Wistar大鼠乳鼠与成年鼠心室肌细胞的分离与性质鉴定

目的 探索和优化稳定的适于电生理实验研究的乳鼠及成年大鼠心室肌细胞分离方法。方法 切碎乳鼠心室肌,胰蛋白酶消化,差速贴壁2 h纯化心室肌细胞,台盼蓝染色判定心肌细胞活力,体外培养48 h后分别行倒置显微镜观察细胞形态,免疫组化鉴定,微电极阵列记录细胞搏动频率和场电位。采用Langendorff灌流成年大鼠心脏,主动脉逆行插管,胶原酶域反复灌流消化约30 min,无钙台氏液冲洗心脏5 min,剪下心室肌组织,台氏液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,将细胞悬液用逐步复钙法复钙后,室温静置1 h,用于膜片钳记录。结果 经4 -6次消化后,乳鼠心室组织消化完全,细胞存活率大于80%。倒置显微镜下观察,细胞呈梭形、多角形。 12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30 - 80次/分。 琢鄄辅肌动蛋白（琢鄄actin）经免疫组化检测,纯度达96%。 Langendorff灌流酶解法可获得形态呈杆状、横纹清晰、膜周边光滑完整、立体感强的单个成年鼠心肌细胞,存活率85%,复钙后存活率50%,可用于膜片钳记录。结论 采用本方法可以获得高产量与高质量的用于电生理检测的心室肌细胞。

Isolation and identification of ventricular cardiomyocytes from neonatal and adult Wistar rat

Objective To explore and improve a practical and reliable technique of isolation single ventricular myocytes from neonatal and adult Wistar rat for electrophysiological measurement. Methods The ventricular myocytes from neonatal rat were minced and digested in trypsin,the purified ventricular cardiomyocytes were obtained by differential adhesion about 2 hour. The survival rate was assessed by trypan staining. Ventricular myocytes can be observed through the methods of inverted phase contrast microscope after cultured 48 h,and identified through immunocytochemistry and MEA which investigated the beat rhythm and field potential in vitro. Adult rats were intubated through aorta and perfused for 30 min by the Langendorff apparatus with typeⅡcollagenase,then with Ca2 ＋-free Tyrode＇s solution for 5 min,after which the ventricular were minced into small pieces in Tyrode＇s solution,dispersed and filtered,incubated 5 min,filted with 200 screen mesh. The isolated myocytes were filtered and recovered to normal calcium concentration gradually,and stored in keeping solution at room temperature for 1 h and before patch clamp experiments. Results The ventricular tissues of neonatal rat were almost completely digested through 4- 6 times. Trypan staining showed that the survival rate of the cardiocytes cultured was more than 80%. Under the inverted phase contrast microscope,we found that the cells were fusiform or polyhedron and some cells began beat after 12 hours. The cells monolayer formed a network and beated spontaneously at the 30- 80 times / min after 48 hours. The cell purity was up to 96% by α-actin of strepta- vidin peroxidase conjunction method. The survival rate of freshly isolated adult rat ventricular myocytes were 85%,and decreased to 50% after recovered to normal calcium concentration,rod-shaped with clear cross-striations and complete membrane,which can be used for patch clamp. Conclusion High yield and high quality single rat ventricular myocytes could be obtained and are suitable for the measurement of elec- trophysiological technique.