人胚胎干细胞源神经球条件培养液对大鼠施万细胞增殖和迁移的影响

Effect of human embryonic stem cell-derived neurospheres conditioned medium on the proliferation and migration of rat Schwann cells

目的:探讨人胚胎干细胞源神经球条件培养液(human embryonic stem cell-derived neurospheres conditioned medium,h ESCN-CM)对施万细胞增殖和迁移的影响及其潜在的机制。方法:取3~5 d SD大鼠双侧坐骨神经,分离纯化施万细胞,用免疫荧光染色法进行鉴定。分别用0%、50%和100%h ESCN-CM刺激施万细胞,CCK-8实验检测细胞增殖,划痕实验检测细胞迁移,实时荧光定量PCR检测Nt3、Vegf、Ngf、Bfgf、Igf-1等mRNA的表达。结果:免疫荧光染色结果显示,施万细胞S100特异性表达可达99%。CCK-8实验显示,与0%h ESCN-CM相比,50%h ESCN-CM刺激后施万细胞增殖明显增强,100%h ESCN-CM刺激后施万细胞增殖明显降低(P均<0.05)。划痕实验显示,刺激6 h,与0%h ESCN-CM刺激后施万细胞相比,50%和100%h ESCN-CM刺激后施万细胞迁移明显增强(P<0.05);刺激12 h,50%h ESCN-CM刺激后施万细胞迁移明显增强(P<0.01),100%h ESCN-CM差异无统计学意义。荧光定量PCR结果显示,与0%h ESCN-CM相比,50%h ESCN-CM刺激后施万细胞Nt3、Vegf、Ngf、Bfgf等mRNA表达明显增加(P<0.05)。结论:h ESCN-CM可能通过上调Nt3、Vegf、Ngf、Bfgf等mRNA表达促进施万细胞增殖和迁移。

Objective:To investigate the effect of human embryonic stem cell-derived neurospheres conditioned medium(hESCN-CM)on the proliferation and migration of Schwann cells and its potential mechanisms.Methods:The bilateral sciatic nerves of 3-5 days old SD rats were isolated for Schwann cells,which were identified by immunofluorescence.Schwann cells were stimulated with 0%,50%and100%hESCN-CM and the effect of h ESCN-CM on the proliferation of Schwann cells was measured by CCK-8 assay.The migration of Schwann cells was measured by would healing assay.The mRNA expression of Nt3,Vegf,Ngf,Bfgf and Igf-1 was determined by quantitative real-time PCR(qRT-PCR).Results:Immunofluorescence staining showed that specific marker S100 expression was detected in 99%of all cells.The CCK-8 assay showed that 50%h ESCN-CM promoted the proliferation of Schwann cells and inhibited proliferation at 100%concentration.Wound healing assay suggested that 50%hESCN-CM and 100%h ESCN-CM promoted migration in condition of culturing for 6 h and 50%hESCN-CM promoted migration at 12 h;while showed no significance between 0%h ESCN-CM and 100%hESCN-CM at 12 hours.qRT-PCR demonstrated that compared with 0%hESCN-CM,50%hESCN-CM showed higher mRNA expression levels of Nt3,Vegf,Ngf,Bfgf.Conclusion:hESCN-CM could promote the proliferation and migration of rat Schwann cells,which may be attributed to up-regulation of mRNA expression of Nt3,Vegf,Ngf and Bfgf.