酪氨酸蛋白激酶抑制剂对缺氧复氧时内皮细胞连接通讯功能的影响

Change in gap junctional intercellular communication in cultured human endothelial cells with hypoxia/reoxygenation and effect of tyrosine kinase inhibitor on its change

目的:缺氧复氧刺激能损伤内皮细胞间隙连接通讯功能,本研究观察了酪氨酸蛋白激酶抑制剂genistein对这种损伤的影响。方法:将人脐静脉内皮细胞株ECV304分3组给予不同处理。①常氧对照组:常氧状态下培养12～18小时;②缺氧复氧组:氧浓度低于1％状态下培养12～14小时,再置于常氧状态复氧0～6小时;③genistein组:加入不同浓度genistein(2μM、10μM、50μM)后,给予缺氧复氧组相同的处理。在试验各时段应用荧光漂白回复技术对细胞间隙连接通讯功能进行分析,结果用荧光回复率表示。结果:不同浓度genistein对复氧2小时的内皮细胞荧光回复率作用不同。低浓度genistein(2μM)对荧光回复率没有作用;中、高浓度genistein(10μM、50μM)能使荧光回复率上升(P<0.05)。结论:gensetein能保护缺氧复氧时内皮细胞间隙连接通讯功能。提示缺氧刺激通过酪氨酸蛋白激酶途径损伤内皮细胞的间隙连接通讯功能。

Objective: To study the tyrosine kinase inhibitor (genistein) prevents the gap junctional intercellular communication (GJIC) abnormality induced by hypoxia/reoxygenation. Methods: After human umbilical vein endothelial cells (ECs) had grown to confluence, they were divided to three groups and exposed to different conditions. ECs in group Ⅰ were incubated in normal oxygenic condition. ECs in group Ⅱ were exposed to hypoxia (PO2<1%) for 12-14 hours and then were returned to normal atmospheric condition for reoxygenation. ECs in group Ⅲ were incubated with genistein and were exposed to hypoxia/reoxygenation. GJIC was measured by means of a fluorescence recovery after photobleaching (FRAP) technique and was represented by the fluorescence recovery rate. Results: The genistein (10μM. 50μM) completely blocked the fluorescence recovery rate decrease of ECs after two hours reoxygenation (P<0. 05). Conclusion: The tyrosine kinase inhibitor genistein protect the GJIC from hypoxia/reoxygenation injury. This study suggests that hypoxia/reoxygenation induce a transient endothelial GJIC abnormality through a tyrosine kinase pathway.