Rap1會受到T細胞抗原受體 (TCR) 的活化，並在細胞中扮演調控黏著分子integrin的角色。然而Rap1在T細胞活化上所扮演的角色及影響黏著分子integrin時受到的調控機制，並無明確的結論。在本論文中，我們將分三個方面探討Rap1的角色。第一，探討CD28的刺激對於Rap1的活化。第二，利用表現抑制型Rap1N17及持續活化型Rap1V12的DO11.10細胞株，與建立Rap1V12轉殖基因小鼠來探討Rap1在T細胞活化時的影響。第三，了解p38 MAPK是否參與Rap1的訊息傳遞。
從我們的研究中發現，T細胞株EL4受到CD28的刺激會增加TCR對於Rap1的活化。而在表現Rap1V12的轉殖小鼠內，不影響胸腺T細胞與脾臟T細胞之發育(包含細胞數目、胸腺T細胞的正向選擇、CD4與CD8細胞之比例、TCR的表現)。而胸腺T細胞與脾臟T細胞受到活化後之細胞增生及IL-2的產量則較對照組高，顯示Rap1在T細胞內為正向調節分子。但在T 細胞株DO11.10內表現持續活化型Rap1V12及抑制型Rap1N17，其活化所產生的IL-2則未受到影響，也不影響DO11.10與抗原呈現細胞在有抗原存在下之結合比率。
在探討TCR活化Rap1的訊息傳遞上，我們發現T細胞株EL4以p38 MAPK特異抑制劑SB203580的預先處理後，會促進Rap1受到TCR刺激後的活性。但小鼠脾臟T細胞以SB203580的預先處理，增加了未活化T細胞內Rap1的活性，但經TCR及CD28刺激後則Rap1之活性並未明顯增加。因此，p38 MAPK調控Rap1的活化因不同細胞而異。
總結來說，我們的研究支持Rap1在正常細胞內扮演正向調節的角色。CD28的訊息會促進Rap1在T細胞內的活化。此外，p38 MAPK可能參與在TCR活化Rap1的訊息傳遞路徑中。我們同時也發現在不同的細胞中有相異的實驗結果，顯示調控Rap1的活化會因為在不同的細胞、細胞內的分布及活化量上的不同而有相當之複雜性。所以Rap1在T細胞活化及訊息傳導上所扮演的真正角色，需要更多研究來進一步釐清。Rap1 is activated by TCR signaling and is participated in integrin activation. The exact role of Rap1 in T cell activation and the mechanism on how Rap1 regulates integrin activation have not been precisely defined. In this study, we explored the role of Rap1 in three different aspects. First, we examined the effect of CD28 co-stimulation on Rap1 activation. Second, we elucidated the role of Rap1 in T cell activation by expressing the dominant negative Rap1N17 and constitutive active Rap1V12 in DO11.10 hybridoma and Rap1V12 in transgenic mice. Third, we examined the possible involvement of p38 MAPK in Rap1 activation.
Results from our study illustrated CD28 co-stimulation increased Rap1 activation both in EL4 T lymphoma and splenic T cell, supporting a positive role of CD28. The T cell-specific transgenic expression of Rap1V12 did not affect T cell development, including cell number, CD4/CD8 ratio, positive selection and TCR levels. T cell proliferation and IL-2 production were increased in Rap1V12-transgenic mice, suggesting a positive role of Rap1 in T cell activation. In contrast, expression of Rap1V12 and Rap1N17 in DO11.10 T hybridoma did not affect CD3 or antigen peptide induced IL-2 production nor was antigen peptide-mediated T cell-antigen presenting cell conjugation.
In the study of the TCR signals that activate Rap1, we found that pretreatment with p38 MAPK specific inhibitor SB203580 increased TCR-stimulated Rap1 activation in EL4 cell. Pretreatment of splenic T cell with SB203580 resulted in increase Rap1 basal level not TCR-stimulated Rap1 activation. Therefore, the involvement of p38 MAPK in Rap1 activation is cell-type dependent.
In summary, our results clearly support a positive role of Rap1 in the activation of normal T cells. CD28 co-stimulation promoted the Rap1 activation. In addition, p38 MAPK may couple TCR activation to Rap1 activation. However, we also found exceptional results in different types of T cells, suggesting a complicated regulation of Rap1 which may depend on cell type, distribution, and quantity of Rap1. Further studies are required to delineate the exact role of Rap1 in T cell activation and signal coupling.中文摘要 i
英文摘要 iii
縮寫對照表 viii
第一章 簡介
一、Rap1的發現 1
二、Rap1在細胞內之弁?
1.Rap1與Ras的交互作用 2
2.Rap1影響細胞型態與integrin的活化 4
3.Rap1與p38 MAPK的交互作用 6
研究目的 7
第二章 材料與方法
壹、細胞株與細胞培養
一、細胞株 8
二、小鼠胸腺與脾臟細胞 8
三、細胞培養 9
貳、藥品與試劑 9
參、抗體 9
肆、質體構築
一、pcDNA3-myc-Rap1 N17及pcDNA3-myc-Rap1 V12
質體 10
二、pGC-myc-Rap1 N17-YFP及pGC-myc-Rap1V12-YFP
質體 11
三、CD2-myc-Rap1 V12質體 11
伍、在DO11.10細胞表現Rap1突變株
一、以calcium phosphate轉染法將質體送入Phoenix細胞 11
二、以離心法感染DO11.10細胞 12
陸、基因轉殖鼠之建立
一、Genomic DNA測試 12
二、mRNA測試
1.以TRIzol試劑抽取細胞全RNA 13
2. RNA反轉錄反應 14
3. 反轉錄聚
