一氧化氮对大鼠肠神经干细胞增殖与分化的影响

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

目的通过增加外源性一氧化氮或减少内源性一氧化氮，观察一氧化氮对大鼠肠神经干细胞（ENSCs）增殖与分化的影响。方法从孕15d胚鼠肠道提取肠神经干细胞，传代后肠神经干细胞分为DETA／NO50timol／L组、-NAME100p．mol／L组、DETA／N050μmol／L＋L-NAME100／μmol／L组及空白对照组继续培养。培养48h后，硝酸盐还原酶法检测各组培养液中一氧化氮浓度；形态学观察各组神经球大小；流式细胞仪检测各组肠神经干细胞Nestin、BrdU标记的增殖细胞、子代细胞神经元Tuj-1染色阳性细胞百分比及各组细胞的凋亡率。结果与对照组（30．27±18．52）p．mol／L相比，传代培养48h后DETA／NO50umol／L组一氧化氮浓度明显增加（51．94±12．43）umol／L（P〈0．05）、L-NAME100umol／L组明显减少（16．24±12．25）／~mol／L（P〈0．05），DETA／NO50umol／L＋L-NAMEl00~mol／L组无明显差异（38．73±7．95）umol／L（P^0．05）。形态学发现DETA／NO50“mol／L组神经球直径较小，~NAME100p．mol／L组神经球直径较大。流式检测发现DETA／NO50umol／L组肠神经干细胞Nestin百分比（16．57±1．45）％明显降低（P〈0．05），Br（1U标记的增殖细胞百分比（5．33±0．55）％明显降低（P〈0．05），神经元Tuj-1百分比（25．59±2．78）％明显增加（P〈0．05），而L-NAME100／μmol／L组则效应相反，DETA／N050μmol／L＋L_NAME100μmol／L组与对照组无明显差异（P〉0．05）。各组细胞凋亡率无显著性差异。结论DETA／NO在细胞培养液中能分解、释放一氧化氮，可作为合适的外源性一氧化氮的体外供体之一；外源性一氧化氮能抑制肠神经干细胞增殖、促进其向神经元分化，而L-NAME则效应相反。

Objective To study the effects o{ 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 # groups according to the treatment： DETA/NO group （cells were treated with 50 Gmol/L DE＇A/NO） ; L-NAME group （cells were treated with i00 ~mol/L L-NAIV[E; DETA/NO ＋ L-NA^d~ group（cells were treated ~th 50 ~mol/L DETA/NO, and 100 pmol/L L-NAME）. The control group was treated with vehicles. NO concentrations were measured by nitrate reduetase analysis 48 hours after treatment. Neurosphere size was measured by morphological observation. The percentages of the cells which were positively immu- noreactive to Nestin, BrdU, Tuj-I were measured. The apoptosis was analyzed by flow cytometry. Re- s~ Forty-eight hours after treatment, compared with the control group, DETA/NO treatment sta- tistically increased the NO concentration （51.94 -＋ 12. 43 ~mol/L vs 30. 2＂7 -＋ 18. 52 pmol/L, P〈 0. 05）, and L-NAME treatment decreased NO concentration （16. 24 -＋ 12. 25 Gmol/L vs 30. 27 -＋ 18. 52 ~mol/L, P〈0. 05）. 50 Gmol/L DETA/NO ＋ I00 Gmol/L L-NAME was not statistical signifi- cance （38. ＂73 ＋- 7. 95）~mol/L vs （30. 27 ＋ 18. 52）pmol/L（P〈0.05）. The DETA/NO increased neuro- sphere size of neurons, but L-NA_E could decrease the size of ENSCs. Flow cytometer analysis showed the Nestin （15.57 ＋ I. 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 treat- ment. 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 pro- motes differentiation of enteric neural stem cells in rat.