吗啡对培养新生大鼠心肌细胞凋亡影响的实验研究

Studies on Effect of Morphine on Apoptosis in Cardiomyocyte of Neonatal Rats

目的利用体外原代培养的新生大鼠心肌细胞,研究吗啡对血清饥饿诱导的心肌细胞凋亡的影响。方法体外原代培养新生大鼠心肌细胞,分组给药后,用MTT法测心肌细胞的活力;用流式细胞仪分析心肌细胞周期;用AnnexinV-FITC/PI双标记法测心肌细胞的凋亡率;用Westernblot法测Caspase-3蛋白的表达;用Fura-2双波长荧光法测心肌细胞内游离钙。结果体外原代培养的新生大鼠心肌细胞经无血清饥饿作用48h后,心肌细胞可出现明显的凋亡现象;给予不同浓度的吗啡作用于心肌细胞后,心肌细胞的这种凋亡现象可被抑制,其在1μmol·L-1处最为明显,表现为:心肌细胞凋亡率降低、Caspase-3蛋白表达减少、细胞内Ca2+离子浓度降低;给予10μmol·L-1纳洛酮(Naloxone)后,吗啡的这种抑制心肌细胞凋亡作用可被完全阻断;并且给予1μmol·L-1纳曲吲哚(Naltrindole)后,吗啡抑制心肌细胞凋亡的作用亦在一定程度上降低。结论吗啡可激活心肌细胞膜上的阿片受体,对血清饥饿诱导的心肌细胞凋亡具有抑制作用,且可被Naloxone阻断。

Objective To study effect of morphine on serum deprivation - induced apoptosis in cardiac myocytes. Methods Myocardial ceils of neonatal rats were cultured in vitro. After 48 hours, different agents were added to cardiac myocytes. The cellular survival energy was determined with MTT colormeteric assay ; Apoptosis rates were determined by Annexin V - FITC/PI; the expression of Caspase - 3 was investigated by Western Blotting. Fluorescent indicator fura - 2 was used to measure the changes of [ Ca^2＋ ] i response. Resuits Free - serum induced apoptosis may occur in cardiac myocytes after 48 hours; Different concentration of morphine may inhibit myocardial cell apoptosis and 1umol·L^-1 morphine is the most obvious concentration, apoptosis rates were decreased, Caspase- 3 experssion was decaeased, [ Ca^2＋] i was decreased; Naloxone at 10 umol·L^-1 can completely inhibit the effect of morphine on apoptosis in cardiomyocyte of neonatal rats. 1 umol·L^-1 Naltrindole can inhibit the effect of morphine on apoptosis in eardiomyocyte of neonatal rats, too. Conclusion Morphine may activate opioid receptor of cardiac myocytes, which inhibited serum deprivation - induced apoptosis in cardiac myocytes and Naloxone can block it.