臺灣博碩士論文加值系統

ATM (Ataxia-Telangiectasia Mutated) 基因的突變與造成人類隱性遺傳疾病 A-T (Ataxia-Telangiectasia) 有關。ATM會參與 DNA破壞時的感應。A-T病患和 ATM缺失的老鼠會出現神經性疾病、胸腺細胞發育不全、較高的癌症罹患率和離子輻射敏感等現象。近期報告顯示ATM可抑制造血幹細胞的氧化壓力，維持其自我新生的能力。ATM還參與 T細胞發育階段 V(D)J基因重組時 DNA斷裂處的有效接合反應。但在成熟 T細胞中，ATM所扮演的角色目前並不清楚。報告顯示 T細胞在受到 PHA、EGF或 superantigen等物質的刺激時，其蛋白質含量及基因的表現上會有不同程度的變化。表示 ATM也可能在 T細胞的活化反應上扮演著某些調控功能。為了解 ATM在 T細胞活化過程中所可能扮演的角色，我們利用慢病毒 shRNA系統下調 Jurkat T細胞內生性 ATM，以探討此問題。

下調 Jurkat T細胞內生性 ATM，會顯著增加T細胞活化後所產生的 IL-2。進一步分析已知的幾條 T細胞活化訊息傳遞路徑，我們發現細胞活化早期 PKC-theta 的磷酸化會有部分增加。但其下游 NFkB活化後轉位到細胞核的程度並不明顯。對LAT、Akt、ERK及鈣離子相關訊息等都沒有顯著的影響。在 T細胞活化時，ATM蛋白質量與 Ser1981的磷酸化也都沒改變。歸結以上實驗得知，在 T細胞短暫活化過程中 PKC-theta 的磷酸化會部份受到ATM下調的影響，但其詳細的分子調控機制尚不清楚。此研究有助於我們更進一步了解除了 DNA斷裂的反應外，ATM也在 T細胞活化上可能扮演抑制性的角色。但對於 ATM在 T細胞活化的調控機制，還有待進一步的研究加以釐清。

The Ataxia-Telangiectasia Mutated (ATM) protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia (A-T), mediates responses to DNA damage in mammalian cells. A-T patients and Atm-deficient mice display neurologic dysfunction, defects in T lymphocyte maturation, cancer predisposition and radiation sensitivity. Recent study showed ATM protects bone marrow hemotapoietic stem cells (HSC) from oxidative stress and is required for sustained adult lymphopoiesis. Moreover, ATM routine surveillance of intermediates in V(D)J recombination, which is essential for TCR rearrangement. However, the role of ATM in mature T cells function is still unclear recently. Some reports showed that stimulation of T cells by using PHA, EGF or superantigen is able to alter ATM protein or gene expression level to some extent. Therefore, a general hypothesis emerges that ATM is involved in regulation of T cell activation. To test this hypothesis, we used lentiviral shRNA system to knockdown endogenous ATM in Jurkat T cells.

Downregulation of ATM in Jurkat T cell cause dramatically increase in IL-2 production after stimulated with either OKT-3 plus anti-CD28 or SEE superantigen. To further study the molecular mechanisms, we found a partial effect on PKC-theta activation at early time point. However, the nuclear translocation of NFkB is not affected. LAT, Akt, ERK and calcium-related signals show no significant difference. Moreover, there is no change either in ATM protein expression level or ATM Ser1981 phosphorylation during T cell activation. In summary, knockdown ATM partially activate PKC-theta upon short-term stimulation, but the detail mechanisms are still unclear. This study provides information other than respondses to DNA damage, ATM may play a suppresive role in T cell activation, but the regulatory mechanism need to be clarified extensively.

中文摘要............................................ i
英文摘要............................................ ii
目錄................................................ iv
第一章 簡介........................................ 1
1.1 Ataxia-Telangiectasia與Ataxia-Telangiectasia Mutated
簡介................................................. 1
1.1.1 Ataxia-Telangiectasia (A-T)........... 1
1.1.2 Ataxia-Telangiectasia Mutated (ATM)... 1
1.2 T細胞的發育 (T cell development)................. 4
1.3 ATM與T細胞的關係................................. 6
1.4 T細胞的活........................................ 8
1.5 研究方向與目的.................................. 10
第二章 材料與方法................................. 11
2.1 細胞株與培養.................................... 11
2.2 藥品配製與試劑.................................. 11
2.3 抗體............................................ 12
2.4小分子干擾核醣核酸下調 (shRNA knockdown)......... 12
2.4.1小分子干擾核醣核酸設計與質體構築...... 13
2.4.2 Lentivirus反轉錄病毒製備............. 14
2.4.3 Lentivirus反轉錄病毒效價測定......... 15
2.4.4 Lentivirus反轉錄病毒感染............. 16
2.5T細胞的活化...................................... 16
2.5.1固定式 anti-CD3/anti-CD28活化刺激..... 16
2.5.2SEE (Staphylococcal Enterotoxin E) 活化刺
激.................................... 17
2.6IL-2產量分析 (IL-2 assay)........................ 17
2.7流式細胞儀分析 (Flow cytometry analysis)細胞表面染
色............................................... 18
2.8細胞萃取液的製備................................. 18
2.8.1全細胞萃取液的製備.................... 18
2.8.2細胞核萃取液的製備.................... 19
2.9西方點墨法 (Western Blot)........................ 19
2.10鈣離子訊號的偵測 (Measurement of Ca2+ influx) .. 20
第三章 結果....................................... 22

3.1 ATM shRNA 成功下調JE6.1細胞內生性 ATM........... 22
3.2下調ATM使 T細胞活化 IL-2分泌量增加............... 23
3.3下調ATM對 T細胞活化訊息傳遞分子的影響............. 25
3.3.1 ATM下調不影響 LAT的活化.............. 25
3.3.2 ATM下調影響 PKC-theta活化早期的磷酸化 26
3.3.3 ATM下調不影響 NFκB細胞核轉位......... 26
3.3.4 ATM下調對 Akt活化的影響不明顯........ 27
3.3.5 ATM下調對 JNK、ERK MAPK活化的影響... 28
3.3.6 ATM下調對鈣離子訊息及下游分子 NFATc活化之分
析................................... 29
3.4 T細胞活化不影響 ATM表現......................... 30
3.5 ATM Ser1981磷酸化不參與T細胞的活化.............. 31
3.6 ATM的下調不影響T細胞表面 CD3分子的表現.......... 32
第四章 討論....................................... 33
圖表................................................ 38
參考文獻............................................ 59
附錄................................................ 69

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