一氧化氮和氧自由基诱导缺氧再给氧心肌细胞凋亡的bcl-2和p53信号通路研究
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p53 սΞ֘؆ȥ ֘؆
֘؆ȥ(Ԯ [7], ێХ NO 3 Ɩ
(EDRF)Ǚғ , ǒ
(ONOO−), (t֘؆QǙғ [8].
cGMP 9 ֘؆ȥ(Ҳ ƪΞҲȥ( ̣Ԯ
[17]
NO ǫ.ؕ
.
ҲǙғ
NOS (t NO
֘؆ȥ( [11].
< ȥ( ̫
2001-10-10 Է , 2002-01-04 PԷ * ̒ZХԟ (IȢ? : 29935080)D ** ףƣ , E-mail: zhaobl@sun5.ibp.ac.cn
zХԟ
ރ
ӈ
5
:Ⱥժ : NO D
Қ
Ξ
4֤
֘؆ȥ(Ҳ bcl-2 D p53
?
ԉ
437
438
̒
ԟ
(C
)
32
1.3
̽
DNA ܈ȴ 4֤ ʉ ֘؆ɪj c
[21]Ҳ $˸ jӍ .
DNA q ؖu
ȴ
,
ʽ N ,
ռ
֘؆ȥ( Annexin V-PI = ݊ ֘؆Ʒ $ [22,23] ғ P Vermes , 106 ֘؆ PBS 2 , / ׇ5 C (10 mmol/L Hepes ȀA Annexin &-Flous D PI Ҳ ׇ / NaOH, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2), 5 C 4j 20 min, ȴ Ɂ 488 nm ϯ= D 560 nm Ƶ EPICSElite ֘؆ Ʒ Λj , $ Annexin & D PI ݊ Ҳ֘؆ , Ƶ A Annexin & D PI Ҳ ׇ5 CҲ ֘؆Ǚ ݂ ȥ(D ?֘؆ Ұ ȴ я . 1.4 1.5
bcl-2 ۤ
,
.
8 ֘؆ȥ(Ԯ ֘؆ NO ?=tҲ3
ӈ:Λ
Lj8֘؆ȥ(ҲY
R , Λ4Ϲ+4֤ 8
؆ȥ(=3!9Ҳԉ \ , Υ D . Қ
bcl-2, p53 D p21/waf1/cip1 ǙғҲY
. M
,
ԉ [ܨ
4֤
Ҳ(tƵ= bcl-2, p53 D p21/waf1/cip1
[14]
M bcl-2 D p53 ̘ȴ< , ̘ Y ǔ ̘
¶M ֘؆ ̘ Ǭލ
[16]
, ӈ:Λ bcl-2 D p53 ̘
9 [15]. ʇ
p53 =3
M p21/waf1/cip1
, ǒ
,
p53 Ҳ֘؆ȥ(
,
֘؆ȏ bcl-2
֘؆ȥ(=tҲ ̘ȴ< , ̣ Y C̎ ܧNO D
(SNP)̫
ǔ
ȉƪݳ ȉƪݳ
− NO− 2 / NO3
,
bcl-2 ۤ
p53 D p21/waf1/cip1 ۤ
, ̫ NO− 2
− / NO3
DNA
܈ȥ(=
?֘؆я. L-NAME D SOD/CAT ȴɁ
D TBARS , + ؕ bcl-2 ۤ p53 D p21/waf1/cip1 ۤ , 9 DNA ܈ȥ(= ?֘؆я, D-NAME 8 Ǚғ Ƶ , ̘ bcl-2 ۤ D 4֤ 8 ֘؆?(Ԯ , NO D Қ ɪ , Ӎ ̟ϯ=֘؆ȥ(Ԯ , = p53 D p21/waf1/cip1 ۤ Қ Ξ ֘؆ȥ(Ҳ!9 bcl-2, p53 D p21/waf1/cip1 ? 3.
(LDH)
, ห้องสมุดไป่ตู้֤
ǔ
ȉƪݳ
NO D
NO− 2
/
− NO3
, ̫
TBARS
. bcl-2, p53 D p21/waf1/cip1 ۤ , 4֤ bcl-2 , p53 D p21/waf1/cip1 ۤ . ʇ , ̫ ֘؆ȥ(
̫ ֘؆ ?я. NO ǫ.
