【作者】 李智；

【导师】 肖传国； Xi He；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分Dicer决定神经元极性的发育神经元极性是指神经元有且只有一根细长的轴突和多根较短的树突,是构成复杂的神经网络的结构基础。本部分研究通过敲除Dicer基因,海马神经元极性发生明显改变,多轴突神经元显著增多,证明Dicer在神经元极性发育中起不可或缺的作用。第二部分影响神经元极性发育的Kinase筛选本部分研究通过对海马神经元逐一电穿孔转染75种在神经发育中表达的Kinase,筛选影响神经元极性发育的Kinase.我们目前筛选出6种蛋白激酶在神经元极性发育中有影响：转染LKB1、CERK、CAMK1D、PTK2、PRKCZ、FRKACB显著导致海马神经元多轴突的发生,和GFP控制组有统计学差异(p<0.01)。第三部分影响神经元极性发育的基因筛选最近研究表明,16p11.2上的部分基因影响神经发育。本部分研究通过筛选16p11.2上的八个基因,逐一电穿孔转染MAPK3/MAZ/DOC2A/ TAOK2/TBX6/KCTD13/PPP4C/KIF22基因的shRNA(short hairpinRNA)。研究表明,DOC2A基因、TBX6基因和TAOK2影响神经元极性的发育。更多还原

【Abstract】 PART 1 Dicer is essential for neuronal polarityDicer, an RNaseⅢendonuclease, is the enzyme which cleaves microRNA (miRNA) and small interfering RNA (siRNA) precursors into 21-25 nucleotide species. This cleavage is an essential step in the biogenesis of these small noncoding RNA molecules. In their mature forms, siRNA and miRNAs function to regulate gene expression through different mechanisms1. To investigate the role of Dicer and micro RNAs in neuronal polarity development, we used mice in which the RNaseⅢdomain of Dicer was conditionally floxed. To knockout Dicer gene, hippocampal neurons were electroporated with Cre together with pmaxGFP(?) plasmid by Amaxa(?) Mouse Neuron Nucleofector(?) Kit. Neuronal polarity was analyzed at 3 days in vitro (DIV).Neurons expressing pmaxGFP(?) showed normal polarity. In contrast, the majority (68%) of neurons transfected with Cre developed multiple axons. we found multiple axons were significantly increasing. Here we explore Dicer function in neuronal polarity by inactivating it in the hippocampal neuron using the Cre/loxP approach. Neurons which lack Dicer have multiple axons, demonstrating that Dicer is essential for neuron polarity, providing evidence that Dicer function is required to neuronal development. Key words:Dicer; microRNA; neuronal polarity; multiple axonsPART 2 Kinase screening for neuronal polarityWe electroporated 75 kinases which are expressed in neuronal development to screen kinase which is required for neuronal polarity.We domenstrate that 6 kinases are essential for neuronal polarity development-LKB1、CERK、CAMK1D、PTK2、PRKCZ、FRKACB. PART 3 Gene screening for neuronal polarityRecent study showed that subset of 16p11.2 influences neuronal development. We screened eight genes on 16p11.2. We electroporated MAPK3/MAZ/DOC2A/TAOK2/TBX6/KCTD13/PPP4C/KIF22 shRNA (short hairpin RNA) into hippocampal neurons. The results show that DOC2A, TBX6 and TAOK2 affect neuronal polarity development.更多还原