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目的:建立一种稳定的适合膜片钳技术的逼尿肌细胞急性酶分离方法,为排尿相关障碍性疾病的研究提供必要的技术平台。方法:采用H型胶原酶和木瓜蛋白酶相混合的鸡尾酒酶液对新鲜离体的大鼠膀胱逼尿肌条在37℃条件下振荡消化,α-actin免疫荧光染色对分离并培养的原代细胞进行鉴定,在膜片钳工作台上分别对其进行L型钙电流和BKca钾电流的全细胞记录。结果:可获得大量的单个逼尿肌细胞。经过免疫荧光染色证实为平滑肌细胞。分离细胞活性良好,在膜片钳实验系统上可记录到多种通道电流。结论:建立了一种操作简单、成功率高、活性好的逼尿肌细胞急性酶分离方法并成功应用于膜片钳技术。
OBJECTIVE: To establish a stable method of acute enzyme separation of detrusor muscle cells which is suitable for patch clamp technique and provide the necessary technical platform for the study of urinary-related disorders. Methods: Fresh dendritic rat bladder detrusor strips were shaken and digested with H-type collagenase and papain at 37 ℃. The α-actin immunofluorescence staining of primary cells Were identified in the patch clamp workstation were respectively L-type calcium current and BKca potassium current of whole-cell recording. Results: A large number of individual detrusor cells are available. After immunofluorescence staining showed smooth muscle cells. Separation of cell activity is good, in the patch clamp experimental system can record a variety of channel current. CONCLUSION: A simple method for the separation of acute detrusor cells from patients with simple and high success rate and high activity has been established and successfully applied to patch-clamp technique.