一氧化氮合酶和5-羟色胺在体外培养的胚鼠脑干神经元内的共存研究

COEXISTENCE OF NITRIC OXIDE SYNTHASE AND SEROTONIN IN THE EMBRYONIC RAT BRAIN STEM NEURONS IN VITRO

采用 NADPH-d组织化学和 5 -羟色胺免疫组织化学双重反应技术 ,探讨在体外培养条件下 E14 d SD胚鼠脑干神经元中5 -羟色胺与一氧化氮合酶阳性神经元的体外生长发育过程以及 5 -羟色胺与一氧化氮合酶的共存。将 E14 d SD胚鼠脑干细胞悬液接种于 2 4孔培养板 ,实验按培养龄分 4组分别在接种后 5 d、10 d、15 d和 2 1d终止培养 ,先进行 NADPH-d组织化学反应 ,再行 5 -羟色胺免疫组织化学反应。结果显示 :各实验组均可观察到三种神经元 ,即 5 -羟色胺免疫阳性神经元、一氧化氮合酶阳性神经元和两者的双重阳性神经元 ,其中双重阳性神经元占多数 ,并以梭形神经元为主 ,胞体呈三角形、多角形、梭形或卵圆形。 10 d组的双重阳性神经元突起最长可达 60 0μm以上 ,末端可见明显的生长锥 ,轴突终末分支上分布有大量的膨体 ,并与其它神经元的胞体或突起之间形成接触。本研究结果证明一氧化氮合酶和 5 -羟色胺可以在体外培养的胚鼠脑干神经元内共存 ,提示一氧化氮合酶可能与 5 -羟色胺共同参与胚脑的发育。

By using the method of NADPH diaphorase histochemistry combined with 5 HT immunocytochemistry, we explored the coexistence of NOS and 5 HT in E14 rat brian stem neurons in vitro. Brain stem cell suspension obtained from E14 rat was plated on 24 well culture plates. The experiment was divided into four groups according to the culture day, cultures were terminated respectively at 5 d, 10 d, 15 d, and 20 d after plating. Each group was first stained by NADPH d histochemistry method, then by 5 HT immunocytochemistry method. It was observed that three kinds of cells were found in each group. They were NOS positive neurons, serotonic like immunoreactive cells and the cells that contained both serotonin like immunoreactivity and NADPH d activity. Among those cells, numerous neurons were double staining cells, and belonged to fusiform neurons majorly. The cell bodies were triangular, polygon, and fusiform. The longest neurites of the double stained neurons in the 10 d cultures were longer than 600 μm, at the end of the neurites growth cone could be clearly seen. The axons had many buttons and contacted with the neurites and cell bodies of other neurons. The study proves that NOS and 5 HT can coexist in the embryonic rat stem neurons in vitro, suggesting that NO and 5 HT may be involved in the development of embryonic brain. (Figures 1～6 on plate 47)