本文選題：鈣激活氯通道(CaCCs) + ANO1　； 參考：《河北醫(yī)科大學(xué)》2015年博士論文

【摘要】：鈣激活氯通道(calcium-activated chloride channels,CaCCs)是一類陰離子通道,具有鈣離子和電壓依賴性。其組織分布廣泛,參與了眾多生理過程,包括調(diào)節(jié)上皮細(xì)胞的分泌,參與嗅覺、視覺的信號(hào)轉(zhuǎn)導(dǎo),調(diào)節(jié)平滑肌、心肌、神經(jīng)元的興奮性,還與腫瘤的發(fā)生發(fā)展存在密切的聯(lián)系。其生理病理學(xué)意義十分重要,是重要的藥物的研究靶點(diǎn)。CaCCs的分子基礎(chǔ)一直備受爭(zhēng)議,直到近些年大家才逐漸公認(rèn)兩大家族蛋白,八跨膜蛋白ANO1和四跨膜蛋白Bestrophin1可能是CaCCs的分子基礎(chǔ)。當(dāng)兩者表達(dá)于表達(dá)系統(tǒng)時(shí)都呈現(xiàn)CaCCs特性,如對(duì)鈣離子和電壓的敏感性。因?yàn)槿狈x擇性藥理學(xué)調(diào)節(jié)劑,對(duì)特定組織所觀察到的CaCCs究竟是由ANO1或是Bestrophin1構(gòu)成難以判斷。氯通道調(diào)節(jié)劑眾多,但缺乏選擇性,且研究使用較為混亂。本研究第一部分就針對(duì)此現(xiàn)狀,在建立穩(wěn)定表達(dá)鈣激活氯通道ANO1及Bestrophin1的中國倉鼠卵母(CHO)細(xì)胞系的基礎(chǔ)上觀察比較7種氯通道調(diào)節(jié)劑對(duì)ANO1及Bestrophin1通道的效能、效價(jià)及動(dòng)力學(xué)特征,為進(jìn)一步深入研究?jī)?nèi)源性CaCCs以及設(shè)計(jì)研發(fā)特異性通道調(diào)節(jié)劑提供強(qiáng)有力的支持。容積調(diào)節(jié)氯通道(volume regulated chloride channel,VRCC)是一類組織分布廣泛,對(duì)維持細(xì)胞容積穩(wěn)定具有重要生理學(xué)意義的陰離子通道。VRCC還與細(xì)胞增殖、細(xì)胞周期和細(xì)胞凋亡等生理過程密切相關(guān),并且已經(jīng)發(fā)現(xiàn)在心律失常、心肌缺血再灌注損傷以及充血性心力衰竭等病理狀態(tài)中發(fā)揮重要作用。VRCC的分子基礎(chǔ)研究一直進(jìn)展緩慢,研究者曾認(rèn)為P-糖蛋白、核酸敏感性氯通道蛋白、電壓依賴性氯通道ClC-2和ClC-3以及鈣激活氯通道ANO1和Bestrophin等可能是參與構(gòu)成VRCC的分子基礎(chǔ)。2014年,兩個(gè)研究小組幾乎同時(shí)發(fā)現(xiàn)LRRC8蛋白家族參與構(gòu)成了VRCC。本研究第二部分主要利用人胚腎上皮(HEK)以及中國倉鼠卵母(CHO)細(xì)胞觀察VRCC的分子基礎(chǔ)及藥理學(xué)特征,重點(diǎn)集中在ANO1和LRRC8A上。VRCC受細(xì)胞腫脹激活的機(jī)制尚不清楚,且其激活機(jī)制和調(diào)節(jié)因素在不同的細(xì)胞中有所不同。有研究報(bào)道稱細(xì)胞內(nèi)鈣、酪氨酸介導(dǎo)的蛋白磷酸化、絲裂原激酶和酪氨酸激酶、小g蛋白rhoa以及活性氧(ros)均可能都參與了vrcc的激活過程。本研究第三部分主要研究hek293a細(xì)胞內(nèi)源性vrcc的激活機(jī)制,重點(diǎn)研究細(xì)胞內(nèi)鈣離子在vrcc激活中的作用。論文具體內(nèi)容如下:第一部分氯通道調(diào)節(jié)劑對(duì)鈣激活氯通道ano1和bestrophin1作用的研究目的:研究比較幾種常見氯通道調(diào)節(jié)劑對(duì)鈣激活氯通道ano1和bestrophin1的作用。方法:1建立穩(wěn)定表達(dá)ano1和bestrophin1的cho細(xì)胞系;2利用膜片鉗技術(shù)觀察比較不同氯通道調(diào)節(jié)劑對(duì)ano1及bestrophin1通道的藥理學(xué)作用及對(duì)通道動(dòng)力學(xué)影響的特征。結(jié)果:1westernblot實(shí)驗(yàn)結(jié)果顯示穩(wěn)定轉(zhuǎn)染ano1的cho細(xì)胞系(cho-ano1)和穩(wěn)定轉(zhuǎn)染bestrophin1的cho細(xì)胞系(cho-best1)細(xì)胞膜上目的蛋白表達(dá)都明顯增加。