(3.238.250.105) 您好!臺灣時間:2021/04/18 19:29
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:王家凱
研究生(外文):Wang, Jia-kae
論文名稱:脂多醣體在巨噬細胞及神經膠質細胞引發一氧化氮釋放之研究
論文名稱(外文):Studies on Lipopolysaccharide-Induced Nitric Oxide Release in RAW 264.7 Macrophages and Astrocytes: Role of Protein kinase C Isoforms and MAP kinases
指導教授:陳青周
指導教授(外文):Ching-Chow Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:6
中文關鍵詞:脂多醣體一氧化氮蛋白激脢C一氧化氮合成脢巨噬細胞神經膠質細胞
外文關鍵詞:lipopolysaccharidenitric oxideprotein kinase CNO synthasemacrophageastrocyte
相關次數:
  • 被引用被引用:0
  • 點閱點閱:189
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:2
本論文在RAW 264.7 macrophages和astrocytes上探討LPS引發NO釋放的
訊息傳遞。在RAW 264.7 macrophage 發現LPS 引發之NO 釋放及iNOS 表
現均為dose- 及time-dependent,且均受到蛋白轉錄抑制劑actinomycin
D和蛋白轉譯抑制劑cycloheximide 之抑制。tyrosine kinase 抑制劑
genestein 可抑制LPS引發之NO釋放、iNOS 表現、IP蓄積及PKC 活性之增
加,PI-PLC 抑制劑U73122和PC-PLC 抑制劑D609可抑制LPS 引發之NO 釋
放 和iNOS 表現,而phosphatidate phosphohydrolase 抑制劑
propranolol則無影響。Staurosporine、calphostein C、Ro 31-8220 和
Go6976 四種PKC 抑制劑以及TPA前處理24小時都可抑制LPS5 iNOS 表現和
NO 釋放。表示LPS 可經由tyrosine phosphorylation 活化PLCgamma及
PC-PLC 再活化PKC 而使NO 釋放。Western blot 分析發現RAW cells 含
有PKC alpha, betaI, delta, eta 及zeta,除了zeta之外其他isoforms
均受TPA 10分鐘之刺激而有轉位的現象,alpha、betaI 和delta 可受TPA
處理24小時down-regulation,而eta 則無,表示可能是PKC alpha,
betaI 和delta 參與LPS 之作用,進一步已PKC alpha, betaI 和delta之
antisense 證明的確是PKC alpha, betaI 和delta 三種isoforms 參與
LPS 之iNOS 表現和NO 釋放作用。MEK 抑制劑PD 98059 和p38 MAPK 抑制
劑SB203580亦可抑制LPS 之iNOS 表現和NO 釋放,而LPS 刺激10分鐘可活
化p44/42 MAPK、p38 MAPK 和JNK,但60分鐘活性及下降或消失,TAP 刺
激10分鐘亦可活化p44/42 MAPK 和p38 MAPK。Genestein 可抑制LPS 活化
p44/42 MAPK 和P38 mapk,而TPA 24 小時處理、Ro 31-8220 和
calphostin C,皆可抑制p44/42 和p38 MAPK。而PD 98059 可完全抑制
p44/42 MAPK 之活化,但對p38 MAPK 只有少許影響;SB 203580 則只會
抑制P38 MAPK之活化,對p44/42 MAPK 卻無影響。LPS 作用10 分鐘可使
p65 NF-kappaB 由細胞質轉位到細胞核,而p50 NF-kappaB 則無此現象,
亦會造成IkB-alpha 和IkB-beta 的degredation,前者於1小時後即恢復
,但後者至24小時才有回復現象。EMSA 的結果也發現LPS 可使NF-kappaB
的活性隨作用時間而增加,此外TPA 24 小時處理或geenstein、
calphostin C、PD 98059、SB 203580 或PDTC 皆可抑制NF-kappaB 的轉
位和其與DNA binding 的能力,而PKC antisense 之處理只有alpha、
betaI 和delta antisense 可抑制LPS 引發之NF-kappaB 與DNA 之
binding,因此LPS 活化p44/42 MAPK和p38 MAPK 後,可進一步造成NF-
kappaB 之活化而使iNOS 表現增加,因而NO 釋放增加。DbcAMP 24 小時
處理可隨濃度增加而引發NO 釋放遞增,PKA 抑制劑KT5720可抑制LPS 之
iNOS 表現和NO 釋放,而DbcAMP 和forskolin 皆可增加LPS 釋放NO 及
iNOS 表現之作用。CaMKII 抑制劑KN-62、calcium chelator BAPTA 或
PTX,皆對LPS之 NO釋放和iNOS表現無影響。 在astrocytes,LPS 引
發之NO 釋放及iNOS 表現亦為dose- 及timedependent也受actinomycin D
和cycloheximide 之抑制。Genestein、D609 和propranolol 可抑制LPS
之NO 釋放,而U73122 和U73343則無影響。Staurosporine、calphostinC
、Ro31-8220 和Go6976皆可抑制,而TPA處理24 小時不影響LPS 之NO 釋
放及iNOS表現,因此在astrocytes 中可能是PKC eta 參與LPS引發之iNOS
