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[摘要]
目的 通过比较不同浓度聚山梨酯80溶液对RBL-2H3、P815、Ku812细胞脱颗粒的影响,探究聚山梨酯80的量与过敏反应的关系,进而筛选出一套灵敏、稳定的体外肥大细胞脱颗粒模型。方法 体外培养RBL-2H3、P815和Ku812细胞,测定3株细胞的生长曲线,在细胞生长对数期,以不同浓度(0.04、0.20、1.00、5.00、10.00、20.00、40.00、80.00 mg/mL)聚山梨酯80分别刺激3株细胞,采用中性红染色法显微镜下观察不同浓度聚山梨酯80溶液对3株细胞脱颗粒的影响,并计算脱颗粒百分率,同时以化学发光法分别检测组胺和β-氨基己糖苷酶释放率,以酶联免疫吸附法测定类胰蛋白酶的释放量;并进一步检测聚山梨酯80对人的肥大细胞IgE释放的影响。结果 3株肥大细胞脱颗粒模型中,各细胞的脱颗粒指标均随聚山梨酯80浓度的升高而升高。相同浓度聚山梨酯80对人源、大鼠、小鼠肥大细胞的类胰蛋白酶释放量无较大统计学差异;与RBL-2H3细胞系比较,P815细胞和组胺释放率显著降低(P<0.05、0.01),Ku812细胞组胺释放率和β-氨基己糖苷酶释放率显著升高(P<0.05、0.01),Ku812细胞脱颗粒最灵敏。但在实验过程中发现Ku812细胞模型稳定性、可重复性较差,而RBL-2H3细胞稳定性、可重复性均优于Ku812和P815细胞。0.04~80.00 mg/mL的聚山梨酯80溶液作用于Ku812细胞产生的IgE均低于检测限。结论 聚山梨酯80诱导的过敏反应可能是不经过IgE介导的类过敏反应,随着聚山梨酯80浓度的增大,3个细胞模型脱颗粒现象显著,相比于Ku812和P815细胞,RBL-2H3细胞更适合作为体外肥大细胞脱颗粒检测模型。
[Key word]
[Abstract]
Objective To investigate the relationship between the quantity of Tween 80 and allergic reaction and select a sensitive and stable model of mast cell degranulation in vitro by comparing the effects of different concentrations of Tween 80 solution on the degranulation of RBL-2H3,P815 and Ku812 cells.Methods The RBL-2H3,P815,and Ku812 cells were cultured in vitro,and the growth curves of three cell lines were determined.Stimulated cells which were in the logarithmic phase with different concentrations of Tween 80 solution (0.04,0.2,1,5,10,20,40,80 mg/mL),were observed under microscope to study the effect of different concentrations of Tween 80 solution on degranulation of three cell lines by neutral red staining method,and calculated the percentage of degranulation.Meanwhile,the release rate of histamine and β-hexosaminidase was detected by chemiluminescence assay,and the release amount of trypsin was detected by enzyme-linked immunosorbent assay (ELISA).Furthermore,the IgE release of human mast cells was also examined.Results In the three mast cell degranulation models,the degranulation index of each cell increased with the increase of Tween 80 concentration.There was no significant difference in tryptase release from human,rat and mouse mast cells at the same concentration of Tween 80.Compared with RBL-2H3 cell line,the degranulation rate and histamine release rate of P815 cells were significantly decreased (P< 0.05,0.01),and the histamine release rate and β-hexosaminidase release rate of Ku812 cells were significantly increased (P< 0.05,0.01).The degranulation effect of Ku812 cells was the most sensitive,but the stability and repeatability of Ku812 cells model were poor.However,the sensitivity of RBL-2H3 cells was inferior to Ku812 cells,and its stability and reproducibility were superior to Ku812 and P815 cells.In addition,the IgE produced by 0.04-80.00 mg/mL Tween 80 solution acting on Ku812 cells cannot be detected.Conclusion The anaphylaxis induced by Tween 80 may be the anaphylactoid reaction without mediated by IgE.With the increase of Tween 80 concentration,three models were all degranulated significantly.Compared with Ku812 and P815 cells,RBL-2H3 cells were more suitable for degranulation detection model of mast cells in vitro..
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[基金项目]
天津市中药注射剂关键技术校企协同创新实验室建设项目(17PTSYJC00090)