药理调节剂及细胞体积调节氯通道的作用特征及机制研究

发布时间：2018-06-03 20:19

  本文选题：钙激活氯通道(CaCCs) + ANO1　； 参考：《河北医科大学》2015年博士论文

【摘要】：钙激活氯通道(calcium-activated chloride channels,CaCCs)是一类阴离子通道,具有钙离子和电压依赖性。其组织分布广泛,参与了众多生理过程,包括调节上皮细胞的分泌,参与嗅觉、视觉的信号转导,调节平滑肌、心肌、神经元的兴奋性,还与肿瘤的发生发展存在密切的联系。其生理病理学意义十分重要,是重要的药物的研究靶点。CaCCs的分子基础一直备受争议,直到近些年大家才逐渐公认两大家族蛋白,八跨膜蛋白ANO1和四跨膜蛋白Bestrophin1可能是CaCCs的分子基础。当两者表达于表达系统时都呈现CaCCs特性,如对钙离子和电压的敏感性。因为缺乏选择性药理学调节剂,对特定组织所观察到的CaCCs究竟是由ANO1或是Bestrophin1构成难以判断。氯通道调节剂众多,但缺乏选择性,且研究使用较为混乱。本研究第一部分就针对此现状,在建立稳定表达钙激活氯通道ANO1及Bestrophin1的中国仓鼠卵母(CHO)细胞系的基础上观察比较7种氯通道调节剂对ANO1及Bestrophin1通道的效能、效价及动力学特征,为进一步深入研究内源性CaCCs以及设计研发特异性通道调节剂提供强有力的支持。容积调节氯通道(volume regulated chloride channel,VRCC)是一类组织分布广泛,对维持细胞容积稳定具有重要生理学意义的阴离子通道。VRCC还与细胞增殖、细胞周期和细胞凋亡等生理过程密切相关,并且已经发现在心律失常、心肌缺血再灌注损伤以及充血性心力衰竭等病理状态中发挥重要作用。VRCC的分子基础研究一直进展缓慢,研究者曾认为P-糖蛋白、核酸敏感性氯通道蛋白、电压依赖性氯通道ClC-2和ClC-3以及钙激活氯通道ANO1和Bestrophin等可能是参与构成VRCC的分子基础。2014年,两个研究小组几乎同时发现LRRC8蛋白家族参与构成了VRCC。本研究第二部分主要利用人胚肾上皮(HEK)以及中国仓鼠卵母(CHO)细胞观察VRCC的分子基础及药理学特征,重点集中在ANO1和LRRC8A上。VRCC受细胞肿胀激活的机制尚不清楚,且其激活机制和调节因素在不同的细胞中有所不同。有研究报道称细胞内钙、酪氨酸介导的蛋白磷酸化、丝裂原激酶和酪氨酸激酶、小g蛋白rhoa以及活性氧(ros)均可能都参与了vrcc的激活过程。本研究第三部分主要研究hek293a细胞内源性vrcc的激活机制,重点研究细胞内钙离子在vrcc激活中的作用。论文具体内容如下:第一部分氯通道调节剂对钙激活氯通道ano1和bestrophin1作用的研究目的:研究比较几种常见氯通道调节剂对钙激活氯通道ano1和bestrophin1的作用。方法:1建立稳定表达ano1和bestrophin1的cho细胞系;2利用膜片钳技术观察比较不同氯通道调节剂对ano1及bestrophin1通道的药理学作用及对通道动力学影响的特征。结果:1westernblot实验结果显示稳定转染ano1的cho细胞系(cho-ano1)和稳定转染bestrophin1的cho细胞系(cho-best1)细胞膜上目的蛋白表达都明显增加。膜片钳实验结果显示,在cho-ano1以及cho-best1细胞系均能成功记录到明显的钙离子依赖性的外向整流电流。2膜片钳研究结果发现7种常用氯通道阻断剂中,对于+80mv下的外向电流,dids对bestrophin1(ic50of3.93±0.73μm)的选择性远高于ano1(ic50of548.86±25.57μm);而nfa对ano1(ic50of7.40±0.95μm)的选择性远高于bestrophin1(ic50of102.19±15.05μm)。caccinh-a01对ano1和bestrophin1通道的作用都比较强且效价相近,ic50分别是7.84±0.62μm和7.15±0.65μm。t16ainh-a01可抑制部分ano1电流,而对bestrophin1电流几乎无作用。鞣酸(tannicacid)、nppb和ffa对两者的抑制作用接近。3nfa、ffa以及nppb对-80mv下记录的ano1内向电流有双向作用,低浓度(100μm)可增大内向电流,而高浓度(100μm)呈现抑制作用。nfa,ffa及nppb可明显改变ano1的通道动力学特征,使其去活变慢。结论:1成功建立了稳定表达ano1和bestrophin1的cho细胞系;2七种常用氯通道阻断剂中,dids对bestrophin1的选择性远高于ano1;而nfa对ano1的选择性远高于bestrophin1。caccinh-a01对两者的作用都比较强且效价相近。3nfa,ffa及nppb对ano1内向电流具有双向作用;且可明显改变ano1的通道动力学特征,使其去活变慢。第二部分钙激活氯通道ano1参与构成容积调节氯通道分子基础的研究目的:记录稳定转染ano1的hek293或cho细胞及基因敲除lrrc8a或ano1的hek293细胞中容积调节的氯电流并分析ano1是否参与构成vrcc的分子基础。方法:1使用420mosm的cscl电极内液与320mosm的nacl细胞外液,利用全细胞膜片钳技术记录cho或hek细胞以及稳定转染ano1的cho或hek细胞中vrcc电流;2利用药理学抑制剂观察药物对vrcc电流的作用;3利用crispr/cas9技术构建lrrc8a基因以及ano1基因敲除的hek293a细胞系,观察敲除细胞中vrcc电流;4在lrrc8a敲除细胞中外源转染ano1后,观察记录vrcc。结果:1当电极内液为420mosmcscl,细胞外液为320mosmnacl时,随着细胞容积的增大,可记录到明显的vrcc电流。当hek(cho)细胞膜电位钳制在-60mv时,平均电流密度约为64pa/pf(cho约为48pa/pf)。给予-100mv到+100mv的ramp电压钳制程序时,vrcc呈现明显的外向整流特性。而稳定转染ano1的细胞(hek-mano1)电流密度在-60mv时约为113pa/pf(cho-mano1约为63pa/pf),明显大于未转染ano1细胞的电流。另外,当使用跃阶(step)去极化电压刺激模式时,hek或cho细胞的vrcc电流呈现快速激活且快速去活的特征,当膜电位高于+60mv时出现失活现象。而稳定转染ano1的细胞的vrcc电流则呈现慢激活无失活的电流特征。2第一部分研究发现nfa作为ano1电流的强效抑制剂,100μmnfa可抑制约90%的ano1电流。此部分实验中发现100μmnfa对稳定表达ano1的细胞的vrcc电流呈现部分抑制作用,所抑制的电流表现为慢激活非失活的cacc样电流;10μmdcpib是vrcc电流的相对特异性阻断剂,对稳定表达ano1细胞的vrcc电流呈现部分抑制作用,所抑制的电流呈现快激活且失活的lrcc8a样电流。3基因敲除hek细胞内源性ano1基因后,vrcc电流明显减小。而敲除内源性lrrc8a基因后,vrcc电流基本消失。然而在敲除lrrc8a的细胞中外源转染ano1后,高渗内液和等渗条件下的gtpγs可部分恢复vrcc电流,呈现ano1电流特征,表现为慢激活非失活,以及明显外向整流特征。结论:1稳定转染ano1后的细胞vrcc电流明显增加;敲除内源性ano1基因后的细胞vrcc电流明显降低。2敲除lrrc8a基因后,vrcc电流基本消失,再外源表达ano1可部分恢复,说明ano1可被细胞肿胀激活,可能是参与构成vrcc的分子基础。第三部分容积调节氯通道vrcc的激活机制目的:研究hek293a细胞内源性vrcc的激活机制。