目的 基于 RBL-2H3及 P815細胞系，選擇 Compound 48/80（C48/80）為陽性藥構(gòu)建類過敏反應(yīng)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(zhì)體（ABL）導(dǎo)致類過敏反應(yīng)的潛在風(fēng)險。方法 應(yīng)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(jié)合中性紅染色形態(tài)學(xué)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(biāo)記率；ELISA法檢測β-氨基己糖苷酶（β-Hex）及組胺釋放量。結(jié)果 中性紅染色結(jié)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(yīng)，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(guān)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(biāo)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(guān)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(biāo)記率和組胺、β-Hex釋放量顯著升高（P＜0.05、0.01）。結(jié)論 聚山梨酯80、VK₁I高濃度具有導(dǎo)致類過敏反應(yīng)產(chǎn)生風(fēng)險，ABL 還需進一步研究。

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

上海市科委實驗動物研究領(lǐng)域?qū)ｍ棧?9140901300）