第 2章 ゲル培養基板を用いた細胞の力 学応答の解析

2
2-1
2-1-1
ECM
ECM
2
2
(Substrate rigidity):
(Stress)
(Strain)
(Rigidity sensing
mechanism)
(Pelham and Wang., 1997)
al., 2004b)
2006)
(Engler et al., 2004a; Engler et
(Lo et al., 2000; Peyton et al., 2005; Khatiwala et al.,
(Engler et al., 2004b; Engler et al., 2006)
(Ligand density):
RGD
RGD
(Reinhart-King et al., 2003)
31
(Loftis et al., 2003)
Gaudet
(Gaudet et al.,
2003)
vs
:
(Peyton et al., 2005;
Khatiwala et al., 2006)
Engler
(Engler et al.,
2004a)
2
1
7
3
ECM
PAAm
2
2-1-2
(Trepat and Fredberg, 2011)
in vitro
(Poujade et al., 2007)
32
(Adherens junction)
(Vaezi et al., 2002)
(Fenteany et al., 2000)
MAPK (Mitogen-activated protein kinase)
ERK1/2
(Matsubayashi et al., 2004, Nikolic et al., 2006)
ERK
-
ERK
-
P(NIPAAm-co-NAA)
P(AAm-co-NAA)
(PNIPAAm/PAAm
)
PNIPAAm
37ºC
(Bae et al., 1991)
P(NIPAAm-co-NAA)
ERK
(Yamaki et al., 2009)
PNIPAAm/PAAm
ERK
ERK
PNIPAAm/PAAm
Col-P(NIPAAm-co-NAA)
NIPAAm
PAAm
33
Col-P(AAm-co-NAA)
2-2
2-2-1
N-acryloyl-6-aminocaproic acid (ACA)
ACA
Pless
(Pless et al., 1983) 2.5 M NaOH
(Wako, 10 g/100 ml)
mmol)
6-amino-n-caproic acid (Wako, 10.02 g, 76
(500 ml)
Chloroform, dehydrated (Wako, 8 ml)
Industry, 8 ml, 99 mmol)
Acryloyl chloride(Tokyo Chemical
1
2
HCl (Wako, 10 M)
pH
Acetic
acetate (Wako)
HCl
Acetic
acetate
NaCl
(Wako)
15 min
Sodium sulfate, anhydrous (Wako)
n-hexane (Wako)
DMSO-d6 (Sigma)
1
H NMR (400 MHz, Varian)
Fig.2-2-1 N-acryloyl-6-aminocaproic acid (ACA)
34
2-2-2
Acrylamide-ACA
(P(AAm-co-ACA)
Yamaki
)
(Yamaki et al,. 2009) 2
(Matsunami, 76 mm x 52 mm)
(Tigers
Polymer, Thickness: 0.3 mm)
(25ºC, 1 h)
Phosphate buffered saline (PBS: 137 mM NaCl (Wako), 2.7 mM KCl (Wako), 10
mM Na2HPO4 (Wako), 1.76 mM KH2PO4 (Wako))
MES buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid (MES,
Dojindo), 0.5 M NaCl (Wako), pH 6.1)
TEMED
(N,N,N’,N’-tetramethylethylendiamin, Wako)
(Ammonium persulfate, Wako)
(Polyscience,
APS
0.05 %
: 2.0 µm)
(15 min)
-20ºC
0.1 %
10 % APS (MilliQ
)
!
AAm (40 % Acrylamide / MilliQ, Wako)
!
BIS (2 % N,N’-methylenbisacrylamide / MilliQ, Wako)
!
