PD-L1转基因小鼠的建立及其脊髓损伤后的运动功能恢复

Establishment of PD-L1 transgenic mouse model and recovery of the motion after spinal cord injury

目的:建立广泛表达PD-L1的转基因小鼠,并且评价转基因小鼠脊髓损伤后的运动功能恢复情况。方法:克隆小鼠PD-L1基因编码区的cDNA,经测序正确后,将PD-L1基因cDNA插入到pCAGGS质粒中,构建pCAG-PD-L1转基因载体;经C57BL/6小鼠受精卵原核注射,建立PD-L1转基因小鼠;PCR鉴定转基因小鼠的基因型;以流式细胞术(FCM)检测转基因小鼠脾脏中T、B淋巴细胞PD-L1的表达情况;免疫组织化学检测转基因小鼠周围组织PD-L1表达情况;RT-PCR检测转基因小鼠神经组织内PD-L1表达;BBB评价转基因小鼠脊髓损伤后运动功能恢复。结果:获得PD-L1转基因小鼠,外源PD-L1基因可以顺利遗传给子代;免疫组织化学、RT-PCR和FCM发现:PD-L1转基因小鼠的神经组织、淋巴组织以及生殖器官中高表达PD-L1;PD-L1转基因小鼠脊髓重度夹伤后,其BBB运动学评分明显高于野生型小鼠(P<0.05)。结论:成功地建立了C57BL/6背景的PD-L1转基因小鼠,且PD-L1基因的高表达促进脊髓损伤后的运动功能恢复。

AIM：To establish a ubiquitously expressed PD-L1 transgenic mouse model and evaluate its recovery of motor function after spinal cord injury.METHODS： Clone and sequence the complete mouse PD-L1 cDNA and construct the pCAG-PD-L1 transgenic vector by inserting the PD-L1 cDNA into the pCAGGS vector.The PD-L1 transgenic（TgPD-L1） mice were established by pronuclear micro-injection with fertilized eggs from C57BL/6 mice and the genotypes were confirmed by PCR with tail genomic DNA.The expression of PD-L1 on T and B lymphocytes from mouse spleen were detected by flow cytometry.The expression of PD-L1 in peripheral tissues was displayed by immunohistochemistry.The expression level of PD-L1 in spinal tissue was evaluated by RT-PCR.The recovery of motor function was analyzed by Basso-Beattie-Bresnahan（BBB） locomotion testing system at 3,7,14,21,28 and 35 day after spinal severe crush with forceps in mice.RESULTS： Three lines of TgPD-L1 mice in C57BL/6 background were generated and the exogenous PD-L1 gene can be heritable steadily to offsprings.PD-L1 was highly exppressed in spinal tissue,peripheral tissues,T and B lymphocytes using RT-PCR,immunohistochemistry and flow cytometry respectively in TgPD-L1 mice.The BBB scores were obviously higher at 21 day post-injury in TgPD-L1 than those of in WT mice（P0.05）.CONCLUSION： The TgPD-L1 mice whose background are C57BL/6 were established successfully and high expression level of PD-L1 in tissues promotes locomotion recovery after spinal cord injury in TgPD-L1 mice.