Transcription Factor POU3F3 High Sugar Schwann Cells And Its Regulation Of Expression Of DLL1

Objective:To establish a primary Schwann cell culture model, and observe the growth pattern in high glucosecircumstance.Area of cells, neurite length,andproliferative activity were observed.Methods:10SPF Sprague-Dawleyrats,1-3days after birth were decapitated. Thenharvest the bilateral sciatic nerve aseptically, divestiture off the epineurium. Then cut the sciatic nerve into pieces, and digest the nerve tissue with low concentration enzyme. With the application of two hours of the differential adhesion method and cytarabine the fibroblast cells can be removed. We use anti-S-100antibody to identify schwann cells.Cells are divided into two groups when subcultured to the third generation. One of the two make use of5.5mmol/L D-glucose, the other is25mmol/L D-glucose to culture the schwann cells. We observed neuritelength,cell size and proliferation of the two group of schwann cells.Results:1, We established primary Schwann cellculture method and high glucose model. With the combination of differential adhesion method,enzymatic digestion and chemical methods, we can get pure schwann cells.2, Cultured in high glucose conditions, the primary Schwann cell body area decreased, neurite length werelonger, proliferative activity increased than cells cultured in low glucose conditions.Conclusion:1, Dual enzyme digestion and differential adhesion and cytarabine methods can got relatively high purity of Schwann cells.2, Cultured in high glucose conditions, the primary Schwann cell body area decreased, neurite length were longer, proliferative activity increased than cells cultured in low glucose conditions. This model can be used to research the molecular mechanism of the effect of schwann cells cultured in high glucose. Objective:Schwann cells were cultured in high glucose, and a few factors that may cause SC injury in high glucose or may help SC to adapt to the high glucose environment were amplified with a method of RT-PCR. Any change was observed and we study the function and its possible regulation of the gene.Methods:When SC cultured in high glucose for48hours, RNA was extracted and factors such as BDNF,CNTF,EGF,GMF,IGF-1, EGR-2, MMP9, NF-KB,NGF,NRG-1, POU3F3, SLC2a1,SOX-10,TrkA,TrkB were amplified with RT-PCR. We focus on POU3F3, make use of lipofection to transfectchemically synthesized POU3F3siRNA into schwann cell line RSC96cells. The cellular protrusions, size and the ultrastructure were observed. Using bioinformatical methods to find information, we find that DLL1is regulated by POU3F3. And we want to know if there is a change in high glucose cultured schwann cell line RSC96cell.Results:1,There were not any significant change of the mRNAs of BDNF,CNTF,EGF,GMF,IGF-1, EGR-2, MMP9, NF-KB,NGF,NRG-1, SLC2al,SOX-10,TrkA,TrkB extracted from RSC96cells cultured in25mmol/L glucose compared with cells cultured in5.5mmol/L glucose,P>0.05. But POU3F3mRNA has a significant increase between the two groups,P<0.05. However,all of these genes have no significant change when cultured for96hours.2, With the use of bioinformatical query, we find that POU3F3is closely related to DLL1. And we find that POU3F3is able to activate the DLL1transcription.3, We make a use of RT-PCR to amplify DLL1gene to observe whether its mRNA change or not when RSC96cells cultured in high glucose for48hs and96hs. And we find that its mRNA increase for3.4fold in high glucose compare with a low-glucose group. But it has a1.5fold increase and there is no statistically significant change, P>0.05.4, Chemically synthesized POU3F3siRNA was transfected into RSC96cells with the help of lipofection reagent, then we find there are no significant change between treatment group and the negative group, both neurite length and cell area, after administration of POU3F3siRNA and Negtive siRNA for24h.And there is no change of neurite length when the time lasts to48h, but the cell area has a significantly decrease in the negative siRNA group and the blank compare with the normal group, and there is a further reduce in the interference group.5, POU3F3gene positively influence DLL1mRNA, and the related coefficient (?)=999,P<0.05.Conclusion:1, When RSC96cells cultured in high glucose, we use RT-PCR to detect mRNA of15factors, it is the only one that POU3F3mRNA increase. Suggest this gene play an critical role in schwann cells cultured in high glucose.2, POU3F3may regulate the expression of Dlll. And Dlll mRNA elevate followed POU3F3mRNA increase in high glucose condition.3, Chemically synthesized POU3F3siRNA was transfected into RSC96cells with the help of lipofection reagent, then we find that there are no significant change between treatment group and the negative group,while the cell area reduce.