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研究生:曹瓊文
研究生(外文):Tsao Chiung-Wen
論文名稱:多巴胺在免疫細胞中所扮演的角色
論文名稱(外文):Role of dopamine in the immune cells
指導教授:鄭瑞棠鄭瑞棠引用關係
指導教授(外文):Cheng Juei-Tang
學位類別:博士
校院名稱:國立成功大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:1997
畢業學年度:86
語文別:中文
論文頁數:132
中文關鍵詞:多巴胺神經毒素神經傳導物之合成酵素細胞增殖作用細胞凋亡
外文關鍵詞:dopamineproliferationcell apoptosistyrosine hydroxylaseneurotoxin(6-OHDA)
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本論文目的欲探究多巴胺 (dopamine)在神經與免疫之間的角色。首
先,以神經毒素使神經受損,了解它對免疫細胞所產生之直接作用。
並且,直接投予多巴胺及其刺激劑來探討對免疫細胞的增生反應所
造成的影響,以及確立免疫細胞含有交感神經傳導物之形成所使用
的合成酵素,進一步地,去瞭解神經和免疫系統之間的關係。
6-Hydroxydopamine (6-OHDA)是一種神經毒素,能使交感神經受
損,並造成免疫反應的改變。本實驗發現腹腔注射 6-OHDA會誘發
小鼠胸腺的萎縮,且重量和細胞數目至第三天和第五天減少至最低,
爾後,隨著天數逐漸回升。經流體免疫螢光法 (flow cytometry)分析
測知帶有表面抗原 CD4+CD8+的次胸腺細胞群之變化較
CD4+CD8-, CD4-CD8-和 CD4-CD8+來得明顯,且與重量或細胞數之
改變呈正相關。抽取胸腺細胞的 DNA,發現在給予小鼠 6-OHDA
之後,可由電泳分析看見 DNA呈現180~200 base pairs的片斷 (DNA
fragmentation)。為了證明當神經受損後,是否影響胸腺萎縮,進而使
細胞造成凋亡 (apoptosis)。因此,在投予 6-OHDA前,先以腹腔注
射一種交感神經回收抑制劑 (catecholamine uptake blocker,
desipramine),它能降低 6-OHDA進入細胞內。結果發現 6-OHDA
所導致胸腺重量和細胞數之減少,甚至所造成的細胞凋亡,皆可被
阻斷。由此可知,在影響胸腺細胞凋亡過程中,神經系統受損亦扮
演著相當重要的角色。接著,經由細胞培養的體外實驗得知, 6-
OHDA亦可直接造成細胞凋亡。 Desipramine可直接地抑制 6-OHDA
所造成的細胞凋亡。因此,不論在體內或是體外實驗, 6-OHDA皆
能造成小鼠胸腺細胞的凋亡。
進一步地,藉由胸腺細胞培養,探討 6-OHDA誘發細胞凋亡之
機制。流體免疫螢光法測得 CD4+CD8+次細胞群,如體內實驗一般,
對 6-OHDA最為敏感。經由 6-OHDA處理後, Bcl-2蛋白的表現亦
隨之減少。另外, VAD-FMK是一種廣效性的 interleukin-1 b
converting enzyme (ICE)類蛋白鋂 (ICE-family proteases)的抑制劑。
DEVD-CHO則可選擇性地阻斷ICE蛋白鋂 CPP32 (Caspase-3)。此二
者皆可阻礙 6-OHDA誘發胸腺細胞凋亡。然而,另一種對 ICE
(Caspase-1)本身較具有選擇性的蛋白鋂抑制劑 YVAD-CMK卻沒有
作用。同時,凋亡過程中,亦能被蛋白質合成抑制劑 (protein synthesis
inhibitor, cycloheximide)或是抗氧化劑 (N-acteylcysteine和 butylated
hydroxyanisole)所阻斷。因此, 6-OHDA誘發胸腺細胞進行凋亡,乃
因神經毒素進入細胞中,使 Bcl-2蛋白表現量降低,活化 ICE類蛋
白鋂(如 CPP32),需要合成新的蛋白,並產生自由基 (reactive oxygen
species, ROS)所致。
多巴胺是由交感神經傳導物速率限制鋂 (tyrosine hydroxylase,
TH)所合成的產物,並擔任交感神經傳導物質生合成的前驅物。同
時,它本身即是一種在中樞和週邊神經組織的神經傳導物。為了瞭
解多巴胺在免疫反應中扮演的角色。實驗結果顯示給予小鼠腹腔注
射 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)會降低體內組
織的多巴胺含量,並使 Lipopolysaccharide (LPS) 和 Concanavalin A
(Con A) 等 mitogens刺激脾臟細胞的增殖反應受到抑制。另一方
面,給予小鼠靜脈注射多巴胺受體刺激劑(第一型受體 SKF38393或
第二型受體 LY171555)皆可增強 LPS或 Con A對脾臟細胞的增殖
反應。同時,體外實驗也發現 SKF38393和 LY171555可直接增強
LPS或 Con A對細胞之增生。 LPS和 Con A分別可刺激免疫細胞 B
和 T細胞之生長。因此,不論是體內和體外實驗指出外給的多巴胺
