乳兔窦房结细胞的分离及鉴定

Isolation and culture of sinus node cells and identification of patch clamp technique in neonatal rabbits

目的乳兔窦房结细胞(sinus node cell,SNC)的分离、纯化、培养与鉴定。方法选用新生新西兰乳兔5只,采用双酶解法对细胞进行消化分离,差速贴壁结合5-BrdU对分离的细胞进行纯化培养,观察SNC形态变化并采用全细胞膜片钳技术对SNC进行动作电位的记录。结果培养得到的SNC主要有3种形态:梭形、三角形与不规则形,而梭形细胞最多,搏动频率最快,符合窦房结细胞的特征。采用膜片钳技术记录10个梭形细胞的动作电位中,平均最大舒张电位为(-50.9±5.3)mV,动作电位幅度为(61.9±4.8)mV。结论采用双酶解、差速贴壁及BrdU对乳兔窦房结细胞进行消化、分离可得到纯化的SNC,此种方法得到的SNC状态活性良好,且具有典型特征的动作电位。

Objective To summarize the isolation, purification, culture and identification of sinus node cells （ SNC ） in neonatal rabbits. Methods New Zealand neonatal rabbits （ n=5 ） were selected and from them SNC were digested and isolated by using dual anzymolysis. The isolated SNC were purified and cultured by using differential adherence combining 5-BrdU. The morphological changes of SNC were observed and action potential was recorded by using patch clamp technique. Results The morphological forms of cultured SNC were mainly spindle cells, triangular cells and irregular cells and fusiformis was the most with the fastest beating rate, which was accorded with the characteristics of SNC. Among action potential recorded from 10 spindle cells by patch clamp technique, the average maximum diastolic potential was （ -50.9 ± 5.3 ） mV and amplitude of action potential was （ 61.9 ± 4.8 ） mV. Conclusion The active condition of SNC digested, isolated and purified by using dual enzymolysis, differential adherenc and 5-BrdU are quite good with typical action potential.