Babuška V, Kulda V, Houdek Z, Pešta M, Cendelín J, Zech N, Pacherník J, Vožeh F, Uher P, Králíčková M
Charles University in Prague, Faculty of Medicine in Plzeň, Department of Medical Chemistry and Biochemistry, Plzeň, Czech Republic.
Prague Med Rep. 2010;111(4):289-99.
The aim of our study was to characterize mouse embryonal carcinoma (EC) cells P19 in different stages of retinoic acid induced neurodifferentiation by two methods, immunocytochemistry and RT qPCR. The characterization of the cells is crucial before any transplantation into any model, e.g. in our case into the mouse brain with the aim to treat a neurodegenerative disease. Specific protein markers (MAP-2, OCT-4, FORSE-1) were detected by immunocytochemistry in the cell cultures. The mRNA expression levels of PAX-6, MASH-1, Brachyury, GATA-4 and AFP were determined by RT qPCR method. HPRT was used as a housekeeping gene. The degree of differentiation can be characterized by expression of analyzed genes. The presence of OCT-4 and FORSE-1 proteins in undifferentiated pluripotent cells and the presence of dendrite specific MAP-2 in neuroprogenitors was detected. The expression levels of PAX-6 and MASH-1 increased and expression of Brachyury decreased during the neurodifferentiation process. The expression levels of GATA-4 and AFP were the highest after induction of differentiation with retinoic acid. Detailed characterization of cells before transplantation experiments can contribute to better understanding of their effect.
我们研究的目的是通过免疫细胞化学和实时定量聚合酶链反应（RT qPCR）这两种方法，对维甲酸诱导神经分化不同阶段的小鼠胚胎癌细胞P19进行表征。在将任何细胞移植到任何模型中之前，例如在我们的案例中移植到小鼠大脑以治疗神经退行性疾病之前，对细胞进行表征至关重要。通过免疫细胞化学在细胞培养物中检测特定的蛋白质标志物（微管相关蛋白2（MAP-2）、八聚体结合转录因子4（OCT-4）、叉头框E1（FORSE-1））。采用RT qPCR方法测定配对盒基因6（PAX-6）、肌萎缩侧索硬化症相关蛋白1（MASH-1）、短尾相关蛋白（Brachyury）、GATA结合蛋白4（GATA-4）和甲胎蛋白（AFP）的mRNA表达水平。次黄嘌呤磷酸核糖转移酶（HPRT）用作看家基因。分化程度可以通过所分析基因的表达来表征。在未分化的多能细胞中检测到OCT-4和FORSE-1蛋白的存在，在神经祖细胞中检测到树突特异性MAP-2的存在。在神经分化过程中，PAX-6和MASH-1的表达水平升高，Brachyury的表达水平降低。在用维甲酸诱导分化后，GATA-4和AFP的表达水平最高。在移植实验前对细胞进行详细表征有助于更好地了解它们的作用。
