| ObjectiveFrom the perspective of "Quyu Shengxin" in TCM,based on the biological characteristics of neural stem cells,we explored the injury role and mechanism of NSCs in vivo and transplantation after ischemic injury.Methods1 Hippocampal NSCs were isolated from the brain of 15-17 days SD pregnant rat embryos in vitro.Immunofluorescence was used to identify NSCs.Nestin was labeled as the specific marker of NSCs.BrdU was labeled as proliferation marker of NSCs.Tuj-1 was labeled as neural progenitor cell specific marker and vimentin was labeled as astrocytes progenitor cell specific markers.2 Male SD rats were randomly divided into 5 groups:sham group,model group,ginsenoside treatment group,NSCs transplantation group(NSCs group)and ginsenoside treatment combined with neural stem cell transplantation in the treatment group(ginsenoside+NSCs group).Focal cerebral ischemia model was established.NSCs transplantation group was treated with NSCs transplantation in paracele after ischemic.Neurological function and cerebral infarction after ischemia 7 d were evaluated.3 With the same experimental group above,blood and brain tissue were collected 7d after ischemia for preparation of serum and brain tissue homogenate.Concentrations of inflammatory related cytolines were determined with ELISA kit in serum and tissue homogenates,including IL-1 beta,IL-6,TNF-alpha,IL-8,MCP-1,CAM-1,IL-4,IL-10.This study was to explore provide the treatment mechanism of neural stem cell transplantation on ischemic stroke,and the mechanism of ginsenoside against stroke.4 NSCs were transfected by lentiviral vector carrying GFP gene and the positive rate of GPF was observed to determine the transfection rate of NSCs.The optimal transfection index MOI value and the optimal transfection time of lentivirus transfected NSCs were screened.The experiment was divided into 4 groups:Sham group,Model group,NSCs group and LV-NSCs group.NSCs and lentivirus transfected NSCs were injected in ischemic lateral ventricle after cerebral ischemia.Serum and brain homogenate was used to determine its inflammatory related factors with ELISA kit.It is to explore the possible role of HIF-1 alpha in the repair of brain injury in lateral ventricle NSCs and its correlation with inflammatory.5 The CD1 mice were randomly divided into 4 groups:Sham group,HI group,2ME2 treatment group and DMOG group.The right carotid artery was permanently ligated and 30min hypoxic treatment was performed to establish a hypoxic-ischemic model.2ME2 was palyed as a HIF-1a protein inhibitor.DMOG was paly ed as active inhibitors of PHD.Contents of HIF-la and VEGF in brain tissues of neonatal rats after hypoxic ischemia were measured using western bloting.6 Nestin,the specific markers of NSCs,was used to identify NSCs,and PCNA was to mark the NSCs proliferation in the SVZ region of the cerebral ventricle after HI.Based on the groups above,double-labelled immunofluorescence method to detect the NSCs proliferation in the SVZ of the lateral ventricle after hypoxia ischemia,and explore the effects of HIF-la and VEGF protein levels on the proliferation of SVZ NSCs.Based on the groups above,western blotting was used to detect the expression of Caspase 3 in the brain tissue after HI.The effects of DMOG and 2ME2 on the apoptosis of cerebral cortex in neonatal rats and the intervention effect of HIF-1 alpha on neurons were further explored.Results1 Primary culture and identification of NSCs,astrocytes and brain microvascular endothelial cellsNSCs,astrocytes and brain microvascular endothelial cells were isolated and cultured in vitro successfully,and the purity was over 95%.2 Improvement of nerve function,brain edema and infarction in rats after 7d cerebral ischemia(1)The nerve behavior score of model group was significantly higher than that of Sham group,indicating that the model was successful.Ginsenoside treatment group and NSCs transplantation group could significantly improve the nerve injury(p<0.05)at 7 days after focal cerebral ischemia.(2)There were obvious ischemic regions in model group.Ginsenoside and transplanted neural stem cells could significantly reduce the area of cerebral ischemia and significantly reduce the infarct volume(p<0.01).(3)Compare with sham group,the rate of brain edema in model group was significantly high after 7 d cerebral ischemia(p<0.05),while compared with model group,ginsenosides and transplanted NSCs could significantly improve the brain edema(p<0.05).3 Determination of inflammatory cytokines in serum and tissue homogenate by ELISA method(1)the level of inflammatory factors in the homogenate of brain tissueThe inflammatory cytokines(IL-1?,IL-6,TNF-a)in the brain homogenate of model group rats were significantly increased(p<0.05)after 7d cerebral ischemia.The level of IL-1?in the lateral ventricle NSCs transplantation group decreased,but there was no significant difference(p>0.05).The ginsenoside treatment group and the NSCs transplantation and ginsenoside treatment group had significantly decreased the level of IL-1?(p<0.05).The level of IL-6 in each group decreased,but there was no significant difference(p>0.05).TNF-a level in the NSCs transplantation group,ginsenoside treatment group,and NSCs transplantation and ginsenoside treatment group were significantly decreased(p<0.05).