多疣壁虎降钙素基因相关肽基因的克隆及表达分析

Cloning and expression analysis of calcitonin gene related peptide(CGRP) in Gekko japonicus

目的克隆多疣壁虎降钙素基因相关肽(CGRP)基因的全长序列;研究CGRP在壁虎断尾损伤后脊髓再生过程中的表达变化。方法应用cDNA末端快速扩增(RACE)技术获得壁虎CGRP的cDNA全长序列;制备壁虎断尾损伤模型,应用RT-PCR和原位杂交方法检测壁虎断尾后该基因在中枢神经系统的表达变化。结果 RACE方法获得CGRP cDNA全长序列1 002bp;RT-PCR检测结果表明,壁虎断尾损伤1d后断端近侧脊髓中mRNA表达呈明显上升趋势且转录水平最高(P<0.05);脊髓原位杂交结果显示,CGRP mRNA阳性信号主要分布于脊髓灰质中,白质中少量表达;断尾损伤后1 d阳性信号明显增强(P<0.05)。结论该试验克隆得到了壁虎CGRP cDNA的全长序列;壁虎断尾损伤后1 d CGRP在断端近侧脊髓中的表达明显上调。神经系统合成分泌的CGRP可能参与调节免疫反应或伤口愈合。

Objective To clone the full length of calcitonin gene related peptide（CGRP） in Gekko japonicus,and investigate the expression changes of CGRP in the spinal cord regeneration after tail amputation.Methods Full length of CGRP cDNA was obtained from Gekko by rapid amplification of cDNA ends（RACE）.Then the expression and distribution of CGRP in the proximal spinal cord were compared at different time points after tail amputation（P0.05） by in situ hybridization（ISH） and RT-PCR.Results CGRP cDNA spans 1 002 bp.RT-PCR results showed that CGRP mRNA was remarkably up-regulated in the spinal cord proximal to the amputation at 1d after tail amputation（P0.05）.In situ hybridization showed that CGRP mRNA was expressed mainly in the gray matter of spinal cord and scarcely in white matter.After tail amputation,the expression of CGRP was significantly increased in the proximal spinal cord of injury,at 1d（P0.05）.Conclusion Full length of CGRP cDNA was obtained.CGRP was remarkably up-regulated in the spinal cord proximal to the amputation at 1d after tail amputation.CGRP synthesized and released from neural system may participate in regulating immunoreaction or wound healing.