摘要
目的探讨肿瘤坏死因子相关凋亡诱导配体基因(TRAIL)和顺铂联合应用对人RD胚胎型横纹肌肉瘤细胞的生长抑制和凋亡的诱导作用。方法将重组腺病毒载体(Ad)介导的TRAIL基因作用于人RD胚胎型横纹肌肉瘤细胞,通过电镜、MTT比色法、RT-PCR及流式细胞仪方法,观察和分析其对RD胚胎型横纹肌肉瘤细胞作用的效果,并与联合应用顺铂的效果进行对比,分析其作用机制。结果MTT法显示顺铂浓度为1.0μg/ml、5.0μg/ml和10.0μg/ml对RD细胞生长的抑制率分别为9.8%、23.4%和43.8%;按MOI值1000转染腺病毒Ad/GT-TRAIL对RD细胞生长的抑制率为41.2%,再联合Ad/PGK-GV16后,生长抑制率上升到52.5%;Ad/GT-TRAIL联合Ad/PGK-GV16再加用顺铂后的生长抑制率为72.7%。顺铂作用3d后的凋亡率为7.41%;Ad/GT-TRAIL联合Ad/PGK-GV16作用3d后的凋亡率为12.95%;Ad/GT-TRAIL联合Ad/PGK-GV16再加用顺铂作用3d后的凋亡率为23.24%,而1640对照组的凋亡率为2.19%。顺铂作用后,细胞的cFLIPmRNA表达下调,与细胞凋亡增加的改变相一致。结论Ad/GT-TRAIL能有效诱导横纹肌肉瘤细胞的凋亡,从而抑制横纹肌肉瘤细胞的生长,联合应用顺铂后能显著提高疗效。
Objective To study the synergistic induction of apoptosis by the combination of TNF-related apoptosis-inducing ligand (TRAIL) gene and cisplatin in rhabdomyosarcoma cell line. Methods Human rhabdomyosarcoma cells were transfected with adenovirus-mediated TRAIL gene (Ad/GT-TRAIL). The morphological changes, cell growth and apoptosis were measured by electron microscope, MTT method and flow cytometry respectively. The expression of cFLIP mRNA was shown by RT-PCR. Results Obvious apoptosis morphological changes in rhabdomyosarcoma cells were induced by Ad/GT-TRAIL and Ad/PGK-GVl6. The suppression percentage of cisplatin on rhabdomyosarcoma cell at 1.0 μg/ml, 5.0 μg/ml and 10.0 μg/ml was 9.8%, 23.4% and 43.8% respectively, however, the percentage of Ad/GT-TRAIL was 41.2%, and the percentage of Ad/GT-TRAIL combined with Ad/PGK-GVl6 and cispltin increased to 72.7%. The apoptotic rate of cispltin at 5.0 μg/ml was 7.41%, the rate of Ad/GT-TRAIL combined with Ad/PGK-GVl6 was 12.9%, and the rate of combination of cispltin, Ad/GT-TRAIL and Ad/PGK-GVl6 was 23.24%. The expression of cFLIP was down regulated with cisplatin, which is paralleled by the apoptotic rates. Moreover, the expression of cFLIP decreased more significantly when the cells were cultured with cispltin and TRAIL. The obvious apoptosis morphological changes, which were shrinking cellular morphology, chromatin condensation, cytoplasmic vacuoles in rhabdomyosarcoma cells, were shown by electron microscope when the cells were co-cultured with cispltin, Ad/GT-TRAIL and Ad/PGK-GVl6. Conclusion Ad/GT-TRAIL is able to induce apoptosis of rhabdomyosarcoma cells and inhibit its growth. TRAIL gene and cisplatin have synergistic effect on rhabdomyosarcoma cells. This is a potential and valuable proposal for clinical treatment of rhabdomyosacoma.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2004年第7期422-426,共5页
Chinese Journal of Orthopaedics
关键词
横纹肌肉瘤
肿瘤坏死因子
基因抑制
肿瘤
Rhabdomyosarcoma
Tumor necrosis factor
Genes, suppressor, tumor