(2)the level of anti-inflammatory cytokines in the homogenate of brain tissueThe anti inflammatory cytokines(IL-4,IL-10)in the homogenate of brain tissue of model rats were significantly improved(p<0.05)by 7d cerebral ischemia.The level of IL-4 significantly increased in each treatment group(p<0.05).The level of IL-10 in NSCs transplantation group,ginsenoside treatment group,and NSCs transplantation and ginsenoside treatment group increased significantly(p<0.01).(3)the level of adhesion factor in rat serumThe levels of chemokine(ICAM-1)in serum of model group rats were significantly improved(p<0.05)after 7d cerebral ischemia.The level of chemokine ICAM-1 decreased in all the treatment group significantly(p<0.05).(4)the level of chemokines in the homogenate and serum of the brainMCP-1 in brain tissue homogenate and CXCL-1 in serum of model group increased significantly after 7d cerebral ischemia(p<0.05).MCP-1 level in NSCs transplantation group and ginsenoside treatment group decreased,but there was no significant difference(p>0.05).The level of IL-1 beta in the NSCs transplantation and ginsenoside treatment group was significantly decreased(p<0.05).The level of chemokine CXCL-1 in NSCs transplantation group,ginsenoside treatment group,lateral ventricle NSCs transplantation and ginsenoside treatment group were decreased significantly(p<0.05).4 The possible role of HIF-1 alpha in the repairment of brain injury by transplantation of NSCs in the lateral ventricle(1)the best MOI value of NSCs transfected by lentivirus was 10,and the best transfection time was 144 h after transfection.(2)neurologic score:NSCs transplantation could improve the nerve injury symptoms significantly 7 days after focal cerebral ischemia(p<0.01).Compared with model group,LV-NSCs transplantation could improve nerve function,but the difference was not statistically significant(p>0.05).(3)the effect of NSCs transplantation on inflammatory related factors in brain homogenate and serum? The levels of inflammatory cytokines IL-1?,IL-6 and TNF-? were decreased in the LV-NSCs transplantation group,but the difference was not statistically significant(p>0.05).? The level of IL-10 significantly increased in the LV-NSCs transplantation group(p<0.05).? The level of MCP-1 in the LV-NSCs transplantation group decreased,but there was no significant difference(p>0.05).The level of CXCL-1 in the LV-NSCs transplantation group decreased significantly(p<0.05).? The level of chemokine ICAM-1 decreased in the LV-NSCs transplantation group significantly(p<0.05).5 Determination of HIF-la and VEGF in the cortex of newborn mice with Western BlotUnder the condition of ischemic and anoxia,the expression of HIF-la and VEGF protein was up-regulated.DMOG significantly up-regulated the expression of HIF-1? and VEGF protein in the cerebral cortex of neonatal rats after hypoxia ischemia,while 2ME2 down-regulated the expression of HIF-1 alpha and VEGF protein.6 Effect of DMOG and 2ME2 on cerebral cortex apoptosis in neonatal mice after.ischemia and hypoxiaUnder normal condition,caspase 3 was low expressed.At the early stage of ischemia,the level f caspase 3 increased,while ischemia and hypoxia for 24 h,the expression of caspase 3 increased significantly.the overexpression of HIF-1? in cerebral cortex could induce apoptosis.7.Effect of HIF-1 alpha and VEGF protein on the proliferation of NSCs in SVZ of neonatal mice after ischemia and hypoxiaThe number of HIF-1?/Nestin positive and VEGF/Nestin positive cells increased significantly in the SVZ area of the lateral ventricle after hypoxia ischemia.2ME2 effectively reduced HIF-la/Nestin and VEGF/Nestin labelled positive cells,and DMOG significantly increased the HIF-1 a/Nestin and VEGF/Nestin labeled positive cells after hypoxia ischemia.8 The effect of HIF-la and VEGF protein levels on the differentiation of NSCs in the subependymal subventricular zone of newborn mice after hypoxia ischemiaUnder normal condition,the expression of DCX/PCNA and NeuN/PCNA was low,and the expression of DCX/PCNA increased at the early stage of ischemia and hypoxia.After 2ME2 intervention,the expression level of DCX/PCNA decreased.The DCX/PCNA positive cells in DMOG intervention group increased significantly.The expression of NeuN/PCN increased significantly after ischemia and hypoxia for 2 W.The expression of NeuN/PCN decreased after 2ME2 intervention and increased significantly in DMOG intervention group.9 Effect of DMOG and 2ME2 on cerebral cortex apoptosis in neonatal mice after ischemia and hypoxiaUnder normal condition,caspase 3 was low expressed.At the early stage of ischemia,the level f caspase 3 increased,while ischemia and hypoxia for 24 h,the expression of caspase 3 increased significantly.the overexpression of HIF-la in cerebral cortex could induce apoptosis.ConclusionFrom the two perspectives of allogeneic neural stem cell transplantation and in vivo neurogenesis,we studied the role and mechanism of neural stem cells in repairing injury after the occurrence of ischemic injury.Based on the inflammatory system,it has been confirmed that allograft neural stem cells can play an important role in the treatment of cerebral ischemia by regulating the levels of inflammatory related factors.Ginsenosides plays a synergistic role during this process.With immunofluorescence double labeling and western blotting method,we confirmed that the high expression of HIF-1 and VEGF protein can promote the proliferation and differentiation of NSCs and neurogenesis after ischemia and hypoxia. |
PDF Full Text Request