摘要
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、BcbxL、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±0．12VS．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±0．23VS．1．32±0．15，P〈0．05），Bid表达增高（1．53±0．07VS．0．69±0．04，P〈O．05），Bcl-2表达降低（1．66±0．09VS．1．97±0．11，P〈0．05）、Bcl-xL表达降低（1．57±0．08VS．1．93±0．12，P〈0．05），Bak（1．51±0．07VS．1．53±0．08）、Bax（1．35±0．06VS．1．32±0．06）表达无变化（P〉0．05）。MYCN表达增强24h后，长春新碱（19．55±1．99VS．18．42±1．98）、阿霉素（11．65±0．93VS．6．22±0．43）、顺铂（8．70±0．84VS．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。
Objective To investigate the impact of MYCN expression on apoptosis and ehemo- sensitivity 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,vincris- tine, 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 trans- fection of pcDNA3.1 （ ＋ ）-MYCN could increase the expression of MYCN gene; the DNA fragmenta tion 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. l. 93 ± 0. 12,P%O. 05 ） was de creased,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 ap- optosis 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.
出处
《中华小儿外科杂志》
CSCD
北大核心
2012年第10期778-781,共4页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金青年科学基金项目（30801200）
