Objective To investigate the effects of 13-cis retinoic acid (13-cis RA) in inducing differentiation of 3 types of human neuroblastoma (NB) cells in vitro.Methods The status of MYCN gene amplification of cultured SH-SY5Y,SK-N-SH and SK-N-BE2 cells was detected by fluorescence in situ hybridization.After treatment with different concentrations of 13-cis RA,morphological changes were observed by phase-contrast microscope,and neuron-specific enolase (NSE) concentrations were determined by enzyme linked immunosorbent assay.The cell viability was measured through cell counting kit-8 assay,and the cell apoptosis was assayed with flow cytometry (FCM).Results The morphological changes in differentiation were observed in all 3 types of NB cells after 13-cis RA treatment.MYCN amplification was detected in SK-N-BE2 cells even after 13-cis RA treatment,while the other 2 cell lines were amplification-null.After different concentrations of 13-cis RA treatment,NSE concentration increased with prolonged time,especially for SK-N-BE2 cell(F =27.00,P ＜ 0.000 1).13-cis RA stimulated cell proliferation within 48 hours of treatment,and then inhibited cell growth.FCM indicated that the degree of apoptosis in SH-SY5Y cell was significantly higher after 13-cis RA treatment of 10 μmol/L concentration for continuous 96 h and 120 h as compared to the control group (F =16.21,P =0.011;F =16.04,P =0.016).Cell apoptosis of SK-N-SH cell after 13-cis RA treatment of 1 μ mol/L and 10 μ mol/L concentration for 48 h,were significantly higher than those of the control group (F =15.05,P =0.012;F =31.18,P =0.005);while SK-N-BE2 cell with different concentrations of 13-cis RA(1 μmol/L,5 μmol/L,10 μ mol/L) for 120 h were significantly higher than those of the control group(F =9.05,P =0.030;F =11.38,P =0.028;F =7.88,P =0.041).Conclusions The present study showed that 13-cis RA could induce differentiation of human NB ceils in vitro.It induces cell proliferation within 48 hours of 13-cis RA,and thereafter suppresses cell growth.No improvement was found in MYCN amplification cells with the detection of DNA level after 13-cis RA treatment,which suggests that combined treatment is possibly needed.%目的 探讨13-顺式维甲酸(13-cis RA)体外对3种人神经母细胞瘤(NB)细胞株生长的影响及其作用机制.方法 常规培养SH-SY5Y、SK-N-SH和SK-N-BE2细胞株,荧光原位杂交技术(FISH)检测3种细胞MYCN基因扩增情况.不同浓度13-cis RA作用后,采用相差显微镜观察细胞形态变化,酶联免疫吸附法测定细胞培养液神经元特异性烯醇化酶(NSE),细胞增殖毒性检测法检测细胞增殖,流式细胞仪检测细胞凋亡率.结果 13-cis RA作用后,3种细胞均伸出多个轴突树突状突起,呈极性化改变,出现细胞分化.FISH检测结果显示,SK-N-BE2细胞为MYCN扩增阳性,13-cis RA作用后MYCN仍扩增阳性,另2种细胞株未见扩增.3种细胞经不同浓度13-cis RA作用后NSE水平随药物作用时间延长而升高,其中SK-N-BE2细胞NSE水平显著升高,差异均有统计学意义(F =27.00,P＜0.000 1).13-cis RA作用48 h内均促进细胞增殖,随后转为抑制细胞生长.SH-SY5Y细胞经10 μmol/L 13-cis RA作用96 h及120 h显著增加细胞凋亡(F=16.21,P=0.011;F=16.04,P=0.016);SK-N-SH细胞经1μmol/L及10 μmol/L 13-cis RA作用48 h,细胞凋亡均显著增加(F=15.05,P=0.012;F=31.18,P=0.005);SK-N-BE2细胞经不同浓度13-cis RA(1 μmoL/L、5μmol/L、10 μmol/L)作用120 h细胞凋亡均显著增加(F=9.05,P=0.030;F=11.38,P=0.028;F=7.88,P=O.041).结论 13-cis RA体外能显著诱导NB细胞分化,作用48 h内促进细胞增殖,随后表现为抑制细胞生长,促进细胞凋亡. 13-cis RA对MYCN扩增阳性的细胞在DNA检测水平无改善,提示可能需要联合用药.
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