臺灣博碩士論文加值系統

　　在胸腺發育的過程，有大部分的胸腺細胞其接受體 (T cell receptor, TCR) 未成功完成重組 (rearrangement) 或與自身MHC/peptide親合力太低，因而缺乏經由TCR所提供的保護訊息，則胸腺細胞會進行凋亡 (apoptosis) 而予以清除。經由體外培養 (in vitro) 的方式，胸腺細胞會發生自發性凋亡 (spontaneous thymocyte apoptosis, STA) 也許可以模擬胸腺細胞在體內發生的情況。從我們實驗結果得知不同品系的小鼠，包括BALB/c、C3H/HeN以及B57BL/6的胸腺細胞發生自發性凋亡是一個普遍的過程。鋰鹽 (lithium) 是用於治療躁鬱症的臨床藥物，在neurodegenerative的刺激下可以保護神經細胞，具有抗細胞凋亡的功能。本研究希望探討STA發生的分子機轉以及鋰鹽在STA模式下抗凋亡的角色。首先我們將胸腺細胞處理不同caspase的抑制劑，包括caspase-1、-2、-3、-8以及廣效性caspase抑制劑都可以阻斷STA，然而，STA的發生卻不會受到caspase-9抑制劑以及維持粒線體膜通透性穩定的藥物bongkrekic acid、cyclosporin A所抑制，此外，直至12小時才偵測到粒線體膜電位有降低的情形。從以上結果得知早期是需要caspase-2、-8以及-3的活化而且是經由mitochondria-independent的路徑導致。我們也觀察到STA的發生伴隨acid sphingomyelinase (ASM) 表現以及ceramide產生，另外，利用chlorpromazine hydrochloride抑制ASM也可以降低STA和caspase-3的活性。有趣的是，鋰鹽可以抑制STA和降低caspase活性，然而對於ASM的表現並沒有任何影響。另一方面，鋰鹽被視為是GSK-3�狶磻蹌砥A若使用GSK-3�狶磻蹌特B415286對STA同樣有抑制的作用，證明STA發生是GSK-3�狳抰鄔吽C進一步的實驗觀察到GSK-3�狾b鋰鹽作用下Ser-9磷酸化增加而降低活性。有趣的是，鋰鹽保護胸腺細胞凋亡在加入p38 MAPK抑制劑SB203580後被阻礙，但是加入MEK/ERK、PI3K/Akt以及JNK/SAPK抑制劑對於鋰鹽的保護作用並無影響。鋰鹽的處理可以增加p38 MAPK的磷酸化，進一步的研究顯示p38 MAPK抑制劑SB203580可以阻斷鋰鹽去調控GSK-3�畛C酸化的作用。根據這些結果我們推測鋰鹽可以經由調控p38 MAPK使GSK-3�猁漪〝坉飢C以及抑制caspase cascade的活化來防止STA發生。綜合本論文的研究結果，我們認為STA發生和ASM/ceramide的產生以及caspase活化的參與有關，而鋰鹽保護細胞免於凋亡的機制是透過活化p38 MAPK以及降低GSK-3�猁漪〝囧蚢F成。

