构建稳定表达胞外信号调节激酶的大鼠骨髓间充质干细胞及其对大鼠脑卒中的神经保护作用

摘要

目的:构建稳定表达细胞外信号调节激酶(ERK1/2)的骨髓间充质干细胞(MSCs)及探讨其对脑卒中的神经保护作用.方法:制备ERK1/2重组病毒和空病毒(对照),并转染大鼠MSCs,分别构建ERK1/2/MSCs和BV/MSCs(对照).用RT-PCR和免疫印迹检测ERK1/2/MSCs的ERK1/2表达水平.制备大鼠脑卒中模型,移植ERK1/2/MSCs、BV/MSCs和生理盐水后1、2周,观察动物的神经行为症状和TTC染色观察脑梗死体积.结果:构建了ERK1/2的重组慢病毒质粒,并转染MSCs,RT-PCR和免疫印迹检测显示ERK1/2/MSCs细胞系过表达ERK1/2.大鼠脑卒中后移植ERK1/2/MSCs、BV/MSCs和生理盐水(对照组),ERK1/2/MSCs和BV/MSCs组的大鼠1、2周时,神经功能的恢复显著好于对照组,而ERK1/2/MSCs比BV/MSCs有更好的效果；ERK1/2/MSCs和BV/MSCs的大鼠脑梗死的体积比对照组显著降低,而ERK1/2/MSCs和BV/MSCs两组之间没有差异；移植后2周,ERK1/2/MSCs组的大鼠移植细胞存活数比BV/MSCs组的多.结论:本实验成功构建了稳定表达ERK1/2的MSCs,其对脑卒中的神经保护效果优于MSCs.%Objective:To construct marrow stroma cells stably overexpressing extracellular singal-regulated kinase 1/2 (ERK1/2/MSCs) and explore whether transplantation of ERK1/2/MSCs provides a better therapeutic for rat stroke.Methods:After the resultant ERK1/2 recombinant viruses were amplificated in colibacillus and were packaged in 293FT,ERK1/2 and blank virus solution was got.After MSCs were transfected by ERK1/2 and blank virus solutions (ERK1/2/MSCs,BV/ MSCs),RT-PCR and Western blotting were used to detect the ERK1/2 expression.The ischemic stroke model was established by occluding the middle cerebral artery.At 3 days after reperfusion,ERK1/2/MSCs,BV/MSCs and physiological saline (control) were infused into ipsilateral ventricle for 1 and 2 weeks.Neural behavior and infarct volume was detected.Results:The ERK1/2 recombinant virus was successfully constructed,and then MSCs were transfected by ERK1/2 recombinant virus and blank virus (ERK1/2/MSCs,BV/MSCs).RT-PCR and Western blotting showed that ERK1/2/MSCs over-expressed ERK1/2 compared to BV/MSCs and native MSCs.At 1 and 2 weeks after transplantation,ERK1/2/MSCs and BV/ MSCs groups significantly improved neural behavioral deficits compared to the rats treated with physiological saline (control),and ERK1/2/MSCs showed more effective effects than BV/MSCs.ERK1/2/MSCs and BV/MSCs significantly decreased infarct volume compared to the control rats at the given time,while ERK1/2/MSCs did not show significant difference as compared to BV/MSCs.The survival of grafted cells in ERK1/2 group was significantly increased as compared to BV/MSCs group at 2 weeks after reperfusion.Conclusion:The present study successfully constitutes MSCs stably over-expressing ERK1/2,which provide better neuroprotection than BV/MSCs against stroke.