博尔纳病病毒核蛋白调控神经干细胞存活增殖及ERK1/2信号通路的实验研究

The experimental study of borna disease virus nucleoprotein affecting survival,proliferation and ERK1/2 signal pathway of neural stem cells

目的观察博尔纳病病毒核蛋白(Borna disease virus p40,BDV p40)对大鼠海马源性神经干细胞(Neu-ral stem cells,NSCs)增殖、存活、分化及ERK1/2信号通路的影响,揭示BDV引起神经精神疾病的部分发病机制。方法(1)分别用pEGFP-N1-p40及pEGFP-N1质粒转染NSCs,观察转染效率并鉴定BDV p40在NSCs中的表达。(2)实验设置3组:未转染组、pEGFP-N1空转对照组及pEGFP-N1-p40转染组,用CCK-8试剂盒、BrdU摄入实验及免疫组化分别检测细胞存活、增殖及分化为神经元、星型胶质细胞、少突胶质细胞的比例的变化,并经Western Blot检测ERK1/2磷酸化的改变。结果 (1)成功建立表达BDV p40的NSCs模型;PCR结果显示只有pEGFP-N1-p40转染组细胞有BDV p40基因表达。(2)BDV p40抑制NSCs的存活、增殖,但对于转染后贴壁分化14 d时3组细胞分化为神经元、星型胶质细胞、少突胶质细胞的比例未见显著差异。Western Blot结果显示BDV p40下调了磷酸化ERK1/2在蛋白水平的表达。结论 BDV p40抑制NSCs的存活、增殖,但是对NSCs的分化方向没有明显的影响。BDV p40有可能通过下调磷酸化ERK1/2活性对NSCs的存活、增殖起抑制作用。

Objective To observe the variation of survival, proliferation, differentiation and ERK1/2 signal cascade of neural stem cells （NSCs）, derived from hippocampus of new-boru Sprague-Dawley （SD） rat after being transfected with the eukaryotic expression plasmid containing Borua Disease Virus p40 （BDV p40） gene and pEGFP-NI-p40, and investigate part of pathogenesis of BDV inducing neurological and psychiatric disorders. Method （ 1 ） pEGFP-Nl-p40 and pEGFP-N1 plasmids were transfected into NSCs respectively. Transfection efficiency was observed and BDV p40 expression in NSCs was identified. （2） Three groups were set up： non-transfection group, pEGFP-N1 transfection group and pEGFP-NI-p40 transfection group. Survival of NSCs was detected by CCK-8 kit, proliferation by BrdU uptaking experiment, while the ratios of differentiation to neuro, astrocyte and oligodendrocyte by immunohistochemical staining. Westemblot was used to detect the activation of ERK1/ 2 of NSCs. Result 1. NSCs expressing BDV p40 were formed. It was indicated that BDV p40 genetic fragment was transcribed only in NSCs of pEGFP-NI-p40 traiasfection group by PCR result. 2. BDV p40 restrained the survival and proliferation of NSCs. The sigificant difference of ratios of differentiation to neuro, astrocyte and oligodendrocyte in the 14th day after adherent differentiation wasn＇t indicated. BDV p40 down-regulated ERK1/2 protein expression of rats NSCs. Conclusion BDV p40 could restrain the survival and proliferation of NSCs, but have no significant effect on the direction of differentiation. BDV p40 could down-regulate ERK1/2 protein expression of NSCs. It would be possible that BDV p40 restrained the survival and proliferation of NSCs by down-regulating ERK1/2 activation of rats NSCs.