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研究生:黃彥銘
研究生(外文):Yan-MingHuang
論文名稱:帝盟多 (Temozolomide)對多型性膠質母細胞瘤細胞上中型電導鈣離子活化鉀離子通道的抑制作用
論文名稱(外文):Evidence for Inhibitory Actions of Temozolomide, an Alkylator of the Imidazotetrazine Family, on Intermediate-Conductance Ca2+-Activated K+ Channels in U373-MG Glioblastoma Cells
指導教授:吳勝男
指導教授(外文):Sheng-Nan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:48
中文關鍵詞:帝盟多中型鈣離子活化鉀離子通道鉀離子電流多型性膠質母細胞瘤膜電位
外文關鍵詞:Temozolomideintermediate-conductance Ca2+-activated K+ channelK+ currentglioma cellmembrane potential
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帝盟多 (Temozolomide) ,是一種四氮雜苯咪唑 (imidazotetrazine) 所衍生而來的口服烷化劑藥物。目前來說,對於治療多型性膠質母細胞瘤而言為一種最有效的抗腫瘤藥物。根據前人的藥理學研究與報告,帝盟多會造成去氧核醣核酸 (DNA) 上鳥嘌呤 (guanine) 結構中第六號位置氧的甲基化,進而造成胸腺嘧啶 (thymine) 的錯誤配對以及受影響細胞的細胞凋亡。然而,帝盟多是否會對多型性膠質母細胞瘤細胞細胞膜上的離子通道產生任何作用目前還是未知的,需進一步探討。因此,在我們的研究當中主要是要探討這項藥物針對多型性膠質母細胞瘤細胞 ̶ U373-MG細胞的細胞膜上離子通道產生之電流造成的可能影響。首先,我們利用完整細胞模式記錄 (whole-cell recordings) 的方式作觀察,加入帝盟多後U373-MG細胞上電壓依賴性鉀離子電流 (voltage-dependent K+ currents ,IK) 的振幅強度下降,而這種帝盟多所誘導的電壓依賴性鉀離子電流抑制作用可由加入離子黴素 (ionomycin) 或1-EBIO來逆轉。接下來以細胞連接片紀錄 (cell-attached current recordings) 的實驗中觀察到暴露於帝盟多下所造成的中型電導鈣離子活化鉀離子通道活性的抑制效果呈現濃度依賴性關係,其通道蛋白活性下降一半時的藥物濃度 (IC50) 為9.2 μM,加入Chlorzoxazone 或1-EBIO可以逆轉帝盟多誘導的中型電導鈣離子活化鉀離子通道抑制效果。儘管帝盟多不會造成中型電導鈣離子活化鉀離子通道的電導改變,但這個藥物的抑制效果具有微弱電壓依賴性並伴隨著通道平均關閉時間慢組成的延長。當分別以大型電導鈣離子活化鉀離子通道 (large-conductance Ca2+-activated, BKCa) 抑制劑 - Paxillin及內向整流型鉀離子通道 (inwardly rectifying K+, Kir) 抑制劑 - BaCl2所進行的鉀離子通道抑制測試顯示大型電導鈣離子活化鉀離子通道或內向整流型鉀離子通道的活性皆不會受到帝盟多的影響所改變。最後,在電壓-膜片箝制紀錄 (current-clamp recordings) 中顯示帝盟多會造成細胞膜的去極化,而1-EBIO可以逆轉帝盟多誘導的去極化作用。此外,在反轉錄聚合酶鏈鎖反應 (reverse transcription-PCR) 實驗中顯示U373-MG細胞中KCNN4 (KCa3.1) mRNA的表現不會受到暴露於帝盟多的影響。因此,帝盟多誘導的抗腫瘤作用除了造成DNA的受損之外,對於中型電導鈣離子活化鉀離子通道的抑制作用並伴隨細胞膜的去極化作用可能是額外但卻重要的藥理機制。故中型電導鈣離子活化鉀離子通道對多型性膠質母細胞瘤的治療可能是一個有效的替代目標。
Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is currently the most effective antineoplastic drug in the treatment of glioma cells. According to pharmacological report, TMZ causes alkylation of O6 position of guanine and lead to mispairing with thymine and apoptosis of the affected cells. However, whether this drug has any effects on membrane ion channels in glioma cells remain largely unclear. This study was conducted to investigate the possible effects of this drug on ionic currents present in U373-MG glioma cells. In whole-cell recordings, addition of TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373-MG cells. TMZ-induced inhibition of IK was reversed by further addition of ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached current recordings, cell exposure to TMZ decreased the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels in a concentration-dependent manner with an IC50 value of 9.2 M. Chlorzoxazone or 1-EBIO counteracted TMZ-induced inhibition of IKCa channels. Despite the inability of TMZ to modify single-channel conductance, the inhibition by this drug of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neither large-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels in these cells became altered in the presence of TMZ. Paxilline and BaCl2 was effective at suppressing BKCa and Kir channels, respectively. Under current-clamp recordings, TMZ depolarized cell membrane and 1-EBIO reversed TMZ-induced depolarization. The mRNA expression of KCNN4 (KCa3.1) detected in U373-MG glioma cells was unaltered during exposure to TMZ. Therefore, besides its DNA damage, this inhibitory effect on IKCa channels accompanied by membrane depolarization could be an additional but important mechanism underlying TMZ-induced anti-neoplastic actions, provided these actions occur in vivo. The IKCa channel could be an alternative target for therapy of glioblastomas.
Abbreviation Ⅳ
Figure contents Ⅴ
Abstract in Chinese 1
Abstract 3
Acknowledgement 5
Introduction 7
Materials and Methods 10
Drugs and Solutions 10
Cell Preparations 11
RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) 11
Western blot analysis 12
Electrophysiological Measurements 12
Data Recordings 13
Single-Channel Analyses 14
Results 16
Effect of TMZ on the Amplitude of Voltage-Dependent K+ Current (IK) in U373-MG Cells 16
Pharmacological Properties of IKCa Channels in U373-MG Cells 17
Identification of the presence of BKCa and IKCa channels in U373-MG Cells 18
Effect of TMZ on the Activity of IKCa Channels in U373-MG Cells 18
Lack of Effect of TMZ on Single-Channel Conductance of IKCa Channels in U373-MG Cells 19
Voltage Dependency of TMZ-Mediated Inhibition of IKCa Channels in U373-MG Cells 19
Effect of TMZ on the kinetic behavior of IKCa channels in U373-MG cells 20
Inability of TMZ to Modify Large-Conductance Ca2+-Activated K+ (BKCa) Channels in U373-MG Cells 20
Lack of TMZ Effect on Inwardly Rectifying K+ (Kir) Channels in U373-MG Cells 21
Effect of TMZ on the Resting Membrane Potential (Vrest) in U373-MG Cells 22
Discussion 24
References 28
Figures 32
Figures Legends 42
Appendix 47
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