本文關(guān)鍵詞： p38激酶 硫氧還蛋白 鉀通道 Ito電流 心肌梗死　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:p38激酶是細(xì)胞凋亡的關(guān)鍵調(diào)節(jié)因子,尤其在因?yàn)椴±硇詰?yīng)激所導(dǎo)致的心肌肥厚及壞死中,但是它對(duì)病理狀態(tài)下心肌離子通道尤其是電壓門控鉀通道(Kv)的調(diào)控機(jī)制還并不清楚。所以本研究目的是進(jìn)一步明確大鼠心梗(MI)6-8周后p38信號(hào)通路在電壓門控鉀通道(Kv)調(diào)控中的作用機(jī)理。方法:從河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心購(gòu)得雄性Sprague-Dawley(SD)大鼠。稱得體重在180~200g的雄性Sprague-Dawley(SD)大鼠按照文獻(xiàn)方法建立MI模型,在手術(shù)后6-8周,對(duì)大鼠進(jìn)行麻醉,開(kāi)胸分離出心臟,并使用Langendoff法通過(guò)冠狀動(dòng)脈進(jìn)行膠原酶灌流,獲得單一分離的心室肌細(xì)胞,在CO2培養(yǎng)箱中孵育4.5小時(shí)后,應(yīng)用細(xì)胞膜片鉗試驗(yàn)對(duì)樣本電壓門控鉀通道(Kv)及Ito進(jìn)行分析。本實(shí)驗(yàn)使用非放射性p38激酶檢測(cè)試劑盒檢測(cè)(細(xì)胞信號(hào)技術(shù))p38激酶活性。結(jié)果:1使用免疫印跡法檢測(cè)顯示MI后心肌p38激酶活性明顯增強(qiáng),與對(duì)照組相比增加大約2-6倍(Control組:2.0±1.1,n=6;MI組:7.2±1.7,n=6;P0.05)。2把實(shí)驗(yàn)組分離提取的組織標(biāo)本經(jīng)過(guò)與p38激酶阻斷劑SB20358015?M混合后4-5個(gè)小時(shí),應(yīng)用電生理技術(shù)檢測(cè)Ito電流強(qiáng)度,可見(jiàn)其明顯增高(SB203580+MI:25.6±1.0 p A/p F,n=10;MI組:15.9±2.1 p A/p F,n=10;P0.05)。3硫氧還蛋白(Trx)還原酶阻斷劑金諾芬(AF)試劑明顯阻斷了SB203580對(duì)MI老鼠心肌Ito電流的明顯的增強(qiáng)效果(MI+AF+SB203580:0.5±0.1,n=10),而AF對(duì)對(duì)照組Ito無(wú)明顯影響,Trx能夠顯著增加Ito電流的強(qiáng)度,另外胰島素樣生長(zhǎng)因子能夠增加Trx的作用,能夠增強(qiáng)Ito電流的強(qiáng)度。4免疫蛋白印記法顯示經(jīng)過(guò)p38阻滯劑SB203580溶解混合后的MI心肌細(xì)胞的Kv4.2通道蛋白表達(dá)量顯著增加,即使并沒(méi)有恢復(fù)到未處理組的水平,但是這與既往在MI心肌相關(guān)的研究中所了解到的Ito電流密度的改變是一致的。經(jīng)過(guò)p38激酶阻斷劑SB203580處理過(guò)的對(duì)照組的相關(guān)心肌的Kv4.2通道蛋白表達(dá)含量沒(méi)有表現(xiàn)出明顯增多的跡象,而基本上保持平衡。結(jié)論:缺血壞死導(dǎo)致心梗的心肌鉀通道改變是能夠調(diào)節(jié)的,能夠通過(guò)激活p38信號(hào)通路促進(jìn)鉀通道的改變導(dǎo)致Ito改變。這一過(guò)程可被Trx、IGF-1調(diào)控。本研究結(jié)果表明,在MI心肌中,p38激酶活性明顯增強(qiáng),進(jìn)一步降低Kv通道介導(dǎo)的Ito電流強(qiáng)度;抑制p38信號(hào)通路能夠明顯提高Kv通道蛋白表達(dá),進(jìn)而提高Ito電流強(qiáng)度,這一過(guò)程能被Trx、IGF-1系統(tǒng)調(diào)控。
[Abstract]:Objective: p38 kinases are key regulators of apoptosis, especially in hypertrophy and myocardial necrosis because of pathological stress caused by, but it is of cardiac ion channels under pathological conditions especially voltage-gated potassium channels (Kv) of the regulation mechanism is not clear. So the purpose of this study is to further clarify the rat myocardial infarction (MI) after 6-8 weeks of p38 signaling pathway in the voltage-gated potassium channel (Kv) in the regulation mechanism. Methods: male Sprague-Dawley purchased from the experimental animal center of Hebei Medical University (SD) in rats. Called weight in male Sprague-Dawley 180~200g (SD) rat MI model was established according to the methods of literature, in the 6-8 weeks after surgery, the rats were anesthetized, open chest isolated heart, and collagenase perfusion through coronary artery using the Langendoff method to obtain a single isolated ventricular myocytes, cultured in the CO2 box after 4.5 hours of incubation by cell membrane Clamp voltage gated potassium channel on the test sample (Kv) and Ito were analyzed. The experiments using non radioactive p38 kinase assay kit (cell signaling technology) p38 kinase activity. Results: 1 using immunoblotting assay showed that after MI myocardial p38 kinase activity increased significantly, compared with the control group increased about 2-6 times (group Control: 2 + 1.1, n=6; group MI: 7.2 + 1.7, n=6; P0.05).2 the experimental group extracted from tissue samples with p38 kinase inhibitor SB20358015? M 4-5 hours after mixing, the electrophysiological technique was used to detect Ito current intensity, can see the increased (SB203580+MI:25.6 + 1 p A/p F, n=10; group MI: 15.9 + 2.1 P A/p F, n=10; P0.05).3 thioredoxin reductase inhibitor (Trx) Jin Nuofen (AF) SB203580 of MI reagent significantly blocked the mouse myocardial Ito current obvious enhancement effect (MI+AF+ SB203580:0.5 + 0.1, n=10), while the AF of control group Ito no Obviously, Trx can Ito the current intensity increased significantly, and insulin-like growth factor can increase the effect of Trx, can enhance the strength of.4 Western blot method showed that after Ito current MI myocardial p38 blocker SB203580 dissolving the Kv4.2 channel protein expression was significantly increased, even if did not return to the untreated group the level of Ito, but the current density in the MI are aware of this and previous myocardial related research in the change is consistent. After blocking the Kv4.2 channel protein kinase p38 expression in myocardial control group treated by agent SB203580 showed no signs of a marked increase, and basically balance. Conclusion: ischemic necrosis leading to myocardial infarction is the change of potassium channel can be adjusted, can activate p38 signaling pathway to promote potassium channel changes lead to changes in Ito. This process can be Trx, IGF-1 Regulation and control. This study shows that in MI myocardium, p38 kinase activity is significantly enhanced, further reducing Kv channel mediated Ito current intensity. Inhibition of p38 signaling pathway can significantly improve Kv channel protein expression, and further enhance Ito current intensity, which can be regulated by Trx and IGF-1 system.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.22

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