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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 observe the changes of permeability and cytoskeleton of rat glomerular epithelial cells (GECs) induced by doxorubicin in vitro and to explore the possible role of epidermal growth factor (EGF) in them. Methods Exogenous EGF was administered with or without 24 h before adriamycin-induced GEC. The amount of bovine serum albumin (BSA) was measured by Millicell-PCFInserts, and the viability of GEC was evaluated. Cell immunofluorescence and laser scanning confocal microscopy were used to observe and measure the expression of cytoskeletal molecules such as actin and actinin. Results After adriamycin stimulated GEC for 24 hours, the amount of BSA filtration significantly increased while the F-actin rearrangement rate increased, disordered and partially disaggregated, and the cytoplasmic stress fibers decreased significantly. Α-actinin tended to aggregate perinuclear. Adriamycin before induction of EGF, the BSA filtration significantly reduced, the cytoskeleton restored to normal assembly, α-actinin evenly distributed in the cytoplasm. However, the EGF receptor (EGFR) inhibitor AG1478 or the phospholipase C (PLC) γ inhibitor U73122 was administered before EGF, then the improvement effect of EGF on doxorubicin-induced GEC was weakened, but before EGF-induced doxorubicin induction Application of AG1478 or U73122 did not change. Conclusion Adriamycin induced the rearrangement and destruction of GEC cytoskeleton, resulting in increased epithelial permeability. However, EGF may block the effects of doxorubicin on the cytoskeleton of GEC by EGF-EGFR-PLCγ signaling pathway and protect GEC epithelial barrier.