丙泊酚通过促进蛋白sumo化表达增加抑制神经母细胞系 钙内流水平 .docVIP

丙泊酚通过促进蛋白SUMO化表达增加抑制神经母细胞系 钙内流水平变化 常晶 陆菡 薛庆生 于布为 [摘要] 目的：立足于SUMO化修饰这一蛋白质翻译后修饰的全新角度，通过研究丙泊酚对细胞蛋白SUMO化修饰及钙内流的变化探讨丙泊酚的全麻机制。 方法：采用western方法观察丙泊酚对神经母细胞瘤细胞（SHSY5Y）蛋白SUMO化水平变化的影响。并通过脂质体转染法将SENP1导入3×Flag-SUMO1-SHSY5Y，降低SUMO化水平以及采用SENP抑制剂处理细胞，过表达SUMO水平，以观察丙泊酚对细胞钙内流变化的影响。细胞内钙离子浓度测定选用Fluo-3AM钙离子荧光测定法。 结果：丙泊酚诱导3×Flag-SUMO1-SHSY5Y细胞系SUMO化水平呈时间依赖性及浓度依赖性增加，临床相关浓度的丙泊酚（50μmol/L）处理60min变化显著。丙泊酚引起细胞内钙内流呈双向性变化：在加入丙泊酚的最初数分钟，细胞内钙离子迅速增加，约10-15min达到峰值，后逐渐降低，以45-60min左右为转折点，随后逐渐低于对照组（p0.05），与细胞内SUMO1水平变化趋势相同。在转入野生型SENP1(WT)细胞中，丙泊酚对其钙内流影响的双向性消失，作用60min及其以后的时间，并不出现对钙内流的抑制。而在表达突变体SENP1(mutate)及空载对照组细胞中，丙泊酚对钙内流的双向性影响仍然存在。给予SENP抑制剂后，可以增加丙泊酚引起的3×Flag-SUMO1-SHSY5Y细胞SUMO化水平升高，同时丙泊酚引起钙内流抑制相时间提前。 结论：丙泊酚通过诱导3×Flag-SUMO1-SHSY5Y细胞SUMO化水平表达增加抑制细胞钙内流变化，这也许是丙泊酚发挥抑制效应的全麻机制之一。 关键词：丙泊酚，SUMO化修饰，神经母细胞瘤细胞系，钙内流，全麻机制 Propofol inhibit calcium influx by promoting protein sumoylation in neuroblastoma cell lines Chang Jing Lu Han Xue Qingsheng Yu Buwei [Abstract] Objective Posttranslational modi?cation by small ubiquitin-like modi?er (SUMO) proteins is emerging as an important regulatory mechanism for neuronal function .Here we investigate the effect of propofol on protein sumoylation and calcium influx in neuroblastoma cell lines (SHSY5Y). Methods Western blot was used to detect the effects of propofol on protein SUMOylation in 3×Flag-SUMOs-SHSY5Y cell lines. To reduce the SUMO1 modification, cells were transfected with wide type SENP (WT) or mutant SENP (mutant) or vehicle as control. The intracellular calcium was monitored using a well-established fluo-3AM continuous fluorometry. Results Propofol induced 3×Flag-SUMO1- SHSY5Y protein sumoylation increased in a time-dependent and concentration–dependent manner. Propofol have dual effect on the calcium influx. With the increasing of protein sumoylation mediated by propofol ,the calcium influx diminished. Decreasing protein SUMOylation by entrapping recombinant SENP1 in SHSY5Y-3flag SUMO1 cell line increased calcium influx incubated with propofol in 60 min