模拟缺血预处理对原代培养乳鼠窦房结细胞Cx及I_（Cl,swell）表达的影响
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摘要
背景和目的
病态窦房结综合征是一种严重危害人类健康的常见心血管病。明确其发病机理并找出有效的预防和治疗措施一直是心血管领域最重要的课题之一。在我们先前的研究中发现,模拟缺血/再灌注(I/R)能引起在体或离体窦房结细胞出现结构和功能的紊乱,导致心律或频率的异常。在几乎所有动物细胞上都发现缺血预处理(IP)是最强的内源性保护机制,它可以减轻I/R引起的各种心肌损伤。但IP信号转导非常复杂,目前这方面知之不多,而窦房结细胞中的IP研究更少。最近研究显示缝隙连接蛋白(Cx)可能是预处理保护作用的终末效应子之一。大量的研究显示肿胀激活氯电流(ICl,swell)在心肌细胞生理或病理过程中参与细胞电活动和细胞容积调节,在I/R时细胞肿胀激活该电流,该电流在心肌细胞电-机械反馈中起调节作用。本研究利用原代培养乳鼠窦房结细胞研究模拟IP对细胞的保护作用;研究Cx在预处理时的改变及可能机理;研究在培养窦房结细胞中是否存在ICl,swell及其特点,以及模拟IP对其的影响,希望能为将来窦房结细胞缺血性损伤的保护提供新的思路。
方法
1.通过MTT比色和PI染色检测细胞存活率比较不同模拟预处理模型。
2.利用激光共聚焦显微镜和免疫细胞化学检测窦房结细胞Cx的表达。3.用全细胞膜片钳记录给予不同干预的乳鼠窦房结细胞的ICl,swell。
结果
1.成功建立了原代培养乳鼠窦房结细胞模拟IP模型。通过研究不同时间组合的模拟IP方案,在IP诱导启动过程中起决定作用的应该是预缺血的总时程,在培养乳鼠窦房结细胞中IP作用的预缺血时程为5min-30min,而15min的累计预缺血时间获得了最显著的保护效果,故筛选出5min模拟缺血+5min模拟再灌注,反复三次,后再行3h模拟缺血+4h模拟再灌注的模拟IP方案对培养乳鼠窦房结细胞的保护作用最强。
2.通过药物干预研究,证实了PKC及线粒体KATP通道的激活介导了模拟IP对乳鼠窦房结细胞的保护作用。氯通道阻断剂对原代培养培养窦房结细胞的IRI具有一定的保护作用,提示IP可能通过氯通道发挥保护作用。
3.用免疫荧光检测方法发现乳鼠窦房结细胞中有Cx45、Cx43和Cx40免疫荧光
Background and objective
Sick sinus syndrome (SSS) is a common cardiovascular disease which is greatly harmful to human health. To elucidate the pathogenesis of SSS and find out the possible measures of prevention and radical cure is always an important part of the angiocardiopathy study. In the previous study, we have found that simulated ischemia/reperfusion(I/R) could cause structural and functional damages to sinoatrial node cells in vivo and in vitro, leading to electrophysiological disturbance by affecting the regular rhythm and frequency.
Myocardium ischemic preconditioning(IP), which is considered as the most powerful protective inherence found in almost all kinds of animals and human, can relief various kinds of cardiac damages caused by I/R. But the signal transduction of IP is very complicated and redundant, only a little has been known about its protective mechanism and the study of effect of IP on the sinoatrial node cells is rarely seen. Connexin has also been given more and more concern about it’s contribution to the IP recently due to an end effector in IP. Substantial evidence indicates that swell-induced chloride current (ICl,swell) contributes to cardiac electrical activity and cellular volume regulation in both physiologic and pathophysiologic situations. Swelling occurs during ischemia and reperfusion, and it is postulated that this activates ICl,swell. Studies have revealed ICl,swell is essential for the IP due to a regulating role in mechanoelectrical feedback of myocardium. Based on the primarily cultured sinoatrial node pacemaker cells in neonatal rat, the purpose of our investigation is to observe the effects of simulated IP on the changes of cell viability after severe simulated I/R, detect the role of connexins in IP and discuss the mechanism, observe whether ICl,swell is present in primarily cultured sinoatrial node cells in neonatal rat, and discuss the signaling cascades participated in its activation, providing experimental evidence for further prevention of sinoatrial node injuries caused by I/R.
