臺灣博碩士論文加值系統

中 文 摘 要
鳳梨酵素是從鳳梨之莖幹部分離之粗萃取物，具有多種藥理效能，如抗血液栓塞、抗水腫、抗發炎及抗癌等效能，但其抗發炎途徑尚未被人們所確定。因此本實驗之目的，在利用人類周邊血液單核細胞及人類血癌細胞株THP-1細胞來研究鳳梨酵素藉由抑制何種路徑，來抑制發炎反應。本實驗以不同濃度之鳳梨酵素(1、10、30、50、70、100 μg/ml)處理經內毒素刺激之人類周邊血液單核細胞及THP-1細胞，首先分析鳳梨酵素及內毒素對細胞毒性之影響。然後以免疫酵素分析法(ELISA)分析這些細胞經內毒素刺激所產生發炎因子TNF-α、IL-1β及IL-6之濃度，並進一步以反轉錄聚合酶連鎖反應(RT-PCR)偵測這些細胞的介白質基因mRNA之變化。結果發現鳳梨酵素在50-100 μg/ml之濃度下，有顯著抑制發炎因子之產生。由於此作用可能經酵素作用產生，因此利用重組蛋白研究並證實，鳳梨酵素亦可水解發炎因子TNF-α及IL-1β兩種基因重組製成蛋白質。此外，經ELISA分析細胞培養上清液，發現鳳梨酵素可以有效地降低THP-1細胞所產生的前列腺素(PGE2)，以RT-PCR分析其上游基因COX-2，發現鳳梨酵素亦可抑制此基因表現。再以西方點墨法(Western blot)分析其發炎信號傳導途徑，在LPS與鳳梨酵素作用30秒後，發現鳳梨酵素在50-100 μg/ml之濃度下，顯著的抑制MAPK (p38、ERK1/2及JNK1/2)的磷酸化。經ELISA分析，鳳梨酵素亦可以降低轉錄因子nuclear-factor kappa B (NF-κB)在細胞核內之含量。最後在抑制鳳梨酵素其酵素活性後，再以西方點墨法分析MAPK磷酸化，發現其抑制效果大幅降低，只可部分的抑制ERK-2及JNK1/2的磷酸化。綜合上述結果，當巨噬細胞受刺激而活化時，鳳梨酵素能抑制發炎因子TNF-α、IL-1β、IL-6及PGE2之產生，其作用機轉可能是經由抑制MAPK的磷酸化及NF-κB的活化。而其酵素活性為抑制發炎反應的一重要因子。

Abstract
Bromelain is a crude extract from the pineapple stem. Bromelain has a lot of physiological effects, such as anti-thrombotic, anti-edematous, anti-inflammatory, and anti-cancer activities. It has been shown that oral therapy with proteolytic enzymes including bromelain produces certain analgesic and anti-inflammatory effect in certain patients with rheumatic diseases. However, the molecular mechanism of bromelain on anti-inflammatory response is not clear. Therefore, this study was aimed to dissect this anti-inflammatory mechanism. Peripheral blood mononuclear cells (PBMC) from healthy donors and THP-1 cells were used for this study. Cells were cultured with lipopolysaccharides (LPS) (1μg/ml) and or different concentration of bromelain (1-100μg/ml) for 24hr, and found that bromelain was not toxic to cell by MTT assay. The cultured supernatants were collected and assayed for TNF-α, IL-1β, and IL-6 by ELISA methods, the cytokine gene expression from the stimulated cells was also analyzed by RT-PCR. The results showed that the bromelain significantly inhibited pro-inflammatory factors released from the LPS-stimulated PBMC and THP-1 cells. Since this effect could be due to the enzymatic activity of bromelain, we then test this possibility and found that bromelain could digest the recombinant TNF-α, and IL-1β protein structure by its enzymatic activity. Since PGE2 is an important inflammatory mediator, we also found that bromelain significantly reduced PGE2 and COX-2 gene expression from THP-1 cells. Because these factors were induced by MAP kinase signaling pathway, we found that bromelain significantly inhibited MAPK (p38, ERK1/2, and JNK1/2) phosphorylation after 30 sec treatment with LPS. Moreover, bromelain reduced the nuclear-factor kappa B (NF-κB) activation from the LPS-activated cells. Finally we found bromelain could slightly but significantly inhibited ERK-2 and JNK1/2 phosphorylation without its enzymatic activity. Taken together, we concluded that bromelain inhibited inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, might be through the inhibition of the MAPK and NF-κB cascades and the enzymatic activity of bromelain was an important factor to inhibit inflammatory reaction.

