氯马斯汀对急性辐射损伤后OPCs细胞分化和髓鞘形成的影响

Clemastine promotes oligodendrocyte precursor cell differentiation and myelination after acute radiation injury

构建大鼠辐射损伤模型,蛋白质免疫印迹(Western blot,WB)检测髓鞘碱性蛋白(Myelin basic protein,MBP)表达,从SD大鼠中分离原代少突胶质前体细胞(Oligodendrocyte precursor cells,OPCs)和神经元细胞,并通过免疫荧光检神经丝蛋白(Neurofilament protein,NF)和OPCs标志蛋白硫酸软骨素蛋白多糖(Chondroitinsulphate peoteoglycan,CSPGs/NG 2)的表达以鉴定分离的原代细胞。CCK 8(Cell counting kit-8)实验用于检测不同浓度的氯马斯汀对OPCs细胞增殖的影响。通过慢病毒构建5组毒蕈碱型受体(Muscarinic cholinergic receptor,CHMR)干扰表达载体,并通过实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)和WB检测转染效率。此外,为探究氯马斯汀对OPCs细胞分化和髓鞘形成的影响,通过WB和免疫荧光检测MBP和OLs表达少突胶质细胞系转录因子2(Oligodendrocyte lineage transcription factor 2,Olig 2)的表达。结果表明,氯马斯汀能够促进大鼠MBP形成,抑制OPCs细胞增殖,且具有浓度效率。与对照组相比,CHRM 1-CHRM 5干扰均显著降低了mRNA和蛋白的表达水平,其中CHRM 2干扰效率最佳。与对照组相比,CHRM 2干扰显著促进了MBP的表达,而氯马斯汀处理后进一步促进了MBP的表达;此外,免疫双荧光结果与上述推论一致,CHRM 2干扰显著促进了MBP和OLIG 2的表达,而氯马斯汀处理进一步的促进了MBP和OLIG 2的表达。氯马斯汀促进OPCs细胞分化和髓鞘形成。

A rat radiation injury model was constructed,in which myelin basic protein(MBP)expression was detected via western blot.Primary oligodendrocyte precursor cells(OPCs)and neuronal cells were isolated from Sprague-Dawley rats,and expression of neurofilament protein and chondroitin sulphate proteoglycan was detected via immunofluorescence to identify isolated primary cells.Cell counting kit-8(CCK 8)experiments were used to assess the effects of different concentrations of clemastine on OPC proliferation.Five groups of lentiviral shRNA vector targeting muscarinic receptor were constructed,and their transfection efficiencies were quantified using RT qPCR and western blot.To explore the effect of clemastine on OPC differentiation and myelination,MBP and OLIG 2 expression were detected via immunofluorescence and western blot.Clemastine was found to promote MBP formation in rats and inhibit OPC proliferation in a concentration-dependent manner.Compared with the control group,CHRM 1,CHRM 2,CHRM 3,CHRM 4,and CHRM 5 shRNA expression significantly decreased mRNA and protein expression levels(p<0.01).Among these,CHRM 2 displayed the best interference efficiency.CHRM 2 interference(M 2 R)significantly increased MBP and OLIG 2 expression in a manner that was further enhanced by clemastine treatment(p<0.05).Clemastine enhances the antimuscarinic effect,promotes the differentiation of OPCs into OLs,and plays an important role in myelination by up-regulating the expression of MBP.