【摘要】：心肌肥厚、心力衰竭是冠心病、心瓣膜病、高血壓等多種心血管病的常見合并癥。心肌肥厚、心力衰竭時發(fā)生的病理性電重構使心臟的電不穩(wěn)定性增加，常伴發(fā)各種心律失常。心衰病人在泵功能穩(wěn)定的情況下半數(shù)以上因發(fā)生惡性心律失常而猝死（即心源性猝死Sudden Cardiac Death,SCD）。因此，闡明病理情況下心律失常產(chǎn)生機制，尋找藥物作用靶點具有重要意義。 已知心肌肥厚、心力衰竭病理發(fā)展過程中全身和局部的腎素-血管緊張素系統(tǒng)（renin-angiotensin system, RAS）活性增加是病理性組織重構重要的原因，該系統(tǒng)的關鍵成分血管緊張素II （AngII）主要通過激活AT1受體從而激活多種細胞內(nèi)信號通路，對心血管功能發(fā)揮廣泛的調(diào)節(jié)作用。實驗證明，過表達人AngII基因的大鼠常因嚴重室性心動過速而猝死；在急性分離的犬和大鼠的心室肌細胞、以及轉染編碼Ito通道基因Kv4.3哺乳細胞上，AngII均可使Ito電流密度減小。上述研究結果提示，AngII通過刺激AT1受體直接引起離子通道的改變導致病理性電重構是心律失常發(fā)生的重要原因。 哺乳類動物心室肌細胞延遲整流鉀電流在動作電位復極化過程中發(fā)揮關鍵作用，其中快激活成份（IKr）通道孔區(qū)亞單位由human‘ether-a-go-go’ related gene基因(簡稱hERG)編碼，此種hERG鉀通道由于特殊的動力學特征對動作電位形狀、時程具有重要影響。疾病狀況下該通道功能下調(diào)引發(fā)動作電位時程延長和/或形狀改變是室性心律失常發(fā)生的重要原因。hERG鉀通道由于本身的缺陷或者藥物的影響而發(fā)生變異或功能異常，產(chǎn)生先天性LQTS和獲得性LQTS，均有較高尖端扭轉型室性心律失常和猝死的危險。 在前期的研究我們觀察到AngII對豚鼠心室肌細胞IKr和表達的IhERG呈現(xiàn)急性抑制作用，提示AngII可能通過直接下調(diào)IKr參與病理條件下電生理重構。已知AngII可激活多條細胞內(nèi)信號通路引發(fā)急性和慢性生物效應，慢性效應發(fā)生在數(shù)小時甚至更晚時間，常涉及蛋白基因轉錄及轉錄后的改變。我們已證明AngII長時間作用（12h）可使表達的hERG鉀通道成熟膜蛋白明顯減少。那么，AngII下調(diào)通道膜蛋白含量潛在的機制尚不清楚。 新近實驗證明，慢性刺激一些G蛋白偶聯(lián)受體對hERG鉀通道發(fā)揮明顯的轉錄后調(diào)節(jié)作用。因此，本研究主要利用分子生物學的方法，在前期研究AngII作用的基礎上又在異源表達系統(tǒng)上進一步研究AT1受體刺激對hERG鉀通道的轉錄后調(diào)節(jié)作用（包括如何影響hERG鉀通道蛋白正向轉運及膜蛋白入胞降解等轉錄后調(diào)節(jié)過程而改變細胞膜蛋白含量）并對AngII下調(diào)hERG鉀通道膜蛋白量的信號傳導機制進行分析，這對理解離子通道病理重構機制從而解釋心律失常發(fā)生機制具重要理論意義。第一部分血管緊張素II對hERG鉀通道轉錄后調(diào)節(jié)作用 目的：研究AngII對hERG鉀通道轉錄后的調(diào)節(jié)作用。 方法：（1）Lipofectamine2000瞬時轉染：將AT1受體質(zhì)粒轉染到穩(wěn)態(tài)轉染的hERG-HEK293細胞上，或將AT1受體質(zhì)粒與hERG質(zhì)粒以1:1的比例瞬時轉染到HEK293細胞上。 （2）細胞免疫熒光：將細胞用4%多聚甲醛固定后，，用0.2%TritonX-100透化處理（不做透化處理省略這一步），之后用PBS配制含5%BSA的封閉液進行封閉，加入特異性一抗和FITC標記的二抗，用共聚焦顯微鏡觀察顯色情況。 （3）Western blot：將提取的細胞總蛋白進行SDS-PAGE電泳，電轉移到NC膜上，用TBST配制含5%脫脂牛奶進行封閉，加入特異性一抗和紅外熒光標記的二抗，用雙色紅外激光成像系統(tǒng)儀器掃描。 （4）In-cell western：將細胞用4%多聚甲醛固定后用TBST配制含5%脫脂牛奶封閉，加入特異性一抗和紅外熒光標記的二抗，用雙色紅外激光成像系統(tǒng)儀器掃描。 結果：（1）Western blot檢測到目的條帶約在65kDa出現(xiàn)，與AT1受體分子量大小基本一致。提示AT1受體在hERG-HEK293細胞中成功表達。 （2）AngII長時間孵育對hERG鉀通道蛋白含量影響:AngI（I100nM）孵育24小時后In-cell western檢測顯示膜上成熟蛋白減少為（69%±4%，P0.01）；細胞免疫熒光染色共聚焦顯微鏡下觀察通道蛋白在細胞膜和細胞內(nèi)分布情況，發(fā)現(xiàn)AngII使未透化處理過的細胞膜上的熒光明顯減少，透化處理過的細胞膜熒光減少，胞漿熒光變化不明顯。 （3）AngII對hERG鉀通道蛋白正向轉運的影響: AngII孵育24h后western blot檢測到成熟蛋白的表達顯著減少為（68%±4%，P0.01），胞漿未成熟蛋白表達沒有明顯變化（P0.05）。而已知能夠引起hERG鉀通道轉運障礙的Fluoxetine（3μM）或在瞬時轉染的hERG鉀通道突變體A422T和H562P，均可檢測到成熟蛋白表達明顯減少，同時伴未成熟蛋白表達的明顯增加。