醛固酮快速调控肾上皮细胞钠通道作用的研究
发布时间:2018-02-04 22:54
本文关键词: 醛固酮 EaNC mpkCCD TEER SICM 出处:《中国人民解放军军事医学科学院》2012年博士论文 论文类型:学位论文
【摘要】:研究目的:醛固酮在调节细胞外液,维持电解质平衡以及控制血压稳定的过程中发挥着重要的作用,醛固酮重要的生理功能有赖于通过调控肾上皮钠通道(ENaC)对钠的重吸收,目前已知相关的作用机制分为基因组作用和非基因组作用。其中,研究较清楚的是醛固酮的基因组作用方式:醛固酮进入集合管主细胞后,与胞浆内的盐皮质激素受体(mineralocorticoid receptor,MR)结合,形成激素-受体复合体,后者进入细胞核与核中DNA特异性结合位点相互作用,调节特异性mRNA转录,最终合成多种醛固酮诱导蛋白(aldosterone-induced protein, AIP),使管腔膜(apicalside)ENaC活性增强,基侧膜(basolateral side)钠钾泵的活性增加,从而促进跨上皮细胞的Na+重吸收。醛固酮基因组作用的特点是作用起效较慢,作用时间长,对MR阻断剂敏感。此外,醛固酮还可发挥快速的非基因组作用来调控Na+的重吸收,此作用的特点为起效迅速,对MR阻断剂不敏感,但其内在机制还知之甚少。因此研究醛固酮非依赖MR快速调控ENaC的作用及其相关的作用机制,有助于进一步全面了解醛固酮调节ENaC作用的生理、病理意义,同时也可促进新型醛固酮拮抗药物的研发,为醛固酮快速作用提供可能的应对措施。 主要内容: 1建立具有ENaC活性的哺乳动物肾上皮细胞模型并进行评价。 2研究醛固酮快速调控ENaC的作用。 3探讨醛固酮快速调控ENaC的可能机制。 研究方法: 1.实验细胞模型的建立 建立具有ENaC活性的哺乳动物肾上皮细胞模型-选择MDCK(Madin-Darbycanine kidney)、mpkCCD(mouse principle cell of kidney in cortical collecting duct)两种细胞株,培养成具有完整膜,有极性,高阻抗,阿米洛利敏感的细胞模型,最后通过观察细胞形态结构、测量跨膜电阻值、测量细胞单通道记录三个方面来评估,择优选出适合进一步实验的理想细胞模型。 2.醛固酮对ENaC的快速调控作用 给予醛固酮(10-6M/L)作用于mpkCCD细胞模型,观察醛固酮对细胞形态结构、阿米洛利敏感的跨膜电阻、胞内钙离子浓度、单通道离子电流的影响。使用MR阻断剂螺内酯以及醛固酮转录、蛋白合成抑制剂,验证醛固酮对ENaC快速(<3小时)调节作用主要是非依赖MR的非基因组作用。 3.醛固酮对ENaC作用机制的研究 (1)通过使用PI3-K通路特异性的阻断剂LY294002(50um/L)验证PI3-K通路在醛固酮快速调控ENaC过程中的作用。 (2)通过使用钙离子载体A23187(1um/L)快速增加胞内钙离子浓度来探讨胞内钙离子在醛固酮调控快速调控ENaC过程中的作用。 (3)通过使用细胞松弛素D(Cyt D)打断细胞骨架F-actin,探讨细胞形态与ENaC功能活性之间的关系。 4.主要的指标及测量方法 (1)细胞形态学观察:扫描离子电导显微镜(SICM) (2)跨膜电阻值:跨膜电阻仪(EVOM2) (3)单通道离子电流:膜片钳cell-attached单通道记录 (4)胞内钙离子:高速比率钙离子浓度测量荧光显微技术 结果: 1.建立了具有ENaC活性哺乳动物肾上皮细胞模型-mpkCCD细胞模型。 2.醛固酮作用于mpkCCD细胞,细胞发生横向收缩纵向伸展,胞内钙离子浓度升高,整体膜和单通道水平的ENaC活性增强,表现为阿米洛利敏感的跨膜电阻升高和离子通道开放概率增加。 3. LY294002能够阻断醛固酮对ENaC的激活作用,表现为细胞恢复原貌,阿米洛利敏感的跨膜电阻降低和离子通道开放概率减少。 4. A23187能迅速增加胞内钙离子浓度,并增强ENaC活性,表现在阿米洛利敏感的跨膜电阻升高和离子通道开放概率增加。 5. Cyt D能使细胞发生横向收缩纵向伸展的形变,ENaC活性增强,表现为阿米洛利敏感的跨膜电阻升高和离子通道开放概率增加。 结论: 1.醛固酮能够快速增强ENaC活性。 2.醛固酮快速增强ENaC活性作用机制可能是通过快速增加胞内钙离子浓度,,激活PI3-K通路,使细胞发生横向收缩纵向伸展的形变,从而增加的离子通道的开放概率。 3. Cyt D能使细胞发生形变(横向收缩纵向伸展),ENaC活性增强,说明细胞骨架F-actin解聚有利于提高离子通道开放概率。
[Abstract]:Research purposes: aldosterone in the regulation of extracellular fluid, plays an important role in maintaining electrolyte balance and control of blood pressure, the physiological function of aldosterone depends on important through the regulation of renal epithelial sodium channel (ENaC) on sodium reabsorption, currently known mechanisms related to genomic and non genomic effects into effect. The research is clear, the mode of action of aldosterone genome: aldosterone into the collection of the main cell, mineralocorticoid receptor and intracellular (mineralocorticoid receptor, MR) with the formation of hormone receptor complex, which enters the nucleus and nuclear DNA specific binding site interaction, transcriptional regulation of specific mRNA, the final the synthesis of a variety of aldosterone induced protein (aldosterone-induced protein, AIP), the luminal membrane (apicalside) enhanced ENaC activity, the basolateral membrane (basolateral side) sodium potassium pump The activity increased, thereby promoting trans epithelial cells Na + reabsorption. Characteristics