中华实用儿科临床杂志
中華實用兒科臨床雜誌
중화실용인과림상잡지
Journal of Applied Clinical Pediatrics
2015年
15期
1152-1156
,共5页
邵静波%吴伟%李玲玲%吕志葆
邵靜波%吳偉%李玲玲%呂誌葆
소정파%오위%리령령%려지보
神经母细胞瘤%诱导分化%13-顺式维甲酸%MYCN基因
神經母細胞瘤%誘導分化%13-順式維甲痠%MYCN基因
신경모세포류%유도분화%13-순식유갑산%MYCN기인
Neuroblastoma%Differentiation%13-cis retinoic acid%MYCN gene
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.
目的 探討13-順式維甲痠(13-cis RA)體外對3種人神經母細胞瘤(NB)細胞株生長的影響及其作用機製.方法 常規培養SH-SY5Y、SK-N-SH和SK-N-BE2細胞株,熒光原位雜交技術(FISH)檢測3種細胞MYCN基因擴增情況.不同濃度13-cis RA作用後,採用相差顯微鏡觀察細胞形態變化,酶聯免疫吸附法測定細胞培養液神經元特異性烯醇化酶(NSE),細胞增殖毒性檢測法檢測細胞增殖,流式細胞儀檢測細胞凋亡率.結果 13-cis RA作用後,3種細胞均伸齣多箇軸突樹突狀突起,呈極性化改變,齣現細胞分化.FISH檢測結果顯示,SK-N-BE2細胞為MYCN擴增暘性,13-cis RA作用後MYCN仍擴增暘性,另2種細胞株未見擴增.3種細胞經不同濃度13-cis RA作用後NSE水平隨藥物作用時間延長而升高,其中SK-N-BE2細胞NSE水平顯著升高,差異均有統計學意義(F =27.00,P＜0.000 1).13-cis RA作用48 h內均促進細胞增殖,隨後轉為抑製細胞生長.SH-SY5Y細胞經10 μmol/L 13-cis RA作用96 h及120 h顯著增加細胞凋亡(F=16.21,P=0.011;F=16.04,P=0.016);SK-N-SH細胞經1μmol/L及10 μmol/L 13-cis RA作用48 h,細胞凋亡均顯著增加(F=15.05,P=0.012;F=31.18,P=0.005);SK-N-BE2細胞經不同濃度13-cis RA(1 μmoL/L、5μmol/L、10 μmol/L)作用120 h細胞凋亡均顯著增加(F=9.05,P=0.030;F=11.38,P=0.028;F=7.88,P=O.041).結論 13-cis RA體外能顯著誘導NB細胞分化,作用48 h內促進細胞增殖,隨後錶現為抑製細胞生長,促進細胞凋亡. 13-cis RA對MYCN擴增暘性的細胞在DNA檢測水平無改善,提示可能需要聯閤用藥.
목적 탐토13-순식유갑산(13-cis RA)체외대3충인신경모세포류(NB)세포주생장적영향급기작용궤제.방법 상규배양SH-SY5Y、SK-N-SH화SK-N-BE2세포주,형광원위잡교기술(FISH)검측3충세포MYCN기인확증정황.불동농도13-cis RA작용후,채용상차현미경관찰세포형태변화,매련면역흡부법측정세포배양액신경원특이성희순화매(NSE),세포증식독성검측법검측세포증식,류식세포의검측세포조망솔.결과 13-cis RA작용후,3충세포균신출다개축돌수돌상돌기,정겁성화개변,출현세포분화.FISH검측결과현시,SK-N-BE2세포위MYCN확증양성,13-cis RA작용후MYCN잉확증양성,령2충세포주미견확증.3충세포경불동농도13-cis RA작용후NSE수평수약물작용시간연장이승고,기중SK-N-BE2세포NSE수평현저승고,차이균유통계학의의(F =27.00,P＜0.000 1).13-cis RA작용48 h내균촉진세포증식,수후전위억제세포생장.SH-SY5Y세포경10 μmol/L 13-cis RA작용96 h급120 h현저증가세포조망(F=16.21,P=0.011;F=16.04,P=0.016);SK-N-SH세포경1μmol/L급10 μmol/L 13-cis RA작용48 h,세포조망균현저증가(F=15.05,P=0.012;F=31.18,P=0.005);SK-N-BE2세포경불동농도13-cis RA(1 μmoL/L、5μmol/L、10 μmol/L)작용120 h세포조망균현저증가(F=9.05,P=0.030;F=11.38,P=0.028;F=7.88,P=O.041).결론 13-cis RA체외능현저유도NB세포분화,작용48 h내촉진세포증식,수후표현위억제세포생장,촉진세포조망. 13-cis RA대MYCN확증양성적세포재DNA검측수평무개선,제시가능수요연합용약.
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.
