全反式维甲酸诱导神经干细胞向神经元的分化

Differentiation of neural stem cells into neurons induced by all-trans-retinoic-acid

背景：目前研究证实全反式维甲酸在调控多种组织和细胞的增殖、生长发育、代谢以及维持内环境稳定等方面具有广泛的生物学作用。目的：探讨全反式维甲酸诱导神经干细胞分化为神经元过程中的作用及其分子机制。设计、时间及地点：对照观察。体外细胞学实验，于2006-07／2007-06在上海同济大学附属医院实验室完成。材料：普通级孕13-14d雌性SD大鼠6只。取胎鼠用于制备神经干细胞。诱导剂全反式维甲酸为Sigma公司产品。方法：取第3代神经干细胞，以2×10^8L^-1密度接种，分为全反式维甲酸诱导1d，3d,7d，10d组和空白对照组，各诱导组均加入1μmol／L全反式维甲酸。空白对照组仅加入等量生理盐水。主要观察指标：免疫荧光染色鉴定结果，计数分化为神经元的比例。应用半定量RT-PCR法检测维甲酸受体αmRNA的表达。结果：全反式维甲酸诱导后神经元特异性烯醇化酶染色呈阳性，细胞有多个细长突起。胞体呈绿色荧光，胞核呈蓝色荧光。与空白对照组比较。诱导3d,7d,10d组神经元特异性烯醇化酶染色阳性细胞率均明显升高（P〈0．05）。且诱导7d组达峰值。与空白对照组比较，各诱导组维甲酸受体αmRNA的表达量均明显升高（P〈0．01）。结论：全反式维甲酸能显著提高大鼠胚胎神经干细胞分化为神经元的比例，同时发现细胞维甲酸受体αmRNA的表达增加。

BACKGROUND： Present studies verified that all-trans-retinoic-acid （ATRA） has a wide biological effect in regulating the proliferation, growth development, metabolism and maintaining homeostatic equilibrium in multiple tissues and cells. OBJECTIVE： To explore the effect and molecular mechanism of ATRA in inducing neural stem cell differentiation into neurons. DESIGN, TIME AND SETTING： The in vitro cytology control experiment was performed at the Laboratory of Hospital Affiliated to Tongji University from July 2006 to June 2007. MATERIALS： Six female Sprague Dawley （SD） rats pregnant for 13-14 days were used in this study. Fetal rats were used to collect neural stem cells. ATRA was purchased from Sigma, USA. METHODS： At the third passage, neural stem cells were incubated at a density of × 10^ 8L^-1. There were ATRA 1-day, 3-day, 7-day, 1G-day groups and a blank control group. Neural stem cells in each experimental group were induced with 1 μ mol/L ATRA, whereas those in the blank control group received an equal volume of saline. MAIN OUTCOME MEASURES： Immunofluorescence staining was performed to identify the percentage of neural stem cell differentiation into neurons. Ratinoic acid receptor α mRNA expression was tested by semi-quantitative reverse transcriptase-polymerase chain reaction assay. RESULTS： After ATRA induction, neural stem cells were positive for neuron specific enolase, with many thin processes, cell body with green fluorescence, whereas nuclei showed blue fluorescence. Compared with the bland control group, the rate of neuron specific enolase-positive cells significantly increased in the ATRA 3-day, 7-day and 10-day groups （P 〈 0.05）, and reached a peak in the ATRA 7-day group. Compared with the blank control group, ratinoic acid receptor α mRNA expression significantly increased in each group （P 〈 0.01）. CONCLUSION： ATRA can greatly increase the percentage of neurons differentiated from rat embryonic neural stem cells. It is found that the expression of ratinoic acid receptor α mRNA increases.