[關鍵詞]
[摘要]
目的 基于 RBL-2H3及 P815細胞系,選擇 Compound 48/80(C48/80)為陽性藥構建類過敏反應的體外評價模型,初步評價聚山梨酯80、維生素K1注射劑(VK1I)、注射用兩性霉素B脂質體(ABL)導致類過敏反應的潛在風險。方法 應用 CCK-8 法檢測 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)對 RBL-2H3、P815 細胞活性的影響,計算半數(shù)抑制濃度(IC50),陽性藥及受試物均選擇<IC50濃度進行后續(xù)實驗;結合中性紅染色形態(tài)學觀察,研究陽性藥及受試物的細胞毒性;流式細胞儀檢測Annexin V陽性細胞率及Fluo-4AM標記率;ELISA法檢測β-氨基己糖苷酶(β-Hex)及組胺釋放量。結果 中性紅染色結果顯示,在C48/80作用下RBL-2H3、P815細胞部分皺縮或者偶有破損,但大部分細胞仍維持完整細胞形態(tài);在聚山梨酯 80、ABL、VK1I高濃度的作用下,RBL-2H3、P815細胞大部分仍能保持正常形態(tài)。C48/80在<IC50濃度時能夠引起較強的細胞脫顆粒反應,模型建立成功。聚山梨酯80、VK1I在<IC50濃度時均出現(xiàn)了濃度相關性細胞脫顆?,F(xiàn)象,與對照組比較,2種細胞聚山梨酯80 2.5、5.0 mg·mL-1組及VK1I 0.75、1.50、3.00 mg·mL-1組Annexin V陽性率、Fluo-4AM標記率均顯著升高(P<0.05、0.01);聚山梨酯 80 2.5、5.0 mg · mL-1 組及 VK1I 1.5、3.0 mg·mL-1組的組胺、β-Hex釋放量顯著升高(P<0.05、0.01)。ABL 組 Annexin V 陽性率濃度相關性升高趨勢不明顯 ,與對照組比較 ,僅 P815 細胞 ABL 2.00 mg·mL-1組顯著升高(P<0.05),且升高幅度不大 ;與對照組比較 ,2種細胞ABL 1、2 mg·mL-1組的Fluo-4AM標記率和組胺、β-Hex釋放量顯著升高(P<0.05、0.01)。結論 聚山梨酯80、VK1I高濃度具有導致類過敏反應產生風險,ABL 還需進一步研究。
[Key word]
[Abstract]
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.
[中圖分類號]
R965.3
[基金項目]
上海市科委實驗動物研究領域專項(19140901300)