Construction Of Controllable Neurod2 Gene Vector And Study On Directional Differentiation Of MSCs

Some results indicate that BMSCs had beneficial effects on locomotor improvement as well as on axonal regeneration in SCI rats.This study main point is constructing the eukaryotic expression vector hHSP70-ND2,and investigate the effects of different induction time on differentiation of mouse mesenchymal stem cells(MSCs)into neuron like cells.1.The Neurod2 gene was cloned,and analysed the gene sequence,different structures,functional domains,cell localization,homology with other species and constructed pIRES2-AcGFP1-ND2 with pIRES2-AcGFP1 to transfect mouse bone marrow mesenchymal stem cells(MSCs)and divided into control group(pIRES2-AcGFP1 transfected group)and experimental group(pIRES2-AcGFP1-ND2 transfected group),observe the expression of green fluorescent protein AcGFP1 and the expression level of Neurod2 with the help of real-time PCR,immunofluorescence and western blotting.2.The CMV promoter was replaced by hHsp70 under the help of enzyme Nde I and Bgl II,and verified the biological activity by thermal induction.The cloning gene Neurod2 would be recombined into hHsp70-AcGFP1.Then transfected the purified plasmid into mouse MSCs.These cells belong to different group including control group(induced by 0 h)and experimental group(0.5 h,1 h,2 h,3 h).The expression of Ac GFP1 green fluorescent protein was observed by fluorescence microscope.3.Analyzing changes of cell morphology and expression of Neurod2,myelin basic protein(MBP),microtubule-associated protein-2(MAP2),myelin protein zero(MPZ),epidermal growth factor(EGF),glial fibrillary acidic protein(GFAP),neurofilament(NF),synaptophysin(Syn)and 200 kD neurofilament heavy(200KD NFH)in eukaryotic cells were identified by real-time PCR,western blotting and immunofluorescence staining.The result shows: 1.The CDS region of Neurod2 gene in rats is 1 149 bp,compare with Norway rats in same area,finding these sites including 672,703,747,770 and 794 have cytosine mutation,and this area would formed protein containing 382 amino acids,belonging to the bHLH family.The amino acid sequence have high homology with mouse(99%),rhesus monkey(98%)and human(97.7%).The second structure was mainly about ?-helix and random coil,spatial structure presents the " helix loop helix l(HLH)structure,and mainly distributed in the nucleus;After transfection,real-time PCR showed the expression of Neurod2 gene in transfected group was significantly higher than that in control group(P<0.05).Immunofluorescence and western blotting find the same result,expression of NeuroD2 protein in the transfected group as well as significantly difference than control group(P<0.05).2.After double digestion,the length of hHsp70 promoter was presented,the expression of AcGFP1 was 39% after transfection,and connected with Neurod2,the expression of AcGFP1 from high to low were 0.5 h,1 h,control group,2 h,3 h.Real-time PCR analysis that the expression of Neurod2 in 0.5 h showed significant higher than control group,2 h and 3 h(P<0.05),western blotting analysis showed that the expression of NeuroD2 protein in 2 h had significant lower than 0.5 h induction(P<0.05).3.Immunofluorescence staining showed that the expression of MBP,GFAP and NF protein was up-regulated,and morphology of cells changed significantly compared with before,even more cells with long spindle shaped,real-time PCR and western blotting analysis showed that after 0.5 h and 1 h,the expression of NF was the highest,and compared with the other induction group,the difference was significant(P<0.01);After 1 h,he expression of MBP gene was significantly different from the other groups(P<0.05),the protein in 2 h and 3 h was significantly higher(P<0.01);GFAP gene was significantly different from 2 h and 3 h to 0.5 h and 1 h(P<0.05),and the expression of protein was significantly lower than the other group after induction of 2 h(P<0.01);So the above results showed that this study have successfully constructed the recombinant vector hHsp70-ND2,and under the 42? could regulated the mouse MSCs differentiate into neuron by heat shock,and at 0.5 h and 1 h have the highest differentiation efficiency.