Fluoxetine使成熟蛋白減少為（64%±2%，P0.01），未成熟蛋白增加為（114%±5%，P0.05）。提示AngII不影響通道蛋白的正向轉運。 （4） AngII對hERG鉀通道膜蛋白降解的影響：已知Brefeldin A1(BFA)能夠阻斷通道正向轉運，加入BFA（10μM）使用western blot觀察不同時間成熟蛋白的降解情況，對照組在2h、4h、8h、12h、24h下降為82%±8%、70%±1%、63%±9%、55%±8%、53%±8%（P0.05），AngII組在2h、4h、8h、12h、24h下降為77%±7%、64%±9%、55%±11%、44%±12%、40%±12%（P0.05），提示AngII加速通道膜蛋白降解。Lactacystin（5μM，蛋白酶體抑制劑）明顯能夠抑制AngII介導的成熟蛋白減少（67±4%增加為89%±3%，P0.05），而Bafilomycin（1μM，溶酶體抑制劑）對AngII的作用無明顯影響（P0.05），提示AngII加速蛋白降解主要通過蛋白酶體途徑。 結論：在異源表達系統(tǒng)上，AngII長時間孵育可顯著減少hERG鉀通道膜上成熟蛋白含量，其原因可能是由于膜蛋白以泛素-蛋白酶體途徑降解加速引起。第二部分血管緊張素II下調(diào)hERG鉀通道膜蛋白含量的細胞內(nèi)信號傳導機制 目的：分析AngII下調(diào)hERG鉀通道膜蛋白含量的細胞內(nèi)信號傳導機制。 方法：（1）Lipofectamine2000瞬時轉染：將AT1受體質(zhì)粒轉染到穩(wěn)態(tài)轉染的hERG-HEK293細胞上。 （2）Western blot：方法同第一部分。 結果：Western blot結果顯示:（1）PKC抑制劑Bis I（100nM）明顯能夠抑制AngII介導的成熟蛋白減少(64%±4%增加到90%±4%,P0.01)。 （2）PKC激動劑PMA（100nM）或OAG（100nM），也明顯能夠抑制AngII介導的成熟蛋白減少（59%±5%增加到101%±5%，P0.01；58%±4%增加到100%±5%, P0.05）。 （3）PKA抑制劑H-89（1μM）不能抑制AngII介導的成熟蛋白減少（P0.05）。 結論：在異源表達系統(tǒng)上，AngII長時間孵育減少hERG鉀通道膜上成熟蛋白的表達主要由PKC信號通路介導。
[Abstract]:Cardiac hypertrophy and heart failure are common complications of coronary heart disease, valvular heart disease, hypertension and other cardiovascular diseases. Cardiac hypertrophy and pathological electrical remodeling during heart failure increase electrical instability of the heart and often accompany various arrhythmias. More than half of patients with heart failure develop malignant arrhythmias when the pump function is stable. Sudden cardiac death (SCD). Therefore, it is of great significance to elucidate the mechanism of arrhythmia in pathological conditions and find the target of drug action.
It is known that the increased activity of renin-angiotensin system (RAS) is an important cause of pathological tissue remodeling in cardiac hypertrophy and heart failure. AngII has been shown to reduce Ito current density in acute isolated canine and rat ventricular myocytes and in transfected mammalian cells encoding the Ito channel gene Kv4.3. Pathological electrical remodeling is an important cause of arrhythmia by stimulating AT1 receptor.
