中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2012年
10期
778-781
,共4页
吕凡%邬文杰%张弛%严文波%吴晔明
呂凡%鄔文傑%張弛%嚴文波%吳曄明
려범%오문걸%장이%엄문파%오엽명
神经母细胞瘤%基因,肿瘤%细胞凋亡
神經母細胞瘤%基因,腫瘤%細胞凋亡
신경모세포류%기인,종류%세포조망
Neuroblastoma%Gene,neoplasm%Apoptosis
目的 以MYCN基因正常扩增神经母细胞瘤细胞株为研究对象,增强其MYCN基因的表达,观察其后细胞凋亡改变,及对常用化疗药物敏感性的影响,为临床治疗神经母细胞瘤开辟新思路.方法 克隆MYCN基因,构建MYCN高表达载体；转染神经母细胞瘤细胞株SK-N-SH,增强MYCN表达后,使用ELISA法检测细胞凋亡情况；Western-blot法检测Bcl-2、Bcl-xL、Bak、Bax、Bid 表达变化；ELISA法检验MYCN表达增强后长春新碱、阿霉素、顺铂对肿瘤细胞凋亡的诱导情况.结果 构建pcDNA3.1(+)-MYCN载体可表达N-Myc蛋白.Western-blot显示被转染SK-N-SH 细胞中MYCN基因表达与对照组比较显著强化(1.26±0.12 vs.0.58±0.06,P＜0.05).表达增强后与对照组比较肿瘤细胞凋亡增加(2.11±0.23 vs.1.32±0.15,P＜0.05),Bid表达增高(1.53±0.07 vs.0.69±0.04,P＜0.05),Bcl-2表达降低(1.66±0.09 vs.1.97±0.11,P＜0.05)、Bcl-xL表达降低(1.57±0.08 vs.1.93±0.12,P＜0.05),Bak (1.51±0.07 vs.1.53±0.08)、Bax( 1.35±0.06vs.1.32±0.06)表达尢变化(P＞0.05).MYCN表达增强24 h后,长春新碱(19.55±1.99 vs.18.42±1.98)、阿霉素(11.65±0.93 vs.6.22±0.43)、顺铂(8.70±0.84 vs.6.83±0.65)诱发肿瘤细胞凋亡显著增加(P＜0.05).结论 外源性增强神经母细胞瘤细胞株SK-N-SH中MYCN基因的表达引起肿瘤细胞凋亡增强；对阿霉素,长春新碱,顺铂的敏感性增强；MYCN表达增强可能是通过抑制Bcl-2,Bcl-xL,增强Bid表达促进肿瘤细胞凋亡.
目的 以MYCN基因正常擴增神經母細胞瘤細胞株為研究對象,增彊其MYCN基因的錶達,觀察其後細胞凋亡改變,及對常用化療藥物敏感性的影響,為臨床治療神經母細胞瘤開闢新思路.方法 剋隆MYCN基因,構建MYCN高錶達載體；轉染神經母細胞瘤細胞株SK-N-SH,增彊MYCN錶達後,使用ELISA法檢測細胞凋亡情況；Western-blot法檢測Bcl-2、Bcl-xL、Bak、Bax、Bid 錶達變化；ELISA法檢驗MYCN錶達增彊後長春新堿、阿黴素、順鉑對腫瘤細胞凋亡的誘導情況.結果 構建pcDNA3.1(+)-MYCN載體可錶達N-Myc蛋白.Western-blot顯示被轉染SK-N-SH 細胞中MYCN基因錶達與對照組比較顯著彊化(1.26±0.12 vs.0.58±0.06,P＜0.05).錶達增彊後與對照組比較腫瘤細胞凋亡增加(2.11±0.23 vs.1.32±0.15,P＜0.05),Bid錶達增高(1.53±0.07 vs.0.69±0.04,P＜0.05),Bcl-2錶達降低(1.66±0.09 vs.1.97±0.11,P＜0.05)、Bcl-xL錶達降低(1.57±0.08 vs.1.93±0.12,P＜0.05),Bak (1.51±0.07 vs.1.53±0.08)、Bax( 1.35±0.06vs.1.32±0.06)錶達尢變化(P＞0.05).MYCN錶達增彊24 h後,長春新堿(19.55±1.99 vs.18.42±1.98)、阿黴素(11.65±0.93 vs.6.22±0.43)、順鉑(8.70±0.84 vs.6.83±0.65)誘髮腫瘤細胞凋亡顯著增加(P＜0.05).結論 外源性增彊神經母細胞瘤細胞株SK-N-SH中MYCN基因的錶達引起腫瘤細胞凋亡增彊；對阿黴素,長春新堿,順鉑的敏感性增彊；MYCN錶達增彊可能是通過抑製Bcl-2,Bcl-xL,增彊Bid錶達促進腫瘤細胞凋亡.
