细胞骨架蛋白基因转录与小分子促小鼠胚胎干细胞定向分化神经元样细胞的相关性

Transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem cells induced by small molecules

目的:探索细胞骨架蛋白基因转录是否涉及全反式维甲酸(RA)促胚胎干(ES)细胞早期定向分化神经元样细胞,以此构建快速评价药物对ES细胞定向分化神经元的促进或抑制作用。方法:采用悬滴培养法模拟体内胚胎发育状态,形成拟胚体,进而贴壁向神经细胞分化。利用RT-PCR法评价神经元呈特征性表达的一类细胞骨架蛋白基因微管相关蛋白(Mtap2)、神经丝(Nefm)和微管蛋白(β-tubulin)转录水平,作为RA促ES细胞定向分化神经元指标,结合光镜和免疫荧光法鉴定神经细胞的形成,由此构建快速评价体系。并在此基础上进一步评价合成化合物库内6个小分子对细胞骨架蛋白基因转录的影响。结果:RA诱导ES细胞定向分化神经元过程,Mtap2和Nefm转录水平均呈上调趋势,其中以Mtap2为甚,增加了1.27倍,并与同期细胞表型改变相一致,但β-tubulin无明显变化。6个合成小分子均使Mtap2转录受到抑制,但Nefm转录水平有不同程度上调,1号和3号化合物尤甚,分别增加了1.4和1.2倍,该上调作用与同期细胞表型改变不尽一致。结论:RA诱导ES细胞定向分化神经元早期,Mtap2和Nefm转录水平均呈上调趋势,其中Mtap2的表达与否与早期细胞表型改变相一致。

Objective： To investigate the transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem （ES） cells induced by all-trans retinoic acid （RA）, and to explore the possibility of setting up a method to screen small molecules with promoting or inhibiting effect. Methods： The body formation to mimic embryo development performed to investigate mRNA expression of Mtap2, Nefm and β-tubulin Ⅲ which were hanging drop method was employed for embryonic in vivo. Reverse transcriptase PCR （RT-PCR） was the neuron-specific cytoskeleton proteins including regarded as the inducing effect indexes of RA.Morphological evaluation and immunocytochemistry staining were conducted to identify the neural derivatives. Moreover, the inducing effects of six synthetic molecules were further evaluated. Results ： RA up-regulated the mRNA expression of Mtap2 and Nefm ,especially Mtap2 increased by 1.27 times,which was consistent with the morphological alteration. However,there was no significant changes of β-tubulin Ⅲ expression. With addition of the six syntheticmolecules,the transcription of Mtap2 was inhibited,while the Nefm mRNA expression was up regulated in some degree,especially for molecule 1 and 3 that was increased by 1.4 and 1.2 times, which,however ,was not parallel to the morphological changes. Conclusions： The transcriptional levels of Mtap2 and NeJm are both up-regulated in the RA induced differentiation of ES cells towards neurons. The up-regulation of Mtap2 is consistent with the morphological alteration, which might be the key landmark in the RA-induced differentiation of ES ceils into neurons.