RT-PCR法检测神经源性分化因子mRNA在大鼠脑发育过程中的表达

A semi-quantitative RT-PCR analysis for NeuroD mRNA expression in brain during rat development

目的观察大鼠不同发育时期脑内神经源性分化因子(NeuroD)mRNA表达量的变化,探讨其对神经细胞的分化和成熟发挥的作用。方法提取不同发育阶段大鼠不同脑组织中的mRNA,RT-PCR法反转录扩增NeuroD和β-actin,利用凝胶成像系统检测其相对表达量。结果 NeuroD在受孕7.5d(E7.5)时出现表达,在E10.5和E21.5时分别达到两次高峰,平均吸光度值为20437.88±598.28和14482.23±1134.49,表达水平显著高于其他各组(P<0.01),NeuroD/β-actin比值在E12.5、E18.5和E21.5分别为1.59±0.09、1.61±0.07和1.70±0.11,显著高于其他时间段(P<0.01)。结论在大鼠脑发育过程中,NeuroD mRNA的表达呈明显时间特异性,在E10.5左右大鼠各原始脑泡的形成过程中,NeuroD mRNA明显增高;在胚胎发育的晚期,脑组织形态进一步完善和成熟的过程中NeuroD达到第二次表达高峰,与鼠脑的发育过程相符。推测NeuroD对鼠脑的早期神经细胞分化以及晚期细胞的成熟均起一定作用。

Objective To observe the NeuroD mRNA expression during rat neural system development and to investigate its possible roles in neural development and differentiation. Methods Total mRNA was abstracted from different parts of brains in different development stages with Trizol kit in rats. The mRNA of NeuroD and β-actin was reversely transcripted and amplificated by semi-quantitative reverse transcription-polymerase chain reaction method （ RT- PCR）. The quantity of NeuroD and 13-actin mRNA expression was detected by gel imaging system. Results NeuroD mRNA existed in E7.5. The mRNA expression of NeuroD and β-actin peaked in El0.5 and E21.5, which average gray values were 20437.88 ± 598.28 and 14482.23 ± 1134.49, respectively. The mRNA expression of NeuroD and β-actin in EIO. 5 and E21.5 was significantly higher than other time points （P 〈0. 01 ）. The ratios of NeuroD to β-actin in El2. 5, El8.5 and E21.5 were 1.59 ＋0. 09,1.61±0.07 and 1.70 -50. 11 respectively, which were higher than other time points （P 〈 0. O1 ）. Conclusion The expression of NeuroD mRNA showed an apparent time-specific pattern during the development of the rat brain. The expression level of neuroD increased obviously in two phases. One was in El0. 5, when the original rat brain came into being; the other was the anaphase in neural development, when brain form made the perfection, brain tissue stepped into its maturation. Thus, our results suggested the functions of NeuroD： 1. NeuroD influenced the generation and differentiation of nerve cells ; 2. NeuroD played its role in the maturation of nervous system.