交链孢毒素在Caco-2细胞中的转运与吸收

Transportation and absorption of Alternaria mycotoxins in Caco-2 cells

目的利用构建的结肠腺癌细胞系（Caco-2）细胞模型,探讨交链孢毒素经Caco-2细胞模型的跨膜转运与吸收特征。方法将Caco-2细胞接种于Transwell培养板的聚碳酯膜上培养21 d,用跨上皮细胞电阻（TEER）、碱性磷酸酶（ALP）活性、荧光素表观渗透系数（Papp）和细胞形态学等指标评价所构建的Caco-2细胞模型。通过测定细胞单层顶侧和基底膜侧交链孢毒素的含量,研究不同浓度和转运时间交链孢酚（AOH）、交链孢酚单甲醚（AME）、交链孢菌酮酸（Te A）溶液的转运与吸收。结果培养至第21天的Caco-2细胞其TEER超过（300±15）Ω·cm2,且细胞顶侧和基底膜侧ALP活性差异有统计学意义（P〈0.01）。透射电镜下可见清晰的微绒毛和细胞间的紧密连接。AOH、AME和Te A的Papp范围为（1~10）×10-5cm/s,不同浓度和转运时间的毒素吸收率范围为1.5%~63.0%。结论建立的模型形成了完整且紧密的细胞单层。AOH和AM E的Papp大小与毒素浓度和转运时间有关,AOH、AME和Te A为易于吸收或中等易吸收的毒素。

Objective To develop and evaluate the Caco-2 cell model and to explore the transportation and absorption of 3 Alternaria mycotoxins. Methods Caco-2 cell model was established by inoculating Caco-2 cell on the polycarbonate membrane attached to the transwell plates and evaluated on the basis of transepithelium electrical resistance（TEER）,activity of alkaline phosphatase（ALP）,apparent permeability coefficient（Papp） of fluorescein and cytomorphology. Transportation and absorption of 3 Alternaria toxins including alternariol（ AOH）,alternariol methyl ether（AME） and tenuazonic acid（Te A） in different concentrations and at different time points were studied by determining the difference in toxin concentrations between apical side and basolateral side of the cell line. Results The TEER exceeded 300 ± 15 Ω·cm2and ALP activity was statistically different（ P 0. 01） between apical side and basolateral side after 21 days＇ incubation. Clear microvilli and tight intercellular junctions were observed with transmission electron microscope. Papps of AOH,AME and Te A varied between 1 × 10-5cm / s and 10 × 10-5cm / s. Transportation and absorptionof 3 Alternaria toxins at different concentrations ranged from 1. 5% to 63. 0%. Conclusion Intact and tight cell model was developed for Alternaria toxins transportation and absorption. AOH,AME and Te A were toxins easily or moderately absorbed by human beings.