本文選題：3-磷酸肌醇依賴(lài)性蛋白激酶1 + 叉頭框蛋白O　； 參考：《南京醫(yī)科大學(xué)》2015年博士論文

【摘要】：背景慢性心力衰竭(chronic heart failure,CHF)是一種復(fù)雜的臨床疾病,發(fā)病率高,有臨床癥狀患者的5年存活率與惡性腫瘤相仿,這類(lèi)患者中約一半因惡性室性心律失常的發(fā)生而猝死。雖然心律失常是發(fā)生心臟性猝死(SCD)的重要原因,與多種離子通道有關(guān),但是其中的潛在的電生理機(jī)制仍舊不清楚。3-磷酸肌醇依賴(lài)性蛋白激酶1(PDK1)是蛋白激酶家族AGC中的一員,它位于該信號(hào)通路的上游,參與調(diào)控AGC家族下的多種調(diào)節(jié)因子,其中包括蛋白激酶B(PKB/Akt),磷酸化核糖體S6蛋白激酶(P70S6K),血清和糖皮質(zhì)激素誘導(dǎo)的蛋白激酶(SGK)以及叉頭框蛋白O(FoxO)等。PDK1參與多種細(xì)胞生理的調(diào)控,諸如代謝、生長(zhǎng)及生存等。此外,目前多個(gè)研究發(fā)現(xiàn),該信號(hào)通路的多種上游及下游因子參與心臟心力衰竭和病理性重構(gòu)的發(fā)生與發(fā)展。例如在小鼠上,當(dāng)心臟特異性敲除PDK1后,可以發(fā)現(xiàn)心肌細(xì)胞體積縮小,心臟結(jié)構(gòu)改變以及嚴(yán)重的心力衰竭,且于出生后的11周左右突然死亡。其中是否有心律失常的參與還不清楚。AGC家族蛋白激酶的上游因子磷脂酰肌醇3-激酶(PI3K)的特異性阻斷劑在臨床上常常用于抗腫瘤的治療,但是心律失常是其中的副作用之一。最近的研究就發(fā)現(xiàn)PI3K特異性阻斷劑可以影響多種離子通道,延長(zhǎng)動(dòng)作電位時(shí)程(APD)。Fox O1是AGC家族蛋白激酶的主要下游調(diào)控因子之一,最近的研究發(fā)現(xiàn)它可能是鈉通道負(fù)向調(diào)控因子。而且,鈉通道功能的下降與臨床上傳導(dǎo)阻滯(AVB),3型長(zhǎng)QT間期綜合征(LQT3)和Brugada綜合征(Brs)有關(guān),這類(lèi)患者的主要死亡原因之一就是心律失常。因此,PDK1作為該信號(hào)通路的主要上游因子,是否參與心臟鈉通道以及其他離子通道的調(diào)控,乃至多種病理情況下心律失常的發(fā)生,值得進(jìn)一步的研究。因此,本研究構(gòu)建心臟特異性敲除pdk1的小鼠模型,運(yùn)用全細(xì)胞膜片鉗和westernblot技術(shù),研究心臟動(dòng)作電位,多種離子通道之間變化及可能原因,探討pdk1參與心律失常發(fā)生的潛在機(jī)制。實(shí)驗(yàn)內(nèi)容1.心臟特異性敲除pdk1小鼠的鑒定與電生理改變的初步探索目的:心臟特異性敲除pdk1小鼠模型的鑒定。方法:采用westernblot方法,檢測(cè)小鼠心室肌細(xì)胞pdk1的表達(dá)情況;運(yùn)用體表心電圖檢測(cè)基因敲除后小鼠心率、qrs、qtc的變化。結(jié)果:基因敲除后的8周,心室肌pdk1的表達(dá)顯著下降。且心電圖表現(xiàn)為心率下降(362.22±12.69vs.422.31±20.10,p0.05),qrs(12.81±0.30msvs.18.93±1.17ms)、qtc(82.69±4.08msvs.113.91±8.20ms)時(shí)程的延長(zhǎng),11周即出現(xiàn)了一定程度的心臟傳導(dǎo)異常。結(jié)論:心臟特異性敲除pdk1小鼠模型構(gòu)建是有效的,且出現(xiàn)了初步的電生理異常。2.小鼠心肌細(xì)胞的分離目的:建立穩(wěn)定的急性分離小鼠心室肌細(xì)胞的方法。方法:采用酶解法分離小鼠心室肌細(xì)胞,使用改良langendorff裝置和灌流液,經(jīng)主動(dòng)脈逆行性灌流ii型膠原酶消化心肌,得到單個(gè)心室肌細(xì)胞。結(jié)果:經(jīng)過(guò)ii型膠原酶的消化,在pdk1基因敲除以及正常小鼠上均可獲得長(zhǎng)桿狀,邊緣及橫紋清晰的存活心室肌細(xì)胞。結(jié)論:穩(wěn)定的急性分離小鼠心室肌細(xì)胞的方法已構(gòu)建成功。3.pdk1對(duì)小鼠心臟鈉通道的調(diào)控目的:探索心臟pdk1敲除后對(duì)于心室肌細(xì)胞鈉通道的影響以及可能的機(jī)制方法:運(yùn)用全細(xì)胞膜片鉗方法,比較pdk1基因敲除后心室肌鈉通道電流以及相關(guān)動(dòng)力學(xué)的變化。運(yùn)用westernblot技術(shù),檢查pdk1下游信號(hào)通路的改變。結(jié)果:心臟特異性敲除pdk1后,小鼠心室肌細(xì)胞鈉電流密度下降(-23.86±1.10pa/pfvs.-36.34±1.45pa/pf,p0.05),且11周大小鼠有著類(lèi)似的改變(-24.11±1.23pa/pfvs.-36.76±2.07pa/pf,p0.05),但是鈉通道動(dòng)力學(xué)僅有輕度變化。westernblot檢測(cè)發(fā)現(xiàn)小鼠pdk1信號(hào)通路下游308位點(diǎn)akt磷酸化下降,24位點(diǎn)foxo1磷酸化下降,且細(xì)胞核中foxo1表達(dá)上升,鈉通道蛋白表達(dá)下降。結(jié)論:pdk1敲除可以通過(guò)抑制鈉通道蛋白的表達(dá)來(lái)抑制心室肌細(xì)胞鈉通道電流的密度,潛在的機(jī)制可能是通過(guò)調(diào)控下游信號(hào)通路的磷酸化,通過(guò)提高鈉通道負(fù)向調(diào)控因子foxo1在細(xì)胞核的表達(dá)來(lái)調(diào)控的。4.pdk1對(duì)乳大鼠心室肌鈉通道的調(diào)控目的:排除pdk1敲除對(duì)心功能的影響因素,進(jìn)一步證實(shí)pdk1敲除導(dǎo)致鈉電流下調(diào)的機(jī)制。方法:在培養(yǎng)的乳大鼠心室肌細(xì)胞上,運(yùn)用pdk1特異性阻斷劑gsk2334470,akt特異性阻斷劑mk2206,foxo1特異性阻斷劑as1842856或聯(lián)合使用,分別運(yùn)用全細(xì)胞膜片鉗技術(shù)和westernblot技術(shù),觀察鈉通道電流的變化以及相關(guān)信號(hào)通路的變化。結(jié)果:運(yùn)用pdk1特異性阻斷劑gsk2334470和akt特異性阻斷劑mk2206后,乳大鼠心室肌細(xì)胞的鈉電流密度下降從-36.23±1.25pa/pf分別下降至-27.52±1.49pa/pf和25.48±2.06pa/pf。