【作者】 赵欢欢；

【导师】 张建琼；

【作者基本信息】 东南大学 ， 生物学， 2017， 硕士

【摘要】 近年报道多种在免疫系统发现并起重要作用的蛋白质,在发育及成年神经系统也有表达。其中,经典MHCⅠ类分子在中枢神经系统的表达和功能逐渐成为神经发育及神经免疫领域的研究热点。该分子在中枢神经系统呈时空特异性的表达,提示该分子可能存在重要功能。目前已知该分子可改变突触的可塑性。本实验室前期研究证实:小鼠出生后早期(P4天),即突触发育高峰期前,大脑皮层神经元存在MHCⅠ类分子的表达;与野生型相比,MHCⅠ类分子敲除鼠体外培养的皮层神经元突起数目增加。这些结果表明MHC Ⅰ类分子在突触形成前已在小鼠皮层神经元中表达,且可能参与神经突起的形成。基于前期的研究结果,选取C57BL/6野生小鼠和MHCⅠ类分子基因敲除小鼠,运用原代培养的皮层神经元,通过病毒感染、抗体阻断、免疫荧光、Western Blot、RT-qPCR等方法探究经典MHC Ⅰ类分子与神经元突起形成的相关性及调控机制。主要研究结果如下:1.在C57BL/6野生型小鼠原代培养的皮层神经元突起形成的早期(1-2DIV)、树突和轴突分化的高峰期(4-5 DIV)以及树突生长的高峰期(7-10 DIV),利用MHCⅠ类分子特异性的抗体(OX18)和细胞骨架F-actin特异性的染料(鬼笔环肽)进行免疫荧光双标检测,结果显示在神经突起生长的三个关键期,经典MHCⅠ类分子表达于神经元的胞体、树突以及生长锥部位,且在神经突起形成处和生长锥部位与细胞骨架分子F-actin共标。提示在突触形成前,经典MHCⅠ类分子已在小鼠皮层神经元中表达,且可能参与神经突起的形成。2.原代培养C57BL/6野生型和MHCⅠ类分子敲除小鼠(H2-DbKb-/-小鼠)的皮层神经元。通过病毒载体在敲除小鼠的皮层神经元过表达MHCⅠ类分子,可抑制神经突起的形成。在C57BL/6野生型小鼠原代培养的神经元中加入MHC Ⅰ类分子的特异性抗体(OX18)、PirB的抗体和胞外段。Sholl analysis定量分析显示,在阻断信号通路后,皮层神经元突起数目和总长度明显增加。提示在原代培养的皮层神经元中,经典MHCⅠ类分子可抑制神经突起的生长。3.原代培养C57BL/6野生型小鼠的皮层神经元,利用MHCⅠ类分子的特异性抗体(OX18)阻断MHCⅠ类分子相关信号通路,利用Western Blot检测细胞骨架相关分子的表达。结果显示细胞骨架调控蛋白Cofilin的磷酸化水平下调,调控该蛋白的蛋白激酶LIMK1的磷酸化水平也同步下调。加入PirB的抗体或其胞外段阻断相关信号通路与加入MHC Ⅰ类分子的抗体阻断信号通路后对Cofilin的影响相似,间接表明MHC Ⅰ可能与PirB相互作用引起细胞骨架相关分子的变化,从而改变神经突起的形态。4.原代培养C57BL/6野生型和MHC Ⅰ敲除小鼠的皮层神经元,通过RT-qPCR检测神经突起发育相关基因GAP43、GPRIN1、STMN2在mRNA的表达水平。结果显示在MHC Ⅰ敲除小鼠体外培养6天的神经元中,这三个基因在mRNA的表达水平明显升高。提示调控MHCⅠ类分子的表达可影响神经突起发育相关基因的表达,可能间接改变神经突起的形态。5.利用无Mg2+培养液诱导体外细胞癫痫模型,在诱导3h后换成正常的神经元培养液继续培养。Western blot结果显示换上正常的培养液培养16h后,MHCⅠ类分子的表达升高。且免疫荧光的结果显示,在用无Mg2+培养液诱导后,用正常的神经元培养液继续培养8h、16h、24h后,树突分支数目和总长度均增加,在16h时,树突分支增加的幅度最明显。但在MHC Ⅰ敲除小鼠体外培养的皮层神经元,用无Mg2+培养液诱导后,树突分支的数目和总长度不改变。上述结果表明,在皮层神经元发育过程中,经典MHCⅠ类分子可抑制神经突起的形成,且可能与一些受体相互作用影响细胞骨架相关蛋白的活性。在体外诱导的细胞癫痫模型中,树突分支数目的异常形成与MHCⅠ类分子的表达上调相关。我们的研究为深入阐明MHC Ⅰ类分子抑制神经突起形成的分子机制及在疾病状态下如何发挥功能提供实验基础,有助于理解MHC Ⅰ类分子在神经环路形成过程及相关疾病中的作用。更多还原

