大鼠羊水与RBL-2H3肥大细胞共培养后脱颗粒的检测

Analysis of Degranulation After Cultivation of RBL-2H3 Cell with Rat Amniotic Fluid

[目的]探讨大鼠羊水与 RBL-2H3肥大细胞共培养后是否有脱颗粒现象发生。[方法]取大鼠不同稀释度的羊水与大鼠来源的RBL-2H3肥大细胞系共培养,用酶联免疫法测定不同时间样品上清的组胺、自发释放的组胺和总组胺,计算组胺释放率。对细胞用阿利新蓝染色,计算脱颗粒指数。用流式细胞仪测定RBL-2H3细胞Annexin Ⅴ标记后的阳性细胞率。[结果]与不同稀释度的羊水共培养后,细胞的组胺释放率有显著差异,组胺的释放和羊水浓度正相关。阿利新蓝染色后计算的脱颗粒指数与组胺释放率测定结果一致。细胞Annexin Ⅴ标记后的阳性率在羊水刺激后明显上升,进一步证明了脱颗粒的发生。[结论]羊水中的物质可以通过直接作用导致RBL-2H3肥大细胞脱颗粒,为羊水栓塞时肥大细胞脱颗粒机制的研究提供了一定的实验依据。

[Objective] To investigate the effect of rat amniotic fluid on the degranulation function of REL-2H3 cell. [Methods] RBL-2H3 cells were cultivated with different dilutions of rat amniotic fluid. The histamine from the supernatants, the spontaneously released histamine and the total his-tamine were all determined by ELISA. The percentage of histamine release was calculated from these data. The RBL-2H3 cells were stained with alcian blue, then degranulation index was calculated after counting the positive cells. The percentage of Annexin V positive cells were also obtained by flow cy-tometry analysis. [Results] RBL-2H3 cells released histamine in a concentration-dependent manner upon challenge with rat amniotic fluid, the level of histimine was directly correlated with the concentration of rat amniotic fluid. Degranulation index was 23 % , and the percentage of Annexin V positive cells increased upon the stimulation with rat amniotic fluid. The results of degranulation index and the percentage of Annexin V positive cells were all in accord with the percentage of histamine release. [Conclusion] Rat amniotic fluid may induce degranulation of RBL-2H3 cell in a direct way. This fact supports the postulate that degranulation is involved in amniotic fluid embolism.