星形胶质细胞重编程诱导为神经细胞的实验研究

Experimental study on reprogramming astrocytes into neurons

目的研究在体外星形胶质细胞重编程诱导为神经细胞的方法。方法在体外培养出生1~4 d大鼠幼仔皮层脑组织来源的星形胶质细胞。在24孔板中将提纯、鉴定过的第三代星形胶质细胞培养在聚D-赖氨酸涂层的盖玻片上,并分为诱导组与对照两组。诱导组:将Y-27632(0.5μmol/L)、VPA(0.5μmol/L)、SB431542(94 nmol/L)、Rep Sox(25μmol/L)这4种小分子溶入二甲基亚砜(DMSO),再加入培养基中,最终达到0.5μmol/L,0.5μmol/L,94 nmol/L,25μmol/L的终浓度,孵育星形胶质细胞7 d;对照组:加入相同体积的DMSO。诱导组细胞使用小分子物质孵育7 d后,更换含有生长因子BDNF、NT-3的神经元培养基培养14 d,对照组细胞于7 d后,更换同样含有生长因子BDNF、NT-3的神经元培养基培养14 d。14 d后光镜下观察各组细胞的形态变化;并采用细胞免疫荧光检测神经元标志物TUJ1、MAP2及Neu N的表达。结果诱导组星形胶质细胞经过小分子物质重编程诱导7 d并加入神经元培养基14 d后,光镜下观察发现大部分细胞形态发生改变,饱满的胞质变得细长,有多个突起伸出且突起较长;免疫荧光检测示,神经元标志物TUJ1、MAP2及Neu N表达阳性细胞的比例(15.6%±0.7%)明显高于对照组(3.2%±0.3%),差异有统计学意义(P<0.05)。结论小分子化合物Y-27632、VPA、SB431542、Rep Sox可以将星形胶质细胞直接重编程诱导为神经细胞。

Objective reprogramming in vitro.Methods Astrocytes were cultured in vitro from the cerebral cortex of rats 1 to 4 days old.The purified and identified third generation astrocytes were cultured on poly-D-lysine coated cover slips in 24 well plates and divided into induction group and control group.Induction group:Y-27632(0.5μmol/L),VPA(0.5μmol/L),SB431542(94 nmol/L)and Rep Sox(25μmol/L)were dissolved in DMSO,and the mixture of these four small molecules was added into 24 well plate.Control group:the same volume of DMSO was added.After 7 days of continuous stimulation with small molecular substances,the cells in the induction group were cultured in the medium containing BDNF and NT-3 for 14 days to make immature neurons transform into mature neurons.After 7 days,the cells in the control group were cultured in the medium containing BDNF and NT-3 for 14 days.After 14 days,the morphological changes of cells were observed under light microscope.The expression of TUJ1,MAP2 and NeuN were detected by immunofluorescence.Results In the induction group,the astrocytes were reprogrammed with small molecular substances for 7 days and then added to the neuron culture medium for 14 days.Under light microscope,most of the astrocytes changed in morphology,the full cytoplasm became slender,and many processes extended and the processes were longer.Immunofluorescence detection showed that the proportion of positive cells of neuron markers TUJ1,MAP2 and NeuN(15.6%±0.7%)was significantly higher than that of the control group(15.6%±0.7%)(P<0.05).Conclusion Small molecule Y-27632、VPA、SB431542 andRepSox compounds can directly reprogram astrocytes into neurons.