吐温80诱导RBL-2H3细胞脱颗粒作用研究

Effect on Tween 80 on the degranulation of RBL-2H3 cells

通过观察吐温80对RBL-2H3细胞的诱导脱颗粒作用,以初步探讨吐温80导致类过敏反应的可能机制。采用不同浓度的吐温80溶液与RBL-2H3细胞共孵育,ELISA和生化法测定过敏活性物质组胺和β氨基己糖苷酶释放率,AnnexinV-FITC荧光抗体染色法(流式细胞仪检测和荧光显微镜观察)检测细胞膜磷脂酰丝氨酸外翻情况;结果250、1250、6250mg/L吐温80溶液均可以导致RBL-2H3细胞组胺和β氨基己糖苷酶释放率增加(P<0.05或P<0.01),呈浓度依赖性;流式细胞仪检测和荧光显微镜观察发现此3个浓度吐温80可以导致RBL-2H3细胞膜磷脂酰丝氨酸外翻增加(P<0.01),且呈浓度依赖性;使用含钙与不含钙缓冲体系进行孵育。结果显示,β氨基己糖苷酶释放率没有显著性差异(P>0.05)。吐温80可以在不依赖细胞外游离钙离子的情况下直接诱导肥大细胞脱颗粒,释放活性介质,这可能是其导致类过敏反应的重要原因之一。

To investigate the effect of Tween 80 on the mast cell degranulation in RBL-ZH3 cells and to find out the possible mechanism of the anaphylactoid reaction induced by Tween 80, different concentrations of Tween 80 solution were incubated with RBL-2H3 cells. The releasing rate of the anaphylaxis activating substances, such as histamine and β-P-nitrophyenyl-N-acetyl-β-D-glucosaminide, were detected by ELISA and biochemical method and the transloeation of phosphatidylserine （PS） in cell membrane was assayed by Annexin-V-FITC fluorescent antibody staining （ flow cytometry and fluorescent microscopy）. It was demonstrated that the releasing rates of these anaphylaxis activating substances were increased after co incubation of the cells with 250, 1 250 and 6 250 mg/L concentrations of Tween 80 in a dose-dependent manner （P〈0.01 or 0.05）. Mean while, the releasing rate of PS translocation in cell membrane after treatment with Tween 80 increased also in a dose-depend ent manner （P〈0. 01）. However, the degranutation induced by Tween 80 independent on the extracellular calcium; no difference could be detected upon addition of calcium solution or without addition （P〈0.05）. From these observations, it is concluded that Tween 80 can induce degranulation of mast cells directly to release active mediators of anaphylaxis and this may be one of the cause of the anaphylactoid reaction induced by Tween 80.