臺灣博碩士論文加值系統

中文摘要
乙胺丁醇 ( Ethambutol ) 是臨床上初期經常用來治療肺結核病
患的藥物，副作用之一是造成眼睛毒性。在視力電位圖分析發現到服
用乙胺丁醇的病人視網膜與視神經有異常的現象。本篇論文便是觀察
乙胺丁醇造成人類視網膜色素上皮細胞生長抑制的分子機轉。
首先，我們發現到乙胺丁醇在24-72 小時中，會隨著濃度的提高
造成細胞的生長抑制以及在細胞質形成空泡。實驗結果發現乙胺丁醇
也會造成細胞生長週期終止在G1 時期。利用西方墨點法也觀察到乙
胺丁醇會造成P-ERK 量的上升。但是當我們使用ERK 的抑制劑
PD98059 時，卻不能回復乙胺丁醇所造成的細胞生長抑制。
此外，實驗發現到PKC 的抑制劑 Bisindoyllmaleimides I 可以部
分回復經由乙胺丁醇所造成的細胞生長抑制、空泡形成以及細胞週期
G1 時期中止的現象。而在西方墨點法結果也顯示乙胺丁醇會造成
PKCa, bII 以及 e 從細胞質中轉移至細胞膜上，另外在PKC 活性分
析中證實PKC 確實有被乙胺丁醇活化。接下來我們利用PKC 活化劑
TPA 處理RPE 細胞，亦發現造成RPE 細胞生長抑制，但是無空泡形
成。
經由以上的結果，我們推想乙胺丁醇可以經由活化 PKC 訊息傳
導路徑來造成細胞生長抑制以及空泡的產生。

Abstract
Ethambutol, used as a first-line anti-Mycobacterial agent, may easily
induces ocular toxicity. Electrophysiological tests of the patients using
ethambutol implicate that certain retina cells as well as optic neuropathy
were affected by the drug. In this study, the effect of ethambutol on
human retinal pigmented epithelium (RPE), which is a master cell in the
retina for regulation of photosensitivity, was investigated.
Firstly, we discovered that EMB can induce cell growth inhibition as
well as vesicles formation in the cytosol in a time and dose dependent
manner at 24-72 hr. this result were consistent with G1 cell cycle arrest
induced by EMB. Western blot analysis showed EMB induced elevation
of p-ERK, the active form of ERK. However, PD98059, the inhibitor of
ERK, do not prevent EMB-induced cellular toxicity of RPE. Furtherly,
we found that Bisindoyllmaleimides I, which is a PKC inhibitor, can
partially prevent EMB-induced cell growth inhibition, vesicles formation
and G1 cell cycle arrest. Also, Western blot analysis demonstrated that
EMB can induce translocation of PKCa, bII and e from cytosol to cell
membrane, indicating that PCK might be activated by EMB. In addition,
we found that PKC was activated by EMB. We also observed that
treatment of the PKC activator, TPA (12-O-tetradecanoyl-phorbol
13-acetate) resulted in growth inhibition of RPE but without vacuoles
formation. Take together our results suggest that EMB may activate PKC
pathway to suppress cell growth and induce vacuole formation of RPE.

目錄
目錄.......................................................................................................................... 1
壹、緒論.................................................................................................................. 2
貳、實驗材料與方法............................................................................................... 6
一、人類網膜色素上皮細胞的培養................................................................ 6
二、細胞生長與藥物反應................................................................................ 6
三、細胞週期分析........................................................................................... 7
四、轉染細胞, 暫時過量表現......................................................................... 8
五、細胞膜上蛋白質的萃取............................................................................ 9
六、西方點墨法............................................................................................. 10
七、PKC 蛋白質活性分析............................................................................. 14
參、實驗結果......................................................................................................... 15
一、乙胺丁醇會造成細胞週期終止以及產生空泡....................................... 15
二、乙胺丁醇會造成ERK 活化.................................................................... 16
三、乙胺丁醇可經由PKC 訊息傳導路徑來造成細胞生長抑制.................. 17
肆、結論與討論..................................................................................................... 21
伍、參考文獻......................................................................................................... 24
陸、附錄圖表......................................................................................................... 28
圖 1. Ethambutol 可以造成RPE 細胞生長抑制........................................... 28
圖 2. Ethambutol 會造成RPE 細胞質內產生空泡狀物質............................ 29
圖 3. Ethambutol 會造成ERK 活化............................................................... 30
圖 4. BIS (PKC inhibitor)可以阻止Ethambutol 所造成的細胞生長抑制.... 31
圖 5. BIS (PKC inhibitor)可以抑制Ethambutol 所造成的空泡形成............ 32
圖 6. Ethambutol 可以誘導RPE 細胞之PKC 活性..................................... 33
圖 7. Ethambutol 可以造成PKC isozymes 從細胞質轉移至細胞膜........... 34
圖 8.Anti-sense PKC 可以抑制EMB 所造成的RPE 生長抑制................. 35
圖 9. TPA 可以造成RPE 細胞生長抑制但不會形成空泡.......................... 36
表 1. Ethambutol 會造成RPE 細胞週期終止於G1 時期............................. 37
表 2. BIS 可以抑制Ethambutol 所造成的G1 時期細胞週期終止............... 37

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