重组人促红细胞生成素对成年大鼠脊髓中央管膜下神经干细胞增殖能力的影响

摘要

Objective To investigate the effect of erythropoietin(EPO)on the proliferation of neural stem cells(NSCs)derived from central canal of adult rat spinal cord in vitro ,so as to provide a theoretical basis for clinical treatment of spinal cord injury by autotransplanting or allograft transplanting of adult spinal cord NSCs. Method NSCs were isolated from the central canal of the adult rats spinal cord by microsurgical method,and Nestin(nestin)and Sox2 immunofluorescence stain were used to identify the cells. After cells were treated with different dose of EPO,5,10,20 and 40 U/mL,respectively,the optical treatment concentration and time were determined by CCK8 assay. The effect of EPO on the cell count and the expression of Cyclin D1 in NSCs were detected at the treatment time 96 h. Result The NSCs derived from the central canal of adult SD rats spinal cord could stably express protein Nestin and transcription factor Sox2. As the results of CCK8 test,cell counts and real-time quantitative PCR showed the optimal treatment of concentration and time maybe 20 U/mL and 96 h. Conclusions This study shows that EPO can promote the proliferation of NSCs derived from central canal of adult rat spinal cord,and the optimal treatment of concentration and time for proliferation might be 20 U/mL and 96 h.%目的 探讨促红细胞生成素(EPO)对成年大鼠脊髓中央管膜下神经干细胞(NSCs)体外增殖能力的影响,为临床进行成体脊髓中央管膜下NSCs自体移植或异体移植治疗脊髓损伤提供实验室理论依据.方法 通过显微外科方法分离成年大鼠脊髓中央管膜下NSCs行体外培养,利用CCK8检测筛选EPO促进NSCs增殖的适宜处理时间,并检测96 h的EPO对NSCs的细胞计数和Cyclin D1表达的影响.结果 成年SD大鼠脊髓中央管膜下可提取出稳定表达Nestin蛋白和Sox2转录因子的NSCs;CCK8结果示20 U/mL、96 h可能为EPO促进NSCs增殖的适宜浓度和处理时间,细胞计数及荧光定量PCR结果示EPO处理NSCs 96 h后可明显促进细胞增殖.结论 EPO可促进行体外培养的成年大鼠脊髓中央管膜下NSCs的增殖,且20 U/mL和96 h可能为EPO促进NSCs体外增殖的适宜浓度和适宜处理时间.