相反,這種抑制效應(yīng)可以被foxo1特異性阻斷劑所抵消。westernblot實(shí)驗(yàn)中運(yùn)用了pdk1特異性阻斷劑和foxo1特異性阻斷劑探索了鈉通道的表達(dá)變化,此外細(xì)胞核中foxo1的變化與pdk1敲除的小鼠類(lèi)似。結(jié)論:實(shí)驗(yàn)結(jié)果進(jìn)一步說(shuō)明PDK1可以通過(guò)對(duì)于下游調(diào)控因子的FoxO1的調(diào)控來(lái)調(diào)節(jié)心室肌細(xì)胞鈉通道功能。5.PDK1對(duì)小鼠心室肌細(xì)胞電生理的影響目的:進(jìn)一步探索PDK1在心律失常的發(fā)生中發(fā)揮的作用。方法:運(yùn)用全細(xì)胞膜片鉗技術(shù),檢測(cè)小鼠心室肌細(xì)胞動(dòng)作電位時(shí)程的變化,EAD的誘發(fā),以及復(fù)極相關(guān)主要離子通道的功能變化。結(jié)果:心臟特異性敲除PDK1后,小鼠心室肌細(xì)胞APD顯著延長(zhǎng),L型鈣電流(49%)以及Ito電流都顯著抑制(89%),但EAD誘發(fā)幾率和正常小鼠相比無(wú)顯著差異。結(jié)論:PDK1敲除延長(zhǎng)動(dòng)作電位時(shí)程主要是通過(guò)抑制瞬時(shí)外向鉀電流來(lái)完成的,它可能在心臟心律失常發(fā)生發(fā)展中發(fā)揮了重要作用。
[Abstract]:Background chronic heart failure (chronic heart failure, CHF) is a complex clinical disease with high incidence and 5 year survival rates in patients with clinical symptoms similar to malignant tumors. About half of these patients die of sudden death due to malignant ventricular arrhythmia. Subchannel related, but the potential electrophysiological mechanism remains unclear..3- phosphoric acid inositol dependent protein kinase 1 (PDK1) is a member of the protein kinase family AGC, which is located upstream of the signal pathway and participates in the regulation of a variety of regulatory factors under the AGC family, including protein kinase B (PKB/Akt), phosphorylated ribosome S6 protein kinase (P). 70S6K), serum and glucocorticoid induced protein kinase (SGK) and fork head frame protein O (FoxO), such as.PDK1, are involved in a variety of cell physiological regulation, such as metabolism, growth and survival. In addition, a number of studies have found that a variety of upstream and downstream signals of the signal pathway participate in the development and development of heart failure and pathological remodeling. For example, in mice, when cardiac specific knockout of PDK1, myocardial cell volume, cardiac structural changes, and severe heart failure can be found and suddenly died around 11 weeks after birth. The involvement of arrhythmia is not clear about the specificity of the upstream factor of the.AGC family protein kinase, phosphatidylinositol 3- kinase (PI3K) Sexual blockers are often used in clinical antitumor treatment, but arrhythmia is one of the side effects. Recent studies have found that PI3K specific blockers can affect a variety of ion channels, prolonging action potential time (APD).Fox O1 is one of the main downstream regulators of AGC family protein kinase. Recent studies have found that it is possible It can be a negative regulator of sodium channel. Moreover, the decrease of sodium channel function is associated with clinical conduction block (AVB), type 3 long QT interval syndrome (LQT3) and Brugada syndrome (Brs). One of the main causes of death in these patients is arrhythmia. Therefore, PDK1 is the main upstream factor of the