小鼠胚胎心肌细胞ATP敏感性钾通道（K_（ATP））的发育依赖性研究
【摘要】 目的:探讨小鼠胚胎心肌细胞ATP依赖性钾通道(KATP)随着胚胎的发育在心肌细胞上的表达、电生理学特点及其功能的变化。方法:分离第10.5天和第17.5天的小鼠胚胎,采用胶原酶B消化获得单个胚胎心肌细胞并培养24-36小时。以Pinacidil和Cromacalim作为KATP通道的特异性开放剂,Glibenclamide作为通道的特异性抑制剂,采用电流钳模式记录细胞的动作电位观察IKATP功能的发育依赖性变化;使用全细胞电压钳模式(细胞外液K+浓度为140mM,钳制电位0mV,测试脉冲为-100到+40mV,步阶为20mV,持续时间500ms),记录心肌细胞的KATP电流(IKATP)观察其电生理学特点的发育依赖性变化。采用RT-PCR的方法分析KATP亚单位Kir6.1和Kir6.2以及调节亚单位SUR1、SUR2A在早晚期心肌细胞上的表达量的发育依赖性变化。结果:电流钳的数据表明,IKATP特异性开放剂Pinacidil使早期的大部分心房和心室肌的动作电位时程缩短,对动作电位的最大舒张期电位没有影响;但是使晚期胚胎心肌细胞的动作电位的膜电位产生超极化。电压钳的数据表明,早晚期胚胎心肌细胞的IKATP的电流密度没有显著差异,但是早期心肌细胞IKATP的内向整流特性明显强于晚期心肌细胞。Kir6.1、Kir6.2、SUR1和SUR2在mRNA水平的表达随着发育均有上调趋势,发育晚期以Kir6.2和SUR2表达占优势。结论:随着小鼠胚胎的发育,其心肌细胞的KATP通道亚单位在mRNA水平表达发育依赖性增加,但电流密度没有明显变化,并伴随KATP通道的内向整流特性降低。IKATP的电生理学特点和分子生物学水平表达的差异可能来自于通道亚单位组合不同,亦或由于蛋白质翻译水平的差异。
【Abstract】 Objective: ATP-sensitive K+ channels (KATP) might be involved in the hypoxia resistance of fetal cardiomyocytes, therefore we aimed to investigate the developmental change of KATP in mouse embryonic myocardial cells, including the electrophysiological characteristics and the functional contributions.Methods: Mouse embryos of 10.5 days and 17.5 days were taken as early (EDS) and late (LDS) developmental stage, respectively. The single mouse embryonic cardiomyocytes were dissociated enzymatically using collagenase B and cultured for 24-36h before electrophysiological recordings. The specific openers of KATP channel Pinacidil and Cromakalim, as well as the specific inhibitor Glibenclamide, were used to indentify IKATP. The current-clamp was used to record the action potentials and observe the functional contributions of IKATP during development, the whole-cell voltage-clamp (Extracellular K+ 140 mM, holding potential 0mV, test pulse from -100 to 40mV, step 20mV, duration 500ms) was applied to study the electrophysiological characteristics of IKATP. Finally, RT-PCR was performed investigate the expression levels of KATP subunits Kir6.1 , Kir6.2 and the regulatory subunits SUR1, SUR2A .Results: Data acquired from action potential recordings showed that the specific opener Pinacidil shortened the action potential duration (APD) of most working cardiomyocytes while no effects on maximum diastolic potential (MDP). However, it strongly hyperpolarized the MDP of LDS working cardiomyocytes. Whole cell recordings revealed that there was no significant difference of the IKATP current density between EDS and LDS fetal cardiomyocytes, although the expression of Kir6.1, Kir6.2, SUR1 and SUR2 at mRNA level were all increased with the development. Moreover, the IKATP inward rectifier characteristics of the EDS cardiomyocytes was stronger than LDS cells.Conclusions: With maturation, the expression of KATP channel subunits upregulated whereas the current density did not change significantly but the inward rectifier characteristics of the KATP channels reduced. The conflictions of IKATP electrophysiological characteristics and the levels of molecular expression may rise from the different combination of each subunits, or else the different levels of protein translation.
【关键词】 胚胎心肌细胞； KATP； 全细胞膜片钳； 发育；
【Key words】 embryonic cardiomyocytes； KATP； whole-cell patch clamp； development；
小鼠胚胎心肌细胞ATP敏感性钾通道（K_（ATP））的发育依赖性研究
【目录】中文摘要 5-6
英文摘要 6-7
前言 8-11
材料与方法 11-18
结果 18-21
讨论 21-23
结论 23-24
附图 24-30
参考文献 30-33
综述 33-44
参考文献 39-44
致谢 44
【参考文献】[1] 王剑. 急性低氧下甘肃鼢鼠心脏抗氧化酶及ATP酶活性研究[D]. 陕西师范大学 2010
[2] 王伟霞. 异三聚体G蛋白调控的离子流动参与了细胞外ATP调控的拟南芥气孔运动[D]. 河北师范大学 2010
[3] 楼凯凯. ATP生物发光法检测微生物总量及其自动原位分析的研究[D]. 浙江大学 2006
[4] 张玉辉. ATP诱导大鼠海马神经元神经毒性及对GAP-43表达的影响[D]. 大连医科大学 2009 [5] 王灵敏. 细胞外ATP对拟南芥根向重力性的影响及其作用机制初探[D]. 河北师范大学 2011
[6] 杨吉文. 机体及细胞内ATP动态变化过程模型及其模拟试验[D]. 苏州大学 2010 [7] 高巍. 异三聚体G蛋白在细胞外ATP调控拟南芥离子通道过程中的作用[D]. 河北师范大学 2011
[8] 毛玉婷. 生后发育中NR2B在C57BL/6小鼠听皮层脑片LTP诱导中的作用[D]. 华东师范大学 2004 [9] 臧绍云. 生后发育中NR2B受体在C57BL/6小鼠听皮层LTP诱导中的作用[D]. 华东师范大学 2004
[10] 刘亚利. 小鼠肾近曲小管上皮细胞顶侧膜钾通道的电生理学特性及环孢素A的影响[D]. 第三军医大学 2005