白藜芦醇诱导HepG2细胞凋亡的线粒体机制
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(A):crude mitochondria;(B):pm矗ed mitochonda-ia
附嘲
A B
圈4-3线粒体蛋白的双向电泳图谱f银染)。(A):对照;(B):1009mol/L白藜芦醇24h
Fig.4-32-DE map ofmitochondrial proteome in H雌G2cells(sliver-staining)
Fig.2—2The time-dependent effects ofresveratrol Oil MPTP opening.HepG2cells were co-loaded with TMRE and calcein after exposure to resveratrol(1001.maol/L),as.described in Mamials and Methods.Red-fluorescing TMRE monitored mitochondrial depolarization and green-fluorescing ealcein monitored the MPTP werg monitored simultaneously in living cells by confocal microscopy.The MPTP began to occur within lOb of treatment.as mdieated by decre∞e of red TMRE fi'om mitochondria and i)ermeabilization of green calcein from the eytosol into mitochondria.One experiment wag repeated three times.
博士学住论文
图1.3白藜芦醇作用下HepG2细胞的形态学观察.A:对照组光镜图片;B:100ttmoFL白藜芦醇处理24h后的光镜图片;C和G.对照组电镜图片;D和H:100pmol/L白藜芦醇处理24h后的电镜图片;E.对照组荧光图片;F:100tanoFL白藜芦醇处理24h后荧光图片Fig.1-3Morphological changes ofHepG2cells after treatment with resveralr01.Cells were treated With100p.mol/L ofresveralzol裙described and obselved by phase conlxast microscope(A∞d B) and transmission electron microscope(C’G and D'I{).Cells we∞also stained with Hoechst33342 dyetoeofamineapoptoticnuclearmorphology饵and F).A:Phaseconlrastimageofcontrol cells; B:Phase contrast image of cells after100ttmo儿resveratrol treatment;C and G:Electron microscope image ofcontrol cells;D and H:Electron microscope image ofcells after100ttmoFL resveratrol Ireatment;E:Fluorescence picture ofcontrol cells;F:Fluorescence image ofceils after 100￥tmo儿resveratroi treatment.
Fig.1-5Resveratzol induccxt Hcl哦32cdl death via硼Ⅺptosis.‘Al HepG2cells WCfe treated with DMSO(contr01)aand resveratrol(100)tmol/L and 200)tmol/L).AR胃24h,cells we∞harvested for Annexin v.Fn℃and PI analysis.(B)Hel0G2cells Wel'e舡∞ted with resveratrol(100lamol/L)for 24h and live cells analyzed for phosphatidylserine extermalization using
(A):Control;(B):t∞atmcntofl00pmoFLresvcratrolfor24h
博士学住论文
图4巧蛋白点的表达变化(考染).左图:对照;右图:100岬。儿白藜芦醇处理24h Fig.4.5Expressive Mterafons ofpfotein sopS.The arrow8showed恤si卸i6c柚t differel3cc血
.t≈F
.‘’:!==:_■凝缸§:·‘1.1%4一懈xIM
l。。。Resvemrol(1∞I·岣fC蛔ng
图l-5白藜芦醇诱导Hep(32细胞凋亡的流式细胞和荧光显微镜分析。(A)HepG2细胞经DMSO(对照)和自藜芦醇(100]mlol/L aad 200panoFL)的处理。24小时后,收集细胞进行Annatin V-FITC和PI标记,流式细胞仪分析.(B)HqoG2细胞经1001anoFL自藜芦醇处理24小时后,标记Annexin V和hoedm03342,进行荧光显微镜分析。
60 O
附图
Alexa Fluor488Fh町“埔∞n∞
Oh4h8h121116h
Time∞
A
∞{寻
博士学位论文
B C
博士学位论文
∞
∞
萋柏∞Βιβλιοθήκη OARc_tl州
B^PL“AM
肼^
B
C
苦§目o
博士学O-fe文
A B
图禾l提取HepG2细胞线粒体的电镜观察(A)和Jenu's Green染色观察(B)。
FigA-I Electron microscope image ofmitochondria(A)and stained with JellU’s Green(13).
A B
图4.2线粒体蛋白的双向电泳.(A):线粒体粗提物的蛋白质组图;(B):纯化后线粒体
的蛋白质组图。
Fig.4-2Two-dimensional electrophorefic(2-DE)map ofmitochondrial proteome in HepG2cells.
(A):Control;(B):t*eatmentofl00tnnoFLrvsvemtrol for24h
A B
图4_4线粒体蛋白的双向电泳图谱(考染)。(A);对照;(B):100¨mol/L自藜芦醇24h 糍14-42一DE map ofmitochondrial prot∞me in He'p02cells(Coomassie brilliant blue staining)
博士学住论文
图2-3CsA阻断白藜芦醇诱导MFFP的开放。在白藜芦醇处理细胞前30min加入21tmoFL CsA。细胞同时负载TMRE和calcein并进行共聚焦显微镜的成像分析.
