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研究生:徐麗君
研究生(外文):Li-Jin Hsu
論文名稱:金黃色葡萄球菌腸毒素誘發之免疫抑制作用
論文名稱(外文):Immunosuppression Induced by Staphylococcal Enterotoxins
指導教授:林以行
指導教授(外文):Yee-Shin Lin
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:2002
畢業學年度:90
語文別:中文
論文頁數:157
中文關鍵詞:金黃色葡萄球菌腸毒素超抗原免疫抑制作用
外文關鍵詞:staphylococcal enterotoxinsuperantigenimmunosuppression
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施予細菌性超抗原會造成特定的T細胞活化,其後T細胞則會對後來的抗原刺激出現低反應的現象。利用細胞共同培養的實驗模式,我們發現注射過金黃色葡萄球菌B型腸毒素(staphylococcal enterotoxin B; SEB)之BALB/c與MRL+/+小鼠的脾臟細胞會抑制同品系naïve小鼠脾臟細胞受到SEB、SEA、或者是Con A刺激後的增殖反應,實驗結果也顯示免疫抑制活性是由注射過SEB之小鼠的脾臟CD4+、或者是CD8+ T細胞居間傳遞。使用Fas基因發生缺陷之MRL-lpr/lpr小鼠進行的實驗也可見到顯著的免疫抑制現象。當我們使用半通透膜將naïve responder cells和注射過SEB之BALB/c小鼠的脾臟細胞隔開培養時,抑制作用仍然相當地明顯。這些naïve responder cells受到抗原刺激後出現低反應的現象並不是因為大量細胞進行凋亡所致,而是細胞在進入細胞週期的S和G2/M phases受到了抑制。當naïve responder cells與注射過SEB之BALB/小鼠的脾臟細胞共同培養時,細胞培養上清液中IL-2的含量很低,然而外加IL-2只能部分地逆轉naïve responder cells增殖反應受到抑制的現象。在利用半通透膜將注射過SEB之BALB/c小鼠的脾臟細胞隔開培養的過程中,naïve responder cells細胞表面CD25與CTLA-4分子的表現情形與控制組相較之下都受到了抑制。將anti-CD28 mAb加入細胞混合共同培養的實驗中,可以使naïve responder cells增殖反應受到抑制的現象消失。此外,與SEB在BALB/c (H-2d haplotype)、MRL+/+以及MRL-lpr/lpr (H-2k)等I-E+品系小鼠體內誘發免疫抑制現象的實驗相互對照之下,注射過SEB之B6、B10、A.BY (H-2b)以及A.SW (H-2s)等I-E-品系小鼠的脾臟細胞無法抑制同品系naïve responder cells細胞表面CD25分子的表現與細胞增殖反應。進一步的實驗也發現,注射過SEB之BALB/c小鼠的脾臟細胞可以抑制來自BALB/c或B6小鼠之naïve responder cells細胞表面CD25分子的表現及細胞增殖反應,但注射過SEB之B6小鼠的脾臟細胞則無法抑制這兩種品系小鼠的responder cells,顯示effector cells具有決定性的角色。不同於SEB的實驗結果,SEA注射至BALB/c與B6小鼠體內以後,都可以誘發明顯的免疫抑制作用。另外,CD28基因缺失之BALB/c小鼠在注射過SEB以後,其脾臟細胞所具有的免疫抑制活性明顯減低。總結而言,SEB在小鼠體內誘發具有抑制活性的調控性細胞時,須要I-E分子和CD28 costimulation的參與,在利用半通透膜將調控性細胞與target cells隔開培養的實驗中,我們發現target cells細胞表面CD25分子的表現與細胞增殖反應均受到了抑制,這可能是SEB在動物體內誘發免疫低反應的機制之一。

Administration of bacterial superantigen results in clonal activation of T cells followed by a state of hyporesponsiveness to subsequent antigen stimulation. Using a coculture system, we showed that the splenocytes from staphylococcal enterotoxin B (SEB)-injected BALB/c and MRL+/+ mice suppressed the proliferative response of naive syngeneic splenocytes to SEB, SEA, or Con A stimulation, and the suppressive activity was mediated by either CD4+ or CD8+ T cells. The suppressive effect also occurred in Fas-deficient MRL-lpr/lpr mice. When naive responder cells were separated by a semipermeable membrane from SEB-primed effector cells, the suppressive effect remained apparent. The hyporesponsiveness of responder cells did not result from excessive induction of apoptosis, but rather, prevention of entering the S and G2/M phases of the cell cycle. The IL-2 levels in culture supernatants were low with the presence of SEB-primed effector cells. However, addition of IL-2 to the cocultures only partially reversed the inhibitory effect. Further studies revealed reduced levels of CD25 and CTLA-4 expression in responder cells when cultured in the transwells with the presence of SEB-primed effector cells as compared to that with saline-primed controls. The addition of anti-CD28 mAb could reverse the suppressive effect on naive cell proliferation in the coculture experiments. Furthermore, in contrast to the suppressive effect induced by SEB in BALB/c (H-2d haplotype), MRL+/+, and MRL-lpr/lpr (H-2k) mice, SEB-primed splenocytes from I-E- strains such as B6, B10, A.BY (H-2b), and A.SW (H-2s) mice failed to inhibit the CD25 expression and the proliferative activity of their syngeneic naive responder splenocytes. Additional studies showed that the SEB-primed cells from BALB/c, but not B6, mice inhibited the CD25 expression and proliferation of naive responder cells from either BALB/c or B6 mice, indicating the critical regulatory role of the effector cells. Unlike SEB, SEA induced profound suppression in both BALB/c and B6 mice. Moreover, the suppressive competence of SEB-primed splenocytes was diminished in CD28-deficient BALB/c mice. Taken together, when SEB is used as a stimulator in vivo, both I-E molecule and CD28 costimulation are required for the induction of the regulatory cells bearing suppressive activity. Using a transwell culture system, we show in this study an inhibition of CD25 expression and cell cycle arrest in target cells, which may serve at least in part the mechanisms of SEB-induced hyporesponsiveness.

中文摘要
英文摘要
表目錄
圖目錄
縮寫索引
背景
材料與方法
A、 實驗動物
B、 細胞株
C、 免疫抑制作用之誘導與小鼠脾臟細胞之分離
D、 Lymphocyte細胞亞群之分離(Fractionation of
Lymphocyte Subsets)
E、 以細胞混合培養的實驗模式偵測Responder
Cells之增殖反應
F、 細胞培養上清液中IL-2含量之測定
G、 Transwell實驗
H、 流體細胞螢光分析儀(Flow Cytometer)測定細
胞內DNA含量與表面抗原之表現
I、 Ex Vivo [3H]Thymidine Incorporation Assay
J、 反轉錄-聚合酵素連鎖反應(Reverse
Transcription-Polymerase Chain Reaction;
RT-PCR)
K、 儀器
結果
討論
參考文獻



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