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研究生:張翰林
研究生(外文):Han-Lin Chang
論文名稱:雙萜類衍生物與C18脂肪酸影響人類嗜中性白血球釋放超氧自由基和彈性蛋白酶的機轉探討
論文名稱(外文):Effect of clerodane diterpenoid derivatives and C18 fatty acids on superoxide anion and elastase release in human neutrophils
指導教授:黃聰龍黃聰龍引用關係
指導教授(外文):Tsong-Long Hwang
學位類別:碩士
校院名稱:長庚大學
系所名稱:天然藥物研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:114
中文關鍵詞:人類嗜中性白血球C18脂肪酸垂枝長葉暗羅超氧自由基彈性蛋白酶鈣離子
外文關鍵詞:human neutrophilsC18 fatty acidscalcium16-Hydroxycleroda-313E-dien-15-oic acidClerodane diterpenoidsCalciumtore-operated
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摘要
人類嗜中性白血球分泌活性氧屬、彈性蛋白酶與類風濕關節炎、局部缺血性再灌流損傷、慢性肺阻塞疾病、氣喘和其它發炎性疾病皆有重要關聯。本論文利用分離出的人類嗜中性白血球來評估16-hydroxycleroda-3,13 (E)-dien-15-oic acid PL3S(第一單元)以及18碳脂肪酸(第二單元)的抗發炎作用。
第ㄧ單元:實驗證實PL3S能以濃度相關性的方式抑制fMLP活化的人類嗜中性白血球 O2.- 的生成和elastase的釋放,其IC50 值經統計分別為3.06  0.20 和 3.30  0.48 μM。而這抗發炎現象不是經由毒殺細胞,因為PL3S不會造成細胞LDH釋放。並且 PL3S不具有抗氧化作用和直接清除O2.- 的能力。另外,PL3S不會改變NADPH oxidase活性,也不會影響cAMP/PKA路徑。在formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) 刺激下,PL3S 有效抑制鈣離子第一個 peak,但不影響Ca2+-ATPase 抑制劑thapsigargin 誘導的鈣離子變化。此外PLC活化劑m-3M3FBS 誘導人類嗜中性白血球elastase 的釋放也會被PL3S 抑制。總結以上結果顯示PL3S抑制fMLP 誘導人類嗜中性白血球產生O2.- 和 elastase的作用可能和阻斷細胞內鈣離子有關,而和cAMP路徑無關。
第二單元:從基礎到臨床,以及流行病學的調查,都觀察到脂肪酸或脂肪酸衍生物對細胞很重要。一般認為,維持脂肪酸恆定的做法會是治療心血管疾病與發炎性疾病的有效方法。儘管如此,C18脂肪酸調節人類嗜中性白血球的生理功能仍未瞭解。實驗結果顯示,cis型態不飽和肪酸和linoelaidic acid有濃度梯度抑制fMLP誘發人類嗜中性白血球釋放超氧自由基和彈性蛋白酶與加速其鈣離子回復的現象,但是飽和脂肪酸和其餘的trans結構的C18脂肪酸則不具有作用。隨著雙鍵數目增加不飽合脂肪酸抑制效果逐漸減弱,同時若不飽和脂肪酸具有酯類其抑制效果喪失。這些脂肪酸不具有抗氧化作用和直接清除O2.- 的能力。此外,這些抑制效果不是經由影響cAMP/PKA路徑而來。cis型態不飽和脂肪酸會抑制fMLP, A23187以及thapsigargin所引起的細胞內Ca2+排空而打開的通道(store-operated calcium entry)。综合以上結果顯示,脂肪酸抑制嗜中性白血球功能的作用可能是經由阻斷細胞內Ca2+排空而打開的通道來影響細胞內Ca2+ 離子濃度。
Abstract
Reactive oxygen species and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, a cellular model in isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, asthma and other inflammatory diseases, was established to elucidate the anti-inflammatory functions of 16-hydroxycleroda-3,13 (E)-dien-15-oic acid (PL3S) (Part I) and C18 fatty acids (Part II).
Part I: PL3S suppressed the generation of superoxide anion and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-activated human neutrophils in a concentration-dependent fashion with IC50 values of 3.06  0.20 and 3.30  0.48 μM, respectively. The anti-inflammatory effects were not due to cytotoxicity because culturing with PL3S did not cause lactate dehydrogenase release. PL3S did not display antioxidant and superoxide anion-scavenging ability. Additionally, neither subcellular NADPH oxidase activities nor cAMP-dependent pathways were altered by PL3S. PL3S significantly inhibited rapid calcium release from internal calcium stores caused by fMLP but not thapsigargin. Furthermore, PL3S suppressed phospholipase C activator-elicited elastase release in human neutrophils. In summary, these results suggest that the inhibitory effects of PL3S on respiratory burst and degranulation of human neutrophils may be through the inhibition of cytosolic calcium mobilization, but not via the cAMP-dependent pathways.
Part II: There is considerable evidence from clinical, experimental, and epidemiologic observations that fatty acids (FAs) and FA-derived compounds play a critical role in cellular viability and manipulation of FA homeostasis may open novel therapeutic avenues to against cardiovascular diseases and inflammatory diseases. In spite of this, the physiological role of C18 FAs in regulating neutrophil functions remains poorly understood. In this study, cis unsaturated (UST)FAs and linoelaidic acid, but not saturated (ST)FA nor other trans USTFAs, significantly inhibited the generation of O2•− and the release of elastase, as well as the accelerated resequestration of cytosolic calcium in fMLP-activated human neutrophils in a concentration-dependent fashion. As increase in number of double bound, the inhibitory actions of USTFAs were weakened. Additionally, cis USTFA with methyl ester did not share the inhibitions. These FAs did not display significant antioxidant and superoxide anion-scavenging ability. Furthermore, these inhibitory effects were not mediated by the cAMP-dependent pathways. cis USTFAs inhibited store-operated calcium entry in human neutrophils in response to fMLP, calcium ionophore A23187, and endomembrane calcium-ATPase inhibitor thapsigargin. In summary, the inhibition of human neutrophil functions by cis USTFAs and trans linoelaidic acid in fMLP-activated human neutrophils can probably be attributed to the blockade of calcium mobilization via the modulation of store-operated calcium entry.
目錄
縮寫表………………………………………………………………....1
中文摘要.……………….……………………..................5
英文摘要……….……………………………………………….......7
序論……………………………………………………………………...9
表1………………………………………………………………………..23
實驗材料與方法………………………………………………………...25
PartⅠ :
雙萜類衍生物影響人類嗜中性白血球釋放超氧自由基和彈性蛋白酶的機轉探討……………………………………………................32
結果及討論……………………………………..............33
圖表…………………………………………………….……....39
PartⅡ :
C18 fatty acids 對嗜中性白血球功能之探討.…………......57
結果及討論………..…………………….....................58
圖表……………………………………………………..........….68
參考文獻……….......................................99
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