Fig.2-3CsA blocks mitochondrial depolarization and MPTP opening induced by resveratr01. HepG2cells Wel"e treated with resveratrol(100ttmol/L)and CsA(2)tmol/L),co-loaded with calcein and TMRE.and analyzed∞described for Fig.2-2.One experiment was repeated three times.
abun妇of印。忸.
Left.C∞缸咄鼬ght:Irea衄蛐tofloo舯口。儿∞svera咖l f研24h
附图
博士学位论文
图4.6质谱鉴定蛋白斑点
Fig.zI-6Identification ofprotein sopb with ma路spech‰
B:S9{D臼,sFS
圃;髓;眨a ~Rrn由峨i腱12}∞。一Rho由嚏i憎12孑啪..Rhod蚺I憎12孑啪
B
C l : !
pr;ou
附图
图2-2白藜芦醇诱导MIrrP的开放。1001m'lol/L的白藜芦醇处理Hep02细胞,8h后同时负载TlvlRE和calcein,激光扫描共聚焦显微镜实时观察MPTP的状态。10h后,观察到MPTP 的开放(红色TMRE荧光强度降低和绿色的calcein从胞质进入线粒体内)。实验重复3次,结果一样。
and1001amol/L resveratrol for24h.
博士学位论文
12
9.,%
.·‘',+.q』
:
:·:9.■‘
!.÷鞠鬻:j;静i矗他4
l 2甜慨.j辫i :;;彰::;:;:’
!’.::f≈薹:;‘r渊黔乏7崩4一斛￡xIH 1。。。Resveamra(200IIM)A B
l 2
03%
a commercial Annexin V kitandhoechst33342staining
asdescribedundermaterialsmdmethods(scaleb%30wn).
附图
6:S争.LoBv5PS6:SS,--I.06,vsFS6:SS-L06v5FS 厦a;圜;魉3一Rho如ninel矗。∞
附图
40
O
图1_4白藜芦醇对HepC2细胞周期分布的影响(分别以DMSO,251.unoFL,50￥u'noFL和
lOOpmol/L白藜芦醇处理细胞24h)。
Fig.14Cdl cycle analysis ofHepG2cells after treatment with O.2%DMSO,25pmol/L,50￥maol/L
附嘲
A B
圈4-3线粒体蛋白的双向电泳图谱f银染)。(A):对照;(B):1009mol/L白藜芦醇24h
Fig.4-32-DE map ofmitochondrial proteome in H雌G2cells(sliver-staining)
Fig.2—2The time-dependent effects ofresveratrol Oil MPTP opening.HepG2cells were co-loaded with TMRE and calcein after exposure to resveratrol(1001.maol/L),as.described in Mamials and Methods.Red-fluorescing TMRE monitored mitochondrial depolarization and green-fluorescing ealcein monitored the MPTP werg monitored simultaneously in living cells by confocal microscopy.The MPTP began to occur within lOb of treatment.as mdieated by decre∞e of red TMRE fi'om mitochondria and i)ermeabilization of green calcein from the eytosol into mitochondria.One experiment wag repeated three times.
博士学住论文
图1.3白藜芦醇作用下HepG2细胞的形态学观察.A:对照组光镜图片;B:100ttmoFL白藜芦醇处理24h后的光镜图片;C和G.对照组电镜图片;D和H:100pmol/L白藜芦醇处理24h后的电镜图片;E.对照组荧光图片;F:100tanoFL白藜芦醇处理24h后荧光图片Fig.1-3Morphological changes ofHepG2cells after treatment with resveralr01.Cells were treated With100p.mol/L ofresveralzol裙described and obselved by phase conlxast microscope(A∞d B) and transmission electron microscope(C’G and D'I{).Cells we∞also stained with Hoechst33342 dyetoeofamineapoptoticnuclearmorphology饵and F).A:Phaseconlrastimageofcontrol cells; B:Phase contrast image of cells after100ttmo儿resveratrol treatment;C and G:Electron microscope image ofcontrol cells;D and H:Electron microscope image ofcells after100ttmoFL resveratrol Ireatment;E:Fluorescence picture ofcontrol cells;F:Fluorescence image ofceils after 100￥tmo儿resveratroi treatment.
