【作者】 卢连军；

【导师】 王锦玲；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2001， 博士

【摘要】 头颈部外伤和手术常累及面神经，而面神经损伤后的功能恢复很差。因此研究面神经损伤再生过程中相关分子的变化对进一步了解神经损伤再生机制具有重要意义。乙酰胆碱（Ach）作为运动神经元支配骨骼肌的重要神经递质，由胆碱乙酰转移酶（ChAT）合成并由囊状乙酰胆碱转运体（VAChT）包装至突触囊泡。对于合成并释放乙酰胆碱作为神经递质的初级运动神经元来说，ChAT已被作为一功能性和特异性的标志。由于编码VAChT的基因位于ChAT基因的第一个内含子内，提示ChAT和VAChT在胆碱能神经元可能在很大程度上被一些因子协同调节。神经营养因子（NTs）家族在神经系统的发育、维持及重塑中发挥重要作用。NTs通过与其受体酪氨酸激酶（trk）结合而发挥生物学作用。IL-6类的细胞因子（IL-6 type cytokines）属于生神经细胞因子家族（neuropoietic cytokinefamily），与神经营养因子一样，具有类似的神经营养作用。已经证明这些细胞因子的信号转导通过激活JAK-STAT途径来完成。NTs和细胞因子的信号转导过程中涉及多步的蛋白磷酸化，可逆的蛋白磷酸化和去磷酸化在 第 四 军 医 大 学 博 土 学 位 论 文 一 细胞的信息传递中占有极重要的地位。面神经损伤后，在神经再支配过程 中经常不可避免地出现面肌联带运动＊ynkinesis），而这一面神经麻痹后 遗症的形成机制尚不清楚。 本研究分别以外周面神经切断和切断后的吻合作为神经是再生和神 经再生模型，分别采用免疫组织化学染色、原位杂交和 RT－PCR方法，观 察成年大鼠面神经外周切断和即刻端端吻合后面神经核ChAT与VAChT、 trkB与trkC、STAT3以及蛋白质磷酸化水平的变化；通过逆行荧光双标技 术观察大鼠面神经颊支和下颁缘支神经元的分布及神经再支配后的变化。 研究结果如下： 第一部分 大鼠面神经切断及修复后面神经核内ChAT和 VAChT的变化 通过对大鼠触须节律性拂动的观察，发现面神经的再生出现在损伤后 的2．5周，术后35天与未损伤侧相比，损伤侧触须节律性拂动已经恢复 正常。免疫组化结果显示面神经损伤后面神经核 ChAT和 VAChT阳性神 经元的数量和免疫染色强度明显下降。在试验观察期问，面神经核ChAT 和 VAChT的下调在面神经单纯切断组未能恢复，而在面神经切断后吻合 组，CM和VAChT阳性神经元的数量和免疫染色强度却可恢复至正常。 两组面神经核 ChAT和 VAChT免疫染色强度在术后 14、ZI和 35大相差 显著（尸＜0刀5）。 上述结果提示面神经吻合并不能阻止损伤侧面神经核ChAT 4［ VA＊hT免疫阳性产物的下降，伴随着面神经再支配的发生，面神经核 ChAT和VAChT免疫阳性产物逐渐恢复iE常。 第二部分大鼠面神经切断及修复后面神经核内trkB和trkC的 变化 3 第 四 军 医 大 学 博 士 学 位 论 文 一 采用原位杂交、RT－PCR和免疫组织化学染色方法，观察成年大鼠面 神经外周切断和即刻端端吻合后面神经核 trkB mRNA和 trkC mRNA以及 蛋白表达的时程变化。结果表明在面神经单纯切断组，术后第3天厂始损 伤侧面神经核 trkB mRNA信号强度开始增加，7天增加最为明显，14天 厂始下降，但刀与35天损伤侧汀kB mRNA信号强度仍高于未损伤侧的 12倍。在面神经切断后端端吻合组，损伤侧面神经核 trkB mRNA信号强 度的变化趋势与面神经单纯切断组基本相同，不同之处在于吻合组汀kB mRNA信号强度在14天以后下降比较明显，术后35天与未损伤侧相比无 明显差别。神经损伤后 tfkC的变化与 tfkB相反，面神经核 tfkC mRNA信 号强度下降。在面神经单纯切断组，术后第 3天损伤侧 trkC mRNA信号 强度开始降低，7天时降低最为明显，以后略有回升，但术后 3 5天 trkC mRNA信号强度仍低于未损伤侧。在面神经切断后端端吻合组，术后7 天以前也表现为 trkC mANA信号强度降低，但 3 5天与未损伤侧相比已 无明显差别。神经损伤后trkB和trkC在蛋白水平的变化趋势与mRNA水 平相同。以上结果提示面神经切断后即刻行端端吻合并不能对抗或消弱神 经损伤所诱导的 trkB和 trkC的 变化，神经元trkB和 trkC完全恢复到损 伤前水平则有赖于面神经的成功再生。 第三部分大鼠面神经切断及更多还原

