小鼠肠神经嵴干细胞体外神经元亚型分化的研究

The differentiation ability of murine neural crest stem cells

目的研究胚鼠（E14-17）、新生鼠（P1、P5）肠神经嵴干细胞（GNCSCs）在体外培养中经诱导向一氧化氮合酶（NOS）和血管活性肠肽（VIP）神经元分化的特点；研究标本取材和细胞消化分离间隔时间对P5鼠收获GNCSCs克隆球数量和诱导分化能力的影响。方法分离并培养胚鼠、新生鼠GNCSCs，采用血清联合多聚赖氨酸或单纯多聚赖氨酸促分化，SABC法进行nNOS和VIP免疫细胞化学染色；将P5鼠按标本取材和细胞分离的间隔时间分为0、15、30、45、60min组，对培养72hGNCSCs克隆球进行计数，再采用血清联合多聚赖氨酸促分化，SABC法进行nNOS和VIP免疫细胞化学染色。结果血清联合多聚赖氨酸对胚鼠、新生鼠GNCSCs克隆球诱导分化作用明显强于单纯使用多聚赖氨酸（P〈0．05）；胚鼠、新生鼠GNCSCs经体外培养、血清联合多聚赖氨酸诱导，均可分化出nNOS和VIP阳性细胞；P5鼠0min组获得的GNCSCs克隆球数和经联合诱导分化出的阳性细胞数明显高于其他各组（P〈0．05），15、30min均明显高于45、60min组（P〈0．05）。结论血清联合多聚赖氨酸对GNCSCs向NOS和VIP神经元分化的诱导作用明显优于单纯使用多聚赖氨酸；胚鼠和新生鼠GNCSCs经体外培养、血清联合多聚赖氨酸的诱导，可分化出NOS和VIP神经元；P5鼠肠管获得GNCSCs数和NOS、VIP神经元分化能力随标本取材、细胞分离间隔时间延长而降低。

Objective To investigate the abilities of gut neural crest stem cells（GNCSCs） acquired from embryo and neonatal mice to differentiate into nitric oxide synthase（NOS） and vasoactive intestinal peptide（VIP） positive neurons under inductive condition. Methods We had isolated and cultured GNCSCs from embryo and neonatal mice. The derivations of GNCSCs induced by serum with poly-L-lysine or unique poly-L-lysine were investigated by immunocytochemistry （SABC）. P5 mice were divided into five groups （0min, 15min, 30min, 45min, and 60min） according to time interval between reserving specimen and manipulation. We counted the number of neurospheres on the third day after primary culture, and NOS as well as VIP neurons were identified by SABC following induction by serum with poly-L-lysine. Results The serum with poly-L-lysine could significantly promote the GNCSCs to differentiate into subsets of neurons in contrast of using poly-L-lysine alone （P〈0. 05 ）. Either neurospheres from embryo or from neonatal mice in vitro could differentiate into nNOS and VIP positive cells under inductive condition. There were no significant differences of nNOS and VIP positive cells between the 2 groups （P〉0. 05）. The numbers of nNOS and VIP positive cells and neurospheres in group 0 min were significantly higher than those in other 4 groups（P〈0. 05） on the third day. And the numbers of nNOS and VIP positive cells and neurospheres in group 15 and 30 min groups were higher than those in 45 and 60 min groups（P〈0. 05）. Conclusions The serum with poly-L-lysine can significantly promote GNCSCs to differentiate into NOS and VIP positive neurons. GNCSCs from embryo and neonatal mice can differentiate into NOS and VIP positive neurons under inductive condition in vitro. Ability of neurospheres acquirement and NOS and viP positive neurons differentiation might be decreased with prolongation of time interval between reserving specimen and manipulation.