实时监测GHRP对心肌细胞内[Ca2+]i和NO调控的双标记方法研究

目的探索实时监测GHRP对心肌细胞内[Ca2 ]i和NO调控的双标记方法,为研究GHRP对心脏直接保护效应机制提供新方法。方法用改良的Langendorff恒流灌注仪系统和酶解法急性分离SD成年大鼠心肌细胞,在LSCM下分别以Ca2 探针Rhod-2/AM和NO探针DAF-FM/DA对心肌细胞内Ca2 和NO进行双标记并观察GHRP即时刺激对两者的调控影响。结果在LSCM下心肌细胞内Ca2 呈红色荧光,NO呈绿色荧光,两者双标记后呈现黄绿色荧光,GHRP使红色荧光出现瞬时增强后又迟缓地回降,绿色荧光强度没有明显变化,双标记下GHRP对两者的调控同单标记结果。结论LSCM下实验中设计的双标记方法能实时监测成年大鼠心肌细胞内Ca2 和NO存在及GHRP对两者同一时相调控作用,引起[Ca2 ]i两个时相的变化,而对NO信号系统未见明显影响。

To Explore the Double-labeling Method of Monitoring the GHRP Regulatory Function on [Ca2+] i and NO on Real Time in Cardiomyocytes Under LSCM

Objective To explore the double-labeling method of monitoring the GHRP regulatory function on [Ca2 ]i and NO in cardiomyocytes of rats on real time under LSCM.Methods The reformed constant-flow Langendorff system and enzyme-dissociated was used to isolate cardiomyocytes.[Ca2 ]i and NO in the cardiomyocytes of SD rats were double-labeled by their molecular probe Rhod-2/AM and DAF-FM/DA,respectively to monitor the regulatory function of GHRP on [Ca2 ]i and NO on real time by LSCM.Results Ca2 signal showed a red fluorescence and NO showed a green fluorescence while the overlapping of the two signals showed a yellow-green fluorescence by this system,and the similar effect presents in both double-labeled state and the single labeled one:GHRP induced a transient[Ca2 ]i increase then followed by a plateau phase while there was not significant change in NO signal system after GHRP stimulation under the LSCM in the cardiomyocytes of rats.Conclusions After having established the double-labeling method we monitored the GHRP regulatory function on [Ca2 ]i and NO on real time in cardiomyocytes of rats under LSCM causing the [Ca2 ]i biphasic increase while no significant change in NO signal system.