实时定量RT-PCR法检测健侧C7神经移位前后神经生长相关蛋白43的变化

Altered growth-associated protein-43 expression before and after contralateral C_7 nerve transfer detected using real-time quantitative PCR

背景:已有研究证实,周围神经损伤后大脑皮质会出现功能可塑性变化;神经生长相关蛋白43在发育中神经系统的表达特征提示,它与神经发育中突起延伸和突触形成有关,参与突触重建过程。目的:实时定量RT-PCR方法检测实验动物健侧颈7神经根移位前后,不同阶段相应大脑皮质组织神经生长相关蛋白mRNA的相对表达量及动态变化,探讨周围神经修复与中枢神经可塑性的关系。设计、时间及地点:随机对照动物实验,于2007-09/2008-12在上海市周围神经重点实验室和复旦大学实验动物科学部完成。材料:成年SD大鼠108只,分为臂丛损伤未修复组(n=48)、臂丛损伤修复组(n=48)及对照组(n=12)。方法:大鼠左侧为实验侧,臂丛损伤未修复组在大鼠左侧取锁骨下横形切口,找到臂丛各神经根并造成全臂丛根性撕脱伤。臂丛损伤修复组同法将大鼠全臂丛神经根性撕脱后,取右侧锁骨下切口,显露健侧颈C7神经根备用;取左侧前臂尺神经通过皮下隧道桥接健侧颈7神经根与正中神经端端吻合。对照组为成年雌性正常大鼠,不进行任何处理。主要观察指标:于术后1d,3d,1周,2周,1个月,3个月,6个月,10个月共8个时段及对照组取材,采用SYBRGreenⅠ实时定量RT-PCR法检测健侧颈7神经根移位术前后对侧(右侧)大脑前肢投射区域皮质组织生长相关蛋白43mRNA相对表达量及动态变化。结果:臂丛损伤未修复组对侧(右侧)大脑前肢投射区域皮质组织生长相关蛋白43mRNA的相对表达量变化规律为术后第1天开始升高,第3天达到高峰,然后逐渐降低。术后1d,3d和1周与对照组比较差异具有显著性意义(P<0.05,P<0.01)。臂丛损伤修复组对侧(右侧)大脑前肢投射区域皮质组织生长相关蛋白43mRNA的相对表达量变化规律为术后第1天开始升高,第3天达到高峰,然后逐渐降低,至术后3个月再次升高,6个月达到高峰,然后逐渐降低。术后1d,3d,1周和6个月与对照组比较差异具有显著性意义(P<0.05,P<0.01)。结论:臂丛损伤健侧颈7移位术前后相应大脑皮质生长相关蛋白表达变化与临床现象一致,提示生长相关蛋白在大脑皮质及突触可塑性方面发挥作用。

BACKGROUND： Study has confirmed that there will be functional changes of cerebral cortex plasticity following peripheral nerve injury; Expression of growth associated protein-43 （GAP-43） in the developing nervous system suggests that it takes part in the synapse formation and synapse reconstruction in neural development. OBJECTIVE： To detect expression of GAP-43 mRNA in cerebral cortex using real-time quantitative PCR before and after contralateral C7 root transfer, and to reveal the relation between the central nervous system plasticity and the peripheral nervous system injury. DEISNG, TIME AND SETTING： A randomized controlled animal study was performed at the Shanghai Municipal Key Laboratory of Peripheral Nerve, and Department of Laboratory Animal of Fudan University from September 2007 to December 2008. MATERIALS： A total of 108 adult female SD rats were divided into three groups： BPI （brachial plexus injury） without contralateral C7 transfer group （n=48）, BPI with contralateral C7 transfer group （n=48） and control group （n=12）. METHODS： Rat left limb was experimental side. Rats in the BPI without contralateral C7 transfer group were operated through the left subclavian transverse incision to find brachial plexus roots, then resulting in total brachial plexus root avulsion. Rats in the BPI with contralateral C7 transfer group were operated the same as BPI group, contralateral C7 nerve root was excised through right subclavian transverse incision for further use; using subcutaneou tunnel bridge, the ulnar nerve on the affected limb was processed into end-to-end anastomosis with median nerve on the affected limb. There was no intervention given to normal adult female rats, serving as control group. MAIN OUTCOME MEASURES： Drawing cerebral cortex （right side） at 8 different time stages （1 day, 3 days, 7 days, 2 weeks, 1 month, 3 months, 6 months, 10 months） post-surgery and control stage, SYBR Green 1 quantitative real-time PCR was used to detect the mRNA levels of GAP-43 gene on the right forelimb projection areas of cerebral cortex in BPI with or without contralateral C7 transfer groups. RESULTS： Expression of GAP-43 mRNA in the right forelimb projection areas of cerebral cortex in BPI without contralateral C7 transfer group was up-regulated at 1 day, peaked at 3 days, and then gradually reduced, there were significantly differences compared with the control group at 1 day, 3 days and 1 week （P 〈 0.05, P 〈 0.01 ）. While expression of GAP-43 mRNA in the BPI with contralateral C7 transfer group was up-regulated at 1 day, peaked at 3 days, then gradually reduced, again up-regulated at 3 months, peaked at 6 months and then gradually reduced. There were significant differences compared with the control group at 1 day, 3 days, 1 week and 6 months （P〈 0.05, P〈 0.01）. CONCLUSION： GAP-43 expressions in cerebral cortex of rat brain are consistent with clinical phenomenon following contralateral C7 root transfer of BPI, which supports the concept that GAP-43 may play an important role in the cerebral cortex and the synaptic plasticity.