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系統識別號 U0026-0908201810370500
論文名稱(中文) 利用光遺傳學揭示不同鈣離子振盪波所誘導之細胞死亡
論文名稱(英文) Revealing of cell death induced in different Ca2+ oscillations by optogenetics
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 106
學期 2
出版年 107
研究生(中文) 陳永裕
研究生(英文) Yong-Yi Chen
學號 P86055036
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 口試委員-湯銘哲
口試委員-楊德明
口試委員-林錫慧
口試委員-李純純
指導教授-邱文泰
中文關鍵字 光遺傳學  鈣離子震盪  細胞死亡  細胞壞死  自噬 
英文關鍵字 optogenetics  Ca2+ oscillations  apoptosis  necrosis  autophagy 
學科別分類
中文摘要 鈣離子在細胞的訊息傳遞中扮演了非常重要的角色,因為它參與了細胞的生命及死亡的訊息,例如受精,轉錄因子激活,細胞增殖,細胞遷移,ATP合成,細胞凋亡等。過去的研究顯示細胞內鈣離子濃度的恆定對於維持生命途徑非常重要;但是如果鈣離子濃度處於病理水平,它將刺激細胞死亡途徑,從而導致細胞凋亡。光遺傳學技術是一種具有空間和時間精度的新方法,已被開發並應用於許多生物醫學研究中的細胞活動之調控。因此,我們建立了具有470 nm藍光照明的光遺傳平台,並可以調控光學參數(頻率,光能量和作用時間)。一種穩定表達光明感通道 (calcium translocating channelrhodopsin, CatCh) 的人類骨肉瘤細胞 (U2OS),通過照射470 nm藍光產生鈣離子波流入細胞內。在我們的研究中,我們通過光遺傳學研究了細胞死亡中鈣離子振盪的模式。結果顯示,當細胞在10、30和60分鍾光照處理中並培養6小時以上,高頻率(1 Hz)誘導細胞腫脹並形成變形。此外,光刺激後的sub G1 群體的數量會隨著照光後培養的時間增加,其現象代表細胞壞死。另一方面,在較低之頻率(0.1 Hz)之下,當細胞分別在30和60分鐘的光刺激後進行48小時和12小時培養,會誘導細胞皺縮。我們發現0.1 Hz光刺激之下的細胞死亡主要是凋亡細胞,因為sub G1群體遠低於1 Hz。最後,在0.1 Hz光刺激之下,我們發現30分鐘組別中的自噬活性高於60分鐘組別,這使得降低了30分鐘組別中細胞死亡率。
英文摘要 Calcium (Ca2+) plays an important and ubiquitous role of intracellular signaling as it involved in life and death signals of the cell, for example, fertilization, transcription factors activation, cell proliferation, cell migration, ATP synthesis, cell apoptosis, etc. Previous studies mentioned that the homeostasis of Ca2+ concentrations in the cell is important to maintain cell life pathway; however if the concentration of Ca2+ was in the pathological level, it will activate cell death pathway, which leads to cell apoptosis. Optogenetic technology, a new approach with spatial and temporal precision, have been developed and applied to control cell activity in many biomedical studies. Therefore, we set up optogenetic platforms with blue light (470 nm) illumination that can modulate the optical parameters (frequency, power, duty cycle, and duration). The stable U2OS cell line overexpressing Ca2+ translocating channelrhodopsin (CatCh), a point-mutated form of channelrhodopsin 2 (ChR2), generates Ca2+ influx by illuminating 470 nm blue light. In our study, we investigated the patterns of Ca2+ oscillations in cell death by optogenetics. The results showed that a higher frequency (1 Hz) induced cell swelling and round up of the morphological changes when the cells incubated over 6 hours in 10, 30, and 60 minutes of light treatment. In addition, the sub G1 population increases during incubation after light treatment, which represented cell necrosis. On the other hand, a lower frequency (0.1 Hz) induced cell shrinkage when the cells incubated over 48 and 12 hours in 30, and 60 minutes of light stimulation, respectively. Furthermore, we found that the cell death in 0.1 Hz treatment was predominantly apoptotic cell as the sub G1 population was far lower than 1 Hz. And, in 0.1 Hz stimulation, we found that the autophagy activity in the 30-minute group is higher than the 60-minute group, which lowered down the percentage of cell death in the 30-minutes group.
論文目次 Abstract............................................................................................................ i
中文摘要......................................................................................................... iii
Contents.......................................................................................................... iv
Figure contents................................................................................................vi Chapter 1. Introduction.................................................................................. 1
1.1 Ca2+ homeostasis and signaling ........................................................... 1
1.2 Mitochondrial physiology ....................................................................2
1.3 Cell apoptosis .......................................................................................3
1.4 Ca2+ in the regulation of mitochondrial function .................................4
1.5 Ca2+ mediated cell apoptosis ................................................................5
1.6 Optogenetics......................................................................................... 6
1.7 The specific aim of the study ............................................................... 8
Chapter 2. Materials and Methods ............................................................... 9
2.1 Cell culture .......................................................................................... 9
2.2 Optogenetic platform............................................................................ 9
2.3 Fluorescence microscopy imaging....................................................... 9
2.4 Live cell staining.................................................................................. 9
2.5 PI staining for cell death analysis........................................................ 10
2.6 Immunofluorescence staining............................................................ 10
2.7 Statistical analysis...............................................................................11
Chapter 3. Results
3.1 Ca2+-induced morphological change after light illumination..............12
3.2 Light-stimulated Ca2+ oscillations induced immediate cell death after light illumination ..............................................................................................12
3.3 Light-stimulated Ca2+ oscillations induced morphological change during long-term incubation......................................................................13
3.4 Light-stimulated Ca2+ oscillations induced cell death during long-term incubation. ................................................................................................14
3.5 Different frequencies of light stimulation induced apoptosis and necrosis .................................................................................................... 15
3.6 Autophagy rates affect cell apoptosis after light illumination............ 16
Chapter 4 Discussion .................................................................................... 17
References ..................................................................................................... 20
Figures ........................................................................................................... 26


Figure 1. Morphological change observed in U2OS-CatCh-Venus at a higher frequency of light illumination........................................................................ 26
Figure 2. Live and death analysis of the U2OS-CatCh-Venus after light illuminations ...................................................................................................28
Figure 3. Live and death analysis of the U2OS-WT after light illuminations ...................................................................................................30
Figure 4. The effect of Ca2+-induced cell death in 0.01 Hz of light illumination .....................................................................................................32
Figure 5. The effect of Ca2+-induced cell death in 0.1 Hz of light illumination .................................................................................................... 35
Figure 6. The effect of Ca2+-induced cell death in 1 Hz of light illumination .................................................................................................... 38
Figure 7. Effects of the duration of light illumination in Ca2+-induced cell death................................................................................................................ 41
Figure 8. The effect of DNA content in lower frequency (0.1 Hz) treatment......................................................................................................... 43
Figure 9. The effect of DNA content in higher frequency (1 Hz) treatment......................................................................................................... 45
Figure 10. The investigation of autophagy after light-induced Ca2+ oscillations by optogenetics............................................................................................... 47
Figure 11. Fluorescence probes for mitochondrial functions evaluation........................................................................................................ 50
Figure 12. Mitochondrial functions evaluation by live cell flow cytometry........................................................................................................ 52
Figure 13. Mitochondrial functions evaluation by live cell fluorescence imaging........................................................................................................... 54
Figure 14. Schematic diagram of Ca2+-induced cell death by optogenetics ................................................................................................... 56
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