方法:1利用全细胞膜片钳技术记录hek293a细胞中的vrcc电流;2利用激光扫描共聚焦显微镜观察细胞内钙信号的变化;3利用plc通路中相关分子的阻断剂,以及经sirna沉降plc后,观察对vrcc电流的影响;4利用激光扫描共聚焦显微镜观察细胞膜pip2的水解情况。结果:1使用高渗(420mosmcscl)的电极内液,在hek293a细胞记录到的vrcc电流有震荡的现象;vrcc电流的失活与电流幅度有关,表现为电流越小失活特征越明显。使用等渗内液,低渗(220mosm)外液记录到的vrcc电流有与上述描述相似特征。2低渗外液可诱导细胞内震荡性钙升高,且不被细胞外液中加入egta(2mm,降低游离钙)或cdcl2(100μm,阻断钙内流)所影响。3高渗电极内液中加入20mmegta可抑制vrcc电流的震荡现象,但不影响最大电流幅度;而加入20mmbapta,可明显抑制vrcc电流;同样使用thapasigagin预处理细胞使细胞内钙耗竭后,vrcc电流基本消失。43‰的血清可诱发一过性氯电流,此电流可被dcpib或caccinh-a01基本完全抑制,同时血清还可引起一过性的细胞内钙浓度升高。5使用plc阻断剂u73122(5μm)处理细胞,vrcc的电流会显著减小;而u73122的无效结构类似物u73343(5μm)对vrcc电流无明显作用。6hek293a细胞转染pip2特异性荧光探针tubby-yfp,进行激光共聚焦实验,结果显示低渗细胞外液使荧光强度明显减弱。7ip3阻断剂xe-c(1μm)可明显抑制vrcc电流,pkc抑制剂bis-1(200nm)则对vrcc电流无明显作用。高渗内液中加入gdp-βs(500μm)不会影响vrcc电流。8使用sirna干扰技术,使plcβ4、plcγ1以及plcδ3表达降低之后,vrcc电流明显减小;plcβ3和plcε1表达降低对vrcc电流无明显作用。结论:1hek293a细胞的容积调节氯电流存在震荡现象,且随着电流的增大,电流失活变慢。2 HEK293A细胞中容积调节氯通道的开放依赖于细胞内钙,尤其是细胞内局部钙离子的升高。3 HEK293A细胞中容积调节氯通道的激活依赖于PLC的激活,且PLCγ1、PLCβ4以及PLCδ3亚型可能参与其中。4 G蛋白和PKC不参与HEK293A细胞中容积调节氯通道的激活。
[Abstract]:Calcium activated calcium-activated chloride channels (CaCCs) is a class of anionic channel, which has calcium ion and voltage dependence. Its tissue is widely distributed and participates in many physiological processes, including regulating the secretion of epithelial cells, participating in the olfactory, visual signal transduction, regulating the excitability of the smooth muscle, myocardium, neuron, and tumor. There is a close relationship between development and development. Its physiological and pathological significance is very important. The molecular basis of the target.CaCCs is controversial. Until recently, two large family proteins have been recognized, and eight transmembrane protein ANO1 and four transmembrane protein Bestrophin1 may be the molecular basis of CaCCs. The system presents CaCCs characteristics, such as the sensitivity to calcium ions and voltages. Because of the lack of selective pharmacological regulators, it is difficult to judge whether the CaCCs observed by specific tissues is composed of ANO1 or Bestrophin1. There are many chlorine channel regulators, but lack of selectivity, and the research is confused. The first part of this study is aimed at On the basis of establishing a Chinese hamster egg (CHO) cell line that expresses calcium activated chloride channel ANO1 and Bestrophin1, the efficacy, potency and kinetic characteristics of 7 chlorine channel regulators on ANO1 and Bestrophin1 channels are compared and compared. Strong support. Volume regulated chloride channel (VRCC) is a wide range of tissue distribution. The anionic channel.VRCC, which has an important physiological significance for maintaining cell volume stability, is also closely related to cell proliferation, cell cycle and apoptosis, and has been found in arrhythmia and myocardial deficiency. The molecular basis of.VRCC, which plays an important role in the pathological state of blood reperfusion injury and congestive heart failure, has been progressing slowly. Researchers have thought that P- glycoprotein, nucleic acid sensitive chloride channel protein, voltage dependent chloride channel ClC-2 and ClC-3, and calcium activated chloride channel ANO1 and Bestrophin may be part of the component of VRCC. The sub base.2014, two research teams almost simultaneously