The Study On Neural Stem Cells By Lentivirus Mediated UCP2 Lower Expression

Objectives:1.Isolation,cultivation and identify C57 mice primary neural stem cells,providing a cell model to study the mechanism of neural tube defects caused by low expression of UCP2.2.Construct an effective UCP2 RNA interference vector,and establish an efficient method to transfect neural stem cells,to support the study of gene function latter.3.Testing the change of ROS level and cell apoptosis after UCP2 experssion decrease in NSCs,exploring the pathogenesis of NTDs caused by UCP2 lower expression.Methods:1.Neural stem cells extraction,cultivation and identification: Through mechanical method,isolating neural stem cells from 14.5 days fetal rat's brain tissue,Neural stem cells were identified by characteristic protein nestin immunofluorescence staining.DMEM/F12 culture medium containing 10% fetal calf serum was used to induce NSCs to differentiate,neuron,astrocyte and oligodendroglia were identified through characteristic proteins NF-H,GFAP and GALC immunofluorescence staining.2.Construct UCP2 RNA interfere lentiviral vector and virus packing: Targeting UCP2 gene specific sequence were designed to link with pMagic4.1 lentiviral vector(pNL-EGFP/CMV/WPREdU3-sh mUCP2).Then virus packing recombinant lentiviral vectors were harvested from 293 T cells with recombinant lentiviral vector,second generation virus packing plasmid pCD/NL-BH*DDD and membrane protein expression plasmid pLTR-G co-transfection.3.Construct electroporation UCP2 RNA interfere vector: The same as UCP2 RNA interfere lentiviral vector pNL-EGFP/CMV/WPREdU3-sh mUCP2 above.4.Compare lentiviral transfection and electroporation: NSCs were transfected by lentiviral transfection and electroporation,transfection efficiency was compared Byobserving the green fluorescent protein expression of NSCs;The interference maintenance time was compared by green fluorescent intensity of NSCs after transfection 48 h and 72 h.Western Blot and QPCR were used to test NSCs UCP2 expression after lentiviral transfection.5.ROS level detection: Testing NSCs ROS level of blank group,negative control group and UCP2 low expression vector transfection experimental group by DCFH-DA respectively,Comparing ROS level of each group.6.Cells apoptosis detection: Dividing NSCs into three groups: blank group,negative control group and UCP2 low expression vector transfection experimental group.Caspase3 activation fluorescent staining was used to test the rate of caspase3 activation NSCs,Western Blot was used to test caspase3 expression.7.Statistical analysis method: SPSS 17.0 was used to analyze data,and data was showed as ` x±s;Analyzing data by t-test or rank test,P<0.05,then the difference was statistically significant;Graphpad Prism v 5.01 was used to draw maps.Results:1.Primary NSCs were suspended and agglomerate growth,and had a good state.NSCs characteristic protein nestin Immunofluorescence positive staining.After induced differentiation,Neuron,astrocyte and oligodendroglia characteristic protein NF–H,GFAP and GALC Immunofluorescence positive staining.2.After 48 h transfection,lentiviral transfection efficiency was 77.6%±6.6%,and electroporation efficiency was 29.2%±4.8%,lentiviral transfection efficiency was higher,the difference was statistically significant(P<0.01);After 72 h transfection,70.1%±5.8% of lentiviral transfection NSCs express green fluorescence,10.5%±5.1% of electroporation NSCs express green fluorescence,lentiviral transfection NSCs have a longer transfection effect and more brighter green fluorescence.Compared with blank group and negative control group,UCP2 gene lentiviral transfection experimental group had a lower UCP2 expression,the difference was statistically significant(P<0.05).3.UCP2 gene lentiviral transfection experimental group had a higher ROS level than blank group and negative control group,the test value of blank group,negative group and experimental group were 11130±1338,6128±524.2,18560±983.9 respective,the difference was statistically significant(P<0.01).4.Caspase3 activation fluorescent staining result: Compared with blank group and negative control group,experimental group had a higher positive cells rate,The positive cells rate of blank group,negative group and experimental group were 5.7%±1.8%,9.7%±1.3 and 28%±6.1% respective,the difference was statistically significant(P<0.01).Western Blot result: experimental group had a higher caspase 3 level than blank group and negative control group,the difference was statistically significant(P<0.01).Conclusions:1.Successfully isolated,cultured and identified C57 mice primary neural stem cells,providing a reliable cell model.2.Successfully construct UCP2 RNA interfere lentiviral vector,For NSCs,lentiviral transfection had a higher effect and maintained longer time than electroporation,to support follow–up gene function study.3.The NSCs ROS level rising caused by UCP2 low expression may be related to NSCs apoptosis,and too much NSCs apoptosis is related to NTDs.