微激光照射肥大细胞对胞内钙离子浓度的影响

The Effect of Micro-laser on Intracellular Free Calcium Concentration of Mast Cell

目的:研究850 nm波长微激光对肥大细胞照射后胞内钙离子浓度的影响。方法:实验前12 h,将肥大细胞接种于激光共聚焦专用培养皿中,然后用D-Hank’s液洗涤3次,加入2 m L D-Hank’s液,按照照射时间不同共分六组(Control,1 min,2 min,2.5 min,3 min,4 min),其中,空白对照组不进行激光照射处理;激光照射后,弃去D-Hank’s液,加入含有Fluo-3/AM的D-Hank’s液1 m L,放入培养箱中孵育30 min后,用D-Hank’s液洗涤3次去除胞外多余的Fluo-3/AM,再加入含有10%FBS的D-Hank’s液2 m L,置激光扫描共聚焦显微镜检测。结果:空白对照组中细胞内未见荧光,说明此时胞内无钙离子,但从1 min开始在细胞中发现荧光,而且发现随着照射时间的加长,其荧光强度越来越强,这可能与微激光照射时间越长从而刺激胞内出现更多的钙离子有关。从上面的实验说明850 nm微激光能作为仿灸仪器的光源。

Objective： To investigate the effects of micro-laser irradiation on intracellular calcium concentration（ [Ca2 ＋]i） of mast cell. Methods： 12 hours before the experiment,The mast cells were seeded into paltes,which were used on the laser scanning confocal microscopy（ LSCM）. Then the cells were washed three times with D-hank＇s solution,and added 2 m L D-hank＇s solution to each group. There were six groups,which were irradiated at different times（ Control,1 min,2 min,2. 5 min,3 min,4 min） by micro-laser 850 nm. The control group was irradiated 0 min. After micro-laser irradiation,the cells were removed the D-hank＇s solution,and loaded with 1 m L Fluo-3 / AM at 37° C for 30 min. The cells were washed again for another three times with D-hank＇s solution to remove the unloaded Fluo-3 / AM in the medium,and then added 2 m L D-hank＇s solution with 10% FBS. The images of Ca2 ＋concentration in each group cells were monitored by LCSM. Result： The results have shown that there were no fluorescence in the control group. It clearly indicated that there were no Ca2 ＋in the control group cells. But in the irradiated 1 min group,there was significant fluorescence. Following the time of irradiation increasing,the fluorescence intensity were enhanced. The intracellular calcium concentration become higher,which may be related to the micro-laser irradiation time increase. It has shown that the 850 nm micro-laser could be the optical source of moxibustion instrument.