found that the LRRC8 protein family was involved in the formation of VRCC., the second part mainly used the human embryonic renal epithelium (HEK) and the Chinese hamster egg mother (CHO) cells to observe the molecular basis and pharmacological characteristics of VRCC. The mechanism of.VRCC activated by the.VRCC cell swelling on ANO1 and LRRC8A is not clear. It is reported that intracellular calcium, tyrosine mediated protein phosphorylation, mitogen kinase and tyrosine kinase, small G protein RhoA and reactive oxygen species (ROS) may all participate in the activation process of VRCC. The third part of this study mainly studies the endogenous hek293a cell origin. The activation mechanism of sexual VRCC is focused on the role of intracellular calcium in the activation of VRCC. The main contents of this paper are as follows: the first part of the study is the effect of chlorine channel regulator on calcium activated chlorine channel Ano1 and bestrophin1. The purpose of this study is to compare the effects of several common chloride channel regulators on calcium activated chloride channel Ano1 and bestrophin1. Methods: 1 The CHO cell lines that stably express Ano1 and bestrophin1; 2 the pharmacological effects of different chloride channel regulators on the Ano1 and bestrophin1 channels and the characteristics of channel dynamics are compared with the patch clamp technique. Results: the results of 1westernblot experiment showed the stable transfection of Ano1 CHO cell line (cho-ano1) and Cho transfection bestrophin1 Cho The expression of the target protein on the cell line (cho-best1) cell membrane was significantly increased. The patch clamp results showed that the cho-ano1 and cho-best1 cell lines could be successfully recorded to the obvious calcium dependent extroverted rectifying current.2 patch clamp research results found in 7 commonly used chlorine channel blockers, for the extroverted current under +80mv, DIDS to bes The selectivity of trophin1 (ic50of3.93 + 0.73 m) is much higher than that of Ano1 (ic50of548.86 + 25.57 m), while the selectivity of NFA to Ano1 (ic50of7.40 + 0.95 micron m) is far higher than bestrophin1 (ic50of102.19 + 15.05 micron), which is stronger and more effective than 7.84 + 0.62 Mu and 7.15 + 0.65 Mu respectively. The partial Ano1 current can be suppressed, but the bestrophin1 current is almost no effect. The inhibitory effect of tannic acid (tannicacid), NPPB and FFA on both is close to.3nfa. FFA and NPPB have two-way action on the Ano1 inward current recorded in -80mv, and the low concentration (100 mu m) can increase the inward current, while the high concentration (100 mu m) exhibits a inhibition effect. Change the dynamic characteristics of Ano1 channel to make it slow. Conclusion: 1 the CHO cell line that stably expresses Ano1 and bestrophin1 is successfully established, and 2 of seven common chlorine channel blockers, the selectivity of DIDS to bestrophin1 is far higher than that of Ano1, and NFA's selectivity to Ano1 is much higher than that of bestrophin1.caccinh-a01. Near.3nfa, FFA and NPPB have bi-directional effect on the Ano1 inward current, and can obviously change the channel dynamics of Ano1 and make it deactivate. The second part of calcium activated chloride channel Ano1 participates in