甲基-β-环糊精及脂筏对细胞膜磷脂PIP2代谢的影响及分子机制研究
发布时间：2018-09-04 11:30
【摘要】：细胞膜是细胞进行物质交换和信息传递的重要通道，细胞在接受刺激后，众多的信号通路同时在细胞膜上发生，细胞膜上必然存在着协调不同通路转导的区域。脂筏就是参与信号转导的平台之一。脂筏是细胞膜上富含胆固醇和鞘磷脂的微结构域，与膜的信号转导以及蛋白质分选均有密切关系。脂筏中的小窝区域是一个去污剂不溶性膜区域，以存在小窝蛋白分子（caveolin）为特征，可参与细胞胆固醇运输，细胞膜合成，信号传导和肿瘤生成等多种细胞生命活动。目前已知的小窝蛋白有三类：caveolin-1（包括两种变异体α和β），，caveolin-2和caveolin-3。研究表明，caveolin-1具有与胆固醇结合能力，并参与了细胞内胆固醇稳态的维持和胆固醇的转运过程。甲基-β-环糊精(M-β-CD)是一种去污剂，能够将细胞膜上的胆固醇从细胞膜抽提出来，从而破坏脂筏的完整性。 细胞膜电位是由细胞膜两侧存在的离子浓度差和细胞膜对离子的通透性（离子通道）不同而形成的。不同离子通道在细胞膜上的表达和功能的不同构成了复杂而又规律的细胞电活动。跨细胞膜的电活动可以影响许多细胞活动及功能。在细胞膜有不同的感觉跨膜电活动的蛋白分子，这些分子除显而易见的离子通道蛋白分子外，还有其他一些非生电蛋白分子例如一些酶类。 在之前的研究中发现，在卵母细胞中细胞膜去极化可导致PIP2水平升高，这依赖于PKC、PI4K和PLC等酶活性的增强，在这些酶的参与下，磷脂酰肌醇（PIP2）代谢加快并在细胞膜上维持高水平状态，可以使卵母细胞中表达的、功能依赖于PIP2的KCNQ2/Q3钾电流增大。 本课题研究开始于我们观察到高渗外液能阻断细胞膜去极化增大KCNQ2/Q3通道电流的作用。细胞膜去极化导致的细胞膜PIP2合成代谢增加、KCNQ2/Q3功能增强的机制可能与高渗外液改变细胞膜物理性状而影响细胞功能的机制类似。因此，我们针对细胞膜完整性，具体是脂筏在细胞膜电位调节PIP2代谢中的作用及机制展开研究。具体研究内容如下： 目的：研究甲基-β-环糊精（M-β-CD）对KCNQ2/Q3电流的影响，由此推测M-β-CD对细胞膜PIP2代谢的影响并对其分子机制进行探索研究；了解细胞膜脂筏在细胞膜电位调节PIP2代谢中作用及分子机制。 方法：体外转录的方法制备KCNQ2和KCNQ3通道的cRNA并使之表达于爪蟾卵母细胞；使用双电极电压钳记录表达在卵母细胞上的KCNQ2/Q3电流，并观察不同实验条件下电流的变化；Western blot检测不同处理组卵母细胞中PI4K和Caveolin-1表达情况。 结果：(1) M-β-CD对表达于卵母细胞的KCNQ2/Q3电流有很强的增大作用，其无效类似物α-CD没有增加KCNQ2/Q3电流；(2)PLC阻断剂U73122，PI4K阻断剂PIK93，PKC阻断剂Bis均能阻断M-β-CD增大KCNQ2/Q3电流的的作用；(3) M-β-CD能完全阻断细胞膜去极化以及PMA对KCNQ2/Q3电流的增大作用；(4)NaCl及糖类高渗外液能阻断PMA以及M-β-CD对KCNQ2/Q3电流的增大作用，且对KCNQ2/Q3电流有轻微抑制；(5)细胞外高浓度胆固醇能阻断M-β-CD对KCNQ2/Q3电流的增大作用，且对KCNQ2/Q3电流有强的抑制作用；(6)Western blot结果显示，M-β-CD处理组细胞中PI4K含量显著高于正常对照组，而细胞中Caveolin-1比例显著减少。细胞膜去极化处理组细胞中PI4K含量显著增多但Caveolin-1比例与对照组无显著性差异。 结论：实验结果表明M-β-CD明显增大表达在卵母细胞上的KCNQ2/Q3电流，表明M-β-CD使卵母细胞膜PIP2含量增加。细胞膜去极化导致的细胞膜PIP2合成代谢增加、KCNQ2/Q3功能增强可能与M-β-CD作用的机制类似。细胞膜脂筏结构的完整性可能在细胞膜去极化导致的细胞膜PIP2合成代谢的作用中发挥重要作用。
[Abstract]:Cell membrane is an important channel for substance exchange and information transmission. After stimulation, many signal pathways occur on the cell membrane at the same time. There must be areas on the cell membrane that coordinate different pathways. Lipid raft is one of the platforms involved in signal transduction. Lipid raft is rich in cholesterol and sphingomyelin on the cell membrane. Microstructure domains are closely related to membrane signal transduction and protein sorting. The litter region in the lipid raft is a detergent-insoluble membrane region characterized by caveolin molecules, which can participate in cell cholesterol transport, cell membrane synthesis, signal transduction and tumor formation. Caveolin-1 (including two variants alpha and beta), Caveolin-2 and caveolin-3. Studies have shown that caveolin-1 binds to cholesterol and is involved in the maintenance of intracellular cholesterol homeostasis and cholesterol transport. Methyl-beta-cyclodextrin (M-beta-CD) is a detergent that removes cholesterol from cell membranes. The cell membrane is extracted to destroy the integrity of lipid rafts.
