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目的观察钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)抑制剂KN93对新生大鼠心房肌细胞钙负荷的影响,并对细胞CaMKⅡ的表达变化进行检测。方法新生大鼠心房肌细胞原代培养96h,应用钙离子导入剂ionomycin(1.0μmol/L)建立心房肌细胞钙超载模型,并在KN933种浓度(0.25、0.5、1.0μmol/L)的干预下,以钙离子指示剂Fluo-3/AM负载心房肌细胞,激光共聚焦显微镜观察心房肌细胞内游离钙的变化;应用Western blot法检测CaMKⅡ表达的变化。结果①细胞培养至第4天,免疫组织化学染色90%以上细胞α-肌动蛋白抗体阳性。②与对照组比较,钙离子导入剂ionomycin明显增加细胞内钙离子荧光值[(660.16±108.47)vs(376.12±57.57),P<0.01];KN93对细胞内钙离子荧光值无明显影响(P>0.05)。③预先加入3种不同浓度KN93可显著降低ionomycin导致的细胞内钙离子荧光强度的增加幅度(P<0.01),与对照组比较差异也有统计学意义(P<0.01)。④钙超载组细胞CaMKⅡ表达较对照组明显增加(P<0.01);而KN93对细胞CaMKⅡ表达影响不明显。⑤不同浓度KN93预处理后,钙超载组细胞CaMKⅡ的表达显著降低(P<0.01)。结论 CaMKⅡ抑制剂KN93可降低ionomycin引发的大鼠心房肌细胞钙负荷,并下调细胞CaMKⅡ表达。
Objective To observe the effects of calcium / calmodulin-dependent protein kinase Ⅱ (KN93) inhibitor KN93 on the calcium load of atrial myocytes in neonatal rats and to detect the expression of CaMKⅡ. Methods Primary cultured atrial myocytes of neonatal rats were cultured for 96 hours. A calcium overload model of atrial myocytes was established by ionomycin (1.0 μmol / L). Under the action of KN933 (0.25, 0.5 and 1.0 μmol / L) The acellular cardiomyocytes were loaded with Fluo-3 / AM, and the changes of intracellular free calcium in atrial myocytes were detected by laser confocal microscopy. The expression of CaMKⅡ was detected by Western blot. Results ①The cells were cultured until day 4, and more than 90% of the cells were positive for α-actin antibody by immunohistochemical staining. Compared with the control group, ionomycin increased the intracellular calcium fluorescence [(660.16 ± 108.47) vs (376.12 ± 57.57), P <0.01]; KN93 had no significant effect on intracellular calcium fluorescence (P > 0.05). ③ Pretreatment with three different concentrations of KN93 significantly reduced the increase of intracellular calcium fluorescence intensity induced by ionomycin (P <0.01), and there was also a significant difference compared with the control group (P <0.01). ④ CaMKⅡexpression in Ca2 + overload group was significantly higher than that in control group (P <0.01), but KN93 had no obvious effect on CaMKⅡexpression. (5) After pretreatment with different concentrations of KN93, the expression of CaMKII in Ca2 + overload group was significantly decreased (P <0.01). CONCLUSION: KN93, a CaMKⅡ inhibitor, decreases ionomycin-induced calcium load in rat atrial myocytes and down-regulates CaMKⅡ expression.