the study of the molecular basis of the volume regulated chloride channel: recording the stable transfection of Ano1 HEK293 or CHO cells and gene knockout lrrc8a or Ano1 HEK293 Volume regulated chlorine currents in cells and analysis of whether Ano1 participates in the molecular basis of VRCC. Methods: 1 using 420mosm CSCL electrode internal liquid and 320mosm NaCl extracellular fluid, full cell patch clamp technique is used to record Cho or HEK cells as well as VRCC currents in CHO or HEK cells that stably transfect Ano1, and 2 use pharmacological inhibitors to observe drug pairs. The effect of VRCC current; 3 using crispr/cas9 technology to construct lrrc8a gene and Ano1 gene knockout hek293a cell line and observe the VRCC current in knockout cells. 4 after transfection of Ano1 in lrrc8a knockout cells, the results of vrcc. are observed: 1 when the liquid in the electrode is 420mosmcscl, and the extracellular fluid is 320mosmnacl, as the cell volume increases, An obvious VRCC current is recorded. When the HEK (CHO) cell membrane potential is clamped at -60mv, the average current density is about 64pa/pf (CHO is about 48pa/pf). When the ramp voltage clamp program is given to -100mv to +100mv, the VRCC appears extroverted rectifying characteristics. About 63pa/pf), obviously greater than the current of untransfected Ano1 cells. In addition, when the step (step) depolarizing voltage stimulation mode is used, the VRCC current of the HEK or CHO cells is characterized by rapid activation and rapid deactivation. When the membrane potential is higher than +60mv, the deactivation phenomenon occurs. The VRCC current of the cells that stabilize the transfected Ano1 presents slow activation and inactivation. The first part of the current characteristic.2 found that NFA is a powerful inhibitor of Ano1 current, and 100 mu mnfa can inhibit the Ano1 current of about 90%. This part of the experiment shows that 100 mu mnfa has a partial inhibitory effect on the VRCC current that stably expressed Ano1, and the suppressed current is a slow excited non inactivated CACC sample current; 10 mu mdcpib is a VRCC electricity. The relative specific blocking agent of the flow showed a partial inhibitory effect on the VRCC current of the stable expression of Ano1 cells. The suppressed current presented fast activation and the deactivated lrcc8a like current.3 gene knocked out the endogenous Ano1 gene of HEK cells, and the VRCC current decreased obviously. The VRCC current disappeared basically after knocking out endogenous lrrc8a gene. However, the current was knocked out of lrrc. After exogenous transfection of Ano1 in 8A cells, the hypertonic internal fluid and GTP gamma S under the isoosmotic condition could partially restore the VRCC current, showing the characteristics of Ano1 current, showing the slow activation non inactivation, and the obvious outward rectifying characteristics. Conclusion: 1 the VRCC current after the stable transfection of Ano1 obviously increases, and the VRCC current of the cells after the knockout of the endogenous Ano1 gene is obviously reduced. After.2 knockout lrrc8a gene, the VRCC current basically disappeared, and then the exogenous expression of Ano1 could be partially restored, indicating that Ano1 could be activated by cell swelling