全反式维甲酸诱导神经母细胞瘤分化及TrkA剪接异构体表达的研究

目的：通过对NB细胞系SH-SY5Y进行体外实验,研究ATRA对NB细胞的增殖抑制与诱导分化作用,进而探讨TrkA剪接异构体在不同分化程度的SH-SY5Y细胞中的表达情况。方法：将SH-SY5Y细胞取对数生长期细胞培养分组,利用台盼蓝拒染计数活细胞,倒置相差显微镜观察细胞形态学变化。采用SYBR GreenⅠ实时荧光定量PCR方法检测TrkA三种剪接异构体的表达水平。结果：对照组活细胞数随着培养时间延长而增多,实验组中ATRA对SH-SY5Y的生长抑制作用在（0.1-10）μmol/L浓度范围内有剂量依赖关系。不同浓度的ATRA均可诱导SH-SY5Y细胞产生形态学上的变化,对照组细胞分化百分率随时间延长无明显变化,各时间段间比较,差异不显著（F=2.889,P=0.079）。TrkAⅠ/ⅡmRNA表达水平增加与ATRA浓度及作用时间呈正相关。TrkAⅢmRNA表达与ATRA浓度及作用时间呈负相关。结论：在SH-SY5Y细胞中,ATRA对细胞生长抑制作用呈明显的时间和剂量依赖关系;TrkAⅠ/Ⅱ表达与ATRA作用时间、剂量呈正相关;随着ATRA作用浓度及作用时间的增加,TrkAⅢmRNA表达降低。

Study on ATRA-induced differentiation of neuroblastoma and the expression of TrkA splicing isoforms

Objective：Human SH-SY5Y NB cells response to all-trans-retinonic acid（ATRA）by growth inhibition and cellular differentiation would be studied in vitro,and alternative splicing isoforms of TrkA expression in different differentiation phase of SH-SY5Y cell would be evaluated.Methods： After 24h cultured,logarithmic growth phase of SH-SY5Y cells were divided into experimental and control group,the experimental group was treated with 0.1,1.0 and 10μmol /L ATRA,the control group treated without any drugs.Using trypan blue dye counting living cells on the 1,3,5 and 7 d of the cell culture,morphological changes were observed with inverted phase contrast microscope.Extracted total RNA,reaction system was disposed as 20μl to synthesize cDNA;primers were designed and synthesized by Takara company,SYBR GreenⅠ fluorescent real-time quantitative PCR method was applied to detect expression level of TrkAⅠ/Ⅱand TrkAⅢ.Results： In control group,the number of viable cells increased as the incubation time,different groups with different concentrations of ATRA could inhibit the proliferation of SH-SY5Y cells.The ATRA-induced growth inhibition to SH-SY5Y cells had dose-dependent relationship in（0.1-10）μmol/L concentration range.Different concentrations of ATRA could induce the SH-SY5Y cells morphological changes,the percentage of cells in control group was no significant change with time,there was no significant difference between different time（F=2.889,P=0.079）.Real-time quantitative PCR results showed that TrkAⅠ/Ⅱ mRNA expression increased with ATRA concentration and time positively.TrkA Ⅲ mRNA expression increased with ATRA concentration and time negatively.Conclusion： ATRA on growth inhibition of SH-SY5Y cells showed a time and dose dependent,1.0μmol/L ATRA have significant effects on morphology,with the time past,the cells showed significant differentiation characteristics.Increase the concentration of ATRA,the function of cell growth inhibition and induction of differentiation was no significant difference;in SH-SY5Y cells,TrkAⅠ/Ⅱ expression had a positive correlation with the ATRA acting time and dose;with the role of concentration of ATRA and time increase,TrkA Ⅲ mRNA expression decreased and showed a certain correlation.