膜片鉗實(shí)驗(yàn)結(jié)果顯示,在cho-ano1以及cho-best1細(xì)胞系均能成功記錄到明顯的鈣離子依賴性的外向整流電流。2膜片鉗研究結(jié)果發(fā)現(xiàn)7種常用氯通道阻斷劑中,對(duì)于+80mv下的外向電流,dids對(duì)bestrophin1(ic50of3.93±0.73μm)的選擇性遠(yuǎn)高于ano1(ic50of548.86±25.57μm);而nfa對(duì)ano1(ic50of7.40±0.95μm)的選擇性遠(yuǎn)高于bestrophin1(ic50of102.19±15.05μm)。caccinh-a01對(duì)ano1和bestrophin1通道的作用都比較強(qiáng)且效價(jià)相近,ic50分別是7.84±0.62μm和7.15±0.65μm。t16ainh-a01可抑制部分ano1電流,而對(duì)bestrophin1電流幾乎無作用。鞣酸(tannicacid)、nppb和ffa對(duì)兩者的抑制作用接近。3nfa、ffa以及nppb對(duì)-80mv下記錄的ano1內(nèi)向電流有雙向作用,低濃度(100μm)可增大內(nèi)向電流,而高濃度(100μm)呈現(xiàn)抑制作用。nfa,ffa及nppb可明顯改變ano1的通道動(dòng)力學(xué)特征,使其去活變慢。結(jié)論:1成功建立了穩(wěn)定表達(dá)ano1和bestrophin1的cho細(xì)胞系;2七種常用氯通道阻斷劑中,dids對(duì)bestrophin1的選擇性遠(yuǎn)高于ano1;而nfa對(duì)ano1的選擇性遠(yuǎn)高于bestrophin1。caccinh-a01對(duì)兩者的作用都比較強(qiáng)且效價(jià)相近。3nfa,ffa及nppb對(duì)ano1內(nèi)向電流具有雙向作用;且可明顯改變ano1的通道動(dòng)力學(xué)特征,使其去活變慢。第二部分鈣激活氯通道ano1參與構(gòu)成容積調(diào)節(jié)氯通道分子基礎(chǔ)的研究目的:記錄穩(wěn)定轉(zhuǎn)染ano1的hek293或cho細(xì)胞及基因敲除lrrc8a或ano1的hek293細(xì)胞中容積調(diào)節(jié)的氯電流并分析ano1是否參與構(gòu)成vrcc的分子基礎(chǔ)。方法:1使用420mosm的cscl電極內(nèi)液與320mosm的nacl細(xì)胞外液,利用全細(xì)胞膜片鉗技術(shù)記錄cho或hek細(xì)胞以及穩(wěn)定轉(zhuǎn)染ano1的cho或hek細(xì)胞中vrcc電流;2利用藥理學(xué)抑制劑觀察藥物對(duì)vrcc電流的作用;3利用crispr/cas9技術(shù)構(gòu)建lrrc8a基因以及ano1基因敲除的hek293a細(xì)胞系,觀察敲除細(xì)胞中vrcc電流;4在lrrc8a敲除細(xì)胞中外源轉(zhuǎn)染ano1后,觀察記錄vrcc。結(jié)果:1當(dāng)電極內(nèi)液為420mosmcscl,細(xì)胞外液為320mosmnacl時(shí),隨著細(xì)胞容積的增大,可記錄到明顯的vrcc電流。當(dāng)hek(cho)細(xì)胞膜電位鉗制在-60mv時(shí),平均電流密度約為64pa/pf(cho約為48pa/pf)。給予-100mv到+100mv的ramp電壓鉗制程序時(shí),vrcc呈現(xiàn)明顯的外向整流特性。而穩(wěn)定轉(zhuǎn)染ano1的細(xì)胞(hek-mano1)電流密度在-60mv時(shí)約為113pa/pf(cho-mano1約為63pa/pf),明顯大于未轉(zhuǎn)染ano1細(xì)胞的電流。另外,當(dāng)使用躍階(step)去極化電壓刺激模式時(shí),hek或cho細(xì)胞的vrcc電流呈現(xiàn)快速激活且快速去活的特征,當(dāng)膜電位高于+60mv時(shí)出現(xiàn)失活現(xiàn)象。而穩(wěn)定轉(zhuǎn)染ano1的細(xì)胞的vrcc電流則呈現(xiàn)慢激活無失活的電流特征。2第一部分研究發(fā)現(xiàn)nfa作為ano1電流的強(qiáng)效抑制劑,100μmnfa可抑制約90%的ano1電流。