Construction Of Lentiviral Vector Targeting Neuritin Gene By RNA Interference And The Investigation Of Neuritin Gene Function

Objective:1. To construct the neuritin shRNA silencing vector and observe its neuritin silencing effect. To establish in vitro high glucose induced Schwann cells apoptosis model.2. To observe the effect of neuritin gene deficiency on survival of Schwann cells and investigate the relationship between neuritin gene expression and survival of Schwann cells.3. To determine if the IGF-1can protect Schwann cells from apoptosis induced by high glucose via neuritin expression Methods:1. We designed and synthesized four RNA interference targeting neuritin gene sequences cloned into pGCL-GFP vector. The vector, pHelper1.0plasmid and pHelper2.0plasmid were cotransfected into293T cells, packing lentiviral vector particles to293T cell GFP expression level of the determination of virus titers.2. Schwann cells were cultured and purified in vitro and stained with antibody against S-100by immunocytochemistry.3. We used the lentiviral vector to transfect the Schwann cells in different groups, which were divided by the multiplicity of infection, the transfection reagent and the transfection conditions. Then, we observed the GFP expression of Schwann cells under the fluorescence microscope at different time to obtain the transfection efficiency by calculating the rate of cells expressing green fluorescence protein. We detected the changes of neuritin expression in Schwann cells by RT-qPCR and western blot, and the effect of silencing on the apoptosis of Schwann cell by Annetin V-PE/7-ADD.4. After controlling for the hypertonic effect, we observed the effect of high glucose on Schwann cell viability and expression of neuritin. Cells were used for detecting apoptosis rate with flow cytometry assay in different groups:normal glucose group (5.6mM GS), high glucose group I (125mM GS), high glucose group II (150mM GS). And the three groups were treated for the different length of time:Oh,12h,24h,30h,36h,48h, respectively.5. We observed the apoptosis of neuritin down-regulated Schwann cells treated with100ng/mL IGF-1in normal glucose for36h. We also examined the preventive effect of various levels of IGF-1on the Schwann cells cultured in high glucose for36h: IGF-1treated I group (125mM GS+0.1ng/mL IGF-1), IGF-1treated Ⅱ group (125mM GS+1ng/mL IGF-1), IGF-1treatedⅢ group (125mM GS+10ng/mL IGF-1), IGF-1treated Ⅳ group (125mM GS+100ng/mL IGF-1), in contrast to non-IGF-1treatment control group(125mM GS).Results:1. The sequencing results showed shRNA neuritin specific lentiviral interference vector was constructed correctly with the virus titers were higher than1x108Tu/mL.2. Schwann cells were isolated, cultured and identified. Schwann cells of90%purity were generated and passed on from generation to generation.3. Multiplicity of infection affected the transfection efficiency in varying degrees but transfection reagent and the transfection conditions, and the lentivirus-mediated gene transfer of green fluorescent protein into the cells remained stable. We detected the expression level of neuritin by RT-QPCR and Western blot. Compared with the normal control group and nonsense vector control group, the expression of neuritin was significantly decreased in the Schwann cells transfected by the lentiviral vector particles targeting neuritin(P<0.05). We found the apoptosis rate of neuritin RNAi silenced Schwann cells significantly high. The results show that the silenced neuritin induced more Schwann cells apoptosis(P<0.05).4. Compared with normal glucose group, the apoptosis rate of Schwann cells cultured in highly hypertonic group was not statistically significant(P>0.05). High glucose group had the increased apoptosis rate than normal and highly hypertonic groups (P<0.05). We observed the effect of different high glucose on Schwann cell viability at different time. The cell viability in the125mM GS (glucose) group decreased significantly after48h or36h compared with the normal glucose group(P<0.05), and more significantly in the150mM GS group (P<0.05), the difference was also shown between treatments for48h and36h.5. Compared with control group, the apoptosis rate of neuritin RNAi silenced Schwann cells was not decreased by IGF-1treatment(P>0.05). However, the apoptosis rate in IGF-1+high glucose group was lower than non-IGF-1treatment group (P<0.05).Conclusion:1. Lentiviral vectors targeting neuritin gene by RNA interference were constructed successfully. The recombinant neuritin lentivirus showed significantly inhibiting effects on neuritin expression in Schwann cells. Neuritin was connected to SCs survival2. We obtained a large number of high-purified normal Schwann cells to satisfy the need of the experiments.3. After controlling for the hypertonic effect, the viability of Schwann cells was inhibited significantly under high glucose.4. IGF-1did not prevent apoptosis of neuritin RNAi silenced Schwann cells. However, it prevented, to some extent, apoptosis of the Schwann cell in the high glucose conditions. Therefore, neuritin may mediate the effects of IGF-1in reducing apoptosis of Schwann cell exposed to high glucose in this study.