　Large number of cells which have not successfully completed TCR (T cell receptor) rearrangement or have low affinity for self MHC/peptide are eliminated by apoptosis during thymus development. Spontaneous thymocyte apoptosis (STA) may, at least in part, mimic this process in vitro. Results showed that STA was a common process in different strains of mice, including BALB/c, C3H/HeN, and B57BL/6. Lithium, a common drug used for bipolar disorder, confers cell protection in response to neurodegenerative stimuli. An anti-apoptotic role of lithium has been suggested. In this study, the molecular mechanisms of STA and the anti-apoptotic effects of lithium were studied. Mouse thymocytes treated with caspase inhibitors, including caspase-1 (zYVAD-fmk), -2 (zVDVAD-fmk), -3 (zDEVD-fmk), -8 (zIETD-fmk), and pan caspase inhibitor zVAD-fmk, could prevent STA. However, STA was not inhibited by caspase-9 inhibitor zLEHD-fmk and mitochondrial transmembrane potential pore stabilizers, bongkrekic acid and cyclosporin A. In addition, the reduction of mitochondrial transmembrane potential could not be detected until 12 h. Our results suggested the dependence of caspase-2, -8, and -3 activation and the independence of mitochondrial pathway in early stage of STA. We had also observed acid sphingomyelinase (ASM) expression and ceramide generation in STA. Consistent with this result, inhibition of ASM by chlorpromazine hydrochloride could reduce STA and caspase-3 activation. Interestingly, lithium was able to inhibit STA and decrease caspase activity. Nevertheless, lithium could not reduce ASM production. To mimic the GSK-3�� inhibition of lithium, specific GSK-3�� inhibitor SB415286 was used. Results confirmed the dependence of GSK-3�� in STA. We observed GSK-3�� phosphorylation on Ser-9 and inactivation by lithium. Interestingly, results showed that only p38 MAPK inhibitor SB203580 could suppress lithium-conferred protection from STA, but MEK/ERK, PI3K/Akt, and JNK/SAPK inhibitors had no effect. Lithium treatment was able to phosphorylate p38 MAPK. Further study indicated that treatment with p38 MAPK inhibitor SB203580 blocked lithium-mediated effect of GSK-3�� phosphorylation. Based on these results, we suggest that lithium could regulate p38 MAPK-mediated GSK-3�� inactivation and caspase cascade activation to prevent the induction of STA. The findings in the present study may reveal the production of ASM/ceramide and the involvement of caspase activation in STA and that lithium protects cells from death is mediated by a mechanism of p38 MAPK activation and GSK-3�� inactivation.

中文摘要 I
英文摘要 III
致謝 V
目錄 VI
圖目錄 VIII
緒論 1
細胞粒腺體凋亡 1
Ceramide的凋亡角色 2
胸腺細胞自發性凋亡 (Spontaneous thymocyte apoptosis) 2
鋰鹽 (Lithium chloride, LiCl) 的抗凋亡角色 3
GSK-3�� (Glycogen synthase kinase-3��) 訊息調控 5
p38 MAPK訊息傳遞路徑 6
實驗材料方法
A 材料 9
A-1 實驗動物 9
A-2 胸腺細胞的培養 9
A-3 試劑藥品 9
A-4 抗體 11
A-5 耗材 11
A-6 儀器 12
B 方法 13
B-1 製備細胞培養液 13
B-2 老鼠胸腺細胞之分離 14
B-3 細胞濃度與存活率之測定 15
B-4 流體細胞分析儀 (Flow Cytometer) 偵測凋亡細胞 15
B-5 萃取細胞蛋白 17
B-6 定量蛋白質濃度 17
B-7 蛋白質電泳與西方墨點法 18
B-8 粒腺體膜電位測定 20
B-9 蛋白酶活性偵測 20
B-10 免疫螢光染色觀察ASM及ceramide表現 21
實驗結果
1.胸腺細胞發生自發性凋亡 (Spontaneous thymocyte apoptosis) 23
2.胸腺細胞凋亡過程引發caspase cascade的活化 23
3.胸腺細胞凋亡發生早期是經由mitochondria-independent路徑 24
4.胸腺細胞凋亡發生需要ASM及ceramide參與 24
5.鋰鹽 (Lithium) 抑制胸腺細胞自發性凋亡 25
6.鋰鹽增加GSK-3�� Ser-9的磷酸化而抑制其活性 26
7.p38 MAPK抑制劑SB203580干擾鋰鹽促進胸腺細胞存活的作用 26
8.鋰鹽增加p38 MAPK蛋白質磷酸化 27
9.鋰鹽增加GSK-3�� Ser-9磷酸化的作用可被SB203580抑制 27
討論
STA與caspase及粒線體的關係 28
STA與caspase-8的關聯性 29
STA的發生機制 30
鋰鹽抑制STA的分子機制 31
總結 33
參考文獻 34
圖附錄 42

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