应用两种方法诱导SH-SY5Y细胞分化的研究

The Study of Differentiation of SH-SY5Y Cells Induced by Two Methods

目的:观察全反式维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法:应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果:ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论:维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。

Objective： To investigate the effects of All trans-retinoic acid（ATRA） and ATRA followed by phorbol ester 12-o-te-tradecanoylphorbol-13-acetate（TPA）（ATRA/TPA） on growth and morphological differentiation of human neuroblastoma SH-SY5Y cell line.Methods： SH-SY5Y cells were differentiated with 10 μM ATRA for 6 days and 10 μM ATRA for 3 days followed by 80 nM TPA for another 3 days（RA/TPA）.Morphological differentiation was detected by using the inverse microscope.And MTT assay was used to detect the anti-proliferative effect of ATRA and ATRA/TPA on SH-SY5Y cells.Resulls： Both ATRA and ATRA/TPA could induce mor-phological differentiation and inhibit growth of SH-SY5Y cells.Differentiated cells underwent striking morphological changes and result-ed in neuronal phenotype.ATRA-differentiated SH-SY5Y cells exhibited a spindle-shaped morphology with elongated processes extend-ing from opposing ends.Whereas ATRA/TPA-differentiated SH-SY5Y cells showed polygon-shaped morphology with numerous elon-gated processes extending from cell body.The cell viability decreased to 78.7%±2% when exposured to ATRA at the concentration of 10μmol/ L for 6 days.After the removal of ATRA,cell viability increased to 89%±0.2% and 86.3%±1.4%,respectively,when cul-tured for another one day and two days.The cell viability decreased to 75.9±0.4% when differentiated with 10 μM ATRA for 3 days followed by 80 nM TPA for another 3 days.The cell viability was 75.5±0.7% and 74.9±1%,respectively,when SH-SY5Y cells were stoped treating with ATRA/TPA and cultured for another one day and two days.Conclusion： ATRA and ATRA/TPA induced SH-SY5Y cell differentiation.The two kind of differentiated SH-SY5Y cell provided excellent in vitro cell model for neurological studies,especial-ly RA/TPA differentiated SH-SY5Y cells.