NO
2 /NO 3 $
ғ P Yu ժƣ [26]
, 200 µL
ȉ
U
90 mU/mL
y<
, 0.28 mmol/L 25j ȇ ʇ
NADPH, 35 mmol/L FAD ʂ 100 µL , 0.1 mol/L pH 7.5 Ҳ 1 h, j
Ȣ
y ׇ5 C 200 µL
3 min, NO− 2 /
< / (SOD/CAT, ʂ 100 U/mL)ȴɁ ȉ. : Ȁ ȉ . Λj֖ ֘؆ 95% c/5% CO2 nP , D D LDH ۤ я, 4֤ D TBARS 24 h ̫ؕ ȉƪݳ
− NO, NO − 2 / NO3 , Ƴ
Sͤ
=
(
(TBARS)
¶<M ̘
, bcl-2 ؕ
Ҳ
NO ƪΞҲ֘؆ȥ(D
֘؆ȥ( [18], ӈ:Λ [19], ǒ ֘
[20]
poly(ADP
)Rץ
Ҳܵ܈ ̣̘
. bcl-2 ؕ
̣ ֖֙ Қ
9 p53 ƪΞҲ bcl-2 ̫ Λ̘ 4֤
bcl-2 Ҳ +
Ҳ[ $
405 nm
bcl-2, p53 D p21/waf1/cip1 ۤ $ ɪj c
[24] , ֘؆ 70% ̑ , 4j R , Ƶ A 4%mU ۤ Ҳ y ׇ5 C (PBS/BSA) 2 , ȴ Ɂ Ȁ 1ƅ 100 Ԫ Ҳ P bcl-2 Rǽ P. (N-19) p53 Rǽ P. (CM-1)D p21/waf1/cip1 Rǽ P. (C-19)ʂ 60 min, Ƶ PBS/BSA ɧ R ǖ P. , Ȁ 1ƅ 50 Ԫ ҲP FITC P , ֘؆ / A 10 µg/mL PI D 70 µg/mL RNA Ҳ PBS , Ƶ EPICSElite ֘؆ Ʒ $ . P ݂_ 5000 ֘؆ , Ƶ A P.Ҳ ׇ5 C ֘؆Ǚ Λj , ݂ bcl-2, p53 D p21/waf1/cip1 ۤ Ұ ȴ я . 1.6 LDH $ ғ ̒ԟ ̽ ȴ ǼǼ ݂$ 1.7 1.8
tt
Ԯ ȃ? ; $
LDH ݟɊ y , Ƶ Beckman DU-640
ռ
.
NO $ ғ 3 mL A 1%FCS, 5 mL DETC, 1 mmol/L FeCl2 Ҳ DMEM ׇ5 CɭƳ ȉC , ̘ 37j = , ғ 1ƅ 5 .SҲ ؖȀ ݩ. , ¥ 3 min, ְ֫ ȉ 30 min. Х ʉȜ 10000Cg ԛ 6 min. ȴ ԛ γ ! C , Ǽ D̘ ER-200 Ʒ $ ESR [25]. ESR $ ݟ.Ƹ : X N , 100 kHz 9 , 9 3.2C10−4 T, я 20 mW, O −4 −4 3380C10 T, OZ 400C10 T, _ 0.3 s. 1.9
ٜ
֘؆ȥ( ELISA $ Ƶ Boehringer Mannheim ȃ? ֘؆ȥ( ELISA ݟɊ y $ ֘؆ Ǚ ɪjݟɊ y . Ƶ Bio-Rad 450 DR ٜ \ . , ǼΑ , ݂ ȥ(֘؆ Γ M (enrichment factor, EF).
DNA
Ʒ
܈.