【Abstract】 It has been reported that many kinds of proteins,which play an important role in immune system,can be expressed in the developing and adult brain.Among them,studies on the expression and function of classical MHC class Ⅰ molecules in the central nervous system(CNS)have become hot spots in the field of brain development and neuroimmunology.The temporal and spatial patterns of classical MHC class Ⅰ molecules expression suggest that MHC class Ⅰ plays an important role in manipulation of CNS synase plasticity.A recent paper has reported that basal dendrites of L2/3 cortical neurons are more highly branched in the MHC class Ⅰ knockout mice.In our previous study,we found that classical MHC class Ⅰmolecules are expressed by neurons in mouse cerebral cortex during the early postnatal period before development of synaptic connections.We have also found that neurons cultured from MHC class Ⅰ knockout mouse displayed faster neurite outgrowth and developed more primary neuritis than neurons from wild type mouse.The results suggest that classical MHC class Ⅰ molecules can modulate neuritogenesis before development of synaptic connections.In the present study,we aim to elucidate the roles and molecular mechanisms of classical MHC class Ⅰ in neurite outgrowth by viral infection,immunostaining,western-blot and RT-PCR in primary cultured cortical neurons.The results were shown as follows:1.In the critical period of neurite growth in primary cultured neuron,double-staining experiments with MHC class Ⅰ antibody(0X18)and TRITC-phalloidin that visualized the F-actin was performed on 2 DIV,4 DIV and 7 DIV neurons.At 1-2 DIV,a stage corresponds to the emergence of few minor neuritis.At 4-5 DIV,a stage was characterized by continuing axonal growth and differentiation of minor neurites into dendrites.By 7-10 DIV,a stage was marked by continued maturation of axonal and dendritic arbors.The results showed that MHC class Ⅰ were expressed in cell bodies,dendrites and growth cones of neurons during the three periods of neurite outgrowth.Furthermore,MHC class Ⅰ immunoreactivity largely overlapped with the F-actin in the neurite formation site and growth cone.2.Overexpression of MHC class Ⅰ by viral vectors in primary cultured cortical neurons of C57BL/6 MHC class Ⅰ knockout mouse(H2-DbKb-/－ mouse)inhibited the formation of neurite.The specific antibodies(0X18)of MHC class Ⅰ,the antibody and ectodomain of PirB were added to the primary cultured neurons of C57BL/6 wild type mouse.Quantitative analysis showed that the number of dendritic arborization increased significantly after blocking the signal pathway,suggesting that classical MHC class I molecules can inhibit the neurite outgrowth in primary cultured cortical neurons.3.The classical MHC class Ⅰ-related signal pathway was blocked by the specific antibody(0X18)in the primary cortical neurons of C57BL/6 wild-type mouse.The expression of cytoskeletal related molecules was detected by western blot.The results showed that the phosphorylation level of Cofilin and LIMK1 was down-regulated.4.The expression of GAP43,GPRIN1 and STMN2 were detected by RT-qPCR in the primary cortical neurons of C57BL/6 wild type and MHC class Ⅰ knockout mouse.Results showed that the mRNA expression levels of the three genes were significantly increased in MHC class Ⅰ knockout neurons cultured in vitro for 6 days.5.A cultured developing cortical neuronal epilepsy model was established by exposing the neurons to Mg-free media for 3 h and then returning to regular media.After 16h with regular media,the expression of MHC class I was up-regulated.The results of immunofluorescence showed that the number of dendritic branches increased after induction with Mg-free media and the increase of dendritic branch was most obvious after 16 h with regular media.However,the number of dendritic branches did not change after induction with Mg-free media in the primary cultured cortical neurons of MHC class Ⅰ knockout neurons.These results suggest that classical MHC class Ⅰ molecules can inhibit the formation of neurite processes during the development of cortical neurons and may interact with some receptors to influence the activity of cytoskeletal proteins.In the cultured developing cortical neuronal epilepsy model,the abnormal formation of dendritic arborization is related to the up-regulation of classical MHC Ⅰ molecules.Our study provides an experimental basis for elucidating deeply the molecular mechanism of MHC class Ⅰ molecules in inhibiting neurite outgrowth and the function of MHC class Ⅰ molecules in the disease state.It will provide new insights into the physiological and pathological functions of classical MHC class Ⅰmolecules in CNS.更多还原