研究电压门控性钠离子通道的3种细胞处理方法比较

Comparison of three cellular processing approaches for studying voltage-gated sodium ion channels

目的 建立适用于膜片钳技术记录电压门控性钠离子通道的3种细胞处理方法并进行比较.方法 采用急性分离技术、分离脑片技术、pCMV_SCN1A质粒钙转染HEK293细胞3种方式处理细胞,镜下观察细胞形态,膜片钳记录钠通道激活情况.结果 急性分离的神经元形态正常,有较长突起；膜片钳记录封接成功率达50％以上,记录到电流的细胞膜电容为（7.56±3.47）pF（n=20）,串联电阻为（10.18±4.82）MΩ（n=20）.分离脑片的神经元形态正常饱满；膜片钳记录封接成功率达30％以上,记录到电流的细胞膜电容为（9.45±4.26）pF（n=10）,串联电阻为（12.53±3.87）MΩ（n=10）.钙转染处理的细胞完整,有立体感；膜片钳记录封接成功率约20％,记录到电流的细胞膜电容为（8.79±4.54）pF（n=10）,串联电阻为（14.12±4.26）MΩ（n=10）.3种细胞处理方法均能提供研究所需的钠通道电流.结论 3种细胞处理方法均可以简单、快速记录电压门控性钠离子通道,各有优缺点,适用于不同的研究需求.

Objective To establish and compare three cellular processing approaches for voltagegated sodium ion channels in which patch clamp technique is suitable to apply.Methods Cells were processed with acute isolation technique,brain slices separating technique and pCMV-SCN1A plasmid calcium-transfected HEK293 cells.Morphology was observed under microscopic vision and activation of sodium ion channel was monitored using patch clamp technique.Results The acute isolated neurons had normal morphology and relatively long processes.The success rate of patch clamp technique was over 50%,with the recorded cellular membrane capacity of （7.56± 3.47）pF （n=20） and serial resistence of （10.18±4.82） MΩ （n=20）.Isolated neurons in brain slices were normal and plump.The success rate of patch clamp technique was over 30%,with the recorded cellular membrane capacity of （9.45±4.26）pF （n=10） and serial resistence of （12.53 ± 3.87）MΩ （n=10）.Calcium-transfected cells were integrated and stereo.The success rate of patch clamp technique was approximately 20%,with the recorded cellular membrane capacity of （8.79±4.54） pF （n=10） and serial resistence of （14.12±4.26） MΩ （n=10）.All these approaches could provide the current of sodium channel required for research.Conclusion These three cellular processing approaches,which are feasible for distinct researches due to different strengths and weaknesses,can record voltage-gated sodium ion channels conveniently and rapidly.