< (SOD), (CAT), (SNP), N- -L қ (L-NAME), ؆ٜD ¾ Sigma; 3*ݟɊ ̒(ȴ . 1.1
DMEM
ݟɊ
4֤ 8 ֘؆ Lj [2] ɪj Tanaka , t 2~3 d Wistar ̾ ( ȓ ̾
) ɭ7 , 0.06%ۤ , 9̸ ֘؆ C, ̘ 37jӍΛ 100%Ҳ CO2 ȉ 15 min, չ(50 U/mL)D չ(50 µg/mL)Ҳ DMEM ̰ݹҲ ֘؆Ԟ A 10%mU −6 ȉ 6 h, ХʉƵA 10 mol/L ؆ٜ(Ara C)Ҳ DMEM ȉ ȉ 48 h Ƶ6\ ȉ 24 h, $ NO, TBARS D ֘؆S0, ХʉƵA 1%mUҲ DMEM ȉ LDH, :ƵA 5%mU A քҲǔ DMEM ȉ Ƴ, ֘؆ 95% N2/5% CO2 nP ȉ 24 h, Хʉ 95% O2 /5% CO2 nP ȉ 4 h 4֤ ֘؆ Lj . ֖ ֘؆ 95% N2/5% CO2 nP ȉ 24 h ǒ 4֤ . SOD/CAT (ʂ 100 U/mL), SNP (5 µmol/L), L-NAME (100 µmol/L)D D-NAME (100 µmol/L)ȴɁ : Ȁ ȉ , ܨ Λj֖ ֘؆ 95% c/5% CO2 nP ȉ. [3Λ ֘؆ Lj+ XǙғ. 1.2
Rԉ ȏԟ ΅,
ѩԟ,
c;
Ɲ 100101; c)
t
ѩԉ 8ؘ
, ܮܫ
¶ [ܨ 4֤ LjҲ (nitric oxide, NO)D Қ !9. t Wistar ̾ ֘؆ 95% N2/5% CO2 nP ȉ 24 h, Хʉ 95% O2 /5% CO2 nP ȉ 4 h 4֤ ֘؆ Lj . ֖ ֘؆ 95% N2/5% (SNP, 5 µmol/L) NO ʄ 9Ɋ NωCO2 nP ȉ 24 h, ǒ 4֤ . NO ǫ. -L- қ (L-NAME, 100 µmol/L) D 3 8 . Nω- -D- қ (D-NAME, 100 µmol/L)Ƶ=Â
(7 , ȥ(D ϯ
?
ɪ D
̣Ҳ֘؆?(Ԯ [4]. ¶<M , ϯ t M
[10] [12,13]
֘؆ȥ(=tҲ )Ԩ
Ҳ
[5,6]
Қ
ɹƪΞ DNA
Lj, ϯ
poly-(ADPɪ t (
[5,9]
p53 ̫
, Ξ֘؆ȥ(
ȏx֘؆5 Y
. ʇ
,
(nitric oxide, NO) NO ɹ O− 2=
(C
32 5
)
2002 10
SCIENCE IN CHINA ( Series C )
D Қ Ξ 4֤ ֘؆ ȥ(Ҳ bcl-2 D p53 ? ԉ *
:Ⱥ
̒ԟ ȓ ̾
t W ț Ɛ ޛ
¹Ӻԟ ԉ , 510515; c̾
**
3 ݘ ֘؆ȥ( Қ bcl-2 p53 p21/waf1/cip1
֘؆?( ȴ 8t
?Dȥ( D.
Ԟ!9 . Ԟ
[1]
?֘؆Ҳع MԂҲԮ
[2]
a
ͅ , ֘؆
Dԓ ܈, ֘؆ ȏ
[3]
R9C֖̎֙ , ȥ(֘؆ ɯ
?(Ԯ ,
. ̘
֘؆ D4Ϲ
ɹ=і֘؆ȥ
֘؆ȥ(D
1
¦ ȉ ¾ GIBCO; mU¾ Kibbutz Beit Haemek (Iserl); HEPES ¾ Farco; bcl-2 Rǽ P. (N-19)¾ Santa Cruz, p53 Rǽ P. (CM-1) ¾ Novo; p21/waf1/cip1 Rǽ P.(C-19) ¾ Santa Cruz; P FITC ¾ Doko; ۤ K ¾ Merk; Annexin V-Flous ¾ ʽ(PI), NADPH, FAD, y< , Nŧ ؓ, Ƴ Sͤ Boehringer Mannheim; , ̈ Л , ؓ , Â
1.10
Ƴ Sͤ =
( ݳTBARS)$ ȉ U TBARS A $ Ƶ Ohkawa ժƣ [15]Ҳ
5
:Ⱥժ : NO D
Қ
Ξ
4֤
֘؆ȥ(Ҳ bcl-2 D p53
− NO3
Ȁժ .Ҳ Griess ݟɊ (2% N- Ʒ 550 nm \P $ Ƶ֎
ؓ ƅ 0.2 % ؓ 3ʚ
1ƅ 1 Qʄ ),
60j
10 min, Ƶ Bio-Rad 450
, Ƶ
(0~20 nmol/mL)
,
(nmol/mL) . 532 nm ғ Beckman DU-640
֘؆ȥ(Ԯ [7], ێХ NO 3 Ɩ
(EDRF)Ǚғ , ǒ
(ONOO−), (t֘؆QǙғ [8].
cGMP 9 ֘؆ȥ(Ҳ ƪΞҲȥ( ̣Ԯ
[17]
NO ǫ.ؕ
.
