摘要
目的研究etoposide诱导视网膜神经节细胞RGC-5细胞死亡的分子机制。方法建立etoposide诱导的RGC-5细胞死亡模型,应用APOPercentageTM及原位TUNEL染色确定RGC-5细胞的死亡方式,并进而利用Westernblot检测细胞内活化caspase-3及PARP-1的变化情况,并通过广谱caspase抑制剂间接证明是否有caspase依赖途径的激活。结果相对高浓度的etoposide(1～10μmol/L)可以迅速降低RGC-5细胞的活性并诱导死亡;APOPercentageTM及原位TUNEL染色确定etoposide是以凋亡的方式诱导RGC-5细胞死亡;Westernblot显示etoposide诱导后的RGC-5细胞中caspase-3被激活并伴有PARP-1的降解片段出现;广谱caspase抑制剂Z-VAD-fmk可以保护etoposide诱导的RGC-5细胞,提高细胞活性。结论Etoposide经由caspase依赖途径诱导RGC-5细胞凋亡。
Objective The apoptosis of retinal ganglion cell（RGC）is the common feature of many ophthalmic diseases.To rescue dying neurons,the study on cell death pathway is very important.The current projective was to demonstrate the molecular mechanism of RGC death induced by etoposide.Methods RGC-5 was cultured in DMEM medium containing 10% fetal bovine serum and treated with etoposide firstly.100 nmol/L,1 μmol/L,10 μmol/L of etoposide dissolved in DMSO was added in medium in different experimental groups respectively,and only equivalent amount of DMSO was used in the control group.The death pattern of RGC-5 was then confirmed with APOPercentage^TM assay and in-situ TUNEL.Activated caspase-3 and cleaved PARP-1 were further measured with Western blot.The pan-caspase inhibitor was used to indirectly identify whether caspase-dependent pathway was involved in this cell death cascade.Results Etoposide at relatively high concentration（1-10 μmol/L）was able to rapidly reduce RGC-5 cell viability to 60% and 30% respectively in 24 hours,and 10 μmol/L of etoposide caused cell death in 48 hours.The positive results of APOPercentage^TM and in-situ TUNEL assay indicated that etoposide-induced RGC-5 death via the pathway of apoptosis.Moreover,both of activated caspase-3 and cleaved PARP-1 were detected by Western blot in etoposide-treated RGC-5 cells,but they were not found in normal control cells.At last,Z-VAD-fmk,as a pan-caspase inhibitor,was able to effectively block the cell death caused by etoposide.Conclusion Etoposide induces RGC-5 apoptosis through caspase-dependent pathway,and Z-VAD-fmk exhibites protection on RGC-5 against the damage of etoposide.
出处
《眼科研究》
CSCD
北大核心
2008年第8期598-601,共4页
Chinese Ophthalmic Research
