摘要
目的探讨建立新生大鼠原代心房肌细胞钙超载模型的方法。方法用消化法分离新生大鼠心房肌细胞并进行原代培养。纯化后随机分为对照组(C组)、实验组(E1、E2、E3组),依次加入不同浓度(0.5、1.0、2.0μmol/L)伊屋诺霉素(ionomy-cin),以钙离子指示剂Fluo-3/AM负载心房肌细胞,激光扫描共聚焦显微镜检测心房肌细胞游离钙离子浓度([Ca2+]i),建立新生大鼠心房肌细胞不同程度钙超载模型。结果 (1)培养至第4天,细胞生长密度可以达到瓶底70%左右;免疫组织化学染色鉴定:90%以上培养细胞α-肌动蛋白抗体染色呈阳性;(2)对照组心房肌细胞活性率为(73.00±2.37)%,与各实验组心房肌细胞活性率比较,差异无统计学意义(P>0.05);(3)经不同浓度ionomycin处理l h后,各实验组心房肌细胞[Ca2+]i明显增高,与对照组比较,差异有统计学意义(431.54±20.97、705.87±28.34、1 305.05±53.69 vs 257.08±18.63,P<0.05),且各实验组间心房肌细胞[Ca2+]i水平随ionomycin浓度的增加而上升,实验组间比较,差异有统计学意义(P<0.05)。光镜下形态学观察表明,随ionomycin浓度的增加,荧光强度逐渐加强,呈浓度依赖性。结论应用钙离子载体ionomycin能够建立可靠的心房肌细胞钙超载模型。
Objective To investigate the modeling of calcium overload with primary atrial myocytes of neonate rats.Methods The atrial myocytes were disassociated from neonate rats with digestion method and cultured for 96 hours.Co-incubated with the ionomycin（0,0.5,1.0,2.0 μmol/L,respectively）,the cells were divided into control group and experiment group（E1,E2 and E3） and loaded with Ca^2＋ indicator Fluo-3 /AM.The levels of intracellular Ca^2＋（i） were measured with laser confocal scanning microscope.Results（1）More than 90% of cultured cells were positive to α-actin antibody.（2）The activity ratio of cells in control group was（73.00±2.37）%.Compared with each subgroup of experiment,there was no significant difference（P〉0.05）.（3）One hour after treated with different concentrations of ionomycin,the i of atrial muscle cell was significantly increased in all the experimental groups（431.54±20.97,705.87±28.34,1 305.05±53.69 vs 257.08±18.63,respectively,P〈0.05）.There was significant difference between each subset of the experimental group.i level and the fluorescence intensity of the myocytes increased gradually in a dose-dependent manner with the concentration of ionomycin.Conclusion Calcium overload model with primary atrial myocytes of neonate rats can be built by ionomycin.
出处
《重庆医学》
CAS
CSCD
北大核心
2010年第11期1331-1333,F0003,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(30700315)
关键词
大鼠
伊屋诺霉素
心肌细胞
钙超载
激光共聚焦
rats
ionomycin
atrial myocytes
calcium overload
laser confocal scanning microscopy