ҲǙғ
NOS (t NO
֘؆ȥ( [11].
< ȥ( ̫
2001-10-10 Է , 2002-01-04 PԷ * ̒ZХԟ (IȢ? : 29935080)D ** ףƣ , E-mail: zhaobl@sun5.ibp.ac.cn
zХԟ
ރ
ӈ
5
:Ⱥժ : NO D
Қ
Ξ
4֤
֘؆ȥ(Ҳ bcl-2 D p53
?
ԉ
437
438
̒
ԟ
(C
)
32
1.3
̽
DNA ܈ȴ 4֤ ʉ ֘؆ɪj c
[21]Ҳ $˸ jӍ .
DNA q ؖu
ȴ
,
ʽ N ,
ռ
֘؆ȥ( Annexin V-PI = ݊ ֘؆Ʒ $ [22,23] ғ P Vermes , 106 ֘؆ PBS 2 , / ׇ5 C (10 mmol/L Hepes ȀA Annexin &-Flous D PI Ҳ ׇ / NaOH, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2), 5 C 4j 20 min, ȴ Ɂ 488 nm ϯ= D 560 nm Ƶ EPICSElite ֘؆ Ʒ Λj , $ Annexin & D PI ݊ Ҳ֘؆ , Ƶ A Annexin & D PI Ҳ ׇ5 CҲ ֘؆Ǚ ݂ ȥ(D ?֘؆ Ұ ȴ я . 1.4 1.5
bcl-2 ۤ
,
.
8 ֘؆ȥ(Ԯ ֘؆ NO ?=tҲ3
ӈ:Λ
Lj8֘؆ȥ(ҲY
R , Λ4Ϲ+4֤ 8
؆ȥ(=3!9Ҳԉ \ , Υ D . Қ
bcl-2, p53 D p21/waf1/cip1 ǙғҲY
. M
,
ԉ [ܨ
4֤
Ҳ(tƵ= bcl-2, p53 D p21/waf1/cip1
[14]
M bcl-2 D p53 ̘ȴ< , ̘ Y ǔ ̘
¶M ֘؆ ̘ Ǭލ
[16]
, ӈ:Λ bcl-2 D p53 ̘
9 [15]. ʇ
p53 =3
M p21/waf1/cip1
, ǒ
,
p53 Ҳ֘؆ȥ(
,
֘؆ȏ bcl-2
֘؆ȥ(=tҲ ̘ȴ< , ̣ Y C̎ ܧNO D
(SNP)̫
ǔ
ȉƪݳ ȉƪݳ
− NO− 2 / NO3
,
bcl-2 ۤ
p53 D p21/waf1/cip1 ۤ
, ̫ NO− 2
− / NO3
DNA
܈ȥ(=
?֘؆я. L-NAME D SOD/CAT ȴɁ
D TBARS , + ؕ bcl-2 ۤ p53 D p21/waf1/cip1 ۤ , 9 DNA ܈ȥ(= ?֘؆я, D-NAME 8 Ǚғ Ƶ , ̘ bcl-2 ۤ D 4֤ 8 ֘؆?(Ԯ , NO D Қ ɪ , Ӎ ̟ϯ=֘؆ȥ(Ԯ , = p53 D p21/waf1/cip1 ۤ Қ Ξ ֘؆ȥ(Ҳ!9 bcl-2, p53 D p21/waf1/cip1 ? 3.
(LDH)
, ห้องสมุดไป่ตู้֤
ǔ
ȉƪݳ
NO D
NO− 2
/
− NO3
, ̫
TBARS
. bcl-2, p53 D p21/waf1/cip1 ۤ , 4֤ bcl-2 , p53 D p21/waf1/cip1 ۤ . ʇ , ̫ ֘؆ȥ(
̫ ֘؆ ?я. NO ǫ.