病态窦房结综合征是一种严重危害人类健康的常见心血管病。明确其发病机理并找出有效的预防和治疗措施一直是心血管领域最重要的课题之一。在我们先前的研究中发现,模拟缺血/再灌注(I/R)能引起在体或离体窦房结细胞出现结构和功能的紊乱,导致心律或频率的异常。在几乎所有动物细胞上都发现缺血预处理(IP)是最强的内源性保护机制,它可以减轻I/R引起的各种心肌损伤。但IP信号转导非常复杂,目前这方面知之不多,而窦房结细胞中的IP研究更少。最近研究显示缝隙连接蛋白(Cx)可能是预处理保护作用的终末效应子之一。大量的研究显示肿胀激活氯电流(ICl,swell)在心肌细胞生理或病理过程中参与细胞电活动和细胞容积调节,在I/R时细胞肿胀激活该电流,该电流在心肌细胞电-机械反馈中起调节作用。本研究利用原代培养乳鼠窦房结细胞研究模拟IP对细胞的保护作用;研究Cx在预处理时的改变及可能机理;研究在培养窦房结细胞中是否存在ICl,swell及其特点,以及模拟IP对其的影响,希望能为将来窦房结细胞缺血性损伤的保护提供新的思路。
方法
1.通过MTT比色和PI染色检测细胞存活率比较不同模拟预处理模型。
2.利用激光共聚焦显微镜和免疫细胞化学检测窦房结细胞Cx的表达。3.用全细胞膜片钳记录给予不同干预的乳鼠窦房结细胞的ICl,swell。
结果
1.成功建立了原代培养乳鼠窦房结细胞模拟IP模型。通过研究不同时间组合的模拟IP方案,在IP诱导启动过程中起决定作用的应该是预缺血的总时程,在培养乳鼠窦房结细胞中IP作用的预缺血时程为5min-30min,而15min的累计预缺血时间获得了最显著的保护效果,故筛选出5min模拟缺血+5min模拟再灌注,反复三次,后再行3h模拟缺血+4h模拟再灌注的模拟IP方案对培养乳鼠窦房结细胞的保护作用最强。
2.通过药物干预研究,证实了PKC及线粒体KATP通道的激活介导了模拟IP对乳鼠窦房结细胞的保护作用。氯通道阻断剂对原代培养培养窦房结细胞的IRI具有一定的保护作用,提示IP可能通过氯通道发挥保护作用。
3.用免疫荧光检测方法发现乳鼠窦房结细胞中有Cx45、Cx43和Cx40免疫荧光
Background and objective
Sick sinus syndrome (SSS) is a common cardiovascular disease which is greatly harmful to human health. To elucidate the pathogenesis of SSS and find out the possible measures of prevention and radical cure is always an important part of the angiocardiopathy study. In the previous study, we have found that simulated ischemia/reperfusion(I/R) could cause structural and functional damages to sinoatrial node cells in vivo and in vitro, leading to electrophysiological disturbance by affecting the regular rhythm and frequency.
Myocardium ischemic preconditioning(IP), which is considered as the most powerful protective inherence found in almost all kinds of animals and human, can relief various kinds of cardiac damages caused by I/R. But the signal transduction of IP is very complicated and redundant, only a little has been known about its protective mechanism and the study of effect of IP on the sinoatrial node cells is rarely seen. Connexin has also been given more and more concern about it’s contribution to the IP recently due to an end effector in IP. Substantial evidence indicates that swell-induced chloride current (ICl,swell) contributes to cardiac electrical activity and cellular volume regulation in both physiologic and pathophysiologic situations. Swelling occurs during ischemia and reperfusion, and it is postulated that this activates ICl,swell. Studies have revealed ICl,swell is essential for the IP due to a regulating role in mechanoelectrical feedback of myocardium. Based on the primarily cultured sinoatrial node pacemaker cells in neonatal rat, the purpose of our investigation is to observe the effects of simulated IP on the changes of cell viability after severe simulated I/R, detect the role of connexins in IP and discuss the mechanism, observe whether ICl,swell is present in primarily cultured sinoatrial node cells in neonatal rat, and discuss the signaling cascades participated in its activation, providing experimental evidence for further prevention of sinoatrial node injuries caused by I/R.
引文
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