多疣壁虎Fstl1基因表达及其在断尾损伤中的变化

Expression of Fstl1 in the spinal cord and regenerating tail of Gekko japonicus

目的:研究多疣壁虎卵泡抑制素相似蛋白1(Fstl1)基因的表达及其在壁虎断尾损伤再生中的变化。方法:通过对多疣壁虎断尾损伤cDNA文库克隆测序获得Fstl1基因序列,并采用ClustalX软件比较GenBank同源蛋白进行系统进化分析。用Northern blot与原位杂交分析多疣壁虎Fstl1转录本大小及其在脊髓中的表达定位。通过RT-PCR方法研究Fstl1基因在壁虎主要组织中的表达分布与壁虎断尾损伤模型中变化。结果:基因系统进化分析表明,多疣壁虎Fstl1与人类Fstl1同源性较高,为96.5%;与爪蟾同源性较低,为62.9%。Northen blot分析表明,成年多疣壁虎Fstl1脊髓转录本大小约为3.7kb。原位杂交结果显示,多疣壁虎脊髓中Fstl1的阳性信号主要存在于灰质神经元细胞及室管膜细胞;而在断尾再生2～3周尾芽中,Fstl1阳性信号广泛存在于再生尾芽的新生区未分化细胞及间充质细胞,3周尾芽中,阳性信号还存在于软骨生成区。RT-PCR结果显示,多疣壁虎Fstl1基因在壁虎脑、脊髓、心、肝、肺、肾、卵巢、尾巴、肌肉中均有表达,其在脊髓中呈高表达。在断尾损伤模型中,多疣壁虎Fstl1表达呈动态变化,其在近端脊髓组织中,损伤3天时Fstl1转录表达增高,并随后开始下降恢复;在断尾再生尾芽中,3周时Fstl1转录表达低于2周表达量。结论:Fstl1基因主要分布于脊髓灰质神经元细胞中,Fstl1在壁虎断尾损伤后近端脊髓的表达呈动态变化,在再生尾芽表达3周下调。

Objective：To analyze expression of Follistatin-like1（Fstl1） in the spinal cord and regenerating tail of Gekko japonicus.Methods：A large scale sequencing of the Gekko japonicus rengeneration cDNA library was performed to obtain Fstl1 cDNA sequence.Comparison against the GenBank protein database was performed using the BLAST network and Phylogenetic analysis was conducted by applying the Clustal method.The expression of Gekko Fstl1 was analyzed by northern blot,in situ hybridization（ISH） and reverse transfer polymerase chain reaction（RT-PCR）.Results：Phylogenetic analysis revealed that Gekko Fstl1 shared 96.5% with human and 62.9% with X.tropicalis Fstl1 mRNA.Northern blot shown a 3.7 kb band of Fstl1 transcript detected in spinal cord of adult geckos.Section in situ hybridization demonstrated that the Fstl1 transcripts in cervico-lumbar spinal cord were distributed in the gray matter neuro-cells and ependymal cells.hybridization signals were present widely in dedifferentiated cells and mesenchyme cells at 2 wks and 3 wks while also in cartilaginous cells at 3 wks of regenerating blastema after tail amputation.RT-PCR results showed that Fstl1 gene was expressed in brain,heart,liver,lung,kidney,muscle,tail,ovary and highly in spinal cord.The expression of Fstl1 was increased at 3 d and then returned to control levels at 1 wks in cervico-lumbar segment of the spinal cord.However,Fstl1 expression was significantly lower at 3 wks than control values at 2 wks in regenerating blastema.Conclusion：Fstl1 transcripts in cervico-lumbar spinal cord were distributed in the gray matter neuro-cells and ependymal cells.It＇s expression level was dynamic change in the proximal spinal cord and regenerating blastema after tail amputation.