和其受體刺激劑 (exogenous dopamine and its agonists) 能調節 B和
T細胞的增生反應。
此外,本實驗也證實內生性多巴胺 (endogenous dopamine) 與細
胞生長有關。 Haloperidol是一種常用的多巴胺受體拮抗劑。小鼠 T
細胞株 (T cell hybridoma, 10I) 和神經細胞 (rat pheochromocytoma
cells, PC12) 兩種細胞株,在經 haloperidol處理後,可降低細胞生長
速率。利用流體免疫螢光分析也指出各種免疫細胞含有 TH,如, T
細胞:小鼠胸腺細胞 (thymocytes),脾臟中的 T細胞 (splenic T
cells),以及 T細胞株(小鼠: 10I cells; 人類: Jurkat-T cells和
MOLT-4 cells); B細胞株 A20 cells;單核球/巨噬細胞株(小鼠:
P388D1 cells; 人類: U937 cells)。實驗中,以 PC12為控制組。
在細胞持續飢餓狀態 (serum starvation) 下, TH的表現會受到抑
制。細胞隨著時間不斷地生長中,不論是 10I或 PC12細胞株, TH
表現也隨之增加。同時,這變化會被 cycloheximide和 actinomycin D
所抑制。在培養的神經細胞株和免疫細胞株中,給予 TH酵素抑制
劑 (a-methyl-p-tyrosine和 MPTP)可降低 TH的含量,並呈現劑量相
關性。經由這些結果指出 TH的表現與細胞生長有正相關,而其所
製造的多巴胺可從免疫細胞釋出,並作用至受體,藉由 autocrine或
paracrine的方式,進一步調節免疫細胞的生長。
The present study is designed to characterize the role of dopamine in
neuroimmunomodulation of mice. The neurotoxin-induced
neurodegeneration was used to investigate the mechanism in lymphoid
tissue atrophy. In addition, effects of the dopamine and its agonists on
lymphocyte proliferation were assessed. The presence of tyrosine
hydroxylase in immune cells was identified. Correlation of tyrosine
hydroxylase expression with cell growth was also elucidated.
6-Hydroxydopamine (6-OHDA) was reported to induce the
degeneration of noradrenergic nerves and alter the immune responses.
In this study, intraperitoneal administration of 6-OHDA induces thymus
atrophy in mice. The lowest levels of thymus weight and cell number
were reached between day 3 to 5 in mice receiving 6-OHDA treatment;
they gradually recovered thereafter. On flow cytometry analysis, the
most substantial reductions were recorded for CD4+CD8+ thymocytes,
although the numbers of other subpopulations such as CD4+CD8-, CD4-
CD8+, CD4-CD8- cells were also reduced. DNA fragmentation, a
characteristic of apoptosis, was detected in the thymocytes following 6-
OHDA injection. Pretreatment with desipramine, a catecholamine
uptake inhibitor, greatly blocked the effects of 6-OHDA on reduction in
thymus size and thymocyte number, the changes in thymocyte
subpopulations, the percentage of apoptotic cells and the appearance of
DNA fragmented bands. These results indicate that 6-OHDA induces
mouse thymocytes to undergo apoptosis in vivo.
