【作者】 丁悦敏；

【导师】 夏强；

【作者基本信息】 浙江大学 ， 生理学， 2014， 博士

【摘要】 背景：目前针对脊髓损伤的临床治疗仍然缺乏有效手段,如何促进损伤后神经再生,提高临床治疗效果,是神经科学研究者的研究重点。轴突内细胞骨架是脊髓损伤后微环境中多种细胞外部信号在细胞内的整合位点,越来越多的研究均证明通过调控细胞骨架蛋白的重组可综合细胞外部因素和内在因子的作用,显著改善轴突再生的能力并促进功能的恢复。细胞骨架蛋白则受细胞内多种蛋白调控因子调控,因而这些能作用于轴突内细胞骨架蛋白的调控因子对脊髓损伤的修复有着举足轻重的作用。Nischarin是2000年发现的一种分布于多种细胞胞浆的蛋白质。在过去的十年中关于Nischarin蛋白的功能研究主要集中于它对肿瘤细胞迁移能力的抑制作用上Alahari及同事发现Nischarin在胞浆内可与Rho GTPase家族的重要成员Rac1、PAK结合并直接抑制它们的活性,从而减弱细胞的活动能力。随着研究的深入,又发现Nischarin不仅能抑制PAK,还能直接抑制PAK下游的关键激酶LIMK的活性,从而阻碍细胞骨架相关蛋白cofilin的磷酸化,进而影响细胞运动能力。这些研究表明,肿瘤细胞的内源性Nischarin可通过调控RhoGTPase/PAK/LIMK/cofilin信号通路,影响细胞骨架蛋白的重组。如前所述,能调控神经元轴突内细胞骨架蛋白重组的因子对脊髓损伤修复有极其重要的意义,Nischarin若在神经元中表达,且也能通过Rho GTPase信号通路调控轴突内细胞骨架蛋白重组,则应该有理由成为脊髓损伤治疗中的一个新靶标。为此,本课题的主要研究目的为：探讨新蛋白Nischarin在正常成年大鼠中枢神经系统的表达与分布；研究Nischarin蛋白在脊髓损伤后轴突再生过程中的作用及其可能机制。获得以上两块内容的实验结果并加以分析,可望拓展对新蛋白Nischarin功能的研究范围,并可能为脊髓损伤的基因或分子治疗提供新的思路和靶点。方法：1取正常成年大鼠组织,用RT-qPCR、western blot及免疫荧光染色方法检测大鼠各主要器官和中枢神经系统各部位Nischarin的表达情况。2建立大鼠脊髓损伤模型,探讨损伤后不同阶段Nischarin蛋白在脊髓组织的表达情况；在细胞水平通过RNAi技术抑制内源性Nischarin蛋白表达,采用活细胞成像技术观察神经细胞突起生长的变化。3采用免疫共沉淀技术研究神经元内源性Nischarin与PAK1、LIMK1的互作情况；探讨Nischarin对PAK1、LIMK1及cofilin的磷酸化调控作用及其对细胞骨架蛋白F-actin的调控；活细胞成像法观察PAK1的特异性抑制剂IPA3对Nischarin-shRNA调控神经突起生长作用的影响。结果：1Nischarin蛋白在正常成年大鼠的心、肺、肝、肾、胃、小肠、脑和脊髓组织中均有表达,在肝脏和脑组织中最为丰富；其mRNA及蛋白广泛表达于脑的各个部位,在大脑皮层神经元上表达量最高,海马区神经元其次,脑干和嗅球部位则较低,且只表达于神经元而非胶质细胞；在神经细胞内,Nischarin的表达集中在细胞的核周及细胞突起的前缘。2在正常成年大鼠脊髓组织上,Nischarin主要分布在前角运动核神经元的核周胞浆中；与正常大鼠脊髓组织比较,Nischarin mRNA的表达从SCI后1d起显著增高,损伤后7d达到顶峰(P<0.01),随后下降；与之相符,SCI后1d起Nischarin蛋白的表达即有显著增高(P<0.05),损伤后7d达到最高(P<0.05),此后下降；免疫荧光染色结果提示SCI后3d,脊髓组织结构紊乱,除神经元有Nischarin表达外,胶质细胞上也一定量的表达；损伤后7d,损伤区Nischarin高度表达,大量胶质细胞增生；用aggrecan、CSPGs或TNF-α三种脊髓损伤局部产物处理Neuro-2a细胞,孵育2h后对Nischarin的表达影响不大(P>0.05),孵育24h和48h后三者均显著促进Nischarin的表达(P<0.05)。3①用Nis-siRNA转染Neuro-2a细胞能有效抑制内源性Nischarin的表达,抑制率达到86%(P<0.01),Neuro-2a细胞的神经突起形态发生明显变化,有突起细胞百分比较control-siRNA组细胞增加3.3倍(P<0.001),最长突起平均值增长5.4倍(P<0.001),而平均细胞突起数目增加了1.57倍(P<0.01)；②将4条Nis-shRNA克隆到慢病毒质粒载体,用脂质体转染Neuro-2a细胞评价其对Nischarin的内源性表达的抑制效果,实时定量PCR结果显示Neuro-2a细胞上的转染效率达到80%以上,与未处理细胞和对照shRNA比较,4个Nis-shRNA质粒均能有效抑制Nischarin的表达,其中Nis-shRNA-3的抑制效率到达60%(P<0.05),Western Blotting实验结果与RT-qPCR实验结果相符,4个Nis-shRNA质粒中以3号质粒的抑制效果为最佳,抑制率为81.3%(P<0.01)；③用慢病毒包装体系对3号质粒及其scramble对照质粒进行包装,收获病毒液,感染PC-12细胞和原代培养神经元,荧光显微镜下发现抑制内源性Nischarin表达显著促进PC-12细胞神经突起的生长,细胞最长神经突起的平均值增加140%(P<0.01),每个细胞的平均突起数目增加56%(P<0.05),神经突起生长速度明显增快；抑制内源性Nischarin表达也促进原代培养大脑皮层神经元突起的生长,细胞最长神经突起的平均值增加51%(P<0.05),但对神经元的平均突起数目影响不显著(P>0.05)；④胶质瘢痕抑制物aggrecan或CSPGs使Neuro-2a细胞的最长神经突起平均值和突起数目均显著下降,而当内源性Nischarin被抑制后,不仅使得PDL基质上的神经细胞的突起生长加速,而且还促使细胞克服CSPGs或aggrecan对神经生长的抑制作用(P<0.05)。4①免疫共沉淀结果显示,神经元内源性Nischarin可以与PAK1、LIMK1结合；②用Nischarin-shRNA转染Neuro-2a细胞使其内源性Nischarin表达量降低75%(P<0.01),于此同时,p-PAK1/PAK1比值、p-LIMK1/LIMK1比值及p-cofilin/cofilin比值分别增加了41.5%(P<0.05)、40%(P<0.05)及57.5%(P<0.01)；而在SCI大鼠模型上,Nischarin的表达先增高再下降,在损伤后一周达到峰值,比sham组增高196%(P<0.01),而p-PAK1/PAK1、p-LIMK1/LIMK1及p-cofilin/cofilin比值均呈现先降低再增高的趋势,并且均在损伤后一周达到最低值,分别比sham组下降了40.2%(P<0.05)、56.3%(P<0.01)及61.8%(P<0.01)；③免疫荧光染色结果显示,在感染Nischarin-shRNA病毒的EGFP阳性神经元上,神经突起生长锥处F-actin的含量显著增高；④抑制内源性Nischarin的表达可促使抑制性环境下神经细胞的突起生长,但这种促进作用在加入PAK1的抑制剂IPA3之后被完全逆转(P<0.05)。结论：1Nischarin在正常成年大鼠的中枢神经系统呈区域化分布,主要表达于神经元胞浆核周部位及神经突起前缘。2脊髓损伤可引起Nischarin的表达时间依赖性增高,其表达上调可能与脊髓损伤局部高浓度的TNF-α、抑制性物质CSPGs和aggrecan有关。3用RNAi技术抑制神经细胞内源性Nischarin的表达后有效促进神经细胞在正常及抑制性环境中神经突起的生长。4神经元内源性Nischarin与PAKl及LIMK1存在互作关系,直接抑制PAK1的磷酸化,并影响LIMK1和cofilin的磷酸化过程；这一机制可能介导了Nischarin蛋白对神经突起生长的抑制作用。更多还原

