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系統識別號 U0026-1507201722203600
論文名稱(中文) Curcuminoids(類薑黃素)對胰島素分泌細胞上延遲修正性鉀離子電流之抑制作用
論文名稱(英文) Evidence for the Inhibitory Actions of Curcuminoids on Delayed-Rectifier K+ Currents in Insulin-Secreting (INS-1) Cells
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 105
學期 2
出版年 106
研究生(中文) 楊佳容
研究生(英文) Chia-Jung Yang
學號 S36044096
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-吳勝男
召集委員-劉彥青
口試委員-郭賓崇
中文關鍵字 類薑黃素  胰島素分泌細胞  延遲修正性鉀離子電流  電流不活化作用  動作電位  蛋白激酶B  磷酸化蛋白激酶B 
英文關鍵字 curcuminoids  insulin-secreting cell  delayed-rectifier K+ current  current inactivation  action potential  protein kinase B  phosphorylated protein kinase B 
學科別分類
中文摘要 類薑黃素(Curcuminoids)主要是由薑黃素(curcumin)、去甲氧基薑黃素(demethoxycurcumin)以及二去甲氧基薑黃素(bisdemethoxycurcumin)所組成。 薑黃素則是類薑黃素中最主要的成分,近年來被認為會影響細胞信號分子(signaling molecules)以及胰臟B細胞中胰島素的分泌。 第二型糖尿病的發生通常是因為胰臟B細胞分泌的胰島素不足。 這會使得血糖升高,且若不治療將可能導致多種併發症。 然而,類薑黃素是如何影響胰島素分泌細胞上細胞膜的離子通道現在仍然所知甚少。 在過去的研究當中,ATP敏感性鉀離子通道(ATP-sensitive K+ channels)、電壓門控的鈣離子通道(voltage-gated Ca2+ channels)以及電壓門控的鉀離子通道(voltage-gated K+ channels)被認為是調控胰臟B細胞電訊號傳遞中最主要的三種離子通道。 其中,延遲修正性鉀離子通道(delayed-rectifier K+ channel)的功能為使細胞動作電位再極化進而阻斷葡萄糖所刺激的胰島素分泌。 在本篇研究中我們利用細胞膜箝制(patch-clamp)的技術來評估類薑黃素在胰島素分泌細胞(INS-1 cell)的電生理特性,特別是在於延遲修正性鉀離子通道上的表現,以及利用西方點墨法(Western blot)觀察其蛋白質的表現量。 我們的研究結果顯示將不同濃度的類薑黃素作用在INS-1細胞上,其細胞死亡率及延遲性修正性鉀離子通道的蛋白質表現量並無影響,但薑黃素作用在INS-1細胞上抑制了延遲修正性鉀離子電流,並且有時間、狀態及濃度的依賴,而這些抑制作用無法被氯甲苯噻嗪(diazoxide)、喜革脈錠(nicorandil)及蠍氯毒素(chlorotoxin)等藥物逆轉。 透過計算可得到薑黃素在INS-1細胞的延遲修正性鉀離子電流上的解離常數為1.26 uM。 然而儘管薑黃素對於延遲修正性鉀離子電流的活化速率(activation rate)較無反應作用,但對於不活化作用(inactivation)有加速的作用。 增加薑黃素的濃度使延遲修正性鉀離子電流的不活化作用曲線往過極化電位偏移且使得延遲修正性鉀離子電流的不活化作用復原變慢。 薑黃素、去甲氧基薑黃素以及二去甲氧基薑黃素同樣都抑制了相類似的延遲修正性鉀離子電流大小,雖然去甲氧基薑黃素及二去甲氧基薑黃素對於不活化作用的電流反應較不明顯。 另外,也發現薑黃素可以抑制電壓門控的鈉離子電流(voltage-gated Na+ current)。 薑黃素、去甲氧基薑黃素以及二去甲氧基薑黃素也會使得靜止膜電位去極化並增加動作電位的頻率。而不同濃度的薑黃素、去甲氧基薑黃素及二去甲氧基薑黃素在蛋白激酶B (Akt)及磷酸化蛋白激酶B (pAkt)蛋白質的表現上並無顯著差異。 綜合以上的實驗,透過類薑黃素對INS-1細胞延遲修正性鉀離子電流的抑制作用,可以對活體中胰臟B細胞的功能活性有更進一步的了解。
英文摘要 Curcuminoids are mainly composed of curcumin (CUR), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC). CUR, a principal constituent of the curcuminoids, has been recently demonstrated to modulate various cellular signaling molecules and induce insulin release from pancreatic b-cells. Diabetes mellitus occurs when the pancreatic b-cells produce insufficient amounts of the hormone insulin. This causes high blood glucose levels, which can lead to a number of complications if untreated. However, how curcuminoids exert any possible effects on membrane ion currents in insulin-secreting cells remains largely unclear. There were recognized to be three major ion currents, that is, ATP-sensitive K+ channels, voltage-gated Ca2+ channels and voltage-gated K+ channels, operating in pancreatic b-cells and the most important ionic events in b-cell signaling. The delayed-rectifier K+ (IK(DR)) channel can function as brake for glucose-stimulated insulin secretion. The effects of curcuminoids on ion currents especially in IK(DR) in rat INS-1 insulinoma cells were therefore investigated in this study by using patch-clamp technique and the protein expression were detected by western blot. The results showed