NO
2 /NO 3 $
ғ P Yu ժƣ [26]
, 200 µL
ȉ
U
90 mU/mL
y<
, 0.28 mmol/L 25j ȇ ʇ
NADPH, 35 mmol/L FAD ʂ 100 µL , 0.1 mol/L pH 7.5 Ҳ 1 h, j
Ȣ
y ׇ5 C 200 µL
3 min, NO− 2 /
< / (SOD/CAT, ʂ 100 U/mL)ȴɁ ȉ. : Ȁ ȉ . Λj֖ ֘؆ 95% c/5% CO2 nP , D D LDH ۤ я, 4֤ D TBARS 24 h ̫ؕ ȉƪݳ
− NO, NO − 2 / NO3 , Ƴ
Sͤ
=
(
(TBARS)
¶<M ̘
, bcl-2 ؕ
Ҳ
NO ƪΞҲ֘؆ȥ(D
֘؆ȥ( [18], ӈ:Λ [19], ǒ ֘
[20]
poly(ADP
)Rץ
Ҳܵ܈ ̣̘
. bcl-2 ؕ
̣ ֖֙ Қ
9 p53 ƪΞҲ bcl-2 ̫ Λ̘ 4֤
bcl-2 Ҳ +
Ҳ[ $
405 nm
bcl-2, p53 D p21/waf1/cip1 ۤ $ ɪj c
[24] , ֘؆ 70% ̑ , 4j R , Ƶ A 4%mU ۤ Ҳ y ׇ5 C (PBS/BSA) 2 , ȴ Ɂ Ȁ 1ƅ 100 Ԫ Ҳ P bcl-2 Rǽ P. (N-19) p53 Rǽ P. (CM-1)D p21/waf1/cip1 Rǽ P. (C-19)ʂ 60 min, Ƶ PBS/BSA ɧ R ǖ P. , Ȁ 1ƅ 50 Ԫ ҲP FITC P , ֘؆ / A 10 µg/mL PI D 70 µg/mL RNA Ҳ PBS , Ƶ EPICSElite ֘؆ Ʒ $ . P ݂_ 5000 ֘؆ , Ƶ A P.Ҳ ׇ5 C ֘؆Ǚ Λj , ݂ bcl-2, p53 D p21/waf1/cip1 ۤ Ұ ȴ я . 1.6 LDH $ ғ ̒ԟ ̽ ȴ ǼǼ ݂$ 1.7 1.8
tt
Ԯ ȃ? ; $
LDH ݟɊ y , Ƶ Beckman DU-640
ռ
.
NO $ ғ 3 mL A 1%FCS, 5 mL DETC, 1 mmol/L FeCl2 Ҳ DMEM ׇ5 CɭƳ ȉC , ̘ 37j = , ғ 1ƅ 5 .SҲ ؖȀ ݩ. , ¥ 3 min, ְ֫ ȉ 30 min. Х ʉȜ 10000Cg ԛ 6 min. ȴ ԛ γ ! C , Ǽ D̘ ER-200 Ʒ $ ESR [25]. ESR $ ݟ.Ƹ : X N , 100 kHz 9 , 9 3.2C10−4 T, я 20 mW, O −4 −4 3380C10 T, OZ 400C10 T, _ 0.3 s. 1.9
ٜ
֘؆ȥ( ELISA $ Ƶ Boehringer Mannheim ȃ? ֘؆ȥ( ELISA ݟɊ y $ ֘؆ Ǚ ɪjݟɊ y . Ƶ Bio-Rad 450 DR ٜ \ . , ǼΑ , ݂ ȥ(֘؆ Γ M (enrichment factor, EF).
DNA
Ʒ
܈.