Delayed rectifier potassium currents play a key role in action potential repolarization in mammalian ventricular myocytes. The pore region subunit of the fast-activating component (IKr) channel is encoded by the human `ether-a-go-go'related gene (hERG) gene. This kind of hERG potassium channel has special dynamic characteristics for action potential shape and duration. Important effects. The prolongation of action potential duration and/or shape changes caused by the downregulation of the channel function are important causes of ventricular arrhythmias in disease conditions. The risk of arrhythmia and sudden death.
In previous studies, we observed the acute inhibitory effect of AngII on IKr and IhERG expression in guinea pig ventricular myocytes, suggesting that AngII may be involved in electrophysiological remodeling through direct down-regulation of IKr under pathological conditions. AngII is known to activate multiple intracellular signaling pathways leading to acute and chronic biological effects, with chronic effects occurring for hours or even hours. Later, it is often involved in the transcriptional and post-transcriptional changes of protein genes. We have shown that long-term action of AngII (12 hours) can significantly reduce the expression of mature membrane proteins of hERG potassium channels. The underlying mechanism of AngII down-regulation of channel membrane proteins remains unclear.
Recent studies have shown that chronic stimulation of some G-protein-coupled receptors plays a significant posttranscriptional role in the regulation of hERG potassium channels. The signal transduction mechanism of AngII down-regulating the membrane protein content of hERG potassium channel was analyzed, which is important for understanding the mechanism of ion channel pathological remodeling and explaining the mechanism of arrhythmia. Significance. Part one: angiotensin II regulates post transcriptional regulation of hERG potassium channel.
Objective: To study the regulatory effect of AngII on the transcription of hERG potassium channel.
Methods: (1) Lipofectamine 2000 transient transfection: AT1 receptor plasmid was transfected into stable transfected hERG-HEK293 cells, or AT1 receptor plasmid and hERG plasmid were transfected into HEK293 cells in 1:1 ratio.
(2) Cell immunofluorescence: Fixed with 4% paraformaldehyde, the cells were treated with 0.2% Triton X-100 dialysis (without dialysis omitting this step), then blocked with 5% BSA blocking solution prepared with PBS. Specific primary antibody and FITC-labeled secondary antibody were added to the blocking solution, and the pornography was observed by confocal microscopy.
