臺灣博碩士論文加值系統

腸病毒71型(EV71)，是一種會造成神經性疾病的病毒，也曾在世界各地由於中樞神經系統的感染而引起大量的死亡及致病的病例。許多的研究已指出，病毒的感染會引起發炎反應並會誘發iNOS還有COX-2的表現。因此在這個論文裡，我們將會使用大鼠腦部星狀細胞(rat brain astrocyte cells；RBA cells)來當一個模式，讓我們可以探討p42/p44 MAPK、PDGFR、PI3K/Akt、PKC、NF-κB及AP-1在大鼠腦部星狀細胞中由腸病毒71型誘導iNOS和COX-2的表現所扮演的角色。我們利用西方點漬分析法(Western blotting)、Reverse transcriptase-polymerase chain reaction (RT-PCR)及Immunofluorescent staining (IF) 探討。在EV71的感染下的確可以刺激iNOS和COX-2的表現及p42/p44 MAPK、PDGFR與Akt的磷酸化現象。所以進一步利用專一性的藥理抑制劑PD98059 (MEK1/2)、PP1 (c-Src)、AG1296 (PDGFR)、wortmannin (PI3K)、helenalin (NF-kB)、curcumin (AP-1)、Gö6976 (Ca2+-dependent PKC)及rottlerin (PKC-δ)先行處理，可以發現對於iNOS和COX-2的表現有抑制現象。在mRNA表現方面也可以看到相同的抑劑情形。在NF-B的活化方面利用免疫螢光的方法來分析，發現只有Gö6976前處理會抑制NF-B的translocation。對於AP-1的參與，由RT-PCR的結果發現其c-jun 與 c-fos 的mRNA表現會明顯的受到專一性抑制劑curcumin所抑制。Curcumin 也會抑制iNOS與COX-2的表現。另外為了更進一步確定PDGFR、Akt及c-Src的上下游關係，我們使用抗體免疫沉澱的方法，發現c-Src是PDGFR的上游蛋白，而Akt則是位於兩者的下游蛋白。最後利用NO release detection的方法，發現p42/p44 MAPK、PDGFR及Akt抑制劑也會降低EV71誘導NO釋放的情形。所以根據我們的實驗結果可以推測EV71誘導大鼠腦部星狀細胞中iNOS及COX-2的表現，會透過p42/p44 MAPK的磷酸化、 receptor tyrosine kinase的轉活化和NF-B的translocation；另外PKC及AP-1的參與也是重要的訊號傳遞調控路徑。這些實驗結果建立了EV71感染引起發炎反應的新模式，並可以增加我們對於EV71誘導大鼠腦部星狀細胞中iNOS及COX-2表現的了解，進而可以對我們在治療EV71的疾病上有更多的幫助。

Enterovirus 71 (EV71), a neurotropic virus with undefined pathogenesis, has caused significant morbidity and mortality worldwide. The central nervous system (CNS) is the most vulnerable target of EV71 infection. Many reports have indicated that viral infection induced iNOS and COX-2 expression. Therefore, we used rat brain astrocyte cells (RBA cells) as a model to investigate p42/p44 MAPK, PDGFR, PI3K/Akt, PKC, NF-κB and AP-1 involved in EV71-induced iNOS and COX-2 expression using Western blotting, Reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescene (IF). EV71 stimulated the expression of iNOS and COX-2 and the phosphorylation of p42/p44 MAPK, PDGFR, and Akt. EV71-induced responses were attenuated by pretreatment with selective pharmacological inhibitors PD98059 (MEK1/2), PP1 (c-Src), AG1296 (PDGFR), wortmannin (PI3K), helenalin (NF-B), curcumin (AP-1), Gö6976 (Ca2+-dependent PKC), and rottlerin (PKC-δ). iNOS and COX-2 mRNA expression were inhibited by these inhibitors. The translocation of NF-kB was proved by IF and cytosolic and nuclear fractions prepared from RBA cells exposed to EV71. Only Gö6976 and helenalin inhibited NF-kB tranlocation. AP-1 mRNA expression induced by EV71 in a time-dependent manner. To establish the relationship between PDGFR, Akt, and c-Src, we used immunoprecipitation to investigate this purpose. We demonstrated that c-Src was the upstream of PDGFR and Akt was downstream of both c-Src and PDGFR. Finally, NO release was determined by Griess reagent. The activation of p42/p44 MAPK, PDGFR, and Akt was associated with the expression of iNOS induced by EV71. The signaling pathways involved in RBA cells have been demonstrated that the expression of iNOS and COX-2 induced by EV71 was mediated through the phosphorylation of p42/p44 MAPK, the transactivation of receptor tyrosine kinase, the translocation of NF-B. In addition, PKC and AP-1 may play an important role in EV71-induced iNOS and COX-2 expression. These results established a new model that EV71 induced inflammatory responses and increased the understanding of EV71-induced iNOS and COX-2 expression. These results may provide a potential therapeutic value in the treatment of EV71 infection.

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Contents---------------------------------------------------------------------------- i
Abbreviations-------------------------------------------------------------------- iii
Inhibitors------------------------------------------------------------------------- vi
Abstract in Chinese------------------------------------------------------------ vii
Abstract in English------------------------------------------------------------- ix
CHAPTER I Introduction------------------------------------------------------- 1
1. Enterovirus 71 (EV71)--------------------------------------------------- 1
2. Neuroglia------------------------------------------------------------------ 3
3. The role of iNOS in virus----------------------------------------------- 5
4. NO and NOS in virus infection----------------------------------------- 6
5. The role of COX in virus infection------------------------------------- 9
6. COX (Cyclooxygenase)------------------------------------------------- 9
7. NF-κB in virus infection------------------------------------------------12
8. Intracellular signaling cascades and virus infection---------------- 13
CHAPTER II Appended Figures and Tables-------------------------------- 16
CHAPTER III Specific Aims-------------------------------------------------- 26
CHAPTER IV Materials and methods--------------------------------------- 28
1. Materials------------------------------------------------------------------ 28
2. Cell culture--------------------------------------------------------------- 28
3. Preparation of cell extracts and western blot analysis-------------- 29
4. EV71 propagation------------------------------------------------------- 30
5. Determination of the virus titer via plaque assay------------------- 31
6. Purification of EV71 viral particles---------------------------------- 32
7. Total RNA extraction and RT-PCR analysis------------------------- 32
8. NF-kB translocation---------------------------------------------------- 33
9. Immunoprecipitation assay-------------------------------------------- 34
10. Immunofluorescence staining----------------------------------------- 35
11. Cytokine array----------------------------------------------------------- 35
12. Measurement of NO levels-------------------------------------------- 36
13. Plasmids and transfection---------------------------------------------- 36
14. Data analysis------------------------------------------------------------- 37
CHAPTER V Result------------------------------------------------------------ 38
1. PART I-------------------------------------------------------------------- 38
2. PART II------------------------------------------------------------------- 44
3. PART III------------------------------------------------------------------ 47
4. PART IV------------------------------------------------------------------ 50
CHAPTER VI Discussion----------------------------------------------------- 51
CHAPTER VII Figures of Results------------------------------------------- 57
CHAPTER VIII References--------------------------------------------------- 94

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