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抑制 HO-1 增强 As2O3 对胶质瘤疗效的实验研究. 刘耀华 陈晓丰 梁元 郑秉杰 赵世光. 哈尔滨医科大学附属第一医院 神经外科. It was proved that subarachnoid injection of As 2 O 3 is safe. Intraoperative transplantation of Ommaya device (ACNU or ACNU+As 2 O 3 ). Preoperation. 47 male, Grade Ⅲ-Ⅳ ： - PowerPoint PPT Presentation
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抑制 HO-1 增强 As2O3 对胶质瘤疗效的实验研究
刘耀华 陈晓丰 梁元 郑秉杰 赵世光
哈尔滨医科大学附属第一医院 神经外科
It was proved that subarachnoid injection of As2O3 is safe.
Intraoperative transplantation of Ommaya device(ACNU or ACNU+As2O3)
Preoperation
One year after operation
47 male, Grade -Ⅲ Ⅳ：
Ommaya device (ACNU+As2O3), Arterial chemotherapy (As2O3), and Radiotherapy
Recurrent glioma
6 months after the second operation
54 female, Grade :Ⅲ
Ommaya device (ACNU+As2O3), Arterial chemotherapy (As2O3), and Radiotherapy
47 female, Grade - :Ⅲ Ⅳ
Ommaya device (ACNU+As2O3), Arterial chemotherapy (As2O3), and Radiotherapy
Preoperation
2 years after operation
Before operative MRI
Preoperative CTCT one week after operative and the first chemotherapy
Case3
MRI before operation
CT one and half a year after operation
44 male, Grade :Ⅲ
Ommaya device (ACNU+As2O3), Arterial chemotherapy (As2O3), and Radiotherapy
国内外研究发现 As2O3 在实体肿瘤治疗受到限制，小剂量药物不能达到治疗效果，而增大剂量往往增加药物毒性；
As2O3 作用于细胞后不仅引起细胞毒性效应，同时还激活细胞内一些保护性分子和信号通路；
As2O3 诱导细胞凋亡的机制被认为主要是通过活性氧（ ROS ）的诱导而实现；
ROS 可以诱导细胞保护性分子血红素加氧酶 -1(HO-1); HO-1 在胶质瘤中广泛表达； 抑制 HO-1 是否可以提高 As2O3 对胶质瘤的治疗效果？
三氧化二砷（ As2O3 ）与肿瘤治疗
研究背景
实验设计胶质瘤细胞系 U251MG 用 As2O3 处理，检测细胞活性、线粒体膜电位（ MMP ），并观察 ROS 清除剂 LNAC 对细胞是否有保护作用；
检测 As2O3 诱导胶质瘤细胞 ROS 生成、 MAPK 信号通路的活化， HO-1 的诱导以及 LNAC 对其影响；
使用 HO-1 的诱导剂和抑制剂处理细胞，观察其对 As2O3 效应的影响；
Nrf2 是 HO-1 表达的重要转录因子， SiRNA 技术干扰 Nrf2 表达后对比细胞对 As2O3 的敏感性。
Cell Viability
1 1 1 1 10
10
20
30 without LNAC
with LNAC
Ce
ll d
ea
th (
% o
f to
tal)
0 2.5 5 10 10ATO concentrations (uM)
**
**
▼
1 1 1 1 10
10
20
30 without LNAC
with LNAC
Ce
ll d
ea
th (
% o
f to
tal)
0 2.5 5 10 10ATO concentrations (uM)
**
**
▼
** P<0.01 compared with control;▼P<0.01 compared with LNAC untreated cells.
Mitochondrial Membrane Potential
100 10 1 10 2 10 3 10 4100 10 1 10 2 10 3 10 4
9.22
LNAC/ATO
10 0 10 1 10 2 10 3 10410 0 10 1 10 2 10 3 104
3.38
Control
10 0 10 1 10 2 10 3 10 410 0 10 1 10 2 10 3 10 4
33.25
ATO (10uM)
Sub-G1 (PI Staining)
0 200 400 600
FL2-A
0.94
Control ATO
0 200 400 600
FL2-A
21.48
ATO (10uM)
0 200 400 600
FL2-A
3.02
LNAC/ATO
ROS induction
100
101
102
103
104
4.74
Control
100
101
102
103
104
4.74
Control
100
101
102
103
104
8.68
4h
100
101
102
103
104
8.68
4h
100
101
102
103
104
4.98
LNAC/ATO 8h
100
101
102
103
104
4.98
LNAC/ATO 8h
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
8h
20.31
100
101
102
103
104
9.69
12h
100
101
102
103
104
9.69
12h
Downstreams of ROS
0h 4h 8h 12h 0h 4h 8h 12h
HO-1
P-P38
P-JNK
JNK-1
P38
-actin
- - - - + + + +LNAC
ATO
B
0h 3h 6h 9h 0h 3h 6h 9h
Cytoplasm Nuclear
ATO
-actin
Nrf-2
Lamin-B
C
Nrf2 Translocation
C CoPPIX C CoPPIX0
10
20
30 without ATO
ATO (10uM)
Ce
ll d
ea
th (
% o
f to
tal)
HO-1
-actin
- 3h 6h 12h
CoPPIX
Effects of HO-1 inducer
* P<0.01
C CoPPIX C CoPPIX0
2
4
6 without ATO
ATO (10uM)R
OS
(fo
ld i
nc
rea
se
)
Effects of HO-1 inducer
* P<0.01
C ZnPPIX C ZnPPIX0
10
20
30
40
50 without ATO
ATO (5uM)
Ce
ll d
ea
th (
% o
f to
tal)
C ZnPPIX C ZnPPIX0
2
4
6
8 without ATO
ATO (5uM)
RO
S (
fold
in
cre
as
e)
Effects of HO-1 inhibitor
* P<0.01 * P<0.01
Nrf2
GAPDH
Random Nrf2
HO-1
-actin
0h 3h 6h
Random
0h 3h 6h
Nrf2
ATO (5uM)
Knockdown of Nrf2
random Nrf2 random Nrf20
20
40
60 without ATOATO (5uM)
cell
dea
th (
% o
f to
tal)
Knockdown of Nrf2
* P<0.01
100
101
102
103
104
FL1-H10
010
110
210
310
4
FL1-H
100
101
102
103
104
FL1-H10
010
110
210
310
4
FL1-H
FL2
Control
ATO (5uM)
Si-random Si-Nrf2
7.99 8.32
15.44 59.73
Knockdown of Nrf2
random Nrf2 random Nrf20
2
4
6
8 without ATOATO (5uM)
RO
S (
fold
incr
ease
)
Knockdown of Nrf2
* P<0.01
ATO ROS Nrf2 activation
Cell death
HO-1 induction
