發(fā)布時(shí)間：2018-02-04 22:54

  本文關(guān)鍵詞： 醛固酮 EaNC mpkCCD TEER SICM　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文　論文類型：學(xué)位論文

【摘要】：研究目的：醛固酮在調(diào)節(jié)細(xì)胞外液，維持電解質(zhì)平衡以及控制血壓穩(wěn)定的過(guò)程中發(fā)揮著重要的作用，醛固酮重要的生理功能有賴于通過(guò)調(diào)控腎上皮鈉通道（ENaC）對(duì)鈉的重吸收，目前已知相關(guān)的作用機(jī)制分為基因組作用和非基因組作用。其中，研究較清楚的是醛固酮的基因組作用方式：醛固酮進(jìn)入集合管主細(xì)胞后，與胞漿內(nèi)的鹽皮質(zhì)激素受體(mineralocorticoid receptor，MR)結(jié)合，形成激素-受體復(fù)合體，后者進(jìn)入細(xì)胞核與核中DNA特異性結(jié)合位點(diǎn)相互作用，調(diào)節(jié)特異性mRNA轉(zhuǎn)錄，最終合成多種醛固酮誘導(dǎo)蛋白(aldosterone-induced protein, AIP)，使管腔膜(apicalside)ENaC活性增強(qiáng)，基側(cè)膜(basolateral side)鈉鉀泵的活性增加，從而促進(jìn)跨上皮細(xì)胞的Na＋重吸收。醛固酮基因組作用的特點(diǎn)是作用起效較慢，作用時(shí)間長(zhǎng)，對(duì)MR阻斷劑敏感。此外,醛固酮還可發(fā)揮快速的非基因組作用來(lái)調(diào)控Na+的重吸收，此作用的特點(diǎn)為起效迅速,對(duì)MR阻斷劑不敏感,但其內(nèi)在機(jī)制還知之甚少。因此研究醛固酮非依賴MR快速調(diào)控ENaC的作用及其相關(guān)的作用機(jī)制，有助于進(jìn)一步全面了解醛固酮調(diào)節(jié)ENaC作用的生理、病理意義，同時(shí)也可促進(jìn)新型醛固酮拮抗藥物的研發(fā)，為醛固酮快速作用提供可能的應(yīng)對(duì)措施。 主要內(nèi)容： 1建立具有ENaC活性的哺乳動(dòng)物腎上皮細(xì)胞模型并進(jìn)行評(píng)價(jià)。 2研究醛固酮快速調(diào)控ENaC的作用。 3探討醛固酮快速調(diào)控ENaC的可能機(jī)制。 研究方法： 1．實(shí)驗(yàn)細(xì)胞模型的建立 建立具有ENaC活性的哺乳動(dòng)物腎上皮細(xì)胞模型-選擇MDCK（Madin-Darbycanine kidney）、mpkCCD（mouse principle cell of kidney in cortical collecting duct）兩種細(xì)胞株，培養(yǎng)成具有完整膜，有極性，高阻抗，阿米洛利敏感的細(xì)胞模型,最后通過(guò)觀察細(xì)胞形態(tài)結(jié)構(gòu)、測(cè)量跨膜電阻值、測(cè)量細(xì)胞單通道記錄三個(gè)方面來(lái)評(píng)估，擇優(yōu)選出適合進(jìn)一步實(shí)驗(yàn)的理想細(xì)胞模型。 2．醛固酮對(duì)ENaC的快速調(diào)控作用 給予醛固酮(10-6M/L)作用于mpkCCD細(xì)胞模型，觀察醛固酮對(duì)細(xì)胞形態(tài)結(jié)構(gòu)、阿米洛利敏感的跨膜電阻、胞內(nèi)鈣離子濃度、單通道離子電流的影響。使用MR阻斷劑螺內(nèi)酯以及醛固酮轉(zhuǎn)錄、蛋白合成抑制劑，驗(yàn)證醛固酮對(duì)ENaC快速（＜3小時(shí)）調(diào)節(jié)作用主要是非依賴MR的非基因組作用。 3．醛固酮對(duì)ENaC作用機(jī)制的研究 （1）通過(guò)使用PI3-K通路特異性的阻斷劑LY294002(50um/L)驗(yàn)證PI3-K通路在醛固酮快速調(diào)控ENaC過(guò)程中的作用。 （2）通過(guò)使用鈣離子載體A23187（1um/L）快速增加胞內(nèi)鈣離子濃度來(lái)探討胞內(nèi)鈣離子在醛固酮調(diào)控快速調(diào)控ENaC過(guò)程中的作用。 （3）通過(guò)使用細(xì)胞松弛素D(Cyt D)打斷細(xì)胞骨架F-actin,探討細(xì)胞形態(tài)與ENaC功能活性之間的關(guān)系。 4．主要的指標(biāo)及測(cè)量方法 （1）細(xì)胞形態(tài)學(xué)觀察：掃描離子電導(dǎo)顯微鏡（SICM） （2）跨膜電阻值：跨膜電阻儀（EVOM2） （3）單通道離子電流：膜片鉗cell-attached單通道記錄 （4）胞內(nèi)鈣離子:高速比率鈣離子濃度測(cè)量熒光顯微技術(shù) 結(jié)果： 1.建立了具有ENaC活性哺乳動(dòng)物腎上皮細(xì)胞模型-mpkCCD細(xì)胞模型。 2.醛固酮作用于mpkCCD細(xì)胞，細(xì)胞發(fā)生橫向收縮縱向伸展，胞內(nèi)鈣離子濃度升高，整體膜和單通道水平的ENaC活性增強(qiáng)，表現(xiàn)為阿米洛利敏感的跨膜電阻升高和離子通道開放概率增加。 3. LY294002能夠阻斷醛固酮對(duì)ENaC的激活作用，表現(xiàn)為細(xì)胞恢復(fù)原貌，阿米洛利敏感的跨膜電阻降低和離子通道開放概率減少。 4. A23187能迅速增加胞內(nèi)鈣離子濃度，并增強(qiáng)ENaC活性，表現(xiàn)在阿米洛利敏感的跨膜電阻升高和離子通道開放概率增加。 5. Cyt D能使細(xì)胞發(fā)生橫向收縮縱向伸展的形變，ENaC活性增強(qiáng)，表現(xiàn)為阿米洛利敏感的跨膜電阻升高和離子通道開放概率增加。 結(jié)論： 1.醛固酮能夠快速增強(qiáng)ENaC活性。 2.醛固酮快速增強(qiáng)ENaC活性作用機(jī)制可能是通過(guò)快速增加胞內(nèi)鈣離子濃度，，激活PI3-K通路，使細(xì)胞發(fā)生橫向收縮縱向伸展的形變，從而增加的離子通道的開放概率。 3. Cyt D能使細(xì)胞發(fā)生形變（橫向收縮縱向伸展），ENaC活性增強(qiáng)，說(shuō)明細(xì)胞骨架F-actin解聚有利于提高離子通道開放概率。
[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錛

本文編號(hào)：1491384