目 錄
頁 次
目錄 I
圖目錄 VI
表目錄 VIII
中英及縮寫目對照表 IX
中文摘要 XII
英文摘要 XIII





頁次
第一章 前言 1
一 、 發炎反應之概述 1
(一) 急性發炎反應 1
(二) 慢性發炎反應 2
二 、 發炎反應的調控 5
(一) MAPKs (mitogen-activated protein kinase)路徑 5
(二) 環狀氧化酶 (cyclooxygenase) 8
(三) NF-kB 路徑 11
三 、 鳳梨酵素之簡介 14
四 、 鳳梨酵素之藥理作用 15
(一) 抗血小板凝集 15
(二) 血纖維蛋白溶解作用 15
(三) 改善心血管及循環系統 15
(四) 抗發炎作用 16
(五) 調節細胞激素及免疫 16
(六) 抗腫瘤作用 17
(七) 增進傷口癒合 17
五 、 研究目的 18
第二章 材料與方法 19
一、常用藥品與試劑配製 19
(一) 化學藥品 19
(二) 試劑配製 19
二、人類周邊血液單核細胞之製備 21
三、細胞培養及分化 22
(一) 細胞培養液的配製 22
(二) 完全培養液 22
(三) 細胞株的培養 21
(四) 巨噬細胞分化 23
四、淋巴球及THP-1細胞存活率之測定 23
五、免疫酵素法測定細胞激素TNF-α、IL-1β與IL-6之濃度 25
六、反轉錄聚合酶鏈鎖反應半定量細胞激素mRNA之表現 25
(一) RNA濃度之測定 26
七、反轉錄聚合酶鏈鎖反應 26
(一) 反轉錄聚合酶反應 26
(二) 引子之製備 27
(三) 聚合酶鏈鎖反應 27
八、細胞內蛋白質萃取及西方點墨法 27
(一) 蛋白質的萃取 27
(二) 聚丙烯醯胺膠體電泳法 28
(三) 西方轉漬法 28
九、PGE2測定 29
十、TXB2測定 29
十一、NF-κB活性測試 30
十二、鳳梨酵素酵素活性分析 30
十三、統計分析 31
第三章 結果 33
一 、 不同濃度之鳳梨酵素及LPS對人類周邊血液單核細胞(PBMC)及THP-1細胞存活率之影響 33
二 、 由ELISA方式測定不同濃度之鳳梨酵素對經由LPS所刺激之人類周邊血液單核細胞及THP-1細胞細胞激素分泌之影響 33
三 、 利用反轉錄聚合酶連鎖反應測定不同濃度之鳳梨酵素對經由LPS所刺激之THP-1細胞之TNF-α及IL-1β mRNA表現之影響 34
四 、 利用考馬式亮藍(coomassie blue)染色法測定不同濃度之鳳梨酵素對TNF-α及IL-1β之影響 34
五 、 用ELISA測定不同濃度之鳳梨酵素對由LPS所刺激之THP-1細胞之PGE2及TXA2分泌之影響 35
六 、 用反轉錄聚合酶連鎖反應測定不同濃度之鳳梨酵素對經由LPS所刺激之THP-1細胞之COX-1及COX-2表現之影響 35
七 、 利用西方點墨法檢視不同濃度之鳳梨酵素對經LPS所刺激之THP-1細胞 MAPK(mitogen-activated protein kinase)路徑之影響 36
八 、 用ELISA方式測定不同濃度之鳳梨酵素對經由LPS所刺激之THP-1細胞之NF-κB活化之影響 36
九 、 利用西方點墨法檢視失去酵素活性之不同濃度之鳳梨酵素對經LPS所刺激之THP-1細胞 MAPK路徑之影響 37
第四章 討論 51
一 、 鳳梨酵素與LPS對細胞存活率之影響 51
二 、 鳳梨酵素對經LPS刺激分泌之發炎細胞激素之影響 51
三 、 鳳梨酵素對PGE2與TXB2分泌及其基因之影響 53
四 、 鳳梨酵素對經LPS所誘發之MAPK路徑之關係 54
五 、 鳳梨酵素對經LPS所活化之NF-κB的影響 54
六 、 鳳梨酵素之酵素活性對其抑制經內毒素所引起之發炎反應之影響 55
第五章 結論 57
第六章 參考文獻 58












圖 目 錄

頁 次
Figure 1-1 Overview of the cells and mediators involved in local acute inflammatory response. 3
Figure 1-2 Overview of the organs and mediators involved in a systemic acute-phase response. 4
Figure 1-3 Redundant and pleiotropic effects of IL-1, TNF-α, and IL-6. 4
Figure 1-4 LPS stimulation of monocytes activates signaling pathways and transcription factors. 7
Figure 1-5 COX-1 and COX-2 are enzymes for PG biosynthesis. 9
Figure 1-6 Schematic model of the pathways and transcription factors involved in induction of the COX-2 promoter by LPS in macrophage. 10
Figure 1-7 Overview of the NF-κB and I-κB homology sequences. 12
Figure 1-8 A schematic representation of signaling cascades for LPS, IL and TNF-α stimulation and activation of NF-κB (p50/65). 13
Figure 3-1 Effect of LPS (1 μg/ml) and bromelain (1-100 μg/ml) on cell viability of human PBMC and THP-1 cells. 38
Figure 3-2 Effect of bromelain (1-100 μg/ml) on cytokines TNF-α, IL-1β, and IL-6 of human PBMC induced by LPS (1μg/ml). 39
Figure 3-3 Effect of bromelain (1-100 μg/ml) on cytokine TNF-α, and IL-1β of THP-1 cells induced by LPS (1 μg/ml). 40
Figure 3-4 Effect of bromelain (1-100 μg/ml) on TNF-α and IL-1β mRNA expression of THP-1 cells induced by LPS (1μg/ml) 41

