表皮生长因子对肾小球上皮细胞骨架的影响

Effect of epidermal growth factor on glomerular epithelial cells cytoskeleton

目的观察阿霉素作用下体外培养的大鼠肾小球上皮细胞(GEC)通透性和细胞骨架变化并研究表皮生长因子(EGF)对它们的作用及可能途径。方法在阿霉素作用GEC24h之前给予和不给予外源性EGF,利用Millicell—PCFInserts检测牛血清白蛋白(BSA)滤过量,同时评价GEC细胞活力。细胞免疫荧光法和激光共聚焦显微镜观察和测定细胞骨架分子F-肌动蛋白(actin)、α-辅肌动蛋白(actinin)的表达。结果阿霉素刺激GEC24h后,BSA滤过量明显增加,而F-actin重排率增加,排列紊乱,部分解聚,胞浆内应力纤维明显减少;α-actinin趋向核周聚集。阿霉素诱导前先给予EGF,则BSA滤过量明显减少,细胞骨架恢复正常组装状态,α-actinin在胞浆内均匀分布。但在EGF之前给予EGF受体(EGFR)抑制剂AG1478或磷脂酶C(PLC)γ抑制剂U73122,则EGF对阿霉素诱导下GEC的改善效应减弱,而在无EGF的阿霉素诱导之前应用AG1478或U73122,均未产生改变。结论阿霉素诱导了GEC细胞骨架的重排和破坏,致使上皮通透性增高,而EGF可能通过EGF-EGFR-PLCγ这条信号转导途径阻止了阿霉素对GEC细胞骨架的影响,保护了GEC上皮屏障。

Objective To study the changes of adriamycin-induced glomerular epithelial cells (GECs) permeability and cytoskeleton, and to explore the role and possible mechanism of epithelia growth factor (EGF) on adriamycin-induced glomerular epithelial barrier function. Methods Rat GECs on Milicell-PCF Inserts were exposed to adriamycin (0.5 μmmol/L) 24 hours in the absence or presence of EGF. The paracelluar permeability to BSA and cell viability were evaluated. The expression of F-actin and alpha-actinin were analyzed by immunofluorescence and Leica laser scan confocal microscopy. Results After induced by adriamycin for 24 hours, paracelluar permeability to BSA and F-actin reorganization rate increased. Disassembling of cortical cytoskeleton architecture was observed. Stress fiber in cytoplasm disappeared. Alpha-actinin staining showed perinuclear enhancement, which is different from normal cytosolic pattern. EGF significantly reduced adriamycin induced effect. Higher level of BSA passed through GEC monolayer in a dose-dependent manner. EGF prevented adriamycin-induced cytoskeletal reorganization. Disassembled cortical cytoskeleton was recovered, and stress fiber appeared again. Alpha-actinin staining mainly exhibited cytosolic uniform distribution as control GEC. In addition, administration of either AG1478, a specific inhibitor of EGF-receptor tyrosine kinase, or U73122, a specific inhibitor of PLCγ before EGF treatment attenuated the EGF-mediated effect, whereas neither AG1478 nor U73122 exerted influence in the absence of EGF. Conclusions Adriamycin increases GEC paracellular permeability to BSA as a result of adriamycin-induced cytoskeleton disassembling and disruption. EGF may prevent adriamycininduced cytoskeleton reorganization and maintain glomerular epithelial barrier function through EGF mediated EGF-EGFR-PLCγ signal pathway.