of aldosterone genomic effects is slow onset, long duration of action, sensitive to MR blocking agent. In addition, aldosterone can also play rapid nongenomic effects to control the reabsorption of Na+, the characteristics of this effect is not sensitive to the rapid onset of agent the MR block, but its mechanism is still poorly understood. So the study of aldosterone independent MR rapid regulation of ENaC activity and its related mechanisms, physiology, help to further understand the role of ENaC in regulating aldosterone pathological significance, but also can promote the development of new aldosterone antagonist drugs, provide the possible measures to respond to rapid effects of aldosterone.
Main contents:
1 the model of mammalian renal epithelial cells with ENaC activity was established and evaluated.
2 study the effect of aldosterone on the rapid regulation of ENaC.
3 to investigate the possible mechanism of aldosterone regulation for ENaC.
Research methods:
The establishment of 1. experimental cell model
The establishment of the mammalian kidney epithelial cell model with ENaC activity MDCK (Madin-Darbycanine kidney), mpkCCD (mouse principle cell of kidney in cortical collecting duct) two cell lines, cultured with complete membrane polarity, high impedance, amiloride sensitive cell model, finally through the observation of cell morphology, measurement of membrane the resistance value measuring cell single channel recording in three aspects to evaluate, select the ideal cell model for further experiments.
The effect of 2. aldosterone on the rapid regulation of ENaC
Administration of aldosterone (10-6M/L) in mpkCCD cells on the cell structure model, we observed the effects of amiloride sensitive, membrane resistance, intracellular calcium concentration, influence of single channel current. The use of MR antagonist spironolactone, aldosterone transcription, protein synthesis inhibitor, verification of aldosterone on ENaC fast (< 3 hours) regulation is the main non genomic effects independent of MR.
Study on the mechanism of the action of 3. aldosterone to ENaC
(1) the use of the PI3-K pathway specific blocking agent LY294002 (50um/L) to verify the role of the PI3-K pathway in the rapid regulation of ENaC by aldosterone.
(2) by using calcium ionophore A23187 (1um/L) to rapidly increase intracellular calcium concentration, we explored the role of intracellular calcium in aldosterone fast regulation of ENaC.
(3) the relationship between cell morphology and ENaC functional activity was explored by interrupting cytoskeleton F-actin by using cytochalasin D (Cyt D).
4. main indexes and measurement methods
(1) morphological observation of cell: scanning ion conductance microscope (SICM)
(2) transmembrane resistance value: transmembrane resistor (EVOM2)
(3) single channel ion current: single channel recording of patch clamp cell-attached
(4) intracellular calcium ion: high speed ratio calcium ion concentration measurement fluorescence microscopy
Result锛
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