中 文 摘 要
本論文研究目的在於探討由內毒素引起的未分化的PC12 細胞死亡過程中，熱休克蛋白 60、70 及90 所扮演的角色。
將 1 x 105 cells/ml 的 PC12 細胞培養於 poly-L-lysine 修飾過的 3.5 公分的培養皿中，以內毒素 (1 mg/ml；血清型 O55:B5) 分別投予 3、6、12 及 24 小時，利用 Trypan blue 檢測細胞的存活率，並以 Western blot 偵測細胞內熱休克蛋白 60、70 及 90 的表現情形。結果顯示投予內毒素於 PC12 細胞，12 小時後即會造成顯著的細胞死亡，24 小時後其存活率僅剩下約 30%；隨著 PC12 細胞存活率的降低，熱休克蛋白 70 及 60 的蛋白質表現量亦會增加。熱休克蛋白 70在內毒素處理 12 小時後即有顯著增加，24 小時後更增加為對照組的 10 倍以上；熱休克蛋白 60 在內毒素處理 24 小時後增加為對照組的 2 倍左右。相對於熱休克蛋白 60 及 70，熱休克蛋白 90 在 PC12 細胞中的表現量並不會受到內毒素的影響而改變。
為了進一步探討熱休克蛋白 60、70 及 90 在內毒素引起的 PC12 細胞死亡過程中所扮演的角色，預先投予熱休克蛋白專一的反義寡核苷酸，處理 24 小時，抑制細胞內熱休克蛋白的表現，再投予內毒素；同樣計數細胞的存活率並偵測細胞內熱休克蛋白 60、70 及 90 的表現情形。結果顯示抑制熱休克蛋白 60 及 70 的蛋白質表現，PC12 細胞受內毒素影響下的存活率會降低；但抑制熱休克蛋白 90 的蛋白質表現並不會影響處理內毒素後 PC12 細胞的存活率。
本研究結果顯示未分化的 PC12 細胞會隨著內毒素處理的時間增加而造成存活率的漸趨下降，並且伴隨著熱休克蛋白 60 及 70 的蛋白質表現增加，但並不會影響熱休克蛋白 90 的表現情形；更進而發現熱休克蛋白 60 及 70 均扮演保護 PC12 細胞的角色，降低內毒素所造成的 PC12 細胞死亡；而熱休克蛋白 90 則不參與內毒素所引起的 PC12 細胞死亡過程。

英 文 摘 要
We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced naïve pheochrommocytoma cell (PC12) death.
PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect.
The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

目 錄
中文摘要……………………………………………………………………………...1
英文摘要……………………………………………………………………………...3
緒 論…………………………….………………….…………………………….5
研究源起與目的………………………….……………………………………14
研究方法…………………………….……………………………………………...15
實驗結果…………………………….……………………………………………...28
討 論…………………………….……………………………………………...33
結 論…………………………….……………………………………………...42
參考文獻…………………………….……………………………………………...43
附 圖…………………………….……………………………………………...55

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