Figure 3-5 Recombinant protein of TNF-α and IL-1β were treated with bromelain (1-100 μg/ml) for a certain period of time (0.5-16 min). TNF-α and IL-1β were digested by the enzymatic activity of bromelain. 42
Figure 3-6 Effect of bromelain (1-100 μg/ml) on PGE2 expression of THP-1 cells induced by LPS (1 μg/ml) 43
Figure 3-7 Effect of bromelain (1-100 μg/ml) on (TXB2) expression of THP-1 cells induced by LPS (1 μg/ml) 44
Figure 3-8 Effect of bromelain (1-100 μg/ml) on COX-1 and COX-2 mRNA expression of THP-1 cells induced by LPS (1μg/ml) 45
Figure 3-9 Effect of bromelain (70 μg/ml) on p38, ERK1/2, and JNK1/2 phosphorylation of THP-1 cells induced by LPS (1 μg/ml) for a certain period of time. 46-47
Figure 3-10 Effect of bromelian (1-100 μg/ml) on p38, ERK1/2, and JNK1/2 phosphorylation of THP-1 cells induced by LPS (1 μg/ml) 48
Figure 3-11 Effect of bromelain (70 μg/ml) on NF-κB activation of THP-1 cells induced by LPS (1 μg/ml) 49
Figure 3-12 Effect of non-enzymatic activity bromelian (10-100 μg/ml) on p38, ERK1/2, and JNK1/2 phosphorylation of THP-1 cells induced by LPS (1 μg/ml) 50












表 目 錄

頁 次
Table 2-1 The sequence of oligonucleotide primers and the condition of PCR. 32



























中 英 對 照 表
英文縮寫目 英文全文 中文翻譯
A.A. arachidonic acid 花生四烯酸
COX cyclooxygenase 環狀氧化酶
FBS Fetal Bovine Serum 胎牛血清
IFN-γ Interferon-γ 干擾素-γ
IL-1β Interleukin-1 β 介白質1-β
IL-6 Interleukin-6 介白質-6
iNOS induced NO-synthase 誘發性NO合成酶
LPS Lipololysaccharide 內毒素
LTB4 leukotriene B4 白三烯素 B4
PBMC Peripheral blood mononuclear cells 人類周邊血液單核細胞
PGE prostaglandin 前列腺素
PLA2 phospholipase A2 磷酸酯酶 A2
TNF-α Tumor necrosis factor-α 腫瘤壞死因子-α
TXA thromboxane 凝血酶
VCAM-1 Vascular adhesion molecular 1 血管附著因子-1
VEGF vascular endothelial cells growth factor 血管內皮細胞生長因子


ATF-2 Activation transcription factor 2
Bcl-3 B-cells leukemia-3
C/EBPβ CCAAT enhancer-binding protein β
CHOP C/EBP homologous protein
CRE/E-box cAMP responsive element overlapping a non-canonical E-box
DLK Localization of the mixed-lineage kinase
Elk-1 ets related protein-1
ERK Extra-cellular signal-regulated kinase
GCK germinal center kinase
hPAK1 human p21-activated protein kinase-1
ICAM-1 Inter-cellular adhesion molecular 1
I-κB Inhibitory-kappa B
JNK c-jun N-terminal kinase
MAPK Mitogen-activated protein kinase
MEF2C myocyte-enhancer factor 2C
MEKK1/4 MAPK kinase kinase-1/4
MK2 Map kinases activated kinases-2
MKK3 mitogen-activated protein kinase kinase 3
MKK4 mitogen-activated protein kinase kinase 4
MKK6 mitogen-activated protein kinase kinase 6
MMP-9 matrix metalloprotenase-9
MNK1/2 Map kinases-interacting kinases 1 and 2
mPAK3 mouse p21-activated protein kinase-3
MSK1 mitogen and stress activated protein kinase-1
MUK MAPK-upstream protein kinase
NF-κB Nuclear factor-kappa B
NLS Nuclear-localization sequences
PRAK p38-regulated and -activated kinase
RHD Rel homology domain
Sap1a stress-activated protein-1a
SAPK stress-activation protein kinase
SRE serum response element
SRF serum response factor
ZPK zipper protein kinase

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