体外诱导小鼠胎肝间充质细胞向神经元样细胞分化的研究

Differentiation of mesenchymal cells derived from mouse fetal liver into neuronal cells in vitro

目的 :探讨胎肝间充质细胞在体外向神经元样细胞分化的潜能。 方法 :无菌条件下获取胎龄 1 4 .5d的胎肝细胞 ,DMEM/HEPES/F1 2 (2 0 %FCS)培养得到贴壁细胞并传代 ,第 5代细胞经 β 巯基乙醇 (β mercaptoethanol,β ME)与全反式维甲酸 (all trans rentinoicacid ,ATRA)程序化诱导处理后 5h、5d ,用免疫细胞化学鉴定神经元抗原表达 ,同时用半定量RT PCR检测神经元相关基因mRNA表达情况。 结果 :胎肝间充质细胞经 β ME、ATRA诱导处理后 ,约 80 %细胞呈现典型的神经元样的形态表型。免疫细胞化学显示诱导分化后的细胞表达神经元特异蛋白如神经元核特异性抗原 (neuron specificnuclearpro tein ,NeuN)、神经元特异性管蛋白Ⅲ型 (neuronc specifictubulinⅢ ,TuJ 1 )、神经丝蛋白中链 (neurofilament M ,NF M )及神经元特异性烯醇化酶 (neuron specificenolase ,NSE) ,但是无成熟神经元微管相关蛋白Tau(microtubule associatedproteintau ,Tau 1 )、微管相关蛋白 2 (microtubule associatedprotein 2 ,MAP 2 )表达。半定量RT PCR结果显示诱导分化后的细胞酪氨酸羟化酶 (tyrosinehydroxylase ,TH)mRNA有表达 ,脑因子 1基因 (brainfactor 1 ,BF 1 )、脑因子 3基因 (brain 3,Brn 3)、神经丝蛋白轻链基因

Objective:To investigate potency of mouse fetal liver mesenchymal cells differentiating into neural cells in vitro .Methods: Sterile cells from 14.5 day old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20%FCS and adherent cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β mercaptoethanol (β ME) and all trans retinoic acid (ATRA). The characteristics of treated cells were assayed by immunocytochemistry staining and semi quantitative RT PCR method on 5 h and 5 d after induction. Results: After induction by β ME and ATRA, approximately 80% of the cells exhibited typical neuronal morphology;most cells expressed neuronal phenotype such as neuron specific nuclear protein(NeuN), neuron specific tubulin Ⅲ(TuJ 1), neurofilament M(NF M),and neuron specific enolase(NSE).However, microtubule associated protein tau(Tau 1)and microtubule associated protein 2 (MAP 2) as indicators of mature neuronal phenotype were undetectable. Semi quantitative RT PCR showed increased mRNA expression of brain factor 1(BF 1), brain 3(Brn 3), and neurofilament L(NF L) in treated cells versus untreated cells ( P < 0.01), and tyrosine hydroxylase (TH) mRNA expression was also found in treated cells. Conclusion:These results indicate that mesenchymal cells derived from mouse fetal liver can differentiate into immature neuronal cells after induction with β ME and ATRA in vitro .