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研究生:林秋烽
研究生(外文):Chiou-Feng Lin
論文名稱:金黃色葡萄球菌B型腸毒素導致小鼠胸腺細胞凋亡的過程中黏著分子所扮演之角色
論文名稱(外文):Thymocyte Apoptosis Induced by Staphylococcal Enterotoxin B in Mice: Role of Adhesion Molecules
指導教授:林 以 行
指導教授(外文):Yee-Shin Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:82
中文關鍵詞:金黃色葡萄球菌B型腸毒素超抗原細胞凋亡細胞激素黏著分子淋巴球功能抗原-1細胞間黏著分子-1
外文關鍵詞:Staphylococcal enterotoxin BSuperantigenApoptosisCytokineAdhesion moleculeLeukocyte function-associated antigen-1Intercellular adhesion molecule-1
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中文摘要
金黃色葡萄球菌B型腸毒素 (簡稱SEB) 是一種細菌性超抗原,可以刺
激具有特定TCR V*7、8.1-3 的T細胞群並且造成細胞增生、免疫低反應或
者細胞凋亡等現象。根據本實驗室先前的研究顯示,靜脈注射給予小鼠
SEB會導致BALB/c (I-E+) 小鼠的胸腺細胞進行凋亡,但在B6 (I-E-) 小
鼠則無此影響。如果預先給予BALB/c小鼠anti-MHC class II單株抗體,
發現anti-I-Ed能抑制SEB引發之胸腺細胞凋亡,而anti-I-Ad則較無影響。
利用I-E+及I-E-小鼠對SEB的反應性之不同,我們想研究有哪些因素參與
SEB引發小鼠胸腺細胞凋亡的過程中,並藉以對細胞死亡機制做進一步的
探討。我們有興趣探討的因素包括在胸腺產生的細胞激素及細胞與細胞間
的交互作用。本實驗室先前的結果顯示SEB引發胸腺細胞凋亡的過程中與
IL-2R 及其他細胞激素受體的表現及其所傳遞的訊息有關。至於細胞與細
胞間的交互作用包括胸腺細胞本身或與thymic stroma cells間的作用,
在這其中,黏著分子扮演的角色是我們所感到興趣的。淋巴球功能抗原-1
(LFA-1) 與其相對受體間的交互作用已知會參與T細胞的發育過程及免疫
反應中。然而,其所扮演的真正角色卻不是十分清楚。於本實驗中,我們
研究LFA-1和細胞間黏著分子-1 (ICAM-1) 的交互作用在SEB引發小鼠胸腺
細胞凋亡過程中所扮演的角色。利用細胞流體分析儀偵測細胞表面黏著分
子的表現,實驗結果顯示,在BALB/c及B6兩種小鼠胸腺細胞的表面皆表現
有高量的LFA-1,且其表現量不會受到SEB的刺激所影響。另一方面,
ICAM-1在正常BALB/c小鼠胸腺細胞有低量表現而受到SEB刺激後6到24小時
之間表現量增加,這樣的結果在B6小鼠並沒有看到。以免疫組織化學染色
法觀察胸腺組織ICAM-1的表現,BALB/c小鼠在SEB刺激後胸腺組織ICAM-1
表現量增加的時間比B6小鼠可以更早出現,約在早期2小時即可看到。另
外,由此染色的結果亦顯示出ICAM-1的表現主要是在非胸腺細胞。綜合以
上的結果,ICAM-1的表現會受到SEB刺激的影響。為了進一步探討在SEB刺
激所引發胸腺細胞凋亡的過程中LFA-1和ICAM-1之間的交互作用所扮演的
角色,我們給予BALB/c小鼠anti-ICAM-1或anti-LFA-1單株抗體來觀察。
有趣的是,當給予BALB/c小鼠anti-ICAM-1或anti-LFA-1單株抗體後,SEB
所引發胸腺細胞凋亡的程度都明顯地受到抑制,這樣的結果顯示出LFA-1
和ICAM-1的交互作用參與了胸腺細胞凋亡的過程。另一方面,實驗結果顯
示在SEB引發小鼠胸腺細胞凋亡的過程中,伴隨有細胞激素IL-2 mRNA以及
細胞激素受體CD25 (IL-2Ra) 的表現量增加。當給予BALB/c小鼠anti-LFA-1
單株抗體後,SEB所引發IL-2 mRNA以及IL-2R的表現量都明顯地受到抑制,
這樣的結果在給予anti-ICAM-1的實驗組較不明顯,顯示出LFA-1和ICAM-1
可能經由不同的機制調控胸腺細胞凋亡。根據這些的結果顯示,經由SEB
的刺激造成ICAM-1分子的表現量增加,進而藉由ICAM-1和LFA-1交互作用
以及IL-2和IL-2R的共同影響可能參與了胸腺細胞凋亡的過程。

英文摘要
Staphylococcal enterotoxin B (SEB), a bacterial superantigen,
stimulates specific T cells bearing T cell receptor (TCR) Vb7,
8.1-3 domains and causes cell proliferation, anergy, or
apoptosis. Previous studies in our laboratory showed that in
vivo administration of SEB induced thymocyte apoptosis in BALB/c
(I-E+) but not in B6 (I-E-) mice. Further experiments by
treatment with anti-MHC class II monoclonal antibodies (mAb) to
BALB/c mice indicated that anti-I-Ed pretreatment inhibited SEB-
