摘要
目的 观察心脏营养素-1（CT-1）对培养乳鼠心肌细胞存活率的影响以及蛋白激酶C（PKC）信号转导通路在调控CT-1诱导心肌细胞存活中的作用。方法 分离、纯化并差速贴壁培养新生大鼠心肌细胞，MTT比色法测定各孔心肌细胞存活率。结果 ①无血清DMEM培养心肌细胞24h后加入4个剂量组CT-1（0.1～10nmol／L），随着剂量的增加MTT计数随之增高，呈明显的剂量依赖性；②在培养介质中预先加入蛋白激酶C（PKC）激动剂佛波酯（PMA），30min后再加入CT-1，MTT计数明显增加，而用PKC抑制剂Calphostin C（Cal）则使MTT计数明显降低；③用丝裂原激活蛋白激酶（MAPK）亚型细胞外信号调节激酶（ERK）选择性抑制剂PD098059进行预处理，再加入CT-1，心肌细胞存活率明显降低，与单纯CT-1组相比有显著性差异；单纯用ERK抑制剂PD098059对心肌细胞存活率无明显影响；预先加入ERK抑制剂PD098059，再加入PMA和CT-1，MTT计数明显降低，与（PMA＋CT-1）组相比有极显著性差异。结论 CT-1对心肌细胞存活率具有明显的剂量依赖性；PKC可上调CT-1促进的心肌细胞存活率，PKC可能成为CT-1促进心肌细胞存活率的信号转导通路之一；CT-1诱导心肌细胞存活率的胞内信号转导通路可能是先激活PKC，然后诱导MAPK亚型ERK的激活。
AIM To observe the effects of cardiotrophin-1 （ CT-1 ）, an interleukin 6-related cytokine, on the survival rate of cultured neonatal rat myocardial cell and the effects of protein kinase C （PKC） signal transduction pathway in regulating the survival rate of CT-1-induced cells. METHODS The cardiomyocytes of neonatal rats were isolated, purified and cultured with method of pre-plating for different times and the cell survival rate of each well was measured with MTT chromatometry approach. RESULTS ① The survival rate of cardiac myocytes increased by CT-1 in a dose-dependent manner （0.1 - 10 nmol/L） in cultured neonatal rat cardiomyocytes. ②This effect was strengthened by the phorbol 12-myristate 13- acetate （ PMA, 10 μmol/L）, a PKC activator. In contrast, pretreatment of Calphostin C （ Cal, 10 μmol/ L）, a PKC inhibitor, significantly decreased the survival rate of CT-1-induced cardiac myocytes. ③The pretreatment of PD098059 （50 μmol/L）, a mitogen-activated protein kinase （MAPK, ERK1/2 ）specific blocker, also significantly decreased the survival rate of cardiomyocytes promoted by CT-1 and abolished the effect of PMA on CT-1-induced cardiac myocytes survival in vitro. CONCLUSION CT-1 is a potent factor of promoting myocardial survival and significantly increases the survival rate of cardiomyocytes in a dose-dependent manner in cultured myocardial cells of neonatal rats. PKC can upregulate CT-1-induced myocardial survival. MAPK signaling transduction pathway may be a downstream target site of PKC signaling molecule in CT-1 induced cardiomyocyte survival in vivo.
出处
《心脏杂志》
CAS
2006年第4期406-410,共5页
Chinese Heart Journal
基金
国家自然科学基金项目资助(No.30260032)
关键词
心肌细胞培养
心脏营养素-1
蛋白激酶C
丝裂原激活蛋白激酶
存活率
neonatal rat myocardial cell culture
cardiotrophin-1
protein kinase C
mitogen-activated protein kinase
survival rate
