| Objective The effects of growth differentiation factor 11(GDF11)on the proliferation of neural stem cells(NSCs)isolatited from embryonic mice were studied.Then the expression of Smad2/3,Smad4 and ?-catenin were tested to investigate the effect of GDF11 on TGF-?/Smads signaling pathway and Wnt/?-Catenin signaling pathway in NSCs.Methods NSCs used in this study were derived from the subventricular zone(SVZ)of E14 d CD1 mice and were confirmed by Immunofluorescence assays.CCK-8 method was used to study the effect of GDF11 on the uptake of NSCs by a certain concentration gradient and to determine the subsequent experimental concentration.The third generation cells were chosen and randomly divided into two groups when in their exponential phase: experimental group was added GDF11 to make the final concentration 50ng/ml while control group equal amount of culture fluid.The proliferation of the cells in two groups was detected by 5-Ethynyl-2'-deoxyuridine(Ed U)kits and protein expression of Smad2/3,Phospho(P)-Smad2/3,Smad4,?-Catenin at 1h and 6h after treatment was measured by western blotting analysis.Learning using SPSS19.0 software,the comparison between groups using independent sample t test,the results of P<0.05 for the difference was statistically significant.Results 1.After the stem cells were isolated and cultured in vitro,the cells were inoculated in the culture flask.Under inverted microscope,a large number of circular translucent cells showed lethal growth and gradually formed multiblocks with the passage of time.After about 3-5 days,the centers of the cells were dark,suggesting that passage was required.2.Immunofluorescence results suggested that more than 90% of the cells cultured in this experiment were positive for Nestin and SOX2.After induction of differentiation,some cells were Neu N and GFAP positive,which proved its potential of differentiation into neurons and astrocytes,so the cells we extracted could be identified as NSCs which met the experimental requirements.3.The results of CCK-8 test showed that the growth rate in 12ng/ml,25ng/ml,50ng/ml,100ng/ml and 200ng/ml groups were higher than that in the control group,but the difference was not obvious.Cells in 400ng/ml group and 800ng/ml group were significantly inhibited.We decided to use 50 ng / ml as the experimental group of experimental treatment concentration in this experiment.4.The results of Ed U test showed that GDF11 could increase the cell proliferation rate(Ed U+/DAPI+)in the experimental group at 6h after adding GDF11,indicating that GDF11 promoted the enhancement of neural stem cells in the experimental group and the difference was statistically significant(P<0.05).5.Western Blotting results showed that the relative expression of Smad2/3 protein in the experimental group was not statistically different between the two groups at 1h and 6h.The relative expression of P-Smad2/3 protein in the experimental group was higher than that in the control group at both time points.The results were statistically significant(P<0.05).The expression of Smad4 in the experimental group was significantly higher than that in the control group at both time points(1h group P<0.01;6h group P<0.05).The expression of ?-Catenin protein in the experimental group was higher than that in the control group at both two time points.The difference was statistically significant(P<0.01).The phosphorylation of Smad2/3 and the expression of Smad4 and ?-Catenin were enhanced under the action of GDF11.Conclusions In this study,we isolated the neural stem cells from SVZ of E13-day mouse embryos and added GDF11 to confirm that a certain concentration of GDF11 could promote the proliferation of neural stem cells in vitro for a short time period.The TGF-? signaling pathway and Wnt/?-Catenin may be involved in the process. |
PDF Full Text Request