(3) Western blot: SDS-PAGE electrophoresis was carried out on the extracted total cell proteins, which were transferred to NC membrane, sealed with 5% skimmed milk prepared by TBST, and scanned with dual-color infrared laser imaging system.
(4) In-cell western: Fixed cells with 4% paraformaldehyde and sealed them with 5% skimmed milk by TBST, added specific antibodies and infrared fluorescent labeled antibodies, and scanned them with dual-color infrared laser imaging system.
Results: (1) Western blot showed that the target band appeared at about 65 kDa, which was consistent with the molecular weight of AT1 receptor, suggesting that AT1 receptor was successfully expressed in hERG-HEK293 cells.
(2) The effect of long incubation with AngII on the content of potassium channel protein in hERG: In-cell Western assay showed that the mature protein on the membrane decreased to (69%+4%, P 0.01) 24 hours after incubation with AngI (I100nM); the distribution of channel protein in the cell membrane and in the cell was observed by confocal microscopy with immunofluorescence staining, and it was found that AngII made the unpermeable treatment fine. The fluorescence on the cell membrane decreased significantly, the fluorescence of the permeable treated cell membrane decreased, and the cytoplasmic fluorescence did not change significantly.
(3) The effect of AngII on the forward transport of hERG potassium channel protein: Western blot showed that the expression of mature protein was significantly reduced to (68% + 4%, P 0.01) after incubation for 24 hours, and the expression of immature protein was not changed significantly (P 0.05). However, fluoxetine (3 mu M) or transient transfection of hERG potassium channel were known to cause the hERG potassium channel transport disorder. Fluoxetine reduced the expression of mature protein to (64%+2%, P 0.01) and increased the immature protein to (114%+5%, P 0.05). AngII did not affect the positive transport of channel protein.
(4) Effects of AngII on the degradation of hERG potassium channel membrane proteins: Brefeldin A1 (BFA) was known to block the forward transport of the channel. The degradation of mature proteins at different time points was observed by Western blot after adding BFA (10 mu M). In the control group, the degradation rate was 82% + 8%, 70% + 1%, 63% + 9%, 55% + 8%, 53% + 8% (P 0.05) at 2h, 4h, 8h, 12h, 24h, 12h and 24h, respectively. Lactacystin (5 mu M, proteasome inhibitor) significantly inhibited the decrease of mature protein mediated by AngII (67.4% increased to 89% + 3%, P 0.05), while Bafilomycin (1 mu M, lysosomal inhibitor) had no significant effect on AngII (P 0.05). It suggested that AngII accelerated protein degradation mainly through proteasome pathway.
CONCLUSIONS: AngII prolonged incubation can significantly reduce the content of mature proteins on hERG potassium channel membranes in the heterologous expression system, possibly due to the accelerated degradation of membrane proteins via ubiquitin-proteasome pathway. Part II Intracellular signal transduction mechanism of angiotensin II down-regulating the content of hERG potassium channel membrane proteins.
Objective: to analyze the intracellular signal transduction mechanism of AngII down regulating the protein content of hERG potassium channel membrane.
Methods: (1) Lipofectamine 2000 transient transfection: AT1 receptor plasmid was transfected into stable transfected hERG-HEK293 cells.
(2) Western blot: method is the same as the first part.
Results: Western blot showed that: (1) PKC inhibitor Bis I (100nM) could significantly inhibit AngII-mediated decrease of mature protein (64%+4% increased to 90%+4%, P 0.01).
(2) PKC agonists PMA (100nM) or OAG (100nM) also significantly inhibited AngII-mediated decrease of mature protein (59% + 5% to 101% + 5%, P 0.01, 58% + 4% to 100% + 5%, P 0.05).
(3) PKA inhibitor H-89 (1 M) can not inhibit AngII mediated maturation protein decrease (P0.05).
CONCLUSION: Long-term incubation of AngII reduces the expression of mature proteins on hERG potassium channel membranes, which is mediated by PKC signaling pathway.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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相關博士學位論文 前1條

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