Membrane potential is formed by the difference of ion concentration on both sides of the cell membrane and the difference of ion permeability (ion channel) between the two sides of the cell membrane. Cell membranes have different protein molecules that sense transmembrane electrical activity. These molecules, in addition to the obvious ion channel proteins, have other non-electrogenic proteins such as enzymes.
Previous studies have shown that depolarization of cell membrane in oocytes leads to an increase in PIP2 levels, which is dependent on enzymatic activities such as PKC, PI4K and PLC. With the participation of these enzymes, phosphoinositide (PIP2) metabolism is accelerated and maintains a high level on the cell membrane, enabling oocytes to express and function dependent on PIP2 KCNQ2/Q. 3 potassium current increases.
This study began with our observation that hypertonic exudates could block the depolarization of cell membranes and increase the current of KCNQ2/Q3 channels. The depolarization of cell membranes resulted in the increase of PIP2 synthesis and metabolism. The mechanism of KCNQ2/Q3 enhancement may be similar to that of hypertonic exudates which altered the physical properties of cell membranes and affected cell functions. The role and mechanism of lipid rafts in the regulation of PIP2 metabolism by membrane potential were studied.
AIM: To study the effect of methyl-beta-cyclodextrin (M-beta-CD) on KCNQ2/Q3 current, and to speculate the effect of M-beta-CD on the metabolism of cell membrane PIP2 and explore its molecular mechanism.
Methods: KCNQ2 and KCNQ3 channel cRNA were transcribed in vitro and expressed in Xenopus oocytes. The KCNQ2/Q3 currents were recorded by two-electrode voltage clamp, and the changes of current under different experimental conditions were observed. The expressions of PI4K and Caveolin-1 were detected by Western blot.
Results: (1) M-beta-CD had a strong effect on KCNQ2/Q3 currents in oocytes, but its ineffective analogue alpha-CD did not increase KCNQ2/Q3 currents; (2) PLC blocker U73122, PI4K blocker PIK93 and PKC blocker Bis could block the effect of M-beta-CD on KCNQ2/Q3 currents; (3) M-beta-CD completely blocked cell membrane depolarization and P-Q3 currents. MA can increase KCNQ2/Q3 current; (4) NaCl and carbohydrate hypertonic solution can block the increase of PMA and M-beta-CD on KCNQ2/Q3 current, and slightly inhibit KCNQ2/Q3 current; (5) extracellular high concentration of cholesterol can block the increase of M-beta-CD on KCNQ2/Q3 current, and has a strong inhibition on KCNQ2/Q3 current; (6) Western blot The results showed that the content of PI4K in M-beta-CD treated cells was significantly higher than that in the normal control group, while the proportion of Caveolin-1 in cells was significantly decreased.
