CAJ | 학술논문

目的探讨蛋白激酶C(PKC)在异氟醚诱导原代培养大鼠心肌细胞分泌血管内皮生长因子(VEGF)中的作用.方法 1～3 d SD新生大鼠,分离培养原代心肌细胞,随机分为6组(n=6):对照组(C组)培养后的细胞不经任何处理;不同浓度异氟醚组(Ⅰ1组～Ⅰ3组)细胞分别经0.7%、1.4%、2.1%异氟醚处理6 h;PKC抑制剂组(P组)细胞培养液中给予PKC抑制剂--calphostin C,终浓度50 nmol/L;PKC抑制剂+异氟醚组(PI组)心肌细胞培养液中加入calphostin C 50 nmol/L后,置入无菌密闭容器,持续输入1.4%异氟醚6 h.采用ELISA法测定细胞培养液VEGF浓度,Western blot法测定心肌细胞PKC亚型的表达.结果与C组比较,Ⅰ2组和Ⅰ3组细胞培养液VEGF浓度升高,Ⅰ2组胞浆PKCε表达下调,胞膜PKCε表达上调(P＜0.01),胞浆和胞膜PKCα、PKCδ和PKCζ表达差异无统计学意义(P＞0.05),P组上述指标差异无统计学意义(P＞0.05).与胞膜比较,C组和Ⅰ2组胞浆PKCα、PKCδ和PKCζ表达上调(P＜0.05).随异氟醚浓度升高细胞培养液VEGF浓度升高(P＜0.05).与Ⅰ2组比较,PI组细胞培养液VEGF浓度降低(P＜0.05).结论异氟醚可通过PKCε从胞浆转位到胞膜的途径诱导心肌细胞分泌VEGF,是异氟醚心肌保护作用的机制之一.
목적 탐토단백격매C(PKC)재이불미유도원대배양대서심기세포분비혈관내피생장인자(VEGF)중적작용.방법 1～3 d SD신생대서,분리배양원대심기세포,수궤분위6조(n=6):대조조(C조)배양후적세포불경임하처리;불동농도이불미조(Ⅰ1조～Ⅰ3조)세포분별경0.7%、1.4%、2.1%이불미처리6 h;PKC억제제조(P조)세포배양액중급여PKC억제제--calphostin C,종농도50 nmol/L;PKC억제제+이불미조(PI조)심기세포배양액중가입calphostin C 50 nmol/L후,치입무균밀폐용기,지속수입1.4%이불미6 h.채용ELISA법측정세포배양액VEGF농도,Western blot법측정심기세포PKC아형적표체.결과 여C조비교,Ⅰ2조화Ⅰ3조세포배양액VEGF농도승고,Ⅰ2조포장PKCε표체하조,포막PKCε표체상조(P＜0.01),포장화포막PKCα、PKCδ화PKCζ표체차이무통계학의의(P＞0.05),P조상술지표차이무통계학의의(P＞0.05).여포막비교,C조화Ⅰ2조포장PKCα、PKCδ화PKCζ표체상조(P＜0.05).수이불미농도승고세포배양액VEGF농도승고(P＜0.05).여Ⅰ2조비교,PI조세포배양액VEGF농도강저(P＜0.05).결론 이불미가통과PKCε종포장전위도포막적도경유도심기세포분비VEGF,시이불미심기보호작용적궤제지일.
Objective To investigate the role of protein kinase C (PKC) in induction of vascular endothelial growth factor (VEGF) secretion by isoflurane in primary cultured rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups ( n = 6 each): control group (group C), 3 different concentration isoflurane groups (group Ⅰ1-3 ), PKC inhibitor calphostin C group (group P), and PKC inhibitor + isoflurane group (group PI). The cells were exposed to 0.7%, 1.4% and 2.1% isoflurane for6 h in group Ⅰ1-3 respectivly. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L in group P. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L, then the cells were exposed to 1.4% isoflurane for 6 h in group PI. VEGF concentrations and expression of PKC isoforms were determined by ELISA and Western blot respectively. Results Compared with group C, the VEGF concentration was significantly increased in group Ⅰ2 and Ⅰ3, and PKCε expression was down-regulated in the cytoplasm while upregulated in the cytomembrane in group Ⅰ2 ( P ＜ 0.01 ), but no significant change was found in the parameters mentioned above in group Ⅰ2 ( P ＞ 0.05). PKCα, PKCδ and PKCζ expression was significantly higher in the cytoplasm than in the cytomembrane in group C and Ⅰ2. VEGF concentrations were gradually increased with the increase in isoflurane concentrations ( P ＜ 0.05). VEGF concentrations were significantly lower in group PI than in Ⅰ2 ( P ＜0.05) .Conclusion Isoflurane induces VEGF secretion in primary cultured rat cardiomyocytes through translocation of PKCε from the cytoplasm to the cytomembrane, suggesting that it is a mechanism of the cardioprotective effects of isoflurane.