13-顺式维甲酸体外诱导3种神经母细胞瘤细胞株分化研究

Study on 13 - cis retinoic acid inducing differentiation of three types of human neuroblastonm cell lines in vitro

目的探讨13-顺式维甲酸（13-cisRA）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-cisRA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-cisRA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-cisRA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．cisRA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〈0．0001）。13-cisRA作用48h内均促进细胞增殖，随后转为抑制细胞生长。SH-SY5Y细胞经10μmol／L13-cisfLA作用96h及120h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10μmol／L13-cisRA作用48h，细胞惆亡均显著增加（F=15．05，P=0．012；F=31．18，P=0．005）；SK-N-BE2细胞经不同浓度13-cisRA（1μmol／L、5μmol／L、10μmol／L）作用120h细胞啊亡均显著增加（F=9．05，P=0．030；F=11．38，P=0．028；F=7．88，P=0．041）。结论13-cisRA体外能显著诱导NB细胞分化，作用48h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-cisRA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。

Objective To investigate the effects of 13 -eis rctinoie acid （13 -cis RA） in inducing differen- tiation 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,morphologieal changes were observed by phase -contrast micro= scope, and neuron - specific enolase （NSE） concentrations were determined by enzyme linked immunosorhent assay. The cell viability was measured through cell counting kit -8 assay, and the cell apoptosis was assayed with flow cytome- try （FCM）. Results The morphological changes in differentiation were observed in all 3 types of NB ceils after 13 - eis 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 - SYSY 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 μmol/L and 10 μmol/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 concentra- tions of 13 -cis RA（ 1 μmol/L,5 μmoL/L, 10 μmol/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 cells 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 de- tection of DNA level after 13 -cis RA treatment,which suggests that combined treatment is possibly needed.