Ozanimod(RPC1063)在少突胶质前体细胞分化中的作用和机制

Role and Mechanism of Ozanimod(RPC1063)in Oligodendrocyte Precursor Cell Differentiation

目的:研究Ozanimod(RPC1063)在少突胶质前体细胞(oligodendrocyte precursor cell,OPC)分化中的作用,并初步探讨其分子机制。方法:利用免疫吸附法直接分离OPC诱导培养,使用免疫荧光染色、实时荧光定量PCR(quantitative real time-PCR,qRT-PCR)对细胞进行鉴定。qRTPCR法检测大脑皮层发育、OPC分化过程中的S1pr家族基因mRNA水平变化。OPC经不同浓度RPC1063处理后,使用MTT法、ATP细胞活性检测法、免疫荧光染色、qRT-PCR和Western blot检测RPC1063对OPC分化细胞数目、mRNA或蛋白水平的影响。结果:利用免疫吸附法可获得高纯度的OPC;而MTT、ATP检测结果显示在0、0. 1、1、5、50、100nmol/L浓度下,RPC1063对细胞活性无明显影响。进一步研究发现,RPC1063处理OPC后,O4、MBP阳性细胞形态舒展,MBP蛋白表达量增加,OPC分化相关基因Mbp、Mag、Sox10、Cnp的mRNA水平增加。机制上,少突胶质细胞系Oli-neu或OPC经RPC1063处理5min后,AKT-mTOR信号通路相关蛋白p-AKT、p-mTOR、p-4EBP1显著增加,OPC经RPC1063处理48h后,p-AKT、p-mTOR蛋白水平增加;而抑制m TOR活性,RPC1063作用减弱。结论:RPC1063通过AKT-mTOR信号通路促进少突胶质前体细胞的分化。

Objective:This study aims to pinpoint the role of Ozanimod(RPC1063)in the differentiation of oligodendrocyte precursor cell(OPC)and investigate the underlying molecular mechanism.Methods:OPC was directly isolated by immune adsorption and indentified through immunofluorescence and quantitative real time-PCR.Firstly,the mRNA level of S1pr family genes was detected by qRT-PCR during the cerebral cortex development and OPC differentiation.Then,viability of OPC treated with different concentrations of RPC1063 was measured by MTT assay and ATP cell viability assay.Moreover,by means of immunofluorescence,qRT-PCR,and Western blot,we analysed the effects of RPC1063 on the differentiation rate of OPC,and the mRNA or protein level of OPC-differentiation associated genes.Results:High purity of OPC could be obtained by the method of immune adsorption from the cortex of mice.There was no significant effect of RPC1063 on cell viability at concentrations of 0,0.1,1,5,50,100nmol/L.Additionally,up-regulation of O4+or MBP+cells,and protein level of MBP and mRNA level of Mbp,Sox10,Cnp,or Plp were observed in OPC treated with RPC1063.Moreover,in RPC1063-treated Oli-neu cells,the protein levels of pAKT,p-mTOR and p-4EBP1 were significantly increased at 5min.Besides,the proteins related to p-AKT-mTOR signaling pathway were also significantly increased in OPC.The promotion of RPC1063 on OPC differentiation was weakened by the inhibition of mTOR pathway.Conclusion:RPC1063 promotes the differentiation of oligodendrocyte precursor cell through activating the AKT-mTOR signaling pathway.