The mechanism of 6-OHDA-induced thymocyte apoptosis were
further investigated in vitro. Results showed that 6-OHDA-induced
thymocyte apoptosis could be detected in vitro and blocked by
desipramine treatment. Flow cytometry analysis indicated that
CD4+CD8+ cells were most susceptible to 6-OHDA, and Bcl-2 expression
was reduced after 6-OHDA treatment. Both VAD-FMK, a broad
spectrum interleukin-1b-converting enzyme (ICE)-family protease
inhibitor, and DEVD-CHO, a potent inhibitor of CPP32 (caspase-3),
blocked 6-OHDA-induced thymocyte apoptosis. However, the ICE
(caspase-1) inhibitor YVAD-CMK failed to cause such effect. This cell
death process was prevented by the treatment of protein synthesis
inhibitor, cycloheximide, and by the antioxidants such as N-
acetylcysteine or butylated hydroxyanisole. These results suggest that
down-regulation of Bcl-2, activation of ICE-like proteases such as CPP32
but not ICE, generation of reactive oxygen species, and synthesis of new
proteins are involved in the apoptotic mechanism of 6-OHDA in
thymocytes.
Dopamine is known as the precursor of catecholamine and one of the
neurotransmitters in brain and peripheral tissues. Recent studies
implicated the important role of dopamine in immune responses.
Intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) at dose sufficient to lower endogenous
dopamine suppressed splenocyte proliferation in response to mitogens
such as lipopolysaccharide (LPS) or concanavalin A (Con A). Moreover,
intravenous injection of the specific agonists for dopamine DA-1 receptor
(SKF38393) or DA-2 receptor (LY171555) into mice enhanced the
splenocyte proliferation stimulated by LPS or Con A. In the in vitro
cultures, dopamine, SKF38393 and LY171555 all promoted cell
proliferation in the presence of LPS or Con A. These results indicate
that dopamine has an ability to regulate B- and T-cell proliferation both in
vivo and in vitro.
The role of endogenous dopamine in immune cell growth was further
investigated. Haloperidol, a general antagonist of dopamine receptor,
reduced cell growth rate of T cell hybridoma (10I) and rat nervous
pheochromocytoma cells (PC12). Tyrosine hydroxylase (TH) is the
initial rate-limiting enzyme of catecholamine biosynthesis in nervous
system. Flow cytometric analysis indicated TH also expresses in
various immune cells including T-cell lineage (murine thymocytes, spleen
T cells, T cell hybridoma 10I, human Jurkat-T and MOLT-4 cells), B-cell
lineage (A20 cells), and monocyte/macrophage lineage (P388D1 and
U937 cells). The presence of TH in PC12 cells was used as control.
TH expression was down-regulated in low serum condition in which cell
growth rate was reduced. Temporal studies indicated that the expression
of TH increased during 10I cell growth. Both a-methyl-p-tyrosine and
MPTP reduced TH expression and cell growth in a dose-dependent
manner. These results suggest that immune T cells expresses TH which
is correlated to cell growth, and that dopamine released from immune
cells may bind to the receptors to act in an autocrine or paracrine way.
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中文摘要
英文摘要
縮寫指引
表目錄
圖目錄
第一篇 緒論
第二篇 材料和方法
第三篇 神經毒素 6-hydroxydopamine誘發小鼠胸腺細胞凋亡之機制
緒言
結果
第一章 神經毒素 6-hydroxydopamine造成小鼠胸腺細胞凋亡
第二章 神經毒素 6-hydroxydopamine在誘發經培養的胸腺細胞進行細胞凋亡的過程中,會使Bcl-2蛋白的表現減少,活ICE蛋白□以及涉及了自由基的產生
討論
第四篇 免疫細胞中tyrosine hydroxylase和其產物多巴胺與細胞生長有關
諸言
結果
第三章 多巴胺對小鼠免疫細胞增殖之作用
第四章 免疫細胞中tyrosine hydroxylase的表現與細胞生長有關
討論
第五篇 結論
第六篇 參考文獻
表附錄
圖附錄
自述
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