CONCLUSION: The results showed that M-beta-CD significantly increased KCNQ2/Q3 currents on oocytes, indicating that M-beta-CD increased the content of membrane PIP2, increased the synthesis and metabolism of membrane PIP2 induced by cell membrane depolarization, and the enhancement of KCNQ2/Q3 function may be similar to the mechanism of M-beta-CD. The integrity of membrane lipid raft structure may be fine. Membrane depolarization plays an important role in the role of cell membrane PIP2 metabolism.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
本文编号：2221964
[Abstract]:Cell membrane is an important channel for substance exchange and information transmission. After stimulation, many signal pathways occur on the cell membrane at the same time. There must be areas on the cell membrane that coordinate different pathways. Lipid raft is one of the platforms involved in signal transduction. Lipid raft is rich in cholesterol and sphingomyelin on the cell membrane. Microstructure domains are closely related to membrane signal transduction and protein sorting. The litter region in the lipid raft is a detergent-insoluble membrane region characterized by caveolin molecules, which can participate in cell cholesterol transport, cell membrane synthesis, signal transduction and tumor formation. Caveolin-1 (including two variants alpha and beta), Caveolin-2 and caveolin-3. Studies have shown that caveolin-1 binds to cholesterol and is involved in the maintenance of intracellular cholesterol homeostasis and cholesterol transport. Methyl-beta-cyclodextrin (M-beta-CD) is a detergent that removes cholesterol from cell membranes. The cell membrane is extracted to destroy the integrity of lipid rafts.
Membrane potential is formed by the difference of ion concentration on both sides of the cell membrane and the difference of ion permeability (ion channel) between the two sides of the cell membrane. Cell membranes have different protein molecules that sense transmembrane electrical activity. These molecules, in addition to the obvious ion channel proteins, have other non-electrogenic proteins such as enzymes.
Previous studies have shown that depolarization of cell membrane in oocytes leads to an increase in PIP2 levels, which is dependent on enzymatic activities such as PKC, PI4K and PLC. With the participation of these enzymes, phosphoinositide (PIP2) metabolism is accelerated and maintains a high level on the cell membrane, enabling oocytes to express and function dependent on PIP2 KCNQ2/Q. 3 potassium current increases.
This study began with our observation that hypertonic exudates could block the depolarization of cell membranes and increase the current of KCNQ2/Q3 channels. The depolarization of cell membranes resulted in the increase of PIP2 synthesis and metabolism. The mechanism of KCNQ2/Q3 enhancement may be similar to that of hypertonic exudates which altered the physical properties of cell membranes and affected cell functions. The role and mechanism of lipid rafts in the regulation of PIP2 metabolism by membrane potential were studied.
AIM: To study the effect of methyl-beta-cyclodextrin (M-beta-CD) on KCNQ2/Q3 current, and to speculate the effect of M-beta-CD on the metabolism of cell membrane PIP2 and explore its molecular mechanism.
Methods: KCNQ2 and KCNQ3 channel cRNA were transcribed in vitro and expressed in Xenopus oocytes. The KCNQ2/Q3 currents were recorded by two-electrode voltage clamp, and the changes of current under different experimental conditions were observed. The expressions of PI4K and Caveolin-1 were detected by Western blot.
Results: (1) M-beta-CD had a strong effect on KCNQ2/Q3 currents in oocytes, but its ineffective analogue alpha-CD did not increase KCNQ2/Q3 currents; (2) PLC blocker U73122, PI4K blocker PIK93 and PKC blocker Bis could block the effect of M-beta-CD on KCNQ2/Q3 currents; (3) M-beta-CD completely blocked cell membrane depolarization and P-Q3 currents. MA can increase KCNQ2/Q3 current; (4) NaCl and carbohydrate hypertonic solution can block the increase of PMA and M-beta-CD on KCNQ2/Q3 current, and slightly inhibit KCNQ2/Q3 current; (5) extracellular high concentration of cholesterol can block the increase of M-beta-CD on KCNQ2/Q3 current, and has a strong inhibition on KCNQ2/Q3 current; (6) Western blot The results showed that the content of PI4K in M-beta-CD treated cells was significantly higher than that in the normal control group, while the proportion of Caveolin-1 in cells was significantly decreased.
CONCLUSION: The results showed that M-beta-CD significantly increased KCNQ2/Q3 currents on oocytes, indicating that M-beta-CD increased the content of membrane PIP2, increased the synthesis and metabolism of membrane PIP2 induced by cell membrane depolarization, and the enhancement of KCNQ2/Q3 function may be similar to the mechanism of M-beta-CD. The integrity of membrane lipid raft structure may be fine. Membrane depolarization plays an important role in the role of cell membrane PIP2 metabolism.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
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1 张熙东;甲基-β-环糊精及脂筏对细胞膜磷脂PIP2代谢的影响及分子机制研究[D];河北医科大学;2014年
本文编号：2221964
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