【摘要】 目的 探究全氟壬烯氧基苯磺酸钠（OBS）对人源结肠癌细胞系（CaCo2细胞）的毒性以及引起细胞通透性和屏障改变的可能因素。方法 将对数期生长的CaCo2细胞分别暴露于含0.0（对照）～25.0 mg/L OBS低糖DMEM培养基染毒24 h和48 h，采用CCK-8法测定细胞活性。将对数期生长的CaCo2细胞分别暴露于含0.0（对照）～5.0 mg/L OBS低糖DMEM培养基染毒24 h和48 h，采用实时荧光定量PCR(qRT-PCR)法测定细胞中黏蛋白2(MUC2)、紧密连接蛋白1(TJP1)、闭合蛋白1(CLDN1)、咬合蛋白（OCCLUDIN）、凋亡相关基因和炎性相关基因的m RNA表达水平，采用蛋白印迹（WB）法检测TJP1、CLDN1、OCCLUDIN和含半胱氨酸的天冬氨酸蛋白水解酶9(CASPASE9)蛋白的表达水平。结果 与对照组相比，5.0～25.0 mg/L OBS染毒组CaCo2细胞的存活率较低，差异有统计学意义（P<0.05）；而0.01～1.0 mg/L OBS染毒组CaCo2细胞的存活率均无明显改变。与对照组相比，5.0 mg/L OBS染毒24 h时CaCo2细胞中MUC2、CLDN1 mRNA的表达水平和0.1、1.0 mg/L OBS染毒48 h时细胞中OCCLUDIN mRNA的表达水平及各浓度OBS染毒48 h时细胞中MUC2mRNA的表达水平较低，而各浓度OBS染毒48 h时细胞中CLDN1 m RNA的表达水平及5.0 mg/L OBS染毒48 h时细胞中OCCLUDIN mRNA的表达水平较高，差异均有统计学意义（P<0.05）。与对照组相比，各浓度OBS染毒48 h时细胞中CLDN1蛋白表达水平和0.1 mg/L OBS染毒24 h、48 h时TJP1蛋白表达水平较高，而5.0 mg/L OBS染毒24 h、48 h时细胞中TJP1蛋白表达水平较低，差异均有统计学意义（P<0.05）。与对照组相比，仅0.1和1.0 mg/L OBS染毒48 h时CaCo2细胞中基因CASPASE9的表达mRNA表达水平下降，差异有统计学意义（P<0.05）。与对照组相比，1.0 mg/L OBS染毒24 h时CaCo2细胞中基因肿瘤坏死因子α(TNFα)和白细胞介素-22(IL-22)的mRNA表达水平、0.1和1.0 mg/L OBS染毒48 h时白细胞介素-1β(IL-1β)的mRNA表达水平、以及5.0 mg/L OBS染毒48 h时细胞中基因核因子κB(NFκB)、TNFα、toll样受体4(TLR4)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、IL-22的mRNA表达水平均增加，差异有统计学意义（P<0.05）。结论 OBS在一定染毒浓度或者时间内，可以显著降低细胞活性，减少黏蛋白MUC2的正常表达以破坏细胞间的紧密连接，并且激活细胞中炎性相关基因，表明OBS对细胞通透性和细胞屏障的影响可能与细胞中免疫调控激活有关。更多还原

【Abstract】 Objective To explore the toxicity of sodium ρ-perfluorousnonenoxybenzene sulfonate(OBS) to CaCo2 cells and the possible factors that cause changes in cell permeability and barrier. Methods CaCo2 cells grown in logarithmic phase were exposed to DMEM-L medium containing 0.0(control,Con)-25.0 mg/L OBS for 24 h and 48 h respectively. The cell activity was measured by CCK-8 method. The mRNA levels of mucin MUC2,tight junction protein TJP1,CLDN1,OCCLUDIN,apoptosis related genes and inflammation related genes in cells were measured by qRT-PCR,and the protein levels of TJP1,CLDN1,OCCLUDIN and CASPASE9 were detected by western blot(WB). Results Compared with the control,the survival rates of CaCo2 cells in 5.0-25.0 mg/L OBS groups were significantly lower(P<0.05),while those were no significant change in0.01-1.0 mg/L OBS groups. Compared with the control,the mRNA levels of MUC2 and CLDN1 after 24 h of 5.0 mg/L OBS exposure,the mRNA levels of OCCLUDIN after 48 h of 0.1 and 1.0 mg/L OBS exposure,and the mRNA levels of MUC2 after48 h of OBS exposure were downregulated in CaCo2 cells. However,the mRNA levels of CLDN1 after 48 h of OBS exposure and the m RNA level of OCCLUDIN after 48 h of 5.0 mg/L OBS exposure were upregulated in CaCo2 cells,with significant differences(P<0.05). Compared with the control,the protein levels of Cldn1 after 48 h of exposure in all OBS groups and the protein levels of TJP1 after 0.1 mg/L OBS exposure at 24 h and 48 h were higher,while the protein levels of TJP1 in cells were lower at 24 h and 48 h of 5.0 mg/L OBS exposure,with significant differences(P<0.05). Compared with the control,the m RNA level of CASPASE9 in CaCo2 cells significantly decreased only in 0.1 and 1.0 mg/L OBS exposure for 48 h groups(P<0.05).Compared with the control,the mRNA levels of TNFα and IL-22 in CaCo2 cells after 1.0 mg/L OBS exposure for 24 h,the mRNA level of IL-1β at 48 h exposure to 0.1 and 1.0 mg/L OBS and the mRNA levels of NFκB,TNFα,TLR4,IL-6,IL-10 and IL-22 after 5.0 mg/L OBS exposure for 48 hours increased significantly(P<0.05). Conclusion At a certain concentration or time of exposure,OBS can significantly inhibit cell activity,reduce the normal expression of MUC2 to disrupt tight junctions between cells,and activate the expression of inflammatory related genes,indicating that the effect of OBS on cell permeability and cell barrier may be related to the activation of immune regulation.更多还原