表現和NO 釋放,由PKC eta antisense 之實驗證明確實是PKC eta 參與
LPS 之作用。因此在astrocytes,LPS 可能經由PC-PLC 和PC-PLD 來活化
PKC eta 而使NO 釋放。PD 98059 和SB 203580 亦可抑制LPS之iNOS 表現
和NO 釋放,而LPS 刺激10分鐘可活化p42 MAPK、p38 MPAK 和JNK,且持
續至1小時。RO 31-8220可抑制而tpa 處理24小時則不影響LPS活化p42
MAPK 作用,而此兩項處理均不影響p38 MAPK 之活化。PD 98059 完全抑
制p42 MAPK 之活化,對p38 MAPK 則只有少許影響。由EMSA 可知LPS 作
用10分鐘即可使NF-kappaB 活化,1小時刺激活性更大,Ro 31-8220、
genestein和PDTC皆可抑制,而TPA 處理24 小時則不影響NF-kappaB 之
DNA binding。另外PKC eta 和eta-C antisense 之處理,只有eta
antisense 可抑制LPS 引發之NF-kappa與DNA之binding。DbcAMP處理24小
時可隨濃度增加使NO 釋放遞增 和iNOS 表現。KT5720、H8或RpcAMP 可抑
制LPS 之NO 釋放,且H8 可抑制iNOS 表現但對p42 MAPK、p38 MAPK 和
NF-kappaB 的活化皆無影響。DbcAMP 可增加LPS 之NO 釋放與iNOS 表現
KN-62、BAPTA 或PTX 皆對LPS 之NO 釋放無影響。 LPS在這兩種細胞
引發NO 釋放的訊息傳遞中,其活化PKC5 DAG 來源可能不同,在RAW
cells 可能為PI-PLC gamma 和PC-PLC,而astrocytes 則可能為PC-PLC
和PC-PLD,所參與之PKC isoforms 也不同,RAW cells 為PKC alpha,
betaI 和delta,而astrocytes 只有PKC eta。此外NF-kappaB在
astrocytes 上對LPS 引發iNOS 表現之重要性可能不及RAW cells。而兩
者的起始訊息可能都是tyrosine phosphorylation,且p44/42 MAPK、p38
MAPK 和pka 亦都有參與,但calcium-CaMKII 和PTX sensitive G
protein 可能不重要。

The siganl transduction pathwayof LPS-induced NO release
wasstudied in RAW 264.7 macrophages and astrocytes. In RAW
cells,LPS-induced a dose- and time-dependent increase of NO
release andiNOS expression, and both effects were inhibited by
actinomycin D andcycloheximide. Tyrosine kinase inhibitor
genestein attenuated the NOrelease, iNOS expression, PI
hydrolysis as well as the increase in PKCactivity stimulated by
LPS. The LPS-elicited NO release and iNOS expression were also
inhibited by PI-PLC inhibitor U73122 and PC-PLCinhibitor D609,
but not phosphatidate phosphohydrolase inhibitorpropranolol. PKC
inhibitors staurosporine, calphostin C, Ro 31-8220 and Go 6976
and long-term treatment of the cells with TPA inhibitedthe NO
release and iNOS expression induced by LPS as well,
indicatingthat LPS might activate PLC-gamma and PC-PLC through
tyrosinephosphoylation to elicit PKC activation and eventully
release of NO.Western blot showed the expression of PKC alpha,
betaI, delta, eta andzeta in RAW cells. Except zeta, evry
isoforms was translocated by 10 min treatment with TPA and down-
regulation of PKCalpha, betaI and delta but not eta was seen
after long-term treatmet, indicating the possible involvement
of PKC alpha, betaI and delta, PKC isoforms antisense
oligonucleotide were used. The results did show the involvement
of PKCalpha, betaI and delta but not eta in LPS-mediated NO
release and iNOS expression. LPS-mediated effects were also
inhibited by both MEK inhibitorPD 98059 and p38 MAPK inhibitor
SB 203580, and 10 min treatment of RAW cellswith LPS did
stimulated activation of p44/42 MAPK, p38 MAPK and JNK.
