CAJ | 학술논문

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/NO 50 μ.mol/L组、L-NAME 100μmol/L组、DETA/NO50 μmol/L+L-NAME100 μmol/L组及空白对照组继续培养.培养48 h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)μmol/L相比,传代培养48 h后DETA/NO 50 μmol/L组一氧化氮浓度明显增加(51.94±12.43)μmol/L(P＜0.05)、L-NAME 100 μmol/L组明显减少(16.24±12.25)μmol/L (P＜0.05),DETA/NO 50 μmol/L+ L-NAME100mol/L组无明显差异(38.73±7.95)μmol/L(P＞0.05).形态学发现DETA/NO 50 μmol/L组神经球直径较小,L-NAME 100 μmol/L组神经球直径较大.流式检测发现DETA/NO 50 μmol/L组肠神经干细胞Nestin百分比(16.57±1.45)％明显降低(P＜0.05),BrdU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-NAME 100μmol/L组则效应相反,DETA/NO 50μmol/L+L-NAME 100μmol/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.
목적 통과증가외원성일양화담혹감소내원성일양화담,관찰일양화담대대서장신경간세포(ENSCs)증식여분화적영향.방법 종잉15d배서장도제취장신경간세포,전대후장신경간세포분위DETA/NO 50 μ.mol/L조、L-NAME 100μmol/L조、DETA/NO50 μmol/L+L-NAME100 μmol/L조급공백대조조계속배양.배양48 h후,초산염환원매법검측각조배양액중일양화담농도；형태학관찰각조신경구대소；류식세포의검측각조장신경간세포Nestin、BrdU표기적증식세포、자대세포신경원Tuj-1염색양성세포백분비급각조세포적조망솔.결과 여대조조(30.27±18.52)μmol/L상비,전대배양48 h후DETA/NO 50 μmol/L조일양화담농도명현증가(51.94±12.43)μmol/L(P＜0.05)、L-NAME 100 μmol/L조명현감소(16.24±12.25)μmol/L (P＜0.05),DETA/NO 50 μmol/L+ L-NAME100mol/L조무명현차이(38.73±7.95)μmol/L(P＞0.05).형태학발현DETA/NO 50 μmol/L조신경구직경교소,L-NAME 100 μmol/L조신경구직경교대.류식검측발현DETA/NO 50 μmol/L조장신경간세포Nestin백분비(16.57±1.45)％명현강저(P＜0.05),BrdU표기적증식세포백분비(5.33±0.55)％명현강저(P＜0.05),신경원Tuj-1백분비(25.59±2.78)％명현증가(P＜0.05),이L-NAME 100μmol/L조칙효응상반,DETA/NO 50μmol/L+L-NAME 100μmol/L조여대조조무명현차이(P＞0.05).각조세포조망솔무현저성차이.결론 DETA/NO재세포배양액중능분해、석방일양화담,가작위합괄적외원성일양화담적체외공체지일；외원성일양화담능억제장신경간세포증식、촉진기향신경원분화,이L-NAME칙효응상반.
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.