Effects Of A Lentiviral Vector With SiRNA To Silence Glol Gene Of Rat Bilateral Hippocampus On Spatial Learning And Memory Ability

Objective:Glyoxalase 1(Glo1)is an important part of the glyxalase system, which is present in the cytosol of all cells.The glyoxalase system catalyses the conversion of reactive,acyclicα-oxoaldehydes into the correspondingα-hydroxyacids. Physiological function of Glo1 is to detoxify dicarbonyl metabolites,mostly methylglyoxal into D-lactate in coordination with glyoxalase2.The level of Glo1 expression and activity decreased in brain may be important to normal aging of the nervous system such as Alzheimer's disease.Glo1 also linkes to other neurological disorders.Virus vectors provide attractive gene delivery vehicles currently.Lentiviral vector(LV)genetic systems and adenovirus vectors genetic systems are advanced in these virus vectors.Lentiviral vectors can deliver and express siRNA(Short interference RNA)in a wide variety of dividing and nondividing cell,including terminally differentiated neurons.We packaged siGlo1 recombinant lentiviral vector in 293T cells(Human embryonic kidney 293 cells derived cell lines)by three-plasmid system,and it can functionally silence Glo1 gene of rat bilateral hippocampus.Morris watermaze is an.important tool of evaluating hippocampal-dependent spatial learning and memory ability.In the study,the rats' reference memory ability were measured by swimming rats to search the submerged platform.The main purpose of this paper is to study the effect of siGlo1 on spatial learning and memory ability,and to provide a basis for further study of the lentiviral vectors with siRNA or overexpression RNA transfecting definite part of animal in vivo.The study can also provide a new animal model for electrophysiological patch clamp research,and provide experiment reference for the appling gene therapy.Method:1.Packaging LVs.Vector plasmids(siGlo1 and sihp53)and packaging plasmids (pMDL,pMD.G and pREV)were transformed into competent E.coli.After extracting and purifying plasmids,we measured the density of plasmids.293T cells were co-transformed by the three-plasmid system.The LVs in cell culture medium were collected,concentrated and quantified. 2.Infection with LVs in vivo.27 rats were random divided into three groups: blank,siGlo1 and sihp53.After fixing the animal on a stereotaxic frame,LVs were injected into bilateral hippocampus to silence Glo1.And the same treating measure was taken in the sihp53 group.A week later,these two groups were taken to Morris watermaze test.3.Morris watermaze test.The test includes place navigation test and spatial probe test.In the place navigation test,the escape latencies of rats were compared.In the spatial probe test,we compared the percentage of the time in each quadrant,the percentage of the time in the second quadrant and the times passing though platform in the three groups(blank,siGlo1 and sihp53).4.The result of infection in vivo.After watermaze task,siGlo1group and sihp53 group rats were perfused and fixated.The brain was cut into slices,then choosed brain slices randomly and DAPI afterstained.The fluorescence of brain slices were observed by laser scaning confocal microscope.Result:1.Density of plasmid DNA:siGlo1(1.2μg/μl),sihp53(0.6μg/μl),pMDL(1.259μg/μl),pMD.G(1.512μg/μl),pREV(0.66μg/μl)。2.The titer of LV:sihp53 LV(2.22×10~8),siGlo1 LV(7.22×10~7)。3.The place navigation test:The ability of memory in rats of three groups increased with the increasing times of Morris watermaze training.ANOVA showed significant differences in three groups(P<0.05).siGlo1 group studied more quickly than others in initial 2 days,but fell behind of other groups in following 3days.4.Spatial probe test:The statistical result showed that the rats of three groups mostly stayed in the sencond quadrant,and siGlo1 group markedly spended less percentage of the time in the second quadrant than other groups(vs.blank P＜0.01, vs.sihp53 P＜0.05).The times of passing though platform of siGlo1 group were less than other groups,but the differences were not remarkable.5.EGFP expressioned very well in SD rats bilateral hippocampus.Itproved that siGlo1 lentiviral vector had high infection rate in vivo.Coclusion:1.Successfully packaged and quantified the LV transfercting in vivo and showed effective expression in rat bilateral hippocampus.2.The silence of Glo1 gene in the rat bilateral hippocampus displayed that rats' spatial learning and memory decreased in the Morris watermaze.But they were eventually able to take the task with more training.