The effects of nitric oxide on proliferation and differentiation of enteric neural stem cells in rats

Abstract

Objective To study the effects of nitric oxide on differentiation and proliferation of enteric neural stem cells (ENSCs) in rats.Methods ENSCs were isolated from the gut of rat embryos at 15 days,and cultured ex vivo.The ENSCs were sub-cultured and divided into 4 groups according to the treatment:DETA/NO group (cells were treated with 50 μmol/L DETA/NO) ; L-NAME group (cells were treated with 100 μmol/L L-NAME; DETA/NO + L-NAME group(cells were treated with 50 μmol/L DETA/NO,and 100 μmol/L L-NAME).The control group was treated with vehicles.NO concentrations were measured by nitrate reductase analysis 48 hours after treatment.Neurosphere size was measured by morphological observation.The percentages of the cells which were positively immunoreactive to Nestin,BrdU,Tuj-1 were measured.The apoptosis was analyzed by flow cytometry.Results Forty-eight hours after treatment,compared with the control group,DETA/NO treatment statistically increased the NO concentration (51.94-± 12.43 μmol/L vs 30.27 ± 18.52μmol/L,P＜0.05),and L-NAME treatment decreased NO concentration (16.24 ± 12.25μmol/L vs 30.27 ±18.52 μmol/L,P＜0.05).50 μmol/L DETA/NO + 100 μmol/L L-NAME was not statistical significance (38.73-± 7.95)nol/L vs (30.27 ± 18.52)μmol/L(P＜0.05).The DETA/NO increased neurosphere size of neurons,but L-NAME could decrease the size of ENSCs.Flow cytometer analysis showed the Nestin (15.57 ± 1.45 ％) and BrdU (5.33 ±-0.55 ％) significantly decreased (P＜0.05),while tuj-1 positive neurons increased significantly (25.59 ±-2.78％,P＜0.05) after DETA/NO treatment.L-NAME changes the opposite expression of Nestin,BrdU and tuj-1.Every group did not changed cell death vs.control group.Conclusions Nitric oxide decreases the proliferation and promotes differentiation of enteric neural stem cells in rat. Key words: Neural stem cells; Nitric oxide; Cell proliferation; Cell differentiation