亚油酸对大鼠胰腺β细胞电压依赖性钾电流的影响及其机制探讨

摘要

本研究应用全细胞电流记录、分子生物学、激素测定等方法，在原代培养的大鼠胰腺β细胞上记录并分离出电压依赖性钾电流的亚型，即迟发性外向整流型钾电流(IK)和一过性外向钾电流(IA)，同时在体外检测长链脂肪酸亚油酸对原代培养的大鼠胰腺β细胞上电压依赖性钾电流和胰岛素分泌的影响，并探讨其可能的信号转导途径。 研究采用半定量RT-PCR检测GPR40mRNA在大鼠胰岛等组织细胞上的表达，GAPDH为内对照；采用制霉菌素穿孔的全细胞电流记录方法，此方法在形成封接后，通过制霉菌素穿孔；放射免疫测定方法检测细胞内cAMP的浓度；采用试剂盒ELISA方法测定β细胞胰岛素的分泌。　　研究结果表明，大鼠胰腺β细胞上电压依赖性钾电流有IK和IA两种亚型，其中主要成分是IK.亚油酸短期处理胰腺β细胞上，呈浓度依赖性增加β细胞胰岛素的分泌。

目录

文摘

英文文摘

论文说明：Abbtrevoatons

原创性声明及关于学位论文使用授权说明

Chapter 1 Introduction

1.1 Insulin

1.1.1 Structure and Synthesis

1.1.2 Actions

1.1.3 Insulin secretion and regulation

1.2. Ion channels in insulin secreting β-cells

1.2.1 Voltage-gated K+ channels(Kv)

1.2.2Regulation of Kv channel activity inβ-cells

1.3.GPR40

1.4.Aim of the project

Chapter 2 Materials and Methods

2.1 Chemical Reagents

2.2 Cell culture and preparation

2.2.1 Rat pancreatic β-cells

2.2.2 MIN6 cells

2.3 Molecular Biology

2.3.1 RNA isolation

2.3.2 RT-PCR analysis

2.4 Electrophysiological recording

2.5 Intracellular cyclic AMP and insulin secretion

2.5.1 Radioimmunoassay for intracellular cyclic AMP assay

2.5.2 ELISA for insulin assay

2.6 Statistical analysis

Chapter 3 Results

3.1 Identification of GPR40 mRNA expression

3.2 Characterization of the voltage-gated K+ currents

3.3 Effect of FFAs on the voltage-gated K+ currents

3.4 Effect of methyl linoleate on the voltage-gated K+ currents

3.5 Involvement of protein kinase in the K+ current response to FFAs

3.6 cAMP assay in MIN6 cell line after treatment with FFAs

3.7 Effect of linoleic acid on insulin secretion

Figures

Chapter 4. Discussion

4.1 Characterization of the voltage-gated K+ currents

4.2 FFAs reduce the voltage-gated K+ currents via GPR40

4.3 Involvement of protein kinase in the K+ current response to FFAs

4.4 Conclusion

Reference

Chapter 5 综述脂肪细胞分秘因子对糖尿病中β细胞功能的影响

致谢

Publication and achivementsz(2001-2005)