【Abstract】 The facial nerve is the most frequently affected nerve in head-neck trauma and surgery, but the functional recovery remains poor. So studies of signal transduction and associated factors during facial nerve injury and regeneration are important in understanding its mechanism. Acetylcholine (Ach) is a crucial neurotransmitter for the motoneurons that innervate skeletal muscles. It is synthesized by choline acetyltransferase (ChAT) and transported into synaptic vesicles by vesicular acetylcholine (VAChT). ChAT is presently the most specific indicator for monoitoring the functional state of cholinergic neurons in the central and peripheral nervous systems. It was found the entire VAChT gene is located within the first intron of the ChAT gene in the same transcriptional orientation. The expression of ChAT and VAChT appear to be coordinately regulated by multiple factors in the cholinergic neurons. Neurotrophic factors(NTs) play an important role in the development, maintenance and plasticity of the nervous system. They bind, dimerize, and thereby activate protein tyrosine kinase receptor of the trk family. The interactions appear to mediate the major biological function of NTs. IL-6 type cytokines belong to neuropoietic cytokine family, have a similar role as NTs in nervous system. It was found that these cytokines signal via the activation of Janus kinascs (Jaks) and transcription factors of the STAT family. The signaltransfer of NTs and cytokines involved several steps of protein phosphoration. The reversible phosphoration and dephosphoration play important roles in singnal transduction. While facial nerve reinnervation, the synkinesis always inevitable occur, but its mechanism is still unknown.In present studies, we employed the facial nerve transection and immediate end-to-end anastomosis as models for facial nerve degeneration and regeneration in the rat and attampted to examine the time course of the changes of ChAT, VAChT, trkB, trkC, STATS in motoneurons of the facial nucleus following peripheral nerve transection and end-to-end anastomosis by using in situ hybridization, RT-PCR and immunohistochemical staining, to examine the distribution of motoneurons innervated buccall and marginal mandibular branches, and its reorganization after facial nerve reinnervation.Part 1 Changes of ChAT and VAChT in facial motoneurons after axotomy and end-to-end anastomsis in the ratThe facial nerve regeneration occurs between 2 and 5 weeks in group of anastomsis by observing the restoration of rhythmical whisking. By 35 days symmetrical whisking activity was attained bilaterally. Transection of the facial nerve resulted in a striking reduction in both number of ChAT/VAChT immunoreactive neurons and optical density of ChAT/VAChT immunostaining in the facial nucleus ipsilateral to the operation. A maximal decrease in these values was observed 7 days after operation. The patterns of reduction in both number of ChAT/VAChT immunoreactive neurons and optical density of ChAT/VAChT immunostaining following an immediate end-to-end anastomosis within 7 days post-operation were similar to that of transection group. These values began to increase at the time point of 14 days afteranastomosis. Thirty-five days after anastomosis, ChAT/VAChT immunostaining intensity return to normal level. Our results indicate that an immediate end-to-end anastomosis of the transected facial nerve is unable to attenuate the maximal decrease of ChAT/VAChT immunoreactivity within 7 days post-operation. The successful return of axotomy-induced decrease of ChAT/VAChT immunoreactivity in facial motoneurons occurs following facial nerve to sprout and reinnervate the denervated facial muscles. The expression of ChAT and VAChT in the facial motoneurons appear to be coordinately regulated after facial nerve transection.Part 2 Changes of trkB and trkC in facial motoneurons after axotomy and end-to-end anastomsis in the ratTransection of the facial nerve resulted in a upregulation of trkB, but a downregulation of trkC in both mRNA and pro更多还原