【Abstract】 IntroductionThere is still a lack of effective means of clinical treatment for spinal cord injury at present. How to promote nerve regeneration and to improve the clinical treatment effect is still the focus of neuroscience researchers. The axon cytoskeleton is the intracellular site where a variety of the outside cell signals in microenvironment integrated after spinal cord injury. More and more studies have proved that reorganization of cytoskeletal proteins, resulted from regulation by both the intracelluar and extracellular factors, improve the ability of axonal regeneration and promote functional recovery. Cytoskeletal proteins are affected by many intracellular protein regulators. Thus, these regulatory factors acting on the cytoskeleton in the axon play a decisive role in repair of spinal cord injury.Nischarin is a novel protein found in2000and located in the cytoplasm of a variety of cells. In the past decade, functional studies of Nischarin protein mainly focus on its inhibitory effect on the migration of tumor cells. Alahari and colleagues found that Nischarin bind to Racl and PAK, important members of and Rho-GTPase family, and directly inhibit their activity in the cytosol, which suppresses the cell motility. With the deepening of the study, it was also found that Nischarin can not only inhibit PAK, but also directly inhibit the activity of LIMK, a key downstream kinase of PAK, thereby negatively regulate the phosphorylation of cofilin, a cytoskeleton associated protein, and finally affect cell motility. As mentioned above, factors which can regulate cytoskeletal protein reorganization in axon play an important role on the repair of spinal cord injury. If Nischarin is expressed in neurons, and also regulates the cytoskeleton reorganization via Rho-GTPase signaling pathway in the axon, it should have become a novel target in the treatment of spinal cord injury.Therefore, the main purposes of the study are to investigate the expression and distribution of Nischarin protein in the nervous system of normal adult rats, especially in the spinal cord, and to study the function of Nischarin protein on axonal regeneration after spinal cord injury and its possible mechanism. Based on these experimental results and analysis, the scope of functional study of Nischarin protein is expected to expand, and it may provide new ideas and targets for gene or molecular treatment of spinal cord injury.Methods1The expression of Nischarin in different parts of organs and central nervous system in the normal adult rats was detected by real-time quantitative reverse transcription-PCR (RT-qPCR), Western blot assay and immunofluorescence staining.2A rat model of spinal cord injury was established and the expression of Nischarin protein in different stages of SCI was evaluated by RT-qPCR and Western blot. By inhibiting the expression of Nischarin with RNAi technique in neuronal cells and primary cortical neurons, changes in neurite growth of neuronal cells were