摘要
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 investigate the effect of calcium/calmodulin-dependent protein kinaseⅡ （CaMK[KG-＊6]Ⅱ） inhibitor,KN93 on calcium overloading of atrial muscle cells in neonatal rats and detect the expression of CaMK[KG-＊6]Ⅱ. Methods The atrial muscle cells from neonatal rats were primarily cultured for 96 h and then divided into 6 group,control,calcium overloading group,KN93 group （0.5 μmol/L）,low-,moderate-and high-dose of KN93＋ calcium overloading group. A model of calcium overloading for atrial muscle cells was established by using calcium ionophore （ionomycin,1.0 μmol/L）. For the later 3 groups,KN93 at doses of 0.25,0.5 and 1.0 μmol/L was added into the culture medium for 30 min followed by 1.0 μmol/L ionomycin treatment for another 30 min. The identification of α-actin was performed by immunofluorescence staining. In the present of Fluo-3/AM （an indicator of calcium）,intracellular calcium and the expression of CaMK[KG-＊6]Ⅱ were detected under the intervention of KN93 with laser cofocal microscopy and Western blotting respectively. Results More than 90% of cultured cells were positive to α-actin antibody. Compared with the control group,the fluorescence intensity of intracellular Ca2＋ was increased significantly by ionomycin （660.16±108.47 vs 376.12±57.57,P0.01）,but KN93 alone had no effect on it （389.00±64.01,P0.05）. In presence of KN93 pretreatment （0.25,0.5 and 1.0 μmol/L）,the fluorescence intensity of intracellular Ca2＋ induced by ionomycin was significantly decreased （558.00±102.56,499.58±64.99,494.25±78.77 vs 660.16±108.47,P0.01）. The expression of CaMK[KG-＊6]Ⅱ was increased in calcium overloading group compared with control （P0.01） and KN93 alone had no effect on it. But KN93 pretreatment significantly reduced the expression of CaMK[KG-＊6]Ⅱ compared with in cells of calcium overloading group （P0.01）. Conclusion KN93,an inhibitor of CaMK[KG-＊6]Ⅱ,can reduce the calcium loading of atrial muscle cells induced by ionomycin,and downregulate the expression of CaMKⅡ.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第12期1275-1277,共3页
Journal of Third Military Medical University
基金
国家自然科学基金(30700315)~~
关键词
钙-钙调素依赖性蛋白激酶2型
肌细胞
心脏
钙
大鼠
calcium-calmodulin-dependent protein kinase type 2
myocytes
cardiac
calcium
rats
