臺灣博碩士論文加值系統

先前的研究發現細菌內毒素（Bacterial lipoplysaccharide, LPS）及其毒性中心脂質A（Lipid A）會透過不同的訊息傳遞路徑刺激T細胞NF-κB mRNA的表現暨NF-κB蛋白之活化，和促成T細胞表面抗原蛋白CD25、CD28和CTLA-4（CD152）表現量增加。Lipid A（LA）可能是透過活化PTK和PI3K之路徑刺激細胞內鈣離子濃度增加；而LPS可能是透過活化PTK和PKCθ之路徑引發細胞鹼化。但LPS及LA並不會刺激引發T細胞的增生，因此本研究將進一步探討LPS及LA的早期訊息對於下游基因的表現有什麼影響？本實驗以人類T細胞為研究材料，分別觀察LPS及LA對T細胞內鈣離子濃度、PLD、NF-κB 、TLR2和TLR4 mRNA的表現及T細胞表面抗原變化的影響。且以不同的訊息抑制劑如：phophoinisitide 3-kinases（PI3K）抑制劑，LY294002及Wortmanin；PKCθ抑制劑，Rottlerin；protein tyrosine kinase（PTK）抑制劑，Genistein；探討LA是透過何訊息路徑刺激T細胞不同基因及表面抗原的表現。本研究以Fura-2螢光染劑測量細胞內鈣離子濃度的變化。以反轉錄聚合脢連鎖反應（RT-PCR）的方式分析mRNA之變化。用流式細胞儀（flow cytometry）測定不同細胞表面抗原的表現量。最後以T細胞對[3H]-thymidine攝取至DNA之多寡來評估T細胞增生能力。實驗結果發現（一）Lipid A所刺激之胞內鈣離子濃度上升之現象會受到LY294002及Wortmanin的抑制，因此PI3K可能參與細胞外鈣離子流入T細胞之機轉，而不影響內質網鈣離子之釋放。（二）由RT-PCR的結果顯示LPS可使PLD mRNA及TLR4 mRNA 在刺激30分鐘後之表現量分別上升2倍及1倍，而NF-κB及TLR2之mRNA在刺激1小時內表現增加到達高峰，分別上升1.2倍及3倍，但Lipid A對於TLR2及PLD之 mRNA 的表現沒有影響，但對於NF-κB在刺激30分鐘後之表現量分別上升1倍，在4小時表現增加到達高峰上升3 倍，TLR4 之mRNA在1小時內表現增加到達高峰上升1.6倍。（三）EGTA及PI3K抑制劑Wortmanin抑制Lipid A刺激T細胞NF-κB 之表現而TLR4 mRNA之表現則不受此影響。（四）由flow cytometry結果顯示LA及LPS刺激T細胞後，並不影響其細胞表面抗原CD40L的表現量，而Lipid A對於細胞表面抗原TLR2有明顯增加，對TLR4則沒有影響；而LPS對於細胞表面抗原TLR2及TLR4皆有顯著增加的現象。（五）當以LA與PMA合併刺激T細胞並不會刺激T細胞增生。總結發現LPS可顯著誘發T細胞PLD，NF-κB，TLR2，TLR4之mRNA表現增加暨細胞表面抗原TLR2及TLR4之增加。而LA僅可顯著刺激T細胞NF-κB，TLR4之mRNA表現增加暨細胞表面抗原TLR2之增加。而 LPS 及LA刺激T細胞其下游 mRNA 之表現差異主要不同在 PLD 及 TLR2，且LA並不能增強T細胞TLR4表面抗原之表現，故本研究推論LPS比LA更能刺激T細胞產生較多之先天免疫配備以增強其先天免疫力。

Previous works indicated that Bacterial lipoplysaccharide (LPS) and it's toxically center, lipid A (LA), stimulated NF-κB mRNA expression and NF-κB protein activation, but not the proliferation in T cells. The increasing in intracellular calcium was through phosphoinositide 3-kinase (PI3K) pathway by Lipid A, whereas, the increases in intracellular pH (pHi) was through protein kinase C (PKC)/phospholipase D (PLD) pathway in T cells. Therefore, the aim of this study was to investigate whether gene expression by LPS or LA were regulated by differential signals stimulated in human peripheral T cells. The changes in intracellular calcium ([Ca2+]i) were measured by fluorescent dye, Fura-2, and the mRNA expression in Toll-like receptors (TLRs, TLR2 and TLR4), NF-κB and PLD were measured by reverse transcriptional-polymerase chain reaction (RT-PCR). The T cell proliferation was determined by [3H]-thymidine incorporation. The appearance of cell marker CD40L、TLR2、TLR4 on T cells were analyzed by flow cytometry. Inhibitors of PI3K, LY294002 and Wortmanin, inhibitor of PKCθ, Rottlerin, and inhibitor of protein tyrosine kinase (PTK), Genistein were used to explore the alternation on signals and the expression by lipid A. The results indicate that 1) The Ca2+ influx, but not intracellular calcium release by LA and was suppressed by Wortmanin and LY294002; 2) The mRNA expression on PLD、TLR4 was induced by LPS at 30 min and reached a peak at 1h. In addition, the mRNA expression on NF-κB and TLR2 was induced by LPS and reached a peak at 1 h. In contrast, LA did not stimulate PLD and TLR2 expression but increased the expression of NF-κB at 1h and reached a peak at 4 h; 3) The mRNA expression on NF-κB by LA was suppressed by pretreate cells with EGTA and Wortmanin, however, the expression on TLR4 was not affected; 4) Both LPS and LA did not affect the expression of cell surface protein marker CD40L. LPS increased the expression of cell surface protein markers, TLR2 and TLR4. Whereas, LA increased the expression of TLR2 but not TLR4; 5) Combinations of LA and PMA did not alter [3H]-thymidine incorporation into T cells. In summary, LPS could induce the mRNA expression on PLD, NF-κB, TLR2 and TLR4 as well as the cell surface protein on TLR2 and TLR4. In contrast, LA only increased mRNA expression of NF-κB and TLR4 as well as the cell surface protein on TLR2. In conclusion, the mRNA expression on PLD and TLR2 was the only differential signal that found by LPS and LA, thus, LPS had better enhancement than LA on T cell natural immunity.

中文摘要 ------------- 2
英文摘要 ------------- 4
緒 言 ------------- 6
材料方法 ------------- 17
結 果 ------------- 30
討 論 ------------- 38
參考文獻 ------------- 48
圖表說明 ------------- 52
附 錄 ------------- 73
誌 謝 ------------- 75

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