臺灣博碩士論文加值系統

槲寄生Viscum coloratum是一種半寄生植物，用於中草藥可治療發炎疾病。研究顯示，PPE-SVC (槲寄生的部份純化萃取物)與其純化成分viscolin可抑制FMLP刺激嗜中性球之超氧化陰離子的產生和彈性蛋白酶的活化。此抑制作用被證實是經由抑制PDE4的活性進而增加細胞內cAMP所致。PDE4為分解cAMP的酵素，其家族包含四個基因，分別為PDE4A、PDE4B、PDE4C和PDE4D，專一性PDE4抑制劑如rolipram可抑制這些酵素的活性。已知PDE4抑制劑可降低多種免疫細胞的發炎反應，故本研究欲探討viscolin以及其結構相似的合成分子RX78與RX116是否如rolipram一樣也可抑制多種免疫細胞的發炎反應。結果顯示，LPS刺激Raw 264.7巨噬細胞株及小鼠腹腔巨噬細胞可釋放大量TNF-?，而viscolin、RX78和RX116均可有效抑制TNF-?的釋放，唯其抑制程度不如rolipram。進一步利用野生型與PDE4剔除小鼠樹突細胞進行實驗，結果顯示viscolin等三種藥劑可降低LPS誘導TNF-?的釋放，其中以viscolin的抑制效果較佳，RX116次之，但其抑制能力遠不及rolipram，同時我們也發現三種PDE4亞型皆參與樹突細胞LPS/TNF-?的反應，以PDE4B與PDE4D為主要調控者。在小鼠脾臟T細胞實驗中，結果顯示單獨以CD3抗體或CD3與CD28抗體共同刺激細胞都可使IL-2釋放，但CD3/CD28抗體刺激IL-2的釋放量比單獨CD3抗體刺激增加約20倍，此等增加作用可被三種藥劑所抑制，且抑制程度與rolipram相當。此外結果也顯示，PDE4D會直接影響TCR的訊息傳遞，而PDE4B可參與CD28輔助TCR的作用。利用PDE4KO小鼠進行PDE酵素活性檢測顯示，小鼠大腦皮質中PDE4活性佔總PDE活性約50%，其中以PDE4B與PDE4D為主。同時以重組PDE4A與PDE4D進行酵素活性抑制分析，結果顯示viscolin、RX78與RX116等藥劑對PDE4抑制的能力分別為51%、31%與45%。綜合結果證實三種藥劑均可抑制PDE4的活性，雖然其抑制能力不及rolipram，然而皆可有效抑制免疫細胞如T細胞的發炎反應。

Viscum coloratum is a semiparasitic plant, used in traditional Chinese medicine to treat inflammatory diseases. It has been reported that its active component viscolin can inhibit FMLP-activated superoxide anion production and elastase release in human neurtophils. This inhibition is due to a decrease in the activity of type 4 phosphodiesterase (PDE4) activity, and thereby increasing in intracellular cAMP levels. The family of this cAMP-hydrolyzing enzymes consists of four genes named PDE4A, PDE4B, PDE4C and PDE4D, and inhibition of PDE4 is known to produce a wide range of anti-inflammatory response. Thus, the goal of this study is to examine whether viscolin and its structurally similar compounds RX78 and RX116 are similar to rolipram in attenuating inflammatory responses. The results showed that in Raw 264.7 cells and mouse peritoneal macrophages viscolin, RX78 and RX116 significantly inhibit LPS-induced TNF-??release, although the levels of inhibition are not as compelling as that of rolipram. Experiments with bone marrow-derived dendritic cells of wild-type and PDE4KO mice also showed that three compounds exhibited inhibitory effects on LPS-induced TNF-? release. However, the potency was much less than that of rolipram. In addition, three PDE4 isoforms were found to be involved in the LPS/TNF-? response in dendritic cells, with PDE4B and PDE4D the predominant regulators. Using mouse spleen T cells, we observed that IL-2 release in response to CD3 antibody or CD3/CD28 antibody co-stimulation was significantly increased, and the increase with CD3/CD28 antibody co-stimulation was ～20 folds over that with CD3 antibody alone. Such increases could be suppressed by the three compounds to the extents similar to the effect of rolipram. Moreover, the results from PDE4KO T cells indicated that PDE4D is involved in the TCR signaling, while PDE4B contributes to the CD28 co-stimulatory effects. Measurements of cAMP-hydrolyzing activity with recombinant PDE4A and PDE4D revealed that the potency of viscolin, RX78, and RX116 in inhibiting PDE4 activity is 51%, 31% and 45%, respectively. Take togather, these results indicate that the three compounds possess the PDE4 inhibition activity, which is thought to be associated with their anti-inflammatory activites in different cells, in particular of T cells.