analyzed by live cell imaging technique.3The interaction between endogenous Nischarin and PAK1, Nischarin and LIMK1were examined by co-immunoprecipitation. The phosphorylation regulation of Nischarin on PAK1, LIMK1and cofilin in Neuro-2a cells was detected by Western blot assay. The regulation effect of Nischarin on cytoskeletal protein F-actin was observed after immunofluorescence staining. Finally, the effect of IPA3, the specific inhibitor of PAK1, against Nischarin-shRNA was assessed by live cell imaging method.Results1Nischarin protein is differentially expressed in heart, lung, liver, kidney, stomach, intestine, brain and spinal cord tissues from normal adult rats, with a higher expression in the liver, brain and spinal cord tissue. It’s mRNA and protein is widely expressed in brain, strongest staining for Nischarin was present in the pyramidal neurons of the cerebral cortex and hippocampus, while weak in brain stem and the olfactory bulbs. In addition, Nischarin is only expressed in neurons but not glial cells. The subcellular distribution of Nischarin in neurons or Neuro-2a and PC-12cells were concentrated in the perinuclear and the leading cytoplasmic processes of cells.2In the spinal cord of normal adult rats, Nischarin was distributed mainly in the perinuclear cytoplasm of the neurons in lateral anterior horn motor nucleus. Compared with normal rats, the Nischarin mRNA level was significantly increased1d after injury, and reached the peak by7d after SCI (P<0.01), then decreased. Consistently, the expression of Nischarin protein were significantly increased at1d after injury (P<0.05) and reached the highest at7d after SCI (P<0.05), then dropped briefly and began to increase from3w after SCI (P<0.05). Immunofluorescence staining results showed that in addition to neurons, glial cells also have a certain amount of expression of Nischarin at3d after SCI, the expression of Nischarin in the damage zone strongly expressed at7d after injury. With the treatment of aggrecan, CSPGs or TNF-a, the local products in the injury sites of the spinal cord, the expression of Nischarin did not changed after2h-incubation (P>0.05), but were significantly promoted after24h-or48h-incubation (P<0.05).3①Expression of the endogenous Nischarin in Neuro-2a cells transfected by Nis-siRNA was effectively inhibited and the inhibition rate reached86%(P<0.01). In the meantime, the neurite morphology of Neuro-2a cells changed obviously. Compared to cells in control-siRNA group, the percentage of neurite-bearing cells increased by3.3times (P<0.001), the mean length of the longest neurite increased by5.4times (P<0.001), while the number of neurites per cell increased by1.57times (P<0.01).②The4Nis-shRNA was cloned into the lentiviral vector, and the inhibitory effect was evaluated by checking the expression of endogenous Nischarin in Neuro-2a cells transfected with the vectors using lipofectamine. Real-time quantity PCR results revealed that the transfection efficiency reaches above80%. Compared to cells of the untreated and control shRNA groups, all of the4Nis-shRNA plasmids effectively knocked down Nischarin expression, amone which, the inhibition efficiency of Nis-shRNA-3reached60%(P<0.05). Consistently, Western Blotting results indicated that plasmid Nis-shRNA-3had the highest inhibitory rate of81.3%(P<0.01).