一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

Nitric oxide promotes the differentiation of neural stem cells in vitro derived from the subventricular zone of neonatal rats

目的:探讨一氧化氮(NO)对新生大鼠体外培养的神经干细胞(NSCs)分化的作用。方法:采用常规方法分离新生大鼠脑室下区(SVZ)组织,进行NSCs体外培养。用DETA/NO作为NO供体,用L-NAME作为一氧化氮合酶(NOS)抑制剂。免疫荧光法检测NSCs标志物—巢蛋白(nestin)、神经元标志物—βIII型微管蛋白(Tuj-1)和星型胶质细胞标志物—胶质原纤维酸性蛋白(GFAP)的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果:培养的神经球均为nestin阳性、BrdU阳性和nNOS阳性。NSCs和40μmol/L、50μmol/L、60μmol/L DETA/NO共培养5d,实验组培养液中NO浓度较对照组显著增高(P<0.01),相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加(P<0.01和P<0.05)。NSCs和100μmol/L、150μmol/L、200μmol/L L-NAME共培养5d,实验组培养液中NO浓度较对照组降低(P<0.05),相应实验组分化的神经元数和星型胶质细胞数也较对照组减少(P<0.05)。结论:NO能直接促进大鼠SVZ体外培养的NSCs分化。

Objective： To obsevve the effect of nitric oxide （NO） on the differentiation of neural stem cells （NSCs） derived from subventricular zone （SVZ） of neonatal rats in vitro. Methods： Conventional method was used to isolate and culture the NSCs from SVZ. Diethylene-triamine/ NO（DETA/ NO）was used as NO donor and Nitro-L-arginine methylester（L-NAME）was used as inhibitor of nitric oxide synthase （NOS）. The immunofluorescence was used to identify the expression of nestin （a marker of NSCs）, ~IlI- tubulin（Tuj-1, a marker of neu- rons）, glial fibrillary acidic protein（GFAP, a marker of astrocytes） and nNOS. The concentration of NO in medium was measured by Greiss assay. Results： Cultured neurospheres were nestin-, BrdU- and nNOS-positive. After treatment with 40μmol/L,50 μmol/L and 60 μmol/L of DETA/NO for 5 days, the concentration of NO released was increased significantly（ P 〈 0.01 ） as compared with that of the control group. The percentage of both differentiated neurons and astrocytes was increased significantly（ P 〈 0.01 and P 〈 0.05 ） as compared with that of the control group. After treatment with 100 μmol/L, 150μmol/L and 200 μmol/L of L-NAME for 5 days, the concentration of NO released was decreased as compared with that of the control group （ P 〈 0.05） . The percentage of both differentiated neurons and astrocytes were decreased as compared with that of the control group（ P 〈 0.05 ）. Conclusion： NO could directly promote the differentiation of NSCs derived from rat subventricular zone in vitro.