论文标题:神经突起导向因子NETRIN-4与NNAT的相互作用及其表达相关性研究
Comparative Transcriptome Analysis of Yersinia Pestis in Response to Various Environmental Stresses
论文作者 余利红
论文导师 孙志贤；张成岗,论文学位 博士,论文专业 生物化学与分子生物学
论文单位 中国人民解放军军事医学科学院,点击次数 92,论文页数 103页File Size11750k
2005-05-30论文网 http://www.lw23.com/lunwen_344312092/ Natrin-4;酵母双杂交;Neuronatin;神经发育;突触形成
Netrin-4; yeast two hybrid system; neuronatin; developing nervous system; formation of synaptic connections
神经突起与其靶细胞之间精确联系的建立是由多种导向因子共同作用完成的。Netrin基因家族是此类因子的代表之一。本研究主要围绕该家族成员之一Netrin-4及其相互作用蛋白NNAT(Neuronatin)的结构、功能和表达变化特征进行系统分析。 本研究首先构建Netrin-4及其三个结构域的原核表达载体,用IPTG诱导得到相应的融合蛋白,并进一步采用合成肽制备抗Netrin-4特异性抗体。为了探讨Netrin-4发挥作用的分子基础,利用酵母双杂交技术从人胎脑cDNA文库中筛选并通过GST-pull down等实验鉴定出NNAT是其强相互作用蛋白,且证明Netrin-4的EGF重复结构域是Netrin-4与NNAT相互作用的重要结构。为了阐明功能尚属未知的NNAT蛋白与Netrin-4发生相互作用的意义,将NNAT与GFP融合表达,观察到NNAT蛋白在真核细胞中的表达存在核定位的现象,而流式细胞仪检测结果表明融合蛋白的过表达可导致细胞周期改变。进一步通过对所构建的能够稳定表达NNAT的工程细胞株的研究,发现NNAT蛋白仍然存在核定位和细胞周期阻滞现象。这些结果提示NNAT通过定位到细胞核中发挥其生物学功能,可能与细胞的增殖与分化的控制有关。利用RT-PCR技术检测Netrin-4和Nnat在11种细胞系和原代培养神经元中的表达情况,发现二者仅在神经元中有共表达,且在大脑发育的不同时期中Netrin-4和Nnat mRNA的表达特点不同,大鼠出生前后是Nnat表达的高峰期,Netrin-4出生后表达量变化不大。Netrin-4和Nnat在出生前后的高表达提示二者之间的相互作用可能与该时期神经发育过程及神经可塑性密切相关。 综上所述,本研究发现Netrin-4和Nnat都是在大脑发育过程中发挥重要作用的基因。Netrin-4除在发育初期发挥神经突起导向功能外,在大脑发育过程有可能通过与NNAT蛋白相互作用,共同影响突触及特定神经回路的形成。本研究为理解神经发育与分化过程中神经突起导向、神经可塑性的分子机制提供了新的数据。
During development of nervous system for establishing neuronal connections, several guidance factors including ephrin, slit, netrin and semaphorin were critically important for mediating accurate guidance of developing axons to right targets. Here we show the structure, function and character of expression of netrin-4, a novel member of netrin family and its interaction with a novel protein neuronatin (NNAT).At first, we prepared the prokaryotic expression vectors of netrin-4 and its three domains (Laminin N-terminal, EGF repeat, Netrin C-terminal). The fusion proteins with GST were successfully induced with IPTG treatment. For studying the function of Netrin-4, we prepared the special antibody of netrin-4 using synthesized peptide. To better understanding the molecular mechanism of netrin-4, the yeast two-hybrid system was used to identify netrin-4 interaction protein from human fetal brain cDNA library. A novel protein, neuronatin (NNAT) was found to have high affinity with netrin-4 and confirmed by GST-pull down test. In addition, we found that the EGF repeat domain was an important structure for netrin-4-NNAT interaction. To clarify the significance of interaction between netrin-4 and NNAT which function was currently unknown, we further checked the sub-cellular localization of NNAT fused with GFP protein. Interestingly, the NNAT protein was found to be located at nucleus. Using flow cytometer, we also found that excessive expression of fusion protein would induce the change of cell cycle. After creating the engineering cell lines (CHO cells) which could steadily express NNAT following routine protocol, we found that NNAT protein still localized at the nucleus and cell cycle was affected by NNAT. These results indicate that NNAT might control the proliferation and differentiation of neuronal cells through its localization in nucleus. We further checked the expression of netrin-4 and Nnat in 11 cell lines and primary cultured neuron using RT-PCR. The results showed that netrin-4 and Nnat could only co-express in neuronal cells. The expression level of netrin-4 mRNA and Nnat mRNA were different during the development of brain. Nnat mRNA
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