2+ level in HeLa cells............................ 63

2.3.4

Effects of nsPEFs on the intracellular Ca2+ level in CHO cells ............................ 68

2.3.5

Detection of the size of nsPEFs-induced nanopores ............................................. 71

2.4
3

Discussion ..................................................................................................................... 74

Effects of nsPEFs on temperature ........................................................................................ 82
3.1

Introduction................................................................................................................... 82

3.2

Material and methods ................................................................................................... 86

3.2.1

Experiments of Raman image ............................................................................... 86

3.2.1.1 Treatment of nsPEFs ............................................................................................... 86
3.2.1.2 Measurement on multi-confocal Raman microscope ............................................. 87
3.2.1.3 Data analysis of Raman images .............................................................................. 87

3.2.2

Experiments of continuous measurements of Raman images ............................... 88

3.2.2.1 Treatment of nsPEFs ............................................................................................... 89
3.2.2.2 Continuous measurement on multi-confocal Raman microscope .......................... 89
3.2.2.3 Data analysis of Raman images .............................................................................. 90

3.3

Results........................................................................................................................... 91

3.3.1

Temperature rise induced by continuous treatments of nsPEFs ........................... 91

3.3.2

Detection of the temperature change induced by a single treatment of nsPEFs . 103

3.4

Discussion ................................................................................................................... 105

4

Conclusion and Perspective ............................................................................................... 109

5

Appendix ............................................................................................................................ 111
5.1

Cell culture.................................................................................................................. 111

5.1.1

Thawing frozen cells ........................................................................................... 111

5.1.2

Subculturing cells ................................................................................................ 112

5.1.3

Freezing cells ...................................................................................................... 114

5.2

Measurement of Raman images for single cells to detect volume changes ............... 116

5.3

Data process of Raman images to detect volume changes ......................................... 118

5.4

Measurement of fluorescence videos for single cells to detect volume changes........ 119

5.5

Data process of fluorescence videos to detect volume changes ................................. 120

5.6

Measurement of Raman images for single cells to detect volume changes. .............. 121

2

5.7

Data process of Raman images to detect temperature rise ......................................... 121

6

References .......................................................................................................................... 123

7

Acknowledgements ............................................................................................................ 137

3

Abbreviations
CCD

charge-coupled device

CHO

Chinese hamster ovrary

CICR

calcium-induced calcium release

DAG

diacylglycerol

DMEM

Dulbecco's modified eagle's medium

DMSO

dimethyl sulfoxide

DNA

deoxyribonucleic acid

EGTA

ethylene glycol tetraacetic acid

FBS

fetal bovine serum

FCCP

carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

HBSS

Hank's balance salt solution

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

NADH

nicotinamide adenine dinucleotide hydrate

NADPH

nicotinamide adenine dinucleotide phosphate

nsPEF

nanosecond pulsed electric field

PBS

phosphate-buffered saline

PI

phosphatidyl inositol

PS

phosphatidylserine

ROS

reactive oxygen species

4

RVD

regulatory volume decrease

SVD

singular value decomposition

TRP

transient receptor potential

VGCC

voltage-gated calcium channel

VGNC

voltage-gated Na+ channel

VSOR

volume-sensitive outwardly rectifying

YP

YO-PRO-1

5

1 General background
1.1

NsPEFs and Electroporation

Physically stimulating cells and tissues is a technology that has been studied and
improved for a long time. By studying the effects of physical stimuli, such as pressure,
tension, cold plasma, and electric fields, on biological systems, significant progress has
been made in the fields of biology and medicine1–5. For example, cold plasma has been
used in medicine to treat cancers5–8, care for wounds8,9, and sterilize or disinfect10–12.
Electric fields have been applied to cells as physical stimuli for a long time, capable of
inducing transient loss of semi-permeability of cell membrane called electroporation13. ...

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