Abstract
Bax is predominantly a cytosolic monomer in healthy cells. Upon receiving
apoptotic signal, it performs a series of biological processes, which include
conformational changes, translocation to mitochondria as well as oligomerization,
consequently leading to the event of mitochondrial outer membrane permeabilization
(MOMP) and then cell death. However, little is known about the molecular basis of
the driving forces that direct the translocation of Bax to bring about the MOMP.
Localization of Bax to the mitochondrial outer membrane is a key step to the
activation ability of Bax to modulate mitochondrial membrane integrity. In this thesis,
we used giant unilamellar vesicle (GUV) along with confocal fluorescence
microscope to investigate how this Bax translocation, upon receipt of apoptotic
signaling, depend on the lipid compositions and properties. The activation of Bax was
induced with the addition of BimBH3 peptides, which is known to be useful to
promote the oligomerization of Bax (o-Bax). This study prepared GUVs of several
different lipid compositions using PC (POPC), PE (POPE), PG (POPG), and CL
(Cardiolipin). Quantitative analyses regarding the membrane permeability and pore
size were obtained by the time-dependent fluorescence intensity observed within
GUVs from three fluorescence probes of different molecular size. In PC-only GUVs,
some leakages due to the associations of Bax with membrane surface could be
observed. The leakages were found to be mostly caused by small pores (> 0.45 nm in
radius), although there existed small number of large pores (> 6 nm) that appeared
transiently during the observations. In GUVs composed of PC/PG, both the membrane
surface association of Bax and the pore permeability were largely abolished. The
charged property of PG, though similar to CL, does not seem to be important. In the
PC-based GUVs containing of PE or CL, stable large pores (> 6 nm) could only be
II
observed in the presence of o-Bax. The membrane permeability and the stability of
the large pores were found to be higher in the PC/CL than in the PC/PE vesicles,
indicating that the greater the negative curvature of the doped lipid is, the greater the
number of the large pores is observed. Collectively, our study indicates that the
formation and stability of large pores, which are essential to the MOMP process,
depend critically on the lipid compositions. Inactive Bax monomers could easily
cause disturbances to membrane surface. The disturbances result in some accidental
breaks for small probes to flow in and, therefore, are considered as a measure of the
membrane surface association of Bax monomers. O-Bax was found to effectively
induce large pores on PC-based GUVs containing either CL or PE but rather than PG.
Nevertheless, only in the presence of CL could the large pores remain stable over a
long time. Our studies demonstrate the importance of the curvature property of
lipids in the MOMP process.

目錄
第一章 緒論..................................................................................................................1
1.1 細胞凋亡(Apoptosis) ................................................................................................ 1
1.2 Bcl-2 (B-cell lymphoma)家族蛋白 .......................................................................... 1
1.3 Bax蛋白質(Bcl-2-associated X protein)與Cardiolipin (CL) .................................. 3
1.4 巨型單層微脂體(Giant Unilamellar Vesicle, GUV) ............................................... 5
1.5 研究動機與目的 ...................................................................................................... 5
第二章 儀器介紹與原理..............................................................................................7
2.1 共軛焦螢光顯微鏡(Confocal Fluorescence Microscopy) ....................................... 7
2.1.1 背景 .................................................................................................................. 7
2.1.2 現今的共軛焦顯微鏡 ...................................................................................... 7
2.1.3 螢光 .................................................................................................................. 7
2.1.4 螢光顯微鏡 ...................................................................................................... 9
2.1.5 共軛焦顯微鏡 .................................................................................................. 9
2.1.6 共軛焦顯微鏡的運作 .................................................................................... 11
2.1.7 三維影像的建構 ............................................................................................ 12
2.1.8 總結 ................................................................................................................ 12
第三章 樣品製備、儀器測量方法與藥品................................................................13
3.1 實驗流程 ................................................................................................................ 13
3.1.1 蛋白質的表現與純化 .................................................................................... 13
3.1.2 巨型單層微脂體(GUV)的製作 ..................................................................... 15
3.1.3 樣品配製 ........................................................................................................ 17
3.2 儀器測量方法 ........................................................................................................ 18
3.3 藥品與儀器 ............................................................................................................ 20
3.3.1 實驗藥品 ........................................................................................................ 20
3.3.2 實驗儀器 ........................................................................................................ 21
第四章 結果與討論....................................................................................................22
4.1 研究策略 ................................................................................................................ 22
VI
4.2 利用小分子螢光染料5(6)-CF觀察Bax對於GUV，產生之滲入現象 ............ 22
4.2.1 測試不同細胞膜成分的GUV於含鹽緩衝溶液之穩定性 ......................... 22
4.2.2 Bax加入BimBH3後對GUV之作用 ......................................................... 24
4.3 利用小分子螢光染料Alexa 647觀察Bax對於含有CL的GUV，產生之滲入現
象 ............................................................................................................................ 26
4.3.1 測試含有CL的GUV於含鹽緩衝溶液之穩定性 ...................................... 26
4.3.2 Bax對含有CL的GUV之作用 ................................................................... 27
4.4 利用大分子螢光染料FITC-Dextran 70檢驗於不同細胞膜成分的GUV下 Bax
之功能性 ................................................................................................................ 28
4.4.1 測試含有CL的GUV於含鹽緩衝溶液之穩定性 ...................................... 28
4.4.2 Bax加入BimBH3後對GUV穿孔之功能性 ............................................. 29
4.4.3 o-Bax對不含CL的GUV穿孔之功能性 ................................................... 30
4.5 同時利用小分子螢光染料Alexa 647及大分子螢光染料FITC-Dextran 70檢驗於
不同細胞膜成分的GUV下o-Bax之功能性 ...................................................... 31
4.5.1 o-Bax對含有CL的GUV穿孔之功能性 ................................................... 31
4.5.2 o-Bax對含有PG的GUV穿孔之功能性 ................................................... 33
4.6 定性討論 ................................................................................................................ 33
4.6.1 PC - 純粹PC的細胞膜並不適合Bax於其上表現穿孔之功能性............ 33
4.6.2 PC/PE (75:25) - 抑制寡聚化，但負曲率利於Bax穿孔 ............................ 34
4.6.3 PC/PG (75:25)&(90:10) - 不利於Bax貼附上亦不適合於其上工作......... 35
4.6.4 PC/CL (90:10) - 具有增進BimBH3活化Bax的能力，負曲率利於Bax工
作 .................................................................................................................... 35
4.7 利用模型對於Bax產生之孔洞大小作定量分析 ................................................ 36
4.7.1 計算穿透率P值(Permeability) ..................................................................... 36
4.7.2 使用模型並透過穿透率P值推算孔洞大小 ................................................ 38
4.7.3 不同條件下分析所得之孔洞半徑 ................................................................ 40
第五章 結論................................................................................................................53
參考文獻......................................................................................................................55

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