应用激光共聚焦扫描显微镜测定缺氧对大鼠脑微血管内皮细胞Ca2＋含量的影响

Use Confocal Microscopy to Study Changes of Ca 2+ in Response to Anoxia in Endothelial Cells from Cerebral Microvessels of Rat/

应用Ｕｌｔｉｍａ型激光共聚焦扫描显微镜、ｆｌｕｏ－３荧光探针，检测大鼠脑微血管内皮细胞在正常体外培养及缺氧培养时游离钙含量的动态变化。缺氧由培养液中加入２ｍｍｏｌ连二亚硫酸钠（Ｎａ２Ｓ２Ｏ４）同时充入Ｎ２（９５％）、ＣＯ２（５％）气体造成。实验结果发现，当缺氧１～２ｍｉｎ时，内皮细胞Ｃａ２＋含量迅速增加，２～３ｍｉｎ达高峰。此后，尽管内皮细胞仍处于缺氧条件，荧光强度却逐渐减弱。停止缺氧后，荧光强度曲线可趋于稳定。应用Ｎｉｋｏｎ倒置相差显微镜观察内皮细胞形态学变化，发现缺氧３～５ｍｉｎ，内皮细胞发生肿胀。缺氧８～１０ｍｉｎ，细胞表面有明显受损迹象。

In has been known that anoxia can cause accumulation of free cytosolic calcium (Ca 2+ ) in central neurons in rat, but we do not know whether endothelial cell (EC) from cerebral microvessels increase Ca 2+ in response to anoxic condition. In order to determine whether anoxia increase Ca 2+ in EC in culture, intracellular calcium were measured using the calcium sensitive probe fluo 3/AM and confocal microscopy. To induce anoxia, the perfusing buffer PBS was equilibrated with N 2 (95%), CO 2 (5%), and 2mmol Na 2S 2O 4 was added. The present study demonstrates that anoxia caused a rapid response in Ca 2+ in EC from cerebral microvessels of rat. Within 2￣3min of anoxia exposure, these ECs showed an increase in free intracellular Ca 2+ . Following this increase, EC exhibited a decrease in fluorescence even though these cells still exposed to anoxic condition. After stopping anoxia, fluorescence level trend to stable. A Nikon inverted phase contrast microscope was used for observation in morphology of EC. Within 5min of anoxic condition, ECs showed swelling appearance. It is suggested that anoxia can caused a increase in Ca 2+ in EC from cerebral microvessels of rat, followed by swelling and being damaged.