ACA (500 mM N-acryloyl-6-aminocaproic acid (Tokyo Chemical
Industry) / 350 mM NaOH (Wako), pH 6 ~ 7)
Fig.2-2-2a P(AAm-co-ACA)
Fig.2-2-2b 35 mm dish
35
P(AAm-co-ACA)
2-2-3
(Col-P(AAm-co-ACA)
P(AAm-co-ACA)
Jiang
(Jiang et al., 2004) MES buffer
Wako)
0.5 M NHS (N-hydroxysuccinimide,
0.2 M EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride, Dojindo)
min)
h)
)
P(AAm-co-ACA)
60 % Methanol (PBS
ACA
15 mM
(25ºC, 30
, Wako)
(4ºC, 2
40% Methanol
HEPES buffer (0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (Dojindo), pH 9.0)
0.2 mg/ml Type I Native Collagen (KOKEN)
(4ºC, Over night)
HEPES buffer
ethanolamine hydrochloride (Sigma)
h)
P(AAm-co-ACA)
HEPES buffer
(25ºC, 30 min, 3
10% FBS-DMEM
0.5 M
(4ºC, 30 min, 3
)
(4ºC, 1
)
PBS
UV
(4ºC, over night)
Fig.2-2-3 P(AAm-co-ACA)
36
2-2-4
Col-P(AAm-co-ACA)
Col-P(AAm-co-ACA)
Tris buffered saline (TBS: 137 mM NaCl (Wako),
2.7 mM KCl (Wako), 25 mM tris(hydroxymethyl)aminomethane (Wako), pH 7.4)
Blocking one® (Nacalai)
(4ºC, over night) 1
Purified rabbit anti-bovine Type I Collagen (Cedarlane, TBS
1000
)
(25ºC, 1 h) TBS
(20 min, 3
Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen, TBS
(25ºC, 1 h) TBS
(20 min, 3
) 2
5000
)
)
Typhoon 9400 (Amersham Biosciences)
ImageJ (National
Institute of Health, USA)
P(AAm-co-ACA)
48 well plate
TBS
Alexa Fluor 555 purified goat anti-rabbit IgG
Typhoon 9400
ImageJ
Zhu
Sirius red
(Zhu, 2007) Type I Native Collagen (KOKEN, bovine skin,
5 mg/ml)
(
HCl
(pH 3.0, Wako)
24 well plate
: 200 µl/well)
(25ºC, 1 h) Sircol®
(Biocolor)
250 µl
(60ºC, 6 h) 200 µl
0.5 M NaOH
HCl
3
(Wako)
AA: 7.5 %-BIS: 0.02 %-ACA: 240 mM
14.6 mm
(25ºC, 1 h)
1 ml
Sircol®
Col-P(AAm-co-ACA)
12 well plate
Sircol®
1 ml
250 µl
1 ml
Sircol®
HCl
0.5 M NaOH
(25ºC, 1 h)
100 µl
96 well assay plate
(Beckman)
(OD: 550 nm)
P(AAm-co-ACA)
37
2-2-5
P(AAm-co-ACA)
(Penetration method)
2 mm
2
PBS
3
f
h
2
h
h
f
(Landau and Lifshiz, 1986)
1
2
3
( " 1 1 %+ 3
h = f * D2 $ + ') # R R' &,
R R’
2
D
3 $1 − σ 2 1 − σ'2 '
D= &
+
)
4% E
E' (
€
E E’
’
E’
E €
G
E=2G(1+
)
D
3(1 − σ)
8G
D=
1/2
1/2
€
E=3G
D=
9
16E
R
€
38
h = bf
2
3
2+
(
1 %3
"
*
2
**b = $#9 /16ER' '& -)
,
h:
€
f:
€
E:
R’:
(
: 6 mm)
f
2/3
h
2-2-6
(Swiss3T3)
DMEM (Dulbecco’s modified Eagle’s medium, Nacalai)
100x PSN
(0.5 % Potassium penicillin G (Sigma), 0.5 % Streptomycin
sulfate (Sigma), 1.0 % Neomycin sulfate (Nacalai))
serum, Nichirei)
FBS-DMEM
1
10 v/v%
37ºC 5 % CO2
Trypsin-EDTA
1 v/v% FBS (Fetal bovine
Swiss3T3
10 %
DMEM
PBS
(0.25 % Trypsin (Sigma), 0.05 % EDTA
(Ethylendiaminetetraacetic acid, Dojindo), PBS)
5.4x103 cells/cm2 (12 well plate: 3.5x104
2.6x103
cells/well)
cells/cm2 (35 mm dish: 2.5x104 cells/well)
39
2-2-7
Swiss3T3
Katz
®
Mildform 20N (Wako)
)
1:1
(Katz et. al., 2000)
PBS (10 % sucrose (Nacalai), 1 % Triton X (Wako)
(
: 4 % formaldehyde, 5 % sucrose, 0.5 %
Triton X)
(25ºC, 3 min)
(25ºC, 20 min)
137
mM
NaCl
(Wako),
Tris buffered saline (TBS:
2.7
mM
KCl
tris(hydroxymethyl)aminomethane (Wako), pH 7.4)
one® (Nacalai)
one
(25ºC, 30 min
®
TBS
Triton X
(Wako),
3
(10 min, 3
)
)
4ºC, Over night) Blocking
2
(25ºC, 1 h)
(25ºC, 1 h) TBS
(Prolong® Gold (Invitrogen))
1
!
Monoclonal Anti-Vinculin (Sigma): 400
2
!
