Study On The Mechanism Of Exogenous Neurotrophin - 3 (NT - 3) Promoting Neuronal Cell Proliferation Through MAPK Signaling Pathway

SECETION 1 DETERMINATION OF THE OPTIMAL NT-3 TREATMENT OF HNObjective:Determination of the optimal NT-3 treatment of HNMethods:To determine the optimal concentration and duration of NT-3 treatment of HN, NT-3 was added to the cell culture media at varying concentrations, and cell growth was monitored for 8 days.Results:the growth and survival of HN treated with 10,20,40 and 80 nM NT-3 after 2,4,6 and 8 days. The NT-3 treatment that yielded the maximal growth rate was 20 nM for 4 days.Conclusion:The NT-3 treatment that yielded the maximal growth rate was 20 nM for 4 days.SECTION 2 NT-3 PROMOTES THE GROWTH OF HN VIA THEERK1/2 PATHWAYObjective:To explore the NT-3 promotes the growth of HN via theERK1/2 pathwayMethods:To identify the signaling pathway through which NT-3 acts on HN, cells were treated with 20 nM NT-3 for 4 days (the optimal conditions as determined above) and the phosphorylation state of the MAPKs ERK1/2, JNK and p38 was examined at 0,2,4,6 and 12 h following treatment by western blot. Following siRNA knockdown of ERK1/2, cells were again treated with NT-3, and growth was compared to untransfected cells on days 2,4,6, and 8 by MTT.Results:the phosphorylation of ERK1/2 increased to a greater extent than either JNK or p38 after 6h. To verify that NT-3 promotes the growth of HN via the ERK1/2 pathway, siRNA was used to inhibit ERK1/2 activity. Following siRNA knockdown of ERK1/2, cells were again treated with NT-3, and growth was compared to untransfected cells on days 2,4,6, and 8. Following ERK1/2 knockdown, phosphorylation of p90rsk was decreased compared to controls, demonstrating the effectiveness of the knockdown. Inhibition of ERK1/2 by siRNA eliminated the ability of 20 nM NT-3 to increase the growth of HN, even after 8 days of treatment.Conclusion:NT-3 promotes the growth of HN via theERK1/2 pathwaySECTION 3 THE EFFECT OF TRKC KNOCKDOWN BY SIRNA IN HNObjective:To explore the effect of TrkC knockdown by siRNA in HNMethods:The NT-3 receptor TrkC was silenced by RNA interference. The growth rate of HN effected by NT-3 with 10,20,40 and 80nM at 2,4,6 and 8day was detected by MTT. The expression level of ERK1/2,JNK,P38 and phosphorylation of ERK1/2,JNK,P38 was detected by westren blot.Results:TrkC protein levels were reduced, blocking the stimulation of growth by NT-3. This effect persisted under all NT-3 treatment conditions (10,20,40 and 80 nM NT-3 for 2,4,6 and 8 days). Furthermore, the phosphorylation of ERK1/2 and p90rsk was also blocked by TrkC knockdown, showing no significant difference when compared to cells that were not treated with NT-3.Conclusion:NT-3 promoted the growth of HN by TrkC...