that the INS-1 cells treated different concentrations of curcuminoids had no effect on the cell viability and KDR channels protein expression. CUR suppressed the amplitude of IK(DR) in a time-, state- and concentration-dependent manner in these cells and the inhibition was not reversed by diazoxide, nicorandil or chlorotoxin. The value of dissociation constant for CUR-induced suppression of IK(DR) in INS-1 cells was 1.26 uM. Despite the inability of CUR to alter the activation rate of IK(DR), it accelerated current inactivation elicited by membrane depolarization. Increasing CUR concentrations shifted the inactivation curve of IK(DR) to hyperpolarized potential and slowed the recovery of IK(DR) inactivation. CUR, DMC, and BDMC exerted depressant actions on IK(DR) amplitude to a similar magnitude, although DMC and BDMC did not increase current inactivation clearly. CUR suppressed the peak amplitude of voltage-gated Na+ current. CUR, DMC and BDMC depolarized the resting potential and increased firing frequency of action potentials. There was no significant difference in the protein expression of protein kinase B (Akt) and phosphorylated protein kinase B (pAkt) among different concentrations of CUR, DMC, BDMC. Taken these results together, these effects can significantly contribute to their actions on functional activities of insulin-secreting cells if similar findings are found in vivo.
論文目次 中文摘要 I
Abstract III
誌謝 V
Table of Contents VI
List of figures IX
Abbreviation XI
1. Introduction 1
1.1. Curcuminoids 1
1.2. Diabetes Mellitus 2
1.3. Model of Glucose-Stimulated Insulin Release 2
1.4. Curcuminoids Might Affect The Membrane Ion Channels 2
1.5. Insulinoma Cell Line (INS-1) 3
2. Materials and methods 5
2.1. Extraction and Fractionation of Medicinal Plants 5
2.2. Cell Preparation 6
2.3. Trypan Blue Exclusion Assay 7
2.4. Western Blot Analysis 7
2.5. Electrophysiological Measurements 8
2.6. Data Recordings 9
2.7. Data Analyses 9
2.8. Drugs and Solutions 10
2.9. Statistical Analyses 11
3. Results 12
3.1. Inhibitory Effect of CUR on Delayed-Rectifier K+ Current (IK(DR)) Measured from INS-1 Cells 12
3.2. Comparison among Effects of CUR, CUR plus Diazoxide, CUR plus Nicorandil, CUR plus Chlorotoxin and CUR plus Nonactin on IK(DR) Amplitude in INS-1 Cells 13
3.3. Kinetic Constants of Block by CUR 13
3.4. Effect of CUR on Steady-State Inactivation of IK(DR) in INS-1 Cells 15
3.5. Recovery of IK(DR) Block in the Presence of CUR 15
3.6. Concentration-Dependent Effect of CUR, DMC and BDMC on IK(DR) Amplitude 16
3.7. The Viability of INS-1 Cells to Different Concentrations of CUR, DMC and BDMC 16
3.8. Protein Expression of Delayed-Rectifier Potassium Channels in INS-1 Cells with Curcuminoids of Different Concentrations 17
3.9. Effect of CUR on Voltage-Gated Na+ Current (INa) in INS-1 Cells 17
3.10. Effect of CUR, DMC, BDMC on Spontaneous Action Potentials (APs) in INS-1 Cells 18
3.11. Identification the Akt and Phospho-Akt Protein Expression in INS-1 Cells with Curcuminoids at Different Concentrations 18
4. Discussion 19
5. Conclusion 25
6. Figures 26
7. Figure Legends 44
8. References 50

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