中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
圖目錄 vii
表目錄 viii
縮寫檢索表 ix
一 緒論 1
1-1 cAMP之訊息傳導 1
1-2 cAMP調節免疫反應之功能 1
1-3 環狀核苷酸磷酸二酯酶(Cyclic Nucleotide Phosphodiesterase；PDE) 2
1-3-1 PDE之結構 2
1-3-2 PDE4與其活性調節 3
1-3-3 cAMP/PDE4與免疫細胞之關係 3
1-3-3-1 PDE4/cAMP/單核球與巨噬細胞 3
1-3-3-2 PDE4/cAMP/樹突細胞 4
1-3-3-3 PDE4/cAMP/T細胞 5
1-3-4 PDE4抑制劑作用與副作用 6
1-3-5 Rolipram抑制PDE4分子之機制 6
1-4 槲寄生/Viscolin/RX78/RX116 7
二 研究動機與目的 9
三 材料與方法 10
3-1 材料 10
3-1-1 實驗小鼠 10
3-1-2 實驗細胞株 10
3-1-3 實驗藥材 10
3-2 方法 11
3-2-1 巨噬細胞之培養及處理方法 11
3-2-1-1 Raw 264.7細胞株的培養 11
3-2-1-2 Raw 264.7細胞株的處理 11
3-2-1-3 收取小鼠腹腔巨噬細胞 11
3-2-1-3-1 備製B cell panning培養皿 11
3-2-1-3-2 小鼠腹腔巨噬細胞之收取 12
3-2-1-3-3 小鼠腹腔巨噬細胞之處理 12
3-2-2 小鼠骨髓幹細胞活體外分化及處理 12
3-2-2-1 小鼠股骨與脛骨之取得 12
3-2-2-2 小鼠腿骨骨髓細胞之取得 13
3-2-2-3 骨髓幹細胞分化成樹突細胞 13
3-2-2-4 樹突細胞之藥物處理 14
3-2-3 PDE4A、PDE4B和PDE4D轉染(transfection)至HEK-293細胞 14
3-2-3-1 HEK-293細胞之培養 14
3-2-3-2 氯化鈣轉染作用(CaCl2 transfection) 14
3-2-3-3 轉染細胞粗萃取液(cell crude extracts)之備製 15
3-2-4 小鼠大腦皮質細胞粗萃取液(cell crude extracts)之備製 15
3-2-5 蛋白質定量 15
3-2-6 PDE酵素活性檢測 16
3-2-7 小鼠脾臟T細胞之培養及處理 16
3-2-7-1 備製anti-CD3於96孔盤 16
3-2-7-2 小鼠脾臟T 細胞準備 17
3-2-7-3 脾臟T細胞之藥物處理 17
3-2-8 ELISA 17
四 實驗結果 19
4-1 LPS刺激巨噬細胞釋放TNF-?之影響 19
4-1-1 Viscolin、RX78和RX116對小鼠腹腔巨噬細胞釋放TNF-?之影響 19
4-1-2 Viscolin、RX78和RX116對Raw 264.7細胞株釋放TNF-?之影響 20
4-2 Viscolin、RX78和RX116對LPS刺激樹突細胞釋放TNF-?之影響 20
4-3 Viscolin、RX78和RX116對anti-CD3及anti-CD3/CD28刺激小鼠
T細胞釋放IL-2之影響 22
4-4 Viscolin、RX78和RX116抑制PDE4活性之能力 24
4-5 Viscolin、RX78和RX116對小鼠大腦皮質之PDE活性抑制作用 25
五 討論 27
5-1 Viscolin、RX78和RX116對不同細胞之抗發炎作用 27
5-1-1 Raw 264.7巨噬細胞株與小鼠腹腔巨噬細胞 27
5-1-2 小鼠樹突細胞 27
5-1-3 小鼠T細胞 28
5-2 Viscolin、RX78和RX11對PDE4亞型之抑制能力 29
六 圖與圖解 31
七 表 42
參考文獻 44
附錄 52

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