induced thymocyte apoptosis whereas anti-I-Ad exerted less
effect. Taking advantage of the differential effects of SEB in
I-E+ and I-E- mice, one may investigate the factors which are
involved in thymocyte apoptosis and further the mechanism of
apoptosis. The parameters of interest for the studies include
cytokines produced in the thymus and cell-to-cell interaction.
Previous studies in our laboratory indicated that SEB-induced
thymocyte apoptosis is related to the expression and signal
transduction of IL-2R or other cytokine receptors. Cell-to-cell
interaction involves both the interaction among thymocytes
themselves and between thymocytes and thymic stromal cells, and
the role that adhesion molecules may play is of interest for
studies. The interactions between leukocyte function-associated
antigen-1 (LFA-1) and its ligands have been suggested to be
involved in T-cell development and immune response. However,
the precise role of these molecules is not clear. In this study,
we investigated in vivo the involvement of LFA-1 and
intercellular adhesion molecule-1 (ICAM-1) in thymocyte
apoptosis induced by SEB. By flow cytometric analysis, a high
level of LFA-1 expression was observed on thymocytes from both
BALB/c and B6 mice, and its expression remained unchanged
following SEB treatment. The expression of ICAM-1 on thymocytes
was low in naive mice and slightly increased between 6 and 24 h
after SEB stimulation in BALB/c but not in B6 mice. Detection
of ICAM-1 expression in thymic organs by immunohistochemistry
revealed an earlier onset (2 h) in BALB/c mice than that in B6
mice. The ICAM-1 molecules were predominantly expressed in
nonthymic cells. To further investigate the role of LFA-1
/ICAM-1 interaction in SEB-induced thymocyte apoptosis,
anti-ICAM-1 or anti-LFA-1 mAb were given to BALB/c mice before
SEB administration. Treatment with anti-ICAM-1 or anti-LFA-1
mAb both caused an inhibition on SEB-induced cell death,
suggesting the involvement of LFA-1/ICAM-1 interaction during
thymocyte apoptosis. During the process of SEB-induced
thymocyte apoptosis, there was an increased expression of IL-2
mRNA and CD25 (IL-2Ra). The upregulation of IL-2 mRNA and CD25
expression was diminished by treatment with anti-LFA-1 mAb.
However, anti-ICAM-1 mAb treatment caused less effect. These
results suggest the differential regulation caused by LFA-1 and
ICAM-1. In summary, ICAM-1 expression is upregulated after SEB
stimulation, and the interaction of ICAM-1/LFA-1 and IL-2/IL-2R
are involved in thymocyte apoptotic process.

