肌球蛋白轻链激酶介导缺氧后肠上皮屏障功能紊乱的研究

目的 了解肌球蛋白轻链激酶(MLCK)在缺氧后肠上皮屏障功能损害中的作用.方法 建立单层肠上皮细胞缺氧模型,根据缺氧时间不同将细胞分为正常对照组(缺氧0 h),缺氧2、6、8、12、24 h组,(5-氯萘-1-磺酰)-1-六氢-1,4-双苯妥英钠(ML-9)处理组(缺氧6 h并用终浓度为100μmol/L的MLCK特异性抑制剂ML-9处理).用电阻仪测定单层肠上皮细胞跨细胞电阻(TER);免疫荧光法检测单层肠上皮细胞紧密连接蛋白ZO-1的变化;蛋白质印迹法检测肠上皮细胞磷酸化肌球蛋白轻链(p-MLC)及MLCK蛋白表达.结果 缺氧6、8、12、24 h组单层肠上皮细胞TER值分别为(422±17)、(427±27)、(403±40)、(426±22)Ω,均低于正常对照组[(451±27)Ω,P<0.05],ML-9处理组为(558±110)Ω,显著高于各单纯缺氧组(P<0.01).缺氧6 h组单层肠上皮细胞紧密连接蛋白ZO-1发生明显改变,主要表现为不规则,出现明显皱褶、圆滑排列程度减弱,甚至有断裂及锯齿等不连续现象.ML-9组肠上皮细胞紧密连接蛋白ZO-1近似于正常对照组,排列较规则,皱褶及锯齿状改变较单纯缺氧组减少或消失.各单纯缺氧组肠上皮细胞MLCK、p-MLC蛋白表达水平均较正常对照组增加.结论 MLCK可介导缺氧引起的肠上皮屏障功能紊乱.

The role of myosin light chain kinase in intestinal epithelial barrier dysfunction due to hypoxia

Objective To study the role of myosin light chain kinase (MLCK) in intestinal epithe-lial barrier dysfunction after hypoxia. Methods The Caco-2 monolayers developed with Transwell inserts were exposed to hypoxia for 0 h(NC group), 2, 6, 8, 12 and 24 h(H group), and 6 h hypoxic specimens were treated with 100 mul/L ML-9 (T group). The transepithelial electrical resistance (TER) of monolayers was measured with an ohmmeter. The tight junction protein ZO-1 of monolayers was analyzed by immunofluo-rescence assay. The protein expressions of phosphorylated myosin light chain (p-MLC) and MLCK were de-tected by Western blotting. Results The TER of monolayers in H group at 6, 8, 12 and 24 h was 422± 17, 427±27,403±40 and 426±22Ω respectively, which was significantly lower than that of NC group (451±27Ω, P < 0.05). The TER of monulayers in T group was 558±110Ω, which was significantly high-er than that in H group at each time point (P <0.01). The ZO-1 of monolayers in H group at 6 h was ir-regular in arrangement, with interruptions and rugae, and sawtooth. These abnormalities were amelioated in T group(regular in arrangement, with little or without ruga and sawtooth). The protein expressions of p-MLC and MLCK in H group at each time point were higher than those in NC group. Conclusions Intestinal ep-ithelial barrier dysfunction after hypoxia can be mediated by MLCK.