用于膜片钳研究的乳鼠窦房结细胞的分离与鉴定  

A method for isolating and identifying newborn rat sino-atrial node cells for patch-clamp study

作　　者：汪艳丽[1] 刘如秀[1] 刘宇[1] 李泱[2] 

机构地区：[1]中国中医科学院广安门医院心内科,北京100053 [2]解放军总医院老年心血管病研究所,北京100853

出　　处：《南方医科大学学报》2013年第3期397-400,共4页Journal of Southern Medical University

基　　金：北京市自然科学基金(7102135)

摘　　要：目的比较"酶解+差速贴壁"法和"酶解+差速贴壁+5-溴脱氧尿嘧啶核苷(常规培养)"法体外分离培养乳鼠窦房结细胞,寻找可行的用于膜片钳实验的分离方法。方法取Wistar乳鼠的窦房结组织,分别采用"酶解+差速贴壁"和常规培养法分离培养窦房结细胞,观察细胞形态结构、搏动频率、膜片钳实验难易程度及成功率。同样方法分离培养心房肌细胞作为对照。结果培养的窦房结细胞中梭形细胞最多、心房肌细胞中三角形细胞最多。两种方法分离的窦房结细胞中均为梭形细胞最多并均可记录到自发性动作电位,其中"酶解+差速贴壁"法分离的细胞活性更好,进行膜片钳实验的成功率更高。结论"酶解+差速贴壁"法能成功分离出乳鼠窦房结细胞,操作步骤简单,细胞损伤小、活性好,能更好地进行膜片钳实验研究。Objective To compare the effects of enzyme digestion with differential attachment and enzyme digestion with differential attachment plus 5＇-bromodeoxyuridine （5-BrdU） （conventional） method for isolating sino-atrial node cells from newborn rats for patch clamp studies. Methods The sino-atrial node tissue were removed from neonatal Wistar rats, digested and separated into single sino-atrial node cells with the above two methods. The obtained ceils were observed for morphology, structure, beat frequenc~ degree of difficulty for patch clamp experiment and the success rate. The atrial cells isolated with enzyme digestion with differential attachment served as the control ceils. Results The spindle-shaped cells constituted the greatest proportion of the cultured sino-atrial cells, and the cultured atrial cells showed mostly a triangular morphology. Both of the sino-atrial cells separated with the two methods contained mostly spindle-shaped cells, in which spontaneous action potential was recorded. The spindle-shaped cells isolated with enzyme digestion and differential attachment showed a greater viability with a higher success rate in patch clamp studies. Conclusion The sino-atrial node cells can be successfully isolated with enzyme digestion and differential attachment, which requires simple procedures and well preserves the cell viability for better performance in patch clamp experiment.

关 键 词：窦房结细胞 分离 膜片钳 实验研究 

分 类 号：R363[医药卫生—病理学]