Alexa Fluor 555 goat anti-mouse IgG (Invitrogen): 5000
!
Alexa Fluor 488 Phalloidin (Invitrogen): 50
!
DAPI (Invitrogen): 1000
40
mM
Blocking
1
(10 min, 3
25
2-2-8
Swiss3T3
Col-P(AAm-co-ACA)
35 mm dish
3
(22 mm
22 mm, Matsunami)
Swiss 3T3
(Cardiac fibroblast)
2
4
2.6x10 cells/cm (35 mm dish: 2.5x10 cells/dish)
24
(Vinculin)
(Olympus, oil)
60
10
(Swiss3T3)
(Olympus)
(Cardiac fibroblast)
(
20
)
ImageJ (National Institute of Health, USA)
RGB color
2
8 bit gray scale
2
(Projected cell area)
(Form factor)
2-2-9
Swiss3T3
Time-lapse imaging
ImageJ
Manual Tracking
18 h~22 h
(Othmer et al., 1988; Gail et al., 1970; Lo et al., 2000;
Faassen et al., 1993)
(Mean squared distance (MSD): <d2(t)> (µm2))
(Migration speed: S [µm/min])
(min))
(Time persistence: P
(Time: t (min))
[
]
d 2 (t) = 2S 2 P t − P (1 − e −t / P )
(1)
€
t
(1)
3
0
* $
'-3
t
t2
t3
S 2t 3
2 2
5
.
d 2 (t) = 2S 2 P 2 t − P+1 − &1 − +
−
+⋅⋅⋅
=
S
t
−
)
2
6P 3
3P
21
(/54
, % P 2P
€
41
(2)
1
1
# 2 2 S 2 t 3 & − 2 1 # 3P & 2
= %S t −
(
( = %
3P '
St $ 3P − t '
d 2 (t) $
1
(3)
P
€
(t/P)~0
1
2 +
5
/
−
1 4 - 1 $ 3P ' 2
3P 7
0 ⋅ t +⋅⋅⋅
≅ 41+ , &
) ⋅
2
7
2
%
(
St
2
3P
−
t
(3P
−
t)
-1
d (t)
43 -.
76
t =0
'
1$ 1 1
= &1+ ⋅
⋅ t +⋅⋅⋅ )
(
St % 2 3P
1
1 1
1
∴
= ⋅ +
S t 6SP
d 2 (t)
1
(4)
(1/RMSD (root of mean
squared distance))
€
1/t
1
t (0<t<=Tfinal)
n=(Tfinal-t+1)
d
T=0
T= Tfinal
n
d
2
n
d 2 ( t) =
∑( d( t) )
2
k
k =1
(5)
n
MSD
t
€
2
3
9
42
2-2-10
PNIPAAm/PAAm
N-acryloyl alanine (NAA)
Yamaki
(Yamaki et al.,
2009) PNIPAAm/PAAm
(
0.3 mm)
Matsunami)
2
(76 mm x 52 mm,
1.5 mm,
P(AAm-co-NAA)
(1 h, 25ºC)
P(NIPAAm-co-NAA)
(1 h, 25ºC) (Fig.2-3-10A)
(
2µm, Polyscience)
Phosphate buffered saline (PBS: 137 mM
NaCl (Wako), 2.7 mM KCl (Wako), 10 mM Na2HPO4 (Wako), 1.76 mM KH2PO4
(Wako))
(Fig.2-3-10B) MES buffer (0.1 M
2-(N-morpholino)ethanesulfonic acid (MES, Dojindo), 0.5 M NaCl (Wako), pH
6.1)
2
2
3
P(AAm-co-NAA)
!
950 mM: Acrylamide (AAm)
!
10 mM: N,N’-methylenebisacrylamide (BIS)
!
50 mM: N-acryloyl alanine (NAA)
!
50 mM: 2-amino-2- hydroxymethyl-1,3-propanediol (Tris)
!
10 µl/ml: N,N,N’,N’-tetramethylethyenediamine (TEMED)
!
5 µl/ml: 10% ammonium persulfate (APS)
P(NIPAAm-co-NAA)
!
970 mM: N-isopropylacrylamide (AAm)
!
10 mM: N,N’-methylenebisacrylamide (BIS)
!
30 mM: N-acryloyl alanine (NAA)
!
30 mM: 2-amino-2- hydroxymethyl-1,3-propanediol (Tris)
!
1 µl/ml: N,N,N’,N’-tetramethylethyenediamine (TEMED)
!
