一种有效记录成骨细胞 L 型钙通道电流的穿孔全细胞膜片钳方法

摘要

[ ABSTRACT] AIM:To establish a perforated whole-cell patch-clamp technique withβ-escin to record L-type cal-cium current (ICa,L) in osteoblasts.METHODS:ROS 17/2.8 is a rat osteoblast-like osteosarcoma cell line.β-escin was applied to the pipette solution to permeabilize the cell membrane and the perforated patch recording mode was obtained. RESULTS:β-escin at concentration of 50μmol/L easily permeabilized the cell membrane and obtained a perforated patch recording mode in 2~7 min.This technique prevented ICa,L rundown and preserved cytoplasmic signaling pathways.CON-CLUSION:β-escin may be used as an alternative ionophore for perforated patch-clamp studies in osteoblasts and results in minimal rundown that could facilitate recordings of ICa,L in osteoblasts.%目的：探讨一种有效记录成骨细胞L型钙通道电流的穿孔全细胞膜片钳方法。此方法使玻璃电极与细胞内液保持电化学连续性，使细胞内环境保持稳定，改善常规全细胞膜片钳破膜造成的破膜难和封接不稳，以及常规全细胞膜片钳破膜后造成的封接不稳。方法：以成骨细胞为研究对象，采用β－七叶素穿孔全细胞膜片钳技术，记录骨细胞L型钙通道电流。结果：采用这种穿孔全细胞膜片钳技术，能有效记录到成骨细胞上L型钙通道电流。结论：穿孔膜片钳技术是一种简便而有效的记录全细胞电流的实验方法，该方法的建立为今后更深入的研究疾病中成骨细胞的病理生理机制提供实验基础。