摘要
目的:研究利用聚酰胺(polyamidoamine,PAMAM)树形分子递送survivin基因反义寡核苷酸(antisense oligodeoxy- nucleotide,asODN)抑制人肝癌细胞株HepG2细胞增殖的效应。方法:采用第1至第5代的聚酰胺树形分子室温下与反义survivin寡核苷酸混合制备树形分子与反义寡核苷酸的复合物(PAMAM-asODN),应用琼脂糖凝胶电泳及原子力学显微镜观察复合物的形态结构。PAMAM-asODN复合物转染HepG2细胞,同时设survivin反义寡核苷酸转染细胞作对照。共聚焦荧光显微镜检测复合物细胞转染效果;RT-PCR分析转染后细胞survivin mRNA的表达水平;MTT法检测复合物对HepG2细胞增殖的抑制效应。结果:琼脂糖凝胶电泳分析表明,树形分子与survivin反义寡核苷酸具有高效络合作用,形成了大小为25 nm左右的复合物;共聚焦荧光显微镜检测显示,与对照相比,PAMAM-asODN复合物转染细胞的效率显著提高;RT-PCR的结果表明,转染PAMAM-asODN复合物的肿瘤细胞中survivin mRNA表达显著降低;MTT结果表明,树形分子递送survivin反义寡核苷酸进入细胞后,HepG2细胞增殖明显受抑制,增殖抑制率随培养时间、复合物浓度、树形分子代数的增加而增加,6.0μmol/L G4.0 PAMAM-asODN与细胞培养96h可使抑制率达55%以上。结论:PAMAM树形分子能高效递送survivin asODN进入细胞,并抑制HepG2肿瘤细胞的增殖。树形分子可能是一种高效基因药物递送载体,在肿瘤治疗中具有潜在应用价值。
Objective： To use polyamidoamine （PAMAM） dendrimer as gene delivery system for survivin gene antisense oligodeoxynucleotide （asODN） transfection for inhibition of HepG2 cancer cell growth. Methods： The first to the fifth generation of PAMAM and asODN were used to prepare a complex： PAMAM-asODN. The morphology of PAMAM- asODN was observed using agrose electrophoresis and atomic force microscope （AFM）. PAMAM-asODN was then used to transfect HepG2 cells and cells transfected with asODN served as control. The transfection efficacy of PAMAM-asODN into HepG2 cells was observed under confocol microscope, the surviving mRNA expression was analyzed by RT-PCR, and the inhibition of HepG2 cell growth was determined by MTT assay. Results： Agrose electrophoresis showed strong complexing action between PAMAM and asODN and they formed a complex with a diameter of 25 nm. Confocol microscope showed the transfection efficacy of PAMAM-asODN was higher than that of asODN. RT-PCR showed a decreased expression of survivin mRNA in PAMAM-asODN transfected cells. MTT results demonstrated that the growth of HepG2 cell was obviously inhibited after transfection of PAMAM-asODN and the inhibition rate increased with culture time, concentration of complex, the generation of PAMAM. PAMAM-asODN at 6.0 μmoL/L G4.0 resulted in a 55% inhibition of HepG2 cells 96 h after culture. Conclusion： PAMAM dendrimers can efficiently mediate the entry of survivin asODN into HepG2 cells, resuiting in inhibition of HepG2 cells. PAMAM might be a promising gene carrier for potential molecular therapy of cancer.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2007年第5期411-416,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金(No.30471599)
国家重点基础研究发展(973)计划资助项目(No.2005CB724300-G)
上海科技发展基金(No.03ZR14057)
Supported by the National Natural Science Foundation of China(No.30471599)
the Major State Basic Research Development(973)Program of China(No.2005CB724300-G)(03DZ14025)
the Science and Technology Development Program of Shanghai(No.03ZR14057)