5 µl/ml: 10% ammonium persulfate (APS)
43
2-2-11
PNIPAAm/PAAm
2.5 ml
35 mm dish
37ºC
PNIPAAm/PAAm
12 h
32ºC
(Cytomotion, Altair Corporation)
(IX70, OLYMPUS)
CCD
(Penguin 600CL,
Pixera)
(
50 µm)
2
Image J (National Institute of Health)
PNIPAAm
PNIPAAm
PAAm
5
2-2-12
(Mardin-Darby canine kidney cell
(MDCK
))
DMEM (Dulbecco’s modified Eagle’s medium, Nacalai)
100x PS
(0.5 % Potassium penicillin G (Sigma), 0.5 % Streptomycin
sulfate (Sigma))
1 v/v% FBS (Fetal bovine serum, Nichirei)
MDCK
10 v/v%
37ºC 5 % CO2
5.0x105 cells/cm2
PNIPAAm/PAAm
(24 h)
2-2-13
PNIPAAm/PAAm
37ºC
MDCK
(IX70, OLYMPUS)
(32ºC, Cytomotion, Altair Corporation)
CCD
(Penguin 600CL, Pixera)
(WACOM)
ImageJ (National
Institute of Health)
factor: 4
x(
)/(
(Form
2
))
44
2-2-14
PNIPAAm/PAAm
5 min
(Wako) (PBS
)
15 min
4 % (v/v) Formaldehyde
20 min
Triton X-100 (Wako) (PBS
30 min
MDCK
0.5% (v/v)
)
3 min
®
Blocking one (Nacalai)
1
1h
2
Tris buffered saline (TBS: 137 mM
NaCl (Wako), 2.7 mM KCl (Wako), 25 mM tris(hydroxymethyl)aminomethane
(Wako), pH 7.4)
(A1 Nikon,
: CFI Plan DLL 10x, 40x,
Nikon)
Intensity profile
ImageJ
8-bit gray scale
1
!
Purified Rat anti-E-cadherin antibody (DECMA-1) (Sigma): 1600
!
Purified mouse anti-phospho-ERK antibodies (Thr202/Tyr204)
(E-10) (Cell Signaling Technology): 1000
2
!
Alexa Fluor 448-conjugated secondary antibody
!
Alexa Fluor 555-conjugated secondary antibody
!
Alexa Fluor 488-conjugated phalloidin (Invitrogen)
45
2-3
2-3-1
ACA
H 1 NMR
H1 NMR (Varian, 400 MHz)
(ACA)
(Fig.2-3-1) a: 6.15 (q, 1H), b: 6.0 (d, 1H), c: 3.06 (d,
1H), d: 2.15 (q, 2H), e: 1.2-1.4 (t, 2H)
Fig.2-3-1 ACA
68%
H1 NMR
46
2-3-2
P(AAm-co-ACA)
ACA
P(AAm-co-ACA)
0 mM 15 mM 30 mM 60 mM 120 mM
ACA
ACA: 120 mM
ACA
(Fig.2-3-2)
AAm
5
AAm: 7.5% BIS:
0.64%
Fig.2-3-2
ACA
ACA
Fig.2-3-2 ACA
47
2-3-3
ACA
ACA
ECM
PAAm
ACA
Col-P(AAm-co-ACA)
Swiss3T3
6h
Tissue culture dish
ACA
(Fig.2-3-3a)
(ACA: 0 mM)
ACA
(Fig.2-3-3b,c,d,e) Scale bar: 500 µm
15 mM
Swiss3T3
ACA
Col-P(AAm-co-ACA)
Fig.2-3-3 ACA
Swiss3T3
48
2-3-4
Col-P(AAm-co-ACA)
ECM
Khatiwala et al.,
2006; Peyton and Putnam, 2005; Engler et al., 2004
BIS: 0.02 % 0.08 % 0.64 %
ACA: 15 mM
ACA: 240 mM
2
Table.2-3-4
BIS:
0.02 %-ACA: 240 mM
2
4
Fig.2-3-4
(Fig.2-3-4)
2
240 mM
4
AAm: 7.5 %
Col-P(AAm-co-ACA)
3.14 ± 0.40 µg/cm2
49
BIS: 0.02 % ACA:
Table.2-3-4
Fig.2-3-4
50
2-3-5
Swiss3T3
Col-P(AAm-co-ACA)
6
Tissue culture dish (TC-dish)
Swiss 3T3
7
103 cells/cm2, 30 min, 60 min)
(5.4
DAPI
Fig.2-3-5
(Fig.2-3-5) n.s.: p>0.05
Fig.2-3-5 Swiss3T3
51
60 min
TC-dish