and may be a molecular basis for the formation of VRCC. Third part of the activation mechanism of VRCC in the volume regulated chloride channel: study the activation mechanism of endogenous VRCC in hek293a cells. Method: 1 using whole cell patch clamp Technology recorded the VRCC current in hek293a cells; 2 the changes in intracellular calcium signals were observed by laser scanning confocal microscopy; 3 the effect of the VRCC current was observed with the blockers of the related molecules in the PLC pathway and PLC after siRNA sedimentation; 4 the hydrolysis of the cell membrane PIP2 was observed by laser scanning confocal microscopy. Results: 1 use The VRCC current recorded by the hek293a cell is oscillatory in the electrode of hypertonic (420mosmcscl) electrode; the inactivation of the VRCC current is related to the amplitude of the current, the more obvious the inactivation characteristic of the current is, the less the current is, the VRCC current recorded by the isosotic (220mosm) external liquid has the same characteristics as the above description.2 hypotonic liquid can induce the cells. The increase of internal concussion calcium, and the addition of EGTA (2mm, free calcium) or CdCl2 (100 mu m, blocking calcium internal flow) to the internal liquid of the.3 hypertonic electrode can inhibit the oscillation of the VRCC current in the.3 hypertonic electrode, but does not affect the maximum current amplitude, but the addition of 20mmbapta can obviously inhibit the VRCC current. The same thapasigagin preconditioning is also used. After the cells make the intracellular calcium depletion, the VRCC current basically disappears.43 per thousand serum can induce an excessive chlorine current, which can be completely suppressed by dcpib or caccinh-a01, and the serum can also cause an excessive intracellular calcium concentration,.5 using PLC blocker u73122 (5 u m) at u73122 (5 u m), the current will decrease significantly; and u73122 no The effect structure analogue u73343 (5 mu m) had no obvious effect on the VRCC current,.6hek293a cells transfected with PIP2 specific fluorescent probe tubby-yfp, and the laser confocal experiment was carried out. The results showed that the fluorescence intensity of the hypotonic cells weakened the.7ip3 blocker xe-c (1 u m), and the VRCC current was obviously inhibited. The PKC Inhibitor BIS-1 (BIS-1) had no obvious current. The addition of gdp- beta s (500 u m) in the hypertonic liquid does not affect the VRCC current.8 using siRNA interference technology, and the VRCC current decreases obviously after the expression of PLC beta 4, PLC gamma 1 and PLC delta 3, PLC beta 3 and PLC epsilon 1 have no obvious effect on the current. Enlargement, inactivation of current in.2 HEK293A cells, the opening of volume regulated chlorine channels depends on intracellular calcium, especially intracellular calcium ions, and the activation of volume regulated chloride channels in.3 HEK293A cells depends on the activation of PLC, and PLC gamma 1, PLC beta 4, and PLC delta 3 subtypes can participate in.4 G protein and PKC do not participate in HEK293A cells. Medium volume regulates the activation of the chloride channel.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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