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中文摘要：
目的探讨转录因子cAMP反应元件结合蛋白（cAMP response element binding protein，CREB）的磷酸化是否介导了缓激肽刺激大鼠c6胶质瘤细胞内白细胞介素-1β（IL-1β）的表达。方法利用RNA干扰（RNAi）技术，以CREB为靶基因，将体外化学合成的CREB序列特异性双链小干扰RNAfsiRNA）和无关序列的siRNA分别与Lipofectamine2000结合后转染细胞，用Western blot分析CREB蛋白的表达水平。利用逆转录-聚合酶链反应（RT—PCR）半定量技术和放射免疫法分别检测CREB siRNA转染前后，缓激肽诱导C6胶质瘤细胞IL-1β mRNA及细胞培养上清液中IL-1β的表达水平。结果CREB序列特异性siRNA转染细胞72h后，可较强的抑制CREB蛋白的表达，而无关序列的siRNA对CREB蛋白表达水平无明显的抑制作用。RT—PCR和放射免疫法结果均显示CREB siRNA干预C6细胞后，并未改变缓激肽刺激IL-1β表达的能力。结论化学合成的siRNA可有效抑制C6胶质瘤细胞中CREB的表达，CREB可能未参与缓激肽刺激C6细胞IL-1β表达的信号转导过程。
英文摘要：
Objective To determine whether IL-1β expression stimulated by bradykinin in C6 glioma cells is mediated by the phosphorylation of cAMP response element binding protein （CREB） by RNAi. Methods C6 glioma cells were transfected with chemically synthesized CREB-specific siRNA and random sequence siRNA formulated with lipofectamine 2000 respectively. The expression of CREB was determined by Western blot. Semi-quantitive RT-PCR and radioimmunoassay were used to detect the expression of IL-1β mRNA in C6 glioma cells and IL-1β protein in the supernate of culture media transfected with CREB-specific siRNA prior to stimulation with 1 μmol · L^-1 bradykinin. Results Sequence specific siRNA targeting CREB downregulated CREB expression significandy compared with the blank control group, and no obvious suppression of CREB expression was observed in C6 glioma cells transfected with random sequence siRNA. RT-PCR and radioimmunoassay revealed that downregulated expression of IL-1β mRNA and protein was not observed in C6 glioma cells and the supernate of culture media transfected with CREB-specific siRNA prior to stimulation with bradykinin. Conclusion Chemically synthetic siRNA targeting CREB was capable of suppressing CREB expression in C6 glioma, CREB was not implicated in the secretion of IL-1β stimulated by bradykinin.
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