目的：探究miR-200 b对肠上皮紧密连接的影响及作用机制。方法利用阴性对照慢病毒及表达miR-200 b的慢病毒感染Caco-2细胞形成NC株和200 b株，以10 ng/mL肿瘤坏死因子（TNF-α）处理建立体外肠上皮损伤模型，并分为NC组、NC+TNF-α组、200 b组和200 b+TNF-α组。测量4组细胞对肠上皮跨膜电阻抗（TEER）及对异硫氰酸荧光素-右旋糖酐（FITC-dextran）的通透性；Western blot检测肌球蛋白轻链激酶（MLCK）及磷酸化肌球蛋白轻链（P-MLC）表达。结果与NC组相比，NC+TNF-α组TEER值降低、对FITC-dextran通透量显著增高、MLCK和P-MLC表达上调，差异均有统计学意义（P< 0.05）。与NC+TNF-α组相比，200b+TNF-α组TEER显著升高、FITC-dextran通透量显著降低、MLCK和P-MLC表达显著下调，差异均有统计学意义（P< 0.05）。结论 miR-200 b可调控MLCK/P-MLC信号通路修复TNF-α导致的肠上皮紧密连接损伤。%Objective To explore the impact and mechanism of miR-200 b on intestinal epithelial tight junction. Methods The negative-lentivirus and human-miR-200 b-lentivirus were employed to infect the Caco-2 cell thus establishing two stable cell lines which were then stimulated by 10 ng/mL human tumor necrosis factor-α(TNF-α) to establish the model of the intestinal epithelial injury. Those Caco-2 cells were divided into NC, NC+TNF-α, 200b, and 200b+TNF-αgroups.The tight junction permeability was detected by transepithelial electrical resistance (TEER) and Fluorescein isothiocyanate-labeled dextran (FITC-dextran). The protein alterations myosin light chain kinase (MLCK)/phosphorylated myosin light chain (P-MLC) pathways were measured by Western blot analysis. Results Compared to NC group, NC+TNF-αgroup had lower TEER, higher FITC-dextran, and up-regulated expressions of MLCK and P-MLC proteins (P< 0 . 05 ). Compared to NC+TNF-αgroup, 200 b+TNF-αgroup had higher TEER, lower FITC-dextran and down-regulated expressions of MLCK and P-MLC proteins (P<0 . 05 ). Conclusion miR-200 b ameliorated TNF-α-induced intestinal epithelial tight junction disruption via regulation MLCK/P-MLC pathway.
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