signal pathway, whether it is involved in the heart sodium channel. The regulation of the other ion channels and the occurrence of arrhythmia in a variety of pathological conditions is worth further study. Therefore, this study constructs a mouse model of cardiac specific knockout PDK1, using whole cell patch clamp and Westernblot technique to study the cardiac action potential, the changes of multiple ion channels and possible causes, and discuss the PDK1 parameter. Potential mechanisms for the occurrence of arrhythmias. Experimental content 1. identification of PDK1 mice with cardiac specific knockout and preliminary exploration of electrophysiological changes: identification of cardiac specific knockout PDK1 mouse models. Methods: Westernblot method was used to detect the expression of PDK1 in ventricular myocytes of mice; the detection of gene knockout with body surface electrocardiogram was used to detect gene knockout. The changes of heart rate, QRS, QTc in mice. Results: the expression of PDK1 in ventricular muscle decreased significantly at 8 weeks after knockout, and the electrocardiogram showed a decrease of heart rate (362.22 + 12.69vs.422.31 + 20.10, P0.05), QRS (12.81 + 0.30msvs.18.93 + 1.17ms), QTc (82.69 + 4.08msvs.113.91 + 8.20ms) duration, and a certain degree of cardiac conduction in the 11 week. Conclusion: the construction of cardiac specific knockout PDK1 mouse model is effective, and the primary electrophysiological abnormalities of.2. mouse cardiomyocytes are isolated: a stable and acute method for acute isolation of murine ventricular myocytes is established. Method: the isolated mouse ventricular myocytes were separated by enzymatic method, and the improved Langendorff device and perfusion fluid were used. Pulse retrograde perfusion II collagenase digested the myocardium and obtained single ventricular myocytes. Results: through the digestion of II collagenase, the survival of the ventricular myocytes with long rod-shaped, marginal and transverse lines could be obtained on PDK1 gene knockout and normal mice. Conclusion: a stable and acute method of separating ventricular myocytes from mice has been constructed successfully.3.pdk1 The aim of the control of sodium channel in the heart of mice: To explore the effect of PDK1 knockout on the sodium channel of ventricular myocytes and the possible mechanism: using the whole cell patch clamp method to compare the changes of ventricular sodium channel current and related kinetics after PDK1 knockout. Westernblot technique was used to examine the downstream signal pathway of the PDK1. Results: after the cardiac specific knockout of PDK1, the sodium current density of ventricular myocytes in mice decreased (-23.86 + 1.10pa/pfvs.-36.34 + 1.45pa/pf, P0.05), and the 11 weeks of mice had similar changes (-24.11 + 1.23pa/pfvs.