线粒体DNA对Caco-2细胞通透性的影响及机制

Effects of mitochondrial DNA on permeability of Caco-2 cells and its mechanism

目的探讨线粒体脱氧核糖核酸(mtDNA)对Caco-2细胞单层通透性的影响及作用机制。方法利用Transwell培养板制备Caco-2细胞模型,从C57BL/6J小鼠肝脏中提取mtDNA,然后在Transwell培养板顶侧加入不同浓度的mtDNA(0、1、5、10μg/ml)和异硫氰酸荧光素-葡聚糖(FITC-dextran),检测不同时间跨上皮电阻(TEER)和底室中FITC-dextran含量的改变,确定最佳mtDNA浓度;再将实验分为4组,分别加入FITC-dextran、mtDNA+FITC-dextran、ODN TTAGGG+FITC-dextran和mtDNA+ODN TTAGGG+FITC-dextran,检测不同时间TEER和底室中FITC-dextran含量的改变。同时分别用mtDNA、ODN TTAGGG、mtDNA+ODN TTAGGG预处理Caco-2细胞,采用qRT-PCR方法检测Toll样受体9(TLR9)mRNA表达水平,ELISA检测上清液中肿瘤坏死因子-ɑ(TNF-ɑ)、白细胞介素-6(IL-6)、IL-1β和IL-18的水平。结果与对照组相比,随着mtDNA作用时间的延长和作用浓度的增加,Caco-2细胞单层的TEER降低,底室中FITC-dextran含量增加,其中mtDNA浓度为10μg/ml时,TEER降低明显;以10μg/ml的mtDNA为对照,加入ODN TTAGGG可减少TEER下降程度,底室中FITC-dextran含量降低,同时TLR9 mRNA表达水平和炎症因子水平降低。结论mtDNA增加Caco-2细胞单层的通透性,机制可能与激活TLR9信号通路有关。

Objective To investigate the effect of mtDNA on monolayer permeability of Caco-2 cells and its mechanism.Methods The Caco-2 cell model was prepared by using Transwell culture plate.The mtDNA was extracted from the liver of C57BL/6J mice.Then,different concentrations of mtDNA(0,1,5,10μg/ml)and FITC-dex-tran were added to the top of the Transwell culture plate to detect the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate at different time,which determined the best mtDNA concentration.Then,the experiment was randomized into four groups:FITC-dextran,mtDNA+FITC-dextran,TLR9 antagonists(ODN TTAGGG)+FITC-dextra,and mtDNA+ODN TTAGGG+FITC-dextran,and the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate were detected at different time.Meanwhile,Caco-2 cells were pretreated with mtDNA,ODN TTAGGG,mtDNA+ODN TTAGGG,respectively.The expression of TLR9 mRNA was detected by qRT-PCR,and the levels of TNF-lα,IL-6,IL-1βand IL-18 in the supernatant were detected by ELISA.Results Compared with the control group,with the prolongation of the action time and the increase of the concentration of mtDNA,the TEER of the monolayer of Caco-2 cells decreased and the content of FITC-dextran in the bottom chamber increased,and the TEER decreased significantly when the concentration of mtDNA was 10μg/ml.Compared with 10μg/ml mtDNA group,the addition of ODN TTAGGG significantly reduced the falling speed of TEER,and reduced the content of FITC-dextran in the bottom chamber,meanwile,the expression of TLR9 mRNA and the levels of inflammatory factors also declined.Conclusion The mtDNA significantly increases the permeability of Caco-2 monolayer,which may be related to the activation of TLR9 signal pathway.