Staurosporine诱导RGC-5细胞分化作用的研究  

Staurosporine-induced differentiation of retinal ganglion cell

作　　者：李光宇[1] 范斌[1] 李亚萍[1] 崔极哲[1] 

机构地区：[1]吉林大学第二医院眼科,长春130041

出　　处：《眼科研究》2008年第10期721-725,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金(30801271);长春市社会发展计划项目(2009-2011)资助

摘　　要：目的观察Staurosporine是否可以诱导视网膜神经节细胞-5(RGC-5)的分化。方法正常培养RGC-5细胞,应用500nmol/L Staurosporine诱导RGC-5细胞分化,并在诱导后不同时间段连续观察细胞形态的变化。应用免疫荧光检测视网膜神经节细胞(RGCs)阳性标志因子Thy-1和Brn-3的表达,应用Western Blot、RT-PCR分别定量检测诱导分化后RGC-5细胞中的Thy-1和Brn-3蛋白表达及mRNA的转录情况。结果应用500nmol/L Staurosporine诱导后的RGC-5形态上表现出增生停止,胞体周围纤长轴突生长,轴突末端可见类似突触结构。500nmol/L Staurosporine可以诱导RGC-5细胞中RGCs阳性标志因子Thy-1和Brn-3转录及表达上调。结论500nmol/L Staurosporine可以有效地诱导RGC-5细胞向成熟RGCs分化。Objective The retinal ganglion cell-5,a new developed cell line,can be used as an in vitro model to explore pathological features of real retinal ganglion cells.However,whether RGC-5 cells can be differentiated into mature neurons is still unknown.The purpose of current project was to induce differentiation of RGC-5 cells with a pan-protein-kinase inhibitor,staurosporine.MethodsRGC-5 cells were cultured in DMEM containing 10% fetal bovine serum.500nmol/L staurosporine was added to the cultured cells to induce the differentiation of RGC-5.The morphology of induced RGC-5 cells was observed at different time points under the inverted microscope,and the positive markers of retinal ganglion cells,Thy-1 and Brn-3,were qualitatively labeled in RGC-5 cells with immunofluorescence assay and quantitatively detected with Western blot and RT-PCR.ResultsCultured RGC-5 presented the spindle in shape with short axons.The morphology of RGC-5 appeared small and the longer axons with synapse-like structure at the end outgrew from the cell body in 48 hours after treated by staurosporine,and further exhibited the shape like mature RGCs in 72 hours.Western blot and RT-PCR displayed that the mRNA and protein of Thy-1 and Brn-3 were significantly up-regulated in RGC-5 cells after exposure of 500nmol/L staurosporine for 2 or 3 days （P〈0.01）.Immunofluorescence assay also showed the enhanced green or red fluorescence of Thy-1 and Brn-3 respectively in nuclear and cytoplasm.Conclusion500nmol/L staurosporine is able to effectively induce differentiation of RGC-5 cells into mature RGCs.

关 键 词：RGC-5细胞 STAUROSPORINE 细胞分化 THY-1 Brn-3 

分 类 号：R774.1[医药卫生—眼科]