Effect Of Bdnf Rna Interference On The Pathogenesis Of Parkinson's Disease

Part I The identification of the culture of PC12 cells and the efficiency of BDNFgene RNA interferenceObjective To approach the proliferation activity of PC12 cell line, the efficiency of brain derived neurotrophic factor (BDNF) gene RNA interference and the effect of BDNFgene RNA interference on BDNF gene expression. Methods Specific small double-stranded interfering RNA (siRNA) against BDNF mRNA was designed, synthesized and transfected into PC12 cell line using lipofectamine 2000 with or without BDNF protein protection. Using the CCK-8 to exam cell multiplication, using the immunofluorescence method to examine the efficiency of BDNF RNA interference, using real-time PCR assay to exam the influence on BDNF gene RNAi on BDNF mRNA expression. Results The proliferation of PC12 cell is well, the efficiency of BDNF gene RNAi achieves 95%, BDNF mRNA reduced more than 70% in the siRNA transfected cells as compared with the control group, but not in the scrambled siRNA. Conclusion The present study showed that that BDNF RNA interference could downregulate the expression of BDNF mRNA on PC12 cell while cell apoptosis was alleviated when post-incubated with BDNF protein.Part II Effect of BDNF RNA interference on the apoptosis of PC12 cell and the Study of apoptosis signal transduction pathwayObjective To approach the exact role of BDNF in Doparminergic PC12 cell apoptosis. Methods siRNA against BDNF mRNA was transfected into PC12 cell line using lipofectamine 2000 with or without BDNF protein protection. Flow cytometry assay was used to detect the effect of siRNA on cell apoptosis. Western blotting was used to to see whether the effect of BDNF RNAi on the apoptosis of Doparminergic PC12 cell through the bcl-2 signal transduction pathway. Results BDNF RNAi induced cell apoptosis of PC12 cell coincide with 6-Hydroxydopamine (6-OHDA) neurotoxicity. Cell apoptosis was alleviated when post-incubated with BDNF protein. BDNF RNAi reduced the expression of bcl-2 protein and enhanced the expression of bax protein. Conclusion The present study showed that downregulation of BDNF could induce PC12 cell apoptosis through the bcl-2 signal transduction pathway. Part III Effect of BDNF RNA interference on PC12 cell Oxidative stressObjective To approach the exact role of BDNF RNAi in PC12 cell oxidative stress. Methods siRNA against BDNF mRNA was transfected into PC12 cell line using lipofectamine 2000 with or without BDNF protein protection. Reactive Oxygen Species Testing Kit was used to check the expression of the level of reactive oxygen species. Maleic Dialdehyde (MDA) Testing Kit was used to check the expression of the level of expression levels of lipid peroxides. Results BDNF RNAi increased ROS and MDA in PC12 cell obviously, the 6-OHDA group Could also increase ROS and MDA as compared with the control group, but not in the scrambled siRNA transfection group. The change was alleviated when post-incubated with BDNF protein. Conclusion The present study showed that downregulation of BDNF could enhance the Oxidative stress of PC12 cell, but alleviated after post-incubated with BDNF protein.