细胞特异miR-29对缺血后星形细胞的调控机制

One of the most feared disabilities in survivors of cardiac arrest is neurological impairment. Even brief global cerebral ischemia causes delayed loss of CA1 hippocampal neurons while sparing nearby hippocampal dentate gyrus (DG) neurons. Lack of consideration of the role of astrocytes is thought to be a factor in the failure of many potential stroke therapies aimed only at neuronal survival. But why CA1 hippocampal astrocytes are more vulnerable and life/death decision of astrocytes after ischemais are not well understood. MicroRNAs (miRNAs) are a novel, abundant class of ~22-nucleotide RNAs that control gene expression post-transcriptionally. Although hundreds of miRNAs have been cloned, little is known about their real targets and functions. Numerous miRNAs are expressed in a cell-specific manner and the miR-29 family has been suggested to be astrocyte related. From computational miRNA target prediction algorithms, we surprisingly found that miR-29 could potentially target messenger RNAs of several Bcl-2 family members,both pro- and anti-apoptotic.We suppose that the interaction between miR-29 and Bcl-2 family members regulate astrocyte reaction after ischemia.In this study we will investigate the role of miR-29 in brain ischemia and neuroprotection, assess the targets of miR-29 in the hippocampus, and evaluate manipulation of miR-29 as a novel target for neuroprotection. Aim 1 will assess the role of miR-29 in outcome from ischemic brain injury in vitro and forebrain ischemia in vivo. Changes in miR-29 family member levels in response to ischemia will be assessed, including cell type specific changes. Aim 2 will assess endogenous targets of miR-29 in the Bcl-2 family in adult brain. The effects of miR-29 on mitochondrial function, ATP production, cytosolic calcium and oxidative stress will be assessed. Aim 3 will focus on the role of one of those target genes, Puma in ischemic forebrain injury and in the role of astrocytes in this paradigm. Overall this proposal will test the hypothesis that specific miRNAs are a novel and effective target for protection from ischemic brain injury, as well as advancing our understanding of the contributions of astrocyte regulation and impairment to neurological outcome following cardiac arrest and resuscitation.

神经功能障碍是心脏骤停患者复苏后致残的重要原因。即使轻微的脑缺血也会在海马DG区神经元功能正常的情况下，导致CA1区神经元死亡。仅关注神经元而缺少对星形细胞的重视是导致许多中风治疗策略失败的主要原因。但海马CA1区缺血敏感机制及缺血后星形细胞的存/亡机制仍不清。miRNA调控基因的转录后表达，但其真正作用靶点及生物功能仍需阐明。我们前期工作发现，miR-29是星形细胞特异的一种miRNA，可以与多个Bcl-2家族成员，包括促凋亡和抗凋亡蛋白的mRNA 3′UTR相结合。据此提出miR-29与Bcl-2家族蛋白相互作用调节星形细胞对缺血的反应机制。我们将使用大鼠前脑缺血模型及培养星形细胞探讨miR-29在脑缺血及神经保护中的重要作用，分析miR-29在海马的作用靶点。本研究对阐明细胞特异miRNA作为一种脑缺血后新型有效神经保护靶点有重要意义，并为星形细胞功能保护改善复苏后神经功能提供思路。

神经功能障碍是心脏骤停患者复苏后致残的重要原因。即使轻微的脑缺血也会在海马DG区神经元功能正常的情况下，导致CA1区神经元死亡。仅关注神经元而缺少对星形细胞的重视是导致许多中风治疗策略失败的主要原因。miRNA调控基因的转录后表达，但其真正作用靶点及生物功能仍需阐明。我们发现，miR-29是星形细胞特异的一种miRNA，可以与多个Bcl-2家族成员，包括促凋亡和抗凋亡蛋白的mRNA 3´UTR相结合。据此提出miR-29与Bcl-2家族蛋白相互作用调节星形细胞对缺血的反应机制。.我们利用大鼠前脑缺血模型，得到miR-29在海马CA1区和DG区的变化规律，发现miR-29a表达质粒可以保护前脑缺血CA1区神经细胞，同时伴随Puma蛋白表达减少，而miR-29a体内抑制剂使CA1区神经细胞死亡增加，Puma蛋白表达增加。在使用miR-29a表达质粒研究星形细胞缺血变化规律的基础上，进一步使用表达更为稳定、高效的miR-29a模拟剂和抑制剂，发现miR-29a模拟剂对体外培养星形细胞缺血应激时具有保护作用，增加线粒体膜稳定性，抑制ROS产生，并且星形细胞内Puma蛋白表达受到明显抑制；另一方面，miR-29a抑制剂增加缺血应激时星形细胞的死亡率，降低线粒体膜稳定性，ROS产生增多，且Puma蛋白表达相应增加。进一步通过将大鼠特异Puma siRNA 及非特异的siRNA对照导入星形细胞，发现大鼠特异Puma siRNA明显抑制星形细胞内Puma蛋白表达。在GD及OGD处理后，星形细胞存活率较转染非特异的siRNA对照组星形细胞明显升高。利用大鼠前脑缺血模型，获得了大鼠前脑缺血再灌注1hr和5hr时脑片标本，荧光双免疫染色检测得到Puma蛋白及星形细胞标志物GFAP蛋白的表达关系。进一步，将大鼠特异Puma siRNA注射入大鼠海马组织，观察得到大鼠特异Puma siRNA对大鼠海马组织CA1及DG区Puma表达的调控作用。.本研究使用大鼠前脑缺血模型及培养星形细胞探讨miR-29在脑缺血及神经保护中的重要作用，分析miR-29在海马的作用靶点。对阐明细胞特异miRNA作为一种脑缺血后新型有效神经保护靶点有重要意义，并为星形细胞功能保护改善复苏后神经功能提供思路。