转录因子ZNF580 在缺血-再灌注脑损伤中的作用及分子机制

Human ZNF580 gene was cloned by our research group. It encodes a novel C2H2 zinc finger transcriptional factor. Our previous studies showed that ZNF580 might be involved in the TGF-β1 signal transduction pathway, by which it played the role of regulating the vascular endothelium homeostasis. ZNF580 might be interacted with activity-dependent neuroprotective protein (ADNP). Many studies confirm that both of ADNP and TGF-β1 have important protective effects on ischemia-reperfusion cerebral injury. Furthermore, both of them play their protective roles on the same mechanisms. But more importantly, both of them have close correlation with zinc finger transcriptional factor ZNF580. We propose that the activation of TGF-β1 signaling pathway might up-regulate the transcription of ZNF580 and further activate the transcription of ADNP in ischemia-reperfusion cerebral injury, which promotes ADNP secretion from neurogliocytes and protects the microvascular endothelium and neurocytes. In this study,we will try to reveal the role and the signal transduction pathway of ZNF580 involved in neurogliocytes by some of experiments, such as co-immunoprecipitation, immunofluorescence labeling, luciferase report essay, siRNA essay, microarray-based gene expression profiles. We will further investigate the possible neuroprotective role and the molecular mechanism of ZNF580 played in ischemia-reperfusion cerebral injury rat models. Real-time PCR,Western blot and immunohistochemistry assay will be used in this part of research. Our study may provide some theory and experimental data for searching the new target in the clinical treatments of ischemia-reperfusion cerebral injury.

人ZNF580基因由本课题组首先克隆，其编码的蛋白为锌指核转录因子家族的新成员。研究证实：ZNF580可能参与TGF-β1信号转导通路，并与活性依赖的神经保护蛋白（ADNP）间可能存在相互作用。文献显示：TGF-β1和ADNP对缺血-再灌注脑损伤都具有重要的保护作用，且作用机制相似。重要的是二者都与ZNF580之间存在密切联系。本课题拟应用免疫共沉淀、免疫荧光标记、荧光素酶报告载体实验、RNA干扰技术、基因芯片筛选等方法初步阐明ZNF580在神经胶质细胞中发挥的作用及其参与的信号转导通路。并在脑缺血-再灌注大鼠模型中应用Real-time PCR、Western blot、免疫组化等实验方法进一步研究ZNF580可能的神经保护作用及其分子机制，为临床寻找治疗脑缺血-再灌注损伤的新靶点提供理论和实验依据。

目的：研究转录因子ZNF580 对缺血-再灌注脑损伤的保护作用及相关机制。方法与结果：第一部分：应用免疫共沉淀和免疫荧光双标验证了ZNF580与ADNP间的相互作用。第二部分：原代培养大鼠星形胶质细胞，TGF-β1诱导大鼠神经胶质细胞，ZFP580和ADNP在mRNA和蛋白水平的表达具有时间和剂量依赖性。TGF-β1/ALK5/ Smad2 /3通路特异性抑制剂SB431542诱导神经胶质细胞，ZFP580和ADNP在mRNA和蛋白水平的表达受到不同程度抑制，而且同样具有时间和剂量依赖性。TGF-β1诱导大鼠神经胶质细胞，细胞迁移和增殖活性明显增强；干扰ZFP580表达及应用TGF-β1/ALK5/Smad2/3通路特异性抑制剂SB431542诱导的神经胶质细胞的迁移和增殖活性明显减弱。第三部分：复制大鼠大脑中动脉缺血-再灌注损伤模型，再灌注增加了ZNF580 mRNA和蛋白的表达，同时上调了TGF-β、Smad2和ADNP的表达。应用ZNF580 siRNA处理的MACO模型组， ADNP的表达降低。此外，其细胞凋亡数量较对照组明显增加。第四部分：应用基因芯片技术检测了转染携带有空载体和ZNF580 siRNA 慢病毒的大鼠星形胶质细胞基因表达谱改变。获得表达相差1.5倍以上差异基因248个，其中上调基因22个，下调基因226个，进一步分析部分差异基因功能发现，ZNF580基因表达变化与水通道蛋白2（AQP2）、某些炎症因子（IL12、IL10、IL17）、某些神经营养因子相关基因（CNTFR、ADNP）的表达密切相关。另外，还与某些神经退行性疾病相关基因（PARK2、APOER）、某些细胞增殖分化相关基因（CTXN3、VRTN）某些参与细胞代谢的酶类基因（CH25H、ADH1A、STK19）表达关系密切。结论：本研究初步阐明了转录因子ZNF580在缺血-再灌注脑损伤及调节神经胶质细胞的增殖和迁移中发挥的重要作用，从而揭示了其促进脑损伤后神经组织的修复和再生的可能机制，并为进一步研究其在神经退行性疾病中的作用奠定了实验基础。