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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 investigate the effect of antisense oligodeoxy-nucleotides (asODN) on the proliferation of human hepatocellular carcinoma cell line HepG2 induced by polyamidoamine (PAMAM) dendrimer. METHODS: Polyamide dendrimers from 1st to 5th generation were mixed with antisense survivin oligonucleotides at room temperature to prepare a complex of dendrimer and antisense oligonucleotides (PAMAM-asODN). The morphological structure of the complex was observed by gel electrophoresis and atomic force microscopy. The PAMAM-asODN complex was transfected into HepG2 cells, and the survivin antisense oligonucleotide transfected cells were used as controls. Confocal fluorescence microscopy was used to detect the transfection efficiency of the complex cells; the expression of survivin mRNA was detected by RT-PCR; the inhibitory effect of the complex on the proliferation of HepG2 cells was detected by MTT assay. Results: Agarose gel electrophoresis analysis showed that the dendrimer and survivin antisense oligodeoxynucleotides had high efficiency complexation and formed a complex size of about 25 nm. Confocal fluorescence microscopy showed that compared with the control, The efficiency of transfected cells with PAMAM-asODN complexes was significantly improved; the results of RT-PCR showed that the expression of survivin mRNA was significantly decreased in tumor cells transfected with PAMAM-asODN complex; MTT results showed that dendritic molecules delivered survivin antisense oligonucleotides After glucuronide enters the cells, the proliferation of HepG2 cells is significantly inhibited, and the inhibition rate of proliferation increases with the increase of culture time, complex concentration, and number of dendrograms. The inhibition of 6.0 μmol/L G4.0 PAMAM-asODN and 96 h can inhibit the proliferation of HepG2 cells. The rate of more than 55%. Conclusion: PAMAM dendrimer can efficiently deliver survivin asODN into cells and inhibit the proliferation of HepG2 tumor cells. The dendrimer may be a highly efficient gene drug delivery vehicle and has potential application value in the treatment of tumors.