目的 基于 RBL-2H3及 P815细胞系，选择 Compound 48/80（C48/80）为阳性药构建类过敏反应的体外评价模型，初步评价聚山梨酯80、维生素K₁注射剂（VK₁I）、注射用两性霉素B脂质体（ABL）导致类过敏反应的潜在风险。方法 应用 CCK-8 法检测 C48/80（10、30、60、100 μg·mL^-1）、聚山梨酯 80（2.5、5.0、10.0、50.0 mg·mL^-1）、VK₁I（0.625、1.250、2.500、5.000、10.000 mg·mL^-1）、ABL（0.125、0.250、0.500、1.000、2.000 mg·mL^-1）对 RBL-2H3、P815 细胞活性的影响，计算半数抑制浓度（IC₅₀），阳性药及受试物均选择＜IC₅₀浓度进行后续实验；结合中性红染色形态学观察，研究阳性药及受试物的细胞毒性；流式细胞仪检测Annexin V阳性细胞率及Fluo-4AM标记率；ELISA法检测β-氨基己糖苷酶（β-Hex）及组胺释放量。结果 中性红染色结果显示，在C48/80作用下RBL-2H3、P815细胞部分皱缩或者偶有破损，但大部分细胞仍维持完整细胞形态；在聚山梨酯 80、ABL、VK₁I高浓度的作用下，RBL-2H3、P815细胞大部分仍能保持正常形态。C48/80在＜IC₅₀浓度时能够引起较强的细胞脱颗粒反应，模型建立成功。聚山梨酯80、VK₁I在＜IC₅₀浓度时均出现了浓度相关性细胞脱颗粒现象，与对照组比较，2种细胞聚山梨酯80 2.5、5.0 mg·mL^-1组及VK₁I 0.75、1.50、3.00 mg·mL^-1组Annexin V阳性率、Fluo-4AM标记率均显著升高（P＜0.05、0.01）；聚山梨酯 80 2.5、5.0 mg · mL^-1 组及 VK₁I 1.5、3.0 mg·mL^-1组的组胺、β-Hex释放量显著升高（P＜0.05、0.01）。ABL 组 Annexin V 阳性率浓度相关性升高趋势不明显 ，与对照组比较 ，仅 P815 细胞 ABL 2.00 mg·mL^-1组显著升高（P＜0.05），且升高幅度不大 ；与对照组比较 ，2种细胞ABL 1、2 mg·mL^-1组的Fluo-4AM标记率和组胺、β-Hex释放量显著升高（P＜0.05、0.01）。结论 聚山梨酯80、VK₁I高浓度具有导致类过敏反应产生风险，ABL 还需进一步研究。

Objective To establish an in vitro pseudoallergy model based on RBL-2H3 and P815 cell lines and select Compound 48/ 80 as positive drug to evaluate potential of tween 80, Vitamin K1 Injection (VK₁I) and Amphotericin B Liposomes (ABL) to induce pseudoallergy.Methods Applying CCK-8 method to detect the effects of C48/80 (10, 30, 60, 100 μg·mL^-1), polysorbate 80 (2.5, 5.0, 10.0, 50.0 mg·mL^-1), VK₁I (0.625, 1.250, 2.500, 5.000, 10.000 mg·mL^-1), ABL (0.125, 0.250, 0.500, 1.000, 2.000 mg·mL^-1) on the activity of RBL-2H3 and P815 cells, and the half inhibitory concentration (IC₅₀) were calculated. The positive drug and test substance were selected at concentrations < IC 50 for subsequent experiments. Combining neutral red staining with morphological observation to study the cytotoxicity of positive drugs and test substances. The percentage of Annexin V positive cells and the labeling rate of Fluo-4AM were detected by Flow cytometry staining, the release of β-hexosaminidase (β-Hex) and histamine were detected by ELISA.Results The results of neutral red staining showed that under the action of C48/80, RBL-2H3 and P815 cells were partially shrunk or occasionally damaged, but most cells still maintained intact cell morphology. Under the action of high concentrations of polysorbate 80, ABL, and VK₁I, most RBL-2H3 and P815 cells can still maintain their normal morphology. C48/ 80 can induce strong cell degranulation reaction at concentrations below IC₅₀, and the model was successfully established. Both polysorbate 80 and VK₁I showed concentration dependent cell degranulation at concentrations below IC₅₀. Compared with the control group, the 2.5, 5.0 mg·mL^-1 group of polysorbate 80 and the 0.75, 1.50, and 3.00 mg·mL^-1 group of VK₁I showed a significant increase in the positive rate of Annexin V and the labeling rate of Fluor-4AM (P < 0.05, 0.01). The release of histamines and β-Hex in the groups of polysorbate 80 2.5, 5.0 mg·mL^-1 and VK₁I 1.5, 3.0 mg·mL^-1 significantly increased (P < 0.05, 0.01). The concentration correlation of Annexin V positive rate in the ABL group showed no significant increase. Compared with the control group, only P815 cells in the ABL 2.00 mg·mL^-1 group showed a significant increase (P < 0.05), and the increase was not significant. Compared with the control group, the labeling rate of Fluo-4AM and the release of histamine and β-Hex in the ABL 1 and 2 mg·mL^-1 groups increased significantly (P < 0.05, 0.01).Conclusion Some concentrations of Tween-80 and VK₁I have the risk of pseudoallergy, and ABL need further study.

R965.3

上海市科委实验动物研究领域专项（19140901300）