氯胺酮对荷包牡丹碱诱导PC12细胞内Ca^(2+)浓度波动方式的影响

The effects of ketamine on bicuculline-induced calcium fluctuation in PC12 cell

目的研究氯胺酮对荷包牡丹碱诱导PC12细胞内Ca2+浓度波动方式的影响。方法使用含25ng/LNGF的DMED培养基在多聚赖氨酸包被的培养皿中培养PC12细胞;与终浓度10μmol/L的Ca2+指示剂Fluo-3AMester共孵育30min洗涤后,加入终浓度50μmol/L荷包牡丹碱;在激光共聚焦显微镜选定多个细胞分别测定荧光强度的变化;随后加入氯胺酮,记录细胞荧光强度的改变。在试验结束前依次加入TritonX-100和EGTA分别记录单个细胞最大荧光强度(Fmax)和最小荧光强度(Fmin),以计算细胞内Ca2+的相对强度。结果氯胺酮不改变荷包牡丹碱诱导PC12细胞内Ca2+浓度波动的基线,但抑制细胞内Ca2+浓度升高的幅度(P<0.05),缩短相邻波峰间的时间间隙(P<0.05)。结论氯胺酮不仅改变荷包牡丹碱诱导PC12细胞内Ca2+浓度升高的幅度,而且改变Ca2+浓度波动的周期。

Objective To observe the effects of ketamine on calcium fluctuation in PCl2 cell induced by bicuculline. Methods PCl2 cells were cultured on culture plate coated with Poly-L-lysine （1 mg/ml） in DMEM containing 15% fetal calf serum, 25 ng/L NGF for 4 days. PCl2 cells were loaded with 10 μmol/L Fluo 3 AM for 30 min, and then washed with dye free DMEM. Fluo-3 fluorescence（F） images were taken before and after bicuculline or ketamine. To calibrate the images, Fluo-3 was saturated by adding 50% Triton X-100 and then quenched with 0.2 tool EGTA for getting maximal and minimal fluorescence （Fmax and Fmin）, respectively. Calcium concentrations were expressed as a function of the Fluo-3 fluorescence[（F-Fmin）/（Fmax-F）]. Results Ketamine did not alert the basic line of calcium concentration fluctuations in PCl2 cells resulting from bicuculline. But it not only inhibited the increase of calcium concentration （P 〈0.05）, also decreased the duration between two calcium peaks in PCl2 cells induced by bicuculline（P〈0.05）. Conclusion Ketamine not only inhibits the increase of calcium concentration, but also decreases the cycle time of calcium concentration fluctuations in PCl2 cells induced by bicuculline.