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目的建立膜片钳-激光扫描共聚焦显微镜同步实时控制系统,并将其应用于体外心肌细胞膜钙离子通道的研究,验证其应用效果。方法通过在激光扫描共聚焦显微镜上加装膜片钳装置,采用计算机自动控制技术建立膜片钳与激光扫描共聚焦显微镜的同步实时控制系统;将建立好的装置应用于观察体外雄性大鼠心肌细胞膜钙离子通道,并分析观察结果。结果成功建立膜片钳-激光扫描共聚焦显微镜同步实时控制系统;当刺激心肌细胞时,激光扫描共聚焦显微镜-膜片钳同步实时控制系统在通过激光扫描共聚焦显微镜观察钙火花的同时可通过膜片钳记录心肌细胞膜钙离子通道电流信号。定量分析结果表明相邻两个钙火花之间的时间间距分别为(10.055±0.021)、(10.079±0.021)、(10.087±0.021)s,符合膜片钳设定的刺激间隔(10 s);单个钙火花在空间上均局限于2μm直径范围,在时间上平均经历了约30 ms,从出现至达到最高浓度平均需10 ms,从达到最高浓度到消失平均需20 ms,与钙火花理论吻合。结论成功建立膜片钳-激光扫描共聚焦显微镜同步实时控制系统,在心肌细胞实现了在利用膜片钳进行全细胞记录观测和测定钙离子通道电流及其开闭时程的同时,利用激光扫描共聚焦显微镜获得了钙火花的显微结构形态图像,测定钙离子的位点变化,有助于进一步了解质膜钙离子通道的内部机制。
Objective To establish a synchronous real-time control system of patch clamp and laser scanning confocal microscopy and to apply it to the study of calcium channels in myocardial cell membrane in vitro to verify its application effect. Methods Patch clamp device was installed on the laser scanning confocal microscope, and the automatic real-time control system of patch clamp and laser scanning confocal microscope was established by computer automatic control technology. The established device was applied to observe the in vitro rat cardiac muscle Cell membrane calcium channels, and analyze the observations. Results Patch clamp-laser scanning confocal microscopy synchronized real-time control system was successfully established. When stimulating cardiomyocytes, laser scanning confocal microscope-patch-clamp synchronized real-time control system can be used to detect the calcium spark through laser scanning confocal microscope Patch clamp recording myocardial cell membrane calcium channel current signal. The results of quantitative analysis showed that the time interval between two adjacent calcium sparks was (10.055 ± 0.021), (10.079 ± 0.021) and (10.087 ± 0.021) s, respectively, which accorded with the stimulation interval (10 s) set by patch clamp. A single calcium spark is spatially confined to a diameter range of 2 μm with an average of about 30 ms in time, an average of 10 ms from the time it reaches the highest concentration, an average of 20 ms from the time it reaches the highest concentration until it disappears, in agreement with the calcium spark theory . Conclusion Simultaneous real-time control system of patch clamp and laser scanning confocal microscope was successfully established. In the process of cardiomyocytes, whole-cell recording and measurement of calcium channel current and opening and closing time were carried out. At the same time, Confocal microscopy obtained calcium spark microstructure morphological images, determination of calcium site changes, help to further understand the plasma membrane calcium channel internal mechanism.