目錄
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 IX
符號及縮寫 XI
壹、 緒論 1
貳、 材料與方法
I、材料
A、實驗動物 9
B、試劑 9
C、耗材 12
D、儀器 13
II、方法
A、小鼠體內抗原或抗體的注射 15
A-1 SEB的處理
A-2抗體的給予
A-2a. 抗小鼠MHC class II分子抗體的給予
A-2b. 抗小鼠LFA-1或ICAM-1抗體的給予
B、小鼠胸腺細胞數目的測定 16
C、DNA片段之純化及膠體電泳分析 16
C-1 DNA片段之純化
C-2 DNA片段之膠體電泳分析
D、免疫螢光法測定細胞表面特定分子的表現 18
D-1直接免疫螢光法
D-2間接免疫螢光法
E、免疫組織化學染色法測定ICAM-1的表現 19
E-1 玻片的前處理與組織切片的製備
E-2 組織切片的染色
F、免疫組織化學染色法觀察細胞死亡的情形 20
F-1 玻片的前處理與組織切片的製備
F-2 組織切片的染色與TUNEL反應
G、免疫螢光法定量細胞死亡的程度 21
G-1 細胞的前處理
G-2 細胞的染色與TUNEL反應
H、以反轉錄聚合酵素連鎖反應測定細胞內 22
IL-2 mRNA的表現
H-1. RNA之抽取
H-2. RNA純度的確定
H-3. cDNA的合成
H-4. PCR的進行
H-5. PCR產物的膠體電泳分析
參、 結果
I、I-E+及I-E-小鼠對SEB之不同反應 27
A、SEB對小鼠胸腺細胞片段化的影響
B、免疫組織化學染色法觀察SEB誘發小鼠胸腺
細胞死亡的情形
C、免疫螢光法定量SEB誘發小鼠胸腺細胞死亡
的程度
II、抗小鼠MHC class II分子抗體對於SEB誘發
小鼠胸腺細胞凋亡的影響 28
A、小鼠胸腺細胞數目的影響
B、小鼠胸腺細胞凋亡的影響
III、SEB影響小鼠胸腺細胞上 LFA-1的表現 30
IV、抗小鼠LFA-1抗體影響小鼠胸腺細胞上
LFA-1的表現 30
V、抗小鼠LFA-1抗體對於SEB誘發小鼠胸腺細胞
凋亡的影響 31
A、免疫組織化學染色法觀察小鼠胸腺細胞
死亡的情形
B、免疫螢光法定量小鼠胸腺細胞死亡的程度
VI、SEB影響小鼠胸腺ICAM-1的表現 32
A、免疫螢光法測定胸腺細胞上ICAM-1的表現
B、免疫組織化學染色法觀察胸腺組織ICAM-1
的表現
VII、抗小鼠ICAM-1抗體影響小鼠胸腺ICAM-1
在SEB作用下的表現 33
A、免疫螢光法測定胸腺細胞上ICAM-1的表現
B、免疫組織化學染色法觀察胸腺組織ICAM-1
的表現
VIII、抗小鼠ICAM-1抗體對於SEB誘發小鼠
胸腺細胞凋亡的影響 34
A、免疫組織化學染色法觀察小鼠胸腺細胞
死亡的情形
B、免疫螢光法定量小鼠胸腺細胞死亡的程度
IX、抗小鼠LFA-1或抗小鼠ICAM-1抗體影響小鼠
胸腺細胞在SEB作用下細胞激素及受體的表現 35
A、以反轉錄聚合酵素連鎖反應測定細胞內IL-2
mRNA的表現
B、免疫螢光法測定胸腺細胞上CD25的表現
肆、 討論 38
伍、 參考文獻 43
陸、 作者簡歷 52
柒、 授權書 53
捌、 圖表 54

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