臺灣博碩士論文加值系統

熱休克蛋白早期是由於溫度刺激的研究所發現的，隨著陸陸續續的研究發現熱休克蛋白是一群維持細胞生理機能的保護性蛋白質，並且也涉及細胞凋亡的機制。而熱休克蛋白平時就會表現來維持基本的細胞生化功能，在遇到溫度、氧化壓力、化學性毒素、紫外光或特定波長光線刺激時，熱休克蛋白會大量表現來穩定細胞的生理現象來保護細胞。
高溫熱療運用在各種腫瘤治療已經有二十多年的歷史，而熱療所引發的細胞反應包括核糖核酸(RNA)及去氧核糖核酸(DNA)的生成、免疫反應、細胞骨架重組、細胞凋亡及熱休克蛋白的表現。除此之外，光動力療法由於造成單態氧的生成，使細胞受到氧化壓力，也會引發熱休克蛋白的表現以及細胞凋亡。
在本研究中，我們利用光動力療法與高溫熱療刺激細胞中熱休克蛋白的表現，觀察細胞中熱休克蛋白RNA與DNA以及熱休克蛋白質量的表現，比較不同溫度、不同劑量、不同時間以及此兩種不同治療的方式，對於細胞生化合成的影響。我們的研究結果顯示，高溫熱療與光動力療法都能引發熱休克蛋白的表現，而高溫熱寮的熱休克蛋白表現呈現溫度依存性(temperature-dependant)，並且以43℃、30分鐘的熱療為最佳條件。熱療後的蛋白質表現量隨著時間增加，但以HSP70較明顯，而熱療對於HSP60的表現則效果不佳。
在光動力療法方面，熱休克蛋白的表現也呈現劑量依存性(dose-dependant)。隨著時間的增加，光動力療法誘發的熱休克蛋白量也逐漸升高，達到高峰後漸漸減低回到正常表現量。相較於HSP60與HSP27而言，HSP70在較短的時間表現量即增加，但是也在較短的時間內降低。此外，對於HSP60的誘發，光動力療法效果較高溫熱療好。
以上研究使我們對高溫熱療與光動力療法對於熱休克蛋白的調控影響有更近一步的了解，幫助我們未來研究系統的設計上能有更好的改進。

Heat shock proteins (HSPs) serve as the chaperones, antiaggregants and cytoprotectants in both normal and stress conditions [Matthew et al. 2004]. Under the stress stimuli, such as the temperature, chemical toxins, heavy metals and also UV or specific light illumination, HSPs will be induced to protect living cell from damage.
Both hyperthermia and Photodynamic therapy (PDT) can induce cellular apoptosis and the expression of heat shock proteins as well. It is noticeable that several families of heat shock proteins are involved in cell apoptosis pathway [Steven et al, 2000].
In this study, we investigated the gene expression of RNA and protein level of HSPs after the treatment of PDT and hyperthermia. We discovered that the expression of HSPs induced by hyperthermia was temperature-dependant, especially for HSP70, and the optimal condition was 43℃ for 30 minutes. The RNA and protein expression of HSPs increased in concordance with the duration after hyperthermia. HSP70 had strongest expression after hyperthermia. Hyperthermia, however, did not change HSP60 expression significantly.
For PDT, the RNA expression of HSPs was slightly increased by different irradiation energy. And the protein expression of HSPs was show to be dose-dependant. Similar to the study of hyperthermia, the expression of HSPs increased after PDT and reached the peak at 12 hours (2 hours for RNA level). HSP70 was induced more rapidly and began to decrease after 9 hours. On the other hand, HSP60 and HSP27 remained relative stable all the time. Generally PDT was more effective than hyperthermia in inducing HSP60 expression.
The result of this study revealed the effect on PDT and hyperthermia of gene expression and helped us to understand the regulation of HSP expression.

CONTENT
中文摘要 1
ABSTRACT 2
INTRODUCITON 3
MATERIALS AND METHODS 6
◆ Cell culture
◆ Hyperthermia
◆ Photodynamic therapy
◆ SDS-Polyacryamide Gel Electrophoresis and Western blotting
◆ Image quantity analysis
RERULTS 9
◆ The RNA expression of HSPs of HeLa cells at different temperature after hyperthermia
◆ The proteins expression of HSPs of HeLa cells at different temperatures after hyperthermia
◆ The RNA expression of HSPs of HeLa cells at different time periods after hyperthermia
◆ The protein expression of HSPs of HeLa cells at different time periods after hyperthermia
◆ The RNA expression of HSPs of HeLa cells at different illumination times for PDT

◆ The protein expression of HSPs of HeLa cells at different illumination times for PDT
◆ The RNA expression of HSPs of HeLa cells at different time period after PDT
◆ The protein expression of HSPs of HeLa cells at different time period after PDT

FIGURES
Fig. 1 The diagram illustrate the proposed mechanism of cell injure by reactive oxygen species. 4
Fig. 2 The scheme of the speculative pathway in apoptosis. 5
Fig. 3 The agarose analysis of RT-PCR after hyperthermia at different temperature. 14
Fig. 4 The statistical graph of quantified image data for Fig. 3. 14
Fig. 5 The Western blotting of the HSPs expression after hyperthermia at different temperature. 15
Fig. 6 The statistical graph of quantified image data for Fig. 5 15
Fig. 7 The agarose analysis of RT-PCR after 43℃ hyperthermia at different time periods. 16
Fig. 8 The statistical graph of quantified image data for Fig. 7. 16
Fig. 9 The Western blotting of the HSPs expression after hyperthermia at different time periods. 17
Fig. 10 The statistical graph of quantified image data for Fig. 9. 17
Fig. 11 The agarose analysis of RT-PCR 2 hours after PDT with different illumination times. 18
Fig. 12 The statistical graph of quantified image data for Fig. 11. 18
Fig. 13 The Western blotting of the HSPs expression 4hours after PDT with different illumination times. 19
Fig. 14 The statistical graph of quantified image data for Fig. 13. 19
Fig. 15 The agarose analysis of RT-PCR after PDT at different time periods. 20
Fig. 16 The statistical graph of quantified image data for Fig. 15. 20
Fig. 17 The Western blotting of the HSPs expression after PDT for different time periods. 21
Fig. 18 The statistical graph of quantified image data for Fig. 17. 21

DISCUSSION 22
REFERENCES 26

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