此部分實(shí)驗(yàn)中發(fā)現(xiàn)100μmnfa對(duì)穩(wěn)定表達(dá)ano1的細(xì)胞的vrcc電流呈現(xiàn)部分抑制作用,所抑制的電流表現(xiàn)為慢激活非失活的cacc樣電流;10μmdcpib是vrcc電流的相對(duì)特異性阻斷劑,對(duì)穩(wěn)定表達(dá)ano1細(xì)胞的vrcc電流呈現(xiàn)部分抑制作用,所抑制的電流呈現(xiàn)快激活且失活的lrcc8a樣電流。3基因敲除hek細(xì)胞內(nèi)源性ano1基因后,vrcc電流明顯減小。而敲除內(nèi)源性lrrc8a基因后,vrcc電流基本消失。然而在敲除lrrc8a的細(xì)胞中外源轉(zhuǎn)染ano1后,高滲內(nèi)液和等滲條件下的gtpγs可部分恢復(fù)vrcc電流,呈現(xiàn)ano1電流特征,表現(xiàn)為慢激活非失活,以及明顯外向整流特征。結(jié)論:1穩(wěn)定轉(zhuǎn)染ano1后的細(xì)胞vrcc電流明顯增加;敲除內(nèi)源性ano1基因后的細(xì)胞vrcc電流明顯降低。2敲除lrrc8a基因后,vrcc電流基本消失,再外源表達(dá)ano1可部分恢復(fù),說明ano1可被細(xì)胞腫脹激活,可能是參與構(gòu)成vrcc的分子基礎(chǔ)。第三部分容積調(diào)節(jié)氯通道vrcc的激活機(jī)制目的:研究hek293a細(xì)胞內(nèi)源性vrcc的激活機(jī)制。方法:1利用全細(xì)胞膜片鉗技術(shù)記錄hek293a細(xì)胞中的vrcc電流;2利用激光掃描共聚焦顯微鏡觀察細(xì)胞內(nèi)鈣信號(hào)的變化;3利用plc通路中相關(guān)分子的阻斷劑,以及經(jīng)sirna沉降plc后,觀察對(duì)vrcc電流的影響;4利用激光掃描共聚焦顯微鏡觀察細(xì)胞膜pip2的水解情況。結(jié)果:1使用高滲(420mosmcscl)的電極內(nèi)液,在hek293a細(xì)胞記錄到的vrcc電流有震蕩的現(xiàn)象;vrcc電流的失活與電流幅度有關(guān),表現(xiàn)為電流越小失活特征越明顯。使用等滲內(nèi)液,低滲(220mosm)外液記錄到的vrcc電流有與上述描述相似特征。2低滲外液可誘導(dǎo)細(xì)胞內(nèi)震蕩性鈣升高,且不被細(xì)胞外液中加入egta(2mm,降低游離鈣)或cdcl2(100μm,阻斷鈣內(nèi)流)所影響。3高滲電極內(nèi)液中加入20mmegta可抑制vrcc電流的震蕩現(xiàn)象,但不影響最大電流幅度;而加入20mmbapta,可明顯抑制vrcc電流;同樣使用thapasigagin預(yù)處理細(xì)胞使細(xì)胞內(nèi)鈣耗竭后,vrcc電流基本消失。43‰的血清可誘發(fā)一過性氯電流,此電流可被dcpib或caccinh-a01基本完全抑制,同時(shí)血清還可引起一過性的細(xì)胞內(nèi)鈣濃度升高。5使用plc阻斷劑u73122(5μm)處理細(xì)胞,vrcc的電流會(huì)顯著減小;而u73122的無效結(jié)構(gòu)類似物u73343(5μm)對(duì)vrcc電流無明顯作用。6hek293a細(xì)胞轉(zhuǎn)染pip2特異性熒光探針tubby-yfp,進(jìn)行激光共聚焦實(shí)驗(yàn),結(jié)果顯示低滲細(xì)胞外液使熒光強(qiáng)度明顯減弱。7ip3阻斷劑xe-c(1μm)可明顯抑制vrcc電流,pkc抑制劑bis-1(200nm)則對(duì)vrcc電流無明顯作用。高滲內(nèi)液中加入gdp-βs(500μm)不會(huì)影響vrcc電流。8使用sirna干擾技術(shù),使plcβ4、plcγ1以及plcδ3表達(dá)降低之后,vrcc電流明顯減小;plcβ3和plcε1表達(dá)降低對(duì)vrcc電流無明顯作用。結(jié)論:1hek293a細(xì)胞的容積調(diào)節(jié)氯電流存在震蕩現(xiàn)象,且隨著電流的增大,電流失活變慢。2 HEK293A細(xì)胞中容積調(diào)節(jié)氯通道的開放依賴于細(xì)胞內(nèi)鈣,尤其是細(xì)胞內(nèi)局部鈣離子的升高。3 HEK293A細(xì)胞中容積調(diào)節(jié)氯通道的激活依賴于PLC的激活,且PLCγ1、PLCβ4以及PLCδ3亞型可能參與其中。4 G蛋白和PKC不參與HEK293A細(xì)胞中容積調(diào)節(jié)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R96

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