新生大鼠原代心房肌细胞钙超载模型建立的实验研究※.docVIP

新生大鼠原代心房肌细胞钙超载模型建立的实验研究※ 丁 翔 仝识非 秦 瑶 宋治远▲ 第三军医大学西南医院心内科 重庆 400038 [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：消化法分离新生大鼠心房肌细胞，并行原代培养。第96小时不同浓度ionomycin（00.5、1.0、2.0 μmol/L）和心房肌细胞共孵育，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%左右免疫化学鉴定90%以上培养细胞α-肌动蛋白抗体染色阳性。②对照组心房肌细胞活性比率为73.00士2.37 %，各间心房肌细胞活性无显著性差异（0.05）。③经不同浓度ionomycin处理l h后，实验心房肌细胞[Ca2+]i明显增高，与对照组有显著性差异（0.05），且各实验组间心房肌[Ca2+]i水平随ionomycin浓度的增加而上升（0.05）。光镜下形态学观察表明，随ionomycin浓度的增加，荧光强度，呈浓度依赖性。结论：ionomycin能够建立可靠的心房肌细胞钙超载模型。 The study of calcium overload model with primary atrial myocytes of neonate rats Ding Xiang Tong Shifei Qin Yao Song-Zhiyuan▲ Department of Cardiology, Southwest Hospital, Chongqing 400038 [Abstract] 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. The cells were co-incubated with the ionomycin（0、0.5、1.0、2.0 μmol/L）loaded with Ca2+ indicator Fluo-3 /AM. The level of intracellular Ca2+ ([Ca2+]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士2.37%. Compared with the each subgroup of experiment, there was no significant difference(p0.05). ③ One hour after treated with different concentrations of ionomycin, the [Ca2+]i of atrial muscle cell was significantly increased in all the experimental groups(vs control group, p0.05). There was significant difference between each subset of the experimental group. [Ca2+]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. [Keywords] rats; ionomycin; atrial myocytes; calcium overload; laser confocal scannin