基于RBL-2H3与P815细胞体外模型的聚山梨酯80、维生素K_(1)注射剂、注射用两性霉素B脂质体类过敏反应潜在风险评价

Evaluation of tween 80,Vitamin K_(1) Injection and Amphotericin B Liposomes pseudoallergy in vitro based on RBL-2H3 and P815 cell models

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K_(1)注射剂(VK_(1)I)、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、100μg·mL^(−1))、聚山梨酯80(2.5、5.0、10.0、50.0 mg·mL^(−1))、VK_(1)I(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、VK_(1)I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、VK_(1)I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802.5、5.0 mg·mL^(−1)组及VK_(1)I 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802.5、5.0 mg·mL^(−1)组及VK_(1)I 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、VK_(1)I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 K_(1) Injection(VK_(1)I)and Amphotericin B Liposomes(ABL)to induce pseudoallergy.Methods Applying CCK-8 method to detect the effects of C48/80(10,30,60,100μg·mL^(−1)),polysorbate 80(2.5,5.0,10.0,50.0 mg·mL^(−1)),VK_(1)I(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<IC50 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 ofβ-hexosaminidase(β-Hex)and histamine weredetected 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 VK_(1)I,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 VK_(1)I 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 VK_(1)I 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 andβ-Hex in the groups of polysorbate 802.5,5.0 mg·mL^(−1) and VK_(1)I 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 andβ-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 VK_(1)I have the risk of pseudoallergy,and ABL need further study.