< (SOD), (CAT), (SNP), N- -L қ (L-NAME), ؆ٜD ¾ Sigma; 3*ݟɊ ̒(ȴ . 1.1
DMEM
ݟɊ
4֤ 8 ֘؆ Lj [2] ɪj Tanaka , t 2~3 d Wistar ̾ ( ȓ ̾
) ɭ7 , 0.06%ۤ , 9̸ ֘؆ C, ̘ 37jӍΛ 100%Ҳ CO2 ȉ 15 min, չ(50 U/mL)D չ(50 µg/mL)Ҳ DMEM ̰ݹҲ ֘؆Ԟ A 10%mU −6 ȉ 6 h, ХʉƵA 10 mol/L ؆ٜ(Ara C)Ҳ DMEM ȉ ȉ 48 h Ƶ6\ ȉ 24 h, $ NO, TBARS D ֘؆S0, ХʉƵA 1%mUҲ DMEM ȉ LDH, :ƵA 5%mU A քҲǔ DMEM ȉ Ƴ, ֘؆ 95% N2/5% CO2 nP ȉ 24 h, Хʉ 95% O2 /5% CO2 nP ȉ 4 h 4֤ ֘؆ Lj . ֖ ֘؆ 95% N2/5% CO2 nP ȉ 24 h ǒ 4֤ . SOD/CAT (ʂ 100 U/mL), SNP (5 µmol/L), L-NAME (100 µmol/L)D D-NAME (100 µmol/L)ȴɁ : Ȁ ȉ , ܨ Λj֖ ֘؆ 95% c/5% CO2 nP ȉ. [3Λ ֘؆ Lj+ XǙғ. 1.2
Rԉ ȏԟ ΅,
ѩԟ,
c;
Ɲ 100101; c)
t
ѩԉ 8ؘ
, ܮܫ
¶ [ܨ 4֤ LjҲ (nitric oxide, NO)D Қ !9. t Wistar ̾ ֘؆ 95% N2/5% CO2 nP ȉ 24 h, Хʉ 95% O2 /5% CO2 nP ȉ 4 h 4֤ ֘؆ Lj . ֖ ֘؆ 95% N2/5% (SNP, 5 µmol/L) NO ʄ 9Ɋ NωCO2 nP ȉ 24 h, ǒ 4֤ . NO ǫ. -L- қ (L-NAME, 100 µmol/L) D 3 8 . Nω- -D- қ (D-NAME, 100 µmol/L)Ƶ=Â
(7 , ȥ(D ϯ
?
ɪ D
̣Ҳ֘؆?(Ԯ [4]. ¶<M , ϯ t M
[10] [12,13]
֘؆ȥ(=tҲ )Ԩ
Ҳ
[5,6]
Қ
ɹƪΞ DNA
Lj, ϯ
poly-(ADPɪ t (
[5,9]
p53 ̫
, Ξ֘؆ȥ(
ȏx֘؆5 Y
. ʇ
,
(nitric oxide, NO) NO ɹ O− 2=
(C
32 5
)
2002 10
SCIENCE IN CHINA ( Series C )
D Қ Ξ 4֤ ֘؆ ȥ(Ҳ bcl-2 D p53 ? ԉ *
:Ⱥ
̒ԟ ȓ ̾
t W ț Ɛ ޛ
¹Ӻԟ ԉ , 510515; c̾
**
3 ݘ ֘؆ȥ( Қ bcl-2 p53 p21/waf1/cip1
֘؆?( ȴ 8t
?Dȥ( D.
Ԟ!9 . Ԟ
[1]
?֘؆Ҳع MԂҲԮ
[2]
a
ͅ , ֘؆
Dԓ ܈, ֘؆ ȏ
[3]
R9C֖̎֙ , ȥ(֘؆ ɯ
?(Ԯ ,
. ̘
֘؆ D4Ϲ
ɹ=і֘؆ȥ
֘؆ȥ(D
1
¦ ȉ ¾ GIBCO; mU¾ Kibbutz Beit Haemek (Iserl); HEPES ¾ Farco; bcl-2 Rǽ P. (N-19)¾ Santa Cruz, p53 Rǽ P. (CM-1) ¾ Novo; p21/waf1/cip1 Rǽ P.(C-19) ¾ Santa Cruz; P FITC ¾ Doko; ۤ K ¾ Merk; Annexin V-Flous ¾ ʽ(PI), NADPH, FAD, y< , Nŧ ؓ, Ƴ Sͤ Boehringer Mannheim; , ̈ Л , ؓ , Â
1.10
Ƴ Sͤ =
( ݳTBARS)$ ȉ U TBARS A $ Ƶ Ohkawa ժƣ [15]Ҳ
5
:Ⱥժ : NO D
Қ
Ξ
4֤
֘؆ȥ(Ҳ bcl-2 D p53
− NO3
Ȁժ .Ҳ Griess ݟɊ (2% N- Ʒ 550 nm \P $ Ƶ֎
ؓ ƅ 0.2 % ؓ 3ʚ
1ƅ 1 Qʄ ),
60j
10 min, Ƶ Bio-Rad 450
, Ƶ
(0~20 nmol/mL)
,
(nmol/mL) . 532 nm ғ Beckman DU-640