缺氧血管内皮细胞Rho激酶信号通路活化与通透性的关系

Relationship between activation of Rho kinase signal pathway and permeability of hypoxic vascular enduthelial cells

目的探讨缺氧时血管内皮细胞Rho激酶信号通路活化与通透性的关系。 方法 （1）将人血管内皮细胞株VE细胞接种于6孔板Transwell小室上，按随机数字表法分为对照组（未行缺氧处理）及缺氧1、2、3、6、12 h组（分别缺氧处理相应时间），每组5孔。用蛋白质印迹法检测细胞中Rho激酶Ⅰ、Ⅱ及肌球蛋白轻链磷酸酶靶亚单位1（MYPT1）、磷酸化MYPT1（ p-MYPT1）、肌球蛋白轻链（ MLC）、p-MLC的蛋白表达，计算p-MYPT1/MYPT1、p-MLC/MLC比值。（2）将VE细胞接种于24孔板Transwell小室上，按随机数字表法分为对照组（未行缺氧处理）与缺氧6h组（缺氧处理6h），每组5孔，用荧光分光光度计检测单层细胞通透性。对数据进行单因素方差分析或￡检验，多组间两 两比较采用Newman-Keuls法。 结果 （1）对照组及缺氧1、2、3、6、12 h组细胞Rho激酶I蛋白表达量分别为0. 63 ＋0. 14及0.36±0.08、1.25±0.21、1.98±0.16、1.49±0.38、0.79±0.24（F=36. 52， P 〈0.01），其中缺氧2、3、6h组显著高于对照组【P值均小于0．01）。各组细胞Rho激酶Ⅱ蛋白表达量比较，差异具有统计学意义（F= 17. 84，P〈0.01），其中缺氧2h组为1.33±0.17，显著高于对照组的1.05 ＋0.04（P〈0.01）。对照组及缺氧1、2、3、6、12 h组细胞p-MYPT1/MYPT1比值分别为0. 62±0.13及0.62±0.11、0.65±0.10、1.06±0.23、1.37±0.16、1.91 ＋0.32（F=37.41,P〈0.01），其中缺氧3、6、12 h组显著高于对照组（P值均小于0.01）。对照组及缺氧1、2、3、6、12 h组细胞p-MLC/MLC比值分别为0.72±0.19及0.83±0.17、0.91±0.15、1.39±0.16、2.02±0.15、0.90±0. 25（F=36. 92，P〈0．01），其中缺氧3、6h组显著高于对照组（P值均小于0.01）。（2）缺氧6h组单层细胞通透性为36.1 ＋8．0，显著高于对照组的9.1 ＋2.1（z=7.30，P〈0.01）。 结论 Rho激酶信号通路活化可能参与了缺氧引起的血管内皮通透性增加的发生。

Objective To investigate tile relationship between activation of Rho kinase （ROCK） signal pathway and permeability of hypoxic vaseularendolhelial cells. Methods （1） Human vascular endothelial cell line VE cells were phanted onto 6-well plates Transwell and divided into control group （ without hypoxia treatment） and hypoxia for 1, 2, 3, 6, 12 h groups （exposed to 1% O2, 5% CO2, and 94%N2, for corresponding time） according Io the random number tahle, with 5 wells in each group. The expression levels of ROCK Ⅰ , ROCKⅡ , myosin light chain phosphatase target suhuni/ 1 （MYPT1） and phosphorylated MYPTI （p-MYPTI）, myosin light chain （MLC）, p-MLC in cells were detected by Western blotting. The ratios of p-MYPTI/MYPTI and p-MLC/MI.C were cah＇ulated. （2） VE cells were planted onto 24-well plates Transwell, and the monolayer cells were divided into control group （ without hypoxia treatment） and hypoxia for h group （exposed to 1%,, 5%C0,, and 94%N, for 6 h） according to the random number lable, with 5 wells in each group. Permeability of monolayer cells was determined by fluoreseencespectropho- Iometer. Dala were processed with one-way analysis of variance or t test, and Newman-Keuls method was used in paired comparison among groups. Results （ I ） ROCK [ prolein expression in eonlrol and hypoxia for 1, 2, 3, 6, 12 h groups was obviously 0.63 ±0.14 and 0.36 ±0.08, 1.25 ±0.21, 1.98 ±0. 16, 1.49 ±0.38, 0.79 ±0.24 （ F =36.52, P 〈0.01）. ROCK I protein expression in hypoxia for2, 3, 6 h groups were obviously higher than that in control group （with P values all below 0.01 ）. （2） There was significant statistical difference among all groups in ROCK II protein expression （ F = 17.84, P 〈 0.01 ）. ROCK Ⅱ protein expression in hypoxia for 2 h group （ 1.33 ± 0. 17） was significantly higher than that in control group （1.05 ±0.04, P 〈 0.01 ）. （3） p-MYPT1/MYPT1 ratio in control and hypoxia for 1, 2, 3, 6, 12 h groups was respectively0.62 ±0.13 and0.62±0. 11, 0.65 ±0. 10, 1.06±0.23, 1.37 ±0. 16, 1.91 ±0.32 （ F =37.41,P 〈0.01）. p-MYPT1/MYPT1 ratio inhypoxiafor3, 6, 12 hgroupswereobviously higher than that in control group （ with P values all below 0. 01 ）. （4） p-MLC/MLC ratio in control and hypoxia for 1, 2, 3, 6, 12 h groups was respectively0.72±0. 19 and 0.83 ±0. 17, 0.91 ±0. 15, 1.39 ± 0.16, 2.02±0.15, 0.90±0.25 （ F =36.92,P 〈0.01）. p-MLC/MLC ratio inhypoxiafor3, 6 hgroups were obviously higher than that in control group （with P values all below 0.01 ）. （5） Permeability of VE monolayer in hypoxia for 6 h group （36. 1 ± 8.0） was obviously higher than that in control group （9. 1 ± 2. 1 , t = 7.30, P 〈 0.01 ）. Coneluslons Activation of ROCK signal pathway may be involved in the pathogenesis of vascular endothelial cell hyperpermeability induced by hypoxia.