Effect And Mechanism Of Diethylstilbestrol And GDNF On Proliferation And Differentiation Of Rat NSCs

INTRODUCTION Parkinson's disease(PD)is a common neurological disorder in aged people, the pathological hallmark of PD is a gradual loss of nigro dopamine-containing neurons, but degeneration also occurs in systems of non-dopaminergic neurons. All current therapies do not stop the progression of the disease. Steady progress supports the hope that NSCs-based therapies to offer the real possibility for treatments and cures for PD. Estrogen has been the focus of increased attention over recent years because of its protection of neurons. Estrogen receptors have been found at cellular membrane of the NSCs and neural precursor cells. Furthermore estrogen has been shown to play important roles in NSCs proliferation and neuronal differentiation ; It also can encourage human fetal NSCs to differentiate dopaminergic neurons and survive when they are transplanted in host brain. GDNF is expressed extensively in the CNS and it also contributes efficiently to protection of neurons by neurotrophy and anti- apoptosis. Furthermore, it increases the generation of dopaminergic neurons in the differentiation of NSCs. However,the influence and mechanism of them in mesencephalon development is currently poorly reported. The development of fetal mesencephalic NSCs provides a useful model of mesencephalon neurogenesis, So we studied artificial estrogen- diaethylstilbestrol and GDNF,effect on mesencephalic NSCs to obtain more materials for therapy of PD.AIM To study the effect and mechanism of diaethylstilbestrol and GDNF on proliferation and differentiation of NSCs and the relationship between diaethylstilbestrol and GDNF to obtain more materials for therapy of PD. METHODS The mesencephalon of fetal rat of 14 days old were cultured in vitro and treated with diethylstilbestrol , the cells were divided into four groups according to the concentration of diethylstilbestrol : high dosage group(B group:1x10-7 mol·L-1), middle dosage group(C group:1x10-8 mol·L-1) ,low dosage group(D group:1x10-9 mol·L-1) and blank control group(A group).All cells were processed for NSE and c-fos immunocytochemistry to detect the differentiation of expression and determine the effects of diethylstilbestrol on mesencephalic neurons . The NSCs were cultured in vitro in the same method. The experiment was divided into three stages: (i)we observed three different dosage(high dosage group:ⅠB group-1x10-7 mol·L-1; middle dosage group:ⅠC group-1x10-8 mol·L-1; low dosage group:ⅠD group:1x10-9 mol·L-1) diethylstilbestrol,s roles in proliferation and differentiation of rat NSCs ,then we determined an effective dosage through comparison with blank control group(ⅠA group ) . ( ii ) we observed two different dosage ( low dosage group:ⅡB group-10ng/ml; high dosage group:ⅡC group-20ng/ml ) GDNF , s roles in proliferation and differentiation of rat NSCs ,then we determined an effective dosage through comparison with blank group(ⅡA group).(iii)We observed combination groups,s influence on proliferation and differentiation of rat NSCs .We presented the effective dosage diethylstilbestrol in media involving high dosage GDNF(ⅢB group: the effective dosage diethylstilbestrol +20ng/ml GDNF) and low dosage GDNF(ⅢC group: the effective dosage diethylstilbestrol +10ng/ml GDNF) respectively, while we set up blank control group (ⅢA group) to compare with combination groups.Nestin,NSE and GFAP proteins were examined by immunofluoresence staining,and we analyzed roles in development of NSCs and discussed the potential mechanism.RESULTS Diethylstilbestrol protected mesencephalic neurons in vitro, especially the middle dosage(1x10-8 mol·L-1) . Diaethylstilbestrol enhanced proliferation of fetal rat mesencephalic NSCs and increased neuronal differentiation, Furthermore diaethylstilbestrol played important roles in neurite development.The middle dosage (1x10-8 mol·L-1)is the most efficiently. High dosage GDNF(20ng/ml) increased the generation of neurons in the differentiation of NSCs,and promoted its proliferation. The middle dosage diaethylstilbestrol and low dosage GDNF ( 1x10-8 mol·L-1 diaethylstilbestrol+10ng/mlGDNF) had synergistic effect on promotion of NSCs proliferation, moreover they enhanced significantly neuronal differentiation.CONCLUSIONS The results suggest that estrogen promotes mesencephalon growth and development in dose dependent manner; Estrogen and GDNF can play a synergistic role in Proliferation and Differentiation of mesencephalic NSCs in defined condition.