목적 이MYCN기인정상확증신경모세포류세포주위연구대상,증강기MYCN기인적표체,관찰기후세포조망개변,급대상용화료약물민감성적영향,위림상치료신경모세포류개벽신사로.방법 극륭MYCN기인,구건MYCN고표체재체；전염신경모세포류세포주SK-N-SH,증강MYCN표체후,사용ELISA법검측세포조망정황；Western-blot법검측Bcl-2、Bcl-xL、Bak、Bax、Bid 표체변화；ELISA법검험MYCN표체증강후장춘신감、아매소、순박대종류세포조망적유도정황.결과 구건pcDNA3.1(+)-MYCN재체가표체N-Myc단백.Western-blot현시피전염SK-N-SH 세포중MYCN기인표체여대조조비교현저강화(1.26±0.12 vs.0.58±0.06,P＜0.05).표체증강후여대조조비교종류세포조망증가(2.11±0.23 vs.1.32±0.15,P＜0.05),Bid표체증고(1.53±0.07 vs.0.69±0.04,P＜0.05),Bcl-2표체강저(1.66±0.09 vs.1.97±0.11,P＜0.05)、Bcl-xL표체강저(1.57±0.08 vs.1.93±0.12,P＜0.05),Bak (1.51±0.07 vs.1.53±0.08)、Bax( 1.35±0.06vs.1.32±0.06)표체왕변화(P＞0.05).MYCN표체증강24 h후,장춘신감(19.55±1.99 vs.18.42±1.98)、아매소(11.65±0.93 vs.6.22±0.43)、순박(8.70±0.84 vs.6.83±0.65)유발종류세포조망현저증가(P＜0.05).결론 외원성증강신경모세포류세포주SK-N-SH중MYCN기인적표체인기종류세포조망증강；대아매소,장춘신감,순박적민감성증강；MYCN표체증강가능시통과억제Bcl-2,Bcl-xL,증강Bid표체촉진종류세포조망.
Objective To investigate the impact of MYCN expression on apoptosis and chemosensitivity of neuroblastoma cells.Methods MYCN gene was cloned using RT-PCR and pcDNA3.1 ( + )-MYCN was constructed via biological delivery system.Then the vector pcDNA3.1 ( + )-MYCN was transfected into BMMSC SK-N-SH cells with the method of liposome mediated.Western blotting was conducted to investigate the expressions of Bcl-2,Bcl-xL,Bak,Bax,and Bid.Furthermore,vincristine,cisplatin and doxorubicine were added to the transfected cells.Apoptosis was measured by ELISA assay.Results MYCN gene was successfully cloned and inserted into the pcDNA3.1 ( + ).The transfection of pcDNA3.1 ( + )-MYCN could increase the expression of MYCN gene; the DNA fragmentation of transfected cells was higher than that of control group(2.11±0.23 vs.1.32±0.15,P＜0.05 ) ;The expression of Bid (1.53±0.07 vs.0.69±0.04,P＜0.05 were increased,the expression of Bcl-2(1.66±0.09 vs.1.97±0.11,P＜0.05 ) and Bcl-xL( 1.57±0.08 vs.1.93±0.12,P＜0.05 ) was decreased,while the expression of Bax( 1.35±0.06 vs.1.32±0.06)and Bak (1.51±0.07 vs.1.53±0.08)had no significant change.The Increased expression of MYCN gene could also increased the apoptosis induced by vincristine(19.55±1.99 vs.18.42±1.98,P＜0.05 ),cisplatin(8.70±0.84 vs.6.83±0.65,P＜0.05 ) and doxorubicine ( 11.65±0.93 vs.6.22±0.43,P＜0.05 ) compared with control groups.Conclusions The increased MYCN gene expression induces apoptosis and increases chemo-sensitivity of neuroblastoma cells.
