Effects And Mechanisms Of Growth Differentiation Factor 11 On Proliferation And Differentiation Of Neural Cell

ObjectiveGrowth differentiation factor 11(GDF11)and C-C motif chemokine ligand 11(CCL11)were studied.Firstly,we detecte,d the specificity of growth differentiation factor 11(GDF11)antibody from R&D Systems.Secondly,we measured the concentrations of GDF11 and CCL11 in plasma from blood donors.And then,we evaluated the influences of age,gender and ABO blood group on plasma GDF11 and CCL11 levels in Chinese blood donors.Finally,we studied the effect and mechanisms of GDF11 on HT22 or C17.2 cell lines,to explore the potential medical value of GDF11 in age-related diseases and to provide some references for translational medicine in future.Methods1.SDS-PAGE and Western blot were used to detect the specificity of GDF11 antibody from R&D Systems.2.Fresh frozen plasma donated by about 300 normal blood donors was collected,frozen at-80℃ refrigerator,avoiding repeated freezing and thawing.Plasma GDF11 and CCL11 levels were dectected by using ELISA.3.HT22 cells were fed with complete medium(high-glucose DMEM supplemented with 5%horse serum(HS),10%fetal bovine serum(FBS),penicillin(100 U/mL)and streptomycin(100 mM/mL))and cultured in 5%CO2 at 37℃ overnight.Then,the cells were cultured in serum-starved medium(high-glucose DMEM supplemented with 0.5%HS and 1%FBS)for another 6h.Finally,in treatment groups of HT22,GDF11 was added at different concentrations(0,12.5,25,50 and 100ng/mL,respectively)and in treatment groups of C17.2,100 ng/mL NGF,100 ng/mL GDF11 and 25ng/mL GDF11 were added;in control group,the same volume serum-starved medium as the treatment groups were added and cultured 6h,24h or 48h.The following experiments were carried out.4.The morphology and apoptosis of HT22 and C17.2 cells were observed using inverted fluorescence microscope.The cell viability and proliferation were measured by using enhanced cell counting kit-8.5.The HT22 cell monolayer in 24-well plate was wounded by manually scraping the cells with a blue pipette tip to analyze the effect of GDF 11(12.5,25,50,100ng/mL)on migration of HT22 cell line.6.The mRNA expression of CyclingD2,nestin,βⅢ-tubulin,GFAP,EGFR,Notchl etc.were detected by real-time quantitative PCR(qRT-PCR)when the cells were stimulated for 6h by GDF11.7.Western blot was used to analyze the effects of GDF11 on the phosphorylation of Smad2/3 in TGF-β signaling pathway and the phosphorylation of cAMP responsive element binding protein(Creb).8.Statistical analysis methods:The graphs and results were analyzed using GraphPad Prism 5 software and Image J was used to analysis gray scale of western blot bands.Levels of phosphorylated protein were normalized by total protein and the ratio in normal control group was set to 1.Paired t-test was used to compared differences between two groups and one way ANOVA was used to compare differeces between multiple groups.Differences were considered significant when P<0.05.Results1.GDF11 monoclonal antibody from R&D Systems can bind GDF8,but does not have cross-reactivity with immunoglobin single light chain.2.The results of ELISA showed that the levels of GDF11 in plasma were decreased with age and the concentration of CCL11 has a significant positive correlation with age.Moreover,the CCL11 levels in female were obviously lower than that in male(P<0.01).3.GDF11 showed no effect on the poptosis of HT22 cells using immunofluorescence.4.The results of enhanced CCK8 showed when compared with the control group,the viability of HT22 and C17.2 cells that was treated by GDF11 significantly increased(P<0.01).5.The number of cells in treatment groups migrated to wound surface is lower than control group.6.Compared with the control group,the mRNA expression of(3111-tubulin was significantly increased(P<0.01,n=3)in the treatment groups.Nestin showed a light decrease and GFAP displayed an increase,but the changes were not statistically significant.CyclinD2,EGFR and Notch1 were all significantly does-dependent decreased(P<0.05,n=3).7.Western blot results indicated that in the treatment groups(24h),compared with the control group,Smad2/3 and Creb levels of the treatment groups had no significant change,but p-Smad2/3 and p-Creb were significantly increased,consistent with activation of TGF-β-Smad2/3 and Creb signal pathways.However,no significant dose-dependence was found after treated by different concentrations of GDF11.Conclusions1.GDF11 monoclonal antibody was not specific for GDF115 and it also have the cross-reactivity with GDF8.2.The levels of GDF11 in human plasma decreased with age.The concentrations of CCL11 had a positive correlation with age,and the CCL11 levels in female were significantly lower than that in male.3.GDF11 showed no effect on the poptosis of HT22 cells,and played important roles in the proliferation and differentiation of HT22 and C17.2 cells and inhibited the migration of HT22 cell line.Furthermore,GDF11 can activate the TGF-β/smad and CREB signal pathways and inhibite Notch and EGFR signal pathways.Therefore,GDF11 may regulate the migration,proliferation and differentiation of neural cells through the effects of cross-talk among TGF-β/smad,CREB,Notch and EGFR pathways.