目的 通過(guò)比較不同濃度聚山梨酯80溶液對(duì)RBL-2H3、P815、Ku812細(xì)胞脫顆粒的影響，探究聚山梨酯80的量與過(guò)敏反應(yīng)的關(guān)系，進(jìn)而篩選出一套靈敏、穩(wěn)定的體外肥大細(xì)胞脫顆粒模型。方法 體外培養(yǎng)RBL-2H3、P815和Ku812細(xì)胞，測(cè)定3株細(xì)胞的生長(zhǎng)曲線，在細(xì)胞生長(zhǎng)對(duì)數(shù)期，以不同濃度（0.04、0.20、1.00、5.00、10.00、20.00、40.00、80.00 mg/mL）聚山梨酯80分別刺激3株細(xì)胞，采用中性紅染色法顯微鏡下觀察不同濃度聚山梨酯80溶液對(duì)3株細(xì)胞脫顆粒的影響，并計(jì)算脫顆粒百分率，同時(shí)以化學(xué)發(fā)光法分別檢測(cè)組胺和β-氨基己糖苷酶釋放率，以酶聯(lián)免疫吸附法測(cè)定類(lèi)胰蛋白酶的釋放量；并進(jìn)一步檢測(cè)聚山梨酯80對(duì)人的肥大細(xì)胞IgE釋放的影響。結(jié)果 3株肥大細(xì)胞脫顆粒模型中，各細(xì)胞的脫顆粒指標(biāo)均隨聚山梨酯80濃度的升高而升高。相同濃度聚山梨酯80對(duì)人源、大鼠、小鼠肥大細(xì)胞的類(lèi)胰蛋白酶釋放量無(wú)較大統(tǒng)計(jì)學(xué)差異；與RBL-2H3細(xì)胞系比較，P815細(xì)胞和組胺釋放率顯著降低（P<0.05、0.01），Ku812細(xì)胞組胺釋放率和β-氨基己糖苷酶釋放率顯著升高（P<0.05、0.01），Ku812細(xì)胞脫顆粒最靈敏。但在實(shí)驗(yàn)過(guò)程中發(fā)現(xiàn)Ku812細(xì)胞模型穩(wěn)定性、可重復(fù)性較差，而RBL-2H3細(xì)胞穩(wěn)定性、可重復(fù)性均優(yōu)于Ku812和P815細(xì)胞。0.04~80.00 mg/mL的聚山梨酯80溶液作用于Ku812細(xì)胞產(chǎn)生的IgE均低于檢測(cè)限。結(jié)論 聚山梨酯80誘導(dǎo)的過(guò)敏反應(yīng)可能是不經(jīng)過(guò)IgE介導(dǎo)的類(lèi)過(guò)敏反應(yīng)，隨著聚山梨酯80濃度的增大，3個(gè)細(xì)胞模型脫顆?，F(xiàn)象顯著，相比于Ku812和P815細(xì)胞，RBL-2H3細(xì)胞更適合作為體外肥大細(xì)胞脫顆粒檢測(cè)模型。

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..

天津市中藥注射劑關(guān)鍵技術(shù)校企協(xié)同創(chuàng)新實(shí)驗(yàn)室建設(shè)項(xiàng)目（17PTSYJC00090）