Fig.1-5Resveratzol induccxt Hcl哦32cdl death via硼Ⅺptosis.‘Al HepG2cells WCfe treated with DMSO(contr01)aand resveratrol(100)tmol/L and 200)tmol/L).AR胃24h,cells we∞harvested for Annexin v.Fn℃and PI analysis.(B)Hel0G2cells Wel'e舡∞ted with resveratrol(100lamol/L)for 24h and live cells analyzed for phosphatidylserine extermalization using
(A):Control;(B):t∞atmcntofl00pmoFLresvcratrolfor24h
博士学住论文
图4巧蛋白点的表达变化(考染).左图:对照;右图:100岬。儿白藜芦醇处理24h Fig.4.5Expressive Mterafons ofpfotein sopS.The arrow8showed恤si卸i6c柚t differel3cc血
.t≈F
.‘’:!==:_■凝缸§:·‘1.1%4一懈xIM
l。。。Resvemrol(1∞I·岣fC蛔ng
图l-5白藜芦醇诱导Hep(32细胞凋亡的流式细胞和荧光显微镜分析。(A)HepG2细胞经DMSO(对照)和自藜芦醇(100]mlol/L aad 200panoFL)的处理。24小时后,收集细胞进行Annatin V-FITC和PI标记,流式细胞仪分析.(B)HqoG2细胞经1001anoFL自藜芦醇处理24小时后,标记Annexin V和hoedm03342,进行荧光显微镜分析。
60 O
附图
Alexa Fluor488Fh町“埔∞n∞
Oh4h8h121116h
Time∞
A
∞{寻
博士学位论文
B C
博士学位论文
∞
∞
萋柏∞Βιβλιοθήκη OARc_tl州
B^PL“AM
肼^
B
C
苦§目o
博士学O-fe文
A B
图禾l提取HepG2细胞线粒体的电镜观察(A)和Jenu's Green染色观察(B)。
FigA-I Electron microscope image ofmitochondria(A)and stained with JellU’s Green(13).
A B
图4.2线粒体蛋白的双向电泳.(A):线粒体粗提物的蛋白质组图;(B):纯化后线粒体
的蛋白质组图。
Fig.4-2Two-dimensional electrophorefic(2-DE)map ofmitochondrial proteome in HepG2cells.
(A):Control;(B):t*eatmentofl00tnnoFLrvsvemtrol for24h
A B
图4_4线粒体蛋白的双向电泳图谱(考染)。(A);对照;(B):100¨mol/L自藜芦醇24h 糍14-42一DE map ofmitochondrial prot∞me in He'p02cells(Coomassie brilliant blue staining)
博士学住论文
图2-3CsA阻断白藜芦醇诱导MFFP的开放。在白藜芦醇处理细胞前30min加入21tmoFL CsA。细胞同时负载TMRE和calcein并进行共聚焦显微镜的成像分析.
Fig.2-3CsA blocks mitochondrial depolarization and MPTP opening induced by resveratr01. HepG2cells Wel"e treated with resveratrol(100ttmol/L)and CsA(2)tmol/L),co-loaded with calcein and TMRE.and analyzed∞described for Fig.2-2.One experiment was repeated three times.
abun妇of印。忸.
Left.C∞缸咄鼬ght:Irea衄蛐tofloo舯口。儿∞svera咖l f研24h
附图
博士学位论文
图4.6质谱鉴定蛋白斑点
Fig.zI-6Identification ofprotein sopb with ma路spech‰
B:S9{D臼,sFS
圃;髓;眨a ~Rrn由峨i腱12}∞。一Rho由嚏i憎12孑啪..Rhod蚺I憎12孑啪
B
C l : !
pr;ou
附图
图2-2白藜芦醇诱导MIrrP的开放。1001m'lol/L的白藜芦醇处理Hep02细胞,8h后同时负载TlvlRE和calcein,激光扫描共聚焦显微镜实时观察MPTP的状态。10h后,观察到MPTP 的开放(红色TMRE荧光强度降低和绿色的calcein从胞质进入线粒体内)。实验重复3次,结果一样。
and1001amol/L resveratrol for24h.
博士学位论文
12
9.,%
.·‘',+.q』
:
:·:9.■‘
!.÷鞠鬻:j;静i矗他4
l 2甜慨.j辫i :;;彰::;:;:’
!’.::f≈薹:;‘r渊黔乏7崩4一斛￡xIH 1。。。Resveamra(200IIM)A B
l 2
03%
a commercial Annexin V kitandhoechst33342staining
asdescribedundermaterialsmdmethods(scaleb%30wn).
附图
6:S争.LoBv5PS6:SS,--I.06,vsFS6:SS-L06v5FS 厦a;圜;魉3一Rho如ninel矗。∞
附图
40
O
图1_4白藜芦醇对HepC2细胞周期分布的影响(分别以DMSO,251.unoFL,50￥u'noFL和
lOOpmol/L白藜芦醇处理细胞24h)。
Fig.14Cdl cycle analysis ofHepG2cells after treatment with O.2%DMSO,25pmol/L,50￥maol/L