大鼠雪旺细胞系RSC96条件培养液对少突胶质前体细胞的体外增殖作用

Conditioned medium from rat RSC96 cells promotes proliferation of oligodendrocyte progenitor cells in vitro

目的观察大鼠雪旺细胞系RSC96细胞条件培养液(RSC96-CM)对少突胶质前体细胞(OPCs)增殖的影响,探讨其作用机制。方法应用免疫粘附法从胚胎15d的SD大鼠脊髓分离OPCs,应用Brd U掺入实验检测OPCs的增殖,应用RT-PCR技术检测PDGF-AA和bFGF在RSC96细胞的表达,应用ELISA技术检测RSC96-CM中PDGF-AA和bFGF蛋白的浓度,以特异性抑制剂阻断观察PDGF-AA和bFGF在RSC96-CM诱导OPCs增殖中的作用,以特异性抑制剂观察ERK和JNK信号途径在RSC96-CM诱导OPCs增殖中的作用。结果不同浓度的RSC96-CM培养的OPCs,其BrdU+细胞的比例与对照组相比均显著增加(P<0.05),其中,在含50%RSC96-CM的培养液中培养的OPCs的BrdU+细胞的比例达高峰。RT-PCR证实RSC96细胞显著表达PDGF-AA和bFGF的mRNAs,ELISA结果发现,RSC96-CM中PDGF-AA和bFGF的蛋白浓度分别是我们前期报道的B104CM中PDGF-AA和bFGF的蛋白水平的0.87倍和0.92倍。PDGFR的特异性抑制剂AG1295和bFGFR特异性抑制剂PD173074均显著降低RSC96-CM诱导的OPCs增殖;此外,Erk1/2特异性抑制剂U0126和JNK特异性抑制SP600125的预孵也显著降低RSC96-CM诱导的BrdU+OPCs的比例(P<0.01)。结论 RSC96-CM显著诱导OPCs的增殖,其通过RSC96分泌的PDGF-AA和bFGF激活Erk1/2和JNK信号途径而发挥作用;RSC96-CM也可以作为OPCs常规培养的扩增剂。

Objective To investigate the effect of conditioned medium from rat RSC96 cells(RSC96-CM) on the proliferation of oligodendrocyte progenitor cells(OPCs) and explore the underlying mechanism. Methods OPCs isolated from the spinal cords of SD rats of embryonic day 15 using immunopanning were treated with RSC96-CM. The proliferation of OPCs was detected using 5-bromo-2'-deoxyuridine(Brd U) incorporation assay. The m RNA expressions of PDGF-AA and b FGF in RSC96 cells were detected using RT-PCR, and their protein concentrations in RSC96-CM were detected with enzyme-linked immunosorbent assay(ELISA). The effects of PDGF-AA and b FGF in RSC96-CM on OPC proliferation and the roles of ERK and JNK signaling pathways in RSC96-CM-induced OPC proliferation were determined by application of their specific inhibitors. Results The percentage of Brd U + OPCs was significantly increased in response to treatment with RSC96-CM(P<0.05), reaching the peak level when 50% RSC96-CM was added in the cell culture. RSC96 cells expressed a substantial amount of PDGF-AA and b FGF m RNAs, and PDGF-AA and b FGF protein concentrations in RSC96-CM were higher than those in a conditioned medium(B104CM) we used previously by 0.87 and 0.92 folds, respectively. Both the specific inhibitor of PDGFR signal pathway(AG1295) and the specific inhibitor of b FGFR signal pathway(PD173074) significantly attenuated RSC96-CM-induced OPC proliferation. The specific inhibitors of ERK signal pathway(U0126) and JNK signal pathway(SP600125) significantly decreased the percentage of Brd U + cells in RSC96-CM-induced OPCs(P<0.01).Conclusion RSC96-CM can effectively promote OPC proliferation, possibly as a result of PDGF-AA and b FGF secretion by RSC96 cells to activate ERK1/2 and JNK signaling pathways. RSC96-CM can be used as a routine stimulator for promoting OPC proliferation.