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研究生：	賴怡旬 Lai, Yi-Shyun
論文名稱：	光遺傳學對於鈣離子依賴性轉錄因子與細胞爬行之調控 Modulation of Ca2+-dependent transcription factors and cell migration by optogenetics
指導教授：	邱文泰 Chiu, Wen-Tai
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	65
中文關鍵詞：	光遺傳學、鈣離子依賴性轉錄因子、細胞爬行
外文關鍵詞：	optogenetics, Ca2+-dependent transcription factors, cell migration
相關次數：	點閱：100 下載：2
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細胞調節中鈣離子的訊號傳導扮演廣泛的角色，包括:授精、細胞增生與分化、肌肉收縮或者是細胞學習與死亡。鈣離子作用也表現於細胞爬行以及鈣離子依賴性轉錄因子活化(NFAT、NFκB和CREB)。然而，在現今的研究中無法精準的在時間與空間上調控細胞內的鈣離子。因此，我們假設不同鈣離子振盪波會影響鈣離子依賴性轉錄因子與細胞行為。本研究利用光遺傳學平台來創造出不同的鈣離子振盪波，藉由調控相關光參數，包含光密度、頻率與作用時間，並利用470 nm藍光活化表達於人骨肉瘤細胞上之光敏感通道(CatCh)使鈣離子流入細胞內。結果顯示，低頻率鈣離子振盪會使NFκB活化，反之高頻率鈣離子振盪會使NFAT活化，另一方面，不管鈣離子振盪頻率高低CREB皆活化。此外，利用傷口癒合分析得到在低頻率鈣離子振盪下會促進細胞爬行。然而，進一步也探討細胞爬行的相關蛋白發現ERK和AKT皆有活化。最後，得知低頻率鈣離子振盪會使NFκB活化與細胞爬行速度增加。本研究探討不同鈣離子振盪波如何去影響鈣離子相關訊息的傳遞和細胞行為。

Calcium (Ca2+) signaling can regulate a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, cell learning, and death. Cell migration and activation of several transcription factors are also shown in a Ca2+-dependent response, such as NFAT (nuclear factor of activated T-cells), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell), and CREB (cAMP response element binding protein). However, control of intracellular Ca2+ with spatial and temporal precision is a limitation in the current studies on these topics. Here, we hypothesize that different Ca2+ oscillations can affect the Ca2+-dependent gene transcription and cell function profiles. In this study, an optogenetic platform was used for the purpose of creating different Ca2+ oscillation patterns through manipulation of lighting parameters, including density, frequency, duty cycle, and duration. U2OS cell overexpression of Ca2+ translocating channelrhodopsin (CatCh) can be activated by 470 nm blue light to induce Ca2+ influx. Our results showed that activation of NFκB required low-frequency Ca2+ oscillations, whereas high-frequency Ca2+ oscillations tended to activate NFAT. On the other hand, CREB activation occurred regardless of the frequency of Ca2+ oscillations. In addition, the upregulation of cell migration upon low-frequency Ca2+ oscillations was analyzed using a wound healing assay. However, further exploration is needed to elucidate the relevant proteins, ERK and AKT, if they are activated during cell migration. Finally, the activation of NFκB and increased ability cell migration were found under low-frequency Ca2+ oscillations. This study investigated how different Ca2+ oscillators affect Ca2+-related transmission and cellular functions.

Contents
Abstract	i
中文摘要	ii
Acknowledge	iii
Contents	iv
Figure contents	vi
Chapter 1 Introduction	1
1.1 The important role of calcium (Ca2+) in cells	1
1.2 Ca2+ dependent transcription factors	3
1.2.1 CREB	4
1.2.2 NFAT	4
1.2.3 NFκB	5
1.3 Cell migration	6
1.4 Optogenetics	7
1.5 The specific aims of the study	8
Chapter 2 Materials and methods	10
2.1 Cell culture	10
2.2 Optogenetic system	10
2.3 Live and dead assay	10
2.4 Cell proliferation assay	10
2.5 Western blotting	11
2.6 Immunofluorescence staining	12
2.7 Migration assay	12
2.8 NFκB, ERK and AKT inhibitors	13
	2.9 Statistical analysis	13
Chapter 3 Results	14
3.1 The optogenetic tool.	14
3.2 Manipulating Ca2+ frequencies to evaluate cell viability.	14
3.3 Manipulating Ca2+ oscillation affects cell proliferation..	15
3.4 Ca2+ oscillation frequencies affect cell migration.	15
3.5 Amplitude and duty cycle of Ca2+ oscillation affects cell migration..	16
3.6 Regulation of Ca2+ oscillations induced transcription factors activation (CREB, NFAT, and NFκB)..	17
3.7 Ca2+ oscillations affect cell migration-related signaling pathway.	17
3.8 Different inhibitors inhibit Ca2+-related cell migration.	18
Chapter 4 Discussion	19
References	22
Figures	27
 
Figure contents
Figure 1. The optogenetic system and molecular tool – CatCh.	27
Figure 2. Modulation of Ca2+ oscillations to affect cell viability in U2OS-CatCh-Venus.	29
Figure 3. Modulation of Ca2+ oscillations to affect cell viability in U2OS-WT.	31
Figure 4. Modulation of Ca2+ oscillations by different frequency affects cell proliferation.	33
Figure 5. Modulation of Ca2+ oscillations by low frequency affects cell proliferation.	35
Figure 6. Modulation of Ca2+ oscillations by low frequency and high cell density affects cell proliferation. 	37
Figure 7. Modulation of Ca2+ oscillations by different frequencies affects cell migration.	39
Figure 8. Modulation of Ca2+ oscillations by low frequencies and different light treatment times affects cell migration.	41
Figure 9. Modulation of Ca2+ oscillations with different power affects cell migration.	43
Figure 10. Modulation of Ca2+ oscillations by different duty cycles affects cell migration.	45
Figure 11. Activating of transcription factor CREB using optogenetics.	47
Figure 12. Activating of transcription factor NFAT using optogenetics.	49
Figure 13. Activating of transcription factor NFκB using optogenetics.	51
Figure 14. Ca2+ oscillations affected migration-related proteins.	53
Figure 15. NFκB inhibitor decreased the potency of migration in U2OS-CatCh-Venus cells using blue light stimulation.	55
Figure 16. ERK inhibitor decreased the potency of migration in U2OS-CatCh-Venus cells using blue light stimulation.	57
Figure 17. AKT inhibitor decreased the potency of migration in U2OS-CatCh-Venus cells using blue light stimulation.	59
Figure 18. AKT inhibitor decreased the potency of migration in U2OS-CatCh-Venus cells using blue light stimulation.	61
Figure 19. Proof of inhibitory effect of inhibitors.	63
Figure 20. The schematic of this study.	65
 

                                    

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