Research On MSCs Transfected With ECSOD Gene For Treatment Of Ischemic Stroke In Rats

Part I Construction of ECSOD gene expression vectors and ECSOD gene in vitro transfection bone marrow mesenchymal stem cellObjective:After ECSOD gene expression vectors were constructed, extracellular superoxide dismutasc(ECSOD)gene, packaged by lentivirus, transfect bone marrow mesenchymal stem cell(MSCs) of male Sprague-Dawley(SD) rats, to observe the efficiency of ECSOD gene transfection and the effect of ECSOD gene transfection on the cell vitality; ECSOD gene expression,in the MSCs, were detected after transfetion.Methods:Extract total RNA from MSCs in use of gene engineering technology, ECSOD gene fragment were got after PCR and restriction enzyme digestion, connect ECSOD gene fragment to GV230-EGFP vectors to construct GV230-EGFP-ECSOD expression vectors. Positive clones of ECSOD gene were identified by PCR and then sequenced. GV230-EGFP-ECSOD, packaged by lentivirus, transfect MSCs of male Sprague-Dawley (SD) rats, number of positive cells were detected under fluorescence microscopy, mRNA in the transfection MSCs were analysised by quatitative PCR, effect on proliferation and cell vitality after transfection ECSOD genetic were determined by MTT and EDU, ECSOD protein expression at the cellular level were detected by Western Blot.Results:RNA extraction product connect the target gene to the eukaryotic expression vector CV230-EGFP after PCR amplification and restriction enzyme. The size of target gene which got by colony PCR was the same as the positive control group, by sequencing analysis, colony PCR positive transformation sequences was completely matched the target gene sequences; GV230-EGFP-ECSOD packaged by lentivirus transfect MSCs, positive cells were detected under fluorescence Microsc-opy after48h, the transfection rate was (75±3.6)%, using real-time quantitative PCR to calculate mRNA, mRNA content after transfection is significantly higher than the control group. ECSOD transfection had no effect on the cell vitality. Western Blot test proved that ECSOD protein positive expression in the MSCs after ECSOD transfection.Conclution:GV230-EGFP-ECSOD eukaryotic expression vector was constructed successfully, there was functioning ECSOD gene protein expression after it transfection to MSCs by lentivirus packaging transfection technology, to establish the experiment basis for further research on genetically modified MSCs to treat cerebral infarction animal model. Part II Effect of MSCs transplantation with ECSOD genetic modification on cerebral infarction in ratsObjective:To observe the nerve protective effect of acute cerebral infarction in rats by transplant MSCs transfected with ECSOD gene.Methods:80male adult Sprague DaWley (SD) rats were randomly divided into6h group (n=40) and12h group (n=40),6h,12h group were divided into four subgroups:the Model group (n=10), the PBS group (n=10), MSCs group (n=10), ECSOD-MSCs group (n=10); the left middle cerebral artery ischemia model was make according to the modified Zea Longa line plug method, then ischemia/reperfusion in6h or12h, the model group was not disposed, the other three groups of cerebral ischemia Model injected10mu LPBS liquid, MSCs, ECSOD-MSCs, respectively, by transcranial three-dimensional positioning. Assessment of prognosis using mNSS evaluation method in1d,3d,7d,2w,4w after Ischemia/reperfusion, MRI determination of infarction volume changes in rats in1day,28day respectively after ischemia/reperfusion. Took the brain tissue out after4weeks, to determine the expression of BAX, Bcl-2and ECSOD around brain infarction by immunohistochemical method.Results:2weeks,4weeks after cerebral infarction transplantation, Modified Neurological Severity Score (mNSS) evaluation of the group ECSOD-MSCs and group MSCS were obviously higher than that of the group PBS and group model, the difference was statistically significant (P <0.05); compared with group MSCs, mNSS evaluation of group ECSOD-MSCs was lower. Cerebral infarction volume of group ECSOD and group MSCs were obviously higher than that of the group PBS and group model, the difference was statistically significant (P<0.05); The ECSOD protein expression of group ECSOD-MSCs was higher than group MSCs, group PBS and group model, the difference was statistically significant (P<0.05) number of Bax positive cells of group ECSOD-MSCs and group MSCs were lower than that of the group PBS and group model(P<0.05), group ECSOD-MSCs also lower than group MSCs; number of Bcl-2positive cells of group ECSOD-MSCs and group MSCSs were higher than that of the group PBS and group model(P <0.05), group ECSOD-MSCs also higher than group MSCs.Conclusion:ECSOD gene transfection MSCs can prevent from cerebral ischemia reperfusion injury, improve neurologic deficits in rats and save the brink of brain infarction. This paper contains23Figures,20tables and38Reference.