利用brainbow研究Foxp1基因在小鼠C5神经根损伤后短固有束重塑过程中的作用

Postoperative C5 palsy is well known as a common complication after decompression surgery at the cervical spine，70% of the C5 palsy can recover without medical care. So far, the mechanism of C5 palsy recovery has not yet been illustrated. Being the previous national scientific foundation support, the number of BDA fibers in the propriospinal tract area in the gray matter region increased. The RT-PCR show the Foxp1 expression increased in the C3-C7 gray matter. However, with the limitation of the BDA tracing, the proriospinal tract increased can not be confirmed. In this application, we try to illustrate the short propriospinal tract remolding after the C5 nerve root injury, the brainbow1.0 mice be bought from the Jackson Lab, modify the brainbow to Foxp1-brainbow and Foxp1silence-brainbow mice by flanked the Foxp1 terminator and Foxp1extron between the LoxP sequence, and construct the Foxp1-loxP sequence to the pPk-CMV expression vector alternatively . The phenotype of brainbow mice is examined by laser confocal microscopy, the gene sequence of brainbow mice is checked by PCR. Following the one side C5 nerve root excision, the short proriospinal tract remolding is evaluated by the three-dimensional digital reconstruction of the laser confocal pictures, and the expression of Foxp1 in the promotion remolding process is evaluated by RT-PCR and western blot. The motor function of forelimb is examined by gait analysis.

70%C5神经根损伤可自行恢复。探讨其恢复过程有利于研究颈椎病相关瘫痪的治疗。前一项国家基金研究发现，C5神经根损伤周围短固有束区BDA阳性纤维增多，伴Foxp1高表达。但不能确定BDA纤维增多即短固有束重塑。因brainbow具有显示神经网络和制作转基因和基因沉默动物的空前优势。申报拟引入brainbow1.0小鼠，并修饰成为Foxp1-brainbow和Foxp1沉默-brainbow小鼠。首先用PCR和激光共具焦检测三种小鼠的基因型和表现型。在此基础上，切断三种小鼠的单侧C5神经根，分别在损伤前、后12周内，用步态分析仪评价肢体功能恢复。利用可计数轴突的共聚焦技术、3D重建技术和单轴突示踪技术确定短固有束重塑，观察和计数短固有束再生数量和突触连接情况，用RT-PCR和westernblot判断Foxp1的表达水平。从而，评价C5神经根损伤后短固有束重塑和Foxp1对重塑的促进作用。

本研究依计划分为临床试验部分和动物试验部分。目前临床试验部分已发表相关SCI文章一篇。基础动物试验通过B6.Cg-Tg(Thy-brainbow1.0)HLich/J小鼠和B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J小鼠，构建出brainbow1.0-Cre-ERTM目标小鼠及C5神经根损伤模型，通过肌容积、 BBB (Basso, Beattie, and Bresnahan) 功能评分和步态分析等指标及激光共聚焦显微镜和western blot技术，发现，Thy-brainbow1.0小鼠和UBC-cre/ERT2小鼠，通过交配繁殖，他莫昔芬诱导、筛选，可以得到brainbow1.0-Cre-ERTM目标小鼠。C5神经根损伤后C3-7脊髓节段短固有束所在区域短固有束纤维生长大致分为三个阶段：第一阶段是短固有束纤维开始生长阶段，为损伤后1周内；第二阶段为短固有束生长迅速阶段，为损伤后2周至4周；第三阶段为短固有束生长稳定阶段，为损伤后6周至12周。Foxp1基因的表达量与短固有束纤维生长情况相对应，提示可能对短固有束纤维生长有促进作用。术前和术后小鼠肌容积、BBB功能评分评价神经功能及Footprint test分析步态形态学分析等指标，可以从形态上反映神经根损伤与恢复情况。上述结果证实C5神经根损伤后短固有束的重塑作用及Foxp1基因对重塑有促进作用。