Tenminutes treatment with TPA stimulated the activation of both
p44/42 MAPK and p38 MAPK as well. Genestein attenuated the
activation of both p44/42and p38 MAPK. Ro 31-8220, calphostin
and long-term treatment with TPAinhibited the LPS-stimulated
activation of p44/42 MAPK, and activation of p38 MAPK was also
slightly inhibited, indicated that LPS mediated PKC activation
culd induced activation of these two MAPKs. Treatment of RAW
cells with LPS for 10 min resulted in the translocation of p65
NF-kappaBfrom cytosol to the nucleus as well as the degredation
of IkB-alpha and IkB-beta in the cytosol. NF-kappaB DNA-protein
binding activity was also enhanced by LPS. Genestein, calphostin
C, long-treatment with TPA, PD 98059, SB 203580 and PDTC all
inhibited the translocation of NF-kappaB and DNA-binding
activity stimulated by LPS. PKC alpha, betaI and delta antisense
inhibited the LPS-induced DNA-protein binding in nuclear
extracts as well.Therefore, LPS-induced p44/42 MAPK activation
results in the stimulationof NF-kappaB DNA-protein binding, then
initiated the expression of iNOS and in turn NO was release.
In astrocytes, LPS also induced a dose- and time-dependent
increaseof NO release and iNOS expression, and both effects were
inhibited by actinomycin D and cycloheximide. Genestein, D609
and propranolol but not long-term treatment with TPA attenuated
LPS-stimulated NO release andiNOS expression, indicating the
possible involvement of PKC eta in astrocytes to mediated LPS
effects. The PKC eta, eta-C and delta antisense study confirmed
the role of PKC eta in LPS elicited NO release and iNOS
expression. Both effectsof LPS were inhibited by PD 98059 and SB
203580 as well and LPS did stimulate p42 MAPK, p38 MAPK and JNK
after 10 min treatment. Ro 31-8220 but not long-term TPA
treatment inhibited LPS-induced activation of p42 MAPK, while
the activationof p38 MAPK was not affected by these two
treatments. These results indicatingthat LPS acts through PC-PLC
and PC-PLD pathways to activate PKC eta, in turn p42 MAPK was
then activated,leading to iNOS expression and NO release in
astrocytes. Treatment of astrocytes with LPS for 10 min results
in thestimulation of NF-kappaB DNA-protein binding activity in
nuclear extracts and more stimulation were seen after 1 hr
treatment. Ro 31-8220, genestein and PDTC but not long term TAP
treatment inhibited LPS-stimulated NF-kappaB DNA-protein binding
and PKC eta antisense inhibited this effect as well. In
summary, PKC activation mediated by PI-PLC and PC-PLC pathways
in RAW cells while that mediated by PC-PLC and PC-PLD pathways
in astrocytes was involved in the LPS-elicited signal
transduction of NO release and iNOSexpression. The PKC isoforms
involved were PKC alpha, betaI and delta in RAWcells and PKC eta
in astrocytes. Both p44/42 and p38 MAPK were also involved in
LPS-induced signal transduction pathways in these two types of
cells.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊
 
系統版面圖檔 系統版面圖檔