-36.76 + 2.07pa/pf, P0.05), but the sodium channel dynamics only slightly changed.Westernblot to detect the PDK1 signal of mice. Akt phosphorylation at the 308 loci of the channel decreased, the 24 site FoxO1 phosphorylation decreased, and the expression of FoxO1 in the nucleus increased, and the expression of sodium channel protein decreased. Conclusion: PDK1 knockout can inhibit the density of sodium channel current in ventricular myocytes by inhibiting the expression of sodium channel protein. The potential mechanism may be through the regulation of phosphorus in the downstream signal pathway. Acidification, by improving the regulation of.4.pdk1 on the sodium channel of ventricular myocardium in milk rats by improving the expression of the negative control factor FoxO1 in the nucleus: exclude the influence factors of PDK1 knockout on the cardiac function, and further confirm the mechanism of the downregulation of sodium current caused by PDK1 knockout. Method: the use of PDK1 in cultured neonatal rat ventricular myocytes. Heterosexual blockers gsk2334470, Akt specific blocker mk2206, FoxO1 specific blocker as1842856 or combined use. Whole cell patch clamp technique and Westernblot technology were used to observe changes in sodium channel current and related signal pathways. Results: PDK1 specific blocker gsk2334470 and Akt specific blocker mk2206 The decrease of sodium current density in ventricular myocytes of milk rats decreased from -36.23 + 1.25pa/pf to -27.52 + 1.49pa/pf and 25.48 + 2.06pa/pf., respectively. This inhibitory effect could be counteracted by FoxO1 specific blockers in.Westernblot experiments and the PDK1 specific blockers and FoxO1 specific blockers were used to explore the expression of sodium channels. In addition, the change of FoxO1 in the nucleus is similar to that of PDK1 knockout mice. Conclusion: the experimental results further indicate that PDK1 can regulate the effect of.5.PDK1 on ventricular myocyte electrophysiology in ventricular myocytes by regulating the FoxO1 of the downstream regulatory factors in order to further explore the occurrence of PDK1 in the occurrence of arrhythmia. Methods: whole cell patch clamp technique was used to detect the changes of action potential time of ventricular myocytes in mice, the induction of EAD and the function changes of major ion channels related to repolarization. Results: after the specific knockout of PDK1, the APD of ventricular myocytes in mice was significantly longer, L type calcium current (49%) and Ito current were significantly inhibited (89%). However, there is no significant difference in EAD induced probability compared with normal mice. Conclusion: PDK1 knockout prolongs the action potential mainly by inhibiting transient outward potassium current, which may play an important role in the development of cardiac arrhythmias.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R541.6

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