③The No.3plasmid and the scramble control were packaged into the virus using lentiviral packaging system and the virus liquid was harvested and infected into the PC-12cells and primary cultured neurons. The fluorescence microscopy results indicated that knockdown of endogenous Nischarin expression significantly promote neurite outgrowth in PC-12cell. The mean length of the longest neurite increased by140%(P<0.01) and the average number of neurites per cell increased by56%(P <0.05). It also promotes neurites outgrowth in the cultured cortical neurons. The average length of the longest neurite cells increased by51%(P<0.05), but the average numbers of neurites did not changed (P<0.05).④The length of the longest neurites and the number of average neurites per cell were significantly decreased when Neuro-2a cells were treated by myelin inhibitors aggrecan or CSPGs. When the expression of the endogenous Nischarin was inhibited, the neurite outgrowth was accelerated not only on the PDL matrix, but also on the CSPGs or aggrecan by promoting cells overcome the inhibitors (P<0.05).4①Co-immunoprecipitation data suggested that the endogenous Nischarin in neurons interacted with PAK1and LIMK1.②The expression of endogenous Nischarin in Neuro-2a cells decreased by75%by Nischarin-shRNA transfection (P<0.01). At the same time, p-PAK1/PAK1, p-LIMK1/LIMKl and p-cofilin/cofilin ratio were increased by41.5%(P<0.05),40%(P<0.05) and57.5%(P<0.01), respectively. In the model of SCI rats, the expression of Nischarin increased with time, reached the peak in one week after injury with a196%increase over control (P <0.01), and then decreased. On the contrary, p-PAK1/PAKl, p-LIMK1/LIMK1and p-cofilin/cofilin ratio were decreased firstly and then increased, and reached the minimum in one week after injury, decreased by40.2%(P<0.05),56.3%(P<0.01) and61.8%(P<0.01), respectively.③mmunofluorescence staining revealed that F-actin increased significantly in the growth cones of the neurons infected by Nischarin-shRNA lentivirus.④The neurite outgrowth was promoted by inhibition of endogenous Nischarin expression in the neuronal cells, however, this promoting effect was completely reversed after adding IPA3, a PAK1inhibitor (P<0.05).Conclusions1Nischarin is regionally distributed in the central nervous system of the normal adult rats, mainly in the perinuclear region and the leading edges of the protrusions of the neurons.2The expression of Nischarin increased as a time-dependent manner after spinal cord injury. The upregulation may be associated with TNF and the inhibitory substances CSPGs and aggrecan.3Inhibiting expression of endogenous Nischarin using RNAi techniques effectively promote neurite outgrowth of the neuronal cells in both normal and inhibitory environments. 4The interaction between endogenous Nischarin and PAK1or LIMK1in neurons directly inhibits phosphorylation of PAK1, and regulates LIMK1and cofilin phosphorylation activity. The mechanism may mediate the inhibitory effect of Nischarin protein on the neurite outgrowth.更多还原